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007afdec-bed4-405d-873d-c355ba9add0e.267	2.5.1. Proximal Analyses, Physicochemical Analysis and Color Proximate composition (moisture, ash, protein, and fat) was evaluated following the official methods AOAC 19,927.05, AOAC 945.46, AOAC 991.22, and AOAC 905.02, respectively [27]. Carbohydrate content was estimated by difference. Protein content was estimated by nitrogen determination using the factor of 6.25. The pH was measured by a digital pH-meter (Methrom 827, Herisau, Switzerland), previously calibrated, at room temperature. The titratable acidity (TA) of the yogurts was determined by titrating 9 g of the sample with 0.1 M NaOH using phenolphthalein as an indicator, which was expressed as % lactic acid. Both determinations were performed in triplicate. The color of the yogurts was measured in a Konica Minolta CM-3500d spectrophotometer (Konica Minolta Sensing, Inc., Osaka, Japan) from ten measurements of each sample, and the CIELAB color space was used to obtain the color coordinates. The color was expressed by the parameters L\, a\ and b\*. The whiteness index (WI) was determined according to Equation (1): $$\text{WI} = 100 - \sqrt{\left(100 - \text{L}\right)^2 + \text{a}^2 + \text{b}^2} \tag{1}$$	doab	2025-04-07T03:56:59.224294	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 267 }
007afdec-bed4-405d-873d-c355ba9add0e.268	2.5.2. Syneresis The syneresis of the yogurts was determined according to Santillán-Urquiza et al. [28]. Approximately 10 g of yogurt was centrifuged at 176× g for 20 min at 10 ◦C. The syneresis, expressed as percentage, was performed in triplicate and estimated as the weight of the supernatant released over the weight of the initial yogurt × 100.	doab	2025-04-07T03:56:59.224391	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 268 }
007afdec-bed4-405d-873d-c355ba9add0e.269	2.5.3. Texture Analysis Firmness and cohesiveness parameters were determined using a texture analyzer (TA.XTplus, Stable Micro Systems) as described by Santillán-Urquiza et al. [28], with modifications. Briefly, the compression force (N) in 50 mL of yogurt was measured using a 36 mm diameter × 50 mm high cylindrical body (P36R), with a speed of 0.5 mm/s and reaching a depth of 20 mm. Three different yogurt cups were measured for each lot and the firmness results were expressed in N.	doab	2025-04-07T03:56:59.224433	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 269 }
007afdec-bed4-405d-873d-c355ba9add0e.270	2.5.4. Rheological Analyses Oscillatory shear measurements were performed on a Bohlin CVO rheometer (Bohlin Instruments Ltd., Gloucestershire, UK) using a cone-plate geometry (4◦ angle, 40 mm diameter, 0.15 mm gap). The temperature in the lower plate was 5 ◦C. Frequency sweep tests were carried out over a range of angular frequencies between 0.63 and 63 rad/s with an oscillation strain of 5%, selected from the linear viscoelastic region (LVER). The storage modulus (G ) and loss modulus (G) were plotted as a function of angular frequency (ω). To understand the viscoelastic properties of the different yogurts, flow and viscoelastic properties of the additive aqueous suspensions were examined. Thus, three aqueous suspensions at the same concentration (1.5% w/v) were prepared: maltodextrin (MD), free hydrolysate (H), and microencapsulated hydrolysate (HEn). For determining the viscoelastic moduli, time sweeps at 20 ◦C for 1800 s at 0.05 Hz and small stress (σ = 1.5 Pa) were performed to minimize structural changes. Flows were characterized by the step test, with three intervals starting with a preshear interval to homogenize the suspensions (100 s−<sup>1</sup> for 300 s). The three steps were: (1) reference interval (150 s−<sup>1</sup> for 90 s); (2) high shear-rate interval (1000 s−<sup>1</sup> for 45 s) to damage the internal structure; (3) regeneration interval (150 s−1, 600 s) at 20 ◦C. Each rheological test was repeated five times.	doab	2025-04-07T03:56:59.224485	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 270 }
007afdec-bed4-405d-873d-c355ba9add0e.271	2.6. Microbiological Analysis Yogurts were evaluated for microbiological analysis according to the method described by Arancibia et al. [29]. Aliquots of approximately 10.0 ± 0.2 g were weighed and transferred to sterile polyethylene bags (Sterilin, Stone, UK) with 90 mL of 0.1% (w/v) sterile peptone water (Oxoid, Unipath Ltd., Basingstoke, UK) and homogenized for 1 min medium speed in a Stomacher (Colworth 400, Seward, UK) at room temperature. Then, appropriate dilutions were prepared for the following bacteriological determinations: (i) Streptococcus thermophilus, on spread plates of ESTY agar + lactose (0.5%) (Pronadisa, Spain) and (ii) Lactobacillus delbruckii sp. bulgaricus, on spread plates of MRS Agar (Pronadisa, Spain) + tween 80 (0.1%) (Sigma-Aldrich, Darmstadt, Germany) + Cysteine (0.05%) (Sigma-Aldrich), both incubated at 42 ◦C for 24 h in an anaerobic jar (Oxoid), (iii) Enterobacteriaceae, on pour double-layered plates of Violet Red Bile Glucose Agar—VRBG (Oxoid), incubated at 30 ◦C for 48 h, and (iv) molds and yeasts on spread plates of potato dextrose agar (Scharlab, Spain) incubated at 25 ◦C for 72 h. These analyses were carried out on the day the yogurts were prepared to confirm that a suitable product was obtained. All determinations were conducted in duplicate.	doab	2025-04-07T03:56:59.224592	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 271 }
007afdec-bed4-405d-873d-c355ba9add0e.272	2.7. Yogurt Bioactivity For the analysis of antioxidant and antihypertensive activities, the supernatant of yogurt was used, which was prepared according to Zhang et al. [30]. For that, 10 g of yogurt samples were centrifuged at 4330× g for 5 min at 4 ◦C, and then the supernatants were recentrifuged under the same conditions. #### 2.7.1. Antioxidant Activity ABTS Radical Scavenging Activity The 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was determined using the methods detailed by Zheng et al. [31], with modifications. The stock solution of the ABTS radical was generated by incubating 7 mM ABTS with 140 mM potassium persulfate in the dark for 16 h at room temperature. Before use, the stock solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. A 50 μL aliquot of the yogurt supernatant (diluted 1:5 in PBS) was homogenized with 150 μL of ABTS, and after reaction at 30 ◦C for 6 min in the dark, the absorbance was measured at 734 nm. The ABTS radical scavenging activity was calculated as follows: [(Ac − As)/Ac] × 100, where Ac is the absorbance of the control, reagents without sample, and As is absorbance with sample. The determination was carried out in triplicate.	doab	2025-04-07T03:56:59.224687	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 272 }
007afdec-bed4-405d-873d-c355ba9add0e.273	Reducing Power The reducing power was assessed as described by Canabady-Rochelle et al. [32], with modifications. An aliquot of 70 μL of yogurt supernatant (diluted 1:5 in phosphate buffer pH 6.6, 200 mM) was homogenized with 35 μL of potassium ferricyanide solution (1% w/v), and incubated at 50 ◦C for 20 min. Subsequently, 135 μL of distilled water, 33 μL of trichloroacetic acid (10% w/v) and 27 μL of ferric chloride (0.1% w/v) were added and after 10 min, the absorbance was measured at 700 nm. The reducing power of the sample was shown as the absorbance at 700 nm after subtracting the absorbance value from the blank, where a higher absorbance value indicates greater reducing power. The determination was carried out in triplicate.	doab	2025-04-07T03:56:59.224785	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 273 }
007afdec-bed4-405d-873d-c355ba9add0e.274	2.7.2. ACE Inhibitory Activity The ability of the sample to inhibit the angiotensin-converting enzyme (ACE) was determined according to Alemán et al. [33], with some modifications. The reaction was composed of 50 μL of 5 mM Hipuryl-histidyl-leucine (HHL), 80 μL of ACE (0.025 U/mL) and 20 μL of the yogurt supernatant (diluted 1:5 in 100 mM potassium phosphate buffer, containing 300 mM NaCl, pH 8.3). The determination was performed by reverse phase High Performance Liquid Chromatography (RP-HPLC) (model SPE-MA10AVP, Shimadzu, Kyoto, Japan). The injection volume was 50 μL and the flow rate 0.8 mL/ min, using an acetonitrile gradient from 20% to 60% in 0.1% trifluoroacetic acid (TFA) (v/v) for 26 min. The results were expressed as % of ACE-inhibitory activity. #### 2.8. Sensory Analysis The sensory analysis of the formulated yogurts was conducted at the Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC, Madrid, Spain) with 10 semi-trained judges of both sexes, selected from among students and researchers of the Institute. The specific attributes that the panel was asked to classify in the yogurts were the following: color, flavor, odor, texture and acidity. The scale values ranged from 0 to 9. Number 9 was assigned to the most positive terms, while number 0 was assigned to the least desirable attributes/characteristics. Regarding texture and acidity, number 9 was assigned to firm and very acid, respectively. Sensory tests were performed in individual booths with lighting control; samples were served in disposable plastic cups, encoded with 3-digit numbers obtained from a table of random numbers. Mineral water was also provided for cleansing the palate between the evaluations of the different samples of yogurt.	doab	2025-04-07T03:56:59.224842	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 274 }
007afdec-bed4-405d-873d-c355ba9add0e.275	2.9. Statistical Analysis The data were submitted to analysis of variance (ANOVA) and the means compared by the t-Student test (to compare between day 1 and day 7) or the Tukey test with a significance level of 5% using Statistica software (StatSoft, Inc., Tulsa, OK, USA). ## 3. Results and Discussion ## 3.1. Visual Appearance, Proximal Analyses and Physicochemical Properties of Yogurts Figure 1 shows the visual appearance of the different yogurts. To the naked eye, all yogurts presented a similar color, with similar gel-like appearance and no evidence of whey separation. The proximate composition revealed that the yogurts were similar, with significant differences (p < 0.05) mainly in carbohydrate and protein content due to the incorporation of maltodextrin and hydrolysate in some yogurts. Figure 1. Visual appearance and proximate composition of the different yogurts. YC: control yogurt; YMD: yogurt with addition of maltodextrin; YH: yogurt with addition of free protein hydrolysate; YHEn: yogurt with addition of microencapsulated protein hydrolysate. Different letters (a,b,c) indicate significant differences between samples within the same line (p < 0.05). The pH, titratable acidity (TA), color and syneresis of the yogurts after 1 and 7 days of storage under refrigeration, are shown in Table 1. The lowest pH value after 1 and 7 days of storage was observed in the yogurt with the addition of the microencapsulated hydrolysate (YHEn) while the highest pH observed was in the yogurt with the addition of MD (YMD), (p < 0.05). The pH values of MD, the free hydrolysate and microencapsulated hydrolysate were 5.7, 7.03 and 6.91, respectively. Hence, it seems that the pH of the yogurts was probably not affected by the addition of these ingredients. Moreover, a decrease in pH was observed in all yogurts throughout storage, which can be mostly attributed to the production of microbial metabolites. Similarly, a decrease in pH was also observed by Abdel-Hamid et al. [9] when supplementing probiotic yogurt with 1% and 2% monk fruit extract (Siraitia grosvenorii). These authors reported that supplementation with the extract may have stimulated bacterial growth. Notably, fermented products with pH in the range of 4.2 and 4.4 are preferred by consumers [34]. In the present work, these pH ranges were achieved in yogurts after 7 days of storage (Table 1). The titratable acidity of the yogurts varied from 0.73% to 1.09% (Table 1), the YH and YHEn yogurts showing the highest values, both after 1 and 7 days of storage. In addition, with the exception of YC, there was an increase in the titratable acidity with storage time (p < 0.05). Similar results were reported by Córdova-Ramos et al. [35] when studying the addition of jumbo squid powder (Dosidicus gigas) using maltodextrin as an encapsulating agent by spray drying (MD with different DE (11) and drying conditions) in yogurt. The authors verified an increase in acidity as squid powder concentration increased (1%, 3%, 5%, 7% and 10%), ranging from 0.76% to 1.05% in the yogurt without squid powder and with the highest concentration, respectively. An increase in titratable acidity was also observed in yogurts supplemented with pineapple peel powder or inulin after 28 days of storage [36]. According to the authors, the supplementation increased the acidifying capacity of the starter cultures during storage. Color is the first characteristic perceived by consumers, and it can influence their preference, so it is an important attribute to be evaluated [37]. The color parameters (L\, a\ and b\) of the different formulated yogurts are shown in Table 1. A decrease in lightness values (L\) was observed for yogurts with the addition of the different tested ingredients, and therefore, the highest values observed for YC also reflected a higher whiteness index. As also observed by Silva et al. [5] such a phenomenon may have occurred because this batch did not have any powder ingredients. Conversely, on the same evaluation day, the YH and YHEn yogurts tended to show higher values for yellowness (b\) (p* < 0.05), probably due to the color of the hydrolysate. Carmona et al. [38] verified that encapsulating yellow– orange cactus pear Opuntia ficus-indica pulp, using maltodextrin (MD) as an encapsulating agent, protected the quality of the pigment (thus allowing its use as a yellow colorant for yogurt) and that, additionally, maltodextrin did not negatively affect b\* and a\* parameters in the yogurt when compared with other treatments. Table 1. Physicochemical properties of yogurts during storage. TA: titratable acidity; L\: lightness; a\: red-green axis; b\: blue-yellow axis; WI: whiteness index; YC: control yogurt; YMD: yogurt with addition of maltodextrin; YH: yogurt with addition of free protein hydrolysate; YHEn: yogurt with addition of microencapsulated protein hydrolysate. Different lowercase letters (a,b,c,d) indicate a significant difference between samples for the same day (p* < 0.05). Different uppercase letters (A,B) indicate a significant difference for the same sample on different days (p < 0.05). > The YMD sample showed differences (p < 0.05) in a\* values compared with YH after 1 day of storage and with YH and YHEn after 7 days of storage, but was similar to YC (p > 0.05). Regarding storage, in general the L\* parameter showed a slight increase over time, while a\* and b\* values did not change, except in YHEn, which did not present any change in relation to the L\* parameter and showed a slight decrease in a\* values. The whiteness index (WI) was in the same range in all samples, decreasing less than 2% compared with the control (p < 0.05). > Syneresis is an important parameter in yogurt since it can affect its quality during storage through the accumulation of serum on the surface, influencing the acceptability of the product [4,28,30]. The addition of the different ingredients provided a decrease in syneresis, with the lowest values observed in YHEn, independently of storage time (p < 0.05) (Table 1). In general, syneresis was not influenced by storage time, with the exception of YH, which showed a small decrease after day 7 (p < 0.05). Córdova-Ramos et al. [35] reported a decrease in syneresis in yogurts with the addition of different concentrations (1, 3, 5, 7 and 10 g/100 mL) of jumbo squid powder (Dosidicus gigas) obtained by spray drying using MD as encapsulating agent. The decrease in syneresis was associated with the functional properties of the protein, since protein and MD facilitate water retention, thus preventing this phenomenon. These authors reported that the lowest syneresis values were observed in yogurts with 7 and 10 g/100 mL of the powder (4.20% and 1.0%, respectively) while the addition of 3 g/100 mL presented a syneresis percentage (9.10%) similar to that obtained in the present study in YHEn (Table 1). In addition, the lowest syneresis was found in YHEn. This result was similar to that reported by Demirci et al. [6] in yogurts with the addition of different concentrations of rice bran (1, 2 and 3%), which ranged from 9.79% to 8.80%, while the control yogurt presented 10.29%. The authors related the decrease in syneresis to the water-retention capacity of rice bran dietary fibers. In the present work, the reduction in the syneresis of formulated yogurts may be associated with the water-retention capacity of the protein hydrolysate by ion-dipole interactions. This fact may be due to the amino acid profile, which has a predominance of hydrophilic amino acids (57.4%) and negatively charged amino acids (64.4%). Moreover, the reduction in syneresis was enhanced in the yogurt to which the microencapsulated hydrolysate was added, possibly because MD also contributed to increase water retention by hydrogen bonding and dipole–dipole interactions.	doab	2025-04-07T03:56:59.224960	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 275 }
007afdec-bed4-405d-873d-c355ba9add0e.276	3.2. Textural and Viscoelastic Properties Texture is an important attribute regarding the quality of yogurt [37]. The firmness and cohesiveness of the different yogurts after 1 and 7 days of storage are shown in Table 2. The addition of the hydrolysate to the yogurts, both free and microencapsulated, resulted in a slight decrease in firmness compared to the YC sample (p < 0.05), representing approximately 11% (YH) and 10% (YHEn) after 1 day, and 30% (YH) and 19% (YHEn) after 7 days of storage. On the other hand, YH and YHEn showed increases in cohesiveness (p < 0.05) of approximately 12% and 15%, respectively, after 1 day and ~92% and 18%, respectively, after 7 days of storage compared with YC. After 7 days YH showed the highest cohesiveness values (p < 0.05). In general, the firmness and cohesiveness of the different yogurts were not significantly influenced by storage time, except for YC and YH, which showed greater firmness and greater cohesiveness, respectively, after 7 days of storage. Table 2. Textural and viscoelastic parameters (Equations (2) and (3)) of yogurts during refrigerated storage. YC: control yogurt; YMD: yogurt with addition of maltodextrin; YH: yogurt with addition of free protein hydrolysate; YHEn: yogurt with addition of microencapsulated protein hydrolysate. Different lowercase letters (a,b,c) indicate a significant difference between samples for the same day (p < 0.05). Different uppercase letters (A,B) indicate a significant difference for the same sample on different days (p < 0.05). > Barkallah et al. [4] studied the addition of different concentrations of Spirulina platensis (0.25%, 0.5%, 0.75% and 1.0%) to yogurt and observed greater firmness in the control yogurt and with the addition of 0.25% of Spirulina platensis (0.67 and 0.62 N, respectively). The addition of higher concentrations resulted in yogurts with lower firmness values which, according to the authors, was due to the interruption of gel formation as concentrations of microalga were increased. In the same study, the authors found no differences in relation to cohesiveness for the samples. It is worth noting that both firmness and cohesiveness values were lower than those observed in the present study. Additionally, Öztürk et al. [39], when studying the fortification of set-type yogurts with peeled or unpeeled oleaster (Elaeagnus angustifolia L.) flours (1% and 2%), reported a reduction in firmness and an increase in cohesiveness on the first day of storage compared to the control yogurt. According to the authors, the control yogurt showed a longer fermentation time resulting in greater firmness, while the increase in cohesiveness would occur due to the water retention capacity of the flours added into the protein matrix. During fermentation, the formation of lactic acid by the action of microorganisms occurs, and the pH drop induces the aggregation of casein and the formation of disulfide bonds between denatured whey proteins and k–casein, resulting in the characteristic gel formation, texture and properties of yogurt [37,40,41]. In the present study, the yogurts with the addition of the free and microencapsulated hydrolysate required less time to ferment (about 60–90 min) compared with the control yogurt or that with MD; this factor may have influenced the lower firmness values found. Moreover, the addition of these ingredients may have caused an interruption of gel formation, reducing firmness. Parallel to this, the greater cohesiveness may be associated with the water retention capacity, mainly due to the functional properties of protein hydrolysates [42]. The viscoelastic properties of the yogurt samples were evaluated by small-amplitude oscillatory shear (SAOS) tests. Mechanical spectra of the different yogurts after 1 and 7 days of storage showed typical gel-like behaviour, with G > G along the whole frequency range (Figure 2). The power law model may be used to fit both G and G with the angular frequency ω (Equations (2) and (3)): $$G' = G\_0' \cdot \omega^{\mathbb{N}'} \tag{2}$$ $$G'' = G\_0'' \cdot \omega^{\text{n}''} \tag{3}$$ where G<sup>0</sup> and G<sup>0</sup> are the respective storage and loss moduli at 1 rad/s, and n and n exponents denote the viscoelastic response in terms of the time stability of both G and G at short time scales. The viscoelastic parameters resulting from Equations (2) and (3) (G<sup>0</sup> and G<sup>0</sup> ) are both a measurement of the gel strength of samples since they provide the complete (elastic and viscous) deformation resistance [43]. After day 1, the yogurts with the free hydrolysate (YH) and microencapsulated hydrolysate (YHEn) exhibited the lowest values for G<sup>0</sup> and G<sup>0</sup> (Table 2). This result indicates that the hydrolysate, in both forms, reduced the gel strength of the casein matrix, maintaining a similar degree of viscoelasticity as was evidenced by the similar values for the loss factor at 1 rad/s (tanδ) (Table 2). This result could be explained by the fact that the hydrolysate introduces a negative electric charge, consequently increasing the electrostatic repulsive forces within the micellar structure, which expand the network, enhancing the hydration of the gel network [44]. This result is consistent with the observed decrease in firmness in YH and YHEn as compared with YC from textural analysis. The yogurt with maltodextrin (YMD) exhibited slightly higher values for G<sup>0</sup> and G<sup>0</sup> than those in YC after day 1. The electrostatic neutral character of these glucose polymers would favor the mutual associations between maltodextrin and casein by hydrogen bonding and dipole–dipole interactions, showing a certain binder role for MD in the yogurt matrix. This result is consistent with the lower tanδ value for YMD vs. YC (Table 2), which suggests greater strength in the intermolecular interactions and consequently a longer bond lifetime in the gel matrix [45]. Figure 2. Mechanical spectra of yogurts during storage at 5 ◦C. Storage modulus −G at 1 day (a); loss modulus −G at 1 day (b); storage modulus −G at 7 days (c); loss modulus −G at 7 days (d). YC: control yogurt; YMD: yogurt with addition of maltodextrin; YH: yogurt with addition of free protein hydrolysate; YHEn: yogurt with addition of microencapsulated protein hydrolysate. As regards n and n exponents, it might be observed that for all samples, n > n , this means that the rate of decrease of G with decreasing ω, was higher than that for G , resulting in a shear-induced gelation at lower frequencies (higher oscillation times) [46]. This fact shows a shear-induced increase in the energy stabilization of network bonds at lower frequencies, compatible with the decrease in the gel strength at higher oscillation times [47]. After 7 days of storage, G<sup>0</sup> and G<sup>0</sup> values increased in YC and YMD compared with those found after day 1. This result was consistent with the observed increase in firmness from textural analysis, indicating considerable gel reinforcement during storage, which would be explained by the natural strengthening of the dipolar interactions and hydrogen bonds in the casein matrix induced by cool storage [44]. This effect was partially mitigated by the presence of maltodextrin, which would stabilize the structural rearrangements in the casein matrix during storage. In addition, both G<sup>0</sup> and G<sup>0</sup> were scarcely modified in YH and YHEn during the storage period, showing the stabilizing role of the hydrolysate in the casein matrix, especially in microencapsulated form. The increase in both G<sup>0</sup> and G<sup>0</sup> after 7 days was consistent with the observed increase in L\* (Table 1). L\* shows the light scattered by various structural elements (casein aggregates, molecular fragments, etc.). After 7 days, the gel strength of the different networks increased moderately, so that denser matrices were formed, enhancing the diffuse reflection of electromagnetic waves and consequently increasing L\* [45]. In general, n and n values decreased after day 7 of storage, maintaining a similar positive difference between n and n (Table 2). Therefore, the lower exponents (n and n) indicate an improvement in stability in the four yogurt networks over time, maintaining a similar stabilization energy at lower frequencies than on day 1. In order to gain insight into the rheological differences among the three ingredients (H, MD and HEn) in model systems, i.e., outside the protein gel matrix environment at the same concentration, the flow behaviour and the viscoelastic characteristics of diluted aqueous solutions (1.5%, w/v) were also analyzed (Figure 3). Such a low concentration was selected to resemble the low concentration of the hydrolysate in the yogurt matrix. The dissolved maltodextrin (MD) and the microencapsulated hydrolysate (HEn) presented a similar trend (shear thinning flow) and similar viscosity values during the three-step flow, while the opposite was observed in the free hydrolysate, which exhibited the lowest viscosity and virtually Newtonian behaviour, as was evidenced by the stationary values of viscosity in the three steps (Figure 3a). The dynamic oscillatory test showed an evident fluid-like response in the three samples based on noticeably higher values for G compared with those for G (Figure 3b). Both MD and HEn solutions showed considerably higher G values compared with the H aqueous solution, while showing no significant differences between each other. This result was consistent with the decrease in viscosity in the second step, and their regeneration ability in the third step, attributed to the contribution of maltodextrin (Figure 3a). In contrast, G decreased in H, and more evidently in HEn, with respect to plain MD (Figure 3b). These findings reflect a lower level of intermolecular association at small oscillatory shear in HEn, and suggest that microcapsules may be less prone to interacting with each other and also with the surrounding medium, explaining why this preparation in yogurt resulted in more stable samples, which behaved differently compared with plain MD yogurts.	doab	2025-04-07T03:56:59.225476	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 276 }
007afdec-bed4-405d-873d-c355ba9add0e.277	3.3. Microbiological Analysis To confirm that the process of obtaining the yogurts was properly carried out, specific counts of the two major bacterial species present in yogurt were determined. The counts of S. thermophilus were YC: 8.72 ± 0.10 log CFU/g; YMD: 8.71 ± 0.10 log CFU/g; YH: 8.82 ± 0.04 log CFU/g; YHEn: 9.20 ± 0.01 log CFU/g (p < 0.05). Thus, the addition of the microencapsulated hydrolysate seems to have favored the growth of S. thermophilus, which could explain the lower pH and higher acidity compared with the YC sample. Regarding L. bulgaricus, counts were slightly higher in YH (YC: 6.42 ± 0.11; YMD: 6.92 ± 0.06; YH: 7.25 ± 0.09; YHEn: 6.94 ± 0.09 log UFC/g) (p < 0.05), indicating that the incorporation of the hydrolysate favored the growth of the starter culture in some way. YHEn registered higher values than the control yogurt, and similar values to those of YMD (p < 0.05), which indicated that maltodextrin, as a glucose supply, could also promote in some way the growth of this group of microorganisms. The viable counts of S. thermophilus were significantly more numerous than those of L. bulgaricus in all samples (p < 0.05). Zhao et al. [48] verified the same tendency in yogurts elaborated with α-lactalbumin hydrolysatecalcium (α-LAH-Ca) complexes. The relatively low pH of yogurt and other fermented dairy products is a result of the fermentation of milk lactose into lactic acid, caused by the activity of lactic acid bacteria usually added as starter cultures [49,50]. In this study, as shown in Table 1, the pH of the yogurts evaluated ranged from 4.35 to 4.61. Furthermore, the yogurt starter cultures contained Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, which largely out-compete other bacteria present in milk due the inhibitory effect of lactic acid and the utilization of the primary carbohydrate source (lactose), as well as the production of other inhibitory compounds [49,50]. Figure 3. Evolution of viscosity values for the three-step flow (a) and viscoelastic parameters (b) for aqueous solutions at 1.5% concentration of maltodextrin (MD), hydrolysate (H) and microencapsulated hydrolysate (HEn) at 20 ◦C. Different letters (a,b) or (A,B) indicate a significant difference between samples (p < 0.05). In this study, the presence of Enterobacteriaceae, molds and yeasts was not found after 1 and 7 days of storage (data not shown). There is growing evidence that the Enterobacteriaceae family can accurately reflect the hygienic conditions of processing milk and its derivatives, even during storage, despite the widespread use of coliforms as an indicator of hygienic– sanitary conditions. In addition, the presence of molds and yeasts in yogurt is indicative of unsatisfactory sanitary practices in manufacturing or packaging [49]. Accordingly, Barkallah et al. [4] did not observe the presence of the mentioned microorganisms in yogurt with the addition of Spirulina platensis. #### 3.4. Bioactivity ## 3.4.1. Antioxidant Activity Antioxidant compounds can contribute to health promotion by eliminating free radicals, such as reactive oxygen species, as well as to increase the shelf life of foods, slowing down the process of lipid oxidation [36]. Yogurt per se, in general, may present antioxidant activity in view of its content of bioactive peptides, as reported in previous studies [6,9,36,39]. The antioxidant activity of the different yogurts after 1 and 7 days of storage was evaluated by assessing the ABTS radical scavenging activity and reducing power (Figures 4A and 4B, respectively). The addition of the free and microencapsulated hydrolysate to yogurts (YH and YHEn, respectively) increased the ABTS radicals scavenging activity (p < 0.05) in comparison with the YC and YMD samples, which showed a similar behavior (p > 0.05). The increase in antioxidant activity was probably due to peptides with antioxidant activity present in the protein hydrolysate of stripped weakfish, as previously reported by Lima et al. [25]. The activity of the hydrolysates was maintained, both in the free and microencapsulated forms in the yogurt matrix. These findings are in line with those reported by Abdel-Hamid et al. [9] and Demirci et al. [6], who found an increase in the antioxidant activity of yogurts with the addition of the fruit extracts Siraitia grosvenorii and rice bran, respectively. According to the authors, this was due to the composition of added phytochemicals with antioxidant activity. Additionally, Silva et al. [5] evaluated the antioxidant activity of yogurts functionalized with Spirulina platensis in free form and microencapsulated with MD, or with MD cross-linked with citric acid obtained by spray drying, reporting greater antioxidant activity (DPPH assay) for both functionalized yogurts compared with the control yogurt (especially in the case of the sample with MD cross-linked with citric acid). According to the authors, the addition of Spirulina platensis in the encapsulated form presents advantages such as masking the unpleasant fishy odor. Similarly, in the mentioned study, no differences in antioxidant activity were observed in terms of storage time (4 and 7 days). Francisco et al. [7] reported a decrease in antioxidant activity during storage (0–7 days) for yogurts with added mushroom extracts (Agaricus bisporus) in free form and encapsulated by spray drying with MD cross-linked with citric acid (thermally untreated forms), while the opposite behavior was observed in thermally treated microencapsulated extracts after atomization. This decrease throughout storage time may be associated with the degradation of the extract when incorporated in free form by direct contact with the food matrix, and when thermally untreated, by the rapid release of the microencapsulated extract to the food matrix with the subsequent degradation. In the present study there was no degradation of bioactive peptides, possibly because the antioxidant activity (ABTS and reducing power) remained stable throughout storage (p > 0.05). It should be noted that YH and YHEn did not differ significantly in either evaluated property (p > 0.05) (Figure 3). Thus, the incorporation of the microencapsulated hydrolysate may be an alternative for increasing the antioxidant activity of yogurts while possibly masking any potential changes to odor and flavor.	doab	2025-04-07T03:56:59.226016	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 277 }
007afdec-bed4-405d-873d-c355ba9add0e.278	3.4.2. ACE Inhibitory Activity Angiotensin-I converting enzyme (ACE) plays an important role in regulating blood pressure, as it produces the potent vasoconstrictor angiotensin II and degrades a vasodilator called bradykinin, causing an increase in blood pressure [51,52]. Fermented dairy products can provide beneficial health effects by releasing peptides with ACE inhibitory activity as a result of the proteolysis of milk proteins during fermentation [8,53]. Figure 4C shows the ACE inhibitory activity after 1 and 7 days of storage. According to these results, the activity presented by YH and YHEn was more than threefold greater compared with that of YC or YMD (p < 0.05). In addition, the effect of the storage time can be considered negligible in these yogurts since the ACE inhibitory activity remained stable throughout the 7 days of storage (p > 0.05). The results obtained in the present study are consistent with those observed by Abdel-Hamid et al. [9] who reported a significant increase in the ACE inhibitory activity of probiotic yogurts supplemented with Siraitia grosvenorii fruit extract (73.36–81.39% compared to the 62.05% control). The mentioned authors correlated this activity with the degree of hydrolysis (proteolysis) of the supplemented yogurt, in which the release of small peptides occurs. In another study, Wulandani et al. [10] evaluated the ACE inhibitory activity of yogurts added with Ficus glomerata Roxb fruit extract (5% and 10%) during cold storage for 28 days, reporting the greatest activity on the seventh day of storage in yogurt with the addition of 10% F. glomerata Roxb extract (69.11 ± 0.50%) compared with yogurt without F. glomerata Roxb extract (53.47 ± 1.07%). Shori et al. [8] also found an increase in the ACE inhibitory activity in freshly made yogurts to which fish collagen was added compared with the control yogurt, presenting approximately 53% and 40% more inhibition, respectively. It is worth mentioning that the reported values in yogurts with the addition of fruits were lower than those observed in the present study. Thus, the results in the present study suggest that the hydrolysate, both free and microencapsulated, is a good source of ACE inhibitor peptides, providing yogurt with increased ACE inhibitory activity after at least 7 days of storage. Figure 4. ABTS radical scavenging activity (A), reducing power (B) and ACE inhibitory activity (C) of yogurts during storage. Different lowercase letters (a,b) indicate a significant difference between samples for the same day (p < 0.05). Different uppercase letters (A,B) indicate a significant difference for the same sample on different days (p < 0.05).	doab	2025-04-07T03:56:59.226416	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 278 }
007afdec-bed4-405d-873d-c355ba9add0e.279	3.5. Sensory Analysis The addition of new ingredients to yogurt can cause possible interferences, mainly in regard to taste and texture. Figure 5 shows the sensory profile of the different yogurts as evaluated by the panelists. According to the results, there were no significant differences among yogurts for color and acidity (p > 0.05). Moreover, the presence of MD did not affect the organoleptic properties of yogurt in any of the evaluated attributes. Figure 5. Sensory evaluation of different yogurts. The addition of the free and microencapsulated protein hydrolysate (YH and YHEn) affected the texture, odor and flavor compared with the control yogurt (p < 0.05). Although they presented a significant difference in relation to YC, YH and YHEn showed an intermediate score in relation to texture; the panelists described the texture of YH and YHEn as slightly softer than that of the control but without losing the characteristic gel-like consistency of yogurt. Regarding odor, a slightly fishy or peculiar smell was detected, especially in YH. Among the sensory attributes evaluated, the lowest scores obtained corresponded to flavor. However, YHEn showed a significant difference compared with YH (p < 0.05), presenting a higher score, and thus suggesting that encapsulation masked the fishy flavor. In the observations, the panelists indicated that the yogurts with the free and microencapsulated hydrolysates had a different flavor compared with that of a traditional yogurt (control), but were also different from each other. A more intense fishy flavor was identified in the yogurt with the free hydrolysate (YH), while YHEn was perceived as having a different flavor from that of the control, which was, however, undefined. Taking into account the sensory attributes of a traditional yogurt, a very familiar product to consumers, it is frequently and well documented that the incorporation of ingredients (other than fruits) modifies or is detrimental to the organoleptic characteristics. Accordingly, the addition of Allium sativum to yogurt lowered the score for wateriness, aroma and taste (in terms of sourness) compared with the control yogurt [8]. Demirci et al. [6] reported that the addition of 1%, 2% and 3% rice bran negatively affected the appearance, texture, taste and odor of the yogurt. In the formulation of yogurt with different concentrations of jumbo squid powder (1%, 3%, 5%, 7% and 10%), significant differences in relation to color, taste, and texture were reported, while the addition of 3% did not differ statistically from the controls [35]. Barkallah et al. [4] stated that there was no significant difference in flavor between the control yogurt and that containing 0.25% Spirulina platensis, however, the addition of 1% had the lowest score for flavor. In the present study, although there were differences in texture and flavor, the addition of microencapsulated hydrolysate was positive, as it masked the fish flavor while maintaining its bioactivity. There are many types of yogurt on the market; the consumer chooses among the available options, including fortified yogurt with bioactive products, which constitutes a new product. Thus, while the judges were able to differentiate the yogurts containing hydrolysates, this did not imply that the quality was worse, but rather that the yogurts were different with respect to the control; therefore, the acceptability parameter was excluded in the evaluation so as to prevent a biased judgement based on familiarity. Nevertheless, more studies should be conducted in order to improve these sensorial attributes in supplemented yogurt with fish protein hydrolysates.	doab	2025-04-07T03:56:59.226577	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 279 }
007afdec-bed4-405d-873d-c355ba9add0e.280	4. Conclusions Quality yogurts were made with the incorporation of a protein hydrolysate, free or microencapsulated with maltodextrin. These ingredients led to a reduction in syneresis, especially for the yogurt with the added microencapsulated hydrolysate. Yogurts with hydrolysates showed a slight reduction in firmness, while they were slightly more cohesive compared with the control yogurt. Both free and microencapsulated hydrolysates caused a reduction in the viscoelastic parameters, maintaining the characteristic gel-like structure of yogurt, and provided greater rheological stability after one week of storage, especially in the microencapsulated form. In addition, the incorporation of hydrolysates in both forms resulted in yogurts with greater antioxidant and antihypertensive activities, which were maintained after 7 days of storage. The antioxidant and antihypertensive activities shown in vitro by a functional food, such as yoghurt made with probiotic bacteria and further enhanced by the incorporation of protein hydrolysates, provide such a product with a potential health effect, although in vivo studies are needed to demonstrate this effect (yogurt behavior once digested, bioavailability studies, etc.). If the health effect is proven, the inclusion of yogurt in a diet could reduce the need for medication to regulate, for example, blood pressure. Although there were differences in texture, odor and flavor, the incorporation of the hydrolysate in microencapsulated form showed advantages over the free form in relation to the masking of the fish flavor, without altering the bioactive properties, and thus a yoghurt containing this addition may constitute an acceptable fortified food. Author Contributions: Conceptualization, K.O.L., M.E.L.-C., P.M. and C.P.; methodology, K.O.L. and A.A.; software, K.O.L. and M.d.R.; validation, M.E.L.-C., M.C.G.-G., C.A.T. and P.M.; formal analysis, K.O.L., M.E.L.-C., M.C.G.-G., C.A.T. and P.M.; investigation, K.O.L., M.d.R., A.A., C.A.T. and M.E.L.-C.; resources, K.O.L., P.M., M.C.G.-G. and C.P.; data curation, K.O.L., M.E.L.-C., P.M., M.C.G.-G. and C.A.T.; writing—original draft preparation, K.O.L. and M.E.L.-C.; writing—review and editing, K.O.L., A.A., M.E.L.-C., P.M., M.C.G.-G. and C.A.T.; visualization, M.E.L.-C., P.M. and M.C.G.-G.; supervision, M.E.L.-C., P.M., M.C.G.-G. and C.A.T.; project administration, M.E.L.-C., P.M. and M.C.G.-G.; funding acquisition, K.O.L., M.E.L.-C., M.C.G.-G., P.M. and C.P. All authors have read and agreed to the published version of the manuscript. Funding: Rio Grande do Sul State Research Foundation (FAPERGS) (17/2551-0000916-9). This work was supported by the Spanish Ministry of Economy and Competitiveness project NANOALI-VAL AGL2017-84161, cofounded with ERDF (European Regional Development Fund) and CSIC -202070E218. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data relevant to the study are included in the article. Acknowledgments: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. We also thank the Rio Grande do Sul State Research Foundation (FAPERGS) (17/2551-0000916-9), the Spanish Ministry of Economy and Competitiveness project NANOALIVAL AGL2017-84161, cofounded with ERDF (European Regional Development Fund) and CSIC -202070E218. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.226858	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 280 }
007afdec-bed4-405d-873d-c355ba9add0e.282	Article Tamarillo Polyphenols Encapsulated-Cubosome: Formation, Characterization, Stability during Digestion and Application in Yoghurt *Tung Thanh Diep 1,2, Michelle Ji Yeon Yoo 1,2,\ and Elaine Rush 2,3 Abstract: Tamarillo extract is a good source of phenolic and anthocyanin compounds which are well-known for beneficial antioxidant activity, but their bioactivity maybe lost during digestion. In this study, promising prospects of tamarillo polyphenols encapsulated in cubosome nanoparticles prepared via a top-down method were explored. The prepared nanocarriers were examined for their morphology, entrapment efficiency, particle size and stability during in vitro digestion as well as potential fortification of yoghurt. Tamarillo polyphenol-loaded cubosomes showed cubic shape with a mean particle size of 322.4 ± 7.27 nm and the entrapment efficiency for most polyphenols was over 50%. The encapsulated polyphenols showed high stability during the gastric phase of in vitro digestion and were almost completely, but slowly released in the intestinal phase. Addition of encapsulated tamarillo polyphenols to yoghurt (5, 10 and 15 wt% through pre- and post-fermentation) improved the physicochemical and potential nutritional properties (polyphenols concentration, TPC) as well as antioxidant activity. The encapsulation of tamarillo polyphenols protected against pH changes and enzymatic digestion and facilitated a targeted delivery and slow release of the encapsulated compounds to the intestine. Overall, the cubosomal delivery system demonstrated the potential for encapsulation of polyphenols from tamarillo for value-added food product development with yoghurt as the vehicle. Keywords:** tamarillo extract; yoghurts; cubosome; polyphenols; encapsulation; in vitro digestion	doab	2025-04-07T03:56:59.227097	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 282 }
007afdec-bed4-405d-873d-c355ba9add0e.283	1. Introduction Inverse bicontinuous liquid crystalline nanoparticles, termed cubosomes, have advantageous properties that may be suitable for the delivery of bioactive compounds to the small intestine. Amphiphilic lipids such as the monoglyceride monoolein can selfassemble in water to produce dispersions of cubosomes. The basic structure of a cubosome is a honeycomb-like structure with two non-intersecting internal aqueous channels separated by lipid bilayers. The internal hydrophilic (aqueous) areas are separated by lipid bilayers that are twisted into a tightly packed three-dimensional honeycomb structure that has a high internal surface area to volume. Within this structure, encapsulation of diverse hydrophilic, hydrophobic and amphiphilic compounds of small to large molecular weights, such as proteins, peptides, amino acids and nucleic acids, is possible [1]. Within cubosomes, hydrophobic molecules can be located within the lipid bilayers, hydrophilic components in the aqueous channels or around the polar head of the lipid, and amphiphilic molecules can be located at the lipid–water interface. This structure generally maintains the efficacy—stability of actives (vitamins and proteins) without adverse effects Citation: Diep, T.T.; Yoo, M.J.Y.; Rush, E. Tamarillo Polyphenols Encapsulated-Cubosome: Formation, Characterization, Stability during Digestion and Application in Yoghurt. Antioxidants 2022, 11, 520. https:// doi.org/10.3390/antiox11030520 Academic Editors: Li Liang and Hao Cheng Received: 8 February 2022 Accepted: 7 March 2022 Published: 8 March 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/). on the recipient [2]. Both polar and non-polar compounds can be solubilized in the water channel and the bilayers, respectively; therefore, both can be loaded into these particles. According to Meikle et al. [3], cubosomes are relatively non-toxic, stable over a broad range of biologically relevant environmental conditions, and can be formulated using a wide array of lipids and stabilizers. Their size, surface charge, and bilayer structure can be tuned through adjustments in their composition. Moreover, previous studies have demonstrated the ability of cubosomes to deliver drugs to both the eukaryotic and prokaryotic cells [4–8] found in the human digestive system. Cubosome encapsulation has been used to deliver quercetin in vitro [9] and curcumin to the skin [10] and demonstrated improved solubility and availability with an entrapment of 84% and 82%, respectively. Improved anti-inflammatory/antioxidant effects and controlled diffusion of encapsulated curcumin through the skin were observed [10]. Cubosome encapsulated has also enhanced the stability and antibacterial activity of curcumin [11]. Another study had reported successful encapsulation of both piperine and curcumin in the interior of the cubosome particles [12]. Tamarillo (Solanum betaceum Cav.), a common fruit of New Zealand, is a good source of polyphenols compounds including anthocyanins, hydroxy benzoic acids, hydroxycinnamic acids, flavonols, flavanols and flavonol glycosides. The main polyphenols in the dried pulp of Laird's Large tamarillo cultivar were identified in our previous study [13] as delphinidin rutinoside (255 mg/100 g), pelargonidin rutinoside (201 mg/100 g DW), chlorogenic acid (66 mg/100 g), kaempferol rutinoside (50 mg/100 g) and cyanidin rutinoside (26 mg/100 g). These polyphenols are strong antioxidants possessing many potential health benefits such as preventing lipid oxidation and are associated with reduced risk for certain cancers, cardiovascular diseases and type 2 diabetes mellitus [14]. Tamarillo fruit therefore has the potential to be an ingredient in functional food products [15]. Evaluation of the stability of polyphenol compounds is important as these compounds are often degraded by oxidation during digestion, resulting in reduced biological activity [16]. For example, anthocyanins, present in high amounts in the nutrient-dense tamarillo, are oxidized into quinones, reducing the biological power of these molecules during digestion [16]. To overcome this obstacle, new generation–functional foods often use encapsulation technologies to protect polyphenols from degradation as well as maintain their bioavailability [16]. This study aimed to investigate the morphology, entrapment efficiency, and particle size of cubosome-encapsulated tamarillo extract (CUBTAM) and test the stability and antioxidant activity and release of the CUBTAM before and after in vitro digestion. In vitro digestion of yoghurt fortified with CUBTAM was similarly investigated for the stability and release of polyphenol compounds.	doab	2025-04-07T03:56:59.227243	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 283 }
007afdec-bed4-405d-873d-c355ba9add0e.285	2.1. Materials The yoghurt ingredients included standard milk (Anchor™ blue top, Fonterra) from a local supermarket (Auckland, New Zealand) and starter culture containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (YoFlex® Express 1.1 powder) from CHR Hansen (Hoersholm, Denmark). Dimodan® MO 90/D (monoolein) was kindly provided by Danisco (Auckland, New Zealand). Pluronic F127 (PEO99–PPO67–PEO99) was purchased from Sigma-Aldrich (Auckland, New Zealand). The analytical grade standards of phenolics and anthocyanins were obtained from Sigma-Aldrich (Auckland, New Zealand) or Extrasynthese (Genay Cedex, France). Purite Fusion Milli-Q water purifying machine (Purite Limited, Thame, Oxon, UK) was used to produce Milli-Q water. All chemicals and reagents used were AnalaR grade or purer.	doab	2025-04-07T03:56:59.227689	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 285 }
007afdec-bed4-405d-873d-c355ba9add0e.286	2.2. Tamarillo Extract (EXT) Preparation and Identtification of Polyphenol Components Fresh fruits of Laird's Large (red) tamarillo cultivar were collected from growers in the Northland region of New Zealand with commercial maturity of 21–24 weeks from anthesis. The fruits were cleaned and peeled; then, the pulp was packed in polyethylene bags, sealed and frozen at −18 ◦C and defrosted (15 min) immediately before use. The extraction process was based on the method of Piovesana and Noreña [17] with some modifications. Aqueous citric acid (2% w/v) was used for extraction in the ratio of 1:5 (tamarillo: aqueous citric acid, w/w) and homogenised in a blender, and agitated (1000 rpm) at 55 ◦C in a water bath (1000 rpm) (Heidoplph, LABOROTA) for one hour after the addition of (20 μL/100 mL) pectin lyase (Novozym 33095), to improve the extraction of the bioactive compounds. Parafilm and tightly sealed lids of container and centrifuge tube were used to prevent entry of air and oxidation of polyphenols. The mixture was centrifuged at 10,000 RPM for 10 min and the supernatant (tamarillo extract) was separated and stored at −20 ◦C until being injected into LC-MS for identification of polyphenol component as reported in our previous study [13].	doab	2025-04-07T03:56:59.227765	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 286 }
007afdec-bed4-405d-873d-c355ba9add0e.287	2.3. Preparation of Cubosome and Cubosome Containing Tamarillo Extract The lyotropic liquid cubic phase was prepared and dispersed into cubosomes (CUB) as previously described in previous studies [18–20]. Briefly, molten monoolein (50 ◦C) was mixed with MilliQ water at a mass ratio of 60:40 to form the cubic phase. The cubic phase gel was equilibrated at room temperature for at least 48 h. Then, 25 mg of cubic phase gel was added to 5 mL of Pluronic F127 solution (2% (w/v)), and sonicated using a probe sonicator (BEM-150A, Bueno Biotech Ltd., Nanjing, China) (50% amplitude, pulsing 5 s on, 5 s off, for 7 min total run time) to obtain cubosomes (CUB). In parallel, the water was replaced by the tamarillo extract with a mass ratio of 60:40 (monoolein: tamarillo extract) to produce tamarillo polyphenol loaded-cubosomes (CUBTAM). #### 2.4. Polarized Light Microscopy (PLM) and Scanning Electron Microscopy (SEM) Microstructure and morphology of CUB and CUBTAM particles were observed using a polarized light microscope (DM750, Leica, Wetzlar, Germany) equipped with a digital camera (ICC50HD, Leica, Wetzlar, Germany). Dried CUB and CUBTAM were coated by using Platinum (Pt) using a sputtering technique with a Sputter Coater (Hitachi E-1045, Hitachi, Tokyo, Japan) for 60 s at room temperature. The particle morphology was then observed by a scanning electron microscope (Hitachi SU-70 Schottky, Hitachi, Tokyo, Japan) at 25 mA and 10 kV [21].	doab	2025-04-07T03:56:59.227885	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 287 }
007afdec-bed4-405d-873d-c355ba9add0e.288	2.5. Dynamic Light Scattering (DLS) Each sample of the CUB and CUBTAM diluted 20× in MilliQ was measured in triplicate using disposable plastic cuvettes at 25 ◦C. Particle size and its distribution were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). The mean z-average diameter and polydispersity indices (PDI) were obtained from cumulative analysis using the Malvern software version 7.13 (Malvern Panalytical Ltd., Worcestershire, UK) [18]. #### 2.6. Determination of the Entrapment Efficiency (EE) The sample preparation procedure was applied according to Zhou et al. [22] with minor modifications. Briefly, 20 μL of CUB or CUBTAM were transferred into a 1 mL Eppendorf microcentrifuge tube and made up to volume with methanol. Then, the sample solutions were centrifuged at 5000 RPM for 5 min, and the supernatant was separated and transferred to an amber 1.8 mL glass vial, then injected into the LC-MS system. The phenolic and anthocyanin compounds were analysed by using LC-MS according to our previous study [13] without further modification. According to Patil, Pawara, Gudewar and Tekade [23], the entrapment efficiency (EE%) is calculated as follows: $$\text{EE\%} = \frac{\text{A} - \text{B}}{\text{A}} \times 100$$ where A is the polyphenol concentration added into cubosome and B is the polyphenol concentration present in the supernatant	doab	2025-04-07T03:56:59.227993	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 288 }
007afdec-bed4-405d-873d-c355ba9add0e.289	2.7. Yoghurt Fermentation and Fortification with Tamarillo Polyphenol Loaded-Cubosome CUBTAM suspensions from Section 2.3 were snap frozen by liquid nitrogen, and then lyophilized for 36 h (Alpha 1–2 LD plus Freeze Dryer, Martin Christ, New Zealand). The tamarillo bioactive loaded-cubosome (CUBTAM) powder was stored at −20 ◦C until use. Kitchen yoghurt makers (Davis & Waddell, Stevens, New Zealand) were purchased to produce the yoghurt. For the control yoghurt, starter culture and milk in the ratio of 0.1:100 (w/w), were placed in the yoghurt maker. Incubation was implemented at 45 ◦C for 8 h until the pH of below 5.0 was obtained. The yoghurt was stored at 4 ◦C overnight and then homogenized at 4000 RPM using laboratory mixer (Silverson, Waterside, UK) for 2 min [24]. The yoghurt was stored at 4 ◦C until further analyses within 24 h. The CUBTAM powder with concentration of 5, 10 and 15% were fortified into the yoghurt either before (PRE) or after (POS) the fermentation process. For PRE, CUBTAM powder was added to the mixture of milk and starter culture at the start of yoghurt making process, prior to fermentation. For POS, CUBTAM was added to yoghurt in the final homogenization step. #### 2.8. Determination of Physicochemical Properties of Fortified Yoghurts The pH was measured with a digital pH meter to one decimal place. Syneresis index of yoghurt was identified based on the method of Wang et al. [24]. Some modifications were made from Kristo, Miao and Corredig [25] for rheological measurements, using a rheometer (RST-SST, Brookfield Ametek, Middleboro, MA, USA). The viscosity profile (viscosity, consistency coefficient, flow behavior index) was determined with a concentric cylinder with the diameters of cup and bob of 28.92 and 26.66 mm, respectively. Elastic modulus was determined with a vane spindle (SSVANE-) at a speed of 0.5 rpm for 5 min. TA-XT plus texture analyser (Texture Technologies Corp., New York, NY, USA) was used to perform textural analysis with a backward-extrusion test based on the method of Wang et al. [24] with some modifications. The parameters of test included cylinder probe diameter of 50 mm, test speed of 1.0 mm/s, penetration distance of 25 mm and surface trigger force of 10 g.	doab	2025-04-07T03:56:59.228107	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 289 }
007afdec-bed4-405d-873d-c355ba9add0e.290	2.9. In Vitro Digestion The EXT, CUB, CUBTAM and yoghurt samples fortified with CUBTAM were subjected to in vitro digestion to identify the bioavailability of bioactive contents using method of Zhang et al. [26] without further modification. The sample (2 mL) was collected before digestion and after oral (5 min), gastric (120 min) and intestinal phases (180 min). The samples were snap frozen using liquid nitrogen to stop enzyme activity and centrifuged at 10,000 RPM at 4 ◦C for 10 min. The supernatants were collected and stored at −20 ◦C before being injected into the LC-MS for phenolic and anthocyanins identification as well as total phenolic content (TPC) and antioxidant activity analysis [13].	doab	2025-04-07T03:56:59.228271	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 290 }
007afdec-bed4-405d-873d-c355ba9add0e.291	2.10. Total Phenolic Content (TPC) and Antioxidant Activity The TPC of extracts and digests at each stage of digestion was determined using a Folin–Ciocalteu assay as described in our previous study [13]. Two different methods were used to determine the antioxidant activity, namely cupric ion reducing antioxidant capacity (CUPRAC) and ferric-reducing antioxidant power (FRAP) assays, which were mentioned in our previous study [13]. Results of TPC and antioxidant activity are presented as mg gallic acid equivalent per 100 g of tamarillo or yoghurt (mg GAE/100 g) and μmol Trolox equivalent antioxidant capacity per 100 g of tamarillo or yoghurt (μmol TEAC/100 g), respectively. #### 2.11. Statistical Analysis Measurements of all the analytes were undertaken in triplicate, and the results are presented as mean ± standard deviation (SD). For comparison among different samples, one-way analysis of variance (ANOVA) was applied using SPSS 25.0 (IBM Corp., Armonk, NY, USA). Fisher's (LSD) multiple comparison tests were used to determine the magnitude of differences between means. A p-value of <0.05 was considered statistically significant. #### 3. Results and Discussion #### 3.1. Characterization of Cubosomal Suspensions Containing Tamarillo Extract (CUBTAM) Adapting the temperature–composition phase diagram of a monoolein/water system [27], pure monoolein cubosomes (CUB) and tamarillo polyphenols loaded-cubosomes (CUBTAM) were prepared in a top–down approach. This method allows the formation of reproducible, stable cubosomes without adding solvents. Therefore, it is unnecessary to reinvestigate phase behaviour, and there are no solvent concerns for cellular toxicity [2]. The concentration of the surfactant pluronic F127 was chosen to be 2 wt%, which yields stable cubosome dispersions [28]. The CUB dispersion appeared homogenously milky white and CUBTAM appeared semi-opaque pink (picture not shown). Figure 1 shows the PLM and SEM photos of CUB and CUBTAM while their particle size distribution (PSD) is summarized in Figure S1 and Table S1. The addition of tamarillo extract did not significantly affect the morphology of cubosome particles. The initial cubic periodicity can be clearly visualized for both samples using PLM and SEM. Because tamarillo extracts are mainly water-soluble compounds (phenolics and anthocyanins), they should be dispersed in the water channel of the cubosome and should minimally affect the structure of the nanoparticles [19]. Figure 1. (A) PLM and (B) SEM micrographs of CUB (left) and CUBTAM (right). For particle size distribution, CUBTAM had a unimodal curve and its polydispersity index (PDI) was quite small (below 0.3), as shown in Figure S1. The mean hydrodynamic diameter of liquid crystal particles increased significantly (from 270 to 327 nm) with the addition of the tamarillo extract. In general, this parameter depends on several factors such as the concentration of amphiphile (lipid and polymer), the presence of charged lipids, the ionic strength and the interactions between groups [23]. For CUBTAM, the addition of hydrophilic groups contributed to increasing electrostatic interaction as well as the coalescence between colloidal particles resulting in a bigger average particle size. However, most of the cubic particles in CUBTAM were still limited to a sub-micron range (100–1000 nm). According to Danaei et al. [29] the small particle size and the narrow size distribution (small PDI) create a large surface area that benefits cellular uptake. Entrapment efficiency (EE%) of bioactive compounds from CUBTAM ranged from 19.8 (catechin) to 87.7% kaempferol rutinoside (Figure 2). Twelve of the fourteen tamarillo bioactive compounds had an EE of more than 50%. In addition, it is noteworthy that we show high EE% (>69%) for the major polyphenols in tamarillo (chlorogenic acid, kaempferol rutinoside, delphinidin rutinoside, cyanidin rutinoside and pelargonidin rutinoside). The high EE in the CUBTAM could be attributed to the fact that polyphenols in tamarillo extract are water-soluble compounds which embed in the water channels. The EE difference between polyphenols encapsulated by cubosome might also depend on the number of -OH groups in molecular structure. For example, the hydroxycinnamic acids chlorogenic acid, caffeic acid and ferulic acid (with >2 -OH groups) showed higher EE than p-coumaric acid (which has only 1 -OH group). More -OH groups will more easily attach in the aqueous channel of cubosome particles. Furthermore, different polyphenol classes showed different EE. For instance, the hydroxycinnamic acids (chlorogenic acid, caffeic acid and ferulic acid), hydroxybenzoic acid (gallic acid), flavonol glycosides (rutin, kaempferol rutinoside, isorhamnetin rutinoside) and anthocyanins (delphinidin rutinoside, cyanidin rutinosid, pelargonidin rutinoside) showed higher EE than flavanols (catechin, epicatechin). According to Patil et al. [23], the EE is dependent on particle size rather than the amount of poloxamer (pluronic F127) used to stabilise the cubosome. The larger the nanoparticles, the higher entrapment efficiency for the polyphenols. This is because surface area to volume ratio of large particles is less than that of smaller particles and exposure to water of active compounds also decreased. Thus, the active compound loss due to diffusion also decreased in larger particles. Figure 2. Entrapment efficiency of polyphenols from tamarillo extract using lyotropic liquid crystalline nanoparticles. Data are presented as mean and error bar (standard deviation) (n = 3).	doab	2025-04-07T03:56:59.228332	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 291 }
007afdec-bed4-405d-873d-c355ba9add0e.292	3.2. TPC and Antioxidant Activities of Encapsulated and Non-Encapsulated Extracts during In Vitro Digestion The impact of digestion on TPC and antioxidant activities of EXT and CUBTAM is shown in Figure 3. The TPC and antioxidant activities decreased significantly after digestion for both non-encapsulated and encapsulated in comparison to the undigested samples. Figure 3. Changes in the total phenolic content and antioxidant activities of tamarillo extract (A) and tamarillo polyphenol loaded-cubosomes (B) before and after in vitro digestion. The units of TPC (-), CUPRAC (-) and FRAP (-) were mg GAE/100 g tamarillo, μmol TEAC/100 g tamarillo and μmol TEAC/100 g tamarillo, respectively. Data are presented as mean and error bar (standard deviation) (n = 3). Different alphabets indicate statistical difference (p < 0.05) for each assay. For both EXT and CUBTAM, there were significant differences (p < 0.05) between the amounts of TPC in the supernatant after each stage of digestion. Gastric digests recorded the highest TPC for EXT, while, in the oral and intestinal phases, no significant differences were observed (Figure 3A). Low values of TPC in the supernatant of the oral digest (after 2 min of digestion) are related to the short time for diffusion and low solubility of polyphenols. The loss of polyphenols during digestion could be explained by physicochemical transformations, such as oxidation or the presence of yoghurt molecules (fats and proteins) in the digestion mixture. Furthermore, the decrease of bioactive content could arise from precipitation of several compounds with proteins or enzymes in the digest [30]. However, for the CUBTAM, the release of polyphenols increased during the digestion and was greater in the intestinal phase than the gastric phase (Figure 3B), which was not seen for the non-encapsulated extract, demonstrating a protective effect of the cubosome encapsulation technique against digestive enzymes and pH changes during gastric digestion. Results obtained from LC-MS (Table 1) further support the protective effect of the cubosomes on polyphenols. Similar findings have been reported on release properties of encapsulated blueberry extract [31] and carob pulp extract [32] during simulated gastrointestinal digestion. Both studies showed that TPC in the supernatant increased throughout gastric to intestinal digestion. The materials used for lipid bilayer and stabilisation of cubosomes determine susceptibility of polyphenols to digestive enzymes as well as pH at each stage [33]. The reduction in TPC of tamarillo extract during in vitro digestion might be associated to sensitivity of phenolic compounds to higher pH (>6), since, at that pH, monomers obtained by hydrolysis from larger molecules might be less stable [34]. The increase in the TPC of CUBTAM could be related to the release of complexed bioactive compounds as a result of the digestive process [35]. Table 1. Phenolics and anthocyanins in tamarillo extract and tamarillo polyphenol loaded-cubosome released during three stages of in vitro digestion. Results are expressed as % with respect to the initial concentration. Data are expressed as Mean ± SD (n = 3). Different letters of the alphabet superscripts indicate statistical difference (p < 0.05) across each row. The antioxidant activity of tamarillo fruit phenolic extracts is mainly linked to their anthocyanins, chlorogenic acid and kaempferol rutinoside compounds. However, due to the chemical transformations from different mechanisms, the antioxidant properties of these compounds might change during digestion. Thus, the influence of digestion on the antioxidant capacity of tamarillo pulp extracts in non-encapsulation and encapsulation form was assessed by using CUPRAC and FRAP assays (Figure 3). All activities tested significantly (p < 0.05) decreased after digestion in comparison to the raw material, which coincide with the decrease in bioactive compounds, mainly polyphenols, after digestion (Table 1). There were substantial and significant differences in CUPRAC values between the non-encapsulated and encapsulated extracts (p < 0.05) throughout the process of in vitro digestion. Tamarillo extract had the highest CUPRAC value in the gastric phase, whereas, for CUBTAM, the highest supernatant activity was noted in the intestinal medium. EXT and CUBTAM presented significant differences (p < 0.05) in FRAP values during the digestion process. The highest FRAP value was observed in the oral phase for non-encapsulated extract, whereas, for encapsulated samples, this activity increased with the progress of digestion with the highest FRAP activity of the supernatant was recorded at the end of the intestinal phase. In other studies, an increase in the FRAP with digestion was most pronounced at the intestinal phase for both encapsulated blueberry extract [31] and carob pulp extract [32]. The difference in FRAP and CUPRAC activities, in the oral and intestinal phases, respectively, may not be due to the content of phenolics and anthocyanins, but rather to the diversity and characteristics of the polyphenols present. However, the highest activities (FRAP and CUPRAC) after the gastric phase might be due to the higher release of phenolics and anthocyanins content and the quenching and reducing properties of the acidic medium of the sample. The effect of the pH could also be different among various polyphenols. At neutral pH, some polyphenols have exhibited pro-oxidant activities, whereas, at lower pH, others have demonstrated antioxidant activities [32]. Furthermore, the difference might be related to in vitro digestion conditions used and/or change of polyphenol availability related to the release of matrix associated compounds [36]. In fact, free polyphenols have shown higher antioxidant activity than iron-phenol chelates. Together with the enzymatic action, the pH influence within the gastrointestinal digestion and the presence of compounds that were not analysed in this study (e.g., peptides or complex polyphenols) enhance antioxidant activity [35]. The increase in antioxidant power of the supernatant between the acidic gastric phase and alkaline intestinal phase environments, as seen in this study, can be partially explained by the deprotonation of the hydroxyl groups on the aromatic rings of the polyphenols [36]. Under the intestinal conditions, the decrease in antioxidant activity (CUPRAC and FRAP) for the non-encapsulated extract would be related to the lower TPC alongside transformation of some polyphenols into conformations related to the neutral pH (Figure 3A). Meanwhile, the highest antioxidant activities for CUBTAM supernatant, in the intestinal phase (Figure 3B), could be explained by their release from the microcapsules as they are degraded in the neutral pH. The weak activities recorded in oral and gastric phases of digestion might be due to a small amount of polyphenol release from the microcapsule surface and/or via the penetration of salivary and gastric fluids into the microcapsules through their surface pores.	doab	2025-04-07T03:56:59.228929	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 292 }
007afdec-bed4-405d-873d-c355ba9add0e.293	3.3. Release of Tamarillo Polypehnols from Cubosomes during Digestion In order to evaluate the stability of individual polyphenol compounds during digestion, a total of fourteen compounds were evaluated by LC-MS (Table 1). The CUB was also analysed as a control. The results showed that 11 phenolic compounds and three anthocyanins were released from the microcapsules after the digestion process, demonstrating that these phytochemical compounds were well encapsulated by the cubosomes. The phenolics presented different behaviours during the simulated digestion (Table 1). Analysis of phenolics released from EXT during digestion showed a significant instability for the major phenolic acids (gallic acid, chlorogenic acid and p-coumaric acid), other phenolics (epicatechin, rutin and kaempferol rutinoside) as well as all anthocyanins after oral and gastric phases. For gallic acid, the concentrations in oral phase remained stable and only a significant (p < 0.05) increase was observed in the gastric phase (46.49%) when compared to the initial undigested EXT. Then, the concentration of this acid dropped down to 20.4%. Tagliazucchi, Verzelloni, Bertolini and Conte [37] reported the degradation (43%) of pure gallic acid after gastrointestinal digestion, while the total degradation for gallic acid from grape extract and carob pulp extract had been explored by Jara-Palacios et al. [38] and Ydjedd et al. [32], respectively. Meanwhile, caffeic acid showed insignificant changes during the digestion (24.13% at the oral phase, 31.42% after the gastric phase and 21.94% at the end of the intestinal phase). According to Wojtunik-Kulesza et al. [39], the remaining percentage of caffeic acid decreased to 75% and 78% after oral and gastric phases, respectively. Some studies have reported that the gastric phase has increased the bioaccessibility of some phenolic acids, while, during the intestinal phase, their levels could be decreased. This behaviour has been closely related to the stability and structural changes that each type of polyphenolic acid undergoes [37]. Due to its low molecular weight, gallic acid has been better absorbed in humans compared to other phenolic acids, which makes it highly bioaccessible [36]. For chlorogenic acid, the highest concentration was detected in the gastric phase; then, the concentration of this compound reduced by 63% in the intestinal phase (Table 1). According to Tagliazucchi, Helal, Verzelloni and Conte [40], the degradation of chlorogenic acid during gastro-pancreatic digestion might be due to the oxidation and polymerization to form quinone in an alkaline environment. Significant reductions of free phenolic acids (gallic, chlorogenic, caffeic, p-coumaric acids) during in vitro digestion have been reported in previous studies [41–43]. These decreases in phenolic acids could be related to changes in pH and the presence of bile salts in the intestinal phase, which may lead to the formation of precipitates [42], which may explain the reductions observed at the end the intestinal phase of this study. The concentrations of kaempferol rutinoside in EXT remained stable after the oral and gastric phases but decreased significantly (p < 0.05) at the end of the intestinal phase (Table 1). A similar trend for kaempferol rutinoside during in vitro digestion of the Cactus Cladodes plant had been observed [44]. Hydrolysis of glycoside flavonoids starts in the mouth by means of β-glycosidase action, but the degree of hydrolysis depends on the types of sugars present in the flavonoid compounds. For example, polyphenol compounds with more hydrophobic properties often interact more strongly with proteins [39]. Degradation of polyphenols with high molecular weights (such as kaempferol rutinoside) may be related to their strong affinities with human salivary proline- and histidine-rich proteins to form non-covalent and covalent associations [39]. All of the anthocyanins, especially delphinidin rutinoside and pelargonidin rutinoside, showed the same releasing behavior during in vitro digestion (Table 1). For these main anthocyanins in tamarillo extract, a significantly (p < 0.05) higher proportion of anthocyanins (43 to 76%) was released after the intestinal phase when compared to the undigested samples. The instability of anthocyanins at neutral or slightly basic pH has been observed for polyphenols from grape and chokeberry [37,45]. The instability can be explained by the formation of a colourless chalcone pseudo-base, resulting in the destruction of the anthocyanin chromophore [46]. The current results support these previous findings, suggesting that anthocyanins are stable in the acidic conditions of the gastric phase but are degraded in the alkaline/neutral conditions of the intestinal phase. The reduction of anthocyanins may also be related to the fact that, in aqueous solution in response to changes in pH, anthocyanins undergo structural rearrangements, change colour, may form complexes with proteins in food and digestate and be degraded to phenolic acids [42]. The quantity of individual bioactive compounds from the CUBTAM at the end of each digestive phase varied by compound (Table 1). Catechin, epicatechin, isorhamnetin rutinoside and all anthocyanins (delphinidin rutinoside, cyanidin rutinoside and pelargonidin rutinoside) were released after the gastric phase in acidic medium; gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, rutin, kaempferol and kaempferol rutinoside were released into the neutral medium after oral and intestinal phases. It is worth noting that the percentage of free polyphenols was lower in CUBTAM (encapsulated) than in EXT (non-encapsulated) ones, and remained fairly constant along different in vitro digestion phases. These results were also expected because the initial amount of polyphenols in the encapsulated sample was lower owing to the encapsulated efficiency (over 50%). According to Ydjedd et al. [32], the properties of encapsulating material play a significant role in enhancing the entrapment efficiency and controlled release of the core compounds. They reported a slow release of some phenolics (gallic acid, p-coumaric acid, and kaempferol) from the microcapsules and a period of more than 3 h in the intestinal phase (neutral medium) has been necessary for complete release of these compounds, when the encapsulating material was completely degraded [32]. The present study is the first to report the proportion of cubosome encapsulated polyphenols released after each phase of in vitro digestion, demonstrating the potential of cubosomes to protect bioactive compounds in their matrix. Similarly, reduction of the degradation in cubosome encapsulated bioactive antimicrobial peptide has been reported, showing resistance towards the enzymatic degradation [18]. Cubosomes have a high viscosity which hinders the diffusion of polyphenols into the release medium and slows the entry of water, which sustains the slow release profile [47]. The rate of release controlled by the structure also depends both on the partition coefficient and on the diffusion of the drug through the hydrocarbon tail region [48]. #### 3.4. Physicochemical Properties of Yoghurt Fortified with CUBTAM The addition of 5%, 10% and 15% CUBTAM to yoghurt was associated with a small but statistically significant fall in pH and reduced syneresis (Table 2). This can be explained by the use of the freeze-drying treatment to prepare the powder for the cubosome, which would result in an increase in total dry solids, which in turn would increase the water holding capacity, reduce porosity and reduce the syneresis. Table 2. Physicochemical properties of yoghurt fortified with CUBTAM (5, 10 and 15%) in PRE and POS. Control yoghurt contained no CUBTAM. N/A: not applicable. Data are expressed as Mean ± SD (n = 3). Different alphabetic superscripts indicate statistical difference (p < 0.05) across each row. CUBTAM: tamarillo polyphenols loaded-cubosomes. POS5, POS10, POS15: addition of 5%, 10%, 15% of CUBTAM post to fermentation process, respectively. PRE5, PRE10 and PRE15: addition of 5%, 10%, 15% of CUBTAM prior to fermentation process, respectively. Viscosity of yoghurt increased with the increase of the concentration of CUBTAM, showing significant (p < 0.05) differences across the yoghurt samples (Table 2). Within the same % CUBTAM fortification, there was no significant (p > 0.05) difference in viscosity between PRE and POS. Based on the Oswald–de Waele power law model, yoghurts fortified with CUBTAM made from both fermentation processes can be considered as non-Newtonian fluids with shear-thinning behaviour due to the flow behaviour index (n) below 1. The breakage of bonds between the protein aggregates as a consequence of shear stress led to the pseudoplastic behaviour of the yoghurt samples [49]. The consistency index (K) and flow behaviour index (n) of yoghurts were not significantly influenced by the fermentation process, whereas the increase of encapsulated powder concentration led to the increase of K and decrease of n values. The increase of K value might be attributed to the water holding capacity, caused by the addition of powder. The elastic modulus of all yoghurts was very low, indicating the same relatively weak structure with or without CUBTAM (Table 2).	doab	2025-04-07T03:56:59.229318	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 293 }
007afdec-bed4-405d-873d-c355ba9add0e.294	3.5. Total Phenol Content, Antioxidant Activity and Release of Polyphenol Compounds in Yoghurt Fortified with CUBTAM during Digestion In a yoghurt matrix, catalase and super oxidase enzymes, casein as well as lactic acid bacteria which have antioxidant properties are present [50]. Without digestion, as expected, the addition of CUBTAM led to a dose-dependent increase in TPC and total antioxidant capacity; i.e., as expected, a higher level of fortification led to a higher TPC as well as antioxidant activity (p < 0.05). Furthermore, fortification in PRE resulted in higher TPC and antioxidant activity than in POS at the same concentration (Figure 4A). Figure 4. Total phenolic content (TPC) and antioxidant activity (CUPRAC and FRAP assay) of yoghurts fortified with cubosome containing tamarillo extract before digestion (A), after oral (B), gastric (C) and intestinal (D) phases of in vitro digestion. The units of TPC (-), CUPRAC (-) and FRAP (-) were mg GAE/100 g yoghurt, μmol TEAC/100 g yoghurt and μmol TEAC/100 g yoghurt, respectively. Data are presented as mean and error bar (standard deviation) (n = 3). Different alphabets indicate statistical difference (p < 0.05) for each assay. After the oral phase, TPC and total antioxidant capacity determined using CUPRAC and FRAP assays in both POS and PRE samples at all fortified concentrations reduced by 7.6–8.9%, 12.9–15.5% and 7.8–9.4%, respectively, compared to undigested samples (Figure 4B). After the gastric phase, significant increases in TPC and total antioxidant capacity were obtained compared to those of the oral phase (up to 3-, 2- and 3-fold, respectively), (p < 0.05) (Figure 4C). After simulated intestinal digestion, further significant increases in TPC (64–71%) were observed (p < 0.05). Furthermore, total antioxidant activity resulted in an additional 39–50% and 63–70% increase for CUPRAC and FRAP, respectively (Figure 4D). These results were in line with the measures of TPC and antioxidant activity of encapsulated tamarillo extracts (CUBTAM) (Figure 3) as well as polyphenol concentration determined by LC-MS (Table 1), in which the TPC and antioxidant activity increased greatly during the gastric and intestinal phases of in vitro digestion. Previous research showed that the antioxidant activity of the yoghurt samples containing encapsulated phenolics was increased due to the controlled release of the phenolic components from the encapsulation network [50]. Some researchers have considered the Folin–Ciocalteau assay as an antioxidant capacity test since this assay not only measures phenolic compounds but also the total reducing capacity of a sample and hence [42]. This may explain the difference in polyphenols content measured by LC-MS and by the colorimetric tests. Considering the pH conditions of the total antioxidant capacity assays performed in this study, the CUPRAC assay (pH 7.0) could be more appropriate for evaluating total antioxidant activity after the oral and intestinal phases, while the FRAP assay (pH 3.6) could be more suitable to assess total antioxidant activity after the gastric phase. The data in Table 3 are described as a percentage of each digestion phase in the supernatant compared to the content of the undigested sample. The TPC and antioxidant activities measured by CUPRAC and FRAP between CUBTAM and yoghurt fortified with CUBTAM were relatively similar at each phase of the digestion simulation (Table 3), demonstrating that yoghurt was a suitable carrier of cubosome without significant interference. For the oral phase, the TPC released and antioxidant activities in CUBTAM fortified yoghurt were higher than that for the CUBTAM itself by 1.5–1.7, 1.2–1.5 and 1.1–1.4 times, respectively. For the gastric phase, the amount of TPC released for CUBTAM was slightly higher than the fortified yoghurts, whereas the opposite trend was observed for the antioxidant activities. For the intestinal phase, the TPC and FRAP of CUBTAM were lower than that for CUBTAM fortified yoghurts, whereas, for the CUPRAC, the CUBTAM showed a similar value to PRE5, which were both much higher compared to the rest of the samples. Overall, cubosomes containing tamarillo extract showed effective protection for polyphenols in the oral and gastric phases. The differences in the LC-MS profiles between PRE and POS were significant (p < 0.05). Major polyphenols in tamarillo were also detected in CUBTAM fortified yoghurts (both from PRE and POS) before digestion (Table S2). Thus, the yoghurt matrix as well as encapsulation had helped to retain these individual polyphenols during processing. Encapsulation of bioactive compounds promoted lower loss of polyphenols under refrigerated conditions [51]. At the same concentration of CUBTAM yoghurt, addition of tamarillo polyphenol loaded-cubosomes prior to fermentation was associated with a higher concentration of major polyphenols than addition post fermentation. The concentrations of chlorogenic acid, kaempferol rutinoside and delphinidin rutinoside (accounted for over 65% of the total polyphenol content in yoghurts) were higher for pre-fermentation versus the post fermentation approach (Table S2). Delphinidin rutinoside was the dominant anthocyanin in fortified yoghurts that was in agreement with the main anthocyanin in Laird's Large tamarillo pulp reported in our previous study [13]. The results showed that the fermentation process appeared to have little impact on anthocyanins present in both yoghurts, which might be due to the encapsulation of anthocyanins. The yields of polyphenols were associated with the extractability of polyphenols from the original tamarillo extract. According to Sun-Waterhouse, Zhou and Wadhwa [52], during fermentation, the yoghurt starter cultures could transform the polyphenols into other forms/types of compounds, e.g., via flavonoid glycosides hydrolysis or C-ring cleavage. Such a conversion may result in the deactivation of bioactive compounds or activation of previously inactive compounds, e.g., polyphenol glycosides are hydrolyzed into their aglycones of higher free radical scavenging ability, and procyanidins break down to flavan-3-ols or to smaller molecular phenolic acids. Acidity of yoghurt may have induced acid hydrolysis of polyphenols, hence this could explain an increased amount of hydroxycinnamates such as caffeic acid, chlorogenic acid, ferulic acid and p-coumaric acid in the fortified yoghurts. Sun-Waterhouse, Zhou and Wadhwa [53] stated that the yields of hydroxycinnamic acids and flavonols detected might be dependent on the extractability of these polyphenols from different product matrices. Table 3. Measures of total phenolic content and antioxidant activity of supernatant of cubosome encapsulated tamarillo and yoghurt fortified with encapsulated tamarillo during in vitro digestion. Data are presented as % of the sample before digestion. CUBTAM: tamarillo polyphenols loaded-cubosomes. POS5, POS10, POS15: addition of 5%, 10%, 15% of CUBTAM post to fermentation process, respectively. PRE5, PRE10 and PRE15: addition of 5%, 10%, 15% of CUBTAM prior to fermentation process, respectively. Despite the importance of the recovery in each digestion phase, bioactive compounds will need to be released from their food matrix and reach the intestine where they can be absorbed and be metabolised [54]. The release rate during in vitro digestion has been considered as an indicator to assess the effectiveness of compound carriers [55]. In general, non-encapsulated phenolic compounds in drinking yoghurt were highly degraded after digestion [56] while microencapsulated formulation showed the ability to preserve the antioxidant activity of extract in yoghurt when compared with the free form [51,57]. The amount of individual polyphenol in yoghurt samples was significantly different (p < 0.05) after each phase of in vitro digestion. The quantity of polyphenols released at each stage is dependent on the time at each phase, pH and the concentration of CUBTAM (Table S3). Most polyphenol components were detected in both PRE and POS in each digestion phase. The oral digestion lasts for a few minutes; the encapsulated polyphenol in yoghurts release from oral digestion was significantly lower than the gastric (post to 2 h) and intestinal (post to 3 h) simulated digests. These data are in line with the findings from Section 3.3 that encapsulating polyphenol in cubosome particles could effectively protect the bioactive compounds from the gastric enzymes and facilitate the utilization of polyphenols in the human body. Studies on the metabolism of bioactive compounds in the humans have shown that bioactives are mainly metabolised by a large number of small and large intestinal bacteria, and the metabolites are absorbed into the human blood [58]. Therefore, for the polyphenols to be absorbed by the human body or to be active in the microbiome of the small and large intestines, the polyphenols should be protected in encapsulated form until completely released in the intestinal tract. When the polyphenol capsules were present in the simulated oral phase, the concentration of all bioactive compounds was low, indicating good retention in the cubosomes. Furthermore, compared to undigested samples, a significantly (p < 0.05) lower amount of most polyphenols (percentage loss < 10%) were released during the oral phase (except for epicatechin, rutin and kaempferol rutinoside). The findings provide evidence that cubosomes protect bioactive compounds from interaction with, for example, milk proteins and digestive enzymes, reducing the risk of polyphenol degradation. To our knowledge, most of reports of the loads of cubosomes are for proteins or small molecules such as drugs while research about encapsulation of hydrophilic polyphenols, mainly presented in tamarillo, using the cubosome is limited. Effects of the digestion process on properties and stability of cubosome as well as application of cubosome in food are still scarce. Hence, a strength of this study is it is the first attempt to encapsulate polyphenols from tamarillo and has demonstrated the proof of principle that this technique can be used to fortify yoghurt with a fruit extract. Entrapment efficiency was greater than 50% and, together with enhancement of antioxidant effects, stability and bioavailability of polyphenols in vivo and in vitro [2], the results showed the potential of cubosome to minimize degradation of polyphenols and contribute to controlled release of these and other bioactives during digestion. Application of encapsulated polyphenols into yoghurt did not significantly change texture and rheology of yoghurts when compared to the control, except for 15% fortification, where higher texture and rheology values were observed. However, there are still challenges about applications of cubosomes, including a deeper understanding of the stabilizer and possible cytotoxicity. In the future interaction between yoghurt components (mainly protein), starter culture and encapsulated bioactives should be evaluated with longer intestinal digestion and possible effects on the microbiota explored. According to Wei et al. [21], monoolein has been easily hydrolysed to free oleic acid due to the presence of pancreatic lipase and bile salt; therefore, it can be assumed that cubosome particles (with 60% of monoolein in components) can only be degraded in intestinal phase. However, degradation or stability of cubosome after each phase of digestion should be evaluated to ensure the safety, effectiveness and acceptability to the consumer of this approach. It is a limitation that we did not validate the correct encapsulation of the tamarillo polyphenols in the cubosome. In future work, we could confirm the encapsulation with confocal Raman/FTIR microscopy.	doab	2025-04-07T03:56:59.229957	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 294 }
007afdec-bed4-405d-873d-c355ba9add0e.295	4. Conclusions This study demonstrated the proof-of-principle that tamarillo polyphenols could be effectively encapsulated by cubosome nanoparticles with relatively high loading efficiency and preservation of high antioxidant activity. Compared to the unencapsulated extract, cubosomal encapsulation provided a protective effect to the tamarillo polyphenols under simulated gastrointestinal conditions, exhibiting good free polyphenol concentrations at the end of the intestinal phase. A cubosomal system was employed for the delivery of tamarillo polyphenols via yoghurt, and the addition of encapsulated bioactive improved the physicochemical and nutritional properties of yoghurt. The addition of CUBTAM at increasing concentrations successfully increased the concentration of polyphenols, TPC and antioxidant activity of yoghurts, with controlled stability during digestion, suggesting that polyphenols with enhanced bioavailability could be delivered in a dose-controlled manner. This research informs application of cubosome encapsulation to fortification of food products, for example both water-soluble and lipid-soluble vitamins and carotenoids (β-carotene). However, although the components of cubosomes (monoolein and Pluronic F127) are listed as "generally recognised as safe" (GRAS) by the FDA and approved in principle, further investigations should be carried out before sensory testing or consumption by humans as a food. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11030520/s1, Table S1: Particle size and polydispersity index of CUB and CUBTAM, Table S2: Concentrations (μg/g FW) of individual polyphenols in yoghurts fortified with CUBTAM before in vitro digestion, Table S3: Concentrations (μg/g FW) of individual polyphenols in yoghurts fortified with CUBTAM after each step of in vitro digestion, Figure S1: Size distribution of CUB () and CUBTAM (-), Figure S2: Concentration of polyphenols added into cubosome ((-) and concentration of polyphenols present in the supernatant (-). Author Contributions: Conceptualization, M.J.Y.Y., T.T.D.; Methodology, T.T.D., M.J.Y.Y.; Software, T.T.D.; Validation, T.T.D., M.J.Y.Y.; Formal Analysis, T.T.D.; Investigation, T.T.D.; Resources, M.J.Y.Y., E.R.; Data Curation, T.T.D., M.J.Y.Y.; Writing—Original Draft Preparation, T.T.D.; Writing—Review and Editing, M.J.Y.Y., E.R.; Visualization, T.T.D., M.J.Y.Y.; Supervision, M.J.Y.Y., E.R.; Project Administration, M.J.Y.Y., E.R.; Funding Acquisition, M.J.Y.Y., E.R. All authors have read and agreed to the published version of the manuscript. Funding: This research received a Performance Based Research Fund and internal postgraduate research project funds from the Auckland University of Technology. A PhD scholarship for the first author and internal project fund from Riddet Institute was also received. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data are contained within the article and supplementary material. Acknowledgments: The authors would like to acknowledge the Auckland University of Technology for the Performance Based Research Funding and postgraduate research project funds received for the project. The authors would like to thank the Riddet Institute for the Doctoral Scholarship provided to the first author, and the internal project fund to support the research. We also thank DuPont Nutrition and Bioscience (Danisco Ltd.) for supplying Dimodan®. We thank Azelis New Zealand for supplying Novozym to our research. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.230675	17-11-2022 17:23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007afdec-bed4-405d-873d-c355ba9add0e", "url": "https://mdpi.com/books/pdfview/book/6198", "author": "", "title": "Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036554563", "section_idx": 295 }
007e4761-4bfc-4411-b0b4-7cd4adcff760	# Between Kafka and Gogol'. 'De-territorialising' National Narrative(s) in Post-Soviet Ukrainian Literature in Russian Marco Puleri 356 Alessandro Achilli Vasyl Stus' Ukrainian Culture Between Europe and Russia European Modernism, Intertextuality. The poetical works and the critical writings of Vasyl' Stus (1938-1985) offer an interesting example of certain typical cultural dynamics of the Ukrainian literary civilisation, divided among attraction for Europe and the constant need to reaffirm its own belonging to Europe, the autoreferential temptation, and the Russian model – in its turn marked by the traditional dichotomy between Westernism and Slavophilism. In his literary writings as well as in his poetical production, both intrinsically linked to the cultural climate of the Thaw, Stus reflects upon the nature of Ukrainian literature in relation to its own romantic tradition, European modernism and Russian literature, often coming to critical conclusions and literary approaches that may appear unexpected and paradoxical for an intellectual usually deemed a model of traditional patriotism. Keywords: Vasil' Stus, Ukrainian poetry, Russian-Ukrainian Literary Intersections, Abstract Alessandro Achilli In Kafka (as much as in Gogol) the boundary of the awakening assumes an even more important role: there it is not only a new chronological piece of time that is about to begin, but also a new framework of interrelations and connections, which comes out to be completely different from anything that was 'yesterday.' A new reality begins: an 'unreal reality.' (Mann 1999: web)<sup>1</sup> In his essay entitled A Meeting in The Labyrinth. Franz Kafka and Nikolaj Gogol' (Vstreča v labirinte. Franz Kafka i Nikolaj Gogol', 1999), Jurij Vladimirovič Mann reflects upon the opportunity of finding symbolic points of intersection between the artistic experiences of the authors of such works as, respectively, The Trial (Der Process, 1925) and Dead Souls (Mertvye Duši, 1842). Reading the Russian literary critic's insights, their evocative 'meeting' lies mainly in the creation of a literary world split into two intersecting faces: both authors aimed to rewrite the romantic duality (dvoemirie) in a reduced form, crossing the line between the 'real world' and the 'world of the imagination" (Mann 1999: web)<sup>2</sup> . In both authors' works, the 'eternal discord between dream and reality,' as cried out by Piskarev in Gogol's Nevskij Prospekt (1835), can only be overcome in a purely literary dimension. Following these lines, it is the existential fracture experienced by both Kafka and Gogol' in their respective historical and cultural environments that underlies their programmatic will to trespass the limits of the 'awakening:' the first author, a German-speaking Jewish writer, experienced Prague at the fall of the Austro-Hungarian Empire, while the second one, writing in Russian from the core of the Empire, embodied the duality of the Ukrainian cultural experience. For this reason, their peculiar representation of the complex ‒ and conflicting ‒ relations between the figure of the artist with the changing surroundings goes beyond the stylistic, geographical, and temporal distance between their artistic worlds (Ibid.) 3 . Accordingly, Mann has wondered whether the strict ideological Firenze University Press <sup>1</sup> Unless otherwise indicated, translations from Russian are mine. <sup>2</sup> "And here we come to the most important trend leading from Gogol to Kafka; I would define it as the reduction of dvoemirie. The Romantic writer, as stated by Liliana Furst, always maintained a specific understanding of the parallelism between two worlds: 'the world of reality […] was always close to the world of imagination'". <sup>3</sup> "And here we need to mention another motive bringing together both the authors […] This is the motif of space, or ‒ rather ‒ the relationship of man to space, or ‒ to FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup\_best\_practice) Russia, Oriente slavo e Occidente europeo. Fratture e integrazioni nella storia e nella civiltà letteraria, a cura di Claudia Pieralli, Claire Delaunay, Eugène Priadko, ISBN 9788864535074 (online), CC BY 4.0, 2017 Marco Puleri, Between Kafka and Gogol'. 'De-territorialising' National Narrative(s) in Post-Soviet Ukrainian Literature in Russian, pp. 357-376, © 2017 Author(s), CC BY 4.0 International, DOI 10.36253/978-88-6453-507-4.26 [...] Ukraine came to its independence with a considerably distorted language situation. Russian as the language spread throughout the former empire ousted the national language on the vast territory of Ukraine, primarily in large industrial centers. Ukrainian-speaking communities in the Eastern and Southern regions of the country and to a lesser extent in the Central regions are limited to rural localities. Thus, nowadays Ukrainian is not the language of the country's absolute territorial or ethnic circulation, yet Russian has not completely superseded the Ukrainian lan- The ideologization of the language plays an important role in Post-soviet Ukraine, where the historical transition experienced in the early Nineties produced a complete overthrow of the benchmark values related to the language categories that had existed before national independence (see Bilaniuk 2009). In the national context, this has been played out in the contrast between exclusive language ideologies and inclusive cultural practices, giving rise to a complex model of self-positioning, particularly in the case of the Ukrainian Russophone community. The alternative outlooks on the configuration of the 'Ukrainian nation' lay on the different historical narratives of the area, leading to the institu- The case of Ukraine after the fall of Soviet power [...] presents a vivid example of a system in which both linguistic and social values have been shifting. The Ukrainian language, which had been marginalized and denigrated relative to Russian, has become increasingly used in public urban contexts and by political and cultural leaders, some of whom had themselves been marginalized in the Soviet system [...] In choices of language use and in debates about language, the previously dominant discourses clash with new discourses and practices elevating Ukrainian. Following these lines, the language issue still represents a contested benchmark even in defining what belongs ‒ and what does not ‒ to the national literary canon. Contextualising the Ukrainian cultural legacy, it is worth wondering about the specific cultural positioning of authors such as Nikolaj Gogol', Taras Ševčenko, Hryhorij Skovoroda and others who worked 'between' languages, traditions, and cultures. As Oleh Ilnitskyj (2003: 322) has stressed, "these individuals were products of a cross-cultural experience generally unfamiliar to ethnic Russians, but typical for members of Ukrainian society." This experience was "essentially liminal" and "dualistic in terms of language and institutions" (Ibid.). Especially throughout the nineteenth century, the reconceptualization of the Imperial cultural system into distinct national models was an ongoing and ever-changing process, establishing new ideological frontiers between the emerging literary phenomena. The rise of the Ukrainian literary system within the 'All-Russian' cultural context was thus 'filtered' by the use of the Imperial lingua franca. This phenomenon gave birth to a large body of literature in Russian written by Ukrainian authors, which emerged in a composite self-positioning guage even in the most assimilated regions of the country either. tionalization of cultural standards (ivi: 337): pattern (Ilchuk 2009: 21): interpretation of their literary roles, especially concerning the authors' identity dimension at the crossroads between different national cultural canons, could perhaps be an obstacle to a new understanding of their artistic relations, affecting their cultural legacy even at the eve of the New Millennium (Ibid.): "What can they have in common? On the one side there is an unconventional decadent, detached from the national soil, who does not believe in progress and in the creative forces of the people; on the other, there is literature full of mental health and 'looking to the future'". Between Kafka and Gogol'. This suggestive title reflects the path that will be undertaken in this paper, approaching the two authors as privileged interlocutors in order to analyze the specific artistic dimension of the newest category of writers that has emerged in Ukraine following the post-Soviet historical fracture. Through the lenses of the peculiar features characterizing Franz Kafka's and Nikolaj Gogol's literary experiences, we will be able to highlight the nuances characterizing the specific cultural positioning of contemporary Ukrainian literary production in Russian. Following Mann, we will also deal with a 'labyrinthine' spatiotemporal dimension, as stressed by the literary critic in his study, which gives birth not only to "a new chronological piece of time," but also to "a new framework of interrelations and connections, which comes out to be completely different from anything that was 'yesterday'" (Ibid.). A 'new reality' thus comes out of this process as well: precisely as the Russian scholar stressed, an 'unreal reality' emerges. Meeting with Gogol' and Kafka in the 'post-Soviet labyrinth,' we will be able to arrive at a new understanding of the cultural condition experienced by contemporary Ukrainian Russian-language writers. Moreover, by reading and interpreting Aleksej Nikitin's works, we will offer a new artistic attempt to recompose the fragments of the existential mosaics left unbound in the aftermath of the Soviet collapse. ### Two Worlds, Two Souls: Gogol' and The Ukrainian Dvoedušie4 Ukraine represents an interesting case study in highlighting the 'hybrid' condition experienced by the Russophone literary communities in the post-Soviet space (see Puleri 2015, 2016). Our analysis is concerned with the recent developments within the cultural identity process, showing the main dynamics that characterized the area before the so-called 'Ukrainian Crisis' (2014-15). Nowadays, Ukraine has the largest ethnic Russian minority in the post-Soviet area, but what is worth stressing is that a large part of the Ukrainian population is actually Russophone (Masenko 2008: 101-102): be even more precise ‒ the relationship of space to man […]". <sup>4</sup> An earlier version of the following sections was included in Puleri 2016. and 'looking to the future'". 'unreal reality' emerges. of the Soviet collapse. interpretation of their literary roles, especially concerning the authors' identity dimension at the crossroads between different national cultural canons, could perhaps be an obstacle to a new understanding of their artistic relations, affecting their cultural legacy even at the eve of the New Millennium (Ibid.): "What can they have in common? On the one side there is an unconventional decadent, detached from the national soil, who does not believe in progress and in the creative forces of the people; on the other, there is literature full of mental health Between Kafka and Gogol'. This suggestive title reflects the path that will be undertaken in this paper, approaching the two authors as privileged interlocutors in order to analyze the specific artistic dimension of the newest category of writers that has emerged in Ukraine following the post-Soviet historical fracture. Through the lenses of the peculiar features characterizing Franz Kafka's and Nikolaj Gogol's literary experiences, we will be able to highlight the nuances characterizing the specific cultural positioning of contemporary Ukrainian literary production in Russian. Following Mann, we will also deal with a 'labyrinthine' spatiotemporal dimension, as stressed by the literary critic in his study, which gives birth not only to "a new chronological piece of time," but also to "a new framework of interrelations and connections, which comes out to be completely different from anything that was 'yesterday'" (Ibid.). A 'new reality' thus comes out of this process as well: precisely as the Russian scholar stressed, an Meeting with Gogol' and Kafka in the 'post-Soviet labyrinth,' we will be able to arrive at a new understanding of the cultural condition experienced by contemporary Ukrainian Russian-language writers. Moreover, by reading and interpreting Aleksej Nikitin's works, we will offer a new artistic attempt to recompose the fragments of the existential mosaics left unbound in the aftermath Ukraine represents an interesting case study in highlighting the 'hybrid' condition experienced by the Russophone literary communities in the post-Soviet space (see Puleri 2015, 2016). Our analysis is concerned with the recent developments within the cultural identity process, showing the main dynamics that characterized the area before the so-called 'Ukrainian Crisis' (2014-15). Nowadays, Ukraine has the largest ethnic Russian minority in the post-Soviet area, but what is worth stressing is that a large part of the Ukrainian population 4 An earlier version of the following sections was included in Puleri 2016. Two Worlds, Two Souls: Gogol' and The Ukrainian Dvoedušie4 is actually Russophone (Masenko 2008: 101-102): be even more precise ‒ the relationship of space to man […]". [...] Ukraine came to its independence with a considerably distorted language situation. Russian as the language spread throughout the former empire ousted the national language on the vast territory of Ukraine, primarily in large industrial centers. Ukrainian-speaking communities in the Eastern and Southern regions of the country and to a lesser extent in the Central regions are limited to rural localities. Thus, nowadays Ukrainian is not the language of the country's absolute territorial or ethnic circulation, yet Russian has not completely superseded the Ukrainian language even in the most assimilated regions of the country either. The ideologization of the language plays an important role in Post-soviet Ukraine, where the historical transition experienced in the early Nineties produced a complete overthrow of the benchmark values related to the language categories that had existed before national independence (see Bilaniuk 2009). In the national context, this has been played out in the contrast between exclusive language ideologies and inclusive cultural practices, giving rise to a complex model of self-positioning, particularly in the case of the Ukrainian Russophone community. The alternative outlooks on the configuration of the 'Ukrainian nation' lay on the different historical narratives of the area, leading to the institutionalization of cultural standards (ivi: 337): The case of Ukraine after the fall of Soviet power [...] presents a vivid example of a system in which both linguistic and social values have been shifting. The Ukrainian language, which had been marginalized and denigrated relative to Russian, has become increasingly used in public urban contexts and by political and cultural leaders, some of whom had themselves been marginalized in the Soviet system [...] In choices of language use and in debates about language, the previously dominant discourses clash with new discourses and practices elevating Ukrainian. Following these lines, the language issue still represents a contested benchmark even in defining what belongs ‒ and what does not ‒ to the national literary canon. Contextualising the Ukrainian cultural legacy, it is worth wondering about the specific cultural positioning of authors such as Nikolaj Gogol', Taras Ševčenko, Hryhorij Skovoroda and others who worked 'between' languages, traditions, and cultures. As Oleh Ilnitskyj (2003: 322) has stressed, "these individuals were products of a cross-cultural experience generally unfamiliar to ethnic Russians, but typical for members of Ukrainian society." This experience was "essentially liminal" and "dualistic in terms of language and institutions" (Ibid.). Especially throughout the nineteenth century, the reconceptualization of the Imperial cultural system into distinct national models was an ongoing and ever-changing process, establishing new ideological frontiers between the emerging literary phenomena. The rise of the Ukrainian literary system within the 'All-Russian' cultural context was thus 'filtered' by the use of the Imperial lingua franca. This phenomenon gave birth to a large body of literature in Russian written by Ukrainian authors, which emerged in a composite self-positioning pattern (Ilchuk 2009: 21): . section between three cultural paradigms: the Ukrainian tradition, the Russian model, and the Imperial paradigm. This entails a positioning 'between cultures' that, as observed by Yulia Ilchuk (2009), implies an artistic experience moving in an intermediate space 'between languages'. It is the presence of Ukrainian and hybrid Russo-Ukrainian forms that confer a 'defamiliarizing effect' onto Gogol's literary language: "Positioned on the 'interstices' of two cultures, Gogol existed in the in-between space of cultural ambivalence that diluted the imaginary essence of the Russian nation through a "'distorted' Russian language" (Ilchuk 2009: 19). Thus, Gogol' gives birth to a 'transcultural' identity model, which lies outside the rigid parameters of 'national canonization' (Gogol' 1952: […] I only know that I would grant primacy neither to a Little Russian over a Russian nor to a Russian over a Little Russian. Both natures are generously endowed by god, and as if on purpose, each of them in its own way includes in itself that which the other lacks – a clear sign that they are meant to complement each other5 Gogol's/Hohol's dvoedušie reflects the duality of the Ukrainian cultural experience. The impracticable way to 'univocal canonization' lay in the fact that in the author's epoch, as stated by Grabowicz (1992: 224), "the very idea of what is to be a Ukrainian writer (and indeed a 'Ukrainian') was in a state of becoming". Actually, even in post-Soviet times, the ideological legacy of the Imperial and Soviet experience has refrained from an assimilation of the featuring duality of the national culture (see Shkandrij 2009: web)6. Nonetheless, nowadays it is just this kind of duality that could open the way to a new epistemological and cultu- Russian-language culture [...] has its representative throughout Ukraine, including in the West [...] They can no doubt identify with strange, in-between linguistic and cultural space [...] The vantage point of this space affords a perspective on culture and literature as phenomena that are never easy to define, since they are the product of complex histories, linguistic hybrids and entangled identities. These are things that are not always embraced in Ukraine or in Russia; they are rarely perceived by casual observers of Ukraine. Yet they are there, and they are part of the <sup>5</sup> "[...] никак бы не дал преимущества ни малороссиянину перед русским, <sup>6</sup> "Even though it is clear to all that there is a vast difference between a forced or imposed hybridity and a freely-assumed one, the imperial-Soviet experience has made this issue a painful one for Ukrainian intellectuals [...] To the 'anti-colonialists' hybrid- ни русскому пред малороссиянином. Обе природы слишком щедро одарены Богом, и как нарочно каждая из них порознь заключает в себе то, чего нет в другой, ‒ явный знак, что они должны пополнить одна другую". ral understanding of the post-Soviet area (Blacker 2014: web): everyday lives of millions in the country. ity damages the idea of a core tradition [...]". 418): Some writers, like Vasilii Kapnist, Somov, Narezhnyi and Gogol, maintained their regional Ukrainian identities while embracing Russian national identities; some, like Hryhorii Kvitka-Osnovianenko, Mykola Markevych, and Mykhail Drahomanov, existed as 'all-Russian'; and others, like Taras Shevchenko, Panteleimon Kulish, Marko Vovchok and Mykola Kostomarov, enjoyed a more or less separate Ukrainian identity. This artistic phenomenon arose from the contact between the different cultural and identity affiliations held by Ukrainian in-between literary actors. As observed by G. Grabowicz (1992: 232), this literary production "should indeed be considered part of Ukrainian literature," even if "there was an inescapable sense for virtually all these writers that Ukrainian literature was a subset of Imperial, All Russian literature." Nonetheless, the Ukrainian writers who gained success in the 'center' of the Empire played the important role of cultural mediators between the Russian and Ukrainian societies. In their literary depictions, the Ukrainian 'periphery' was transformed and adapted to make it accessible to Russian readership: "Implicitly if not explicitly, their work tended to minimize or aestheticize the differences between Russia and Ukraine, thus discounting the inherent autonomy or 'otherness' of the Ukrainian historical and cultural experience" (Andriewsky 2003: 184). The case of Nikolaj Gogol'/Mykola Hohol' (1809-1852) definitely embodies the fluid cultural dynamics of his epoch. The definition of his national identity has been at the core of intellectual and political debates in Russia and Ukraine, where his literary experience has been included in both the Russian canon (as Nikolaj Gogol') and in the Ukrainian one (as Mykola Hohol'). Reading his works, critics have mainly categorised it according to two different periods: the Ukrainian one (1829-1836), including the works devoted to 'national' themes, and the Imperial one (1836-1852). Nevertheless, throughout the last decades a huge body of literature on Gogol' has been issued, focusing especially on the hybrid aspects of the literary figure (see Grabowicz 1994; Luckyj 1998; Ilnytzkyj 2002; Bojanowska 2007). E. M. Bojanowska (2007: 6), in her study entitled Nikolaj Gogol. Between Ukrainian and Russian Nationalism, stresses how the author's national identity "cannot be framed as an either/or question [...] Whether Gogol was a Russian or a Ukrainian is thus the wrong question to ask." The periodization of Gogol's literary production into two distinct 'artistic phases' seems to address the complex duality of the author's experience by means of abstract ideological terms, ignoring the extraordinary patchwork of language, cultural and political elements involved in his identity formation. Gogol's 'inbetween' positioning underlies the ambivalence of the 'literary space' imagined by the author. As stressed by Myroslav Shkandrij (2001: 115), "Gogol brought a Ukrainian consciousness to St. Petersburg, that is, structures of thought and feeling that were deeply critical of Russian society, which he drew upon throughout his creative life". O. S. Ilnytzkyj (2002), moreover, has tried to define the artistic experience undertaken by Gogol'/Hohol' as the outcome of the inter- Ukrainian identity. rience" (Andriewsky 2003: 184). Some writers, like Vasilii Kapnist, Somov, Narezhnyi and Gogol, maintained their regional Ukrainian identities while embracing Russian national identities; some, like Hryhorii Kvitka-Osnovianenko, Mykola Markevych, and Mykhail Drahomanov, existed as 'all-Russian'; and others, like Taras Shevchenko, Panteleimon Kulish, Marko Vovchok and Mykola Kostomarov, enjoyed a more or less separate This artistic phenomenon arose from the contact between the different cultural and identity affiliations held by Ukrainian in-between literary actors. As observed by G. Grabowicz (1992: 232), this literary production "should indeed be considered part of Ukrainian literature," even if "there was an inescapable sense for virtually all these writers that Ukrainian literature was a subset of Imperial, All Russian literature." Nonetheless, the Ukrainian writers who gained success in the 'center' of the Empire played the important role of cultural mediators between the Russian and Ukrainian societies. In their literary depictions, the Ukrainian 'periphery' was transformed and adapted to make it accessible to Russian readership: "Implicitly if not explicitly, their work tended to minimize or aestheticize the differences between Russia and Ukraine, thus discounting the inherent autonomy or 'otherness' of the Ukrainian historical and cultural expe- The case of Nikolaj Gogol'/Mykola Hohol' (1809-1852) definitely embodies the fluid cultural dynamics of his epoch. The definition of his national identity has been at the core of intellectual and political debates in Russia and Ukraine, where his literary experience has been included in both the Russian canon (as Nikolaj Gogol') and in the Ukrainian one (as Mykola Hohol'). Reading his works, critics have mainly categorised it according to two different periods: the Ukrainian one (1829-1836), including the works devoted to 'national' themes, and the Imperial one (1836-1852). Nevertheless, throughout the last decades a huge body of literature on Gogol' has been issued, focusing especially on the hybrid aspects of the literary figure (see Grabowicz 1994; Luckyj 1998; Ilnytzkyj 2002; Bojanowska 2007). E. M. Bojanowska (2007: 6), in her study entitled Nikolaj Gogol. Between Ukrainian and Russian Nationalism, stresses how the author's national identity "cannot be framed as an either/or question [...] Whether Gogol was a Russian or a Ukrainian is thus the wrong question to ask." The periodization of Gogol's literary production into two distinct 'artistic phases' seems to address the complex duality of the author's experience by means of abstract ideological terms, ignoring the extraordinary patchwork of language, cultural and political elements involved in his identity formation. Gogol's 'inbetween' positioning underlies the ambivalence of the 'literary space' imagined by the author. As stressed by Myroslav Shkandrij (2001: 115), "Gogol brought a Ukrainian consciousness to St. Petersburg, that is, structures of thought and feeling that were deeply critical of Russian society, which he drew upon throughout his creative life". O. S. Ilnytzkyj (2002), moreover, has tried to define the artistic experience undertaken by Gogol'/Hohol' as the outcome of the intersection between three cultural paradigms: the Ukrainian tradition, the Russian model, and the Imperial paradigm. This entails a positioning 'between cultures' that, as observed by Yulia Ilchuk (2009), implies an artistic experience moving in an intermediate space 'between languages'. It is the presence of Ukrainian and hybrid Russo-Ukrainian forms that confer a 'defamiliarizing effect' onto Gogol's literary language: "Positioned on the 'interstices' of two cultures, Gogol existed in the in-between space of cultural ambivalence that diluted the imaginary essence of the Russian nation through a "'distorted' Russian language" (Ilchuk 2009: 19). Thus, Gogol' gives birth to a 'transcultural' identity model, which lies outside the rigid parameters of 'national canonization' (Gogol' 1952: 418): […] I only know that I would grant primacy neither to a Little Russian over a Russian nor to a Russian over a Little Russian. Both natures are generously endowed by god, and as if on purpose, each of them in its own way includes in itself that which the other lacks – a clear sign that they are meant to complement each other5 . Gogol's/Hohol's dvoedušie reflects the duality of the Ukrainian cultural experience. The impracticable way to 'univocal canonization' lay in the fact that in the author's epoch, as stated by Grabowicz (1992: 224), "the very idea of what is to be a Ukrainian writer (and indeed a 'Ukrainian') was in a state of becoming". Actually, even in post-Soviet times, the ideological legacy of the Imperial and Soviet experience has refrained from an assimilation of the featuring duality of the national culture (see Shkandrij 2009: web)6. Nonetheless, nowadays it is just this kind of duality that could open the way to a new epistemological and cultural understanding of the post-Soviet area (Blacker 2014: web): Russian-language culture [...] has its representative throughout Ukraine, including in the West [...] They can no doubt identify with strange, in-between linguistic and cultural space [...] The vantage point of this space affords a perspective on culture and literature as phenomena that are never easy to define, since they are the product of complex histories, linguistic hybrids and entangled identities. These are things that are not always embraced in Ukraine or in Russia; they are rarely perceived by casual observers of Ukraine. Yet they are there, and they are part of the everyday lives of millions in the country. <sup>5</sup> "[...] никак бы не дал преимущества ни малороссиянину перед русским, ни русскому пред малороссиянином. Обе природы слишком щедро одарены Богом, и как нарочно каждая из них порознь заключает в себе то, чего нет в другой, ‒ явный знак, что они должны пополнить одна другую". <sup>6</sup> "Even though it is clear to all that there is a vast difference between a forced or imposed hybridity and a freely-assumed one, the imperial-Soviet experience has made this issue a painful one for Ukrainian intellectuals [...] To the 'anti-colonialists' hybridity damages the idea of a core tradition [...]". . His literature turns out to be Through the lenses of Kafka: A Minor Perspective on Ukrainian There has been much discussion of the questions "What is a marginal literature?" and "What is a popular literature, a proletarian literature?" The criteria are obviously difficult to establish if one doesn't start with a more objective concept – that of minor literature. Only the possibility of setting up a minor practice of major language from within allows one to define popular literature, marginal literature, and so on. Only in this way can literature really become a collective machine of expression and really be able to treat and develop its contents. Kafka emphatically declares that a minor literature is much more able to work over its material (De- As observed by Gilles Deleuze and Félix Guattari, throughout his literary experience Franz Kafka (1883-1924) reflected upon "the problem of expression" in art, especially "in relation to those literatures that are considered minor, for example, the Jewish literature of Warsaw and Prague" (Deleuze, Guattari 1986: 16). In their work devoted to the analysis of Kafka's literary production, entitled Kafka: Toward a Minor Literature (Kafka. Pour une littérature mineure, 1975), the French philosophers strove to theorize the characterizing features of the 'minor' artistic paradigm. In their view, a minor literature comes to be defined as the one "that a minority constructs within a major language" (Ibid.). This is the condition experienced by Kafka, a Czech Jew writing in German: it is exactly "the situation of the German language in Czechoslovakia, as a fluid language intermixed with Czech and Yiddish" that "will allow Kafka the possi- "something impossible" due to "the impossibility of not writing, the impossibility of writing in German, [and] the impossibility of writing otherwise" (ivi: 16). In these conditions, art becomes the main 'line of escape,' "because national consciousness, uncertain or oppressed, necessarily exists by means of literature" (Ibid.). Thus, in search for a new self-positioning, a minor writer's main instru- There is nothing that is major or revolutionary except the minor. To hate all languages of masters. Kafka's fascination for servants and employees (the same thing in Proust in relation to servants, to their language). What interests him even more is the possibility of making of his own language – assuming that it is unique, <sup>8</sup> In this light it is worth mentioning, in particular, how complex was Kafka's relation to Yiddish, as stressed by Deleuze and Guattari (ivi: 25): "What fascinates him in Yiddish is less a language of a religious community than that of a popular theatre […] Yiddish is a language that frightens more than it invites disdain […] it is a language that is lacking a grammar and that is filled with vocables that are fleeting, mobilized, emigrating, and turned into nomads that interiorize 'relations of force.' It is a language that is grafted onto Middle High German and that so reworked the German language from within that one cannot translate it into German without destroying it". Literature in Russian leuze, Guattari 1986: 18-19). bility of invention" (Deleuze, Guattari 1986: 20)<sup>8</sup> ment is his own language (ivi: 26): We are dealing here with those authors who belong to "the millions of people" who live outside of the political borders of the Russian Federation and "who consider Russian to be their mother tongue," as stressed by Čuprinin (2008: 6) in his study Russian Literature Today: Abroad (Russkaja literatura segodnja: zarubež'e). In this case, the use of strict geographical or language criteria would clearly be inadequate. Such an approach would dismiss the composite nature of these literary practices: language rewords the peculiar patchwork made of heterogeneous cultural strata, undertaking artistic routes that can diverge from literary references pertaining to a single national model. Nowadays, as stressed by Michail Nazarenko (2005: 117-118), professor at the Taras Ševčenko National University of Kiev, it is the 'marginality' of the Ukrainian literature in Russian, as compared with both the Ukrainian and Russian cultural systems, that gives birth to an 'experiential-expressive' model that privileges narratives focusing on 'the man at the crossroads between languages, cultures and epochs': The Russian Literature of Ukraine reveals marginal features when compared to both its 'sisters' […] Its most interesting and valuable trait consists in its point of view on both cultures from the inside and the outside – simultaneously […] Russian literature in Ukraine is in need of finding its own characterising attributes […] in order to understand its unique, distinct and original nature […] and it is in need of understanding the tasks to be faced. This is needed in order to realize that its main object is the man at the crossroads between languages, cultures and epochs. Furthermore, the peculiar positioning of this literary phenomenon on the 'interstices' has allowed the cultural actors to follow different kinds of interactions with Ukrainophone and with Russian literatures. As the Russian critic Il'ja Kukulin stated in the introduction to his edited issue on Ukraino-Russian relations in Novoe Literaturnoe Obozrenie, "actually, the measure of the Ukraino-Russian relations is, first of all, the individual writer, and only then the literary groups, the periodicals, and so on" (Kukulin 2007: web)<sup>7</sup> . Thus, the unsystematic character of the Ukrainian literature in Russian does not let us define either its autonomy from or dependence on one of the respective cultural systems. Following these lines, an analytical description of these 'marginal' literary practices can only be undertaken by the recognition of their 'minor' nature. <sup>7</sup> "[...] Russian literature in Ukraine consists of several so-called 'sub-literatures,' which establish different kinds of interrelations with the literature in both Russian and Ukrainian: some authors are oriented towards the European postmodern style, others towards the uncensored traditionalist poetry of the 1970's, and others still towards the 'derevenskaja' prose […] Each author comes to be included immediately in several contexts, both literary and extraliterary: those who write in Russian in their everyday life face documents in Ukrainian, join everyday conversations in Ukrainian or 'surzhyk,' and so on. Actually, the measure of the Ukraino-Russian relations is, first of all, the individual writer, and only then the literary groups, the periodicals, and so on". We are dealing here with those authors who belong to "the millions of people" who live outside of the political borders of the Russian Federation and "who consider Russian to be their mother tongue," as stressed by Čuprinin (2008: 6) in his study Russian Literature Today: Abroad (Russkaja literatura segodnja: zarubež'e). In this case, the use of strict geographical or language criteria would clearly be inadequate. Such an approach would dismiss the composite nature of these literary practices: language rewords the peculiar patchwork made of heterogeneous cultural strata, undertaking artistic routes that can diverge from literary references pertaining to a single national model. Nowadays, as stressed by Michail Nazarenko (2005: 117-118), professor at the Taras Ševčenko National University of Kiev, it is the 'marginality' of the Ukrainian literature in Russian, as compared with both the Ukrainian and Russian cultural systems, that gives birth to an 'experiential-expressive' model that privileges narratives focusing on The Russian Literature of Ukraine reveals marginal features when compared to both its 'sisters' […] Its most interesting and valuable trait consists in its point of view on both cultures from the inside and the outside – simultaneously […] Russian literature in Ukraine is in need of finding its own characterising attributes […] in order to understand its unique, distinct and original nature […] and it is in need of understanding the tasks to be faced. This is needed in order to realize that its main object is the man at the crossroads between languages, cultures and epochs. Furthermore, the peculiar positioning of this literary phenomenon on the 'interstices' has allowed the cultural actors to follow different kinds of interactions with Ukrainophone and with Russian literatures. As the Russian critic Il'ja Kukulin stated in the introduction to his edited issue on Ukraino-Russian relations in Novoe Literaturnoe Obozrenie, "actually, the measure of the Ukraino-Russian relations is, first of all, the individual writer, and only then the literary tic character of the Ukrainian literature in Russian does not let us define either its autonomy from or dependence on one of the respective cultural systems. Following these lines, an analytical description of these 'marginal' literary prac- <sup>7</sup> "[...] Russian literature in Ukraine consists of several so-called 'sub-literatures,' which establish different kinds of interrelations with the literature in both Russian and Ukrainian: some authors are oriented towards the European postmodern style, others towards the uncensored traditionalist poetry of the 1970's, and others still towards the 'derevenskaja' prose […] Each author comes to be included immediately in several contexts, both literary and extraliterary: those who write in Russian in their everyday life face documents in Ukrainian, join everyday conversations in Ukrainian or 'surzhyk,' and so on. Actually, the measure of the Ukraino-Russian relations is, first of all, the individual writer, and only then the literary groups, the periodicals, and so on". tices can only be undertaken by the recognition of their 'minor' nature. . Thus, the unsystema- 'the man at the crossroads between languages, cultures and epochs': groups, the periodicals, and so on" (Kukulin 2007: web)<sup>7</sup> ## Through the lenses of Kafka: A Minor Perspective on Ukrainian Literature in Russian There has been much discussion of the questions "What is a marginal literature?" and "What is a popular literature, a proletarian literature?" The criteria are obviously difficult to establish if one doesn't start with a more objective concept – that of minor literature. Only the possibility of setting up a minor practice of major language from within allows one to define popular literature, marginal literature, and so on. Only in this way can literature really become a collective machine of expression and really be able to treat and develop its contents. Kafka emphatically declares that a minor literature is much more able to work over its material (Deleuze, Guattari 1986: 18-19). As observed by Gilles Deleuze and Félix Guattari, throughout his literary experience Franz Kafka (1883-1924) reflected upon "the problem of expression" in art, especially "in relation to those literatures that are considered minor, for example, the Jewish literature of Warsaw and Prague" (Deleuze, Guattari 1986: 16). In their work devoted to the analysis of Kafka's literary production, entitled Kafka: Toward a Minor Literature (Kafka. Pour une littérature mineure, 1975), the French philosophers strove to theorize the characterizing features of the 'minor' artistic paradigm. In their view, a minor literature comes to be defined as the one "that a minority constructs within a major language" (Ibid.). This is the condition experienced by Kafka, a Czech Jew writing in German: it is exactly "the situation of the German language in Czechoslovakia, as a fluid language intermixed with Czech and Yiddish" that "will allow Kafka the possibility of invention" (Deleuze, Guattari 1986: 20)<sup>8</sup> . His literature turns out to be "something impossible" due to "the impossibility of not writing, the impossibility of writing in German, [and] the impossibility of writing otherwise" (ivi: 16). In these conditions, art becomes the main 'line of escape,' "because national consciousness, uncertain or oppressed, necessarily exists by means of literature" (Ibid.). Thus, in search for a new self-positioning, a minor writer's main instrument is his own language (ivi: 26): There is nothing that is major or revolutionary except the minor. To hate all languages of masters. Kafka's fascination for servants and employees (the same thing in Proust in relation to servants, to their language). What interests him even more is the possibility of making of his own language – assuming that it is unique, <sup>8</sup> In this light it is worth mentioning, in particular, how complex was Kafka's relation to Yiddish, as stressed by Deleuze and Guattari (ivi: 25): "What fascinates him in Yiddish is less a language of a religious community than that of a popular theatre […] Yiddish is a language that frightens more than it invites disdain […] it is a language that is lacking a grammar and that is filled with vocables that are fleeting, mobilized, emigrating, and turned into nomads that interiorize 'relations of force.' It is a language that is grafted onto Middle High German and that so reworked the German language from within that one cannot translate it into German without destroying it". that it is a major language or has been – a minor utilization. To be a sort of stranger within his own language. Between Kafka and Gogol' 365 There we have the most important question to face: why is it all the same Ukrainian, and not a Russian enclave in Ukraine? The issue is not simple at all. But to answer this question as simply as possible [...] then, this is because the Germanlanguage literature of Prague is also not German, and neither is Austrian literature, while at the same time the Irish English-language literature is not English [...] And what can be said about the American, the Canadian and the Australian literatures? The Belgian literature is not French. The Latin-American literature is neither Spanish nor Portuguese. But what, then, is the determinant? It is the theme, the mentality, the traditions or the local color: it is this we need to understand and contest. These marginal voices have a collective value in terms of textualising (and recomposing) the post-Soviet historical fracture. Thus, Russian-language literature seeks mainly to re-discuss and to reword a 'history with holes:' narrative strategies built on the privileged representation of metamorphosis, identity transformation, and symbolic 'interstitiality' emerge within the frame of the Russophone perspective. Moreover, a peculiar kind of duality emerges in the literary production created by those authors who position themselves 'outside of' the contemporary national canons. This happens to be exactly because, borrowing Deleuze's and Guattari's words, "talent isn't abundant in a minor literature, that is, there are no possibilities for an individuated enunciation that would belong to this or that 'master' and that could be separated from a collective enunciation" (Deleuze, Guattari 1986: 17). Accordingly, meeting in the post-Soviet labyrinth with the Ukrainian Russian-language author Aleksej Nikitin (b. 1967, Kiev), we will be able to approach his literary mosaics by analysing his minor perspective on the 'new framework of interrelations and connections' that arose in the 'un- Of Other Spaces (and Of Other Times): Aleksej Nikitin's Literary In his essay entitled Of Other Spaces (Des Espaces Autres, 1984), published just after his death, Michel Foucault studied the interactions between the human being and her/his space perception. According to the French philosopher, "we live inside a set of relations that delineates sites that are irreducible to one another and absolutely not superimposable on one another" (Foucault 1984: 3). This coordinate system defines our consciousness of the space in which we live. However, quoting Foucault, "there are also in every culture, in every civilization, real places […] that are something like counter-sites, a kind of effectively enacted utopia in which the real sites that can be found within the culture are simultaneously represented, contested, and inverted" (Ibid). Thus, Foucault discerns the dimension of the utopia, a site devoid of any spatial references, from <sup>10</sup> For further information on Aleksej Nikitin and other contemporary Ukrainian real reality' of the post-Soviet epoch. Russian-language authors, see also Puleri 2014, 2016. Heterotopias10 Affected with a "high coefficient of deterritorialization" (ivi: 16), in minor literatures language comes to be the most effective 'political' vehicle in order "to express another possible community and to forge the means for another consciousness and another sensibility" (ivi: 17). This has also been the case of the Irish writers James Joyce (1882-1941) and Samuel Beckett (1906-1989), particularly "the use of English and of every language" by the novelist, and "the use of English and French" by the playwright (ivi: 19). According to the French philosophers, "we might as well say that the term minor no longer designates specific literatures but the revolutionary conditions for every literature within the heart of what is called great (or established) literature" (ivi: 18). Thus, on one hand, the 'major' writer is honoured with the role of 'canonical mirror' of the human passions; on the other hand, "the primary feature of any literature that is defined as minor is its exclusion from the canon, an exclusion that may on the face of it be as much on the grounds of purely aesthetic judgments as on those of racial or sexual discrimination" (Lloyd 1987: 20). In his works, the minor writer does not long for the recognition of the grades of 'representative authority' of the "human experience" (ivi: 20)<sup>9</sup> , precisely because of his 'marginal' perspective. This 'lack of representativeness' lies in "the oppositional relationship of the canon and the state" (ivi: 21) and is "the product of the biographical alienation of a German-speaking Czech Jew or of a Creole woman in the post-colonial Caribbean" (ivi: 22). It is the symbolic representation of 'non-identity' that becomes the key feature of this literary paradigm. Minor literatures focus on the crisis of the 'hegemonic' narratives on identity, rewriting the expressive forms of tradition by means of parody and frequent intertextual references. Thus, the authorial voice interacts with the dynamics of major literature and, at the same time, seeks to subvert them. Following this frame of reference, post-Soviet Ukrainian literature in Russian could be read as a minor perspective on the identity and artistic categories pertaining to both the Russian and the Ukrainian traditions. In a recent article published in the Novyj Mir's September 2015 issue, the Ukrainian writer Andrej Krasnjaščich (b. 1970) endeavours to address exactly the question concerning the 'canonization' of the Russophone literary phenomena emerging in post-Soviet Ukraine. By using the acronym Rusukrlit ‒ Russian Ukrainian Literature ‒ the author wants to stress its tight connections with both of the national cultural contexts. According to Krasnjaščich (2015: 174), language is not the only factor to be considered in such an analysis, which can also be noticed in the case of other 'minor' literary traditions: <sup>9</sup> "For it is exactly insofar as the writer represents not only his own private experience but 'elementary passions' that he becomes both representative and canonical". within his own language. the "human experience" (ivi: 20)<sup>9</sup> time, seeks to subvert them. other 'minor' literary traditions: that it is a major language or has been – a minor utilization. To be a sort of stranger Affected with a "high coefficient of deterritorialization" (ivi: 16), in minor literatures language comes to be the most effective 'political' vehicle in order "to express another possible community and to forge the means for another consciousness and another sensibility" (ivi: 17). This has also been the case of the Irish writers James Joyce (1882-1941) and Samuel Beckett (1906-1989), particularly "the use of English and of every language" by the novelist, and "the use of English and French" by the playwright (ivi: 19). According to the French philosophers, "we might as well say that the term minor no longer designates specific literatures but the revolutionary conditions for every literature within the heart of what is called great (or established) literature" (ivi: 18). Thus, on one hand, the 'major' writer is honoured with the role of 'canonical mirror' of the human passions; on the other hand, "the primary feature of any literature that is defined as minor is its exclusion from the canon, an exclusion that may on the face of it be as much on the grounds of purely aesthetic judgments as on those of racial or sexual discrimination" (Lloyd 1987: 20). In his works, the minor writer does not long for the recognition of the grades of 'representative authority' of tive. This 'lack of representativeness' lies in "the oppositional relationship of the canon and the state" (ivi: 21) and is "the product of the biographical alienation of a German-speaking Czech Jew or of a Creole woman in the post-colonial Caribbean" (ivi: 22). It is the symbolic representation of 'non-identity' that becomes the key feature of this literary paradigm. Minor literatures focus on the crisis of the 'hegemonic' narratives on identity, rewriting the expressive forms of tradition by means of parody and frequent intertextual references. Thus, the authorial voice interacts with the dynamics of major literature and, at the same Following this frame of reference, post-Soviet Ukrainian literature in Russian could be read as a minor perspective on the identity and artistic categories pertaining to both the Russian and the Ukrainian traditions. In a recent article published in the Novyj Mir's September 2015 issue, the Ukrainian writer Andrej Krasnjaščich (b. 1970) endeavours to address exactly the question concerning the 'canonization' of the Russophone literary phenomena emerging in post-Soviet Ukraine. By using the acronym Rusukrlit ‒ Russian Ukrainian Literature ‒ the author wants to stress its tight connections with both of the national cultural contexts. According to Krasnjaščich (2015: 174), language is not the only factor to be considered in such an analysis, which can also be noticed in the case of <sup>9</sup> "For it is exactly insofar as the writer represents not only his own private experience but 'elementary passions' that he becomes both representative and canonical". , precisely because of his 'marginal' perspec- There we have the most important question to face: why is it all the same Ukrainian, and not a Russian enclave in Ukraine? The issue is not simple at all. But to answer this question as simply as possible [...] then, this is because the Germanlanguage literature of Prague is also not German, and neither is Austrian literature, while at the same time the Irish English-language literature is not English [...] And what can be said about the American, the Canadian and the Australian literatures? The Belgian literature is not French. The Latin-American literature is neither Spanish nor Portuguese. But what, then, is the determinant? It is the theme, the mentality, the traditions or the local color: it is this we need to understand and contest. These marginal voices have a collective value in terms of textualising (and recomposing) the post-Soviet historical fracture. Thus, Russian-language literature seeks mainly to re-discuss and to reword a 'history with holes:' narrative strategies built on the privileged representation of metamorphosis, identity transformation, and symbolic 'interstitiality' emerge within the frame of the Russophone perspective. Moreover, a peculiar kind of duality emerges in the literary production created by those authors who position themselves 'outside of' the contemporary national canons. This happens to be exactly because, borrowing Deleuze's and Guattari's words, "talent isn't abundant in a minor literature, that is, there are no possibilities for an individuated enunciation that would belong to this or that 'master' and that could be separated from a collective enunciation" (Deleuze, Guattari 1986: 17). Accordingly, meeting in the post-Soviet labyrinth with the Ukrainian Russian-language author Aleksej Nikitin (b. 1967, Kiev), we will be able to approach his literary mosaics by analysing his minor perspective on the 'new framework of interrelations and connections' that arose in the 'unreal reality' of the post-Soviet epoch. ## Of Other Spaces (and Of Other Times): Aleksej Nikitin's Literary Heterotopias10 In his essay entitled Of Other Spaces (Des Espaces Autres, 1984), published just after his death, Michel Foucault studied the interactions between the human being and her/his space perception. According to the French philosopher, "we live inside a set of relations that delineates sites that are irreducible to one another and absolutely not superimposable on one another" (Foucault 1984: 3). This coordinate system defines our consciousness of the space in which we live. However, quoting Foucault, "there are also in every culture, in every civilization, real places […] that are something like counter-sites, a kind of effectively enacted utopia in which the real sites that can be found within the culture are simultaneously represented, contested, and inverted" (Ibid). Thus, Foucault discerns the dimension of the utopia, a site devoid of any spatial references, from <sup>10</sup> For further information on Aleksej Nikitin and other contemporary Ukrainian Russian-language authors, see also Puleri 2014, 2016. literary production lies his hometown, Kiev (see Krasnjaščich 2015: 177)11. According to Nikitin, this focus is rooted in the need for "a proper narrative of the capital in the late Eighties and in the Nineties" (Puleri 2016: 192). The marginal position of such an historical period in the 'major' narratives devoted to Kiev makes the author's textualisation an important practice of resignification. In order to fill this blank space in the collective memory of his community, Nikitin symbolically chooses to follow the game dynamics. Thus, in Istemi (2011), the 'invention' of history lies in the creation of a world made of new imaginary states: in 1984, five students of Kiev University invent a fictional role-playing game based on historical events and set in the territories of the former Soviet Union. Within the time frame, which goes until 2004, the borders between past In the novel, the synthesis of real and fictional elements works on different narrative levels. Furthermore, Istemi is also the outcome of a 'rewriting' process: "Do you remember The Black Book and Shwambraniya? That's where we got the idea. Lev Kassil…12" admits the protagonist Davydov during an interrogation in the KGB's offices. This passage refers to the Soviet novel written in 1928-1931 by Lev Kassil' (1905-1970) and based on an autobiographical subject. In The Black Book and Shwambraniya (Konduit i Švambranija), likewise, two boys 'invent' their history, setting it in an imaginary country: Švambranija. Also in this case, the developments occurring in the game reflect the advent of the Revolution in real life, mixing historical characters and settings with fantasy. By intersecting different temporal strata and constructing 'other' spaces, Nikitin has the textual instruments apt to recompose the fragmented identity of his characters. In Istemi, the Russian-language author aims to represent "history as a black hole […] rather than a utopian repository of Truth" (Chernetsky 2007: 93-94). Nikitin's characters gain awareness of their precarity by means of a constant dialogue with the Ukrainian capital space, which embodies the true and present come to be blurred in the Kiev heterotopic space. etherotopic mirror of their existential condition (Nikitin 2011: 122-123): No metaphysics13. most Kievan' contemporary writer". tin 2011: 53). Translated by Anne Marie Jackson (2013). In the intervening years nothing had changed here. Everything was the same, the street, Castle Hill, the heaviness of the raw evening sky […] Here Was Borichev, the Church of the Mother of God that they'd finished rebuilding ten years earlier. It was a dead place. Here it seemed that everything was the same as it had ever been: the howling dogs, the old snow at the beginning of spring, the incredible colours of the evening sky. Even the smells were the same. Even Castle Hill. But the bridge to the cosmos had been destroyed. It was gone. There was no cosmos. <sup>11</sup> "Everything is about Kiev. Kiev is everywhere [...] No doubt, Nikitin is 'the <sup>12</sup> "Кондуит и Швамбрания. Помните? Идея – оттуда. Лев Кассиль…" (Niki- <sup>13</sup> "Здесь ничего не изменилось за прошедшие годы, все осталось таким же: улицы, Замковая, тяжесть сырого вечернего неба […] Вот Боричев, вот церковь Успенья Богородицы, заново отстроенная десять лет назад. Мертвое место. Здесь, the heterotopia, which describes those spaces "that are absolutely different from all the sites that they reflect or speak about" (ivi: 4). In order to understand their peculiar functioning, the French philosopher metaphorically describes heterotopias as mirrors, which are able to re-signify reality in other, 'unreal' spatial dimensions (Foucault 1984: 4): The mirror is, after all, a utopia, since it is a placeless place [...] But it is also a heterotopia in so far as the mirror does exist in reality, where it exerts a sort of counteraction on the position that I occupy. From the standpoint of the mirror I discover my absence from the place where I am since I see myself over there. Starting from this gaze that is, as it were, directed toward me, from the ground of this virtual space that is on the other side of the glass, I come back toward myself; I begin again to direct my eyes toward myself and to reconstitute myself there where I am. The mirror functions as a heterotopia in this respect: it makes this place that I occupy at the moment when I look at myself in the glass at once absolutely real, connected with all the space that surrounds it, and absolutely unreal, since in order to be perceived it has to pass through this virtual point which is over there. Heterotopic mirroring enacts a process of metamorphosis: an identity transformation that involves both the man and the space. Thus, borrowing Foucault's words, it is possible to envision this spatial trope also as a different kind of textualisation in order to reword the 'major' cultural narratives: "Heterotopias are disturbing, probably because they secretly undermine language […] as they destroy 'syntax' in advance, and not only the syntax with which we construct sentences, but also that less apparent syntax that causes words and things […] to 'hold together'" (Foucault 1970: xxviii). By calling a text heterotopic, we mean that "it is preoccupied with an exploration of those topoi ‒ cultural, social, linguistic ‒ that lie on the margins of the traditionally privileged literary discourses" (Chernetsky 2007: 91). It is exactly by means of his depiction of 'other spaces' and 'other times' that Aleksej Nikitin endeavours to recompose the compensatory illusion of his epoch. In an attempt to de-territorialize the post-Soviet experience, the Ukrainian Russian-language writer constructs his texts as 'literary heterotopias.' The duality of the spatial dimension portrayed by the Russophone author 'mirrors' the ongoing metamorphosis of the people who experience this space, a recurring theme in Nikitin's works that is emblematically described by Krasnjaščich (2015: 177) as the 'mystery of binary human nature:' The question that lies at the core of Nikitin's entire literary production concerns the mystery of binary human nature. This is a mystery that cannot be solved, but that we nevertheless need to try to answer, because our attempts at least reconcile with the fact that anyone who today is a friend-comrade-brother or beloved will imperceptibly turn into something foreign and hostile tomorrow. In his novels, the author focuses on the late Soviet years, retracing the period of transition that preceded the 'historical catastrophe.' At the core of his dimensions (Foucault 1984: 4): discourses" (Chernetsky 2007: 91). (2015: 177) as the 'mystery of binary human nature:' imperceptibly turn into something foreign and hostile tomorrow. the heterotopia, which describes those spaces "that are absolutely different from all the sites that they reflect or speak about" (ivi: 4). In order to understand their peculiar functioning, the French philosopher metaphorically describes heterotopias as mirrors, which are able to re-signify reality in other, 'unreal' spatial The mirror is, after all, a utopia, since it is a placeless place [...] But it is also a heterotopia in so far as the mirror does exist in reality, where it exerts a sort of counteraction on the position that I occupy. From the standpoint of the mirror I discover my absence from the place where I am since I see myself over there. Starting from this gaze that is, as it were, directed toward me, from the ground of this virtual space that is on the other side of the glass, I come back toward myself; I begin again to direct my eyes toward myself and to reconstitute myself there where I am. The mirror functions as a heterotopia in this respect: it makes this place that I occupy at the moment when I look at myself in the glass at once absolutely real, connected with all the space that surrounds it, and absolutely unreal, since in order to be perceived it has to pass through this virtual point which is over there. Heterotopic mirroring enacts a process of metamorphosis: an identity transformation that involves both the man and the space. Thus, borrowing Foucault's words, it is possible to envision this spatial trope also as a different kind of textualisation in order to reword the 'major' cultural narratives: "Heterotopias are disturbing, probably because they secretly undermine language […] as they destroy 'syntax' in advance, and not only the syntax with which we construct sentences, but also that less apparent syntax that causes words and things […] to 'hold together'" (Foucault 1970: xxviii). By calling a text heterotopic, we mean that "it is preoccupied with an exploration of those topoi ‒ cultural, social, linguistic ‒ that lie on the margins of the traditionally privileged literary It is exactly by means of his depiction of 'other spaces' and 'other times' that Aleksej Nikitin endeavours to recompose the compensatory illusion of his epoch. In an attempt to de-territorialize the post-Soviet experience, the Ukrainian Russian-language writer constructs his texts as 'literary heterotopias.' The duality of the spatial dimension portrayed by the Russophone author 'mirrors' the ongoing metamorphosis of the people who experience this space, a recurring theme in Nikitin's works that is emblematically described by Krasnjaščich The question that lies at the core of Nikitin's entire literary production concerns the mystery of binary human nature. This is a mystery that cannot be solved, but that we nevertheless need to try to answer, because our attempts at least reconcile with the fact that anyone who today is a friend-comrade-brother or beloved will In his novels, the author focuses on the late Soviet years, retracing the period of transition that preceded the 'historical catastrophe.' At the core of his literary production lies his hometown, Kiev (see Krasnjaščich 2015: 177)11. According to Nikitin, this focus is rooted in the need for "a proper narrative of the capital in the late Eighties and in the Nineties" (Puleri 2016: 192). The marginal position of such an historical period in the 'major' narratives devoted to Kiev makes the author's textualisation an important practice of resignification. In order to fill this blank space in the collective memory of his community, Nikitin symbolically chooses to follow the game dynamics. Thus, in Istemi (2011), the 'invention' of history lies in the creation of a world made of new imaginary states: in 1984, five students of Kiev University invent a fictional role-playing game based on historical events and set in the territories of the former Soviet Union. Within the time frame, which goes until 2004, the borders between past and present come to be blurred in the Kiev heterotopic space. In the novel, the synthesis of real and fictional elements works on different narrative levels. Furthermore, Istemi is also the outcome of a 'rewriting' process: "Do you remember The Black Book and Shwambraniya? That's where we got the idea. Lev Kassil…12" admits the protagonist Davydov during an interrogation in the KGB's offices. This passage refers to the Soviet novel written in 1928-1931 by Lev Kassil' (1905-1970) and based on an autobiographical subject. In The Black Book and Shwambraniya (Konduit i Švambranija), likewise, two boys 'invent' their history, setting it in an imaginary country: Švambranija. Also in this case, the developments occurring in the game reflect the advent of the Revolution in real life, mixing historical characters and settings with fantasy. By intersecting different temporal strata and constructing 'other' spaces, Nikitin has the textual instruments apt to recompose the fragmented identity of his characters. In Istemi, the Russian-language author aims to represent "history as a black hole […] rather than a utopian repository of Truth" (Chernetsky 2007: 93-94). Nikitin's characters gain awareness of their precarity by means of a constant dialogue with the Ukrainian capital space, which embodies the true etherotopic mirror of their existential condition (Nikitin 2011: 122-123): In the intervening years nothing had changed here. Everything was the same, the street, Castle Hill, the heaviness of the raw evening sky […] Here Was Borichev, the Church of the Mother of God that they'd finished rebuilding ten years earlier. It was a dead place. Here it seemed that everything was the same as it had ever been: the howling dogs, the old snow at the beginning of spring, the incredible colours of the evening sky. Even the smells were the same. Even Castle Hill. But the bridge to the cosmos had been destroyed. It was gone. There was no cosmos. No metaphysics13. <sup>11</sup> "Everything is about Kiev. Kiev is everywhere [...] No doubt, Nikitin is 'the most Kievan' contemporary writer". <sup>12</sup> "Кондуит и Швамбрания. Помните? Идея – оттуда. Лев Кассиль…" (Nikitin 2011: 53). Translated by Anne Marie Jackson (2013). <sup>13</sup> "Здесь ничего не изменилось за прошедшие годы, все осталось таким же: улицы, Замковая, тяжесть сырого вечернего неба […] Вот Боричев, вот церковь Успенья Богородицы, заново отстроенная десять лет назад. Мертвое место. Здесь, and intimate gaze of its last great narrators in the twentieth century: Michail Bulgakov (1891-1940) and Viktor Nekrasov (1911-1987). Both Bulgakov in The White Guard (Belaja Gvardija, 1924) and Nekrasov in his Notes of an Idler (Zapiski Zevaki, 1976) recorded the familiar historical memory of a 'lost city.' It is however through a deeper glance at Nikitin's Kiev, where "life has never been snuffed out" (Nikitin 2011: 179)16, that we can understand how the Russian narrative of the city, which describes Kiev as fallen in an 'eternal dream', and the Ukrainian one, which depicts it as a 'holy city', 'out of time', can intersect Somewhere in here we witness the main crossroads between the Russian and the Ukrainian images of Kiev (Gogol, as always, lies at the intersection). If in the Russian tradition Kiev is frozen in an absolute past, has fallen in an eternal sleep (golden or nightmarish), and has turned into a sacred graveyard, then in the Ukrainian tradition the sacred and ancient image of the city instead remains timeless: from this point of view, the modern Kiev, fussy and profane, is just another link in that unbroken chain that began, following Nestor the Chronicler, already in Apostle Andrew's times. This duality of Kiev's image is primarily due to the real history of the city, where periods of rapid rise were followed by decades of decline and The duality embodied by Kiev reflects the dvoedušie experienced by Davydov-Istemi. The protagonist's 'duplicity' finds its 'heterotopic mirror' in the textual space created by Nikitin. If in the last pages of Istemi Davydov can still glimpse the Zamkova hill, which is "already disappearing into the night" (Nikitin 2011: 197)17, it is in Nikitin's second novel that the Kiev hills are there to convey a warning to its inhabitants. In Mahjong (Madžong, 2012), in the urban space imagined by the Russophone author, the remaining bastions of the national culture are eroded by the new post-Soviet 'winds of change' (Nikitin <sup>16</sup> "It's not for no reason that human beings have lived for thousands of years on these high clay banks, not wishing to leave them. Whatever the circumstances – and at times the curcumstances were gut-wrenching and life grew utterly unbearable – life has never been snuffed out. Something keeps us here, replenishing us with the force of life. Come what may, the force of life has always been abundant in the Kiev hills [Все-таки не зря последнюю пару тысяч лет на этом крутом и глинистом берегу реки суетятся люди, не желая его оставлять. Как бы ни складывались обстоятельства, а временами они складывались очень кисло и жизнь здесь становилась невыносимой, полностью, все же, она не пресекалась никогда. Чтото держит нас на этом месте, наполняя жизненной силой. Чего-чего, а жизненной силы на киевских холмах всегда было в избытке]". Translated by Anne Marie Jack- <sup>17</sup> "Я стоял напротив Замковой, но гора уже ушла в ночь. Я различал только ее силуэт [...]". Translated by Anne Marie Jackson (2013). (Kochanovskaja, Nazarenko 2012: web): immersion into an ahistorical stillness. 2012: 358): son (2013). In this excerpt from the novel, Kiev is portrayed as the victim of its different historical narratives. Davydov's reflections recall the Soviet past and how it strove for the creation of a 'bridge to the cosmos,' that is, the artificial alternative to real life under the regime. On the other hand, Istemi, the last lord of the Zaporižian Khanate, is Davydov's alternative, the alter ego chosen by the protagonist in the historical game played with his friends (ivi: 67-68): Later, sitting up on Castle Hill and looking down at Kiev in May, I knew, with a distinct and vivid certainty, that our biggest problems were behind us and nothing worse would happen. Could there really be something worse than the prison inside the KGB building? […] I haven't been back up Castle Hill since then. Probably for no good reason. The view from there is marvellous. Marvellous and very precise ‒ no aberrations, no distortions. Now, twenty years on, I can see that the hill was right and I was wrong. But what can you take from me now? […] I'm now a peddler of fizzy drinks, and my affairs no longer take me to Castle Hill. But back then…Then, Istemi was behind me, and we were equals. Not in everything, but in some ways we were. And Castle Hill knew it14. Throughout the novel, Kiev is represented as a universal place. The holy hills of the capital preserve Kiev's historical prominence, as conveyed by the traditional textualisation of the city's secular image (see Kochanovskaja, Nazarenko 2012: web)15. Nevertheless, Nikitin's narrative on Kiev recovers and integrates different traditions. On a first reading, it seems to recall the nostalgic <sup>14</sup> "Тогда, сидя на Замковой горе и глядя на майский Киев, я понимал отчетливо и ясно, что самые серьезные неприятности позади, и хуже чем было ‒ не будет. Может ли быть что-то хуже внутренней тюрьмы КГБ? […] С тех пор я не поднимался на Замковую. Наверное, зря. С нее открывается удивительный вид. Удивительный и очень точный. Никаких аберраций, никаких искажений. Сейчас, двадцать лет спустя, я понимаю: права тогда была гора, а я ошибался. Но, что теперь с меня возьмешь? […] Теперь я торговец водой, и мне больше нечего делать на Замковой. А тогда... Тогда за мной был Истеми, и мы были равны. Пусть не во всем, но в чем-то были. И Замковая признавала это равенство". Translated by Anne Marie Jackson (2013). <sup>15</sup> "The brightest example is Gogol [...] and philologists have began to argue about the chthonic and magical nature of Gogol's Kiev. Meanwhile, it is enough to look without prejudice at Mirgorod and Dikanka in order to see that to Gogol Kiev, as Dikanka itself, is the heart of an ordered existence, a safely protected place [...] And this is not Gogol's individual perspective. Kiev was perceived in a similar way by Shevchenko, and not only in his poetry, but also in his prose [...] It is the Ukrainian variant of one of the main ideologemes related to Kiev. 'Kiev is the second Jerusalem:' it is a holy city that, by definition, stands on the hill in the center of the world. This image, which has been secularised for obvious reasons, survived the Soviet power and, becoming a cliché, has come into our days [...]". вроде бы, все, как всегда: лай собак, старый снег в начале весны, невообразимые цвета вечернего неба. Даже запахи не изменились. Даже Замковая. Но мост в космос разрушен. Его нет. Никакого космоса. Никакой метафизики". Translated by Anne Marie Jackson (2013). we were. And Castle Hill knew it14. Anne Marie Jackson (2013). Anne Marie Jackson (2013). has come into our days [...]". In this excerpt from the novel, Kiev is portrayed as the victim of its different historical narratives. Davydov's reflections recall the Soviet past and how it strove for the creation of a 'bridge to the cosmos,' that is, the artificial alternative to real life under the regime. On the other hand, Istemi, the last lord of the Zaporižian Khanate, is Davydov's alternative, the alter ego chosen by the Later, sitting up on Castle Hill and looking down at Kiev in May, I knew, with a distinct and vivid certainty, that our biggest problems were behind us and nothing worse would happen. Could there really be something worse than the prison inside the KGB building? […] I haven't been back up Castle Hill since then. Probably for no good reason. The view from there is marvellous. Marvellous and very precise ‒ no aberrations, no distortions. Now, twenty years on, I can see that the hill was right and I was wrong. But what can you take from me now? […] I'm now a peddler of fizzy drinks, and my affairs no longer take me to Castle Hill. But back then…Then, Istemi was behind me, and we were equals. Not in everything, but in some ways Throughout the novel, Kiev is represented as a universal place. The holy hills of the capital preserve Kiev's historical prominence, as conveyed by the traditional textualisation of the city's secular image (see Kochanovskaja, Nazarenko 2012: web)15. Nevertheless, Nikitin's narrative on Kiev recovers and integrates different traditions. On a first reading, it seems to recall the nostalgic вроде бы, все, как всегда: лай собак, старый снег в начале весны, невообразимые цвета вечернего неба. Даже запахи не изменились. Даже Замковая. Но мост в космос разрушен. Его нет. Никакого космоса. Никакой метафизики". Translated by <sup>14</sup> "Тогда, сидя на Замковой горе и глядя на майский Киев, я понимал отчетливо и ясно, что самые серьезные неприятности позади, и хуже чем было ‒ не будет. Может ли быть что-то хуже внутренней тюрьмы КГБ? […] С тех пор я не поднимался на Замковую. Наверное, зря. С нее открывается удивительный вид. Удивительный и очень точный. Никаких аберраций, никаких искажений. Сейчас, двадцать лет спустя, я понимаю: права тогда была гора, а я ошибался. Но, что теперь с меня возьмешь? […] Теперь я торговец водой, и мне больше нечего делать на Замковой. А тогда... Тогда за мной был Истеми, и мы были равны. Пусть не во всем, но в чем-то были. И Замковая признавала это равенство". Translated by <sup>15</sup> "The brightest example is Gogol [...] and philologists have began to argue about the chthonic and magical nature of Gogol's Kiev. Meanwhile, it is enough to look without prejudice at Mirgorod and Dikanka in order to see that to Gogol Kiev, as Dikanka itself, is the heart of an ordered existence, a safely protected place [...] And this is not Gogol's individual perspective. Kiev was perceived in a similar way by Shevchenko, and not only in his poetry, but also in his prose [...] It is the Ukrainian variant of one of the main ideologemes related to Kiev. 'Kiev is the second Jerusalem:' it is a holy city that, by definition, stands on the hill in the center of the world. This image, which has been secularised for obvious reasons, survived the Soviet power and, becoming a cliché, protagonist in the historical game played with his friends (ivi: 67-68): and intimate gaze of its last great narrators in the twentieth century: Michail Bulgakov (1891-1940) and Viktor Nekrasov (1911-1987). Both Bulgakov in The White Guard (Belaja Gvardija, 1924) and Nekrasov in his Notes of an Idler (Zapiski Zevaki, 1976) recorded the familiar historical memory of a 'lost city.' It is however through a deeper glance at Nikitin's Kiev, where "life has never been snuffed out" (Nikitin 2011: 179)16, that we can understand how the Russian narrative of the city, which describes Kiev as fallen in an 'eternal dream', and the Ukrainian one, which depicts it as a 'holy city', 'out of time', can intersect (Kochanovskaja, Nazarenko 2012: web): Somewhere in here we witness the main crossroads between the Russian and the Ukrainian images of Kiev (Gogol, as always, lies at the intersection). If in the Russian tradition Kiev is frozen in an absolute past, has fallen in an eternal sleep (golden or nightmarish), and has turned into a sacred graveyard, then in the Ukrainian tradition the sacred and ancient image of the city instead remains timeless: from this point of view, the modern Kiev, fussy and profane, is just another link in that unbroken chain that began, following Nestor the Chronicler, already in Apostle Andrew's times. This duality of Kiev's image is primarily due to the real history of the city, where periods of rapid rise were followed by decades of decline and immersion into an ahistorical stillness. The duality embodied by Kiev reflects the dvoedušie experienced by Davydov-Istemi. The protagonist's 'duplicity' finds its 'heterotopic mirror' in the textual space created by Nikitin. If in the last pages of Istemi Davydov can still glimpse the Zamkova hill, which is "already disappearing into the night" (Nikitin 2011: 197)17, it is in Nikitin's second novel that the Kiev hills are there to convey a warning to its inhabitants. In Mahjong (Madžong, 2012), in the urban space imagined by the Russophone author, the remaining bastions of the national culture are eroded by the new post-Soviet 'winds of change' (Nikitin 2012: 358): <sup>16</sup> "It's not for no reason that human beings have lived for thousands of years on these high clay banks, not wishing to leave them. Whatever the circumstances – and at times the curcumstances were gut-wrenching and life grew utterly unbearable – life has never been snuffed out. Something keeps us here, replenishing us with the force of life. Come what may, the force of life has always been abundant in the Kiev hills [Все-таки не зря последнюю пару тысяч лет на этом крутом и глинистом берегу реки суетятся люди, не желая его оставлять. Как бы ни складывались обстоятельства, а временами они складывались очень кисло и жизнь здесь становилась невыносимой, полностью, все же, она не пресекалась никогда. Чтото держит нас на этом месте, наполняя жизненной силой. Чего-чего, а жизненной силы на киевских холмах всегда было в избытке]". Translated by Anne Marie Jackson (2013). <sup>17</sup> "Я стоял напротив Замковой, но гора уже ушла в ночь. Я различал только ее силуэт [...]". Translated by Anne Marie Jackson (2013). as well as History, is subject to fate. Man is bound to go through in his desperate search for an absolute and an unambiguous narrative: a search that can never be satisfied. At the end of the 'game,' the different narrative levels symbolically intertwine in the "last unnumbered chapter," a spatial dimension where Ženja becomes the shaman Kara Gerzen. Under these guises, the protagonist finally What's the manuscript? ‒ "Dead Souls." Nikolaj Vasil'evič Gogol'. Part Three. ‒ Great. I sincerely congratulate you. ‒ Why? ‒ Because you are in good company now. – With Gogol'? Thanks. ‒ If it was just with him…well, what's wrong with your manuscript? – It does not exist. Ok, it existed before. I read some pages. ‒ But then, when it came the time to place Hen Tamgan, it turned out that there was no manuscript, didn't it? ‒ Yes. That's how it turned out [...] ‒ That's all right. Actually, the manuscript does not exist. It does not and never did exist. You have not written it yet. ‒ Me? That's me who did not write it? [...] ‒ No one wrote it. Neither "More than everything else, that story looked like a game" (ivi: 370), comments the narrator in the last pages of Madžong. In addition to the well-structured stylisation of Gogol's prose in the imaginary passages from the third volume of Dead Souls, Nikitin injects his authorial intrusions and historical digressions to consolidate his rewriting of tradition. Thus, the writer can disguise himself as Old Kačalov, one of the Mahjong players, and re-appropriate his voice to formu- [...] The Russian language was created by Ukrainians and it should be recognised abroad as Ukrainian property. Kačalov began with the Primary Chronicle, which was written only three hundred meters from his office, and did not forget anyone. In his list it was not only the theologians from Mohyla Academy, who were invited by Patriarch Nikon to Moscow to put in order the church books, that needed to be included, but also all the renown Ukrainian nobles and raznochincy <sup>20</sup> "Что за рукопись? – 'Мертвые души'. Николай Васильевич Гоголь. Том третий. – Прекрасно. От души тебя поздравляю. – С чем? – Ты попал в хорошую компанию. – К Гоголю? Спасибо. ‒ Если бы только к нему... Ладно. Что же не так с твоей рукописью? – Ее нет. Прежде она была. Ее читали, держали в руках. Я сам видел несколько страниц. – А когда пришло время наложить хэн тамган, оказалось, что рукописи нет. Правильно? – Да. Именно так и оказалось [...] – Тогда все в порядке. Дело в том, что этой рукописи действительно нет. Нет и никогда не было. Ты ее еще не написал. – Я? Не написал? [...] – Никто не написал. Ни ты, ни <sup>21</sup> "[...] русский язык создан украинцами и его следует признать собственностью Украины за границей. Качалов начал с "Повести временных лет", написанной в трехстах метрах от его офиса, и не забыл никого. В записке были перечислены все богословы Могилянской Академии, которых Патриарх Никон has the power to change the course of 'History' (Nikitin 2012: 381): you nor Gogol. Nor anybody else20. late his 'historical truth' (ivi: 72-73): who wrote in Russian<sup>21</sup>. Гоголь. Ни кто-то другой". A gust of wind coming from Dnipro can rip the hat off the careless passer-by in Marinsky Park. It can tear away the child's balloon, taking it over the crystalline skies of Kiev. Don't cry, baby. Don't cry. Get used to it. Not far from here, there are other winds, other hurricanes, which echo. And don't compare the winds coming from Dnipro with the dry and dusty ones that are eroding the country, which are rising from the bustle coming from Hrushevsky and Bankova Streets […] in Kiev, as some are wearing away and building up the soil of the historical hill of Shchekavytsa, while Sofia and Lavra are trying to hold on with all their remaining energies. How long is it going to last? […] and what can be said of us, disunited and weak, who vivaciously argue over trivial matters, invented out of a whole cloth?18. In Madžong, Kiev is crowded with failed writers, ambitious bukinisty, and unscrupulous billionaires, all striving to seize a 'priceless manuscript.' In this novel as well, the game dynamics underlie Nikitin's literary world, employed in order to re-write tradition and to re-appropriate History. It is the trilogy of Mertvye duši as planned by Nikolaj Gogol', the contested father of Ukrainian literature in Russian, that is to be the target of a complex rewriting. Gogol's project was to write a great 'epic poem in prose' on the Russian Empire, which was to be structured in three parts. It would narrate the journey of the protagonist, Pavel Ivanovič Čičikov, following lines of development close to Dante's Comedy. Legend has it that the second part of the trilogy was destroyed by Gogol' shortly before his death, while the drafting of the third part never even started. In Madžong, the main plot concerns the fortuitous finding of the fragments of a supposed Dead Souls third volume19. On the one hand, the protagonist is a failed philologist, Ženja L'vov, whose 'unfinished' doctoral dissertation was devoted to studying the 'evolution of Čičikov's developments' in Gogol's 'missing' trilogy. On the other, the demiurges of the story come to be the four players of Mahjong: throughout the novel, their matches open the chapters and their moves upset the balance of the exhausting search for Gogol's volume. As stressed by the literary critic V. Toporov (2012), we witness a "Russian prose ‒ built on a Ukrainian subject ‒ and clearly oriented towards Western models (Borges, Cortázar, Umberto Eco, Pérez-Reverte ‒ here on this line)." Literature, <sup>18</sup> "Порыв свежего ветра с Днепра может сорвать шляпу с неосторожного прохожего в Мариинском парке, может выхватить шарик у ребенка и унести его в ясно-голубое киевское небо. Не плачь, детка. Не плачь. Привыкай. Совсем рядом ревут другие ветры, другие ураганы. И не сравниться ветру с Днепра с иссушающими страну самумами, поднятыми шелестом на улицах Грушевского и Банковой ...срезают и застраивают в Киеве историческую Щекавицу, из последних сил держатся София и Лавра. Долго ли продержатся? […] то что же говорить о нас, разъединенных и слабых, радостно грызущихся из-за выдуманных, из пальца высосанных пустяков?". <sup>19</sup> In this light it is worth mentioning that also in The Good Angel of Death (Dobryj Angel Smerti, 1998) by Andrej Kurkov (b. 1961), a Ukrainian Russian-language writer based in Kiev, the plot concerns the search of a mysterious 'treasure' belonging to one of the fathers of modern Ukrainian literature: Taras Ševčenko. For further information, see Puleri 2015a. высосанных пустяков?". tion, see Puleri 2015a. A gust of wind coming from Dnipro can rip the hat off the careless passer-by in Marinsky Park. It can tear away the child's balloon, taking it over the crystalline skies of Kiev. Don't cry, baby. Don't cry. Get used to it. Not far from here, there are other winds, other hurricanes, which echo. And don't compare the winds coming from Dnipro with the dry and dusty ones that are eroding the country, which are rising from the bustle coming from Hrushevsky and Bankova Streets […] in Kiev, as some are wearing away and building up the soil of the historical hill of Shchekavytsa, while Sofia and Lavra are trying to hold on with all their remaining energies. How long is it going to last? […] and what can be said of us, disunited and weak, who vivaciously argue over trivial matters, invented out of a whole cloth?18. In Madžong, Kiev is crowded with failed writers, ambitious bukinisty, and unscrupulous billionaires, all striving to seize a 'priceless manuscript.' In this novel as well, the game dynamics underlie Nikitin's literary world, employed in order to re-write tradition and to re-appropriate History. It is the trilogy of Mertvye duši as planned by Nikolaj Gogol', the contested father of Ukrainian literature in Russian, that is to be the target of a complex rewriting. Gogol's project was to write a great 'epic poem in prose' on the Russian Empire, which was to be structured in three parts. It would narrate the journey of the protagonist, Pavel Ivanovič Čičikov, following lines of development close to Dante's Comedy. Legend has it that the second part of the trilogy was destroyed by Gogol' shortly before his death, while the drafting of the third part never even started. In Madžong, the main plot concerns the fortuitous finding of the fragments of a supposed Dead Souls third volume19. On the one hand, the protagonist is a failed philologist, Ženja L'vov, whose 'unfinished' doctoral dissertation was devoted to studying the 'evolution of Čičikov's developments' in Gogol's 'missing' trilogy. On the other, the demiurges of the story come to be the four players of Mahjong: throughout the novel, their matches open the chapters and their moves upset the balance of the exhausting search for Gogol's volume. As stressed by the literary critic V. Toporov (2012), we witness a "Russian prose ‒ built on a Ukrainian subject ‒ and clearly oriented towards Western models (Borges, Cortázar, Umberto Eco, Pérez-Reverte ‒ here on this line)." Literature, <sup>18</sup> "Порыв свежего ветра с Днепра может сорвать шляпу с неосторожного прохожего в Мариинском парке, может выхватить шарик у ребенка и унести его в ясно-голубое киевское небо. Не плачь, детка. Не плачь. Привыкай. Совсем рядом ревут другие ветры, другие ураганы. И не сравниться ветру с Днепра с иссушающими страну самумами, поднятыми шелестом на улицах Грушевского и Банковой ...срезают и застраивают в Киеве историческую Щекавицу, из последних сил держатся София и Лавра. Долго ли продержатся? […] то что же говорить о нас, разъединенных и слабых, радостно грызущихся из-за выдуманных, из пальца <sup>19</sup> In this light it is worth mentioning that also in The Good Angel of Death (Dobryj Angel Smerti, 1998) by Andrej Kurkov (b. 1961), a Ukrainian Russian-language writer based in Kiev, the plot concerns the search of a mysterious 'treasure' belonging to one of the fathers of modern Ukrainian literature: Taras Ševčenko. For further informaas well as History, is subject to fate. Man is bound to go through in his desperate search for an absolute and an unambiguous narrative: a search that can never be satisfied. At the end of the 'game,' the different narrative levels symbolically intertwine in the "last unnumbered chapter," a spatial dimension where Ženja becomes the shaman Kara Gerzen. Under these guises, the protagonist finally has the power to change the course of 'History' (Nikitin 2012: 381): What's the manuscript? ‒ "Dead Souls." Nikolaj Vasil'evič Gogol'. Part Three. ‒ Great. I sincerely congratulate you. ‒ Why? ‒ Because you are in good company now. – With Gogol'? Thanks. ‒ If it was just with him…well, what's wrong with your manuscript? – It does not exist. Ok, it existed before. I read some pages. ‒ But then, when it came the time to place Hen Tamgan, it turned out that there was no manuscript, didn't it? ‒ Yes. That's how it turned out [...] ‒ That's all right. Actually, the manuscript does not exist. It does not and never did exist. You have not written it yet. ‒ Me? That's me who did not write it? [...] ‒ No one wrote it. Neither you nor Gogol. Nor anybody else20. "More than everything else, that story looked like a game" (ivi: 370), comments the narrator in the last pages of Madžong. In addition to the well-structured stylisation of Gogol's prose in the imaginary passages from the third volume of Dead Souls, Nikitin injects his authorial intrusions and historical digressions to consolidate his rewriting of tradition. Thus, the writer can disguise himself as Old Kačalov, one of the Mahjong players, and re-appropriate his voice to formulate his 'historical truth' (ivi: 72-73): [...] The Russian language was created by Ukrainians and it should be recognised abroad as Ukrainian property. Kačalov began with the Primary Chronicle, which was written only three hundred meters from his office, and did not forget anyone. In his list it was not only the theologians from Mohyla Academy, who were invited by Patriarch Nikon to Moscow to put in order the church books, that needed to be included, but also all the renown Ukrainian nobles and raznochincy who wrote in Russian<sup>21</sup>. <sup>20</sup> "Что за рукопись? – 'Мертвые души'. Николай Васильевич Гоголь. Том третий. – Прекрасно. От души тебя поздравляю. – С чем? – Ты попал в хорошую компанию. – К Гоголю? Спасибо. ‒ Если бы только к нему... Ладно. Что же не так с твоей рукописью? – Ее нет. Прежде она была. Ее читали, держали в руках. Я сам видел несколько страниц. – А когда пришло время наложить хэн тамган, оказалось, что рукописи нет. Правильно? – Да. Именно так и оказалось [...] – Тогда все в порядке. Дело в том, что этой рукописи действительно нет. Нет и никогда не было. Ты ее еще не написал. – Я? Не написал? [...] – Никто не написал. Ни ты, ни Гоголь. Ни кто-то другой". <sup>21</sup> "[...] русский язык создан украинцами и его следует признать собственностью Украины за границей. Качалов начал с "Повести временных лет", написанной в трехстах метрах от его офиса, и не забыл никого. В записке были перечислены все богословы Могилянской Академии, которых Патриарх Никон the Failure of the 'Little Russian Solution', 1782-1917, in: A. Kappeler et al. (eds), Culture, Nation and Identity: The Ukrainian-Russian Encounter, 1600-1945, Ed- ‒ utrata dlja strany, "Šo", IX-X, 2013, pp. 62-69. of the linguistic marketplace in Ukraine, in: L.M.L. Zaleska Onyshkevych, M. G. Rewakowicz (eds), Contemporary Ukraine on the Cultural Map of Europe, New Cultural Diversity in Ukraine, "The Calvert Journal", http://calvertjournal.com/comment/show/2176/russian- nian and Russian Nationalism, Cambridge and London culture-in-ukraine-literature, 17/03/2014. You are looking for a meaning, but you cannot find it in this. Once it was, but it has weathered a long time ago. Only tradition has remained. For example, in most countries they write from left to right. In others it is from right to left, while in others still they write from top to bottom, vertically. And when in a country where they write from left to right you start writing from right to left, they will not be able to understand you. And here they don't understand you. A tradition is often irrational: do not look for logic in it. Over time it loses all meaning, you need only to observe it. Because crossing the borders of the unintelligible, it is not only you who doesn't know whether the breach is great or not, but also those who follow the strict observance of the rules will not be able to make sense of it23. Andriewsky 2003: O. Andriewsky, The Russian-Ukrainian Discourse and monton 2003, pp. 182-214. Besedin 2013: P. Besedin, Aleksej Nikitin: Isčeznovenie ljubogo jazyka Bilaniuk 2009: L. M. Bilaniuk, Criticism, confidence, and the reshaping York 2009, pp. 336-358. Blacker 2014: U. Blacker, Blurred Lines: Russian Literature and Bojanowska 2007: E. M. Bojanowska, Nikolaj Gogol: Between Ukrai- <sup>23</sup> "Ты ищешь смысл, а смысла в этом нет – когда-то он был, но давно выветрился. Осталась традиция. Например, в большинстве стран пишут слева направо. Но в некоторых – справа налево, а в некоторых – сверху вниз. И если в стране, где пишут слева направо, ты начнешь писать справа налево, то тебя могут не понять. Вот и здесь тебя не понимают. А традиция часто иррациональна, не стоит искать в ней логику. Со временем она теряет всякий смысл, ее нужно просто соблюдать, потому что, пересекая границу непонятного, не только ты не знаешь, велико ли нарушение, но и те, кто следит за точным соблюдением правил, ничего 2007. References в этом не смыслят" . The sarcastic comment made by Kačalov reflects Nikitin's 'minor position' on the language and identity issues in Ukraine. According to the author, the Ukrainian contribution to Russian culture throughout the centuries is an "indisputable matter:" it is not to be envisioned as an Imperial "cultural expansion," but rather as an "exchange of authors" (Puleri 2016: 186). The Russophone writer considers those arguments recognising the presence of the Russian language in Ukraine as the outcome of a kind of 'colonial domination' to be inappropriate. Instead, Nikitin asserts that we witness a mutual influence between the Russian and Ukrainian systems, which later gave rise to the development of two different cultural models (see Serebrjakova 2011: web)22. Furthermore, framing the contemporary "Russian literature of Ukraine" as an active element of the Ukrainian artistic production would not limit it from being considered as a "constitutive category of Russian literature" (Meležik 2013: web). As stressed by Nikitin, to writers, self-identification is an "intimate question" and is affected by multiple factors that have nothing to do with the discourses of tradition (Besedin 2013: 65): Actually, self-identification is quite an intimate question. It touches pretty deep in one's heartstrings. Moreover, people change over time. They can switch from Russian to Ukrainian, and then again from Ukrainian to Russian, or vice versa. And, eventually, they can also write in both languages. They can change their country of residence, and they can do that more than once. An objective criterion does not exist […]. In constant search of a solution for the 'eternal discord' between ambiguous narratives of the past, as well as of the present, the writer can only unmask the precarity of all human attempts to find a proper answer to the game dynamics of History. Thus, in his last novel Victory Park (2014), Nikitin portrays the 'lyric dimension' of the fragmented lives experienced by veterans coming back from the Afghan war, by old revolutionists and inveterate smugglers. However, the world in miniature contained in Victory Park, set on the left bank of the Dnipro river at the edges of Kiev, also belongs to other spaces: it mirrors the search of an answer to the "ideological void" (Sochareva 2014) of the Ukrainian capital on the eve of the Soviet collapse. Eventually, in the post-Soviet labyrinth, it is exactly the epistemological crisis of tradition that reconciles both the simple man and the writer with that unreal image reflected on the mirror of an epoch (Nikitin 2014: 197): пригласил в Москву приводить в порядок церковные книги, а также все известные украинские дворяне и разночинцы, писавшие по-русски" (Nikitin 2012: 72-73). <sup>22</sup> "Literature is an open system: if something is missing, then this gap, as a rule, comes to be quickly filled. The exceptions come in those periods when someone – the church or the state – tries to regulate the literary processes from the outside, allowing some forms and banning others". You are looking for a meaning, but you cannot find it in this. Once it was, but it has weathered a long time ago. Only tradition has remained. For example, in most countries they write from left to right. In others it is from right to left, while in others still they write from top to bottom, vertically. And when in a country where they write from left to right you start writing from right to left, they will not be able to understand you. And here they don't understand you. A tradition is often irrational: do not look for logic in it. Over time it loses all meaning, you need only to observe it. Because crossing the borders of the unintelligible, it is not only you who doesn't know whether the breach is great or not, but also those who follow the strict observance of the rules will not be able to make sense of it23. ### References 372 Marco Puleri 65): does not exist […]. (Nikitin 2014: 197): some forms and banning others". The sarcastic comment made by Kačalov reflects Nikitin's 'minor position' on the language and identity issues in Ukraine. According to the author, the Ukrainian contribution to Russian culture throughout the centuries is an "indisputable matter:" it is not to be envisioned as an Imperial "cultural expansion," but rather as an "exchange of authors" (Puleri 2016: 186). The Russophone writer considers those arguments recognising the presence of the Russian language in Ukraine as the outcome of a kind of 'colonial domination' to be inappropriate. Instead, Nikitin asserts that we witness a mutual influence between the Russian and Ukrainian systems, which later gave rise to the development of two different cultural models (see Serebrjakova 2011: web)22. Furthermore, framing the contemporary "Russian literature of Ukraine" as an active element of the Ukrainian artistic production would not limit it from being considered as a "constitutive category of Russian literature" (Meležik 2013: web). As stressed by Nikitin, to writers, self-identification is an "intimate question" and is affected by multiple factors that have nothing to do with the discourses of tradition (Besedin 2013: Actually, self-identification is quite an intimate question. It touches pretty deep in one's heartstrings. Moreover, people change over time. They can switch from Russian to Ukrainian, and then again from Ukrainian to Russian, or vice versa. And, eventually, they can also write in both languages. They can change their country of residence, and they can do that more than once. An objective criterion In constant search of a solution for the 'eternal discord' between ambiguous narratives of the past, as well as of the present, the writer can only unmask the precarity of all human attempts to find a proper answer to the game dynamics of History. Thus, in his last novel Victory Park (2014), Nikitin portrays the 'lyric dimension' of the fragmented lives experienced by veterans coming back from the Afghan war, by old revolutionists and inveterate smugglers. However, the world in miniature contained in Victory Park, set on the left bank of the Dnipro river at the edges of Kiev, also belongs to other spaces: it mirrors the search of an answer to the "ideological void" (Sochareva 2014) of the Ukrainian capital on the eve of the Soviet collapse. Eventually, in the post-Soviet labyrinth, it is exactly the epistemological crisis of tradition that reconciles both the simple man and the writer with that unreal image reflected on the mirror of an epoch пригласил в Москву приводить в порядок церковные книги, а также все известные украинские дворяне и разночинцы, писавшие по-русски" (Nikitin 2012: 72-73). <sup>22</sup> "Literature is an open system: if something is missing, then this gap, as a rule, comes to be quickly filled. The exceptions come in those periods when someone – the church or the state – tries to regulate the literary processes from the outside, allowing <sup>23</sup> "Ты ищешь смысл, а смысла в этом нет – когда-то он был, но давно выветрился. Осталась традиция. Например, в большинстве стран пишут слева направо. Но в некоторых – справа налево, а в некоторых – сверху вниз. И если в стране, где пишут слева направо, ты начнешь писать справа налево, то тебя могут не понять. Вот и здесь тебя не понимают. А традиция часто иррациональна, не стоит искать в ней логику. Со временем она теряет всякий смысл, ее нужно просто соблюдать, потому что, пересекая границу непонятного, не только ты не знаешь, велико ли нарушение, но и те, кто следит за точным соблюдением правил, ничего в этом не смыслят" . Istorija s geografiej. Kievskie gory i nory, "Novyj Mir", 2012, 10, <http://magazines.russ.ru/novyi\_mi/ 2012/10/ zrenie", LXXXV, 2007, <http://magazines.russ.ru/ Clarence Mangan and The Emergence of Irish Cultural Nationalism, Berkeley-Los Angeles-London 1987. Gogol', "Voprosy literarury", II, 1999, <http://magazi- guistic Analysis, in: J. Besters-Dilger (ed), Language Policy and Language Situation in Ukraine, Frankfurt dentifikacija pisatelja vne Rossii, "Svobodnaja Pressa", <http://svpressa.ru/culture/article/76791/?rss=1>, Self-Identification in Post-Soviet Ukrainian Literature in Russian, "Ab Imperio", 2014, 2, pp. 367-397. letterario ucraino, "Between", V, 2015, 9, <http://ojs. unica.it/index.php/between/article/view/1395>. rie nel contesto ucraino post-sovietico, in: I. Both et al., Innesti e ibridazione tra spazi culturali, Firenze 2015, nes.russ.ru:8080/voplit/1999/2/mann.html>. Kochanovskaja, Nazarenko 2012:T. Kochanovskaja, M. Nazarenko, Ukrainskij Vektor: Krasnjaščich 2015: Krasnjaščich, Rusukrlit kak on est', "Novyj Mir", IX, 2015, pp. 173-197. Kukulin 2007: Kukulin, Ot redaktora, "Novoe Literaturnoe Obo- nlo/2007/85/ik18.html>. Lloyd 1987: D. Lloyd, Nationalism and Minor Literature. James Luckyj 1998: G. S. N. Luckyj, The Anguish of Mykola Hohol a.k.a. Nikolaj Gogol, Toronto 1998. Mann 1999: J.V. Mann, Vstreča v labirinte. Franz Kafka i Nikolaj Masenko 2008: L. Masenko, Language Situation in Ukraine: Sociolin- Nikitin 2011: A. Nikitin, Istemi, Moskva 2011 (en. ed. di Anne Marie Jackson, Istemi, London 2013). Puleri 2014: M. Puleri, Ukraïns'kyi, Rosiis'komovnyi, Rosiis'kyi: Puleri 2015: M. Puleri, L'ibrido assente: 'Immaginando' lo spazio Puleri 2015a: M. Puleri, Uno sguardo al passato. Dinamiche identita- pp. 65-86. 04/11/2013. Nazarenko 2005: M. Nazarenko, Real'nost' čuda, Kiev 2005. Nikitin 2012: A. Nikitin, Madžong, Moskva 2012. Nikitin 2014: A. Nikitin, Victory Park, Moskva 2014. am Main 2008, pp. 101-137. Meležik 2013: Leonid Meležik, Russkij ili Russkoiazyčnyj: samoi- u21.html>. Chernetsky 2007: V. Chernetsky, Mapping Postcommunist Cultures: Rus- Čuprinin 2008: S. Čuprinin, Russkaja literatura segodnja: Zarubež'e, Deleuze, Guattari 1986: G. Deleuze, F. Guattari, Kafka. Toward a Minor Lite- Foucault 1970: Michel Foucault, The Order of Things: An Archaeology Foucault 1984: M. Foucault, Of Other Spaces: Utopias and Heteroto- Gogol' 1952: N. V. Gogol', Polnoe sobranie sočinenij, XII, Moskva Grabowicz 1992: G. G. Grabowicz, Ukrainian-Russian Literary Rela- Grabowicz 1994: G. G. Grabowicz, Hohol' i mif Ukrajiny, "Sučasnist'", Ilchuk 2009: Y. Ilchuk, Gogol's Hybrid Performance: The Creation, Ilnytzkyj 2002: O. S. Ilnytzkyj, Cultural Indeterminacy in the Russian Ilnytzkyj 2003: O. S. Ilnytzkyj, Modeling Culture in the Empire: Ukrai- monton 2003, pp. 298-324. IX-X, 1994, pp. 77-92; 137-149. cault1.pdf>. 1937-1952. pp. 214-244. bia 2009. 171. treal 2007. Moskva 2008. sia and Ukraine in the Context of Globalization, Mon- rature, tr. D. Polan, Minneapolis-London 1986 (ed. or. Kafka. Pour une littérature mineure, Paris 1975). of Human Sciences, tr. A.M. Sheridan Smith, New York 1970 (ed. or. Les Mots et les Choses, Paris 1966). pias, tr. J. Miskowiec, (ed. or. Des Espaces Autres, "Architecture/Mouvement/Continuité", 5, October 1984, pp. 46-49), <http://web.mit.edu/allanmc/www/fou- tions in the Nineteenth Century: A Formulation of the Problem, in: P. J. Potichnyj et al. (eds), Ukraine and Russia in Their Historical Encounter, Edmonton 1992, Reception and Editing of Vechera na Khutore Bliz Dikanki (Evenings on a farm near Dikan'ka, 1831-32). A dissertation presented to the Faculty of the USC Graduate School, University of Southern Carolina, Colum- Empire: Nikolai Gogol as a Ukrainian Post-Colonial Writer, in: P. D. Morris (ed), A World of Slavic Literatures. Essays in Comparative Slavic Studies in Honour of Edward Mozejko, Bloomington-Indiana 2002, pp. 153- nian Modernism and the Death of the All-Russian Idea, in: A. Kappeler et al. (eds), Culture, Nation and Identity: The Ukrainian-Russian Encounter, 1600-1945, Ed- ### Abstract Marco Puleri ### Tra Kafka e Gogol'. Modelli di 'deterritorializzazione' nella letteratura ucraina di lingua russa Nell'Ucraina post-sovietica è emersa una nuova categoria di scrittori: il loro ricorso alla lingua russa si muove all'interno di una dimensione 'minore', tra la tradizione culturale sovietica e l'odierno radicamento nel contesto nazionale. Avremo modo di osservare le strategie narrative adottate da Aleksej Nikitin (1967, Kiev) attraverso la lente delle categorie d'analisi elaborate da G. Deleuze e F. Guattari per lo studio della produzione letteraria di F. Kafka (Kafka. Pour une littérature mineure, 1975). Nel tentativo di 'deterritorializzare' la 'frattura storica' sovietica, Nikitin costruisce 'specchi eterotopici', riuscendo a recuperare modelli narrativi occidentali all'interno della propria esperienza artistica 'minore'. L'analisi di alcuni brani tratti da Istemi (2011) e Madžong (2012) di Nikitin ci darà la possibilità di individuare la nascita di nuove testualizzazioni volte a ristabilire un continuum nell'esperienza storica ed artistica post-sovietica, i cui principali interlocutori sembrano essere proprio gli strumenti epistemologici e letterari occidentali. Keywords: Ukrainian russophone literature, post-soviet Literature, A. Nikitin, deterritorialization. > Russia, Oriente slavo e Occidente europeo. Fratture e integrazioni nella storia e nella civiltà letteraria, a cura di Claudia Pieralli, Claire Delaunay, Eugène Priadko, ISBN 9788864535074 (online), CC BY 4.0, 2017 > Alessandro Achilli è Lecturer in Ukrainian Studies presso la Monash University, Melbourne. Dopo essersi addottorato nel 2015 con una tesi sul poeta ucraino Vasyl' Stus, è stato Research Fellow presso lo Harvard Ukrainian Research Institute e docente a contratto all'Università degli Studi di Milano. I suoi interessi vertono principalmente sulla poesia ucraina e russa moderna e contemporanea, la storia culturale ucraina, i rapporti letterari interslavi e slavo- > Claire Delaunay si è laureata in Lingua, Letteratura e civiltà russa, ed è attualmente dottoranda di ricerca presso l'Università Paris IV-Sorbonne, dove dal 2013 insegna Letteratura russa e traduzione letteraria russo-francese. La Tesi di Dottorato che sta completando sotto la direzione scientifica di Luba Jurgenson riguarda la scrittura dell'angoscia nell'opera di L. Tolstoj, autore al quale ha dedicato la sua tesi di laurea e diversi studi, pubblicati in francese e in russo. > Catherine Depretto è Professore di Letteratura Russa presso l'Università Paris IV-Sorbonne e Direttore della Revue des études slaves. È specializzata nello studio della teoria letteraria in Russia, in particolare del formalismo. Ha tradotto e curato il maggiore testo storico-letterario di Ju. Tynjanov, Formalisme et histoire littéraire (1991), curato l'opera L'héritage de Bakhtine (1995) e pubblicato Le formalisme en Russie (2009; trad. ru: Formalizm v Rossii, Мoskva, NLO, 2015). Le sue ricerche riguardano anche la storia culturale del periodo > Alessandro Farsetti ha conseguito il titolo di Dottore di ricerca in Lingue e Letterature Slave all'Università Ca' Foscari di Venezia con una tesi sul futurista Ivan Aksenov (2015). I suoi campi di indagine comprendono la poetica dell'avanguardia russa di inizio Novecento, la cultura popolare dell'URSS, la letteratura di viaggio. Recentemente ha curato l'edizione italiana del romanzo di E.L. Voynich The Gadfly (1897), opera di culto della Russia sovietica (Il figlio del cardinale, 2013), e ha collaborato al commento dell'edizione russa delle me- germanici, la comparatistica, la teoria letteraria e la teoria della lirica. sovietico e le forme non-fiction dell'auto-narrazione. morie di N.P. Anciferov sull'Italia (Otčizna moej duši, 2016). Firenze University Press Profilo degli autori	doab	2025-04-07T03:56:59.233138	2-6-2022 06:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "007e4761-4bfc-4411-b0b4-7cd4adcff760", "url": "https://library.oapen.org/bitstream/20.500.12657/55843/1/26512.pdf", "author": "Puleri, Marco", "title": "Chapter Between Kafka and Gogol’. ‘De-territorialising’ National Narrative(s) in Post-Soviet Ukrainian Literature in Russian", "publisher": "Firenze University Press", "isbn": "", "section_idx": 0 }
008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67.0	Edited by Burak Pamukçu Atherosclerotic cardiovascular disease is still the most common cause of death among adults. Its prevalence is increasing in developing countries and despite all advances in both diagnostic tools and treatment modalities, it is still very common in the developed world. Obesity, diabetes mellitus, hypercholesterolemia, and overuse of dietary salt play a pivotal role in increased cardiovascular morbidity and mortality worldwide. Current clinical efforts are mainly focused on the diagnosis and treatment of myocardial infarction. In this book we provide epidemiological data on myocardial infarction and atherosclerotic cardiovascular disease, current diagnostic biochemical tests, and management strategies. A specific patient group, children, experiencing myocardial infarction is also addressed. Current advancements in the management of myocardial infarction have decreased the morbidity and mortality from atherosclerotic cardiovascular disease and especially myocardial infarction; however, further progress can be achieved by the prevention of atherosclerotic processes by focusing on the early stages of the disease. ISBN 978-1-83880-089-5 Angiography Published in London, UK © 2019 IntechOpen © 1001gece / iStock	doab	2025-04-07T03:56:59.253635	1-12-2023 18:50	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67", "url": "https://mts.intechopen.com/storage/books/7055/authors_book/authors_book.pdf", "author": "", "title": "Angiography", "publisher": "IntechOpen", "isbn": "9781838800901", "section_idx": 0 }
008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67.2	ANGIOGRAPHY #### Angiography http://dx.doi.org/10.5772/intechopen.73817 Edited by Burak Pamukçu #### Contributors Abdel-Razzak Mohammad Saeed Al-Hinnawi, Daniel Emilio Dalledone Siqueira, Ana Terezinha Guillaumon, Behzad Alizadeh, Yakup Balaban, Vincent Dangoisse, Antonio Jesus Guerra, Joaquim Ciurana, Iveta Tasheva, Ivo Petrov, Jivka Stoykova, Liubomir Dosev, Petar Polomski, Burak Pamukçu #### © The Editor(s) and the Author(s) 2019 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, 2019 by IntechOpen IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, The Shard, 25th floor, 32 London Bridge Street London, SE19SG – 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] Angiography, Edited by Burak Pamukçu p. cm. Print ISBN 978-1-83880-089-5 Online ISBN 978-1-83880-090-1 eBook (PDF) ISBN 978-1-78985-625-5	doab	2025-04-07T03:56:59.253844	1-12-2023 18:50	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67", "url": "https://mts.intechopen.com/storage/books/7055/authors_book/authors_book.pdf", "author": "", "title": "Angiography", "publisher": "IntechOpen", "isbn": "9781838800901", "section_idx": 2 }
008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67.3	We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists 4,200+ Angiography Contributors Notice Edited by Burak Pamukçu http://dx.doi.org/10.5772/intechopen.73817 © The Editor(s) and the Author(s) 2019 Stoykova, Liubomir Dosev, Petar Polomski, Burak Pamukçu at http://www.intechopen.com/copyright-policy.html. rights and permissions department ([email protected]). Violations are liable to prosecution under the governing Copyright Law. use of any materials, instructions, methods or ideas contained in the book. First published in London, United Kingdom, 2019 by IntechOpen A catalogue record for this book is available from the British Library Additional hard and PDF copies can be obtained from [email protected] 11086078, The Shard, 25th floor, 32 London Bridge Street London, SE19SG – United Kingdom British Library Cataloguing-in-Publication Data Angiography, Edited by Burak Pamukçu Print ISBN 978-1-83880-089-5 Online ISBN 978-1-83880-090-1 eBook (PDF) ISBN 978-1-78985-625-5 Printed in Croatia p. cm. Abdel-Razzak Mohammad Saeed Al-Hinnawi, Daniel Emilio Dalledone Siqueira, Ana Terezinha Guillaumon, Behzad Alizadeh, Yakup Balaban, Vincent Dangoisse, Antonio Jesus Guerra, Joaquim Ciurana, Iveta Tasheva, Ivo Petrov, Jivka 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 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 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 IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: Open access books available 116,000+ International authors and editors 125M+ 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)	doab	2025-04-07T03:56:59.254096	1-12-2023 18:50	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67", "url": "https://mts.intechopen.com/storage/books/7055/authors_book/authors_book.pdf", "author": "", "title": "Angiography", "publisher": "IntechOpen", "isbn": "9781838800901", "section_idx": 3 }
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008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67.5	Meet the editor Burak Pamukçu (MD) obtained a doctorate degree in Cardiology from Istanbul University Faculty of Medicine, Istanbul, Turkey. Dr. Pamukcu made his postdoctorate fellowship (European Society of Cardiology Atherothrombosis Research Fellowship) in the University Department of Medicine, Centre for Cardiovascular Sciences, City Hospital, Birmingham, England, UK. He is mainly inter- ested in atherothrombosis, atherosclerotic heart vessel disease, antithrombotic therapy, and interventional cardiology. Currently, Dr. Pamukcu is working as a consultant cardiologist and associate professor of cardiology in Acibadem Mehmet Ali Aydinlar University. He has published 99 scientific publications and served as a referee for more than 30 different medical journals indexed in SCI and SCI expanded. He is serving as associate editor and editorial board member for peer-reviewed medical journals. Contents Preface VII Interventions 3 Burak Pamukçu Interventions 7 Yakup Balaban Prospects 41 Chapter 5 Dedicated Bifurcation Stents 61 Stankov and Petar Polomski Transradial Access 9 Vincent Dangoisse Chapter 3 The Role of Catheter Reshaping at the Angiographic Success 27 Chapter 1 Introductory Chapter: An Overview of Angiographic Vascular Chapter 2 Minimally Invasive Cardiology for Everyone: Challenging the Section 2 Coronary Angiography and Percutaneous Coronary Chapter 4 Stent's Manufacturing Field: Past, Present, and Future Ivo Petrov, Iveta Tasheva, Jivka Stoykova, Liubomir Dosev, Zoran Daniel Emilio Dalledone Siqueira and Ana Terezinha Guillaumon Antonio J. Guerra and Joaquim Ciurana Section 3 Angiography for Peripheral Arterial Diseases 85 Chapter 6 Angiography for Renal Artery Diseases 87 Section 1 Introduction 1 ## Contents Daniel Emilio Dalledone Siqueira and Ana Terezinha Guillaumon Preface neously (TAVR) with great success. intravascular ultrasound, and optical coherence tomography. mina Skomersic for their contributions to the project. interventional therapies for patients with vascular and structural diseases. Conventional angiography is still the gold standard diagnostic procedure for the majority of coronary and peripheral vascular diseases, and therapeutic interventions have advanced dramatically in the last few decades. The evolution of both diagnostic and therapeutic inter‐ ventional techniques and implementation of new devices (catheters, wires, balloons, stents, valves, etc.) into clinical practice have been important successes in angiography, cardiac catheterization, angioplasty, and modern catheter-based interventions used for the treat‐ ment of structural heart disease in recent years. Fifty years after the first angioplasty proce‐ dure realized by Dr. Gruentzig, we now have the ability to make successful angioplasty and stenting for left main coronary artery stenosis, bifurcation lesions, chronic total occlusions, and peripheral arterial diseases, including carotid, subclavian, iliac, femoral, and even small extremity vessel lesions. Advancements in the treatment of structural heart and vessel dis‐ eases by catheter-based techniques are astonishing and it is possible to close septal defects, to treat aortic aneurysms (EVAR, TEVAR) by devices, or to implant an aortic valve percuta‐ Coronary angiographic interventions evolved from basic left heart catheterization to com‐ plex interventions with the aid of a huge amount of medical industrial material, including differently shaped catheters, guidewires, microcatheters, balloons, stents (bare metal, drug eluting), absorbable biovascular scaffolds, etc. Vascular access sites and techniques have also evolved within the last few decades, and radial access has become the preferred access site for the majority of coronary interventions. Several new techniques are provided for the eval‐ uation of lesion severity, including fractional flow reserve, instantaneous wave-free ratio, In this book we aim to overview developments and innovations in diagnostic, technical, and therapeutic angiographic procedures. Despite all developments in the field, morbidity and mortality from vascular diseases are still very high. On the other hand, we need further ad‐ vancements for practical treatment of structural heart diseases. Our perspective for the next few decades should be based on preventing vascular disease and providing more successful I hope that this book will support our practical and theoretical knowledge in the field. I would like to acknowledge all contributing authors and our author service manager Ms. Ro‐ Consultant Cardiologist & Associate Professor of Cardiology Acibadem Mehmet Ali Aydinlar University Vocational School of Health Sciences Department of Emergency and First Aid Burak Pamukcu, MD Istanbul, Turkey ## Preface Section 4 Interventions in Structural Heart Diseases 101 Chapter 8 Computer-Aided Detection, Pulmonary Embolism, Behzad Alizadeh VI Contents Arterial Diseases 113 Current Status 115 Abdel-Razzak M. Al-hinnawi Chapter 7 Transcatheter Closure of Congenital VSDs: Tips and Tricks 103 Section 5 Computed Tomography Angiography in Coronary and Periferal Computerized Tomography Pulmonary Angiography: Conventional angiography is still the gold standard diagnostic procedure for the majority of coronary and peripheral vascular diseases, and therapeutic interventions have advanced dramatically in the last few decades. The evolution of both diagnostic and therapeutic inter‐ ventional techniques and implementation of new devices (catheters, wires, balloons, stents, valves, etc.) into clinical practice have been important successes in angiography, cardiac catheterization, angioplasty, and modern catheter-based interventions used for the treat‐ ment of structural heart disease in recent years. Fifty years after the first angioplasty proce‐ dure realized by Dr. Gruentzig, we now have the ability to make successful angioplasty and stenting for left main coronary artery stenosis, bifurcation lesions, chronic total occlusions, and peripheral arterial diseases, including carotid, subclavian, iliac, femoral, and even small extremity vessel lesions. Advancements in the treatment of structural heart and vessel dis‐ eases by catheter-based techniques are astonishing and it is possible to close septal defects, to treat aortic aneurysms (EVAR, TEVAR) by devices, or to implant an aortic valve percuta‐ neously (TAVR) with great success. Coronary angiographic interventions evolved from basic left heart catheterization to com‐ plex interventions with the aid of a huge amount of medical industrial material, including differently shaped catheters, guidewires, microcatheters, balloons, stents (bare metal, drug eluting), absorbable biovascular scaffolds, etc. Vascular access sites and techniques have also evolved within the last few decades, and radial access has become the preferred access site for the majority of coronary interventions. Several new techniques are provided for the eval‐ uation of lesion severity, including fractional flow reserve, instantaneous wave-free ratio, intravascular ultrasound, and optical coherence tomography. In this book we aim to overview developments and innovations in diagnostic, technical, and therapeutic angiographic procedures. Despite all developments in the field, morbidity and mortality from vascular diseases are still very high. On the other hand, we need further ad‐ vancements for practical treatment of structural heart diseases. Our perspective for the next few decades should be based on preventing vascular disease and providing more successful interventional therapies for patients with vascular and structural diseases. I hope that this book will support our practical and theoretical knowledge in the field. I would like to acknowledge all contributing authors and our author service manager Ms. Ro‐ mina Skomersic for their contributions to the project. #### Burak Pamukcu, MD Consultant Cardiologist & Associate Professor of Cardiology Acibadem Mehmet Ali Aydinlar University Vocational School of Health Sciences Department of Emergency and First Aid Istanbul, Turkey Section 1 Introduction Section 1 ## Introduction Chapter 1 Provisional chapter Introductory Chapter: An Overview of Angiographic Introductory Chapter: An Overview of Angiographic Coronary heart disease is the most common cause of death among adults in developed countries, and its prevalence is increasing in developing world [1, 2]. Physical inactivity, obesity, metabolic disturbances including diabetes mellitus, hypercholesterolemia, and hypertension Angiography is still the gold standard diagnostic technique in a majority of vascular diseases. The histories of angiography, cardiac catheterization, angioplasty, and modern catheterbased interventions start in the early eighteenth century. Hales made the first documented biventricular catheterization in a horse in 1711 [3]. Then, Forssmann's right heart selfcatheterization in 1929 became a milestone in the history of heart catheterization [3]. After success in right heart catheterization, researchers focused on left heart, and Zimmerman, Cope, Ross, and colleagues achieved left heart catheterization too [3]. The start for coronary angiography was given by Sones in 1958 and was followed and developed by Judkins and Amplatz via femoral access in angiography [3]. In 1963, Dotter accidentally caused recanalization of a peripheral artery with the catheter. In the 1970s, Gruentzig started balloon angioplasty and unlocked the door of vascular interventions era [3]. Several technical and industrial developments allowed us to perform vascular interventions frequently and with great success today. Current catheter-based interventions are frequently used in cardiology, interventional radiology, and neuroradiology for treatment of aortic, coronary, cerebrovascular, and other peripheral arterial occlusive and nonocclusive diseases. Interventional therapies are also available for a long time in structural heart diseases (e.g., closure of; atrial or ventricular septal defects, and salty diet, and cigarette smoking are leading to increased vascular diseases [1, 2]. © 2016 The Author(s). Licensee InTech. 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. © 2018 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. DOI: 10.5772/intechopen.81768 Vascular Interventions Vascular Interventions http://dx.doi.org/10.5772/intechopen.81768 patent foramen ovale, or left atrial appendage). Additional information is available at the end of the chapter Burak PamukçuAdditional information is available at the end of the chapter Burak Pamukçu 1. Introduction #### Introductory Chapter: An Overview of Angiographic Vascular Interventions Introductory Chapter: An Overview of Angiographic Vascular Interventions DOI: 10.5772/intechopen.81768 Burak Pamukçu Additional information is available at the end of the chapter Burak PamukçuAdditional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.81768 #### 1. Introduction Coronary heart disease is the most common cause of death among adults in developed countries, and its prevalence is increasing in developing world [1, 2]. Physical inactivity, obesity, metabolic disturbances including diabetes mellitus, hypercholesterolemia, and hypertension and salty diet, and cigarette smoking are leading to increased vascular diseases [1, 2]. Angiography is still the gold standard diagnostic technique in a majority of vascular diseases. The histories of angiography, cardiac catheterization, angioplasty, and modern catheterbased interventions start in the early eighteenth century. Hales made the first documented biventricular catheterization in a horse in 1711 [3]. Then, Forssmann's right heart selfcatheterization in 1929 became a milestone in the history of heart catheterization [3]. After success in right heart catheterization, researchers focused on left heart, and Zimmerman, Cope, Ross, and colleagues achieved left heart catheterization too [3]. The start for coronary angiography was given by Sones in 1958 and was followed and developed by Judkins and Amplatz via femoral access in angiography [3]. In 1963, Dotter accidentally caused recanalization of a peripheral artery with the catheter. In the 1970s, Gruentzig started balloon angioplasty and unlocked the door of vascular interventions era [3]. Several technical and industrial developments allowed us to perform vascular interventions frequently and with great success today. Current catheter-based interventions are frequently used in cardiology, interventional radiology, and neuroradiology for treatment of aortic, coronary, cerebrovascular, and other peripheral arterial occlusive and nonocclusive diseases. Interventional therapies are also available for a long time in structural heart diseases (e.g., closure of; atrial or ventricular septal defects, patent foramen ovale, or left atrial appendage). © 2016 The Author(s). Licensee InTech. 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. © 2018 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. Coronary angiographic interventions evolved from basic left heart catheterization to complex interventions including multivessel, bifurcation, left main, and chronic total occlusion therapies. There is a huge amount of industrial materials including differently shaped catheters, guidewires, balloons, stents (bare metal and drug eluting), absorbable biovascular scaffolds, etc. used in modern catheter laboratories. artery disease patients in patients with non-STEMI (after stabilization of the patient), use of radial artery grafts over saphenous vein grafts in patients with severe coronary stenosis, and to prefer CABG surgery for patients with coronary artery disease, heart failure, and left Introductory Chapter: An Overview of Angiographic Vascular Interventions http://dx.doi.org/10.5772/intechopen.81768 5 In this book, we aimed to overview our current diagnostic and therapeutic abilities while using angiography in patients with different vascular diseases. Recent developments in interventional therapies, drugs, and devices provided us great success in the treatment of vascular diseases but we have to learn and progress more. In the future, researchers and developers will keep on fighting against atherosclerotic vascular diseases with the aim of decreasing morbidity and mortality, providing people a healthy life and protecting the well-being of subjects. However, we should not underestimate the value of preventive medicine to achieve more success. Acibadem Mehmet Ali Aydinlar University, Vocational School of Health Sciences, Istanbul, [1] Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, et al. Heart disease and stroke Statistics-2017 update: A report from the American Heart Association. Circulation. [2] Global burden of disease (GBD) 2010 study. The Lancet (2012). Available from: http:// [3] Mueller RL, Sanborn TA. The history of interventional cardiology: Cardiac catheterization, angioplasty, and related interventions. American Heart Journal. 1995;129(1):146-172 [4] Matsuo H, Kawase Y. FFR and iFR guided percutaneous coronary intervention. Cardio- [5] Hebsgaard L, Nielsen TM, Tu S, Krusell LR, Maeng M, Veien KT, et al. Co-registration of optical coherence tomography and X-ray angiography in percutaneous coronary intervention. The Does Optical Coherence Tomography Optimize Revascularization [6] Pyxaras SA, Tu S, Barbato E, Barbati G, Di Serafino L, De Vroey F, et al. Quantitative angiography and optical coherence tomography for the functional assessment of nonobstructive coronary stenoses: Comparison with fractional flow reserve. American (DOCTOR) fusion study. International Journal of Cardiology. 2015;182:272-278 ventricular ejection fraction <35% [9]. Address all correspondence to: [email protected] www.thelancet.com/themed/global-burden-of-disease vascular Intervention and Therapeutics. 2016;31(3):183-195 Heart Journal. 2013;166(6):1010-1018.e1 Author details Burak Pamukçu Turkey References 2017;135(10):e146-e603 On the other hand, the more we understand the coronary physiology, the more we use the evidence-based techniques. For example, fractional flow reserve (FFR) allows us to evaluate the severity of a coronary stenosis (does the stenosis cause ischemia or not) during coronary angiography and guides us whether revascularization is needed [4]. The superiority of FFRguided revascularization strategy has been shown in recent trials (DEFER, FAME, and FAME 2) over angiography guided strategy [4]. Furthermore, newer techniques not requiring hyperemic stimulation, for example, instantaneous wave free ratio (iFR) can be used today for the evaluation of coronary stenosis [4]. Intracoronary imaging modalities (intravascular ultrasound (IVUS) and optical coherence tomography (OCT)) aim to provide accurate lesion delineation and precise measurements for use during angioplasty. It can provide valuable information while choosing the stent size and best position for implantation [5, 6]. Nowadays, both IVUS and OCT are more frequently used especially during complex coronary interventions (e.g., left main and bifurcation lesions). Implementation of interventional therapies in daily clinical practice has made the most determinant changes in clinical results of coronary heart disease. Angioplasty and stenting especially in patients with ST-segment elevation myocardial infarction (STEMI) provided important clinical benefits when compared to conventional medical or thrombolytic therapies. At the beginning, angiography was performed via brachial access, then femoral access became the access standard, and now radial access is recommended as the most convenient access site by the current revascularization guidelines [7–9]. In recent years, scientific researches accumulated more evidence to prefer radial access [7, 8] and drug eluting stents (DES) in primary percutaneous interventions [8]. Complex revascularizations during primary percutaneous intervention (PCI) were accepted as contraindicated (class III indication) for a long time, but now, the 2017 European Society of Cardiology (ESC) revascularization guidelines recommend complete revascularization during index primary PCI in STEMI patients in shock with class IIa indication [8]. Another issue, aspiration of blood cloth from the occluded vessel during primary angioplasty (thrombus aspiration) is no more recommended during primary PCI according to the new guidelines. The use of enoxaparin and early hospital discharge are encouraged in patients with STEMI with class IIa indication, while former guidelines were recommending it as class IIb indication [8]. Current advancements in lipid lowering therapy have also affected our practice, and additional lipid lowering therapy is now recommended (class IIa) if low density lipoprotein levels are over 70 mg/dL despite maximum tolerated statin therapy [8]. Current European revascularization guidelines also recommend radial access as standard approach in both angiography and PCI, use of DES instead bare metal stents (BMS) in any PCI, use of Syntax score in revascularization procedures involving left main coronary artery or multivessel disease, use the revascularization strategy preferred among stable coronary artery disease patients in patients with non-STEMI (after stabilization of the patient), use of radial artery grafts over saphenous vein grafts in patients with severe coronary stenosis, and to prefer CABG surgery for patients with coronary artery disease, heart failure, and left ventricular ejection fraction <35% [9]. In this book, we aimed to overview our current diagnostic and therapeutic abilities while using angiography in patients with different vascular diseases. Recent developments in interventional therapies, drugs, and devices provided us great success in the treatment of vascular diseases but we have to learn and progress more. In the future, researchers and developers will keep on fighting against atherosclerotic vascular diseases with the aim of decreasing morbidity and mortality, providing people a healthy life and protecting the well-being of subjects. However, we should not underestimate the value of preventive medicine to achieve more success. ## Author details Coronary angiographic interventions evolved from basic left heart catheterization to complex interventions including multivessel, bifurcation, left main, and chronic total occlusion therapies. There is a huge amount of industrial materials including differently shaped catheters, guidewires, balloons, stents (bare metal and drug eluting), absorbable biovascular scaffolds, On the other hand, the more we understand the coronary physiology, the more we use the evidence-based techniques. For example, fractional flow reserve (FFR) allows us to evaluate the severity of a coronary stenosis (does the stenosis cause ischemia or not) during coronary angiography and guides us whether revascularization is needed [4]. The superiority of FFRguided revascularization strategy has been shown in recent trials (DEFER, FAME, and FAME 2) over angiography guided strategy [4]. Furthermore, newer techniques not requiring hyperemic stimulation, for example, instantaneous wave free ratio (iFR) can be used today for the Intracoronary imaging modalities (intravascular ultrasound (IVUS) and optical coherence tomography (OCT)) aim to provide accurate lesion delineation and precise measurements for use during angioplasty. It can provide valuable information while choosing the stent size and best position for implantation [5, 6]. Nowadays, both IVUS and OCT are more frequently used especially during complex coronary interventions (e.g., left main and bifurcation lesions). Implementation of interventional therapies in daily clinical practice has made the most determinant changes in clinical results of coronary heart disease. Angioplasty and stenting especially in patients with ST-segment elevation myocardial infarction (STEMI) provided important clinical benefits when compared to conventional medical or thrombolytic therapies. At the beginning, angiography was performed via brachial access, then femoral access became the access standard, and now radial access is recommended as the most convenient In recent years, scientific researches accumulated more evidence to prefer radial access [7, 8] and drug eluting stents (DES) in primary percutaneous interventions [8]. Complex revascularizations during primary percutaneous intervention (PCI) were accepted as contraindicated (class III indication) for a long time, but now, the 2017 European Society of Cardiology (ESC) revascularization guidelines recommend complete revascularization during index primary PCI in STEMI patients in shock with class IIa indication [8]. Another issue, aspiration of blood cloth from the occluded vessel during primary angioplasty (thrombus aspiration) is no more recommended during primary PCI according to the new guidelines. The use of enoxaparin and early hospital discharge are encouraged in patients with STEMI with class IIa indication, while former guidelines were recommending it as class IIb indication [8]. Current advancements in lipid lowering therapy have also affected our practice, and additional lipid lowering therapy is now recommended (class IIa) if low density lipoprotein levels are over 70 mg/dL Current European revascularization guidelines also recommend radial access as standard approach in both angiography and PCI, use of DES instead bare metal stents (BMS) in any PCI, use of Syntax score in revascularization procedures involving left main coronary artery or multivessel disease, use the revascularization strategy preferred among stable coronary etc. used in modern catheter laboratories. 4 Angiography evaluation of coronary stenosis [4]. access site by the current revascularization guidelines [7–9]. despite maximum tolerated statin therapy [8]. #### Burak Pamukçu Address all correspondence to: [email protected] Acibadem Mehmet Ali Aydinlar University, Vocational School of Health Sciences, Istanbul, Turkey ### References [7] Kiemeneij F, Laarman GJ, de Melker E. Transradial artery coronary angioplasty. American Heart Journal. 1995;129(1):1-7 Section 2 Coronary Angiography and Percutaneous Coronary Interventions Coronary Angiography and Percutaneous Coronary Interventions [7] Kiemeneij F, Laarman GJ, de Melker E. Transradial artery coronary angioplasty. [8] Ibanez B, James S, Agewall S, Antunes MJ, Bucciarelli-Ducci C, Bueno H, et al. ESC guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The task force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of [9] Neumann FJ, Sousa-Uva M, Ahlsson A, Alfonso F, Banning AP, Benedetto U, et al. 2018 ESC/EACTS guidelines on myocardial revascularization. European Heart Journal. 2018. Cardiology (ESC). European Heart Journal. 2017, 2018;39:119-177 American Heart Journal. 1995;129(1):1-7 6 Angiography DOI: 10.1093/eurheartj/ehy394 Chapter 2 Provisional chapter Minimally Invasive Cardiology for Everyone: Minimally Invasive Cardiology for Everyone: DOI: 10.5772/intechopen.82765 Transradial access is now well established as the safest route for percutaneous coronary intervention. Nevertheless, its use is often restricted to "easy" cases, switch to the transfemoral route being too rapidly advocated/mandated. We will discuss the different challenges associated with a "TRA for everybody" strategy. (1) The vascular access per se is challenging. TRA failure is most of the time an operator failure to cannulate this vessel. There are some ways to overcome the technical problems and to improve the operator skill and his success rate. (2) TRA is systematically denied for some patient populations: patients with previous coronary artery bypass graft surgery are particularly at risk of not being catheterized by TRA despite excellent performance of this route for diagnostic or intervention. In the same way, MI patients in unstable condition are also at risk to be catheterized by TFA although, most of the time, their condition is addressable through TRA and will largely benefit from this route. (3) Frailty and small body-sized ill patients are also at risk of TFA for PCI when proximal coronary artery disease must be treated. There are alternatives to the use of large and very large catheters for treatment of proximal coronary artery disease. (4) The radial occlusion is a manageable problem, with Keywords: transradial access, coronary angiography and percutaneous coronary Transradial access (TRA) is now well established as the safest route for percutaneous coronary intervention (PCI) and must be attempted at first whenever possible. In real world, too many cases today are still performed though transfemoral access (TFA). In a case-to-case > © 2016 The Author(s). Licensee InTech. 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. © 2018 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. Challenging the Transradial Access Challenging the Transradial Access Additional information is available at the end of the chapter Vincent DangoisseAdditional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.82765 simple and effective solutions. interventions 1. Introduction Vincent Dangoisse Abstract #### Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access DOI: 10.5772/intechopen.82765 #### Vincent Dangoisse Additional information is available at the end of the chapter Vincent DangoisseAdditional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.82765 #### Abstract Transradial access is now well established as the safest route for percutaneous coronary intervention. Nevertheless, its use is often restricted to "easy" cases, switch to the transfemoral route being too rapidly advocated/mandated. We will discuss the different challenges associated with a "TRA for everybody" strategy. (1) The vascular access per se is challenging. TRA failure is most of the time an operator failure to cannulate this vessel. There are some ways to overcome the technical problems and to improve the operator skill and his success rate. (2) TRA is systematically denied for some patient populations: patients with previous coronary artery bypass graft surgery are particularly at risk of not being catheterized by TRA despite excellent performance of this route for diagnostic or intervention. In the same way, MI patients in unstable condition are also at risk to be catheterized by TFA although, most of the time, their condition is addressable through TRA and will largely benefit from this route. (3) Frailty and small body-sized ill patients are also at risk of TFA for PCI when proximal coronary artery disease must be treated. There are alternatives to the use of large and very large catheters for treatment of proximal coronary artery disease. (4) The radial occlusion is a manageable problem, with simple and effective solutions. Keywords: transradial access, coronary angiography and percutaneous coronary interventions #### 1. Introduction Transradial access (TRA) is now well established as the safest route for percutaneous coronary intervention (PCI) and must be attempted at first whenever possible. In real world, too many cases today are still performed though transfemoral access (TFA). In a case-to-case © 2016 The Author(s). Licensee InTech. 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. © 2018 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. confrontation, "believers" in the radial way [1] would easily refute common arguments still actually developed by TFA proponents to skip the TRA attempt (or to dash the attempt off): planned intervention better managed/proceeded through femoral access (angiography for a patient with previous coronary artery bypass graft (CABG) surgery being a frequent one), planned interventions requiring large materials/catheters, anticipated lack of support during intervention (a true but often hidden fear for several operators), poor/small/not palpable radial artery, previous attempt failure(s), lack of time ("urgency"), "fast" intervention required for a frailty patient, negative Allen test (it is the most hilarious reason for a radial believer), and finally a failed but often too short attempt. to go through; the vessel may flee away the tip of the needle or may simply "disappear" when a so frequently—and wrongly—invoked "spasm" occurs after a first unsuccessful stick (see discussion below). Although permeable and functional, a radial artery may be unpalpable due to a thick arterial wall or a low flow state. As any other arterial bed, the radial artery is not spared by the inverse remodeling process associated with diabetes, arteritis, and aging. Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 11 So, the first step to optimize the TRA success rate and therefore "TRA for everybody" is Data clarifying the causes of TRA failure will now be presented, and secondly, some clues A prospective study about the TRA, conducted by two TRA "believers" and with two major objectives in mind, started in 2015 [4]: firstly, defining the rate of conversion to transfemoral access when the operator must first attempt (and fail) both radial arteries before converting to transfemoral and secondly, the study was designed to record daily and on a case-by-case basis the causes/location of radial artery attempt failure and to define their relative occurrences. The protocol also required grading (1) the ease for cannulation (before catheterization, as assessed at the bedside), (2) the real ease for the attempt, and (3) the ease for the catheteriza- By protocol, every single consecutive left heart cardiac catheterization, diagnostic or interventional, elective or urgent, has to be first attempted by TRA (right or left or both). Only non-palpable radial artery (on both sides) or abnormal Allen test (Barbeau type D on both sides) and patient refusal were excluded. Let us say immediately that the two operators never encountered a patient with a Barbeau type D present on both sides and that non-palpable radial artery may be permeable and functional as easily assessed with a Doppler probe. Thus, basically, no patient was excluded from the study for such reasons: "TRA for everybody" is As designed by the protocol, all sorts of patient populations were attempted by radial artery, including post-CABG surgery patients, even patients grafted with both left and right internal mammary artery. Some local surgeons also used the gastroepiploïc artery as graft (and not as From January 2005 to June 2007, both operators successively and prospectively proceeded to catheterize 1826 patients, starting from right or left TRA, at the operator discretion. PCI accounted for 40% of the procedures. The study was published as an original contribution in feasible at the level of the initial assessment for an arterial access. free graft). Such patients were also included. Even shock patients were included in the study. the Journal of Invasive Cardiology in September 2010 [4]. Finally, the patient may be hypotensive with a more difficult vascular access. helping in the problem's resolution will be explored. 3. TRA failure's causes tion itself (catheter manipulation). investing in solving the problem of the puncture/cannulation of the radial artery. What is the truth in the background of this resistance? The truth is TRA is not and will never be an easy way to perform cardiac catheterization, the learning curve is long (and never ends), and TRA requires from the operator a true personal investment. Hopefully—and it has to be written somewhere—the return on investments in the TRA technique is large, and succeeding in a difficult case is usually even more gratifying for the physician and his patient when the procedure involved only TRA. "TRA for everybody" is a personal crusade, and it lets the author to succeed a "non-femoral" vascular route for 1019 of his last 1023 procedures (from mid-March 2017 to mid-November 2018). The 1023 procedures included primary PCI for 152 ST elevation myocardial infarct (STEMI) patients—femoral access for 2 of them—coronary angiography (and ad hoc PCI when requested) for 108 post-CABG patients, and a total of 568 PCI were performed (73% with 5 Fr guide catheters). Common challenges associated with a "TRA for everybody" strategy and possible solutions must be considered. Subjects being discussed will be: - The CABG population. - Myocardial infarct (MI) patients in unstable condition. - Frailty and small body-sized ill patients. #### 2. Access to the radial artery: the vascular access Cannulation failure, which is the main cause of TRA failure, is disgracefully received by the majority of interventional cardiologists and stays subsequently hidden. And yet it is not a shame to fail the radial artery cannulation: it is a difficult task and so for many reasons. First, the artery is usually small-sized, its diameter being around 2–3 mm [2] depending of the individual body height as demonstrated in one recent study [3]. The arterial wall may be difficult to go through; the vessel may flee away the tip of the needle or may simply "disappear" when a so frequently—and wrongly—invoked "spasm" occurs after a first unsuccessful stick (see discussion below). Although permeable and functional, a radial artery may be unpalpable due to a thick arterial wall or a low flow state. As any other arterial bed, the radial artery is not spared by the inverse remodeling process associated with diabetes, arteritis, and aging. Finally, the patient may be hypotensive with a more difficult vascular access. So, the first step to optimize the TRA success rate and therefore "TRA for everybody" is investing in solving the problem of the puncture/cannulation of the radial artery. Data clarifying the causes of TRA failure will now be presented, and secondly, some clues helping in the problem's resolution will be explored. #### 3. TRA failure's causes confrontation, "believers" in the radial way [1] would easily refute common arguments still actually developed by TFA proponents to skip the TRA attempt (or to dash the attempt off): planned intervention better managed/proceeded through femoral access (angiography for a patient with previous coronary artery bypass graft (CABG) surgery being a frequent one), planned interventions requiring large materials/catheters, anticipated lack of support during intervention (a true but often hidden fear for several operators), poor/small/not palpable radial artery, previous attempt failure(s), lack of time ("urgency"), "fast" intervention required for a frailty patient, negative Allen test (it is the most hilarious reason for a radial believer), and What is the truth in the background of this resistance? The truth is TRA is not and will never be an easy way to perform cardiac catheterization, the learning curve is long (and never ends), and TRA requires from the operator a true personal investment. Hopefully—and it has to be written somewhere—the return on investments in the TRA technique is large, and succeeding in a difficult case is usually even more gratifying for the physician and his patient when the "TRA for everybody" is a personal crusade, and it lets the author to succeed a "non-femoral" vascular route for 1019 of his last 1023 procedures (from mid-March 2017 to mid-November 2018). The 1023 procedures included primary PCI for 152 ST elevation myocardial infarct (STEMI) patients—femoral access for 2 of them—coronary angiography (and ad hoc PCI when requested) for 108 post-CABG patients, and a total of 568 PCI were performed (73% Common challenges associated with a "TRA for everybody" strategy and possible solutions Cannulation failure, which is the main cause of TRA failure, is disgracefully received by the majority of interventional cardiologists and stays subsequently hidden. And yet it is not a shame to fail the radial artery cannulation: it is a difficult task and so for many reasons. First, the artery is usually small-sized, its diameter being around 2–3 mm [2] depending of the individual body height as demonstrated in one recent study [3]. The arterial wall may be difficult finally a failed but often too short attempt. must be considered. Subjects being discussed will be: • Frailty and small body-sized ill patients. 3. The radial artery occlusion problem. 2. Some patient populations "at risk" to be catheterized by TFA: • Myocardial infarct (MI) patients in unstable condition. 2. Access to the radial artery: the vascular access procedure involved only TRA. 10 Angiography with 5 Fr guide catheters). 1. The vascular access. • The CABG population. A prospective study about the TRA, conducted by two TRA "believers" and with two major objectives in mind, started in 2015 [4]: firstly, defining the rate of conversion to transfemoral access when the operator must first attempt (and fail) both radial arteries before converting to transfemoral and secondly, the study was designed to record daily and on a case-by-case basis the causes/location of radial artery attempt failure and to define their relative occurrences. The protocol also required grading (1) the ease for cannulation (before catheterization, as assessed at the bedside), (2) the real ease for the attempt, and (3) the ease for the catheterization itself (catheter manipulation). By protocol, every single consecutive left heart cardiac catheterization, diagnostic or interventional, elective or urgent, has to be first attempted by TRA (right or left or both). Only non-palpable radial artery (on both sides) or abnormal Allen test (Barbeau type D on both sides) and patient refusal were excluded. Let us say immediately that the two operators never encountered a patient with a Barbeau type D present on both sides and that non-palpable radial artery may be permeable and functional as easily assessed with a Doppler probe. Thus, basically, no patient was excluded from the study for such reasons: "TRA for everybody" is feasible at the level of the initial assessment for an arterial access. As designed by the protocol, all sorts of patient populations were attempted by radial artery, including post-CABG surgery patients, even patients grafted with both left and right internal mammary artery. Some local surgeons also used the gastroepiploïc artery as graft (and not as free graft). Such patients were also included. Even shock patients were included in the study. From January 2005 to June 2007, both operators successively and prospectively proceeded to catheterize 1826 patients, starting from right or left TRA, at the operator discretion. PCI accounted for 40% of the procedures. The study was published as an original contribution in the Journal of Invasive Cardiology in September 2010 [4]. The first and major contribution of the study was to offer strong data for the cornerstone of an effective politic of "TRA for everybody": to have to attempt both radial arteries before converting to a femoral access. The study succeeded in offering a "TRA only" procedure for 98.8% of the study population (Figure 1). This high success rate was obtained after attempting the second radial artery for the 6.2% missed first radial artery, 4.4% when excluding the patients with previous CABG surgery. Attempting the second radial artery was successful in about 80% of the cases. As illustrated in Figure 1, the study did not identify a special population who will not benefit from TRA. It also dismisses some apprehensions regarding TRA: rate of truly difficult cannulation was half less than anticipated, and difficult catheterizations were 50% less frequent than anticipated (and this rate stays stable at 6%), Figure 2. Multivariate analysis (GEE, RMGEE program, K.Y. Liang and S.I. Zeger, Longitudinal data analysis using generalized linear models, Biometrika 73, 1986) strongly proved the learning curve existence and identified some of the major factors playing in the TRA world, namely, the artery size and a diffusely diseased arterial bed (peripheral artery disease). Four predictive variables for a first radial attempt failure emerged in the study: The second major contribution was to reveal that the main cause of TRA failure was not at all related to difficult anatomies as commonly reported and taught (Figure 3). At the start of a TRA business, the radial artery cannulation is accounting for about 75% as the main cause to fail. It accounts for 90% of attempt failures when operator had gained more experience. There are several ways to fail an artery cannulation, reflecting the three steps involved: (1) the puncture itself, (2) wiring the needle or the plastic cannula when they sit in the lumen vessel, and (3) after needle/cannula removal, pushing the sheath over the wire. When analyzing the three different steps, the far most prevalent problem arises at the wiring step: operator reaches the lumen artery, usually with a good blood's backflow, but he cannot forward the wire. Identifying this problem lets to develop better strategies and material (see below). Thereafter 2007, enrollment in the study protocol was extended, allowing the creation of a large radial access database. With time and experience, more expertise arose [5], and looking at the same data in 2010, failures related to anatomical consideration were actually avoided: failure to cannulate the artery emerged as the cause for 92% of failed attempts (again mainly because of the wiring problem). Crossover to femoral access declined to 0.9% (2010) and is now 0.4% (2017–2018). Figure 2. TRA: Real versus anticipated difficulties. Figure 1. TRA success rate for 1826 consecutive procedures. Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 13 Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 13 Figure 1. TRA success rate for 1826 consecutive procedures. The first and major contribution of the study was to offer strong data for the cornerstone of an effective politic of "TRA for everybody": to have to attempt both radial arteries before converting to a femoral access. The study succeeded in offering a "TRA only" procedure for 98.8% of the study population (Figure 1). This high success rate was obtained after attempting the second radial artery for the 6.2% missed first radial artery, 4.4% when excluding the patients with previous CABG surgery. Attempting the second radial artery was successful in about 80% of the As illustrated in Figure 1, the study did not identify a special population who will not benefit from TRA. It also dismisses some apprehensions regarding TRA: rate of truly difficult cannulation was half less than anticipated, and difficult catheterizations were 50% less frequent Multivariate analysis (GEE, RMGEE program, K.Y. Liang and S.I. Zeger, Longitudinal data analysis using generalized linear models, Biometrika 73, 1986) strongly proved the learning curve existence and identified some of the major factors playing in the TRA world, namely, the artery size and a diffusely diseased arterial bed (peripheral artery disease). Four predic- • Year of the procedure, variable related to the learning curve, and operators getting better • Pre-procedure clinical evaluation for a difficult access, OR 2.5, 95% C.I. 1.3–4.9, p 0.006. • Small radial artery size as assessed clinically before catheterization, OR 2.6, 95% C.I. 1.4–5.0, The second major contribution was to reveal that the main cause of TRA failure was not at all related to difficult anatomies as commonly reported and taught (Figure 3). At the start of a TRA business, the radial artery cannulation is accounting for about 75% as the main cause to fail. It accounts for 90% of attempt failures when operator had gained more experience. There are several ways to fail an artery cannulation, reflecting the three steps involved: (1) the puncture itself, (2) wiring the needle or the plastic cannula when they sit in the lumen vessel, When analyzing the three different steps, the far most prevalent problem arises at the wiring step: operator reaches the lumen artery, usually with a good blood's backflow, but he cannot forward the wire. Identifying this problem lets to develop better strategies and material (see Thereafter 2007, enrollment in the study protocol was extended, allowing the creation of a large radial access database. With time and experience, more expertise arose [5], and looking at the same data in 2010, failures related to anatomical consideration were actually avoided: failure to cannulate the artery emerged as the cause for 92% of failed attempts (again mainly because of the wiring problem). Crossover to femoral access declined to 0.9% (2010) and is than anticipated (and this rate stays stable at 6%), Figure 2. tive variables for a first radial attempt failure emerged in the study: • Presence of peripheral artery disease, OR 1.8, 95% CI 1.1–2.8, p 0.016. and (3) after needle/cannula removal, pushing the sheath over the wire. and better with time, OR 0.6, 95% C.I. 0.4–0.8, p < 0.001. cases. 12 Angiography p 0.003. below). now 0.4% (2017–2018). Figure 2. TRA: Real versus anticipated difficulties. of the small cannula within the first mm of the radial artery greatly facilitates the radial wiring and subsequent sheath insertion. Exchange for any hydrophilic or coronary dedicated angioplasty wires is possible and may help and saves many attempts failing with standard wires. The technique is in the author's mind more successful addressing radial arteries of small diameter. Using a 22 gauge system, the vessel injury at the tip of the needle is minimal and allows a through-and-through puncture. The technique is easy to standardize and thus to teach: beginners are instructed to push more deeply the over-the-needle cannula system once the needle reaches the artery lumen (backflow at the hub): doing so, the bevel of the needle is now lying beyond the arterial's posterior wall. Keeping firmly the cannula in place, the metallic part of the system is removed. Then the plastic cannula is gently and mm per mm withdrawn until the blood flows again: the cannula is now perfectly lying in the arterial lumen, and wiring is easier (with the standard or optional wires). For wiring attempts, it is easier to secure in position a plastic cannula than a bore metallic needle. The drawback of the technique is quite minimal: the bevel of the needle is usually not well sharpened for crossing an arterial wall (stiffer and thicker than a venous one); the echogenicity is less than the bore metallic needle (smaller size); some plastics are not well supportive for the standard metallic wire when it has to enter the arterial lumen (all brands of intravenous cannula are not equiva- Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 15 Investing in the over-the-needle cannula system, a needle dedicated for the radial access was designed, several prototypes were successfully tested, and the needle is now patented in the USA and Japan and patent pending in the EU [6, 7]. The invention lies in a small distal aperture near the metallic needle tip combined with at least one reinforcing shoulder at the inner surface of the overlying plastic cannula: the system allows a very fast visualization of the tip needle entering the vessel lumen, faster than when you have to wait until the blood flow reaches the more distant needle hub: with the invention, the operator sees first the blood entering the needle's body and then reaching the hub. This feature helps for the first step of sticking the vessel, shortens the cannulation time, and enhances the success rate (no need to re-puncture, less chance to have "spasm" after a first unsuccessful stick). The needle is wait- So, the first recommendation for resolution of the cannulation problem is to invest in the overthe-needle cannula technique for puncturing, giving attention to the choice of the puncture The radial artery is a quite superficial structure easily assessed by ultrasound. The technique requires only a small-sized probe, and setup is easy and fast: puncturing while viewing the artery is obviously easier, and in the most recent author's practice, its use allowed to succeed when blinded attempt had failed. Since its introduction in January 2018 as a bailout technique, and for 439 consecutive procedures, the ultrasound-guided puncture accounted for 43 patients. The visualization of an artery just attempted blindly offered the explanation for the probably most frequent mechanism in play when reattempting unsuccessfully to re-puncture or rewire the vessel: hematoma arising around and within the wall of the vessel, reducing lent for that purpose). ing investors/manufacturers to get in production. material and to the alternative wires. 4.2. Ultrasound-guided puncture Figure 3. Causes of TRA failure at first attempt. ### 4. What are the proposed solutions for improving the success rate of radial artery cannulation? #### 4.1. Utilizing the best techniques and materials for puncturing Let us talk about sticking the artery, first. Two techniques for radial artery cannulation are at works in the cardiac catheterization world and both gets around 50% of the actual "market." The first one is derived from the femoral world and is the Seldinger technique. It uses a bore metal needle (usually a 21 Gauge) wired with a short metallic 0.021″ straight or J wire. Its advantage is a sharper bevel, more able to penetrate a stiff arterial wall and a better echogenicity when puncturing with ultrasound guidance. Using the needle with a small body length is recommended: backflow at the hub, signing the puncture success, will happen quicker with a short needle like the Cook® needle 3.5 cm 21 G. There is a major drawback with the bore metal needle technique: when the standard wire does not progress (either at the needle's entry in the vessel or further away within the artery), the choice for an alternative wire is limited, and particularly all kind of hydrophilic or angioplasty wires may not be used (possible "peeling" of the coating). When you know that more than 75% of artery cannulation is missed because of the wiring problem, it is annoying to lose this possibility. The second method is derived from the nursing world: it uses an over-the-needle cannula system (needles designed for puncturing veins and intravenous cannula insertion). Insertion of the small cannula within the first mm of the radial artery greatly facilitates the radial wiring and subsequent sheath insertion. Exchange for any hydrophilic or coronary dedicated angioplasty wires is possible and may help and saves many attempts failing with standard wires. The technique is in the author's mind more successful addressing radial arteries of small diameter. Using a 22 gauge system, the vessel injury at the tip of the needle is minimal and allows a through-and-through puncture. The technique is easy to standardize and thus to teach: beginners are instructed to push more deeply the over-the-needle cannula system once the needle reaches the artery lumen (backflow at the hub): doing so, the bevel of the needle is now lying beyond the arterial's posterior wall. Keeping firmly the cannula in place, the metallic part of the system is removed. Then the plastic cannula is gently and mm per mm withdrawn until the blood flows again: the cannula is now perfectly lying in the arterial lumen, and wiring is easier (with the standard or optional wires). For wiring attempts, it is easier to secure in position a plastic cannula than a bore metallic needle. The drawback of the technique is quite minimal: the bevel of the needle is usually not well sharpened for crossing an arterial wall (stiffer and thicker than a venous one); the echogenicity is less than the bore metallic needle (smaller size); some plastics are not well supportive for the standard metallic wire when it has to enter the arterial lumen (all brands of intravenous cannula are not equivalent for that purpose). Investing in the over-the-needle cannula system, a needle dedicated for the radial access was designed, several prototypes were successfully tested, and the needle is now patented in the USA and Japan and patent pending in the EU [6, 7]. The invention lies in a small distal aperture near the metallic needle tip combined with at least one reinforcing shoulder at the inner surface of the overlying plastic cannula: the system allows a very fast visualization of the tip needle entering the vessel lumen, faster than when you have to wait until the blood flow reaches the more distant needle hub: with the invention, the operator sees first the blood entering the needle's body and then reaching the hub. This feature helps for the first step of sticking the vessel, shortens the cannulation time, and enhances the success rate (no need to re-puncture, less chance to have "spasm" after a first unsuccessful stick). The needle is waiting investors/manufacturers to get in production. So, the first recommendation for resolution of the cannulation problem is to invest in the overthe-needle cannula technique for puncturing, giving attention to the choice of the puncture material and to the alternative wires. #### 4.2. Ultrasound-guided puncture 4. What are the proposed solutions for improving the success rate of Let us talk about sticking the artery, first. Two techniques for radial artery cannulation are at works in the cardiac catheterization world and both gets around 50% of the actual "market." The first one is derived from the femoral world and is the Seldinger technique. It uses a bore metal needle (usually a 21 Gauge) wired with a short metallic 0.021″ straight or J wire. Its advantage is a sharper bevel, more able to penetrate a stiff arterial wall and a better echogenicity when puncturing with ultrasound guidance. Using the needle with a small body length is recommended: backflow at the hub, signing the puncture success, will happen There is a major drawback with the bore metal needle technique: when the standard wire does not progress (either at the needle's entry in the vessel or further away within the artery), the choice for an alternative wire is limited, and particularly all kind of hydrophilic or angioplasty wires may not be used (possible "peeling" of the coating). When you know that more than 75% of artery cannulation is missed because of the wiring problem, it is annoying to lose The second method is derived from the nursing world: it uses an over-the-needle cannula system (needles designed for puncturing veins and intravenous cannula insertion). Insertion 4.1. Utilizing the best techniques and materials for puncturing quicker with a short needle like the Cook® needle 3.5 cm 21 G. radial artery cannulation? 14 Angiography Figure 3. Causes of TRA failure at first attempt. this possibility. The radial artery is a quite superficial structure easily assessed by ultrasound. The technique requires only a small-sized probe, and setup is easy and fast: puncturing while viewing the artery is obviously easier, and in the most recent author's practice, its use allowed to succeed when blinded attempt had failed. Since its introduction in January 2018 as a bailout technique, and for 439 consecutive procedures, the ultrasound-guided puncture accounted for 43 patients. The visualization of an artery just attempted blindly offered the explanation for the probably most frequent mechanism in play when reattempting unsuccessfully to re-puncture or rewire the vessel: hematoma arising around and within the wall of the vessel, reducing further its lumen (and the pulse). So the "spasm" frequently invoked is in reality hematoma compression/expansion: it explains why the artery is no more palpable and more difficult to re-stick. Furthermore, the ultrasound guidance lets the operator decides to stick at another position or to skip to the ulnar or the contralateral radial/ulnar artery. The ultrasound-guided puncture allows the operator to stick more successfully the artery, but it does not resolve the problem of wiring. way to further improve operator and mainly patient comfort and safety, at least when starting from the left hand. Large series looking at the ease and effectiveness of the hemostasis together with vessel patency and avoidance of ischemic/sensitive problems are mandatory. Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 17 The last recommendation will be to become familiar with ulnar artery cannulation, and Patients with previous CABG surgery are difficult to angiography by comparison with non-CABG surgery patients. They are older and have advanced coronary artery disease for many years. Peripheral artery disease and other comorbid conditions such as some degree of chronic kidney failure and chronic obstructive pulmonary disease are frequently found. Graft assessment is an additional task after coronary angiogram and may be tricky due to nonstandard vein graft's ostial location and unavailable dedicated efficient pre-shaped catheters. Arterial grafts are also uneasy to reach: both mammary arteries are originating at a sharp angle from their subclavicular arteries that are to be engaged: the right subclavicular artery may particularly be tricky to reach, except when starting from the right upper extremity. Finally, the gastroepiploic artery, a branch of the coeliac trunk artery, used by some surgeons to graft the right posterior descending artery, may also be difficult to adequately angiography. The additional task of graft angiography and the higher-risk profile of these patients let to more catheters use, 5. Some patient populations "at risk" to be catheterized by TFA more manipulations, and therefore a greater risk of neurological complications [9]. Due to the anticipated complexity of this procedure and concerns about possible greater X-ray exposition, TRA—and its associated clinical benefits—is often denied to this population. Louvard et al. [10] and Yabe et al. [11] reported in 1998 the feasibility of graft angiography and particularly left internal mammary artery (LIMA) angiography through a right radial artery approach. Kim [12] and Kwang [13] described bilateral selective internal mammary artery angiography via the right radial approach as early as 2001. Sanmartin et al. published in 2006 their feasibility analysis and comparison with transfemoral approach [14]. They concluded that there is no excessive delay or greater radiation exposure and that the TRA appears at least as safe as TFA. Their study was retrospective, excluded patients with bilateral mammary grafted, and the left radial access was predominant (133 of the 151 TRA compared to the 154 TFA). They reported four failures of cannulation, one puncture failure, one LIMA, and one saphenous vein graft (SVG) not reached. Only 15% of ad hoc PCI were carried out. In 2008, Burzotta [15] and experienced TRA operators described tips and tricks available for addressing post-CABG patients and already pointed out the right radial access as the best first option In 2009, Rathore et al. [16] reported a similar technical TRA success rate for SVG-PCI compared to TFA. Periprocedural MI, access-site bleeding-related complications, and large hematomas were higher in the TFA group. They reported a 5.8% crossover to the femoral route. ultrasound-guided puncture should be a must. 5.1. The population with previous CABG surgery in case of bilateral mammary artery grafts. Since many years, as soon as a Doppler signaled the presence of an arterial flow, cannulation of unpalpable radial arteries was attempted. With the ultrasound guidance, it is now far easier to perform this task (and to confirm that the operator may attempt the cannulation, the views allowing the diagnosis of an occluded artery). With the help of ultrasound, the operator may also decide to attempt a less disease or a larger vessel (ulnar or contralateral). Clearly, the use of ultrasound-guided puncture has a major role to play in a modern strategy of "transradial (or transulnar) access for everybody," and it is the second recommendation for solving the cannulation problem. #### 4.3. Utilizing all individual resources of the nursing/technologist permanent cath lab staff You may be an excellent PCI operator and be quite "ordinary" regarding the puncture task (and some hate this fundamental step). On another hand, in every catheterization laboratory, there are individuals very efficient for sticking vessels, and there are individuals well trained for surface ultrasound. A few years ago, nurses interested in the cannulation task were trained for radial artery puncture. The over-the-needle cannula technique was taught, nurses being well customized with this kind of needle and technique. The fundamental difference between their usual way of working with an over-the-needle cannula and for intravenous cannulation is that they absolutely need to go through and through the vessel for a successful radial artery cannulation. They also have to learn the use of the different wires at (good) works. Recently, the same teaching program was successfully offered to technicians in radiology working in the catheterization laboratory: it provided the advantage of adding peoples trained for ultrasound techniques. Actually, the trained nurses and technologists perform 70% of the author's artery cannulations without any undesired crossover to a femoral access. So, the next recommendation is to train willing and well-skilled nurses and technologists to perform the cannulation task. #### 4.4. Using forearm artery alternatives (the ulnar artery or the left distal radial artery) The ulnar artery has been shown to be a safe alternative route for left heart catheterization [8]. It is anyway a safer route than the femoral access and is sometimes larger than the radial artery. It seats deeper and a sensitive nerve is present along the vessel at the puncture level. Hemostasis is easily performed with the material dedicated for the radial artery compression. So, when a radial attempt fails, the ulnar artery cannulation may be a good alternative even at the same wrist. Of course, ultrasound-guided puncture is also an excellent way to optimize the success cannulation rate. Left distal radial TRA is actually in evaluation as a possible way to further improve operator and mainly patient comfort and safety, at least when starting from the left hand. Large series looking at the ease and effectiveness of the hemostasis together with vessel patency and avoidance of ischemic/sensitive problems are mandatory. The last recommendation will be to become familiar with ulnar artery cannulation, and ultrasound-guided puncture should be a must. #### 5. Some patient populations "at risk" to be catheterized by TFA #### 5.1. The population with previous CABG surgery further its lumen (and the pulse). So the "spasm" frequently invoked is in reality hematoma compression/expansion: it explains why the artery is no more palpable and more difficult to re-stick. Furthermore, the ultrasound guidance lets the operator decides to stick at another position or to skip to the ulnar or the contralateral radial/ulnar artery. The ultrasound-guided puncture allows the operator to stick more successfully the artery, but it does not resolve the Since many years, as soon as a Doppler signaled the presence of an arterial flow, cannulation of unpalpable radial arteries was attempted. With the ultrasound guidance, it is now far easier to perform this task (and to confirm that the operator may attempt the cannulation, the views allowing the diagnosis of an occluded artery). With the help of ultrasound, the operator may Clearly, the use of ultrasound-guided puncture has a major role to play in a modern strategy of "transradial (or transulnar) access for everybody," and it is the second recommendation for You may be an excellent PCI operator and be quite "ordinary" regarding the puncture task (and some hate this fundamental step). On another hand, in every catheterization laboratory, there are individuals very efficient for sticking vessels, and there are individuals well trained for surface ultrasound. A few years ago, nurses interested in the cannulation task were trained for radial artery puncture. The over-the-needle cannula technique was taught, nurses being well customized with this kind of needle and technique. The fundamental difference between their usual way of working with an over-the-needle cannula and for intravenous cannulation is that they absolutely need to go through and through the vessel for a successful radial artery cannulation. They also have to learn the use of the different wires at (good) works. Recently, the same teaching program was successfully offered to technicians in radiology working in the catheterization laboratory: it provided the advantage of adding peoples trained for ultrasound techniques. Actually, the trained nurses and technologists perform 70% of the author's So, the next recommendation is to train willing and well-skilled nurses and technologists to The ulnar artery has been shown to be a safe alternative route for left heart catheterization [8]. It is anyway a safer route than the femoral access and is sometimes larger than the radial artery. It seats deeper and a sensitive nerve is present along the vessel at the puncture level. Hemostasis is easily performed with the material dedicated for the radial artery compression. So, when a radial attempt fails, the ulnar artery cannulation may be a good alternative even at the same wrist. Of course, ultrasound-guided puncture is also an excellent way to optimize the success cannulation rate. Left distal radial TRA is actually in evaluation as a possible 4.4. Using forearm artery alternatives (the ulnar artery or the left distal radial artery) also decide to attempt a less disease or a larger vessel (ulnar or contralateral). artery cannulations without any undesired crossover to a femoral access. 4.3. Utilizing all individual resources of the nursing/technologist permanent cath problem of wiring. 16 Angiography lab staff solving the cannulation problem. perform the cannulation task. Patients with previous CABG surgery are difficult to angiography by comparison with non-CABG surgery patients. They are older and have advanced coronary artery disease for many years. Peripheral artery disease and other comorbid conditions such as some degree of chronic kidney failure and chronic obstructive pulmonary disease are frequently found. Graft assessment is an additional task after coronary angiogram and may be tricky due to nonstandard vein graft's ostial location and unavailable dedicated efficient pre-shaped catheters. Arterial grafts are also uneasy to reach: both mammary arteries are originating at a sharp angle from their subclavicular arteries that are to be engaged: the right subclavicular artery may particularly be tricky to reach, except when starting from the right upper extremity. Finally, the gastroepiploic artery, a branch of the coeliac trunk artery, used by some surgeons to graft the right posterior descending artery, may also be difficult to adequately angiography. The additional task of graft angiography and the higher-risk profile of these patients let to more catheters use, more manipulations, and therefore a greater risk of neurological complications [9]. Due to the anticipated complexity of this procedure and concerns about possible greater X-ray exposition, TRA—and its associated clinical benefits—is often denied to this population. Louvard et al. [10] and Yabe et al. [11] reported in 1998 the feasibility of graft angiography and particularly left internal mammary artery (LIMA) angiography through a right radial artery approach. Kim [12] and Kwang [13] described bilateral selective internal mammary artery angiography via the right radial approach as early as 2001. Sanmartin et al. published in 2006 their feasibility analysis and comparison with transfemoral approach [14]. They concluded that there is no excessive delay or greater radiation exposure and that the TRA appears at least as safe as TFA. Their study was retrospective, excluded patients with bilateral mammary grafted, and the left radial access was predominant (133 of the 151 TRA compared to the 154 TFA). They reported four failures of cannulation, one puncture failure, one LIMA, and one saphenous vein graft (SVG) not reached. Only 15% of ad hoc PCI were carried out. In 2008, Burzotta [15] and experienced TRA operators described tips and tricks available for addressing post-CABG patients and already pointed out the right radial access as the best first option in case of bilateral mammary artery grafts. In 2009, Rathore et al. [16] reported a similar technical TRA success rate for SVG-PCI compared to TFA. Periprocedural MI, access-site bleeding-related complications, and large hematomas were higher in the TFA group. They reported a 5.8% crossover to the femoral route. The Transradial Committee of the SCAI in a 2011 publication concluded that TRA for CABG patients might safely be integrated into routine practice as experience increases [17]. The published 2010 prospective study of 1826 consecutive procedures [4] looking at the conversion rate from TRA to TFA when both radial arteries have to be attempted before the crossover to femoral included 187 patients with previous CABG surgery. The study was extended for the CABG population until 2012, and results were presented at the 2012 ACC meeting [18]. The study differs from the previously reported series: it prospectively addressed patients with previous CABG surgery; the choice of the radial artery to be attempted at first, right versus left, was free, but both radial arteries had to be attempted before converting to femoral access. Patients grafted with both mammary arteries were not excluded, and the study also included patients with gastroepiploïc arteries used as graft. Ad hoc and elective PCI were performed. Importantly, all causes of failed attempts were analyzed and classified. This study reinforces the previous conclusions about TRA feasibility. When considering angiography for the CABG population, particularly when only one mammary artery is grafted, TRA performs as well as for the general population. A success rate above 98% was obtained with a very low requirement for a second artery access (7.2%) in case of only one internal mammary artery (IMA) grafted. Of course, the radial artery to be attempted at first must be ipsilateral to the utilized IMA, and it is better to start with in hand the description of the performed surgery. In case of bilateral IMA, the strategy of attempting the right radial artery at first enhances the chance of completing the procedure through one arterial access (actual chance of success is around 60%). To be noted, in the published series, about 35.5% of procedures included angioplasty and stenting (mainly ad hoc). Tables 1–3 describe the CABG population (from 2007 to 2012) compared to the non-CABG population of the 2010 publication. To summarize the published statistics, for a general population and excluding patients with previous CABG surgery, cannulation failed in 4.9% (requiring crossover to the second radial artery or to an ulnar artery). For the CABG population when the surgeon protocol is available and when only one IMA is grafted, the ipsilateral to the IMA radial cannulation fails for 5.4% of patients and requires use of the ipsilateral ulnar artery. When both IMA are grafted, starting from the right TRA succeeds for about 60% of patients; 40% will further need cannulation of the left TRA (mainly for an adequate LIMA graft patency assessment). In terms of patient safety and avoidance of vascular access-related complications and hemorrhage, a bilateral radial cannulation is far less dangerous than one femoral access, particularly in case of coronary/graft angioplasty. By the way and as reported [19], the PCI success rate stays unaltered by the vascular access. high anticoagulation level. Performing through a radial artery access in these circumstances removes the fear of "collateral damages" related to intense anti-clot treatments and allows retaining the benefits linked to their use. When the situation requires the use of intra-aortic counterpulsation, the TRA PCI saves at least this access from hemorrhage, the intra-aortic balloon being usually removed some days later, when the degree of anticoagulation is far less intense. Door-to-balloon time is only one of the important lifesaving parameters and must not serve as a pretext to skip a well-performed TRA attempt: speed may not lead to haste! Acute MI patients usually maintain initially a decent radial pulse, and cannulation may succeed as in stable condition. Particularly in the setting of acute coronary syndrome (ACS), a failed first attempt must lead to attempting the second radial artery (or the ulnar artery) before crossing over to the less safe femoral route. The author's most recent statistics in STEMI patients will Since mid-March 2017 to mid-November 2018, from a total of 1023 procedures, 152 STEMI patients were primarily addressed by PCI. A grand total of two primary PCI were performed through a femoral access: one MI patient had previously a thoracic vascular repair after a illustrate the TRA feasibility "in real world." CABG, coronary artery bypass graft; IMA, internal mammary artery. Table 1. TRA and CABG vs. non-CABG populations. Population Any Peripheral vascular disease (%) Radial artery looks not easy to puncture Volume of contrast Percutaneous coronary Intervention (ad hoc + elective) (ml) previous CABG surgery Previous CABG surgery N 1639 507 320 187 Age 65 ± 11 71 ± 9 <0.001 72 ± 10 <0.001 70 ± 9 <0.001 Female (%) 31% 18% <0.001 20% <0.001 14% <0.001 Diabetes (%) 17% 29% <0.001 28% <0.001 32% <0.001 HTN (%) 44% 56% <0.001 54% 0.001 58% <0.001 Weight (kg) 79.0 ± 16 81.6 ± 15 0.001 80.8 ± 15 0.037 83.0 ± 16 0.001 Height (cm) 168 ± 9 169 ± 8 0.26 168 ± 9 0.82 170 ± 8 0.021 BMI 27.8 ± 5 28.6 ± 9 0.001 28.6 ± 6 0.009 28.6 ± 5 0.017 BMI ≤ 22 (%) 10.5% 4.3% <0.001 4.1% <0.001 4.8% 0.014 p Previous CABG surgery ≤1 IMA 21% 41% <0.001 43% <0.001 38% <0.001 13.9% 12.6% 0.46 13.8% 0.94 10.7% 0.224 156 ± 78 226 ± 92 <0.001 218 ± 87 <0.0001 239 ± 97 <0.0001 40.5% 35.5% 0.04 41% 0.8 26% <0.001 p (vs. no previous CABGs) Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access Previous CABG surgery 2 IMA http://dx.doi.org/10.5772/intechopen.82765 p (vs. no previous CABGs) 19 #### 5.2. MI patients in unstable condition The TRA lifesaving benefit is directly linked to the illness severity: the STEMI and the unstable non-ST elevation myocardial infarct (NSTEMI) patients are the more likely to require a high level of several anticoagulants/antiplatelet therapies paving the road for serious hemorrhagic events mainly at the vascular (femoral) access site [20]. Vascular closure devices have not been demonstrated to be effective in reducing these vascular complications in the setting of ACS. On the contrary, hemostasis is easily obtained after TRA, even in situation of CABG, coronary artery bypass graft; IMA, internal mammary artery. Table 1. TRA and CABG vs. non-CABG populations. The Transradial Committee of the SCAI in a 2011 publication concluded that TRA for CABG The published 2010 prospective study of 1826 consecutive procedures [4] looking at the conversion rate from TRA to TFA when both radial arteries have to be attempted before the crossover to femoral included 187 patients with previous CABG surgery. The study was extended for the CABG population until 2012, and results were presented at the 2012 ACC meeting [18]. The study differs from the previously reported series: it prospectively addressed patients with previous CABG surgery; the choice of the radial artery to be attempted at first, right versus left, was free, but both radial arteries had to be attempted before converting to femoral access. Patients grafted with both mammary arteries were not excluded, and the study also included patients with gastroepiploïc arteries used as graft. Ad hoc and elective PCI were This study reinforces the previous conclusions about TRA feasibility. When considering angiography for the CABG population, particularly when only one mammary artery is grafted, TRA performs as well as for the general population. A success rate above 98% was obtained with a very low requirement for a second artery access (7.2%) in case of only one internal mammary artery (IMA) grafted. Of course, the radial artery to be attempted at first must be ipsilateral to the utilized IMA, and it is better to start with in hand the description of the performed surgery. In case of bilateral IMA, the strategy of attempting the right radial artery at first enhances the chance of completing the procedure through one arterial access (actual chance of success is around 60%). To be noted, in the published series, about 35.5% of procedures included angioplasty and stenting (mainly ad hoc). Tables 1–3 describe the CABG population (from 2007 to 2012) compared to the non-CABG population of the 2010 publication. To summarize the published statistics, for a general population and excluding patients with previous CABG surgery, cannulation failed in 4.9% (requiring crossover to the second radial artery or to an ulnar artery). For the CABG population when the surgeon protocol is available and when only one IMA is grafted, the ipsilateral to the IMA radial cannulation fails for 5.4% of patients and requires use of the ipsilateral ulnar artery. When both IMA are grafted, starting from the right TRA succeeds for about 60% of patients; 40% will further need cannulation of the left TRA (mainly for an adequate LIMA graft patency assessment). In terms of patient safety and avoidance of vascular access-related complications and hemorrhage, a bilateral radial cannulation is far less dangerous than one femoral access, particularly in case of coronary/graft angioplasty. By the way and as reported [19], the PCI success rate stays unaltered by the vascular access. The TRA lifesaving benefit is directly linked to the illness severity: the STEMI and the unstable non-ST elevation myocardial infarct (NSTEMI) patients are the more likely to require a high level of several anticoagulants/antiplatelet therapies paving the road for serious hemorrhagic events mainly at the vascular (femoral) access site [20]. Vascular closure devices have not been demonstrated to be effective in reducing these vascular complications in the setting of ACS. On the contrary, hemostasis is easily obtained after TRA, even in situation of 5.2. MI patients in unstable condition 18 Angiography patients might safely be integrated into routine practice as experience increases [17]. performed. Importantly, all causes of failed attempts were analyzed and classified. high anticoagulation level. Performing through a radial artery access in these circumstances removes the fear of "collateral damages" related to intense anti-clot treatments and allows retaining the benefits linked to their use. When the situation requires the use of intra-aortic counterpulsation, the TRA PCI saves at least this access from hemorrhage, the intra-aortic balloon being usually removed some days later, when the degree of anticoagulation is far less intense. Door-to-balloon time is only one of the important lifesaving parameters and must not serve as a pretext to skip a well-performed TRA attempt: speed may not lead to haste! Acute MI patients usually maintain initially a decent radial pulse, and cannulation may succeed as in stable condition. Particularly in the setting of acute coronary syndrome (ACS), a failed first attempt must lead to attempting the second radial artery (or the ulnar artery) before crossing over to the less safe femoral route. The author's most recent statistics in STEMI patients will illustrate the TRA feasibility "in real world." Since mid-March 2017 to mid-November 2018, from a total of 1023 procedures, 152 STEMI patients were primarily addressed by PCI. A grand total of two primary PCI were performed through a femoral access: one MI patient had previously a thoracic vascular repair after a Table 2. Radial artery cannulation: failures at first attempt. thoracic trauma (car accident): his right arm was unavailable for left heart catheterization, and cannulation of the left radial artery led to an occluded left subclavicular artery. The second femoral case was a small-sized lady, and the attempts to wire radial (and ulnar) arteries failed at both wrists. This case happened before the availability of the ultrasound technique for rescuing the failed attempts. Seven STEMI cases required simultaneous use of the femoral artery for left ventricular assistance (intra-aortic balloon pump). The hemorrhage-saving TRA feature lets to more liberal use of potent antiplatelet drugs and high heparin doses: it may lead to less distal thrombus embolism and less no-reflow post-reperfusion states. As already puncture/wiring the radial artery; stated, femoral access accounted for only 4 cases of the last 1023 author's procedures. There is another category of patients likely to get catheterized by the femoral route: the small body-sized ill patients, particularly if a frailty condition is associated. Excuses to skip the radial access stay similar: anticipated—but not objectively assessed by ultrasound—too small radial artery, need of a large guide catheter for a quicker intervention, speed required by the degree of illness, etc. Nevertheless, this kind of patient will very badly recover from any hemorrhagic event, and their condition increases greatly the vascular risk [20]. The reader is invited to look at the way a cohort of such frailty; old and severely diseased patients were successfully TRA managed through a double radial route for addressing distal left main or proximal left coronary artery disease [21]. It allowed to position and to work simultaneously with two 5 French-sized guide catheters at no cost of vascular-related complication (Table 4). 5.3. Frailty and small body-sized ill patients Non-CABG patients CABG, coronary artery bypass graft; IMA, internal mammary artery. Table 3. TRA CABG vs. non-CABG, causes of failed attempt. N R (% patients) failures) ± L radial failed N "route to aorta" failed (% total failures) N "coronary or SVG ostium" failed (% total N "IMA contra not reached" (% total failures) TRA for right IMA). Causes of radial attempt failure, saphenous vein graft (SVG) ostia cannulation; N puncture/wiring failed (% total failures) CABG patients N 1639 507 320 187 p CABG patient ≤1 p (vs. no CABG) Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access catheter, route to aorta failure; catheter, contralateral IMA not cannulated (right TRA for left IMA or left CABG patient 2 IMA http://dx.doi.org/10.5772/intechopen.82765 p (vs. no CABG) 21 catheter, coronary/ IMA 90 (5.5%) 119 (23.5%) <0.001 25 (7.8%) 0.106 94 (50.3%) <0.001 67 (74.4%) 14 (11.8%) <0.001 12 (48%) 0.012 2 (2.1%) <0.001 12 (13.3%) 5 (4.2%) 0.017 2 (8%) 0.471 3 (3.2%) 0.012 11 (12.2%) 12 (10.0) 0.625 5 (20%) 0.32 7 (7.5%) 0.276 0 (0%) 88 (74.0%) <0.001 6 (24%) <0.001 82 (87.2%) <0.001 CABG, coronary artery bypass graft; IMA, internal mammary artery. Causes of radial attempt failure, puncture/wiring the radial artery; catheter, route to aorta failure; catheter, coronary/ saphenous vein graft (SVG) ostia cannulation; catheter, contralateral IMA not cannulated (right TRA for left IMA or left TRA for right IMA). Table 3. TRA CABG vs. non-CABG, causes of failed attempt. thoracic trauma (car accident): his right arm was unavailable for left heart catheterization, and cannulation of the left radial artery led to an occluded left subclavicular artery. The second femoral case was a small-sized lady, and the attempts to wire radial (and ulnar) arteries failed at both wrists. This case happened before the availability of the ultrasound technique for rescuing the failed attempts. Seven STEMI cases required simultaneous use of the femoral artery for left ventricular assistance (intra-aortic balloon pump). The hemorrhage-saving TRA feature lets to more liberal use of potent antiplatelet drugs and high heparin doses: it may lead to less distal thrombus embolism and less no-reflow post-reperfusion states. As already stated, femoral access accounted for only 4 cases of the last 1023 author's procedures. #### 5.3. Frailty and small body-sized ill patients All > N Patients TRA success (%) N radial attempted N of radial attempts/patient Right radial as first attempt Right radial failed at first 139 (8.9%) 68 (5.0%) 71 (35.3%) <0.001 10 (16.1%) <0.001 61 (43.9%) <0.001 4(9.7%) 0.175 attempt Left radial as first attempt Left radial failed at first 56 (9.6%) 12 (4.3%) 44 (14.4%) <0.001 13(5.0%) 0.678 31 (64.6%) <0.001 12 (4.7%) 0.83 attempt All failures (first attempt) One (first) failure/patient Table 2. Radial artery cannulation: failures at first attempt. 9.1% 4.9% 22.7% <0.001 CABG, coronary artery bypass graft; IMA, internal mammary artery; TRA, transradial access; TRA-IMA same side, transradial access for ipsilateral IMA. 7.2% 0.091 49.2% <0.001 5.4% 0.721 195 80 115 23 92 16 586 280 306 258 48 257 1560 1359 201 62 1.084 2327 1711 1.044 1.215 <0.001 1.059 0.23 1.481 139 <0.001 1.047 41 0.981 616 2146 2118 98.7 98.9 98 0.13 98.1 339 0.247 97.9 277 0.216 98.3 311 0.395 1621 497 1639 507 Non-CABG CABG p CABG ≤1 p CABG 2 p TRA-IMA p 20 Angiography same side 298 293 IMA 187 183 IMA 320 314 patients patients There is another category of patients likely to get catheterized by the femoral route: the small body-sized ill patients, particularly if a frailty condition is associated. Excuses to skip the radial access stay similar: anticipated—but not objectively assessed by ultrasound—too small radial artery, need of a large guide catheter for a quicker intervention, speed required by the degree of illness, etc. Nevertheless, this kind of patient will very badly recover from any hemorrhagic event, and their condition increases greatly the vascular risk [20]. The reader is invited to look at the way a cohort of such frailty; old and severely diseased patients were successfully TRA managed through a double radial route for addressing distal left main or proximal left coronary artery disease [21]. It allowed to position and to work simultaneously with two 5 French-sized guide catheters at no cost of vascular-related complication (Table 4). VT, ventricular tachycardia; HF, heart failure; NSTEMI, non-ST elevation myocardial infarction; COPD, chronic obstructive pulmonary disease (severity as assessed by the GOLD classification); CVA, cerebrovascular accident; CAD, coronary artery disease; Ef, left ventricular ejection fraction; LMCA, left main coronary artery; RCA, right coronary artery; 3VD, triple vessel disease; LAD, left anterior descending (artery); CX, circumflex (artery); R TUlnA, right transulnar artery; L TRA, left transradial access; 6. How to avoid the radial artery occlusion problem ing a well-patent radial artery may be lifesaving later. Journal of Cardiology [ patency. 7. Conclusion Coronary artery disease (CAD) is a progressive disease, and many patients "enjoying" a first successful TRA PCI will require in the future one or more interventions, evenly in emergency (subacute stent thrombosis, new STEMI or new acute coronary syndrome, etc.). So, preserv In 2016, 1 year before the CRASOC studies were accepted for publication in the American hemostasis role in radial artery occlusion (RAO). Rashid et al. published a well-documented systematic review and meta-analysis [22] about the TRA-related radial artery occlusion. It is easy to summarize the problem and to understand the different ways we must follow to reduce the RAO rate. RAO is the direct consequence of the vessel injury associated with any TRA. Injury happens at three levels, and we have to minimize the trauma at each of these levels: first, the puncture; second, the sheath insertion and catheter manipulations (within the sheath); and finally, the compression/hemostasis following the catheterization. Mention for selecting the best technique and material for artery puncture and cannulation was already made. The author makes the hope that, someday, TRA operators will get the chance to handle the specifically designed and actually patented radial artery needle: it should be the best way to reduce as far as possible the puncture-related aggression. In the same way, it is obvious that reducing the size of the sheath is another excellent way to reduce the related harm against the artery wall. Not only should the size be reduced as far as possible, but also the material must be hydrophilic: non-hydrophilic sheath should be banished from a good TRA practice. The "slippery" problem of the hydrophilic sheath is the best proof of the appropriately reduced parietal stress. This problem may be easily "fixed" at the skin level: a simple "opsite" film placed over the sheath does the job. The introduction of the Terumo® "Glidesheath" family of radial introducers represents a welcomed improvement: The company cleverly worked to offer a reduced outside diameter of the sheath (what the artery" feels") together with a normal inside diameter. It allows operators working predominantly in 5 French (including for PCI), to offer a "virtual 4F" TRA procedure for the majority of their patients. The post-catheterization hemostasis step contributes to the global artery's damage: as proven effective thanks to the 3616 analyzed patients in the CRASOC studies, a gentle and short hemostasis with pneumatic compression (TR Band® compression device, 10 cc of air/90 min utes) represents today the best and most elegant way to minimize the RAO rate. A TRA operator has to be reminded that the hemostasis step is his last chance to save the radial artery "TRA for everybody" is highly desirable for patient safety and comfort. This strategy is achievable, and solutions for more complex populations are provided. Ways to maintain the artery patency are described. Better-suited materials for easier TRA are already offered, 3] as the largest randomized and prospective study analyzing the Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 23 R TRA, right transradial access; DAP, dose area product (Gy/cm2); CPK, creatine phosphokinase. Table 4. TRA PCI and frailties, adapted from ACC, 18;3, 45-52. ### 6. How to avoid the radial artery occlusion problem Coronary artery disease (CAD) is a progressive disease, and many patients "enjoying" a first successful TRA PCI will require in the future one or more interventions, evenly in emergency (subacute stent thrombosis, new STEMI or new acute coronary syndrome, etc.). So, preserving a well-patent radial artery may be lifesaving later. In 2016, 1 year before the CRASOC studies were accepted for publication in the American Journal of Cardiology [3] as the largest randomized and prospective study analyzing the hemostasis role in radial artery occlusion (RAO). Rashid et al. published a well-documented systematic review and meta-analysis [22] about the TRA-related radial artery occlusion. It is easy to summarize the problem and to understand the different ways we must follow to reduce the RAO rate. RAO is the direct consequence of the vessel injury associated with any TRA. Injury happens at three levels, and we have to minimize the trauma at each of these levels: first, the puncture; second, the sheath insertion and catheter manipulations (within the sheath); and finally, the compression/hemostasis following the catheterization. Mention for selecting the best technique and material for artery puncture and cannulation was already made. The author makes the hope that, someday, TRA operators will get the chance to handle the specifically designed and actually patented radial artery needle: it should be the best way to reduce as far as possible the puncture-related aggression. In the same way, it is obvious that reducing the size of the sheath is another excellent way to reduce the related harm against the artery wall. Not only should the size be reduced as far as possible, but also the material must be hydrophilic: non-hydrophilic sheath should be banished from a good TRA practice. The "slippery" problem of the hydrophilic sheath is the best proof of the appropriately reduced parietal stress. This problem may be easily "fixed" at the skin level: a simple "opsite" film placed over the sheath does the job. The introduction of the Terumo® "Glidesheath" family of radial introducers represents a welcomed improvement: The company cleverly worked to offer a reduced outside diameter of the sheath (what the artery" feels") together with a normal inside diameter. It allows operators working predominantly in 5 French (including for PCI), to offer a "virtual 4F" TRA procedure for the majority of their patients. The post-catheterization hemostasis step contributes to the global artery's damage: as proven effective thanks to the 3616 analyzed patients in the CRASOC studies, a gentle and short hemostasis with pneumatic compression (TR Band® compression device, 10 cc of air/90 minutes) represents today the best and most elegant way to minimize the RAO rate. A TRA operator has to be reminded that the hemostasis step is his last chance to save the radial artery patency. #### 7. Conclusion Case 1 > Age M/F BMI/height (cm)/ 25/174/75 weight (kg) Frailty (0–3+) Symptoms at New chest pain and SOB Progression of chest Chest pain and SOB Acute pulmonary edema Acute pulmonary edema NSTEMI VT, persistent HF HBP, diabetes, low Ef (recent) NSTEMI VT, persistent HF HBP, paroxysmal atrial fibrillation Low Ef (recent) CCVS Class 4 pain, SOB CCVS Class 3 Severe COPD Gold III Valvular aortic stenosis, severe (< 1 transient AV block distal LMCA, medina LMCA equivalent, 100% RCA Prox. and mid-LAD a. 75% CX a., 95% RCa. lesion 70%, syntax score 35 Syntax score 28 1,1,1 occluded RCA (3VD), Syntax score 48 cm2), COPD, Past CVA and CAD CCVS Class 3 presentation Associated medical Severe COPD Gold IV, permanent O2 therapy conditions CAD, syntax score Distal LMCA Medina 1,1,1 Distal LMCA Medina 1,1,1 Syntax score 23 Left dominance Syntax score 30 Addressed vessel(s) Arterial access Fluoroscopy time/ 44′/290 ml/228 Gy/cm2 CPK (−) volume of contrast/ DAP/CPK 24 h Follow-up Alive > 1 pneumonia at 1 month SOB, shortness of breath; CCVS, Canadian Cardiovascular Society classification; the GOLD classification); CVA, cerebrovascular accident; CAD, coronary artery disease; CX, circumflex (artery); R TUlnA, right transulnar artery; L TRA, left transradial access; R TRA, right transradial access; DAP, dose area product (Gy/cm2); CPK, creatine phosphokinase. Table 4. TRA PCI and frailties, adapted from ACC, 18;3, 45-52. year, acute Alive > 1 angina year, no Alive > 1 year, aortic Alive > 1 year, class 1, Alive > 1 year, class 1, normalized LV function (had 2 weeks re-hosp for HF) normalized LV function valve stenosis said moderate VT, ventricular tachycardia; HF, heart failure; NSTEMI, non-ST elevation myocardial infarction; COPD, chronic obstructive pulmonary disease (severity as assessed by Ef, left ventricular ejection fraction; LMCA, left main coronary artery; RCA, right coronary artery; 3VD, triple vessel disease; LAD, left anterior descending (artery); R TUlnA/L TRA, 5F R+ L TRA, 5F R+ L TRA, 5F Glidesheath R+ L TRA, 5F Glidesheath Glidesheath 17′/212 ml/143 Gy/cm2 20′/148 ml/114 Gy/cm2 15′/140 ml/73 Gy/cm2 CPK (−) 20′/128 ml/173 Gy/cm2 CPK (−) CPK (−) CPK (−) Glidesheath LMCA LMCA LMCA Ostial-LAD/ostial CX arteries Prox. to distal LAD-Diag a. R+ L TRA, 5F Glidesheath (SS) 2+ 1+ 3+ 3+ 3+ M 81 Case II 70 F 33/159/85 30/160/78 22/160/56 Case III 86 M Case IV 87 F Case V 22 Angiography 89 F 22/160/56 "TRA for everybody" is highly desirable for patient safety and comfort. This strategy is achievable, and solutions for more complex populations are provided. Ways to maintain the artery patency are described. Better-suited materials for easier TRA are already offered, and innovations will continue [23, 24]. The author still believes that in year 2018 the radial way requires a gentleman attitude (at least for the radial artery), demands a rebel inclination—to discard all the negative thinking about the TRA feasibility—and is best served by a "believer" behavior: a believer always will try to find indication rather than contraindication for TRA. [8] Fernandez et al. Safety and efficacy of ulnar artery approach for percutaneous cardiac catheterization: Systematic review and meta-analysis. Catheterization and Cardiovascular Minimally Invasive Cardiology for Everyone: Challenging the Transradial Access http://dx.doi.org/10.5772/intechopen.82765 25 [9] Nilsson T, Lagerqvist B, Tornvall P. Coronary angiography of patients with a previous coronary by-pass operation is associated with a three times increased risk for neurological complications. A report from the Swedish Coronary Angiography and Angioplasty [10] Louvard Y. Graft angiography via the right transradial approach. In: Marco J, Fajadet J, editors. Endovascular Therapy Course Coronary and Peripheral: Ninth Complex Coronary [11] Yabe J, Hirose Y, Kouchi N, Ohmura M, Lijima S, Tanaka K, et al. Is LIMA angiography possible with right radial approach? The use of left Judkins 1.0 (BABY Judkins). Japanese [12] Kim MH, Cha KS, Kim HJ, Kim JS. Bilateral selective internal mammary artery angiography via right radial approach: Clinical experience with newly designed Yumiko [13] Cha KS, Kim MH. Feasibility and safety of concomitant left internal mammary arteriography at the setting of the right transradial coronary angiography. Catheterization and [14] Sanmartin M, Cuevas D, Moxica J, Valdes M, Esparza J, Baz JA, et al. Transradial cardiac catheterization in patients with coronary bypass grafts: Feasibility analysis and comparison with transfemoral approach. Catheterization and Cardiovascular Interventions. [15] Burzotta F, Trani C, Hamon M, Amoroso G, Kiemeneij F. Transradial approach for coronary angiography and interventions in patients with coronary bypass grafts: Tips and [16] Rathore S, Roberts E, Hakeem AR, Pauriah M, Beaumont A, Morris JL. The feasibility of percutaneous transradial coronary intervention for saphenous vein graft lesions and comparison with transfemoral route. Journal of Interventional Cardiology. 2009;22: [17] Caputo RP, Tremmel JA, Rao S, Gilchrist IC, Pyne C, Pancholy S, et al. Transradial arterial access for coronary and peripheral procedures: Executive summary by the transradial committee of the SCAI. Catheterization and Cardiovascular Interventions. [18] Dangoisse V, Guédès A, Gabriel L, Jamart J, Marchandise B, Chenu P, et al. Transradial approach for evaluation of post Coronary Artery Bypass Graft surgery patients: Left or Right radial artery when both mammary arteries are grafted? Journal of the American College of Cardiology. 2012;59(13s1):E187-E187. DOI: 10.13140/2.1.3654.1440 tricks. Catheterization and Cardiovascular Interventions. 2008;72:263-272 catheter. Catheterization and Cardiovascular Interventions. 2001;54:19-24 Registry (SCAAR). Scandinavian Cardiovascular Journal. 2009;43(6):374-379 Angioplasty Course Book. Paris: Europa Edition; 1998. pp. 325-327 Journal of Interventional Cardiology. 1998;13(Suppl I):169 Cardiovascular Interventions. 2002;56:188-195 2006;67:580-584 336-340 2011;78(6):823-839 Interventions. 2018;91:1273-1280 ## Author details Vincent Dangoisse1,2\* \Address all correspondence to: [email protected] 1 CSPQ, Université de Montréal, Canada 2 Catheterization Laboratory, CIUSSS-MCQ, Hôpital Ste-Marie, Trois-Rivières, Québec, Canada ### References* [8] Fernandez et al. Safety and efficacy of ulnar artery approach for percutaneous cardiac catheterization: Systematic review and meta-analysis. Catheterization and Cardiovascular Interventions. 2018;91:1273-1280 and innovations will continue [23, 24]. The author still believes that in year 2018 the radial way requires a gentleman attitude (at least for the radial artery), demands a rebel inclination—to discard all the negative thinking about the TRA feasibility—and is best served by a "believer" behavior: a believer always will try to find indication rather than contraindica- 2 Catheterization Laboratory, CIUSSS-MCQ, Hôpital Ste-Marie, Trois-Rivières, Québec, [1] Dangoisse V. Gentleman, rebel and believer: The radial way. Indian Heart Journal. [2] Saito S, Ikei H, Hosokawa G, Tanaka S. Influence of the ratio between radial artery inner diameter and sheath outer diameter on radial artery flow after transradial coronary [3] Dangoisse V, Guédès A, Chenu P, Hanet C, Albert C, Robin V, et al. Usefulness of a gentle and short hemostasis using the transradial band device after transradial access for percutaneous coronary angiography and interventions to reduce the radial artery occlusion rate (from the prospective and randomized CRASOC I, II, and III studies). The [4] Guédès A, Dangoisse V, Gabriel L, et al. Low rate of conversion to transfemoral approach when attempting both radial arteries for coronary angiography and percutaneous coronary intervention: A study of 1,826 consecutive procedures. Journal of Invasive [5] Dangoisse V, Guédès A. Radial artery cannulation and transradial access for percutaneous coronary angiography and interventions: From experience to expertise of a single [6] Dangoisse V. Vascular Needle System 2016; United States Patent, No.: US 9,408,999 B2, [7] Dangoisse V. Vascular Needle System, JPN, Patent, No.: 6043301, Date of Patent: Nov cardiac centre. J Anesthe Clinic Res. 2012;4:322. DOI: 10.4172/2155-6148.1000322 intervention. Catheterization and Cardiovascular Interventions. 1999;46:173-178 tion for TRA. 24 Angiography Canada References 2010;62(3):202e205 Author details Vincent Dangoisse1,2\* \Address all correspondence to: [email protected] American Journal of Cardiology. 2017;120:374e379 Cardiology. 2010;22(9):391-397 Date of Patent: Aug 9 18, 2016 1 CSPQ, Université de Montréal, Canada [19] Dangoisse V, Guédès A, Gabriel L, Jamart J, Chenu P, Marchandise B, et al. Full conversion from transfemoral to transradial approach for percutaneous coronary interventions results in a similar success rate and a rapid reduction of in-hospital cardiac and vascular major events. EuroIntervention. 2013;9:345e352 Chapter 3* Provisional chapter The Role of Catheter Reshaping at the Angiographic The Role of Catheter Reshaping at the Angiographic In coronary angiography, inability to selectively visualize anomalous coronary arteries is one of the major problems, which a cardiologist encounters during angiography. The process we expect to finish in 15 min can take hours. The angiography times exceeding 1 h are not uncommon. On 2.6% of coronary angiograms, anomalous origin of coronary arteries is encountered. In 0.58% of the cases, the left anterior descending artery (LAD) arises from a separate ostium. The absence of the left main coronary artery (LMCA) can be discerned directly on coronary angiograms obtained using selective visualization of the left circumflex (Cx) or LAD because in most of the cases, the LAD and circumflex artery (Cx) arise from separate ostia. In such situations and where it seems impossible to achieve imaging of anomalous coronary artery, catheter reshaping can be accepted as a solution. This method can be used looking for the carotid and vertebral arteries at the angiography performed via the radial or femoral route also the right and left coronary ostia with anomalous origin, that easily and safely. Keywords: catheter reshaping, angiographic success, anomalous coronary origins For arteries that cannot be selectively visualized frequently, the presence of anomalous origins is looked for, and if they cannot be displayed for the second time, diagnosis of single or atresic coronary arteries is made [1–5]. The most common coronary artery abnormalities are follows; the right coronary artery (RCA) arising from a superoanterior position or left coronary sinus (0.65%), the LAD and Cx arising from separate ostia (0.48%), the Cx arising from the right coronary ostium (0.20%), the LAD arising from the right coronary sinus (0.20%), the single coronary artery is the LMCA (0.02%), the single coronary artery is the RCA (0.11%), and the DOI: 10.5772/intechopen.79210 © 2016 The Author(s). Licensee InTech. 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. © 2018 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. Success Success Yakup Balaban Abstract Yakup Balaban Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.79210 1. Coronary anomalies and frequency #### The Role of Catheter Reshaping at the Angiographic Success The Role of Catheter Reshaping at the Angiographic Success DOI: 10.5772/intechopen.79210 #### Yakup Balaban Yakup Balaban [19] Dangoisse V, Guédès A, Gabriel L, Jamart J, Chenu P, Marchandise B, et al. Full conversion from transfemoral to transradial approach for percutaneous coronary interventions results in a similar success rate and a rapid reduction of in-hospital cardiac and vascular [20] Mehran R, Pocock SJ, Nikolsky E, Clayton T, Dangoas GD, Kirtane AJ, et al. A risk score to predict bleeding in patients with acute coronary syndromes. Journal of the American [21] Dangoisse V, Schroeder E, Hanet C, Guédès A, Pancholy S. Double guide double wrist 5F left coronary artery transradial percutaneous coronary intervention and the X-Kiss technique. Acute Cardiac Care. 2017;18(3):45-52. DOI: 10.1080/17482941.2017.1369126 [22] Rashid M, Kwok CS, Pancholy S, Chugh S, Kedev SA, Bernat I, et al. Radial artery occlusion after transradial interventions: A systematic review and meta-analysis. Journal of [23] Dangoisse V. New 6F guiding catheter for right transradial RCA-percutaneous coronary interventions: First report of performance. JACC: Cardiovascular Interventions. [24] Dangoisse V. A new 5F guiding catheter for right transradial LCA-percutaneous coronary interventions: First report of performance. Catheterization and Cardiovascular College of Cardiology. 2010;55:2556-2566. DOI: 10.1016/j.jacc.2009.09.076 (http://www.tandfonline.com/doi/full/10.1080/17482941.2017.1369126) Interventions. 2013;81(S1):S93-94-D024. DOI: 10.1002/ ccd.24919 major events. EuroIntervention. 2013;9:345e352 26 Angiography the American Heart Association. 2016;5:e002686 2013;6(2\_S):S27. DOI: 10.1016/j.jcin.2012.12.098 Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.79210 #### Abstract In coronary angiography, inability to selectively visualize anomalous coronary arteries is one of the major problems, which a cardiologist encounters during angiography. The process we expect to finish in 15 min can take hours. The angiography times exceeding 1 h are not uncommon. On 2.6% of coronary angiograms, anomalous origin of coronary arteries is encountered. In 0.58% of the cases, the left anterior descending artery (LAD) arises from a separate ostium. The absence of the left main coronary artery (LMCA) can be discerned directly on coronary angiograms obtained using selective visualization of the left circumflex (Cx) or LAD because in most of the cases, the LAD and circumflex artery (Cx) arise from separate ostia. In such situations and where it seems impossible to achieve imaging of anomalous coronary artery, catheter reshaping can be accepted as a solution. This method can be used looking for the carotid and vertebral arteries at the angiography performed via the radial or femoral route also the right and left coronary ostia with anomalous origin, that easily and safely. Keywords: catheter reshaping, angiographic success, anomalous coronary origins #### 1. Coronary anomalies and frequency For arteries that cannot be selectively visualized frequently, the presence of anomalous origins is looked for, and if they cannot be displayed for the second time, diagnosis of single or atresic coronary arteries is made [1–5]. The most common coronary artery abnormalities are follows; the right coronary artery (RCA) arising from a superoanterior position or left coronary sinus (0.65%), the LAD and Cx arising from separate ostia (0.48%), the Cx arising from the right coronary ostium (0.20%), the LAD arising from the right coronary sinus (0.20%), the single coronary artery is the LMCA (0.02%), the single coronary artery is the RCA (0.11%), and the © 2016 The Author(s). Licensee InTech. 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. © 2018 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. LAD arising from the pulmonary artery (0.02%, ALPACA syndrome). The LAD originated from the LIMA can also be present rarely in cases (Balaban Syndrome) (Figures 1 and 2 and Table 1). In most of these cases during angiography, standard catheters fail to selectively visualize the anomalous coronary arteries [1–4] (Figures 1 and 2 and Table 1). In publications on anomalous origins and their incidence rates, detection of anomaly has been made in autopsy or using computed tomography. Currently, there are no encountered comprehensive and detailed publications related to angiographically defined coronary arteries of anomalous origin or their incidence. In patients with anomalous coronary arteries, by shaping catheter, angiographic visualization of anomalous coronary arteries may be enabled Figure 2. (A, B) The LAD arising from the pulmonary artery is a very rarely encountered anomaly (0.02% ALPACA syndrome). (C, D) The LAD is originated from the LIMA in the very rarely encountered cases (Balaban Syndrome). The Role of Catheter Reshaping at the Angiographic Success http://dx.doi.org/10.5772/intechopen.79210 29 If the physician does not suspect the presence of another coronary artery, this negligence may lead to catastrophic consequences during a potential attack of myocardial infarction in cases with anomalous origin of coronary arteries, inability to visualize the coronary artery [6]. In nearly 97.4% of the patient population, selective visualization of coronary arteries can be achieved with the aid of catheters available in most of the catheterization laboratories. However, in the remaining 2.6% of the cases, these catheters cannot aid in visualization. Otherwise, if an infarction-related coronary artery could not be selectively detected, this con- within a short time and using lesser amount of opaque material [6–8]. dition may lead to fatal consequences for the patient [1–8]. Figure 1. (A) Normal variation is 97.4% of the population. (B) The RCA arising from a superoanterior position or left coronary sinus (0.65%). (C) The LAD and Cx arising from separate ostia (0.48%). (D) The Cx arising from the right coronary ostium (0.20%). (E) The single coronary artery is the LMCA (0.02%). (F) The single coronary artery is the right coronary artery (0.11%). (G) The LAD arising from the right coronary sinus (0.20%). (H) The LAD arising from the pulmonary artery is a very rarely encountered anomaly (0.02% ALPACA syndrome). (I) The LAD is originated from LIMA in the very rarely encountered cases. LAD arising from the pulmonary artery (0.02%, ALPACA syndrome). The LAD originated from the LIMA can also be present rarely in cases (Balaban Syndrome) (Figures 1 and 2 and Table 1). In most of these cases during angiography, standard catheters fail to selectively visu- In publications on anomalous origins and their incidence rates, detection of anomaly has been made in autopsy or using computed tomography. Currently, there are no encountered Figure 1. (A) Normal variation is 97.4% of the population. (B) The RCA arising from a superoanterior position or left coronary sinus (0.65%). (C) The LAD and Cx arising from separate ostia (0.48%). (D) The Cx arising from the right coronary ostium (0.20%). (E) The single coronary artery is the LMCA (0.02%). (F) The single coronary artery is the right coronary artery (0.11%). (G) The LAD arising from the right coronary sinus (0.20%). (H) The LAD arising from the pulmonary artery is a very rarely encountered anomaly (0.02% ALPACA syndrome). (I) The LAD is originated from LIMA in the very rarely encountered cases. alize the anomalous coronary arteries [1–4] (Figures 1 and 2 and Table 1). 28 Angiography Figure 2. (A, B) The LAD arising from the pulmonary artery is a very rarely encountered anomaly (0.02% ALPACA syndrome). (C, D) The LAD is originated from the LIMA in the very rarely encountered cases (Balaban Syndrome). comprehensive and detailed publications related to angiographically defined coronary arteries of anomalous origin or their incidence. In patients with anomalous coronary arteries, by shaping catheter, angiographic visualization of anomalous coronary arteries may be enabled within a short time and using lesser amount of opaque material [6–8]. If the physician does not suspect the presence of another coronary artery, this negligence may lead to catastrophic consequences during a potential attack of myocardial infarction in cases with anomalous origin of coronary arteries, inability to visualize the coronary artery [6]. In nearly 97.4% of the patient population, selective visualization of coronary arteries can be achieved with the aid of catheters available in most of the catheterization laboratories. However, in the remaining 2.6% of the cases, these catheters cannot aid in visualization. Otherwise, if an infarction-related coronary artery could not be selectively detected, this condition may lead to fatal consequences for the patient [1–8]. Coronary arteries of anomalous origin, and their incidence rates. LAD: left anterior descending artery; CX: circumflex artery; RCA: right coronary artery; LMCA: left main coronary artery; CS: coronary sinus. > Figure 3. The catheter reshaping method for carotid angiography via transradial approach: (A) the distal tip of the 0.035 inch guidewire we used during angiography was inserted through the distal tip of the catheter and advanced for 10 cm. With the aid of the guidewire, the required shaping was then performed. (B) Afterwards, the end of the catheter was held 10 cm away from a heat gun and exposed to hot air (450°C) for 4–6 s. Soon after the tip of the heated and reshaped catheter was immersed in water. (C) Using a plastic injector, the catheter with the guidewire inside was irrigated from the opposite end using a sterile isotonic saline solution. Before completion of the cooling process, the guidewire was removed, and The Role of Catheter Reshaping at the Angiographic Success http://dx.doi.org/10.5772/intechopen.79210 31 Figure 4. The catheter reshaping for carotid angiography via femoral way: (A) the distal tip of the 0.035 inch guidewire we used during angiography was inserted through the distal tip of the catheter and advanced for 10 cm. With the aid of the guidewire, the required shaping was then performed. (B) Afterwards, the end of the catheter was held 10 cm away from a heat gun and exposed to hot air (450°C) for 4–6 s. Soon after the tip of the heated and reshaped catheter was immersed in water. (C) Using a plastic injector, the catheter with the guidewire inside was irrigated from the opposite end using a sterile isotonic saline solution. Before completion of the cooling process, the guidewire was removed, and water cooling water cooling was continued. (D) Final viewing of the reshaped catheter. was continued. (D) Final viewing of the reshaped catheter. Table 1. Incidence rates of coronary arteries of anomalous origin. In these circumstances a physician who is both an explorer and an inventor in cases of MI may save the life of his/her patient. In cases with coronary arteries with anomalous origin, which are difficult or even impossible to visualize in catheterization laboratory, after determination of approximate origin and angle of exit of the coronary artery, it is possible to design a catheter that can negotiate anomalous origin and angle of exit. Shaping catheter may be an effective, reliable, and in cases with acute MI, life-saving procedure in the achievement of visualization and decreasing the duration of the procedure, radiation dose, and opaque material used [4, 8]. #### 2. The catheter reshaping method The standard diagnostic catheters that we use in angiography can be reshaped with the standardized methods that we explain as below: The distal tip of the 0.035 inch guidewire that we use during angiography is inserted through the distal tip of the catheter and advanced for 10 cm. Then, with the aid of the guidewire, the required shaping can be performed. Afterwards, the end of the catheter is held 10 cm away from a heat gun and exposed to hot air (450°C) for 4–6 s. Soon after the tip of the heated and reshaped catheter is immersed in water. Using a plastic injector, the catheter with the guidewire inside is irrigated from the opposite end using a sterile isotonic saline solution. Before completion of the cooling process, the guidewire is removed, and water cooling is continued. When the catheter is completely cooled, the catheter is cannulated with the aid of a 0.38-inch guidewire using rotational movements through the carotid ostium or anomalous coronary ostium to visualize the carotid or coronary arteries (Figures 3 and 4) [4, 8–12]. Figure 3. The catheter reshaping method for carotid angiography via transradial approach: (A) the distal tip of the 0.035 inch guidewire we used during angiography was inserted through the distal tip of the catheter and advanced for 10 cm. With the aid of the guidewire, the required shaping was then performed. (B) Afterwards, the end of the catheter was held 10 cm away from a heat gun and exposed to hot air (450°C) for 4–6 s. Soon after the tip of the heated and reshaped catheter was immersed in water. (C) Using a plastic injector, the catheter with the guidewire inside was irrigated from the opposite end using a sterile isotonic saline solution. Before completion of the cooling process, the guidewire was removed, and water cooling was continued. (D) Final viewing of the reshaped catheter. In these circumstances a physician who is both an explorer and an inventor in cases of MI may Coronary arteries of anomalous origin, and their incidence rates. LAD: left anterior descending artery; CX: circumflex Anomalous origin Incidence (%) High take off RCA or from left CS (Figure 1B) 0.65 LAD and CX arising from separate ostia (Figure 1C) 0.48 CX arising from right CS (Figure 1D) 0.20 Single coronary artery RCA (Figure 1E) 0.11 Single coronary artery LMCA(Figure 1F) 0.02 LAD arising from the right coronary sinus (Figure 1G) 0.20 LAD arising from pulmonary artery(Figure 1H) 0.02 LAD arising from LIMA(Figure 1I) 0.001 In cases with coronary arteries with anomalous origin, which are difficult or even impossible to visualize in catheterization laboratory, after determination of approximate origin and angle of exit of the coronary artery, it is possible to design a catheter that can negotiate anomalous origin and angle of exit. Shaping catheter may be an effective, reliable, and in cases with acute MI, life-saving procedure in the achievement of visualization and decreasing the duration of The standard diagnostic catheters that we use in angiography can be reshaped with the stan- The distal tip of the 0.035 inch guidewire that we use during angiography is inserted through the distal tip of the catheter and advanced for 10 cm. Then, with the aid of the guidewire, the required shaping can be performed. Afterwards, the end of the catheter is held 10 cm away from a heat gun and exposed to hot air (450°C) for 4–6 s. Soon after the tip of the heated and reshaped catheter is immersed in water. Using a plastic injector, the catheter with the guidewire inside is irrigated from the opposite end using a sterile isotonic saline solution. Before completion of the cooling process, the guidewire is removed, and water cooling is continued. When the catheter is completely cooled, the catheter is cannulated with the aid of a 0.38-inch guidewire using rotational movements through the carotid ostium or anomalous coronary ostium to visualize the carotid or coronary arteries (Figures 3 and 4) [4, 8–12]. the procedure, radiation dose, and opaque material used [4, 8]. artery; RCA: right coronary artery; LMCA: left main coronary artery; CS: coronary sinus. Table 1. Incidence rates of coronary arteries of anomalous origin. save the life of his/her patient. 30 Angiography 2. The catheter reshaping method dardized methods that we explain as below: Figure 4. The catheter reshaping for carotid angiography via femoral way: (A) the distal tip of the 0.035 inch guidewire we used during angiography was inserted through the distal tip of the catheter and advanced for 10 cm. With the aid of the guidewire, the required shaping was then performed. (B) Afterwards, the end of the catheter was held 10 cm away from a heat gun and exposed to hot air (450°C) for 4–6 s. Soon after the tip of the heated and reshaped catheter was immersed in water. (C) Using a plastic injector, the catheter with the guidewire inside was irrigated from the opposite end using a sterile isotonic saline solution. Before completion of the cooling process, the guidewire was removed, and water cooling was continued. (D) Final viewing of the reshaped catheter. #### 3. Types of aortic arches and their incidence rates In cases where selective angiography is not successful, it is necessary to know the anomaly in order to solve the problem. The identification of aortic type may be helpful to determine the most appropriate shape of catheter. At first, the physician should know and form an estimate aortic arch and coronary origin anatomy. 4. The role of catheter reshaping in the success of carotid It is difficult to perform transradial carotid angiography using conventional multipurpose catheters, which suffer from lower success rates and longer procedural times. Therefore, physicians do not prefer to perform transradial carotid angiography. We thought that among conventional catheters, the Simpson catheter and the 3.5 JL catheter were the most suitable for transradial visualization. To this end, the reshaped catheters can be more effective than available conventional catheters in carotid angiography. There are a few new publications in this regard, reaching 1500 participants. However, one study has examined the use of the modified Simmons catheter in the right transradial route carotid angiography. The Simpson catheter has an angled distal tip, which opens far from the long axis of the catheter. This configuration decreases the right transradial procedural success rate of selective visualization of the left carotid artery in cases where the left carotid artery stems from a point near the brachiocephalic trunk. Therefore, performing selective angiography with a Simpson catheter requires special experience and manual dexterity. The retroflexed tip of the 3.5 JL catheter provides improved cannulation of the left common carotid artery and is one of the most frequently The Role of Catheter Reshaping at the Angiographic Success http://dx.doi.org/10.5772/intechopen.79210 33 Even though transradial carotid visualization can be achieved using standard catheters, carotid arteries cannot be selectively cannulated, and the images obtained are far from satisfactory Figure 6. The reshaped catheter types using at the different angiographies: (A) right transradial carotid angiography with hooklike reshaped catheter; (B) the right transradial carotid angiography in the arteria lusoria case (type IV aorta); (C) the "S"-shaped catheter using with femoral access; and (D) the left transradial carotid angiography with the angiography by radial pathway available catheters in every laboratory [9–11, 16]. "hooklike" reshaped catheter. In some publications, various classifications for various types of aortic arches have been proposed. Most of such classification schemes fail to improve the transradial angiography procedure. We have recently proposed a classification system. Type I aortic arches, also known as normal aortic arches, are the most frequently encountered (86%). In this arch type, the right carotid artery arises from the right brachial trunk, and the left common carotid artery originates from the aortic arch 1–2 cm to the left of the brachiocephalic trunk. In 9% of cases in which a Type II aortic arch is observed, the right and left common carotid arteries originate from the brachial trunk. In Type III aortic arches (2%), the right and left common carotid arteries directly originate from the aortic arch as a one unit or separately. In Type IV aortic arches (3%), which also include arteria lusoria, the right subclavian artery stems from the aorta near the descending aorta after the carotid arteries [9, 10, 13–15] (Figure 5). Figure 5. RSCA, right subclavian artery; LCCA, left common carotid artery; RCCA, left common carotid artery; LSCA, left subclavian artery. Types I, II, III, and IV are encountered in 86, 9, 2, and 3% of cases, respectively. ## 4. The role of catheter reshaping in the success of carotid angiography by radial pathway 3. Types of aortic arches and their incidence rates the descending aorta after the carotid arteries [9, 10, 13–15] (Figure 5). aortic arch and coronary origin anatomy. 32 Angiography In cases where selective angiography is not successful, it is necessary to know the anomaly in order to solve the problem. The identification of aortic type may be helpful to determine the most appropriate shape of catheter. At first, the physician should know and form an estimate In some publications, various classifications for various types of aortic arches have been proposed. Most of such classification schemes fail to improve the transradial angiography procedure. We have recently proposed a classification system. Type I aortic arches, also known as normal aortic arches, are the most frequently encountered (86%). In this arch type, the right carotid artery arises from the right brachial trunk, and the left common carotid artery originates from the aortic arch 1–2 cm to the left of the brachiocephalic trunk. In 9% of cases in which a Type II aortic arch is observed, the right and left common carotid arteries originate from the brachial trunk. In Type III aortic arches (2%), the right and left common carotid arteries directly originate from the aortic arch as a one unit or separately. In Type IV aortic arches (3%), which also include arteria lusoria, the right subclavian artery stems from the aorta near Figure 5. RSCA, right subclavian artery; LCCA, left common carotid artery; RCCA, left common carotid artery; LSCA, left subclavian artery. Types I, II, III, and IV are encountered in 86, 9, 2, and 3% of cases, respectively. It is difficult to perform transradial carotid angiography using conventional multipurpose catheters, which suffer from lower success rates and longer procedural times. Therefore, physicians do not prefer to perform transradial carotid angiography. We thought that among conventional catheters, the Simpson catheter and the 3.5 JL catheter were the most suitable for transradial visualization. To this end, the reshaped catheters can be more effective than available conventional catheters in carotid angiography. There are a few new publications in this regard, reaching 1500 participants. However, one study has examined the use of the modified Simmons catheter in the right transradial route carotid angiography. The Simpson catheter has an angled distal tip, which opens far from the long axis of the catheter. This configuration decreases the right transradial procedural success rate of selective visualization of the left carotid artery in cases where the left carotid artery stems from a point near the brachiocephalic trunk. Therefore, performing selective angiography with a Simpson catheter requires special experience and manual dexterity. The retroflexed tip of the 3.5 JL catheter provides improved cannulation of the left common carotid artery and is one of the most frequently available catheters in every laboratory [9–11, 16]. Even though transradial carotid visualization can be achieved using standard catheters, carotid arteries cannot be selectively cannulated, and the images obtained are far from satisfactory Figure 6. The reshaped catheter types using at the different angiographies: (A) right transradial carotid angiography with hooklike reshaped catheter; (B) the right transradial carotid angiography in the arteria lusoria case (type IV aorta); (C) the "S"-shaped catheter using with femoral access; and (D) the left transradial carotid angiography with the "hooklike" reshaped catheter. compared with the selective images. The size and track of the plaque, in particular, cannot be clearly evaluated. Only the degree of stenosis may be estimated. However, determining the morphology and fragility of the plaque, and the presence of vascularity or plaque dissection and erosion, is required to predict the probability of experiencing a cerebrovascular event in the future and for planning the appropriate treatment modality and decreasing procedural complications. The new visualization method that is catheter reshaping is more useful to detect the presence of plaque fractures, erosion, and dissection due to good quality images. These satisfactory images aid us in selection of appropriate treatment methods and prediction of complications [10] (Figure 6). with coronary artery stenosis of 50% or more, the likelihood of a significant carotid artery stenosis exceeds 60%. This ratio can be interpreted; if carotid angiography is performed in the patients with a significant coronary stenosis, carotid artery disease can be shown at least among 60% of the patients. Therefore, in patients with planned coronary angiography, it would be useful to look at carotids in in the presence of severe coronary atherosclerosis at the same session without preliminary investigation of carotid stenosis. Also the opposite can also be considered. In other words, that is very useful to perform coronary angiography at the same procedure in the patient with carotid stenosis. At least 60% of these patients with carotid The Role of Catheter Reshaping at the Angiographic Success http://dx.doi.org/10.5772/intechopen.79210 35 In the patients with carotid stenosis by Doppler ultrasonography, invasive angiography may be more advantageous than MR and CT angiography, because if a person has carotid stenosis, there is a high probability of coronary artery disease. If we focus on carotid stenosis and ignore potential coronary artery disease, the patient may become vulnerable to myocardial infarction. Furthermore, the image quality of noninvasive techniques is not as detailed as in the invasive angiography. The conventional angiography can show the character of carotid plaque, the cracks in the plaque, whether the plaque is vulnerable or not. Knowing plaque features give valuable information to physicians for treatment approach [9, 10, 18–27]. Cardiologists who perform carotid angiography followed by coronary angiography perform carotid angiography often with the right Judkins diagnostic catheter. There are no clinical studies conducted to determine which of the HN4-5, CK1, and MAN catheters are more effective and reliable in carotid angiography [21, 22]. The cardiologists performing coronary angiography can make a very successful and reliable carotid angiography by giving the "S" shape to "right diagnostic catheter" at the transfemoral way or by giving "hooklike" shape to right diagnostic catheter at the transradial way, without having an additional catheter in the Catheter shaping is done as an amateur in many coronary angiography laboratories. There are clinical trials that standardize catheter reshaping. The reliability and success of catheter shaping by hand have been proven with these clinical studies reaching over 1500 participants. All experiences and discoveries are creating new percutaneous intervention areas. Twenty years ago, transcatheter aortic valve replacement was a dream. The retrograde chronic total occlusion intervention was almost impossible with techniques and instruments of 15 years ago. All these developments have been made possible by the transformation of new methods The initial experience of angiography was performed with brachial pathway, but the femoral route was considered safer and simpler at the beginning. Therefore, the development of angiography has been initiated using the femoral route. However, in the last 15 years, radial route has been used again and even has been shown to have advantages, so it is used now all over the world. Today, coronary and peripheral invasive angiography can be performed as fast and reliable as MR and CT angiography. Invasive angiography allows for real-time imaging and immediate intervention allowing physicians to better identify condition of patient Teaching and implementing this method in daily practice may be useful [9, 10, 12, 17]. artery disease have also significant coronary artery disease [9, 10, 20–22]. catheterization laboratory (Figures 3, 4, and 6) [9, 10]. into scientific publications [28, 29]. The visualization with new reshaped catheter requires a shorter procedural time and less opaque material usage. Since selective engagement of catheters in carotid arteries is achieved with this method, higher-quality images can be obtained. The new catheter greatly contributes to the determination of appropriate treatment [10, 17]. ### 5. Following coronary angiography performed by femoral approach, the carotid angiography, and the contribution of the catheter reshape to success The relationship between peripheral artery disease and coronary artery disease is a known fact. This relationship is often predictive of stroke, renal insufficiency, and extremity artery disease in patients with coronary artery disease. At the moment of coronary angiography procedure, the visualisable of carotids seems to be advantageous for the patient. The carotids can be displayed noninvasively by MR angiography and CT angiography. But it can only be displayed at low resolution according to invasive angiography. This noninvasive imaging may be the first choice, for isolated cases. However, in patients already undergoing coronary angiography, carotid imaging application following this procedure is a more appropriate choice. The quality of the invasive carotid image is still better than the MR and CT angiography. Invasive imaging is still the gold standard in neck malformations, carotid tumors, neck injuries, and carotid artery stenosis. In still today, cardiovascular surgeons, neurologists, and neurosurgeons, if they see lesions in the carotid artery at the MR and CT angiography, require invasive carotid angiography when they propose an additional intervention for these lesions [9, 10, 12–18]. It is known that physicians who perform coronary angiography use "the right diagnostic catheter" for their desired carotid imaging. Simon catheters and HN4–5, CK1, and MAN catheters can be used, especially if carotid interventional procedures are going to be performed. However, these catheters are not commonly used by cardiologists. They can often be supplied by special order, and they are not available in most coronary angiography laboratories. For these reasons, the reshaping of catheter in the coronary angiography laboratory can be a snapshot solution to the problem of imaging anomalous vascular origins and for carotid and other peripheral arterial imaging [4, 9, 10, 12, 19–21]. There is a strong relationship between carotid artery disease and coronary artery disease. According to recent studies, in patients with coronary artery stenosis of 50% or more, the likelihood of a significant carotid artery stenosis exceeds 60%. This ratio can be interpreted; if carotid angiography is performed in the patients with a significant coronary stenosis, carotid artery disease can be shown at least among 60% of the patients. Therefore, in patients with planned coronary angiography, it would be useful to look at carotids in in the presence of severe coronary atherosclerosis at the same session without preliminary investigation of carotid stenosis. Also the opposite can also be considered. In other words, that is very useful to perform coronary angiography at the same procedure in the patient with carotid stenosis. At least 60% of these patients with carotid artery disease have also significant coronary artery disease [9, 10, 20–22]. compared with the selective images. The size and track of the plaque, in particular, cannot be clearly evaluated. Only the degree of stenosis may be estimated. However, determining the morphology and fragility of the plaque, and the presence of vascularity or plaque dissection and erosion, is required to predict the probability of experiencing a cerebrovascular event in the future and for planning the appropriate treatment modality and decreasing procedural complications. The new visualization method that is catheter reshaping is more useful to detect the presence of plaque fractures, erosion, and dissection due to good quality images. These satisfactory images aid us in selection of appropriate treatment methods and prediction The visualization with new reshaped catheter requires a shorter procedural time and less opaque material usage. Since selective engagement of catheters in carotid arteries is achieved with this method, higher-quality images can be obtained. The new catheter greatly contrib- 5. Following coronary angiography performed by femoral approach, the carotid angiography, and the contribution of the catheter reshape The relationship between peripheral artery disease and coronary artery disease is a known fact. This relationship is often predictive of stroke, renal insufficiency, and extremity artery disease in patients with coronary artery disease. At the moment of coronary angiography procedure, the visualisable of carotids seems to be advantageous for the patient. The carotids can be displayed noninvasively by MR angiography and CT angiography. But it can only be displayed at low resolution according to invasive angiography. This noninvasive imaging may be the first choice, for isolated cases. However, in patients already undergoing coronary angiography, carotid imaging application following this procedure is a more appropriate choice. The quality of the invasive carotid image is still better than the MR and CT angiography. Invasive imaging is still the gold standard in neck malformations, carotid tumors, neck injuries, and carotid artery stenosis. In still today, cardiovascular surgeons, neurologists, and neurosurgeons, if they see lesions in the carotid artery at the MR and CT angiography, require invasive carotid angiography when they propose an additional intervention for these It is known that physicians who perform coronary angiography use "the right diagnostic catheter" for their desired carotid imaging. Simon catheters and HN4–5, CK1, and MAN catheters can be used, especially if carotid interventional procedures are going to be performed. However, these catheters are not commonly used by cardiologists. They can often be supplied by special order, and they are not available in most coronary angiography laboratories. For these reasons, the reshaping of catheter in the coronary angiography laboratory can be a snapshot solution to the problem of imaging anomalous vascular origins and for carotid and other peripheral arterial imaging [4, 9, 10, 12, 19–21]. There is a strong relationship between carotid artery disease and coronary artery disease. According to recent studies, in patients of complications [10] (Figure 6). to success 34 Angiography lesions [9, 10, 12–18]. utes to the determination of appropriate treatment [10, 17]. In the patients with carotid stenosis by Doppler ultrasonography, invasive angiography may be more advantageous than MR and CT angiography, because if a person has carotid stenosis, there is a high probability of coronary artery disease. If we focus on carotid stenosis and ignore potential coronary artery disease, the patient may become vulnerable to myocardial infarction. Furthermore, the image quality of noninvasive techniques is not as detailed as in the invasive angiography. The conventional angiography can show the character of carotid plaque, the cracks in the plaque, whether the plaque is vulnerable or not. Knowing plaque features give valuable information to physicians for treatment approach [9, 10, 18–27]. Cardiologists who perform carotid angiography followed by coronary angiography perform carotid angiography often with the right Judkins diagnostic catheter. There are no clinical studies conducted to determine which of the HN4-5, CK1, and MAN catheters are more effective and reliable in carotid angiography [21, 22]. The cardiologists performing coronary angiography can make a very successful and reliable carotid angiography by giving the "S" shape to "right diagnostic catheter" at the transfemoral way or by giving "hooklike" shape to right diagnostic catheter at the transradial way, without having an additional catheter in the catheterization laboratory (Figures 3, 4, and 6) [9, 10]. Catheter shaping is done as an amateur in many coronary angiography laboratories. There are clinical trials that standardize catheter reshaping. The reliability and success of catheter shaping by hand have been proven with these clinical studies reaching over 1500 participants. Teaching and implementing this method in daily practice may be useful [9, 10, 12, 17]. All experiences and discoveries are creating new percutaneous intervention areas. Twenty years ago, transcatheter aortic valve replacement was a dream. The retrograde chronic total occlusion intervention was almost impossible with techniques and instruments of 15 years ago. All these developments have been made possible by the transformation of new methods into scientific publications [28, 29]. The initial experience of angiography was performed with brachial pathway, but the femoral route was considered safer and simpler at the beginning. Therefore, the development of angiography has been initiated using the femoral route. However, in the last 15 years, radial route has been used again and even has been shown to have advantages, so it is used now all over the world. Today, coronary and peripheral invasive angiography can be performed as fast and reliable as MR and CT angiography. Invasive angiography allows for real-time imaging and immediate intervention allowing physicians to better identify condition of patient and treatment of the disease. All interventional physicians should strive to publish the useful and practical methods that they have recently discovered. These publications are very important because they can shed light on the development of new techniques and devices [30–32] (Figures 3, 4, and 6). [5] Villa AD, Sammut E, Nair A, Rajani R, Bonamini R, Chiribiri A. Coronary artery anomalies overview: The normal and the abnormal. World Journal of Radiology. 2016;8(6):537-555 The Role of Catheter Reshaping at the Angiographic Success http://dx.doi.org/10.5772/intechopen.79210 37 [6] Frescura C, Basso C, Thiene G, Corrado D, Pennelli T, et al. Anomalous origin of coronary arteries and risk of sudden death: A study based on an autopsy population of con- [7] Gräni C, Benz DC, Schmied C, Vontobel J, Possner M, et al. Prevalence and characteristics of coronary artery anomalies detected by coronary computed tomography angiography in 5634 consecutive patients in a single Centre in Switzerland. Swiss Medical [8] Kim JY, Yoon GS, Doh HJ, Choe HM, Kwon SU, Namgung J. Two cases of successful primary percutaneous coronary intervention in patients with an anomalous right coronary artery arising from the left coronary cusp. Korean Circulation Journal. 2008;38(3):179-183 [9] Balaban Y. New developed femoral-S carotid catheter' is very effective and safe in carotid [10] Balaban Y. Effectiveness of a handmade "new carotid catheter" in transradial carotid angiography: A comparison with conventional multipurpose catheters. Journal of Interventional Cardiology. 2018;31(1):94-105. DOI: 10.1111/joic.12454. Epub: October 11, 2017 [11] Cha KS, Kim MH, Kim YD, Kim JS. Combined right transradial coronary angiography and selective carotid angiography: Safety and feasibility in unselected patients. [12] Erden I, Golcuk E, Bozyel S, Erden EC, Balaban Y, Yalın K, et al. Effectiveness of handmade "Jacky-like catheter" As a single multipurpose catheter in transradial coronary angiography: A randomized comparison with conventional two-catheter strategy. [13] Huapaya JA, Trujillo KC, Trelles M, Carbaja RD, Espadin RF. Anatomic variations of the branches of the aortic arch in a Peruvian population. Medwave. 2015;15(6):e6194. DOI: [14] Ergun E, Simsek B, Kosar P, Yilmaz BK, Turgut AT. Anatomical variations in branching patterns of arcus aorta: 64-slice CTA appearance. Surgical and Radiologic Anatomy. [15] Cummings MS, Kuo BT, Ziada KM. A rare anomaly of the aortic arch: Aberrant right subclavian artery associated with common carotid trunk. The Journal of Invasive [16] Park JH, Kim DY, Kim JW, Park YS, Seung WB. Efficacy of transradial cerebral angiography in the elderly. Journal of Korean Neurosurgical Association. 2013;53(4):213-217. [17] Fang Y, Yang C, Wang X, Zhou L, Wang H, Zeng C. Feasibility and application of single 5F multipurpose catheter in coronary and peripheral angiography via a transradial 2014;36(10):1105-1106. DOI: 10.1007/s00276-014-1288-4. Epub: March 27, 2014 angiography: A retrospective study OMICS. Journal of Radiology. 2017;6:6 Catheterization and Cardiovascular Interventions. 2001;53(3):380-385 Journal of Interventional Cardiology. 2017;30(1):24-32 DOI: 10.3340/jkns.2013.53.4.213. Epub: April 30, 2013 approach. International Journal of Cardiology. 2011;151:182-118 10.5867/medwave.2015.06.6194 Cardiology. 2011;23(10):E241-E243 genital heart disease. Human Pathology. 1998;29(7):689-695 Weekly. 2016;146:w14294 #### 6. Conclusion The newly obtained catheter by reshaping of the plastic catheters used in angiography by cooling after reshaping with heat can be more effective at the displaying vessels with anomalous origin than existing catheters. First, the aortography must be performed to determine the location of the coronary artery ostium in the aorta at the anomalous coronary events. Thus, the appropriate catheter shape can be estimated. The selective angiography can be quite easy if the current catheter can be successfully shaped. This method can also be used to image the carotid and vertebral arteries by radial and femoral routes. The carotid and vertebral artery angiography can be performed more easily with the use of reshaped catheters in some cases including anomalous coronary cases. #### Author details Yakup Balaban Address all correspondence to: [email protected] Department of Cardiology, VM Medical Park Kocaeli Hospital, Turkey #### References [5] Villa AD, Sammut E, Nair A, Rajani R, Bonamini R, Chiribiri A. Coronary artery anomalies overview: The normal and the abnormal. World Journal of Radiology. 2016;8(6):537-555 and treatment of the disease. All interventional physicians should strive to publish the useful and practical methods that they have recently discovered. These publications are very important because they can shed light on the development of new techniques and devices [30–32] The newly obtained catheter by reshaping of the plastic catheters used in angiography by cooling after reshaping with heat can be more effective at the displaying vessels with anomalous origin than existing catheters. First, the aortography must be performed to determine the location of the coronary artery ostium in the aorta at the anomalous coronary events. Thus, the appropriate catheter shape can be estimated. The selective angiography can be quite easy if the current catheter can be successfully shaped. This method can also be used to image the carotid and vertebral arteries by radial and femoral routes. The carotid and vertebral artery angiography can be performed more easily with the use of reshaped catheters in some cases [1] Graidis C, Dimitriadis D, Karasavvidis V, Dimitriadis G, Argyropoulou E, et al. Prevalence and characteristics of coronary artery anomalies in an adult population undergoing multidetector-row computed tomography for the evaluation of coronary artery [2] Mavi A, Ayalp R, Serçelik A, Pesţemalci T, Batyraliev T, Gümüşburun E. Frequency of the anomalous origin of the left main coronary artery with angiography in a Turkish [3] Ayalp R, Mavi A, Serçelik A, Batyraliev T, Gümüsburun E. Frequency of the anomalous origin of the right coronary artery with angiography in a Turkish population. [4] Balaban YHA, Gümrükçüoğlu D, Güngör SC. Left anterior descending artery of anomalous origin; native LAD arises from left internal mammarian artery. A case report and article review. Cardiovascular Revascularization Medicine. 2018;19(2):209-214 (Figures 3, 4, and 6). 36 Angiography 6. Conclusion Author details Yakup Balaban References including anomalous coronary cases. Address all correspondence to: [email protected] disease. BMC Cardiovascular Disorders. 2015;15:112 population. Acta Medica Okayama. 2004;58(1):17-22 International Journal of Cardiology. 2002;82(3):253-257 Department of Cardiology, VM Medical Park Kocaeli Hospital, Turkey [18] Ledwoch J, Staubach S, Segerer M, Strohm H, Mudra H. Incidence and risk factors of embolized particles in carotid artery stenting and association with clinical outcome. International Journal of Cardiology. 2017;227:550-555. DOI: 10.1016/j.ijcard.2016.10.103. Epub: November 2, 2016 [30] Morgan JH, Johnson JH, Brown RB, Harvey RL, Rizzoni WE, Tyson CS. Initial experience with routine selective carotid arteriography by vascular surgeons. The American The Role of Catheter Reshaping at the Angiographic Success http://dx.doi.org/10.5772/intechopen.79210 39 [31] Louvard Y, Lefèvre T, Allain A, Morice M. Coronary angiography through the radial or the femoral approach: The CARAFE study. Catheterization and Cardiovascular [32] Oren O, Oren M, Turgeman Y. Transradial versus Transfemoral approach in peripheral arterial interventions. International Journal of Angiology. 2016;25(3):148-152. DOI: Surgeon. 2006;72(8):684-686; Discussion 687 10.1055/s-0035-1563607. Epub: September 7, 2015 Interventions. 2001;52(2):181-187 [30] Morgan JH, Johnson JH, Brown RB, Harvey RL, Rizzoni WE, Tyson CS. Initial experience with routine selective carotid arteriography by vascular surgeons. The American Surgeon. 2006;72(8):684-686; Discussion 687 [18] Ledwoch J, Staubach S, Segerer M, Strohm H, Mudra H. Incidence and risk factors of embolized particles in carotid artery stenting and association with clinical outcome. International Journal of Cardiology. 2017;227:550-555. DOI: 10.1016/j.ijcard.2016.10.103. [19] Rafailidis V, Chryssogonidis I, Tegos T, Kouskouras K, Charitanti-Kouridou A. Imaging of the ulcerated carotid atherosclerotic plaque: A review of the literature. Insights into [20] Rigatelli G. Screening angiography of supraaortic vessels performed by invasive cardiologists at the time of cardiac catheterization: Indications and results. The International [21] Seo WK, Yong HS, Koh SB, Suh SI, Kim JH, Yu SW, et al. Correlation of coronary artery atherosclerosis with atherosclerosis of the intracranial cerebral artery and the extracranial carotid artery. European Neurology. 2008;59(6):292-298. DOI: 10.1159/000121418. [22] Bae HJ, Yoon BW, Kang DW, Koo JS, Lee SH, Kim KB, et al. Correlation of coronary and cerebral atherosclerosis: Difference between extracranial and intracranial arteries. [23] Rigatelli G, Gemelli M, Zamboni A, Docali G, Rossi P, Grazio M. Significance of selective carotid angiography during complete cardiac catheterization in patients candidates to combined aortic valve and carotid surgery. Minerva Cardioangiologica. [24] Rigatelli G. Diagnosis of carotid artery occlusive disease in patients scheduled for cardiac or vascular surgery: Is this a place for invasive selective carotid angiography? [25] Korotkikh NG, Ol'shanskiĭ MS, Stepanov IV.Multidisciplinary approach to diagnostics of extensive vascular head and neck malformations. Stomatologiia (Mosk). 2012;91(1):40-45 [26] Korotkikh NG, Ol'shanskiĭ MS, Stepanov IV, Mashkova TA, Nerovnyĭ AI, Panchenko IG. The multidisciplinary approach to diagnostics and treatment of hypervascular [27] Arslan F1, Yılmaz S, Özer F, Andıç C, Canpolat T, Yavuz H. Surgical treatment of carotid body tumors. Kulak Burun Boğaz Ihtisas Dergisi. 2013;23(6):336-340. DOI: 10.5606/ [28] Kaneda H, Takahashi S, Saito S. Successful coronary intervention for chronic total occlusion in an anomalous right coronary artery using the retrograde approach via a collat- [29] Cribier A, Eltchaninoff H, Tron C, Bauer F, Agatiello C. Early experience with percutaneous transcatheter implantation of heart valve prosthesis for the treatment of endstage inoperable patients with calcific aortic stenosis. Journal of the American College of Cerebrovascular Diseases. 2006;21(1-2):112-119. Epub: December 9, 2005 Stroke. 2004;35(5):e89-e90; Author reply: e89-90. Epub: March 25, 2004 masses in ENTstructures. Vestnik Otorinolaringologii. 2013;5:44-47 eral vessel. The Journal of Invasive Cardiology. 2007;19(1):E1-E4 Imaging. 2017;8(2):213-225. DOI: 10.1007/s13244-017-0543-8 Journal of Cardiovascular Imaging. 2005;21(2-3):179-183 Epub: November 2, 2016 38 Angiography Epub: April 11, 2008 2003;51(3):305-309 kbbihtisas.2013.28159 Cardiology. 2004;43(4):698-703 Chapter 4 Provisional chapter Stent's Manufacturing Field: Past, Present, and Future Stent's Manufacturing Field: Past, Present, and Future From the introduction of stents, nobody was able to predict the advances that will occur in stent technology over the upcoming decades. Since their appearances, it became evident that this device had significant limitations, such as vessel occlusion and/or restenosis. Despite that, this medical device is the best clinical solution for cardiovascular vessel occlusions. Stents require a deep analysis, in terms of thrombogenicity, manufacturing process, geometrical aspects, and mechanical performance, among many other characteristics. The surface quality obtained in their manufacture process is crucial to blood compatibility, prevents the activation process of thrombosis, and improves the healing efficiency. The forecast stent market makes necessary continuous studies on this field, which help to solve the medical and engineering problems of this device, which are in constant development. Stents have been the center of many research lines over the last decades. The present chapter aims to summarize the state of the art of this medical device in the last years in the fields of design, manufacturing, and materials. Keywords: stent, BMS, DES, BRS, ETS, permanent, fully absorbable, design, FEA, In medicine, atherosclerotic vascular diseases are common and life-threatening diseases that narrows the vessels and reduces the blood flow in the arteries. Nowadays, angioplasty, also known as percutaneous coronary intervention (PCI), or peripheral artery balloon dilation and stenting are frequently used interventional therapeutic methods, in which a special tubing > © 2016 The Author(s). Licensee InTech. 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. © 2018 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. DOI: 10.5772/intechopen.81668 Prospects Abstract 1. Introduction Prospects Antonio J. Guerra and Joaquim Ciurana Antonio J. Guerra and Joaquim Ciurana http://dx.doi.org/10.5772/intechopen.81668 manufacturing, processes, material (stent) is usually placed to open the narrowed arterial vessel. Additional information is available at the end of the chapter Additional information is available at the end of the chapter #### Stent's Manufacturing Field: Past, Present, and Future Prospects Stent's Manufacturing Field: Past, Present, and Future Prospects DOI: 10.5772/intechopen.81668 Antonio J. Guerra and Joaquim Ciurana Antonio J. Guerra and Joaquim Ciurana Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.81668 #### Abstract From the introduction of stents, nobody was able to predict the advances that will occur in stent technology over the upcoming decades. Since their appearances, it became evident that this device had significant limitations, such as vessel occlusion and/or restenosis. Despite that, this medical device is the best clinical solution for cardiovascular vessel occlusions. Stents require a deep analysis, in terms of thrombogenicity, manufacturing process, geometrical aspects, and mechanical performance, among many other characteristics. The surface quality obtained in their manufacture process is crucial to blood compatibility, prevents the activation process of thrombosis, and improves the healing efficiency. The forecast stent market makes necessary continuous studies on this field, which help to solve the medical and engineering problems of this device, which are in constant development. Stents have been the center of many research lines over the last decades. The present chapter aims to summarize the state of the art of this medical device in the last years in the fields of design, manufacturing, and materials. Keywords: stent, BMS, DES, BRS, ETS, permanent, fully absorbable, design, FEA, manufacturing, processes, material #### 1. Introduction In medicine, atherosclerotic vascular diseases are common and life-threatening diseases that narrows the vessels and reduces the blood flow in the arteries. Nowadays, angioplasty, also known as percutaneous coronary intervention (PCI), or peripheral artery balloon dilation and stenting are frequently used interventional therapeutic methods, in which a special tubing (stent) is usually placed to open the narrowed arterial vessel. © 2016 The Author(s). Licensee InTech. 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. © 2018 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. From the introduction of PCI, nobody was able to predict the advances that will occur in stent technology over the upcoming decades [1]. Since PCI appearances, it became evident that this approach has significant limitations, such as vessel occlusion and/or restenosis [2]. To overcome these problems, bare metal stents (BMS) were introduced, and despite reducing the vessel occlusion, however, high rates of restenosis constituted their major limitation [3]. To surmount this hurdle, the metallic stent coated with antiproliferative drug was conceived, the drug-eluting stents (DES). With the introduction of DES, the antiproliferative drug over the struts prolonged vessel wall healing, reduced neointima hyperplasia, and consequently decreased the target lesion revascularization (TLR). The most important limitation of the first generation of DES was related to the lack of biocompatibility of the drug-eluting polymer leading to a persistent inflammatory response after the drug-eluting period. Although permanent stents (BMS and DES) are effective, in most cases, the role of stent is temporary and is limited to the intervention, and shortly thereafter, until healing and re-endothelialization are obtained [4]. Bioresorbable stents (BRS) were introduced to overcome these limitations with important advantages: complete bioresorption, mechanical flexibility, etc. The BRS concept introduced the use of polymers in stenting procedures for the first time. With the inclusion of polymeric materials in the field of stents, a new and promising idea makes its way, electrospun tubular scaffolds (ETS) for stenting process. Unlike current stents, ETS theoretically could present some advances such as (I) better longitudinal flexibility to help the placement of the stent and (II) their surface mimics body tissue to help to obtain a best proliferation rates [5] and thus a rapid endothelialization. Nevertheless, ETS could have some disadvantages such as their radial flexibility. The radial expansion of ETS will occur by elongation of its fibers, in contrast to current stents in which it occurs by elongation of its radial cells. This fact could make correct vessel support difficult, which would restrict the use of ETS for only peripheral applications. With the author's best knowledge, this new idea has been overlooked, but it presents a promising approach to solve cardiovascular problems. Once we have decided the mechanical properties, we give way to the manufacturing process step. The manufacturing process mainly depends on the materials and stent type chosen: Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 43 Manufacturing process = f (material, stent type) (2) Finally, the additional properties step. Although it constitutes the final layer of the stent pyramid, additional properties cover a range of modifications to stent designs. This last step Additional properties = f (stent type, application) (3) The next sections present the main issues to consider in the abovementioned decision pyra- Stent's mechanical properties are interrelated and sometimes contradictory, requiring careful Following the classification done by Stoeckel et al. in their manuscript "A survey of stent indirectly relates to the stent type and its medical application: Figure 1. Pyramid of stent manufacturing process design. mid steps with special attention to the manufacturing process step. compromise between geometrical and material aspects. designs," stents can be classified into five categories [7]. 3. Stent mechanical properties 3.1. Geometrical aspects Stents can be used for a wide range of indication: de novo lesions, small vessel disease (SVD), bifurcation lesions, and tortuous and narrows lesions. Stents can improve the clinical outcomes for all of these indications as well as quality of life for patients suffering from this debilitating disease. In 2013, sales of DES and BMS in the 10 major markets were \$ 4.89 billion. Global data [6] estimates that by 2020, sales of stents will grow to \$ 5.65 billion. #### 2. Stent manufacturing process design Regardless of the stent choice, BMS, DES, BRS, or ETS, the challenges associated to this medical device remain similar. Figure 1 shows the pyramid of stent manufacturing process design. It represents the main issues to consider at the time to design and manufacture this medical device. The mechanical properties of the stent govern the decision process. This important property will be in charge of providing the correct longitudinal and radial behavior. The mechanical properties of the stents mainly depend on the material, geometry, and medical application of the stent: $$\text{Mechanical properties} = \text{f} \left( \text{material}, \text{geometry}, \text{application} \right) \tag{1}$$ Figure 1. Pyramid of stent manufacturing process design. From the introduction of PCI, nobody was able to predict the advances that will occur in stent technology over the upcoming decades [1]. Since PCI appearances, it became evident that this approach has significant limitations, such as vessel occlusion and/or restenosis [2]. To overcome these problems, bare metal stents (BMS) were introduced, and despite reducing the vessel occlusion, however, high rates of restenosis constituted their major limitation [3]. To surmount this hurdle, the metallic stent coated with antiproliferative drug was conceived, the drug-eluting stents (DES). With the introduction of DES, the antiproliferative drug over the struts prolonged vessel wall healing, reduced neointima hyperplasia, and consequently decreased the target lesion revascularization (TLR). The most important limitation of the first generation of DES was related to the lack of biocompatibility of the drug-eluting polymer leading to a persistent inflammatory response after the drug-eluting period. Although permanent stents (BMS and DES) are effective, in most cases, the role of stent is temporary and is limited to the intervention, and shortly thereafter, until healing and re-endothelialization are obtained [4]. Bioresorbable stents (BRS) were introduced to overcome these limitations with important advantages: complete bioresorption, mechanical flexibility, etc. The BRS concept introduced the use of polymers in stenting procedures for the first time. With the inclusion of polymeric materials in the field of stents, a new and promising idea makes its way, electrospun tubular scaffolds (ETS) for stenting process. Unlike current stents, ETS theoretically could present some advances such as (I) better longitudinal flexibility to help the placement of the stent and (II) their surface mimics body tissue to help to obtain a best proliferation rates [5] and thus a rapid endothelialization. Nevertheless, ETS could have some disadvantages such as their radial flexibility. The radial expansion of ETS will occur by elongation of its fibers, in contrast to current stents in which it occurs by elongation of its radial cells. This fact could make correct vessel support difficult, which would restrict the use of ETS for only peripheral applications. With the author's best knowledge, this new idea has been overlooked, but it presents a promising approach to solve cardiovascular problems. 2. Stent manufacturing process design 42 Angiography Stents can be used for a wide range of indication: de novo lesions, small vessel disease (SVD), bifurcation lesions, and tortuous and narrows lesions. Stents can improve the clinical outcomes for all of these indications as well as quality of life for patients suffering from this debilitating disease. In 2013, sales of DES and BMS in the 10 major markets were \$ 4.89 billion. Regardless of the stent choice, BMS, DES, BRS, or ETS, the challenges associated to this medical device remain similar. Figure 1 shows the pyramid of stent manufacturing process design. It represents the main issues to consider at the time to design and manufacture this medical device. The mechanical properties of the stent govern the decision process. This important property will be in charge of providing the correct longitudinal and radial behavior. The mechanical properties of the stents mainly depend on the material, geometry, and medical application of the stent: Mechanical properties = f (material, geometry, application) (1) Global data [6] estimates that by 2020, sales of stents will grow to \$ 5.65 billion. Once we have decided the mechanical properties, we give way to the manufacturing process step. The manufacturing process mainly depends on the materials and stent type chosen: $$\text{Manufacturing process = f(material, sent type)}\tag{2}$$ Finally, the additional properties step. Although it constitutes the final layer of the stent pyramid, additional properties cover a range of modifications to stent designs. This last step indirectly relates to the stent type and its medical application: $$\text{Additional properties = } f \text{(sent type, application)}\tag{3}$$ The next sections present the main issues to consider in the abovementioned decision pyramid steps with special attention to the manufacturing process step. #### 3. Stent mechanical properties Stent's mechanical properties are interrelated and sometimes contradictory, requiring careful compromise between geometrical and material aspects. #### 3.1. Geometrical aspects Following the classification done by Stoeckel et al. in their manuscript "A survey of stent designs," stents can be classified into five categories [7]. #### 3.1.1. Coil Most common in nonvascular applications, the coil design allows for retrievability after implantation (Figure 2a). These designs are extremely flexible, but their strength is limited, and their low expansion ratio results in high-profile devices. The main materials currently used or which are being investigated are briefly presented in In the field of mechanical properties of stents, many authors have focused their investigations in the study of the effects of the geometry and the material that this medical device have. Due to the high cost of these medical devices, much of the works have been performed by finite In 2007, Kiousis et al. [8] proposed a methodology to identify optimal stents for specific clinical criteria. They presented a numerical study to understand the interaction between the stent and a patient-specific atherosclerotic human lesion of type V. Mortier et al. [9] compared three different second-generation drug-eluting stents (DES) using a parametric modeling approach, when being implanted in the curved main branch of a coronary bifurcation with the aim of providing better insights into the related changes of the mechanical environment. Hopkins et al. [10] carried out a study which provided insight into the critical factors governing coating delamination during stent deployment and offered a predictive framework that can be used to improve the design of coated stents. They concluded that delamination initiation is governed by coating thickness and stiffness, interface strength, and hinge curvature. Augsburger et al. [11] proposed an alternative strategy, which is based on the modeling of the device as porous medium. Results predicted by the porous medium approach compare well with the real stent Nondegradable 316 stainless steels (SS316) It is also referred to as marine-grade stainless steel; is Fully degradable Magnesium (Mg) Magnesium is the third most commonly used a chromium, nickel, and molybdenum alloy of steel that exhibits relatively good strength and corrosion resistance; and is a common choice for biomedical Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 45 the two elements are present in roughly equal atomic percentages. Nitinol alloys exhibit two closely related and unique properties—shape memory (SME) and superelasticity (SE)— perfect to self-expandable structural metal. Magnesium is used in super strong, lightweight materials and alloys. Magnesium alloys have been historically used by the magnesium tendency to corrode, creep at high temperature, and polyester derived from renewable resources, such as corn starch. Degradation is produced by hydrolysis of its ester linkages in physiological conditions melting point (60°C) and a glass transition of about −60°C. Degradation is produced by hydrolysis of its ester linkages in physiological conditions and has therefore received a great deal of attention implants, such as stents Nitinol (NiTi) Nitinol is a metal alloy of nickel and titanium, where stents combust Poly-l-lactide acid (PLLA) PLLA is a biodegradable thermoplastic aliphatic Polycaprolactone (PCL) PCL is a biodegradable polyester with a low Type Material Description Table 1. element analysis (FEA). Table 1. Stent materials. #### 3.1.2. Helical spiral These designs are generally promoted for their flexibility. With no or minimal internal connection points, they are very flexible but also lack longitudinal support. As such, they can be subject to elongation or compression during delivery and deployment and, consequently, irregular cell size. With internal connection points, some flexibility is sacrificed in exchange for longitudinal stability and additional control over cell size. #### 3.1.3. Woven Woven designs are often used for self-expanding structures. While these designs offer excellent coverage, they typically shorten substantially during expansion. The radial strength of such a woven structure is also highly dependent on axial fixation of its ends. #### 3.1.4. Individual rings These are commonly used to support grafts or similar prostheses. This design is not typically used as vascular stents by itself. #### 3.1.5. Sequential rings This design is the most common in the market and includes two different categories: (I) closed cell, made of sequential ring construction wherein all internal inflection points of the structural members are connected (Figure 2b) and (II) open cells, a stent wherein some or all the internal inflection points of the structural member are not connected by bridging elements (Figure 2c). #### 3.2. Material aspects Since the introduction of the first stainless steel devices, the materials used for stents have evolved and diversified rapidly. In the drive to obtain a share of what was becoming a vast and growing market, manufacturers invested heavily in research and development to gain continuous. Figure 2. Designs: (a) coil EsophaCoil, (b) closed cell Palmaz-Schatz, and (c) open cell SMART. The main materials currently used or which are being investigated are briefly presented in Table 1. In the field of mechanical properties of stents, many authors have focused their investigations in the study of the effects of the geometry and the material that this medical device have. Due to the high cost of these medical devices, much of the works have been performed by finite element analysis (FEA). In 2007, Kiousis et al. [8] proposed a methodology to identify optimal stents for specific clinical criteria. They presented a numerical study to understand the interaction between the stent and a patient-specific atherosclerotic human lesion of type V. Mortier et al. [9] compared three different second-generation drug-eluting stents (DES) using a parametric modeling approach, when being implanted in the curved main branch of a coronary bifurcation with the aim of providing better insights into the related changes of the mechanical environment. Hopkins et al. [10] carried out a study which provided insight into the critical factors governing coating delamination during stent deployment and offered a predictive framework that can be used to improve the design of coated stents. They concluded that delamination initiation is governed by coating thickness and stiffness, interface strength, and hinge curvature. Augsburger et al. [11] proposed an alternative strategy, which is based on the modeling of the device as porous medium. Results predicted by the porous medium approach compare well with the real stent Table 1. Stent materials. 3.1.1. Coil 44 Angiography 3.1.2. Helical spiral 3.1.3. Woven 3.1.4. Individual rings 3.1.5. Sequential rings 3.2. Material aspects continuous. used as vascular stents by itself. low expansion ratio results in high-profile devices. for longitudinal stability and additional control over cell size. Most common in nonvascular applications, the coil design allows for retrievability after implantation (Figure 2a). These designs are extremely flexible, but their strength is limited, and their These designs are generally promoted for their flexibility. With no or minimal internal connection points, they are very flexible but also lack longitudinal support. As such, they can be subject to elongation or compression during delivery and deployment and, consequently, irregular cell size. With internal connection points, some flexibility is sacrificed in exchange Woven designs are often used for self-expanding structures. While these designs offer excellent coverage, they typically shorten substantially during expansion. The radial strength of These are commonly used to support grafts or similar prostheses. This design is not typically This design is the most common in the market and includes two different categories: (I) closed cell, made of sequential ring construction wherein all internal inflection points of the structural members are connected (Figure 2b) and (II) open cells, a stent wherein some or all the internal inflection points of the structural member are not connected by bridging elements (Figure 2c). Since the introduction of the first stainless steel devices, the materials used for stents have evolved and diversified rapidly. In the drive to obtain a share of what was becoming a vast and growing market, manufacturers invested heavily in research and development to gain Figure 2. Designs: (a) coil EsophaCoil, (b) closed cell Palmaz-Schatz, and (c) open cell SMART. such a woven structure is also highly dependent on axial fixation of its ends. geometry model and allow predicting the main effects of the device on intra-aneurismal flow, facilitating thus the analysis. Hsiao et al. [12] proposed to apply the parametric design concept onto the stent design and integrate it with the developed FEA and CFD models to evaluate these key clinical attributes as a function of the stent design parameters. They concluded that the most critical parameter for the equivalent plastic strain and the expansion recoil was the crown radius. Grujicic et al. [13] investigated the fatigue-controlled service life of the selfexpanding nitinol vascular stents. Praveen Kumar et al. [14] provided a simple, fast, and costeffective tool to quantitatively determine the fatigue resistance of stent components. Their results showed that the stent model passed the fatigue test under the aforementioned loading conditions. Nowadays the main efforts are being done in simulating polymeric materials in order to understand the mechanical behavior of the new polymeric BRS [15, 16]. the cyclic uniaxial strain region degraded at the same rate as unstressed and constant stress controls. In 2000, Tamai et al. [21] evaluated the feasibility, safety, and efficacy of the PLLA stent in humans. Fifteen patients electively underwent PLLA Igaki-Tamai stent implantation for coronary artery stenosis. The results were promising. Zilberman et al. [22] focused their studies on the mechanical properties of bioresorbable fibers. PLLA, PDS, and PGACL were studied in vitro. The three fibers combined a relatively high initial strength and modulus together with sufficient ductility. Venkatraman et al. [23] reported, for the first time, the development of a fully biodegradable polymeric stent that can self-expand at body temperatures. Ajili et al. [24] reported new self-expanding polymer made from polyurethane/polycaprolactone (PU/PCL). The results showed that the blend supported cell adhesion and proliferation, which indicated good biocompatibility. Their results suggested that this blend might be a potential material as a stent implant. Xue et al. [25] designed a biodegradable shape-memory block copolymer (PCTBV-25) for fast self-expandable stents. The stent made from PCTBV-25 film showed nearly complete self-expansion at 37°C within only 25 s, which is much better and faster than the best-known self-expandable stents. Vieira et al. [26] studied the evolution of mechanical properties during degradation based on experimental data. The decrease of tensile strength followed the same trend as the decrease of molecular weight. Weinandy et al. [27] designed a new viable stent structure (BioStent) to overcome in-stent restenosis. Despite the advances, many concerns still remain; one of the most important is the investigation on Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 47 the cell proliferation of the material that helps to induce a rapid endothelialization. Five technique has been used to manufacture stents: etching, micro-electro discharge machin- Etching method is based upon the photolithography process (Figure 3a). In this process, the desired mask pattern is first projected on the plain sheet coated with photoresist, which after 4. Traditional stent manufacturing processes Figure 3. Stent manufacturing processes: (a) etching and (b) laser cutting. 4.1. Etching ing, electroforming, die-casting, and, nowadays, laser cutting [28]. exposure can be developed and etched for the desired pattern [29, 30]. Stent materials have been investigated in-depth in the last decades. Understand how the manufacturing processes affect the material properties; develop new materials, analyses for new coating and its effect on the biological aspects, etc.; and have been the main research lines. In 2010, Liu et al. [17] analyzed the inhibition of bacterial adherence on the surface of biliary stent made of 316L SS modified with chitosan. Bacterial infection plays an important role in the initiation of biliary sludge formation. Bacterial adherence and biofilm formation on the surface of a material have been considered as one of the main factors of stent re-occlusion in clinic. Results suggested that that chitosan could be applied to biliary stent in clinical setting because of its antimicrobial activities. In 2011, Man et al. [18] used a Nd:YAG laser to cut NiTi alloy employing air and argon environment, respectively. The corrosion resistance improved for samples treated in air. In 2012, Ye [19] coated WE43 magnesium alloy with phytic acid (PA) by immersion. Authors aim to study the effect of PA's pH on the microstructure. Results showed that PA can enhance the corrosion resistance of WE43 magnesium especially when the pH value of the modified solution is 5 and the cytotoxicity of the PA-coated WE43 magnesium alloy is much better than that of the bare WE43 magnesium alloy. Moreover, all the hemolysis rates of the PA-coated WE43 Mg alloy were lower than 5%, indicating that the modified Mg alloy met the hemolysis standard of biomaterials. Therefore, PA coating is a good candidate to improve the biocompatibility of WE43 magnesium alloy. The inclusion of BRS concepts was made that many author started to study bioresorbable materials. In the design of biodegradable stent, it is advantageous to consider materials that have received regulatory approval for other applications such as PLA. Other synthetic biodegradable polymers with regulatory approval have been attempted as stent materials, such as PCL, PGA, and P4HB. Regardless of polymer choice, the challenges associated with material formulation, polymerization process, material processing, and material property characterization remain similar. Most importantly, it is not only necessary to know the characteristics of the material at its initial non-degraded stage but also how do these evolve with degradation. The vast majority of studies of polymer degradation were performed without mechanical loading. The few studies that have included mechanical loading indicate that degradation is accelerated, depending on the specific type of loading. Wiggins et al. [20] found that the degradation rate of polyurethane increased with cyclic strain rate, whereas strain magnitude has essentially no effect. In a separate study, the same group demonstrated that polymer from the cyclic uniaxial strain region degraded at the same rate as unstressed and constant stress controls. In 2000, Tamai et al. [21] evaluated the feasibility, safety, and efficacy of the PLLA stent in humans. Fifteen patients electively underwent PLLA Igaki-Tamai stent implantation for coronary artery stenosis. The results were promising. Zilberman et al. [22] focused their studies on the mechanical properties of bioresorbable fibers. PLLA, PDS, and PGACL were studied in vitro. The three fibers combined a relatively high initial strength and modulus together with sufficient ductility. Venkatraman et al. [23] reported, for the first time, the development of a fully biodegradable polymeric stent that can self-expand at body temperatures. Ajili et al. [24] reported new self-expanding polymer made from polyurethane/polycaprolactone (PU/PCL). The results showed that the blend supported cell adhesion and proliferation, which indicated good biocompatibility. Their results suggested that this blend might be a potential material as a stent implant. Xue et al. [25] designed a biodegradable shape-memory block copolymer (PCTBV-25) for fast self-expandable stents. The stent made from PCTBV-25 film showed nearly complete self-expansion at 37°C within only 25 s, which is much better and faster than the best-known self-expandable stents. Vieira et al. [26] studied the evolution of mechanical properties during degradation based on experimental data. The decrease of tensile strength followed the same trend as the decrease of molecular weight. Weinandy et al. [27] designed a new viable stent structure (BioStent) to overcome in-stent restenosis. Despite the advances, many concerns still remain; one of the most important is the investigation on the cell proliferation of the material that helps to induce a rapid endothelialization. #### 4. Traditional stent manufacturing processes Five technique has been used to manufacture stents: etching, micro-electro discharge machining, electroforming, die-casting, and, nowadays, laser cutting [28]. #### 4.1. Etching geometry model and allow predicting the main effects of the device on intra-aneurismal flow, facilitating thus the analysis. Hsiao et al. [12] proposed to apply the parametric design concept onto the stent design and integrate it with the developed FEA and CFD models to evaluate these key clinical attributes as a function of the stent design parameters. They concluded that the most critical parameter for the equivalent plastic strain and the expansion recoil was the crown radius. Grujicic et al. [13] investigated the fatigue-controlled service life of the selfexpanding nitinol vascular stents. Praveen Kumar et al. [14] provided a simple, fast, and costeffective tool to quantitatively determine the fatigue resistance of stent components. Their results showed that the stent model passed the fatigue test under the aforementioned loading conditions. Nowadays the main efforts are being done in simulating polymeric materials in Stent materials have been investigated in-depth in the last decades. Understand how the manufacturing processes affect the material properties; develop new materials, analyses for new coating and its effect on the biological aspects, etc.; and have been the main research lines. In 2010, Liu et al. [17] analyzed the inhibition of bacterial adherence on the surface of biliary stent made of 316L SS modified with chitosan. Bacterial infection plays an important role in the initiation of biliary sludge formation. Bacterial adherence and biofilm formation on the surface of a material have been considered as one of the main factors of stent re-occlusion in clinic. Results suggested that that chitosan could be applied to biliary stent in clinical setting because of its antimicrobial activities. In 2011, Man et al. [18] used a Nd:YAG laser to cut NiTi alloy employing air and argon environment, respectively. The corrosion resistance improved In 2012, Ye [19] coated WE43 magnesium alloy with phytic acid (PA) by immersion. Authors aim to study the effect of PA's pH on the microstructure. Results showed that PA can enhance the corrosion resistance of WE43 magnesium especially when the pH value of the modified solution is 5 and the cytotoxicity of the PA-coated WE43 magnesium alloy is much better than that of the bare WE43 magnesium alloy. Moreover, all the hemolysis rates of the PA-coated WE43 Mg alloy were lower than 5%, indicating that the modified Mg alloy met the hemolysis standard of biomaterials. Therefore, PA coating is a good candidate to improve the biocom- The inclusion of BRS concepts was made that many author started to study bioresorbable materials. In the design of biodegradable stent, it is advantageous to consider materials that have received regulatory approval for other applications such as PLA. Other synthetic biodegradable polymers with regulatory approval have been attempted as stent materials, such as PCL, PGA, and P4HB. Regardless of polymer choice, the challenges associated with material formulation, polymerization process, material processing, and material property characterization remain similar. Most importantly, it is not only necessary to know the characteristics of the material at its initial non-degraded stage but also how do these evolve with degradation. The vast majority of studies of polymer degradation were performed without mechanical loading. The few studies that have included mechanical loading indicate that degradation is accelerated, depending on the specific type of loading. Wiggins et al. [20] found that the degradation rate of polyurethane increased with cyclic strain rate, whereas strain magnitude has essentially no effect. In a separate study, the same group demonstrated that polymer from order to understand the mechanical behavior of the new polymeric BRS [15, 16]. for samples treated in air. 46 Angiography patibility of WE43 magnesium alloy. Etching method is based upon the photolithography process (Figure 3a). In this process, the desired mask pattern is first projected on the plain sheet coated with photoresist, which after exposure can be developed and etched for the desired pattern [29, 30]. Figure 3. Stent manufacturing processes: (a) etching and (b) laser cutting. #### 4.2. Micro-EDM In micro-EDM, the material removal takes place by electro-erosion due to electric discharge generated between closely spaced electrodes in the presence of a dielectric medium. The shape of the machined feature is the mirror image of electrode [31]. of AZ31 magnesium alloy sheet, and differences in cutting efficiency between fiber and CO<sup>2</sup> laser were studied. They investigate the effect of processing parameters in a laser cutting of 1 and 3.3-mm-thick sheets on the cutting quality. Their results showed that productivity, process efficiency, and cutting edge quality obtained using fiber lasers outperform CO<sup>2</sup> Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 performances. Teixidor et al. [39] carried out an experimental study of fiber laser cutting of 316L stainless steel thin sheets. They analyzed the effect of laser parameters on the cutting quality for fixed nitrogen assistance gas. Besides that, they presented a mathematical model Notwithstanding, avoiding the thermal damages in laser processing is impossible by itself. Another alternative would be to solve it during the laser ablation process itself. This can be accomplished by laser machining under liquid. Laser processing in the presence of liquid has been studied for more than 40 years for various applications [40, 41]. Nevertheless underwater laser micromachining for tubes specifically for coronary stent applications received less attention. Work by Muhammad et al. [42] directed a water flow through the tubes during the fiber laser micromachining to reduce HAZ, as well as protecting the opposite surface of the tube. Yang et al. [43] reported underwater machining of deep cavities in alumina ceramic. They found that underwater machining has the capability of preventing crack initiation, reducing heat damage, and giving an insignificant recast layer. In other works, Muhammad and Li [44] Despite the advances, the inclusion of the BRS concept should make us wonder about the applicability of the current laser-cutting manufacturing process for making BRS due to the new materials that have to be used, mostly polymers. There are several works about laser processing of biodegradable materials, both polymers and metals, trying to answer this question. and physicochemical properties of polyhydroxybutyrate. The result showed that cells preferred laser-machined areas. They concluded that not only were the material properties altered as a result of processing but also the biological response was affected. Grabow et al. [46] studied the effect of laser cutting on poly-l-lactide (PLLA). The results showed the dramatic influence of the plasticizer content and sterilization procedure on the mechanical properties of the material. Laser cutting had a lesser effect. Hence the effects of processing and sterilization must not be overlooked in the material selection and design phases of the development process leading to clinical use. Tiaw et al. [47] studied the effect of Nd:YAG laser on microdrilling and microcutting of thin PCL films. Melting and tearing of the thin polymer film were not much of an issue for the thin spin-cast film, but a slight extent of melting was observed in the thicker biaxial drawn film. Baer et al. [48] described the fabrication of a laser-activated shape memory polymer (SMP) stent and demonstrated photothermal expansion of the stent in an in vitro artery model. In materials. Their results showed that HAZ increases with the laser power and decreases with the cutting velocity. Yeong et al. [50] analyzed the effect of femtosecond laser micromachining on poly-E-caprolactone (PCL). Ortiz et al. [51] examined the picosecond laser ablation of PLLA as a function of laser fluence and degree of crystallinity. High-quality microgrooves were produced in amorphous PLLA, revealing the potential of ultra-fast laser processing technique. Demir et al. [52] demonstrated the feasibility of laser micro-cutting to produced magnesium BRS. By Q-switched fiber laser authors cut AZ31 magnesium alloy. The cutting process was studied the underwater femtosecond laser micromachining of thin nitinol tubes. Lootz et al. [45] analyzed the influence of laser-cutting process (CO<sup>2</sup> 2008, Davim et al. [49] realized some experimental studies on CO<sup>2</sup> based on energy balance for the dross dimensions. laser 49 laser) in morphological laser cutting of polymeric #### 4.3. Electroforming In this process, electroplating is performed on a mandrel in a given pattern. When the desired thickness has been reached, the mandrel is etched away from the electroformed stent, leaving a free standing structure, a fully functional stent [32, 33]. #### 4.4. Die-casting This is another technique in which the stent can also be formed by subjecting one or more. The metal may be cast directly in a stent-like form or cast into sheet or tubes from which the inventive stents are produced by using any of the method mentioned here. #### 4.5. Laser cutting A high energy density laser beam is focused on workpiece surface; the thermal energy is absorbed which heats and transforms the workpiece volume into a molten, vaporized, or chemically changed state that can easily be removed by flow of high pressure assist gas jet [34, 35] (Figure 3b). Currently, this is the technology in the market. Different types of lasers have been used in stent manufacture including CO<sup>2</sup> lasers, Nd:YAG lasers, fiber lasers, excimer lasers, and ultra-short pulse lasers. Fiber lasers have advantages compared to other laser technologies such as better beam quality, reliability, and process efficiency with lower acquisition cost and maintenance. Laser cutting is a thermal process which results in thermal damage such as heat-affected zone (HAZ), striation, recast layer, microcracks, tensile residual stress, and dross. To overcome the thermal damages, basically the following post-processing techniques are applied: pickling techniques, soft etching, annealing, and electropolishing. All these post-processing techniques raise the manufacture cost and could affect the mechanical properties of stents. There are several works, which study how the process parameters affect the quality, trying to reduce the thermal problems and thus reduce the cost of the stent manufacturing process. Kathuria [36] described the precision fabrication of metallic stent from stainless steel by using short pulse Nd:YAG laser. They conclude that the processing of stent with desired taper and quality shall still be preferred by the short pulse and higher pulse repetition rate of the laser. Meng et al. [35] analyzed the cut parameters with a fiber laser system. They concluded that the high-quality coronary stent has been cut with the power of 7 W, pulse length of 0.15 ms, frequency of 1500 Hz, scanning speed of 8 mm/s, and oxygen gas at 0.3 MPa as assistance gas. Muhammad et al. [37] studied the capability of picosecond laser micromachining of nitinol and platinum-iridium alloy in improving the cut quality. Process parameters used in the process have achieved dross-free cut and minimum extent of HAZ. Scintilla and Tricarico [38] analyzed the influence of processing parameters and laser source type on cutting edge quality of AZ31 magnesium alloy sheet, and differences in cutting efficiency between fiber and CO<sup>2</sup> laser were studied. They investigate the effect of processing parameters in a laser cutting of 1 and 3.3-mm-thick sheets on the cutting quality. Their results showed that productivity, process efficiency, and cutting edge quality obtained using fiber lasers outperform CO<sup>2</sup> laser performances. Teixidor et al. [39] carried out an experimental study of fiber laser cutting of 316L stainless steel thin sheets. They analyzed the effect of laser parameters on the cutting quality for fixed nitrogen assistance gas. Besides that, they presented a mathematical model based on energy balance for the dross dimensions. 4.2. Micro-EDM 48 Angiography 4.3. Electroforming 4.4. Die-casting 4.5. Laser cutting In micro-EDM, the material removal takes place by electro-erosion due to electric discharge generated between closely spaced electrodes in the presence of a dielectric medium. The In this process, electroplating is performed on a mandrel in a given pattern. When the desired thickness has been reached, the mandrel is etched away from the electroformed stent, leaving This is another technique in which the stent can also be formed by subjecting one or more. The metal may be cast directly in a stent-like form or cast into sheet or tubes from which the A high energy density laser beam is focused on workpiece surface; the thermal energy is absorbed which heats and transforms the workpiece volume into a molten, vaporized, or chemically changed state that can easily be removed by flow of high pressure assist gas jet lasers, fiber lasers, excimer lasers, and ultra-short pulse lasers. Fiber lasers have advantages compared to other laser technologies such as better beam quality, reliability, and process efficiency with lower acquisition cost and maintenance. Laser cutting is a thermal process which results in thermal damage such as heat-affected zone (HAZ), striation, recast layer, microcracks, tensile residual stress, and dross. To overcome the thermal damages, basically the following post-processing techniques are applied: pickling techniques, soft etching, annealing, and electropolishing. All these post-processing techniques raise the manufacture cost and There are several works, which study how the process parameters affect the quality, trying to reduce the thermal problems and thus reduce the cost of the stent manufacturing process. Kathuria [36] described the precision fabrication of metallic stent from stainless steel by using short pulse Nd:YAG laser. They conclude that the processing of stent with desired taper and quality shall still be preferred by the short pulse and higher pulse repetition rate of the laser. Meng et al. [35] analyzed the cut parameters with a fiber laser system. They concluded that the high-quality coronary stent has been cut with the power of 7 W, pulse length of 0.15 ms, frequency of 1500 Hz, scanning speed of 8 mm/s, and oxygen gas at 0.3 MPa as assistance gas. Muhammad et al. [37] studied the capability of picosecond laser micromachining of nitinol and platinum-iridium alloy in improving the cut quality. Process parameters used in the process have achieved dross-free cut and minimum extent of HAZ. Scintilla and Tricarico [38] analyzed the influence of processing parameters and laser source type on cutting edge quality lasers, Nd:YAG inventive stents are produced by using any of the method mentioned here. [34, 35] (Figure 3b). Currently, this is the technology in the market. could affect the mechanical properties of stents. Different types of lasers have been used in stent manufacture including CO<sup>2</sup> shape of the machined feature is the mirror image of electrode [31]. a free standing structure, a fully functional stent [32, 33]. Notwithstanding, avoiding the thermal damages in laser processing is impossible by itself. Another alternative would be to solve it during the laser ablation process itself. This can be accomplished by laser machining under liquid. Laser processing in the presence of liquid has been studied for more than 40 years for various applications [40, 41]. Nevertheless underwater laser micromachining for tubes specifically for coronary stent applications received less attention. Work by Muhammad et al. [42] directed a water flow through the tubes during the fiber laser micromachining to reduce HAZ, as well as protecting the opposite surface of the tube. Yang et al. [43] reported underwater machining of deep cavities in alumina ceramic. They found that underwater machining has the capability of preventing crack initiation, reducing heat damage, and giving an insignificant recast layer. In other works, Muhammad and Li [44] studied the underwater femtosecond laser micromachining of thin nitinol tubes. Despite the advances, the inclusion of the BRS concept should make us wonder about the applicability of the current laser-cutting manufacturing process for making BRS due to the new materials that have to be used, mostly polymers. There are several works about laser processing of biodegradable materials, both polymers and metals, trying to answer this question. Lootz et al. [45] analyzed the influence of laser-cutting process (CO<sup>2</sup> laser) in morphological and physicochemical properties of polyhydroxybutyrate. The result showed that cells preferred laser-machined areas. They concluded that not only were the material properties altered as a result of processing but also the biological response was affected. Grabow et al. [46] studied the effect of laser cutting on poly-l-lactide (PLLA). The results showed the dramatic influence of the plasticizer content and sterilization procedure on the mechanical properties of the material. Laser cutting had a lesser effect. Hence the effects of processing and sterilization must not be overlooked in the material selection and design phases of the development process leading to clinical use. Tiaw et al. [47] studied the effect of Nd:YAG laser on microdrilling and microcutting of thin PCL films. Melting and tearing of the thin polymer film were not much of an issue for the thin spin-cast film, but a slight extent of melting was observed in the thicker biaxial drawn film. Baer et al. [48] described the fabrication of a laser-activated shape memory polymer (SMP) stent and demonstrated photothermal expansion of the stent in an in vitro artery model. In 2008, Davim et al. [49] realized some experimental studies on CO<sup>2</sup> laser cutting of polymeric materials. Their results showed that HAZ increases with the laser power and decreases with the cutting velocity. Yeong et al. [50] analyzed the effect of femtosecond laser micromachining on poly-E-caprolactone (PCL). Ortiz et al. [51] examined the picosecond laser ablation of PLLA as a function of laser fluence and degree of crystallinity. High-quality microgrooves were produced in amorphous PLLA, revealing the potential of ultra-fast laser processing technique. Demir et al. [52] demonstrated the feasibility of laser micro-cutting to produced magnesium BRS. By Q-switched fiber laser authors cut AZ31 magnesium alloy. The cutting process was followed by a subsequent chemical etching to clean the kerf and surface finish. Stepak et al. [53] presented the impact of the KrF excimer laser irradiation above the ablation threshold on physicochemical properties of biodegradable PLLA. It could be concluded that the usage of the 248 nm wavelength resulted in simultaneous ablation at the surface and photodegradation within the entire irradiated volume due to high penetration depth. Furthermore, the thermal activation originating from relaxation of excited chromophores to vibrationally excited ground states enhances the degradation process. Stepak et al. [54] fabricated a polymer-based biodegradable stent using a CO<sup>2</sup> laser. They noted that the high-temperature gradient during the process altered the properties of the material within the heat-affected zone (HAZ). Guerra et al. [55, 56] demonstrated the feasibility of fiber lasers of 1.8 μm of wavelength to cut polycaprolactone sheet with higher precisions. The process is barely affected by the material properties. Nowadays there are many authors that carry on this field trying to give an answer to the current problems. Nevertheless, the inclusion of BRS concept has motivated most of the researchers to move to new technologies. The next section presents the most promising manufacturing techniques under investigation nowadays for producing BRS. computer-controlled platform (Figure 4a). It can differentiate two FFF processes for stent Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 51 Electrospinning (SE) is a fiber production method which uses electric force to draw charged threads of polymer solution or polymer melts up to fiber diameter in the order of some hundred nanometers (Figure 4c). Traditionally, this technique has been used for coating perma- In this new context, in the AM processes, some authors have focused their efforts to produce stents mainly focused on the new BRS. The novel use of polymeric materials joined with these new manufacturing methods opens a window to a novel concept, electrospun tubular bioabsorbable scaffold for stenting application. This new idea could reduce the problems derived Regarding the traditional BRS, the first document that presented a stent produced by AM was reported in 2015 when Park et al. [57] evaluated the properties of a 3D-printed BRS drug-coated stent in animals. Their results were promising in animals (20.7% restenosis). van Lith et al. [58] BMS design, they analyzed the effect of UV intensity, UV absorber, and wall thickness. Although results were promising, there are some limitations since the best-performing stent had a fabrication of 70 min. Misra et al. [61] performed an in silico analysis to optimize the stent design for printing and its prediction of sustainability under force exerted by the coronary artery or blood flow. Ware et al. [63] reported the process development in manufacturing high-resolution bioresorbable stents using the μCLIP system. They employed 26.5 min to manufacture a 2 cm stent. Cabrera et al. [62] manufactured a BRS stent employing a traditional 3D printer. Although the results were promising, the traditional 3D printer presents some limitations to manufacture tubular devices such as stent. Guerra et al. [59, 64] design and implement a novel 3D tubular printer to BRS manufacture, the first full research paper that applies 3D-printing process based on FFF to manufacture a BRS stent with nontraditional printer. Their approach presented a new and interesting method to produce stents based on polymers in less than 2 min (Figure 5). . Employing a Cipher manufacturing, (I) traditional Cartesian FFF or (II) novel tubular FFF. from the current stents, such as its placements or rapid endothelialization. built a novel μCLIP setup capable of speed ranging from 2.5 to 100 μm s−<sup>1</sup> Figure 4. Promising manufacturing processes: (a) FFF, (b) SL, and (c) electrospinning. 5.4. Electrospinning (SE) nent stents to produce DES. #### 5. Promising stent manufacturing processes Despite the proved feasibility of laser cutting to produce BRS, both metallic and polymeric, there is a need to develop new technologies to produce these medical devices. In this context, additive manufacturing (AM) could be a more economical solution. AM refers to processes used to create a 3D object in which layers of material are formed under computer control. The use of this technology for stent manufacture is recent [57–64] and could be a really interesting method to produce stent. Mainly, there are four different AM technologies in the stent field. #### 5.1. Stereolithography (SL) processes Stereolithography (SL) (Figure 4b) works by focusing an ultraviolet (UV) light or visible light onto a vat of photopolymerizable resin. It can differentiate three types of SL technologies, laserbased stereolithography (SLA), digital light processing (DLP), and very recently liquid crystal display (LCD). #### 5.2. Selective laser sintering (SLS) Selective laser sintering (SLS) uses a laser as the power source to sinter powdered material (metals, polymers, etc.), aiming the laser automatically at points in space defined by a 3D model, binding the material together to create a solid structure. #### 5.3. Fused filament fabrication (FFF) Also known as fused deposition modeling (FDM), here a hot thermoplastic is extruded from a temperature-controlled print head to produce fairly robust objects to a high degree of accuracy. The filament is melted into the extruder, which deposited the material onto a computer-controlled platform (Figure 4a). It can differentiate two FFF processes for stent manufacturing, (I) traditional Cartesian FFF or (II) novel tubular FFF. #### 5.4. Electrospinning (SE) followed by a subsequent chemical etching to clean the kerf and surface finish. Stepak et al. [53] presented the impact of the KrF excimer laser irradiation above the ablation threshold on physicochemical properties of biodegradable PLLA. It could be concluded that the usage of the 248 nm wavelength resulted in simultaneous ablation at the surface and photodegradation within the entire irradiated volume due to high penetration depth. Furthermore, the thermal activation originating from relaxation of excited chromophores to vibrationally excited ground states enhances the degradation process. Stepak et al. [54] fabricated a polymer-based ing the process altered the properties of the material within the heat-affected zone (HAZ). Guerra et al. [55, 56] demonstrated the feasibility of fiber lasers of 1.8 μm of wavelength to cut polycaprolactone sheet with higher precisions. The process is barely affected by the material properties. Nowadays there are many authors that carry on this field trying to give an answer to the current problems. Nevertheless, the inclusion of BRS concept has motivated most of the researchers to move to new technologies. The next section presents the most promising Despite the proved feasibility of laser cutting to produce BRS, both metallic and polymeric, there is a need to develop new technologies to produce these medical devices. In this context, additive manufacturing (AM) could be a more economical solution. AM refers to processes used to create a 3D object in which layers of material are formed under computer control. The use of this technology for stent manufacture is recent [57–64] and could be a really interesting method to produce stent. Mainly, there are four different AM technologies in the stent field. Stereolithography (SL) (Figure 4b) works by focusing an ultraviolet (UV) light or visible light onto a vat of photopolymerizable resin. It can differentiate three types of SL technologies, laserbased stereolithography (SLA), digital light processing (DLP), and very recently liquid crystal Selective laser sintering (SLS) uses a laser as the power source to sinter powdered material (metals, polymers, etc.), aiming the laser automatically at points in space defined by a 3D Also known as fused deposition modeling (FDM), here a hot thermoplastic is extruded from a temperature-controlled print head to produce fairly robust objects to a high degree of accuracy. The filament is melted into the extruder, which deposited the material onto a model, binding the material together to create a solid structure. manufacturing techniques under investigation nowadays for producing BRS. 5. Promising stent manufacturing processes 5.1. Stereolithography (SL) processes 5.2. Selective laser sintering (SLS) 5.3. Fused filament fabrication (FFF) display (LCD). laser. They noted that the high-temperature gradient dur- biodegradable stent using a CO<sup>2</sup> 50 Angiography Electrospinning (SE) is a fiber production method which uses electric force to draw charged threads of polymer solution or polymer melts up to fiber diameter in the order of some hundred nanometers (Figure 4c). Traditionally, this technique has been used for coating permanent stents to produce DES. In this new context, in the AM processes, some authors have focused their efforts to produce stents mainly focused on the new BRS. The novel use of polymeric materials joined with these new manufacturing methods opens a window to a novel concept, electrospun tubular bioabsorbable scaffold for stenting application. This new idea could reduce the problems derived from the current stents, such as its placements or rapid endothelialization. Regarding the traditional BRS, the first document that presented a stent produced by AM was reported in 2015 when Park et al. [57] evaluated the properties of a 3D-printed BRS drug-coated stent in animals. Their results were promising in animals (20.7% restenosis). van Lith et al. [58] built a novel μCLIP setup capable of speed ranging from 2.5 to 100 μm s−<sup>1</sup> . Employing a Cipher BMS design, they analyzed the effect of UV intensity, UV absorber, and wall thickness. Although results were promising, there are some limitations since the best-performing stent had a fabrication of 70 min. Misra et al. [61] performed an in silico analysis to optimize the stent design for printing and its prediction of sustainability under force exerted by the coronary artery or blood flow. Ware et al. [63] reported the process development in manufacturing high-resolution bioresorbable stents using the μCLIP system. They employed 26.5 min to manufacture a 2 cm stent. Cabrera et al. [62] manufactured a BRS stent employing a traditional 3D printer. Although the results were promising, the traditional 3D printer presents some limitations to manufacture tubular devices such as stent. Guerra et al. [59, 64] design and implement a novel 3D tubular printer to BRS manufacture, the first full research paper that applies 3D-printing process based on FFF to manufacture a BRS stent with nontraditional printer. Their approach presented a new and interesting method to produce stents based on polymers in less than 2 min (Figure 5). Figure 4. Promising manufacturing processes: (a) FFF, (b) SL, and (c) electrospinning. Figure 5. 3D-printed PCL stent [65]. Demir and Previtali [60] produced CoCr stent through SLM as an alternative method to the conventional manufacturing cycle. Results showed that SLM can be considered as a substitute operation to laser micro-cutting. Prototype stents with acceptable geometrical accuracy were achieved, and surface quality could be improved through electrochemical polishing. The chemical composition remained unvaried, with a marginal increase in the oxide content. AM processes have proved their effectiveness, and clearly they present a novel method to produce stents that could replace the current laser-cutting process. Depending on the AM process use, the manufacturer will have some advantages and disadvantages. Table 2 sum- Costs Speeds Precisions Radial Longitudinal Biological Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 53 AM process Manufacturing issues Final stent properties Despite the few works about AM of stents, results have proved that these techniques could be promising. Further studies with new materials and technologies become crucial to understand the effect the process parameters has on stent properties and enclose the best material Although it constitutes the final layer of the stent manufacturing pyramid, additional properties cover a range of modifications to stent designs that sometimes become crucial. This section presents briefly the most common additional properties that are added to the stents. Stents made from stainless steel, nitinol, or polymers are sometimes hard to see fluoroscopically, particularly if they are small and/or have thin and narrow struts. To improve X-ray visibility, markers are often attached to the stents. These additions are typically made from It is the main additional property usually added to stents in the current clinic practice. The main stent currently used in clinic is DES. There are a variety of coating techniques to incor- Multiple variables can be adjusted to optimize drug release for cardiovascular stent applications, including biocompatible polymers and antirestenotic drugs. While the presence of a porate drugs to the stent [68]. Table 3 briefly presents the most common techniques. gold, platinum, or tantalum and can be sleeves crimped around a strut [7]. marized some on them. , poor property; , good property; , medium property Table 2. Additive manufacturing (AM) process advantages and disadvantages. 6. Additional properties 6.1. Radiopacity markers 6.2. Drug release and process. SL SLS ES Traditional FFF Tubular FFF In recent studies, Guerra et al. [66] manufactured a PCL/PLA composite stent by FFF. They suggested that composite stents could improve the mechanical behavior of polymeric materials. Also, their results suggested that composite stents could improve the biological requirements, namely, high proliferation external surface, to help the endothelialization with low proliferation internal surface to avoid the adhesion and proliferation of the cell in charge of restenosis. Regarding the novel electrospun tubular scaffolds (ETS) for stenting applications, some authors have been trying to produce them by electrospinning. Scaffold for stenting applications presents some advances as regards the traditional closed-cell stents. The longitudinal flexibility of scaffolds is higher than traditional stents, making their placement easy and reducing the risk during the stenting protocol. Also, scaffolds, produced by electrospinning, mimic the human tissue being able to increase the cell proliferation and helping a better and faster endothelialization of the vessel. However, scaffolds, due to their geometrical aspects can have worse radial behavior making it difficult for the vessel support. As a new idea, scaffold for stenting application has been barely studied. Bakola et al. [67] manufactured a drug-eluting bioabsorbable stent with PCL and PLA by electrospinning process. Authors focus their work in its application as coating to produce DES; nevertheless, results showed good morphology and topography. Nevertheless authors did not perform radial tests to check the feasibility of scaffold for stenting purposes. ETS present a new research field into the stent world. Further works that get into the manufacturing process and mechanical behavior of this new concept are completely necessary. Table 2. Additive manufacturing (AM) process advantages and disadvantages. AM processes have proved their effectiveness, and clearly they present a novel method to produce stents that could replace the current laser-cutting process. Depending on the AM process use, the manufacturer will have some advantages and disadvantages. Table 2 summarized some on them. Despite the few works about AM of stents, results have proved that these techniques could be promising. Further studies with new materials and technologies become crucial to understand the effect the process parameters has on stent properties and enclose the best material and process. #### 6. Additional properties Demir and Previtali [60] produced CoCr stent through SLM as an alternative method to the conventional manufacturing cycle. Results showed that SLM can be considered as a substitute operation to laser micro-cutting. Prototype stents with acceptable geometrical accuracy were achieved, and surface quality could be improved through electrochemical polishing. The chemical composition remained unvaried, with a marginal increase in the In recent studies, Guerra et al. [66] manufactured a PCL/PLA composite stent by FFF. They suggested that composite stents could improve the mechanical behavior of polymeric materials. Also, their results suggested that composite stents could improve the biological requirements, namely, high proliferation external surface, to help the endothelialization with low proliferation internal surface to avoid the adhesion and proliferation of the cell in charge of restenosis. Regarding the novel electrospun tubular scaffolds (ETS) for stenting applications, some authors have been trying to produce them by electrospinning. Scaffold for stenting applications presents some advances as regards the traditional closed-cell stents. The longitudinal flexibility of scaffolds is higher than traditional stents, making their placement easy and reducing the risk during the stenting protocol. Also, scaffolds, produced by electrospinning, mimic the human tissue being able to increase the cell proliferation and helping a better and faster endothelialization of the vessel. However, scaffolds, due to their geometrical aspects can have worse radial behavior making it difficult for the vessel support. As a new idea, scaffold for stenting application has been barely studied. Bakola et al. [67] manufactured a drug-eluting bioabsorbable stent with PCL and PLA by electrospinning process. Authors focus their work in its application as coating to produce DES; nevertheless, results showed good morphology and topography. Nevertheless authors did not perform radial tests to check the feasibility of scaffold for stenting purposes. ETS present a new research field into the stent world. Further works that get into the manufacturing process and mechanical behavior of this new concept oxide content. 52 Angiography Figure 5. 3D-printed PCL stent [65]. are completely necessary. Although it constitutes the final layer of the stent manufacturing pyramid, additional properties cover a range of modifications to stent designs that sometimes become crucial. This section presents briefly the most common additional properties that are added to the stents. #### 6.1. Radiopacity markers Stents made from stainless steel, nitinol, or polymers are sometimes hard to see fluoroscopically, particularly if they are small and/or have thin and narrow struts. To improve X-ray visibility, markers are often attached to the stents. These additions are typically made from gold, platinum, or tantalum and can be sleeves crimped around a strut [7]. #### 6.2. Drug release It is the main additional property usually added to stents in the current clinic practice. The main stent currently used in clinic is DES. There are a variety of coating techniques to incorporate drugs to the stent [68]. Table 3 briefly presents the most common techniques. Multiple variables can be adjusted to optimize drug release for cardiovascular stent applications, including biocompatible polymers and antirestenotic drugs. While the presence of a manufacturing process field should work to increase the knowledge about the process parameters to obtain a more accurate result in the case of tubular FFF process and faster production in the case of SL processes that nowadays are presented as the most promising technique under investigation. Also, further works about ETS are necessary. ETS present a new research field into the stent world that could solve some current medical problems. Finally, the sterilization process should be noted. This last but mandatory manufacturing step could be critical for the final properties of stents. As Grabow et al. [46], or more recently Guerra et al. [65], have demonstrated, sterilization processes can change the final properties. Further works that get into the effect the sterilization processes has on stent properties are completely necessary. Stent's Manufacturing Field: Past, Present, and Future Prospects http://dx.doi.org/10.5772/intechopen.81668 55 Authors acknowledge the financial support from the Ministry of Economy and Competitiveness (MINECO, DPI2016-77156-R), the University of Girona (UDG, MPCUdG2016/036), and the Department of Mechanical Engineering and Civil Construction, Universitat de Girona, [1] Grüntzig A. Transluminal dilatation of coronary artery stenosis. Lancet. 1978;1(C):263. Available from: http://scholar.google.com/scholar?hl=en&btnG=Search&q=intitle:Tra [2] Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, et al. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. The New England Journal of Medicine. 1994;331:489-495 [3] Schwartz RS, Topol EJ, Serruys PW, Sangiorgi G, Holmes DRJ.Artery size, neointima, and remodeling: Time for some standards. Journal of the American College of Cardiology. Acknowledgements Conflict of interest Author details Girona, Spain References 1998;32(7):2087-2094 Catalan government (2017SGR00385). The authors declare no conflict of interest. Antonio J. Guerra and Joaquim Ciurana\* \Address all correspondence to: [email protected] nsluminal+dilatation+of+coronary+artery+stenosis#0 Table 3.* Most common coating techniques in stent manufacturing industry. polymer coating on a DES is not required, the process to choose one can be difficult because it has been previously shown that most polymers listed as biocompatible and used in medicine can cause vascular inflammation. #### 7. Summary Stents have been the center of many research lines in the last decades. The challenges associated with this medical device are numerous: geometry, material, manufacturing process, biocompatibility, etc. Finite element analysis (FEA) studies have been trying to understand the role of the different geometrical and material aspects in the mechanical behavior of stents. Material studies have been developing new material that accomplishes with the stent requirements in terms of mechanical and medical properties, both in the permanent field and in the biodegradable field. Finally, in the manufacturing research field, authors have been focused in improving the process by analyzing the parameters to obtain a stent with the higher precision and the minimal costs. In this research line, there are noteworthy lines: laser micro-cutting and, more recently, additive manufacturing. Additive manufacturing studies are showing their huge potential in the field of polymeric BRS. There is a need to develop techniques for evaluating the ability of biodegradable stents to provide not only acute support but also reliable structural integrity for an appropriate period of time. In all these properties, the material and manufacturing process plays an important role. To achieve this, the previously mentioned research fields should be converged. The FEA field is developing new geometries with bioabsorbable polymer such as polycaprolactone that accomplishes with the stent mechanical requirements. The material field is developing new materials that accomplish the strict stent requirements. In the case of metallic alloys, develop material degraded with an appropriate rate. In the case of polymers, develop material accomplish with the mechanical and manufacturing properties of metallic ones. Lastly, the manufacturing process field should work to increase the knowledge about the process parameters to obtain a more accurate result in the case of tubular FFF process and faster production in the case of SL processes that nowadays are presented as the most promising technique under investigation. Also, further works about ETS are necessary. ETS present a new research field into the stent world that could solve some current medical problems. Finally, the sterilization process should be noted. This last but mandatory manufacturing step could be critical for the final properties of stents. As Grabow et al. [46], or more recently Guerra et al. [65], have demonstrated, sterilization processes can change the final properties. Further works that get into the effect the sterilization processes has on stent properties are completely necessary. #### Acknowledgements Authors acknowledge the financial support from the Ministry of Economy and Competitiveness (MINECO, DPI2016-77156-R), the University of Girona (UDG, MPCUdG2016/036), and the Catalan government (2017SGR00385). #### Conflict of interest polymer coating on a DES is not required, the process to choose one can be difficult because it has been previously shown that most polymers listed as biocompatible and used in medicine Dip coating Coating by submerging the stent in a solution of drug and/or polymer in Plasma-treated coating Plasma coating (plasma activation) is a method of surface modification modification of the topography Spray coating These techniques use apparatuses that spray polymer and drug solutions or their homogeneity Electro-treated coating The drug/polymer particles are suspended in a liquid medium migrate (stent) Table 3. Most common coating techniques in stent manufacturing industry. a solvent. The stent is then left to dry, allowing for evaporation, in the air/ oven. In this technique the drug/polymer can vary their concentration and/ under the influence of an electric field and are deposited onto an electrode which improves surface adhesion properties. Plasma produce a reduction of metal oxides, surface cleaning from organic contaminants, and (using various solvents) onto a stent, enabling consistent deposit of a uniform drug release layer(s) onto the stent surface Stents have been the center of many research lines in the last decades. The challenges associated with this medical device are numerous: geometry, material, manufacturing process, biocompatibility, etc. Finite element analysis (FEA) studies have been trying to understand the role of the different geometrical and material aspects in the mechanical behavior of stents. Material studies have been developing new material that accomplishes with the stent requirements in terms of mechanical and medical properties, both in the permanent field and in the biodegradable field. Finally, in the manufacturing research field, authors have been focused in improving the process by analyzing the parameters to obtain a stent with the higher precision and the minimal costs. In this research line, there are noteworthy lines: laser micro-cutting and, more recently, additive manufacturing. Additive manufacturing studies are showing There is a need to develop techniques for evaluating the ability of biodegradable stents to provide not only acute support but also reliable structural integrity for an appropriate period of time. In all these properties, the material and manufacturing process plays an important role. To achieve this, the previously mentioned research fields should be converged. The FEA field is developing new geometries with bioabsorbable polymer such as polycaprolactone that accomplishes with the stent mechanical requirements. The material field is developing new materials that accomplish the strict stent requirements. In the case of metallic alloys, develop material degraded with an appropriate rate. In the case of polymers, develop material accomplish with the mechanical and manufacturing properties of metallic ones. Lastly, the can cause vascular inflammation. Technique Brief description their huge potential in the field of polymeric BRS. 7. Summary 54 Angiography The authors declare no conflict of interest. #### Author details Antonio J. Guerra and Joaquim Ciurana\* \Address all correspondence to: [email protected] Department of Mechanical Engineering and Civil Construction, Universitat de Girona, Girona, Spain #### References* [4] Waksman R. Update on bioabsorbable stents: From bench to clinical. 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Acta Biomaterialia. 2010;6(5):1843-1851 Materials Processing Technology. 2005;170(3):545-550 10.1016/j.optlastec.2012.04.026 Processing. 2012;107(4):849-861 Biomaterials. 2001;22(18):2447-2452 1200-1206 58 Angiography [60] Demir AG, Previtali B.Additive manufacturing of cardiovascular CoCr stents by selective laser melting. Materials & Design. 2017;119:338-350. DOI: 10.1016/j.matdes.2017.01.091 Chapter 5 Provisional chapter Dedicated Bifurcation Stents Dedicated Bifurcation Stents Ivo Petrov, Iveta Tasheva, Jivka Stoykova, Ivo Petrov, Iveta Tasheva, Jivka Stoykova, http://dx.doi.org/10.5772/intechopen.82573 Abstract 1. Introduction Additional information is available at the end of the chapter Additional information is available at the end of the chapter Liubomir Dosev, Zoran Stankov and Petar Polomski Liubomir Dosev, Zoran Stankov and Petar Polomski DOI: 10.5772/intechopen.82573 Bifurcations still remain one of the most challenging lesions to be treated in the modern PCI era. They are associated with lower procedural success rates, higher rates of periprocedural complications, and complicated long-term outcomes. Their incidence is assessed to be approximately 15–20%. There is still debate on how should they be treated—one-stent versus two-stent techniques, whether there is a need for obligatory proximal optimization or kissing balloons. Multiple clinical trials have tested different PCI strategies. We will cover theoretical basics of treating bifurcations and describe different types of dedicated bifurcation stents—Nile PAX, Nile SIR, BiOSS Expert, BiOSS LIM, Stentys, Tryton, and Axxess Plus. We will discuss the data from studies comparing these bifurcation devices and will show our own experience and results working with these devices. There will be a discussion, tips, and tricks treating bifurcation lesions with Bifurcations still remain one of the most challenging lesions to be treated in the modern PCI era. They are associated with lower procedural success rates, higher rates of periprocedural complications, and complicated long-term outcomes. Their incidence is assessed to be approximately 15–20%. There is still debate on how should they be treated—one-stent versus two-stent techniques, whether there is a need for obligatory proximal optimization or kissing The conventional stents are not designed for bifurcations as there is huge variation in vessel anatomy, mostly discrepancy in proximal and distal diameter of the treated vessel and a need dedicated devices—most common pitfalls and how to deal with them. balloons. Multiple clinical trials have tested different PCI strategies. Keywords: bifurcation, left main, new devices © 2016 The Author(s). Licensee InTech. 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. © 2019 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. Chapter 5 Provisional chapter #### Dedicated Bifurcation Stents Dedicated Bifurcation Stents Ivo Petrov, Iveta Tasheva, Jivka Stoykova, Liubomir Dosev, Zoran Stankov and Petar Polomski Ivo Petrov, Iveta Tasheva, Jivka Stoykova, Liubomir Dosev, Zoran Stankov and Petar Polomski Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.82573 #### Abstract [60] Demir AG, Previtali B.Additive manufacturing of cardiovascular CoCr stents by selective laser melting. Materials & Design. 2017;119:338-350. DOI: 10.1016/j.matdes.2017.01.091 [61] Misra SK, Ostadhossein F, Babu R, Kus J, Tankasala D, Sutrisno A, et al. 3D-printed multidrug-eluting stent from graphene-nanoplatelet-doped biodegradable polymer composite. [62] Cabrera MS, Sanders B, Goor OJGM, Driessen-Mol A, Oomens CW, Baaijens FPT. Computationally designed 3D printed self-expandable polymer stents with biodegradation capacity for minimally invasive heart valve implantation: A proof-of-concept study. 3D Printing and Additive Manufacturing. 2017;4(1):19-29. DOI: 10.1089/3dp. [63] Ware HOT, Farsheed AC, van Lith R, Baker E, Ameer G, Sun C. Process development for high-resolution 3D-printing of bioresorbable vascular stents. Biomedical Engineering Mechanical Engineering. 2017;10115:101150N. Available from: http://proceedings.spiedig- [64] Guerra A, Ciurana J. 3D-printed bioabsordable polycaprolactone stent: The effect of process parameters on its physical features. Materials & Design. 2018;137:430-437. Available [65] Guerra AJ, Cano P, Rabionet M, Puig T. Effects of different sterilization processes on the properties of a novel 3D-printed polycaprolactone stent. 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DOI: 10.1016/j.matpr.2017.07.016 2016.0052 60 Angiography Bifurcations still remain one of the most challenging lesions to be treated in the modern PCI era. They are associated with lower procedural success rates, higher rates of periprocedural complications, and complicated long-term outcomes. Their incidence is assessed to be approximately 15–20%. There is still debate on how should they be treated—one-stent versus two-stent techniques, whether there is a need for obligatory proximal optimization or kissing balloons. Multiple clinical trials have tested different PCI strategies. We will cover theoretical basics of treating bifurcations and describe different types of dedicated bifurcation stents—Nile PAX, Nile SIR, BiOSS Expert, BiOSS LIM, Stentys, Tryton, and Axxess Plus. We will discuss the data from studies comparing these bifurcation devices and will show our own experience and results working with these devices. There will be a discussion, tips, and tricks treating bifurcation lesions with dedicated devices—most common pitfalls and how to deal with them. DOI: 10.5772/intechopen.82573 Keywords: bifurcation, left main, new devices #### 1. Introduction Bifurcations still remain one of the most challenging lesions to be treated in the modern PCI era. They are associated with lower procedural success rates, higher rates of periprocedural complications, and complicated long-term outcomes. Their incidence is assessed to be approximately 15–20%. There is still debate on how should they be treated—one-stent versus two-stent techniques, whether there is a need for obligatory proximal optimization or kissing balloons. Multiple clinical trials have tested different PCI strategies. The conventional stents are not designed for bifurcations as there is huge variation in vessel anatomy, mostly discrepancy in proximal and distal diameter of the treated vessel and a need © 2016 The Author(s). Licensee InTech. 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. © 2019 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. for free access in the side branch. One major problem in bifurcation PCI is the high rate of SB ostium restenosis. There are also several procedural difficulties such as maintaining access to the side branch, irregular overlapping, and uneven distribution of struts at the carina. The final result and long-term success rates are highly variable and are operator dependent. balloon; finally, kissing balloons should be inflated. In culotte, SB stent also covers some of the MB and this part in MB is overexpanded with POT, leaving double strut layer at the proximal MB; final kissing is a must. More modern minicrush and miniculotte use shorter stent overlapping, thus decreasing strut burden. In Tap, the SB stent is left to protrude a bit proximal to carina, while leaving a balloon in MB for simultaneous kissing, thus making a neostrut carina. Dedicated Bifurcation Stents 63 http://dx.doi.org/10.5772/intechopen.82573 Sometimes, if there is a large and diseased SB prone to ischemia and deterioration in LV function (mostly in left main), a two-stent strategy is chosen instead of provisional stent. It is operator's decision which part MB or SB to be stented first and which of two-stent strategies to choose. Bioresorbable stents, besides their potential advantage to dissolve with time, have a lot of limitations—they are thicker (some techniques require multiple struts layers and you cannot always go with provisional one-stent strategy) and have limited expansion property and increased rates of thrombosis. Absorb BRS (Abbott Vascular USA) is already taken down from market. DESolve BRS (Elixir Medical USA) and Magmaris (BIOTRONIK Germany) could be One of them is the dedicated bifurcational stents, Tryton and Tryton SHORT stents. The delivery system of the stent is based on rapid-exchange catheter with single-wire tracking (no risk of wire wrap or bias); there is no need for rotational orientation. The stent has low profile and is 5-Fr guide compatible with two central markers delineating central transition The deployment sequence of the stent consists of wiring the side branch and predilatation. Then, Tryton is positioned and is deployed from main branch to the side branch, after which the main branch is wired and a DES is positioned 1 mm proximal to the Tryton proximal edge. After deploying the stent in the main branch, rewiring of side branch and kissing is performed. There are several drawbacks of Tryton system. It is a bare metal stent with consistently <5% TLR in all clinical studies in over 1.800 pts. It requires adequate proximal landing zone and accurate positioning, but the learning curve is short. It is not indicated for <2.25 mm vessels TRYTON randomized 704 patients with bifurcation coronary lesions at 58 centers (30 from Europe and 28 from the United States). At 9 months, TVF was 4.6% higher in the bifurcation stent group compared with the provisional group (p = 0.11). TVF was mainly presented by higher rates of periprocedural myocardial infarction rate (13.6 vs. 10.1%). The SB in-segment diameter stenosis was lower in the bifurcation stent group compared with the provisional group (31.6 vs. 38.6%), with no difference in binary restenosis rates (diameter stenosis ≥50%) a choice, but still there are a few data. 3. Tryton and Tryton SHORT stents zone used to precisely position the stent (Figures 1–3). and is tied to two-stent strategy as intention to treat. at 9 months follow-up (22.6 vs. 26.8%) [2]. There are several randomized studies as far as this stent is concerned. There are several devices dedicated to bifurcation treatment. Closure of side branch bigger than 1 mm often is associated with 14% incidence of periprocedural myocardial infarction [1]. The dedicated bifurcation stents are produced to tackle most of these problems. Dedicated bifurcation stents give hope for technically straightforward and fast success rate; they aim to protect the SB and allow permanent SB access as well as optimal MB and SB scaffolding and coverage. They also tend to limit multiple layering of stent struts, limit gaps in support, and prevent restenosis reducing SB risk, optimizing immediate and long-term outcomes. #### 2. Anatomy, physiology, and conventional stent treatment Coronary bifurcation lesions should be viewed as three major parts—proximal main branch, distal main branch, and a side branch. Significant side branch is defined on the subjective judgment of the operator, which warrants its diameter, length, size of myocardial mass supplied, its viability, and left ventricular function. Treating a bifurcation comes with the risk of losing SB patency; thus, the operator should thoroughly review the plaque burden at the carina, its angle, and diameter of MB and SB. The MADS classification presents various techniques used in bifurcation stenting. There is one-stent (provisional) or two-stent strategy. Provisional stent strategy is done in majority of cases; it is recommended by the European Bifurcation Club as KISSS principle (Keep It Simple, Swift and Safe). At first, the MV is stented, the stent diameter is 1:1 according to distal MB diameter, and then operator should perform POT (proximal optimization technique) and eventually kissing balloon inflation. POT presents short oversized balloon dilation at proximal part of the stent (from proximal edge of the stent to proximal carina edge). This technique apposes stent struts to the proximal MV and enhances scaffolding at the SB ostium. After this, if operator decides based on SB ostium stenosis and flow, kissing balloon inflation could be done. Kissing inflation involves rewiring of the SB from the distal strut at SB ostium, and getting a balloon through the stent struts could be the most challenging part of bifurcation treatment. After rewiring, two NC balloons are simultaneously inflated in MB and SB. After POT and kissing, if there is a need for SB stenting because of low flow or SB dissection, there are basically few strategies—T stenting, TAP (T stenting and protrusion), culotte, and crush techniques. In all two-stent techniques, kissing balloon dilation is mandatory to achieve full stent expansion at both ostia; sometimes, final POT should be considered. T-stenting strategy is chosen when there is scaffolding at the SB ostium from the POT and when SB angulation is nearly 90°. If SB is not covered by POT technique and the angle is acute, crush (minicrush), culotte, and TAP should be considered as they all cover SB ostium with struts. In crush (minicrush), the stent in SB is placed overlapping the MB and is crushed with oversized balloon; finally, kissing balloons should be inflated. In culotte, SB stent also covers some of the MB and this part in MB is overexpanded with POT, leaving double strut layer at the proximal MB; final kissing is a must. More modern minicrush and miniculotte use shorter stent overlapping, thus decreasing strut burden. In Tap, the SB stent is left to protrude a bit proximal to carina, while leaving a balloon in MB for simultaneous kissing, thus making a neostrut carina. Sometimes, if there is a large and diseased SB prone to ischemia and deterioration in LV function (mostly in left main), a two-stent strategy is chosen instead of provisional stent. It is operator's decision which part MB or SB to be stented first and which of two-stent strategies to choose. Bioresorbable stents, besides their potential advantage to dissolve with time, have a lot of limitations—they are thicker (some techniques require multiple struts layers and you cannot always go with provisional one-stent strategy) and have limited expansion property and increased rates of thrombosis. Absorb BRS (Abbott Vascular USA) is already taken down from market. DESolve BRS (Elixir Medical USA) and Magmaris (BIOTRONIK Germany) could be a choice, but still there are a few data. There are several devices dedicated to bifurcation treatment. #### 3. Tryton and Tryton SHORT stents for free access in the side branch. One major problem in bifurcation PCI is the high rate of SB ostium restenosis. There are also several procedural difficulties such as maintaining access to the side branch, irregular overlapping, and uneven distribution of struts at the carina. The final result and long-term success rates are highly variable and are operator dependent. Closure of side branch bigger than 1 mm often is associated with 14% incidence of periproce- Dedicated bifurcation stents give hope for technically straightforward and fast success rate; they aim to protect the SB and allow permanent SB access as well as optimal MB and SB scaffolding and coverage. They also tend to limit multiple layering of stent struts, limit gaps in support, and prevent restenosis reducing SB risk, optimizing immediate and long-term outcomes. Coronary bifurcation lesions should be viewed as three major parts—proximal main branch, distal main branch, and a side branch. Significant side branch is defined on the subjective judgment of the operator, which warrants its diameter, length, size of myocardial mass sup- Treating a bifurcation comes with the risk of losing SB patency; thus, the operator should thoroughly review the plaque burden at the carina, its angle, and diameter of MB and SB. The MADS classification presents various techniques used in bifurcation stenting. There is one-stent (provisional) or two-stent strategy. Provisional stent strategy is done in majority of cases; it is recommended by the European Bifurcation Club as KISSS principle (Keep It Simple, Swift and Safe). At first, the MV is stented, the stent diameter is 1:1 according to distal MB diameter, and then operator should perform POT (proximal optimization technique) and eventually kissing balloon inflation. POT presents short oversized balloon dilation at proximal part of the stent (from proximal edge of the stent to proximal carina edge). This technique apposes stent struts to the proximal MV and enhances scaffolding at the SB ostium. After this, if operator decides based on SB ostium stenosis and flow, kissing balloon inflation could be done. Kissing inflation involves rewiring of the SB from the distal strut at SB ostium, and getting a balloon through the stent struts could be the most challenging part of bifurcation treatment. After rewiring, two NC balloons are simultaneously inflated in MB and SB. full stent expansion at both ostia; sometimes, final POT should be considered. After POT and kissing, if there is a need for SB stenting because of low flow or SB dissection, there are basically few strategies—T stenting, TAP (T stenting and protrusion), culotte, and crush techniques. In all two-stent techniques, kissing balloon dilation is mandatory to achieve T-stenting strategy is chosen when there is scaffolding at the SB ostium from the POT and when SB angulation is nearly 90°. If SB is not covered by POT technique and the angle is acute, crush (minicrush), culotte, and TAP should be considered as they all cover SB ostium with struts. In crush (minicrush), the stent in SB is placed overlapping the MB and is crushed with oversized The dedicated bifurcation stents are produced to tackle most of these problems. 2. Anatomy, physiology, and conventional stent treatment plied, its viability, and left ventricular function. dural myocardial infarction [1]. 62 Angiography One of them is the dedicated bifurcational stents, Tryton and Tryton SHORT stents. The delivery system of the stent is based on rapid-exchange catheter with single-wire tracking (no risk of wire wrap or bias); there is no need for rotational orientation. The stent has low profile and is 5-Fr guide compatible with two central markers delineating central transition zone used to precisely position the stent (Figures 1–3). The deployment sequence of the stent consists of wiring the side branch and predilatation. Then, Tryton is positioned and is deployed from main branch to the side branch, after which the main branch is wired and a DES is positioned 1 mm proximal to the Tryton proximal edge. After deploying the stent in the main branch, rewiring of side branch and kissing is performed. There are several drawbacks of Tryton system. It is a bare metal stent with consistently <5% TLR in all clinical studies in over 1.800 pts. It requires adequate proximal landing zone and accurate positioning, but the learning curve is short. It is not indicated for <2.25 mm vessels and is tied to two-stent strategy as intention to treat. There are several randomized studies as far as this stent is concerned. TRYTON randomized 704 patients with bifurcation coronary lesions at 58 centers (30 from Europe and 28 from the United States). At 9 months, TVF was 4.6% higher in the bifurcation stent group compared with the provisional group (p = 0.11). TVF was mainly presented by higher rates of periprocedural myocardial infarction rate (13.6 vs. 10.1%). The SB in-segment diameter stenosis was lower in the bifurcation stent group compared with the provisional group (31.6 vs. 38.6%), with no difference in binary restenosis rates (diameter stenosis ≥50%) at 9 months follow-up (22.6 vs. 26.8%) [2]. After TRYTON study, a subanalysis of it examines the benefit of the Tryton compared with provisional stenting in treatment of complex bifurcation lesions involving large SB. Among the 704 patients enrolled in the TRYTON trial, 289 patients (143 provisional and 146 Tryton stent) Dedicated Bifurcation Stents 65 http://dx.doi.org/10.5772/intechopen.82573 Figure 5. QCA outcomes in TRYTON subanalysis (Genereux et al. Catheter Cardiovasc Interv. 2015). Figure 4. TRYTON subanalysis results. Figure 1. Tryton structure. It is cobalt-chromium balloon expandable stent. The strut thickness is 85 mkm and the total length of the stent is 19 mm. The transition zone can flare and rotate in order to accommodate the atomic variations of the side branch ostium and scaffolds like a main vessel stent at the ostium. Figure 2. Tryton and Tryton SHORT stents. The proximal part of the main branch zone was designed to accommodate larger expansion, and the side branch zone is with additional links in order to increase radial force of the stent. The SHORT stent has 3 mm shorter landing zone, and the position of central markers is optimized for large vessels and has improved delivery system. Figure 3. Tryton deployment scheme. The stent is positioned and deployed after predilatation (secures and protects side branch). The next pictures show treatment of main vessel with approved DES through main vessel portion of Tryton. The final step is kissing balloon postdilatation to ensure complete lesion and ostium coverage. Source: Genereux [3]. After TRYTON study, a subanalysis of it examines the benefit of the Tryton compared with provisional stenting in treatment of complex bifurcation lesions involving large SB. Among the 704 patients enrolled in the TRYTON trial, 289 patients (143 provisional and 146 Tryton stent) Figure 4. TRYTON subanalysis results. Figure 3. Tryton deployment scheme. The stent is positioned and deployed after predilatation (secures and protects side branch). The next pictures show treatment of main vessel with approved DES through main vessel portion of Tryton. The final step is kissing balloon postdilatation to ensure complete lesion and ostium coverage. Source: Genereux [3]. Figure 2. Tryton and Tryton SHORT stents. The proximal part of the main branch zone was designed to accommodate larger expansion, and the side branch zone is with additional links in order to increase radial force of the stent. The SHORT stent has 3 mm shorter landing zone, and the position of central markers is optimized for large vessels and has Figure 1. Tryton structure. It is cobalt-chromium balloon expandable stent. The strut thickness is 85 mkm and the total length of the stent is 19 mm. The transition zone can flare and rotate in order to accommodate the atomic variations of the side branch ostium and scaffolds like a main vessel stent at the ostium. improved delivery system. 64 Angiography Figure 5. QCA outcomes in TRYTON subanalysis (Genereux et al. Catheter Cardiovasc Interv. 2015). The goal of stent placement is to cover the proximal lesion segment as well as the ostium of the side branch and distal patent vessel without compromising access to the side branch. It is accomplished if two markers - in 1 branch and 1 is in the other. Axxess provides convenient Dedicated Bifurcation Stents 67 http://dx.doi.org/10.5772/intechopen.82573 Stent implantation—the AXXESS stent is advanced so it is astride the carina and is pushed further forward as far as it will go. Implantation steps: a wire is placed in each branch; the stent is advanced to the most angulated branch, and the distal stent markers are aligned with the carina; the self-expanding distal struts partially expand as the sheath is partially withdrawn after which the partially expanded stent in the main branch is advanced to cover the There is a growing body of literature that supports the use of the AXXESS system in the treatment of coronary bifurcation lesions. The first-in-man AXXESS Plus trial reported results at 6 months in 139 patients who underwent implantation across 13 centers, with low rates of TLR (7.5%) and late-lumen loss (0.09 mm). There was a low rate of periprocedural complications (MACE rate 5% (n = 7), non-Q wave MI 4.3% (n = 6)), with a late-stent thrombosis rate of 2.1% (three patients, two The following DIVERGE Trial was a prospective, single-arm, multicenter trial. Any type of bifurcation lesion was included with significant SB larger than 2.25 mm and PV-SB angulation About 302 patients in 16 clinical sites in Europe, Australia, and New Zealand were included. Clinical follow-up at the first, sixth, ninth month, and after that yearly up to fifth year was completed. First endpoint was MACE at 9 months, with secondary end points and 12 months and 2-, 3-, 4- and 5-year death, cardiac death, MI-Q and non-Q, TLR, TVR, stent thrombosis at 30 days, 6, of whom associated with premature cessation of antiplatelet therapy) [6]. placement marker for additional distal stents (Figure 6). bifurcation (Figure 7). 4.1. Clinical studies <70° (Figure 8). Figure 6. Axxess structure. Source: Rizik et al. [5]. Table 1. Procedural and 30-day follow-up. had an SB ≥2.25 mm. The primary endpoint of TVF was numerically lower in the Tryton group compared with the provisional group (11.3% vs. 15.6%, P = 0.38). No difference among the rates of clinically driven target vessel revascularization or cardiac death was seen. In-segment percent diameter stenosis of the SB was significantly lower (10.2%) in the Tryton group compared with the provisional group. In conclusion, TRYTON trial cohort of SB ≥2.25 mm supports the safety and efficacy of the Tryton SB stent compared with a provisional stenting strategy in the treatment of bifurcation lesions involving large SBs [4] (Figures 4 and 5). TRYTON confirmatory study rationale was to prospectively confirm the safety (periprocedural MI) of the Tryton dedicated bifurcation stent in the treatment of true bifurcation lesions involving large side branches (>2.25 mm by QCA analysis). The angiographic inclusion criteria were the same. The study included 28 investigational centers with 12-month enrolment. Procedural and 30-day follow-up are given in Table 1. #### 4. AXXESS stent Another bifurcation stent is AXXESS. Axxess is self-expanding nickel-titanium alloy; it has conical shape and proximal and distal gold markers, facilitating implantation. The device is Biolimus A9 coated using a bioabsorbable polymer matrix in a dose of 22 mg/mm of stent length. The strut thickness is 0.15 mm and the drug release rate is 70% in 30 days, remaining 30% released in <6 months with polymer absorption 6–9 months. There are three models of the stent—11 and 14 mm in length with reference diameter 2.75– 3.25 mm. The maximal proximal and distal stent diameters are 3.75 and 6 mm, respectively. For reference diameter 3.25–3.75, the maximal proximal and distal stent diameters are 4.25 and 6.5 mm. The delivery system is covered sheath, that is, rapid-exchange delivery catheter. It is 5 Fr of higher compatible. The goal of stent placement is to cover the proximal lesion segment as well as the ostium of the side branch and distal patent vessel without compromising access to the side branch. It is accomplished if two markers - in 1 branch and 1 is in the other. Axxess provides convenient placement marker for additional distal stents (Figure 6). Stent implantation—the AXXESS stent is advanced so it is astride the carina and is pushed further forward as far as it will go. Implantation steps: a wire is placed in each branch; the stent is advanced to the most angulated branch, and the distal stent markers are aligned with the carina; the self-expanding distal struts partially expand as the sheath is partially withdrawn after which the partially expanded stent in the main branch is advanced to cover the bifurcation (Figure 7). #### 4.1. Clinical studies had an SB ≥2.25 mm. The primary endpoint of TVF was numerically lower in the Tryton group compared with the provisional group (11.3% vs. 15.6%, P = 0.38). No difference among the rates of clinically driven target vessel revascularization or cardiac death was seen. In-segment percent diameter stenosis of the SB was significantly lower (10.2%) in the Tryton group compared with the provisional group. In conclusion, TRYTON trial cohort of SB ≥2.25 mm supports the safety and efficacy of the Tryton SB stent compared with a provisional stenting strategy in the TRYTON N = 146 Provisional N = 143 TRYTON confirmatory study rationale was to prospectively confirm the safety (periprocedural MI) of the Tryton dedicated bifurcation stent in the treatment of true bifurcation lesions involving large side branches (>2.25 mm by QCA analysis). The angiographic inclusion criteria were the same. The study included 28 investigational centers with 12-month enrolment. Axxess is self-expanding nickel-titanium alloy; it has conical shape and proximal and distal gold markers, facilitating implantation. The device is Biolimus A9 coated using a bioabsorbable polymer matrix in a dose of 22 mg/mm of stent length. The strut thickness is 0.15 mm and the drug release rate is 70% in 30 days, remaining 30% released in <6 months with polymer There are three models of the stent—11 and 14 mm in length with reference diameter 2.75– 3.25 mm. The maximal proximal and distal stent diameters are 3.75 and 6 mm, respectively. For reference diameter 3.25–3.75, the maximal proximal and distal stent diameters are 4.25 and 6.5 mm. The delivery system is covered sheath, that is, rapid-exchange delivery catheter. treatment of bifurcation lesions involving large SBs [4] (Figures 4 and 5). Confirmatory study Randomized trial > = 2.25mm Procedural 0% (0.0) 0% (0.0) 0% (0.0) 30-day 0% (0.0) 0% (0.0) 0% (0.0) Procedural (3xCKMB) 10.5% (14/133) 9.2% (13/141) 12.1% (17/141) Procedural (5× CKMB) 4.5% (7/133) 3.4% (4/118) 6.8% (7/103) 30-day 10.8% (14/130) 8.2% (12/146) 11.9% (17/143) Stent thrombosis 0% (0.0) 0.7% ()1/146 0.0% (0/143) N = 133 Endpoints % TRYTON Table 1. Procedural and 30-day follow-up. Death 66 Angiography Myocardial infarction Procedural and 30-day follow-up are given in Table 1. 4. AXXESS stent absorption 6–9 months. It is 5 Fr of higher compatible. Another bifurcation stent is AXXESS. There is a growing body of literature that supports the use of the AXXESS system in the treatment of coronary bifurcation lesions. The first-in-man AXXESS Plus trial reported results at 6 months in 139 patients who underwent implantation across 13 centers, with low rates of TLR (7.5%) and late-lumen loss (0.09 mm). There was a low rate of periprocedural complications (MACE rate 5% (n = 7), non-Q wave MI 4.3% (n = 6)), with a late-stent thrombosis rate of 2.1% (three patients, two of whom associated with premature cessation of antiplatelet therapy) [6]. The following DIVERGE Trial was a prospective, single-arm, multicenter trial. Any type of bifurcation lesion was included with significant SB larger than 2.25 mm and PV-SB angulation <70° (Figure 8). About 302 patients in 16 clinical sites in Europe, Australia, and New Zealand were included. Clinical follow-up at the first, sixth, ninth month, and after that yearly up to fifth year was completed. First endpoint was MACE at 9 months, with secondary end points and 12 months and 2-, 3-, 4- and 5-year death, cardiac death, MI-Q and non-Q, TLR, TVR, stent thrombosis at 30 days, 6, Figure 6. Axxess structure. Source: Rizik et al. [5]. Figure 7. Axxess deployment sequence. Nine-month QCA results are presented in Table 2 [7, 8]. At follow-up Parent vessel Late loss (mm) In-stent LL (Axxess only) 0.18 ± 0.49 - Restenosis per vessel In-stent LL (Axxess only) 0.7% - In-lesion restenosis (all stents + edges) Overall bifurcation restenosis In-stent PV+SB 5% (7/140) N = 140 3.6% 4.3% In-stent LL (all stents) 0.29 ± 0.50 0.29 ± 0.45 In-lesion LL 0.20 ± 0.41 0.17 ± 0.34 In-stent (Cypher) 2.3% 4.8% In-stent or edges PV+SB 6.4% (9/140) Side branch N = 140 Dedicated Bifurcation Stents 69 http://dx.doi.org/10.5772/intechopen.82573 9 – month QCA results Table 2. QCA results. Figure 9. COBRA trial results at 9 months. Another study evaluating Axxess in bifurcation treatment is COBRA [8]. It compares Axxess + DES versus culotte with EES. OCT stent and lumen area at ninth month are compared. The percentage of uncovered struts in each bifurcation segment at 9 months (primary endpoint) was similar between groups. Five-year clinical follow-up was available for all patients and included major adverse cardiac events [MACE; a composite of cardiac death, myocardial infarction (MI) and ischemia-driven target lesion revascularization (TLR)], target-vessel (TVR) and non-target-vessel revascularization (non-TVR), non-TLR, and stent thrombosis. At 9, and 12 months and 2, 3, 4, and 5 years. The angiographic secondary end points were in-stent restenosis and late loss at 9 months. DAPT for 12 months was recommended. About 77.4% of all patients were with true bifurcations with predominating Medina 1:1:1. Axxess only was used in 12.3%; Axxess with PV stenting in 17.7%, Axxess with SB stenting in 4%, and stenting of the two branches was present in 64.7%. In general, side branch stent was used in 68.7%. Table 2. QCA results. #### Nine-month QCA results are presented in Table 2 [7, 8]. Another study evaluating Axxess in bifurcation treatment is COBRA [8]. It compares Axxess + DES versus culotte with EES. OCT stent and lumen area at ninth month are compared. The percentage of uncovered struts in each bifurcation segment at 9 months (primary endpoint) was similar between groups. Five-year clinical follow-up was available for all patients and included major adverse cardiac events [MACE; a composite of cardiac death, myocardial infarction (MI) and ischemia-driven target lesion revascularization (TLR)], target-vessel (TVR) and non-target-vessel revascularization (non-TVR), non-TLR, and stent thrombosis. At Figure 9. COBRA trial results at 9 months. Figure 8. DIVERGE trial: 5-year outcomes. Figure 7. Axxess deployment sequence. 68 Angiography used in 68.7%. 9, and 12 months and 2, 3, 4, and 5 years. The angiographic secondary end points were in-stent restenosis and late loss at 9 months. DAPT for 12 months was recommended. About 77.4% of Axxess only was used in 12.3%; Axxess with PV stenting in 17.7%, Axxess with SB stenting in 4%, and stenting of the two branches was present in 64.7%. In general, side branch stent was all patients were with true bifurcations with predominating Medina 1:1:1. 5 years, in the culotte group, one patient had undergone TLR and another suffered a clinical MI, resulting in 10% MACE versus none in the Axxess group. TVR (5 vs. 10%, P = 0.54) and non-TVR (5 vs. 20%, P = 0.39) rates were similar between the Axxess and culotte groups, respectively. There was no stent thrombosis (Figure 9). In conclusion, Axxess is a self-expanding coated device with positive results in bifurcation lesions, including complex morphology. It has relatively high profile, and frequently multiple stents are used in cases of double stenting. There is some proof or larger luminal gain in the carina segment. Prospective data support its use in complex coronary bifurcations; however, its use has yet to be studied in a large-scale randomized controlled trial. #### 5. BiOSS stent The BiOSS stent (Balton, Warsaw, Poland)—Bifurcation Optimisation Stent System was created and firstly used in 2008. It has three generations. At first, it started as bare metal BiOSS stent, then drug-eluting stents—BiOSS Expert in 2010; then in 2012, BiOSS LIM was introduced. Recently, in 2018, the last generation BiOSS LIM C was tested in man. #### 5.1. The stent design All generations of BiOSS stent have the same primary design. As it is delivered by one 0.014 guidewire through rapid-exchange system with five French guiding catheter, it makes BIOSS no different than the other conventional stents. The difference comes with the strut and balloon design. drug that is eluted paclitaxel in BiOSS Expert and sirolimus in LIM and LIM C as it was shown Dedicated Bifurcation Stents 71 http://dx.doi.org/10.5772/intechopen.82573 The stent is made of two parts—proximal and distal with two small connecting struts at the middle zone (0.9–1.5 mm in length). The proximal part of the stent has a larger diameter than the distal one—the ratio is 1.15–1.3. Proximal diameters vary from 3.25 to 4.5 mm and the distal ones from 2.5 to 3.75; the stent length is 15, 18, and 23 mm. The proximal part is always a bit shorter than the distal one (average 1 mm). So, for instance, the smallest stent is 2.5 × 3.25 mm in diameter—distal and proximal part and the largest one is 3.75 × 4.5 mm, and there are four varieties between them with 0.5–0.75 difference in proximal and distal diameters (2.75 × 3.5 mm; 3.0 × 3.5 mm; 3 × 3.75 mm; 3.5 × 4.25 mm). So with three different lengths, it makes a total of 18 combinations. The stent is delivered on a bottle-shaped balloon (Bottle, Balton, Poland), allowing bigger proximal and smaller distal diameter when inflated. It is semicomplaint balloon with nominal pressure of 10 atm and burst pressure of 18 atm (Figures 11 and 12). The stent has three markers: two at both ends and one in the middle. The markers at both ends are like every other stent—they show proximal and distal ends of the device. The midmarker shows the proximal end of the distal (smaller) part of the stent—it should be positioned exactly at the carina as shown in Figure 13. So this placement helps to keep the natural anatomic proportions (bigger proximal part, smaller distal) and keep same carina confirmation—it has no carina displacement because the lateral force from proximal stretching counterbalances with the medial force from distal stretching. The other important design virtue is that there are no struts at the opening of the side branch. that sirolimus decreased the rates of MACE and TLR [9, 10]. Figure 10. Visualization of Murray's law. 5.2. How the stent design of BiOSS comes to tackle these problems? We know from the Murray's law (see Figure 10) of bifurcations that there is difference between main vessel and side branch diameters. In plain words, the bigger the side branch, the bigger the difference of proximal and distal part of the main vessel. When we plan to stent a bifurcation, we should choose a stent that has the diameter of the distal part of the main vessel, and after implantation, we do a proximal optimization—to dilate a bigger balloon at the proximal part (above carina) to keep the stent conformation as the natural anatomic structure. In case we choose stent size 1:1 in diameter to the proximal part of the main vessel it will overstretch the distal part thus moving the carina and plaque to the orifice of the side branch and compromising the blood flow. Secondly, the conventional stents, even with open cell design, will have struts at the side branch orifice. Main difference between those different generation of stents are the material (BiOSS Expert and LIM being the same design stainless steel 140-μm struts and BiOSS LIM C made from cobalt-chromium alloy 70-μm struts) and different drug (first-generation BiOSS Expert eluted paclitaxel and the next BiOSS LIM and LIM C eluted sirolimus). So there is huge difference between them as 140 μm may provoke arterial wall injury and lead neointimal proliferation and thinner struts may facilitate endothelialization [8]. The other important difference is the Figure 10. Visualization of Murray's law. 5 years, in the culotte group, one patient had undergone TLR and another suffered a clinical MI, resulting in 10% MACE versus none in the Axxess group. TVR (5 vs. 10%, P = 0.54) and non-TVR (5 vs. 20%, P = 0.39) rates were similar between the Axxess and culotte groups, In conclusion, Axxess is a self-expanding coated device with positive results in bifurcation lesions, including complex morphology. It has relatively high profile, and frequently multiple stents are used in cases of double stenting. There is some proof or larger luminal gain in the carina segment. Prospective data support its use in complex coronary bifurcations; however, The BiOSS stent (Balton, Warsaw, Poland)—Bifurcation Optimisation Stent System was created and firstly used in 2008. It has three generations. At first, it started as bare metal BiOSS stent, then drug-eluting stents—BiOSS Expert in 2010; then in 2012, BiOSS LIM was intro- All generations of BiOSS stent have the same primary design. As it is delivered by one 0.014 guidewire through rapid-exchange system with five French guiding catheter, it makes BIOSS no different than the other conventional stents. The difference comes with the strut and bal- We know from the Murray's law (see Figure 10) of bifurcations that there is difference between main vessel and side branch diameters. In plain words, the bigger the side branch, the bigger the difference of proximal and distal part of the main vessel. When we plan to stent a bifurcation, we should choose a stent that has the diameter of the distal part of the main vessel, and after implantation, we do a proximal optimization—to dilate a bigger balloon at the proximal part (above carina) to keep the stent conformation as the natural In case we choose stent size 1:1 in diameter to the proximal part of the main vessel it will overstretch the distal part thus moving the carina and plaque to the orifice of the side branch and compromising the blood flow. Secondly, the conventional stents, even with open cell Main difference between those different generation of stents are the material (BiOSS Expert and LIM being the same design stainless steel 140-μm struts and BiOSS LIM C made from cobalt-chromium alloy 70-μm struts) and different drug (first-generation BiOSS Expert eluted paclitaxel and the next BiOSS LIM and LIM C eluted sirolimus). So there is huge difference between them as 140 μm may provoke arterial wall injury and lead neointimal proliferation and thinner struts may facilitate endothelialization [8]. The other important difference is the respectively. There was no stent thrombosis (Figure 9). 5. BiOSS stent 70 Angiography 5.1. The stent design anatomic structure. design, will have struts at the side branch orifice. loon design. its use has yet to be studied in a large-scale randomized controlled trial. duced. Recently, in 2018, the last generation BiOSS LIM C was tested in man. drug that is eluted paclitaxel in BiOSS Expert and sirolimus in LIM and LIM C as it was shown that sirolimus decreased the rates of MACE and TLR [9, 10]. #### 5.2. How the stent design of BiOSS comes to tackle these problems? The stent is made of two parts—proximal and distal with two small connecting struts at the middle zone (0.9–1.5 mm in length). The proximal part of the stent has a larger diameter than the distal one—the ratio is 1.15–1.3. Proximal diameters vary from 3.25 to 4.5 mm and the distal ones from 2.5 to 3.75; the stent length is 15, 18, and 23 mm. The proximal part is always a bit shorter than the distal one (average 1 mm). So, for instance, the smallest stent is 2.5 × 3.25 mm in diameter—distal and proximal part and the largest one is 3.75 × 4.5 mm, and there are four varieties between them with 0.5–0.75 difference in proximal and distal diameters (2.75 × 3.5 mm; 3.0 × 3.5 mm; 3 × 3.75 mm; 3.5 × 4.25 mm). So with three different lengths, it makes a total of 18 combinations. The stent is delivered on a bottle-shaped balloon (Bottle, Balton, Poland), allowing bigger proximal and smaller distal diameter when inflated. It is semicomplaint balloon with nominal pressure of 10 atm and burst pressure of 18 atm (Figures 11 and 12). The stent has three markers: two at both ends and one in the middle. The markers at both ends are like every other stent—they show proximal and distal ends of the device. The midmarker shows the proximal end of the distal (smaller) part of the stent—it should be positioned exactly at the carina as shown in Figure 13. So this placement helps to keep the natural anatomic proportions (bigger proximal part, smaller distal) and keep same carina confirmation—it has no carina displacement because the lateral force from proximal stretching counterbalances with the medial force from distal stretching. The other important design virtue is that there are no struts at the opening of the side branch. Figure 11. (A) Expanded stent; (B) stent balloon only; (C) stent system not expended; (D) expanded stent system. Source: Gil et al. [11]. 5.3. What are the results when comparing BiOSS stent versus conventional DES in drug-eluting stents—POLBOS I (2010–2013) and POLBOS II (2012–2013). They have published results at 12 and 48 months of follow-up. Figure 13. Visualization of stent positioning. Source: Gil et al. [12]. Two randomized multicenter trials compared BiOSS Expert and LIM versus conventional Dedicated Bifurcation Stents 73 http://dx.doi.org/10.5772/intechopen.82573 POLBOS I (n = 243) study compared BiOSS Expert (paclitaxel) stent versus conventional DES paclitaxel, sirolimus, everolimus, zotarolimus, and tacrolimus stents (LucChopin2, Xience, Promus, Cypher, Prolim, Orsiro, Biomime, Biomatrix, Resolute Integrity, and Optima). POT balloons were advised but left to operator discretion. Kissing was left to operator discretion in BiOSS group, and in DES group, there was second randomization for kissing balloons. At 12 months of follow up, the cumulative incidence of MACE was similar in both groups 13.3% in BiOSS vs. 12.2% in DES. There were also no significant differences in cardiac-related death (0 in BiOSS vs. 1.6% in DES), myocardial infarctions (1.6% in BiOSS vs. 3.2% in DES), and stent thrombosis (0.8% in BiOSS vs. 0 in DES). Target vessel revascularizations were significantly higher in BiOSS group 15.8% vs. 9.7% in DES group. Target lesion revasculariza- POLBOS II (n = 202) compared the next-generation BiOSS LIM (sirolimus) stent vs. regular DES (same as above). POT and kissing were left to operator discretion in BiOSS group and POT to operator discretion but kissing randomized in DES group. At 12 months, there were no significant differences in MACE (11.8% in BiOSS vs. 15% in DES), death (1% in BiOSS vs. 3% DES), MI (1.9 vs. 3%), TLR (9.8% in BiOSS vs. 9% in DES), and TVR (13.7% in BiOSS vs. tions were also significantly higher in BiOSS group 11.5% vs. 7.3% in DES group. treating bifurcation lesions? Figure 12. BIOSS Lim and BIOSS Lim C stents. Source: Gil et al. [12]. An IVUS study showed that BiOSS stent provides better access to the SB in comparison with the conventional drug-eluting stents. There was significantly bigger orifice length found in the BiOSS group—a parameter, which represents the access to the SB [13]. The analysis of the plaque, lumen, and vessel areas shows that BiOSS stent design spares the proximal optimization technique (POT), which is strongly recommended by the European Bifurcation Club [14]. But this imposes the question whether no struts at the carina could predispose to restenosis. This issue was addressed in the IVUS study and showed that the carina actually had the least neointimal burden during follow-up [15]. This study also showed that sirolimus was better than paclitaxel in rates of neointimal hyperplasia. Figure 13. Visualization of stent positioning. Source: Gil et al. [12]. An IVUS study showed that BiOSS stent provides better access to the SB in comparison with the conventional drug-eluting stents. There was significantly bigger orifice length found in the BiOSS group—a parameter, which represents the access to the SB [13]. The analysis of the plaque, lumen, and vessel areas shows that BiOSS stent design spares the proximal optimization technique (POT), which is strongly recommended by the European Bifurcation Club [14]. But this imposes the question whether no struts at the carina could predispose to restenosis. This issue was addressed in the IVUS study and showed that the carina actually had the least neointimal burden during follow-up [15]. This study also showed that sirolimus was better Figure 11. (A) Expanded stent; (B) stent balloon only; (C) stent system not expended; (D) expanded stent system. Source: than paclitaxel in rates of neointimal hyperplasia. Figure 12. BIOSS Lim and BIOSS Lim C stents. Source: Gil et al. [12]. Gil et al. [11]. 72 Angiography #### 5.3. What are the results when comparing BiOSS stent versus conventional DES in treating bifurcation lesions? Two randomized multicenter trials compared BiOSS Expert and LIM versus conventional drug-eluting stents—POLBOS I (2010–2013) and POLBOS II (2012–2013). They have published results at 12 and 48 months of follow-up. POLBOS I (n = 243) study compared BiOSS Expert (paclitaxel) stent versus conventional DES paclitaxel, sirolimus, everolimus, zotarolimus, and tacrolimus stents (LucChopin2, Xience, Promus, Cypher, Prolim, Orsiro, Biomime, Biomatrix, Resolute Integrity, and Optima). POT balloons were advised but left to operator discretion. Kissing was left to operator discretion in BiOSS group, and in DES group, there was second randomization for kissing balloons. At 12 months of follow up, the cumulative incidence of MACE was similar in both groups 13.3% in BiOSS vs. 12.2% in DES. There were also no significant differences in cardiac-related death (0 in BiOSS vs. 1.6% in DES), myocardial infarctions (1.6% in BiOSS vs. 3.2% in DES), and stent thrombosis (0.8% in BiOSS vs. 0 in DES). Target vessel revascularizations were significantly higher in BiOSS group 15.8% vs. 9.7% in DES group. Target lesion revascularizations were also significantly higher in BiOSS group 11.5% vs. 7.3% in DES group. POLBOS II (n = 202) compared the next-generation BiOSS LIM (sirolimus) stent vs. regular DES (same as above). POT and kissing were left to operator discretion in BiOSS group and POT to operator discretion but kissing randomized in DES group. At 12 months, there were no significant differences in MACE (11.8% in BiOSS vs. 15% in DES), death (1% in BiOSS vs. 3% DES), MI (1.9 vs. 3%), TLR (9.8% in BiOSS vs. 9% in DES), and TVR (13.7% in BiOSS vs. 12% in DES). Final kissing was associated with significantly less TLR only in BiOSS group (5.9 vs. 11.8%, p < 0.05), but POT technique was associated with less TLR in both groups (in BiOSS 5.3% vs. 12.5% p < 0.05 and in DES group 2.9% vs. 25% p < 0.01). Implanting the stent beyond the carina will compromise the side branch ostium; in the other case, implanting the stent too much proximal to the carina will not have these adverse effects Dedicated Bifurcation Stents 75 http://dx.doi.org/10.5772/intechopen.82573 In conclusion, BiOSS stent should be considered a good choice for treating bifurcation lesions, Minvasys offers one of the current stent solutions for bifurcation stenting. First approved stent from Nile family is Nile CroCo, which takes CE brand in 2005. Nile CroCo presents the basic Minvasys concept of bifurcation stenting. Future generations of dedicated stents are Nile CroCo stent is based on cobalt-chromium bare metal platform. The stent consists of three segments. Proximal segment includes 7–9 cells (depending on stent size), the medial segment includes 8–10 cells, and the distal segment includes 6–8 cells (Figure 15). This distribution ensures same metal to artery ratio (between 10.3 and 14.6%) [16] along entire bifurcation, which is important for optimal stent apposition. Stent thickness of Nile CroCo platform is 73 μm. Two stent length are offered—18 and 24 mm; three diameters for main branch balloon (2.5, 3.0, and 3.5 mm) and three diameters for side branch balloon (2.0, 2.5, and 3.0 mm) are offered. There are seven stents with different MB and SB diameters (as follows: 2.5–2.0, 3.0– 2.0, 3.0–2.5, 3.5–2.5, 3.5–3.0, 2.5–2.5, and 3.0–3.0 mm), each of them proposed in two lengths Stent delivery system integrates two monorail balloons: one for the side branch and one for the main branch. It requires 6.0F guiding catheter for stent delivery, and two 0.014-inch guidewires, previously positioned in MB and SB. Both balloons have similar characteristics with Cobalt-chromium stent platform is mounted on main branch balloon. The system also contains side branch catheter tip. Side branch balloon is located proximally from the stent. Both balloon catheters are rapid exchange. In order to prevent entanglement of two balloon shafts, during system delivery, there is autorelease sheath. The system is equipped with five roentgen-positive markers—three of them located on MB balloon—proximal, distal, and medial. Medial marker is located exactly on the bifurcation. There are two additional markers, point- Despite the fact that every dedicated bifurcation stent proposes a unique approach to bifurcation lesion, Nile system allows provisional T-stenting in the A category, according to MADS classification. Provisional stenting strategy is recommended as the first-line approach by nominal pressure 8 atm and rated burst pressure 14 atm (Figure 16). ing proximal and distal parts of SB balloon. many modern consensuses. in the side branch but will leave struts at the carina. intended for one- or two-stent strategy. 6. Nile concept based on Nile CroCo. 6.1. Nile CroCo (18 and 24 mm). When analyzed POLBOS I and II at 48 months, there were no statistical differences in terms of MACE (DES vs. BiOSS: 18.8 vs. 19.8%, p = 0.64), TLR (12.1 vs. 15.3%, p = 0.34), MI (4.5 vs. 2.1%, p = 0.72), or cardiac death (2.2 vs. 1.8%, p = 0.81) between DES and BiOSS groups. #### 5.4. Why one should use BiOSS stent if the long-term results are the same? Implanting BiOSS stent will not spare you the proximal optimization or the kissing balloons if there is a need for such techniques. In theory, it will leave the SB ostium open, thus making it easier to reach with a wire and go through it with a balloon for a kissing. In case you need twostent technique, there will be less struts at the carina and easier to go through with a second stent. So in our opinion, BiOSS stent makes treating of bifurcation lesion simpler and sometimes cheaper (sparing a smaller balloon for opening the struts to the side branch in order to reach with a bigger one for kissing). #### 5.5. What are the pitfalls in implanting BiOSS stent? In our opinion, there are two main issues when implanting BiOSS stent. The first is when you have long lesion that incudes bifurcation. Consider that longest BiOSS is 23 mm in length with approximately 46/50% length in proximal and distal length, so having a longer than 12 mm proximal or distal lesions means that if you are planning to use BiOSS, you will need at least two stents, one regular DES and one BiOSS; in this case, implanting just one long regular DES and doing proximal optimization is more suitable. The second is that when implanting BiOSS stent, it is of crucial matter to find very good projections where carina is seen, because you have to place the middle marker at the ostium of the main branch (Figure 14). Figure 14. Correctly positioned stent (angio). Implanting the stent beyond the carina will compromise the side branch ostium; in the other case, implanting the stent too much proximal to the carina will not have these adverse effects in the side branch but will leave struts at the carina. In conclusion, BiOSS stent should be considered a good choice for treating bifurcation lesions, intended for one- or two-stent strategy. #### 6. Nile concept 12% in DES). Final kissing was associated with significantly less TLR only in BiOSS group (5.9 vs. 11.8%, p < 0.05), but POT technique was associated with less TLR in both groups (in BiOSS When analyzed POLBOS I and II at 48 months, there were no statistical differences in terms of MACE (DES vs. BiOSS: 18.8 vs. 19.8%, p = 0.64), TLR (12.1 vs. 15.3%, p = 0.34), MI (4.5 vs. 2.1%, Implanting BiOSS stent will not spare you the proximal optimization or the kissing balloons if there is a need for such techniques. In theory, it will leave the SB ostium open, thus making it easier to reach with a wire and go through it with a balloon for a kissing. In case you need twostent technique, there will be less struts at the carina and easier to go through with a second stent. So in our opinion, BiOSS stent makes treating of bifurcation lesion simpler and sometimes cheaper (sparing a smaller balloon for opening the struts to the side branch in order to reach In our opinion, there are two main issues when implanting BiOSS stent. The first is when you have long lesion that incudes bifurcation. Consider that longest BiOSS is 23 mm in length with approximately 46/50% length in proximal and distal length, so having a longer than 12 mm proximal or distal lesions means that if you are planning to use BiOSS, you will need at least two stents, one regular DES and one BiOSS; in this case, implanting just one long regular DES The second is that when implanting BiOSS stent, it is of crucial matter to find very good projections where carina is seen, because you have to place the middle marker at the ostium p = 0.72), or cardiac death (2.2 vs. 1.8%, p = 0.81) between DES and BiOSS groups. 5.4. Why one should use BiOSS stent if the long-term results are the same? 5.3% vs. 12.5% p < 0.05 and in DES group 2.9% vs. 25% p < 0.01). with a bigger one for kissing). 74 Angiography of the main branch (Figure 14). Figure 14. Correctly positioned stent (angio). 5.5. What are the pitfalls in implanting BiOSS stent? and doing proximal optimization is more suitable. Minvasys offers one of the current stent solutions for bifurcation stenting. First approved stent from Nile family is Nile CroCo, which takes CE brand in 2005. Nile CroCo presents the basic Minvasys concept of bifurcation stenting. Future generations of dedicated stents are based on Nile CroCo. #### 6.1. Nile CroCo Nile CroCo stent is based on cobalt-chromium bare metal platform. The stent consists of three segments. Proximal segment includes 7–9 cells (depending on stent size), the medial segment includes 8–10 cells, and the distal segment includes 6–8 cells (Figure 15). This distribution ensures same metal to artery ratio (between 10.3 and 14.6%) [16] along entire bifurcation, which is important for optimal stent apposition. Stent thickness of Nile CroCo platform is 73 μm. Two stent length are offered—18 and 24 mm; three diameters for main branch balloon (2.5, 3.0, and 3.5 mm) and three diameters for side branch balloon (2.0, 2.5, and 3.0 mm) are offered. There are seven stents with different MB and SB diameters (as follows: 2.5–2.0, 3.0– 2.0, 3.0–2.5, 3.5–2.5, 3.5–3.0, 2.5–2.5, and 3.0–3.0 mm), each of them proposed in two lengths (18 and 24 mm). Stent delivery system integrates two monorail balloons: one for the side branch and one for the main branch. It requires 6.0F guiding catheter for stent delivery, and two 0.014-inch guidewires, previously positioned in MB and SB. Both balloons have similar characteristics with nominal pressure 8 atm and rated burst pressure 14 atm (Figure 16). Cobalt-chromium stent platform is mounted on main branch balloon. The system also contains side branch catheter tip. Side branch balloon is located proximally from the stent. Both balloon catheters are rapid exchange. In order to prevent entanglement of two balloon shafts, during system delivery, there is autorelease sheath. The system is equipped with five roentgen-positive markers—three of them located on MB balloon—proximal, distal, and medial. Medial marker is located exactly on the bifurcation. There are two additional markers, pointing proximal and distal parts of SB balloon. Despite the fact that every dedicated bifurcation stent proposes a unique approach to bifurcation lesion, Nile system allows provisional T-stenting in the A category, according to MADS classification. Provisional stenting strategy is recommended as the first-line approach by many modern consensuses. Stenting procedure (Figure 17). The stenting procedure begins with placement of two guidewires distally in the main branch and side branch (step 1). Predilatation of MB and SB (if stenosis seems to be significant) is crucial for seamlessly stent delivery. Optimal stent size must be selected according to distal diameters of MB and SB. The system must be introduced on the both rapid-exchange balloon catheters. After that, the system is pushed through the guiding catheter. During the advancement, autorelease sheath must be released. Two monorail balloon catheters are advanced until distal part of the stent crosses through stenosis in the main branch. When the midsegment of the stent is positioned on the carina, and the tip of SB balloon catheter is in the proximal part of the SB (step 2), the system will stop and the operator will feel resistance. At this moment, the middle roentgen positive marker must be exactly at the level of carina (shown with a narrow at step 2). In this way, the operator is maximally facilitated in optimal positioning the stent because it can use guidewires as well as the radiopaque marker and tactile sensation. In about 40% of cases, twisting of guidewires may occur. This can be easily solved by retrieving the system in the guiding catheter and repositioning one of the guidewires. After optimal stent positioning, the MB balloon is inflated, providing stent implantation in the main branch (step 3). After this step, the medium "strutless" part of the stent is positioned exactly at the level of bifurcation. Next step is advancement of the SB balloon catheter into SB ostium (step 4). This can be achieved easily, because of two facts. First of all, the SB guidewire is not jailed, so there is no need to recross wires. Furthermore, there is a "defect" in the struts, at the level of carina that allows balloon advancement. The next step (step 5) is kissing, using both balloons, integrated in the system. Because of the special SB balloon shape, overextension of the proximal segment of the bifurcation is avoided. After the removal of delivery system (step 6), both guidewires stay positioned into main branch and side branch. This made implantation of additional stent (only if necessary) very easy. This concept of stent implantation is preserved in next two generations of Nile stents—Nile PAX and Nile SIR. Bruno Garcia Del Blanco et al. reported the results of a series of 151 patients treated successfully by implantation of Nile CroCo stent [18]. Success in stent positioning and implantation was reported in 99% of patients. Radial approach was used in 75% of patients. In more than 80% of patients, 6F introducer and guiding catheter was enough. Significant lesion at the ostium of the SB was reported in 73% of patients. A kissing inflation with the system was performed in almost all (95%). In 19% of cases, additional stent was implanted in MB, while in 10% of cases, additional stent was implanted in SB. Among 138 patients with a complete follow-up at 6 months, the MACE rate was 14% and the TLR was 7.2%, despite the fact that the patient profile being relatively severe, including 55 patients with myocardial infarction <24 h. Guidewire twisting—as mentioned earlier—requiring repositioning was noted in 33% Dedicated Bifurcation Stents 77 http://dx.doi.org/10.5772/intechopen.82573 of cases. Figure 16. Scheme of delivery system. Source: [17]. Figure 17. Stenting procedure step-by-step. Source: [17]. Figure 15. Nile CroCo structure. Source: [17]. Figure 16. Scheme of delivery system. Source: [17]. Stenting procedure (Figure 17). The stenting procedure begins with placement of two guidewires distally in the main branch and side branch (step 1). Predilatation of MB and SB (if stenosis seems to be significant) is crucial for seamlessly stent delivery. Optimal stent size must be selected according to distal diameters of MB and SB. The system must be introduced on the both rapid-exchange balloon catheters. After that, the system is pushed through the guiding catheter. During the advancement, autorelease sheath must be released. Two monorail balloon catheters are advanced until distal part of the stent crosses through stenosis in the main branch. When the midsegment of the stent is positioned on the carina, and the tip of SB balloon catheter is in the proximal part of the SB (step 2), the system will stop and the operator will feel resistance. At this moment, the middle roentgen positive marker must be exactly at the level of carina (shown with a narrow at step 2). In this way, the operator is maximally facilitated in optimal positioning the stent because it can use guidewires as well as the radiopaque marker and tactile In about 40% of cases, twisting of guidewires may occur. This can be easily solved by retriev- After optimal stent positioning, the MB balloon is inflated, providing stent implantation in the main branch (step 3). After this step, the medium "strutless" part of the stent is positioned exactly at the level of bifurcation. Next step is advancement of the SB balloon catheter into SB ostium (step 4). This can be achieved easily, because of two facts. First of all, the SB guidewire is not jailed, so there is no need to recross wires. Furthermore, there is a "defect" in the struts, at the level of carina that allows balloon advancement. The next step (step 5) is kissing, using both balloons, integrated in the system. Because of the special SB balloon shape, overextension of the proximal segment of the bifurcation is avoided. After the removal of delivery system (step 6), both guidewires stay positioned into main branch and side branch. This made This concept of stent implantation is preserved in next two generations of Nile stents—Nile ing the system in the guiding catheter and repositioning one of the guidewires. implantation of additional stent (only if necessary) very easy. sensation. 76 Angiography PAX and Nile SIR. Figure 15. Nile CroCo structure. Source: [17]. Figure 17. Stenting procedure step-by-step. Source: [17]. Bruno Garcia Del Blanco et al. reported the results of a series of 151 patients treated successfully by implantation of Nile CroCo stent [18]. Success in stent positioning and implantation was reported in 99% of patients. Radial approach was used in 75% of patients. In more than 80% of patients, 6F introducer and guiding catheter was enough. Significant lesion at the ostium of the SB was reported in 73% of patients. A kissing inflation with the system was performed in almost all (95%). In 19% of cases, additional stent was implanted in MB, while in 10% of cases, additional stent was implanted in SB. Among 138 patients with a complete follow-up at 6 months, the MACE rate was 14% and the TLR was 7.2%, despite the fact that the patient profile being relatively severe, including 55 patients with myocardial infarction <24 h. Guidewire twisting—as mentioned earlier—requiring repositioning was noted in 33% of cases. #### 6.2. Nile PAX In 2009, next-generation (Figure 18) bifurcation stent gets CE brand approval. Stent structure, stent sizes, and delivery system are the same as in Nile CroCo. The stent was made by same cobalt-chromium alloy with similar metal to artery ratio (between 10 and 15%). Strut thickness is 73 μm, but in Nile PAX, there are additional 5-μm polymer-free coatings with paclitaxel only on the abluminal surface in crystallized form. Drug concentration is 2.5 μg/mm2 , and based on manufacturer's data, whole drug is released 45 days after stent implantation. Stent delivery system and the steps of stent implantation are actually the same as described for Nile CroCo stents. Because of that, we will repeat the steps, using our real clinical case for visualization. We present 68-year-old female with stable angina pectoris and bifurcation LM stenosis (Medina 0:1:1) (Figure 19). We placed two 0.014 inch guidewires in MB and SB and made predilatation of MB with NC balloon 2.0/20 mm (please note that LCx is chosen for MB). Stent positioning with central marker at the level of carina is shown in Figure 20. The balloon for the SB postdilatation may be seen in guiding catheter on this step. After angiographic verification of optimal stent apposition, MB balloon was inflated (Figure 21). Final kissing was made, using both balloons incorporated in system, and the final result is visualized in Figure 22. Figure 19. Stenosis before the beginning of the procedure. Source: Owned by Acibadem City Clinic. Dedicated Bifurcation Stents 79 http://dx.doi.org/10.5772/intechopen.82573 Figure 20. Stent positioning. Source: Owned by Acibadem City Clinic. Figure 18. Nile PAX stent. Source: Owned by Acibadem City Clinic. Figure 19. Stenosis before the beginning of the procedure. Source: Owned by Acibadem City Clinic. 6.2. Nile PAX 78 Angiography 2.5 μg/mm2 implantation. visualization. (Medina 0:1:1) (Figure 19). visualized in Figure 22. marker at the level of carina is shown in Figure 20. Figure 18. Nile PAX stent. Source: Owned by Acibadem City Clinic. In 2009, next-generation (Figure 18) bifurcation stent gets CE brand approval. Stent structure, stent sizes, and delivery system are the same as in Nile CroCo. The stent was made by same cobalt-chromium alloy with similar metal to artery ratio (between 10 and 15%). Strut thickness is 73 μm, but in Nile PAX, there are additional 5-μm polymer-free coatings with paclitaxel only on the abluminal surface in crystallized form. Drug concentration is Stent delivery system and the steps of stent implantation are actually the same as described for Nile CroCo stents. Because of that, we will repeat the steps, using our real clinical case for We present 68-year-old female with stable angina pectoris and bifurcation LM stenosis We placed two 0.014 inch guidewires in MB and SB and made predilatation of MB with NC balloon 2.0/20 mm (please note that LCx is chosen for MB). Stent positioning with central The balloon for the SB postdilatation may be seen in guiding catheter on this step. After angiographic verification of optimal stent apposition, MB balloon was inflated (Figure 21). Final kissing was made, using both balloons incorporated in system, and the final result is , and based on manufacturer's data, whole drug is released 45 days after stent Figure 20. Stent positioning. Source: Owned by Acibadem City Clinic. In 2012, results from a big trial with Nile PAX stent were published [19]. The trial is multicenter, including 101 patients from Europe and Brazil. Our team is part of this study. Procedural success and 30-day follow-up data of the paclitaxel-eluting version (Nile PAX) were presented. In 80% of cases, the LAD-diagonal branch bifurcation was the target lesion with 62% also having an SB lesion. In total, 102 lesions were treated, of which 62% had a stenosis on each of the main or side branches. Procedural success was achieved in 97% of cases. An additional stent was implanted in the SB in 25% of cases. Longer-term (9-month) clinical follow-up data of this study were presented at EuroPCR 2011 (by Bruno Garcia). The rate of restenosis was 13.9% in the MB, 12.8% in the SB, and a total of 18.6% in the bifurcation, which led to a TLR of 12.6% In order to reduce the rate of SB restenosis (12.8%) found in the Nile PAX trial, a new Nile PAX study with systematic use of a paclitaxel-eluting balloon (Danubio; Minvasys) in the SB at the Last generation of Minvasys Nile family is Nile SIR dedicated bifurcation stent. It is based on the same conception as Nile CroCo and Nile PAX but covered by sirolimus as antiproliferative drug. Sirolimus is deposited with biodegradable polymer on abluminal stent surface with The active drug is deposed on abluminal stent surface on two layers (Figure 23). First layer (A) contains sirolimus, poly-L lactic acid, poly(lactic-co-glycolic) acid, and polyvinylpyrrolidone. It is located between stent strut and layer B. Layer B is water soluble, contains polyvinylpyr- Between 2013 and 2014 in India, 37 patients with symptomatic bifurcation stenosis were treated by implantation of Nile SIR and followed up. Most of the patients had LAD-DIAGONAL stenosis (74%), and 26% had LCx-OM stenosis. Stable angina at admission was reported in 64.9%, and the rest was with unstable angina pectoris. There were no procedural complications or adverse cardiac events till discharge. In 27% of patients, there was additional stent implanted at the MB level, implantation, and complete reversion to BMS is expected within 180 days. . About 85% of the drug is expected to be eluted within 48 days after Dedicated Bifurcation Stents 81 http://dx.doi.org/10.5772/intechopen.82573 with only one myocardial infarction reported. rolidone, and had only protective function. 6.3. Nile SIR concentration 1.4 μg/mm2 Figure 23. Drug deposal on the stent. end of the procedure shows only 2% rate of restenosis. Figure 21. Inflation of MB balloon. Source: Owned by Acibadem City Clinic. Figure 22. Final result. Source: [20]. In 2012, results from a big trial with Nile PAX stent were published [19]. The trial is multicenter, including 101 patients from Europe and Brazil. Our team is part of this study. Procedural success and 30-day follow-up data of the paclitaxel-eluting version (Nile PAX) were presented. In 80% of cases, the LAD-diagonal branch bifurcation was the target lesion with 62% also having an SB lesion. In total, 102 lesions were treated, of which 62% had a stenosis on each of the main or side branches. Procedural success was achieved in 97% of cases. An additional stent was implanted in the SB in 25% of cases. Longer-term (9-month) clinical follow-up data of this study were presented at EuroPCR 2011 (by Bruno Garcia). The rate of restenosis was 13.9% in the MB, 12.8% in the SB, and a total of 18.6% in the bifurcation, which led to a TLR of 12.6% with only one myocardial infarction reported. In order to reduce the rate of SB restenosis (12.8%) found in the Nile PAX trial, a new Nile PAX study with systematic use of a paclitaxel-eluting balloon (Danubio; Minvasys) in the SB at the end of the procedure shows only 2% rate of restenosis. #### 6.3. Nile SIR Figure 21. Inflation of MB balloon. Source: Owned by Acibadem City Clinic. Figure 22. Final result. Source: [20]. 80 Angiography Last generation of Minvasys Nile family is Nile SIR dedicated bifurcation stent. It is based on the same conception as Nile CroCo and Nile PAX but covered by sirolimus as antiproliferative drug. Sirolimus is deposited with biodegradable polymer on abluminal stent surface with concentration 1.4 μg/mm2 . About 85% of the drug is expected to be eluted within 48 days after implantation, and complete reversion to BMS is expected within 180 days. The active drug is deposed on abluminal stent surface on two layers (Figure 23). First layer (A) contains sirolimus, poly-L lactic acid, poly(lactic-co-glycolic) acid, and polyvinylpyrrolidone. It is located between stent strut and layer B. Layer B is water soluble, contains polyvinylpyrrolidone, and had only protective function. Between 2013 and 2014 in India, 37 patients with symptomatic bifurcation stenosis were treated by implantation of Nile SIR and followed up. Most of the patients had LAD-DIAGONAL stenosis (74%), and 26% had LCx-OM stenosis. Stable angina at admission was reported in 64.9%, and the rest was with unstable angina pectoris. There were no procedural complications or adverse cardiac events till discharge. In 27% of patients, there was additional stent implanted at the MB level, Figure 23. Drug deposal on the stent. and 3% of patients had additional stent implanted in SB. During 6-month clinical follow-up, there was no MACE reported. Three of the patients were died from noncardiac reason. Angiographic follow-up was done in 15 of these patients. In-stent late-lumen loss was reported as follows: on the proximal MB level 0.15 ± 0.15, on the distal MB level 0.16 ± 0.29, and on the SB level 0.26 ± 0.41. [5] Rizik DG, Klassen KJ, Hermiller JB. Bifurcation coronary artery disease: Current techniques and future directions (Part 2). The Journal of invasive cardiology. 2008;20(3):135-141 Dedicated Bifurcation Stents 83 http://dx.doi.org/10.5772/intechopen.82573 [6] Rawlins J, Din J, Talwar S, O'Kane P. AXXESS™ stent: Delivery indications and outcomes. Interventional Cardiology. 2015;10(2):85-89. DOI: 10.15420/icr.2015.10.2.85 [7] Verheye S, Agostoni P, Dubois CL, et al. 9-month clinical, angiographic, and intravascular ultrasound results of a prospective evaluation of the Axxess self-expanding biolimus A9-eluting stent in coronary bifurcation lesions: The DIVERGE (drug-eluting stent intervention for treating side branches effectively) study. Journal of the American College of [8] Pache J, Kastrati A, Mehilli J, et al. Intracoronary stenting and angiographic results: Strut thickness effect on restenosis outcome (ISAR-STEREO-2) trial. Journal of the American [9] Kong J, Liu P, Fan X, et al. Long-term outcomes of paclitaxel-eluting versus sirolimuseluting stent for percutaneous coronary intervention: A meta-analysis. Journal of the [10] Minvasys Website. Available from: http://www.minvasys.com/fr/ bifurcation-stents/ [12] Gil RJ, Bil J, Kern A, Pawłowski T. First-in-man study of dedicated bifurcation cobaltchromium sirolimus-eluting stent BiOSS LIM C®—Three-month results. Kardiologia [13] Gil RJ, Michalek A, Bil J. Comparative analysis of lumen enlargement mechanisms achieved with bifurcation dedicated stent BiOSS® and classical coronary stent implantations by means of provisional side branch stenting strategy—The intravascular ultrasound study. The International Journal of Cardiovascular Imaging. 2013;29(8):1667-1676. [14] Lassen JF, Holm NR, Banning A, Burzotta F, Lefèvre T, Chieffo A, et al. Percutaneous coronary intervention for coronary bifurcation disease: 11th consensus document from the European Bifurcation Club. EuroIntervention. 2016;12(1):38-46. DOI: 10.4244/ [15] Gil RJ, Bil J, Costa RA, Gil KE, Vassiliev D. 12-month intravascular ultrasound observations from BiOSS® first-in-man studies. The International Journal of Cardiovascular [16] Ughi GJ, Verjans J, Fard AM. Dual modality intravascular optical coherence tomography (OCT) and near-infrared fluorescence (NIRF) imaging: A fully automated algorithm for the distance-calibration of NIRF signal intensity for quantitative molecular imaging. The Imaging. 2016;32(9):1339-1347. DOI: 10.1007/s10554-016-0926-9 International Journal of Cardiovascular Imaging. 2015;31(2):259-268 College of Physicians and Surgeons–Pakistan. 2017;27(7):432-439. DOI: 2659 [11] Gil RG, Bil J, Vassilev D. The BiOSS stent. EuroIntervention. 2015;11:V153-V154 Cardiology. 2009;53(12):1031-1039. DOI: 10.1016/j.jacc.2008.12.012 College of Cardiology. 2003;41(8):1283-1288 nile-croco.php [Accessed: December 14, 2018] DOI: 10.1007/s10554-013-0264-0 EIJV12I1A7 Polska. 2018;76(2):464-470. DOI: 10.5603/KP.a2017.0226 In our center, 42 patients with bifurcation LM stenosis were treated in the past 2 years. About 52.94% of patients had stable angina and 47.06% with acute MI (including one patient with STEMI). Based on the Medina classification, bifurcation lesions were type 1:1:1 in 33.82%, type 1:0:1 in 20.59%, and type 0:1:1 in 11.76% patients. Procedural success was achieved in all patients. According to center protocol, clinical follow-up was done on the first month. Control angiography with intravascular ultrasound was done on the sixth month. Twenty-four (57.14%) patients were followed up angiographically. We observed two MACE (4.76%)—one patient died before FU and one in-stent restenosis at the SB level. The measures of the late-lumen loss were 0.22 ± 0.38, 0.29 ± 0.34, and 0.18 ± 0.31 mm for the main branch proximal, distal, and side branch, respectively. #### Conflict of interest There is no conflict of interest to declare. ### Author details Ivo Petrov, Iveta Tasheva\, Jivka Stoykova, Liubomir Dosev, Zoran Stankov and Petar Polomski \Address all correspondence to: iveta\[email protected] Acibadem City Clinic Cardiovascular Center, Sofia, Bulgaria #### References [5] Rizik DG, Klassen KJ, Hermiller JB. Bifurcation coronary artery disease: Current techniques and future directions (Part 2). The Journal of invasive cardiology. 2008;20(3):135-141 and 3% of patients had additional stent implanted in SB. During 6-month clinical follow-up, there was no MACE reported. Three of the patients were died from noncardiac reason. Angiographic follow-up was done in 15 of these patients. In-stent late-lumen loss was reported as follows: on the proximal MB level 0.15 ± 0.15, on the distal MB level 0.16 ± 0.29, and on the SB level 0.26 ± 0.41. In our center, 42 patients with bifurcation LM stenosis were treated in the past 2 years. About 52.94% of patients had stable angina and 47.06% with acute MI (including one patient with STEMI). Based on the Medina classification, bifurcation lesions were type 1:1:1 in 33.82%, type 1:0:1 in 20.59%, and type 0:1:1 in 11.76% patients. Procedural success was achieved in all patients. According to center protocol, clinical follow-up was done on the first month. Control angiography with intravascular ultrasound was done on the sixth month. Twenty-four (57.14%) patients were followed up angiographically. We observed two MACE (4.76%)—one patient died before FU and one in-stent restenosis at the SB level. The measures of the late-lumen loss were 0.22 ± 0.38, 0.29 ± 0.34, and 0.18 ± 0.31 mm for the main branch proximal, distal, and side branch, respectively. Conflict of interest 82 Angiography Author details and Petar Polomski References ccd.26240 There is no conflict of interest to declare. \Address all correspondence to: iveta\[email protected] the American College of Cardiology Acibadem City Clinic Cardiovascular Center, Sofia, Bulgaria Ivo Petrov, Iveta Tasheva\, Jivka Stoykova, Liubomir Dosev, Zoran Stankov [1] Arora RR et al. Catheterization and Cardiovascular Diagnosis. 1989;18:210-212 [3] Genereux P. TRYTON Pivotal: Randomized Trial and Confirmatory Study [2] Généreux P, Kumsars I, Lesiak M. A randomized trial of a dedicated bifurcation stent versus provisional stenting in the treatment of coronary bifurcation lesions. Journal of [4] Généreux P, Kini A, Lesiak M. Outcomes of a dedicated stent in coronary bifurcations with large side branches: A subanalysis of the randomized TRYTON bifurcation study. Catheterization and Cardiovascular Interventions. 2016;87(7):1231-1241. DOI: 10.1002/ Section 3 Angiography for Peripheral Arterial Diseases Angiography for Peripheral Arterial Diseases [17] http://www.minvasys.com/fr/bifurcation-stents/nile-pax.php trial. EuroIntervention. 2012;7:1301-1309 [20] https://www.minvasys.com/nile-sir-technology.php Book; 2011. p. H45 84 Angiography [18] Garcia del Blanco G, Bellera Gotarda N, Martí G, et al. Clinical and procedural evaluation of the Nile Croco dedicated stent for bifurcation. Results of one center initial experience with the first 151 consecutive non-selected patients. EuroIntervention. Abstract [19] Costa RA, Abizaid A, Abizaid AS, Garcia del Blanco B, et al. BIPAX investigators. Procedural and early clinical outcomes of patients with de novo coronary bifurcation lesions treated with the novel Nile PAX dedicated bifurcation polymer-free paclitaxel coated stents: Results from the prospective, multicentre, non-randomised BIPAX clinical Chapter 6 Provisional chapter Angiography for Renal Artery Diseases Angiography for Renal Artery Diseases DOI: 10.5772/intechopen.79232 Renal Artery disease is one of the main causes of systemic arterial hypertension. Among its etiologies are atherosclerosis, fibromuscular dysplasia, Takayasu arteritis, among others. These diseases may evolve into stenosis, occlusion or aneurysms of the renal arteries. In the last decades, technological advances in both imaging diagnosis and treatment, have improved prognosis of patients, leading to earlier medical interventions. For the identification of renal vascular diseases, the adequate angiography technique as well as the capture of quality images are of utter importance. This chapter aims to address the main Keywords: angiography, renal, atherosclerosis, nephropathy, fibromuscular dysplasia, renal aneurysm, Takayasu arteritis, kidney transplantation, diagnostic catheter The correct identification of renal vascular diseases by means of imaging techniques, among them the renal angiography, enables physicians to perform an appropriate morphological study and an efficient treatment [1]. Currently, new diagnostic and therapeutic methods have been developed and employed in the treatment of patients with renal vascular disease [2–4]. The present chapter is divided into three sections. The first section introduces the renal vascular anatomy, anatomical variations, renal angiography and its techniques. The second section examines the renal vascular diseases and their imaging findings. The third section presents aspects of the renal artery diseases and their arteriographic findings. other methods for the diagnosis of the renal vascular disease. © 2016 The Author(s). Licensee InTech. 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. © 2019 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. Daniel Emilio Dalledone Siqueira and Daniel Emilio Dalledone Siqueira and http://dx.doi.org/10.5772/intechopen.79232 Additional information is available at the end of the chapter Additional information is available at the end of the chapter Ana Terezinha Guillaumon Ana Terezinha Guillaumon Abstract 1. Introduction #### Angiography for Renal Artery Diseases Angiography for Renal Artery Diseases Daniel Emilio Dalledone Siqueira and Ana Terezinha Guillaumon Daniel Emilio Dalledone Siqueira and Ana Terezinha Guillaumon Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.79232 #### Abstract Renal Artery disease is one of the main causes of systemic arterial hypertension. Among its etiologies are atherosclerosis, fibromuscular dysplasia, Takayasu arteritis, among others. These diseases may evolve into stenosis, occlusion or aneurysms of the renal arteries. In the last decades, technological advances in both imaging diagnosis and treatment, have improved prognosis of patients, leading to earlier medical interventions. For the identification of renal vascular diseases, the adequate angiography technique as well as the capture of quality images are of utter importance. This chapter aims to address the main aspects of the renal artery diseases and their arteriographic findings. DOI: 10.5772/intechopen.79232 Keywords: angiography, renal, atherosclerosis, nephropathy, fibromuscular dysplasia, renal aneurysm, Takayasu arteritis, kidney transplantation, diagnostic catheter #### 1. Introduction The correct identification of renal vascular diseases by means of imaging techniques, among them the renal angiography, enables physicians to perform an appropriate morphological study and an efficient treatment [1]. Currently, new diagnostic and therapeutic methods have been developed and employed in the treatment of patients with renal vascular disease [2–4]. The present chapter is divided into three sections. The first section introduces the renal vascular anatomy, anatomical variations, renal angiography and its techniques. The second section examines the renal vascular diseases and their imaging findings. The third section presents other methods for the diagnosis of the renal vascular disease. © 2016 The Author(s). Licensee InTech. 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. © 2019 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. ## 2. Renal angiographic morfology #### 2.1. Renal angiography Renal angiography, despite the technological advances of the imaging methods, is still considered the final diagnostic method of the renal artery diseases. This method provides an anatomical visibility of the renal arteries when correct techniques are applied. Moreover, treatment decisions are made based on the lesion morphological aspects at arteriography. Another relevant aspect is that arteriography can provide images for immediate diagnostic and endovascular treatment during the procedure. A correct optimization of the arteriographic images provides an adequate assessment of the renal arteries, as well as of their segmental and subsegmental branches. Some technical details must be considered for the correct visibility of the renal parenchyma images. The side holes of the catheter must be positioned at the L1–L2 level. The correct positioning is necessary to prevent the contrast agent from filling the superior mesenteric artery, Angiography for Renal Artery Diseases http://dx.doi.org/10.5772/intechopen.79232 89 In some cases, depending on the quality of the images captured during aortography, it is necessary to perform selective catheterization of the renal arteries in order to better visualize the ostium and the distal branches, without the overlapping of the aorta and the branches. Selective catheterization must be performed with a preformed catheter such as Cobra, Renal Double Curved (RDC), Omni Selective, Simmons, among others. The renal intravenous contrast may be administered manually or with an injection pump. Volume of contrast agent is Renal selective catheterization presents advantages in relation to aortography, as a smaller amount of contrast material is necessary to capture renal vessel images. Additionally, it offers the possibility to carry out a hemodynamic study of the stenosis by measuring the blood pressure before and after the lesion. Pressure gradient values cannot be obtained with other Renal angiography may be performed by means of films or digital subtraction. In film-based angiography, X-ray images are recorded after the intravenous contrast injection. On the other hand, in digital subtraction angiography (DSA), the fluoroscopic images are produced in a sequence by means of an image intensifier (Figure 2). The captured images are electronically filed in digital format. The initial image, produced before contrast medium injection, is subtracted, generating a mask. All subsequent images are subtracted from this mask image, and Figure 2. Hemodynamic room – School of Medical Sciences at Universidade Estadual de Campinas – UNICAMP. causing overlapping of images. 2.3. Selective catheterization of renal arteries usually 3–6 mL/s, administered for 1–2 minutes. 2.4. Registration methods of the angiographic images non-invasive diagnostic methods. only the contrast medium is visualized. #### 2.2. The renal angiography technique Arterial access is obtained by inserting a 5F or 6F sheath into the femoral or brachial artery, using the modified Seldinger technique [5]. Ultrasound-guided arterial puncture can also be performed. A 5F catheter is positioned inside the aorta using the femoral or brachial artery access. Renal arteriography must start with the aortography, which aims to identify ostial stenosis of the renal arteries (Figure 1). Contrast material is injected manually or by means of an automatic injection pump, providing the sequence of images. The contrast volume, infusion speed, as well as the acquisition characteristics of the images, depend on each patient. Usual parameters include a 10–20 mL/K injection of contrast agent for 2 seconds. Images are usually registered for 3–5 seconds, about 1–2 images per second. However, this procedure may be extended for 3–5 additional seconds, depending on the images of interest, providing assessment of the intra-parenchymatous and venous arterial phases. Figure 1. Aortography showing – (1) abdominal aorta; (2) superior mesenteric artery; (3) right renal artery, with atherosclerotic ostial stenosis; (4) left renal artery. Some technical details must be considered for the correct visibility of the renal parenchyma images. The side holes of the catheter must be positioned at the L1–L2 level. The correct positioning is necessary to prevent the contrast agent from filling the superior mesenteric artery, causing overlapping of images. #### 2.3. Selective catheterization of renal arteries 2. Renal angiographic morfology mental and subsegmental branches. 2.2. The renal angiography technique atherosclerotic ostial stenosis; (4) left renal artery. Renal angiography, despite the technological advances of the imaging methods, is still considered the final diagnostic method of the renal artery diseases. This method provides an anatomical visibility of the renal arteries when correct techniques are applied. Moreover, treatment decisions are made based on the lesion morphological aspects at arteriography. Another relevant aspect is that arteriography can provide images for immediate diagnostic and endovascular treatment during the procedure. A correct optimization of the arteriographic images provides an adequate assessment of the renal arteries, as well as of their seg- Arterial access is obtained by inserting a 5F or 6F sheath into the femoral or brachial artery, using the modified Seldinger technique [5]. Ultrasound-guided arterial puncture can also be performed. A 5F catheter is positioned inside the aorta using the femoral or brachial artery access. Renal arteriography must start with the aortography, which aims to identify ostial stenosis of the renal arteries (Figure 1). Contrast material is injected manually or by means of an automatic injection pump, providing the sequence of images. The contrast volume, infusion speed, as well as the acquisition characteristics of the images, depend on each patient. Usual parameters include a 10–20 mL/K injection of contrast agent for 2 seconds. Images are usually registered for 3–5 seconds, about 1–2 images per second. However, this procedure may be extended for 3–5 additional seconds, depending on the images of interest, providing Figure 1. Aortography showing – (1) abdominal aorta; (2) superior mesenteric artery; (3) right renal artery, with assessment of the intra-parenchymatous and venous arterial phases. 2.1. Renal angiography 88 Angiography In some cases, depending on the quality of the images captured during aortography, it is necessary to perform selective catheterization of the renal arteries in order to better visualize the ostium and the distal branches, without the overlapping of the aorta and the branches. Selective catheterization must be performed with a preformed catheter such as Cobra, Renal Double Curved (RDC), Omni Selective, Simmons, among others. The renal intravenous contrast may be administered manually or with an injection pump. Volume of contrast agent is usually 3–6 mL/s, administered for 1–2 minutes. Renal selective catheterization presents advantages in relation to aortography, as a smaller amount of contrast material is necessary to capture renal vessel images. Additionally, it offers the possibility to carry out a hemodynamic study of the stenosis by measuring the blood pressure before and after the lesion. Pressure gradient values cannot be obtained with other non-invasive diagnostic methods. #### 2.4. Registration methods of the angiographic images Renal angiography may be performed by means of films or digital subtraction. In film-based angiography, X-ray images are recorded after the intravenous contrast injection. On the other hand, in digital subtraction angiography (DSA), the fluoroscopic images are produced in a sequence by means of an image intensifier (Figure 2). The captured images are electronically filed in digital format. The initial image, produced before contrast medium injection, is subtracted, generating a mask. All subsequent images are subtracted from this mask image, and only the contrast medium is visualized. Figure 2. Hemodynamic room – School of Medical Sciences at Universidade Estadual de Campinas – UNICAMP. #### 2.5. Normal anatomy The kidneys are a pair of retroperitoneal organs, located parallel to the spinal column, on the psoas major muscle, generally between L1 and L4 vertebrae level, in an orthostatic position. There may be a change in the position of both kidneys during the respiratory cycle, that is, during the inhalation and exhalation processes. This breathing variation may represent from 1 to 7 cm in the cranial-caudal position. In the supine position, the kidneys generally lie at the T12-L13 spinal segment level. The right kidney may be found slightly more caudal than the left kidney, due to the liver location. Both kidneys represent 0.4% of the total body weight and are approximately 11–13 cm long, the left kidney slightly longer than the right one. three collateral arterial systems, capsular, peripelvic and periureteral, which are supplied by lumbar arteries, abdominal aorta, internal iliac arteries, inferior adrenal arteries and other vessels [13]. In renal vein occlusion, blood flow is provided through ureteric, gonadal, adrenal, ascending Angiography for Renal Artery Diseases http://dx.doi.org/10.5772/intechopen.79232 91 Stenosis of the renal arteries may be defined as a multifactorial disease, of several etiologies, which affects the renal artery vasculature, at unilateral or bilateral level, determining various stenosis degrees, from its origin to the renal hilum [1, 14]. Clinically, it is presented as a renovascular hypertension and a renal ischemic disease and is associated with a higher cardiovascular risk and an increase in the mortality rate [15–18]. For research and treatment purposes, several The natural history of the renal atherosclerotic disease is still to be fully clarified. However, it is known that there is a progressive stenosis, associated with a decrease in the arterial flow, leading to an ultimate kidney failure, directly related to the stenosis degree of the renal artery [20, 21]. It is estimated that renovascular disease induces kidney failure in 5–15% of dialysis Prevalence of renal artery stenosis is controversial, as few population-based studies have been conducted to relate the disease to race, age and gender. Nevertheless, some research studies have demonstrated that renal artery stenosis affects 1–5% of patients with systemic arterial hypertension, representing the main cause of the secondary hypertension [1]. The disease is believed to account for 1% of the cases of mild to moderate hypertension and for 10–40% of the acute, severe and refractory hypertension cases [3]. Additionally, some population-based studies suggest that prevalence of the disease in patients older than 65 years of age is higher than 7% [1]. Random autopsy studies conducted in patients whose death was caused by other etiologies reveal that 4–50% of these patients presented renal arterial stenosis, 40% with no Renal artery stenosis may present several etiologies, among them atherosclerosis, fibromus- Atherosclerotic renal artery occlusion is more frequent and accounts for 70–80% of the cases. It is more prevalent in men, over 40 years of age, induces stenosis in the proximal segments of the renal arteries and is characterized by the presence of stenotic lesions at the proximal third of the renal arteries [25]. Morphologically, a renal artery atherosclerotic disease resembles eccentric atherosclerotic plaques, evolving towards the arterial lumen, with no precise aortic boundaries (Figure 3). It may affect the renal arteries at unilateral or bilateral levels, as well as the polar renal arteries, when they are present. Progression of the disease is observed in 50% of the cases, which may lead to bilateral stenosis, arterial occlusion, with or without renal infarction [20, 21]. authors suggest that renal artery stenosis is critical when it is greater than 60–70% [19]. lumbar and capsular veins [13]. 3.1. Renal occlusive disease patients every year [22]. history of a systemic arterial hypertension [23]. 3.1.1. Renal occlusive disease: atherosclerosis cular dysplasia (FMD) and Takayasu's arteritis (TAK) [24]. 3. Renal diseases and arteriographic morphology Kidneys are related with the suprarenal gland, located above them, encapsulated by the renal fascia. Posteriorly situated are the diaphragm muscle, psoas major and quadratus lumborum muscles, the branches of the lumbar plexus, the 12 costal arch and the lateral edge of the erector spinae muscle. Anteriorly, they are covered by the peritoneum, the right kidney related to the posterior border of the liver and small intestine, whereas the left kidney is related to the stomach, spleen and small intestine. At the medial border of the kidneys is a vertical fissure, called renal hilus, through which the renal vessels, nerves and ureter pass. Each kidney is structurally divided into cortex and medulla. Renal arteries branch off the aorta, at the lateral side, right below the superior mesenteric artery, next to the L1–L2 vertebrae level [6, 7]. The right renal artery passes behind the inferior cava vein. Renal arteries are single in approximately 65% of the population and multiple in 35%. They are subdivided into segmental, interlobar and arcuate arteries. Venous drainage takes place in the renal vessels, located anteriorly to the ipsilateral renal artery. Both veins drain into the inferior cava vein. Left renal vein is longer and receives tributary veins such as left gonadal vein, suprarenal vein, phrenic vein, among others. Right renal vein, on the other hand, is shorter, and generally does not receive tributaries. #### 2.6. Anatomical variations Accessory renal arteries may be present in one or in both kidneys, corresponding to 25–35% of the general population [8]. Most accessory renal arteries supply the inferior pole of the kidney, arising from the suprarenal aorta to iliac arteries. Kidney anatomical alterations, such as ectopia, fusion and rotation, are associated with vascular alterations related to variations in origin and the number of renal arteries. In the horseshoe kidney, arterial irrigation is generally provided by three or more arteries, arising from the aorta or the iliac, or from both [9, 10]. Alterations in renal veins are usual. It is possible to observe the circumaortic left renal vein with retroaortic and pre-aortic segments, draining into the inferior cava vein. Additionally, multiple renal veins to the right, the retroaortic left renal vein and the right gonadal vein are observed, draining into the right renal vein [11, 12]. #### 2.7. Renal collateral circulation Renal arteries, despite being described as terminal branches of the abdominal aorta, present systems of collateralization, in cases of stenosis or occlusion. Intra-renal blood flow is provided by three collateral arterial systems, capsular, peripelvic and periureteral, which are supplied by lumbar arteries, abdominal aorta, internal iliac arteries, inferior adrenal arteries and other vessels [13]. In renal vein occlusion, blood flow is provided through ureteric, gonadal, adrenal, ascending lumbar and capsular veins [13]. #### 3. Renal diseases and arteriographic morphology #### 3.1. Renal occlusive disease 2.5. Normal anatomy 90 Angiography stomach, spleen and small intestine. 2.6. Anatomical variations 2.7. Renal collateral circulation observed, draining into the right renal vein [11, 12]. The kidneys are a pair of retroperitoneal organs, located parallel to the spinal column, on the psoas major muscle, generally between L1 and L4 vertebrae level, in an orthostatic position. There may be a change in the position of both kidneys during the respiratory cycle, that is, during the inhalation and exhalation processes. This breathing variation may represent from 1 to 7 cm in the cranial-caudal position. In the supine position, the kidneys generally lie at the T12-L13 spinal segment level. The right kidney may be found slightly more caudal than the left kidney, due to the liver location. Both kidneys represent 0.4% of the total body weight and Kidneys are related with the suprarenal gland, located above them, encapsulated by the renal fascia. Posteriorly situated are the diaphragm muscle, psoas major and quadratus lumborum muscles, the branches of the lumbar plexus, the 12 costal arch and the lateral edge of the erector spinae muscle. Anteriorly, they are covered by the peritoneum, the right kidney related to the posterior border of the liver and small intestine, whereas the left kidney is related to the At the medial border of the kidneys is a vertical fissure, called renal hilus, through which the renal vessels, nerves and ureter pass. Each kidney is structurally divided into cortex and medulla. Renal arteries branch off the aorta, at the lateral side, right below the superior mesenteric artery, next to the L1–L2 vertebrae level [6, 7]. The right renal artery passes behind the inferior cava vein. Renal arteries are single in approximately 65% of the population and multiple in Venous drainage takes place in the renal vessels, located anteriorly to the ipsilateral renal artery. Both veins drain into the inferior cava vein. Left renal vein is longer and receives tributary veins such as left gonadal vein, suprarenal vein, phrenic vein, among others. Right renal Accessory renal arteries may be present in one or in both kidneys, corresponding to 25–35% of the general population [8]. Most accessory renal arteries supply the inferior pole of the kidney, arising from the suprarenal aorta to iliac arteries. Kidney anatomical alterations, such as ectopia, fusion and rotation, are associated with vascular alterations related to variations in origin and the number of renal arteries. In the horseshoe kidney, arterial irrigation is generally provided by three or more arteries, arising from the aorta or the iliac, or from both [9, 10]. Alterations in renal veins are usual. It is possible to observe the circumaortic left renal vein with retroaortic and pre-aortic segments, draining into the inferior cava vein. Additionally, multiple renal veins to the right, the retroaortic left renal vein and the right gonadal vein are Renal arteries, despite being described as terminal branches of the abdominal aorta, present systems of collateralization, in cases of stenosis or occlusion. Intra-renal blood flow is provided by 35%. They are subdivided into segmental, interlobar and arcuate arteries. vein, on the other hand, is shorter, and generally does not receive tributaries. are approximately 11–13 cm long, the left kidney slightly longer than the right one. Stenosis of the renal arteries may be defined as a multifactorial disease, of several etiologies, which affects the renal artery vasculature, at unilateral or bilateral level, determining various stenosis degrees, from its origin to the renal hilum [1, 14]. Clinically, it is presented as a renovascular hypertension and a renal ischemic disease and is associated with a higher cardiovascular risk and an increase in the mortality rate [15–18]. For research and treatment purposes, several authors suggest that renal artery stenosis is critical when it is greater than 60–70% [19]. The natural history of the renal atherosclerotic disease is still to be fully clarified. However, it is known that there is a progressive stenosis, associated with a decrease in the arterial flow, leading to an ultimate kidney failure, directly related to the stenosis degree of the renal artery [20, 21]. It is estimated that renovascular disease induces kidney failure in 5–15% of dialysis patients every year [22]. Prevalence of renal artery stenosis is controversial, as few population-based studies have been conducted to relate the disease to race, age and gender. Nevertheless, some research studies have demonstrated that renal artery stenosis affects 1–5% of patients with systemic arterial hypertension, representing the main cause of the secondary hypertension [1]. The disease is believed to account for 1% of the cases of mild to moderate hypertension and for 10–40% of the acute, severe and refractory hypertension cases [3]. Additionally, some population-based studies suggest that prevalence of the disease in patients older than 65 years of age is higher than 7% [1]. Random autopsy studies conducted in patients whose death was caused by other etiologies reveal that 4–50% of these patients presented renal arterial stenosis, 40% with no history of a systemic arterial hypertension [23]. Renal artery stenosis may present several etiologies, among them atherosclerosis, fibromuscular dysplasia (FMD) and Takayasu's arteritis (TAK) [24]. #### 3.1.1. Renal occlusive disease: atherosclerosis Atherosclerotic renal artery occlusion is more frequent and accounts for 70–80% of the cases. It is more prevalent in men, over 40 years of age, induces stenosis in the proximal segments of the renal arteries and is characterized by the presence of stenotic lesions at the proximal third of the renal arteries [25]. Morphologically, a renal artery atherosclerotic disease resembles eccentric atherosclerotic plaques, evolving towards the arterial lumen, with no precise aortic boundaries (Figure 3). It may affect the renal arteries at unilateral or bilateral levels, as well as the polar renal arteries, when they are present. Progression of the disease is observed in 50% of the cases, which may lead to bilateral stenosis, arterial occlusion, with or without renal infarction [20, 21]. Figure 3. Atherosclerotic renal artery stenosis, located at the proximal third of the artery. #### 3.1.2. Renal occlusive disease: fibromuscular dysplasia (FMD) Renal artery stenosis secondary to fibromuscular dysplasia accounts for 20–25% of the cases [26]. Fibromuscular dysplasia is a non-atherosclerotic and a non-inflammatory disease, which affects medium-sized arteries, rarely involving small-sized ones [27]. It is more prevalent in the Caucasian population, 15- to 50-year old women, affecting the more distal segments of the renal arteries and the intra-parenchymal segments. It may affect both renal arteries in 60% of the cases but is rarely observed in patients older than 60. Recent studies reveal that approximately 2% of renovascular hypertension are related to fibromuscular dysplasia [28]. general, the clinical lab findings reveal elevated inflammatory markers, such as, C-reactive protein and hemosedimentation rate. Normocytic and normochromic anemia, and hypoalbuminemia reveal the chronic nature of the disease. Diagnostic criteria, defined by the American School of Rheumatology, include the development of signs and symptoms related to Takayasu's arteritis before the age of 40; claudication of the extremities, weakness; extremities discomfort and fatigue, more commonly in the upper limbs; decrease in the pulse rate amplitude in one or both brachial arteries; difference in the blood pressure of the upper limbs of at least 10 mmHg; murmur in one or both subclavian arteries; abdominal murmur; alterations in the arteriography: narrowing or occlusion of the aorta and /or its main branches or of the large proximal arteries in the lower and upper limbs, not caused by atherosclerosis or fibromuscular dysplasia. Three of the above criteria confirm the diagnosis, sensibility of 90.5% Angiography for Renal Artery Diseases http://dx.doi.org/10.5772/intechopen.79232 93 Figure 4. Morphological aspect of the fibromuscular dysplasia at selective angiography of the renal artery. Renal arteries play an important role in the development of the disease in 50–60% of the cases [30–32]. It is clinically characterized by the renal occlusive disease, inducing renovascular hypertension or kidney failure. Treatment of patients affected by renovascular hypertension has been proven difficult. Generally, arteriography is required to confirm the diagnosis of the disease, characterized by its location in the proximal aorta and its branches (Figure 5). Nevertheless, due to the method limitation, angiography does not provide the identification of the arterial wall thickening. Other imaging methods for clinical investigation, such as com- The renal aneurysmal disease is uncommon and asymptomatic in most cases. Populationbased studies show an incidence of 0.1% of the total population, representing 25% of the visceral aneurysm cases [33]. The actual prevalence is unknown, considering that it is a multifactorial disease and depends on the hereditary aspects of the studied population. Its puted angiotomography or angioresonance, are recommended. and specificity of 97.8%. 3.2. Renal aneurysmal disease Among the fibromuscular dysplasia types, the medial one is the most prevalent, the medial fibroplasia subtype accounting for 70–95% of dysplasia cases and 85% of the renovascular lesions [28]. Lesions predominantly affect the medial and distal third of the renal and polar arteries and their branches [26]. Arteriography may provide a high degree of diagnostic accuracy. The usual angiographic profile of medial fibroplasia resembles a string of bead (Figure 4). However, the gold standard procedure for the diagnosis of fibromuscular dysplasia is the histopathologic exam. The disease may evolve to renal artery stenosis or aneurysms and its diagnosis is usually an exam incidental finding, as fibromuscular dysplasia does not present alterations of inflammatory expressions such as hemosedimentation rate and C-reactive protein [29]. #### 3.1.3. Renal occlusive disease: Takayasu's arteritis (TAK) Takayasu's arteritis is a chronic, inflammatory and granulomatous disease, a vasculitis that affects large and middle-size arteries [30]. It is prevalent in women (80–90-% of the cases), starting between the age of 10 and 40 [31]. Initial symptoms are not specific, and include fever, general feeling of being unwell, weight loss and joint pains. There might be a vascular problem, which at the initial phase involves the thoracic and abdominal aorta and its main branches [32]. The inflammatory process induces the thickening of the arterial wall, leading to stenosis, occlusion or dilation of the affected arterial segments, at several stages [30]. In Figure 4. Morphological aspect of the fibromuscular dysplasia at selective angiography of the renal artery. general, the clinical lab findings reveal elevated inflammatory markers, such as, C-reactive protein and hemosedimentation rate. Normocytic and normochromic anemia, and hypoalbuminemia reveal the chronic nature of the disease. Diagnostic criteria, defined by the American School of Rheumatology, include the development of signs and symptoms related to Takayasu's arteritis before the age of 40; claudication of the extremities, weakness; extremities discomfort and fatigue, more commonly in the upper limbs; decrease in the pulse rate amplitude in one or both brachial arteries; difference in the blood pressure of the upper limbs of at least 10 mmHg; murmur in one or both subclavian arteries; abdominal murmur; alterations in the arteriography: narrowing or occlusion of the aorta and /or its main branches or of the large proximal arteries in the lower and upper limbs, not caused by atherosclerosis or fibromuscular dysplasia. Three of the above criteria confirm the diagnosis, sensibility of 90.5% and specificity of 97.8%. Renal arteries play an important role in the development of the disease in 50–60% of the cases [30–32]. It is clinically characterized by the renal occlusive disease, inducing renovascular hypertension or kidney failure. Treatment of patients affected by renovascular hypertension has been proven difficult. Generally, arteriography is required to confirm the diagnosis of the disease, characterized by its location in the proximal aorta and its branches (Figure 5). Nevertheless, due to the method limitation, angiography does not provide the identification of the arterial wall thickening. Other imaging methods for clinical investigation, such as computed angiotomography or angioresonance, are recommended. #### 3.2. Renal aneurysmal disease 3.1.2. Renal occlusive disease: fibromuscular dysplasia (FMD) Figure 3. Atherosclerotic renal artery stenosis, located at the proximal third of the artery. fibromuscular dysplasia [28]. 92 Angiography arteries and their branches [26]. Renal artery stenosis secondary to fibromuscular dysplasia accounts for 20–25% of the cases [26]. Fibromuscular dysplasia is a non-atherosclerotic and a non-inflammatory disease, which affects medium-sized arteries, rarely involving small-sized ones [27]. It is more prevalent in the Caucasian population, 15- to 50-year old women, affecting the more distal segments of the renal arteries and the intra-parenchymal segments. It may affect both renal arteries in 60% of the cases but is rarely observed in patients older than 60. Recent studies reveal that approximately 2% of renovascular hypertension are related to Among the fibromuscular dysplasia types, the medial one is the most prevalent, the medial fibroplasia subtype accounting for 70–95% of dysplasia cases and 85% of the renovascular lesions [28]. Lesions predominantly affect the medial and distal third of the renal and polar Arteriography may provide a high degree of diagnostic accuracy. The usual angiographic profile of medial fibroplasia resembles a string of bead (Figure 4). However, the gold standard procedure for the diagnosis of fibromuscular dysplasia is the histopathologic exam. The disease may evolve to renal artery stenosis or aneurysms and its diagnosis is usually an exam incidental finding, as fibromuscular dysplasia does not present alterations of inflammatory Takayasu's arteritis is a chronic, inflammatory and granulomatous disease, a vasculitis that affects large and middle-size arteries [30]. It is prevalent in women (80–90-% of the cases), starting between the age of 10 and 40 [31]. Initial symptoms are not specific, and include fever, general feeling of being unwell, weight loss and joint pains. There might be a vascular problem, which at the initial phase involves the thoracic and abdominal aorta and its main branches [32]. The inflammatory process induces the thickening of the arterial wall, leading to stenosis, occlusion or dilation of the affected arterial segments, at several stages [30]. In expressions such as hemosedimentation rate and C-reactive protein [29]. 3.1.3. Renal occlusive disease: Takayasu's arteritis (TAK) The renal aneurysmal disease is uncommon and asymptomatic in most cases. Populationbased studies show an incidence of 0.1% of the total population, representing 25% of the visceral aneurysm cases [33]. The actual prevalence is unknown, considering that it is a multifactorial disease and depends on the hereditary aspects of the studied population. Its Figure 5. Aortography revealing renal artery occlusion induced by Takayasu's arteritis. diagnosis usually represents an incidental finding during regular exams. It is more prevalent in women, in the right renal artery and rarely bilateral [34]. In most cases, they are solitary aneurysms, mostly saccular-shaped (Figure 6), accounting for 75% of the cases, but with some fusiform-shaped aneurysms as well, most often developed in the renal artery bifurcation, 90% of the cases extra-parenchymal and only 10% intra-parenchymal [35]. Among the various etiologies, the most prevalent is fibromuscular dysplasia. Other causes for the development of the renal aneurysmal disease include trauma, congenital diseases (Ehlers-Danlos syndrome and neurofibromatosis), inflammatory diseases, among others [35]. Renal artery aneurysms may be classified according to their location: Type 1 in the main renal artery; Type 2 in the bifurcation and beginning of the segmental branches; Type 3 in the distal: Angiography for Renal Artery Diseases http://dx.doi.org/10.5772/intechopen.79232 95 Figure 6. Selective arteriography of the renal artery revealing a saccular aneurysm. Currently, several endovascular treatment methods have been developed to preserve renal function. Treatment indications reported by Henke et al., exhaustively revised, include: (1) renal artery aneurysms of over 1.0 cm in diameter, with a difficulty-to-control hypertension; (2) all renal artery aneurysms of over 2.0 cm in diameter; (3) most aneurysms of 1.5–2.0 cm in diameter. Some authors consider the possibility to treat all symptomatic renal artery Renal transplant represents a treatment option for the terminal renal disease. In this case, arteriography before and after the transplant provides better and thorough technical Vascular complications are rare in renal transplants; however, they may lead to the loss of the transplanted kidney. Among the most common vascular complications are the renal artery stenosis (Figure 7), renal artery thrombosis, vascular lesions after biopsy, pseudoaneurysms intra-parenchymal artery. 3.3. Kidney transplantation aneurysms [35]. information. and hematomas [38]. Renal arterial aneurysm presents low complication rates. Main complications are renovascular hypertension, renal artery thrombosis, renal infarction, distal embolization, formation of the arteriovenous fistula, dissection and rupture, the latter more common during pregnancy, mainly in the third trimester [36]. Maternal and fetal mortality rates associated with rupture during pregnancy are 55 and 85%, respectively [37]. Figure 6. Selective arteriography of the renal artery revealing a saccular aneurysm. Renal artery aneurysms may be classified according to their location: Type 1 in the main renal artery; Type 2 in the bifurcation and beginning of the segmental branches; Type 3 in the distal: intra-parenchymal artery. Currently, several endovascular treatment methods have been developed to preserve renal function. Treatment indications reported by Henke et al., exhaustively revised, include: (1) renal artery aneurysms of over 1.0 cm in diameter, with a difficulty-to-control hypertension; (2) all renal artery aneurysms of over 2.0 cm in diameter; (3) most aneurysms of 1.5–2.0 cm in diameter. Some authors consider the possibility to treat all symptomatic renal artery aneurysms [35]. #### 3.3. Kidney transplantation diagnosis usually represents an incidental finding during regular exams. It is more prevalent in women, in the right renal artery and rarely bilateral [34]. In most cases, they are solitary aneurysms, mostly saccular-shaped (Figure 6), accounting for 75% of the cases, but with some fusiform-shaped aneurysms as well, most often developed in the renal artery bifurcation, 90% of the cases extra-parenchymal and only 10% intra-parenchymal [35]. Among the various etiologies, the most prevalent is fibromuscular dysplasia. Other causes for the development of the renal aneurysmal disease include trauma, congenital diseases (Ehlers-Danlos syndrome and neurofibromatosis), inflammatory diseases, among Figure 5. Aortography revealing renal artery occlusion induced by Takayasu's arteritis. Renal arterial aneurysm presents low complication rates. Main complications are renovascular hypertension, renal artery thrombosis, renal infarction, distal embolization, formation of the arteriovenous fistula, dissection and rupture, the latter more common during pregnancy, mainly in the third trimester [36]. Maternal and fetal mortality rates associated with rupture during pregnancy are 55 and 85%, respectively [37]. others [35]. 94 Angiography Renal transplant represents a treatment option for the terminal renal disease. In this case, arteriography before and after the transplant provides better and thorough technical information. Vascular complications are rare in renal transplants; however, they may lead to the loss of the transplanted kidney. Among the most common vascular complications are the renal artery stenosis (Figure 7), renal artery thrombosis, vascular lesions after biopsy, pseudoaneurysms and hematomas [38]. 4.3. Computed tomography (CT scan) 10–20 mL, respectively. software programs, providing a tridimensional view (Figure 8). Figure 8. Computed angiotomography of the aorta and its branches, tridimensional view. Computed angiotomography of the aorta and its branches is performed by administering an iodine-based contrast dye injection into a peripheral vein [4]. After the administration of the contrast material and the time necessary for each type of study, several sequential images are captured with a rotation platform. The images of the renal arteries are obtained at axial level, in thin slices, and then processed and reconstructed at several other levels with specific Angiography for Renal Artery Diseases http://dx.doi.org/10.5772/intechopen.79232 97 The advantages of computed angiotomography include the possibility to measure the renal dimensions, the cortical thickness, the renal perfusion and vascular alterations in the aorta and its branches. On the other hand, the use of iodinated contrast media, known to be nephrotoxic, represents a risk factor mainly to nephropathic patients. The contrast volume used at angiotomography is larger than the one at digital subtraction angiography, 120–150 and Figure 7. Selective arteriography of the transplanted kidney showing anastomotic stenosis. #### 4. Other diagnostic methods The imaging study of the renal arteries may be performed by other diagnostic methods. In the last years, several imaging techniques have been developed in an attempt to precisely display the renal vascular anatomy, and obtain good quality of images, reproducibility and a lower rate of complications. Each method presents its advantages and disadvantages; however, in general, when compared, these exams are less invasive than renal angiography, considered a standard gold procedure [4]. #### 4.1. Eco-Doppler Eco-Doppler of renal arteries and veins has been widely used as an initial method in the investigation of vascular diseases. It combines the visualization of B-mode images and the measurement of blood flow velocities of renal arteries and veins as well as of specific indexes [39, 40]. Moreover, Eco-Doppler provides information related to renal anatomy, intra-renal vasculature, and kidney size. #### 4.2. Magnetic angioresonance Magnetic angioresonance (MAR) has been largely used to complement Eco-Doppler investigation of the renal arteries. It is a less invasive method than angiography and provides images of a quality similar to that obtained at angiography. However, it requires neither arterial puncture nor nephrotoxic contrast medium to capture images [4]. The images generated by means of an electromagnetic field are compiled as multiple thin slices, adjacent and transversal. Additionally, tri-dimensional data from the images captured through magnetic angioresonance may be projected in multiple levels, providing better and thorough anatomic understanding of the images. #### 4.3. Computed tomography (CT scan) 4. Other diagnostic methods standard gold procedure [4]. vasculature, and kidney size. 4.2. Magnetic angioresonance 4.1. Eco-Doppler 96 Angiography The imaging study of the renal arteries may be performed by other diagnostic methods. In the last years, several imaging techniques have been developed in an attempt to precisely display the renal vascular anatomy, and obtain good quality of images, reproducibility and a lower rate of complications. Each method presents its advantages and disadvantages; however, in general, when compared, these exams are less invasive than renal angiography, considered a Figure 7. Selective arteriography of the transplanted kidney showing anastomotic stenosis. Eco-Doppler of renal arteries and veins has been widely used as an initial method in the investigation of vascular diseases. It combines the visualization of B-mode images and the measurement of blood flow velocities of renal arteries and veins as well as of specific indexes [39, 40]. Moreover, Eco-Doppler provides information related to renal anatomy, intra-renal Magnetic angioresonance (MAR) has been largely used to complement Eco-Doppler investigation of the renal arteries. It is a less invasive method than angiography and provides images of a quality similar to that obtained at angiography. However, it requires neither arterial The images generated by means of an electromagnetic field are compiled as multiple thin slices, adjacent and transversal. Additionally, tri-dimensional data from the images captured through magnetic angioresonance may be projected in multiple levels, providing better and puncture nor nephrotoxic contrast medium to capture images [4]. thorough anatomic understanding of the images. Computed angiotomography of the aorta and its branches is performed by administering an iodine-based contrast dye injection into a peripheral vein [4]. After the administration of the contrast material and the time necessary for each type of study, several sequential images are captured with a rotation platform. The images of the renal arteries are obtained at axial level, in thin slices, and then processed and reconstructed at several other levels with specific software programs, providing a tridimensional view (Figure 8). The advantages of computed angiotomography include the possibility to measure the renal dimensions, the cortical thickness, the renal perfusion and vascular alterations in the aorta and its branches. On the other hand, the use of iodinated contrast media, known to be nephrotoxic, represents a risk factor mainly to nephropathic patients. The contrast volume used at angiotomography is larger than the one at digital subtraction angiography, 120–150 and 10–20 mL, respectively. Figure 8. Computed angiotomography of the aorta and its branches, tridimensional view. #### Author details Daniel Emilio Dalledone Siqueira1,2\* and Ana Terezinha Guillaumon1,3,4 \Address all correspondence to: siq\[email protected] 1 School of Medical Sciences, UNICAMP, Brazil #### References* [1] Cooper CJ, Murphy TP, Cutlip DE, Jamerson K, Henrich W, Reid DM, et al. Stenting and medical therapy for atherosclerotic renal-artery stenosis. The New England Journal of Medicine. 2014;370(1):13-22 [11] Trigaux JP. Vandroogenbroek S, deWispelaere JF, et al: Congenital anomalies of the inferior vena cava and left renal vein: Evaluation with spiral CT. Journal of Vascular and Angiography for Renal Artery Diseases http://dx.doi.org/10.5772/intechopen.79232 99 [12] Aljabri B, MacDonald PS, Satin R, et al. Incidence of major venous and renal anomalies relevant to aortoiliac surgery as demonstrated by computed tomography. Annals of [13] Abrams HL, Cornell SH. Patterns of collateral flow in renal ischemia. 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Circulation. 2002;106:1572-1585 \Address all correspondence to: siq\[email protected] 2 Brazilian Society of Vascular Surgery – SBACV, Curitiba, Brazil 4 Brazilian Society of Vascular Surgery – SBACV, Campinas, Brazil 1 School of Medical Sciences, UNICAMP, Brazil Medicine. 2014;370(1):13-22 Radiologica. 1953;39:368-376 2001;344:431-442 15:2219-2229 1995;6:721 [26] Persu A, Giavarini A, Touze E, Januszewicz A, Sapoval M, Azizi M, et al. European consensus on the diagnosis and management of fibromuscular dysplasia. Journal of Hypertension. 2014;32:1367-1378 Section 4* Interventions in Structural Heart Diseases Interventions in Structural Heart Diseases [26] Persu A, Giavarini A, Touze E, Januszewicz A, Sapoval M, Azizi M, et al. European consensus on the diagnosis and management of fibromuscular dysplasia. Journal of [27] Olin JW, Gornik HL, Bacharach JM, Biller J, Fine LJ, Gray BH, et al. 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Journal of Vascular Surgery. 2011;53(3):826-836 Hypertension. 2014;32:1367-1378 100 Angiography 2012;125(18):e636-e639 Surgery. 2004;39:749-757 Surgery. 1975;110:1327-1333 Vascular Surgery. 1986;6:51-59 Rheumatology (Oxford). 2007;46:6-15 Cardiovascular Surgery. 1996;4:185-189 nosis. Annals of Surgery. 1983;197:348-352 American Society of Nephrology. 2004;15:134-141 Journal of Kidney Diseases. 1994;24:674-678 Chapter 7 Provisional chapter Transcatheter Closure of Congenital VSDs: Tips and Transcatheter Closure of Congenital VSDs: Tips and Nowadays transcatheter device closure of ventricular septal defects (VSDs) is an attractive and feasible alternative to surgical closure of congenital VSDs. Isolated congenital VSDs constitute the most common form of congenital heart disease (CHD) in infants and children and account for 20–30% of all types of cardiac malformations. Most of the VSDs are located in the membranous portion of the ventricular septum (perimembranous VSDs). There are also less common types of VSDs located in the muscular portion (muscular VSDs), below the pulmonary valve (subpulmonary or supracristal VSDs), and near the junction of the tricuspid and mitral valves (inlet type VSDs). Indications for closure of VSDs include a hemodynamically significant left to right shunt and prevention of long-term complications, including pulmonary hypertension, progressive ventricular dilatation, aortic insufficiency, double-chambered right ventricle, and endocarditis. In this chapter, we review the technical details for achieving a successful procedure, as well as some tips and tricks Ventricular septal defect (VSD) is the most common congenital heart defect and accounts for approximately 20% of all forms of congenital heart disease as an isolated lesion with incidence increasing up to 40% in case of multiple congenital heart defects [1]. Perimembranous VSDs are the most common form (70%), and muscular (15–20%) and sub arterial (5%) are less common (Figure 1). The size of the defect determines the size of the left to right shunt, which affects the hemodynamic state from negligible to cardiac failure and mild to severe on using off-label devices during transcatheter approach in VSD closure. Keywords: congenital VSD, transcatheter device closure © 2016 The Author(s). Licensee InTech. 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. © 2019 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. DOI: 10.5772/intechopen.83641 Tricks Tricks Behzad Alizadeh Behzad Alizadeh Abstract 1. Introduction Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.83641 #### Transcatheter Closure of Congenital VSDs: Tips and Tricks Transcatheter Closure of Congenital VSDs: Tips and Tricks DOI: 10.5772/intechopen.83641 #### Behzad Alizadeh Behzad Alizadeh Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.83641 #### Abstract Nowadays transcatheter device closure of ventricular septal defects (VSDs) is an attractive and feasible alternative to surgical closure of congenital VSDs. Isolated congenital VSDs constitute the most common form of congenital heart disease (CHD) in infants and children and account for 20–30% of all types of cardiac malformations. Most of the VSDs are located in the membranous portion of the ventricular septum (perimembranous VSDs). There are also less common types of VSDs located in the muscular portion (muscular VSDs), below the pulmonary valve (subpulmonary or supracristal VSDs), and near the junction of the tricuspid and mitral valves (inlet type VSDs). Indications for closure of VSDs include a hemodynamically significant left to right shunt and prevention of long-term complications, including pulmonary hypertension, progressive ventricular dilatation, aortic insufficiency, double-chambered right ventricle, and endocarditis. In this chapter, we review the technical details for achieving a successful procedure, as well as some tips and tricks on using off-label devices during transcatheter approach in VSD closure. Keywords: congenital VSD, transcatheter device closure #### 1. Introduction Ventricular septal defect (VSD) is the most common congenital heart defect and accounts for approximately 20% of all forms of congenital heart disease as an isolated lesion with incidence increasing up to 40% in case of multiple congenital heart defects [1]. Perimembranous VSDs are the most common form (70%), and muscular (15–20%) and sub arterial (5%) are less common (Figure 1). The size of the defect determines the size of the left to right shunt, which affects the hemodynamic state from negligible to cardiac failure and mild to severe © 2016 The Author(s). Licensee InTech. 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. © 2019 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. Lock et al. used Rashkind double disc PDA umbrella in human subsequently. Six of the seven devices were implanted successfully in his series, while the seventh embolized into the pulmonary artery. Goldstein used clamshell occluder to close the VSDs. Gianturco coils, Amplatzer membranous and muscular devices, buttoned device, wireless devices (detachable steel coils, detachable balloon, and transcatheter patch), cardioSEAL/STARFlex devices, Nitocclud (Nickel-Titanium Spiral Coil), and Amplatzer Duct Occluder I and Amplatzer Duct Transcatheter Closure of Congenital VSDs: Tips and Tricks http://dx.doi.org/10.5772/intechopen.83641 105 Postmyocardial infarction VSDs were also closed percutaneously using Rashkind double disc PDA umbrella, clamshell, CardioSEAL or STARFlex, Amplatzer septal occluder, Amplatzer Amplatzer muscular VSD and PIMVSD occluders, Qwik-Load and STARFlex Septal Occlusion System, and CardioSEAL Septal Occlusion System are now approved by FDA, but there are multiple devices that have been used to close VSDs so far, which include VSD Le Coils, Muscular and Membranous VSD devices of Occlutech Co., and many Chinese symmetrical and Asymmetrical VSD occluders and the Amplatzer Duct occluder with Duct Occluder II (St. Jude Medical, Inc.) as well. The use of the Chinese devices is most commonly reported from China itself. Although the Amplatzer membranous VSD occluder was found useful, development of heart block precipitated its removal from clinical trials in the USA. Other devices are Different patients with VSDs may present with different scenarios. Most muscular VSDs and a few perimembranous type may close spontaneously in early month of life. Some large unrestrictive VSDs may cause heart failure and failure to thrive in small babies. These infants need a large device for interventional closure, which makes the procedure difficult and unsafe. So Although closure of VSDs in patients with aortic valve prolapse or AI remains controversial and most of this patients with trivial AI are now referred to surgeons, Le VSD Coil (pfm medical ag, Koln, Germany) occluder has recently used in such condition with good results [2]. VSDs are considered eligible for transcatheter device closure in the presence of one or more , unresponsive to oxygen Occluder II devices were used for transcatheter occlusion of VSDs subsequently. duct occluder, and Amplatzer post-infarct muscular VSD (PIMVSD) successfully. in clinical trials in either the USA or abroad [1]. this group of patient often refers to surgeons. • Evidence of left heart volume over load • History of previous endocarditis 3.1. Contraindications • body weight less than 6 kg • Evidence of heart failure not controlled by medical therapy • pulmonary vascular resistance index greater than 7 WU/m<sup>2</sup> • Pulmonary to systemic blood flow ratio greater than 1.5 (Qp/Qs > 1.5) of the following indications: 3. Indications Figure 1. Prevalence of different forms of VSDs (picture courtesy: Patrick J Lynch; C. Carl Jaffe Yale University). pulmonary hypertension. Although most of the larger defects persist through adulthood, some smaller defects have a high likelihood of spontaneous closure. There are some longterm complications of VSDs including prolapse of aortic cusps with regurgitation, infective endocarditis, arrhythmias, and pulmonary hypertension, which may lead to pulmonary vascular obstructive disease or Eisenmenger syndrome. Traditionally, closure of ventricular septal defects (VSDs) has been a surgical procedure for over 50 years with a low operative mortality and postoperative morbidity. However, in 1988, Lock et al. [2] reported the results of transcatheter VSD closure using the Rashkind double umbrella device in six patients with congenital and acquired VSDs. Transcatheter closure of VSD as an alternative to surgery has now gained increasing acceptance due to a comparable success rate and low risk of complications. This approach has several advantages, such as avoidance of sternotomy and cardiopulmonary bypass, with less pain and no scar and shorter hospital stay, as well. There are also some disadvantages like the need for X-ray and contrast media injections. Although surgical treatment remains the standard approach for VSDs, percutaneous device closure has brought hope to be a safe and effective treatment with a high rate of success. #### 2. Historical aspects Transcatheter closure of ventricular septal defects was first described by Rashkind when he used a single-disc device to perform this in dogs. Lock et al. used Rashkind double disc PDA umbrella in human subsequently. Six of the seven devices were implanted successfully in his series, while the seventh embolized into the pulmonary artery. Goldstein used clamshell occluder to close the VSDs. Gianturco coils, Amplatzer membranous and muscular devices, buttoned device, wireless devices (detachable steel coils, detachable balloon, and transcatheter patch), cardioSEAL/STARFlex devices, Nitocclud (Nickel-Titanium Spiral Coil), and Amplatzer Duct Occluder I and Amplatzer Duct Occluder II devices were used for transcatheter occlusion of VSDs subsequently. Postmyocardial infarction VSDs were also closed percutaneously using Rashkind double disc PDA umbrella, clamshell, CardioSEAL or STARFlex, Amplatzer septal occluder, Amplatzer duct occluder, and Amplatzer post-infarct muscular VSD (PIMVSD) successfully. Amplatzer muscular VSD and PIMVSD occluders, Qwik-Load and STARFlex Septal Occlusion System, and CardioSEAL Septal Occlusion System are now approved by FDA, but there are multiple devices that have been used to close VSDs so far, which include VSD Le Coils, Muscular and Membranous VSD devices of Occlutech Co., and many Chinese symmetrical and Asymmetrical VSD occluders and the Amplatzer Duct occluder with Duct Occluder II (St. Jude Medical, Inc.) as well. The use of the Chinese devices is most commonly reported from China itself. Although the Amplatzer membranous VSD occluder was found useful, development of heart block precipitated its removal from clinical trials in the USA. Other devices are in clinical trials in either the USA or abroad [1]. #### 3. Indications pulmonary hypertension. Although most of the larger defects persist through adulthood, some smaller defects have a high likelihood of spontaneous closure. There are some longterm complications of VSDs including prolapse of aortic cusps with regurgitation, infective endocarditis, arrhythmias, and pulmonary hypertension, which may lead to pulmonary vas- Figure 1. Prevalence of different forms of VSDs (picture courtesy: Patrick J Lynch; C. Carl Jaffe Yale University). Traditionally, closure of ventricular septal defects (VSDs) has been a surgical procedure for over 50 years with a low operative mortality and postoperative morbidity. However, in 1988, Lock et al. [2] reported the results of transcatheter VSD closure using the Rashkind double umbrella device in six patients with congenital and acquired VSDs. Transcatheter closure of VSD as an alternative to surgery has now gained increasing acceptance due to a comparable success rate and low risk of complications. This approach has several advantages, such as avoidance of sternotomy and cardiopulmonary bypass, with less pain and no scar and shorter hospital stay, as well. There are also some disadvantages like the need for X-ray and contrast Although surgical treatment remains the standard approach for VSDs, percutaneous device closure has brought hope to be a safe and effective treatment with a high rate of success. Transcatheter closure of ventricular septal defects was first described by Rashkind when he cular obstructive disease or Eisenmenger syndrome. used a single-disc device to perform this in dogs. media injections. 104 Angiography 2. Historical aspects Different patients with VSDs may present with different scenarios. Most muscular VSDs and a few perimembranous type may close spontaneously in early month of life. Some large unrestrictive VSDs may cause heart failure and failure to thrive in small babies. These infants need a large device for interventional closure, which makes the procedure difficult and unsafe. So this group of patient often refers to surgeons. Although closure of VSDs in patients with aortic valve prolapse or AI remains controversial and most of this patients with trivial AI are now referred to surgeons, Le VSD Coil (pfm medical ag, Koln, Germany) occluder has recently used in such condition with good results [2]. VSDs are considered eligible for transcatheter device closure in the presence of one or more of the following indications: #### 3.1. Contraindications Pulmonary hypertension is defined as a mean pulmonary artery pressure of 20 mm Hg or greater based on the original natural history studies [3]. In some selected older children with some difficulties, the VSD may be approached retrogradely from the femoral artery. Activated clotting time is maintained above 200 seconds Transcatheter Closure of Congenital VSDs: Tips and Tricks http://dx.doi.org/10.5772/intechopen.83641 107 • Device selection: the device is usually selected 1–2 mm larger than the maximal diameter of the defect as assessed by TEE and angiography. Balloon sizing is hardly ever used, since the inter-ventricular septum is regarded to be a nonstretchable structure. There are some other important considerations in device selection but paying attention to the location, shape, morphology, and length and thickness of the edges of defect are of the most impor- • Crossing the VSD: normally, the VSD is crossed from the left ventricle using a Judkins right coronary artery catheter, but a variety of tip angled catheters can be used based on the defect location and shape like Bern, Cobra or a cutoff Pigtail. An exchange guidewire like Terumo or Noodle wire is then placed into the left or right pulmonary artery via the VSD Figure 2. (A) Amplatzer membranous VSD device, (B and C) Amplatzer Duct Occluder and Amplatzer Duct Occluder II devices. (D and G) Amplatzer and Occlutech muscular VSD device. (F) Nit-Occlud (Nickel-Titanium Spiral Coil) and (E) asymmetric membranous VSD device were used for transcatheter occlusion of VSDs. throughout the procedure. tant factors (Figures 2 and 3). crossed catheter (Figure 4B). There are some other considerations like distance from the edge of the VSD to the semilunar valves: #### 4. Preprocedure assessment Echocardiography can provide valuable information on the number, the location, the size, and the relationship of the VSD to the adjacent structures. In addition, transesophageal and 3D-echocardiography are now widely available and may provide additional information of unusual VSDs. #### 5. Technique #### 5.1. General considerations Compared to patent ductus and atrial septal defect closure, VSD closure is considerably more complicated. It is thus important to recognize that this procedure should only be undertaken in well-equipped units with sufficient skill, knowledge, and surgical backup. In some selected older children with some difficulties, the VSD may be approached retrogradely from the femoral artery. Activated clotting time is maintained above 200 seconds throughout the procedure. • PMVSD extending to the inlet • <4 mm—for the Nit-Occlud VSD 4. Preprocedure assessment valves: 106 Angiography unusual VSDs. 5. Technique can be used. 5.1. General considerations and regurgitation as well. • when parents prefer surgical intervention. greater based on the original natural history studies [3]. • <2 mm—for the Amplatzer membranous VSD occlude • the presence of additional lesions requiring surgical intervention (ToF) Pulmonary hypertension is defined as a mean pulmonary artery pressure of 20 mm Hg or There are some other considerations like distance from the edge of the VSD to the semilunar Echocardiography can provide valuable information on the number, the location, the size, and the relationship of the VSD to the adjacent structures. In addition, transesophageal and 3D-echocardiography are now widely available and may provide additional information of Compared to patent ductus and atrial septal defect closure, VSD closure is considerably more complicated. It is thus important to recognize that this procedure should only be undertaken • Anesthesia: the procedure may be performed under general anesthesia, although sedation • Imaging: most of the centers use continuous transesophageal echocardiography (TEE), but • Catheterization: a comprehensive evaluation should be done during angiography to obtain different views at multiple angles of the VSD and a complete study for valvular function • Access: although in some certain muscular VSDs, the right internal jugular vein access may be used, and femoral artery and vein are usually gained as the main access. Alternatively, in small patients who making an arteriovenous circuit may cause hemodynamic instability when long stiff sheath is placed across the tricuspid valve, a hybrid approach can be used. skilled echo cardiographers may use transthoracic echo during the procedure. in well-equipped units with sufficient skill, knowledge, and surgical backup. Figure 2. (A) Amplatzer membranous VSD device, (B and C) Amplatzer Duct Occluder and Amplatzer Duct Occluder II devices. (D and G) Amplatzer and Occlutech muscular VSD device. (F) Nit-Occlud (Nickel-Titanium Spiral Coil) and (E) asymmetric membranous VSD device were used for transcatheter occlusion of VSDs. Figure 3. Schematic depictions of conventional technique. 1—Transaortic access into LV chamber retrogradely. 2—Crossing a guide wire from LV to RV chamber retrogradely. 3—Snaring the trans-aortic guide wire from the pulmonary artery. 4—Making an arteriovenous loop to provide a way to deliver the VSD delivery sheath. 5—The delivery sheath is advanced antegradely across the VSD. 6—The device is fixed in position and released. (Courtesy of Lydia Kibiuk, NIH Medical Arts and Photography Branch). Figure 4. Stepwise percutaneous VSD closure technique using CaridoFix Muscular VSD Occluder. (A) Small upper muscular VSD. (B) Crossing the VSD and putting the wire in pulmonary artery. (C) Snaring the pulmonary wire via venous access. (D) Making the AV loop from arterial and venous access. (E) Crossing the VSD by long sheet and into the descending aorta. (F) Releasing the first disc of the device. (G) Pulling back the device. (H) Fixing the device to the septum. (I) Releasing the second disc of the device. (J) Complete deploying and Transcatheter Closure of Congenital VSDs: Tips and Tricks http://dx.doi.org/10.5772/intechopen.83641 109 releasing of the device. • Arteriovenous guide wire loop: to make an arteriovenous wire loop, the guide wire is then snared and pulled out via the right femoral vein. A delivery sheath will be then advanced via the femoral vein access into right ventricle into the aorta carefully. Undue tension on the arteriovenous loop and VSD may cause rhythm disturbances. To avoid direct contact of the guide wire with the VSD when crossed by the sheath, a "kissing catheter technique" should be used. The delivery sheath is then positioned to a suitable position in the left • The device: the proper size and suitable shape device is then screwed to the tip of the delivery cable and advanced to the cavity of left ventricle or descending aorta via the long sheet with special care to avoid the occurrence air embolism. The LV disc is first deployed within the LV chamber or descending aorta and gently pulled back to the intra-ventricular VSD under echo or angiographic guidance. The waist of the device and right ventricular disc are then deployed, respectively. Careful attention should be paid to good positioning and stability of the device and any potential compromising of the adjusting structures. In the case of any impingent of valves or other structures, the device can still be recaptured into the sheath and repositioned gently. Once proper positioning has been achieved, the device may be released by unscrewing it counter clockwise using the pin vice. After release, confirmation of correct positioning should be established using TEE or TTE and angiography (Figures 4F–I and 5). Figure 3. Schematic depictions of conventional technique. 1—Transaortic access into LV chamber retrogradely. 2—Crossing a guide wire from LV to RV chamber retrogradely. 3—Snaring the trans-aortic guide wire from the pulmonary artery. 4—Making an arteriovenous loop to provide a way to deliver the VSD delivery sheath. 5—The delivery sheath is advanced antegradely across the VSD. 6—The device is fixed in position and released. (Courtesy of ventricle or descending aorta (Figure 4C,D). 108 Angiography Lydia Kibiuk, NIH Medical Arts and Photography Branch). Figure 4. Stepwise percutaneous VSD closure technique using CaridoFix Muscular VSD Occluder. (A) Small upper muscular VSD. (B) Crossing the VSD and putting the wire in pulmonary artery. (C) Snaring the pulmonary wire via venous access. (D) Making the AV loop from arterial and venous access. (E) Crossing the VSD by long sheet and into the descending aorta. (F) Releasing the first disc of the device. (G) Pulling back the device. (H) Fixing the device to the septum. (I) Releasing the second disc of the device. (J) Complete deploying and releasing of the device. • Early postprocedure care: three doses of an antibiotic should be given to the patient within the 24 hours of observation with ECG monitoring. Endocarditis prophylaxis and a low Transcatheter Closure of Congenital VSDs: Tips and Tricks http://dx.doi.org/10.5772/intechopen.83641 111 The complications may occur immediately after the procedure or late during the follow up. Device embolization: both systemic and right heart embolization of the device may occur and have been reported in up to 2% of cases. Continues TEE or intermittent TTE during the procedure and especially before the releasing of the device is crucial and very helpful. Operators should be familiar with retrieval techniques, and all necessary equipment for retrieval should Dysrhythmias: during catheter and device manipulation, temporary dysrhythmias, usually ventricular, are common. Right bundle branch block occurred in only 6% of patients (5) compared to up to 64% of reported surgical series. Complete atrioventricular block (cAVB) is also a potential complication of transcatheter or surgical VSD closure. The reported cases of cAVB are mostly attributed to direct compression trauma, the pressure of radial forces (shape memory of the device), clamping forces, inflammatory processes, and/or the use of Valvular malfunction: the device may influence function of any of the adjacent valves, but especially with the membranous device, aortic and tricuspid regurgitation should actively be Hemolysis: blood scape through a small residual shunt after VSD device closure may lead to hemolysis. To be aware of this potential complication especially when there is a residual shunt is very important. Checking the patient's urine color change in early hours after the procedure is a simple way to detect the hemolysis. Some form of little hemolysis may be self-limited and could be managed conservatively but in the form of massive hemolysis blood Pediatric and Congenital Cardiology Department, Mashhad University of Medical Sciences, [1] Balzer D. Current status of percutaneous closure of ventricular septal defects. Pediatrics dose of an anti-platelet agent like aspirin are recommended for 6 months [4]. be available. Surgical backup is also considered essential [5]. looked for with TTE or TEE prior to release of the device. Address all correspondence to: [email protected] Imam Reza University Hospital, Mashhad, Iran and Therapeutics. 2012;2:112 transfusion and surgical removal of the device should be considered. 5.2. Complications oversized devices [6]. Author details Behzad Alizadeh References Figure 5. Post operation residual VSD closure technique in a patient with TOF using Occutech Duct Occluder. (A) Small residual VSD around the patch. (B) Main pulmonary injection showing free PI (lateral view). (C) Main pulmonary injection showing free PI (AP view). (D) Snaring the pulmonary wire via venous access making the AV loop. (E) Crossing the VSD into the ascending aorta and releasing the first disc of the device. (F) Pulling back the device and fixing the device to the septum. (G) Aortic root injection, checking the device position and probable AI. (H) Releasing the device. • Early postprocedure care: three doses of an antibiotic should be given to the patient within the 24 hours of observation with ECG monitoring. Endocarditis prophylaxis and a low dose of an anti-platelet agent like aspirin are recommended for 6 months [4]. #### 5.2. Complications The complications may occur immediately after the procedure or late during the follow up. Device embolization: both systemic and right heart embolization of the device may occur and have been reported in up to 2% of cases. Continues TEE or intermittent TTE during the procedure and especially before the releasing of the device is crucial and very helpful. Operators should be familiar with retrieval techniques, and all necessary equipment for retrieval should be available. Surgical backup is also considered essential [5]. Dysrhythmias: during catheter and device manipulation, temporary dysrhythmias, usually ventricular, are common. Right bundle branch block occurred in only 6% of patients (5) compared to up to 64% of reported surgical series. Complete atrioventricular block (cAVB) is also a potential complication of transcatheter or surgical VSD closure. The reported cases of cAVB are mostly attributed to direct compression trauma, the pressure of radial forces (shape memory of the device), clamping forces, inflammatory processes, and/or the use of oversized devices [6]. Valvular malfunction: the device may influence function of any of the adjacent valves, but especially with the membranous device, aortic and tricuspid regurgitation should actively be looked for with TTE or TEE prior to release of the device. Hemolysis: blood scape through a small residual shunt after VSD device closure may lead to hemolysis. To be aware of this potential complication especially when there is a residual shunt is very important. Checking the patient's urine color change in early hours after the procedure is a simple way to detect the hemolysis. Some form of little hemolysis may be self-limited and could be managed conservatively but in the form of massive hemolysis blood transfusion and surgical removal of the device should be considered. #### Author details Behzad Alizadeh Address all correspondence to: [email protected] Pediatric and Congenital Cardiology Department, Mashhad University of Medical Sciences, Imam Reza University Hospital, Mashhad, Iran #### References Figure 5. Post operation residual VSD closure technique in a patient with TOF using Occutech Duct Occluder. (A) Small residual VSD around the patch. (B) Main pulmonary injection showing free PI (lateral view). (C) Main pulmonary injection showing free PI (AP view). (D) Snaring the pulmonary wire via venous access making the AV loop. (E) Crossing the VSD into the ascending aorta and releasing the first disc of the device. (F) Pulling back the device and fixing the device to the septum. (G) Aortic root injection, checking the device position and probable AI. (H) Releasing the device. 110 Angiography [1] Balzer D. Current status of percutaneous closure of ventricular septal defects. Pediatrics and Therapeutics. 2012;2:112 [2] Lock JE, Block PC, McKay RG, Baim DS, Keane JF. Transcatheter closure of ventricular septal defects. Circulation. 1988;78:361-368 Section 5 Computed Tomography Angiography in Coronary and Periferal Arterial Diseases Computed Tomography Angiography in Coronary and Periferal Arterial Diseases [2] Lock JE, Block PC, McKay RG, Baim DS, Keane JF. Transcatheter closure of ventricular [3] Weidman WH, Blount SG Jr, Dushane JW, Gersony WM, Hayes CJ, Nadas AS. Clinical [4] Brown SC, Bruwer AD, Smit FE. Percutaneous closure of ventricular septal defects in [5] Sanz AP, Álvarez-Fuente M, Centella T, del Cerro MJ. Early complete atrioventricular block after percutaneous closure of a perimembranous ventricular septal defect with a [6] Ratnayaka K, Raman VK, Faranesh AZ, Sonmez M, Kim JH, Gutiérrez LF, et al. Antegrade percutaneous closure of membranous ventricular septal defect using X-ray fused with magnetic resonance imaging. JACC: Cardiovascular Interventions. 2009;2(3):224-230 Nit-Occlud (®) Lê VSD coil. Progress in Pediatric Cardiology. 2018;49:81-83 course in ventricular septal defect. Circulation. 1977;56(1 Suppl):I56 septal defects. Circulation. 1988;78:361-368 112 Angiography childhood. SA Heart Journal. 2017;4(1):26-31 Chapter 8 Provisional chapter Computer-Aided Detection, Pulmonary Embolism, Computer-Aided Detection, Pulmonary Embolism, Current Status Abstract angiography 1. Introduction Current Status Abdel-Razzak M. Al-hinnawi Abdel-Razzak M. Al-hinnawi http://dx.doi.org/10.5772/intechopen.79339 Additional information is available at the end of the chapter Additional information is available at the end of the chapter Computerized Tomography Pulmonary Angiography: Angiography (mostly computed tomography, but in some cases, conventional) is still the gold diagnostic standard in the clinical diagnosis of pulmonary embolism (PE). Computeraided detection (CAD) is software that alerts radiologists the presence of PE during computerized tomography pulmonary angiography (CTPA) examinations. Interpreting CTPA scans with the aid of commercially available CTPA-CAD has improved the detectability of PE patients. This chapter aims to complete the scope of this book by explaining the clinical evidences of PE, the CTPA technology, the role of CTPA-CAD software in improving the diagnostic abilities of CTPA and the role of conventional pulmonary angiography in daily clinical practice. The reader will be introduced to the performance of diagnosing PE with or without the aid of CTPA-CAD algorithms. Differences among CTPA-CAD's output will be compared and tabled according to "per patient," "per clot," "first reader," and "second reader" basis. This includes, but not limited to, the CTPA-CAD's sensitivity and specificity in comparison to human observer performance (i.e., radiologist). These topics cover the current status practice at the pulmonary angiography clinic. Keywords: computer-aided detection, computerized tomography pulmonary angiography, pulmonary embolism, digital image processing, conventional pulmonary Computer sciences have reached medicine [1]. This includes developing algorithms to participate in the clinical interpretation of medical images acquired from various medical Computerized Tomography Pulmonary Angiography: © 2016 The Author(s). Licensee InTech. 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. © 2018 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. DOI: 10.5772/intechopen.79339 #### Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary Angiography: Current Status Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary Angiography: Current Status DOI: 10.5772/intechopen.79339 Abdel-Razzak M. Al-hinnawi Abdel-Razzak M. Al-hinnawi Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.79339 #### Abstract Angiography (mostly computed tomography, but in some cases, conventional) is still the gold diagnostic standard in the clinical diagnosis of pulmonary embolism (PE). Computeraided detection (CAD) is software that alerts radiologists the presence of PE during computerized tomography pulmonary angiography (CTPA) examinations. Interpreting CTPA scans with the aid of commercially available CTPA-CAD has improved the detectability of PE patients. This chapter aims to complete the scope of this book by explaining the clinical evidences of PE, the CTPA technology, the role of CTPA-CAD software in improving the diagnostic abilities of CTPA and the role of conventional pulmonary angiography in daily clinical practice. The reader will be introduced to the performance of diagnosing PE with or without the aid of CTPA-CAD algorithms. Differences among CTPA-CAD's output will be compared and tabled according to "per patient," "per clot," "first reader," and "second reader" basis. This includes, but not limited to, the CTPA-CAD's sensitivity and specificity in comparison to human observer performance (i.e., radiologist). These topics cover the current status practice at the pulmonary angiography clinic. Keywords: computer-aided detection, computerized tomography pulmonary angiography, pulmonary embolism, digital image processing, conventional pulmonary angiography #### 1. Introduction Computer sciences have reached medicine [1]. This includes developing algorithms to participate in the clinical interpretation of medical images acquired from various medical © 2016 The Author(s). Licensee InTech. 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. © 2018 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. imaging systems [2, 3]. These algorithms are categorized into two main groups. They are the computer-aided detection (CAD) and the computer-aided diagnosis (CADx) [2, 3]. Both of them employ principles of digital image processing (DIP). Pulmonary embolism (PE) is the partial or complete blockage of one or some of pulmonary arteries; it is a life threatening disease with a challenging diagnosis [4]. In Europe and the USA, it leads to high incidence of mortality, morbidity, and hospitalization [4, 5]. Internationally, PE is expected to become the third leading cause of death by 2030 according to clinical projections on disease mortality [6]. Computerized tomography pulmonary angiography (CTPA) has become the first-line imaging examination to detect the occurrence of PE [4, 7–9]. Clinical trials of CTPA examination, without the aid of CAD, reported that the sensitivity and specificity CTPA scan may not reach 100% [8, 10]. This indicates that misdiagnoses, which are a prospective health burden and potential life threatening, may occur. This chapter describes the current state of PE diagnosis in CTPA clinic, with and without the use of CAD algorithms. The chapter is divided into three main sections. Section 2 presents all clinical evidences about the PE as a disease that threaten lives. This covers the PE epidemiology, incidence rate, characterizations, load scores, diagnosis, and treatment. Section 3 is dedicated to explain the CTPA physics and technology, image appearance, PE radiographic features, clinical trials, and common artifacts. Section 4 explains the art of computer-assisted detection and its applications in diagnosing PE. This demonstrates the role of CAD software in improving the PE diagnosis. In general, this chapter provides the up-to-date knowledge of PE diagnosis in angiography clinics. > variation is likely attributed to the type and accuracy of the diagnostic procedure rather than the actual incidence of the disease itself [6, 15–18]. Annually, there are 430,000 and 300,000 to 600,000 PE conditions in Europe and the USA, respectively [15, 16]. Globally, the clinical projections of mortality estimate that the incidence of PE will be the third major cause of death in year 2030 [6]. In Europe and the USA, autopsy investigations on hospitalized patients showed a PE prevalence from 60 to 70% [4, 5]. Therefore, the precise detection and diagnosis Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 117 Embolus is characterized as central or peripheral, based on the site of the affected blood vessel branch [4]. Central vascular regions include the left and right main pulmonary arteries (PA), the right and left interlobar arteries, the right and left lobar arteries, and right and left lobar veins (Figure 1). Peripheral vascular regions include the remaining blood vessels in the upper, middle, and lower lobes of the right lung; and the upper and lower lobes in the left lung. This includes all the segmental, subsegmental, and intralobular arteries and veins including the Once a thrombus has been identified in the PA, it is further characterized as acute or chronic, as illustrated in Figure 2 [4]. In some cases, there is doubt between those two classes, depending on diagnosis procedure and experience of the observer. A clot in PA is usually considered as being "acute" if it is located centrally within the vascular lumen. This may lead to vessel dilation. However, a clot in PA is characterized as "chronic" when it appears contiguous to are highly desirable [4, 5]. Figure 1. Pulmonary embolism pathophysiology. culmen and the lingual (Figure 1). 2.3. Characterization #### 2. Pulmonary embolism #### 2.1. Definition Pulmonary embolism (PE) occurs when a blood clot, also known as a thrombus or embolus, arrives to pulmonary arteries. The source of thrombus is likely to be large veins of the lower extremity before it migrates through venous system to reach first the right heart chambers and later the lungs (Figure 1). Once a clot arrives to pulmonary arterial tree, it travels in the arteries of the lung until it blockades vessel/s that is/are too narrow to continue further. Thus, PE happens (Figure 1) leading to pulmonary blood flow shortage. Consequently, this condition associates with rise in the artery pressure due to the increased resistance to the bloodstream, shortness of breath, chest pain, and breathing difficulties; it can also lead to infarct or decrease in cardiac output, which in turn can cause hemodynamic disturbances, heart failure or even death [4, 5, 11, 12]. The common risk factors for PE are immobility or inactivity, hypertension, surgery or trauma, cigarette smoking, obesity, heart failure, cancer, chronic obstructive lung disease, hormone therapy, pregnancy, and advanced age and family members with thrombosis or embolism [4, 5, 12]. #### 2.2. Incidence Clinical surveys showed that the PE exhibits the highest incidence of mortality, morbidity, and hospitalization [4, 5, 13, 14]. The incidence of PE vary from one country to other but this Figure 1. Pulmonary embolism pathophysiology. variation is likely attributed to the type and accuracy of the diagnostic procedure rather than the actual incidence of the disease itself [6, 15–18]. Annually, there are 430,000 and 300,000 to 600,000 PE conditions in Europe and the USA, respectively [15, 16]. Globally, the clinical projections of mortality estimate that the incidence of PE will be the third major cause of death in year 2030 [6]. In Europe and the USA, autopsy investigations on hospitalized patients showed a PE prevalence from 60 to 70% [4, 5]. Therefore, the precise detection and diagnosis are highly desirable [4, 5]. #### 2.3. Characterization imaging systems [2, 3]. These algorithms are categorized into two main groups. They are the computer-aided detection (CAD) and the computer-aided diagnosis (CADx) [2, 3]. Both of Pulmonary embolism (PE) is the partial or complete blockage of one or some of pulmonary arteries; it is a life threatening disease with a challenging diagnosis [4]. In Europe and the USA, it leads to high incidence of mortality, morbidity, and hospitalization [4, 5]. Internationally, PE is expected to become the third leading cause of death by 2030 according to clinical projections on disease mortality [6]. Computerized tomography pulmonary angiography (CTPA) has become the first-line imaging examination to detect the occurrence of PE [4, 7–9]. Clinical trials of CTPA examination, without the aid of CAD, reported that the sensitivity and specificity CTPA scan may not reach 100% [8, 10]. This indicates that misdiagnoses, which are a This chapter describes the current state of PE diagnosis in CTPA clinic, with and without the use of CAD algorithms. The chapter is divided into three main sections. Section 2 presents all clinical evidences about the PE as a disease that threaten lives. This covers the PE epidemiology, incidence rate, characterizations, load scores, diagnosis, and treatment. Section 3 is dedicated to explain the CTPA physics and technology, image appearance, PE radiographic features, clinical trials, and common artifacts. Section 4 explains the art of computer-assisted detection and its applications in diagnosing PE. This demonstrates the role of CAD software in improving the PE diagnosis. In general, this chapter provides the up-to-date knowledge of Pulmonary embolism (PE) occurs when a blood clot, also known as a thrombus or embolus, arrives to pulmonary arteries. The source of thrombus is likely to be large veins of the lower extremity before it migrates through venous system to reach first the right heart chambers and later the lungs (Figure 1). Once a clot arrives to pulmonary arterial tree, it travels in the arteries of the lung until it blockades vessel/s that is/are too narrow to continue further. Thus, PE happens (Figure 1) leading to pulmonary blood flow shortage. Consequently, this condition associates with rise in the artery pressure due to the increased resistance to the bloodstream, shortness of breath, chest pain, and breathing difficulties; it can also lead to infarct or decrease in cardiac output, which in turn can cause hemodynamic disturbances, heart failure or even death [4, 5, 11, 12]. The common risk factors for PE are immobility or inactivity, hypertension, surgery or trauma, cigarette smoking, obesity, heart failure, cancer, chronic obstructive lung disease, hormone therapy, pregnancy, and advanced age and family Clinical surveys showed that the PE exhibits the highest incidence of mortality, morbidity, and hospitalization [4, 5, 13, 14]. The incidence of PE vary from one country to other but this them employ principles of digital image processing (DIP). prospective health burden and potential life threatening, may occur. PE diagnosis in angiography clinics. members with thrombosis or embolism [4, 5, 12]. 2. Pulmonary embolism 2.1. Definition 116 Angiography 2.2. Incidence Embolus is characterized as central or peripheral, based on the site of the affected blood vessel branch [4]. Central vascular regions include the left and right main pulmonary arteries (PA), the right and left interlobar arteries, the right and left lobar arteries, and right and left lobar veins (Figure 1). Peripheral vascular regions include the remaining blood vessels in the upper, middle, and lower lobes of the right lung; and the upper and lower lobes in the left lung. This includes all the segmental, subsegmental, and intralobular arteries and veins including the culmen and the lingual (Figure 1). Once a thrombus has been identified in the PA, it is further characterized as acute or chronic, as illustrated in Figure 2 [4]. In some cases, there is doubt between those two classes, depending on diagnosis procedure and experience of the observer. A clot in PA is usually considered as being "acute" if it is located centrally within the vascular lumen. This may lead to vessel dilation. However, a clot in PA is characterized as "chronic" when it appears contiguous to (V/Q) scan. This scan remained as the traditional preferred imaging technique before the shift toward CT [4]. A mismatch of ventilated but not perfused lung tissue was considered as indicator for pulmonary embolism. Thus, it is indirect detection of an embolus by looking at the effects of an occlusion. A normal perfusion scan securely excluded pulmonary embolism, but was found in a minority of the patients that are suspected of PE, and thus, often further testing was needed [4]. The advantages of V/Q scan are that it is not invasive and less irradiant than CT, and may be more suitable for patients that are allergic to iodinated agents (CT). Its disadvantage is that the obtained image determines only regions of the lungs that are not correctly vascularized, nonobstructing "small" clots remaining invisible [4]. Moreover, the duration of the exam is in the order of 20 min, which is slower than other modalities. Several reports showed that the CT scan outweigh the V/Q scan by performing both lower rate of false-negative scans and lower number of "indeterminate scans" not yielding a definite Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 119 The sequence of chest X-ray projections during the administration of contrast agent directly into the target vessels, also called as pulmonary angiography, is a reliable test for diagnosing PE. In this imaging scan, a catheter is inserted into a femoral vein and navigated through the heart toward the pulmonary arteries. Amounts of contrast media is injected several times at various locations of pulmonary vessels, and sequence of X-ray planner projections are obtained. It is used to provide a definitive diagnosis when other imaging tests fail [4]. Although conventional pulmonary angiography has great value in PE diagnosis, it suffers from being expensive, invasive with serious side effect, requiring expertise and supporting Vascular Doppler ultrasound is a quick, noninvasive, and reliable technique [23]. It is painless and carries no risk. However, it provides less clinical information (e.g., number of clots and amount of obstruction) than other imaging techniques and very dependent on the experience Finally, several reports showed promising results for the assessment of PE with magnetic resonance imaging (MRI) [24, 25]. This modality is promising because images can be generated without radiation, and because it allows a combination of morphological and functional imaging (e.g., perfusion). However, MR has a lower spatial resolution than CT and much Untreated PE can be fatal with high mortality rate that can be decreased under rapid detection [6]. There are ranges of different types of treatment procedures [4]. They include hemodynamic and respiratory support, anticoagulation medications, thrombolytic therapy, surgical embolectomy, percutaneous catheter-directed therapy, and venous filter intervention, which are not without complications. The selection of treatment depends on the PE severity and prognosis. Generally, the obstruction is mild when only a few subsegmental vessels are blocked and it is severe when multiple segmental or a few lobar vessels are blocked. Mild PE is managed with clot-dissolving medication. Severe PE requires additional medical intervention, such as placement of a filter in the inferior vena cava, or clot removal with either a catheter or surgery. longer acquisition times (around 30 min as opposed to seconds in CT). diagnosis [4, 22]. of the examiner [4, 23]. 2.6. Treatment staff, and not readily available in most hospitals. Figure 2. Illustration of acute and chronic PE. the vessel wall substantially reducing the arterial diameter. A clot that exhibits caves or canals within itself is also considered as "chronic." #### 2.4. Load scores Several scoring systems have been introduced to measure the severity of the PE clinic. For central PE, physicians utilize the Walsh, Miller, Qanadli, or Mastora score. While for peripheral PE, they use Marder, Arnesen, Mewissen (American Venous Registry), Porter, Ouriel, or Bjorgell scores. They are well summarized by Ghaye et al. [19, 20]. All these scores depend on the number of clots, location, and the percentage of obstruction. They all verified appropriate for assessing the severity of PE and treatment effectiveness, but are not much used in angiography clinics due to time it takes to manually assess them. #### 2.5. Diagnosis Diagnosing PE remains a challenge to physicians because the symptoms are unspecific and may not be present in all patients. The PE symptoms and risk factors (Section 2.1) are used to determine the probability of PE. Although biomarkers and laboratory tests are crucial to estimate the probability of PE, such as the electrocardiogram (ECG) and the measurement of percentage of cross-linked fibrin in the blood (D-dimer), the diagnostic decision is always based on radiographic findings from medical imaging procedures [4]. Different medical imaging techniques exist to "rule in" or "rule out" the presence of the PE. Each technique exhibits its strength and weakness and a shift toward the computerized tomography (CT) has been approved. A planner chest X-ray remains the first imaging step because it can rule out the conditions that mimic PE (e.g., a pneumothorax can cause chest pain similar to pain caused by acute PE), but it cannot exclude PE. Another X-ray imaging test is the lower extremity venous angiography, in which contrast media is injected via a foot vein, and several X-ray projections contrastfilled leg veins are taken [4]. The leg veins are "opacified" with contrast media, indicating the site of blood flow obstruction due to thrombosis. In both diagnosis situations, that is venous thrombosis is ruled in or out, further medical imaging tests are required to assess the decision of PE, which implies burden and probably additional radiation dose to patient. Nuclear medicine techniques (e.g., pulmonary ventilation perfusion scintigraphy) permit the visualization of the distribution of a radioactive substance (i.e., radiopharmaceutical) through planner gamma camera or single photon emission computerized tomography (SPECT) [21, 22]. In this technique, after the administration of gamma ray isotope tracer, the observation of airways and pulmonary blood vessels activity is made, hence the name, ventilation/perfusion (V/Q) scan. This scan remained as the traditional preferred imaging technique before the shift toward CT [4]. A mismatch of ventilated but not perfused lung tissue was considered as indicator for pulmonary embolism. Thus, it is indirect detection of an embolus by looking at the effects of an occlusion. A normal perfusion scan securely excluded pulmonary embolism, but was found in a minority of the patients that are suspected of PE, and thus, often further testing was needed [4]. The advantages of V/Q scan are that it is not invasive and less irradiant than CT, and may be more suitable for patients that are allergic to iodinated agents (CT). Its disadvantage is that the obtained image determines only regions of the lungs that are not correctly vascularized, nonobstructing "small" clots remaining invisible [4]. Moreover, the duration of the exam is in the order of 20 min, which is slower than other modalities. Several reports showed that the CT scan outweigh the V/Q scan by performing both lower rate of false-negative scans and lower number of "indeterminate scans" not yielding a definite diagnosis [4, 22]. The sequence of chest X-ray projections during the administration of contrast agent directly into the target vessels, also called as pulmonary angiography, is a reliable test for diagnosing PE. In this imaging scan, a catheter is inserted into a femoral vein and navigated through the heart toward the pulmonary arteries. Amounts of contrast media is injected several times at various locations of pulmonary vessels, and sequence of X-ray planner projections are obtained. It is used to provide a definitive diagnosis when other imaging tests fail [4]. Although conventional pulmonary angiography has great value in PE diagnosis, it suffers from being expensive, invasive with serious side effect, requiring expertise and supporting staff, and not readily available in most hospitals. Vascular Doppler ultrasound is a quick, noninvasive, and reliable technique [23]. It is painless and carries no risk. However, it provides less clinical information (e.g., number of clots and amount of obstruction) than other imaging techniques and very dependent on the experience of the examiner [4, 23]. Finally, several reports showed promising results for the assessment of PE with magnetic resonance imaging (MRI) [24, 25]. This modality is promising because images can be generated without radiation, and because it allows a combination of morphological and functional imaging (e.g., perfusion). However, MR has a lower spatial resolution than CT and much longer acquisition times (around 30 min as opposed to seconds in CT). #### 2.6. Treatment the vessel wall substantially reducing the arterial diameter. A clot that exhibits caves or canals Several scoring systems have been introduced to measure the severity of the PE clinic. For central PE, physicians utilize the Walsh, Miller, Qanadli, or Mastora score. While for peripheral PE, they use Marder, Arnesen, Mewissen (American Venous Registry), Porter, Ouriel, or Bjorgell scores. They are well summarized by Ghaye et al. [19, 20]. All these scores depend on the number of clots, location, and the percentage of obstruction. They all verified appropriate for assessing the severity of PE and treatment effectiveness, but are not much used in angiog- Diagnosing PE remains a challenge to physicians because the symptoms are unspecific and may not be present in all patients. The PE symptoms and risk factors (Section 2.1) are used to determine the probability of PE. Although biomarkers and laboratory tests are crucial to estimate the probability of PE, such as the electrocardiogram (ECG) and the measurement of percentage of cross-linked fibrin in the blood (D-dimer), the diagnostic decision is always based on radiographic findings from medical imaging procedures [4]. Different medical imaging techniques exist to "rule in" or "rule out" the presence of the PE. Each technique exhibits its strength and weakness and a shift toward the computerized tomography (CT) has A planner chest X-ray remains the first imaging step because it can rule out the conditions that mimic PE (e.g., a pneumothorax can cause chest pain similar to pain caused by acute PE), but it cannot exclude PE. Another X-ray imaging test is the lower extremity venous angiography, in which contrast media is injected via a foot vein, and several X-ray projections contrastfilled leg veins are taken [4]. The leg veins are "opacified" with contrast media, indicating the site of blood flow obstruction due to thrombosis. In both diagnosis situations, that is venous thrombosis is ruled in or out, further medical imaging tests are required to assess the decision Nuclear medicine techniques (e.g., pulmonary ventilation perfusion scintigraphy) permit the visualization of the distribution of a radioactive substance (i.e., radiopharmaceutical) through planner gamma camera or single photon emission computerized tomography (SPECT) [21, 22]. In this technique, after the administration of gamma ray isotope tracer, the observation of airways and pulmonary blood vessels activity is made, hence the name, ventilation/perfusion of PE, which implies burden and probably additional radiation dose to patient. within itself is also considered as "chronic." Figure 2. Illustration of acute and chronic PE. raphy clinics due to time it takes to manually assess them. 2.4. Load scores 118 Angiography 2.5. Diagnosis been approved. Untreated PE can be fatal with high mortality rate that can be decreased under rapid detection [6]. There are ranges of different types of treatment procedures [4]. They include hemodynamic and respiratory support, anticoagulation medications, thrombolytic therapy, surgical embolectomy, percutaneous catheter-directed therapy, and venous filter intervention, which are not without complications. The selection of treatment depends on the PE severity and prognosis. Generally, the obstruction is mild when only a few subsegmental vessels are blocked and it is severe when multiple segmental or a few lobar vessels are blocked. Mild PE is managed with clot-dissolving medication. Severe PE requires additional medical intervention, such as placement of a filter in the inferior vena cava, or clot removal with either a catheter or surgery. #### 2.7. Summary This section introduced clinical facts of pulmonary embolism. This includes concise of PE's epidemiology, predisposing factors, pathophysiology, classifications, and treatments procedures. The available diagnostic medical imaging systems, which are the decision-makers of the presence and severity of PE, were explained with their strengths and limitations, rationalizing the shift toward the use of CT. cone beam rotates 360 degrees around the patient. At each angle, the penetrated X-ray beam from patient strikes rings of X-ray digital detectors. The detectors measure the amount of X-ray absorption and then fed to computer which, in turn, processes these data to reconstruct a CTPA three dimensional (3D) volumetric radiographic data of the chest's tissues. The CTPA image quality is governed by set of technical parameters; the interested readers may revise Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 121 The volume of contrast media depends on patient weight, and may vary in [40 mL, 140 mL], also based on the dye concentration. Iodine is commonly used because of its relatively harmless interaction with the body and its solubility; the concentration is usually 250–350 mg/mL. Generally, a 4 or 5 mL/s is injected through a catheter in an antecubital vein. The rate of injection may increase in the advanced generations of MDCT, when acquisition time decreases in order to maintain sufficient iodine concentration within the vessels. The contrast peak happens after 10–25 s depending on the patient. Ideally, the scan should be complete before the radiographic dye reaches the left ventricle in the heart, as this may mean contrast has drained The CTPA 3D radiographic data are displayed in three orthogonal views (i.e., Digital images); these are the two dimensional (2D) axial, sagittal, and coronal cross sections (Figure 4). In each CTPA view, the pixel brightness (2D picture element) is proportional to amount of X-ray Understanding the radiographic CTPA appearance is important during the detection of PE. The lung parenchyma exhibits the lowest X-ray absorption so it appears as "black" regions clearly delineating the borders of lungs in the CTPA view. The soft tissues (i.e., the heart, muscles, fat, and upper constituents of the abdomen) appear as a radiolucent area (lower X-ray absorption). These regions appear in a form of various radiographic shades of gray levels (i.e., various optical intensities). On contrast, the regions of bones and the contrast agent in the pulmonary vessels are radio-opaque (higher X-ray absorption) and appear as bright (i.e., white) regions. Figure 4 demonstrates the radiographic appearance of these different tissues where the contrast agent (indicated by red letters "CA") looks brighter than the surrounding lung parenchyma, while remaining thorax constituents look with various intensities. Figure 4. The three CTPA orthogonal views radiographic appearance (axial, sagittal, coronal). one of the MDCT books such as the book by Seeram in Ref. [26]. from the pulmonary arteries, or require a larger dose of contrast media. absorption at tiny 3D cube (i.e., voxel) in the patient's chest. 3.2. CTPA radiographic appearance #### 3. Computerized tomography pulmonary angiography (CTPA) Computerized tomography pulmonary angiography (CTPA) is a multidetector computerized tomography (MDCT) scanner that acquires cross-sectional chest images during the administration of contrast agent [10]. This permits the visualization of the blood flow in pulmonary veins and arteries. This section describes the CTPA technology and its impact in diagnosing PE. Aspects covered include: (1) the basic of CTPA technology, (2) the CTPA radiographic appearance, (3) the radiographic features of suspicious PE, and (4) the clinical performance of CTPA scans including sensitivity, specificity, and pitfalls. #### 3.1. CTPA technology On CTPA, the patient's chest is exposed by a calibrated X-ray cone beam during the injection of contrast media into patient, as shown in Figure 3. The X-ray photons are absorbed (i.e., attenuated) by various structures within the chest. The amount of X-ray attenuations varies with accordance to the type and density of the tissues in the chest. The maximum absorption occurs in the dense bone and sites of contrast agent (i.e., pulmonary vessels); while the minimum absorption happens in the lung (i.e., air); the other thorax tissues (i.e., the heart, the muscles, and the upper parts of abdomen) lay in between those two structures. The X-ray Figure 3. Four rings MDCT technology (there are 256 rings in modern MDCT). cone beam rotates 360 degrees around the patient. At each angle, the penetrated X-ray beam from patient strikes rings of X-ray digital detectors. The detectors measure the amount of X-ray absorption and then fed to computer which, in turn, processes these data to reconstruct a CTPA three dimensional (3D) volumetric radiographic data of the chest's tissues. The CTPA image quality is governed by set of technical parameters; the interested readers may revise one of the MDCT books such as the book by Seeram in Ref. [26]. The volume of contrast media depends on patient weight, and may vary in [40 mL, 140 mL], also based on the dye concentration. Iodine is commonly used because of its relatively harmless interaction with the body and its solubility; the concentration is usually 250–350 mg/mL. Generally, a 4 or 5 mL/s is injected through a catheter in an antecubital vein. The rate of injection may increase in the advanced generations of MDCT, when acquisition time decreases in order to maintain sufficient iodine concentration within the vessels. The contrast peak happens after 10–25 s depending on the patient. Ideally, the scan should be complete before the radiographic dye reaches the left ventricle in the heart, as this may mean contrast has drained from the pulmonary arteries, or require a larger dose of contrast media. #### 3.2. CTPA radiographic appearance 2.7. Summary 120 Angiography izing the shift toward the use of CT. 3.1. CTPA technology This section introduced clinical facts of pulmonary embolism. This includes concise of PE's epidemiology, predisposing factors, pathophysiology, classifications, and treatments procedures. The available diagnostic medical imaging systems, which are the decision-makers of the presence and severity of PE, were explained with their strengths and limitations, rational- Computerized tomography pulmonary angiography (CTPA) is a multidetector computerized tomography (MDCT) scanner that acquires cross-sectional chest images during the administration of contrast agent [10]. This permits the visualization of the blood flow in pulmonary veins and arteries. This section describes the CTPA technology and its impact in diagnosing PE. Aspects covered include: (1) the basic of CTPA technology, (2) the CTPA radiographic appearance, (3) the radiographic features of suspicious PE, and (4) the clinical performance of On CTPA, the patient's chest is exposed by a calibrated X-ray cone beam during the injection of contrast media into patient, as shown in Figure 3. The X-ray photons are absorbed (i.e., attenuated) by various structures within the chest. The amount of X-ray attenuations varies with accordance to the type and density of the tissues in the chest. The maximum absorption occurs in the dense bone and sites of contrast agent (i.e., pulmonary vessels); while the minimum absorption happens in the lung (i.e., air); the other thorax tissues (i.e., the heart, the muscles, and the upper parts of abdomen) lay in between those two structures. The X-ray 3. Computerized tomography pulmonary angiography (CTPA) CTPA scans including sensitivity, specificity, and pitfalls. Figure 3. Four rings MDCT technology (there are 256 rings in modern MDCT). The CTPA 3D radiographic data are displayed in three orthogonal views (i.e., Digital images); these are the two dimensional (2D) axial, sagittal, and coronal cross sections (Figure 4). In each CTPA view, the pixel brightness (2D picture element) is proportional to amount of X-ray absorption at tiny 3D cube (i.e., voxel) in the patient's chest. Understanding the radiographic CTPA appearance is important during the detection of PE. The lung parenchyma exhibits the lowest X-ray absorption so it appears as "black" regions clearly delineating the borders of lungs in the CTPA view. The soft tissues (i.e., the heart, muscles, fat, and upper constituents of the abdomen) appear as a radiolucent area (lower X-ray absorption). These regions appear in a form of various radiographic shades of gray levels (i.e., various optical intensities). On contrast, the regions of bones and the contrast agent in the pulmonary vessels are radio-opaque (higher X-ray absorption) and appear as bright (i.e., white) regions. Figure 4 demonstrates the radiographic appearance of these different tissues where the contrast agent (indicated by red letters "CA") looks brighter than the surrounding lung parenchyma, while remaining thorax constituents look with various intensities. Figure 4. The three CTPA orthogonal views radiographic appearance (axial, sagittal, coronal). #### 3.3. PE radiographic features The contrast medium opacifies the bloodstream in the lung. In case of PE, the pulmonary vessel is either completely blocked, or passes around it. Thus, on CTPA view, the veins and arteries appear white where the bloodstream is present, and a thrombus can be observed as a dark spot inside the white mass. Figure 5 (left) illustrates examples of acute and chronic PE affecting the left main PA and right lobar artery, respectively. Acute thrombi appear as a hole, or concavity, in the vessel, while chronic clots are found on the edge of the vessel, with no concavity. Figure 5 (right) also shows example of peripheral clot (segmental). It is calculated as the ratio of TPs to the sum of FNs and TPs as illustrated in Eq. (1). On the other hand, the proportion of TN responses to the total number of patients, which do not have PE, is called the true-negative fraction (TNF) or specificity (the ability to exclude patients without PE). It Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… Consequently, sensitivity measures the reader's performance in detecting patients with clots, whereas specificity measures the reader's ability to avoid producing false responses. Specificity is usually derived from the false-positive fraction (FPF) as shown in Eq. (3). Also, the predictive value of a positive test PV(+) and the predictive value of false test PV(−) can be evaluated as in Eqs. (4) and (5). These are also alternatively referred as positive predictive value (PPV) and negative predictive value (NPV). As FP and FN increase (i.e., increment in Early investigations were reported in the 1990s regarding the impact of CTPA examinations in detecting PE. The results of these reports were reviewed by Rathbun in 2000 [27] and Hiorns in 2002 [28], showing that the sensitivity and specificity of CTPA may vary between the range of 51–100 and 81–100%, respectively. These initial results revealed the possibility of MDCT to Over the last decade, the clinical role of CTPA examination has undergone extensive scientific investigations [7–10]. The largest and most significant collaborative clinical trial was conducted in 2006 [10]. This study is well-known as PIOPED II (Prospective investigation of pulmonary TP <sup>+</sup> FN (1) http://dx.doi.org/10.5772/intechopen.79339 123 TN <sup>+</sup> FP (2) TP <sup>+</sup> FP (4) TN <sup>+</sup> FN (5) TN <sup>+</sup> FP = 1 − specificity (3) is calculated as the ratio of TNs to the sum of FPs and TNs as illustrated in Eq. (2). CTPA truth information Abnormal True positive (TP) False positive (FP) Normal False negative (FN) True negative (TN) Radiologist decision Abnormal (PE exists) Normal sensitivity <sup>=</sup> TPF <sup>=</sup> \_\_\_\_\_\_ TP specificity <sup>=</sup> \_\_\_\_\_\_ TN Table 1. Outcomes of radiologist interpretation of CTPA examinations. interpretation mistakes), the PPV and NPV decrease. PV(+) = \_\_\_\_\_\_ TP PV(−) <sup>=</sup> \_\_\_\_\_\_ TN diagnose PE. 3.4.2. CTPA's sensitivity, specificity, and negative and positive predictive value FPF <sup>=</sup> \_\_\_\_\_\_ FP #### 3.4. Clinical performance of CTPA scan The radiologist navigates the CTPA slices searching the presence of a clot. The diagnosis of PE is categorized in a yes-or-no decision, independent of the location and severity of emboli. When a defect (clot) is found in one slice, the adjacent neighbors are analyzed. The radiologist tracks the clot to the point where she/he knows for sure its anatomical location in the pulmonary vascular veins and arteries and assesses his/her conclusion. Sensitivity and specificity are two statistical parameters commonly used to evaluate any diagnostic test. In this subsection, these two parameters are introduced in association with CTPA examination. This leads to the discussion of the CTPA artifacts. #### 3.4.1. Terminology background Let us suppose that a population of patients, pathologically proved to have or not to have PE, was asked to undergo CTPA examination. Then, radiologists are asked to interpret these CTPA scans. The correct interpretation of a CTPA scan can be either a true-positive (TP) response (i.e., the correct detection of clots) or a true-negative (TN) response (i.e., the correct decision that there is no clot). In contrast, the false interpretation of a CTPA scan is described as either a false-positive (FP) response (i.e., the false suggestion of PE that does not exist) or a false-negative (FN) response (i.e., the PE is missed). These four possible categories of CTPA interpretation are illustrated in Table 1. The proportion of TP responses to the total number of pathologically proven PE patients is referred as the true-positive fraction (TPF) or the sensitivity (the ability to detect patients with PE). Figure 5. Examples of chronic and acute PE (left), and segmental PE (right) appearance on CTPA image. Table 1. Outcomes of radiologist interpretation of CTPA examinations. 3.3. PE radiographic features 122 Angiography 3.4. Clinical performance of CTPA scan to the discussion of the CTPA artifacts. interpretation are illustrated in Table 1. 3.4.1. Terminology background The contrast medium opacifies the bloodstream in the lung. In case of PE, the pulmonary vessel is either completely blocked, or passes around it. Thus, on CTPA view, the veins and arteries appear white where the bloodstream is present, and a thrombus can be observed as a dark spot inside the white mass. Figure 5 (left) illustrates examples of acute and chronic PE affecting the left main PA and right lobar artery, respectively. Acute thrombi appear as a hole, or concavity, in the vessel, while chronic clots are found on the edge of the vessel, with no The radiologist navigates the CTPA slices searching the presence of a clot. The diagnosis of PE is categorized in a yes-or-no decision, independent of the location and severity of emboli. When a defect (clot) is found in one slice, the adjacent neighbors are analyzed. The radiologist tracks the clot to the point where she/he knows for sure its anatomical location in the pulmonary vascular veins and arteries and assesses his/her conclusion. Sensitivity and specificity are two statistical parameters commonly used to evaluate any diagnostic test. In this subsection, these two parameters are introduced in association with CTPA examination. This leads Let us suppose that a population of patients, pathologically proved to have or not to have PE, was asked to undergo CTPA examination. Then, radiologists are asked to interpret these CTPA scans. The correct interpretation of a CTPA scan can be either a true-positive (TP) response (i.e., the correct detection of clots) or a true-negative (TN) response (i.e., the correct decision that there is no clot). In contrast, the false interpretation of a CTPA scan is described as either a false-positive (FP) response (i.e., the false suggestion of PE that does not exist) or a false-negative (FN) response (i.e., the PE is missed). These four possible categories of CTPA The proportion of TP responses to the total number of pathologically proven PE patients is referred as the true-positive fraction (TPF) or the sensitivity (the ability to detect patients with PE). Figure 5. Examples of chronic and acute PE (left), and segmental PE (right) appearance on CTPA image. concavity. Figure 5 (right) also shows example of peripheral clot (segmental). It is calculated as the ratio of TPs to the sum of FNs and TPs as illustrated in Eq. (1). On the other hand, the proportion of TN responses to the total number of patients, which do not have PE, is called the true-negative fraction (TNF) or specificity (the ability to exclude patients without PE). It is calculated as the ratio of TNs to the sum of FPs and TNs as illustrated in Eq. (2). $$sensitivity = TPF = \frac{TP}{TP + FN} \tag{1}$$ $$\text{specific} = \frac{\text{TN}}{\text{TN} + \text{FP}} \tag{2}$$ $$FPF = \frac{FP}{TN + FP} = 1 - \text{specificity} \tag{3}$$ Consequently, sensitivity measures the reader's performance in detecting patients with clots, whereas specificity measures the reader's ability to avoid producing false responses. Specificity is usually derived from the false-positive fraction (FPF) as shown in Eq. (3). Also, the predictive value of a positive test PV(+) and the predictive value of false test PV(−) can be evaluated as in Eqs. (4) and (5). These are also alternatively referred as positive predictive value (PPV) and negative predictive value (NPV). As FP and FN increase (i.e., increment in interpretation mistakes), the PPV and NPV decrease. $$PV(\text{+}) = \frac{TP}{TP + FP} \tag{4}$$ $$PV(-) = \frac{TN}{TN + FN} \tag{5}$$ #### 3.4.2. CTPA's sensitivity, specificity, and negative and positive predictive value Early investigations were reported in the 1990s regarding the impact of CTPA examinations in detecting PE. The results of these reports were reviewed by Rathbun in 2000 [27] and Hiorns in 2002 [28], showing that the sensitivity and specificity of CTPA may vary between the range of 51–100 and 81–100%, respectively. These initial results revealed the possibility of MDCT to diagnose PE. Over the last decade, the clinical role of CTPA examination has undergone extensive scientific investigations [7–10]. The largest and most significant collaborative clinical trial was conducted in 2006 [10]. This study is well-known as PIOPED II (Prospective investigation of pulmonary embolism diagnosis, second study). The dataset consisted of 824 patients who had enrolled for CTPA examination in the period 2001–2003 using 4, 8, and 16 rows MDCT devices. These CTPA scans were interpreted by different radiologists at remote clinical centers (i.e., hospitals) in the USA and Canada. The study reported sensitivity (i.e., the proportion of correct diagnosis of patients with PE) of 83% and specificity (i.e., the proportion of the correct diagnosis of patients without PE) of 96%. The high value of sensitivity means high TP and low FN (Eq. (1)), while high value of specificity means high TN and low FP (Eq. (2)). Based on the PE probability is low, intermediate, or high, the PPV and NPV was 58 and 96%, 92 and 89%, and 96 and 60%, respectively. On the other hand, based on the PE location is lobar, segmental, or subsegmental vessels, the PPV was 97, 68, and 25%, respectively. The defects at extreme sites of pulmonary vascular branches (segmental and subsegmental vessels) exhibit less observability, and consequently more challenging to radiologists, than lobar and main PA clots. or cardiac motion leading to inhomogeneous intravascular contrast, streak-artifacts near the superior vena cava due to beam hardening, incorrect timing resulting in insufficient intravascular contrast, or artifacts due to edge-enhancing image reconstruction. Further details of these artifacts are explained in MDCT technical books such as the reference number [26]. Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 125 This section explained the CTPA practice. The CTPA basic physics, technology, examination, and radiographic appearance of PE and other thorax tissues were explained. The sensitivity, specificity, and PPV and NPV of CTPA are outlined based on clinical trials and surveys in the last two decades. These reports concluded that that CTPA remains, at the time, as the first line diagnostic procedure providing the less invasive procedure, and highest sensitivity, specificity, and NPV among other imaging techniques. The CTPA artifacts, which may contribute to It is difficult and time consuming for a radiologist to navigate all CTPA orthogonal slices and find all emboli, this also depends on the radiologist experience. In PIOPED II, which was held at well-estimated clinical institutes, the average of 9.3% FP and 2.4 FN responses were reported [10]. This means that among 1000 suspicious PE patients, which is a daily small number of CTPA examination in any developed country, 93 patients may incorrectly diagnosed as having PE and may be asked to undergo further clinical tests, which is a clinical burden and probably additional X-ray radiation dose. Also, which is more critical, 24 patients may be incorrectly excluded of having PE and would leave the hospital without considering medication; these patients would be left under serious medical consequences that may be fatal. It is important to note again that FP and FN depend on radiologist experience, for example, in PIOPED II, it is possible for the NPV and PPV to be 58 and 60%, respectively. This lack of imperfection diagnosis may attribute to many reasons as explained in Section 3.4.3. Therefore, a computer-assisted detection (CAD) for the diagnosis of PE is desirable. This section aims to describe the CAD technology then explains the current state of how PE-CAD can contribute in improving patient health in CTPA clinic. This leads to explain the comments on CAD and the necessary recommendations. Comprehensive 3.5. Summary misdiagnosis, were mentioned. 4.1. CTPA-CAD definitions 4. Computer-aided detection of PE on CTPA views information on CAD technology in medical imaging can be found in Ref. [31]. A computer-aided detection (CAD) algorithm is an architecture of computer image analysis processes that yield, when applied to a CTPA examination, to the prompting of regions of suggestive pulmonary obstruction such as the presence of clots. Such prompting is often used as a "second opinion" to alert the radiologist to structures that, otherwise, might be overlooked [2, 3]. Figure 6 shows a CTPA slice (left) and the responses of the CTPA-CAD (right), which are indicated as red overlay. They are also called as CAD stimuli, candidates, or outputs. These CTPA-CAD responses are categorized to one of four possible groups, similarly to those explained in Section 3.4.1 describing the radiologist accuracy in interpreting CTPA A recent report, in 2015, was published by Dogan et al. in the Netherlands; this study reviewed different CTPA clinical trials and reported that the sensitivity and specificity of CTPA scans may vary between the range of 83–100% and 89–96%, respectively [8]. The NPV was 96–99% showing the high CTPA scan's certainty in ruling out PE; a negative CTPA can safely exclude PE. Estrada-Y-Martin and Oldham supervised a survey regarding the clinical practice in the diagnosis of PE in USA [9]. The survey included members (i.e., Intervention Radiologists) of the Society of Thoracic Radiology (524 members) and the Society of Interventional Radiologists (389 members). The surveyed members believed that CTPA examination is the gold standard to diagnose PE. This conclusion sustained previous study emphasizing that CTPA is the firstline imaging for the evaluation of PE [7]. These clinical trials and surveys have resulted in the worldwide acceptance of CTPA as the best method for the detection of PE. #### 3.4.3. CTPA artifacts Although the clinical reports accepted CTPA as best-reliable method with high sensitivity for diagnosing PE, their results also showed that FNs diagnosis, which are potentially life-threatening or a prospective health burden, may occur. For a radiologist, it can be difficult to detect all PE in the CTPA data [4, 10] for several reasons. In CTPA clinic, the radiologist is asked to examine stack of high resolution 2D CTPA images for single patient. Each 2D CTPA image is a 512 by 512 pixels. The stack builds a 3D CTPA volume of voxels (volume pixels), of which the size, in modern MDCT devices, is approximately 0.6 mm in every direction. Thus, a CTPA scan consists of millions of voxels have to be reviewed. Furthermore, the segmental and subsegmental vascular branches are quit complex; it is impossible to visualize all vascular structures within one CTPA image at a time. Therefore, radiologists usually revise the 3D CTPA volume several times examining only parts of the vascular system in the attempt not to miss an intravascular (sometimes very small) black dot indicating PE. A secure detection or exclusion of PE is therefore quite time-consuming and dependent on the experience of the radiologist. Additionally, diagnostic pitfalls may occur due to CTPA artifacts [29, 30]. Some artifacts leads to defects that imitate PE; this may include a poorly filled vein with contrast media, lymphoid tissue around the vessels, impacted bronchi mimic dark tubular structures, or parenchymal diseases altering pulmonary perfusion. Technical factors may also lead to artifact hampering the correct PE diagnosis; this include image noise due to low dose or obese patients, respiratory or cardiac motion leading to inhomogeneous intravascular contrast, streak-artifacts near the superior vena cava due to beam hardening, incorrect timing resulting in insufficient intravascular contrast, or artifacts due to edge-enhancing image reconstruction. Further details of these artifacts are explained in MDCT technical books such as the reference number [26]. #### 3.5. Summary embolism diagnosis, second study). The dataset consisted of 824 patients who had enrolled for CTPA examination in the period 2001–2003 using 4, 8, and 16 rows MDCT devices. These CTPA scans were interpreted by different radiologists at remote clinical centers (i.e., hospitals) in the USA and Canada. The study reported sensitivity (i.e., the proportion of correct diagnosis of patients with PE) of 83% and specificity (i.e., the proportion of the correct diagnosis of patients without PE) of 96%. The high value of sensitivity means high TP and low FN (Eq. (1)), while high value of specificity means high TN and low FP (Eq. (2)). Based on the PE probability is low, intermediate, or high, the PPV and NPV was 58 and 96%, 92 and 89%, and 96 and 60%, respectively. On the other hand, based on the PE location is lobar, segmental, or subsegmental vessels, the PPV was 97, 68, and 25%, respectively. The defects at extreme sites of pulmonary vascular branches (segmental and subsegmental vessels) exhibit less observability, and conse- A recent report, in 2015, was published by Dogan et al. in the Netherlands; this study reviewed different CTPA clinical trials and reported that the sensitivity and specificity of CTPA scans may vary between the range of 83–100% and 89–96%, respectively [8]. The NPV was 96–99% showing the high CTPA scan's certainty in ruling out PE; a negative CTPA can safely exclude PE. Estrada-Y-Martin and Oldham supervised a survey regarding the clinical practice in the diagnosis of PE in USA [9]. The survey included members (i.e., Intervention Radiologists) of the Society of Thoracic Radiology (524 members) and the Society of Interventional Radiologists (389 members). The surveyed members believed that CTPA examination is the gold standard to diagnose PE. This conclusion sustained previous study emphasizing that CTPA is the firstline imaging for the evaluation of PE [7]. These clinical trials and surveys have resulted in the Although the clinical reports accepted CTPA as best-reliable method with high sensitivity for diagnosing PE, their results also showed that FNs diagnosis, which are potentially life-threatening or a prospective health burden, may occur. For a radiologist, it can be difficult to detect all PE in the CTPA data [4, 10] for several reasons. In CTPA clinic, the radiologist is asked to examine stack of high resolution 2D CTPA images for single patient. Each 2D CTPA image is a 512 by 512 pixels. The stack builds a 3D CTPA volume of voxels (volume pixels), of which the size, in modern MDCT devices, is approximately 0.6 mm in every direction. Thus, a CTPA scan consists of millions of voxels have to be reviewed. Furthermore, the segmental and subsegmental vascular branches are quit complex; it is impossible to visualize all vascular structures within one CTPA image at a time. Therefore, radiologists usually revise the 3D CTPA volume several times examining only parts of the vascular system in the attempt not to miss an intravascular (sometimes very small) black dot indicating PE. A secure detection or exclusion of PE is therefore quite time-consuming and dependent on the experience of the radiologist. Additionally, diagnostic pitfalls may occur due to CTPA artifacts [29, 30]. Some artifacts leads to defects that imitate PE; this may include a poorly filled vein with contrast media, lymphoid tissue around the vessels, impacted bronchi mimic dark tubular structures, or parenchymal diseases altering pulmonary perfusion. Technical factors may also lead to artifact hampering the correct PE diagnosis; this include image noise due to low dose or obese patients, respiratory quently more challenging to radiologists, than lobar and main PA clots. worldwide acceptance of CTPA as the best method for the detection of PE. 3.4.3. CTPA artifacts 124 Angiography This section explained the CTPA practice. The CTPA basic physics, technology, examination, and radiographic appearance of PE and other thorax tissues were explained. The sensitivity, specificity, and PPV and NPV of CTPA are outlined based on clinical trials and surveys in the last two decades. These reports concluded that that CTPA remains, at the time, as the first line diagnostic procedure providing the less invasive procedure, and highest sensitivity, specificity, and NPV among other imaging techniques. The CTPA artifacts, which may contribute to misdiagnosis, were mentioned. #### 4. Computer-aided detection of PE on CTPA views It is difficult and time consuming for a radiologist to navigate all CTPA orthogonal slices and find all emboli, this also depends on the radiologist experience. In PIOPED II, which was held at well-estimated clinical institutes, the average of 9.3% FP and 2.4 FN responses were reported [10]. This means that among 1000 suspicious PE patients, which is a daily small number of CTPA examination in any developed country, 93 patients may incorrectly diagnosed as having PE and may be asked to undergo further clinical tests, which is a clinical burden and probably additional X-ray radiation dose. Also, which is more critical, 24 patients may be incorrectly excluded of having PE and would leave the hospital without considering medication; these patients would be left under serious medical consequences that may be fatal. It is important to note again that FP and FN depend on radiologist experience, for example, in PIOPED II, it is possible for the NPV and PPV to be 58 and 60%, respectively. This lack of imperfection diagnosis may attribute to many reasons as explained in Section 3.4.3. Therefore, a computer-assisted detection (CAD) for the diagnosis of PE is desirable. This section aims to describe the CAD technology then explains the current state of how PE-CAD can contribute in improving patient health in CTPA clinic. This leads to explain the comments on CAD and the necessary recommendations. Comprehensive information on CAD technology in medical imaging can be found in Ref. [31]. #### 4.1. CTPA-CAD definitions A computer-aided detection (CAD) algorithm is an architecture of computer image analysis processes that yield, when applied to a CTPA examination, to the prompting of regions of suggestive pulmonary obstruction such as the presence of clots. Such prompting is often used as a "second opinion" to alert the radiologist to structures that, otherwise, might be overlooked [2, 3]. Figure 6 shows a CTPA slice (left) and the responses of the CTPA-CAD (right), which are indicated as red overlay. They are also called as CAD stimuli, candidates, or outputs. These CTPA-CAD responses are categorized to one of four possible groups, similarly to those explained in Section 3.4.1 describing the radiologist accuracy in interpreting CTPA groups. The first group describes the marketable CTPA-CAD available from famous vendors such as SIEMENS, PHILIPS, and GE. The second group describes underconstruction CTPA-CAD software. In general, the review focuses on the main image analysis aspects implemented in the CAD algorithm (if it was disclosed), the method's performance, size of dataset, and the characteristics of CTPA images, particularly the slice thickness that has direct impact on diagnosing PE. Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 127 These are the CTPA-CAD methods that were developed at famous medical imaging vendors, for example, Philips (Philips Healthcare, Best, The Netherlands), Siemens (Siemens Medical Solutions, Germany), and GE (General Electric Healthcare, USA). They have been FDA approved and tested in a clinical environment. Since they are offered in the medical imaging market, the methodology (i.e., sequence of image analysis aspects) is not disclosed. This subsection presents For CTPA-CAD made by Philips, two clinical trials by Wittenberg et al. [33, 34] and one by Lahiji et al. [35] were reported. The first trial by Wittenberg et al. was in 2010; they tested the CAD output on 225 negative and 67 positive CTPA scans (292 retrospective scans) acquired from 16 and 64 MDCT devices with 0.9 or 1 mm slice thickness [33]. For "first reader" basis, the results showed 94 and 21% sensitivity and specificity, in turn. The rate of FP stimuli was 4.7 per examination. The NPV was 92% indicating possibility to serve as reassurance for less experienced readers. The CAD also found seven FN scans, two at segmental and five at subsegmental vessels. The second trial was published in 2012 [34]. They examined the performance of six radiologists with and without the CTPA-CAD on 158 negative and 51 positive retrospective CTPA scans, which were obtained from 16 and 64 MDCT devices with 0.9 or 1 mm slice thickness. For "second reader" basis, there was no significant change in specificity, but the sensitivity increased in the range from 12% (expert reader) to 12 (radiologist-in-training or less expert). The rate of FP was 4.9 per scan. Lahiji et al., in 2014 [35], evaluated 26 negative and 40 positive CTPA scans from 256 MDCT device with 0.9 mm slice thickness. Although their study was to compare two different CTPA image reconstruction algorithms (the iterative and filtered back projection techniques), a CTPA-CAD software was used for the assessment. For "first reader" basis, the reported sensitivity and specificity for both image reconstruction techniques were in the reports explaining their clinical performance "first reader" or "second reader" basis. the range 85–97.2 and 26.9–61.5, respectively. The rate of FP was in the range 1.5–3.6. increased in the range 3–7%. The FP rate was 4.1 per scan. For CTPA-CAD made by Siemens, Lee et al. studied 16 negative and 21 positive CTPA scans acquired from dual energy CT angiography (DCTA) with 1.2 mm slice thickness [36]. When both readers used the CAD prototype, the sensitivity was improved by approximately of 5% without significant loss in specificity. The rate of FP was 3.5 per examination. Blockmon et al. evaluated 79 CTPA scans (36 positive and 43 negative) from 16 and 64 CTPA devices at 1 mm slice thickness [37]. The radiologists, without the aid of CTPA-CAD, scored 84.4 and 92.6 sensitivity and specificity, in turn. For "first reader" basis, the CTPA-CAD achieved 93.8% sensitivity and 14.9% specificity; while it achieved 92.2% and 88.3% for "second reader" basis. The FP rate was 3.5 per scan. Earlier study was by Engelke et al. in 2008 [38]. They studied 56 positive CTPA scans obtained from 64 MDCT device with 0.6-mm slice thickness. On "second reader" basis, the four readers reported no significant loss of specificity while sensitivity 4.2.1. Commercial CTPA-CAD Figure 6. Types of assessment of CTPA-CAD stimuli on CTPA image. scans. They are: the TP, FP, TN, FNs groups. Figure 6 (right) illustrates TP (i.e., correct prompt of clot) and FP (incorrect prompt of clot) stimuli, indicated with red and green arrows, respectively. There are two clots that were not prompted, so they are FN stimuli, which are indicated with blue arrow. The remaining pulmonary vessels, which were not prompted as PE, are the TN stimuli. As with radiologist, it is desirable that a CTPA-CAD algorithm leads to the highest TP and TN, while it yields the lowest possible FP and FN stimuli. The performance of the CAD software can be tested "per clot" or "per patient" basis. For "per clot" basis, the CTPA-CAD responses are counted in comparison to truth of all actual PE occurrences (i.e., all clots) in the CTPA examination. While for "per patient" basis, it is not important for the CTPA-CAD to find all thrombi. Additionally, the CTPA-CAD responses can be evaluated on "first reader" or "second reader" basis. The "first reader" analysis, which is also called as "standalone performance," refers to the outcomes of the CTPA-CAD software in a defined dataset of clinical CTPA scans without interference of radiologists. The "second reader" performance means that the CTPA-CAD output is utilized to support the radiologist decision after he/she has assessed the examination primarily unassisted and uses the results of CTPA-CAD only to refine his/her judgment. As explained in Section 3.4, in current clinical CTPA practice, the diagnosis of PE is divided in a yes-or-no decision, regardless to the colts' number, location, and severity of emboli [4]. It is, therefore, less important that a CTPA-CAD system finds all emboli in a CTPA scan. More significant tasks of CTPA-CAD seem to be: to increase the radiologist's certainty to rule in or out PE (i.e., improve sensitivity and specificity), to reduce the CTPA interpretation time, and to decrease inter-reader variability [2, 3]. Thus, most researchers prefer the "per patient" basis to evaluate the performance of CTPA-CAD because it is more clinically relevant than the "per clot" basis. #### 4.2. CTPA-CAD performance The first study on a CTPA-CAD algorithm was published in 2002 by Masutani et al. [32] for a group of 19 high-quality CTPA examinations; they reported a sensitivity on a per clot basis of 100% with 7.7 false-positive findings per examination. Since then, numerous methods, utilizing various image analysis concepts, have been tested by different vendors and image analysis groups. Some CTPA-CAD algorithms have attained clinical merit, while some are still under development. This section reviews the performance of these methods, dividing them into two groups. The first group describes the marketable CTPA-CAD available from famous vendors such as SIEMENS, PHILIPS, and GE. The second group describes underconstruction CTPA-CAD software. In general, the review focuses on the main image analysis aspects implemented in the CAD algorithm (if it was disclosed), the method's performance, size of dataset, and the characteristics of CTPA images, particularly the slice thickness that has direct impact on diagnosing PE. #### 4.2.1. Commercial CTPA-CAD scans. They are: the TP, FP, TN, FNs groups. Figure 6 (right) illustrates TP (i.e., correct prompt of clot) and FP (incorrect prompt of clot) stimuli, indicated with red and green arrows, respectively. There are two clots that were not prompted, so they are FN stimuli, which are indicated with blue arrow. The remaining pulmonary vessels, which were not prompted as PE, are the TN stimuli. As with radiologist, it is desirable that a CTPA-CAD algorithm leads to the high- The performance of the CAD software can be tested "per clot" or "per patient" basis. For "per clot" basis, the CTPA-CAD responses are counted in comparison to truth of all actual PE occurrences (i.e., all clots) in the CTPA examination. While for "per patient" basis, it is not important for the CTPA-CAD to find all thrombi. Additionally, the CTPA-CAD responses can be evaluated on "first reader" or "second reader" basis. The "first reader" analysis, which is also called as "standalone performance," refers to the outcomes of the CTPA-CAD software in a defined dataset of clinical CTPA scans without interference of radiologists. The "second reader" performance means that the CTPA-CAD output is utilized to support the radiologist decision after he/she has assessed the examination primarily unassisted and uses the results As explained in Section 3.4, in current clinical CTPA practice, the diagnosis of PE is divided in a yes-or-no decision, regardless to the colts' number, location, and severity of emboli [4]. It is, therefore, less important that a CTPA-CAD system finds all emboli in a CTPA scan. More significant tasks of CTPA-CAD seem to be: to increase the radiologist's certainty to rule in or out PE (i.e., improve sensitivity and specificity), to reduce the CTPA interpretation time, and to decrease inter-reader variability [2, 3]. Thus, most researchers prefer the "per patient" basis to evaluate the performance of CTPA-CAD because it is more clinically relevant than the "per The first study on a CTPA-CAD algorithm was published in 2002 by Masutani et al. [32] for a group of 19 high-quality CTPA examinations; they reported a sensitivity on a per clot basis of 100% with 7.7 false-positive findings per examination. Since then, numerous methods, utilizing various image analysis concepts, have been tested by different vendors and image analysis groups. Some CTPA-CAD algorithms have attained clinical merit, while some are still under development. This section reviews the performance of these methods, dividing them into two est TP and TN, while it yields the lowest possible FP and FN stimuli. Figure 6. Types of assessment of CTPA-CAD stimuli on CTPA image. of CTPA-CAD only to refine his/her judgment. clot" basis. 126 Angiography 4.2. CTPA-CAD performance These are the CTPA-CAD methods that were developed at famous medical imaging vendors, for example, Philips (Philips Healthcare, Best, The Netherlands), Siemens (Siemens Medical Solutions, Germany), and GE (General Electric Healthcare, USA). They have been FDA approved and tested in a clinical environment. Since they are offered in the medical imaging market, the methodology (i.e., sequence of image analysis aspects) is not disclosed. This subsection presents the reports explaining their clinical performance "first reader" or "second reader" basis. For CTPA-CAD made by Philips, two clinical trials by Wittenberg et al. [33, 34] and one by Lahiji et al. [35] were reported. The first trial by Wittenberg et al. was in 2010; they tested the CAD output on 225 negative and 67 positive CTPA scans (292 retrospective scans) acquired from 16 and 64 MDCT devices with 0.9 or 1 mm slice thickness [33]. For "first reader" basis, the results showed 94 and 21% sensitivity and specificity, in turn. The rate of FP stimuli was 4.7 per examination. The NPV was 92% indicating possibility to serve as reassurance for less experienced readers. The CAD also found seven FN scans, two at segmental and five at subsegmental vessels. The second trial was published in 2012 [34]. They examined the performance of six radiologists with and without the CTPA-CAD on 158 negative and 51 positive retrospective CTPA scans, which were obtained from 16 and 64 MDCT devices with 0.9 or 1 mm slice thickness. For "second reader" basis, there was no significant change in specificity, but the sensitivity increased in the range from 12% (expert reader) to 12 (radiologist-in-training or less expert). The rate of FP was 4.9 per scan. Lahiji et al., in 2014 [35], evaluated 26 negative and 40 positive CTPA scans from 256 MDCT device with 0.9 mm slice thickness. Although their study was to compare two different CTPA image reconstruction algorithms (the iterative and filtered back projection techniques), a CTPA-CAD software was used for the assessment. For "first reader" basis, the reported sensitivity and specificity for both image reconstruction techniques were in the range 85–97.2 and 26.9–61.5, respectively. The rate of FP was in the range 1.5–3.6. For CTPA-CAD made by Siemens, Lee et al. studied 16 negative and 21 positive CTPA scans acquired from dual energy CT angiography (DCTA) with 1.2 mm slice thickness [36]. When both readers used the CAD prototype, the sensitivity was improved by approximately of 5% without significant loss in specificity. The rate of FP was 3.5 per examination. Blockmon et al. evaluated 79 CTPA scans (36 positive and 43 negative) from 16 and 64 CTPA devices at 1 mm slice thickness [37]. The radiologists, without the aid of CTPA-CAD, scored 84.4 and 92.6 sensitivity and specificity, in turn. For "first reader" basis, the CTPA-CAD achieved 93.8% sensitivity and 14.9% specificity; while it achieved 92.2% and 88.3% for "second reader" basis. The FP rate was 3.5 per scan. Earlier study was by Engelke et al. in 2008 [38]. They studied 56 positive CTPA scans obtained from 64 MDCT device with 0.6-mm slice thickness. On "second reader" basis, the four readers reported no significant loss of specificity while sensitivity increased in the range 3–7%. The FP rate was 4.1 per scan. Wittenberg et al. compared the performance of three different CTPA-CAD systems made by Philips, GE, and Siemens [39]. They studied three groups of CTPA scans from 64 MDCT devices made in Philips, GE, and Siemens with 0.6, 0.9, and 1.5 mm slice thickness, in order. The three groups of dataset contained 38, 39, and 38 positive CTPA scans; it also contained 40, 40, 37 negative CTPA scans, respectively, according to each CTPA group. For "first reader" basis, the comparison yielded a sensitivity of 100, 97, and 92, specificity of 18, 15, and 13, and FP rate of 4.5, 6.2, and 3.7, respectively, to each group. Author CTPA PIOPED II [10] Wittenberg [34] Blackmoon [37] Wittenberg [33] Blackmoon [37] Wittenberg [39] Wittenberg [39] Wittenberg [39] Wittenberg [34] Blackmoon [37] interpretation protocol CTPA-CAD Philips, Siemens, Consensus between Radiologist and CTPA-CAD dataset used in the clinical trials. or GE CTPA Dataset size Sensitivity Radiologists 192 632 83 96 Not Lee [36] 21 16 90.9 93.3–100 1.2 Engelke [38] 56 — 77–93 — 0.6 Lahiji [35] 40 26 85–97.5 26.9–61.5 1.5–3.6 0.9 Lee [36] 21 16 95.5 93.3–100 3.5 1.2 Engelke [38] 56 — 84–96 — 4.1 0.6 Table 2. "Per patient" CTPA interpretation by the radiologist, "first reader" marketable CTPA-CAD software, and "second reader" CTPA-CAD software, showing sensitivity, specificity, rate of FP responses, and the characteristics of Negative CTPA (%) Specificity (%) device Positive Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… 51 158 78–94 89–98 0.9–1.0 36 43 84.4 92.6 1.0 67 225 94 21 4.7 0.9–1.0 36 43 93.8 14.9 3.5 1.0 38 40 100 18 4.5 0.6 39 40 97 15 6.2 0.9 38 37 92 13 3.7 1.5 51 158 90–96 91–98 4.9 0.9–1 36 43 92.2 88.3 3.5 1.0 FP rate per scan http://dx.doi.org/10.5772/intechopen.79339 applicable Slice thickness (mm), MDCT 129 4, 8, 16 rows MDCT 16, 64 rows MDCT 16, 64 rows MDCT 64 rows MDCT 16, 64 rows MDCT 256 rows MDCT 16, 64 rows MDCT 64 rows MDCT 64 rows MDCT 64 rows MDCT 16, 64 MDCT 16, 64 rows MDCT 64 rows MDCT DCTA DCTA 1.25 Table 2 summarizes the results of previous trials of different CTPA-CAD systems from different vendors. For comparison, the table also includes the results from PIOPED II (the largest clinical trial) explained in Section 3.4.2. The table illustrates the CTPA clinical performance once CTPA images are interpreted by the radiologist, the CTPA-CAD software (CAD first Reader basis), consensus between radiologist and the CTPA-CAD software (CAD second reader basis). The table consists of 7 main columns. The first column shows the author with the reference number. The second column indicates the interpretation procedure type (radiologist, the CTPA-CAD software, consensus of Radiologist and the CTPA-CAD software). The third column demonstrates the dataset's size indicating the number of positive and negative CTPA scans. The fourth, fifth, and sixth column indicate the performance in term of sensitivity, specificity and rate of FP, in order. Finally, the last column shows slice thickness used to acquire CTPA image. Table 3 shows the range values of the sensitivity, specificity, and FP rate for each diagnosis protocol, which are reported in Table 2. The findings listed in Tables 2 and 3 indicate the following: Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 129 Wittenberg et al. compared the performance of three different CTPA-CAD systems made by Philips, GE, and Siemens [39]. They studied three groups of CTPA scans from 64 MDCT devices made in Philips, GE, and Siemens with 0.6, 0.9, and 1.5 mm slice thickness, in order. The three groups of dataset contained 38, 39, and 38 positive CTPA scans; it also contained 40, 40, 37 negative CTPA scans, respectively, according to each CTPA group. For "first reader" basis, the comparison yielded a sensitivity of 100, 97, and 92, specificity of 18, 15, and 13, and Table 2 summarizes the results of previous trials of different CTPA-CAD systems from different vendors. For comparison, the table also includes the results from PIOPED II (the largest clinical trial) explained in Section 3.4.2. The table illustrates the CTPA clinical performance once CTPA images are interpreted by the radiologist, the CTPA-CAD software (CAD first Reader basis), consensus between radiologist and the CTPA-CAD software (CAD second reader basis). The table consists of 7 main columns. The first column shows the author with the reference number. The second column indicates the interpretation procedure type (radiologist, the CTPA-CAD software, consensus of Radiologist and the CTPA-CAD software). The third column demonstrates the dataset's size indicating the number of positive and negative CTPA scans. The fourth, fifth, and sixth column indicate the performance in term of sensitivity, specificity and rate of FP, in order. Finally, the last column shows slice thickness used to acquire CTPA image. Table 3 shows the range values of the sensitivity, specificity, and FP rate 1. If CTPA scan is interpreted by radiologist only, the sensitivity and specificity may not reach 100%. The sensitivity range from 77.7% (for radiologist-in-training or less experience) to 94% (expert radiologist). While the specificity varies in the range 89–98% indicating almost perfect performance in excluding PE. The 100% specificity reported by Lee et al. [36] (Table 2) was reported on small number of negative CTPA scans, so it cannot be 2. For CTPA-CAD "first reader" basis, the marketable CTPA-CAD methods can score reliable high sensitivity, which can exceed the performance of an expert radiologist. However, the specificity is low (~20% in all reports in Table 2) due to 3.4–4.9 FP stimuli per CTPA scan. The 61.5 specificity reported by Lahiji et al. [35] is concluded from applying iterative 3. For CTPA-CAD "second reader" basis, the CAD can improve radiologists' sensitivity up to 7%. This increment in sensitivity coincides with no significant change in specificity. This enhancement can be substantial for inexperienced radiologist as reported by Wittenberg et al. [34], which one of the radiologists scored 90% with the aid of CTPA-CAD in compari- 4. The results were obtained on different MDCT devices, thus the performance of CTPA-CAD is independent of scanner type. However, it is relevant to image quality and scanning protocols such as slice thickness. Actually, the slice thickness has significant impact on PE diagnosis. For example, Jung et al. [40] analyzed 15 positive and 25 negative CTPA scans acquired with slice thicknesses of 0.625, 1.3, and 2.5 mm from 64 MDCT device. As FP rate of 4.5, 6.2, and 3.7, respectively, to each group. for each diagnosis protocol, which are reported in Table 2. The findings listed in Tables 2 and 3 indicate the following: generalized. 128 Angiography reconstruction that is under research. son to 78% without the CAD assistance. Table 2. "Per patient" CTPA interpretation by the radiologist, "first reader" marketable CTPA-CAD software, and "second reader" CTPA-CAD software, showing sensitivity, specificity, rate of FP responses, and the characteristics of dataset used in the clinical trials. superfluous sensitivity of CTPA-CAD. To reduce the FP rate, Tajbakhsh [45] employed 3D presentation of PE and blood vessels, coupled to neural network, to produce 2 FP per CTPA scan (Table 4). Al-hinnawi et al. [46] suggested another 3D technique, but simpler than neural networks that requires training and calibration on dataset characteristics, to reduce the FP rate. In the final step of their CTPA-CAD system, the stimuli from three CTPA views were orthogonally recombined to produce a single interactive 3D display of PE candidates from the CTPA case, as illustrated in Figure 7. Thus, this would permit, in a single analysis instead of slice by slice analysis, the assessment of CAD performance on the aggregated CAD responses on the three CTPA views of each patient. This could reduce time, and consequently reduce burden to radiologist. Clots Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 131 the CAD system can be tuned in accordance with the variations in CTPA acquisition settings due to patient differences, which was not employed in previous marketable CTPA-CAD systems or underconstruction CTPA-CAD prototypes. This reduces the variations in CAD outputs due to variation in patient preparation such as slice thickness. They reported that this approach would reduce the FP rate of from CAD systems, such as those in Tables 2 and 4, by 30% while it CTPA-CAD systems are image quality dependent [2, 3, 47, 48]. As for any X-ray imaging technique, the CTPA image quality is ruled by factors related to subject variance, acquisition parameters, patient preparation, and dose management [4, 49]. The subject variances such as Figure 7. Three dimensional visualization of CTPA-CAD output (courtesy from radiological physics and technology no. increases or ascertains the correct rate of CTPA-CAD's TP stimuli as much as 27%. were retained based on the voxel size of the CTPA scan. Thus, that are bigger in size than 1 mm3 JSMP30–180024). 4.3. Factors affecting CTPA-CAD performance Table 3. "Per patient" range of the values of sensitivity, specificity, rate of FP responses, based on each protocol of CTPA interpretation. slice thickness increases, there was significant decrease of PE diagnosis of lobar, segmental, subsegmental clots on both the axial and coronal CTPA views. They concluded that a slice thickness of 1 mm is a must to achieve high sensitivity, particularly the subsegmental PE. This impact is applicable to CTPA-CAD and must be considered in any CAD prototype [2, 3]. This matter, among other variables, is further described in Section 4.3. Consequently, the marketable CTPA-CAD software can increase reader sensitivity for the detection of PE, particularly the segmental and subsegmental pulmonary clots, and enforce reader confidence for the diagnosis of PE without significant loss of specificity. This rise in sensitivity means less FN CTPA scans (Eq. (1)), thus improving patient health. #### 4.2.2. Underconstruction CTPA-CAD There are nonmarketable CAD systems under construction by researchers. They share the employment of CTPA pulmonary vessels segmentation, candidate clot detection, and texture and feature computation with/without morphology analysis on 2D and 3D levels. However, they explored recent advances in computer sciences to reduce the FP rate such as complex mathematical classifier [41–43] and artificial intelligence (e.g., neural networks) [44, 45]. Thus, they reported their results on "per clot" basis. The results in Table 4 demonstrate that the "per clot" CTPA-CAD's sensitivity is in the range from 63–80%. This qualifies those CAD prototypes to be tested on "per patient" basis. However, the rate of FP stimuli is still high, so a low specificity value, as those in Table 2, is again very likely to happen. False-positive stimuli are the main burden hindering radiologist from accepting the Table 4. "Per clot" sensitivity and FP rate for underconstruction CTPA-CAD prototypes. superfluous sensitivity of CTPA-CAD. To reduce the FP rate, Tajbakhsh [45] employed 3D presentation of PE and blood vessels, coupled to neural network, to produce 2 FP per CTPA scan (Table 4). Al-hinnawi et al. [46] suggested another 3D technique, but simpler than neural networks that requires training and calibration on dataset characteristics, to reduce the FP rate. In the final step of their CTPA-CAD system, the stimuli from three CTPA views were orthogonally recombined to produce a single interactive 3D display of PE candidates from the CTPA case, as illustrated in Figure 7. Thus, this would permit, in a single analysis instead of slice by slice analysis, the assessment of CAD performance on the aggregated CAD responses on the three CTPA views of each patient. This could reduce time, and consequently reduce burden to radiologist. Clots that are bigger in size than 1 mm3 were retained based on the voxel size of the CTPA scan. Thus, the CAD system can be tuned in accordance with the variations in CTPA acquisition settings due to patient differences, which was not employed in previous marketable CTPA-CAD systems or underconstruction CTPA-CAD prototypes. This reduces the variations in CAD outputs due to variation in patient preparation such as slice thickness. They reported that this approach would reduce the FP rate of from CAD systems, such as those in Tables 2 and 4, by 30% while it increases or ascertains the correct rate of CTPA-CAD's TP stimuli as much as 27%. #### 4.3. Factors affecting CTPA-CAD performance slice thickness increases, there was significant decrease of PE diagnosis of lobar, segmental, subsegmental clots on both the axial and coronal CTPA views. They concluded that a slice thickness of 1 mm is a must to achieve high sensitivity, particularly the subsegmental PE. This impact is applicable to CTPA-CAD and must be considered in any CAD prototype Table 3. "Per patient" range of the values of sensitivity, specificity, rate of FP responses, based on each protocol of CTPA 84–96 88.3–100 3.5–4.9 CTPA interpretation protocol Sensitivity (%) Specificity (%) FP rate per scan Radiologists 77–94 89–98 — CAD (Philips, Siemens, or GE) 85–100 13–61.5 1.5–6.2 Consequently, the marketable CTPA-CAD software can increase reader sensitivity for the detection of PE, particularly the segmental and subsegmental pulmonary clots, and enforce reader confidence for the diagnosis of PE without significant loss of specificity. This rise in There are nonmarketable CAD systems under construction by researchers. They share the employment of CTPA pulmonary vessels segmentation, candidate clot detection, and texture and feature computation with/without morphology analysis on 2D and 3D levels. However, they explored recent advances in computer sciences to reduce the FP rate such as complex mathematical classifier [41–43] and artificial intelligence (e.g., neural networks) [44, 45]. Thus, The results in Table 4 demonstrate that the "per clot" CTPA-CAD's sensitivity is in the range from 63–80%. This qualifies those CAD prototypes to be tested on "per patient" basis. However, the rate of FP stimuli is still high, so a low specificity value, as those in Table 2, is again very likely to happen. False-positive stimuli are the main burden hindering radiologist from accepting the Author Number of clots in CTPA cases Sensitivity (%) FP rate per scan Bouma [41] 318 in 57 positive CTPA 63 4.9 Zhou [42] 595 in 59 positive CTPA 80 22.6 Zhou [43] 537 in 50 positive CTPA (PIOPED II) 80 8.6 Park [44] 44 in 18 positive CTPA 63.2 18.4 Tajbakhsh [45] 326 in 121 positive CTPA 83 2 Table 4. "Per clot" sensitivity and FP rate for underconstruction CTPA-CAD prototypes. [2, 3]. This matter, among other variables, is further described in Section 4.3. sensitivity means less FN CTPA scans (Eq. (1)), thus improving patient health. 4.2.2. Underconstruction CTPA-CAD they reported their results on "per clot" basis. Consensus between radiologist and CAD (Philips, Siemens, GE) 130 Angiography interpretation. CTPA-CAD systems are image quality dependent [2, 3, 47, 48]. As for any X-ray imaging technique, the CTPA image quality is ruled by factors related to subject variance, acquisition parameters, patient preparation, and dose management [4, 49]. The subject variances such as Figure 7. Three dimensional visualization of CTPA-CAD output (courtesy from radiological physics and technology no. JSMP30–180024). age, sex, race, presence of risk factors, prevalence and morphology of the clots, they do not have impact on CTPA image quality; so they should not influence the CTPA-CAD outcome. Thus, there is no need to calibrate the CAD system based on different patients or countries. specificity drops dramatically and become much less than the radiologist performance in case of "first reader" basis. Studies showed that the best operating scenario is the "second reader" basis because it improves sensitivity, which means less FN results, while it guarantees no significant change in specificity, in comparison to human observer performance. The factors affecting the CTPA-CAD output were described at the end of this section, this yielded to sug- Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 133 This chapter presents comprehensive current status knowledge in the PE angiography clinic. The clinical proofs of thrombosis are explained in Section 2. Knowledge of PE epidemiology, predisposing factors, pathophysiology, classifications, diagnostic medical imaging modalities, and treatments procedures are described. Then, in Section 3, concentration is focused on describing the CTPA technology that is the clinically accepted as the best first-line imaging procedure in PE diagnosis because it is fast, lowest invasive, and the highest sensitivity procedure, among other medical imaging modalities. Issues covered are CTPA physics, technology, examination, radiographic appearance, and PE features, the clinical trials in term of sensitivity, specificity, PPV, and NPV, are explained. Section 4 provides demonstration of the art of CAD system and their influence on improving the PE diagnoses by being eligible to reduce miss diagnosis by radiologist. Advantages and disadvantages of assessing CTPA-CAD performance with regard to "per patient", "per clot", "first reader", and "second reader" basis are explained. Results suggested that the "second reader" along with "per patient" basis is the best scenario to utilize PE CAD systems, because this raises the sensitivity without effect on specificity of radiologist performance. Precautions and recommendations of optimal practice of PE CAD prototypes are described indicating the necessity to follow the operation manual specifications, particularly the CTPA acquisition parameters and patient preparation, which the CAD relies on. Thanks with appreciation are due to Mrs. Haneen Kharayseh and Ms. Ayah Bittar, Architecture Engineering Department at the Hashemite University, for their cooperation in the production Faculty of Allied Health Sciences, Medical Imaging Department, The Hashemite University, gest recommendation. Acknowledgements of Figures 1–3. Author details Zarqa, Jordan Abdel-Razzak M. Al-hinnawi Address all correspondence to: [email protected] 5. Conclusion However, the acquisition and patient preparation variables are fundamental factors in CTPA image quality. They may lead to bias in the CTPA-CAD output [2, 3, 47–49]. Acquisition parameters include KV, mA, type of reconstruction algorithm, accurate MDCT window, and any post processing filtration, among other technical parameters, they all affect CTPA image contrast. On the other hand, patient preparation include mainly the slice thickness, correct contrast agent dose and rate of injection, and accurate timing to acquire CTPA images during the pulmonary vessels are filled with contrast agent not after it drained to heart, they all affect the precise depiction of clots with variable sizes and locations [4, 7, 10, 40]. While radiologist, and the clinical physicist or radiographer, may be relatively unaware to such factors, it is believed for sure that they have direct impact on CAD systems because they lead to alterations in the representation of radiographic features on which CAD relies [2, 3, 48, 50]. Dose management also has crucial role on any CAD output. As reducing X-ray dose is main concern in imaging, different vendors provide imaging protocols and reconstruction techniques to reduce patient dose without substantially risking the image quality. These ultra-low dose, or even low dose, settings lead to lower contrast to noise ratio, which in turn leads to higher possibility to CT artifacts and lower capability to spot diseases [51–53]. These parameters are relevant to any CAD system [2, 3, 47, 48], so can greatly affect the PE CAD performance. #### 4.4. CTPA-CAD recommendations Therefore, subsequently to the discussion mentioned earlier, the CTPA-CAD manufactures should clearly describe the operating characteristics of their PE CAD prototypes. This include, the supported range of equipment, CTPA image acquisition settings, patient preparation requirements, reconstruction algorithms, and patient special cases such as those with additional lung defects, among other relevant factors, that radiologist need to be aware during the use of CTPA-CAD. Additionally, in case of CTPA-CAD comparison with findings from previous CTPA scans is used to assess decisions on diagnosis, disease progression, and/or treatment effectiveness, care must be considered to match the acquisition conditions accordingly to CTPA-CAD operating manual [2, 3, 47–53]. #### 4.5. Summary This section described the current status of utilizing PE CAD prototypes in CTPA clinic. The necessity of CAD systems in PE diagnosis is highlighted, and different possible CAD group of outputs are explained. Then, according to research centers from different countries, the performance of marketable and underdevelopment CTPA-CAD systems is elucidated. The PE CAD performance can be described according to "per clot" or "per patient" basis, and "first reader" or "second reader" basis. The "per patient" basis is more relevant to CTPA clinical practice than "per clot". Both the "first reader" and "second reader" basis lead to high sensitivity that can reach 100%, outweighing the radiologist performance. However, the specificity drops dramatically and become much less than the radiologist performance in case of "first reader" basis. Studies showed that the best operating scenario is the "second reader" basis because it improves sensitivity, which means less FN results, while it guarantees no significant change in specificity, in comparison to human observer performance. The factors affecting the CTPA-CAD output were described at the end of this section, this yielded to suggest recommendation. #### 5. Conclusion age, sex, race, presence of risk factors, prevalence and morphology of the clots, they do not have impact on CTPA image quality; so they should not influence the CTPA-CAD outcome. Thus, there is no need to calibrate the CAD system based on different patients or countries. However, the acquisition and patient preparation variables are fundamental factors in CTPA image quality. They may lead to bias in the CTPA-CAD output [2, 3, 47–49]. Acquisition parameters include KV, mA, type of reconstruction algorithm, accurate MDCT window, and any post processing filtration, among other technical parameters, they all affect CTPA image contrast. On the other hand, patient preparation include mainly the slice thickness, correct contrast agent dose and rate of injection, and accurate timing to acquire CTPA images during the pulmonary vessels are filled with contrast agent not after it drained to heart, they all affect the precise depiction of clots with variable sizes and locations [4, 7, 10, 40]. While radiologist, and the clinical physicist or radiographer, may be relatively unaware to such factors, it is believed for sure that they have direct impact on CAD systems because they lead to altera- tions in the representation of radiographic features on which CAD relies [2, 3, 48, 50]. 4.4. CTPA-CAD recommendations 4.5. Summary 132 Angiography ingly to CTPA-CAD operating manual [2, 3, 47–53]. Dose management also has crucial role on any CAD output. As reducing X-ray dose is main concern in imaging, different vendors provide imaging protocols and reconstruction techniques to reduce patient dose without substantially risking the image quality. These ultra-low dose, or even low dose, settings lead to lower contrast to noise ratio, which in turn leads to higher possibility to CT artifacts and lower capability to spot diseases [51–53]. These parameters are relevant to any CAD system [2, 3, 47, 48], so can greatly affect the PE CAD performance. Therefore, subsequently to the discussion mentioned earlier, the CTPA-CAD manufactures should clearly describe the operating characteristics of their PE CAD prototypes. This include, the supported range of equipment, CTPA image acquisition settings, patient preparation requirements, reconstruction algorithms, and patient special cases such as those with additional lung defects, among other relevant factors, that radiologist need to be aware during the use of CTPA-CAD. Additionally, in case of CTPA-CAD comparison with findings from previous CTPA scans is used to assess decisions on diagnosis, disease progression, and/or treatment effectiveness, care must be considered to match the acquisition conditions accord- This section described the current status of utilizing PE CAD prototypes in CTPA clinic. The necessity of CAD systems in PE diagnosis is highlighted, and different possible CAD group of outputs are explained. Then, according to research centers from different countries, the performance of marketable and underdevelopment CTPA-CAD systems is elucidated. The PE CAD performance can be described according to "per clot" or "per patient" basis, and "first reader" or "second reader" basis. The "per patient" basis is more relevant to CTPA clinical practice than "per clot". Both the "first reader" and "second reader" basis lead to high sensitivity that can reach 100%, outweighing the radiologist performance. However, the This chapter presents comprehensive current status knowledge in the PE angiography clinic. The clinical proofs of thrombosis are explained in Section 2. Knowledge of PE epidemiology, predisposing factors, pathophysiology, classifications, diagnostic medical imaging modalities, and treatments procedures are described. Then, in Section 3, concentration is focused on describing the CTPA technology that is the clinically accepted as the best first-line imaging procedure in PE diagnosis because it is fast, lowest invasive, and the highest sensitivity procedure, among other medical imaging modalities. Issues covered are CTPA physics, technology, examination, radiographic appearance, and PE features, the clinical trials in term of sensitivity, specificity, PPV, and NPV, are explained. Section 4 provides demonstration of the art of CAD system and their influence on improving the PE diagnoses by being eligible to reduce miss diagnosis by radiologist. Advantages and disadvantages of assessing CTPA-CAD performance with regard to "per patient", "per clot", "first reader", and "second reader" basis are explained. Results suggested that the "second reader" along with "per patient" basis is the best scenario to utilize PE CAD systems, because this raises the sensitivity without effect on specificity of radiologist performance. Precautions and recommendations of optimal practice of PE CAD prototypes are described indicating the necessity to follow the operation manual specifications, particularly the CTPA acquisition parameters and patient preparation, which the CAD relies on. #### Acknowledgements Thanks with appreciation are due to Mrs. Haneen Kharayseh and Ms. Ayah Bittar, Architecture Engineering Department at the Hashemite University, for their cooperation in the production of Figures 1–3. #### Author details Abdel-Razzak M. Al-hinnawi Address all correspondence to: [email protected] Faculty of Allied Health Sciences, Medical Imaging Department, The Hashemite University, Zarqa, Jordan #### References [1] Litjens G, Kooi T, Babak Bejnordi E, Setio AA, Ciompi F, Ghafoorian M, van der Laak JAWM, Ginneken B, Sánchez CI. A survey on deep learning in medical image analysis. Medical Image Analysis. 2017;42:60-88 [12] Goldhaber SZ, Morrison RB. Pulmonary embolism and deep vein thrombosis. Circu- Computer-Aided Detection, Pulmonary Embolism, Computerized Tomography Pulmonary… http://dx.doi.org/10.5772/intechopen.79339 135 [13] Temgoua1 MN, Tochie JN, Noubiap JJ, Agbor VN, Danwang C, Endomba1 FTA, Nkemngu NJ. Global incidence and case fatality rate of pulmonary embolism following major surgery: A protocol for a systematic review and meta-analysis of cohort studies. [14] Akgüllü C, Ömürlü IK, Eryılmaz U, Avcil M, Dağtekin E, Akdeniz M, Güngör H, Zencir C. 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Medical Physics. 2016;43(8):4854-4865 ## Edited by Burak Pamukçu Atherosclerotic cardiovascular disease is still the most common cause of death among adults. Its prevalence is increasing in developing countries and despite all advances in both diagnostic tools and treatment modalities, it is still very common in the developed world. Obesity, diabetes mellitus, hypercholesterolemia, and overuse of dietary salt play a pivotal role in increased cardiovascular morbidity and mortality worldwide. Current clinical efforts are mainly focused on the diagnosis and treatment of myocardial infarction. In this book we provide epidemiological data on myocardial infarction and atherosclerotic cardiovascular disease, current diagnostic biochemical tests, and management strategies. A specific patient group, children, experiencing myocardial infarction is also addressed. Current advancements in the management of myocardial infarction have decreased the morbidity and mortality from atherosclerotic cardiovascular disease and especially myocardial infarction; however, further progress can be achieved by the prevention of atherosclerotic processes by focusing on the early stages of the disease. Published in London, UK © 2019 IntechOpen © 1001gece / iStock Angiography Angiography	doab	2025-04-07T03:56:59.254279	1-12-2023 18:50	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "008df3d5-f5cd-4cb9-b407-5ac6d6ac3e67", "url": "https://mts.intechopen.com/storage/books/7055/authors_book/authors_book.pdf", "author": "", "title": "Angiography", "publisher": "IntechOpen", "isbn": "9781838800901", "section_idx": 5 }
008e5806-622f-4253-b2ea-b5ff3bc68b10	# OME-WIDE STUDIES OF GRAPEVINE FRUIT COMPOSITION AND RESPONSES TO AGRO-ENVIRONMENTAL FACTORS IN THE ERA OF SYSTEMS BIOLOGY EDITED BY : José Tomás Matus, Simone Diego Castellarin and Giovanni Battista Tornielli PUBLISHED IN : Frontiers in Plant Science #### Frontiers eBook Copyright Statement The copyright in the text of individual articles in this eBook is the property of their respective authors or their respective institutions or funders. The copyright in graphics and images within each article may be subject to copyright of other parties. In both cases this is subject to a license granted to Frontiers. The compilation of articles constituting this eBook is the property of Frontiers. Each article within this eBook, and the eBook itself, are published under the most recent version of the Creative Commons CC-BY licence. The version current at the date of publication of this eBook is CC-BY 4.0. 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For further information please read Frontiers' Conditions for Website Use and Copyright Statement, and the applicable CC-BY licence. ISSN 1664-8714 ISBN 978-2-88963-211-4 DOI 10.3389/978-2-88963-211-4 #### 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. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too. #### Dedication to Quality Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews. Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation. #### What are Frontiers Research Topics? Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! 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] # OME-WIDE STUDIES OF GRAPEVINE FRUIT COMPOSITION AND RESPONSES TO AGRO-ENVIRONMENTAL FACTORS IN THE ERA OF SYSTEMS BIOLOGY Topic Editors: José Tomás Matus, Institute for Integrative Systems Biology, Spain Simone Diego Castellarin, University of British Columbia, Canada Giovanni Battista Tornielli, University of Verona, Italy Network representation of a grape cluster as a group of modules of highly-connected nodes. Image: José Tomás Matus. Fruits play a substantial role in the human diet as a source of vitamins, minerals, dietary fiber and a wide range of molecules relevant to health promotion and disease prevention. The characterization of genes involved in the accumulation of these molecules during fruit development and ripening, and in the overall plant's response to the environment, constitutes a fundamental step for improving yield- and quality-related traits, and for predicting this crop's behavior in the field. This is certainly the case for grapevine (Vitis vinifera L.), one of the most largely cultivated fruit crops in the world. The cultivation of this species is facing challenging scenarios driven by climate change – including increases in atmospheric carbon dioxide (CO2 ), solar radiation, and earth surface temperature, and decreases of water and nutrient availability. All these events will potentially affect the grapevine phenology, physiology, and metabolism in many growing regions and ultimately affect the quality of their fruits and of the most important derived product, the wine. The sequencing of the grapevine genome has given rise to a new era, characterized by the generation of large-scale data that requires complex computational analyses. Numerous transcriptomic and metabolomic studies have been performed in the past fifteen years, providing insights into the gene circuits that control the accumulation of all sorts of metabolites in grapevines. From now on, the integration of two or more 'omics' will allow depicting gene-transcript-metabolite networks from a more holistic (i.e. systems) perspective. This eBook attempts to support this new direction, by gathering innovative studies that assess the impact of genotypes, the environment, and agronomical practices on fruits at the 'ome'-scale. The works hereby collected are part of a Research Topic covering the use of 'omics'-driven strategies to understand how environmental factors and agronomical practices – including microclimate modification (e.g. sunlight incidence or temperature), water availability and irrigation, and postharvest management – affect fruit development and composition. These studies report well-settled transcriptomic and metabolomic methods, in addition to newly-developed techniques addressing proteome profiles, genome methylation landscapes and ionomic signatures, some of which attempt to tackle the influence of terroir, i.e. the synergic effect of (micro) climate, soil composition, grape genotype, and vineyard practices. A few reviews and opinions are included that focus on the advantages of applying network theory in grapevine research. Studies on vegetative organs in their relation to fruit development and on fruit-derived cell cultures are also considered. Citation: Matus, J. T., Castellarin, S. D., Tornielli, G. B., eds. (2019). Ome-wide Studies of Grapevine Fruit Composition and Responses to Agro-environmental Factors in the Era of Systems Biology. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-211-4 # Table of Contents ## SECTION 1 #### INTEGRATED RESPONSES TO FLUCTUATING ENVIRONMENTAL CONDITIONS 1.1 SUNLIGHT AND ULTRAVIOLET RADIATION Run-Ze Sun, Guo Cheng, Qiang Li, Yan-Nan He, Yu Wang, Yi-Bin Lan, Si-Yu Li, Yan-Rong Zhu, Wen-Feng Song, Xue Zhang, Xiao-Di Cui, Wu Chen and Jun Wang Stefan Czemmel, Janine Höll, Rodrigo Loyola, Patricio Arce-Johnson, José Antonio Alcalde, José Tomás Matus and Jochen Bogs #### 1.2 TEMPERATURE Gianluca Allegro, Gabriele Valentini, Ilaria Filippetti and Giovanni Battista Tornielli #### 1.3 NUTRITIONAL STATE 123 Constraint-Based Modeling Highlights Cell Energy, Redox Status and a-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation Eric Soubeyrand, Sophie Colombié, Bertrand Beauvoit, Zhanwu Dai, Stéphanie Cluzet, Ghislaine Hilbert, Christel Renaud, Lilly Maneta-Peyret, Martine Dieuaide-Noubhani, Jean-Michel Mérillon, Yves Gibon, Serge Delrot and Eric Gomès #### SECTION 2 #### EFFECT OF AGRICULTURAL AND POST-HARVEST PRACTICES AND RELATION TO TERROIR #### 137 Dissecting the Variations of Ripening Progression and Flavonoid Metabolism in Grape Berries Grown Under Double Cropping System Wei-Kai Chen, Xian-Jin Bai, Mu-Ming Cao, Guo Cheng, Xiong-Jun Cao, Rong-Rong Guo, Yu Wang, Lei He, Xiao-Hui Yang, Fei He, Chang-Qing Duan and Jun Wang 157 Multi-Omics and Integrated Network Analyses Reveal New Insights Into the Systems Relationships Between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L.) Exposed to Water Deficit Stefania Savoi, Darren C. J. Wong, Asfaw Degu, Jose C. Herrera, Barbara Bucchetti, Enrico Peterlunger, Aaron Fait, Fulvio Mattivi and Simone D. Castellarin 176 Transcriptional Responses to Pre-flowering Leaf Defoliation in Grapevine Berry From Different Growing Sites, Years, and Genotypes Sara Zenoni, Silvia Dal Santo, Giovanni B. Tornielli, Erica D'Incà, Ilaria Filippetti, Chiara Pastore, Gianluca Allegro, Oriana Silvestroni, Vania Lanari, Antonino Pisciotta, Rosario Di Lorenzo, Alberto Palliotti, Sergio Tombesi, Matteo Gatti and Stefano Poni 197 Global DNA Methylation Patterns Can Play a Role in Defining Terroir in Grapevine (Vitis vinifera cv. Shiraz) Huahan Xie, Moumouni Konate, Na Sai, Kiflu G. Tesfamicael, Timothy Cavagnaro, Matthew Gilliham, James Breen, Andrew Metcalfe, John R. Stephen, Roberta De Bei, Cassandra Collins and Carlos M. R. Lopez 213 Prospect on Ionomic Signatures for the Classification of Grapevine Berries According to Their Geographical Origin Youry Pii, Anita Zamboni, Silvia Dal Santo, Mario Pezzotti, Zeno Varanini and Tiziana Pandolfini 220 The Induction of Noble Rot (Botrytis cinerea) Infection During Postharvest Withering Changes the Metabolome of Grapevine Berries (Vitis vinifera L., cv. Garganega) Stefano Negri, Arianna Lovato, Filippo Boscaini, Elisa Salvetti, Sandra Torriani, Mauro Commisso, Roberta Danzi, Maurizio Ugliano, Annalisa Polverari, Giovanni B. Tornielli and Flavia Guzzo # SECTION 3 #### GENOTYPE AND DEVELOPMENT-RELATED FACTORS 232 Metabolite Profiling Reveals Developmental Inequalities in Pinot Noir Berry Tissues Late in Ripening Amanda M. Vondras, Mauro Commisso, Flavia Guzzo and Laurent G. Deluc 246 System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism Lei Wang, Xiaoliang Sun, Jakob Weiszmann and Wolfram Weckwerth Darren C. J. Wong and José Tomás Matus Stefania Pilati, Giorgia Bagagli, Paolo Sonego, Marco Moretto, Daniele Brazzale, Giulia Castorina, Laura Simoni, Chiara Tonelli, Graziano Guella, Kristof Engelen, Massimo Galbiati and Claudio Moser 354 Insights Into the Role of the Berry-Specific Ethylene Responsive Factor VviERF045 Carmen Leida, Antonio Dal Rì, Lorenza Dalla Costa, Maria D. Gómez, Valerio Pompili, Paolo Sonego, Kristof Engelen, Domenico Masuero, Gabino Ríos and Claudio Moser 371 Sequence Polymorphisms and Structural Variations Among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture Luca Mercenaro, Giovanni Nieddu, Andrea Porceddu, Mario Pezzotti and Salvatore Camiolo #### SECTION 4 #### SUMMING-UP AND PERSPECTIVE VIEWS OF 'OME'-WIDE RESEARCH IN GRAPEVINE 384 Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated With Endogenous Processes and Environmental Responses Alejandra Serrano, Carmen Espinoza, Grace Armijo, Claudio Inostroza-Blancheteau, Evelyn Poblete, Carlos Meyer-Regueiro, Anibal Arce, Francisca Parada, Claudia Santibáñez and Patricio Arce-Johnson 399 How Single Molecule Real-Time Sequencing and Haplotype Phasing Have Enabled Reference-Grade Diploid Genome Assembly of Wine Grapes Andrea Minio, Jerry Lin, Brandon S. Gaut and Dario Cantu 405 A Concise Review on Multi-Omics Data Integration for Terroir Analysis in Vitis vinifera Pastor Jullian Fabres, Cassandra Collins, Timothy R. Cavagnaro and Carlos M. Rodríguez López 413 Plant Stress Responses and Phenotypic Plasticity in the Epigenomics Era: Perspectives on the Grapevine Scenario, a Model for Perennial Crop Plants Ana M. Fortes and Philippe Gallusci # The Transcriptional Responses and Metabolic Consequences of Acclimation to Elevated Light Exposure in Grapevine Berries Kari du Plessis <sup>1</sup> , Philip R. Young<sup>1</sup> , Hans A. Eyéghé-Bickong1, 2 and Melané A. Vivier <sup>1</sup> \* 1 Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch, South Africa, <sup>2</sup> Institute for Grape and Wine Sciences, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch, South Africa An increasing number of field studies that focus on grapevine berry development and ripening implement systems biology approaches; the results are highlighting not only the intricacies of the developmental programming/reprogramming that occurs, but also the complexity of how profoundly the microclimate influences the metabolism of the berry throughout the different stages of development. In a previous study we confirmed that a leaf removal treatment to Sauvignon Blanc grapes, grown in a highly characterized vineyard, primarily affected the level of light exposure to the berries throughout their development. A full transcriptomic analysis of berries from this model vineyard details the underlying molecular responses of the berries in reaction to the exposure and show how the berries acclimated to the imposing light stress. Gene expression involved in the protection of the photosynthetic machinery through rapid protein-turnover and the expression of photoprotective flavonoid compounds were most significantly affected in green berries. Overall, the transcriptome analysis showed that the berries implemented multiple stress-mitigation strategies in parallel and metabolite analysis was used to support the main findings. Combining the transcriptome data and amino acid profiling provided evidence that amino acid catabolism probably contributed to the mitigation of a likely energetic deficit created by the upregulation of (energetically) costly stress defense mechanisms. Furthermore, the rapid turnover of essential proteins involved in the maintenance of primary metabolism and growth in the photosynthetically active grapes appeared to provide precursors for the production of protective secondary metabolites such as apocarotenoids and flavonols in the ripening stages of the berries. Taken together, these results confirmed that the green grape berries responded to light stress much like other vegetative organs and were able to acclimate to the increased exposure, managing their metabolism and energy requirements to sustain the developmental cycle toward ripening. The typical metabolic consequences of leaf removal on grape berries can therefore now be linked to increased light exposure through mechanisms of photoprotection in green berries that leads toward acclimation responses that remain intact until ripening. Keywords: grape, microclimate, photosynthesis, RNAseq analysis, acclimation to stress #### Edited by: Giovanni Battista Tornielli, University of Verona, Italy #### Reviewed by: Claudio Moser, Fondazione Edmund Mach, Italy Etti Or, Agricultural Research Organization, Volcani Center, Israel > \Correspondence: Melané A. Vivier [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 10 April 2017 Accepted: 04 July 2017 Published: 20 July 2017 #### Citation: du Plessis K, Young PR, Eyéghé-Bickong HA and Vivier MA (2017) The Transcriptional Responses and Metabolic Consequences of Acclimation to Elevated Light Exposure in Grapevine Berries. Front. Plant Sci. 8:1261. doi: 10.3389/fpls.2017.01261 # INTRODUCTION Plants show remarkable adaptability to environmental factors and/or stresses to ultimately ensure that their core metabolic functions are maintained. Although these aspects have been intensively studied in model plants under controlled conditions to establish the basic principles and underlying pathways, as technologies developed, our ability to study and understand crop plants in their cultivated natural environments are yielding important information regarding the processes of stress protection and specifically the concept of acclimation. In plant biology, stress is generically defined as any unfavorable conditions that affect metabolism, growth and/or development (Lichtenthaler and Burkart, 1996). The relative tolerance/sensitivity of the affected plant subsequently determines if a stress factor will have a positive (eustress) or negative (distress) outcome (Kranner et al., 2010). Acclimation refers to the short-term responses of plants to adapt to unfavorable (stress) factors in their immediate environment (Lichtenthaler and Burkart, 1996; Lichtenthaler, 1998); whereas adaptation refers to plants' long-term survival strategy to stress factors that occurs via genetic changes such as mutations and subsequent natural selection over many generations within a population. When compared to adaptation, acclimation is a rapid response, occurs within individuals, is reversible, and does not involve any permanent genetic changes. Acclimation can involve transcriptional, metabolic and/or physiological responses to improve the performance and survival of the individual to the stress. The ability of biennial plants (e.g., onions, cabbages, and carrots) to survive winter (Andrews, 1996) and the accumulation of phenolic compounds in response to increased light exposure (Caldwell et al., 1983), are examples of acclimation to low temperature and UV-B, respectively. In grapevine, acclimation to climatic conditions is particularly important and the plasticity of grapevine responses have been highlighted in a number of publications (overviewed recently in Kuhn et al., 2014). The transcriptomic and metabolic reprogramming occurring during grape berry development has been well studied (Zenoni et al., 2010; Sweetman et al., 2012; Palumbo et al., 2014; Pilati et al., 2014; Wong et al., 2016). Research on abiotic stress factors has focused on the dominant environmental factors either individually: temperature (Carbonell-Bejerano et al., 2013; Rienth et al., 2014), light (Wu et al., 2014; Reshef et al., 2017; Sun et al., 2017), UV (Martinez-Luscher et al., 2014; Suzuki et al., 2015; Matus, 2016), and water deficit (Ghan et al., 2015; Santo et al., 2016; Savoi et al., 2016) or collectively as terroir or vintage studies (e.g., Santo et al., 2013; Anesi et al., 2015). Light has long been recognized as central to plant metabolism through photosynthesis, but recent studies have highlighted the importance of light as a source of information for plants (reviewed in Apel and Hirt, 2004; Eberhard et al., 2008; Li et al., 2009 and references within). In viticulture, many canopy management practices are performed to optimize light exposure to drive photosynthesis of the canopy (reviewed in Smart, 1985; Clingeleffer, 2010). Apart from leaves, other plant organs including the stems, flowers, tendrils and fruits contain functional chloroplasts, and are capable of photosynthesis (reviewed in Blanke and Lenz, 1989). The conditions under which photosynthesis occurs in these non-foliar organs, however, are markedly different to their foliar counterparts. In fruits, for example, the gradual disappearance of stomata and/or the development of an impermeable waxy cuticle during development results in an internal environment that is characterized by high CO<sup>2</sup> and low O<sup>2</sup> (hypoxic) levels (Blanke and Leyhe, 1987, 1988; Kyzeridou et al., 2015). Decreased photosynthesis in green fruits can be attributed to these physical/anatomical features, rather than a decrease in the photosystems. Kyzeridou et al. (2015) demonstrated that in comparison to leaves, the green fruits of Nerium oleander and Rosa sp. had higher Car/Chl ratio due to increased xanthophyll cycle components (violaxanthin, antheraxanthin and zeaxanthin) and a lower chlorophyll content. This resulted in a photoprotective xanthophyll cycle that is more functional under high light in green fruits than in leaves. This has also been reported for apple (Cheng and Ma, 2004) and grapevine (Young et al., 2016) and it is speculated that this exists in nonfoliar photosynthetic organs to reflect a common strategy for photosynthetic green tissues under similar low oxygen conditions (Kyzeridou et al., 2015). Some canopy manipulations, such as leaf removal in the fruiting zones are, however, utilized to increase light penetration to the berries (reviewed in Reynolds, 2010). A significant number of studies have investigated the impacts of leaf removal on berry development and ripening. Depending on the cultivar, the objectives range from improving the acid balance (Hunter and Visser, 1990; Toda et al., 2013; Baiano et al., 2015); improving anthocyanin/color stability (Chorti et al., 2010; Sternad Lemut et al., 2011; Lee and Skinkis, 2013; Baiano et al., 2015; Song et al., 2015; Guan et al., 2016; Yu et al., 2016; Pastore et al., 2017); increasing specific secondary metabolites such as volatile aroma precursors (Staff et al., 1997; Tardaguila et al., 2010; Feng et al., 2015; Song et al., 2015; Suklje et al., 2016; Young et al., 2016) or lowering of metabolites that are perceived negatively in the grapes/wines (Sala et al., 2004; reviewed in Sidhu et al., 2015). One of the main outcomes of leaf removal in the bunch zones is the accumulation of protective phenolic compounds i.e., anthocyanins (Lee and Skinkis, 2013; Guan et al., 2016; Lee, 2017) and flavonols (Yu et al., 2016; Pastore et al., 2017), as well as changes to volatile aroma compounds i.e., the norisoprenoid, β-damascenone (Feng et al., 2015; Young et al., 2016) and monoterpenes (Song et al., 2015; Young et al., 2016). These studies have all highlighted the adaptability of the grapevine berries to the changed microclimate and have also provided scope to investigate mechanisms of perceiving and adapting to the stresses linked to changes in microclimate. Taking advantage of a validated experimental setting where light exposure (to the bunch zone) was the major environmental factor significantly altered by a classic leaf removal treatment in a model Sauvignon Blanc vineyard, the mechanism of berry acclimation to increased light exposure (Young et al., 2016) was targeted in this study. A pertinent result from the phenotyping and metabolite profiling was that none of the parameters and metabolites measured indicated a compromised primary growth/development and ripening of the berries under the increased exposure. Metabolically, the berries responded to increased light exposure by producing specific secondary metabolites that have photo-protective and/or antioxidant functions. The data generated in the targeted metabolite profiling of the berries lead to the conclusion that the berries mitigated the stress with metabolite reprogramming to acclimate to the increased exposure and that the response was strongly influenced by developmental stage. Although sugars, organic acids, chlorophylls and major photosynthetic pigments (β-carotene and lutein) were not affected by the increased light exposure; specific monoterpenes and photoprotective xanthophylls (zeaxanthin, antheraxanthin, and lutein epoxide) were shown to be increased (Young et al., 2016). These results raised an important question: How were primary metabolism and developmental patterns maintained, despite the light stressresponse and metabolic reorganization activated in the exposed berries? Our primary approach toward achieving these aims was to take a global transcriptional snapshot of gene expression at various berry developmental stages using RNA Sequencing (RNASeq) to thereby create an overview of the effects of elevated light exposure on berry development and ripening. Using this global overview, we were able to target specific metabolic pathways of which gene expression was most significantly affected by the treatment. We could further explore what affects these alterations in gene expression could have on accumulation of metabolites involved in these affected pathways to ultimately determine how berry growth and primary metabolism was maintained despite the activation of stress response mechanisms previously reported (Young et al., 2016). #### MATERIALS AND METHODS #### Experimental Design, Agronomical Treatments, and Sampling Strategy The Vitis vinifera cv. Sauvignon blanc grapes that were the research materials for this study were harvested from an experimental vineyard located in Elgin region of South Africa during the 2010/2011-harvest season. The complete details pertaining to the climatic measurements, vineyard layout, viticultural practices and sampling strategy of the relevant samples have been performed according to an established fieldomics workflow (Alexandersson et al., 2014) and are available in Young et al. (2016). Briefly, grapes were sampled from twelve biological replicates (or panels with six panels per row; and six panels per treatment) in two adjacent vineyard rows (NW-SE row orientation). Each individual biological replicate (panel) consisted of four consecutive vines. The leaf-removal treatment included leaf and lateral shoot removal applied in the bunch zone on the SE-facing side of the canopy at EL29. This leafremoval treatment was applied to every alternate panel creating a "checkerboard" plot layout where a control panel was always adjacent to an exposed panel (both within a row, and between rows) (Young et al., 2016). The berries were sampled at green- (pea-sized) (EL31) (Eichhorn and Lorenz, 1977), pre-véraison- (EL33), véraison- (EL35), and the ripe-stage (EL38; corresponding to the commercial harvest date) from control (shaded) and exposed vine panels after which it was frozen in liquid nitrogen in the field. The seeds were removed from the frozen berries in the laboratory and the whole berries, including skins and pulp, were kept at −80◦C until subsequent analyses were performed. # Transcriptional Analysis #### RNA Extraction and Sequencing Total RNA was extracted from three out of the six biological replicates sampled at four developmental stages under both exposed and control conditions according to an established protocol (Reid et al., 2006). Each of the 24 samples was subjected to DNAse1 treatment (Sigma-Aldrich, Saint-Louis, MO, USA) to eliminate contamination with genomic DNA. The concentration and purity of the extracted RNA samples were established using a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the integrity of the samples were confirmed through analysis of a Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions. After achieving each of the quality control criteria, poly(A) mRNA was prepared for each of the RNA samples and sequenced through an Illumina HiSeq 1000 sequencer according to manufacturing protocols (Illumina Inc., San Diego, CA, USA). The reads generated from the sequencing procedure were aligned to the V1 version of the V. vinifera genome (PN40024) using version 2.0 of the TopHat software (Trapnell et al., 2012), allowing a maximum of two nucleotide mismatches. Cufflinks software (version 2.0) was subsequently used in order to assemble transcripts from generated sequence reads (Trapnell et al., 2012), hereby calculating the transcript abundance of each gene in the form of an FPKM value (expected fragments per kilobase of transcript per million fragments mapped). For the purpose of determining which transcripts show differential expression between treatments, CuffDiff (version 2.0) was used after transcript abundances were determined (Trapnell et al., 2012). #### RNASeq Expression Data Analysis When comparing the entire transcriptomes of each of the samples included in this study, Pearson correlations were calculated using R (version 3.3.1) in RStudio (version 0.99.903) and the visualization of the results in the form of a correlation matrix were performed using Microsoft Excel (version 14.1.0). Gene Ontology (GO) Enrichment analyses of the entire gene lists that showed non-significant differential expression between exposed and control samples at each phenological stage were performed in the BiNGO application in Cytoscape (version 3.4.0) using the Benjamini and Hochberg False Discovery Rate Correction metric. These genes will be further referred to as "unaffected." GO terms were considered significant with a pvalue smaller than 0.05. In order to evaluate genes that were significantly affected by elevated light, the results generated from the differential expression analysis were implemented in a three step process according to different selection criteria. The first step was to perform GO enrichment analysis of all the genes that were significantly differentially expressed (q ≤ 0.05) under exposed conditions at each developmental stage in order to evaluate the effect that the treatment had on the berry transcriptome throughout development. Next, two distinct thresholds were chosen based upon the number of genes generated that would be most appropriate for subsequent analyses. The first threshold was set to include all differentially expressed genes with a log<sup>2</sup> fold change greater than 1.5 and smaller than −1.5 when comparing the expression of exposed to control genes in order to generate a large list of highly significantly affected genes for the purpose of clustering analysis. This would allow for the identification and evaluation of the most prominent expression profiles of the genes affected by increased exposure without specifically focusing on individual genes. The second threshold was set to include only differentially expressed genes with a log<sup>2</sup> fold change greater than 2 and smaller than −2 for the purpose of focusing on the individual genes that were most affected by increased exposure. GO enrichment analysis of significantly enriched expression profile clusters of genes expressed at a log<sup>2</sup> fold change (log2FC) greater than 1.5 between exposed and control grapes during at least one of the phenological stages were performed using the online analysis tool, AgriGO (Du et al., 2010) using the Fisher statistical method with the Yekutieli False Discovery Rate multitest adjustment metric. Significantly enriched GO terms (p < 0.05) were further visualized and summarized using the Reduce + Visualize Gene Ontology Web Server (http://revigo. irb.hr; Supek et al., 2011). For the purpose of performing clustering analysis to infer which genes conform significantly to predetermined gene expression profiles, the Short Time-Course Expression Miner (STEM) was implemented (Ernst et al., 2006). Visualizations of the abovementioned differential expression analyses were performed using Microsoft Excel and Powerpoint (version 14.1.0). The putative developmental biomarkers were identified and further explored in a three step process. Firstly, the molecular biomarkers of the control grapes representing the two most distinct developmental phases (i.e., green stages vs. ripening stages) were identified by implementing a previously established method (Zamboni et al., 2010). Putative biomarkers that represent the transcriptional difference between the green and the ripening grape berry stages were identified. A two-class OPLS-DA model was generated by representing the expression of green, control berry samples (EL31 and EL33) as its own class as a reference against expression of ripening, control berry samples (EL35 and EL38) set as the second class using SIMCA (version 14.0). An S-plot was subsequently generated to identify the loading correlation coefficient of each gene as described by Zamboni et al. (2010; Wiklund et al., 2008). The aim of this investigation was to generate a broad overview of the developmental progression of the grapes included in this study and therefore, a less stringent correlation cut-off was implemented than in previous studies to identify genes with a loading correlation coefficient higher than 0.8 (positive biomarkers) and lower than −0.8 (negative biomarkers). The expression of positive biomarkers were significantly higher in ripening berries compared to green berries, whereas negative biomarker expression was significantly lower in ripening berries compared to green berries (according to the nomenclature adopted by Zamboni et al., 2010). Secondly, to establish whether these identified control grape berry developmental biomarkers were comparable to those already established for grape developmental progression, molecular biomarkers identified in this investigation were compared to those published from two previous investigations. The first set of biomarkers included in this comparison was published by Zamboni et al. (2010) in which transcriptional elements unique to early berry development (EL33 and EL35) and late berry development (EL36 and EL38) were identified and named Class a and Class b genes, respectively. These biomarkers will be referred to as early and late developmental markers in subsequent sections of this publication. The second set of genes used to compare the development of the grapes included in this study was published by Palumbo et al. (2014) in which they identified so-called "switch genes" that are considered to characterize the unique transcriptional switch that occurs when grape berries transition from being green, photosynthesizing organs to becoming ripening, sink organs. This aforementioned study utilized transcriptional data generated from five red Italian grape cultivars as well as data generated from the grapevine transcription atlas (Fasoli et al., 2012). A Venn diagram was constructed using the Bioinformatics and Evolutionary Genomics platform (http://bioinformatics.psb. ugent.be/webtools/Venn/) by comparing the genes from the abovementioned studies and the molecular biomarkers identified in this study. Finally, using the identified developmental biomarkers, the effect of the treatment on the progression of berry development was further explored. This was achieved by determining which of the identified biomarkers shared between this and previous studies were significantly affected by the leaf-removal treatment (and increased exposure) by evaluating the differential expression of these genes. In order to determine how photosynthesis is affected on a transcriptional level by elevated light exposure, the appropriate gene accessions encoding proteins of PSI and PSII of the thylakoid membranes were obtained from the KEGG Pathway database for V. vinifera (http://www.kegg.jp/kegg-bin/highlight\_ pathway?scale=1.0&map=vvi00195&keyword). #### Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) In order to validate the accuracy of the gene expression patterns observed in the results generated through RNASeq analysis, RT-PCR was performed using the Applied Biosystems 7500 Real-time PCR System. For these verification assays, total RNA was extracted from three of the six biological replicates originally harvested for metabolic and RNA Seq analyses using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, Saint-Louis, MO, USA). cDNA was synthesized from the total RNA using the SensiFASTTM cDNA Synthesis Kit (Bioline, London, UK) and RT-PCR was performed using the KAPA SYBR <sup>R</sup> FAST qRT-PCR Kit according to the manufacturer's instructions (Kapa Biosystems, Cape Town, South Africa). Six genes were selected as targets for the PCR reactions based on their expression patterns in response to the treatment as reported by the RNASeq analysis. Four of these target genes were upregulated in response to the treatment by a log2FC greater than 2 at various developmental stages (VIT\_10s0116g00410, VIT\_18s0001g03470, VIT\_05s0020g04110, VIT\_02s0025g04060). The other two of the target genes were related to photosynthesis and were significantly upregulated by elevated light exposure in the green berries (VIT\_01s0010g03620, VIT\_19s0014g00160). Appropriate primers were designed using QuantPrime (Arvidsson et al., 2008). These primers, their sequences and their characteristics are summarized in Table S1. All PCR reactions were performed in triplicate. The normalization and absolute quantification of the expression levels of each of the six genes were performed using the Linear Regression Efficiency (LRE) method using LRE Analyzer software (Rutledge and Stewart, 2008; Rutledge, 2011). #### Metabolite Analysis Extractions and subsequent metabolite analyses were performed from three out of the six available biological repeats that represented the biological triplicates sampled at four developmental stages under both exposed and control conditions. #### Amino Acid Analysis The extraction and HPLC analysis of amino acids in berry samples was performed as described in Antalick et al. (2010), with minor changes. Frozen homogenized berry tissue (200 ± 10 mg) was weighed into 2 mL microfuge tubes and 0.5 mL of 70% (v/v) methanol [containing 25 mg/L of each of the two internal standards (IS), sarcosine and norvaline] was added. Samples were briefly vortexed and sonicated for 10 min at room temperature. After sonication, the samples were centrifuged at 1,250 rpm for 5 min and 200 µL of the supernatant was transferred to amber vials, crimp-sealed and if not analyzed immediately stored at −4 ◦C. Each biological replicate was extracted and analyzed in triplicate. The extracted amino acids were derivatized before analysis on HPLC as described in Suklje et al. (2016). Major amino acids (AAs) were identified based on their retention times with respect to authentic standard elution and quantified using external standard calibration based on standard curves plotted using the peak areas vs. the standard concentrations. Concentrations were normalized to the IS amount and the sample fresh weight (FW) to obtain the AA concentrations per fresh berry weight (mg/g FW). #### Quantification of Phenolic Compound Contents All authentic standards namely quercetin-glucoside; catechin, epicatechin as well as caftaric acid and caffeic acid as well as the HPLC grade solvents used for sample extraction and separation such as methanol (MeOH, 99.0%), acetonitrile (99.0%), hydrochloric acid (HCl), and the orthophosphoric acid (H2PO4, 99.0%) were acquired from Sigma Aldrich (Steinheim, Germany). Homogenized grapevine berries (200 ± 10 mg) were weighed and 0.5 ml of acidified MeOH (70%; adjusted to pH 1.5 with HCl) was added to each vial, which was then vortexed and sonicated for 15 min at room temperature. After sonication, the samples were centrifuged at 1400 rpm for 5 min and 200 µL of the supernatant was collected and added into amber vials, crimp-sealed for HPLC analysis. Extraction was done in triplicate, in a dark room away from direct light. Extracted flavonoids and phenolic acids in berries were separated and quantified using an Agilent 1100 series HPLC system (Agilent Technologies©, Palo Alto, California, USA) equipped with a diode array detector (DAD) and controlled by a ChemStation Rev. A.10.02 software (Agilent Technologies©). The column used was a Phenomenex Prodigy ODS-2 (4.6 × 150 mm, 5 µm) preloaded with Phenomenex Prodigy guard cartridge (2.1 mm × 100 mm, 1.7 um). The mobile phases were composed of 15% (v/v) H2PO<sup>4</sup> (A) and 80% acetonitrile containing 20% A (B) and the flow rate was 1 mL/min. The gradient elution conditions started with a linear gradient from 6 to 31% B for 68 min following with another linear gradient from 31 to 65% B for 5 min. Then, the gradient was kept constant at 65% B for 5 min and was decreased from 65%, back to the starting conditions at 6% B for 5 min. The system was re-equilibrated at 6% B for another 10 min before the next injection. The injection volume was set at 20 µL and the column temperature at 40◦C. The major flavonoids and phenolic acids in grapevine berry samples were identified based on their retention times with respect to authentic standard elution and quantified using external standard calibration based on standard curves plotted using the peak areas vs. the standard concentrations. These chromatographic peaks were obtained using the following DAD wavelengths: 280 nm for flavan-3-ols; 360 nm for flavonols and 320 nm for the phenolic acids. Compounds without available standards were quantified using the calibration parameters from quercetin-glucoside (all flavonols) and caftaric acid. The concentrations in samples were normalized to the sample fresh weight (FW) to obtain the sample amount per berry FW (µg/gFW). Table S2* summarizes the retention time and calibration parameters of all standards used in this analysis. #### Lipophilic-Oxygen Radical Absorbance Capacity (L-ORAC) Assay L-ORAC analysis was performed by the Antioxidant Research Unit (Cape Peninsula University of Technology, South Africa) on three biological replicates (in triplicate) harvested at EL33 and EL38, respectively. #### Statistical Analysis The concentrations generated from the analysis of amino acids and phenolic compounds of the grapes were subjected to multivariate data analysis using Statistica (version 13.0). A repeated measures analysis of variance (ANOVA) was performed to identify the relationship between the increased exposure treatment and the concentrations of the measured compounds (AAs and Phenolic compounds). A Fisher LSD Post-Hoc test was conducted for each compound to confirm whether the concentration of the compound was statistically significantly affected by the treatment (q-value). Basic statistical analysis of data generated from the L-ORAC assay was conducted in Microsoft Excel (version 14.1.0) using a paired t-test to determine whether exposed grapes had significantly higher lipophilic antioxidant capacity than control grapes at EL33 and EL38. #### RESULTS #### Overview of the Transcriptional Data Generated In this study, RNASeq was performed with 24 Sauvignon blanc berry samples representing grapes from shaded (control) and exposed (treatment) microclimates at four developmental stages from a highly characterized vineyard. A summary of the parsed reads from each of the samples and the number of reads that mapped onto the V. vinifera cv. Pinot noir reference genome (PN40024) are included in Table S3. The complete RNASeq dataset is available in the NCBI's GEO under the series accession, GSE98873. In order to compare the complete transcriptomes generated for the 24 grape samples, a correlation matrix was generated by implementing a Pearson's correlation coefficient as a distance metric (Figure S1). The resulting matrix revealed that one sample harvested at EL38 did not correlate strongly to the rest of the EL38 samples, but rather to samples taken at EL33. Not only were the other 23 samples closely grouped according to their specific developmental stage, targeted metabolite profiling of the same grape samples previously confirmed the close grouping of all the EL38 samples (Figures 3, 4 in Young et al., 2016). This sample was treated as an outlier (anomaly) and excluded from all subsequent analyses. The Pearson correlation matrix was reconstructed including only the 23 remaining samples and is presented in Figure 1; the matrix shows a strong correlation between grapes from the same developmental stage, regardless of the viticultural treatment implemented. Furthermore, gene expression of green berries was more closely correlated between EL31 and EL33 stages than with the two consecutive ripening stages, EL35 and EL38. The correlation matrix also provided confidence in the experimental design and sampling strategy since the biological replicates of the control and exposed treatments confirmed the repeatability of the effect that the leaf removal treatment had on the berry transcriptome at each developmental stage. Out of the 29,970 genes represented in this version (V1) of the grapevine genome, the expression of 5,050 genes (16.5%) could not be detected across any of the observed developmental stages and treatments and the enriched GO terms representing these genes are summarized as Figure S2 (as represented by Revigo). A further 4,715 genes with FPKM expression values lower than the recommended reliable RNASeq threshold of an FPKM = 1 (Warden et al., 2013; Massonnet, 2015) throughout all developmental stages and treatments were excluded from further analyses. RT-PCR analysis of six genes that showed significant upregulation in response to the exposure treatment was conducted and validated the accuracy of the RNASeq results (Figure S3). Predominantly, the general expression trend throughout development of each of the genes was similar when comparing the RNASeq and RT-PCR results for control and exposed grapes. These initial analyses not only established confidence in the experimental design and the repeatability among biological replicates, but it further established the accuracy of the RNASeq method and subsequent results generated. #### Developmental Biomarker Analysis In total, the expression of 4,975 genes was identified as developmental phase-specific biomarkers responsible for the greatest transcriptional differences between the green and ripening developmental stages. 2,242 and 2,733 of these genes were positively and negatively correlated (Correlation value ≥ 0.8) to the separation, respectively (Table S4). The expression of these markers was comparable to previously established markers for grape berry development (Zamboni FIGURE 1 \| Pearson correlation matrix of the entire transcriptomes of 23 samples representing exposed and control grapes at four developmental stages (EL31, EL33, EL35, and EL38). et al., 2010; Palumbo et al., 2014). Furthermore, the expression of 81% of these shared markers developmental markers were not affected by the treatment. The remaining nine genes responsible for the 19% of developmental biomarkers that were affected by the treatment included an auxin-responsive gene (SAUR29; VIT\_16s0098g01150), two genes encoding protein subunits of photosystem I and II (VIT\_12s0028g01080, VIT\_05s0020g03180) and a calmodulin-binding heat shock protein (VIT\_14s0006g01030). The results are summarized in Figure S3. #### Transcriptional Response of the Berries to Increased Exposure #### Transcripts That Were Unaffected The number of annotated genes that were either not expressed, unaffected by the leaf removal treatment or differentially expressed when comparing exposed to control grapes at each of the phenological stages are summarized in Figure 2. GO enrichment analysis of the genes statistically unaffected by the light treatment revealed that GO terms associated with growth and development were enriched throughout development. Among these were GO terms related to "Biosynthetic process," "Signal transduction," "Protein metabolic process," "Translation," "Transport," and "Response to external stimulus" (Figure 2). Furthermore, during the developmental stages in which the berries were photosynthetically active and growing in size (EL31, EL33, and EL35), genes associated with the GO terms "Growth" and "Multicellular organismal development" were unaffected by the treatment at EL31 and EL33 as well. #### Transcripts That Were Differentially Expressed as a Consequence of the Treatment By implementing Cuffdiff software, transcripts that were significantly differentially expressed (q ≤ 0.05) when comparing exposed to control grapes could be identified. For each of the FIGURE 2 \| Pie charts representing the number of genes in the grapevine genome either not expressed, significantly unaffected (q ≥ 0.05) and significantly differentially expressed in response to elevated light (q ≤ 0.05) at the four phenological stages, respectively. The GO terms significantly enriched representing the genes unaffected by the treatment at each phenological stage are summarized in tables. Gray shading represents GO terms that were commonly unaffected throughout berry development. four developmental stages being evaluated, the percentage of differentially expressed genes (DEGs) were calculated and the genes that were either significantly up or downregulated in response to the leaf removal treatment could be explored by implementing GO enrichment analysis. The results of these analyses are summarized in Figure 3. These results revealed that grape berries were most significantly affected by the treatment on a transcriptional level during the early developmental stages (EL31 and EL33) and the global description of the biological processes these gene groups were involved with, shared a high degree of similarity between EL31 and EL33 grapes. GO terms associated with photosynthesis and the generation of precursor metabolites and energy were very highly upregulated in exposed grapes until véraison. In the green grapes, especially during EL33, genes associated with the GO terms "cell death" and "response to stress" were among the most significantly downregulated functional groups, exclusively representing genes associated with disease and nematode resistance. Although ripe berries had the highest number of DEGs in response to the treatment, the enrichment of the GO terms affected by the treatment were lower in comparison to the preceding developmental stages. These enriched GO terms were further associated with genes that were significantly downregulated in response to the treatment as opposed to the preceding stages that were dominated by upregulation in response to increased exposure. Out of the 29970 genes included in the grapevine genome, 723 genes showed either significant up or downregulation with a factor greater than 1.5 (log2FC) during at least one developmental stage in response to the elevated light treatment. Clustering analysis revealed that the expression of 431 of these genes could be grouped to seven expression profile clusters as predetermined by the STEM software (Figure 4A), with the GO subcategories provided in Figure 4B and the genes within each cluster summarized in Table S6. Two of these identified expression clusters (clusters 1 and 2) were represented by genes that followed the predicted developmental progression whilst simultaneously being affected by the treatment. Cluster 1 (p = 5E−90; total of 128 genes) represented genes that were simultaneously FIGURE 3 \| Grape berry transcripts that are significantly differentially expressed in response to elevated light exposure at four phenological stages. Significantly enriched GO categories (q ≤ 0.05) at each phenological stage. Significance is represented as log10 P-values of each GO category with positive values indicating upregulation and negative values indicating downregulation. in response to elevated light exposure during at least one of the four phenological stages. (A) The seven expression profiles to which a significant amount of genes aligned. Shaded columns (gray) indicate the expression of the genes in that specific cluster under control conditions and white columns indicate expression of the same genes under exposed conditions at the developmental stage indicated in the X-axis below. Significance is indicated in each cluster profile representation in the form of a P-value. (B) Functional GO subcategories of each significantly enriched expression cluster summarized within representative GO terms as summarized by ReviGO. Significance is represented as −log10 P-values of each subcategory; the size of each data circle indicates the number of genes that is represented within each enriched GO term. driven by the increased exposure treatment as well as developmental cues. Several of the functional annotations were associated with the progression of grape berry development, but also secondary metabolic processes linked to abiotic stress responses. Examples of genes within cluster 1 included three Ethylene-responsive transcription factors (VIT\_07S0031G01980, VIT\_01S0150G00120, VIT\_14S0108G00050), a 2-oxoglutarate/malate carrier protein (UCP5; VIT\_18S0001G07320) that has been proposed to be involved with acid regulation in grape berries (Chen et al., 2015), a Galactinol synthase (GolS4; VIT\_01S0127G00470) involved in the synthesis of the osmoprotectant oligosaccharide, raffinose, a gene encoding a Gamma-aminobutrytic acid transporter (VIT\_13S0074G00570), two genes encoding enzymes involved in the phenylpropanoid/flavonoid pathway (anthocyanidin 3-O-glucosyltransferase, VIT\_12S0034G00130; Flavanone 3-hydroxylase, VIT\_16S0098G00860), as well as the early light-inducible protein (ELIP1, VIT\_05S0020G04110) involved in the inhibition of chlorophyll biosynthesis. Interestingly, 64 of the genes represented by cluster 1 were also identified as developmental biomarkers (Figure S4) of which five were shared with the analyses of Zamboni et al. (2010) and Palumbo et al. (2014). One of these genes is a 9-cis-epoxycarotenoid dioxygenase encoding gene (NCED; VIT\_02S0087G00930) responsible for the degradation of carotenoids synthesized during the early developmental phases to produce the plant hormone, abscisic acid (ABA) that further plays a pivotal role in plant adaptation to stress. Cluster 2 represented 80 genes that showed significant downregulation throughout development under exposed conditions, while simultaneously following the same developmental progression. Among the GO terms associated with this cluster were "lipid metabolic process" that represented two senescence-associated genes (SAG101, VIT\_14S0066G01830, VIT\_14S0066G01820) involved in stress-related signaling, as well as the GO terms "photosynthesis" and "generation of precursor metabolites and energy" that both represented genes that encode a photosystem II PsbO protein (VIT\_18S0001G11710), an LHB1B1 light harvesting protein (VIT\_12S0028G00320) and another a polyphenol oxidase chloroplast precursor (VIT\_10S0116G00560). Cluster 2 also contained an Alanine-glyoxylate aminotransferase encoding gene (Alanine-glyoxylate aminotransferase 2 3, mitochondrial, VIT\_08S0058G00930) that plays a central role in the photorespiratory pathway and a gene encoding a trehalose-6-phosphate phosphatase (VIT\_00S0304G00080) that is known to have an indispensible role in normal plant growth and development. Furthermore, 18 of the genes represented by cluster 2 have been identified as negative biomarkers in this study (Figure S4). Clusters 3 and 4 (Figure 4) contained genes that were highly responsive to the elevated light exposure treatment regardless of the developmental profile. The genes represented by these clusters show strong functional associations to the activation of several protection mechanisms of the photosynthetic machinery, activated at either the first (EL31) or the second green developmental stage (EL33). Several heat shock protein (HSP) encoding genes, including the well-known abiotic stress signaling regulator, heat shock factor 2A (VIT\_04S0008G01110), alongside its putative co-activator, Multiprotein-bridging factor 1 (VIT\_11S0016G04080), as well as small HSPs formed part of these clusters. Cluster 3 further represented several genes that contributed to the GO term, "photosynthesis." These included a gene encoding a chloroplastic carbonic anhydrase (VIT\_14S0066G01210) critical in the maintenance of the rate of photosynthetic CO<sup>2</sup> fixation, and a photosystem II protein encoding gene (PsbP, VIT\_13S0019G00320) that forms part of the oxygen evolving complex of PSII, specifically contributing toward its stabilization. Furthermore, a WUSCHEL encoding gene (VIT\_18S0001G10160) was present in this cluster that represents a member of a transcription factor gene family involved in reproductive organ development, hormone signaling and abiotic stress response in several plant species. The 30 genes represented by expression cluster 4 show significantly higher expression from EL33 until véraison after which the expression of these genes was unaffected in ripe berries in response to the treatment. Among the 24 genes within this cluster that had been functionally annotated, an FtsH protease encoding gene (VIT\_14S0108G00590), known to be involved in the efficient turnover of the D1 protein of PSII in response to photooxidation, as well as a Calmodulin encoding gene (VIT\_18S0122G00180) known to be involved in stress perception and signaling related to cellular calcium ion (Ca2+) concentration in plants were included. Furthermore, this cluster represented genes encoding a galactinol synthase (VIT\_07S0005G01970), a Methyl jasmonate esterase (VIT\_00S0253G00150) and a 2-oxoglutarate-dependent dioxygenase (VIT\_05S0049G00220) among others. Clusters 3 and 4 therefore point toward the activation and maintenance of light stress mitigation strategies during the green developmental stages. The remaining three clusters (clusters 5, 6, and 7) represented genes that were differentially affected by elevated light exposure according to neither a unique developmental pattern nor consistently by the treatment (Figure 4; Table S6). Due to the random and complex nature of their transcriptional responses, these gene clusters were not further investigated for the purpose of this study. In the second step taken to elucidate which transcriptional elements are the most significantly affected by elevated light exposure at each individual stage, genes that show a Log<sup>2</sup> fold change (Log2FC) either higher than 2 or lower than −2 in exposed compared to control grapes were further explored. In total, 245 and 157 genes were up and downregulated in exposed compared to control grapes according to these criteria, respectively. These genes are listed in Table S5 and their functional associations are summarized in Figure 5. Among these 245 significantly upregulated genes, 185 were uniquely upregulated at very high levels at each developmental stage investigated with 12, 47, 61, and 65 genes upregulated (Log2FC ≥ 2) at EL31, EL33, EL35, and EL38, respectively. Out of the 157 genes that were most significantly downregulated (Log2FC ≤ −2), 156 of these genes were uniquely downregulated at either EL31 (28 genes), EL33 (29 genes), EL35 (12), or EL38 (87) in response to elevated light exposure. Several genes were similarly upregulated in various developmental stages (Figure 5). These genes, their functional annotations and the significance of their differential expression (q-values) are summarized in Table S7. ### Metabolic Processes Most Affected by Elevated Light Exposure The global transcriptional analysis of Sauvignon blanc grape berries yielded insights into which metabolic processes are most affected by elevated light exposure. Gene expression involved in photosynthesis and the synthesis of flavonoid compounds were most significantly activated by the light treatment, which warranted further investigation into how subsequent primary and secondary metabolism of the grape berries was affected by the treatment. In order to investigate these metabolic processes, the synthesis and degradation of the amino acid transcription and composition was further investigated and explored in the context of how this AA metabolism may affect secondary metabolism in response to elevated light exposure in the berry bunch zone. #### Protection of the Photosynthetic Machinery The 24 genes included in the investigation of PSI and PSII, their functional annotations and the Log<sup>2</sup> fold change of the expression of each gene when comparing exposed to control grapes at each developmental stage is summarized in Figure 6. Every gene included in this analysis was significantly upregulated (q ≤ 0.05) in response to the leaf removal treatment at EL31. Similarly, during EL33 and EL35, most of the genes of PSI and PSII remained significantly upregulated with the exception of one LHCA gene (LHCA5, VIT\_18s0001g10550), two Psb encoding genes (PsbP, VIT\_13s0019g00320; PsbZ, VIT\_12s0059g01810) that were unaffected from véraison onwards and a PsbQ (VIT\_19s0014g05080) that was unaffected by elevated light exposure from EL33 onwards. Thereafter, at EL38, with the exception of one CAB encoding gene (LHCII type I CAB-1, VIT\_19s0014g00160), all of the genes evaluated became either FIGURE 5 \| Venn diagram summarizing the functional associations of the genes up or downregulated by a Log2 fold-change greater than 2 and smaller than −2 when comparing exposed to control grapes at four phenological stages. The number of upregulated genes is indicated in bold and the number of downregulated genes is italicized in the Venn diagram and GO annotations of the genes uniquely highly differentially regulated at each developmental stage. GO descriptions indicate GO terms that were representative of each gene group, percentages indicate the percentage of genes that are represented by each GO description. unaffected by the treatment or significantly downregulated in response to the treatment. The genes putatively encoding enzymes involved in photoprotection mechanisms in grapevine have been acquired from Arabidopsis orthologs and the log2FC of their expression when comparing exposed to control grapes at each developmental stage and is also summarized in Figure 6. At EL31, all the genes encoding the enzymes of both nonphotochemical quenching (NPQ) and reversible photoinhibition were significantly upregulated with exception of one FtsH protease-encoding gene (VIT\_14S0108G00590). Although the abovementioned FtsH protease appeared to be highly upregulated (Log2FC = 7.32), it's expression proved to be highly variable among the biological replicates in this study and was therefore not significantly different when comparing exposed to control berries at EL31. The genes encoding NPQ associated proteins that include PsbS (VIT\_18s0001g02740) and one violaxanthin deepoxidase enzyme (VDE) encoding gene (VIT\_04s0043g01010) were strongly upregulated by the treatment at EL31. At EL33, however, the FtsH protease-encoding gene (VIT\_14S0108G00590), putatively responsible for the degradation of damaged copies of the D1 protein, was most significantly and highly upregulated until the berries were ripe (EL38). #### The Effect of Elevated Light Exposure on Amino Acid Metabolism of Developing Grape Berries HPLC analysis was performed that yielded the concentrations of 23 amino acids at the four developmental stages. The amino acid (AA) concentrations generated for each of the samples generated are included in Table S8. The log2FC values and statistical significance between exposed and control grapes are summarized in Table 1. Among these 23 amino acids, the concentrations of eight of these were not affected by the leaf removal treatment at any of the developmental stages evaluated. The only amino acid that was affected by the leaf removal treatment throughout the entire berry development was Gly that was present at significantly higher concentrations from EL31 until EL38. Taken together these results revealed that, with the exception of Gly, most of the AA concentrations remained unaffected by the treatment until the onset of ripening, followed by the accumulation of significantly altered AA concentrations when comparing exposed to control grapes. thylakoid membrane within the chloroplasts. (B) A table of representative candidate genes involved in photosystem I and II and two mechanisms of photoprotection in the form of non-photochemical quenching and reversible photoinhibition (RPI), their accessions and the log2 fold-change when comparing their expression levels (FPKM) between exposed and control grapes at each developmental stage. Significant differences in expression between exposed and control grapes are indicated in bold. At véraison (EL35) 10 out of the 23 AAs measured were present at significantly lower concentrations in exposed grapes, including the four key nitrogen assimilation AAs, Asp, Asn, Glu, Gln, as well as Ala, Arg, Cys, Met and two aromatic AAs, Phe, and Trp. When the berries achieved ripeness at EL38, GABA, Met, Pro, and Val were present at significantly higher concentrations TABLE 1 \| The fold change (Log2) of the amino acid concentrations (mg/gFW) of developing grapes when comparing exposed to control berries at four phenological stages. Values that are statistically different between exposed and control grapes (q ≤ 0.05) are colored according to either higher or lower concentrations. These colors indicate higher or lower log<sup>2</sup> fold changes between exposed and control grapes based on their concentrations but are not indicative of higher or lower concentrations themselves. along with Gly whereas Arg, Asp, Phe, and Trp remained present at lower concentrations in exposed grapes. At this stage, His concentrations were also significantly lower when comparing exposed to control grapes. To explore the transcriptional regulation of the synthesis and degradation of several of the AAs that were present at altered concentrations in response to the leaf removal treatment, four metabolic pathways including several of the altered AAs were targeted for further investigation. These metabolic pathways included Gly metabolism (Figure 7A), the superpathway of Lys, Met and Thr metabolism (Figure 7B), the superpathway of Trp, Phe, and Tyr metabolism (Figure 7C) and the pathway that involved Pro, Arg, and GABA metabolism (Figure 7D). The genes putatively involved in these metabolic pathways according to the current available gene annotation collection are indicated by numbers in the appropriate diagrams and are summarized in Table S9. By evaluating these four AA metabolic pathways it became clear that transcription of the biosynthetic enzyme encoding genes were only marginally affected by the leaf removal treatment, whereas the genes encoding enzymes responsible for the degradation of many of the evaluated AAs were transcriptionally far more reactive to the treatment in the ripening period. The pathway depicting Trp, Phe, and Tyr metabolism (Figure 7C) is one example of this upregulation of AA catabolic enzyme encoding genes where the genes responsible for the synthesis of Phe and Tyr were not significantly affected by elevated light exposure at any of the berry developmental stages. The Phe ammonia lyase (PAL) encoding genes (VIT\_06s0004g02620, VIT\_08s0040g01710, VIT\_13s0019g04460) and the Tyr aminotransferase encoding genes (VIT\_00s0225g00230, VIT\_00s0394g00040) respectively responsible for the degradation of Phe and Tyr were, however, significantly differentially expressed in response to the increased exposure at various stages of berry development. Increased exposure had distinctly different consequences on grape AA metabolism when comparing green to ripening berries. An example of this developmental, stage-specific metabolism was evident in the upregulation of AA catabolic enzymes in the pathways involved in Gly synthesis (Figure 7A) whereby Gly synthesis from the catabolism of both Ser and Glyoxylate were higher in exposed grapes during the green berry stages under elevated light conditions. Conversely, during the berry ripening stages, the synthesis of Gly from the degradation of Ser and Thr by the upregulation of catabolic enzyme encoding genes were higher in exposed grapes. The degradation of several of these AAs will make their constituents, whether secondary compounds or other AAs, available as substrates to secondary metabolic processes that warranted further investigation. #### Metabolic Shifts between Primary and Secondary Metabolism in Response to Elevated Light Exposure throughout Berry Development For the purpose of determining how elevated light exposure could shift developing grape primary and secondary metabolism, a summarized diagram was constructed to evaluate several metabolic branch points by integrating transcriptomic and metabolomic data generated from the same developing grape berries (Figure 8). The diagram overlays the concentrations of AAs, phenolic acids and flavonoid compounds in developing grapes with the expression levels of the transcripts known to be responsible for the enzymatic steps in the metabolic pathway between primary and secondary metabolism (Table S10). This integrated metabolic pathway focused on the branch point at which Shikimic acid could be either utilized toward the synthesis of hydrolysable tannins or toward the synthesis of chorismate, which serves as substrate for multiple downstream metabolic processes that include the synthesis of auxin from Trp or the synthesis of Tyr or Phe. Tyr in turn serves as a substrate for either the synthesis of the lipophilic antioxidants, tocopherol, or the synthesis of hydroxycinnamic acids from tyramine. Phe on the other hand is an aromatic AA that serves as a precursor for the synthesis of several secondary metabolites such as phenolic acids and flavonoid compounds that could serve as antioxidant molecules under abiotic stress conditions. The synthesis of higher levels of hydroxycinnamic acids in green grapes were facilitated by both the upregulation of genes encoding the catabolism enzymes of Tyr (VIT\_07s0005g04480, FIGURE 7 \| A summarized schematic representation of the four amino acid metabolite and transcriptomic networks analyzed in this study. Enzymatic steps are indicated as black arrowed lines, spontaneous (non-enzymatic) metabolic processes are indicated by gray arrowed lines. (A) The network representing the various (Continued) #### FIGURE 7 \| Continued pathways involved in Gly synthesis. (B) The superpathway of Lys, Met, and Thr synthesis from Asp. (C) The superpathway of Trp, Phe and Tyr synthesis from chorismate. (D) The superpathway of Pro, Arg, and GABA metabolism. Dotted lines represent feedback inhibition loops, whereas striped lines represent catabolic pathways of amino acids not included in this diagram. Blocks indicate the mean-centered log2 fold change of the FPKM expression value of the specific transcript encoding the particular enzymatic step at each berry developmental stage when comparing exposed to control samples. Significant differences between FPKM expression values between exposed and control grapes at a particular developmental stage is indicated by a bold frame around the specific gene. Amino acid concentrations [mg/g fresh weight (FW)] are represented as ANOVA line-plots where significant differences (q ≤ 0.05) between exposed and control grapes are indicated by an asterisks (\). Line graphs representing exposed and control samples are staggered along the x-axis representing the respective developmental stages. The genes represented by numbers are listed in Table S9. VIT\_13s0019g04540) and Phe (VIT\_06s0004g02620, VIT\_08s0040g01710, VIT\_13s0019g04460) while upregulation of the same Phe catabolism genes facilitated the accumulation of higher levels of flavonols. The upregulation of a different set of Tyr catabolic enzyme genes (VIT\_00s0394g00040, VIT\_00s0225g00230, VIT\_10s0116g01660, VIT\_12s0028g00710, VIT\_16s0039g01410) simultaneously contributed to the transcription of tocopherols that subsequently lead to the accumulation of elevated lipophilic antioxidant levels (L-ORAC) in green grapes exposed to elevated light (Figure 8). ## DISCUSSION Molecular profiling tools provide sensitive and comprehensive snapshots of how a plant/organ/tissue is responding at a specific point in time. It is quite obvious that the value of these molecular snapshots is amplified if they are framed by an accurate understanding of the environmental cues, the developmental stage and general plant status of the plant. This has lead to a renewed focus on integrating accurate measurements of environmental impact factors with grapevine phenotypes observed, specifically in grapevine berries. Several recent studies have advanced our understanding of berry development, ripening and reactions to stress signals and have convincingly shown that berries throughout their growth curve react to their microclimatic environments, but with different responses (Zenoni et al., 2010; Sweetman et al., 2012; Palumbo et al., 2014; Pilati et al., 2014; Wong et al., 2016). Interestingly, many of these studies also showed the resilience of berries to mitigate mild stresses (Carbonell-Bejerano et al., 2013; Martinez-Luscher et al., 2014; Rienth et al., 2014; Wu et al., 2014; Ghan et al., 2015; Suzuki et al., 2015; Joubert et al., 2016; Santo et al., 2016; Savoi et al., 2016; Young et al., 2016; Sun et al., 2017) leading to minimal impacts on overall berry growth and development. How this is orchestrated/managed was the focus of this study, and an experimental system that was previously proven to render grape berries more exposed to light, with minimal changes in berry temperatures, was used (validation of light as the main experimental parameter in the vineyard experiment was described in Young et al., 2016). #### The Grape Berry Developmental Profile Remained the Strongest Transcriptional Driver Despite Elevated Light Exposure Our data confirmed that development remained the strongest driver for the statistical separation of the grape samples based on their transcriptomes, regardless of viticulture treatment implemented. On average, not more than 8% of the berry transcriptome was affected by the elevated exposure at any of the developmental stages evaluated. As expected, berries in the green developmental stages were transcriptionally more similar in the global sense to each other than to berries from the ripening stages. Developmental phase-specific biomarkers were identified as genes that were responsible for the greatest transcriptional differences observed between green and ripening grape berries. Not only were 48 of the biomarkers identified in this study (Figure S4) also previously established as biomarkers by other research groups (Zamboni et al., 2010; Palumbo et al., 2014), but all, except nine of these genes, were unaffected by elevated light exposure at the stages when the berries were either green, ripening or throughout development. #### Green Grapes Maintain Growth and Development by Protecting the Photosynthetic Machinery under Light Stress Conditions It was previously shown that the exposed grape berries were not different from their control counterparts in terms of size and weight, sugar accumulation and acid degradation patterns (Young et al., 2016) and the transcriptional data also showed that gene expression associated with growth and development, and primary metabolism was not altered by the leaf removal treatment (this study). Despite this fact, photosynthesis-related gene expression, that forms part of primary metabolism, proved to be (the most) significantly affected by the treatment in green grapes. Our data confirmed that the green berries responded to the increased exposure to try and mitigate the light stress—the first line of defense against potentially damaging effects of photodamage, was the simultaneous activation of several avoidance strategies. One of the strong reactions was the transcription and synthesis of phenolic compounds and tocopherols that were activated, presumably to maintain the redox balance. Among the phenolic compounds that accumulated at higher levels in response to elevated light were hydroxycinnamic acids and flavonols. Both hydroxycinnamic acids and flavonols can limit photodamage through their ability to scavenge free radicals and ROS, thereby contributing to the maintenance of oxidative homeostasis (Tattini et al., 2005; Agati et al., 2007, 2012, 2013). Flavonols, however, additionally possess the ability to act as sunscreen molecules themselves. They FIGURE 8 \| A summarized overview of the branch points between primary berry metabolism toward the phenylpropanoid pathway overlaying transcriptomic and metabolomic data generated from exposed and control grapes harvested at each phenological stage. Blocks indicate the mean-centered log2 fold change of (Continued) #### FIGURE 8 \| Continued the FPKM expression value of the specific transcripts and metabolites involved in the particular enzymatic step at each berry developmental stage when comparing exposed to control samples. Significant differential expression (q ≤ 0.05) of genes and compounds are indicated by a bold contour (frame). Total concentrations (µg/g FW) of phenolic acids, flavonols and flavan-3-ols are represented by ANOVA line-plots where significant differences (q ≤ 0.05) between exposed and control samples are indicated by an asterisks (\). Line graphs representing exposed and control samples are staggered along the x-axis representing the respective developmental stages. Gray circles represented compounds that were not measured, whereas black circles represent various possible compounds at the same enzymatic step. Striped gray arrows represent regulatory steps by associated transcription factors. The genes represented by numbers are listed in Table S10. achieve this by absorbing highly energetic solar wavelengths, thereby limiting the generation of ROS due to photooxidation. Although flavonol levels have been found to be negligibly low in developing grape berries, the transcription and subsequent accumulation of these compounds in both a light-dependent and development-independent manner have been reported and extensively characterized in grapes (reviewed by Downey et al., 2006; Czemmel et al., 2009; Matus et al., 2009; Malacarne et al., 2016; Yu et al., 2016; Pastore et al., 2017). The other avoidance mechanism activated in the exposed berries was non-photochemical quenching, the process by which a large part of excitation energy generated by excessive light exposure can be dissipated as heat (via carotenoids). Through this process, the xanthophyll cycle is activated in which the xanthophyll pigment, violaxanthin, is de-epoxidized to zeaxanthin through the activity of the violaxanthin de-epoxidase (VDE) enzyme, thereby limiting energy transfer from LHCII to PSII. Although the carotenoid metabolites and their ratio's, as well as the transcriptional activation and elevated synthesis of the VDE enzyme already confirmed that the green berries have activated the xanthophyll cycle pigments (Young et al., 2016), the transcriptional mechanism of NPQ activation could be further explored in this study. The PsbS subunit of PSII has been established as the enzyme responsible for "sensing" the impending light stress and initiating NPQ (Li et al., 2000; Gregan and Jordan, 2016). The gene encoding the grapevine PsbS enzyme was found to be significantly upregulated by the leaf removal treatment from the onset of green berry development, potentially linking to the activation and upregulation of the VDE enzyme and subsequent increase of the xanthophyll pool as reported in Young et al. (2016). However, as high levels of light exposure were maintained throughout the season, it appears that damage to the photosynthetic machinery could no longer be avoided through NPQ alone. At the second green developmental stage (EL33); the process of reversible photoinhibition (RPI) was subsequently activated in an attempt to no longer avoid, but rather acclimate to the continuous light stress, while the synthesis of other antioxidant molecules such as tocopherol and flavonols remained transcriptionally and metabolically upregulated (Figure 8, Table S10). RPI is the process in which photodamage is actively concentrated to the reaction-center binding D1 protein that forms part of Photosystem II (Kyle et al., 1984; Powles, 1984). In doing so, the rapid and ongoing turnover of the D1 protein is ensured through the disorganization of the PSII-LCHII supercomplex in order to remove and replace the damaged D1 protein with a newly synthesized copy. This results in the protection of the photosynthetic machinery from photooxidative stress. These photoprotective strategies have been well characterized and extensively reported in vegetative tissues (leaves and stems) of numerous plant species (Li et al., 2000; Crouchman et al., 2006; Kato et al., 2012; Niyogi and Truong, 2013; Gorecka et al., 2014). To our knowledge, NPQ and RPI have not been thoroughly investigated in the context of green grape development. Young et al. (2016) showed higher carotenoid levels (especially xanthophylls) in the exposed berries, yet chlorophyll a: chlorophyll b and total carotene: chlorophyll ratios were maintained in the earlier stages (up until véraison). Total chlorophyll, and the levels of the major photosynthetic carotenoids (β-carotene and lutein) were also not significantly affected. The authors concluded that a pool of carotenoids (predominantly xanthophylls) were responsive to the treatment and increased in response to the increased exposure (light). Since the major carotenoids and chlorophylls were seemingly unaffected, the authors concluded that the increased pool of xanthophylls were able to protect the photosynthetic machinery for normal development to proceed (without damage). The data presented here shows that on a transcriptional level the structural proteins of photosynthesis were significantly upregulated and indicated that there was a higher demand for these proteins possibly due to an increased turnover (damage and repair cycle). Kyzeridou et al. (2015) demonstrated the green fruits of Nerium oleander and Rosa sp. have a higher cyclic electron flow activity around PSI, when compared to leaves. Kotakis et al. (2006) further showed that cyclic electron flow is enhanced (at the expense of the linear photosynthetic electron flow) in twig collenchyma to adjust potential ATP/NADPH ratios and/or to counteract the detrimental effects of hypoxia. This, combined with the increased activity of non-photochemical quenching via the xanthophyll cycle observed in apple (Cheng and Ma, 2004) and grapevine (Young et al., 2016), suggest that non-foliar photosynthesis is possibly required to produce ATP in organs where gas exchange is prevented (Kalachanis and Manetas, 2010). In this study, the sequential and simultaneous transcriptional activation of light stress mitigation mechanisms proved to be effective in avoiding irreversible photoinhibition and maintaining the development and growth of grapes. This was evident in the global transcriptional responses and the accumulation of AAs that remained predominantly unaffected by the treatment in the green berries. Furthermore, the AAs considered as oxidative stress markers, Pro and GABA, remained unaffected by the treatment in the green grapes despite elevated exposure to light. This combination of NPQ, RPI and developmentindependent flavonol synthesis, although effective in stress mitigation and acclimation, appeared to be energetically costly to the developing green grapes. Firstly, hydroxycinnamic acids were no longer differentially accumulated in response to elevated light, although the transcription and accumulation of flavonols remained dramatically higher in exposed grapes (Figure 8, Table S10). This might be explained by the fact that these compounds compete for the same aromatic AAs, Phe and Tyr, as precursors. Results to this effect were previously reported in tomato leaves exposed to various abiotic stresses (Martinez et al., 2016). The authors demonstrated that flavonols were more effective in the maintenance of oxidative homeostasis than hydroxycinnamic acids when precursors were limited. Furthermore, the MYB transcription factors known to regulate the transcription of several enzymatic steps involved in flavonoid synthesis (Czemmel et al., 2009) were significantly upregulated at each of the developmental stages (Figure 8). Secondly, the upregulation of several AA catabolic enzymes were further testament to the limitations placed on grape berry energetic resources as a consequence of photoprotection. AAs are involved in highly regulated metabolic networks and are crucial for the synthesis of proteins whilst also acting as precursors for a myriad of downstream metabolic processes. AAs have not only been implicated in normal growth and development but also in stress tolerance as their degradation may provide energetic advantage to maintain stress response mechanisms which prove to be energetically expensive to plant metabolism under suboptimal growing conditions. In Arabidopsis, evidence exist that transcription of AA catabolic enzymes, with the exception of Pro catabolic enzymes, were more sensitive to abiotic stresses than that of the enzymes responsible for AA synthesis (Less and Galili, 2008). Caldana et al. (2011) showed that amino acid catabolism serves as the main cellular energy supply under adverse environmental conditions as inferred by high-density kinetic analysis. The activity of these catabolic enzymes could therefore provide metabolic energy generated from the breakdown of AAs for utilization toward maintaining plant primary metabolism under stressful biotic and abiotic conditions. Additionally, it has been proposed that excessive accumulation of the branched chain amino acids, or rapid protein turnover induced by adverse environmental conditions could potentially lead to cellular apoptosis as a result of respiratory oxidation. The catabolic breakdown of these AAs is seen as a necessary detoxification mechanism under these conditions, as observed in Arabidopsis cell cultures (Taylor, 2004). Since, the branched chain AAs did not accumulate differentially in our investigation (Figure S5) we, however, did not consider it the likely metabolic driver for the differential transcription of AA catabolic enzyme encoding genes. Genes characterized in one of the aforementioned studies (Less and Galili, 2008) were utilized to identify homologous grapevine genes and their expression analysis in our investigation yielded similar results to previous reports. Transcription of the enzymes responsible for AA synthesis was predominantly unaltered by the elevated light exposure treatment whereas genes encoding the AA catabolic enzymes were far more sensitive to the treatment in comparison (Figure 7). The catabolism of AAs during the green berry developmental stages therefore could have provided the green grapes with substrates necessary for downstream metabolic reactions when energetically costly abiotic stress protection mechanisms were simultaneously activated. These included the maintenance of nitrogen fixation that lead to slightly shifted substrate utilization and lower accumulation of Asn, Asp, and Gln levels. The accumulation of lower levels of Phe that serves as the precursor for flavonols necessarily synthesized to protect the grapes against elevated light, were also evident, similar to the mechanisms implemented by vegetative plant organs. Significantly higher concentrations of Gly in response to the light treatment further substantiate the notion that green grapes respond to light stress as vegetative, source organs. Gly and the enzymes responsible for its decarboxylation, Gly decarboxylase complex (GDC) play an integral part in the successful functioning of photorespiration system. Increased photosynthesis and subsequent elevated levels of electron flow through the photosystems as a means to protect the photosynthetic machinery from light stress, is proposed to cause an altered redox state that ultimately influences the rate of photorespiration (Hutchison et al., 2000; Wingler et al., 2000; Voss et al., 2013). Despite elevated expression levels of the GDC encoding genes reported in our investigation (Figure 7A), the GDC themselves are prone to oxidation, hereby causing the accumulation of Gly under high light. Furthermore, Gly is considered to be the rate-determining compound in the synthesis of the antioxidant, glutathione, that might contribute to maintaining the oxidative homeostasis within the developing grape berry. This effect that elevated light exposure had on photorespiration and subsequent high Gly accumulation were previously reported in Arabidopsis (Caldana et al., 2011; Florian et al., 2014). To further support this proposed link between Gly and protection of the photosynthetic machinery in green grapes, the difference in the concentration of Gly when comparing exposed to control grapes become less significant as photosynthetic activity declines throughout berry development. These findings established that green grapes responded to elevated light exposure by activating and refining stress mitigation strategies to predominantly protect the photosynthetic machinery similar to vegetative plant organs. In an attempt to prioritize growth and development, green grapes utilized and combined several precursor substrates and mechanisms to maintain photoprotection and the synthesis of flavonols, regardless of limited energetic resources. # Ripening Berries Do Not Effectively Mitigate the Effects of Light Stress Véraison is the grape developmental stage during which the berry begins to transition from being a photosynthesizing, organ toward becoming a senescing organ while it retains metabolic characteristics of both berry developmental phases on a transcriptional level, as reported here. Véraison has further been extensively characterized by an oxidative burst that includes the production of ROS (particularly H2O2) that serves as a signaling molecule to signify the initiation of the ripening (Pilati et al., 2007). It would be reasonable to expect that the production of low-levels of H2O<sup>2</sup> as a consequence of light stress along with this developmentally driven oxidative burst could culminate toward a redox imbalance in berries exposed to elevated light. In contrast, the grapes that were exposed to elevated light at véraison did not accumulate higher levels of the known stress markers, Pro and GABA, however, at EL38, when the grapes were no longer photosynthetically active, these stress markers did accumulate at higher levels in exposed grapes. It would therefore be reasonable to speculate that this could be a reflection of the berries' successful limitation of the accumulation of ROS through the combination of NPQ, RPI and flavonol production until véraison (Figures 4, 6, 8). The rapid accumulation of both Pro and the non-protein AA, GABA, have been extensively reported in plants exposed to abiotic stresses and the metabolism of these AAs are intimately linked (Figure 7). Pro has been shown to enhance primary photochemical activity of thylakoid membranes by limiting photoinhibition and its synthesis is highly sensitive to light (Alia et al., 1997). Furthermore, in grapevine leaves, it has been reported that Pro has the ability to limit inactivation of some antioxidant enzymes while further being capable of stimulating the expression of others (Agudelo-Romero et al., 2013). Therefore, the importance of Pro homeostasis, as opposed to its accumulation, in response to oxidative stress has gained particular interest in the context of plant abiotic stress response (Kavi Kishor and Sreenivasulu, 2014). The homeostasis of Pro levels was found to be imperative to actively dividing plant cells to sustain growth despite exposure to long-term stress. GABA, on the other hand, is capable of either contributing to plant abiotic stress response through its involvement as either a stress signal amplifier or in the maintenance of the carbon: nitrogen ratio under stressful conditions (Barbosa et al., 2010; Kinnersley and Turano, 2010). The accumulation of elevated levels of both Pro and GABA can therefore be associated with plants experiencing abiotic stress symptoms. Similar to the earlier green developmental stages, the maintenance of photoprotective mechanisms throughout most of the berry development comes at an energetic cost to the grapes that are at this stage no longer accumulating precursors and energy at the rate that photosynthesizing organs are able to. This energetic strain on the grapes are reflected in lower levels of almost half of the AAs measured in these grapes as well as lower total AA concentrations overall measured in the grapes exposed to elevated light. The transcription and accumulation of flavonols remained elevated in an attempt to protect the berries from light damage and at this stage, the antioxidant pool available to the ripe berries were additionally supplemented by higher levels of apocarotenoid accumulation as reported earlier (Young et al., 2016). Due to significantly higher transcription involved in photosynthesis-related proteins during the early developmental stages, combined with increased carotenoids provides a larger pool of substrates for the degradation via carotenoid cleavage enzymes (CCDs). This leads to an increased apocarotenoid pool in the later stages. Although these compounds are thought of as mere degradation products or volatile impact odorants; they also function as antioxidants and it is speculated that apocarotenoids may play an important signaling role in plant development and in responses to environmental stimuli (Avendaño-Vázquez et al., 2014; Hou et al., 2016). Similarly, we hypothesize that higher concentrations of several AAs at EL38 (Table 1) in response to elevated light exposure may not be a consequence of transcription of the related biosynthetic enzyme genes at this late developmental stage, but rather due to the systematic degradation of higher protein levels synthesized during early development. The degradation of higher protein levels could therefore liberate higher levels of the respective AA constituents. The dramatic and consistent upregulation of numerous heat shock proteins throughout berry development (Table S7) further supports this hypothesis because of their wellestablished role as molecular chaperones associated with protein recycling in response to abiotic stress in other plant models as reviewed in Wang et al. (2004). This systematic shut-down of the protection strategies as the grapes reach maturity were further evident by the fact that the lipophilic antioxidant capacity (L-ORAC) of these grapes were no longer elevated significantly and that Pro and GABA levels were significantly higher in exposed compared to control grapes at this stage. Although the oxidative homeostasis of these grapes were no longer entirely intact (as evident by elevated Pro and GABA levels), it is however important to consider that despite the lightinduced stress status of these grapes at EL38, the sole purpose of the fruit had been achieved in the successful development and maturation of the grape seed. The redox-balance and stress responses of the grape berry were no longer of critical importance to the final development of the fruit as evident by the fact that the exposed and control grapes were not physically distinguishable when they were ripe. # CONCLUSION In this study, we aimed to determine how developing Sauvignon blanc grapes manage to maintain primary metabolism and development despite being exposed and responding to nonlethal light stress. Our approach was to explore the global transcriptional response of grapes sampled from a highly characterized vineyard to determine how these grapes acclimated to light stress on a transcriptional level and to elucidate the metabolic consequences of these transcriptional changes. This approach allowed us to demonstrate that a leaf removal treatment in the berry bunch zone of developing Sauvignon blanc grape berries lead to the activation and refinement of several stress avoidance and tolerance strategies in parallel for the purpose of mitigating the effects of light stress whilst maintaining the normal developmental program of the grapes. These results revealed that photosynthetically active berries are successful at mitigating the effects of light stress much like other vegetative plant organs by potentially limiting the synthesis and distribution of potentially harmful ROS through the continuous turnover of the photosynthetic machinery and the production of light-absorbing flavonoid compounds as well as higher levels of carotenoids in green berries and subsequent apocarotenoids in ripe berries. These grapes achieved a state of acclimation through the redistribution of energy resources in the form of AA catabolism that provided energy precursors and substrates that contributed to the maintenance of these energetically costly stress mitigation mechanisms. To this end, green, photosynthesizing grapes maintain growth and development at all costs to protect the development and maturation of the grape seed. #### AUTHOR CONTRIBUTIONS MV and PY conceptualized and planned the study. PY implemented and maintained the viticultural treatments and was responsible for the berry sampling. KdP performed RNA processing and RNASeq data analysis. HE performed HPLC analysis for AAs and phenolic compounds. KdP, PY, and MV drafted the original manuscript and all authors contributed and finalized the publication. #### ACKNOWLEDGMENTS The authors would like to acknowledge the following people for their invaluable contributions to this research: Dr. Dan Jacobson, Dr. Erik Alexandersson, Ms. Zelmari Coetzee, Mr. Cobus Steyn for assistance with the viticultural treatments and/or sampling, Dr. Liezel Gouws, Ms. Varsha Premsagar, and Dr. Jay Belli-Kullan for assistance with sample processing; Prof. Mario Pezzotti, Prof. Massimo Delledonne, and Dr. Alessandra Dal Molin for her contribution with analysis of the raw RNASeq data and differential expression analysis; Dr. Marianna Fasoli for guidance and support with the RNASeq data analysis; Mr. Fanie Rautenbach for assistance with L-ORAC assays; Ms. Chandré Joubert for her assistance with statistical analyses. This research was made possible by the financial contributions from Wine Industry Network for Expertise and Technology (Winetech), The South African Department of Science and Technology (DST), the Technology and Human Resources for Industry Program (THRIP) and the South African National Research Foundation (NRF). #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 01261/full#supplementary-material Figure S1 \| Pearson correlation matrix representing the entire transcriptomes of the initial 24 samples representing 3 biological replicates from control and exposed grapes at four phenological stages. Figure S2 \| ReviGO analysis output of GO enrichment data generated from the 5050 genes in the grapevine genome that was not expressed whatsoever in the grapes investigated in this study. Figure S3 \| Summarized results generated from Real-time PCR analysis. Figure S4 \| Venn diagram comparing the molecular biomarkers generated in this study to previously published biomarkers from Zamboni et al. (2010) and Palumbo et al. (2014) and differential expression analysis of biomarkers shared between this investigation and previously published biomarkers. Figure S5 \| The amino acid super pathway of Ile, Val, and Leu biosynthesis. Table S1 \| Primers used for Real-time PCR. Table S2 \| A table summarizing the retention times of phenolic compounds measured. Table S3 \| Summary of RNASeq reads and mapping. Table S4 \| List of positive and negative molecular biomarkers separating green (EL31 and EL33) from ripening (EL35 and EL38) berries. Table S5 \| Table listing the genes most significantly up and downregulated at each developmental stage (−2 > Log2FC > 2). Table S6 \| Table listing all significantly differentially expressed genes (q ≤ 0.05; 1.5 ≤ Log2FC ≤ −1.5) significantly correlated to predetermined gene expression clusters according to STEM analysis. Table S7 \| Functional annotation (Grimplet et al., 2012) of each of the genes that were highly upregulated (2 ≤ Log2FC ≤ −2) between two or more phenological stages indicated in color as represented in Figure 5. Q-values represent the level of significant difference between the expression of each indicated gene at the specific developmental stage. Asterisks (∗) indicate multiple genes represented by the same functional annotation with Q-values in this case indicative of the average value of the multiple genes sharing the same function. Table S8 \| The amino acid concentrations of all the exposed and control grapes sampled from EL31, EL33, EL35, and EL38. D.N.Q. refers to AA concentrations that were detected but were at concentrations below the limit of quantification. Table S9 \| Genes involved in amino acid synthesis and catabolism as indicated by the numbers assigned in Figure 7. Table S10 \| The metabolite concentrations and gene expression levels as indicated by the numbers assigned in Figure 8. #### REFERENCES important role of polyamine catabolism. Plant Physiol. Biochem. 67, 105–119. doi: 10.1016/j.plaphy.2013.02.024 on Cabernet Sauvignon, Nero d'Avola, Raboso Piave and Sangiovese Vitis vinifera L. cultivars. Sci. Hortic. (Amsterdam). 218, 147–155. doi: 10.1016/j.scienta.2017.01.048 L.) wines: a chemical and sensory investigation. J. Sci. Food Agric. 96, 915–926. doi: 10.1002/jsfa.7165 for identification of biochemically interesting compounds using OPLS class models. Anal. Chem. 80, 115–122. doi: 10.1021/ac0713510 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 du Plessis, Young, Eyéghé-Bickong and Vivier. This is an openaccess 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. # Light-induced Variation in Phenolic Compounds in Cabernet Sauvignon Grapes (Vitis vinifera L.) Involves Extensive Transcriptome Reprogramming of Biosynthetic Enzymes, Transcription Factors, and Phytohormonal Regulators Run-Ze Sun1,5, Guo Cheng1,6, Qiang Li1,7, Yan-Nan He<sup>3</sup> , Yu Wang1,2, Yi-Bin Lan1,2 , Si-Yu Li1,2, Yan-Rong Zhu<sup>1</sup> , Wen-Feng Song<sup>1</sup> , Xue Zhang<sup>1</sup> , Xiao-Di Cui<sup>1</sup> , Wu Chen<sup>4</sup> and Jun Wang1,2 \* #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics, Spain #### Reviewed by: Rodrigo Loyola, Pontificia Universidad Católica de Chile, Chile Chiara Pastore, University of Bologna, Italy #### \Correspondence: Jun Wang jun\[email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 01 December 2016 Accepted: 27 March 2017 Published: 19 April 2017 #### Citation: Sun R-Z, Cheng G, Li Q, He Y-N, Wang Y, Lan Y-B, Li S-Y, Zhu Y-R, Song W-F, Zhang X, Cui X -D, Chen W and Wang J (2017) Light-induced Variation in Phenolic Compounds in Cabernet Sauvignon Grapes (Vitis vinifera L.) Involves Extensive Transcriptome Reprogramming of Biosynthetic Enzymes, Transcription Factors, and Phytohormonal Regulators. Front. Plant Sci. 8:547. doi: 10.3389/fpls.2017.00547 <sup>1</sup> Center for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China, <sup>2</sup> Key Laboratory of Viticulture and Enology, Ministry of Agriculture, Beijing, China, <sup>3</sup> College of Enology, Northwest A&F University, Yangling, China, <sup>4</sup> CITIC Guoan Wine Co. Ltd., Xinjiang, China, <sup>5</sup> Key Laboratory of Plant Resources and Beijing Botanical Garden, Institute of Botany, Chinese Academy of Science, Beijing, China, <sup>6</sup> Grape and Wine Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China, <sup>7</sup> Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China Light environments have long been known to influence grape (Vitis vinifera L.) berry development and biosynthesis of phenolic compounds, and ultimately affect wine quality. Here, the accumulation and compositional changes of hydroxycinnamic acids (HCAs) and flavonoids, as well as global gene expression were analyzed in Cabernet Sauvignon grape berries under sunlight exposure treatments at different phenological stages. Sunlight exposure did not consistently affect the accumulation of berry skin flavan-3-ol or anthocyanin among different seasons due to climatic variations, but increased HCA content significantly at véraison and harvest, and enhanced flavonol accumulation dramatically with its timing and severity degree trend. As in sunlight exposed berries, a highly significant correlation was observed between the expression of genes coding phenylalanine ammonia-lyase, 4-coumarate: CoA ligase, flavanone 3-hydroxylase and flavonol synthase family members and corresponding metabolite accumulation in the phenolic biosynthesis pathway, which may positively or negatively be regulated by MYB, bHLH, WRKY, AP2/EREBP, C2C2, NAC, and C2H2 transcription factors (TFs). Furthermore, some candidate genes required for auxin, ethylene and abscisic acid signal transductions were also identified which are probably involved in berry development and flavonoid biosynthesis in response to enhanced sunlight irradiation. Taken together, this study provides a valuable overview of the light-induced phenolic metabolism and transcriptome changes, especially the dynamic responses of TFs and signaling components of phytohormones, and contributes to the further understanding of sunlight-responsive phenolic biosynthesis regulation in grape berries. Keywords: Vitis vinifera, sunlight exposure, phenolic compounds, transcriptome, transcription factor, phytohormone signaling ## INTRODUCTION fpls-08-00547 April 19, 2017 Time: 17:49 # 2 Phenolic compounds, mainly hydroxycinnamic acids (HCAs) and flavonoids, are one of the most abundant secondary metabolites in grape (Vitis vinifera L.) berries and important to wine quality. HCAs accumulated in grape berry skin and flesh are p-coumaric, caffeic, ferulic, sinapic acid and their derivatives, usually in the form of esters (Baderschneider and Winterhalter, 2001). Three major classes of flavonoid compounds found in grapes include proanthocyanidins (PAs), anthocyanins and flavonols. PAs, also named condensed tannins, are polymers of flavan-3-ol monomeric units (such as catechin, epicatechin, epicatechin-3-O-gallte, and epigallocatechin) which located in both the grape skins and the seeds, with trace amounts also accumulated in the vasculature of berries, whereas flavonols and anthocyanins are detected only in berry skins (Downey et al., 2006). All these compounds have important physiological functions in diverse aspects of grape berry development, such as free radical scavenging, pigmentation and co-pigmentation, ultraviolet (UV) radiation protection and defense against microbial and fungal infections (Harborne and Williams, 2000; Winkel-Shirley, 2001). Furthermore, their contribution to the color, bitterness, astringency, and antioxidant properties of red wine and potential benefits for human health have gained much attention on elucidating the regulatory mechanism of phenolic biosynthesis in grapes over the years (Santos-Buelga and Scalbert, 2000; Conde et al., 2007). Phenolic compounds are derived from multiple branches of the phenylpropanoid biosynthetic pathway, one of the secondary metabolic routes well-characterized in diverse plant species (Hahlbrock and Scheel, 1989; Ferrer et al., 2008). The genes encoding the enzymes of the phenolic biosynthesis pathway in grapes have been isolated (Sparvoli et al., 1994) and also predicted from the complete genome sequence (Velasco et al., 2007; Da Silva et al., 2013), nearly all of which are composed of small gene families. The transcriptional regulation of some structural genes is mainly controlled by a ternary complex (MBW) involving transcription factors (TFs) from the R2R3-MYB, MYC-like basic helix-loop-helix (bHLH), and tryptophan-aspartic acid repeat (WDR, also known as WD40) proteins in several plant species, including V. vinifera (Hichri et al., 2011). Additional potential regulators of the phenolic biosynthesis pathway have also been identified in model and crop plants, such as the Arabidopsis WRKY, MADS (MCM1, Agamous, Deficiens, serum response factor) box and bZIP (basic domain/leucine zipper) TFs, as well as the maize R-Interacting Factor 1 (RIF1), an EMSY-related protein interacted with a certain bHLH protein, ZmR (Hichri et al., 2011). Furthermore, several other R2R3-MYB and single-repeat R3-MYB proteins, such as Arabidopsis AtMYBL2 and CPC (CAPRICE), gentian GtMYB1R1 and GtMYB1R9 and strawberry FaMYB1, act as transcriptional repressors which negatively regulate the biosynthesis of anthocyanins or PAs in plants (Aharoni et al., 2001; Matsui et al., 2008; Zhu et al., 2009; Nakatsuka et al., 2013). In the case of grapevine, VviMYB4a and its close homolog VviMYB4b have been characterized as important negative regulators of small-weight phenolic biosynthesis, whereas two other repressors, VviMYBC2-L1 and VviMYBC2-L3, were shown to fine tune flavonoid levels additionally (Huang et al., 2014; Cavallini et al., 2015). Environmental factors (light, temperature, water status, and nutrients, etc.) and viticulture practices have been acknowledged to influence the development, ripening and phenolics composition of grape berries, and could thereby affect wine quality (Jackson and Lombard, 1993; Downey et al., 2006). Bunch shading and exposure treatments are regarded as influential practices that alter the accumulation and composition of phenolics and the expression of the corresponding biosynthetic genes by directly affecting the incidence of light on grape clusters and also changing other microclimatic aspects, such as temperature and humidity (Ristic et al., 2007; Koyama and Goto-Yamamoto, 2008; Chorti et al., 2010). Many studies have shown that artificial bunch shading resulted in greatly decreased flavonol concentrations, while the levels of PAs and anthocyanins were not significantly changed at harvest (Downey et al., 2004; Fujita et al., 2005; Cortell and Kennedy, 2006; Koyama and Goto-Yamamoto, 2008). On the other hand, enhanced sunlight exposure induced by basal leaf removal generally led to increased accumulation of flavonols, but did not alter anthocyanin concentration compared with the control, which might be correlated with the negative effects of elevated berry skin temperature (Downey et al., 2004; Chorti et al., 2010). In addition, grapes from sunlight exposure bunches had a higher proportion of B-ring trihydroxylation subunits within PAs and anthocyanins in comparison with normal and bunch shading fruit, which agree with the relative increase of flavonoid 3<sup>0</sup> ,50 -hydroxylase (VviF3<sup>0</sup> 5 <sup>0</sup>H) expression (Cortell and Kennedy, 2006; Koyama and Goto-Yamamoto, 2008). In recent years, a considerable amount of effort has been devoted to investigating the impact of cluster sunlight exposure treatments during specific stages of berry development and ripening on the detailed phenolic profiles as well as the expression of related structural and regulatory genes in different grape varieties (Matus et al., 2009; Chorti et al., 2010; Lemut et al., 2011; Kotseridis et al., 2012; Lee and Skinkis, 2013; Matsuyama et al., 2014; Wu et al., 2014; Friedel et al., 2015). For instance, the expression of flavonol synthase 1 (VviFLS1, also known as VviFLS4) and its specific transcriptional activator VviMYB12 (also named VviMYBF1) was drastically increased following leaf removal treatment, which ultimately resulted in the quickly increased flavonol synthesis (Matus et al., 2009). Leaf removal also up-regulated anthocyanin synthesis related structural genes and regulators in grape skins, such as chalcone synthase (VviCHS), uridine diphosphate (UDP)-glucose:flavonoid 3-Oglucosyltransferase (VviUFGT), anthocyanin-O-methyltransferase (VviAOMT), flavonoid 3<sup>0</sup> -hydroxylase (VviF30H), VviF3<sup>0</sup> 5 0H, VviMYBA1, and VviMYB5a (Matus et al., 2009; Matsuyama et al., 2014; Wu et al., 2014). More recently, two bZIP TFs elongated hypocotyl 5 protein (HY5) orthologs, VviHY5 and VviHYH, were characterized as constituents of the UV-B response pathway in grapevine and mediated flavonol accumulation in response to high radiation exposure (Loyola et al., 2016; Matus, 2016). However, there are still pending questions regarding the complex underlying molecular mechanism of the phenolic metabolism regulation network involved in light response. In the present study, accumulation and compositional changes of HCAs, flavan-3-ols, anthocyanins and flavonols were determined in V. vinifera L. cv. Cabernet Sauvignon grape berries from different fruit-zone light-exposure treatments in multiple phenological stages under field conditions over three successive seasons. To understand the regulation of phenolic biosynthesis under different irradiation conditions, the influences of light exposure on the transcription of phenolic biosynthetic genes and their putative upstream regulators, as well as the relationship between metabolism and transcription in grapes throughout berry development were also examined. # MATERIALS AND METHODS # Plant Material and Sunlight Exposure Treatment Field experiments were conducted in a commercial vineyard of V. vinifera L. Cabernet Sauvignon located in Manas Country (44◦ 17<sup>0</sup> North, 86◦ 12<sup>0</sup> East, 475 m above sea level), the wine-producing region of Xinjiang province, China, for three consecutive growing seasons (2011, 2012, and 2013). The ownrooted vines in this vineyard were planted in 2000, managed on a modified Vertical-Shoot-Positioned (M-VSP) trellis system with a spur-pruned cordon retaining 15 nodes per linear meter, arranged in north-south rows with 2.5 m × 1 m vine spacing and equipped with a furrow irrigated system. Nutrition and pest management was carried out according to industry standards for this cultivar and the region as previously described (Cheng et al., 2014). Sunlight exposure treatments were carried out as described by Matus et al. (2009), with some modifications. In three consecutive years, eight fruit-zone light exposure levels were established in the vines through artificial leaf removal, half leaf removal, or leaf moving (Figure 1): leaf removal at berry pepper-corn size (LR-PS); leaf removal at véraison (LR-V); leaf removal after véraison (LR-AV); half leaf removal at véraison (HLR-V); half leaf removal after véraison (HLR-AV); leaf moving at véraison (LM-V); leaf moving after véraison (LM-AV); and non-treated control (C). Leaf removal and half leaf removal treatments were carried out by removing the first one to six basal leaves from the main shoots with clusters and three basal leaves from the first, third, and fifth of each shoot with clusters, respectively. For leaf moving treatment, the first one to six basal leaves of each shoot with clusters were moved aside by the use of nylon zipties, in order to increase the sunlight exposure of grape clusters without affecting the photosynthetic carbon assimilation to the fruit. Each treatment was arranged in a completely randomized experimental design with three biological replicates. In each biological replicate, treatment was applied to 15 vines randomly selected from the vineyard's south and north sites. The meteorological data during berry development in 2011, 2012, and 2013, including sunlight duration (h), growing degree days (◦C), temperature (◦C), rainfall (mm) and relative humidity (%), were gathered from the local meteorological administration (Supplementary Table S1). To determine the influence of sunlight exposure on canopy microclimatic conditions, photosynthetically active radiation (PAR) sensor (model S-LIA-M003, Onset Computer Corporation, Bourne, MA, USA) and total radiation sensor (model S-LIB-M003, Onset Computer Corporation, Bourne, MA, USA) were positioned parallel with the cordon at the bunch zone on the defoliated side (west) of the canopy of both exposure and control groups during grape berry development in 2012 and 2013. The air temperature and relative humidity (RH) inside the canopy of each group were also monitored via a Hobo temp/RH smart sensor (model S-THB-M002, Onset Computer Corporation, Bourne, MA, USA) placed at the fruit zone. Each measurement was performed at 5-min intervals (Supplementary Table S2). Berries from each treatment and control group were sampled at the following developmental time points: 3 weeks after flowering (waf) (berry pepper-corn size; E-L 29), five waf (berry pea-size, E-L 31), seven waf (berry still hard and green, E-L 33), early-véraison (berries begin to color, E-L 35), mid-ripening stage (berries with intermediate Brix values, E-L 36), end of véraison (berries not quite ripe, E-L 37) and complete ripening stage (E-L 38) (Coombe, 1995). For each biological replicate, 600 berries were randomly separated from both sunny and shade sides of at least 100 clusters within 15 vines. The sampling time was fixed at 10:00 to 11:00 am, and three biological replicates were collected with the same method at each sampling date. After being washed with distilled water, a sub-sample of 100 berries from each biological replicate was subjected to the physiological measurements, including berry fresh weight, total soluble solids (TSS) content and titratable acidity (TA), the rest were frozen in liquid nitrogen immediately and transported to the lab in dry ice for the subsequent metabolites determination or transcriptional analysis. TSS concentrations of the juices were measured with digital pocket handheld refractometer (Digital Hand-held Pocket Refractometer PAL-1, Atago, Tokyo, Japan), and TA was determined by titration with NaOH to the end point of pH 8.2 and expressed as tartaric acid equivalent (Cheng et al., 2014). # Isolation and Identification of Compounds Phenolic acids were extracted from berries and analyzed as described by Song et al. (2013, in Chinese with English abstract). In detail, a sub-sample of 100 frozen berries randomly selected from each biological replicate was first ground into powder under liquid nitrogen after weighing and removing the seeds. For the analysis of monomeric phenolics, 5 g of ground powder was extracted with 25 mL of 1% (v/v) ascorbic acid and 10 mM EDTA in 4 M NaOH. The extraction mixture was then sonicated for 3 min and shaken in incubator shakers in dark for 8 h under a nitrogen atmosphere at 35◦C. After acidification to pH 2 using 6 M HCl and centrifugation at 8,000 rpm for 20 min, the clear supernatant was extracted four times with diethyl ether to obtain the free phenolic acids released from the soluble ester. The combined supernatant was evaporated to dryness, dispersed in 0.5 mL of methanol, and filtered through a 0.45 µm Millipore membrane filter (Millipore Co. Ltd, Billerica, MA, USA) prior to high performance liquid chromatography (HPLC) analysis. Skin flavonoids were extracted from a sub-sample of 100 berries randomly selected from each biological replicate as described by Li et al. (2014), with some modifications. For flavan-3-ols preparation, 0.1 g sub-sample of skin powder was extracted in a solution of 50 g/L phloroglucinol (1 mL) containing 0.3 N HCl and 0.5% (v/v) ascorbic acid in darkness at 50◦C for 20 min. After terminating the reaction by addition of 1 ml NaAc (50 mM), the extraction mixture was centrifuged at 10,000 rpm for 15 min, and the clear supernatant was collected. The residues were re-extracted three times, and all the supernatants were mixed and stored at −40◦C. To extract anthocyanins, 0.5 g sub-sample of skin powder was extracted in 10 mL of methanol solution containing 1% formic acid under sonication for 10 min at room temperature, and then shaken in incubator shakers in dark at 25◦C for 30 min at a rate of 200 rpm. The extraction mixture was centrifuged at 8,000 rpm for 20 min and the clear supernatant was collected. The residues were re-extracted four times, and all the supernatants were pooled and evaporated to dryness in a rotary evaporator at 30◦C, and then dissolved in 10 mL of 10.8% (v/v) acetonitrile aqueous solution with 2% formic acid. All the extracts obtained above were filtered through 0.22 µm nylon membrane filters before HPLC analysis. For flavonols extraction, 5 g subsample of skin powder was immersed in 15 mL of 50% ethanol solution containing 1% acetic acid with the aid of ultrasonic vibrations for 35 min at room temperature and then centrifuged at 8,000 rpm for 10 min. The residues were re-extracted four times, and the pooled supernatants were macerated with 50 mL of distilled water and then extracted in 40 mL of ethyl acetate three times. The organic phase was collected and evaporated to dryness in a rotary evaporator at 30◦C, and then suspended in 2 mL of 25% methanol. Three independent extractions from three biological repeats were conducted for either the berry or the skin of each sample. Phenolic acids were monitored on an Agilent 1100 series HPLC-MSD trap VL (Agilent, Santa Clara, CA, USA), equipped with a diode array detector (DAD) and a reversed phase column (Zorbax SB-C18, 250 × 4 mm, 5 µm). The injection volumes were 10 µL and the column thermostat was set at 30◦C. Mobile phase A consisted of methanol/acetic acid/water (10:2:88, v/v/v), and mobile phase B consisted of methanol/acetic acid/water (900:15:85, v/v/v). The gradient was from 0 to 3.6% B for 7 min, from 3.6 to 15% B for 19 min, from 15 to 25.5% B for 6 min, from 25.5 to 29.7% B for 3 min, from 29.7 to 45.5% B for 10 min, from 45.5 to 0% B for 8 min, at a flow rate of 1 mL/min. All phenolic acid compounds were identified by matching the retention time and their spectral characteristics against those of standards (Sigma, St. Louis, MO, USA). Chlorogenic acid and caffeic acid were quantified at 325 nm while p-coumaric acid, ferulic acid and sinapic acid at 275 nm. The conditions for the mass spectrometry (MS) were as follows: electrospray ionization (ESI) interface; negative ion model; nebulizer pressure, 241.3 kPa; dry gas flow rate, 10 L/min; dry gas temperature, 350◦C; Trap ion charge control (ICC), 30,000 units; collision-induced dissociation (CID) voltage, 1.00 V; scan at m/z 100–1,000. Qualitative and quantitative analyses of flavonoids were carried out on an Agilent 1200 series HPLC-MSD trap VL linked simultaneously to a DAD (for flavan-3-ols and anthocyanins) or a variable wavelength detector (for flavonols) as described previously (Cheng et al., 2014; Li et al., 2014; Zhu et al., 2014). Flavan-3-ols, anthocyanins and their derivatives were analyzed as in Cheng et al. (2014) and Li et al. (2014), respectively. Flavonols and their derivatives were eluted by using a selection of reverse phase column (Zorbax SB-C18, 50 × 3 mm, 1.8 µm) and binary gradient elution with mobile phase A consisted of acetonitrile/formic acid/water (50:85:865, v/v/v), and mobile phase B consisted of acetonitrile/methanol/formic acid/water (250:450:85:215, v/v/v/v), which was in accordance with Zhu et al. (2014, in Chinese with English abstract) with minor revision. Proportions of solvent B varied as follows: from 0 to 14.2% for 24.2 min, from 14.2 to 15.7% for 2.8 min, from 15.7 to 18.8% for 6.4 min, from 18.8 to 23.5% for 5.4 min, from 23.5 to 26% for 6 min, from 26 to 27.4% for 2 min, from 27.4 to 32% for 4.6 min, from 32 to 40% for 10.2 min, from 40 to 100% for 6 min, from 100 to 0% B for 10.6 min, at a flow rate of 1 mL/min. The injection volumes were 50 µL and the column thermostat was set at 40◦C. All flavonol compounds were identified by the UV spectrum and retention time of quercetin-3-O-glucoside (Sigma, St. Louis, MO, USA). The detector wavelength was 360 nm. The ESI parameters were as follows: negative ion model, nebulizer pressure, 30 psi; dry gas flow rate, 10 mL/min; dry gas temperature, 325◦C; Trap ICC, 30,000 units; CID voltage, 1.00 V; scan at m/z 100–1,000. Quantitative determination of flavonoids was performed using the external standard method with commercial standards. All analyses were run in replicate and averaged for each biological replicate. One-way ANOVA followed by the Duncan's new multiple range test was performed using SPSS 20.0 for windows (SPSS Inc., Chicago, IL, USA) to determine significant differences of the physicochemical indexes and phenolic accumulations among treatments at each sampling time point. #### RNA Isolation, Sequencing, and Data Analysis Based on biochemical parameters and metabolite profiles, we selected the berries of three developmental stages (E-L 36, 37, and 38) from the LR-V and LM-V treatments and the control group during the 2012 growing season to conduct the transcriptome profiling analysis. A sub-sample of 50 berries were randomly selected from each biological replicate for RNA extraction. Total RNAs for RNA-seq analysis were isolated from frozen deseeded berries using a Plant Total RNA Extraction Kit (Sigma, St. Louis, MO, USA), and further purified by DNase I (Promega, Madison, WI, USA) digestion. RNA integrity and concentration were analyzed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and the Aglient 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Following quality assessment, cDNA libraries constructed from three biological replicates of each sample were sequenced by Illumina HiseqTM2000 sequencer (Illumina Inc., San Diego, CA, USA) with a 50-bp single read module RNA-seq reads and then aligned against the reference grapevine genome V2<sup>1</sup> using the alignment software Bowtie (Langmead et al., 2009), allowing no more than two nucleotides mismatched. The FPKM (expected fragments per kilobase of transcript per million fragments mapped) method was used for calculating the transcript abundance of each gene (Trapnell et al., 2010). Transcripts were mapped to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG)<sup>2</sup> as described previously (Sun et al., 2015a). Prediction of TFs was performed by using the HMMsearch program. Identification of differentially expressed genes (DEGs) between samples was performed with R package 'NOISeq' (Tarazona et al., 2011). A threshold of fold-change ≥ 2 and divergence probability ≥ 0.8 was used for filtering the significance of the gene expression difference. Heatmap visualizations were performed using the R package 'pheatmap' (Kolde, 2012). Pearson correlation evaluation was conducted with R package 'Hmisc' using the rcorr function (Harrell and Dupont, 2012) and co-expression networks were visualized with the Cytoscape software version 3.2.0 (Shannon et al., 2003). #### Quantitative Real-time PCR Validation of the transcript quantification from the RNA-seq data was carried out through quantitative real-time PCR (qRT-PCR). For extraction of skin RNAs, berry skins from another subsample of 50 berries randomly selected from each biological replicate were manually separated from pulps. Total RNA from berry skins was isolated using the same method mentioned above. The subsequent cDNA synthesis and qRT-PCRs were performed as described by Sun et al. (2015a). Gene-specific primers used for qRT-PCR are listed in Supplementary Table S3 (Downey et al., 2003; Castellarin et al., 2006; Fujita et al., 2006; Reid et al., 2006; Bogs et al., 2007; Czemmel et al., 2009; Shimazaki et al., 2011; Azuma et al., 2012; Sun et al., 2015b, 2016). All reactions were run in triplicate, and the normalized relative expression levels of target genes were calculated by 2−δCt (1Ct = CtTarget – CtControl, Ct: cycle threshold). VviUbiquitin1 and Vviβ-Actin genes were selected as endogenous controls for normalization and CtControl was the geometric mean of their threshold cycles. <sup>1</sup>http://genomes.cribi.unipd.it/grape/ <sup>2</sup>http://www.genome.ad.jp/kegg/ # RESULTS AND DISCUSSION fpls-08-00547 April 19, 2017 Time: 17:49 # 6 ### Berry Development and Ripening Changes in berry fresh weight, TSS and TA of Cabernet Sauvignon grape berries collected from E-L 31 stage until harvest during the first growing season (2011) and from E-L 29 stage until harvest during the subsequent two growing seasons (2012 and 2013) are shown in Supplementary Figure S1. The date of véraison and harvest were set at approximately eight and sixteen waf, respectively, during all three seasons. Previous studies on several grape species and cultivars reported little or no effect of leaf removal treatments on fruit weights and juice soluble solid contents at harvest (Haselgrove et al., 2000; Main and Morris, 2004; Chorti et al., 2010; Kotseridis et al., 2012). In this study, weight of berries for LR-PS was significantly lower than that of the control at harvest in the 2011 season, while an opposite result was observed in the 2012 season. LM-V and LM-AV significantly increased berry weights from 1 week after each treatment until berry ripening during the 2011 and 2012 seasons, however, there were no discernible differences between each of the two treatments and the control during the third experimental season (Supplementary Table S4). Overall, there were no consistent trends in the differences of berry fresh weight between each of the light-exposure treatment group and the control group during berry development in all the three experimental seasons. Similarly, the differences in the level of juice soluble solids between the exposed and shaded berries during berry development were also inconsistent among different seasons. No treatment differences were found in soluble solid contents at harvest in all the 3 years, except for those of berries from LR-PS, which was higher than that of the control in the 2011 season (Supplementary Figure S1). Specifically, ripening berries from the control and all light-exposure treated clusters have lower soluble solid contents in the 2013 season compared with the other two seasons, which could be attributed to the relative lower level of flowering-to-harvest growing degree days (GDD) accumulation in 2013 than those in the other 2 years (Supplementary Table S1), similar with the results from a previous study (Spayd et al., 2002). The decrease of the TA in the berries from LR-PS treatment during véraison was slightly faster than that of the control during the 2011 and 2012 seasons. However, the influence of LR-PS treatment on TA was negligible during the 2013 season. At harvest, there was no significant difference in the TA among treatments in all the three seasons (Supplementary Figure S1), which was inconsistent with previous studies that leaf removal treatments reduced TA (Zoecklein et al., 1992; Percival et al., 1994), suggesting that TA was differently affected by sunlight exposure depending on cultivar and climate. #### Influence of Cluster Sunlight Exposure Treatments on Phenolic Concentration and Composition Of the many environmental factors that affect the phenolic biosynthesis in many plants, light has been regarded as one of the major influences (Downey et al., 2004; Cominelli et al., 2008; Wang et al., 2012; Jaakola, 2013). The present study shows that the concentration of total HCAs was significantly increased in the sunlight exposure berries in comparison with the control berries at both véraison and harvest in all the three experimental seasons, except for berries from LR-PS and LR-AV treatments, in which the level of HCAs was slightly higher during véraison while lower at the harvest stage compared with the control in both the 2011 and 2012 seasons (Figure 2A* and Supplementary Table S4). The results obtained in our study are inconsistent with a previous study conducted on Pinot Noir, which showed that the concentration of HCAs throughout maturation was effectively enhanced by leaf removal at berry set but slight influenced by leaf removal at véraison (Lemut et al., 2011). Of the three classes of flavonoids, flavan-3-ols are present in the greatest proportion in grapes, followed by anthocyanins, with flavonols being present at relatively low levels (Downey et al., 2004). Sunlight exposure did not consistently affect the accumulation of total flavonoids or flavan-3-ols in the skins throughout berry development or at harvest among different seasons (Figures 2B, 3A). The concentration of total flavonoids and flavan-3-ols in the skins was slightly lower in LR-PS, LR-V, LR-AV, and HLR-V, while the former was higher in HLR-AV and LM-V treated berries than those from control berries at harvest in the 2011 season. Each of the seven sunlight exposure treatments resulted in decrease in concentration of skin total flavonoids as well as flavan-3-ols at harvest in the 2012 season, while an opposite result was observed in the 2013 season, except for berries from LR-PS, in which the concentration of total flavonoids and flavan-3-ols showed a significant decrease and no discernible difference compared with the control, respectively (Supplementary Table S4). The seasonal variations in the level of flavan-3-ol compounds might be caused by differences of temperature, GDD or the level of PAR during berry ripening among seasons (Supplementary Tables S1, S2), which suggests an ambiguous effect of light-exposure on the flavan-3-ol biosynthesis in grape berry skins. Anthocyanin biosynthesis has been found to be variably regulated in response to light conditions in a number of sunlight exposure studies, which is possibly confounded by varying experimental settings and other factors, such as cultivar, vineyard location, timing of leaf removal, and growing season. For instance, leaf removal at berry set increased skin anthocyanins in grapes of Merlot, Cabernet Sauvignon and Pinot noir (Lemut et al., 2011; Kotseridis et al., 2012), and pre-bloom leaf removal also substantially increased anthocyanin concentration in Barbera, Lambrusco salamino, Graciano, and Carignan grapes when compared with no leaf removal (Poni et al., 2009; Tardaguila et al., 2010). However, another exposure study indicated that no significant differences among treatments were observed in anthocyanin levels in Nebbiolo grapes at harvest, although leaf removal caused a temporary acceleration of anthocyanin accumulation throughout ripening (Chorti et al., 2010). In the present study, sunlight exposure increased the level of skin anthocyanins at the initiation of grape coloration compared to the control in at least two of the three experimental seasons, while no consistent differences among treatments were observed in the concentration of anthocyanins at harvest over the three experimental seasons (Figure 3B). In the 2011 season, almost all exposure treatments could significantly (LR-V, LR-AV, biological replicates. Light gray background represents the phenological phase of véraison from 5 to 100% of colored berries. HLR-AV, and LM-V) or slightly (LR-PS and HLR-V) increase skin anthocyanin amount at harvest, whereas no significant differences in the concentration of skin anthocyanins at harvest among treatments were observed in the 2013 season. On the contrary, slight or significant decreases in the skin anthocyanin amount were observed from all of the light exposed berries in the 2012 season (Supplementary Table S4). The diametrically opposed result was possibly due to increased levels of PAR, solar radiation and average temperature for exposed berries during coloration and the maturation phase (Supplementary Table S2), accompanied with a relative high temperature during berry ripening in this experimental season (Supplementary Table S1), which could lead to the inhibit formation or induce degradation of anthocyanins (Yamane et al., 2006; Tarara et al., 2008; Pastore et al., 2013). Among the three major classes of flavonoid compounds, the accumulation of flavonols was most drastically affected in berry skins under the sunlight exposure treatments. Flavonols have been found to accumulate in sun-exposed tissue of grapes and are thought to act as UV protectants and free radical scavengers (Price et al., 1995; Downey et al., 2004). Leaf removal at all three phenological stages resulted in a dramatic increase in flavonol concentration in the grape skin throughout berry development during the three experimental seasons, similar to the results conducted on Sangiovese berries previously (Pastore et al., 2013), but the degree of their effect was variable among seasons in our experiments (Figure 3C). The level of flavonol compounds was also moderately increased by HLR-AV and LM-V treatments, except that there was no significant difference between the control and HLR-AV in the 2011 season. HLR-V and LM-AV did not significantly influence the concentration of skin flavonols at harvest compared with the control, although there was a temporary acceleration of flavonol accumulation throughout ripening in HLR-V treated berries (Figure 3C and Supplementary Table S4). However, opposite results regarding changes in flavonol contents under different sunlight exposure treatments have been found previously, in which leaf moving at véraison increased flavonol synthesis greater than leaf removal treatment (Matus et al., 2009), suggesting that the enhanced accumulation of flavonols under treatments is strongly associated with the severity degree of sunlight exposure, and also climate divergences in different years and regions. In the flavonoid biosynthesis pathway, two metabolic branches leading to the biosynthesis of B-ring dihydroxylated (3,3<sup>0</sup> -OH) and trihydroxylated (3,3<sup>0</sup> ,50 -OH) subunits were reported to have different sensitivities in response to various lighting conditions in many previous researches (Downey et al., 2004; Azuma et al., 2012; Guan et al., 2015). Our results showed that the molar ratio of dihydroxylated to trihydroxylated flavonoids was continuously decreased throughout berry ripening, while changes of the ratio among treatments were inconsistent during the three seasons (Figure 3D). In the first experimental season, the ratio of dihydroxylated/trihydroxylated flavonoids in berries from almost all of the sunlight exposure treatments was decreased compared with the control across development, except for LR-PS at E-L 33 stage, LR-V at E-L 35 stage and LM-AV at harvest. In contrast, however, light-exposure caused a slight or marked increase in the ratio of dihydroxylated/trihydroxylated flavonoids during berry ripening in the 2012 growing season. In the last experimental season, no significant differences in the ratio of dihydroxylated/trihydroxylated flavonoids were observed among treatments except for berries from the leaf removal treatments, which was increase in LR-V and LR-AV treated berries at harvest and in LR-PS treated berries during coloration (Figure 3D and Supplementary Table S4). In contrast to the results obtained from cluster shading studies (Koyama and Goto-Yamamoto, 2008; Guan et al., 2015), the effect of light-exposure was possibly influenced by the temperature or other climate variables of the year. # Light-induced Transcriptional Changes of Phenolic Biosynthetic Pathway Genes To investigate the responses of phenylpropanoid/flavonoid biosynthetic pathway related structural and regulatory genes to different light-exposure treatments, berries of three distinct development stages (E-L 36, 37, and 38) from the LR-V and LM-V treated and the control groups during the 2012 growing season were selected to characterize the changes in gene expression at the transcript level by RNA-seq. Results showed that the general structural genes of phenylpropanoid and flavonoid metabolic pathways, including some members of phenylalanine ammonialyase (PAL, EC 4.3.1.24), 4-coumarate: CoA ligase (4CL, EC 6.2.1.12) and flavanone 3-hydroxylase (F3H, EC 1.14.11.9), were significantly or moderately (fold-change ≥ 2 while divergence probability ≤ 0.8) up-regulated in berries from the LR-V and LM-V treatment groups at E-L 36 and 38 stages while downregulated at E-L 37 stage. Furthermore, almost all members of CHS (EC 2.3.1.74) and chalcone isomerase (CHI, EC 5.5.1.6) were moderately down-regulated in sunlight exposed grapes in comparison with those from the control group across the three developmental stages. The expression of members of specific structural genes required for HCA and flavonol biosynthesis, including cinnamyl-alcohol dehydrogenase (CAD, EC 1.1.1.195) and FLS (EC 1.14.11.23) across the three developmental stages as well as bifunctional UDP-glucose/UDP-galactose:flavonol-3- O-glucosyltransferase/galactosyltransferase (GT6, EC 2.1.1.76) at E-L 38 stage, and members of dihydroflavonol reductase (DFR, EC 1.1.1.219) and UFGT (EC 2.4.1.115) required for anthocyanin biosynthesis at E-L 36 and 37 stages were significantly or moderately up-regulated in LR-V and LM-V treated berries, leading to the increased accumulation of corresponding phenolic products. The lower contents of flavan-3-ols in LR-V and LM-V treated berry skins compared with the control group at E-L 37 and 38 stages is supported by the moderately down-regulation of members of leucoanthocyanidin reductase (LAR, EC 1.17.1.3) and anthocyanidin reductase (ANR, EC 1.3.1.77), which are directly involved in flavan-3-ol biosynthesis (Figures 3, 4A). It was previously reported that VviFLS4 was the sole member of the grapevine FLS family which specifically responded to different light regimes and showed a clear expression pattern corresponding to the accumulation of flavonols in the berry skins (Fujita et al., 2006; Matus et al., 2009; Koyama et al., 2012; Pastore et al., 2013). In our study, the transcription of several other members of FLS family in addition to VviFLS4 (VIT\_218s0001g03470) was also drastically induced (fold-change ≥ 2) by LR-V and/or LM-V treatments in different developmental stages, such as VIT\_202s0012g00390, VIT\_202s0012g00400 and VIT\_202s0012g00450 in LR-V treated berries at E-L 36 stage, as well as VIT\_208s0007g00750 and VIT\_213s0067g01020 in LR-V and LM-V treated berries at both E-L 36 and 38 stages (Figure 4A). These results were consistent with the increased accumulation of flavonols in lightexposure berries, which indicates that grapevine FLS gene family may be functionally redundant in response to light signal. The hydroxylation pattern of flavonoids is known to be mediated by the enzyme activity of F30H (EC 1.14.13.21) and F3<sup>0</sup> 5 <sup>0</sup>H (EC 1.14.13.88), which catalyze the hydroxylation of naringenin and dihydrokaempferol at the 3<sup>0</sup> and 3<sup>0</sup> 5 <sup>0</sup> positions of the B-ring, respectively (Bogs et al., 2006; Guan et al., 2015). Our results showed that no significant differences in the expression level of genes encoding F30H among treatments were detected at each sampling point, but the transcription abundance of several F30 5 <sup>0</sup>H family members was significantly down-regulated by LR-V and moderately down-regulated by LM-V at both E-L 36 and 37 stages (Figure 4A), which were in fair agreement with the higher ratio of dihydroxylated/trihydroxylated flavonoids observed in berries from LR-V and LM-V treatments, in comparison with those in berries from the control group (Figure 3D). To identify additional genes that might contribute to alterations in phenolic metabolism in berries grown under different light conditions, the transcription profile of phenolic biosynthesis-related genes was compared with the HCA, total flavonoid, flavonol, flavan-3-ol, and anthocyanin profiles of all samples, respectively. The correlation analysis based on pearson's coefficient revealed that the expression of four members of PAL (VIT\_216s0039g01100, VIT\_216s0039g01110, VIT\_216s0039g01120, and VIT\_216s0039g01130), the first committed enzyme in phenylpropanoid metabolism (Sparvoli et al., 1994), was highly significantly (p-value ≤ 0.01) correlated with the accumulation of flavonoids in berries from different light treated groups. The transcript of two other genes (VIT\_213s0047g00210 and VIT\_206s0061g00450) belonging to the 4CL and F3H families was also significantly (p-value ≤ 0.05) correlated with the changes of flavonoid content in each samples. In addition, there was a significant correlation (p-value ≤ 0.05) between flavonol accumulation and the expression of two members of FLS family (VviFLS4 and VIT\_208s0007g00750) mentioned above (Supplementary Table S5). However, no specific structural genes for phenolic biosynthesis pathway were found to be correlated with the accumulation of HCAs, flavan-3-ols or anthocyanins, which indicates that the biosynthesis or degradation of these compounds in berries under different light regimes might be controlled by the cooperation of multiple enzymes from the entrance to branches. ## Expression Analysis of Flavonoid Biosynthesis-related Transcription Factors In grapes, some members of R2R3-MYB TF family and their co-activators belonging to other TF families (bHLH and WDR) which could regulate the transcription of downstream target genes required for phenolic biosynthesis pathways have been isolated and characterized recently (Hichri et al., 2011). The expression of these TFs involved in flavonoid metabolism has also been reported to be induced or suppressed by many environmental factors, such as light quality, temperature and water deficit conditions (Castellarin et al., 2007; Cominelli et al., 2008; Azuma et al., 2012). The transcript level of VviMYBF1, which acts as a direct regulator of VviFLS4 expression (Czemmel et al., 2009; Matus et al., 2009), was moderately greater in berries after light-exposure treatments than those in the control berries, except for a slight decrease in LM-V treated berries at E-L 37 stage compared with the control (Figure 4B). Expression of two UV-B-inducible grapevine flavonol synthesis regulators, VviHY5 and VviHYH (Loyola et al., 2016), was also significantly and moderately up-regulated in LR-V and LM-V treated berries at postvéraison berry developmental (E-L 38) stage, respectively. These results were well consistent with an increase in the transcript abundances of members of FLS as well as flavonol concentrations after light-exposed treatments, which indicates that light affects flavonol biosynthesis through transcript activation of a series of TFs and structural genes. Several regulators of the general branch and different branches of flavonoid synthesis, including VviMYB5a, VviMYB5b, VviMYBPA1, VviMYBPA2, VviMYBPAR, VviMYBC2-L1, VviMYBC2-L2, VviMYBC2-L3, and a TTG2-like homolog protein VviWRKY26 (Amato et al., 2016), showed large divergent changes in the transcript levels during berry development or under different light-exposure treatments (Figure 4B). Transcript abundances for VviMYB5a and VviMYB5b, VviWRKY26, as well as the negative regulator of PA accumulation VviMYBC2-L1 (Huang et al., 2014) in grape berries presented a high level during the three development stages but did not respond to changing light conditions, while low levels of VviMYBPA2, VviMYBPAR, VviMYBC2-L2, and VviMYBC2-L3 transcripts were detected in berries from all treatment groups. In addition, the expression of VviMYBPA1 was significantly and slightly down-regulated in LR-V and LM-V treated berries FIGURE 4 \| Effects of sunlight exposure on the transcript profile of the (A) enzymes and (B) regulatory factors involved in phenolic biosynthesis in grape berries. PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-monooxygenase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl-alcohol dehydrogenase; COMT, caffeic acid 3-O-methyltransferase; 4CL, 4-coumarate: CoA ligase; CHS:,chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F30H, flavonoid 3<sup>0</sup> -hydroxylase; F305 <sup>0</sup>H: flavonoid 3<sup>0</sup> ,50 -hydroxylase; FLS, flavonol synthase; GT5, uridine diphosphate (UDP)-glucuronic acid:flavonol-3-O-glucuronosyltransferase; GT6, bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase; DFR, dihydroflavonol reductase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase. The number of expressed family members for each enzyme is indicated in green box. Each square in the heatmap located beside their gene names corresponds to the average FPKM value of the gene in each sample as illustrated in the legend. Genes with significant expression changes compared with the control groups in each developmental stage are indicated by asterisks (<sup>∗</sup> ) in the squares. Expression profiles of specific structural genes required for the biosynthesis of HCAs, flavonols, flavan-3-ols and anthocyanins are shown in pink, yellow, and violet dotted boxes, respectively. C, control group; LR-V, leaf removal at véraison; LM-V, leaf moving at véraison. at E-L 38 stage, respectively, which correlated well with the changes of LDOX expression and total flavan-3-ol contents at this stage. Therefore, it may be speculated that the transcript level of VviMYBPA1 might lead to the difference in the flavan-3-ol contents of grape berries growing under different light conditions. Furthermore, transcript abundances of VviMYBA1 and VviMYBA2, two regulators of the anthocyanin branch (Walker et al., 2007), were slightly up-regulated at E-L 36 stage while down-regulated at E-L 37 or 38 stage in light exposed berries, correlating with the anthocyanin levels responded to light conditions in the grape skin. However, no significant changes in the expression of two bHLH factors, VviMYC1 and VviMYCA1, as well as two WDR proteins VviWDR1 and VviWDR2 among different light-exposure groups were observed (Figure 4B), although they have been reported to be involved in anthocyanin and/or PA synthesis (Hichri et al., 2010; Matus et al., 2010) and differentially modulated by different light qualities in other plant species (Sompornpailin et al., 2002; Cominelli et al., 2008). # Validation of RNA-seq by Quantitative Real-time PCR To validate the expression profiles obtained from the RNA-seq data, 15 genes relating to our biological focus were selected to subject to qRT-PCR analysis. They included 12 phenylpropanoid/flavonoid biosynthetic pathway related structural genes (VviPAL1, VviPAL2, VviPAL7, VviPAL15, VviF3H1, VviF3H2, VviF3'H, VviF3<sup>0</sup> 5 <sup>0</sup>H, VviFLS1, VviFLS2, VviFLS3, and VviFLS4), as well as VviMYBF1, VviMYBPA1 and VviMYBA1 TF genes involved in the regulation of flavonol, flavan-3-ol and anthocyanin biosynthesis, respectively (Supplementary Figure S2). Two housekeeping genes in V. vinifera, VviUbiquitin1 and Vviβ-Actin were used as endogenous controls for normalization as their relatively constant expression throughout grape berry development as well as in berries under various stress conditions (Downey et al., 2003; Reid et al., 2006). The results showed that the expression of 15 genes determined by qRT-PCR was significantly correlation with those from the RNA-seq data at the 0.01 level (r = 0.54), thus verifying the method. ### Co-expression Analysis between Metabolic Pathway Genes and Transcription Factor Genes Transcriptome co-expression analysis, which is based on the assumption that genes with similar expression patterns are most likely to be functionally associated, has proven to be a powerful tool for revealing regulatory networks of genes involved in linked processes (Persson et al., 2005). In plants, this strategy has been applied to identify factors regulating several metabolic pathways, such as two Arabidopsis MYB TFs regulating aliphatic glucosinolate biosynthesis and a rice AP2/EREBP (APETALA 2/ethylene responsive element binding protein) family TF involved in starch biosynthesis (Hirai et al., 2007; Fu and Xue, 2010). To systemically identify unknown putative regulators that control the phenolic biosynthesis in grape berries in response to different light regimes, a genomewide co-expression analysis was employed between metabolic pathway genes and TF genes. Eight phenolic synthesis genes screened previously, including genes encoding PALs, 4CL, F3H, and FLSs, were selected as "guide genes" to identify co-expression relationships specific to the light-induced differentially expressed TF genes using expression data of all light treated and control samples from RNA-seq. Any two genes with an absolute value of the Pearson Correlation Coefficient (PCC) greater than 0.7 (or 0.8) and p-value less than or equal to 0.05 (or 0.01) between their expression profiles were considered as significant (or highly significant) co-expressed genes (Fu and Xue, 2010). The results showed that a total of 120 and 59 TFs were highly co-expressed with the six total flavonoid biosynthesis-related (group I) and the two flavonol biosynthesis-related (group II) guide genes, respectively (Figure 5). Among the identified group I co-expressed TFs, the most abundant positively correlated TFs were members of the MYB, WRKY, C2C2, AP2/EREBP, bHLH, and MADS-box families (p-value ≤ 0.01), whereas the most abundant negatively correlated TFs belonging to MYB, NAC (No apical meristem, ATAF 1,2, Cup-shaped cotyledon 2), Cys2/His2 (C2H2) type and CCCH type (C3H) zinc finger protein families (p-value ≤ 0.01). Similarly, specific members of MYB, AP2/EREBP and C2C2 families were also found to be the most abundant significantly positively co-expressed TFs with group II (p-value ≤ 0.05). In all plant species analyzed to date, MYB TFs, together with bHLH and WDR proteins, act as common denominators in the regulation of flavonoid accumulation under various biotic or abiotic signals, such as high-light, UV, drought, and extreme temperatures (Koes et al., 2005; Xu et al., 2015). Some additional potential transcriptional regulators that belong to WRKY, MADS-box, and bZIP TF families have also been reported to be involved in specific branches of the phenylpropanoid metabolism (Hichri et al., 2011). Besides, several negative regulators of flavonoid synthesis, such as R2R3-MYB, single domain R3-MYB repressors and truncated bHLH, inhibit the formation of MBW complex or modify it, thereby actively repress transcription in plants (Liu et al., 2015). The positively or negatively co-expression of multiple members of MYB, bHLH, WRKY, and MADS-box TF families with those key genes in the present result suggests that light-regulated flavonoid biosynthesis in grape berries is maintained by a complex regulatory network involves both positive and negative feedback loops. By using over-expressing and antisense transgenic plant strategies, it was shown that some DOF (DNA-binding One Zinc Finger) genes from the C2C2 zinc finger-containing TF superfamily putatively involved in regulation of enzymes of the phenylpropanoid and flavonoid pathways in Arabidopsis (Noguero et al., 2013). The plant-specific AP2/EREBP TF family, which was composed of AP2, DREB (cis-acting dehydration responsive element-binding protein), RAV (related to ABI3/VP1), ERF (ethylene responsive factor) and other subfamilies, plays a major role in several developmental processes, and also participates in plant hormone signal transduction as well as plant's responses to pathogens and various environmental stresses (Dietz et al., 2010). Recent studies revealed that a class of repressor-type ERF-subfamily TFs act as active or passive repressors of transcription via their ERF-associated amphiphilic repression (EAR) domain, which was also found in some C2H2 type zinc-finger proteins and R2R3-MYB repressors of flavonoid synthesis-related genes in various plant species (Aharoni et al., 2001; Ciftci-Yilmaz and Mittler, 2008; Huang et al., 2014). NAC proteins are another plant-specific TFs which have been shown to play an essential role in regulating senescence, cell division, and wood formation, and also participate in plant response to pathogens, viral infections, and various environmental stresses (Nakashima et al., 2012). It was also reported that a NAC protein in Arabidopsis, ANAC078, positively regulates the expression of genes related to the biosynthesis of flavonoids, subsequently leading to the accumulation of anthocyanins in response to high-light (Morishita et al., 2009). Furthermore, VviNAC29, a protein belonging to the grapevine NAC TF superfamily, was demonstrated to act as a cooperative regulator controlling the stress-responsive expression of VviF30H in our previous study (Sun et al., 2015b). However, their roles in the negative regulation of the flavonoid synthesis-related genes have not been investigated previously, thus, the conclusion that whether they could directly or indirectly regulate the light-response of phenolic biosynthesis still needs to be further characterized. # Light Response of Plant Hormone Signal Transduction Related Genes Phytohormones have been implicated in controlling various aspects of grape berry development, in particular, the important processes of ripening and adaptation to adverse environmental conditions, including harmful UV radiation (Jeong et al., 2004). In some cases, hormone pathways act downstream of the light signal pathways to regulate growth, whereas in other cases they interact with each other reciprocally (Alabadí and Blázquez, 2009). In LR-V treated berries, the transcript abundances of some members of PYR/PYL (VIT\_208s0058g00470 and VIT\_210s0003g01335) and abscisic acid (ABA) responsive element binding factor (ABF; VIT\_208s0007g03420) involved in ABA signal transduction (Klingler et al., 2010), xyloglucan:xyloglucosyl transferase TCH4 (VIT\_211s0052g01190) involved in BR signal transduction (Clouse, 2015), jasmonate ZIM domain-containing protein (JAZ; VIT\_201s0146g00480) and MYC2 TF (VIT\_211s0052g00100) involved in jasmonic acid (JA) signal transduction (Fernández-Calvo et al., 2011), as well as TGA TF (VIT\_207s0031g02670 and VIT\_208s0007g06160) and pathogenesis-related protein 1 (PR-1; VIT\_203s0088g00780, VIT\_203s0088g00810, VIT\_203s0088g00910, and VIT\_203s0097g00700) involved in salicylic acid (SA) signal transduction (Eulgem, 2005) were significantly or moderately higher compared with that of the control at nearly all the three developmental stages (Figure 6). In addition, the transcription of some proteins involved in other plant hormone signal transductions was also significantly or moderately up-regulated in LR-V treated berries at specific developmental stages, such as members of the histidine kinase receptors CRE1 (VIT\_213s0019g01180 and VIT\_217s0000g04920) and histidine-containing phosphotransfer protein (AHP; VIT\_211s0016g03170) required for cytokinin (CTK) signal transduction (Choi and Hwang, 2007) and gibberellin receptor GID1 (VIT\_213s0084g00130) required for Gibberellin (GA) signal transduction (Hirano et al., 2008) at E-L 36 stage, members of the auxin influx carrier AUX1 (VIT\_208s0007g02030) and auxin-responsive protein IAA (AUX/IAA; VIT\_211s0016g03540) required for auxin signal transduction (Lau et al., 2008) at E-L 38 stage. Notably, a member of serine/threonine-protein kinase CTR1 (VIT\_218s0001g07700), a negative regulator of the ethylene (ETH) response pathway (Chen et al., 2005), was up-regulated in LR-V treated berries at E-L 38 stage. Meanwhile, a member of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO, EC 1.14.17.4; VIT\_211s0016g02380), the last enzyme in the ETH production pathway which controlled the biosynthesis of ETH in plants (Chervin et al., 2004), was significantly down-regulated in the same developmental stage. In berries from LM-V treatment, a member of TCH4 (VIT\_211s0052g01190) was significantly up-regulated, while members of AUX/IAA (VIT\_214s0030g02310) and PR-1 (VIT\_203s0088g00710) were significantly down-regulated at E-L 37 stage. No significant differences in the expression of plant hormone signal transduction related genes between berries from LM-V treatment and the control group were detected at both E-L 36 and 38 stages. Nevertheless, a member of 9-cis-epoxycarotenoid dioxygenase (NCED, EC 1.13.11.51; VIT\_219s0093g00550), which limits the level of ABA in the biosynthesis pathway (Zhang et al., 2009), was up-regulated in LM-V treated berries at E-L 36 stage, while a member of ACO (VIT\_211s0016g02380) was down-regulated at E-L 38 stage (Figure 6). Previous studies indicate that the accumulation of phenolics in berry skin during the ripening stage, as well as the expression of structural genes and their transcriptional regulators considered to be involved in the phenylpropanoid and flavonoid pathways, were enhanced by exogenous ABA and ETH treatments, while suppressed by synthetic auxins, NAA (Ban et al., 2003; El-Kereamy et al., 2003; Jeong et al., 2004; Fujita et al., 2006; Koyama et al., 2010). The acceleration of berry ripening and flavonoid accumulation in LR-V treated grape berries was well correlated with the enhancement of ABA signal transduction, which might act as a protective mechanism induced by enhanced light irradiation (Berli et al., 2011), while the increased auxin signal transduction and decreased biosynthesis of ETH might result in the suppression of flavonoid biosynthesis, especially flavan-3-ols and anthocyanins at harvest. Moreover, the different expression of metabolic enzymes of phytohormones at E-L 36 and 38 stages, and the transcriptional changes of AUX/IAA at E-L 37 stage, were shown to be perfectly correlated with changes in the accumulation of flavonoids in LM-V treated berries. A number of studies have indicated that light signaling affects the biosynthesis and/or signaling of multiple phytohormones such as auxin, GA, CTKs, ETH, and BRs (Carvalho et al., 2011). The interactions between light and hormones pathways operate through distinct molecular mechanisms in plants and play an important role in the adjustment of developmental programs and behavior of the plants to the environment (Alabadí and Blázquez, 2009). Taken together, our results suggest that phenolic metabolic in berry skins of the Cabernet Sauvignon grape is precisely controlled by a series of phytohormones in response to exchanged light irradiation. # CONCLUSION In the present study, the transcriptional profiles and metabolite profiles of phenolic biosynthesis pathway were analyzed in Cabernet Sauvignon grapes under different sunlight exposure treatments during berry development. Leaf removal or leaf moving at different berry development stages did not show consistent effects on the accumulation of flavan-3-ol, anthocyanin or total flavonoids in grape berries over three seasons. However, the concentrations of HCAs and flavonols were moderately and drastically increased in sunlight exposed grape berries, respectively, which is well correlated with changes in transcriptional abundance of PAL, 4CL, F3H, and FLS family members as well as large amounts of regulatory genes. Furthermore, the transcriptional changes of genes required for the biosynthesis and signal transduction of auxin, ETH and ABA were found to be exactly in accordance with the accumulation of phenolics in light exposed berries during development, confirmed the importance of phytohormones on berry phenolic biosynthesis of grapes in response to light environment. Taken together, our results provide new valuable insights into understanding of the complex regulatory network of sunlightresponsive phenolic biosynthesis in grape berries, as well as theoretical foundations for cultivation management and wine production. #### AUTHOR CONTRIBUTIONS JW and WC conceived and designed the experiments. GC and Y-NH did the field experiments. R-ZS, GC, and QL analyzed the transcriptome sequencing data. GC, QL, YW, Y-RZ, W-FS, XZ, and X-DC performed the HPLC quantification. Y-BL and S-YL provided technical support. R-ZS and JW wrote the paper. All the authors revised it critically for important #### REFERENCES intellectual content and approved the final version of this manuscript. #### FUNDING This work was supported by China Agriculture Research System (CARS-30). #### ACKNOWLEDGMENTS The authors thank to Prof. Chang-Qing Duan, Prof. Qiu-Hong Pan, Dr. Fei He (China Agricultural University) and Dr. Bao-Qing Zhu (Beijing Forestry University) for information support. The authors are also grateful to CITIC Guoan Wine Co. Ltd for technical assistance. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00547/ full#supplementary-material of white-fleshed and teinturier grape berries. Planta 243, 23–41. doi: 10.1007/ s00425-015-2391-4 fpls-08-00547 April 19, 2017 Time: 17:49 # 16 of public microarray data sets. Proc. Natl. Acad. Sci. U.S.A. 102, 8633–8638. doi: 10.1073/pnas.0503392102 fpls-08-00547 April 19, 2017 Time: 17:49 # 17 heterozygous grapevine variety. PLoS ONE 2:e1326. doi: 10.1371/journal.pone. 0001326 during abscisic acid biosynthesis from peach and grape fruits. J. Plant Physiol. 166, 1241–1252. doi: 10.1016/j.jplph.2009.01.013 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 © 2017 Sun, Cheng, Li, He, Wang, Lan, Li, Zhu, Song, Zhang, Cui, Chen and Wang. 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. # Sunlight Modulates Fruit Metabolic Profile and Shapes the Spatial Pattern of Compound Accumulation within the Grape Cluster #### Noam Reshef, Natasha Walbaum, Nurit Agam\* and Aaron Fait\* French Associates Institute for Agriculture and Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer, Israel Vineyards are characterized by their large spatial variability of solar irradiance (SI) and temperature, known to effectively modulate grape metabolism. To explore the role of sunlight in shaping fruit composition and cluster uniformity, we studied the spatial pattern of incoming irradiance, fruit temperature and metabolic profile within individual grape clusters under three levels of sunlight exposure. The experiment was conducted in a vineyard of Cabernet Sauvignon cv. located in the Negev Highlands, Israel, where excess SI and midday temperatures are known to degrade grape quality. Filtering SI lowered the surface temperature of exposed fruits and increased the uniformity of irradiance and temperature in the cluster zone. SI affected the overall levels and patterns of accumulation of sugars, organic acids, amino acids and phenylpropanoids, across the grape cluster. Increased exposure to sunlight was associated with lower accumulation levels of malate, aspartate, and maleate but with higher levels of valine, leucine, and serine, in addition to the stress-related proline and GABA. Flavan-3-ols metabolites showed a negative response to SI, whereas flavonols were highly induced. The overall levels of anthocyanins decreased with increased sunlight exposure; however, a hierarchical cluster analysis revealed that the members of this family were grouped into three distinct accumulation patterns, with malvidin anthocyanins and cyanidin-glucoside showing contrasting trends. The flavonol-glucosides, quercetin and kaempferol, exhibited a logarithmic response to SI, leading to improved cluster uniformity under high-light conditions. Comparing the within-cluster variability of metabolite accumulation highlighted the stability of sugars, flavan-3-ols, and cinnamic acid metabolites to SI, in contrast to the plasticity of flavonols. A correlation-based network analysis revealed that extended exposure to SI modified metabolic coordination, increasing the number of negative correlations between metabolites in both pulp and skin. This integrated study of micrometeorology and metabolomics provided insights into the grape-cluster pattern of accumulation of 70 primary and secondary metabolites as a function of spatial variations in SI. Studying compound-specific responses against an extended gradient of quantified conditions improved our knowledge regarding the modulation of berry metabolism by SI, with the aim of using sunlight regulation to accurately modulate fruit composition in warm and arid/semi-arid regions. Keywords: solar irradiance, microclimate, spatial heterogeneity, grape composition, phenylpropanoids, metabolite profiling, climate change, shading #### Edited by: Giovanni Battista Tornielli, University of Verona, Italy #### Reviewed by: Laurent Deluc, Oregon State University, USA Marianna Fasoli, E & J Gallo Winery, USA #### \Correspondence: Nurit Agam [email protected] Aaron Fait [email protected]* #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 01 November 2016 Accepted: 12 January 2017 Published: 01 February 2017 #### Citation: Reshef N, Walbaum N, Agam N and Fait A (2017) Sunlight Modulates Fruit Metabolic Profile and Shapes the Spatial Pattern of Compound Accumulation within the Grape Cluster. Front. Plant Sci. 8:70. doi: 10.3389/fpls.2017.00070 # INTRODUCTION Plasticity is the ability of an organism to adapt its phenotype in response to changes in the environment. For plants, it is an important adaptive strategy to cope with environmental variability (Svanback and Eklov, 2006) and facilitates the use of the same cultivar in a wide range of growing conditions (Dai et al., 2011). However, at the single-field scale, phenotypic plasticity may be regarded as a significant cause for within-field variation. Field systems, such as vineyards experience a large range of microclimate conditions manifested in significant spatial variation of SI and temperature (Oyarzun et al., 2007; Matese et al., 2014). Grape and wine quality reflects the levels and composition of a large number of primary and secondary metabolites that shape the overall sensory experience of its derived product. Primary metabolites are largely accumulated in the pulp and include sugars, organic acids, and amino acids. Grape secondary metabolites predominantly include the phenylpropanoids, typically found in the berry skin, and comprise flavonoids, phenolic acids, stilbenes, and viniferins (Anesi et al., 2015). While sugars are largely imported to the fruit through the plant vascular tissues (Ollat et al., 2002), the overall levels and composition of organic acids, amino acids, and secondary metabolites at harvest are determined by the sum of complex metabolic processes occurring in the fruit during its lifetime (Conde et al., 2007; Deluc et al., 2007; Dai et al., 2013). Indeed, metabolic shifts caused by changes in climatic conditions in the immediate vicinity of the fruit (i.e., microclimate), such as light and temperature, are well-known to affect fruit composition (Jackson and Lombard, 1993; Downey et al., 2006). Studies exploring the effect of microclimate on fruit primary metabolites largely agree that sunlight, and specifically an increase in the temperature load of the fruit, highly modulate malic acid levels, leading to a corresponding decrease in overall fruit acidity (Kliewer, 1965; Jackson and Lombard, 1993; Conde et al., 2007; Sweetman et al., 2014). However, results regarding the effect on the relatively stable tartaric acid are still controversial (Rienth et al., 2016; Young et al., 2016). Discrepancies also exist regarding the effect on amino acids, owing in part to the differential response of cultivated varieties. For example, filtering UV-B irradiance was found to increase the overall levels of amino acids in the juice of Riesling cv. (Schultz et al., 1998), but had no effect on the levels and composition of amino acids in Sauvignon Blanc cv. grapes at harvest (Gregan et al., 2012). With regards to grape specialized metabolism, studies underline the flavonoids as a highly responsive group to light and temperature perturbations (Chalker-Scott, 1999; Winkel-Shirley, 2002). For instance, flavonol glucoside concentrations in the fruit positively correlated with increased SI on the cluster (Haselgrove et al., 2000; Spayd et al., 2002), and were found to be the most significant metabolites distinguishing the metabolic profile of shaded berries from that of exposed berries (Pereira et al., 2006). Fruit sunlight exposure was found to modulate the composition of anthocyanins, such as the proportion of acylated and coumarylated forms, the ratio between di-hydroxylated and tri-hydroxylated anthocyanins (Haselgrove et al., 2000; Spayd et al., 2002; Downey et al., 2004; Tarara et al., 2008; Chorti et al., 2010), and the proportion of ortho-di phenol anthocyanins (Rustioni et al., 2011). However, the overall accumulation of anthocyanins showed higher dependency on temperature conditions than on SI (Downey et al., 2006), with the effect of the interaction between incoming SI and fruit temperature on grape anthocyanin levels exhibiting a synergistic or antagonistic character, depending on the ranges of both factors (Tarara et al., 2008; Azuma et al., 2012). While the overall environmental effect on grape has been investigated, few studies have addressed the within-cluster spatial profile of compound accumulation and variability. Pisciotta et al. (2013) characterized berry anthocyanin content considering its vertical position on the rachis and external and internal faces of the cluster (i.e., facing the inter-row or the canopy). However, no data regarding micrometeorological conditions were presented, hampering the interpretation of the results with respect to environmental conditions. Zhang et al. (2015) found greater polyphenol content in regions of the cluster characterized by lower berry surface temperature in Shiraz cv. More recently, by using model-generated microclimate data, Pieri et al. (2016) related the overall levels of berry flavonols, anthocyanins, and amino acids to cluster exposition and its impact on the estimated levels of SI and berry surface temperature (BST). Taken together, these works indicate that micro-scale environments play an active role in generating the spatial patterns of metabolite accumulation that affect fruit composition, as well as the uniformity of the crop. Optimizing crop uniformity is generally regarded as an important aspect in the overall improvement of wine quality (Keller, 2010), owing to its significant role in the composition of the harvested grape, and final wine (Kontoudakis et al., 2011; Liu et al., 2016). However, recent attempts to minimize field heterogeneity through the use of common vineyard management techniques, such as deficit irrigation and cluster thinning, were found inefficient (Calderon-Orellana et al., 2014). This stresses the importance of exploring the effectiveness of other potential techniques, including the modulation of clusterzone microclimate, to form more homogeneous conditions. As a key factor determining fruit intercepted irradiance and temperature (Smart and Sinclair, 1976), practical interventions to ameliorate fruit microclimate generally involve the regulation of sunlight exposure. Practices, such as basal leaf removal, or shading, whether by the use of artificial nets or by the use of Abbreviations: GABA, gamma-Aminobutyric acid; Cyan-3-glu, Cyanidin-3- O-glucoside; Pet-3-glu, Petunidin-3-O-glucoside; Peo-3-glu, Peonidin-3-Oglucoside; Mal-3-glu, Malvidin-3-O-glucoside; Delph-3-glu, Delphindin-3-Oglucoside; Delph-3-acet, Delphinidin-3-O-(6′′-acetyl-glucoside); Cyan-3-acet, Cyanidin-3-O-(6′′-acetyl-glucoside); Pet-3-acet, Petundin-3-O-(6′′-acetylglucoside); Mal-3-acet, Malvindin-3-O-(6′′-acetyl-glucoside); Peo-3-acet, Peonidin-3-O-(6′′-acetyl-glucoside); Delph-3-coum, Delphinidin-3-O-(6′′ p-coumaroyl-glucoside); Mal-3-caffe, Malvidin-3-O-(6′′-caffeoyl-glucoside); Cyan-3-coum, Cyanidin-3-O-(6′′-p-coumaroyl-glucoside); Pet-3-coum, Petunidin-3-O-(6′′-p-coumaroyl-glucoside); Peo-3-coum, Peonidin-3-O-(6′′-pcoumaroyl-glucoside); Mal-3-coum, Malvidin-3-O-(6′′-p-coumaroyl-glucoside); Myr-3-glr, Myricetin-3-O-glucuronide; Rutin, Quercetin-3-O-rutinoside; Myr-3-glu, Myricetin-3-O-glucoside; Quer-3-glr, Quercetin-3-O-glucuronide; Quer-3-glu, Quercetin-3-O-glucoside; Kaemp-3-glr, Kaempferol-3-Oglucuronide; Kaemp-3-glu, Kaempferol-3-O-glucoside; Narin-chalc-glu, Naringenin-chalcone-4-O-glucoside; Hex., Hexoside. extended canopies, are routinely used in the industry. However, integrated studies encompassing a gradient of sunlight exposure, profiling of metabolic data, and a quantitative characterization of microclimate conditions are lacking. Hence, our understanding of how sunlight regulation influences the spatial heterogeneity of fruit microclimate and its effect on fruit metabolic processes and coordination is currently limited. In this study, we performed a spatial characterization of micrometeorological conditions and metabolic profiles within individual grape clusters subjected to differing levels and directions of sunlight exposure. The extended range of SI intensities allowed us to explore the responses of a large number of primary and secondary (phenylpropanoids) metabolites to a range of microclimate conditions typically found in the field. This study's insights will aid in defining strategies for sunlight regulation aimed at improving fruit composition and uniformity in challenging environments. ## MATERIALS AND METHODS #### Site Description The experiment was conducted during the 2014 and 2015 growing seasons, in a vineyard located in the heart of the Negev Desert, Israel (30◦ 36′ 55.22′′N, 34◦ 45′ 12.00′′E, 800 m altitude). This is an arid region with an average annual precipitation rate of 70 mm (Israel Meteorological Service), which, during the growing season, is characterized by stable meteorological conditions including high SI and elevated midday temperatures (Supplementary Figure 1). The vineyard was planted in 2007 with Vitis Vinifera L. cv. Cabernet Sauvignon grafted on 140 Ruggeri rootstock, irrigated using a covered drip-irrigation system as is common in the region. Rows are orientated north-south with a 30◦ angle to the north-east\south-west, and are trained in vertical shoot positioning (VSP). Three experimental rows were selected, with one border row between every two experimental rows. In each row, three groups of nine adjacent vines were marked as field repetitions, and the basal leaves in the vicinity of the clusters (up to 30 cm above the cordon) were completely removed at the onset of veraison. Each field repetition was further assigned one of three shading treatments: fully exposed, i.e., no net (Exposed), 30% shading net (30% shaded) and 60% shading net (60% shaded), using UV-stabilized woven mesh shading nets for agriculture (Ginegar, Israel) with the percentage representing the PAR filtering capacity as published by the manufacturer. The nets were placed using thin metal support wires, one next to each vine, in a manner that created a shading tunnel with a diameter of about 80 cm around the cluster zone in order to facilitate wind flow and prevent an increase in relative humidity (Supplementary Figure 2). Shading was placed from the onset of veraison (the day of basal leaf removal) until harvest date. Treatments were repeated once on each row in a way that represented all locations along the row to minimize effects of spatial differences both between and within the rows. #### Meteorological Measurements During the 2015 growing season, a detailed study of clusterscale micrometeorological conditions was performed. Incoming SI, air temperature, relative humidity (RH%), and wind speed and direction were continuously monitored in 15-min intervals from veraison to harvest by placing a multi-sensor (WS501- UMB, Lufft, Fellbach, Germany) connected to a data logger (CR200, Campbell Scientific, Utah, USA) 1 m above the canopy, positioned in the field to allow for a maximum fetch in the direction of the prevailing winds (northwest). The distance between the sensor and the most distant experimental vine was approximately 90 m. Cluster-zone air temperature and RH% were measured continuously every 15 min from veraison to harvest by placing sensors equipped with an internal data logger (Hobo ProV2, Onset, MA, USA) at a shaded spot in the immediate vicinity of the clusters. Two sensors were placed at each treatment site to verify repeatability. The SI intensity on the clusters was measured simultaneously at four horizontal axes: parallel and perpendicular to the vine row, and vertically, to the sky (Supplementary Figure 3). This was done by constructing a box positioning all five pyranometers (LI200R, Li-Core, NE, USA) and connecting them to a single data logger (21X, Campbell Scientific, UT, USA). The sensors were stabilized and leveled on a tripod and placed at the cluster zone, with representative locations and distances from adjacent clusters verified to simulate cluster conditions. This system was continuously rotated every 3 days between field treatments and normalized to the SI measured above the canopy to allow for a comparison between measurements made on different dates. Measurements were done on the east side of the canopy for the shaded treatments and on both sides of the canopy for the exposed treatment. Berry surface temperature (BST) was measured on 28 July (post-veraison) and 19 August (pre-harvest) 2015, during two diurnal field campaigns from 6:00 to 20:00 using an infrared camera (T640, FLIR systems, OR, USA). Every hour, photos were taken from two representative clusters on each treatment, from each side of the canopy, for a total of 12 clusters. Each cluster was divided into three faces, north- and south-facing, and facing the inter-row (east for east-located clusters and west for westlocated clusters). Data analysis included a careful selection of berries located in the middle section of the cluster's vertical axis (i.e., middle height) in each photo and the exclusion of pixels representing non-grape background data. # Berry Sampling Sampling during both seasons was done several days prior to harvest date scheduled at 24◦Brix, to represent final berry composition and the potential of accumulated spatial differences. Samples were taken at pre-dawn in order to prevent any differences between sunlit and shaded berries at the time of sampling. Four vines per shading treatment were sampled, located in two out of the three field repetitions. Selected vines for sampling were at least four vines apart (Figure 1A). In each vine, four to six exterior-located clusters (furthest from the trunk toward the inter-row) were selected from each canopy side, to avoid possible shading by the canopy apart from midday hours. Each cluster was dissected into four horizontal orientations (Figure 1B), and samples were taken from the middle height of each orientation. Berries from a single vine and canopy side, located on the same cluster plane, were pooled together and immediately frozen in liquid nitrogen. In the lab, skin, pulp and seeds were carefully separated while kept frozen on dry ice, placed in Eppendorf tubes, and stored at −80◦C until further processing. Analysis included grape skin tissues sampled during both seasons and pulp tissues sampled during the 2015 season. # Analysis of Skin Phenylpropanoids Grape skin samples were analyzed using Ultra Performance Liquid Chromatography coupled to a Quadrupole Time-of-Flight Mass-Spectrometer (UPLC QTOF-MS, Waters, MA, USA), following an extraction protocol for metabolite profiling as described in Weckwerth et al. (2004). Skin tissues were lyophilized and ground under liquid nitrogen using a RETSCH-mill (Retsch, Haan, Germany) with prechilled holders and grinding beads. The powder was weighed (40 mg), and metabolites were extracted in a 1-ml pre-chilled methanol:chloroform:water extraction solution (2.5:1:1 v/v). Internal standards, i.e., 0.2 mg/ml ribitol in water, 1 mg/ml ampicillin in water, 1 mg/ml corticosterone in methanol, were subsequently added. The mixture was then briefly vortexed, and 100 µl of methanol was added; the mixture was then placed on a horizontal shaker for 10 min at 1000 rpm. The samples were later sonicated for 10 min (Elmasonic S30, Elma, Singen, Germany) and centrifuged for 10 min (20,817 × g, microcentrifuge 5417R, Eppendorf, Hamburg, Germany). The supernatant was decanted into new tubes, mixed with 300 µl of chloroform and 300 µl of MiliQ water (Millipore, MA, USA), vortexed for 10 s and then centrifuged at 20,817 × g for 5 min. Next, the water/methanol phase was separated, filtered (0.22 µm Millipore, MA, USA) and transferred to UPLC vials for analysis. ## LC/MS Conditions UPLC-QTOF-MS conditions were exactly as described previously by Hochberg et al. (2013). ### LC/MS Annotation MassLynxTM software (Waters) version 4.1 was used for system control and data acquisition. Metabolite annotation was validated using the standard libraries described in Arapitsas et al. (2012), based on retention time order, given in Degu et al. (2014). Metabolites were also annotated based on fragmentation patterns searched against the Chemspider metabolite database (http:// www.chemspider.com/), the consistency of their retention times with those of identified metabolites, and comparison with the data in the current scientific literature. #### Analysis of Pulp Primary Metabolites Pulp samples were manually crushed with a mortar and pestle while kept frozen with liquid nitrogen. Next, 100 mg of crushed, frozen powder was weighed and extracted by adding a methanol:chloroform:water solution (2.5:1:1), similar to the aforementioned phenylpropanoid extraction. Then, 70 µl of the extract were dried using a Concentrator Plus (Eppendorf, Hamburg, Germany) and derivatized exactly as described in Hochberg et al. (2015) with sorbitol as the internal standard. Glucose, fructose, tartaric and malic acids were quantified using a calibration curve of standards (Sigma-Aldrich, MO, USA) with 10 concentration points from 50 to 900 ng for glucose and fructose and 2.5 to 45 ng for malic and tartaric acids. A split ratio of 50:1 was used to correctly determine the levels of glucose, fructose, malic and tartaric acids, due to their relatively high abundance in the pulp. The GC-MS conditions were exactly as described previously by Hochberg et al. (2013). The Xcalibur data system V2.0.7 was used for system control and data acquisition. Annotation was based on spectral searching supported by the National Institute of Standards and Technology (NIST, Gaithersburg, MA, USA) against RI libraries from the Max-Plank Institute for Plant Physiology (Golm, Germany). #### Statistical Analysis Statistical analysis was performed using R v3.3.1 in RStudio. A profile analysis, from the "profileR" package, was used in order to compare the spatial profiles of clusters from exposed and shaded treatments and both canopy sides. This analysis tested the multivariate spatial data with two separate null hypotheses: a) the multivariate profile between groups is parallel; and b) the multivariate levels between groups are equal. This was done separately for each of the 70 annotated metabolites. In addition, within-cluster differences were tested for significance using the aov() function for ANOVA and the post-hoc Tukey test using the "agricolae" package. The same method was used for comparing the within-cluster coefficient of variance for each metabolite between different shading treatments, to highlight significant differences in cluster homogeneity caused by shading. Differences in malate/tartaric acid levels were tested for significance by using the built-in t-test() function. The Pearson correlation of the "corrplot" package was used in order to construct separate metabolite correlation matrices for fully exposed and 60% shaded clusters, based on the entire set of samples from each treatment. A regression analysis of each metabolite with daily incoming SI levels was done using the entire set of samples obtained from all treatments and cluster orientation, for which irradiance data was available. Linear and logarithmic regressions were assessed by the built-in lm() function, using non-transformed and log-transformed irradiance data, respectively. The R 2 -values of metabolites found to have significant regressions were then calculated by using the mean of four biological replicates, in order to minimize the effect of within-group variability on this coefficient. Heatmap figures were generated using TMeV v4.9, using the mean of four biological replicates. Hierarchical clustering of the heatmaps was based on the Pearson correlation. ### Network Analysis Correlation networks were constructed based on the data obtained from Pearson correlation analyses performed in R (detailed above), separately for exposed and 60% shaded treatments, and for metabolites detected in pulp and skin tissues (i.e., primary and secondary metabolites). Visualization and computation of network properties were performed using the "MetScape" application and the NetworkAnalyzer tool, respectively, available in Cytoscape V3.4.0. Correlations were incorporated into the network if the r-value was r > 0.5 or r < (−0.5). # RESULTS ### The Spatial Distribution of Micrometeorological Conditions across the Cluster Differed in Response to Shading and Position Cluster-zone shading successfully filtered the incoming SI as shown in Table 1. Clusters shaded with 30% and 60% shading nets received 64% and 34%, on average, of the incoming SI measured for fully exposed clusters, respectively. This did not lead to measured differences in the cluster-zone air temperature and relative humidity between treatments (data not shown). Comparing the different orientations in the east-facing cluster, the east-south orientation (orientation 1) received the largest amount of daily SI, followed by east-north (orientation 2) and finally west-south (orientation 3). The higher SI recorded for east-north compared to west-south may be attributed to the sun's zenith relative to the row orientation at the time of direct sunlight exposure on the corresponding sensors. Interestingly, a comparison between the exposed and the 30% shaded clusters, and the 30% shaded and the 60% shaded clusters revealed that incoming SI differences within a cluster exceeded the differences between treatments. Finally, while the percentage of SI intercepted by the different cluster orientations, compared to the reference of each treatment, remained unaffected, lowering the overall incoming SI levels to the cluster, by shading, effectively minimized the within-cluster heterogeneity in SI. Shading reduced the incoming energy differences between orientations 1 and 3 by 3-fold, from 6.8 to 2.3 MJ/m<sup>2</sup> /day for exposed and 60% shaded clusters, respectively. As expected, filtering the incoming SI caused a reduction in BST (Figure 2A). As shown by the percentiles, during direct sun exposure hours (e.g., morning for east-facing clusters), shading reduced the maximum BST, while having no effect on the minimum values, which represent the temperature of the shaded cluster faces. Notably, during the afternoon, when the east-facing clusters were exposed only to diffused radiation, the minimum BST of the 60% shaded clusters increased, while it remained stable for the 30% shaded and fully exposed clusters. Shading clusters with 60% shading nets decreased their maximum temperature by up to 7.1◦C and daytime (6:00–20:00) mean BST by 0.7◦C (Table 2). Examining the distribution of BST within a cluster, presented in histograms (Figure 2B), revealed that shaded clusters were more homogeneous than exposed ones. While maximum differences in BST within an exposed cluster Sums of daily irradiance, measured within the cluster-zone to represent the different cluster orientations.Values marked by \* are estimated based on the mean filtering efficiency of each shading net. Relative sums of daily irradiance values represent the percentage compared to the daily irradiance in orientation 1 of each treatment separately. Values enclosed in parentheses represent the percentage compared to the daily irradiance of the exposed treatment for each orientation separately. Data were not collected for orientations 4 and 5. TABLE 2 \| Berry surface temperature by cluster orientation in the three shading treatments. Berry surface temperature (BST) calculations based on hourly spatial measurements of representative clusters acquired on August 19, 2015. Data were not collected for orientations 4 and 5. reached 17◦C (33 to 50◦C at 11:00), they were 9.6◦C in 60% shaded clusters (34.3 to 43.9◦C at 12:00). ## Cluster Spatial Profile of Metabolite Level Was Affected by SI Intensity and Cluster Position The spatial profile analysis, i.e., the levels and pattern of metabolite accumulation in response to the berry position/orientation, was conducted separately for the primary (Figure 3) and secondary metabolites (Figure 4), and summarized in Supplementary Table 1. #### The Impact of Cluster Shading on the Spatial Pattern and Overall Levels of Fruit Metabolites #### Pulp primary metabolites Shading affected the spatial pattern (i.e., the pattern of accumulation in the different cluster orientations) of leucine, beta-alanine, and citrate in east-located clusters, increasing from internal to external orientations in exposed clusters, while shaded clusters had the opposite trend. Shading increased the levels of maleate and aspartate by 1.6- and 1.8-folds, respectively, and decreased the levels of tartaric acid, valine, and leucine by 1.3-, 2.2-, and 2.2-folds, respectively, in east-located clusters. In westlocated clusters, shading decreased the levels of phosphoric acid, erythritol, beta-alanine, valine, leucine, and GABA by 1.3-, 1.4-, 1.3-, 1.9-, 1.6-, and 2.1-folds, respectively. #### Skin phenylpropanoids Shading significantly affected the levels of 11 and 12 phenylpropanoids in the east- and west-located clusters, respectively, in 2015. Shading increased the levels of procyanidin B1 and epicatechin by 1.5- and 1.2-folds, respectively, and decreased the levels of the anthocyanins cyan-3-glu and cyan-3-acet (1.8- and 2-folds, respectively), phenylalanine and narin-chalc-glu (2.1- and 3-folds), and the flavonols myr-3-glu, myr-3-glr, quer-3-glu, kaemp-3-glu, and kaemp-3-glr by 1.7-, 1.4-, 2.1-, 4-, and 2.5-folds, respectively, in east-located clusters. In west-located clusters, shading significantly increased the levels of both mal-3-acet and mal-3-coum by 1.3-fold, and decreased the levels of cyan-3-glu, phenylalanine, narin-chalc-glu, and hydroxybenzoate hex (1.8-, 1.8-, 3-, and 1.5-folds), the stilbenes delta-viniferin and piceid (1.8- and 4.2-folds), and the flavonols myr-3-glu, quer-3-glu, kaemp-3-glu and kaemp-3-glr by 1.6-, 2.6-, 5.6-, and 2.8-folds, respectively. #### The Impact of Cluster Canopy Side on the Spatial Pattern and Overall Levels of Fruit Metabolites Pulp primary metabolites The canopy side of the cluster significantly affected the spatial pattern of malate, galactarate, maleate, threonine, glucose, and fructose in shaded clusters, while no significant effect was found in exposed clusters (Figure 3B). All mentioned metabolites followed a trend in which external cluster orientations had lower values than the internal ones, resulting in an opposite eastnorth-south-west pattern between the two sides of the canopy, corresponding with SI. In addition, canopy side significantly affected the overall levels of glucose and fructose in shaded clusters, and of lumichrome, beta-alanine, and GABA in exposed clusters. With the exception of lumichrome, all were found to be higher in clusters from the west side of the canopy than in those from the east. #### Skin phenylpropanoids Canopy side affected the spatial profile of 15 and 24 phenylpropanoid metabolites in exposed and shaded clusters, respectively, in the 2015 season (Figure 4). These included all the anthocyanins, with the exception of cyan-3-glu and vitisin A, the flavan-3-ols epigallocatechin and catechin, narin-chalcglu and phenylalanine, and the entire set of flavonols, with the exception of myr-3-glu. In the 2014 season, canopy side significantly affected the spatial profile of only four and five phenylpropanoid metabolites, in exposed and shaded clusters, respectively; of these, mal-3-glu and peo-3-coum, in exposed clusters, and the flavonols rutin, quer-3-glr, quer-3-glu, and kaemp-3-glu, in shaded clusters, repeated in both years. Canopy side had no significant effect on the levels of phenylpropanoids FIGURE 3 \| Cluster spatial level results for primary metabolites detected by GC-MS in berry pulp tissues sampled from different cluster orientations and shading treatments. (A) Illustrations of the daily irradiance levels in each cluster orientation including estimations based on diffusive light measurements and the measured mean filtering capacity of the shading nets. (B) Organic acids, amino acids, sugars, sugar alcohols, and other compounds found to be significantly affected by SI levels, cluster position and/or cluster orientation. Levels represent relative abundance based on ion count. Numbers on the X axis represent cluster orientations. Yellow bars represent fully exposed clusters, and dark gray bars represent 60% shaded clusters. Error bars are standard error (n = 4). Bars of the same cluster location and treatment, marked by different letters, represent significantly different values (α < 0.05). Information given in the boxes details the significant effects of treatment (upper box) for clusters located on the east (E) and the west (W) side of the canopy, as well as canopy side (lower box), on the spatial pattern and profile levels of metabolite accumulation in fully exposed (Ex) and 60% shaded (Sh) clusters. Asterisks in upper box indicate a significant effect of treatment on the spatial pattern of compound accumulation. Yellow and gray boxes indicate a significantly higher overall compound level in the exposed and 60% shaded treatments, respectively. Asterisks in lower boxes indicate a significant effect of canopy side on the spatial pattern and overall compound levels. FIGURE 4 \| Cluster spatial level results for phenylpropanoid metabolites detected by UPLC-QTOF-MS in berry skin tissues sampled from different cluster orientations and shading treatments. Included are pivotal phenylpropanoids and stilbenes, flavan-3-ols, flavonols, and anthocyanins found to be significantly affected by SI levels, cluster position and/or cluster orientation. Levels represent relative abundance based on ion count. Numbers on the X axis represent cluster orientations. Yellow bars represent fully exposed clusters, and dark gray bars represent 60% shaded clusters. Error bars are standard error (n = 4). Bars of the same cluster location and treatment, marked by different letters, represent significantly different values (α < 0.05). Information given in the boxes details the significant effects of treatment (upper box) for clusters located on the east (E) and the west (W) side of the canopy, as well as canopy side (lower box), on the spatial pattern and profile levels of metabolite accumulation in fully exposed (Ex) and 60% shaded (Sh) clusters. Asterisks in upper box indicate a significant effect of treatment on the spatial pattern of compound accumulation. Yellow and gray boxes indicate a significantly higher overall compound level in the exposed and 60% shaded treatments, respectively. Asterisks in lower boxes indicate a significant effect of canopy side on the spatial pattern and overall compound levels. Illustrations of the daily irradiance levels in the corresponding cluster orientation are given in Figure 3A. in 2015, while in 2014, mal-3-glu was higher in the eastlocated clusters than in the west; the opposite was found for phenylalanine. Overall, the proportion of metabolites showing significant responses to canopy side and shading were higher in the phenylpropanoids than in the primary metabolites, with sugars and hydroxy-cinnamates being the least affected chemical groups in pulp and skin tissues, respectively. #### Common and Differential Responses of Metabolite Groups to SI Hierarchical clustering of primary metabolites (Figure 5A) highlighted three major compound groups. The first group was composed mainly of nitrogenous compounds, including the amino acids leucine, valine, alanine, serine, and GABA, the biogenic amines putrescine and ethanolamine, and two sugar alcohols, galactinol, and erythritol. This group's compounds showed an increase from shaded to fully exposed clusters. In addition, in 30% shaded and fully exposed clusters, a generally higher abundance was found in external cluster orientations (i.e., facing the inter-row), especially for clusters located at the west side of the canopy, than in the internal orientations (i.e., facing the canopy), as seen for orientations 7 and 8 (external) compared to 4 and 5 (internal). The second group included metabolites associated with the TCA cycle, such as citrate, fumarate, malate, as well as the closely linked aspartate and maleate, in addition to glycolate, gluconate, and raffinose. These compounds showed a gradual decrease from the densely shaded to the fully exposed samples. In addition, their content was greater in the internal cluster orientations than in the external ones, irrespective of the treatment, as seen for orientations 4 and 5 compared to 1 and 8, respectively. The third group, comprising tartaric acid, the amino acids proline, beta-alanine and threonine, sucrose, glucose-6-phosphate, phosphoric acids, galactarate, malonate, transcaffeate, pyroglutamate, anhydro-gluopyranose, myo-inositol, and lumichrome, had a less pronounced pattern of change between treatments and cluster orientations. Clustering the phenylpropanoids using the 2015 season data (Figure 5B) grouped together compounds according to their chemical properties and biochemical pathways, such as those belonging to the flavonols, flavan-3-ols and hydroxy-cinnamates. In general, the flavonols showed a strong increase from the densely shaded to the exposed clusters, while the flavan-3 ols and cinnamates had the opposite trend. In contrast to the mentioned groups, the anthocyanins showed metabolitespecific trends. Cyanidin (glycosylated and acetylated) and peonidin (glycosylated) increased from the densely shaded to the exposed clusters and were grouped with the flavonols, as well as phenylalanine and the stilbene piceid, exhibiting an opposite trend to that of the malvidin metabolites. The rest of the anthocyanins were grouped together and exhibited an optimum in the 30% shaded cluster samples. The 2014 season data (Supplementary Figure 4), based on fully exposed and 60% shaded clusters, yielded similar trends. However, in contrast to the 2015 results, in the 2014 season, the coumaroylated forms of cyaniding, delphinidin, and petunidin clustered with the flavonols, while the glycosylated cyanidin and peonidin, together with the majority of the anthocyanins, formed a separate group. # Within-Cluster/within-Vine Heterogeneity of Metabolite Abundance Was Affected by Exposure to SI To study how the within-cluster heterogeneity of metabolite content was affected by shading, metabolite abundance values in samples taken from different orientations of an individual cluster were normalized to that specific cluster median. A hierarchical clustering of the samples (i.e., based on cluster orientations) from the 2015 season (Supplementary Figure 5) was used as a qualitative assessment of cluster heterogeneity where the magnitude of change from the cluster median is represented by the intensity of red and blue colors. Spatial patterns common to all treatments were evident, as samples from the internal orientations (i.e., samples 4 and 5) were clustered together and apart from the external ones (i.e., samples 1 and 8). Among the primary metabolites (Supplementary Figure 5A), organic and amino acids were the most heterogeneous compounds in all treatments, in contrast to the sugars glucose, fructose, and sucrose that were relatively uniform across cluster orientations. Finally, a subtle gradual increase in cluster heterogeneity was visible from the more uniform 60% shaded clusters to the more heterogeneous exposed clusters for the nitrogenous compounds putrescine, pyroglutamate, and ethanolamine, and the compounds trans-caffeate, lumichrome, and erythritol. Among the different phenylpropanoid groups (Supplementary Figure 5B), the flavonols were the most heterogeneous; furthermore, their within-cluster variability clearly increased with increasing levels of shading. To further verify this trend, the coefficient of variance of each metabolite was calculated per vine (i.e., within all samples originating in a single vine). Values were compared between treatments, and a significant treatment effect on the coefficient of variance was found for five out of the total 70 metabolites (Supplementary Figure 6). Among the primary metabolites, putrescine had a significantly lower coefficient of variance in the 60% shaded clusters than in the 30% shaded and fully exposed clusters, showing an improvement in uniformity with shading. In the phenylpropanoid group, the glucuronide and glycosylated forms of the flavonols quercetin and kaempferol had a significantly higher coefficient of variance in the 60% shaded clusters than in the 30% shaded and fully exposed clusters, showing an improvement in cluster uniformity with an increasing degree of sunlight exposure. ### Modulation of Metabolite-Specific Abundance by Sunlight The metabolite profiles of berries exposed to a gradient of SI intensity resulted in a significant linear regression between 33, out of 70, metabolites and the sum of daily SI. Figure 6 shows the regression of 12 metabolites of interest that obtained R <sup>2</sup> > 0.5, highlighting major metabolic shifts in the fruit in response to SI. Among the primary metabolites (Figure 6A), the two most abundant organic acids, malate and tartaric acid, were found to have contrasting responses to incoming SI levels, showing negative and positive regressions with incoming SI levels, respectively. This caused a significant increase in the ratio of tartaric acid/malate in fully exposed clusters compared to shaded ones (Supplementary Figure 7). As with malate, fumarate and aspartate showed similar negative responses. In contrast, a large number of nitrogenous compounds, including betaalanine, the branch-chained amino acids, valine and leucine, stress-related proline and GABA, and the biogenic amines, putrescine and ethanolamine, had positive regressions with SI levels (Figure 6A and Supplementary Table 2). Among the phenylpropanoids (Figure 6B and Supplementary Table 2), the flavan-3-ols catechin, epicatechin, epigallocatechin, and procyanidin B1 had negative regressions with SI levels. A similar negative trend was found for malvidin anthocyanins, and acetylated and coumaroylated peonidin, while cyanidinglucoside showed a contrasting response, verifying the preceding results shown in Figure 5B. As expected, the flavonols showed positive regressions with incoming SI. In addition, the logarithmic trend that best fitted the response of the glycosylated kaempferol and quercetin was in accordance with the higher variability of these metabolites found by within-cluster hierarchical clustering (Supplementary Figure 5), and calculated as an increase in the coefficient of variance, observed under low-light conditions (i.e., 60% shaded clusters) in Supplementary Figure 6. #### Metabolic Coordination in Response to SI Differed between Exposed and Shaded Clusters Correlation matrices were separately constructed for primary and phenylpropanoid metabolites based on samples obtained during the 2015 season, from all cluster orientations and canopy sides of the same treatment. This allowed a comparison of the metabolite coordination of exposed vs. 60% shaded clusters in response to changes in SI (Figures 7A–D). For both metabolite groups, exposed clusters had a higher number of negative correlations between metabolites and a lower number of positive correlations compared to the 60% shaded clusters. The same trend was found in the phenylpropanoid data obtained in 2014. Primary metabolites (Figures 7A,B) had 136 negative correlations with r < −0.5 in exposed clusters compared to 36 in the 60% shaded clusters, and 270 positive correlations with r > 0.5 compared to 384, respectively. In the exposed clusters, the amino acids serine, alanine, leucine and valine, the biogenic amine putrescine, and galactinol were found to negatively correlate with glucose-6-phophate, glycolate, myo-inositol, glucopyranose, trans-caffeate, and gluconate, as well as with malate, fumarate, maleate, aspartate, and threonine. Fumarate, gluconate and aspartate were also negatively correlated with tartaric acid, glucose, fructose, GABA, beta-alanine, erythritol, and raffinose. Tartaric acid and galactarate were negatively correlated in addition to a strong negative correlation of proline and ethanolamine with malate. GABA, beta-alanine and erythritol were strongly positively correlated, as well as glucose and fructose, fumarate and gluconate and the group of glucose-6-phosphate, malate, glycolate, maleate, myoinositol, and threonine. In the 60% shaded clusters, alanine was negatively correlated with the associated citrate, malate and maleate, the amino acids GABA and proline and trans-caffeate. Pyroglutamate had a strong negative correlation with leucine, while gluconate was negatively correlated with raffinose. In contrast, a large group of metabolites, comprising serine, threonine, aspartate, GABA, myo-inositol, fumarate, phosphoric acid, maleate, glycolate, citrate, malonate, galactarate, sucrose, glucopyranose, and erythritol, were positively correlated, in addition to a strong positive correlation between tartaric acid, malate and glucose. The phenylpropanoids (Figures 7C,D) shared 88 negative correlations with r < −0.5 in exposed clusters compared to 74 in the 60% shaded clusters and 506 positive correlations with r > 0.5 compared to 610, respectively. In the exposed clusters, phenylalanine was found to negatively correlate with a large number of metabolites, including all the annotated flavan-3-ols and anthocyanins, and to positively correlate with the flavonols of kaempferol, as well as quercetin conjugates (excluding quer-3-glr). The latter flavonols, in addition to hydroxy-benzoate, were negatively correlated with a large number of anthocyanins, including all malvidin metabolites and the acetylated forms of peonidin, petunidin and delphinidin, but not with their glycosylated and coumaroylated forms, nor with any of the cyanidin metabolites. In contrast, in the 60% shaded clusters, phenylalanine showed only few, weak negative correlations, and correlations between flavonols and anthocyanins were strongly positive. Instead, epicatechin and coumarate hex showed differing degrees of negative correlations with the entire set of annotated anthocyanins. Finally, correlations between the stilbene delta-viniferin and flavonols, which were slightly positive in the exposed clusters, were strongly negative in the shaded clusters. Four networks were created based on the correlation matrices of the four datasets (Supplementary Figures 8A–D), two treatments (exposed and 60% shaded clusters), and two metabolic groups (primary and phenylpropanoid metabolites), across the different cluster locations and orientations. At r > 0.5 and r < −0.5, shading slightly increased the number of edges in the primary metabolite network from 157 to 169, the network density from 0.28 to 0.301, the clustering coefficient from 0.486 to 0.537 and the average node degree from 9.24 to 9.94. In the phenylpropanoid networks, shading increased the number of edges from 303 to 340, the network density from 0.481 to 0.54, the clustering coefficient from 0.71 to 0.77 and the average node degree from 16.83 to 18.89. Shading caused an at least 2-fold increase in the nodal degree of the primary metabolites glycolate, galactarate, phosphoric acid, maleate, malonate, beta-alanine, and fructose, and an at least 2-fold decrease in the nodal degree of the nitrogenous compounds valine, serine, leucine, proline, putrescine, ethanolamine and pyroglutamate, as well as glycerate and tartaric acid. In the phenylpropanoids, shading caused an at least 2-fold increase in the nodal degree of the flavonols quer-3-glu, rutin, kaemp-3-glu, and kaemp-3-glr, the flavan-3 ols catechin and Epicatechin, and the stilbene delta-viniferin, and an at least 2-fold decrease in the nodal degree of phenylalanine, the hydroxy-cinnamates p-coumarate and coutarate, hydroxybenzoate hex, procyanidin B1, vitisin A, and the stilbene piceid. #### DISCUSSION The accumulation levels of the major primary and secondary metabolites that determine a grape berry's sensorial profile vary as a function of the micrometeorological conditions in its immediate vicinity (Jackson and Lombard, 1993; Downey et al., 2006). Nevertheless, few studies have investigated how these conditions vary within a vine and across a single grape cluster, and how their variation affects the spatial pattern of the fruit metabolic profile in the vineyard and crop uniformity at harvest. This study provided a detailed spatial characterization of metabolite abundance in clusters located on both canopy sides and subjected to different degrees of sun exposure. The coupling of this sampling layout with high resolution measurements of both SI and BST was used to assess the role of sun exposure as a determining factor in the spatial pattern and variability in grape clusters' chemical composition, and to expand current knowledge regarding compound-specific responses and plasticity to SI. SI filtering was proven to be an efficient tool to minimize the within-cluster variability of both incoming irradiance and BST. Artificial shading resulted in decreased BST of sunlit berries and an increased BST of shaded berries. This phenomenon could not be explained by diffusive light or air temperature, since the diffusive light intensity was lower in the shaded treatment and air temperature within the cluster-zone showed no differences (data not shown). For both SI and BST, differences between the orientations of the same cluster exceeded those between treatments, stressing the extent of within-cluster variability and the range of conditions that can be investigated within a single grape cluster. Finally, due to the strong correlation between SI and BST, as previously suggested (Smart and Sinclair, 1976) and found here, and since SI plays an important role in both the direct triggering of fruit-located photoreceptors and in fruit thermal balance, SI was selected as the explanatory variable in this work. Elevated fruit temperatures have previously been suggested to increase the metabolic flux toward the TCA cycle and to modify its regulation, utilizing malate to enhance its anaplerotic capacity in grapevine berries (Sweetman et al., 2014; Rienth et al., 2016). The former work related the flux increment to the enhanced biosynthesis of pyruvate (valine, leucine, serine and glycine), oxaloacetate (aspartate, threonine and isoleucine) and 2-oxoglutarate-driven compounds (GABA, proline and putrescine). Indeed, increased TCA flux and the accumulation of all or part of the mentioned metabolites have been described in response to a wide array of abiotic stresses in a number of plant species, as summarized by Krasensky and Jonak (2012) and Obata and Fernie (2012). Here, SI conditions (i.e., cluster location and sun exposure levels) triggered responses in malate and oxaloacetate-associated compounds (malate, fumarate and aspartate) that contrasted with those in pyruvate (valine, leucine and serine) and 2-oxoglutarate-derived (proline, GABA and putrescine) nitrogenous compounds. Increased SI levels involve a concomitant increase in tissue temperature, and their combination is expected to exert oxidative stress (Foyer et al., 1994). Indeed, redox homeostasis was recently suggested to be the link between the metabolic modulation of grapevine berries, found in different leaf-removal studies (Young et al., 2016). This may explain the increase in the levels of proline, GABA and the polyamine putrescine, which share a role in mitigating oxidative stress (Krasensky and Jonak, 2012). Furthermore, the decrease in TCA cycle metabolites and the increase in specific amino acids may also have resulted from an arrest in glycolytic activity coupled with protein degradation, generating amino acids in a non-biosynthetic manner (Araujo et al., 2011; Lehmann et al., 2012; Obata and Fernie, 2012). Unfortunately, current knowledge is lacking regarding the direct effect of SI on fruit primary metabolism. Hence, such metabolic shifts are currently attributed to the secondary effects of temperature, UV-B irradiance and combined stress responses. Future research on the photo-receptor-mediated modulation of primary metabolism can greatly contribute to our understanding. When considering the results of the network analysis, a higher coordination of the metabolic processes in the central metabolism was observed in this study in shaded grapes. Compared to fully exposed grapes, glycolysis, the TCA cycle, and amino acid metabolism shared a greater number of correlations, and the relations were mainly positive. The fact that the acclimation of the exposed and shaded clusters to perturbations in SI levels resulted in opposite types of correlations between nitrogen and carbon metabolites points to a differential coordination, possibly resulting from a comparison between non-stress (shaded clusters) and stress-related (fully exposed) responses. As described above, and as shown previously in potted grapevines and grape cell cultures subjected to shortterm heat treatment (Sweetman et al., 2014; Ayenew et al., 2015), and berries of vines exposed to water deficit (Hochberg et al., 2013), a combined stress response could lead to the specific accumulation of nitrogen metabolites. In contrast, a positive coordination was maintained between glycolysis sugars and TCA cycle intermediates in both conditions, emphasizing the tight co-regulation existing between these two metabolic pathways. The induction of the flavonoids biosynthesis by fruit-located photo-receptors was shown in a number of species, including grapevine, as reviewed in Zoratti et al. (2014). Summarizing a large number of studies, it can be concluded that while the R2R3 MYB transcription factors family includes both positive and negative regulators of the flavonoid biosynthesis, light seemed to strictly induce the expression of positive ones. However, a more complex interaction is evident, considering the consequent increase in fruit temperature. As found by a number of studies (Spayd et al., 2002; Mori et al., 2007; Tarara et al., 2008; Azuma et al., 2012), above a certain threshold, temperature causes a reduction in the anthocyanin levels, possibly through degradation, whether enzymatic or non-enzymatic (Vaknin et al., 2005; Mori et al., 2007; Chassy et al., 2015). This results in an expected antagonistic effect of elevated BST and strong SI conditions, as found in the exposed berries. Under these assumptions, the response of anthocyanins to increasing SI levels is expected to reflect the metabolite turnover between biosynthesis and degradation. The negative linear effect of SI on the levels of acylated peonidin and malvidin and mal-3 glu, measured in this study, might not be solely attributed to the consequent increase in temperature, as the proportion of acylated anthocyanins was found to increase with rising temperatures (Downey et al., 2004; Tarara et al., 2008), and malvidin metabolites were found to be exceptionally stable to high temperatures in Mori et al. (2007). Instead, considering the contrasting linear increase in the levels of cyanidin-glucoside, it is possible that the combined heat and light modulation of the biosynthesis-related genes' expression patterns, as found in Azuma et al. (2012), was involved in the preferential biosynthesis of upstream metabolites as was observed for cyanidin-glucoside. This may explain the overall positive net turnover of cyanidinglucoside, while downstream metabolites, such as acylated peonidin and malvidin, showed a clear negative result. The induction of flavonol accumulation by increasing SI levels, measured here, is in accordance with a large number of studies regarding light-induced flavonol biosynthesis in grape berries (Czemmel et al., 2009; Matus et al., 2009; Carbonell-Bejerano et al., 2014; Liu et al., 2015) and overall accumulation (Haselgrove et al., 2000; Downey et al., 2004; Cortell and Kennedy, 2006; Pereira et al., 2006; Matus et al., 2009; Azuma et al., 2012), yet the regression between SI and flavonol-glucoside accumulation in grape skins has been found so far to be purely linear (Haselgrove et al., 2000). The extensive gradient and relatively high SI intensity present in our study may be the cause for this discrepancy, yet whether this trend is due to a stagnation in biosynthesis or to an increase in temperaturedriven degradation remains unclear. The accumulation of monomeric and condensed Flavan-3 ols in grape skin peaks around veraison, followed by a decrease during the final stages of ripening (Downey et al., 2004; Fujita et al., 2007; Cohen et al., 2012). Increased SI exposure was found to increase flavan-3-ols levels at veraison, yet, since sunlit berries showed a faster decline during ripening, differences were no longer evident at harvest (Downey et al., 2004; Fujita et al., 2007). In this study, conducted between veraison and harvest, the linear negative effect of SI on the levels of flavan-3-ols metabolites was in accordance with the cited literature. A possible explanation could be a delay in the typical post-veraison decrease in the expression of the biosynthesis-related genes LAR and ANR under shading, as found in Fujita et al. (2007). The stilbenes, which play a role in antifungal activity (Langcake, 1981; Pont and Pezet, 1990; Pezet et al., 2004) and offer potential health promoting effects (Bradamante et al., 2004; Baur et al., 2006; Gresele et al., 2011), accumulate in grapevine tissues in response to biotic and abiotic stresses, such as mold development, wounding, and UV-C irradiation treatments (Fritzemeier and Kindl, 1981; Bais et al., 2000; Vannozzi et al., 2012). Attempts to understand the impact of environmental factors on the accumulation of stilbenes in grape skin tissues have yielded confounding results, possibly owing to the importance of genetic, developmental, and pedological factors (Versari et al., 2001; Bavaresco et al., 2007, 2008, 2012; Berli et al., 2008; Carbonell-Bejerano et al., 2014; Degu et al., 2016). In this study, no significant correlation was found between SI and the levels of the stilbenes piceid and delta-viniferin. However, their levels were significantly higher in the exposed, compared to the shaded, clusters that were positioned on the west side of the canopy but not the east, an opposite trend to that of the major anthocyanins Mal-3-acet and Mal-3-coum. Considering the UV-B induction of stilbene synthase (STS) expression and stilbene accumulation (Versari et al., 2001; Carbonell-Bejerano et al., 2014), and the fact that chalcone synthase and STS compete for the same substrate (Jeandet et al., 1995; Vannozzi et al., 2012), the measured increase in stilbene accumulation in the exposed, compared to the shaded, berries may have depended on a combination of increased STS expression and higher substrate availability, provided by a possible inhibition of anthocyanin biosynthesis. The differences in metabolic coordination between the exposed and shaded clusters in phenylpropanoid metabolism are intriguing. Exposing clusters to high SI generated a strong negative association between the precursor of the polyphenol pathway, phenylalanine, and the anthocyanins, which was not evident in the shaded clusters. In addition, in the exposed clusters, anthocyanins were more strongly correlated with narinchalc-glu, but less with flavonols, and the correlation between flavonols and stilbenes shifted from highly negative (shaded) to partially positive (exposed). Anthocyanin accumulation in grape-tissue culture was recently shown to be decoupled from phenylalanine under conditions in which biosynthesis-related gene expression was down-regulated (Manela et al., 2015). As a result, the tissue accumulated higher levels of flavonols and stilbenes. It is possible that a similar phenomenon occurred in the fully exposed clusters, as biosynthetic gene expression may have been hampered under conditions that included elevated temperatures (Azuma et al., 2012). Taken together, these lines of evidence are in support of a repartitioning of carbon precursors of the polyphenol pathway from anthocyanin biosynthesis to that of stilbenes and flavonols. This hypothesis should be confirmed in future studies by implementing stable isotopes based flux analysis in detached berries. Our results revealed that shifting the intensity and direction of solar irradiance (SI) significantly modulated the spatial patterns of the accumulation of organic and amino acids, the main sugars glucose and fructose, and the majority of skin phenylpropanoid metabolites, across the grape-cluster, while hydroxy-cinnamates were not affected. In addition, filtering the irradiance intensity significantly affected the levels of 24 metabolites across the spatial locations, including organic and amino acids, flavonols, flavan-3-ols, anthocyanins and stilbenes. The within-cluster spatial heterogeneity was characterized by large variations in the flavonol levels, found to be significantly affected by sunlight exposure. Overall, the effect of SI conditions on skin phenylpropanoids was comprehensive, while it was found to be more specific in the case of pulp primary metabolites. Together, these findings suggest that sunlight plays a major role in shaping the spatial accumulation of quality-related compounds within a single grape cluster. The calculated fold changes in metabolite accumulation, across the spatial dataset presented here (Supplementary Table 3), summarizes the compound plasticity of 70 primary and secondary metabolites to light and temperature perturbations, revealing the potential modulating effect of sunlight regulation on fruit composition. Considering that a single layer of grapevine leaves absorbs at least 60–70% of the visible wavelengths (Schultz, 1996), the gradient of SI intensities and metabolite fold change values found in this study may represent, yet possibly underestimate, the range found within a single, non-defoliated, commercial vine. # CONCLUSIONS Grape berries' acclimation to their surrounding environment involves local metabolic shifts, which affect their chemical composition and quality at harvest. Sunlight exposure triggers a complex response through both irradiance-mediated signaling and accumulated heat. Here, we show that this metabolic acclimation to sunlight drives the spatial variability of chemical composition between berries on a single cluster, and that it involves the interaction and modulation of partitioning between several biochemical processes in both pulp and skin. These include the accumulation of pyruvate and 2-oxoglutarate-derived nitrogenous compounds, at the expense of malate, fumarate and aspartate in the pulp, and the accumulation of phenylalanine, narin-chalc-glu, cyan-3-glu and the flavonols, accompanied with a decrease in flavan-3-ols, hydroxy-cinnamates and malvidin anthocyanins, in the skin. Figure 8 illustrates the main findings of the study. clusters, while mesh trapezoids represent 60% shaded clusters. This study characterized, yet did not isolate, the accompanying climatic components, such as temperature, that are known to influence fruit metabolic processes. Therefore, our findings may apply to a defined climate, namely warm and arid to semi-arid regions. The existing knowledge gap regarding the intriguing interaction between (SI) and berry temperature, as well as the contribution of temperature-driven processes to the overall fruit metabolic response, may hinder the extrapolation of our findings to systems experiencing different climatic conditions. At the practical level, (SI) is the most easily and readily controlled climatic factor. In the context of climate change, these results will aid in designing a knowledgebased use of sunlight regulation as tool to improve grape composition, under the conditions that are expected to prevail in an ever-expanding number of commercial vineyards worldwide. # AUTHOR CONTRIBUTIONS NR, NA, and AF conceived and planned the study. NR and NW applied the viticultural treatments, and collected the meteorological data and berry samples in the field. NR and NW analyzed the meteorological data and prepared the berry samples for extraction. NW processed and analyzed the thermal images, and NR performed the sample extraction and analysis using the UPLC-QTOF-MS and GC-MS devices. NR integrated the data and performed the data analysis. NR wrote the body of the paper with AF and NA. All authors reviewed and approved the manuscript. #### FUNDING This work was partially funded by the Koshland Foundation for the Support of Interdisciplinary Research in Combating Desertification, and the Frances and Elias Margolin Trust. #### ACKNOWLEDGMENTS The authors would like to thank the owner of the Nana Farm, Eran Raz, for his professional maintenance of the experimental parcel, and Noga Sikron, Mariela Leiderman, Talya Samani and #### REFERENCES Biruk Ayenew for their technical guidance and support in the lab and field. #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00070/full#supplementary-material stable isotopes. Food Chem. 166, 448–455. doi: 10.1016/j.foodchem.2014. 06.002 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 Reshef, Walbaum, Agam and Fait. 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. # Transcriptome-Wide Identification of Novel UV-B- and Light Modulated Flavonol Pathway Genes Controlled by VviMYBF1 Stefan Czemmel1,2, Janine Höll<sup>2</sup> , Rodrigo Loyola3,4, Patricio Arce-Johnson<sup>3</sup> , José Antonio Alcalde<sup>4</sup> , José Tomás Matus<sup>5</sup> and Jochen Bogs2,6,7 \* <sup>1</sup> Quantitative Biology Center, University of Tübingen, Tübingen, Germany, <sup>2</sup> Centre for Organismal Studies Heidelberg, Heidelberg, Germany, <sup>3</sup> Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile, <sup>4</sup> Departamento de Fruticultura y Enología, Pontificia Universidad Católica de Chile, Santiago, Chile, <sup>5</sup> Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Barcelona, Spain, <sup>6</sup> Dienstleistungszentrum Ländlicher Raum Rheinpfalz, Viticulture and Enology Group, Neustadt/W, Germany, <sup>7</sup> Fachhochschule Bingen, Bingen am Rhein, Germany #### Edited by: Claudio Bonghi, University of Padua, Italy #### Reviewed by: Zhanwu Dai, INRA, France Ilaria Filippetti, Università di Bologna, Italy > \Correspondence: Jochen Bogs [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 02 March 2017 Accepted: 06 June 2017 Published: 22 June 2017 #### Citation: Czemmel S, Höll J, Loyola R, Arce-Johnson P, Alcalde JA, Matus JT and Bogs J (2017) Transcriptome-Wide Identification of Novel UV-B- and Light Modulated Flavonol Pathway Genes Controlled by VviMYBF1. Front. Plant Sci. 8:1084. doi: 10.3389/fpls.2017.01084 Flavonols constitute a group of flavonoids with important photoprotective roles in plants. In addition, flavonol content and composition greatly influences fruit quality. We previously demonstrated that the grapevine R2R3-MYB transcription factor (TF) VviMYBF1 promotes flavonol accumulation by inducing the expression of flavonol synthase (VviFLS1/VviFLS4), a key step of the initial flavonol pathway. Despite this, gene networks underlying flavonol modification in grapevine including both structural and regulatory genes remain poorly understood. In order to identify flavonol modifying genes and TFs acting downstream of VviMYBF1 a microarray-based transcriptome analysis was performed on grapevine hairy roots ectopically expressing VviMYBF1 or a Green Fluorescent Protein as control. VviFLS1 was induced in VviMYBF1 transgenic roots and glycosylated flavonols accumulated significantly compared with control lines. Among the differentially expressed genes, potential flavonol-modifying enzymes with predicted rhamnosyltransferase (e.g., RhaT1) or glycosyltransferase (e.g., GT3) activities were identified. In addition, important TFs of the MYB and bZIP families such as the proanthocyanidin regulator VviMYBPA1 and the UV-B light responsive HY5 homolog VviHYH were significantly altered in their expression pattern by overexpression of VviMYBF1. Co-temporal expression analysis demonstrated positive correlation of VviMYBF1 with VviFLS1, VviGT3, and VviRhaT1 during berry development and in fruits ripened with different light and UV-B radiation conditions at field. These results show that VviMYBF1 overexpression led to the identification of novel genes of the flavonol pathway and that the flavonol modifying machinery can be influenced by agricultural practices to optimize flavonol composition in grapes. Keywords: flavonols, MYB, gene regulation, HY5, UV-containing light, grapevine # INTRODUCTION fpls-08-01084 June 20, 2017 Time: 17:4 # 2 Flavonols are the most ubiquitous flavonoids found in dietary plant-based foods (Middleton et al., 2000) and provide the second most abundant group of flavonoids in grapevine (Vitis vinifera L.) fruits (Georgiev et al., 2014). Flavonols largely accumulate in grape berry skins and show a remarkable facet of chemical diversity. In skins of the cultivar (cv.) 'Shiraz', modification of flavonols were mainly identified as 3-, 7-, and 4<sup>0</sup> -O-glycosylations of the basic flavonol scaffold (Downey and Rochfort, 2008; Figure 1). Flavonol profiles strongly depend on grapevine cultivars (Zhu et al., 2012), but in general, the main representatives in red grapes are quercetin-3-O-glucosides followed by myricetin, whereas quercetin and kaempferol derivatives constitute the most prominent flavonol compounds in white grapes (Mattivi et al., 2006; Castillo-Muñoz et al., 2007). The quality of red wines can be influenced by flavonols as the color of the wines is positively influenced by the copigmentation phenomenon, which is due to molecular associations between anthocyanins and flavonols or other uncolored phenolic compounds in solutions (Boulton, 2001). In recent years, it has been shown that biomedical activities of flavonols are tightly linked to their chemical diversity. The efficiency of flavonols as antioxidant compounds greatly depends on their chemical structure demonstrating a decrease in antioxidant and antibacterial capacity of flavonol glycosides compared to flavonol aglycones (Mori et al., 1987; Vinson et al., 1999; Burda and Oleszek, 2001; Montoro et al., 2005). Although flavonols are produced as secondary metabolites, the relationship between their biological function and modification in planta still remains elusive. In grapevine a boost in flavonol accumulation is often observed in response to any agronomical practice that favors the exposure of grape brunches to sunlight (Teixeira et al., 2013). Consequently, flavonol biosynthesis has been extensively studied in response to its induction by UV-containing light in grapes (Price et al., 1995; Czemmel et al., 2009; Matus et al., 2009). In Arabidopsis it was shown that the tt4 and tt5 mutants, which are defective in the gene CHALCONE SYNTHASE and CHALCONE ISOMERASE, respectively, are sensitive to high-irradiance UV-containing light (Li et al., 1993). exclusively glycosylated in position 3 of the C ring. the flavan skeleton are abbreviated as follows: Glc, glucose; Gal, galactose; Rha, rhamnose; Me, methyl group. Note that flavonols detected in grape so far are The authors showed that this effect was correlated with low content or even absence of kaempferol derivatives in leaves (Li et al., 1993), suggesting an important role of flavonols as UV screens in planta. Outside its role in UV protection, modified flavonols were found to act as endogenous flavonol inhibitors of polar auxin transport in the model plant Arabidopsis (Yin et al., 2014). Conversely the expression or activity of flavonol glycosyltransferases have been shown to be influenced by auxin levels (Santelia et al., 2008). Moreover flavonol aglycones but not flavonol 3-O-glycosides were able to restore pollen fertility in conditionally male-fertile petunia pollen both in vivo and in vitro (Vogt and Taylor, 1995). Induction of flavonol biosynthesis by solar radiation can be attributed to an upregulation of flavonol biosynthetic genes leading to an increase in glycosylated flavonols. This process is negatively affected in the grape berry skin in response to light depletion (Matus et al., 2009) or UV-B filtering (Carbonell-Bejerano et al., 2014). As seen from these studies and many others, visible light and UV-B strongly affect the expression of the R2R3-MYB transcription factor (TF) VviMYBF1 and its target, the first flavonol branch gene FLAVONOL SYNTHASE 1 (VviFLS1, also known as FLS4 by Fujita et al., 2006; Figure 1), whose relation was demonstrated in grape suspension cells treated with UV-B light (Czemmel et al., 2009). Besides this well-described regulatory mechanism, only two structural genes have been identified so far involved in flavonol modification, namely VviGT5 and VviGT6, which encode a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase and a bifunctional UDP-glucose/UDP-galactose:flavonol-3-Oglucosyltransferase/galactosyltransferase, respectively (Ono et al., 2010). From these two genes, GT5 (in addition to MYBF1 and FLS1) has been directly associated with the UV-B signaling pathway in grapevine (Loyola et al., 2016). In addition to the above-mentioned VviMYBF1, other R2R3-MYB TFs have been described as regulators of the phenylpropanoid pathway (reviewed by Czemmel et al., 2012; Matus, 2016). Similar to other plants, these TFs provide the common denominators in the regulation of structural genes of all flavonoid branches whereas co-factors encoding the basic helix–loop–helix (bHLH) domains (also referred to as MYC proteins) and conserved WD repeats (WDR) have been so far exclusively associated to the regulation of anthocyanin/PA accumulation and not to flavonol biosynthesis (Czemmel et al., 2012). The main players in grapevine are VviMYB5A and VviMYB5B proteins that are considered to be general regulators of the flavonoid biosynthetic pathway while VviMYBPA1, VviMYBPA2, and VviMYBPAR are PA biosynthesis regulators and VviMYBA1, VviMYBA2, VviMYBA6, and VviMYBA7 are specific for anthocyanin biosynthesis (Kobayashi et al., 2002; Bogs et al., 2006, 2007; Walker et al., 2007; Deluc et al., 2008; Terrier et al., 2009; Koyama et al., 2014; Matus et al., 2017). In Arabidopsis the VviMYBF1 homologes AtMYB12, AtMYB11, and AtMYB111 regulate flavonol biosynthesis in a tissue-specific manner (Stracke et al., 2007). In both grape and Arabidopsis, flavonol related MYBs are regulated by the bZIP TF ELONGATED HYPOCOTYL 5 (AtHY5 and VviHY5) contributing to the establishment of UV-B tolerance in these species (Stracke et al., 2010; Loyola et al., 2016). This is in line with findings that the hy5 mutant shows flavonol-deficient roots and downregulation of AtMYB12 expression (Lee et al., 2007; Stracke et al., 2010). Besides the well-established control mechanism of the initial flavonol pathway gene VviFLS1 by VviMYBF1, it is unclear how the immense biodiversity of flavonol compounds found in grapes is achieved. In order to elucidate the regulation in biosynthesis and modification of flavonols in grapevine, VviMYBF1 was ectopically expressed in grapevine hairy roots (HRs). Follow up experiments on these HRs allowed to identify novel structural and regulatory genes of the flavonol branch. The hypothesis that genes involved in flavonol modification were under similar transcriptional control by VviMYBF1 and environmental factors such as UV-B light was tested. Microarray analysis revealed several differentially regulated candidate genes, which are potentially involved in flavonol modification, transcriptional regulation and response to light. We selected two genes encoding for a putative flavonol rhamnosyltransferase (VviRhaT1) and a glycosyltransferase (VviGT3), which is closely related to the previously characterized flavonol modifying enzymes GT5 and GT6 (Ono et al., 2010), for downstream analysis. Expression of these candidates correlated with flavonol accumulation and VviMYBF1 and VviFLS1 expression in VviMYBF1 overexpressing grapevine HRs and also in response to UV-containing light in field and greenhouse experiments. These genes are promising VviMYBF1 target structural genes that can be further investigated in relation to their impact on flavonol biosynthesis in response to common agronomical practices in the vineyard. # MATERIALS AND METHODS #### Cloning of MYBF1 Construct The cloning of VviMYBF1 from the cultivar (cv.) 'Shiraz' (GenBank locus accession: FJ948477) into the vector pART7 (Gleave, 1992) to give the construct pART7MYBF1 was described previously (Czemmel et al., 2009). For generation of HRs overexpressing VviMYBF1, pART7MYBF1 was NotI digested and the insert (VviMYBF1 under control of CaMV35S) was cloned into pART27 (Gleave, 1992) to give pART27MYBF1. The binary vector pART27 contains spectinomycin resistance for bacterial selection and kanamycin resistance for selection in planta. # Plant Material Collection for Developmental Series The collection of the samples for the grapevine developmental series of V. vinifera cv. 'Pinot Noir' was described before (Höll et al., 2013). In short, sampling was performed from the start of floral initiation until harvest with early flower and later berry samples collected from a commercial vineyard during the 2007 to 2008 season in Schriesheim near Heidelberg, Germany. The berry samples derived from ∼100 berries from 20 different plants and bunches and were sampled at weekly intervals, as described previously (Downey et al., 2003). All samples were frozen in liquid nitrogen upon collection on the field and stored at −80◦C until analyzed. # Hairy Root Culture and Transformation Before HR transformation, pART27-MYBF1 or pKGWFS7 control plasmid expressing GFP-GUS were transferred by electroporation into the Agrobacterium rhizogenes strain ATCC 15834, carrying the Ri plasmid pRi15834 (neither the chromosome nor the plasmid ATCC 15834 of this A. rhizogenes strain carry an antibiotic marker). Transformation and induction of transgenic VviMYBF1 or GFP-GUS expressing grapevine HRs derived from leaves from in vitro-grown cv. 'Chardonnay' plants was performed exactly as described in Höll et al. (2013). After transformation and induction, transgenic HRs were routinely kept on HR) medium (Höll et al., 2013) and transferred to fresh medium after 4 weeks. HR tissues used for RNA extraction and high performance liquid chromatography (HPLC) analysis were rapidly frozen in liquid nitrogen, ground to a fine powder using a Retchmill (MM20, Retch) and stored at −80◦C until further use. # RNA Extraction and Quantitative PCR (qPCR) Analysis Total RNA was extracted from grinded material from the developmental series of V. vinifera cv. 'Pinot Noir' as previously described in Höll et al. (2013). RNA of MYBF1 and GFP transgenic grapevine HRs was isolated following the protocol of the EURx GeneMATRIX Universal RNA purification kit (Roboklon). Prior to RT reactions, RNA samples were heated to 65◦C for 10 min and immediately placed on ice to destroy secondary structures. Synthesis of cDNA was performed with SuperScriptTM III First-Strand Biosynthesis System (Invitrogen, catalog no. 18080-051) according to the instructions in the manual for RT using oligo dT primers. Typically, 1–2 µg of RNA were reverse transcribed in a volume of 20 µl for 1 h at 50◦C followed by termination of the enzyme reaction at 85◦C for 5 min. After digest of the residual RNA strands using RNase H, 30 µl of RNase free H2O were added. This 50 µl solution served as cDNA stock for further application and was stored at −20◦C. PCR reactions were carried out for flower and berry skin samples from the cv. 'Pinot Noir' developmental series as well as HR samples by using 0.5 µl of 10 µM forward and 0.5 µl of 10 µM reverse primer, 5 µl cDNA (diluted 1:20), 7.5 µl of 2x ABsoluteTM QPCR SYBR <sup>R</sup> Green Fluorescein Mix (ABgene) and 1.5 µl of H2O in a final volume of 15 µl (for all set of qPCR primers used in this work, see Supplementary Table 1). Thermal cycling conditions were identical for all primer pairs: 96◦C for 15 min followed by 96◦C for 30 s, 58◦C for 30 s, and 72◦C for 30 s for 35 cycles, followed by a melt cycle from 50 to 96◦C. QPCR was carried out using SYBR green method for detection of double stranded PCR products on an iCyclerTM optical Module real-time cycler (Bio-Rad). UBIQUITIN1 (previous TIGR database id: TC32075, VIT\_16s0098g01190) was used for normalization of target gene levels in grapevine HRs and light experiments while the three reference genes GAPDH (VIT\_17s0000g10430), UBIQUITIN1 and EF1-alpha (VIT\_06s0004g03220) were used to normalize gene expression during cv. 'Pinot Noir' berry development as done previously (Höll et al., 2013). The primers MYBintF and MYBintR2 were used to detect the transcript level of VviMYBF1 in grapevine by amplification of a 214 bp PCR-fragment from the 3 0 region of the gene. The primers VviFLS1 and VviFLS2 were used to detect a 154 bp amplicon of the VviFLS1/FLS4 gene (for other primer pairs see Supplementary Table 1). The efficiency of the primers was tested in preliminary experiments with dilutions of plasmid or purified PCR products maintaining an r 2 value ≥ 0.96. With all cDNAs used, the primer set gave a single PCR product, which was verified by agarose gel electrophoresis and by determination of the melt curves for the product at the end of each run. # Flavonol Staining Fresh cross sections from 3 to 4 weeks old HRs were transferred in a freshly prepared solution of 0.25% (w/v) diphenylboric acid 2-amino-ethyl ester (DBPA, Naturstoffreagenz A, Roth) and 0.00375% (v/v) Triton X-100. Fluorescence was visualized with either an inverted epifluorescence microscope (Leica DM IRB, Leica) or a stereomicroscope (Leica MZ FL III, Leica) using a DAPI fluorescence filter with an excitation wavelength of 340–380 nm and an emission wavelength of 425 nm. Images were captured using the Leica Image Manager (IM) 50 software and handled with Adobe Photoshop version 8.0.1 without changing the color parameters. ## Microarray Analysis Microarray raw data generation was produced at the Centro Nacional de Biotecnologia (CNB, Madrid) by using the Grapegen GeneChip, which is an Affymetrix custom made chip containing 23,096 probe sets corresponding to ∼15,800 different annotated genes (Pontin et al., 2010) and covering ∼52% of the genome. Quality assessment of microarray data and differential expression (DE) analysis were carried out in the statistical language R (version 3.2.1) mainly using the affy (version 1.48.0) and LIMMA packages (version 3.26.9) by the Quantitative Biology Center (QBiC) in Tübingen<sup>1</sup> . Based on PCR analysis for the overexpressed TF VviMYBF1 and its target VviFLS1, four VviMYBF1 (lines no. 32, 33, 34, and 62) and four GFP overexpressing lines (lines no. 77, 79, 83, and 97) out of 10 stable transformants were selected. During raw data control analysis on all eight lines (four GFP and four VviMYBF1 lines each) several sensitive measures to assess array quality such as Normalized Unscaled Standard Error (NUSE) and Relative Log Expression (RLE) confirmed low quality of array corresponding to the GFP control line 79. Since the array for line 79 was detected as an outlier, DE analysis has been performed on four VviMYBF1 and three GFP lines excluding GFP line 79. Raw data (CEL files) and meta-information (e.g., experimental design, sample names) of microarray data were deposited to NCBI's Gene Expression Omnibus (GEO) and can be accessed under the GEO identifier GSE95532. <sup>1</sup>https://portal.qbic.uni-tuebingen.de/portal/ ### Light and UV-B Radiation Treatments Four different sunlight or UV-B radiation experiments were conducted in cv. 'Cabernet Sauvignon' commercial plants (either treated at field or uprooted and transferred to a greenhouse). #### Experiment 1 Three sunlight depletion treatments were conducted at field in commercial cv. 'Cabernet Sauvignon' vines during the 2006–2007 ripening season, as described by Matus et al. (2009). In this study, different sunlight exposures were generated: (i) full shading of fruits by the plant's own canopy (0% exposure), (ii) full sunlight exposure from veraison (ripening onset) onward, generated by displacement of leaves around the cluster region (100% exposure), and (iii) 25% exposure, by shading from veraison until 6 weeks after which leaves were displaced for cluster illumination. #### Experiments 2 and 3 High (∼0.3 Wm−<sup>2</sup> ) and Low (∼0.1 Wm−<sup>2</sup> ) UV-B exposure treatments were applied to clusters from 9 year old potted vines in a UV-free greenhouse during 2011–2012 and 2012–2013 growing seasons, respectively, as described by Loyola et al. (2016). #### Experiment 4 A UV-B filtering radiation treatment (here called -UV-B) was applied in cv. 'Cabernet Sauvignon' plants from a commercial vineyard in Maipo Valley, Chile (33◦ 430 28<sup>00</sup> S, 70◦ 450 9.71<sup>00</sup> W) during 2011–2012 growing season. The filtering treatment consisted in blocking solar UV-B radiation by installing a 100 µm clear polyester film at the position of grape clusters. Grape clusters from both east and west side of the rows were treated, but only grape clusters from the east side of each experimental row (exposed to sunlight during the morning until midday) were sampled. The experimental design consisted in four blocks with five plants each (biological replicates n = 4). Three berries per cluster (randomly sampled) and four clusters per plant were used for each sample. The treatments started at 6 weeks before veraison. Weeks −3, 0, 3, and 6 after veraison (WAV) were considered for RNA extraction and gene expression quantification while weeks −2, 1, 4, and 7 after veraison were used for HPLC analysis. A total of 60 berries were sampled for each biological replicate per condition at each sampling date. Berries were immediately peeled and deseeded. Berry skins were frozen in liquid nitrogen and stored at −80◦C until RNA was extracted. Total flavonols were measured as quercetin equivalents and environmental parameters such as solar UV-B irradiance, temperature, solar radiation at cluster level and total solar radiation were measured around veraison from 08.00 to 19.45 h in the east side of the row. ### Separation of Hairy Root and Berry Skin Flavonols by HPLC High performance liquid chromatography analysis was performed on methanolic extracts of HR lines using a reverse phase HPLC (Kontron Instruments 322 pump system/360 autosampler/335 HPLC detector; Kontron) with a Symmetry C18 column (3,5 µm, 4,6 mm × 150 mm, catalog no. WAT200632, Waters) protected by a guard column (Czemmel et al., 2009). Liquid nitrogen-frozen and subsequently homogenized sample aliquots of 20–50 mg were used to extract flavonoids by adding 200 µl of 50% (v/v) methanol (HPLC grade) in H2O. Samples were then sonificated for 20 min in an ice water bath and centrifuged for 10 min at 13000 rpm. 200 µl of the clear supernatant were loaded for HPLC analysis. Separation was carried out with a binary gradient of solvent A [10% formic acid (v/v) in H2O] to solvent B [100% (v/v) methanol, HPLC grade]. The gradient conditions were 0 min, 17% solvent B; 15 min, 35% solvent B; 40 min, 37% solvent B; 42 min, 100% solvent B; 50 min, 100% solvent B; 51 min, 17% solvent B; 58 min, 17% solvent B. The column was maintained at 40◦C and the flow rate was 1 mL/min. Data acquisition and processing was performed by Kroma System 2000 software (Kontron). Data are presented as HPLC chromatogram peak areas over time and expressed as milli-absorbance units (mAU) at 520 nm for anthocyanins and at 353 nm for flavonols. Concentrations were calculated from calibration curves prepared from commercial standards (Phytolab) and expressed as quercetin-3-O-glucoside equivalents for flavonols and malvidin-3-O-glucoside equivalents for anthocyanins. Acidic hydrolysis was performed to identify the compounds as flavonols by cleaving the glycosyl group for a shift to the aglycone form by the addition of a 3 N HCL solution, 50% (v/v) methanol in water and incubation for 3 h at 95◦C. All HPLC separation experiments were performed in three independent extractions of from the same biological material. High performance liquid chromatography quantification of flavonols in berry skins from light experiments was different to flavonol measurements from HRs and berry skin and flower samples from the developmental series of cv. 'Pinot Noir.' For light experiments, flavonol measurements were conducted as described in Matus et al. (2009). #### Statistics For statistical evaluation of microarray data, the LIMMA package was used (Ritchie et al., 2015). First, its function lmFit() fitted linear models on the quality controlled and RMA normalized expression values of each gene across the seven samples with the factor genotype (with the two levels MYBF1 and GFP-GUS). Then the functions eBayes() and topTable() with the argument adjust.method = "BH" were used to compute moderated t-statistics and extract a table of the top-ranked genes from a linear model fit with p-values corrected for multiple testing by applying the false discovery rate (FDR) from Benjamini and Hochberg (1995), respectively. Genes were considered differentially expressed (DE) with a FDR corrected p < = 0.05. No log fold change (logFC) cutoff was applied to assess DE of a gene. To statistically assess differences in expression of genes and flavonol accumulation between VviMYBF1 and GFP control HR lines, an unpaired two-tailed t-test was performed with assumption of equal standard deviation (SD) between both sample groups. Results were considered statistically different at p < 0.05. For each of the four UV/light experiments, differential gene expression was assessed using a two-way analysis of variance factors time (in WAV) and treatment (+UV-B, −UV-B) and an interaction term between those two factors. To analyze at what time points the treatment effects differed, a post hoc analysis was performed on the model using Tukey's test. Genes were considered statistically DE at this time point at p < 0.05. Note that for accumulations of individual flavonol in response to light no statistical test was performed. #### Accession Numbers Microarray data were submitted in the Gene Expression Omnibus (GEO) under accession number GSE95532. # RESULTS #### Selection of Hairy Roots for Transcriptome Analysis Previous studies indicate that the grapevine HR system provides a suitable tool to identify novel target genes regulated by an ectopically expressed MYB TF (Cutanda-Perez et al., 2009; Terrier et al., 2009; Khater et al., 2012; Höll et al., 2013). Transgenic HRs were generated by infiltration of V. vinifera cv. 'Chardonnay' leaves with A. rhizogenes carrying either VviMYBF1 or GFP (control) cDNA. Presence of VviMYBF1 transgene was verified by PCR, whereas control lines expressing GFP were selected by PCR and fluorescence microscopy. Four VviMYBF1 (lines no. 32, 33, 34, and 62) and four GFP overexpressing lines (lines no. 77, 79, 83, and 97) out of 10 stable transformants were analyzed for transcript amount of VviMYBF1 and its known target VviFLS1 (Czemmel et al., 2009). During microarray quality control analysis, line no. 79 was detected as an outlier (for details, see Materials and Methods) and downstream analysis has been performed on four VviMYBF1 and three GFP lines excluding GFP line 79. Additionally, HR flavonols were stained in vivo with diphenylboric acid-2-aminoethylester (DPBA, Figure 2A) and quantified using HPLC (Figure 2B). By using fluorescence microscopy, we observed orange like bodies (indicative of the accumulation of quercetin-derivatives) exclusively in VviMYBF1 overexpressing HRs (Figure 2A). These bodies were prominent in epidermal cell layers while endodermal cells and parenchymatic cells of the cortex accumulate only trace amounts or did not show any orange staining indicative for flavonol accumulation. Flavonols, mainly quercetin 3-glucoside, quercetin 3-galactoside and a so far unknown flavonol derivative accumulated significantly higher in VviMYBF1 overexpressing lines as compared to GFP control lines when measured quantitatively using HPLC (Figure 2B). To show that MYBF1 induces only the flavonol branch of the flavonoid pathway anthocyanins were measured in the HR lines. Neither in the VviMYBF1 nor in the GFP overexpressing lines anthocyanins were detected. With the aim to decipher the mechanisms that underlie the phenotypic changes observed in HRs ectopically expressing VviMYBF1, qPCR analysis was deployed. Expression of VviMYBF1 in selected VviMYBF1 lines exceeded significantly its expression compared to GFP control lines (Figure 2C), FIGURE 2 \| Selection of transgenic hairy root (HR) lines. Lines for microarray analysis were selected under the following criteria: flavonol accumulation as visualized by fluorescence microscopy on in situ diphenylboric acid-2 aminoethylester (DPBA) stained root cross sections (A), flavonol content as shown by high performance liquid chromatography (HPLC) analysis (B) and gene expression analysis to verify transcript levels of the VviMYBF1 transgene and its well-characterized target VviFLS1 (C,D). DBPA staining analysis shown in (A) was conducted with sections of the VviMYBF1 line 33 (left) and control line 77 (right). HPLC results in (B) are the mean values of three independent extractions from the same biological material. Gene expression analysis in (C,D) is shown relative to the expression of UBIQUITIN1 gene (previous TIGR database id: TC32075, VIT\_16s0098g01190) with standard deviations derived from three qPCR runs with three technical replicates each. Stars indicate significant differences (p < 0.05) between MYBF1 and control HRs based on t-test. names are indicated on the x-axis with MYBF1 samples on the left and control samples on the right. (B) Principal component analysis (PCA) of gene expression in hairy roots expressing MYBF1 (blue) or GFP control (red). The plot shows the first two principal components (PC1 and PC2) that account for 38% and 22% of the total variation of the data. which resulted in a pronounced induction of its target, VviFLS1 (Figure 2D). Results from this homologous expression experiment clearly support the role of VviMYBF1 in regulation of flavonol biosynthesis by induction of VviFLS1 transcription. These results showed that selected VviMYBF1 and GFP HR lines were suitable candidates for a differential transcriptome analysis approach to identify novel target genes of VviMYBF1 involved in flavonol biosynthesis and its regulation. # Transcriptome Analysis to Identify VviMYBF1 Target Genes To identify novel target genes of VviMYBF1, total RNA was isolated from the four VviMYBF1 and the three GFP overexpressing HR lines to perform a Microarray-based transcriptome analysis. Principal component analysis (PCA) clearly separated both, VviMYBF1 and GFP HR lines in distinct groups (Figure 3B) indicating differences between underlying gene expression pattern in VviMYBF1 and GFP control groups which is also visible using a heatmap (Figure 3A). To test the a priori hypothesis that genes will have significantly different mean expression values between VviMYBF1 and GFP lines, pairwise comparison between the two groups VviMYBF1 and GFP was performed and moderated t-statistics and associated p-values were generated, which were further adjusted for multiple testing using Benjamini and Hochberg's method to control the FDR (Benjamini and Hochberg, 1995). In total, 548 probesets corresponding to 484 differentially expressed genes (DEGs; that is because several oligonucleotides hybridize to the same gene) were DE based on an adjusted p-value smaller than 0.05 (Table 1* and Supplementary Table 1). These 548 probesets represent ∼2% of the probes on the chip (23,096 probesets in total). 279 probesets corresponding to 230 genes have been called positive DE, while the other 269 probes, corresponding to 254 genes, show significant negative regulation in VviMYBF1 compared to GFP HR lines. Amongst the DEGs, three candidate genes were found which could modify flavonol scaffold structures (Table 1): a putative UDP-sugar flavonoid/flavonol glycosyltransferase [VIT\_11s0052g01580, VviGT3 (Ono et al., 2010)], a putative UDP-rhamnose:rhamnosyltransferase (VIT\_00s0218g00170, VviRhaT1) and a putative flavonol modifying sulfotransferase (VIT\_17s0000g04930, VviST1). An investigation of the positive DEGs for TFs revealed VviHYH (VIT\_05s0020g01090) and VviMYB4A (VIT\_03s0038g02310), which showed 1.5 and a 1.6 fold inductions in gene expression in VviMYBF1 versus control HRs, respectively (Supplementary Table 1). Amongst the pool of significantly downregulated transcripts several genes of the PA biosynthetic pathway were identified: the PA regulatory TF gene VviMYBPA1 (VIT\_15s0046g00170) and genes under genetic control of VviMYBPA1 including two chalcone synthase isoforms (VviCHS3 and VviCHS1, VIT\_05s0136g00260, VIT\_14s0068g00930) and anthocyanidin reductase (VviANR, VIT\_00s0361g00040). Microarrays also indicate that transcript abundance of a yet uncharacterized FLAVONOL SYNTHASE isoform, VviFLS5 [(VIT\_18s0001g03430 (Fujita et al., 2006)] was negatively affected by overexpression of VviMYBF1 (Table 1). With the aim to validate the microarray experiment, expression of the three candidates flavonol-modifying genes VviST1, VviRhaT1, and VviGT3 and the PA regulator VviMYBPA1 were analyzed by qPCR (for primers used see Supplementary Table 1). Quantitative analysis revealed that VviGT3, VviST1 and VviRhaT1 were indeed induced in VviMYBF1 lines compared to GFP controls (Figures 4A–C), whereas transcript abundance of VviMYBPA1 was negatively influenced by ectopic expression of the flavonol regulator VviMYBF1 (Supplementary Figure 1). Expression of the previously characterized flavonol modifying genes VviGT5 and VviGT6 were not checked in the HR samples, as probesets representing these genes were not present on the microarray chip design used here. The probesets correspond to significantly upregulated and downregulated genes that passed the filter criteria of a multiple adjusted p-value of 0.05. Note that Table 1 is sorted from highest to lowest log fold change (logFC). The respective probesets correspond to the 12Xv1 genome accession. Note that the microarray chip used here does not contain a probe corresponding to the expressed transgene VviMYBF1. The probeset VVTU16103 for the known VviMYBF1 target gene VviFLS1/VviFLS4 was therefore used as a positive control for DE analysis. ## Candidate Gene Expression during Grape Berry Development For correlative analysis of gene expression during grape berry ripening, the same V. vinifera cv. 'Pinot Noir' developmental series was used, in which gene expression related to stilbene biosynthesis was measured previously (Höll et al., 2013). Expression pattern correlation between VviMYBF1 and VviFLS1 transcripts was highest at eleven-to-ten weeks prior to beginning of berry ripening (veraison), correlating with previous results on flavonol accumulation and VviFLS1 and VviMYBF1 expression pattern (Downey et al., 2003; Czemmel et al., 2009). Monitoring expression pattern of the putative VviMYBF1 target genes in developing berries showed that VviST1 and VviGT3 are low expressed during berry ripening (Figures 4D,E) though showing correlation with VviFLS1 and VviMYBF1 expressions at some early developmental stages (Downey et al., 2003; Czemmel et al., 2009). There is correlation of VviRhaT1 expression with flavonol accumulation throughout all stages of development as VviRhaT1 transcript amounts gradually increase from early to late stages of development (Figure 4F), which is similar to flavonol accumulation in grapevine (Downey et al., 2003). #### Candidate Gene Expression and Flavonol Analysis under Changes in UV-Containing Light Exposure Fruit zone leaf removal is a common viticultural practice that exposes grape berry clusters to sunlight, reducing humidity and improving the accumulation of various secondary metabolites important for fruit quality (Reynolds et al., 1995; Teixeira et al., 2013). In order to evaluate whether gene expression of candidate flavonol-modifying genes is influenced by agronomical practices that alter the exposure of berries to UV-containing light, four experiments were conducted: Experiment 1: sunlight depletion generated by leaf displacement around clusters of fieldgrowing plants (Figures 5A–D), Experiments 2–3: control versus 'High UV-B' and 'Low UV-B' radiation in clusters of 9 yearold plants growing in a greenhouse (Supplementary Figure 2); and Experiment 4: UV-B filtering in clusters of field-growing plants (Figures 5E–H and Supplementary Figure 3A). For Experiment 4 not only gene expression levels were measured (Figures 5E–H), but also total (Supplementary Figure 3B) and individual flavonol levels determined (Supplementary Figure 4). Furthermore, environmental parameters such as solar UV-B irradiance, temperature, solar radiation at cluster level and total solar radiation were measured (Supplementary Figure 5). For further experimental details for these four light experiments, refer to Section "Light and UV-B Radiation Treatments". The MYB TF VviMYBF1 was strongly upregulated in all developmental stages at 100% exposure in the sunlight field experiment compared with 0% exposure (Figure 5A). Its regulation was also higher in the +UV-B condition of the filtering experiment at 0 and +3 WAV (Figure 5E). In the greenhouse experiments, VviMYBF1 expression was more strongly induced by UV-B irradiation (+UV-B) at low intensities but not at high intensities (Supplementary Figures 2A,E). The induction at low intensities was prominent at early stages of development (−3 WAV and time point 0, Supplementary Figure 2B). Consequently, VviFLS1, the target gene of VviMYBF1, was also upregulated by sunlight and UV-B, most prominently at all time points of 100% exposure in the sunlight experiment compared with 0% exposure (Figure 5B), and at 0 and +3 WAV in the UV-B filtering field experiment (Figure 5F). VviFLS1 was already induced at −3 WAV, although not statistically significant (Figure 5F). Expression of VviFLS1 was also affected in the greenhouse experiments under UV-B irradiance, especially at +3 WAV and +6 WAV (Supplementary Figures 2B,F). VviRhaT1 expression was induced by +UV-B in the UV filtering experiment around veraison (Figure 5G) but showed no significant response to 25 or 100% sun exposure in the light field experiment except at +7 WAV (Figure 5C) or to low UV-B irradiance conditions in the greenhouse (Supplementary Figure 2G). In contrast to VviMYBF1 and VviFLS1, VviRhaT1 expression profile throughout development was similar in the 25 and 100% exposure treatments in the sunlight field experiment (Figure 5C). Materials and Methods, D–F). Data points in (D–F) are given as weeks from the onset of ripening (time point 0). Note that from 6 weeks before veraison (-6), berry skin has been separated from seeds. Flowering occurred 8 weeks before veraison. Each sample of the developmental series from –6 on corresponds to a pool of >100 berries collected from ∼20 plants, as described in Höll et al. (2013). Values and standard deviations are derived from two PCR runs with triplicate PCR reactions each. Stars in (A–C) indicate significant differences (p < 0.05) between MYBF1 and control HRs based on t-test. No statistical analysis was performed for data in (D–F). Expression of the three genes MYBF1 and FLS1 and RhaT1 are indicative of a rapid induction to late sunlight exposure (stage +7 WAV in the 25% exposure treatment), as this is the stage when leaves are replaced and clusters are exposed to light (see diagram at the bottom of Figure 5 and see Materials and Methods) which is supported by a similar expression level of the respective gene at the same stage in the 100% exposure treatment (Figures 5A–C). Regarding VviGT3, there is a consistent two-to-three-fold induction of its expression in response to UV-B at 3 weeks before veraison in the greenhouse UV-B irradiance or filtering FIGURE 5 \| Transcript profiles of known and putative flavonol biosynthetic genes in the berry skin in response to light and UV-B-modifying field conditions. Expression data results for VviMYBF1 (A,E), VviFLS1 (B,F), VviRhaT1 (C,G), and VviGT3 (D,H) are shown in two field radiation experiments: cluster sunlight-depletion generated by leaf displacement at field (A–D) and UV-B radiation filtering experiment at field (E–H, see Materials and Methods). Results from the greenhouse experiments are shown in Supplementary Figure 2. Data points in the UV-B filtering field experiments are given as weeks from the onset of ripening (veraison, time point 0). Sunlight percentages for each treatment refer to the range of time under sunlight exposure: 0% corresponds to full shading of fruits by plant leaves, 100% corresponds to full sunlight exposure from veraison onward, generated by movement of leaves around the cluster region, and 25% shading from veraison until 6 weeks after which leaves were displaced for cluster illumination. Each of the two experiments is exemplified with a diagram at the bottom of the Figure. The experimental design consisted in four blocks with five plants each (biological replicates n = 4). Three berries per cluster (randomly sampled) and four clusters per plant were used for each sample. Gene expression in cv. 'Cabernet Sauvignon' berries is shown relative to UBIQUITIN1 expression. Values and standard deviations derived from one PCR run with duplicate PCR reactions on each of the four biological replicates. Stars indicate significant differences at indicated time points across treatments (p < 0.05) based on a two-way ANOVA followed by Tukey's post hoc test. Note that the lines in (B) indicate that at each time point in 0% versus 100% sun exposure differences in gene expression of VviFLS1 were statistically significant. experiments (Supplementary Figures 2D,H and Figure 5H). In contrast, VviGT3 gene expression was not detectable in post-veraison samples such as in the light field experiment (Figure 5D), which correlates well with its low expression at late stages of development (Figure 4E). While VviST1 transcript levels were detectable in the HR lines overexpressing VviMYBF1, in none of the light experiments expression of VviST1 was detectable, also correlating to its low expression found in the cv. 'Pinot Noir' developmental series (Figure 4D). In order to correlate gene expression levels with flavonol accumulation, flavonols were quantified in the UV-B filtering field experiment. Flavonols show a significant increase in response to UV-B at different time points (Supplementary Figure 3B). When analyzing for individual flavonol compounds it was found that many flavonols are 3-O-glycosylated, as reported before (Carbonell-Bejerano et al., 2014) and the mostly affected were 3-O-glucosides of quercetin, kaempferol, and isorhamnetin (Supplementary Figure 4). #### DISCUSSION ### Selection of VviMYBF1 Transgenic Hairy Root Lines for Microarray Analysis Several studies have shown that target gene specificity of TFs may differ when introduced into heterologous hosts (Bovy et al., 2002; Luo et al., 2008). Therefore microarray-based transcriptome analysis was performed on the homologous system V. vinifera cv. 'Chardonnay' HRs overexpressing VviMYBF1 and compared to GFP expressing control roots. For microarray analysis, suitable HR lines were pre-selected by qPCR to detect the presence of significant amounts of VviMYBF1 and its known target gene, VviFLS1 (Czemmel et al., 2009). In situ staining of transgenic VviMYBF1 HRs for flavonol compounds clearly supported qPCR analysis and demonstrated that flavonols accumulated in VviMYBF1 transgenic roots but not in GFP (control) lines. Flavonol compounds were visible in yellow vacuolar-like cytoplasmic inclusion bodies but not uniformly located in all HR tissues. Interestingly, similar structures named anthocyanin vacuolar inclusions (AVIs) were observed after overexpression of the anthocyanin regulator VviMYBA1 (Cutanda-Perez et al., 2009). The sectors in VviMYBF1 root cross sections accumulating highest levels of flavonols include skin layers (epidermis, hypodermis), endodermis and vascular bundles, whereas no flavonols could be observed in the cortical parenchyma cells. As flavonol regulation by VviMYBF1 is not dependent on the presence of bHLH proteins (Czemmel et al., 2012), a lack of flavonol accumulation in HR tissues such as cortical parenchyma cells might result from either the presence of inhibitors (e.g., transcriptional repressors) or the lack of WD-repeat proteins or other yet unknown activators in these tissues. Main known flavonols found in VviMYBF1 HR extracts were quercetin-3-O-glucoside and also quercetin-3-Ogalactoside. Both of these quercetin-derived compounds have been reported as abundant compounds found in grape leaves and berries (Castillo-Muñoz et al., 2007; Downey and Rochfort, 2008). ### Overexpression of VviMYBF1 in Hairy Roots Identifies Novel Putative Flavonol Pathway Genes Controlled by UV-Containing Light It is well-known that flavonols, in a similar way as anthocyanins, mainly exist as glycosylated forms whereas corresponding aglycones rarely exist in planta (Mattivi et al., 2006). Conjugation with glucose leads to increased water solubility, bioavailability and reduced toxicity, as respective reaction products can be subsequently stored in the vacuole. For example, kaempferol-diglucoside was suggested as one substrate for the tonoplast localized MATE protein Flower Flavonoid Transporter (FFT) in Arabidopsis (Thompson et al., 2010). Sugar moieties at the flavonol core skeleton also influence antioxidant potential (Vinson et al., 1999; Burda and Oleszek, 2001; Montoro et al., 2005) and they are able to protect polyphenols from enzymatic oxidation by plant peroxidases (Regev-Shoshani et al., 2003). These flavonol modifications, also in addition to their content and composition, can be remarkably different among grape varieties, during berry development (Downey et al., 2003; Mattivi et al., 2006; Castillo-Muñoz et al., 2007) and in response to UV-containing light (Price et al., 1995; Czemmel et al., 2009; Matus et al., 2009). Still, major knowledge gaps exist in the regulatory network underlying flavonol biosynthesis under developmental and environmental conditions that determine visible and UV-B light qualities. Microarray analysis of VviMYBF1 overexpressing HR lines was therefore centered toward the identification of novel structural and regulatory genes involved in flavonol biosynthesis. Promising candidates, namely two glycosyltransferases (VviGT3, VviRhaT1) and a putative flavonol-sulfotransferase (VviST1) were identified using high throughput DE profiling. qPCR analysis of transgenic HRs overexpressing VviMYBF1 confirmed the microarray results and showed that VviGT3, VviRhaT1, and VviST1 are significantly upregulated (Figure 4). Results in this work clearly support the idea that VviGT3 is under the control of VviMYBF1 to produce glycosylated flavonols in young developing berries. Despite the many existences of glycosylated flavonols in planta and their high importance as sunscreen pigments, when comparing grapevine to the model plant Arabidopsis (Yonekura-Sakakibara et al., 2007, 2008), only a few genes involved in flavonol glycosylation have been characterized in vitro (Ono et al., 2010). The same holds true for the identification of only a few structural genes involved in glycosylation of other flavonoids (PAs, anthocyanins) in grapes (Ford et al., 1998; Hugueney et al., 2009; Khater et al., 2012). VviGT3 is located in the same orientation on chromosome 11 in close vicinity to VviGT5 and VviGT6 (Ono et al., 2010). This tandem organization suggests that VviGT3 arose by tandem duplication and might provide another flavonol modifying gene. Sequence similarity between VviGT3 and VviGT5 (64%) or VviGT6 (64%) is lower compared to the sequence identity between VviGT5 and VviGT6 (88%), which implies functional differences. Although the GT3 protein has not been functionally categorized yet, co-expression analysis reveals some spatial and potential functional differences compared to GT5/GT6. VviGT5 and VviGT6 were shown to be co-expressed with VviFLS1 in berries, leaves and petioles (Ono et al., 2010) with higher GT5 expressions after the onset of ripening (Loyola et al., 2016). Results from light experiments and berry development show that VviGT3 transcripts are more profoundly expressed and inducible at early stages of development (Figure 4E), which correlates well with peaks of VviMYBF1 and VviFLS1 expression in grapes (Downey et al., 2003; Czemmel et al., 2009). Furthermore, VviGT3 is light inducible only at early stages of development (Figure 5H) where it is expressed (Figure 4E). The absence of induction in the light field experiment (Figure 5D) can be explained by the fact that all samples used there correspond to post-veraison stages where VviGT3 is low expressed, as it was also demonstrated in the cv. 'Pinot Noir' developmental series (Figure 4E). VviGT3 is therefore regulated by light in a similar way as VviGT5 and VviGT6 and the flavonol marker genes VviFLS1 and VviMYBF1 (Carbonell-Bejerano et al., 2014). Neither in the cv. 'Corvina' gene atlas (Fasoli et al., 2012) nor in the cv. 'Pinot Noir' series VviGT3 expression was observed in skin samples of berries toward ripening (Figure 4E). These results indicate that at very early stages of development VviMYBF1 could regulate a cascade of GT expression profiles including VviGT3, VviGT5 and VviGT6 to attach sugar moieties [mainly of the class of 3-O-galactosides and 3-O-glucosides (Supplementary Figure 4)] to flavonol skeletons produced by the co-induced VviFLS1 gene. Later in berry development, flavonol modifications attached by VviGT3 might not be needed in ripening berry skins, in which the transcriptional network of VviMYBF1 activates the expression of other flavonol structural genes except VviGT3. All UV radiation-increased flavonols found in this study were glycosylated, corroborating the results of Carbonell-Bejerano et al. (2014). The structural similarities between GT5, GT6, and GT3 and the pronounced accumulation of 3-O-galactosides and 3-O-glucosides in the field experiment render the possibility that GT3, besides GT5 and GT6, contribute to the glycosylation of flavonols under UV-light regimes to provide non-toxic UV screens to young berries before veraison. In conjunction with its albeit modest but present induction by UV-containing light during late stages of development, the VviRhaT1 gene was induced in VviMYBF1 HRs when using microarray analysis and confirmative qPCR analysis. The Grape Gene Atlas data showed that cDNA of VviRhaT1 is present ubiquitously in 50 out of 54 measured samples (Fasoli et al., 2012). It is expressed in all tissues where VviGT3 is expressed but in addition in many berry and fruit related tissues and additionally in leaves where VviRhaT1 expression correlates with VviMYBF1 and VviFLS1 transcript abundances. This correlates well with the uniform expression of this gene across the cv. 'Pinot Noir' developmental series (Figure 4F). VviRhaT1 shows an expression pattern very similar to flavonol accumulation on a per berry basis (Downey et al., 2003). This indicates its involvement in flavonol modification throughout rather than at the beginning of berry ripening at which stage VviMYBF1 and VviFLS1 expression peaks (Czemmel et al., 2009). This also supported by a co-induction of quercetin-rhamnoside (+1 WAV) and VviRhaT1 (0 and +3 WAV) gene expression around or post-veraison in the UV-B filtering field experiment (Figure 5G and Supplementary Figure 4H) indicating a putative role of VviRhaT1 as flavonol modifying enzyme. This function is further corroborated by the high sequence similarity to At2g22590 (UGT91A1) from Arabidopsis, which presumably encodes a rhamnosyltransferase under transcriptional control of the flavonol regulators MYB12, MYB11, and MYB111 (Stracke et al., 2007). This regulatory link was identified by transcriptome analysis on a flavonol deficient mutant (myb11-myb12-myb111) showing strong downregulation of the transcripts encoding UGT91A1. Transient expression analysis in Arabidopsis protoplasts confirmed that the promoter of UGT91A1 was responsive to flavonol regulators (Stracke et al., 2007). Whereas O-glycosylation of flavonoids, which may be influenced by VviGT3 and VviRhaT1, has been at least partially studied in grapes, almost nothing is known regarding flavonoid sulfurylation in developing grape berries. In plants, flavonol sulfotransferases (STs) have been initially described in Flaveria spp. (Varin and Ibrahim, 1989) and sulfurylated flavonols have often been associated with the nucleus (Grandmaison and Ibrahim, 1995; Naoumkina and Dixon, 2008). In addition, another study suggests that sulfurylated flavonols might play a positive role in the regulation of polar auxin transport by acting as antagonist to quercetin (Faulkner and Rubery, 1992). A detailed examination of grapevine chromosome 11 revealed that genes encoding GTs alternate with two putative flavonol sulfotransferase genes with one of them, VviST1, being significantly overexpressed in VviMYBF1 transgenic root lines but not induced by sunlight or UV-B. Expression data regarding flavonol STs in grapes are sparse. The Grape Gene Atlas shows that VviST1 is only present in 13 out of 54 tissue samples and only in berry pericarp and berry flesh when considering fruit samples (Fasoli et al., 2012). VviST1 is expressed in roots but not in flowers, which contrasts the expression of VviMYBF1 in these samples. The low expression, as reported in the Grape Gene Atlas, is supported here when studying expression pattern of ST1 in developing berries (Figure 4D) but thereby shows some correlation with early expression of VviMYBF1 in developing berries (Czemmel et al., 2009). Taken together, these results confirm a regulative effect of VviMYBF1 on VviFLS1, VviRhaT1, and VviGT3 gene expression in VviMYBF1 HRs, during berry development and at particular time points in response to sunlight and UV-B radiation. In contrast, greenhouse and UV-B field experiments did not gain any insight on a light-dependent regulation of the putative flavonol sulfotransferase gene VviST1. These results extend our knowledge about flavonol glycosylation in response to light and leave open the question whether flavonol sulfurylation can be modified by agronomical practices influencing light. # VviMYBF1 Is Part of a Regulatory Cascade Involving VviHY5/VviHYH and the PA Regulator VviMYBPA1 Microarray analysis on VviMYBF1 HRs identified the grape HY5 homolog (VviHYH) and VviMYBPA1 as target of VviMYBF1 (Table 1 and Supplementary Table 1). While the bZIP factor VviHY5 could not be analyzed in this work because it is not represented on the Grapegen GeneChip, transient expression of VviHY5 in grapevine plantlets resulted in an increase of the expression of VviMYBF1 (Loyola et al., 2016). Furthermore, the presence of HY5 binding elements typical for bZIP TFs in the promoters of VviMYBF1 and VviFLS1 genes was proven by in silico approaches (Czemmel et al., 2009). These results suggest a positive feedback loop of VviMYBF1 and VviHYH/VviHY5 in grapevine. A similar relationship is found between the VviMYBF1 and VviHYH homologes AtMYB12 and AtHY5 in Arabidopsis. Lee et al. (2007) identified HY5 binding sites (ACEs) in the promoters of numerous Arabidopsis genes including AtCHS, AtFLS and most prominently AtMYB12 (Lee et al., 2007). This information was used by Stracke et al. (2010) to demonstrate that in Arabidopsis HY5 is required for the transcriptional activation of the AtMYB12 and AtMYB111 genes under UV-B and visible light. This is consistent with the observation that the hy5 mutant shows flavonoid-deficient roots (Sibout et al., 2006) and downregulation of AtMYB12 transcripts (Stracke et al., 2010). Taken together, these results support the theory that the UV-B response machinery (including the TFs VviHY5 and VviMYBF1) exist in grapes to propel flavonol accumulation through the activation of the regulatory network consisting of both, MYB and bZIP TFs (Malacarne et al., 2016; Matus, 2016). Our transcriptome study also demonstrated a downregulation of structural and regulatory genes of the proanthocyanidin (PA) pathway by VviMYBF1 and therefore indicated a competition between the flavonol and PA branch of the pathway (Table 1). Indeed, qPCR analysis on VviMYBF1 HRs showed that there is a negative influence of the flavonol regulator VviMYBF1 on the expression of the PA regulatory TF VviMYBPA1 (Table 1 and Supplementary Figure 1). Concomitantly, genes which were shown to be under control of VviMYBPA1 such as two CHALCONE SYNTHASE isoforms, ANR and CHI (Terrier et al., 2009) were also downregulated. These results are in line with previous findings that also proposed a competition between flavonol and PA/anthocyanin biosynthesis (Czemmel et al., 2009). Interestingly, an inverse relationship – a differential regulation of VviMYBF1 expression by VviMYBPA1 – seems not to exist as the expression of the key target of VviMYBF1, VviFLS1 was not altered in VviMYBPA1 HRs (Terrier et al., 2009). In context with the contrasting expression profiles of VviMYBF1 and VviMYBPA1 in developing cv. 'Shiraz' berries (Bogs et al., 2007; Czemmel et al., 2009), the data presented here imply that VviMYBF1 is a positive regulator of flavonol biosynthesis at the expense of PA accumulation via downregulation of VviMYBPA1 transcription during initial berry ripening stages. # CONCLUSION In this study the grapevine flavonol regulator VviMYBF1 was overexpressed in the homologous model system of cv. 'Chardonnay' HRs. Microarrays, qPCR analysis and light/UV exclusion experiments identified promising novel flavonol biosynthetic genes, such as a flavonol glucosyltransferase (VviGT3) and a rhamnosyltransferase (VviRhaT1). These structural genes in context with regulatory genes such as VviHYH, both being under control by VviMYBF1, could play a role in the biosynthesis of flavonols, acting downstream of VviFLS1 and playing a role in UV light responses. Subsequent biochemical characterization of the substrate specificity of the structural candidate genes and the transcriptional potential of the identified VviMYBF1 targeted TFs will foster our understanding of flavonol biodiversity in fruit and thrive the development of molecular tools and agronomical practices to optimize flavonol biosynthesis in response to UV light. As flavonols are more and more explored as important quality determinants of fruit-derived products and have been suggested as quality markers for different grape varieties (Ritchey and Waterhouse, 1999; Hermosín-Gutiérrez et al., 2011), genes involved in flavonol biosynthesis could be implemented as molecular traits during marker-assisted breeding approaches. These techniques will provide tools to the wine industry to optimize fruit quality by adaptation of flavonol content and composition using viticultural practices such as optimization of light regimens. # AUTHOR CONTRIBUTIONS JB conceived and designed the study. SC wrote the manuscript, performed the experiments related to HRs except the qPCR work, analyzed all the data and run the bioinformatics workflow for microarray analysis at QBiC. JH did the qPCR analysis experiments in HRs and developmental series. JM, RL, PA-J, JA performed the sunlight and UV-B greenhouse and field experiments. JM revised the study critically for important intellectual content and together with JH and JB revised the manuscript. # FUNDING We acknowledge financial support for this research from the Bundesministerium für Bildung und Forschung and its initiative Genomanalyse im Biologischen System Pflanze, Comisión Nacional de Investigación Científica y Tecnológica, Chile (CONICYT; Ph.D. grant no. 21120255 to RL), Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT grants no. 1150220, 1040551 and FONDECYT postdoctoral grant no. 3150578 to RL) and Programs ECOS-CONICYT (grant no. C11B01) and Millennium Nucleus for Plant Synthetic Biology and Systems Biology NC130030. # ACKNOWLEDGMENT We especially thank Cornelia Walter for excellent technical lab assistance and Pablo Carbonell-Bejerano and José Martínez-Zapater to obtain high quality microarray data with the Affymetrix custom-made Grapegen GeneChip. # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01084/ full#supplementary-material # REFERENCES fpls-08-01084 June 20, 2017 Time: 17:4 # 14 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 Czemmel, Höll, Loyola, Arce-Johnson, Alcalde, Matus and Bogs. 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. # Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries Fatma Lecourieux 1‡, Christian Kappel 2 †‡, Philippe Pieri <sup>2</sup> , Justine Charon<sup>2</sup> , Jérémy Pillet 2 † , Ghislaine Hilbert <sup>2</sup> , Christel Renaud<sup>2</sup> , Eric Gomès <sup>3</sup> , Serge Delrot <sup>3</sup> and David Lecourieux <sup>3</sup> \* <sup>1</sup> Centre National de la Recherche Scientifique, Institut des Sciences de la Vigne et du Vin, UMR Ecophysiologie et Génomique Fonctionnelle de la Vigne, Villenave d'Ornon, France, <sup>2</sup> Institut National de la Recherche Agronomique (INRA), Institut des Sciences de la Vigne et du Vin, UMR Ecophysiologie et Génomique Fonctionnelle de la Vigne, Villenave d'Ornon, France, <sup>3</sup> Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, UMR Ecophysiologie et Génomique Fonctionnelle de la Vigne, Villenave d'Ornon, France Reproductive development of grapevine and berry composition are both strongly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries response to high temperatures are poorly understood. Unlike recent data that addressed the effects on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat treatment (HT). In the context of climate change, this work focusing on temperature effect at the microclimate level is of particular interest as it can help to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HT (+ 8 ◦C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, veraison and middle ripening). Samples were collected 1, 7, and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HT delayed the onset of veraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g., phenylalanine, γ-aminobutyric acid and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodeling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories "stress responses," "protein metabolism" and "secondary metabolism," highlighting the intrinsic capacity of grape berries to perceive HT and to build adaptive responses. Additionally, important changes in processes related to "transport," "hormone" and "cell wall" might contribute #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics (CRAG), Spain #### Reviewed by: Alessandro Vannozzi, University of Padua, Italy Claudio Pastenes, University of Chile, Chile #### \Correspondence: David Lecourieux [email protected] #### † Present Address: Christian Kappel, Institut für Biochemie und Biologie, Universität Potsdam, Potsdam, Germany; Jérémy Pillet, Laboratorio de Bioquímica y Biotecnología Vegetal, Universidad de Málaga, Málaga, Spain ‡ These authors have contributed equally to this work. #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 27 October 2016 Accepted: 10 January 2017 Published: 31 January 2017 #### Citation: Lecourieux F, Kappel C, Pieri P, Charon J, Pillet J, Hilbert G, Renaud C, Gomès E, Delrot S and Lecourieux D (2017) Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries. Front. Plant Sci. 8:53. doi: 10.3389/fpls.2017.00053 84* to the postponing of veraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HT-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation. Keywords: grapevine, berry development, microclimate, high temperature, microarrays, metabolomics/metabolite profiling, climate change ## INTRODUCTION Grapevine is probably the fruit species whose economical value depends the most on climatic conditions. Besides the soil and local viticultural practices, which are components of the socalled "terroir" effect, grapevine and wine quality are determined by vintage that integrates the different climatic parameters experienced by the plant during growth and ripening. Among these environmental factors, temperature is a major regulator affecting both grapevine phenology and fruit composition (Schultz, 2000; Jones et al., 2005). The Intergovernmental Panel on Climate Change (IPCC) predicts a significant increase of mean temperatures ranging between 1.8 and 4◦C by the end of the 21st century, depending on the scenario (IPCC, 2013). Given that grapevine varieties have been ranked according to their thermal preferences (Huglin, 1978), this may lead to change the varieties traditionally grown in AOC areas, or even threaten the sustainability of some Mediterranean viticultural areas (Hannah et al., 2013; Fraga et al., 2016). However, mean temperature perceived by the plant may be altered by common viticultural practices that may be envisaged as a mean to cope with the effects of climate change (Van Leeuwen et al., 2013). For example, the extent of leaf removal directly affects light exposure and temperature of the berries. Berry temperatures depend on row orientation and significantly differ between east and west-exposed sides in the case of north-south row orientation. The temperature also interacts with the leaf/fruit (source/sink) relationships to determine yield (Sadras and Moran, 2013) and with the circadian clock to determine transcriptomic responses (Rienth et al., 2014). Moderate increases of berry temperatures obtained in open-top chambers uncouple the anthocyanin and sugar contents in red varieties (Sadras and Moran, 2012) and affect berry sensory traits (Sadras et al., 2013). Because of the direct irradiative effects, the temperature of reproductive organs may differ very significantly from that of the whole plant or surrounding air (Spayd et al., 2002; Pieri and Fermaud, 2005; Cola et al., 2009). Secondary metabolites, which are among the most prominent discriminating compounds for berry quality, are synthesized in situ under the direct influence of the local micro-environment (mostly temperature and light conditions). In this context, it is important to investigate the effects of high temperatures on berry metabolism and composition. These effects depend on the imposed temperature gradient, and on the time and duration of the heat stress. During flowering, high temperatures inhibit berry set and reduce yield (Greer and Weston, 2010). After fruit set, high temperatures stimulate sugar accumulation at the expense of other qualitative compounds (Greer and Weston, 2010) such as organic acids (Champagnol, 1984), flavonols, anthocyanins, amino acids (Spayd et al., 2002; Pereira et al., 2006; Mori et al., 2007; Tarara et al., 2008; Cohen et al., 2012) and aromas (Schultz, 2000). Together with other parameters such as the carbohydrate source-sink ratio (Ollat and Gaudillère, 1998; Parker et al., 2014; de Toda and Balda, 2015), ambient air temperature is a wellknown factor that influences berry growth and veraison timing (Duchene et al., 2010), but the impact of heat treatment (HT) at the berry level on ripening onset has not been described extensively. In the present work, we compare the effects of HT (+8 ◦C, 14 days) imposed on berry clusters at 3 different stages (green, veraison, ripening) of berry development. This experimental setup avoids the complexity of the responses involving both light and heat effects, and allows a detailed study of the relevance of developmental stages in the response to HT. Biochemical analysis and gene expression studies were conducted at 3 time points (1, 7, and 14 days) after the beginning of HT, which also allowed us to study the time course of the responses. Our results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. Heating strongly altered the berry concentration of amino acids and organic acids and decreased the anthocyanin content at maturity. When applied at the herbaceous stage, HT delayed the onset of veraison. More than 7000 genes were deregulated in at least one of the nine experimental conditions contributing to the postponing of veraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HT-induced decrease in anthocyanin content may result from a combination of transcript abundance and metabolite degradation. # MATERIALS AND METHODS #### Plant Material Fruiting cuttings of Vitis vinifera L. cv Cabernet Sauvignon (Ollat and Gaudillère, 1998) were grown in a greenhouse, in 0.5 L pots containing a mixture of perlite, sand and vermiculite (1:1:1). A drip irrigation system supplied water and a complete nutrient solution to the roots five times a day all along the experiment, avoiding any water or nutrient shortage. All fruiting cuttings bore only one single cluster and lateral shoots were removed as soon as they appeared during growth. Before the experiment, the tip of each shoot was removed as soon as 16 leaves per plant were produced to maintain approximately the same leaf area in all plants and a high leaf to fruit ratio (Ollat and Gaudillère, 1998). Therefore, all bunches were assumed to experience neither water nor assimilate limitation. In addition, the fruit cuttings were selected on the basis of similar vegetative growth and vigor as well as size and compactness of bunches. ## Heat Treatment, Temperature Measurements, and Sampling Both control and heat treatments were applied for 14 d periods, at 3 phenological stages, namely middle-green (30 days after fruit set), veraison (50% of berries turning to a visible red color) and middle-ripening (80 days after fruit set). Total soluble solids (TSS) content in berries was determined before, during and after each treatment using a digital refractometer (Atago, Tokyo). Before treatments, middle-green, veraison and middleripening berries displayed a TSS of 3.9 ± 0.3, 8.9 ± 1.4 and 14.1 ± 1.1◦Bx, respectively (mean of replicates ± SD). Three sets of 25 fruiting cuttings were used as controls [GC (Green Control), VC (Veraison Control) and RC (Ripening Control)]; the temperature of their bunch closely followed greenhouse ambient air temperature. Three sets of 25 bunches from other fruiting cuttings were submitted to heat treatment: GHT (Green Heat Treatment), VHT (Veraison Heat Treatment) and RHT (Ripening Heat Treatment). The clusters were submitted to an elevated temperature airflow produced by fan heaters (common domestic models, used at 1000 W). Only bunches were heated since shoots, leaves and roots were all protected from the heated airflow by extruded polystyrene foam deflectors. Air heating was applied repeatedly during 14 d, from 7:00 a.m. to 7:00 p.m. every day to mimic the usual diurnal temperature course of exposed berries. To avoid any differential effect linked to airflow and possible mechanical stress, simple fan blowers were used to create a continuous airflow around the control clusters during 14 d, from 7:00 a.m. to 7:00 p.m. every day. The flesh temperatures in control and heated berries (10 replicates for each treatment) were monitored continuously by copper-constantan thermocouples inserted into the berries, connected to a Campbell datalogger (Campbell Scientific). After treatments, each set of fruiting cuttings was replaced in control conditions until harvest. The experiment was conducted over 3 years. The samples collected the first year were used for biochemical and transcriptomic analysis. Samples from years 2 and 3 were used for biochemical and RT-qPCR analysis. #### Sampling In order to analyse short- and long-term responses for each treatment, 3 control and 3 heat-stressed berries per cluster (25 clusters per condition) were sampled in the evening (7:00 p.m.) at 3 different time points after treatment (1, 7, and 14 d), immediately frozen in liquid nitrogen and stored at −80◦C. To obtain 3 experimental replicates, each of the 3 berries collected per cluster was used to constitute 3 independent groups of 25 berries for each condition. The pool of deseeded berries from each group was used as a biological replication and underwent independent RNA extractions. For the biochemical analysis conducted around harvest, 3 biological replicates were prepared as described above, with berries collected from the same 25 fruiting cutting used for each treatment. Then, frozen berries (5 berries per replicate) were slightly thawed and separated quickly into skin, pulp, and seed. The skin and pulp were immediately ground into fine powder in liquid nitrogen using mortar and pestle. # Metabolites Quantification #### Sugars, Organic Acids, and Amino Acids Contents An aliquot of 500 mg fine powder of pulp was extracted sequentially with ethanol (80 and 50%), dried in a Speed-Vac, and re-dissolved in 2.5 mL de-ionized water. Glucose and fructose content were measured enzymatically with an automated micro-plate reader (Elx800UV, Biotek Instruments Inc., Winooski, VT, USA) according to the method of Gomez et al. (2007). Tartaric acid content was assessed by using the colorimetric method based on ammonium vanadate reactions (Pereira et al., 2006). Malic acid was determined using an enzyme-coupled spectrophotometric method that measures the change in absorbance at 340 nm from the reduction of NAD+ to NADH (Pereira et al., 2006). The amino acid content was determined after derivatization (Cohen and Michaud, 1993) using a Waters 2695 HPLC system equipped with Waters 474 fluorescence detector (Waters, Milford, MA, USA). Twenty amino acids were identified and quantified as described by Pereira et al. (2006). The results were expressed in µmol.L−<sup>1</sup> juice. #### Anthocyanins Quantification An aliquot of 500 mg of berry skin powder was freeze-dried for 72 h and the dried powder (50 mg) was extracted in 1.0 mL methanol containing 0.1% HCl (v/v). Extracts were filtered through a 0.45 µm polypropylene syringe filter (Pall Gelman Corp., Ann Harbor, MI, USA) for HPLC analysis. Each individual anthocyanin was analyzed with HPLC as described in Soubeyrand et al. (2014). Quantification was carried out by peak area integration at 520 nm. The concentration of individual anthocyanins was calculated in milligrams per gram (mg. g−<sup>1</sup> ) of dry skin weight (DW) using malvidin 3-O-glucoside (Extrasynthese, Genay, France) as external standard. # RNA Extraction and cDNA Production Berries collected from Cabernet Sauvignon fruit cuttings were quickly frozen in liquid nitrogen, ground to a fine powder with a Dangoumau blender, and stored at −80◦C prior to use. Total RNA from deseeded berries was extracted according to Lecourieux et al. (2010). RNA isolation was followed by DNase I treatment. The purity and quantity of the RNA were determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA integrity was determined using a Bioanalyzer 2100 (Agilent) with RNA 6000 Nano Kit I (Agilent). For each sample, reverse transcription was performed from 2 µg of purified RNA using the Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. The cDNA obtained was diluted (1:10) in distilled water. # Microarray Hybridization and Data Processing Total RNAs extracted from 3 biological replicates per condition were hybridized with 60-mer oligoarrays bearing a set of probes for 29,582 unigenes (NimbleGen Gene Expression 12 × 135K Arrays). Labeling, hybridization and scanning were carried out at the GeT-transcriptomic platform (GenoToul-Toulouse, France). Microarray data were analyzed using the R and R/Bioconductor software (Gentleman et al., 2004; R Core Team, 2013). Quality control was done using the arrayQualityMetrics package (Kauffmann et al., 2009), one identified outlier (GHS14\_1) was excluded from further analyses. Data were normalized using the Robust Multi-array Average algorithm (RMA) (Irizarry et al., 2003). Principal component analysis was done using the prcomp function, visualizations were made using ggplot2 (Wickham, 2009). Differentially expressed genes between treatment and control samples for all stages and treatment durations were identified using Limma package (Smyth, 2005). Differentials with absolute fold changes above 2 and BH (Benjamini and Hochberg, 1995) corrected P-values below 0.05 were considered significant. Significantly affected gene categories based on the MapMan Ontology (Thimm et al., 2004; Usadel et al., 2005) were identified using a Chi-square test. MapMan mappings for the Cribi 12X grapevine genome were based on closest homologs regarding to the Arabidopsis thaliana genome. Empirical cumulative distribution function plots for selected categories were made using the latticeExtra package. Gene annotations were taken from Grimplet et al. (2012). The raw microarray data were submitted to Gene Expression Omnibus (NCBI) and are accessible through GEO accession number GSE86551. # RESULTS ### Heat Treatment and Berry Temperature Recording HT was applied locally on clusters of Cabernet Sauvignon fruiting cuttings at 3 developmental stages, namely middle-green, veraison and middle-ripening (Figure 1A). Three different sets of 25 plants per condition (control or HT) were used for each developmental stage. HT was applied every day during 14 d from 7:00 a.m. to 7:00 p.m. to mimic usual daytime temperature course of sunlight-exposed berries. Under control conditions, average daytime berry flesh temperature measured were 26.5◦C ± 1.4 (control green stage, GC clusters), 25.8◦C ± 2.2 (control veraison stage, VC clusters), and 26.2◦C ± 2.5 (control ripe stage, RC clusters). HT increased the average daytime pulp temperature to 34.7 ± 1.4◦C, 33.7 ± 1.9◦C, and 35.2 ± 2.5◦C for GHT, VHT, and RHT clusters respectively (Figure 1B). Depending on the developmental stage, this experimental set-up led to average pulp temperature differences of 8.2◦C ± 1.3 (GHT vs. GC), 7.9◦C ± 1.1 (VHT vs. VC) and 9.0◦C ± 0.8 (RHT vs. RC) between heat-treated and control berries (Figure 1B). The HT imposed during the herbaceous stage delayed the onset of anthocyanin accumulation by 2 to 3 weeks (GHT vs GC berries), (Figure 2A). Furthermore, for control clusters 4 weeks were needed to reach complete color turning stage (100% clusters (n = 25) while at least one additional week was required to reach the same stage in the heated clusters. However, even at the last stage, the percentage of uncolored berries per cluster (46%) remained much higher than in control clusters (8%) (Figure 2C). A similar delay in the increase in total soluble solids (TSS) content was also observed in stressed berries (Figure 2B) while the average berry weight was not affected by the treatments (data not shown). # Biochemical Analysis of the Treated Berries The biochemical content of berries collected at harvest from control fruiting cuttings was compared with that of berries exposed to a 2-week period HT at 3 different developmental stages (Table 1). The berry hexose content (glucose and fructose) at harvest was not affected whenever the fruit-localized HT took place. By contrast, applying HT at veraison and to a lesser extent at ripening stage decreased malate content while tartrate content was slightly higher in VHT clusters than in other samples. In the present study, 20 amino acids including the non-proteinogenic ones were quantified. The concentration of 7 amino acids (THR, ARG, TYR, PHE, CYS, LYS, GABA) was significantly increased by a HT applied at veraison or ripening stages. Conversely, the PRO amount decreased dramatically after mid-ripening treatment (Table 1). The total anthocyanin concentration (TSA) in berry skin at harvest was reduced by about 50% in VHT and RHT when compared to control and GHT berries (Table 1). Interestingly, the GHT berries that colored later (Figure 1, Supplementary data) displayed a TSA comparable to control berries at harvest. Cabernet Sauvignon berries contained higher amounts of tri-hydroxylated anthocyanins than dihydroxylated ones in both control and heated conditions. Heat exposure preferentially decreased the proportion of dihydroxylated anthocyanins, regardless of the period of HT treatment (Table 1). Acylated anthocyanins represented a higher proportion of the total anthocyanin pool in HT berries (Table 1). The concentration of malvidin-3-O-glucoside, which is the most abundant anthocyanin in Cabernet Sauvignon berries (Dimitrovska et al., 2011; Lorrain et al., 2011) remained unaffected at harvest whereas the amount of one of its acylated forms (malvidin-3-O-(6′ -acetyl)-glucoside) was significantly increased in GHT clusters. By contrast, the concentrations of dephinidin, cyanidin, petunidin, peonidin and of their 3-acetylglucoside derivatives were severely reduced by heat irrespectively of the period of treatment in GHT, VHT and RHT berries. The inhibiting effect of HT on the corresponding 3-coumaroylglucoside derivatives was less pronounced. #### Alteration of Global Berry Transcriptome in Response to Localized Heat Treatment Principal component analysis of the whole normalized gene expression dataset showed that the 3 replicates of each experimental condition are well-grouped and therefore adequate for further analysis (Figure 3; Supplementary Figure 1). Principal component 1 (PC1) and PC2 explained 26% of the total variance in gene expression and can be attributed to development. PC1 (18.9%) clearly separated the green stage from both veraison and ripening stages, whereas separation between veraison and the two other stages can be distinguished on PC2 (7.1%) (Supplementary Figure 1). PC3 explained 6.2% of the total variance and splits HT from control samples in a similar proportion whatever the developmental stage. Finally, the samples were clearly separated according to the treatment duration, potentially reflecting a developmental dependent response (Figure 3). A total of 7518 transcripts were differentially expressed (fold change > 2, p-value adj. < 0.05) in at least one of the 9 conditions (Supplementary Table 1), corresponding to 25.4% of the unigenes represented on the microarray slide. The overlaps in DEGs under these nine conditions were depicted with 3-way Venn diagrams, according to developmental stage (Figure 4A) or stress duration (Figure 4B). The strongest HT effect was observed for the herbaceous stage with 5287 DEGs, compared to 4122 and 5061 DEGs identified after HT exposure at veraison and ripening stage, respectively (Figure 4A; Supplementary Table 2). A total number of 4141, 6612, and 3717 genes were deregulated after 1, 7, and 14 days of HT, respectively (Figure 4B; Supplementary Table 2). Except for one condition (V14D), the number of up-regulated genes was always higher than the number of down-regulated genes. The strongest HT effect was observed at G7D with 4024 DEGs (2135 up-regulated and 1889 downregulated genes, 7 and 6% of the grapevine unigenes, respectively) whereas the smallest effect (577 DEGs) was observed in G1D. The comparison of all 9 conditions showed that only 36 genes were steadily induced under HT condition whereas continuously Metabolite concentrations were determined around harvest. Means ± SE are shown (n = 3). Different letters indicate significant differences between control and stress conditions within a given compound at P < 0.05 accoring to a Tukey's test. CT, control; GHT, heat treatment applied during the herbaceous stage; VHT, heat treatment applied during veraison stage; RHT, heat treatment applied during the ripening stage. nd, not detected. down-regulated transcripts were not found (Supplementary Table 3). These genes will be further considered in the discussion section below. ### Identification of Significantly Altered Functional Categories According to the Stage and the Treatment Duration To gain insight into the functional categories impacted by HT, the 7518 DEGs were distributed into 35 MapMan functional categories, the so-called BINs (Usadel et al., 2005). The proportion of DEGs representing each category was determined for each of the nine experimental conditions (Figure 5). Functional enrichment analysis was performed to better identify the significantly altered functional categories according to the stage and the stress duration. HT triggered a wide range of effects on the berry transcriptome (Figure 5; Supplementary Table 4). Twenty-seven of the 35 MapMan BINs were significantly altered in HT berries in at least one condition, but only 3 functional categories, namely "Stress" (BIN 20), "Protein" (BIN 29) and "Secondary metabolism" (BIN 16), were profoundly affected in all 9 conditions. The 24 other categories were differentially affected according to the developmental stage and the stress duration, and particularly correspond to Photosynthesis (BIN 1), Cell wall (BIN 10), Hormone metabolism (BIN 17), RNA (BIN 27), DNA (BIN 28), Signaling (BIN 30), and Transport (BIN 34). #### "Stress" -Associated BIN Four hundred twenty seven DEGs were related to the functional category "Stress" and mainly belonged to the "abiotic/heat stress" cluster (Supplementary Table 5; 98 out of the 427 "Stress" DEGs). Most of the transcripts associated to "heat stress" category were predominantly up-regulated (Supplementary Figure 2A) and belong to the HSP family (Heat Shock Protein). Transcripts encoding universal stress proteins (USPs; VIT\_17s0000g04260, VIT\_04s0079g00610, VIT\_08s0032g00590) also accumulated in response to HT. #### "Protein" -Associated BIN HT deeply affects protein homeostasis. Indeed, numerous HSP and chaperones genes were up-regulated in HT berries (Supplementary Tables 5, 6). These proteins play an important role in protein-protein interactions (Kotak et al., 2007a; Bokszczanin and Fragkostefanakis, 2013). The abundance of several FK506-binding protein (FKBP) related transcripts was also increased by HT (VIT\_19s0015g01100, VIT\_07s0031g01150, VIT\_08s0007g04340, VIT\_13s0064g00580, VIT\_01s0011g00930, VIT\_00s0260g00070). These proteins belong to the large family of peptidyl prolyl cis–trans isomerases that can function as chaperones. Protein synthesis and degradation were also strongly affected in heated berries as suggested by the 60 DEGs linked to protein synthesis category and the 348 genes belonging to proteolysis (Supplementary Tables 5, 6). Protein degradation through the ubiquitin-proteasome system (UPS) plays an essential role in diverse cellular pathways, cell-cycle progression, DNA repair, and degradation of damaged proteins as well as in signal transduction. Particularly, UPS components are major players in plant acclimation to abiotic stresses (Stone, 2014; Guerra et al., 2015). In the present study, 167 transcripts potentially linked to the ubiquitin machinery were deregulated after HT (102 up-, 56 down-, 9 both up- and down-regulated transcripts; Supplementary Table 6). Most of these DEGs encode different putative ubiquitin ligases (E3). Orthologs of each of the 3 major E3 classes (namely RING-type (Really Interesting New Gene); HECT-type (Homology to E6-Associated Carboxyl-Terminus), and U-box-type) were up-regulated in heatstressed berries. While some E3-like transcripts accumulated whatever the developmental stage (VIT\_09s0002g00220, VIT\_12s0034g01390, VIT\_05s0124g00230, VIT\_06s0009g03670, VIT\_08s0040g02600, VIT\_19s0027g00320), most of these were transiently or specifically deregulated at a particular stage. For instance, some E3-related transcripts were only HT upregulated during the herbaceous stage (VIT\_17s0000g09790, VIT\_18s0041g01090, VIT\_14s0068g02150, VIT\_14s0066g02580, VIT\_08s0056g01410) whereas others responded to HT during veraison or ripening stages (VIT\_19s0015g00660, VIT\_01s0011g02950, VIT\_01s0026g00300, VIT\_07s0005g01360, VIT\_08s0007g04790, VIT\_18s0001g02280, VIT\_00s0160g00270, VIT\_18s0001g06220). Furthermore, the CRL group (Cullin based Ring E3 ligases), corresponding to the largest class of ubiquitin ligases (Stone, 2014), and especially the CUL1 based E3s, [also referred to as Skp1-Cullin-F-box (SCF)] were strongly impacted by HT. Indeed, an ortholog of the Arabidopsis adaptor protein S-Phase kinase-associated protein (SKP; VIT\_03s0038g02480) and many F-box proteins are up-regulated at the transcriptional level upon HT. #### "Secondary Metabolism" -Associated BIN The secondary metabolism produces compounds of critical importance for berry quality and wine bitterness and astringency (Lund and Bohlmann, 2006; Ali et al., 2010; Kuhn et al., 2014; Robinson et al., 2014). Three hundred twenty seven DEGs were related to "Secondary metabolism" and mainly belonged to the subcategories "isoprenoids," "phenylpropanoid-lignin," and "flavonoids" (Supplementary Table 7). Numerous genes affected by HT correspond to aroma and aroma-precursor related gene. Terpenes (predominantly eucalyptol, β-caryophyllene, and α-humulene) are usually present at low levels in Cabernet Sauvignon grapes, accumulating during the preveraison stage whereas benzene derivatives (2 phenylethanol and 2-phenylethanal) appear at late ripening (Kalua and Boss, 2009; Robinson et al., 2014). The strong repression of genes encoding the 1-deoxy-D-xylulose-5 phosphate synthase (VIT\_05s0020g02130, VIT\_09s0002g02050, VIT\_11s0052g01730, VIT\_11s0052g01780) suggests that volatile terpenoids biosynthesis may be decreased by HT (Supplementary Table 7). The 1-deoxy-D-xylulose-5-phosphate synthase catalyzes the synthesis of isopentenylpyrophosphate (IPP), which is the precursor of all terpenes. The condensation of IPP and its isomer DMAPP (dimethylallylpyrophosphate) forms geranyl diphosphate (GPP) that is used by terpene synthase (TPS) to produce monoterpenes and derivatives. The Pinot Noir reference genome contains 89 putative TPS, among which half have been functionally characterized (Martin et al., 2010). Among the 55 probe sets giving reliable results and representing transcripts of functional, partial and pseudo TPS on the NimbleGen array (Cramer et al., 2014), 16 transcripts showed differential abundance in HT berries (Supplementary Table 8). Thirteen TPS were transiently repressed in GHT, VHT and or RHT clusters whereas only three TPS were upregulated by HT (VviTPS25, VviTPS26, VviTPS50). From the literature, most of these TPS accumulate at the late stages of ripening in vineyard conditions (Cramer et al., 2014). Finally, the transcript abundance of several terpene-related genes decreased in berries directly exposed to HT, in a similar way to that observed after exposing the whole vine to HT (Rienth et al., 2014). These repressed genes encode geraniol 10-hydroxylase, (-)-germacrene D synthase and linalool synthase (Supplementary Table 8). Within the terpene family, carotenoids are a complex subgroup of isoprenoid pigments playing diverse roles in plants and providing nutritional value. Carotenoids also lead to C13-norisoprenoids which contribute the characteristic aromas of Vitis vinifera varieties (Mendes-Pinto, 2009). Among the 42 putative grape carotenoid metabolic genes (Young et al., 2012), 21 transcripts showed differential abundance after HT in at least one of the 9 conditions, most of these being down-regulated (Supplementary Tables 7, 9). The abundance of 2 out of 3 phytoene synthase transcripts (VviPSY, VIT\_12s0028g00960, VIT\_06s0004g00820), encoding the enzyme that catalyzes the first step committed to carotenoid biosynthesis, decreased upon HT. This down-regulation was also observed for downstream genes, namely phytoene dehydrogenase (VIT\_04s0023g01790), carotene desaturase (VIT\_14s0030g01740), carotenoid isomerase (VIT\_08s0032g00800, VIT\_12s0035g01080), lycopene cyclase (VIT\_06s0080g00810, VIT\_11s0016g01880) and carotene hydroxylase (VIT\_04s0023g00080). The β-carotene hydroxylase 2 transcripts (VviBCH2, VIT\_16s0050g01090) were the only ones strongly accumulating under HT, and especially at the herbaceous stage. Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that participate to the distinct herbaceous/ bell pepper characters of some wines such as Cabernet Sauvignon (Dunlevy et al., 2009; Darriet et al., 2012; Kuhn et al., 2014). Isobutyl methoxypyrazine (IBMP) is the predominant MP in Cabernet Sauvignon berries, accumulating throughout the pre-veraison stage before declining during the ripening phase. The last step of its biosynthesis was recently deciphered in grapevine, through the unambiguous identification of VviOMT3 (VIT\_03s0038g03090), an O-methyltransferase capable of converting the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP (Dunlevy et al., 2013; Guillaumie et al., 2013). By contrast to VviOMT4, VviOMT1 and VviOMT2 are active with a broad range of substrates but methylate IBHP in vitro, although with poor affinity (Dunlevy et al., 2010). Interestingly, VviOMT3 transcripts were strongly repressed in green berries exposed to HT (Supplementary Table 7). A similar decrease was also observed in GHT berries for VviOMT4 (VIT\_03s0038g03080) and VviOMT1 (VIT\_12s0059g01790) transcripts, whereas VviOMT2 (VIT\_12s0059g01750) remained unaffected. Grape berry phenolics derived from the phenylpropanoid pathway participate to sensory properties, color and protection against environmental stress (Teixeira et al., 2013). Our biochemical analysis highlighted the dramatic effects of local warming on the onset of veraison and on the final anthocyanin contents in berries at harvest (Table 1; Figure 2). MapMan analysis (Figure 6) shows the contrasted heat responses of DEGs related to the general phenylpropanoid and to the flavonoid biosynthesis pathways (Figure 6; Supplementary Figure 3; Supplementary Table 10). The transcriptomic responses of GHT samples clearly differed from both VHT and RHT samples. During the herbaceous stage, genes involved in the general phenylpropanoid pathway (phenylalanine ammonialyase PAL, cinnamate 4-hydroxylase C4H, 4-coumarate-CoA ligase 4CL) were induced by HT regardless to the stress duration. By contrast, in VHT and RHT samples, the same genes were up-regulated after 1 day, but strongly repressed after 7 and 14 days of treatment. Many transcripts related to the flavonoid biosynthesis pathway were repressed in GHT clusters. Two chalcone synthase transcripts (VviCHS1: VIT\_14s0068g00920, VviCHS2: VIT\_14s0068g00930) encoding the first committed enzyme in flavonoid biosynthesis (Parage et al., 2012) were repressed after 7 days. A significant repression by HT was also observed for many flavonoid transcripts of the late biosynthetic pathway, such as flavonoid 3 ′ -hydroxylase (VviF3′H, VIT\_11s0016g01020, VIT\_11s0016g01030, VIT\_09s0002g01090), flavonoid 3′ 5 ′ -hydroxylase (VviF3′ 5 ′H), dihydroflavonol 4-reductase (VviDFR, VIT\_16s0039g02350, VIT\_18s0001g1280) and leucoanthocyanidin dioxygenase (VviLDOX, VIT\_08s0105g00380), whereas the flavanone 3-hydroxylase gene (VviF3H, VIT\_16s0098g00860) was upregulated (Figure 6; Supplementary Figure 3; Supplementary Table 10). The HT led to contrasted effects on the same set of genes in VHT and RHT clusters when compared with GHT. VviF3H transcript was significantly less abundant whereas VviCHI (chalcone isomerase, VIT\_19s0014g00100) and VviF3′H genes were mostly up-regulated. Moreover, the repressive effect of HT on VviF3′ 5 ′H isoforms was not observed in VHT clusters and only transiently detected in RHT berries. In addition, the structural genes VviANR (anthocyanidin reductase, VIT\_00s0361g00040), and VviLAR (leucoanthocyanidin reductase, VIT\_01s0011g02960, VIT\_17s0000g04150) involved in proanthocyanidins (PA) synthesis were significantly repressed under HT, according to the stage and/or duration of the stress. The anthocyanidin aglycones are further modified through glycosylation, methylation and acylation events, leading to the production of a wide variety of anthocyanin compounds. Glycosylation of flavonoids is catalyzed by enzymes from the large glycosyltranferase (GT) family, represented by 240 genes in the grape genome (Ono et al., 2010). Glycosylation enhances the structural diversity and modifies the functional properties of these secondary metabolites. For instance, the expression of UFGT [UDP-glucose:flavonoid 3-O-glycosyltransferase, renamed VviGT1, VIT\_16s0039g02230 (Parage et al., 2012)], catalyzing the 3-O-specific glycosylation of anthocyanidin is critical for the coloration of grape skin (Boss et al., 1996). Twenty-nine putative VviGT were deregulated in berries exposed to HT (Figure 6; Supplementary Figure 3; Supplementary Table 10). Despite the loss of anthocyanins upon HT, VviGT1 expression remained unaffected by HT. By contrast, VviGT5 (VIT\_11s0052g01600) and VviGT6 (VIT\_04s0023g01290) transcripts were strongly down-regulated in VHT and RHT clusters. VviGT5 and VviGT6 drive the glycosylation of flavonols, which increase their water solubility and their accumulation (Ono et al., 2010). In grapevine, anthocyanins can also be modified through the action of O-methyltransferases (AOMTs) and acyltransferases (ACTs) before being transported into the vacuole (Fournier-Level et al., 2011; Rinaldo et al., 2015). These modifications modulate berry color by reducing anthocyanin reactivity and increasing their stability and solubility in water. None of the two grape AOMTs (VviAOMT1; VIT\_01s0010g03510, VviAOMT2; VIT\_01s0010g03490) that were previously described as effective anthocyanin 3′ - and 3′ ,5′ -O-methyltransferase (Hugueney et al., 2009; Lücker et al., 2010; Fournier-Level et al., 2011) was significantly deregulated by HT (Supplementary Table 10). However, other putative VviAOMTs transcripts (VIT\_11s0016g02610, VIT\_07s0031g00350, VIT\_03s0063g00140, VIT\_12s0028g03110), were transiently up- or down-regulated by HT depending on the ripening stage and stress duration. HT also modified the transcript amounts of various ACTs (Supplementary Table 10). Indeed, 5 putative ACTs were transiently repressed in GHT berries (VIT\_12s0134g00590, VIT\_12s0134g00630, VIT\_12s0134g00600, VIT\_12s0134g00650, VIT\_12s0134g0660), whereas 3 ACTs (VIT\_03s0017g00870, VIT\_12s0134g00590, VIT\_12s0134g00630) were up-regulated in both VHT and RHT berries. VIT\_03s0017g00870 corresponds to Vvi3AT, an enzyme recently associated to the production of the common grape berry acylated anthocyanins (Rinaldo et al., 2015). Stilbenes and lignins represent branching points in the phenylpropanoid pathway. Stilbenes are a small family of phytoalexins synthesized by plants in response to biotic and abiotic stresses. Under normal growth conditions, berries stilbene content increases from veraison to ripening, with significant differences among V. vinifera varieties (Gatto et al., 2008). Fortyeight stilbene synthases (STSs) genes catalyzing the biosynthesis of the stilbene backbone were found in grapevine genome. This represents an unusual example of functional redundancy (Parage et al., 2012). Most of these (39 genes) were impacted in berries exposed to HT, displaying a quite similar and noticeable expression profile (Figure 6; Supplementary Figure 3; Supplementary Table 10). In GHT clusters, 18 STS transcripts accumulated after 1 and 7 d of treatment before being repressed at 14 d. In VHT and RHT fruits, the STSs were transiently induced at 1 d and then strongly repressed over the 2 weeks of experiment. Lignification can also be induced as a response to various biotic and abiotic stresses, as shown in Citrus fruit after postharvest HT (Yun et al., 2013). Conversely, the cell wall lignin content was reduced in the skin of grape mature berries experiencing a water stress (Vannozzi et al., 2012; Fernandes et al., 2015). In the present study, numerous transcripts (39) potentially involved in the lignin biosynthetic pathway differentially accumulated in HT berries (Figure 6; Supplementary Figure 3; Supplementary Table 10). Two cinnamoyl-CoA reductase (VviCCR) gene isoforms were affected in an opposite way by HT, VIT\_14s0066g01150 being induced, and VIT\_09s0070g00240 being repressed. In GHT and VHT clusters, the transcript levels of 16 cinnamyl alcohol dehydrogenase (VviCAD) genes were mainly repressed whereas a few ones accumulated in RHT berries. The content of 3 ferulate 5-hydroxylase transcripts (VviF5H) was reduced in both VHT and RHT berries. Finally, HT led to contrasted effects on caffeic acid O-methyltransferase (VviCOMT) and peroxidase transcripts (catalyzing the polymerization of monolignols into lignins). While many structural genes were transcriptionally affected in HT berries, the positive regulatory genes identified so far in grapevine were either weakly (VviMYBPA1, VviMYB14, VviMYB15, VviMYBF1) or not deregulated (VviMYBA1, VviMYBA2, VviMYBA3, VviMYC1, VviMYCA1, VviMYBPA2) in HT berries (Supplementary Table 11). Accordingly, HT did not affect the amount of VviGT1 transcript (VIT\_16s0039g02230) that encodes the anthocyanidin glycosyltransferase catalyzing the limiting step for anthocyanin accumulation and are a direct target of VviMybA1 transcription factor (TF; VIT\_02s0033g00410) (Cutanda-Perez et al., 2009). For comparison, no effect of HT was observed on VviMYBA1 transcripts in berries from fruiting cuttings (Carbonell-Bejerano et al., 2013), whereas a VviMYBA1 repression was reported in two others studies (Yamane et al., 2006; Rienth et al., 2014). It is also noteworthy that both VviMYBC2-L3 (VIT\_14s0006g01620) and VviMYB4b (VIT\_04s0023g0371) transcripts were transiently enhanced in VHT and RHT berries, respectively. These two TFs were recently described as negative regulators of the phenylpropanoid pathway in grape (Cavallini et al., 2015). While HT did not significantly impact the expression of genes known to act in the transport of flavonoids (reviewed by Zhao (2015), either directly (VviAM1, VviAM3, VviABCC1, VviMATE1, VviMATE2) or indirectly (VviGST1, VviGST4), the present work pointed out a significant deregulation of many putative ABC or MATE transporters and Glutathion S-transferase family members (Supplementary Tables 1, 11). It cannot be excluded that these proteins could be involved in the control of flavonoid transport in HT grape berries, as well. In addition to synthesis, stabilization and vacuolar sequestration, the anthocyanin content can also be modulated through degradation that may involve enzymes such as laccases, polyphenol oxidases, class III peroxidases, and β-glucosidases (Oren-Shamir, 2009). Our experiments showed a strong HT effect on the expression of laccase genes, potentially impacting the polymerization rate of various phenolic compounds. Out of the 93 laccase-annotated genes in the grapevine genome, 33 transcripts were deregulated by HT (Supplementary Table 12). Interestingly, the laccase TT10 (Transparent Testa 10, VIT\_18s0075g00600) showed a maintained up-regulation by HT whatever the stage of development. In Arabidopsis, TT10 was proposed to participate in the oxidative polymerization of phenolic compounds (Pourcel et al., 2005). In a similar way, numerous genes encoding putative polyphenol oxidases (4), peroxidases (25) and β-glucosidases (16) were impacted by HT (Supplementary Table 12). In the present study, HT induced several peroxidase genes including class III type (VIT\_07s0130g00220, VIT\_18s0001g06850) whereas others were repressed (VIT\_18s0001g06840, VIT\_18s0001g06890). Recently, Movahed et al. (2016) showed that the expression of 3 of the 5 peroxidase genes (VIT\_14s0066g01850, VIT\_06s0004g07770, VIT\_07s0191g00050, VIT\_11s0016g05320 and VIT\_18s0072g00160) that were most strongly expressed in grape berry pericarp during ripening was influenced by temperature elevation. In agreement with these results, the present data also pinpoints the alteration of 4 of these genes (VIT\_14s0066g01850, VIT\_06s0004g07770, VIT\_11s0016g05320 and VIT\_18s0072g00160) in response to HT but with different kinetics and intensities. # DISCUSSION #### Locally Applied HT Delays the Onset of veraison and Alters Berry Composition at Maturity The temperature differences used in this experiment (Figure 1) are within the range of values obtained in vineyards between sun-exposed and shaded berries. For instance, the temperature of exposed Merlot berry can exceed air temperature by more than 10◦C above ambient temperature, especially for clusters directly exposed to solar radiation (Pieri and Fermaud, 2005). The present work highlights the strong inhibiting effects of HT (∼ 35◦C) locally imposed to clusters, on fruit-specific processes required for the onset of veraison. Conversely, damping diurnal berry temperature fluctuations (daytime cooling and night-time heating of clusters starting from fruit set) advanced the onset of ripening (Cohen et al., 2008; Tarara et al., 2008), as well as separated treatments (daytime cooling or night-time heating) even though the effects were less marked in the latter case (Cohen et al., 2012). High temperatures promote the decrease of organic acid content observed after veraison, by exacerbating the malic acid breakdown (Ford, 2012; Rienth et al., 2016). Elevated temperatures accelerate the utilization of malate, enhancing the anaplerotic capacity of the TCA cycle for amino acid biosynthesis (Sweetman et al., 2014). By contrast to previous reports describing an increase in berry concentrations of various amino acids after exposure of whole grapevines to HT, the present data were obtained after berry-localized treatments. After a 14 d HT of whole grape fruiting cuttings, Carbonell-Bejerano et al. (2013) observed in fruits an increase in the content of 5 amino acid including TYR and PHE. Similarly, Sweetman et al. (2014) reported a boost in the berry concentration of 10 amino acids, among which THR, PRO and GABA, after applying at veraison an 11 d HT to potted Shiraz vines. The accumulation of amino acids is a widespread phenomenon of higher plants response to various abiotic stresses, including HT (Krasensky and Jonak, 2012; Bokszczanin and Fragkostefanakis, 2013). This increase may result from an up-regulation of amino acid synthesis, a decreased amino acid catabolism, and/or of an enhanced of stress-induced protein breakdown or to a change in amino acids imported from the plant. In grape berries exposed to HT, Sweetman et al. (2014) suggested that the amino acid accumulation was rather due to de-novo biosynthesis than to proteolysis. Among others, the increased levels of PRO and GABA may have a beneficial effect upon exposure to environmental cues (Krasensky and Jonak, 2012; Bokszczanin and Fragkostefanakis, 2013). PRO, which might act as a compatible osmolyte, a free radical scavenger and a protein chaperone, has been reported to have a protective role against abiotic stresses in many plant species. By contrast, tobacco and Arabidopsis plants do not accumulate PRO under HT and an excess of PRO reduces thermotolerance in Arabidopsis (Lv et al., 2011). PRO hyper-accumulated in Shiraz berries submitted to warming (Sweetman et al., 2014), but not in heat- treated Muscat Hambourg berries (Carbonell-Bejerano et al., 2013). PRO content was even strongly reduced in Cabernet Sauvignon fruits as shown in the present study (Table 1). These differences may be due to different experimental procedures (local vs whole plant treatments) and/or result from variety-dependent responses. Therefore the exact role of PRO in grape berry adaptation to HT remains to be determined. The amount of the osmolyte GABA also increased in RHT berries (Table 1). This compound may act as a signaling molecule and affect different processes, including the control of the carbon–nitrogen balance and the protection against oxidative stress (Fait et al., 2007; Krasensky and Jonak, 2012). Recently, Sweetman et al. (2014) showed that a GABA shunt was up-regulated in warmed grape berries. This shunt is important for stress tolerance when the TCA cycle is down-regulated (Fait et al., 2007). PHE and TYR whose concentration also increased in VHT and RHT berries (Table 1) are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway. Beside their role as building blocks of proteins, PHE and TYR serve as precursors for a wide range of secondary metabolites. Particularly, PHE is the substrate of PAL, the key enzyme of phenolic biosynthesis. The accumulation of PHE in HT berries may result from the strong repression of PAL genes observed during ripening, and to a larger extent, to the repression of genes encoding enzymes involved in the general phenylpropanoid pathway (Figure 6; Supplementary Table 7). Dai et al. (2014) reported that the up-regulation of the phenylpropanoid pathway genes and the increased accumulation of anthocyanins in response to high sugar supply is paralleled by a decrease in berry PHE content. Heat exposure reduced the concentration of total anthocyanins and particularly the amount of dihydroxylated anthocyanins, and increased the proportion of acylated anthocyanins (Table 1). This result agrees with previous studies showing that application of elevated temperatures (>30◦C) to fruits results in anthocyanin degradation and inhibition of their accumulation. The intensity of this phenomenon depends on the type of anthocyanin derivative and on the grape variety (Kliewer and Torres, 1972; Spayd et al., 2002; Mori et al., 2007; Cohen et al., 2008; Tarara et al., 2008; Azuma et al., 2012). Altogether, the experimental design set up with Cabernet Sauvignon fruiting cuttings to address the direct impact of elevated temperatures on berry development provided reliable biochemical results that are comparable to those observed in vineyards or using potted grapevines. Moreover, the alterations observed in heated berries directly result from the cluster exposure to HT. Finally, while veraison is delayed in GHT berries, their biochemical contents at harvest are comparable to that observed in control fruits. ## Locally Applied HT Deeply Affects Grape Berry Transcriptome and Triggers Adaptative Responses Our results indicate that a local HT application deeply affected the berry transcriptome whatever the stage and the stress duration (Figure 4). The amplitude of the response is consistent with previous studies reporting that about 5% of the plant transcriptome is up-regulated 2-fold or more in response to HT (Mittler et al., 2012). The berry transcriptome varied with a similar amplitude when microvines were subjected to a short 2 h stress period during the day, whereas gene expression changes were more pronounced when this 2 h-HT was applied during the night (Rienth et al., 2014). The present work also revealed that only few genes were heatderegulated whatever the developmental stage and the stress duration. The comparison of all 9 conditions showed that only 36 genes were steadily induced under HT condition whereas continuously down-regulated transcripts were not found (Supplementary Table 3). Not surprisingly, 24 genes encoding various HSPs are listed among the permanently up-regulated genes. Other genes correspond to transcripts encoding putative transcription factors (TFs) from the MYB (VIT\_08s0007g06180) and the AP2/ERF (VIT\_15s0046g01390) families. These TFs contrast with many other TFs that are deregulated in at least one experimental condition (602 DEGS, Supplementary Table 13), and take part in the observed deep remodeling of the berry transcriptome under heat. Especially, the function of AP2/ERF members in plant abiotic stress responses is clearly established (Mizoi et al., 2012) and few recent works suggested a similar role for this TF family in grapevine (Carbonell-Bejerano et al., 2013; Zhu et al., 2013; Rocheta et al., 2014). Two others transcripts coding for a sterol O-acyltransferase (VIT\_00s2300g00010) and a truncated receptor-like kinase (VIT\_00s0437g00010) respectively, showed disrupted expression levels in all the nine conditions (Supplementary Table 3). The closest ortholog of VIT\_00s2300g00010 in Arabidopsis corresponds to AtASAT1 (Acyl-CoA Sterol Acyltransferase1), an enzyme catalyzing the phytosterol ester biosynthesis in seeds (Chen et al., 2007). The precise role of this enzyme in the context of heat-induced responses remains to be determined. Finally, VviGOLS1 (VIT\_07s0005g01970) encoding a galactinol synthase (GOLS) was consistently up-regulated by HT, confirming our previous work (Pillet et al., 2012). #### Heat-Induced Adaptive Responses in Berries Essential for cell growth and viability, HSPs function as molecular chaperones in maintaining protein quality and folding, and are required for the acquisition of the thermotolerance (Bokszczanin and Fragkostefanakis, 2013). A massive up-regulation of HSP genes is a highly conserved response in heat-exposed plants (Finka et al., 2011). This response is a result of the complex signaling cascade, whose final steps consist in the activation of heat shock TFs (HSFs) and their binding to the HSP promoters (Mittler et al., 2012). HSF activity is regulated at the transcriptional, post-transcriptional and post-translational levels (Mittler et al., 2012) and 21 Arabidopsis HSF transcripts are upregulated in response to various environmental stress conditions (Miller and Mittler, 2006). Data mining of the 12X version of the grape genome allowed the identification of 19 putative VviHSFs (Pillet et al., 2012; Scharf et al., 2012) among which 6 were up-regulated in HT berries (Supplementary Table 14). Among these, VIT\_04s0008g01110 corresponds to VviHSFA2 that was recently reported as involved in the regulation of heat responses in stressed berries (Pillet et al., 2012). HSFA2 is a key player for basal and acquired thermotolerance, extending the effect of heat acclimation in both tomato and Arabidopsis (Charng et al., 2007). AtHSFA2 which is the most highly HT-induced HSF (Busch et al., 2005), serves as a regulatory amplifier of a subset of genes (Schramm et al., 2008) and is required for the induction and maintenance of HT memory-related genes (Lämke et al., 2016). The transcriptional regulation of the heat-induced responses may also be due to VviMBF1c (Multiprotein bridging factor 1c; VIT\_11s0016g04080) that showed enhanced expression pattern in HT berries (Supplementary Table 14), in good agreement with two recent studies (Carbonell-Bejerano et al., 2013; Rienth et al., 2014). In Arabidopsis, AtMBF1c acts as a thermotolerance mediator through the heat-dependent regulation of 36 different transcripts (Suzuki et al., 2011). The existence of a similar MBF1c regulon was proposed in ripening berries of microvines submitted to HT (Rienth et al., 2014). In the present study, the relationship between VviMBF1c and putative members of this regulon was not so tight, probably due to a much longer heat exposure (14 days vs. few hours). Several transcripts encoding putative USPs also accumulated in response to HT (Supplementary Table 5). The USP family plays an important role in stress resistance in bacteria (Kvint et al., 2003). Based on expression profiles, a similar role in abiotic stress tolerance was suggested for various plant USPs (Kerk et al., 2003; Maqbool et al., 2009; Isokpehi et al., 2011). Recently, the Arabidopsis AtUSP was shown to exhibit a redox-dependant chaperone function and to enhance plant tolerance to heat shock and oxidative stress (Young Jun et al., 2015). The role of USPs in grapevine remains to be elucidated. #### Protein Homeostasis in Heated Berries The maintenance of protein homeostasis, which includes the control of synthesis, intracellular sorting, folding, the function and degradation of proteins, is fundamental to ensure growth and development of plants under normal and stressful environmental conditions. In Arabidopsis, FKBP62 (ROF1) and FKBP65 (ROF2) are involved in acquired thermotolerance through the interaction with HSP90.1 and HSFA2 (Meiri and Breiman, 2009; Meiri et al., 2010). ROF1 contributes to the transcription activity of HSFA2 but ROF2, in the presence of ROF1, abolishes this activity. This suggests that ROF2 acts as a HT modulator through a negative feedback regulation of HSFA2 (Meiri et al., 2010). While expression of the grapevine VviROF1 ortholog (VIT\_00s0769g00010) was not affected in our experiments (Supplementary Table 6), VviROF2 transcripts (VIT\_00s0260g00070) accumulated upon HT at green and ripening stages (G7D, R1D and R7D). Half of the DEGs linked to the functional category "protein synthesis" encode ribosomal proteins known to prevent inhibition of protein synthesis under HT (Muñoz and Castellano, 2012). A massive heat-dependent deregulation of ribosomal protein genes was also described in wheat (Qin et al., 2008). Interestingly, one of the constantly and strongly up-regulated genes encodes a chloroplast ribosomal protein (RPS1; VIT\_13s0101g00050), a heat responsive protein involved in retrograde activation of heat responses in Arabidopsis (Yu et al., 2012). Particularly, RPS1 was proposed as a critical factor in the activation of HSFA2 and of its target genes. Many genes related to protein targeting (34 DEGs) were also affected in HT berries and mainly up-regulated during the green stage (Supplementary Table 6). HT-induced protein degradation is a complex process involving a multitude of proteolytic pathways, with many DEGs encoding cysteine protease, serine protease, arginine protease, subtilase or metalloprotease activities. For instance, 24 transcripts encoding AAA/FtsH metalloproteases were deregulated in heated berries (Supplementary Table 6). This protease family is essential for the protein quality control of mitochondrial and chloroplastic membranes, and to prevent damages caused by stress conditions (Janska et al., 2013). For example, VviFtsH6 transcripts (VIT\_14s0108g00590) strongly accumulated in berries when the HT was applied during the herbaceous stage. In Arabidopsis, the FtsH6 protease is involved in the degradation of the light-harvesting complex II (LHC II) during high light acclimatization and dark-induced senescence (Zelisko et al., 2005). The involvement of FtsH proteases in the quality control of the photosystem II was also described under moderate HT (Yoshioka et al., 2006) and a similar role may be attributed to FtsH6 in green photosynthetic berries exposed to HT. Another metalloprotease encoding gene (VIT\_03s0088g00320) was highly up-regulated by HT at all stages. This transcript encodes an Arabidopsis EGY3 ortholog (At1g17870) belonging to the EGY family (ethylene-dependent gravitropism-deficient and yellow-greenlike 3). AtEGY3 is an AtHSFA2 target gene (Nishizawa et al., 2006). The involvement of this putative membrane and plastidial metalloprotease in heat responses was recently pinpointed (Laranjeira and coworkers, unpublished, PhD thesis; http:// hdl.handle.net/1822/18268). Both FtsH6 and EGY3 genes were also recently identified as heat responsive genes in Brassica napus siliques (Yu et al., 2014). The deregulation of numerous E3 ligase genes in HT berries may reflect an important role in triggering adaptive responses. Indeed, recent findings highlighted the role of various E3 ligases in mediating abiotic stress tolerance in Arabidopsis and in different crop species (Stone, 2014; Guerra et al., 2015). Most stress-related E3 ligases identified so far facilitate responses to environmental stimuli by modulating the abundance of key downstream stress-responsive TFs, thus affecting stress-related changes in gene expression. A non-proteolytic function of ubiquitin modification has also been reported in abiotic stress tolerance (Stone, 2014). For example, the rice E3 ligase OsHCI1 (Heat and Cold induced 1) monoubiquitinates some nuclear proteins in plants exposed to HT, which results in their translocation to the cytoplasm, and promotes HT tolerance (Lim et al., 2013). In apple, the E3 ligases MdCOP1s negatively regulate the peel anthocyanins content of fruits by modulating the degradation of the MdMYB1 protein (Li et al., 2012). A similar role can be envisaged in heat exposed berries to control the stability of flavonoid associated TFs. Many E2 conjugating enzyme genes are stressinducible (Stone, 2014; Guerra et al., 2015). Two E2 transcripts were up-regulated when HT was applied on ripening berries. The first one (VIT\_06s0004g08200) is an E2 enzyme ortholog of the Arabidopsis ubiquitin-conjugating enzyme 28 (UBC28, At1g64230). The second one (VIT\_18s0001g10100) encodes a SUMO Conjugating Enzyme (SCE1a) binding small ubiquitinlike modifiers (SUMOs) to a wide range of cellular proteins. SUMOylation of AtHSFA2 represses its transcriptional activity and SUMO overexpression decreases sHSPs accumulation after HT (Cohen-Peer et al., 2010). #### Heat Treatment Potentially Decrease Berry Phenolic Quality and Aromatic Potential Low temperatures are beneficial to aroma production in the cool climate white grape cultivars (Duchêne and Schneider, 2005). Conversely, the aromatic potential of berries exposed to HT may be reduced (Belancic et al., 1997; Falcão et al., 2007), possibly due to heat-induced transcriptional changes (Rienth et al., 2014). Cabernet Sauvignon berries contain various aromatic molecules such as terpenes and C13-norisoprenoids (Robinson et al., 2014) and are typified by specific volatile thiols and methoxypyrazines (Bouchilloux et al., 1998). Our transcriptomic data indicate that direct exposure of Cabernet Sauvignon berries to HT may decrease their aromatic potential through deregulation of numerous aroma and aroma precursor-related genes (Supplementary Tables 7, 9). Indeed, volatile terpenoids biosynthesis may be decreased by HT, as suggested by the heat repression of many key enzymes of the biosynthetic pathway (1-deoxy-D-xylulose-5-phosphate synthase, terpene synthase, geraniol 10-hydroxylase, (-)-germacrene D synthase and linalool synthase). Comparable results were obtained after exposure of the whole vine to HT (Rienth et al., 2014). Our data also revealed that most of 21 DEGs linked to carotenoid metabolism were down-regulated after HT, with the exception of VviBCH2. In Arabidopsis, overexpression of BCH2 improved tolerance to high light and HT by catalyzing the conversion of β-carotene to zeaxanthin and therefore preventing membranes from oxidative damage (Davison et al., 2002). While a constant decline in carotenoid abundance is generally observed after veraison in various cultivars including Cabernet Sauvignon (Deluc et al., 2009), our results suggest that HT may contribute to the decrease of carotenoid (except zeaxanthin) concentration before veraison, and/or accelerate its decrease after veraison. However, the possible consequences in term of aroma potential are not clear since only one type of CCD transcripts (VviCCD4a, VIT\_02s0087g00910) was decreased by HT. Another gene (VviCCD4b, VIT\_02s0087g00930) was transiently up-regulated by HT at veraison. The CCD enzymes convert their carotenoid substrates to C13-norisoprenoids, which encompass desirable flavor and aroma compounds in grapes and wine. In normal growth conditions, up-regulation of VviCCD4a and VviCCD4b was observed during ripening in Sauvignon Blanc and Pinotage (Young et al., 2012; Lashbrooke et al., 2013), whereas VviCCD4a expression pattern during berry development was dependent on the growth temperature regime applied to Cabernet Sauvignon fruit cuttings (Guillaumie et al., 2011). Carotenoid concentration in grape berries is influenced by microclimate through an effect of light exposure on the cluster (Kwasniewski et al., 2010; Young et al., 2015) and possibly through an impact of the temperature as suggested by our results. Finally, the present work highlights the HT repressive effects on 3 out of 4 VviOMTs (Supplementary Table 7), including VviOMT3, responsible for the synthesis of the predominant methoxypyrazine IBMP (Guillaumie et al., 2013). Natural (climate, soil) and viticultural factors impact on IBMP concentration in grapes and wines, and grapes ripened under HT produce wines with reduced IBMP contents (Darriet et al., 2012). Considering the strong correlation between VviOMT3 expression and berry MP content, our data implies that HT lead to a strong reduction in IBMP synthesis during the herbaceous stage, resulting from the repression of the key gene VviOMT3, thus drastically reducing IBMP content in ripe berries. Grape berry phenolic compounds contribute to organoleptic properties, color and protection against environmental cues. Our data show that a local warming of developing berries strongly affects the phenylpropanoid metabolic pathway. The effects depend on the stress duration and on the developmental stage (Figure 6; Supplementary Figures 2B, 3). Interestingly, the repression of VviPAL genes that correlated well with the accumulation of its substrate PHE and the deregulation of genes involved in anthocyanin stabilization (VviAOMTs and VviACTs) may contribute to the significant decrease of anthocyanin contents in HT berries at harvest (Table 1). Therefore, the delayed onset of veraison observed for GHT clusters could be considered at the transcriptional level. Additionally, together with the repression of VviFLS1 (flavonol synthase, VIT\_18s0001g03470; also named FLS4, Fujita et al., 2006; Czemmel et al., 2009), the down-regulation of VviUFGT genes (VviGT5 and VviGT6) may contribute to the decrease of the flavonol content in heated berries. This result agrees with a recent work reporting the repression of VviGT5 in detached berries subjected to elevated temperatures (Loyola et al., 2016). Since previous work reported little or no effect on skin flavonol amounts (Spayd et al., 2002), the consequences of a locally applied HT on flavonol content still have to be determined. The transcriptional regulation of many flavonoid structural genes under HT suggests a control through the associated TFs. Since no or weak effect was observed on the expression of the positive TFs identified so far (Supplementary Table 11), several hypothesis can be proposed among which an up-regulation of negative regulators such as VviMYBC2-L3 and VviMYB4b (Supplementary Table 11; Cavallini et al., 2015), and a post-transcriptional control of the flavonoid associated genes as observed for the lightcontrolled stability of MYB1 protein in apple (Li et al., 2012). A relationship between increased anthocyanin catabolism and elevated temperatures was proposed for grape berries (Mori et al., 2007). Although it is poorly known, anthocyanin degradation may involve enzymes such as laccases, polyphenol oxidases, class III peroxidases, and β-glucosidases (Oren-Shamir, 2009). In plants, the role of laccases remains largely unknown but their involvement in different steps of phenylpropanoid metabolism has been suggested. Fang et al. (2015) identified a vacuolar located ADE/LAC protein (anthocyanin degradation enzyme/laccase) responsible for the epicatechin-mediated anthocyanin degradation in litchi fruit during pericarp browning. Likewise, a gene encoding a putative rice laccase plays an important role in responses to abiotic stress (Cho et al., 2014). Our work strengthens the idea that the decrease in berry flavonoid content induced by HT involves an enzymaticmediated degradation of these molecules in the vacuolar compartment. Indeed, laccases, polyphenol oxidase, class III peroxidase, and β-glucosidase genes were deregulated in local HT conditions (Supplementary Table 12). Particularly, the role of vacuolar class III peroxidase in anthocyanin degradation has been highlighted in different plant species including grape (Calderon et al., 1992; Vaknin et al., 2005; Zipor and Oren-Shamir, 2013; Zipor et al., 2015). For instance, a transcriptional up-regulation of VviPrx31 (VIT\_14s0066g01850) was observed in berries exposed to HT (Movahed et al., 2016; and Supplementary Table 1). In Petunia, its overexpression reduced anthocyanin contents in petals exposed to heat. Finally, genes involved in stilbenes and lignins biosynthesis were also specifically altered by HT during berry development. These alterations may modify berry texture and tolerance to the environment. ### Heat Deregulation of Green Berry Transcriptome Can Contribute to Delay veraison During the herbaceous stage, heating significantly affected categories associated with stress and secondary metabolism (as discussed above), as well as categories associated with transport, cell wall, or hormones (Figure 5; Supplementary Table 15). The deregulation of these categories could explain, at least in part, the delayed veraison typified by the late accumulation of sugars and anthocyanins in GHT clusters (Figure 2). #### Membrane Transporters Many genes encoding proteins related to transport processes were enriched in GHT conditions (Supplementary Table 15). These genes were mainly repressed in GHT berries, in agreement with previous reports indicating that HT exposure led to a decrease in micronutrient transport. Genes involved in the calcium homeostasis were also downregulated by applying HT to green berries, and particularly CNGCs genes. CNGCs are nonspecific cation channels that are regulated by cyclic nucleotides such as cAMP or cyclic GMP (Ward et al., 2009) and that contribute to Ca2<sup>+</sup> signaling in the context of developmental processes, biotic and abiotic stress responses (Jha et al., 2016). Calcium may act through facilitating developmental and stress response signaling, stabilizing membranes, influencing water relations and modifying cell wall properties through crosslinking of de-esterified pectins (Hocking et al., 2016). Beside a sum of DEGs potentially linked to calcium homeostasis, the HT also impacted numerous genes related to signaling (protein kinase, transcription factors, Ca2+-binding proteins) (Supplementary Table 1). Potassium transporter gene expression was also altered after GHT exposure. As potassium is required for cell expansion, alteration in the expression of its transporters may affect berry growth. Additionally, as K<sup>+</sup> transporters and channels are known targets of ABA (that play an important role in berry ripening) (Davies et al., 2006), the alteration of their expression during the herbaceous stage may contribute to delay fruit ripening. MATE efflux transporters and ABC transporters were specifically affected by HT. For instance, while members of subfamily B were up-regulated at GHT, MATE, and ABCC and G were down-regulated. Interestingly, ABCG, the most affected subfamily, is involved in fruit maturation and exhibits a protective role (detoxification, vacuolar transport of ABA and glucosyl ester, anion transport) (Andolfo et al., 2015). It is the largest plant ABC transporter subfamily divided in two groups: WBCs and PDRs. The first group is involved in the extrusion of cuticular lipids and the second, in resistance to pathogens, antimicrobial terpenoids and auxinic herbicides, and in transport of signaling molecules or in secretion of volatile compounds (Kretzschmar et al., 2011; Andolfo et al., 2015). The repression of most of these transporters could potentially contribute to decrease anthocyanin accumulation. The down-regulation of sugar transporter genes by HT fits well with earlier reports showing that HT results in the delay or arrest of the ripening process and of the accumulation of sugars (Greer and Weston, 2010; Greer and Weedon, 2013). However, in our experimental conditions, the postponing of the ripening process appears only when HT is directly applied on green clusters (GHT). At later stages, most of these sugar transporters were either down-regulated or not affected by HT (Supplementary Table 15). Altogether, these modifications in the amount of transporter transcripts may affect the berry physiology by modifying the pH and the nutrient content. As a consequence, amounts of certain primary and secondary metabolites were also affected in heated berries, resulting in the delayed onset of veraison. #### Cell Wall Metabolism Genes involved in cell wall composition were strongly altered in green berries during HT, highlighting the importance of cell wall adjustment (Supplementary Table 15). Heating effects on cell wall metabolism were also observed in VHT and RHT clusters. The fine-tuning of the cellulose-hemicellulose networks appears to be crucial for tolerance to heat (Tenhaken, 2014; Le Gall et al., 2015). The present transcriptomic data suggest differential cell wall synthesis and remodeling in heat-exposed berries, potentially affecting the overall fruit growth even if the exact consequences remain to be determined. In vineyard conditions, Dal Santo et al. (2013) found a correlation between the season climate and some differentially expressed genes encoding enzymes involved in cell wall structural modifications (especially CESs, PAEs, and XETs). XETs contribute to cell wall expansion and loosening, through their action on xyloglucan frame. After whole grape exposure to heat, an increased level of XETs transcripts was measured in warmed green berries. It was postulated that enhanced expression could be related to the adaptation of berry volume to temperature and the need for more flexible cell walls (Rienth et al., 2014). Our data reinforce these observations with 19 up-regulated XETs in GHT clusters. Cell wall loosening through increase level of expansin might help to maintain cellular functions during HT (Le Gall et al., 2015). #### Hormone Homeostasis and Signaling Fruit development and responses to environmental cues are controlled by plant hormones (Davies and Böttcher, 2009; Kuhn et al., 2014) and particularly by ABA. The present work revealed that berry heating significantly affects the expression of genes involved in ABA metabolism and signaling (Supplementary Table 9). The deregulation of ABA biosynthesis and signaling by applying HT during the herbaceous stage may contribute to the postponing of veraison and/or help to face HT. Interestingly, NCED expression and particularly VviNCED2 and VviNCED4 was impacted in GHT but also in VHT and RHT clusters. The down regulation of these 2 genes contrasts with data from Carbonell-Bejerano et al. (2013) reporting an HT-induction of these two genes after veraison and an increased ABA level in full ripe heated berries. The differences between both studies might be due to differences in the genotype and/or in the experimental conditions (temperature regime, microenvironment versus Lecourieux et al. Local Heating Impacts Berry Development whole plant exposure, stress kinetic). Genes involved in ABA signaling were also affected. For instance, VviABI3 (VIT\_07s0005g05400) strongly accumulated in GHT berries (Supplementary Table 9). ABI3 is a B3-domain TF that is a part of the core ABA signaling network and the corresponding gene is up-regulated in Cabernet Sauvignon berries treated with ABA (Rattanakon et al., 2016). In normal growth conditions, VviABI3 transcripts accumulated during the lag phase prior veraison in Cabernet Sauvignon (Deluc et al., 2007). Thus, heating seems to accelerate the expression of VviABI3 during the herbaceous stage. In Arabidopsis, ABI3 is exclusively expressed in seeds, controlling gene expression programs that are essential to achieve seed maturation (Baud et al., 2016). Whereas the role of ABI3 in nonseed tissues remains unknown, a functional connection was demonstrated between ABI3 and HSFs in sunflowers embryos and during seed development in Arabidopsis (Rojas et al., 1999; Kotak et al., 2007b). Rattanakon et al. (2016) identified others TFs showing modified expression level after ABA treatment of grape organs, these TFs belonging to the AP2/ERF, NAC, bZIP/ABRE, and MYC/MYB families. In berries exposed to heat, more than 20 transcripts from this set of TFs were deregulated what can be potentially due to ABA action (Supplementary Table 15). The strongest deregulated TF of this list corresponds to the bZIP/ABRE Abscisic Acid Insensitive protein 5 (VviABI5; VIT\_06s0080g00340, VIT\_08s0007g03420) that significantly accumulated in GHT clusters whatever the treatment duration (Supplementary Table 15). ABI5 is depicted as a key regulator in the ABA signaling pathway, and a recent work highlighted its contribution in the ABA-dependent stimulation of plant thermotolerance (Lee et al., 2015). After interaction with the plant-specific RNA-binding protein FCA, ABI5 enhanced antioxidant activity under HT conditions. Particularly, ABI5 promotes the expression of genes encoding antioxidants, including 1-CYSTEINE PEROXIREDOXIN 1 (PER1). In agreement with these data, the heat-induced expression profiles of VviABI5 and VviPER1 (VIT\_05s0020g00600) were closely related in berries, suggesting a conserved mechanism in the fruit (Supplementary Table 1). Peroxiredoxins, which are thiol-based peroxidases, enhance plant tolerance to oxidative and heat stresses. More broadly, in this work, 70 DEGs were identified in heated berries as ROS (reactive oxygen species) scavenging/detoxifying enzymes and various antioxidants (Supplementary Table 1). This kind of adaptative response, described in different plants exposed to HT (de Pinto et al., 2015), may help the berries to manage potential damages due to oxidative burst. Indeed, ROS mainly attributable to NADPH oxidase activity accumulate under stress conditions including HT (Miller et al., 2009). Accordingly, two VviRBOH (respiratory burst oxidase homolog; VIT\_14s0060g02320, VIT\_01s0150g00440) genes were up-regulated in heat-exposed berries (Supplementary Table 1). Besides ABA, our transcriptomic data (Supplementary Table 1) also suggest that HT affects the metabolism of auxin, ethylene and jasmonic acid. These hormones are involved in the control of grape berry development (Davies and Böttcher, 2009) and in the activation of key genes responsible for HT response (Bokszczanin and Fragkostefanakis, 2013; Sharma and Laxmi, 2015). For instance, indole-3-acetic acid (IAA) inhibits berry growth, sugar and anthocyanin accumulation (Kuhn et al., 2014) and prevents ripening. Indeed, the decrease in IAA content and the increase of its conjugated form are needed to induce ripening (Davies et al., 1997; Böttcher et al., 2010). Interestingly, our work showed that application of a HT to green berries resulted in the reduction of IAA-amido synthetase (VviGH3, VIT\_07s0005g00090) transcript abundance and in an increase in the expression of two transcripts encoding IAA-amino acid hydrolases (VIT\_11s0016g02700, VIT\_08s0007g02740), thus suggesting that heating is disrupting IAA conjugating process in GHT clusters and therefore postponing the onset of ripening. Further work is needed to address the respective role of each hormone and their interactions in the context of grape berry grown under HT. # CONCLUSIONS This work provides the first molecular data describing the effect of HT at the microclimate level and brings important information on the consequences of temperature elevation in the context of leaf removal practice. HT effects depend both on the developmental stage and on the stress duration. Heating delayed the onset of veraison and strongly altered the berry biochemical composition at harvest. These physiological modifications could be partly explained by the deep remodeling of heated berry transcriptome. The intrinsic capacity of grape berries to perceive heat stress and to build adaptive responses is suggested by the deregulation of categories such as "stress responses," "protein metabolism" and "secondary metabolism." Additionally, important changes in processes related to "transport," "hormone" and "cell wall" might contribute to the postponing of veraison. Furthermore, opposite effects depending on heat duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HT-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation. However, one cannot exclude that this process could also be regulated at the protein level as HT strongly affects protein homeostasis related genes. Finally, the data reported here provide a rich transcriptomic resource for functional characterization of the genes that potentially control HT response and/or adaptation in grapevine. The functional characterization of some putative candidates is in progress. # AUTHOR CONTRIBUTIONS DL and PP designed the research; DL oversaw the research; JP, PP, and DL performed the greenhouse experiments; FL, JC, and DL carried out the RNA extraction for transcriptomic analysis and performed RT-QPCR; CK performed the bioinformatics analysis; JC, JP, GH, and CR did the metabolic analysis; FL, CK, and DL analyzed and interpreted the data; FL, CK, SD, and DL drafted the manuscript; PP and EG critically revised the manuscript. All authors read and approved the final manuscript. #### FUNDING This research received funding from the Agence Nationale de la Recherche for the project "DURAVITIS" (grant no. ANR-2010- GENM-004-01). #### ACKNOWLEDGMENTS The authors would like to thank Pr. Laurent Torregrosa, Dr. Charles Romieu, Dr. Markus Rienth, Dr. ZhanWu Dai and Pr. Grant Cramer for valuable discussions and scientific #### REFERENCES interactions. The authors also thank Dr. Pablo Carbonell Bejerano for critical reading of the manuscript. For the production of the fruiting cuttings and technical assistance during greenhouse experiments, we express our gratitude to Jean Pierre Petit and Guillaume Pacreau. ## SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00053/full#supplementary-material anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiol. 160, 1011–1022. doi: 10.1104/pp.112.199703 in response to several types of environmental stress. Plant J. 48, 535–547. doi: 10.1111/j.1365-313X.2006.02889.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 Lecourieux, Kappel, Pieri, Charon, Pillet, Hilbert, Renaud, Gomès, Delrot and Lecourieux. 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. # Whole Plant Temperature Manipulation Affects Flavonoid Metabolism and the Transcriptome of Grapevine Berries Chiara Pastore<sup>1</sup>† , Silvia Dal Santo<sup>2</sup>† , Sara Zenoni<sup>2</sup> , Nushin Movahed<sup>1</sup> , Gianluca Allegro<sup>1</sup> , Gabriele Valentini<sup>1</sup> , Ilaria Filippetti<sup>1</sup> \* and Giovanni Battista Tornielli<sup>2</sup> <sup>1</sup> Department of Agricultural Sciences, University of Bologna, Bologna, Italy, <sup>2</sup> Department of Biotechnology, University of Verona, Verona, Italy #### Edited by: Ashraf El-kereamy, University of California, United States #### Reviewed by: Ramesh Katam, Florida A&M University, United States David Lecourieux, University of Bordeaux 1, France Maria Carmen Gomez-Jimenez, University of Extremadura, Spain > \Correspondence: Ilaria Filippetti [email protected] †These authors have contributed equally to this work. #### Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science > Received: 24 February 2017 Accepted: 17 May 2017 Published: 06 June 2017 #### Citation: Pastore C, Dal Santo S, Zenoni S, Movahed N, Allegro G, Valentini G, Filippetti I and Tornielli GB (2017) Whole Plant Temperature Manipulation Affects Flavonoid Metabolism and the Transcriptome of Grapevine Berries. Front. Plant Sci. 8:929. doi: 10.3389/fpls.2017.00929 Among environmental factors, temperature is the one that poses serious threats to viticulture in the present and future scenarios of global climate change. In this work, we evaluated the effects on berry ripening of two thermal regimes, imposed from veraison to harvest. Potted vines were grown in two air-conditioned greenhouses with High Temperature (HT) and Low Temperature (LT) regimes characterized by 26 and 21◦C as average and 42 and 35◦C as maximum air daily temperature, respectively. We conducted analyses of the main berry compositional parameters, berry skin flavonoids and berry skin transcriptome on HT and LT berries sampled during ripening. The two thermal conditions strongly differentiated the berries. HT regime increased sugar accumulation at the beginning of ripening, but not at harvest, when HT treatment contributed to a slight total acidity reduction and pH increase. Conversely, growing temperatures greatly impacted on anthocyanin and flavonol concentrations, which resulted as strongly reduced, while no effects were found on skin tannins accumulation. Berry transcriptome was analyzed with several approaches in order to identify genes with different expression profile in berries ripened under HT or LT conditions. The analysis of whole transcriptome showed that the main differences emerging from this approach appeared to be more due to a shift in the ripening process, rather than to a strong rearrangement at transcriptional level, revealing that the LT temperature regime could delay berry ripening, at least in the early stages. Moreover, the results of the in-depth screening of genes differentially expressed in HT and LT did not highlight differences in the expression of transcripts involved in the biosynthesis of flavonoids (with the exception of PAL and STS) despite the enzymatic activities of PALs and UFGT being significantly higher in LT than HT. This suggests only a partial correlation between molecular and biochemical data in our conditions and the putative existence of post-transcriptional and post-translational mechanisms playing significant roles in the regulation of flavonoid metabolic pathways and in particular of anthocyanins. Keywords: grape, ripening, flavonoid, transcriptome, temperature # INTRODUCTION fpls-08-00929 June 1, 2017 Time: 19:43 # 2 Agriculture, and in particular viticulture, is highly dependent upon climatic conditions during the growing season. The predicted climate change therefore presents a major challenge for wine production. Although Vitis vinifera shows large variations in terms of tolerance to abiotic summer stresses, i.e., high temperature (HT) and radiation and low water availability (Palliotti and Poni, 2016), the effects of a temperature increase on berry composition have been widely studied, in terms of both extreme heatwaves or mild-to-moderate increase during ripening. Since berry temperature and solar radiation often act synergistically and sun exposure of grape bunches can be modified by viticulture practices, several researches have focused on the effect of both parameters simultaneously (Bergqvist et al., 2001; Spayd et al., 2002; Downey et al., 2004; Cortell and Kennedy, 2006; Tarara et al., 2008; Azuma et al., 2012; Movahed et al., 2016) and only a few computed the precise role of temperature in these multi-factor studies (Mori et al., 2005, 2007; Yamane et al., 2006; Cohen et al., 2012; Sadras et al., 2013a,b; Rienth et al., 2016). High sugar concentration at harvest is often associated with thermal increase, as indicated by the trend observed in the last decades (Petrie and Sadras, 2008; Mira de Orduña, 2010; Sadras and Petrie, 2011). However, several experiments showed that sugar accumulation is not or only slightly affected (Spayd et al., 2002; Mori et al., 2005; Mori et al., 2007; Sadras and Denison, 2009; Movahed et al., 2016), or sometimes even reduced (Greer and Weston, 2010; Greer et al., 2010; Carbonell-Bejerano et al., 2014; Rienth et al., 2016) by air temperature increase. The different results can probably be ascribed to variation in diurnal temperature levels, since temperatures over 30◦C may lead to the stopping of soluble solids transport from leaves to berry, but may sometimes indirectly cause a higher concentration by evaporative loss (Keller, 2010). Temperature has been known for some time to have significant effects on berry acidity, accelerating the breakdown of malic acid (Rienth et al., 2016) and decreasing the titratable acidity the greater the heat summation (Tarara et al., 2008). Intriguingly, other studies suggested a cultivar-dependent thermal response of acidity and pH (Bergqvist et al., 2001; Sadras et al., 2013a,b; Movahed et al., 2016). Of particular interest are the effects of temperature on the phenylpropanoid biosynthetic pathway, involved in the biosynthesis of flavonoids (anthocyanins, flavonols, and tannins) that play a crucial role in grape and wine composition with regards to color, bitterness and stability, and also in the biosynthesis of non-flavonoid compounds (i.e., stilbenes). A negative correlation between elevated temperature during the day (over 30◦C) and anthocyanin concentrations has recently been explored (Mori et al., 2007; Carbonell-Bejerano et al., 2013; Movahed et al., 2016). Some authors pointed out the effect of increasing temperatures on the reduction of the enzymatic activity of some key enzymes involved in flavonoid biosynthesis as phenylalanine ammonia-lyase (PAL), which presides the first step of general phenylpropanoid biosynthesis, and UDPglucose:flavonoid 3-O-glucosyltransferase (UFGT), which is involved in the last and specific step of anthocyanin biosynthesis (Mori et al., 2007; Movahed et al., 2016). Flavonols are known to behave as UV-protectants and to play a role in co-pigmentation with anthocyanins. Flavonols in the berry can be affected by sunlight exposure, which usually promotes strong enhancement in concentrations and in the expression of flavonol biosynthesis-related genes (Spayd et al., 2002; Downey et al., 2004; Czemmel et al., 2009). On the contrary, temperature seems to have less effect than light in flavonol synthesis control and under thermal increase flavonols can be unaffected (Tarara et al., 2008) or slightly reduced (Azuma et al., 2012). Temperature appears to have little impact on tannins (Cohen et al., 2012) whose accumulation in skins and seeds occurs predominantly before veraison (Downey et al., 2004). Grapevine transcriptomic analysis has provided a wealth of data concerning the mechanisms responsible for the temperature effects on berry composition, especially on sugars, acidity and anthocyanin concentrations (Carbonell-Bejerano et al., 2013; Pastore et al., 2013; Rienth et al., 2014, 2016; Lecourieux et al., 2017). Several authors reported that the loss of anthocyanin synthesis following HT is due to the reduced expression of anthocyanin biosynthetic genes (Yamane et al., 2006; Azuma et al., 2012; Lecourieux et al., 2017). Sometimes, however, despite a sharp reduction in terms of anthocyanin concentration, a concomitant reduction in anthocyanin biosynthetic genes expression was not found, as reported for Muscat Hamburg berries on fruiting cuttings (Carbonell-Bejerano et al., 2013) and in Cabernet Sauvignon and Sangiovese vines grown under increasing temperature (Mori et al., 2007; Movahed et al., 2016). In these cases, also an involvement of anthocyanin degradation, implying the action of peroxidases should be hypothesized (Mori et al., 2007; Movahed et al., 2016) as it was previously seen in other plant species, as Brunfelsia flower petals (Vaknin et al., 2005), litchi (Zhang et al., 2005), and strawberry fruits (Chisari et al., 2007). Despite recent progress, the direct and indirect effects of temperature on the grape ripening process, and specifically on flavonoid composition, are far from being completely unraveled. In particular, there is growing interest in charting the impact of temperature in specific viticultural areas and different seasons on flavonoid composition. Here, we analyzed the grapevine cultivar Sangiovese, the most cultivated Italian variety, comparing the effects of two thermal regimes on the berry skin biochemical composition, flavonoid-related enzymatic activity, and whole transcriptome during ripening. # MATERIALS AND METHODS # Grapevine Plant Material and Growing Conditions Experiments on grapevine berries were conducted in 2012 on 6-year-old uniformly potted plants (V. vinifera cv. Sangiovese). The vines, grafted on SO4 rootstocks, were grown in 30-liter pots containing a 1:1 mixture of sand and soil (27% sand, 46% silt and 27% clay, clay loam soil). The number of shoots was standardized to nine per vine. In addition, to achieve a uniform leaf area on all the vines, the tip of each shoot was removed and 15 main leaves were maintained before the experiment started. At the beginning of bunch closure [BBCH 77, (Lorenz et al., 1995)], 10 vines were selected and bunch numbers were adjusted to 11–12 per vine. The vines were assigned to two treatments: low temperature (LT) and HT. Five LT vines were placed from 1 week before veraison to harvest in a plastic greenhouse (20 m<sup>3</sup> ) where the air temperature was controlled by a cooler and a fan was used to homogenize environmental conditions in the greenhouse (Supplementary Figure 1). During the night, the tunnel was opened. Five HT vines were placed in an identical plastic greenhouse, without fan, whose basal segment was open but all the canopies were covered to maintain similar illumination to the LT vines. The average, maximum and minimum air temperatures were recorded using air temperature sensors (TL20, 3M, Milan, Italy) in both greenhouses during the ripening period (Table 1). Both greenhouses were made of polyethylene film (MOP, Bologna, Italy) that did not alter the spectral composition of light. The incident light during the day, which outside ranged from 500 to 2000 µmol·m<sup>2</sup> ·s −1 , was reduced by the polyethylene film up to 12% within the visible range. The humidity recorded during the experiment was comparable between LT and HT greenhouses. All vines were automatically watered daily and were well supplied with nutrients. #### Berry Temperature Monitoring Berry temperature was monitored in 10 bunches from each treatment using 10 T-type thermocouples (RS component, Milan, Italy) positioned in the sub-cuticular tissues of the berry skin. Each probe was then connected to a CR10X data logger (Campbell Scientific Ltd, Leicestershire, United Kingdom), registering temperature data every 20 min during the development period. #### Berries Sampling Berries were sampled before the treatment (T0, 1 week before veraison), at veraison (T1) and 10 (T2), 20 (T3), 32 (T4), and 45 (T5) days after veraison, corresponding to harvest. The berries were collected at the same time of day (9–10 am). At T0 five berries from each of the ten vines were sampled and pooled and this procedure was repeated 4 times to create four independent biological replicates of 50 berries each. Upon thermal treatment imposition, nine berries were randomly selected from each of the five treated vines and pooled. The same sampling procedure was repeated four times to create four independent pools of 45 Average, maximum, and minimum air temperature recorded during the experiment under the two different growing regimes. LT = low temperature, HT = high temperature. berries per each sampling date/treatment combination. In total, the experiment entailed the collection and the analysis of 44 berry samples [4 control samples + (2 thermal regimes × 5 stages × 4 biological replicates)]. From each biological replicate about 20 berries were weighed and directly tested for the evaluation of soluble solids (◦Brix), titratable acidity and pH. The remaining berries were peeled and the skins were immediately frozen in liquid nitrogen and stored at −80◦C for subsequent metabolic analyses, enzyme activity and expression analysis. ## Soluble Solids, Titratable Acidity, and pH Measurements The sampled berries were crushed and the must was sieved and used for soluble solids analysis with a temperature-compensating CR50 refractometer (Maselli Misure Spa, Parma, Italy). We then diluted 5 ml of the same must seven times with bi-distilled water for titration using a Crison Compact Titrator (Crison, Barcelona, Spain) with 1 N, 0.5 N or 0.25 N NaOH (Sigma-Aldrich, St. Louis, MO, United States), according to the stage of berry ripening to obtain pH and titratable acidity data (expressed as g L−<sup>1</sup> of tartaric acid equivalents). #### Analysis of Grape Berry Anthocyanins and Flavonols Total anthocyanins and flavonols were analyzed in all 44 samples by soaking 2–3 grams of peeled skins, depending on the berry phenological stage, per each sampling date/treatment combination in 50 mL methanol for 24 h (Mattivi et al., 2006), then storing the extracts at −20◦C. To analyze the total concentrations of each flavonol aglycone, an aliquote of 5 ml of methanolic extract was completely dried under vacuum. To achieved the acid hydrolization of flavonol glucosides, the pellet was resuspended in 2.5 ml of methanol and 2.5 ml of 2M trifluoroacetic acid (Sigma–Aldric, Saint Louis, MO, United States) in milliQ water. The reaction was conducted at 100◦C in a boiling hot water bath, with a condenser, for 2 h. The reactions product was then completely dried under vacuum and the pellet obtained resuspended in 1 ml of methanol until HPLC analyses (Mattivi et al., 2006). HPLC separation and quantifications of anthocyanins and flavonols (Mattivi et al., 2006) were performed on a Waters 1525 HPLC (Waters, Milford, MA, United States) equipped with a diode array detector (DAD) and a Phenomenex (Castel Maggiore, Bologna, Italy) reversed-phase column (RP18, 250 mm × 4 mm, 5 µM). Anthocyanins were quantified at 520 nm using an external calibration curve with malvidin-3-glucoside chloride as the standard (Sigma-Aldrich). Flavonols were quantified at 370 nm with the corresponding external standards (myricetin, quercetin, and kaempferol) purchased from Extrasynthese (Genay, France). #### Analysis of Skin Berry Tannins Skin tannins extraction was performed following the procedure proposed by Downey et al. (2003): About 100 mg of skins per each sampling date/treatment combination were ground to a fine powder separately, extracted with a solution containing 70% acetone for 24 h in dark room and measured by HPLC using the same equipment used for anthocyanins analysis. After free monomers were removed, the tannin content was determined by acid-catalyzed cleavage in the presence of excess phloroglucinol as described by Kennedy and Jones (2001). Individual reversedphase HPLC separations were used to determine the abundance of free monomers and cleaved proanthocyanidins by measuring absorbance at 280 nm (Downey et al., 2003). The concentrations of free monomers and hydrolyzed terminal subunits were determined from standard curves prepared with commercial standards of catechin, epicatechin, epicatechin-gallate and epigallocatechin (Extrasynthese, France). #### Enzymatic Activity Assays Berry skins (0.2 gr) were ground with a mortar and pestle in liquid nitrogen to a fine powder. For PAL and UFGT activity assays, the protein extraction was performed according to the methods of Mori et al. (2005, 2007), respectively. Peroxidase activity was instead measured on berry skin after protein extractions as described by Ushimaru et al. (1997). For PAL activity measurement, the reaction mixture consisted of 0.5 ml of phenylalanine and 0.5 ml of protein extract. The assay mixture was incubated at 37◦C for 60 min. The reaction was terminated by adding 0.5 ml of HCl acid (18%). The quantity of the product, trans-cinnamic acid, was calculated using its extinction coefficient of 9630 M−<sup>1</sup> cm−<sup>1</sup> at 290 nm. One unit (U) of PAL activity, expressed on berry skin fresh weight, was defined as the production of 1 mol of trans-cinnamic acid per minute. The UFGT assay was performed on the protein extract using either cyanidin or delphinidin as substrate in 200 mM Tris-HCl (pH 7.5), containing 0.1 mM cyanidin or delphinidin and 10 mM UDP-glucose. After incubation for 5 min at 37◦C, the reaction was stopped by adding 150 µl 5% HCl. The concentration of cyanidin-3-glucoside and delphinidin-3-glucoside was calculated at 520 nm and pH 1, using extinction coefficients of 26,900 M−<sup>1</sup> cm−<sup>1</sup> and 26,000 M−<sup>1</sup> cm−<sup>1</sup> , respectively. One unit (U) of UFGT activity, expressed on berry skin fresh weight, was defined as the production of 1 mol of cyanidin-3-glucoside or delphinidin-3-glucoside per second. Guaiacol peroxidase activity was determined in the ripening berry skin as described by Ushimaru et al. (1997), using pyrogallol as the electron donor. The reaction mixture comprised the protein extract in 50 mM sodium phosphate buffer (pH 7.0), 0.1 mM H2O<sup>2</sup> and 50 mM pyrogallol (H2O<sup>2</sup> and pyrogallol were freshly prepared just before use). The absorbance at 430 nm was recorded immediately after the addition of pyrogallol and after 7 min at room temperature, and was compared to a blank with no protein extract added. One unit (U) of peroxidase activity, expressed on berry skin fresh weight, was defined as the amount of enzyme that catalyzes the oxidation of 1 µmol of pyrogallol per minute. #### Statistical Analyses The experiment had a completely randomized design and the agronomic parameters and biochemical data were submitted to analysis of variance (ANOVA) using SAS statistical software (SAS Institute, Cary, NC, United States) with four replications for each treatment. The temperature data were analyzed with 10 replications for each treatment. # RNA Extraction and Microarray Analyses Total RNA was isolated from approximately 400 mg of pulverized berry skins from three biological replicates sampled at all dates except harvest, for a total of 27 berry samples [3 control samples + (2 thermal regimes × 4 stages × 3 biological replicates)], using the Spectrum Plant Total RNA kit (Sigma–Aldrich), with modifications as described in Dal Santo et al. (2016). RNA quality and quantity were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, United States) and a Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, CA, United States). We hybridized 5 µg of total RNA per sample to a NimbleGen microarray 090818\_Vitus\_exp\_HX12 chip (Roche, NimbleGen Inc., Madison, WI, United States), according to the manufacturer's instructions (Dal Santo et al., 2013). Statistical analysis of the microarray data was conducted using TMeV v4.8<sup>1</sup> . Statistical analysis of microarrays (SAM) was performed with a false discovery rate (FDR) of 0.1% and ANOVA using α = 0.05 and standard Bonferroni correction. Heat maps were created using log2-transformed expression values and then median-centered by transcript. Cluster analysis was performed by the k-means method (KMC) with Pearson's correlation distance. Principal component analysis (PCA) was conducted using SIMCA P+ v13 (Umetrics, United States). Gene Ontology (GO) annotation was applied using the BiNGO v2.3 plug-in tool in Cytoscape v2.6 with PlantGOslim categories, as described by Maere et al. (2005). Overrepresented PlantGOslim categories were identified using a hypergeometric test with a significance threshold of 0.05. STEM v1.3.8 was used for clustering, comparing and visualizing gene expression data (Ernst et al., 2005). ### Reverse Transcription (RT) and Real Time qPCR One microgram of extracted RNA was treated with 2 units (U) of Turbo DNase (TURBO DNA-free kit—Ambion) according to the instructions provided with the commercial kit. DNase- treated RNA was then used for cDNA synthesis using the SuperScriptIII Reverse Transcriptase kit (Invitrogen) following the producer's indications. In order to assess if the cDNA had been properly produced, an amplification with primers designed on the 30UTR of an actin coding gene (VIT\_12s0178g0020, (Pastore et al., 2011) was performed. Real Time qPCR was performed using GoTaq <sup>R</sup> GreenMaster Mix kit (Promega) to amplify a specific region of target genes (UFGT, VvUFGT – VIT\_16s0039g02230; PAL – VIT\_00s2849g00010 and the peroxidase VvPrx31 – VIT\_14s0066g01850) with previously described primer pairs (Movahed et al., 2016). Primers and cDNA were mixed with the Power SYBR <sup>R</sup> Green PCR Master Mix (Applied Biosystems, Foster City, CA, United States) and the reaction was carried out on an ABI PRISM StepOne Sequence Detection System (Applied Biosystems, Foster City, CA, United States) using the following cycling conditions: 95◦C hold for 10 min followed by 45 cycles <sup>1</sup>mev.tm4.org/ at 95◦C for 30 s, 55◦C for 30 s and 72◦C for 20 s. 95◦C hold for 2 min followed by 40 cycles at 95◦C for 15 s, 55◦C for 30 s, 60◦C for 30 s, and 95◦C for 15 s. Non-specific PCR products were identified by the dissociation curves. Amplification efficiency was calculated from raw data using LingReg PCR software (Ramakers et al., 2003). The mean normalized expression (MNE)-value was calculated for each sample referred to the ubiquitin expression according to the Simon equation (Simon, 2003). Standard error (SE) values were calculated according to Pfaffl et al. (2002). #### Accession Numbers Grape berry microarray expression data are available in the Gene Expression Omnibus under the series entry GSE92864<sup>2</sup> . #### RESULTS ### Two Different Thermal Regimes Differently Affect Ripening Parameters We set up an experimental design to impose two different thermal regimes in potted grapevine plants over the course of grape ripening. Five vines were placed in a plastic greenhouse where the air temperature was artificially cooled whereas other five vines were placed in an identical plastic greenhouse with only the basal segment open. These two conditions were named LT and HT, respectively, with HT representing the closest condition to the 2012 thermal regime. HT and LT berry temperature were strongly differentiated in the two greenhouses (Table 2* and Figure 1). Indeed, the average berry temperature during the treatment period was ∼21.8◦C in the LT greenhouse and 26.5◦C in the HT greenhouse (Table 2 and Figure 1A). Maximum temperatures were higher in HT during ripening, with several heatwaves, sometimes reaching values of around 40◦C in HT berries, corresponding to 7–8◦C higher than the maximum temperatures detected in LT berries (Figure 1B). Overall, HT berries accumulated an additional 238 Degree Days (DD) compared to LT berries (Table 2). The number of hours with berry temperature exceeding 30◦C was four-fold greater in the HT greenhouse, while berry temperature exceeding 35◦C was registered only in the HT greenhouse (Table 2 and Figure 1B). <sup>2</sup>http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE92864 TABLE 2 \| Average Berry Temperature, accumulated Degree Days (DDs), and heatwaves hitting the berries (calculated as the number of hours with average berry temperature > 30◦C and > 35◦C) under the two different growing regimes. Values represent means of ten replicates. LT = low temperature, HT = high temperature. Asterisks indicate significant differences between HT and LT using ANOVA (∗P < 0.05). On the contrary, the minimum temperatures in HT and LT were aligned (Figure 1B). The LT thermal regime slightly delayed technological ripening in berries, which showed a lower level of ◦Brix and pH and higher values of titratable acidity compared to HT. However, at harvest, the soluble solids reached comparable values in HT and LT berries (Figure 2). Until 10 days after veraison, there was no detectable difference in total anthocyanin concentration between the two thermal regimes (Figure 3A). Starting from 10 days after veraison, total anthocyanin accumulation rate began to accelerate in the skin of LT berries compared to HT. This different behavior was maintained at harvest, when the concentration of total anthocyanins in the skin of LT grapes was almost doubled compared to HT (Figure 3A). The most abundant individual anthocyanin in Sangiovese berry skin was malvidin-3-glucoside, in both LT and HT treatments (Figure 3A). However, its percentage was significantly lower in HT than LT (Figure 3A). This decrease of malvidin-3-glucoside was counterbalanced in HT by an increase in the percentage of petunidin-3-glucoside, delfinidin-3-glucoside, and especially of cyanidin-3-glucoside, albeit non-significant. The accumulation trend of flavonols was similar to that of anthocyanins in both HT and LT. At harvest, flavonol concentration in LT berry skin was thus three-times of that found in HT berry skin (Figure 3B). The analyses of the single flavonol compounds at harvest showed a higher quercetin and lower myricetin percentage in LT berries compared to HT (Figure 3B). Kaempferol was instead unaffected by different growing temperature regimes (Figure 3B). Berry skin tannins decreased during ripening without significant differences between HT and LT in any developmental stage analyzed. Likewise, regardless of thermal regime, the percentage, measured at harvest, of the four individual flavan-3-ol monomers catechin, epicatechin, epicatechin-gallate and epigallocatechin did not differ between HT and LT berries, epicatechin being the tannin compound present in the greatest concentration in both treatments (Figure 3C). Our results suggest a different effect of LT and HT thermal regimes, to a less extent, on technological berry ripening and, more strongly, on flavonoid increase, with the enhancement of anthocyanin and flavonol accumulations under LT conditions. #### The Effect of Temperature on Berry Skins Whole Transcriptome The transcriptome of Sangiovese berry skins under LT and HT growing temperature regimes was assessed at five sampling times (i.e., before the treatments (T0), at veraison (T1) and 10 (T2), 20 (T3) and 32 (T4) days after veraison). The dataset was initially screened by Significance Analysis of Microarrays (SAM, 9 groups, FDR = 0.1%) to select genes that were differentially modulated under our experimental conditions. Analysis of Variance (ANOVA, 9 groups, α = 0.05, standard Bonferroni correction) was applied to transcripts positive in the SAM in order to retrieve the most significantly modulated transcripts (6441 genes, Supplementary File 1). To verify the uniformity of biological replicates and investigate the transcriptomes of LT and HT berries, we performed a PCA, obtaining a significant model (8 PCs, R2X = 0.941, Q2 (cum) = 0.848, Figure 4A). PC1 explained ∼60% of the total dataset variability and mostly reflected differences among the four sampling dates (Figure 4A and Supplementary Figure 2), suggesting a slight delay in LT berry ripening at stages 3 and 4 in comparison to berries ripened under HT conditions. PC2 accounted for 13.3% of the total variability and mainly described differences between first and last sampling times, and intermediate ones (Supplementary Figure 2). Notably, PC3, explaining ∼8% of total dataset variability reflected differences among T0, LT and HT samples (Figure 4A). Indeed, the expression profiles of the first and last percentile PC3 loadings showed more expression in LT and HT samples, respectively (Figures 4B,C). The GO enrichment analysis revealed that the positive PC3 loadings were significantly enriched in the functional categories Cellular process, Biosynthetic process and Secondary metabolic process (Figure 4E). In particular, the first percentile PC3 loadings, more expressed in LT, comprised several genes involved in the metabolism of phenylpropanoids (5 Stilbene Synthase, STS, and 4 PAL), in the plant response to abiotic and biotic stresses (several ascorbate oxidases and glutaredoxines as well as many R proteins), in carbohydrate metabolism (one alcohol dehydrogenase and one trehalose-6 phosphate phosphatase). The transcript of ethylene response factor VvERF075, which belongs to the AP2/ERF superfamily and is usually upregulated in berry skin during ripening (Licausi et al., 2010), was also found more abundant in LT. Furthermore, we found among the positive PC loadings the transcription factor VvNAC60 already described as a putative master regulator of the transition between unripe and ripe red berries (Palumbo et al., 2014). No significant GO enrichment could be found in the negative loadings of the PC3, more expressed under HT regime (Figure 4D). The last percentile of the PC3 loadings showed higher level of transcript under HT regime of several cell-wall related transcripts (including two cellulose synthases, a pectinacetylesterase and a xyloglucan endotransglucosylase/hydrolase), of regulatory and structural genes involved in cytokinin metabolism (two isopentenyltransferases) and in ethylene metabolism (the 1-aminocyclopropane-1-carboxylate oxidase), in protein degradation and biosynthesis (including several proteases and amino acid transporters) and in the metabolism of carbohydrates and lipids (including an aldose 1-epimerase and a desaturase). Two genes involved in flavonoid biosynthesis and regulation, the dihydroflavonol-4-reductase and the transcription factor VvMybPA1, previously reported as a regulator of proanthocyanidin biosynthesis (Bogs LT at the same date using ANOVA (∗P < 0.05). ns, not significant. et al., 2007) were also more expressed in HT compared to LT. In summary, the comparison of berry skin transcriptomes highlighted the effect of the temperature on gene expression during fruit ripening. In particular, LT condition was characterized by a higher expression of transcripts associated with the metabolism of phenylpropanoids. ### Gene Expression Profiles Inspection Highlights a Strong Effect of Temperature on Early Phenylpropanoid Pathway We focused on changes in expression profiles of genes scoring an absolute value of fold change \|FC\| ≥ 2, identifying 2,257 of genes positively (right) and negatively (left) correlated to the third principal component were selected within the first (positive) and last (negative) percentile of the third component loadings. Enriched GO terms for the genes negatively (D) and positively (E) correlated to the PC3. The network graphs show BiNGO visualizations of the overrepresented GO terms. Categories in GoSlimPlants (Maere et al., 2005) were used to simplify this analysis. Colored nodes represent GO terms that are significantly overrepresented (p < 0.05). T0 = plants before treatment, LT = low temperature, HT = high temperature. Sample names are composed by temperature treatment abbreviation followed by the indication of the developmental stage (1, 2, 3, or 4), and by the description of the biological replicate (A–C). Gray, blue, and purple indicate samples of control, LT-treated and HT-treated, respectively. annotated genes (Supplementary File 2). Differences in the timing of activation/repression of the differentially expressed genes during ripening in HT and LT was investigated in more detail by applying the short time-series expression miner (STEM) clustering method (Ernst et al., 2005) to the 2,257 identified genes (Supplementary File 3). Figure 5 reports the most significant results of the STEM approach. Some of the transcriptional trends changes were also confirmed by Real Time qPCR analysis (Supplementary Figure 3). Notably, many PAL and STS genes resulted as much more expressed in LT berry skins after veraison (Figure 5A), whereas they showed a slower rate of activation in HT berry skins (HT-2 → LT-10 in STEM analysis – Supplementary File 3), corroborating the PCA results (Figure 4A). Interestingly, a similar trend of expression was also detected for a protondependent oligopeptide transporter (POT), which contains a PTR2 domain that characterizes both nitrate and peptide transporters (Widhalm et al., 2015). We found transcripts showing a peak of expression 10 days after veraison in HT, while their expression generally increased during the whole ripening period in LT (Figure 5B; HT-23 → LT-22 in STEM analysis – Supplementary File 3). Some genes involved in anthocyanin biosynthesis and transport (VvUFGT and VvGST4) and other phenylpropanoid/flavonoid related genes (one PAL, VvCHS3 and VvF3H1), were found to belong to this group, together with transcripts involved in hormone metabolism, such as one jasmonate O-methyltransferase and one gibberellin 20 oxidase, and with one gene that showed high homology with a cold-induced wall associated kinase (Cao et al., 2009). We instead found that in HT berry skins, starting from 10 days after veraison, there was an earlier activation (HT-22 → LT-14 in STEM analysis – Supplementary File 3) of genes involved in the biosynthesis of volatile aromas [the terpene synthases VvTPS25 and VvTPS26, (Martin et al., 2010)], in cell wall metabolism and in DNA metabolism (Figure 5C). The same trend was shared by one peroxidase transcript (peroxidase 50). Several genes that were downregulated in both HT and LT berries during ripening showed a different expression profile, i.e., a rapid and progressive decrease of expression starting from veraison in HT berries, and a much slower decrease in LT berries (Figure 5D; HT-4 → LT-12 in STEM analysis – Supplementary File 3). Cell wall-related transcripts, including genes linked to chlorophyll degradation and response to biotic stresses showed this trend. VvPrx31, a gene coding for a peroxidase putatively associated with anthocyanin degradation (Movahed et al., 2016) was also found in this group. In order to point out the genes differentially expressed between LT and HT, we decided to consider just genes with a fluorescence expression threshold value ≥ 100 and transcripts in which the \|FC\| between LT and HT was ≥ 2 in at least one stage of development. By this approach, we obtained 417 differentially expressed genes (Supplementary File 4) that were over-represented in the GO functional categories of Secondary metabolic process, Generation of precursor metabolites and energy, Response to biotic stimulus, and Biosynthetic process (Supplementary Figure 4). These genes were grouped into five different FC clusters by K-means clustering (KMC) analysis highlighting the times with the greatest difference in gene expression between LT and HT (Figure 6). At a glance, the genes in each cluster showed a peak of expression at a given stage in LT. In the other ripening stages, expression of the same genes may be higher in HT, however, the FC of the LT/HT ratio at the peaking stage was generally much higher compared to the HT/LT values in the other stages. The most represented cluster included genes with high FC between LT and HT at 10 days after veraison (Figure 6B). The remaining differentially expressed genes were almost equally divided in the other four clusters (Figure 6). In the first cluster (Figure 6A), collecting genes more promptly activated by the LT regime at veraison (stage 1), we found a glycerol-3-phosphate acyltransferase 3 (AtGPAT3) to be the most differentially expressed gene (Supplementary File 4). Furthermore, several ERF/AP2 transcription factors (Licausi et al., 2010) were among the most differentially expressed genes belonging to this cluster (Figure 6A and Supplementary File 4). STSs were the most differentially expressed genes in LT regime at 10 and 20 days after veraison (Figures 6B,C), confirming the strong involvement of such genes in association with LT regime, as previously observed with the STEM approach (Figures 4C, 5A). In these clusters, we also found genes involved in the response to biotic stimuli, such as wound induced proteins, beta 1–3 glucanase, and pathogenesis-related proteins, indicating a general activation of defense mechanisms under LT conditions. The cluster in Figure 6D is characterized by high FC in LT/HT 20 days after veraison (Supplementary File 4). Interestingly, this cluster included three ABC transporters and three isoforms of LRR receptor kinase CLAVATA1 (CLV1), which seem to be able to confer resistance to various abiotic stresses (Grzeskowiak et al., 2013). Lastly, several genes involved in volatile compounds synthesis were present in the last cluster, which groups genes showing a peak of expression in LT at 32 days after veraison (Figure 6E and Supplementary File 4). Two galactinol synthases, were also included in this cluster. Overall, using this FC clustering approach we were able to validate expression of genes related to phenylpropanoid biosynthesis identified by STEM analysis, but also to retrieve other genes involved in the metabolism of volatile compounds, lipids and hormones that showed high sensitivity to thermal changes. #### Temperature Affects the Activity of Enzymes Involved in Anthocyanin Metabolism In LT treatment, growing temperatures positively impacted on the anthocyanins accumulation compared to HT (Figure 3A). However, such an accumulation was not fully explained by differences in transcription of genes related to phenylpropanoid/flavonoid metabolism. We therefore evaluated the enzymatic activity of PAL, the key enzyme of phenylpropanoid biosynthesis pathway (Zhang and Liu, FIGURE 5 \| Growing temperature regime affects specific clusters of genes. Four selected significant profiles (<5% Bonferroni correction method) of the 2,257 genes modulated in different growing temperature, from among 25 profiles obtained by STEM analysis. For each cluster, the average gene expression trend (top panels) and heat map of all the genes' expression profiles (bottom panels) are depicted. See Supplementary File 3 for the complete comparison profile table and clusters numbering. (A) HT 2 → LT 10, (B) HT 23 → LT 22, (C) HT 22 → LT 14, (D) HT 4 → LT 12. T0 = plants before treatment, LT = low temperature, HT = high temperature. Sample names are composed by temperature treatment abbreviation followed by the indication of the developmental stage (1, 2, 3 or 4). Data are the average of the three biological replicates. Gray, blue, and purple indicate samples of control, LT-treated and HT-treated, respectively. 2015), of UFGT, which catalyzes the last step of anthocyanin biosynthesis and is considered the key enzyme for this pathway (Kobayashi et al., 2001), and of peroxidases, which are supposed to degrade anthocyanins under elevated temperature growing conditions (Mori et al., 2007; Movahed et al., 2016). PAL activity gradually decreased in both temperature regimes until 10 days after veraison and was steady thereafter in HT, whereas it abruptly increased in LT, especially between 10 and 20 days after veraison. Afterward, high levels of PAL activity still persisted in LT berries. Thus, from the end of veraison to harvest, PAL activity under HT conditions was significantly lower in berry skins in comparison to LT (Figure 7A). In order to elucidate a possible effect of the two thermal regimes on the affinity of UFGT for delphinidin or cyanidin, the enzymatic assay was performed twice using both substrates (Figure 7B and Supplementary Figure 5). After an initial fluctuating trend, in both cases the activity of UFGT showed an increasing trend from 10 days after veraison to harvest in HT and LT berries (Figure 7B and Supplementary Figure 5) and a strong and significant reduction of UFGT activity was observed in HT compared to LT from 20 days after veraison to harvest. No differences were found in terms of higher or lower affinity of the UFGT enzyme for delphinidin or cyanidin substrates as the activity of the enzyme showed comparable values between the two assays after veraison (Figure 7B and Supplementary Figure 5). The peroxidase activity of HT and LT berry skin showed a similar trend during ripening, with a significantly higher activity in HT than LT berries from veraison to harvest (Figure 7C), supporting a role of peroxidases in anthocyanins degradation under HT conditions. Overall, the enzymatic activity analyses indicated the presence of a different balance between anthocyanin biosynthesis and degradation under LT and HT thermal regimes due to an increased action of PAL and UFGT enzymes in LT and of peroxidases in HT. # DISCUSSION # Increased Temperature Accelerates Sugar Accumulation and Acidity Depletion during Ripening The increasing temperature associated with climate change is expected to modify air and land temperatures in most vine growing regions, which will undergo a warming of 2 to 4◦C in the next decades (Hannah et al., 2013). Mild to moderate temperature increases were shown to cause phenological changes in grapevine, accelerating the vegetative development and fruit maturation, ultimately affecting the berry composition (Duchene and Schneider, 2005; Jones et al., 2005). The two regimes analyzed in this study (LT and HT) can be considered representative of the inter-seasonal thermal variability occurring in recent years in the typical Sangiovese growing area of north-central Italy (Filippetti et al., 2013; Teslic et al., 2016 ´ ), with a tendency toward the thermal level of HT. The experiment design aimed to affect temperature regimes, without modifying other environmental parameters. The main differences between HT and LT regimes during preveraison-to-harvest period were mainly associated to an increase in maximum temperature under HT conditions. There was a general acceleration of sugar accumulation and total acidity reduction in the HT thermal regime. The effect of elevated temperature on sugar accumulation may depend on the amount of temperature variation as it has been reported that HTs (≥40◦C) could impact on the photosynthetic supply of sugar to the berry, causing a significant reduction in sugar accumulation (Greer and Weston, 2010; Greer and Weedon, 2013). On the contrary, the milder conditions of our experiment, in which temperatures ≥ 40◦C were seldom recorded under HT regime, led to an increased sugars accumulation, compared to LT, in all developmental stages except at harvest time. Indeed, a decrease in acidity and increase in pH associated with HT have been reported in other grape varieties (i.e., Shiraz, Chardonnay and Cabernet Franc) grown under warm conditions (Sadras and Moran, 2012; Bonada and Sadras, 2015). However, other studies registered no effect of temperature increase on either titratable acidity or pH, nor on both simultaneously (Sadras and Moran, 2012; Greer and Weedon, 2013; Movahed et al., 2016) corroborating a previous hypothesis that grape berry acidity and pH depend on the interaction between cultivar and the amount of temperature increase (Sadras and Moran, 2012). # Increased Temperature Negatively Affects Anthocyanin and Flavonol Concentrations The influence of temperature on the flavonoid concentration of grape berry has been extensively reviewed (Downey et al., 2006; Teixeira et al., 2013). The biosynthesis of tannin and flavonols is high at flowering and in the berry skin the accumulation increases from fruit set until 1 to 2 weeks after veraison (Kennedy and Jones, 2001; Downey et al., 2003; Bogs et al., 2005). Anthocyanin accumulation, on the contrary, starts from veraison and reaches its maximum in the latest phases of fruit maturation, when their synthesis ceases (Boss and Davies, 2001). In our study, no significant relationship could be verified between temperature increase and total skin tannin concentration in Sangiovese berries (Figure 3). These results, in line with the findings by Cohen et al. (2012), suggested that increasing temperature from veraison to harvest has little impact on tannin accumulation, probably because these compounds have already been synthesized. Furthermore, skin tannins are the most stable flavonoids under diverse growing conditions, due to their chemical structure which is widely variable in size, ranging from dimers to polymers with more than 40 units (Teixeira et al., 2013). This could cause less susceptibility to potential degradative processes induced by temperature. The HT thermal regime induced a similar decrease in the concentration of flavonols and anthocyanins (Figure 3). Flavonols are very sensitive to changes in environmental conditions. For example, sunlight is known to enhance flavonols accumulation in berries (Downey et al., 2006), reflecting their role as UV protectants (Spayd et al., 2002; Pastore et al., 2013). Since we maintained the same light intensity and quality between the two greenhouses, our data highlighted the strong temperature effect on this class of compounds. The reduced accumulation of anthocyanins in Sangiovese berries during ripening under increased temperature has been already reported for various genotypes in different conditions (Spayd et al., 2002; Mori et al., 2005, 2007; Tarara et al., 2008; Movahed et al., 2016). Anthocyanins and flavonols changed also their composition, in berry skins. In particular, HT berries had a lower percentage of the anthocyanin malvidin 3-G and the flavonol quercetin. In grapevine, growing temperatures have been associated with increased proportions of highly hydroxylated and methylated anthocyanins (Mori et al., 2007; Tarara et al., 2008; Cohen et al., 2012). Conversely, in a recent study an increase in the degradation of petunidin and malvidin glucoside at elevated temperatures was observed in Cabernet Sauvignon grapes exposed to labeled phenylalanine (Chassy et al., 2015). The relationship between berry temperature and flavonols profiles has been less extensively studied. Consistently with our results, a higher proportion of flavonols with di-hydroxylation, as quercetin, was detected in Merlot berries when temperature was reduced by approximately 8◦C in comparison with control temperature (Cohen et al., 2008). Overall, this evidence supports the high susceptibility of anthocyanins and flavonols to air temperature, and the critical role of experimental conditions in this kind of assessment. Accumulation of these classes of secondary compounds is the result of complex and interconnected processes such as synthesis, degradation, hydroxylation, methylation, acylation and transport, thus it is not always possible to determine general univocal relations. ## Transcriptomic Analysis Highlights Processes Affected by Temperature in Berry Skin We analyzed the entire transcriptome of the berry skin in cv. Sangiovese during ripening exploiting a combination of complementary statistical approaches to retrieve those transcripts mostly associated with each of the two temperature regimes. In particular, the PCA analysis suggested that, beyond a slight transcriptional hastening of ripening in a few developmental stages of HT samples, a clear rearrangement in the skin transcriptome (∼8% total variability of the dataset) can be ascribed to the imposition of different thermal regimes, throughout the course of the experiment. The inhibitory effects of HT on stilbene biosynthetic pathway have already been described by Rienth et al. (2014) and in our conditions many members of the STS and PAL gene families were induced under LT regime suggesting a clear activation of stilbene biosynthesis. A coordinated gene expression of PAL and STS was observed in grape berry, suggesting that several enzymatic steps in the stilbene biosynthetic pathway are co-regulated (Zenoni et al., 2016). Interestingly, several ERF transcription factors resulted as promptly higher expressed under LT compared to HT regime, whereas few were less expressed under LT regime, suggesting that members of this family of transcription factors, known regulators of thermotolerance in plants (Carbonell-Bejerano et al., 2013) play a central role in the response to temperature variation in grapevine berry skin as reported for Arabidopsis (Cheng et al., 2013), Chickpea (Cicer arietinum) (Deokar et al., 2015) and tomato (Severo et al., 2015). Moreover, the presence of ACO1 and ACS among genes more expressed in LT suggests that the ethylene signaling is required in the regulation of the temperature-driven ripening processes of grapevine berries. Another response observed under LT regime was associated to the reaction toward environmental solicitations. This included the glycerol-3-phosphate acyltransferases (GPAT) that catalyze the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA), an important intermediate for the formation of different types of acyl-lipids (Chen et al., 2011) and two galactinol synthases, whose involvement in multiple abiotic stresses responses (Zhuo et al., 2013) and in particular in heat stress responses (Pillet et al., 2012) has been reported. Lastly, some genes involved in the biosynthesis of aromas, such as linalool synthase, delta-cadinene synthase, vetispiradiene synthase and a germacrene synthase, were found more expressed under LT conditions. These results well corroborate previous reports regarding the reduction in aromatic potential in grapevine berries exposed to HT (Rienth et al., 2014). # Transcriptional and Post-transcriptional Regulation of Genes Belonging to the Phenylpropanoid/Flavonoid Pathway The accumulation of flavonoids and anthocyanins in Sangiovese berries skin was strongly affected by the two thermal regimes. However, a clear correlation between the upregulation of genes involved in anthocyanin and flavonol biosynthesis and transport and the higher levels of these compounds under LT conditions was not revealed. The slight difference of the VvUFGT and VvGST4 expression profile in LT compared to HT seems insufficient to support the significant difference in anthocyanin accumulation. Similar results were obtained by other authors reporting that mRNA accumulation of anthocyanin biosynthetic genes was not reduced under HT growing conditions (Mori et al., 2007; Carbonell-Bejerano et al., 2013; Rienth et al., 2014). Moreover, the expression of VvMYBA1, the transcription factor that activates VvUFGT and VvGST4 in grape berry skin, was not significantly affected by the thermal regime, under our experimental conditions. A first hypothesis to explain the strong differences observed in terms of flavonoid accumulation between LT and HT, based on the expression profiles of other genes involved in such metabolism, suggests the involvement of the proanthocyanidin regulator VvMybPA1 in the activation of an alternative branch under HT regime, competing with anthocyanins pathway. Another hypothesis arose from the high expression of several PAL members in LT conditions, which could supply substrates to both stilbenes and, presumably, flavonoid accumulation. An additional possibility derives from the observation that the enzymatic activities of PALs and VvUFGT were significantly higher in LT thermal regime. This was consistent with the transcriptomic data for PALs, whereas clearly discordant with the VvUFGT expression profile. These enzymatic assays clearly support the higher levels of anthocyanins and flavonols observed in berry skins under LT conditions, and that a posttranscriptional mechanism may be crucial in the regulation of the late steps of anthocyanin biosynthesis. Recent researches have revealed that post-translational mechanisms may play significant roles in the regulation of phenylpropanoid/flavonoid metabolic pathways. In Arabidopsis and apple (Malus domestica), where MYB transcriptions factors are required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway, it has been demonstrated that the repression of anthocyanin accumulation in darkness requires an interaction between a proteasome protein complex and MYBs (Li et al., 2012; Maier et al., 2013). Furthermore, a post-translational regulation mediated by the ubiquitin/26S proteasome system of the anthocyanin-related TRANSPARENT TESTA 8 (TT8) transcription factor has been detected in Arabidopsis (Patra et al., 2013). The possibility of a posttranscriptional regulation of the enzymatic steps of the pathway has also been described, showing that a multiple-level control governs the enzymatic activity of PAL (Zhang et al., 2013; Zhang and Liu, 2015). The analysis of peroxidase activity revealed a significant increase under HT conditions, supporting that this class of enzymes may trigger the temperature-dependent degradation of anthocyanins (Mori et al., 2007). Our transcriptomic survey did not reveal a significant increase in the gene expression of the recently characterized VvPrx31 (Movahed et al., 2016) in HT conditions compared to LT, suggesting that new peroxidases isoforms (as peroxidase 50) may be involved in anthocyanins degradation following increasing temperature. Overall, it is possible to hypothesize that different environmental conditions could influence anthocyanin biosynthesis and degradation in grapevine through posttranscriptional modifications of key structural genes of the pathway, such as UFGT and peroxidases. #### CONCLUSION Our results provide valuable insights into the understanding of the mechanisms that underlie Sangiovese berries response to changes in growing temperatures that could be useful to identify the most suitable areas for Sangiovese cultivation under current climate change, given the great sensitivity of this cultivar to the increasing temperature. Two thermal regimes characterized by difference of 5◦C in average and 7◦C in maximum air temperature, specifically affected berry ripening. The LT regime delayed ripening in the early phases and had great impact on grape phenolic composition and on the activity of some enzymes involved in flavonoid biosynthesis, enhancing the accumulation of anthocyanins and flavonols. Conversely, berries grown at HT showed an increase in peroxidase activity, which could concur to the reduced accumulation of flavonoids found in these conditions. Transcriptional analyses identified the existence of a strong effect of both thermal regimes on the whole transcriptome, but the partial correlation between biochemical and molecular data requires further research to elucidate the existence of posttranscriptional and post-translational mechanisms involved in #### REFERENCES the balance between biosynthesis and degradation of flavonoids and in particular of anthocyanins. ## AUTHOR CONTRIBUTIONS CP performed the RNA extraction, contributed to the enzymatic and HPLC analyses and wrote the manuscript. SDS conducted the microarray experiments, interpreted the microarray data and wrote the manuscript. SZ and GT designed the microarray experiments and critically revised the manuscript. NM, GA, and GV sampled the material and contributed to the enzymatic and HPLC analysis. IF conceived and supervised the study, wrote and critically revised the manuscript. # FUNDING CP was granted by Alma Mater Studiorum – University of Bologna research fellowship. SDS was financially supported by the Italian Ministry of University and Research FIRB RBFR13GHC5 project "The epigenomic plasticity of grapevine in genotype per environment interactions." ### ACKNOWLEDGMENT We thank Emilia Colucci for her help in setting up the greenhouses. # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00929/ full#supplementary-material the Grapevine, ed. K. A. Roubelakis-Angelakis (Dordrecht: Kluwer Academic Publishers), 1–33. different stress signals. Plant Physiol. 162, 1566–1582. doi: 10.1104/pp.113. 221911 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 Pastore, Dal Santo, Zenoni, Movahed, Allegro, Valentini, Filippetti and Tornielli. 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. # Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation #### Edited by: José Tomás Matus, Universitat Autònoma de Barcelona, Spain #### Reviewed by: Maria Pedreno, Universidad de Murcia, Spain Roque Bru-Martinez, University of Alicante, Spain #### \Correspondence: Eric Gomès [email protected] †These authors have contributed #### Specialty section: equally to this work. This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 07 January 2018 Accepted: 16 March 2018 Published: 17 May 2018 #### Citation: Soubeyrand E, Colombié S, Beauvoit B, Dai Z, Cluzet S, Hilbert G, Renaud C, Maneta-Peyret L, Dieuaide-Noubhani M, Mérillon J-M, Gibon Y, Delrot S and Gomès E (2018) Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation. Front. Plant Sci. 9:421. doi: 10.3389/fpls.2018.00421 Eric Soubeyrand<sup>1</sup>† , Sophie Colombié<sup>2</sup>† , Bertrand Beauvoit<sup>2</sup>† , Zhanwu Dai<sup>3</sup> , Stéphanie Cluzet<sup>4</sup> , Ghislaine Hilbert<sup>3</sup> , Christel Renaud<sup>3</sup> , Lilly Maneta-Peyret<sup>5</sup> , Martine Dieuaide-Noubhani<sup>6</sup> , Jean-Michel Mérillon<sup>4</sup> , Yves Gibon<sup>2</sup> , Serge Delrot<sup>1</sup> and Eric Gomès<sup>1</sup> \ <sup>1</sup> UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne, Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, Bordeaux, France, <sup>2</sup> UMR 1332 Biologie du Fruit et Pathologie, INRA-Bordeaux, IBVM, Bordeaux, France, <sup>3</sup> UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne, INRA-Bordeaux, Institut des Sciences de la Vigne et du Vin, Bordeaux, France, <sup>4</sup> EA 3675 GESVAB, Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, Bordeaux, France, <sup>5</sup> UMR 5200 Laboratoire de Biogenèse Membranaire, Université de Bordeaux, Bordeaux, France, <sup>6</sup> UMR 1332 Biologie du Fruit et Pathologie, Université de Bordeaux, IBVM, Bordeaux, France Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an "energy escape valve," helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3β-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars. Keywords: anthocyanins, grapevine, cell redox status, energy escape valve hypothesis, constraint-based modeling #### INTRODUCTION Flavonoids are naturally occurring secondary metabolites belonging to the group of polyphenols, which are ubiquitous in all land plants, with currently over 9,000 compounds identified (Buer et al., 2010). Among polyphenols, flavonoids encompass over 6000 distinct molecules, divided into aurones, flavones, flavonols, flavanols, anthocyanins, phlobaphenes, and isoflavonoids, the last two being almost exclusively synthesized in maize and leguminous plants (Hichri et al., 2011). They exhibit a large variety of biological roles in plants. They control pollen fertility in many species (Taylor and Jorgensen, 1992) and influence auxin transport (Peer and Murphy, 2007). Light absorbing pigments such as anthocyanins and aurones color flower petals and fruit epicarp, thus facilitating pollinator attraction and seed dispersal (Mol et al., 1998). With regard to human health, the consumption of grapes or grape-derived products, has been correlated with a reduced incidence of a number of chronic illnesses (Iriti and Faoro, 2009; Kozlowska and Szostak-Wegierek, 2014), and flavonoids have been proposed as major contributors of these health-promoting effects (Butelli et al., 2008; Qin et al., 2011). Anthocyanins, which are key compounds for premium red wine making, are present in the skin (epicarp) of the red grape berries, and sometimes, in the case of the so-called "teinturier" cultivars also in the pulp (mesocarp) (Petrussa et al., 2011; Guan et al., 2016). Hence, in order to optimize anthocyanin content in the berries, it is important to understand the molecular regulation of the anthocyanin production by environmental factors and viticultural practices. Anthocyanins are synthesized through the phenylpropanoid and flavonoid pathways, starting with phenylalanine as a precursor, and splitting into two branches to produce the di- and tri-hydroxylated flavonoids (Figure 1; Tanaka et al., 2008; He et al., 2010). The accumulation and the proportion of these compounds in the berry skin depends on genetic, developmental, and environmental factors (Rio Senegade et al., 2008; He et al., 2010; Dai et al., 2011) as well as on viticultural practices (Downey et al., 2006). Light, temperature, irrigation, and nitrogen supply have been shown to impact grape berry anthocyanin content (Dai et al., 2011; Berdeja et al., 2015; Keller, 2015; Habran et al., 2016). In several crops like tomato or grapevine and model plants such as Arabidopsis, nitrogen depletion increases the concentration of phenolics in general and of anthocyanins in particular (Keller and Hrazdina, 1998; Hilbert et al., 2003; Fritz et al., 2006; Feyissa et al., 2009; Løvdal et al., 2010; Soubeyrand et al., 2014; Habran et al., 2016). The transcriptional regulation of the phenylpropanoid and flavonoid pathways in response to low nitrogen availability has been extensively studied in Arabidopsis (Lillo et al., 2008), tomato (Løvdal et al., 2010), tobacco (Fritz et al., 2006), or grapevine (Soubeyrand et al., 2014). These studies shed light on the molecular mechanisms underlying the regulation of the flavonoid metabolism by nitrogen depletion, pointing out the responsiveness of the pathway's positive and negative regulators, i.e., R2-R3MYB and LBD transcription factors. They do not, however, address the pending question of the existence of a metabolic "driver" that would fuel the increase in flavonoid biosynthesis. According to Hernández and Van Breusegem (2010), a plausible candidate for such a metabolic driver could be the cell energetic status. High carbon-to-nitrogen ratio leads to an energy excess, both in terms of high ATP and high reducing power (NADH and NADPH); and an increase in flavonoid biosynthetic metabolic fluxes would act as an "energy escape valve," helping plant cells to cope with that energy excess. Indeed, the flavonoid pathway consumes ATP and NADPH reducing equivalents in several of its enzymatic steps particularly when the shikimate pathway, that links the central metabolism to the phenylpropanoid pathway, is also taken into account (Figure 1). Constraint-based modeling can be used in order to test that hypothesis by comparing maps of metabolic fluxes in the two contrasted situations, i.e., in nitrogen limiting condition compared to the control condition. Mathematical modeling of metabolism is a particularly promising tool as it offers a systems approach to analyze the structure, dynamics, and behavior of complex metabolic networks. In plant research, the issue of modeling metabolism is increasingly gaining attention, and several mathematical modeling approaches applied to plant metabolism exist (for reviews, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007). Constraint-based modeling such as flux balance analysis (FBA, Orth et al., 2010) allows the prediction of metabolic fluxes at steady-state by applying mass balance constraints to a stoichiometric model. Mass-balance information, such as growth rate, biomass composition, and substrate consumption rate, are used to fix boundaries on the flux solutions space (Reed and Palsson, 2003) and an objective function is used to identify the optimal flux distribution among all possible steady-state flux distributions. This modeling has the advantage of not requiring the knowledge of enzyme kinetic parameters. The present work aims to investigate the metabolic flux reorganization that is involved in the response of anthocyanin accumulation to nitrogen supply, taking advantage of a grapevine red cell suspension system, the GT3 Vitis vinifera L., cv. Gamay Teinturier (syn. Freaux or Gamay Freaux Tintorier, Vitis International Variety Catalogue #4382, Decendit and Mérillon, 1996). To this end, the cells were cultivated in control or nitrogen limiting conditions. Then we generated and compared flux maps of plant cell metabolism by coupling the network of heterotrophic metabolism previously described (Colombié et al., 2015) with the overall reactions to phenolic compounds production (anthocyanins, flavonols, tannins, and stilbenes), paying special attention to energetic processes by balancing cofactors. The results are consistent with excessive ATP and reducing equivalent (NADPH mostly) as well as α-ketoglutarate availability acting as "pushers" that increase anthocyanin and more broadly polyphenol biosynthesis in nitrogen-depleted cells. #### MATERIALS AND METHODS ## Grapevine Cell Culture Growth and Sampling Vitis vinifera cv. "Gamay Fréaux" var. teinturier GT3 cell suspensions were sub-cultured on a modified Gamborg B5 medium, supplemented with 20 g L−<sup>1</sup> sucrose, 250 mg L−<sup>1</sup> casein hydrolysate, 0.1 mg mL−<sup>1</sup> 1-naphthalene acetic acid and 0.2 mg mL−<sup>1</sup> kinetin (Saigne-Soulard et al., 2006). Cells were routinely sub-cultured every 7 days in 250 mL Erlenmeyer flasks containing 50 mL of culture medium. For experimental purpose, 7-days old cells were inoculated, with a 1:6 (v/v) ratio in 200 mL of the same medium but containing either 5 mM (final concentration, low nitrogen, N−) or 25 mM (final concentration, control, N) KNO3, in 1 L Erlenmeyer flasks. The ammonium concentration was identical in both N<sup>−</sup> and N treatments (2 mM). For each sampling point, three replicate flasks of cell culture were harvested at 0 (or 1 for experiment 1), 4, 6, 8, and 11 days post-inoculation by vacuum filtration, quickly washed twice with ice-cold distillated water, weighed and quick-frozen in liquid nitrogen. Frozen cells were then reduced to fine powder in a liquid nitrogen-cooled MM200 ball grinder (Retsch, Haan, Germany), and stored at −80◦C until further analysis. #### Cell Biomass and Metabolites Content Analysis #### Phenolic Compounds Anthocyanins and flavonols were analyzed on powdered freezedried cells, which were extracted and analyzed according to Soubeyrand et al. (2014). Tannins and stilbenes were extracted from 40 mg of freezedried cells with 4 mL of methanol (100%) overnight at +4 ◦C. The samples were centrifuged at 6,000 × g for 10 min. Two milliliters of supernatant were vacuum-dried using a SpeedVac SC 110 plus (Thermo Fisher Scientific, Saint-Herblain, France) for the analysis of stilbenes and 50 µL were used for the analysis of the total phenolic content. Then, 100 µL MeOH (100%) and 1 mL H2O were added to the vacuum-dried samples and filtered through an Ion Exchange Resin (Dowex 50 WX 4-400) to remove anthocyanins. Extracts were vacuum-dried using a SpeedVac SC 110 plus and the dry pellet was re-suspended in 800 µL of MeOH/H20 50/50 (v/v) for the HPLC analysis. Extracts were then filtered through a 0.45 µm polypropylene syringe filter (Pall Gelman Sciences Corp., Ann Arbor, MI, United States). Stilbenes analysis was performed with a Summit HPLC System consisting of P680 pump, ASI-100TTM autosampler and UVD 340U UV-Vis detector operating at 320 nm (Dionex Corporation, Sunnyvale, CA, United States) (Ramirez-Lopez and DeWitt, 2014). After injecting 20 µL, separation was achieved at ambient temperature on a reverse-phase Ultrasphere ODS column 25 cm × 4.6 mm, 5 µm particle size with an Ultrasphere ODS guard column 4.5 cm × 4.6 mm (Beckman Instruments Inc., Fullerton, CA, United States). All reagents were of analytical grade. Separation was performed according to Saigne-Soulard et al. (2006). Total phenolic content was assessed by the Folin–Ciocalteu method (Singleton and Rossi, 1965). The assay mixture (3 mL) contained 50 µL of extract, 450 µL MeOH/H2O (50/50, v/v), 250 µL Folin–Ciocalteu reagent and 2.25 mL of ultrapure water. After 3 min, 2 mL Na2CO<sup>3</sup> (75g L−<sup>1</sup> ) were added and the samples were incubated at 50◦C for 5 min and absorbance was read at 760 nm. Calculation of phenolics was based on a standard curve prepared using gallic acid, and the results were expressed as mg gallic acid equivalents per liter (mg GAE L−<sup>1</sup> ). #### Sugars and Amino Acids Five hundred milligrams of cell powder (FW) were extracted from cell suspension samples using decreasing concentrations of ethanol: ethanol 80%, ethanol 50% (v/v) and ultrapure water. All three supernatants were pooled, vacuum-dried using a Speed Vac SC 110 plus (Thermo Fisher Scientific, Saint-Herblain, France). The dry pellet was re-suspended in 2 mL of ultrapure water and stored at −20◦C before further analysis. Amino-acid content was analyzed by the method described by Cohen and Michaud (1993), modified according to Martínez-Lüscher et al. (2014). Briefly, after derivatization with 6 aminoquinolyl-N-hydroxysuccinimidyl-carbamate, amino acids were analyzed using a Waters 2695 HPLC system equipped with a Waters 474 fluorescence detector (Waters, Milford, MA, United States). Separation was performed on a Nova-Pack C18 AccQ-Tag column (Waters, Milford, MA, United States) at 37◦C with elution at 1 mL min−<sup>1</sup> with a 67 min linear gradient (eluent A, sodium acetate buffer, 140 mM at pH 5.7; eluent B, acetonitrile 60% in water (v/v)). Chromatograms corresponding to excitation at 250 nm and emission at 395 nm were recorded and quantified with chemical standards purchased from Sigma (St. Louis, MO, United States). Soluble sugars (glucose, fructose, and sucrose) were measured enzymatically with a microplate reader (ELx800UV, BioTek Instruments Inc., Winooski, VT, United States) as described by Gomez et al. (2007). #### Malic Acid, Total Starch, and Proteins Malate, starch, and protein content were measured as described in Biais et al. (2014). #### Cell Wall Total Polysaccharides Total cell wall polysaccharides quantification was subcontracted to the BIBS platform of INRA-Nantes<sup>1</sup> , using 100 mg of freezedried cell powder, as described in Colombié et al. (2015). #### Lipids Five hundred milligrams of cell powder (FW) were extracted by 1 mL of MeOH:H2SO<sup>4</sup> (40:1, v/v), supplemented with 2 µg of heptadecanoic acid (internal standard) and incubated 60 min at 80◦C in screw-capped tubes. Then, 400 µL hexane and 1.5 mL of ultrapure water were added, vigorously mixed and centrifuged at 3,000 g for 5 min. The organic phase was collected and transferred to injection vial to analyze fatty acids by GC-FID (Gas Chromatography coupled to Flame Ionization Detection), as described by Maneta-Peyret et al. (2014). #### Total Carbon and Nitrogen Content Cell total carbon and nitrogen contents were determined by Dumas' combustion method, with a Flash EA 112 autoanalyzer (Thermo fisher, Courtaboeuf, France), following the manufacturer's instructions and using 8 mg of freeze-dried cell powder. In the case of culture medium analysis, 250 µL of freeze-dried medium were used instead. #### Total Nucleic Acid The total DNA content was measured using the deoxyribosespecific diphenylamine reaction, using 15 mg of freeze-dried cell powder as starting material, and salmon sperm DNA for calibration (Colombié et al., 2015). #### Enzyme Capacity Determinations Phenylalanine ammonia-lyase (PAL) activity was measured according to Gagné (2007). Approximately 250 mg of cell powder were extracted by vigorous shaking with 40 mg polyvinylpolypyrrolidone (PVPP) and 2.5 mL extraction buffer composed of 0.1 mM Tris–HCl (pH 8.8), 5 mM EDTA, 0.05% spermidine (w/v), 4 mM β-mercaptoethanol, <sup>1</sup>http://www.bibs.inra.fr and 1 mM phenylmethylsulfonyl fluoride (PMSF, added just prior extraction). The samples were centrifuged for 20 min at 16,000 × g at 4◦C. The protein extract was desalted on a PD-10 column (Sephadex resin G-25, PD-10 column, GE Healthcare) equilibrated with 25 mL of 0.1 mM Tris–HCl (pH 8.8). Aliquots of desalted proteins were frozen in liquid nitrogen and stored at −80◦C. Spectrophotometric assays contained 300 µL of protein extract in 30 mM L-phenylalanine in 0.1 M Tris–HCl (pH 8.8) and 150 µL of 30 mM L-phenylalanine in 0.1 M Tris–HCl (pH 8.8). Reactions were incubated for 15 to 180 min at 37◦C. The amount of trans-cinnamic acid formed in the assay was measured spectrophotometrically at 290 nm. PAL activity was expressed as µg of cinnamic acid formed per µg FW−<sup>1</sup> . For all other enzyme measurements [glucose-6-phosphate dehydrogenase (G6PDH), phosphoglucomutase (PGM), fructose hexokinase (FK), glucose hexokinase (GK), shikimate dehydrogenase (SD), enolase (ENO), pyruvate kinase (PK) and glutamine synthetase (GS)], aliquots of 20 mg frozen FW cell powder were extracted by vigorous mixing with extraction buffer (Nunes-Nesi et al., 2007). FK, GK, G6PDH, PK, SD, and GS were assayed as described in Gibon et al. (2004). ENO was assayed as described by Biais et al. (2014), PGM was assayed according to Gibon et al. (2009). #### Respiration Measurements Oxygen consumption rates of cells were measured with Clark's electrode at 25◦C in a 1 mL thermostatically controlled chamber. Respiration assays of growing cells were performed in the GT3 cell suspension medium under stirring. Seven hundred and fifty microliters of cell suspension were centrifuged (1,500 × g for 5 min) and the resulting pellet gently re-suspended in 1 mL of cell culture medium. Respiration rates were initially expressed in nmol O<sup>2</sup> min−<sup>1</sup> g <sup>−</sup><sup>1</sup> FW. #### Coenzyme Analysis All extractions were performed at 4◦C with 200 mg of frozen powder cell. For the assays of NAD<sup>+</sup> and NADP+, aliquots of frozen cells were extracted with 500 µL of 0.2 N HCl then incubated for 5 min at 80◦C. Fifty microliters of 0.2 M NaH2PO<sup>4</sup> (pH 5.6) was added and the extract was neutralized to a final pH in the range from 5.5 to 6.5 with 0.2 M NaOH. To quantify NADH and NADPH, other aliquots of frozen cells were extracted as for NAD<sup>+</sup> and NADP<sup>+</sup> except that the extraction medium was 0.2 M NaOH and the heated sample was neutralized with 0.2 N HCl to a final pH in the range from 7.5 to 8.5. Coenzyme content was quantified by adapting methods described by Wilhelm (2009). The reaction buffer was composed by Tris/KOH (pH 7.7) qsp 350 µL, 100 µL of 10 mM methylthiazolyldiphenyl-tetrazolium (MTT) and 50 µL of 4 mM phenazine ethosulfate (PES). For the NAD<sup>+</sup> and NADH assay, the reaction was started by adding 3.5 U of alcohol dehydrogenase (ADH, Roche, Melan, France) and 10 µL ethanol (99%). For the NADP<sup>+</sup> and NADPH assay, 1.6 U of glucose-6-phosphate dehydrogenase (Roche) and 0.5 M glucose-6-phosphate were added to the assay. Absorbance was read at 570 nm for 10 min, and the results were expressed in nmol g−<sup>1</sup> FW. #### Modeling Concentrations of accumulated metabolites and biomass components were converted from gram-to mole-basis and then multiplied by the specific growth rate calculated at day 4 and 6 in order to calculate the corresponding fluxes used as constraints in the flux balance model. Stoichiometric network reconstruction encompassing central and polyphenol metabolism (model in sbml format, Supplementary Presentation 2) and mathematical problems were implemented using MATLAB (Mathworks R2012b, Natick, MA, United States) and the optimization toolbox, solver quadprod with interior-point-convex algorithm for the minimization. #### Statistical Analysis Statistical analyses were done using the statistical package of the "R" software (R Development Core Team, 2010). A oneway analysis of variance (ANOVA) was used. Unless otherwise stated, the mean of the 3 biological replicate treatments was used in data analysis. Unless otherwise stated, comparisons of means were performed using HSD.r multiple comparisons function of Tukey's post hoc test at P < 0.05. ### RESULTS #### Anthocyanin Accumulation in Cells Cultivated Under Control and Low Nitrogen Conditions Dry biomass accumulation kinetics were nearly identical for cells cultured in either control or low nitrogen conditions, increasing from about 1.6 g DW−<sup>1</sup> of cells per L at culture initiation to about 10 g DW−<sup>1</sup> at the 8th day of culture, and starting to decline afterwards (Figure 2A). Conversely, total anthocyanin accumulation patterns were strikingly different in control and low nitrogen condition cultures (Figure 2B). In control cells (N, 25 mM NO<sup>3</sup> <sup>−</sup>), total anthocyanin content was fairly stable during the culture (around 2–3 mg g DW−<sup>1</sup> ), whereas in cells cultivated at low nitrogen (N−, 5 mM NO<sup>3</sup> <sup>−</sup>) total anthocyanin cell content strongly increased from the 4th day of culture to the 12th day, reaching a maximum of about 20 mg g DW−<sup>1</sup> at the end of the culture. This result has been reproduced with three cell cultures (named exp. 1, exp. 2, and exp. 3), that were conducted in the same conditions. Similar growth profiles were observed with about 10 g DW−<sup>1</sup> of cells per L at the 11th day of culture (Supplementary Figure 1). Plotted in log-scale, the cell biomass concentration increased linearly up to the 6th day of culture. Maximal specific growth rate was reached at the 4th day of culture, in the exponential growth phase. Consequently, the steady state was assumed for modeling, i.e., the state when there was no accumulation of internal metabolites of the network, at day 4. Nevertheless, all data have also been collected and analyzed at the 6th day of culture to assess the evolution of the metabolic fluxes from day 4 to 6. For each experiment, the specific growth rate (µ in day−<sup>1</sup> ) was determined as the growth rate (g DW day−<sup>1</sup> ) related to the biomass (g DW) and the average was: 0.288 ± 0.013 day−<sup>1</sup> and 0.160 ± 0.045 day−<sup>1</sup> at day 4 and 6 of the culture, respectively. ## Nitrogen and Carbon Consumption in Culture Medium Both total nitrogen and total carbon have been determined in the medium (for experiments 2 and 3, Supplementary Figure 2). Only in the case of low nitrogen condition (N−), nitrogen was limiting and even fully depleted since the 6th day of culture. The total carbon concentration in the medium was stable since the beginning up to the 8th day of culture, implying that there was no carbon limitation. #### Glucose and Fructose as Metabolized Sugars Sucrose (20 g L−<sup>1</sup> ) was the carbon source supplied in the culture medium, but it is generally cleaved to form hexoses by cell wall invertase activity (Atanassova et al., 2003; Chen et al., 2013). Measurements of sucrose, glucose, and fructose in the medium during 2 cultures (exp. 2, Supplementary Figure 3; exp. 3, Supplementary Figure 4) showed that hexose concentrations were higher than sucrose concentration at the 4th of culture. Thus glucose and fructose were assumed to be the main sugars metabolized by the cells. #### Metabolic Fluxes Modeling Flux-Balance Model The flux-balance model was constructed by integrating biochemical and physiological knowledge about the stoichiometry of reactions and the boundary conditions, i.e., the definition of external compounds. The model describes one cell and assumes that the suspension is homogeneous. The model combines the central metabolism previously described (Beurton-Aimar et al., 2011; Colombié et al., 2015) dedicated to breakdown and transformation of extracellular nutriments to produce energy and metabolic precursors (amino acids, proteins, cell wall, . . .) and the secondary metabolic pathway to produce the main polyphenols (anthocyanins, flavonols, tannins, and stilbenes). This network of reactions (schematized in Supplementary Figure 5, and the list of the stoichiometric reactions in Supplementary Table 1) includes the glycolysis, the tricarboxylic acid cycle (TCA), the pentose phosphate pathway, starch metabolism, and sucrose metabolism. The carbon source was described through glucose and fructose uptake (Vglc-up, Vfru-up). The inorganic nitrogen source was nitrate (Vno3-up) involving enzymes of the nitrogen assimilation pathway [nitrate reductase (Vnr), glutamine synthetase (Vgs), and glutamate synthase (Vgogat)]. Ammonium, with a low concentration (2 mM), was neglected as nitrogen source. For the phenolic pathway, three reversible reactions involving naringenin, dihydroquercetin, and leucocyanidin (Vnar, Vdhq, and Vlcc) and two irreversible reactions involving cinnamate and coumaroyl coenzyme A (Vpal and Vcoum) were connected to central metabolism via phenylalanine. The fluxes directed toward the main phenolic compounds, i.e., anthocyanins, flavonols, tannins, and stilbenes were described by four overall reactions (Vanthoc, Vflav, Vtannins, and Vstilb, respectively). The main biosynthetic processes were described with overall reactions: (1) cell wall polysaccharides from UDP-glucose (Vcw), (2) protein synthesis (Vprotein) according to the measured amino acid composition of proteins (Supplementary Table 2), (3) fatty acids synthesis (diacyl glycerol, Vdag) from pyruvate and trioses phosphate according to total fatty acid biomass measurement (Supplementary Table 3), and (4) nucleotides synthesis (DNA and RNA, Vnucleotides) from ribose-5-phosphate by using plant metabolic pathway databases<sup>2</sup> . All other accumulated compounds were described as a simple accumulation: (1) malate (Vac-mal), (2) soluble sugars, i.e., glucose (Vac-glc), fructose (Vac-fru), and sucrose (Vac-suc), and (3) four groups of free amino acids, glutamate (Vac-Glu), aspartate (Vac-Asp), alanine (Vac-Ala), and phenylalanine (Vac-Phe). It has been checked that no metabolites were excreted in the medium (data not shown). <sup>2</sup>http://www.plantcyc.org Energy intermediates, both ATP and NAD(P)H, were explicitly taken into account. The cofactors NADP/NADPH were linked to biomass and the phenolic compounds production, and the cofactors NAD/NADH and FAD/FADH were linked to ATP synthesis via two essential reactions of oxidative phosphorylation (Vnrj1 and Vnrj2), which are associated to the mitochondrial respiration. Recycling of AMP by adenylate kinase is described by Vadk. The portion of synthesized ATP that is not used for growth has been balanced by the model as an ATP hydrolyzing reaction (Vnga-ATPm) that physiologically represents cellular maintenance (Amthor, 2000). Finally, all the cofactors were defined as internal metabolites, which means that they were balanced, thus constraining the metabolic network not only through the carbon and nitrogen balance but also through the redox and energy status. In summary, the model of the metabolic network describes the main growth components of the cell through a set of n reactions involving m metabolites whose mint were internal metabolites. At steady-state, the mass balance equation is expressed by $$\frac{dX\_{\text{int}}}{dt} = NV = 0\tag{1}$$ With Xint the vector of mint internal metabolites, V = (vi) t i=1...n the flux vector composed by the rates of n reactions of the network, and N = (nij) <sup>i</sup>=1...mint, <sup>j</sup>=1...<sup>n</sup> the stoichiometry matrix where nij is the stoichiometric coefficient of metabolite xint,<sup>i</sup> in reaction j. To solve the system, a lower and an upper bound constrained each flux. #### Constraints Limiting the Flux Space and Resolution The first type of constraints applied to limit the flux space to flux directions was inferred from thermodynamic properties of reversibility or irreversibility. Thus, among the internal reactions of the metabolic network, 33 were irreversible as indicated by unidirectional arrows on Supplementary Figure 5, which meant that their lower bounds were set to zero. The second type of constraints was the maximal enzyme capacities. Experimentally determined activities of enzymes of central metabolism and flavonoid pathway, considered as maximal enzyme capacities (converted in mmol g DW−<sup>1</sup> day−<sup>1</sup> , Supplementary Table 4) were used to limit each corresponding flux in the metabolic network. The same values, but negative, were used as lower bounds of reversible enzymes. When the capacity of a given enzyme was not known, the bounds were set to infinity. The third type of constraint concerned the respiration rate (see Section "Materials and Methods"). The sum of the two reactions of ATP synthesis by oxidative phosphorylation (Vnrj1 and Vnrj2) was constrained by the respiration measurements: 3.54 ± 0.18 and 2.86 ± 0.24 mmol g DW−<sup>1</sup> day−<sup>1</sup> in control and low nitrogen conditions at day 4 of culture, respectively. Finally, the essential constraints required to set up the system were the external fluxes, also called exchange fluxes. Assuming steady state, these 16 fluxes (rates) were calculated from experimental data (Supplementary Table 5) and used as both lower and upper bounds. Also, respiration rates (Supplementary Table 6) were used to constrained ATP synthesis fluxes. The mass balance of accumulated metabolites and biomass components covered an average of 81 and 91% of the dry biomass in control and nitrogen-limiting conditions, respectively (Supplementary Table 5). The accumulation of phenolic compounds in nitrogen-limiting condition was followed by an increase in sugar accumulation in cells at the expense of proteins synthesis and malate accumulation, especially at day 6 (Figure 3). Flux minimization, which leads to a unique solution (Holzhutter, 2004), was used as the objective function to solve the system and generate flux maps in both N and N− conditions (Figure 4). Unsurprisingly, flux maps obtained with low nitrogen cultured cells compared to control condition showed higher fluxes in primary than in secondary metabolism both at 4 and 6 days of culture. Also a lower flux of ATP synthesis is pointed in low nitrogen condition. More than the absolute values of the calculated fluxes, we were interested in the relative changes in the fluxes to look for cell metabolism reprograming under low nitrogen condition (Supplementary Table 7 and Figure 5). Concerning external fluxes (4th day) the main changes in nitrogen-limiting condition were the increase in accumulation of phenolic compounds (except flavonols), hexoses and starch (Figure 5A). Conversely protein synthesis, sucrose accumulation, and respiration were decreased. Consequently, the calculated fluxes in the main pathways (glycolysis, TCA, PPP. . .) were decreased of about 20 to 30%, except the fluxes of the phenolic pathway which were increased: Vmacl, Vshik, Vpal, and Vcoum by 38% and, Vnar, Vdhq, and Vlcc by 26%. More surprisingly, two internal fluxes, the PPi-dependent phosphofructokinase (Vpfp) and the pyruvate kinase (Vpk) were strongly increased (80 and 63%), and also glucose and fructose uptakes, were also slightly increased (7 and 6%) at day 4. Finally, fluxes corresponding to enzymatic steps of the flavonoid biosynthesis that use α-ketoglutarate (α-KG) as a reducing agent and convert it to succinate, namely Vdhq and Vanthoc were increased by 25 and 104%, also at day 4. Conversely, α-KG conversion to succinate the TCA cycle (Vkgdh) was decreased by 25% in low nitrogen culture conditions. The global behavior of external fluxes was exacerbated at day 6 and resulted in similar observations than at day 4, i.e., a global diminution of all fluxes (40–50%) but here without significant change in Vpfp and Vpk and sugar uptake (Figure 5B and Supplementary Table 7). Changes in fluxes for Vdhq and Vanthoc were further enhanced by low nitrogen culture conditions, compared to control, with an increase of 129 and 518%, respectively. A third α-KG-dependent flavonoid biosynthetic flux was also strongly enhanced by 320%. TCA-linked metabolic flux that converts α-KG into succinate (Vkgdh) was reduced by 50%. The internal metabolite concentrations were not accessible with the flux-balance model. Then complementary analyses have been done to determine the total contents of redox metabolism coenzymes (NAD+, NADH, NADP+, and NADPH). While NADH was slightly affected, NADPH significantly increased in low nitrogen condition compared to the control (Supplementary Table 8). Thus, the NADP+/NADPH ratio was significantly lower in low nitrogen condition at day 4 (Figure 6). The same trend was observed at day 6, but was not deemed statistically significant according to Student's t-test (Figure 6). These results clearly showed an excess of NADPH, concomitant with the accumulation of anthocyanin (at day 4 and 6) and stilbene compounds (at day 4). # DISCUSSION #### Low Nitrogen Stimulates Anthocyanin Biosynthesis in GT3 Grapevine Cells Under nitrogen limitation, an increase of the total anthocyanin content, especially peonidin and petunidin derivatives was noted. Moreover, the low phenylalanine concentration in cells cultivated in limiting nitrogen condition is in agreement with an increase in the phenylpropanoid catabolic flux, supported by the increase in PAL activity. Stimulation of anthocyanin biosynthesis in vineyard-grown grape berries by low nitrogen availability has been well documented in the literature, with an increase of around 30% in berry anthocyanin content (see for example Keller and Hrazdina, 1998; Hilbert et al., 2003; Soubeyrand et al., 2014). Similar studies using grapevine cell suspensions are much more scarce, however. In our experiments, GT3 grapevine cell suspension responded to low nitrogen in the culture media by a c.a. 700%, in average, increase total anthocyanin accumulation. This result is in the same order of magnitude as the results previously obtained on strawberry (Mori and Sakurai, 1994), or Gamay Fréaux grapevine (Do and Cormier, 1991), thus validating the GT3 cell suspension culture used in this work for acquiring the dataset that allowed us to perform FBA modeling. intensity. # Building a FBA Model That Links Central Primary Metabolism and the Polyphenol Secondary Metabolism The metabolic model utilized in this work was sufficiently detailed to describe the global functioning of the cell. The originality of this work was to couple both primary and secondary metabolism, including the flavonoid biosynthetic pathway. As far as we know, in plant science only few models take into account secondary metabolic pathways. A genome-scale metabolic model of maize has already been reconstructed (Saha et al., 2011). Bekaert et al. (2012) described their updated mathematical model of Arabidopsis thaliana Columbia metabolism, which adds the glucosinolates, an important group of secondary metabolites, to the reactions of primary metabolism. In a recent review, Collakova et al. (2012) showed that metabolism can be modeled mathematically by using models and genomescale models (GEMs) predicting the combination of flux values of a defined metabolic network given the influence of internal and external signals. Nevertheless plant GEMs tend to be accurate in predicting only qualitative changes in selected aspects of central carbon metabolism, while secondary metabolism is largely neglected mainly due to the missing (unknown) genes and metabolites. As such, these models are suitable for exploring metabolism in simplified models such cell cultures in plants grown in favorable (controlled) conditions, but not in field-grown plants that have to cope with environmental changes in complex ecosystems (Collakova et al., 2012). ## Cell Energy and Reducing Power as a Driver for Anthocyanin Biosynthesis in Grape Cells The question of the existence of a metabolic driver that would fuel the increase in anthocyanin (and more generally in flavonoid) biosynthesis in such situation remains open. One emerging property of the FBA-generated flux maps is the fact that in low nitrogen conditions (N−), several enzymatic steps that consume ATP and reducing power (NADPH or NADH) have their metabolic flux either maintained or increased (i.e., starch synthase, phosphofructokinase, enzymes of the pentosephosphate pathway, all the enzymes of the phenylpropanoid and flavonoid biosynthetic pathway, as well as the stilbene biosynthetic pathway). Conversely, most of the metabolic fluxes that lead to ATP, NADH, or NADPH formation were decreased by low nitrogen conditions (i.e., phosphoglycerate kinase and pyruvate kinase in the lower part of the glycolytic pathway, the p < 0.05. malic enzyme, most of the TCA cycle enzymes with noticeable exception of the malate dehydrogenase which has its metabolic flux slightly increased). This strongly advocates for a link between cell energy status (i.e., excess of ATP and reducing power) and secondary metabolism, confirming an hypothesis made by Hernández and Van Breusegem (2010). Recently, redox-dependent modulation of the anthocyanin pathway has been reported in Arabidopsis leaves during exposure to high light intensity (Page et al., 2011; Viola et al., 2016), or in Citrus callus (Cao et al., 2015). FBA modeling results strongly support that hypothesis, as well as actual NADP+/NADPH ratio measurements, pointing toward ATP and NADPH excess as a metabolic driver for flavonoid (and particularly anthocyanin) biosynthesis in grapevine GT3 cells. In the same review from Hernández and Van Breusegem (2010) also hypothesized that flavonoid biosynthesis could also constitute a carbon sink in situations of high carbon-to-nutrient ratio. Indeed in leaves from plants such as Rosemary or Tea trees, flux analysis suggests that up 20% of the fixed carbon would flow through the phenylpropanoid pathway, leading to a phenolic content accounting for up to 30% of dry matter, making it the main non-structural carbon sink of the plant, and thus an efficient mechanism to deal with carbon excess, without mobilizing any nitrogen (Hernández et al., 2004; Rippert et al., 2004; Yao et al., 2005). In the case of GT3 grape cells, however, model flux calculations and biomass composition analysis demonstrated that anthocyanins, and more broadly flavonoids, represent only a marginal storage sink for non-structural carbon (0.49 and 1.5% of total dry matter, at day 4 and 6, respectively, in N− condition), ruling out a role of anthocyanin (and more broadly flavonoids) biosynthesis as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations. Instead, FBA model output and biomass analysis indicate that carbon excess is dealt with by diverting embolic flux to vacuolar storage of soluble sugars (hexose and to a lesser extent sucrose) and malic acid. This discrepancy could be linked to fact that cell suspensions and whole organs such as leaves obviously differ in their behavior in term of carbon management. Leaves can act both as source and sinks for carbon, whether cultured cells only acts as carbon sink. The comparison is thus limited, but nevertheless points out two potentially different strategies for leaves and grape cells to cope with carbon excess. #### α-Ketoglutarate Levels as a Potential Regulator of Anthocyanin Biosynthesis in Grape Cells Besides the fact that low nitrogen culture conditions might lead to an altered cell energy status (i.e., an excess of ATP and NADPH), another output of the FBA-generated flux maps is that three fluxes of the flavonoid pathway that use α-KG were strongly up-regulated in low nitrogen cultured cells. α-KG has emerged in the past decade as a signal molecule in plants, linking TCA cycle to secondary metabolism, including the flavonoid pathway (Araújo et al., 2014). Indeed, three enzymatic steps of the flavonoid pathway use α-KG as reducing agent in their catalytic cycle: the flavanone 3β-hydroxylase, the flavonol synthase and the anthocyanidin synthase (Turnbull et al., 2004). Under low nitrogen culture conditions, consumption of α-KG by GOGAT for glutamate synthesis is bound to decreased. This is advocated by model output that predicts a diminution of 21 and 50% at day 4 and 6, respectively, potentially leading to an increase of cell α-KG level, which would be used to fuel anthocyanin and more generally flavonoid biosynthesis. Thus, α-KG availability would be part of the metabolic driver that lead to enhanced flavonoid biosynthesis high carbon-to-nitrogen ratio conditions. Actual α-KG level measurements would be required to further advocate this hypothesis. #### CONCLUSION Flux balance analysis modeling was used to investigate metabolic flux reprogramming in grapevine cells in response to low nitrogen culture conditions and revise the well-known upregulation of anthocyanin biosynthesis in response to low nitrogen availability. Model outputs unambiguously point toward cell energy excess and increased α-KG availability as the metabolic drivers of anthocyanin synthesis (and more broadly flavonoid synthesis) under high carbon-tonitrogen ratio conditions. This work was conducted in a cell suspension culture, and the next obvious question is whether such a metabolic driver effect is also occurring in ripening berries of red grape varieties, which accumulate anthocyanins to high levels in their exocarp cells, a key feature for high quality red wine making. Further modeling and biochemical work is needed to address that question. ## AUTHOR CONTRIBUTIONS ES, GH, CR, StC, LM-P, and BB performed the experiments and the analytical work. SoC performed model construction and calculations, participated to data analysis and manuscript writing. MD-N generated the flux maps. YG, J-MM, ZD, and SD discussed the results and performed manuscript critical reading. EG led the project and designed the experimental flowchart, discussed the results and coordinated the manuscript writing and critical reading. #### REFERENCES #### FUNDING ES was supported by a Ph.D. grant from the Ministère de l'Éducation Nationale, de l'Enseignement Supérieur et de la Recherche and the work was partially funded by a grant from the FR BIE (Fédération de Recherche Biologie Intégrative et Ecologie) of University of Bordeaux. #### ACKNOWLEDGMENTS We thank the Metabolome Facility of Bordeaux Functional Genomics Centre. We are indebted to the BIBS Facility [IBISA/BioGenOuest Biopolymers, Interactions, Structural Biology platform (BIBS), UR 1268 BIA, INRA Angers-Nantes, F-44300 Nantes, France] for cell wall analyses. #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00421/ full#supplementary-material flavonoid/anthocyanin accumulation in citrus. BMC Plant Biol. 15:27. doi: 10. 1186/s12870-015-0426-4 Yao, L., Caffin, N., D'arcy, B., Jiang, J., Shi, R., Singanusong, X., et al. (2005). Seasonal variations of phenolic compounds in Australia-grown Tea (Camellia sinensis). J. Agric. Food Chem. 53, 6477–6483. doi: 10.1021/jf050382y 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 Soubeyrand, Colombié, Beauvoit, Dai, Cluzet, Hilbert, Renaud, Maneta-Peyret, Dieuaide-Noubhani, Mérillon, Gibon, Delrot and Gomès. 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. # Dissecting the Variations of Ripening Progression and Flavonoid Metabolism in Grape Berries Grown under Double Cropping System Wei-Kai Chen1,2, Xian-Jin Bai<sup>3</sup> , Mu-Ming Cao<sup>4</sup> , Guo Cheng<sup>4</sup> , Xiong-Jun Cao<sup>4</sup> , Rong-Rong Guo<sup>5</sup> , Yu Wang1,2, Lei He1,2, Xiao-Hui Yang1,2, Fei He1,2, Chang-Qing Duan1,2 and Jun Wang1,2 \* <sup>1</sup> Center for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China, <sup>2</sup> Key Laboratory of Viticulture and Enology, Ministry of Agriculture, Beijing, China, <sup>3</sup> Guangxi Academy of Agricultural Sciences, Nanning, China, <sup>4</sup> Grape and Wine Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China, <sup>5</sup> Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Nanning, China #### Edited by: Soren K. Rasmussen, University of Copenhagen, Denmark #### Reviewed by: José Tomás Matus, Universitat Autònoma de Barcelona, Spain Chonghuai Liu, Zhengzhou Fruit Research Institute (CAAS), China > \Correspondence: Jun Wang jun\[email protected] #### Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 05 June 2017 Accepted: 23 October 2017 Published: 10 November 2017 #### Citation: Chen W-K, Bai X-J, Cao M-M, Cheng G, Cao X-J, Guo R-R, Wang Y, He L, Yang X-H, He F, Duan C-Q and Wang J (2017) Dissecting the Variations of Ripening Progression and Flavonoid Metabolism in Grape Berries Grown under Double Cropping System. Front. Plant Sci. 8:1912. doi: 10.3389/fpls.2017.01912 A double cropping system has been commercially adopted in southern China, where there is abundant sunshine and heat resources. In this viticulture system, the first growing season normally starts as a summer cropping cycle; then, the vine is pruned and forced, resulting in a second crop in winter. Due to climate differences between the summer and winter growing seasons, grape ripening progression and flavonoid metabolism vary greatly. Here, the metabolites and transcriptome of flavonoid pathways were analyzed in grapes grown under two growing seasons at different stages. Notably, the winter cropping cycle strongly increased flavonoid levels by several times in comparison to summer grapes, while the summer season took a major toll on anthocyanin and flavonol accumulation, since the winter cropping greatly triggered the expression of upstream genes in the flavonoid pathway in a coordinated expression pattern. Moreover, the ratio of VviF305 <sup>0</sup>Hs (flavonoid 305 0 -hydroxylase) to VviF30Hs (flavonoid 3<sup>0</sup> -hydroxylase) transcript levels correlated remarkably well with the ratio of 3 05 0 -substituted to 3<sup>0</sup> -substituted flavonoids, which was presumed to control the flux of intermediates into different flavonoid branches. On the other hand, the phenological phase also varied greatly in the two crops. Compared to summer cropping, winter growing season accelerated the duration from budburst to veraison, therefore advancing the onset of ripening, but also prolonging the duration of ripening progression due to the purposes to harvest high-quality grapes. The differential expression pattern of hormonerelated genes between the two cropping cycles might explain this phenomenon. Keywords: transcriptome, secondary metabolism, subtropical climate, double cropping system, Vitis vinifera, flavonoid biosynthesis, ripening # INTRODUCTION The growing season of grapevine is the time of year during which local climatic conditions (i.e., temperature, sunlight, and rainfall) permit its normal growth. In most temperate regions, grapevines undergo dormancy from late fall to early spring, and a single pruning and harvest is the conventional grapevine practice. However, in southern China, which shows extremely high 137* temperature and concentrated rainfall during July and August, this traditional cultivation pattern does not adapt to local climate, and it takes a major toll on crop and grape quality, since these rainy months increase the occurrence of fungal diseases (Bai et al., 2008). In conventional practices, the compound buds remain in a stationary state during the current growing season, and they break dormancy in the following spring (Lavee and May, 1997). However, if these compound buds are forced out of dormancy early during the current season, a double cropping viticulture system can be achieved (Lin et al., 1985; Bai et al., 2008; Gu et al., 2012). Two crops of table grapes per year were achieved in Taiwan by a combination of pruning, defoliation, and chemical treatments (Lin et al., 1985). Similarly, in the Brazilian Southeast, the making of good wines was also attained by a double pruning approach (Favero et al., 2011). Therefore, the farmers in southern China introduced this strategy to overcome all detrimental environment under conventional practices. With the benefit of high total yield and good quality, the double cropping system has great potential in subtropical viticulture regions (Lin et al., 1985). Recently, the winter grapes in Brazil showed physicochemical characteristics more suitable than those from the summer growing season for winemaking purposes, since the summer grapes were featured by higher cluster weight and titratable acidity while the winter crops were characterized by higher total soluble solids (TSS) content and pH value (Júnior et al., 2017). This viticulture system (Figure 1) has been commercially adopted in southern China, in which the first growing season normally starts in March to July; then, the vine is pruned and forced by a cyanamide solution in August, resulting in a second bud break in August, and a second crop until January of the following year (Lin et al., 1985; Bai et al., 2008). In some vineyards, the purpose of the first cropping is not to harvest grapes in summer, but to have the inflorescence primordia differentiated into latent buds (Dias et al., 2017). The second cropping resembles the extending of fruit ripening from summer to the autumn-winter of the growing season (Gu et al., 2012). This double cropping system could minimize the negative impact of a rainy and hot climate with the help of rain-shelter treatment, and it could maximize the use of sunshine and heat resources in subtropical regions. Flavonoids are a group of natural compounds that share a common polyphenolic flavan skeleton. The biosynthetic pathways for anthocyanins, flavonols, and flavan-3-ols in plants share a common upstream step through phenylalanine ammonia-lyase (PAL) to flavanone 3-hydroxylase (F3H). Flavonol synthase (FLS) catalyzes dihydroflavonols to form their corresponding flavonol aglycones, and leads the flavonoid flux into the flavonol branch. On the other hand, leucoanthocyanidins and anthocyanidins can be converted into flavan-3-ols by leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), respectively. As for anthocyanin synthesis, UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) is considered as the key enzyme that determines the formation of anthocyanidin-3-O-glucosides. Due to internal and external differences between grapes, the flux through the flavonoid pathway toward downstream anthocyanins, flavonols, and flavan-3-ols varies significantly (Castellarin et al., 2007; Mouradov and Spangenberg, 2014). The climate of Nanning County in southern China is characterized by long growing seasons with higher temperature months from June to September. Many studies have shown that the flavonoid compounds of grape berries are greatly influenced by climate conditions (reviewed by Downey et al., 2006). High temperature often increases the degradation of flavonoid compounds, resulting in poor coloration (Yamane et al., 2006; Mori et al., 2007; Gu et al., 2012). Due to climate differences between the summer and winter growing seasons, especially the reverse temperature evolution pattern during grape berry development, flavonoid composition and content are often distinct between the two crops (Xu et al., 2011; Zhu et al., 2017). It is understandable that these variances in metabolites have much to do with the transcriptomic difference of grapes between the two growing seasons. Since the first report of the draft genome sequence of the Pinot Noir grapevine (Jaillon et al., 2007), genomewide transcriptome research revealing the regulation of berry ripening and its associated metabolic processes has become a hot topic (Fabres et al., 2017; Wong and Matus, 2017). Transcriptomic analyses also provide a comprehensive approach to study the transcriptional responses of grapes to changing environment conditions (Sun et al., 2017). Water deficit activates the expression of phenylpropanoid pathway transcripts, which increases flavonoid content in wine grape (Grimplet et al., 2007; Deluc et al., 2009; Savoi et al., 2016). Solar ultraviolet radiation triggers regulatory responses through the UV-B radiation-specific signaling pathway, which also results in the activation of phenylpropanoid biosynthesis (Carbonell-Bejerano et al., 2014). The transcriptional expression patterns of flavonoid biosynthesis in wine grapes grown in two regions with distinct climates also showed regional differences (Li et al., 2014). Furthermore, several mRNA expression profiling studies have been reported that show a detailed analysis of gene expression during grape berry development (Deluc et al., 2007, 2009; Sweetman et al., 2012; Degu et al., 2014). However, to the best of our knowledge, the transcriptomic analysis of seasonal variation on grape flavonoid compounds has not been reported within a double cropping system. Thus, in the present study, we performed transcriptome and metabolite analysis of flavonoid biosynthesis both in summerand winter-grapes of Vitis vinifera L. cv. "Cabernet Sauvignon" (CS) and V. vinifera cv. "Riesling" by RNA-sequencing and HPLC-ESI-MS/MS. Comparative analysis of the transcript and metabolite profiles revealed season- or cultivar-specific patterns of flavonoid biosynthesis. This paper provides insights into the mechanisms of growing season influence on flavonoid accumulation. #### MATERIALS AND METHODS ### Experimental Vineyard and Double Cropping Viticulture Practices The experiment was conducted in 2014 and 2015 on 7-year-old grapevines grafted onto SO4 rootstock in the vineyards of the Guangxi Academy of Agricultural Sciences, located in southern China (22◦ 360 6400N, 108◦ 140 1300E, altitude 104 m), where it is typically a subtropical humid monsoon climate with abundant sunshine and heat resources. The vines were planted in east-west rows under a rain-shelter treatment with an inter- and intra-row vine spacing of 3.5 m × 1.5 m and were managed in a closing Y-shaped training system with 2 × 4/5 shoots per meter and 1.0 m cordon above ground. Two widely planted wine grape cultivars, "CS" and "Riesling," were selected for research. The grape viticulture regime in the subtropical region is dominated by double cropping systems (Lin et al., 1985; Bai et al., 2008). In these cultivation practices, the vines are pruned twice and the grapes are harvested twice per year. To produce the first crop, the vines were pruned and enforced with 2.5–3.0% hydrogen cyanamide in mid-February when the temperature was maintained above 10◦C. The terminal bud was not sprayed to avoid apical dominance. In addition, the soil was kept moist to accelerate germination. Then, the grapevines were in full bloom around mid-April, while the veraison stage ranged from mid-June to early July, followed by the harvest stage (summer grape). This whole period was termed the summer cropping cycle. Then, the vines were pruned and forced again in August, leading to the second crop (winter grape) in early January of the following year. In detail, the grapevines were pruned on approximately 20th August, were manually defoliated, and an average of 5–10 buds were left for each cane. Hydrogen cyanamide was smeared only at the terminal bud and in the vicinity of the pruning-wound surface. Then, the bud burst 5–8 days later and initiated the winter cropping cycle. Grape berries in three biological replicates were collected at four E-L stages (Coombe, 1995) according to berry color and TSS (◦Brix) for each crop as follows: pea-size berries (E-L 31), the onset of veraison (E-L 35), the end of veraison (E-L 36), and the harvest stage (E-L 38). In brief, 300 berries were selected from both the sunny and shady sides of at least 50 whole vine selections, among which 100-berry sub-samples were processed immediately to determine the physicochemical parameters. The remaining samples were frozen in liquid nitrogen and stored at −80◦C for subsequent RNA and flavonoid extraction. TSS of the juices was determined with digital pocket handheld refractometer (PAL-1, Atago, Japan), and titratable acidity was measured by acid–base titration. # Extraction of Flavonoid Compounds The fresh skins were peeled from the grapes and were immediately ground into powder in liquid nitrogen. Afterward, the skin powder was lyophilized at −50◦C and used for extraction of anthocyanin, flavan-3-ol, and flavonol. The anthocyanins and flavonols were simultaneously extracted in two analytical replicates according to a previous report (Downey et al., 2007). Three aliquots of grape skin powder (0.10 g) were immersed in 1.0 ml of 50% methanol in water, were ultrasound sonicated for 20 min, and were centrifuged. Then, the supernatant was collected and the residues were re-extracted again. All the supernatants were mixed, filtered through a 0.22-µm nylon membrane, and transferred to HPLC auto-sampler vials. The extraction of flavan-3-ol was also conducted twice for each sample according to method described by Liang et al. (2012). Grape skin powder (0.10 g) was mixed with 1 ml of phloroglucinol buffer (0.5% ascorbate, 300 mM HCl, and 50 g/l phloroglucinol in methanol), incubated at 50◦C for 20 min, neutralized with 1 ml of sodium acetate (200 mM, pH 7.5), and finally centrifuged at 8000 × g for 15 min. This procedure was repeated three times, and the supernatants were combined and filtered for HPLC analysis. #### Analysis of Flavonoid Compounds Analysis of flavonoid compounds was carried out using an Agilent 1200 Series HPLC–MSD trap VL equipped with a variable wavelength detector (for flavonol) or a diode array detector (for flavan-3-ol and anthocyanin). The mass spectrometric acquisition parameters were as follows: ESI interface, positive ion mode (for anthocyanin) or negative ion mode (for flavonol and flavan-3-ol), 35 psi nebulizer pressure, 10 ml/min drying N<sup>2</sup> flow rate, 350◦C drying N<sup>2</sup> temperature, capillary voltage 3000 V, and scans at m/z 100–1000. Anthocyanin extract was injected onto a Kromasil C18 column (250 mm × 4.6 mm, 5 µm). The mobile phases A and B were aqueous 2% formic acid and acetonitrile containing 2% formic acid, respectively. The flow rate was 1.0 ml/min, and the solvent gradients were as follows: from 6 to 10% B over 4 min, from 10 to 25% B over 8 min, isocratic 25% B for 1 min, from 25 to 40% B over 7 min, from 40 to 60% B over 15 min, from 60 to 100% B over 5 min, and from 100 to 6% B over 5 min. Other conditions were as follows: injection volume, 30 µl; detection wavelength, 525 nm; and column temperature, 50◦C. Flavan-3-ol compounds were separated on a reversed phase Zorbax SB-C18 column (250 mm × 4.6 mm, 5 µm) using mobile phase A of aqueous 0.2% acetic acid and mobile phase B of acetonitrile: 0.2% acetic acid (4:1) at a flow rate of 1 ml/min and were monitored at 280 nm at 25◦C. The elution gradients of solvent B were as follows: 0 min, 10% B; 20 min, 10% B; 30 min, 15% B; 40 min, 20% B; 50 min, 33% B; 55 min, 40% B; 58 min, 100% B; 63 min, 100% B; and 64 min, 10% B. For flavonol separation, mobile phase A was a mixture of formic acid:acetonitrile:water (85:50:865) and B was formic acid:acetonitrile:water:methanol (85:250:215:450). The column selected was a Zorbax SB-C18 (4.6 mm × 250 mm, 5 µm), with the temperature maintained at 40◦C. The gradient conditions were as follows: 0% B over 7 min; 24.2 min, 14.2% B; 27 min, 15.7% B; 39 min, 23.5% B; 45 min, 26% B; 51.6 min, 32% B; 61.8 min, 40% B; 62.3 min, 60% B; 67.8 min, 100% B; and 78.4 min, 0% B. The flow rate was 0.63 ml/min, and the detector wavelength was 360 nm. # Transcriptome Sequencing and Data Analysis Three biological replicates for each sample were performed. A sub-sample of 50 berries were randomly selected from each biological replicate for RNA extraction. Total RNA was extracted from the frozen deseeded berries (whole pericarp) at three development stages (E-L 35, 36, and 38) using a SpectrumTM Plant Total RNA Kit (Sigma–Aldrich, Carlsbad, CA, United States) to conduct transcriptome analysis on the Illumina HiSeqTM 2000 platform with 50-bp single reads and were then aligned against the reference grapevine genome 12×V2, allowing no more than two mismatches. Transcriptome de novo assembly was conducted using the short reads assembling program Trinity with a fixed k-mer length of 25. To determine gene expression levels, the longest transcript was chosen to calculate the fragments per kilobases per million reads (FPKM) value when more than one transcript was obtained for a single gene (Mortazavi et al., 2008). For the functional annotation, unigene sequences were aligned to databases as described previously (Li et al., 2014). Differentially expressed genes (DEGs) between the samples were identified by the R package called "DESeq2." A false discovery rate ≤0.01 and a fold change ≥2 were set as the threshold to judge the significance of gene expression differences. Gene Ontology (GO) and KEGG enrichment analysis of DEGs was used to select candidate genes. The data have been deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible through GEO accession GSE103226. # Statistical Analysis Heatmap visualizations were performed using the R package "pheatmap." Principal component (PC) analysis was done using MetaboAnalyst 3.0<sup>1</sup> . A one-way analysis of variance (ANOVA) was used to measure the differences between flavonoid contents employing Duncan's multiple range tests at p < 0.05. The column plots were prepared using OriginPro 9.2 (OriginLab Corporation, Northampton, MA, United States). # RESULTS ### Meteorological Data and Phenological Characteristics The environmental condition in southern China corresponds to a typically subtropical humid monsoon climate, which is characterized by a hot and humid summer and a mild to cool winter (Supplementary Table S1). The climatic conditions displayed great differences between the two growing seasons (Figure 2). Basically, the daily temperature showed reverse <sup>1</sup>http://www.metaboanalyst.ca/faces/home.xhtml evolution patterns in the two growing seasons from budbreak to harvest. In the summer cropping cycle, the temperature was low at an early stage, then it increased gradually until the veraison and ripening stages. The mean temperature in the summer season approached 30◦C, and the daily maximum temperature frequently exceeded 35◦C, which has been considered as detrimental to plant growth and anthocyanin accumulation (Cohen et al., 2012; de Rosas et al., 2017). While in the second cropping cycle, the temperature dropped from the point of flowering until the ripening process, with a mean temperature of approximately 20◦C during the entire winter cropping. In addition, the extremely high temperature hours in the winter season during grape berry ripening was almost negligible, only 2 h versus 210 h in the summer. Another important difference in climate was rainfall. Southern China always experiences abundant and concentrated rainfall in the summer or, to a lesser extent, in the autumn. The heavy rainfall in June and July could greatly reduce grape quality, making the double cropping practice appealing, as this approach harvested double crops and shifted fruit ripening from the hot and rainy summer (July and August) to the mild and cool winter (January) in each growing season (Bai et al., 2008). However, the sunshine hours during berry development, ranging from fruit-set to harvest, were distinctly higher in summer cropping in both years, and this was also the case for the growing degrees days and photosynthetically active radiation. Diurnal temperature between day and night was another important climatic index (Cohen et al., 2012), and it was 1–4◦C lower in winter cropping. Due to climate difference in the two growing seasons, the phenological phase also varied greatly in the two crops (Supplementary Table S1). The entire period of berry development in winter cropping, from fruit-set to full-ripeness, was longer than or equal to that of the summer cycle, but the duration of the early phenological phase was hastened and the late progression was delayed. The winter cropping was characterized by shorter green and veraison stages, but a longer berry ripening stage. Compared to stage durations of cv. "CS" described in single cropped vineyards (Li et al., 2014), both crops showed shorter berry development periods partially due to higher temperature. The grapes of summer crop showed a longer veraison stage than that from single cropped vineyards, which was nearly equal to that in winter season. Comparing the ripening duration, the single cropped grapes ranged between the summer crop and winter crop herein (Li et al., 2014). With regard to whole duration from flowering to harvest, the years of 2014 and 2015 showed opposite results. The difference on the ripening duration in the winter cropping was behind this phenomenon, resulting from distinct differences in rainfall. It was noted that "CS" and "Riesling" demonstrated similar flowering, veraison, and harvest dates under the double cropping system. To determine the impact of the cropping cycles on berry development and flavonoid metabolism, we collected berries at four development stages for each crop. The berries in summer cropping showed higher TSS at E-L 31, but showed an opposite result at E-L 38, and no significant difference was found at E-L 35 and E-L 36 (Figure 3). Berries sampled from both crops in both cultivars showed similar patterns of physicochemical characteristics during grape berry development (Supplementary Figure S1). Berry weight increased along with berry development, but the berries in the winter cropping were significantly smaller than those of the summer cropping at E-L 35, 36, and 38. The same phenomenon also occurred in berries from two separate years, with smaller berries in 2014 than that in 2015, which coincided with rainfall. #### Impact of Cropping Season on Flavonoid Metabolites across Berry Development The flavonoid compounds were analyzed to explore the effect of the double cropping system on grape berries in a subtropical region and to compare the metabolites with the transcriptome. A total of 14 and 8 flavonol glycosides were identified in "CS" and "Riesling," respectively, which corresponded to six types of free aglycone in "CS" and three types in "Riesling" (Supplementary Table S2). The content of total flavonols in both "CS" and "Riesling" was significantly higher in winter grapes than the content in summer grapes, except for a single point of E-L 31 of "Riesling" in 2015 (Figure 4), in agreement with the data reported by Zhu et al. (2017). The total flavonol content showed an increasing trend for "CS," but a declining trend for "Riesling." It has been suggested that kaempferol-type, quercetin-type, and isorhamnetin-type flavonols are generally present in both red and white grapes, while myricetin-type flavonol and its derivatives are only accounted for in V. vinifera red grapes, which is in agreement with the present study (Castillo-Muñoz et al., 2007, 2010). For "CS" grapes, quercetin and myricetin were the most abundant flavonols, the proportion of which showed an inverse relationship during berry development. Quercetin accounted for more than 90% of the flavonol at stage E-L 31, while at E-L 38, its proportion reduced to 60–70% in summer grapes and 40–50% in winter grapes. Similarly, the proportion of glucoside and glucuronide also varied with the development stage and growing season. The green stage featured 3-O-glucuronides, while the harvest stage was characterized by 3-O-glucosides. In addition, the proportion of 3-O-glucosides at E-L 38 was 10% lower in winter grapes than in summer grapes. However, in "Riesling" grapes, the flavonol content was dominated by quercetin and 3- O-glucuronide, and their proportion showed no consistent trend versus the developmental stage. The anthocyanins in "CS" displayed progressive accumulation in both crops across grape development. The content of total anthocyanins at E-L 38 in winter grapes, which was 2047.4 mg/kg FW in 2014 and 1682.3 mg/kg FW in 2015, was almost 10-fold higher than the content in summer grapes (Figure 4). In addition, the individual accumulation patterns of delphinidin-3- O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, and malvidin-3-O-glucoside, and their acylated derivatives all showed similar trends (Supplementary Table S2). Malvidin-3-O-glucoside and its acylated derivatives accounted for 80% of the anthocyanins in summer grapes, while in winter grapes, the proportion of malvidin was significantly lower, dropping to 45% in 2014 and 60–70% in 2015. In addition, there was an increase in the percentages of delphinidin and petunidin in winter grapes in both years, but an increase in the proportion of cyanidin and peonidin occurred only in 2014. Thus, the content of 3<sup>0</sup> 5 0 -substituted anthocyanins increased drastically in winter grapes relative to summer grapes, but its proportion sometimes decreased. Additionally, the ratios of methylated/non-methylated and acylated/non-acylated anthocyanins also showed significant differences between the two growing seasons, with higher methylation and acylation levels in summer grapes. In fact, the proportion of acylated anthocyanins was previously shown to greatly decrease with exposure to sunlight and high temperatures (Mori et al., 2007; Tarara et al., 2008). Flavan-3-ols, the immediate competitors of the precursors for flavonol and anthocyanin synthesis, showed a mild reduction in total content as the stage progressed in the summer crop and maintained a relatively stable level in winter grapes, with the exception of "Riesling" in the 2015 winter crop, which displayed a transient peak at E-L 35, indicating the relatively limited effects of the cropping cycle on flavan-3-ols (Figure 4). The content of flavan-3-ols was higher in "CS" berries than "Riesling" berries, as well as flavonol. Comparing the total flavan-3-ol content between the two cropping cycles, the result in 2014 showed significant differences, but no significant difference was found in 2015. It seemed that the summer grapes had more flavanol at E-L 31, after which were reduced to lower levels than that in winter crops at later stages. Zhu et al. (2017) also found higher flavan-3-ol levels in winter grapes of cv. "Muscat Hamburg" at maturity, but no consistent trend was found in cv. "Khoyo." In regard to flavanol profiles, epicatechin, as expected, was the most abundant fraction in both varieties, and its proportion was slightly but significantly higher in winter grapes at E-L 38. Epicatechin-3-O-gallate was the second main constituent, and it accounted for approximately 20% of the total composition, but showed no detectable differences between the two growing seasons. Dihydrokaempferol represents the branching node in the flavonoid pathway, and it converts to dihydroquercetin and dihydromyricetin with the catalysis of F30H (flavonoid 3 0 -hydroxylase) and F3<sup>0</sup> 5 <sup>0</sup>H (flavonoid 3<sup>0</sup> 5 0 -hydroxylase), respectively, giving rise to the 3<sup>0</sup> -substituted and 3<sup>0</sup> 5 0 -substituted flavonoid compounds (Degu et al., 2014). Since almost no 3 0 5 0 -substituted flavonoids existed in "Riesling" grapes, we only analyzed the proportion of 3<sup>0</sup> -hydroxylated and 3<sup>0</sup> 5 0 hydroxylated flavonoids in "CS" (Figure 4). At E-L 31, there was no significant difference among the four groups in the proportion of 3<sup>0</sup> -substituted flavonoids (approximately 85%). Then, their proportion gradually decreased and displayed significant differences at E-L 38, approaching 65–80% in summer grapes and 35–50% in winter grapes at E-L 38, due to an abundant accumulation of 3<sup>0</sup> 5 0 -substituted anthocyanins and flavonols. # Transcriptomic Changes of Flavonoid Pathway under Double Cropping System The genetic control of the flavonoid pathway is well known, but the mechanisms of seasonal influence in the double cropping system are poorly understood. Therefore, one of the goals in this study was to explore the molecular changes in the berries under different cropping cycles and to correlate these changes with FIGURE 5 \| Principal component analysis of the whole normalized gene expression dataset. Four different colors (one per each type of sample: CS-W, CS-S, R-W, R-S) and then for each color three different tones (35, 36, and 38 EL stages) were used to represent different samples. Ellipses encircle the three replicates of each sample subjected to the same stage. CS/R, "Cabernet Sauvignon"/"Riesling"; S/W, summer/winter crop. metabolite accumulation. Berries of three selected developmental stages (E-L 35, 36, and 38) from summer and winter cropping in 2014 were chosen to perform transcriptome analyses by RNA-Seq. PC analysis for the whole normalized gene expression was performed and most three biological replicates of each sample were well-grouped (Figure 5). PC1 explained 25.4% of the total variance in gene expression and separated the ripening stage of winter crop from both green and veraison stages of "Riesling" sample. PC2 explained 15.7% of the total variance and clearly separated "CS" at ripening stage of winter crop from other "CS" samples, and separation between two varieties can also be distinguished on PC2 (Figure 5). The expression patterns of the transcripts involved in the phenylpropanoid and flavonoid pathways were depicted as FPKM values across three developmental stages in Supplementary Table S3. In addition, the influences of cropping season on the expression of flavonoid genes were expressed as the log2-fold change of the transcript abundance in winter cropping compared to summer cropping (Figure 6). A subset of DEGs participating in multiple branches of flavonoid metabolism was identified in two varieties. The early phenylpropanoid pathway, acting upstream of the flavonoid pathway branch, provides the precursors of p-coumaroyl-CoA for subsequent phenolic biosynthesis. The winter cropping displayed a distinct and significant upregulation of most upstream genes during ripening, among which one unigene of Vvi4CL (4-coumarate: CoA ligase, VIT\_206s0061 g00450) was downregulated in both cultivars. Interestingly, a flavonoid pathway transcripts are depicted in dotted red rectangle. The complete data set can be accessed in Supplementary Table S3. recent study conducted on red blotch-infected berries found 25 suppressed genes in phenylpropanoid metabolism, while this unique Vvi4CL was induced by the disease during berry ripening (Blanco-Ulate et al., 2017). It is noteworthy that p-coumaroyl-CoA is a branch point toward flavonoid and stilbene synthesis. In the present study, the transcripts encoding stilbene synthase (STS) were increased in "CS" during the ripening process, while they decreased in "Riesling" across the three developmental stages (Supplementary Table S3). The VviSTS unigenes were significantly modulated by the growing season, and their transcripts were distinctly induced as the berry developed from veraison to post-veraison in both cultivars, which coincided with the accumulation of stilbenes at the onset of ripening (Deluc et al., 2011). In parallel, the cropping season greatly triggered the expression of upstream genes in the flavonoid pathway, the transcripts of which showed coordinated expression patterns with genes in the phenylpropanoid pathway, ensuring a sufficient quantity of precursors for the subsequent synthesis of flavonoid compounds. The flavonoid metabolic pathway shares a common upstream route, and then the flux is diverted to 3<sup>0</sup> -hydroxylated or 3<sup>0</sup> 5 0 hydroxylated flavonoids separately via the enzyme F30H or F3<sup>0</sup> 5 <sup>0</sup>H. Similar with upstream genes, including two VviF3H (VIT\_204s0023g03370, VIT\_218s0001g14310), two VviF30H (VIT\_203s0063g01690, VIT\_209s0002g01090), and six VviF3<sup>0</sup> 5 <sup>0</sup>H (VIT\_206s0009g02810, VIT\_206s0009g02830, VIT\_206s0009g02840, VIT\_206s0009g02970, VIT\_206s0009 g03000, VIT\_206s0009g03010), were significantly upregulated around the veraison stage in the winter berries versus the summer berries. Of particular interest were the transcripts encoding the F3<sup>0</sup> 5 <sup>0</sup>H enzyme, which were mainly expressed in the red grapes of "CS." The transcript levels of VviF3<sup>0</sup> 5 0H were extremely low in "Riesling," suggesting that a different transcriptional regulation mechanism for VviF3<sup>0</sup> 5 <sup>0</sup>H existed in white and red varieties (Matus et al., 2017). The simultaneous upregulation of both VviF30H and VviF3<sup>0</sup> 5 <sup>0</sup>H transcripts in winter berries compared to summer berries of "CS" also modulated the relative abundance of the different flavonoid forms. In the current study, the ratio of VviF3<sup>0</sup> 5 <sup>0</sup>Hs to VviF30Hs levels in "CS" was significantly and distinctly higher in winter cropping berries during ripening, in parallel with their higher ratio of 3<sup>0</sup> 5 0 -substituted to 3<sup>0</sup> -substituted flavonoids, which was presumed to control the flavonoid composition of grape berries. Similarly, a previous study also found that the temporal and variety-specific expression of VviF30H and VviF3<sup>0</sup> 5 <sup>0</sup>H in grapes occurred in coordination with the accumulation of the respective hydroxylated metabolites (Bogs et al., 2006; Sun et al., 2016). The downstream flux of flavonoid metabolism in grape berries included multiple branches, and the related genes showed divergent expression patterns across the three developmental stages in the two cropping cycles. Contrary to the coordinated upregulation of upstream genes, a subset of transcripts involved in the late biosynthetic pathway was significantly repressed at some stages, such as VviDFR (dihydroflavonol 4-reductase, VIT\_215s0048g01010, VIT\_219 s0014g04980, VIT\_202s0025g01260), VviFLS (VIT\_218s0001g 03430), VviLDOX (leucoanthocyanin dioxygenase, VIT\_208 s0105g00380), and VviGT5 (UDP-glucuronic acid: flavonol 3-Oglucuronosyltransferase, VIT\_211s0052g01600), whereas other transcripts, such as VviDFR (VIT\_216s0039g02350, VIT\_218 s0001g12800), VviFLS (VIT\_211s0118g00390, VIT\_218s000 1g03470), VviLDOX (VIT\_202s0025g04720), and VviLAR (VIT\_201s0011g02960, VIT\_217s0000g04150), were significantly upregulated in many cases. Of the five known VviFLS genes, the enzyme of which commits to flavonol biosynthesis, only two transcripts of VviFLS4 (VIT\_218s0001g03470) and VviFLS5 (VIT\_218s0001g03430) were expressed, and they showed differential expression patterns in the two cropping cycles (Fujita et al., 2006). In this study, the transcript level of VviFLS4 was extremely low, and it was only upregulated by winter cropping at E-L 38 in "CS" and at E-L 35 in "Riesling." Genes encoding two previously characterized flavonol glycosyltransferases, VviGT5 and VviGT6 (UDP-glucose/UDP-galactose: flavonol-3- O-glucosyltransferase, VIT\_211s0052g01630), were co-expressed with VviFLS4, which is in agreement with a previous report (Malacarne et al., 2015). Czemmel et al. (2017) also demonstrated positive correlation of VviMYBF1 with novel genes of the flavonol pathway, VviGT3 (flavonol glycosyltransferase, VIT\_211s0052g01580) and VviRhaT1 (flavonol rhamnosyltransferase, VIT\_200s0218g00170), during berry development. In the present study, the transcript of the former was detected at low levels (FPKM < 0.1), while the latter was expressed with a FPKM ranging from 5 to 15 (Supplementary Table S3). Additionally, VviRhaT1 was significantly upregulated at E-L 35 in "CS," which might also account for the higher flavonol in winter grapes. LAR and ANR are key regulators of flavan-3-ol and proanthocyanidin biosynthesis. The expression of VviANR (VIT\_200s0361g00040) was higher in winter grapes at E-L 35, but no significant difference was found. Winter cropping significantly upregulated VviLAR1 (VIT\_201s0011g02960) at E-L 36 and VviLAR2 (VIT\_217s0000g04150) at E-L 38 in "CS," while in "Riesling," only VviLAR2 was significantly affected, with an upregulation at E-L 36 and a downregulation at E-L 38. Comparing the expression patterns between the two seasons, VviLAR2 in "Riesling" kept a moderate and stable level in summer cropping, while the transcript in winter cropping increased to twofold higher at veraison, and then it dropped sharply at harvest. Hence, it may be speculated that the transcript of VviLAR2 leads to the peak flavan-3-ol content at E-L 35 in winter grapes. Furthermore, there is a range of anthocyanin biosynthetic enzymes relevant to the glycosylation, methylation, and acylation events of anthocyanin, such as UFGT (Boss et al., 1996), anthocyanin O-methyltransferase (AOMT; Fournier-Level et al., 2011), and anthocyanin acyltransferase (3AT; Rinaldo et al., 2015). The expression patterns of these genes involved in anthocyanin modification across three developmental stages were similar with the expression patterns of the upstream metabolic genes, the transcripts of which peaked at E-L 36, with higher levels in the winter cropping than in the summer cropping. The expression of VviUFGT, catalyzing the formation of anthocyanin-3-O-glucosides, is critical for berry coloration (Boss et al., 1996). The constantly higher expression of VviUFGT in winter cropping correlated remarkably well with the greater abundance of anthocyanins in winter grapes than summer grapes. The two AOMTs responsible for anthocyanin methylation (Fournier-Level et al., 2011) were also significantly upregulated in winter cropping, which failed to explain the methylation variation, since the proportion of malvidin was dramatically decreased in winter grapes. It was noticed that two transcripts, VIT\_205s0062g00300 and VIT\_205s0062g00310, exhibited similar patterns in the two cultivars, but they differed in their magnitude of abundances; these transcripts might encode a UDP-glucose: anthocyanidin 5,3-O-glucosyltransferase (53GT) with homology to a flavonol glucosyltransferase-like protein. The winter cropping significantly downregulated the expression of Vvi53GT in "Riesling" at E-L 31, and then upregulated it at E-L 38. In fact, the real role of VIT\_205s0062g00300 and VIT\_205s0062g00310 in grapes still needs further research, and the reason for their high abundances in "Riesling" is unclear. # Transcriptional Modulation of Flavonoid Biosynthesis Two classes of genes are required for flavonoid biosynthesis in grapes, the first class is structural genes that encode enzymes in the metabolic pathway, and the second class includes regulatory genes that control the transcription of these biosynthetic genes. The flavonoid pathway genes are known to be coordinately controlled by the interactions of R2R3–MYB, basic helix-loophelix (bHLH), and WD40-repeat transcription factors (TFs) in response to developmental cues or external stress factors, with MYB being central to the transcriptional complexes (Heppel et al., 2013). The ternary complex of MYB/bHLH/WD40 binds to responsive elements in the promoters of biosynthesis genes, activating transcription of genes in the pathway. While many structural genes were significantly upregulated in winter cropping berries, many previously identified regulatory genes were either weakly affected or were not affected by cropping cycle (Supplementary Table S3). VviMYBF1 regulates a narrow set of genes involved in flavonol biosynthesis, potentially comprising the genes VviCHS (chalcone synthase) and VviFLS4 (also named VviFLS1; Czemmel et al., 2009, 2017). The expression of VviMYBF1 was very low in both varieties, and its transcript abundance decreased across the three developmental stages, similar to a previously reported expression pattern in developing Shiraz berries (Czemmel et al., 2009). The present study showed an upregulation of VviMYBF1 in the winter cropping cycle at E-L 38/E-L 35 in "CS"/"Riesling" (Figure 6), that was not well correlated with the expression pattern of VviFLS4. With respect to the regulation of proanthocyanidin-specific biosynthesis, many regulators have been recently characterized, including VviMYBPA1, VviMYBPA2, and VviMybPAR (Bogs et al., 2007; Terrier et al., 2009; Koyama et al., 2014). In addition, VviMYB5a and VviMYB5b, as well as the negative repressors VviMYBC2-L1 and VviMYBC2-L3, regulate the genes involved in proanthocyanidin biosynthesis and several steps in the upstream pathway (Deluc et al., 2006, 2008; Cavallini et al., 2015). The genes encoding the above TFs were expressed at some stage during ripening, and some of them showed similar expression patterns between the two varieties. It has been reported that MYB5a and MYB5b tightly exert their regulation in a temporal way during berry development, with MYB5a predominantly acting in the early stages and MYB5b acting near the later ripening process (Deluc et al., 2008; Matus, 2016). Similarly, the transcript profiles of VviMYB5a and VviMYB5b were distinct during ripening, with peak levels of VviMYB5a at E-L 35 and peak levels of VviMYB5b at E-L 38. The transcript levels of VviMYBPA2, VviMYBPAR, and VviMYBC2-L2, on the other hand, remained low throughout berry development, and they were not differentially expressed between the two cropping cycles. The expression of VviMYBPA1 was significantly downregulated in winter cropping berries at E-L 35 and E-L 38 in both varieties, which correlated well with the expression of two VviDFR transcripts (VIT\_219s0014g04980, VIT\_202s0025g01260). Interestingly, the expression of two negative regulators, VviMYBC2-L1 and VviMYBC2-L3, was significantly upregulated at E-L 36 by winter cropping in "CS"; VviMYB4a was significantly upregulated as well, and it might negatively regulate flavonoid or phenolic acid synthesis (Cavallini et al., 2015). MYB14 and MYB15 were two TFs demonstrated to specifically activate STS genes (Höll et al., 2013). The expressions of VviMYB14 and VviMYB15 matched well with STSs profiles in "CS" rather than in "Riesling," suggesting different transcriptional regulation of stilbene biosynthesis in two varieties. In V. vinifera grapes, two VviMYBA genes in a single gene cluster are responsible for berry color variation and anthocyanin accumulation, among which are the VviMYBA1 and VviMYBA2 genes encoding putative regulators of anthocyanin biosynthesis in red grapes (but these genes are non-functional in white grapes), while VviMYBA3 is only statistically associated with berry color without functional validation (Walker et al., 2007; Fournier-Level et al., 2009). These three VviMYBAs showed similar expression patterns in "CS," namely, they were significantly upregulated by winter cropping, especially at E-L 35 and E-L 36, in parallel with the expression profiles of VviUFGT, VviAOMT, and Vvi3AT, and the evolution of anthocyanin levels. More recently, two bHLH proteins of VviMYC1 (VIT\_207s0104g00090) and VviMYCA1 (VIT\_215s0046g02560) were demonstrated to promote anthocyanin accumulation in cooperative interaction with VviMYBA1 (Matus et al., 2010; Hichri et al., 2011), the transcripts of which showed no significant variation between the two cropping cycles. VviWDR1 (VIT\_216s0098g00870), which contributed positively to the accumulation of anthocyanins, was downregulated by winter cropping at E-L 38, contrary to the expression change of VviMYBA1. In fact, apart from the widely acknowledged MYB, bHLH, and WD40 TFs, some regulators that belong to the WRKY, AP2/ERF, MADS-box, GATA, and bZIP families were also positively or negatively involved in flavonoid metabolisms (reviewed by Hichri et al., 2011). A recently identified TF of VvibZIPC22 is involved in the regulation of flavonoid biosynthesis, the expression of which was induced by UV light, paralleled by accumulation of the VviFLS4 transcript and flavonol compounds (Malacarne et al., 2016). In the present research, VvibZIPC22 was significantly upregulated by winter cropping in both varieties, in good agreement with the accumulation of flavonols. However, no correlation was found between the expression profiles of VviFLS4 and VvibZIPC22. It is relevant to mention the photomorphogenic factors from the bZIP family, which play important roles in mediating light-dependent flavonoid regulation, especially the biosynthesis of flavonols (Loyola et al., 2016; Matus, 2016). Many components in the UV-B signaling pathway that specifically perceive UV-B radiation have been identified in V. vinifera, including UV-B RECEPTOR 1 (UVR1, VIT\_207s0031g02560), two ELONGATED HYPOCOTYL 5 grape homologs (HY5, VIT\_204s0008g05210; HYH, VIT\_205s0020g01090), and two CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1-1, VIT\_212s0059g01420; COP1-2, VIT\_210s0523g00030), which could mediate flavonol biosynthesis in grapes under UV-B exposure (Carbonell-Bejerano et al., 2014; Liu et al., 2015; Loyola et al., 2016). In southern China, UV-B radiation was shown to be higher in the summer, due to stronger sunlight and a longer duration of sunlight, although we did not detect the precise daily dose of UV-B. Concomitantly, most genes involved in the UV-B response pathway were significantly repressed in the winter cropping, except for VviCOP1-1 (Supplementary Table S3), which aligns with the climatic conditions. Solar UV radiation was shown to enhance flavonol accumulation (Carbonell-Bejerano et al., 2014; Loyola et al., 2016). However, the changes in VviUVR1, VviHY5, and VviHYH expression could not match the flavonol variation between the two cropping cycles, which might be resulted from temperature-responsive changes in HY5 levels, since HY5 protein has been shown to degrade at high temperature (Kim et al., 2017; Park et al., 2017). #### Seasonal Response of Plant Hormone-Related Genes Based on many years of cultivation experiences, grape farmers found the progression of ripening was different between the two cropping cycles. Since plant hormones play important roles in the ripening process of fruit, the expression of ABA and ethylene associated genes was analyzed. It was thought that VviNCED (9-cis-epoxycarotenoid dioxygenase) encodes the key enzyme for the bulk of ABA biosynthesis, and its expression correlated well with ABA accumulation (Sun et al., 2010; Young et al., 2012). The transcript abundances of VviNCED2 (VIT\_210s0003g03750) and VviNCED3 (VIT\_219s0093g00550) peaked at E-L 35 in both varieties, and they decreased after veraison. It was clear that the winter cropping upregulated three VviNCEDs in "CS," especially VviNCED3, which showed massive fold-changes in all of the berry stages. In "Riesling," VviNCED3 was upregulated at E-L 35 while VviNCED6 was significantly promoted at E-L 36 (Figure 7 and Supplementary Table S4). The relative expression of genes involved in ABA catabolism, such as ABA 8 0 -hydroxylase (VIT\_202s0087g00710), was also significantly increased in both varieties. ABA plays a crucial role in response to a variety of abiotic stresses, such as drought, salinity, and extreme temperature. Thus, the drought due to low rainfall in the winter cropping had a more pronounced influence on ABA induction. In the ABA signaling pathway, the transcript abundances of VviPYR1/PYL/RCAR (VIT\_205s0077g01550, VIT\_202s0012g01270, VIT\_215s0046g01050) were significantly increased in winter cropping berries, while the expressions of VviPP2Cs (protein phosphatase 2C) were also significantly upregulated, except for two transcripts (VIT\_201s0011g03910, VIT\_206s0004g06840), which showed significant declines after veraison. For VviSnRK2s (sucrose-non-fermenting1-related kinase 2), both upregulated and downregulated transcripts were identified between two cropping cycles. ABA responsive element binding factors play a crucial role in ABA-dependent gene activation. There were no significant changes in the gene expression of ABFs in "Riesling," while significant downregulation was observed in "CS," except for VviABF2 (VIT\_218s0001g10450), which was confirmed to modulate ABA-dependent berry ripening processes, such as the promotion of phenolic synthesis and cell wall softening (Nicolas et al., 2014). Considering the important role of ethylene in the ripening process and its cross-talk with ABA (Sun et al., 2010; Cramer et al., 2014), the expression changes in the ethylene biosynthesis and signaling pathways were also analyzed (Supplementary Table S4). Similar to the role of NCED in ABA biosynthesis, 1-aminocyclopropane 1-carboxylate oxidase (ACO) is known to determine the production of ethylene. The transcript accumulation of VviACO1 was suggested to match the occurrence of the ethylene peak in "CS" clusters (Chervin et al., 2004). The study here also showed decreasing expression levels of VviACO1 (VIT\_200s2086g00010) as berry ripening progressed from veraison, except for a sudden rise in the summer grapes of "Riesling" at harvest. The expression of putative VviACO1 and three other candidate genes (VIT\_205s0049g00280, VIT\_205s0049g00390, VIT\_205s0049g00420) were significantly repressed in winter grapes in both varieties in comparison to their expression in the summer cropping. The VviACO2 gene was expressed at high levels throughout berry development, with a significant upregulation in the winter berries of "CS" at postveraison. Only one transcript of VviACS (1-aminocyclopropane 1-carboxylate synthase, VIT\_202s0025g00360) was differentially expressed between the two cropping cycles, which peaked at E-L 35 and had higher levels in "Riesling" berries. Interestingly, winter cropping repressed the ethylene biosynthesis pathway, while the ethylene signaling pathway was greatly induced. Particularly, many genes involved in ethylene signaling, such as the ethylene receptors ethylene response 2 (ETR2) and ethylene insensitive 4 (EIN4), the negative regulators constitutive triple response 1 (CTR1), EIN3-binding F-box 1 and 2 (EBF1/2), and ethylene response factor (ERF), were significantly upregulated in winter cropping at different stages in both varieties. Auxin plays positive roles in plant growth and delays ripening-associated processes. In grape berries, auxin is produced through the combined action of tryptophan aminotransferase related (TAR) and YUCCA (YUC) proteins in a two-step biosynthesis pathway (Böttcher et al., 2013). As expected, many transcripts in the auxin biosynthesis and signaling pathways were expressed in developing berries, peaking at E-L 35. Gene expression of the putative auxin biosynthesis members from the TAR and YUC families was analyzed. Four members of VviTAR were downregulated in "CS," but only VviTAR1 and VviTAR2 were differentially expressed. Comparing the three developmental stages, the transcript of VviTAR1 peaked at E-L 36 in summer cropping, while it increased gradually until E-L 38 in winter cropping. Both varieties showed similar expression patterns, but the winter cropping downregulated VviTAR1 in "CS" while it upregulated the transcript in "Riesling." The auxin-influx transporter AUX1 mediates the uptake of auxin, and many transcripts encoding AUX1 were upregulated by winter cropping. Auxin/indole acetic acid (AUX/IAA) proteins are a family of transcriptional repressors that play a central role in auxin response. Most VviAUX/IAA genes were downregulated at E-L 36 in "CS" while in "Riesling," the effects mainly occurred at E-L 38. In addition, there were many transcripts that also showed upregulation at E-L 38 in "Riesling," including VviTAR1 and VviAUX1. Gretchen Hagen 3 (GH3) plays a role in the conjugation of indole-3-acetic acid to aspartate at the onset of grape berry ripening (Böttcher et al., 2011); one putative transcript of FIGURE 7 \| Expression profiles of DEGs related to hormone biosynthesis and signaling pathway during grape development in "CS" and "Riesling" in 2014. The log2-transformed FPKM values (Winter/Summer) are represented by the color map. Blue and red boxes indicate downregulated and upregulated transcripts, respectively, in winter berries versus large berries. The numbers indicate different components. The complete data set can be accessed in Supplementary Table S4. VIT\_203s0091g00310 was upregulated at E-L 35 in winter versus summer grapes, and another of VIT\_219s0014g04690 was promoted at E-L 38. The SMALL AUXIN UP RNA (SAURs) act as positive or negative regulators of auxin synthesis, and their transcription depended on the level of active auxin (Hagen and Guilfoyle, 2002). Downregulation was found to dominate the differential expression of VviSAURs in winter versus summer berries. Among the 10 VviSAURs, two (VIT\_209s0002g00650, VIT\_208s0040g01390) showed consecutive upregulation along three stages in "CS," but in "Riesling," the upregulation only occurred at E-L 38. #### DISCUSSION The metabolism of flavonoids in grapevine is closely correlated with environmental stresses, variety and tissue, developmental cues, and phytohormone regulation. There have been many transcriptomic studies showing the dynamics of gene expression and flavonoid accumulation in response to internal and external stimuli (Degu et al., 2014; Carbonell-Bejerano et al., 2014; Li et al., 2014; Savoi et al., 2016; Blanco-Ulate et al., 2017; Lecourieux et al., 2017; Sun et al., 2017). Here, a comparative parallel analysis was conducted on grapes grown under a double cropping system in two cultivars. To the best of our knowledge, this is the first systemic research of double cropping berries from both transcriptomic and metabolic views during the course of berry development, which enables a comprehensive description of the cropping cycle controlling flavonoid synthesis in grape during ripening. # Winter Cropping Promotes Phenylpropanoid–Flavonoid Metabolism during Berry Ripening Considering that the environmental conditions vary greatly in the two growing seasons, we propose that the climate factors of temperature, light, and rainfall exert great contributions to the variation of flavonoid compounds between the two cropping cycles. Investigations into the influences of climate factors on flavonoid biosynthesis in a vineyard have various approaches at different stages (reviewed by Kuhn et al., 2014). These cultural practices or environmental factors resulted in a range of differences in the levels and profiles of flavonoid compounds in grape berries. The effects of the cropping cycle on flavonoid accumulation are the result of a combination of many climate factors across the whole life cycle in the same vineyard. Notably, these three classes of flavonoids: anthocyanins, flavonols, and flavan-3-ols, respond differently to the climatic stimulus. In grapes, flavonols are important ultraviolet light protectants and play pivotal roles in fresh fruit and the resulting wine. Flavonol accumulated in berries across the entire berry development process, showing an increase from pre-veraison to harvest in cv. "CS" but remaining relatively constant during ripening in "Riesling," which resulted in a different content and profile of flavonol products between the two cropping cycles. It has been reported that flavonols are the flavonoids that are most drastically affected by different light levels, correlating with the expression pattern of VviFLS4 but not with that of VviFLS5 and coinciding with its putative transcriptional regulator, VviMYBF1 (Czemmel et al., 2009; Matus et al., 2009; Carbonell-Bejerano et al., 2014). Contrarily, the berries in the summer cropping, which had abundant sunshine hours and high illuminance, accumulated lower amounts of flavonols than the winter grapes in both varieties. What was more interesting was the expression profiles of VviFLS4 and VviMYBF1; the former was upregulated at the harvest stage of "CS" versus post-veraison of "Riesling," while the latter was upregulated in "CS" but downregulated in "Riesling," and both were expressed at low transcript levels. The VviFLS5 gene was expressed at a level several times higher than VviFLS4 and showed a downregulation in winter cropping in both cultivars. The fact that there was discrepancy in flavonol biosynthesis and the expression profiles of VviFLS and VviMYBF1 in the present study indicated the involvement of post-transcriptional control or other regulators, which require further research. Notably, some differences not correlating well could be due to the fact that both shaded and sunny berries were sampled together. Besides, the deseeded berries were used to extract RNA while the flavonols were isolated from the skin, which also had an effect. Several studies have shown that light radiation plays a profound effect on flavonol synthesis, while temperature has no effect or a weak effect on their content (Spayd et al., 2002; Cohen et al., 2012; Azuma et al., 2015). Thus, the induction of flavonols in response to climatic stresses is a complex process, and the promotion by some factors in the winter cropping was suggested to have a greater effect than the offset from lower light radiation. Downey et al. (2003) displayed the two distinct periods of flavonol synthesis; the first occurred around flowering and the second occurred during berry ripening, which all coincided with the expression of VviFLS4. Therefore, the green berries in the winter cropping accumulated more FLS around fruit-set in response to strong light, which might have also impacted the veraison stage, contributing to larger amounts of flavonol. Additionally, the active upstream pathway in winter cropping could provide a greater abundance of precursors for the production of flavonols in grape skin. UV-B radiation is a key environmental signal, exerting a strong effect on flavonol synthesis in grape berries. Briefly, UV-B is specifically perceived by UVR8, which in turn, function together with a positive COP1 to activate a range of signaling cascades mediated by HY5 and HYH; then, they finally regulate transcription of target genes, resulting in downstream responses (Loyola et al., 2016; Matus, 2016). Flavonol biosynthesis in grapes is suggested to be predominantly stimulated by the UV-B response pathway through the activation of HY5 and HYH (Liu et al., 2015; Czemmel et al., 2017). In addition, VviMYBF1 could act on the UV-B signaling cascade by activating HYH (Czemmel et al., 2017), thus inducing the direct and indirect activation of VviFLS4 and several targets in the phenylpropanoid pathway, such as VviCHS3 and VviGT5 (Carbonell-Bejerano et al., 2014; Loyola et al., 2016). The expression pattern of the UV-B response machinery in the two cropping cycles was correlated with light radiation, but it failed to explain the variations in flavonol content in the current study, since enhanced levels of flavonols normally coincide with the upregulation of genes in the light-signaling pathway (Loyola et al., 2016). Flavan-3-ols are monomeric subunits of condensed tannins in grape skin and seeds, contributing greatly to the body and mouthfeel of wines being produced. However, relatively little is known about the mechanism of environmental impact on flavan-3-ol production in grape skins, despite the fact that it shares common upstream steps with flavonol and anthocyanin. The grape skin had high levels of flavan-3-ols compared with other flavonoids; they accumulated from fruitset until veraison and then declined afterward, in parallel with the expression patterns of VviANR and VviLAR (Bogs et al., 2005). In the present study, the content of total flavan-3 ol in skin was determined in two consecutive years, but it varied only in 2014. The evolution pattern of total flavan-3-ols in grape skins also varied in two cropping cycles. Since the massive production of flavan-3-ols occurred at the green stage, it was hard to correlate the gene expression profiles with its accumulation, as we only conducted RNA-Seq from the onset of ripening. However, a higher expression of VviLAR1 or VviLAR2 might explain the higher levels of flavan-3-ols in the winter cropping. To date, several regulatory TFs related to flavan-3-ol biosynthesis have been found in grape: the MYB positive regulators of VviMYBPAR and VviMYBPA1/PA2 and the MYB C2 repressors of VviMYBC2- L1/L3 (Bogs et al., 2007; Terrier et al., 2009; Koyama et al., 2014; Cavallini et al., 2015), many members of which also worked beyond the phenylpropanoid pathway in addition to VviMYB5a/5b (Deluc et al., 2006, 2008). According to the regulatory mechanisms of flavonoid biosynthesis in grapes, VviMYBPAR, VviMYBPA1, VviMYBPA2, and VviMYB5a are particularly involved in regulation before the onset of ripening, while VviMYB5b also controlled the general flavonoid pathway after veraison (Deluc et al., 2008; Matus, 2016). However, VviMYB5b is more related to anthocyanins in the ripening stage rather than proanthocyanidins (Cavallini et al., 2014). Thus, it was hard to correlate the flavanol evolution pattern with gene expression profiles during ripening. Additionally, Carbonell-Bejerano et al. (2013) also found that elevated temperature promoted the expression of VviMYBPA1, but repressed VviMYB5a, indicating that these regulators responded differently to external stimuli during grape development and that the regulatory network was tightly organized in a spatial and temporal way. As specific metabolites of the flavonoid pathway, anthocyanins are synthesized only in red grapes under the strict control of multiple regulatory factors. A rapid accumulation of anthocyanins in red varieties begins from veraison until harvest, leading to more assimilated carbon flux into the production of anthocyanins rather than flavan-3-ols. The positive regulators VviMYBA1 and VviMYBA2 are specific to controlling the biosynthesis of anthocyanin in red V. vinifera grapes mediated through VviUFGT, while in white grapes, a retrotransposon of Gret1 inserted in the promoter region of the VviMYBA1 gene and two non-synonymous mutations that occurred in the VviMYBA2 coding region disrupt their regulatory function (Boss et al., 1996; Kobayashi et al., 2004; Walker et al., 2007). In the winter cropping of "CS," both VviMYBA1 (fold change > 1.5) and VviMYBA2 (fold change approximately 4.0) were significantly promoted at pre- and post-veraison, they then significantly upregulated the key gene of VviUFGT (fold change > 2.0) at post-veraison, and finally, they contributed to the drastic accumulation of anthocyanins (fold change approximately 10) during ripening. As far as we know, little research has been conducted on double cropping grapes in subtropical climates, except for two contrasting works. One also found higher flavan-3-ol and anthocyanin levels in winter berries at harvest (Xu et al., 2011), while the other one showed no difference between the two cropping cycles at maturity (Chou and Li, 2014). Differences in grape genotype, environmental conditions, and cultural practices might explain this conflicting result. Recent studies also confirmed that high temperature (35◦C) strongly reduced anthocyanin synthesis and enhanced its degradation (Carbonell-Bejerano et al., 2013; Lecourieux et al., 2017). Moreover, the repressors VviMYB4a and VviMYBC2- L1/L3 were significantly upregulated in the winter cropping at post-veraison, in accordance with the potential roles of balancing the inductive effects of activators (Cavallini et al., 2015). Here, it is worth discussing the competition between F30H and F3<sup>0</sup> 5 <sup>0</sup>H in grapes, since the profiles of flavonoids varied significantly in the two cropping cycles, especially for anthocyanins and flavonols. In contrast to the high expression of VviF30H and VviF3<sup>0</sup> 5 <sup>0</sup>H in red grapes, the expression of the VviF3<sup>0</sup> 5 <sup>0</sup>H transcript in white grapes during ripening was extremely low, suggesting a different regulatory mechanism (Bogs et al., 2006). Thus, it was interesting to note an accumulation of the 3<sup>0</sup> 5 0 -substituted compounds of flavan-3 ol (i.e., epigallocatechin), but not that of flavonol (myricetin), in the white "Riesling" grapes. Considering the great "loss" of the F3<sup>0</sup> 5 <sup>0</sup>H pathway in "Riesling" during ripening, we only discussed the competitive relation of F30H and F3<sup>0</sup> 5 <sup>0</sup>H in "CS." The expression patterns of VviF30H and VviF3<sup>0</sup> 5 <sup>0</sup>H coordinated with the upstream pathway genes, peaking around post-veraison, but varying in magnitude. Concurrent with higher the expression ratio of VviF30H to VviF3<sup>0</sup> 5 <sup>0</sup>H in summer grapes, there was an increase in the proportion of 3<sup>0</sup> -substituted flavonoids in the summer cropping, which is similar to a previous result induced by water deficits (Castellarin et al., 2007). Clearly, the prevalence of VviF3<sup>0</sup> 5 <sup>0</sup>H over VviF30H would lead to more dihydromyricetin, the precursor of myricetin, epigallocatechin, and delphinidin, and in contrast, it would yield less dihydroquercetin, the precursor of quercetin, catechin, and cyanidin (Kuhn et al., 2014). The flavonol composition was almost all quercetintype compounds (95%) at the green stage, and this fraction decreased during ripening and dropped to a low level (70% in summer versus 40% in winter) at harvest, while the flavan-3-ols were almost exclusively catechin-type compounds (85%), and its proportion showed no difference regardless of the sample stage or cropping cycle (Supplementary Table S2). With respect to anthocyanin, the effects of the cropping cycle were somewhere in between. The anthocyanin profile showed no consistent difference in 2015, whereas in 2014, the percentage of cyanidin-type compounds was significantly higher in the winter cropping cycle (15% in summer versus 25% in winter). Hence, the work here confirmed that F30H acted as an early flavonoid gene, while F3<sup>0</sup> 5 <sup>0</sup>H worked at later stages (Degu et al., 2014). The higher fraction of these 3<sup>0</sup> -substituted flavonoids at harvest in summer cropping resembled the metabolic profiles of the grapes under heat or sunlight exposure (Guan et al., 2014; Lecourieux et al., 2017). A recent work showed that VviMYBA1 could specifically induce VviF3<sup>0</sup> 5 <sup>0</sup>H and promote tri-hydroxylated fractions (Matus et al., 2017). Therefore, in winter cropping, the induced F3<sup>0</sup> 5 <sup>0</sup>H might divert more flux to the 3<sup>0</sup> 5 0 -substituted sub-branch and might provide more precursors to synthesize anthocyanins and flavonols. However, the production of 3<sup>0</sup> 5 0 -substituted flavan-3-ol reached a plateau in spite of a large amount of precursors, which is likely due to flavonol-specific control or the low substrate activity of LAR for leucodelphinidin. ## Winter Cropping Advances the Onset of Veraison and Accelerates Ripening Progression In fruits, ripening is a complex event that involves major physiological and metabolic changes controlled by plant hormones, which are also signals in response to developmental and environmental cues (Kuhn et al., 2014). However, to date, the ripening mechanism of non-climacteric fruits is still poorly understood, especially with respect to the regulation of phytohormones. Since the climatic conditions varied greatly in the two cropping cycles, it inevitably affected the initiation of veraison and the progression of ripening, which was a good opportunity to learn about the ripening mechanism in grapevines. Understanding how plants respond to environmental stimuli is crucial to improving yield and grape quality in the field. Berry ripening is affected by multiple climatic factors, of which water deficit (Castellarin et al., 2007), light exposure (Matus et al., 2009), and damping diurnal temperature range (Cohen et al., 2012) advance the onset of ripening, while high temperature (Lecourieux et al., 2017) and shading treatment (Matus et al., 2009) delay it. In the current study, the phenological phase varied greatly between the two growing seasons. Winter cropping accelerated the duration from budburst to post-veraison, therefore advancing but also prolonging the subsequent ripening process. The difference in ambient temperature accounted for most of this phenomenon. On the one hand, the optimum temperature range between 25 and 30◦C was associated with higher rates of plant growth (Buttrose, 1969; Cohen et al., 2008), and this was the case for processes from bud-break to flowering in the winter cycle. On the other hand, with respect to grape berries, low temperature and damping diurnal temperature fluctuation in the winter cropping hastened their development and ripening processes (Kliewer and Torres, 1972; Cohen et al., 2008), while the extremely high temperature in the summer might inhibit fruit growth and berry ripening. The photoperiod or the daylength is also considered a fundamental environmental signal that affects phenological development (Sreekantan et al., 2010). The phases of flowering, onset of ripening, bud dormancy, and leaf senescence are modulated by day length, together with other stress factors (Keller, 2010). This seasonal decrease in the photoperiod and temperature might also be associated with the temporal variation in ripening progression. Moreover, berry weight was negatively correlated with the onset of veraison, since the larger berry seemed to be a dilution of the smaller one, needing more time to achieve the accumulation of the primary and secondary metabolites. The reason for the advancement of veraison in wintering cropping might be, at least in part, due to the smaller berry size. More importantly, the berries in winter cropping showed a hastened increase in TSS, especially before or around veraison, and then the rate dropped to a similar level to summer berries during ripening. Another consequence of high temperature is a hastened decline of acidity in berries (Carbonell-Bejerano et al., 2013). The sugar:acid balance at harvest is an important trait of fruit quality, and a gradual fall in acidity is also coupled with a sugar increase. During the ripening process in winter, the temperature was extremely low (approximately 10◦C), so it took a long time to complete the degradation of acid, resulting in more sugar accumulation, and this was the reason why winter cropping prolonged the ripening duration. Thus, the longer duration of ripening stage in winter versus summer cropping was due to the purposes to harvest high-quality grapes. The regulation of grape development and ripening in response to external or internal cues involves a dynamic interplay among hormones. Albeit unclearly, the functional reciprocity among plant hormones was suggested to control ripening transitions and progression (Sun et al., 2010; Böttcher et al., 2013; Pilati et al., 2017). The transition of ripening, termed as veraison, is accompanied by the modulation of hormones, concurrently with many physical, chemical, and physiological changes (Castellarin et al., 2016). Here, most genes encoding NCED/ACO/TAR, the key enzymes of ABA/ethylene/auxin biosynthesis, showed coordinated expression patterns, peaking around pre-veraison, in spite of their different expression levels. Particularly, during early berry development, the level of endogenous auxin was high and peaked around veraison, then its rapid decline occurred from pre-veraison, followed by sequential increases in ethylene and ABA content (Böttcher et al., 2013; Kuhn et al., 2014). Several studies confirmed the ethylene peak preceded the ABA peak at pre-veraison, and they suggested the trace ethylene could induce the expression of VviNCED and therefore the biosynthesis of ABA (Chervin et al., 2004; Sun et al., 2010). In turn, the exogenous ABA treatment on pre-veraison berries also triggered ethylene biosynthesis, suggesting a functional and positive interaction between ABA and ethylene (Sun et al., 2010; Pilati et al., 2017). In the present study, genes in ABA-related pathway were significantly upregulated in winter cropping cycle, which might explain the advance of ripening initiation. The applications of ABA and ethylene before veraison seemed to accelerate the onset of berry ripening, whereas the synthetic auxins treatment delayed the ripening initiation (Sun et al., 2010; Ziliotto et al., 2012). Ziliotto et al. (2012) found an "antagonistic" effect between auxin and ethylene, and a "synergistic" effect between auxins and ABA. The genes involved in ABA biosynthesis and perception were repressed by auxin spraying at pre-veraison, while the ethylene biosynthetic pathway was triggered (Ziliotto et al., 2012). Ziliotto et al. (2012) found a peak of ethylene biosynthesis genes coincided with high expression levels of auxin biosynthesis genes by a pre-veraison auxin analogs treatment, leading the berries back to the preveraison stage. Similarly, when the pre-veraison fruits were treated with ethylene-releasing compound, the transient increase of auxin specifically induced by ethylene would counteract the positive effect of excess auxin, thus delaying the initiation of ripening phase (Böttcher et al., 2013). In this context, VviACO1, the transcript that was confirmed to be consistent with the ethylene peak (Sun et al., 2010), was induced by summer cropping, together with VviTAR in "CS," probably resulting from the strong antagonism between auxin and ethylene. As regards to auxin degradation, it has been shown that GH3 determined this process by conjugation of auxin with amino acids at pre-veraison, and it positively responded to the application of exogenous ABA and ethylene, representing a signal of the berry ripening (Böttcher et al., 2010). The decrease of auxin levels after veraison was probably due to the formation of IAA-Aspartate, which was closely correlated with the VviGH3 transcript levels (Böttcher et al., 2010; Corso et al., 2016). And GH3-1 was claimed to be responsible for the auxin homeostasis at pre-veraison (Böttcher et al., 2010). Here, the VviGH3-1 (VIT\_203s0091g00310) expression was peaked at E-L 35 and remained at high levels at E-L 36 and E-L 38, in agreement with previous report (Böttcher et al., 2010). The transcript of VviGH3-1 was upregulated at E-L 35 in winter grapes compared to summer grapes, with a 0.5-fold higher in "CS" and a 1.2-fold higher in "Riesling" (Supplementary Table S4). This behavior of VviGH3-1 might be associated to the earlier onset of veraison in winter cropping cycle, since the action of GH3 could potentially control of berry ripening rate (Böttcher et al., 2010, 2013; Corso et al., 2016). Taken together, the differential expression pattern of hormone-related transcripts between the two cropping cycles explained the ripening variation at a hormone level. In general, ABA is acted as a stress-stimulated signal, and the water deficit and low temperature in winter cropping acted as a positive regulator of ABA production (Deluc et al., 2009). Furthermore, coloring by the synthesis of pigments is another major change during the onset of ripening (Castellarin et al., 2016). The deeper color with more anthocyanins in winter cropping also coincided with the transcript levels of VviNCED, reproducing the major effects of ABA on the rise of anthocyanins, together with a sugar increase (Wheeler et al., 2009). #### Final Remarks Climate conditions have far-reaching implications for grape cultivation in the field. The extremely high temperature and frequent, intense precipitation in the conventional grapevine growing cycle take a major toll on grape yield and quality in southern China. Thus, the development of a double cropping system per year is a great breakthrough, not only minimizing the impact of bad weather in a subtropical monsoon climate but also improving the quality and yield of out-of-season grapes. Our research here was mainly focused on the flavonoid metabolism, since the grapes grown under two cropping cycles showed distinctly different phenolic compounds in addition to their differences in skin coloration. In addition, we demonstrated that the winter cropping cycle promoted the biosynthesis of flavonoids by (i) avoiding many types of detrimental weather events and making good use of the abundant heat and light resources in southern China; (ii) prolonging the duration of ripening stage to give the berry more time to accumulate flavonoid compounds; (iii) altering the expression patterns of flavonoid-related TFs, particularly with the upregulation of VviMYBA1, VviMYBA2, VviMYBF1, and VviMYB5a and the downregulation of VviMYBPA1, which in turn, greatly induced the flavonoid biosynthetic genes; (iv) triggering the ABArelated ripening processes, which also positively coincided with anthocyanin accumulation; and (v) correlating with their smaller berries and higher sugars. Thus, the alterations in ripening regulatory networks and the flavonoid biosynthetic pathway probably mainly occurred at pre-veraison, leading to a great increase in metabolic gene expression around post-veraison and subsequent flavonoid accumulation. # AUTHOR CONTRIBUTIONS JW, X-JB, and C-QD designed the experiments on vineyard samples. M-MC, GC, and X-JC promoted the double cropping system and sampled the grapes. W-KC processed the samples for RNA isolation for RNA-seq analysis and flavonoid extraction for HPLC-MS/MS analysis, and drafted the manuscript. X-HY participated in the process of flavonoid extractions. R-RG, YW, and LH provided statistical and bioinformatics analysis. FH and JW revised the manuscript and provided suggestions. All authors contributed to discussion of the results and approved the final manuscript. # FUNDING This work was supported by China Agriculture Research System (CARS-29). # ACKNOWLEDGMENTS We thank the staff from the Grape and Wine Research Institute in the Guangxi Academy of Agricultural Sciences for sampling and pruning, particularly Li Chen, Guo-Pin Chen, and Shu-Yu Xie for providing technical support. # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.01912/ full#supplementary-material FIGURE S1 \| The evolution of berry fresh weight of 'Cabernet Sauvignon' and 'Riesling' under double cropping system in 2014 and 2015. # REFERENCES fpls-08-01912 November 8, 2017 Time: 17:51 # 18 under double pruning management. Sci. Agric. 74, 134–141. doi: 10.1590/1678- 992x-2015-0384 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 Chen, Bai, Cao, Cheng, Cao, Guo, Wang, He, Yang, He, Duan and Wang. 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. #### Edited by: Ashraf El-kereamy, University of California, United States #### Reviewed by: M. Teresa Sanchez-Ballesta, Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC), Spain Jerome Grimplet, Instituto de Ciencias de la Vid y del Vino (ICVV), Spain #### \Correspondence: Simone D. Castellarin [email protected] #### †Present address: Stefania Savoi and Jose C. Herrera, Division of Viticulture and Pomology, Department of Crop Sciences, University of Natural Resources and Life Sciences Vienna, Tulln, Austria ‡These authors have contributed equally to this work. #### Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science > Received: 04 April 2017 Accepted: 12 June 2017 Published: 10 July 2017 #### Citation: Savoi S, Wong DCJ, Degu A, Herrera JC, Bucchetti B, Peterlunger E, Fait A, Mattivi F and Castellarin SD (2017) Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L.) Exposed to Water Deficit. Front. Plant Sci. 8:1124. doi: 10.3389/fpls.2017.01124 Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L.) Exposed to Water Deficit Stefania Savoi1,2†‡, Darren C. J. Wong<sup>3</sup>‡ , Asfaw Degu<sup>4</sup> , Jose C. Herrera<sup>1</sup>† , Barbara Bucchetti<sup>1</sup> , Enrico Peterlunger<sup>1</sup> , Aaron Fait<sup>4</sup> , Fulvio Mattivi2,5 and Simone D. Castellarin<sup>3</sup> \ <sup>1</sup> Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Udine, Italy, <sup>2</sup> Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy, <sup>3</sup> Wine Research Centre, The University of British Columbia, Vancouver, BC, Canada, <sup>4</sup> The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer, Israel, <sup>5</sup> Center Agriculture Food Environment, University of Trento, San Michele all'Adige, Italy Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and genemetabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stressresponsive metabolites. Enrichment of known and novel cis-regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction 157 pathways may be critical to achieve a balance between the regulation of the stressresponse and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress. Keywords: abiotic stress, central metabolism, drought, grapevine, fruit quality, ripening, RNA-sequencing, specialized metabolism #### INTRODUCTION fpls-08-01124 July 6, 2017 Time: 16:47 # 2 Drought is considered one of the major threats for crops in the predicted future climatic scenarios. In fruit crops, drought can impact the accumulation of metabolites that determine fruit quality (Ripoll et al., 2014). Generally classified as drought tolerant, grape is often not irrigated or minimally irrigated to improve the berry composition (Chaves et al., 2007). Previous research showed that water deficit causes large changes on the specialized metabolism of the grape berry, promoting the synthesis of specific volatile organic compounds (Bindon et al., 2007; Savoi et al., 2016), carotenoids (Deluc et al., 2009), and phenolics (Castellarin et al., 2007a,b; Hochberg et al., 2015). The grape berry central metabolism is also affected by water deficit. Deluc et al. (2009) reported an increase of proline concentration in Cabernet Sauvignon berries, and the increase paralleled the induction of key genes for the proline synthesis such as the one codifying for the pyrroline-5-carboxylate synthase. Hochberg et al. (2015) reported that also other central metabolites, including sucrose, several amino acids and organic acids, ascorbate, and raffinose can take part in the metabolic response of the grape berry to water deficit. These results indicate that a complex regulation of several metabolic pathways, possibly determined by common or specific molecular signals, underlay the metabolic response of the grape berry to water deficit. In the model plant Arabidopsis thaliana, the transcriptional response to drought is modulated by both ABAdependent and ABA-independent signal transduction pathways (Yamaguchi-Shinozaki and Shinozaki, 2006; Singh and Laxmi, 2015). In the ABA dependent pathway, the accumulation of ABA is sensed by PYR/PYL/RCAR-PP2C receptor complex, and activates a class III SnRK2s that phosphorylates four transcription factors (TFs), ABA-responsive element (ABRE) binding protein 1 (AREB1), AREB2, ABRE binding factor 3 (ABF3), and ABF1. These TFs regulate several downstream genes by binding to the ABRE cis-regulatory element (CRE) present in the promoter region of these genes. Drought induced ABA also regulates the activity of MYB/MYC, NAC, WRKY, and NF-Y TFs. In the ABA-independent signaling pathway, the DREB2A, a protein that belongs to the AP2/ERF family, plays a pivotal role in the transcriptional response under drought stress (Yoshida et al., 2014). Recently, a cross-talk between ABA-dependent and ABA-independent pathways has been hypothesized under drought stress, with AREB/ABFs inducing DREB2A (Nakashima et al., 2014; Singh and Laxmi, 2015). In grape, ABA participates in the drought response of multiple organs and in the process of fruit ripening. In the xylem sap of shoots and roots, ABA levels negatively correlate with stem and root water potential, and increases of ABA concentration correspond to decreases of leaf transpiration (Rossdeutsch et al., 2016). ABA related genes were shown to be involved in the transcriptional response of the leaf to drought (Dal Santo et al., 2016; Hopper et al., 2016) and, in the grape berry, water deficit increased ABA concentration and ABA related transcripts, but not consistently between varieties (Deluc et al., 2009). Among fruit crops, grapes are the most considered crop in studies on the impact of drought on fruit composition. The adoption of large-scale metabolite and transcript analyses (Deluc et al., 2009; Hochberg et al., 2015; Savoi et al., 2016) has strongly enhanced our understanding of the metabolic response of the grape berry to water deficit; however, systems approaches considering the analysis of primary and secondary metabolism, the molecular pathways, and the network relationships between genes and metabolites involved in the response to water deficit are still missing in grape, as well as in other fleshy fruits. Furthermore, the modulation of the volatile organic compounds (VOCs) under water deficit has been poorly investigated in wine grapes (Savoi et al., 2016), although they represent a key component of the final wine flavor. Recent studies have successfully considered a multi-omics approach in several annual herbaceous crops, such as Medicago truncatula (Zhang et al., 2014), Oryza sativa (Maruyama et al., 2014), and Zea mays (Opitz et al., 2016). In these studies, RNA-sequencing and large-scale metabolite analyses were adopted and revealed several commonalities in the metabolic response between these crops and A. thaliana. Network-based analyses using multi-omics data provide a powerful tool for discovering links between and within the many layers of biological complexity that governs plant functions such as the coordinated regulation of genes and metabolic pathways. This approach has been performed in a few fruit crop studies to prioritize candidate genes involved the control of fruit development and composition (Mounet et al., 2009; Zamboni et al., 2010; Osorio et al., 2011, 2012; Savoi et al., 2016). We aimed to apply a similar approach to deeply characterize the molecular and metabolite response to water deficit in the grape berry, to uncover the relationships between water deficit-responsive genes and the metabolite accumulation during berry development, and to identify the key putative molecular regulators that underlay the metabolic response to water deficit. For that purpose, we conducted a two season experiment where two contrasting water regimes were applied and the levels of transcripts and metabolites were analyzed at several berry developmental stages. Transcripts and metabolites that were modulated by water deficit were used for constructing molecular networks to investigate their relationships and to identify the major molecular pathways that underlay the response of grape berry metabolism to this abiotic stress. # MATERIALS AND METHODS ## Field Experiment, Physiological Measurements, and Sample Preparation Field experiments were conducted in 2011 and 2012 at the University of Udine experimental farm on 18/19 years old Vitis vinifera L. 'Merlot' (clone R3 onto SO4 rootstock) vines. The vines were planted with 2.5 m × 1.0 m spacing and trellised to a spur cordon system. To ensure a proper management of the water regime during the experimental trial and consistent treatments across seasons, four rows were covered with an ethylene-vinylacetate (EVA) film at the beginning of the seasons, as described in Herrera et al. (2015). Two irrigation treatments were imposed at approximately 25 days after anthesis (DAA): control (CT) vines where weekly irrigated maintaining the midday stem water potential (9Stem) above −0.6 MPa and water deficit (WD) vines were not irrigated from fruit set until 9Stem was lower than −1.4 MPa, whereupon irrigation was managed to maintain 9Stem between −1.0 and −1.4 MPa. Each treatment was replicated four times in plots of 10 vines each in a completely randomized design. Plant water status was monitored weekly by measuring midday 9Stem (Savoi et al., 2016). Berries were randomly sampled for analyses seven times during each season: three times before the beginning of ripening (30, 44, and 60 DAA in 2011, and 26, 40, and 53 DAA in 2012), at the beginning of ripening (74 DAA in 2011 and 67 DAA in 2012), and three times during berry ripening (87, 100, and 115 DAA in 2011, and 81, 95, and 106 DAA in 2012). These developmental stages corresponded to E-L 31, 32, 33, 35, 36, 37, and 38 in the E-L system, respectively. The onset of ripening (veraison, defined as the day at which 50% of the berries had changed color from green to red) was recorded at 69 and 60 DAA in 2011 and 2012, respectively. In both seasons, the last sampling point coincided with the harvest date. At each date, two sets of berries were randomly collected from each plot, for a total of four biological replicates per irrigation treatment. The first set of 60 berries was used for measuring berry weight, total soluble solids (TSS), titratable acidity (TA), and pH as described in Herrera et al. (2015). The second set of 40 berries was used for the metabolite and transcript analyses; samples were snap frozen with liquid nitrogen, and stored at −80◦C. For metabolite and transcript analyses, whole berries, without pedicel, were grinded to a fine powder under liquid nitrogen using an analytical mill (IKA-Werke GMbH & Co.). The frozen powder was aliquoted for metabolite and RNA extraction as described below. Moreover, a quality control (QC) sample for metabolite analysis was prepared by pooling together aliquots of all the samples. Large-scale targeted metabolite analyses were undertaken in both seasons, while transcriptome analyses were performed only in 2012. #### Metabolite Analyses Primary metabolites were extracted from 100 mg of frozen powder, derivatized for GC-MS analysis and analyzed in a Trace GC Ultra gas chromatograph coupled to a DSQII quadrupole mass spectrometer (Thermo Scientific) as described in Degu et al. (2014). XCalibur software was used for the mass spectra identification using the NIST library (United States) and the RI libraries from the Max-Planck Institute for Plant Physiology (Germany). The QC sample was used for data normalization. Metabolites were determined as described in Savoi et al. (2016) unless specified. Phenylpropanoids, stilbenoids, flavonols, flavan-3-ols, and proanthocyanidins chromatographic analysis was carried out using a Waters Acquity UPLC system (Milford) coupled to a Waters Xevo triple-quadrupole mass spectrometer detector (Milford). Compounds were identified with TargetLynx software based on their reference standard, retention time, and qualifier and quantifier ion, and were accurately quantified by their calibration curve and expressed as mg/Kg of grapes. Anthocyanins were analyzed as described in Sivilotti et al. (2016) using a HPLC (Shimadzu) equipped with a diode array detector. The concentration of individual anthocyanins was expressed in oenin chloride equivalents and expressed as mg/Kg of grapes. Carotenoids chromatographic analysis was performed in a 1290 Infinity Binary UPLC (Agilent) equipped with an RP C30 3 µm column Spectra components and elution profiles were determined with the R package 'alsace' 3.0. Compounds were quantified from linear calibration curves built with standard solutions and expressed as mg/Kg of grapes. Free (non-glycosylated) VOCs analysis was performed with a Trace GC Ultra gas chromatograph (Thermo Scientific) coupled to a TSQ Quantum Tandem mass spectrometer. XCalibur software was used for the peaks identification. VOCs were identified by comparing the retention times of individual peaks with the retention times of their reference standards, and by identifying the mass spectra using the NIST library. The ratio of each VOC area to the d8-acetophenone internal standard area was considered to reduce technical variability among extractions and chromatographic runs and VOCs quantity were expressed as µg/Kg of grapes of d8-acetophenone equivalents. Extractions and injections of the samples were performed in a random sequence and QC samples were injected at the beginning of the sequence and every six sample injections. ### RNA Extractions and RNA Sequencing Analysis Transcriptome analyses were performed on the samples collected at 26, 53, 67, 81, and 106 DAA in 2012. Three out of the four biological replicates per treatment were considered. RNA extraction, RNA quality and quantity determination, library preparation and quantification, sequencing and QC analysis were performed as described in Savoi et al. (2016). Reads were aligned against the reference grape genome V1 PN40024 12X (Jaillon et al., 2007) using the software TopHat version 2.0.6 (Trapnell et al., 2012) with default parameters. Aligned reads were counted with htseq-count (version 0.6.0), in intersectionnon-empty mode for overlap resolution (Anders et al., 2015). Differentially expressed (DE) genes [false discovery rate (FDR) less than 0.05] analysis was performed with the R package 'DESeq2' (Love et al., 2014). Annotation of gene functions was done according to Grimplet et al. (2012) and Naithani et al. (2014) and retrieved from recent literature. Gene ontology analyses for each sampling were carried out as described in Savoi et al. (2016). ## Quantitative Real-Time Polymerase Chain Reaction The validation of RNA-Seq data was carried out on a set of DE genes using the quantitative real-time polymerase chain reaction (qPCR) technique. The reverse transcription of RNA samples was performed with the QuantiTect Reverse Transcription Kit (Qiagen); specific primers for 12 selected genes were designed with Primer3web version 4.0.0 (Supplementary Table S1). Real-Time PCR experiments were performed on a Bio-Rad CFX96TM using SsoFastTM EvaGreen <sup>R</sup> Supermix. qPCR run condition were as per instructions with annealing temperature of 58◦C. VviAP47 (VIT\_02s0012g00910) was used as a reference gene (Reid et al., 2006). In order to validate the technical and biological reliability of the transcriptome dataset, qPCR analysis was carried out on samples collected at different stages of development (starting from the sampling that preceded the onset of ripening) in 2011 and 2012. #### Statistical and Network Analyses A one-way ANOVA was performed using JMP 7 (SAS Institute Inc.) to detect significant differences (P < 0.05) between irrigation treatments at each sampling date for the several physiological and compositional parameters considered. Heatmaps representing the log<sup>2</sup> fold change (log2FC) of metabolite concentrations between treatments (WD/CT) were drawn with R software. Metabolites were clustered with Person correlation and a complete link. Principal component analyses (PCAs) on metabolite and transcriptome datasets were performed using the R software. Co-expression analysis was performed using weighted correlation gene co-expression network analysis (WGCNA) package in R (Langfelder and Horvath, 2008) using the log<sup>2</sup> fold change (WD/CT) [dataset1] and the variance stabilized transformed (VST) counts [dataset2] of DE genes at 26, 53, 67, 81, and 106 DAA, separately to identify highly correlated genes sharing similar water deficit co-response and development accumulation patterns, respectively. Empirical P-value of Pearson Correlation Coefficient (PCC) values (statistical significance) of dataset1 and dataset2 were estimated by 1,000 permutations using the 'rsgcc' package (Ma and Wang, 2012). Enrichment for CRE in the gene promoters (1 kb upstream of the 5<sup>0</sup> UTR or TSS) of each WGCNA co-response modules was conducted as described previously (Savoi et al., 2016; Wong et al., 2016). Known CREs of 6-, 7-, 8-mers (222 in total) were analyzed. Enrichment of CREs was validated with the hypergeometric test adjusted with FDR correction. Putative CREs were deemed significantly enriched under FDR < 0.01. Stress co-response and development-regulated submodules were created using PCC threshold > 0.8 with an empirical P-value < 0.01 and visualized using Cytoscape software (version 3.1.1) (Shannon et al., 2003). #### Accession Number All raw sequence reads have been deposited in NCBI Sequence Read Archive<sup>1</sup> . The BioProject accession is PRJNA348618. # RESULTS #### Impact of Water Deficit on Fruit Development In 2011, differences in 9Stem between treatments started from 50 DAA and the lowest values in WD vines (<−1.4 MPa) were measured at late stages of fruit development (95 and 110 DAA). Whereas in 2012, differences between the treatments started 10 days earlier (39 DAA), and the lowest values in WD vines (<−1.4 MPa) were measured at 75 and 88 DAA (Figure 1). At this stage, the 9Stem in WD vines was −0.8 and −1.2 MPa in 2011 and in 2012, respectively. In both seasons, 9Stem of WD vines was consistently lower than −1.0 MPa for the entire ripening period. Water deficit significantly reduced berry weight at 74, 100, and 115 DAA in 2011 (Figure 1) and at 67, 81, 95, and 106 DAA in 2012 (Figure 1). The reduction in berry weight was also reflected in lower vine productivity in both seasons (Supplementary Table S2). Significant differences in TSS and TA were observed only in 2012 (Figure 1). In this season, water deficit increased TSS before (40 and 53 DAA) and after (95 and 106 DAA) the onset of fruit ripening, while TA was observed to be lower and higher in WD than in CT at 26 and 53 DAA, respectively. # Impact of Water Deficit on Berry Metabolites A total number of 101 compounds, belonging to the central (primary) and specialized (secondary) metabolism, were identified (standard annotation level 1) and quantified. A complete list of the compounds identified, with their machine readable chemical identifier, is reported in Supplementary Table S3, and the kinetics of the accumulation of these compounds in CT and WD berries during development in both vintages is reported in Supplementary Figure S1. Principal component analyses were used to compare the metabolite profiles of CT and WD berries during development in both seasons considering the whole set of metabolites and the central and specialized metabolism separately (Figure 2). The first two principal components represent from 64.17 to 67.67% of the variance in the datasets. In all the PC1-PC2 score plots, clear separation among samples was observed based on the berry developmental stage. This separation was consistent between the two seasons; samples collected at similar developmental stages in the two seasons clustered together in the PC1-PC2 score plots. The PC2 and PC1 separated the developmental stages before and after the beginning of fruit ripening, respectively. <sup>1</sup>http://www.ncbi.nlm.nih.gov/sra Separation between irrigation treatments was observed from 100 DAA in 2011 and from 81 DAA in 2012. This large and coordinated change in the whole composition of the berry was more evident when the whole set of metabolites was included in the analysis than when central and specialized metabolism were analyzed independently (Figure 2). In both seasons, differences in the berry metabolite profiling between CT and WD berries were maximized at the last sampling stage. Among the 34 central – amino acids, sugars, organic acids, polyols, and polyamines – metabolites analyzed, a total of 11 and 15 metabolites were significantly modulated by water deficit at one or more developmental stages in 2011 and 2012, respectively (Figure 3 and Supplementary Figure S1). Most differences were observed during berry ripening, when in WD berries the level of leucine, valine, isoleucine, threonine, proline, and putrescine increased consistently in the two seasons. On the contrary, raffinose decreased before the beginning of ripening in both seasons as a result of water deficit, as well as malate decreased at late stages of ripening. The specialized metabolites analyzed in this study were phenolics, carotenoids, and free VOCs. Among the 39 phenolics detected, 22 and 28 were significantly modulated by water deficit in 2011 and 2012, respectively (Figure 4 and Supplementary Figure S1). Eighteen of them were consistently modulated between the two seasons. Water deficit increased the concentration of benzoic and cinnamic acids. Specifically, gallic acid was increased by water deficit at harvest in both seasons, while trans-caftaric, trans-coutaric, and trans-fertaric acids were increased only in 2012. Water deficit strongly increased the concentration of most anthocyanins detected. Stilbenoids, such as trans-resveratrol, piceatannol, and pallidol, were decreased by water deficit in both seasons at harvest, while cis- and trans-piceid were significantly decreased by water deficit only in 2011 even though they had a similar trend but showed no significance in 2012. Monomeric flavan-3-ols, such as catechin and epicatechin gallate, were decreased by water deficit in both seasons during berry ripening. Conversely, proanthocyanidins B1, B2+B4 were generally increased by water deficit. Flavonols were not consistently affected by water deficit; quercetin-3-O-rutinoside was significantly increased by water deficit, but only at 95 DAA in 2012. However, the method used for detecting and quantifying flavonols was unable to detect the glycosylated myricetin, one of the main flavonols produced in red grapes (Mattivi et al., 2006). Carotenoids such as violaxanthin, neoxanthin, and lutein 5-6-epoxide, decreased in WD berries during ripening, while zeaxanthin increased (Figure 5 and Supplementary Figure S1). A strong increase of free VOCs was consistently observed between the two seasons at late developmental stages under water deficit. Remarkably, eight free VOCs were consistently modulated between seasons by water deficit (Figure 5 and Supplementary Figure S1). C6 VOCs, such as (E)-2-hexenal and 3-hexenol, increased in WD berries at the onset of ripening, while the C6 VOC hexanol increased at 74 DAA in 2011 and at 40 DAA in 2012, but decreased at 26 DAA in 2012. Furthermore, C5, C7, C8, and C9 VOCs, such as 1-penten-3-ol, (E)-2-heptenal, (E)-2 octenal, 1-octen-3-ol, and nonanol increased in WD berries at late ripening stages. ### Impact of Water Deficit on Berry Transcriptome Larger metabolic changes occurred in 2012, when water deficit started earlier and was overall more severe during the season (Figure 1), thus transcriptome analysis was undertaken on berry samples collected during this season at five berry developmental stages (before the onset of ripening, 26 and 53 DAA; at the onset of ripening, 67 DAA; and during ripening, 81 and 106 DAA). The average number of unique reads that mapped the 12X V1 version of the grape genome (Jaillon et al., 2007) was 27.1 M (Supplementary Table S4). Among the 29,971 genes of the grape genome, 23,253 (77.6%) genes were expressed at 26 DAA, 23,220 (77.5%) at 53 DAA, 21,997 (73.4%) at 67 DAA, 22,453 (74.9%) at 81 DAA, and 22,162 (73.9%) at 106 DAA. A PCA was performed to compare the transcriptome profiles of the 30 independent samples analyzed (2 treatments × 5 developmental stages × 3 biological replicates) (Figure 6). The first two principal components explain 61.9 and 15.8%, of the variance among samples, respectively. Berry transcriptome were clearly separated accordingly to the developmental stage; moreover, within developmental stages, the berry transcriptomes of WD berries grouped together and were separated from the transcriptomes of CT berries. The total number of DE genes between CT and WD was 5,167 (Supplementary Table S5). Water deficit modulated the expression of 214 genes (175 up-regulated; 39 down-regulated) at 26 DAA, 90 genes (38 up-regulated; 52 down-regulated) at 53 DAA, 1,290 genes (662 up-regulated; 628 down-regulated) at 67 DAA, 2,900 genes (1,569 up-regulated; 1,331 downregulated) at 81 DAA, and 2,925 genes (1,431 up-regulated; 1,494 down-regulated) at 106 DAA (Figure 6). Several of them were differentially regulated at more than one developmental stage (Figure 6). Thirty GO categories (slim biological processes) were significantly overrepresented among the DE genes as presented in Supplementary Figure S2. The expression of 12 selected DE genes was tested at several developmental stages in both seasons with a qPCR (Supplementary Figure S3). This analysis indicates that differences in the gene expression level between treatments remained consistent regardless the platform or the season considered. #### Transcriptional Regulatory Networks of Berries under Water Deficit Transcription factors are central in regulating many plant biological processes; including developmental processes and response to the environment. A total of 447 TFs out of 2,211 possible TFs encoded in the grape genome (Grimplet et al., 2012) were modulated in the berry in response to water deficit. In this study, emphasis was given on the ripening stages (67, 81, and 106 DAA), when large metabolite and transcriptome responses to water deficit occurred. A large proportion of the water deficit-modulated TFs belongs to the MYB (33 genes), bHLH (33 genes), AP2-ERF (28 genes), C2H2 (27 genes), WRKY (26 genes), NAC (23 genes), C3H (20 genes), HB (18 genes), GRAS (16 genes), and bZIP (14 genes) families (Supplementary Table S5). A selection of these genes is reported in Figure 7. Central components of the ABA-independent drought response pathway are the AP2/ERF TFs encoding dehydrationresponsive element binding proteins (DREBs) that bind the dehydration-responsive element/C-repeat (DRE/CRT) sequence (Mizoi et al., 2012). Several of these genes, were significantly modulated by water deficit. For example, VviERF1, an upstream component of the jasmonic acid and ethylene signaling pathway, induced by high salinity and drought stress in Arabidopsis (Cheng et al., 2013), was strongly up-regulated by water deficit during fruit ripening. Another AP2/ERF-DREB (VviRAP2.4) was up-regulated by water deficit at late ripening. In Arabidopsis this TF confers drought tolerance by activating drought-responsive genes (Lin et al., 2008). Our data indicate that basic leucine zipper (bZIP) proteins – components of the ABA-dependent signaling pathway – might also be implicated in the regulation of the drought response in fruits. MYB TFs are key modulators of plant metabolism and development, and have been shown to be involved in the drought response. Several MYB TFs were strongly modulated by water deficit. Among them, we noted VviMYB14 (VIT\_07s0005g03340) and VviMYB15 (VIT\_05s0049g01020), VviMYB5B (VIT\_06s0004g00570), VviMYBA1 (VIT\_02s0033g00410), VviMYBC2-L1 (VIT\_01s0011g04760), and VviMYBPA1 (VIT\_15s0046g00170) that are involved in regulating various branches of the phenylpropanoid, stilbenoid, and flavonoid metabolism. In this study, most of the DE NACs were up-regulated by water deficit. Among the other TFs modulated by water deficit, two auxin response factors (VviARF3 – VIT\_10s0003g00420; VviARF2 – VIT\_17s0000g00320) – possibly implicated in fruit development and ripening (Kumar et al., 2014) – encoding orthologous genes for AtARF3/ETTIN and AtARF2, were down- and up-regulated during ripening, respectively. Example of grape ARFs that may integrate multiple signaling pathways includes, VviARF2 (VIT\_17s0000g00320), the homolog of tomato ARF2, and VviARF5 (MONOPTEROS, indicates the class of metabolites. VIT\_18s0001g13930). Arabidopsis ARF2 and ARF5 are master regulators of auxin hormone responses, largely targeting the genes implicated in hormone-mediated signaling pathway, growth, and tissues development, among others (O'Malley et al., 2016). Two homeobox-leucine zipper proteins (VviHB7 – VIT\_15s0048g02870, and VviHB12 – VIT\_16s0098g01170), encoding for orthologous genes of AtHB7 and AtHB12 (Valdés et al., 2012), were significantly up-regulated under water deficit. Particularly, VviHB12 was up-regulated from 53 DAA onward, with the highest induction at 81 DAA when water deficit reached the highest severity. Also, two of the WRKYs (VviWRK18 – VIT\_04s0008g05760, and VviWRKY40 – VIT\_09s0018g00240) involved in the ABA signaling pathway (Geilen and Böhmer, 2015) were down-regulated under water deficit during ripening. Vice versa, one WRKY TF (VviWRKY71 – VIT\_12s0028g00270), that has been shown to be involved in the oxidative stress process, as well as in the salicylic acid and jasmonic acid signaling pathways (Guo and Qin, 2016), was up-regulated at 81 DAA by water deficit. #### Modulation of Central and Specialized Pathway Genes under Water Deficit Many genes DE under water deficit codify for enzyme involved in major central and specialized pathways. Detailed description of how these major pathways were modulated is presented and discussed in Supplementary Figures S4–S7. Water deficit affected the expression of genes involved in 9 steps out of 10 of the glycolysis metabolic pathway (Supplementary Figure S4). Significant differences in the gene expression were observed from 67 DAA onward, and the majority of these genes were up-regulated by water deficit. Few genes of the TCA cycle were moderately modulated at 81 or 106 DAA. Moreover, several sugar transporters, possibly involved in the monosaccharide, sucrose, and polyol and hexose intake into the berry cells were modulated. Hexoses are the major sugars accumulated in the grape berry during ripening and, interestingly, most of the hexose transporters were downregulated. Water deficit increased the concentration of the short branched-chain amino acids leucine, valine, and isoleucine in both seasons (Figure 3) and several genes involved in valine and leucine biosynthesis were modulated during berry ripening (Supplementary Figure S5). Genes that underlay the synthesis of proline including glutamate dehydrogenases (GluDH), glutamate synthases (GluS), and a pyrroline-5-carboxylate synthase (P5CS) were strongly up-regulated during the final stages of ripening. Furthermore, genes that promote the decarboxylation of arginine or ornithine into polyamines were up-regulated. Consistently with the differences observed in the phenylpropanoid, stilbenoid, and flavonoid accumulation (Figure 4), Water deficit strongly modulated most steps of the related biosynthetic pathways (Supplementary Figure S6); it promoted the expression of the branch of the flavonoid pathway which leads to the production of tri-hydroxylated anthocyanins (Castellarin et al., 2007b) and down-regulated 28 out of 45 stilbene synthases (STSs). Water deficit also affected the expression of several genes of the carotenoid pathway (Supplementary Figure S7), mostly by up-regulating them. Carotenoids such as neoxanthin and violaxanthin can be cleaved by 9-cis-epoxycarotenoid dioxygenase (NCED) and further modified to produce the drought and ripening related hormone ABA. Three VviNCEDs were up-regulated in WD berries during ripening. The molecular pathways that underlay the VOC production in fruits, as well as their modulation under water deficit remain largely unknown. Several VOCs detected in this study are produced from the peroxidation of free C18 polyunsaturated fatty acids, such as linolenic and linoleic acids, which lead to the production of C6, C9 (Kalua and Boss, 2009) and putatively C5 (Shen et al., 2014) VOCs. The fatty acid degradation involves lipoxygenases (LOX), hydroperoxide lyase (HPL), hexenal isomerases (HI), and alcohol dehydrogenases (ADH) (Schwab et al., 2008). We found that a 13-LOX, a HPL and five ADHs were up-regulated in WD berries during berry ripening. The induction of VviLOX (VIT\_06s0004g01510) in WD berries at 106 DAA, may explain the higher accumulation of C5, C8, and C9 VOCs observed. In addition, we identified two grape genes codifying for (Z)-3:(E)-2-hexenal isomerases, recently identified paprika and tomato (Kunishima et al., 2016), consistently up-regulated in WD berries from 67 DAA onward. Finally, we observed a strong modulation of many transcripts involved in the reactive oxygen species (ROS) related pathways (production, scavenging, and signaling) involved in the plant responses to abiotic stresses (Supplementary Table S5) (Dal Santo et al., 2016). ## Predicted Water Deficit-Regulated Modules Link Central Players in the Metabolic Response To determine the correlation pattern among DE genes and analyze their regulation during berry development and in response to water deficit, WGCNA was performed (Langfelder and Horvath, 2008). Eleven co-response gene modules (clusters) of highly correlated genes based on the water deficit-modulation were identified, with each module containing up to six generalized development-based accumulation patterns (Figure 8 and Supplementary Table S6). In seven modules (named WD1, 2, 5, 6, 8, 9, and 11) we observed a general upregulation of the gene expression under water deficit; on the contrary, in four modules (named WD3, 4, 7, and 10) we observed a general down-regulation of the gene expression. Based on the fact that water deficit was stronger on metabolite and transcript abundance during fruit ripening, we focused on the DE that decreased in expression during ripening (sub-module DEV1 and DEV4), the ones that peaked at the onset of ripening (sub-module DEV5), the ones which were generally highly expressed from the onset of ripening to harvest (sub-module DEV3), and the ones which showed a steady increase in transcripts during ripening before peaking at harvest (sub-module DEV2). In order to gain more insights into the regulatory control of water deficit-induced genes, the promoters of module genes were evaluated for CRE enrichment (Figure 8 and Supplementary Table S6). We evaluated 222 CREs, 6- to 8 mer in length, given that these lengths usually define the primary DNA element recognized by various plant TFs (Franco-Zorrilla et al., 2014). We have recently shown that, in grape, many of these CREs are indeed bona-fide CREs with large biological relevance (Wong et al., 2016, 2017). The ACGTGKC CRE was highly enriched in general water deficit-induced modules, such as in WD2, WD9, and WD11 modules. Similarly, CACGTG CRE was highly enriched in the WD2 and WD11 modules. Although the core DRE element (RCCGAC) was not enriched in the modules, the 7-mer DRE, RCCGACA, and other DRE-related CREs (e.g., RYCGAC and MACCGMCW) involved in drought responses were enriched in the WD9 and WD11 modules. Examples of genes containing ABRE and G-box CREs in promoters include all three water deficit upregulated VviNCEDs (VIT\_05s0051g00670, VIT\_10s0003g03750, and VIT\_19s0093g00550) – whose homologs in Arabidopsis (e.g., AtNCED3) regulate ABA biosynthesis during drought stress (Yamaguchi-Shinozaki and Shinozaki, 2006). Other CREs enrichment in these DE genes have been associated with droughtinduced transcriptional pathways, including NACR (CATGTG enriched in the WD11 module and TTRCGT in the WD2 and WD11 modules), GCC-box (GCCGCC enriched in the WD1 and WD11 modules), and MYB (CCGTTA enriched in the WD2 and WD11 modules), while others may be novel, such as AuxRE/ETT (TGTCGG enriched in the WD2 and WD11 modules) and ZAT6 (ACACTA enriched in the WD8 module). Module-metabolite co-response was first determined for each module satisfying a PCC > 0.8, as a preliminary step to understand the coordinated regulation of modules with target metabolites (Supplementary Table S6). For instance, the WD2 module was correlated with 11 of 15 anthocyanin compounds profiled, in agreement with the many flavonoid pathway genes (e.g., four flavonoid 3<sup>0</sup> 5 0 -hydroxylases –VviF3<sup>0</sup> 5 <sup>0</sup>Hs, the UDP-glucose:flavonoid-3-O-glucosyltransferase –VviUFGT, one leucoanthocyanidin dioxygenase –VviLDOX, and one flavanone 3-hydroxylases –VviF3H) that were localized in that module. To a lesser extent, the WD11 module was correlated with four different anthocyanin compounds coinciding with fewer flavonoid pathway genes (e.g., three VviF3<sup>0</sup> 5 <sup>0</sup>Hs, two chalcone synthases –VviCHSs, and one anthocyanin-acylglucoside transporter –VviAnthoMATE2). The WD9, WD2, and WD11 modules were correlated with various amino acids. For example, the WD11 module correlated to valine, threonine, and proline, and contains key structural genes involved in the formation of precursors for proline (such as VviGluDH, VviGluS, and VviP5CS) and threonine (e.g., threonine synthase – VviTS). Correlations between stilbenoid compounds with the modules considered were generally weak (PCC < 0.8), despite a large number of VviSTS genes being localized into WD3 (8 VviSTSs) and WD4 (19 VviSTSs) modules. As regards the correlation of modules with VOCs concentration, the WD2 module was correlated with 1-octen-3-ol and nonanol, and contained FIGURE 8 \| Functional overview of biological modules modulated by water deficit in the grape berry. (A) Violin plots represent the distribution of log2FC (WD/CT) of all DE genes in stress (WD) co-response gene modules. Box plots represent the distribution of variance stabilized transformed expression values of DE genes in development (DEV) co-regulated gene modules. (B) Overview of selected enriched cis-regulatory elements (CREs) in stress co-response genes modules. Colors of circles representative the TF family that recognize a designated CRE. Sizes or circles represent the number of genes containing a designated CRE in their promoter region. Opacity of circles (in color) represent the enrichment score, –log10(FDR), of CREs. Only enriched CREs (FDR < 0.01) in designated modules are depicted. Network representation of WD2 and WD11 modules, having ripening-associated expression patterns (DEV2/3/5), centered on significantly correlated TF and/or structural genes with (C) anthocyanin (pink, lavender blue, cyan, blue, and purple), and (D) amino acid compounds (orange, red, and green). Structural genes, TF, and metabolites are represented by circle, square, and triangle nodes, respectively. Thick edges in light red and gray edges represent associations between genes/TFs (PCC > 0.8; P < 0.01) for (C,D), respectively. Gene-metabolite (PCC > 0.8; P < 0.01) are depicted in thinner edges and the different colors denote different categories, as reported in (C,D). VviHPL1. Interestingly, several transcripts involved in the degradation of storage lipids such as triacylglycerol lipase and phospholipase A2 were also found in this module. Finally, strong correlations were observed between the WD5 module and zeaxanthin; consistently, the module included several structural carotenoid genes such as phytoene desaturase and ζ-carotene desaturase. In order to discover new links between and within the many layers of biological complexity that governs the metabolic response of the grape berry to water deficit, the coordination between metabolite and gene patterns and water deficit responses was further investigated. For the metabolitegene centered subnetwork, significant correlations between metabolites (anthocyanin and amino acids) and annotated structural pathway genes and TFs were investigated further for sub-modules DEV2, DEV3, and DEV5 in WD2 and WD11. The WD2 and WD11 modules were considered for anthocyanin (Figure 8), while only module WD11 was considered for amino acid metabolite-gene correlations (Figure 8). In addition, only regulatory genes from the bZIP, AP2/ERF, MYB, and NAC families were mainly considered given a consistent enrichment of the associated CREs in the promoters of both modules. Focusing on the anthocyanin subnetwork, we observe frequent correlations of bZIP (VviABF4 and VviGBF3), AP2/ERF (VviRAP2.1 and VviRAP2.4), and NAC (VIT\_02s0012g01040 and VIT\_10s0003g00350) TFs with various anthocyanin in the WD2 module. Consistently, frequent correlations of AP2/ERF (VviERF62) and NAC (VviNAC87 and VviRD26) TFs were also observed with anthocyanin in the WD11 module. As expected, frequent correlations of VviMYBA1 (WD11) to three VviF3<sup>0</sup> 5 <sup>0</sup>Hs, and to eight anthocyanins further corroborate its role in regulating various anthocyanin pathway genes during berry development (Walker et al., 2007; Rinaldo et al., 2015) and under water deficit (Castellarin et al., 2007b). Interestingly, the ABRE, DRE/CRT and GCC, and/or NACR were also found in the promoter of VviMYBA1and of various flavonoid genes in WD11 module (Supplementary Table S6); this might provide additional clues into the regulation of anthocyanin biosynthesis under water deficit. In the amino acid subnetwork, VviERF1 and VviNAC1/VviNAC33 (VIT\_19s0027g00230) were among the top five highly correlated TFs with proline in this module, and were also connected with VviP5CS in the network. ERF1 is involved in the regulation of P5CS during normal growth and abiotic stresses in Arabidopsis (Cheng et al., 2013). Reinforcing the observed gene and metabolite correlations, VviP5CS promoter also contains CRE signatures potentially relevant for VviERF1 binding, via the GCC-box (GCCGCC) and DRE (MACCGMCW) CRE (Supplementary Table S6) suggesting a conserved regulatory mechanism regulating proline via VviP5CS in the berries in response to water deficit stress. Other TF binding sites such as NAC (TTRCGT and TTACGTGT) (Supplementary Table S6) in P5CS promoter highlight its potential regulation via VviNAC1/VviNAC33. In addition, VviERF62 and VviRD26 were also correlated with VviGluS and CRE signatures related to DRE, GCC-box, and NACR are found within VviGluS promoter indicating potential regulation of these TFs on VviGluS. # DISCUSSION Our results represent the first multi-omics study of the metabolic changes induced by water deficit in grape berry and, to the best of our knowledge, in any fruit. We revealed that both the ABAdependent and ABA-independent signal transduction pathways are modulated by water deficit during fruit ripening, highlighting their central role in a reproductive organ such as the berry, in addition to their widely accepted roles in vegetative tissues of many plants (Yamaguchi-Shinozaki and Shinozaki, 2006; Shinozaki and Yamaguchi-Shinozaki, 2007). Gene-gene, genemetabolite network analyses, as well as gene promoter analysis shed light on the poorly understood systems relationships between regulators, structural genes, and metabolites in the fruit response to water deficit not only in grapes but also in other fruits. The integrated network analysis associated genes involved in amino acid, phenylpropanoid, and flavonoid pathways with the stress-responsive TFs (bZIPs, AP2/ERFs, MYBs, and NACs) that took part in the water deficit-stress signal into tightly interconnected modules. Enrichment of specific CREs (ABRE, DRE, NACR, GCC-box, MYB, AuxRE/ETT, and ZAT6) in DE genes of specific modules was consistent with the module membership of TFs that recognize these sites. This provides strength into the role of these TFs in modulating water deficit-responsive genes. The analysis of gene-metabolite co-response networks in ripening-associated sub-modules led us to propose several VviAP2/ERF and VviNAC members as putative regulators of the amino acid and anthocyanin accumulation in the grape berries under water deficit. Network and promoter analysis also revealed that the participation of VviARFs and AuxRE, involved in the auxin signaling pathway, may be an important component of the fruit response to water deficit. The coordinated response of genes and metabolites (Figure 8 and Supplementary Table S6) to water deficit in a fleshy fruit is similar to what observed in various tissues of A. thaliana (Harb et al., 2010), M. truncatula (Zhang et al., 2014), and Z. mays (Opitz et al., 2016). Several studies have shown that both ABA-dependent and ABA-independent pathways are indispensable for water deficit response in vegetative tissues (Yamaguchi-Shinozaki and Shinozaki, 2006; Shinozaki and Yamaguchi-Shinozaki, 2007). Here, we show that similar mechanisms apply in a reproductive organ such as the grape berry. In regards to the ABA-dependent signaling pathway, several VviAREB/ABFs and VvibZIP genes were modulated by water deficit during berry ripening when deficit reached its highest severity. In regards to the ABA-independent pathway, VviAP2/ERFs-DREBs TFs were highly induced by water deficit (Mizoi et al., 2012) as reported in Arabidopsis (Lin et al., 2008; Cheng et al., 2013). The relevance for the observed modulation of these pathways in the grape may be attributed with higher ABA sensitivity, reduced transpiration rate, and improved drought tolerance (Kang et al., 2002; Fujita et al., 2005). In our dataset, one TF in particular (VviHB12) was upregulated by water deficit at 53, 67, 81, and 106 DAA. The induction of VviHB12 at 53 DAA, just before the onset of ripening and when major effects of water deficit on the level of transcripts and metabolites were not observed yet, suggests that VviHB12 may be involved in one of the earliest responses to water deficit. The Arabidopsis AtHB12 is strongly induced by water deficit and ABA, and participates in the regulation of ABA signaling through the regulation of PP2C and ABA receptor gene expression (Olsson et al., 2004; Valdés et al., 2012). Similarly to what was found in Arabidopsis, the overexpression of this gene is also related with the higher expression of a VviPP2C highly expressed during fruit ripening (Supplementary Figure S3). The involvement of multiple stress regulons (Nakashima et al., 2014) might be critical to orchestrate the balance between stress-responsive regulation and the berry ripening program. These examples suggest that increased osmotic stress signals induced by water deficit may further fine-tune the ripening program through regulating multiple interacting TFs, possibly accelerating ripening (Castellarin et al., 2007b; Herrera and Castellarin, 2016). Further supporting this, several TF 'switch' genes that may be master regulators of berry ripening, such as VviMYBA1-2, VviNAC1/VviNAC33- 47-71, and VviLBD15-18-38 (Palumbo et al., 2014), are induced under water deficit from the onset of ripening onward. Furthermore, this study confirmed several metabolic reprogramming patterns previously reported in grapevine as well as in other plants. WD increased the accumulation of proline and branch chain amino acids such as leucine, valine, and isoleucine, confirming their role in drought response in grapevine (Deluc et al., 2009; Hochberg et al., 2015), A. thaliana (Nambara et al., 1998; Urano et al., 2009) and O. sativa (Maruyama et al., 2014). In the case of proline increase a parallel up-regulation of the key biosynthetic gene VviP5CS and of VviGluDH was observed. A study in tobacco and grape has shown that abiotic stress-induced ROS activate GluDH expression and enhances GluDH activity to produce glutamate for proline biosynthesis (Skopelitis et al., 2006). The coordinative induction of VviGluDH with VviP5CS under water deficit, observed in WD berries during ripening, may be a conserved mechanism necessary for maintaining a large amount of glutamate available for proline accumulation. NAC binding sites in VviP5CS promoter and the CRE signatures related to DRE, GCCbox, and NACR found within VviGluS promoter indicate potential regulation of these TFs on the proline accumulation. Direct implication of NAC1 on P5CS, or RD26 and ERF62 on GluS regulation has not been shown in plants, but some evidences show that other plant NAC and AP2/ERF members, such as JUNGBRUNNEN1/ANAC042 (Wu et al., 2012), OsNAC5 (Song et al., 2011), or GmERF3 (Zhang et al., 2009) are directly implicated in drought stress-mediated proline accumulation. Several transcripts involved in phenylpropanoid and flavonoid biosynthesis, including VviMYB5b – a generic regulator of this pathway (Deluc et al., 2008; Cavallini et al., 2014), were enhanced by water deficit in parallel with a higher accumulation of related metabolites, such as benzoic and cinnamic acids, and anthocyanins. Previous studies have already reported a modulation of these pathways in grape berries exposed to water deficit (Deluc et al., 2009; Savoi et al., 2016). Nonetheless, our gene-metabolite network analysis identified correlations between specific structural and regulatory flavonoid genes (e.g., CHSs, LDOX, UFGT, AOMT, F30 5 <sup>0</sup>Hs, and MybAs) and anthocyanin modulated by water deficit. The large demand for precursor for anthocyanins production possibly determines the observed impairment of stilbenoid production which decreased both in biosynthesis and concentration, indicating a redirection of phenylpropanoids to the flavonoid pathway instead to the stilbenoid one. However, the two MYBs, VviMYB14 and VviMYB15, that regulate stilbene biosynthesis in grapevine (Höll et al., 2013) do not correlate with the transcripts levels of VviSTSs modulated in WD berries, suggesting that other TFs might contribute to the stilbenoid regulation (Wong et al., 2016). One candidate could be VviMYBC2-L1, a negative regulator of flavonoid (anthocyanin and proanthocyanidin) and stilbenoid biosynthesis (Huang et al., 2014; Cavallini et al., 2015), that was up-regulated at 67 and 81 DAA, potentially repressing VviSTS transcripts. Stilbenoid production increased in Cabernet Sauvignon berries exposed to water deficit (Deluc et al., 2011) but were not significantly affected in Tocai Friulano (Savoi et al., 2016), indicating that the degree of water deficit and the genotype may be key factors for stilbenoid accumulation under drought events. Analysis of gene-metabolite co-response networks in ripening-associated sub-modules, revealed a strong coordinated response between structural pathway genes and metabolite whilst identifying known regulators for grape anthocyanin biosynthesis (e.g., VviMYBA1-2). Gene-metabolite correlation networks has been successfully applied to prioritize candidate genes involved the control of fruit composition and development in tomato (Mounet et al., 2009) and in grapes (Zamboni et al., 2010; Savoi et al., 2016). In this study, we identified new candidate regulators for anthocyanin compounds, including several VviNACs that may play direct and/or indirect roles in regulating structural genes or specific pathway regulators, respectively. A recent study demonstrated that NAC TFs (PpBL, PpNAC1, and PpNAC2) can trans-activate PpMYB10.1 (homologs to VviMYBA1-2) promoter and that the silencing of PpBL inhibits anthocyanin pigmentation in peach fruits (Zhou et al., 2015). Little is known on the regulation of grape amino acid metabolism. The WD11 metabolite-gene subnetwork centered on proline, valine, and threonine correlated with the expected pathway genes such as VviP5CS, VviGluDH, VviGluS, VviTS, and other amino acid metabolism genes, and with TFs, such as VviERF1, VviNAC1, and VviERF62. Functional validation of these regulatory modules is necessary to confirm their role in the regulation of critical anthocyanin and amino acid biosynthetic genes in response to water deficit. Many ABRE, DRE/CRT, and NACR CREs involved in stress-responsive transcription in vegetative tissues (Yamaguchi-Shinozaki and Shinozaki, 2006) were found in promoters of DE genes and were also enriched in water deficitinduced modules. This highlights the conserved role of these stress-responsive CREs in modulating water deficit-responsive genes in reproductive tissues such as berries. Nonetheless, our analysis also show additional CRE pertaining to auxin responses may be an important component of the fruit response to water deficit, an observation/role that has not been implicated before in other fruit systems. Enrichment of the auxin response element (AuxRE/ETT – TGTCGG) in water deficit-induced modules (e.g., WD2 and WD11) suggests that ARFs that bind to these sites may play an important role in regulating water deficit-induced genes in berries, potentially via auxin signaling. ARFs have relevant function in drought-stress responses in plants like Glycine max (Ha et al., 2013) and regulate many aspects of fruit development and ripening (Kumar et al., 2014). Some of them [e.g., SlARF2, Hao et al. (2015)] are central components of fruit (tomato) development and ripening regulatory network. These observations reinforces that stress-responsive CREs may serve a critical role in ripening regulatory networks and fruit maturation. Many fruit ripening-associated TFs bind to these elements (Karlova et al., 2014; Kumar et al., 2014; Leng et al., 2014), of which some (e.g., VviABF2 and tomato SlNAC4 – homologs of Arabidopsis ATAF1 and VviATAF1) are known to regulate both abiotic stress responses and fruit ripening (Nicolas et al., 2014; Zhu et al., 2014). Finally, limited water availability affects VOCs production in several plant organs (Nowak et al., 2010; Griesser et al., 2015), including fleshy fruits, such as apple (Behboudian et al., 1998; Hooijdonk et al., 2007), and tomato (Veit-Köhler et al., 1999). We have recently reported that water deficit modulates the synthesis of monoterpenes in white grapes (Savoi et al., 2016) and, interestingly, at the end of ripening several volatiles such as 1-octen-3-one, (E)-2-heptenal, (E)-2-octenal, and nonanol were commonly up-regulated by water deficit in Merlot as well as in Tocai Friulano. The higher accumulation of VOCs compounds under water deficit may be the result of complex modulation of fatty acid degradation pathway genes, such as VviLOX and VviHPL1 observed here (Supplementary Table S5). Furthermore, consistent up-regulation of triacylglycerol lipase and phospholipase A2 transcripts suggests an additional role for storage lipid degradation on various VOC accumulation. In cucumber, phospholipase A2 has been demonstrated increase the susceptibility of lipid body membrane to lipolytic enzymes such as LOX and lipases via partial degradation of membrane proteins and associated phospholipid monolayer (Rudolph et al., 2011). Indeed, silencing of TomloxC in tomatoes has been shown to increase C8 and C10 compounds (Orzaez et al., 2009) and decrease C5 and C6 ones in ripe fruits. This is probably due to higher precursor availability to other LOXs that may catalyze the production of C8 and C9 precursors (Senger et al., 2005). Shen et al. (2014) also demonstrated that the modulation of tomato HPL correlates with changes in C5 and C6 volatiles in fruits. Consistent induction of VviHPL1 from the onset of ripening onward may explain higher accumulation of VOC aldehydes under water deficit. Moreover, a role of LOXs and HPLs in the drought adaptation process has been already suggested in Arabidopsis (Grebner et al., 2013; Savchenko et al., 2014). While various chain length volatiles impart important flavor attributes (Baldwin et al., 2004; Ripoll et al., 2014), these compounds may also act as powerful signaling molecules activating abiotic stress response (Alméras et al., 2003; Yamauchi et al., 2015) and potentially hasten the developmental program in berries during water deficit. Both abiotic stress and ripening processes involves oxidative stress signals. Recent studies have demonstrated that (E)-2 hexenal, 2-butenal, and 3-hepten-2-one treatment in Arabidopsis seedlings can trigger large transcriptome changes involving abiotic stress genes and TFs, such as DREBs (Yamauchi et al., 2015). The possibility of VOCs in modulating maturationrelated transcriptome changes (e.g., senescence and ripening) in fruit tissues should not be discounted in light of these observations. # CONCLUSION Our results confirm several previously reported modulation of the primary and specialized metabolism and also provide new insight into the stilbenoid and volatile compounds response to water deficit. The integration with network analysis revealed major water deficit-regulated gene modules that are strongly linked to central and specialized metabolites as well as multiple signal transduction pathways (e.g., regulation of anthocyanin and amino acids via members of VviAP2/ERF and VviNAC TF families). Activation of both ABA-dependent and ABAindependent signaling pathway may also be critical to achieve a balance between the regulation of the stress response and the berry ripening program. Further functional analyses are needed to characterize the putative identified modulators of this metabolic response. This study represents a first step into understanding the transcriptional control and their downstream regulatory cascades in grapes or other fruits while providing an important resource for breeding opportunities, irrigation management, and comparative studies centered on reproductive tissue metabolism under abiotic stress in fruit crops. # AUTHOR CONTRIBUTIONS SS participated in the design of the study, carried out the specialized metabolite analyses, RNA extractions, part of the transcriptome data analysis, and drafted part of the manuscript; DCJW carried out part of the transcriptome data analysis, the network analysis, and drafted part of the manuscript; AD carried out the central metabolite analysis; JCH carried out the anthocyanin analysis; BB performed the field experiment; EP coordinated the field experiments; AF supervised the central metabolite analysis and critically revised the manuscript; FM participated in the design of the study, supervised the metabolite analysis, and critically revised the manuscript; SDC conceived the study, coordinated the experiments, supervised the field experiment, transcriptome analysis, and network analysis, interpreted the results, and drafted part of the manuscript. All authors read and approved the final manuscript. #### FUNDING This study was funded by the European Territorial Cooperation program (Sustainable viticulture and improvement of the territorial resources of the grape and wine industry), the Fondazione Edmund Mach (GMPF Program), the COST Action FA1106 Quality Fruit, Genome British Columbia (10R21188), and the Natural Sciences and Engineering Research Council of Canada (10R23082). #### ACKNOWLEDGMENTS We would like to thank Panagiotis Arapitsas, Georg Weingart, Silvia Carlin, and Domenico Masuero for technical support in the metabolite analyses, and Federica Cattonaro and Mara Miculan of the Institute of Applied Genomics (Udine, Italy) for technical support in the RNA-sequencing analysis. #### REFERENCES #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01124/ full#supplementary-material TABLE S1 \| List of genes and primer sequences assayed for expression by qPCR. TABLE S2 \| Impact of water deficit on grape productivity. TABLE S3 \| List of compounds identified in this study using GC-MS, UHPLC-MS/MS, HPLC-DAD, and HS-SPME-GC-MS platforms. TABLE S4 \| RNA sequencing analysis metrics. TABLE S5 \| Summary of differentially expressed genes and associated information at 26 (A), 53 (B), 67 (C), 81 (D), and 106 (E) days after anthesis. (F) TFs differentially expressed. (G) Information on TFs reported in Figure 7. (H) List of differentially expressed genes involved in ROS production, scavenging, and signaling pathways and associated information. TABLE S6 \| (A) Weighted correlation gene co-expression network analysis (WGCNA). (B) CRE sequence, length, and description, number of genes containing each CRE, total occurrence in the genome, enrichment value, and the list of genes containing each CRE. (C) Module-metabolite co-response matrix determined for each module. to the production of the leaf aldehyde in plants. J. Biol. Chem. 291, 14023–14033. doi: 10.1074/jbc.M116.726687 rin) and ethylene receptor (Nr) mutants reveals novel regulatory interactions. Plant Physiol. 157, 405–425. doi: 10.1104/pp.111.175463 expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 18, 2767–2781. doi: 10.1105/tpc. 105.038323 SlNAC4, functions as a positive regulator of fruit ripening and carotenoid accumulation. Plant Cell Physiol. 55, 119–135. doi: 10.1093/pcp/ pct162 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 Savoi, Wong, Degu, Herrera, Bucchetti, Peterlunger, Fait, Mattivi and Castellarin. 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. , # Transcriptional Responses to Pre-flowering Leaf Defoliation in Grapevine Berry from Different Growing Sites, Years, and Genotypes Sara Zenoni<sup>1</sup> \, Silvia Dal Santo<sup>1</sup> , Giovanni B. Tornielli<sup>1</sup> , Erica D'Incà<sup>1</sup> , Ilaria Filippetti<sup>2</sup> Chiara Pastore<sup>2</sup> , Gianluca Allegro<sup>2</sup> , Oriana Silvestroni<sup>3</sup> , Vania Lanari<sup>3</sup> , Antonino Pisciotta<sup>4</sup> , Rosario Di Lorenzo<sup>4</sup> , Alberto Palliotti<sup>5</sup> , Sergio Tombesi5,6 , Matteo Gatti<sup>6</sup> and Stefano Poni<sup>6</sup> <sup>1</sup> Department of Biotechnology, University of Verona, Verona, Italy, <sup>2</sup> Department of Agricultural Science, University of Bologna, Bologna, Italy, <sup>3</sup> Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università Politecnica delle Marche, Ancona, Italy, <sup>4</sup> Department of Agricultural and Forest sciences, University of Palermo, Palermo, Italy, <sup>5</sup> Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università di Perugia, Perugia, Italy, <sup>6</sup> Dipartimento di Scienze delle Produzioni Vegetali Sostenibili, Università Cattolica del Sacro Cuore, Piacenza, Italy #### Edited by: Giuseppe Ferrara, Università degli Studi di Bari Aldo Moro, Italy #### Reviewed by: Ana Margarida Fortes, Instituto de Biossistemas e Ciências Integrativas (BioISI), Portugal Massimiliano Corso, Université libre de Bruxelles, Belgium Christine Böttcher, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia > \Correspondence: Sara Zenoni [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 20 January 2017 Accepted: 07 April 2017 Published: 02 May 2017 #### Citation: Zenoni S, Dal Santo S, Tornielli GB, D'Incà E, Filippetti I, Pastore C, Allegro G, Silvestroni O, Lanari V, Pisciotta A, Di Lorenzo R, Palliotti A, Tombesi S, Gatti M and Poni S (2017) Transcriptional Responses to Pre-flowering Leaf Defoliation in Grapevine Berry from Different Growing Sites, Years, and Genotypes. Front. Plant Sci. 8:630. doi: 10.3389/fpls.2017.00630 Leaf removal is a grapevine canopy management technique widely used to modify the source–sink balance and/or microclimate around berry clusters to optimize fruit composition. In general, the removal of basal leaves before flowering reduces fruit set, hence achieving looser clusters, and improves grape composition since yield is generally curtailed more than proportionally to leaf area itself. Albeit responses to this practice seem quite consistent, overall vine performance is affected by genotype, environmental conditions, and severity of treatment. The physiological responses of grape varieties to defoliation practices have been widely investigated, and just recently a whole genome transcriptomic approach was exploited showing an extensive transcriptome rearrangement in berries defoliated before flowering. Nevertheless, the extent to which these transcriptomic reactions could be manifested by different genotypes and growing environments is entirely unexplored. To highlight general responses to defoliation vs. different locations, we analyzed the transcriptome of cv. Sangiovese berries sampled at four development stages from pre-flowering defoliated vines in two different geographical areas of Italy. We obtained and validated five markers of the early defoliation treatment in Sangiovese, an ATP-binding cassette transporter, an auxin response factor, a cinnamyl alcohol dehydrogenase, a flavonoid 3-O-glucosyltransferase and an indole-3-acetate beta-glucosyltransferase. Candidate molecular markers were also obtained in another three grapevine genotypes (Nero d'Avola, Ortrugo, and Ciliegiolo), subjected to the same level of selective pre-flowering defoliation (PFD) over two consecutive years in their different areas of cultivation. The flavonol synthase was identified as a marker in the pre-veraison phase, the jasmonate methyltransferase during the transition phase and the abscisic acid receptor PYL4 in the ripening phase. The characterization of transcriptome changes in Sangiovese berry after PFD highlights, on one hand, the stronger effect of environment than treatment on the whole berry transcriptome rearrangement during development and, on the other, expands existing knowledge of the main molecular and biochemical modifications occurring in defoliated vines. Moreover, the identification of candidate genes associated with PFD in different genotypes and environments provides new insights into the applicability and repeatability of this crop practice, as well as its possible agricultural and qualitative outcomes across genetic and environmental variability. Keywords: grapevine, pre-flowering defoliation, berry transcriptome, flavonoid, secondary metabolite #### INTRODUCTION fpls-08-00630 April 29, 2017 Time: 12:26 # 2 Viticulture is still strongly bound to the concept of terroir, relating the sensory attributes of wine to the environmental conditions in which the grapes are grown (Van Leeuwen and Seguin, 2006). Though a shared definition of terroir is still hard to find, there is general consensus that the main factors composing terroir are climate, soil, cultivar/rootstock and human practices and that these factors strongly interact (Renouf et al., 2010). Quantifying the relative importance of each factor influencing terroir is an extremely difficult task since the variability of all factors involved must be represented. A quite considerable effort was made by van Leeuwen et al. (2004) who concluded that climate, soil, and cultivar had a decreasing importance in influencing performance of the cultivars Merlot, Cabernet franc, and Cabernet Sauvignon grown in three different soil environments and observed over 5 years. A first important consequence of the complex climate × soil × cultivar interactions is that the same cultivar grown in different environments can originate products of different composition and market value. The capacity of a genotype to modulate its phenotype under different environmental conditions is defined phenotypic plasticity, a phenomenon of considerable interest in plant physiology. Over the last decade, a number of studies exploring metabolomic and transcriptomic bases of phenotypic plasticity in Vitis vinifera have been conducted in local cultivars such as Corvina and Garganega (Dal Santo et al., 2013a, 2016b; Fernie and Tohge, 2013; Anesi et al., 2015). These works demonstrated the direct effect of growing conditions on gene expression during berry ripening, allowing several environmentally modulated genes to be identified, including many belonging to the phenylpropanoid/flavonoid pathway (Dal Santo et al., 2013a, 2016b). The existence of a terroir-specific effect on berry transcriptome and metabolome was also revealed, which persists over several vintages (Anesi et al., 2015), and specific plastic transcripts were associated with groups of vineyards sharing common viticulture practices (Dal Santo et al., 2013a). If it is agreed that human practices are an important component of the terroir concept (Renouf et al., 2010), forecasting their effects on grape composition and wine can greatly benefit from associating omics tools to traditional agronomic assessment. Among the different operations pertaining to grapevine canopy management, pre-flowering leaf removal is likely the one that has received the greatest interest from the scientific community over the last decade. Starting with the original work (Poni et al., 2006), a number of subsequent studies, representing a broad array of cultivars and environments (Diago et al., 2012; Gatti et al., 2012, 2015; Palliotti et al., 2012; Lee and Skinkis, 2013; Pastore et al., 2013; Risco et al., 2014; Komm and Moyer, 2015; Sternad Lemut et al., 2015; Sivilotti et al., 2016), have confirmed the technique to be valuable and repeatable for: (i) reducing vine yield through a decrease in fruit-set and/or berry size; (ii) decreasing cluster compactness, hence susceptibility to rot diseases, and (iii) improving grape composition in terms of total soluble solids (TSSs), phenolic and aroma compounds. However, it was also observed that the outcome of pre-flowering defoliation (PFD) could be quite variable between consecutive years (Gatti et al., 2015) and that different cultivars or the same cultivar in different environments could be influenced differently by the treatment (Kuhn et al., 2014). Although the practice is widely used in viticulture, very little molecular information is available and, as a consequence, the definition of common mechanisms linking the impact of leaf removal to berry physiological and metabolic responses, is far from complete. The pioneer study conducted on genome-wide expression analysis in cv. Sangiovese vines subjected to either pre-flowering or late season (i.e., at veraison) defoliation revealed a general delay in transcriptional ripening following both treatments (Pastore et al., 2013). Moreover, a more extensive transcriptome rearrangement in berries subjected to PFD was observed, which reflects the uncoupling of metabolic processes, in particular anthocyanin and flavonol synthesis, from the general ripening program (Pastore et al., 2013). A very recent study performed on Sauvignon blanc shed more light on grapevine response to an altered microclimate due to early leaf removal (Young et al., 2016). When main and lateral leaves were removed from the cluster zone at fruit-set in order to induce and maintain berry light exposure, higher levels of carotenoids and volatile terpenoids were found in the berries, in two consecutive years. The study also clearly demonstrated that the main physiological responses occur in the early stages of berry development, when berries are still photosynthetically active (Palliotti and Cartechini, 2001), and that the key response is the change in pigment levels and metabolite pools that have photoprotective and/or antioxidant functions (Young et al., 2016). Overall, it is clear that early defoliation combined with environmental conditions affects berry composition through changes in gene expression. The complexity involved in the reprogramming of berry transcriptome, proteome, and metabolome during development has been progressively described in different grapevine varieties (Deluc et al., 2007; Zamboni et al., 2010; Fasoli et al., 2012; Agudelo-Romero et al., 2013; Dal Santo et al., 2013a; Anesi et al., 2015), demonstrating that a large part of metabolic changes characterizing berry formation and ripening are under transcriptional control. It is also known that grape berry development involves the integration of multiple hormonal signals, with some hormones acting as promoters and others as repressors. In particular, in non-climacteric fruits, such as grape, where no burst in ethylene production is observed during ripening, the abscisic acid (ABA) seems to play a stronger role during ripening and its crosstalk with other growth regulators has been proposed at different berry stages (Davies and Bottcher, 2009; McAtee et al., 2013; Fortes et al., 2015). Despite the amount of information already reported, hormonal control in grape ripening is still poorly understood (Fortes et al., 2015). In this context, the identification of molecular markers, addressing the question of how stable and replicable is the link of PFD to favorable physiological and metabolic changes in berry, represents a huge challenge. In this work, a comparative study of the agronomic and molecular berry responses to PFD was performed in four genotypes grown in different areas of cultivation over two consecutive years, with the aim of identifying genes whose expression could be attributable to this viticulture practice, regardless of site, year, and genotype. Molecular responses in Sangiovese berries during development from defoliated and untreated control vines in two different growing sites were investigated by a genome wide expression analysis. The expression profiles of selected candidate genes were assessed by qPCR in all experimental conditions and integrated with agronomic and ripening parameters, to unveil developmental and metabolic processes commonly affected in berries after PFD. ## MATERIALS AND METHODS #### Plant Material, Experimental Layout, and Berry Sampling for Gene Expression Analyses Berry samples for subsequent transcriptomic analyses and real time qPCR analyses were taken from mature and healthy vineyards located in Emilia Romagna (cvs Sangiovese and Ortrugo), Umbria (cv. Ciliegiolo), Marche (cv. Sangiovese), and Sicily (cv. Nero d'Avola), Italy. Sangiovese plots in Emilia Romagna and Marche shared the same rootstock (S.O.4.), whereas clones were different: clone R24 and clone SG12T, respectively. All vineyards were standard either cane or spur pruned vertically shoot positioned (VSP) trellises. Single vine spacing within row varied between 0.8 and 1.5 m, whereas between-row spacing was between 2.5 and 3.3 m, resulting in a vine density varying from 2020 to 5000 vines/hectare. More details regarding trellis structure, bud load, soil characteristics, climate trends and canopy and vineyard management practices can be found in Filippetti et al. (2011), Alagna et al. (2014), Gatti et al. (2015), and Silvestroni et al. (2016). In 2012 and 2013, in each site × cultivar combination, two treatments were compared consisting of PFD performed at the "separated closed flowers" stage (Baggiolini, 1952) by removing the six basal main leaves of all shoots on each test vine (varying from 6 to 12 according to site; Supplementary Figure 1A) while any lateral shoot emerging from the same basal nodes at the time of defoliation was retained. PFD was compared with a non-defoliated control treatment (C). Using an identical sampling protocol, berry sampling at each site × cultivar combination was performed on both control and PFD treatments at four development stages as follows: 20 days after leaf removal (Stage 1); hard and green berries at veraison (i.e., 1–5% slightly colored berries in a cluster) (Baggiolini, 1952) (Stage 2); soft, yet still not colored berries at veraison (Stage 3), berries at a TSS concentration of about 18◦Brix (Stage 4). On each sampling date, a batch of 60 berries was collected. In detail, three independent pools of 20 berries each were collected from clusters of different vines in order to create three biological replicates that represent almost the entire variability of the experimental design. The sampling was performed by carefully cutting each berry at the pedicel with scissors in order to avoid any damage or juice loss. Berries were immediately frozen in liquid nitrogen and then shipped to labs at the University of Verona (northern Italy) for transcriptome processing and expression analyses. Given the typical asynchrony in individual berry ripening, berry sampling for Stages 2 and 3 were performed on the same date for both treatments. In total for the transcriptomic analysis on Sangiovese, the experiment entailed the collection and analysis of 48 berry samples (4 stages × 2 treatments × 2 sites × 3 biological replicates). For the real time qPCR on all site × cultivar combinations, the experiment entailed the collection and analysis of 240 berry samples (4 stages × 2 treatments × 5 sites × 3 biological replicates × 2 years). ### Vegetative Growth and Yield Components For every site × cultivar combination vegetative growth capacity was expressed by estimated total final leaf area per vine and measured pruning weight. Total leaf area per vine was estimated by node counts and surface area of fully expanded main and lateral leaves (Lopes and Pinto, 2015), whereas 1-year pruning weight per vine was taken soon after leaf shedding in fall was completed. At harvest, each year, total yield and cluster number per vine were recorded, and mean cluster weight calculated accordingly. Single berry weight was taken on the samples then processed for must analyses and total berry number calculated from mean cluster weight. For more details on sample size and sampling procedures, please refer to the papers cited above. Source-to-sink balance was expressed as leaf area-to-yield ratio. ### Must Composition and Phenolic Compounds Analyses Four-to-six 100-berry samples were taken pre-harvest from each genotype by treatment combination in the different test sites. The 100-berry sample was composed by five berries taken for a total of 20 clusters; two berries were sampled from the top portion or wings, two from the middle and one from the tip of the cluster in order to account for within cluster variability in ripening. TSS concentration, pH, and titratable acidity (TA) were determined on must samples according to standard methods described in Iland et al. (2011). Total anthocyanin concentration (mg/kg of fresh berry mass) was determined according to Iland (1988). Flavonol compounds were extracted from grape skins as reported by Downey and Rochfort (2008). In brief: 0.100 g of lyophilized grape skins were extracted in 1.0 mL of 50% (v/v) methanol in water for 20 min with sonication. The extracts were centrifuged (5 min at 10000 × g at 4 ◦C), filtered through a 0.22 µm polypropylene syringe for HPLC analysis and transferred to HPLC auto-sampler vials. The chromatographic method was developed using an Agilent 1260 Infinity Quaternary LC (Agilent Technology, Santa Clara, CA, USA) consisting of a G1311B/C quaternary pump with inline degassing unit, G1329B autosampler, G1330B thermostat, G1316B thermostatted column compartment and a G4212B diode array detector fitted with a 10 mm path, 1 µL volume Max-Light cartridge flow cell. The instrument was controlled using Agilent Chemstation software version A.01.05. Separation was achieved on a reverse-phase C-18 Synergi Hydro RP 80A, 250 mm × 4.6 mm, 4 µm (Phenomenex, Torrance, CA, USA). The solvents used were 5% (v/v) formic acid (solvent A) and acetonitrile (solvent B). The flow rate was 0.5 mL/min, with a linear gradient profile consisting of solvent A with the following proportions (v/v) of solvent B: 0–10 min, 2–10% B; 10–25 min, 10–12% B; 25–35 min, 12–30% B; 35–43 min, 30% B; 43–48 min, 30–40% B; 48–52 min, 40–50% B; 52–55 min, 50–60% B; 55–58 min, 60–98% B; 58–63 min, 98% B; 63–66 min, 98–2% B; 66–72 min 98% B. The column temperature was maintained at 40 ± 0.1◦C. Five microliters of sample extract was injected. The elution was monitored at 200–700 nm, detection by UV-Vis absorption with DAD scanning between 280, 320, and 370 nm. Anthocyanins and flavonols were identified using authentic standards and by comparing the retention times. Quantification was based on peak areas and performed by external calibration with standards. ### Statistical Analyses A completely randomized block design was used and the agronomic parameters and must composition were subjected to analysis of variance (ANOVA, SAS statistical software, SAS Institute, Cary, NC, USA) and mean separation performed by t-test. In other cases, variability across treatments was expressed as mean ± standard error (SE). #### RNA Extraction Total RNA was isolated from approximately 400 mg of berry pericarp tissue (i.e., entire berries without seeds) using the SpectrumTM Plant Total RNA kit (Sigma–Aldrich), with modifications as described in Dal Santo et al. (2016a). Seeds were manually removed from the 20 berries of each biological replicate before the liquid nitrogen grinding procedure. RNA quality and quantity were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and a Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, CA, USA). # Microarray Analyses and Statistical Approaches We hybridized 5 µg of total RNA per sample to a NimbleGen microarray 090818\_Vitus\_exp\_HX12 chip (Roche, NimbleGen Inc., Madison, WI, USA), representing 29,549 predicted genes on the basis of the 12X grapevine V1 gene prediction version. The hybridization was performed according to the manufacturer's instructions (Dal Santo et al., 2016a). Statistical analysis of the microarray data was conducted using TMeV v4.8 (mev.tm4.org/). Statistical analysis of microarrays (SAM) was conducted with a false discovery rate (FDR) of 0.1% and ANOVA using α = 0.01 and standard Bonferroni correction in order to skim off genes that showed a high variability among the three biological replicates. Correlation matrixes were prepared using R software and Pearson's correlation coefficient as statistical metric to compare the values of the whole transcriptome (29,549 genes) in all analyzed samples. Correlation values were converted into distance coefficients to define the height scale of the dendrogram. Principal component analysis (PCA) was conducted using SIMCA P+ v13 (Umetrics, USA) and applied to the significantly modulated transcripts dataset (18,771 genes). Differentially modulated genes at each developmental stage in both sites were retrieved by performing a between-subjects (C vs. PFD samples) t-test (α = 0.01), assuming equal variance among samples. Gene Ontology (GO) enrichment analysis was performed with the AgriGO online software<sup>1</sup> , using Singular Enrichment Analysis (SEA) tool and Fisher's as statistical test method (Du et al., 2010). Heat maps were created using log2 transformed expression values and then median-centered by transcript. Cluster analysis was conducted on transcript mediancentered fluorescent values by the k-means method (KMC) with Pearson's correlation distance. We used the Figure of Merit (FOM) statistic to determine the optimal number of clusters (n = 10). ## Reverse Transcription (RT) and Real-Time qPCR One microgram of total RNA was treated with DNase I (Promega) according to the instructions provided with the commercial kit. DNase treated RNA was then used for cDNA synthesis using the Improm-II TMReverse Transcriptase (Promega) following the producer's indications. The transcriptional profile was analyzed by real-time RT-PCR as described by Zenoni et al. (2011), using the SYBR Green PCR master mix (Applied Biosystems) and a Mx3000P real-time PCR system (Stratagene). Each expression value, relative to VvUBIQUITIN1 (VIT\_16s0098g01190), widely used as a suitable and robust reference gene during berry development (Chen et al., 2013; Dal Santo et al., 2013b, 2016b; Cramer et al., 2014), was determined in triplicate. Non-specific PCR products were identified by the dissociation curves. Amplification efficiency was calculated from raw data using LingRegPCR software (Ramakers et al., 2003). The mean normalized expression (MNE)-value was calculated for each sample referred to the ubiquitin expression <sup>1</sup>http://bioinfo.cau.edu.cn/agriGO Zenoni et al. Grape Responses to Defoliation according to the Simon equation (Simon, 2003). Standard error (SE)-values were calculated according to Pfaffl et al. (2002). The primer sequences used in qPCR analysis are listed in Supplementary Table 1. # Analysis of Correlation between Microarray and qPCR Correlation between the microarray and qPCR results was performed for the six putative molecular markers of the PFD treatment in Sangiovese for the year 2012, and the statistical significance of this correlation determined. For the NimbleGen microarray, the data input into the correlation analysis was the Log2 value of the average of the three biological replicates for each gene × site × treatment combination. For qPCR, we used the mean Log2 ratio value reported by qPCR from all replicate. Prior to performing correlation analyses, the data were tested for normality using the Shapiro–Wilk test, as indicated by Morey et al. (2006). Because the data were not normally distributed, Spearman's Rho, instead of Pearson's correlation, was computed using R software. The calculated correlation coefficient was 0.4617597 (Spearman's Rho, p = 2.185e-06, n = 96). By normal standards [n = (96 – 2) = 94], the correlation between the NimbleGen microarray data and qPCR data for the indicated six genes, would be considered statistically significant (p < 0.005). #### Accession Numbers Grape berry microarray expression data are available in the Gene Expression Omnibus under the series entry GSE92980<sup>2</sup> . # RESULTS #### Impact of Pre-flowering Defoliation on Agronomic Parameters and Berry Transcriptome of cv. Sangiovese under Two Growing Conditions In order to provide a preliminary evaluation of site × early defoliation interaction, the cv. Sangiovese was subjected in 2012 to the pre-flowering defoliation treatment (PFD) using the same protocol in Ancona (AN) and Bologna (BO) and, within each site, a non-defoliated control treatment (C) was also included. Daily maximum, minimum, and mean air temperatures (T) as well as daily rainfall for the two locations evaluated from the 1 April until 30 September, showed some common features for the two sites, with a quite cool spring and long summer (Supplementary Figure 1B). Agronomic parameters showed that the two sites shared significant differences for total leaf area and yield per vine, cluster weight, and berries per cluster between PFD and C treatments, with PFD showing lower values than C vines in all cases (Table 1). The remaining parameters, including the source–sink balance expressed as leaf area-to-yield ratio were either unchanged or slightly enhanced in AN, as concerns TA and total anthocyanins concentration. To investigate the molecular changes that take place after defoliation during berry development, Sangiovese berry transcriptomes of PFD and C vines were compared at the four berry developmental stages in both sites (Figure 1A). A dendrogram of the global transcriptomic data revealed enough uniformity among the three biological replicates in the C and PFD samples at each time point (Figure 1B). The main separation among samples is related to berry stage, with Stage 1 resulting as the most divergent. Indeed, Stage 2 showed more similarity at the transcriptional level to Stages 3 and 4, suggesting that, despite berries still being hard and green at Stage 2, many molecular processes related to ripening were already activated. Interestingly, the second variable that strongly influenced sample association was site. In fact, except for Stage 1, AN and BO samples were characterized by distinctive berry transcriptomes during ripening. This evidence highlights the strong effect of growing conditions on berry transcriptome in Sangiovese and suggests that this effect is more evident when the ripening program is initiated. Concerning the effect of the defoliation treatment on berry transcriptome we found that PFD and C vines were distinguishable only at specific combinations of berry stage and site. In fact, the separation between PFD and C is evident at Stage 1 in AN, at Stage 2 in BO, at Stage 3 in AN and at Stage 4 in BO. These results suggest that, in Sangiovese, PFD has a weaker effect on berry transcriptome than growing conditions. To retrieve genes differentially modulated under our experimental conditions, the berry transcriptome dataset was screened by significance analysis of microarrays (SAM, 16 groups, FDR = 0.1%). Analysis of variance (ANOVA, 16 groups, α = 0.01, standard Bonferroni correction) was applied to transcripts positive in the previous SAM experiment in order to skim off the most significantly modulated transcripts. We obtained a reduced dataset of 18,771 genes (Supplementary File 1), which was inspected by PCA analysis. The two principal components, explaining 56.5% of the total dataset variability, allowed berry samples to be clearly separated on the basis of their developmental stage (Figure 1C). Sample distribution confirmed that Stage 2 is more similar to Stage 3 at both sites; moreover, it can be observed that, at transcriptional level, the ripening process (Stages 3 and 4) at AN is slightly advanced in comparison to BO (Figure 1C). Interestingly, principal component 3 (PC3), explaining 11.8% of the total dataset variability, clearly separated AN from BO samples, in particular after veraison, again evidencing the strong effect of growing conditions on the berry transcriptome rearrangement during ripening in cv. Sangiovese (Supplementary Figure 2). At no stage nor in either site were principal components found that separated PFD samples from C ones. Notwithstanding the small effect of PFD on berry transcriptome, we focused on the identification of differentially expressed genes (DEGs) after PFD, regardless of the growing site. We then compared the PFD and C berry transcriptomes at each time point using a t-test (between subjects t-test, α = 0.01) for both sites separately. The number of DEGs identified between PFD and C vines at each stage was different in the two sites <sup>2</sup>https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE92980 #### TABLE 1 \| Agronomic and ripening parameters 2012. Vegetative growth, yield parameters, and grape composition recorded in 2012 on cv. Sangiovese grapevines grown in Ancona and Bologna and subjected, within each location, to a pre-flowering defoliation (PFD) or undefoliated (Control). <sup>∗</sup>P < 0.05 and ∗∗P < 0.01, respectively. (Figure 2A and Supplementary File 2). In particular, at Stage 1 and 3 a higher number of DEGs characterized AN, whereas at Stage 2 and 4 a higher number of DEGs was found in BO. These differences well mirrored the behavior observed in the dendrogram analysis. A total of 1746 and 1041 DEGs in at least one stage between PFD and C vines, were found in BO and AN, respectively. By comparing the list of DEGs from BO and AN, only 125 genes were identified as differentially expressed at both sites (Figure 2B and Supplementary File 3). The GO enrichment analysis performed on the three group of DEGs, i.e., BO-specific, AN-specific and common, revealed a significant overrepresentation of the "response to stimulus" functional category in all groups of DEGs. Genes belonging to this functional category may be involved in the detection and response to external and endogenous stimuli and also to many stresses, such as biotic and abiotic stress, redox state, and others. Regarding the common DEGs, another two functional categories resulted as significantly overrepresented, the "biosynthetic process," and "cellular amino acid and derivative metabolic process," represented by an aspartate aminotransferase, two glycine hydroxymethyltransferases, a glutamine synthetase, and a serine hydroxymethyltransferase. #### Identification of Berry Molecular Markers Associated to Pre-flowering Defoliation in Sangiovese In order to identify putative molecular markers associated to the PFD in cv. Sangiovese, independently of site, we focused on the 125 DEGs shared by BO and AN. We checked the expression profile of the 125 genes during berry development on C vines in the two sites, in order to identify genes whose expression was not or slightly influenced by the growing conditions. A KMC clustering analysis identified 10 expression clusters and revealed a very high expression variability of these genes in C vines growing in the two sites, with only 38 genes belonging to same clusters of expression during development in BO and AN (Figure 3A and Supplementary File 4). Subsequently, we evaluated the pattern of Fold change (FC) between PFD and C at each stage in both sites, and we found that only 11 genes were affected by the PFD in a similar manner throughout berry development in BO and AN (Figure 3B). Among these genes we selected six characterized by an upregulation at all stages after the PFD treatment or by an upregulation till Stage 3 and a downregulation at Stage 4 (Figure 3C). The selected genes were represented by the ATP-binding cassette (ABC) transporter VvPDR20/VvABCG50 (VIT\_06s0061g01490), auxin response factor (ARF) 10 (VIT\_13s0019g04380), cinnamyl alcohol dehydrogenase (CAD; VIT\_00s0615g00020), flavonoid 3-O-glucosyltransferase (G3T; VIT\_11s0052g01630), geraniol 10-hydroxylase (G10H; VIT\_02s0012g02820), and indole-3 acetate beta-glucosyltransferase (IND; VIT\_13s0019g03040). The expression of these genes were validated by qPCR on PFD and C berries in the two sites at the four berry developmental stages (Figures 3D–I). The VvPDR20/VvABCG50 showed a peak of expression at Stage 2 in C berries throughout development, whereas in PFD berries its expression significantly increased at Stage 3 in both sites (Figure 3D). Although very few functional studies have been performed on ABC transporters activity in grape, a role in the vacuolar localization and transport of glucosylated anthocyanidins was demonstrated for the member VvABCC1 (Francisco et al., 2013). The expression profiles obtained by our analysis suggest a role of VvPDR20/VvABCG50 at the onset of ripening and evidenced that PFD delays its expression during berry development. In the case of the ARF the increase in expression at Stage 4 in C condition is significantly hastened by the PFD treatment at Stage 3 in both sites (Figure 3E). The CAD gene is characterized by a significant increase of expression at Stage 3 in PFD condition instead of the Stage 4 observed in C vines in AN, and by a significant increase at Stage 2 in PFD in both sites, particularly in BO (Figure 3F). This FIGURE 1 \| Whole transcriptome analysis of Sangiovese berries subjected to PFD treatment in two different sites. (A) Schematic representation of the sampling design used. (B) Cluster dendrogram of the whole transcriptome dataset in all analyzed samples. Pearson's correlation values were converted into distance coefficients to define the height of the dendrogram. Samples are colored according to the developmental stage of sampling. (C) Score scatterplot (PC1 vs. PC2) of the PCA model (9 Principal Components, R 2 (cumulative) = 0.903, Q<sup>2</sup> (cumulative) = 0.848) applied to the significantly modulated transcripts dataset. Samples are colored according to the developmental stage of sampling. Different treatments are indicated by different symbols, "✩" = Control and "O" = Pre-flowering defoliation. behavior well mirrored the trend observed in the dendrogram, showing that the separation between PFD and C is more evident at Stage 2 in BO, and at Stage 3 in AN. The G3T was characterized by an increase in expression at Stage 4 in C condition in both growing sites. For this gene the PFD treatment led to a significant increase of expression level throughout berry development, in particular at Stage 2 in BO and Stage 3 in AN, similarly to the CAD (Figure 3G). The G10H showed a peak of expression at Stage 3 in C vines in both growing sites. The PFD treatment enhances the expression level of this gene at Stage 3 but also significantly hastens its induction at Stage 2 in both BO and AN (Figure 3H). Concerning the IND, involved in the regulation of auxin levels by IAA conjugation (Bottcher et al., 2010; Fortes et al., 2015), a flat expression trend during the first stages of berry development with a slight downregulation at Stage 4 was found in BO C vines, whereas a high expression was observed at Stages 1 FIGURE 3 \| PFD treatment molecular markers of Sangiovese cultivar selection and real-time qPCR validation in 2012. (A) Heat map representing the fluorescence intensity of C vines in the 125 commonly modulated genes. KMC analysis was used to determine the transcripts with unaltered expression between Bologna and Ancona sites (highlighted as same cluster) and those with different expression (highlighted as different cluster). (B) Schematic representation of the Fold Change (FC), calculated between C and PFD vines at each developmental stage in Ancona and Bologna, in the 38 same cluster transcripts found in (A). The black arrows indicate the 11 genes showing a similar trend of FC. (C) FC between C and PFD vines at each developmental stage in Ancona and Bologna in a selection of six transcripts. (D–I) Real-time qPCR validation of the (D) ABC transporter VvPDR20-VvABCG50 (ABC; VIT\_06s0061g01490), (E) Auxin response factor 10 (ARF; VIT\_13s0019g04380), (F) Cinnamoyl alcohol dehydrogenase (CAD; VIT\_00s0615g00020), (G) Flavonoid 3-O-glucosyltransferase (G3T; VIT\_11s0052g01630), (H) Geraniol 10-hydroxylase (G10H; VIT\_15s0048g01490) and (I) Indole-3-acetate beta-glucosyltransferase (IND; VIT\_13s0019g03040) expression profiles in PDF and C Sangiovese vines during berry development in 2012. The mean normalized expression (MNE)-value was calculated for each sample referred to the VvUBIQUITIN1 (VIT\_16s0098g01190) expression according to the Simon equation (Simon, 2003). Bars represent means ± SE of three biological replicates. The significant modulation (t-test, p < 0.05) of gene expression between C and PFD berries at each stage per each site is indicated by an asterisk, red for BO and green for AN. and 2 in AN, followed by a decrease in expression after veraison. The PFD induced a significant increase of IND expression in both sites but at Stage 2 in BO and Stage 3 in AN (Figure 3I). Overall real-time qPCR analysis confirmed the microarray expression profiles for all the selected genes, demonstrating that these genes could be considered putative molecular markers of PFD in berry throughout development in cv. Sangiovese in 2012. In order to investigate the influence of year on the PFD effect, pre-flowering leaf removal was also applied at both sites in 2013 using the same protocol. Seasonal weather data as daily air temperature and rainfall are shown in Supplementary Figure 1B. Leaf area and yield per vine, cluster weight, and berries per cluster were again significantly lower in PFD vines, whereas pruning weight per vine and leaf area-to-yield ratio were not modified (Table 2). A striking berry size reduction was recorded in BO, while TSS were notably higher in PFD in both locations. However, while increased TSS did not achieve a concurrent increase in anthocyanins at the AN site, total anthocyanins were significantly higher in the defoliated vines at BO (Table 2). Berry samples were collected following the same protocol used in 2012. The expression of the six genes identified as markers of PFD was analyzed on C and PFD berries collected during 2013 by the qPCR approach. The ABC transporter gene (ABC) showed the same expression profile as that revealed in 2012 in C and PFD vines, with a with a significant induction at Stage 3 in both PFD vines and a very high expression during PFD berry development in AN (Figure 4A). The ARF expression was confirmed in 2013 in C and PFD berries in both sites (Figure 4B), as well as the effect of PFD on CAD expression (Figure 4C). The expression of CAD in C berries was instead slightly different from 2012 in BO, with a clear decrease of expression from Stage 1 throughout berry development not observed the year before. The effect on G3T expression in PFD berries also resulted as the same in the 2 years, again with the significant and stronger effect at Stage 2 in BO and Stage 3 in AN (Figure 4D). On the contrary, the expression of GH10 gene was strongly affected by year in C berries and not influenced by PFD treatment in either site (Figure 4E). Lastly, beside the different expression in C vines at Stage 2 in AN, IND gene showed the same trend of significant induction as in 2012 due to the PFD treatment in both sites (Figure 4F). Overall, the five genes, ABC, ARF, CAD, G3T, and IND resulted as being putative molecular markers of PFD treatment in Sangiovese during berry development, independently of growing site and year. #### Identification of Putative Molecular Markers of Pre-flowering Defoliation in Different Genotypes In order to evaluate if the five putative Sangiovese marker genes could also represent molecular markers of PFD for other genotypes cultivated in different environments, we applied PFD using the same protocol adopted for Sangiovese, on Nero d'Avola (ND), Ortrugo (OR), and Ciliegiolo (CI) cultivars, cultivated during 2012 and 2013in three different Italian areas, Palermo (PA-Sicily), Perugia (PE-Umbria) and Piacenza (PI-Emilia Romagna), respectively. The environmental parameters recorded during 2012 and 2013 at the three sites are reported in the Supplementary Figure 3. In general, for sites located in north and central Italy (PI and PE) yearly weather patterns and rainfall showed some common features while the Sicilian location (PA) had a quite different trend. As observed for BO and AN, 2012 was marked by a quite cool spring and long summer (mid-June till end of August) in PI and PE, with hot spells reaching 40◦C and very limited rainfall (Supplementary Figures 1, 3). In PA, both seasons showed a more progressive increase in air temperature peaking around 40◦C in 2012 with basically no rainfall. 2013 was slightly cooler with some rain falling at the end of the season. In 2012, yield per vine was significantly reduced by PFD regardless of cultivar, although responsiveness of some yield components showed variability (i.e., unchanged berry weight in Ciliegiolo and unchanged cluster weight and berries per cluster in Ortrugo) (Table 3). TSS at harvest were always increased by PFD in all cultivars and the same response was seen for total anthocyanins in cvs Ciliegiolo and Nero d'Avola. Except for the white cv. Ortrugo, must pH and TA were less responsive overall, whereas final LA/yield ratio was in general slightly enhanced in PFD. In 2013, yield per vine and cluster weight were reduced in PFD regardless of location (Table 4). In agreement with the 2012 response, TSS and total anthocyanins were higher in the defoliated vines across all cultivars. Must pH and TA confirmed their relatively low sensitivity to the applied treatment, whereas moderately higher LA/yield ratios were again found in the PFD vines. Berry samples for gene expression analysis were collected from C and PFD vines at the same four phenological stages used for Sangiovese. The real-time qPCR of the five Sangiovese PFD molecular markers performed on berries collected during 2012 showed that the five genes were not differentially modulated nor showed different expression profiles during berry development in Nero d'Avola, Ortrugo, and Ciliegiolo subjected to PFD (data not shown). These results demonstrated that these genes could not be considered molecular markers of PFD for other genotypes and/or for other environmental conditions. The transcriptomic dataset of 18771 genes obtained for Sangiovese was therefore inspected again with the aim of finding genes consistently modulated by PFD at the same stage in both sites, without necessarily showing a similar expression profile throughout the entire berry development. By using a threshold of \| FC \| > 2 between C and PFD we found that 6 genes were consistently modulated by PFD at Stage 1, 28 genes at Stage 2, 39 at Stage 3, and 19 at Stage 4 in both sites (Supplementary File 5). Among genes modulated at Stage 1, 5 resulted as upregulated by PFD and only 1, an unknown protein, downregulated. The flavonol synthase (VIT\_18s0001g03470) resulted as the most induced by the treatment in both sites and was therefore selected for further transcriptional investigation in other terroirs, years, and genotypes (Supplementary File 5). At Stage 2, 16 genes resulted as commonly upregulated by PFD, including the already analyzed geraniol 10-hydroxylase (G10H), some genes involved in carbohydrate metabolism, the MADS-box AGL20 (VIT\_15s0048g01240) and jasmonate #### TABLE 2 \| Agronomic and ripening parameters 2013. fpls-08-00630 April 29, 2017 Time: 12:26 # 11 Vegetative growth, yield parameters, and grape composition recorded in 2013 on cv. Sangiovese grapevines grown in Ancona and Bologna and subjected, within each location, to a PFD or undefoliated (Control). <sup>∗</sup>P < 0.05 and ∗∗P < 0.01, respectively. O-methyltransferase (VIT\_18s0001g12890). Among the 12 genes downregulated by the treatment we found three chitinases, one pathogenesis-related protein and the indol-3-acetic acid amino synthetase (Supplementary File 5). We selected the MADS-box PFD berries at each stage per each site is indicated by an asterisk, red for BO and green for AN. AGL20 and jasmonate O-methyltransferase for further analysis. Interestingly, at Stage 3 all the 39 commonly modulated genes resulted as upregulated by PFD. Among these genes at least 10 terpene synthases were found, together with three multidrug equation (Simon, 2003). Bars represent means ± SE of three biological replicates. The significant modulation (t-test, p < 0.05) of gene expression between C and #### TABLE 3 \| Agronomic and ripening parameters 2012. fpls-08-00630 April 29, 2017 Time: 12:26 # 12 Vegetative growth, yield parameters, and grape composition recorded in 2012 on cv. Nero d'Avola, Ortrugo, and Ciliegiolo grapevines grown in Sicily, Emilia Romagna, and Umbria, respectively, and subjected, within each location, to a PFD or undefoliated (Control). <sup>∗</sup>P < 0.05 and ∗∗P < 0.01, respectively. #### TABLE 4 \| Agronomic and ripening parameters 2013. Vegetative growth, yield parameters, and grape composition recorded in 2013 on cv. Nero d'Avola, Ortrugo, and Ciliegiolo grapevines grown in Sicily, Emilia Romagna, and Umbria, respectively, and subjected, within each location, to a PFD or undefoliated (Control). <sup>∗</sup>P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, respectively. resistance-associated proteins, three serine carboxypeptidases and two genes involved in jasmonate metabolism, the VvJAZ2 and jasmonate O-methyltransferase, upregulated also at Stage 2 (Supplementary File 4). For further investigation, we chose one multidrug transporter (ABC- VIT\_09s0020g05380), the linalool synthase VvTPS62 (VIT\_00s0572g00020) and jasmonate O-methyltransferase. Finally, the 19 genes commonly modulated by PFD at Stage 4 all resulted as downregulated in comparison to the C vine. Among these genes we found three kinases, two NAC transcription factors and the ABA receptor PYL4 (VIT\_08s0058g00470) that were the most downregulated (Supplementary File 5). The latter was chosen for the next investigation. The expression of the seven selected genes was investigated by qPCR in C and PFD berries of Nero d'Avola, Ortrugo, and Ciliegiolo during 2012, only at the corresponding stage. We found that flavonol synthase at Stage 1, jasmonate O-methyltransferase at Stages 2 and 3 and the ABA receptor PYL4 at Stage 4 confirmed the modulation of expression obtained by microarray analysis in Sangiovese in BO and AN (Figure 5). Instead, the MADS-box AGL20, ABC transporter and VvTPS62 were not commonly modulated by PFD in all genotypes (data not shown). The expression of flavonol synthase, jasmonate O-methyltransferase and the ABA receptor PYL4 was then evaluated by qPCR in C and PFD berries of all genotypes during 2013. They all showed the same modulation after treatment in all genotypes and growing conditions (Figure 6), resulting as good candidates for molecular markers of the PDF treatment. Concerning the common upregulation of flavonol synthase, we determined main berry flavonols at harvest in both 2012 TABLE 5 \| Main flavonols concentration. expression according to the Simon equation (Simon, 2003). Bars represent means ± SE of three biological replicates. All genes in all genotypes resulted significantly Concentration of quercetin, myricetin, and kaempferol recorded at harvest in berries of cvs Ortrugo and Sangiovese (Bologna site) vines subjected to PFD or undefoliated (control) in 2012 and 2013. Data are given as mean ± standard error (SE). Mean separation within row and parameter by t-test, P < 0.05. Absence of letters means ns. <sup>1</sup>Data are given on a skin dry weight basis. <sup>2</sup>Data are given on a berry fresh weight basis. and 2013 on cvs Ortrugo and Sangiovese, evidencing a clear pattern of a significant increase in PFD treatment in the two sites and genotypes (Table 5). This trend was overwhelming vs. year-to-year variability and especially marked for quercetin 3-O glucuronide + quercetin 3-O glucoside and kaempferol 3-O glucoside. modulated (t-test; p < 0.05) between C and PFD berries. The † indicates no significance. These data strongly support the involvement of flavonol synthase in the PFD response in berry independently of growing site, year, and genotype. #### DISCUSSION #### Influence of Growing Site, Year, and Genotype on the Defoliation Response The testing of four different genotypes over two consecutive years and having also compared a cultivar (Sangiovese) in the same year under two growing conditions (BO and AN) permits a proper discussion about the interactive effects between the above factors on vine response to PFD applied in all instances with the same protocol (i.e., six main basal leaves removed at the "separated closed flowers" stage with retention of any laterals). Growing conditions (BO vs. AN) exerted an overall moderate effect on vine response variability to early leaf removal. It is notable that, in 2012, although AN vines were clearly under-cropped as compared to BO, variations of significantly modified parameters were always the same, while the same also held true for unmodified parameters, with the exception of TA and total anthocyanins (Table 1). In 2013, the influence of growing conditions was greater, albeit essentially limited to berry weight that was greatly reduced at BO site, while it was unchanged at AN. Since the vine balance given as LA/yield did not change in the two locations, it is likely that the improved total anthocyanins concentration at harvest in BO grapes results from inherently smaller berry size, hence higher skin-to-pulp ratio. This hypothesis is well supported by results obtained in a previous study showing that berries from pre-flowering defoliated Sangiovese vines, characterized by a significant increase of anthocyanin concentration in comparison to the control, also demonstrated a significant increase of berry skin thickness (Pastore et al., 2013). Variability in the response to PFD attributable to year was, in AN and BO sites, more pertinent to must composition than to vegetative growth and yield parameters. In particular, TSS was more responsive in 2013, showing a large increase in PFD vines, whereas TSS at both sites was unchanged in 2012. The reasons for this difference are not easy to distinguish; however, the quite high TSS reached in AN in 2012 likely reflects the high LA/yield (>1.9 m<sup>2</sup> /kg) placing no limitations on the sugar accumulation process; in confirmation of this, PFD was more effective in increasing TSS in 2013 at quite low LA/yield ratio, suggesting that, in both sites, PFD might have benefitted from higher foliage "quality" due to lower canopy age from veraison onward (Poni et al., 2006). In the other sites, response to PFD over the 2 years was more consistent for all the parameters considered. Comparing four genotypes over 2 years yielded a total of 10 C vs. PFD comparisons. All genotypes showed high responsiveness to the technique and no "recalcitrant" varieties could be discriminated. In more detail, in all cases yield per vine was significantly reduced after the early defoliation; among the main yield components cluster weight was reduced in 9 out of 10, berry weight in 5 out of 10, and berries per cluster in 6 out of 10. This confirms previous studies that PFD is extremely effective and consistent at reducing yield through either lower fruit set or smaller berry size or a combination of both, in turn resulting in looser clusters. This is not a surprising finding since the physiological background on which the technique relies is quite robust; it is well-known from the literature (Coombe, 1962; Hardie and Considine, 1976) that a calibrated source limitation imposed pre-flowering constrains the carbohydrate pool available to support flowering and fruit-set which, inherently, become limited. In terms of grape composition, it is likewise confirmed that, with very few exceptions, all genotypes subjected to PFD show a significant increase in TSS at harvest (7 cases out of 10) and, for red cultivars, in total anthocyanins (6 out of 8). The physiological bases for such response are also quite solid: (i) in PFD, ripening benefits from non-limiting or even higher final LA/yield ratios since induced yield limitation is often greater than the amount of leaf area removed "per se" with defoliation; (ii) as reported in Poni et al. (2006), in PDF the amount of carbohydrate supply per unit of grape fresh mass is higher from veraison onward due to an overall younger, hence more efficient canopy, and compensation mechanisms in either leaf area development or maximum photosynthetic rates and (iii) TSS and total anthocyanins can also be enhanced due to smaller berry size. Another quite remarkable and consistent feature of the PDF practice was that, despite the large increase in TSS, TA was reduced only in the Ortrugo trial, in five out of eight cases it was unchanged and on one occasion (AN, 2012) it was increased. This suggests that the technique is quite effective at decoupling the sugar/acid ratio and if TSS are increased, TA is not necessarily concurrently decreased. This feature is of special interest within a global warming scenario (Palliotti et al., 2014), where maintenance of adequate acidity in warm environments is an increasing concern. #### Common Molecular Responses to Pre-flowering Defoliation in Sangiovese Berries Involved Genes Related to Secondary and Hormone Metabolism Berry molecular responses to PFD were initially investigated by global gene expression analysis performed on berries at four developmental stages in defoliated and control Sangiovese vines, grown in BO and AN in 2012. Statistical analysis clearly revealed that the environment has a stronger effect than defoliation treatment on the transcriptome rearrangement during berry development in Sangiovese. Indeed, the correlation dendrogram showed a very clear distinction between BO and AN berry transcriptomes at each developmental stage and a weak and variable separation between berries from PFD and C vines. In addition, a PCA analysis revealed that no principal components were able to distinguish, at transcriptional level, berries from PFD and C vines. PCA analysis also showed that the ripening process was slightly advanced in AN in comparison to BO, reflecting the higher level of TSS, lower values of TA and higher anthocyanin content observed in ripe berries grown in AN, independently of the PFD effect (Table 1). It is very unlikely that inter-clonal variation within the same genotype might have interfered in the transcriptional responses. Albeit some slight differences in berry transcriptome during development can be attributable to small variations in the sampling procedure adopted in the two locations, the growing site seems to strongly affect berry gene expression during development in Sangiovese, evidencing that a large part of its transcriptional ripening program is plastic. Previous attempts to quantify the transcriptomic plasticity in grapevine berry were recently reported for the red cultivar Corvina and white cultivar Garganega (Dal Santo et al., 2013a, 2016b). These studies demonstrated a wide berry phenotypic plasticity in both cultivars, in particular affecting the secondary metabolism, suggesting that this phenomenon, which allows the production of different wines from the same cultivar and the adaptation of the same cultivar to diverse growing regions, is still scarcely characterized in grapevine. Sangiovese is the top red variety grown in Italy with about 70.000 hectares (ISTAT, 2015), and is cultivated in several regions (i.e., Tuscany, Emilia Romagna, Marche, Umbria). It is well known that Sangiovese wine made with grapes from Emilia Romagna usually reaches a price tag that is 5–10 times lower than any Brunello di Montalcino label, which is likewise made with 100% Sangiovese grapes. These differences might reflect consumer perception and expectations as well as marketing strategies. In this context our transcriptomic survey, reflecting the growing site effect of two distinct Italian regions (Emilia Romagna and Marche), should be further explored to unveil the genotype × environment interactions of this important Italian grapevine cultivar. The impact of the environment on Sangiovese berry transcriptome was further highlighted by the analysis of DEGs in berries from C and PFD vines in the two sites. Among the 1746 and 1041 DEGs found in BO and AN, respectively, only 125 were commonly differentially expressed, strongly suggesting that the effect of PFD on berry gene expression is mainly affected by growing conditions. The GO enrichment analysis performed on these commonly DEGs revealed that "response to stimulus" and "cellular amino acid and derivative metabolic process" functional categories were significantly over-represented. The same two categories were previously found overrepresented in the list of genes differentially expressed at the end of veraison in Sangiovese berries subjected to both pre-flowering and veraison defoliation treatment (Pastore et al., 2013). The role in stress resistance of many genes involved in amino acid metabolism, such as the aspartate aminotransferase, glutamine synthetase, and serine hydroxymethyltransferase, was previously described (Moreno et al., 2005; Singh and Ghosh, 2013; de la Torre et al., 2014; Wu et al., 2016), strongly suggesting that stress response induction is one of the principal effects of PFD on berry transcriptome, independently of the environment. Interestingly, other functional categories were found among the 125 genes, predominantly the "secondary metabolic process," mainly represented by genes belonging to the phenylpropanoid pathway, and "hormone stimulus," with several genes related to auxin, ethylene, and ABA metabolism. These results support previous observations regarding the direct effect on the expression of genes involved in berry ripening exerted by leaf removal (Pastore et al., 2013). Moreover, the modulation of phenylpropanoidrelated genes in defoliated berries could represent a stress response, as previously observed in grapevine upon various stresses (Matus et al., 2009; Rienth et al., 2014; Corso et al., 2015). In order to identify putative berry molecular markers of the PFD treatment, we focused only on genes characterized by a low plasticity during berry development. Among these, six were selected as putative markers of the treatment, being similarly modulated throughout berry development by PFD in the two sites and for their possible role in berry formation and ripening. The expression profiles of these candidates were analyzed by qPCR in berries from PFD and C vines in BO and AN in 2012, in order to validate microarray data, and in 2013 in order to assess the vintage effect on their expression modulation after treatment. Five out of six selected genes demonstrated a consistent modulation of expression induced by PFD throughout berry development in both years and in both sites, emerging as putative molecular markers for this treatment in the cultivar Sangiovese. The ABC transporter VvPDR20/VvABCG50 showed a delay in its peak of expression at Stage 3, when berries are softening and start to accumulate pigments, instead of Stage 2, when berries are still green and firm. ABC transporters superfamily constitutes one of the largest families of transmembrane proteins that plays important roles in the vacuolar accumulation of secondary metabolites, such as flavonoids, in detoxification and heavy metal sequestration, in chlorophyll catabolite transport and ion channel regulation (Klein et al., 2006; Kang et al., 2010). In grapevine, 135 putative ABC proteins were identified and classified (Cakir and Kilickaya, 2013). The VvPDR20/VvABCG50 gene belongs to the PDR subfamily in V. vinifera, the largest ABC transporter subfamily. In grapevine, no PDR-related ORF has been cloned in its entirety and characterized (Cakir and Kilickaya, 2013). However, in other species, members of this family confer resistance to various biotic and abiotic stresses (Moons, 2003; Lee et al., 2005; Stukkens et al., 2005). Interestingly, it was recently shown in Arabidopsis that PDR12 is a plasma membrane ABA uptake transporter that mediates cellular uptake of the phytohormone ABA in guard cells (Kang et al., 2010). VvPDR20/VvABCG50 gene is an interesting molecular marker directly affected by the PFD treatment in Sangiovese, and is a good candidate for future functional studies. The indole-3-acetate beta-glucosyltransferase and the auxin responsive factor 10, involved in auxin metabolism and signaling, respectively, were also identified as PFD molecular biomarkers. It is generally acknowledged that auxin plays a role in fruit growth. However, the change in auxin levels during berry development and the dynamics of auxin transport and signaling are still under debate (Fortes et al., 2015). It has been demonstrated that auxin treatment of pre-veraison grape berries delays ripening and alters the expression of developmentally regulated genes, suggesting that low auxin levels are required to trigger the onset of ripening (Davies et al., 1997; Bottcher et al., 2011; Ziliotto et al., 2012). However, it has been also hypothesized that high auxin levels at pre-veraison stages are required for the induction of genes involved in the ripening inception (Ziliotto et al., 2012; Corso et al., 2016). The regulation of auxin levels is associated with the conjugation of indole acetic acid (IAA) by the indol-3-acetate beta-glucosyltransferase, which allows an increase in the conjugated form of auxin after veraison. The decrease of expression after veraison of the indol-3 acetate beta-glucosyltransferase gene, obtained in C Sangiovese vines in the 2 years, corroborates previous results obtained in three Portuguese varieties (Agudelo-Romero et al., 2013) and supports the role of this gene in auxin level regulation during ripening. Interestingly, an increase in expression level of this gene was found as a consequence of PFD treatment, which delays the gene downregulation, possibly affecting the IAA homeostasis and, hence, the regulation of the onset of ripening. Auxin response factors regulate the auxin-mediated gene expression (Tiwari et al., 2003). In grapevine, 19 ARF genes were recently identified (Wan et al., 2014; Corso et al., 2016) but no functional studies have been performed to date. The ARF 10 exhibited a peak of expression at the ripening stage in C vines, whereas the peak of expression was hastened at the end of veraison in PFD vines (Stage 3), strongly suggesting that the treatment can interfere with the ripening progress by affecting the auxin level at the end of veraison. Biochemical analyses aimed to quantify the free and conjugate auxin form in berries from PFD and C vines will be necessary to thoroughly characterize the possible impact on hormonal regulation exerted by the PFD. The last two putative PFD molecular markers identified in Sangiovese were a CAD gene, involved in the last step of the synthesis of the monomeric precursors of lignin, and a G3T, involved in the glycosylation of flavonols. Both genes showed an increase of expression in berries from PFD vines, more pronounced at Stage 2 in Bologna and at Stage 3 in Ancona. The increase in expression of CAD was already observed in Sangiovese berries from PFD vines (Pastore et al., 2013). It was previously proposed that the increase in sunlight exposure of berries on defoliated vines induced the expression of genes involved in cell wall metabolism that allow an increase in berry skin thickness, providing more epidermal layers for protection against sunburn and storage of anthocyanin compounds (Pastore et al., 2013). The G3T coincides with the previously characterized VvGT6, contributing to flavonol glycosylation (Ono et al., 2010). The upregulation of this gene obtained in berries from PFD vines is consistent with the higher flavonol content that berries subjected to PFD manifested (Pastore et al., 2013; this work). Among the six genes, only the expression of geraniol 10-hydroxylase, a cytochrome P450 monooxygenase involved in the biosynthesis of terpenoids (Collu et al., 2001) and phenylpropanoids (Sung et al., 2011), showed an inconsistent modulation of expression between the 2 years. Differences in expression trend of this gene and its responsiveness to the defoliation treatment could be linked to the differences in weather patterns between 2012, marked by a long summer with hot spells, and the slightly cooler 2013. It is well known that terpenoids metabolism contributes to plant adaptation to the environment, in particular solar exposure, UV-B radiation (Gil et al., 2013; Zhang et al., 2014) and drought (Deluc et al., 2009; Selmar and Kleinwachter, 2013; Savoi et al., 2016). #### General Molecular Markers of the Pre-flowering Defoliation Expression analysis of the PFD Sangiovese putative markers was performed on another three genotypes subjected to the same treatment in different environments in 2012. A stronger influence of genotype and/or growing site than PFD treatment on these genes' expression was clearly revealed during berry development, indicating that they should not be considered as PFD markers for genotypes other than Sangiovese. We therefore selected new candidates by focusing on DEGs at each berry developmental stage, without evaluating the entire expression profile. The inclusion in our experimental plan of an early stage of berry development (Stage 1), allowed previous transcriptomic results, focused only on the PFD effect on berry ripening phase, to be greatly improved (Pastore et al., 2013). Indeed, several DEGs in PFD berries in comparison to C were found at Stage 1 in both sites. This strongly supports recent results obtained in defoliated Cabernet Sauvignon berries showing that earlier berry stages react to leaf removal distinctly from the later developmental stages (Young et al., 2016). A flavonol synthase resulted as one of the most upregulated genes in PFD Sangiovese berries at Stage 1 in both sites and its upregulation was confirmed by qPCR in all genotypes, environments, and years. Interestingly, the upregulation of the same gene was previously observed in both pre-flowering and veraison defoliated Sangiovese berries at the end of veraison (Pastore et al., 2013) and the expression of two isoforms of flavonol synthase was affected in defoliated berries of Cabernet Sauvignon (Matus et al., 2009). Quantification of the main berry flavonols at harvest in Ortrugo and Sangiovese in both years evidenced a significant increase in berries from PFD vines, in the two sites, genotypes and years, strongly supporting transcriptomic data. In Sangiovese PFD treated berries, the concentration of quercetin, the main flavonol in red grapes (Mattivi et al., 2006), and kaempferol, was more than twice that in control, in both years, whereas the increase of myricetin at harvest was less intense, as previously observed (Pastore et al., 2013). This shift in flavonol composition was not observed in Ortrugo, in which the abundance of all flavonol compounds increased after PFD treatment in both years, as previously reported for Merlot (Spayd et al., 2002). It was demonstrated that the induction of flavonols synthesis is positively correlated to sunlight exposure, reflecting their role as UV protectants (Price et al., 1995; Haselgrove et al., 2000; Downey and Rochfort, 2008; Matus et al., 2009). Our data suggested that the PFD-induced expression of flavonol synthase gene at an early stage of berry development is due to an increase in cluster sunlight exposure, causing a significant accumulation of flavonols in berries at harvest, and that this effect is shared across different environments, years, and genotypes. A jasmonate O-methyltransferase gene resulted as being another common molecular marker of the defoliation for both Stage 2 and Stage 3 of berry development, being positively modulated by PFD in all tested conditions. The role of methyl jasmonate (MeJA) in the response to biotic and abiotic stresses was widely discussed in the past (Cheong and Choi, 2003; Wasternack and Song, 2016). In non-climacteric fruits such as strawberry and grape, JA levels are reported to be high in early development and decreasing to lower values in riper fruits, enabling the onset of ripening to occur (Kondo and Fukuda, 2001). In grapevine, the gene coding for jasmonate O-methyltransferase was found downregulated in ripe fruits of three grape varieties (Agudelo-Romero et al., 2013). Interestingly, the downregulation of jasmonate O-methyltransferase during berry development was revealed by transcriptomic analysis on Sangiovese berries from C vines in both Bologna and Ancona in 2012. As a consequence, the upregulation observed at Stages 2 and 3 in berries from PFD vines corresponded to a delay in its downregulation and not to a genuine induction. It was recently demonstrated that JA plays an important role in grape berry coloring and softening by inducing the transcription of several ripening-related genes, such as phenylpropanoid genes, cell wall metabolism-related genes and genes involved in aroma accumulation (Jia et al., 2016). Consistently, we observed that at Stage 2 and Stage 3 several terpenoid synthase genes (e.g., one geraniol 10-hydroxylase and several different terpene synthases), involved in berry aromatic compounds accumulation, was found commonly upregulated by PFD in Bologna and Ancona in 2012, together with jasmonate O-methyltransferase. Intriguingly, at Stage 3 also a jasmonate ZIM-Domain VvJAZ2 gene, involved in JA signaling cascade (Pauwels and Goossens, 2011; Wasternack and Song, 2016), resulted as commonly modulated by PFD in Sangiovese berries in both sites. Although the expression of this gene was not assessed in all conditions, we could hypothesize that the PFD impacts on berry ripening, possibly affecting the JA metabolism at veraison, when the JA level has recently been proposed to play a crucial role in ripening regulation (Jia et al., 2016). The last identified berry PFD marker was the ABA receptor PYL4, which resulted as being the most commonly downregulated gene at Stage 4 in Sangiovese in 2012. Interestingly, all genes commonly modulated in PFD Sangiovese berries at Stage 4 in that year resulted as downregulated, suggesting that the treatment could affect many metabolisms by hastening their normal shutdown. This hypothesis also holds true for the ABA receptor PYL4, showing a decreasing expression trend during ripening in C conditions with a peak of expression at veraison, and an accelerated downregulation in PFD berries. The ABA receptor PYL4 belongs to the PYR/PYL/RCAR receptors family that, together with the PP2Cs and SnRK2s kinases, constitutes the complex molecular machinery involved in the ABA-mediated signaling pathway (Boneh et al., 2012). Many studies indicated that ABA, together with other phytohormones like brassinosteroids (BRs) and ethylene may play an important role in several ripening-associated processes of grape berry (Davies and Bottcher, 2009; Kuhn et al., 2014). It was observed that free ABA levels increase around veraison, concurrently with sugar accumulation, berry coloration, and softening, whereas during ripening ABA levels may be controlled mainly by conjugation to glucose. Nevertheless, it was found that a set of genes involved in the ABA-mediated signaling pathway, including the ABA receptors PYL8 RCAR3 and PYL9 RCAR1, were upregulated at the mid-ripening phase in three Portuguese varieties (Fortes et al., 2011; Agudelo-Romero et al., 2013). This expression trend was consistent with the expression of PYL4 found in C vines, suggesting that later in ripening, ABA synthesis is not induced, but ABA-regulated processes are instead activated. The lower expression of PYL4 observed in PFD berries at harvest, which corresponds to a faster downregulation in comparison to C, suggests that PFD treatment hastened the ABAmediated ripening signaling possibly by inducing abiotic stress response early during development. This faster downregulation of PYL4 is particularly marked in Ortrugo cultivar, in which the highly significant increase in ripening parameters in both years suggests that the ripening process in this cultivar started much earlier than in the other cultivars in the berries after treatment. # CONCLUSION A comparison was made of physiological and molecular responses to PFD in four grapevine cultivars grown in different Italian geographical areas and during two consecutive years to evaluate the interactive effects between these factors on vine response to PFD and to determine the common effect of defoliation in berry at transcriptional level. All genotypes were highly responsive to the technique, the yield per vine being significantly reduced in all conditions. In terms of grape composition, a significant increase in sugar content and total anthocyanins at harvest was obtained in most genotypes. Sangiovese resulted as being the cultivar with the stronger variability in must composition in the response to defoliation. Global gene expression analysis performed on Sangiovese berries from defoliated and untreated control vines grown in two different sites highlighted, on the one hand, the strong effect of environment on the berry transcriptional ripening program in this cultivar and, on the other, allowed genes commonly regulated by selective leaf removal to be identified. The differential expression of these putative marker genes, mainly related to secondary metabolism and hormone signaling, could link the defoliation treatment to physiological and metabolic changes found in treated berries. These new insights greatly improve previous knowledge about molecular mechanisms on the basis of the qualitative outcomes of an important and widely used management technique in viticulture, allowing physiological responses in berry to the selective PFD practice to be precisely defined across genetic and environmental variability. # AUTHOR CONTRIBUTIONS SZ designed the microarray experiments, interpreted the micro array data, and wrote the manuscript, SDS performed the statistical analyses, SDS and GBT interpreted the microarray data and helped in drafting the manuscript, ED performed the RNA extraction, microarray experiments, and real-time qPCR analysis, IF, CP, GA, OS, VL, APi, RDL, APa, ST and MG conducted defoliation experiments, sampled the material and processed the data, CP, GA and MG performed the HPLC analysis, SP conceived and supervised the study, wrote and critically revised the manuscript. All authors contributed to the revision of the manuscript. #### FUNDING The research leading to these results received funding from the Italian Ministry of University and Research PRIN 2009P3B89K\_003 Project "Characterization of genes involved in berry ripening after pre-bloom defoliation" and by the European INNOVINE Project FP7-311775 "Combining innovation in vineyard management and genetic diversity for a sustainable European viticulture." SDS was financially supported by the Italian Ministry of University and Research FIRB RBFR13GHC5 #### REFERENCES Project "The epigenomic plasticity of grapevine in genotype per environment interactions." #### ACKNOWLEDGMENT We thank Martina Albarello for technical assistance in real-time qPCR analyses. #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00630/ full#supplementary-material auxin-related genes in cabernet sauvignon berries. Front. Plant Sci. 7:69. doi: 10.3389/fpls.2016.00069 responses in green and ripening grapevine (Vitis vinifera) fruit. BMC Plant Biol. 14:108. doi: 10.1186/1471-2229-14-108 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 Zenoni, Dal Santo, Tornielli, D'Incà, Filippetti, Pastore, Allegro, Silvestroni, Lanari, Pisciotta, Di Lorenzo, Palliotti, Tombesi, Gatti and Poni. 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. # Global DNA Methylation Patterns Can Play a Role in Defining Terroir in Grapevine (Vitis vinifera cv. Shiraz) Huahan Xie1,2† , Moumouni Konate1,2† , Na Sai1,2,3, Kiflu G. Tesfamicael1,2 , Timothy Cavagnaro<sup>2</sup> , Matthew Gilliham2,3, James Breen4,5, Andrew Metcalfe<sup>6</sup> , John R. Stephen<sup>7</sup> , Roberta De Bei<sup>2</sup> , Cassandra Collins<sup>2</sup> and Carlos M. R. Lopez1,2 \* <sup>1</sup> Environmental Epigenetics and Genetics Group, University of Adelaide, Adelaide, SA, Australia, <sup>2</sup> The Waite Research Institute and The School of Agriculture, Food and Wine, University of Adelaide, Adelaide, SA, Australia, <sup>3</sup> The ARC Centre of Excellence in Plant Energy Biology, University of Adelaide, Adelaide, SA, Australia, <sup>4</sup> Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia, <sup>5</sup> Bioinformatics Hub, School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia, <sup>6</sup> School of Mathematical Sciences, University of Adelaide, Adelaide, SA, Australia, <sup>7</sup> Plant Genomics Centre, Australian Genome Research Facility Ltd., Adelaide, SA, Australia #### Edited by: José Tomás Matus, Centro de Investigación en Agrigenómica, Universitat Autònoma de Barcelona, Spain #### Reviewed by: Philippe Gallusci, Université de Bordeaux, France Melane Alethea Vivier, Stellenbosch University, South Africa #### \Correspondence: Carlos M. R. Lopez [email protected] > †These authors have contributed equally to this work. #### Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 18 April 2017 Accepted: 11 October 2017 Published: 30 October 2017 #### Citation: Xie H, Konate M, Sai N, Tesfamicael KG, Cavagnaro T, Gilliham M, Breen J, Metcalfe A, Stephen JR, De Bei R, Collins C and Lopez CMR (2017) Global DNA Methylation Patterns Can Play a Role in Defining Terroir in Grapevine (Vitis vinifera cv. Shiraz). Front. Plant Sci. 8:1860. doi: 10.3389/fpls.2017.01860 Understanding how grapevines perceive and adapt to different environments will provide us with an insight into how to better manage crop quality. Mounting evidence suggests that epigenetic mechanisms are a key interface between the environment and the genotype that ultimately affect the plant's phenotype. Moreover, it is now widely accepted that epigenetic mechanisms are a source of useful variability during crop varietal selection that could affect crop performance. While the contribution of DNA methylation to plant performance has been extensively studied in other major crops, very little work has been done in grapevine. To study the genetic and epigenetic diversity across 22 vineyards planted with the cultivar Shiraz in six wine sub-regions of the Barossa, South Australia. Methylation sensitive amplified polymorphisms (MSAPs) were used to obtain global patterns of DNA methylation. The observed epigenetic profiles showed a high level of differentiation that grouped vineyards by their area of provenance despite the low genetic differentiation between vineyards and sub-regions. Pairwise epigenetic distances between vineyards indicate that the main contributor (23–24%) to the detected variability is associated to the distribution of the vineyards on the N–S axis. Analysis of the methylation profiles of vineyards pruned with the same system increased the positive correlation observed between geographic distance and epigenetic distance suggesting that pruning system affects inter-vineyard epigenetic differentiation. Finally, methylation sensitive genotyping by sequencing identified 3,598 differentially methylated genes in grapevine leaves that were assigned to 1,144 unique gene ontology terms of which 8.6% were associated with response to environmental stimulus. Our results suggest that DNA methylation differences between vineyards and sub-regions within The Barossa are influenced both by the geographic location and, to a lesser extent, by pruning system. Finally, we discuss how epigenetic variability can be used as a tool to understand and potentially modulate terroir in grapevine. Keywords: environmental epigenetics, DNA methylation, terroir, MSAP, msGBS, Vitis vinifera, Shiraz, Barossa #### INTRODUCTION fpls-08-01860 October 27, 2017 Time: 16:50 # 2 The ability of plants to produce alternative phenotypes in response to changing environments is known as phenotypic plasticity (Pigliucci, 2005). Genotypes with this characteristic are able to produce a variety of phenotypes including improved growth and reproduction (Lacaze et al., 2009). Grapevine (Vitis vinifera L.) is a highly plastic crop that exhibits large differences in fruit composition from a given variety depending upon the environmental conditions of the vineyard of origin (Dal Santo et al., 2016). Fruit traits that affect wine quality are thought to be largely driven by the interaction of a grapevine's genetic characteristics with environmental factors (i.e., climate, soil, and topography) and vineyard management (Robinson et al., 2012). The grapevine cycle extends for two seasons, with buds formed in a specific year giving rise to shoots that will carry fruit the next year (Keller et al., 2010). Environmental cues over two seasons can impact on yield (fruit quantity) and fruit composition by influencing the formation of the inflorescence primordia (Buttrose, 1974), flowering and fruitset (Petrie and Clingeleffer, 2005). Temperature and water availability are also known to influence sugar concentration, acidity, pH, color, and other characteristics in the fruit (Adams, 2006; Downey et al., 2006). Moreover, climate change predictions of elevated CO<sup>2</sup> and rising temperature are also likely to have an effect on the grapevine reproductive cycle and on fruit composition (Parra et al., 2010). All these variables, in conjunction with the wine making process, give a wine its distinctive character. The impact of the environment on grape composition and subsequent wine excellence has given rise to the concept of 'terroir,' a French term referring to terre, "land" (Fanet and Brutton, 2004). Terroir is defined as the interaction between the physical and biological environment and applied viticultural and oenological practices that lead to unique characteristics in a final wine (Seguin, 1986). Extensive studies have been published on terroir, but generally, these focus on a single parameter such as climatic factors, soil structure, or soil microbiology (Harrison, 2000; Tonietto and Carbonneau, 2004). However, studying only one environmental parameter does not provide an entire understanding of how wine quality is influenced by terroir (van Leeuwen et al., 2004). A significant amount of work has also been published on the genetic basis of fruit composition in grapevines (e.g., Doligez et al., 2002). Despite these insights, further research is required on the molecular changes that are involved in the vine interaction with its environment. One of the molecular changes worth investigating relates to environmentally induced epigenetic modifications. In fact, phenotypic plasticity has been previously associated to epigenetic variation (Vogt, 2015). Interestingly, analysis of epigenetic diversity has been shown to be more effective in discriminating inter-clonal variability in grapevine than the use of purely genetic molecular markers such as simple sequence repeats (SSRs) or amplified fragment length polymorphisms (AFLPs) (Imazio et al., 2002; Schellenbaum et al., 2008; Ocaña et al., 2013). Epigenetic mechanisms refer to potentially heritable (via mitosis or meiosis) molecular changes that affect gene expression leading to differences in phenotype without changing the organism DNA sequence (Jaenisch and Bird, 2003; Haig, 2004). Such mechanisms are involved in the control of a range of plant processes, including developmental control (Daccord et al., 2017), genomic imprinting (Köhler et al., 2012), and response to stress (Yaish et al., 2011; Tricker et al., 2012). It is now also widely accepted that epigenetic mechanisms have been the source of useful variability during crop varietal selection (Amoah et al., 2012; Bloomfield et al., 2014; Rodríguez López and Wilkinson, 2015). Multiple environmental cues have been shown to induce persistent changes in epigenetic modifications, resulting in an epigenetic priming that can act over multiple vegetative (Kumar et al., 2016) or sexual generations (Tricker et al., 2012). Although whether environmentally induced epialleles have any effect on the phenotypes of future generations remains a matter of debate, such priming is considered by some as an adaptive strategy by which plants use their memory of the environment to modify their phenotypes to adapt to subsequent conditions (Kelly et al., 2012; Tricker et al., 2013a,b; Vogt, 2015). It is commonly accepted that DNA methylation constitutes an adaptation strategy to the environment (YunLei et al., 2009), and that changes in DNA methylation can produce altered phenotypes (Zhang et al., 2006; Herrera and Bazaga, 2011; Iqbal et al., 2011). Moreover, epigenetic mechanisms are now considered as potential drivers of rapid adaptation to the environmental variability (Bräutigam et al., 2013). These processes facilitate adaptation by regulating the expression of genes controlling phenotypic plasticity (Richards, 2006; Bossdorf et al., 2008) early in adaptive walks (Kronholm and Collins, 2016) but also by releasing cryptic genetic variation and/or facilitating mutations in functional loci over longer-term timescales (O'Dea et al., 2016). To this extent, there have been extensive studies establishing a link between DNA methylation in plants and environmental conditions both in wild (Fonseca Lira-Medeiros et al., 2010; Herrera and Bazaga, 2010; Alonso et al., 2016) and cultivated species (Zheng et al., 2017). All major epigenetic mechanisms, DNA methylation, histone modifications, and RNA interference, are present in plants (Pikaard and Mittelsten Scheid, 2014; Holoch and Moazed, 2015; Wendte and Pikaard, 2017). In plants, DNA methylation (5mC) occurs at different cytosine contexts (CpG, CpHpG, or CpHpH) (H = A, T or C) (Richards, 1997; Baulcombe and Dean, 2014; Niederhuth and Schmitz, 2017) and it is induced, maintained or removed by different classes of methyltransferase in conjunction with environmental and developmental cues (Law and Jacobsen, 2010; Baulcombe and Dean, 2014). The contribution of DNA Abbreviations:* AMOVA, analysis of molecular variance; BAM file, Binary Alignment/Map file; BIC, Bayesian Information Criterion; CPM, counts per million; DAPC, discriminant analysis of principal components; DMG, differentially methylated gene; DMM, differentially methylated marker; FDR, false discovery rate; gDNA, genomic DNA; GeoD, geographic distance; GO, gene ontology; kb, kilobase; log2FC, logarithm 2 of fold change; MSAPs, methylation sensitive amplified polymorphisms; msGBS, methylation sensitive genotyping by sequencing; MSL, methylation sensitive polymorphic loci; NML, non-methylated polymorphic loci; PCA, principal component analysis; PCoA, principal coordinate analysis; PhiPT, measurement of epi/genetic diversity among populations; SI, Shannon's diversity index; SNP, single nucleotide polymorphism; TES, transcription end site; TSS, transcription start site. methylation to plant performance has been extensively studied in model organisms and some annual crops (Rodríguez López and Wilkinson, 2015). However, we are only beginning to understand how long-living plants, such as grapevines, use epigenetic mechanisms to adapt to changing environments (Fortes and Gallusci, 2017). Effects of environmental conditions on non-annual crops performance can be very difficult to evaluate since many environmental factors interact over the life of the plant to ultimately contribute toward the plant's phenotype (Fortes and Gallusci, 2017). Although epigenetic mechanisms have been shown to act as a memory of the organism's growing environment during mitotic division (Morao et al., 2016), even after vegetative propagation (Raj et al., 2011; Guarino et al., 2015), very few studies have focussed on DNA methylation changes in grapevine. The few known studies in this field used MSAPs (Reyna-López et al., 1997) for the detection of in vitro culture induced epigenetic somaclonal variability (Baránek et al., 2015), and the identification of commercial clones (Imazio et al., 2002; Schellenbaum et al., 2008; Ocaña et al., 2013). In this study, we hypothesize that DNA methylation can play a role in defining terroir. To test this hypothesis, we investigated the effect of environmental and management conditions on DNA methylation variation in grapevine cultivar Shiraz across 22 vineyards representative of The Barossa wine zone (Australia) (Robinson and Sandercock, 2014) using MSAPs. Finally, we used msGBS to characterize the genomic context of the observed regional genetic and epigenetic variability. # MATERIALS AND METHODS #### Vineyard Selection and Plant Material Vines from 22 commercial vineyards located in the iconic Barossa wine zone (The Barossa hereafter) (Australia) were included in this study. Vineyards were chosen based on the knowledge that they produce premium Shiraz wines that are representative of the climate, soil and management practices that are used in the Barossa sub-regions as described by the Barossa Grounds Project (Robinson and Sandercock, 2014) [i.e., Eden Valley (three vineyards) and Barossa Valley (19 vineyards) which included vineyards in the five distinctive sub-regions within the Barossa Valley Region: Northern Grounds (four vineyards), Central Grounds (four vineyards), Eastern Edge (four vineyards), Western Ridge (four vineyards), Southern Grounds (three vineyards)] (Supplementary Table S1). To simplify the nomenclature, the Eden Valley region, Northern, Central, Southern Grounds, Eastern Edge, and Western Ridge will be defined as sub-regions hereafter. All vineyards were planted with own-rooted vines of the cv. Shiraz. Ten vineyards were planted with clone SA 1654 (Whiting, 2003), one with clone BVRC30 (Whiting, 2003), one with clone PT15 Griffith (Farquhar, 2005) and 10 of 'unknown' clonal status (Supplementary Table S1). Nine vines from three rows from each vineyard were selected and vines adjacent to missing vines, end of row vines and border rows were excluded, to prevent differences in competition effects between plants. Also, rows containing sampled plants were selected from each vineyard after discussion with vineyard managers to capture the variability in each vineyard. A total of 198 plants (nine plants per vineyard) were selected to capture the diversity from each vineyard. Leaf samples (first fully expanded leaf at bud burst, E-L 7) (Coombe, 1995) were collected from three nodes per plant and pooled into a single sample per plant. All samples were taken before dawn (between 10:00 pm and sunrise) to minimize variability associated with differences in plant water status (Williams and Araujo, 2002). Samples were immediately snap-frozen in liquid nitrogen in the vineyard and stored at −80◦C until DNA extraction. #### DNA Isolation Genomic DNA extractions from all 198 samples were performed using the three pooled leaves per plant powdered using an automatic mill grinder (Genogrinder). The obtained frozen powder was used for DNA extraction using the Oktopure automated DNA extraction platform (LGC Genomics GmbH) according to the manufacturer's instructions. Isolated DNA was quantified using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). DNA final concentrations were normalized to 20 ng/µl using nanopure water (Eppendorf, Germany). ### Analysis of Genetic/Epigenetic Variability Using MSAP Methylation sensitive amplified polymorphism analysis was performed as described by Rodríguez López et al. (2012). In brief, gDNA from 88 plants (four plants per vineyard) was digested with a combination of the restriction enzymes EcoRI and one of two DNA methylation sensitive isoschizomers (HpaII or MspI). Double stranded DNA adapters (See Supplementary Table S2 for the sequence of all oligonucleotides used) containing co-adhesive ends complementary to those generated by EcoRI and HpaII/MspI were ligated to the digested gDNA and then used as a template for the first of two consecutive selective PCR amplifications in which the primers were complementary to the adaptors but possessed unique 3<sup>0</sup> overhangs. The second selective PCR amplification used primers containing 3<sup>0</sup> overhangs previously tested on grapevine (Baránek et al., 2015). HpaII/MspI selective primer was 5<sup>0</sup> end-labeled using a 6-FAM reporter molecule for fragment detection using capillary electrophoresis on a ABI PRISM 3130 (Applied Biosystems, Foster City, CA, United States) housed at the Australian Genome Research Facility Ltd., Adelaide, SA, Australia. Generated electropherograms were visualized using GeneMapper Software v4 (Applied Biosystems, Foster City, CA, United States). A binary matrix containing presence (1) absence (0) epilocus information was generated for each enzyme combination (i.e., EcoRI/HpaII and EcoRI/MspI). MSAP fragment selection was limited to allelic sizes between 95 and 500 bp to reduce the potential impact of size co-migration during capillary electrophoresis (Caballero et al., 2008). Different levels of hierarchy were used to group the samples. Samples were first grouped according to vineyard of origin. Then, samples were divided into their sub-regions of origin. Finally, samples were further separated into groups according to clones and the vineyard management systems (i.e., pruning system used in their vineyard of origin) (Supplementary Table S1). HpaII and MspI binary matrices were then used to compute Shannon's Diversity Index implemented using msap R package (v. 1.1.8) (Pérez-Figueroa, 2013) and PCoA was estimated in all regions to determine and visualize the contribution to the observed molecular variability within regions of NML and of MSL (genetic and epigenetic variability, respectively) (Smouse et al., 2015). GenAlex v 6.5 software (Peakall and Smouse, 2012) was used for PCoA in order to visualize the molecular differentiation between Barossa sub-regions detected using MSAP profiles generated after the restriction of gDNA with HpaII or MspI. We then used AMOVA to determine the structure of the observed variability using PCoA. Molecular differences between vineyards and regions was inferred as pairwise PhiPT distances (Michalakis and Excoffier, 1996). Mantel test analysis (Hutchison and Templeton, 1999) was used to estimate the correlation between the calculated pairwise molecular distances with (1) the GeoD [i.e., Log(1 + GeoD (Km)] and (2) differences in environmental variables among vineyards (i.e., vineyard altitude, regional average annual rainfall, regional growing season rainfall, regional mean January temperature, regional growing season temperature, and growing degree days). Mantel test was implemented in Genalex v 6.5 as described by Róis et al. (2013) and significance was assigned by random permutations tests (based on 9,999 replicates). ### Characterization of Genetic/Epigenetic Variability Using msGBS Methylation sensitive genotyping by sequencing was performed as described by Kitimu et al. (2015). In brief, 200 ng of gDNA from nine samples from Northern, Central, and Southern Grounds (vineyards 1–4, 5–8, and 13–15, respectively) were digested using 8 U of HF-EcoRI and 8 U of MspI (New England BioLabs Inc., Ipswich, MA, United States) in a volume of 20 µl containing 2 µl of NEB Smartcut buffer at 37◦C for 2 h followed by enzyme inactivation at 65◦C for 10 min. Sequencing adapters were ligated by adding 0.1 pmol of the MspI adapters (uniquely barcoded for each of the 198 samples) and 15 pmol of the common EcoRI Y adapter (See Supplementary Table S2 for the sequence of all oligonucleotides used), 200 U of T4 ligase and T4 ligase buffer (New England BioLabs Inc., Ipswich, MA, United States) in a total volume of 40 µl at 24◦C for 2 h followed by an enzyme inactivation step at 65◦C for 10 min. Excess adapters were removed from ligation products using Agencourt AMPure XP beads (Beckman Coulter, Australia) at the ratio of 0.85 and following manufacturer's instructions. Single sample msGBS libraries were then quantified using Qbit 3 (Thermo Fisher). A single library was generated by pooling 25 ng of DNA from each sample. Library was then amplified in eight separate PCR reactions (25 µl each) containing 10 µl of library DNA, 5 µl of 5x Q5 high fidelity buffer, 0.25 µl polymerase Q5 high fidelity, 1 µl of each forward and reverse common primers at 10 µM, 0.5 µl of 10 µM dNTP and 7.25 µl of pure sterile water. PCR amplification was performed in a Bio-Rad T100 thermocycler consisting of DNA denaturation at 98◦C (30 s) and 10 cycles of 98◦C (30 s), 62◦C (20 s), and 72◦C (30 s), followed by 72◦C for 5 min. PCR products were then re-pooled and DNA fragments ranging between 200 and 350 bp in size were captured using the AMPure XP beads following manufacturer's instructions. Libraries were sequenced using an Illumina NextSeq High Output 75 bp pair-end run (Illumina Inc., San Diego, CA, United States) at the Australian Genome Research Facility (AGRF, Adelaide, SA, Australia). #### msGBS Data Analysis Analysis of genetic diversity between regions was performed by SNP calling using TASSEL 3 (Bradbury et al., 2007) on msGBS sequencing results. Only SNPs present in at least 80% of the samples were considered for analysis. PCA was implemented on TASSEL 3 using the selected SNPs. To identify any possible geographical genetic structure, the optimal number of genetic clusters present in the three regions were computed using BIC as effected by DAPC using adegenet 2.0.0<sup>1</sup> . Identification of significant DMMs between regions was then computed using the package msgbsR<sup>2</sup> (accessed on 26/08/2016). In brief, raw sequencing data was first demultiplexed using GBSX (Herten et al., 2015) and filtered to remove any reads that did not match the barcode sequence used for library construction. Following demultiplexing, paired-end reads were merged using bbmerge in bbtools package (Bushnell, 2016). Merged reads were next aligned to the 12X grapevine reference genome<sup>3</sup> . Alignment BAM files where then used to generate a read count matrix with marker sequence tags, and used as source data to perform subsequent analyses using msgbsR in the R environment (R Core Team, 2015). Finally, the presence of differential methylation between regions was inferred from the difference in the number of read counts from all sequenced MspI containing loci that had at least 1 CPM reads and present in at least 15 samples per region. Significance threshold was set at Bonferroni adjusted P-value (or FDR) < 0.01 for difference in read CPM. The logFC (logarithm 2 of fold change) was computed to evaluate the intensity and direction of the region specific DNA methylation polymorphism. To determine how the observed changes in DNA methylation between sub-regions were associated to protein coding genes, the distribution of DMMs was assessed around such genomic features, as defined in Ensembl database<sup>4</sup> , by tallying the number of DMMs between the TSS and the TES and within five 1 kb windows before the TSS and after TES of all V. Vinifera genes, using bedtools (Quinlan and Hall, 2010). Genes within 5 kb of a DMM were referred to as DMGs. DMGs in each pairwise regional comparison were grouped into those showing hypermethylation or hypomethylation, and were next used separately for GO terms enrichment, using the R packages: GO.db (Carlson, 2016) and annotate (Gentleman, 2016). Significant GO terms were selected based on Bonferroni adjusted P-values at significance threshold of 0.05. Finally, GO <sup>1</sup>http://adegenet.r-forge.r-project.org/ <sup>2</sup>https://github.com/BenjaminAdelaide/msgbsR <sup>3</sup>http://plants.ensembl.org/Vitis\_vinifera/ <sup>4</sup>http://plants.ensembl.org/biomart/martview/ terms containing DMGs in all three pairwise comparisons were visualized using REViGO (Supek et al., 2011). # RESULTS Analysis of MSAP profiles obtained from DNA extractions of the first fully expanded leaf of 88 individual vines selected from 22 commercial vineyards within the six Barossa sub-regions (Figure 1 and Supplementary Table S1) yielded 215 fragments comprising 189 from MspI and 211 from HpaII, of which 80 and 84%, respectively, were polymorphic (i.e., not present in all the analyzed samples/replicates when restricted with one of the isoschizomers). Comparison of the HpaII and MspI banding patterns showed that in average, 42.1% of analyzed bands represented fully methylated or SNP containing loci, 22.3% represented hemimethylated loci, 19.6% represented unmethylated loci, and 18.1% represented loci containing internal cytosine methylation (Supplementary Table S3). ## Analysis of Genome/Methylome Differences within Wine Sub-regions in the Barossa Principal coordinate analysis of the MSAP profiles generated from NML (genetic variability) and by MSL (epigenetic variability) (Pérez-Figueroa, 2013) revealed a higher separation between vineyards when using epigenetic information than when using genetic (Supplementary Figure S1). The capacity of both types of variability to differentiate between vineyards was more evident on vineyards in the Southern Grounds (Supplementary Figures S2G,H). Both PCoA analysis and Shannon's diversity index showed significantly higher epigenetic than genetic diversity for all sub-regions (Supplementary Figure S2 and Table 1). Among sub-regions, Southern Grounds had the highest epigenetic diversity (0.581 ± 0.124) and Western Ridge the lowest (0.536 ± 0.143). Genetic diversity showed the highest value in the Southern Grounds (0.374 ± 0.143) and the lowest in the Northern Grounds (0.240 ± 0.030). ## Analysis of Genome/Methylome Differences between Wine Sub-regions in the Barossa We used AMOVA (Table 2) to obtain an overview of the molecular variability between all the studied sub-regions. Overall, MSAP profiles generated using restriction enzyme MspI achieved better separation between sub-regions than those generated using HpaII. Of all 30 calculated molecular pairwise distances between sub-regions (PhiPTs), 25 were significant (P < 0.05) (Table 2). Calculated PhiPT values ranged from 0.115 (PhiPT of Northern Grounds vs Southern Grounds calculated using MspI) and 0.012 (PhiPT of Central Grounds vs Eastern Edge calculated using HpaII). Analysis of molecular variance on MSAP profiles indicates that the majority of the observed variability is explained by differences within vineyards (81% using profiles generated with MspI and 91% with HpaII). A significant proportion of the total variability detected was associated to differences between vineyards (17% with MspI and 8% with HpaII) and 2 and 1% was due to differences between sub-regions (MspI and HpaII, respectively). # Effect of Vineyard Location on Methylome Differentiation To determine if environmental differences between vineyards influenced the observed epigenetic differences we studied the vineyards' pairwise geographic and molecular distances correlation. Vineyards located on the North–South axis of the Barossa Valley [i.e., vineyards 1, 2, 3, and 4 (Northern Grounds), 5, 6, 7, and 8 (Central Grounds), and 13, 14, and 15 (Southern Grounds)] (Figure 2A) were selected as Northern and Southern Grounds showed the greatest epigenetic differentiation (Table 2). PCoA analysis showed that Central Grounds samples occupied an intermediate Eigen space between Northern and Southern Grounds samples with coordinate 1 (24% of the observed variability) representing the North– South axis (Figure 2B). Moreover, Mantel test showed a significant (P = 0.0003) positive correlation (R <sup>2</sup> = 0.3066) between pairwise vineyard epigenetic and GeoDs (Figure 2C). Then, Mantel test analysis was implemented to compare the observed molecular differences against environmental variables. Differences in vineyard altitude showed a small but significant positive correlations (R <sup>2</sup> = 0.1615, P = 0.013) with PhiPT values between vineyards (Supplementary Figure S3). We then investigated if clone and vineyard management systems could be contributing to this correlation, by comparing the epigenetic/GeoDs correlation of 10 vineyards planted with clone 1654 [vineyards 1 and 4 (Northern Grounds), 7 (Central Grounds), 9 and 12 (Eastern Ridge), 15 (Southern Grounds) 16, 17, 18, and 19 (Western Ridge) (Figure 3A)] and of six vineyards Columns %Polym MSL and %Polym NML show the percentage of polymorphic loci of each class per region. Shannon diversity indices are reported as mean (± SD). Wilcoxon rank test provides statistical support for all Shannon diversity indices (P < 0.0001). TABLE 2 \| Molecular distances (PhiPT) between Barossa Valley wine producing sub-regions. PhiPT values were calculated using MSAP profiles generated from 88 grapevine plants (four individual plants per vineyard) using restriction enzyme combinations MspI/EcoRI (above diagonal) and HpaII/EcoRI (below diagonal). P-values (shown in parenthesis) were calculated based on 9,999 permutations. Pairwise regional comparisons showing not significantly different PhiPT values are highlighted in bold. A total of 22 vineyards were included in the analysis: Northern Grounds (4), Central Grounds (4), Southern Grounds (3), four vineyards in Eastern Edge (4), four vineyards in Western Ridge (4) and Eden valley (3). planted with the same clone (1654) and trained using the same pruning system (i.e., spur pruning) [vineyards 1 (Northern Grounds), 7 (Central Grounds), 9 (Eastern Ridge), 15 (Southern Grounds), 16 and 19 (Western Ridge) (Figure 4A)]. Again, PCoA shows that the main contributor (23–24%) to the detected variability is associated to the distribution of the vineyards on the N–S axis. Mantel test showed a positive correlation for both epigenetic/GeoD comparisons, however, although both correlations were significant (P < 0.05), the correlation among vineyards pruned using the same system (Figures 4B,C) was higher than that observed when all pruning systems were incorporated in the analysis (Figures 3B,C). #### msGBS Analysis of Genome/Methylome Differentiation between Northern, Central, and Southern Grounds TASSEL 3 was then implemented on msGBS data for SNP calling from 99 samples collected in 11 vineyards in the Northern, Central, and Southern Grounds sub-regions. This generated a total of 8,139 SNPs of which 4,893 were present in at least 80% of the sequenced samples. PCA analysis using filtered SNPs showed very low level of genetic structure, with only five plants from vineyard 3 (Northern Grounds) separating from the rest (Supplementary Figure S4A). However, this clustering was FIGURE 3 \| Analysis of the correlation between molecular differentiation and geographic distance (Km) of vineyards planted with clone 1654 in the Barossa region. (A) Location of the selected Barossa Valley vineyards from the three sub-regions distributed along the Barossa Valley North–South axis Northern Grounds (blue), Central Grounds (green), Eastern Edge (red), Southern Grounds (yellow), and Western Ridge (purple). Arrow indicates the direction of geographic North. (B) PCoA representing genetic/epigenetic differences between leaf samples collected from four plants/vineyard. Percentage of the variability captured by each PC is shown in parenthesis. (C) Correlation between pairwise genetic/epigenetic distance (MspI PhiPT) and geographical distance [Log(1 + GeoD) (Km)] between vineyards. Shown equations are the correlation's R <sup>2</sup> and the Mantel test significance (P-value was estimated over 9,999 random permutations tests). PCoA and PhiPT for Mantel test were based on presence/absence of 215 loci obtained from MSAP profiles generated using MspI. not supported by DAPC (i.e., the optimal clustering solution should correspond to the lowest BIC) which indicated the optimal number of clusters for this data set is 1 (Supplementary Figure S4B) suggesting a lack of genetic structure in the vineyards/regions analyzed. Principal components-linear discriminant analysis (PC-LDA) was then used to visualize differences in DNA methylation detected using msGBS. DNA methylation profiles clustered samples by their sub-region of origin, with Northern and Central Grounds being separated by differential factor (DF1) from Southern Grounds while DF2 separated Northern from Central Grounds (Figure 5). These results were supported by the higher number of DMMs found when comparing samples from Southern to samples from Central or Northern Grounds than when comparing Northern to Central (Table 3). We next investigated the association of the detected DMMs to annotated protein-coding genes in the grapevine genome by surveying their location and density within and flanking such genomic features. A total of 3,598 genes were deemed differentially methylated (i.e., presented one or more DMMs within 5 kb of the TSS or the TSE) or within genes (Table 3). Quantification of such DNA methylation changes showed that, in average, methylation levels are higher in the northern most region in each comparison (i.e., NG > CG > SG) (Figure 6A). The majority of detected DMMs associated to a gene were present in the body of the gene and the number of DMMs decreased symmetrically with distance from the TSS and the TES (Figure 6B and Supplementary Tables S4–S6). Finally, as observed with all DMMs, the comparison between Northern and Central Grounds samples showed the lowest number of DMGs (Table 3, Figure 6C, and Supplementary Tables S4–S6). To gain further insight into the functional implications of the DNA methylation differences detected between sub-regions, we used GO.db (Carlson, 2016) and annotate (Gentleman, 2016) to assign 1,144 unique GO terms to the observed DMGs (adjusted P-value < 0.05). As observed with DMMs and DMGs the comparison between Northern and Central Grounds samples showed the lowest number of GO terms containing DMGOs (Table 3, Figure 6C, and Supplementary Tables S4–S6). REViGO semantic analysis of GO terms shared by all three pairwise regional comparisons (Figure 7) showed an increase of gene enrichment (i.e., a decrease in adjusted P-values) with GeoD (e.g., see Figure 7 for comparisons between Northern Grounds and Southern Grounds (A,B) and Central Grounds and Northern Grounds (C,D). Three hundred and eleven DMGs (8.6% of the total) were allocated in GO terms associated to response to environmental stimulus (161 and 150 abiotic and biotic challenges, respectively) (Figure 7 and Supplementary Tables S7, S8), which included GO terms in the semantic space of plant response to light, temperature, osmotic/salt stress and defense to biotic stimulus. # DISCUSSION In this study, we analyzed the effect of growing region on the methylation profiles of Shiraz plants using MSAP and msGBS. Both techniques use methylation sensitive enzymes to discover DNA methylation polymorphisms between samples. Although the use of restriction enzymes has the obvious limitation of being capable of detecting such polymorphisms only on the context of their recognition sequence, the technology has been extensively validated over the last 20 years and is considered highly reliable (Yaish et al., 2014; Li et al., 2015; Guevara et al., 2017). # Grapevine DNA Methylation Patterns Are Region Specific Analysis of HpaII and MspI generated MSAP profiles showed that the methylation profiles of the six different sub-regions were significantly different (P < 0.05) in 25 of the 30 possible pairwise comparisons (Table 2). Variability among vineyards and sub-regions was higher in MspI generated profiles (17 and 2%) than in HpaII profiles (8 and 1%), indicating that the TABLE 3 \| Identification of DMMs, DMGs, and GO terms (DMGOs) between sub-regions in Barossa Shiraz. Cells contain the number of DMMs, DMGs, or DMGOs detected in each pairwise comparison. Differential methylation between sub-regions was calculated using msGBS data from nine Shiraz plants per vineyard [Northern Grounds (NG): four vineyards, Central Grounds (CG): four vineyards and Southern Grounds (SG); three vineyards]. Directionality of methylation (i.e., hyper/hypomethylation) indicates an increase or decrease in DNA methylation in the second region compared to the first region in each pairwise comparison. FIGURE 6 \| Analysis of DMGs and GO terms (DMGOs) among three wine sub-regions in Barossa Shiraz. Genes were considered differentially methylated if located within 5 kb of at least one DMM (FDR < 0.01). DMMs were generated using msGBS on nine plants per vineyard (Northern Grounds: four vineyards, Central Grounds: four vineyards, and Southern Grounds; three vineyards). (A) Directionality of methylation differences between regions. Boxplots show the distribution of the intensity of changes in DNA methylation level between regions, represented here as the fold-change (2 power log2FC) in read counts for a given msGBS markers between two regions. Median shows the direction of the methylation flux at a whole genome level in each region comparison (i.e., positive medians indicate a global increase in DNA methylation (hypermethylation) while negative medians indicate a global decrease in DNA methylation (hypomethylation) in the second region in the (Continued) #### FIGURE 6 \| Continued comparison (e.g., Northern Grounds is hypermethylated compared to Southern Grounds). (B) Distribution of 3598 region specific DMMs around genes. Columns –5 to –1 and 1 to 5 represents the number of DMMs per kb around V. vinifera genes. Column 0 indicates the number of DMMs within the coding sequence (i.e., between the transcription start and end sites) of V. vinifera genes. (C) Shared DMGs and DMGOs between regional comparisons. Venn diagrams show the number of unique and shared DMGs and DMGOs between each regional pairwise comparison (i.e., Blue: hyper/hypomethylated genes and GOs in Northern Grounds compared to Southern Grounds; Yellow: in Central Grounds compared to Southern Grounds; and Green: in Central Grounds compared to Northern Grounds). detected regional epigenetic differences are, at least partially, sequence context specific (Tricker et al., 2012; Meyer, 2015). Calculated PhiPT values showed low levels of molecular differentiation between sub-regions, even when those differences were statistically significant (Table 2). This could be explained by the high proportion of the total variability associated to differences between individual plants (81–91%) compared to 1–2% associated to differences between sub-regions. Such high levels of molecular differentiation between individuals could be due to the random accumulation of somatic variation with age, which can be genetic or epigenetic in nature. A specific limitation of MSAPs is its inability to distinguish between a fully methylated site from a site containing a genetic mutation (Yaish et al., 2014). PCA of genetic polymorphisms detected using msGBS results showed a high level of genetic variability between plants (Supplementary Figure S4A) which is characteristic of long living plants in general (Baali-Cherif and Besnard, 2005) and in grapevine in particular (Torregrosa et al., 2011). However, DAPC did not detect any sample clustering associated to their sub-region of origin (Supplementary Figure S4B) indicating that genetic diversity is not structured in a geographic manner. Although both genetic and epigenetic somatic variation can be random (Vogt, 2015), different growing conditions will differentially affect the DNA methylation profiles of otherwise genetically identical individuals (Consuegra and Rodríguez López, 2016) as previously shown on clonally propagated Populus alba (Guarino et al., 2015). It is, therefore, not surprising to find that epigenetic profiling was a better predictor of sample origin than genetic profiling alone both using MSAP data (Table 2 and Supplementary Figure S1) or msGBS data (Figures 5–7 and Supplementary Figure S4). This suggests that although genetic differences between regions or vineyards can partly contribute to the observed molecular differentiation between vineyards/subregions, epigenetic differences are the major driver of such differentiation. Previous studies have shown that in some instances clonal variability in grapevine is better explain by epigenetic than genetic differences (Imazio et al., 2002; Schellenbaum et al., 2008; Ocaña et al., 2013). It is therefore possible, that the epigenetic differences observed here are not associated to regional environmental differences but that they were present since the time of planting or due to environmental variations that may have occurred at the time of the sampling. For this reason, further research including information from more than one season is needed to validate the DNA methylation differences between of differentiation when inter-vineyard variability was analyzed (Supplementary Figures S2G,H), suggesting a major contributor to the observed molecular variability between vines in the Southern Grounds is linked to the vineyard of origin. Taken collectively, these results suggest that the specific growing conditions from each subregion impose DNA methylation patterns on grapevine plants specific for each region as previously shown both in cultivated (Guarino et al., 2015) and wild plant populations (Fonseca Lira-Medeiros et al., 2010). Not surprisingly, and contrary to what has been shown in natural plant populations (Fonseca Lira-Medeiros et al., 2010; Róis et al., 2013), no clear negative correlation between genetic and epigenetic diversity was observed in the studied vineyards. This is most probably due to the intensive phenotypic selection to which grapevine cultivars have been under since domestication and the relative low levels of environmental disparity to which vines growing in the same vineyard are exposed to. ## Differences in Altitude and Pruning System Correlate with Vineyard Epigenetic Differentiation Principal coordinate and Mantel test analysis showed that the correlation between epigenetic and GeoD between vineyards on the North–South axis of the Barossa Valley (Figure 2A) was significant (P = 0.0003) (Figure 2C) and that the main contributor to the observed epigenetic differences was the position of the studied vineyards along the N–S axis (Figure 2B). This suggests that environmental differences between locations could be contributing to the observed molecular differences between sub-regions or vineyards (Figure 3). Moreover, the correlation (R <sup>2</sup> = 0.3066) between epigenetic and GeoD among vineyards planted with clone 1654 on the N–S axis (Figure 2) supports the Shannon diversity analysis that indicate that the different genetic backgrounds used in this study do not greatly affect the epigenetic differences observed between regions (Table 1). Conversely, differences in vineyard altitude appear to be a contributor to the detected epigenetic differentiation between vineyards (Supplementary Figure S3). Previous work FIGURE 7 \| REViGO semantic analysis of differentially methylated GO terms shared by all three regional pairwise comparisons. Functional enrichment of GO-terms was carried out for the genes deemed differentially methylated (DMGs) hypermethylated (185) (A,C) or hypomethylated (211) (B,D) in Northern Grounds compared to Southern Grounds (A,B) and Central Grounds compared to Northern Grounds (C,D) using GO.db and annotate and summarized using REViGO. Bubble color indicates the p-value for the FDRs (the first 10 terms are labeled with legends in black. A detailed list of all GO terms containing DMGs has been supplied as a Supplementary Tables S7 and S8); circle size indicates the frequency of the GO term in the underlying GO database (bubbles of more general terms are larger). fpls-08-01860 October 27, 2017 Time: 16:50 # 11 has shown that sun exposure can have significant effects both in berry metabolomic profiles (Son et al., 2009; Tarr et al., 2013) and on the epigenetic profiles of plants growing in different environments (Guarino et al., 2015). Although altitude does not necessarily affect sun exposure, it can have a profound effect on the UV levels experienced by plants (approximately 1% increase every 70 m gain in altitude). Our results suggest that, although DNA methylation in and around genes changes in both directions (hyper- and hypo-methylation), on average, it increases with altitude (i.e., NG > CG > SG; vineyard average altitude 301, 277, and 236 m, respectively) (Figure 6A). Due to the nature of the msGBS approach used here, all sequenced DMMs are in the CHG context. Global methylation levels on this context varies widely between plant species (9.3% in Eutrema salsugineum to 81.2% in Beta vulgaris) (Niederhuth et al., 2016). In V. vinifera, genome-wide weighted CHG methylation level is 20.4%, more than double than that found in A. thaliana (Niederhuth et al., 2016). Although the analysis of DNA methylation has traditionally focus on the CG context, CHG differential methylation has been reported to be more prominent than CG differential methylation in other perennial crops (i.e., Apple, Malus domestica) (Daccord et al., 2017). In this study, the majority of detected DMMs associated to a gene were present in the body of the gene (Figure 6B). The function of gene body methylation (GbM) is not yet well understood (Zilberman, 2017) and recent studies have shown that GbM can be lost over evolutionary time with no deleterious consequences, suggesting that it might not be required for plant viability (Bewick et al., 2016). However, plant accessions with higher average GbM have been shown to have higher average expression of gene body methylated genes (Dubin et al., 2015; Wang et al., 2015). Moreover, GbM has also been proposed as a regulator of alternative splicing (Wang et al., 2016) and suppressor of intragenic cryptic promoters and transposable element (Maunakea et al., 2010). In particular CHG GbM has been associated to the silencing of genes lacking CG GbM (Niederhuth and Schmitz, 2017) and to the repression of splicing in maize (Regulski et al., 2013). Remarkably, global methylation levels of the CG, CHG, and CHH contexts has been proposed to be an adaptive trait to environmental variables such as latitude, aridity and photosynthetically active radiation temperature, respectively (Dubin et al., 2015). It is, therefore, tempting to speculate that the differences in GbM observed between regions reflect plant adaptation to their local environments that could be affecting alternative splicing, which has been itself been proposed as an adaptive mechanism (Ast, 2004). Functional analysis of the DMGs between sub-regions generated GO terms associated to plant response to light stimulus (Supplementary Table S8). More importantly, the number of genes associated to such GO terms was higher in comparison between regions with bigger differences in altitude [74 and 46 genes in comparison SG vs. NG (65 m difference) and SG vs. CG (41 m), respectively] than in the pairwise comparison with lower difference in altitude (6 genes NG vs. CG (24 m)]. Although this positive polynomial grade 2 correlation (R <sup>2</sup> = 1) was generated using only three data points, it is tempting to speculate that differences in light incidence due to differences in altitude are triggering the observed changes in DNA methylation in response to light stimulus genes. Especially when previous work has shown that, in grapevine leaves, increased UV levels trigger the synthesis of non-flavonoid phenolics such as resveratrol (Sbaghi et al., 1995; Teixeira et al., 2013). Interestingly, DNA methylation has been previously linked to the regulation of the gene VaSTS10, which controls the synthesis of resveratrol in Vitis amurensis (Kiselev et al., 2013; Tyunin et al., 2013). The correlation between epigenetic and GeoDs observed between vineyards planted with clone 1654 and pruned with the same method (spur pruning) (Figure 4) was reduced when all vineyards planted with clone 1654 were considered irrespectively of the pruning system used (Figure 3). This suggests that differences in pruning system, in conjunction with environmental conditions, might be contributing to the epigenetic differences observed between vineyards and subregions in this study. However, further research on the effect of the observed change in DNA methylation with vineyard altitude and pruning on gene expression are needed to validate the hypothesis that such changes might be regulating plant adaptation to such environmental cues. # CONCLUSION Vintage, geographic location, and vineyard management have been shown to influence both vegetative growth (Jackson and Lombard, 1993) and fruit composition in grapevine (Roullier-Gall et al., 2014). In light of the results shown here, we propose that epigenetic processes in general and DNA methylation in particular, could constitute an important set of molecular mechanisms implicated in the effect that provenance and vintage has, not only on plant vegetative growth, but also on fruit and wine quality. It is important to stress that since global patterns of DNA methylation are tissue/organ specific (Rodríguez López et al., 2010), the observed differences in DNA methylation profiles between plants growing in different regions can only be taken as indicative of those occurring in leaves. However, the effect of the environment on the epigenetic profiles of different tissues in plants reflects their mode of development. That is, unlike mammals, plants growth and organ formation occurs from stem cell populations in the meristems (Pikaard and Mittelsten Scheid, 2014). Previous studies (e.g., Verhoeven et al., 2010; Tricker et al., 2013a) have shown that environmentally induced markers detected in leaf tissue can be found on subsequent generations. This indicates that the DNA methylation markers observed in leaves were also present in the meristematic tissue that ultimately produced the reproductive organs. For this reason, it is plausible to expect that region-specific markers detected in grapevine leaves, could also be present in other organs such as berries since these are originated from the same shoot apical meristems as leaves. Although preliminary, our results open the door to speculate that epigenetic priming (Tricker et al., 2013b) could act as a form of epigenetic memory of the vineyard's environment that would ultimately contribute, at least partially, to the uniqueness of wines produced in different regions. Testing this hypothesis will require the integrative analysis of fruit DNA methylation, gene expression, and metabolite composition data from multiple seasons to account for the effect of inter-annual climatic variations on fruit composition (Fabres et al., 2017). ## AUTHOR CONTRIBUTIONS fpls-08-01860 October 27, 2017 Time: 16:50 # 13 HX and MK carried out the experiments and contributed to data analysis. NS performed gene ontology analysis on msGBS data. KT performed TASSEL analysis on msGBS data. TC, MG, JB, AM, and JS contributed to the design of the research project. RDB and CC contributed to the design of the research project, site selection and collection of material. CL contributed to the design of the research project, data analysis and drafted the manuscript. All authors read and contributed to the final manuscript. #### FUNDING This study was funded through a Pilot Program in Genomic Applications in Agriculture and Environment Sectors jointly supported by the University of Adelaide and the Australian Genome Research Facility Ltd. MK was supported by the Australian Agency for International Development (AusAID) Ph.D. scholarship. CL is supported by a University of Adelaide Research Fellowship. MG is supported by the Australian Research Council through Centre of Excellence (CE1400008) and Future Fellowship (FT130100709) funding. #### ACKNOWLEDGMENTS The authors would like to gratefully acknowledge the Barossa Grounds Project and in particular the growers that allowed us to sample material from their properties and supplied information about their vineyards and management strategies. Dr. Kendall R. Corbin performed DNA extractions from all samples used in this study. Personnel in the viticulture group, Dr. Sandra Milena Mantilla, Annette James, and Valentin Olek contributed to collection of material. # REFERENCES # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.01860/ full#supplementary-material FIGURE S1 \| Analysis of genetic and epigenetic differences within Barossa Valley sub-regions. PCoAs represent variability of NML (genetic variability) (A,C,E,G,I,K) and of MSL (epigenetic variability) (B,D,F,H,J,L) as classified by the msap (v. 1.1.8) software (Pérez-Figueroa, 2013) of leaf samples in vineyards from Northern Grounds (A,B), and the Barossa Valley's Western Ridge Central Grounds (C,D), Eastern Edge (E,F), and Southern Grounds (G,H), Western Ridge (I,J), and Eden valley (K,L). Coordinates 1 and 2 are shown with the percentage of variance explained by them. Points represent individuals from each vineyard. Vineyard code (NG, CG, EE, SG, WR, and EV) is shown as the centroid for each vineyard. Ellipses represent the average dispersion of those poinst around their center. The long axis of the ellipse shows the direction of maximum dispersion and the short axis, the direction of minimum dispersion. FIGURE S2 \| Analysis of regional genetic and epigenetic diversity. Red symbols indicate samples analyzed using genetic information only, black symbols represent samples analyzed using epigenetic information only according to the R package for statistical analysis of MSAP data "msap." PCoAs were calculated using MSAP profiles generated from gDNA extracted from E-L 7-stage leaves (Coombe, 1995) of 88 grapevine plants grown in vineyards located in the six Barossa Valley wine sub-regions (A, Northern Grounds; B, Central Grounds; C, Southern Grounds; D, Eastern Edge; E, Western Ridge; F, Eden valley) (four individual plants per vineyard) using restriction enzyme combinations MspI/EcoRI and HpaII/EcoRI. FIGURE S3 \| Analysis of the correlation between epigenetic differentiation and environmental differences among vineyards planted along the Barossa Valley North–South axis: Mantel test analysis of the correlation between pairwise epigenetic distance (MspI PhiPT) and differences in altitude between vineyards. Shown equations are the correlation's R <sup>2</sup> and the Mantel test significance (P-value was estimated over 9,999 random permutations tests). PhiPT values were based on presence/absence of 215 loci obtained from MSAP profiles generated using MspI. FIGURE S4 \| Analysis of the grapevine genetic diversity in vineyards planted along the Barossa Valley North–South axis. (A) PCA representing genetic structure calculated using 4,893 high quality SNPs (i.e., present in at least 80% of the samples) in genomic DNA collected from 11 vineyards [Northern Grounds (blue) four vineyards, Central Grounds (green) four vineyards, and Southern Grounds (yellow) three vineyards (nine plants/vineyard)]. (B) Identification of the optimal number of genetic clusters present within the three sub-regions compared using BIC as implemented by DAPC using adegenet 2.0.0 (i.e., the optimal clustering solution should correspond to the lowest BIC). complex traits in diverse samples. Bioinformatics 23, 2633–2635. doi: 10.1093/ bioinformatics/btm308 fpls-08-01860 October 27, 2017 Time: 16:50 # 14 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 Xie, Konate, Sai, Tesfamicael, Cavagnaro, Gilliham, Breen, Metcalfe, Stephen, De Bei, Collins and Lopez. 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. # Prospect on Ionomic Signatures for the Classification of Grapevine Berries According to Their Geographical Origin Youry Pii<sup>1</sup> \, Anita Zamboni<sup>2</sup> , Silvia Dal Santo<sup>2</sup> , Mario Pezzotti<sup>2</sup> , Zeno Varanini<sup>2</sup> and Tiziana Pandolfini<sup>2</sup> <sup>1</sup> Faculty of Science and Technology, Free University of Bozen-Bolzano, Bolzano, Italy, <sup>2</sup> Department of Biotechnology, University of Verona, Verona, Italy The determination of food geographical origin has been an important subject of study over the past decade, with an increasing number of analytical techniques being developed to determine the provenance of agricultural products. Agricultural soils can differ for the composition and the relative quantities of mineral nutrients and trace elements whose bioavailability depends on soil properties. Therefore, the ionome of fruits, vegetables and derived products can reflect the mineral composition of the growth substrate. Multi-elemental analysis has been successfully applied to trace the provenance of wines from different countries or different wine-producing regions. However, winemaking process and environmental and cultural conditions may affect a geographical fingerprint. In this article, we discuss the possibility of applying ionomics in wines classification on a local scale and also by exploiting grape berry analyses. In this regard, we present the ionomic profile of grapevine berries grown within an area of approximately 300 km<sup>2</sup> and the subsequent application of chemometric methods for the assignment of their geographical origin. The best discrimination was obtained by using a dataset composed only of rare earth elements. Considering the experiences reported in the literature and our results, we concluded that sample representativeness and the application of a preliminary Principal Component Analysis, as pattern recognition techniques, might represent two necessary starting points for the geographical determination of the geographical origin of grape berries; therefore, on the basis of these observations we also include some recommendations to be considered for future application of these techniques for grape and wines classification. Keywords: grape, wine, ionomic profile, traceability, rare earth elements, ICP-MS #### INTRODUCTION The geographical origin and the authenticity of food products are often related to the overall perception that consumers have in terms of quality, thus having a strong impact on the commercial value of the goods. In the last decades, fingerprinting techniques based on the chemical analyses of agricultural products followed by multivariate statistical approaches have been developed, aiming at identifying and classifying products according to their geographical origin (Versari et al., 2014). These methods assume that the chemical composition of the food product under study (e.g., mineral elements, stable isotopes ratios, and metabolites) is depending on the provenance #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics, Spain #### Reviewed by: Jose Antonio Alcalde, Pontifical Catholic University of Chile, Chile Andreas Zitek, University of Natural Resources and Life Sciences, Vienna, Austria > \Correspondence: Youry Pii [email protected] #### Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science > Received: 16 January 2017 Accepted: 10 April 2017 Published: 24 April 2017 #### Citation: Pii Y, Zamboni A, Dal Santo S, Pezzotti M, Varanini Z and Pandolfini T (2017) Prospect on Ionomic Signatures for the Classification of Grapevine Berries According to Their Geographical Origin. Front. Plant Sci. 8:640. doi: 10.3389/fpls.2017.00640 213 environment (Versari et al., 2014). The fingerprinting methods based on mineral element composition of food stuff have been largely adopted in the last years to trace the geographical origin of wine, olive oil, honey, cheese, coffee, vegetable, fruits, and spices (Danezis et al., 2016). One of the most popular techniques adopted for these analyses is the inductively-coupled plasma mass spectrometry (ICP-MS), which can be used for the determination of both the ionomic profile and the isotope ratios (Baxter et al., 1997; Rebolo et al., 2000; Wieser et al., 2001; Castiñeira et al., 2004; Coetzee et al., 2005; Šelih et al., 2014; Capici et al., 2015; Mimmo et al., 2015; Popescu et al., 2015; Scampicchio et al., 2016). In the particular case of agricultural products, it is postulated that the presence and the concentration of the mineral elements might reflect their geographical provenance (Almeida and Vasconcelos, 2003). Considering that the natural diffusion of mineral elements follows a pathway starting from the rocks, going through the soil and, finally, reaching the plant, it is thus conceivable that ionomic profile of plant organs and tissues is dependent on the geochemistry of the soil on which crops are cultivated (Geana et al., 2013). In addition, also anthropogenic activities, including the soil management, the use of fertilizers and phytochemicals, might determine alterations in the ionomic signature of agricultural products (Pepi et al., 2016a). #### TRACEABILITY OF WINES Among a wide variety of experiences dedicated to the geographical tracing of food products, a large number of studies, aiming at finding out reliable fingerprinting methods, have been carried out on wines traceability, most probably due to their relatively high commercial value. For these reasons, the elemental composition of different type of wines have been investigated with the aim of correlating them to the provenance soil for geographical tracing purposes (for an extensive review see Versari et al., 2014) (Table 1). However, the critical reading of the scientific literature published in this field of research demonstrates that the determination of the chemical descriptors for the origin of wines are strongly dependent on a plethora of factors, as for instance the number of samples used in the analyses, the type of wine (i.e., white, red, or rosè), the pattern recognition technique applied for the statistical analysis [e.g., Discriminant Analysis, Principal Component Analysis (PCA), Cluster Analysis, Stepwise Linear Discriminant Analysis and similar] and, most importantly, the geographical origin (Baxter et al., 1997; Díaz et al., 2003; Marengo and Aceto, 2003; Castiñeira et al., 2004; Jos et al., 2004; Thiel et al., 2004; Coetzee et al., 2005, 2014; Angus et al., 2006; Capron et al., 2007; Galgano et al., 2008; Serapinas et al., 2008; Forina et al., 2009; Fabani et al., 2010; Catarino et al., 2011; Rodrigues et al., 2011; Martin et al., 2012; Zou et al., 2012; Azcarate et al., 2013; Geana et al., 2013; Šelih et al., 2014). As also shown in Table 1, the majority of geographical tracing studies explores the analytical dataset by means of unsupervised pattern recognition analyses (e.g., PCA) and, once the most discriminant variables have been found, ad hoc statistical analyses, specifically supervised methods, are run in order to exacerbate the clusterization and to extract further information from the dataset. In addition, it could also be inferred that the power of the technique exploited for the chemical analysis (i.e., AAS, ICP-OES, ICP-AES, and ICP-MS) might be determinant for the tracing purposes. Therefore, the use of multi-elemental profile of wines as a fingerprinting technique requires the careful identification of suitable elements that generally reflect the characteristic features of the provenance soil. Thus, the correlation between chemical composition of wine and provenance soil is usually considered an important prerequisite for classification of wines according to geographical origin. In wine analyses, the choice of the indicator elements should also take into account possible distortions due to agricultural practices, environmental conditions and winemaking process. Winemaking is a complex process that involves multiple stages, as for instance blending, fermentation, rectification, and clarification, which have shown to influence element concentration in the final product. Early studies have shown that the concentration of elements may either increase (e.g., Al, Cd, Cr, Fe, Pb, and V) (Kristl et al., 2002; Almeida and Vasconcelos, 2003) or decrease (i.e., Al, Cd, Co, Cr, Fe, Pb, and V) (Eschnauer et al., 1989) in the processes of must fermentation and wine fining. More recently, Aceto et al. (2013) carried out a geographical tracing study on Moscato wines and demonstrated that the concentration of lanthanides, used as chemical markers, is conserved from soil to must, whilst the fingerprinting was affected by the treatments with bentonites. These observations led to the conclusion that wine traceability could be only pursued if the fining treatments were alternative to the bentonites ones (Aceto et al., 2013). #### GEOGRAPHICAL ORIGIN OF GRAPE BERRIES USING RARE EARTH ELEMENTS AS CHEMICAL DESCRIPTORS: THE VERONA REGION CASE Some of the limitations in wine fingerprinting, as discussed above, may be overcome by analyzing the chemical composition of berries. In particular, this would circumvent the problems associated with the chemical changes caused by winemaking, in particular for Rare Earth Elements (REEs). It is widely accepted that the composition in terms of REEs in the rocks is reflected also in the soil and in the plant tissues, even though a certain degree of variability is observed depending on the plant species (Wang et al., 1997; Wyttenbach et al., 1998; Zhang et al., 2002; Oddone et al., 2009). In the case of Vitis vinifera, the distribution of REEs within the berries has been studied in different cultivars (e.g., Chardonnay, Cabernet Sauvignon, Italian Riesling) by ICP-MS techniques (Bertoldi et al., 2009; Yang et al., 2010). In particular, Bertoldi et al. (2009) were able to show that Europium was accumulated in grape berries seed. Collectively, the results obtained within these studies prompted other authors to exploit these features (i.e., REEs) to study the geographical origin of wines, also considering the recent evidence demonstrating that different rootstocks do not significantly affect the REE content in the grape berries (Pisciotta et al., 2017). For instance, the afore-mentioned Moscato tracing work represents a comprehensive study in which the wine production chain has been investigated for geographical discrimination purposes and the correlation between the soil composition in terms of REEs and their concentration of berries and musts has been examined (Aceto et al., 2013). The unsupervised pattern recognition analyses carried out on musts did not highlight any difference between samples collected in the Moscato DOCG geographical region (Aceto et al., 2013). On one hand, these results indeed highlighted the power of REEs to assess the belonging of Moscato samples to the DOCG area; on the other, no striking distinctions between the different vineyards were found. Further insight in the use of REEs for the determination of geographical provenance was obtained with a study considering the REEs profile in berries of the "Glera" cultivar sampled in five different vineyards in the Veneto region, Italy (Pepi et al., 2016b). The authors established a correlation between the REEs concentration in the berries and REEs available fraction in the soil; this indeed allowed the discrimination of the provenance (Pepi et al., 2016b), provided that the geological origin of the soil in the vineyard considered across the Veneto region was fairly diverse. Few examples also indicated the possibility to use the trace element composition for classification of wines produced in wine growing regions located in small geographical area (Coetzee et al., 2005; Šelih et al., 2014). In this context, we decided to investigate whether the ICP-MS multi-elemental analysis followed by multivariate statistical analyses could be effective in the distinction of grape samples originating from neighboring vineyards (within an area of 300 km<sup>2</sup> ). We harvested berries of V. vinifera cv. Corvina (clone 48) at full ripening stage (Brix degree ranging between 18 and 24) from eleven vineyards located in the three most important wine production macroareas of the Verona region, namely Bardolino, Valpolicella, and Soave. The sampling rationale and procedures, as well as the sampling sites, were previously described by Dal Santo et al. (2013) and Anesi et al. (2015). The samples were homogenized, mineralized, and the concentration of 34 mineral elements in grapevine berries was determined by ICP-MS. These data were used as chemical descriptors to establish, through chemometric methods, criteria for assigning their geographical origin. In order to obtain a comprehensive view of the whole dataset, the concentration of mineral elements was used to build a heat map, in which each value has been calculated as the log2 of the ratio between the element concentration in the sample and the average concentration of that element in all the samples (Figure 1A). Within the heat map, it was used for the scatter plot accounts for 59.50% of the total variance. (C) The PCA analysis was carried out considering only micro-, macro- and beneficial elements generating a model composed of five components, which described 96.97% of the total variance. The scatter plot was obtained by combining the first two components, accounting for 74.92% of the variance. (D) The PCA analysis was carried out considering only REEs generating a model composed of two components, which describe 98.65% of the total variance. (E,F) Scatter plot obtained by PCA of the sub-data set encompassing only micro-, macro- and beneficial elements. The age of the vineyards (E) and the rootstock genotype (F) are highlighted. possible to differentiate two big groups of elements: a first group, encompassing mostly plant macro- and micronutrients (except for Ga and Rb), which showed strong variations, both positive and negative, and a second group that was formed mostly by REEs, which showed milder fluctuations between samples (Figure 1A). Micro- and macronutrients, as well as beneficial elements (Marschner, 2011), are actively taken up, accumulated, and differentially allocated in tissues and organs, and their homeostasis is tightly regulated in order to avoid nutritional imbalances (Williams and Salt, 2009). Therefore, substantial variations in the micro- and macronutrient concentrations are to be expected, depending on the soil type and on the rootstock genotype. On the other hand, REEs are not essential to plants; still they can be absorbed following the route of Ca, with which they share a similar ionic radius (Pickard, 1970; Hu et al., 2004). This is supported by the observations that Ca can be replaced by REEs in several biochemical and physiological functions (Pickard, 1970; Hu et al., 2004; Liu and Hasenstein, 2005; Babula et al., 2008; Xiaoqing et al., 2009; Carpenter et al., 2015; Yang et al., 2015). Besides the natural variations of element concentrations due to soil characteristics and origin, it is noteworthy that Cu, in 5 samples out of 11, was more abundant as compared to the global average value (Figure 1A). This behavior, also documented by Geana et al. (2013) in Romanian wine samples, might be due to Cu accumulation in soil, following the agronomical practice of using Cu-based fungicides for the protection of grapevine plants against downy mildew. Pattern recognition analyses were carried out on the whole dataset in order to highlight possible differences and similarities among the samples considered, finally aiming at the geographical origin discrimination. The PCA generated a six-component model, accounting for a total variance of 97.66%. The first two components, which together explained about 59.50% of the total variance, have been used to graphically represent the model (Figure 1B). The validity of the PCA models were assessed by the cross-validation approach previously described Bro et al. (2008) and Pii et al. (2015). Despite accounting for more than half of the total variance, the model obtained failed in describing the geographical provenance of samples, since they resulted randomly scattered across the diagram, except for three samples belonging to the Bardolino area that closely clustered together in the same quarter (Figure 1B). According to differences in element behavior displayed in the concentration heat map (Figure 1A), the whole dataset was split into two sub-datasets, the first encompassing micro-, macro- and beneficial elements, and the second comprising REEs, and they were subjected to PCA (Figures 1C,D). The multivariate analysis of the first sub-dataset (i.e., micro-, macro- and beneficial elements) generated a five components model, accounting on the whole for 96.96% of the total variance. The scatter plot obtained combining the first two components, which represented 74.92% of the variance, showed neither the separation of samples according with the geographical origin nor any other clear clustering (Figure 1C). Indeed, the distribution along the first component was mainly driven by Cu and, to a lower extent, by Fe, Na and B (Data not shown). As previously discussed, the differential accumulation of Cu could be due to agronomical practices (Geana et al., 2013); nonetheless, it has also been observed that the element composition of berries is also dependent on the rootstock genotype (Ga¸stoł and Domagała-Switkiewicz, ´ 2013). For this reason, both the age of the vineyard and the rootstock genotype have been highlighted within the PCA model discussed above (Figures 1C,E,F). In spite of this, any clear clustering regarding the classification of the samples considered (i.e., vineyard age and rootstock genotype) was obtained (Figures 1E,F). On the other hand, when only the REEs were considered for PCA, a two-components model accounting for 98.65% of the total variance has been obtained (Figure 1D). The scatterplot showed the separation of samples into two distinct clusters along the first component of the model, one encompassing samples from Bardolino and Valpolicella vineyards and the other comprising Soave vineyards. Nevertheless, two outliers from the other vineyards clustered with Soave samples (Figure 1D). According to the loading plot, the separation along the first component was mainly driven by Lu, whereas the other REEs contributed to the separation of samples along the second component (data not shown). This behavior might be due to the fact that Lu showed the strongest variation in concentration among the REEs group (Figure 1A). To the best of our knowledge, Lu has not emerged as discriminant element yet. ## CONCLUSION AND RECOMMENDATIONS FOR FUTURE STUDIES In conclusion, the new data presented here showed that the whole ionomic signature of the grape berries did not fully allow the discrimination of their geographical origin, most likely due to the heterogeneity in the characteristics (i.e., vineyards age, rootstock genotypes and agricultural practices) of the vineyards and the limited number of samples analyzed. Nonetheless, our data confirm that the multielemental analyses based on REEs of agricultural products might be a powerful technique to trace the geographical origin of foodstuff, and, in this specific case, of grapevine berries and musts. Furthermore, our data indicate that the ionomic signature can be suitable even for agricultural products originating from neighboring regions. On the bases of this experience and the pieces of research published in the literature, we suggest making the following recommendations, which may be considered in the experimental design, aiming at improving the efficacy and the resolution of the predictive tool: The commercial value of wines greatly depends on the authentication of their geographical origin, which represents a benefit for both consumers and wine producers. The ionomic signature appears as a powerful and flexible method to trace wine provenance even at the level of wine-producing sub-regions. Its flexibility relies on the availability of multiple elemental markers, different types of samples (wine, must, grape) for chemical analysis and numerous analytical and statistical methods. The optimization of these parameters, as well as the application of a sufficiently large number of variables, may allow tailoring the experimental set up for each wine-making area. #### AUTHOR CONTRIBUTIONS YP, AZ, ZV, and TP: Designed the experiments. YP and AZ: Samples and Data Analyses. SD and MP: Provided the samples #### REFERENCES and information about the vineyards. YP, AZ, ZV, and TP: Critical discussion of the data. YP and TP: Paper preparation. TP: Research coordination. #### FUNDING This project was financed by "Fondo Sociale Europeo nel Veneto" Project Number 1695/1/1/1103/2010, Project Title "Sviluppo di metodi analitici per la tracciabilità e l'autenticazione dei vini" with the partnership of Unione Italiana Vini. 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 Pii, Zamboni, Dal Santo, Pezzotti, Varanini and Pandolfini. 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. # The Induction of Noble Rot (Botrytis cinerea) Infection during Postharvest Withering Changes the Metabolome of Grapevine Berries (Vitis vinifera L., cv. Garganega) Stefano Negri 1 †, Arianna Lovato1 †, Filippo Boscaini <sup>1</sup> , Elisa Salvetti <sup>1</sup> , Sandra Torriani <sup>1</sup> , Mauro Commisso<sup>1</sup> , Roberta Danzi <sup>2</sup> , Maurizio Ugliano<sup>1</sup> , Annalisa Polverari <sup>1</sup> , Giovanni B. Tornielli <sup>1</sup> \* and Flavia Guzzo<sup>1</sup> \* <sup>1</sup> Biotechnology Department, University of Verona, Verona, Italy, <sup>2</sup> Unione Italiana Vini Soc. coop, Verona, Italy #### Edited by: Alma Balestrazzi, University of Pavia, Italy #### Reviewed by: Michaela Griesser, University of Natural Resources and Life Sciences, Austria Claudio Pastenes, Universidad de Chile, Chile #### \Correspondence:* Giovanni B. Tornielli [email protected] Flavia Guzzo [email protected] † These authors have contributed equally to this work. #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 06 March 2017 Accepted: 26 May 2017 Published: 21 June 2017 #### Citation: Negri S, Lovato A, Boscaini F, Salvetti E, Torriani S, Commisso M, Danzi R, Ugliano M, Polverari A, Tornielli GB and Guzzo F (2017) The Induction of Noble Rot (Botrytis cinerea) Infection during Postharvest Withering Changes the Metabolome of Grapevine Berries (Vitis vinifera L., cv. Garganega). Front. Plant Sci. 8:1002. doi: 10.3389/fpls.2017.01002 The natural or induced development of noble rot caused by the fungus Botrytis cinerea during the late stages of grapevine (Vitis vinifera L.) berry ripening is used in some traditional viticulture areas to produce high-quality wines such as Sauternes and Tokaji. In this research, we wanted to verify if by changing the environmental conditions during post-harvest withering we could induce the noble rot development on harvested berries in order to positively change the wine produced from withered Garganega berries. Therefore, we exposed the berries to postharvest withering under normal or artificially humid conditions, the latter to induce noble rot. The presence of noble rot symptoms was associated with the development of B. cinerea in the berries maintained under humid conditions. The composition of infected and non-infected berries was investigated by untargeted metabolomics using liquid chromatography/mass spectrometry. We also explored the effects of the two withering methods on the abundance of volatile organic compounds in wine by yeast-inoculated micro-fermentation followed by targeted gas chromatography/mass spectrometry. These experiments revealed significant metabolic differences between berries withered under normal and humid conditions, indicating that noble rot affects berry metabolism and composition. As well as well-known botrytization markers, we detected two novel lipids that have not been observed before in berries infected with noble rot. Unraveling the specific metabolic profile of berries infected with noble rot may help to determine the compounds responsible for the organoleptic quality traits of botrytized Garganega wines. Keywords: postharvest withering, Garganega grapes, noble rot induction, metabolomics, VOCs ### INTRODUCTION The necrotrophic ascomycete Botrytis cinerea has been described as a 'Jekyll and Hyde' fungus because it causes devastating gray mold disease in grapevine plants but is also responsible for noble rot in ripe and overripe berries, which allows the production of high-quality sweet wines such as Sauternes and Tokaji (Fournier et al., 2013). Gray mold caused by B. cinerea is one of the 220 most severe grapevine diseases, reducing both the quality and quantity of berries. The resulting wines are poor because the infected berries have an unfavorable composition and the pathogen also produces toxic compounds that affect yeast and thus inhibit the fermentation process (Bocquet et al., 1995; Hong et al., 2011; Agudelo-Romero et al., 2015). However, the development of B. cinerea as noble rot (botrytization) is a favorable process lasting 10–20 days and is typical of particular wine productions. The berry is transformed by the penetration of fungi through stomata, wounds or microfissures on the fruit surface (the pourri plein stage), the permeabilization of the fruit skin encouraging water loss and sugar concentration, and finally enzymatic maceration (the pourri rôti stage) (Ribéreau-Gayon et al., 2006). At the end of this process, further fungal development is arrested by the high sugar concentration and, if still on the plants, the botrytized berries can be harvested individually. Noble rot confers a berry composition which is distinct from that of berries with gray rot and uninfected berries and is potentially associated with desirable aroma characters of the resulting wine. In addition to directly producing potent odorants such as phenylacetaldehyde, lactones and vanillin (Lopez Pinar et al., 2016), noble rot infection can indeed stimulate production, in the berry, of cysteine and glutathione conjugates which can be transformed by the yeast into the powerful aroma compound 3-mercaptohexanol (Thibon et al., 2009, 2011). The developmental transition between gray rot and noble rot is influenced by environmental conditions and soil characteristics. Moist nights, foggy mornings and dry, sunny days promote the slow infection that results in noble rot, whereas strong rainfall and high humidity facilitate the more aggressive gray mold (Ribéreau-Gayon et al., 1980; Gubler et al., 2013). Recioto di Soave is an Italian passito wine (i.e., a wine produced from dehydrated grapes) made from the white-skinned berries of the cultivar Garganega. Grape dehydration (known as withering) takes place after harvest in a dedicated room known as the fruttaio. Slow postharvest withering can favor noble rot development, induced by particular environmental conditions and/or artificial B. cinerea inoculation, thus allowing botrytization to be implemented in regions where natural noble rot is uncommon (Lorenzini et al., 2012; Tosi et al., 2013). The metabolomic and transcriptomic changes that occur in black-skinned grape berries of various cultivars during traditional postharvest withering in fruttaio have recently been described (Zenoni et al., 2016). The behavior of withering berries is strongly cultivar dependent. The berries of slow-withering cultivars are more metabolically active during the process, showing both de novo synthesis of various metabolites (especially stilbenes) and a higher number of differentially expressed genes. Transcriptomics and metabolomics have also been used to investigate the response of white-skinned Sémillon berries during noble rot infections (Blanco-Ulate et al., 2015). The fruits of this cultivar respond to B. cinerea infection by upregulating genes involved in the response to pathogens and stress, fruit ripening, and hormone metabolism, and by accumulating certain secondary metabolites such as phenylpropanoids and terpenes. Here we used untargeted metabolomics based on liquid chromatography/mass spectrometry (LC-MS) to characterize the metabolites of Garganega berries during postharvest withering in fruttaio, under standard conditions and with artificial humidification used to induce B. cinerea colonization. We also used targeted gas chromatography/mass spectrometry (GC-MS) to characterize the volatile organic compounds (VOCs) in wines produced from berries exposed to the two postharvest withering treatments. # MATERIALS AND METHODS #### Withering Methods and Berry Sampling Approximately 170 kg of Garganega berries was harvested at the commercial ripening stage (soluble solids content = 18.5 ± 0.25◦Brix) in Monteforte d'Alpone (Verona, Italy) at the beginning of October and transported to the Pasqua Vigneti e Cantine winery. The berries were placed in perforated plastic boxes (plateaux, ∼5 kg in each) in a ventilated withering facility under natural conditions (17–20◦C, 78–82% relative humidity) and sampled (T0). Brix degrees were measured weekly in three randomly selected replicates using a DBR35 digital refractometer (Giorgio Bormac, Carpi, Italy). Three boxes were also weighed weekly using a CH50K50 electronic balance (Kern, Balingen, Germany) in order to determine the weight loss of berry bunches during withering. After 29 days, when the weight loss was ∼30% of the initial weight, grape clusters were sampled (T1) and half of the plateaux were covered with plastic film at 15–17◦C. Water-filled trays were placed inside to increase the relative humidity (88–94%) and encourage B. cinerea development. The remaining plateaux were left under normal withering conditions (15–17◦C, 68–75% relative humidity). The two different environmental conditions were imposed for 32 days before the final samples were taken obtaining T2-n (normal withering, ventilated) and T2-i (induced noble rot) samples. The total duration of dehydration was 61 days. The relative humidity and temperature inside and outside the covering were monitored using Hobo Pro v2 sensors connected to data loggers (Onset Computer Corporation, Bourne, MA, USA). Three independent pools of 3 kg of berries each were collected for each treatment and used in the following analysis. For T0, T1 and T2-n grape berries were randomly sampled whereas for T2-i berries were visually selected for noble rot symptoms. Each sample was used to determine the average berry weight, the number of B. cinerea colony forming units (CFUs) and for LC-MS analysis. The remaining of T0, T2-n, and T2-i berry samples (about 2 Kg for each biological replicates) were also pressed and the resulting musts were micro-fermented followed by GC-MS analysis of the wine. A simplified experimental workflow is reported in Figure 1. # Enumeration of B. cinerea CFUs We randomly selected 100 g of berries from each biological replicate and crushed them independently. The juices were serially diluted in 25% Ringer's solution (Oxoid, Basingstoke, UK) and 100-µl aliquots were spread on triplicate plates of Botrytis Selective Medium (Edwards and Seddon, 2001). The B. cinerea CFUs were counted on the plates after 5–7 days of incubation at 20◦C in the dark. ## Extraction of Metabolites and LC-MS Analysis 250 g of frozen berries for each pool was ground in liquid nitrogen and 300 mg of berry powder was extracted in three volumes of cold LC-MS-grade methanol. After mixing, the samples were sonicated for 15 min at 4◦C and centrifuged (16 000 rcf, 10 min, 4◦C). The supernatants were diluted 1:2 in LC-MS-grade water, passed through 0.2µm Minisart RC4 filters (Sartorius-Stedim Biotech, Göttingen, Germany) and analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Gold 127 HPLC System (Beckman Coulter, Brea, CA, USA) equipped with a C18 guard column (7.5 × 2.1 mm, 5µm particle size) and an Alltech (Nicholasville, KT, USA) RP C18 column (150 × 2.1 mm, 3µm particle size). A gradient between solvent A (0.5% formic acid and 5% acetonitrile in water) and solvent B (100% acetonitrile) was set as follows: 0–10% B in 2 min, 10–20% B in 10 min, 20–25% B in 2 min, 25–70% B in 7 min, isocratic for 5 min, 70–90% B in 1 min, isocratic for 14 min, 90–0% B in 1 min, and 20 min equilibration. For each sample, 20µl was injected at a flow rate of 0.2 ml min−<sup>1</sup> . The HPLC instrument was coupled on-line to an Esquire 6000 ion trap mass spectrometer equipped with an electrospray ionization (ESI) source (Bruker Daltonik, Bremen, Germany). Mass spectra were recorded in alternating positive and negative ionization mode within the range 50–1,500 m/z with a target mass of 400 m/z. Nitrogen was used as the nebulizing gas (50 psi, 350◦C) and drying gas (10 L min−<sup>1</sup> ) and the vacuum pressure was 1.4 × 10−<sup>8</sup> bar. For fragmentation analysis, MS/MS and MS<sup>3</sup> spectra were recorded in positive and negative ionization modes in the range 50–1,500 m/z. Helium was used for collision induced dissociation (amplitude = 1 V). MS data were collected using Esquire Control v5.2 software and processed using Esquire Data Analysis v3.2 software (both provided by Bruker Daltonik). Metabolites were identified by comparing retention times, m/z values and fragmentation patterns with those of commercial standards in our in-house library. When no authentic standard compounds were available, identification relied on the fragmentation patterns in online databases such as MassBank (www.massbank.jp) or reported in the literature. Neutral losses of 132, 146, and 162 Da were considered diagnostic of the loss of pentose, deoxyhexose, and hexose sugars, respectively. ## Lipid Extraction and LC-MS Analysis Botrytis cinerea strain B05.10 (Amselem et al., 2011) was inoculated into flasks containing 125 ml potato dextrose broth (Formedium, Hunstanton, UK) at a concentration of 7 × 10<sup>6</sup> conidia/ml, and incubated for 7 days at 22◦C, shaking at 120 rpm. B. cinerea mycelia were recovered by filtration and ground in liquid nitrogen. We resuspended 300 mg of frozen berry powder or B. cinerea mycelia in 300µl LC-MS-grade water and then mixed the suspension with 3 ml glacial chloroform/methanol (2:1). The samples were vortexed for 30 s, stored on ice for 1 h, sonicated for 15 min and centrifuged (25 min, 4,500 rcf, 4 ◦C). The chloroform phases (∼2 ml) were recovered, placed in 2-ml plastic tubes and centrifuged again (10 min, 16,000 rcf, 4 ◦C). The supernatants were recovered, partially evaporated in a Heto Holton Maxi-Dry Plus Vacuum (Thermo Fisher Scientific, Waltham, MA, USA) and tubes containing the same extract were pooled. Finally, the solvent was completely evaporated, the residue was resuspended in three volumes (w/v) of LC-MS-grade methanol and sonicated for 3 min. One sample, arbitrary selected as Quality Control 1 (QC1), and a methanolic solution including 1µg/µl palmitic acid as QC2 (Sigma-Aldrich, St Louis, MO, USA) were analyzed at the beginning and end of the experiment, respectively. Finally, the solutions were passed through a Minisart RC4 0.2-µm filter and 20µl was injected into the abovementioned LC-MS system. The solvents were 0.5% (v/v) formic acid in LC-MS-grade water (A) and 100% acetonitrile (B). A gradient was established from 50 to 100% B in 10 min, followed by 65 min under isocratic conditions and then from 100 to 50% B in 1 min. The column was finally equilibrated for 15 min. MS analysis was carried out by equipping the mass spectrometer with an atmospheric pressure chemical ionization (APCI) source and using the same parameters described above for medium-polar metabolites. #### Micro-Vinification Fermentation trials were carried out using musts from T0, T2-n, and T2-i berry samples. The musts were separated from the pomace and mixed with 0.3 g/l activating agent (Apapiù Mix, Tebaldi, Colognola ai Colli, Italy) and 15 mg/l sodium metabisulfite (Sigma-Aldrich) before transferring 500 ml of each must carefully into sterile bottles. After measuring the Brix degrees, the musts were inoculated with 10<sup>6</sup> CFU/ml of the commercial wine strain Saccharomyces cerevisiae Mycoferm CRU 69, previously activated by following the manufacturer's instructions (Ever, Pramaggiore, Italy). Each fermentation experiment was performed in triplicate at a controlled temperature of 18◦C. We monitored the fermentation kinetics for 14 days by gravimetric analysis to determine the loss of weight due to the production of CO2. ### GC-MS Analysis of Micro-Vinificated Wines Volatiles were analyzed by gas chromatography–mass spectrometry (GC-MS) after solid–phase extraction (SPE). SPE was performed using ENV<sup>+</sup> cartridge (1 g, 40–140µm; Isolute, IST Ltd., Mid Glamorgan, UK) and an Aspec XL Sample Processor for SPE (Gilson Inc. Middleton, WI, USA). The cartridges were sequentially conditioned with methanol (9.5 ml) and distilled water (19 ml). A total of 38 ml of wine sample diluted 1:2 with distilled water, and 1–heptanol added as internal standard (500µg/l) was loaded onto the cartridge. The residue was washed with 19 ml of distilled water. The free aroma compounds were eluted with 9 ml of dichloromethane. The solution was dried with Na2SO<sup>4</sup> and concentrated to 0.4 ml by nitrogen flow stream. GC–MS analysis was performed with 6980N Network GC System coupled with a 5975 XL EI/CI MSD (Agilent Technologies, Santa Clara, CA, USA), equipped with DB–WAX Bonded PEG fused silica capillary column (60 m × 320µm i.d. × 0.25µm film thickness; Agilent Technologies). Instrumental conditions were: electron impact (EI) mode 70 eV; injector temperature 200◦C; He carrier flow 1.5 ml/min; column temperature 50◦C for 4 min, rising to 240◦C at 4◦C/min, then 20 min at 240◦C; and injection volume 2.0µL in splitless mode. The analyses were performed in SCAN mode. NIST data bank and co-injection of pure reference standards were used to identify the compounds. # Statistical Analysis of Samples Statistical significance between samples analyzed for percentage of weight loss, soluble solid content, average berry weight, B. cinerea enumeration and fermentation kinetics was evaluated by t-student. For LC-MS and GC-MS data, raw chromatograms were converted to netCDF files for peak alignment and area extraction using MZmine software (http://mzmine.sourceforge.net/) and multivariate statistical analysis was applied to the resulting dataset using SIMCA v.13.0 (UmetrixAB, Umeå, Sweden). Pareto scaling was applied to all analytical methods. Unsupervised principal component analysis (PCA) was used to identify the major clusters defined by the samples, and two supervised methods, namely partial least squares discriminant analysis (PLS-DA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA/O2PLS-DA), were used to compare classes in order to identify metabolites that characterize different withering stages. PLS-DA models were validated by a permutation test (200 permutations) and the corresponding OPLS-DA/O2PLS-DA models were cross-validated by analysis of variance (ANOVA) with a threshold of p < 0.01. ## RESULTS # Artificial Humidification of Berries Increases the Prevalence of B. cinerea Colonization The berries maintained under standard conditions showed a linear weight loss during the first 40 days of withering followed by slower weight loss toward the end of the process, whereas the berries maintained under artificially high humidity lost weight more slowly (p < 0.01, Figure 2A). The Brix degrees increased during withering due to the loss of water and consequently the concentration of sugars, hence the profile was complementary to the loss of weight, increasing more slowly in the covered berries (Figure 2B). At the end of the withering process, only the covered berries showed the typical symptoms of noble rot, with roughly 70% of the berries visually appearing as chocolate-brown colored, more shriveled and dehydrated compared to control berries (Figures 2C,D). These berries were visually selected for further analysis. The comparison between the average berry weight of visually selected T2-n and T2-i berries revealed that T2-i weight was slightly lower than the control berries (p < 0.05, Figure 2E). This was also associated with a higher prevalence of B. cinerea colonization as determined by counting the number of CFUs on selective medium, confirming that the conditions used for induction supported B. cinerea growth in its latent form (p < 0.05, Figure 2F). No gray mold symptoms were observed among the covered berries. # Untargeted Metabolomics Reveals Fungal Metabolites, Plant Phytoalexin Accumulation, and Plant Metabolite Depletion Berries were sampled for metabolomic comparison at the beginning of the experiment (T0), 29 days later just before the two different withering conditions were applied (T1), and at the end of the experiment, separately for the berries exposed to conventional withering (T2-n) and the high humidity conditions (T2-i). Representative LC-MS chromatograms obtained in negative and positive ionization modes are shown in Figure 3. The blue zones highlight metabolites that increased in abundance during dehydration but were largely consumed in T2-i berries in comparison with T2-n berries. These metabolites included caffeoyl tartaric acid (caftaric acid), the amino acids leucine/isoleucine, phenylalanine, and tryptophan (together with its caffeic acid derivative), flavan-3-ols and flavonols. The red zones indicate metabolites that became more abundant or appeared de novo in the artificially humidified berries (T2-i) in comparison with T2-n berries. These included uridine 5'-diphospho-N-acetylglucosamine, a resveratrol tetramer and 13-keto-9Z,11E-octadecadienoic acid/13-oxo-9Z,11Eoctadecadienoic acid (13-KODE). The metabolites present in all samples are summarized in Table 1. The chromatograms were used to build two data matrices (negative and positive ionization mode, respectively). The negative ionization data matrix contained 257 m/z features, 74 of (B) soluble solids (◦Bx) during normal withering (T2-n) and induced noble rot (T2-i). (C) Appearance of sampled berries withered under natural conditions or (D) under higher humidity to induce noble rot. (E) Average berry weight in the T2-n and T2-i samples at the end of the withering process. (F) Enumeration of Botrytis cinerea colony forming units (CFUs) in samples T0, T1, T2-n, and T2-i. Vertical bars represented standard deviations (SD) of means (n = 3). Asterisks refer to t-student p-values obtained from T2-n and T2-i comparison (\*p < 0.05, \\p < 0.01). which were tentatively identified. They included 52 metabolites plus adducts and fragments (Supplementary File 1, Datasheet 1). The positive ionization data matrix contained 356 m/z features, 33 of which were identified, corresponding to 22 different metabolites (Supplementary File 1, Datasheet 2). The two data matrices were explored by multivariate analysis (O2PLS-DA). The results obtained for the negative ionization data matrix are shown in Supplementary Figures 1A,B. The O2PLS-DA loading plot was expressed as a pq(corr) value, representing the correlation between the p part of the model (the class of samples) and the q part of the model (the metabolites). The spatial closeness among the metabolites (black triangles) and the samples (blue squares) reflects their relationships, revealing the concentration effect from T0 to T1 and T2, but also specific effects in the botrytized samples that differ substantially from the non-botrytized controls (Supplementary Figure 1B). To overcome concentration effects and focus on concentrationindependent effects, the data matrices were normalized for weight loss, and the signal intensities were expressed relative to the weight at the beginning of the experiment (Supplementary File 1, Datasheets 3, 4). The results following data normalization are shown for the negative data matrix (Figures 4A,B). The enrichment of metabolites previously observed in the T2-n samples is now effectively shared among the T0, T1, and T2 n samples (light blue circle) confirming these metabolites are characteristic of the entire traditional withering process. Despite this normalization effect, a few metabolites were typical of the T0 and T2-n control samples (green and orange circles, respectively). Interestingly, the botrytized berries (brown circles) were strongly characterized by a group of specific metabolites which correlate negatively with the traditional withering process. The metabolites are listed in Supplementary File 2, Datasheet 1. OPLS-DA analysis was also applied to both the negative and positive ionization mode data matrices for the normal withering (T2-n) and botrytized (T2-i) berry samples (Figures 4C,D) better highlighting differences between T2-n and T2-i. The set of strongly characteristic metabolites identified by this analysis (Supplementary File 2, Datasheet 2) reflects the depletion and de novo production of metabolites in T2-i as already highlighted in Figure 3 and reported in Table 1 (e.g., resveratrol derivatives, 13-KODE and uridine 5′ -diphospho-N-acetylglucosamine). The depleted compounds represent diverse metabolite classes (e.g., sugars, amino acids, flavonoids and some stilbenes). Other metabolites accumulate rapidly in the botrytized berries, including pantothenic acid, some stilbenes (dimers, trimers and tetramers but not the monomers), glucose-6-phosphate, uridine 5 ′ -diphospho-N-acetylglucosamine, a lipid putatively annotated as 13-KODE, and other unidentified metabolites. The presence of the N-acetylglucosamine donor uridine 5′ -diphospho-Nacetylglucosamine indicates an active fungal metabolism because this sugar is a precursor of the chitin found in the fungal cell wall. Some of the molecules accumulating in botrytized berries were also clearly detectable in the chromatograms as major signals (Figure 3), including one resveratrol tetramer (Figure 3) and two metabolites with retention times of 30 and 32 min respectively (not shown). These two metabolites showed similar behavior, suggesting similar chemical properties. Both showed the chloride and formic adducts as main signals in negative ionization mode, and the molecular ions in positive ionization mode, and both generated fragments at m/z 355, 337, and 206. Although we were unable to identify these molecules, the higher retention time suggested they are lipids. We therefore re-extracted the methanol extracts with chloroform, and the resulting lipid fractions were analyzed by LC-APCI-MS using a method optimized for lipid analysis. The lipid profiles of T2-n and botrytized (T2-i) samples are shown in Supplementary Figure 2. This analysis confirmed the lipid-like nature of the two unidentified T2-i metabolites (highlighted in the figure) and showed that the general lipid profile is otherwise similar between the T2-n and T2-i samples. The same peaks could not be detected using the same LC-APCI-MS approach following the extraction of lipids from B. cinerea strain B05.10 grown in vitro, indicating that the two unidentified lipids might not be typical constituents of the fungus (data not shown). On the other side, we cannot exclude that the wild type B. cinerea strains developed in this experiments have different composition than the used reference B05.10 strain (Amselem et al., 2011). ## The Wines Produced by Botrytized and Conventionally Withered Berries Show Different Aromatic Profiles Micro-scale vinification was performed on fresh berries (T0), withered botrytized berries (T2-i) and conventionally withered berries (T2-n). The ◦Brix values of the musts from the T0, T2-i and T2-n berries were 18.67 ± 0.25, 31.13 ± 0.11, and 32.87 ± 0.06, respectively. The must fermentation rate (calculated as grams of CO2/100 ml of must) was generally higher in the musts from withered grapes (Figure 5A). Moreover, musts from the conventionally withered grapes showed a more vigorous fermentation compared to the musts from botrytized berries (p < 0.05). The aromatic profile of the three wines was analyzed by GC-MS 14 days after the beginning of the vinification. The O2PLS-DA model of the entire GC-MS data matrix revealed that the three wines showed distinct aromatic compositions (Figures 5B,C). The compounds characterizing the three wines are listed in Table 2, and their pq(corr) values are shown in Supplementary File 3. Wines from T2-n grapes were mainly characterized by fruity aromas, whereas botrytized wines were characterized by spicy aromas (Figure 5C). When the two withering processes were compared, wine from the naturally withered berries was strongly characterized by the presence of ethyl-4-hydroxy butanoate, as well as benzyl alcohol, eugenol, guaiacol, homovanillic alcohol, homovanillic acid, trans-3-hexenol, β-damascenone, and methyl vanillate. In contrast, the botrytized wine was strongly characterized by the presence of N-(3-methylbutyl)acetamide, as well as sherry lactone 1, benzaldehyde, 1-octen-3-ol, trans-8-dihydroxylinalool, Peak numbers refer to the chromatogram profiles in Figure 3. Rt, retention time (min); Nf, not fragmented. Asterisks refer to those metabolites which average peak areas was significantly different between T2-n and T2-i as assessed by t-student test. \*p < 0.05, \\p < 0.01. ethyl vanillate, ethyl isoamyl succinate, diethyl succinate, p-cresol, ho-diendiol, 4-terpineol, γ-nonalactone, and ethyl phenylacetate (Figure 5D and Supplementary File 3). When considering only the more abundant VOCs (more than 500 ppb) showing at least a two-fold difference in abundance between the two samples, the wine produced from naturally-withered berries was characterized by isovalerianic acid, isoamylacetate, decanoic acid, and homovanillic acid, whereas the botrytized wine was characterized by N-(3-methylbutyl)-acetamide, sherry lactones 1 and 2, benzaldehyde and 4-terpineol. #### DISCUSSION #### The Natural Development of Noble Rot Can Be Strongly Induced in Garganega Berries Undergoing Postharvest Dehydration The postharvest induction of noble rot could be used for the production of botrytized wines in regions with climates unsuitable for natural botrytization and also in those with suitable climates, to overcome the unpredictability of natural botrytization. However, a controlled widespread noble rot development on dehydrating grapes is not easy to achieve because the natural or forced ventilation of cases to accelerate dehydration makes the berries less susceptible to infection with B. cinerea (Barbanti et al., 2008). Fedrizzi et al. (2011) investigated natural botrytization in Corvina berries during withering, but in this case it was necessary to discard rotten berries developing gray mold and to manually separate the botrytized and non-botrytized fruit. Lorenzini et al. (2012) showed that noble rot can be induced under postharvest laboratory conditions by inoculating Garganega and Corvina berries with the fungus. The ability to achieve widespread noble rot development during natural withering has been reported anecdotally (Ferrarini et al., 2009; Vannini and Chilosi, 2013). Here we demonstrated the ability to induce noble rot development in Garganega berries without the concomitant development of gray mold by implementing a special management strategy during postharvest withering, comprising an initial period of normal withering to allow partial berry dehydration (which prevents the development of gray mold by ensuring the adequate concentration of sugars) followed by a The metabolites which strongly characterize each sample are highlighted with colored circles and are listed in Supplementary File 2 (Datasheet 1). The light blue circle comprises all metabolites that characterize the natural withering process and negatively correlate with berries infected with noble rot (T2-i). Correlation loading plots for the OPLS-DA models of negative (C) and positive (D) data matrices show the distribution of metabolites between T2-n and T2-i berries. All metabolites with pq(corr) values > 0.7 or < −0.7 are considered highly characteristic of T2-n (highlighted in yellow) or T2-i (highlighted in brown) berries and are listed in the Supplementary File 2 (Datasheet 2). period of increased humidity achieved by covering the berries in the presence of water-filled trays. This simple procedure increased the humidity without affecting the temperature, slightly reduced the rate of berry dehydration, and encouraged the development of B. cinerea infection without the need for artificial inoculation because the fungus is commonly present in the vineyard and in cellars as an environmental contaminant. Noble rot induction was confirmed by berry characteristics and the enumeration of B. cinerea CFUs in selective medium. We characterized the changes in the metabolite profile of grapes and wines attributable to the proliferation of the fungus. However, the possibility that the modified air humidity could be the cause of part of the differences between T2-n and T2-i cannot be completely ruled out. # B. cinerea Growth and Plant Defence Can Be Monitored by Untargeted Metabolomics Untargeted metabolomics based on LC-MS revealed metabolites associated with the infection of berries by B. cinerea. Some of these metabolites were biochemical markers of the fungus, including structural components and products of fungal metabolism, while others were derived from the berries and represent the onset of plant defense mechanisms during withering. The LC-MS data matrix revealed many imprints of fungal metabolism, including the presence of the N-acetylglucosamine donor uridine 5′ -diphospho-N-acetylglucosamine, which is utilized by fungi including B. cinerea as a substrate for the enzyme chitin synthase (Causier et al., 1994). The declining levels of many grape metabolites in botrytized fruits suggests they were degraded by fungal metabolism, including sucrose, hydroxycinnamic acids (coutaric, caftaric, and fertaric acids), amino acids, lignans, and many flavonoids (including flavan-3 ols and flavonols). The loss of polyphenols has been reported in other white-berry cultivars infected with noble rot, including Chenin Blanc (Carbajal-Ida et al., 2016) and Chardonnay (Hong et al., 2012) although there was a specific increase in the abundance of flavan-3-ols in Chenin Blanc, in contrast to other polyphenols (Carbajal-Ida et al., 2016). However, cultivars such as Sémillon accumulated high levels of phenylpropanoids following the onset of noble rot (Blanco-Ulate et al., 2015). Therefore, the impact of B. cinerea on phenylpropanoid metabolism appears to be cultivar dependent. The stilbenes are phytoalexins that are known to accumulate during botrytization (Landrault et al., 2002; Blanco-Ulate et al., 2015). The observed decline in the abundance of stilbene monomers (resveratrol and resveratrol glucoside) could reflect the consumption of these metabolites by the fungus, but the concomitant increase in the levels of stilbene dimers, trimers and tetramers suggests that botrytization causes the aggregation of stilbene monomers into oligomers. The accumulation of the oxylipin 13-KODE could also represent a plant defense response because this metabolite is induced as a defense molecule in soybean (Glycine max) in response to fungi such as Aspergillus niger, A. oryzae, Rhizopus oligosporus, and A. niger wry (Feng et al., 2007). To the best of our knowledge, this is the first report describing the induction of 13-KODE in grapevine berries in response to noble rot. However, octadecadienoic acids, the precursors of KODE oxylipins, have been proposed as potential positive metabolic markers of gray mold (Agudelo-Romero et al., 2015). The botrytized fruits also accumulated large amounts of pantothenic acid, D-glucose-6-phosphate and two unannotated lipids. ### The Postharvest Induction of Noble Rot Influenced the Accumulation of Wine Aroma Compounds The proliferation of B. cinerea induced remarkable changes in the accumulation of VOCs, affecting several aroma compounds that may contribute to the sensory characters of white wines. From a quantitative perspective, N-(3-methylbutyl)acetamide was the strongest marker of botrytized wine in agreement with previous studies of botrytization in Recioto di Soave (Azzolini et al., 2013; Tosi et al., 2013), Amarone (Fedrizzi et al., 2011), and Fiano (Genovese et al., 2007) wines. From a qualitative perspective, several VOCs detected at lower concentrations but with a potentially higher impact on aroma (Francis and Newton, 2005) were also influenced by noble rot. The sherry lactone isomers and γ-nonalactone were detected at higher concentrations in botrytized wines, in agreement with previous reports (Genovese et al., 2007; Sarrazin et al., 2007; Azzolini et al., 2013; Tosi et al., 2013). Although lactones do not contribute directly to the aroma of botrytized wines, they are involved in perceptive interaction phenomena resulting in an enhanced sensory contribution, e.g., synergy between γ-nonalactone and eugenol can enhance the overripe orange aroma notes typical of noble rot wines (Stamatopoulos et al., 2014). Eugenol is mostly derived from contact between the wine and oak wood, so the storage in oak barrels of Garganega wines from berries infected with noble rot could enhance these overripe orange aromas. Terpenes such as citronellol, ho-diendiol, hydroxylated linalool derivatives and 4-terpineol became more abundant during dehydration in the berries with noble rot, especially in the case of 4-terpineol. Likewise, the norisoprenoid 3-oxo-α-ionol (which gives rise to the tobacco aroma compound megastigmatrienone) accumulated to higher levels during withering. Terpenes and norisoprenoids are two important groups of aroma compounds that contribute the floral, fruity and tobacco-like attributes of wines. They accumulate in the berries as free molecules and as glycosylated precursors, which can be revealed by the action of yeast during fermentation or by acid hydrolysis during wine aging (Ugliano #### TABLE 2 \| Aroma compounds highlighted in Figure 5C characterizing T0, T2-n, and T2-i musts. The corresponding pq(corr) values are reported in Supplementary File 3. Items marked with an asterisk have to be multiplied by 10<sup>3</sup> . et al., 2006). Dehydration can favor their accumulation, and the presence of B. cinerea can facilitate their release from precursors by means of the pool of enzymes released into the must (Donèche, 1993). Several volatile benzenoids, generally characterized by sweet/spicy aroma notes, were shown to increase in response to noble rot and/or simple dehydration, including benzaldehyde, vanillin, cresols, guaiacols and eugenol. Although benzaldehyde is often associated with the development of B. cinerea (Genovese et al., 2007; Fedrizzi et al., 2011), the behavior of volatile benzenoids has not been investigated in detail. However, Genovese et al. (2007) also observed the accumulation of eugenol and vinyl guaiacol in white wines prepared from berries infected with noble rot. The mushroom-like aroma compound 1-octen-3-ol is found at significantly higher concentrations in wines produced from botrytized berries. B. cinerea and other pathogens such as Uncinula necator (powdery mildew) produce 1-octen-3-ol, which at high concentrations can reduce the quality of berries and introduce mushroom off-odors in the finished wine (Darriet et al., 2002). Interestingly, several aroma compounds arising from yeast metabolism, in particular the powerful fruit-smelling esters isoamyl acetate, ethyl butanoate, ethyl hexanoate and ethyl octanoate, were present at lower concentrations in the botrytized wines compared to wines from either the fresh or dehydrated berries. When comparing fresh and dehydrated berries, it was clear that dehydration without noble rot infection favored the accumulation of these metabolites during fermentation, probably due to the higher concentrations of nitrogen available to the yeast (Ugliano et al., 2006; Vilanova et al., 2007). The lower concentrations of esters in the botrytized wines could therefore reflect the depletion of nitrogen or the release of esterases by B. cinerea. In conclusion, the analysis of the volatile fraction of wines and evaluation of the potential odor contribution of different volatiles indicated that wines from dehydrated berries were generally characterized by higher content of fresh fruit-smelling compounds (esters), whereas noble rot induced the accumulation of several spicy aroma compounds such as lactones, combined with compounds with floral attributes such as 4-terpineol and the mushroom smelling compound 1-octen-3-ol. #### REFERENCES #### AUTHOR CONTRIBUTIONS GT: designed the experiments; SN and FB: performed the LC-MS-based untargeted metabolomics; SN and RD: performed the GC-MS based metabolomics; MC: performed the lipidomic experiment; ST and ES: did the bacterial counts and analyses; AP and AL: projected and executed the sampling and the agronomical analyses; SN and FG: analyzed the metabolomics data; FG: wrote the manuscript; SN, AL, MU, and GT: contributed to the draft writing; MU, AP, and ST: critically revised the manuscript; all authors read and approved the final manuscript. #### FUNDING This work was supported by Regione Veneto - POR - Fondo Sociale Europeo 2007–2013 – Ob. Competitività Regionale e Occupazione - Reg. 1081/2006. Asse IV "Capitale Umano." This work benefited from the networking activities coordinated within the EU-funded COST ACTION FA1106 "An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine." #### ACKNOWLEDGMENTS We are grateful to the winery "Pasqua Vigneti e Cantine SpA" for providing Garganega samples and for allowing us to set up the experiment in their withering facility. #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 01002/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 © 2017 Negri, Lovato, Boscaini, Salvetti, Torriani, Commisso, Danzi, Ugliano, Polverari, Tornielli and Guzzo. 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. # Metabolite Profiling Reveals Developmental Inequalities in Pinot Noir Berry Tissues Late in Ripening Amanda M. Vondras<sup>1</sup> , Mauro Commisso<sup>2</sup> , Flavia Guzzo<sup>2</sup> and Laurent G. Deluc<sup>1</sup> \* <sup>1</sup> Deluc Laboratory, Department of Horticulture, Oregon State University, Corvallis, OR, United States, <sup>2</sup> Guzzo Laboratory, Department of Biotechnology, University of Verona, Verona, Italy Uneven ripening in Vitis vinifera is increasingly recognized as a phenomenon of interest, with substantial implications for fruit and wine composition and quality. This study sought to determine whether variation late in ripening (∼Modified Eichhorn-Lorenz stage 39) was associated with developmental differences that were observable as fruits within a cluster initiated ripening (véraison). Four developmentally distinct ripening classes of berries were tagged at cluster véraison, sampled at three times late in ripening, and subjected to untargeted HPLC-MS to measure variation in amino acids, sugars, organic acids, and phenolic metabolites in skin, pulp, and seed tissues separately. Variability was described using predominantly two strategies. In the first, multivariate analysis (Orthogonal Projections to Latent Structures-Discriminant Analysis, OPLS-DA) was used to determine whether fruits were still distinguishable per their developmental position at véraison and to identify which metabolites accounted for these distinctions. The same technique was used to assess changes in each tissue over time. In a second strategy and for each annotated metabolite, the variance across the ripening classes at each time point was measured to show whether intra-cluster variance (ICV) was growing, shrinking, or constant over the period observed. Indeed, berries could be segregated by OPLS-DA late in ripening based on their developmental position at véraison, though the four ripening classes were aggregated into two larger ripening groups. Further, not all tissues were dynamic over the period examined. Although pulp tissues could be segregated by time sampled, this was not true for seed and only moderately so for skin. Ripening group differences in seed and skin, rather than the time fruit was sampled, were better able to define berries. Metabolites also experienced significant reductions in ICV between single pairs of time points, but never across the entire experiment. Metabolites often exhibited a combination of ICV expansion, contraction and persistence. Finally, we observed significant differences in the abundance of some metabolites between ripening classes that suggest the berries that initiated ripening first remained developmentally ahead of the lagging fruit even late in the ripening phase. This presents a challenge to producers who would seek to harvest at uniformity or at a predefined level of variation. #### Edited by: Simone Diego Castellarin, University of British Columbia, Canada #### Reviewed by: Amnon Lichter, The Volcani Center, Israel Dario Cantu, University of California, Davis, United States \Correspondence: Laurent G. Deluc [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 03 April 2017 Accepted: 08 June 2017 Published: 30 June 2017 #### Citation: Vondras AM, Commisso M, Guzzo F and Deluc LG (2017) Metabolite Profiling Reveals Developmental Inequalities in Pinot Noir Berry Tissues Late in Ripening. Front. Plant Sci. 8:1108. doi: 10.3389/fpls.2017.01108 Keywords: uneven ripening, crop heterogeneity, metabolomics, HPLC-MS, fruit composition, Vitis vinifera # INTRODUCTION fpls-08-01108 June 28, 2017 Time: 18:39 # 2 That producers seek to define and pursue optimal levels of enologically important metabolites in grapes is understood. However, intra-cluster variation is an important consideration as well, given the link between fruit uniformity and crop quality (Carroll et al., 1978; Selvaraj et al., 1995; Barbagallo et al., 2011; Kontoudakis et al., 2011; Liu et al., 2016). This contrasts the prevalent paradigm wherein the mean amount of a metabolite for a population of berries, rather than the variability inherent to that population, influences harvesting decisions. Whether optimal levels of traditional markers that influence harvest decisions (sugars, pigments, tannins, and organic acids) coincide with desirable levels of heterogeneity is largely unexplored. Several studies have examined the ways in which fruits within a cluster vary, why fruits may initiate ripening unevenly, and means of managing heterogeneity (Cawthon and Morris, 1982a,b; Coombe, 1992; Fernandez et al., 2006; Friend et al., 2009; Gray and Coombe, 2009; Pagay and Cheng, 2010; Calderon-Orellana et al., 2014b; Gouthu and Deluc, 2015). The uneven onset of ripening in a cluster (véraison) has been attributed to fruits' seed content, weakly to flowering time, and the interplay of hormones (Böttcher et al., 2010; Gouthu and Deluc, 2015; Vondras et al., 2016). Then, between véraison and harvest, intracluster variance (ICV) is reduced in terms of gene expression, ◦Brix, color index, and size (Gray and Coombe, 2009; Pagay and Cheng, 2010; Gouthu et al., 2014). However, differences at harvest are still observed and not without consequences. Although one study found no significant relationship between crop price and crop heterogeneity (Calderon-Orellana et al., 2014a), Carroll et al. (1978) showed that wines from fruits belonging to the least and most advanced berries had the lowest sensory scores. They observed differences in sugar, pH, titratable acidity, wine tannins and color between different classes of berries. In Syrah, larger berries at commercial harvest had lower quality characteristics and a yellow–green color indicative of incomplete maturity and possibly higher seed catechin extractability (Barbagallo et al., 2011). In recognizing that substantial variation at harvest limits accurate determination of phenolic maturity, Kontoudakis et al. (2011) also showed that wines from higher density (high sugar) berries were associated with higher ethanol content, pH, color intensity, total phenolic indexes, anthocyanins, and polymerization of proanthocyanidins and lower titratable acidity and bitterness; the resulting wines were higher quality and better balanced. In another study, less-dense grapes contributed fewer anthocyanins and more seed tannins than skin tannins, detrimentally affecting wine composition, while denser berries had the highest total phenolic content (Liu et al., 2016). Whether or not this variability is predominantly due to developmental differences is unexplored, though previous reports have demonstrated variation associated with other factors, like fruit position within clusters (Kasimatis et al., 1975; Tarter and Keuter, 2005; Pagay and Cheng, 2010; Pisciotta et al., 2013). If fruits are developmentally equals, then dynamic tissues should undergo key developmental transitions, like véraison and dehydration, uniformly. Therefore, perhaps the most appropriate time to make such assessments is as those transitions occur. Late ripening, which we define here as the period of extended ripening immediately following ripeness (Coombe, 1995), is a period during which fruits dehydrate. Distinctive wines are produced using both on- (Rolle et al., 2009; Bowen and Reynolds, 2015; Khairallah et al., 2016; Lukic et al., 2016 ´ ) and off-vine (Bellincontro et al., 2004; Costantini et al., 2006; Moreno et al., 2008; Toffali et al., 2011; Zenoni et al., 2016) dehydration strategies (Figueiredo-González et al., 2013). Both practices have similar effects on sugars, secondary metabolism, and cell integrity (Zamboni et al., 2008) and desiccation can produce responses analogous to those of water stress (Bellincontro et al., 2009). This developmental window is not only important for winemakers because of the dramatic metabolic changes that occur, but also because it might be used to better appreciate developmental inequality within a cluster. The purpose of this study was to determine whether intracluster variation late in ripening was linked to differences in developmental progress that are observable as fruits unevenly being ripening at véraison. Toward this objective, fruits were tagged as members of qualitative developmental categories or "ripening classes" based on their color at véraison and collected as fruits passed what would be considered commercial harvest into a stage that could be described as on-the-vine withering. If developmental differences persisted between fruits in a cluster, they might be best captured (1) as fruits transition into this stage and (2) in "dynamic" tissues (tissues that demonstrate they are changing within the window observed). A multivariate technique called Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) was used to clarify the extent to which different berry tissues remained dynamic in the late ripening period and to determine if and due to which metabolites berries late in ripening could be segregated based on their developmental category at véraison. It was concluded that (1) overall, fruits that were developmentally distinct at véraison remain distinguishable late in ripening, (2) skin, pulp, and seeds were not equally dynamic in the late ripening period, and (3) this period was marked by both reductions and expansions in variation for many metabolites, though most annotated metabolites showed no significant changes in ICV over the period. ## MATERIALS AND METHODS #### Experimental Design This study was conducted in 2011 at the Oregon State University Woodhall experimental vineyard in Alpine, Oregon. Pommard grapevines, clones of Vitis vinifera L. cv. Pinot noir, grown on 101-14 rootstock, and trained in a double Guyot system with vertical shoot positioning were used. The five vines used for this study were managed using standard viticultural techniques. On each plant, six primary clusters were chosen on both the east (three clusters) and west (three clusters) side of plants. A non-invasive tagging technique was used to label four qualitatively distinct ripening classes of fruits at véraison (Lund et al., 2008; Gouthu et al., 2014) on September 10th. Here, véraison is defined as when ∼50% of the cluster remains green, while ∼50% has visibly initiated ripening. Among these fruits, Green Hard (GH), Green Soft (GS), Pink (PS), and Red (RS) fruits were randomly selected and tagged throughout each of the selected clusters. GH and GS were completely green with no evidence of color-change. GH and GS were distinguished by touch, with GH having no perceptible deformation. PS often exhibited green and pink marbling or were light pink in color. RS berries were dark pink or red. Within each cluster, representatives of each ripening class were tagged at véraison using different colored strings. Then, six berries from each ripening class were sampled from each plant 34, 41, and 48 days after véraison: October 14th (t1), 21st (t2), and 28th (t3). In this study, one biological replicate is equal to six berries of a particular ripening class and from one of the plants used. Sampled berries were immediately frozen on dry ice and then stored at −80◦C. These sampling dates corresponded approximately to stage 39 (overripe) in the modified Eichhorn-Lorenz system for classifying grapevine growth stages (Coombe, 1995). #### Berry Measurements Total soluble solids and color were measured per berry (n = 5). A SPER Scientific digital refractometer (Scottsdale, AZ, United States) was used to measure total soluble solids in units of degrees Brix (◦Bx) and a Konica Minolta CR-300 chroma meter (Minolta Corp, Osaka, Japan) was used to quantitatively measure color [lightness (L), hue angle (h), and chroma (C)]. The color index of each berry was calculated as previously described (Carreño et al., 1995) and computed as (180 − h)/(L + C). #### Metabolite Extraction Approximately 40 mg of lyophilized material were weighed and extracted with 20 and 40 volumes (w/v) of cold, 90% methanol for pulp and seed, respectively. Skin tissues were subjected to 40 volumes (w/v) of cold 89.9% methanol acidified with 0.1% (v/v) of formic acid (Toffali et al., 2011). The extracts were vortexed, sonicated in an ice-filled ultrasonic bath (Falc Instruments, Bergamo, Italy) for 20 min at 40 kHz, kept in darkness for 2h at 4◦C and finally centrifuged at 13000 rpm for 10 min at 4◦C. Supernatants were collected and stored at −20◦C. The 200 µL of each extract were diluted 1:2 with LC-MS-grade water and filtered with Minisart RC 4 membrane filters (0.2 µm diameter pores, Sartorius) prior to injection into the HPLC-MS system. #### Metabolite Separation, Detection, and Annotation Twenty microliters of each diluted sample were drawn through a 508 Autosampler (Beckman Coulter, Fullerton, CA, United States) system and injected to a Beckman Coulter Gold 127 HPLC system (Beckman Coulter, Fullerton, CA, United States) equipped with a C18 guard column (7.5 mm × 2.1 mm) in front of an Alltima HP C18 column (150 mm× 2.1 mm, particle size 3 µm; Alltech Associates Inc, Derfield, IL, United States). Samples were analyzed randomly and in technical duplicate. The chromatographic solvents, conditions, and gradient are described in Anesi et al. (2015). Metabolite detection was carried out with a Bruker ion trap Esquire 6000 (Bruker Daltonics GmbH, Bremen, Germany) equipped with an ESI ion source with the following specifications: 10 L/min for N<sup>2</sup> drying gas and 50 psi for the N<sup>2</sup> nebulizing gas heated at 350◦C. The analyses were performed in negative and positive alternate modality, setting a target mass of 400 m/z and a scan range of 50–3000 m/z. Metabolite fragmentation was performed up to MS<sup>3</sup> by using Helium gas and setting the fragmentation amplitude at 1 V. Chromatographic data were recorded up to 55 min with Esquire Control v5.2 software and the.d generated files were processed with the proprietary Data Analysis v3.2 (Bruker Daltonics), converted in net.cdf files and analyzed with open-source MZmine 2.10<sup>1</sup> software to create a data matrix reporting feature peak areas. After peak deconvolution, alignment and gap filling performed by MZmine can result in few missing values in the data matrix; such values were considered "missing" and not as zero (Commisso et al., 2017). The subsequent multivariate statistical analyses were carried out with SIMCA v13.0 (Umetrix AB, Umea, Sweden). Metabolite annotation were made by comparing the m/z, retention time and fragmentation pattern (MS/MS and MS<sup>3</sup> ) of the detected signals with an in-house library of authentic commercial standards or, in their absence, with data reported in literature or online databases<sup>2</sup>,<sup>3</sup> . The confidence of each metabolite annotation was classified as prescribed by Sumner et al. (2007) and is defined in Data Sheet 1. #### Statistical Analyses Several statistical methods were used to explore intra-cluster differences (differences among the ripening classes) and changes within the cluster over time. Pareto scaling was applied to all analytical methods (van den Berg et al., 2006; Toffali et al., 2011). PCA was used to identify and remove 20 probable outliers from the 180 samples. The data from the remaining 160 samples (n = 3–5) were analyzed by PCA and OPLS-DA using SIMCA 13.0 (Umetrix AB, Umea, Sweden) to identify metabolites or biomarkers that accounted for differences between ripening groups and distinguished time points. OPLS-DA models were cross validated by ANOVA (p-value < 0.05) and equivalent PLS-DA models were fit and tested by permutation (200 permutations) to avoid overfitting (Triba et al., 2014). In addition to meeting these criteria, only models with high, crossvalidated predictability (Q2 > 0.50) were considered as high confidence (Triba et al., 2014; Anesi et al., 2015). All features were used in SIMCA 13 analyses; only annotated metabolites are shown in figures and discussed. ICV was estimated by averaging the five biological replicates within a ripening class and calculating the variance across the four ripening classes at a single time point for each annotated metabolite. Unidentified features/metabolites were not included in this analysis. An F-test for variance was used to test for significant changes in ICV between t1, t2, and t3 for each metabolite. In addition, a Tukey-test was used to test for <sup>1</sup>http://mzmine.github.io/ <sup>2</sup>http://www.massbank.jp <sup>3</sup>http://www.hmdb.ca significant differences in relative metabolite amount between ripening classes. #### Interactive Figures Figures were constructed using Prism (GraphPad Software Inc., San Diego, CA, United States), SIMCA 13.0, and plot.ly, an online graphing resource. Though still images are presented in this manuscript, the interactive equivalents of plot.ly-generated figures are provided as supplemental HTML files for an enriched exploration of the results (Presentation 1). Given the interactive files, readers can include or exclude groups of metabolites by clicking on them in the figure legends, zoom in and out of specified regions of the plots, rotate three-dimensional figures, and identify individual data points which have not been labeled in the still images provided herein. These figures are helpful for visualizing the described trends in the data. #### RESULTS #### Developmental and Metabolic Inequality We sought to determine whether intra-cluster variation late in ripening was associated with developmental inequalities that were apparent at véraison. This was enabled by tagging developmentally distinct "ripening classes" of fruits at véraison (GH, GS, PS, RS) and then sampling them late in ripening. In terms of total soluble solids (Figure 1A), the four ripening classes trended toward uniformity at t2, but this uniformity was short-lived. Green fruits (GH and GS) had significantly lower ◦Brix than more advanced fruits (PS and RS) at t3. No significant differences were observed among the ripening classes at t1 or t2 (Figure 1A). With respect to berry color, GH showed significantly lower color index than PS and RS at every collection time and lower color index than GS at t2 and t3 (Figure 1B). The color index of GS was similar to PS at t1 and indistinguishable from PS and RS at t2 and t3. Together, Figures 1A,B* examined separately present seemingly distinct narratives concerning fruit development and changes in ICV. ICV as defined by total soluble solids (◦Brix) describes clusters trending toward uniformity from t1 to t2, with inequalities reappearing after t2. Alternatively, ICV as defined by color index suggests that at and after harvest, differences between the berries that were the most and least advanced at véraison persist without an obvious point of relatively high uniformity. These initial observations indicate that (1) perception of cluster uniformity depends on the metabolites being measured, (2) developmental inequalities persist, though they may be temporarily masked, and (3) metabolite uniformity does not necessarily suggest developmental uniformity. This also suggests limits to how much developmental inequality observed at véraison is actually mitigated by harvest. Importantly, Figure 1A shows that fruits that were relatively advanced at véraison also initiated the dehydration stage first, as by this time increases in sugars are linked to dehydration rather than import. Untargeted HPLC-MS was used to further assess ICV of metabolites separately in berry seeds, skin, and pulp. Following data acquisition, 139 metabolites were annotated using an inhouse library (Data Sheet 1). Including all features, annotated and unknown, Orthogonal Projections to Latent Structures – Discriminant Analysis (OPLS-DA) was used to determine whether the ripening classes were distinguishable late in ripening, whether fruits from t1, t2, and t3 were distinguishable overall and if so, which metabolites account for the segregation of different groups. Prior to this, however, Principal Component Analysis (PCA) of all samples (including all annotated metabolites and unidentified features) revealed that the three berry tissues were remarkably distinct in their metabolite profiles (Supplementary Figure S1), with anthocyanins and stilbenes highest in and positively correlated with skin and with proanthocyanidins and flavanols in seed. Most flavonols and other flavonoids were associated with skin tissues. The distinct metabolic profiles of each tissue warranted analyzing each tissue separately to resolve any differences among the groups of interest. Interestingly, a high degree of similarity among the GH and GS berries and among the PS and RS berries was observed such that we were unable to model their differences with high predictability (Q2). This might indicate that the ripening classes are more similar to one another late in ripening than they were at véraison, but this is impossible to say conclusively without equivalent measures at véraison. The four ripening classes were aggregated into two (B–D) are provided as in Presentation 1. groups which could be reliably well-modeled by OPLS-DA in each tissue (Figure 2)—Lagging (GH + GS) and Advanced (PS + RS). Still, the original ripening classes are colored in Figure 2. For each tissue, OPLS-DA was used to identify metabolites that define the ripening groups (Figure 2 and Supplementary Figure S2) and define the intra-cluster metabolic changes during some of the latest stages of ripening (Figure 3). Model parameters are summarized in Data Sheet 2. Score plots (Figure 2A and Supplementary Figure S2) were used to visualize segregation among the samples, with the predictive component describing between-group differences and the orthogonal component describing within-group differences. For each tissue, the metabolic profiles of Lagging and Advanced berries were distinct, with no clear trends in within-group variance that could clearly be attributed to the original ripening classes (GH vs. GS and RS vs. PS). Next, S-plots with VIP integration were used to identify metabolites that best explain the segregation of ripening groups in each tissue (Figures 2B–D). S-plots show the covariance and correlation structure between the metabolites and predictive score. In other words, they show the reliability and influence of the metabolites on group segregation. The VIP score, also considered, is an additional metric that describes the extent to which any metabolite drives group distinctions. Metabolites with high VIP (>2) and relatively high \|p (corr)\| and \|p\| are putative biomarkers that define berries late in ripening that were Lagging or Advanced at véraison (Figure 2). The seeds of Lagging and Advanced berries were defined by their high levels of proanthocyanidins and sugars versus leucine/isoleucine, respectively (Figure 2B). The skins of Lagging and Advanced berries were distinguished by high anthocyanins and sugars, respectively (Figure 2C), and the pulps of Lagging berries were high in and defined by leucine/isoleucine (Figure 2D), in contrast to the high levels of leucine/isoleucine found in Advanced berries' seeds. Unsurprisingly, Advanced berry pulp was distinguishable by high levels of sugars (Figure 2D). The score plots in Figures 3A,C also visualize segregation among the samples, but per their collection date and irrespective of their ripening class or group. No model that passed all acceptable thresholds upon cross-validation could be established to describe metabolic differences in seed or skin over time (seed, Q2 = 0.25, CV-ANOVA p > 0.05; skin, Q2 = 0.27, CV-ANOVA p = 0.022). Because the OPLS-DA skin-by-time model was valid, we have included it here despite low Q2. Metabolites with high scores in the corresponding S-plot (Figures 3B,D) indicate why the late ripening stages observed were distinctive. The skins of t1 and t3 berries were distinguished by their high levels of flavonols and other flavonoids versus phenylalanine and sugars, respectively (Figure 3B), and the pulp of t1 and t2/t3 berries importance (VIP > 2) are indicated with a cross symbol. Putative biomarkers are labeled. Interactive versions of B and D are provided in Presentation 1. could be segregated on the basis of high phenolic acids versus amino acids and sugars, respectively (Figure 3D). Overall, biomarkers that define differences between ripening groups and were shared across all three tissues were exclusively sugars, specifically sucrose species and a di-hexose derivative. Leucine/isoleucine was an in-common biomarker between seed and pulp that distinguished Lagging from Advanced fruits (Figure 4). Taken together, these results indicate that (1) variability late in ripening is associated with the developmental inequalities present at the ripening onset, (2) the metabolome remains dynamic post-harvest for pulp (and less for skin), with markers that define points during this late ripening period, and (3) that for seeds, the differences associated with ripening group at véraison exceeded those associated with change over the period observed. ### Trends in Intra-cluster Variance during Late Ripening Next, trends in ICV late in ripening and the amount of ICV for metabolites with constant ICV were measured. The log<sup>10</sup> fold-change in variance was plotted for each metabolite between pairs of sequential time points. This allows the visualization of ICV patterns for each annotated metabolite in the data (Figure 5). The ICV patterns characteristic of each region are summarized in Figure 5A. How metabolite variance behaved as fruits enter this late phase should provide evidence regarding whether the ripening classes were developmentally uniform or not. Increasing ICV might suggest developmental inequality (Quadrants 2, 3, and 4), whereas decreasing ICV suggests migration toward developmental uniformity (+y/+x-axis and Quadrant 1). Metabolites with constant ICV would require further examination (Center). None of the annotated metabolites, in any tissue, significantly and exclusively increased or decreased in variance (Figures 5B–D). Though, several metabolites did demonstrate a significant change in ICV between a single pair of time points in each tissue. Further, some non-significant but observable trends appear upon examining classes of metabolites. In seed (Figure 5B), amino acids, anthocyanins, flavonols, and organic acids predominantly localized on the right-hand side of the plot. Most metabolites that showed significant changes in ICV fell in quadrants 2 or 4. Several, however, experienced significant reductions in variation, characteristic of the +y-axis and +x-axis regions including several pigments, a P2-type proanthocyanidin, and quercetin-o-glucuronide. In skin (Figure 5C), leucine showed significant changes in ICV characteristic of quadrant 2 over the time-course and most amino acids fell in quadrants 2 and 3. Anthocyanins and sugars localized predominantly in quadrant 2, and phenolic acids, flavonols and other flavonoids in quadrants 1 and 2. Stilbenes mostly fell in quadrants 1 and 4, and proanthocyanidins into 2 and 3. Like seed, most metabolites in skin that showed significant changes in ICV fell in quadrants 2 or 4, and few metabolites showed no increase in ICV. In skin, this included quercetin aglycone, citric acid, and a glucoside of cis-resveratrol. For pulp (Figure 5D), stilbenes and amino acids occurred in quadrants 2 and 3, anthocyanins and other flavonoids predominantly in quadrant 2, phenolic acids in 1 and 2, and flavanols in 1 and 4. Like seed and skin, most metabolites in pulp that showed significant changes in ICV over the time course fell in quadrants 2 and 4, and several exhibited reductions in ICV either between t1 and t2 or t2 and t3. In pulp, these included sucrose and a hexose fragment, several proanthocyanidins, flavanols, and flavonols. Overall, though, most metabolites did not show significant changes in ICV over the time-course (Figures 5B–D). Taking these metabolites, the magnitude of variance that persisted in the cluster was explored (Figure 6). This group could include metabolites with persistently high or low variance across the ripening classes throughout this study; in other words, the amount of difference between ripening classes did not significantly change over time for these metabolites, and that difference could be either large or small. The magnitude of ICV also provides insight into the developmental uniformity of berries in the cluster. High, constant ICV might suggest persistent developmental differences, whereas low, constant ICV suggests developmental uniformity. Classes of metabolites tended to exhibit similar levels of ICV, so ICV was summarized per metabolite class in Table 1. In all tissues, amino acids had comparatively high levels of persistent ICV. Some of the most contextually important metabolites for each tissue tended to be among the most persistently variable– proanthocyanidins and flavanols in seed (Figure 6A), anthocyanins in skin (Figure 6B), and sugars in pulp (Figure 6C). In addition, flavonols and phenolic acids in seed (Figure 6A) and flavanols and proanthocyanidins in skin (Figure 6B) were among the least variable groups of metabolites. These results indicate that the tendency toward reduced ICV is rare late in ripening, that variability will remain constant over this period for most metabolites (at least those considered here), and that high variability was often observed for the most spatiocontextually relevant metabolites. #### DISCUSSION This study predominantly examined the extent and behavior of intra-cluster variation late in the ripening phase and, similar to others who observed variation among berries, we observed variability between ripening classes (Carroll et al., 1978; Barbagallo et al., 2011; Kontoudakis et al., 2011; Rolle et al., 2011; Liu et al., 2016). In contrast to earlier studies which used berry density, color or weight classes to classify fruits and assess metabolite differences, we directly assessed variation associated with uneven ripening onset and, therefore, developmental inequality. If fruit uniformity at harvest is desirable, then understanding how metabolites accumulate in a developmentally diverse cluster of fruits, particularly late in ripening, should aid the identification of biomarkers to improve harvest decisions. We propose an approach to identify markers in the future and, given our data, the features that make this change between both pairs of time points, cross. Significance threshold, p-value < 0.05. Interactive versions of (B–D) are provided in Presentation 1. challenging. First, our inability to model four distinct ripening classes (instead, modeling Lagging vs. Advanced), which at véraison were distinguishable, does support some reduction in developmental variation across the classes as was observed in terms of gene expression and berry size by others (Gray and Coombe, 2009; Gouthu et al., 2014). However, developmentassociated differences among the ripening groups over the lateripening period were still identifiable, and there were diverse patterns in ICV except a significant, continual reduction in variation. Fruits within a cluster are strong, competitive sinks during ripening (Coombe, 1988; Davies et al., 1999). However, changes in sugar concentration are more associated with dehydration than sugar import into berries near commercial harvest (Coombe and McCarthy, 2000). This change delineates the majority of the ripening phase from that observed in this study. Fruits may indeed undergo a reduction of intra-cluster variation during ripening (Gray and Coombe, 2009; Gouthu et al., 2014), but if fruits were truly developmentally uniform or were approaching uniformity, spatio-contextually relevant metabolites would have low ICV or only have exhibited reductions in ICV as fruits enter this late stage. Instead, the re-divergence of the ripening classes in terms of ◦Brix and other metabolites, as well as persistently high variance in others indicates that the fruits remain developmentally distinct. Metabolic uniformity does not necessarily imply developmental uniformity. This contrasts the conclusions of Gray and Coombe (2009), wherein fruits must developmentally synchronize to proceed into subsequent growth stages. In the interest of improving harvest decisions, trends in ICV may be a worthy consideration, given that if one waits longer to harvest, for instance, there is no guarantee that variation will continually reduce. In each of the tissues studied, amino acids were among the most variable overall. Most amino acids showed no significant changes in ICV over this experiment, except for leucine/isoleucine hexose in seed and skin. In addition, leucine/isoleucine was able to distinguish ripening groups in seed and pulp. Significant differences between ripening classes were observed at one or more time points for arginine, phenylalanine, leucine/isoleucine, leucine/isoleucine hexose, and tryptophan; these amino acids and proline were also able to distinguish t1 from t3 berry pulp (Supplementary Figure S3). Together, proline and arginine constitute 90% of the Nitrogen content in grape juice and influence the perception of acidity in wine (Gerós et al., 2012). Arginine and phenylalanine are both sources of Yeast Assimilable Nitrogen (YAN), and phenylalanine specifically is the precursor for the phenylpropanoid pathway giving rise to flavonoids and stilbenes. As the most abundant yeast-assimilable, N-containing metabolite in juice, arginine content is one factor in the production of fruity and floral wine aromas (Gutiérrez et al., 2015). Lagging berries had significantly higher levels of arginine and phenylalanine than pink and red berries at one or more times in and pulp tissues (Supplementary Figure S3). skin More specifically, in skin, the level of arginine in GH berries was significantly higher than in PS and RS berries at times 1 and 3; arginine was significantly higher in GS than PS and RS at t2. Likewise, the level of phenylalanine in skin was TABLE 1 \| Tukey HSD tests comparing variances of metabolites classes within individual sampling dates. In each metabolite class, only metabolites with persistent variance (as in Figure 6) were included in comparisons. Groups that do not share letters in common are significantly different, p-value < 0.05. significantly higher in GS than in PS and RS at t2. In pulp, the level of phenylalanine was significantly higher for GH berries than the other classes at t2 and RS at t3. Similarly, the pulp levels of arginine in GH berries were higher than PS berries at t2 and RS berries at t3. Developmental differences in either arginine or phenylalanine could be important determinants of the differences we observed among downstream secondary metabolites. Leucine/isoleucine was among the metabolites best able to distinguish the seeds and pulp of less from more advanced berries. Phenylalanine and leucine participate in the production of higher alcohols during fermentation, namely 2-phenylethanol and isoamyl alcohol, the most abundant higher alcohols found in wine; these higher alcohols affect the aromas of wine and model solutions (Yoshizawa et al., 1961; Äyräpää, 1967; Vilanova et al., 2013; Noguerol-Pato et al., 2014; Vidal et al., 2014; Cameleyre et al., 2015). Previous work has implicated 2-phenylethanol in Pinot noir aroma, the variety used in this study (Miranda-Lopez et al., 1992; Girard et al., 2001). Finally, the potential implications of developmental inequalities in tryptophan are also interesting. Tryptophan is a precursor of auxin, a major regulator of fruit development and suspected precursor of 2-aminoacetophenone (AAP), an off-aroma described in white wines and the production of which can vary with harvest time (Hoenicke et al., 2001, 2002; Maeda and Dudareva, 2012; Schneider, 2014). Future studies could shed more light on how amino acid inequalities between individual berries originate and possibly propagate other metabolite inequalities, for better or worse. Although ripening groups were distinguishable in each of the tissues examined, t1 vs. t3 fruits were only reliably differentiable in pulp and per their sugar and amino acid content. These observations add to previous reports which also demonstrate pulp continues to undergo metabolism and transcriptomic changes during dehydration on and off the vine (Bellincontro et al., 2004; Costantini et al., 2006; Moreno et al., 2008; Rolle et al., 2009; Toffali et al., 2011; Bowen and Reynolds, 2015; Khairallah et al., 2016; Lukic et al., 2016 ´ ; Zenoni et al., 2016). For seed, the OPLS-DA and ICV patterns present seemingly contrasting results. OPLS-DA was unable to define seeds by their collection date, though was able to segregate ripening groups, and significant changes in ICV were observed for some metabolites between pairs of time points in seed. This suggests that the change in ICV over time in seed was not sufficiently large so as to define one time point versus another even though the difference between ripening groups may have expanded or contracted for some metabolites. Véraison for an individual berry marks the onset of ripening and coincides with the initiation of seed maturation, tannin oxidation, a cessation of seed growth, and seed dehydration (Ristic and Iland, 2005). The lack of or inability to observe changes is not entirely unexpected, given that the seed has matured by this period (Ristic and Iland, 2005) and changes that occur over time in seed might only be observed over longer time scales than in this study. Perhaps underlying factors that contributed to differences in seeds early in their development also influence seed composition after seeds have completed maturation such that they are distinguishable past the largest phases of seed development. Tartaric and malic acids compose ∼90% of total berry acidity. Both acids are considered in harvesting decisions and impact final wine composition and perception. Typically, levels increase in the berry up to 4 weeks after anthesis and decline during ripening (Kliewer et al., 1967; Lamikanra et al., 1995). Consistent with this expectation, tartaric acid was consistently highest in and able to distinguish t1 versus t3 fruits in pulp. Furthermore, tartaric acid was significantly higher in GH fruits' pulp than in GS, PS, and RS (Supplementary Figure S4); tartaric acid was probably an unsuitable marker of Lagging fruits (by OPLS-DA) because GS was indistinguishable from the red berries over the time course (Supplementary Figure S4). Although OPLS-DA analyses indicate that changes in organic acids late in ripening among berries have more to do with a dynamic pulp over time, this observation upon close inspection suggests that developmental differences do persist such that GH is perpetually laggard if defined by its tartaric acid content. Further examination could reveal how these inequalities in primary metabolites occur in the first place, and the extent to which they, plus environmental and physiological factors, contribute to wine quality and ICV among secondary metabolites. Among the secondary metabolites are berry phenolics, which contribute to the color, astringency, and bitterness of wines. Because of their importance, there is substantial interest in characterizing phenolic composition in a way to better inform harvest times and anticipate wine composition (Cagnasso et al., 2008; Tian et al., 2009; Kontoudakis et al., 2011). Persistently variable ripening classes would include, then, fruits at different levels of phenolic maturity. The color of red wines is influenced by anthocyanins and other phenolics (copigmentation). Anthocyanins were highest in Lagging fruits' skin, were able to distinguish Lagging from Advanced berries, and were among the most variable group of metabolites in skin tissues. Interestingly, developmental differences among the ripening classes were well-described by anthocyanins; being developmentally delayed and possibly having passed into the period of pigment decline (Hilbert et al., 2015), respectively, GH and RS fruits generally had lower anthocyanin levels, whereas the intermediate classes (GS and PS) tended to exhibit higher pigment levels (Supplementary Figure S5). Bautista-Ortín et al. (2006) showed anthocyanin extraction was improved in laterharvested fruits, even though they have lower pigment levels overall, to such a degree that slight over-maturation of fruit is perhaps desirable for determining harvest times (Bautista-Ortín et al., 2006); the developmental distance between the ripening classes may not only impact the abundance of anthocyanins, but their extractability as well. Proanthocyanidins make significant contributions to wine bitterness and astringency in addition to their co-pigmentation activities. Kontoudakis et al. (2011) showed that underripe berries will differ from high density counterparts in their contribution of seed versus skin tannins, degree of polymerization, and resultant wine quality. Proanthocyanidins and flavanols were among the most variable metabolites in seeds, but only proanthocyanins were highest in and distinguished Lagging seeds (Figure 2B and Supplementary Figure S6). Per their seeds' proanthocyanidin content, pink and red berries could be considered developmentally ahead of green fruits. Again, however, changes in the seeds over time were not sufficient to define time points over this period. This result is consistent with Ristic and Iland (2005), who showed tannin accumulation closely tied to fruit development and ripening, peaking at véraison and declining in seeds as they dry, mature, and brown. Curiously, catechin accumulation also peaks at véraison in seeds and skin and declines during ripening (Kennedy et al., 2000; Downey et al., 2003a; Ristic and Iland, 2005), though flavanols did not define ripening groups in seeds as did the proanthocyanidins. Furthermore, proanthocyanidins and flavanols were the least persistently variable in skin tissues; the same trends in abundance (green > red) were not observed in skin even though they are contextually relevant within that tissue. Unlike anthocyanins and proanthocyanidins, hydroxycinnamic acids were neither distinctive features of a ripening group, nor at remarkable levels of ICV. Upon closer examination, no significant differences were observed among the ripening classes for caftaric or coutaric acids in pulp, though their abundance did change over time (Supplementary Figure S4). However, significant differences in gallic acid (a hydroxybenzoic acid derivative) were observed among the ripening classes' seeds, with higher concentrations observed in pink and red fruits than in green fruits' seeds. This result is somewhat consistent with Tian et al. (2009), who showed higher levels of gallic acid in must for later harvested fruits and is additional evidence that RS and PS remained "ahead" of green fruits late in ripening. Unlike other metabolite classes which provide evidence that fruits are developmentally distinct, stilbenes did not define either of the ripening groups, though many stilbenes showed relatively high levels of ICV in skin and changed in ICV between at least one pair of time points in several tissues. High levels of stilbenes are found in berry skin, with variability between cultivars (Sun et al., 2006), can be induced to resist pathogens, and increase from véraison to harvest in Pinot noir (Gatto et al., 2008). Sources of variation besides developmental differences may contribute to the high ICV observed for stilbenes. Similarly, flavonols were not effective markers of developmental differences, but did distinguish t1 from t3 fruit overall, and were highly variable in a contextually relevant tissue—skin (Downey et al., 2003b). If either uniformity or minimal heterogeneity is optimal, then when metabolites of interest are at a desired level and uniformity across the cluster would be pertinent to harvest decisions and identifying measurable biomarkers for this purpose would be valuable. This, of course, is complicated by developmental inequalities within the cluster that persist. An ideal marker would (1) have persistently low ICV such that any berry, regardless of developmental stage, could be used as a representative individual and (2) change over time so that a particular level of the marker might be associated with low ICV for a set of metabolites of interest. Although sugars are important in harvest decisions, those annotated here would be unsuitable markers by this definition. The sugars in our metabolite library were the best indicators for segregating ripening groups and several were indicative of their diversity, not of their uniformity. Identifying such a marker would at least require a longer time scale, more frequent sampling intervals than used in this study, thorough field testing, and would benefit from expanding grape-specific metabolite libraries (Aretz and Meierhofer, 2016). This study contributes to the growing body of work and interest in uneven ripening, its implications, and is pertinent to an ever-evolving paradigm in harvest decision-making. To better understand heterogeneity within the cluster and its impacts, future studies should continue to segregate variation associated with inherently unequal "starting positions" as well as other factors, such as microclimate and position in and of the cluster (Reshef et al., 2017). #### AUTHOR CONTRIBUTIONS All authors assisted in critical revision of this manuscript, approve of the version submitted for publication, and have agreed to be accountable for all aspects of the work, including ensuring that all questions regarding the accuracy or integrity of the work are appropriately investigated and resolved. This work was conceived by AV, FG, and LD. The experimental design and metabolite extraction were performed by AV and MC. The LC-MS instrumentation and metabolite identification were performed by MC and FG. The description of the LC-MS methodology was provided by MC. The statistical analyses were performed and manuscript was composed by AV. # FUNDING This work was funded by the Grape Research Coordination Network (NSF 0741876). ## ACKNOWLEDGMENTS The authors are thankful for financial support for this project from the Grape Research Coordination Network and ongoing support from Oregon State University's College of Agriculture, the Oregon Wine Research Institute, and University of Verona's Department of Biotechnology. ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01108/ full#supplementary-material FIGURE S1 \| PCA bi-plot of all samples [p (corr)] and associated loadings [metabolites, t (corr)], plotted using components 1 and 2. FIGURE S2 \| Score plots from OPLS-DA analysis, by ripening group, of skin (A), and pulp (B) which show separation of samples. Hotelling's T2 Ellipse (95%) is not shown, but samples outside the ellipse are denoted with an asterisk. Samples are distributed along a predictive component (x-axis) and orthogonal component (y-axis) and are colored per their ripening class: GH, green; GS, light green; PS, pink; RS, red. FIGURE S3 \| Selected amino acid dynamics over the time course. Tukey HSD tests were used to compare ripening classes at individual time points. Significant differences between the ripening classes at single time points are denoted with different letters, p-value < 0.05. For visual ease, the points may be shifted slightly to the right. However, there are only three time points (t1, t2, and t3) at which measurements were taken. FIGURE S4 \| Selected organic and phenolic acid dynamics over the time course. Tukey HSD tests were used to compare ripening classes at individual time points. Significant differences between the ripening classes at single time points are denoted with different letters, p-value < 0.05. For visual ease, the points may be shifted slightly to the right. However, there are only three time points (t1, t2, and t3) at which measurements were taken. FIGURE S5 \| Selected anthocyanins over the time course. Tukey HSD tests were used to compare ripening classes at individual time points. Significant differences between the ripening classes at single time points are denoted with different letters, p-value < 0.05. For visual ease, the points may be shifted slightly to the right. However, there are only three time points (t1, t2, and t3) at which measurements were taken. FIGURE S6 \| Selected proanthocyanidins over the time course. Tukey HSD tests were used to compare ripening classes at individual time points. Significant differences between the ripening classes at single time points are denoted with different letters, p-value < 0.05. For visual ease, the points may be shifted slightly to the right. However, there are only three time points (t1, t2, and t3) at which measurements were taken. DATA SHEET 1 \| Metabolite annotation. DATA SHEET 2 \| SIMCA OPLS-DA model parameters and score plot axes details. PRESENTATION 1 \| Interactive figures. characteristics of Vitis vinifera L. cv. Syrah. S. Afr. J. Enol. Vitic 32, 129–136. Bautista-Ortín, A. B., Fernández-Fernández, J. I., López-Roca, J. M., and Gómez-Plaza, E. (2006). The effect of grape ripening stage on red wine color. J. Int. Sci. Vigne Vin. 40, 15–24. doi: 10.20870/oeno-one.2006.40.1.879 Anesi, A., Stocchero, M., Santo, S. D., Commisso, M., Zenoni, S., Ceoldo, S., et al. (2015). Towards a scientific interpretation of the terroir concept: plasticity of the grape berry metabolome. BMC Plant Biol. 15, 191. doi: 10.1186/s12870-015- Aretz, I., and Meierhofer, D. (2016). Advantages and pitfalls of mass spectrometry based metabolome profiling in systems biology. Int. J. Mol. Sci. 17, 632. Äyräpää, T. (1967). Formation of higher alcohols from 14C-labelled valine and leucine. J. Inst. Brew. 73, 17–30. doi: 10.1002/j.2050-0416.1967.tb03012.x Barbagallo, M. G., Guidoni, S., and Hunter, J. J. (2011). Berry size and qualitative 0584-4 REFERENCES doi: 10.3390/ijms17050632 fpls-08-01108 June 28, 2017 Time: 18:39 # 13 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 Vondras, Commisso, Guzzo and Deluc. 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. # System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism #### Lei Wang<sup>1</sup> , Xiaoliang Sun<sup>1</sup> , Jakob Weiszmann1, 2 and Wolfram Weckwerth1, 2 \* <sup>1</sup> Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, Austria, <sup>2</sup> Vienna Metabolomics Center, University of Vienna, Vienna, Austria #### Edited by: Simone Diego Castellarin, University of British Columbia, Canada #### Reviewed by: Darren Wong, Australian National University, Australia Flavia Guzzo, University of Verona, Italy > \Correspondence: Wolfram Weckwerth [email protected]* #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 23 November 2016 Accepted: 02 June 2017 Published: 30 June 2017 #### Citation: Wang L, Sun X, Weiszmann J and Weckwerth W (2017) System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism. Front. Plant Sci. 8:1066. doi: 10.3389/fpls.2017.01066 Grapevine is a fruit crop with worldwide economic importance. The grape berry undergoes complex biochemical changes from fruit set until ripening. This ripening process and production processes define the wine quality. Thus, a thorough understanding of berry ripening is crucial for the prediction of wine quality. For a systemic analysis of grape berry development we applied mass spectrometry based platforms to analyse the metabolome and proteome of Early Campbell at 12 stages covering major developmental phases. Primary metabolites involved in central carbon metabolism, such as sugars, organic acids and amino acids together with various bioactive secondary metabolites like flavonols, flavan-3-ols and anthocyanins were annotated and quantified. At the same time, the proteomic analysis revealed the protein dynamics of the developing grape berries. Multivariate statistical analysis of the integrated metabolomic and proteomic dataset revealed the growth trajectory and corresponding metabolites and proteins contributing most to the specific developmental process. K-means clustering analysis revealed 12 highly specific clusters of coregulated metabolites and proteins. Granger causality network analysis allowed for the identification of time-shift correlations between metabolite-metabolite, protein- protein and protein-metabolite pairs which is especially interesting for the understanding of developmental processes. The integration of metabolite and protein dynamics with their corresponding biochemical pathways revealed an energy-linked metabolism before veraison with high abundances of amino acids and accumulation of organic acids, followed by protein and secondary metabolite synthesis. Anthocyanins were strongly accumulated after veraison whereas other flavonoids were in higher abundance at early developmental stages and decreased during the grape berry developmental processes. A comparison of the anthocyanin profile of Early Campbell to other cultivars revealed similarities to Concord grape and indicates the strong effect of genetic background on metabolic partitioning in primary and secondary metabolism. Keywords: Vitis vinifera, berry development, mass spectrometry, primary metabolism, secondary metabolism, flavonoids, systems biology, data integration # INTRODUCTION Grapevine (Vitis vinifera L.) is one of the most important and widely cultivated economic crops. Grape berries are consumed either as fresh fruit or processed to raisins, juice and wine. Besides its enormous economical and nutritional values, grapes and grape products possess a wide variety of health benefits, such as antioxidation (Sánchez-Moreno et al., 1999; Doshi et al., 2006; Sánchez-Alonso et al., 2007; Sáyago-Ayerdi et al., 2009; Anastasiadi et al., 2010), cardiovascular protection (Tebib et al., 1994; Adisakwattana et al., 2010; Razavi et al., 2013), neuroprotection (Feng et al., 2007), anti-obesity (Kim et al., 2013; Zhang et al., 2013), etc. The grape berry is a non-climacteric fruit. From fruit set to ripening, grape berries undergo three main developmental phases including two sigmoidal growth phases with an intermediate lag phase (Kennedy, 2002). The performance of grape berry development is characterized by dramatic changes in both physiology and biochemistry, including increases in volume and weight, changes in texture, color, aroma, acidity, sugar contents, susceptibility to disease, etc. The first growth phase (phase I) is characterized by fruit formation and enlargement due to the active cell division and expansion. In this phase, a notable accumulation of organic acids, especially malic and tartaric acid has been observed (Conde et al., 2007). Phase II, which is defined as a lag phase features a slow enlargement of berry volume caused by a stop in cell division. The grape berry is still green and hard at this phase. Organic acids continuously accumulate until veraison, which marks the beginning of phase III. During the last phase, the grape berries undergo a second sigmoidal growth accompanied by a decrease in acidity and increase in sugar content (Conde et al., 2007; Deluc et al., 2007; Fortes et al., 2011; Liang et al., 2011; Dai et al., 2013; Degu et al., 2014; Fraige et al., 2015; Cuadros-Inostroza et al., 2016). The peel of red varieties colors as a result of the accumulation of anthocyanins (Boss et al., 1996; Ali M. B. et al., 2011; Degu et al., 2014; Fraige et al., 2015). The grape berry becomes soft in the final phase and is ready to be harvested. Another generally adopted descriptive system is the E-L system which was proposed firstly by Eichhorn and Lorenz (1978) with a more detailed description of grape berry development stages. Fruit development is an intricate process, featuring complex regulation and fine-tuned changes in metabolism. Its analysis requires the use of sensitive methods, which allow high sample throughput to cope with the amount of samples necessary to examine a time continuous process. Since the release of the grapevine genome sequence in 2007 (Jaillon et al., 2007; Velasco et al., 2007), studies of developing grape berry based on transcriptomic (Deluc et al., 2007; Palumbo et al., 2014), proteomic (Giribaldi et al., 2007; Negri et al., 2008; Martinez-Esteso et al., 2011; Fraige et al., 2015) and metabolomic (Ali K. et al., 2011; Dai et al., 2013; Degu et al., 2015) techniques contributed extensively to our understanding of berry growing and ripening process. These studies not only enhanced and supplemented the morphological and physiological descriptions but also promoted the work to molecular level. Exploring the developmental process basing on a single level data results in a partial view of the progress. Several studies described the developmental process by combining transcriptomic and metabolomic profiles (Fortes et al., 2011; Agudelo-Romero et al., 2013; Degu et al., 2014). Considering that the proteome is the active part of the metabolic phenotype, integration and complex statistical correlation network analysis of those data will provide crucial information for the understanding of the metabolic and physiological changes (Weckwerth et al., 2004b; Morgenthal et al., 2005; Wienkoop et al., 2008; Valledor et al., 2013, 2014; Nukarinen et al., 2016; Wang et al., 2016a,c). Nonetheless, systematic analysis of integrated metabolome and proteome profiles of developing grape berries is still less covered. It is also problematic to schematize the metabolic dynamics of developing grape berry by summarizing or comparing those studies due to the coverage limitation of either developmental stages or metabolism branches. For instance, some studies only focus on primary metabolism (Dai et al., 2013) whereas others target flavonoid accumulation during grape berry ripening (Ali M. B. et al., 2011). Zamboni and coworkers integrated the transcriptomic, proteomic and secondary metabolite data of four developmental and three postharvest time points of Corvina grape berry into a complex statistical correlation network analysis for the identification of putative, stage-specific biomarkers (Zamboni et al., 2010). In addition, some studies worked on individual parts of grape berries, such as skin (Negri et al., 2008; Ali M. B. et al., 2011; Degu et al., 2014, 2015; Wu et al., 2014) or berries depleted of seed or peel (Martinez-Esteso et al., 2011; Fang et al., 2013; Fraige et al., 2015). In this study, we harvested samples according to the modified E-L system (Coombe, 1995) from fruit set to ripening at 12 time points. Mass spectrometry based high-throughput platforms were applied for the metabolomic and proteomic analysis of both primary and secondary metabolism dynamics of developing grape berries. Multivariate statistical analysis of the dynamics of metabolites and proteins involved in primary metabolism i.e., glycolysis, tricarboxylic acid (TCA) cycle, amino acid metabolism as well as secondary metabolism i.e., flavonol, flavan-3-ols, anthocyanins and lignin unveiled metabolism interactions during the berry growing period. # MATERIALS AND METHODS #### Sample Collection Berries at 12 developmental stages corresponding to EL 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37.5 (to distinguish with the samples at early EL 37 stage), 38 (Figure 1) were harvested according to the modified Eichhorn-Lorenz system (E-L system) (Eichhorn and Lorenz, 1978; Coombe, 1995) from V. vinifera (Early Campbell) growing in the plant garden of University of Vienna (48◦ 13′ 50.2′′N 16◦ 21′ 28.2′′E) during the 2014 growing year. The plants did not receive any specific training system. Three biological replicates each containing 5 to 10 berries were collected for each developmental phase. The harvested berries were frozen in liquid nitrogen immediately and stored at −80◦C. #### Metabolite and Protein Extraction An integrative extraction of metabolites and proteins was performed according to a universal extraction protocol (Weckwerth et al., 2004b) with some modifications. The grape berries were ground to fine powder in liquid nitrogen using mortar and pestle. 50 to 100 mg of material was extracted with 750µl of extraction solution (methanol: water: formic acid = 70:28:2) and 250µl of hexane. The mixture was homogenized by vigorous vortexing and incubated 30 min on ice. Then the mixture was centrifugated at 20,000 g for 8 min to separate the lipophilic and hydrophilic phases which were subsequently transferred into new tubes, respectively. The extraction procedure was repeated once with the lipophilic and hydrophilic phases pooled together with those from the first extraction, respectively. The extracts were dried under vacuum. Proteins were extracted from the residue pellets according to a previous protocol (Noah et al., 2013). #### Metabolite Measurement, Identification and Quantification The dried hydrophilic phases were re-dissolved in 400 µl of 50% methanol. For the primary metabolite analysis, 25µl of this re-dissolved hydrophilic phase was dried under vacuum and subsequently derivatized according to a modified protocol (Weckwerth et al., 2004b; Mari et al., 2013). Agilent <sup>R</sup> 6890 gas chromatograph coupled to a LECO Pegasus <sup>R</sup> 4D GC × GC-TOF spectrometer was used for the primary metabolite measurement. Instrument parameters were set as described previously (Doerfler et al., 2013). GC separation was performed at a constant flow 1 mL min−<sup>1</sup> helium. Initial oven temperature was set to 70◦C and hold for 1 min, followed by a ramp to 76◦C at 1◦C min−<sup>1</sup> and a second ramp at 6◦C min−<sup>1</sup> to 350◦C hold for 1 min. Transfer line temperature was set to 340◦C and post run temperature to 325◦C for 10 min. The metabolite identification and quantification was performed with LECO Chroma TOF <sup>R</sup> . Retention times (RTs) of the peaks were converted to retention indices (RIs) according to the RTs of spiked alkanes (C12-C40). Metabolites were annotated by comparing their RIs and mass spectra to those of standards in the GMD Golm database (Kopka et al., 2005) with a minimum match factor set to 700. The peak areas of the annotated metabolites corresponding to specific masses were extracted and used for relative quantification. Mixtures of standard compounds were measured under the same conditions at different concentrations to calculate the standard curves for absolute quantification. For the secondary metabolite analysis, 10µl of the redissolved hydrophilic phase was mixed with 2.5µl of reserpine (5 mg l−<sup>1</sup> ) as an internal standard, 10µl of 1.0% formic acid (FA) solution and 77.5µl of water. After centrifugation at 20,000 g for 8 min, 5µl of the supernatant were loaded on Waters ACQUITY UPLC HSS T3 nanoACQUITY Column (particle size 1.8µm, dimension 100µm × 100 mm) via a HTC PAL Autosampler device coupled to an Eksigent nano LC pump and eluted with a non-linear gradient (Mari et al., 2013) at a constant flow rate of 500 nl min−<sup>1</sup> . The LC conditions were 5% B during 0– 3 min, a linear increase from 5 to 20% B during 3–25 min, from 20 to 40% B during 25–40 min and from 40 to 50% B during 40–55 min, finally from 50 to 95% B during 55–63 min followed by 15 min of maintenance with a flow rate of 500 nl min−<sup>1</sup> . Ionization was performed by a nano ESI source (Thermo Scientific, USA) in positive mode with the masses analyzed by a LTQ Orbitrap XLTM mass spectrometer (Thermo, Germany). Each full scan was followed by one MS/MS scan with the most abundant precursor ion fragmented by collision induced dissociation (CID) under 35% of the normalized collision energy during 90 ms activation time. The minimum signal threshold was set to 50,000. Before measurement, the machine was calibrated and standards were measured to check the condition of measurement. We also ensured linearity of the spiked internal standard in different concentrations. The combination of a very low flow rate (500 nL/min) and a gradient that minimized co-elution was chosen to minimize matrix effects. For secondary metabolite identification, accurate precursor masses, sum formula RTs together with mass accuracy were exported from Xcalibur (Thermo Xcalibur 2.2 SP1.48) and compared with the information from literature or standard compounds. The annotation levels were marked according to a standard proposed by the Metabolomics Standards Initiative (Sumner et al., 2007). LCquan (Thermo, v2.6.6.1128) was used for peak extraction and peak area integration. #### Protein Digestion and Analysis Protein concentration was determined by the Bradford method (Bradford, 1976) with a BSA standard curve. 100µg of protein were firstly reduced with dithiothreitol (DTT, 5 mM, 37◦C, 45 min); then alkylated with iodoacetamide (IAA, 10 mM, 23◦C, dark, 60 min) and finally 5 mM of DTT was added (23◦C, dark, 15 min). Endoproteinase LysC and trypsin were applied for digestion based on a previous protocol (Hoehenwarter et al., 2008). After digestion, samples were desalted with C18-SPEC-96 well plate (15 mg, Agilent, USA) according to the manufacturer's instruction. The eluted peptides were dried under vacuum and dissolved in 500µl of start gradient solution (4.5% acetonitrile, 0.1% FA). 1µg of the digested protein was loaded on an Ascentis Peptide ES-C18 column (particle size 2.7µm, dimension 15 cm × 100µm, Sigma-Aldrich, USA) and eluted with a 90 min linear gradient from 5 to 40% of mobile phase B (90% acetonitrile, 0.1% FA; phase A, 0.1% FA in water) at a constant flow rate of 400 nl min−<sup>1</sup> . The same ESI-LTQ-Obitrap equipment used for metabolite analysis was applied for peptide measurement. Each full scan was followed by 10 MS/MS scans in which the 10 most abundant ions were selected and fragmented by CID with 35% of the normalized collision energy during a 30 ms activation time. Minimum signal threshold was set to 10,000. The obtained raw files containing peptide information were searched against a grape fasta file including 65,448 protein sequences from UniProt with the SEQUEST algorithm in Proteome Discoverer (v 1.3, Thermo Scientific). Searching parameters were set as below: maximum two missed cleavage sites, acetylation for N-terminal modification, oxidation of methionine for dynamic modification and carbamidomethylation of cysteine for static modification were allowed. Mass tolerance for precursors was set to 10 ppm and for fragment masses to 0.8 Da. False discovery rate (FDR) was set to 0.01. Protein candidates were defined by at least two peptides with high confidence. The obtained raw files and sequence information of the identified proteins were submitted to the public repository ProteomeXchange (Vizcaino et al., 2014) with the dataset identifier PXD003769 (http://www.proteomexchange. org/) as well as to the PROMEX database (http://promex.pph. univie.ac.at/promex/). Normalized spectral abundance factors (NSAFs) were calculated (Zybailov et al., 2006) for relative quantification. The protein candidates that are present in all the three biological replicates of at least one stage were considered for the statistical analysis. For the functional analysis, the identified protein sequences were blasted against a protein database of Arabidopsis thaliana (from PLAZA with 27,407 protein sequences) and Theobroma cacao (from PLAZA with 44,404 protein sequences) with the BLASTP function in NCBI (v 2.2.31, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). A cacao mapping file from GoMapMan ("tca\_Phytozome9.1\_ transcript\_2015-01-09\_mapping.xlsx," http://www.gomapman. org/export/current/mapman) was applied for the functional analysis. #### Statistical Analysis The obtained metabolite data were normalized to fresh weight and dodecane (C12 alkane, GC-MS data) or total ion intensity (LC-MS data). Analysis of variance (ANOVA) and k-means clustering were performed within Matlab <sup>R</sup> (V8.4.0 R2014b; http://www.mathworks.com). The significant levels of the candidates were presented with lower case letters according to results of the Duncan's test (Duncan, 1955). K-means clustering analysis was repeated 100 times and finally the result with minimal total distance was selected. Principal component analysis (PCA), hierarchical clustering analysis and Granger causality analysis were performed with COVAIN under Matlab environment (Sun and Weckwerth, 2012). Granger causality analysis was performed on all the identified metabolites and proteins as well as the clusters after k-means clustering analysis with their time lag was set to 1, 2, 3, respectively. The correlations with p-values less than 0.05 were recorded. The network of Granger result was visualized in Cytoscape (http:// www.cytoscape.org/). The Venn diagram was drawn with Venny 2.0.2 (http://bioinfogp.cnb.csic.es/tools/venny/). # RESULTS ### Metabolomic Profiles of Developing Grape Berry The GC-TOF-MS platform allowed the annotation of 87 candidates including sugars, amino acids, organic acids together with simple amine and phenolic compounds, according to their RIs and mass spectra. Additionally, 49 flavonoids were annotated from LC-Orbitrap-MS data according to the accurate precursor masses, sum formula and their fragmentation patterns (Table 1). The detailed information (RI and RTs, quantification masses or (Continued) #### TABLE 1 \| Continued Api, Apigenin; Caff, Caffeoyl; Cou, Coumaryl; Cy, Cyanidin; DHQ, Dihydroquercetin (taxifolin); Dp, Delphinidin; Glc, Glucose; Glu, Glucuronide; Kae, Kaempferol; Lar, Laricitrin; Lut, Luteolin; MDHQ, methyldihydroquercetin; Mv, Malvidin; Myr, Myricetin; Pg, Pelargonidin; Pn, Peonidin; Pt, Petunidin; Que, Quercetin; Rha, Rhamnoside; Tri, Tricetin. MS2 fragments and the integrated peak area, one way ANOVA results) of the annotated candidates is listed in Table S1. The dynamic patterns of the annotated metabolites were visualized by a hierarchical bi-clustering color map (Figure 2A). Sugars (including sugar alcohols, sugar acids), amino acids, organic acids and flavonoids were further shown in Figures 2B–E. #### Sugars The main sugars in grape berries are fructose, glucose and sucrose. In the present study, fructose constantly accumulated during development with significant increases before veraison and during ripening (Figure 2B). Glucose also significantly accumulated around veraison but declined afterwards (Figure 2B). The content of sucrose fluctuated during grape development (Figure 2B) with four inflection points at EL 30, 32, 34, and 36 respectively. Other sugars, sugar alcohols and sugar acids either decreased during development (ribose, xylose, myo-inositol, arabinose, rhamnose, galactaric acid) or showed the highest level at EL 32 (threonic acid, galactonic acid, gluconic acid, erythrirol) (Figure 2B). #### Amino Acids Amino acids showed distinct dynamics during grape berry development (Figure 2C). Arginine and asparagine were the most abundant amino acids in young berries, alanine and glutamine in mature berries (Table S2). Lysine, tyrosine, arginine, ornithine and phenylalanine increased significantly from EL 27 to EL 29, however, decreased dramatically until the end of lag phase (EL 34) and remained at a relatively low level during the second sigmoidal growth period (Figure 2C). Asparagine was in high abundance at the first two developing stages followed by a dramatic decline from EL 29 to EL 30 then stayed in low level until the end. Other amino acids fluctuated during grape berry developing and all showed a turning point at EL 34 which is the end of the lag phase and the beginning of the veraison (Figure 2C). #### Organic Acids The predominant organic acids detected in grape berry were malic acid, tartaric acid and citric acid which increased before veraison (EL 35) and decreased afterwards (Figure 2D). Other organic acids showed similar changing pattern except pyruvic acid, gallic acid and caffeic acid which were highest in the young berries and then decreased throughout the developmental process (Figure 2D). #### Flavonoids Grape and its products are rich in polyphenolics. These secondary metabolites, especially flavonoids, play multiple roles in grape and attract more and more attentions due to their health benefits (Anastasiadi et al., 2010; Kim et al., 2013; Zhang et al., 2013). During grape berry development, the detected flavonoids presented two distinct changing patterns (Figure 2E). All the anthocyanins accumulated during ripening whereas most of the candidates in the other subfamilies like proanthocyanins, flavan-3-ol, flavonol, flavanonol and their glycosides were abundant in young berries and decreased during the time course of development (Figure 2E). The synthesis of anthocyanins splits into three branches, i.e., the monohydroxylated (pelargonidin, Pg), the dihydroxylated (cyanidin, Cy), and the trihydroxylated (delphinidin, Dp) branch. Cy and peonidin (Pn) glycosides which belong to the dihydroxylated branch were detected from stage EL 37 or even EL 36 whereas derivatives of the other two branches started to appear one stage later. Furthermore, in mature berries (EL 38), the relative abundance of Cy- and Dp- derivatives were higher than the corresponding derivatives of other aglycones (Figure S1). For instance, Cy-Cou-diGlc (30) and Dp-Cou-diGlc (25) were more abundant than petunidin- (Pt, 29), malvidin- (Mv, 32) and Pg- (33) coumaroyl-diglucoside; Cy-Glc (2) and Dp-Glc (8) were in higher level than glucoside of other aglycones (Figure S1). #### Protein Profiles of Developing Grape Berry In total, 1313 proteins were identified from all the samples (for sequences information see in Table S3). 848 candidates prevalent in all replicates of at least one stage were used for further statistical analysis. NSAFs and the ANOVA analysis result can be found in Table S4. The functions of all the protein candidates were annotated by blasting against protein sequences of A. thaliana and T. cacao. Blast results were summarized in Table S4. The matching with the T. cacao database yielded a higher amount of hits with a better blast quality and was therefore used for functional analysis. Subsequently, the identified protein candidates were assigned to corresponding functional bins according to the cacao mapping file from GoMapMan (Table S4). Hierarchical bi-clustering analysis was applied to visualize the dynamic proteome profiles of developing grape berry (Figure S2). Samples of 12 developing stages were clustered into 3 groups indicated with color blue, red and green (Figure S2). Samples from stage EL 27, 29, 30 and 31 were assigned to group 1; EL 32, 33, 34, 35 group 2 and EL 36, 37, 37.5, 38 group 3. The Venn diagram (Figure 3A) shows 394 proteins were common to all groups and 157, 43 and 80 protein candidates are specific to group 1, 2, and 3, respectively. The functional distribution of these group specific proteins was summarized with pie charts (Figure 3A). There were 18 amino acid metabolism related proteins detected accounting for 5.83% of all the group 1 specific proteins whereas only 1 and 2 proteins were accounting for 1.61 and 1.43% respectively of group 2 and 3 (Figure 3A) indicating active amino acid metabolism at phase I on proteome level. There were 8 transport related proteins accounting for 2.59% of group 1 specific proteins and 5 accounting 3.57% of group 3 specific proteins whereas there was no transport related protein exclusive to group 2 (Figure 3A). Another notable point is that more proteins associated with secondary metabolism existed exclusively in group 1 (12, 3.88%) and 3 (12, 8.57%) than in group 2 (2, 3.23%) (Figure 3A) indicating the synthesis of secondary metabolites was more active in the beginning and the end of berry developmental stages than in the middle. The annotated protein candidates were assigned to 50 functional bins (Table S4). The majority of functional categories include candidates involved in protein synthesis (11.41%), protein degradation (7.11%), RNA regulation of transcription (6.09%), signaling (4.38%), and abiotic stress response (4.30%) (Figure 3B). The changing patterns of the proteins in these functional categories were summarized into four groups by hierarchical clustering analysis (Figure 3C) with their summarized changing patterns shown on the right side. Proteins in 30 out of 50 functional bins were in lowest content around veraison (Figure 3C in red and green). In contrast, proteins involved in 7 functional groups were with highest abundance just before veraison (EL 33) (Figure 3C in blue). The functional bins in purple group (Figure 3C) involve proteins constantly accumulating during grape berry development. Proteins related to abiotic and biotic stresses (4.30 and 2.03%, respectively) showed high abundance at early developmental stages or/and during ripening (Table S3, Figure 3C in red) indicating high resistance ability of grape berry against environmental and developmental stresses during these developmental phases. A larger amount of oxidative stress responsive proteins in young, green berries as well as increasing expression of pathogen responsive proteins after veraison have been previously reported (Giribaldi et al., 2007). Additionally, another study reported a parallel transcript profile of stress/pathogens responsive gene strongly expressed in ripening berries (Davies and Robinson, 2000). Photosynthesis (6.1%) is another major functional category including candidates involved in light reaction (2.97%), photorespiration (0.86%), and calvin cycle (2.27%) (Figure 3B). Proteins involved in light reaction were in higher abundance in the earliest stage and around veraison (Figure 3C in blue) whereas those involved in photorespiration and calvin cycle were more abundant in young green berries. The levels of proteins in all of these three subgroups declined after veraison. The decrease in abundance of photosynthesis related proteins throughout grape berry development especially after veraison was consistent with previous proteomic studies (Martinez-Esteso et al., 2011; Fraige et al., 2015) and the physiological situation (Pandey and Farmahan, 1977) of developing grape berries. Proteins associated with lipid metabolism were observed with high frequency and (3.1%, Figure 3B) showed increasing expression after veraison (Figure 3C). Proteins associated with secondary metabolism showed distinct changing patterns. Those involved in phenylpropanoid synthesis were in high abundance in young berries and then decreased during development (Figure 3C in red) whereas those related with later steps of flavonoid and isoprenoid biosynthesis were strongly accumulated after veraison (Figure 3C in purple). Proteins associated with synthesis of N and S containing metabolites were highly expressed around veraison. The distinct arrangement of protein expression reflected the metabolic adjustment during grape berry development. #### Metabolome and Proteome Data Integration #### Multivariate Statistical Analyses Reveal the Trajectory of Grape Berry Development The PCA plot (Figure 4A) of the integrated metabolomics and proteomic dataset revealed a continuous trajectory during grape berry development. The separation of various developmental stages indicated a distinction of metabolism on metabolite and protein levels. The first principal component (PC 1) accounting for 47.44% (Figure 4A) of the total variance characterized metabolic and proteomic specificities of grape berries at different developmental stages. Candidates with high absolute loading scores (Table S5) included metabolites, especially flavonoids, caffeic acid, gallic acid, lysine, asparagine, arginine and methionine together with protein candidates involved in development, lipid metabolism, cell wall construction, TCA cycle and protein degradation which accounted for most of the separation among developmental stages. Further, k-means clustering analysis was applied to group candidates according to their changing patterns. Figure S3 presents the 12 clusters with a bold red line indicating the averaged pattern of all the candidates in each cluster. Cluster 2 and 6 with 86 and 124 candidates, respectively, present candidates with higher abundance at early developmental stages (EL 27 to 31) (Figure 4B, Figure S3). Candidates in these two clusters include amino acids (methionine, phenylalanine, asparagine, ornithine, arginine, lysine, tyrosine), organic acids (caffeic acid, pyruvic acid, gallic acid), sugars and sugar alcohols (arabinose, rhamnose, myo-inositol), (dihydro)flavonol derivatives (epicatechin, 3, 20, 36, 49, 12, 31, 38, 39, 43, 45, compound number see in Table 1) and proteins involved in amino acid metabolism (Table S6). The candidates in cluster between clusters that resulted from k-means clustering analysis with time lag set as 1, 2, and 3, respectively. 10 and 9 are with highest abundance at EL 32 and veraison (EL 34 and 35), respectively. The most abundant organic acids, i.e., malic acid and citric acid, together with some proteins involved in photosynthesis in cluster 5 were in a relatively higher level during the lag phase and veraison than during the two sigmoidal growth phases. In contrast, those candidates in cluster 3 and 4 showed opposite dynamic patterns including organic acids (shikimic acid, ascorbic acid, fumaric acid, maleic acid), amino acids (valine, leucine, proline isoleucine) and proteins involved in stress response (Figure S3, Table S6). All the annotated anthocyanins and four flavonol derivatives (5, 10, 21, 26, compound number see in Table 1), together with a high abundance of proteins involved in secondary metabolism and lipid metabolism in cluster 1, 8, and 12 mainly accumulated after veraison (EL 36 to 38) (Figure S3, Table S6). The candidates in cluster 7 show a continuous elevation in their abundance whereas those in cluster 11 decreased (Figure S3, Table S6). #### Granger Causation Analysis of an Integrated Metabolomic/Proteomic Network during Grape Berry Development Correlation network analysis has been widely applied in omics studies to investigate molecular correlations and connections in metabolism (Steuer et al., 2003; Weckwerth, 2003; Weckwerth et al., 2004a; Sun and Weckwerth, 2012). Those biomolecules with a higher node degree have more connections with other molecules and are therefore regarded as essential connection points in a metabolic network (Weckwerth, 2003; Weckwerth et al., 2004a). The connection degree of nodes might also vary in grape berries at different developmental stages, different cultivars (Cuadros-Inostroza et al., 2016) or under distinct growth conditions (Hochberg et al., 2013; Savoi et al., 2016). Thus, correlation network analysis is an appropriate method for searching important biomolecules involved in specific metabolism processes. Time-lagged correlation analysis by Granger causation represents an advanced level of correlation network analysis (Doerfler et al., 2013). Granger causality analysis was initially introduced by Granger (1969) to predict events based on time series data and time-lagged correlations in economics. It was also applied to some biological studies to interpret directed and nonlinear correlations between metabolites, transcripts and proteins (Walther et al., 2010; Doerfler et al., 2013; Valledor et al., 2014). To further extend the understanding of the dynamic correlations of all the identified metabolites and proteins during the developmental time course, Granger causality analysis was applied to all the candidates and clusters discussed above. The results indicated significant metabolite-metabolite, metabolite-protein and protein-protein correlations. Figure 4C shows the directed correlation of phenylalanine and phloretin (p-value = 0.02187) which indicated a strong effect of phenylalanine concentration on phloretin synthesis. Not only precursors but also enzymes showed significant correlation to their product synthesis. One example is the accumulation of anthocyanin synthase (ANS, A2ICC9) prior to anthocyanins (Figure 4D, Table S7). Figure 4E presents a significant granger correlation (p-value = 0.00538) between protein E0CSB6 (malate dehydrogenase) and D7T300 (ATPase) indicating the close relationship between TCA cycle and ATP production. Other time series correlations with pvalues less than 0.05 were summarized in Table S7. The Granger causation network (Figure S4) includes 674 nodes (Table S7) with 21 neighbors in average. Many amino acids (ornithine, arginine, phenylalanine, lysine, tyrosine, asparagine), organic acids (acid\_like2, shikimic acid) and their metabolism related proteins (Table S7) show highest node degrees revealing them as potential biochemical hub during grape berry development. The application of Granger analysis to the 12 clusters obtained from the k-means clustering analysis revealed significant time lagged correlations between these clusters. Significant directed connections among these clusters are shown in Figures 4F–H with the time lag set as 1, 2, and 3, respectively (one, two or three time points shifted). The Granger causality correlations from clusters 2 to 6, 6 to 10, 10 to 9, 9 to 12, and 12 to 8 (Figure 4G indicated with red arrows) were visualized as a line chart (Figure 4B) clearly showing their dynamic shift over the developmental time course, especially a significant correlation between cluster 9 and 12. Such combined Granger causality analysis with clustering analysis indicated a general metabolic shift from the metabolism of amino acids, sugars and some flavonoids to organic acid accumulation and finally to lipid and anthocyanin synthesis during grape berry development. PCA and k-means clustering analysis presented the systemic dynamics of the metabolites and proteins during grape berry development. To understand the metabolism progress of developing grape berry in a biochemical context, we mapped the sugars, amino acids, organic acids, flavonoids and the related proteins on their corresponding biosynthetic pathways (Figure 5). The integration of dynamics of metabolites as well as proteins involved in both primary and secondary metabolisms presents metabolic checkpoints during grape berry development. This is further discussed below. # DISCUSSION # Primary Metabolism Dynamics Primary metabolism plays an essential role in grape berry development. The products from primary metabolism pathways are not only crucial for grape survival but also endow grape berry specific characters which are further decisive of its market value. #### Sugar Metabolism Sugars, especially fructose, glucose and sucrose determine the sweetness of grapes, moreover, the alcohol concentration of wine. In grape berries, sucrose is mainly imported via phloem from source organs. Subsequently, the imported sucrose is either hydrolyzed to glucose and fructose by invertase or converted to glycolysis substrates via sucrose synthase (Susy) and UDP-glucose pyrophosphorylase (UGPase). The fluctuation of sucrose content during grape berry development might be caused by a disproportionate ratio of the import to the consumption. Furthermore, synthesis of sucrose from malate via the gluconeogenic pathway (Ruffner et al., 1975; Dai et al., 2013) might also contribute to the fluctuation of sucrose concentration, especially after veraison. The accumulation of glucose and fructose during grape berry development was reported previously (Wu et al., 2011; Dai et al., 2013). In the present study, fructose accumulated throughout the developmental process whereas glucose concentration did not continue to rise after veraison. Similar glucose dynamics were also observed in some table grape varieties i.e., "Thompson Seedless," "Crimson Seedless," and "Red Globe" (Muñoz-Robredo et al., 2011). In contrast to these findings, both glucose and fructose concentration constantly increased during grape berry development in some grape varieties and cultivars (Wu et al., 2011; Dai et al., 2013). The discrepancy in glucose accumulation patterns could be explained by the differences in the ripening process among varieties. The distinct expression pattern of invertase and Susy might explain the unequal accumulation of glucose and fructose. The decline FIGURE 5 \| Visualization of metabolite and protein dynamics on their biosynthetic pathways. Metabolites are written in black letters with blue line charts indicating their changing patterns whereas proteins are written in red letters with red line charts. Relative abundance of metabolites and proteins were averaged over three biological replicates. Bars represent standard errors. Susy, sucrose synthase; UGPase, UDP-glucose pyrophosphorylase; PFP, pyrophosphate-fructose 6-phosphate 1-phosphotransferase; FBPase, fructose 1, 6-bisphosphatase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PK, pyruvate kinase; PDC, pyruvate (Continued) #### FIGURE 5 \| Continued dehydrogenase complex; IDH, isocitrate dehydrogenase; OGDC, oxoglutarate dehydrogenase complex; SCS, succinyl coenzyme A synthetase; SDH, succinate dehydrogenase; MDH, malate dehydrogenase; AspAT, aspartate aminotransferase; AS, asparagine synthetase; ASADH, aspartate-semialdehyde dehydrogenase; MetH, methionine synthase; MAT, methionine adenosyltransferase; PHGDH, phosphoglycerate dehydrogenase; PSAT, Phosphoserine transaminase; SHMT, serine hydroxymethyltransferase; OASTL, O-acetylserine (thiol)-lyase; GLDH, Glutamate dehydrogenase; GS, Glutamine synthetase; ASS, argininosuccinate synthase; ALAT, alanine aminotransferase; AGT, alanine-glyoxylate transaminase; CS, chorismate synthase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4-coumarate-CoA ligase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl-alcohol dehydrogenase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′ -hydroxylase; F3′5 ′H, flavanoid 3′ ,5′ -hydroxylase; FLS, flavonol synthase; DFR, dihydroflavanol 4-reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; 3-GT, anthocyanidin 3-O-glucosyltransferase. in abundance of invertase since EL 31 caused a decrease in the production of glucose and fructose whereas the increasing expression of Susy ensured the continuous accumulation of fructose. #### Glycolysis The substances generated from sugar metabolism are subsequently incorporated into glycolysis. Metabolism along this process generates energy (ATP), reducing equivalents (NADH) as well as intermediates for amino acid biosynthesis, lipids and secondary metabolite production. The abundance of most glycolytic enzymes increased through the development and ripening process (Figure 5). Phosphoglycerate kinase (PGK) was the only glycolytic enzyme, which declined in abundance (Figure 5). The concentration of pyrophosphate-fructose 6 phosphate 1-phosphotransferase (PFP), enolase and pyruvate kinase (PK) in the cytosol strongly increased after veraison (Figure 5). Phosphoglycerate mutase (PGM) was increased during the first sigmoidal growing phase (EL 27 to EL 32) and maintained a relatively constant level afterward. The increase in abundance of glycolytic proteins was consistent with some former reports that studied the proteomic profile of grape skins and berry tissue without seeds (Negri et al., 2008; Kambiranda et al., 2014). However, some studies reported a decrease in abundance of glycolytic enzymes (Davies and Robinson, 2000; Giribaldi et al., 2007; Martinez-Esteso et al., 2011) or glycolytic intermediates (Dai et al., 2013) during berry ripening. Variety and differences in growth conditions might explain these different observations. Additionally, isoforms of enzymes may play different roles at a particular developmental stage (Chaturvedi et al., 2013; Ischebeck et al., 2014; Wang et al., 2016a,b), thus displaying varying dynamics during development. For instance, Fraige et al reported three isoforms of UDPase, of which two candidates decreased in abundance after veraison whereas one increased (Fraige et al., 2015). #### TCA Cycle The tricarboxylic acid cycle (TCA cycle) generates energy, reducing power and carbon skeletons, which makes it a central hub in metabolism. The Pyruvate dehydrogenase complex (PDC) converts pyruvate to acetyl-CoA which serves as fuel to the TCA cycle. During the grape berry development, PDC was in high abundance during the first growing phase (EL 27-32), followed by a significant decline at the lag phase (EL 32-34) and with a subsequent increase (Figure 5). Similar to PDC, isocitrate dehydrogenase (IDH), oxoglutarate dehydrogenase complex (OGDC), and succinyl coenzyme A synthetase (SCS) were in high abundance during the sigmoidal growth phases whereas in low abundance during the lag phase (Figure 5). The high abundance of these enzymes in the young and ripening berries was consistent with the great demand for energy and building blocks at these two phases. Aconitase and succinate dehydrogenase (SDH) were strongly expressed after veraison. In a former report, a sharp expression of aconitase was observed in ripening grape skin (Negri et al., 2008). Fumarase is the only enzyme whose expression gradually declined throughout the berry development (Figure 5). Malate dehydrogenase (MDH), catalyzing a reversible reaction between oxaloacetate and malate, was concentrated at a lower level at phase I whereas it showed a progressive increase in expression during ripening which was in agreement with previous reports (Martinez-Esteso et al., 2011; Kambiranda et al., 2014). The transcript levels of MDH and malic enzyme were reported to increase during grape berry ripening which might contribute to the decline of malate concentration after veraison (Deluc et al., 2007). The contents of the intermediates, citrate, succinate, fumarate and malate gradually increased in early stages of development, up to a peak in concentration at EL 31 (fumarate and succinate), EL 33 (malate) or around veraison (citrate) with a subsequent decline. Similar dynamic patterns were observed in developing Cabernet Sauvignon berries with a peak in accumulation of most TCA cycle intermediates at veraison (Dai et al., 2013). The accumulation of these organic acids before veraison was parallel to the high abundance of enzymes at phase I. However, the gradually increasing expression of TCA cycle associated enzymes was accompanied by a decrease in the intermediates during grape berry ripening (phase III). The discrepancy between the increase in the abundance of the enzymes and the decrease in the content of the intermediates during grape berry ripening indicates a high metabolic flux through this pathway with an efficient incorporation of the intermediates in the synthesis of amino acids, lipids and secondary metabolites. In grape, organic acids are responsible for the titratable acidity which is an index for fruit quality. High amounts of organic acids endow young berries a sour taste for defense against herbivores. The organic acids in mature berries are essential for wine production as they protect the fermentation process from bacterial contamination. In wine they are responsible for the sour part of the taste. They are also essential for the color of wine by contributing to the stabilization of anthocyanins (Clemente and Galli, 2011). The changing patterns of those dominant organic acids i.e., malic acid, tartaric acid and citric acid, were consistent with previous reports (Deluc et al., 2007; Ali K. et al., 2011; Muñoz-Robredo et al., 2011; Fraige et al., 2015). #### Amino Acids Metabolism Amino acids are major transportable nitrogenous compounds in grape. In source organs, intermediates from glycolysis and TCA cycle can be utilized as precursors for the synthesis of amino acids. For instance, phosphoenolpyruvate is the precursor of aromatic amino acids that derive from the shikimate pathway; α-ketoglutarate and oxaloacetate are the precursors of glutamate and aspartate family amino acids, respectively. Asparagine and glutamine were the major amino acids in young and mature grape berries respectively. Both of them carry an extra amide group making them efficient nitrogen-carriers. They play important roles in the nitrogen assimilation, transportation and storage in plants. Asparagine and glutamine can be converted to Asp and Glu to serve as precursors for biosynthesis of many other amino acids, e.g., proline, arginine (from glutamate); methionine, threonine and lysine (from aspartate). Several enzymes involved in amino acid metabolism were detected and mapped on Figure 5. Aspartate aminotransferase (AspAT) catalyzes the reversible transfer of an amino group between aspartate and glutamate thus plays an important role in nitrogen distribution. In concordance with a previous report (Martinez-Esteso et al., 2011), AspAT abundancy gradually decreased during grape berry development. Methionine synthase (MetH), catalyzing the synthesis of methionine, is another essential amino acid of the aspartate family. The abundance of MetH increased until veraison, then stayed relatively constant during ripening in the present study. However, it decreased during green developmental stages and increased during ripening in the study of Martinez-Esteso et al. (2011). Glutamine synthetase (GS) is another crucial enzyme involved in nitrogen assimilation. GS, which catalyzes the condensation of glutamate and ammonia to generate glutamine, gradually declined in abundance from EL 27 to EL 36 and slightly increased afterwards. The transcript level of GS was shown to be significantly higher in phase I in a previous study (Deluc et al., 2007). Marinez-Esteso et al reported a decline in the level of GS before veraison (Martinez-Esteso et al., 2011) which is consistent with our result. However, the changing trend of GS after veraison was absent in their study. #### Secondary Metabolism Dynamics Grape is rich in polyphenolic compounds that derive from the phenylpropanoid pathway. These bioactive secondary metabolites play essential roles in protecting grape berry against abiotic stresses such as UV radiation (Pontin et al., 2010), high light and high temperature stresses (Ayenew et al., 2015), drought stress (Król et al., 2014) as well as biotic stresses (Gutha et al., 2010; Wallis and Chen, 2012). In addition, they also contribute to organoleptic features of grape berry and wine (Schmidtke et al., 2010; Gutiérrez-Capitán et al., 2014). #### Lignin Simple phenolic compounds that are synthesized from phenylalanine can be polymerized to lignin which is an essential component of the cell wall. In the present study, three enzymes, i.e., cinnamoyl-CoA reductase (CCR), cinnamylalcohol dehydrogenase (CAD) and ferulate 5-hydroxylase (F5H, Q9M4H8) involved in lignin synthesis were annotated. CCR and CAD, which catalyze the last two steps of monolignol synthesis not only impact lignification but also plant development. Absence of CCR and CAD resulted in dwarfism and sterility in Arabidopsis (Thevenin et al., 2011). One protein candidate was annotated as CCR (A5AXM6) and detected after veraison. Four candidates were annotated as CAD. The averaged expression pattern of CAD showed high levels in both young and ripening berries and low level around veraison (Figure 5). Aharoni et al. (2002) reported a comparable pattern of the transcription level of CAD in developing strawberries which are also non-climacteric fruits and undergo color turning phases. In their study, CAD expression level was high in green strawberries followed by a decreasing during the white and turning stages and finally increased again in red strawberries (Aharoni et al., 2002). #### Flavonoids Flavonoids are another class of secondary metabolites derived from the phenylpropanoid pathway and share common precursors with lignin. The detected flavonols and flavan-3-ols showed distinct changing patterns with anthocyanins. Parallel phenomena were observed before in other varieties, i.e., Cabernet Sauvigon (Ali M. B. et al., 2011; Degu et al., 2014), Shiraz (Degu et al., 2014) and Norton (V. aestivalis) (Ali M. B. et al., 2011) and supposed to be caused by the competition of precursors between anthocyanins and other subfamily members of flavonoids (Ali K. et al., 2011; Degu et al., 2014). The anthocyanin profile obtained from the present data was consistent with that of "Concord" (Liang et al., 2011) and "Pink Sultana" (Boss et al., 1996) in which Cy and Dp derivatives were the dominant anthocyanins. In contrast, Mv derivatives were the most abundant anthocyanins in the other varieties in these two studies (Boss et al., 1996; Liang et al., 2011) and other reports (an overview of all varieties is provided in Table S8) (Mazza et al., 1999; He et al., 2010; Papini et al., 2010; Ali M. B. et al., 2011; Degu et al., 2014, 2015). Noticeably, Early Campbell is a hybrid of Vitis vinifera and Vitis labrusca by crossing Moore Early with (Belvidere × Muscat of Hamburg). Both Moore Early and Belvidere are seedlings of Concord. The fruit taste and disease resistance of Early Campbell are similar to Concord (Robinson et al., 2012). The similar anthocyanin profile of Early Campbell with that of Concord is probably also due to its genetic background. The annotated proteins that were involved in the flavonoid pathway include chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavanol 4-reductase (DFR) and anthocyanidin synthase (ANS). CHS catalyzes the condensation of p-coumaroyl-CoA with malonyl-CoA to generate chalcone which is further isomerized to naringenin by CHI. Two proteins, annotated as CHS (A2ICC5, G4XGW2), were detected after veraison and increased in abundance during ripening (Table S4). In a previous study, three copies of Chss were found in the grape genome. The mRNA levels of Chs2 and Chs3 significantly coincided with anthocyanin and Chs1 and Chs2 with flavonol biosynthesis (Jeong et al., 2008). The expression of A2ICC5 and G4XGW2 were in accordance with anthocyanin accumulation indicating the involvement of these two CHSs in the coordination of anthocyanin synthesis. The expression level of CHI (A5ANT9) gradually increased before veraison then underwent a sharp decline at veraison (EL 35) with a subsequent recovery to the level before veraison. F3H catalyzes the hydroxylation of flavanones at 3-position to form dihydroflavonols. Both protein candidates annotated as F3H (A2ICC8, A2ICC8) were detected from EL 32 and showed increasing levels during grape berry development. The transcription of F3hs appeared to be induced for the biosynthesis of flavonols and anthocyanins (Jeong et al., 2008). Dihydroflavonols are further converted to either flavonols via flavonol synthase (FLS) or to leucoanthocyanidins via DFR. The competition between FLS and DFR influences the contents of flavonols and anthocyanins (Tian et al., 2015) which further affects the color (Lou et al., 2014) and the abilities of plants to cope with stress (Hua et al., 2013; Wang et al., 2013). DFR is a crucial enzyme in the flavonoid pathway involved in the synthesis of anthocyanins, proanthocyanins and tannins (Moyano et al., 1998; Zhang et al., 2008; Hua et al., 2013; Wang et al., 2016c). In the present study, two protein candidates (A5BGJ0, A5BIY8) were annotated as DFR. Their combined expression pattern indicated a progressive increase in abundance of DFR before veraison and a slight decrease afterwards (Figure 5). ANS catalyzing the conversion of colorless leucoanthocyanins to colored anthocyanidins was detected in ripening berries. The abundance of ANS (A2ICC9) significantly increased after veraison (Figure 5) in accordance with the anthocyanin accumulation. MYBrelated transcription factors (TFs) are involved in regulation of flavonoid synthesis (Czemmel et al., 2009). It was further reported that VvMYB5b was highly expressed after veraison and the anthocyanin synthesis was enhanced in transgenic tobacco due to ectopic expression of VvMYB5b (Deluc et al., 2008). In our study, four proteins i.e., A5ADL7, A5AHA8, F6GTT4, F6I581 were annotated as "MYB-related transcription factor". Their summarized content was lowest around veraison whereas the highest level was observed before veraison and during ripening (Table S4 sheet 2). This pattern was neither directly correlated to the amount of anthocyanins in the developing grape berries nor to the protein levels of ANS (A2ICC9). Further we detected 7 bZIP family members (A5B427, A5BZF5, D7SUP9, D7TNE5, F6GTA6, F6GUN1, F6HBQ3). bZIP family member are thought to be involved in the regulation of flavonoid biosynthesis (Malacarne et al., 2016). In a recent study Loyola et al. propose that HY5 and HYH are involved in UV-Bdependent flavonol accumulation in grapevine (Loyola et al., 2016). The concentrations of the bZIP proteins in our study showed an increase toward veraison and a decrease afterwards. Because there is often not a direct dependency between transcriptional and translational/posttranslational control (Nukarinen et al., 2016) it is difficult to compare gene expression levels from other studies with protein levels from our study. Furthermore, the analysis of TFs requires in most cases a specific enrichment step before proteomic analysis. Future investigations will focus more on the discussed transcription factors and their control on developmental processes and flavonoid biosynthesis. #### Stilbenes Stilbenes are also derived from the phenylpropanoid pathway and were reported to be enriched in grape. However, we did not detect any stilbene or related enzymes in this study. This might be due to different growth and stress conditions or a different genetic background of the variety we studied. Two publications reported stilbene content in the peel of Early Campbell at veraison stage (Islam et al., 2014; Ahn et al., 2015). They also found that hairy vetch and ryegrass extracts and red and blue LED light induced stilbene accumulation as well as the expression of genes involved in stilbene synthesis. We used the whole berry as the study object which is different from using isolated peel. Furthermore there is evidence that support a competition between the synthesis of stilbenes and flavonoids. One evidence is the negative correlation between resveratrol and anthocyanin accumulation in 5 Vitis species at different developmental species (Jeandet et al., 1995). The other evidence is observed in transgenic strawberries. Hanhineva et al transformed a stilbene synthase gene to strawberries (35S:NS-Vitis3 line). While the STS gene was highly expressed in the transgenic strawberry line, CHS expression was down regulated (Hanhineva et al., 2009). These are two examples indicating the competition relationship between flavonoid and stilbene synthesis pathway. It is thus of interest to compare the flavonoid and stilbene content of this variety under different growth conditions and with other grape varieties that produce stilbenes to investigate the competition of stilbene and flavonoid biosynthesis. # CONCLUSION In summary, the analysis of grape berry development from fruit set to mature fruit by mass spectrometry based platforms revealed intimate correlations between the metabolome and the proteome at the interface of primary and secondary metabolism. The broad coverage of developmental stages included in the present study enabled a dense correlation network analysis of these dynamic processes covering central carbon metabolism such as sugar metabolism, glycolysis, TCA cycle, amino acid metabolism as well as secondary metabolism, especially the flavonoid pathway. Multivariate statistical analysis such as PCA, clustering analysis and Granger causality analysis provides a convenient data mining approach for the interpretation of the integrated metabolome and proteome dataset and revealed the systemic associations between metabolites and proteins during grape berry development. The application of Granger causality analysis is helpful in revealing time-lagged correlations between metabolites and proteins which is especially important for understanding the molecular timeshifts during developmental processes of grape berries. Together with other studies this work provides a reference point for future investigations of grape berry development in a variety of different genotypes. #### AUTHOR CONTRIBUTIONS LW and WW conceived and designed the experiments. LW performed the experiments. LW, XS, JW, and WW analyzed the data. WW provided the reagents, materials and analytical tools. LW wrote the manuscript. WW and JW revised the manuscript. All the authors approved the final manuscript. #### FUNDING LW was supported by a Ph.D. scholarship provided by China Scholarship Council (CSC) (Grant number: 201206220134). #### ACKNOWLEDGMENTS We would like to thank the gardeners for their great maintenance of the grapevine. We would like to thank all the MoSys members for fruitful discussions. We would like to thank Reinhard Turetschek for suggestions in protein blast analysis. #### REFERENCES #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 01066/full#supplementary-material Table S1 \| Metabolite information. Table S2 \| Absolute quantification of 29 metabolite candidates. Table S3 \| Protein sequencing information. Table S4 \| NSAFs, ANOVA, BLAST results of protein candidates. Table S5 \| Loading scores. Table S6 \| K-means clustering analysis (12 clusters). Table S7 \| Granger causality analysis of the integrated metabolomic and proteomic data sets. Table S8 \| Anthocyanin profiles of grape berries of different cultivars. Figure S1 \| Relative abundance of anthocyanins in mature berries (EL 38). Figure S2 \| Hierachical bi-clustering analysis of protein candidates. Figure S3 \| K-means clustering analysis of the integrated dataset. Figure S4 \| Granger causality based network. sports differing in berry skin colour. Aust. J. Grape Wine R. 2, 163–170. doi: 10.1111/j.1755-0238.1996.tb00104.x Conde, C., Silva, P., Fontes, N., Dias, A. C. P., Tavares, R. M., Sousa, M. J., et al. (2007). Biochemical changes throughout grape berry development and fruit and wine quality. Food 1, 1–22. pathway in grapevine and mediate flavonol accumulation in response to the environment. J. Exp. Bot. 67, 5429–5445. doi: 10.1093/jxb/erw307 cultivars (Vitis vinifera) in which anthocyanin synthesis is sunlight-dependent or -independent. PLoS ONE 9:e105959. doi: 10.1371/journal.pone.0105959 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 Wang, Sun, Weiszmann and Weckwerth. 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. # Drawing Links from Transcriptome to Metabolites: The Evolution of Aroma in the Ripening Berry of Moscato Bianco (Vitis vinifera L.) Laura Costantini <sup>1</sup> \, Christian D. Kappel <sup>2</sup> , Massimiliano Trenti <sup>1</sup> , Juri Battilana<sup>1</sup> , Francesco Emanuelli <sup>1</sup> , Maddalena Sordo<sup>1</sup> , Marco Moretto<sup>3</sup> , Céline Camps <sup>2</sup> , Roberto Larcher <sup>4</sup> , Serge Delrot <sup>2</sup> and Maria S. Grando1, 5 <sup>1</sup> Grapevine Genetics and Breeding Unit, Genomics and Biology of Fruit Crop Department, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy, <sup>2</sup> UMR Ecophysiology and Grape Functional Genomics, Institut des Sciences de la Vigne et du Vin, University of Bordeaux, Villenave d'Ornon, France, <sup>3</sup> Computational Biology Platform, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy, <sup>4</sup> Experiment and Technological Services Department, Technology Transfer Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy, <sup>5</sup> Center Agriculture Food Environment, University of Trento, San Michele all'Adige, Italy #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics, Spain #### Reviewed by: Philip Richard Young, Institute for Wine Biotechnology, South Africa Patricio Hinrichsen, Instituto de Investigaciones Agropecuarias, Chile > \Correspondence: Laura Costantini [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 10 February 2017 Accepted: 25 April 2017 Published: 16 May 2017 #### Citation: Costantini L, Kappel CD, Trenti M, Battilana J, Emanuelli F, Sordo M, Moretto M, Camps C, Larcher R, Delrot S and Grando MS (2017) Drawing Links from Transcriptome to Metabolites: The Evolution of Aroma in the Ripening Berry of Moscato Bianco (Vitis vinifera L.). Front. Plant Sci. 8:780. doi: 10.3389/fpls.2017.00780 Monoterpenes confer typical floral notes to "Muscat" grapevine varieties and, to a lesser extent, to other aromatic non-Muscat varieties. Previous studies have led to the identification and functional characterization of some enzymes and genes in this pathway. However, the underlying genetic map is still far from being complete. For example, the specific steps of monoterpene metabolism and its regulation are largely unknown. With the aim of identifying new candidates for the missing links, we applied an integrative functional genomics approach based on the targeted metabolic and genome-wide transcript profiling of Moscato Bianco ripening berries. In particular, gas chromatography-mass spectrometry analysis of free and bound terpenoid compounds was combined with microarray analysis in the skins of berries collected at five developmental stages from pre-veraison to over-ripening. Differentially expressed metabolites and probes were identified in the pairwise comparison between time points by using the early stage as a reference. Metabolic and transcriptomic data were integrated through pairwise correlation and clustering approaches to discover genes linked with particular metabolites or groups of metabolites. These candidate transcripts were further checked for co-localization with quantitative trait loci (QTLs) affecting aromatic compounds. Our findings provide insights into the biological networks of grapevine secondary metabolism, both at the catalytic and regulatory levels. Examples include a nudix hydrolase as component of a terpene synthase-independent pathway for monoterpene biosynthesis, genes potentially involved in monoterpene metabolism (cytochrome P450 hydroxylases, epoxide hydrolases, glucosyltransferases), transport (vesicle-associated proteins, ABCG transporters, glutathione S-transferases, amino acid permeases), and transcriptional control (transcription factors of the ERF, MYB and NAC families, intermediates in light- and circadian cycle-mediated regulation with supporting evidence from the literature and additional regulatory genes with a previously unreported association to monoterpene accumulation). Keywords: grapevine, Muscat, monoterpene, development, berry skin, metabolic and transcript profiling, integration, candidate gene # INTRODUCTION A great deal of the consumer interest in wine derives from its aroma characteristics. The major aroma-impact compounds in grape and wine are terpenoids (monoterpenes, sesquiterpenes, and in a wider acception also C13-norisoprenoids), phenylpropanoids/benzenoids, fatty acid derivatives, sulfur compounds, and methoxypyrazines (Dunlevy et al., 2009; Ebeler and Thorngate, 2009; Panighel and Flamini, 2014; Robinson et al., 2014; Black et al., 2015). The typical floral and citrus attributes of Muscat varieties are primarily determined by a combination of linalool, geraniol and nerol (Ribéreau-Gayon et al., 2000). The same monoterpenes contribute to the varietal aroma of Riesling in association with the linalool oxides, hydroxy-linalool, α-terpineol, citronellol, terpendiol I and hydroxy-trienol (Rapp, 1998). Likewise rose oxide, which is highly correlated with Muscat score in grapes (Ruiz-García et al., 2014), is also a potent odorant in Scheurebe and Gewürztraminer (Guth, 1997; Ong and Acree, 1999; Luan et al., 2005). The terpene biosynthetic pathway is generally well known (Dudareva et al., 2013), even though a number of alternative non-canonical reactions may occur (Sun et al., 2016). Of the two systems responsible for the production of plant isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the primarily cytosolic mevalonic acid (MVA) and the plastidial methylerythritol phosphate (MEP) pathway, the latter has been suggested as the dominant route for monoterpene biosynthesis in grape berries (Luan and Wüst, 2002). Several lines of evidence (Battilana et al., 2009, 2011; Duchêne et al., 2009; Emanuelli et al., 2010; Martin et al., 2012; Wen et al., 2015) support the existence of at least two rate-limiting enzymes in the grapevine MEP pathway, namely the first (1-deoxy-Dxylulose 5-phosphate synthase, VvDXS1) and the last (4-hydroxy-3-methylbut-2-enyl diphosphate reductase, VvHDR). Both IPP and DMAPP are substrates for short-chain prenyltransferases, which produce prenyl diphosphate precursors for the large family of terpene synthases (TPSs). To date around 40 fulllength VvTPSs out of 53–89 predicted functional enzymes have been biochemically characterized (Martin et al., 2010) and some major players in grape Muscat aroma have been identified, like the α-terpineol synthase VvTer, the linalool synthase Lis, the linalool/nerolidol synthase VvPNLinNer1 and the geraniol synthase VvPNGer (Ebang-Oke et al., 2003; Martin and Bohlmann, 2004; Martin et al., 2012; Matarese et al., 2013; Zhu et al., 2014; Wen et al., 2015). Once a terpenoid alcohol skeleton has been produced, extensive modifications determine the final monoterpene composition of grapes and wines (Ribéreau-Gayon et al., 1975; Williams et al., 1989; Luan et al., 2004, 2005, 2006a,b; Mathieu et al., 2009). These secondary transformations are (at least in part) catalyzed by enzymes (Luan et al., 2006a; D'Onofrio et al., 2016) that in most cases have not been identified. The only exceptions are the three grape monoterpenol β-D-glucosyltransferases VvGT7, VvGT14 and VvGT15 and the cytochrome P450 CYP76F14 (Bönisch et al., 2014a,b; Ilc et al., 2017). The main reason for this gap is that such enzymes belong to large families with broad substrate tolerance and overlapping activities (Schwab, 2003; Nelson et al., 2008; Schwab and Wüst, 2015). A better knowledge of the missing enzymes might allow us to manipulate the formation of grape aroma compounds. For example, limiting the reactions responsible for the depletion of key odorants (e.g., through the selection of genotypes with low monoterpene glycosyltransferase or oxygenase activities in breeding programs) could be an alternative approach for the improvement of grape/wine flavor (Bönisch et al., 2014a; Hjelmeland and Ebeler, 2015). The grapevine terpenoid pathway is intricately regulated by endogenous and environmental factors that enable spatially and temporally controlled metabolite production (Ebeler and Thorngate, 2009; Robinson et al., 2014). In other plant species a network of transcription factors (TFs) is involved in the regulation of this pathway, including members of the AP2, AP2/ERF, bZIP, MYB, MYC, NAC, WRKY, and YABBY families (De Geyter et al., 2012; Patra et al., 2013; Nieuwenhuizen et al., 2015; Wang et al., 2016). A tight regulation of terpene biosynthesis is additionally exerted at the post-transcriptional level involving both structural enzymes and transcription factors (Vom Endt et al., 2002; Hemmerlin, 2013; Rodríguez-Concepción and Boronat, 2015), as observed also in Vitis vinifera (Bönisch et al., 2014a; Matarese et al., 2014). A number of transcription factors that might control terpene synthesis have been recently predicted in grapevine through gene co-expression network analysis (Wen et al., 2015), though none of them has been yet demonstrated to regulate the expression of relevant terpene pathway genes. Similarly, the reasons of the differential accumulation of the main monoterpenes in grape berry tissues across development (Günata et al., 1985; Wilson et al., 1986; Park et al., 1991; Luan and Wüst, 2002), which is reflected in the identification of specific QTLs for linalool and geraniol/nerol (Doligez et al., 2006; Battilana et al., 2009), are still unknown. This work aims at a better understanding of aroma determination in grapevine and at the identification of candidate genes for further functional analysis. To this purpose, we integrated gas chromatography/mass spectrometrybased quantitative analysis of selected metabolites with microarray-based transcriptomic analysis in Moscato Bianco (Vitis vinifera L.) ripening berries. According to the observed associations between metabolite and transcript profiles, we report several genes that may control the accumulation of free and glycosidically bound monoterpenes and additional aroma-related compounds. # MATERIALS AND METHODS #### Plant Material For Metabolic and Microarray Analyses Berries of the cultivar Moscato Bianco (Vitis vinifera L.) were collected from pre-veraison to over-ripening in 2005, 2006, and 2007 (Figure 1A and Table 1). At each sampling date, ten bunches were taken from ten adult plants out of the ∼ 250 grown on Kober 5BB rootstocks in the experimental fields of FEM (Fondazione Edmund Mach, San Michele all'Adige, Italy). Care was taken to sample from different vines and positions within each vine. In the lab, berries were pooled in order FIGURE 1 \| Acidity, sugars (A) and monoterpene content (B–F) of the Moscato Bianco samples collected during berry development in 2005, 2006, and 2007. The five stages assayed by microarray analysis in 2006 are highlighted with red in (A). Exemplar monoterpenes with a major contribution to the total free monoterpene profile (B) during PV, R1, and R2 are shown in (D–F), respectively. A single biological replicate was considered at each stage in each season; bars in (D–F) correspond to the standard error calculated from six technical replicates, as described in Supplementary Data1: Method S1. The metabolites were quantified by using solid SPE-HRGC-MS and referring to the internal standard 1-heptanol. The lines connecting data points were smoothed through the specific option provided by Excel. E-L stage, growth stage according to the modified Eichhorn-Lorenz scheme (Coombe, 1995); PV, pre-veraison; V, veraison; R1, ripening (till technological maturity or stage E-L 38); R2, over-ripening (after technological maturity). The decimal E-L stages were arbitrarily assigned by the authors of the present study to facilitate the alignment of the sampling dates from the three different seasons. The five stages used for microarray analysis are in boldface. E-L stage, growth stage according to the modified Eichhorn-Lorenz scheme (Coombe, 1995). The decimal E-L stages were arbitrarily assigned by the authors of the present study to facilitate the alignment of sampling dates from three different seasons (Figure 1). The weeks from veraison were established with a maximum tolerance of 2 days from the exact date. to minimize environmental effects and then divided into two (2005 and 2007) or three (2006) batches. Berries from the first batch were homogenized to juice (80 mL) and analyzed for titratable acidity and soluble solids content by FT-IR (Fourier Transform InfraRed) spectroscopy with a FOSS instrument (FOSS NIRSystems, Oatley, Australia). Berries from the second batch were stored at −80◦C till metabolic analysis. Berries from the third batch were hand-peeled, the skins were immediately frozen in liquid nitrogen and stored at −80◦C pending RNA extraction. #### Metabolic Analysis Thirty-two aroma-active compounds were quantified in their free and glycosidically bound form by using solid phase extraction (SPE) and high-resolution gas chromatography-mass spectrometry (HRGC-MS; Supplementary Data1: Method S1 and Supplementary Table S1) in the growing seasons 2005, 2006, and 2007. Network analysis for 2006 metabolic data included pairwise correlation, hierarchical clustering with bootstrapping (Pvclust with 10,000 resamplings, see Suzuki and Shimodaira, 2006) and principal component analysis (PCA) and was applied to different data sets (free and glycosidically bound metabolites, absolute quantities and differentials, 5 and 13 time points). #### Microarray Analysis Based on monoterpene accumulation during berry development in 2006 (Figure 2), five time points were chosen along this season (Figure 1 and Table 1). Total RNA was extracted from grape skins using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, St. Louis, Missouri, USA). RNA quantity and quality were evaluated with a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, Ontario, Canada). Microarray experiments were carried out with a 70-mer oligoarray containing all 14,562 probes from the Array-Ready Oligo SetTM (AROS) for the Grape (Vitis vinifera) Genome version 1.0 (Operon Biotechnologies, Huntsville, Alabama, USA). At the time this platform represented a good compromise between genome coverage, cost and computational effort required for data analysis. RNA from points 2 to 5 was hybridized competitively with RNA from point 1 (pre-veraison), following the dye-swap experiment design (Churchill, 2002). A total of sixteen slides were used (four comparisons: 2 vs. 1, 3 vs. 1, 4 vs. 1, 5 vs. 1; two biological and two technical replicates). The biological and technical replicates corresponded to two subgroups from the unique pool of berries (third batch) and to the dye swaps, respectively. Details for probe synthesis, FIGURE 2 \| Evolution of monoterpenoids in their free (solid red line) and glycosidically bound (dashed blue line) form during Moscato Bianco berry ripening in 2006. A single biological replicate was considered at each stage; bars correspond to the standard error calculated from six technical replicates, as described in Supplementary Data1: Method S1 (technical replication is not available for 7-hydroxy-nerol, 7-hydroxy-citronellol, 4-terpineol, rose oxide I and II). The metabolites were quantified by using solid SPE-HRGC-MS and referring to the internal standard 1-heptanol. The lines connecting data points were smoothed through the specific option provided by Excel. hybridization and scanning are described in Supplementary Data1: Method S2. Spot intensities were quantified with the software MAIA 2.75 (Novikov and Barillot, 2007). After excluding poor quality spots due to bad spotting (e.g., spots with irregular shapes or highly unequal intensity distributions), median intensity gene expression data without background subtraction were normalized by a global lowess method followed by a print-tip median method with a modified version of the Goulphar script version 1.1.2 (Lemoine et al., 2006). Differentially expressed probes (DEPs) with a false discovery rate (FDR) < 1% and a cutoff of 2-fold change (FC) were identified with the R/Bioconductor Limma package using linear models (Smyth, 2004) and taking into account biological and technical replicates by doing a twofactor analysis. The earliest sample was used as the reference to whom all the other samples were compared. A multiple testing correction (Benjamini and Hochberg, 1995) was applied to adjust the FDR. The full raw expression dataset is available at the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE76834. #### Probe Functional Annotation The 70-mer probes spotted on the Grape AROS V1.0 array represent 14,562 transcripts from The Institute for Genomic Research (TIGR) Grape Gene Index (VvGI), release 3 (August 13, 2003). The corresponding annotation is based upon a match between each oligo and the gene set of the 12X version of the grape genome at CRIBI (http://genomes.cribi.unipd.it/grape/) and is publicly available at GEO (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GPL15453. Since this annotation provides every oligo with a text description but doesn't associate it to any gene prediction identifier, we independently achieved this information by blastN alignment against the grape gene sets at CRIBI (http://genomes.cribi.unipd.it/grape/, 12X version of the genome, V1 gene prediction, annotation from Grimplet et al., 2012) and IASMA (Velasco et al., 2007), as fully detailed in Supplementary Table S2. For the alignment against the CRIBI gene set, the following parameters were used: sequence identity ≥90%, minimum alignment length of 95%, maximum number of mismatches of 5 and maximum number of gaps of 5. The aligned 70-mers were found to correspond to 7,162 and 8,260 unique gene predictions at CRIBI and IASMA, respectively. The 14,562 probes were also grouped into main functional categories according to the Mapman BIN structure (Rotter et al., 2009; Supplementary Table S2). #### Microarray Validation via Real-Time PCR Real-time quantitative reverse transcription-PCR (qRT-PCR) was used to validate the microarray results. Since RNA from berries collected in 2006 was no more available, new samples (with three biological replicates from pooled berries) as closest as possible to those analyzed in 2006 were obtained in 2016 by adopting the same sampling procedure and the same protocol for RNA extraction. Primers for the amplification of unique PCR products from 70 to 250 bp were designed on 15 Vitis vinifera gene predictions perfectly matching with the microarray 70-mers by using Primer3 (Untergasser et al., 2012), as reported in Supplementary Table S3. Details for the amplification reaction and expression analysis are described in Supplementary Data1: Method S3. The relationship between microarray and qRT-PCR data was established through Pearson correlation. #### Discovery of a Link between Transcriptome and Metabolome in Aroma Development Integration of 2006 Transcriptomic and Metabolic Data Different approaches were tested in order to discover transcripts linked with the accumulation of one or more metabolites. In a first identification step of candidates, the most stable expression changes were preferred to the biggest ones, hence the microarray probes with adjusted p-values < 0.05 in all comparisons were considered, irrespective of their fold change (4,450). Working at probe level instead of gene level was chosen for two main reasons: (1) different probes supposedly matching to the same gene (especially long genes) often show different expression values, which might be an indicator of alternative transcription and (2) the sequence specificity to CRIBI 12X gene predictions is not optimal for a number of probes spotted on the AROS array (this is especially true for probes related to secondary metabolism); for a detailed assessment of probe specificity, Moscato Bianco genome and transcriptome assembly would be required, which is out of the scope of this work. #### Pairwise correlation Pearson pairwise correlation was calculated between transcripts and metabolites across all the time points (log2-transformed differentials in the pairwise comparisons 2 vs. 1, 3 vs. 1, 4 vs. 1, and 5 vs. 1). With the goal of identifying aroma regulatory genes we also tested a two-step strategy, which was based on (1) search for candidate metabolism and transport genes by direct correlation with metabolites and (2) expression pairwise correlations between these enzyme/transporter-coding genes and any regulatory gene within the microarray. For this aim, Pearson correlations were computed both between differential gene expression ratios (n = 4) and microarray channel intensities (n = 32, when considering technical replicates separately). #### Correlation biclustering Based on the assumption that a gene might regulate the accumulation of a metabolite only at specific stages during ripening, correlation biclustering between transcripts and metabolites was achieved with QUBIC (Li et al., 2009) (log2 transformed data, Pearson correlation). Compared to the traditional clustering methods, biclustering algorithms discover local co-expression patterns (groups of genes/metabolites that show similar patterns under a specific subset of the experimental conditions) (Madeira and Oliveira, 2004). We manually inspected our biclustering data only in a few exemplar cases. #### Soft clustering Soft clustering of the metabolite and transcript differentials was performed by using the R/Bioconductor Mfuzz package (Kumar and Futschik, 2007) with the default value 1.25 for the fuzzy parameter m. A membership value in the range of 0–1 was assigned to each metabolite and probe. Soft clustering offers several advantages with respect to hard clustering; in particular, it has been suggested to be more suitable for time course microarray data in which expression patterns are often not well separated (Futschik and Carlisle, 2005; Kumar and Futschik, 2007). The biological significance of the clusters was analyzed by enrichment analysis of the MapMan functional categories assigned to the probes in each cluster. Specifically, Chi square and Fisher statistical tests were employed to search for significant differences (p-value < 0.05) between the observed number of probes within each MapMan functional category per cluster and the expected number of probes in that category based on the overall AROS genome array expression distribution. #### Selection of candidate genes From the whole set of transcripts with a potential association to monoterpenes (based on their correlation with metabolites and membership to soft clusters/biclusters harboring metabolites) we selected a subset of genes with significant expression changes and/or supporting evidence from the literature, like a relevant function in other plant species, co-localization with QTLs for monoterpene content and coexpression with genes involved in the terpene pathway. In particular, the QTL co-localization was stated when the V1 gene predictions fell into the 1-LOD confidence intervals of the QTLs for linalool, geraniol and nerol reported by Doligez et al. (2006) and Battilana et al. (2009) based on the analysis of different segregating progenies in 2– 3 seasons (depending on the progeny). The genomic region corresponding to each QTL confidence interval was determined from the physical position of the two neighboring markers, while the V1 gene prediction physical position was retrieved from Grimplet et al. (2012). #### Integration of Transcriptomic and Metabolic Data Over Multiple Seasons to Verify a Subset of Candidate Genes For the candidate genes assessed by both microarray and real-time PCR analyses (in 2006 and 2016, respectively), the association between expression and metabolic profiles was further tested by employing a general monoterpene quantification that considers the three seasons (2005, 2006, and 2007) as replicates. To this purpose, the average concentration among these seasons was computed for each metabolite at each developmental stage. Pearson and Spearman correlations were calculated between the transcriptional and metabolic data expressed as log<sup>2</sup> fold changes at the stages 2-5 (E-L 34.5, 36, 38, and 39) with respect to the first stage (E-L 31). #### RESULTS AND DISCUSSION Our study gives an example of the systems biology approach. Systems biology has been successfully applied to the discovery of regulatory and biosynthetic genes involved in the control of metabolite production (Yuan et al., 2008; Liberman et al., 2012), including examples from grape (Zamboni et al., 2010; Fortes et al., 2011; Agudelo-Romero et al., 2013; Costantini et al., 2015; Malacarne et al., 2015; Suzuki et al., 2015; Wen et al., 2015; Savoi et al., 2016). #### Metabolic Analysis The present work provides a temporal profiling of aromatic compounds in the Moscato Bianco ripening berry. The protocol used for the chemical analysis was optimized for molecules belonging to the monoterpenoid class, however it allowed the simultaneous quantification of additional metabolites. In particular, the content of 21 monoterpenoids, 3 C13 norisoprenoids, 5 phenylpropanoids/benzenoids and 3 C<sup>6</sup> aliphatic compounds was quantified from pre-veraison to overripening in 2005, 2006, and 2007. For several compounds a coherent accumulation trend was observed in the different years (Figure 1 and Supplementary Figure S1A). The most significant correlation between seasons was observed for free linalool, nerol, α-terpineol, hydroxy-diendiol I + hydroxy-trienol, hydroxydiendiol II, hexanol, cis-3-hexen-1-ol, bound linalool, geraniol, nerol, trans-furan linalool oxide and benzyl alcohol. Other metabolites appeared instead to be more sensitive to seasonal effects, like rainfall and temperature (Supplementary Figure S1B). Hereafter, we will refer to 2006, which is the year assayed by microarray analysis. The most abundant metabolites were monoterpenes (hydroxy-diendiol I, trans-geranic acid, linalool, geraniol and nerol), with concentrations higher than 600 µg/kg of berries (Figure 2 and Supplementary Figure S2). The majority of monoterpenoids reached the highest amount in their glycosidically bound form. The main exceptions are represented by high oxidation state monoterpenes, like the two pyran linalool oxides, the two diendiols and rose oxide I. A clear prevalence of the free form was also observed for the C<sup>6</sup> aliphatic compounds, while the most abundant C13-norisoprenoids and phenylpropanoids/benzenoids were glycosidically bound, in agreement with previous analyses (Sánchez Palomo et al., 2006; D'Onofrio et al., 2016). The quantity of many metabolites was significantly (at least 2-fold) altered during ripening. The compounds that changed most with respect to pre-veraison were linalool, geraniol, nerol, cis/trans-8-hydroxy-linalool, hydroxy-diendiol I and II in both forms; cis-furan linalool oxide, trans-geranic acid, 7 hydroxy-geraniol, 7-hydroxy-citronellol, hydroxy-trienol in their free form; trans-furan linalool oxide, 3-oxo-α-ionol, methyl salicylate, hexanol and cis-3-hexen-1-ol in their bound form (Supplementary Figures S3A,D, Supplementary Table S4). The pattern of accumulation along berry development varied with the metabolite (Figure 2 and Supplementary Figure S2). The concentration of the three compounds mainly contributing to Muscat aroma (linalool, geraniol, and nerol) was from low to moderate before veraison (August 17 or stage E-L 34.5 in this work) and then increased during ripening. Free linalool reached its maximum on September 13 (technological maturity or stage E-L 38) and decreased during over-ripening. A similar behavior was observed in 2005 and 2007, even though the peak corresponded to slightly earlier stages (Figure 1E), proving that technological and aroma ripening might not occur at the same time (Vilanova et al., 2012). Otherwise, free geraniol and nerol as well as the three bound forms showed a steady increase in their content during the sampling period. These results confirm previous findings (Günata et al., 1985; Ebang-Oke et al., 2003; Piazzolla et al., 2016). Several additional patterns were observed. For example, the four linalool oxides could be detected at berry onset; their concentration reached a minimum between July 31 (stage E-L 31.5 in this work) and August 8 (stage E-L 33) and then increased in at least one of the two forms to peak on September 13 (stage E-L 38) in their free form. While the glycosidically bound forms of the two diendiols showed a similar pattern of accumulation, free hydroxy-diendiol I and hydroxytrienol were highly concentrated before veraison and decreased over the course of berry ripening, with a trend opposite to that of free hydroxy-diendiol II (Figures 1, 2). The high content of free hydroxy-diendiol I and hydroxy-trienol at berry onset, when free linalool was not yet produced, may indicate that their accumulation is regulated independently from that of their precursor. In the attempt of simplification, metabolite network analysis was performed on a total of 52 (26 free and 26 glycosidically bound) compounds. Metabolite grouping was obtained through hierarchical clustering and principal component analysis by using different metabolic data sets (Figure 3 and Supplementary Figures S3, S4). It is clearly evident that most monoterpenes are tightly correlated, which is indicative of their common metabolic origin and in agreement with previous findings (Ilc et al., 2016b). In particular, when considering the absolute amount of free metabolites at 13 time points, three main clusters (AU > 95%) could be identified: (1) cis-pyran linalool oxide (OxD), transpyran linalool oxide (OxC), trans-furan linalool oxide (OxA) and cis-furan linalool oxide (OxB); (2) geraniol, nerol, cis-8 hydroxy-linalool, benzyl alcohol, 2-phenylethanol, trans-geranic acid, citronellol, hydroxy-diendiol II, trans-8-hydroxy-linalool, linalool, hexanol, trans-3-hexen-1-ol, rose oxide I, rose oxide II and α-terpineol; (3) 4-terpineol and hydroxy-diendiol I + hydroxy-trienol. Additionally, within the second cluster a clear separation could be noticed between linalool on one side, geraniol and nerol on the other side (Figure 3A and Supplementary Figure S4A). Oppositely, when considering the absolute amount of bound metabolites at 13 time points a single significant cluster was obtained, which included most of the analyzed compounds. It can be easily noticed that bound cis-furan linalool oxide (OxB) has a peculiar behavior with respect to the other three linalool oxides (Figure 3B and Supplementary Figure S4C). #### Microarray Analysis Gene expression in Moscato Bianco berry skin at stages 2–5 was compared to stage 1 (pre-veraison). The two biological replicates assayed at each stage were confirmed to perfectly cluster together (Supplementary Figure S5). The total number of differentially expressed probes (DEPs) in at least one comparison was 2,228, which corresponds to 15% of the chip probes (Supplementary Table S5). As expected, the highest number of DEPs was observed in stage 5 vs. stage 1 (616 up-regulated and 1,132 down-regulated probes), whereas the lowest number was recorded in stage 2 vs. stage 1 (452 up-regulated and 506 down-regulated probes). A number of DEPs were common among comparisons (21, 19, and 28% of common DEPs among 2, 3, and 4 comparisons, respectively), whereas 32% of the DEPs were regulated at only one time point (data calculated from Supplementary Table S5). ### Microarray Validation via Real-Time PCR Specific primers were designed for 15 candidate genes and the change in their expression during berry development was analyzed in skin tissues by qRT-PCR to validate the microarray dataset (Figure 4). A strong relationship was found between the microarray and qPCR fold changes in the expression levels of the 15 genes (overall Pearson correlation coefficient r = 0.84, with individual values ranging from 0.47 to 1), indicating the reliability of the whole transcriptome assay (Figure 4 and Supplementary Table S3). #### Discovery of a Link between Transcriptome and Metabolome in Aroma Development Potential links between transcripts and metabolites were established based on correlation and clustering approaches, though they do not necessarily imply causation. To this purpose, 4,450 probes with adjusted p-values < 0.05 in all comparisons were considered, which included 1,906 out of the 2,228 DEPs with a cut-off of 2 fold-change and adjusted p-value < 0.01. #### Integration of 2006 Transcriptomic and Metabolic Data #### Pairwise correlation Significant (at the 0.05 or 0.01 level) Pearson correlations could be established only in the absence of Benjamini and Hochberg (1995) correction for multiple testing (Supplementary Table S6). Consequently, this result was employed as criterium in the following candidate gene selection only in combination with additional supporting evidence. In the two-step strategy, positive pairwise expression correlations were discovered between 33 enzyme/transporter-coding genes correlated to metabolites and several regulatory genes within the microarray (Supplementary Table S6). #### Correlation biclustering Correlation clustering between transcripts and metabolites resulted in the identification of 419 biclusters, that are groups of probes with a common behavior toward a group of metabolites. The clustered probes and metabolites were found to belong to a number of biclusters ranging from 1 to 10 and from 3 to 182, respectively (Supplementary Table S7). #### Soft clustering Based on their expression profile across stages 1–5, the selected 4,450 probes and 52 metabolites were clustered into nine distinct Mfuzz groups (Figure 5 and Supplementary Table S8). The distribution of probes per cluster within each MapMan functional category is shown in Supplementary Figure S6 and the enriched categories within each cluster are indicated in Figure 5. Probes annotated with the Mapman functional category "Secondary metabolism" were not found to be significantly over-represented in any cluster. Free geraniol and nerol were attributed to a distinct cluster (cluster 6) with respect to free linalool (cluster 9), which reflects their Pvclust clustering (Figure 3A and Supplementary Figure S4A). This separation is mainly due to the decrease of free linalool, but not geraniol and nerol, from technological maturity onwards (Figure 2). The highly similar accumulation trend of geraniol and nerol likely reflects a common chemical origin (nerol is a geometrical isomer of geraniol), while their relationship with linalool is less clear. Oppositely, the bound forms of the three monoterpenoids accumulated to a similar extent (cluster 4 in Figure 5), suggesting dynamic changes in the distribution and concentration of these compounds. #### Selection of Candidate Genes Several genes with a potential association to aroma-related compounds were identified from the probes correlated and clustered with those metabolites (Supplementary Table S9, Supplementary Discussion in Supplementary Data1 and Supplementary Table S10). In particular, the contrasting behavior of free linalool and free geraniol/nerol encouraged us to search for genes specifically related to one or the other profile. The existence of linalool-specific metabolic pathways is even more intriguing if we consider that the prevalence of the linalool class on the geraniol one clearly distinguishes Moscato Bianco from other aromatic varieties (D'Onofrio et al., 2016). From this broad gene set, the most promising candidates for monoterpene biosynthesis and its regulation were further selected (Table 2) based on supporting evidence from the literature, e.g., a relevant role for the homolog gene in other plant species, the co-localization with QTLs for monoterpene content (with a special attention to the linalool-specific QTLs on chromosomes 2 and 10, which were also detected in the Moscato Bianco genetic background by Battilana et al., 2009), or the coexpression from public transcriptomic databases with genes involved in the metabolic pahways under study, which may indicate functional association according to the "guilt-byassociation" principle. Hereafter, we discuss the most interesting findings from the present work; obvioulsy, we can't exclude that additional genes not included in this microarray platform may participate in monoterpene biosynthesis, as well as we can't know a priori whether our findings will be reproduced in other Muscat varieties with a genetic background different from Moscato Bianco. #### Monoterpene skeleton biosynthesis Early terpenoid pathway genes. The role of VvDXS isoforms in the development of aroma was previously investigated by realtime PCR on the same samples of Moscato Bianco analyzed here (Battilana, 2009), for which reason we did not repeat the analysis. In that study a significant up-regulation of VvDXS1 was found to precede the peak of linalool, geraniol and nerol resulting in a positive correlation between VvDXS1 expression profile and monoterpenoid accumulation. On the Grape AROS V1.0 array no probe could be found for VvDXS1, whereas four probes corresponding to other DXS isoforms (VIT\_04s0008g04970 and VIT\_00s0218g00110) were not differentially expressed during Moscato Bianco berry ripening. Several pieces of evidence from different plant species suggest that flux control in the MEP pathway does not converge on a single rate-limiting enzyme, such as DXS, but may involve other enzymes like DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) and HDR (Vranová et al., 2012; Hemmerlin, 2013). The lack of significant modulation and the decreasing correlation between the fold changes (log2) in the expression levels of the 15 genes obtained by microarray and qRT-PCR analyses is shown in the last chart. trend during berry ripening observed for VvDXR in our study (VIT\_17s0000g08390 in Figure 4) do not support a regulatory role, in agreement with Rodríguez-Concepción et al. (2001) and Mendoza-Poudereux et al. (2014). Oppositely, the expression of VvHDR (VIT\_03s0063g02030 in Figure 4) is consistent with the veraison-initiated accumulation of monoterpenes, as reported by Martin et al. (2012) and Wen et al. (2015) (Table 2 and Supplementary Table S9). Middle and late terpenoid pathway genes. In other plant species GPPS works as a heterodimeric complex; in particular, the levels of GPPS small subunit, but not GPPS large subunit, might play a key role in regulating monoterpene biosynthesis (Tholl et al., 2004). Consistently, the AROS probes for GPPS large subunit genes (VIT\_04s0023g01210 and VIT\_18s0001g12000) were neither differentially expressed during Moscato Bianco berry ripening nor correlated to any monoterpene. No probe could be identified for the GPPS small subunit. Only three probes for terpene synthases are present on the Grape AROS V.1 array, which are not specific to any single gene prediction. One of them, showing the best match to the sesquiterpene synthases VIT\_18s0001g04280 and VIT\_18s0001g04530, was up-regulated during Moscato Bianco berry ripening (Table 2 and Supplementary Table S9). It is worth noting that the same genes were reported to correlate with linalool and α-terpineol (Savoi et al., 2016). An interesting candidate gene for the biosynthesis of monoterpenes is a nudix hydrolase (VIT\_10s0003g00880), whose expression increases along berry development (Figure 4). The corresponding probe belongs to cluster 4, which also harbors several monoterpenes (Table 2, Supplementary Tables S8, S9). Recently, a rose nudix hydrolase was reported to convert geranyl diphosphate to geranyl monophosphate, which in turn is hydrolyzed to geraniol by a phosphatase activity (Magnard et al., 2015). This alternative and completely new terpene synthase-independent route for monoterpene production might play a role also in other plants, including grapevine. #### Secondary monoterpene transformations Extensive oxidative monoterpene metabolism has been reported in grapes and wine, with a percentage of linalool oxygenation ranging from 52 to 97% (Ilc et al., 2016b). The main linalool oxidation products are trans/cis-8-hydroxy-linalool (by hydroxylation), trans/cis pyranoid/furanoid linalool oxides and polyhydroxylated derivatives or polyols like the odorless hydroxy-diendiol I and II (by epoxidation and hydrolysis). Similarly, C-8 oxygenated geraniol and citronellol derivatives can be formed through hydroxylation, whereas the oxidation to geranial and neral (altogether named citral) is supposedly mediated by alcohol dehydrogenases. Geranic acid is another oxidation product of geraniol. Rose oxide is generated from citronellol by allylic hydroxylation and acid-catalyzed cyclization. Citronellol in turn arises from the reduction of geraniol and nerol (hydrogenation). Members of the cytochrome P450 (CYP) 71 and 76 families were recently shown to metabolize linalool in Arabidopsis thaliana (Ginglinger et al., 2013; Höfer et al., 2014; Boachon et al., 2015). Interestingly, the CYP76 gene family has encountered an evident expansion in the grape genome (Nelson et al., 2008). In order to identify genes potentially implicated in grape monoterpenoid metabolism we looked (Continued) Frontiers in Plant Science \| www.frontiersin.org TABLE 2 \| Summary of the most significant candidate genes for monoterpene biosynthesis. TABLE 2 \| Continued (Continued) TABLE 2 \| Continued Supplementary genes involved in the terpene pahway), profile consistency between microarray and qRT-PCR analyses (the genes assayed by both techniques are marked with an asterisk). The symbols # and § indicate references for grapevine and other plant species, respectively. For details, including the correspondence between microarray probes and V1 gene predictions, see Supplementary Table S9. in VTCdb database (http://vtcdb.adelaide.edu.au/Home.aspx) for CYP genes coexpressed with linalool synthases, as in Ginglinger et al. (2013). This information was then added to our transcriptomic and metabolic integrated datasets. On this base, we propose some genes (VIT\_15s0048g01490, VIT\_15s0048g01590, VIT\_18s0001g13790, VIT\_00s0389g00030, VIT\_00s0389g00040) and eventually additional ones as potential CYPs involved in linalool metabolism (Table 2 and Supplementary Table S9). Most of these candidates have been never reported elsewhere, and thus deserve further attention. Conversely, VIT\_15s0048g01490 and VIT\_18s0001g13790 were recently characterized by Ilc et al. (2017) but their biochemical activity was only tested on a limited number of compounds. Our findings suggest instead that these genes might play a role in the production of a broader set of hydroxylated and/or epoxidized products as in other species (Meesters et al., 2007; Ginglinger et al., 2013; Höfer et al., 2013, 2014; Boachon et al., 2015) and, even if the need for further oxidoreductases can not be excluded (Ilc et al., 2016a), they encourage to check this hypothesis by analyzing additional substrates (geraniol, nerol, citronellol) and products (e. g. pyranoid/furanoid linalool oxides, hydroxydiendiols, geranic acid, rose oxides) in CYP enzymatic assays. We also propose an epoxide hydrolase (VIT\_04s0023g02610) to be assessed for involvement in monoterpene oxidative metabolism (Table 2 and Supplementary Table S9). Based on their sequence similarity with terpenoid glucosyltransferases from different plant species and on their membership in biclusters harboring some glucosylated monoterpenes, we propose that VIT\_03s0180g00200, VIT\_03s0180g00320 (Table 2) and eventually other genes reported in Supplementary Table S9 (VIT\_03s0091g00040, VIT\_03s0180g00280, VIT\_05s0062g00430, VIT\_05s0062g00520, VIT\_05s0062g00630, VIT\_05s0062g00640) might code for enzymes that glucosylate monoterpenes along with additional metabolites. Most of these genes have been investigated in previous works but they were not considered as candidates for monoterpene glucosylation in view of their decreasing expression during berry development (Khater et al., 2011; Bönisch et al., 2014a,b). However, they might be involved in the production of glucosylated monoterpenes with a similar trend, like the high oxidation state monoterpenoids sharing the same biclusters (Table 2 and Supplementary Table S9), which were not quantified in those papers. This hypothesis is not contradicted by the lack of gene annotation referring to the "Monoterpenoid biosynthesis" pathway and of positive correlation between transcript and monoterpenyl glucoside accumulation, as the same holds for the biochemically characterized monoterpenol glucosyltransferase VvGT7 (Table 2) and may be explained by the broad substrate tolerance and overlapping enzymatic activities of the large GT family. Monoterpenyl glucosides are only intermediates within the glycosylation pathway and post-transcriptional control is additionally involved (Bönisch et al., 2014a). #### Monoterpene transport Terpene transport within the cell and into the apoplast is an almost unexplored field. It may engage multiple pathways, e.g., (1) insertion of the hydrophobic terpenes into vesicle membranes TABLE 2 Continued followed by transport and fusion to the plasma membrane, (2) carrier proteins (like GSTs, glutathione S-transferases, and ABC, ATP-binding cassette transporters) that conduct these molecules to the (plasma) membrane, and (3) direct diffusion between the endoplasmic reticulum and/or plastidial (stromule) membranes and the plasma membrane (Ting et al., 2015). The fusion of vesicles with target membranes is mediated by a group of proteins called SNAREs (soluble NSF attachment protein receptors). Surprisingly and still without a clear underlying mechanism, both sesquiterpenes and monoterpenes were boosted when vesicle fusion was inhibited in Nicotiana benthamiana (Ting et al., 2015). Moreover, two Arabidopsis linalool synthases were detected in vesicular structures associated with the plastids (Ginglinger et al., 2013). Based on these findings, we included among our candidates a gene coding for a SNARE associated Golgi protein (VIT\_02s0012g01630). Plant ABCG transporters play a role in the flux of secondary metabolites, particularly of terpenoid origin (Kang et al., 2011). Interestingly, we found an ABCG gene (VIT\_16s0039g00010) that shows a profile consistent with monoterpene accumulation and is coexpressed with several monoterpene synthases in VTCdb. We also selected a glutathione S-transferase (VIT\_08s0040g03040) and two amino acid permeases (VIT\_06s0009g01140 and VIT\_08s0007g05210), which are coexpressed with monoterpene biosynthetic genes in VTCdb and positively correlated to several monoterpenes in the present study (Table 2 and Supplementary Table S9). #### Monoterpene biosynthesis transcriptional regulation Recent works (Cramer et al., 2014; Wen et al., 2015) suggested that a group of ERF6-type transcription factors clustered on chromosome 16 are involved in aroma accumulation, based on the correlation of their transcript abundance and the transcript abundance of several terpenoid pathway genes. For some of these regulatory genes, e.g., the orthologs of CrORCA2, CrORCA3, and AaERF1 (De Geyter et al., 2012), no probe was found among the 4,450 probes used for our integrative analysis. Other ERF genes reported in the mentioned papers (VIT\_16s0013g00950, VIT\_16s0013g00980, VIT\_16s0013g00990, VIT\_16s0013g01030, VIT\_16s0013g01050, VIT\_16s0013g01060, not listed in Supplementary Table S9) belonged to clusters 1, 2, 7 and did not show any relevant positive correlation with monoterpenes. However, some of the AROS probes had only a partial match with these genes, as a consequence they might correspond instead to ERF gene isoforms not involved in flavor determination. Conversely, the genes VIT\_16s0100g00400 and VIT\_18s0001g05250 showed an expression profile consistent with the accumulation of monoterpenes in Moscato Bianco ripening berry (Table 2 and Supplementary Table S9). We also observed an interesting behavior (Figure 4, Table 2, and Supplementary Table S9) for TFs of the MYB (VIT\_14s0066g01090) and NAC (VIT\_19s0014g03300) families that promote mono- and sequiterpene production in other plant species (Reeves et al., 2012; Nieuwenhuizen et al., 2015). In particular, VIT\_14s0066g01090 (MYB24) has been proposed as a candidate transcriptional regulator of (mono)terpene biosynthesis also in grapevine (Matus, 2016; Savoi et al., 2016), for which reason it deserves further attention. Finally, based on the negative effect of GBF1 (G-box binding factor 1) and ZCT (zinc-finger Catharanthus transcription factor) proteins on the expression of the TIA (terpenoid indole alkaloid) biosynthetic genes Str (strictosidine synthase) and Tdc (tryptophan decarboxylase) (Sibéril et al., 2001; Pauw et al., 2004), we selected two genes (VIT\_15s0046g01440 and VIT\_18s0001g09230) negatively correlated with monoterpene accumulation during Moscato Bianco berry ripening (Table 2 and Supplementary Table S9). One of the signals dramatically impacting isoprenoid biosynthesis in higher plants is light, which activates the MEP pathway at the transcriptional and post-transcriptional level (Rodríguez-Concepción, 2006; Cordoba et al., 2009; Vranová et al., 2012; Mannen et al., 2014). Sunlight exclusion limits the synthesis and accumulation of terpenes also in grape berries (linalool and the bound forms being the most responsive) by especially affecting DXS and TPS genes (Zhang et al., 2014; Friedel et al., 2016; Joubert et al., 2016; Matus, 2016; Sasaki et al., 2016). Our findings (Figure 4, Table 2, and Supplementary Table S9) are consistent with a role, among others, for HY5 (LONG HYPOCOTYL5, VIT\_04s0008g05210) in the regulation of light-induced terpenoid biosynthesis in grapes, in agreement with other evidences (Carbonell-Bejerano et al., 2014a,b; Zhou et al., 2015; Loyola et al., 2016). The isoprenoid pathway has also been reported to be under the circadian clock control. In particular, the emission of volatile terpenoids follows a diurnal rhythm and genes encoding enzymes involved in IPP biosynthesis (especially those from the MEP pathway) and downstream pathways are coexpressed with circadian clock genes and show typical circadian expression profiles (Cordoba et al., 2009; Vranová et al., 2012; Pokhilko et al., 2015). Some probes on the AROS array correspond to a gene of the circadian oscillator (VIT\_15s0048g02410) and fall into clusters harboring several monoterpenes (Table 2 and Supplementary Table S9). The expression profile of a number of additional transcription factors (including master regulators) and genes potentially involved in the post-transcriptional regulation (Hemmerlin, 2013) overlaps monoterpene accumulation during Moscato Bianco berry ripening, which supports a role in the control of monoterpene biosynthesis for VIT\_01s0026g01970, VIT\_02s0012g01040, VIT\_02s0012g01240, VIT\_02s0234g00100, VIT\_03s0038g02500, VIT\_04s0023g00130, VIT\_04s0023g01250, VIT\_04s0023g02950, VIT\_06s0004g07550, VIT\_07s0031g01320, VIT\_07s0031g01930, VIT\_07s0104g01050, VIT\_08s0007g05880, VIT\_09s0054g01780, VIT\_10s0003g03190, VIT\_12s0028g03860, VIT\_00s0214g00090, VIT\_00s0463g00020 (Figure 4, Table 2, Supplementary Table S9). To our knowledge, these genes represent new regulatory candidates for the production of several (cluster 4) or specific metabolites, like linalool (cluster 9) and geraniol/nerol (cluster 6), as suggested by their co-localization with QTLs and their correlation with enzyme/transporter genes correlated to metabolites. (Continued) TABLE 3 \| Continued (Continued) correlation; A, microarray analysis; R, qRT-PCR analysis. was gray at #### Integration of Transcriptomic and Metabolic Data Over Multiple Seasons to Verify a Subset of Candidate Genes In order to confirm the above links between transcriptome and metabolome in aroma development, the 15 genes assessed by both microarray and real-time analyses were also tested for correlation with the metabolic profile over three seasons, which were considered as three biological replicates (Table 3 and Supplementary Figure S1A). Significant correlations were found for all the genes except VvDXR (confirming the results from 2006 data) and VvHDR, which probably precedes monoterpene accumulation. Several compounds were affected, especially in their glycosidically bound form. Unsurprisingly, most of the metabolites with no correlation showed an inconsistent profile among seasons (e.g., free OxA and citronellol, bound α-terpineol) or a decreasing trend along berry ripening (e.g., free HOdiendiol I + HO-trienol and bound OxB). Though not ensuring a punctual conformity to the observations from a single year (Table 2), the findings from multiple seasons (Table 3) prove the general consistency of the outcomes of different techniques and years and argue for the reliability of the whole set of results based on the integration of 2006 transcriptomic and metabolic data. #### CONCLUSION Understanding the origin of grape aromatic compounds is essential in the breeding of new varieties and in the management of high-quality crops in a changing climate. In this work, previously undescribed gene-to-metabolite networks with a possible association to grape flavor were deduced by integrating the expression profiles of 4,450 gene tags and the accumulation profiles of 52 metabolites. Pairwise correlation and clustering methods pointed to several structural and regulatory genes potentially involved in the biosynthesis of monoterpenes, which #### REFERENCES paves the way for locating candidates for at least some of the missing links in the underlying pathway. Our collective findings contribute toward understanding the regulation of secondary metabolism in Muscat-type grape cultivars through the formulation of testable hypotheses regarding the function of specific genes. #### AUTHOR CONTRIBUTIONS LC, CK, JB, FE, SD, and MG contributed to the project design; LC, MT, JB, FE, MS, and CC took part in the experimental work; RL provided the metabolic analysis; LC, CK, and MM performed the statistical and bioinformatic analyses; LC and CK were involved in data interpretation; LC wrote the manuscript. All the authors approved the final version of this text. #### FUNDING This research was sustained by a Short Term Scientific Mission grant awarded to LC by the Institute of Vine and Wine Sciences (Bordeaux, France) and with the financial support provided by the Autonomous Province of Trento (Accordo di Programma). #### ACKNOWLEDGMENTS We thank Silvia Lorenzi for sample collection, Sergio Moser for metabolic analysis and Pietro Franceschi for statistical support. #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00780/full#supplementary-material information regarding the dynamics of grape ripening. BMC Plant Biol. 11:149. doi: 10.1186/1471-2229-11-149 (Syrah×Pinot Noir) mature grapes: integration of transcriptional profiling and metabolic quantitative trait locus analyses. J. Exp. Bot. 66, 4441–4453. doi: 10.1093/jxb/erv243 pathway in white grapes (Vitis vinifera L.). BMC Plant Biol. 16:67. doi: 10.1186/s12870-016-0760-1 synthase gene QH6 in modulating its rhythmic expression. Front. Plant Sci. 6:304. doi: 10.3389/fpls.2015.00304 Zhu, B. Q., Cai, J., Wang, Z. Q., Xu, X. Q., Duan, C. Q., and Pan, Q. H. (2014). Identification of a plastid-localized bifunctional nerolidol/linalool synthase in relation to linalool biosynthesis in young grape berries. Int. J. Mol. Sci. 15, 21992–22010. doi: 10.3390/ijms1512 21992 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 Costantini, Kappel, Trenti, Battilana, Emanuelli, Sordo, Moretto, Camps, Larcher, Delrot and Grando. 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. # Constructing Integrated Networks for Identifying New Secondary Metabolic Pathway Regulators in Grapevine: Recent Applications and Future Opportunities #### Darren C. J. Wong<sup>1</sup> and José Tomás Matus <sup>2</sup> \* <sup>1</sup> Ecology and Evolution, Research School of Biology, Australian National University, Acton, ACT, Australia, <sup>2</sup> Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Barcelona, Spain #### Edited by: Ashraf El-kereamy, University of California, USA #### Reviewed by: Sara Zenoni, University of Verona, Italy Jose A. Casaretto, University of Guelph, Canada \Correspondence: José Tomás Matus [email protected] #### Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science > Received: 16 February 2017 Accepted: 22 March 2017 Published: 12 April 2017 #### Citation: Wong DCJ and Matus JT (2017) Constructing Integrated Networks for Identifying New Secondary Metabolic Pathway Regulators in Grapevine: Recent Applications and Future Opportunities. Front. Plant Sci. 8:505. doi: 10.3389/fpls.2017.00505 Representing large biological data as networks is becoming increasingly adopted for predicting gene function while elucidating the multifaceted organization of life processes. In grapevine (Vitis vinifera L.), network analyses have been mostly adopted to contribute to the understanding of the regulatory mechanisms that control berry composition. Whereas, some studies have used gene co-expression networks to find common pathways and putative targets for transcription factors related to development and metabolism, others have defined networks of primary and secondary metabolites for characterizing the main metabolic differences between cultivars throughout fruit ripening. Lately, proteomic-related networks and those integrating genome-wide analyses of promoter regulatory elements have also been generated. The integration of all these data in multilayered networks allows building complex maps of molecular regulation and interaction. This perspective article describes the currently available network data and related resources for grapevine. With the aim of illustrating data integration approaches into network construction and analysis in grapevine, we searched for berry-specific regulators of the phenylpropanoid pathway. We generated a composite network consisting of overlaying maps of co-expression between structural and transcription factor genes, integrated with the presence of promoter cis-binding elements, microRNAs, and long non-coding RNAs (lncRNA). This approach revealed new uncharacterized transcription factors together with several microRNAs potentially regulating different steps of the phenylpropanoid pathway, and one particular lncRNA compromising the expression of nine stilbene synthase (STS) genes located in chromosome 10. Application of network-based approaches into multi-omics data will continue providing supplementary resources to address important questions regarding grapevine fruit quality and composition. Keywords: Vitis vinifera, stilbenes, flavonoids, ripening, systems biology, genome-wide, multi-omics, data integration ## NETWORK-BASED APPROACHES INTO OMICS DATA Complex biological processes can be studied from a "multiomics" perspective thanks to the recent improvements in genome-wide techniques and systems biology approaches. Each omics data type is particularly useful in elucidating the constituents and function of a particular cellular domain. Together, they constitute layers of biological complexity. Genomic data generated from genome sequencing projects are commonly used to ascribe molecular function and biological processes based on sequence similarity, while transcriptomics and metabolomics data typically provide a global "snapshot" of gene expression and metabolite dynamics in various biological contexts. For many omics data, interactions/associations between molecules can be represented as networks, where nodes (genes, proteins, metabolites) are connected by edges. These edges denote an association often inferred from correlational and informational theoretic measures such as Pearson correlation coefficient (PCC) and mutual information (MI), respectively. In the case of gene co-expression networks (GCNs), edges represent similar gene expression behaviors. Based on the "guilt by association" principle, genes involved in related processes share similar gene expression dynamics across a wide range of experiments (Wolfe et al., 2005). However, as functional information could be delimited to a reduced number of interactions within a gene network (Gillis and Pavlidis, 2012), subsequent targeted gene characterizations are needed to prove these relationships. Whether the function of a network is dependent or not on specific interactions, GCN analysis have proven to be a powerful tool for inferring gene function and coordinated biological processes related to plant metabolism (Persson et al., 2005; Itkin et al., 2013). Other forms of networks constructed from omics datasets do not necessarily rely on abundance or expression levels to establish node relationships. For example, protein-protein interaction networks describe physically interacting protein pairs identified from high-throughput yeast two-hybrid screens (e.g., Arabidopsis Interactome Mapping Consortium, 2011). Also, genome-wide location studies (i.e., by using ChIP-Seq) allow determining regulatory networks for transcription factors (TF) and other DNA-binding proteins. These TF-binding networks have led to the identification of novel components and of new connections that alter the network diagrams originally drawn by genetic and molecular analyses (reviewed by Ferrier et al., 2011). #### RECENT APPLICATION OF METABOLITE NETWORK-BASED APPROACHES IN GRAPES Studies utilizing networks constructed from omics data profiled in the berry are continuously increasing (Table 1). Network analyses involving metabolite datasets (primary and/or secondary metabolites) are by far the most reported. These studies have included networks inferred from single contexts such as berry development and ripening (Zamboni et al., 2010; Dai et al., 2013; Wang et al., in press), or in combination with other factors including environmental influence (Guan et al., 2016; Savoi et al., 2016; Reshef et al., 2017), and/or cultivar differences (Degu et al., 2014; Cuadros-Inostroza et al., 2016). Network topology has also been investigated in detail to reveal critical metabolites and their regulation. For instance, Cuadros-Inostroza et al. (2016) showed that an increase in network connectedness and density (especially regarding primary metabolites) became prevalent at specific berry developmental stages such as fruit set and veraison (i.e., the onset of ripening). The same study, in concordance with Degu et al. (2014), highlighted that berry-metabolite networks from different cultivars could possess contrasting network topologies, albeit with overall network connections generally maintained. Metabolite networks from the cultivars cv. "Merlot" (Cuadros-Inostroza et al., 2016) and cv. "Shiraz" (Degu et al., 2014) were consistently denser to that off cv. "Cabernet Sauvignon." Rewiring of berry metabolite networks under different environmental conditions or perturbations such as drought (Savoi et al., 2016) and sunlight exposure (Reshef et al., 2017) have also been reported. These studies have shown that higher network connectivity is commonly observed in perturbed networks. Such property could be associated to a tighter metabolic control of the metabolic pathways under investigation. Such is seen for phenylpropanoid and volatile organic compounds (VOC) in berries under prolonged drought compared to non-stress berries (Savoi et al., 2016). Similarly, primary metabolite networks encompassing compounds related to glycolysis, the TCA cycle, and amino acid metabolism showed higher network connectivity in shaded berries compared to fully exposed berries (Reshef et al., 2017). Some metabolic-network studies have shown that certain metabolites (or classes) could act as important switches in the developmental regulation of metabolism during berry growth and ripening, given their high centrality (number of connections) or degree scores in their network. Dai et al. (2013) showed that trehalose-6-phosphate appeared to be the most connected compound in the primary metabolite network of cv. "Cabernet Sauvignon" grapes, with significant partial correlations to sugar metabolism, glycolysis, and TCA cycle intermediates. Altogether, these compounds may be implicated in coordinating metabolite dynamics during berry development. One recent study highlighted fucose as critical for coordinating metabolic regulation in a stage-specific manner, thus deprioritizing the importance of sugars such as glucose, fructose, and sucrose as a function of network centrality measure (Cuadros-Inostroza et al., 2016). These findings further demonstrate the complexity of berry metabolic regulation during development and ripening. # GENE CO-EXPRESSION NETWORKS TO STUDY GRAPE BERRY RIPENING The increased ease of transcriptome profiling, combined with availability of datasets shared by the grapevine research community in public repositories, has led to increased attention #### TABLE 1 \| Studies of grape berry development and composition involving molecular networks approaches. G, genes; M, metabolite; Prot, protein; Prom, promoter; + R, includes resource/database; I, integrated (G, M, Prot, Prom, miRNA inclusive). in the use of gene co-expression networks (GCNs) in the study of berry development and metabolism. GCNs can be classified into "condition-dependent" and "condition-independent" categories (Usadel et al., 2009). In grapes, several studies have focused on condition-independent GCNs (encompassing different cultivars, tissues, developmental stages, stress and vineyard management treatments) as it provides a more convenient and representative (albeit "static") relationship overview (Table 1). This approach has been useful for ascribing the most representative biological functions of the 134 grapevine R2R3-MYB transcription factors based on their top 100 co-expressed genes (Wong et al., 2016), where VviMYB13 (close homolog of VviMYB14 and VviMYB15) was identified as an additional STILBENE SYNTHASE regulator acting in a tissue- and/or stress-dynamic manner. Platforms such as the ViTis Co-expression DataBase (VTCdb; Wong et al., 2013) and VESPUCCI (Moretto et al., 2016) have been successfully exploited to study the extent of transcription factor regulatory networks, providing support for targeted functional studies. Such is the case for the bZIP TF VvibZIPC22, which is involved in the regulation of flavonoid biosynthesis in grapes and may be also implicated in carbohydrate and amino acid metabolism, as inferred from VESPUCCI (Malacarne et al., 2016). Two other bZIP TFs (VviHY5 and VviHYH) were shown to co-operatively mediate flavonol accumulation in grapes in response to sunlight and ultraviolet radiation exposure (Loyola et al., 2016). As inferred from VTCdb and GCN analysis, these regulators were potentially implicated in carbohydrate and isoprenoid metabolism in addition to the control of the flavonoid pathway. Similarly, the involvement of the grapevine VviWRKY26 in the regulation of vacuolar transport and flavonoid biosynthesis was demonstrated using a combination of transcriptomic approaches including GCNs (Amato et al., 2017). Condition-dependent GCNs have been constructed from tissue- or stress-specific datasets, including berry (Zamboni et al., 2010; Palumbo et al., 2014) or abiotic and biotic stresses (Wong et al., 2017). These GCNs provide several advantages over condition-independent networks as inferring gene function is largely simplified, providing a more "dynamic" overview of gene relationships that otherwise could be enhanced or lost in certain conditions (Obayashi et al., 2011). One example of a condition-specific GCN involves the study of the transcriptomes of five black-skinned cultivars across four berry phenological stages (Palumbo et al., 2014). The authors identified "fight-club" nodes and "switch" genes, having the latter unique expression profiles and network topological properties, such as a marked negative correlation connectivity to both neighboring genes and genes grouped to other modules in the network. Genes associated with transcription factor activity; cell wall modification and carbohydrate and secondary metabolism were found as candidate master regulators, potentially inducing large-scale transcriptome reprogramming during berry development (Palumbo et al., 2014). Finally, miRNA and siRNA-mediated gene regulatory networks have also been constructed from high-throughput small RNA and degradome sequencing and computational target prediction methods (Zhang et al., 2012; Belli Kullan et al., 2015). These networks (not relying in abundance or expression levels) revealed novel modules such as miR156/miR172 regulatory circuits and VviTAS3/4 regulatory cascades, which are implicated in regulating plant growth and development and in the control of flavonoid biosynthesis, respectively. # TOWARD THE INTEGRATION OF MULTI-OMICS DATA IN GRAPES Although individual omic network methods have been widely used, a shift toward multi-omics data and integration is increasingly being adopted in plant biology (Proost and Mutwil, 2016), including grapevine (Table 1). Integration approaches allow building complex maps of molecular regulation and interaction. By these means, complex traits from these networks can be assessed (e.g., plasticity and evolution). The first systems level study in grapes leveraged transcriptomic, metabolomic, and proteomic technologies to understand berry development and the postharvest withering process (i.e., controlled dehydration) in cv. "Corvina" grapes (Zamboni et al., 2010). Using a combination of hypothesis-free and -driven integration approaches, the authors were able to tease out putative berry stage-specific functional networks. As an outcome, a fully integrated network related to the withering process revealed key phenylpropanoid and stressresponsive genes (i.e., biotic, osmotic, and oxidative), together with proteins involved in oxidative- and osmotic-stress, and secondary metabolites such as acylated anthocyanins and stilbenes. Recently, integration of berry metabolome (primary and secondary) and proteome networks encompassing 12 developmental stages revealed a greater propensity of an energylinked metabolism in berries prior to veraison (Wang et al., in press). These observations corroborated earlier studies (Dai et al., 2013; Cuadros-Inostroza et al., 2016), demonstrating that pronounced changes in the berry occurs before veraison, characterized by a reduction of many early accumulating primary metabolites. Interestingly, the integrated network also revealed several modules with high node degree for many metabolites (amino acids and organic acids) and corresponding enzymes catalyzing their synthesis (Wang et al., in press). Characterizing genes that regulate the accumulation of secondary metabolites throughout fruit ripening is key for improving quality traits and for predicting plant behavior in response to the environment. In this sense, transcript-metabolite associations have been used to prioritize candidate genes important for determining berry quality parameters under adverse environmental conditions (Savoi et al., 2016). Integrated transcript-metabolite networks encompassing monoterpenes that are both ripening-related and drought-modulated (e.g., linalool, nerol, α-terpineol) revealed many highly co-regulated transcripts to be involved in terpene and lipid metabolism. The authors further highlighted VviMYB24 as a promising regulatory candidate for monoterpene biosynthesis given consistent correlations with all three monoterpenes in their study. Cis-regulatory element-driven networks have been recently constructed using integrated information of promoter CRE structure and network connectivity (Wong et al., 2017). Numerous CRE-driven modules inferred using conditiondependent GCNs (development-dynamic and stress-specific) highlighted roles in stress response (e.g., to drought and pathogens) and developmental processes (e.g., fruit ripening). For example, GCC-core sub-modules contained many genes that were highly induced in berries and leaves infected with fungi (Wong et al., 2017). Cis-regulatory element enrichment maps or transcript information for miRNA target enrichment analysis can be easily integrated into plant GCNs. This approach has been used to prioritize target genes of the entire grape R2R3-MYB family (Wong et al., 2016) and also to explain the expression responses of module genes under prolonged drought stress in berries (Savoi et al., 2016). Enrichment for miRNA targets within GCNs has suggested a pivotal role of these molecules in regulating the expression of "switch" genes in a stage-specific manner (Palumbo et al., 2014). Finally, aggregating several networks into a community network can also be advantageous to effectively reveal discrepancies between individual networks while highlighting associations common across individual networks (Proost and Mutwil, 2016). This approach has been used by Loyola et al. (2016) to identify a set of high confidence targets of HY5 and HYH given by the combination of microarray and RNA-Seq data with genome-wide promoter inspections. It is noteworthy that "condition-independent" and "condition-dependent" approaches are still useful for providing a preliminary insight into co-expression relationships in grapes. ## AN ILLUSTRATION FOR THE INTEGRATION OF MULTILAYERED NETWORKS FOR DISSECTING THE COMPLEXITY OF THE BERRY'S PHENYLPROPANOID COMPOSITION In grapes, phenylpropanoids influence their organoleptic properties and beneficial attributes to human health, highlighting the importance of their study. Several reports have demonstrated the complex nature of secondary metabolism in grapevine, both at the level of chemical composition and genetic regulation (Dal Santo et al., 2013; Costantini et al., 2015; Malacarne et al., 2015). Among the many phenylpropanoid compounds that influence the quality of grapes and wines, some of the most important are flavonoids (anthocyanins, flavonols and tannins) and stilbenes. These compounds accumulate in a temporal and compartmentalized manner and numerous regulators of their accumulation have been characterized to date (Reviewed by Kuhn et al., 2014; Matus, 2016). One strikingly relevant feature of the grapevine genome is that wine quality-related gene families are expanded in gene number (Martin et al., 2010; Vannozzi et al., 2012), including those related with transcription factor activity (Matus et al., 2008; Wong et al., 2016). Genomics and transcriptomics data originated from these and others studies suggest that the regulation of secondary metabolism in grape is a much more complex trait compared to plant model species. As large-scale omics data are periodically accumulating; there is an enormous potential for gene discovery in relation to grape secondary metabolic pathways. To demonstrate how various biological networks can be integrated to study berry's phenylpropanoid composition, we gathered networks generated from gene co-expression analyses, predicted miRNA-gene and long non-coding RNA (lncRNA) gene interactions. First, we re-analyzed a comprehensive berry ripening RNA-Seq transcriptome dataset (five black-skinned cultivars sampled at four developmental stages; Palumbo et al., 2014) and constructed a ripening-specific gene co-expression network (PCC > \|0.8\|). This ripening-specific GCN was then used as a basis for lncRNA-gene network, which consisted of predicted lncRNAs (Vitulo et al., 2014) that showed strong correlation with a putative "interacting" gene (PCC > \| 0.8 \|) that was co-located within 100 kb flanking the lncRNA position. Using a comprehensive catalog of grapevine miRNAs (Belli Kullan et al., 2015; Pulvirenti et al., 2015), we also reanalyzed potential miRNA-mRNA interactions using psRNATarget with default parameters (Dai and Zhao, 2011). As the interpretation of each network at a global scale is out of the scope of this perspective, we focused our attention on the early phenylpropanoid and flavonoid (ePP and Fla) pathways and on the potential regulatory genes and their interactions (among genes, miRNAs, and lncRNAs). The resulting network is composed of 112 ePP/Fla pathway genes (differentially expressed during berry development and ripening) together with five miRNA and 14 predicted grapevine lncRNAs (Figure 1). GCN analysis revealed a strong co-regulation within early phenylpropanoid and flavonoid pathway genes maintaining few connections between both sub-pathway genes during the course of berry development and ripening. Three clusters (I, II and III) were observed for Fla pathway genes sharing many positive correlations within each group (Figure 1). Cluster I contained genes mainly involved in the regulation of anthocyanin accumulation such as five flavonoid-3′ ,5′ -hydroxylases (F3′ 5 ′H), two anthocyanin-o-methyltransferases (AOMT1-2), the UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UFGT) and ANTHOCYANIN-3-O-GLUCOSIDE-6′′-O-ACYLTRANSFERASE (3AT). Cluster II consisted of genes encoding proanthocyanidin biosynthesis genes including three predicted galloyl glucosyltransferases, ANTHOCYANIDIN REDUCTASE (ANR), and LEUCOANTHOCYANIDIN REDUCTASE (LAR), as well as upstream flavonoid pathway genes such as CHALCONE SYNTHASE (CHS) and CHALCONE ISOMERASE (CHI). One predicted antisense lncRNA (VIT\_203s0180n00020) collocated (within 50 kB) and positively correlated with one galloyl glucosyltransferase gene (VIT\_03s0180g00200). This cluster also contained genes encoding one 4-coumarate-Co-A ligase (4CL), two flavonol synthases (FLS4-5), one flavanone-3-hydroxylase (F3H), and one caffeic acid 3-o-methyltransferase (COMT), all of which were negatively correlated with genes from cluster I. Furthermore, grapevine miRNAs miR169r/t and grape-m0534 were predicted to target 4CL and FLS4, respectively. Several genes belonging to cluster II and I shared negative correlations (light blue solid edges, Figure 1). This separation is evident whereby the majority interaction and lncRNA-gene co-location (within 100 kb). Clusters I, II and III connect genes belonging to the early phenylpropanoid and flavonoid (ePP and Fla) sub-pathways. Cluster IV shows a dense group containing predominantly PHENYLALANINE AMMONIA-LYASE (PAL) and STILBENE SYNTHASE (STS) genes that are largely co-expressed with positive co-expression correlations. Purple edges represent positive co-expression correlations between TFs and early phenylpropanoid pathway genes. Pie chart colors represent the presence of selected TF-binding sites (based on cis-regulatory element enrichment analysis) in promoter regions of the corresponding enzyme-coding genes. Light blue border edges depict STS genes located in chromosome 10. of genes from cluster I are ripening-specific (i.e., upregulated from veraison onwards), while many genes from cluster II are mostly expressed during the early-to-mid stages of berry development (and subsequently downregulated as ripening progresses). As there is much less evidence in the regulation of the early phenylpropanoid and stilbene sub-pathways compared to the regulation of flavonoid biosynthesis, we focused our attention on a fourth, highly connected cluster (IV) holding strong positive correlations within and between the two large PHENYLALANINE AMMONIA-LYASE (PAL) and STILBENE SYNTHASE (STS) gene families (Figure 1). Two cinnamate-4-hydroxylases (C4H) also shared many strong positive correlations with PAL genes and one 4CL was positively correlated with many STS encoding genes. Gene expressions within this cluster were mainly late-ripening specific, with many of them peaking at harvest. Promoters from cluster IV were highly enriched for cis-regulatory elements including those for R2R3-MYB, AP2/ERF, WRKY, bHLH, and bZIP TF binding. In particular, the MYB binding site CCWACC was present in one CCoAMT, two C4H, 10 PAL, and 27 STS genes. The potential regulation of these genes by MYB transcription factors is supported by recent studies showing that several grapevine MYBs may have regulatory roles controlling the levels of small weight phenylpropanoids and stilbenes (Höll et al., 2013; Cavallini et al., 2015). Our approach is novel in suggesting the regulatory roles by other TF families such as WRKY and AP2/ERF. For example, strong co-regulation of nine WRKY TF to 11 PAL and 44 STS genes coincided with the presence of WRKY cis-regulatory elements in many PAL and STS genes. Interestingly, one of the four predicted intergenic lncRNAs (VIT\_210s0042n00100) was co-located and strongly co-regulated with all nine STS positioned on chromosome 10. Recent evidence from several functionally characterized lncRNAs in animals and plants suggest that lncRNAs could operate as decoys, guides, signals, and scaffolds, acting as single molecules or complexes regulating pre- and post-transcriptional processes (Wang and Chang, 2011). As such, our observation raises the plausibility of a large-scale regulatory function between this lncRNA and STS genes. This STS-associated lncRNA may fulfill combinatorial roles for the fine-regulation of multiple STS, as signals for transcription activity in a stage-specific way or as guides for chromatin modifiers to the cluster of tandem-positioned STS of chromosome 10, potentially modulating DNA accessibility. #### CONCLUSION Multi-omics studies incorporating systems biology approaches in grapevine have facilitated the identification of new grape secondary metabolism regulators and have helped in the characterization of genome-wide responses to environmental factors. These studies have brought knowledge and new tools to understand how to modify and improve grape's quality. #### REFERENCES Additional efforts will still be needed to map protein-DNA and protein-protein landscapes at a large scale. Also, DNAse I hypersensitivity mapping could be useful to identify pioneering transcription factors controlling grape and wine quality traits. # AUTHOR CONTRIBUTIONS JTM conceived the article and planned its structure. DW and JTM searched and discussed the literature and wrote the manuscript. DW generated new network data. All authors have read and approved the manuscript. #### ACKNOWLEDGMENTS The authors wish to acknowledge Dr. Jason Argyris (Centre for Research in Agricultural Genomics, CRAG) for critically reviewing this work. 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 Wong and Matus. 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. # Cultivar Diversity of Grape Skin Polyphenol Composition and Changes in Response to Drought Investigated by LC-MS Based Metabolomics #### Edited by: Simone Diego Castellarin, University of British Columbia, Canada #### Reviewed by: Jose Carlos Herrera, University of Natural Resources and Life Sciences, Vienna, Austria Markus Keller, Washington State University, United States > \Correspondence: Véronique Cheynier [email protected] #### Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science > Received: 23 May 2017 Accepted: 10 October 2017 Published: 27 October 2017 #### Citation: Pinasseau L, Vallverdú-Queralt A, Verbaere A, Roques M, Meudec E, Le Cunff L, Péros J-P, Ageorges A, Sommerer N, Boulet J-C, Terrier N and Cheynier V (2017) Cultivar Diversity of Grape Skin Polyphenol Composition and Changes in Response to Drought Investigated by LC-MS Based Metabolomics. Front. Plant Sci. 8:1826. doi: 10.3389/fpls.2017.01826 Lucie Pinasseau<sup>1</sup> , Anna Vallverdú-Queralt <sup>1</sup> , Arnaud Verbaere<sup>1</sup> , Maryline Roques 1, 2, 3 , Emmanuelle Meudec<sup>1</sup> , Loïc Le Cunff <sup>3</sup> , Jean-Pierre Péros <sup>4</sup> , Agnès Ageorges <sup>2</sup> , Nicolas Sommerer <sup>1</sup> , Jean-Claude Boulet <sup>1</sup> , Nancy Terrier <sup>2</sup> and Véronique Cheynier 1, 2 \ <sup>1</sup> Plateforme Polyphénols SPO, INRA, Montpellier SupAgro, Université de Montpellier, Montpellier, France, <sup>2</sup> SPO, INRA, Montpellier SupAgro, Université de Montpellier, Montpellier, France, <sup>3</sup> IFV Pôle national matériel végétal, UMT Génovigne, Montpellier, France, <sup>4</sup> AGAP, INRA, CIRAD, Montpellier SupAgro, Université de Montpellier, Montpellier, France Phenolic compounds represent a large family of plant secondary metabolites, essential for the quality of grape and wine and playing a major role in plant defense against biotic and abiotic stresses. Phenolic composition is genetically driven and greatly affected by environmental factors, including water stress. A major challenge for breeding of grapevine cultivars adapted to climate change and with high potential for wine-making is to dissect the complex plant metabolic response involved in adaptation mechanisms. A targeted metabolomics approach based on ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode has been developed for high throughput profiling of the phenolic composition of grape skins. This method enables rapid, selective, and sensitive quantification of 96 phenolic compounds (anthocyanins, phenolic acids, stilbenoids, flavonols, dihydroflavonols, flavan-3-ol monomers, and oligomers…), and of the constitutive units of proanthocyanidins (i.e., condensed tannins), giving access to detailed polyphenol composition. It was applied on the skins of mature grape berries from a core-collection of 279 Vitis vinifera cultivars grown with or without watering to assess the genetic variation for polyphenol composition and its modulation by irrigation, in two successive vintages (2014–2015). Distribution of berry weights and δ <sup>13</sup>C values showed that non irrigated vines were subjected to a marked water stress in 2014 and to a very limited one in 2015. Metabolomics analysis of the polyphenol composition and chemometrics analysis of this data demonstrated an influence of water stress on the biosynthesis of different polyphenol classes and cultivar differences in metabolic response to water deficit. Correlation networks gave insight on the relationships between the different polyphenol metabolites and related biosynthetic pathways. They also established patterns of polyphenol response to drought, with different molecular families affected either positively or negatively in the different cultivars, with potential impact on grape and wine quality. Keywords: grape berry, Vitis vinifera, phenolic compounds, UHPLC-QqQ-MS, metabolomics, water deficit, large-scale studies ## INTRODUCTION In the context of climate change, it is of prime importance to anticipate and predict the response of the different biota to the changes in environmental conditions, especially for plants, that are devoid of motility. Climate change is expected to affect plant composition and consequently, in the case of crop species such as grapevine, the quality of plant derived products. Among plant metabolites, secondary metabolites, including phenolic compounds, have been recognized as playing multiple roles in plant response to a wide range of biotic and abiotic stresses and in particular to water stress (Baker and Orlandi, 1995; Dixon and Paiva, 1995; Caldwell et al., 2003). They are also essential components of plant derived foods and beverages, responsible for major organoleptic properties such as color and taste and contributing health benefit (Manach et al., 2004). Grape phenolic compounds comprise several families, divided between non flavonoids (hydroxybenzoic acids, hydroxycinnamic acids, and stilbenes) and flavonoids, based on the same C6-C3-C6 skeleton (flavonols, dihydroflavonols, flavan-3-ols, and anthocyanins). Each family is represented by several compounds differing by their hydroxylation level and by substitution of the hydroxy groups (methylation, glycosylation, acylation). For example, anthocyanins, the red grape pigments, are based on six aglycones which can be mono- or di-glucosylated and further acylated with acetic, p-coumaric, and caffeic acid, giving rise to a large number of compounds (Favretto and Flamini, 2000; Heier et al., 2002; Vidal et al., 2004a). Moreover, various anthocyanin derivatives such as anthocyanin dimers and flavan-3-ol anthocyanin adducts have been detected in grape skin extracts (Vidal et al., 2004b). Grape flavan-3-ols also show high diversity. They include several monomers (catechin, epicatechin, gallocatechin, epigallocatechin, and epicatechin 3-gallate) that are the constitutive units of oligomers and polymers (proanthocyanidins or condensed tannins), with degrees of polymerization ranging from 2 to over 100 in grape skin (Souquet et al., 1996). The impact of water stress on grape berry composition has already been investigated (reviewed in Downey et al., 2006; Teixeira et al., 2013). However, those studies were performed on a few elite cultivars (e.g., Cabernet-Sauvignon, Chardonnay, Syrah, Merlot. . . ) analyzed for a limited number of phenolic metabolites, most often anthocyanins. In addition, results are hardly comparable between studies since differences in water regime were not applied at the same developmental stage and with the same intensity, and amounts of phenolic compounds were not expressed in the same units. Since water stress induces a decrease of berry size, and given that most of phenolic compounds are stored in external cell layers of the cells, an increase of phenolic concentration expressed as mg/g of fresh weight can be measured without any increase of content expressed in mg/berry (Bucchetti et al., 2011). As a general trend, water stress was shown to induce an increase of anthocyanin content and a qualitative modification of the anthocyanin pool, when fine analysis was performed (Castellarin et al., 2007; Bucchetti et al., 2011; Ollé et al., 2011; Hochberg et al., 2015). In contrast, conflicting results were obtained on other classes of phenolic compounds. For example, no (Kennedy et al., 2002; Ollé et al., 2011) or slight (Ojeda et al., 2002) modifications in flavan-3-ol composition and a reduction (Hochberg et al., 2015; Savoi et al., 2017) or increase (Deluc et al., 2011; Herrera et al., 2017) of stilbene accumulation have been observed in response to water deficit. Cultivar specificity of these responses has been reported by comparing cv. Chardonnay (Deluc et al., 2009) or cv. Syrah (Hochberg et al., 2015) to cv. Cabernet Sauvignon. This may be related to hydraulic behavior or to differences in phenological stages (Hochberg et al., 2015) as early and late water deficit affect phenolic composition in different ways (Ojeda et al., 2002; Ollé et al., 2011; Casassa et al., 2015). Nevertheless, a major challenge for breeding of grapevine cultivars adapted to climate change and with high potential for wine-making is to describe and dissect the complex global phenolic response involved in adaptation mechanisms on a wide range of genotypes. The aim of the present study was to investigate the polyphenol composition and its modification in response to water deficit on a large panel of cultivars reflecting the genetic diversity of grapevines. #### MATERIALS AND METHODS #### Plant Material and Experimental Design The diversity panel (DP) of 279 V. vinifera cultivars described by Nicolas et al. (2016) was used for this study. It is composed of three subgroups of 93 cultivars representing the three main genetic pools, which differ in use and geographical origin: wine West (WW), wine East (WE), table East (TE). Each cultivar was over-grafted in 2009 on 6-years old vines of cultivar Marselan in a complete randomized block design with five blocks and one plant of each cultivar per block. The trial was located at the Domaine du Chapitre of Montpellier Supagro (Villeneuve-les-Maguelonne, France), maintained under classical local training system (double cordon, 4,000 plants/ha). A drip irrigation was installed in two blocks in order to create a water contrast with the other three blocks. In 2014 and 2015, irrigation was applied 2 days per week from the last third of June to the end of the berry sampling period (October 6th and October 16th, in 2014 and 2015, respectively). The quantity of supplied water was approximately of 10 mm per 10-day period. Data on total rainfall per 10-day period were obtained for the nearest climatic station. ## Sampling Grape berries were collected at ripeness when sugar concentration reached 20◦Brix. To determine this sampling stage, regular measurements (three times a week from week 30) were performed with an optical refractometer using a few berries per cultivar/treatment. Three clusters were sampled per cultivar/treatment, their end parts were discarded and 100 berries randomly sampled to estimate mean berry weight. Thirty berries were then randomly selected and their skins isolated, frozen in liquid nitrogen, and stored at −80◦C until extraction and analysis. The remaining berries were crushed and the juice was filtered. An aliquot of 1 mL was prepared for the analysis of the 13C/12C ratio (δ13C). #### δ <sup>13</sup>C Analysis δ <sup>13</sup>C or carbon isotope discrimination is expressed compared to a standard and ranges at maturity stage from −27 p. 1000 (no water deficit) to −20 p. 1000 (severe water deficit stress, Van Leeuwen et al., 2001). Its measurement was subcontracted. Samples were freeze-dried, pre-weighed, encapsulated, and then sent to OEA Laboratories Limited (Cornwall, UK). They were analyzed by a Sercon 20-20 dual turbo pumped Continuous Flow Isotope Ratio Mass Spectrometer (CF/IRMS) linked to a Thermo EA1110 Elemental Analyzer (EA) NC dual tube configured fitted with a high performance Carbosieve G separation column. Samples and references were weight optimized for δ13C analysis according to elemental composition. IRMS calibration was scale normalized using isotope references USGS-40 and USGS-41a as lower and upper scale anchors with random QC sample checks within sample sequences. Absolute weights of carbon in samples were determined from the IRMS total beam values relative to the elemental composition of the references. References were weighed from bulk material to 6 decimal places using a Mettler UMX5 microbalance. Standard deviations for isotope reference materials was typically better than 0.15 for carbon. #### Extraction and Sample Preparation for Polyphenol Analysis Extraction The extraction procedure was adapted from that of Mané et al. (2007), as described by Pinasseau et al. (2016). Briefly, frozen skins were ground with liquid nitrogen with a Mortar Grinder Pulverisette 2 (Fritsch, Idar-Oberstein, Germany). One hundred milliliters of of powder were weighed and 500 µL of methanol was immediately added. Then 3.5 mL of acetone/H2O 70/30 (v/v) 0.05% trifluoroacetic acid were added. The mixture was crushed with Precellys (Bertin Technologies, Montigny-le-Bretonneux, France) during three cycles (3 × 40 s each). 3.5 mL were centrifuged with a Heraeus Multifuge X3R Centrifuge (ThermoFischer Scientific, Waltham, USA) (21,320 g, 5 min, 4 ◦C). Aliquots (1 mL) of the supernatant were dried with Genevac (SP Scientific, Warminster, PA, USA). #### Sample Preparation for Determination of Polyphenol Composition Five hundred microliters of methanol/H2O 50/50 (v/v) 1% formic acid were added on the solid obtained after evaporation with Genevac (SP Scientific, Warminster, PA, USA). After solubilization using an Ultrasonic Cleaner (VWR, Fontenay-sous-Bois, France) (30 min), the solution obtained was centrifuged with Hettich Mikro 220R (Hettich Lab Technology, Tuttlingen, Germany) (15,000 rpm, 15 min, 4◦C). Dilutions 1/20 were prepared. Pure and diluted samples were injected in triplicate for UHPLC-QqQ-MS analysis. The phloroglucinolysis reaction was carried out on the solid obtained after evaporation with Genevac (SP Scientific, Warminster, PA, USA), following the procedure described in Pinasseau et al. (2016). #### Instrumentation Analyses were carried out using an Acquity UPLC system (Waters, Saint-Quentin-en-Yvelines, France) hyphenated to a triple quadrupole (QqQ) TQD mass spectrometer (Waters, Saint-Quentin-en-Yvelines, France). The UPLC system included a binary pump, a cooled autosampler maintained at 7◦C and equipped with a 5-µL sample loop, a 100-µL syringe and a 30 µL needle, and a diode array detection (DAD). The DAD spectra were recorded in the range of 210–600 nm (resolution 1.2 nm). MassLynx software was used to control the instruments and to acquire the data which were then processed with the TargetLynx software. #### Chromatographic Conditions The column used for chromatographic separation was a reversedphase Acquity HSS T3 1.8µm 1.0 × 100 mm (Waters, Saint-Quentin-en-Yvelines, France) protected by a 0.2µm in-line filter and maintained at 40◦C. The mobile phase consisted of 1% (v/v) formic acid in deionized water (solvent A) and 1% (v/v) formic acid in methanol (solvent B). The flow rate was 0.170 mL/min. Samples were injected into the column by using the Partial Loop with Needle Overfill injection mode with an injection volume of 1 µL. #### UPLC analysis of polyphenol composition Isocratic 1%B from 0.0 to 2.0 min, linear 1–5%B from 2.0 to 2.1 min, linear 5–10%B from 2.1 to 8.0 min, linear 10–28%B from 8.0 to 12.0 min, isocratic 28%B from 12.0 to 18.0 min, linear 28–45%B from 18.0 to 22.0 min, linear 45–99%B from 22.0 to 23.5 min, isocratic 99%B from 23.5 to 26.5 min. At the end of this sequence, the column was brought back to initial conditions with linear 99–1%B from 26.5 to 27.0 min, then re-equilibrated with isocratic 1%B from 27.0 to 30.0 min. #### UPLC Analysis of tannin Units after phloroglucinolysis Isocratic 2%B from 0.0 to 1.5 min, linear 2–7%B from 1.5 to 3.0 min, linear 7–40%B from 3.0 to 5.0 min, linear 40–99%B from 5.0 to 6.0 min, isocratic 99%B from 6.0 to 6.5 min. As the end of this sequence, the column was brought back to initial conditions with linear 99–2%B from 6.5 to 7.0 min, then re-equilibrated with isocratic 1%B from 7.0 to 10.0 min. #### Mass Spectrometry Conditions The mass spectrometer was operated in MRM mode with electrospray ionization (ESI) either in positive or negative ionization mode. The source and desolvation temperatures were respectively set at 120 and 450◦C. Nitrogen was used as desolvation (500 L/h) and cone (50 L/h) gas. Argon was used as collision gas at a flow rate of 0.16 mL/min. Capillary voltage was set at 3.5 kV in positive mode and 2.8 kV in negative mode. ### Polyphenol Composition Data Lower molecular weight phenolic compounds including phenolic acids, stilbenes, anthocyanins, flavonols, dihydroflavonols, flavan3-ol monomers, dimers and trimers, and derived pigments and tannins, were analyzed by UHPLC-QqQ-MS in the MRM mode, using a method adapted from that described by Lambert et al. (2015). A few additional phenolic compounds detected in the grape extracts were identified and included in the method as detailed below. Glutathione in its reduced and oxidized forms was analyzed by UHPLC-QqQ-MS in the MRM mode as described by Vallverdú-Queralt et al. (2015). Flavan-3 ol units released after phloroglucinolysis were analyzed by UHPLC-QqQ-MS in the MRM mode (Lambert et al., 2015). MRM transitions parameters of added target compounds that are commercially available were optimized by using the Intellistart tool of the Masslynx software which consists in automatically detecting the major fragments and optimizing cone voltages and collision energies. 1-galloyl-β-D-glucose (glucogallin) was characterized by the loss of glucose (−162Th). The main fragment (m/z 139Th) of (-)-epigallocatechin was the result of a Retro-Diels-Alder (RDA) fragmentation. Piceatannol was characterized by the loss of a diphenol (−110Th). These three molecules and quercetin-3-O-glucuronide were included in the calibration standards. For new target analytes that are not commercially available, MRM parameters were optimized directly in grape extracts and compared to data reported in the literature. Pelargonidin 3-glucoside was characterized by the loss of glucose (−162Th) (Arapitsas et al., 2012) while pelargonidin 3-acetylglucoside and pelargonidin 3-coumaroylglucoside were characterized by the loss of the acetylglucose (−204Th) and coumaroylglucose (−308Th), respectively. These fragmentation patterns were specifically targeted in accordance with those of the other anthocyanins described by Lambert et al. (2015). Transitions of (epi)gallocatechin-malvidin 3-glucoside and (epi)gallocatechinpeonidin 3-glucoside were specifically targeted in accordance with their non galloylated equivalents described in Lambert et al. (2015). They are characterized by the loss of glucose (−162Th). Analysis of anthocyanin-tannin (A-T) bicyclic A-type adducts, namely peonidin 3-glucoside-(epi)catechin (m/z 753Th), petunidin 3-glucoside-(epi)catechin (m/z 769Th), malvidin 3-glucoside-(epi) catechin (m/z 783Th), malvidin 3-glucoside- (epi)gallocatechin (m/z 799Th), was optimized in the same way. The main fragments detected at m/z 313, 329, and 343Th, respectively for peonidin, petunidin, and malvidin derived A-T adducts result from a retro Diels-Alder (RDA) fragmentation (−168Th), the loss of the anthocyanin A-ring (−126Th) and that of the glucose substituent (−162Th) (Remy-Tanneau et al., 2003). Malvidin 3-glucoside dimer and malvidin 3-glucosidepeonidin 3-glucoside were characterized by the loss of the two glucose moieties (−324Th) (Vidal et al., 2004b). Glucosylated flavonols such as isorhamnetin 3-glucoside, kaempferol 3 glucoside, and syringetin 3-glucoside were qualified by the loss of the glucose (−162Th) (Vrhovsek et al., 2012). Fragmentation of laricitrin 3-glucoside (−162Th) was specifically targeted in accordance with fragmentation patterns of the other glucosylated flavonols (Lambert et al., 2015). Kaempferol 3-glucuronide was qualified by the loss of the glucuronide (−176Th) (Vrhovsek et al., 2012). Fragmentations of isorhamnetin 3-glucuronide, laricitrin 3-glucuronide, and syringetin 3-glucuronide (loss of the glucuronide −176Th) were optimized in accordance with the fragmentation pattern of the other glucuronidated flavonols (Lambert et al., 2015). Piceatannol 3-glucoside was characterized by the loss of the glucose (−162Th) (Vrhovsek et al., 2012). Fragmentation of (+)-gallocatechin (fragment at m/z 139Th after a RDA fragmentation) was optimized in accordance with (–)-epigallocatechin which is commercially available. Anthocyanins were expressed as equivalent malvidin 3-O-glucoside. Flavonol glucosides and flavonol glucuronides were expressed as equivalent quercetin 3-glucoside and quercetin 3-glucuronide, respectively. Piceatannol glucoside was expressed as equivalent piceid. Quantitative data on 105 compounds was thus obtained. In addition, 17 variables have been calculated, including quantitative variables, namely total concentrations of native anthocyanins (s\_AN\_n), flavonols (s\_FO), stilbenes (s\_ST), hydroxycinnamic acid derivatives (s\_HC), hydroxybenzoic acid derivatives (s\_HB), flavan-3-ols (i.e., sum of tannin units released after phloroglucinolysis, s\_FA), and qualitative variables, %acylated anthocyanins (p\_AN\_acyl), %B-ring trihydroxylated anthocyanins (p\_AN\_tri), %B-ring methylated anthocyanins (p\_AN\_met), %B-ring monohydroxylated flavonols (p\_FO\_mono), %B-ring dihydroxylated flavonols (p\_FO\_di), %B-ring trihydroxylated flavonols (p\_FO\_tri), %Bring methylated flavonols (p\_FO\_met), %flavonol glucuronides (p\_FO-glucur), %B-ring trihydroxylated flavan-3-ol units (p\_FA\_tri) %galloylated flavan-3-ol units (p\_FA\_gall), mean degree of polymerization (dp\_FA), calculated as the molar ratio of total released units to total terminal units. The list of variables is given in Table 1, along with their codes and abbreviations. #### Chemometrics For the 2 years of sampling (2014 and 2015), chemometrics treatments were performed on the MRM data for the 105 compounds, sorted by families (same order in 2014 and 2015) anthocyanins, flavanols, stilbenes, etc. For each observation, the 105 compounds were associated to the 17 calculated parameters, and the three parameters from the vineyard: δ <sup>13</sup>C, refractive index, berry weight. Only cultivars for which both irrigated and non-irrigated observations were available were considered in each vintage. Samples with missing berry weight values were also eliminated. For the 105 MRM parameters, values below the quantification threshold were automatically replaced with a value corresponding to half of the threshold value. #### TABLE 1 \| list of variables, variable codes, and abbreviations. (Continued) (Continued) #### TABLE 1 \| Continued PPt,Pn,Mv = AN4+AN5+AN6+AN9+AN10+AN11+AN15+AN16+AN17+AN21 +AN22+AN23+AN26+AN27+AN28 Pkaempf = FO9+FO10 Pquerc + Isorham = FO3+FO4+FO11+FO12 Pmyric+laric+syring = FO1+FO2+FO5+FO6+FO7+FO8 Pisorham+laric+syring = FO1+FO2+FO7+FO8+FO11+FO12 Pglucuronides = FO1+FO3+FO3+FO7+FO9+FO11. One-way analysis of variance and principal component analysis were performed using the Fact toolbox of the Scilab software. Correlation networks were processed using Cytoscape. Hierarchical clustering of phenolic compounds and genotypes was performed using EXPANDER V6 (Sharan et al., 2003). The distance measurement used in the algorithm is (1-Pearson Correlation)/2, with complete linkage. #### Reagents and Chemicals Formic acid, HPLC grade methanol, acetone, hydrochloric acid, trifluoroacetic acid, ammonium formiate, L-ascorbic acid, and phloroglucinol were purchased from Sigma Aldrich (St Louis, MO, USA). Deionized water was obtained from a Milli-Q purification system (Millipore, Molsheim, France). Standards of (+)-catechin, (–)-epicatechin, (–)-epicatechin 3-O-gallate, reduced L-glutathione, oxidized L-glutathione, piceatannol, p-coumaric acid, protocatechuic acid, syringic acid, trans-caftaric acid, and trans-resveratrol were purchased from Sigma-Aldrich (St Louis, MO, USA). Standards of (–)-epigallocatechin, gallic acid, hydroxytyrosol, malvidin 3-O-glucoside chloride, malvidin code formula Full name (Continued) 3,5-di-O-glucoside chloride, procyanidin B2, quercetin 3-Oglucuronide, and taxifolin were purchased from Extrasynthese (Geney, France). Standards of caffeic acid, ferulic acid, and vanillic acid were purchased from Fluka (Buchs, Switzerland). Standards of 1-O-Galloyl-β-D-glucose and quercetin 3-Oglucoside were purchased from PlantMetaChem, Transmit GmbH (Gießen, Germany). Standard of trans-piceid was purchased from Selleckchem (Houston, TX, USA). ## RESULTS ## Genetic Diversity of Polyphenol Composition After elimination of cultivars for which both samples were not available and/or essential data such as berry weight was missing, complete data was obtained for 208 cultivars in 2014, for 161 cultivars in 2015, and for 147 cultivars in both years. The list of samples collected in 2014 and 2015 and their harvest dates is provided in Table S1. Data for all cultivars in both vintages are available in Pinasseau et al. (2017). Large differences in the phenolic composition were observed between cultivars. Tannins were very abundant in all cultivars with concentrations ranging from 0.4 to 7.5 mg berry−<sup>1</sup> in 2014, and over 12 mg berry−<sup>1</sup> in 2015. Anthocyanin contents ranged from less than 1µg berry−<sup>1</sup> in white cultivars to 8.5 and 14.7 mg berry−<sup>1</sup> , respectively in 2014 and 2015. Flavonols, and especially quercetin derivatives (quercetin 3-glucoside and quercetin 3-glucuronide), were also abundant, with concentrations ranging from 0.04 to over 6 mg berry−<sup>1</sup> in 2014 and from 0.06 to over 5 mg berry−<sup>1</sup> in 2015. Other polyphenol classes were hydroxycinnamic acids (8–2,000µg berry−<sup>1</sup> ) mostly represented by caftaric and coutaric acids, stilbenes (1–745µg berry−<sup>1</sup> ), among which cis- and trans- piceid and trans-resveratrol were the most abundant, dihydroflavonols (trace amounts to 196µg berry−<sup>1</sup> ), and hydroxybenzoic acids (trace amounts to 25µg berry−<sup>1</sup> ). A number of anthocyanin derivatives were also detected. Most of them (i.e., carboxypyranoanthocyanins; e.g., carboxypyranomalvidin 3-glucoside, called vitisin A, caftaric anthocyanin adducts, and series of flavanolanthocyanin, anthocyanin-flavanol, anthocyanin-ethyl-flavanol, and flavanol-ethyl flavanol adducts), were present in low amounts, except pyranoanthocyanins resulting from reaction of acetaldehyde with anthocyanins, especially pyranomalvidin 3-glucoside (vitisin B), detected at concentrations up to 400µg berry−<sup>1</sup> . Table 2 shows the correlation coefficients between irrigated and not irrigated populations, in 2014 and 2015, and between vintages for irrigated and not irrigated samples, for each of the 17 calculated polyphenol composition variables and for berry weight. Berry weight was highly correlated across all four conditions, as expected. The contents (per berry) and concentrations (per g of berry) of all polyphenol families, except flavonols and stilbenes, in irrigated and not-irrigated berries were highly correlated in 2014 but not in 2015. Correlations between years were low under both conditions. In contrast, for all qualitative variables, correlations between irrigated and not irrigated conditions were very high and correlations between years were only slightly lower. Correlation networks established from the phenolic composition data showed several clusters. Correlations >0.8 are presented in Figure 1. The content of malvidin 3 glucoside was correlated on one hand with those of delphinidin 3-glucoside, petunidin-3-glucoside, and of their coumaroyl and caffeoyl derivatives and, on the other hand, with those of some anthocyanin derivatives [pyranomalvidin 3-glucoside, carboxypyranomalvidin 3-glucoside, (epi)gallocatechinmalvidin 3-glucoside, (epi)catechin-malvidin 3-glucoside, and (epi)catechin-petunidin 3-glucoside] (Figure 1, A). Pelargonidin, cyaniding, and peonidin 3-glucosides were correlated together and with peonidin derivatives, namely pyranopeonidin 3-glucoside and (epi)catechin–peonidin 3 glucoside (B) while their caffeoyl and p-coumaroyl esters formed another group (C). All acetylated anthocyanins were correlated together in a separate cluster (D). Other types of anthocyanin pigments, namely anthocyanin 3,5-di-O-glucosides (E), anthocyanin dimers (F), phenylpyranoanthocyanins correlated between them and with caftaric-anthocyanin adducts (G) and the different isomers of (epi)catechin-ethyl-peonidin−3 glucoside and (epi)catechin-ethyl-malvidin 3-glucoside (H) formed additional groups. Flavonols clustered in three different groups consisting of kaempferol and quercetin 3-glucosides (I), myricetin, laricitrin, and syringetin 3-glucosides (J), and laricitrin and syringetin 3-glucuronides (K), respectively. Stilbene glucosides (cis- and trans- piceids and piceatannol glucoside) formed another correlation network (L). Flavan-3-ol variables formed three clusters: (epi)catechin monomers and terminal units (M), (epi)gallocatechin terminal units (N), and (epi)catechin phloroglucinol derivatives (extension and upper units in the tannin structures) (O). # Vine Water Status in 2014 and 2015 Information from the rain and irrigation data and from the measures of δ <sup>13</sup>C and berry weight was combined to characterize the vine water status during the vegetative seasons 2014 and 2015. Bar plots showing water quantities supplied by rainfall and irrigation are provided in Figure 2, showing that the total quantity of rainfall received within the plot trial the preceding winter and spring was very different After including data from 2013 (data not shown), the total rainfall received from November to the second third of June (before irrigation started) was 187.5 and 460.5 mm for 2014 and 2015, respectively. Another notable difference between the two vegetative seasons was the earlier occurrence of summer rainfall in 2015 as compared to 2014 (Figure 2). #### Cultivar Response to Water Stress in Vintages 2014 and 2015 A first round of statistical analysis was performed with oneway ANOVA analysis on the four data sets (irrigated and notirrigated, 2014 and 2015) available for 147 cultivars (Table S2). The absence of significant differences (at p = 0.05) in refractive index values between conditions in both years confirmed that TABLE 2 \| Stability of polyphenol composition data and berry weight; correlations between irrigated and not irrigated berries in 2014 (2014 I/NI) and 2015 (2015 I/NI), and between 2014 and 2015 berries, under irrigated (I\_2014/2015) and not irrigated (NI\_2014/2015) conditions. \Calculated with colored (black, red, and pink) cultivars only. berries were actually collected at the same developmental stages, while differences between years indicated a slight vintage effect. However, large phenotypic diversity was observed on berry weight (Figure 3). Water deficit induced a slight shift toward smaller berry size in 2014, with the major class below 1.5 g and between 1.5 and 2.5 g per berry, respectively, in non-irrigated and irrigated berries. Distribution of berry sizes was not impacted by irrigation in 2015. Large variations were also observed for δ <sup>13</sup>C values within the collection (Figure 4). Irrigation induced larger shifts in 2014 than in 2015 and the whole population showed much lower values in 2015 than in 2014, regardless of the irrigation regime. Berry weight was significantly lower in not-irrigated berries in 2014 but not in 2015. Irrigation induced significant differences on the δ <sup>13</sup>C values in both vintages, but water stress was much lower in 2015with δ <sup>13</sup>C values significantly higher than in 2014. Taken together, these data indicate that irrigation induced a marked contrast in 2014 but a very limited one in 2015. ANOVA analysis performed on the 105 polyphenol variables expressed in mg per g of berry (Table S2) showed that most tannins and flavonols and of their total concentrations were significantly reduced by irrigation in 2014 but not in 2015. In 2015, the concentrations of cis-resveratrol and piceatannol were significantly increased by irrigation and that of glucogallin was significantly reduced. Significant vintage effect was also found on over 50 compounds, with significantly higher levels in 2015 for the majority of them, except gallocatechin and epigallocatechin which were more abundant in 2014. When the analysis was performed on the data expressed per berry (Table S2), no significant difference was found in the levels of phenolic compounds between irrigated and not irrigated conditions in 2015 whereas seven compounds from the flavan-3-ol family and oxidized glutathione were significantly increased by irrigation in 2014. Numerous compounds, distributed within all polyphenol families, were significantly higher in 2015 than in 2014, as well as total flavan-3-ols, flavonols, hydroxybenzoic acids, and hydroxycinnamic acids. One way ANOVA was also performed separately on the complete 2014 and 2015 data sets (Table 3). There was no statistically significant difference between irrigated and not-irrigated conditions (at p = 0.05) in 2015 on polyphenol composition. In contrast, in 2014, irrigation induced significant changes in the content (per berry) of 16 polyphenols and in the concentration (per g of berry) of 47 compounds. Taken together, these results indicate that berries were probably not exposed to any sufficient water stress regime in 2015 to induce changes in their phenolic composition. Consequently, data from 2015 were not further explored in this study. # Impact of Water Stress on Polyphenol Composition Principal component analysis (PCA) was performed on the phenolic composition data of all berry skin samples collected in 2014, expressed in mg per g of fresh berry. Projection of the samples on the first two principal components, accounting together for 37% of the variance, showed large cultivar differences, as well as a strong impact of irrigation (Figure 5A). White and red cultivars were separated along the first axis which was negatively correlated with the concentrations of most phenolic compounds, including anthocyanins, especially delphinidin, petunidin, and malvidin 3-glucosides, myricetin, laricitrin, and syringetin glycosides, hydroxybenzoic acids, especially gallic and syringic acids, and epigallocatechin, both in the free form and as terminal units of proanthocyanidins (Figure 5B). Non-irrigated samples generally appeared shifted negatively along the first axis, indicating that they contained higher levels of these molecules. ANOVA analysis of variance performed on the polyphenol composition data set expressed per g berry (Table 3) indicated that berries from irrigated vines contained significantly lower concentrations of the cis isomers of resveratrol and piceid, of all tannin units determined after phloroglucinolysis, and of most benzoic acids, hydroxycinnamic acids, and flavonols. The concentrations of some anthocyanins, namely 3-glucosides of pelargonidin, delphinidin, petunidin and malvidin, cyanidin 3,5 diglucoside and petunidin 3,5-diglucoside were also significantly decreased, as well as those of some anthocyanin derivatives, namely pyranoanthocyanins, tannin-anthocyanin adducts, and caftaric anthocyanin adducts. Other variables such as the concentrations of flavan-3-ol monomers were not significantly modified. When PCA was performed on the phenolic composition data expressed per berry (Figure 6A), most samples appeared shifted along the first and/or second axis, but in different directions. Again, white cultivars were separated from red cultivars along the first axis, which was negatively associated with the same phenolic compounds as in the previous PCA (Figure 6B). When the data was expressed per berry, 16 compounds were significantly increased in berries from irrigated samples (Table 3). Thus, water stress induced a significant decrease of the biosynthesis of catechin and epicatechin, both as flavan-3-ol monomers and as constitutive units of proanthocyanidins, total flavan-3-ols, phenyl- and catechyl-pyranoanthocyanins, caftaricanthocyanin adducts, (epi)catechin-ethyl-malvidin-3- glucoside, caffeic acid, and piceatannol. Moreover, some qualitative flavan-3-ol variables, namely tannin mDP, and % trihydroxylated tannin units were significantly reduced by irrigation. Unsupervised hierarchical clustering of metabolites and cultivars affected by drought was performed on the response of polyphenol composition to water status, with data expressed as log (irrigated/non-irrigated), with polyphenol contents expressed per berry. The resulting plot (Figure 7) shows different response patterns for different cultivars and for the different groups of analytical variables. Groups of compounds whose content varies in the same direction in response to irrigation can be distinguished. Cluster a* contained mainly mono- and di-hydroxylated flavonols and dihydroflavonols (astilbin and engeletin, respectively mono- and dihydroxylated on the Bring). The most abundant flavanol subunits (and also the sum of tannins) were grouped in cluster b, and linked with cluster c containing anthocyanin-flavanol derivatives linked with an ethyl-bridge. Cluster d grouped hydroxycinnamates and several of their reaction products with anthocyanins (pyranoanthocyanins and caftaric-anthocyanins). Most of the flavanol monomers and terminal units are clustered in the close e1 and e2. Clustersf1 and f2 contained respectively mono-and dihydroxylated anthocyanins along with some of their derivatives and trihydroxylated anthocyanins. The latter encompassed g1 and g2, containing trihydroxylated flavonols. It is also noticeable that β-glucogallin was included in f2. All stilbenes shared the same response to irrigation and were clustered in cluster h. The same data (log (irrigated/non-irrigated), calculated from polyphenol concentrations expressed in mg berry−<sup>1</sup> ) was used to establish the correlation network shown in Figure 8. Only the correlations >0.8 are presented. Major clusters corresponded to stilbenes (A), native anthocyanins derived from delphinidin, petunidin, and malvidin (B), from peonidin (C), and from pelargonidin (D), caftaric and coutaric acids (E), kaempferol and quercetin 3-glucosides (F), catechin and gallocatechin monomers (G), (epi)catechin units of tannins (H), (epi)gallocatechin units of tannins (I), and anthocyanin derivatives, especially phenylpyranoanthocyanins and caftaric-anthocyanin adducts (J). Two additional clusters consisted of pyranopeonidin 3 glucoside, cyanidin 3-acetylglucoside and cluster D (K) and pyranomalvidin 3-glucoside with the 3-acetylglucosides of delphinidin and petunidin (L). Berry anthocyanin, flavonol, hydroxycinnamic acid, and stilbene contents were increased or decreased under irrigated conditions in some cultivars. Groups of cultivars whose composition varies similarly in response to irrigation are clustered together (Figure 7). For example, irrigation resulted in increased and decreased stilbene levels in most cultivars of group 1 and group 2, respectively. The opposite pattern was observed for flavan-3-ols. The distribution of colored (i.e., black, red, and pink) cultivars and white cultivars, and that of the three genetic groups (WW, WE, TE) in some of the subgroups defined by unsupervised hierarchical clustering has been compared to that of the whole population (Figure 9). Chi2 tests performed on each subgroup showed that colored cultivars are overrepresented in subgroups 1-2-1 and 2-1-1 and underrepresented in subgroup 2-2-2 and cultivars from WW origin are overrepresented in subgroup 2-1-2 (Table S3). Although both contain mostly colored cultivars, subgroups 1-2-1 and 2-1-1 show different response to irrigation, with decreased anthocyanins (Figure 7, f1,f2), Bring trihydroxylated flavonols (g1, g2) and stilbenes (h) in the latter, and reduced tannins (b, e1, e2) and increased stilbenes (h) in the former. Distributions of the harvest dates for each group under irrigated and not-irrigated conditions were also examined (Figure 10). Chi-2 tests (Table S3) performed on the entire population showed that the distribution of harvest dates was similar for all subgroups under irrigated conditions but significantly different under not irrigated conditions. Chi-2 test values calculated for each subgroup indicated that the distribution of harvest dates in some of them was significantly different from that of the whole population, although the difference was significant at p = 0.05 only for 2-2-1. Thus, not irrigated cultivars of subgroups 1-1-2 and 1-2-1 and cultivars of subgroups 2-1-1, 2-2-1, and 2-2-2 were harvested earlier and later, respectively, and cultivars of subgroup 1-2-1 were shifted toward later harvest dates under irrigated conditions. # DISCUSSION ## Cultivar Differences in the Polyphenol Composition of Grape Berry Skins Major polyphenol families detected in berry skin samples were flavan-3-ols, including monomers and proanthocyanidins, anthocyanins, flavonols, hydroxycinnamic acids, and stilbenes, along with lower amounts of dihydroflavonols and benzoic acids, as classically reported. All families showed wide ranges of concentrations across the diversity panel. Anthocyanin contents enable distinction between white, pink, and red cultivars (Castellarin and Di Gaspero, 2007; Pelsy, 2010) although white grape berries also contain trace amounts of anthocyanin pigments (Arapitsas et al., 2015). Red cultivars were also characterized by the presence of flavonols with trihydroxylated B-rings, i.e., derived from myricetin, laricitrin, and syringetin glycosides, which are known to be specific of red cultivars (Mattivi et al., 2006). Cultivar differences in hydroxycinnamic acid contents have also been reported and related to differences in cultivar susceptibility to enzymatic browning (Cheynier et al., 1990). Genetic determinism of flavonols has been studied through QTL analysis (Malacarne et al., 2015), but this information is still lacking for stilbenes. However, their concentrations are also believed to highly depend on environmental factors as they are involved in plant defense against UV exposure and fungal attacks (Teixeira et al., 2013). It is noteworthy that some of the samples contained very high levels of flavonols and especially of quercetin and kaempferol derivatives compared to values reported earlier (Mattivi et al., 2006). This may be related to environmental conditions as berry concentration of quercetin glycosides have been shown to increase dramatically following sunlight exposure (Price et al., 1995; Spayd et al., 2002; Downey et al., 2004). Similarly, the lack of correlation between the flavan-3-ol contents of berries collected from a population of 141 grapevines cultivars over 2 successive years indicated that tannin accumulation is mostly driven by environmental factors rather than genetically determined (Huang et al., 2012b). In contrast, qualitative profiles within the different polyphenol groups are known to be cultivar characteristics. Thus, chemotaxonomic approaches based on grape anthocyanin profiles (Roggero et al., 1988; Mazza, 1995; Fournier-Level et al., 2009) or hydroxycinnamic acid profiles (Boursiquot et al., 1986) have been proposed. Skin flavan-3 ol composition also appeared highly conserved between years, meaning that it is mostly linked to genetic factors (Huang et al., 2012b). Our data, showing high correlations between years and between irrigation regimes for qualitative polyphenol variables and low correlations as well as strong vintage effect for quantitative variables (Table 2), confirm that the polyphenol profiles depend on cultivar while contents are affected by environmental factors, as reported in the above cited literature. In addition to the expected native anthocyanins, several anthocyanin derivatives were detected. Among them, caftaric anthocyanin adducts were present only in trace amounts. As these adducts result from enzymatic oxidation catalyzed by grape polyphenoloxidase (Sarni-Manchado et al., 1997), this indicates that no enzymatic oxidation took place during sample preparation. Pyranoanthocyanins and carboxypyranoanthocyanins resulting from reaction of anthocyanins respectively with acetaldehyde (Cheynier et al., 1997) and pyruvic acid (Fulcrand et al., 1998) have been reported in grape (Arapitsas et al., 2015). Anthocyanin dimers have also been isolated from grape skins (Vidal et al., 2004b). Strong correlations between the levels of malvidin-3-glucoside and peonidin 3 glucoside and those of vitisin B and pyranopeonidin 3-glucoside, respectively (Figure 1), substantiate the hypothesis that these compounds are formed in vivo. Moreover, the high level of vitisin B detected in some cultivars indicates that acetaldehyde is present in subcellular compartments in rather large amounts together with anthocyanins. Two major groups of tannin-anthocyanin reaction products were also detected. Flavanol-anthocyanin adducts resulting from cleavage of tannins followed by addition with anthocyanins have been detected in wine (Salas et al., 2004) and in various fruits including grapes (Gonzalez-Paramas et al., 2006). Flavanol-ethyl-anthocyanins resulting from condensation of anthocyanins and flavanols with acetaldehyde are well known to occur in wine (Timberlake and Bridle, 1976; Arapitsas et al., 2012) and have been detected in cranberry extracts (Tarascou et al., 2011). Reactions of (epi)catechin, anthocyanins, and acetaldehyde yield complex mixtures of TABLE 3 \| Results of the ANOVA performed on the data of irrigated (I) and non-irrigated (NI) vines separately on 2014 and 2015; polyphenol composition data in and microgram per berry and microgram per g of berry; variable codes are provided in Table 1. (Continued) #### TABLE 3 \| Continued (Continued) #### TABLE 3 \| Continued <sup>a</sup>Variable codes as in Table 1. \SNK: results of the Student-Newman-Keuls grouping (p < 0.05). <sup>b</sup>A few missing values have been removed from the calculation. products, including pyranoanthocyanin, (epi)catechin-ethylanthocyanin, and (epi)catechin-ethyl-(epi)catechin derivatives (Vallverdú-Queralt et al., 2017a,b). Molecules of the last group have been detected in wine (Cheynier et al., 1997) but this is the first report of their presence in grape. Flavanol-anthocyanins correlated with their anthocyanin precursors (Figures 1, A* and B) while flavanol-ethyl-anthocyanins formed a specific cluster (Figure 1, H). Although these molecules could also form during sample preparation, the levels reported here and the relatively long reaction rates compared to the duration of our extraction procedure suggest that they were present in planta. Other correlations networks established for the 2014 data only (not shown) showed additional relationships between malvidin-3-glucoside and anthocyanin dimers (malvidin 3-glucoside and malvidin 3-glucoside–peonidin 3-glucoside), and syringic acid that arises from degradation of malvidin (Furtado et al., 1993; Vallverdú-Queralt et al., 2016) and between peonidin 3-glucoside and vanillic acid. Formation of syringic and vanillic acids respectively from malvidin 3-glucoside–peonidin 3-glucoside can be promoted by light and heat exposure (Furtado et al., 1993). Correlations between variables (Figure 1) can be interpreted in terms of biosynthetic pathways. Indeed, anthocyanin and FIGURE 5 \| PCA of the MRM phenolic composition data of berry skin samples collected in 2014 (mg g−<sup>1</sup> ); (A), projection of the samples on PC1 and PC2; red and white cultivars are represented in red and in green, respectively; IR, irrigated, NI, not-irrigated. (B), loadings of the variables (coded as in Table 1) on PC1. AN, native anthocyanins+dimers; AP, pyrano anthocyanins; AF, anthocyanin-flavanol adducts; AC, caftaric-anthocyanin adducts; HF, dihydroflavonols; FO, flavonols; ST, stilbenes; FA, flavanols (tannins); HB, hydroxybenzoic acids; HC, hydroxycinnamic acids; OT, others. FIGURE 6 \| PCA of the MRM phenolic composition data of berry skin samples collected in 2014 (mg berry−<sup>1</sup> ); (A), projection of the samples on PC1 and PC2; red and white cultivars are represented in red and in green, respectively; IR, irrigated; NI, not-irrigated. (B), loadings of the variables (coded as in Table 1) on PC1. AN, native anthocyanins+dimers; AP, pyrano anthocyanins; AF, anthocyanin-flavanol adducts; AC, caftaric-anthocyanin adducts; HF, dihydroflavonols; FO, flavonols; ST, stilbenes; FA, flavanols (tannins); HB, hydroxybenzoic acids; HC, hydroxycinnamic acids; OT, others. flavonol variables clustered together according to their B-ring substitution (trihydroxylated/dihydroxylated) and/or acylation pattern. Clustering of those compounds according to their B-ring hydroxylation pattern is consistent with the ability of F3'H and F3'5'H to use both anthocyanin and flavonols as substrate (Bogs et al., 2006). Flavonols clustered in three correlation networks corresponding to B-ring trihydroxylated compounds (myricetin, laricitrin, and syringetin derivatives) and other (mono or dihydroxylated) flavonols and substitution by glucose or glucuronic acid. This probably reflects the high sugar specificity of the already described Vitis flavonol glycosyltransferases: when VvGT5 is quite exclusively a glucuronyl donor, VvGT6 catalyzes both flavonol glucosylation and galactosylation (Ono et al., 2010). Flavan-3-ols also clustered following their B-ring hydroxylation pattern, (epi)-catechin flavanol units, and (epi)gallocatechin units forming different groups. Strong correlations between terminal units and the corresponding monomers likely reflect the analytical method as monomers contribute to terminal units. Moreover, upper units, detected as the corresponding phloroglucinol adducts, were separated from terminal units, suggesting that both types of units have different precursors, as already suspected (Stafford et al., 1982; Huang et al., 2012a). Acetylated anthocyanin derivatives were correlated, regardless of the anthocyanin B-ring substitution while glucosylated, coumaroylated, and caffeoylated anthocyanins derived from malvidin, petunidin, and delphinidin (trihydroxylated) and from other anthocyanidins formed distinct groups. Although the already characterized acyltransferase Vv3AT is able to use both aliphatic and aromatic acyl-CoA as substrate (Rinaldo et al., 2015), this suggests that anthocyanin acylation with acetic acid and with hydroxycinnamic acids could involve alternative biosynthetic mechanisms (Bontpart et al., 2015). Finally, correlations of anthocyanins with their derivatives and degradation products (Figure 1, clusters A and B), and clustering of molecules such as anthocyanin dimers (Figure 1, F), flavanol-ethyl anthocyanins (Figure 1, H) and hydroxyphenyland catechyl-pyranoanthocyanins, resulting from anthocyanin reactions with p-coumaric and caffeic acid (Figure 1, G) reflect their formation from the same precursors and/or through identical reaction mechanisms. ### Impact of Water Deficit on the Polyphenol Composition of Grape Berry Skins Not-irrigated vines suffered water stress in 2014 but not in 2015. Indeed, water stress classically induces a decrease in berry weight (Roby et al., 2004; Bucchetti et al., 2011). In 2015, irrigation had no significant impact on berry weight, which indicates that berries were not exposed to any sufficient water stress regime to induce phenotypic changes. Corroborating this hypothesis, in 2015, berry weights were higher and δ <sup>13</sup>C values were lower than those of berries from irrigated vines in 2014. Accordingly, none of the polyphenol variables showed significant differences between berries from irrigated and not irrigated vines (Table 3). In contrast, in 2014, water stress induced significant loss of berry weight as well as significant differences on the concentration of several polyphenols. This confirms that polyphenols are part of the chemical arsenal allowing adaptive response to abiotic stress, being protective molecules against oxidative damages by scavenging Reactive Oxygen Species (ROS) produced during stress (Rontein et al., 2002). Not irrigated berries contained higher levels of most phenolic compounds when expressed in mg g−<sup>1</sup> fresh berry weight (Table 3, Figure 5). This concentration effect can be attributed to reduced berry size under water stress, as observed earlier (Roby et al., 2004; Bucchetti et al., 2011). However, shifts between irrigated and non-irrigated samples on the PCA performed on the phenolic composition data expressed in mg per berry (Figure 6) showed that water status affected polyphenol biosynthesis. Water deficiency is known to impact berry development, decrease berry weight, and modulate accumulation of secondary metabolites including polyphenols (Kennedy et al., 2002; Roby et al., 2004). Data available on a limited number of genotypes suggest that the response to moderate water stress differs depending on the level of irrigation and/or water stress, on the berry development stage when water deficit occurs and on the cultivar (Ojeda et al., 2002; Teixeira et al., 2013). Thus, several studies have shown an increase in the accumulation of stilbenes, flavonols, and anthocyanins and enhanced transcription of genes involved in these pathways following moderate water deficiency while other studies failed to observe these effects or even observed a decrease. In Syrah, water deficit applied before or after veraison resulted in an increase of total anthocyanin contents and differences in the anthocyanin profiles (Ollé et al., 2011). Data on the effect of environment on tannin biosynthesis is still scarce: water deficiency in Cabernet Sauvignon (Kennedy et al., 2002; Castellarin et al., 2007) or in Syrah (Ollé et al., 2011), or thermic variation in Merlot (Cohen et al., 2012) did not affect tannin accumulation. In another study, a decrease or increase in tannin accumulation was reported in Syrah exposed respectively to early (between anthesis and veraison) or late (after veraison) water stress (Ojeda et al., 2002). However, tannin accumulation might also be related to biotic stress exposure (Dixon et al., 2005). As well, the significant increase of cis-resveratrol and piceatannol concentrations observed in 2015 under irrigated conditions may be due to plant response to increased fungal pressure as stilbenes are known to be involved in defense against fungi (Jeandet et al., 2002). ANOVA performed on the 2014 samples (Table 3) showed that the levels of 16 MRM variables expressed per berry were significantly higher in irrigated berries. Tannins were the major family affected by irrigation, with both quantitative (increase of catechin and epicatechin, detected as monomers, and as terminal and upper units of tannin chains, and of total flavan-3-ol levels) and qualitative (decrease of % B-ring trihydroxylated units and mean DP) variations. A decrease of tannin DP in irrigated vines has been reported earlier (Ojeda et al., 2002) and the proportion of B-ring trihydroxylated units was reduced in shaded berries (Cortell and Kennedy, 2006). Other affected compounds included piceatannol, caffeic acid, and pigments resulting from reactions of anthocyanins with hydroxycinnamic acids, i.e., caftaric-anthocyanin adducts, phenylpyranoanthocyanins and catechyl-pyranoanthocyanins. Several groups of variables were affected by irrigation in the same way and formed clusters on the correlation networks established from the response of MRM variables to irrigation (log; irrigated/non-irrigated of the concentrations expressed in mg berry−<sup>1</sup> , Figure 8). Thus, clustering of phenylpyranoanthocyanins, catechylpyranoanthocyanins, and caftaric-anthocyanin adducts indicated that they were simultaneously increased upon irrigation. Stilbenes formed another cluster, indicating that they were not only closely related, as shown by clustering of their concentrations (Figure 1, L) but also impacted in the same way by water deficit (Figures 7, h, 8, A). Transcription of genes involved in the biosynthesis of stilbene precursors has been shown to increase and decrease in response to water stress in Cabernet Sauvignon and Chardonnay, respectively (Deluc et al., 2011). Other clusters grouped together members of the different flavonoid families (anthocyanins, flavan-3-ols, and flavonols) which were further sorted according to their B-ring substitution pattern. Thus, malvidin, delphinidin, and petunidin derivatives (trisubstituted on the B-ring) and peonidin derivatives (disubstituted on the B-ring) formed different groups (Figures 7, f1 and f2, 8, B and C). Water deficit has been shown to enhance expression of flavonoid 3′ ,5′ -hydroxylase (F3′ 5 ′H), involved in B-ring trihydroxylation, relative to that of flavonoid 3 ′ -hydroxylase (F3′H), involved in B-ring dihydroxylation, and, consequently, to increase the proportion of B-ring trihydroxylated anthocyanins (Castellarin et al., 2007). An increase of O-methyltransferase expression also correlated with accumulation of malvidin and peonidin derivatives (Castellarin et al., 2007). However, in another study, water deficit applied before and after veraison affected anthocyanin composition differently, enhancement of malvidin accumulation being observed only with post-veraison stress (Ollé et al., 2011). Among flavonols, drought responses of kaempferol and quercetin glucosides (respectively mono and dihydroxylated on the B-ring) were correlated (Figures 7, a, 8, F). Quercetin glycosides have been shown to accumulate following UV exposure of the berry (Price et al., 1995) and are believed to play a role in UV protection. Interestingly, expression of F3′ 5 ′H and biosynthesis of B-ring trihydroxylated anthocyanins were reduced in tissues protected from light exposure by shading of the berries or accumulation of phenolic compounds acting as UV screens in external tissues (Guan et al., 2014). The presence of β-glucogallin in the same response group to water status as trihydroxylated flavonols and anthocyanins is unexpected. This compound is suspected to be an intermediate in the flavanol galloylation pathway (Bontpart et al., 2015). However, glucose ester of hydroxybenzoic acid was already described as glucose donor for flavonoid glucosylation (Nishizaki et al., 2013). In the flavanol family, (epi)catechin based upper tannin units clustered with terminal catechin (Figure 8, H) and (epi)gallocatechin tannin units formed a different cluster (Figure 8, I), again indicating different responses of tannins based on trihydroxylated and dihydroxylated B-rings. However, catechin and gallocatechin monomers formed another group (Figure 8, G), suggesting that biosynthesis of flavan-3-ol monomers and of proanthocyanidins are differently regulated. The last two clusters (L and K) were similarly based on dihydroxylated and trihydroxylated anthocyanin B-rings, respectively. However, unexpected correlations of these molecules with pyranoanthocyanins derived from reaction of acetaldehyde with other anthocyanins require further investigation. # Genetic Diversity of Grapevine Response to Drought Shown by Metabolomics Grouping of polyphenols according to their drought response across the diversity panel provided confirmation of the results of variance analysis (e.g., for flavan-3-ols). Additional correlation networks between molecules that had not been detected in the global ANOVA treatment suggest different responses of the molecular clusters in different cultivars. Unsupervised hierarchical clustering of cultivars and metabolites (including MRM data and calculated variables) affected by drought (Figure 7) was performed to explore this hypothesis. Molecules clustered by family and, within some families, by B-ring substitution pattern, confirming the impact of irrigation on some specific branches of the biosynthetic pathway shown by the correlation networks. Clustering of cultivars confirmed cultivar differences in the molecular response to drought. Some of these differences may be related to differences in polyphenol metabolism as some cultivars accumulate specific polyphenol classes (e.g., colored vs. white cultivars). Three cultivar subgroups comprised an excess of colored or white cultivars, compared to the whole population (Figure 9). However, color did not fully explain the clustering based on polyphenol response to drought. Genetic groups did not appear as a major factor although cultivars from genetic group WW were overrepresented in subgroup 2-1-2. Moreover, cultivars from group 1 were generally harvested earlier under not irrigated conditions than those of group 2 (Figure 10). Differences in precocity may also induce different polyphenol response to irrigation as some compounds such as flavan-3-ols and hydroxycinnamic acids are biosynthesized at early development stages while anthocyanins and flavonols accumulate after veraison. Finally, some of the observed responses may be indirect responses, for instance due to differences in cultivar responses to other types of stresses, such as UV-stress since water regime also impacts canopy (for instance for flavonols), or biotic stress (for stilbenes). This preliminary study is based on only two vintages, with no treatment replicate for individual cultivars. However, some cultivars with contrasted responses to water stress have been identified and could be used in future more detailed studies. In particular, it will be of interest to also characterize the plant physiological status, to determine if these contrasted behaviors are related to the near iso/anisohydric phenomenon, analyze their stability and explore potential interferences with phenological stages and environmental factors. ### Large Scale Metabolomics Studies Shedding Light on Polyphenol Composition The MRM method used in this study was targeted on a large number of polyphenolic compounds, including 96 molecules analyzed directly and 9 additional compounds released after acid-catalyzed depolymerization of proanthocyanidins in the presence of pholoroglucinol. Such experiments had never been performed at a large scale, in terms of number of targeted molecules and of number of studied cultivars. The large data collected made it possible to establish correlation networks that confirmed previous knowledge and provided new information on grape polyphenol metabolism. Conversely, the patterns established validate interpretation of mass spectrometry data for most of the compounds analyzed. However, a few compounds appeared as outliers in some of the clusters, raising questions on their attribution. For example, the signal attributed to petunidin-3,5-diglucoside clustered with pigments derived from reactions of anthocyanins with phenolic acids, suggesting confusion or contamination with another molecule of this group. This will be explored further, potentially leading to the discovery of new compounds. Similarly, clustering of β-glucogallin with B-ring trihydroxylated anthocyanins and flavonols is surprising. This may be related to a role of glucogallin in their biosynthesis, as proposed above. However, formation of β-glucogallin may also reflect degradation of delphinidin and/or myricetin, followed by glucosylation of the resulting gallic acid. In this case, other anthocyanins are expected to follow the same catabolic process. For example, degradation of malvidin or syringetin and of peonidin or isorhamnetin should similarly yield syringoyl- and vanilloyl-glucose which have not been included in the molecular targets of the MRM method. Detection of these new molecules would help validate this hypothesis. # CONCLUSIONS A large scale experiment involving cultivation of an association panel of 279 V. vinifera cultivars designed to represent the genetic and phenotypic variation encountered in cultivated grapevine and metabolomics analysis targeted to a large number of polyphenolic compounds (polyphenomics) was performed in 2014 and 2015. Chemometrics analysis of the data showed large differences in polyphenol composition related to genetic factors, environmental factors (i.e., water stress), and genetic x environment interactions. Correlation networks shed light on the relationships between the different polyphenol metabolites and related biosynthetic pathways. In addition, detailed polyphenomics analysis confirmed that polyphenol reactions described in wine take place in the berries. Finally, this paper reports the first large scale study demonstrating an influence of water stress on the different classes of polyphenols but also cultivar differences in the types and extents of drought responses, with different molecules affected either positively or negatively and different impacts on grape and wine quality. This work will be the foundation for identifying the genetic basis of the drought differential response of the cultivars in term of polyphenol composition, through Genome-Wide Association Study. # AUTHOR CONTRIBUTIONS LP and AV developed the MRM methods and performed the analyses, MR developed and applied the extraction protocols, EM and AVQ interpreted the mass spectrometry data, NS supervised the metabolomics analysis, JB and NT performed the chemometrics analysis, LL, JP, AA, NT, NS, and VC conceived the designed research and interpreted the results. All authors contributed to drafting and/or critical revision of the work and approved the manuscript. #### FUNDING The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under the grant agreement no FP7-311775, Project Innovine. Financial support from GIS IBiSA (Infrastructures en Biologie Santé et Agronomie), Région Languedoc Roussillon, and INRA CNOC for funding of the UPLC–MS equipment and funding from Alfonso Martín Escudero Foundation for the postdoctoral fellowship of AVQ are also acknowledged. #### REFERENCES #### ACKNOWLEDGMENTS The authors gratefully acknowledge Gilles Berger, Yves Bertrand, Jean-Luc Guiraud, Thérèse Marlin, and Léa Ollier for technical assistance. #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017. 01826/full#supplementary-material Table S1 \| List of cultivars collected in 2014 and/or 2015, with their codes, genetic groups, colors, and harvest dates under irrigated (I) and not irrigated (NI) conditions. affected by interseason weather variability. J. Agric. Food Chem. 65, 5868–5878. doi: 10.1021/acs.jafc.7b01466 in grape skins. J. Agric. Food Chem. 52, 7144–7151. doi: 10.1021/jf0 48939h Vrhovsek, U.,Masuero, D., Gasperotti, M., Franceschi, P., Caputi, L., Viola, R., et al. (2012). Versatile targeted metabolomics method for the rapid quantification of multiple classes of phenolics in fruits and beverages. J. Agric. Food Chem. 60, 8831–8840. doi: 10.1021/jf2051569 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 Pinasseau, Vallverdú-Queralt, Verbaere, Roques, Meudec, Le Cunff, Péros, Ageorges, Sommerer, Boulet, Terrier and Cheynier. This is an openaccess 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. # Differences in Flower Transcriptome between Grapevine Clones Are Related to Their Cluster Compactness, Fruitfulness, and Berry Size #### Jérôme Grimplet\, Javier Tello † , Natalia Laguna and Javier Ibáñez Departamento de Viticultura, Instituto de Ciencias de la Vid y del Vino (Consejo Superior de Investigaciones Científicas, Universidad de La Rioja, Gobierno de La Rioja), Logroño, Spain #### Edited by: Giovanni Battista Tornielli, University of Verona, Italy #### Reviewed by: Gregory Alan Gambetta, Ecole Nationale Supérieure des Sciences Agronomiques de Bordeaux-Aquitaine, France Silvia Dal Santo, University of Verona, Italy #### \Correspondence: Jérôme Grimplet [email protected] #### † Present Address: Javier Tello, Division of Viticulture and Pomology, Department of Crop Sciences, University of Natural Resources and Life Sciences Vienna (BOKU), Tulln, Austria #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 16 January 2017 Accepted: 07 April 2017 Published: 27 April 2017 #### Citation: Grimplet J, Tello J, Laguna N and Ibáñez J (2017) Differences in Flower Transcriptome between Grapevine Clones Are Related to Their Cluster Compactness, Fruitfulness, and Berry Size. Front. Plant Sci. 8:632. doi: 10.3389/fpls.2017.00632 Grapevine cluster compactness has a clear impact on fruit quality and health status, as clusters with greater compactness are more susceptible to pests and diseases and ripen more asynchronously. Different parameters related to inflorescence and cluster architecture (length, width, branching, etc.), fruitfulness (number of berries, number of seeds) and berry size (length, width) contribute to the final level of compactness. From a collection of 501 clones of cultivar Garnacha Tinta, two compact and two loose clones with stable differences for cluster compactness-related traits were selected and phenotyped. Key organs and developmental stages were selected for sampling and transcriptomic analyses. Comparison of global gene expression patterns in flowers at the end of bloom allowed identification of potential gene networks with a role in determining the final berry number, berry size and ultimately cluster compactness. A large portion of the differentially expressed genes were found in networks related to cell division (carbohydrates uptake, cell wall metabolism, cell cycle, nucleic acids metabolism, cell division, DNA repair). Their greater expression level in flowers of compact clones indicated that the number of berries and the berry size at ripening appear related to the rate of cell replication in flowers during the early growth stages after pollination. In addition, fluctuations in auxin and gibberellin signaling and transport related gene expression support that they play a central role in fruit set and impact berry number and size. Other hormones, such as ethylene and jasmonate may differentially regulate indirect effects, such as defense mechanisms activation or polyphenols production. This is the first transcriptomic based analysis focused on the discovery of the underlying gene networks involved in grapevine traits of grapevine cluster compactness, berry number and berry size. #### Keywords: Vitis vinifera, cluster architecture, phenotyping, transcriptomics, somatic variation # INTRODUCTION Grapevine (Vitis vinifera L.) is one of the most valuable horticultural crops in the world, with a total grape production of 77 million ton (2013, http://faostat3.fao.org). The value of any table grape, grape juice, or wine product relies fundamentally on disease-free and high quality fruits. Cluster compactness, an issue specific to grapevine, directly impacts fruit quality and disease susceptibility: 321 Berries in compact clusters tend to ripe more asynchronously, impacting quality at harvest and compact cluster are also more susceptible to diseases, such as Botrytis cinerea (Molitor et al., 2012b). Cluster compactness is a complex trait, resulting from the interaction of parameters related to cluster architecture and berry morphology, each contributing differently within a cultivar. Shavrukov et al. (2004) indicated the internode length of inflorescence rachis is the major trait responsible for inflorescence openness in four grape cultivars. However, a smaller berry size is responsible for loose cluster in Albariño (Alonso-Villaverde et al., 2008), while in other study, cluster density is correlated with the number of seeds per berry in the progeny of two wine grape cultivars (Bayo-Canha et al., 2012). More recently, our group has dissected the cluster compactness trait on a large set of table and wine cultivars (Tello et al., 2015). This exhaustive survey indicates that the berry number and the length of the rachis main axes (cluster architecture) are the most critical parameters for cluster compactness, followed by berry size. Each of these cluster compactness features is specific to different development stages. (i) Architecture related parameters are defined early. At the end of the first season summer, the primary latent bud contains a compressed shoot with inflorescence meristems, tendril and leaf primordia. In the second season, during initial stages of bud swelling, the inflorescence branch meristems can additionally ramify to form further inflorescence branch meristems that divide into a group of flower meristems (normally three). At that point, the inflorescence/cluster architecture is essentially set, as rachis elongation is limited after flowering (Coombe, 1995; Shavrukov et al., 2004). (ii) Final berry number in the cluster depends on the initial number of flowers and the fruit set rate that occurs after anthesis, although a compensation effect does exist (May, 2004). The initial number of flowers in the inflorescence is determined early in the second season, before bud burst, and it is noted that high temperatures at this stage decrease the number of flowers eventually formed (Ezzili, 1993). The availability of carbohydrate reserves in the trunk and roots (from the previous season) may also be a limiting factor (Bennett et al., 2002). Fruit set rate depends on the success of the pollination and fertilization processes, and also on the competition with other sink organs, mainly growing shoots. (iii) Two main factors are responsible for the size of the ripe berry at harvest: the cell number and their volume. Cell division is particularly active before anthesis and stops when the berry reach the lag phase, at the beginning of ripening (véraison). From that point only growth by cell enlargement occurs (Harris et al., 1968; Dokoozlian, 2000). Little is known about the molecular basis or genetic factors responsible for differences in cluster compactness among grapevine cultivars and clones. Experimental treatments to reduce cluster compactness involve enlarging inflorescence main axes, reducing fruit set, and/or reducing berry size. Plant hormones control grapevine reproductive development and flowering timing through the gibberellin:cytokinin balance. Gibberellins mediate the formation of the inflorescence axis, while cytokinins regulate the differentiation into flowers and are specifically needed for the growth of pistil (Pool, 1975). ABA concentration is high before anthesis, and auxin transport is needed to avoid abscission and promote fruit set (Kühn et al., 2014). The application of the gibberellins inhibitor prohexadione-Ca causes a loosening effect by reducing berry size and/or number of berries, likely through disturbing pollination and cell division processes (Molitor et al., 2011; Schildberger et al., 2011). The application of gibberellic acid pre-bloom promotes the growth of the inflorescence (Hed et al., 2011; Molitor et al., 2012a), while gibberellin treatments during bloom reduce fruit set and increase berry size (Ben-Tal, 1990). The availability of the grapevine genome sequence (Jaillon et al., 2007; Velasco et al., 2007) allowed high throughput studies of the grapevine that are leading to an increased knowledge of the molecular events occurring behind physiological processes. In this work we performed transcriptomic analyses of Garnacha Tinta clones, with stable differences in specific compactnessrelated parameters (berry number, berry size), to identify genes and gene networks involved in cluster compactness characteristics. From this transcriptomics study, 183 candidate genes were selected for an association analysis in a collection of grapevine varieties (Tello et al., 2016). # METHODS #### Plant Material In the early 2000s, Gobierno de La Rioja prospected the entire Rioja region and collected hundreds of grapevine (Vitis vinifera L.) plants of different cultivars, usually old plants and/or plants with particular characteristics. Each of these plants was multiplied by cuttings and grafted on Richter 110 rootstock. Five clonal grafted vines per original plant were planted together in a single plot at the experimental vineyard of La Grajera (Logroño, La Rioja). This clone collection includes 501 clones from Garnacha Tinta, which were screened for cluster compactness, in sequential steps. First, the compactness of all the clones was visually assessed. Then, nine clones were selected for phenotyping during the next season and six of these were also phenotyped during a second and third season. Finally, four of these clones, two with compact clusters ("compact clones") and two with loose clusters ("loose clones") were selected for transcriptomic analysis. #### Phenotyping In three successive seasons, six selected Garnacha Tinta clones were phenotyped for several variables related to cluster compactness using five clusters per clone as described by Tello and Ibáñez (2014). All the clones were subjected to pair-wise comparisons for phenotypic variables grouped in four categories: plant (e.g., fertility), cluster architecture (e.g., cluster length), fruitfulness (berry number and seed number) and berry size (Supplementary Table 1). Clone pairs differing only in one category were favored, but the most selective criterion was consistency over the seasons for the observed significant pair-wise differences, and some clone comparisons with non-consistent differences were discarded. Finally, four clones (368, 906, 1134, and 1154) were used for transcriptome analysis (Supplementary Table 2). # Experimental Design and Sampling for Transcriptome Analysis The experimental design was determined in accordance with the significant pair-wise differences consistently observed between the selected clones of Garnacha Tinta over the three seasons (Supplementary Table 1, Figure 1). Organs and stages were sampled based on specific differential parameters: berry number, seed number and berry size (Table 1). For berry number, flowers were sampled at the end of flowering (E-L 26, Coombe, 1995) before possible abscission or set (Table 1; comparisons G1-26, G2-26, G3-26, and G4-26). Seed number is also determined at that step since it depends on the success of pollination. Spring buds at budburst were sampled in two clones to study the initial number of flowers (E-L 3: comparison G4-03), when flowers start to differentiate (Pouget, 1981; Dunn and Martin, 2000). Berry size is determined by cell division and cell expansion. So, analyses for berry size were carried out on flowers at the end of flowering, when cell division is active (E-L 26; comparison G1-26), and on green berries at the beginning of véraison (E-L 34), when cell division is complete and berry enlargement by cell expansion begins (Dokoozlian, 2000; comparison G1-34). As berries are in different developmental stages within the same cluster at a given time, sampled berries were classified according to their density by flotation on NaCl solutions (Carbonell-Bejerano et al., 2016). Green berries floating in a solution of 80 g/l NaCl and sinking in a solution of 60 g/l NaCl were selected. Three replicate samples were collected from different vines. After collecting, samples were immediately frozen in liquid nitrogen, and then kept in the laboratory at −80◦C until RNA extraction. # RNA Extraction and Microarray Hybridization Total RNA was extracted from samples using the Spectrum plant total RNA kit (Sigma, www.sigmaaldrich.com) as recommended by manufacturer. DNase I digestion was carried out with the RNase-Free DNase Set (QIAGEN). RNA integrity and quantity were assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific) and an Agilent's Bioanalyzer 2100. Microarray hybridizations were performed at the Genomics Unit of the National Centre for Biotechnology (CNB-CSIC, Madrid). Synthesis of cDNA, labeling, hybridization, and washing steps were performed according to the NimbleGen arrays user's guide. Each sample was hybridized to a NimbleGen microarray 090818 Vitis exp HX12 (Roche, NimbleGen), which contains probes targeted to 29,549 predicted grapevine genes and 19,091 random probes as negative controls. Images were analyzed using NimbleScan v2.6 software (Roche), which produces.xys files containing the raw signal intensity data for each. TABLE 1 \| Experimental design for each of the comparisons performed between Garnacha Tinta clones. Organs and sampling stages for transcriptomics analyses were chosen based on the stable phenotypic differences found in three seasons. C, Compact clone; L, Loose clone. #### Microarray Data Processing The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE67708 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67708). Raw intensity values were processed using the R package oligo (Carvalho and Irizarry, 2010). Individual probes raw expression values were computed from.xys files and the in house pd info builder package pd.vitus.exp.vitnames designed to fit the 12Xv1 annotation nomenclature. Normalization was performed with Robust Multi-Array Average (RMA; Irizarry et al., 2003). Resulting RMA expression values were log2 transformed. Distributions of expression values processed via RMA of all arrays were very similar with no apparent outlying arrays. #### Microarray Data Analysis Each condition (clone × stage/organ) was performed in three biological replicates. Differential expression analyses of the comparisons presented in Table 1 were performed with the ebayes (Smyth, 2004) method from the package limma in R. The cutoff of differentially expressed genes was set to a p < 0.05 after Benjamini-Hochberg correction with at least a 2-fold ratio difference of expression. Principal component analysis (PCA) was performed in R using the pca package with the ppca method. Hierarchical clustering was performed using MultiExperiment Viewer (Saeed et al., 2003) based on Pearson's correlation and using the average linkage option and optimal gene ordering. The stringent set was obtained by clustering genes with a distance threshold <0.05. The tolerant set was obtained by clustering genes with a distance threshold <1.5. To identify the biological functions over-represented within selected probe sets, functional enrichment analyses were performed using FatiGO (Medina et al., 2010; P < 0.05). Functional categories were based on manual annotation of 12Xv1 grape genome assembly, described in Grimplet et al. (2012). #### Cytoscape/VitisNet Analysis Expression data were uploaded in Cytoscape version 3 (Shannon et al., 2003) and analyzed with VitisNet (Grimplet et al., 2012). According to FatigoGO analysis, networks related to enriched categories were selected for manual inspection. The visual style in the figures was designed to best represent changes in flower by including notifications of the genes over-expressed in compact or loose clones. A color gradient was used depending on the presence of the differentially expressed gene (DEG) in 2 or 3 (light color) to 4 (dark color) comparisons, to have a visual representation of the DEG degree of recurrence in the comparisons. Network ID corresponds to the VitisNet ID (Grimplet et al., 2009). # RESULTS AND DISCUSSION Phenotyping and Comparison of the # Clones In a multi-cultivar framework, our group identified the major morphological factors influencing the cluster compactness trait (Tello and Ibáñez, 2014; Tello et al., 2015). Different variables, classified within four major groups (plant, cluster architecture, fruitfulness (berry and seed number) and berry size), were phenotyped in a large set of diverse cultivars, and it was concluded that the length of the cluster main axes and berry number were the main discriminant variables for cluster compactness, followed by the berry size. In the present work, a similar set of variables was used to study Garnacha Tinta clones and only clone pairs consistently differing in selected variables were used for analyses. Cluster compactness remained consistent through the seasons, but some of the significant differences observed the first season were not stable over the three seasons. Therefore, some clone comparisons were discarded. Finally, four clones of Garnacha Tinta, two loose (368 and 1154) and two compact clones (906 and 1134) were used for transcriptome analysis (Table 1, Supplementary Table 2). Similar to cluster compactness, the berry number showed very consistent differences through the seasons in the four clone comparisons (Table 1, Figure 1, Supplementary Table 1). The compact clones produced significantly tighter clusters than loose clones and carried a significantly greater number of berries in all the comparisons studied during the three seasons. Berry number was the only differential variable in comparison G3, but in the remaining comparisons additional seasonally stable differences appeared in other traits. Thus, comparison G1 was selected to examine the transcriptional changes observed between a loose clone and a compact clone with significant differences in berry number and berry size. The compact clone (1134) produced more and larger berries than the loose clone (368) (Figure 1, Supplementary Table 1). TABLE 2 \| Number of differentially expressed genes (DEG) at every time point for every comparison Compact vs. Loose clone. Finally, comparisons G2 and G4 were selected to examine differences in global gene expression related to the two variables included in the fruitfulness category: berry number and seed number. In these comparisons, the compact clones always had more berries per cluster and more seeds per berry than the loose clones (Figure 1, Supplementary Table 1). This was expected, as both the number of seeds and fruit set are related to pollination and fertilization, and flower fate (abscission or berry set) greatly depends on the existence of at least one fertilized ovule in the flower (Kassemeyer and Staudt, 1982). #### Global Gene Expression Data The greatest number of differentially expressed genes (DEG) between clones was in flower at the end of flowering (E-L 26), while very few differences could be seen in spring buds (G4-03) (Table 2). Figure 2 represents the first two axes of a PCA of the expression data obtained for the four studied clones at the end of bloom, E-L 26. The first component of the PCA represented 73% of the total variation and seemed related to compactness. Component 2 accounted for 9% of the total variability, separating genotypes. The replicates from compact and loose clones were clearly separated, however clones of the same compactness presented a large variation. There were differences in the sampling dates to match physiological state but they did not seem to be related to the variation, since more variability could be observed between some replicates sampled the same day (data not shown). It is known that, within the same inflorescence, there are flowers in different stages of development, including those with already fertilized ovules, others with fertilization in progress, and others that have not been fertilized and probably will drop (Kühn et al., 2014). These flower stages are not visually distinguishable during sampling, but their transcriptomic profiles are probably different, because there are evidences in grapevine indicating that pollination rapidly modifies gene expression (Kühn and Arce-Johnson, 2012). The proportion of flowers in each of those stages would vary differently between clones, partially explaining the consistent differences in the number of berries observed in the four comparisons. This is probably the major cause for the gene expression differences observed in the four pair-wise comparisons at the end of flowering (Table 2, E-L 26). At that stage, a greater number of DEG was observed in comparisons involving the compact clone 1134 (G1-26 and G2- 26, 5320 and 4446 genes, Table 2) than in comparisons with the other compact clone, 906 (2413 and 665 genes). That variation may be the result of an asynchronous floral development in clone 1134, which would lead to the sampling of slightly different flower stages in the compared clones. This would be supported by the high number of DEG (1607 genes) observed at E-L 26 between compact clone 906. Red dot: Garnacha Tinta compact clone 1134. clones 1134 and 906 (Table 2). This difference is reduced later, as illustrated in comparison G1-34, where the two clones involved (1134 and 368) reached similar transcriptomes, with a minimal number of differentially expressed genes between them at E-L 34. In comparison G1, in addition to a different number of berries, a consistent difference in berry size was observed, unlike in G2. So, the differentially expressed genes found in G1-34 and (partly) in G1-26 could be related to the fact that clone 368 showed a smaller berry size than 1134 during the three studied years. The number of significant DEG obtained for the stage end of flowering (E-L 26) was much lower in loose clones (368 + 1154) than in compact clones (1134 + 906) when all comparisons are considered. Only 70 gene transcripts were more abundant in the loose clones and 400 in the compact clones (Table 2). Many genes, however, were differentially expressed between the compact clone 1134 and any loose clone (2051 genes showed a greater expression in loose clones, 1683 in the compact clone). # Functional Categories Analysis Functional categories enrichment analysis was performed in order to identify the main mechanisms impacted in cluster compactness and their related traits. Since PCA showed greatest differences in expression pattern in clones 1134 and 368, analyses were performed considering several situations: group 1 includes the genes differentially expressed in all comparisons at E-L 26 (Table 3) that are specifically related to compactness independent of the clone (400 over-expressed genes in compact clones, 70 in loose clones); group 2 contains the genes specifically regulated in the most extreme compact clone 1134, i.e., differentially expressed in G1 and G2: genes expressed in clone 1134 (2051 over-expressed genes) vs. all the loose (1703 over-expressed genes) (Table 4); and group 3 comprises the genes specifically regulated in the most extreme loose clone 368, i.e., differentially expressed in G1 and G3 comparisons: over-expressed genes in #### TABLE 3 \| Over-represented functional categories in all E-L 26 comparisons with P < 0.05. Values are expressed as log2 ratio group/genome. clone 368 (1400) vs. all the compact (560) (Table 5). If a category is enriched in both the compact and loose clones, this means that there are distinct genes from that category represented in a larger proportion in both sets than in the whole transcriptome. As indicated in 4.2, for group 1 few genes were differentially expressed in the loose clusters considering all comparisons at E-L 26; therefore, only two categories were enriched in the loose clustered type (Table 3). Overall, several patterns of expression emerged from the three enrichment analyses. Within the functional categories related to the metabolism, several functional categories indicate a dramatic shift of expression of genes involved in the metabolism. The category related to cell growth and death was over-represented in all the clones with compact clusters (Tables 3–5). Cytoskeleton, chromosome organization and biogenesis and DNA metabolism were also over-abundant in the compact clones (Tables 3–5), indicating a possible greater cellular replication activity in the compact clones. Categories related to cell wall showed clear specificity of transcript expression in either compact or loose clones. Pectinrelated categories were only over-represented in the compact clones and cellulose biosynthesis in the loose clones (Tables 4, 5). Phenylpropanoids-related categories showed dramatic changes in gene expression, the phenylpropanoid metabolism category was over-represented in both the compact and loose clones (Tables 4, 5). The lignin biosynthesis category seems more abundant in the compact clone (1134) when compared with both loose clones (Table 4) and the loose clone 368 when compared with both compact clones (Table 5). Terpenoids and alkaloids categories also seemed to be over-represented in the loose clones (Tables 4, 5). In addition the plant-pathogen interaction category was also over-represented in the loose clones vs. 1134 (Table 4). Several categories related to hormone signaling were also over-represented in the loose clones, such as Auxin, brassinosteroids, cytokinins, jasmonate, and ethylene signaling (Tables 4, 5). Transporters showed a balanced pattern; however, oxygen transport was more abundant in the loose clones (Tables 4, 5). Ion transport-related categories were also over-represented in both types of clones. #### VitisNet Analysis Indicates Metabolic Pathways Related to Cluster Compactness Networks were manually inspected to find those that presented relevant changes. These analyses allowed us to identify key networks and possible causes for cluster compactness as well as important information on early fruit development that will be discussed along this section (Figures 3–6, Supplementary Images 1–10). We observed changes in gene expression between compact and loose clones in flowers, and the clone with more and bigger berries (clone 1134) showed more differences with the loose clones than the other compact clone. It was however difficult to clearly distinguish if differences in cell replication or timing impacted fruit set (and thus berry number), the number of cells (berry size), or both. We identified four main categories of genes showing differential expression, related to: cellular activity, pathogens interaction, hormonal response and phenylpropanoids biosynthesis. #### Loose and Compact Clones Show Great Difference in Flower Transcriptome Indicating a Distinct Cell Division Rate and/or Asynchronous Development Comparison between flowers of clones producing tight clusters and clones producing loose clusters indicated a distinct cell division rate and/or asynchronous development. Most noticeably genes related to a greater activity in production of cellular material were more abundant in the compact clones. Evidences were specifically gathered at the level of carbohydrate and nucleic acid metabolism as well as the regulation of cell cycle and cell division. #### Carbohydrate metabolism. Cell wall The composition and size of the fruits as they grow are very dependent of the efficiency of the flower as a nutrient sink (Bihmidine et al., 2013) and significant differences were #### Grimplet et al. Cluster Compactness Transcriptomics in Grapevine #### TABLE 4 \| Over-represented functional categories in G1 and G2 comparisons with P < 0.05. (Continued) #### TABLE 4 \| Continued Values are expressed as log2 ratio group/genome. observed between compact and loose clones in the carbohydrate metabolism in flower. Important regulators of the sucrose metabolism (Figure 3) were seen to have isogenes specifically expressed in flower. Most noticeably, cell wall invertase (VIT\_04s0008g01140) had greater expression in the loose clones than in clone 1134 and a vacuolar form was more expressed in the compact clones than in clone 368 (VIT\_16s0022g00670). The cell-wall forms have been associated with rapidly growing tissues (Eschrich, 1980), they were induced by wounding and pathogenic attack (Sturm and Chrispeels, 1990), and have been implicated in phloem unloading and source/sink regulation (Eschrich, 1980; Roitsch et al., 1995). Gene expression in flower also indicated that starch seems to be preferentially catabolized into dextrin and maltodextrin with the increase of expression of several isogenes of alpha-(7 isoforms) and beta-amylases (2 isoforms) in the loose clones with respect to clone 1134. Higher expression of starch synthase (VIT\_00s1488g00020) might indicate greater starch production in loose clones. Additionally a possible regulator of amylases (Liu and Thornburg, 2012), a transcript homologous to Myb305, was more abundant in the loose clone 368 vs. compact clones (VIT\_14s0083g01060, Figure 3). However, the change of carbohydrate and cell osmolarity might be reminiscent of the #### TABLE 5 \| Over-represented functional categories in G1 and G3 comparisons with P < 0.05. (Continued) #### TABLE 5 \| Continued Values are expressed as log2 ratio group/ genome. flower opening mechanism (van Doorn and Van Meeteren, 2003) thus it would maintain turgor in the flowers of the loose clone, indicating a slight difference in the timing (delay) in loose against compact clones. As mentioned above, this difference could not be phenotyped since the samples were in an equivalent external stage: flowers were sampled at the end of flowering, with fallen stamen. The next step was to identify the potential fate of the carbohydrates that would be produced from the DEG in the compact clones. In plants, most of the carbon fixed by photosynthesis is incorporated into cell wall carbohydrates. Compact clones showed an increase of expression of several transcripts involved in the biosynthesis of compounds that might be related to an increase of cell wall material. Starting from the fructose, all the enzymes that are involved in the biosynthetic pathway of both D-mannose and GDP mannose (Figure 3) presented at least an isoform over-expressed in flowers of the compact FIGURE 4 \| Adapted Cytoscape networks including transcripts differentially expressed in flowers between loose and compact clones related to nucleic acid metabolism. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 10230 and 10240 from Grimplet et al. (2009). clones. These included fructokinases (VIT\_18s0089g01230, VIT\_05s0102g00710), hexokinases (VIT\_18s0001g14230), mannose-6-phosphate\_isomerase (VIT\_01s0011g03750), phosphomannomutase (VIT\_01s0011g03750, only G1). Other genes that might be involved in the biosynthesis of the predominant cell wall components arabinose or UDP-xylose (Seifert, 2004) (Figure 3), such as UDPglucuronate 4-epimerase (VIT\_15s0048g00330, only G1), UDP-glucuronate decarboxylase (VIT\_05s0077g02300) and alpha-N-arabinofuranosidase (VIT\_08s0032g00890, VIT\_12s0055g01180) were also more abundant in compact clones. In cell wall (Supplementary Image 1) many differences between the expression levels of isogenes were observed. There were a few families that seem to be specific to one or the other cluster type. One of them, the pectinacetylesterase, which is involved in the regulation of pectin acetylation, had three isogenes over-expressed in flowers of the compact clones vs. clone 368, as well as four isogenes of the fasciclin-like arabinogalactan proteins involved in cell adhesion (Johnson et al., 2003). They might also be involved in cell expansion, since a mutant was observed causing swelling in roots (Shi et al., 2003). To complete the picture related to cell wall, several major families of cell wall related proteins showed differential expression between isogenes in a large amount but were evenly represented between the loose and compact clones, amongst them, the pectin methylesterase inhibitors, the pectinesterases, the pectate lyases or the xyloglucan endotransglycosylases. These centrally important aspects of expansion are also mediated by auxin, which is critical for skin strength in the earliest stages in flowers (Reeves et al., 2012). Overall while the gene expression is contrasted between clones, no routes leading to specific cell wall metabolites emerged as specific in any cluster type probably because substrate specificity of isogenes is not yet well-described. In summary, there are differences between loose and compact clones in the expression of genes related to carbohydrate and cell wall metabolism. It can be hypothesized that cells in the compact clones were dividing more actively, triggering a large cascade of events that would explain the high number of differentially expressed genes but will likely complicate the identification of the primary genetic factors initiating the events. #### Purine and pyrimidine biosynthesis Transcripts involved in the metabolism of nucleic acid components were another indicator of differences in cellular activity between compact and loose clones. Several genes related to purine metabolism (Figure 4) tended to be upregulated in flowers of the compact clones. Genes coding for enzymes involved in the next part of the pathway were more clearly over-expressed in the flowers of the compact clones indicating greater production of deoxynucleotides. The ribonucleoside-diphosphate reductase presented three isogenes (VIT\_07s0031g01990, VIT\_14s0068g02000, VIT\_07s0031g02000) over-expressed in the compact clones. The nucleoside-triphosphatases (VIT\_19s0015g01800, VIT\_10s0003g01720 over-expressed in compact clone 1134) were involved in the conversion of ATP and GTP into ADP and GDP for RNA biosynthesis. Adenine phosphoribosyltransferase (VIT\_00s1847g00010), which is involved in the purine salvaging, was also over-expressed in flowers of the compact clones. As observed for the purine metabolism pathway, an increase of the gene expression in compact clones was observed in the pyrimidine pathway (Figure 4) with few changes affecting the earlier biosynthesis steps. The changes specifically affected transcripts for the interconversion of nucleotides triphosphate toward the production of deoxynucleotide diphosphates. Activity related to DNA repair was observed by the increase of dUDP pyrophosphatase (Dubois et al., 2011) and then production of dUMP. These observations might be linked to processes in the replication and repair related networks. In addition, transcripts of genes coding for enzymes involved in the pyrimidine salvaging pathways were also more abundant in compact clones, such as cytidine deaminase (VIT\_17s0000g01370) and thymidine kinase (VIT\_12s0057g00500). The VitisNet analysis indicates significant differences in the nucleotide metabolism pathway between flowers of compact and loose clones. It suggests that these differences were not affecting the de novo biosynthesis of the nucleotides but may be related to interconversion and salvaging. These results shall be put in perspective with observations in the networks related to genetic regulation, more specifically DNA repair. #### Regulation of DNA replication and repair mechanisms The rate of DNA repair is dependent on many factors, including cell type, cell age and extracellular environment. In the studied clones at E-L 26 we observed a greater activity of this pathway in compact clones. Most of the DEG involved in DNA replication (twenty; Figure 5) were more abundant in the compact clones, like other pathways related to replication and repair. One of the six identified MCM genes related to DNA replication has previously been shown to be up-regulated after fertilization (Dresselhaus et al., 2006). MCM6 is essential for both vegetative and reproductive growth and development in plants (Dresselhaus et al., 2006). Base excision repair (Figure 5) is the predominant DNA damage repair pathway for the processing of small base lesions. A large portion of the genes (10 over 30 genes) belonging to this network was more abundant in the compact clones. The differentially expressed transcripts were found either in the network common branches or in the mechanism of reparation of segments of 2–13 nucleotides. DNA mismatch repair (Figure 5) is a highly conserved biological pathway that plays a key role in maintaining genomic stability (Li, 2008). Several of the over-expressed genes in the compact clones also belonged to the DNA polymerase complex. Homologous recombination (Figure 5) is essential for the accurate repair of DNA double-strand breaks (potentially lethal lesions), and acts before the cell enters mitosis. Once again, over-expression of genes related to DNA replication and repair in flowers of compact clones is another marker of a more active cell division compared to loose clones. #### Cell cycle and regulation of actin cytoskeleton Transcripts of about a third of the genes (97/322) involved in the cell cycle network (Supplementary Image 2) were more abundant in compact clones. The observation of a greater expression of these genes, combined with transcript abundance related to DNA processing mechanisms was another indicator of a more active division state in the compact clones. In addition, the expression of the genes involved in the network of regulation of actin cytoskeleton (Supplementary Image 3) was more abundant in flowers of the compact clones (82 of 343 genes). Overall, differences were observed between compact and loose clones of Garnacha Tinta in the expression of cell divisionrelated genes (carbohydrate and nucleic acid metabolism as well as regulation of cell cycle and cell division). This could be due to a difference in the rate of fecundated flowers in the two clone types, resulting in the comparison of a pool with a greater ratio of fecundated flowers (in the compact clones) vs. a pool with a lower ratio (in the loose ones) that would be in different cell division states. At this point of the development, cell division is in an exponential phase (Harris et al., 1968; Ojeda et al., 1999) and slight differences could significantly be reflected in the transcriptome. This would eventually lead to the greater number of berries observed in the compact clones. In addition, the differential activity in terms of cell replication could lead to a differential final cell number in the berries, and ultimately to a different berry size. In comparison G1-26, this could explain the differences in berry size between clone 1134 and clone 368 (Supplementary Table 1). Since few differences were observed in networks related to cell expansion (the other cell growth mechanism in berries), it would be the differential number of cells what affect final berry size in this case. The differences observed in the cell division-related gene expression could also be due to a slight delay in the development progress in the loose clone with respect to the compact one. Given that the berries derived from flowers that opened first ("first berries") have less probability to abscise than the later opening flowers (Kühn et al., 2014), a delay in the development could produce a greater berry abscission rate, thus affecting berry number. Unfortunately it was not possible to determine if the transcriptome differences were due to only one or the two possible causes proposed. #### Plant Pathogen Interaction and Relation to the Jasmonate/Methyl Jasmonate Interconversion DEG involved in the mRNA surveillance pathway were predominantly expressed in the loose clones. They belonged more specifically to pre-mRNA 3′ -end processing machinery and non-sense-mediated decay (NMD). Some of them, such as SMG7 (VIT\_00s0527g00010, VIT\_00s0640g00020) appear to regulate the expression of the genes involved in pathogen response in Arabidopsis (Rayson et al., 2012). Therefore, the expression of genes related to the plant-pathogen interaction was further examined. Salicylic acid (SA) is a signal molecule involved in interactions between plants and pathogens. Enzymes potentially involved in its biosynthesis pathway did not exhibit differential expression of their corresponding transcripts. However, some genes involved in SA signaling were differentially expressed in flowers and that are known to be involved in pathogen response. Homologous to EDS1 (4 adjacent isogenes on the genomic sequence) were over-expressed in flowers of the loose clones vs. clone 1134. These genes were known to be involved in R proteinmediated signaling (Dempsey et al., 2011). Twenty R proteins (Supplementary Image 4) presented more abundant transcripts in flowers of the loose clones. In the plant-pathogen interaction network (Supplementary Image 5), several isoforms of BAK1 (8 over 19) and EIX1/2 (7/20) genes were more abundant in flowers of the loose clones vs. clone 1134. These genes are known to act together in the plant defense against pathogens induced by ethylene (Bar et al., 2010). Differences in expression of genes potentially regulated by ethylene (Supplementary Image 6) were observed in both compact and loose clones but members of Ethylene Response Factor subfamily were clearly more abundant in the loose clones. These genes corresponded to the subfamily IX or B-3 according to Nakano et al. (2006). The genes in group IX have often been linked in defensive gene expression in response to pathogen infection (Berrocal-Lobo et al., 2002) and this group contains PTI genes (Gu et al., 2002) that were known to be regulated by EDS1 (Dempsey et al., 2011). The WRKY transcription factors can also play a role in the defense mechanism (Rushton et al., 2010) and many of them were overexpressed in loose clones (20 genes). Transcript level-related evidence of differential accumulation of jasmonic acid was unclear since expression of different transcripts coding for proteins involved in its biosynthesis in the alpha-linolenic acid metabolic pathway was increased in compact or loose clones. The isoforms of jasmonate O methyltransferase/VIT\_04s0023t03810 VIT\_04s0023t03800 VIT\_04s0023t03790) were over-expressed in the flowers of the compact clones and the methyl jasmonate esterases (VIT\_00s0253t00170, VIT\_00s0253t00160 VIT\_00s0253t00150) were preferentially expressed in the loose clones. The first enzyme catalyzes the conversion of jasmonate to methyl-jasmonate (MEJA) and the esterase catalyzes the demethylation of methyljasmonate. Jasmonate needs to be in the demethylated form to trigger defense response to herbivores (Wu et al., 2008), while MEJA is most likely involved in plant morphology. There is no obvious reason explaining the greater expression of genes potentially involved in pathogen interaction in loose clusters, but both (pathogen-related gene expression and cluster loosening) could be consequences of the flower abscission process. The activation of different defense responses at flower abscission zones was described in tomato (Meir et al., 2011). Grapevine inflorescences treated to increase flower abscission showed up-regulation of pathogenesis-related genes (Domingos et al., 2016). #### Gibberellin and Auxin Biosynthesis and Signaling Were Likely to Play a Role in Compact Clones Gibberellins Application of gibberellins (GA) on the clusters is widely used in the table grape industry to control fruit set, elongate rachis or increase berry size (Coombe, 1960). It has different effects depending on the treatment concentration and timing. When applied at bloom, gibberellins affect fruit set and berry size (Dokoozlian and Peacock, 2001). We hypothesize that differences in the gibberellins metabolism or signaling would be observed at flowering between compact and loose clones in flowers of clones differing in berry number (and berry size in G1). Several transcripts coding for enzymes involved in GA biosynthesis (diterpenoid biosynthesis, Figure 6) were more abundant in flowers of the compact clones in the comparison G1-26, such as copalyl diphosphate synthase (VIT\_07s0151g01070 loose clones vs. 1134), ent-kaurene synthase (VIT\_07s0151g01040 loose clones vs. 1134), gibberellin-20 oxidase (VIT\_04s0044g01650 loose clones vs. 1134, VIT\_04s0044g02010) and the regulator BME3 (VIT\_13s0019g04390 only G1). Moreover, flowers of the loose clones showed higher expression of transcript coding for the enzyme converting active GAs (GA1, GA3, GA4, and GA7) to inactive GAs (GA34, GA8): GA2-oxidase (VIT\_10s0116g00410, VIT\_19s0140g00120 loose clones vs. 1134). These findings are in agreement with Giacomelli et al. (2013) proposing that the pool of bioactive GAs in grapevine flowers during flowering and fruit set is controlled by a fine regulation of the abundance and localization of GA oxidase transcripts. Genes involved in GA signaling (Figure 6) did not show differential expression between compact and loose clones in flowers but several genes known to be regulated by GA showed greater expression in the compact clones. Interestingly, several GASA-like transcripts showed preferential expression in the compact clones (VIT\_08s0007g05860, VIT\_18s0072g01110, VIT\_14s0066g01790, VIT\_03s0091g00390, and VIT\_14s0108g00740). GASA proteins are involved in diverse processes, and GASA4 in Arabidopsis is present in flower and involved in the seed development and yield (Roxrud et al., 2007). One of the transcripts (VIT\_03s0091g00390) corresponds to the SNAKIN subfamily which is known to be an antimicrobial (Segura et al., 1999) but more recently its role in the cell division was described (Nahirñak et al., 2012). #### Auxin In grapevine, auxin is a growth factor required for fruit growth. No significant observation could be made on auxin biosynthesis related transcripts to identify a possible greater production in compact or loose clones. More significantly, transcripts involved in the auxin transport (Figure 6) were more abundant in flowers of the compact clones, such as PINOID (VIT\_11s0016g04910, VIT\_13s0074g00730) and the auxin efflux carriers PIN3 (VIT\_08s0040g01230, VIT\_17s0000g02420) PIN6 (VIT\_18s0001g15420) PIN5 (VIT\_04s0023g00320) PIN10 (VIT\_08s0040g01220), and AUX1 (VIT\_03s0038g02140). As mentioned above, it has been recently shown that berries derived from flowers that open first have less probability to abscise than the flowers that open later, and that this ability requires decreased ethylene-related gene expression dependent on polar auxin transport (Kühn et al., 2014). Later, Kühn et al. (2016) found that polar auxin transport and transcripts of four putative PIN genes decreased in conjunction with increased abscission, and the inhibition of polar auxin transport resulted in fruit drop. In this context, over-expression of auxin transporter genes could be related to a greater final number of berries in the cluster by contributing to lower the number of abscised flowers or fruitlets. In the auxin regulation pathway (Figure 6), transcripts coding for proteins related to the early response to auxin were upregulated in flowers of the compact clones, including six transcripts for AUX/IAA and seven transcripts for SAUR. Quantitatively, ARF6 was one of the most differentially expressed genes in the G4 comparison (G4-03 and G4-26). ARF6 is known to be present in the flower and embryo, and in Arabidopsis it was specifically localized in the lower tier of the embryo and suspensors (Rademacher et al., 2011). Recently, Su et al. (2016) found that ARF6 and ARF8 are required in Arabidopsis for gradient auxin response and can mediate auxin-induced gene activation in somatic embryogenesis induction. In tomato, down-regulation of ARF6 and ARF8 by microRNA 167 led to floral development defects and female sterility (Liu et al., 2014). ARF4 was the second Auxin Response Factor over-expressed in the compact clone 1134 (in G1-26). It has been characterized in tomato fruit (Sagar et al., 2013), where lowers chloroplast production and starch and is down-regulated by presence of sugars. The expression of ARF4 in tomato increases between anthesis and 4 days post-anthesis and might be involved in fruit set (Zouine et al., 2014). In grapevine ARF4 is more abundant in high seed content berries at ripening (Gouthu and Deluc, 2015). Cross-talk between GAs and auxins has proven to play an important role during fruit set in tomato via the activation of GA biosynthtetic enzyme GA20 oxydase by auxin (de Jong et al., 2009) two transcripts coding for GA20ox are over-expressed in compact clones (Figure 6). In grapevine crosstalk beween these two hormones is also critical in flower set initiation and parthenocarpy (Jung et al., 2014; Lu et al., 2016). #### Genes Involved in Phenylpropanoids Biosynthesis Show That Important Secondary Metabolites Might Be Specifically Expressed within Clones A significant number of genes involved in the biosynthesis of phenylpropanoids, flavonoids and anthocyanins were differentially expressed between cluster types, although most of them showed isogenes preferentially expressed in either compact or loose clones. There were also differences in the transcript abundance of genes affecting the production of several important secondary metabolites. All but three of the 46 stilbene synthase genes were preferentially expressed in flowers of the loose clones vs. clone 1134 (Supplementary Image 7). It was shown that over-expression of grapevine stilbene synthase VIT\_16s0100g00910 can induce parthenocarpy in tomato (Ingrosso et al., 2011) and thus this gene might be related to the control of berry number. These authors also observed that greater amounts of stilbene were related to pollen sterility. Several transcripts coding for enzymes potentially involved in the anthocyanin biosynthesis showed preferential expression in flowers of compact clones (Supplementary Image 9), including three Anthocyanidine rhamnosyltransferase (VIT\_00s0820g00020, VIT\_15s0046g01950, VIT\_00s0218g00140) and three Anthocyanidin 3-Oglucoside-6"-O-malonyltransferase (VIT\_12s0134g00660, VIT\_12s0134g00620, VIT\_12s0134g00640). The latter two were up-regulated in clusters with small berries. The earlier steps in the phenylalanine biosynthesis (Supplementary Image 10) also showed a greater gene expression in the flowers of the compact clones, including shikimate dehydrogenase (VIT\_14s0030g00650, VIT\_14s0030g00660), shikimate kinase (VIT\_18s0001g01730), and prephenate dehydratase (VIT\_10s0116g01670). ### CONCLUSIONS The characterization of the differential expression in clones of Garnacha Tinta presenting phenotypic differences in traits related to cluster compactness allowed us to identify networks and candidate genes potentially involved in those traits. The flowers at the end of bloom seem to be an organ and developmental stage of crucial importance for the traits studied, while much less differences were observed in spring buds and young berries. Our study focused on the end of flowering which is a particularly active period of rapid changes but other stages could also play important role in compactness and a fine monitoring of the flowering stages would improve our knowledge. In the case of the analysis on berry, the microclimate caused by different compactness levels may also influence the genes expression and make more difficult the discrimination between genetics and environmental factors. All the stable differential traits considered (berry number, seed number and berry size), are potentially affected by the magnitude of cell division rate, and many related gene networks showed different expression levels, indicating a greater division rate in compact clones with more berries (and eventually more seeds or larger berries). Differential expression of transcripts involved in hormone signaling and transport support that auxin and gibberellins play a central role in fruit set, and some identified key genes have been noted. Other hormones, such as ethylene and jasmonate may differentially regulate potential indirect effects, such as the activation of some defense mechanism or polyphenols production. # AUTHOR CONTRIBUTIONS JG performed the gene expression analysis and interpretation. JT and NL performed phenotyping analysis. JI designed the study. JG and JI drafted the manuscript. All authors read and approved the final manuscript. ## FUNDING This work was financially supported by the projects AGL2014- 59171R (co-funded by FEDER) and AGL2010-15694 and the Ramon y Cajal grant RYC-2011-07791, all from the Spanish MINECO. JT was the recipient of a predoctoral fellowship from MINECO (Grant: BES-2011-047041). ### ACKNOWLEDGMENTS The authors acknowledges R. Aguirrezábal, S. Hernáiz, B. Larreina, M. I. Montemayor, and E. Vaquero for their technical assistance. We acknowledge Gobierno de La Rioja for the collection and maintenance of the clones. The authors would also like to thanks Anne Fenell for critical review of the manuscript. # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00632/full#supplementary-material Supplementary Image 1 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to cell wall metabolism. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 40006 from Grimplet et al. (2009). Supplementary Image 2 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to cell cycle. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 44110 from Grimplet et al. (2009). Supplementary Image 3 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to regulation of actin cytoskeleton. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 44810 from Grimplet et al. (2009). Supplementary Image 4 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to R proteins from plant–pathogen interaction. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 34627 from Grimplet et al. (2009). Supplementary Image 5 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to plant–pathogen interaction. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 34626 from Grimplet et al. (2009). Supplementary Image 6 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to ethylene signaling. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 30008 from Grimplet et al. (2009). Supplementary Image 7 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to phenylpropanoids biosynthesis. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 10940 from Grimplet et al. (2009). Supplementary Image 8 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to flavonoids biosynthesis. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact #### REFERENCES clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 10941 from Grimplet et al. (2009). Supplementary Image 9 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to anthocyanin biosynthesis. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 10942 from Grimplet et al. (2009). Supplementary Image 10 \| Adapted Cytoscape networks including transcript differentially expressed in flowers between loose and compact clones related to ethylene signaling. Genes over-expressed in compact clones in all comparisons are in dark red. Genes over-expressed in compact clones in 2 or 3 comparisons are in red. Genes over-expressed in loose clones in all comparisons are in dark green. Genes over-expressed in loose clones in 2 or 3 comparisons are in green. Figure is adapted from networks 10400 from Grimplet et al. (2009). #### Supplementary Table 1 \| Phenotypic data and pair-wise t-tests of the selected clones during three seasons. Supplementary Table 2 \| Gene expression values data. Sheet 1: normalized data for each samples, value are expressed as log2 of intensity. Sheet 2: functional annotation. Sheet 3: significance of the differential expression, ratio of transcripts expression in each comparison, genes meeting the cutoff of fold change >2 and P < 0.05 are reported as 1 for compact clones and −1 for loose clones. for grapevine bunch compactness. Aust. J. Grape Wine Res. 21, 277–289. doi: 10.1111/ajgw.12121 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 SDS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review. Copyright © 2017 Grimplet, Tello, Laguna and Ibáñez. 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. # Abscisic Acid Is a Major Regulator of Grape Berry Ripening Onset: New Insights into ABA Signaling Network Stefania Pilati<sup>1</sup> \, Giorgia Bagagli<sup>1</sup> , Paolo Sonego<sup>1</sup> , Marco Moretto<sup>1</sup> , Daniele Brazzale<sup>1</sup> , Giulia Castorina<sup>2</sup>† , Laura Simoni<sup>2</sup> , Chiara Tonelli<sup>2</sup> , Graziano Guella3,4, Kristof Engelen<sup>1</sup> , Massimo Galbiati<sup>2</sup> and Claudio Moser<sup>1</sup> #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics (CRAG), Spain #### Reviewed by: Fatma Lecourieux, Centre National de la Recherche Scientifique (CNRS), France Javier Ibáñez, Instituto de Ciencias de la Vid y del Vino, Spain Alonso Gastón Pérez-Donoso, Pontificia Universidad Católica de Chile, Chile \Correspondence: Stefania Pilati [email protected] #### †Present address: Giulia Castorina, Dipartimento di Scienze Agrarie e Ambientali – Produzione, Territorio, Agroenergia, Università degli Studi di Milano, Milan, Italy #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 03 March 2017 Accepted: 06 June 2017 Published: 21 June 2017 #### Citation: Pilati S, Bagagli G, Sonego P, Moretto M, Brazzale D, Castorina G, Simoni L, Tonelli C, Guella G, Engelen K, Galbiati M and Moser C (2017) Abscisic Acid Is a Major Regulator of Grape Berry Ripening Onset: New Insights into ABA Signaling Network. Front. Plant Sci. 8:1093. doi: 10.3389/fpls.2017.01093 <sup>1</sup> Research and Innovation Centre, Fondazione Edmund Mach, San Michele all0Adige, Italy, <sup>2</sup> Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy, <sup>3</sup> Department of Physics, Bioorganic Chemistry Lab, University of Trento, Trento, Italy, <sup>4</sup> Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Trento, Italy Grapevine is a world-wide cultivated economically relevant crop. The process of berry ripening is non-climacteric and does not rely on the sole ethylene signal. Abscisic acid (ABA) is recognized as an important hormone of ripening inception and color development in ripening berries. In order to elucidate the effect of this signal at the molecular level, pre-véraison berries were treated ex vivo for 20 h with 0.2 mM ABA and berry skin transcriptional modulation was studied by RNA-seq after the treatment and 24 h later, in the absence of exogenous ABA. This study highlighted that a small amount of ABA triggered its own biosynthesis and had a transcriptome-wide effect (1893 modulated genes) characterized by the amplification of the transcriptional response over time. By comparing this dataset with the many studies on ripening collected within the grapevine transcriptomic compendium Vespucci, an extended overlap between ABAand ripening modulated gene sets was observed (71% of the genes), underpinning the role of this hormone in the regulation of berry ripening. The signaling network of ABA, encompassing ABA metabolism, transport and signaling cascade, has been analyzed in detail and expanded based on knowledge from other species in order to provide an integrated molecular description of this pathway at berry ripening onset. Expression data analysis was combined with in silico promoter analysis to identify candidate target genes of ABA responsive element binding protein 2 (VvABF2), a key upstream transcription factor of the ABA signaling cascade which is up-regulated at véraison and also by ABA treatments. Two transcription factors, VvMYB143 and VvNAC17, and two genes involved in protein degradation, Armadillo-like and Xerico-like genes, were selected for in vivo validation by VvABF2-mediated promoter trans-activation in tobacco. VvNAC17 and Armadillo-like promoters were induced by ABA via VvABF2, while VvMYB143 responded to ABA in a VvABF2-independent manner. This knowledge of the ABA cascade in berry skin contributes not only to the understanding of berry ripening regulation but might be useful to other areas of viticultural interest, such as bud dormancy regulation and drought stress tolerance. Keywords: Abscisic acid (ABA), grapevine (Vitis vinifera), berry ripening, RNA sequencing, promoter analysis, AREB/ABF # INTRODUCTION fpls-08-01093 June 21, 2017 Time: 11:34 # 2 Grape is a traditional world-wide cultivated crop, whose fruit is consumed fresh or dried as table grapes, fermented to produce wines, spirits and vinegar or transformed into pharmaceutical health-promoting products. The process of fruit development has been intensively studied initially to improve quality of production and more recently to maintain high quality under changing climatic conditions. Grape berry development can be divided into two phases of berry growth: an initial phase from fruit set until green hard berries, characterized by embryo maturation in the seeds, pericarp intense cell duplication, malic and tartaric acid accumulation and proanthocyanidin synthesis, and a final phase of ripening, characterized by fruit softening, mesocarp cell enlargement and sugar and aroma accumulation and skin coloring. The onset of ripening, that is the transition from the first to the second phase, implies an extensive reprogramming of the berry transcriptome, as observed in several "-omic" studies (Deluc et al., 2007; Pilati et al., 2007; Fasoli et al., 2012; Lijavetzky et al., 2012). An integrated network analysis recently highlighted that this transition occurs via an extensive gene down-regulation driving the suppression of the vegetative growth metabolism and the activation of maturation-specific pathways (Palumbo et al., 2014). Such transcriptional and metabolic reprogramming is orchestrated by numerous signals, such as hormones, in particular ABA, ethylene and brassinosteroids, reviewed in (Kuhn et al., 2014), physiological modifications, such as cell turgor and elasticity (Castellarin et al., 2011), and metabolic factors, such as sugar and reactive oxygen species accumulation (Gambetta et al., 2010; Pilati et al., 2014). However, their reciprocal influence on ripening inception has not been disentangled so far, due to the complexity of the system. Recently, Castellarin et al. (2015) proposed a timeline of events leading to the onset of ripening: an initial fall of elasticity and turgor pressure in the berry is followed by ABA and sugar accumulation and then skin coloring. Yet, an ABA sharp increase at ripening onset has been reported in numerous studies (Deluc et al., 2007; Wheeler et al., 2009; Gambetta et al., 2010; Sun et al., 2010). The fast accumulation of ABA in the cells is due the its typical positive feedback loop, triggered by a small amount of hormone coming possibly from the leaves (Antolìn et al., 2003) or by diffusion from the mature, dormancy-acquiring seeds (Kondo and Kawai, 1998). This ripening-specific accumulation is reported also in peach, sweet cherry and tomato (Zhang et al., 2009; Sun et al., 2012; Tijero et al., 2016). Abscisic acid regulates a variety of plant developmental processes, such as leaf senescence, seed maturation and dormancy, bud dormancy and adaptive responses to abiotic and biotic stresses, in particular drought and salinity, by means of stomata closure, osmotic potential regulation and/or wax deposition (Nambara and Kuchitsu, 2011). During plant evolution, ABA conserved its ancestral role in cellular responses modulation to stimuli affecting the cell water status, but acquired new functions in the regulation of different processes, sometimes also in a species specific way (Takezawa et al., 2011; Wanke, 2011). In fleshy fruit ripening, the relationship between ABA and sugar accumulation and turgor pressure, which together determine water uptake and cell enlargement, could represent an example of acquired functionality of ABA in angiosperms (Wheeler et al., 2009; Gambetta et al., 2010). In tomato, both a transgenic line blocked in ABA synthesis and one overexpressing a transcription factor activating ABA response demonstrated the effect of ABA on tomato texture and firmness and primary metabolites accumulation (Sun et al., 2012; Bastías et al., 2014). A similar decrease in fruit firmness was observed transforming tomato with the Vitis homolog of this transcription factor (Nicolas et al., 2014). ABA effect on berry skin coloring was highlighted in studies focused on seedless varieties of table grapes, where the absence of seeds correlated with low amounts of ABA and low color development, recovered by ABA treatments (Kondo and Kawai, 1998; Ferrara et al., 2013). Nonetheless, studies focused on the effects of adverse external conditions highlighted that anthocyanin accumulation in condition of water stress required not only ABA, but also sugar accumulation and possibly other stimuli, suggesting that ABA is not the only necessary signal for color development, or it can exert an indirect effect (Castellarin et al., 2007; Wheeler et al., 2009; Gambetta et al., 2010; Ferrandino and Lovisolo, 2014). Abscisic acid signaling network encompasses genes involved in its biosynthesis, degradation, conjugation and transport, whose reciprocal transcription and enzymatic activities determine ABA cellular content, and genes involved in its perception and signaling cascade. The knowledge of this network has burst recently, taking advantage of the combination of molecular, biochemical, forward and reverse genetic studies in Arabidopsis, reviewed in (Finkelstein, 2013) and the availability of the genome sequence of Vitis vinifera (Jaillon et al., 2007; Velasco et al., 2007), which accelerated the transfer of knowledge from the model plant to this crop. The early steps of ABA biosynthesis take place in the plastid as part of the MEP pathway leading to carotenoids production. NCED catalyzes the first committed step in ABA biosynthesis, and is rate-limiting, reviewed in (Nambara and Marion-Poll, 2005). NCEDs are encoded by multigene families and the expression of the specific family members is tightly regulated in response to stress or developmental signals contributing to ABA synthesis in different contexts. In addition to ABA synthesis, catabolism is a major mechanism for regulating ABA levels: ABA can be irreversibly hydroxylated at the 8<sup>0</sup> position by P-450 type monooxygenases to give an unstable intermediate (8<sup>0</sup> -OH-ABA) isomerized to phaseic acid; or reversibly esterified to ABA-glucose ester (ABA-GE), which can accumulate in vacuoles or apoplast as storage. Transporters of the G subfamily of the ATP-binding cassette (ABC) family mediate the import and export of ABA through the plasmalemma (Jarzyniak and Jasinski, 2014). Abbreviations : ABA, abscisic acid; ABRE, ABA-responsive element; AREB/ABF, ABRE-binding proteins/ABRE-binding factors; CIPK, CBL-interacting protein kinase; CPK, calcium-dependent protein kinase; NCED, 9-cis-epoxycarotenoid dioxygenase; PP2C, protein phosphatase 2C; PYR/PYL/RCAR, pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors; RBOH, respiratory burst oxidase homolog; SnRK2, sucrose-non-fermenting1-related kinase 2. Abscisic acid perception and signaling in grapevine has been recently elucidated in root and leaf (Boneh et al., 2011, 2012) and in fruit (Gambetta et al., 2010), by identifying and partially characterizing ABA receptors, PP2Cs and SnRK2 kinases. The best characterized ABA receptors in Arabidopsis are soluble proteins of the family PYR (pyrabactin resistant), PYL (PYR-like) or RCAR (regulatory component of ABA receptor). Eight RCARs, four of which were induced by ABA in leaf, were identified in grapevine (Boneh et al., 2011). ABA binds to PYR/PYL/RCAR proteins, resulting in a conformational change that enhances stability of a complex with clade A PP2Cs, which in Arabidopsis are all induced by ABA and include the ABA insensitive mutants abi1 and abi2 (Merlot et al., 2001). In grapevine, nine PP2Cs have been identified (Gambetta et al., 2010; Boneh et al., 2011): six are induced by ABA in leaf, and five are induced at véraison in the berry. In the absence of ABA, the PP2Cs keep SNF1 related kinases (SnRKs) inactive through physical interaction and dephosphorylation. When ABA binds to its receptors, they recruit PP2C, thus releasing the inhibition of SnRKs which become active by autophosphorylation and activate more than 50 target proteins, which include transcription factors as well as other targets. In grapevine, seven SnRKs were identified and they appeared differently modulated in leaf, root and fruit upon abiotic stresses and development. PP2Cs may also dephosphorylate other classes of kinases, e.g., the ABA-stimulated calcium-dependent protein kinase (ACDK), linking ABA to calcium signaling (Yu et al., 2006) and widening the cascade. Among the transcription factors activated by ABA, a subgroup of the bZIP family, called AREB/ABF (Liu et al., 2014), are directly activated by SnRKs (Yoshida et al., 2010): in grapevine 11 putative ABAresponsive bZIPs have been identified by sequence homology and one of them, VvABF2, has been recently characterized (Nicolas et al., 2014). The present work analyzes the early transcriptional events occurring in green hard, pre-véraison berry skin treated with exogenous ABA showing the dramatic effect of this hormone on ripening onset and identifying candidates targets of VvABF2, thus expanding our knowledge on ABA network in the fruit. # MATERIALS AND METHODS # Plant Material, Biochemical Analyses, ABA Treatment During 2011, two clusters of V. vinifera cv. Pinot Noir ENTAV115 were collected almost daily between 9 am and 10 am at 6–7 weeks post-flowering (wpf), corresponding to EL-33 and EL-34, at the FEM study site (San Michele all0Adige-TN Italy). Each cluster represented one biological replicate. Each cluster was divided in smaller bunches and then half of them, at random, were pressed for must analysis by means of Fourier transform infrared spectroscopy (FTIR) using the instrument WineScanTM Type 77310 (Foss Electric, Denmark) while the remaining small bunches were rapidly frozen. Frozen berries were peeled with a scalpel and the skins were ground to obtain a fine powder. Skin anthocyanin concentration was measured after methanol extraction (1 g berry skin powder in 10 mL methanol) according to the double pH differential method (Cheng and Breen, 1991). Lipid extraction and analysis were performed as described in Pilati et al. (2014). ABA detection and quantification was carried by LC-UV-MS technique. In particular we used as stationary phase a column Kinetex C18 (5 µm, 150 mm × 4.6 mm, flow 0.8 mL/min) and as mobile phase an A:B gradient elution (A = H2O + 0.5% formic acid; B = MeOH + 0.5% formic Acid) with B changing from 40 to 55% in 10 min. 10 µL of pure ABA solution or raw skin berry extracts (solution prepared in MeOH/CHCl<sup>3</sup> 9:1) were injected in every chromatographic run. The retention time of ABA was 8.9 min. ABA was detected both by ion-positive mode ESI-MS analysis (characteristic ions at m/z 287, 265, 247, 229, 201, 187, and 173) and by photodiode array detector. ABA was quantified by interpolation on a working curve (absorbance vs. concentration) built on three ABA solutions at concentration 1.9, 19, and 190 ng/µL and the corresponding absorbance measured at fixed wavelength of 262 nm (R <sup>2</sup> = 0.998). During 2013, three clusters of V. vinifera cv. Pinot Noir ENTAV115 were collected between 9 and 10 am at 7 wpf, corresponding to EL-33, at the FEM study site (San Michele all0Adige-TN Italy). Each cluster represented one biological replicate. Berries were detached from each cluster by cutting the petiole, at 2–3 mm distance from the berry. Berries were washed in 0.1% Plant Preservative Mixture (PPM, Duchefa) water solution for 30 min. 20 berries per cluster were put in a 100 mL water solution containing 0.2 mM ABA (Sigma) and 0.5% methanol (used to dissolve ABA) or 0.5% methanol as control. After 20 h mild shaking, the berries were extensively rinsed and then eight berries were frozen in liquid nitrogen. The remaining berries, both treated and not treated with ABA, were plated on solid medium in Petri dishes (0.9% agar, 10% sucrose, 0.1% PPM) for 24 h, and then eight were frozen as described above. ABA and sucrose concentrations were taken from Gambetta et al. (2010). # RNA Extraction and Expression Analysis by qPCR Total RNA was extracted from the skin powder samples using Spectrum Total Plant RNA kit (Sigma) and was quantified using a Nanodrop 8000 (Thermo Scientific). The integrity was checked using Bioanalyzer 2100 (Agilent) and RNA Nano Chips. For Real-time PCR analyses, first strand cDNA was synthesized from 2 µg RNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. The cDNAs were mixed with Fast SYBR Green Master Mix (Applied Biosystems) and amplified on a ViiA 7 Real Time PCR System (Applied Biosystems) using an initial heating step at 95◦C for 20 min, followed by 40 cycles of 95◦C for 1 min and 60◦C for 20 s, using the primers reported in Supplementary Table S1. Raw fluorescence data were extracted using Viia 7 Software v1.0. Ct and reaction efficiency were calculated using LinRegPCR (Ruijter et al., 2009). Relative expression was calculated according to (Pfaffl, 2001) by centering expression values for each gene on the mean value. Three reference genes (Actin, SAND and GAPDH) were used for normalization with geNorm (Vandesompele et al., 2002). For RNA sequencing, 2 µg RNA for each sample were shipped in dry ice to Genomicx4life (Salerno, Italy). # RNA-Seq Analysis and Identification of Differentially Expressed Genes Sequencing has been performed on Illumina Hi-seq 1500, producing 100 nt directional single-end reads. Raw reads were pre-processed for quality using fastqc v.0.11.2<sup>1</sup> and cleaned with cutadapt v.1.12 (Martin, 2011). The resulting reads were aligned to the grape (12x v1<sup>2</sup> ) genome using the Subread aligner (Liao et al., 2013). Raw read counts were extracted from the Subread alignments using the featureCount read summarization program (Liao et al., 2014). The summarized read count data was used to identify DEGs among various treatments by using the voom method (Law et al., 2014), which estimates the mean-variance relationship of the log-counts, generating a precision weight for each observation that is fed into the limma empirical Bayes analysis pipeline (Smyth, 2004). A Volcano Plot generated using the ShinyVolcanoPlot Web App<sup>3</sup> was used to select sets of DEGs for each comparison based on both p-value and expression fold change. In the present work, a maximum p-value of 0.05 and a minimum absolute fold change of 2 were imposed. Raw sequences were deposited at the Sequence Read Archive of the National Center for Biotechnology<sup>4</sup> under BioProject accession number PRJNA369777. ## Gene Annotation and Promoter Analysis Vitisnet gene annotation has been used (Grimplet et al., 2009), except for the transcription factors gene families already characterized in grapevine, for which the specific published annotation has been used (Licausi et al., 2010; Wang N. et al., 2013; Liu et al., 2014; Wang et al., 2014; Wong et al., 2016). Functional class enrichment was performed on GO (Gene Ontology) terms (annotation<sup>5</sup> ) using the TopGO Bioconductor package (Alexa and Rahnenfuhrer, 2016) and on Vitisnet gene annotation (Grimplet et al., 2009) taking advantage of the VESPUCCI grape compendium (Moretto et al., 2016). Promoter analysis has been performed on the 1-kb promoters of the genes modulated at 20 and 44 h using DREME software (Bailey, 2011). Statistically enriched motifs were annotated using the "DAP motifs" database for Arabidopsis (O'Malley et al., 2016). The enriched motifs were searched and counted in the dataset using Patmatch software (Yan et al., 2005). #### Plasmid Constructs Two type of constructs were prepared for transient expression, using the Gateway system (Karimi et al., 2002). The coding sequence of ABF2 (VIT\_18s0001g10450) was amplified from Pinot Noir berry cDNA using Phusion DNA polymerase (Finnzymes) and the primers ABF2Fw and ABF2rev and cloned into pENTR-D-TOPO vector (Invitrogen), sequenced and transferred into pK7WG2, under the control of 35S promoter. The 1-kb promoters of VvNAC17 (VIT\_19s0014g03290), Armadillo-like (VIT\_17s0000g08080), Xerico-like (VIT\_01s0137g00780), VvMYB143 (VIT\_00s0203g00070) and HB5 (VIT\_04s0023g01330) were amplified from PN40024 genomic DNA using Phusion DNA polymerase and the pairs of primers indicated in Supplementary Table S1. These DNA fragments were cloned in pENTR-D-TOPO, sequenced and transferred into PHGWFS7, upstream of EGFP and GUS reporter genes. #### Transient Expression Assay in N. benthamiana Promoter activation assays were performed in 5-week-old Nicotiana benthamiana plants agroinfiltrated as described in Li (2011). Three leaves from four tobacco plants, representing four biological replicates, were co-infiltrated with the activating plasmid pK7WG2:CaMV35S:ABF2 and individual pHGWFS7:promoter:GUS target constructs. Leaves co-infiltrated with the pK7WG2 empty vector and each of the pHGWFS7:promoter:GUS plasmids were used as a control for the trans-activation assay. 48 h after the first infiltration, leaves were infiltrated with 50 µM ABA dissolved in 10 mM MgCl<sup>2</sup> and 0.07% EtOH, or with a mock solution (0.07% EtOH in 10 mM MgCl2). Leaf samples were collected at 15 min, 1 and 3 h after the beginning of the ABA treatment. qPCR analysis of GUS expression was performed as previously described for the grape samples, using the primers qPCR\_GUSF1 and qPCR\_GUSR1 (Supplementary Table S1). GUS expression was normalized using the Elongation factor 1α (EF-1α) gene (AF120093), amplified with primers pEFfw and pEFrev (Schmidt and Delaney, 2010). # RESULTS #### Treatment of Pre-véraison Berries with ABA In order to identify the exact moment preceding the onset of ripening, we focused our attention on the 2 weeks preceding color break in Pinot Noir berries. Samples were collected daily during season 2011 and analyzed for biochemical and molecular parameters which are known from both literature and our experience to change dramatically at ripening inception (Pilati et al., 2014). These include biochemical profiles, such as total acidity, sugar and anthocyanins content (Figure 1A), galactolipid peroxidation state and ABA content in berry skins (Figure 1B) and gene expression profiles, such as those of lipoxygenase A (LOXA, VIT\_06g0004s01510) – responsible of the enzymatic galactolipid peroxidation – and of NCED1 (VIT\_19s0093g00550) – first committed enzyme in ABA biosynthesis (Figure 1C). This preliminary analysis showed that in the 48 h preceding anthocyanins accumulation (on July 14th), all these parameters undergo a transition, which marks the beginning of a distinct developmental phase, i.e., the ripening. This discontinuity implies an extensive regulation occurring within the cells to trigger all the metabolic pathways characterizing the biochemical changes of fruit ripening. To study the role of ABA as a trigger of ripening, in 2013 we collected berries at the hard green pre-véraison stage (E-L 33) <sup>1</sup>https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ <sup>2</sup>http://genomes.cribi.unipd.it/ <sup>3</sup>https://github.com/onertipaday/ShinyVolcanoPlot <sup>4</sup>www.ncbi.nlm.nih.gov/sra <sup>5</sup>http://genomes.cribi.unipd.it/DATA/V1/ANNOTATION/ and treated them with exogenous ABA. The treatment was performed on detached berries with short petioles in an aqueous medium containing 0.2 mM ABA for 20 h, which allowed for both homogenous ABA diffusion into the berry skin trough functional stomata and accurate experimental reproducibility. After 20 h, eight berries were collected, rinsed with water and frozen while the remaining were rinsed and plated for additional 24 h on solid medium containing 10% sucrose, in the absence of ABA, and then frozen (Supplementary Figure S1). expression in the skin of berries treated with 0.2 mM ABA for 20 h and then cultured on solid 10% sucrose medium without ABA for 24 h (light gray) and controls (dark gray). Normalized relative quantities were obtained by RT-PCR analysis using the two best reference genes. Data represent the mean of three biological replicates. Sucrose was added for avoiding osmotic stress according to (Gambetta et al., 2010) in order to mimic berry sugar content at véraison (6–7◦Brix).Treatment efficacy has been verified by two independent approaches. Firstly, ABA was quantified in skin samples of control and ABA treated berries by HPLC-UV-MS showing an average accumulation of 1.62 micrograms ABA/gr FW skin powder at 20 h and 0.55 µg/gr FW at 44 h in treated samples (Figure 2A). The latter value was similar to physiological values measured at véraison in other studies, such as 300 ng/gr FW in Wheeler et al. (2009) and 200 ng/gr FW in Sun et al. (2010), whereas higher ABA level at 20 h could be explained by ABA direct uptake. The very low ABA measured in control samples is consistent with berries at the green hard pre-véraison stage (Figure 1B). Secondly, the expression of the two genes known to be up-regulated at véraison, LOXA and NCED1, has been measured by qPCR (Figure 2B). NCED1 was up-regulated 66 times by ABA treatment at 20 h and 198 times at 44 h; LOXA showed a similar behavior, as it was up-regulated 4 and 13 times at 20 and 44 h, respectively. These profiles are consistent with those observed in 2011 in the transition from E-L 33 to E-L 34. Moreover, the induction of NCED1 can explain the intracellular ABA level measured at 44 h. # ABA Extensively Modulates the Berry Skin Transcriptome Transcriptomes of treated and control samples were analyzed by RNA-sequencing (Supplementary Table S2). Principal component analysis (PCA, Figure 3) shows that the four conditions are well-separated, while the biological replicates are grouped together. Major variance (41.5%) distinguished the two time-points (20 vs. 44 h), while treated vs. untreated berries were neatly separated along the second principal component, which explained 19.5% of the variance. It appeared that ABA treatment extensively impacted on pre-véraison berry skin transcriptome. Treated vs. untreated samples within each time-point were statistically compared to extract the lists of significantly modulated genes and a further restriction on fold change (greater than 2) was applied. 871 genes resulted modulated at 20 h by ABA and 1512 at 44 h; 490 genes were modulated at both time-points, with a coherent trend (Figure 4; and Supplementary Table S3). Interestingly, ABA-induced transcriptional modulation increased over time, regardless of the absence of the external stimulus. This amplification in the response suggests that ABA likely acted as a primer of a broad cellular program. According to RNA-seq analysis, NCED1 was induced 32 and 64 times at 20 and 44 h, respectively, whereas LOXA was induced 3 and 12 times, thus confirming previous qPCR analysis. Genes have been functionally annotated using Vitisnet (Grimplet et al., 2009) integrated with manual curation. For functional class enrichment analyses both Vitisnet and Gene Ontology were used (Supplementary Table S4). Genes modulated exclusively at 20 h, the less abundant group, were enriched in classes related to stress, cell wall modification, photosynthesis, respiration and translation. They could represent a stress response due to the excess or sudden delivery of ABA in the treatment, which induced a high turn-over of proteins involved in basic energy metabolism and cell wall. The set of ABA-positively modulated genes at both 20 and 44 h was enriched in genes involved in cell regulation: ABA and ethylene networks were over-represented, along with transcription factors related to these hormones, such as members of the large bZIP, APETALA2 and MYB families. At 44 h post-treatment, the number of genes positively modulated equaled that of the negatively modulated and the functional categories related to the metabolic pathways typical of the ripening process were enriched. Lipid and carbohydrate metabolism, cell wall modification, and flavonoid metabolism were over-represented among the up-regulated genes, supporting the role ABA plays in regulation of sugar metabolism and accumulation, cell enlargement and softening and color development. Ethylene, Auxin and ABArelated categories remained over-represented, suggesting that these hormones regulated not only the onset but also the process of grape berry ripening. Photosynthesis was over-represented among the down-regulated genes, suggesting that ABA triggers the switching off of this basal metabolism inducing the transition to a specialized sink organ, such as the ripe berry. ## Most of ABA Responsive Genes Are Involved in Ripening In order to outline the role of ABA at ripening onset, a metaanalysis using the grapevine expression data compendium Vespucci (Moretto et al., 2016) was performed. Seven experiments in which berry ripening transcriptome had been analyzed were selected to visualize how the sets of genes modulated in the skin by ABA at the two time-points were modulated during physiological ripening. These experiments were performed in different berry tissues (seed, pulp, skin, and pericarp) in six different cultivars: Cabernet Sauvignon (GSE11406), Sauvignon Blanc (GSE34634), Corvina (GSE36128), Pinot Noir (GSE49569 and GSE31674), Muscat Hamburg (GSE41206) and Norton (GSE24561). Heatmaps representing the comparisons are shown in Figure 4, while the tables which generated the heatmaps, downloaded from Vespucci website, are available as Supplementary Table S5. Within each comparison, a percentage of genes ranging between 49 and 87% were modulated coherently by ABA and during ripening in all tissues and cultivars (Figure 4). However, the heatmaps showed also variable profiles, likely related to the tissue and/or cultivar specific modulation of these transcripts. While the smallest overlap occurred with genes up-modulated exclusively at 20 h, the sets of genes modulated at both 20 and 44 h and those modulated only after 44 h showed a more extended overlap with ripening, between 64 and 87%, higher when considering the down-regulated genes. Functional categories enrichment analyses were repeated on the restricted sets of genes modulated both by ABA and during ripening (Figure 4 and Supplementary Table S4). In general, the analysis reproduced the results described in the previous section, with small refinements, such as for genes up-modulated at 20 and 44 h in which more specific categories related to carbohydrate metabolism, such as aminosugars, galactose and glycerolipids, appeared enriched or changes, as occurred for genes up-modulated at 44 h, in which the two categories of cell wall and flavonoid biosynthesis were lost while that of fruit ripening and abscission were gained. On a broad scale, we show that green hard berries treated with ABA are not simply responding to a stimulus, rather activating genes that are typical of the ripening program. Palumbo and collaborators highlighted that the transition from immature-to-mature stage in the berry is characterized by an extensive transcriptomic down-regulation, anti-correlated to a small group of 190 so called "switch genes" (Palumbo et al., 2014). Interestingly, we identified in our set of positively modulated genes 80 of such "switch genes" (Supplementary Table S6). This finding is consistent with ABA playing an important role in ripening regulation, partly by switching off typical vegetative pathways, such as those related to photosynthesis. Besides, 13 out of these 80 candidate "ABA-responsive switch genes" were predicted to be regulated post-transcriptionally by miRNA (Palumbo et al., 2014), suggesting that ABA modulation p-value < 0.05) and twofold change threshold. Green bars on the left are proportional to the number of repressed genes, while red bars on the right to the number of induced genes. On left and right sides, heatmaps of the down- and up-ABA modulated genes obtained in Vespucci, selecting 7 experiments on berry ripening performed in six Vitis vinifera cv.: Cabernet Sauvignon (GSE11406), Sauvignon Blanc (GSE34634), Corvina (GSE36128), Pinot Noir (GSE49569 and GSE31674), Muscat Hamburg (GSE41206) and Norton (GSE24561). Genes are clustered according to their expression profiles. The number of the transcripts coherently modulated in ABA treatment and berry ripening is indicated beside the heatmaps by green/red bars. Outer left and right columns, enriched functional categories of each subset of genes coherently modulated by ABA and during ripening are reported. For the complete output of the GO and Vitisnet enrichment analyses, refer to Supplementary Table S4. can occur both via direct targets activation and via posttranscriptional mechanisms. Finally, we compared our results with the list of genes modulated in a grapevine cell culture by a 1-h treatment with 20 µM ABA (Nicolas et al., 2014). Only 55 genes were modulated by ABA in both experiments and they were mostly up-regulated (Supplementary Table S7). They are related to ABA network and cell response to abiotic stresses, such as drought, dehydration, osmotic stress and potentially represent a basal ABA signaling core conserved in any type of cell. #### ABA Network in Berry Skin at the Onset of Ripening Abscisic acid treatment of berries at pre-véraison stage significantly modulated several genes involved in ABA metabolism, perception and signaling, which are summarized in Figure 5. All the gene families involved in ABA metabolism appeared transcriptionally affected by ABA: NCED family, involved in ABA biosynthesis, ABA 8<sup>0</sup> hydroxylase, in its degradation, ABA glucosidase, in ABA conjugation with sugar moieties and ABC transporters of the G subfamily, involved in ABA transport. The participation of the different NCED isoforms in berry ripening has been widely reported (Lund et al., 2008; Wheeler et al., 2009; Sun et al., 2010; Young et al., 2012). Using Vespucci, we could visualize the expression of the five NCEDs present in grapevine genome in at least three cultivars during berry development and post-harvest withering (Supplementary Figure S2). By combining this information with our results, we can state that VIT\_19s0093g00550, called NCED3 in Young et al. (2012) and NCED1 in Sun et al. (2010), is very rapidly induced in the skin by ABA treatment and its upregulation specifically occurs in the pulp and skin tissues at véraison, while the gene is not modulated later in ripening (Supplementary Figure S2). In our results, there are two other NCEDs which are modulated though to a lesser extent and only at 44 h: VIT\_02s0087g00910, which is also modulated during post-harvest withering in Corvina, and VIT\_10s0003g03750, which does not seem related to the process of berry ripening (Supplementary Figure S2). To understand their function, further investigations are needed. Instead, from Vespucci analysis, the isoform VIT\_02s0087g00930 seems induced during the whole berry ripening in all tissues, slightly more in the pulp. Concerning ABA perception and signaling, both ABA receptors of the PYL/PYR/RCAR family and PP2C phosphatases were affected by the presence of exogenous ABA. The two receptors (VIT\_08s0058g00470 and VIT\_02s0012g01270), previously identified as RCAR5 and 7 by Boneh et al. (2011) and as PYL3 and PYL1 by Li et al. (2012), were down-regulated in our experiment. Three PP2C phosphatases were strongly induced at 20 and 44 h: VIT\_11s0016g03180 was identified as AtABI1 homolog by phylogenetic analysis (named PP2C-2 in Gambetta et al., 2010), and was characterized in leaf and root (named PP2C4) by Boneh et al. (2011). VIT\_06s0004g05460 and VIT\_13s0019g02200, corresponding to PP2C-6 and PP2C-3 in Gambetta et al. (2010) and PP2C9 and PP2C8 in Boneh et al. (2011) were found induced at ripening onset in developing berries, anticipated in deficit irrigation conditions and induced by ABA in the skin of in vitro cultured berries. Out of the five identified Snf1-related kinases involved in ABA response, one (VIT\_02s0236g00130) corresponding to SnRK2.1 in Boneh et al. (2012) was up-regulated at 20 and 44 h. In Figure 5, other kinases belonging to the calcium dependent protein kinases family (CPK/CDPK/CDKs) or calcineurin B-like interacting protein kinases (CIPKs) and the three tiers of mitogen-activated protein kinase cascades (MAPKs, MAPKKs, and MAPKKKs) were included, based on Arabidopsis literature reporting their potential involvement in ABA signaling (reviewed in Finkelstein, 2013). In grapevine, some CPK and CDPK have been studied due to their up-regulation at véraison and relationship to ABA or drought stress (Yu et al., 2006; Cuéllar et al., 2013). Interestingly, also a LRK10 receptor kinase was found induced at 20 and 44 h and was included, as it is induced also in ABA-treated cell culture and in Arabidopsis one isoform, AtLRK10L1.2, is implicated in ABA response and drought resistance (Lim et al., 2015). Known direct targets of SnRK2 phosphorylation are NADPH oxidases of the Respiratory Burst Oxidase family (Rboh), leading to the production of hydrogen peroxide, plasma membrane anion and K+-channels, regulating ion transport and stomata opening, and basic domain/leucine zipper (bZIP) transcription factors of the ABA responsive element binding factor subgroup (AREB/ABF), mediating ABA-responsive genes transcription (Wang P. et al., 2013). In our experiment, representatives for all these functional categories were modulated (Figure 5, top). Interestingly, only one member of the grapevine AREB/ABF predicted subgroup (Liu et al., 2014) was modulated, namely the VvAREB/ABF2 (VvbZIP045, VIT\_18s0001g10450), recently characterized by Nicolas et al. (2014). VvAREB/ABF2 likely represents the isoform phosphorylated by SnRK2 in berry skin cells at ripening onset and activating down-stream genes of the ABA signaling cascade. ### Extending the Regulatory Circuit of ABA Signaling With the aim of identifying candidate target genes of VvABF2 activity, promoter regions of the genes modulated at 20 and 44 h were analyzed to find significantly enriched cis-acting motifs. The three most enriched motifs that were found were recognized by bZIP transcription factors, NAC and ABF subgroup of the bZIP family, respectively (Figure 6). Calculating the frequency of refined consensus motifs highlighted that the ABRE motif, "CACGTGT/GC," was about threefold more represented in our ABA-modulated gene set compared to the whole genome set of promoters. The recurrence of ABREs within each promoter was calculated: it ranged from 0 to 4 and is represented in Figure 5. ABREs are present in genes involved in ABA network, like NCEDs, PP2Cs and PYL/RCAR receptors but also in some TFs of the NAC, MYB, HB, bZIP and ERF families. In particular, VvMYB143 and VvNAC17 were the most enriched in ABREs and were also modulated by ABA in cell cultures (Nicolas et al., 2014). VvMYB143 belongs to the subgroup S11 of R2R3-MYB TFs, whose function has not been characterized the genes chosen for in vivo promoter activation assay by VvABF2. promoter analysis. Underlined genes are modulated in the same way in grape berry cell cultures treated with ABA, reported in Nicolas et al. (2014). Arrows indicate FIGURE 6 \| In silico promoter analysis of the genes modulated at both 20 and 44 h. The 1 kb promoter regions have been analyzed in DREME (Bailey, 2011) and motif enrichment has been calculated using the whole genome promoters as reference. The three most enriched motifs and the transcription factor families they are likely recognized by, are reported. A refined search of the most enriched exact sequence motifs has been performed using Patmatch (Yan et al., 2005) and the corresponding frequencies are reported in the last two columns. yet (Wong et al., 2016). VvNAC17 belongs to subgroup III (Wang N. et al., 2013) and has been recently characterized for VvABF2 activation in tobacco protoplasts (Nicolas et al., 2014). Other two genes, VIT\_17s0000g08080 and VIT\_01s0137g00780, were highly modulated by ABA in our experiment and also in grapevine cell culture (Nicolas et al., 2014) and possess 4 and 2 ABREs in their promoters, respectively. The first gene is annotated as Armadillo b-catenin, because it contains the 3D motif Armadillo, involved in binding of large molecules such as proteins or DNA but also the U-box domain, for recruiting E2-adenylated ubiquitin in the protein degradation pathway (Coates, 2003). The gene VIT\_01s0137g00780 was annotated as "unknown." RNA-seq data have been used to refine this gene prediction, revealing an incorrect splicing site (Supplementary Table S8). The improved transcript prediction based on reads mapping was used for homology search by BLAST algorithm in other species, highlighting the presence of a RING Zinc finger domain, as in Xerico (Ko et al., 2006). Interestingly, in Arabidopsis, genes containing these domains are reported to be involved in ABA response modulation and in drought resistance (Ko et al., 2006; Moody et al., 2016). These two genes will be hereafter referred to as Armadillo-like and Xericolike, for brevity. Vespucci analysis showed that VvNAC17 and Armadillo-like were part the genes modulated by ABA at 20 and 44 h and during ripening, while VvMYB143 and Xericolike were characterized by more variable profiles in the different cultivars and tissues and would need more specific investigation to precisely describe their modulation. These four genes were further investigated in a transient transactivation assay in N. benthamiana to verify their dependence on VvABF2 activity. The transcription factor HB5, activated by ABA but lacking ABREs in its promoter, was included as a negative control. Tobacco leaves were co-infiltrated with the target promoters fused to the reporter GUS along with either the VvABF2 activator or the control empty vector. The constitutive over-expression of ABF2 on its own is insufficient to induce expression of the downstream target genes, as ABF2 activity involves the ABA-dependent phosphorylation of its N-terminal domain, as demonstrated in Arabidopsis for AtABF2 (Fujita et al., 2005). Consequently, 2 days after the agro-infiltration, leaves were re-infiltrated either with 50 µM ABA or with a mock solution. GUS gene expression was analyzed by qPCR at 15 min, 1, and 3 h after the ABA treatment (Figures 7A–E). The promoters of VvNAC17 and Armadillo-like showed a remarkable activation by VvABF2 following ABA treatment, in agreement with the presence of four ABREs in their sequences (Figure 7F). Expression of VvMYB143, which contains two ABRE motifs in its promoter, was activated by ABA, irrespectively of the presence of VvABF2. Conversely, despite the presence of two ABF binding domains, the Xerico-like promoter did not show any activation in all the conditions, as observed for the negative control gene HB5. # DISCUSSION This work outlines the importance of ABA in the initial phases of berry skin ripening and describes in detail the molecular components of its network, discriminating ripening-specific isoforms and identifying new elements of the signaling cascade. The positive correlation between the rate of ABA accumulation and the ripening rate of the berry has been firstly reported in 1973 (Coombe and Hale, 1973). Since then, numerous studies highlighted the reciprocal influence and importance of ABA, ethylene, auxins and brassinosteroids at the onset of ripening, based on the observation that variations in the level of an hormone affects the relative concentration of the others and consequently the timing of ripening (Coombe and Hale, 1973; Davies et al., 1997; Symons et al., 2006; Sun fpls-08-01093 June 21, 2017 Time: 11:34 # 10 et al., 2010; Su et al., 2015). A previous work investigated at the transcriptomic level the effect of ABA on berry ripening, by means of the Affymetrix Vitis Genechip (Koyama et al., 2010). More recently two additional works, performed using NimbleGen whole genome arrays, addressed the transcriptional response to ABA in grapevine cell cultures and in different grapevine organs (Nicolas et al., 2014; Rattanakon et al., 2016). These studies highlighted that ABA is a ubiquitous signal raising specific responses according to the cell and tissue type and to the precise developmental stage. In the present work, great attention has been paid to the experimental setting, in the attempt to simulate the molecular events occurring in the berry just before ripening starts. Therefore, the precise developmental stage of green hard berry has been identified by means of a preliminary study in 2011, based on daily sampling during the 2 weeks before color break. This study suggested three molecular markers useful to define the pre-véraison stage, characterized by very low content of ABA and very low expression levels of the two genes LOXA and NCED, and the transition to the ripening stage, characterized by a significant increase in all these values. In order to capture the early events of ABA response in the context of berry skin ripening onset regulation, green hard berries were collected in 2013 and treated with ABA for 20 h in liquid and then cultured for additional 24 h in the absence of exogenous ABA, to assay its role as a trigger. ABA uptake and its effect on berry development have been initially assayed by measuring the values of the three markers, which suggested the occurrence of the transition (Figure 2). Then berry skin transcriptomes have been analyzed by RNA-seq. PCA analysis highlighted that ABA treatment extensively impacted on berry skin cells transcriptome (Figure 3). In fact, 871 genes were modulated by ABA compared to mock treated samples after 20 h, and 1512 after 44 h, indicating that the response to ABA amplified over time, as a signaling cascade (Figure 4). Even though this cascade has been triggered by a small amount of exogenous ABA, we actually know from the up-regulation of the enzyme NCED and the measured intracellular ABA level at 44 h (Figure 2) that ABA triggered its own biosynthesis through a positive feedback loop. As our focus was on the role of ABA in ripening onset, we narrowed our attention on those genes modulated by ABA during physiological ripening. This comparison has been performed using the grapevine expression data compendium Vespucci (Figure 4). Remarkably, 1346 (71%) ABA-skin responsive genes appeared modulated also during ripening in the berry tissues and cultivars considered, strongly supporting the regulatory role this hormone has in this non-climacteric plant. Nonetheless, this analysis highlighted genes characterized by a more variable profile, related to tissue and/or cultivar specific modulation, which will deserve further characterization. Based on functional enrichment analyses, the categories related to signaling were over-represented within the genes induced at 20 and 44 h (Figure 4 and Supplementary Table S4), namely the whole ABA network, from biosynthesis and perception to bZIP transcription factors and the ethylene signaling cascade mediated by AP2/EREBP transcription factors. It is interesting to note that some ethylene responsive factors were rapidly modulated by ABA before ethylene biosynthesis was stimulated, suggesting that they might actually represent points of convergence between the two hormones signaling cascades, explaining the tight interconnection between these two hormones at the onset of ripening (Sun et al., 2010). Even though not statistically over-represented, many genes involved in auxin response (IAA, SAUR, ARF), metabolism (GH3) and transport and five genes involved in brassinosteroids biosynthesis and signaling were modulated by ABA, supporting the previously proposed model of a complex integrated signaling network (Kuhn et al., 2014). Conversely, functional classes related to ripening-specific metabolisms were found over-represented at the 44 h timepoint. In particular, the up-regulated set was enriched in starch and sucrose metabolism, ethylene signaling, including AP2/EREBP transcription factors, whereas the down-modulated genes were mainly related to photosynthesis. The occurrence of this modulation at 44 h might reflect an indirect effect of ABA on these pathways, possibly mediated by other signals, such as ethylene and/or sugars. Besides, the modulation of many genes related to ions and water transport and cell wall modification supports the role of ABA in the fine tuning of sugar accumulation and water uptake to control cell osmosis which, in concert with cell wall structure modifications, drives the process of cell distension in the skin at the onset of berry ripening. The role of ABA in stimulating color accumulation through the activation of regulatory and structural genes of the anthocyanin pathway is still elusive. Treatments with this hormone after véraison were effective in stimulating berry coloring (Peppi et al., 2008; Ferrara et al., 2013; Katayama-Ikegami et al., 2016). Other studies showed that sugars were effective in promoting anthocyanins accumulation both in grapevine cell cultures (reviewed in Lecourieux et al., 2014) and in vitro berry cultures (Dai et al., 2014). In our analysis, we observed the up-regulation of the TF VvMYBA2 and of some structural genes such as flavonoid 3<sup>0</sup> 5 <sup>0</sup> hydroxylases and UDPglucose: anthocyanidin 5,3-O-glucosyltransferases, at 44 h, even though the whole pathway was not statistically enriched and we did not observe berry coloring. This delay was probably due to the fact that other cofactors from the MYB-bHLH-WD40 complex (such as MYC1 or MYCA1) were still not induced, suggesting that other signals beside ABA were required. Interestingly, VvMYBA7, recently characterized as a regulator of the anthocyanins synthesis in vegetative organs, was up-regulated by ABA but only at the 20 h time-point, suggesting its direct induction by ABA, but not its participation to the ripening program (Matus et al., 2017). The role of ABA in downregulating photosynthesis under stressful conditions, such as drought, salinity or low temperature, and during developmental processes, such as senescence (Lee et al., 2004; Yang et al., 2011; Gao et al., 2016), has been extensively described. It is not surprising then that at 44 h the functional enriched categories within the down-regulated genes involve different aspects of the photosynthetic metabolism. The switching off of this metabolism, central in green vegetative tissues, requires a highly regulated and concerted ensemble of reactions in order to avoid dangerous accumulation of reactive oxygen species or metabolic unbalances. The comparison with a previous meta-analysis focused on the genes involved in the transition from immature-to-mature stage in the berries highlighted that a relevant proportion (42%) of these genes were up-regulated by ABA (Palumbo et al., 2014). This observation supports the importance of ABA in triggering the transition to ripening and the down-regulation of the photosynthetic metabolism (Supplementary Table S5). Moreover, this comparison indicated that ABA regulates gene expression not only at the transcriptional level, by means of transcription factors modulation, but also post-transcriptionally, modulating miRNAs transcription. As mentioned above, ABA is an ancient and ubiquitous signaling molecule, evolutionary linked to plant adaptation to dry terrestrial land (Takezawa et al., 2011; Wanke, 2011). It controls transpiration, dehydration tolerance and other water status-associated processes such as seed and bud maturation and dormancy, root growth, leaf morphogenesis and senescence, thus making this compound a key player in integrating plants growth and development with environmental conditions (Nambara and Kuchitsu, 2011). The specificity of cellular ABA response must thus rely on specific isoforms of upstream perception components and on the wide variety of downstream signaling cascades. In this study, such ripening specific isoforms have been identified (Figure 5) and they represent strong candidates for experimental validation. Concerning the ABA biosynthetic enzyme NCED, a véraison specific isoform, VIT\_19s0093g00550 was identified (Supplementary Figure S2). Two ABC transporters of the G subfamily, VIT\_18s0072g01220 and VIT\_01s0011g02730, could be involved in ABA import/export, the former being induced also in ABAtreated grapevine cell cultures (Nicolas et al., 2014). Three PP2C genes out of the nine previously characterized (Boneh et al., 2011), were up-regulated in the berry by ABA and during ripening; one of these, ABI1, is induced also in the study by (Nicolas et al., 2014). Many kinases of the calcium- or calcineurin-dependent families, beside the better characterized SnRK2, are induced by ABA and likely to be related to this signaling cascade. Known transcription factors directly activated by SnRK2 belong to the bZIP family and are named ABA responsive element binding (ABRE/ABF). In grapevine, VvABF2 (VIT\_18s0001g10450, VvbZIP45), initially named GRIP55 due to its induction during ripening (Davies and Robinson, 2000), has been exhaustively characterized, showing its induction by ABA and its prevalent transcription in berry skin and seeds (Nicolas et al., 2014). As we also found VvABF2 induced by ABA in berry skins in our study, we tried to identify its possible targets. By an in silico promoter analysis of the genes modulated at 20 and 44 h, a motif very similar to the ABA responsive elements (ABREs) present in the Arabidopsis database was found as significantly enriched (Figure 6). We focused our attention on four genes that were strongly up-regulated by ABA at both time-points and in cell cultures and enriched in ABREs: two TFs, VvMYB143 and VvNAC17, and two uncharacterized genes possibly involved in proteasome-dependent protein degradation, Armadillo-like and Xerico-like. These genes are modulated during berry ripening, even if MYB143 and Xerico-like need further investigation as they show a tissue and/or cultivar specific behavior. Interestingly enough, all these genes are reported in literature to be related to osmotic stress (Denekamp and Smeekens, 2003), drought stress (Ko et al., 2006) and/or ABA accumulation (Kong et al., 2015). In particular, the transcriptional modulation of Armadillo-like and Xerico-like genes and their involvement in ABA response via protein stability indicates a third level of ABA response regulation, in addition to the transcriptional and miRNAmediated post-transcriptional ones. This translational regulation level could affect either the perception/activation mechanism of ABA signaling, as suggested by Kong et al. (2015) or proteins down-stream in the cascade, as suggested by Liu et al. (2011) and Seo et al. (2012) in Arabidopsis. The trans-activation assay performed in tobacco leaves showed that VvNAC17 and Armadillo-like were strongly activated by VvABF2, consistently with the presence of four ABREs in their promoters (Figure 7). Instead, VvMYB143 was activated by ABA irrespectively of the presence of VvABF2, suggesting that endogenous tobacco transcription factors can mediate its ABA dependent expression. Finally, Xerico-like neither showed significant activation by ABA nor by VvABF2. This is in apparent contrast with the observation that Xericolike expression is activated in ABA-treated grapevine cell cultures constitutively over-expressing VvABF2 (Nicolas et al., 2014). One possible explanation is that other possibly grapespecific factors are required to prime Xerico-like expression in addition to VvABF2 (e.g., additional transcription factors or VvABF2-interacting proteins). Interestingly, an in silico analysis identified Xerico-like among the co-expressed VvMYB143 genes and enriched in MYB-core type I binding motif, suggesting that VvMYB143 might be the regulator of Xerico-like (Wong et al., 2016). # CONCLUSION We proved the importance of ABA signaling to trigger the onset of ripening in the skin of green hard berries, occurring via an extensive gene modulation. Many molecular components of the ABA network, encompassing metabolism, perception and signaling, have been identified and many have been proposed as candidates to be experimentally validated. Four genes have been experimentally characterized showing different behaviors in response to ABA. As these genes are related to ABA/drought tolerance in other species, it will be of interest to functionally characterize them not only at ripening onset, but also under abiotic stress conditions. # AUTHOR CONTRIBUTIONS SP designed the study, performed time-course study in 2011 and ABA treatment in 2013 together with DB, supervised all the analyses and drafted the manuscript, GB performed the RNAseq experiment and worked on data analysis, KE, PS, and MM performed the bioinformatic analyses (RNAseq raw data analysis, gene co-expression and promoter analyses), GC, LS, and MG performed promoter activation assays in tobacco leaves, GG did ABA and lipid peroxidation analyses, CT revised the manuscript, CM contributed to the design of the study and interpretation of the results, supervised all the study and revised the manuscript. All authors revised and approved the final manuscript. # FUNDING The research was supported by the Autonomous Province of Trento (PAT-ADP 2013-2016). It also benefited from the networking activities coordinated under the EU-funded COST ACTION FA1106 "An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine." LS was supported by a fellowship from Fondazione Umberto Veronesi per il Progresso delle Scienze, Milan, Italy. # ACKNOWLEDGMENT We would like to thank Mickael Malnoy for providing the vectors for promoter activation assays in tobacco and Lisa Giacomelli for technical support. # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01093/ full#supplementary-material FIGURE S1 \| Description of the ABA treatment. FIGURE S2 \| Visualization of NCED gene family during berry ripening in three Vitis vinifera cultivars. # REFERENCES fpls-08-01093 June 21, 2017 Time: 11:34 # 14 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 Pilati, Bagagli, Sonego, Moretto, Brazzale, Castorina, Simoni, Tonelli, Guella, Engelen, Galbiati and Moser. 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. # Insights into the Role of the Berry-Specific Ethylene Responsive Factor VviERF045 Carmen Leida<sup>1</sup> \, Antonio Dal Rì<sup>1</sup> , Lorenza Dalla Costa<sup>1</sup> , Maria D. Gómez<sup>2</sup> , Valerio Pompili<sup>1</sup> , Paolo Sonego<sup>3</sup> , Kristof Engelen<sup>3</sup> , Domenico Masuero<sup>4</sup> , Gabino Ríos<sup>5</sup> and Claudio Moser<sup>1</sup> <sup>1</sup> Genomics and Biology of Fruit Crops Department, Research and Innovation Center, Fondazione Edmund Mach, San Michele all'Adige, Italy, <sup>2</sup> Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain, <sup>3</sup> Computational Biology Department, Research and Innovation Center, Fondazione Edmund Mach, Trento, Italy, <sup>4</sup> Food Quality and Nutrition Department, Research and Innovation Centre, Fondazione Edmund Mach, Trento, Italy, <sup>5</sup> Fruit Tree Breeding Department, Instituto Valenciano de Investigaciones Agrarias, Moncada, Spain During grape ripening, numerous transcriptional and metabolic changes are required in order to obtain colored, sweet, and flavored berries. There is evidence that ethylene, together with other signals, plays an important role in triggering the onset of ripening. Here, we report the functional characterization of a berry-specific Ethylene Responsive Factor (ERF), VviERF045, which is induced just before véraison and peaks at ripening. Phylogenetic analysis revealed it is close to the SHINE clade of ERFs, factors involved in the regulation of wax biosynthesis and cuticle morphology. Transgenic grapevines lines overexpressing VviERF045 were obtained, in vitro propagated, phenotypically characterized, and analyzed for the content of specific classes of metabolites. The effect of VviERF045 was correlated with the level of transgene expression, with highexpressing lines showing stunted growth, discolored and smaller leaves, and a lower level of chlorophylls and carotenoids. One line with intermediate expression, L15, was characterized at the transcriptomic level and showed 573 differentially expressed genes compared to wild type plants. Microscopy and gene expression analyses point toward a major role of VviERF045 in epidermis patterning by acting on waxes and cuticle. They also indicate that VviERF045 affects phenolic secondary metabolism and induces a reaction resembling a plant immune response with modulation of receptor likekinases and pathogen related genes. These results suggest also a possible role of this transcription factor in berry ripening, likely related to changes in epidermis and cuticle of the berry, cell expansion, a decrease in photosynthetic capacity, and the activation of several defense related genes as well as from the phenylpropanoid metabolism. All these processes occur in the berry during ripening. Keywords: ERF, RNA-seq, over-expressing transgenic lines, VOCs, wax, Vitis vinifera # INTRODUCTION Fruit ripening is a developmental process whereby mature seed-bearing organs undergo physiological and metabolic changes that promote seed dispersal. These changes affect the nutritional value of fruit and are thus of key relevance for human and animal diet, but it also makes the fruits more susceptible to pathogen attacks, reasons for which the process attracts considerable attention from the scientific community (Giovannoni, 2004). #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics, Spain #### Reviewed by: Ana Margarida Fortes, University of Lisbon, Portugal Kazuya Koyama, National Research Institute of Brewing, Japan > \Correspondence: Carmen Leida [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 17 September 2016 Accepted: 15 November 2016 Published: 09 December 2016 #### Citation: Leida C, Dal Rì A, Dalla Costa L, Gómez MD, Pompili V, Sonego P, Engelen K, Masuero D, Ríos G and Moser C (2016) Insights into the Role of the Berry-Specific Ethylene Responsive Factor VviERF045. Front. Plant Sci. 7:1793. doi: 10.3389/fpls.2016.01793 Grapevine is one of the most important cultivated crops in the world; the fruit is used as a source of fresh fruit, or once fermented, for production of wine and distilled beverages. The beginning of grape ripening, called véraison, coincides with a dramatic metabolic re-arrangement, affecting the accumulation of sugars, metabolism of acids, berry softening and coloring, and fruit growth. Ripening control in non-climacteric fruits, such as grapes, was originally thought to be ethylene independent, but recent evidence demonstrates a common genetic regulatory mechanism between climacteric and non-climacteric fruits (Lin et al., 2009). For example, a small amount of ethylene was measured in non-climacteric strawberries and this production was correlated to the expression of an ACC oxidase 1 gene (Trainotti et al., 2005). Other evidence includes the observation that climacteric, such as tomato, and non-climacteric species, such as grapevine, share common ripening regulators like members of the MADS-box, Zn-fingers, and bZIP transcription factor (TF) families (Fei et al., 2004). There are hints suggesting that ethylene is also affecting grape ripening. The application on grapes of the ethylene releasing compound 2-chloroethylphosphonic acid (CEPA) 3–6 weeks before véraison causes a delay of the ripening process, while treatments 2 weeks before véraison accelerate the start of grape ripening (Coombe and Hale, 1973). Application of the inhibitor of the ethylene receptor 1-methylciclopropene (1-MCP) before véraison delays berry growth, acid degradation, sucrose production, and coloring (Chervin et al., 2004). A peak of endogenous ethylene has also been detected in grapevine berries, although at much lower concentrations than in climacteric fruits, 1 week before véraison (Chervin et al., 2004). The potential role of ethylene in the ripening of non-climacteric fruits is likely to occur via cross-talk with other hormones such as abscisic acid, auxin and brassinosteroids, all of which are known to play a part in grapevine berry ripening (Hale et al., 1970; Coombe and Hale, 1973; Davies et al., 1997; Jeong et al., 2004; Symons et al., 2006). A key step in ethylene signal transduction is the activation of ethylene responsive factors (ERFs) that belong to the large superfamily of AP2/ERF TFs, specific to plants (Nakano et al., 2006). These factors are characterized by the presence of one or more AP2/ERF domains, consisting of 58–59 amino acids folded in one α-helix and a β-sheet, that recognizes the GCC box (5<sup>0</sup> -AGCCGCC-3<sup>0</sup> ) cis-element in the promoter of the target genes (Fujimoto et al., 2000). Based on the number of AP2/ERF domains and presence of other conserved domains, this superfamily can be divided into three families called AP2, ERF and RAV. The ERF family is characterized by one single AP2/ERF domain and it comprises the CBF/DREB and ERF sensu stricto subfamilies (Sakuma et al., 2002). ERF members have been discovered in many plant species due to the high degree of conservation of AP2/ERF domain (Nakano et al., 2006; Zhang et al., 2008; Zhuang et al., 2008), including grapevine, where 132 and 149 AP2/ERF genes have been predicted (Zhuang et al., 2009; Licausi et al., 2010). ERF and DREB factors are often involved in fruit ripening control, and plant response to stress (Nakano et al., 2006). Among ERF proteins involved in fruit ripening are factors characterized in plum, apple and tomato. Seven ERFs have been proposed to regulate plum fruit development and ripening, based on their gene expression patterns (El-Sharkawy et al., 2009). MdERF1 and MdERF2 are regulated by ethylene in apple as suggested by exogenous MCP treatment and varietal studies (Wang et al., 2007). Overexpression and silencing of the tomato LeERF1 gene has revealed an important role in plant development, fruit ripening and softening (Li et al., 2007), and tolerance to drought (Lu et al., 2010). Members of the SHINE clade of ERF factors (Aharoni et al., 2004) are involved in the regulation of lipid biosynthesis and the accumulation of cuticular waxes in tomato, leading to drought tolerance and recovery from water deficit (Shi et al., 2013). In this study we focus on VviERF045, a factor phylogenetically related to the SHINE clade of ERF genes from Arabidopsis (Aharoni et al., 2004) which is specifically induced after véraison in grapevine fruit, and thought to play a role in the ripening process (Pilati et al., 2007; Fasoli et al., 2012; Lijavetzky et al., 2012; Palumbo et al., 2014). Five transgenic lines overexpressing VviERF045 were obtained and used for functional characterization through phenotypic observation and metabolic and transcriptomic analyses. #### MATERIALS AND METHODS #### Plant Material, 1-MCP and Etephon Treatments Fruits were harvested from 'Pinot Noir' grapevine 10-years old plants cultivated in open field at Fondazione Edmund Mach (FEM) in San Michele all'Adige (Italy), following standard cultural practices and disease management. During 2006, three independent clusters were collected weekly starting from 4 to 10 weeks after anthesis (WAA) and at 14 WAA. Seeds, buds, tendrils, adult and young leaves, roots and flowers were also collected. The fruit (10 WAA) was dissected into pulp, skin and seed. 1-MCP and etephon treatments (both at 5 ppm) were performed at 7, 8, 9 WAA for 24 h, in a polyethylene bag wrapped around the cluster. Véraison (berry color change) occurred at 7 WAA. Mock treatments were applied to the control samples. Plant material was immediately frozen at −80◦C and stored until analysis. #### Phylogenetic Analysis The protein sequences of VviERF045, 7 ERFs from Prunus salicina (El-Sharkawy et al., 2009) and the three best blastx matches to VviERF045 from Solanum lycopersicum and Arabidopsis were aligned with MUSCLE (Edgar, 2004). In order to assess the real orthologs, a reciprocal best hit approach was used. Genebank accession numbers are listed in Figure 1F. A distance matrix was constructed according to the PAM model and clustered with the Neighbor-Joining method, using the EMBL-EBI bioinformatic tools framework (Li et al., 2015). The reliability of the phylogenetic grouping was assessed by bootstrapping (1000 replicates). FIGURE 1 \| VviERF045 expression pattern and protein sequence comparative analysis. Left panel: real time RT-qPCR analysis of VviERF045 expression profile in different tissues (A): B, Bud; S, Shoot; ML, Mature Leaf; YL, Young Leaf; F, Flower; R, Root; T, Tendril; 5WAA, berry at 5 weeks after anthesis (WAA); 10 WAA, berry at 10 WAA. ML and YL were used as reference samples. (B) VviERF045 expression in different fruit parts: pulp, skin and seed at 10 WAA. Pulp is taken as calibrator (C) VviERF045 expression at different developmental stages, berry developmental stages are indicated as WAA, (v) indicates véraison. (D) VviERF045 expression after 1-MCP and ethephon treatment: points represent 1-MCP treatment, points and lines represent ethephon treatment and continuous line represents control. Error bars represent SD and are based on three biological and two technical replicates. Data were normalized using ubiquitin and tubulin as reference genes. Different letters in the figure mean significant difference (p < 0.05) according to Tuckey's post hoc test. Right panel: (E) Phylogenetic tree of the ERF amino acid sequences from Prunus salicina [PsERF1a (FJ026009), PsERF1b (FJ026008), PsERF12 (FJ026003), PsERF3a (FJ026005), PsERF3b (FJ026004), PsERF2a (FJ026007), PsERF2b (FJ026006)], Arabidopsis thaliana [AT5G25190 (NP\_197901.1), AT1G15360-SHN1 (NP\_172988.1), AT5G25390-SHN3 (NP\_197921.1), AT5G11190-SHN2 (NP\_196680.1)], Solanum lycopersicum [SlSHN1 (XP\_004235965.1), SlSHN3 (XP\_004240977.1), SlERF1 (NP\_001234848.1)] and Vitis vinifera ERF from clade V [VviERF045, VviERF042, VviERF051, VviERF044, VviERF043, VviERF048, VviERF049, VviERF041, VviERF050, VviERF047, VviERF046]. The aa sequences were selected based on these criteria: (i) Grapevine ERFs belonging to the same clade of VviERF045, (ii) Prunus salicina ERF sequences related to fruit ripening, (iii) Best blastx matches to VviERF045 from A. thaliana and S. lycopersicum. Numbers on the branch represent the percentage for bootstrap value n = 1000. (F) Alignment of the amino acid sequences of clade V of the phylogenetic tree. 'AP2,''mm' and 'cm' conserved domains are represented as blue rectangles. #### Production of Transgenic Lines The complete coding region of VviERF045 (GenBank accession number KX179904) was amplified with Pfu Ultra Hotstart DNA polymerase (Stratagene, San Diego, CA, USA), starting from cDNA from mature berry. The purified PCR product was cloned into pENTR-D TOPO cloning vector (Invitrogen, Carlsbad, CA, USA), sequenced and transferred to pK7WG2 binary vector (Karimi et al., 2002) downstream of the 35SCaMV promoter, by using the Gateway technology (Invitrogen). The Agrobacterium strain EHA105 containing the VviERF045 binary vector and the pCH32 virulence helper plasmid were used for grape transformation. Gene transfer experiments were performed as described in Dalla Costa et al. (2016) on embyogenic calli of Vitis vinifera cv. 'Brachetto'. Transgenic and wild type plants were grown and propagated in vitro. # Expression by Quantitative Real-Time PCR (RT-qPCR) Analysis Each sample was composed of a pool of leaves (first five leaves from the apical meristem) from five different in vitro plants. Total RNA was extracted from 100 mg of leaf powder by using SpectrumTM Plant Total RNA kit (Sigma–Aldrich, St Louis, MO, USA), adding 1% PVP40 in the extraction buffer. Total RNA was quantified with Nanodrop8000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA integrity was checked by agarose gel electrophoresis. Total RNA (1 µg) was treated with Ambion <sup>R</sup> DNA-free DNase Treatment in order to remove contaminating DNA (Life technologies, Carlsbad CA, USA), and subsequently reverse transcribed with SuperScript <sup>R</sup> VILOTM cDNA Synthesis Kit (Invitrogen) in a final volume of 20 µL, according to manufacturer's instructions. One microliter of a 10X diluted first strand cDNA was used for each amplification reaction in a final volume of 20 µL. RT-qPCR was performed in a ViiATM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using the KAPA SYBR Fast qPCR Master Mix (Kapa biosystems, Wilmington, MA, USA). Reaction composition and conditions followed manufacturer's instructions. The cycling protocol consisted of 10 min at 95◦C, then 40 cycles of 30 s at 95◦C and 60 s at 60◦C. Specificity of the PCR was assessed by the presence of a single peak in the dissociation curve after the amplification and through size estimation of the amplified product. The relative standard curve method was used to quantify relative expression genes in case of efficiency less than 90%. Otherwise the 1Ct method was used as described in Applied Biosystems user's manual. Results were calculated as the average of three independent biological replicates for each line, repeated twice, using tubulin and ubiquitin as reference genes (Supplementary Table S2). For the amplification of VviERF045, we used two different primer pairs, namely "VviERF045" and "VviERF045endog" (Supplementary Table S2). Both primers of the first pair anneal on the coding sequence and they measure the total expression of the endogenous and exogenous (transgene) VviERF045 copies. Unlike, the reverse primer of the second pair anneals on the 3 <sup>0</sup>UTR region of the transcript which is present only in the endogenous gene copy but not in the transgenic one. The "VviERF045endog" primers were thus used to amplify specifically endogenous gene expression both in the transgenic lines and in the different grapevine tissues (Figure 1). ### RNA-Seq Analysis and Identification of Differentially Expressed Genes (DEGs) Total RNA was extracted from three independent pools of leaves (1 g) from in vitro grown plants as described above. RNA-Seq for transgenic line L15 and control were performed using an Illumina HiSeq2000 sequencing service (Illumina, Inc., San Diego, CA, USA). Samples were sequenced twice in separated lanes. Pairedend (2 × 100 bp) and raw reads were pre-processed for quality using fastqc 0.11.2<sup>1</sup> and adapter trimming with qtrim v0.94<sup>2</sup> . The resulting pre-processed reads were aligned to the reference transcriptome of Vitis vinifera (V1 grapevine annotation)<sup>3</sup> using the bowtie2 aligner v2.2.3 (Langmead and Salzberg, 2012) and deposited in Gene Expression Omnibus<sup>4</sup> series entry GSE77240. The summarized read count data was used to identify DEGs among various treatments by using the voom method (Law et al., 2014), which estimates the mean-variance relationship of the logcounts, generating a precision weight for each observation that is fed into the limma empirical Bayes analysis pipeline (Smyth et al., 2008). DEGs were identified between OE\_ERF and WT using a P-value of 0.05 and a log2-fold change greater than 1.5 and lower than −1.5 (Figure 4B; Supplementary Table S4; Supplementary Figure S1). # Functional Analysis Differentially expressed genes were analyzed by BLAST2GOv 3.0.9 (Conesa et al., 2005) and TopGO (Alexa et al., 2006). The analysis with TopGO was done by comparing three statistical methods (Fisher's, weight, Kolmogorov–Smirnov), and selecting the best 10 GO terms. # Phenolic Metabolites Determination Leaves from transgenic lines and control were sampled as described above (three biological replicates). Approximately 100 mg of powder from each sample was extracted in sealed glass vials using of a mixture of water/methanol/chlorophorm (20:40:40). Phenolics were extracted following Vrhovsek et al. (2012) method and UPLC chromatography was performed by injecting 2 µL of each sample. The same extract was used to measure anthocyanins by UPLC (Arapitsas et al., 2012). # Lipid Profile Analysis The lipid profile in leaves was determined following Della Corte et al. (2015) protocol, starting from 100 mg of powdered leaves and injecting 5 µL of lipid extracted solution into the LC-MS/MS system. # Chlorophylls and Carotenoids Quantification Leaves from in vitro cultivated transgenic lines and control were collected and powdered with liquid nitrogen (three biological replicates). Total carotenoids and chlorophylls were extracted from 100 mg samples using acetone 80% and read with a spectrophotometer at the wavelengths 470, 646.8, and 663.2 nm. Chlorophylls and carotenoids were determined following Lichtenthaler (1987) method. <sup>1</sup>http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc <sup>2</sup>https://ccb.jhu.edu/software/fqtrim/index.shtml <sup>3</sup>http://genomes.cribi.unipd.it/grape/ <sup>4</sup>http://www.ncbi.nlm.nih.gov/geo/ ### Leaf Area Measurement fpls-07-01793 December 9, 2016 Time: 15:1 # 5 Leaf area was measured with Iris Electronic Eye Analyzer VA300 (iBiosys Solutions Ltd, UK) and calculated with AlphaSoft 12.44 (Alpha MOS, France) using a fixed area as reference. In case of folded leaves the doubled part was cut and pasted aside with GIMP 2.6.12 image manipulation program (GNU GPL) in order to measure the whole leaf area. A non-parametric test was preferred for statistical analysis given the non-normality of data. We used the ggplot2 R package to graphically present these data in form of boxplots, using the geom\_boxplot function (Wickham, 2009). ### PCA Analysis and Heatmaps Principal component analysis (PCA) of the metabolites (Supplementary Figure S4) was obtained with R after scaling and centering the data. Heatmap representation of secondary metabolite content in the transgenic lines (Figures 3, 5, and 6) was calculated for each metabolite. Values were scaled by subtracting the mean value of WT and dividing by the standard deviation. Significance was assessed by ANOVA test followed by Tuckey's test using R<sup>5</sup> . #### Optical Microscopy and CryoSEM For lipid staining, a Sudan IV (Sigma–Aldrich) stock solution (0.1% w/v in isopropyl alcohol) was diluted 1:1 with glycerol, mixed well, and allowed to sit at room temperature for 30 min and syringe filtered to remove precipitates. The fourth or fifth leaves, starting from apex, were taken and cut in little squares of 4–5 mm. Leaves were included in agarose 5%, sectioned in 30 µm slices with a vibratome, stained for 30 min, mounted in distilled water with a cover slip and viewed immediately. Images were captured using a microscope Eclipse E600 (Nikon, Melville, NY, USA). For Cryo-SEM, leaves were harvested, mounted on SEM stubs attached to a CT-1000C Cryo-transfer system (Oxford Instruments, Oxford, UK) and frozen in liquid N2. The frozen leaves were transferred to the cryo-stage of a JEOL JSM-5410 scanning electron microscope (SEM). The samples were then fractured, sublimated by controlled warming to −90◦C, and sputter coated with a thin film of gold. Finally, leaves were viewed at an accelerating voltage of 15 keV and captured at 1000x and 2000x magnification. # RESULTS AND DISCUSSION #### VviERF045 Is a Berry-Specific Transcription Factor Induced at Ripening and Closely Related to the ERFs from the SHINE-Clade Expression analysis via RT-qPCR showed that VviERF045 is highly expressed in fruit at 10 WAA, while its expression is much lower in other tissues such as root, stem, leaf, bud, flower and green berry (Figure 1A). During berry development VviERF045 <sup>5</sup>www.R-project.org/ expression raises starting from 7 WAA and peaks 2–3 weeks later, about at the end of the véraison period, (Figure 1B) at which time, VviERF045 expression is more pronounced in skin and pulp rather than seed (Figure 1C). These observations suggest that VviERF045 might play a regulatory role in the berry ripening process. Although several members of the ethylene response factor family are ethylene inducible (Pirrello et al., 2012), berries treated around véraison (7, 8, and 9 WAA) with 1-MCP or etephon showed no significant differences in the expression of VviERF045 compared with the control (Figure 1D). However, our study could not exclude that treatments done in a prevéraison stage could have led to the same results. Former phylogenetic analysis assigned VviERF045 to clade V of the ten clades identified for the 122 grapevine members of the ERF superfamily (Licausi et al., 2010). In this study we have made a more focused analysis comparing VviERF045 and other VviERFs from cladeV to 15 highly similar and previously characterized ERF protein sequences from Prunus salicina, Arabidopsis thaliana, and Solanum lycopersicum (Figure 1E). Our phylogenetic tree highlights that the seven ERFs related to fruit development and ripening in Prunus salicina (El-Sharkawy et al., 2009) cluster apart from the other ERF sequences. On the other side, VviERF045 and its homologs from tomato and Arabidopsis fall into a single clade together with VviERF046 and VviERF047. Interestingly, one subgroup into this clade contains exclusively wax biosynthesis genes whose overexpression results in a glossy leaf phenotype and increased drought tolerance: AT1G15360 (SHN1), AT5G11190 (SHN2), and AT5G25390 (SHN3) from Arabidopsis, and SlSHN1 and SlSHN3 from tomato (Tournier et al., 2003; Aharoni et al., 2004; Shi et al., 2013). They share a high degree of similarity since in addition to the conserved AP2 domain they display two other conserved motifs located in the middle and the C-terminus of the protein sequence ('mm' and 'cm'; Aharoni et al., 2004). The remaining three sequences, which comprise VviERF045, SlERF1, and the Arabidopsis AT5G25190, form a distinct subgroup within clade V that distinguishes itself most notably by a deletion of six and one amino acid(s) in the 'mm' and 'cm' domains, respectively (Figure 1F). AT5G25190 was reported to be induced by 1 aminocyclopropane-1-carboxylic acid (ACC) and salt (Zhang et al., 2011) as well as by drought (Huang et al., 2008), but it was shown that its overexpression does not lead to a typical leaf shine phenotype (Aharoni et al., 2004). SlERF1 overexpression leads to several phenotypic effects including ethylene triple response on etiolated seedling, leaf development, enhanced fruit ripening and softening (Li et al., 2007) and improved tolerance to drought stress (Lu et al., 2010). # Phenotypic Characterization of VviERF045 Transgenic Lines Fourteen transgenic lines overexpressing VviERF045 from the Vitis vinifera cv. 'Brachetto' were generated (see Materials and Methods). In five of them, the expression of VviERF045 (due to transcription of exogenous and endogenous gene copies) was much higher than in the wild type and they were used for further functional characterization. In lines L6 and L7 the expression of FIGURE 2 \| Characterization of VviERF045 overexpressing lines. (A) Relative expression level of the endogenous + exogenous gene (blue bars), and endogenous gene (orange bars) in the transgenic lines. Error bars are based on data from three biological and two technical replicates. Data were normalized using ubiquitin and actin as reference genes. Relative expressions with the same letter are not significantly different (p < 0.05) according to Tuckey's post hoc test. The ANOVA test refers to the red bars since for blue bars no significant difference was observed. (B) In vitro phenotype of transgenic and WT plants of the same age. VviERF045 was about 100-fold increased; in the other three lines it was increased around 25–30 times (Figure 2A). The overall phenotype of the transgenic lines seemed directly related to the level of expression of the transgene, affecting not only leaf morphology and color, but also root biomass and architecture (Figure 2B) and this was particularly evident in case of L6. Several leaf features correlated strongly with the expression level of VviERF045, such as the leaf blade insertion angle on the petiole (Figure 2B), the leaf area and the leaf margins (Figures 3A,B). In general, L6 leaves displayed an acute insertion angle, a globular and chlorotic surface (almost yellow) and leaf margins curved toward the abaxial surface of the leaf, resembling somehow an 'epinastic' phenotype (Barry et al., 2001). Unlike L6, WT plants carried leaves with an insertion angle ranging between 180◦ and 140◦ , with a plane and bright green surface and an evident dentate margin. L15-19-22 showed an intermediate phenotype with insertion angles of 90–120◦ , a WTlike dentate leaf margin and a light green color, while L7 leaves were more similar to L6 in form and color. The analysis of pigment contents confirmed these phenotypic observations, with L6 showing lower values of chlorophyll a and b and carotenoids (Figure 3C). The transgenic lines showed also a general reduction in leaf number and leaf area. The total leaf area in L6 was about 10 times lower than in WT. L7, 15, and 19 showed comparable leaf areas, about half of the WT, whereas L22 was more similar to WT (Figures 3A,B). The VviERF045 overexpressing lines had a smaller root system with short and thick roots (Figure 2B). This could be due to a defective auxin gradient, which plays a key role in root development (Overvoorde et al., 2010). # Effect of VviERF045 Overexpression on the Transcriptome Three pools of leaves harvested from in vitro plants of the transgenic line L15 and from WT plants were used to compare the two transcriptomes by a RNA-Seq approach. L15 was selected since it showed a high level of VviERF045 expression, while growing sufficiently well in vitro and in vivo. Between 29 and 79 million of paired-end reads of 100 nucleotides were obtained for each replicate, and on average 79% of them were properly aligned in both senses (Supplementary Table S1). A multidimensional scaling approach to the analysis of the expression data highlighted that the three replicates of L15 were well separated from those of WT (Figure 4A). Using a volcano plot, 573 DEGs between L15 and WT were identified in the region with absolute values of log2 fold change greater than 1.5 and a p-value <0.05 (Figure 4B; Supplementary Table S4). To identify over-represented gene categories within the DEGs, we ran an enrichment analysis with both Blast2GO (Conesa et al., 2005) and TopGO (Alexa et al., 2006). Blast2GO found 35 categories (p-value < 0.05) from the 'biological function' subtree enriched with respect to the reference transcriptome (Supplementary Figure S3A). By grouping these GO categories into broader functional categories, phenylpropanoid metabolism, signaling and amino acid metabolism were over-represented. The analysis of GO terms using Blast2GO showed a higher percentage of transferase, protein phosphorylation, protein kinase and receptor activities, which suggests the participation of VviERF045 in complex regulatory pathways. In addition, the presence of genes related to secondary metabolite pathways, such as trihydroxystilbene synthase, naringerin-chalcone synthase, flavonoid biosynthesis, and flavonoid metabolic process suggests the involvement of VviERF045 in the synthesis and metabolism of phenolic compounds (Supplementary Figure S3A). Nine major GO categories came out enriched by applying weight count versus classic count in Fisher's enrichment test by using TopGO (Alexa et al., 2006): protein phosphorylation (Supplementary Table S4), wax biosynthetic process, response to endogenous stimulus, cotyledonal vascular tissue/pattern formation, drug transmembrane transport, jasmonic acid biosynthesis, response to salt stress (Supplementary Table S4), defense response to bacterium (Supplementary Table S4), and negative regulation of endopeptidase activity (Supplementary Figure S3B). #### Analysis of Metabolites We measured 56 phenolic secondary metabolites (Supplementary Figure S4), including chlorophyll a and b and total carotenoids (Figure 3C), hydroxycinnamic acids, benzoic acids, stilbenes, flavonols, flavan-3-ols and anthocyanins (Figure 5A) and lipids (Figure 6A) for the all five transgenic lines and WT (Supplementary Table S3). To have a general idea of the dispersion of the metabolic data, a PCA was computed for all the metabolites (Supplementary Figure S4). In this analysis the WT and the L6 plants were the most separated groups, whereas the other lines showed an intermediate position. Thus, the metabolomic study confirmed the extreme behavior of L6 line observed in the phenotypic characterization. ### VviERF045 Is Involved in Plant Growth and Development Among the DEGs we found a significant number of genes whose Arabidopsis putative orthologs are involved in growth and development, and more specifically are associated to the development of anatomical structures, the formation of cotyledonal vascular pattern, procambial histogenesis and multidimensional cell growth (Supplementary Table S4). Noteworthy was VIT\_04s0008g01970, coding for the putative ortholog of the ERECTA (ER) gene, which appeared strongly down-regulated in L15. ER codes for a leucine-rich repeat receptor-like Ser/Thr kinase that is a major transcriptional regulator with pleiotropic effects on development and plant physiology. It controls plant transpiration efficiency, modulating stomatal opening and CO<sup>2</sup> fixation (Masle et al., 2005), stomatal density and patterning (Lampard et al., 2008), abaxial-adaxial identity (Qi et al., 2004), petal shape and size (Abraham et al., 2013), ethylene induced hyponastic growth and leaf petiole angle (Van Zanten et al., 2010), leaf area and plant biomass during shade avoidance syndrome (SAS) (Kasulin et al., 2013), and resistance against specific pathogens such as fungi (Häffner et al., 2014). Indeed er loss of function mutants show reduced plant size, rounder and shorter leaves, shorter petioles and compact inflorescences in Arabidopsis. These features closely resemble those we observed in the lines overexpressing VviERF045, namely reduced leaf biomass, leaves with globular surface and different leaf margins and changes in leaf-petiole angle (Figure 2B). In L15, VIT\_18s0001g10160, coding for the putative WUSCHEL-RELATED HOMEOBOX4 (WOX4) grapevine ortholog, was induced twofold with respect to WT plants. WOX4 is expressed in the pro-cambium and plays an important role in vascular meristem organization. Recent evidence indicated that ER participates also in vascular development, acting upstream to WOX4 (Uchida and Tasaka, 2013), and our results suggest a similar interplay between these genes in grapevine. # VviERF045 Regulates Lipid Metabolism As Well As Cuticle and Waxes Synthesis Our results show that VviERF045 is functionally linked to lipid metabolism, specifically to the synthesis of cuticle and cuticular waxes. Optical images revealed a different pattern of the lipid distribution on the surface of L6 leaves compared to WT (Figure 7A). In the latter, the reddish color was evenly distributed along the cuticular layer that covers the epidermal cells, while in L6 the stain was observed in groups of intracellular droplets, similar to lipid bodies, in the epidermal layers. Scanning Electron Microscopy (Cryo-SEM) confirmed a striking difference in the structure of the epicuticular waxes between the two (Figure 7B): FIGURE 4 \| RNA-Seq analysis of L15 line. (A) Classical multidimensional scaling showing the percentage of variance explicated by PC1 and PC2. (B) Volcano plot used for selection of differentially expressed genes (DEGs) between L15 and WT lines. Spots represent genes. The red line represents the significance threshold of p-value (0.05) and the blue lines represent log2 fold change values of 1.5 and −1.5. Genes located above the red line and on the right-hand side or the left-hand side of blue lines were selected as DEGs. and highly significant differences (p < 0.01). A legend with the color scale is reported on the top left part. B.A.D., Benzoic acid derivative. (B) Transcripts involved in their biosynthesis. 4.3.1.25 Phenylalanine ammonium lyase (PAL) (VIT\_16s0039g01170, VIT\_16s0039g01240, VIT\_16s0039g01280, VIT\_16s0039g01300, VIT\_16s0039g01360), 1.1.1.195 cinnamyl-alcohol dehydrogenase (CAD), (VIT\_03s0110g00310, VIT\_13s0064g00270), ferulate 5-hydroxylase (F5H) (VIT\_17s0000g03930), 2.3.1.74 Chalcone reductase (CHR) (VIT\_01s0011g06440), 21.1.104 Caffeoyl-CoA O-methyltransferase 1 (VIT\_07s0031g00350), 1.11.1.7 peroxidase (VIT\_12s0028g01830, VIT\_14s0068g01920, VIT\_10s0116g01780, VIT\_18s0001g06890, VIT\_16s0098g00820), 2.3.1.95 STS (stilbene synthase) (VviSTS2, VviSTS3, VviSTS5, VviSTS6, VviSTS7, VviSTS10, VviSTS13, VviSTS15, VviSTS17, VviSTS18, VviSTS19, VviSTST20, VviSTST21, VviSTST25, VviSTS25, VviSTS28, VviSTS29, VviSTS30, VviSTS31, VviSTST37, VviSTST38, VviSTST39, VviSTST42, VviSTS46, VviSTS47 (Vannozzi et al., 2012). 2.3.1.116 Quercetin 3-O-glucoside-6<sup>00</sup> -O-malonyltransferase (VIT\_12s0134g00630). 2.4.1.115 anthocyanidin 3-O-glucosyltransferase (VIT\_03s0017g02110, VIT\_16s0022g01970), 2.4.1- UDP-glucose (VIT\_18s0041g00830, VIT\_18s0041g00840, VIT\_18s0041g00930, VIT\_18s0041g01010, VIT\_16s0050g01680). Green color means down-regulated gene, red color up-regulation. the WT cuticle appeared heavily decorated with wax aggregates, while the L6 leaf surface was smooth. The wax decoration in the other transgenic lines was reduced compared to WT. The lipid composition of leaf samples was analyzed to further understand the observed alterations at the cuticular level. Six lipid compounds belonging to the classes of fatty acids, sterols, glycerolipids, glycerophospholipids and sphingolipids appeared significantly modulated (Figure 6A). The steroid-like lanosterol was at a higher concentration in WT and diminished in transgenic lines proportionally to VviERF045 expression (Figure 6A). Lanosterol is known to be a component of the tomato cuticular waxes. In the lcer6 mutant, an increase of lanosterol together with other sterols and triterpenic cycles, was accompanied by a proportional decrease in long chain alkanes (Leide et al., 2007). This result is in line with the down-regulation in L15 of a squalene monoxigenase (VIT\_00s0441g00020), involved in the oxidation of squalene to squalene epoxide, the precursor for lanosterol biosynthesis (Figure 6B). Cuticular waxes are made of very-long-chain (VLC) fatty acids (FAs), synthesized starting from plastidial C16-C18 FAs, elongated into VLCFAs in the endoplasmic reticulum membrane, and subsequently modified into primary alcohols and wax esthers (Figure 6A, Yeats and Rose, 2013). Several genes involved in FA elongation and wax biosynthesis (VIT\_00s0179g00380, VIT\_01s0011g03490, VIT\_16s0050g00830) were expressed at lower level in the L15 transgenic line (Supplementary Table S4), implying VviERF045 overexpression reduces long FA and wax content. Down-regulation was observed for the putative orthologs of the Arabidopsis CER1 and CER2 genes, the grapevine genes VIT\_15s0021g00050 and VIT\_05s0029g00480. The cer1 mutant of Arabidopsis is blocked in the conversion of stem wax C30 aldehydes to C29 alkanes, leading to a lack of secondary alcohols and ketones. This biochemical impairment results in a reduced wax load on the leaf surface of the cer1 mutants (Bourdenx et al., 2011), resembling the cuticular phenotype we observed in the transgenic lines in this study. The closest sequence to VIT\_05s0029g00480 is CER26, the homologue of CER2, which encodes for an acyl-transferase enzyme involved in the elongation process of C28 FAs (Pascal et al., 2013). VIT\_08s0007g00390, similar to the Arabidopsis PROTEOLYSIS 6 (PRT6), was less expressed in the L15 line. prt6 mutants are impaired in lipid degradation and retain oil bodies in the cells, similar to the ones observed in the external layers of the L6 leaves in our analysis (Supplementary Figure S6) (Holman et al., 2009). The cytochrome P450 genes VIT\_02s0025g03320 and VIT\_07s0031g01680 were found down-regulated in VviERF045 overexpressing lines. VIT\_02s0025g03320 belongs to the CYP86A subfamily, known to have ω-hydroxylase activity on midchain FAs (Yeats and Rose, 2013). The similar Arabidopsis gene LACERATA was reported to be involved in cutin biosynthesis (Wellesen et al., 2001). The most similar sequence to VIT\_07s0031g01680 in Arabidopsis belongs to the CYP96A subfamily, which includes MIDCHAIN ALKANE HYDROXYLASE 1 (MAH1, CYP96A15), an enzyme of the decarbonylation pathway catalyzing the synthesis of cuticular wax secondary alcohols and ketones from VLC alkanes (Greer et al., 2007). L15 plants also displayed lower expression of three lipases belonging to the GDSL family (VIT\_13s0106g00350, VIT\_18s0041g02160, VIT\_18s0086g00220). Members of this large gene family appear to have a broad range of activities in the regulation of plant development, morphogenesis, synthesis of secondary metabolites, and defense response (Chepyshko et al., 2012). Recently, specific members within the family have been shown to play a role in cutin synthesis by catalyzing the formation of cutin ester oligomers (Yeats et al., 2014). The only two genes of the wax biosynthetic pathway which resulted up-regulated in L15 were VIT\_02s0012g02500 and VIT\_15s0046g00490, encoding for a putative stearoyl-acyl carrier protein-desaturase (S-ACP-DES) and a putative wax synthase/diacylglycerol acyltransferase 1 (WSD1), respectively (Figure 6A). In plants, S-ACP-DESs tune the ratio of saturated to monounsaturated FAs (Kachroo et al., 2007). In Arabidopsis, WSD1 is responsible for the esterification of VLC primary alcohols to long chain wax esthers using C16 FAs as substrates (Li et al., 2008). CR, Cuticular Ridge. Lower part: images of the adaxial leaf surface (1000X). WA, Wax Aggregates. As our results pointed toward a role of VviERF045 in regulating cuticle biosynthesis, we compared the effects of its overexpression with those described for the major known regulators, namely the SHINE gene family and some specific MYB TFs. WAX INDUCER1/SHINE1 (WAX1/SHN1) was the first TF identified (Aharoni et al., 2004). It is an ERF sequence of clade V, whose overexpression gives rise to dwarf plants with curved and glossy leaves, lower stomata density, thicker cutin and higher wax density. The cauline leaves of the gain of function mutant shine display cuticular ridges similar to those here reported on the L7 leaves (Aharoni et al., 2004; Kannangara et al., 2007). When SHN1 and the other two closely related members SHN2 and SHN3 were silenced, Arabidopsis plants exhibited, among other phenotypic traits, a decrease in cutin load (Shi et al., 2011), and changes in cuticle structure and lipid composition have also been demonstrated in tomato (Shi et al., 2013). SHINE regulators exert their function by acting on several cuticle- and epidermisassociated genes, including CYTOCHROME P450s, GSDL-type LIPASES, ACYLTRANSFERASES, LONG CHAIN ACYL\_CoA SYNTHASES, CER1 and CER2 (Kannangara et al., 2007; Shi et al., 2011, 2013). Genes with similar functions were down-regulated in L15 transgenic plants, as reported above. AP2-containing TFs can be either activators or repressors depending on the effect on transcription of specific target genes. Transcriptional repressors are further classified as active or passive repressors: active repressors contain a repression domain (RD), which allows these proteins to actively prevent transcription of a target gene; passive repressors do not have an RD and suppress transcription by competing with transcriptional activators for binding to the target sequence (Licausi et al., 2013). VviERF045 can not be classified as an active repressor because its sequence does not display a RD. The cuticular phenotype of the lines, as revealed by microscopical investigation, as well as the negative regulation of the cuticle- and waxrelated genes in L15, are similar to those reported for SHINE silenced lines of Arabidopsis and tomato, indicating VviERF045 as a potential passive repressor. In particular VviERF045 might negatively regulate VviERF042 encoded by VIT\_09s0002g06750 and VviERF044 encoded by VIT\_04s0008g05440, which are down-regulated in L15 (Supplementary Table S4). Interestingly, VviERF042 and VviERF044 are the putative horthologues of the Arabidopsis SHINE1and SHINE3 genes, whose silencing leads to a decrease in cutin load and to changes in cell wall structure (Shi et al., 2011) similar to the ones observed in the transgenic lines of this study. The FA chain α-linolenic acid is also the precursor of the phytohormone Methyl Jasmonate (MeJA) via the action of a lipoxygenase and a jasmonate O-methyltransferase. In L15 we found up-regulated the genes encoding for these two enzymes (VIT\_06s0004g01470 and VIT\_14s0006g02170), as well as for a MeJA esterase (VIT\_00s0253g00090), catalyzing the inverse reaction from MeJA to JA (Supplementary Figure S6). MeJA and JA are considered to be defense-related hormones and they do not seem to play a major role during berry ripening. It is not clear if the observed induction of the MeJA biosynthetic genes in L15 is related to the stress induced by the expression of the transgene or to direct regulatory effect of VviERF045. ### VviERF045 Modulates Genes Involved in Secondary Metabolic Processes Phenolics are a large and complex group of secondary metabolites with chemical properties that contribute to pigmentation and defense against several biotic and abiotic stresses in grapes (Ali et al., 2010). Their biosynthesis starts from the amino acid phenylalanine which is converted into a vast array of molecules belonging to the major classes of the phenylpropanoids (hydroxycinnamic acids, stilbenes and lignins) and of the flavonoids (flavonols, proanthocyanidins and anthocyanins) (Figure 5B). Several DEGs belonging to the phenylpropanoid and flavonoid biosynthetic pathway (Supplementary Table S4) encode for enzymes often positioned at the branching point of the pathway. In agreement with the overall increases in phenolic compound concentration in the transgenic lines, the majority of related DEGs also were up-regulated (Supplementary Table S4, Figure 5B). This includes the induction of five PHENYLALANINE AMMONIA-LYASE (PAL) encoding genes (VIT\_16s0039g01170, VIT\_16s0039g01240, VIT\_16s0039g01280, VIT\_16s0039g01300 and VIT\_16s0039g01360), which catalyze the conversion of L-phenylalanine to trans-cinnamic acid and ammonia. Among the DEGs there are also genes known to affect lignin amount and composition, suggesting that this metabolic class was likely induced as indicated by the high level of vanillin in L6 (Vanholme et al., 2010). In particular, different genes coding for cinnamyl-alcohol dehydrogenases (CADs), ferulate 5 hydroxylase (F5H), caffeoyl-CoA o-methyltransferase (COMT1) and several peroxidases were induced in L15 (Figure 5B). It is interesting to note that SlSHN3 silenced tomato lines showed a thicker cell wall of the epidermal cells, and that Ambavaram et al. (2011) reported that AtSHN2 controls secondary cell wall biosynthesis (lignin and cellulose) acting on CAD genes (Ambavaram et al., 2011), observations that support VviERF045 acting as a SHINE factor. Twenty-three stilbene synthase (STS) genes appeared positively associated to VviERF045 over-expression. STSs form a rather expanded gene family in grapevine, including at least 33 members (Vannozzi et al., 2012), and they produce the basic stilbene structure, trans-resveratrol, from one p-coumaroyl-CoA and three malonyl-CoA molecules. Trans-resveratrol can then be modified by hydroxylation, methylation, glycosylation, or condensation of more units to form the ample class of stilbenoids, which represent the major antimicrobial phenolic compounds in grapevine (Jeandet et al., 2002; Malacarne et al., 2011). These compounds are also produced upon abiotic stresses such as UV-light, salinity stress (Ismail et al., 2012), and during leaf senescence and fruit ripening (Gatto et al., 2008). The only highly accumulated stilbene common to all transgenic lines was the glucoside derivative of t-resveratrol, trans-piceide, but in L15 and L19 also the monomers cis-piceide, isorhapontin, astringin and the dimers pallidol and ampelopsin D exhibited higher levels than in WT plants. Since polymeric forms of resveratrol are usually produced during fungal attacks (Malacarne et al., 2011), this might indicates that VviSTS upregulation in L15 was mainly driven by a more general stress (Cuendet et al., 2000). In grapes, flavan 3-ols are mainly present in skin and seed tissues, where they accumulate before véraison. In vegetative organs, their content constantly increases during leaf development, but their synthesis decreases in old leaves (Bogs et al., 2005). They are found as monomers, namely catechin, epicatechin and epicatechin 3-O-gallate, as well as oligomers, and polymers called proanthocyanidins (PA), also known as condensed tannins. In our transgenic lines, compounds of this class, either in monomeric or dimeric form (procyanidin B), or condensed to caffeic acid, were clearly found at higher concentration than in WT (Figure 5A). As flavan 3-ols appear to function in resistance against various biotic and abiotic stresses, including UV irradiation by decreasing oxidative stress (Hammerbacher et al., 2014), it is likely that the transgenic lines face a more stressful situation than WT plants, due, for example, to cuticle impairment and to reduced photosynthetic capacity. In our experiment, a significant higher content of peonidin p-coumaryl3glu and to a lesser extent of the glycosylated forms of cyanidin, delphinidin and malvidin, were observed in most transgenic lines (Figure 5A). The glycosylated forms of the flavonols quercetin and isorhamnetin displayed a similar behavior. Where the main role of anthocyanins in grapes is the red berry pigmentation to attract animals for seed dispersal, the main function of flavonols is UV-protection. Both classes are antioxidant molecules induced during different stresses, which might be the main reason of their increase in the transgenic overexpression lines. In case of the anthocyanins, the expression data were congruent with the metabolic data for two anthocyanidin 3 o-glucosyltransferases up-regulated in L15 (VIT\_03s0017g02110, VIT\_16s0022g01970, Supplementary Table S4), but less coherent for five MATE genes (VIT\_11s0052g01560, VIT\_11s0052g01540, VIT\_07s0031g00750, VIT\_00s0225g00080, VIT\_11s0052g01500 Supplementary Table S4), which were down-regulated. This grapevine protein family plays a role in the H+-dependent transport of acylated anthocyanins into the vacuole (Gomez et al., 2009), and the observed down-regulation possibly indicates a problem with the vacuolar storage of these molecules. Another important class of secondary metabolites affected in the transgenic over-expressing lines was the photosynthetic pigments, namely chlorophylls and carotenoids. As expected from the pale leaf color, the analysis of chlorophylls and carotenoids confirmed a much lower concentration in the transgenic lines, with a minimum in L6 (Figure 3C). During fruit ripening the photosynthetic apparatus is dismantled (Lijavetzky et al., 2012), and our results suggest that VviERF045 might play such a role in the berries. In Arabidopsis COP1-INTERACTING PROTEIN 7 (CIP7) is involved in light-dependent anthocyanin and chlorophyll accumulation (Yamamoto et al., 1998). The putative CIP7 gene of grapevine (VIT\_00s1306g00010) was down-regulated in our study, as confirmed by RT-qPCR (Supplementary Figure S2). This gene was reported to be down-regulated at véraison in five red Italian varieties (Palumbo et al., 2014), as well as during post harvest withering (Fasoli et al., 2012). Other L15 repressed genes related to chlorophyll metabolism are FERRITINS (VIT\_08s0058g00410, VIT\_08s0058g00430, VIT\_08s0058g00440), iron-storage proteins involved in the regulation of free iron levels in the cells, whose impairment cause rapid natural senescence with leaf yellowing accompanied by accelerated decrease of maximal photochemical efficiency and chlorophyll degradation (Murgia et al., 2007). In the transgenic lines, we observed the up-regulation of sesquiterpene synthase genes encoding for delta-cadinene synthase, alpha-farnesene synthase and valencene synthase (Lücker et al., 2004) (Supplementary Figure S5). Sesquiterpenes are a class of volatile terpenoids enriched in the epicuticular wax layer of the berry fruit. They act as antimicrobial volatile compounds (Petronilho et al., 2014) and they are induced by pathogenic fungi as well as by elicitors and MeJA (Hampel et al., 2005), but they contribute to the typical flavor of aromatic grape varieties too. ### VviERF045 in Fruit Ripening fpls-07-01793 December 9, 2016 Time: 15:1 # 13 Berry ripening is a complex physiological process under tight regulation, which begins about 8 WAA and proceeds for about 5–6 weeks. From ripening onset, the berry undergoes chlorophyll degradation, accumulation of color, sugar and aroma compounds, organic acid catabolism, and an increase in berry size and elasticity (Coombe and McCARTHY, 2000). Among the ERF regulatory factors possibly linked to the berry ripening process, identified previously in a microarray experiment on Pinot Noir berries at three developmental stages (Pilati et al., 2007), we selected VviERF045 for further characterization, since this factor displays a fruit ripening specific expression (Figure 1). An important role for VviERF045, as major switch in berry ripening, was recently also proposed by Palumbo et al. (2014). Although our study was not conducted on berries, but in leaves from in vitro plants, implying that the results cannot be transferred straightforwardly to the fruit system, we have observed the modulation of several processes in the transgenic overexpressing lines, which are also typical of grape ripening: changes in the epidermis and in the cuticle, a decrease in photosynthetic capacity, and the activation of several defense related genes. In this study, we collected clear evidence that VviERF045 regulates wax biosynthesis and the morphology of the cuticle and probably of the cell wall in the epidermal cells by modulating a set of specific genes. The phylogenetic proximity of VviERF045 to the SHYNE clade (Figure 1E) of ERFs, known to function in cuticle and epidermis patterning, further corroborates this conclusion. At ripening onset, three processes take place, all of which imply a modification of the outer structures of the epidermal cells and thus possibly the intervention of VviERF045: berry softening, berry expansion (Coombe, 1992), and a reduction in the thickness of cuticular waxes (Rogiers et al., 2004). In the overexpressing transgenic line L15, genes known to be involved in these berry processes, such as an endo-1,4-betaglucanase (VIT\_04s0008g02010) involved in cell wall disassembly (Libertini et al., 2004), three expansins (VIT\_06s0004g04860, VIT\_06s0004g07970, VIT\_12s0059g00190), a polygalacturonase PG1 (VIT\_07s0005g01550), and a pectinesterase (VIT\_11s0016g00330) related to berry expansion and skin softening (Deytieux-Belleau et al., 2008), are down-regulated compared to the WT plants. These same genes are induced in the berry, at ripening onset. This might suggest that VviERF045 down-regulates these enzymes to counterbalance an excessive cell wall disassembling. The post-véraison development of an amorphous layer of cuticular waxes and the observation that deposition of epicuticular wax ceases at véraison as reported in Shiraz berries (Rogiers et al., 2004), is in line with our microscopic analyses (Figure 7) and the repression of cuticle and wax biosynthetic genes in L15 (Figure 6). With the beginning of berry ripening, the photosynthetic apparatus is dismantled and consequently the photosynthetic capacity of the berry drops dramatically (Pandey and Farmahan, 1977). VviERF045 could contribute to this switch-off in virtue of its effect on chlorophylls and carotenoids content (Figure 3), and the down-regulation of genes important for chlorophyll accumulation, like CIP7 and FERRITINs. Many pathogen-resistance genes appear modulated by VviERF045 (Supplementary Table S4), suggesting its action also increases plant defense via activation of the basic immune defense system. Among the proteins that change their levels of expression during berry ripening, there are many pathogenesis-related proteins (PRs). PRs are highly abundant at ripening and generally lowly expressed or absent in unripe berries. The presence of this class of proteins in healthy fruit suggests that they may play a role in fruit development, or that they are part of a pre-emptive defense when softening and sugar accumulation make fruit attractive targets for pathogens (Davies and Robinson, 2000). To further corroborate the importance of the obtained results in understanding berry ripening regulation, we ran in silico analyses taking advantage of the grapevine gene expression compendium VESPUCCI (Moretto et al., 2016). We looked whether the 563 DEGs modulated in the L15 to WT plants comparison, were expressed in the berry during ripening, in order to gain insights about their role in the process. Five hundred and forty five DEGs (18 genes were not unique in the database) were analyzed in 389 condition contrasts (Supplementary Table S5) mostly derived from samples of berries at different phenological stages, between EL 27 and EL 41. Interestingly, a large fraction (70%) of the DE genes appeared either up- (153 genes) or down-regulated (231 genes) (Supplementary Table S4), indicating that these genes are indeed modulated during ripening. The two groups were also enriched in functional classes characteristic of berry ripening like starch and sucrose metabolism, auxin biosynthesis, ethylene signaling and phenylpropanoid biosynthesis in the case of the up-regulated genes, cell wall and HomeoBox TFs in the case of the down-regulated ones. An important interaction between ethylene and auxin in the control of berry ripening has been recently elucidated (Böttcher et al., 2013). Within the DEGs, we found 7 ERF encoding genes: two SHINE putative horthologues (VviERF042 and VviERF044) that were downregulated, and other five ERFs that were strongly up-regulated. In this last group with the exception of VviERF045, there were four ERF genes (VviERF093, VviERF111, VviERF118, VviERF120), from clade IX or X, previously shown to be induced in the transition from véraison to ripe berries either in skin or in flesh (Licausi et al., 2010). These evidences strongly suggest an involvement of these ERF TFs in the control of berry ripening. # CONCLUSION We have functionally characterized VviERF045 by overexpressing the encoding gene in in vitro grown grapevine plants and by phenotyping them at morphological and molecular level. VviERF045 seems to regulate, in coordination with other ERF factors, including the putative horthologues of the Arabidopsis SHINE1 and 3 genes, different processes such as the structuring of the epidermis and cuticle of the berry, cell expansion, photosynthesis, phenylpropanoid metabolism and the activation of several defense related genes. If this functional role will be confirmed by follow-up studies on the fruits of the transgenic lines, we can predict that having the possibility to adjust the expression of VviERF045 by well-timed viticultural practices (e.g. water stress, hormonal treatments) or by breeding, might allow to improve grape quality and plant resilience. The expression of VviERF045 can be used as an expression marker of the plant resilience status. # AUTHOR CONTRIBUTIONS CL, AD, VP, and DM did the experimental work, CL, LD, MG, and GR assessed the best way to prove the gene function, CL did the phylogenetic trees, PS and KE elaborated RNA-seq data and were involved in data interpretation, CL, GR, and CM substantially contributed to the design of the work. All the authors revised it critically for important intellectual content and approved the final version of this manuscript. ### FUNDING CL was supported by the Marie Curie FP7-PEOPLE-2011-CIG action program-[Graperipe project n. 303907]. Network activities have been supported by COST1106 action. #### ACKNOWLEDGMENTS Authors thank Pietro Franceschi for suggestions in the PCA statistical analysis, Valentino Poletti and Susanna Micheli for helping in in vitro micropropagation, Luca Zulini for chlorophyll and carotenoids measure method, Marisol Gascón Irún and Manuel Josep Planes Insausti for the excellent service with the optic and electronic microscope, Michele Perazzolli for preliminary work on VviERF045 expression analysis and Ivana Gribaudo for 'Brachetto' embryogenic callus. #### REFERENCES # SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.01793/ full#supplementary-material #### FIGURE S1 \| Library size of each RNA-seq sample replicate. FIGURE S2 \| Comparison of RNA-seq and real time RT-qPCR analysis. Expression profiles of VviERF045 VIT\_04s0008g06000, RPT2 VIT\_00s0665g00050, PIR VIT\_00s1306g00010, NAC VIT\_01s0026g02710, ERECTA VIT\_04s0008g01970, SBP VIT\_04s0210g00170, N-acetyltransferase VIT\_05s0020g03680, HB1 VIT\_12s0059g01190, SA VIT\_14s0006g02170, YABBY VIT\_15s0048g00550, Peroxidase VIT\_16s0098g00820, HB1 VIT\_18s0001g10160. Lines represent expression levels (log Fold Change) by RNA-seq analyses in WT and L15, reported as means and standard errors of three independent biological replicates. Histograms represent the relative expression levels (logFC) to the expression of the WT, as assessed by real time RT-qPCR and reported as means and standard errors of three biological and two technical replicates for each plant line. Different letters show significant differences among samples with p < 0.05 and Tuckey's significance test. In case of no significance no letters are reported in the figure. FIGURE S3 \| GO term enrichment in selected DEGs. (A) Blast2GO Fisher's enrichment test analysis for GOterms. Blue bars indicate Test Set (DEGs from L15 vs. WT comparison) while red line indicate the Reference Set (entire reference transcriptome). On the X-axis is reported the percentage of sequences for each GO category (B) Best 9 GOterms by comparing classic with weight method from Fisher's test elaborated with TopGO (Alexa et al., 2006). FIGURE S4 \| PCA score and loading plots of metabolites in the transgenic lines. Different color points represents different samples: black (L15), red (L19), green (L22), blue (L6), light blue (L7), pink (WT). Distribution of the average values in (A) all the analyzed metabolites, (B) phenolic compounds, (C) anthocyanins (D) lipids (E) chlorophylls (Ca and Cb coincide) and total carotenoids (Cxc). The most weighted loadings are represented in each plot. FIGURE S5 \| Transcripts involved in Terpene biosynthesis. 4.2.3.46 alpha-farnesene synthase (AFS1) (VIT\_00s0361g00060, VIT\_00s0392g00030, VIT\_00s0392g00060), 1.14.99.7 (VIT\_00s0441g00020) squalene monoxygenase, 4.2.3.13 (VIT\_18s0001g04710) (+)-delta-cadinene synthase, 4.2.3.75 (-)-germacrene D synthase (VIT\_18s0001g04990, VIT\_18s0001g05240), 4.2.3.119 (VIT\_08s0007g06860) pinene synthase, 1.14.-.- CYP82C4 (VIT\_18s0001g11480), 1.3.3.9 CYP72A1 secologanin synthase (VIT\_19s0135g00150), 1.14.13.72 C-4 sterol methyl oxidase (VIT\_00s2125g00010), CYP724B1 (VIT\_14s0066g00170), CYP90B1 Steroid 22-alpha-hydroxylase (VIT\_04s0023g01630, VIT\_04s0023g01640, VIT\_12s0057g01460). Green color means down-regulated gene, red color means up-regulated gene. FIGURE S6 \| Transcripts involved in alpha-linolenic metabolism. 1.13.11.12 LOX1 (VIT\_06s0004g01470) lipoxygenase 1, 21.1.141 Jasmonate O-methyltransferase (VIT\_14s0006g02170), MJAE MeJA esterase (VIT\_00s0253g00090). Red color means up-regulated gene. cooperatively regulated by ethylene and jasmonate in Arabidopsis thaliana. J. Plant Res. 119, 407–413. doi: 10.1007/s10265-006-0287-x growth in Arabidopsis thaliana is controlled by ERECTA. Plant J. 61, 83–95. doi: 10.1111/j.1365-313X.2009.04035.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 © 2016 Leida, Dal Rì, Dalla Costa, Gómez, Pompili, Sonego, Engelen, Masuero, Ríos and Moser. 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. # Sequence Polymorphisms and Structural Variations among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture Luca Mercenaro<sup>1</sup> , Giovanni Nieddu<sup>1</sup> , Andrea Porceddu<sup>1</sup> , Mario Pezzotti<sup>2</sup> and Salvatore Camiolo<sup>1</sup> \* <sup>1</sup> Dipartimento di Agraria, Università degli Studi di Sassari, Sassari, Italy, <sup>2</sup> Dipartimento di Biotecnologie, Università degli Studi di Verona, Verona, Italy The genetic diversity among grapevine (Vitis vinifera L.) cultivars that underlies differences in agronomic performance and wine quality reflects the accumulation of single nucleotide polymorphisms (SNPs) and small indels as well as larger genomic variations. A combination of high throughput sequencing and mapping against the grapevine reference genome allows the creation of comprehensive sequence variation maps. We used next generation sequencing and bioinformatics to generate an inventory of SNPs and small indels in four widely cultivated Sardinian grape cultivars (Bovale sardo, Cannonau, Carignano and Vermentino). More than 3,200,000 SNPs were identified with high statistical confidence. Some of the SNPs caused the appearance of premature stop codons and thus identified putative pseudogenes. The analysis of SNP distribution along chromosomes led to the identification of large genomic regions with uninterrupted series of homozygous SNPs. We used a digital comparative genomic hybridization approach to identify 6526 genomic regions with significant differences in copy number among the four cultivars compared to the reference sequence, including 81 regions shared between all four cultivars and 4953 specific to single cultivars (representing 1.2 and 75.9% of total copy number variation, respectively). Reads mapping at a distance that was not compatible with the insert size were used to identify a dataset of putative large deletions with cultivar Cannonau revealing the highest number. The analysis of genes mapping to these regions provided a list of candidates that may explain some of the phenotypic differences among the Bovale sardo, Cannonau, Carignano and Vermentino cultivars. Keywords: Vitis vinifera, next generation sequencing, structural variation, SNP, CNV, Run of homozygosity # INTRODUCTION Grapevine berries (Vitis spp.) are marketed worldwide as wine, fresh and dried fruits, and as ingredients for cosmetics and nutraceuticals<sup>1</sup> . These diverse applications are possible due to the broad genetic basis of cultivated grapevine germplasm (Laucou et al., 2011; Emanuelli et al., 2013; Maul et al., 2015), which has been propagated independently by many civilizations throughout history (Imazio et al., 2006; This et al., 2006). There are now thousands of cultivated varieties, many <sup>1</sup>http://www.oiv.int/ #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics (CRAG), Spain #### Reviewed by: Rosa Arroyo-Garcia, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Spain Sergio Lanteri, University of Turin, Italy > \Correspondence: Salvatore Camiolo [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science > Received: 06 March 2017 Accepted: 06 July 2017 Published: 20 July 2017 > > Citation: #### Mercenaro L, Nieddu G, Porceddu A, Pezzotti M and Camiolo S (2017) Sequence Polymorphisms and Structural Variations among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture. Front. Plant Sci. 8:1279. doi: 10.3389/fpls.2017.01279 grown in the traditional wine-producing countries of Europe, which have arisen by spontaneous mutation, hybridization, self-fertilization, and interactions with viruses (Arroyo-Garcìa et al., 2006). There is significant evidence of introgression from wild vine (Vitis vinifera europaea subsp. sylvestris) in current commercial cultivars (Sefc et al., 2003; Crespan, 2004; Myles et al., 2011). The domesticated grapevine is thought to have originated in the Mediterranean (Zohary, 1995; Imazio et al., 2006) although a secondary center of domestication has been proposed in the western Mediterranean (Grassi et al., 2003; Chessa and Nieddu, 2005; Arroyo-Garcìa et al., 2006). The high diversity of local germplasm reflects the domestication of the wild relatives and has been conditioned by vegetative propagation and the repeated introduction of cuttings and plants (Myles et al., 2011). One of the proposed secondary origins is the island of Sardinia, the second largest island in the Mediterranean Sea (Grassi et al., 2003; Chessa and Nieddu, 2005). This ancient civilization was among the first in the western part of the basin to cultivate grapevine and process the berries (Ucchesu et al., 2015, 2016). Wild vines still grow near hundreds of Sardinian rivers, wetlands and commercial vineyards, and the berries are often used to make wine. The extant Sardinian grapevine germplasm includes hundreds of cultivars differing in agronomic performance, berry chemical composition and oenological potential (Castia et al., 1992; Calb et al., 2006; Vacca et al., 2009; Nieddu, 2011). Even so, only 26 traditional cultivars are recorded in the national grape varietal register, although dozens more can be found in older Sardinian vineyards. Despite the large number of cultivars present in Sardinia, few are routinely used for wine production. The amount of land dedicated to viticulture in Sardinia is 26,000 ha, 85% of which is represented by six cultivars. These include the three major red berry varieties Cannonau, Carignano and Bovale sardo, and the major white berry variety Vermentino (Nieddu, 2011). Cannonau is the most important red berry cultivar grown in Sardinia (8000 hectares, 29% of the total) and is considered synonymous with Garnacha tinta cultivated in Spain and Grenache noir cultivated in France (di Rovasenda, 1877; Molon, 1906; Viala and Vermorel, 1991). This relationship has been confirmed by ampelographic analysis and the comparison of isoenzymes and molecular markers (Calb et al., 2006; De Mattia et al., 2007; Cipriani et al., 2010). The Grenache family is cultivated on 185,000 ha of land worldwide (Anderson and Aryal, 2013) and the existence of differentiated locally adapted genotypes has been proposed (Ortiz et al., 2004; Mercenaro et al., 2016b), with the sequence diversity of numerous accessions sampled in France, Spain and Italy clustering according to the sampling area (Meneghetti et al., 2011). A comparison between cultivars form Armenia and Georgia (the first areas of grapevine domestication) with European cultivars showed that Cannonau was more closely related to the transcaucasian varieties than to other Italian cultivars (Crespan, 2010). Carignano is grown mostly ungrafted on 2,000 ha of land in south-western Sardinia characterized by extremely sandy soil (Mercenaro et al., 2014). It is genetically similar to the Spanish varieties Carinena and Mazuelo, and the French variety Carignan noir (Mercenaro et al., 2014). These synonymous varieties are cultivated on 80,000 ha of land worldwide (Anderson and Aryal, 2013). Bovale sardo is cultivated on ∼800 ha in central Sardinia and is characterized by extensive intra-varietal differences (Nieddu, 2011). It probably has a local origin (Grassi et al., 2003). Finally, Vermentino is one of the most promising white wine cultivars (ranking fifth by volume of wine sold in Italy). It is traditionally cultivated in the west Mediterranean region and was recently introduced into Australia, South Africa and Argentina. Sardinia and France each cultivate Vermentino on ∼4,200 ha, with further vineyards in Ligury and West Tuscany. Sardinian Vermentino has been awarded DOCG status (Controlled and Guaranteed Denomination of Origin). The combination of high throughput sequencing technologies and the grapevine reference genome (Jaillon et al., 2007) has facilitated comprehensive sequence analysis in diverse grapevine germplasms. Cultivars with different agronomic and oenological characteristics have been re-sequenced to identify genetic differences underlying the distinct phenotypes (Da Silva et al., 2014; Di Genova et al., 2014; Cardone et al., 2016) and comprehensive sequence variation maps are under construction to link these differences with transcriptomic and metabolomic data, as well as information about grapevine breeding practices (Ray and Satya, 2014). In this regard, availability of NGS data boosted, particularly in the last decade, the identification of candidate genes involved in response to stress (Xu et al., 2014), in the production of essential secondary metabolites (Kim and Buell, 2015) in both model and non-model plants (Unamba et al., 2015). Indeed, Giannuzzi et al. (2011) identified duplications in grapevine cultivar Pinot Noir hosting genes responsible for adaptation and response to environmental changes. The analysis of genomic features in different V. vinifera cultivars will expand our knowledge of the evolution of the grapevine genome and will facilitate breeding programs. Here we report a thorough characterization of genomic sequence variation in four Sardinian cultivars compared to the PN40024 reference genome to determine the genomic characteristics underlying the phenotypic differences among these varieties. SNPs and indels for the four Sardinian cultivars were compared to data from three additional cultivars (Gewurztraminer, Sultanina and Tannat) that are not typical of this island agriculture. The present study aims to characterize the reported cultivars in terms of SNPs/indels, complex structural variations and degree of homozigosity, in order to speculate those features possibly underlying their phenotypic peculiarities. # MATERIALS AND METHODS #### Reference Sequence and Annotation The grapevine reference genome with corresponding annotations and associated gene ontology terms (V. vinifera, cv. Pinot noir, PN40024 12× assembly V1 prediction) was downloaded from the Grapevine Genome CRIBI Biotech Center website<sup>2</sup> . We used gff2sequence (Camiolo and Porceddu, 2013) to generate coding sequences (CDSs) and the 5<sup>0</sup> and 3<sup>0</sup> untranslated regions (UTRs). <sup>2</sup>http://genomes.cribi.unipd.it/grape/ #### DNA Resequencing fpls-08-01279 July 18, 2017 Time: 17:22 # 3 High molecular weight DNA was extracted from nuclei starting form 3–5 g of young leaves of V. vinifera cv. Cannonau, Bovale, Carignano and Vermentino, using the procedure described in Zhang et al. (1995) without embedding the nuclei in agarose plugs, but directly performing the lysis of nuclear walls with detergent and proteinase K. Resequencing with an Illumina HiSeq 2000 instrument at the Istituto di Genomica Applicata (IGA, Udine, Italy) produced paired-end short reads of variable length and number (Supplementary Table S1). The produced reads have been deposited in the SRA database with the accession numbers SRR5803837, SRR5803836, SRR5803839 and SRR5803838 for Bovale, Cannonau, Carignano and Vermentino, respectively. Sequence read datasets were quality filtered using the NGS QC toolkit (Patel and Jain, 2012) with default parameters prior to downstream analysis. Quality filtering together with the entire downstream analysis pipeline described below was also carried out on three additional grapevine varieties (Gewurztraminer, Tannat and Sultanina) for comparison. The resequencing data corresponding to these latter three cultivars were retrieved from the Sequence Read Archive (Gewurztraminer, ERR514999; Sultanina, SRR924200; and Tannat, SRR863595 and SRR863618). #### Alignment of Reads and Single Nucleotide Variation Detection Filtered reads were aligned to the reference genome using Altools (Camiolo et al., 2016) (edit distance = 5% of the read length, base quality cutoff = 10). The embedded Altools Pileup Analyzer module was used to create a pileup formatted file reporting only essential data such as coverage and presence/absence of SNPs/indels at each genomic position. Base information was retrieved only when the corresponding position was covered by at least three reads, whereas SNPs and indels were considered only at positions featuring at least the average coverage and with the polymorphism supported by at least half of this value. The statistical significance of the called SNPs and indels was estimated using VarScan (Koboldt et al., 2012) with default parameters and applying a p-value cutoff of 0.05. In order to avoid possible bias in the detection of polymorphisms we only retained unambiguously mapped reads at this stage. Finally a threshold of 0.2 in the allele frequency was applied to minimize the detection of somatic mutation. In this condition only 0.1% of the called polymorphisms proved to feature an excessive depth of coverage (e.g., 6 times the coverage standard deviation) possibly underlining alignment artifacts. #### Genome-Wide Data Visualization The Altools Sliding Analysis module was used to visualize the alignment statistics along the genome (e.g., coverage, SNPs and indels). Briefly, each chromosome was split into a series of adjacent windows (windows size and step = 20,000 bp) that were investigated in terms of average coverage and polymorphism density. Only positions that were covered by reads were used at this stage. The resulting data were used to plot a genome circular representation using Circos (Krzywinski et al., 2009), with all the reported values normalized to the genome average. #### Depth of Coverage Analysis The Altools Coverage Analyzer tool was used to detect copy number variation (CNV) and presence/absence variation (PAV) in the grapevine cultivar genomes. False CNV due to known genomic repeats was avoided by comparison with the reference genome. Therefore, we first generated a simulated Illumina paired-end reads dataset for the PN40024 reference genome (average coverage 40×) using DWGSIM<sup>3</sup> and repeated the alignment and pileup procedures. Simulated reads were used in place of the real resequencing data to reduce the effect of non-homogeneous coverage and hemizygous loci in the reference genome (although we are aware that the usage of real reference genome resequencing data may take into account possible technical artifacts in the reads generation step). We then computed the coverage ratio between the target (all paired reads properly mapping were considered at this stage) and reference genomes in 500-bp adjacent windows. Significant fluctuations in the ratio identified using the DNAcopy algorithm (Seshan and Olshen, 2010) were deemed to explain the CNV. PAV was called if coverage was detected in the reference genome but not in the target. Only structural variations longer than 1000 bp were retained for downstream analyses. We decided not to use the paired end information to detect smaller structural variations, since the used DNA library featured a short insert. Hereafter we define gains as those genomic regions featuring a significant higher copy number in the target genome compared to the reference. Similarly we define losses as those regions featuring a lower copy number in the target genome (including no copies for zero-coverage areas). It is important to note that such definitions are not intended as statements of phylogenesis because the absence of an outgroup makes it impossible to establish which genome has lost or gained DNA during evolution. For the same reason, no effort was made to infer the copy number of these regions. The Altools Genic Extractor tool was used retrieve the annotated genes within the CNV and PAV regions. #### Large Deletions Analysis Reads mapping onto the reference produced a sam formatted alignment file (Li et al., 2009) that was used to investigate the occurrence of large deletions. We first filtered the initial dataset by removing all reads mapping at multiple positions (only alignments featuring the XT:A:U tag were retained). This step was necessary to exclude the interpretation of genome duplication events within the same chromosome as large deletions. Paired-end reads mapping at a distance between 10,000 and 1,000,000 bp, e.g., incompatible with the estimated insert size, were then considered to reflect a large deletion event. Only structural variations confirmed by at least three paired-end read mates were used for downstream analysis. Such a task was performed by using the Large deletion finder software within the Altools suite. The Genic Extractor tool was used to retrieve the annotated genes within the large deletions. Large deletions <sup>3</sup>https://github.com/nh13/DWGSIM together with CNV were not analyzed for the three outgroup cultivars for the sake of clarity and to keep the focus on the varieties typical of the Sardinian agriculture (although they will be considered for future studies). #### Genic Polymorphism Analysis Single nucleotide polymorphisms and indels were mapped to the genic portion of each genome using the Altools module Polymorphism Analyzer. This estimated the number of events with the potential to modify polypeptide structures, e.g., non-synonymous substitutions responsible for amino acid replacements or premature stop codons, or indels creating a frameshift in the CDS. Transcripts featuring more than five SNPs were aligned to the corresponding reference gene and dn/ds was calculated by using scripts incorporating the Biopython (Cock et al., 2009) library cal\_dn\_ds and using the Maximum Likelihood estimation method. Significance of the selection signal was tested by a Fisher's exact test. ### Regions Characterized by Extensive Homozygosity Regions of homozygosity (ROH) were identified using plink (Purcell et al., 2007) with a sliding window of 500 SNPs and a minimal ROH size of 50 kb with one heterozygous or missing SNP allowed for each window. Because ROHs may arise due to hemizygosity, we excluded all ROHs that overlapped regions identified as losses. ## Gene Ontology Studies The R package topGO was used to carry out single-gene enrichment analysis and to determine ontology codes for biological processes and molecular functions. # RESULTS #### Alignment Statistics Paired-end genomic reads representing cultivars Bovale, Cannonau, Carignano and Vermentino were mapped to the PN40024 reference genome and reads representing cultivars Gewurztraminer, Sultanina and Tannat were downloaded from public databases for comparison. As shown in Table 1, there was significant diversity among the cultivars in terms of several sequence diversity parameters. The SNP density (number of SNPs per Mbp of covered reference genome sequence) varied from a minimum of 5508.0 for Bovale to a maximum of 8522.1 for Vermentino. The indel density (number of indels per Mbp of covered reference genome sequence) ranged from 213.4 for Gewurztraminer to 728.4 for Vermentino. The ratio of the total number of heterozygous/homozygous SNPs varied from 0.6 for Bovale to more than 2 for Cannonau, Gewurztraminer and Tannat. On the other hand, the ratio of the total number of homozygous/heterozygous indels was ∼2 in most cultivars, although Bovale was exceptional with a ratio of 0.7. Among the varieties cultivated in Sardinia, the highest sequence diversity compared to the reference genome was observed for Vermentino, as confirmed by the lowest number of aligned sites. It is important to note that both the total number of reference bases covered by reads and the total number of reads (e.g., the depth of re-sequencing) differed widely for each cultivar. However, it is unlikely that these factors influenced our diversity estimations significantly because we used a conservative approach in which variant calling was restricted to genomic regions covered by a number of reads at least equal to the average genome coverage. Mild tendencies toward a compositional shift emerged from compositional analysis of the polymorphic sites: the average GC content of the polymorphic sites was lower in the reference genome than in the resequenced cultivars, particularly in the case of Cannonau, Gewurztraminer and Tannat (Table 1). To gain insight into the level of sequence diversity at regions presumably subjected to purifying selection, we extrapolated the sequence polymorphisms within the transcripts (CDS and UTRs). As expected, the polymorphism density was much lower in these regions, particularly in the CDS, where sequences are under greater selective pressure due to their role in protein synthesis (Table 2). The density of indels in the CDS was even lower, presumably due to their ability to cause disruptive frameshifts (Table 2). In some cultivars, the sequence variation in genic regions was dissimilar to the variation observed at the whole-genome level. Cannonau showed the least genomic variation but the highest SNP density in transcripts, although this trend was not uniform throughout the transcript. Indeed, Cannonau UTRs (but not CDSs) proved to be more polymorphic than the other cultivars with the exception of Gewurztraminer (Table 2). Bovale, the most similar to the Pinot noir reference genome at the genic level, ranked second in terms of CDS diversity. Indel density in the CDS was uniform in all cultivars with the exception of the two white berry varieties Vermentino and Sultanina, which showed a remarkably higher number of such polymorphisms in CDSs. Many SNPs caused the loss or gain of stop codons (Table 2). Again, Cannonau was distinguished from the other cultivars with the highest number of both premature and new stop codons in both homozygous and heterozygous genomic regions. In contrast, the two white berry varieties Vermentino and Sultanina showed the lowest number of premature stop codons in both homozygous and heterozygous genomic regions. Most of these genes can be considered as pseudogenes because plant transcripts with premature stop codons are usually targeted for degradation via the nonsense mediated decay pathway. We found 1296 putative pseudogenes among the four Sardinian cultivars, 118 of which contained two or more premature stop codons in at least one cultivar. Among these pseudogenes, 75.6% were specific for one cultivar and only 1.3% were shared by all cultivars. #### Homozygosity Islands We next investigated the allelic variability of SNPs along chromosomes, seeking ROHs (chromosome regions featuring uninterrupted runs of consecutive homozygous SNPs) which are common features of many resequenced genomes (Ku et al., 2011; Metzger et al., 2015). We set a minimal ROH size of 50 kb with a sliding window of 500 homozygous SNPs, allowing for one missing or heterozygous SNP per window. TABLE 1 \| Polymorphisms statistics for 7 grapevine cultivars (four grown in Sardinia + 3 outgroups). TABLE 2 \| Polymorphisms statistics for 7 grapevine cultivars (four grown in Sardinia + 3 outgroups). (a) SNPs and Indels percentage are calculated by dividing the number of polymorphism occurrences by the total length of the polymorphic regions. The cultivars could be assigned to two groups, the first with many ROHs (Bovale, Vermentino and Carignano) and the other with few ROHs (Cannonau Sultanina and Tannat) with Gewurztraminer showing intermediate behavior (Table 3). As expected, the proportion of the genome included in ROHs was associated with the ROH number. A large proportion of the genome was found within ROHs in the first group: 17.3% in Bovale (34,847,149 bp), 8.6% in Vermentino (24,538,350 bp) and 5.9% in Carignano (18,274,355 bp). A much smaller proportion was found in the second group: 1.2% in Cannonau (4,674,100 bp), 0.9% in Sultanina (3,816,666) and 0.8% in Tannat (3,593,251 bp). The intermediate cultivar Gewurztraminer showed an intermediate proportion of 3.2% (11,412,631 bp). However, there were only minor differences between the two groups in the frequency of ROH distribution. The cultivars with many ROHs tended also to have larger ROHs, whereas those with fewer ROHs tended to have smaller ROHs (Figure 1A). Interestingly the frequency distribution of SNP density within ROHs distinguished the two groups more clearly: Carignano, Bovale and Vermentino contained more ROHs with densely clustered SNPs, whereas Cannoanu, Sultanina and Tannat contained more ROHs with sparse SNPs (Figure 1B). The ROHs were distributed along all 19 chromosomes, although in a nonuniform manner (Figure 1C). Only 16,402 bp of the ROH sequence was common to all cultivars, and this contained 31 protein-coding genes (Supplementary Table S2). An average of 62.5% ROH sequence in each cultivar was private, i.e., restricted to that variety. #### Structural Variation Copy number variation, PAV and large deletions are complex structural variations that can be inferred by the analysis of coverage variation along chromosomes. The Altools module Sliding Analysis was used to visualize these variations, and TABLE 3 \| Regions of homozygosity (ROH) statistics for the 7 analyzed grapevine cultivars. For the four varieties cultivated in Sardinia, private ROHs are calculated within this group. as reported for other resequenced cultivars (Da Silva et al., 2014; Di Genova et al., 2014; Cardone et al., 2016) we found that the coverage was not homogeneous along chromosomes (Supplementary Figure S1). We therefore used a digital comparative genome hybridization approach to identify duplicated/deleted genomic regions in the Sardinian cultivars. These were identified as regions with a copy number significantly higher (gains) or lower (losses) than the corresponding regions in the reference genome. However, we did not determine the actual copy number of these regions in the reference genome so the terms gain and loss are not intended to indicate the direction of mutational events during evolution. We identified 6526 genomic regions with significant differences in copy number among the four cultivars compared to the reference sequence with 81 regions being shared between all four cultivars and 4953 specific to single cultivars. On average, we found that 4.3% of the reference genome was duplicated and 1.4% was deleted in the Sardinian cultivars. Furthermore, 81 of the CNVs (49 gains and 32 losses) corresponding to 316,000 bp (131,000 bp in gains and 185,000 bp in losses) were common to all Sardinian cultivars, whereas 619.1 CNVs were unique to individual cultivars (Table 4). The common CNVs encompassed 12 protein-coding genes (Supplementary Table S2). The Cannonau genome contained ninefold more duplicated regions than the Vermentino genome and ∼2.5-fold more than the Carignano and Bovale genomes. In contrast, the Bovale genome showed the highest number of low-copy-number regions followed by Vermentino, Carignano and finally Cannonau (Table 4). The length distribution of gains and losses in the Sardinian cultivars is shown in Supplementary Figure S2, and Figure 2* presents a circular genomic map of the distribution of gains and losses along each chromosome. Most of chromosomes 1 and 17 together with the whole of chromosome 10 did not show any gains in any of the cultivars. Vermentino showed the lowest number of chromosomes involved in gain events with chromosomes 14, 3 and 4 featuring only a few such variations. Several common patterns also emerged from the distribution of losses. For example, we observed a common high density of loss events in chromosome 16, but a very low number in chromosome 17. The absence of gains/losses within extended genomic portions must be considered in the light of the DNAcopy algorithm high stringency (e.g., segmentation default p-value < 0.01), which can result in a poorer sensitivity. In this regards, applying a higher p-value during the genome segmentation step actually resulted in the emergence of previously undetected CNVs events (results not shown). Notably, this phenomenon only apparently affects more the detection of gain compared to loss events (Figure 2) due to the losses datasets being enriched also in zero-coverage genomic portions. Transposable elements (TE) are known to play a primary role in shaping the genomic architecture of plants (Carrier et al., 2012; Bai et al., 2016) and may contribute to the occurrence of CNVs. Indeed a relevant, although variable, proportion of the detected CNV proved to overlap annotated TE for all the analyzed cultivars (Supplementary Table S3). We found that, on average, TE overlap 23.2 and 6.7% of the detected gains and losses, respectively. Notably, CNVs proved to host a higher percentage of TE in cultivar Cannonau with a relative abundance almost double than those observed for the other varieties. A more detailed analysis of TE types and distribution within CNV regions was beyond the scope of this manuscript and will be reported elsewhere. The distribution of large deletions clearly differentiated the four Sardinian cultivars, with Cannonau featuring the highest number (1990), Vermentino the lowest (50) and Bovale and Carignano featuring intermediate numbers of 419 and 529, respectively (Figure 2 and Table 5). Approximately 1,100,000 bp included in the large deletions was common to the four Sardinian varieties and this encompassed 44 protein-coding genes (Supplementary Table S2). The proportion of private large deletions ranged from 16.9% in Vermentino to 54.5% in Cannonau (Table 5). #### Functional Diversity We next investigated whether the sequence and structural polymorphisms within genes provided insight into the adaptive and/or artificial selection traits of the cultivars. Gene ontology enrichment analysis was applied to the putative pseudogenes, revealing that several biological process categories such as "defense response" and "apoptotic process" were significantly overrepresented in the Sardinian cultivars (Supplementary Table S4). We calculated the rate of non-synonymous (dn) and synonymous (ds) substitutions at loci featuring more than five SNPs and used the dn/ds ratio to identify the genes under either purifying (dn/ds < 1) or diversifying selection (dn/ds > 1). Gene ontology single-gene enrichment analysis revealed several common features among the Sardinian cultivars. Several genes involved in methionine biosynthesis appeared subject to purifying selection in all the red berry Sardinian varieties. Similarly, a number of genes involved in the regulation of auxin response factor (ARF) signal transduction appeared subject to purifying selection in all the Sardinian varieties with the exception of Carignano (Supplementary Table S5a). Finally, genes involved in apoptosis and other defense processes appeared subject to positive selection in all the cultivars (Supplementary Table S5b). TABLE 4 \| Copy number variations statistics for the four analyzed Sardinian cultivars. Gene ontology enrichment analysis of the genes within ROHs indicated the predominance of primary metabolism, stress response and secondary metabolism categories (Supplementary Table S6). However, within these wide classes each cultivar featured specific biological process or molecular functions. ROHs in Bovale were enriched for genes involved in defense responses and the biosynthesis of salicylic and jasmonic acids. ROHs in Cannonau were enriched for genes involved in solute transport across cellular membranes and responses to biotic and abiotic stress, such as cold, wounding and fungi. Stress response genes were also significantly enriched in the Carignano ROHs, together with genes encoding strictosidine synthetases and those involved in cytoskeletal organization. The ROHs in Vermentino were enriched for genes involved in embryo sac development, trehalose biosynthesis and oxidation/reduction. The ontologies of genes in CNV regions depended on whether the regions were gains or losses. The gained regions were enriched for genes involved in flavonoid synthesis and other secondary metabolic processes, especially in Cannonau (Supplementary Table S7). In contrast, the lost regions were enriched for stressresponse genes (Supplementary Table S8). As stated above, the gain or loss of regions was relative to the reference genome, so a significant enrichment should be interpreted as evidence that mutation (either deletion or duplication) has affected regions hosting specific gene functions rather than enrichment of the function with respect to the gene copy number in the reference sequence. Gene ontology enrichment analysis focusing on genes within large deletions also revealed the prevalence of genes that respond to biotic/abiotic stress. Notably, several ontologies were shared among the Sardinian cultivars, with 11 common genes involved in cycloartenol biosynthesis lost in three of the varieties (Supplementary Table S9). #### DISCUSSION Viticulture and wine-making play a primary role in the Sardinian economy. Indeed, almost 26,000 ha of the island is devoted to grapevine cultivation yielding ∼500,000 hectoliters of wine every year (Nieddu, 2011). Cannonau, Bovale and Carignano are among the most widespread red berry cultivars, and Vermentino is by far the most widely cultivated white berry cultivar. These varieties were resequenced to investigate genomic characteristics potentially associated with their distinct phenotypes. #### Genetic Diversity and Distribution of Sequence Polymorphisms Sequence reads from the Sardinian varieties were aligned to the Pinot noir PN40024 reference genome, allowing the identification of several forms of sequence polymorphism, such as SNPs and indels, as well as structural variations such as CNVs, PAVs and large deletions. The cultivars showed wide variation in several sequence diversity parameters, and 2,421,176 SNPs were discovered by comparing the Sardinian cultivars with three varieties not grown in Sardinia. Cannonau was most similar to the reference genome in terms of the percentage of homozygous SNPs and reference bases covered by reads (Table 1) whereas Vermentino showed the greatest divergence from the Pinot noir genome due to the greater number of genomic positions not covered by reads, and the frequency of SNPs/indels at both the genomic and genic levels. This may reflect the original selection of this cultivar for the production of table grapes (Nieddu, 2011), in accordance with previous studies highlighting marked genomic differences between wine and table varieties (Myles et al., 2011). Indeed, several alignment statistics were common between Vermentino and Sultanina, a well-known table variety, such as the high number of indels within the transcripts and the lower number of mutations producing stop codons (Table 2). The ratio of heterozygous to homozygous SNPs differed substantially among the cultivars, suggesting their breeding histories were also distinct. Bovale showed the lowest ratio of heterozygous to homozygous SNPs, and historical data suggest this cultivar originated by local breeding with the selection of several clones. Based on simple sequence repeat (SSR) polymorphism, several closely related clones have been identified that can be assigned to a cluster of Bovale-like genotypes (Meneghetti et al., 2013). The breeding of these clones may have been characterized by intercrossing and the selection of Bovale-related materials, including Bovale muristellu and Bovale murru (Grassi et al., 2003). Uninterrupted arrays of homozygous SNPs, defined as ROHs, are often considered as signatures of inbreeding. Several ROH parameters are reliable predictors of the breeding histories of carriers, including their size, SNP density and distribution. Using a conservative approach, we identified the extent of ROHs in all the cultivars. Only a small proportion of the total ROH sequence was shared among all the cultivars. Notably, the length distributions of ROHs in each cultivar were similar, with most belonging to the smallest length classes. ROHs have been associated to inbreeding events in several systems and the length distributions of ROHs has been taken as a marker of the timing and extent of inbreeding: large ROHs are associated with recent inbreeding whereas smaller ones are older and thus usually diagnostic of germplasm origin. Following these considerations the rather homogenous ROH distributions we observed may reflect the limited number of sexual reproduction events typical of grape breeding. In Cannonau, the lower number of ROHs together with the higher percentage of heterozygous polymorphisms may suggests a more complex breeding history than the other cultivars grown in Sardinia. This is supported by evidence that Cannonau clusters more closely to varieties cultivated in the near East than with other Italian varieties (Crespan, 2010). However, we urge caution in interpreting these results only in terms of inbreeding because, in species vegetatively propagated, regions with reduced heterozygosity (and thus with high level of homozigosity) may be coincident with mosaic structural variations. Application of dedicated software together with resequencing experiments featuring higher depth of coverage will be needed to discriminate ROH origin in the analyzed cultivars (Marroni et al., 2017). #### Signatures of Selection Selection for desirable traits may have driven the emergence of unique genomic features in each of the cultivars so we searched for genes under purifying and positive selection by calculating the dn/ds ratio at each polymorphic locus. Our data indicated that the selected traits play key roles in the plant life cycle. For example, six genes involved in methionine biosynthesis were found to be under purifying selection in all the Sardinian red berry varieties (Supplementary Table S5a). Methionine metabolism appears to be involved in the ripening of berries given that the derivative S-adenosylmethionine is required for the production of ethylene during maturation (Agudelo-Romero et al., 2013) and methionine precursors differ widely in abundance from veraison onward in diverse grapevine varieties (Giribaldi et al., 2010). Several ARF genes were also found to be under purifying selection, and this family of regulators is also implicated in grapevine berry ripening (Wan et al., 2014). Finally, an enrichment in biological processes involved in the cell shape regulation (e.g., "regulation of cell shape," "microtubule-based movement," "actin filament-based movement") emerged when analyzing genes under purifying selection. In this regards the cytoskeleton of plant cells is believed to play a role in the response to several external stimuli such as heat or cold that are sensed as a mechanical load upon the membrane (Nick, 2013). In contrast, stress-response genes (particularly those involved in apoptosis) were found to be under positive selection, which may provide the genetic variation needed to deal with a wider range of local conditions (Supplementary Table S5b). The plasticity of stress-response genes was also confirmed by the gene ontology enrichment analysis of transcripts featuring premature stop codons in all the cultivars. In this regard, previous reports highlighted a diverse response to abiotic stresses (e.g., water depletion) for cultivars Cannonau, Carignano and Bovale (Mercenaro et al., 2016a) with such a trend being also confirmed when Vermentino was compared to the international cv Chardonnay (Mercenaro et al., 2012). A wide variety of genes proved to be involved in the response to several abiotic stresses (e.g., high light, high heat and drought) also in other grapevine varieties. Interestingly, transcriptomic analyses revealed that the number and type of differentially expressed stress related genes may largely vary when comparing different cultivars resulting in candidate gene sets that are poorly overlapping (Rocheta et al., 2016). # Structural Variation Complex structural variations such as CNVs, PAVs and large deletions contribute to both intraspecies and interspecies genetic variation. CNV polymorphisms are widely studied in humans because they are associated with many severe diseases (Buchanan and Scherer, 2008). CNV has only recently been investigated in plants and CNVs may be more abundant in intergenic regions, although CNVs involving genes have also been reported (Zmie ˙ nko et al., 2013 ´ ). Copy number variation proved to be non-homogeneously distributed along the chromosomes of the analyzed cultivars. Interestingly, the occurrence of gains (and, at a lesser extent, losses) was not detected in extended portions of chromosomes 1, 10 and 17. Although we cannot exclude that technical reasons may have contributed to such a phenomenon (see Result), other causes should be taken into consideration. The presented varieties may share a higher homology with the reference PN40024 cultivar in the highlighted genomic portions. Indeed, chromosomes 1 and 17 featured SNPs frequency values below the average for all the analyzed cultivars (data not shown). Additionally, the unequal distribution of TE (whose presence is highly correlated with the occurrence of CNVs) may contribute to the observed trend (i.e., chromosome 17 contains 4.7 repetitive sequences per Mb, that is the lowest value among the V. vinifera chromosomes). Having identified CNVs in the genomes of the four Sardinian cultivars, we extracted the associated genes and used a single-gene enrichment analysis to investigate their ontologies. We found that biological processes and molecular functions related to stress responses were the most overrepresented categories among these genes in all four cultivars (Supplementary Tables S7, S8). However, each cultivar was also characterized by unique ontologies. For example, among the molecular functions specifically overrepresented in the Cannonau gained genomic regions we found 13 genes involved in the synthesis of naringenin and resveratrol, and 6 of the 12 known genes involved in the synthesis of jasmonate, which enhances the production of resveratrol. This observation seems to be in line with previous reports highlighting a high content of this longevity-linked (Bhullar and Hubbard, 2015) secondary metabolite in Cannonau (Franco et al., 2000; Corder et al., 2006). Genes involved in the synthesis of resveratrol and naringenin-chalcone were also overrepresented in the gained regions of Bovale together with genes involved in the metabolism of hydrogen peroxide, e.g., a molecule whose accumulation proved to vary during the V. vinifera plant cycle (Qsaib et al., 2014). Notably, genes in the gained regions of Carignano and Vermentino shared several biological processes and molecular functions related to redox activity and electron transport. The main processes represented by genes in the lost regions of all four cultivars were related to stress responses, thus confirming the widespread genomic plasticity of this class of genes. In Cannonau, the molecular function "chitinase activity" was also overrepresented in the lost regions, and this is associated with resistance to fungal pathogens (Busam et al., 1997). In Bovale, the molecular function "strictosidine synthase activity" was overrepresented in the lost regions, concurring that the absence of these enzymes in grapevine has no impact on fitness (Zhang et al., 2014). Finally, several genes were lost in all the red berry varieties due to large deletions events. In particular, 11 genes with cycloartenol synthase activity were potentially lost in at least one allele of Cannonau, Bovale and Carignano. Cycloartenol synthase converts 2,3-oxidosqualene to cycloartenol, which is the first step in the biosynthesis of sterols. Arabidopsis thaliana plants with a mutation in this gene failed to produce progeny suggesting a role in male gametophyte function (Babiychuk et al., 2008). Because the grapevine cultivars we investigated have been bred by vegetative propagation for several centuries it is likely that some gene required for pollen development may be lost in large deletions without this phenomenon being counter selected. Notably a significantly higher number of ontologies associated with the synthesis of resveratrol emerged for genes within large deletions in Vermentino. This finding was confirmed in other white grape wines (Gewurztraminer and Sultanina), providing a genetic explanation for the lower resveratrol content of white wines compared to reds (Bavaresco, 2003). #### CONCLUSION We produced a list of CNV, SNP and indels which could be of functional significance and thus contribute to explain agronomic differences among cultivars. Although the reported polymorphisms rely on a mere in silico investigation, the high stringency of the method together with an extensive quality check of our pipeline (see Data Sheet 1) allowed to produce reliable inferences. The integration of such data with transcriptomic and metabolomic analyses under different stress conditions will allow to narrow the number of candidate regions under investigations and construct hypothesis breeding strategies to improve V. vinifera resilience. ### AUTHOR CONTRIBUTIONS SC and AP contributed to the design and conception of the work together to the drafting of the manuscript. LM, GN, and MP contributed to the interpretation of the generated data and to #### REFERENCES the revision of the manuscript. All the authors approved the final draft of the submitted manuscript. #### FUNDING We would like to thank the R.A.S. for funding as a part of the project "Effetto dello stress idrico sulle risposte fisio-metaboliche e genetiche della vite in Sardegna" (L.R. 7 - CRP 7900). ### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01279/ full#supplementary-material FIGURE S1 \| Coverage distribution along 19 chromosomes of Vitis vinifera for the 4 cultivars grown in Sardinia. All possible mapping locations were considered at this stage in order to highlight common patterns due to highly repetitive regions. From outward to inward: 19 Vitis vinifera chromosomes ideogram, Bovale, Cannonau, Carignano, Vermentino. Colors range ascendingly from green (low coverage) to red (high coverage) and represents coverage values that were normalized on the individual average genomic coverage (9 color classes were used, e.g., green, dgreen, vdgreen, vvdgreen, black, vvdred, vdred, dred, red, with color prefixes v, very and d, dark). FIGURE S2 \| Length distribution of gained regions, lost regions and large deletions, in the four analyzed Sardinian grape cultivars. grapevine by means of different PCR-derived marker systems. Mol. Biotechnol. 48, 244–254. doi: 10.1007/s12033-010-9365-3 Molon, G. (1906). Ampelografia. Milan: U. Hoepli. Viala, P., and Vermorel, V. (1991). Ampélographie. Paris: Masson. 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 Mercenaro, Nieddu, Porceddu, Pezzotti and Camiolo. 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. # Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses Alejandra Serrano<sup>1</sup> , Carmen Espinoza<sup>1</sup> , Grace Armijo<sup>1</sup> , Claudio Inostroza-Blancheteau<sup>2</sup> , Evelyn Poblete<sup>1</sup> , Carlos Meyer-Regueiro<sup>1</sup> , Anibal Arce<sup>1</sup> , Francisca Parada<sup>1</sup> , Claudia Santibáñez1,3 and Patricio Arce-Johnson<sup>1</sup> \* <sup>1</sup> Laboratorio de Biología Molecular y Biotecnología Vegetal, Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile, <sup>2</sup> Núcleo de Investigación en Producción Alimentaría, Facultad de Recursos Naturales, Escuela de Agronomía, Universidad Católica de Temuco, Temuco, Chile, <sup>3</sup> Ecophysiology and Functional Genomic of Grapevine, Institut des Sciences de la Vigne et du Vin, Institut National de la Recherche Agronomique, Université de Bordeaux, Bordeaux, France #### Edited by: Simone Diego Castellarin, University of British Columbia, Canada #### Reviewed by: Grant Cramer, University of Nevada, Reno, United States Gregory Alan Gambetta, Ecole Nationale Supérieure des Sciences Agronomiques de Bordeaux-Aquitaine, France > \Correspondence: Patricio Arce-Johnson [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 27 March 2017 Accepted: 10 August 2017 Published: 07 September 2017 #### Citation: Serrano A, Espinoza C, Armijo G, Inostroza-Blancheteau C, Poblete E, Meyer-Regueiro C, Arce A, Parada F, Santibáñez C and Arce-Johnson P (2017) Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses. Front. Plant Sci. 8:1486. doi: 10.3389/fpls.2017.01486 Grapevine fruit development is a dynamic process that can be divided into three stages: formation (I), lag (II), and ripening (III), in which physiological and biochemical changes occur, leading to cell differentiation and accumulation of different solutes. These stages can be positively or negatively affected by multiple environmental factors. During the last decade, efforts have been made to understand berry development from a global perspective. Special attention has been paid to transcriptional and metabolic networks associated with the control of grape berry development, and how external factors affect the ripening process. In this review, we focus on the integration of global approaches, including proteomics, metabolomics, and especially transcriptomics, to understand grape berry development. Several aspects will be considered, including seed development and the production of seedless fruits; veraison, at which anthocyanin accumulation begins in the berry skin of colored varieties; and hormonal regulation of berry development and signaling throughout ripening, focusing on the transcriptional regulation of hormone receptors, protein kinases, and genes related to secondary messenger sensing. Finally, berry responses to different environmental factors, including abiotic (temperature, water-related stress and UV-B radiation) and biotic (fungi and viruses) stresses, and how they can significantly modify both, development and composition of vine fruit, will be discussed. Until now, advances have been made due to the application of Omics tools at different molecular levels. However, the potential of these technologies should not be limited to the study of single-level questions; instead, data obtained by these platforms should be integrated to unravel the molecular aspects of grapevine development. Therefore, the current challenge is the generation of new tools that integrate large-scale data to assess new questions in this field, and to support agronomical practices. Keywords: grapevine fruit development, seed development, biotic and abiotic stresses, transcriptomics, metabolomics ## INTRODUCTION fpls-08-01486 September 6, 2017 Time: 16:34 # 2 The grapevine (Vitis vinifera), one of the most important fruit crops worldwide, provides berries that can be used as fresh fruit, raisins, and for wine making and distillation of liquors. The grapevine has fleshy berries derived from the ovary of the flower, whose development is a complex process that can be divided into three stages with distinctive physiological and biochemical characteristics (Coombe and McCarthy, 2000). During the first stage (stage I) there is an exponential increase in berry size due to rapid cell division and growth, leading to the establishment of the final number of cells (Coombe and Hale, 1973). Some of the principal compounds that are present in the berry at stage I are tartaric and malic acids, which accumulate mainly in skin and flesh and confer acidity to fruits and wine (Sweetman et al., 2009, 2012). The second stage (stage II) is a lag phase in which important physiological and biochemical changes occur, such as softening and coloring. Within this stage, veraison takes place, characterized by the beginning of the synthesis of anthocyanins, soluble flavonoids compounds that provide color to red varieties (Figure 1) (Boss et al., 1996). Sucrose, originating from leaves, reaches the fruits through the phloem, and is then hydrolyzed forming glucose and fructose (Robinson and Davies, 2000; Kennedy, 2002; Vignault et al., 2005; Deluc et al., 2007; Hayes et al., 2007; Fontes et al., 2011). Stage II is thus a transition between an unripe fruit and the third stage of development (stage III or ripening). The latter involves important morphological and physiological changes, like color development (Boss et al., 1996), turgor reduction and berry enlargement (Chervin et al., 2008), and decreased acidity (Costenaro-da-Silva et al., 2010), among others. In addition, hormonal changes that occur throughout development positively or negatively regulate ripening (Figure 1; Gerós et al., 2012). Therefore, during ripening, a large number of complex transcriptional and/or post-transcriptional regulatory processes are triggered. In this review, we focus on the integration of global approaches, including proteomics, metabolomics, and especially transcriptomics, to understand grape berry development and the influence of environmental factors on this process. Thus, we will cover initial fruit development, with emphasis on seed formation; the importance of coloration and hormonal changes during development, especially on ripening; and finally, the effect of environmental factors on this process will be discussed. # GRAPE BERRY DEVELOPMENT FROM AN OMICS PERSPECTIVE #### Seed Development and Seedless Fruits Grape berry development begins after fertilization, when in a process known as fruit set, the ovary changes from a stationary state and experiences an abrupt increase in size that occurs due to cell division and enlargement, leading to rapid pericarp growth. Throughout this period, seed development is an important process, mainly because seeds produce auxins, gibberellins (GAs) and cytokinins, which play multiple roles in grape berry development (Keller, 2010). Seed and berry development are coordinated, and the changes that seeds undergo have an impact on fruit ontogeny. The first stage of berry development is characterized by a rapid increase in seed size, during which embryogenesis and endosperm growth occur. At the second stage, about 10 and 15 days prior to veraison, seeds reach their final size and maximum fresh weight, and at the beginning of the third stage, embryo growth ceases and the endosperm accumulates reserves until the seeds turn dormant (Figure 1; Keller, 2010). Ripe berries usually contain up to four seeds derived from four ovules (Dokoozlian, 2000; Kennedy, 2002). However, seedless grape varieties have been spontaneously found in nature and have been preserved over the years through asexual propagation. Seedless berries develop naturally via two different mechanisms, parthenocarpy and stenospermocarpy, which generate berries without or with rudimentary seeds, respectively (Varoquaux et al., 2000). In order to understand the main differences between parthenocarpy and stenospermocarpy, we will discuss the few available Omics analyses of early stages of berry development and seed formation. #### Parthenocarpy In parthenocarpic fruits, the stimulus of pollination is sufficient to trigger fruit set (Dokoozlian, 2000). Since the ovary is able to enlarge and form a berry without ovule fertilization, there is no seed in the fruit (Varoquaux et al., 2000). Until now, few parthenocarpic grape cultivars have been described. Of these, cv. Corinto Bianco (CB), a somatic variant of the seeded cv. Pedro Ximenez (PX), constitutes a good model to study seed formation (Vargas et al., 2007). To understand the molecular differences between CB and PX genotypes, flowers at 1 and 10 days pre-anthesis were transcriptionally compared using microarray (Royo et al., 2016). The analyses allowed the identification of 1958 differentially expressed genes between CB and PX. Interestingly, several genes that are specifically expressed in reproductive organs were down-regulated in CB. Processes such as cell wall biosynthesis, cytoskeleton biogenesis, vesicular transport, signaling through G proteins or phosphatidylinositol, among others, were enriched. Also, 14 single-nucleotide polymorphisms (SNP) were identified between both genotypes, which could explain the parthenocarpy phenotype (Royo et al., 2016). Considering that microarrays deliver limited information, a suitable approach to analyze the different stages of development in more detail, would be using RNA-seq technologies, in order to Abbreviations:* ABA, abscisic acid; ABF, ABA-response element binding factor; ARF, auxin response factor; ASR, ABA stress responsive element; BR, Brassinosteroids; CAA, carbonic anhydrase; CAB, chlorophyll a/b binding protein; CB, Corinto Bianco; ERF, ethylene response factor; GAs, gibberellins; GLD, grapevine leafroll disease; GLRaV, Grapevine leafroll-associated virus; GVA, Grapevine virus A; HXK, hexokinase; IAA, indole-3-acetic acid; JA, jasmonic acid; LOX, chloroplast lipoxygenase; NCED, 9-cis-epoxycarotenoid dioxygenase; PX, Pedro Ximenez; QTL, quantitative trait locus; ROS, reactive oxygen species; RSPaV, Rupestris Stem Pitting virus; SDI, seed development inhibitor; SNP, singlenucleotide polymorphism; SnRK1, sucrose-non-fermentative related kinase 1; SSH, suppression subtractive hybridization; T6P, trehalose-6-phosphate; UV, ultraviolet. FIGURE 1 \| Fruit development and environmental effects. Scheme of the most important changes that berries and seeds undergo during development, and the main environmental factors affecting this process. (A) Boxes indicate the phase where each stress condition (temperature, water-related stress and UV-B radiation, Botrytis cinerea and viruses) affect grape berry and its development. (B) Changes in size, color, brix degree, and pH during berry ripening and (C) variations in hormonal content during grape berry development. (D) Seed development showing the time in which parthenocarpy and stenospermocarpy can take place. The main events are indicated by open triangles. Bars represent the described changes throughout development, in which gray and white represent the higher and lower estimated referential values for each parameter, respectively. gain further insights into the understanding of seed development and to generate new parthenocarpic varieties. #### Stenospermocarpy fpls-08-01486 September 6, 2017 Time: 16:34 # 4 During stenospermocarpy, pollination and fertilization occur normally, but a few weeks later, the embryo and/or the endosperm abort and the berries that have been generated possess just traces of seed (Varoquaux et al., 2000). It has been demonstrated that stenospermocarpy occurs in several seedless varieties, and is stable and unaffected by environmental factors (Zhang et al., 2013). However, little is known about the molecular mechanisms that underlie stenospermocarpy in grapes. The most accepted hypothesis proposes the existence of a dominant regulator gene called Seed Development Inhibitor (SDI), which could control three other recessive genes (Bouquet and Danglot, 1996). Different studies based on quantitative trait locus (QTL) analysis have reported a main QTL in linkage group 18 (LG18) (Doligez et al., 2002; Cabezas et al., 2006; Mejía et al., 2007; Costantini et al., 2008), which could explain between 50 and 70% of the seedlessness phenotype in grapes; LG18 could be considered as the SDI locus trait. In this context, VvAGL11 (MADs BOX transcription factor) was in silico mapped to the SDI locus and it has been proposed as the main functional candidate gene for seedlessness (Mejia et al., 2011). In fact, it was demonstrated that the silencing of a VvAGL11 homologous gene in tomato (Solanum lycopersicum L. cv Micro-Tom) generates fruits with few or rudimentary seeds (Ocarez and Mejia, 2016). Based on genome sequencing data, it is known that in the stenospermocarpic variety cv. Thompson Seedless, the VvAGL11 gene has an insertion of 15 bp in the 50UTR, which could be the cause of the seedless phenotype (Di Genova et al., 2014). In addition, in cv. Sultanine Monococco, which is also a seeded variety of Thompson, the VvAGL11 transcript level is higher in comparison with the seedless variety (Ocarez and Mejia, 2016), supporting the hypothesis that this gene is one of the main regulators of seed formation in grapes. On the other hand, due to abnormal ovules may be formed during flower development before or after meiosis, through either the abnormal development of the nucellus or ovule integuments, or the degeneration of the egg in the embryo sac (Ebadi et al., 1996). In order to determine the molecular bases of this phenotype, flowers from cv. Thompson Seedless and cv. Thompson Seeded, a spontaneous mutant with seeded berries, were compared through suppression subtractive hybridization (SSH) (Hanania et al., 2007, 2009). The results demonstrated that ch-Cpn21, a gene that encodes for a chloroplastic chaperonin, is repressed in developing flowers of cv. Thompson Seedless. Likewise, the silencing of this gene in tobacco plants (Nicotiana benthamiana) and tomato induces seed abortion (Hanania et al., 2007). The use of somatic variants in combination with current transcriptomic technologies, would be very promising in the study of stenospermocarpy, helping to discover new genes playing important roles in seed abortion. Based on the studies of Costenaro-da-Silva et al. (2010) and Nwafor et al. (2014), several genes have been associated with early stages of grape berry development. These include VvUBP1, a heterogeneous-nuclear ribonucleoprotein, VvFS41, a putative S1-like ribosomal protein involved in mRNA processing and synthesis of proteins related with cell division during the first days of berry development, VvERF1 and VvERF9, which encode for transcription factors related to several developmental processes, VvDOF1, possibly related to seed development, and VvRIP1 and ABI3, which have been related to hormone signaling, among others. Many of these genes have pleiotropic effects, so it is difficult to estimate their specific molecular contribution to the stenospermocarpy phenotype. Some of them could be involved in this process, but their functional characterization is needed to test this hypothesis. #### Non-characterized Mechanisms of Seedlessness A demonstrated way to produce seedlessness is the exogenous application of GAs before bloom or during anthesis. It is believed that GAs promote seedless grapes by inhibiting pollen germination, allowing unfertilized ovules to enlarge and form fruits, as occurs in parthenocarpy (Kimura et al., 1996; Cheng et al., 2013). However, another study suggests that exogenous GAs interfere with seed development, as described in stenospermocarpy (Cheng et al., 2013). So, the mechanism involved in this response is not clear. A transcriptional analysis by RNA-seq was performed in GA3-treated flowers of the seeded cv. Kyoho and a comparison with non-treated flowers was carried out (Cheng et al., 2015). This study demonstrated that GA<sup>3</sup> application modifies the expression profile of genes related to developmental processes, such as cellular metabolism, biosynthesis of different metabolites, stress response, transport, etc. Also, changes in the expression of genes related to flowering, fruit, and embryonic development were found. Within the genes possibly related to seedlessness, the Pelo gene, whose mouse homolog has a role in meiosis and causes embryonic lethality (Adham et al., 2003), was repressed after GA<sup>3</sup> treatment, and was correlated with seedlessness in grapes (Cheng et al., 2015). The Pelo gene probably has conserved roles across several species. However, deeper functional studies are needed to corroborate this information in plants and to determine if this gene does indeed fulfill a role in seed development, and more studies are needed to correlate any transcriptional changes with particular phenotypes. Recent studies have demonstrated that reactive oxygen species (ROS) are present throughout the entire seed's life cycle (Jeevan Kumar et al., 2015). In fact, the oxidative damage induced by an imbalance in plant redox homeostasis can affect normal seed development, leading to abortion (Cheng et al., 2013). Pathways related to ROS scavenging and detoxification are significantly affected after GA<sup>3</sup> treatment (Cheng et al., 2015). So, probably, exogenous GA application generates physiological changes that could induce seedless fruits through a ROS-related mechanism, but further research is needed to understand the role of ROS regarding the presence or absence of seeds. Naturally occurring seedlessness could be the result of a series of coordinated transcriptomic switches that cause a global reprogramming of the cell. To date, little is known about the seedless phenotype in grapevines, presenting a great challenge for researchers. The best model for understanding seedlessness is to compare somatic variants (seeded versus seedless) through global approaches, since they have the same genetic background and could be used to discover new genes involved in this phenotype. Even though somatic variants are rare in nature, it is clear that these comparisons are much more informative than the use of two different varieties. ### First Stage of Grape Berry Development The first stage of grape berry development (stage I) is initiated with fruit set. During the first 2 weeks, berry size increases markedly as auxin and GAs directly promote cell division and enlargement (Ojeda et al., 1999; Bottcher et al., 2010; Fortes et al., 2015). Tartaric, malic, and other organic acids, along with different phenolic precursors such as tannins and hydroxycinnamates, are synthesized, modifying the organoleptic properties of the berries (Deluc et al., 2007). Besides, minerals, micronutrients, and aroma-related compounds are present. Transcriptomic analysis of young berries in cv. Shiraz revealed an enrichment of hormone signaling responsive transcripts, suggesting that hormone-controlled metabolic pathways are highly active in early stages of development (Sweetman et al., 2012). During this stage, GAs are the key regulators of fruit set, cell division and cell expansion (Fortes et al., 2015). RNAseq analysis of cv. Centennial Seedless berries treated with GA<sup>3</sup> (12 days after flowering), revealed a repression of an abscisic acid (ABA)-response element binding factor (ABF) and ethylene response factors (ERFs) (Chai et al., 2014). Showing the occurrence of both GA3–ABA and GA3–ethylene crosstalk. The role of jasmonic acid (JA) in grapes remains unclear, but, as has been demonstrated in potato (Solanum tuberosum) leaves, it might stimulate cell division (Ravnikar et al., 1992). In grape, high levels of JA are present during the first stage, which then decrease in mature berries (Fortes et al., 2015). A proteomic study in cv. Muscat Hamburg has reported abundant levels of chloroplast lipoxygenases (LOX), enzymes that provide intermediates for JA biosynthesis during green berry development, followed by a decrease when berries reach a size of 15 mm (Martinez-Esteso et al., 2011). In the case of auxins, it has been proposed that they have a role in fruit growth delaying ripening (Fortes et al., 2015). In berry flesh of cv. Kyoho, a high concentration of auxins has been reported, in particular of indole-3-acetic acid (IAA) during the beginning of stage I, with a rapid decrease at the end of this stage and throughout stage II, consistent with the high rate of cell division observed in the first stage (Zhang et al., 2003). Considering that the final number of cells in the grape berries is defined in the first stage of development (Dokoozlian, 2000), the interaction between hormones regulating cell division is key to cluster progress, and might be an interesting target in studies aimed at improving yield. # Second Stage of Grape Berry Development #### Main Changes in Metabolites during Stage II The second stage of grape berry development (stage II) is a lag phase, where the rate of increase in both fresh and dry weight is very low. At the end of this stage, veraison occurs, which is the transition from the second to the third stage of berry development, and is considered the onset of ripening. Different physiological and biochemical changes take place during veraison, of which anthocyanin synthesis and sugar accumulation are the most characteristic and important processes. In fact, anthocyanins are one of the main pigments present in colored grape berry skins (Souquet et al., 1996), while sugar content is widely considered one of the most important properties that define ripening (Guelfat-Reich and Safran, 1971; Jayasena and Cameron, 2008). Depending on the cultivar, five types of anthocyanins are frequently found in V. vinifera, which are associated with organoleptic properties such as color (in the case of red wine), bitterness, astringency and also as antioxidant molecules with beneficial effects on human health (Dixon et al., 2005). Anthocyanin biosynthesis occurs through the phenylpropanoid pathway, in which two types of genes are involved: structural genes, encoding for biosynthetic enzymes, and regulatory genes, which are associated with temporal and spatial regulation of the structural genes (Deluc et al., 2007). Both, structural and regulatory genes are present in colorless and colored grapevine cultivars, but in the case of white cultivars, color is not expressed due to multiallelic mutations in MybA1 and MybA2, that prevent the transcription of these two important positive regulators of the phenylpropanoid pathway (Kobayashi et al., 2004). In the case of red cultivars, MYBA1 and MYBA2 transcription factors control anthocyanidin glycosylation through the regulation of flavonoid 3-O-glucosyltransferase (UFGT) expression (Ford et al., 1998). The anthocyanin biosynthesis pathway is not only regulated by MYB transcription factors, as it is also controlled by the critical transcriptional R2R3-MYB/bHLH/WD40 (MBW) complex in grapevine (Wu et al., 2014). Flavonoid 3<sup>0</sup> -hydroxylase (F30H) and flavonoid 3<sup>0</sup> ,50 hydroxylase (F3<sup>0</sup> 5 <sup>0</sup>H) genes seem to be an important regulatory points in anthocyanin biosynthesis (Castellarin and Di Gaspero, 2007; Matus et al., 2016). Their proteins belong to the cytochrome P450 protein family and compete for a common precursor for the biosynthesis of red and blue anthocyanins, respectively (Bogs et al., 2006). Metabolic and transcriptomic analyses determined that in cv. Cabernet Sauvignon and cv. Shiraz, the F3<sup>0</sup> 5 <sup>0</sup>H gene is up-regulated, whilst that of F30H is down-regulated (Degu et al., 2014). However, further studies are necessary to understand the fine regulation of the phenylpropanoid pathway, focusing on the anthocyanin branch. In this case, the use of varieties with different berry skin colors would be informative. None of the aforementioned studies consider pink varieties, which have an intermediate color between red and white varieties, and could be used to complete the overview of anthocyanin biosynthesis in a fuller range of colors. In a recent analysis using Omics approaches to analyze ripe berry skins of five cultivars, Cabernet Sauvignon, Merlot and Pinot Noir (red cultivars), and Chardonnay and Semillon (white cultivars) (Ghan et al., 2015), several transcripts and metabolites were mapped to the phenylpropanoid pathway. A higher transcript abundance for enzymes involved in anthocyanin biosynthesis, such as phenylalanine ammonialyase (PAL), chalcone synthase (CHS), flavanone 3-dioxygenase (F3H), leucoanthocyanidin dioxygenase (LDOX), and UFGT was observed only in red cultivars. Shikimate was the most abundant metabolite in cv. Cabernet Sauvignon, which acts as a precursor for aromatic amino acid biosynthesis within the shikimate pathway (Maeda and Dudareva, 2012). This intermediary is important because it allows the transfer of the carbon skeleton into anthocyanin structures, and could become a crucial point in the study of anthocyanins in different varieties. Sugar accumulation (mainly of glucose and fructose) is another important process that begins in veraison and continues throughout ripening. Sugar sensing mechanisms may play important roles during grape berry ripening, as they do in other aspects of plant development (Smeekens et al., 2010; Wind et al., 2010). Thus, the role of sugar is covered in the context of the third stage of berry development (see Third Stage of Grape Berry Development). #### Hormonal Control during Stage II ABA levels are high in young berries and then fall until veraison. A microarray analysis carried out over seven sequential points of berry development in cv. Cabernet Sauvignon, revealed that the transcript abundance of 9-cis-epoxycarotenoid dioxygenase (NCED1), the enzyme that conducts the limiting step in ABA synthesis (Tattersall et al., 2007), increases during the lag phase and peaks at veraison (Deluc et al., 2007). A similar expression pattern was shown for a gene encoding for the ABA signaling transduction protein phosphatase 2C ABI1, while the gene encoding a transcription factor of the same pathway, ABI3/VP1 (Abscisic acid Insensitive 3/Viviparous 1), showed the highest transcript abundance during lag phase (Deluc et al., 2007). Several studies have highlighted the control that ABA exerts over the biosynthesis of anthocyanins; at the transcriptional level by upregulation of biosynthetic genes, and at the metabolic level by increasing anthocyanin content (Wheeler et al., 2009; Giribaldi et al., 2010; Cramer et al., 2014). In this context, A 2- DE proteomic approach in cv. Cabernet Sauvignon showed that ABA treatment before veraison increases three proteins required for flavonoid biosynthesis: chalcone isomerase, dihydroflavonol-4-reductase, and anthocyanidin reductase (Giribaldi et al., 2010). In non-climacteric fruits, such as grape, the role of ethylene is not fully understood due to the low levels of this hormone during development and the technical difficulties associated with its quantification (Symons et al., 2012). Nevertheless, several reports indicate a possible role of this hormone in grape berry ripening, mainly supported by the consistent presence of a small peak about 2 weeks after veraison (Chervin et al., 2004). These data are consistent with the findings of Pilati et al. (2007), who showed that the expression of the ACC synthase gene, involved in ethylene synthesis, increases just prior to veraison and decreases afterward, together with a peak in expression of ACC oxidase around veraison, which encodes for the enzyme responsible for the last step of ethylene biosynthesis. Brassinosteroids (BR), on the other hand, are steroid hormones that have been implicated in the ripening of nonclimacteric fruits (Symons et al., 2006; Chai et al., 2013). The transcript abundance of the brassinosteroid receptor 1 gene (BRI1) peaks in the entry to lag phase and declines thereafter (Deluc et al., 2007). The expression profile of VvBR6OX1, which encodes for the enzyme that converts 6-deoxocastasterone to castasterone (the bioactive BR in grapes) shows a peak of induction just prior to veraison (Pilati et al., 2007). This evidence is consistent with an increase in BR levels at veraison and the high content observed during ripening in cv. Cabernet Sauvignon berries (Symons et al., 2006). Interestingly, exogenous application of BR increases anthocyanin content leading to premature grape berry coloration, similar to the effect of ABA. The connection between the molecular pathways of BR and ABA that regulate initial events of ripening stages has yet to be clarified. Based on transcriptomic analysis of cv. Merlot berries, it has been hypothesized that BR might be an early signal for ripening, modulating ethylene content (Ziliotto et al., 2012). In this model, the small peak of ethylene could upregulate genes associated with ABA biosynthesis and then initiate all ripening-associated ABA-induced metabolic changes (Ziliotto et al., 2012). It has been well documented that auxin has a negative role during grape berry ripening. In fact, IAA levels (the active form) remain low from veraison throughout ripening, and auxin treatments during pre-veraison inhibit ripening (Davies et al., 1997; Bottcher et al., 2010, 2011; Ziliotto et al., 2012). Two auxin carriers (an AUX1-like and a PIN1-like) are expressed before veraison, while two auxin response factors (ARFs), ARF5 and ARF18, and an auxin receptor of the ABP family are expressed at pre-veraison and are then repressed during ripening (Pilati et al., 2007; Fortes et al., 2011). It has been suggested that ethylene represses auxin biosynthesis and thus regulates the balance between auxin and ABA to initiate ripening (Ziliotto et al., 2012). Probably, a network coordinated by ABA, BR, ethylene, and auxin levels are regulating the ripening stage, however, the master regulators that connect all these pathways are still unknown. #### Third Stage of Grape Berry Development Main Changes in Metabolites during Stage III During the third stage of development (Stage III), berries approximately double in size and there is a marked decrease in organic acid concentration and a dramatic accumulation of glucose and fructose (∼1 M each) in the vacuole of flesh cells (Fontes et al., 2011; Dai et al., 2013). The scientific community has gained understanding about the complexity and diversity of sugar-sensing systems, including hexokinase (HXK), protein kinases, as well as, novel molecular regulators, such as trehalose-6-phosphate (T6P) (Li and Sheen, 2016). It has been shown that the HXK enzyme, responsible for the 6-phosphorylation of glucose and fructose, plays a dual-function with both catalytic and regulatory activities and therefore, links gene expression and metabolism in plants (Moore et al., 2003). HXK-dependent signaling represses photosynthetic related-genes in the presence of hexoses, forming a repressive complex that is directly associated with the promoter regions of several genes including those that encode for chlorophyll a/b binding protein (CAB) and carbonic anhydrase (CAA) (Cho et al., 2006). In grapevine, a genome wide analysis using the completely sequenced V. vinifera genotype PN40024 (cv. Pinot Noir) led to the identification of six members of the HXK family (Çakir, 2014). Four genes that encode for HXKs in cv. Cabernet Sauvignon were analyzed (Gambetta et al., 2010). The authors showed that these genes are highly regulated at the transcriptional level during berry development. Specifically, HXK-1, HXK-2, and HXK-3, which were induced during ripening, while HXK-4 was repressed (Gambetta et al., 2010). Interestingly, under water deficit conditions, HXK-4 was induced during the third stage of berry development compared to the control under well irrigated conditions, indicating that there is both genetic and environmental control of the sugar sensing mechanisms during ripening (Gambetta et al., 2010). Protein kinases are the major components of intracellular signaling and are responsible for rapid responses to changes in the environment. VviSK1, a protein kinases with sugar signaling function during berry development, whose transcript was shown to be accumulated after sucrose treatments in cv. Cabernet Sauvignon suspension cells (Lecourieux et al., 2010), positively affects sugar accumulation in grape cells and controls glucose transport through the regulation of four genes that encode the hexose transporters VvHT3, VvHT4, VvHT5, and VvHT6. Moreover, during berry development, VviSK1 transcripts decrease after the green stage and increase again after veraison, when sugar is accumulated (Lecourieux et al., 2010). Another protein kinase that may participate in sugar signaling during ripening is SnRK1 (Sucrose-non-fermentative Related kinase 1). In plants, SnRK1 receives inputs from hormones, as well as, sugar phosphates, and has been linked to several developmental processes and the control of primary and secondary metabolism, including photosynthesis and anthocyanin biosynthesis (Baena-Gonzalez et al., 2007; Nunes et al., 2013; Tsai and Gazzarrini, 2014). In grapevine, SnRK1 transcripts accumulate continuously in cv. Cabernet Sauvignon berries from the green stage until ripening (Gambetta et al., 2010). Nonetheless, to our knowledge, the abundance and activity of this protein kinase has not been measured in grapevine berries. Phosphate sugars are other members of the sugar signaling landscape. Among them, T6P, which is generated by primary metabolism (Lunn et al., 2014) has been recently uncovered as a signal molecule with major implications in plant growth, development, and metabolism (Van Houtte et al., 2013; Wahl et al., 2013). Transcriptomic studies had uncovered that several genes that control T6P abundance are regulated during berry development. In one of the first gene expression profile analyses using AFLP in berry samples from cv. Corvina, the authors reported a T6P-phosphatase as one of the most upregulated genes in postharvest (Zamboni et al., 2008). Moreover, Deluc et al. (2007), using the first commercially available grapevine Affymetrix, identified different profiles for genes encoding T6Psynthase, which was overexpressed in the early days before veraison, and T6P-phosphatase, which was overexpressed at postharvest. Suggesting that the abundance of T6P is highly controlled during grape berry development. In plants, T6P is linked to sugar signaling and the control of SnRK1, which is linked to developmental processes and control of metabolic pathways, including repression of anthocyanin biosynthesis (Baena-Gonzalez et al., 2007). In grapevine, several transcriptomic studies have shown that orthologs genes of SnRK1 and of the enzymes that control T6P homeostasis are highly regulated during berry development (Deluc et al., 2007; Gambetta et al., 2010). Therefore, the SnRK1/T6P pathway may be an important component of sugar signaling during berry development, and in this context, it remains to be studied whether the activity of SnRK1 protein kinase is actually inhibited by T6P, as found in other plant tissues. The Omics studies shown so far, using mainly transcriptomic and metabolomic approaches, have been useful for the identification of several sugar-signaling components, leading to the proposal that new mechanisms or candidate genes are involved in berry ripening. Nonetheless, specifically in the field of signaling through protein kinases, it is known that transcript accumulation is not the only, or main mechanism that influences their role and activity. In this perspective, it may be necessary to perform more protein-oriented Omics studies such as proteomics or phosphoproteomics. These are powerful technologies and could help to elucidate the importance of protein kinase signaling during berry development. #### Hormonal Control during Stage III Microarray and RNA-seq analyses have uncovered transcriptional reprogramming during ripening (Fasoli et al., 2012). At the onset of ripening in cv. Cabernet Sauvignon, low levels of IAA are required, while the auxin conjugate to aspartate (inactive form) concentration is high (Bottcher et al., 2010). In the case of IAA conjugate formation, the up-regulation of a gene coding for GH3.1 was found at veraison (Bottcher et al., 2010), in contrast to a decrease in GH3.3 expression (Pilati et al., 2007). On the other hand, genes coding for AUX–IAA proteins, transcriptional repressors of auxin-responsive genes, are down-regulated during ripening, while genes coding for IAA19 and IAA16 are up-regulated around veraison. Likewise, a gene homologous to Arabidopsis amidase AtAMI1, that in vitro synthesizes IAA from indole-3-acetamide, decreases its expression during ripening (Pilati et al., 2007) indicating a complex regulation for the maintenance of low levels of active auxin during ripening. Related to the ethylene metabolism, the role of this hormone during ripening has not been clearly established (Chervin et al., 2004). Nevertheless, it is known that the transcript abundance of genes coding for ACC synthase decrease at veraison, while several genes coding for ACC oxidase are down-regulated and only one is up-regulated during ripening (Terrier et al., 2005). The intricate regulation of the ethylene signaling pathway during ripening seems to be more consistent and clearer during the later stage of this process. Cramer et al. (2014) assessed the transcriptome of Cabernet Sauvignon berries in the late stages of ripening using whole-genome microarrays. They reported that several positive regulators of the ethylene pathway are upregulated, including three different ethylene receptors (VviETR1, VviETR2, and VviEIN4) and several members of the ERF family of transcription factors. Moreover, the negative regulator of ethylene signaling, VviCTR1 is downregulated at the transcript level during late ripening in both pulp and flesh (Cramer et al., 2014). Supporting the idea of an active ethylene signaling role during berry ripening. Regarding BRs, it has been shown that exogenous application to grape berries significantly promotes ripening, whilst endogenous BR levels dramatically increase at the onset of ripening and then decrease (Symons et al., 2006). These results coincide with the transcript accumulation of the VvBR6OX1 gene observed by Pilati et al. (2007), responsible for the synthesis of the bioactive BR, castasterone. In addition, a gene coding for an enzyme putatively involved in castasterone catabolism (castasterone 26-hydroxylase), leading to the inactivation of this BR, is down-regulated at ripening (Fortes et al., 2011). On the other hand, a gene related to BR biosynthesis that codes for steroid 5-alpha-reductase (DET2), was less expressed around veraison (Fortes et al., 2011). Diminished expression of biosynthetic genes could be associated with negative feedback regulation by increasing levels of BRs. The ABA concentration increases dramatically during berry ripening (Coombe and Hale, 1973). Several reports suggest that this hormone plays a major role controlling color development (Koyama et al., 2010) and softness (Gambetta et al., 2010). ABA levels are directly related to changes in NCED activity (Wheeler et al., 2009), and indeed, NCED1 transcripts peak around veraison and decrease at advanced ripening (Deluc et al., 2007). A proteomic analysis in berry skins of cv. Barbera at different stages throughout ripening revealed that the most abundant proteins belong to the ABA stress responsive elements (ASR) family, representing nearly 13% of the total protein spot volume in early ripening (Negri et al., 2008). In Arabidopsis, the proposed model of ABA signaling involves the protein kinases SnRK2, which act as positive elements in signaling downstream of ABA. SnRK2s interact with the negative regulators PP2C protein phosphatases that inhibit the activity of SnRK2s. Recently, Liu et al. (2016) identified eight VviSnRK2 genes in the grapevine genome and generated a detailed co-expression network of the ABA signaling components, including transcription factors from the ABF family. They found a high co-expression coefficient of both VviSnRK2.8 and VviSnRK2.11 with VviABF2, which is an important transcriptional regulator of ABA-dependent signaling during grape berry ripening (Nicolas et al., 2014). VviABF2 expression rises from veraison until ripening, and transcriptomic analysis of VviABF2-overexpressing grapevine cells allowed the identification of several co-overexpressed genes regulated by ABA (Wong et al., 2013; Nicolas et al., 2014). The regulation of the ABA signaling pathway is complex; the cellular, physiological and transcriptomic responses to this hormone change dramatically in a tissue-specific manner (Rattanakon et al., 2016), and are also cultivar-dependent (Rossdeutsch et al., 2016). Moreover, as has been mentioned, the gene families that act positively, or negatively downstream of ABA are composed of several genes, and it is plausible that subspecialization of members in these families exists. The complexity mentioned above is a significant challenge for researchers when attempting to extract mechanisms related to ABA signaling. An important issue to address in the coming years is that of understanding the differential sensitivity to ABA across cultivars, and the crosstalk of ABA with other signals that seem to be important in berry development, such as sugars and other hormones. # THE EFFECT OF THE ENVIRONMENT ON GRAPE BERRY DEVELOPMENT Grape berries are constantly exposed to several biotic and abiotic factors that, to some extent, can affect their normal development and trigger positive or negative changes. In most cases, these factors negatively impact grape cultivation at different stages of plant and berry development during pre- and post-harvest (Armijo et al., 2016a). In this review, some of the most relevant grapevine abiotic and biotic stresses are discussed. ## Abiotic Stress Climate change has caused significant warming in most grapegrowing areas, increasing some important abiotic stresses like heat, drought and UV radiation (Teixeira et al., 2013; Keller, 2015). These stresses mainly affect phenolic metabolism and, at the same time, berry composition and development (Figure 1). Changes in temperatures during vegetative grape development are associated with changes in berry harvest date (Meier et al., 2007). Studies of transcripts, metabolites and proteins also show that sugar accumulation and other parameters related to color and aroma could be affected. Moderate warmer temperatures (∼25◦C) lead to higher berry sugar content (Coombe, 1987), while higher temperatures (>30◦C) negatively affect photosynthesis, with consequent reductions in sugar, anthocyanin, and malic acid accumulation, followed by a decrease in berry size and weight (Sadras and Moran, 2012; Teixeira et al., 2013; Rienth et al., 2016; de Rosas et al., 2017). Sugar and organic acid metabolism are desynchronized in ripening grapevine fruits at high temperatures, and secondary metabolism is diminished due to the transcriptional repression of their respective genes (Rienth et al., 2016; de Rosas et al., 2017). Thus, high temperatures are a negative regulator of berry development at ripening, but the mechanism behind this is still not clear. Integrated global analyses are required to identify the possible genes associated with the changes in the corresponding physiological traits. In general, V. vinifera is considered as a salt and drought tolerant species (Tattersall et al., 2007). However, stress caused by water availability is having progressively more impact, due to can generate significant effects on grapevine cultivation. In response to salinity and drought, plants intensify the synthesis of ABA, which is transported to the aerial organs, inducing changes in the expression of genes related to their acclimatization (Tattersall et al., 2007; Cramer et al., 2011). Few studies have addressed salinity and drought stress in berries. For instance, it has been shown that water stress can increase berry flavonol content and affect the expression of genes involved in biosynthesis of stilbene precursors (Teixeira et al., 2013). All these analyses suggest a differential response to water limiting abiotic stresses that is cultivar dependent. Likewise, the optimal growth temperature for grapevines may vary between cultivars, and the activation of ABA and ethylene signaling pathways can differ according to their sensitivity or tolerance to drought. These responses have consequences in grapevine berries, since a common mechanism in response to stress in these organs, is the induction of the anthocyanins accumulation, which act as protective molecules. #### The Effect of UV Radiation in Grape Berry Development Vitis vinifera is often cultivated in Mediterranean climates with varied UV-B radiation dosages (Martinez-Luscher et al., 2013), and it is considered as well adapted to solar radiation due to a variety of physiological responses, mainly based on antioxidant enzyme activities and secondary metabolites. The UV-B spectrum (280–315 nm) can provoke potential damage in macromolecules, including DNA, induce ROS, and disrupt several cellular processes in all living organisms (Frohnmeyer and Staiger, 2003; Jenkins, 2009). In grapevine, several studies have been performed to discover the processes associated with the UV radiation response during berry development. In this context, VviHY5 (ELONGATED HYPOCOTYL 5), VviHYH (VviHY5 HOMOLOGUE), and VviUVR1 (the photomorphogenic factor UV-B RECEPTOR 1) genes were characterized (Loyola et al., 2016). In this work, the authors described that the expression of VviHY5 and VviHYH differs during grape berry and inflorescence development upon exposure to low or high UV-B radiation, while VviUVR1 expression was not regulated by UV-B. Studies performed by Carbonell-Bejerano et al. (2014) indicated that grapevine berries respond to UV-B through the activation of the phenylpropanoid pathway and the production of photoprotective compounds. The accumulation of polyphenolic compounds in the berry involves specific UV-responsive genes that induce the expression of phenylpropanoid pathway related genes and several MYB transcription factors that regulate this pathway (Matus et al., 2009; Berli and Bottini, 2013). Matus et al. (2009) demonstrated that different light conditions increase the accumulation of flavonoid compounds in grape berries, while Loyola et al. (2016) shown that high and low UV-B radiation induce flavonol accumulation in this organ. Carbonell-Bejerano et al. (2014) suggest that UV-B radiation triggers flavonol accumulation in grape berry skin of cv. Tempranillo and induces the expression of VvFLS1 and VvGT5, two flavonol biosynthetic genes. Furthermore, several flavonol biosynthetic genes are regulated by the R2R3-MYB transcription factor VvMYBF1, which triggers flavonol and anthocyanin production in grape berries exposed to solar UV radiation (Czemmel et al., 2009, 2017; Matus et al., 2009; Matus, 2016). Genome-wide microarray studies performed in grape berry skins of cv. Pinot Noir exposed to UV-C light (100–280 nm), showed 238 upregulated genes (more than fivefold), including several genes encoding for proteins related to stilbene synthesis (Suzuki et al., 2015). These authors also reported that UV-C light increases levels of phenolic compounds like resveratrol and its analogs. Similar results were observed in berries of cv. Tempranillo exposed to solar UV radiation (Carbonell-Bejerano et al., 2014). In general, several volatile compounds accumulate in grape berries during ripening, but the amount of these compounds depends on specific irradiance levels and the type of radiation (Joubert et al., 2016). Summarizing, there are numerous studies demonstrating that light can affect anthocyanin accumulation in berry skins. Which can be explained by changes in the expression of structural genes related to the phenylpropanoid pathway, as well as regulatory genes such as those of the MYB, bHLH, and WD40 families (Wu et al., 2014). #### Biotic Stress In addition to abiotic stress, grape berry development can be influenced by biotic factors, such as pathogens, of which fungal and viral diseases are the most common and harmful, negatively affecting fruit quality. #### Fungal Infections: Botrytis cinerea and Its Dual Effect on Berry Development The most important fungal disease affecting grape berry development is gray mould, caused by B. cinerea. Grape berries are resistant to the infection until veraison, but are highly susceptible at the onset of ripening and harvest (Kelloniemi et al., 2015). As a necrotrophic pathogen, B. cinerea secretes lytic enzymes and phytotoxins in order to promote cell degradation (Armijo et al., 2016b). Most of the agronomically relevant grapevine cultivars are susceptible to this pathogen, leading to significant losses worldwide. Different large-scale approaches have been carried out in order to understand the regulatory networks and processes involved in the grape berry–B. cinerea interaction, and to characterize how berry development is affected. Transcriptomic and metabolic analysis of cv. Marselan, comparing B. cinerea berries at veraison with ripe berries, revealed that the former activates an early burst of ROS, together with multiple defense responses, including a salicylate-dependent pathway, resveratrol synthesis and cell-wall strengthening. In contrast, ripe berries activate the JA-dependent pathway against the fungus (Kelloniemi et al., 2015). As a common response, both developmental stages displayed an upregulation of genes encoding WRKY transcription factors, pathogenesis-related proteins, glutathione S-transferase (involved in cellular detoxification), stilbene synthase and PAL (involved in phenylpropanoid biosynthesis), and production of anthocyanins and phytoalexins. Global metabolic changes in cv. Marselan induced by B. cinerea infection correlate the greater resistance of veraison berries with an accumulation of resveratrol and caffeic, ferulic, and chlorogenic acids (Kelloniemi et al., 2015). Also, significantly higher levels of proline, glutamate, arginine, and alanine were detected in B. cinerea-infected ripe berries of cv. Chardonnay, as well as, an accumulation of glycerol, gluconic acid, and succinate, mainly in the berry skin (Hong et al., 2012). A reprogramming of carbohydrate and lipid metabolism toward an increased synthesis of secondary metabolites with antioxidant properties, such as trans-resveratrol and gallic acid, was also observed by Agudelo-Romero et al. (2015) in cv. Trincadeira. During later stages of infection, energy metabolism (photosystem I supercomplex) and secondary metabolism (phenylpropanoid and stilbenoid biosynthesis) also seemed to be downregulated (Agudelo-Romero et al., 2015). Contrary to the effects caused by gray mould on grape berries, some particular cases of B. cinerea infection can generate favorable effects on wine grapes, in an interaction known as noble rot. Botrytized wines are produced from grapes that have been affected by this fungus under specific environmental conditions, which are typically hot and dry. The infection produces berry dehydration, altering metabolic processes and the saprophytic microbiota (Magyar, 2011). The berry–fungus interaction promotes the accumulation of secondary metabolites that enhance wine grape composition in ripe berries. Transcriptomic and metabolic analyses of noble rot in cv. Semillon determined that anthocyanin biosynthesis is the most consistent hallmark of noble rot. In addition, the biosynthesis of terpenes and fatty acid aroma precursors increase during the infection (Blanco-Ulate et al., 2015). APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2-ERF), and NON APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION FACTOR/CUPSHAPED COTYLEDON (NAC) transcription factors, were up-regulated during noble rot (Blanco-Ulate et al., 2015). Early products of the phenylpropanoid pathway are accumulated in noblerotted berries, such as rosmarinic acid (a cinnamic acid derivative with antioxidant and aromatic properties). Also, a significant accumulation of several flavonoid glycosides and flavanones was detected, along with build ups of cyanidin-3 rutinoside, delphinidin-3-rutinoside, cyanidin-3-gentiobioside, and delphinidin-3-gentiobioside, anthocyanins that are normally scarce in white-skinned grape berries. Other aromatic compounds such as acetophenones, benzoic acid derivatives, methoxyphenols, and phenolic glycosides showed increased abundance in noble rot, together with gallic acid, a precursor of tannin biosynthesis (Blanco-Ulate et al., 2015). In conclusion, reprogramming of secondary metabolites and hormonal pathways are common features in B. cinerea-infected grape berries. Additionally, it has been shown that during grape berry development, the fruit undergoes changes that facilitate fungal infection, such as fruit softening, organic acid and sugar level modifications, loss of the preformed defenses and decreased stilbene production, among others. On the other hand, noble rot also alters berry metabolism by inducing stress responses and accelerating ripening to enhance the colonization process. B. cinerea infections can affect the color and sugar concentration, improving wine grape composition. This effect is caused by an imbalance of hormone synthesis and perception, which in turn activates several ripening-associated pathways. However, the mechanism behind this acceleration is still under study. #### Viral Diseases and Their Effect on Grape Berry Development Viral diseases are also common in grapevine plantations. Infections caused by these pathogens are highly complex, due to the large number of viral agents described and the occurrence of multiple infections (Prosser et al., 2007; Martelli, 2014; Jooste et al., 2015; Naidu et al., 2015). Grapevines show no resistance against virus; instead, viruses and host plants establish compatible interactions, where pathogens spread throughout all plant tissues, unimpeded by the resistance responses, generating global cellular stress and developmental defects. However, in compatible interactions, hosts are not passive against the pathogen, and molecular, cellular and physiological responses can be observed (O'Donnell et al., 2003; Ehrenfeld et al., 2005). In general, grapevine viruses infect vegetative organs, but infections also have consequences for berry development, causing a reduction in berry setting, and delayed berry ripening (Martelli, 1993, 2014). Molecular changes during berry ripening in virus-infected grapevine plants have been less characterized than leaf symptomatology. For instance, characterization of the Grapevine leafroll-associated virus 3 (GLRaV-3) infection in the red cv. Cabernet Sauvignon revealed the presence of viral particles in berry tissues together with massive transcriptional changes, which were more pronounced during ripening (Aquea et al., 2011; Vega et al., 2011). Since this virus is restricted to the phloem (Martelli, 2014), GLRaV-3 infection could physically modify sugar accumulation, altering source–sink relationships. Transcript profiling analyses performed in cv. Cabernet Sauvignon berries at veraison and ripening, using the V. vinifera Affymetrix GeneChip, revealed numerous changes in transcripts related either to viral infection or to berry development (Vega et al., 2011). About 400 genes showed differential expression between veraison and ripening, in uninfected tissues. However, only half of these exhibited such differences when the two stages were compared in infected berries. Thus, viral disease greatly modifies the transcript abundance profile during berry development. The number of differentially expressed genes in infected berries was higher during ripening (146 up- and 86 down-regulated genes) than at veraison (41 up- and 14 downregulated genes), suggesting that the former stage could be more dramatically affected by virus infection. Among the transcripts that change in infected berries, is a group related to sugar transport and metabolism, including ATOCT2, a carbohydrate transmembrane transporter; ATSPS4F, a putative sucrose-phosphate synthase; a short-chain dehydrogenase/reductase (SDR) family protein involved in sugar metabolism; SUS2, sucrose synthase 2; and BFRUCT3, a betafructosidase. In agreement with this, glucose and fructose levels also decreased during ripening in infected berries. Several genes from the phenylpropanoid pathway were repressed by viral infection during ripening, such as CHS2 and UFGT, as well as genes that encode for transcription factors MYBPA1 and MYBA (anthocyanin biosynthesis), and FLS1, related to the flavonol biosynthetic pathway. These results were further supported by a decrease in total anthocyanin content and flavonol concentration during ripening in infected berries (Vega et al., 2011). A characterization of berries of the Italian cv. Nebbiolo harboring a mixed infection of GLRaV-1, Grapevine virus A (GVA) and Rupestris Stem Pitting virus (RSPaV), showed significant differences in bud burst index, berry weight, titratable acidity, and resveratrol content when compared with uninfected berries (Giribaldi et al., 2011). In that study, a proteomic analysis revealed that mixed viral infection affects proteins related to cell structure metabolism in pulp, such as pectin methylesterase, N-acetyl-gamma-glutamyl-phosphate reductase, plastid movement impaired 1, phosphoglycerate kinase, polyphenol oxidase and alpha-tubulin, among others (Giribaldi et al., 2011). A thorough study carried out over three seasons, on the effects of grapevine leafroll disease (GLD) on cv. Merlot, showed that infection impacted greatly on yield, as well as on fruit quality (Alabi et al., 2016). For instance, the authors consistently found a lower fruit yield over the seasons evaluated, supporting previous conclusions that GLD negatively affects vine performance. In virus-infected cv. Merlot plants, developing green berries showed minor compositional changes in comparison to uninfected plants. However, after veraison, dramatic variations were observed as a consequence of viral disease, suggesting that the virus can affect ripening-related processes occurring from veraison onward, as previously shown for cv. Cabernet Sauvignon (Vega et al., 2011). In general, transcriptomic and metabolic data support the observation that viral diseases delay grape berry ripening, altering several characteristic parameters associated with this stage, such as sugar accumulation and color, among others. However, more studies should be carried out in order to establish how viruses alter grapevine berry ripening, how cultivars and environmental factors interact to produce the complete symptomatology, and how these multiple cues modify berry ripening. #### CONCLUSION Significant progress has been made toward understanding grape berry development, and how environmental factors can positively or negatively regulate this process. In this field, Omics platforms have been an important tool in the elucidation of the mechanisms underlying these interactions. Due to the lack of transgenic lines and suitable technologies for reverse genetics in grapes, Omics analyses have allowed us to make progress in unraveling the complex mechanisms that take place during berry development. Of the future challenges, the establishment of a robust model to assess biological questions is key. In grapes, the availability of mutant varieties and related cultivars with contrasting phenotypes is an advantage, but differences between cultivars could be more complex than expected. Therefore, global analysis should be carried out. In this context, as Omics provide numerous tools that generate huge data sets from an overall perspective, the integration of this information is the next challenge which needs to be addressed in order to understand the different processes underlying grape berry development. Systems biology deals with the integration of these data sets, advancing the way in which biological processes are studied from gene-by-gene #### REFERENCES studies toward a global perspective, where the different processes are depicted in regulatory networks. Those networks are useful in the prediction of gene function, while providing new insights into the regulatory mechanisms at a global level. The generation of robust networks to identify new regulators and genome-wide responses to environmental factors requires a vast number of data sets and the integration of multi-omics studies. In grape berries, most of the Omics studies are based on transcriptomic and metabolomic profiles; more-integrated networks are hindered by the lack of proteomic and phosphoproteomic studies. Another challenge in the bioinformatic field is the standardization and centralization of the stored data in order to facilitate the access to, and analysis of, Omics studies. Currently in grape, due to the multiple sources of data and gene annotation, there is a lack of consensus in the integrative tools available. For instance, annotation version 1 (V1) and version 2 (V2) differ in the number of annotated genes, with V2 having around 2000 new genes and 3000 putative long non-coding RNAs (lncRNA). The integration of the different annotations is a task that remains unresolved by the scientific community studying grape. Therefore, to improve our current knowledge, further Omics studies are undoubtedly necessary, yet this new data must be integrated with systems biology tools in order to comprehensively depict the associated regulatory networks. ### AUTHOR CONTRIBUTIONS The manuscript was written by AS, CE, GA, CI-B, EP, CM-R, CS, FP, AA, and PA-J. AS was involved in revising the manuscript critically for important intellectual content. Manuscript editing was conducted by AS and CE. Figure design was conducted by CM-R. All authors contributed, read and approved the final manuscript. PA-J made the final approval. # FUNDING This work was supported by FONDECYT postdoctoral research 3150608 (AS), Millennium Nucleus of Plant Systems and Synthetic Biology NC130030 and FONDECYT 1150220. and the promotion of Vitis vinifera L. berry ripening by abscisic acid. Aust. J. Grape Wine Res. 15, 195–204. doi: 10.1111/j.1755-0238.2008.00045.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 Serrano, Espinoza, Armijo, Inostroza-Blancheteau, Poblete, Meyer-Regueiro, Arce, Parada, Santibáñez and Arce-Johnson. 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. # How Single Molecule Real-Time Sequencing and Haplotype Phasing Have Enabled Reference-Grade Diploid Genome Assembly of Wine Grapes Andrea Minio<sup>1</sup> , Jerry Lin<sup>1</sup> , Brandon S. Gaut <sup>2</sup> and Dario Cantu<sup>1</sup> \* <sup>1</sup> Department of Viticulture and Enology, University of California, Davis, Davis, CA, United States, <sup>2</sup> Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA, United States Keywords: heterozygosity, inbreeding depression, cabernet sauvignon, comparative genomics, grape #### Edited by: pan-genome José Tomás Matus, Centre for Research in Agricultural Genomics, Spain #### Reviewed by: Jordi Garcia-Mas, Institute for Research and Technology in Food and Agriculture, Spain Michela Troggio, Fondazione Edmund Mach, Italy #### \Correspondence: Dario Cantu [email protected] #### Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science > Received: 04 April 2017 Accepted: 02 May 2017 Published: 17 May 2017 #### Citation: Minio A, Lin J, Gaut BS and Cantu D (2017) How Single Molecule Real-Time Sequencing and Haplotype Phasing Have Enabled Reference-Grade Diploid Genome Assembly of Wine Grapes. Front. Plant Sci. 8:826. doi: 10.3389/fpls.2017.00826 ## HIGH HETEROZYGOSITY IS A CHALLENGE FOR GRAPE GENOME ASSEMBLY Domesticated grapevines (Vitis vinifera) have relatively small genomes of about 500 Mb (Lodhi and Reisch, 1995; Jaillon et al., 2007; Velasco et al., 2007), which is similar to other small-genomes species like rice (430 Mb; Goff et al., 2002), medicago (500 Mb; Tang et al., 2014), and poplar (465 Mb; Tuskan et al., 2006). Despite their small genome size, the sequencing and assembling of grapevine genomes is difficult because of high levels of heterozygosity. The high heterozygosity in domesticated grapes may be due, in part, to their domestication from an obligately outcrossing, dioecious wild progenitor. Domesticated grapes can be selfed, in theory, because their mating system transitioned to hermaphroditic, self-fertile flowers during domestication. In practice, however, selfed progeny tend to be non-viable, presumably due to a high deleterious recessive load and resulting inbreeding depression. As a consequence of these fitness effects, most grape cultivars are crosses between distantly related parents (Strefeler et al., 1992; Ohmi et al., 1993; Bowers and Meredith, 1997; Sefc et al., 1998; Lopes et al., 1999; Di Gaspero et al., 2005; Tapia et al., 2007; Ibáñez et al., 2009; Cipriani et al., 2010; Myles et al., 2011; Lacombe et al., 2013). One such cultivar is Cabernet Sauvignon, one of the most widely cultivated wine grape cultivars. Cabernet Sauvignon was produced from a cross between Sauvignon Blanc and Cabernet Franc sometime before the seventeenth century in the Aquitaine region of France (Bowers and Meredith, 1997). Whether a spontaneous hybrid or a product of human breeding, all of the Cabernet Sauvignon grown around the world is thought to have resulted from this single hybridization event. Just as the parents of Cabernet Sauvignon have been identified, the genetic origin of many other important wine grape cultivars is known, and they often originate from the direct crossing of common, distantly-related cultivars (Strefeler et al., 1992; Ohmi et al., 1993; Qu et al., 1996; Bowers and Meredith, 1997; Sefc et al., 1998; Lopes et al., 1999; Crespan and Milani, 2001; Vouillamoz et al., 2003, 2004; Di Gaspero et al., 2005; Vouillamoz and Grando, 2006; Lacombe et al., 2007, 2013; Tapia et al., 2007; Boursiquot et al., 2009; Ibáñez et al., 2009; Cipriani et al., 2010; Myles et al., 2011; García-Muñoz et al., 2012). Due to this intraspecific hybridization process, levels of heterozygosity in grape cultivars can easily exceed 11% (Jaillon et al., 2007). High heterozygosity is challenging for genome assembly, because heterozygous genomes typically produce more fragmented sequences than haploid or homozygous genomes of similar size and complexity (Yu et al., 2005; Argout et al., 2011; The Tomato Genome Consortium, 2012). The goal of standard assembly approaches is to collapse homologous regions with sufficient similarity into haploid consensus sequences, but divergent haplotypes in heterozygous regions typically result in multiple, difficult to resolve assembly paths which must then be assembled separately. Additionally, the boundaries between haploid consensus contigs and heterozygous regions cannot be resolved with a unique path; as a result they are left unlinked, which breaks assembly contiguity (Figure 1A). Altogether, elevated heterozygosity increases fragmentation and inflates the size of the total assembly, potentially doubling the genome size if the majority of the two homologous genomes are assembled separately (Huang et al., 2012; Li et al., 2012; Safonova et al., 2015). Fragmentation and retention of redundant regions can also lead to inaccurate gene models, apparent paralogous genes and duplicated blocks, incorrect gene copy number, and synteny breaks. #### INITIAL ATTEMPTS TO SEQUENCE THE GRAPE GENOME Despite the challenges in assembling heterozygous genomes, the commercial and cultural importance of the grapevine has led to several sequencing attempts. Two genome reference drafts for the common grapevine were released in 2007 (Jaillon et al., 2007; Velasco et al., 2007). Remarkably, these were the first genomes of any fruiting crop to be sequenced and only the fourth for flowering plants. These reference genomes, both of which utilized the Pinot Noir cultivar, were assembled using different approaches to address heterozygosity. The first genome by Jaillon et al. reduced heterozygosity by inbreeding a line of Pinot Noir (var. PN40024) to ∼7% heterozygosity (Jaillon et al., 2007). To produce the second genome, Velasco et al. sequenced a Pinot Noir clone (ENTAV115) directly then assembled contigs that represented separate homologous chromosomes (Velasco et al., 2007). Unsurprisingly, these early efforts are poor by current standards. The PN40024 genome had ∼8.4-fold coverage and was assembled into 19,577 contigs with a contig N<sup>50</sup> of only 65.9 kbp. Later sequencing increased coverage to up to 12x and the contig N<sup>50</sup> of the PN40024 genome to 102.7 kb (Figure 1B). The ENTAV115 genome used both Sanger pairedreads and 454 sequencing to achieve a total coverage of ∼4.2x. Although riddled with gaps and potentially omitting large regions of repetitive sequences where genes could be located, the two genomes provided valuable insights into grape genomes. Together they revealed that the Pinot Noir genome features: (i) ∼30,000 protein-coding genes, comparable to Arabidopsis but about 75% of rice and poplar; (ii) a high proportion of repetitive elements comprising an estimated ∼40% of the genome; (iii) complex patterns of gene duplications consistent with one or more paleopolypoidy events; (iv) expansion of gene families that influence the organoleptic properties of the berry; (v) a typical number (∼200) of NBS-LRR genes, which often function in disease resistance, and (vi) a standard complement of genes involved in disease signaling pathways. Despite its limitations, the PN40024 genome assembly has proven to be invaluable to the grape research community. Cited in over 2,000 articles, it has served as a reference in more than 3,000 genome-wide transcriptional analyses. Following the publication of the PN40024 genome in 2007, no genome reference of equivalent or greater quality has been released for V. vinifera. Only a handful of studies have attempted to use bona fide genome-wide approaches to measure diversity within the species (Giannuzzi et al., 2011; Da Silva et al., 2013; Di Genova et al., 2014; Cardone et al., 2016). With the advent of second generation short read sequencing, attempts were made to perform de novo assembly and reference based resequencing of grape cultivars. These attempts failed to provide a high quality representation of the sequenced grape genotypes. A de novo approach was adopted to assemble the genome sequence of Thompson Seedless, a ubiquitous multipurpose cultivar. Despite an enormous sequencing depth (327x), the short fragment size did not permit resolution of repetitive regions, resulting in an extremely fragmented assembly (Di Genova et al., 2014; Figure 1B). For the wine grape cultivar Tannat (Da Silva et al., 2013), the authors applied a reference based assembly approach, which had proved to be effective in assembling multiple Arabidopsis genotypes (Gan et al., 2011). However, reference-based assembly failed to reconstruct genotype specific sequences with Tannat data, demonstrating that large scale resequencing initiatives like the 1,000 Human Genome project (Auton et al., 2015) and the 1,001 Arabidopsis Genomes project (Alonso-Blanco et al., 2016) would not succeed for Vitis. In fact, while the approach supported variant calling with de novo assembly to resolve regions highly divergent in sequence between Tannat and PN40024, it was unable to recover regions absent in the reference but present in Tannat. Consequently, over 10% of the gene space was not represented in the assembly, illustrating that the genomic sequence of one cultivar is insufficient for representing the total variability of the species. To improve representation of the V. vinifera pan-genome and encompass the variability of the species, we need the complete de novo assembled genomes of additional genotypes. Moreover, as grape cultivars are intraspecific hybrids of different genotypes, assembly of each genome should include a diploid representation of the genome to preserve information about the characteristics of each haplotype. # RECENT DEVELOPMENTS IN GRAPE GENOME SEQUENCING Single Molecule Real Time (SMRT) DNA sequencing (Pacific Biosciences) has emerged as a leading technology for characterizing complex structural variations, supporting and refining the assembly of complex genomes in hybrid fashion or alone for reconstructing highly continuous assemblies of both #### FIGURE 1 \| Continued sequences from the Cabernet Sauvignon primary contigs were aligned using GMAP (Wu and Watanabe, 2005) to the Cabernet Sauvignon haplotigs and the PN40024 chromosomes to identify the shared part of the represented gene space. Only alignments with identity ≥80% and coverage ≥66% were considered. In similar fashion, coding sequences from the Cabernet Sauvignon haplotigs were aligned against the primary contigs and the PN40024 chromosomes, and coding sequences from PN40024 were aligned against both primary contigs and haplotigs of Cabernet Sauvignon. small and highly repetitive genomes (Chin et al., 2013; Doi et al., 2014; Huddleston et al., 2014, 2016; Gordon et al., 2016; Ricker et al., 2016; Seo et al., 2016; Vij et al., 2016). The advantage of SMRT technology arises from the delivery of long reads, currently averaging over 30 kbp and potentially approaching 100 kbp. In addition to facilitating assembly of more contiguous genomes, long reads carry the necessary information to phase haplotypes over multiple kilobase distances. The open-source software, FALCON-unzip (Chin et al., 2016), was developed specifically to utilize the long reads generated using SMRT sequencing technology and assemble diploid genomes into highly contiguous and correctly phased diploid genomes. The algorithm first constructs a string graph composed of "haploid consensus" contigs together with bubbles representing structural variant sites between homologous loci. Sequenced reads are then phased and separated for each haplotype on the basis of heterozygous positions. Phased reads are finally used to assemble the backbone sequence (primary contigs) and the alternative haplotype sequences (haplotigs) (Figure 1A). The combination of primary contigs and haplotigs constitute the final diploid assembly with phased single-nucleotide polymorphisms and structural variants between the two haplotypes. We have recently reported the assembly using SMRT technology and FALCON-unzip of the highly heterozygous diploid genome of Cabernet Sauvignon (Chin et al., 2016), one of the most widely cultivated wine grape cultivars. As it is the progeny of Cabernet Franc and Sauvignon Blanc, two cultivars with extremely divergent phenotypical traits, reconstructing the diploid structure of Cabernet Sauvignon is necessary for identifying the alleles inherited from the parent cultivars. We sequenced the Cabernet Sauvignon genome with a coverage depth of ∼140x using SMRT sequencing technology. Sequencing reads were then assembled using FALCON-unzip into a highly contiguous genome that integrated phased haplotype information. FALCON-unzip generated a set of primary contigs (591.4 Mbp in 718 contigs with N<sup>50</sup> = 2.17 Mbp, Figure 1B) that covers one of the two haplotypes, and a set of correlated haplotigs (367.8 Mbp in 2,037 contigs with N<sup>50</sup> = 0.80 Mbp). The assembled sequences exceed PN40024 contigs and Thompson Seedless scaffolds by nearly two orders of magnitude in size (Figure 1B), ranking this assembly not only as the best V. vinifera genome assembly but also among the highest quality plant genomes published to date, including other genomes sequenced with SMRT technology (Sakai et al., 2015; VanBuren et al., 2015; Jiao et al., 2016; The UC Davis Coffee Genome Project, 2017). Symptomatic of the extreme divergence in allele sequences in Vitis, the length of the primary assembly was inflated with respect to the expected genome size, illustrating one of the challenges of sequencing highly heterozygous genomes (Chin et al., 2016). After manual removal of un-phased haplotigs, the primary assembly is an ideal candidate for scaffolding or hybrid assembly with optical maps to produce a genome assembly of even higher quality. Preliminary gene model prediction identified over 34,000 protein coding sequences on the primary assembly of the Cabernet Sauvignon genome and nearly 24,000 on the haplotigs (Chin et al., 2016). Just a few hundred of PN40024 annotated coding genes did not find any suitable alignment on the Cabernet Sauvignon assembly (411 genes; identity ≥80% and coverage ≥66%), but nearly 4,900 Cabernet Sauvignon loci could not be found on the PN40024 genome (Figure 1D). These results are in accordance with other studies that reported presence/absence polymorphisms of gene models between wine grape cultivars (Da Silva et al., 2013; Venturini et al., 2013; Jiao et al., 2015), but the high number of genes not found in PN40024 likely reflects its incompleteness. Moreover, nearly 2,100 coding sequences identified in the Cabernet Sauvignon haplotigs were not found on the primary assembly (Figure 1D). While limited by the preliminary status of the annotation, these observations point to a high degree of structural variation between homologous chromosomes. Moreover, these structural variations are likely to have functional consequences since they encompass coding sequences. The variability between haplotypes may also impact and potentially confound the analysis of RNAseq data. In the worst case, the expression of haplotype-specific loci that are not represented on the reference genome would be assigned to the most similar genomic region of the reference, which is likely to generate expression mismeasurement artifacts. As shown in Figure 1C, in the presence of a diploid reference (primary contigs plus haplotigs), about 10% more RNAseq reads map at ≥99% identity. This observation suggests that when both alleles are represented in the reference reads align to their respective haplotype; RNAseq can therefore be used to determine allelic specific gene expression. #### CONCLUSIONS Genome resequencing projects of both prokaryotic and eukaryotic organisms have clearly shown that one genome sequence is insufficient to properly describe the genetic characteristics of a species (Tettelin et al., 2005; Donati et al., 2010). In order to grasp comprehensive genetic variability and complete gene pools in outcrossing species, such as grape, we also need to go beyond the generation of haploid consensus sequences and focus our efforts to begin assembling diploid genome sequences with phased haplotypes. As discussed in this article, long read sequences and bioinformatic tools that take advantage of them have solved a critical bottleneck in grape genomics. As long-range scaffolding technologies, such as those based on proximity ligation–based methods like Hi-C (Putnam et al., 2016) or optical maps (Hastie et al., 2013; Yoon et al., 2016) are optimized for highly heterozygous plant genomes, we expect that reference-grade genome references will quickly become available for many grape species and cultivars of interest. This genomic information will allow us to identify core sequences that are common to all cultivars, as well as dispensable sequences comprising partially shared and non-shared genes that contribute to inter-cultivar phenotypic variation. This genomic information will also enable the identification of the genetic bases of economically important traits to accelerate the breeding of new cultivars and rootstocks. #### REFERENCES #### AUTHOR CONTRIBUTIONS DC and AM conceived the article. Figure was prepared by AM and DC. AM, JL, BG, and DC wrote the first draft of the manuscript. DC revised and finalized. ### ACKNOWLEDGMENTS The genome sequencing of Cabernet Sauvignon in the Cantu lab is supported by J. Lohr Vineyards and Wines and by E. & J. Gallo Winery. Part of this work is carried out in collaboration with UC Davis Chile and funded by the Chilean Economic Development Agency (CORFO). 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 Minio, Lin, Gaut and Cantu. 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. # A Concise Review on Multi-Omics Data Integration for Terroir Analysis in Vitis vinifera Pastor Jullian Fabres<sup>1</sup> , Cassandra Collins<sup>2</sup> , Timothy R. Cavagnaro<sup>2</sup> and Carlos M. Rodríguez López<sup>1</sup> \* <sup>1</sup> Environmental Epigenetics and Genetics Group, Plant Research Centre, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA, Australia, <sup>2</sup> The Waite Research Institute, The School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA, Australia Vitis vinifera (grapevine) is one of the most important fruit crops, both for fresh consumption and wine and spirit production. The term terroir is frequently used in viticulture and the wine industry to relate wine sensory attributes to its geographic origin. Although, it can be cultivated in a wide range of environments, differences in growing conditions have a significant impact on fruit traits that ultimately affect wine quality. Understanding how fruit quality and yield are controlled at a molecular level in grapevine in response to environmental cues has been a major driver of research. Advances in the area of genomics, epigenomics, transcriptomics, proteomics and metabolomics, have significantly increased our knowledge on the abiotic regulation of yield and quality in many crop species, including V. vinifera. The integrated analysis of multiple 'omics' can give us the opportunity to better understand how plants modulate their response to different environments. However, 'omics' technologies provide a large amount of biological data and its interpretation is not always straightforward, especially when different 'omic' results are combined. Here we examine the current strategies used to integrate multi-omics, and how these have been used in V. vinifera. In addition, we also discuss the importance of including epigenomics data when integrating omics data as epigenetic mechanisms could play a major role as an intermediary between the environment and the genome. #### Edited by: Giovanni Battista Tornielli, University of Verona, Italy #### Reviewed by: Pablo Carbonell-Bejerano, Instituto de las Ciencias de la Vid y del Vino (ICVV), CSIC, Spain Stefania Pilati, Fondazione Edmund Mach, Italy \Correspondence: Carlos M. Rodríguez López [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 08 February 2017 Accepted: 02 June 2017 Published: 20 June 2017 #### Citation: Fabres PJ, Collins C, Cavagnaro TR and Rodríguez López CM (2017) A Concise Review on Multi-Omics Data Integration for Terroir Analysis in Vitis vinifera. Front. Plant Sci. 8:1065. doi: 10.3389/fpls.2017.01065 Keywords: multi-omics, environment, Vitis vinifera, data integration, epigenetics, transcriptomics, metabolomics # INTRODUCTION Grapevine is one of the most economically important fruit crops, and it is largely used for wine production (FAO, 2012). Most the chemical compounds that give its unique characteristics to wine are synthesized during berry development (Conde et al., 2007). However, fruit/wine composition is strongly influenced by the interactions between the plant's genome and the local growing conditions (including the vine management system), and the oenological practices of each winery (Figure 1), which could explain why it is so difficult to replicate a wine from a region outside that area. Terroir is defined as the interactions between the plants, the environment and human factors (Gladstones, 2011) and it is frequently used to relate wine sensory attributes to its geographic origin (Van Leeuwen and Seguin, 2006). Although the relevance of terroir is still under debate (Anesi et al., 2015), a better understanding of how the environment affects grape berry composition can have a 405* significant effect on viticulture. To achieve such an understanding, it is necessary to identify the elements that drive terroir and analyze the interaction between them and the grapevine. #### DECODING Terroir from the copyright holder). Terroir has been long studied, through the characterization of the different environmental factors affecting berry composition and wine quality, and climate exerts the strongest effect on berry composition (Robinson et al., 2012). Soil physicochemical properties as well have been identified as an influential factor defining the uniqueness of berry composition by vines grown in a specific climate (Cheng et al., 2014; Zerihun et al., 2015). Grapevine microbiome community may play an important role determining wine quality (Burns et al., 2015; Bokulich et al., 2016). Efforts have been made to study the grapevine microbiome landscape in relation to the vegetative growth cycle of the plant (Pinto et al., 2014), the post-harvest treatment of berries (Salvetti et al., 2016) and provenance (Bokulich et al., 2016) (For a review on microbiome analysis see Ibrahim and Kumar, 2017). Less work has been done to elucidate the molecular mechanisms involved in the plant response to terroir. A strategy to better understand the genome and environment interaction is to use 'omics' technologies. Omics refers to high throughput technologies that generate a large amount of data for each sample, allowing a deeper insight of the mechanisms regulating biological systems. ## ANALYSIS OF Terroir EFFECT ON GRAPE COMPOSITION USING TRANSCRIPTOMICS Using transcriptomics is possible to study the grape's complete set of RNA transcripts encoded by the genome using high throughput methods (Hale et al., 2005). Dal Santo et al. (2013) performed gene expression analysis in a single Corvina clone cultivated in 11 different vineyards for three consecutive years. Samples strongly clustered by season, known as a vintage effect, rather than common environmental conditions. However, the genes that showed more variation in expression between years were those involved in secondary metabolism, (mainly the biosynthesis of phenylpropanoids). Only when samples from a single vintage (i.e., 2008) were analyzed, it was observed that 5% of the studied annotated coding genes were differentially regulated under different growing conditions and agronomical practices. Anesi et al. (2015) complemented this study by analyzing the transcriptome and metabolome of the same cultivar. They identified metabolites that could describe a terroir signature for each vineyard. Moreover, it was possible to correlate terroir-sensitive metabolites with changes in the transcript level of genes involved in the biosynthesis of these metabolites. Similar results were obtained by Dal Santo et al. (2016) as they identified a clear correlation between gene expression and accumulation of phenylpropanoids and flavonoids in the variety Garganega grown at four different vineyards. Small RNA profiles have been analyzed to understand the interaction between genotype and environment in the varieties Sangiovese and Cabernet Sauvignon. In silico analysis suggests that microRNAs may be involved in berry development and the accumulation of secondary metabolites (Paim Pinto et al., 2016). Transcriptional analysis of berries from different regions has also shown that transcripts from the abscisic acid (ABA) biosynthesis pathway are among the most terroir sensitive genes (Sun et al., 2015). ABA is a plant hormone that regulates important steps in plant growth and development as well as play a key role in plant biotic and abiotic stress response (Cutler et al., 2010). ABA concentrations affect anthocyanin and flavonol accumulation (Koyama et al., 2010), suggesting a possible mechanism through which the environment affects grape berry composition and wine flavor and aroma. ## ANALYSIS OF Terroir EFFECT ON GRAPE COMPOSITION USING METABOLOMICS Metabolomics is defined as the identification and quantification of metabolites using high-throughput techniques (Cevallos-Cevallos et al., 2009). This technology can screen higher numbers of products than more traditional approaches (Pereira et al., 2006; Atanassov et al., 2009; Hong, 2011), while the use of non-targeted metabolomics approaches allows the identification of un characterized metabolites (Panighel et al., 2015). Terroir can be explored by analyzing berry metabolite composition through different analytical methods (For a review in grape and wine metabolomics see Cozzolino, 2016). Son et al. (2009) identified that differences in berry metabolomes associated to environmental regional differences (radiation and rainfall) could explain the observed differences in wine composition. Similar results were obtained by Tarr et al. (2013) who distinguished the metabolic signatures of different grapevine varieties. Metabolomic analysis has also been performed to identify chemical compounds that can be associated to regional wine quality traits (Gambetta et al., 2016, 2017). Roullier-Gall et al. (2014) assessed the metabolomics profiles of two different terroirs, which were just 2 km apart, over three vintages. Although vintage had the greatest effect in the berry's metabolite composition, differences in fruit chemical composition associated to nearby terroirs could be detected when vintages were individually analyzed. This suggests that subtle geographical differences have a significant effect on grape/wine composition even when variability within vineyards can be relatively high (Mulas et al., 2011). # MULTI-OMICS INTEGRATION The aim of integrating multi-omic data is to reduce the gap between data generation and the ability to analyze and understand the biological mechanisms behind an organism's response to environmental cues. The objective of multi-omic data integration is to combine different types of data to construct a model that can be used to predict complex traits and phenotypes (Figure 2). This approach also allows the identification of biomarkers and of previously unknown relationships between the datasets (Rajasundaram and Selbig, 2016). Through the integration of environmental information with genomic, epigenomic, transcriptomic, and metabolomic data, we hypothesize that it will be possible to better understand the effect of terroir at a molecular level. The use of a multi-omic approach will also help reduce the incidence of false positives generated from single source data sets (Aho, 2013; Ritchie et al., 2015). However, integration of multi-omics data is not a trivial task, because the diversity of characteristics of the data generated from the different high throughput technologies (machine sensitivity, error rate, data structure) makes its combination challenging. # APPROACHES TO DATASETS INTEGRATION Analysis of large data sets from different origins has been done using two main approaches: network models (NMs) and pathway analysis (PA). Both share the basic idea of storing the data in a clear and meaningful way. NMs use concepts from mathematical graph theory, to represent biological components (e.g., genes) as nodes and their interactions (physical, genetic or functional) as their links (For a review on NM applied to plant biology see Fukushima et al., 2014). NMs are classified as homogeneous or heterogeneous depending on the number of different levels of information integrated (Gligorijevic and ´ Pržulj, 2015). Homogenous approaches integrate datasets with the same type of nodes and therefore cannot analyze the connectivity between multiple datasets simultaneously. However, complex biological questions such as the molecular regulation of fruit composition in grapevine are increasingly being addressed through the integration of multiple layers of cellular information (Wong and Matus, 2017), including but not restricted to genomics, transcriptomics, proteomics and metabolomics, using heterogeneous methods. Bayesian networks (BNs) and Kernel-based methods (KBMs) are heterogeneous approaches commonly used for data integration (Zhang, 2009; Gligorijevic and Pržulj, 2015 ´ ). BNs are efficient detecting relationships hidden in noisy datasets but they are computationally demanding (Gligorijevic and Pržulj, 2015 ´ ) and are therefore, better suited for the interrogation of small datasets in hypothesis driven questions (Gligorijevic and Pržulj, ´ 2015) (i.e., the analysis of terroir on defined pathways such as those leading to the biosynthesis of metabolites related to fruit quality). KBMs are not as computationally demanding and so can integrate large molecular, structural and phenotypic datasets (Mizrachi et al., 2017), making them ideal for data driven terroir exploratory studies, biomarker discovery or for the reclassification of previously identified drivers of quality (Qi et al., 2008). On the other hand, pathway analysis requires well documented biochemical pathways where omics data is combined to seek overrepresented groups (Wanichthanarak et al., 2015). For example, multiple co-inertia analysis (MCIA) can detect explanatory omic features even when they are not present in all datasets (Meng et al., 2014), which makes it attractive for the integration of terroir data from different studies. Random Forest implemented for pathway analysis (Pang et al., 2006), can be used to predict fruit/wine quality traits associated to terroir integrating multi-omic and phenotypic data as shown recently for potato (Acharjee et al., 2016). Most of these multi-omics analysis approaches are pipelines that perform task sequences which share statistical methods (Bersanelli et al., 2016). Correlation analyses are the most common approaches performed to find relationships between the omics data. Simple correlation analyses, like Pearson or Spearman correlation, are widely used for multi-omics data integration (Rajasundaram et al., 2014; Rajasundaram and Selbig, 2016). Partial least square/projections to latent structures (PLS) and its extension, orthogonal partial least square (OPLS) (Tobias, 1995) have also been used for data integration from multiomics results. Even though their predictive power is similar, OPLS results are much easier to interpret and outliers are quickly detected. OPLS can be used as a discriminate analysis (OPLS-DA), to identify differences between the overall data properties while removing systematic variation (Kirwan et al., 2012). However, these methods provided little insight when they are used in complex biological systems (highly multicollinear systems) (Wanichthanarak et al., 2015). Modifications to these methods have been implemented to facilitate the interpretation of the data, for example, sparse PLS (sPLS) (Chun and Keles˛, 2010) can better predict phenotypes through multi-omics data integration than previous methods (Rajasundaram et al., 2014). Orthogonal 2PLS (O2PLS), capable of dealing with unrelated systematic variation between datasets (Bouhaddani et al., 2016), has been successfully used for data integration of transcriptomics and metabolomics results from aspen under different light treatments (Bylesjö et al., 2007). Srivastava et al. (2013) used orthogonal projections to latent structures (OnPLS), an extension of O2PLS, to integrate transcriptomics, proteomics, and metabolomics data to construct a model that could identify biological relevant events in the oxidative stress response in poplar. #### DATA INTEGRATION IN V. vinifera In plant science, most of data integration of omics results comes from model plants; however, there is an increase in publications on multi-omics data integration in V. vinifera. One of the first publications in multi-omics data integration in V. vinifera was the work of Zamboni et al. (2010). Integrating transcriptome, proteome and metabolome data, they identified stage specific biomarkers for berry development. Data integration was performed using two strategies, one hypothesis driven (i.e., a hypothesis was tested) and the other hypothesis free (i.e., discovery driven), in both cases principal component analysis (PCA), O2PLS and O2PLS-DA were used. Using five different omics technologies and correlation analysis (PCA and Pearson correlation) together with biochemical pathway analysis (KEGG, PlantCyC and VitisCyC), Ghan et al. (2015) could differentiate biochemical characteristics from five different cultivars. Moreover, Anesi et al. (2015) studied the terroir effect in V. vinifera cultivar Corvina in seven different sites over a 3 years period using metabolome and transcriptome data. Using correlation analyses (PCA, PLS-DA and O2PLS-DA) they could identify a terroir signature in the berry metabolome composition for each growing site. Network analyses have been recently adopted to integrate grapevine multi omics results (Wong and Matus, 2017). For example, Palumbo et al. (2014) using network-based methods, identified "fight-club" nodes (genes with negatively correlated profiles) that may be relevant for the control of berry transition between development and ripening. There are also online resources available that can help analyze omic data from V. vinifera. For example, VitisNet (Grimplet et al., 2009, 2012) offers manually annotated molecular networks (16,000 genes and 247 networks) where omics data can be loaded to visualize changes in the transcriptome, proteome and metabolome for a given experiment. VTCdb (Wong et al., 2013) is a gene co-expression database for V. vinifera that allows exploring transcription regulation. With more than 29,000 genes (95% of the predicted grapevine transcriptome) to query co-expression networks, VTCdb offers the possibility to analyze the transcriptional network of grapevine development, metabolism and stress response. VitisCyc (Naithani et al., 2014) is a grapevine metabolic pathway database that also allows omics data to be uploaded (transcriptome, proteome and metabolome) and to analyze changes in metabolic networks in each experiment. VESPUCCI (Moretto et al., 2016) is a manually annotated gene expression compendium exploratory tool that can be used to investigate grapevine's gene expression patterns. #### PHENOTYPIC PLASTICITY THROUGH EPIGENETIC MODIFICATIONS Epigenetics is the study of heritable phenotypes that occur through modifications that alter DNA activity without modifying its basic nucleotide structure (Feil and Fraga, 2012). Many epigenetic mechanisms, acting in an interactive and redundant fashion (Grant-Downton and Dickinson, 2005; Berger et al., 2009), have been described to date, with DNA methylation probably being the best-studied of all (Rapp and Wendel, 2005). DNA methylation affects chromatin condensation in a rapid and reversible manner (Grativol et al., 2012). In turn, the regional level of chromatin condensation affects the transcriptional state of nearby genomic features such as genes and transposable elements (Zhang et al., 2006). Global changes in DNA methylation associated to local environments can be analyze using a myriad of methods (Kurdyukov and Bullock, 2016). Bisulfite modification of genomic DNA combined with whole genome sequencing (BS-Seq) is the gold standard for methylation analysis because it can assess an entire methylome with single base resolution (Krueger et al., 2012). However, due to their lower cost, other approaches such as next generation sequencing following the capture of the methylated fraction of the genome or its fragmentation using methylation sensitive restriction enzymes (Bock et al., 2010; Li et al., 2010; Kitimu et al., 2015) are better suited to study large number of samples. Both generate quantitative and qualitative information of the methylation status of a reduced but significant representation of the total genome. Environmental signals are one of the elements that can have a major effect in modifying the DNA methylation patterns leading to gene expression changes that ultimately affect the plant phenotype (Feil and Fraga, 2012). The idea that the environment could modify the epigenetic status, and these modifications passed to the offspring (Tricker et al., 2013) or maintained as epigenetic memory on long lived organisms (Latzel et al., 2016), has attracted attention from scientists studying mechanisms involved in adaptation to local environments (Consuegra and Rodríguez López, 2016) and how these could be used to enhance crop performance (Rodríguez López and Wilkinson, 2015). There are many reported examples of how the environment affects the epigenome in natural environments and how epigenetic variations in plant populations could help to overcome the lack of genetic diversity (Fonseca Lira-Medeiros et al., 2010; Verhoeven et al., 2010). One of the most well-known examples in which the environment affects the phenotype through epigenetic modifications is vernalization (Feil and Fraga, 2012). Through this process, plants in temperate regions mitigate the deleterious effects of low winter temperatures on flower and fruit development by breaking dormancy only after having been exposed to a cold period (Kumar et al., 2016). Unusual environmental conditions during dormancy such as high winter temperatures have been shown to exert a negative effect on fruit quality and yield on perennial crops requiring a vernalization period (Sugiura et al., 2012). Recent work in apple shows how methylation and expression levels of key genes involved in flowering and fruit set are modified by the level of chill received during bud dormancy (Kumar et al., 2016), indicating that the environmentally induced changes observed in fruit quality could be regulated by DNA methylation. Together these studies suggest that the environment can have a long lasting phenotypic effect in plants through epigenetic changes without the need for genetic variation, and that epigenetic mechanisms could be working as intermediaries between environmental variation and the plant genome, and in this way, potentially contributing to plant phenotypic plasticity. Moreover, this mechanism could give plant populations a way of adapting to the local growing conditions (Platt et al., 2015; González et al., 2016). However, to our knowledge, almost all epigenetic studies done in V. vinifera have focused on the identification of commercial clones (Imazio et al., 2002; Schellenbaum et al., 2008; Ocaña et al., 2013) and on the assessment of in vitro culture on the epigenome (Baránek et al., 2015), there is, therefore, a lack of information of how the environment affects a grapevine's epigenome and to what extent this interaction affects fruit quality. Until now, there are no studies looking at the epigenome to understand the control of gene expression in V. vinifera and how environmental signals can change the regulation of metabolic pathways through epigenetic modifications. In our view, the inclusion of epigenomic data on the analysis of the terroir effect will not only increase the resolution of analysis but will also help us to understand the regulatory mechanisms behind the observed differences. #### CONCLUSION There is no doubt that the elements affecting grapevine growth and fruit composition are complex and multifarious. While the concept of terroir is widely discussed, the underlying mechanisms remain somewhat enigmatic. However, with the recent parallel development of omics technologies and of statistical approaches for their integration, we are reaching a point where it may be possible to overcome this challenge. The geographic delimitation of a terroir is the first challenge to overcome before its molecular characterization. This delimitation could be achieved 1. Empirically, based in the number of significantly different environmental subregions present in the study or/and 2. based on the traditionally defined wine regions. Moreover, the masking effect that environmental inter-annual variations can have over single year measurements demands the incorporation of data from multiple seasons to be able to determine terroirs with enough confidence. Ideally such seasons should be, from a weather perspective, variable within the range characteristic for the region of study to be able to capture its "normal terroir." Understanding how the genome, environment and viticulture practices interact to affect fruit quality will allow us the #### REFERENCES opportunity to implement agricultural practices aimed to obtain the desired fruit characteristics for every climate/cultivar combination (Jones and Davis, 2000), leading to more efficient use of resources and better management of vineyards. In addition, grape growers can maximize the terroir effect on the grapevine to highlight the uniqueness of their vineyards ultimately increasing their industrial competitiveness. We propose that the integration of multi-omic and environmental datasets will contribute to a better understanding of the drivers of the terroir effect in grapevine. Moreover, multiple dataset data integration will increase our understanding of the molecular mechanisms involved in the regulation of multifactorial genome by environment interactions. Finally, it is increasingly recognized that plants are involved in complex interactions their soil and epiphytic microbiomes, which can affect their phenotype (Mueller and Sachs, 2015). The 'omics' era gives us the ability to explore the nature and consequences of biotic/abiotic interactions and so, a future challenge will be to bring the concept of the holobiont (the plant host plus its microbiomes) into the analysis of terroir and its effect on grapevine growth and fruit composition. #### AUTHOR CONTRIBUTIONS PF, TC, CC, and CR designed and wrote the manuscript. #### ACKNOWLEDGMENTS PF was supported by Australian Grape and Wine Authority (AGWA) and University of Adelaide Graduate Research Scholarships. CR is supported by a University of Adelaide Research Fellowship. This study was funded through a Pilot Program in Genomic applications in Agriculture and Environment Sectors jointly supported by the University of Adelaide and the Australian Genome Research Facility Ltd. 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 Fabres, Collins, Cavagnaro and Rodríguez López. This is an openaccess 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. # Plant Stress Responses and Phenotypic Plasticity in the Epigenomics Era: Perspectives on the Grapevine Scenario, a Model for Perennial Crop Plants Ana M. Fortes<sup>1</sup> \* and Philippe Gallusci<sup>2</sup> \* <sup>1</sup> Faculdade de Ciências, Instituto de Biossistemas e Ciências Integrativas, Universidade de Lisboa, Lisboa, Portugal, <sup>2</sup> UMR EGFV, Université de Bordeaux, Institut national de la recherche agronomique, Institut des Sciences de la Vigne et du Vin, Villenave-d'Ornon, France #### Edited by: José Tomás Matus, Centre for Research in Agricultural Genomics, Spain #### Reviewed by: Carlos Marcelino Rodriguez Lopez, University of Adelaide, Australia Patricio Arce, Pontifical Catholic University of Chile, Chile #### \Correspondence: Ana M. Fortes [email protected] Philippe Gallusci [email protected] #### Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 02 November 2016 Accepted: 16 January 2017 Published: 06 February 2017 #### Citation: Fortes AM and Gallusci P (2017) Plant Stress Responses and Phenotypic Plasticity in the Epigenomics Era: Perspectives on the Grapevine Scenario, a Model for Perennial Crop Plants. Front. Plant Sci. 8:82. doi: 10.3389/fpls.2017.00082 Epigenetic marks include Histone Post-Translational Modifications and DNA methylation which are known to participate in the programming of gene expression in plants and animals. These epigenetic marks may be subjected to dynamic changes in response to endogenous and/or external stimuli and can have an impact on phenotypic plasticity. Studying how plant genomes can be epigenetically shaped under stressed conditions has become an essential issue in order to better understand the molecular mechanisms underlying plant stress responses and enabling epigenetic in addition to genetic factors to be considered when breeding crop plants. In this perspective, we discuss the contribution of epigenetic mechanisms to our understanding of plant responses to biotic and abiotic stresses. This regulation of gene expression in response to environment raises important biological questions for perennial species such as grapevine which is asexually propagated and grown worldwide in contrasting terroirs and environmental conditions. However, most species used for epigenomic studies are annual herbaceous plants, and epigenome dynamics has been poorly investigated in perennial woody plants, including grapevine. In this context, we propose grape as an essential model for epigenetic and epigenomic studies in perennial woody plants of agricultural importance. Keywords: DNA methylation, epigenomics, grape, Histone Post-Translational Modifications, small RNAs, Vitis vinifera #### INTRODUCTION Epigenetic mechanisms regulate chromatin structure, gene expression, transposon mobility and DNA recombination (He et al., 2011; Pikaard and Scheid, 2015). They generally refer to modifications of gene expression that can be inherited through mitosis or meiosis yet without changes in the underlying DNA sequences (Eichten et al., 2014) and also include chromatin modifications that may lead to stable alteration of the transcriptional programming of non-dividing cells even after removal of the triggering signals (Avramova, 2015). Epigenetic regulation is mediated by a complex interplay among different molecular actors. These include the DNA methylation/demethylation machinery, enzymes mediating histone post-translational modifications (PTMs), the remodeling of chromatin organization and specific classes of small RNAs and long non-coding RNAs (Lauria and Rossi, 2011; Pikaard and Scheid, 2015; Gallusci et al., 2016). Briefly, in plants 5 methyl-cytosine (m5C) is found in all sequence context, including the CG and CHG (H = A, T, or C) symmetrical motives and the non-symmetrical CHH motif (reviewed in Gehring, 2013). DNA methylation is maintained in a post-replicative way by three classes of DNA methyltransferases: DNA METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3) for CG and CHG contexts, respectively, and by the DOMAIN REARRANGED METHYLTRANSFERASE 2 (DRM2), which requires an siRNA guide and reestablishment after each cycle of DNA replication or by CMT2 for the asymmetric CHH context (Du et al., 2012; Matzke and Mosher, 2014). Finally, DNA methylation can be lost after replication when maintenance of DNA methylation is not functional or actively reversed by DNA Glycosylase-Lyases (Piccolo and Fisher, 2014). Histone PTMs are also essential epigenetic signals that can occur at the N-terminal tail of core histones (H2A, H2B, H3, H4) through acetylation, methylation, phosphorylation and ubiquitination (Berr et al., 2011). Histone acetylation and methylation at lysine residues are established by histone acetyltransferases (HATs) and histone lysine methyltransferases (HKMTs), respectively, which are encoded by complex multigenic families. These epigenetic marks can be removed by histone deacetylases (HDACs) and histone demethylases (HDMs), respectively (Berr et al., 2011; He et al., 2011; Pikaard and Scheid, 2015). The recent development of epigenome profiling has boosted our understanding of the dynamics and function of epigenetic marks in plants. Several approaches have been recently developed (Schmitz and Zhang, 2011; Lee and Kim, 2014<sup>1</sup> ). So far, histone PTM analysis relies on Chromatin Immunoprecipitation (ChIP) using specific antibodies followed by hybridization to tilling arrays (ChIP- chip, Makarevitch et al., 2015) or by high throughput sequencing (ChIP-Seq, Wang et al., 2009). DNA methylation landscape can be studied by making use of methyl sensitive restriction enzyme to enrich DNA in methylated or un-methylated sequences that are subsequently hybridized to tilling arrays or sequenced (Kim et al., 2014). Alternatively, methylated regions can be selected using m5C specific antibodies (MeDIP), and analyzed with tilling arrays (MeDip-ChIP) or by Next Generation Sequencing (Medip Seq). Both approaches were used for methylome analysis for example in Arabidopsis, or poplar (Zhang et al., 2006; Zilberman et al., 2006; Kim et al., 2014). In particular, Medip-Seq was used to analyze the changes in methylation patterns during in vitro culture of cassava (Kitimu et al., 2015). But the golden standard for methylome analysis is the combination of bisulfite conversion of DNA to high throughput sequencing that allows analyzing the methylation landscape at a single base resolution (Whole Genome Bisulfite sequencing: WGBS). The methylomes of Arabidopsis (Cokus et al., 2008; Lister et al., 2008; Stroud et al., 2013), rice (Li et al., 2012; Garg et al., 2015), maize (Eichten et al., 2013), tomato (Zhong et al., 2013), Brassica (Chalhoub et al., 2014) and many others (Niederhuth et al., 2016) have now been described using this approach. In this perspective, we will firstly focus on the analysis of the genome wide distribution of epigenetic marks in plants under stresses. However, most species used for epigenomic studies are annual herbaceous plants and little is known about epigenomes in perennial woody plants. Indeed, omics' approaches have been initiated in grape to understand environmental effects on plant and fruit development (Fortes et al., 2011; Agudelo-Romero et al., 2015). In addition, a few studies have indicated that epigenetic mechanisms might be involved in various aspects of grape development (Aquea et al., 2011). However, knowledge of grape epigenomes and of their variation has remained very limited until now (Niederhuth et al., 2016). Yet, grapevine presents several features that make it a relevant model for the study of epigenetic mechanisms due to the fact that is a perennial woody plant and the fruit maturation is subjected to nonclimacteric molecular and hormonal regulation (Fortes et al., 2015). Grapevine varieties are preserved in their distinct genetic backgrounds through clonal propagation. However, phenotypic diversity exists within clones (Pelsy, 2010) that is unlikely to be solely driven by differences in DNA sequence. These facts contribute to the relevance of grape as a model for epigenetic and epigenomic studies in perennial woody plants of agricultural importance. #### EPIGENETIC REPROGRAMMING DURING ABIOTIC STRESS RESPONSES Recent studies have shown the differential regulation of genes encoding epigenetic regulators (Fang et al., 2014; Li et al., 2014; Su et al., 2015) as well as local chromatin and DNA methylation changes in response to a variety of abiotic stresses including cold, salinity, drought, osmolality, or mineral nutrition, thereby highlighting the relevance of epigenetic regulations in these contexts (Chen et al., 2010; Luo et al., 2012; González et al., 2013; Bocchini et al., 2015; Kim et al., 2015; Liu et al., 2015). Consistent with these results, genome wide analyses of histone PTMs and DNA methylation distribution have revealed global epigenomic reprogramming in plants under abiotic stresses. In a recent study, trimethylation at lysine 4 on histone 3 (H3K4me3), a mark normally associated with gene expression, was analyzed in Arabidopsis plants under drought stress using ChIP-seq and showed to be highly dynamic and positively correlated with the transcription level of drought induced genes in response to stress (Dijk et al., 2010). Similar results were found in rice (Zong et al., 2013) and in moss (Widiez et al., 2014). Osmotic stress also causes an increase in phosphorylated histone H3 threonine 3 (H3T3ph) located at pericentromeric regions where it is thought to help maintaining the heterochromatin structure (Wang et al., 2015). Interestingly, H3T3ph is also present in active genes where it seemed to antagonize H3K4me3, suggesting that H3T3ph may have a repressive function on gene expression during osmotic stress (Wang et al., 2015) a role also suggested for histone deacetylase HDA9 (Zheng et al., 2016). In addition, priming effects in Arabidopsis were <sup>1</sup>https://www.plant-epigenome.org/ shown to be partly mediated by remodeling of the epigenomic landscape, and involves the repressive mark H3K27me3 (Sani et al., 2013). Recently, a specialized histone H1 variant was shown to be required for a substantial part of DNA methylation associated with environmental stress in Arabidopsis (Rutowicz et al., 2015) and two DEAD-box RNA helicases were suggested to be involved in epigenetic silencing of gene expression leading to suppression of Arabidopsis stress response (Khan et al., 2014). In addition, DNA methylation is also critical for the responses of plant to abiotic stresses. This was initially shown by the demonstration that Arabidopsis mutants deficient in various steps of the RdDM pathway or in CHG maintenance methylation are affected in their capacity to modulate the stomatal index under low relative humidity (Tricker et al., 2012), present an hypersensitivity to heat exposure (Popova et al., 2013) or an enhanced sensitivity to phosphate starvation (Yong-Villalobos et al., 2015). These results are consistent with an important function of the DNA methylation dynamics in the regulation of abiotic stress–responsive genes. Indeed drought stress, but also nutrient deprivation cause extensive remodeling of DNA methylation patterns in Arabidopsis (Colaneri and Jones, 2013; Yong-Villalobos et al., 2015; Wibowo et al., 2016), barley (Chwialkowska et al., 2016) or Populus (Liang et al., 2014). In this latter case, modulation of DNA methylation at repetitive elements appeared essential for the control of adjacent gene expression (Liang et al., 2014) a function also suggested in maize where TEs could be used as local enhancers for stress responsive genes (Makarevitch et al., 2015). Similarly Pi deficiency in rice modulates DNA methylation at TEs located close to genes highly induced under this stress (Secco et al., 2015). In this case, however, TEs were hyper-methylated an event that occurred after gene induction most likely to prevent potentially deleterious activity of TEs located in the vicinity of highly induced stress responsive genes. As a conclusion, the results discussed above are consistent with the idea that abiotic stresses cause significant reprogramming of chromatin not only related to gene expression, but also to the control of chromosome organization. In addition, evidence of transgenerational inheritance of plant responses to stress has been provided (Tricker et al., 2013; Migicovsky et al., 2014); although this process appears limited to and mainly mediated by the female gamete (Wibowo et al., 2016). #### EPIGENETIC REPROGRAMMING DURING PLANT BIOTIC STRESS RESPONSES Regarding histone Post-Translational Modifications and DNA methylation occurring upon biotic stress there is lesser information available than for abiotic stress. However, recent findings indicate that chromatin modifications contribute to plant immunity against both necrotrophic and biotrophic pathogens (reviewed by Ding and Wang, 2015). In fact, the expression of R genes which are central regulators of plant immunity was shown to be regulated by Arabidopsis E3 ubiquitin ligase genes HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 (Zou et al., 2014). Histone monoubiquitination at the R gene locus had an impact on immune responses. The loss of- function mutant bon1-1 has enhanced disease resistance to the virulent pathogen Pst DC3000 and both HUB1 and HUB2 mediate its autoimmune responses. In another case, HDA19, an Arabidopsis histone deacetylase, was shown to play a negative role in basal defense mediated by the SA-dependent signaling pathway. Loss of HDA19 causes increased expression of SA biosynthetic genes and defense genes and promotes resistance to the virulent Pst DC3000 (Choi et al., 2012). Dimethylated or trimethylated histone H3 Lys 27 (H3K27me2/3) marks silent or repressed genes involved in stress responses in plants. Li et al. (2013) showed that the rice Jumonji C protein gene JMJ705 encodes a histone Lys demethylase that specifically reverses this mark. An increase in JMJ705 expression in transgenic plants removes H3K27me3 from defense-related genes, induces their expression with involvement of jasmonic acid, and enhances plant resistance to biotic stress. Interestingly, Soyer et al. (2014) showed that chromatin-based transcriptional regulation can also act on effector gene expression in fungi during plant infection. Pathogen infection has been also reported to change histone modifications in some defense response genes (De-La-Pena et al., ´ 2012). The profiling of the DNA methylomes of plants exposed to bacterial pathogen, avirulent bacteria, or salicylic acid revealed numerous stress-induced differentially methylated regions (DMRs) often coupled to differential gene expression (Dowen et al., 2012). Mutant plants globally defective in maintenance of CG methylation (met1-3) or non-CG methylation (ddc, drm1-2 drm2-2 cmt3-11) were markedly resistant to bacterial colonization. DNA demethylation likely primes transposable elements as well as defense gene induction through the concomitant activation of their transactivators and/or the interference with other chromatin marks (Yu et al., 2013). Some immuneresponse genes, containing repeats in their promoter regions, are negatively regulated by DNA methylation. These defense gene loci may lose DNA methylation so that they are more easily activated at the transcriptional level (Yu et al., 2013). This is corroborated by the study of Le et al. (2014); the DNA methylases ROS1, DML2, and DML3 were shown to play a role in fungal disease resistance in Arabidopsis since a triple mutant rdd (ros1 dml2 dml3), presents down-regulation of stress response genes and increased susceptibility to a fungal pathogen. Furthermore, these authors showed that DNA demethylases target promoter transposable elements in stress responsive genes to positively regulate them. #### NATURAL AND INDUCED EPIGENOMIC VARIATION, PHENOTYPIC PLASTICITY AND BREEDING Natural epigenomic variation occurs during species evolution (Hirsch et al., 2013) and together with genetic variation is likely involved in the phenotypic diversity and plasticity of plants. Epigenetic variation is sensitive to environmental inputs; epialleles induced by the environment or experimentally may be formed at a higher rate than alleles generated from genetic variation and may also be inherited leading to better adaptation to the environment (Figure 1; Hirsch et al., 2013). Experimentally induced epialleles have been produced in Arabidopsis by generating Epigenetic Recombinant Inbred Line (EpiRILs) populations derived from decrease in DNA methylation 1-2 (ddm1-2) or the met1 parents (Johannes et al., 2009; Reinders et al., 2009). EpiRILs were subsequently used to identify epiQTL corresponding to DMRs that determine two complex traits, flowering time and primary root length (Cortijo et al., 2014). Interestingly, these EpiRILs present variation in growth capacity (Hu et al., 2015) and are more sensitive to salinity stress than the Col0 parent line suggesting that ddm1 derived epigenotypes limit the ability to adapt to this stress (Kooke et al., 2015). As an alternative approach, a stochastically hypomethylated population was generated by selfing Brassica rapa plants previously treated with the demethylating agent 5-Azacytidine (Amoah et al., 2012). This population was used for forward screening of agronomic traits such as flowering time, seed protein content and fatty acid components. These results suggest that a portion of QTLs that have been used by breeders so far may be due to epigenetic, rather than genetic variation (Springer, 2013). DNA methylation may also have an important role in the long term adaptation of plants (Figure 1; Garg et al., 2015). Two rice cultivars with contrasting sensitivity to drought stress and salinity showed clearly different methylation landscapes; part of the DMRs between cultivars were associated with genes involved in stress responses (Garg et al., 2015). Indeed variation in methylation patterns have been also observed in natural populations and might be associated with specific environmental traits. In a recent study, Dubin et al. (2015) showed by analyzing Arabidopsis accessions from Northern and Southern Sweden that CHH methylation at transposons increases with temperature and this was associated with major genetic variants at the CMT2 locus. In the same study, Gene Body Methylation which was not modified by temperature was shown to be correlated with the latitude of origin; Southern accessions being less methylated than Northern one. This was associated with a lower expression of the targeted genes in Southern accessions consistent with local adaptation of the accessions. Epialleles impacting plant traits have now been identified in many plants (Rodríguez López and Wilkinson, 2015) since the initial characterization of the cycloidea and Cnr epimutations in snapdragon and tomato, respectively (Cubas et al., 1999; Manning et al., 2006; Poole et al., 2006). For example, Vitamin E in tomato is determined by epigenetic variations linked to a SINE retrotransposon located in the promoter region of a gene involved in the vitamin synthesis. This work showed that naturally occurring epialleles may be responsible for regulation of nutritionally important metabolic QTLs and determination TABLE 1 \| Genes involved in epigenetic mechanisms differentially modulated in Trincadeira grapes infected with the fungus Botrytis cinerea at green hard stage (EL33) and véraison stage (EL35). Details on microarray analysis available in Agudelo-Romero et al. (2015). of agronomic traits (Quadrana et al., 2014). In another study, the complex trait of Energy use efficiency was shown to possess an epigenetic component that is stably inherited, allowing the creation of distinct isogenic sublines that can be used in breeding (Hauben et al., 2009). Thus, induced or natural epigenetic diversity may represent an unexplored resource of phenotypical variations that could be used in plant breeding programs, as recently discussed in Rodríguez López and Wilkinson (2015). ## GRAPEVINE EPIGENOMICS AND EPIGENETICS: A MODEL PLANT FOR PERENNIAL CROP PLANT Studies on Arabidopsis revealed functional aspects of epigenetic regulation of gene expression but present limitations since Arabidopsis has only 5% of methylated cytosine in the genome whereas many crops contain more than 20% (Lee and Kim, 2014). In fact, mutations in epigenetic regulators seem to have a higher impact in crops than in Arabidopsis (Mirouze and Vitte, 2014; Gallusci et al., 2016). In addition, Arabidopsis contains very few transposable elements comparing to crops (reviewed by Lee and Kim, 2014). Polymorphisms in transposon insertions and repeats can originate natural epigenetic variation. Furthermore, while the distribution of the genes along the chromosomes of Arabidopsis is fairly homogeneous, this situation may differ in crops. For example, Vitis vinifera genome is characterized by alternation of large regions with high and low gene density (Jaillon et al., 2007). Several studies have already emerged in crops, in particular, recent analyzes carried out in tomato fruits (Zhong et al., 2013; Liu et al., 2015) constitute a relevant background for studies in grape. It is not yet known whether the epigenetic control of ripening is similar in all fleshy fruits or is limited to the tomato and related wild species (reviewed in Gallusci et al., 2016). Nevertheless, the expression patterns of several genes involved in DNA methylation and histones modifications indicate that epigenetic factors are involved in the onset of véraison in grape and a global decrease in DNA methylation may eventually occur during grape ripening (Fortes et al., 2011) as reported for tomato (Zhong et al., 2013; Liu et al., 2015). In this context, the lack of available mutants in grape constitutes a limitation comparing to tomato. However, studies addressing the methylation status of promoters of genes involved in easily identified traits can shed light on epigenetic regulation of gene expression in grape (Figure 1). Chemical treatments that affect DNA methylation patterns could also be utilized to generate epimutations (Amoah et al., 2012) though they may not be as stable as genetic mutants. Several examples of epimutations in crops are mentioned in the review by Zhang and Hsieh (2013). Epimutagenesis may allow the opportunity to explore allelic variation and novel combinations of alleles without relying upon recombination (Springer, 2013). Analysis of the distribution of epi-marks and DNA methylation in grape in relation with gene expression profiles and fruit quality traits would likely identify epialleles that could be used as important new targets for plant breeding (Figure 1). DNA methylation may generate multiple epialleles with various expression levels, thereby leading to continuous quantitative variation of a trait (Zeng and Cheng, 2014). On the other hand, Kitimu et al. (2015) identified candidate epimarks that distinguish between field cuttings and meristem culture cassava samples. Specific methylation signatures may be used in the future for the diagnosis of somaclonal variants and clonal stocks in grapevine. Grape combines several specific features that could make it an appealing model to study epigenetic regulations in woody perennial plants. It is used as one of the main models for nonclimacteric fruits and also flower development is programmed 1 year in advance; the impact of environmental conditions on flower and subsequently fruit development seems to be in part determined by the environmental conditions the year before. Grape also has specific requirements such as grafting, and clonal propagation. In this context, epigenetic variability could add to the genetic diversity of grape to shape the phenotypic variations observed in this plant. Consistent with this view, clonal diversity within V. vinifera varieties has been distinguished using the methylation-sensitive amplified polymorphism technique, highlighting the usefulness of using epigenetic markers in intravarietal diversity studies (Ocaña et al., 2013). Grafting could also impact the epigenetic state of both rootstocks and shoots (scions), Figure 1. Recently, Lewsey et al. (2015) showed that mobile sRNAs regulate the DNA methylation landscape genome wide, and may be an important mechanism of genome defense in crops. They showed that site-specific transmission of epiallelic states from one accession to another can be achieved by grafting and by de novo methylation of unmethylated DNA, consistent with the idea that some effects of grafting are due to the movement of small RNAs. In grapevine, grafting with rootstocks induced the up-regulation of genes associated with DNA methylation and chromatin modification in the shoot apical meristem (Cookson and Ollat, 2013). Clarifying these mechanisms may open doors to innovative applications to enhance grapevine tolerance to stresses and grape quality. In line with these ideas, the recent analysis of the transcriptomic changes associated with grape infection with the necrotrophic pathogen Botrytis cinerea suggested that epigenetic mechanisms are involved in the reprogramming of fruit defense (Agudelo-Romero et al., 2015). Genes coding for histones, DNA (cytosine-5)-methyltransferase, helicases, DICER and ARGONAUTE proteins were modulated during the infection, whereas those associated with TEs mobility were down-regulated (Table 1). Base-resolution methylomes and high-throughput sRNA profilings are already available in more than 34 species (Niederhuth et al., 2016) including V. vinifera. Comparing the epigenomes of wild and cultivated Vitis species with and without biotic and non-biotic stresses will bring insights on the epigenetic basis of grapevine resistance to adverse conditions with potential impact in breeding strategies. Moreover, epigenetic marks may participate in the priming mechanisms to better withstand biotic and abiotic stresses (Crisp et al., 2016), another topic that deserves attention in order to moderate stress susceptibility and increase climate change resilience in grapevine. Interestingly, these epimarks can also be used in the future for distinguishing agronomic practices and terroir certification of wines. Previously, transgenerational systemic acquired resistance, was demonstrated to be a prominent defense mechanism toward downy mildew pathogen and involves DNA methylation (Luna and Ton, 2012). In grapevine, a further layer of complexity can be added since memory in perennial plants is affected every year in meristems committed to flowering. Furthermore, the reason why epigenetic regulation in response to stress can be transient or transgenerational are not clear (Tricker, 2015). It is also not known the contribution of pathogen-responsive siRNAs in transgenerational immune priming and how they drive the selection of new phenotypes especially in perennial plants. A deeper understanding of the molecular mechanisms involving tissue-specific epigenetic changes underlying genotype × environment interactions may be beneficial for long-term improvement of grapevine performance in less predictable climates with new sources of diseases. In a near future, epigenetic marker-assisted breeding strategies will be applied to select for agronomical desirable epigenetic quantitative traits (Figure 1). Crop improvement via locusspecific epigenetic manipulation has become increasingly feasible with TALE- or CRISPR-based genome editing technologies (Mendenhall et al., 2013; Zhang and Hsieh, 2013). Such technologies can be expected to play an important role in grapevine improvement once transgenesis' protocols are optimized for different cultivars. # AUTHOR CONTRIBUTIONS AF and PG designed the perspective and wrote the manuscript. # FUNDING Funding was provided by the Portuguese Foundation for Science and Technology (SFRH/BPD/100928/2014, FCT Investigator FCT050, PEst-OE/BIA/UI4046/2014). # ACKNOWLEDGMENTS The authors would like to thank Prof. Graham Seymour from the University of Nottingham for carefully reading the manuscript and the COST (European Cooperation in Science and Technology) Action FA1106 "Quality fruit." # REFERENCES fpls-08-00082 February 2, 2017 Time: 16:19 # 7 DNA methylation and suppression of genes controlling stomatal development. J. Exp. Bot. 63, 3799–3814. doi: 10.1093/jxb/ers076 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 Fortes and Gallusci. 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	2025-04-07T03:56:59.292106	18-11-2021 17:15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "008e5806-622f-4253-b2ea-b5ff3bc68b10", "url": "https://www.frontiersin.org/research-topics/4675/omics-and-systems-approaches-in-grapevine-fruit-composition-to-understand-responses-to-environmental", "author": "", "title": "Ome-wide Studies of Grapevine Fruit Composition and Responses to the Environment in the Era of Systems Biology", "publisher": "Frontiers Media SA", "isbn": "9782889632114", "section_idx": 0 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.0	Urban Ecosystem Services • Alessio Russo and Giuseppe T. Cirella	doab	2025-04-07T03:56:59.394774	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 0 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.1	Urban Ecosystem Services Printed Edition of the Special Issue Published in Land Alessio Russo and Giuseppe T. Cirella Edited by www.mdpi.com/journal/land Urban Ecosystem Services	doab	2025-04-07T03:56:59.394868	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 1 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.2	Urban Ecosystem Services Editors Alessio Russo Giuseppe T. Cirella MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Alessio Russo University of Gloucestershire UK Giuseppe T. Cirella University of Gdansk Poland 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 Land (ISSN 2073-445X) (available at: https://www.mdpi.com/journal/land/special issues/Urban Ecosystem Services). 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 978-3-0365-0582-4 (Hbk) ISBN 978-3-0365-0583-1 (PDF) Cover image courtesy of Alessio Russo. © 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	2025-04-07T03:56:59.394912	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 2 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.4	About the Editors Alessio Russo is Senior Lecturer and Academic Course Leader in the Master of Landscape Architecture at the University of Gloucestershire, Cheltenham, United Kingdom. Before joining the University of Gloucestershire, he worked in Russia as an Associate Professor at RUDN University in Moscow and Professor and Head of Laboratory of Urban and Landscape Design at Far Eastern Federal University in Vladivostok. He holds a Bachelor in Science in Plant Production from the University of Naples, Post-Graduate Specialization in Healing Garden Design from the University of Milan, and Master in Science in Landscape Design and Planning from the University of Pisa. He received his Ph.D. in Urban Forestry from the University of Bologna. Outside of academia, Dr. Russo has worked as a Landscape Architect in the United Kingdom, Italy, and the United Arab Emirates, dealing with sustainable design and planning. He is a member of the International Scientific Committee on Cultural Landscapes ICOMOS-IFLA, International Federation of Landscape Architects (IFLA) Advisory Circle, and International Union for Conservation of Nature Commission on Ecosystem Management. Giuseppe T. Cirella is Professor of Human Geography at the Faculty of Economics, University of Gdansk, Sopot, Poland. He received his Ph.D. in Environmental Engineering (specialization: Sustainability) from Griffith University, Australia. He is the founder of the Polo Centre of Sustainability and is Director and Head of Research. He has acted as a principal investigator and coordinator in a number of international projects and is a reviewer and member of the editorial board of several reputed international journals on sustainability and the environment. He has extensive interdisciplinary and cross-cultural experience in socioeconomics as well as expertise in landscape architecture, urban planning, and societal development. #### Preface to "Urban Ecosystem Services" This book is inspired by the two decades of exploratory urban research the two editors have ensued, specifically, sustainability and ecosystem services. In particular, the editors believe that ecosystem services should not be underestimated by those involved in city policy and the design and planning of urban environments. We live in an era of societal and environmental issues and challenges that have shaped the way we interact with nature. Homo sapiens have evolved jestingly into "Homo urbanus", where people live in urbanised areas and can regularly spend up to 90% of their time indoors with potential negative impacts on mental health and wellbeing. There are only nine years left to achieve the United Nations Sustainable Development Goals in which several studies point out that the benefits of incorporating ecosystem services into urban design and planning are fundamental. To this end, this book contains 13 thoroughly refereed contributions published within the Special Issue "Urban Ecosystem Services". The contributions underscore key ecosystem service benefits that complement the nutrient cycle, soil formation, habitat provision and biodiversity—aiding in the provision, regulation and cultural services of the human-nature relationship. Best practices in urban greening interrelates the ecological, technological, societal and economic by combining stakeholder values and opinions into action. This book is intended for students, researchers and practitioners who wish to delve deeper and expand their knowledge with trending state-of-the-art research on urban ecosystem services. It is the first publication in the "Urban Ecosystem Services" series and exemplifies the developing readership and importance of the topic for contemporary urban designers, landscape architects and urban futurists. > Alessio Russo, Giuseppe T. Cirella Editors Editorial	doab	2025-04-07T03:56:59.395017	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 4 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.5	Urban Ecosystem Services: New Findings for Landscape Architects, Urban Planners, and Policymakers *Alessio Russo 1,\ and Giuseppe T. Cirella 2 Received: 11 January 2021; Accepted: 15 January 2021; Published: 19 January 2021 More than half of the world's population lives in urban ecosystems. The United Nations has projected that 28% of people worldwide will be concentrated in cities with at least 1 million inhabitants by 2030 [1]. Worldwide megacities are projected to rise from 33 in 2018 to 43 in 2030 [1]. Urbanisation has a profound impact on how we as human beings interact with the world around us [2]. Cities are often described as new ecosystems that did not have a natural analogue before the expansion of the urban population [3]. Cities themselves are microcosms of the kinds of modifications that are occurring, making them informative test cases for understanding the dynamics of the socioecological system and response to change [4]. The concept of urban ecosystems has been defined as "those in which people live at high densities, and where built structures and infrastructure cover much of the land surface" [5]. However, the ecological interpretation of urban systems must also include less densely populated areas due to reciprocal flows and influences between densely and sporadically populated areas [5]. Research of urban ecosystems is a relatively new topic in ecology that dates back to the mid-1970s [6,7]. This concept is discussed across a wide range of science fields, including urban ecology, landscape ecology, environmental science, ecosystem services science, and public health [8]. The concept is also increasingly addressed in sustainability science, landscape architecture, architecture, engineering, urban design, and urban planning [8]. Nature in cities plays a vital role in urbanised systems as the ecological basis for human–nature relations and the production of urban ecosystem services [9]. Several early landscape architects, particularly Fredrick Law Olmsted (1822–1903), attempted not only to improve the aesthetic of the city but also to improve health and provide the crowded urban population with areas for rest and recreation [9]. Olmsted became a leading landscape architect, park builder, and advocate of the 19th century based on an intuitive understanding of the connection between nature and human wellbeing that underpins what we know today as ecosystem services [10]. Researchers define "urban ecosystem services" as "the benefits urban residents derive from local and regional ecosystem functions" that "are co-produced by people and ecosystems" [8,11–13]. Recently, Tan et al. (2020) examined the terminological grouping of "urban ecosystem services" over the past two decades. Two di fferent—but equally valid—interpretations were disseminated: (1) from analogues of natural and seminatural ecosystems within urban boundaries and (2) a much broader definition that incorporates the former as well as urban city-based services [14]. Landscape architects and urban planners recognise urban ecosystem service as a powerful concept that guides the development of urban landscapes towards greater sustainability, resilience, and liveability [14]. It is also evident that urban ecosystem services contribute to the quality of urban life, even though urban citizens still rely on global ecosystem services to survive [15]. The UN Agenda 2030 clearly considers the role of ecosystem services in urban settings [16]. SDG (Sustainable Development Goal) 15 targets the conservation and restoration of using terrestrial ecosystems, reducing the loss of natural habitats and biodiversity, which play an important part in our common future and heritage. Cities, in particular, need to become "more inclusive, safe, resilient and sustainable", as stated in the title of SDG 11 [17], if they are to become "greener" and supportive of current mass urbanisation. Partial solutions to this phenomenon will be the widespread use of urban green technologies via ecosystem service-based features [16]. A number of studies demonstrate linkages between urban ecosystems and public health through a range of benefits such as the mitigation of heat hazards, improvement of mental health and wellbeing through contact with nature, and stormwater management. [18,19]. In particular, green and blue infrastructures provide several ecosystem services, such as pollution removal, carbon storage and sequestration, food production, noise reduction, and recreational and cultural values [15,20,21] (Figure 1). Figure 1. Ecosystem services provided by green and blue infrastructure: (a) regulation of microclimate, (b) noise reduction, (c) food production, (d) carbon storage and sequestration, (e) habitat provision, (f) run-off retention and water filtration, (g) recreational and cultural values, and (h) air purification (image modified from Macrovector/Freepik). However, changes in ecological conditions resulting from human actions in urban environments ultimately impact human health and wellbeing [22]. Sustainability needs complete understanding at all levels of direct and indirect human interventions affecting ecological processes and ecosystem states [23]. According to Gómez-Baggethun and Barton [24], "urban ecosystems are still an open frontier in ecosystem service research", and the interface between economic costs and sociocultural impacts must be taken into account to "enhance resilience and quality of life in cities". This Special Issue contains 12 peer-reviewed papers. The contributions are written by authors from several countries, including Australia, Chile, Ireland, Italy, Norway, Pakistan, Poland, South Africa, Sweden, the Netherlands, the United Kingdom, and the United States. Brzoska and Spa¯ 'ge [25] reviewed urban ecosystem services of different types of green infrastructure. The review identified 40 different ecosystem service classes assessed in relation to different types of green infrastructure. The results show that the majority of the studies focused on assessing regulation ecosystem services classes such as filtration, sequestration, storage, and accumulation by "microorganisms, algae, plants, and animals" and "regulation of temperature and humidity, including ventilation and transpiration" in "urban green spaces" at the city dimension [25]. The results also show that the number of assessments of provisioning ecosystem services has been increasing over the last 6 years. Only a few studies have considered individual small structures, such as green roofs or single gardens; moreover, green spaces are often aggregated into one object of investigation, mostly city-wide, leading to oversimplifications [25]. Colding et al. [26] focused on the incremental change in green spaces—a fate that is largely undetectable for urban residents. The research illustrates the set of drivers resulting in the subtle loss of urban green space and elaborates on the consequences of this for the resilience planning of ecosystem services [26]. Semeraro et al. [27] analysed a case study in Lecce, Italy, applying a top-down and bottom-up approach to dispute resolution at the institutional level in the use of urban space. The research suggests that in the socioecological system, the bottom institutional level can introduce innovation or a new vision in the use of free urban space, and, as a result, bottom-up participation can stimulate or trigger the evolution of the urban ecosystem, while the top institutional level drives a change from top-down to bottom-up participation information in planning actions between decision-makers [27]. Giliani et al. [28] studied the dynamics of urban landscape ecology in the Islamabad Capital Territory in Pakistan during the period of 1976–2016 [28]. The outcomes of their study show a consistent increase in the settlement class, with the highest annual rate of 8.79% during the period of 2000–2010. Tree cover >40% and <40% canopies decreased at annual rates of 0.81% and 0.77%, respectively, between 1976 and 2016. Forest fragmentation analysis indicates that core forests of >500 acres decreased from 392 (i.e., 65.41%) to 241 km<sup>2</sup> (i.e., 55%) and patch forest increased from 15 (i.e., 2.46%) to 20 km<sup>2</sup> (i.e., 4.54%) from 1976 to 2016. SDG indicator 11.3.1 land consumption rate to the population growth rate ratio was 0.62 from 1976 to 2000, increasing to 1.36 from 2000 to 2016 [28]. Holloway and Field [29] composed richness and abundance data for 771 quadrats across three counties, finding a total of 81 species, with 48 species on the groynes and 71 species on the natural rocky shores. Their research found similar degrees across structures for algae, higher diversity and abundance for lichens and mobile animals on natural shores, and higher numbers of sessile animals on groynes. The study points out that groynes host similar ecological communities to those found on natural shores, in which differences do exist, particularly with respect to rock-pool habitats [29]. Combrinck et al. [30] investigated the property values and distance to urban green space in Potchefstroom, South Africa. Potchefstroom residents recognise the social, environmental, and economic value of green spaces; however, fewer residents recognise the economic value of green spaces. Over half of the respondents agreed that green spaces are perceived as crime hotspots and thus as contributing to unsafe neighbourhoods and indicating a related ecosystem disservice. Approximately 60% of those surveyed agreed that they would pay more for a property that is located next to a green space. Professional planners that had been surveyed agreed that unattractive green spaces are due to a lack of maintenance by local authorities and a lack of community engagement. Half of the planners involved in the survey stated that environmental considerations are not prioritised in the planning process, even though environmental managemen<sup>t</sup> is considered a critical component of local urban planning approaches, policy, and legislative frameworks [30]. Parker and Simpson [31] developed a theoretical framework to support human–nature connections and urban resilience via green infrastructure. In particular, they explored how urban resilience theory and human–nature connection theory can inform urban development. The urban resilience theory advocates the improvement of policy and planning frameworks, risk reduction techniques, adaptation strategies, disaster recovery mechanisms, environmentally sustainable alternatives to fossil fuel energy, the building of social capital, and integration of ecologically sustainable urban green infrastructure [31]. In Sweden, to identify key factors for fostering the incorporation of ecosystem services into municipal planning practise, Khoshkar et al. [32] examined and evaluated the views and experiences of practitioners of local spatial planning practises in municipalities in Stockholm County [32]. The practitioners stressed the need to establish legal support and ecosystem services regulation at Swedish and EU policy levels. Moreover, focus was placed on the need for local capacity building and awareness of ecosystem services as well as increased regional support for enhancing local information sharing and learning. In order to fully integrate ecosystem services in urban planning for sustainable development in a decentralised local governmen<sup>t</sup> structure, such as in Sweden, locally adapted practical tools and monitoring procedures were considered important [32]. In Poland, R ˛edzi ´nska and Piotrkowska [33] developed a procedure of building neighbourhood resilience to climate threats, embedded in the planning and design process, and focused on the use of natural adaptive potential. This procedure was applied at the strategic level to the city of Warsaw by drawing up the ranking of districts with a view to prioritising adaptation measures based on climate threats, demographic vulnerability, and the assessment of the potential of Warsaw's green infrastructure [33]. In Oregon, Elderbrock et al. [34] developed a clear method for deciding between alternatives to green infrastructure based on their quantitative and qualitative potential to provide high-priority urban ecosystem services to di fferent stakeholders in a specific area. They used and evaluated this method in Eugene by investigating the potential for increased urban ecosystem services resulting from the conversion of public grass to alternative planting regimes that align with expressed stakeholder priorities [34]. In Norway, Venvik and Boogaard [35] tested the hydraulic infiltration capacity of a rain garden. This research leads to an understanding of the dynamics of infiltration systems, including how a rain garden interacts with the hydrological and hydrogeological aspects of the local urban water cycle [35]. Finally, the contribution from Restemeyer and Boogaard [36] explored how online citizen science platforms, demonstrated by the case of ClimateScan, can stimulate stakeholder participation and encourage nature-based solutions. This has culminated in an illustrated map of over 5000 nature-based solutions initiatives around the world and an average of more than 100 visitors a day within 6 years [36]. As cities are expected to grow rapidly in the coming decades, it is important that urban ecosystem services are understood and valued by city planners and policymakers [15]. We do hope that the results of this Special Issue can be used by landscape architects, urban planners, and policymakers to make cities sustainable, safer, resilient, and adaptable to climate change and other future risks. The survival and wellbeing of the human species in urban environments depend on how we manage to provide ecosystem services for future generations. Author Contributions: Writing—original draft preparation, A.R.; writing—review and editing, A.R. and G.T.C.; visualization, A.R. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments:** We are grateful to the MDPI Land team of academic editors and reviewers for assisting with the Special Issue's academic excellence. Vector imaging was designed by Macrovector/Freepik. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.395314	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 5 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.7	1. Introduction Today, more than half the world's population is living in cities [1]. The continuous growth in urban populations combined with a more extreme urban climate due to global warming are having a detrimental impact on urban ecosystems [2]. In order to maintain the quality of life for the burgeoning numbers of urban residents, it is becoming increasingly important to protect and promote urban ecosystems and their services [3–6]. Concepts such as green and blue infrastructure have been developed in recent years to help tackle the environmental challenges of cities. The strategic planning of urban green structures improves the well-being of inhabitants while simultaneously boosting the resilience of cities to climatic changes [7]. Yet, such strategic planning requires comprehensive insights and information on the multiple functions and services of green infrastructure on different spatial scales. In particular, knowledge and expertise are needed on ecosystem services (ES) on the small spatial scales where planned measures are realized [8–12]. More research into urban ES on small spatial scales will improve our understanding of this planning factor, thereby aiding the integration of the urban ES concept into urban planning as an important factor for sustainable urban development. The correct application of this concept has the potential to better exploit the multiple benefits of urban ecosystems, so that urban planning can be more closely oriented to natural conditions and resources [13–15]. Furthermore, the development of standards and indicators to assess and describe ES in urban contexts can help politicians, urban planners as well as practitioners create ecological and sustainable cities [16]. While we can already point to a few practical examples of the successful integration of ES-related subjects into diverse planning documents and tools, there are still several unresolved problems limiting a more general implementation [17]. One limiting factor is the lack of information on urban ecosystems on di fferent spatial scales and their services. The poor quality of available spatial or other relevant data on small, local scales often complicates the integration of ES into planning frameworks or decision-making processes [13–20]. A further limitation is the lack of suitable methods to assess ES at such spatial scales; hitherto, most assessment methods have referred to the global, national or regional scale. Clearly, if we wish to promote the inclusion of ES in decision making at the urban level, it is necessary to improve our knowledge of this subject at city-wide but most importantly also on local, and thus site-, scales [9,19]. Over the last few decades, urban ES has become a widely investigated topic in di fferent research fields, with scholars recognizing its importance in mitigating climatic extremes and contributing in diverse ways to human well-being [19,21]. In this study, urban ecosystems are defined as areas largely dominated by the built environment and which comprise gray and green infrastructures [4,22,23]. Of course, urban ecosystems only provide a fraction of the ES used by city dwellers – the larger part of these services are provided by widely distributed ecosystems in the city surroundings. Yet in relation to the size of urban ecosystems, they benefit a large number of citizens [24]. Thus, urban ES have a high anthropogenic impact, representing an explicit type of ES that needs to be considered more closely. Locally provided ES generally play an important role in promoting the quality of life of urban residents. Yet, the issue of the ES of urban small-scale structures is an underrepresented research field [14]. The small spaces within cities are designed by urban planners in grea<sup>t</sup> detail, and it is exactly this spatial scale and structure that is directly perceived by residents and thus strongly influences the quality of life in the city. Previous studies on the assessment of urban ES have stressed the importance of the spatial scale of investigations [14,25,26]. Hitherto, many assessments have been conducted on larger spatial scales (city, region, nation-wide) with results often presented in a generalized way. To obtain more realistic results, it is necessary to conduct empirical ES studies of smaller urban structures. In order to ensure the practical implementation of the ES concept, we have to focus on spatial structures and scales that are recognized by existing planning tools, e.g., neighborhoods, small single parks, etc. [26]. Furthermore, previous reviews have revealed a large number of di fferent methods used to assess urban ES [14,26,27]. Most of these involve spatial proxy methods, for example, utilizing land use and land cover data to estimate ES supply capacities. Primary data is rarely collected in urban ES assessments [27]. Another approach to the assessment of ES is to consider the complexity of urban structures [14,26]. In this case, it is important to take account not only of built structures but also urban open spaces, for example, the various types of green open space [14,26]. In particular, Haase et al. [14] found that most previous studies assessed regulative ES in cities, with only a few looking at cultural and provisioning ES. These aspects determine the scope of this review and shape the key questions. We aim to review the current state of knowledge on methods to assess the urban ES of di fferent types of green infrastructures from city to site scales. To this end, we have only considered studies that examine individual spatial structures or forms of land use in cities such as parks, gardens or trees. The review will answer the following three questions: (1) Which urban ES are assessed in relation to green infrastructure types? (2) Which specific spatial structures are the subjects of investigation? (3) Which methods are used to assess ES on larger (city) and smaller (site) spatial scales? Furthermore, we will look at the motivations of studies in assessing the urban ES of di fferent types of green infrastructure types, as well as check which data type (i.e., primary or secondary) has been used by the reviewed studies. #### 2. Materials and Methods #### 2.1. Review Approach The first step was to carry out a systematic quantitative literature review after Pickering and Byrne [28]. In comparison to classical meta-analytical reviews, the methodology after Pickering and Byrne [28] aims to determine general aspects of studies (e.g., numbers, types, and geographical aspects), research trends and gaps as well as methodological patterns. To this end, the literature databases "Web of Science" and "Scopus" were searched for relevant peer-reviewed articles published in international scientific journals. This search was conducted from March to April 2019. Several filter criteria were applied to specify the review but still to identify as many relevant articles as possible: Systematic searches were conducted of the database "Web of Science" for each possible urban green infrastructure type (see Table 1), adding the search terms "urban ecosystem services" and "assessment" or "valuation" (for example, "urban ecosystem services" AND assessment AND park). After completing individual searches for various spatial scales (see examples in the "Scopus" search terms), all results were cross-checked to exclude repeated articles. The search procedure of the database "Web of Science" identified a total of 35 studies. Table 1. Range and definitions of investigated spatial objects. In the database "Scopus", a general search was conducted for all possible green infrastructure types using the following keyword combination: TS = (neighborhood\* OR district\* OR estate\* OR meadow\* OR brownfield\* OR allotment\* OR "community garden\" OR park\ OR woodland\* OR "green space\" OR "green infrastructure" OR residential OR cemeter\ OR wetland\* OR "urban tree\" OR "urban forest\" OR lake\* OR waterbod\* OR river\* OR stream\) AND ("urban ecosystem service\" AND assessment OR valuation). This search identified a total of 31 papers. After cross-checking and combining the results from both databases, two articles were excluded, resulting in a selection of 29 articles. Further articles could be added to this list by screening the bibliographies. This procedure led to a final total of 63 scientific articles. The second step of the review approach was to search the relevant gray literature, such as reports and documents that were compiled by organizations and institutions that do not belong to the "traditional" academic instances (e.g., governmen<sup>t</sup> departments, non-governmental organizations or civil society). For this purpose, an internet search was conducted to identify some initial potential international funding bodies of projects on urban ecosystems across Europe. Their webpages were then screened for relevant projects, after which the project webpages were studied. This search procedure led to a snowball effect, resulting in the identification of 13 relevant project documents, of which six were additional scientific articles drawn from the bibliographies of the project documents. From these two review steps, we were able to identify a total of 76 articles. An overview of the assessed studies is presented in Supplementary Materials (S1). Each publication was analyzed and added in an Excel databank, where specific information was extracted and combined in one table. #### 2.2. Analysis Approach of Included Articles The authors of the various articles made use of a range of different terms and expressions for ES. For the purposes of our study, it was first necessary to consolidate these terms to allow us to summarize and compare the investigated ES (cf. key question 1). For this reason, all identified ES from the articles were classified into the corresponding ES sections, groups and classes of the latest version of the Common International Classification of Ecosystem Services (CICES V 5.1). To provide a comprehensive overview of the investigated differently scaled green infrastructure types (cf. key question 2), the spatial objects in the papers were first assigned to one of two dimensions, i.e,. city- or site-dimension (see Figure 1). This classification was intended to reflect the scope of the investigated objects in each study. Thus, whenever an assessment method was applied to several spatially distributed (yet urban) objects, the dimension of this study was classified as "city" (e.g., ES assessment of various urban parks in a city). Alternatively, if only one object was assessed, e.g., a city park, the dimension was defined as "site". In this case, only situation and location-specific conditions can be said to apply. Figure 1. Schematic illustration of the assignment of studies to city- or site-dimension. Following this initial classification into city- or site-dimension, the urban green infrastructure types in each dimension were summarized and classified into more precise types, e.g., park, garden, forest, etc. An overview of this classificatory system is given in Table 1. Furthermore, we identified seven di fferent categories for the method classification of all reviewed articles (cf. key question 3). These categories are as follows: "spatial proxy methods", "samplings/field mapping and observations", "surveys and questionnaires", "economic valuation methods", "model-based methods", "social media-based methods" and "remote sensing and earth observations" (see Supplementary Materials (S2) for a detailed description of the categories). On the basis of the described classifications, the extracted information from the 76 reviewed articles was then evaluated to answer the key questions.	doab	2025-04-07T03:56:59.396408	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 7 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.8	3. Results #### 3.1. General Overview of Articles In Figure 2, we can see, firstly, that most of the articles focus on European cities, and, secondly, that research on the assessment of the urban ES of di fferently spatial-scaled green infrastructure types is growing. Although the databases were screened for published articles from the year 2000, published articles corresponding to the described search criteria were only identified from 2008. Figure 2. Numbers and geographical distribution of case studies in the articles published between 2008 and April 2019. The figures in brackets beside each year are the total number of articles published in that year; the total numbers of articles from each continent are also indicated. A polynomial trend line illustrates a predicted increase of the number of articles. (Note: One study is excluded due to its analysis of a fictional urban model). There is a clear rise in the number of studies assessing the urban ES of green infrastructure types on di fferent spatial scales, especially over the last two years. The first paper to o ffer a smaller-scale assessment of explicitly urban ES was published in 2008. While the highest number of relevant publications was identified in the year 2018, by April 2019 more than 50% of the total number of articles from 2018 had already been published. The polynomial trend line also draws a predicted increase in the number of articles in this field (see Figure 2). Most of the reviewed publications focus on green infrastructures within European cities (55); a much smaller number deal with cities in Asia (8) or North (7) and South America (4). Only one publication dealt with the assessment of urban ES in an African city, specifically Cape Town (found in the gray literature document TEEB (The Economics of Ecosystems and Biodiversity) [29], referring to [30,31]). The scientific articles were sourced from 32 di fferent journals in diverse fields, such as ecosystem services, environmental science, ecology and urban planning. #### 3.2. Assessed Es of Urban Green Infrastructure Types In total, we identified 40 different ES classes assessed in regard to different green infrastructure types (Table 2, for detailed ES class overview see Supplementary Materials (S3)). The ES section Regulation & Maintenance according to CICES V5.1 specifies 16 different ES classes. The ES section Provisioning examines 12 different ES classes. A total of nine different ES classes were studied within the Cultural ES section. Table 2. Overview of assessed ecosystem services (ES) sections and classes (numbers in brackets) according to CICES V5.1 in regard to urban green infrastructures. With a closer look at the analyzed ES classes (see Supplementary Materials (S3)) we can see the varying frequency of the examined individual ES classes. The most frequently assessed ES classes were "Filtration/sequestration/storage/accumulation by microorganisms, algae, plants, and animals" (2.1.1.2) and "Regulation of temperature and humidity, including ventilation and transpiration" (2.2.6.2). In general, most studies focused on an assessment of ES classes within the ES section Regulation & Maintenance (a total of 177 instances, see Table 2). The ES classes within the Cultural ES section were the second most frequently examined. In total, ES classes were assessed 115 times in this section (see Table 2). Specifically, the most frequently assessed classes were "Characteristics of living systems that enable activities promoting health, recuperation or enjoyment through active or immersive interactions" (3.1.1.1) and "Characteristics of living systems that enable activities promoting health, recuperation or enjoyment through passive or observational interactions" (3.1.1.2). ES were assessed 37 times in the Provisioning ES section (see Table 2). This represents the most rarely assessed ES for urban green infrastructure types. #### 3.3. Investigated Green Infrastructure Types and Dimensions Figure 3 shows the number of investigated green infrastructure types at city- and site-dimensions in the reviewed publications. In general, the articles investigated a wide range of green infrastructure types. It can be seen that the majority of the studies analyzed ES at city-dimension (Figure 3, left side), especially in regard to "urban green spaces" and "structural types" with a much smaller number of studies analyzing single structures in cities (Figure 3, right side). The two dimensions show a similar number of assessments of "parks". Regarding the assessment of ES for green infrastructure types (Table 3), our results show that most of the studies concentrated on assessing the Regulation ES classes "Filtration/sequestration/ storage/accumulation by microorganisms, algae, plants, and animals" (2.1.1.2) and "Regulation of temperature and humidity, including ventilation and transpiration" (2.2.6.2) in "urban green spaces" at city-dimension. This spatial structure type was also the focus of a large number of assessments of the cultural ES classes "Characteristics of living systems that enable activities promoting health, recuperation or enjoyment through active or immersive interactions" (3.1.1.1) and "Characteristics of living systems that enable activities promoting health, recuperation or enjoyment through passive or observational interactions" (3.1.1.2). Within the site-dimension, the ES class "Filtration/sequestration/storage/accumulation by microorganisms, algae, plants, and animals" (2.1.1.2) was most frequently assessed in "neighborhoods" and "parks". Table 3. Assessed ES classes according to CICES V5.1 in relation to the considered green infrastructure types. The figures in brackets indicate how often an ES was assessed for the corresponding spatial structure. Since multiple ES were analyzed in some articles, the sum in brackets is higher and does not represent the total number of articles reviewed. (Note: The specification of the considered ES class is missing in some articles; these were assigned to the ES section "in general, specifications missing" in each ES section.) Table 3. Cont. #### Land 2020, 9, 150 Table 3. Cont. Figure 3. Urban green infrastructure types investigated by the articles, subdivided into city- (left) and site-dimension (right). #### 3.4. Methods Used to Assess the Urban Es of Green Infrastructure Types The majority of the reviewed publications applied "spatial proxy methods" followed by "surveys and questionnaires" (see Figure 4). "Social media-based methods" were the least commonly used method for ES assessment for different green infrastructure types in cities. Our analysis showed that over a quarter of the studies used more than one method to assess urban ES. "Surveys and questionnaires" and "model-based methods" were most frequently combined, followed by "spatial proxy methods" and "model-based methods". Figure 4. Numbers (and % of total share) of methods used in the reviewed articles. Table 4 categorizes the different methods used to assess urban green infrastructure types. At city-dimension, the reviewed studies most frequently applied "spatial proxy methods" and "surveys and questionnaires". The ES of "urban green spaces" were the most studied in this dimension by applying methods that belonged to the categories "spatial proxy methods", "surveys and questionnaires" and "model-based methods". "Structural types" and "trees" were also the focus of many assessments of urban ES. Here, the most common methods were "spatial proxy methods" and "model-based methods". At site-dimension, the most frequently used method was "surveys and questionnaires", largely to assess the urban ES of "parks". Table 4. Numbers of methods identified in the reviewed articles to assess green infrastructure types. The figures in brackets show how often a method was applied to the corresponding structure type. Since multiple methods were used in some articles, the sum of methods is higher than the actual number of articles.	doab	2025-04-07T03:56:59.397198	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 8 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.9	4. Discussion The results show a rise in research interest in this area. In particular, the growing numbers of articles, especially since the year 2018, as well as the trend line confirm the increasing significance of this topic (see Figure 2). Already in the first four months of 2019, more than 50% of the previous year's total number of publications on the investigated topic had been published. The review still confirms the finding of Haase et al. [14] that most studies are carried out in European cities. Due to the higher numbers of publications in the last two years, it can be assumed that the topic is still evolving as a research field and that more studies will be published in the future. Referring to key question (1), we found that Provisioning ES are the least assessed ES section for urban green infrastructure types (see Table 1). This also tallies with the finding of Haase et al. [14]. Unlike the ES sections Regulation & Maintenance and Cultural, Provisioning ES can generally be physically transported (e.g., deliveries of foodstuffs to stores). For this reason, cities mainly import provisioning ES from the surrounding countryside and even further afield. The demand for provisioning ES, however, is very high in densely populated areas. Our review indicates that the number of assessments of provisioning ES has been increasing over the last six years. This trend can perhaps be attributed to new initiatives fostering urban agriculture such as urban gardening, Edible Cities, etc. In today's society, we can identify changes that are serving to highlight the issue of Edible Cities or the role of urban gardening [32]. In the foreseeable future, provisioning ES could become an important element of the urban environment. Moreover, with regard to promoting the sustainable development of cities and the reduction in urban ecological footprints, Gómez Baggethun and Barton [13] have pointed out that ES should not be imported from distant locations. Concurring with the review by Haase et al. in 2014 [14], Regulation & Maintenance were the most frequent forms of ES investigated in the publications (see Table 1). This ES section contains a number of ES classes that play a special role in securing the well-being of inhabitants, although they are generally only perceived when lacking. Such ES are mostly used indirectly and are in grea<sup>t</sup> demand [33]. One reason for the high frequency of these ES assessments may be the current lively debates on climate change and its impact on cities. Cities are facing increasing environmental pressure as well as concerns about human health and the well-being of local residents [34]. Regulation and Maintenance ES can have a considerable impact on human well-being by regulating the microclimate, air pollution or water flows [35]. There is an increasing demand on cities to plan and develop important green structures to counteract the negative impacts of heat islands, heavy rainfall, air pollution, etc. As we also found in our review, more and more research has been conducted over the last few years into these ES classes in urban areas [14]. Our results show that the Cultural ES section is the second most investigated in the reviewed publications (see Table 1). This fact is unsurprising if we remember that the Cultural section contains ES classes, which are commonly referred to as "recreation services". Other studies have shown that ES classes related to recreational aspects (such as 3.1.1.1 and 3.1.1.2) are the most frequently investigated cultural ES within urban areas and especially within urban green spaces or parks, where this ES is mostly directly used [14,36]. Other publications have considered some less obvious small green infrastructure types; Mathey et al. [37] and Pue ffel et al. [38], for instance, have conducted surveys on the recreational ES provided by urban brownfields (Table 2). Contrary to the findings of Haase et al. [14], our results show that many studies assess multiple ES. Those studies that evaluate only one class of ES, such as Takács et al. [39], Marando et al. [40] or Lehmann et al. [41], generally deal with Regulating & Maintenance ES. A closer look at these studies shows that they are largely based on primary data drawn from on-site inspections and include biotope mapping or climatic field measurements. In regard to key question (2), our findings show that previous ES assessments have looked at a wide variety of urban green infrastructure types at larger and smaller spatial scales. The majority of examined urban structures considered the city-dimension (Figure 3). Taking a closer look at the studies dealing with site-dimension, we found that many applied time- and cost-intensive methods, in particular self-collected (primary) datasets (e.g., [42–44]). Consequently, the small number of studies at the site-dimension can be attributed to the requirement for more precise and site-dependent data, usually primary data collected in the field. The high number of publications that assessed ES in "urban green spaces" and "structural types" (Figure 3) can be explained by the aggregation of di fferent spatial structures, in particular treating diverse green spaces as one undi fferentiated study object (e.g., [42,45,46]). On the other hand, this frequency can also be attributed to the data used in the individual articles. Many of the articles took land-use data as a basis for their assessments, resulting in an investigation of di fferent land-use types that usually exist in aggregated classes (e.g., green spaces, streets, buildings, etc.). In addition to this, the frequently examined green infrastructure type "parks" (Figure 3) is one of those structures mostly associated with the urban context; we can thus expect a large number of investigations in such spaces. Many diverse analyses can be carried out in parks, which can also be investigated in regard to direct usage by local residents, especially within the section of Cultural ES. In contrast to parks, for example, which are present in almost all cities, the smaller number of ES assessments of green infrastructure types, such as "forest" or "green roof/wall" (Figure 3), can be attributed to their rarity in many cities. Consequently, ES assessments of such green infrastructure types are less frequent. Based on this review, we identified four main motivations for assessing the ES of urban green infrastructure types on di fferent scales. Most studies based their investigations on the need to successfully implement ES in urban planning and for the development of appropriate measures to preserve and protect these ES (e.g., [46–48]). Others, such as Czembrowski et al. [49], justified their investigations by pointing out the services and value of urban green areas as well as the need to facilitate communication between environmental experts and decision makers. Another stated intention in assessing ES is to ensure the inclusion of user demands in the planning and design of cities and green spaces [33]. Studies such as Mathey et al. [37] aimed to increase the acceptance of less popular green structures such as brownfields by the public as well as by urban planners. This is achieved by highlighting the values of associated ES and striving to prevent the automatic redevelopment of urban brownfields, instead maintaining these as areas of green space. In a large number of publications, green spaces were generally considered in an aggregated form and thus examined in a larger scale study, i.e., defined as "urban green structures" according to our classification (Figure 2). For such green spaces, the studies particularly investigated the Regulative ES. On the other hand, studies examining single and smaller sites (site-dimension) most often focused on spatial structures that have a stronger impact at small-scale levels, such as the usually high perception and significance of parks within a district. While previous studies have particularly highlighted economic valuation methods to assess ecosystem services (e.g., [50,51]), we only identified a small number of articles applying "economic valuation methods" (Figure 4). Regarding key question (3), the most frequently used approach is the "spatial proxy method". This method is particularly applied to the assessment of ES in regard to "structural types", "urban green spaces" as well as "trees". As in the previous section, this observation can be explained by the adopted base data. In most cases, the land-use types of a study area provide the basis for the application of di fferent proxies, as performed in the studies of Speak et al. [52] and Kremer et al. [53], for example. Such studies used proxy variables such as land cover maps and other geoinformation system (GIS) datasets that depict special ecosystem processes as a basis for ES assessments. One reason for this high number of studies applying spatial proxy methods can be limited time and financial resources available to researchers as well as a lack of primary data. However, spatial proxy methods have been criticized for the overgeneralization of obtained results that represent only a fraction of reality [54]. On the other hand, they allow for the investigation of ES in study regions that lack primary data while o ffering the advantage of good comparability of results and generally low costs [54]. Only a few of the reviewed studies applied "social media-based methods" (Figure 4). The inclusion of "modern" (in particular social media-based) datasets such as photographs in Richards and Tunçer [55] is still an under-researched and rather new field in regard to ES assessments. In the near future, this method could potentially become more popular due to the continuous expansion of social media and uptake of mobile technology by the public as well as for research purposes (e.g., citizen science). However, there still exist several limitations in the use of such social-media-based datasets and methods. For example, it can be argued that the real world is not being accurately represented as data generation is currently mainly carried out by young people [55]. The finding that many studies use a mix of methods (Figure 4) agrees with previous work by Seppelt et al. [27]. "Model-based methods" such as "I-tree Eco", a free peer-reviewed software to assess the ES of trees provided by the USDA Forest Service, were applied by some studies, especially for the assessment of Regulating and Maintenance ES. In these cases, the particular issue of interest was indeed "trees", such as in the studies of Parsa et al. [56] and Baró et al. [57]. These models use primary data (in these cases, tree inventories) and apply benefit transfers under location-dependent variables such as climatic parameters. This can also limit the accuracy of results. Therefore, while such investigations can be compared in their underlying methodology, at the same time, they are based on generalizations, especially with regard to ecological parameters, rendering them di fficult to transfer to other geographic patterns. Although the popular "I-tree Eco" model also provides results expressed in monetary values and thus integrates economic valuation methods, it is surprising, that most of the reviewed studies using this model are limited to the presentation of biophysical results [35,52,58–60]. This results in a lower number for "economic valuation methods" in Figure 4. Only a few studies also discuss the economic results provided by "I-tree Eco" [56,57,61]. Regarding our research design, it should be noted that the criteria used in this study placed restrictions on the literature review and thus led to a smaller sample size. In an initial search, in which we searched for the term "ecosystem services" instead of "urban ecosystem services", we received a high amount of results. After reviewing some of the results, we found that a large part did not match our search scope. Therefore, we decided to narrow the search terms to explicitly "urban ecosystem services" in order to reduce the large number of non-relevant articles identified after the initial scan of the databases. We are aware that our narrow selection meant that some potentially relevant studies, which assessed ES in cities but did not explicitly use the term "urban ecosystem services" (in article titles, keywords or abstracts), could not be included. Nevertheless, all studies that refer to "urban ecosystem services" are included in this review. A further restriction results from only searching for the keyword "green infrastructure" (apart from the di fferent green and blue elements) and with this not explicitly integrating other planning concept terms that are sometimes synonymously used especially in the context of regulative ES, e.g., Sustainable Drainage Systems (SuDS), Low Impact Development (LID), or the term Nature-Based Solutions (NBS). An investigation of the underrepresented blue elements and the inclusion of the mentioned concepts represent an interesting field for further research. In this context, it can also be expected that the results are increasingly reflecting studies from European cities, as the term "green infrastructure" is commonly used within the EU member states.	doab	2025-04-07T03:56:59.397723	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 9 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.10	5. Conclusions While the importance of ecosystem services is today widely acknowledged, our results also show that there is still a gap in assessment methods on urban local scales [14,19,27]. The aim of our review was to raise awareness of this issue and provide a framework for further research. Previous reviews on the assessment of urban ES have mainly focused on larger spatial scales within cities. Our findings follow on from here, giving an update as well as checking whether any additional studies have examined even smaller spatial structures in the context of urban ES assessments (such as "green roofs", small garden patches, individual trees, etc.). Our results show that a research gap still exists at this point. The significance of urban ES is readily acknowledged by scientists, and first steps have already been undertaken to adapt this concept for policymaking [17,62]. The high numbers of studies in the last years indicate that this topic is still evolving, especially in Europe. Several published reports have acknowledged that Europe, North America and northeast Asia are the main centers of research into the interrelationship between human well-being and green spaces [34,63,64]. In recent years, multiple ES classes have been investigated in cities as well as in their local spatial structures. Since the review by Haase et al. [14], more studies have been published on ES on the level of small urban structures; yet, the majority of work still focuses on larger spatial structures, mostly applying generalizing methods that provide results with a poor fit to reality [54]. Our results have shown a distinction between the numbers of ES assessments at city- and site-dimension. For a more rigorous implementation of the ES concept in urban planning as well as to develop adapted measures and design clearly additional research is needed, especially in evaluation methods on local urban scales. Improving our understanding of the ecosystem services provided by site-scaled green infrastructure types will constitute an important step towards setting policy objectives and creating suitable measures for sustainable urban development [9,19]. Approaches are needed that require data gathering [19], as these lead to credible and more realistic assessments of urban ES [54]. Furthermore, Beichler et al. [25] have stressed the importance of spatial scale in ES assessments, arguing that the exclusion of settlements and built-up areas from investigations can cause us to overlook ES provided by small ecosystems within such structures. In the urban context, results from spatially comprehensive (natural and built-up areas) approaches ultimately form more convincing arguments for the ecological and sustainable design of future cities with their small green structures and, in this way, could usefully influence the decision-making processes. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-445X/9/5/150/s1, S1. Overview of the studies assessed in this review sorted according to reading order; S2. Short descriptions of method categories; S3. Overview of assessed ES sections and classes according to CICES V5.1 in regard to urban green infrastructures. Author Contributions: P.B. designed the study and conceptualized the methodology. Both P.B. and A.S. conducted the literature search and reviewed the articles. P.B. performed the analysis, visualized the data and wrote the manuscript in consultation with A.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was mainly funded by the Federal Ministry of Education and Research (BMBF) in the joint project "HeatResilientCity" (subproject gran<sup>t</sup> number: 01LR1724A). The promoter of this project is the DLR project managemen<sup>t</sup> agency (DLR-PT). Acknowledgments: The authors would like to thank Derek Henderson for language editing and proofreading. We also thank the three anonymous reviewers for their very constructive comments and valuable hints. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.398626	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 10 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.12	1. Introduction Preserving greenspace quantity and quality in the face of increasing urbanization is a pressing global challenge [1]. Greenspaces provide invaluable ecosystem services to humans that are important to plan for in cities [2]. Economic motives and urban neoliberal policies are liable explanations behind the loss of public space in many cities [3,4]. Public greenspace is an important component of public space [5] and could be defined as "any vegetation found in the urban environment, including parks, open spaces, residential gardens, or street trees" [6] (p. 113). Here, by public space is meant spaces in cities that are "owned by the government, accessible to everyone without restriction, and/or fosters communication and interaction" [7] (p. 9). This definition encapsulates Louis Wirth's notion of urbanism [8], taking into account how individuals interact with one another and with spaces. Public spaces facilitate human exchange and interaction, as in the form of urban squares and market places that traditionally have served as arenas for public communication and social interaction [9]. However, there is a massive shift towards the privatization of public land and resources in many cities today [3,10], a ffecting green spaces in a multitude of ways with repercussion for long-term managemen<sup>t</sup> of ecosystem services. While urbanization causes the direct loss of urban greenspace, comprising habitat fragmentation that involves both loss and/or the breaking apart of habitats [11], there exist more subtle forms of greenspace loss that ultimately are linked to property-rights arrangements. We here refer to such loss as incremental, occurring over a series of gradual declines or small steps but experienced at the cognitive level of urban space as 'baseline shifts' among urban residents. Based on the literature related to institutions, economic geography, urban ecology, and social theory, we present a set of subtle drivers for why public green spaces gradually erode in cities. This gradual erosion, we argue, is often "invisible" in that it can almost exclusively be revealed by high spatial resolution remote sensing data [12] and that their e ffect can be translated into high social-ecological<sup>1</sup> costs that impinge negatively on human wellbeing [13,14]. Based on a set of examples of incremental greenspace change, this paper briefly discusses how urban planning authorities should avoid 'day-to-day planning' [15] and be more long-term oriented to meet an ever-increasing unpredictable future. Property rights theory in relation to natural resource managemen<sup>t</sup> emphasizes institutions at the interface between social and natural systems [16,17], where the term 'institutions' signifies the rules and conventions of society that facilitate coordination among people regarding their behavior [18]. At a more general level, institutions are made up of formal constraints (rules, laws, constitutions), informal constraints (norms of behavior, conventions, and self-imposed codes of conduct), and their enforcement characteristics; thus, they shape incentives in human exchange, whether political, social, or economic [18]. Ecologically oriented scholars define institutions simply as working-rules or rules-in-use, meaning "the set of rules actually used by a set of individuals to organize repetitive activities" [19] (p. 19). Thus, property rights link people to nature, and have the potential to coordinate the social and natural systems in a complementary way for both ecological and human long-term objectives [20]. Property-rights could also be viewed as slow variables in urban transformation; however, their monoculturalization in favor of urban privatization schemes may gradually erode urban resilience (i.e., 'bu ffering capacity' to deal with disturbance and novel events) and make planning bodies and local authorities less pertinent to propel urban growth along more sustainable trajectories that value ecosystem services as risk insurance and as adaptive capacity for responding to known and unknown disturbances. The paper concludes by proposing common property systems as a viable alternative for local governments to survive economic disruptions and in turning public spaces into places that urban residents themselves can manage for improving and protecting greenspace and associated ecosystem services. #### 2. Urban Green Space Dynamics and Reasons for Their Incremental Demise The availability of public green spaces is foremost linked to the geographical location of a city [21]. However, urban expansion in many cities takes place almost exclusively at the expense of farmland [22], with changes in greenspace predominantly occurring in the urban-rural periphery [12]. Urban greenspace consists mainly of semi-natural areas, such as di fferent gardens, road and rail networks and their associated land, airfields, golf courses, parks, allotment areas, urban agriculture, etc. that together with formally protected nature reserves and Natura 2000-sites contribute to the generation of urban ecosystem services [23]. Quantifying spatiotemporal patterns of urban greenspace at more precise levels is reliant upon modern remote sensing techniques. Hence, more steadfast comprehensive assessments of detailed greenspace change are scarce in the ecological literature related to urban systems [24]. Previous studies have also come to di fferent results. For example, Kabisch and Haase [6] could not find a significant change in Western and Southern European cities between 1990 and 2000; but found a significant increase of greenspace in the period from 2000 to 2006. In a study of 386 European cities, Fuller and Gaston [1] found a dramatic drop in per capita green space provision in cities with high population <sup>1</sup> By the term 'social-ecological' is here meant a set of critical natural, socioeconomic, and cultural resources (or, capitals) whose flow and use is regulated by a combination of ecological and social systems. density, likely due to more people being packed into the urban matrix rather than buildings replacing existing green spaces. They also found that access to greenspace rapidly declines as cities grow, decreasing opportunities for people to experience nature. Following post-socialist changes many East-European cities have experienced a decline in greenspace [25,26]. Similarly, McDonald et al. [24] found an open space loss between 1990 and 2000 for all the examined 274 metropolitan areas in the contiguous United States. While many Chinese cities show mixed results, with both increases and decreases, cities in many developing countries are losing green spaces at a rapid pace [12]. Despite the massive shift towards privatization of public land and resources [3,10], comprehensive studies that have examined the relationship between loss of green spaces and ownership regimes are greatly lacking. However, it may not be far-fetched to assume that much of the privatization of public space involves greenspace. While this loss comprises direct habitat loss and the breaking apart of habitats [11], few scholars have addressed more subtle causes behind urban greenspace loss, but see Mensah [27]. In the following we present a set of examples of incremental demise of public urban greenspace. #### 2.1. Lack of Financial Support One of the major reasons behind the privatization of public space is financially strained local governmen<sup>t</sup> budgets, that strain results in the outsourcing and the alienation of land to private interests and the privatization of services that previously were publicly delivered. There exist plenty of examples of how tightening budgets are leading to declines in the quality of green spaces and loss of ecosystem services, due to the lack of staff and maintenance resources [27,28]. The Heritage Lottery Fund—a large funder of heritage in the UK—reported in 2013 that almost half of the local park authorities were considering selling parks and green spaces or transferring their managemen<sup>t</sup> to private entrepreneurs [29]. Considering the current recession due to the Covid-19 pandemic, one can only imagine how this situation will worsen. There has also been an increase in long-term leaseholds to allow the transfer of public land, such as parks and other green spaces, to not-for-profit trusts and to resident-led managemen<sup>t</sup> bodies [30]. While public land may be alienated from the ownership of local governments, privatization predominantly takes place through a mixture of transfers of governance and managemen<sup>t</sup> responsibility from the public sector to a number of other actors in the private, voluntary, and community sectors [7,30], and with the degree of privatization ranging from full to partial outsourcing of responsibilities [4]. Public–private partnerships (PPP) constitute a well-known example of 'contractual governance', which increasingly is used to re-develop and manage public spaces, especially as capital investments [30]. Through a PPP, a local authority or a central-government agency sign a long-term contractual arrangemen<sup>t</sup> with a private supplier for the delivery of services and taking of responsibility for building infrastructure, financing the investment, and managing and upholding the facility [31]. PPPs are increasing across Europe, Canada, and the United States, as well in some developing countries [31]. Business improvement districts (BIDs) are an example of a PPP in which a local authority and a business community together develop schemes to benefit a local district area [32]. The services provided through this type of contractual arrangemen<sup>t</sup> could, for example, include improvements and attractiveness of physical areas [7] and managemen<sup>t</sup> of public parks [33,34]. #### 2.2. The Separation of Attributes A critical fate that can affect underfinanced public spaces is the separation of attributes, which in economic theory can be expected when it is cost-effective and if sufficient demands for this exist [3]. In an overcrowded public domain, markets and governments will strive towards a separation of rights to land according to different attributes. This may be done in order to reduce potential conflicts and to lower transaction costs related to the governance of public space. In this way, the rights to different attributes of a park can be separated and granted to various user groups, such as devising demarcated land for leisure, habitats for wildlife, sporting areas, etc. [35]. Separation of attributes can also involve alienation or leaseholds of bits and pieces of public greenspace. There exist several cases where underfinanced public parks have been opened up to private interests, such as to restaurants, cafés and other social spaces. While local governments can reinvest the revenues from rents and/or property taxes for restoring degraded greenspace, profits are often instead used for other purposes [28,35]. #### 2.3. Increased Private Control Another subtle form of driver linked to urban greenspace change is the increased control and surveillance of public space [7,36]. Increased surveillance and policing over public space will likely intensify with an increased number of terror attacks as witnessed in many parts of the world. Today, much control of public space is outsourced to private corporations, making the boundary between public and private policing complex [37]. New York and Tokyo are telling examples of cities that lose much public space [36]. With increasing fear of terrorism after the Tokyo sarin gas attack in 1995 and the 9/11 attack in New York, many places where people formerly could relax from stress and annoyance have been eradicated [38]. Not only has an increased fear of terrorism acted as a vindication for imposing restrictions on the use of public sidewalks and plazas, but also in the use of natural habitats [38]. During the Covid-19 pandemic, the lack of public space in New York City prompted the banning of cars on certain streets in order to provide more space for pedestrians to upkeep social distancing. In contrast, green spaces in Sweden served as vital areas for social distancing [14]. Control of public space could be in the form of private police and/or surveillance equipment. While it may not directly lead to the loss of public space it can affect public space in more indirect ways, such as making people feel monitored and subsequently avoiding such spaces. The integrity of peoples' personal lives is increasingly also becoming jeopardized as digitalization is increasing in many cities [39]. While control can affect public green spaces the same way as it affects any other type of monitored public space, it especially can affect people's accessibility and use of urban green space. Many dwellers in urban areas display a fear of nature due to cultural reasons [40]. Lush green area habitats may be frightening for people due to lack of safety; hence, such habitats are sometimes replaced for safety reasons, e.g., increasing the width of sidewalks or increasing the occurrence and brightness of street lights, [41] thereby affecting greenspace negatively.	doab	2025-04-07T03:56:59.398941	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 12 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.13	2.4. Under-Utilization Under-utilization of public space represents ye<sup>t</sup> another subtle driver behind greenspace loss. One example is the London Green Belt where green spaces are avoided by people due to poor managemen<sup>t</sup> that have made them less accessible and attractive to be in. Residents may feel insecure and fearful of crime in unmanaged green spaces with short view distances [6]. So called 'boundary parks', located between vastly differing neighborhoods, are particularly susceptible of being underutilized which in turn can contribute to the decline of parks [42]. Accessibility of the public space itself can be argued as being one of the most effective factors and deterrents to utilization of a public space [9]. For example, public space will be less used by people if a user population does not live nearby [43].	doab	2025-04-07T03:56:59.400089	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 13 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.14	2.5. Congestion Direct over-use (congestion) of public greenspace is another indirect reason behind the demise of public urban space [3,35]. Congestion refers to the degree of competition within a public domain, or to "the numbers of individuals who jointly consume it and the range of tastes amongs<sup>t</sup> those individuals (or groups)" [3] (p. 34). When congestion generates excessive transaction costs, such as the costs of queuing or resolving conflicts between different users, economic theory predicts that there is a high probability for pressures to reform the property rights and subdivide public space either into private domains or into smaller public domains (e.g., club goods). When public domains become congested, they must be governed in such a way that use rights to public space are clear and enforceable [3,44]. However, transaction costs for designing, creating, and administering such a system becomes increasingly high the more over-used a space becomes, and if costs for governance become too high in political or financial terms, then public space could become subject to land alienation where the local governmen<sup>t</sup> seek to dispose of the property [3]. #### 2.6. Activity Intensification Activity intensification can also act as a subtle driver behind the transformation of public space [45]. To cut costs and for energy conservation, planners can, for example, resort to multifunctional land use. Haccou et al. [46] distinguish four types of multifunctional land use: interweaving, intensifying, layering, and timing. Interweaving combines di fferent functions on the same piece of land; intensifying increases the e ffectiveness and e fficiency of a certain land use on the same piece of land; layering mixes functions in the vertical dimension if possible; and timing uses the same building or space for di fferent functions at di fferent moments in time [47]. The first two types are also applicable to green spaces. In Stockholm—one of the fastest growing regions in Europe—planners have been taken by surprise by the sudden population boom, with over 300,000 new homes in need of being built in the coming decade. To cope with increased urban densification and population growth, multi-functionality in the use of public parklands has become a quick fix for dealing with overcrowding e ffects [48]. However, such a development may run contrary to planners' aspiration of making cities more resilient to various effects of climate-change and for securing biodiversity that depend on green spaces of a certain size and with certain ecological qualities that do not always rhyme well with social aspirations. #### 3. Baseline Shifts and Benefits of Nature As cities develop over space and time, the di fferent attributes of green spaces also change. Urban residents experience nature most of the time at "the cognitive level of urban space," that is, at the level where "people in the street" experience the city [49]. Human beings also constantly use memories of previous experiences to interpret current experiences [50]. When it comes to experience of urban nature, psychologists talk about baseline shifts, where each generation of humans tends to take the current condition of an ecosystem as the nondegraded state: the 'normal' experience, disregarding the fact that the ecosystem might have changed considerably over time [51]. Baseline shifts can lead to environmental generational amnesia [52], referring to the psychological process whereby each generation perceives the environment into which it is born as the norm, no matter how developed, urbanized, or polluted the environment is [51]. According to Hartig and Kahn [51], baseline shifts can help explain inaction on environmental problems in that people do not feel the urgency of the problems because the experiential baseline has shifted (Figure 1). Experiencing nature during childhood is crucial for shaping sustainable decision-making processes in adulthood [53]. The incremental changes of urban greenspace, previously dealt with herein, likely contribute to baseline shifts. To what extent is hard to determine. There might also be overlaps in the drivers previously dealt with, e.g., under-utilization of greenspace can at many times be explained by a lack of financial support. However, environmental generational amnesia may spread as more and more people are devoid of direct contact with nature in cities [54]. This in turn may make it more di fficult to reach public acceptance of policies to deal with environmental problems, like climate change. Environmental illiteracy may further lead to the erosion of public green spaces since people will not value the ecosystem services they derive from nature (Figure 2). This is especially the case in countries where destruction of ecological sensitive areas and open spaces are rapidly urbanized due to weak urban planning institutions [55]. Figure 1. Schematic illustration of a baseline shift of a hypothetical public park over a 150-year period. Each development stage is perceived as being the nondegraded (normal) state by each new generation of humans—a situation that can contribute to environmental generational amnesia. Figure 2. Ecosystem services from urban greenspaces. These include provisioning services, regulating services, cultural services, and supporting services. Source: Millennium Ecosystem Assessment. Ecosystems and Human Well-Being. Reprinted from Colding and Barthel [56]. #### 4. Long-Term Resilience Planning The incremental changes and loss of urban greenspace elaborated on in this paper fit the pattern of decision making that the economist Alfred E. Kahn once referred to as 'the tyranny of small decisions', representing a situation in which a number of decisions, individually small in size and time, result in non-optimized and socially undesirable outcomes [57]. According to Kahn the 'small decision effects' are common in market economics. In an urban planning context, many small-scale, independent decisions taken over time by a planning unit could culminate in outcomes that are neither intended nor preferred [58]; hence, even well-intended planning decisions in countries with strong state control can result in undesirable social-ecological outcomes that run contrary to the common good of a city and its inhabitants. Nilsson [15] has described how leading politicians often abstain from restricting options in order to handle future planning issues—a phenomenon that often leads to an ad hoc form of planning, termed "day-to-day planning". It is mainly designed to manage acute problems and to resolve problems as soon as possible and is related to a discourse of economic development "in the way in which it tries to satisfy the short-term requirements of industry and commerce, and tends to emphasize the economic dimension of sustainable development" [15] (p. 441). Accessibility to green spaces in cities is ultimately determined by property-rights arrangements that regulate their omission or entry. Institutional scholars have long recognized the role of property rights for linking people to nature, but as institutional research suggests, no single type of ownership regimes (i.e., state, private, or common property rights) can be prescribed as a remedy for resource overuse or environmental degradation [59]. Instead, policy should focus on establishing a multitude of property rights regimes that are designed to fit the cultural, economic, and geographic context in which they are to function [20]. How space and property rights are arranged to ensure access to urban nature will be of direct importance for building general urban resilience to human wellbeing in the long-term [14]. The gradual shift towards privatization of public property in cities, and even of common property systems [60,61] is a worrisome sign since it may negatively a ffect resilience planning, which needs to be long-term oriented. Property-rights could be viewed as slow variables in urban transformation. In resilience science, slow variables—like evolution, coral regrowth, or nutrient transportation through soils—play a substantial role for a system's resilience to change [62]. C.S. Holling's work [63] demonstrated that an ecosystem can absorb an entire chain of disturbances without being adversely a ffected until it suddenly changes to a completely di fferent state where past functions and services no longer can be provided. A gradual loss of the system's slow variables often causes such abrupt change. Critical, slow social variables are characterized by normally being stable or changing slowly over long periods of time, providing continuity of functions. However, even property rights that have existed for millennia may change abruptly, causing changes in land use, social disruption, and even declines in human well-being [60,64]. Lindholm [10] describes how sightlines in cities commonly become blocked by new private developments, with less daylight reaching the ground and with more land areas constantly in shadow, resulting in poorer conditions for plants and biodiversity, and also how people at the eye level of cities experience their local socio-physical environment. New sightlines are often aggravated due to visible landmarks of private interest. Hence, indirect e ffects of private property may negatively impinge on ecosystem services that are nurtured in the public domain. While private building consortia often launch their development projects as "sustainable", they often constitute examples of "green washing" [10]. An often-time neglected aspect of green roofs and green walls that commonly replace natural areas on the ground, is that they only are accessible to a limited set of urban residents. The slow variables, such as the amount and quality of public green spaces, determine how a fast variable, like daily visits to green spaces, is possible when external drivers are in operation, such as excessive heat waves or pandemics. The gradual transfer of public to private property rights and services will in the long run weaken the power of local planning units and local authorities. If left unchecked, the increasing privatization of public space could ultimately become a huge democratic problem. #### 5. Concluding Remarks The ratio of public/private land is diminishing, a ffecting cities in a multitude of ways. The drivers of incremental change of urban greenspace presented herein are ultimately a result of urban densification schemes (spatial and/or population), which seemingly propel a monoculturalization of property-rights regimes towards economic e fficiency and private and semi-private solutions. As elaborated on herein, congestion in the use of public space is but one outcome of urban densification, leading to such phenomena as the separation of attributes, activity intensification, and even to alienation of public space to private interests. Underutilization of greenspace coupled to inadequate funding for greenspace managemen<sup>t</sup> make these spaces more vulnerable to urban encroachment, especially when cities grow inwardly, posing an overall negative e ffect on the maintenance and generation of ecosystem services. To counter shifting baselines that may result from the incremental demise of urban green spaces, Hartig and Kahn [51] propose that cities should provide more opportunities for people to experience more stable, healthy, and even wilder forms of nature. Property rights, however, ultimately determine such opportunities, as no single space should be expected to meet the demands of all users at all times, nor will any single type of property-rights arrangements fulfill the multitude of functions that a vibrant city depends on. Designing urban commons as an alternative to land privatization is no guarantee, however, for halting the demise of urban greenspaces, as the effects of privatization and commodification of commons is also high in some countries, such as in Europe [65]. However, maintaining a well-balanced diversity and mixture of property-rights regimes could be a wise policy for planners to adhere to in order to increase preparedness to meet an increasingly disturbance-prone future. Although common property resource systems have been known to collapse due to overuse, there are promising signs that these systems are experiencing a revival in urban settings and boosting collective environmental action [44,66]. There are important linkages among urban common property systems, social–ecological learning, and managemen<sup>t</sup> of biodiversity and ecosystem services. Urban gardening projects provide not only cultural and provisioning ecosystem services but also regulating ecosystem services to city neighborhoods [44]. There are also examples of whole public parks being managed as common property systems [67]. By granting managemen<sup>t</sup> rights of greenspace to residential collectives and social networks, local governments can still maintain control and ownership of public space. Long-term leaseholds and the establishment of 'user rights contracts' that transfer managemen<sup>t</sup> rights of public space to civil society groups can be a viable interim solution for local governments in order to survive periods of fiscal shortcuts. Studies have shown that urban common property systems can serve a multitude of social-ecological purposes [11] and turn spaces into meaningful places that urban residents themselves can manage to improve and protect urban nature. The transferring of proprietor rights down to local levels can also lower transaction costs for greenspace managemen<sup>t</sup> and governance [44]. To safeguard ecosystem services policy makers and planners should take advantage of these benefits and more meticulously address the incremental demise of public greenspace. Author Contributions: Conceptualization, J.C. Writing—Original Draft Preparation, J.C., S.B., Å.G.; Leading of the writing process, J.C.; Corresponding author, J.C. All authors have read and agreed to the published version of the manuscript. Funding: Johan Colding's and Stephan Barthel's work have partly been funded by University of Gävle. Barthel's work has also been funded by FORMAS/The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning. The project is called Spatial and Experiential Analyses for Urban Social Sustainability (ZEUS) (reference number: 2016-01193). Barthel's work is also funded by the Stockholm Resilience Centre. Johan Colding's and Åsa Gren's work has also been partly funded through a research gran<sup>t</sup> (reference number: 2017-00937) received from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS), and through means provided by the Stockholm County Council and the Stockholm University (SU-SLL Grant 2017: no. 20160884), and from core funding provided by the Beijer Institute of Ecological Economics, Stockholm, Sweden. Acknowledgments: The authors wish to thank Jonas Adner for the nice Figure illustration. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.400179	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 14 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.16	1. Introduction Urban areas are ecosystems characterized by natural and artificial elements such as buildings, roofs, underground pipes, and green areas that are mainly related to human well-being. The urban ecosystem is not self-regulating but is "regulated" by humans [1–3]. The urban area is, therefore, a complex socio-ecological system where various communities can overlap and interact to a greater or lesser extent and co-evolve with their environment through change, instability, and mutual adaptation [2,4]. Therefore, the evolution of an urban ecosystem is influenced by social decisions or needs and by stakeholders' heterogeneity (for instance, culture, education, religion, vision, interest) [5–8]. These institutional levels include decision-makers like administrative and public institutions that plan the socio-ecological system at di fferent scales: "ecosystems in urban areas", "urban areas within ecosystems", and "urban areas within regional/global ecosystems". At di fferent scales, the boundaries of the urban ecosystem are not always well defined or clear, and therefore, boundaries of a survey area are defined considering the topic and interactions to be analyzed and on practical considerations. [5,9]. Institutional levels are hierarchical and consist of vertical relations between actors of the top and bottom of the levels. Therefore, the urban area has to be planned like a socio-ecological system where the urban ecosystems and institutional levels interact with each other in a multi-scale analysis. The urban planning and design have to create a synergy between di fferent institutional levels (Figure 1) [10–13]. Figure 1. Schematic representation of the relationship between urban ecosystem scales and institutional levels in the socio-ecological system [5,6,9]. In many industrialized cities, urban planning must address the phenomenon of "shrinking cities" [14]. These cities have experienced a significant de-urbanization linked to the loss of functionality of some urban areas or buildings due to the decline of the manufacturing industry, migration, and depopulation [15,16]. Consequently, urban areas are characterized by free or temporarily not used urban spaces as a result of technological, economic, and social evolution. In many cases, these urban areas could be brownfield sites: "streets with vacant storefronts, underutilized social and technical infrastructures, and neglected parks and squares" [16,17]. Identifying new functions in urban spaces—either built or otherwise—in a transitioning economy and society is the main focus of resilient thinking, which has to recognize the complex and non-linear dynamic of economic and socio-ecological interactions [18–20]. Currently, the main urban planning and design use the top-down approach, where planners are considered "the experts" who put forward the proposal and then share it with others, mainly the decision-makers that can approve or reject the urban plan [13,21–24]. This generates stakeholders' conflicts in the type of use of the urban space, environmental protection, the interest of residents, labor conditions, economic development, and the identities of urban areas [8,25,26]. The planning of the urban space has to be considered a "public a ffair", aiming to envisage the right use of urban spaces considering the socio-ecological and cultural context of reference and solving conflicts in the choices or preferences in the use of destination of the urban space between stakeholder groups. Being able to evaluate the "awareness, value judgments, behavior, and attitudes" of the citizen in relation to urban space is an important task for a successful plan of urban transformations [27–30]. To create a social and shared vision of possible scenarios that can transform the territory, a prominent role must be given to stakeholders' needs, opinions, and interests, but also fears and doubts, in order to include their vision in the development of the urban space that they use [31–33]. Therefore, urban planning needs to combine bottom-up and top-down approaches, including stakeholder's participation with strategic spatial planning at di fferent urban levels [34]. Public participation helps to understand the aspirations of stakeholders on possible urban development. Moreover, perception stimulates di fferent stakeholders to develop ideas and proposals based on their knowledge, attitudes, and habits, providing greater awareness of their role in urban development. It is an action in urban design useful for increasing the ability to make e ffective planning choices [30]. For this reason, scholars consider stakeholder participation as one of the main aspects to take into consideration in order to guarantee the quality of urban planning [35,36]. On the one hand, there are many examples of bottom-up and top-down approaches in policy activities that are mainly focused on the managemen<sup>t</sup> of natural resources or services (e.g., energy policy, climate change, watershed management, mobility, agricultural, environmental) on municipal, regional, national, and international programs. On the other hand, these approaches are less frequently used on small urban land use planning and design [37–39]. Although these approaches have attempted to include community stakeholders, this has often proved problematic, and planning guidelines do not ye<sup>t</sup> consider design principles that foster social learning, knowledge exchange, and power-sharing [7,40–42]. Mainly, public participation may not always yield a mutually acceptable solution, especially when the interests of stakeholders are diverse and conflicting [38]. Often, top-down and bottom-up urban planning approaches are sometimes considered incompatible because they can produce conflict and fragmentation in the built new environment vision between di fferent urban levels and stakeholders [13,43,44]. This work wants to develop a planning-process of the urban space transformation able to create feedback between di fferent stakeholders at di fferent institutional scales to reduce the mismatch between governance levels and the scales at which people benefit from urban space: from the need of the single individual to the development vision of the decision-makers [5,6,45]. Starting from a practical case study, we propose a combination of a bottom-up and top-down methodology capable of developing a participated urban plan, harmonizing the various stakeholders' interests that act at the di fferent administrative levels and integrating ecological and socio-economic components in the context in which it is inserted [31,45]. Mainly, the study is focused on the Plan of the Urban University Center (PUUC), involving the creation of new university lecture halls in a university urban space that was the research site of tobacco production. Considering that the University represents the main stakeholder, as it is the owner of the urban space, the planners tried to satisfy university needs with the urban transformation vision of the decision-makers (top-down), also taking into account the aspirations of the citizens that act in the context area of the PUUC (bottom-up). We hope to identify the best solutions for the use of urban spaces to integrate the citizens' visions with those of the planners and of the di fferent public institutions that have an administrative role in choosing the final destination of the area. #### 2. Materials and Methods #### 2.1. Study Area The study area is a free space of the university center in the Municipality of Lecce, Apulian region, South of Italy (Figure 2). The presence of the university has greatly influenced the economy of the district, favoring the opening of numerous commercial activities, such as bars, take-aways, restaurants, bookshops, and pubs. Furthermore, the real estate business linked to the rental or sale of student apartments has benefited from the situation. From the cultural and social point of view, the free urban space is located within the former Agricultural Research Center (ex CRA), which was used in the past for tobacco research activities. Currently, the ex CRA is employed for university lectures. The urban free space of interest is characterized by herbaceous vegetation with no ecological value (Figure 2B). Figure 2. (A) Municipality of Lecce and location of the study area; (B) study area with reference to the context of the former Agricultural Research Center (ex CRA). Near the ex CRA, there is a large green urban park, the cemetery of Lecce (classified as a historical asset for its architecture), and the Monastery of the Olivetans (founded at the end of the XII century, and currently used by the Department of Historical Studies of the University). To the west of the ex CRA, there is a main road, the Castle Charles V, and the ancient city walls. The north and south parts of the ex CRA have no significant neighboring elements. #### 2.2. Focus of the Planning Question The Italian Inter-Ministerial Committee for the Economic Planning (CIPE) has identified and allocated resources in favor of interventions of strategic national and regional importance for the implementation of the national plan for the South's strategic priority: "Innovation, research, and competitiveness". One of the projects included in the plan for the South is located within the "Urban University Center of the ex CRA". Mainly, in an area of about 11,186 square meters, the university developed an urban plan involving the construction of a new building of about 3100 square meters for educational activities, a parking area of 1734 square meters, and a recreational green area of 6322 square meters. The new area will be realized near other university buildings, and together they will form a widespread urban university campus. The budget for the execution of the plan is EUR 8,000,000. During the first phase of the planning activity, some regional authorities expressed a favorable opinion of the new PUUC because the plan did not cause negative environmental impact. However, the Ministry of Cultural Heritage, through the Superintendence, was the main institution that opposed the university urban plan. The Superintendence considers that the PUUC will alter the harmony between existing buildings and the identity of the area, and hence it only promotes the development of a green lung. Considering the institutional conflict, this research wanted to develop bottom-up and top-down participation processes approach able to orient future use of free urban spaces. #### 2.3. Design Approach This study was developed considering bottom-up and top-down approaches in the socio-ecological system [46,47]. Mainly, the stakeholders' participation process was designed considering di fferent roles in the transformation of the socio-ecological system. The designers of the University and Superintendence are considered decision-makers that can directly choose the typology of the transformations (top-down). The citizens are considered as users of the urban space on which the choices of the decision-makers are reflected. However, at the same time, the citizens can revolt against the choice of decision-makers and condition the final result (bottom-up). The work is organized as follows (Figure 3): Figure 3. The conceptual work models that we have developed for the top-down and bottom-up approaches. #### 2.4. Bottom-Up Activity: Questionnaire Survey The study was planned by taking into consideration the microscale, which encompasses the urban space, the structure of buildings, the relationship between them, and their interaction with other elements of the neighborhoods [48]. The work used face to face questionnaires to gather information about what citizens "feel, hope, wish, approve, or disapprove" for the future use or transformation of the identified urban space [49,50]. The survey was developed so as to include di fferent types of citizens and users, such as students and people who live or work near the area where the project should be developed. This is important in order to explore their opinions and preferences on the possible uses of the free space of the ex CRA. Questionnaires were administered from 15 April 15 2018 to 30 May 2018, both in the morning and in the afternoon during working days and holidays. This was necessary to better characterize the typology of individuals who frequent the area of interest. The questionnaires were delivered in three di fferent places: the "ex CRA" area, the parking lot in front of the area of interest, and in the urban park "Belloluogo" (Figure 4), as these are the principal hotspot areas of the neighborhood in the context of the urban space of interest. Figure 4. Localization of the points of administration of questionnaires, historical elements, and urban university sites. We used Sierra's formula to detect the sample size of the number of individuals to interview [50]: $$\text{Sample Size} = \frac{4 \ast N \ast p \ast (1 - p)}{E^2 (N - 1) + 4 \ast p \ast (1 - p)}$$ where N is the number of inhabitants; E is tolerated error; p is the portion of the variable in the population" [39].	doab	2025-04-07T03:56:59.401440	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 16 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.17	Questionnaire structure The questionnaire was structured in order to evaluate four main dimensions: the main users of the area; the historical relevance of the place (Items 1–3), the building size and harmonious insertion in the urban context (Items 4–7), and the importance of developing green areas compared to other options (Items 8–9). In particular, considering Item 9, three alternatives in the use of the area were indicated, and respondents could express a preference value. This last aspect was developed after taking inspiration from Directive 2001/42/EC and national law (D.lgs 152/2006), which provide for the analysis of di fferent planning hypotheses in the drafting of the Strategic Environmental Assessment (SEA) to identify the best possible solution [51,52]. The questions were formulated with a simple and clear structure to be filled out quickly by ticking the preference box. Specifically, respondents were asked to express a preference using a five-point for the second section and a ten-point for the third section. In this way, respondents indicate their level of agreemen<sup>t</sup> to a statement. In the present survey, the ten-point scale was used when respondents could express their preference for the di fferent design solutions proposed (e.g., [53–56]). The data analysis was performed with the descriptive statistics for the first seven items, while the last items were analyzed through tables of contingency. The contingency tables are used to represent and to analyze the relationships between two or more variables, through the study of their combined frequencies [57]. Before the survey, a pilot study was conducted, and five questionnaires were administered to people to verify whether the proposed items were adequate and easily understood. #### 2.5. Top-Down Activity: Sharing the Questionnaire Results with the Main Decision-Makers As the final part of this work, we discussed the results of the questionnaires with the decision-makers, who, at that moment, had a di fferent vision for the use of urban space. In this way, we try planning a new hypothesis of the shared urban space transformation. This represents a simple exercise to try to design the new PUUC considering citizens' visions. This was developed with informal appointments with decision-makers like the designers of the University and Superintendence. In these appointments, starting from the original PUUC, we discussed potential new solutions of the PUUC and analyzed how the main results of the questionnaires could be incorporated in the new planning of the urban space. After that, we developed an illustrative conceptual graphic of one of the possible new urban space transformations. For practical reasons of this research, we have not considered the turnover between the various decision makers in time. #### 3. Results and Discussion #### 3.1. Bottom-Up Activities #### 3.1.1. Stakeholders Characterization For the study area, the minimum sample size is 382 individuals. Therefore, our 624 questionnaires can be considered as representative of the population that characterizes the study area. Mainly, 42% of the questionnaires were compiled in the ex CRA area, 33% in the parking area, and 25% in the urban park "Belloluogo" (Figure 4). Table 1 shows the main socio-demographic characteristics of the sample interviewed. It is possible to note that the main users of the area are young people and students. In particular, most of the interviewees were students under 25. This result could be strongly influenced by the presence of two university centers. Table 1. Socio-demographic characteristics of the sample. #### 3.1.2. Historical Relevance of the Place Analyzing the historical relevance of the study area, it is clear how almost all of the respondents considered this area relevant to the historical and cultural point of view. Only 19% said that the area is "not very important" and only 3% that it is "not at all important". However, the surveyed people did not know the history of the ex CRA, or where precisely this building was located. Therefore, the surveyed respondents consider this building historically important for the urban context in which it is located, rather than for the specific history of the structure. The distribution of answers obtained is similar in the different sampled areas (Figure 5). Figure 5. Answers to Questions 3, 4, and 5, concerning the historical relevance of the study area. #### 3.1.3. New construction in urban space Subsequently, the opinion of the surveyed respondents was investigated with reference to the construction of a new building for teaching purposes within the former CRA. In particular, 43% of the respondents answered that the possibility of a new building altering the architectural harmony between existing buildings depends on the type of building that will be built. However, this building will not be a disturbance to the livability of the area (Figure 6). Figure 6. Answers to questions on the possibility of a new building inside the former CRA. According to the surveyed respondents, the width and height of a new building could also be the same as the size of the central building existing within the former CRA, or even wider and higher. In fact, these two options were the second most selected choices in all the three sampling areas (Figure 7). Figure 7. Answers to questions on the possible width and height of the new building that is to be realized within the former CRA. However, the construction of the new building should not lead to the elimination of the currently unused greenhouse. According to the surveyed respondents, the greenhouse should be used for social activities (Figure 8). Figure 8. The question related to the greenhouse. #### 3.1.4. Potential actions that can be developed in the urban space In the final questions of the questionnaire, we investigated the main actions of the plan to be launched within the former CRA (Figure 9). Surprisingly, it emerged that "improving the quality of urban green areas" is not among the main prerogatives indicated by the surveyed respondents. Overall, the main preferences were "finding a right harmony between use, green spaces, and economic activities" and "favoring the development of social activities (concerts, parties, and shows)". However, the surveyed respondents within the ex CRA expressed their preference "to create new classrooms and laboratories to enhance the university's educational offerings and accommodate a larger number of students" and "favoring the development of social activities" (Figure 9). From the analysis of the contingency tables, statistically significant differences emerge. Mainly, there are differences between the alternatives "a", "b", "d", and "i". (p < 0.01). However, considering three sampling points (Figure 9), there are no statistically significant differences in the choice for the alternatives "a", "d", and "i", while there are statistically significant differences for the choice obtained for the alternative "b" in the different areas. Furthermore, the results show statistically significant differences in the choice of the solutions proposed considering the age of the surveyed respondents. The respondents aged 25 years and under expressed their main preferences as "i", "d", and "b", while those over 25 expressed as main preferences "a", "i", and "d" (Figure 9). The numbers in the cells represent the frequencies relative to the appreciation value for each proposed alternative. The answers were analyzed, considering both the single survey areas and the whole (Figure 9a). In Figure 9b, the answers were analyzed considering two age groups: under 25 years and over 25 years. Figure 9. Heat map for the analysis of possible planning actions considering the different survey areas. The colors from red to green represent a gradient of preference from low preference to high preference in relation to the number reported in the cells of the table. (a) The answers were analyzed, considering both the single survey areas and the whole. (b) The answers were analyzed considering two age groups: under 25 years and over 25 years. The last question analyzed the preference of the surveyed respondents with respect to three project alternatives. Option 2, which involves a multifunctional use of the area and the realization of a new building, prevailed over the other two alternative projects. This preference did not show differences, considering the different sampling points or the age of the surveyed (Figure 10). Figure 10. Heat map for the analysis of the results for the three project alternatives, considering the different survey areas and the age of respondents. The colors from red to green represent a gradient of preference from low preference to high preference in relation to the number reported in the cells of the table. (a) The answers were analyzed, considering both the single survey areas and the whole. (b) The answers were analyzed considering two age groups: under 25 years and over 25 years. The numbers in the cells represent the frequencies relative to the satisfaction value for each option. In the first table, the answers were analyzed, considering both the single survey areas and the whole. In the second table, the answers were analyzed considering two age groups: under 25 years and over 25 years. #### 3.1.5. Relevant elements for the development of the new PUUC The results showed that the urban space has to be planned as a social element of the context connected to ecological and economic aspects. Indeed, according to the opinions of the interviewed, in the study area, the green urban spaces were not the principal development vision of urban areas highlighted by citizens. The results showed how the main users emphasized the need to develop an integrated plan between new construction and green area that should favor economic and social development. The urban green space has to be planned as an element integrated within the development of new structures that can increase the social life of the area, including the creation of potential new buildings for economic or educative activities, creating a multifunctional center able to give vitality to the urban context of reference in different moments of the day. From the analysis of the questionnaire, it emerged that the preservation of cultural-historical aspects should not be interpreted as prejudicial to urban transformation. What is important is to create harmony between the new urban elements that will be developed and the cultural–historical value of the area. In this perspective, according to the surveyed respondents, a new building within the former CRA should be designed as a multi-purpose center, without compromising the urban identity in which university activities, but also other social activities, can be developed, e.g., libraries, playrooms, and leisure centers. Through participation, it was also possible to collect suggestions and specific proposals to plan the use and design of the urban space. In the initially proposed PUUC, the elimination of the greenhouse that is currently in a state of neglect was planned so as to make room for the new building. The surveyed respondents expressed their willingness to preserve the greenhouse and use it for recreational activities (Figure 11). An interesting aspect is that many surveyed respondents were unaware of the presence of the greenhouse. Even many students who attend the area have not noticed its presence or thought that the structure was indeed a greenhouse. This probably shows how limiting the use of this area can also compromise the awareness regarding the place and its identity. Therefore, creating a green area without increasing the usability of the place or the ability of people to move within it can limit the cultural value of the area. Figure 11. Photos of the greenhouse. To make this area more active from a social point of view, it is fundamental to ensure greater use of the spaces and movement of individuals within the area. The possibility of an individual to perceive an area of social value and obtain benefits depends on the experience that an individual has in moving within the space, creating occasions for social interactions and enjoyable time [16,18]. The imposing enclosure wall (h = 2.50 m) that surrounds the entire area of the CRA is an obstacle, isolating this urban space from the surrounding area (Figure 12). Figure 12. Photos of the enclosure wall. (<sup>a</sup>,b) are to the south–west side; (<sup>c</sup>,d) are to the north–west side; (e) is to the east side. #### 3.2. Top-Down Activities: Reflective Analysis Using Bottom-Up Information This work represents an experiment of a combination of the top-down and bottom-up planning processes. In particular, in this study, questionnaire activities allowed for the identification of the interests of the main citizens who frequent the area and their social needs that will stimulate the development of the new PUUC of the ex CRA ("bottom-up"). The results of the questionnaire were shared informally with decision-makers. The results were food for thought to hypothesize the development of a plan, which, starting from different visions, could produce a shared urban transformation process. Figure 13 shows an example of possible urban visions that can emerge from the comparison between decision-makers by analyzing the results of the questionnaires (top-down). The representation in Figure 13 is a simple example of potential PUUC visions that can be developed by this process. Other design solutions can also be developed because the focus is the social functionality that has to develop in the urban space independently of the urban elements such as buildings or other elements. Figure 13. (a) schematic representation of the actual state of the area; (b and c) draft of the potential evolution of the area following and interpreting the indications of the respondents and discussion with the citizens, designers, and decision-makers. This figure represents a simple scientific exercise and does not have legal or institutional value or what this area will be in the future. Considering these results, it emerged in these appointments that, in the new PUUC, the demolition of the wall should be further emphasized in order to encourage the development of social activities that may not necessarily be directly related to the university, but also related to leisure of a temporary nature, and, therefore, not pre-planned. Moreover, a greater permeability of this space would facilitate the circulation of people within the urban context of reference, to better harmonize the existing valuable elements and make the buildings of the ex CRA more visible. In addition, the redefinition of the ex CRA perimeter would allow the creation of pedestrian and cycle paths—currently somewhat absent—and new bus stops supporting public transportation The urban space quality is strongly connected with the quality of public transport and the promotion of healthy patterns of walking and cycling as daily activities [58,59]. This solution would increase the type of users of the site, which is currently limited to students, to enhance the cultural and historical value of the area, now restricted within four walls. In this case, the new use of the urban space can increase the contact between citizens and urban elements that influence the cultural identity of urban space, opening the opportunity to new knowledge and new relationships between stakeholder and urban space that was limited from the no-use of urban space. The new PUUC should include the recovery of the greenhouse. The greenhouse could host indoor cultural and social activities, student associations, or a place to set up co-working. Therefore, in the new PUUC, the greenhouse will play a fundamental role in implementing the social activity of the area, increasing the fruition and experiences that the inhabitants can have within the area (Figure 13b). Another point of discussion is in regard to the possible use of the roof of a potential new building as a new social element of the PUUC. In this case, the roof can be designed as a green roof that can be used as an outdoor study area for students or small public events. Green roofs are an important tool used in residential, commercial, government, and public buildings to increase sustainability and biodiversity and decrease energy consumption, urban heat island impacts, and greenhouse gas generation in the city [60]. In this case, it helps to plan the multifunctional use of space. Therefore, green roofs can increase the quality of the buildings by reducing their impact on the urban landscape while also replacing some functions of natural areas and, therefore, assuming an important role in mental health [61] (Figure 13c). However, the realization of a new building would require more discussion among decision-makers. The solutions highlighted can make the area more dynamic, which is an important element of the human experience in urban space that incorporates both the "relationship between the person and the person-to-place relationship", improving the perception of the identity of the urban space [62]. ### 4. Discussion The main inspiration in the urban regeneration of the urban spaces or degraded areas is the realization of green areas, as they are now widely recognized and documented in guaranteeing ecosystem services useful for the wellbeing of the population [62–65]. Indeed, the type of urban space use was the main focus of the conflict between some decision-makers: implementation of the university urban center vs. green areas. As argued by such scholars as Bourdieu, Lefebvre, and Gans [66–68], the result of the bottom-up participation showed that the urban space must be thought of as a "social space", considering the main users and producing transitions able to support good quality of life. Therefore, the challenge going forward will be to apply an increasingly advanced and nuanced understanding of urban ecology in the practice of planning and designing urban ecosystems [69,70]. Urban spaces have to be planned as dynamic areas [71], giving the opportunity to develop new structures and functions to adapt them to new social needs and economic opportunities without upsetting urban identity and ecology quality. The implementation of a university urban center vs. green area is not the main new question of the PUUC, but how this aspect can be combined to implement the social use of the area going beyond the di fferent vision or position of the decision-makers. In socio-ecological systems like an urban ecosystem, the bottom-up and top-down participation approaches can give both a contribution to encourage the evolution in systems and increase the resilience of the area, understood as the ability to adapt their functions and structures to social changes. In particular, the bottom-up approach allows for the identification of the main stakeholders of the area and their social needs, which, in turn, will stimulate the development of the new urban plan of the ex CRA. This is important because it allows us to have a vision of the development of the area that is not conditioned only by the cultural background of the decision-maker, but of those who use the territory to meet the needs of everyday life. The important aspect of this approach is to actively connect the knowledge and information of bottom-up participation to decision-makers that manage the urban space at higher institutional levels. Top-down participation, using the bottom-up information, in this case, can drive the choice and help decision-makers overcome an excessively deep-rooted view of conservation of the urban space that administratively slows down the urban regeneration process. This would arguably help speed up the decision-making process by helping decision-makers become more aware of the transformations that are introduced in the urban context: "doing the right thing in the right place". This can be useful to produce a better acceptance of urban plans reducing the likelihood of conflicts between di fferent experts or people that participate in the processes of planning development [31,72–74]. In this case study, participation activities started after the drafting of a first project by the university in relation to the use of the area that led the decision-makers to express a negative opinion on the project or highlighted specific critical points. From the meetings held with the decision-makers, it emerged that this approach could be useful in the initial phase of the project. However, this approach is also conditioned by the timing of urban planning. Long urban planning times can make this approach di fficult to use because it can be conditioned by the turnover in managemen<sup>t</sup> related to the decision-makers (for instance, the University, the municipality) that may require the need to restart the top-down phase. The vision of the use of urban space by current and future decision-makers can di ffer from previous decision-makers present when this work started. Of course, the techniques used in this work can be improved. An important limitation of this work lies in the number of stakeholders involved in the bottom-up participation. This can a ffect the outcome of participation. To be representative, this method requires a grea<sup>t</sup> deal of e ffort in administering the questionnaires. Questionnaires are not the only tools to carry out the participation activity and cannot be defined as the best tool with respect to other methodologies because this depends on the scale of the investigation and type of activities. On a local level, however, questionnaires can represent valuable tools because they allow for the creation of a face-to-face relationship between planners and stakeholders. Therefore, questionnaires were used as a way to start the dialogue with the population and also to raise the interest of the interlocutors. Regarding the analysis of small transformations, it can be useful to involve citizens and their specific needs and vision in the reference context and try to put them in the final urban space planning. Many participation approaches use the creation of thematic meetings or focus groups and tools such as online questionnaires that can open participation to a larger audience [22–24,37–40]. These actions may attract di fferent citizens who are not necessarily users of the area, and, therefore, they may express a judgment based only on their preference or training and not on needs. This can also open a debate on the weight of the judgments expressed by those who are not familiar with or frequent the urban context of reference. We reckon that the chosen three main areas in which the questionnaires were submitted were the most functional to represent and characterize the typology of users of the urban reference ecosystem and, therefore, to analyze the social, economic, and ecological needs to be developed in the urban plan of the university (Figure 4). However, the characterization of social needs and stakeholders is the main issue of this study that needs improvement. As an alternative, using mixed methods would be an ideal solution, but this is not always feasible because of economic and time issues. For instance, in this work, the top-down activities were conducted with single appointments with the decision-makers. Therefore, in this work, we harmonized the response of decision-makers to social issues. Figure 13 is an example of the result of these activities. In the future, it can be interesting to plan the top-down participation activities by organizing focus groups between all decision-makers that participate in the authorization processes using the social issues derived from bottom-up participation as the starting point of discussion. In a similar focus group, the decision-makers should not limit themselves to expressing an opinion based on their skills and background but should try to produce a draft project, similar to the example in Figure 13, interacting directly with each other. Even if the questionnaire did not provide open answers where citizens could freely express their thoughts, mainly in order not to weigh down the interview, an important aspect during the compilation of the questionnaire was the dialogue established with the interviewee. Often the surveyed was not limited to the simple answers to the questions, but to an open dialogue that went beyond the structure of the questionnaire. In this way, unforeseen or planned information, consideration, and opinions were obtained. The majority of the questionnaires were filled out by young people aged between 18 and 25 years old. The results can be influenced by the greater presence of users under 25 years and students. Probably, this expresses the main current vocation of this area. However, we want to express some "reflections" that did not emerge from the analysis of data but were derived from the dialogue with all respondents during the interviews. People from 18 to 25 years old were more interested in addressing issues concerning the development of the territory in which they live. We can state that no young people refused to complete the questionnaire, a problem that has been found in other age groups, especially men. The students were interested and encouraged to make a contribution, providing their opinion, often critical, on the issues addressed in the questionnaire to try to improve the territory in which they live and project it in the future. In addition, during the interviews, a di fferent attitude among the interviewees emerged. Specifically, the respondents between 18 and 25 years old seemed more cooperative and willing to make a contribution to improve urban quality. Older people sounded more pessimistic about the possibility of the political class favoring actions that could produce a change and were, therefore, less constructive in providing suggestions. The questionnaires showed a di fferent view between young people and adults (Figure 10). In the context of urban planning, those who made the final decisions are mainly adults who occupy managerial positions. Such experts would make final decisions in terms of territorial sustainability, therefore, they are thinking about future generations. However, these managers sometimes have a di fferent vision compared to the generations that they should be protecting. For this reason, participation is an e ffective tool for bridging this gap. #### 5. Conclusions and Recommendation The combination of bottom-up and top-down participation methods can be a tool through which urban planning can drive the transformation or evolution of urban spaces at di fferent institutional levels. It can increase the interactions between citizens in a vision that "unites and inspires" to develop urban quality space helping the decision-makers to identify hypotheses of territorial development that is more suitable on the basis of present and future scenarios of economic, environmental, and social evolution. The study shows that in the socio-ecological system, the bottom institutional levels can introduce innovation or new vision in the use of free urban space and, therefore, bottom-up participation can push or trigger the evolution of the urban ecosystem, while the top institutional level drives the change from the top-down using the bottom-up participation information in planning actions between decision-makers. Therefore, considering the adaptive cycle in social–ecological systems, the bottom-up activity can be considered as a "revolt" process that a ffects the "urban space", changing it from a "conservation (K)" phase, where the urban space can be kept in a state of no-social use contributing in the degradation of identities of urban area, into a "collapse or release" ( Ω) phase that stimulates new social uses of urban space. Therefore, the bottom-up can support the "reorganization" ( α) phase to create situations able to drive innovation, considering economic and social processes. Top-down, in this case, plays a crucial role in determining and designing the new pattern of the urban space. The system will jump into a new adaptive cycle and therefore in new urban use with new environmental, social, and economic characteristics without losing the urban identity (Figure 14). Figure 14. Contribution of the combination of bottom-up and top-down participation approaches in transdisciplinary planning and design of the urban space evolution considered as the socio-ecological system [10–13]. Therefore, the main aspect for the success of the bottom-up and top-down approaches is the creation of feedback between scientific knowledge derived from experiences and studies not directly connected to the characteristics of the study area and non-scientific visions deriving from those who live in the area, who express their opinions and advice based on their own life experiences. The bottom-up and top-down participation approaches can represent the base for transdisciplinary planning and design as they are useful to identify and correlate the ecological urban level and institutional levels integrating di fferent cultural, knowledge, and generational needs, allowing the development of a holistic vision of the evolution of the urban space (Figure 14). This is important because it allows for a vision of the development of the area that is not conditioned only by the cultural background of the decision-makers, but of those who use the territory, in an e ffort to meet the needs of everyday life. (Figure 14). Therefore, this approach can be useful to harmonize the di fferences that can emerge at di fferent institutional levels of the urban space, going from the single individual, the community that uses the area, and the di fferent administrative levels that make the decisions [33,34]. In this way, the participation activities were not seen as an instrument for obtaining maximum consensus, but primarily as an opportunity to take into consideration the di fferent stakeholders' interests and to better deal with urban issues that are not ye<sup>t</sup> well defined. In this paper, the bottom-up and top-down participation approaches are important to combine the need of many stakeholders (single individuals) with the vision of urban development of fewer stakeholders that take the decision (decision-makers). The e ffectiveness of this approach lies precisely in the ability of the decision-makers to review their own position according to the di fferent visions without remaining in a pre-decided position. Without the flexibility of the decision-makers, this approach can fail. Therefore, the main aspect of this approach is not primarily in the techniques used, but in the ability to acquire information and knowledge and make it turn transversely, creating synergy between the various stakeholders who often act and make decisions in isolation. This approach allows for the creation of an urban plan with more "accountability", capable of reinforcing the responsibility of choices, guaranteeing greater insurance to the citizens about the proposed transformations, and giving an account of the choices made to combat the prejudices that accompany urban transformations and making the transformation process more reliable. An aspect of strength of this process is the possibility to analyze conflicts to start an institutional dialogue between decision-makers and final users. In this case, the Superintendence had a central role in starting a productive dialogue, changing critical issues into strong points of the urban plan. Author Contributions: Conceptualization, T.S.; methodology, T.S., R.A., and N.Z., A.L.; software, T.S.; validation, N.Z., A.L., and R.A.; formal analysis, T.S. and N.Z.; investigation, T.S., A.L., and R.A.; resources, T.S. and R.A.; data curation, T.S. and N.Z.; writing—original draft preparation, T.S., R.A., and A.L.; writing—review and editing, T.S., R.A., A.L., and F.S.C.; visualization, T.S., R.A., and A.L.; supervision, F.S.C. and N.Z.; project administration, T.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: I thank all the guys who have given their time to this project. I thank the researcher Sara Invitto for the corrections and suggestions that she made to the paper. I also thank Francesco Dieni for the graphic representation of Figure 13. I thank the Antonie De Vitis and Niko Antonelli for the professionalism and passion with which they worked on the PUUC project. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.402523	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 17 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.19	1. Introduction The conversion of one land cover type to another is one of the most visible and rapid changes that the earth is experiencing and these conversions have profound social and environmental impacts at multiple scales. In the past, the drivers of land cover changes have been classified into two categories: proximate and distant, or indirect [1]. Recently, the phenomenon of tele-coupling between places has been recognized as key to understanding how distant drivers relate to proximate drivers and influence local landscape changes [2–4]. Some of the key drivers of landscape changes include economic, technological, institutional and policy, cultural, and demographic factors [5]. The growth of urban areas leads to land cover change in many parts of the world, especially in developing countries [3,6]. Intense urbanization and increase in anthropogenic activities reflect the scope, intensity, and frequency of human interference, and the changes they cause in ecological processes and systems in the urbanized areas [7]. The urban areas consist of only 1–6% of the earth's land surface, ye<sup>t</sup> they have enormous impacts on the functioning and service of local and global ecosystems, by modifying local climate conditions, eliminating and fragmenting native habitats, generating anthropogenic pollutants, etc. [8]. The spatial pattern of an urban landscape is a result of the interaction between various driving forces including natural and socioeconomic factors [9]. Increasing trends in industrialization and urbanization, along with the migration from rural to urban areas, are the most dominant factors influencing the land cover transformation. In the rural areas, employment opportunities and income are insu fficient, which contributes to large di fferences in income and facility levels between urban and rural areas [10]. In developing countries, new cities are being developed due to human migration, infrastructure development, and growing job opportunities [10,11]. The United Nations (UN) Sustainable Development Goals (SDGs) are a collection of 17 global goals, which include 232 indicators set by the UN General Assembly for the year 2030. These goals are an urgen<sup>t</sup> call for action by all countries—developed and developing—in a global partnership. Pakistan is a signatory to the UN SDGs, and this study analyzes the land use change dynamics of Islamabad Capital Territory (ICT) in the context of the relevant SDGs and indicators. The Goal 11 of the SDGs, "Make cities and human settlements inclusive, safe, resilient and sustainable", with indicator number 11.3.1 "Ratio of Land Consumption Rate to Population Growth Rate (LCRPGR)" [12] is an important parameter for analyzing sustainability of land use and land change with population growth. The LCRPGR parameter is actually based on the previously defined parameters of LCR [13,14] and PGR [15], and several studies of urban areas have been carried out before on the basis of these parameters. The LCRPGR parameter and terminology has been given prime importance now after being linked with the UN defined SDGs. LCRPGR is vital to understand the rate of land change as compared to the population boom, to understand historical land consumption traditions, and to guide decision and policy makers on the planned expansion of the city along with the protection of environmental, social, and economic assets. Another SDG, Goal 15, "Protect, restore and promote sustainable use of terrestrial ecosystems, sustainably manage forests, combat desertification, and halt and reverse land degradation and halt biodiversity loss" emphasizes the protection of tree cover and sustainable managemen<sup>t</sup> of forests. The SDG indicator number 15.2.1, "Progress towards sustainable forest management" [16] calls for sustainable managemen<sup>t</sup> strategies for conserving the forest cover, enhancing environmental education, and engaging a wide range of stakeholder institutions, policies, regulations, and considerations that promote sustainability and utilization of natural resources at multiple spatial scales [17]. Assessment and monitoring of land cover dynamics is essential for the sustainable managemen<sup>t</sup> of natural resources, environmental protection, biodiversity conservation, and developing sustainable livelihoods. Therefore, the development of applicable and systematic methods for producing and updating land cover databases are considered an urgen<sup>t</sup> need [18]. Around the globe, urban land expansion rates are higher than or equal to urban population growth rates [19–21]. Many research studies focus on big cities and metropolises, where increase in population through analysis of census statistics is directly linked with the urban land expansion; however, these statistics do not provide information regarding spatial distribution, pattern, and scale of urban land use change. Multi-temporal land cover change analysis and simulation based on coarse to very high resolution satellite remote sensing images is becoming a well established technique for quantifying changes occurring on the earth's surface, and multi-temporal aerial and satellite datasets are now widely and continuously being used for urban growth mapping, monitoring, and modeling with a focus on the spatial dimension and structures [22–25]. In urban expansion studies, spatiotemporal analysis of land cover and land use changes has helped towards understanding the underlying natural and socio-economic factors and drivers. For instance, Seto et al. [26] have presented a meta-analysis of 326 studies which used temporal satellite images to map urban land conversion. A total of 58,000 km<sup>2</sup> increase in urban land area was reported in thirty years (1970 to 2000) and by 2030, global urban land cover is expected to increase between 430,000 km<sup>2</sup> and 12,568,000 km2, with an estimate of 1,527,000 km<sup>2</sup> more likely. According to Seto et al. [26], across all regions and for all three decades, urban land expansion rates are higher than or equal to urban population growth rates. Yang et al. [27] studied and reported the evidence of urban agglomerations through satellite images in four major bay areas of US (San Francisco and New York), China (Hong Kong-Macau), and Japan (Tokyo), from 1987 to 2017. Clarke et al. [28] proposed a framework to combine remote sensing and spatial metrics for improved understanding and representation of urban dynamics to come up with alternative conceptions of urban spatial structure and change. Particularly with regards to urban forestry, a few studies have focused towards the assessment, mapping, and monitoring of urban forest parameters in fast-growing cities of developing countries. For example, Gong et al. [29] carried out a 30-year forest fragmentation study over Shenzhen Special Economic Zone (SEZ), a city which was established in 1979 in Southern China. Huang et al. [30] utilized satellite images of 77 metropolitan areas in Asia, US, Europe, Latin America, and Australia to calculate and analyze seven spatial metrics (area weighted mean shape, area weighted mean patch fractal dimension index, centrality, compactness index, compactness index of the largest patch, ratio of open space, and density). According to their analysis, the compactness, density, and regularity of urban areas in developing regions generally exceeded the levels reported throughout developed countries [30]. Dewan et al. [31] studied the dynamics of land use/cover changes though landscape fragmentation analysis in Dhaka Metropolitan, Bangladesh, computing and analyzing the following metrics: Number of patches, Patch density, Landscape shape index, Largest patch index, Mean patch size, Area-weighted mean fractal dimension, Interspersion and juxtaposition, Contagion, and Shannon's diversity index. In Pakistan, like other developing countries, most urban development is haphazard, typically lacking appropriate planning strategies [10]. Pakistan's urbanization rate is the highest in South Asia, and by 2030, Pakistan will have more people in cities than in rural areas. Growing population and rapid development is causing prime agricultural land to be encroached and also causing loss of tree cover [32–36]. In the late 1960s, the capital of the Islamic Republic of Pakistan was shifted from Karachi to Islamabad (o fficially named Islamabad Capital Territory (ICT)). The masterplan of ICT was developed by the famous Greek architect and town planner C. A. Doxiadis [37]. In terms of a planned new capital, ICT is similar to planned new post-colonial capitals/relocations as in the cases of Brasilia (Brazil), Nur-Sultan (named as Astana from 1998 to 2019, and Akmola previously) in Kazakhstan, and Canberra (Australia) [38–40]. In the recent decades, with various ongoing development activities, ICT has been struggling with rapid urbanization and gigantic levels of pollution from industrial, residential, and transportation sources. In terms of population, ICT is considered as the most diverse city of Pakistan with a large percentage of immigrants and foreigner population [41]. Unprecedented influx of migrants and population increase has resulted in urban sprawl and conversion of fertile agricultural land and green cover into concrete—a clear deviation from the original ICT master plan [39,40]. Uncontrolled population growth in ICT due to rapid urbanization has deteriorated the living environment, and increased the adverse ecological impacts on human health, flora, and fauna [42]. #### 1.1. Literature Review—ICT Mapping and Monitoring In the last 20 years, several studies have been conducted on the ICT, regarding land cover change, biomass estimation, water quality monitoring, and temperature increase using satellite datasets. In this section, we present a synthesis of published work regarding land cover change dynamics in the ICT. Adeel [43] identified urban growth potential through land use for ICT zone IV (Figure 1), based on SPOT-5 2.5 m panchromatic dataset and population census data, and found that nearly 63% of zone IV carries a 'High' to 'Very High' future growth potential, which is mainly located close to Islamabad Expressway. This work uses satellite imagery and field data from one year (2007) and does not report spatiotemporal change dynamics. Butt et al. [35] studied the metropolitan development in ICT, based on growth direction and expansion trends from the city center, for the period 1972–2009 using Landsat satellite images. Using Principal Components Analysis (PCA), band ratios, and supervised classification methods, they found that the urban development had expanded by 87.31 km<sup>2</sup> in 38 years. Butt et al. [44] conducted a study on land cover change analysis over Simly dam watershed, ICT; the results derived from maximum likelihood supervised classification showed tree cover loss of up to 26% and 6% increase in settlements from 1992–2012, based on Landsat 5 TM and SPOT-5 imagery, respectively. Similarly, the watershed analysis of Rawal dam, ICT using Landsat 5 TM imagery, showed 3% degradation of tree cover and 2% gain of settlement from 1992–2012 [45]. Another ICT land cover change dynamics study conducted by Hassan et al. [46] utilized 30 m Landsat 5 TM data for 1992 and 2.5 m SPOT-5 data for 2012, using the maximum likelihood algorithm for image classification. The study revealed a decrease in forest cover of approximately 49% and over 213% gain of settlement area from 1992–2012. Sohail et al. [47] conducted a study to assess the water quality index and analyze the major change in land cover types, vegetation cover, rate of urbanization and its possible impact on groundwater resources, vegetation, and barren land. They used Landsat images for the years 1993, 1997, 2002, 2007, 2013, and 2017 for the assessment and mapping of land cover dynamics; according to their findings, from 1993 to 2017, vegetation areas decreased by 101.77 km2, surface water was reduced by 1.10 km2, barren land was reduced by 2.90 km2, while built-up lands expanded by 105.77 km2. A comparison of Beijing, China and ICT for the role of vegetation in "controlling the eco environmental conditions for sustainable urban environment" was performed by Naeem et al. [42], where they used Gaofen-1 (GF-1) and Landsat-8 Operational Land Imager (OLI) satellite imagery with 8 m and 30 m spatial resolution, respectively. They evaluated various scenarios and models for future development to predict future spatial patterns in both cities. Another study was conducted by Naeem et al. [48] to study the association between green space characteristics, analyzed through landscape metrics, and land surface temperature for sustainable urban environments comparing Beijing, China and ICT. Khalid et al. [49] conducted a study to quantify the decline of forest reserves and associated temperature variations in a relatively unexplored biodiversity hotspot of ICT, the Margalla Hills National Park (MHNP). In this work, Landsat satellite imagery from 1992, 2000, and 2011 was used to monitor the changes in forest cover and statistical significance tests were used to determine the significance of temperature variation associated with a shift in land cover classes. The study finds that deforestation and forest degradation by local communities is an ongoing practice in MHNP; this necessitates the promotion of conservation practices to minimize ecological disturbances here [49]. Batool and Javaid [50] carried out a study on the assessment of Margalla Hills forest by using Landsat imagery for years 2000 and 2018, and report that the forest cover has decreased from 87% in 2000 to 74% in 2018, whereas built-up area has increased from 5% in 2000 to 7% in 2018, and open land in the study area increased from 2% in 2000 to 7% in 2018. Mannan et al. [51] conducted a study using Landsat imagery, Markov Chain, and Cellular Automata on Margalla Hills, focusing on the quantitative assessment of spatiotemporal land use and land cover changes during 1998, 2008, 2018, and a simulation of 2028. In addition, a forest inventory survey was conducted for biomass and carbon sink estimations. This work shows that the forest area has reduced from 409.36 km<sup>2</sup> to 392.31 km<sup>2</sup> and settlement area has increased from 14.97 km<sup>2</sup> to 39.66 km<sup>2</sup> from 1998 to 2018. The average yearly biomass and carbon losses were 50.34 Gg/ha/yr and 31.33 Gg C/ha/yr, respectively. The ICT is a relatively new and spatially heterogeneous city surrounded by the Himalaya mountainous dense forest as compared to other fast growing and expanding cities like Dhaka, Bangladesh [31,52–55], New Delhi, India [56], Beijing, China [42,48,57,58], Shanghai, China [59–61], Tokyo, Japan [27,62], etc. Based on the literature review, we observed that most studies of the ICT land cover dynamics have used di fferent remotely sensed data, methods, definitions, and classification schemes, and have provided diverse results. Most studies which have analyzed the land change dynamics in the ICT focus on the overall analysis of land-cover and land-use change, and a detailed analysis of the landscape ecology and urban forestry characteristics is missing. There has further been very little focus in these studies towards urban landscape metrics and indicators of sustainable urban growth such as LCRPGR. #### 1.2. Study Objectives In this paper, well established, proven, and articulated research methodology, satellite datasets, and definitions of features were adopted with the goal to systematically achieve the following defined objectives: #### 2. Study Area The ICT is the capital city of Islamic Republic of Pakistan (Figure 1) located in the Potohar plateau. It comprises an area of 906 km<sup>2</sup> including mountains and uneven plains exceeding 1,175 m in height above the mean sea level [63]. According to the 2017 national population census, the total population of ICT is approximately two million, which makes it the ninth largest city of Pakistan. It has a humid and sub-tropical climate with four distinct seasons: autumn, spring, summer, and winter. The temperatures vary from 13 ◦C in January to 38 ◦C in June. ICT consists of five planning zones: Zone I, II, and V are reserved for planned urban development, while the remaining two zones, III and IV, are managed as National Park and the rural fringes. Marble and chemical factories, steel mills, flour mills, oil units, pigments, paints, and pharmaceutical manufacturing plants are some of the main industries in the city [34]. Figure 1. Study area map—Islamabad Capital Territory (ICT), Pakistan. The bounded box inset covering Zone I and Zone II partially is representing the area shown in the Figure in Section 5. #### 3. Materials and Methods	doab	2025-04-07T03:56:59.405290	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 19 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.20	3.1. Datasets To study the land cover dynamics and spatial analysis of ICT, various datasets have been collected from primary and secondary sources. The data collected from the primary sources include Landsat medium resolution satellite imagery and field observations using Geographical Positioning System (GPS) receiver, while the secondary or ancillary data consist of population census and ground truth data from Very High Resolution Satellite (VHRS) imagery. #### 3.1.1. Landsat Satellite and Digital Elevation Model (DEM) Data For land cover mapping, we used 30 m spatial resolution orthorectified and cloud free Landsat Multispectral Scanner System (MSS), Thematic Mapper (TM), Enhanced Thematic Mapper Plus (ETM+), and Operational Land Image (OLI) sensor images. The TM, ETM+, and OLI sensors have the same 30 m spatial resolution, while the MSS has a spatial resolution of 57 m. The overall ICT region is covered within one Landsat scene (185 × 185 km). A total of five images for years 1976, 1990, 2000, 2010, and 2016 were downloaded from the United States Geological Survey (USGS) Earth Resources Observation and Science (EROS) archive. All satellite data was collected for the months of October and November, to avoid cloud cover (Table 1). Table 1. Landsat images used for land cover mapping. A 30 m spatial resolution Shuttle Radar Topography Mission (SRTM) Digital Elevation Model (DEM) from USGS was used to understand the topography of the area and to prepare the study area maps. #### 3.1.2. Population Census Data For the calculation of SDG Goal 11 indicator 11.3.1 LCRPGR, we used the data collected from secondary sources including demographic data (primary census abstracts for the year 1972, 1998, and 2017) from the Pakistan Bureau of Statistics. We used actual census data of 1972 and 1998 to determine the shape of the population curve, which was exponential, as expected. Using the fitted exponential regression function, we derived the growth rate through the exponential function derivative—Equation (1), and then Equation (2) was utilized to determine the approximate population for the year 1976. The population data used for the calculation of LCRPGR is given in Table 2. $$\text{Groupth rate} = (\text{ab})\text{e}^{\text{ac}} \tag{1}$$ $$\text{Pop}\_{1976} = \text{Pop}\_{1972} + 4 \times \text{Groupth rate} \tag{2}$$ where a and b are regression coe fficients, c represents the population variable, POP1976 = Estimated Population in 1976, and POP1972 = Past Population in 1972. Table 2. Population data of ICT for 1972, 1976, 1998/2000, and 2016/2017. #### 3.1.3. Ground Truth Data In this study, we collected ground truth data from two sources: the primary ground data was collected using a Global Positioning System (GPS) receiver in October 2016 during field survey at various locations of ICT, while for the secondary ground data we relied on sub-meter VHRS imagery from Google Earth. A total of 205 samples were gathered, which were utilized as reference data for the satellite image classification and accuracy assessment of results. Using the Google Earth temporal VHRS data, we visually identified and geo-tagged the sites witnessing land degradation, urban development, and tree loss. The geo-tagged dataset helped to validate Landsat-based land cover changes.	doab	2025-04-07T03:56:59.406694	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 20 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.21	3.2. Methodology Our methodology (Figure 2) on land cover assessment and spatial analysis of ICT consists of (i) pre-processing of Landsat images, (ii) classification of Landsat images, (iii) accuracy assessment of land cover products, (iv) temporal land cover assessment, (v) urban landscape metrics analysis, (vi) urban forest fragmentation analysis, and (vii) LCRPGR calculation. Figure 2. Methodological flow chart. #### 3.2.1. Pre-Preprocessing Landsat Images Pre-processing steps for Landsat images included Top of Atmosphere (ToA) reflectance conversion, terrain illumination correction, and layer stacking of spectral bands. A reduction in between-scenes variability was accomplished through normalization for solar irradiance with a two-step process. First, we converted all pixels' Digital Numbers (DNs) to radiance values using the bias and gain values, which were scene-specific and given in the metadata file of the respective scene. Second, we converted radiance data to ToA reflectance [64]. Terrain illumination correction was conducted using an empirical rotation model proposed by Tan et al. [65]. Layer stacking was performed to construct multi-spectral images at 30 m spatial resolution. The false color composite (FCC) of bands 542 from the multi-spectral images was used to visually interpret land cover features. #### 3.2.2. Classification of Landsat Satellite Images The image classification procedure is to instinctively categorize all pixels in an image into land cover classes [66]. In this study, the maximum likelihood classification method was used for land cover-mapping from Landsat images. The maximum likelihood classification method is a supervised classification technique, which works on the basis of multivariate normal probability density function of categories, and utilizes both the variance and co-variance of the spectral response of each unknown pixel to assign it to a particular category [67,68]. In this study, five land cover classes were specified: Tree cover >40% canopy, Tree cover <40% canopy, Settlement, Soil, and Water. Almost 35 training samples were collected for each land cover class to classify the various Landsat images. The classified objects with an area smaller than the Minimum Mapping Unit (MMU) (i.e., 1 ha ~ 3 × 3 pixels) were fused with the neighboring land cover classes [69]. We adopted on-scene digitization technique for land cover change detection. First, we overlaid 2016 base land cover map on the multi-spectral Landsat image of 2010. We traced patches where land changes had occurred, leaving unchanged patches unmodified for consistency [70]. We followed the similar approach to detect land cover change between 2000 and 2010, using 2010 land cover map as a base layer. This process was repeated to generate land cover change maps between 1976–1990, 1990–2000, 2000–2010, and 2010–2016. #### 3.2.3. Accuracy Assessment of Land Cover Products Assessment of classification accuracy of 1976, 1990, 2000, 2010, and 2016 land cover maps was carried out to determine the quality of information derived from the data. Random sampling method was adopted to assess the accuracy of satellite derived land cover products. Accuracy was assessed using 125 reference points, based on ground truth data and satellite visual interpretation. The comparison of classification results and reference data was carried out statistically using error matrices. In addition, the non-parametric kappa statistic was computed for each classified map to measure the accuracy of results, as it not only accounts for diagonal elements but also for all elements in the confusion matrix [71]. #### 3.2.4. Temporal Land Cover Assessment In this study, within the 906 km<sup>2</sup> total area of ICT, the land cover maps (1976, 1990, 2000, 2010, and 2016) were compared in terms of the area. According to Puyravaud [72], the annual rate of change is based on the compound interest law, considering non-linear change across the timeline to estimate the percentage change per year. In this study, the annual rate of change was calculated using Equation (3) proposed by Puyravaud [72]. $$r = \left(\frac{1}{t\_2 - t\_1}\right) \times \left(\ln \frac{A\_2}{A\_1}\right) \times 100\tag{3}$$ where r is the annual rate of change in percentage, and A1 and A2 is the area at earlier time t1 and later time t2, respectively, and ln denotes the natural logarithm function. #### 3.2.5. Urban Landscape Metrics Analysis Landscape metrics are used in this study to quantify the spatial patterns of land cover categories. The landscape metrics can be defined as quantitative and comprehensive measurements showing spatial diversity at a specific scale and resolution [73,74]. In this study, landscape metrics analysis was performed at land cover class level to quantitatively analyze spatial structures and patterns of the topographically and biophysically heterogeneous and diverse landscape of the ICT [74]. Under the three habitat categories Patch Density and Size, Shape and Edge, and Proximity/Isolation, a total of nine landscape metrics were used to quantify structural changes: Number of patches (NP), Mean Patch Size (MPS), Largest Patch Index (LPI), Mean Radius of Gyration (MRG), Mean Shape Index (MSI), Edge Density (ED), Mean Perimeter to Area Ratio (MPAR), Mean Euclidean Nearest Neighbor Distance (MED), and Mean Proximity Index (MPI) (Table 3). The landscape metrics were calculated using the FRAGSTATS v4 software tool. The output of the landscape metric analysis depends upon the spatial resolution of the data [75]; in this particular study, 30 m spatial resolution data has been chosen from 1990 to 2015 and 60 m spatial resolution for 1976. Table 3. Land cover class level description of landscape metrics used in this study. #### 3.2.6. Urban Forest Fragmentation Analysis Forest fragmentation is the splitting up of large contiguous forest fields into smaller or less contiguous areas. A number of events or activities can lead to forest fragmentation including road formations, woodcutting, forest conversion to agriculture, forest fires, and human conflict over forest patches [25]. To assess forest fragmentation in ICT, the land cover map is divided into two major categories: forest and non-forest. Forest class consists of tree cover greater and less than 40% canopy cover while non-forest class comprises of settlement, soil, and water land cover classes. The outcome of forest fragmentation analysis was represented into six categories: Patch, Edge, Perforated, Core (<250 acres), Core (250–500 acres), and Core (>500 acres). These categories are signs of forest ecosystem quality and can be used to estimate the amount of fragmentation present in a landscape and the potential habitat impacts [25,76]. #### 3.2.7. Ratio of Land Consumption Rate to the Population Growth Rate (LCRPGR) Calculation One of the goals of this study is the calculation of the SDGs indicator 11.3.1 Land Consumption Rate to the Population Growth Rate (LCRPGR), which aims at monitoring and measuring urban development by comparing the urban expansion rate with the population growth rate on similar temporal and spatial scales [12,77]. If the LCRPGR ratio value lies between 0 ≤ LCRPGR ≤ 1, it shows the simultaneous increase of population growth rate (PGR) and land consumption rate (LCR), but the land consumption rate is much slower than the population growth rate. On the other hand, if LCRPGR > 1, it reflects the simultaneous increase of PGR and LCR, with a faster LCR than the PGR. To estimate the LCRPGR, satellite data derived land cover maps were used for the years 1976, 1998/2000 and 2016/2017, and census data was used for the specific years (Table 2). The indicator 11.3.1 was assessed at the local level for ICT using the mathematical expressions currently proposed by UN-Habitat, given below in Equations (4)–(6) [77]: $$\text{LCRPGR} = \frac{\text{Land Consumption Rate}}{\text{Population Growth Rate}} \tag{4}$$ $$\text{Land Consumption Rate} = \frac{\ln(\frac{\text{Urb}\_{l+n}}{\text{Urb}\_l})}{\text{n}} \tag{5}$$ $$\text{Population Growth Rate} = \frac{\ln(\frac{\text{Pop}\_{t \to n}}{\text{Pop}\_{t}})}{\text{n}} \tag{6}$$ where, ln = Natural logarithm, Urbt<sup>+</sup>n = Surface occupied by urban areas at the final year (t + n). Urbt = Surface occupied by urban area at the initial year (t), Popt<sup>+</sup>n = Population living in urban areas at the final year (t + n), Popt = Population living in urban areas at the initial year (t), and n = Number of years between the two time intervals.	doab	2025-04-07T03:56:59.406968	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 21 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.22	4. Results #### 4.1. Accuracy Assessment of Land Cover Products According to the confusion matrix analysis, 90% overall accuracy and kappa coefficient value of 0.85 was attained for the 2016 classified map. Similarly, overall classification accuracy levels of 88% with kappa coefficient of 0.84 for 2010, 86% with kappa coefficient of 0.82 for 2000, 85% with kappa coefficient of 0.81 for 1990, and 83% with kappa coefficient of 0.79 for 1976 image classifications were achieved (Table A1 in Appendix A). #### 4.2. Temporal Land Cover Assessment The overall settlement in the ICT increased while tree cover classes (greater and less than 40% tree canopies) decreased in forty years (1976 to 2016). The estimated land cover area for each image is summarized in Table 4 and spatial distributions are presented in Figure 3. Table 4. Land cover 1976, 1990, 2000, 2010, and 2016 assessment based on satellite images. Figure 3. Land cover maps for 1976, 1990, 2000, 2010, and 2016. The settlement class in ICT increased from 29.99 km<sup>2</sup> (3.31%) in 1976 to 170.40 km<sup>2</sup> (18.80%) in 2016. The tree cover >40% canopy class showed a slight increase, 182.87 km<sup>2</sup> (20.18%) in 1976 to 192.19 km<sup>2</sup> (21.21%) in 1990 and then declined to 132.27 km<sup>2</sup> (14.59%) in 2016. Tree cover <40% canopy land cover class faced overall decline from 1976 to 2016: 417.03 km<sup>2</sup> (46.02%) in 1976 to 342.94 km<sup>2</sup> (37.85%) in 1990, increase from 1990 to 2000 to 399.31 km<sup>2</sup> (44.07%), while again decreased from 2000 to 2016 to 306.53 km<sup>2</sup> (35.72%). The other two land cover classes, soil and water, observed fluctuating trends in the forty year period. The soil area of 270.27 km<sup>2</sup> (29.83%) in 1976 increased to 317.91 km<sup>2</sup> (35.08%) in 1990 and then reduced to 257 km<sup>2</sup> (28.37%) in 2000, then further decreased to 254.34 (28.02%) in 2010, and again increased to 289.45 km<sup>2</sup> (33.82%) in 2016. The change in water area was not significant though it has shown fluctuations during the study period; it increased from 5.86 km<sup>2</sup> (0.64%) in 1976 to 8.44 km<sup>2</sup> (0.94%) in 1990, and to 10.51km<sup>2</sup> (1.16%) in 2000, then decreased to 6.29 km<sup>2</sup> (0.69%) in 2010, and then increased slightly to 6.51 km<sup>2</sup> (0.76%) in 2016 (Table 4). Based on the temporal land cover data in the ICT, urban landscape settlement increased at an annual rate of 4.34% since 1976, with the highest annual rate of 8.79% during 2000–2010. The tree cover >40% canopy decreased at an annual rate of 0.81% between 1976 to 2016 and tree cover >40% canopy declined at an annual rate of 0.77% in forty years (1976–2016). The tree cover >40% canopy witnessed the highest loss rate at 2.38% per annum between 2000–2010, and tree cover <40% canopy experienced tree cover loss at 2.72% during 2010–2016 (Table 5). Table 5. Land cover annual rate of change (% change per year) from 1976–1990, 1990–2000, 2000–2010, 2010–2016, and 1976–2016. #### 4.3. Urban Landscape Matrix Analysis Landscape matrix analysis was performed at class level, and the results for each class are reported in Figure 4 and Table A2 in Appendix A. The parameters Number of patches (NP), Largest Patch Index (LPI), Edge Density (ED), and Mean Proximity Index (MPI) for the settlement class overall increased from 1976 to 2016. Largest Patch Index (LPI) increased to 16% in 1990 from 13% in 1976, and then reduced to less than 10% in 2000, 2010, and 2016. For tree cover greater than and less than 40% canopy, Number of patches (NP) and Edge Density (ED) initially increased from 1976 to 2000, and then declined from 2000–2016. For tree cover greater than and less than 40% canopy, soil, and settlement classes, the Mean Patch Size (MPS) and Mean Euclidean Nearest Neighbor Distance (MED) sharply declined from 1976 to 1990 and remained lower. In the Mean Euclidean Nearest Neighbor Distance (MED), tree cover greater than and less than 40% canopy, soil and settlement classes remained less than 250 m throughout from 1976 to 2016. From 1976 to 2010, the Mean Radius of Gyration (MRG) decreased for tree cover greater than and less than 40% canopy, and then slightly increased during the years 2010 to 2016. For tree cover >40%, the canopy Mean Proximity Index (MPI) slightly increased between 1976 to 2016 while for tree cover >40%, the canopy MPI decreased from 1976 to 1990, grew between 1990 to 2010, and then again sharply reduced from 2010 to 2016. In all the land cover classes, the Mean Shape Index (MSI) value remained greater than 1 with slight fluctuations. In the Mean Perimeter to Area Ratio (MPAR), the settlement class increased from 640 in 1976 to 1000 in 1990 with slight increase till 2016. Figure 4. Land cover class level landscape matrix parameters graphs for 1976, 1990, 2000, 2010, and 2016. #### 4.4. Urban Forest Fragmentation Analysis The forest fragmentation analysis results are presented in Figures 5 and 6, and Table A3 in Appendix A. The analysis shows that the overall core forests of >500 acres decreased from 391.98 km<sup>2</sup> (65.41%) in 1976 to 241.44 km<sup>2</sup> (40.29%) in 2016. In the forty years' time span, the patch class increased to 20 km<sup>2</sup> (4.54%) in 2016 from 14.74 km<sup>2</sup> (2.46%) in 1976. The perforated forest fragmentation class was 35.21 km<sup>2</sup> (5.88%) in 1976, which increased 41.79 km<sup>2</sup> (7.23%) in 2000, and then again declined to 22.74 km<sup>2</sup> (5.18%) in 2016. For the edge class, a minor increase was observed from 1976 to 2000 and then a drop in 2016. Figure 5. Forest fragmentation maps for 1976, 1990, 2000, 2010, and 2016. Figure 6. Forest fragmentation graphs for 1976, 1990, 2000, 2010, and 2016. #### 4.5. Ratio of Land Consumption Rate to the Population Growth Rate (LCRPGR) For the years 1976–2000, the LCRPGR ratio (SDG indicator 11.3.1) was obtained to be 0.62, which highlights the simultaneous increase of Population Growth Rate (PGR) and Land Consumption Rate (LCR), but the LCR is much slower than the PGR. For the years 2000–2016, the LCRPGR ratio was obtained to be 1.36, which reflects the simultaneous increase of PGR and LCR, with a faster LCR than PGR.	doab	2025-04-07T03:56:59.407599	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 22 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.23	5. Discussion The present study was conducted in order to quantitatively analyze the landscape change dynamics in the ICT from 1976 to 2016 using medium spatial resolution Landsat images. In this study, we observed an increase in settlements over the last forty years. The dramatic land cover change in settlements is exerting severe pressure on other land cover classes, particularly tree cover and soil. Already existing urban areas can be seen expanding through rapid construction of residential blocks in the form of housing societies, industrial blocks and road expansions, leading to horizontal and vertical developments in the city and development of lavish farm houses in the vicinity areas (Figure 7). This rapid increase in urbanization is linked also with migrations. Urban growth may have positive or negative impacts on the environment but unplanned growth of urban areas has negative e ffects. For the economic development of the country, necessary planning is required to make urbanization helpful, as social, health, and environmental issues often accompany the process of urbanization. In the time span of forty years, an overall decrease has been observed in the area of tree cover classes (i.e., greater than and less than 40% tree canopy) in the ICT, and most of the tree loss has occurred after the year 2000 with a corresponding increase in built-up areas. Apart from tree cutting, another important reason behind rapid tree loss or degradation is forest fires. In the Margalla Hills, forest fires usually take place during dry hot climate conditions when there is no rain for months and temperature goes up to 45 ◦C. According to Khalid and Ahmad [78], a total of 320 forest fires were recorded from 2002 to 2012 and approximately 8 km<sup>2</sup> area go<sup>t</sup> burnt as a result. In the ICT, due to uncontrolled urbanization and lack of awareness, huge tree loss has been observed in the last sixteen years, i.e., 2000–2016. Another factor for forest degradation is uncontrolled grazing of livestock [78]. As such, there are no adequate plans or manageable methods to stop grazing activities. For ecotourism and public awareness drives, a number of jogging and hiking trails have been formed in Margalla Hills National Park, and a large number of visitors has severely a ffected the Margalla Hills National Park by dumping waste. These illegal and unmonitored activities in the forested area cause threats to the forest ecosystem [50,79]. Massive migrations have occurred over the past few years from rural to urban areas, mostly due to low cultivated land output, landlessness, sub-division of land, poor economy, and better educational and health opportunities in urban areas. The rapid increase in population has contributed towards natural resource depletion and rapid deforestation close to settlements [80]. The LCRPGR parameter is an indicator of urban sustainable development, whether urban expansion is in balance with population growth or not. According to literature review, limited scientific peer reviewed studies have been reported on the monitoring and mapping of LCRPGR. Under each SDG, a number of targets and indicators have been defined, which countries have to quantify, but most of the developing countries do not have comprehensive databases through which they can compute, quantify, and report the SDGs indicators. To the best of the authors' knowledge, only Nicolau et al. [77] and Wang et al. [81] have computed and reported scientific results of SDG proposed LCRPGR over urban areas. Nicolau et al. [77] based their study over the mainland of Portugal, while Wang et al. [81] carried out their study over mainland China, using earth observation and population census data, and reported the increase of LCRPGR value from 1.69 in 1990–2000 to 1.78 in 2000–2010. The LCRPGR related research findings from these studies show that in most cities, both horizontal and vertical urban expansions are carried out in an unplanned manner which has already e ffected the equilibrium of land consumption versus population increase to attain e ffective development goals by 2030 [77,81]. In this study over the ICT, the LCRPGR ratio was 0.62 from 1976 to 2000, which increased to 1.36 from 2000 to 2016. Based on studies conducted on the global scale, in the most of the cases LCR is higher than or equal to PGR due to high demand of luxurious occupancies in the urban areas [26,30]. Figure 7. Temporal Landsat satellite images of 1976, 2000, and 2016 in standard False Color Composite (FCC), illustrating an overall increase in built-up areas over the years in the ICT. The area of the ICT shown here represents the bounded box inset covering Zone I and Zone II partially in Figure 1. The Shenzhen SEZ and ICT cities were both developed in late 1970s. In terms of urban forest cover change, Shenzhen SEZ had been restored to ~85% (1973–2005) [29] while in this study, we detected urban forest loss of ~27% (1976–2016) in the ICT. In both cities (SEZ and ICT), urban forest fragmentation results revealed losses in forest patches. In South Asia, the landscape of the ICT resembles strongly with the cities of Kathmandu (the capital of Nepal) and Thimphu (the capital of Bhutan), as they are all cities topographically surrounded by the pine trees. As compared to ICT, Kathmandu and Thimphu cities are older but highly migrant resident populated, vastly encroached, and over grazed. Although many studies have investigated the land cover dynamics of Kathmandu and Thimphu cities using temporal satellite data [82–85], detailed analysis using parameters such as landscape metrics, forest fragmentation, and LCRPGR has not been performed. In this aspect, this study serves as a methodological framework for application and analysis over other similar cities in developing countries. Due to rapid tree loss and urbanization, the ICT has observed rapid spells of dust storms and soil erosion [86]. Soil erosion, dry temperatures, and consequent dust storms have a negative impact in the form of air pollution [87] and land degradation. The soil in ICT and the surrounding areas is shallow and has a clay composition [63]. The alluvial lands and terraces in the area tend to have low agricultural productivity and in the southern and western parts of the Potohar plateau, the soil is thin and infertile [88]. Streams and ravines cut the loose plain and cause erosion and steep slopes. This land is generally unsuitable for cultivation. However, large patches of deep, fertile soil are found in the depressions and sheltered parts of the plateau and these support small forests and agriculture [89]. Butt et al. [45] described the most significant land degradation issues in the Rawal watershed as soil erosion and loss of soil nutrients. They further showed that, between 1992 to 2012, the majority of land that was previously vegetation, bare soil, or water bodies was converted to agriculture and settlements, suggesting increased pressure on natural resources in the Rawal watershed [45]. The results of landscape analysis in this study reveal that the ICT urban landscape has become more heterogeneous, disproportional and diverse, and tree patches have declined. Alarmingly, core forests of >500 acres have declined almost 15% in forty years. Although at the individual level, the residents of ICT, civil society, and the local governmen<sup>t</sup> are trying to recover tree cover loss by planting trees, but these initiatives should be continuous and ongoing on a regular basis to monitor the growth of trees without damaging the existing mature standing trees. The temporal forest fragmentation analysis shows that due to tree cover loss, the three categories of core forest fragmentations (i.e., <250 acres, 250–500 acres, and >500 acres) have decreased in forty years (1976–2016). The loss in forest fragmentation negatively influences the habitat and terrestrial biodiversity of ICT [79,90,91]. Based on landscape metrics analysis over the Dhaka metropolitan, a similar Asian developing city, Dewan et al. [31] revealed that cultivated areas and vegetated lands became highly fragmented with increasing anthropogenic disturbances and urban built up category became aggregated and convoluted. While our analysis of ICT land cover change dynamics in this study is important and unique, there are a few caveats which are worth mentioning. First of all, for the 1976 land cover map, we have relied on approximately 57 m spatial resolution Landsat 3 Multispectral Scanner System (MSS) sensor data which is relatively coarser spatially as compared to Landsat 5 and 8 (i.e., 30 m), which may affect the spatial heterogeneity and accuracy of developed land cover maps. Second, as Landsat is a pioneer Earth Observation (EO) satellite program initiated in 1972, so acquiring remote sensing imagery of the years before that is not possible, and thus we cannot derive land cover maps before the 1960s, when the Islamic Republic of Pakistan's capital shifted from Karachi to Islamabad. Third, from Landsat 30 m medium spatial resolution satellite data, we cannot detect and delineate the boundaries of built-up areas as well as can be done from sub-meter VHRS images available from the 2000s onward. However, the VHRS datasets come with a high cost, especially when acquisitions at multiples times have to be acquired, and may not be therefore feasible for study sites in developing countries. Of course, research in this domain is ongoing with regards to utilization of VHRS data for study of urban areas, using methods like object-based image analysis, machine learning, etc. Fourth, there may be some level of uncertainty for the field measurement data, as there is always a potential for human error especially when ground truth is collected over larger areas. Fifth, in this study, we utilized only temporal optical satellite data, which may cause optical signal saturation in closed canopy forests, and atmospheric effects (i.e., cloud coverage, haze, and smog).	doab	2025-04-07T03:56:59.408038	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 23 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.24	6. Conclusions This study sheds new light on systematic land cover dynamics of ICT over the period of four decades, underlining a major decrease in tree cover along with significant increase in settlements and soil. The findings from the analysis of land cover changes, landscape metrics, forest fragmentation, and LCRPGR, are aligned well and agree overall with previous published work on the regional and global scales. The computation of LCRPGR is vital to understand the rate of land change as compared to the population boom, to understand historical land consumption traditions, and to guide decision and policy makers on planned urban expansion while ensuring the protection of environmental, social, and economic assets. It is indeed a scientifically accepted phenomenon that in most of the cities, the land consumption rate overall is much faster than the population growth rate, due to high demand of luxurious occupancies in the urban areas. Both natural and anthropogenic activities are responsible for land cover changes in ICT. In terms of landscape and fragmentations, the ICT urban landscape ecology is continuously under threat due to the encroachment of housing societies, and influential business community. The findings from urban landscape matrix and forest fragmentation analysis in this study could help ICT Capital Development Authority (CDA) and Islamabad Wildlife Management Board (IWMB) in making strategic decisions to prevent tree loss, forest degradation, and encroachment in the urban landscape of ICT, and also plan future urban growth keeping with the use of remote sensing imagery and geospatial analysis. The methodology outlined in this study is cost-e ffective and could be easily replicable to other countries/regions, especially in developing countries, through integrating freely available satellite images with a 5–10 years interval. Author Contributions: Conceptualization, H.G., W.A.Q.; data analysis, S.A., M.K.; supervision, H.G., visualization, H.G., W.A.Q., S.A., M.K.; writing—original draft preparation, H.G., S.A., W.A.Q., S.M.A.; writing—review and editing, H.G., W.A.Q., S.M.A. All authors have read and approved the final version of this paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This work is an extension of an Undergraduate Final Year Project, which was completed in 2018, and we would like to express our thanks to Institute of Space Technology (IST) managemen<sup>t</sup> and academic sta ff for their support. The authors also would like to acknowledge the editors and three anonymous reviewers for their extensive and critical comments on the paper, which helped to improve the paper immensely. Conflicts of Interest: The authors declare no conflict of interest. The views and interpretations in this publication are those of the authors, and they are not necessarily attributable to their organizations.	doab	2025-04-07T03:56:59.408795	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 24 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.25	Appendix A Table A1. Accuracy assessment of land covers classification for 1976, 1990, 2000, 2010, and 2016. Table A1. Cont. Table A2. Land cover class level landscape matrix parameters results for 1976, 1990, 2000, 2010, and 2016. Table A3. Forest fragmentation results for 1976, 1990, 2000, 2010, and 2016 and percentage assessment based on satellite images).	doab	2025-04-07T03:56:59.409311	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 25 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.27	Article #### Can Rock-Rubble Groynes Support Similar Intertidal Ecological Communities to Natural Rocky Shores? *Paul Holloway 1,2,\ and Richard Field 3 Received: 30 March 2020; Accepted: 24 April 2020; Published: 28 April 2020 Abstract: Despite the global implementation of rock-rubble groyne structures, there is limited research investigating their ecology, much less than for other artificial coastal structures. Here we compare the intertidal ecology of urban (or semi-urban) rock-rubble groynes and more rural natural rocky shores for three areas of the UK coastline. We collected richness and abundance data for 771 quadrats across three counties, finding a total of 81 species, with 48 species on the groynes and 71 species on the natural rocky shores. We performed three-way analysis of variance (ANOVA) on both richness and abundance data, running parallel analysis for rock and rock-pool habitats. We also performed detrended correspondence analysis on all species to identify patterns in community structure. On rock surfaces, we found similar richness and abundance across structures for algae, higher diversity and abundance for lichen and mobile animals on natural shores, and higher numbers of sessile animals on groynes. Rock-pool habitats were depauperate on groynes for all species groups except for sessile animals, relative to natural shores. Only a slight di fferentiation between groyne and natural shore communities was observed, while groynes supported higher abundances of some 'at risk' species than natural shores. Furthermore, groynes did not di ffer substantially from natural shores in terms of their presence and abundance of species not native to the area. We conclude that groynes host similar ecological communities to those found on natural shores, but di fferences do exist, particularly with respect to rock-pool habitats. Keywords:** biodiversity; non-native species; protected species; range expansion; species distributions	doab	2025-04-07T03:56:59.409351	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 27 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.28	1. Introduction Climate change and anthropogenic pressures have fragmented and restricted the distribution of many species worldwide, with significant shifts documented in a vast array of ecological communities [1–4]. In coastal ecosystems, numerous studies have reported a decline in biodiversity [5–12]. For example, Sorte et al. [9] noted a 60% decline in the abundance of the blue mussel Mytilus edulis over the past 40 years along the coastline of Eastern USA and linked this decline with that of several other species within the intertidal community. With at least a billion people expected to live within the lower-elevation coastal zone by 2060 [13] and up to 12.5 million km<sup>2</sup> of natural habitat potentially replaced by 2030 [14,15], there persists a need to identify how novel artificial habitats impact coastal ecology. Introducing hard-engineering structures can negate many of the perceived negative geomorphological and economic impacts of coastal erosion, particularly in urban environs; however, these structures can have significant implications for the configuration of intertidal habitats and biodiversity [16–18]. Studies have predominantly focused on determining whether the communities found across analogous natural shores (largely situated in rural areas) versus artificial structures (largely situated in urban or semi-urban areas) are comparable [18]. The consensus so far is that there is higher diversity on natural shores than analogous artificial rocky sea defences [19–26]. However, investigations have not been conclusive, identifying di fferences across tidal heights [27] and taxa [28,29], while others have found a strong similarity between structures [30,31]. Rock-rubble groynes are commonly implemented hard-engineering structures that run perpendicular to the shoreline, intercepting longshore transport of sediment. The intertidal habitat of these artificial structures o ffers a new rocky habitat that is typically located in predominantly sandy shorelines; however, there are very few studies investigating the ecology of rock-rubble groynes. Studies by Pinn and Rodgers [32] of a natural rocky shore and by Pinn et al. [21] of rock-rubble groynes in Dorset, UK, found higher species richness and abundances of most species on the natural rocky shore at Kimmeridge Bay than on the rock-rubble groynes at the nearby Sandbanks Peninsula. However, the ecological comparison between the structures was not the predominant focus of either study, and there was no statistical analysis beyond the simple comparisons of biodiversity. Similarly, in a study comparing the ecology of eight artificial structures (five of which were groynes) and eight natural rocky-shores in the UK, Firth et al. [23] found higher mean species richness on the natural rocky shores than the artificial structures, with no species unique to the artificial structures. Firth et al. [23] also compared habitats, finding that rock pools supported greater species richness than rock habitats, irrespective of structure. This contrasts with the results of Pinn et al. [21], who found more species on exposed rock than in the pools on the groynes at Sandbanks. The findings of these studies sugges<sup>t</sup> that, while groynes support a lower level of biodiversity than their natural counterparts, they could provide a refuge for intertidal communities found on rocky shores that are under pressure from increasing urbanisation. Conversely, a major criticism of locating hard-bottom artificial structures in soft-bottom areas of coastline is that they can contribute to the decline of barriers (impassable areas of soft-bottom coastline) which isolate distinct regions of rocky shores: removing barriers may enable the dispersal of larvae and propagules of invasive species beyond their natural limits [33–35]. For example, Airoldi et al. [35] found that non-indigenous species were 2–3 times more abundant on artificial structures in part of the North Adriatic Sea, and several other studies have also found artificial structures to support non-indigenous species [36–39]. These studies conclude that there are more non-indigenous species on artificial structures than on nearby rocky shores because they act as points of invasion for many of the non-indigenous taxa. Because of these points of invasion and the resulting creation of stepping-stone dispersal corridors, hard-bottomed artificial structures may pose a serious concern for biodiversity [40]. The UK coastline is one of the most highly human-impacted ecosystems in the world [41]. With the projected coastal urbanisation and climatic changes, there is a pressing need to identify how intertidal communities di ffer between rock-rubble groynes in urban environs and analogous natural rocky shores in rural areas. Theoretically, rock-rubble groynes provide a conservation dilemma. They o ffer an opportunity for the presence of novel rock habitat that could have beneficial implications for populations of under-pressure intertidal species predominantly in highly-impacted urban environments, ye<sup>t</sup> they increase connectivity between isolated rocky shores, which may support populations of native species or increase the potential for non-native species invasion, or both. With only two studies [21,23] having compared the ecology of natural rocky shores with those of rock-rubble groynes (one of these only as an in-passing comment), and with contrasting results found in a single study area (Sandbanks peninsula, Dorset), the questions of whether rock-rubble groynes support ecological communities similar to natural rocky shores, and whether they represent a conservation opportunity or threat, remain open. Furthermore, we still lack basic knowledge of how diverse and abundant the rock-rubble groyne communities are. Here we compare the ecological communities of both exposed rock and rock pools between rock-rubble groynes and nearby rocky shores, using a paired sampling design repeated in three locations around the coast of England. We focus on four main questions: do urban rock-rubble groynes and nearby rural rocky shores (hereafter we refer to the two collectively as rocky 'structure types') di ffer in terms of (1) their species richness and (2) their species' abundances? (3) Are there specific communities found on one of the structure types but not the other? (4) Do the rocky structure types di ffer in terms of presence and abundance of species not native to the area (considering native status at both a country and a within-country level)? In addressing these questions, we investigate the role of rock pools as well as rock surfaces and investigate all macro-organisms found, doing parallel analyses for algae, lichens, sessile animals and mobile animals. #### 2. Materials and Methods #### 2.1. Study Area and Data Collection We selected three stretches of the English coastline that each contained a rural natural rocky shore and urban (or semi-urban) rock-rubble groynes in reasonably close proximity to each other (Figure 1, Table SI1). This gave us three pairs of study sites (Supplementary Information 1), which we sampled in summer 2008. In each site with artificial structures, we randomly selected three rock-rubble groynes of the same age to survey (Table SI1). To control for variations in tidal shore height, we separated each structure into low-, mid- and high-shore sections (we call this variable 'level') based on the mean low- and high-water spring tides. We did not identify tidal height boundaries by the limits of organisms, as often recommended [42] because both physical and biological components influence the zonation of organisms [43], meaning there is the possibility to introduce considerable cross-site error. Figure 1. Location of study sites around the coast of England. Devon, Dorset, and Norfolk are counties. Ipswich and Scarborough are locations mentioned in the text. We used a stratified random sampling technique. With 2 structure types (natural, groyne), 3 counties (Devon, Dorset, Norfolk), 3 replicates of each structure type in each county and 3 levels (high, mid, low) per structure, we had 54 sampling sections in total. Due to the uncertain influence of aspect and exposure on intertidal biological assemblages [21,44], we only surveyed the side of the groynes that did not face the dominant longshore current (therefore sheltered from wave action). Each section of the sampling area was divided up into 0.5 m<sup>2</sup> squares to reduce the chance of recording the same individual twice and selected using a random number generator [42]. On open rock surfaces, we randomly located 12 quadrats in each sampling section, each of 0.25 m × 0.25 m, making 648 rock-surface quadrats in total, and where necessary moved quadrats to avoid sampling very deep crevices or between boulders to allow for consistent surface areas [29]. In each sampling section, we also selected three rock pools (of similar size to the quadrats across structures) in the same way, or all rock pools in the section if fewer than four were present. Ten of the sampling sections (nine of them on groynes) contained no rock pools at all. Overall, we placed quadrats in 123 rock pools: 74 on natural rocky shores and 49 on groynes. In addition, we counted the total number of rock pools present on each entire groyne or equivalent area of natural shore. We did not measure the depth and perimeter of rock pools as is sometimes recommended [42] due to the time constraints associated with the survey, and we acknowledge the limitations of this approach on the analysis in the discussion. We used a non-destructive sampling method, recording species as present if observed within the quadrat. We recorded species richness as the total number of species in each quadrat. For mobile organisms, we recorded abundance as the count of individuals in each quadrat or pool, and for lichens, algae and sessile animals, we recorded abundance as percentage cover using a grid. We grouped the twelve rock quadrats in each sampling section into four sets of three neighbouring quadrats and grouped the three rock-pool quadrats into one set of three rock-pool quadrats (where we had su fficient numbers), with each data value being the arithmetic mean of these quadrats (e.g. the mean number of animal species A across three quadrats). Thus, each unit in the analysis represented a small section of habitat sampled using three quadrats, rather than a single very small quadrat. This was to allow a better representation of the local community in each unit of analysis and to reduce any variation and uncertainty associated with slight di fferences in pool volume and surface area due to the uneven surface of the study areas. Overall, the data analysed included 216 rock surface samples and 46 rock pool samples. #### 2.2. Data Analysis We used a three-way analysis of variance (ANOVA) to explore the di fferences between rock-rubble groynes and natural rocky shores (the 'structure type' explanatory variable). We also included the following factors to account for their expected influence: 'county' (Devon, Dorset, Norfolk—reflecting the pairing of the sites) and [tidal] 'level' (low, mid, high). We used Levene's test to examine the assumption of homogeneity of variance, and we visually examined the model residuals for patterning and tested them for normality using Kolmogorov–Smirnov tests. These diagnostics caused us to square-root transform the response variable in the analyses of species' abundances. We ran parallel analyses for rock and pool habitats. We then used detrended correspondence analysis (DCA) to identify ecological communities and significant environmental centroids within the full species dataset among sites which had species recorded within them. DCA reveals the dispersion of points in ordination space, which reflects species abundances within sampling sites [45]. Due to the high frequency of rare species in pools, analysis was undertaken for both rock and pool habitats together, but rare species were not down-weighted as sometimes suggested [46] due to minimal di fferences in the results when both habitats (rock and pool) were considered together and the importance of the rarer species to ecological communities on natural shores. We chose DCA over other ordination analyses due to the long gradient lengths [47] and the fact that DCA is based on the underlying unimodal model of species distributions [48]—a key foundation of our research questions. We explored the implementation of another method (nonmetric multidimensional scaling: nMDS), but found that the long computation time, coupled with the lack of model convergence and the impact of rare species, made manually exploring model options and subsequent interpretation of the results overly complex. Moreover, results from the nMDS were largely congruen<sup>t</sup> with those of DCA, with the exception of the extreme impact of rare Idotea and Gammarus spp (results not shown). Environmental factors were passively projected on the ordination plot. All analysis was implemented in the open-source software R 3.6.2 [49], with the DCA implemented using the vegan package [46]; see Supplementary Information 2 for data and Supplementary Information 3 for R code. We compared recorded presences in our study with species distribution maps from Gibson et al. [50] and the Marine Life Information Network (MarLIN; [51]) to determine whether we recorded species beyond their distributions, as judged by the two sources. Gibson et al. [50] provide a generalised range of each species, while MarLIN [51] maps are generated from published species records and verified sightings. We chose these sources to correspond with the period of data collection, rather than the most up-to-date distributional data.	doab	2025-04-07T03:56:59.409508	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 28 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.29	3. Results #### 3.1. Species Richness and Abundance We recorded 81 species in total: 27 algae, 6 lichens, 11 sessile animals and 37 mobile animals (Supplementary Information 4). We found 48 species on the groynes and 71 species on the natural rocky shores. Species richness was higher on the natural rocky shores than on the groynes; however, this varied by species group and habitat type (Figure 2a,b). On rock surfaces, species richness on natural shores was significantly higher than that for groynes (Figure 2a, Table 1); however, when species groups were disaggregated, the di fference was not significant for algae and there were significantly more sessile animal species on groynes. In the rock pools, species richness was substantially higher on natural rocky shores than groynes for all species groups except sessile animals (no significant di fference) and lichens (none recorded in pools) (Figure 2b, Table 1). Indeed, the rock pool habitat represents a key di fference between the groynes and the natural rocky shores. Of the 81 species we recorded, 66 were present in rock pools, including 21 only recorded in rock pools. Of these 21, twelve were only in natural rock pools (5 algae, 7 mobile animals), three only in pools on groynes (all were mobile animals) and six in both (5 mobile animals, 1 sessile animal). Species richness varied significantly between counties and across tidal levels, and these factors interacted with structure type to varying degrees to a ffect the magnitude of species richness di fference between groynes and natural rocky shores (Figure 3, Table 1). Figure 2. Comparison of species richness on (a) rock surfaces and (b) rock pools, and of species' abundance on (c) rock surface and (d) rock pools, between rock-rubble groynes and natural rocky shores. Error bars represent 1 standard error of the mean. Abundance for algae, lichen and sessile animals is shown as percentage cover, and for mobile animals is shown as the total count. Table 1. Results of the analysis of variance (ANOVA) testing the e ffects of County, Type, and Level, on average species richness per quadrat, averaged over a set of up to three quadrats. 'Type' is the rocky structure type (natural vs. artificial). 'Level' is the tidal level (low, medium, high). 'County' refers to the three stretches of coastline (Devon, Dorset, Norfolk). \\ p < 0.01, \* p < 0.05. Figure 3. Comparison of species richness on (a) rock surfaces and (b) rock pools, among tidal levels and counties. Error bars represent 1 standard error of the mean. Findings were di fferent when we considered abundance (Figure 2c,d, Table 2). We found no significant di fferences in abundance between structure types for algae or mobile animals in the rock habitat, although in pools the abundances were significantly higher on natural shores. Again, we found a significantly higher abundance of sessile animals on the rock habitat of groynes than natural shores, although this pattern was not significant for pool habitats. Abundance also depended strongly on the tidal level, county, and habitat studied (Supplementary Information 5). For example, in Devon, algal abundances on natural shores increased towards low tide on the rock habitat while it decreased towards low tide in pool habitats. Di fferences also existed across structures, with algal abundances in Norfolk decreasing towards low tide on groynes in pools but increasing on natural structures in pools (Supplementary Information 5). Abundance di fferences between structures were notable for mobile species, particularly in rock pools (F = 56.97; Table 2). The abundance of mobile animals was generally greater on natural rocky shores, with this di fference prominent in Dorset. However, we observed nearly the opposite pattern in Devon, with abundances of mobile animals not significantly di fferent across structures in pools and on rocks at most tidal levels, and abundance higher at low tide for groynes than natural shores. Table 2. Results of the analysis of variance (ANOVA) testing the e ffects of County, Type, and Level, on average species abundance per quadrat, averaged over a set of up to three quadrats. 'Type' is the rocky structure type (natural vs. artificial). 'Level' is the tidal level (low, medium, high). 'County' refers to the three stretches of coastline (Devon, Dorset, Norfolk). \\ p < 0.01, \* p < 0.05. #### 3.2. Species- and Community-Level Analysis Of the 21 species found only in rock pools, most were mobile organisms. Of these mobile species, Idotea granulosa, Gibbula cineraria and Lacuna vincta could potentially survive outside of pools; however, all are sensitive to rapid desiccation or feed on algal species that require water. The other mobile species were either shrimp or fish, which could not survive on open rock. The five species of algae that were only found in rock pools all require sublittoral habitats, except Gelidium pusillum, and were all found only on natural shores. The species found only in pools on groynes were generalist species, which were widely distributed and associated with sandy habitats, such as the brown shrimp Crangon crangon. Species only found on rock surfaces on groynes were the Greenleaf worm Eulalia viridis and the Dahlia anemone Urticina felina, with each only identified at one site: Sidmouth and Sandbanks, respectively. We identified only one Dahlia anemone at Sandbanks, while we found Greenleaf worms (in high abundances) in every low tide quadrat of one groyne at Sidmouth. Both species are habitat-specific, with the anemone requiring crevices and water, and the Greenleaf worm requiring mussel beds. Our dataset contains three species that are of conservation concern (Table 3), with the dog whelk Nucella lapillus occurring in higher abundances on the groynes and the other two at slightly higher abundances on the natural rocky shores. DCA (Figure 4, Table SI4) revealed two main gradients in the di fferentiation of intertidal species composition. The first axis (DCA1) appears to be influenced by type and county and contains the most spread [eigenvalue 0.8114], while the second axis (DCA2) appears to be influenced by tidal level, with less spread [eigenvalue 0.5751]. Distinct communities were observable on the ordination plot. For example, communities found high on axis 1 are dominated by samples from natural shores at high tide and consist of predominantly lichens. Lichens favour areas of high stress and disturbance and are only found on bedrock or boulders [52], so were expected to be found on rocks in the high shore; however, their absence on groynes is notable and is perhaps related to the slow growth rates observed in marine lichens [53]. Species found low on axis 1 are those that occur on groynes in high abundance (e.g., Northern Acorn Barnacle Semibalanus balanoides, Ulva intestinalis) across all sites. Species found at either end of axis 2 include sponges and algae sensitive to desiccation at the lower end and fish and anemones at the higher end. These results sugges<sup>t</sup> that distinct communities of species that are sensitive to desiccation exist at all tidal levels. Overall, this analysis suggested only a slight di fferentiation between communities on groynes versus natural rocky shores, part of which reflects the absence of lichens on the relatively new surfaces of the groynes. Table 3. Total abundance of the three species of conservation concern. For Padina pavonica, the units of abundance are percentage cover averaged across a set of up to three quadrats; for Nucella lapillus and Pomatoschistus minutus they are the total number of individuals per quadrat averaged over a set of up to three quadrats (see Methods). Conservation conventions and legislation: UK Biodiversity Action Plan (UKBAP), the Convention for the Protection of the Marine Environment of the North-East Atlantic (OSPAR), and Bern Convention is the Bern Convention on the Conservation of European Wildlife and Natural Habitats. Figure 4. Ordination plot of detrended correspondence analysis (DCA) results on all species for rock and pool habitats combined. Labels represent species (see Supplementary Information 4 for a full list). Significant (α < 0.01) environmental variables are represented as factor centroids with standard error ellipses. #### 3.3. Species Ranges We observed only two species beyond the ranges recorded for them in the sources we used: the lined top shell Phorcus lineatus at Sandbanks (only just beyond its recorded range [54]) and the small periwinkle Melarhaphe neritoides at Sheringham (closest recorded presence near Ipswich, nearly 140 km away [55]). Our dataset contained only one species not native to Great Britain: Austrominius modestus, identified at Stokes, Sidmouth and West Runton (Table 4). Gibson et al. [50] identified this species as present at all three sites, while Avant [56] recorded this species as only present at Sidmouth and Stokes. When present on groynes, A. modestus was at higher abundances than on the paired natural rocky shores (Table 4). Table 4. Mean abundance of Austrominius modestus on open rock at different shore levels. The unit is the percentage of cover.	doab	2025-04-07T03:56:59.410547	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 29 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.30	4. Discussion The main aim of this investigation was to ascertain the extent to which rock-rubble groynes can support similar ecological communities to natural rocky shores, which several studies have identified as being under pressure [32]. Our findings sugges<sup>t</sup> that although rock-rubble groynes support fewer species of intertidal macro-organisms than natural rocky shores (when measured using small quadrats across groyne-sized extents), these differences are not consistent across habitat (i.e., rock or pool) and tidal level (i.e., high, mid, low). On the exposed rock surfaces, the relatively small difference in species richness between the groynes and the natural rocky shores may reflect no more than a species–area effect over the entire ecosystem, i.e., the total area of rock-surface habitat is relatively small on the groynes compared with the natural rocky shores when viewed at a beach level. On the rock surfaces, the abundance of macro-organisms is, if anything, higher for the groynes. In the pools, it is much lower. Species that only occur on the groynes are associated with the higher abundance of mussels and barnacles (e.g., the Greenleaf worm) or they utilise the sandy habitat that is not present on the rocky shores (e.g., brown shrimp). Alternatively, the species found only on or predominantly on the natural shores appear to be those most sensitive to desiccation (e.g. Desmarestia aculeata). Much of the difference between structures appears to be due to the paucity of rock pools on the groynes, especially complex pools (Table 5). Differences in the complexity of the intertidal zone, particularly in relation to water-retention capacity, may therefore explain some of the results. Natural rocky shores typically have higher complexity, with more microhabitats providing shelter from desiccation and other stresses [43]—for example groves, gullies and cracks, all of which can maintain moisture at low tide. In algae, higher rates of water loss speed up the decline of photosynthesis and respiration [57], and when desiccation past a critical water content occurs, irreversible damage results [58]. Retention of water is also vital for mollusc survival and maintenance [59]. Incorporating rock pool features on artificial structures has been shown to increase the diversity of intertidal taxa, sometimes by four- to nine-fold [24,60–62]. The structure of rock pools on groynes was relatively simple. The predominant formation of pools on groynes was where substrate had become saturated and water had built up against the rocks, with a few pools in the higher tidal level having been formed by chemical weathering. The simplistic structure of these pools meant only certain species (e.g., Palaemon serratus with anti-predatory mechanisms of immobility and cryptic appearance [63]) could survive there. Table 5. Mean numbers of rock pools in the sampling sections (±1 standard error). While clear delineations of communities exist across the tidal levels for rock and pool habitats combined (Figure 4), there was a di fference between generalist species (e.g., P. serratus, Anemonia viridis) at the higher end of axis 2 and specialist species (e.g., Halichondria panicea, Adocia cinerea) at the lower end of axis 2 in the DCA. These results sugges<sup>t</sup> that distinct communities of species that are sensitive to desiccation exist at all tidal levels, but currently the structure of rock pools on groynes only support generalist species. It should be noted that pool depth and perimeter were not measured within this study, following similar approaches used on groynes [21,23], and while precautions were made to minimise variations among pools (e.g., pools of similar size; grouping of quadrats), there is the possibility that di fferences between pools may be due to pool depth and complexity [64,65]. Thus, while our results identified lower biodiversity in rock pool habitats on groynes, there may be potential for artificial rock pool habitats to replicate those found on natural shores, and further research into which species benefit from rock pool presence, as well as which type of rock pools harbour the most species on groynes needs to be undertaken. Furthermore, in times of low amplitude, a characteristic of the diurnal currents in the English Channel (in Dorset) causes the e ffects of the varying topography to become pronounced, causing shallow water harmonics which are responsible for a double-tide [66]. This means that there is a relatively long stand of high water, and for approximately 16 hours of the 24-h cycle, the water level is above the mid-water spring tide level [67]. Consequently, while Sandbanks may not be as complex as a natural shore, it retains water in other ways through this double-tide and the intertidal species are subject to reduced levels of desiccation, which could explain the higher biodiversity observed there (Figure 3, Supplementary Information 5). We felt it was important to include Sandbanks in our study due to the contrasting results found in previous research [21,23], but it should be noted that tidal regimes have a large impact on the ecological communities, and while pairing sites by geographic proximity removed some of this uncertainty, the tidal regime may be a compounding factor within the county variable implemented in the ANOVAs (Tables 1 and 2). The higher algal abundance at Sandbanks may also be explained by the rock surface heterogeneity. Most seaweeds attach to the rock through a combination of etching, gripping or glue, and carbonate rocks tend to support such attachment more than other rock types [68,69], and the physical and chemical substrate properties are known to a ffect organism adhesion and persistence [70]. The Sandbanks groynes are made of limestone, while the groynes at Sheringham and Sidmouth are made of syenite and granite, respectively (Table SI1). However, there was no significant di fference in algal richness or abundance between structures in Devon, although there was a significantly higher abundance of sessile animals on the Sidmouth groynes. The relatively low algal abundance at Sheringham and Sidmouth may be due to the simpler structure of rock material. We paired sample sites based on proximity rather than rock type in order to address questions pertaining to the spread of geographic distributions and compare nearby biological assemblages. Therefore, we are unable to separate possible effects of structure type and rock type; however, our results (Tables 1 and 2, Figures 2 and 3) do sugges<sup>t</sup> that features associated with the structure type may have a stronger influence than rock type alone. While geographic proximity was a key feature of our analysis, the distance between structures in Devon was approximately 100 km. Stokes Beach is publicly-accessible and not characterised by steep cliff faces (as is typical in the region), and represented the closest suitable, safe and accessible rocky shore, rather than necessarily the closest in Euclidean space. Future research should aim to investigate the role of type in the biological assemblages of groynes, as well the conservation value of ensuring that rock type of artificial structures mimics nearby rocky shores (as a form of ecological engineering). The higher abundance of sessile animals on rock-rubble groynes has important conservation implications. The dog whelk Nucella lapillus was recently included in the OSPAR List of threatened and/or declining species and habitats. We found that this species was more prevalent and abundant on the groynes where there was a higher abundance of sessile animals (e.g., mussels and barnacles), which make up a substantial part of N. lapillus' diet [71]. The simple structure of groynes provides refuge for many generalist species, which in turn provide trophic support to the wider ecosystem, resulting in clear communities of dog whelks and barnacles (Figure 4). We also found Pomatoschistus minutus on both natural shores and groynes. While the total abundance was higher on natural shores (Table 3), the presence of this species on groynes suggests that they have the potential to mimic natural shores and rock pools, ye<sup>t</sup> currently do not support equivalent abundances. Interestingly, the DCA identified P. minutus within communities of Sea Oak Halidrys siliquosa and Corallina officinalis (Figure 4). H. siliquosa supports a range of invertebrate epifauna and epifloral (including red algae such as C. officinalis), suggesting that the trophic interactions among these species are vital for the presence of rare species. Moreover, the role of ecosystem engineers [72] in generating microhabitats that provide refuge by reducing the impact of abiotic stresses in coastal ecosystems is important for intertidal communities. The incorporation of biotic interactions in statistical models is a key research frontier in spatial ecology [73,74], and our results (Figure 4) sugges<sup>t</sup> such interactions are key to determining representative communities on artificial structures. However, more research is needed to link the abiotic and biotic factors responsible for intertidal communities on artificial structures and highlight key ecosystem engineering designs that could be introduced to increase biodiversity during construction. Austrominius modestus was the only species identified in our study that is not native to Great Britain. As such, we cannot sensibly comment on the proposition that artificial structures support a higher richness of non-indigenous taxa, beyond stating that this species was located on both structure types in Devon, but only on the natural shore in Norfolk—contradicting the proposition. We did find higher abundances on the groynes in Devon than on the nearby rocky shores (Table 4), which supports other studies that have found higher densities of A. modestus on artificial structures in the UK [75]. However, the indigenous barnacle species were also more abundant on groynes than natural rocky shores, so the non-indigenous species may not differ from the natives in this respect. Possible explanations for the increase in barnacle abundance on groynes could be a function of larval supply, availability of space (i.e., lack of microflora), rock type and post-settlement mortality [76,77], meaning future studies should aim to quantify the possible risk associated with such factors in quantifying non-indigenous taxa spread via artificial structures. Furthermore, we only observed two native British species beyond their previously-recorded ranges: the lined top shell Phorcus lineatus at Sandbanks and the small periwinkle Melarhaphe neritoides at Sheringham. According to Crothers [78], P. lineatus has an Eastern limit around Kimmeridge bay (Figure 1), due to a combination of unsuitable winter temperature and, importantly, lack of suitable habitat to the East. According to Pizzolla [55], M. neritoides is absent between Ipswich (approx. 140 km Southeast of Sheringham) and Scarborough (approx. 400km North of Sheringham). Both species are considered highly-mobile [78,79], and their establishment on the groynes suggests that these artificial structures are providing alternative habitat for species that are limited by a lack of habitat rather than poor dispersal capacity. In sum, while rock-rubble groynes may provide non-indigenous species (whether defined nationally or internationally) with suitable habitat (along with indigenous species), we found no evidence that they enhance the dispersal of these species; more likely is that habitat availability is one of the most important factors limiting intertidal organism distributions [80]. We opted to delineate tidal heights based on the spring tidal levels rather than organism distribution limits for the aforementioned reasons. However, the double high tide at Sandbanks complicates this categorization. A continuous variable measuring distance from a specified point (e.g., the high spring tide mark) may have allowed for more flexibility in investigating the influence of tidal level on the ecological indicators; however, a simple distance metric would not account for variation in the distance and speed of tidal ranges. Discussion on the optimal method of measuring the seaward extent and tidal levels across di fferent study areas remains a priority for studies investigating intertidal ecology. Similarly, we did not measure vertical height. While vertical height influences desiccation rates of species, the range of vertical elevation across tidal levels was quite consistent across study sites, and as such, we are confident that any possible artefacts introduced through the chosen methodology have not biased our results. Similarly, we controlled for aspect through surveying the 'sheltered' side of the structure; while aspect in the sense of compass direction can also influence photosynthetic rates of algae [6,81,82], the predominant aspects of all groynes were East- and West-facing, minimising the influence of this in our study. Finally, it is important to note that the age of an artificial structure will influence the ecological communities found. All groynes surveyed were of a similar age (Table SI1), so therefore, we are confident that cross-site error was minimised. However, this is an important consideration when discussing the ecosystem services provided by groynes, and particularly comparing with natural rocky shores, and when comparing groynes across di fferent studies. Our results sugges<sup>t</sup> di fferent patterns to those identified by Pinn et al. [21] and Firth et al. [23]. The methodological di fferences among all three studies (e.g., deconstruction of the ecological indicators, direct statistical testing, unpaired sites) may be su fficient to explain contradictory results; however, it is important to consider the dynamic nature of ecosystems, and the unlikeliness of a balanced equilibrium. More developed groynes (e.g., older) may explain the higher species richness identified in pool habitats on artificial structures by Firth et al. [23], suggesting that these structures may develop complexity with time and support more diverse ecosystems. Similarly, older groynes o ffer greater opportunity for invasion than younger groynes simply by being around for longer. Therefore, investigating spatiotemporal trends in biodiversity on artificial structures in urban environments should now be a primary avenue of research, to better evaluate their potential as a conservation opportunity, with the data from this study available in Supplementary Information 2 to encourage such studies.	doab	2025-04-07T03:56:59.411116	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 30 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.31	5. Conclusions Rock-rubble groynes in urban environs have provided researchers and practitioners with a conservation dilemma. On the one hand, they can provide much-needed habitat for intertidal species that are decreasing in abundance, while on the other hand, by providing such habitat they may help non-indigenous species expand their ranges. Our results sugges<sup>t</sup> that the rock surfaces of rock-rubble groynes do provide potential benefit for species for which this habitat is suitable, including species that are of conservation concern. We found similar diversity on rock surface habitats between structure types and found higher abundances of sessile animals on groynes. The rock pool habitat represents a key di fference between the groynes and the natural rocky shores, with rock pools on groynes being smaller, fewer and less complex than on natural rocky shores. We identified clear communities of algae and endangered fish species (Figure 4) in pools, suggesting that rock pool creation can result in similar ecological communities on groynes, albeit at lower abundances. We also found very few non-native species on groynes, and those species were also found on corresponding natural rocky shores, o ffering no support for the notion that groynes pose a threat in terms of biological invasions (at least in the UK). While structure age and equilibrium in the ecosystem must be considered, the results of this study sugges<sup>t</sup> that rock-rubble groynes have the potential to support under-pressure intertidal macro-organisms, particularly in urban environments, where the trend to replace natural shoreline with artificial structures is continuing. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-445X/9/5/131/s1: Supplementary Information 1: Details of the study sites. Supplementary Information 2: R Code. Supplementary Information 3: Species Data. Supplementary Information 4: Species name and code. Supplementary Information 5: Extra Analysis. Author Contributions: Conceptualization, P.H. and R.F.; methodology, P.H. and R.F.; data curation, P.H.; formal analysis, P.H. and R.F.; writing—original draft preparation, P.H. and R.F. 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 thank the reviewers and editors for their comments and suggestions. We would also like to thank our field assistants, Julia, Eddie, and Michael. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.412084	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 31 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.33	1. Introduction Urban development often occurs at the expense of urban green spaces (UGSs), separating urbanites from nature [1] and depriving them of the various social and environmental benefits, also known as ecosystem services, that UGSs provide [2–4]. Importantly, UGSs also deliver certain economic benefits. These benefits are often much more di fficult to measure, generally leading to a preference for land uses with an explicit monetary value, including commercial and residential development, above UGSs in property development decisions [5]. Various studies have been conducted using di fferent techniques and methods to quantify the potential economic benefits of UGSs with the majority of these studies focusing on the contexts of the global North [6–10]. Among these, the proximity principle has been widely applied [8,10]. The proximity principle suggests that residential property value increases as the distance to UGSs decreases [11], thus providing potential reciprocal increases in property tax for authorities [6]. The majority of studies in the global North confirmed the proximity principle [8,10], whereas in the global South results have been less conclusive. In 2015, Cilliers and Cilliers disproved the proximity principle in a South African case study, testing the principle in the local context of Potchefstroom. [12]. The current paper returned to the 2015 case study to verify, update and refine the work conducted by Cilliers and Cilliers [12] as a preliminary study to direct future research. The paper followed a quantitative approach investigating municipal property valuations for three zones differentiated based on distance from a green space in five residential areas bordering a UGS in Potchefstroom. Additional data were also gathered by means of a survey on the social, environmental and economic values attributed to green spaces by a sample of Potchefstroom residents; and a second survey of South African urban planners concerning perceptions on planning for green spaces. The paper is initiated with a review of core concepts in a South African context to provide orientation and reference existing studies in the field. This is followed by the quantitative investigation and a discussion of the results, finally arriving at our conclusions and ultimate recommendations. #### 1.1. Green Spaces in the South African Context Urban green spaces (UGSs) include various types and land uses that are located within the urban boundary, primarily covered by permeable surfaces, soil or plant species including grass, shrubs or trees [13]. Zoning classifications may include recreational, institutional, residential, commercial or agricultural categories to accommodate land uses such as sports fields, private gardens, street trees, playgrounds, greenways, urban farms and urban forests [13,14]. UGSs may also include more informal, residual and seemingly abandoned areas, similar to vacant lots, spaces along and under freeways, railways and side streets, sidewalks and derelict properties. Green land uses in urban areas have been studied extensively for their contributions to urban quality of life [1,2,5,15], as part of urban green infrastructure [15]. The majority of studies have been conducted in the contexts of the global North, with scholarship in the global South still being relatively underrepresented [15], with the exception of South Africa. Like most countries, South Africa is experiencing rapid urbanisation as the most urbanised sub-region in Africa [16]. Sub-Saharan Africa along with Asia, is expected to accommodate 90% of the total increase in population over the next four decades [15]. Urban expansion is driven by development in both formal and informal areas, placing green spaces on the expanding urban periphery and within the existing urban envelope in danger of land use conversion [13,17–19]. According to Lubbe et al. [19], the socio-economic status of an urban area determines what resources are available to change the environment, having a tremendous impact on the vegetation types, plant diversity and green space availability [20]. Moreover, South African standards call for a minimum 10% of development area to be allocated to green space, but as in other global areas, UGSs continuously compete with other land uses that may present more explicit monetary values and returns [21,22]. Green spaces also face additional threats from illegal dumping, pollution, invasive species and other social ails that may damage natural ecosystems and diminish biological diversity [23]. To defend existing and promote the development of UGSs, a compelling argumen<sup>t</sup> for the social, environmental and, importantly, the economic benefits UGSs can provide, must be made. However, such arguments must also make balanced reference to the potential disadvantages often related to these green spaces, especially in the contexts of the global South. The following section provides a succinct discussion of these benefits, as ecosystem services, as well as potential disadvantages, as ecosystem disservices. #### 1.2. Ecosystem Services and Disservices Ecosystem services refer to the benefits people derive from any form of ecosystem functions delivered by UGSs (urban green spaces) that have a positive impact on human wellbeing [2]. Ecosystem services may be classified according to two broad categories, distinguishing between direct and indirect benefits [1,5]. Direct benefits include environmental and social benefits or services, whereas indirect benefits refer to economic benefits (Table 1). Sources. [3,5,6,12,15,24–27]. It is imperative that the different categories of UGS and types of ecosystem services and benefits green spaces provide, are well understood and communicated in aid of UGS protection and advancement [1]. A case study in Johannesburg, one of the fastest growing cities in Africa, determined that cities and towns are in desperate need of green planning strategies which are only possible when ecosystem services are recognised in city budgeting and accounting systems and when the "ecological economy" is integrated in urban planning processes [16]. Many of the indirect environmental and social benefits potentially delivered by UGSs may be more readily accepted and anticipated by the public and authorities [28]. The direct economic benefits delivered by UGSs are often less thoroughly understood and accepted, ascribed to the complexities of determining and expressing such values [5]. De Wit et al. [29] determined the monetary value of specific ecosystem services in Cape Town, South Africa, and concluded that these services generated economically valuable services in the context of the metro's economy and should, thus, be considered in its budgeting processes. Cognisance of such economic contributions may incentivise investment in and spending on maintenance and protection of UGSs [6] and may justify expenditure to address possible associated disadvantages (ecosystem disservices). Ecosystem disservices EDS) refer to the same ecosystem functions that provide benefits in social, environmental and economic terms, but recognise related actual or perceived negative impacts on human wellbeing [3,30]. Ecosystem disservices are an especially important consideration in the South African context as [30,31]. This paper highlights the ecosystem disservices that are present in towns and cities in South Africa, similar to Potchefstroom, that pose a threat to human wellbeing and require financial resources and private investment to address these disservices in spatial planning, as seen in the third column of Table 2. The valuation of the economic benefits delivered by UGSs can, thus, be influenced by both ecosystem services and the ecosystem disservices derived from the same spaces. Various approaches and methods have been developed to quantify the economic benefits of UGSs in this regard [6–9,27,32]. A comprehensive review of all existing methods falls beyond the scope of this paper. As this research is based on the work completed by Cilliers and Cilliers [12], only the approach used in that study is recapitulated in Section 1.3 of this paper. #### Table 2. Summary of ecosystem disservices. #### 1.3. The Proximity Principle as A Hedonic Price Analysis Method The proximity principle, also called the proximate principle, suggests that the value of an amenity, like a green space, is determined by the property values of adjacent residential properties [11,12,33]. The proximity principle reflects on the concept of "willingness to pay", as residents are willing to pay more for properties adjacent to or enjoying a view of such an amenity, or green space [33]. As a result, residents pay more property tax leading to higher tax returns available to local municipalities to direct towards planning, developing and maintaining quality UGSs [6]. Hedonic Price Analysis is generally the most commonly employed method to quantify the value of green spaces [8]. Hedonic Price Analysis is used to identify and quantify the different factors and characteristics that influence the value of property by using several regression models and considering property value as a function of measures of proximity to green space [8,33–35]. Thus, the Hedonic Price Analysis method considers that residential properties are not homogeneous, but reflect various factors that influence property value including property size, the physical condition of the property and accessibility [8]. Several studies [6,22] have determined the positive impact of green spaces on property value, thus, testing the proximity principle using Hedonic Price Analysis. Cilliers and Cilliers [12] invoked the proximity principle as a hedonic pricing method in the local context of Potchefstroom, South Africa, to compare local case study findings with case study results proving the proximity principle in international literature. In the Potchefstroom case study, Cilliers and Cilliers [12] rejected the proximity principle, as proximity to nearest UGS exerted a negative impact on residential property value, as elaborated on in Section 2. These contrasting findings underscore the potential impact of ecosystem services and disservices within the parameters of socio-cultural and physical context in South Africa in realising certain economic values derived from UGSs. #### 1.4. The Importance of Context and Planning at Community Level As referenced in Section 1.2 (ecosystem disservices in SA context) and the contrasting findings in the previous section, context is key in eliciting ecosystem services and downplaying potential disservices in the realisation of economic benefits. Context specificities must be acknowledged in planning for UGSs, local level research, data collection and spatial planning [36]. As part of this process, public participation and stakeholder engagemen<sup>t</sup> can render valuable inputs regarding perceptions, experiences and acknowledged benefits and disadvantages related to UGSs at local level. Not all inputs from community members can be incorporated in planning and decision making [36], but such processes at least provide nuanced information related to citizen experience and may highlight previously unknown cultural, biodiversity, recreational values, community needs and preferences [36] that may not be captured in spreadsheets dealing with economic data. A study was conducted in South Africa investigating the inequality of public green space, finding that poorer suburbs are endowed with less public green space area than provided in more a ffluent areas, resulting in poor access to such spaces for the poor [15]. The South African context's severe socio-economic disparities, which are also expressed spatially and in access to amenities, provides fertile ground for community-based participatory planning. Stakeholder engagemen<sup>t</sup> may thus consider the interests and opinions of urban residents in the planning and managemen<sup>t</sup> of public green spaces [15] and provide nuanced understanding of the impacts of such spaces in varied contexts in recognition of generally acknowledged challenges. As mentioned in Section 1.2, UGSs in South Africa are often under-maintained, unattractive and perceived as crime hotspots [37], leading to underutilisation and further dereliction. Such conditions demand continuous e fforts to maintain and improve UGSs, akin to Dempsey and Burton's [38] conceptualisation of 'place-keeping' as a collaborative process that surpasses a focus on mere physical upkeep to include ongoing inputs from public and private stakeholders, as well as communities. Thus, calling for community stewardship and the use of local knowledge and community networks to increase the quality of UGSs and by extension, their potential economic benefits.	doab	2025-04-07T03:56:59.412360	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 33 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.34	2. Methodology This section provides a discussion of the methodologies followed in data acquisition for the two components of quantitative research completed. The results are presented for each component in Section 3. As previously mentioned, this paper presents a refinement of and elaboration on the study conducted by Cilliers and Cilliers (2015) [12] investigating the proximity principle in the case study of Potchefstroom using updated municipal valuations in its analysis (See Section 2.1). The paper tested the proximity principle in the local context in 2019, and compared 2019 findings with 2015 findings, to identify if the proximity principle would still be rejected, as in the 2015 study. The 2019 study was further substantiated with supporting quantitative data collected through two structured questionnaires capturing perceptions amongs<sup>t</sup> a sample of Potchefstroom residents pertaining to green space values; and a questionnaire distributed amongs<sup>t</sup> Professional Planners in South Africa regarding green space planning in the local context (See Section 2.2). Although these questionnaires were limited in sample size, and should be expanded on in future research endeavours, results provide preliminary insights into community and professional perspectives on UGSs in support of the importance of context and community input discussed in Section 1.4. #### 2.1. Data Acquisition Part A: Proximity Principle in Potchefstroom The data were collected in Potchefstroom (26.7145◦ S, 27.0970◦ E), situated in the North-West province of South Africa. Potchefstroom was chosen as the local case study area as international literature were divergent from the results of a study conducted in Potchefstroom by Cilliers and Cilliers in 2015 [12]. This paper refined the previous study by Cilliers and Cilliers, employing the same methods of data collection and data analysis to verify the results study four years later. As such, the same residential areas and respective properties within each residential area included in the 2015 study by Cilliers and Cilliers [12] were reselected in 2019 to determine the impact of green spaces on property value, using 2019 municipal property valuations. These residential areas were originally selected based on their proximity and accessibility to UGSs, and included the following, as seen in Figure 1: Figure 1. The five selected residential areas in Potchefstroom. Within each residential area, the selected properties were divided into three zones to test the proximity principle, as illustrated in Figure 2. Zone 1 bordered a UGS, whereas zone 3 was located furthest away from the green the same UGS. Firstly, the property price per square meter was determined by dividing the municipal property values of June 2019 with the total area (in square meters). Thereafter, the mean square meter value per zone in each selected residential area was calculated. Therefore, in each selected residential area, zone 1, zone 2 and zone 3 were assigned a mean ZAR per square meter value that was used to statistically analyse the data. As verified in the international literature, the proximity principle suggests, that zone 1 should present the highest property value [12]. The data obtained from the 2019 Potchefstroom case study were accordingly analysed using three analytical methods including the Analysis of Variance (ANOVA), the Kruskal–Wallis analysis and the Dependent T-test. Figure 2. Zone 1 to 3 within area B, Van der Hoff Park. #### 2.2. Data Acquisition Part B: Community Survey and Professional Planner Survey As a refinement on the study conducted by Cilliers and Cilliers [12] and gain further insights on the findings derived from the quantitative investigation, two additional surveys were conducted. The data were collected through two structured questionnaires employing a Likert scale (1 = Fully agree, 2 = Agree, 3 = Not sure, 4 = Disagree and 5 = Fully disagree). The first questionnaire, referred to as the Potchefstroom resident survey, focussed on a sample of Potchefstroom residents and their perceptions and appreciation of green space benefits in Potchefstroom. A total of 74 residents completed the survey. The aim was to understand how communities perceive open spaces in Potchefstroom, as the primary users of these spaces. Data were statistically analysed using Cramer's V test and cross-tabulations of selected questions. The second questionnaire focussed on the perspectives of a sample of Professional Planners relating to UGS planning in the context of South Africa. The Professional Planner questionnaire focused on green space aspects including the available financial resources (local budgeting) in green space management, community engagemen<sup>t</sup> in green space planning, environmental considerations in practice and green space typologies. A total of 26 planners completed the survey where 17 were Professional Planners, 8 were Candidate Planners and 1 was not registered as a planner, but did work in planning practice. Both questionnaires were distributed electronically, and consent was granted by virtue of completion of a questionnaire. #### 3. Data Analysis and Results #### 3.1. Data Results Part A: Proximity Principle in Potchefstroom A verdict on the rejection or acceptance of the proximity principle can be made solely based on the mean ZAR per square meter values obtained for the 3 zones within each residential area; however, the statistical evidence provides credence. In observing the 2019 mean ZAR per square metre for each zone in each area the following could be identified. The mean ZAR per square meter values in four of the five residential areas (Grimbeek Park, Van der Hoff Park, Potchefstroom Dam Area and Heilige Akker) indicated that zone 1 represented the lowest value in ZAR per square meter compared to zone 2 and zone 3. Thus, zone 1 that is located closest to the UGS presented the lowest mean ZAR per square meter value instead of the highest value as suggested by the proximity principle. Zone 3 displayed the highest mean ZAR per square meter value in three of the five residential areas (Grimbeek Park, Van der Hoff Park and Potchefstroom Dam area), the zone furthest away from the green space. By using the municipal property values of 2019, the mean ZAR per square meter values of each zone within each selected residential area were calculated as mentioned in the previous section. The latter values were used to complete the ANOVA (mean1 – mean 2/max SD, also referred to as the "standardized difference between the means" and Kruskal–Wallis analyses (Z/sqrt(N). The effect sizes presented by the ANOVA and Kruskal–Wallis analyses were used to verify the significance of the results, as the sample sizes did not provide enough power to test for normality, requiring both parametric and the non-parametric tests. Where results differ, the non-parametric test was preferred. The effect sizes obtained determined whether a practically significant difference was present between zone 1 and zone 2, as well as between zone 1 and zone 3 within each residential area. The effect sizes obtained from analyses were interpreted. A small effect size of 0.2 indicated no practically significant difference, whereas a medium effect size of 0.5 indicated a practically visible difference. A large effect size of 0.8 indicated a practically significant difference [39]. The results showed that the comparison between zone 1 and zone 2 in the respective residential areas delivered an overall medium effect size, thus, a practically visible difference between the mean ZAR per square meter values (≈0.5), as seen in Table 3. Four of the five residential areas indicated a practically visible difference (≈0.5) (Van der Hoff Park, Potchefstroom Dam Area, Heilige Akker and Oewersig), while only one residential area indicated a practically significant difference (≈0.8) (Grimbeek Park). The effect sizes produced by comparing zone 1 and zone 3 in the respective residential areas delivered an overall visible (≈0.5) to a significant difference (≈0.8) (Table 3). Two residential areas presented a practically significant difference (≈0.8) (Grimbeek Park and Van der Hoff Park) and another two residential areas delivered a practically visible difference (≈0.5) (Potchefstroom Dam Area and Oewersig). Only one residential area presented no practically significant difference between zone 1 and zone 3 (≈0.2) (Heilige Akker). The effect sizes indicated an overall medium to large effect size (≈0.5/≈0.8), indicating a visible difference to practical significant difference between the mean ZAR per square meter values of zone 1 and the zones further away from the green space. Zone 1 also presented values lower than the zones further away from the green space, refuting much of the existing research on the positive impact of UGS on property value. The proximity principle was thus rejected, with the statistical analyses supporting the data showing a medium to large differences between values. Thus, in the case study a more proximate location to a UGS did not indicate an increase in property value, but showed an inverse effect. The data from Cilliers and Cilliers (2015) [12] were statistically compared to the data obtained in the 2019 Potchefstroom case study, as captured in Table 4. The effect sizes obtained from the Dependent T-test, once again, indicated whether a practically significant difference existed between the data. The effect sizes presented a practically significant difference between the mean ZAR per square meter values (effect size of ≈ 0.8/0.5). The latter was clear by observing the old and new mean ZAR per square meter values in Table 4. Property values increased between 2015 and 2019 by 68% in Grimbeek Park, 35% in Van der Hoff Park, 55% in the Potchefstroom Dam area, 46% in Heilige Akker and 45% in Oewersig, thus presenting an aggregate increase on average of almost 50%. Various potential factors could have led to the increase in property values identified from 2015 to 2019. Inflation was a potential factor as the Consumer Price Index (CPI) increased from 5.78% in 2013 to 6.59% in 2016 and decreased, thereafter, to 4.25% in 2019 [40]. Other factors influencing property value in South Africa, according to the Absa residential property market database, include migration trends, security issues, income levels, employment, monetary and fiscal policies, investment returns, the condition of the property and foreign property buying of South African properties [41]. Table 4. Dependent T-test statistical analysis. #### 3.2. Data Results Part B: Community Survey and Professional Planner Survey #### 3.2.1. Potchefstroom Resident Survey The results obtained from the survey indicated that the sample of Potchefstroom residents recognise the social, environmental and economic value of green spaces; however, fewer residents recognise the economic value of green spaces. Question 4 focused on the residents' perceptions of green spaces in Potchefstroom, referring to safety matters. A total of 52% of respondents agreed that green spaces in Potchefstroom are perceived as crime hotspots, thus, contributing to unsafe neighbourhoods and indicating a related ecosystem disservice (cross-reference to Section 2.2). A total of 60% of respondents agreed that they would pay more for a property that is located next to a green space in Question 5; however, many residents were unsure or disagreed. Interestingly, some residents who perceived green spaces as crime hotspots in Question 4, still agreed that they would be willing to pay more for a property that is located next to green space (Figure 3). Figure 3. Potchefstroom resident survey results. Question 1: Urban green spaces have environmental value; Question 2: Urban green spaces have social value; Question 3: Urban green spaces have economic value; Question 4: Urban green spaces are perceived as crime hot spots in Potchefstroom; Question 5: I would pay more for a property because it is located next to an urban green space. As a result of the answers to Question 4 and 5, in recognition of the ecosystem disservices linked to the South African and Potchefstroom contexts, a cross-tabulation were conducted as part of the analysis, in an attempt to further clarify findings: Question 4 (Urban green spaces are perceived as crime hot spots) was cross-tabulated with Question 5 (I would pay more for a property because it is located next to an urban green space). The cross-tabulation was completed with 74 valid cases and a medium practical significant difference as the Cramer's V test value was V = 0.287 (V≈0.3) was presented. Thus, findings supported the observation that although certain residents perceived UGSs as crime hotspots (ecosystem disservices), they residents are still willing to pay more for a property is located next to an UGS. #### 3.2.2. Professional Planner Survey The results of Question 1 and Question 2, as shown in Figure 4, both indicated that 88% of the planners agreed that unattractive green spaces are due to a lack of maintenance by local authorities and a lack of community engagement. Question 3 delivered interesting results as 50% of respondent planners agreed that environmental considerations are not prioritised in the planning process; however, the other 50% were either unsure or disagreed that environmental considerations are not prioritised in the planning process Question 4 of the survey focussed on local budgeting for green space planning and indicated that only 62% of respondents agreed with the statement that green spaces are not prioritised in local budgeting. A total of 38% of the planners were either unsure or disagreed that green spaces are not prioritised in local budgeting. The majority of planner respondents reported being familiar with green space typologies (92%). Figure 4. Professional Planner survey results. Question 1: Unattractive urban green spaces are the result of a lack of maintenance by local authorities; Question 2: Unattractive urban green spaces are the result of a lack of community engagement; Question 3: Environmental considerations are not prioritised in the planning process; Question 4: Environmental considerations are not prioritised in local budgeting; Question 5: I am familiar with green space typologies.	doab	2025-04-07T03:56:59.413223	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 34 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.35	4. Conclusions The challenges pertaining to green spaces within urban environments, as captured in the literature review, were also evident from the empirical investigation and local case study. Despite the theoretically recognised values and benefits linked to green spaces, the economic value thereof is still underestimated to a large extent in the South African context [16]. According to international case studies, as discussed in Section 1.3, UGSs can have a positive influence on proximate property value [6]. The proximity principle may hold true in the global North planning context, but was rejected in the Potchefstroom case study in 2015 [12] and 2019. In the Potchefstroom case most properties located further away from a UGS in the purposefully selected residential areas indicated a higher value in ZAR (South African Rand) per square meter in comparison to properties located adjacent to said UGS. Both the 2015 and 2019 case studies thus delivered contrasting results compared to the findings in international literature. From these findings it was evident that the proximity principle could not be applied to all contexts, indicating the need to consider context in the planning of UGSs. The Professional Planner survey investigated perceptions of green spaces, from the perspective of a sample of South African Professional Planners, in attempt to gain insight into planning practice in the South African context. This survey indicated that a lack of maintenance, community engagemen<sup>t</sup> and efficient local budgeting are the core challenges inhibiting UGS planning approaches. Half of the planners included in the survey stated that environmental considerations are not prioritised in the planning process, even though Environmental Management is considered a crucial component of local Urban Planning approaches, policy and legislative frameworks. The latter findings questioned the extent to which the environment is prioritised in mainstream Urban Planning and emphasised the opportunities for more comprehensive, trans-disciplinary planning approaches going forward. Even though the proximity principle was rejected for the Potchefstroom case study, and that Professional Planners indicated that environmental considerations are under-prioritized from a planning perspective, the local resident survey revealed a certain level of recognition of the importance of green spaces. The Potchefstroom resident survey investigated local perceptions towards green spaces, in an attempt to further understand the challenges and needs pertaining to green space planning in Potchefstroom for a participatory perspective. The survey indicated that the majority of respondents recognise the environmental and social values of green spaces, and to a lesser extent the economic value of green spaces. The majority of residents are willing to pay more for a property that is located next to green space, indicating that the residents perceive green spaces benefits as valuable (willingness to pay), regardless of the ecosystem disservices associated with UGSs (poorly maintained green spaces and crime hotspots). Thus, from a community perspective, green space value should be prioritised. Preliminary results indicate a discrepancy between community perspectives, the provision of green spaces within the local context, and economic valuation to support such initiatives.	doab	2025-04-07T03:56:59.414048	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 35 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.36	5. Recommendations #### 5.1. Future In-Depth Research Based on This Paper's Preliminary Findings As the 2019 data confirmed the trends established in 2015 regarding the rejection of the proximity principle in the Potchefstroom case study, it is recommended that comparable research be conducted in various locations around South Africa to provide further clarification on the generalisability of the research premise. Such investigations could consider the influence of specific UGS characteristics and di fferentiation in socio-economic gradients [19] on the proximity principle. The results of the Potchefstroom resident survey, in addition to the emphasis placed on stakeholder engagemen<sup>t</sup> in the literature, further call for future research on the proximity principle to include community surveys to deepen understanding. Such approaches could include qualitative investigations in the form of interviews and focus group discussions with proximate communities to triangulate findings. Participatory planning strategies can be crucial in ensuring long-term returns on investments in UGSs and should thus be included in both research and practice. #### 5.2. Municipal Valuations Should be Reviewed If residents are indeed willing to pay more for properties located next to UGS as the preliminary survey data suggests, local authorities should consider investing in UGSs (following stakeholder engagemen<sup>t</sup> processes) and revise municipal valuation of proximate residential properties in accordance. Such adjustments could result in increases in property tax revenue to reinvest in UGSs to enhance indirect benefits derived from ecosystem services and address ecosystem disservices. The latter may build a compelling argumen<sup>t</sup> to convince local municipalities, private investors and the community to invest in UGSs. This may influence decision-making processes regarding policy formulation to promote environmental protection, green space planning and environmental managemen<sup>t</sup> within cities. #### 5.3. Enhance Both the Quality and Quantity of Green Spaces The quality of green spaces could be enhanced by implementing green planning initiatives as part of broader green infrastructure planning approaches [35]. As dense urban centres do not have adequate space available for development, revitalisation may be considered. The quantity and quality of green spaces within dense urban centres may be increased and improved by compact city and mixed-use planning approaches where green spaces are prioritised as a land use requirement. Local budgeting, maintenance and public participation are also aspects that may ensure that the quality of green spaces is enhanced as the issues regarding ecosystem disservices of green spaces will be addressed. An increase in the quality of green spaces may result in more economic benefits. The latter aspects will be discussed in the next section. Further research may be conducted that investigate the quality and economic benefits of private open spaces, such as gardens in contrast with the quality and economic benefits of public open spaces. The approaches will include "willingness to pay" for amenities, services, green space benefits and properties close to a UGS (stated preference approach) and the impact of the UGS on property value (revealed preference approach). The results will determine whether functional green spaces with high quality have more economic benefits. #### 5.4. Prioritise Environmental Considerations in Mainstream Urban Planning Green space planning should be emphasised in the local context and Professional Planners should collaborate with local authorities to prioritise green space planning in local budgeting. Planning practice should consider participatory planning approaches that include the active involvement of the public specifically in UGS planning. This calls for trans-disciplinary planning approaches. Ecosystem services and disservices should play a more prominent part within urban planning, in an attempt to enhance green space quality and increase the economic benefits. Land Use Schemes (LUS) of local municipal areas could be amended to accommodate, not only the minimum green space provision requirements in applications but also mixed-use zonings. Mixed-use developments may increase the quantity and enhance the quality of green spaces. Mixed-use developments ensure that various land uses similar to residential, business and green spaces are included in a single development [42]. Currently, local authorities follow an approach to development where green spaces are perceived as potential areas for development (cross-reference to Professional Planner survey). Local planning should follow an approach to development that encourages the protection of green spaces and natural systems, rather than the development of these areas. #### 5.5. A Broader Spatial Planning Approach It is crucial to determine and provide evidence of the economic benefits of green spaces to ensure that green space planning is prioritised in the local context. Therefore, it should form part of a broader spatial planning approach. The Spatial Development Frameworks (SDF) of each local municipality should include a section that discusses green spaces in economic terms and what is aimed to be achieved in terms of environmental managemen<sup>t</sup> and green space planning in the future and such should be supported by the Spatial Planning and Land Use Management Act (Act 16 of 2013) [43]. To measure the quality of green spaces, the stated preference and revealed preference approach may be considered. The stated preference approach includes "willingness to pay" that determines what residents are willing to pay for amenities, services, green space benefits and for properties that are located adjacent to green space. The revealed preference approach includes the proximity principle as a hedonic price analysis method, as employed in the 2019 Potchefstroom case study of this research paper (cross-reference to Section 2). The proximity principle may be used as an indicator of the economic benefits of green spaces by determining how green spaces influence property value in di fferent urban areas including public open spaces, private open spaces and neighbourhoods of di fferent social status (the socio-economic gradient). Areas that indicate a green space with a higher economic value (approved proximity principle) may indicate what functions and characteristics should be prioritised to increase the economic benefits of green spaces in other areas. When attempting to valuate green spaces in economic terms, six important aspects should be considered including market value (green space impact on property value), enhancement value (green space influence on adjacent land), production value (contribution to production referring to resources), natural systems value (urban biodiversity and ecosystems), direct and indirect value (social, environmental and economic benefits) and intangible value (how people perceive green spaces referring to ethics, knowledge and opinions) [44]. #### 5.6. Focus on Context-Based Planning Further research may be conducted on the impact of green spaces on property values along a socio-economic gradient as this may influence residents' perceptions of the value of urban green areas, especially where environmental inequity is a reality [19]. Contrasting results may indicate the importance to plan in context. The current research mentioned the di fference in planning contexts of Global North and Global South countries, as the proximity principle holds true in the Global North context, but is rejected in the local context of Potchefstroom, South Africa (cross-reference to Section 2). To plan according to context, a shift in planning approaches will be required. Local communities should participate in planning on a high and empowering level. For local municipalities to shift towards context-/community-based planning, legislative transformation will be required that will support planners in active community engagemen<sup>t</sup> and encourage planners to consider context-based planning. Nature and the environment, basic human rights, should be prioritised in urban planning approaches, in an attempt to reclaim nature in cities. Author Contributions: All authors have read and agree to the published version of the manuscript. Supervision, E.J.C., L.L. and S.C.; Writing—original draft, Z.C. Funding: This research was funded by National Research Foundation South Africa, gran<sup>t</sup> number 116243. Acknowledgments: This research is supported in part by the National Research Foundation of South Africa (Grant Numbers: 116243). Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.414365	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 36 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.38	Article A Theoretical Framework for Bolstering Human-Nature Connections and Urban Resilience via Green Infrastructure #### *Jackie Parker 1,2,\ and Greg D. Simpson 1 #### Received: 9 July 2020; Accepted: 28 July 2020; Published: 29 July 2020 Abstract: Demand for resources and changing structures of human settlements arising from population growth are impacting via the twin crises of anthropogenic climate change and declining human health. Informed by documentary research, this article explores how Urban Resilience Theory (URT) and Human-Nature Connection Theory (HNCT) can inform urban development that leverages urban green infrastructure (UGI) to mitigate and meditate these two crises. The findings of this article are that UGI can be the foundation for action to reduce the severity and impact of those crises and progress inclusive and sustainable community planning and urban development. In summary, the URT promotes improvement in policy and planning frameworks, risk reduction techniques, adaptation strategies, disaster recovery mechanisms, environmentally sustainable alternatives to fossil fuel energy, the building of social capital, and integration of ecologically sustainable UGI. Further, the HNCT advocates pro-environmental behaviors to increase the amount and accessibility of quality remnant and restored UGI to realize the human health benefits provided by nature, while simultaneously enhancing the ecological diversity and health of indigenous ecosystems. The synthesis of this article postulates that realizing the combined potential of URT and HNCT is essential to deliver healthy urban settlements that accommodate projected urban population growth towards the end of the 21st-century. Keywords:** climate change; green infrastructure; human health, human-nature connection theory; urbanization; urban resilience theory	doab	2025-04-07T03:56:59.414930	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 38 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.39	1. Introduction Over the past two centuries, urbanization has changed the relationship that urban dwellers share with the surrounding environment [1,2]. Resultant are adverse effects, with some proving to be devastating to urban communities and indigenous ecosystems [3–10]. Urban centers are now gripped by two crises, exacerbated by a cascade of factors related to the short term doubling of the global human population, rapid urbanization of humanity, unsustainable lifestyle choices, short-term economically focused development, and the resultant changes in the structure of human settlements [5–10]. Those twin crises are anthropogenic climate change (hereafter referred to simply as climate change) and declining human health in urbanized populations [11–13]. Following a short exploration of those crises in the urban context, this article postulates that the Urban Resilience Theory (URT) and the Human-Nature Connection Theory (HNCT) provide complementary opportunities to mitigate and mediate the drivers and impacts of those two crises. Informed by the reviews of Parker [14–18] and others [19,20] and the key research over the past decade delivered by Lovell and Taylor [5], Tzoulas et al. [7], Mathey et al. [8], Norton et al. [9], Meerow, Newell and Stults [10], and Burley [12]; a gap is apparent in the existing literature with respect to applying a complementary combination approach to the implementation of the URT and HNCT in urban centers. The novel approach of integrating the application of those two theories posited in this article addresses this gap in the literary discourse and positively contributes to the refocusing and amendment of the unsustainable development many urban centers are currently pursuing. As such, the conceptual model proffered later in this article postulates that the integration of URT and HNCT to inform the provision of accessible, quality UGI will produce urban centers that can alleviate climate change impacts and declining human health for the betterment of current and future generations. Explicitly defining the URT and the HNCT is problematic given the breadth of disciplines those theories transect, meaning that definitions are highly contested in current literature [19–22]. Both theories move beyond Homo sapiens merely surviving within urban centers, to thriving as humans, communities, and cities in coherence with the surrounds [11]. Recently redefined by Romero-Lankao et al. [11] (p. 2), a combined approach to URT and HNCT seeks to: "develop strategies for environmental protection, economic prosperity, inclusivity, and community wellbeing, while increasing their cities' resilience to both chronic and acute physical, social, and economic challenges." In practice, urban resilience relates to the ability of an urban center to withstand and recover in the event of a shock such as a natural disaster, terrorist attack, economic failure, or pandemic [19]. With the ability to recover from a city-scale shock being one measure for the level of urban resilience, Hobor [19] reports that urban resilience was closely linked to the economic history of a city, as recovery and adaption requires significant financial and infrastructure resources. More recently, urban resilience, in the context of community planning and urban development, is becoming increasingly integrated with higher considerations of human health and wellbeing [23]. Economic factors, social factors, and the intersecting socio-economic factors, play a larger role in recovery efforts, and therefore the resilience of urban centers. Human-nature connection is a cognitive, emotional, spiritual, and biophysical attachment or affinity that humans feel for natural places [20,21]. Humans can connect to nature at local, regional, national, or international scales, or connections may be location non-specific (i.e., a connection to water or nature at large). Two grains exist for human-nature connection reported within the literature; fine and coarse. Fine grain connections arise from personal nature experiences, interactions with features within a natural setting, and/or direct interactions with the land, (i.e., gardening or farming) [20]. Coarse grain connections come from cultural significance, cultural landscapes, and broader place attachments [20,22]. Human-nature connections have changed over the past few decades [2,21,24]. The change in this relationship, within the context of declining human mental and physical health of urbanized human populations, has sparked the interest of researchers across many disciplines [14,25,26]. This high-level synthesis opens dialogue around the overlapping contribution that application of those two theories make to urban planning and building urban communities as humanity looks towards the second half of the 21st-century. To that end, the specific purpose of this article is to: #### 2. Methods and Application The synthesis presented in this article is informed and inspired by the extensive systemic literature reviews and the UGI research that is reported in several earlier articles by the authors [2,14–18,22,26]. In addition, 32 globally focused review articles and 20 geographically specific UGI research articles are directly cited to support the synthesis presented in this article (Tables 1 and 2 below). Further, a summary of the geographic scope and UGI research focus of the case study, empirical, and documentary research reported in those publications is presented in Appendix A (Tables A1–A4). As such, this article is not a systematic. Rather, it is aligned to the "narrative meta-review" of Nieuwenhuijsen [27] (p. 2) that was constructed "around a number of cutting edge and visionary studies on urban ... planning and health reported in the literature over the past few years". However, this article goes beyond providing a meta-review to postulate a new conceptual model for the combined application of URT and HCNT in relation to the provision of accessible, high-quality UGI. As highlighted in the introduction, it is the premise of this article that utilization of that combined model of URT and HCNT can provide a UGI-based response to the crises of climate change and declining human health in the face of the rapid urban population growth. Cognizant of the multifaceted approach that Rist [28] championed for social research undertaken to influence the policy process, the documentary research [29–35] that underpins this article builds on the knowledge reported in the aforementioned literature to scope the key peer reviewed URT and HNCT literature. On the basis of that research, the URT and the HNCT have not previously been connected in other peer reviewed literature. In addition to summarizing the breadth of research that underpins the proposition of this article that an integrated model of the URT and the HNCT can help urban centers to respond to the challenges of climate change and declining human health, Tables A1–A3 also provide evidence that supports the global applicability of that proposition. From the outset, this project was structured to avoid a Euro or Western centric filter, which was aided by the scholarship regarding urban centers reporting the globalization of Western approaches to urban development for creating cities to accommodate increasingly urbanized human populations [34–46], (Tables A1–A3). #### 3. Challenges for Urban Centers #### 3.1. The Climate Change Crisis With most global energy and steel production requiring burning of fossil carbon stores/fuels, the increase in the human population is driving an increase in greenhouse gas emissions [46,47]. As greenhouse gases exceed the natural stable state operating volumes, documented devastating e ffects of climate change are becoming ever more apparent [46,47]. Tipping-point events currently observable include the accelerated melting of the Arctic Tundra, Greenland Ice Sheet, and Polar Icecaps creating feedback loops that further exacerbate global warming [46,47]. While severity in impact is geographically dependent, the rapidly warming climate is: changing global climate patterns of normal and extreme weather events (i.e., rainfall, wind patterns, and storm frequency and tracks); a ffecting the biophysical and ecological function of agricultural and natural systems; and is causing micro and macros scale displacement and extinction of flora, fauna, and human populations [48,49]. #### 3.2. The Crisis for Declining Health in Urban Populations Despite predictions from theorists such as Thomas Malthus in 1798 that humanity would double every 25 years [48], this only occurred during a short window in the late 20th and early 21st centuries [48]. Otherwise, human population growth has not been uniform in either a temporal or a geographical context, nor has it always trended in positive direction. World War One (1914–1918), World War Two and the associated Holocaust (1939–1945), and The Great Famine (1959–1961) are examples of periods when the global human population measurably declined [49–52]. The human population reached 800 million during the Industrial Revolution, but took until the early 1800's to reach 100 million and was 1.6 billion at the start of the 20th-century, had grown to 6 billion at the dawn of the 21st century, currently sits at (pre-COVID-19 pandemic projections) 7.8 billion, and is projected to reach 9.5 billion by 2050 [49–52]. Reflecting the slowing trend of global population growth that has been evident since the 1950s, humanity is predicted to only grow by 1 billion people between 2050 and 2100 to start the 22nd Century at 11.2 billion on pre-COVID-19 pandemic projections [49–52]. While populations of some long-established cities are shrinking because of the demographic transition and/or economic migration [49–52], humanity became an increasingly urbanized species during the 20th-century, and that trend is predicted to continue until 2100. Approximately one quarter of the human population (1.95 billion people) lived in urban centers at the start of the 20th-century [51–54]. Further, urban populations are predicted to more than double by the end of the century, with the current level just over 4 billion people (55% of all humans) growing almost 6.5 billion in 2050 (68% of humanity) to reach to 9.5 billion people (85% of the global human population) by 2100 [51–56]. That rapid change in lifestyle has resulted in human disconnection from nature, with little opportunity for adaptation to those changed circumstances [2,14,56–58]. As a result, significant adverse health impacts, both physiologically and psychologically, are beginning to emerge [14,17,18]. Physiologically: cardiovascular health has decreased; diabetes has increased; obesity has increased; and biological intolerances have increased [14,15]. Psychologically: depression and anxiety have increased; cognition recovery ability has decreased; and stress related conditions have increased [6, 7,12–14]. Human-nature disconnection is likely to have further e ffects that are not ye<sup>t</sup> apparent, the subject of which is an area of growing interest for researchers and practitioners [14–16]. #### 4. Framing of the Urban Resilience Theory #### 4.1. Origins and Perspectives of Urban Resilience Theory Urban Resilience Theory research over the past two decades has been driven by growing environmental, social, and political uncertainty, combined with increased prevalence and severity of risks to urban centers [10]. Tensions arising from the ambiguity and disparity in defining the concept of resilience may go some way in explaining the limited application of the URT principles and teachings by decision makers [10]. To progress the definitional debate and improve wide adoption of the URT propositions, Meerow & Newell [10] (p. 315) proposed the following definition of urban resilience that is appropriate in the context of this article: "Urban resilience refers to the ability of an urban system-and all its constituent socio-ecological and socio-technical networks across temporal and spatial scales-to maintain or rapidly return to desired functions in the face of a disturbance, to adapt to change, and to quickly transform systems that limit current or future adaptive capacity ". The URT emerged from the fields of ecology, engineering, and psychology [59–61]. Summaries in the applications of the term resilience in each of those fields appear below. Each perspective provides a unique contribution to the current understanding of URT. Given the multidisciplinary nature of URT, agreemen<sup>t</sup> on an explicit definition remains elusive, although all the published formulations share the notion of the ability of a system or urban center to bounce-back from external pressures, stresses, or shocks [11]. Ecological resilience has been defined as the amount of disturbance or pressure that an ecosystem is able to withstand without permanently changing self-organizing processes and structures [59]. The greater the capacity of an ecosystem to recover and adapt to stochastic changes in circumstance, the higher the resilience. An allied ecological theory with relevance to the crises reported in this article is the Alternative Stable State Theory [59–62]. This theory holds that a tipping point may be reached whereby the associated feedback that may not allow the system to recover/return to previous state. With respect to engineering, the concept of urban resilience arose from the need to respond to new threats within modern society. Concentrating on infrastructure and networks, Bozza et al. [60] defined resilience to be the ability to recover, absorb, and restore equilibrium after a perturbation. From the engineering perspective, the original formulation for resilience was based on the idea that systems and networks need to realize a post-shock equilibrium position [60]. That perspective has evolved, and the current usage of the term resilience has moved towards a performance-based assessment of recovery and is thus seeking those assets and systems perform in the same capacity as prior to the disturbance or shock [60]. In this context, resilience relates to the recovery of complex systems, which are usually composed of physical subsystems occurring in an urban context [60]. Psychological resilience is the ability to adapt to stress, significant challenges, or adversities, which may include challenging life events, acute trauma, and/or chronic adversity [61]. Those experiences have the potential to substantially impact on brain function and brain structure [61]. A lack of resilience becomes visible through the development of psychological responses such as anxiety, depression, and/or post-traumatic stress disorder [61]. This perspective strongly adheres to the notion of adaptation in pursuit of ongoing health. #### 4.2. Converting Theory to Practice High density urban centers provide access and diversity of services, however also overcrowding, sensory overload, and increased levels of stress [23,62]. Samuelsson et al. [23] posit that greater consideration of resilience principles can improve planning and formation of policy for design and development of urban centers, with respect to densities and provision of services, by assessing opportunities and threats at a city scale. Building on work by the Organization of Economic and Community Development and other stakeholders and researchers, the 2014 researcher of Kim et al. [63] proposed a new Green Growth model to assess the development and growth of urban centers. Kim et al. [63] recommended twelve indicators of sustainable urban development that include measures such as greenhouse gas emissions, energy use, energy sources, water asset usage, portion of land covered by forest, public transportation opportunities, and more [63]. The Green Growth Model is not dissimilar to the Sustainable Development Goals (SDG's) when looking forward to urban development principles and practices. The recommendations and conclusions within this research show the contributions of URT and HNCT making some way to support the SGD's for achievement by 2030. Examples of this are SDG 3 Good Health and Well-being, SDG 11 Sustainability Development and Communities, and SDG 13 Climate Action. In line with the Green Growth Model and the SDG's, this new model lends itself to the approach of urban development that provides a socio-ecological focus, in contrast to traditional socio-economic approaches. In that context, URT proposes that greater concentration be placed on [64]: However, potential problems with implementation of URT that are identified in current literature [62] include the: #### 5. Framing of the Human-Nature Connection Theory #### 5.1. Origins and Perspectives of the Human-Nature Connection Theory Many models and theories attempt to explain the relationship between humans and nature and the resulting impacts or benefits for human health. Models include the Environment of Health, the Mandala of Health, the Wheel of Foundational Health Need, the Healthy Communities, the One Health approach, and more [63]. Each of those models attempt to describe the balance and interactions of the biological, social, and spatial influences of human-nature connections. Through the milestones of urbanization, from ancient city states, through to the 20th-centruy, the connection of humans and nature has been the focus of origin stories and philosophical and scientific writings. Given the ongoing interest in HNCT and similarly the URT, a number of multidisciplinary perspectives inform our current understanding of how exposure to nature influences the health and welfare of human populations. Key perspectives that inform the HNCT are summarized below. The environmentalism perspective suggests that humans traditionally had a relationship with nature being one of power and dominance, which is embedded in the Judeo-Christian belief systems that shaped Western civilizations [63–65]. That relationship has weakened over the past few decades, anticipated to reflect a natural balancing out between humans and the environment [63]. From an evolutionary biology perspective, culture-genetic interactions a ffect our lifestyle choices and thus our health. One example of such an interaction is that humans predominantly gain nutrition derived from food and farming processes [63]. Traditionally, humanity sourced food from farming the land and/or preparing food from natural ingredients that contained varied microflora [63]. For modern human populations living in urban centers, processed and packaged alternatives are now readily available and provide a larger proportion of food intake. Food tolerances and intolerances are suggested to be based largely on food choices, with reduced exposure to microbial activity increasing susceptibility to allergies [63]. An increased incidence of more extreme food-induced allergic reactions is one of the factors contributing to the crisis of declining health in urbanized human populations [63]. The evolutionary psychology perspective is founded on the apparent preference of humans for scenes dominated by nature and natural elements. Emerging only within the past few decades, this perspective suggests that human psychological characteristics are shaped and adapted based on the prevailing ecological and environmental conditions [24,25]. This perspective has delivered concepts that incorporate the inclusions of nature in self, deep ecology, extinction of nature experience, connectedness to nature, and the Biophilic Hypothesis [21,24]. #### 5.2. Converting Theory to Practice Embracing the HNCT to inform community planning and urban development practices can provide diverse opportunities to improve the security and quality of urban life. These opportunities include [20–24]: #### 6. Setting the Scene for Green Infrastructure The term green infrastructure appeared in the peer-reviewed literature and language of urban planning and managemen<sup>t</sup> practices during the 1980s [16]. While initially highly contested, the term is now widely accepted in published literature and has generated significant research interest over the past decade [14,16]. The components of UGI encompass a broad range of assets, including green POS, urban trees, urban stormwater management, green roofs and green-walls, and many other green assets [14,16,67]. A key aspect that distinguishes UGI from other forms of infrastructure (i.e., blue or grey) is the simultaneous delivery of social and environmental services and relief from the hard forms of the built environment [14,16,67]. Urban land managers already rely upon UGI assets, generally in the form of green POS, to deliver opportunities to urban dwellers to connect to nature and mediate the impacts of climate change [14,26]. Arguably the most prominent and widespread occurrence of UGI, green POS, is heavily relied upon to provide public health benefits [14,26]. There is a growing body of published research in which green POS users report and/or have demonstrated improved physiological and psychological health, a better outlook on life, enhanced cognitive recovery, because of the opportunities that quality green POS provides for exercise, recreation, relaxation, and reflection [14,15]. In summary, the opportunities o ffered by UGI are becoming increasingly significant assets that support healthy communities and enhance liveability in urban centers. In addition, UGI has relevance in migrating and mediating the twin crises of climate change (Table 1) and declining human health (Table 2). Table 1. Examples of urban green infrastructure o fferings that could mitigate and/or meditate impacts arising from the crisis of climate change on urban centers. Table 2. Examples of urban green infrastructure offerings that could mitigate and/or meditate impacts arising from the crisis of declining human health in urban centers. #### 7. Concurrent Application of URT and HNCT This section consolidates the information presented above to summarize how the equitable provision of easily accessible quality UGI aligns to both the Urban Resilience and Human-Nature Connection theories. This section provides responses that help mediate and mitigate the impacts from the twin crises of climate change and declining levels of human health within urban centers. Building from this content, Section 9 presents a conceptual model (Figure 1) that shows how the unified application of URT and HCNT can inform community planning and urban development that is preadapted to mitigate and mediate impacts from both climate change and declining human health in this century and beyond. Figure 1. Conceptual model of the interrelationships between the twin crises of climate change and declining human health in urban centers and mitigation and mediation measures offered by the Urban Resilience Theory (URT) and the Human-Nature Connection Theory (HNCT). #### 7.1. Urban Resilience Theory As previously mentioned, URT can contribute to the mediation and mitigation of climate change [77]. In summary, URT provides teachings and direction for practitioners and decision makers that can: improve traditional policy and planning frameworks, stimulate new systems and measures that aim to reduce greenhouse gas emissions, provide tools for climate change adaptation programs, promote capacity building through inter-governmental cooperation, and direct recovery planning for identified crises and emerging challenges. Over the past three decades, natural disasters that impact urban centers have quadrupled [77]. Further, climate change place many urban centers at risk and increases the susceptibility of cities also impacted by natural disasters [77]. Cities were once considered a place of refuge for inhabitants. Urban centers are, however, increasingly seen as hotspots of climate hazards and climate risks [77]. Climate hazards comprise floods, windstorms, droughts, fires, large temperature fluctuations, sea level rise, and landslides [77]. Climate risks represent the likelihood of climate hazards occurring, as well as the likelihood of adverse impacts to human health, green and grey infrastructure assets, and environmental and urban services or even loss of human life [77]. Implementing climate change mediation and mitigation measures informed by the URT, can avoid (mediate) climate hazards or reduce (mitigate) climate risks to: reduce the susceptibility of the affected locations and increase the ability of those locations to withstand imminent hazards, and improve in post-disaster response and post-disaster recovery. Specifically, URT contributes to overcoming climate change hazards and climate change risks in the following ways [77]: For the above strategies to be effective, industry change makers, policy advisors, practitioners, and decision makers need to ensure the following [76]: The application of the URT for mediating and mitigating climate change is strengthened by the scope of the theory and strategic planning of policy creation, infrastructure investment, building and construction, resource extraction and utilization, and environmental asset management. Declining human health in urbanized populations manifests as physiological conditions such as declining cardiovascular health and/or increasing diabetes and obesity and through psychological afflictions such as increases in stress related illness and the associated increase in anxiety and depression [12–15]. The teachings and direction of URT offers three key responses to declining human health among urban dwellers and supports several additional opportunities to stabilize and improve human health within urbanized human populations. Firstly, in addition to other measures, URT advocates and provides frameworks to ensure that urban dwellers have fair and equitable access to the environmental services and benefits provided by UGI assets. In this frame of reference, the UGI may constitute or be incorporated into urban POS that supports exercise and recreation, safe urban spaces suitable for engagemen<sup>t</sup> and connection with other urban dwellers, frameworks for engagemen<sup>t</sup> and deeper involvement of individuals with their local community in order to build social capital and social resilience, and the fair and equitable access to health related services that support and provide care for individuals whose health is compromised [10,65]. Secondly, URT supports and advocates for changes in common practice and approach to the consumption of resources that support modern urban life [10,65]. This is particularly evident in the space of energy production. Application of the teachings and direction of URT in this context supports the implementation of renewable energy sources in conjunction with new technology that proves successful in reducing emissions. Carbon emissions, a large proportion of the waste from traditional energy production, negatively impacts human health in urban centers in directly through the effects of climate change and directly through exposure to atmospheric contaminants [44,45]. Regarding the direct exposure to atmospheric contaminants, energy related emissions reducing air quality precipitates an estimated 3.4 million pre-mature deaths, globally, each year [78] from disease mechanisms linked to adverse respiratory health conditions and cancers [46,47]. Adoption of renewable energy sources and other new technologies aligned to increased urban resilience can significantly reduce emissions and therefore reduce the health risk among urban populations. Further, several policy and legislative changes that can, and already do, contribute to improving public health are aligned to URT principles [78]. Thirdly, briefly returning to the climate change crisis, URT offers frameworks and strategies to support human populations in adapting to the threats and challenges of climate change. This may be in the form of employable technology (i.e., improved heating/cooling, off the grid power back-ups), behavior change (i.e., reduction in waste, reduction in electricity dependence), UGI support (i.e., urban trees, urban POS, revegetation) and implementation, and building and construction changes (i.e., solar passive design, new materials) [10,65]. Those adaptations, informed by the URT, can reduce negative health outcomes, which can manifest in premature death, caused by climate hazards such as extended heatwaves and/or flooding and landslides associated with more frequent extreme weather events [10,65]. #### 7.2. Human-Nature Connection Theory The HNCT provides multiple practical opportunities for modern cities to respond to climate change by cultivating and increasing pro-environmental behaviors, advocating for the creation and implementation more UGI assets, improving practices and approaches to current community planning and urban development regimes, and advocating for the renaturing of urban communities [20–23]. The level of human-nature connection is seen to be a reliable predictor for pro-environmental behavior [20]. Pro-environmental behavior is defined as individuals that display behaviors that contribute to environmental sustainability, such behaviors including limiting waste, limiting energy consumption, improving recycling habits, and more [77]. Pro-environmental behavior represents ground level action in reducing the anthropogenic drivers of climate change. Therefore, the higher the level of human-nature connection, the higher the level of pro-environmental behaviors and the resultant decline in behaviors that contribute to climate change [20–25]. Human-nature connections, in this context, show human-influenced measures of environmental protection and conservation. Urban green infrastructure is at the core of HNCT (and Biophilic Design) principles as it contributes to mediating and mitigating climate change. As highlighted above, the HNCT advocates for increased rates of implementation for UGI assets such as green POS, green walls, green roofs, urban trees, and more. These UGI assets significantly increase the area of spongy surfaces in the built environment. Those spongy surfaces reduce the urban heat island e ffect by absorbing and reducing reflective heat, providing shade that allows for human and fauna refuge, absorbing rainfall and stormwater to help reduce local-scale flooding and erosion, and increasing the amount of biodiversity, all of which increases urban resilience in relation to climate hazards [20–25]. Community planning and urban development strategies play a significant role in shaping how cities are designed and constructed. Improvements to community planning and urban development policies and frameworks greatly influence how humans cohabitate with the environment and each other in urban centers. Planning and development designed around people, and their deep seeded desire to be within and around nature and natural elements can also provide answers to climate change. Urban centers can contribute to the mediation and mitigation of climate through means such as solar passive heating and cooling design principles, providing focus on natural light and natural air flow to reduce the reliance on electricity, employing sustainable building materials to reduce the need for resource extraction, and more [20–25]. Therefore, approaches that align with the HNCT and Biophilic Design principles prove to be essential in addressing the climate change crisis. Similarly, HNCT o ffers some unique contributions to address the crisis of declining human health amongs<sup>t</sup> urban populations. As highlighted by this article, these contributions include the teachings from the ecological health perspective, the direct physiological and psychological benefits available from engaging with nature, the contributions that the presence of quality urban nature make to communities and social capital that supports of public health, and a reduction in vulnerability to adverse health conditions [20–25,46,47]. For the past 50 years, as humans have become an ever more urbanized species, the interrelationships between people and the environment around them, and how these relationships a ffect human health, have been a growing focus for research [14,78,79]. Dating back to the 1980s, the Mandala of Health model was advanced to explain the complexities and holistic influences, systems, and relationships of human interactions with nature. Similarly, Wilson [24] proposed his Biophilic Hypothesis in the 1990s. These anthropocentric models, like others before and after, focus on the benefits that interacting with quality nature spaces provides for mind, body, and spirit of individuals. The Mandala of Health also incorporated the interactions and entanglements at the family level that arise from personal behavior from psycho-social, economic, and environmental factors; from the physical environment, and from human biology [63]. The third layer proposed by Hancock [63] is the human-made environment (community) and fourth an all-encompassing layer of the Mandala is the biosphere (culture). The Mandala of Health proposes that each of these layers affect human health and wellbeing and need cognition to all factors to find the equilibrium required for peak health performance and outcomes. Research over the past few decades has built on ecologic health models, however the notion remains much the same. To achieve good health and wellbeing outcomes, balance must be realized with the built environment and the green infrastructure of urban centers [14,78,79]. A large amount of research now underpins our growing knowledge about the relationship between nature and human physiological and psychological health. A summary of that research is provided below: #### 8. Realigning and Refocusing Towards 2100 and Beyond Due to the dominant impact of humans on the systems of planet Earth, the new geological series of the Anthropocene, also sometimes referred to as the Human Dominated Geological Epoch, has been recognized [80–88]. While somewhat contested, this epoch is proposed to have commenced in the 1950s, the Anthropocene is characterized by trends of urbanism and the depletion and/or contamination of natural resources [1,89]. Despite these negative trends, surveys show that urban dwellers, in the main, consider urban city life as largely positive [14,90]. However, concern is growing about the equity of access and opportunity, a correct power balance, and the current social and environmental crises, which includes the impact of climate change and declining human health addressed in this article. Current approaches to urban development has been seen to be lucrative for a number of industries, which can perpetuate challenges such as poor planning practices and development for financial gain, lack of availability and demand for alterative sustainable practices, lack of demand for different outcomes and community planning values, a perception of a cost premium for the adoption and implementation of alternative practices and measures, and low support and incentives from regulatory bodies [90]. The current dominant paradigm suggests that the primary purpose of nature is the provision of raw materials and environmental services for the benefit and enjoyment of humans. Under this paradigm, humans are not part of nature, but rather nature exists to be conquered for gain on the basis that short-term human growth and progression will provide benefits in the future that are able to compensate for abstraction and destruction now [91]. Also known as the Technocratic Paradigm, this approach is prefaced on most of humanity perceiving that technological advancements will be able to overcome current and future threats to human populations [11,91]. The dominant paradigm suggests that economic growth needs be continuous for communities, cities, and societies to advance [9,11]. Evolving a sustainable dominant paradigm that is focused on equity, opportunity, risk reduction, and resilience is greatly needed. The barriers to adopting a sustainable paradigm need to be investigated, debunked, and worked through to achieve change. #### 9. The Promise of Urban Green Infrastructure The URT and HNCT both show that there are suitable alternatives to the current dominant paradigm, many of which can be realized through the employment and implementation of UGI. While this article only examined the contributions of these theories in the context of climate change and declining human health, it is thought that many other crises may be similarly mediated and mitigated by the application of the URT and HNCT to conserve, protect and reintroduce UGI through inclusive community planning and sustainable development. As reported above, substantial mitigation and mediation of the twin crises can be achieved through the conservation, reintroduction, enhancement, and protection of UGI as a response that addresses both climate change and declining human health, as advocated by the URT and HNCT. Informed by the documentary analysis presented in this article, the four-set Venn diagram conceptual model presented in Figure 1 demonstrates the complex and highly interrelated nexus between the twin crises examined and the combined application of the URT and HNCT. Key points are provided for each interrelationship to provide a starting context to the relationship. Further, the existing literature of this field has a gap with respect to advocating for that combined approach to the implementation of the URT and HNCT in urban centers. The novel approach of integrating the application of those two theories via the equitable provision of easily accessed quality UGI can mediate and mitigate both climate change and declining human health and can positively contribute to the refocusing and reframing of unsustainable development many urban centers are currently pursuing. Adopting the evidence-based approach, advocated in this article and illustrated in the conceptual model provided in Figure 1, is essential for delivering the inclusive community planning and sustainable urban development as humanity recalibrates our focus towards the end of the 21st-century and beyond.	doab	2025-04-07T03:56:59.415101	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 39 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.40	10. Conclusions This article highlights that the URT and the HNCT can both make multifaceted contributions to mitigating and mediating the drivers and impacts of the twin crises of climate change and declining health among urban dwellers. With respect to both theories, conserving, protecting, and restoring quality UGI is the foundation for action to reduce the severity and impact of those crises and for progressing inclusive and sustainable community planning and urban development that focuses beyond 2050. While some UGI can be found within the fabric of most modern cities, the URT, the HNCT, and this article advocate for an increase in the amount, the resourcing, and the perceived value of those UGI assets. Historically, the benefits that UGI provides in terms of mitigating and mediating climate change and providing physiological and psychological health benefits for urban dwellers have been considered as intangibles. As such, UGI has not been valued in terms of the economic return-on-investment those spaces provide with respect to the resources expended to realize the environmental and social value of the ecosystem services that quality UGI delivers for modern urban centers. However, this article highlights how, congruen<sup>t</sup> with the URT and the HNCT, access to quality UGI can assist in changing from "business as usual" to a more sustainable and resilient approach to community planning and urban development in the second half of the 21st-century. Implementing the combined approach to URT and HNCT advocated in this article is likely to have limitations in the global context. The limitations could include geographical and cultural considerations at the local scale, resources that are available to land managers, community demand and expectations for UGI installations, stability of current political environments, and the quality of governments and governmen<sup>t</sup> structures. However, given the previously identified gap in the literature regarding the complementary implementation of URT and HNCT through the medium of UGI, the nature and magnitude of such limitations remains unknown. Clarification of such limitations will most likely be facilitated by the anticipated increase in volume of research URT, HNCT, and UGI. Further, the rapid global changes and growing research interest highlighted in this article, will generate additional insights that are not ye<sup>t</sup> apparent. Further research is therefore required to investigate and understand the contribution that UGI can make in terms of inclusive community planning and sustainable urban development as our increasingly urbanized human population begins to recalibrate and refocus beyond 2100. That research should investigate different forms of UGI to deliver specific and robust findings aligned to the climate change, human health, urban resilience, community planning, and sustainable urban development foci of this article. To that end, two such studies have been completed in support and are under manuscript development: Author Contributions: Conceptualization, J.P.; methodology, J.P.; investigation, J.P.; writing—original draft preparation, J.P.; writing—review and editing, J.P. and G.D.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors thank Richard Harper for his supervision of JPs PhD program and allowing us to progress this article without being listed as an author. We also thank our former and current colleagues and managemen<sup>t</sup> at the City of Belmont and the Towns of Claremont and Mosman Park for their support of the research projects that help to inform this article. The authors also thank the Land editors and four reviewers whose insightful feedback enhanced our article. Conflicts of Interest: The authors declare no conflicts of interest.	doab	2025-04-07T03:56:59.418244	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 40 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.41	Appendix A The tables reported below (Tables A1–A4) summarize the geographic scope and UGI research focus of the case study, empirical, and documentary research reported in the 87 articles analyzed by Parker and Simpson [14], the 171 articles analyzed by Parker and Zingoni de Baro [16], and the 38 UGI related articles cited as evidence in support of the synthesis of the URT and HNCT presented in this article. Table A1. Geographic distribution of case studies and sources of data that informed this article. 1. Data extracted from included articles published in Data Descriptor by Simpson and Parker [17]. 2. Data extracted from included articles published in Data Descriptor by Parker and Simpson [19]. 3. The global-scale analysis of GIS data by Richards and Belcher [43] that covered the five most inhabited continents was coded as a Global Review article. Table A2. Number of countries represented in the case studies and sources of data that informed this article. 1. Data extracted from included articles published in Data Descriptor by Simpson and Parker [17]. 2. Data extracted from included articles published in Data Descriptor by Parker and Simpson [19]. 3. Richards and Belcher [43] conducted a global analysis of GIS data that covered multiple countries on each of the five most inhabited continents. Table A3. Number of cities reported in the case studies and sources of data that informed this article. 1. Data extracted from included articles published in Data Descriptor by Simpson and Parker [17]. 2. Data extracted from included articles published in Data Descriptor by Parker and Simpson [19]. 3. In addition, Richards and Belcher [43] conducted a global review of GIS data that graphically reported UGI data for at least 1000 cities across all five of the most inhabited continents (excluding Antarctica). 4. In addition, Biega ´nska et al. [37] report a GIS-based country-scale comparative analysis of the UGI provided in peri-urban developments associated with approximately 100urbancentersinGermany,Latvia,andPoland. Table A4. Focus for case studies of the aspects of urban communities and development that informed thisarticle. 1. Data extracted from included articles published in Data Descriptor by Simpson and Parker [17]. 2. Data extracted from included articles published in Data Descriptor by Parker and Simpson [19]. NR = Not Reported – NA = Not Assessed.	doab	2025-04-07T03:56:59.418588	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 41 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.43	Ways Forward for Advancing Ecosystem Services in Municipal Planning—Experiences from Stockholm County #### *Sara Khoshkar 1,\, Monica Hammer 2, Sara Borgström 1 and Berit Balfors 1 Received: 4 August 2020; Accepted: 25 August 2020; Published: 26 August 2020 Abstract: This case study from Stockholm County, Sweden, explores practitioners' experiences of barriers and bridges in municipal planning practices to support actions for ecosystem services. This qualitative study is based on information gathered from a focus group, workshops, and semi-structured interviews, which aided in identifying key factors for integrating ecosystem services in municipal planning. We identified 10 key factors divided into three themes: (i) regulatory framework and political support, (ii) local organizational capacity, and (iii) local adaptation of tools and practices. In particular, the practitioners pointed to the need for the development of legal support and regulations for ecosystem services on the national and EU policy levels. Furthermore, the need for local capacity building and understanding of ecosystem services as well as increased regional support to enhance local knowledge exchange and learning was emphasized. Also, in a decentralized local governance system such as in Sweden, to fully implement ecosystem services in urban planning for sustainable development, locally adapted practical tools and monitoring procedures were considered important. Keywords:** capacity building; ecosystem services; municipal planning practice; urban governance	doab	2025-04-07T03:56:59.418751	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 43 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.44	1. Introduction Globally, multiple human drivers, degrading biodiversity and ecosystem functions and services [1] have significantly altered ecosystems. To prevent further decline and restore ecosystems, there is a need for transformation and change for socioeconomic development to strengthen the delivery of ecosystem services (ES) [2]. ES are defined generally as the functions performed by nature that directly or indirectly benefit humankind and contribute to well-being (e.g., water regulation, air regulation, recreation) [1]. Agenda 2030 states that ES and biodiversity are relevant to all sustainable development goals (SDGs) and emphasizes the need for action [3]. Urban areas are rapidly growing in Europe, for example, Berlin, Malaga, and Stockholm [4]. The increasing number and expansion of urban areas underline the importance of integrating ES in spatial planning [5,6]. Strategic decisions on land use allocations made during the planning process can have significant impacts on urban green spaces and their capacity to support the provision of ES. The integration of ES into urban planning processes is considered to provide a more comprehensive understanding of the values at stake and of the synergies and trade-offs that might arise from land-use decisions [7]. Although the knowledge and awareness of the relevance of ES has increased, there remains a gap in the mainstreaming and implementation of ES in practical planning and decision-making [8,9]. Spatial planning in Europe varies amongs<sup>t</sup> the di fferent countries, influenced by the governance arrangements and institutional settings [10,11]. In Sweden, municipalities have the main responsibility for spatial planning [12] and local managemen<sup>t</sup> practices play a key role in sustaining urban ES [13]. Decisions that consider ES at the local planning level can enhance the provision of biodiversity and ES and generate benefits that occur at multiple levels from local to global [14,15]. Also, a ffiliated planning instruments like strategic environmental assessment (SEA) have been recognized as opportunities to enhance decision-making by including the ES approach [16,17]. The importance of governance systems that have the potential to safeguard the long-term delivery and use of ES at di fferent levels of governance is emphasized [6]. Di fferent challenges confronted when implementing ES into practical governance have been identified in the literature related to awareness and interest among practitioners, coordination between planning departments, as well as processes and routines [18,19]. In a study from Berlin, based on qualitative content analysis and semi-structured interviews, several governance challenges for the implementation of ES were identified, including financial constraints, loss of expertise, and insu fficient communication about benefits from urban green spaces to key decision-makers [20]. In Rotterdam, the main challenges included a lack of coordination between planning departments and a lack of evaluation of and learning from pilot projects [21]. The governance challenges for the implementation of ES can be di fferent depending on the countries' governance arrangements. However, current ES approaches often do not take existing governance structures and practices as a starting point, and tend to focus on issues such as the spatial extent of ES [22]. Hence, to assimilate the ES framework in urban planning practice rather than in single projects, it is important to gain an understanding of the governance context including institutional frameworks, existing policies, and planning systems [23]. Furthermore, the views of practitioners and decision-makers involved in the planning process are important for the implementation of ES, particularly on the potentials and added value of using the concept and what tools could be used in practical integration [24]. In light of the transition towards decentralized governance in many European countries [25] and the uniquely strong position of Swedish municipalities in planning [26], the Stockholm case provides an interesting context to examine and contribute to the knowledge on ES implementation in local planning practice. In our study, drawing on empirical experiences of practitioners in Swedish municipal planning, we explored and analyzed local spatial planning practices to identify key factors for advancing ES implementation in planning processes focusing on municipalities in Stockholm County as a case study. The following research questions formed the basis of this paper: #### 2. Theoretical Framework #### Urban Governance and Spatial Planning Governance can be defined as the institutions, structures, and processes that determine who make decisions, how and for whom decisions are made, whether, how, and what actions are taken and by whom [27]. In urban regions, spatial planning is a key component of governance e fforts to guide development processes [28]. To achieve changes for planning [29] emphasizes that the use of knowledge is a central element. More specifically, [30] highlights the role of expert knowledge in order to take planning and development systematically in directions for contributing to the achievement of defined goals. The authors emphasize that it is important to note that various forms of knowledge interplay in planning processes, with actors from di fferent sectors and levels involved. Therefore, even with the existence of the relevant expert knowledge, it may be overruled by the knowledge of actors involved. In the context of planning for ES, new knowledge about ES does not necessarily influence decisions, because complex interactions within and across governance systems may have implications for the actual implementation in policies and plans [31,32]. [33] also discuss that environmental knowledge alone does not shift priorities from unsustainable practices to sustainable development since priority setting is influenced by competing interests and political agendas, power relations and modes of governance, regulatory frameworks, and property rights. Furthermore, multiple governmental and non-governmental actors and institutions are involved in planning for ecosystem services and decision-making, which can lead a heterogeneity of practices dependent on the governance context [34]. This involves that the managemen<sup>t</sup> of ecosystems and landscapes requires an integration of various sources of information and knowledge from various levels and sectors of society [22,35]. Moreover, coordination between involved actors is required to create coherent action and strengthen the role of ES in spatial planning. However, changing land-use patterns, diverse interests, and values of actors on different scales poses challenges for the governance and planning of ES when negotiating the trade-offs in the provision of such ES [36,37]. Lastly, many methods, approaches, and tools have been tested to support decision-making in applying the ES concept [38]. Actions and tools are said to constitute the core of a plan [39], and the probability of success of an action depends on the type of tools used [40]. However, [41] emphasizing that tools and methods alone are not enough to support ES, there remains a need to encourage better ES management. For example, there is a need to provide access to the tools and foster their use through knowledge exchange and application in practice [38–42]. Overall, as highlighted by [43] more attention should be given on how to facilitate change that moves the concept of ES from an "ideal into reality". According to [31], empirical evidence is needed on the governance of ecosystem services, including issues of decision-making and policy implementation. Our paper contributes to the above research gap by providing insight on practices and experiences of ES implementation from a decentralized governance setting and presenting key factors for advancing ES in planning practice. #### 3. Swedish Governance Context In Sweden, ES was introduced in the 2010 Swedish Environmental Policy (Government Bill 2009/10:155) and was further elaborated in 2012 [44]. More specifically, two milestone targets related to the implementation of ES were added in 2012 [44]. Furthermore, in 2013, the Swedish governmen<sup>t</sup> assigned an inquiry (SOU:2013:68) to analyze actions and propose methods and measures to better evaluate ES and integrate the importance of biodiversity and the value of ES into economic plans and other decisions in society [45]. As a result, the ES concept has been implemented to varying degrees in the different parts and at different levels of Swedish society. In 2018, two additional targets regarding ES were added which called for municipalities to (i) have access to a developed method for utilizing and integrating urban greenery and ecosystem services in urban environments, in planning, construction and managemen<sup>t</sup> by 2020, and (ii) shall utilize and integrate urban greenery and ecosystem services in urban environments in planning, construction and managemen<sup>t</sup> by 2025 [46]. Hence, at a national level, there is a strong political will to implement ES approaches as a strategy for urban sustainable development in Sweden. The local level of governance, consisting of 290 municipalities, has an important role in meeting the sustainability targets related to ES. More specifically, the municipalities hold a planning monopoly, where regional and national authorities can intervene when national regulations are at risk of being violated [47]. Spatial planning in Sweden is regulated through the Planning and Building Act (PBA) (National Board of Housing, Building, and Planning) and the Environmental Code (Swedish Environmental Protection Agency) (SEPA) [48]. The PBA mainly regulates exploitation, while the Environmental Code mainly addresses the conservation/protection of land or water [49]. Municipal comprehensive plans (MCP) and detailed development (local) plans are the main statutory planning instruments in Swedish spatial planning [50]. In accordance with the PBA, municipalities have an important role in coordinating their comprehensive plans with plans and policies at other levels of governmen<sup>t</sup> and demonstrating how the international, national, regional, and local goals will be met. A recent analysis of the integration of ES in the MCPs revealed that these plans are increasingly addressing concrete strategies or measures for the provision and/or conservation of ES [51]. #### 4. Materials and Methods #### 4.1. Case Study Area The geographic scope for this study was set as Stockholm County, which is Sweden's largest urbanized region, consisting of 26 municipalities [52]. The planning practices and challenges presented in this study are gathered from six municipalities within Stockholm County: Huddinge, Haninge, Nacka, Stockholm, Täby, and Upplands Väsby (Figure 1). The selection of municipalities represents a mix of rapidly growing urban and peri-urban areas with extensive ongoing urban densification. Moreover, these municipalities have initiated different activities to strengthen ES in their planning practice by, for example, developing approaches and tools for ES mapping, applying for external funding for ES projects, and developing ES strategies. Table 1 provides a brief description of the municipalities studied including population size, projected population size by 2030, percentage of population growth between 2018 and 2030, population density, total land area, and percentage of developed land. Figure 1. Map of Sweden, zoomed to Stockholm County and municipalities comprised in the study. Table 1. Cont. #### 4.2. Research Design The empirical basis of the paper is a qualitative research study, including a mixed set of methods: one focus group, three workshops, and five semi-structured interviews between June 2018 and September 2019. Gathering data and information through different methods provides divergent perspectives, which according to [53] creates a more complex understanding of the phenomena studied. The study began with a focus group, which allowed for in-depth exploration and discussion on ES practice in spatial planning, and aided in the development of the discussions for the workshops in accordance with [54]. With support from the theoretical framework, themes for improved ES practices were derived and refined through an iterative process based on the information and discussions in the focus group, workshops and semi-structured interviews. Throughout the research process, at least two researchers documented discussions as written notes, which were compiled and cross-checked. #### 4.3. Focus Group Discussion An exploratory focus group discussion was organized in accordance with [54] with three experienced municipal practitioners from three municipalities in June 2018, with the aim to discuss future directions for ES in municipal planning. The size of focus groups can vary, however, according to [55], a group size of between 3 and 5 is easy to manage and provides opportunities for all participants to discuss and share views and experiences. The participants were selected based on purposeful sampling, which is a strategy in which particular settings, persons or activities are selected deliberately to provide information that is of relevance to the research questions [56]. The selected participants were three of the most experienced municipal practitioners working with ES in their respective municipalities. More specifically, in their respective municipalities, they had experience of working closely with several research projects regarding ES implementation, as well as in other municipalities and governance levels from which they shared their experiences. The discussions were centered on two questions formulated by the research team: (i) what are the lessons and experiences of ES implementation from ongoing or completed urban development projects? (ii) What are the future directions for ES in municipal planning practice? The outcome of the focus group provided a basis for the topics of discussion for the following workshops, as well as insight into the municipal practitioner's experiences of working with ES.	doab	2025-04-07T03:56:59.418881	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 44 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.45	4.4. Workshops Based on the discussion in the focus group, a sequence of three half-day workshops were organized on three separate occasions (April, May, and June 2019), discussing the same topic and involving different participant constellations. Workshops were chosen to gather information because they provide the opportunity to elicit rich information from participants selected through purposeful sampling [57]. Furthermore, workshops provide the opportunity for stakeholders of different organizations to collaborate with one another and share experiences [58]. The participants of the workshops included representatives from local and regional governance, construction sector, consultants, and academia (Table 2). In general, the same participants did not participate in more than one workshop. The mixed group of participants allowed for the gathering of different perspectives and views. The discussions in the sequence of workshops focused on factors to advance ES practice in municipal planning, along with challenges that may hinder the implementation. Table 2. The constellation of participants attending the workshops in April, May, and June 2019. #### 4.5. Semi-structured Interviews Following the focus group and workshops, five semi-structured interviews (60–90 min) between July and September 2019 were conducted with selected municipal practitioners to validate the researcher's understanding of the gathered information. Prior to the interviews, an interview guide was developed following [59], which summarized the challenges, needs, and key factors identified in the preceding research settings. Identified key factors for advancing ES in planning practice were presented to the participants, providing them the opportunity to elaborate on the researcher's findings. The interviewees were selected based on their involvement in the focus group and workshops, as well as their leading role in working with activities involving ES in the respective municipalities. #### 4.6. Thematic Analysis A thematic analysis in accordance with [54] was conducted based on the theoretical framework and the views and experiences gathered from the focus group, workshops, and interviews. Three main themes were identified: (i) regulatory framework and political support (ii) organizational capacity building for implementation of ES in municipal planning (iii) tools and practices for ES in the local context. The themes were refined through an iterative process in relation to the workshop and interview series. For each of the themes, challenges, and needs were identified. Next, key factors for strengthening ES implementation were linked to the challenges and needs within the three themes.	doab	2025-04-07T03:56:59.420027	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 45 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.46	5. Results Below, the identified key factors for implementing ES in local planning in relation to the practitioner's experiences and views are discussed according to the three themes (see also Table 3). In most cases, general patterns are presented rather than individual viewpoints of the different participants. #### 5.1. Regulatory Framework and Political Support Within the Swedish context, the planning of ES is regulated through both the Planning and Building Act (PBA) and the Environmental Code. According to many participants in our study, ES is still regarded as an aesthetic question, rather than a technical requirement in the legislation. More specifically, the participants pointed out that in accordance with the legislation, the detailed development plans can control what actions cannot be taken, but the possibility of placing stronger demands on developers to conduct specific actions is limited, including implementation of measures to enhance ES. The limitations of PBA regarding ecosystem services are also highlighted by [60], who argue that, in contrast to, for example, parking spaces, there are a lack of clear standards related to the size and number of blue-green space, which places them at risk in land use decision making processes. The participants emphasized that the need arises to develop voluntary municipal policies related to ES in urban development projects in order to provide targets and measures for the inclusion of ES in planning and decision-making. One practitioner highlighted the importance of integrating knowledge from green space plans into the comprehensive plans because then there is a "possibility to really push ecosystem services into the comprehensive plans". Furthermore, in the municipality of Huddinge, ES has been incorporated into the municipality's overarching goals to become a sustainable municipality by 2030. One goal identified was "balanced ecosystem services" (see also [61]), including several sub-goals related to ES (e.g., reduced climate impact, water), which the municipality was working towards. Aligning ES work with existing regulations and municipal organizational goals has been suggested to be important for motivating the integration of ES thinking [62]. The participants also emphasized that to support ES in local policy, there is a need to strengthen the integration of ES in EU policies and regulations. The importance of EU legislation was emphasized by exemplifying how di fferent issues become more or less prioritized due to how they are framed in a juridical sense. For example, in the studied municipalities, regulating ES related to water quality tends to have stronger support in the legislation because of binding instruments at higher governance levels, such as the EU Water Framework Directive (2000/60/EC), with clear indicators and requirements that all EU member states must achieve in relation to ecological and chemical water quality. Even though there is a strong national political support to integrate ES into societal decision-making including urban planning processes, the political support at the local municipal level was identified as a key factor for advancing the implementation of ES. Political shifts were described as directly influencing the available resources, as well as prioritizations among planning objectives and approaches. According to the participants in our study, the benefits of working with ES to achieve national and regional visions must be conveyed to the politicians. More specifically, a participant stated that, "decision-makers are on board when you can translate nature into their conceptions (e.g., storm water, economic benefits)." Hence, communicating the ES concept to local decision-makers was another key factor identified. In Stockholm County, a network of eight southern municipalities in the central urban region employed a development coordinator tasked with communicating within and across municipalities, as well as with municipal decision-making bodies (e.g., local politicians, municipal CEOs). More concretely, in one of the workshops, a local politician suggested using more scenarios to illustrate and serve as a communication tool with the potential to gain interest among politicians. #### 5.2. Organizational Capacity Building for Implementing ES in Municipal Planning In a decentralized planning system like in Sweden, local capacity building is important. There is a need for local knowledge to maneuver confronted challenges in the specific local contexts. According to the participants, an important success factor is the internal basic knowledge of ES. Furthermore, the level of integration of ES in the detailed development plans could vary depending on the basic knowledge of ES of the persons involved in the planning process. Consequently, it was suggested that when the ES knowledge base is lacking, it becomes important to build capacity and learn from others. There is also a need for the knowledge to be maintained and developed within the municipal organization and their daily practices. One format to facilitate knowledge exchange and learning discussed was the development of meeting platforms where municipal practitioners could share their experiences and examples of working with ES. One such national platform exists regarding climate change adaptation in Sweden (Swedish Portal for Climate Change Adaptation). Also, the new online guidance on ES in urban planning, developed by the National Board of Housing, Building, and Planning has ambitions to provide such a platform [63]. Furthermore, the Swedish Environmental Protection Agency (SEPA) has also developed a guide on how to value ES, with step-by-step directions and examples [64]. However, the practitioners discussed that for those municipalities that have already started developing their own routines for integrating ES into planning, these guidance documents were developed too late to provide the needed support. They expressed a need of advisory support for keeping and extending the use of ES within the planning practice, as well as with capacity building within the organization. More specifically, the practitioners emphasized the importance of strengthening the advisory role on the regional level where the County Administrative Board (CAB), is tasked with reviewing the detailed development plans before an application for a construction permit is submitted. According to the practitioners, a limited number of ES are amongs<sup>t</sup> the criteria that are checked in the review process. For example, ES connected to water quality is checked, which has led to water issues gaining momentum and more resources being allocated to studies for handling storm water and flooding within the municipalities. As emphasized by the practitioners, if legislation is strengthened to enable the regulation of a full range of ES in the detailed development plans, the advisory role of the CAB could have the potential of ensuring the implementation of actions related to ES. Additionally, within municipal organizations, there is a frequent change of employees, also confirmed by [65]. This could present challenges, especially if key personnel leading ES initiatives leave a project, process, or the organization. The next person to take over the position may or may not have the same level of knowledge, experience, or motivation to continue with the previous initiatives. Thus, developing processes and routines for transferring knowledge e fficiently and institutionalizing ES knowledge and procedures within municipal organizations is a key factor. Also, the development of systematic monitoring plans with relevant indicators can aid in identifying if ES was integrated in the planning process and actually resulted in implementation as well as contributing to capacity building. Examples provided by the practitioners from the case study municipalities included the municipality of Huddinge, which since 2017, has an annual follow up of their 89 environmental goals in order to monitor the status of implemented actions, initiated measures, delayed actions, and goal completion. The conservation of green areas and biodiversity are amongs<sup>t</sup> these goals. In the municipality of Täby, a practitioner shared the experience of how to structure a follow-up phase. This has been "incorporated into the planning process, which takes place after the adoption of the detailed development plan, and before submission of an application for a construction permit." The follow-up aims to ensure that the quality level of the planned development follows in accordance with what had been agreed by the municipality and developers during the planning process. #### 5.3. Tools and Practices for ES Implementation in the Local Context In Sweden, the 290 municipalities di ffer in land use, population, and financial resources a ffecting the possibilities of implementation of ES in local planning. When working with ES in local municipal planning, the need for incorporating ES into municipal routines was discussed by the participants, with many emphasizing the need to find established ways of working, evaluating practice in the di fferent departments, identifying any gaps and who should fill those gaps. The participants pointed out that several of the progressive municipalities in the Stockholm region have developed their own planning instruments on a voluntary basis as a complement to required municipal comprehensive plans, in order to support planning processes that recognize specific issues such as ES. One example is Upplands Väsby municipality, that has developed extensive planning instruments on ES including "Mapping of Ecosystem Services" and "Development Plan for Ecosystem Services". More recently, the municipality has developed an action plan to support the implementation of the established strategies for ES in municipal activities. Parallel to the voluntary instruments, the participants emphasized the importance of driven individuals to lead the learning process on how to use these planning instruments within the organization for successful implementation. Also, the participants highlighted the importance of specific roles and clear responsibilities for implementing the ES approach. The planning process was described as being long and extensive and the individuals involved in setting the strategic vision are often not involved in the implementation phase. Consequently, the vision set at the start regarding specific ES (e.g., tree planting) may not be implemented. As one of the participants stated, "when developing the detailed development plan in particular, there should be at least one person involved with the competence of ES." However, depending on the resources available in the municipalities, this is not always the case. Furthermore, the material concerning ES was described as being extensive but fragmented. There is an overall lack of clarity regarding who should be responsible for consolidating ES at the municipal level and there are di fferent approaches by di fferent municipalities, which also impact how it is implemented [66]. In the studied municipalities, two potential tools for ES in the planning of new developments were highlighted, Point System 2.0 and the green space factor (GSF). Point System 2.0 was an adaptation of the original Point System to include ES and was developed in Upplands Väsby. The GSF has been implemented in several of the municipalities in Stockholm County. The aim of the Point System is to facilitate collaboration and encourage construction companies to work with sustainability measures at an early stage. Through the application of this tool, the developers will receive points for implementing sustainability measures, which, in turn, generate discounts on the price of the land. In Upplands Väsby, a points system was applied to a large-scale project, Fyrklövern, which has been described as the largest residential building project in the municipality in 30 years, involving 14 di fferent construction companies (see also [67]). As a basis for the points system, a series of dialogue meetings with the citizens and di fferent actors involved in the planning and development process were organized to identify novel ideas for the new development project. The experiences gained from the implementation of the points system were being used to develop an updated version, which includes measures for ES. According to the practitioner, this version has the potential to be a "tool for discussion to keep the ES alive, providing more room to discuss what we want in a particular place". The GSF is a tool for increasing green spaces within development projects while minimizing the extent of sealed or paved surface designs that have been applied in many cities around the world (e.g., Malmö, Seattle, Berlin) [68,69]. In Stockholm, it has also been used in several of the municipalities and was appreciated by the practitioners as a way to facilitate dialogue between municipalities and the developers. GSF enabled concrete measures to increase the proportion of greenery on the land that had been set for development. As a means of building capacity in terms of internal learning and establishing routines, Haninge municipality organized an educational workshop about GSF for the di fferent departments to enhance the usage in detailed development planning processes. Furthermore, the experience from Upplands Väsby was that "maps to identify ES were the starting point to bring ES into the planning process and permitted knowledge to be gained on what exists, what is needed and what means exist for fulfilling the needs." However, a question was raised regarding how long these maps would be relevant and the need to continuously update them was emphasized, although there was often a lack of resources to do so. Table 3. Summary of challenges and key factors for strengthening ES implementation.	doab	2025-04-07T03:56:59.420240	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 46 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.47	6. Discussion Our study explored and analyzed practitioner's views and experiences of local spatial planning practices in municipalities in Stockholm County in order to identify key factors for supporting the integration of ES in municipal planning practice. We found a heterogeneity of practices and experiences amongs<sup>t</sup> municipalities studied. This is expected in light of the strong local planning monopoly and the responsibility of each individual municipality to derive strategies and measures to reach the national and regional goals. Consequently, it may lead to the consideration of relevant goals for ES to be applied differently across municipalities [49]. Also, the diverse development patterns within the municipalities put forth different prerequisites and challenges that each municipality must work with [51]. Similarly, it has been observed in European countries, that the differences in methods, policy mandates and funding mechanisms for ES implementation, results in heterogeneous practices and needs [70]. As discussed in the preceding sections, urban governance systems have the potential to facilitate the use of ES at different levels of governance [6]. However, many factors can influence the uptake of ES, such as political priorities, available knowledge base, and the municipal arrangements [30–34]. In the Swedish local planning context, we identified 10 key factors divided into three themes for supporting the integration of ES in the municipal planning practice (See Table 3). The factors are linked to the identified challenges and needs based on the practitioner's views and experiences. Our findings indicate that regulatory frameworks that set mandatory requirements for a range of ES are important for supporting the implementation of ES in local planning practice. This is in line with [33] emphasis on the importance of integrating ES into EU level regulatory frameworks such as the Thematic Strategy on the Urban Environment and other regulatory frameworks that guide spatial planning and natural resource management. Also, [71] argues that the managemen<sup>t</sup> of biodiversity has binding instruments, for example, the Habitat Directive (92/43/EEC) for specific species and non-binding instruments such as the Biodiversity Strategy 2020, but there remains a need for a specific EU policy devoted to governing urban ES. In line with supporting EU regulation, as a legally required instrument, Strategic Environmental Assessment has been identified to be a good entry point for integrating ES in spatial planning, as it is already established as a widespread (often mandatory) process to assess effects of policies, programs and plans [6,72]. For example, the SEA of spatial plans can ensure that ES is taken into account when evaluating different planning alternatives [73]. Furthermore, although the municipalities have a local planning monopoly, political support and resources are necessary in order to enable local initiatives to be implemented, such as local municipal action plans for ES or the development of new tools. Therefore, communication with local decision-makers is important, also emphasized by [74], who sugges<sup>t</sup> that the values of ES should be communicated to decision-makers in a transparent and viable way. A development coordinator, similar to the example provided in the eight southern municipalities in Stockholm County, can facilitate communication between the municipalities and local politicians. Furthermore, developing and maintaining the knowledge base related to ES in municipal organizations and their daily practices is needed to strengthen the implementation of ES. This is especially important with the frequent transition of employees in municipal organizations. A study of ES planning in Australia [75] highlighted that the turnover of governmen<sup>t</sup> staff combined with factors such as weak regulatory support resulted in stagnation in the development and adoption of ES policies and practices. Measures for organizational capacity building can aid in confronting the challenges associated with the changing knowledge bases. For example, the development of meeting platforms between municipalities can provide a means for knowledge exchange amongs<sup>t</sup> the practitioners working with ES. Also, clearly established roles and responsibilities can aid in maintaining the ES knowledge throughout a long and extensive planning process. Previous studies have also highlighted the importance of individuals who take an active role in promoting the ES approach in municipal organizations [33,62]. Additionally, our findings indicate the important role of regional authorities (CAB) to enhance local knowledge exchange and learning. Adapting existing tools to different contexts and challenges faced is also important for supporting the implementation of ES as emphasized by the practitioners. In Berlin, a governance challenge identified when implementing ES was how to address certain groups of stakeholders (e.g., private property owners), which have the ability to implement strategic goals [20]. In this context, existing tools such as the points system implemented in Upplands Väsby or the green space factor can be adopted and adapted to enable dialogue with the groups of stakeholders. In the Swedish context, these tools aided in facilitating dialogue with the developers to work with sustainability issues in the development projects. Furthermore, the points system invited the local community to contribute to ideas. However, it is important to have an understanding of the limits of the planning tools. For example, a risk that may occur with the use of tools such as the Green Space Factor at the local project scale is the possibility to lose the overall regional perspective on green infrastructure. A common finding in previous studies is the need for structured monitoring and learning from experiences [20,21]. Developing routines for monitoring and evaluation of ES in the planning process following the examples of Täby and Huddinge can provide a way forward for strengthening monitoring and facilitating learning. However, it is important to note that incorporating follow-up procedures in the planning process also requires the process to be evaluated in order to learn from the experiences [76]. According to [74], data and information regarding ecosystem services and the impact of development on them should continuously be gathered and integrated with the goal of learning, adapting and better-informing policy. Overall, our study was directed towards practitioners in the municipalities due to their active role and responsibility for implementing ES in their daily practice. Examining the views and experiences of the practitioners involved in the planning and decision making processes for implementing ES in the local planning context allowed us to gain insight on the bridges and barriers for operationalizing ES. However, future studies directed towards citizens and local actor's perceptions of ES could further contribute to the understanding of challenges for ES implementation by practitioners.	doab	2025-04-07T03:56:59.421188	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 47 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.48	7. Conclusions The case study from Stockholm County provided wide empirical material and experiences of practitioners working under di fferent municipal arrangements in a rapidly growing urban region. Consequently, the findings of this paper can contribute to better understand the factors that can facilitate the integration of ES in local planning practice. Four main conclusions can be drawn from this study. Firstly, there is a need for legal support and regulations for ES on the national and EU policy level. Secondly, local political support is necessary to enable the municipalities to take initiatives. Therefore, there is a need for communication between the municipal practitioners and local politicians so to foster learning concerning ES implementation. Thirdly, knowledge related to ecosystem services and motivation amongs<sup>t</sup> the municipal actors is a key factor for implementing ecosystem services in the municipal planning practice. Capacity building initiatives are necessary within the municipalities to maintain and develop the ES knowledge in the municipal organization. Fourthly, monitoring is important in order to ensure that the visions set strategically are translated into concrete actions and to learn from experiences. Overall, advancing ES in local planning requires a combination of top-down political support and bottom-up planning initiatives from local actors. Author Contributions: Conceptualization, S.K.; methodology, S.K.; writing—original draft preparation, S.K.; writing—review and editing, M.H., S.B. and B.B.; supervision, B.B. and M.H.; funding acquisition, B.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by FORMAS Research Council, gran<sup>t</sup> number 2015-00133. Acknowledgments: We thank Mona Petersson for help with the layout of the map. 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.	doab	2025-04-07T03:56:59.421737	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 48 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.50	1. Introduction Cities and their inhabitants are particularly vulnerable to threats related to climate change (thermal and hydrological in particular), which have a negative impact on human health, quality of life, and urban infrastructure. Creating resilient cities is a major challenge for city builders [1,2]. It applies not only to new development but also to existing urban structures. Creating resilient neighborhoods should be the result of properly implemented urban planning and design [3]; for this to happen, planning documents should contribute significantly, because they shape the natural performance of planned areas. However, this is problematic in terms of the conceptualization of resilience and its implementation in the urban realm. Meerow et al. [4] and Masnavi et al. [5] stated that the concept of resilience related to the urban realm is inconsistent and contested, but crucial in order to develop the adaptive capacity of urban socioecological systems. For a resilient city to be understood as a socioecological system, it should consist of physical and social sub-systems. The physical sub-systems encompass the natural and built components of an urban structure. The social sub-system is built by human societies. Since system structure determines overall system behavior, systems should not be managed only for productivity but also for resilience [6]. Masnavi et al. [5] indicated three conceptual approaches of resilience thinking present in literature: (1) resilience as recovery, (2) resilience as compatibility or adaptation capacity, and (3) resilience as change. There are also two levels of resilience: general and specific. A strategic evaluation of urban resilience focused on general resilience proprieties before considering specific resilience. The authors pointed out the importance of the relationship between urban form and urban resilience. The role of spatial planning in building urban resilience to climate change was demonstrated by Jabareen [7] as one of four drivers in his resilient city planning framework. Adaptive instruments aimed at building urban resilience to climate change should be implemented systemically and strategically, through planning tools to design and technical solutions. The strategic level focuses on a city's general resilience and corresponds to its ecological and social vulnerability [8,9]. The planning and design level relates to the specific urban physical system resilience. According to Aguiar et al. [10], who compared local adaptation strategies in Europe, spatial planning was considered as one of the priority sectors for adaptation. However, there is a little knowledge of how it is implemented in practice at the planning level [11]. Masnavi et al. [5] highlighted the need for further research on spatial morphology and urban spatial structures as tools to build urban resilience. While opportunities at the strategic and technical levels have already been recognized, there is a gap at the planning and design level [5,11–13]. The reason may be internal constraints related to the legal context, for example, available tools and scope of mandatory regulations, or the level of awareness of local authorities and the awareness and skills of designers [11,14]. Such a problem can be observed in Poland, where, at the strategic level, adaptation plans are developed both at the national level [15] in relation to EU policy [16,17] and at the municipal level. Local adaptation plans to climate change were produced in three separate projects including: the Urban Adaptation Plans (MPA44) [18] and the Adaptcity [19], which resulted in plans for 44 cities with over 100,000 residents and the capital city of Warsaw, and the CLIMCITIES [20], which provide training on climate change issues and developing an urban climate adaptation plan for local authorities in cities with populations from 50,000 to 99,000 residents. This paper discusses only the Adaptcity project in more detail as an exemplification. However, even though climate change adaptation is well recognized at the strategic level, Polish spatial planning system does not directly address this issue. Instead, it requires the provision of proper living conditions and the maintenance of biological balance. The key instruments (the study of conditions and directions of spatial development at the municipal scale and local spatial development plans) are accompanied by environmental study and strategic environmental impact assessment (EIA). Environmental study and strategic EIA in general refer to the natural performance and very rarely comprehensively consider the issue of climatic threats. Nevertheless, planning documents must compulsorily set the principles of environmental protection to include: (1) a rational use of the earth's surface, (2) ensuring protection of landscape values of the environment and climatic conditions, and (3) comprehensive solutions for urban development problems with particular emphasis on: (a) water managemen<sup>t</sup> and (b) arranging and shaping green areas. From the climate adaptation perspective, a problem occurs in terms of available tools on the planning level. For instance, even though the biologically active area index is the most important indicator, it lacks su fficient legal clarification and authorization. The index does not reflect the impact of vegetation structure and adopted technical solutions on natural performance. Moreover, stormwater managemen<sup>t</sup> is not mandatory to planning provisions and appears mostly as facultative recommendations. The implementation of adaptation actions to climate change postulated at the strategic level requires the use of urban planning and design tools, but owing to the flawed spatial planning system in Poland as mentioned above, there is a problem in the integration of activities between strategic and planning levels. This article is focused on the issue of urban planning and design as tools for building specific urban resilience to climate change with reference to urban form. Due to the problems indicated in the introduction, it aims to fill the gap in the implementation of adaptation measures that exists at the planning and design level. To fill that gap, we propose the procedure of building neighborhood resilience to climate threats embedded in planning (from the strategic to the local level) and designing while focusing on usage of natural adaptive potential. It will be applicable both when planning new investments and when evaluating the natural condition of existing neighborhoods in order to improve their resilience. The practical implementation of the procedure is described on the example of Warsaw, Poland. To achieve the objective of the paper, a literature review describing resilience implementation with the use of natural adaptation potential and a set of tools for urban planning and design are presented. Next, at the strategic level, a ranking of districts in terms of priority to take adaptation actions has been enumerated, while at the planning and design level a multicriteria analysis to diagnose the natural functioning of the neighborhoods in their existing and planned states has been elaborated. Then, the results and discussion related to the case studies and literature are provided. The paper concludes with proposal of a procedure to integrate the strategic level with the planning and design level. #### 2. Literature Review #### 2.1. Resilience Implementation The first step in dealing with the system is to ge<sup>t</sup> a deep understanding of its structure and behavior [6]. Since cities constitute socioecological systems, the integration of ecology with urban planning and design has been recommended to build urban resilience, particularly to climate change. This way of thinking about urban planning already has a long tradition that fits with existing environmental approaches [1,21–29]. According to McHarg and Steiner [30], the design process should start with a comprehensive ecological inventory focused on natural processes in order to integrate them into planning and design. Ecological factors constitute determinants of the environmental capacity to support human activity and suitability for a particular type of land use. The idea is to use nature as a strategic ally through planning and designing around ecosystems services. To achieve natural and social sub-system compatibility, Pickett et al. [1] indicated understanding and using spatial heterogeneity. Ahern [24] proposed five strategies to build urban resilience capacity: (1) biodiversity, (2) multifunctionality, (3) multiscale networks, (4) modularity, and (5) adaptive design. Nature-based solutions (e.g., green infrastructure) are recommended as best practices in adaptation by the European Commission [31]. Nature could be integrated into built components of urban systems by incorporating its forms and features, natural processes, and entire living systems through planning and design [1,32]. In relation to hydrological and thermal hazards resulting from climate change, two natural processes and their determinants are crucial to build adaptive capacity: hydrological cycle and air circulation. Natural adaptation potential for building adaptive capacity of urban physical sub-systems consists of environmental features of the area such as geology, soils, water, and vegetation. These features enable rainwater managemen<sup>t</sup> based on natural hydrological processes and favorable climatic conditions (in particular, optimal thermal conditions). These properties can be employed to minimize hydrological and climatic hazards. Moreover, entire living systems (ecosystems) like forests or wetlands should be integrated to build natural adaptation potential. A set of the most useful tools for urban planning and design level is presented in the next section. #### 2.2. Tools for Urban Planning and Design There is a wide range of nature-based and technical adaptation solutions to climate change suitable for urban planning and design. First, the proper zoning of the area corresponding to its natural predispositions have to be established [21,25,28]. Next, three types of adaptation tools for urban planning and design should be taken into consideration: (1) urban development indicators, (2) urban structure (morphology), and (3) technical solutions (Table 1). These tools are useful for building resilience to thermal and hydrological threats resulting from climate change; their effectiveness has been supported by numerous published researches (Table 1). Table 1. Tools to build resilience at the planning and design level and its impact on climate threats. Zoning allows the incorporation of the natural ecosystems into building resilient neighborhoods and cities as well as using the natural properties of the areas to create suitable functions of the development. The effectiveness of this ecological approach to urban planning and design has been supported by the Woodlands Neighborhood, designed by McHarg [27]. The urban development indicators support the zoning tool in terms of fitting the development intensity to natural conditions in order to manage natural processes and to provide well-being. The most significant indicators used to shape climatic conditions of the urbanized areas are the biologically active area index (BAAI), the surface runoff indicator, and the maximum building height. According to Szulczewska et al. [33], there is a threshold of 45% of the biologically active area's share to enable proper natural performance in the neighborhoods, especially to provide sustainable stormwater management. The BAAI and surface runoff indicator could be integrated into one indicator as it is implemented in Berlin (Biotope Area Factor), Malmö (Green Space Factor), and Seattle (Seattle Green Factor). Scott et al. [32] stated that one of the methods of adapting cities to future high temperatures is to increase the presence of green spaces. Gill et al. [34] indicated the effectiveness of the increase of BAAI by 10% in his case study of Manchester. The maximum building height in relation to the separation width between buildings shapes the areas' roughness and air circulation conditions in terms of wind flow, velocity, turbulences, and dispersion. The following intervals of building height as obstacles for air circulation can be established: 3–10, 10–15, 15–25, above 25 m [37,67]. Urban morphology is a matter of urban design, which is a crucial tool to incorporate natural adaptive potential into urban composition, and to design natural processes, and adjust them to local environmental performance. The building and vegetation structure and layout, as well as green areas' layout and size, should be considered, in particular, to provide proper climatic conditions. The relationships between both are also important. Stewart and Oke [37] and Krautheim et al. [38] pointed out the width/height ratio (the distance between buildings in relation to their height) as the most useful indicator for climatic conditions in urbanized areas. The best air circulation conditions are in areas where the width/height ratio is above 2.4, between 1.4 and 2.4 air circulation conditions are limited, while below 1.4 they are strongly limited. Vegetation structure modifies not only the roughness, but also evapotranspiration, which is a key process resulting in cooling surface temperature. This is the reason why vegetation structure plays a significant role as a resilience building tool for both thermal and hydrological threats. The areas covered by trees have higher evapotranspiration than grass surfaces and, consequently, a higher cooling e ffect [40,41]. However, since the coverage of trees has higher roughness and limits horizontal air circulation, while improving convection as a thermally contrasting patch, it is more appropriate to introduce it among intensely urbanized areas rather than in ventilation corridors. In contrast, it is di fficult to give precise specifications as to how many, how big, and where the green areas should be established, because there are too many variables determining climatic conditions in cities [29]. Therefore, the configuration should always be considered for each case, using existing natural forces, processes, and features. Nevertheless, some data have been provided. According to Asgarian et al. [43], composition, configuration, and structure of green space patches considerably affect the nearest urban land surface temperature of built areas. They pointed out that the patches should be homogeneously dispersed, stating that the bu ffer zone of lower surface temperature reaches up to 200 m. Stewart and Oke [37] indicated the existence of the thermal transitions zones between thermally contrasting local climate zones like green and built-up areas of 200–500 m, depending on surface roughness, building geometry, and atmospheric stability conditions. The size of green patches is also important: the greater the size, the higher the reduction of surface temperature [43]. Nonetheless, Kensington Garden (100 ha) has the relatively small bu ffer zone of a width of 400 m. Thus, networks of small (2–3 ha) green spaces were recommended by Doick et al. [44] for e ffective cooling of urban environments. Also, Hough [29] stated that a fine net of small green areas, distributed homogenously, is more e ffective than a few large spaces. Apart from size, the crucial features of each green cover patch are: (1) the perimeter-to-area ratio must be minimal—the optimal patches are compact, circular, and rectangular shapes, as well as (2) the core area index—areas with more irregular shapes, which contain more core area, are better than simple, linear shapes [43]. Green areas are also crucial to provide proper hydrological functioning and can be used in stormwater managemen<sup>t</sup> to reduce the risk of flooding in urban areas dominated by impervious surfaces. Generally, green areas minimize runo ff volume; however, the e ffectiveness of the process greatly depends on vegetation structure as well as the layout and size of a patch. Deutscher et al. [42] has shown that areas covered with trees can intercept up to five times as much water as lawns and produce half as much runo ff. Kim and Park [46] indicated that larger and less-fragmented patterns are more likely to decrease peak runo ff. Additionally, the e ffect is amplified by vegetation abundance, especially trees or shrubs, as they increase the storage capacity of an area during flooding. Finally, technical solutions are to be considered. For adaptation to thermal threats, the modification of the albedo is the point. It could be achieved by technical (cool roofs, facades, or pavements) or nature-based solutions (green roofs, facades, infiltration and bioretention basins and trenches, swales, detention and retention ponds, constructed wetlands, etc.). Adaptation to hydrological threats concerns mostly the sustainable storm water management. Nature-based solutions have a positive impact for both hydrological and thermal threats due to the evapotranspiration process. #### 3. Materials and Methods This study consisted of two stages to comply with the aim of the paper, which was the integration of adaptation activities between strategic and planning and design levels. The first stage presents the strategic level of the planning process, and its research area encompasses the city of Warsaw within its administrative borders. The second stage corresponds to the local level of planning and design, and it was conducted on two neighborhoods (existing and planned one) chosen from a district with the most urgen<sup>t</sup> adaptation needs based on the results from the first stage. #### 3.1. Strategic Level The first stage of the study involved exploring adaptation needs of Warsaw's districts in context of their potential to implement nature-based solutions. In order to indicate the priority areas for implementing adaptation actions, a ranking of districts was developed. These areas include districts with climatic and demographic risks as well as limited potential for creating green infrastructure. To assess climatic risk, nine indicators were used according to the Warsaw Adaptation Plan [68]: (1) Flood risk in the Vistula valley (Flood), (2) Risk of local flooding after heavy rainfall (Local Flooding), (3) Urban Heat Island, (4) Number of hot nights with minimal temperature above 18 degrees (Hot Nights), (5) Impervious surface coverage (Impervious Surface), (6) Urban Density, (7) Share of built-up areas (Built-up Areas), (8) Estimated increase in residential units (Projected Development), (9) Green areas and forests share (Green Areas). Demographic vulnerability was estimated based on age structure of inhabitants in districts (percent of the population considered to be vulnerable including people under 4 and over 65 years old) [69]. The potential of green infrastructure was evaluated by eight indicators determining quantitative and spatial potential [70,71]: (1) Share of green infrastructure area in district area (GI Area), (2) Green infrastructure area per inhabitant in district (GI per Inhabitant), (3) Share of recreational green areas in district area (Recreational GI), (4) Share of recreational green areas per inhabitant in district (Recreational GI per Inhabitant), (5) Share of housing areas with recreational green areas within a 500-m distance in district area (Housing with Recreational GI within 500 m), (6) Share of housing areas with recreational green areas above a 500-m distance in district area (Housing with Recreational GI above 500 m), (7) Length of planned bike lanes per 1000 ha of housing area (Planned Bike Lanes Density), (8) Share of potential areas for creating green infrastructure in district area (Potential areas for GI). The indicators were assessed on a point scale, where those increasing climatic risk scored negative points, while those decreasing the risk gained positive points. Each criterion was scored separately to create three sub-rankings; here, districts were assigned to five classes to facilitate comparison of the results. Final summarized ratings of all three criteria determined the priority of taking adaptation actions in the districts. For further analysis, within the district with the highest adaptation priority, two neighborhoods (one existing and one planned) were chosen based on development plans, exposure to climatic risk and similar natural conditions. #### 3.2. Local Planning and Design Level The second stage of the study included determination of natural adaptation potential and an analysis of the existing urban layout and planning provisions of the chosen neighborhoods. We proposed to identify current and future conditions for ventilation, air regeneration and cooling, infiltration, and surface runo ff using multicriteria analysis in which criteria were inspired by planning tools (Table 1) derived from literature review presented in Section 2. The method consists of the following steps: 3. Calculation of factor's values for planning units (units from local spatial development plans) using weighted arithmetic mean (WAM) as in Equation (1): $$\%AMM = \frac{\mathbb{C}\_1 \times A\_1 + \mathbb{C}\_2 \times A\_2 + \dots + \mathbb{C}\_i \times A\_i}{A\_1 + A\_2 + \dots + A\_i} \tag{1}$$ where Ci for i = 1, 2, ... n is the class value assigned separately for each factor in the expert method and Ai for i = 1, 2, ... n is the area of class i in the planning unit. $$\mathcal{WGM} = K\_1^{w\_1} \times K\_2^{w\_2} \times \cdots \times K\_l^{w\_l} \tag{2}$$ where Ki f i = 1, 2, ... n o r is the factor rating (WAM) in the planning unit and wi for i = 1, 2, ... n - the factor weight. This function allows to model synergistic interaction between factors [72]. The step results in average conditions for: ventilation, air regeneration and cooling, infiltration, and average surface runo ff in each planning unit. Next, obtained values (WGM) were classified into four classes of climatic and hydrological conditions (very good, good, moderate, bad). To assess the impact of planned development on hydrological and climatic conditions, the classes in existing and planned state were compared. As a result, three types of changes were distinguished: improvement, no change, deterioration. Criteria values for the existing state were calculated based on data derived from the Warsaw Environmental Atlas [73], topographic objects database (BDOT10k), aerial imagery (orthophoto), and on-site visits. Criteria values for the planned state were estimated according to urban indicators from planning provisions and included biologically active area index, maximum building height, and percentage of impermeable built-up area. All calculations referred to the planning units specific to local spatial development plans. The analysis determined natural performance of case studies as well as their sensitivity to hydrological and thermal hazards. In a further step, the degree of usage of the natural potential in adaptation to climate change was assessed. #### Land 2020, 9, 387 #### 4. Results and Discussion–The City of Warsaw Case Study #### 4.1. Priority of Adaptation to Climate Change in Warsaw Warsaw will be negatively affected by climate change, in particular the increase in number and intensity of hot days [74] and frequency of precipitation that causes local flooding [68]. The ranking of the adaptation priority shows differences among districts. Very-high priority was identified in Wola, Praga Południe, Mokotów, and Zoliborz, while high ˙ priority in Ursus, Ochota, Sr ´ ódmie´scie, Praga Północ (Figure 1, Table 3). These are densely built-up inner parts of the city populated by a vulnerable group of inhabitants, mostly in old age. Climatic threat in those districts comprises urban heat island, high risk of flooding in the event of levee failure in the Vistula valley, and/or inundation due to an overloaded sewage system during heavy rainfall. Simultaneously, the existing amount of greenery and potential areas for creating new green infrastructure is insufficient to compensate the risks. Figure 1. Adaptation priority of Warsaw's districts.	doab	2025-04-07T03:56:59.421916	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 50 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.51	Table 3. Adaptation priority rating of Warsaw's districts. Moderate priority to take adaptation action concerns the outer districts of Bemowo, Targówek, Bielany, Białoł ˛eka, Wawer, and Wilanów, with low or moderate climatic threat characterized by less frequent or no occurrence of urban heat island and only local risk of flooding. This group comprises both older districts with high share of built-up areas and a high amount of well-designed greenery (Bielany, Bemowo, Targówek), and developing districts which lack accessible green infrastructure but have potential areas for creating it (Białoł ˛eka, Wilanów). The former are inhabited predominantly by an older population, the latter by younger people. Low priority was also diagnosed in the outer districts of Włochy, Ursynów, Wesoła and Rembertów, where the climatic threat is very low to moderate due to lower urban density and a larger share of open green areas or forests, and share of vulnerable groups among inhabitants is not significant. For further analysis in the case studies formula, two neighborhoods were chosen from the district of Mokotów (Figure 1): According to existing planning provisions [75,76], the biggest changes will concern the Pod Skoczni ˛a, where, as a result of the development of residential and service buildings, the green area will be significantly reduced and it will take the form of a linear park with a water system consisting of retention ponds and water canals. In Sadyba, the plan allows for more dense housing and the development of service functions within existing housing units. #### 4.2. Neighborhood Resilience in Question Case studies are located on the upper terrace of the Vistula valley. For both analyzed neighborhoods, we diagnosed hydrological threats, such as flooding caused by an overloaded sewage system during heavy rainfall. Moreover, there is a risk of inundation in Sadyba related to levees breaking in the Vistula valley and groundwater ponding in Pod Skoczni ˛a. Thermal threats mainly concern Sadyba due to its location away from the ventilation corridor, land cover albedo, and urban structures that impede air exchange. Whereas in Pod Skoczni ˛a, there are no thermal hazards because of the location in the ventilation corridor and the large share of biologically active areas. According to the Warsaw Environmental Atlas [73] the function of the ventilation corridor is to be maintained. The analysis of natural performance showed that both neighborhoods have high natural adaptive potential, which can be used to minimize thermal and hydrological hazards. In Pod Skoczni ˛a, the adaptive potential is aided by mostly flat relief, water table less than 2 m below ground level, predominantly good soil permeability, good quality soil with water retention capacity (peat), and high proportion of biologically active area. Moreover, the existence of a hydrographic system and location within the ventilation corridor can be considered beneficial. Similarly, the natural adaptive potential of Sadyba comprises flat relief, high proportion of green areas with tree dominance, water table less than 2 m below ground level, and very good soil permeability. As the natural performance analysis indicated, climatic and hydrological disorders occur in Sadyba but not in the undeveloped area of Pod Skoczni ˛a (Figure 2). However, in the case of implementation of planning provisions, the deterioration of the natural performance in terms of climatic and hydrological functioning had been predicted for both areas. Furthermore, the usage of natural adaptive potential of the study areas and technical adaptation solutions in the planning provisions [75,76] had been relatively low and not compulsory. The possibilities of implementing adaptation tools varied between the newly designed housing estate and the modernized one. While in Sadyba, the possibility to engage its natural adaptive potential had been limited; in Pod Skoczni ˛a it had been neglected. In Sadyba, a large proportion of impervious surfaces implied constrained infiltration capacity. In Pod Skoczni ˛a, the existing planning provisions allow for development in areas with organic soils (peats) that are crucial for water retention in the context of adaptation to climate change. Figure 2. Changes in hydrological and climatic performance in neighborhoods: (A) Change of ventilation conditions, (B) Change of air regeneration and heat reduction conditions, (C) Change of infiltration conditions, (D) Change of surface runoff volume. Some adaptation tools identified in Table 1 were introduced in the neighborhoods. These tools included biologically active area index, maximum building height, urban structure, and selected technical solutions (green roofs, permeable pavements) for which optional recommendations were made. Moreover, sustainable rainwater managemen<sup>t</sup> has been introduced as a rule in Pod Skoczni ˛a, while it remains only a recommendation for new investments in Sadyba. #### 4.3. Building Resilient Neighborhoods The implementation of the procedure on the strategic level requires identifying focal areas for adaptive interventions and their spatial distribution. In order to properly locate adaptation actions, the ranking of priority among districts was developed. The method presented in the paper differs from the one in The Warsaw Adaptation Plan [68] (strategy of adaptation to climate change), which identifies priority areas based on data related only to threats resulting from climate change. Our approach, however, also considers demographic vulnerability; its importance was pointed out by Meerow and Newell [8], Shokry et al. [9], and Bła˙zejczyk et al. [69] and the potential to implement green infrastructure [70,71]. Consequently, there was a need to integrate strategic information from different municipal documents [68,71]. The results obtained from both rankings slightly differ, having taken into account the additional criteria allowed for more holistic assessment of districts in terms of their needs and their potential to adapt. Considering evaluation of green infrastructure makes it possible to assign higher priority to districts which lack areas for creating new greenery to sufficiently compensate the climatic risk. With a lesser effect, the social vulnerability index also influenced the position of some districts in the ranking, exposing those inhabited by an older population. The Warsaw Adaptation Plan [68] recommends that adaptation to climate change should be considered during urban planning, particularly in local spatial development plans. Although plans are potentially powerful instruments for building adaptive capacity at the local level, available planning tools in the Polish legal context are deficient. Moreover, it is also a matter of the designer's skills [14] as well as investor and local authorities' awareness [11,27]. In the literature, there are some guidelines and recommendations on how to design with respect to climatic and hydrological processes, but they are dispersed [13], as evidenced by extensive literature (Table 1). The key for selecting the literature for this paper was the e ffectiveness of the planning and design tools in shaping climatic and hydrological conditions proved by empirical studies. Defined tools like land cover height, vegetation vertical structure and size, biologically active area index, surface runo ff volume, and building structure (expressed by local climate zones [37]) impacting terrain roughness were used to design the criteria for the multicriteria analysis. Due to the fact that there are many interdependent tools to build resilience of neighborhoods and that it is desirable to use them simultaneously to obtain the expected e ffect, the multicriteria analysis method was the best to take into account these interdependencies [72]. The multicriteria analysis described in this paper aims to fill the gap of implementing solutions for building urban resilience which exists at the planning and design level. Moreover, it helps to visualize the possible consequences of planning decisions. The results of the analysis were aggregated on the planning unit level which allowed the assessment of the potential impact of specific planning provisions on climatic and hydrological processes in the unit. Conducting such an evaluation enhances the planning process by introducing the issue of adaptation to climate change. Nevertheless, in the analysis, some limitations occur connected with the availability of data. The choice of criteria depended on the possibility of estimating the value of indicators in the selected analysis scale (planning level). Furthermore, the scope and accuracy of spatial development plans which comprise zoning, a set of urban indicators, and building lines for planning units do not allow the extraction of detailed information about future urban composition and planned vertical structure of vegetation. This required making assumptions about those properties of the site that will shape the climatic and hydrological conditions in the future. Consequently, the method has the potential to be further extended. Introducing the floor area ratio to di fferentiate the impact of various development scenarios (for example, a scenario with maximal building coverage ratio or maximal building height) may allow the selection of an optimal combination of built-up and biologically active areas on the site in compliance with the planning provisions. Our analysis of spatial development plans shows that constructing adaptive capacity of the neighborhoods was not the priority. The implementation of planning provisions negatively a ffects the conditions of hydrological and climatic functioning. In case of the Pod Skoczni ˛a neighborhood, where permeable land cover prevailed, the possibility for infiltration will decrease because of an increase in soil sealing. In addition, both housing estates may experience an increase in surface runo ff as a result of fragmentation of green areas and change in the vertical structure of vegetation, which shape the amount of runo ff, as demonstrated by Kim and Park [46] and Deutscher et al. [42]. Moreover, failure to maintain the existing tall trees in the housing estates will result in worse conditions for air regeneration, because, as shown by Zölch et al. [40] and Hertel and Schlink [41], areas covered by trees have a higher cooling e ffect than grass surfaces. Therefore, an update of existing planning provisions in the focal areas is suggested. For Sadyba and Pod Skoczni ˛a, we recommend correction of existing spatial development plans oriented towards the utilization of natural adaptation potential (Figure 3B). In reference to environmental approach recommendations [1,21–26,28], it comprises modifying zoning, increasing the index of the biologically active area, and adjusting technical solutions to the natural conditions. The modification of zoning refers to the units where identified natural potential was ignored and to the units located within the climatic corridor. To sustain its function in the city's natural system, the protection of a 200-m wide strip of open land in the Warsaw Escarpment areas has been proposed. Zoning as green areas has been suggested for all these units [27]. The increase of the index of the biologically active area was motivated by the need to maintain good climatic and hydrological performance in Sadyba and Pod Skoczni ˛a [33]. Technical solutions aimed at stormwater managemen<sup>t</sup> have been suggested in order to reduce runoff. Research-supported guidelines include the application of permeable pavements and combinations of infiltration and bioretention devices [57,58,60]. Figure 3. (A) Existing planning provisions in case studies, (B) Resilience enhancement proposal. #### 5. Summary and Conclusions Redefining cities to build resilience to climate threats in urban neighborhoods should be carefully conducted from the strategic to the local level. While the strategic and technical tools are well developed, the planning and design phase needs to be more considered. The relationship between the strategic and planning levels ought to be strongly established. Polish law related to spatial planning addresses climate adaptation indirectly, providing some deficient tools to implement adaptation actions. The planning provisions aimed to build resilience to climate threats are rather facultative than obligatory. Analyzed case studies have shown that, despite available instruments, local spatial development plans in Sadyba and Pod Skoczni ˛a were not formulated to build neighborhood resilience to climate change. The adaptation of urban areas to climate change by spatial planning and urban design should prioritize the engagemen<sup>t</sup> of natural adaptive potential and the adjustment of adopted solutions to natural conditions. If this is not possible, or if the potential has been limited because of existing or planned development, compensation measures should be implemented (i.e., nature-based solutions, see Table 1—technical solutions). However, we argue that, among a set of adaptation tools provided in this paper, the most important one for building neighborhood resilience is properly conducted urban design. The procedure proposed in this paper could be a useful, simple method within the planning process to build neighborhoods' resilience to climate threats. It is applicable both when planning new investments and when evaluating the natural performance of existing neighborhoods to enhance their resilience. The method utilizes the indicators from literature like land cover height, vegetation vertical structure and size, biologically active area index, building structure, and surface runo ff volume. Still, it has the potential to be further developed considering the local context and tools available. The procedure consists of the following steps: - (a) Identification of needs in response to climatic threats, social vulnerability, and possibilities of implementing adaptation solutions based on ecosystem services. At this stage, rankings of assessment could be helpful, as shown in the paper. - (b) Selection of the most vulnerable areas, which have limited potential to benefit from ecosystem services engagemen<sup>t</sup> and are threatened by rapid urbanization. - (c) Planning and design level - (a) Evaluation of the impact of planning provisions on natural performance (climatic and hydrological functioning) using planning tools embedded in the multicriteria analysis presented in this paper. - (b) Implementing adaptation solutions with reference to diagnosed needs and possibilities. Available planning and design tools comprise zoning, urban development indicators, urban morphology, and technical solutions. The application of this procedure in the Sadyba and Pod Skoczni ˛a neighborhoods in Warsaw has shown various possibilities of using natural adaptation potential and tools. In Sadyba, the natural adaptation potential has already been limited by development, so the possibilities of its incorporation were smaller compared to Pod Skoczni ˛a. This implies the need for compensation measures, including nature-based solutions. However, implementation of these usually requires undertaking renewal actions while adjusting to environmental and technical conditions of existing buildings. In Pod Skoczni ˛a, the range of possibilities was much broader, as the mostly undeveloped area allowed incorporation of natural ecosystems, processes, and features to build a resilient neighborhood. Unfortunately for both neighborhoods, local spatial development plans ignored or neglected natural adaptation potential and only a few solutions to increase the neighborhoods' resilience to climate threats were used. This poor planning will trigger the deterioration of the neighborhoods' natural performance; therefore, enhancement of existing planning provisions is highly recommended. Author Contributions: Conceptualization, K.R.; methodology, K.R. and M.P.; formal analysis, M.P.; investigation, K.R. and M.P.; writing—original draft preparation, K.R. and M.P.; writing—review and editing, K.R. and M.P.; visualization, M.P. All authors have read and agreed to the published version of the manuscript. Funding: The research was carried out as part of the cooperation of the City of Warsaw and the science sector. "The APC was funded by WARSAW UNIVERSITY OF TECHNOLOGY". Acknowledgments: We would like to thank Warsaw City Hall for providing access to data related to Warsaw Green Infrastructure. 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.	doab	2025-04-07T03:56:59.423602	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 51 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.53	1. Introduction Dense networks of streets, buildings, industry, and transportation interfere with numerous ecosystem processes, affecting the local hydrology, quantity and biodiversity of native flora and fauna, biogeochemical cycling, and microclimate stability [1]. Urban ecosystem services (ES), the benefits humans derive from ecological processes in urban and peri-urban areas [2], are therefore often compromised in population centers, resulting in diminished air, water, and soil quality as well as intensified vulnerability to flooding and heatwaves [1,3,4]. As urban populations grow, the importance of urban ES is increasing: over four billion people now live in cities, a 20-fold increase since 1900 [5,6], and by 2050, urban residents are predicted to number six billion [6]. To strengthen urban ES, green infrastructure, or planned networks of urban vegetated land cover ("urban green space"), including parks, right-of-way planting strips, private yards, green roofs, wetlands, and other natural areas, may be deployed [7]. Urban forests, for example, reduce concentrations of air pollutants, including ozone, carbon monoxide, sulfur dioxide, nitrogen oxides, and particulate matter [8,9]; store atmospheric carbon [9,10]; intercept rainfall, thereby reducing stormwater runoff [11]; provide shade and air temperature regulation [12]; increase recreation value [13]; supply diverse nesting and foraging opportunities for birds; diminish soil erosion; and contribute to stormwater purification [14,15]. Recent biophysical, empirical, and GIS-based modeling methods now allow certain urban ES delivery rates to be quantified [16,17], and economic models allow their monetary value to be evaluated (e.g., [18]), facilitating estimation and comparison of urban green space contributions to air quality [9,19], stormwater runoff retention [20], air temperature regulation [21,22], and carbon sequestration [9,23,24]. For example, urban forests removed an estimated 27,000 metric tons of PM2.5, 523,000 metric tons of ozone, 68,000 metric tons of nitrogen dioxide, and 33,000 metric tons of sulfur dioxide from the U.S. urban air in 2010, providing an estimated \$4.7 billion in annual health benefits [8]. Such urban ES quantification and valuation are then directly useful in deciding among urban land-use alternatives [14,16]. Currently, the lawn is the dominant green land cover type throughout urban and suburban areas of Europe, Canada, and the USA [25]; in 2005, lawn accounted for nearly half of all urban land cover in the USA [26], an area comparable to half of the total irrigated cropland in the USA [27,28]. Although lawns are relatively easy and inexpensive to maintain, enjoy widespread acceptance, and provide some urban ES, under typical managemen<sup>t</sup> they consume extensive irrigation water [26] and are treated with fertilizers, pesticides, and herbicides that are harmful to fish, birds, and insects [29]. Additionally, lawns store limited carbon [30], and their mowing leads to both biogenic and fuel-related greenhouse gas emissions [31]. They also contribute less to stormwater retention, air purification, microclimate regulation, and recreation than other vegetative land-cover types [14,25,32,33]. In light of this evidence, urban land use planners face crucial decisions regarding the continuation of public lawn maintenance, complicated by pressures of cost, restrictive land-use codes, and uncertain public support, as well as limited land area with which to provide urban ES [34]. In these decisions, the perspectives of stakeholders such as policymakers, environmental managers, and affected residents are critical [2,16,35–37], revealing ES priorities, design preferences, and barriers to green infrastructure development [34,38–43]. The value of stakeholder input to ES planning was first emphasized by the Millennium Ecosystem Assessment in 2005, and the integration of urban ES quantification with stakeholder-expressed urban ES priorities emerged as a central urban environmental planning prescription [2,37]. The essential nature of stakeholder input in ensuring long-term green infrastructure success, combined with the characteristic urban ES provided by specific land cover types (e.g., woodland, trees, shrubs, native grasses, stormwater filtration facilities, etc.), require effective decision-making processes to integrate several lines of evidence. Specifically, quantitative urban ES delivery potential must be evaluated in the context of a possibly conflicting set of stakeholder perspectives [2,4,16,34,37,44], involving an approach that is widely advocated but has not, to our knowledge, been further investigated. To address this need, here we develop and evaluate such a method. We begin by establishing the urban ES currently provided in the study area and surveying diverse stakeholders to reveal their ES priorities. These data next inform the selection of alternative planting regimes that address individual stakeholder priorities and quantitative urban ES delivery, respectively. Comparison of priority ES delivery among these alternatives then guides their integration, yielding a composite regime that improves upon each initial alternative's likelihood of local acceptance while increasing delivery of the desired ES. Notably, this integrated regime could not have been clearly identified by either stakeholder priorities or quantitative urban ES assessments alone.	doab	2025-04-07T03:56:59.425058	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 53 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.54	2. Methods #### 2.1. Study Area The City of Eugene (Figure 1; population 156,000; median income \$44,859; area 113 km2) sits within the southern Willamette Valley in western Oregon [45], an area with a Mediterranean climate (Köppen Csb) of long cool rainy winters and warm dry summers. The valley surroundings promote winter temperature inversions and summer wildfire smoke collection, causing Eugene to rank among the twenty worst cities in the USA for short-term small particulate (PM2.5) air pollution [46]. The City of Eugene is also currently required by its National Pollution Discharge Elimination System (NPDES) permit to reduce the waterborne discharge of pollutants from the municipal system to the maximum extent possible [47]. The City of Eugene Park system possesses nearly 2000 ha of natural areas and open space, but their aggregation on the outskirts of town [48] limits their contributions to ES in urban neighborhoods. Figure 1. Location of the Friendly Area Neighborhood in Eugene, Oregon. Within the city, the Friendly Area Neighborhood (FAN; Figure 1; population 7000; area 3.7 km2) is zoned primarily (~75%) for low-density residential development (8–10 dwelling units/ha) and consists largely of single-detached units, with a median tax lot parcel area slightly below the USA median (0.073 ha vs. 0.083 ha) [45]. Nearly all streets in the neighborhood contain vegetated planting strips within city right-of-way easements, while sidewalks are intermittent. The FAN median annual household income (\$46,300) is \$7000 below state and \$11,300 below national medians [49,50], but its access to public green space is above average, with >10% of its land devoted to public parks and schoolyards and ~95% of residents living within a five-min walking distance along roads (i.e., <400 m) of a public park or schoolyard (Figure S1); the neighborhood is therefore comparable to top cities in the United States for such access [51]. #### 2.2. Public Green Space Mapping and Urban ES Quantification Although privately owned land is important in providing urban ES [52,53], this study focuses on public green space in which urban ES delivery is managed by the City. To characterize this space, we used multiple mapping strategies to inventory five distinct vegetated land cover types in the neighborhood (Table S1). Each street was traversed on foot in 2017 to identify lawn within the public right-of-way, and lawn without tree canopy cover was geospatially located and measured using a Garmin GPSMAP 62S handheld Global Positioning System (Garmin Ltd., Olathe, KS, USA). In tax lot parcels without adjacent sidewalks, right-of-way boundaries were assumed to extend 3 m on either side of the roadway. The boundaries of woodlands, classified as clustered trees clearly distinguishable from the U.S. Department of Agriculture's 2016 National Agriculture Imagery Program (NAIP) imagery, were assessed visually in ESRI ArcMap 10.7 (ESRI, Redlands, CA, USA) [54] and confirmed in the field. All other vegetation classifications (i.e., trees, tall shrubs, and short shrubs, as well as lawns located in parks) were made using normalized di fference vegetation indices (NDVIs) and height; the NDVI was calculated on a continuum from −1 to +1 using the NAIP four-band imagery with 1 m resolution. The NDVI range for each vegetation class was determined by comparing NDVI and color composite images [55]. The minimum NDVI threshold for all vegetation classes was set at 0.25, with the exception of lawn, which was identified using a minimum NDVI threshold of 0.0 (Table S1). Vegetation height was derived from 2015 light detection and ranging (LiDAR) point-cloud data [56] that were used to generate digital elevation and digital surface models. Digital elevation model values were subtracted from the digital surface model to create a digital height model at 1 m resolution, and vegetation was classified by combining NDVI thresholds with height ranges determined by Derkzen et al. (Table S1) [14]. The accuracy of each NDVI/LiDAR-derived land cover classification was evaluated through a process in which four hundred points, or 100 for each of the four vegetation types classified using NDVIs and LiDAR, were randomly selected and validated visually with NAIP imagery. Air photo interpretation was used to determine land cover type for all points clearly and obviously identifiable from the air photo. Land cover types for all remaining unidentified points were confirmed in the field (Table S2). NDVI/LiDAR-derived public green space land cover quantities were adjusted using validation proportions from Table S2 (see Table S3 footnotes), and five urban ES were quantified from these adjusted spatial data using indicators and supply rates compiled by Derkzen et al. for each of the five green cover types—vegetative ground cover (i.e., lawn), short shrub, tall shrub, tree, and woodland (Table S1) [14]. #### 2.3. Urban ES Supply Rates For the existing land cover, supply rates of five urban ES (air purification, carbon storage, runo ff retention, cooling fraction, and outdoor recreation) provided by the five green cover types described above were estimated according to Derkzen et al. [14], in which urban ES supply rates from numerous studies were integrated for the analogous Mediterranean (Csb) climate of Rotterdam, NL (Table S1). Although numerous modeling techniques exist for urban ES assessment [16], we chose this straightforward approach, consistent with recent recommendations and used by other case studies [57], as one that would be accessible to a wide range of urban planning practices. In exploring potential future alternative planting regimes (Section 4.2.), we included stormwater filtration facilities (e.g., stormwater planters and rain gardens) that are not currently present in the neighborhood, estimating their stormwater reduction potential using the Simplified Approach described in Eugene's Stormwater Management Manual [58]. Impervious surface area, a necessary input, was calculated for the neighborhood using image segmentation and supervised learning in ESRI ArcGIS Pro 2.6 (ESRI, Redlands, CA, USA) [59] based on infrared, red, and blue bands from 2016 NAIP four-band imagery. To assess the accuracy of impervious and pervious surface classification, 100 random points were selected for each land cover type, and every point was validated visually with the NAIP imagery. The overall accuracy of the supervised segmentation classification was 94.5% (Table S9). Urban green space also has the potential to provide ecosystem disservices, including pollen production that exacerbates allergies; a volatile organic compound release that contributes to ground-level ozone formation in the presence of automobile exhaust; and growth of tree limbs that may interfere with electricity lines or fall during storms, causing property damage [60,61]. These may also be estimated quantitatively in some cases (e.g., [62]), but we have not included these considerations here. #### 2.4. Resident Surveys Non-stratified random sample surveys were administered to residents of the FAN to determine their urban ES priorities for public green space and the potential for increased funding for green infrastructure development. A random sample of 500 residential tax lot parcels was selected using county tax lot parcel data for the FAN as a sampling frame. Each selected lot was visited once on a weekday between 5 and 7 PM, and 19.4% of these visits yielded a completed survey (n = 97). The majority of the recorded non-responses resulted from resident absences, suggesting that repeated visits could have increased the response rate, and homes with posted "Do Not Disturb" or "No Soliciting" signs were also recorded as non-responses. Among residents who answered their doors, over half agreed to participate. Surveys were conducted orally in a format approved by the University of Oregon's Institutional Review Board. To minimize the survey's perceived invasiveness, sociodemographic information was not collected, although it could have been informative. Residents were asked to rate 17 randomly ordered urban ES according to their importance for public green space in their neighborhood using a five-point Likert scale from 1 ("very unimportant") to 5 ("very important") (detailed in Supplementary Materials Section S2). They were then asked whether they supported the managemen<sup>t</sup> of public green space to increase urban ES delivery and whether they would be willing to support such e fforts financially, through personal donations or taxes, and through direct contribution of volunteer time. Resident priorities for public green space urban ES were evaluated using Pearson's chi-square tests for both pairwise and aggregate comparisons, and chi-square tests were further used to compare priorities among urban ES classification types (i.e., provisioning, regulating, cultural, and supporting). Results for each urban ES classification type were tested for internal consistency using Cronbach's alpha (α), and values above 0.7 were regarded as acceptable [63]. "Priority" urban ES were defined as those with Likert responses of 4 ("moderately important") or 5 ("very important"), and Likert responses were reclassified as either priority (values 4 and 5) or non-priority (values 1–3) for data analysis. Descriptive statistics were used to compare residents' willingness to support green infrastructure development. All statistical analyses were conducted in R [64]. #### 2.5. Delphi Method We used an iterative survey process, known as a Delphi analysis, to consult with a group of individuals with specific knowledge of the planning and managemen<sup>t</sup> of public green space in Eugene [65,66]. Of the 34 people invited to participate on the basis of their expertise in public policy and green space management, 15 agreed, including nine members of the Eugene Public Works Department (including Parks and Open Space, Stormwater Management, and Urban Forestry), two City Planning and Development members, two local environmental non-profit representatives, one City Council member, and one University of Oregon Landscape Architecture faculty member. In the first survey, participants ranked the 17 urban ES used in the resident survey in order of importance for public green space managemen<sup>t</sup> in Eugene; those urban ES with mean and median rankings below the top 10 were eliminated from the second survey. In addition, seven open-ended questions asked participants to describe and explain their perspectives on urban ES opportunities and barriers further. In the second round, participants were asked to review the collective results and representative responses from the first round before again selecting the urban ES they considered to be priorities and expressing their levels of agreemen<sup>t</sup> with responses to the open-ended questions of the first round. To reflect di fferences in management, safety, and ecological benefit potential, these questions distinguished between parks and right-of-way planting strips (Supplementary Materials Section S3). No particular proportion of agreemen<sup>t</sup> defines "consensus" in the Delphi method, and documented thresholds have varied from a simple majority to 95% [67–70]. Here, we chose a consensus threshold of two-thirds (67%), consistent with practices in many city governments [71,72].	doab	2025-04-07T03:56:59.425479	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 54 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.55	3. Results #### 3.1. Public Green Space Inventory To understand current urban ES delivery rates, we first inventoried public green space in the FAN through a combination of ground survey and NDVI/ LiDAR green cover assessment methods. These revealed that lawn was the dominant green cover type (Figures 2 and 3; Tables S3 and S4), typical of low-density residential development [29]. Of the 57.4 ha of public green space, approximately 55% was covered by lawn without tree canopy, 30% by trees with unidentified understory, 9% by woodland, 4% by tall shrubs, and 3% by short shrubs (Figure 3; Table S4). All woodland and most lawn (>85%) were located in municipal parks and public schoolyards, while ~80% of all non-woodland tree canopy, short shrubs, and tall shrubs were located in right-of-way zones (Table S3). Figure 2. Land cover type and distribution. (a) Lawn size and distribution in right-of-way zones in the Friendly Area Neighborhood, evaluated by ground measurement, and (b) vegetation classes and distributions on all public lands in the neighborhood identified using NDVI and LiDAR data. Figure 3. Urban ecosystem services (ES) provided by existing vegetated land cover. Existing vegetated land cover distribution (a), detailed in Figure 2, and the corresponding provision of urban ES by vegetated land cover type: (b) runoff retention; (c) air purification; (d) carbon storage; (e) cooling fraction; and (f) recreation, as evaluated by supply rates compiled by Derkzen et al. [14], summarized in Table S4. The approach combining LiDAR and NDVI was most accurate in identifying tree cover and lawn (98% and 86% accuracy, respectively), while 66% of the area classified as "tall shrub" was found to be tree canopy cover, and 33% of the area classified as "short shrub" was found to be tall shrubs, lawn, or tree canopy cover (Table S2). Using published supply rates [14] (Table S1), we next estimated that this public green space provides nearly 2900 metric tons of carbon storage, removes over 2000 kg per year of atmospheric particulate matter (PM10), and retains over 4.7 million liters of stormwater during each 12 mm storm event (Figure 3; Table S4). Lawn covers over 50% of the total public green space and provides more than half of the runoff retention and recreation value but less than one-quarter of the air purification services and 2% of the carbon storage (Figure 3; Table S4). By comparison, trees cover less than 30% of the total public green space but supply over half of all air purification and carbon storage, as well as over 40% of cooling services; trees provide runoff retention roughly proportional to their coverage area but only one-fifth of all recreation services. Woodland covers less than one-tenth of the total public green space ye<sup>t</sup> provides over one-tenth of the recreation value and over one-quarter of the carbon storage, or more than 14× that provided by lawn. Tall and short shrubs, in comparison, cover the least area of the total public green space but provide urban ES approximately proportional to their coverage area. #### 3.2. Resident Surveys—Urban ES Priorities To understand residents' urban ES priorities for public green space, we asked a random sample (n = 97) to rate 17 individual urban ES on a scale from 1 ("very unimportant") to 5 ("very important"). Responses showed that outdoor recreation, stormwater quality, air quality, pollinator habitat, and native species were the top priorities (Figure 4; Table S5), showing a clear preference for supporting services; except for outdoor recreation, cultural and provisioning services were rated as relatively unimportant (Table S5). Figure 4. Ratings by Friendly Area Neighborhood residents (n = 97), from 1 (very unimportant) to 5 (very important), of 17 urban ecosystem services (ES). Colors designate urban ES categories (green: supporting; blue: regulating; brown: provisioning; olive: cultural); bubble size designates frequency of the indicated response; outer black line indicates significance (p < 0.05) according to chi-square tests in which responses of 1–3 and 4–5 were binned to compare each individual urban ES to overall urban ES. Data, including Cronbach's alpha values for each urban ES domain, are tabulated in Table S5. This survey also investigated residents' willingness to support public green infrastructure development for urban ES improvement through contributions of time and/or money. Unexpectedly, most respondents (>85%) expressed willingness to contribute financially to urban ES projects in parks, with over one-quarter supporting direct, "out-of-pocket" payments and over 80% supporting tax measures to fund public works projects (Figure 5; Table S6). Support for such projects on right-of-way strips was lower but still substantial, with over 70% stating willingness to contribute financially; again, over one-quarter supported direct payments, but in this case, only 65% supported corresponding tax measures. Additionally, a large majority (>80%) expressed the willingness to volunteer for green infrastructure projects in the neighborhood, and over half stated interest in contributing five or more hours per year (Figure 5; Table S7). #### Figure 5. (a) Residents' stated willingness by in-person survey (n = 97) to financially support green infrastructure development in parks, in the public right-of-way, and on private property that increases urban ecosystem services (ES) through tax measures alone; tax measures combined with personal contributions; personal contributions alone; or none of the above. (b) Residents' stated willingness by in-person survey (n = 97) to volunteer time toward the development of public urban ES projects from 0 to 12+ h per year. Data are provided in Tables S6 and S7. #### 3.3. Delphi Analysis To understand the perspectives of stakeholders involved in the planning, implementation, and managemen<sup>t</sup> of public green space, with the potential to differ substantially from those of residents, we used a Delphi analysis to seek consensus (greater than two-thirds agreement) regarding urban ES priorities, perceived benefits of and concerns regarding lawn cover, benefits of and barriers to green infrastructure development, and strategies for overcoming these barriers. In the first-round survey, six urban ES—noise reduction, community identity, vegetable production, fruit production, improved soil health, and privacy—received sufficiently low rankings that they were excluded from the second round (Supplementary Materials Section S3). In the second-round survey, participants viewed the reduction of stormwater pollution as the top priority for both parks and for right-of-way planting strips, with over 80% agreemen<sup>t</sup> (Table 1; Figure 6; Figure S2). Improving air quality, supporting native species, increasing carbon sequestration, providing natural beauty, and reducing flooding were also consensus priorities for both parks and right-of-way planting strips. Providing shade for cooling was a strong priority for right-of-way strips but did not reach the consensus threshold in parks; instead, parks were most valued for providing habitat and educational opportunities. Outdoor recreation, plant diversity, erosion control, and physical and mental health benefits did not reach the two-thirds consensus threshold and were classified as non-priorities. Table 1. Urban ecosystem services that generated consensus a among Delphi participants (n = 15). a Consensus was defined as a ≥66.7% agreement. b 1 = highest; 17 = lowest. c Data are shown graphically in Figure S2. d Consensus was not reached. Figure 6. Comparison of Delphi stakeholder responses in favor of each urban ecosystem service (ES; vertical axis), detailed in Figure S2, with resident priorities (horizontal axis), detailed in Figure 4 and Table S5. Delphi stakeholder priority was defined as two-thirds or greater consensus approval; residential priority was established by significance of Fisher's exact test at the p < 0.05 level (n = 97); green shaded region represents urban ES prioritized by both stakeholder groups. AQ = air quality; AT = air temperature; BH = bird habitat; CI = community identity; CS = carbon sequestration; FP = fruit production; FR = flood reduction; NB = natural beauty; NS = native species; OR = outdoor recreation; P = privacy; PD = plant diversity; PH = pollinator habitat; SH = soil health; SQ = stormwater quality; VP = vegetable production. Participants viewed the primary benefits of public lawn in parks as providing recreational and gathering space (93% and 73% agreement, respectively) and ease of maintenance (67% agreement) (Table S8); on right-of-way strips, safety and sightlines were the only benefits that reached a consensus, with over 85% agreement. The principal concerns, in turn, both in parks and on right-of-way strips, were lawn's limited ability to provide regulating services (i.e., air and water filtration, carbon sequestration, and flood reduction), irrigation requirements, and lack of biodiversity (Table S8). Additionally, two-thirds agreed that fertilizer, pesticide, and herbicide impacts were a concern for right-of-way planting strips (Table S8). Accordingly, participants agreed that replacing lawn with alternative planting regimes could increase biodiversity and improve the habitat in parks while reducing stormwater runo ff and improving aesthetics along right-of-way planting strips (Table S8). The possibility of impaired sightlines remained a safety concern, however, and emerged as the only consensus barrier to green infrastructure development on right-of-way planting strips (Table S8). While over half agreed that converting lawn to alternative planting regimes would increase maintenance time, complexity, and cost during the transition period, they did not reach a consensus regarding the importance of these barriers. Still, to address them, the consensus recommendation was to install attractive, easily maintained plantings and to implement educational and outreach e fforts to promote support. Overall, a substantial majority (>85%) of participants supported the conversion of at least some lawn to alternative planting regimes both on right-of-way planting strips and in parks. #### 4. Integration of Stakeholder Priorities with Quantitative Urban ES Estimates Although the dual values of stakeholder priorities and quantitative understanding of urban ES potential in municipal decision-making have been widely discussed [2,4,16,34,37], methods to accomplish their integration have not previously been explored. To undertake this integration, we considered a series of questions planners might ask in making urban ES-motivated vegetated land cover decisions; developed a set of alternative planting regimes that responds to these questions in the context of the FAN; and evaluated them according to the local evidence collected, yielding a single integrated result. #### 4.1. Planning Considerations #### 4.1.1. What Urban ES are Available from the Landscape? Comprehensive ES assessments and contemporary literature addressing the location of interest are expected to reveal relevant urban ES for most locations; here, such resources (e.g., [3,37–41,73]) were used to identify the 17 urban ES considered in our survey (Figure 4). Since urban ES vary with climate and biome, however, analogous resources might emphasize very di fferent services for other locations, potentially including insect or disease control, provision of raw materials, production of fresh drinking water, etc. [4,37]. #### 4.1.2. What Land Cover Types Thrive in This Location? Climate, soil, and existing land uses are expected to limit the land cover types eligible for consideration. Here, Eugene's climate and the neighborhood's existing land use and cover types (Figure 2) focused our exploration on combinations of woodlands, dispersed trees, tall shrubs, short shrubs, and grasses, including lawn. #### 4.1.3. What are the Urban ES Priorities of Multiple Stakeholder Groups? Stakeholder perspectives can be revealed through interviews; in-person, mail, or online surveys; focus group discussions; and/or Delphi analyses, each with their own benefits and limitations (e.g., [74–76]). Here, we chose in-person surveys to reveal resident perspectives and to ensure a su fficiently large, random distribution of responses, despite the time-intensive nature of this approach, and we chose Delphi analyses to bring coherence to the input of diverse green space managers. #### 4.1.4. Which Urban ES can be Quantified According to Land Cover Type? Quantitative evidence documenting the ES provided by di fferent land cover types is growing rapidly (e.g., [14,16,77–83]), and where it exists, it can be used to inform decisions among alternatives. Additionally, urban ES delivery without published land cover supply rates may be evaluated qualitatively with guidance from locally or regionally available information (e.g., [84,85]), while others (e.g., natural beauty, pollinator habitat, and native plant species) may still be factored into design decisions, particularly through species choice. Here, the priorities of stormwater quality and air quality were among those with supply rates published by land cover type (e.g., [14,58]), allowing their urban ES to be quantified. Pollinator and native species habitat urban ES had not been similarly quantified, but local guidance existed in the form of a City resolution [86] and in regional lists of recommended native tree, shrub, vine, grass, and forb species (e.g., [87,88]). Using resources such as these, new plantings designed to meet quantifiable urban ES priorities may generally be chosen to meet non-quantifiable priorities as well. #### 4.1.5. What Barriers or Constraints Exist? Finally, various barriers are expected to limit the resulting green infrastructure development options, particularly including lack of funds for establishment, expansion, or maintenance of green infrastructure; insu fficient social support resulting from conflicting stakeholder desires; and safety or accessibility concerns (e.g., [89]). Here, Delphi participants expressed concerns consistent with those found elsewhere, focusing on cost and safety (Table 1). #### 4.2. Alternative Planting Regimes The considerations above guided the following investigation of alternative planting regimes with which to provide urban ES through the conversion of public lawn, illustrating the way in which integration of quantitative urban ES supply rates with stakeholder priorities leads to a di fferent result than that obtained by reliance on any one line of evidence alone. The status quo, to which the others were compared, represents the result of current decision-making processes that have yielded lawn-dominated public spaces, with substantial outdoor playing field area as well as several hectares of dispersed trees and one prominent woodland park. The "Forest and Stream" alternative planting regime maximizes the provision of quantifiable, locally relevant urban ES in the study area, named to reflect the resulting emphasis on woodlands and stormwater filtration facilities. The "Birdland" regime, in contrast, represents Delphi priority urban ES, showing the value placed on bird habitat and air quality; "Flower Sports" represents resident priority urban ES, distinguished by an emphasis on pollinator habitat and outdoor recreation; and "Integration" capitalizes upon the multiple urban ES provided by individual land cover types to address both Delphi and resident priority urban ES with minimal compromise to either one. Urban ES supply rates expected of each alternative planting regime were estimated as described in Methods, with the inclusion of an additional "recreational lawn" metric reflecting the local importance of soccer and other playing fields [90]. The first alternative planting regime, Forest and Stream, maximizes the quantifiable, locally relevant urban ES of air quality, carbon storage, cooling, and runo ff retention and purification, independent of stakeholder priorities. All park and schoolyard lawns are therefore converted to woodlands except for the 0.5 ha devoted to rain gardens, and nearly 4.6 ha of stormwater planters, as well as an additional 0.3 ha of trees, are added to right-of-way planting strips, su fficient to intercept stormwater runo ff pollution from all public and private impervious surfaces in the neighborhood (Figure 7, Tables S10 and S11). Estimated from published supply rates [14,58], this regime would increase air purification by nearly 40%, carbon sequestration by over 150%, and runo ff retention by 3.5%, as well as reduce runo ff pollutant loading by 80% (Table 2). At the same time, Delphi responses sugges<sup>t</sup> that the conversion of such a large area would encounter cost barriers as well as safety concerns associated with dense vegetation. Figure 7. Land cover distributions for alternative planting regimes. Proportions of public green space (57.4 ha total) devoted to dispersed trees, woodland, tall shrubs, short shrubs, lawn or grass, and stormwater facilities, respectively. Status Quo describes the existing condition in the neighborhood (Section 3.1.); Forest and Stream maximizes locally-relevant, quantifiable urban ecosystem services (ES); Birdland maximizes delivery of Delphi respondents' priority urban ES; Flower Sports maximizes delivery of residents' priority urban ES; and Integration maximizes delivery of the urban ES prioritized by both Delphi respondents and residents. Table 2. Urban ecosystem service delivery associated with alternative planting regimes. a Supply rates were calculated according to Derkzen et al. [14] unless otherwise specified. b Retention by woodlands, trees, tall shrubs, short shrubs, and lawn only. c Filtration by stormwater facilities, calculated using the Simplified Approach as described in the City of Eugene Stormwater Manual [58]; accounts for stormwater pollutants from impervious surfaces removed by stormwater planters and rain gardens on both publicly—and privately—owned land (see Table S10). The second planting regime, Birdland, maximizes the response to the Delphi priorities of carbon storage, bird habitat in parks, air temperature regulation (i.e., cooling), and natural beauty, as well as the priorities held in common with residents (i.e., air quality, stormwater quality, and native species throughout the neighborhood, as well as pollinator habitat in parks). Clear sightlines for safety and moderate cost were prominent Delphi concerns, expressed in part as a desire to retain some existing lawn, and Birdland, therefore, converts only about one-quarter as much existing park lawn to woodland as Forest and Stream, or ~8 ha, envisioned as patches of native oak woodland and restoring native willow and ash woodland for bird habitat in the area designated as Westmoreland wetlands [91]. To address air quality and cooling priorities while maintaining ground-level openness, Birdland adds ~8 ha of dispersed trees to parks and schoolyards, capitalizing on the superior air pollutant removal rates of trees near roadways [14]. Like Forest and Stream, this regime adds 0.5 ha of rain gardens and ~5 ha of short and tall shrubs to parks, again removing all recreational lawn (i.e., softball fields) but leaving ~6 ha of other lawn intact, responding to Delphi safety concerns. On right-of-way planting strips, Birdland reduces the ~5 ha of stormwater planters proposed by Forest and Stream to ~2 ha, su fficient to manage the publicly-owned impervious area in the neighborhood (Table S10) and responding to Delphi participants' cost concerns. The remaining ~3 ha of right-of-way lawn are then replaced with dispersed trees for air quality (Table 2). This conversion, involving ~5 fewer ha than Forest and Stream (Table S11), is estimated to increase existing air purification by over 40%, carbon storage by ~100%, and runo ff retention by ~2%, as well as to provide pollutant filtration for about one-third of the neighborhood's total stormwater runo ff (Table 2). The substantial conversion of playing-field lawn found in Forest and Stream and Birdland is reversed in the third planting regime, Flower Sports, which maximizes responses to resident priorities of outdoor recreation and pollinator habitat throughout the neighborhood, while accommodating the priorities of air and water quality held in common with Delphi respondents. A recent survey of Eugene residents showed that outdoor playing fields (i.e., recreational lawn areas) were in especially short supply compared to resident desires, providing specific, local evidence that superseded the outdoor recreation supply rates compiled by Derkzen et al. [14]. In parks and schoolyards, Flower Sports, therefore, converts only half as much lawn to native oak and ash woodland around the Westmoreland wetlands (4 ha) and ~15% less lawn (~7 ha) to dispersed trees as Birdland, while preserving the full 4 ha of existing sports fields (Table S11). Like Birdland, this regime adds 0.5 ha of rain gardens and ~5 ha of tall and short shrubs to parks, as well as ~2 ha of stormwater planters to the right-of-way, for stormwater purification; in contrast, however, it adds 2 ha of flowering shrubs to right-of-way plantings for additional pollinator habitat in place of dispersed trees. This regime converts ~5 fewer ha of lawn than Birdland but still increases air purification over the existing condition by about one-third and carbon storage by 70% while adding the ability to remove about one-third of the neighborhood's stormwater runo ff pollution. The fourth planting regime, Integration, prioritizes the urban ES held in common by both resident and Delphi stakeholders (i.e., stormwater quality, air quality, park pollinator habitat, and native species), using quantitative supply rates to indicate the most e ffective land cover types for each priority and allowing other priorities to be addressed through species selection. Integration, therefore, converts an area of existing park lawn to woodland between those of Birdland and Flower Sports (6 ha), representing a significant compromise that diminishes the outdoor playing field area by one-quarter in the interest of greater air quality, cooling, bird habitat, carbon storage, and native species urban ES. Integration also includes less dispersed tree area in parks (~6 ha) than either stakeholder-driven scheme, accommodating both the outdoor playing field area prioritized by residents and woodland urban ES prioritized by Delphi participants. Like Birdland and Flower Sports, this scheme converts 0.5 ha of park lawn to rain gardens and ~5 ha to flowering shrubs. To compensate for tree loss in parks, Integration increases tree cover and diminishes flowering shrubs relative to Flower Sports on right-of-way strips; stormwater planters are maintained at the level of both stakeholder-driven schemes. Compared to Forest and Stream, which maximizes quantifiable urban ES, Integration converts ~30% less land area but provides 95% of its air quality improvement and over one-third of its stormwater pollutant filtration, while retaining over 3 ha of outdoor playing field area (Table 2). Integration also provides clear but unquantified increases in pollinator habitat and native species diversity through the inclusion of flowering shrubs and woodland, and it addresses concerns of cost and safety raised in the Delphi analysis by converting less total lawn and maintaining greater openness at ground level than Forest and Stream or even Birdland (Table S11).	doab	2025-04-07T03:56:59.426359	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 55 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.56	5. Discussion The investigation above addresses an emerging issue in urban ES development and planning: the integration of stakeholder perspectives with quantitative estimates of urban ES provision to inform decisions regarding future land cover [4,16,35,44]. The new method that results is then applied to an urban neighborhood through the evaluation of existing public green space and current urban ES supply rates, the collection of resident and city-wide stakeholder priorities, and the translation of these lines of evidence into a set of alternative planting regimes. #### 5.1. Public Green Space Inventory The population density and land cover distribution in the study area is broadly representative of urban residential neighborhoods in the USA [92], with lawn as the dominant vegetated land cover type (Tables S3 and S4), consistent with other urban areas studied in the USA [29] and in Europe [93]. Approximately one-eighth of this area is covered by trees, again consistent with tree coverage of European research sites [93]. At the same time, resident access to public green space is substantially above the USA average [51] (Figure S1), an unusual situation in a neighborhood with below-median income [49,50] that may partly explain the high value residents placed on numerous urban ES (Figure 4). In the evaluation of existing vegetated land cover types, the combined LiDAR and NDVI-based analysis identified tree cover with high accuracy but was less accurate in identifying shrubs (Table S2); it also obscured the co-occurrence of tree cover over herbaceous ground cover and shrubs. We, therefore, recommend the incorporation of further image analysis rules and waveform LiDAR processing in future green cover analyses for the accurate distinction of these land cover types (e.g., [94,95]). Currently, lawn provides the majority of runoff retention and recreation value in the neighborhood, while trees and woodlands provide the majority of air purification, carbon storage, and cooling, despite their much smaller area. As a result, the question faced by those responsible for increasing urban ES in Eugene (Section 5.2.) is whether existing public lawn in the neighborhood should be replaced, and if so, with what. #### 5.2. Stakeholder Priorities Alignment with local values is known to be critical to the successful planning, development, and managemen<sup>t</sup> of public urban green space [89,96], and because resident urban ES priorities are locally idiosyncratic (e.g., [40–42,97]), local input is necessary. Accordingly, Eugene's Parks and Open Space Division surveyed thousands of residents and hundreds of city governmen<sup>t</sup> and operations employees from 2015–2018, culminating in a vision and implementation plan for future green space development [48]. Although the plan focused on the recreational importance of public parks, responding to expressed desires for public gathering space and for additional sports fields [48,90], another prominent goal was to "further the parks and recreation system's capacity to serve as critical infrastructure for clean air, clean water, flood control, carbon sequestration, and climate resilience" [48] (p. 42). Strategies to provide these urban ES have not ye<sup>t</sup> been determined, but an economic assessment of current park value has been completed [98], and a \$50 million tax bond was passed in 2018 to support park operations and development in preparation for future green infrastructure development to expand urban ES delivery [99], showing the timeliness and relevance of the decision-making process we contemplate here. #### 5.2.1. Resident Priorities FAN residents valued urban ES highly overall, with most respondents rating 16 of the 17 listed urban ES as "moderately" to "very" important (Table S5). These primarily represented supporting or regulating services, showing the importance of ecological resilience as represented by native plant species, bird and pollinator habitat, stormwater purification, and carbon sequestration [3]. Similarly, the importance of human health and well-being was shown by the priority of air purification. These findings contrasted strongly, however, with results from Paris and Angers, France, and from Porto and Lisbon, Portugal, in which residents valued cultural and provisioning urban ES most highly [41]; Table S5. They also di ffered from the results of six cities in the USA, in which residents viewed native species and pollution mitigation as low priorities [100], and from results of Barcelona, Spain, in which pollination and biodiversity were low priorities as well [97]. Air purification, however, seems to be one of the few urban ES that shows consistently high priority among city dwellers globally, and the high priority of air quality found here is consistent with recent results from China, Portugal, Spain, France, and the Netherlands [39–41,101]. Outdoor recreation was the single cultural urban ES given high priority by FAN residents, consistent with the previous local survey [90] as well as with recent results from Finland and China [42,101]. In contrast, noise reduction and community identity were low priorities among FAN residents, consistent with findings in European cities [41] but unlike those of Guangzhou, China, in which residents ranked noise abatement as a high priority [101]. FAN residents also assigned a low priority to vegetable and fruit production, in contrast with results from cities such as Barcelona [97], revealing an unexpected lack of support given Eugene's promotion of urban farming [102,103]. Still, this result may show that the numerous community and private gardens in the neighborhood have already met this need. #### 5.2.2. Delphi Analysis The perspectives of stakeholders who would either be involved in making and influencing green infrastructure development decisions, or responsible for implementing and maintaining any changes, di ffered from each other substantially in the first round but partly converged during the second round, reflecting the exchange of ideas allowed by the Delphi procedure (Supplementary Materials Section S2; Table S8) and showing the promise of this method in reaching consensus among other diverse stakeholder groups. Among the consensus priorities, Delphi participants valued air and stormwater purification most highly, reflecting current urgen<sup>t</sup> needs for these services at the city level (Section 2.1.). Although residential neighborhoods typically contribute fewer total suspended solids and other stormwater pollutants than do roads with heavy tra ffic [104], low-density development typically contributes greater runo ff volume and a ffects more of its watershed than high-density development [105]. Delphi respondents also assigned high value to carbon sequestration, natural beauty, and native plant species (Table 1), but the prospect of dense vegetation raised safety concerns (Table S8), illustrating an internal conflict in the responses that would require eventual resolution. In contemplating land cover changes, Delphi participants described the recreational and ease-of-maintenance benefits of lawn in neighborhood and city parks; one participant noted that city operations managemen<sup>t</sup> is currently converting long-established landscape beds into lawn to realize these benefits, adding that lawn is viewed as "neat-looking". These views, combined with the acknowledgment of lawn's minimal delivery of regulating and supporting services, are consistent with those found among municipal land managers in three Swedish cities [106]; however, Delphi participants did not express concern about the costs and greenhouse gas emissions of mowing, as Swedish stakeholders did [106]. Instead, most Delphi participants viewed the "cost and complexity" associated with the installation and maintenance of alternative planting regimes as a greater set of barriers, although they did not elaborate (Table S8). Still, most Delphi participants (>85%) supported the conversion of some public lawn to alternative plantings to increase regulating and supporting services, agreeing that the primary limitation would be the preservation of sightlines along right-of-way planting strips. #### 5.2.3. Resident and Delphi Comparison The di fferences between resident and Delphi urban ES priorities appear to have reflected their frames of reference: while Delphi participants contemplated the city scale, residents considered only their own neighborhood, as intended. Illustrating distinctly city-level concerns, one Delphi participant explained: "What drives public agencies such as our Public Works Department is regulatory requirements—meeting the Federal Clean Water Act and our NPDES Permit requirements (e.g., reducing pollution into local waterways, reducing flooding, and improving air quality). Other items are secondary to the basic welfare and safety of the general public." (Supplementary Materials Section S3, p. 17). Consistent with the diversity of stakeholder opinion found in related studies, the partial divergence among Delphi and resident priorities supports the inclusion of neighborhood-level input even in city-wide urban ES planning. A notable discrepancy was found in the stakeholders' view of cost barriers: while Delphi participants agreed that the cost of lawn conversion was an important barrier (Table S7), over 85% of residents expressed willingness to pay for green infrastructure development that would increase urban ES delivery through tax measures or private donations (Figure 5; Table S6). While stated willingness is not a guarantee of future payment, these findings are consistent with survey results regarding willingness to pay for urban ES in Palm Beach, FL, USA, involving biodiversity, outdoor recreation, and flood protection [107], as well as in Wuhan, Changsha, and Nanchang, China, involving climate regulation, cultural services, air quality, erosion prevention, and habitat services [108]. In each of these cases, the willingness to pay exceeded the expected cost per capita despite heterogeneity in the responses. In contrast, residents of Veneto, Italy expressed a willingness to pay for recreational services but felt that biodiversity conservation and landscape quality should be provided without taxes [109], illustrating the importance of personal history and the governmental context in such attitudes [110] and suggesting that urban planners survey their target neighborhoods before assuming particular cost barriers. #### 5.3. Integration of Stakeholder Priorities with Potential for Local Benefit Recent studies have emphasized the need to integrate the ES priorities of multiple stakeholder groups with the quantitative and qualitative potential for the desired ES to be realized [2,16,35,37,44], but none have explored this further. Here, we found that such integration could be readily accomplished by evaluating questions typical of a green space planning process (Section 4.1.) in light of the corresponding stakeholder survey and urban ES supply rate evidence, yielding a set of alternative planting regimes representing each line of evidence. The ability of each vegetated land cover type to provide multiple urban ES then facilitated the creation of an integrated scheme with limited compromise to stakeholder priorities (Section 4.2.). Of central importance, the contrasts among the individual lines of evidence (i.e., quantitative considerations, resident priorities, and Delphi priorities) illustrated the value of consulting all three. In particular, safety and sightline concerns of Delphi participants moderated the extent of woodland required to maximize air and water quality services, while cost concerns limited stormwater planter area; likewise, residents' outdoor recreation priorities limited the conversion of lawn-based playing fields and emphasized widespread inclusion of native flowering shrubs, carrying particular importance because of residents' financial responsibility for any changes as well as their willingness to bear additional costs. As a result, the Integration planting regime is one that could not have been found by any one approach alone, adding strong support to previous recommendations that diverse stakeholder views and quantitative evidence should be considered together in developing urban ES delivery plans [37].	doab	2025-04-07T03:56:59.427727	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 56 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.57	6. Conclusions This study illustrates a straightforward approach for deciding among green infrastructure alternatives based on their quantitative and qualitative potential to provide urban ES of high priority to diverse stakeholders in a particular location. Here, investigating a neighborhood in Eugene, Oregon, we show how a combination of surveys, Delphi analyses, land-cover analysis, and urban ES quantification can be integrated to reveal a clear direction for urban green space development. The importance of consulting multiple stakeholders was emphasized by the prominent areas of agreemen<sup>t</sup> as well as disagreement between residents and decision-makers, consistent with other studies that have queried multiple stakeholder types [111,112]. Since stakeholder support is essential to successful urban ES provision [36], and since these priorities are locally and regionally idiosyncratic (e.g., [38,40,41,101,113]), results here sugges<sup>t</sup> that revealing these consensus priorities is a necessary first step and highlight the value of the Delphi technique as a method for finding consensus among diverse stakeholders. Subsequent evaluation of existing land cover and urban ES delivery showed that, despite the generally high value residents assigned to supporting and regulating urban ES, lawn currently dominates the neighborhood public green space, reflecting the priority that decision-makers have given to ease of maintenance, lawn-related outdoor recreation, and safety perceptions (Supplementary Materials Section S3; Table S8). While lawn prevalence is consistent with that found in other urban settings [29,93], the value residents placed on habitat and regulating urban ES differed from that of European residents, who generally prioritized cultural over environmental urban ES [41]. Additionally, the willingness residents expressed to support urban ES-related changes financially was unexpected, although others have documented similar results (e.g., [107]), showing that resident views should be explored before options are limited for financial reasons alone. Evidence such as this, whether qualitative or quantitative, allows green space development to focus on the priority urban ES that can be meaningfully delivered by the land area of interest; here, air quality and stormwater quality were the clearest stakeholder priorities and had the highest potential for local delivery. Such urban ES priorities can then inform the development and evaluation of alternative planting regimes, as illustrated above (Section 4.2.), to reveal the relative benefits of each. Once large-scale land cover decisions are made, additional urban ES priorities can be addressed through species selection during green space design, as in the accommodation of pollinator habitat and native species coverage priorities above. Together, these steps provide a straightforward, flexible method suitable for widespread application in local planting decisions with the goal of increasing urban ES delivery on public land. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-445X/9/10/391/s1. Supplementary Materials, including Figures S1 and S2, Tables S1–S11, and related discussion are included in a separate file. Survey data, Delphi analysis responses, and green space quantification GIS data are available at doi:10.5281/zenodo.4044203. All other materials are available upon request. Author Contributions: Conceptualization, E.E., A.R.R., C.E., and K.A.L.; methodology, E.E., A.R.R., C.E., and K.A.L.; formal analysis, E.E.; investigation, E.E., A.R.R., and C.E.; resources, A.R.R. and C.E.; data curation, E.E.; writing—original draft preparation, E.E. and A.R.R.; writing—review and editing, E.E., A.R.R., C.E., and K.A.L.; visualization, E.E., A.R.R., and C.E.; supervision, A.R.R., C.E., and K.A.L.; project administration, E.E., A.R.R., C.E., and K.A.L. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: We thank Peg Boulay for lending us the Garmin GPSMAP 62S for field data collection. We thank Scott Altenhoff and Justin Overdevest for early project conceptualization and for their help connecting us with Delphi participants. We also are appreciative of the Friendly Area Neighborhood residents who participated in the survey, as well as the individuals who put their time and effort into responding to the two rounds of Delphi surveys. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.428639	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 57 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.59	1. Introduction Urbanization and climate change e ffect the water balance in our cities, resulting in challenges such as flooding, droughts, and heat stress. The implementation of Sustainable Drainage Systems (SuDS) or small-scale Nature-Based Solutions (NBS) can help to restore the water balance by capturing, retaining, treating, and infiltrating stormwater that runs o ff roofs and impermeable surfaces and potentially into the subsurface [1–5]. This will contribute to minimizing flooding, restoring groundwater levels, increasing soil moisture to alleviate drought impacts, and lowering temperatures by evapotranspiration to mitigate heat stress [2,6–11]. As Wakode et al. [12] point out, the urban water cycle is di fferent from that in non-urban areas, where urbanization can influence natural groundwater recharge due to the restriction of infiltration by impermeable surfaces. Even though leakage from water-wastewater infrastructure is known to recharge the groundwater in cities [13,14] this was not substantial enough to recharge and stabilize the groundwater levels under the UNESCO World Heritage site Bryggen Wharf in Bergen, Western Norway (Figure 1) [15–18]. Therefore, the connected infiltration system at Bryggen was intentionally built for that purpose (Figure 1). Figure 1. The Medieval city in Bergen is located along the shore of Vågen bay. The sustainable infiltration and drainage system that has been implemented within the premises of the Bryggen is the largest in Norway (Figure 3). It was built with the purpose of raising and stabilizing the groundwater level and increasing the soil moisture in the cultural heritage layers in the ground below Bryggen [19–22]. The infiltration system has proven its effectiveness for raising the groundwater level to desired levels for preservation [16,22]. However, the infiltration system has not been full-scale tested for its infiltration capacity and interaction with the groundwater below. Such testing of SuDS is commonly executed with small-scale infiltrometer tests [23–25] and further upscaled in modeling tools. However, small-scale testing, such as Modified Phillipe–Dunne (MPD) infiltration tests, does not give a picture of the overfall infiltration capacity of the SuDS. Therefore, a full-scale methodology was first implemented for testing impermeable pavements [26,27] and further used for other infiltration systems such as rain gardens, swales etc. [28–30]. A full-scale infiltration capacity test at Bryggen will contribute to the understanding of the urban water cycle, by quantifying the hydraulic conductivity and infiltration capacity of SuDS, the connectivity to groundwater levels, and thus the overall effectiveness of this system in a larger hydrological and hydrogeological context. SuDS in cold climates require higher infiltration capacity than warm climates to maintain functionality below 0 ◦C [31,32]. Bryggen is a unique site due to its 35 boreholes with continuous measurements of groundwater level, soil moisture, oxygen content, and other parameters that are essential for the in situ preservation of the cultural heritages below surface. The subsurface data were continuously collected from 2007, gradually expanding with additional boreholes until 2015 and will continue monitoring onwards [19,21]. At present, only a limited part of the catchment area is connected to the infiltration systems (Figure 2). The municipality inquires if the capacity of the rain gardens is acceptable to connect the entire catchment area with stormwater from both roofs and streets to the system. Therefore, the rain gardens were assessed to determine if they work as designed, and if the infiltration capacity and the effectiveness is satisfactory to preserve the cultural layers and thereby enlarge the connected runoff area. The implementation of SuDS at locations where the infiltration of water is a challenge, such as on cultural layers, is a challenge for urban planners, water authorities, and other stakeholders in municipalities. This paper will describe the full-scale infiltration method [26,28] used for testing the hydraulic infiltration capacity of the rain gardens at Bryggen and the response on the groundwater level measured in several monitoring wells. Figure 2. The catchment area and locations of boreholes. #### 1.1. Study Area The infiltration system at Bryggen Wharf is located in the Medieval City center of Bergen, the largest city on the west coast and second largest city in Norway (Figure 1). The average temperatures are 23.8 ◦C in summer and −4.7 ◦C in winter, giving an annual average temperature of 8.6 ◦C. During 61 years of data collected, only 17 winters had temperatures below 0 ◦C [33]. Bergen is one of the wettest places in Europe, with an annual precipitation of 2250 mm/year [33]. The topography is steep hillsides covered with forest vegetation on scares soil, down to flat laying formerly shorelines with thicker natural sediments and anthropogenic layers (Figure 1). The relief goes from 320 m above sea level to 1 m a.s.l. over a distance of 1 km. These natural conditions make surface runoff water abundant. Bryggen is a Hanseatic Wharf where several of the buildings originate from 1702 [34]. The Medieval city, located along the shore of Vågen bay, is to a large degree built on anthropogenic waste including remains from city fires and industrial and household waste. These have accumulated into abundant anthropogenic cultural heritage layers rich on organic content that locally are more than 10 m thick [19,21]. The reduction of soil moisture or lowering of the groundwater level will introduce oxygen into the organic matter. This will accelerate the oxidation and disintegration of the organic material causing collapse and compaction of the organic layers in the subsurface [17,20]. Due to the slow decay causing damage to the Wharf, the Bryggen project was initiated in 2010 [17,20,21]. The abovementioned processes will further cause subsidence of the ground and damage on buildings and infrastructure [18,35]. The ground beneath Bryggen is characterized by a steeply sloping mountain side, with depth to bedrock from 2 to 12 m. The layers consist of up to 10 m of organic, anthropogenic deposit as described above, on top of beach sand and moraine of ca. 2 m thickness. The recharge of the groundwater is primarily by runoff from the uphill catchment area [16,36]. A 3D hydrogeological model of Bryggen and its subsurface has been made to understand the groundwater movement, hydrogeological characteristics of the subsurface layers and processes linked to water, or the lack of water [16,17,20,36]. A monitoring system was established in 2001, which was expanded in 2010 with a total of 35 monitoring wells [37]. Initially, this network of monitoring wells was placed to understand the complex flow system in the area and to identify the causes of the local groundwater levels and observed, increased subsidence rates [16,18,29,35]. An automated groundwater-monitoring system was installed in the wells, for high frequency of measurements. Some boreholes are dedicated to measuring parameters for archaeological purposes [17] while other boreholes are continuously monitored for groundwater levels [21], using equipment such as Schlumberger Micro diver DI 501 [38]. During the Bryggen project a strong link between the level and stability of the groundwater and the decay of the cultural layers was established [16,17,20,22,39]. Therefore, an infiltration system was installed in the ground in and around the Wharf to infiltrate as much surface water as possible into the ground (Figure 3) [18,29,40]. All measures at Bryggen, including monitoring wells and SuDS, were directed towards raising and stabilizing groundwater levels. The long-term goal for the area was to elevate groundwater levels to about 1 m below the surface [16]. Figure 3. Top: Sketch of the infiltration system at Bryggen (Drawing: Multiconsult AS). Bottom: Cross section of rain garden, swale, and permeable pavement (modified from de Beer & Boogaard [40]). #### 1.2. The Rain Garden at Bryggen The rain gardens in Bryggen are a bioretention system that allows runo ff to temporarily pond in a shallow planted depression before filtering through vegetation, roots, and underlying soils for infiltration [2,3,41]. The rain gardens have the following primary functions: infiltration, storage, and purification. The catchment area, indicted in Figure 2, is upstream of the main street "Øvregaten" (Figure 2), which is salted during winter to reduce icing and tra ffic incidence. Plants in the rain gardens are not salt tolerant [29]. To avoid excess salt from the winter salting, water from the watershed is collected in a manhole on the other side of the street and brought in a pipeline underneath the main street to a manhole connected to the rain gardens, as indicated in Figure 3 and with blue points in Figure 2. The infiltration system has two inflow points from the catchment area: into the rain gardens and into the tank, as indicated in Figure 3. Figure 2 shows the current connected area for surface water (blue line) and the total upstream catchment area (red line) for the rain gardens and infiltration system.	doab	2025-04-07T03:56:59.428961	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 59 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.60	2. Methods The full-scale infiltration test was executed on 6 September 2017, with heavy rain that started the day before with a total of 30.5 mm rain fall [33]. Therefore, the soil was moist and the rain garden saturated with water (Table 1). The days 5th to 14th of September were wet with 28.2 mm precipitation on the 9th [33]. The additional contribution of water through precipitation is reflected in the results from the groundwater level monitoring. Table 1. Analysis of soil moisture of samples collected before and after the full-scale test. The results are given in precent (%) of water in the soil. The compartment B of the rain gardens has an area of 180 m2, a depth of 78 cm consisting of three layers: a filter medium consisting of sandy soil (38 cm, 60% soil and 40% sand), a drainage layer of sand and gravel (30 cm) and a bottom layer of silty sand (10 cm), with a nonwoven geotextile on top of intact cultural heritage layers (Figure 4). The average porosity is approximately 35%. The water storage capacity of the rain garden is designed for 30 cm above soil surface at deepest point, giving a storage volume of 54 m<sup>3</sup> [29] (Figure 5). When this level is exceeded the water flows into an outlet and down to the swales below (Figure 3). The compartment A has the same construction and layering as B, but an area of 52 m2. The rain gardens are designed to have an infiltration capacity of 0.5 m/day for a rainfall event with intensity 35–50 mm/day (24-h storm) [28], assuming dry antecedent conditions. In this study, the infiltration capacity of the rain gardens is compared with international guidelines, such as the FAWB in Australia [42], the MPCA in the USA [43,44], and the CIRIA in the UK [41]. The CIRIA SuDS manual [41] is regarded as the most relevant for Norwegian standards and conditions [45]. To evaluate whether the rain gardens at Bryggen qualifies according to the international guidelines, we compared the measured saturated hydraulic conductivity to both the design values and the measured infiltration capacities. Two infiltration tests were used: Modified Phillipe–Dunne (MPD) [23,24] and full-scale infiltration capacity method [26–28]. The full-scale infiltration capacity test was further correlated with continuous monitoring of the groundwater level in several boreholes (Figure 2). Before and after the full-scale infiltration test soil samples were collected in the rain gardens, four in compartment A and four in B (Figure 3). The analysis was executed by Vannlaboratoriet Bergen Vann KF [46], where the samples were dried at 100 ◦C for four days. The samples were analyzed for Land 2020, 9, 520 soil moisture to document the start-up conditions for the full-scale test, and the effect infiltration has on soil moisture. Figure 4. The design of rain garden B. Drawing: Multiconsult AS [21]. Figure 5. Modified Philip–Dunne (MPD) column for infiltration measurements (Photo: Tone M. Muthanna). #### 2.1. Modified Phillip-Dunne Infiltration (MPD) Method The Modified Philip–Dunne (MPD) infiltrometer test, which determines the infiltration capacity for saturated hydraulic conductivity [23,24], was executed at four different locations in compartment A and B in the rain garden. The principle for all small-scale infiltrometer tests is that rings or columns are sealed to the surface and filled with water to provide a positive water head. The column has a diameter of 10 cm and length of 50 cm. A measuring tape is attached to the outside of the column in order to measure the height of the water column, as shown in Figure 5. The time recorded for the water to infiltrate through the permeable surface area is used to estimate an average infiltration rate (usually in mm/h) for the test location. Both the constant head and the falling head methods can be utilized in these testing procedures [23,24,30]. These in-situ field methods are easy to facilitate and are time and cost efficient. An illustration of the MPD is given in Figure 5. The permeability is given by $$p = \mathbb{K} \times \mu / \rho \text{g} \tag{1}$$ where p = permeability (cm2), K = hydraulic conductivity (cm/hr), μ = dynamic viscosity (kg/m\hr), ρ = density of water (kg/m3), and g* = gravitational acceleration (m/s2) [23,24]. #### 2.2. Full-Scale Infiltration Capacity Test at Bryggen For the full-scale infiltration test at Bryggen, the total volume of the rain garden is flooded, and the emptying time is measured [26]. The water source was a fire hydrant, which held a constant water flow of ca. 600 L/minute for 2 h and 10 min (Figures 2 and 6). The water influx was continuously measured by an in-line flowmeter, as shown in Figure 7. This translates to a total water volume of ca. 40 m3, which gives ca. 20,000 L/hour. The water was led through a drainage pipe under the street, Øvregaten, and into a manhole on top of the rain garden (Figure 7) where the water inflow was split into two, into compartment A on the left and compartment B on the right (Figures 3 and 8). All outlets from rain gardens A and B were blocked during the infiltration test to prevent the bypass of water out of the gardens and thus force infiltration. The water influx was kept constant until the infiltration system was completely saturated and water became visible, forming a pond at the surface in both rain gardens and at the swales below (Figures 3, 7 and 8). The rain garden compartment A and B have a confined space which can be filled up to the water level of outflow without any additional restriction to prevent water leaving the rain gardens during the full-scale infiltration test. The maximum water depth at the deepest part of the rain gardens is ca. 30 cm, varying depths due to irregularities of the soil surface (Figure 4). The time from maximum ponded water height to complete infiltration was recorded for both rain garden A and B (Figure 6). To calculate the infiltration rate, $$\mathbf{K} = h\mathbf{\dot{\mu}}\,\mathbf{\dot{\epsilon}}\tag{2}$$ where K is the infiltration rate (cm/min), h = height of the water column (cm), and t = time (duration) of infiltration (min) [26,28]). Figure 6. Description of the full-scale infiltration test at Bryggen on 6th of September 2017. #### 2.3. Continuous Monitoring of Groundwater Level in Boreholes In the boreholes, the automated data collection is set at a frequency of 1 h, and the instrument has an accuracy of measuring the water height within 0.05% [34]. With this detailed measuring frequency, immediate and short-term effects are detectable [16], and therefore able to show the response of the full-scale infiltration capacity test. All loggers are calibrated for measuring depth relative to surface level, showing the correct groundwater level from surface level. The location of boreholes is shown in Figure 2, where the boreholes used in this study are marked. Figure 7. A fire hydrant served as a water source, providing ca. 600 L water per minute. Figure 8. Full-scale infiltration test of the SuDS at Bryggen. #### 3. Results and Discussions #### 3.1. Soil Moisture Results The soil samples had a water content of 28–34% before the infiltration test was executed (Table 1). This saturated condition, ca. 30% water, is explained by the heavy rainfall the previous day. After the full-scale test, four soil samples were collected at the same locations show that the water saturation of the soil had increased to 40–56 percent (%) (Table 1). This is an increase in soil moisture of 12–22 percent (%). Preservation of the organic layers is dependent on the soil moist and prevention of oxygen access, as shown by Matthiesen et al. [17]. An increase in soil moisture by infiltration of water may a ffect the cultural layers below, by preventing exposure to oxygen. A study of repetitive full-scale testing by Boogaard and Lucke [30], on permeable pavements and swales, show that after refilling the storage volume a second time (simulating a stormwater event after a stormwater event) the infiltration capacity is reduced by 39% from unsaturated to saturated soil conditions. This shows that the infiltration of surface water with the aid of SuDS like rain gardens and swales will increase the soil moist, recharge the groundwater, and further contribute to preservation of the cultural layers below. #### 3.2. Small-Scale Results Four MPD infiltration tests were executed 8th of September 2017 in the rain gardens compartment A (tests 3 and 4) and compartment B (tests 1 and 2, Figure 9). The results of the small-scale infiltrometer tests MPD, summarized in Table 2, shows that the infiltration capacity is (1) 245 mm/h, (2) 241 mm/h, (3) 382 mm/h and (4) 404 mm/h (Figure 10). The small-scale infiltration tests verify that the rain gardens qualify according to the international guidelines for SuDS, demanding an infiltration capacity in the range of 100–300 m/h [41–45]. Field tests verify the infiltration capacity, which is a recommendation by the CIRIA guideline [41], due to local variation, if the SuDS is built according to design and possible clogging [41]. The MPD has a small surface, commonly 10 cm diameter (Figures 6 and 9), where the variation of the heterogeneous soil layer has large influence on the measurements. Ahmed et al. [47] show in their study that for the MPD to be representative, a minimum of 20 tests on di fferent locations should be executed to ge<sup>t</sup> a representative average. Unfortunately, only four MPDs are collected in this study and are not statistically representative for infiltration rates in the rain gardens. Since the MPD can be inaccurate because of heterogeneity of the filter soil [26,27], the small-scale tests were compared with a full-scale test (Table 2). Figure 9. MPD infiltration tests where MPD 3 and 4 were measured in the smaller compartment A (picture on the left) and MPD 1 and 2 were measured in the larger compartment B (picture on the right). Photo: Torstein Dalen, Bergen Municipality. Table 2. The results of the small-scale and full-scale infiltration test show different infiltration capacity for the two rain gardens, compartment A and B, tested in this study. Results compared to international guidelines [41–45]. Figure 10. Results of the MPD infiltration test in rain garden A and B. MPD 3 and 4 were measured in compartment A and MPD 1 and 2 in compartment B. #### 3.3. Full-Scale Results The results from the full-scale infiltration test are given in Table 2. Based on ca. 30 cm ponding depth and drainage time, which constitutes infiltrating all visible water, the emptying time of the large compartment B was 11 min and 35 min for the smaller compartment A. The infiltration capacity is ca. 1600 mm/h for the large compartment (B) and 510 mm/h for the smaller compartment (A, Table 2).Therefore, both rain gardens A and B meet the minimum requirement of the international SuDS standards [41–45]. Both rain gardens have considerable infiltration capacity, and the capacity is larger than the amount of water coming from the presently connected watershed upstream. The infiltration time was considerably longer in the smaller rain garden A (Table 2). The total water volume for the full-scale test is of ca. 40 m3, which gives ca. 20,000 L/hour and is larger than any known return period of an extreme event [48]. #### 3.4. Comparison between Small-Scale and Full-Scale Results The hydraulic efficiency of SuDS such as rain gardens and swales rely on two main standards, which are infiltration and retention capacity [41,49]. The infiltration capacity is usually estimated by measuring the rate at which water infiltrates from small test pits, boreholes [50,51] or as ring infiltrometer tests [23–25,52]. International guidelines recommend a design that enables bioretention, such as rain gardens that can infiltrate stormwater at a rate of 100–300 mm/h. These guidelines are based on several factors such as the limited availability of space in urban areas, low native permeability of the soil, shallow groundwater tables, limited public health concerns, and often safety factors, such as mosquitoes and risk of drowning. The guidelines also take into consideration that the infiltration capacity of rain gardens may reduce over time by clogging [53–55]. Vegetation that is resistant to long inundation can prevent clogging of the topsoil, due to root canalling [42]. Further, the implemented SuDS should be tested for its infiltration capacity in the field [1–3,10,41]. The small-scale infiltration test methods are based on the infiltration rate through a very small area that is used to represent the total area of infiltration. Using such small areas for testing could potentially lead to erroneous results as studies have demonstrated a high degree of spatial variability between di fferent infiltration measurements undertaken in the same area [26–28]. A study by Ahmed et al. [47], based on 722 small-scale infiltrations test using the MPD test in five large swales, shows that the hydraulic conductivity has a high spatial variability within the same swale. The full-scale infiltration test shows that both rain gardens have a much higher infiltration capacity than the results from the MDPs indicate (Table 2). The infiltration rate for the full-scale test is increased by a factor of 6.5 for the large compartment B and 0.8 for the smaller compartment A, compared to results from the small-scale tests. The di fference between the large and small compartments may be explained by the coarser material on the surface of the larger and di fferent plants in the smaller compartment (Figures 8 and 9). This reflect the results by Boogaard et al. [26] and Lucke et al. [27] where small-scale (single ring) infiltration test only gives the local condition for the SuDS, independent whether a rain garden, swale or permeable pavement is being tested. Ahmed et al. [47] show that the infiltration capacity or permeability in swales can vary by a factor of 100, giving a large uncertainty if only one or a few small-scale tests are used for testing the infiltration capacity. The full-scale method will demonstrate the infiltration capacity compared to small-scale tests, which is especially important in cold climates [31,32]. #### 3.5. Continuous Monitoring of Groundwater Monitoring wells MB 07 and MB 21 are located <30 m downstream of the swales (purple circles in Figure 4). These show an immediate response in rise of groundwater level to the infiltration test (Figure 11). The infiltration during the full-scale test is helped by the large amount of precipitation the day and night before, which can be seen by the rise in water level before the time of the infiltration test. The increase of ca. 35 cm in MB07 and 42 cm in MB21 in groundwater level, as seen in Figure 11. The changes in the groundwater lever curves show response to the infiltration test in addition to natural fluctuation from precipitation, as shown in Figure 11. Figure 11. Response in groundwater level (cm) in the monitoring well MB07 (green line) and MB21 (blue line). The red dashed line marks the day of the full-scale infiltration test. Precipitation data from Florida metrological station in the center of Bergen, ca. 1.5 km from Bryggen. Data from Norwegian Metrological Institute @ eKlima.no [33]. Figure 12 shows changes in the groundwater level in boreholes MB 02, MB 06 and MB 14 that are located 75–100 m further downstream from the rain gardens (yellow circles in Figure 2). The direct effect of the infiltration test is not as prominent as in borehole MB 07 and MB 21 (Figure 11), due to the distance from the infiltration point. However, the results still show a clear rise in groundwater level on the day of the full-scale infiltration test in addition to a continued increase with the following days, contributed by precipitation. These boreholes show a delay of ca. 2 days in the response, reflecting the travel time of the groundwater from source to monitoring point. The increase in the groundwater level is 20–25 cm in all three boreholes (Figure 12). Combined with additional precipitation, the infiltration peak has a duration of ca. a week. Figure 12. The graph shows variations in the groundwater level (cm) in monitoring wells MB 02 (green line), MB 06 (blue line), and MB 14 (yellow line). The red dashed line marks the day of the full-scale infiltration test. Precipitation data from Norwegian Metrological Institute @ eKlima.no [33]. It could be considered that the rain gardens at Bryggen is constructed with an unnecessarily high infiltration capacity, but if the infiltration capacity were too low, flooding of surface water would occur especially during heavy rainfall of 35–50 mm/day. However, this infiltration system is built for multiple purposes, the main one being to stabilize and increase the groundwater level to protect the cultural layers below Bryggen [16–18]. Other functions of the SuDS are retaining and storing stormwater, filtering pollutants, and increasing the soil moisture [2,9,30]. This study shows good communication between the infiltrated water and the nearby monitoring wells, <30 m distance, with a time delay of ca. 2 days according to the distance from the infiltration point (75–100 m, Figures 2, 11 and 12). A large infiltration capacity is especially important in locations with a cold climate, as winter will mean a reduced infiltration capacity due to freezing and ice [31,32]. The infiltration system at Bryggen was built for a larger catchment area than presently connected (Figure 2). The infiltration capacity of 1600 mm/h is more than sufficient to handle the current runoff surface water. Therefore, the capacity should be sufficient to expand the catchment area from ca. 8500 m<sup>2</sup> to 31,000 m2, as shown in the map in Figure 2. The increased infiltration of surface water will contribute to stabilizes the groundwater and prohibit processes driven by lack of groundwater that causes subsidence [56]. Empirical research from this study will improve the groundwater model for Bryggen, and related models [16,36]. In addition, it strengthens the best practices in cultural heritage managemen<sup>t</sup> that the Bryggen Project has proven to be [21,40]. Bryggen is an example that measures can be taken to infiltrate surface water to restore and stabilize the groundwater, to further delay degradation and subsidence, and as a bonus, prevent flooding. #### 3.6. Lesson Learned When comparing small- and large-scale infiltrations tests the tests should be repeated. For small-scale tests, the statistical representative number is 20, as demonstrated by Ahmed et al. [47]. It is a weakness in this study that only four small-scale tests were executed. The large-scale test should also be repeated for better documentation of the infiltration rate and response on groundwater level. Nordic cold climate has di fferent challenges than warmer climate [31,32] and studies that monitor both the stormwater influx as well as groundwater response is needed. As Prudencio and Null [5] point out, SuDS have positive e ffects as ecosystem services, however, this is not been given any attention in this study at Bryggen. Especially, a lesson learned from Bryggen is documentation of "as built". There are several gaps of knowledge, there among construction deviations, the compilations of soil used, and sketches of design as built are missing. The lack of documentation make testing and follow ups challenging. For future constructions of SuDS, planning for implementation of monitoring systems, both for stormwater and groundwater, is recommended. For interactive dissemination and outreach, the tool Climatescan (www.climatescan.org) is applied for engagemen<sup>t</sup> with stakeholders where open access results on infiltration of stormwater under extreme climate and hydraulic circumstances are displayed [57]. The Bergen Wharf site with its infiltration systems is continuously updated with pictures, information, research results, and open access publications [58].	doab	2025-04-07T03:56:59.429640	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 60 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.61	4. Conclusions This study compared the infiltration capacity of the rain gardens at Bryggen with international guidelines [41–45]. It compares small-scale MPD tests to full-scale infiltrations test and further evaluate if the rain garden is preforming as designed. The full-scale infiltration test showed an increase of the groundwater level in several boreholes at Bryggen. The rain garden and its connected infiltration systems function for the purpose it is built, to infiltrate stormwater into the subsurface to increase the soil moisture as well as groundwater level to protect the cultural layers in the subsurface. The hydraulic conductivity of the SuDS is as designed with an infiltration capacity of 500–1600 mm/h. This study shows that the full-scale infiltration test gives a higher infiltration capacity of the rain gardens, compared to small-scale tests. The e ffect of the infiltrated volume and the natural precipitation influence the groundwater level, with an immediate response in monitoring wells close to the infiltration system (<30 m) and with a time delay of ca. 2 days in wells 75–100 m away from the infiltration point. The infiltration capacity of the rain garden exceeds the amount of available surface water currently connected to the system. The groundwater level would, in dry periods, benefit from more consistent water input to increase soil moisture and thereby preserve cultural layers below. There is excess capacity, and the connected runo ff catchment area can be extended to encompass the total catchment area by 22,500 m<sup>2</sup> or 260 precent (%). There is a need to document the e ffect of SuDS on the urban water cycle. This study shows how monitoring systems can be implemented in designing and planning, which could help stormwater managers with the scheduling of maintenance requirements for rain gardens with more confidence. Author Contributions: Conceptualization, G.V. and F.C.B.; methodology, F.C.B.; validation, G.V. and F.C.B.; formal analysis, G.V. and F.C.B.; investigation, G.V.; data curation, G.V.; writing—original draft preparation, G.V.; writing—review and editing, F.C.B. and G.V.; visualization, G.V. All authors have read and agreed to the published version of the manuscript. Funding: This research is supported through the JPI Water funded INXCES research project "Innovation for eXtreme Climatic EventS" www.inxces.eu. INXCES exchange researchers, methodology and results on an international level, with the aim is to share research results with stakeholders and set up guidelines for design, implementation and maintenance of SuDS to promote sustainable water managemen<sup>t</sup> systems throughout the world. Within the INXCES project knowledge exchange between disciplines and nations is in focus. Acknowledgments: Great appreciation to Torstein Dalen at the Water and Wastewater department at Bergen Municipality for help and contributions. Thanks to the Bryggen project for access to borehole data http: //prosjektbryggen.no. We thank Bergen Municipality and Hanze University of Applied Sciences Groningen, Deltares and the Geological Survey of Norway for support for this work. Thanks to Riksantikvaren (Directorate for Cultural Heritage) and NIKU (Norwegian Institute for Cultural Heritage) for making data freely available. Thanks to Norwegian Hydrology Council for the NHC2018 conference in Bergen, and this special conference issue. Great appreciation to Malin Andersson at the Geological Survey of Norway (NGU) for constructive feedback on this paper. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.431110	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 61 }
0092e559-d8e0-493b-a85b-c4691a50c8ac.63	1. Introduction Online knowledge-sharing platforms could potentially contribute to an accelerated uptake of nature-based solutions (NBS). NBS are considered to be a promising means to address climate change by promoting more green and blue spaces in urban areas. NBS usually have multiple benefits: they increase a city's biodiversity, contribute to more healthy and resilient ecosystems, and provide benefits for health and human well-being [1–3]. However, while advocacy for NBS in the scientific literature is on the rise, implementation in practice is often hampered [4]. Common barriers to implementing NBS are the fear of unknowns (regarding implementation, maintenance, and performance of NBS) and a general lack of understanding the multiple benefits of NBS [4,5]. Moreover, many NBS require the involvement and enthusiasm of a multitude of stakeholders [6]. This is particularly true for small-scale NBS such as bio(swales), green roofs, and green walls, which can be implemented in urban neighbourhoods even if space in a city is scarce. Next to their aesthetical value, they simultaneously help to regulate water flow, prevent floods, and reduce heat stress. However, they often need to be implemented on private land, requiring non-governmental actors such as individual house owners, housing corporations, and property owners of business parks to take action. To build momentum and foster implementation of NBS, Kabisch et al. (2016) [4] have pointed out that it is important to "learn from action that is already taking place" and share existing approaches and experiences among di fferent countries. Everywhere in the world, stakeholders are experimenting with NBS and climate adaptation. However, many of these experiments are on such a small-scale that they do not reach beyond their regional boundaries. An online platform could be an ideal tool to increase international knowledge exchange and raise awareness, with the ultimate goal that a good example of an a ffordable and well-functioning NBS can help actors in other places to move forward in the implementation process. Especially during the current Covid-19 pandemic, we have all come to realize the importance and the opportunities of being connected through the internet. The internet o ffers the advantage that it is accessible to anyone (with an internet connection) at any time [7,8]. This means that an online platform could potentially reach a multitude of stakeholders: those that already have a say in climate adaptation and water governance, and those who were not involved thus far. Many of these platforms exist, the majority of which have been established with the (financial) support of funding agencies and governments. Examples are Climate-ADAPT, OPPLA, NATURVATION Urban Nature Atlas, BISE, DRMKC, Natural Hazards NBS, NBS Initiative, NWRM, PANORAMA, ThinkNature and weADAPT. However, none of these platforms have a citizen science interactive mapping function. ClimateScan (www.climatescan.org), the case in this paper, is built on such an interactive mapping function using citizen science. The platform has been set up without any external resources and using only voluntary e fforts. It was originally developed by Hanze University of Applied Sciences (HUAS) in 2014 to show the locations of Sustainable Urban Drainage Systems (SUDS) in The Netherlands and publish the research results of functionality tests carried out on these locations. In 2016, the platform has been opened up to the public so that anyone could contribute existing best practices of nature-based solutions and climate adaptation on an interactive map. This citizen science approach was chosen for two reasons. First, it increases the geographic range of the ClimateScan database. Second, mapping our own examples is in line with other web 2.0 ideas that makes users active knowledge producers instead of passive knowledge consumers [9]. This can increase the "fun factor" and the commitment of users and thereby help to build a more empowered and more sustainable community of practice. Today, the platform has over 800 registered users, which have uploaded more than 5000 projects assigned to seven main themes (Water, People, Nature, Heat, Energy, Urban Agriculture and Air Quality) all around the globe. In this paper, we discuss how online citizen science platforms such as ClimateScan can stimulate stakeholder engagemen<sup>t</sup> and promote NBS. ClimateScan is a unique case because it has adopted an innovative bottom–up approach. This bottom–up approach entails that ClimateScan is independent from the (financial) support of funding agencies and governments, and that there has not been a top–down induced set of rules about what should be uploaded in what way from the start. Accordingly, the platform relies strongly on the self-organizing capacity of the actual users. One of the authors has been involved with ClimateScan from the outset and throughout. As a result of this, we were able to acquire in-depth and inside knowledge around both the development of the content of the platform and the development of the community behind the platform. We have supplemented these participatory observations with a content analysis of the ClimateScan database and statistical analyses stemming from Google Analytics to analyze what has been uploaded, by whom, and how the content and the community has developed over time. While there have been evaluations of the actual usage and contribution of online decision support tools cf. [9–11], there has not been an evaluation of an online adaptation platform using citizen science. Thereby, we add novel insights regarding the potentials and pitfalls of online citizen science platforms and how they can contribute to climate adaptation and NBS implementation practice. In the following, we will first present the current state of knowledge regarding the potentials and pitfalls of online adaptation platforms to identify research avenues in the current debate (Section 2). Based on these research avenues, we have designed our empirical strategy for evaluating the ClimateScan platform. We will explain this methodological approach in Section 3. Section 4 provides more background information regarding the history of ClimateScan, its key design parameters, and how it uses online and o ffline channels to acquire new users. Section 5 will present the results of our analysis regarding the content and the community of ClimateScan. In Section 6, we will provide concluding remarks on how ClimateScan and similar initiatives could be sustained, with an eye on improving its flaws, without doing harm to its strengths. #### 2. Online Adaptation Platforms—What We Know So Far Climate adaptation is eminently a field where Fischer's request to rethink expertise and his plea to build more cooperative relations between experts and lay citizens applies [12,13]. This is simply because climate adaptation requires a rather radical transformation of the built environment, for which agreemen<sup>t</sup> and action of many di fferent actors, often non-experts, is needed [6]. With democratizing knowledge about climate adaptation, we mean that knowledge about climate adaptation is made accessible to all actors from the quadruple helix (academia, government, business, and civil society), enabling them to negotiate about goals and how to achieve them [14,15]. A key consideration must be that the adaptation process is often undertaken by people for whom climate change is not a primary concern [16]. Eventually, the success of the world's adaptation e fforts will depend on "ordinary" citizens, housing associations, business park owners, and (municipal) civil servants who integrate climate adaptation into the (re-)design of streets, buildings, gardens, parking lots, and other components of our built environment. For them, climate adaptation often has been and still is a voluntary add-on on top of their usual responsibilities. Palutikof et al. (2019) [7] (p. 461) have already pointed out that knowledge adaptation platforms can be a useful and time-saving tool to these typical adaptors, as they can serve as "a comprehensive resource equipping decision-makers with the data, tools, guidance and information needed to adapt to a changing climate". Such platforms are usually online resources, making use of the several benefits the internet o ffers, which can help to democratize knowledge about climate adaptation. The first advantage of online platforms is inclusivity: anyone with an internet access can participate, access is free of charge (except for the internet fee itself), and there is no need to be at a specific time at a specific place [8–10]. Second, the internet o ffers the opportunity to make use of strong visual powers. Videos and photos next to pure text can help in communication [9]. Such visual content is more universal than written text, which can be particularly useful in a field such as climate adaptation where terminology varies strongly between countries and also changes quickly over time (see [17] for an overview of changing terminology in the field of urban stormwater management). Third, web 2.0 developments have made it possible to create interactive platforms, where people can meet and exchange ideas. This increases opportunities to broaden participation and build communities of practice for active knowledge exchange and capacity-building [7,9,10]. However, there are also some common downsides to online adaptation platforms. The downsides of online adaptation platforms are often connected to diverting expectations of users, builders, and funders. A typical problem is that online platforms relate to a specific project and funding horizon; once the project is over and funding stops, information is not updated anymore, and information will ultimately be out of date. Moreover, many platforms need a long time to be designed and built (on average 3-4 years), especially if they aim to function as decision support tools [10]. This can be problematic because the institutional context may have changed by the time the website is up in the air [10]. Particularly in a field such as climate adaptation, regulatory and legislative frameworks are very fluid and can change easily [18]. Users' needs are often not well-understood and hardly evaluated [10]. In one of the few studies focusing on the users' perspective, Hewitson et al. (2017) [11] (p. 16) come to the conclusion that "all climate information websites 'grossly overestimate the ease of use'". A second pitfall relates to the interactive element of online platforms and the strive to build an online community of practice. Building and maintaining such a community is very resource-intensive, as it requires getting to know the users, engaging with them directly, and regularly tracking how this use is evolving [9]. Commonly, online platforms aim at involving practitioners to make the platform's content more relevant to a wide audience, thereby increasing the frequency of visits and active engagemen<sup>t</sup> of users. However, as pointed out above, practitioners often lack the time to actively engage on such a platform because of competing demands. Research has shown that o ffline interactions next to online activities help in advertising the platform, building user confidence and trust in its content, and facilitating the co-production of knowledge [9,10]. A third pitfall relates to the aspect of inclusivity. The inclusivity of online platforms is based on the idea that everyone has access to the internet. However, access to the internet varies in di fferent geographical contexts. Broadly speaking, only one-third of the people in developing countries are online compared to two-thirds in the developed world, and even in developed countries, internet access is much better in urban areas than in rural areas [9,19–21]. The di fference in internet access to climate adaptation platforms between developed and developing countries is particularly striking, bearing in mind that developing countries are likely to su ffer more from climate change than developed countries [6,9,22]. Another important point relates to the actual usage and users of climate adaptation platforms. In one of the few studies carried out about the users of what they call "climate knowledge brokering platforms", Hammill et al. (2013) [9] conclude that research-oriented users dominate on these platforms, and that policy-makers, media representatives, and local level actors are often not actively engaging with these platforms. Therefore, Hammill et al. (2013) [9] (p. 90) warn that online adaptation platforms should not become "essentially online spaces established and managed by researchers for researchers in relatively privileged settings". To conclude, online adaptation platforms are generally held to be a promising tool for knowledge exchange, raising awareness, and building capacity. However, the literature review above also shows that there are three main concerns, and hence research avenues, related to online platforms: (1) Many online platforms are often only "active" for a certain period (i.e., the timeframe of a certain project and related funding), thereby running the risk that the content becomes outdated. (2) Little is known about the actual contributors and users of online platforms and how inclusive these platforms actually are. (3) Creating and maintaining a well-functioning community of practice is a common goal, but it is di fficult to achieve. In this paper, we will shed light on these points by turning to the special case of ClimateScan. The bottom–up approach that ClimateScan has adopted o ffers us the opportunity to analyze what happens if you enable users to create and update content. More specifically, we can analyze what is being uploaded, who contributes to the platform for what reason, and how this could shape an active community of practice. #### 3. Methodological Approach to Evaluate the ClimateScan Platform The research avenues identified above have informed a two-fold evaluation of the ClimateScan platform, analyzing (1) the content of the platform and how it has evolved over time, and (2) the users and the community of practice and how this developed over time. For this analysis, we have applied multiple qualitative methods. First and foremost, we draw on participatory observations from one of the authors, who has initially set up the platform and remained involved throughout. These participatory observations stem from the author's function as a moderator of the website and direct interactions with ClimateScan users. The personal relations with the ClimateScan top users (those who have uploaded 10 or more projects) have helped us to classify which sector they come from (academia, government, business, civil society) and what drives these top users to contribute. That way, we could reflect on the inclusivity of the ClimateScan platform and users' motivations to participate in the ClimateScan community. To ensure reflexivity, the other author, who has not been involved from the beginning, interviewed the participating author on several occasions. To further substantiate the observations, we have used two additional types of data. The first one is the ClimateScan database itself, which provides information about what has been uploaded, by whom, and when. A content analysis of the database has enabled us to distill an overview of the types of NBS that are uploaded and their geographical distribution. Moreover, by creating user profiles analyzing who has uploaded how many projects over time, we could assess the level of involvement of participants. Assessing the level of involvement of participants was important to draw preliminary conclusions on the aspect of community-building, which were then supplemented by the participatory observations mentioned above. Last but not least, we have used data from Google Analytics to better understand how many people visit ClimateScan and how this has developed over time. That way, we could assess the overall popularity of the website and which factors have increased its usage. Moreover, with the data from Google Analytics, we could generate an overview of the projects that are visited most on the ClimateScan platform. By analyzing what these projects have in common, we could better understand what makes a project appealing to a wider audience. As a corollary, we gained insights for the further development and improvement of ClimateScan. #### 4. An Introduction to ClimateScan ClimateScan's history can be described as an evolution from a research-driven website about Sustainable Urban Drainage Systems to a platform where anyone can register and contribute examples of nature-based solutions on an interactive map. ClimateScan was originally set up in 2014 in the context of a PhD thesis called "Stormwater characteristics and new testing methods for certain sustainable drainage systems in The Netherlands" [23]. In order to make the research transparent and publicly available, the website showed the locations of the NBS that have been studied alongside photos and video footage of a new testing method ("full-scale testing", see [24]) which had been used to study the performance of these specific NBS after they have been in place for a few years. This information was shared with other international researchers as a means to achieve 'international knowledge exchange' (Boogaard, 2015: 119). When the website gradually received more attention and positive feedback from practitioners and other researchers, the recognition grew that ClimateScan could be a powerful tool to showcase also other best practices of climate adaptation and urban resilience. This has led to an opening of the website in 2016, so that anyone could register, download an app, and contribute examples of "blue-green" projects around the globe. ClimateScan is not the only platform with such an interactive mapping function (other examples in the same field are ClimateADAPT, OPPLA, and NATURVATION). However, ClimateScan is unique in its organization and focus. Organizationally, ClimateScan has been developed in a "learning by doing way". The platform started out without a clear set of rules about what should be uploaded and how it should be described. Instead, suggestions made by users have been used to improve the platform. To give an example, in the beginning, the entry of a data point could only be assigned to one category (e.g., "Water"). Nowadays, a datapoint can be assigned to several categories (e.g., "Water" and "Heat", which better shows the multiple benefits of NBS. Overall, the focus of ClimateScan is rather small-scale and design-oriented. Many examples on ClimateScan refer to a specific object, e.g., the design of a roundabout with a water storage function. The focus on design implies that ClimateScan does not provide detailed decision support but rather serves as a source of inspiration. ClimateScan showcases many design examples that lend themselves to be easily copied elsewhere. The addition of the function "show related projects" implies that a visitor interested in one example can immediately find similar examples. As such, ClimateScan attempts to be a "solutions broker" [25]. Moreover, because of its origin as a research-driven website, ClimateScan links to location-specific research outcomes wherever possible [26]. Since 2014, several channels—online and offline—have been used to promote and disseminate ClimateScan (see Table 1). ClimateScan makes use of social media, has been linked to other climate adaptation portals, and uses "offline" events called ClimateCafé's to "recruit" new users. ClimateCafés can be characterized as pressure-cooker workshops in which young professionals and practitioners come together for a few days to gather factual data about the vulnerabilities and (potential) solutions of a defined urban or rural area. ClimateScan is a fixed element in each ClimateCafé to scan the study area for existing nature-based solutions. Every ClimateScan workshop results in more datapoints on the map and further stimulates discussions on the type of nature-based solution, its benefits and local characteristics, and governance aspects related to ownership, performance, and maintenance (for an example, see [27]). Table 1. Promotion and dissemination channels of ClimateScan. #### 5. Results from the ClimateScan Evaluation #### 5.1. Content of ClimateScan: From (Bio)swales to Tornado Trails A general overview of the interactive map shows that datapoints have been submitted on every continent, which makes ClimateScan a global platform (see Figure 1). However, due to its origin in The Netherlands, most projects have been submitted in The Netherlands and Western Europe. Other hotspots are Australia, South Africa, and the Philippines. While the magnitude of datapoints in South Africa and the Philippines was actively stimulated through international projects and ClimateCafés taking place there, the Australian hotspot can be explained by a more coincidental meeting. Through presentations about ClimateScan on international conferences, contact was established with someone in Australia who has already had a personal database with Australian projects about rainwater harvesting and bioretention, which he then uploaded to ClimateScan. This coincidental meeting has actually resulted in mutual visits and fieldtrips to see Dutch and Australian nature-based solutions. Although anecdotal, it shows that the idea of mapping nature-based solutions can unite people and strengthen knowledge exchange. Analyzing the types of nature-based solutions that have been submitted (Figure 2) shows that the biggest categories are bioswales, green roofs and walls, and the Australian measures mentioned above. Bioswales are unsurprisingly the biggest category, as this is the type of measure ClimateScan started with (see history) and is a key interest of the founder of the website and his network. A key design principle of ClimateScan is to be as open as possible, which also means that users can create their own categories. The choice for openness over data accuracy has been made consciously, as ClimateScan is more about stimulating people to upload examples instead of creating a perfect database. As mentioned, ClimateScan has a bottom–up approach and offers the opportunity to analyze what happens if you enable users to create and update content, which is illustrated in the next paragraphs. Figure 1. Geographical distribution of ClimateScan datapoints (source: climatescan.org). Figure 2. Overview of nature-based solutions uploaded to ClimateScan, to keep the figure readable only categories with more than 100 projects are depicted (based on ClimateScan database). Moreover, the openness also has the advantage that it leads to surprising content. For example, a more recent example is the category "tornado trails", where users follow the traces of storms and the damage they leave in the landscape in The Netherlands. Looking at the Dutch content in particular also shows that the content uploaded on ClimateScan evolves with the climate adaptation debate. In the beginning, most measures uploaded were related to water storage, as flooding has always been a major concern in The Netherlands. Later, the positive impact of nature-based solutions such as green roofs and green walls on heat stress was emphasized more strongly, as The Netherlands suffered from several heatwaves in a row (visible in the naming and categorization of projects, the request by users to be able to assign more than one category to an example). The new category "tornado trails" shows that storms and wind have gained importance in the debate. The list with the top 15 most visited projects on the ClimateScan platform (see Table 2) includes some very known examples (e.g., waterquares in Rotterdam), but also some rather unknown examples (e.g., swale in Dalfsen). Projects that are uploaded for a longer period have a higher change to be in the top 15 such as the Swale in Haren that was the second project uploaded to the database (last column of Table 2). Hence, ClimateScan also provides a platform for small-scale initiatives and municipalities that usually do not ge<sup>t</sup> so much attention. All the projects have in common that they have good visual content, mostly with photos and video footage. For example, the most visited project, the gully free road in the small town Nieuwleusen, includes a video about how the system functions during a heavy rain event (T100). To sum up, over the years, ClimateScan had to find a balance between being open and low-threshold to stimulate the upload of examples on the one hand and data maturity on the other. Sometimes, datapoints only include a short text description or only a photo. There is a check on the data quality by the admins, scanning aerial photos and searching the web to validate the datapoint and accompanying information. If possible and in reach, the datapoint is also being visited. In recent years, only few datapoints had to be removed because they included false information or were not meant seriously. #### 5.2. Users of ClimateScan: Many Registered Users, But Only a Small, Yet Diverse Active Community Over the years, the attention for ClimateScan has grown, as the timeline with the number of visitors per day shows (see Figure 3). The platform receives on average 100 visits per day, with the highest peak of 1326 (unique) visitors on one day in February 2019 (a day with a university workshop where students were giving an assignment to use ClimateScan as a source for NBS). Figure 3. Daily and total visits of ClimateScan (based on data from Google Analytics). Land 2021, 10, 5 Table 2. Cont. Table 2. Cont. #### Land 2021, 10, 5 On 24 September 2020, Climate Scan had 806 registered users, and the numbers keep growing. Some of these users can be subtracted because their registration details show clear signs of bots. Of the remaining 766 registered users, only 233 have actively contributed content to the ClimateScan platform (see Table 3). The data show that people register after ClimateScan has been presented on a conference or a meeting. Hence, the story of the platform seems to spark people's interest, but they feel not involved enough to actually contribute. Table 3. Level of involvement of registered users (based on data from ClimateScan database). While many users can be linked back to a particular event or project, there are also several users that can be considered unexpected users: for example, a user uploading two green roofs in Moscow, although there have never been any links to Russia. Another unexpected user in The Netherlands contributes a rather unconventional solution from his own property: a trampoline with water storage, with a sketch explaining the technical details of how it works (see Figure 4). The Canadian citizen initiative "Depave Paradise" has reached out through social media and subsequently submitted several projects that they have worked on. These projects concerned a transformation of places dominated by asphalt and stones into green spaces, with a "before" and "after" illustration for each project. The online communication with this initiative revealed that they presented themselves on ClimateScan to ge<sup>t</sup> in touch with similar initiatives in Europe. Figure 4. (a)Trampoline with water storage, technical sketch (source: climatescan.org) and (b) photo of the actual implementation. Depave Paradise belongs to the 25 top users that have uploaded 10 or more projects. For the majority of these top users, there has been offline contact first (e.g., through a conference, a project or a meeting), which has stimulated the person to register and contribute to the map. As a result of this offline contact, we could also classify from which sector these top users are coming (see Figure 5). Figure 5. Classification of 25 top users in quadruple helix, with two people having a double function "academia" and "business", and one person having a double function "government" and "business". Left chart including students, right chart excluding students. Although most of the users can be related to academia, as Hammill et al. (2013) [9] found in their study, there are also quite a few top users that come from business parties or local governments. The distribution between academia, governmen<sup>t</sup> and business parties is actually rather even, when students are excluded who usually contribute to ClimateScan as part of an assignment. Only civil society is still rather underrepresented. The low representation of civil society is not surprising, as they have not ye<sup>t</sup> been targeted directly. For business parties, it is an opportunity to present the exact locations of their own innovations in the field (e.g., water harmonica or a particular permeable pavement). Interestingly, only two of the seven top users on ClimateScan coming from business parties use ClimateScan as such; the others also upload other examples simply because they are interested in the topic and experience it as fun to share these examples with a wider audience. The representatives from governmen<sup>t</sup> mostly come from municipalities and like to share examples of nature-based solutions and climate adaptation from their own city (e.g., Nijmegen, Groningen, and Enschede). While motives such as pride and a certain element of competitiveness certainly play a role, we also know from personal conversations with representatives from municipalities in The Netherlands that ClimateScan gives them the opportunity to create an overview of the nature-based solutions in their municipality. Often, municipalities in The Netherlands do not have such an overview, because measures such as NBS cannot easily be included in the usual sewer asset managemen<sup>t</sup> software, which is being used to calculate storage capacity and floodings in the urban areas. Therefore, local governmen<sup>t</sup> officials also use ClimateScan as a free data repository for themselves and in order to draw more attention to these nature-based solutions. The ClimateScan community that is active and shows a high level of commitment is small ye<sup>t</sup> diverse in their backgrounds. ClimateScan has contributed to intensified contacts between some of the top users. For example, some of the top users have actually made appointments to go "treasure hunting" on the weekends, in which they try to find new and innovative examples of climate adaptation. #### 6. Discussions and Conclusions This paper addresses how online knowledge-sharing platforms can stimulate stakeholder engagemen<sup>t</sup> and promote NBS by providing in depth insights into the case of ClimateScan—an innovative, bottom–up approach to map best practices of NBS around the world using citizen science. Based on an analysis of the content, the actual users, and the community behind the ClimateScan platform, we have identified the following potentials and pitfalls of ClimateScan. A typical concern related to online platforms is that many online platforms are only managed actively for a certain period of time and content becomes quickly outdated [10]. A strength of ClimateScan's citizen science approach is that the database and user numbers only keep growing. Moreover, the content of the platform is "adaptive" and does not easily run the risk of being outdated, as users themselves create and update the content of the website with concrete examples of NBS. Since concrete examples are a manifest of their time, the website can also grow with changing terminology or new developments in the debate (see example "tornado trails"). However, the freedom that is given to users in the way they describe and illustrate examples also leads to varying data maturity and a rather heterogeneous database. Some data points are filled with much information and visual materials, whereas others only contain a dot on the map and a rough description. Although some variation in data maturity will always remain with a citizen science approach, better guidance on the website in the form of video tutorials as well as the training of volunteers would improve the overall data quality. To create a more homogeneous and neater dataset, this training could find inspiration in existing classifications of NBS [1–3]. As visual information seems to be most appealing, the addition of photos and video material could be made a requirement during the data submission process. Another concern related to online adaptation platforms refers to the actual users and the inclusivity of online adaptation platforms. Online platforms often tend to have an overrepresentation of researchers and the Global North [9]. ClimateScan certainly shows similar characteristics, with a dominance of uploaded projects from The Netherlands and Western Europe due to its origin and active promotion in The Netherlands. However, looking at the top users and their background shows that ClimateScan is appealing not only to researchers but also representatives from business parties and local governments. ClimateScan has managed to give a platform to small-scale initiatives that are otherwise undocumented and receive hardly any attention. To further increase its usage, ClimateScan should maintain and further build on its "niche function". The focus on small-scale measures such as (bio)swales, green roofs, and green walls and their design makes it low key and easy for registered users to upload examples, as the imagery can speak for itself, and no detailed knowledge of the example is necessary. Last but not least, online adaptation platforms often struggle with creating and maintaining a well-functioning community of practice [9,10]. The openness of the website and active promotion in online and o ffline fora has resulted in quite a diverse and practice-oriented user group. However, although quite an amount of people have registered over the years, people seem to lose interest after a while. Engaging with the ClimateScan community with workshops and social media updates could stimulate long-term engagemen<sup>t</sup> and recruit new users. Here, we can learn from the motivations of top users, which show that mapping can unite people and that mapping together can be a fun activity. Workshops could address di fferent actor groups. Creating teams and using the element of competitiveness (who finds most nature-based solutions in a short amount of time) could be an idea to grow and foster the ClimateScan community in the future. Author Contributions: Conceptualization, B.R.; methodology, B.R. and F.C.B.; software, B.R. and F.C.B.; validation, B.R. and F.C.B.; formal analysis, B.R. and F.C.B.; investigation, B.R. and F.C.B.; resources, F.C.B.; data curation, F.C.B.; writing—original draft preparation, B.R.; writing—review and editing, B.R. and F.C.B.; visualization, B.R. and F.C.B.; funding acquisition, F.C.B. All authors have read and agreed to the published version of the manuscript. Funding: ClimateScan was applied as a tool in the international project WaterCoG (WaterCo-Governance) co-funded by the North Sea Region Programme 2014–2021. Ref: http://waterjpi.eu/jointcalls/joint-call-2015/ funded-projects-under-the-2015-water-jpi-joint-call and https://northsearegion.eu/watercog/. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data available in a publicly accessible repository. The data presented in this study are openly available in climatescan.org. Acknowledgments: We would like to thank everyone who has contributed to ClimateScan over the years. Without the effort of these volunteers, ClimateScan would not be such a lively platform. Moreover, we would like to thank Koen Salemink for his helpful suggestions during the writing process. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:59.431398	11-1-2022 14:27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0092e559-d8e0-493b-a85b-c4691a50c8ac", "url": "https://mdpi.com/books/pdfview/book/3694", "author": "", "title": "Urban Ecosystem Services", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036505824", "section_idx": 63 }
0094d209-0e07-4730-b988-02f5f7cca9c7	# FISHING FOR HUMAN PERCEPTIONS IN COASTAL AND ISLAND MARINE RESOURCE USE SYSTEMS 2nd Edition EDITED BY : Annette Breckwoldt, Wen-Cheng Wang, Hans von Storch and Beate M. W. Ratter PUBLISHED IN : Frontiers in Marine 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 978-2-88945-903-2 DOI 10.3389/978-2-88945-903-2 #### 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. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too. #### Dedication to Quality Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews. Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation. #### What are Frontiers Research Topics? Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! 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] # FISHING FOR HUMAN PERCEPTIONS IN COASTAL AND ISLAND MARINE RESOURCE USE SYSTEMS, 2nd Edition Topic Editors: Annette Breckwoldt, Alfred-Wegener-Institute, Helmholtz Center for Polar and Marine Research (AWI) & Leibniz Centre for Tropical Marine Research (ZMT), Germany Wen-Cheng Wang, National Taiwan Normal University, Taiwan Hans von Storch, Helmholtz-Zentrum Geesthacht Centre for Materials and Coastal Research, Germany Beate M. W. Ratter, University of Hamburg, Helmholtz-Zentrum Geesthacht Centre for Materials and Coastal Research, Germany Early morning on a coastal seaweed farm on Rote Island, East Nusa Tenggara, Savu Sea, Indonesia. Image: Hauke Reuter (ZMT). Human perceptions, decision-making and (pro-) environmental behaviour are closely connected. This Research Topic focuses on bringing together perceptions and behaviour for sustainable coastal and island marine resource use systems. Management and governance of (large and small-scale) coastal marine resource use systems function in highly complex social and ecological environments, which are culturally embedded, economically interest-led and politically biased. Management processes therefore have to integrate multiple perspectives as well as perception-driven standpoints on the individual as well as the decision-makers' levels. Consequently, the analysis of perceptions has developed not only as part of philosophy and psychology but also of environmental science, anthropology and human geography. It encompasses intuitions, values, attitudes, thoughts, mind-sets, place attachments and sense of place. All of these influence human behavior and action, and are collected or are available within the respective marine resource use system, which may support the livelihood of a large part of the local population. Management and governance are not only about mediating between resource use conflicts or establishing marine protected areas, they deal with people and their ideas and perceptions. Understanding the related decision-making processes on multiple scales and levels hence means much more than economically assessing the available marine resources or existing threats to the associated system. Over the past decade, there has been a growing inter- and transdisciplinary international community becoming interested in research which integrates perceptions of coastal and inland residents, local and regional stakeholder groups, as well as resource and environmental managers and decision-makers. By acknowledging the importance of the individual perspective and interest-led personal views, it became obvious how valuable and important these sources of information are for coastal research. An increase of research effort spent on the link between perceptions and behaviour in marine resource use systems is thus both timely and needed. By offering a diversity of inspiring and comprehensive contributions on the link between perceptions and behaviour, this Research Topic aspires to critically enlighten the discourse and applicability of such research for finding sustainable, locally identified, anchored and integrated marine resource use pathways. Publisher's note: In this 2nd edition, the following article has been updated: Fabinyi M, Barclay K and Eriksson H (2017) Chinese Trader Perceptions on Sourcing and Consumption of Endangered Seafood. Front. Mar. Sci. 4:181. doi: 10.3389/fmars.2017.00181 Citation: Breckwoldt, A., Wang, W.-C., von Storch, H., Ratter, B. M. W., eds. (2019). Fishing for Human Perceptions in Coastal and Island Marine Resource Use Systems, 2nd Edition. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-903-2 # Table of Contents 05 Editorial: Fishing for Human Perceptions in Coastal and Island Marine Resource Use Systems Annette Breckwoldt, Beate M. W. Ratter and Wen-Cheng Wang 08 Complexities and Uncertainties in Transitioning Small-Scale Coral Reef Fisheries Pierre Leenhardt, Matthew Lauer, Rakamaly Madi Moussa, Sally J. Holbrook, Andrew Rassweiler, Russell J. Schmitt and Joachim Claudet 17 Stakeholder-Informed Ecosystem Modeling of Ocean Warming and Acidification Impacts in the Barents Sea Region Stefan Koenigstein, Matthias Ruth and Stefan Gößling-Reisemann 30 Bridging for Better Conservation Fit in Indonesia's Coastal-Marine Systems Samantha Berdej and Derek Armitage Jokim V. Kitolelei and Tetsu Sato 91 Oceans of Discourses: Utilizing Q Methodology for Analyzing Perceptions on Marine Biodiversity Conservation in the Kogelberg Biosphere Reserve, South Africa Kristin Hagan and Samantha Williams Robert E. Katikiro and Jairos J. Mahenge 131 The Role of Perceptions for Community-Based Marine Resource Management Katharina Beyerl, Oliver Putz and Annette Breckwoldt 148 Multiple Drivers of Local (Non-) Compliance in Community-Based Marine Resource Management: Case Studies From the South Pacific Janne R. Rohe, Shankar Aswani, Achim Schlüter and Sebastian C. A. Ferse 162 Chinese Trader Perceptions on Sourcing and Consumption of Endangered Seafood Michael Fabinyi, Kate Barclay and Hampus Eriksson # Editorial: Fishing for Human Perceptions in Coastal and Island Marine Resource Use Systems Annette Breckwoldt <sup>1</sup> \, Beate M. W. Ratter <sup>2</sup> and Wen-Cheng Wang<sup>3</sup> <sup>1</sup> Alfred-Wegener Institute, Helmholtz Centre for Polar and Marine Research (AWI), Bremerhaven, Germany, <sup>2</sup> Department of Human Dimensions in Coastal Areas, Institute of Coastal Research, Helmholtz Zentrum, Geesthacht, Germany, <sup>3</sup> Department of Geography, National Taiwan Normal University, Taipei, Taiwan* Keywords: perceptions, marine resource use, qualitative research, decision-making process, coastal communities, stakeholder interactions #### Editorial on the Research Topic #### Fishing for Human Perceptions in Coastal and Island Marine Resource Use Systems This Research Topic focuses on bringing together human perceptions and activities for sustainable coastal and island marine resource use systems. The initial idea of this topic formed by an increasing awareness that human perceptions, individual decision-making and (pro-) environmental behavior are much closer connected than so far acknowledged by academia and the scientific research community in general. Management and governance of (large and small-scale) coastal marine resource use systems function in highly complex social and ecological environments, which are culturally embedded, value-driven, economically interest-led and politically biased. Local action is not least framed by mental contribution and attribution of coasts as places for living, recreation and resource use. Mental constructs of coasts and marine resources as valuable areas can, in some cases, lead to the protection and preservation by initiatives of collective action, and in other cases, it is difficult to mobilize local communities to adapt and to engage in environmental management strategies (Ratter et al., 2016). Management processes therefore ought to integrate multiple perspectives as well as perception-driven standpoints on the individual as well as the decision-makers' levels. Consequently, the analysis of perceptions has developed not only as part of philosophy and psychology but also of environmental science, anthropology and human geography. It encompasses intuitions, values, attitudes, thoughts, mind-sets, place attachments and sense of place. All of these influence human behavior and action, and can be collected or are available within the respective marine resource use system. Often, these systems support the livelihood of a large part of the local population. The 12 articles in this Research Topic have been authored by 48 researchers from 10 different countries, presenting critical insights from across the globe—from small islands in the South Pacific to Sri Lanka, China, South Africa, Norway or Uzbekistan. The institutions from the submitting authors range from Universities to think tanks, to research centers, or Non-Governmental Organizations—and the authors themselves were at very different stages in their career (from very early to senior researcher). The diversity of the professional backgrounds (Geography, Environmental Psychology, Ecology, Sociology, and Anthropology) shows in essence that researching perceptions in (not only marine) natural resource use systems does not have #### Edited and reviewed by: Annette Cameron Broderick, University of Exeter, United Kingdom > \Correspondence: Annette Breckwoldt [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 23 January 2018 Accepted: 09 February 2018 Published: 22 February 2018 #### Citation: Breckwoldt A, Ratter BMW and Wang W-C (2018) Editorial: Fishing for Human Perceptions in Coastal and Island Marine Resource Use Systems. Front. Mar. Sci. 5:62. doi: 10.3389/fmars.2018.00062 5 a distinctive disciplinary boundary. All of the articles are interdisciplinary, and many also have transdisciplinary approaches, employing mainly qualitative approaches (partly in combination with quantitative methods), to face the challenge of uncovering, exploring and analyzing the perceptions of a variety of stakeholders in marine resource use systems, from traders to conservation managers. The stories told by these articles are indeed just as diverse, from cases of individual meaning-making and highlighting the power of individual perceptions (Beyerl et al.; Siriwardane-de Zoysa and Hornidge) to collective action around the status of fishery resources (Leenhardt et al.) or the persistence of destructive fishing practices (Katikiro and Mahenge). Some of the articles showcase methods for perception research, such as the Q-methodology (Hagan and Williams) or stakeholderinformed modeling (Koenigstein et al.), allowing to quantify these "qualitative subjectivities" in ways readable for machines as well as human decision-makers. All articles have a critically high level of contextuality, and show how perceptions can have an impact on decision-making processes within the science-stakeholder "entanglement." The roles of perceptions in understanding and optimizing the usefulness of management measures, conservation projects and bridging organizations for better and more sustainable conservation solutions are among the less hidden examples (Berdej and Armitage; Beyerl et al.; Hagan and Williams). Understanding aspects of leadership perceived and factual—responsibilities and legitimacies (of persons or activities and functions; Katikiro and Mahenge; Rohe et al.; Sutton and Rudd), very clearly show the requirement of in-depth research on perceptions. Oftentimes, these aspects of leadership, the complexities and confusions linked to it and individual decisions (Gorris), are often based on very tangible/conventional matters such as economic benefit (Fabinyi et al.; Gorris), but are also tightly linked to aspects of transparency, lack of trust (including perceived and real threats; Katikiro and Mahenge), and enforcement (Gorris; Rohe et al.). One aspect that may not be underestimated, but still underresearched, is the role of local (individual and subjective as they may be) perceptions on the transformation and governance of larger coastal areas (Katikiro and Mahenge; Kitolelei and Sato) and even more distant environments (Fabinyi et al.). The articles also show that a strong perception-based impact exists across all levels of such processes, from the individual resource-user, to larger societal and professional networks. This Research Topic show-cases the need for a larger recognition in academia that management and governance are not only about mediating between resource use conflicts or establishing marine protected areas. Moreover, they deal with people and their ideas, knowledge and perceptions (Bennett and Dearden, 2014; Wyles et al., 2014; Bennett, 2016; Gelcich and O'Keeffe, 2016; Ratter et al., 2016; Hoshino et al., 2017). And understanding the related decision-making processes on multiple scales and levels means indeed more than economically assessing the available marine resources or existing threats to the associated system. The editors trust that this Research Topic adds substance and visibility to the growing body of research and literature that presents an integration of perceptions of island, coastal and inland residents, local and regional stakeholder groups, as well as resource and environmental managers and decisionmakers. In conclusion—by acknowledging the importance of the individual perspective and interest-led personal views, it becomes obvious how valuable and important these sources of information are for coastal research. An increase of research effort and academic discourse spent on the link between perceptions and behavior in marine resource use systems is thus both timely and needed, and is in fact observable by the increasing number of perception-related publications. The challenges to find, to analyse and to publish studies on perceptions remains, but by raising their academic profile, authors of articles such as published in this Research Topic should find a different, more visible platform—for a publication- and dialogue-based sharing of questions and findings. By fishing for a diversity of inspiring and comprehensive contributions on the link between perceptions and behavior, this Research Topic shall critically enlighten the discourse and applicability of such research for finding sustainable, locally identified, anchored and integrated marine resource use pathways. ## AUTHOR CONTRIBUTIONS All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. ## ACKNOWLEDGMENTS We would like to express our sincere gratitude to all peerreviewers for their incredible work in effort and time spent to guide these contributions to publication. We would also like to thank our colleague Prof. Hans von Storch for joining us in this effort as a valuable advisor. Finally, we would like to thank the entire editorial team at Frontiers in Marine Science for their accurate, fast and always friendly input, support and feedback. #### REFERENCES impacts, governance and management in Thailand. Mar. Policy 44, 107–116. doi: 10.1016/j.marpol.2013.08.017 resource management in the Kei Islands, Indonesia. Front. Mar. Sci. 4:141. doi: 10.3389/fmars.2017.00141 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 Breckwoldt, Ratter and Wang. 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. # Complexities and Uncertainties in Transitioning Small-Scale Coral Reef Fisheries Pierre Leenhardt 1, 2 , Matthew Lauer <sup>3</sup> , Rakamaly Madi Moussa1, 2, Sally J. Holbrook 4, 5 , Andrew Rassweiler 5, 6, Russell J. Schmitt 4, 5 and Joachim Claudet 1, 2 \* <sup>1</sup> National Center for Scientific Research, Centre de Recherches Insulaires et Observatoire de l'Environnement (CRIOBE), USR 3278 Centre National de la Recherche Scientifique-EPHE-UPVD, Perpignan, France, <sup>2</sup> Laboratoire d'Excellence CORAIL, Perpignan, France, <sup>3</sup> Department of Anthropology, San Diego State University, San Diego, CA, USA, <sup>4</sup> Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA, USA, <sup>5</sup> Marine Science Institute, University of California, Santa Barbara, CA, USA, <sup>6</sup> Department of Biological Science, Florida State University, Tallahassee, FL, USA #### Edited by: Wen-Cheng Wang, National Taiwan Normal University, Taiwan #### Reviewed by: John A. Cigliano, Cedar Crest College, USA Simon Foale, James Cook University, Australia #### \Correspondence: Joachim Claudet [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 04 February 2016 Accepted: 25 April 2016 Published: 13 May 2016 #### Citation: Leenhardt P, Lauer M, Madi Moussa R, Holbrook SJ, Rassweiler A, Schmitt RJ and Claudet J (2016) Complexities and Uncertainties in Transitioning Small-Scale Coral Reef Fisheries. Front. Mar. Sci. 3:70. doi: 10.3389/fmars.2016.00070 Coral reef fisheries support the development of local and national economies and are the basis of important cultural practices and worldviews. Transitioning economies, human development, and environmental stress can harm this livelihood. Here we focus on a transitioning social-ecological system as a case study (Moorea, French Polynesia). We review fishing practices and three decades of effort and landing estimates with the broader goal of informing management. Fishery activities in Moorea are quite challenging to quantify because of the diversity of gears used, the lack of centralized access points or markets, the high participation rates of the population in the fishery, and the overlapping cultural and economic motivations to catch fish. Compounding this challenging diversity, we lack a basic understanding of the complex interplay between the cultural, subsistence, and commercial use of Moorea's reefs. In Moorea, we found an order of magnitude gap between estimates of fishery yield produced by catch monitoring methods ( 2 t km ∼ <sup>−</sup><sup>2</sup> year−<sup>1</sup> ) and estimates produced using consumption or participatory socioeconomic consumer surveys (∼24 t km−<sup>2</sup> year−<sup>1</sup> ). Several lines of evidence suggest reef resources may be overexploited and stakeholders have a diversity of opinions as to whether trends in the stocks are a cause for concern. The reefs, however, remain ecologically resilient. The relative health of the reef is striking given the socio-economic context. Moorea has a relatively high population density, a modern economic system linked into global flows of trade and travel, and the fishery has little remaining traditional or customary management. Other islands in the Pacific in similar contexts in Polynesia such as Hawaii, that continue to develop economically, may have small-scale fisheries that increasingly resemble Moorea. Therefore, understanding Moorea's reef fisheries may provide insight into their future. Keywords: social-ecological systems, small-scale fisheries, coral reef fisheries, transitioning economy, catch monitoring, fishery yield, complexity, resilience ## INTRODUCTION Coral reef fisheries are vital to millions of people dwelling along the world's coasts (Johnson et al., 2013; Teh et al., 2013; Cinner, 2014). They support the development of local and national economies by providing food, income, and employment, and also are the basis of important cultural practices and identities. Yet the coral reefs upon which these fisheries depend are some of the globe's most threatened coastal systems (Mumby and Steneck, 2008). Until recently, coral reefs worldwide demonstrated the capacity to return to coral dominance following perturbations that cause landscape-scale loss of coral, such as cyclones and bleaching (Jackson, 1992; Pandolfi and Jackson, 2006). In the past two decades, however, a growing number of studies have documented cases where major perturbations cause long-lasting and potentially irreversible ecosystem shifts, one of the most common being a shift from a coral-dominated to a macroalgaedominated state (Hughes, 1994; Shulman and Robertson, 1996; Hobbs et al., 2006; Rogers and Miller, 2006; Bruno et al., 2009). The dynamics of these state shifts are fundamental to understanding long-term sustainability of coral reefs and the fisheries that depend on them, yet the interacting human and ecological dynamics, including fisheries, that underpin coral reef resilience are poorly understood (Hughes et al., 2003, 2005, 2010). Most coral reef fisheries are small-scale fisheries in that they involve simple technologies and are either subsistencebased, or supply small local markets, or roadside sellers. Despite their limited technological and economic scope, small-scale fisheries have been identified as a primary threat to coral reefs (Newton et al., 2007). Some studies suggest small-scale coral reef fisheries are experiencing declining fish biomass and size (Cinner J. E. et al., 2009) but the extent, magnitude, and variability of overexploitation is generally not well-documented (Jacquet and Pauly, 2008), and even the number of people involved in such fishing is poorly known (Teh et al., 2013). This paucity of understanding and uncertainty is attributable to the inherent complexity of small-scale coral reef fisheries. They evolve within locally specific social and ecological conditions, making them highly diverse. Variability arises from the diverse set of technologies used for harvesting marine resources, multiple overlapping social, economic, and cultural motivations for fishing, heterogeneous modes of governance, varied stakeholder organization, and complex interactions with other marine-use sectors and governance structures. Here we explore the complexity of coral reef fisheries using Moorea, French Polynesia, as an example. Moorea presents an interesting case study in that economic development and intense exposure to globalization have not undermined the capacity of its coral reefs to recover from perturbations. Extensive, longterm ecological research on Moorea suggests that its reefs are quite resilient to disturbances (Done et al., 1991; Adjeroud et al., 2009; Adam et al., 2011, 2014; Trapon et al., 2011; Lamy et al., 2015, 2016; Galzin et al., 2016). Many Pacific islands have shown declines in the critical adaptive capacities that underpin resilience to environmental variability when they are more exposed to the pressures of globalization and global markets, have higher population densities, and widespread coastal development (Pauly and Chua, 1988; Brewer et al., 2012). Interestingly, Moorea enjoys a higher standard of living than most Pacific islands while its reefs have demonstrated high resilience to environmental perturbation. This may suggest that higher levels of socioeconomic development may reduce dependence on coral reefs and associated human impact (Cinner J. et al., 2009) or may reflect other social or ecological characteristics of the system. We describe Moorea's small-scale coral reef fishery by documenting fishing practices and reviewing uncertainties associated with estimates of effort and landings over the last three decades. Fishing activities on Moorea are widely dispersed, both spatially around the island and temporally throughout the day and the night, making the collection of accurate catch data challenging. Existing statistical data provided by the Territorial government cannot be used for this purpose because they do not effectively assess non-commercial fishing, the most widespread fishing practice on the island. Moreover, we lack a basic understanding of the complex interplay between the cultural, subsistence, and commercial use of Moorea's reefs. Filling these gaps in our knowledge of Moorea's fishery will help enhance the development of marine resource management initiatives that seek long-term sustainability of reef fisheries and foster ecosystem resilience. ## THE SOCIAL-ECOLOGICAL CONTEXT The island of Moorea is surrounded by a barrier reef, broken by 11 passes, enclosing a 49 km<sup>2</sup> lagoon, whose width varies from 500 to 1500 m, with depths of 0.5–30 m (Bell and Galzin, 1984). The island has a marine spatial management plan (Plan de Gestion de l'Espace Maritime, PGEM) initiated in 2004, the first in French Polynesia. The PGEM has four objectives: (1) rational use of resources and sustainable development; (2) managing conflicts for space in the lagoon; (3) controlling pollution and physical damage to marine environments; and (4) protecting marine ecosystems and endangered species. Although the PGEM was carefully developed over a 10 year consultation process, certain segments of the fishing community voiced opposition when it was implemented in 2004 (Aubanel et al., 2013), and it continues to be a source of tension and controversy (Walker, 2001; Gaspar and Bambridge, 2008; Walker and Robinson, 2009). The uses of Moorea's coral reefs have fundamentally changed over the last 100 years. Moorea gradually transitioned during the late nineteenth century from a subsistence economy based on small-scale gardening and fishing to an economy that by the 1940s was based on cash cropping of vanilla and copra. In the 1960s, French military activities drove a burgeoning economy and employment opportunities that drew migrants from other parts of French Polynesia to the capital city, Papeete (Henningham, 1992; Salvat and Pailhe, 2002). These economic changes in French Polynesia influenced Moorea's social-ecological system in several important ways. With regular ferry service established between Moorea and Tahiti, residents of Moorea were able to commute to jobs in Papeete. Moreover, many Papeete residents moved to nearby Moorea or visit the island on weekends and occasionally fish there. Most notably, however, was rapid growth in tourism that progressively became the mainstay of Moorea's economy. In 2011, Moorea was the most visited island in French Polynesia with over 70,000 tourists visiting the island's 22 major hotels and 48 smaller "pensions de famille" (ISPF, 2001). The transition to a tourism-led economy has sustained a level of economic prosperity in French Polynesia, and it continues to be one of the wealthiest Pacific Island nations with a USD 15,272 per capita GDP (Baudchon et al., 2008). An economy that was once dominated by small-scale food production and subsistence fishing was replaced with tourism and service sectors as well as some export-oriented nonindigenous agriculture, such as pineapple. In addition, fishing became just one of many marine-focused activities that include scuba diving and beach and boating activities, each exerting different pressures on the coral reefs and lagoon ecosystem. One important outcome of these transformations was very high population growth, a portion of which was due to immigration from other islands. Census figures indicate that Moorea's population grew from 5058 to 16,893 between 1971 and 2012 (ISPF, 2013)—an annual growth rate of 2.39%, which is higher than the rate for French Polynesia as a whole (1.57%). The effect these demographic changes have had on Moorea's fishery is unknown, but throughout the region fishing pressure appears to be linked to the number of local inhabitants although the relationship is poorly understood (Russ and Alcala, 1989; Jennings and Kaiser, 1998). #### FISHING CATEGORIES Fishing has formed the backbone of Polynesian societies since their initial colonization of the region (Oliver, 1974) and continues to be an integral part of the subsistence economy and Polynesian identity. Today, Moorea's coral reefs directly support two fundamental livelihoods on the island: fishing and tourism. In strict economic terms fishing-based incomes are dwarfed by tourism-based incomes, which stem mostly from tourist accommodations and reef-based recreational activities. A recent economic assessment estimated that recreational activities stemming from Moorea's reefs provided approximately 27 Me/year while fishing activities provided 4Me/year including 2.8 Me value placed on fish not sold but consumed within the fisher's household (Pascal and LePort, 2015). These figures, however, do not capture recreational fishing activities nor the high cultural value of reef fishing in Polynesian society (e.g., enjoyment, identity, prestige, worldview; Cinner, 2014). Polynesian fishing activities can be lumped roughly into three categories: oceanic fisheries, coastal fisheries, and reef (or lagoon) fisheries. Reef fisheries are described as all activities involved in exploiting biological resources and carried out on the fringing and barrier reefs, channels, passes and hoa (small passes not always connected to the ocean) and down to the limits of coral growth (80–100 m depth; Galzin et al., 1989). According to Yonger (2002), Brenier (2009), and Leenhardt (2009) and our own observations, fishing is ubiquitous on Moorea with three broad categories of fishers: commercial fishers, subsistence fishers, and recreational fishers (Table 1). The latter term encompasses fishers not motivated by market imperatives or hunger, but cultural factors. The fisher population is composed of 69% recreational, 20% subsistence, and 11% commercial fishers but the categories are not mutually exclusive (Leenhardt, 2009; Brenier et al., 2014). Over half of the adult population fishes, with the vast majority of households having at least one person who fishes. While subsistence fishers are all Moorea residents, a certain number of commercial and recreational fishers come from the nearby Society Islands, mainly Tahiti (Leenhardt, 2009). It should be noted that nearly 70% of the people who fish on Moorea are recreational fishers, yet none of the catches from this category of fisher appear in the fisheries data collected in market surveys (Figure 1). Moreover, recreational fishing may account for 58% of the catches in the lagoon (Yonger, 2002), yet, a percentage of those catches are never recorded because they are directly destined for home consumption or shared among family or other community members. In addition, roadside sellers reported keeping a very small part of their catch on average for household consumption. ## CAUGHT SPECIES A diverse suite of species is targeted by fishing in Moorea's reefs. More than 40 genera of fishes can be found sold by the roadside. Three groups are caught most frequently: Iihi (soldierfish, Myripristis spp.), Paati (parrotfish, mixed species smaller than 50 cm; mostly Scarus spp. and Chlorurus spp.), and Ume (unicornfish, Naso spp.; Table 2). ## FISHING GEAR TYPES The wide diversity of species caught in part reflects the many fishing techniques that are employed, each adapted to specific suites of organisms. Given the many different techniques, individual fishers often use a multifaceted approach, using several techniques depending on their preferences and resources, on the frequency of fishing, season, weather conditions, target species, and time of day. The main gear types used in the lagoon are spear guns, lines (handlines, hook-and-line), nets (gillnets or nets with pot traps), harpoons, beach seines, and trolling (Yonger, 2002; Leenhardt, 2009; Brenier et al., 2014). Although a wide variety of fishing methods are used, spearfishing dominates the (commercial) roadside catch: sellers reported that a large majority of biomass had been taken by spear gun with the remainder split equally between hook and line and net fishing. Spearfishing occurs both during the day and at night with battery-powered torches. Night spearfishing is very effective, providing high yields per fishing trip. It accounts for about 29% of lagoon fish production in the Windward Islands (which include Moorea) as compared to 18% in the Leeward Islands (SPE, 2007). Night spearfishing is very selective but can lead to local overexploitation of stocks because most targeted species (80%) are non-migratory and tend to be confined to a specific habitat during the night (Lecaillon et al., 2000). Line fishing is done directly from the coastline or from small vessels #### TABLE 1 \| Classification and characteristics of fishers on Moorea. FIGURE 1 \| Reef fish sold along the roadside on Moorea. Boards in background are used by scientists to estimate sizes (Images: R. Madi Moussa). powered by oars or 2–25 hp outboard motors. The different line fishing techniques include trolling, bottom longlining, fishing with artificial lures, using lines with one or more hooks, and fishing with natural and live bait. Nets can take a wide variety of forms: gillnet fishing; beach seine net fishing (used seasonally on bay floors to catch bigeye scad, Selar crumenophthalmus); funnel net fishing that includes a wire net that targets parrotfish, trevallies, surgeonfish, and goatfish; and cast nets and scoop nets, which are used to catch flying fish. Fish traps are widely used in the Tuamotu and Leeward Islands, where they can account for 90% of catches (Galzin et al., 1989), but are not used in Moorea's lagoon. #### YIELD ESTIMATES A variety of studies in the past three decades have attempted to assess fish production (Galzin, 1985) or reef fishery yields (Aubanel, 1993a; Yonger, 2002; Brenier et al., 2014) on Moorea (Table 3). As in other coral island settings, quantifying reef fisheries yield has proven to be a particularly difficult exercise for many reasons. Fishing is often done at night (with or without a boat), is widely dispersed, uses many different types of gear, and landings and sales do not take place at specific sites but rather anywhere along the coast, often on private stretches of coastline (Figure 1). Research methodologies used between the times of Galzin (1985) and Brenier (2009) have also evolved considerably. Over a period of three decades, five different studies attempted to evaluate Moorea's lagoon fishery yield, and only two studies used the same methodology (Aubanel, 1993a; Vieux, 2002), leading to a wide range of production estimates for Moorea's coral reefs. Yield estimates based on catch data provide relatively low figures (from 0.7 to 2.2 t km−<sup>2</sup> year−<sup>1</sup> ), while, by contrast, data from consumption surveys or participatory surveys estimate fishing yields an order of magnitude higher (from 20 to 25 t km−<sup>2</sup> year−<sup>1</sup> ). #### Monitoring Catches, Landings, and Sales Built in 1987, the Paopao market was once the single official point of sales where all fishers from the north side of the island were supposed to sell their fish. The centralized fish market was the result of a regulation that prohibited the sale of fish at roadside stands, although compliance with the law was low and eventually the Paopao market ceased to operate (Aubanel, 1993a). Galzin et al. (1989) estimated catches based on the tax the township levied on the fish sold at the Paopao market. Aubanel (1993a) estimated production and total catch based on fish sold both at roadside stands and at the Paopao market. These estimates were made by counting tuis, a string from which a collection of fish are hung, often of different species and sizes, and the unit by which fish are offered for sale (Figure 1). Vieux (2002) used the same protocol counting only roadside tuis (the market was closed by that time) to assess potential changes in total catch. There were some methodological weaknesses of these assessments that most likely led them to underestimate fishery production. The tax-based approach (Galzin et al., 1989) did not account for non-market based sales (roadside sales and direct sales based on contracts), that were estimated afterwards to Data from: Galzin et al., 1989; Aubanel, 1993b; Vieux, 2002; Yonger, 2002; Brenier, 2009; Kronen et al., 2009; Madi Moussa, 2010. TABLE 3 \| Yield estimates per surface area unit by type of survey. be about 60% of the total catch (Vieux, 2002). Yield estimates based solely on roadside surveys (Aubanel, 1993b; Vieux, 2002) were most likely underestimated for the same reasons. However, and although done 10 years apart, those two assessments based on roadside surveys led to similar yield figures. Surprisingly, the tax-based study done 10 and 20 years earlier, respectively, led to estimates in the same range, suggesting that the market oriented reef catches were similar for the various fishing/selling categories. Although the spatially dispersed nature of landings makes quantifying fish catches difficult, monitoring roadside sales can be an excellent way of discerning spatial patterns of fishing pressure (fish are typically sold in roadside stands near to where the fish were caught) as well as the species and sizes of the fish sold (Figure 1; Madi Moussa, 2010). #### Consumption Surveys An analysis of seafood consumption can be a good alternative for indirectly assessing fishery production (Paddon, 1997; Gilbert, 2006; Labrosse et al., 2006). This method requires a welldefined study area with low quantities of imported or exported reef and lagoon fish. On Moorea, catch exports are limited to recreational fishers who come from Tahiti on the weekends and the importation of fish is negligible, with only small amounts of pelagic fish from Tahiti or the Tuamotu Islands being brought to the island (Leenhardt, 2009; Brenier et al., 2014). On Moorea, annual consumption is nearly 110 kg per inhabitant (Yonger, 2002), far above the 23 kg per inhabitant that is the average annual consumption for the Pacific Islands region (Labrosse et al., 2006; Kronen et al., 2010). The gap between estimates in Moorea and other Pacific Island countries is intriguing and encourages consideration of possible methodological biases. Studies either sampled 5% of Moorea's household population (Yonger, 2002) or concentrated on a village and sampled 12% of its households (Kronen et al., 2010). However these two studies led to similar estimates. We believe a potential source of discrepancy with other similar studies in the region may be due to the fact that residents of Moorea consider leftovers to be a new individual meal (Gilbert, 2006). The one-off nature of the surveys also creates considerable uncertainty in the annual estimates, which were extrapolated from average weekly estimates. The methodology also assumed that eating habits and fishery production remain stable over time (Gilbert, 2006). Fish sizes were generally estimated with gauges, while size and weight conversions were calculated using biometric ratios. Size and weight ratios were not always calculated in a precise manner. In fact, length-weight relationships did not exist for the species studied, so relationships for similar species were used (Gilbert, 2006). Although the information collected from households was quantitative it involved substantial uncertainty because it relied on the short-term memory of the person interviewed and his or her ability to convert an image or a memory into a physical size (Gilbert, 2006). Despite the drawbacks mentioned above, indirect studies based on household seafood consumption surveys offer a good alternative for studying fishery production in small-scale fisheries. In contrast to methods based on landings and sales monitoring, household consumption surveys take into account the catches of all types of fishers, including recreational fishers. They also have been conducted more frequently over the past few years (Yonger, 2002; Lagadec, 2003; Léopold et al., 2004; Kuster et al., 2006). #### Participatory Methods Participatory monitoring of reef fisheries through household surveys can be designed to collect data on consumption and fishing activities from large sample groups and can produce reliable data (Au et al., 2000; Nicholson et al., 2002). On Moorea, fishery production was estimated using surveys by schoolchildren (Brenier, 2009). Surveys consisted of questionnaires designed to gather general information (i) on the household's general fishing activities and fish consumption (including how often fish was eaten, origin of the fish eaten, number of boats and fishers in the household) and (ii) on the number of fishing trips of one fisher in the household over a 2-week period (to cover one spring tide period and one neap tide period) along with (iii) the names, sizes and number of fish eaten at meals over the previous 3 days. These surveys involved 4.4% of total household population and the questionnaire return rate was 68%. The fisher population was estimated at 77 fishers per km<sup>2</sup> , with 1916 ± 530 motorboats and 481 ± 68 fishing trips per km<sup>2</sup> each month (Brenier, 2009). If this calculated fishing pressure is accurate, it is quite high considering that 5 fishers per km<sup>2</sup> is the upper limit at which coral reef resources can be safely exploited (McClanahan et al., 2002). #### PERCEPTIONS OF STOCK STATUS Perception surveys can also serve as a good indicator of fish stock status. On Moorea, perception surveys show mixed results with some indicating that Moorea has experienced a decline in the abundance and size of target fish species, increased scarcity of giant clams, decreased live coral cover, and increased cover of macroalgae (Brenier, 2009), while others suggest heterogeneity in perceptions between communities, with respondents from Afareaitu reporting more marine resource degradation than in southern Ha'apiti and Papetoai. Over the past decade, fishers in most districts report that they are still catching as many fish, yet most agree that their fishing effort has increased (Leenhardt, 2009), although there is some variation between districts. The varied perceptions about the health of fished stocks emphasize the difficulty of using such metrics to infer stock status. Responses are consistent with reefs that are either fully exploited or somewhat overexploited but show no evidence of collapse despite the heavy use. #### DISCUSSION Coral reef fisheries are multifaceted, and when fishers can fish for pleasure, identity, to eat or to sell, yields are very difficult to assess and large uncertainty is common. For instance, in Moorea's reef fisheries, there is considerable uncertainty on the magnitude of the catch or even the status of the stocks being fished. Over a period of 30 years, several studies have attempted to assess fishery production in Moorea's reefs, with nearly every study using a different methodology. Two approaches have yielded an order of magnitude gap between the estimates: ∼2 t km−<sup>2</sup> year−<sup>1</sup> using catch-monitoring methods vs. ∼24 t km−<sup>2</sup> year−<sup>1</sup> using consumption or participatory socioeconomic consumer surveys (Table 2). Market surveys are unable to capture many kinds of fishing activity (e.g., recreational fishing, fishing for household consumption, and contract fishing for private clients), so we expect that methods based on these surveys would underestimate fishery production. Methods involving socioeconomic surveys might potentially give more accurate fishery production estimates as they apply to all fish consumed on the island regardless of source, but they rely on recollections of fish recently consumed, rather than on direct observation, introducing other forms of uncertainty. Similar to many small-scale coral reef fisheries, fishery activities in Moorea's lagoon are quite challenging to monitor and quantify because they vary greatly and are quite dispersed. While estimates of actual production are uncertain, the potential sustainable productivity of Moorea's lagoon fisheries is completely unknown. In fact, there may not be any reliable guidelines for the sustainable yield of many of these fish species, as their biomasses have been shown to change by a factor of five or more over time scales as short as a few years during rapid ecological transitions (Adam et al., 2011, 2014). At present, these variations in biomass are not predictable some are driven by pulse disturbance events (Adam et al., 2014), and there is no infrastructure for monitoring stock status that would permit dynamic estimation of sustainable yield. Adding to the complexity is the fact that Moorea's reefs have been subjected to several large perturbations in the past four decades, including in 2008–2010 (Adjeroud et al., 2009; Trapon et al., 2011; Adam et al., 2014; Lamy et al., 2015, 2016). In all cases, the coral community on the fore reef displayed high resilience to perturbation -returning to predisturbed coral cover (∼40–50%) within about a decade without undergoing a shift to high cover of macroalgae (Adjeroud et al., 2009; Trapon et al., 2011; Adam et al., 2014; Lamy et al., 2016) due in large part to herbivorous fishes preventing the establishment of macroalgae on the fore reef (Adam et al., 2011, 2014). Following the recent disturbances, the relative abundances and biomass of species targeted by Moorea's fishers changed, with several key groups of herbivores experiencing large increases. The complexity of Moorea's coral reef fishery, in many ways, is representative of other small-scale coral reef fisheries around the world. A wide diversity of fish is caught with at least five major gear types and fishing occurs during day or night without any regular schedules or formalized protocols. Moorea's fishery, however, becomes more place-specific when we consider the socio-economic context and the motivations that underlie why people fish. Unlike many other small-scale coral reef fisheries in the Pacific and around the world, French Polynesia is a relatively rich country. For this reason Moorea households are not dependent on marine resources for protein or their livelihoods to the same extent as in poorer countries where necessity motivates the harvesting of marine resources. For this reason, only a small percentage of Moorea households identify fishing as their primary source of income or livelihood. Instead, fishing is vitally important for its non-material benefits. The primary motivation for fishing on Moorea is related to an important cultural factor: eating fresh reef fish. For Moorea's inhabitants the consumption of fresh reef fish is as fundamental to their identity as speaking the Tahitian language. It is pivotal to culturally important events such as Church gatherings, birthdays, Sunday feasts, and other events and continues to dominate the local diet. For these reasons, the three categories of fisher subsistence, commercial, and recreational—that are frequently cited in the literature on small-scale fisheries do not fully capture the nature of fishing on Moorea. Culturally motivated fishing, although most similar to recreational fishing, translates into fishing behavior that cannot easily be analyzed within a costbenefit or profit maximization model where the economic value generated by the activity forms the core of the analysis. To more deeply comprehend fishing on Moorea the non-material benefits related to the Polynesian lifestyle and identity must be considered. Given the social and ecological complexity of Moorea's lagoon fisheries, any attempt to understand their dynamics will likely require integrated methods that consider both systems simultaneously. More integrated fieldwork is required to better evaluate the sustainability of the existing fisheries, in which social science techniques are paired with ecological field surveys to understand how fishing behavior depends on ecological state, livelihood opportunities, non-material benefits, cultural cohesion, and personal identity. #### CONCLUSIONS Understanding Moorea's lagoon fisheries is a major challenge, but also an opportunity. In important ways, Moorea may provide a window into the future of many other islands in the Pacific. Although the influence of globalization and economic development will inevitably vary across the Pacific, many Pacific Island nations are undergoing socio-cultural and economic changes similar to Moorea in that their population densities are increasing, their economies are modernizing and becoming more linked to global flows of trade and travel, and their fisheries are no longer managed through traditional marine tenure practices. If other island nations in the Pacific undergo similar changes, their small-scale fisheries may increasingly come to resemble Moorea's, where fishers are less dependent on the marine environment for subsistence or income and more motivated by non-material factors that sustain personal and cultural identity. Despite these changes on Moorea, its reefs are still superficially healthy with high coral cover and abundant fish. We acknowledge Moorea is unique in that it is economically and geopolitically linked to France, but studying Moorea's system might yield insight into how the processes of globalization can be effectively and sustainably navigated both ecologically and socially in similar contexts in Polynesia such as Hawaii. #### AUTHOR CONTRIBUTIONS PL wrote the first draft and all authors contributed substantially to revisions. #### ACKNOWLEDGMENTS We thank Mark Strother Ashley Bunnell, Terava Atgar, and René Galzin for their help in data collection, and CRIOBE staff and Hinano Murphy for facilitating the project and securing research permits. Two anonymous reviewers made constructive comments on an earlier version of this manuscript. This work was supported by the Fondation de France, The Programme Doctoral International: Modélisation des Systèmes Complexes (PDIMSC) from Université Pierre et Marie Curie, the Agence Nationale de la Recherche (ANR-14-CE03-0001-01), the National Science Foundation (NSF OCE 1325652, OCE 1325554, and OCE 1236905). #### REFERENCES Plan de Gestion de L'espace Maritime (PGEM) de Moorea et de sa Gouvernance. CRIOBE, BEST CORAL Reports, Communication for Policy. 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 © 2016 Leenhardt, Lauer, Madi Moussa, Holbrook, Rassweiler, Schmitt and Claudet. 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. # Stakeholder-Informed Ecosystem Modeling of Ocean Warming and Acidification Impacts in the Barents Sea Region Stefan Koenigstein<sup>1</sup> \, Matthias Ruth<sup>2</sup> and Stefan Gößling-Reisemann<sup>1</sup> <sup>1</sup> Sustainability Research Center (artec) and Department for Resilient Energy Systems, University of Bremen, Bremen, Germany, <sup>2</sup> School of Public Policy and Urban Affairs, Department of Civil and Environmental Engineering, Northeastern University, Boston, MA, USA #### Edited by: Beate M. W. Ratter, University of Hamburg, Germany #### Reviewed by: Carolyn J. Lundquist, National Institute of Water and Atmospheric Research, New Zealand Edward Jeremy Hind, Manchester Metropolitan University, UK \Correspondence: Stefan Koenigstein [email protected] #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 18 March 2016 Accepted: 27 May 2016 Published: 14 June 2016 #### Citation: Koenigstein S, Ruth M and Gößling-Reisemann S (2016) Stakeholder-Informed Ecosystem Modeling of Ocean Warming and Acidification Impacts in the Barents Sea Region. Front. Mar. Sci. 3:93. doi: 10.3389/fmars.2016.00093 Climate change and ocean acidification are anticipated to alter marine ecosystems, with consequences for the provision of marine resources and ecosystem services to human societies. However, considerable uncertainties about future ecological changes and ensuing socio-economic impacts impede the identification of societal adaptation strategies. In a case study from the Barents Sea and Northern Norwegian Sea region, we integrated stakeholder perceptions of ecological changes and their significance for societies with the current state of scientific knowledge, to investigate the marine-human system under climate change and identify societal adaptation options. Stakeholders were engaged through personal interviews, two local workshops, and a web based survey, identifying the most relevant ecosystem services potentially impacted and developing an integrated system dynamics model which links climate change scenarios to the response of relevant species. Stakeholder perceptions of temperature-dependent multiannual fluctuations of fish stocks, interactions among fish, marine mammal, and seabird populations, and ecological processes such as primary production are represented in the model. The model was used for a discourse-based stakeholder evaluation of potential ecosystem changes under ocean warming and acidification scenarios, identifying shifts in ecosystem service provision and discussing associated societal adaptation options. The results pointed to differences in adaptive capacity among user groups. Small-scale fishers and tourism businesses are potentially more affected by changing spatial distribution and local declines in marine species than industrial fisheries. Changes in biodiversity, especially extinctions of polar species, and ecosystem functioning were a concern from an environmental conservation viewpoint. When considering potential additional impacts of ocean acidification, changes observed in the model projections were more uniformly valued as negative, and associated with an increased potential for conflicts among user groups. The stakeholder-informed ecosystem modeling approach has succeeded in driving a discussion and interchange among stakeholder groups and with scientists, integrating knowledge about climate change impacts in the social-ecological system and identifying important factors that shape societal responses. The approach can thus serve to improve governance of marine systems by incorporating knowledge about system dynamics and about societal uses and values. Keywords: participatory modeling, marine ecosystem services, marine systems, climate change adaptation, ocean acidification, Barents Sea #### INTRODUCTION Under global climate change, the oceans are undergoing profound changes. Ocean warming, acidification (decreasing pH values), deoxygenation (insufficient oxygen levels), and other physical and chemical changes are anticipated to affect marine species, drive changes in marine ecosystem structure and dynamics, and impact the productivity of marine ecosystems and the provision of ecosystem services to human societies (Pörtner et al., 2014; Gattuso et al., 2015). Ocean warming is already observed to lead to poleward shifts in the spatial distribution of marine organisms, facilitating species invasions into regional ecosystems, and causing local or regional extinctions by exceeding the thermal tolerance limits of organisms (Poloczanska et al., 2013). Ocean acidification, the decrease in water pH via increasing solution of atmospheric CO2, is anticipated to impact different organism groups in marine ecosystems (Kroeker et al., 2013). Increased mortality and structural damages observed in laboratory experiments with early life stages of fish under future ocean pH values cause concern about the future of fish stocks (Munday et al., 2010; Denman et al., 2011). Nevertheless, considerable uncertainty compounds the analysis of ecosystem-level effects of multiple climate change drivers, and their interactions with anthropogenic impacts and human uses (Gattuso et al., 2015; Riebesell and Gattuso, 2015). Ecological models are increasingly playing an important role in an integrated assessment of these effects in marine social-ecological systems (Perry et al., 2010; Osterblom et al., 2013). A wide range of human uses and activities will be affected by climate change impacts on marine systems (Allison and Bassett, 2015). Economic and nutritional dependence on marine resources, and vulnerability toward change differs strongly among countries (Allison et al., 2009). While societies have a range of options to adapt to changes in marine living resources, e.g., increase of exploitation efforts or economic diversification, these depend on economic, social and cultural conditions (Perry et al., 2011; Haynie and Pfeiffer, 2012). The ecosystem services concept (Millenium Ecosystem Assessment, 2005) can serve as a framework for assessing changes in societal benefits provided by marine ecosystems, like food provision from fisheries and aquaculture, carbon uptake and climate regulation, bioremediation, and nutrient cycling, or recreation and cultural services (Beaumont et al., 2007). To improve the scientific basis for quantifying changes in the provision of these services and important trade-offs among services, assessment methodologies must be equipped to capture the multidimensional nature of the value of ecosystems, to enable better informed individual and institutional decisions and improve governance mechanisms (Daily et al., 2009; Kittinger et al., 2014). Since ecosystem services are ultimately defined by society and governance decisions should be more effective when supported by affected societal groups, there is strong rationale for stakeholder participation as an integral part of ecosystem assessment processes. We present a regional case study on climate change impacts on ecosystem service provision in the Barents Sea and Northern Norwegian Sea area. In Norway, the oceans play an important economic and cultural role. The fisheries sector with a production of 2.3 million tons of fish and 12.800 employees in 2011 makes the country the world's second-largest seafood exporter after China (FAO, 2013), divided between industrial off-shore fishing, aquaculture mostly of Atlantic salmon, and small-scale coastal fisheries. Main capture fish species are Atlantic cod (Gadus morhua), Atlantic herring (Clupea harengus), capelin (Mallotus villosus), Atlantic mackerel (Scomber scombrus), saithe (Pollachius virens), and other whitefish. Norway's national fisheries management is generally seen as well-regulated, sciencedriven, internationally cooperative and sustainable (FAO, 2013, 2014). Fishers participate in management via national and regional fisheries associations and provide catch information (Johnsen, 2013; Jentoft and Mikalsen, 2014). In recent years, good management and favorable environmental conditions under ocean warming have facilitated large fish stock sizes such as of Barents Sea cod (Eide et al., 2013; Kjesbu et al., 2014). Nevertheless, future climate change represents a considerable challenge for Norwegian fisheries management (Harsem and Hoel, 2012), and recent integrated, ecosystem-based coastal zone management plans recognize important knowledge gaps with regard to the impacts of climate change and ocean acidification (Hoel and Olsen, 2012). The Barents Sea is projected to experience rapid ocean warming in the next decades, which together with a reduced extent of Arctic sea ice is already leading to pronounced changes in ecological community composition, spatial distribution and biomass of fish stocks, and thus, fisheries provision (Hollowed and Sundby, 2014; Fossheim et al., 2015; Kortsch et al., 2015). At the same time, Arctic and subarctic areas will be affected by the strongest pH changes expected worldwide until the end of the century, with Arctic waters becoming corrosive to some shellproducing organisms. Thus, changes in food web structure and also direct impacts on fish stocks are expected, but still subject to high scientific uncertainty (AMAP, 2013). The comparatively simple food web in the Barents Sea is expected to be more vulnerable to impacts on certain keystone or bottleneck species than ecosystems with higher species diversity (Wassmann et al., 2006; Duarte et al., 2012). We constructed an ecological model of the expected impacts of ocean warming and acidification on marine ecosystem services in the Barents sea region, based on input from potentially affected stakeholder groups (Costanza and Ruth, 1998; Voinov and Bousquet, 2010). We incorporated stakeholder input regarding which ecosystem elements and processes to include in the model, and used stakeholder perceptions to assess which human uses and societal groups may be impacted by environmental changes in the region. Thus, stakeholders served as representatives of society, to integrate local knowledge and concerns, identify relevant ecosystem elements and services, evaluate projected changes under scientific uncertainties, and identify societal adaptation options (Walker et al., 2002). This integrated social-ecological systems approach is applied with the aim of increasing resilience of marine-human systems and improving adaptive capacity (Hughes et al., 2005), to discover governance options for a more sustainable use of marine resources under climate change. ## MATERIALS AND METHODS #### Stakeholder Consultation For an initial compilation of potential ecosystem changes under climate change in the focus area, the scientific literature was screened for an overview of the problem (reviews on regional ecosystems and on climate change and ocean acidification impacts, reports of expert groups, news, and outreach products produced by regional scientific institutes). To compare these findings to relevant topics of concern for the users, internetbased news portals aimed at regional stakeholders, archives of newspapers of general interest and for user groups (e.g., fisheries magazines) were screened for recent prominent topics. Ten interviews with regional scientific experts with a background in marine ecology, governance of marine resources and areas, oceanography, ecosystem modeling, fisheries science, and other disciplines, further helped to identify potentially affected ecosystem services and stakeholders. Interviews with 25 stakeholders of potentially affected groups from Norway and Russia were conducted in different locations in Norway (Oslo, Bergen, Tromsø, Bodø, Lofoten Islands, Finnmark, Svalbard) or via email between March and September 2013. Stakeholders included representatives from fishing associations and aquaculture companies, individual small-scale fishers, tourism operators (hotels/camps, sport fishing, whale watching), non-governmental organizations (including environmental conservation and indigenous Sami groups), and governmental agencies (Fisheries and Environmental Directorates). The personal interviews aimed at identifying (1) the general socioeconomic situation of participants, (2) perceptions and concerns about regional ecosystem impacts of climate change, (3) the communication between science, politics and stakeholders about expected impacts, (4) societal impacts and adaptation options to climate change, and (5) management options and political adaptation strategies (Supplemental File S1: Interview questionnaire). Participants were also asked for their personal opinion on further potentially affected societal groups, to open up the investigation to ecosystem services and user groups not initially identified. The most frequently mentioned ocean uses, climaterelated concerns, and ecosystem interactions from stakeholder interviews which could be linked to elements of the marine ecosystem, were compiled to form the basis of the model (Table 1). A model-building workshop with stakeholders was held in Bergen, Southern Norway in October 2013, where stakeholders were introduced to the topics of the research project and the current state of scientific knowledge about climate change impacts on marine ecosystems. A draft of the model structure based on the identification of relevant ecosystem services and elements from the interviews was presented. Stakeholder comments on the model structure and requests for further elements and services to be included were collected to inform further model development (Koenigstein and Goessling-Reisemann, 2014). ## Integrative System Dynamics Model A system dynamics model (Costanza and Ruth, 1998) was developed in the modeling software STELLA 9.1, and later converted to STELLA Professional 1.0 (www.iseesystems.com). Its structure was based on the most relevant ecological elements and processes that can be linked and quantified using empirical biological results. Graphical icons for species and ecosystem services were designed and integrated into the model interface to make the model structure more easily accessible to stakeholders. The model was based on a multi-species population structure, with biological processes governing population dynamics of the integrated species, and interactions among species represented by predation and consumption (Figure 1). The model structure incorporates the marine species of high importance to the various stakeholder groups, and the most commonly mentioned biological processes. Some ecologically similar species were aggregated to groups ("other baleen whales," seals, "other seabirds") to limit model complexity, and/or combined in modules (tooth whales, baleen whales, seabirds) in the model interface. Aggregate representations of lower trophic levels (one phytoplankton and three zooplankton groups) were used to base the biomass flow through the food web on a primary production process, integrating stakeholder concerns about primary production and the ecosystem services of carbon uptake and export (Figure 1A). Due to the importance of fish stock recruitment in stakeholder concerns, fish populations were divided into two to four life stages and embedded in a selfenhancing feedback of reproduction and recruitment processes. Ocean warming and acidification were incorporated as changes in fish and zooplankton consumption and growth, based on physiological thermal growth windows (Pörtner and Farrell, 2008) and assuming an increasing loss of metabolic energy under acidification reaching up to 10% of the total energy uptake (Figure 1C). Driver scenarios for temperature and pH were incorporated based on IPCC (Intergovernmental Panel on Climate Change) ensemble earth system model projections for the Barents Sea under the RCP (Representative Concentration Pathway) 8.5 ("business-as-usual") emission scenario (AMAP, 2013; Bopp et al., 2013; Collins et al., 2013). Temperature was additionally adjusted to undergo seasonal fluctuation and an inter-annual oscillation with a period of 8 years (Figure 1B), #### TABLE 1 \| Stakeholder concerns and observations with regard to climate change impacts on marine ecosystems, from personal interviews with stakeholders from the fisheries sector (F), tour providers and other tourism businesses (T), and environmental and other non-governmental organizations (E), ranked by sum over sectors (seven participants for each sector, one additional aquaculture representative for the fisheries sector). Number of instances mentioned across interviews for species of interest or concern (only species mentioned more than once), general ecosystem observations, and observations or concerns explicitly linked to climate change impacts (detailed interview questions given in Supplemental File S1). mimicking the natural fluctuations linked to the North Atlantic Oscillation (Ottersen et al., 2001) as mentioned in the stakeholder interviews. Marine mammal and seabird populations were modeled without direct effects of warming or acidification, as empirically observed changes are mostly indirectly caused through food web changes (Sandvik et al., 2005; Simmonds and Isaac, 2007). Fishing was incorporated as an additional biomass extraction process for fish and minke whales (Balaenoptera acutorostrata), defined as fixed relationships between stock sizes and landings in the following year, based on past quota agreements. Details of model parameterization, calibration and validation will be described in a separate publication, as we focus on the incorporation of stakeholder perceptions into the model and their assessment of the results here. The graphical interface of the model included dynamic displays of the states of the relevant biomass compartments and other indicators in the model over time, grouped in combined graphs aimed at each of the three stakeholder groups in the model valuation workshop (Figure 1D). ## Model Evaluation and Identification of Adaptation Options A web-based online survey was prepared to evaluate the relative importance of socio-economic framing factors identified in the interviews, created in a Norwegian and an English version using the platform Limesurvey by the provider Limeservice (www.limeservice.com). This served to prepare input for the discussions in the second workshop and helped the creation of framing scenarios at a later time. Factors already included in the model (e.g., ocean temperature or marine organism abundances) were not surveyed. primary production and plankton mortality (blue). Icon design for organism and ecosystem services by Leonard Rokita, Bremen. (B) Temperature and pH over model time frame as drivers of the model, affecting biological processes. (C) detailed structure of the species included in the model in system dynamics notation, based on biomass flows (thick arrows) which determine the state of each fish stock and the dynamic links to other stocks in the model (boxes), and are influenced by the environmental drivers temperature and pH, and by fish catches (circles, thin arrows). (D) examples for resulting model projections under "warming only" and "warming and acidification" scenarios (different biomass scales), which qualitatively reproduce natural fluctuations and were evaluated by stakeholders at two time points, the model years 2040 and 2075 (red dotted lines). A second stakeholder workshop for model valuation was held in Tromsø, Northern Norway in June 2015 with representative stakeholders from fisheries, tourism and environmental conservation. Structure and functioning of the parameterized model was explained, model assumptions and scientific uncertainties discussed, and model runs performed under two scenarios, driven by ocean warming alone, and warming and acidification combined, respectively. At two time points in the simulation—the year 2040 and at the end of the simulation in the year 2075—model runs were stopped and stakeholders asked to discuss the developments in stock levels and ecosystem indicators in groups by sector. Stakeholders agreed on a rating in terms of the significance for their business and interests on a scale of +5 to −5, where: +5 refers to a high preference, i.e., the best imaginable event for participants' business or interests; 0 is neutral, and −5 is catastrophic for economic survival or the stakeholders' main interests. Then, stakeholders were asked to decide whether they needed to change their business, take organizational decisions or other steps to adapt to the projected ecosystem changes. Groups discussed and proposed possible adaptation options for their sector. Finally, general societal adaptation options were discussed among all stakeholders, and common policy recommendations developed among the participants of the different sectors. During this process, stakeholders had access to all model variables and indicators (dynamics of species abundance and processes, biodiversity and ecosystem indicators, etc.), which together reflect the complexity of the underlying ecosystem. ## RESULTS #### Stakeholder Perceptions and Concerns Marine species most often mentioned by stakeholders with regard to ecosystem changes in the interview series were the fish species Atlantic cod, mackerel and herring, as well as kelp and seaweeds, king crabs, and followed by other fish species (Table 1). Stakeholders exhibited a high level of ecological knowledge in their observations and concerns about marine organisms. The most prominent environmental changes linked to climate change were distribution range shifts of fish and other marine species, changes in fish abundance or productivity of fish stocks, an increased occurrence of newly immigrated species such as mackerel, and the factor which was attributed as the main cause of these changes, ocean warming. When describing their observations and/or concerns, stakeholders frequently mentioned ecological processes, mainly feeding interactions, e.g., among herring, cod and capelin, fish stock spawning and recruitment, and inter-annual environment-related fluctuations and variability of fish stocks. Ocean acidification as a relatively newly discovered additional factor was known to fewer stakeholders, but was incorporated as a model driver as it was a central topic of the project, and because it could be linked to warming effects via physiological mechanisms. Upon presentation of scientific results from laboratory experiments on ocean warming and acidification at the workshops, acidification was perceived as an additional concern, but uncertainty with regard to effects in the ecosystem was recognized. A compilation of stakeholder statements and backgrounds on the topics in the interviews and the first workshop was published in an open-access report (Koenigstein and Goessling-Reisemann, 2014). Based on these interview results and discussions at the model-building workshop, the ecosystem elements and services of highest relevance to stakeholders, and which were suitable for integration in a foodweb-based model consistent with ecological knowledge, were selected to represent ecosystem service provision in the model. These were the commercially harvested fish stocks Atlantic cod, herring, capelin and halibut, as well as minke whales for food provision via fisheries. Mackerel, which was regularly mentioned in interviews as a newly immigrated fish species in Northern Norway, was not integrated because data on feeding interactions is not yet available. For tourism and recreation services, baleen whales (Humpback whales, Megaptera novaeangliae, and fin whales, Balaenoptera physalus), sperm whales (Physeter macrocephalus), killer whales (Orcinus orca), Atlantic puffin (Fratercula arctica), and other seabirds, as well as Greenland halibut (Reinhardtius hippoglossoides) and Atlantic cod stocks relevant for sports fishing were integrated. To represent a potentially threatened Arctic species dependent on sea ice, and because experimental data on warming and acidification impacts was available, Polar cod (Boreogadus saida) was also included. Lower trophic levels (phyto- and zooplankton) integrated primary production and food availability for fish. These elements thus represent the ecosystem services of food provision to industrial and smallscale fisheries, tourism and recreation as income-generating and cultural services (sports fishing, tours for whale, sea lion, and seabird watching, and other nature-related activities), and regulating and supporting services by carbon uptake via primary production and carbon export via sequestration. Species diversity (Shannon index) was incorporated as an indicator of ecosystem state on demand of stakeholders from the environmental sector in the model valuation workshop. In the interviews, the most prominent socio-economic concern unrelated to climate change was pollution by oil drilling, mining sewage, dumping, or other sources, followed by fish market prices and labor availability. In the web-based survey conducted to gain additional insights on socio-economic factors and to prepare the valuation workshop, participants (12 completely answered surveys) rated sustainability-oriented management of resources, oceanographic changes, fish price, global economic growth and fishing quota as the most important external factors for their business or interest, with differences in importance among sectors (Figure 2). #### Stakeholder Valuation of Model Projections Stakeholder valuations of model projections in the second workshop differed markedly among stakeholder groups (Supplemental File S2: stakeholder valuations). Stakeholders noted that their valuations of the model projections depend on the trend displayed up to the stop in simulation time, i.e., the same stock level was rated more negatively when stock levels had been descending to this level as opposed to when they had ascended. The full development was only revealed after the simulation restarted to complete the run, reflecting uncertainty about the future in decision making in real life. Projected changes in the warming-only scenario included increases in most fish stocks, orcas and "other seabirds," and decreases in sperm whales, seals, krill, and carbon export associated with zooplankton mortality. This scenario was rated as positive for fisheries, but as negative by tourism stakeholders due to decreased sperm whale levels, and caused concern for environmental conservation due to declines in species diversity and the collapse of Polar cod (Table 2). In the "warming and acidification" scenario, most species showed declines due to the energetic loss under ocean acidification incorporated in the model. Stakeholders from the fisheries sector viewed the projections for 2040 as "economically painful," given locally strong socio-economic impacts for fishers, and possible conflicts between large vessels and small-scale fishers. Further decreased stocks and the collapse of the Atlantic cod stock toward the end of the simulation in the year 2075 were perceived as leading to strong socio-economic impacts and a challenge for fisheries policies. At the model valuation workshop, present stakeholders from the fisheries sector noted that haddock, saithe, and the increasingly immigrating mackerel were also important species for regional fisheries (or expected to become important in the future), and should be added to the model. #### Adaptation Options Continued adjustment of fishing quota and intensified regulation of stock management were proposed as an adaptation option to climate change effects for fisheries in the interviews and the valuation workshop. Stricter quotas in times of declining stocks, potentially aided by a diversification of quotas and the regulation of by-catches may support a recovery of stocks. Larger vessels can also respond by moving further out to open waters, following moving fish stocks. In the valuation workshop, switching fisheries to other species (e.g., crab, mackerel, mollusks) was viewed as an additional option for reducing economic losses. Also, in the opinion of the stakeholders, increased fines for illegal fishing and catch limitations for tourist fishing may become necessary. Increased research on and investment in aquaculture as an alternative for food provision was discussed as a further adaptation option with explicit mention of sustainable and multispecies aquaculture, including species such as seaweeds and sea urchins, depending on market demand and cultural acceptance, and research into zoo- and phyto-plankton as a food source. The tourism sector would also suffer from local collapses of small-scale fisheries, and generally decreased fish stock levels. One of the suggested adaptation options was to change marketing, focusing less on marine animals and more on cultural heritage and landscapes, and possibly on winter business to make use of the modest declines projected for orcas, and strengthen networking with small-scale fishers. The potential impacts of aquaculture on tourism and the possible use of aquaculture facilities as a tourist option could be explored. Stakeholders from environmental conservation called for an extension of marine protected areas, e.g., for nursery grounds of polar cod and whales, to mitigate ecological impacts of warming and acidification, and a stricter regulation of additional anthropogenic stressors, e.g., pollution by the deposition of mining wastes in fjords. Commonly agreed policy recommendations of the participants of the second workshop for the projected warming and acidification scenarios were to explore the potential of increased seaweed farming and other alternative aquaculture food. For this, creating training and education, and conserving local economies by appropriate government strategies and incentives would be necessary. Abandoning the consumption TABLE 2 \| Stakeholder concerns about socio-economic impacts on their business or interest, and societal and personal adaptation options to climate change impacts, from personal interviews with stakeholder from the fisheries sector (F), tour providers and other tourism businesses (T), and environmental and other non-governmental organizations (E), ranked by sum over sectors (Σ), seven participants for each sector (one additional aquaculture representative for F). Number of instances mentioned across interviews (detailed interview questions given in Supplemental File S1). of seagull eggs, seals and whales may be advisable, and would necessitate some cultural changes. Under the projected strong stock declines, renegotiations of fishing rights and quotas may become necessary. This would call for the respective political will and actions for conflict resolution among fisheries in Norway. ### DISCUSSION ### Integration of Stakeholder Perceptions about Climate Change and Ocean Acidification Impacts All interviewed stakeholders reported plausible climate change effects on marine species, thus their personal accounts substantiated recent scientific results (Fossheim et al., 2015). However, many participants also pointed to the great variability in marine ecosystems in the region, especially fish stocks, which makes it difficult to distinguish environmental fluctuations from long-term change, and thus increases uncertainty about climate-related trends (Johannesen et al., 2012). Because of the high importance of ecological processes and species interactions for stakeholders in the initial interview series, the ecosystem model was based on the foodweb interactions among pelagic and demersal species in the Barents Sea (Bogstad et al., 2015), explicitly integrating the biological processes of interest (Koenigstein et al., 2016). This enabled the incorporation of a large fraction of the species of interest to the stakeholders into the model, and also allowed us to incorporate fishing quotas as the most important adaptation option and anthropogenic driver initially identified in the interviews. However, this choice of model structure came at the expense of being unable to consider spatially explicit distribution shifts and benthic species such as macroalgae, shellfish, or echinoderms. These are often restricted to coastal and fjord habitats and undergo highly localized conditions, e.g., with regard to freshwater influx or hypoxia. In comparison to mental models or other probabilistic models often used in participatory modeling, the deterministic ecosystem model developed here resolves to some degree the emergent behavior of the ecosystem under different conditions, and enables the integration of scientific knowledge, assessing dynamic trade-offs in effects among species and among biological processes under future climate change conditions (see subsection "Towards ecological realism..."). The main non-climate related concern was pollution, caused by oil and gas exploration, residues from mineral mining along the coasts, or shipping. For whale watching companies, noise pollution from shipping and seismic exploration was a prevalent concern. Due to high scientific uncertainty and highly localized ecological impacts, these concerns could not be incorporated into the model. Also, as aquaculture is not directly linked to marine foodwebs, and was not often mentioned as a factor or concern by the stakeholders in the interview phase, the aquaculture sector was not further considered at this point. Melting of the Arctic sea ice and sea level rise, although of high relevance in the interviews, were also not incorporated due to unclear links to the marine organisms in the model. The scenario-based incorporation of pollution, sea ice and aquaculture is planned for a future extension of the model. Although impacts of marine ecosystem changes on tourism are far less prominently covered in the scientific literature and the media, the relevance of shifts in marine food ecosystems was immediately obvious to most interviewed stakeholders from the tourism sector. Worldwide, biodiversity loss and reduced aesthetic value of landscapes are expected to impact tourism under climate change, among a range of other factors (Simpson et al., 2008). Tourism in our study region is to a high degree dependent on certain locally abundant species (sperm whales, cod, halibut, seals), thus pointing to highly localized climate change impacts on tourism and recreation, and to the necessity for a detailed assessment of local conditions. Biodiversity and cold-water coral reefs were also mentioned as threatened by climate change, and valuation studies point to a very high willingness-to-pay of the Norwegian public to conserve coldwater coral reefs (Aanesen et al., 2015). Stakeholders of all sectors had agreed in the model-building workshop on conserving the protected status of coral reef areas (e.g., prohibition of trawl fisheries) and not including reefs in the model, putatively reflecting the cultural and existence value of this reefs, but also low economic importance of these areas for fisheries. ## Adaptation Options for Stakeholders to Projected Ecosystem Changes Stakeholder valuations and discussions at the valuation workshop showed that small-scale fisheries and tourism businesses have less, or more constrained adaptation options for the ecological changes projected by the model (cf. Table 3). For instance, smallscale fishers often cannot follow moving fish stocks far away from the coast, or have the funds to invest in different gear. The commercially relevant whale-watching and other tour activities in the area are heavily dependent on the sighting probability of certain species (e.g., sperm whales). Adaptation options for sightings decreasing below a critical level would entail drastic changes in the character of tourism activities, with probable reductions in customer numbers and income. Fishing quota adjustments were seen as the primary adaptation option by fisheries and tourism stakeholders. However, small fishing boat owners, often located in more remote areas and with a partial income from sports fishing tours, perceived quota adjustment as less likely to be a sufficient measure for climate change impacts than stakeholders organized in fisheries associations. In a situation with reductions in several co-used fish stocks, as projected under the combined warming and acidification scenario, suggested adaptation options ceased to be sector-exclusive, and conflicts were expected to increase among industrial, small-scale, and sports fishing, when catch efforts would be increasingly concentrated on the remaining stable species (e.g., halibut). Conflict potential among and within sectors led to the recognition of the need for increased cooperation and networking among user groups. Increased investment in aquaculture was a heavily discussed adaptation option at the valuation workshop, which is very relevant worldwide in the context of securing food provision under overfishing of many fish stocks (FAO, 2014). Yet, there was a range of concerns from stakeholders with regard to the ecological impacts of aquaculture (pollution from nutrients and antibiotics, escaped individuals and parasites, spatial use conflicts). Also, the viability of this option depends on economic factors and the continued provision of small pelagic fish by capture fisheries for fish meal production. Stakeholders agreed on the need for increased research on ecological impacts and more sustainable methods of aquaculture production. Environmental conservation stakeholders adopted a broader view on ecosystem functioning, asking for inclusion of a biodiversity indicator during the valuation workshop, and thus brought a precautionary aspect into the discussion. Declines in zooplankton and phytoplankton biomass levels were also negatively rated by stakeholders from the fisheries sector, reflecting concerns about indirect impacts on fish stocks. The discussion among different stakeholder groups was also shaped to some extent by implicit societal values, as e.g., the high cultural importance of the Atlantic cod fishery in Northern Norway and the significance of marine species for the coastal indigenous Sámi were mentioned. In the discussion of societal adaptation options, a focus on options which were undisputed among the workshop participants was observed, while options which would have more potential for conflicts (e.g., total catch bans or area closures for certain uses) were avoided topics. The group evaluation approach thus reproduced certain factors and constellations which govern societal decision-making, e.g., implicit valuing, social agreement, and power balances among stakeholders. These results point to considerable differences in adaptation capacities to climate change impacts among stakeholders in the Northern Norwegian Sea and Barents Sea region, with less resilient small-scale fishers and tourism businesses. Potential food-web mediated impacts e.g., on whales and seabirds or lower trophic levels would thus lead to governance-relevant trade-offs among fish provisioning and other ecosystem services. As model development is ongoing and valuations are based on a preliminary, not finally validated version of the model, projections and societal adaptation options at this stage should be regarded as describing possible paths of system behavior. As framing and limiting conditions for stakeholder decisions have been identified during the valuation workshop, stakeholder decisions will be transferrable to validated projections as these become available. ## Toward Ecological Realism in Assessments of Climate Change Impacts on Ecosystem Services The participating stakeholders' main ecological concerns and the most relevant ecosystem services have been integrated into the developed ecosystem model, considering the scientific knowledge Relevant aspects of impacts, with stakeholder rating on a scale from +5 to –5, adaptation options suggested by stakeholder groups, and conditions or potential drawbacks given for these adaptation options. Stakeholder rating +5 reflects an extremely beneficial effect on stakeholder group, –5 reflects a catastrophic effect. on interactions among ecosystem elements and processes, and helping to build trust in the model. Importantly, this model structure also enables the assessment of indirect ecological climate change impacts (e.g., on marine mammals and seabirds relevant for tourism), thus exploring possible trade-offs among ecosystem services. The process-based structure of the developed model thus enables a more realistic representation of biodiversity (Queirós et al., 2015) and improves the potential for integrating empirical data into climate change projections (Koenigstein et al., 2016). Models used in ecosystem service assessment are usually highly simplified in order to be easily understandable, and it is a challenge to communicate scientific uncertainty (Ruckelshaus et al., 2013). Our stakeholder-informed ecosystem model development represents an intermediate approach between participatory modeling of stakeholder perceptions without a direct empirical basis of ecosystem behavior, and the use of models e.g., in fisheries management, where a predeveloped model is often brought to the stakeholders and explained by scientists. The model developed and used here reproduces the inter-annual variability in ecosystem dynamics and interdependent fluctuations in fish populations observed by the stakeholders, which are governed by climatic fluctuations linked to the North Atlantic Oscillation (Ottersen et al., 2001; Dalpadado et al., 2012). The reflection of their perceptions in the model enabled the participants to "play" with it during the workshop, exploring effects that were in some cases not expected by the model developers, and finding their own explanations for model behavior. Importantly, it was understood and accepted that the model is not a scientifically proven prediction of the future, but has a range of internal uncertainties e.g., in parameter ranges and structural reliability, and depends on uncertain external parameters with regard to climatic and economic factors. The observed influence of the displayed trend in model projections on stakeholder valuations indicates that stakeholders implicitly extrapolate model trends (and fluctuations) into the future, incorporating the perceived uncertainty into their decision. A focus on ecosystem services during model development helped to limit model complexity to ecosystem elements that can be linked to societal uses. The ecosystem service concept promises to improve the participation of stakeholders in the management and conservation of marine areas and resources (Kittinger et al., 2014; Leenhardt et al., 2015). However, ecosystem services have been criticized as being too simplistic and too much focused on monetization (Norgaard, 2010; Silvertown, 2015), and cultural services are often not considered in ecosystem service assessments (Chan et al., 2012). We addressed these issues through the use of a process-based ecosystem model, taking into account ecological complexity and variability, and dynamic trade-offs among ecosystem services. Cultural and ethical values were implicitly considered in the discourse-based valuation, which should improve the perceived legitimacy of the derived recommendations (Wilson and Howarth, 2002). Altogether, our approach to combine stakeholder consultation and ecosystem modeling has been successful in conveying scientific backgrounds and associated uncertainties of climate change processes to stakeholders, motivating stakeholders to participate in the evaluation of impacts and the identification of societally acceptable adaptation options. In a next step, insights on environmental and socio-economic framing factors gained in this study will be integrated into consistent scenarios, and stakeholders will again be involved in finding adaptation options under these scenarios using an extended and validated version of the model. This forms a methodological basis for developing adaptation strategies under scientific uncertainties, that are informed both by knowledge about ecosystem dynamics and by societal uses and values. Characterization of societal responses in connection with the identified properties of the social-ecological system (e.g., species composition, ecological dynamics, human uses and user groups) can yield insights for research in situations with lower data availability and lower level of knowledge of stakeholders, where a comparably high model detail may not be possible. ## CONCLUSIONS Our integrative ecosystem model was designed to consolidate the dynamic simulation of climate change impacts with stakeholder perceptions and concerns. By reflecting the complexity of the biological processes underlying ecosystem dynamics, individual scientific results of ocean warming and acidification research can be integrated and communicated, interactions and uncertainties discussed with affected stakeholders, and trust gained in longterm projections under climate change. Stakeholder-informed ecosystem modeling and discourse-based evaluation are thus useful tools for ecosystem service assessments with multiple user groups, investigating trade-offs and balancing interests under multiple system drivers. Integrative models of intermediate complexity, like the one developed in this work, have the potential to improve understanding of regional social-ecological systems, and help to identify options for adaptive governance of marine systems under climate change and human use. ## REFERENCES ## AUTHOR CONTRIBUTIONS SG conceptualized the work program. SK conducted and evaluated the interviews, SG and SK conducted and evaluated the stakeholder workshops. SK, MR, and SG conceptualized, developed and validated the simulation model. SK drafted the manuscript, MR and SG contributed to the manuscript and reviewed the final version. ## ETHICS STATEMENT All human participants took part voluntarily and gave oral or written informed consent to participate. They also consented to the use of their statements for the study after anonymization. The purpose and background of the study and the planned use of the results were made transparent prior to interviews, surveys and workshops. The confidentiality of personal information and the right to omit uncomfortable questions or withdraw from the interview at any stage were provided. ## FUNDING This work was funded through the research program BIOACID (Biological Impacts of Ocean Acidification, phase II), by the German Federal Ministry of Education and Research (BMBF, FKZ 03F0655J). #### ACKNOWLEDGMENTS The authors thank Jannike Falk-Andersson (UiT), Nicola Beaumont (PML), Annette Breckwoldt (ZMT Bremen), and Christos Zografos (UAB) for advice on and support in preparing and conducting the stakeholder interviews, workshops and web survey. We thank Hauke Reuter (ZMT Bremen) for advice in model development, and Viola Logemann for help with interview transcription. We are indebted to numerous Norwegian colleagues at the Institute of Marine Research (IMR), the Arctic University in Tromsø (UiT) and the Fram Centre, for kindly providing support in workshop logistics, stakeholder contacts, and scientific expertise to make this work possible. ## SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2016.00093 approach. Mar. Pollut. Bull. 54, 253–265. doi: 10.1016/j.marpolbul.2006. 12.003 FAO (2014). The State of World Fisheries and Aquaculture 2014. Rome. 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 © 2016 Koenigstein, Ruth and Gößling-Reisemann. This is an openaccess 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. # Bridging for Better Conservation Fit in Indonesia's Coastal-Marine Systems #### Samantha Berdej <sup>1</sup> \* and Derek Armitage<sup>2</sup> <sup>1</sup> Environmental Change and Governance Group, Department of Geography and Environmental Management, University of Waterloo, Waterloo, ON, Canada, <sup>2</sup> Environmental Change and Governance Group, School of Environment, Resources and Sustainability, University of Waterloo, Waterloo, ON, Canada Efforts to improve the fit between conservation initiatives (e.g., marine protected areas, no-take zones) and the dynamic social dimensions of coastal-marine systems remain underdeveloped. We empirically illustrate here how opportunities to enhance "conservation fit" are influenced by bridging organizations that serve to (1) better align conservation initiatives with characteristics of the social context that influence conservation outcomes (e.g., institutions, culture, values, local practice), (2) foster coordinated and adaptive approaches to conservation that are reflective of multiple perspectives and knowledge, and (3) better connect people and conservation actions across jurisdictional and geographical boundaries. Qualitative methods were used in this research, including semi-structured interviews, observation of key events and meetings, and literature review. We draw from three coastal-marine conservation cases in Bali, Indonesia, that exemplify different approaches to bridging for conservation fit: the Bali MPA Network, the Nusa Penida MPA, and the East Buleleng Conservation Zone. Our synthesis of these cases identifies different strategies used by bridging organizations to deal with conservation fit issues, including their capacity to integrate actors and perspectives using flexible approaches, actualize hybrid forms of decision-making, build capacity and leadership, and foster cross-scale conservation and scale-bridging social networks. We also examine the limitations of bridging organizations and offer direction for future research for coastal-marine conservation in Indonesia specifically, and the Coral Triangle region generally. More broadly, this analysis contributes new insights on emerging forms of governance designed to deliberatively fit conservation initiatives to coastal-marine social-ecological systems experiencing rapid change. Keywords: bridging organizations, conservation, Coral Triangle, fit, governance, Indonesia, marine protected area, social-ecological system ## INTRODUCTION The success of marine conservation in southeast Asia's Coral Triangle (CT) requires modes of governance that deliberately fit conservation initiatives to underlying social dimensions. Insufficient consideration of social dimensions in conservation initiatives has contributed substantially to limited progress in this regard. To this end, we investigate the issue of "conservation fit," which we refer to here as the dynamic alignment of the governing system for conservation and #### Edited by: Annette Breckwoldt, Leibniz Center for Tropical Marine Ecology (ZMT) Bremen, Germany #### Reviewed by: Lydia Chi Ling Teh, University of British Columbia, Canada Carolyn J. Lundquist, National Institute of Water and Atmospheric Research (NIWA), New Zealand \Correspondence: Samantha Berdej [email protected]; [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 07 February 2016 Accepted: 06 June 2016 Published: 27 June 2016 #### Citation: Berdej S and Armitage D (2016) Bridging for Better Conservation Fit in Indonesia's Coastal-Marine Systems. Front. Mar. Sci. 3:101. doi: 10.3389/fmars.2016.00101 the social dimensions of a system that influence the outcomes of conservation policy and practice. Governance is an umbrella term that refers to the "...integrated system of formal and informal rules, rule-making systems, and actor-networks at all levels of human society..." (Biermann et al., 2009, p. 4). For our purposes, governance describes the interactions of different actors and networks that formulate and implement conservation. By social dimensions we refer to the multilevel patterns of interaction between actors and organizations, their values, interests and social customs, and the processes and instruments that drive, support or constrain the practice of conservation (sensu Galaz et al., 2008; Meek, 2013; Epstein et al., 2015). This characterization recognizes that governance systems affect, are affected by, and are also a part of the broader suite of social dimensions that make up coastalmarine social-ecological systems. To examine the issue of conservation fit, we focus on the role of bridging organizations, which are entities that connect social actors or groups through some form of bridging process (Crona and Parker, 2012). These organizations link actors and actions to facilitate coordinated, integrated responses in contexts where resources or capacity are limited. However, few studies have explored their role in developing, implementing and adapting conservation initiatives, or their influence on conservation outcomes (e.g., Jamal et al., 2007; Schultz and Lundholm, 2010; Jacobson and Robertson, 2012; Bodin et al., 2014). Building on previous work in the region (Berdej and Armitage, 2016), this paper empirically demonstrates that bridging organizations can help to better align conservation initiatives with their social context, foster appropriate processes and instruments to pursue coordinated and adaptive conservation, and better connect people and conservation actions across scales and levels. However, as this paper also shows, bridging organizations are not without their limitations, and we identify a number of constraints or barriers that require further consideration. Our focus here is on the congruence of the governing system for conservation and the other crucial social dimensions of a system that influence overall conservation effectiveness—what we term "conservation fit." The concept builds on critiques of conservation initiatives that point to a lack of meaningful engagement with, and integration of, social dimensions such as socioeconomic or cultural context, stakeholder relations, knowledge diversity, or the multiplicity of political scales and domains of action (see CT: Clifton, 2009; Foale et al., 2013; Fidelman et al., 2014; von Heland et al., 2014). Where there is insufficient consideration (or "poor" fit)—as in cases where new conservation policies and rules are introduced without attention to local or indigenous legacies (Majors, 2008), or where trade-offs between biodiversity conservation and development are overlooked (Foale et al., 2013), problems of ineffective and inefficient conservation often result. As such, the concept of conservation fit is a useful frame to understand why certain conservation initiatives may not work as intended and how they might be strengthened via bridging organizations. Enhancing conservation fit in the CT is challenging because of the immense diversity of actors and interests across geographical and jurisdictional scales, and the differing socio-political, cultural and economic contexts (e.g., Mills et al., 2010; Fidelman et al., 2012; Foale et al., 2013; von Heland et al., 2014; Cohen and Steenbergen, 2015). In Indonesia, the partial decentralization of government has afforded greater opportunity for participatory approaches in conservation, but has also contributed to political tensions between levels, governance fragmentation and conflicting government policies (Patlis, 2005; Wiadnya et al., 2011). Further, marine conservation efforts in this region are facing rapidly expanding and increasingly mobile populations, emerging markets for marine commodities, and a limited ability to enforce rules and regulations (Majors, 2008). Many scholars across the CT have stressed the importance of connecting people and conservation practice in ways that communicate knowledge and foster learning, reconcile diverse objectives and views, and which forge relations across domains and governance levels (e.g., Fidelman et al., 2012; von Heland et al., 2014; Pietri et al., 2015). However, until recently, relatively little work has explicitly investigated the influence of bridging organizations in facilitating these needs in the CT, and none has examined their role in the practice of conservation in Indonesia (see Berdej and Armitage, 2016). In the following section, we introduce the concept of conservation fit and examine bridging organizations as an organizational strategy to foster fit. We outline three categories of conservation fit that serve to frame the analysis, and highlight their key challenges in the CT. We then present three cases from Bali, Indonesia, that illustrate the role of bridging organizations in different conservation contexts, and draw on these cases to generate insights about key strategies applied by bridging organizations to influence conservation fit. Finally, we identify a number of constraints or barriers that require further consideration, and speak to commonalities underlying successful bridging approaches that are relevant beyond the particular conservation settings we examine here, recognizing that each case reflects a slightly different social, political and ecological context. ## THEORETICAL BACKGROUND ## Defining the Problem of "Conservation Fit" Our concept of "conservation fit" emerges from a broader discourse on institutional and governance fit. For example, fit has been discussed as part of institutional dimensions of global environmental change (Young, 2002; Ekstrom and Young, 2009), resilience of social-ecological systems (Folke et al., 1998/2007; Galaz et al., 2008; Epstein et al., 2015), and common pool resources (Ostrom, 2007). Much has been written on how well governing systems "fit" ecological dynamics (e.g., Folke et al., 1998/2007; Ekstrom and Young, 2009), and, more recently, on the fit between governing systems and social dynamics (e.g., Brown, 2003; Meek, 2013; Pittman et al., 2015). However, exactly what constitutes a good fit and how such fit can be achieved remains a research puzzle Abbreviations: CI-I, Conservation International Indonesia; CT, Coral Triangle; CTC, Coral Triangle Centre; CTI-CFF, Coral Triangle Initiative on Coral Reefs, Fisheries and Food Security; RC-I, Reef Check Indonesia. (Ekstrom and Young, 2009; Bodin et al., 2014). In particular, limited understanding of the conditions and implications of fit for the practice of marine conservation is a gap in the literature. Conservation initiatives should be more effective in the long-term where the governance system is aligned with, and responsive to, the complexity and dynamism of the social system (e.g., Brown, 2003; Christie et al., 2003; Christie, 2004, 2011; Shackeroff et al., 2009; Ban et al., 2013; Kittinger et al., 2014; von Heland and Clifton, 2015). Our concept of fit responds to calls for more participatory and pluralistic conservation approaches that allow for learning and adapting (Berkes, 2007; Armitage et al., 2012), clarify hard-choices and trade-offs (Hirsch et al., 2011), and which seek social legitimacy and ethical imperatives in conservation (Brechin et al., 2003; Mascia, 2003)—all of which have been difficult to actualize in practice, as detailed below. A "poor" fit, as mentioned, can undermine the effectiveness of conservation initiatives by resulting in inadequate understanding of contentious social issues, unintended negative consequences, missed opportunities for positive change, and an incomplete understanding of the system (Christie et al., 2003; Christie, 2011). Situations of "poor" fit (or misfit) can arise, for example, where governance underplays community norms and livelihood needs (Clifton, 2009; Ferse et al., 2010), or is unable to account for diverse worldviews and belief systems (Majors, 2008; Clifton and Majors, 2012). Alternatively, a "good" fit should contribute to the salience of conservation by generating meaningful benefits, improving perceived legitimacy and sense of ownership, and by reducing the probability of negative impacts. Positive examples include cases where conservation initiatives are hybridized with local or customary practice (Cinner and Aswani, 2007), social networks are built to connect local management to higher-level policy-making (Cohen et al., 2012), or where governance learning networks are created to bridge cultural and jurisdictional boundaries (Pietri et al., 2015). Improved conservation fit alone may be necessary, but not sufficient for conservation success. Even where conservation initiatives are compatible with social dimensions, they may not adequately provide for ecological dimensions or "ecological fit." Although, our focus in this paper is on social dimensions, we join other authors in affirming the importance of engaging both dimensions in the context of developing and ongoing conservation initiatives (e.g., Epstein et al., 2015). There is also no "ideal" conservation fit since social systems and the factors that influence them differ and are constantly changing. Instead, fit is a means to an end, not an end in itself. For analytical purposes, we distinguish three general categories of conservation fit associated with: (1) aligning conservation initiatives with characteristics of the social context (e.g., institutions, culture, values, local practice), (2) enabling governance processes and instruments to bring together and meaningfully engage actors, their interests, norms and knowledge to pursue coordinated and adaptive conservation, and (3) effectively linking conservation initiatives and social actors across scales and levels (Table 1). We make no claim to have articulated all social dimensions influencing conservation policy and practice at this point. Rather, these categories are reflective of the main issues from the literature on fit theory, and which are derived from applicable cases and lessons-learned from across the CT. Attempts to identify strategies to expand the inclusion of social dimensions in conservation in the CT have been plentiful (e.g., Lowry et al., 2009; Mills et al., 2010; Green et al., 2011; Foale et al., 2013; Weeks et al., 2014a,b; Berdej and Armitage, 2016), and a number of relevant conceptual frameworks are proposed (e.g., Ban et al., 2013; Kittinger et al., 2014). All are useful when discussing issues of conservation fit. However, there is limited practice-based guidance on how to move from recognition of the need for greater inclusion of social dimensions to actual operationalization of best practices in different contexts. Practicebased strategies to grapple with conservation fit issues (via e.g., trade-off analysis, ecosystem-based management, integrated coastal zone management) have been slow to emerge and face a range of implementation barriers (e.g., Folke et al., 1998/2007; Christie, 2011; Hirsch et al., 2011; Kittinger et al., 2014). In the next section we introduce bridging organizations as one potential way to help actualize the conditions and processes necessary to enhance conservation fit. ## Bridging Organizations for Fit Bridging organizations can help to improve conservation fit by taking on a number of roles and responsibilities. A bridging organization, as mentioned, is defined as an entity that connects diverse actors or groups through some form of strategic bridging process (Crona and Parker, 2012). These organizations come in many shapes and sizes, as well as levels of formalization. Brown (1991) argued that bridging organizations are central players in an increasingly multi-sectoral paradigm and hold a critical role in liaising actors to solve problems that neither would have been able to solve on their own. These organizations can provide an arena for knowledge co-production, trust building, sense making, social learning, vertical, and horizontal collaboration, and conflict resolution (e.g., Hahn et al., 2006; Olsson et al., 2007; Berkes, 2009; Schultz and Lundholm, 2010; Crona and Parker, 2012). Furthermore, they can fill technical and financial gaps by linking experts and expertise across levels of society, and by mobilizing ideas, resources and leadership. Inherent in bridging different social actors is often a need to overcome some degree of mistrust. Hence, consensus building and conflict resolution are important features in governance, but can be difficult to establish and maintain (Folke et al., 2005). Bridging organizations can facilitate depoliticized arenas that contribute to lowering institutional and cultural barriers between stakeholder groups and aligning their interests (Crona and Parker, 2012). Kowalski and Jenkins' (2015) case study on the science-policy interface of ocean management showed that bridging organization leadership coordinated collective action and resolved group issues within and among scientific and policy communities. Developing neutral space is advantageous for dealing with the ambiguity of multiple objectives, entrenched . #### TABLE 1 \| Categories of conservation fit and their key challenges in the Coral Triangle based on literature review(a) (a)This list is not intended to be inclusive of all issues of fit in the CT. (b)Many of the authors and examples listed here are applicable to multiple fit categories simultaneously. conflicts, and for navigating power differentials among social actors. Important contributors to successful conservation often include government and intermediary non-governmental organizations (NGOs), as well as local actors such as community groups, civil society organizations, and customary decisionmaking bodies. By building linkages to external social actors, bridging organizations help those at the local level to cross geographical and political scales in ways that would have otherwise been difficult, if not impossible. Hahn et al. (2006) showed how a bridging organization linked local actors with other levels of governments to generate legal, political and financial support in a wetlands landscape in Sweden. Through bridging, communities and others are able to gain access to non-local expertise and resources, including technical and financial resources, sources of technology, donors, and alternative trading networks (Folke et al., 2005). Such access can enable capacity building for more engaged or empowered involvement in conservation (e.g., Jamal et al., 2007). However, the literature also suggests a need for a more sophisticated understanding of the influence of bridging organizations on social interactions and social networks for governance generally (Crona and Parker, 2012), and for conservation governance specifically (Berkes, 2007; Jacobson and Robertson, 2012). Despite an increased scholarly interest in bridging organizations, few have empirically addressed their function and implications in conservation contexts (e.g., Hahn et al., 2006; Jamal et al., 2007; Jacobson and Robertson, 2012). This investigation builds on our recent work in the region, in which we report that bridging organizations contribute in several ways to positive governance outcomes by nurturing social networks and interactive processes (Berdej and Armitage, 2016). Here, we seek to further examine their capacity to deal with issues of conservation fit. We also expand the discussion of bridging organizations to assess the different ways through which they develop, implement, and adapt conservation initiatives to fit a broad range of social dimensions associated with conservation of coastal-marine systems (e.g., cultural context, local politics, knowledge systems, multiplicity of scales and levels). ## MATERIALS AND METHODS #### Research Context and Sites The Coral Triangle (CT) comprises marine waters of Indonesia, Philippines, Malaysia, Papua New Guinea, Solomon Islands, and Timor-Leste. The region is globally regarded for its extraordinary marine biodiversity (Allen, 2008) and its exceptional importance to local economies and societies (CTI Secretariat, 2009). As part of efforts to address marine resource decline, the Coral Triangle Initiative on Coral Reefs, Fisheries and Food Security (CTI-CFF) was established in 2009—a collaboration among the six nations to better manage the region's coastal and marine resources. The CTI-CFF sets out a diverse set of goals for the region, from an ecosystem approach to management of fisheries to climate change adaptation. The establishment and effective management of marine protected areas (MPAs) are seen as a key conservation tool in this regard, and comprise the CTI-CFF's third goal. Each of the CT nations has unique ecological, socio-cultural and governance arrangements for defining and establishing MPAs and other conservation initiatives. In Indonesia, the Government has committed to establish 20 million hectares (or 6.5% of territorial waters) of marine conservation area by 2020. MPAs here are declared and administered by national, provincial, and regency or municipal governments, and take on a number of forms (see White et al., 2014). In addition, there are a growing number of community-based conservation areas. Of the 15.7 million hectares of MPAs already designated, however, the majority of MPAs (>85%) offer little to no protection due to budgetary constraints, governance weakness, lack of marine management capacity, and political will (Burke et al., 2012; White et al., 2014). As stated above, these challenges are compounded by a deficit of understanding and incorporation of the social dimensions of conservation (Clifton, 2009; Foale et al., 2013; Fidelman et al., 2014; von Heland et al., 2014). Our research focused on three cases across Bali, Indonesia (Table 2). Cases were selected based on literature review and consultations with Indonesian partners and other experts using geographic and thematic criteria of relevance (e.g., Indonesia, marine, conservation, bridging, coastal-resource management, sharing, learning). Additional details on rationale for selection of bridging organizations can be found in Berdej and Armitage (2016). The use of the term MPA in our cases refers to a type of Indonesian conservation strategy entitled "Kawasan Konservasi Perairan" (literally translated to "aquatic conservation area"), whose definition encompasses both marine and freshwater areas that are managed by a zoning system. #### Data Collection and Analysis Data was collected over eight-months in 2013–2014, with a follow-up visit in January-February 2015. A case study approach (Yin, 2003) was used and included semi-structured interviews (n = 53 Nusa Penida, n = 54 East Buleleng, n = 20 Bali MPA Network), participant observation of key meetings (n = 5) and a literature review. Interviewees included individuals from government (n = 17), NGOs (n = 12), resource user groups (n = 19), other community groups (n = 11), traditional bodies (n = 3), private sector businesses (n = 14), universities (n = 1), and other (n = 1). Some of these organizations were affiliated with more than one study site. A combination of snowball sampling and purposive (or judgmental) sampling methods (Hay 2010) were used to identify participants. Snowball sampling is a technique whereby the current participant nominates subsequent participants (Hay, 2010). The approach is helpful to identify "hidden populations" or key individuals that might have otherwise not been known. Purposive sampling occurs where the researcher purposefully identifies individuals from the population based on her/his own knowledge and judgment. Themes covered in interviews included basic organization details, affiliations and relationships, conservation management and implementation processes, interactions and perceptions of bridging organizations, and constraints and barriers. Interviews were conducted face-to-face in English or Bahasa Indonesia with the aid of a local research assistant. The majority of interviews were recorded by handwritten notes, given that a digital voice recorder was deemed inappropriate to the context. Key public meetings were observed related to each of the cases on the topics of marine planning and MPA socialization. A literature review was conducted to complement and validate data collected, and focused on thematic areas stated above. Documents included annual reports, policy briefs, copies of presentations and newspaper articles. Data analysis was framed around the three conservation fit categories outlined in the previous section (Table 1). These categories were developed from a review of relevant literature on fit theory, and using applicable cases and lessons-learned from across the CT. Analysis of qualitative data from the field (including interviews, participant observation and some document collection) was carried out using an inductive approach to provide insights into emerging patterns of strategies used by bridging organizations. These findings were sorted and grouped, and then linked to one of the three conservation fit categories. We acknowledge that the use of pre-defined categories may overlook or restrict other themes. To counter this, we intentionally chose broad categories to allow for findings to emerge as unrestrained as possible from the raw data, while also linking them to the theoretical base driving the research. This research was carried out with approval from the Office of Research Ethics at the University of Waterloo (Ethics Approval Number 17930). All participants gave verbal consent prior to conducting interviews. An information sheet explaining the purpose of the research and how data would be used was read and/or translated verbally to participants. Participants were made aware of their right to withdraw participation from research at any time. ## CASE STUDIES We introduce three cases below that are illustrative of the diverse ways bridging organizations can influence conservation fit in Bali. This section is organized by case, as opposed to fit #### TABLE 2 \| Study site summaries. (a)The difference between an "established" MPA and a "declared" MPA is the state of its spatial zoning and management plans. category, to give the reader a more holistic understanding of the conservation setting and of how bridging organizations are situated therein. Each sub-section briefly outlines the context, followed by an introduction of the bridging organization or organizations, and an overview of their roles and responsibilities. Results are synthesized according to each of the fit categories of our framework in the section that follows. #### Toward a Bali MPA Network—Crossing Scales, Crossing Boundaries Context The province of Bali is located in the westernmost end of the Lesser Sunda Islands, covers almost 565,000 hectares, and comprises the main island of Bali and a series of satellite islands. High marine biodiversity is documented in the area (Mustika et al., 2012), and important habitats include coral reefs, mangrove forests and seagrass beds. There are over four million people in the province, spread across eight administrative regencies and the capital city of Denpasar. Coastal and marine resources are a cornerstone of Bali's economies and societies, supporting livelihoods such as fisheries, ornamental fish collection, mariculture (e.g., shrimp, fish, seaweed) and a burgeoning marine tourism industry. Partial decentralization, as mentioned earlier, has led to fit challenges associated with poor coordination between levels of government, policy inconsistencies, and non-conformities in the licensing, policing and use of coastal-marine resources between regencies (see Patlis, 2005). The inequitable distribution of assets and access to these resources has fueled ongoing conflicts between villages, between regencies, and between sectors. Together, these have hindered efforts to address pressures from overfishing and destructive fishing practices, marine litter and nutrient run-off, and the rapid development of coastal areas and watersheds (Mustika et al., 2012). In this context, the environmental NGO Conservation International Indonesia has emerged as a key player in the movement toward coordinated, cross-scale conservation practice. #### Conservation International Indonesia (CI-I) Since 2010, Conservation International Indonesia has been a driver behind the development of a Bali MPA Network (hereafter "Network"; Indonesian: Jejaring Kawasan Konservasi Perairan). CI-I has been active in Indonesian seascapes in general since 2004 with a mission of "building upon a strong foundation of science, partnership and field demonstration, [to empower] societies to responsibly and sustainably care for nature, our global biodiversity, for the well-being of humanity" [CI-I (Conservation International Indonesia), 2015: website]. In Bali it has taken on a number of roles and responsibilities, including: biological monitoring to inform Network design; identification and engagement of partners; coordination of activities related to Network planning; and facilitated development of a management planning document (hereafter "Blueprint"). To initiate planning for the Network, CI-I and its partners facilitated a multi-stakeholder workshop in 2010 and together identified 25 sites across Bali for possible inclusion. Site selection was informed by some 66 representatives from government, universities, NGOs, private sector, and community and traditional leaders in attendance from across the province. Marine Rapid Assessments were then carried out by CI-I in each of the proposed sites with data collected about marine biodiversity, coral reef community structure, and current condition of coral reefs and related ecosystems (see Mustika et al., 2012). This was combined with earlier assessments (Allen and Erdman, 2008) and used to inform the evolving design of the Network. Included was the recommendation of nine of the 25 sites for priority as MPAs due to their high ecological, economic and cultural value. The Network was formally initiated in 2013 through a memorandum of understanding signed by all ten heads of marine affairs and fisheries agencies in Bali—comprising nine regency agencies and one provincial agency. Its overall visions is "the creation of harmony and synergy between national, provincial and regency governments in Bali in the management of aquatic resources, with strong support and participation of the community and other institutions, and for the sustainable enhancement of social, economic and cultural benefits" (Gunawan and Dewantama, 2014, p. 7 translated). In practice, the Network is intended to foster cross-boundary coordination to synergistically align all aquatic-related efforts of regencies with the province, while at the same time, respecting the autonomous rights of regencies to manage programs in their territorial waters (CI-I staff, personal communication 2014). A multi-stakeholder, multi-agency task force was established for Network planning, comprising 28 representatives from provincial and regency government (including tourism, environment, planning, and marine and fisheries agencies), existing parks and reserves, traditional councils, and NGOs (see Bali Gov. Decree, 2013). The task force is chaired by the head of the Bali Ministry of Marine Affairs and Fisheries, and network members have described the role of CI-I as project lead and coordinator. Other groups such as local governments and civil society organizations are not members of the Task Force. However, they are expected to contribute to individual working groups on policy-making, spatial planning, and funding as part of the ongoing planning process (which has yet to begin; see Gunawan and Dewantama, 2014). The MPA Network is based on the principle of "One Island, One Management" through which Bali is viewed as a singular ecosystem comprised of terrestrial, marine and aerial space that requires integrated, cross-scale management to deal with conservation challenges. This has been described as a "...need to manage as an island instead of eight or nine separate entities within the island...[where regencies] have to sit down together to talk about general issues and the environment" (anonymous personal communication 2014). Objectives are set for ecological and social connectivity to "...braid cooperation between MPA mangers in Bali for more effective, efficient, comprehensive and sustainable management and conservation" (Gunawan and Dewantama, 2014, p. 21 translated). This is a means for actors to share their experiences, lessons learned and capacities. Three pillars inform the ideology the Network—scientific evidence, rule of law, and culture. A series of Balinese "local wisdoms" have been adopted, including: "Nyegara Gunung" (translates to "ridge to reef " that signify the integration of mountains and sea), "Tri Hita Karana" (a philosophy on sustainability emphasizing interrelation and harmony of human, God and nature), and "Sad Kerti" (six strategies to maintain the balance of nature that are comprised of soul, human, forest, lake or fresh water, sea and the universe). In practice, this translates to a fixed inclusion of local and cultural values, as well as cultural seascapes, in the design and implementation of MPAs. The inclusion of Balinese wisdoms is also intended to uniformly strengthen the "cultural sovereignty of Balinese in conservation" (CI-I staff, personal communication 2014). To support coordination and operation of the Network, a Blueprint document was created to provide consistency in approaches and laws in the planning of aquatic areas across Bali, as well as in setting minimum standards of compliance. These guidelines are to serve in part as reference in developing protected areas (marine or terrestrial) at the level of regency, and include ecological, socio-economic and governance considerations (see Gunawan and Dewantama, 2014). Still, there are numerous challenges facing the actualization of the Bali MPA Network. Cooperation from governments and stakeholders remains problematic given conflicting interests, high turnover of government staff that inhibits relationshipbuilding, and a general lack of trust between groups. An NGO representative was careful to make the distinction between those organizations or agencies in the MPA Network that were "happy" to be included but rarely participate, and those who were "enthusiastic" in moving the process forward by actively participating (local NGO rep., personal communication 2014). Many regencies still do not have dedicated staff, nor sufficient budget, for MPA planning and implementation. In addition, concern has also been raised about the possibility of conflict where the "One Island, One Management" idea could be interpreted by some as an attempt by the province to regain power over coastal-marine decision-making (national NGO rep., personal communication 2014). #### Nusa Penida MPA—Pluralism and Multiple-Use in Conservation Context The Nusa Penida MPA is located southeast of the Balinese coast comprising three islands: Lembongan, Ceningan, and Penida. Its 46,000 inhabitants are distributed across 16 administrative and 46 customary village divisions. Major livelihood activities include capture fisheries (≈850 local fishers in 40 fishers' associations), seaweed production (≈308 ha of farms), and marine tourism (over 200,000 tourists per year; Ruchimat et al., 2013). The area is well known among divers for its large charismatic species such as the ocean sunfish (Mola mola) and manta ray (Manta birostris). Nusa Penida is part of the Klungkung Regency, Bali Province. In addition to regency and village administrative laws, there is customary law implemented by local traditional bodies (Indonesian: Adat) and a Tribes' Council (Indonesian: Majelis Alit). This law is focused on religious and cultural activity, but can also include rules and sanctions associated with natural resources. In Lembongan, for example, customary law forbids logging of mangroves or collection of sea sand. Other regulatory bodies on the islands include a newly formed consortium of diving businesses, and separate fishers' and seaweed farmers' associations through which activities are regulated socially. Intensive utilization of coastal resources and overlapping or competing income-generating activities in a relatively small region such as the one presented here, has posed challenges to fit, and contributed to many ecosystems becoming overexploited (see Welly, 2009). These too have fueled conflicts between various user groups (e.g., tourism and fishers, tourism and seaweed farmers). Here an NGO bridging organization has taken on the central role of facilitating the region's many stakeholders and uses in creating and managing the MPA. #### The Coral Triangle Center (CTC) The Coral Triangle Center, an Indonesian environmental NGO focused on capacity building, has been the lead facilitator of the Nusa Penida MPA since it was initiated in 2008. At the time, CTC was a subsidiary of the US-based NGO The Nature Conservancy, but became an independent foundation in 2010 and now operates in multiple sites across Indonesia. A key objective of CTC is to "...stimulate partnerships with leaders in sectors such as tourism, fisheries, agriculture, and business development, recognizing that holistic and inclusive approaches are necessary for the sustainability of coastal ecoregions and health and economy of local communities" [CTC (Coral Triangle Center), 2011, p. 2]. The major roles of CTC in the MPA include: identification and engagement of local partners; collection of stakeholder inputs and data to inform MPA design; coordination of activities related to MPA planning; and technical advisory and training. Preceding the declaration of the MPA, CTC coordinated a series of 33 public consultations to gather input and mutual agreement on MPA establishment—some 1200 individuals from 16 villages participated between 2009 and 2010 (CTC staff, personal communication 2014). This information would later inform MPA design. In 2010, the Nusa Penida MPA was officially declared by decree of the Head of the Klungkung Regency Government (decree no.12/2010). In an effort to better align benefits to local stakeholders with marine conservation, three objectives were established: (1) biodiversity protection, (2) sustainability of fisheries, and (3) sustainability of marine tourism. A multi-agency, multi-stakeholder working group was created and tasked with disseminating information and undertaking preparations for the MPA. The MPA design process was informed by scientific data (biological assessments and socioeconomic surveys), policy assessments of law and regulation, and stakeholders' input. To be inclusive of the many stakeholder groups, and their interests and knowledge, CTC conducted an additional 30 public stakeholder meetings at the village and regency levels about boundaries and zoning preferences. According to CTC staff, one of its major roles is to "bring people together" (CTC staff, personal communication 2013)—it engaged and included stakeholders from regency (Klungkung Regency) and central governments, NGOs, community groups, tourism operators, traditional leaders, teachers, youth groups, and local fishers' and seaweed farmers' associations. The resulting MPA zoning system consists of four maritime zones and a series sub-zones: (1) core zone for education and research purposes (469 ha), (2) sustainable fisheries zone including traditional fisheries sub-zone (16,916 ha), temporally controlled special use sub-zone (905 ha) (see below), and seaweed farming sub-zone (464 ha), (3) utilization zone—including marine tourism sub-zone (1221 ha) and marine harbor sub-zone (35 ha), and (4) other zone—including traditional sacred subzone (47 ha). This zoning system integrates utilization activities and cultural perspectives alongside biodiversity conservation, and in balance. To ensure impacts on local fishers were minimized, some 80% of MPA waters remain accessible either as prioritized fishing grounds or in multiple use zones. Existing seaweed-farming territories on each island were allocated their own zones. A desire to protect and integrate Balinese culture into planning led to the creation of a "traditional sacred zone," which limits speedboat and tourist access in waters located adjacent to an important temple on the coast. To minimize conflicts between fishers and marine tourism operators in a number of areas along the north coasts of Nusa Penida and Nusa Lembongan, "special use zones" were created to allow temporally controlled access. Between the hours of 4 p.m. and 9 a.m. fishing is permitted in these areas, however, outside of these hours only marine tourism activities are permitted. A pluralist management unit comprised of representatives from various actor groups was formalized in 2013 to allow for representative decision-making, and is supported by a joint patrol team, and biophysical and socioeconomic monitoring teams facilitated by CTC. Team representatives include those from regency government, traditional village police, fishers' associations, the Indonesian Navy, the Indonesian Police Unit, local dive operators, the Tribe's Council, and associated NGOs and community groups. Joint patrols and monitoring are conducted monthly. In addition, CTC coordinates annual reef health monitoring surveys in 12 sites across the islands together with the Management Unit and local partners, and conducts community perception and engagement surveys every two years. These activities are meant to both build skills and capacity for local stewardship (via training and certification of locals by CTC), as well as foster learning that feeds back into the ongoing development of the MPA. In addition to the aforementioned bodies, the process of MPA development has helped to connect several new social networks within different interests in Nusa Penida. For example, an association of local dive operators was founded to link businesses and self-regulate dive tourism practices through agreed codes of conduct. Likewise, a mangrove tourism association to connect local fishers arose out of CTC-led efforts to develop communitymanaged mangrove ecotourism. In addition, a memorandum of understanding was recently signed with the management unit of Nusa Penida MPA to enable CTC to use the area as an "MPA Learning Site" and living laboratory for learning exchanges and training visits among practitioners and sites across the CT region. However, the MPA faces a number of new and ongoing challenges moving forward. Unsurprisingly, building stakeholder relationships is a work-in-progress. Some respondents made note of ongoing tensions between and within groups, particularly between on- and off-island fishers or tourism operators, and between snorkeler and dive operators. Both cultural and language barriers persist between some stakeholder groups. Concern has also been raised about the burgeoning tourism industry and the ability to regulate and enforce tourist carrying capacities on reefs given the number of informal and off-island operators. ## East Buleleng Marine Conservation Zone—Scaling-Up Empowered Community Conservation #### Context The Marine Conservation Zone resides along 26 km of coastline located in northeastern Bali. This is the province's richest area for fish diversity (Mustika et al., 2012) and includes important habitat for marine life such as whale sharks, sea turtles and dolphins. Its 54,000 inhabitants are distributed across ten administrative and 60 customary village divisions that comprise the Tejakula sub-district. Coastal communities rely on fisheries (≈2000 local fishers in 47 fishers' associations), the marine aquarium trade, aquaculture (shrimp, fish, seaweed) and tourism to meet subsistence and livelihood needs [DKP (Dinas Periknanan and Kelautan, Pemerintah Kabupaten Buleleng), 2015]. According to the head of the ornamental fishers association and NGO field staff, there are less than 100 ornamental fishers in the sub-district. Tejakula is part of the Buleleng Regency, Bali Province. Similar to Nusa Penida, coastal-marine regulations here stem from regency and village administrative laws, as well as customary law. Other regulatory bodies include fishers' and ornamental fishers' associations, and community groups responsible for Locally Managed Marine Areas (LMMAs) (Indonesian: Daerah Perlindungan Laut). Major challenges to fit here include intraand inter-community tensions associated with overlapping use and access. For example, the ongoing development of beachfront hotels has meant increasing exclusion of fishers and ornamental fishers from marine spaces. Local people are highly dependent on coastal-marine systems and livelihood alternatives are limited. In addition, capacity to combat environmental threats such as coral mining and pollution, as well as destructive and illegal fishing practices, is limited. Two environmental NGOs have played central, but differing, roles in supporting a transition toward community empowered conservation practice in this region: Reef Check Indonesia and the Indonesian Nature Foundation. #### Reef Check Indonesia (RC-I) Reef Check Indonesia, a chapter of a US-based environmental NGO of the same name, has been active in the Buleleng region since 2006. The NGO embodies a philosophy of "integrated coastal and marine ecosystem management to enhance the welfare of coastal communities" [RC-I (Reef Check Indonesia), 2015: website] and was founded on three pillars of activity: science and technology, collaborative management, and education and awareness. Their main office is located in south Bali, but at the time of data collection a member of RC-I staff was also housed semi-permanently in the office of the Ministry of Marine Affairs and Fisheries, Buleleng. RC-I has taken on a number of roles in the region, including: support of LMMA planning; facilitation of traditional guards; community capacity building and training; and coordination of MPA design and development. Between 2008 and 2009, RC-I worked together with community members and local governments in developing a series of LMMAs in villages across the sub-district, with the aim to curb illegal activities and promote sustainable resource use. LMMA zoning was guided by a mix of local knowledge and scientific data collected by RC-I on coral reef health. According to staff, this involved "sharing sessions" held with different organizations—such as fishers' associations, traditional authorities, community groups, local NGOs and tourism operators—to better understand and integrate their interests in conservation solutions that "accommodate collective importance" (RC-I staff, personal communication 2014). Zoning was undertaken on a village-by-village basis and includes categories for: core zones where extraction activities are prohibited, buffer zones where limited fishing is permitted, and utilization zones where non-destructive activities are permitted. As well as establishing LMMAs, community-based organizations were created for each, and take on the majority of responsibility to implement, manage and monitor these spaces. The head of one such organization described its purpose as helping to create a more sustainable marine environment, while at the same time educating their community and improving community welfare (LMMA rep., personal communication 2014). In this context, RC-I has directed effort to building local capacity—it conducts training on practice and theory of marine ecology and conservation, diving skills (general and scientific), and ecological monitoring techniques (snorkeling and diving). Local fishers are taught and certified to identify and record the health of their coral reefs and fisheries, and have been actively collecting data both independently and alongside RC-I over the last 5 years (LMMA rep., personal communication 2014). Dive training has served the dual purpose of conservation and ecotourism: several LMMA organizations are also tourism dive centers. RC-I has sought to strengthen local stewardship by inaugurating certified diver fishers into community groups called "Pecalang Segara" or "traditional guardians of the sea." The marine-based Pecalang are an extension of the terrestrial-based traditional body (i.e., Adat). Following training, they are tasked with undertaking surveillance and enforcement of regulations in LMMAs. According to the head of an LMMA organization, the enacting of Pecalang strengthens the community's "cultural responsibility" to protect the environment (LMMA rep., personal communication 2014). In 2011, RC-I partnered with the Ministry of Marine Affairs and Fisheries, Buleleng to facilitate the designation of the East Buleleng Marine Conservation Zone, part of a regency-level MPA that would include the already-established LMMAs. The process of scaling-up began in 2013 through a series of public consultations at the village and sub-district levels to gather input and mutual agreement on MPA zones, boundaries, and allowable activities. In attendance were members from fishers' and ornamental fishers' associations, hotels and spas, government, local NGOs, community associations and others. A regency government official explained that MPA zones are meant to align with those in existing LMMAs so that one would strengthen the other (government rep., personal communication 2014). At the time of data collection, substantial progress had been made in zoning, but finalization had yet to take place. The zoning system will include four categories: (1) core zone for protection of ecosystems, traditional cultural sites, and research and education; (2) limited use zone—for tourism and recreational activities, as well as research and education; (3) sustainable fisheries zone—for non-destructive catch and cultivation of fish, tourism and recreational activities, as well as research and education; and (4) other zone—for specific purposes such as port harbors, rehabilitation of specific marine biota or traditional territories. Similar to the Nusa Penida MPA, this zoning system is meant to balance utilization activities and cultural perspectives alongside objectives for biodiversity conservation. However, the creation of LMMAs and subsequent MPA has not been embraced or accepted by all. Numerous fishers and ornamental fishers voiced discontent about their exclusion or the extent of their exclusion from coastal areas. There is also persistent belief among some community members that the word "conservation" implies absolutely no use activities permitted. One business owner explained that it will be difficult for some fishermen to see the benefit of the MPA because they tend to think short term, and MPA benefits will be a long-term gain (business owner, personal communication 2014). #### The Indonesian Nature Foundation (LINI) The Indonesian Nature Foundation has been active in the Buleleng Regency since 2008, with many of its staff having operated in the Regency since 2000. LINI is an NGO from south Bali with a mission to "...work with marginalized coastal communities to reverse the degradation of Indonesian coral reefs and raise awareness about responsible and sustainable marine resource use" [LINI (The Indonesian Nature Foundation), 2015: website]. It works most closely at the community level, particularly with the villages of Les and Penuktukan, to foster a sustainable marine ornamental fishery as part of wider conservation efforts. LINI subscribes to the idea that "...you cannot force people to protect the environment, [rather], you have to start by helping them with livelihoods and understanding (education)" (LINI staff, personal communication 2013). In this respect, it has taken on a number of roles, including: community capacity building and skills training on reef restoration and ornamental fishery; biological and socioeconomic data collection; identification and engagement of local partners and partnerships. LINI has been a leader in building capacity for communitydriven coral reef restoration. It trains local fishers in the production and installation of various types of artificial reef structures, including fish domes, shrimp pods, and "roti buaya" (rough logs of artificial substrate). These are made, deployed and occasionally designed by villagers themselves. With help from LINI, fishers from Les village have taken on stewardship of reef restoration in the area since 2010. As of January 2014, over 100 fish domes and 1000 shrimp pods had been installed on the reef in multiple sites in East Buleleng (ornamental fisher, personal communication 2014). These structures serve the dual purpose of encouraging coral re-growth, and providing nurseries for the marine aquarium trade to fuel local livelihoods. Alongside reef restoration activities, LINI has sought to foster human and institutional capacity in coastal communities for a sustainable ornamental fishery, including sea and land-based aquaculture development. The gathering of ornamental fish has a rich history in the region, but it has tended to come with destructive practices such as cyanide use (e.g., Frey and Berkes, 2014). LINI delivers practical skills training about e.g., marine conservation, fish collection methods, post-harvest handling techniques, fish rearing and mariculture, and diving (general and scientific). It has assisted in the development of an ornamental fish export business by community fishers, including the building of land facilities for a fish rearing program (ornamental fisher, personal communication 2014). Construction has recently been completed on a new Aquaculture and Training Centre in Les village designed to offer skills training, research and work experience in marine conservation and aquaculture. In addition, LINI plays an important role in collecting and distributing information across scales. It has described itself as "...a big knowledge hub, and a trafficker of information" (LINI staff, personal communication 2014). The NGO has established and maintained an extensive database on ornamental fish harvests, fisheries catches, supply chains, and aquaculture data from the village to regional scales. As well, it has been monitoring the progress of reef restoration by recording numbers and species of fish. This information is collected by LINI staff, community members, or with other NGOs such as RC-I. LINI works with regency government on the use of such data to inform fisheries quotas in the region. However, despite strides in the advancement of a sustainable ornamental fishery, some concerns have been raised about its long-term viability in the region. An ornamental fisher explained that many stakeholders in the area—including some local authorities and tourism operators—continue to be suspicious of the activities of ornamental fishers (ornamental fisher, personal communication 2014). It has an unfavorable image, he explained, even though methods have changed significantly. In addition, there are far fewer ornamental fishers than pelagic fishers and, subsequently, their position in the region may not be as strong. ## RESULTS: CONTRIBUTIONS OF BRIDGING ORGANIZATION TO CONSERVATION FIT Results are organized here according to the three main categories of conservation fit outlined in our framework earlier in the paper. These include: (1) aligning conservation initiatives with characteristics of the social context (e.g., institutions, culture, values, local practice), (2) facilitating governance processes and instruments to bring together and meaningfully engage actors to pursue coordinated and adaptive conservation, and (3) effectively linking conservation initiatives and social actors across scales and levels. We identify and discuss in detail the strategies used by bridging organizations to promote and sustain aspects of conservation fit, which are summarized in Table 3. To this end, we draw on specific examples and evidence (e.g., from interviews, document review) from the cases above, as well as surveyed responses from participants about bridging organization contributions (Table 4). As illustrated below, however, not every strategy was employed in every case or to the same degree. ## Alignment with Social Context Integrating Actors and Interests Bridging organizations help to identify and represent multiple social actors and their various and often divergent interests. It is widely acknowledged that the long-term success of a conservation intervention hinges in part on its integration with (local) people, and by association of their needs for livelihood and wellbeing (see Ferse et al., 2010; Glaser et al., 2015). Our cases in Nusa Penida and East Buleleng show how bridging organizations use public meetings, community consultations, and focus group discussions to identify and elicit information about the interests and resource use patterns of affected stakeholder groups. To accommodate this heterogeneity in conservation initiatives, we observed that bridging organizations exercised flexibility in design and implementation. Indeed, all bridging organizations examined in this paper showed some degree of flexibility in their integration of multiple alternative objectives. In East Buleleng, for example, a process of #### TABLE 3 \| Summary of results. (a)CI-I, Conservation International Indonesia; CTC, Coral Triangle Center; RC-I, Reef Check Indonesia; and LINI, Indonesian Nature Foundation. (a)Respondents were asked, "how does [X] bridging organization contribute to marine conservation and management processes in the [region/initiative]?" (b)The initial categories included here were further refined and consolidated in line with the main themes in Table 1. (c)The "other" category included contributions listed such as funding, administrative tasks, technical facilitation, creating new rules, providing checks and balances, and supplying data. multi-use zoning was used in order to represent and integrate the different interests of social actors related to biodiversity protection, sustainable fisheries, ornamental fisheries, marine tourism and culture. A community member here explained, I don't want to do just conservation. I want conservation for all for people, for culture. There needs to be balanced conservation that includes nature, but also people and their needs, their culture, their recreation, and their economic status. There needs to be a balance between nature conservation and social conservation. (community member, personal communication 2014) The CTC similarly orchestrated multi-use spatial and temporal zoning in Nusa Penida to resolve overlapping objectives between fishers, seaweed farmers and marine tourism activities. Other strategies, such as the utilitarian approach applied by LINI, explicitly identified synergies between social and ecological objectives. A representative of LINI stated, Absolutely "no-take" areas are problematic. They are not feasible according to the Balinese way of living. That would mean no fisheries, no tourism. [...] In Indonesia, people have the philosophy that "nature is there for us to use." Conservation must consider this. (LINI staff, personal communication 2014) These actions are in line with calls from across the CT for greater flexibility in conservation, where solutions seek to balance the immediate needs of resource users with conservation or longterm sustainability agendas (see Foale et al., 2013; von Heland et al., 2014; Weeks et al., 2014a). #### Knowledge Diversity Bridging organizations help to integrate knowledge systems and perspectives from different social spheres. Scholars advocate drawing from, and combining, multiple types of knowledge to better understand the conservation context and problem (e.g., Majors, 2008; Clifton and Majors, 2012). A representative from RC-I described this process as finding the "right mix of science and culture" for conservation initiatives (RC-I staff, personal communication 2013). Another interviewee commented on the inseparability of the two: "when we talk about Bali, you cannot avoid the culture...once you talk about marine, you talk about terrestrial, you talk about the people, about culture" (CI-I staff, personal communication 2014). The incorporation of scientific and technical knowledge in our cases was achieved where bridging organizations connected to universities, local research institutes, NGO scientists, and/or managers. Each bridging organization also included its own research-oriented activities to collect scientific data: CI-I undertook marine rapid assessments, CTC carried out biophysical and socioeconomic baseline surveys, and RC-I and LINI collected data on the state of coral reef health and fisheries. The incorporation of local and traditional knowledge in our cases was achieved where bridging organizations involved the expertise of those with long-standing ties to the area—community members, traditional leaders, resource users, teachers, etc. For example, the experience-based knowledge of ornamental fishers in East Buleleng has been used to guide the installation of some artificial reef structures, and traditional custom (i.e., Adat) has been incorporated and reinforced in MPA planning in Nusa Penida through the creation of a sacred zone. Likewise, "local wisdoms" such as "Tri Hita Karana" and "Nyegara Gunung" have been integrated into the Bali MPA Network so as to merge scientific ideas of conservation (e.g., ecological connectivity, social networks) with the Balinese cultural perspective (e.g., "ridge to reef " thinking, harmony between human and nature). A government official added, "If BMN (Bali MPA Network) is applied with awig-awig (customary law), it will work very strongly because most Balinese think of the ocean and beach as sacred place" (government rep., personal communication 2014: translated). #### Facilitating Appropriate Governance Hybrids and Inclusiveness Bridging organizations help actualize hybrid forms of decisionmaking that combine different sets of public, private and civil society actors. Hybrid approaches reflect recognition that many coastal-marine resources are too complex to be governed by a single social actor or agency (Berkes, 2009). One interviewee commented, "we cannot do conservation alone. It requires a long process of negotiation and compromise between many groups of stakeholders" (government rep., personal communication 2014). One way bridging organizations in our cases pursued inclusiveness was to support co-governance arrangements, consisting of collaboration and interplay between diverse representatives from across sectors and scales. In Nusa Penida this took the form of a multi-stakeholder, multi-agency working group (now management unit), and in the Bali MPA Network this was expressed as a 28 member joint Task Force. Hybridizing was also pursued in merging local institutions as part of governance frameworks. In East Buleleng, for example, RC-I helped integrate aspects of customary institutions (i.e., Adat) with conservation governance by extending and incorporating the Pecalang Segara as traditional territorial authorities in LMMAs. This was similarly carried out in the Nusa Penida MPA. A general consensus is that broadening meaningful participation, especially of local communities, is indispensable for the success of marine conservation in the CT and beyond (Christie et al., 2003; Mascia, 2003; Clifton, 2009; Ferse et al., 2010; Glaser et al., 2015). In expressing greater inclusion, a community member in Nusa Penida stated, "...CTC provides a link between government and [us]. They give us a voice" (community rep., personal communication 2014). Opportunities for stakeholder inclusion and input facilitated by bridging organizations in our cases ranged from participatory mapping of resource use, public meetings and focus group discussions on zoning, to membership on monitoring teams, patrol units, and joint committees. In practice, such opportunities become venues for discussion and debate, coordination, sharing information, mobilizing resources, and organizing training activities. #### Capacity Building Bridging organizations aid in building requisite knowledge, skills and capacity for conservation practice and governance, especially where sub-national or local governments lack the capacity (or desire) to fill gaps. Methods observed to foster (local) capacity and leadership ranged from formal to informal. Capacity building activities undertaken by RC-I in East Buleleng, for example, have enabled LMMA managers to actively participate and assume increasing responsibility for planning, implementation, ecological monitoring, and enforcement in their coastal-marine areas. The NGO described an aim of its activities to "...broaden the roles of community members from fishers to tourism operators and reef protectors" (RC-I staff, personal communication 2014). Enlisting resource users in data collection and analysis educates participants, builds capacity and can foster trust (Mascia, 2003). Likewise in Nusa Penida, joint patrol and monitoring teams now perform the tasks of enforcement and data collection following facilitation and training by CTC. In describing their interactions, a representative from a local community organization stated, CTC has provided training to us and have built our capacity to make collaborations and strengthen management. [...] We now serve as a facilitator for the socialization and communication of the MPA and work with various stakeholders about conservation issues in the context of the MPA. (community organization rep., personal communication 2014) Some bridging organizations also advocated local leaders, and not just involvement, in conservation governance. An NGO member expressed the importance of fostering "local champions" to facilitate on-the-group relationships and build stewardship over conservation initiatives (international NGO rep., personal communication 2014). Attempts to decentralize leadership included those where bridging organizations sought to empower locally based organizations (as in the case of LMMAs) and where initiatives were managed and implemented by community members (as in the case of reef restoration). As well, the embedding of key community or traditional leaders in conservation planning and implementation teams, such as working groups, management units or patrol teams, strengthens the overall involvement and conservation leadership of community members. ## Alignment of Scales #### Connectivity As entities that connect others, bridging organizations convene a diversity of social actors to create and hold together scalebridging social networks for conservation. Social networks are important to embrace diversity of perspectives and knowledge representing multiple social actors across seascapes to facilitate adaptive thinking (cf. Folke et al., 2005; Armitage et al., 2009). Through bridging efforts, horizontal linkages have been cultivated across, for example, regency government agencies (as in the case of CI-I) and community groups (as in the case of the CTC). Vertical linkages meanwhile have been fostered between, for example, communities and governments (as in the case RC-I and CTC), and between resource use associations and market actors (as in the case of LINI). Bridging organizations were also the catalyst for the formation of sub-networks of stakeholders focused on particular issues such as dive tourism and mangrove ecotourism in Nusa Penida MPA. Some bridging organizations in our cases have worked collaboratively in the region for upwards of a decade strengthening connectivity between social actors. This is an important pre-condition for coordination, communication, and learning in conservation across the CT (see Lowry et al., 2009; Cohen et al., 2012; Pietri et al., 2015). For example, the CTC connects Nusa Penida MPA to a wider "learning network" of MPAs, which allows managers and practitioners to share knowledge and experiences between sites in the CT and beyond. Similarly, under the guidance of CI-I, a key function of the Bali MPA Network is to connect MPA managers across the province to enable the exchange of experiences and knowledge: There are many, many NGOs and other organizations that work in Bali, and have not always coordinated. [...] The Bali MPA Network is good to share lessons. It serves as an umbrella for multiple organizations to collaborate and connect...it is about sharing knowledge. (national NGO rep., personal communication 2014) Coordination with other stakeholders is difficult because each stakeholder has their own interest, and sometimes this leads to conflicts. BMN (Bali MPA Network) will support information exchange between each regency's DKP (Ministry of Marine Affairs and Fisheries), and conflicts caused by misunderstandings or lack of information could be reduced. (provincial government rep., personal communication 2014) #### Scaling Bridging organizations help foster cooperation to appropriately scale conservation initiatives across geographic and governance boundaries. As urged elsewhere in the CT (Lowry et al., 2009; Green et al., 2011), bottom-up as well as top-down conservation ingenuity is needed. This is shown in the Bali MPA Network, where transboundary conservation is planned to foster coordination across provincial, regency and city units of governance, as well as across sector boundaries (tourism, environment, planning, fisheries). In explaining the challenge, one interviewee stated, Administrative separation by regency has causes differences in managerial decisions and policies between regencies. Bali is a small island, therefore the marine area around Bali is ecologically connected [...]. This means regency management will not work without synchronization with other regencies. This is where BMN (Bali MPA Network) is needed to unite marine management systems in Bali. (NGO rep., personal communication 2014) Here, provincial-level prescriptions are a starting point to identify spatial priorities and provide guidelines for the process of MPA design and implementation, which can be scaled-down and adjusted to accommodate local context and opportunities. Alternatively, under the guidance of RC-I, LMMAs in East Buleleng are being scaled-up and reinforced by higher-level governance units through the development of a regency-level MPA. Aligning conservation initiatives with the regency unit of governance was needed to enforce and implement rules that are beyond the reach of community sanctions, and to resolve inconsistencies and conflicts between LMMAs. ## DISCUSSION: OBSERVATIONS ON BRIDGING AND STRENGTHENING CONSERVATION FIT The cases presented in this paper illustrate that bridging organizations can and do promote and sustain aspects of better conservation fit, although with some limitations. In this regard, conservation fit is a means to an end, not an end to itself. By enacting bridging strategies that integrate actors and interests using flexible approaches, actualize hybrid forms of decision-making, build capacity and leadership, and foster crossscale conservation and scale-bridging social networks, bridging organizations are indeed successfully enhancing aspects of conservation fit. The outputs of these efforts include conservation initiatives that are better aligned with their social contexts, which bring together and empower various public, private and civil society actors, and which better connect people and actions across scales and levels in ways that are locally beneficial. Our findings show that not all bridging organizations made use of the same bridging strategies or did so to the same degree. In part, this is because bridging organizations and the conservation fit issues they seek to address vary with context. Most bridging organizations have distinct identities, priorities and strengths or weaknesses that undoubtedly come into play (see Berdej and Armitage, 2016). This implies that different bridging organizations may have different niches with regards to addressing conservation fit issues. Simultaneously, issues of fit can vary by strength, complexity, urgency and/or scale. Recognizing this variation is important to understand how different bridging organizations can be engaged in different ways to address particular conservation misfits. We observed that bridging organizations share a number of unique features that make them well poised to grapple with conservation fit issues. First, the organizations we studied are able to work across the political or jurisdictional, programmatic and scalar boundaries that tend to serve as organizational barriers to collaboration and information sharing elsewhere. Second, the bridging organizations examined here are positioned at the intersection of diverse actors, and so they are able to draw on broader collections of partners—and their expertise, knowledge and resources—to work together in overcoming barriers and finding common ground. Third, these organizations embody a high degree of organizational flexibility, meaning they tend not to be under the same kind of bureaucratic restrictions or silos as government actors. This allows them to be more nimble in responding to emerging issues, shift programming according to needs, and alter their roles to suit current challenges. Our cases have also brought to light a number of new and ongoing constraints or barriers that indicate the challenges in achieving conservation fit. Social systems in the CT are invariably dynamic and heterogeneous, comprising multiple sub-groups with differing values, interests and priorities that can change and shift over time (see Fidelman et al., 2012, 2014). Bridging strategies that are successful in one place and time and with one set of stakeholders may not be successful elsewhere. By the same token, a bridging organization is subject to competing demands of various stakeholders, not all of whom have equal ability to voice concerns or exert influence. A major obstacle to fit then is overcoming power asymmetries (see also Clement, 2013). In Bali, for example, tourism is a main source of the province's revenue, creating imbalances with other sector interests such as fisheries. As well, corruption remains an ongoing issue (Fidelman et al., 2014), and curbing it is a priority if long-term conservation successes are to be achieved. Differing ideologies and understandings of conservation pose a sizable challenge to bridging organizations in the pursuit of better fit. Social groups embody unique knowledge of marine environments, and can have differing ideas of how resources should be conserved, used, or exploited (e.g., von Heland and Clifton, 2015). A business owner in East Buleleng explained this as: "...a balance between a village life that has been established for centuries, and the rather new and fanciful idea that we need to protect reefs, which has not been understood or grasped in its entirety meaning by the local people" (business owner, personal communication 2014). The integration of differing ideologies can be difficult in the CT given an overreliance on a western conservation narrative (Berdej et al., 2015), general lack of social science data generation, and limited involvement of domestic (social science) academics (Fidelman et al., 2014; von Heland et al., 2014). Bridging organizations may not possess comparable expertise on, for example, economic development, poverty alleviation, or urbanization (cf. Foale et al., 2013). Moreover, bridging organizations themselves, as mentioned, have their own ideologies, agendas and priorities that can favor particular viewpoints and narratives (see Berdej et al., 2015). There is therefore strong need for additional research on the political and ecological dimensions of bridging organizations in the region. Lastly, the pursuit of conservation fit can be time-consuming and costly. There are significant costs associated with bridging activities, including funding, time commitments, staffing, and resource expenses. Funding and capacity for conservation is limited in Indonesia, as elsewhere in the CT, and many government bodies do not have staff or budget to engage sufficiently—plans are often made but not followed on the ground (cf. Mills et al., 2010). Decades of disempowerment have also constrained the capacity of many local institutions and communities to organize, innovate and act. This raises questions about the long-term sustainability of conservation fit outcomes in the absence of bridging organizations. For the time being, a reliance on foreign aid has caused tensions, including those related to implementation of conservation activities based on donor timelines (cf. von Heland et al., 2014). One interviewee voiced frustration over donor timeline expectations that do not align with the reality of building relationships and conducting activities on the ground (anonymous personal communication 2014). ## CONCLUSIONS: FUTURE DIRECTIONS AND INSIGHTS FOR THE CT Efforts to improve the fit between conservation initiatives (e.g., marine protected areas, no-take zones) and the dynamic social dimensions of coastal-marine systems are still rare. This research offers empirical insights for conservation practitioners and policy-makers into the social complexity behind coastal-marine conservation in Bali, and in the CT more broadly, and how bridging organizations can improve navigating this complexity. We contribute understanding of the advantages and limitations of bridging organizations as a governance strategy to foster more robust conservation measures that fit underlying dynamic and shifting social contexts. In Indonesia, decentralized governance has presented both the opportunity and challenge to involve multiple social actors and sectors of society, and work on how bridging organization navigate conservation fit issues such as social context, appropriateness of governance and scale holds promise. Our findings demonstrate key strategies applied by bridging organizations to deliberately address major conservation fit issues faced in the region. These findings have broader relevance to other regions of Indonesia and the CT, who are challenged by similar social and institutional barriers to achieving positive conservation momentum (see Mills et al., 2010; Foale et al., 2013; Fidelman et al., 2014; von Heland et al., 2014; Weeks et al., 2014a). In demonstrating the efficacy of bridging organizations to operationalize conservation fit, we offer the following insights: Moving forward, our findings highlight a need for additional research to understand the implications of bridging organizations for the long-term ecological and social success of conservation initiatives. In many of our cases, for example, the conservation initiatives fostered by bridging organizations are not yet institutionalized and further analysis is needed to understand how that process may evolve under different conditions or in their absence. As such, there is a need to undertake a large "n" comparative analysis of bridging organizations in #### REFERENCES geographically differentiated marine conservation contexts that reflect different social, political and institutional realities. As mentioned, critical political and ecological analysis is needed of how bridging organizations influence social processes such as power, agenda setting and policy narratives that shape conservation (as per Berdej et al., 2015). We do not claim that bridging organizations are guaranteed to enhance conservation fit, but our evidence indicates that they play an important role in leading the conservation process forward, and in fostering multi-actor strategies that meaningfully engage with the social dimensions of marine conservation. ## AUTHOR CONTRIBUTIONS Conception or design of the work (SB, DA); acquisition of data (SB); analysis or interpretation of data for the work (SB, DA); drafting the work or revisiting it critically (SB, DA); final approval of the version to be published (SB, DA). ## FUNDING This research was funded by an International Development Research Centre of Canada Award (no. 107473-99906075-011) to SB, and Social Sciences and Humanities Research Council of Canada Awards to SB (no. 752-2014-1793) and DA (no. 410- 2010-1109). This research was further supported by a Social Sciences and Humanities Research Council of Canada Partnership Grant for the Community Conservation Research Network (no. 895-2011- 1017). ## ACKNOWLEDGMENTS We thank the communities in which we worked for their support and participation in this research project, as well as Conservation International Indonesia, the Coral Triangle Center, Reef Check Indonesia, the Indonesian Nature Foundation, and the Ministry of Marine Affairs and Fisheries, Buleleng Regency. Thank you also to Dr. Arif Satria and his team at Bogor Agricultural University for their guidance and assistance. We thank two reviewers and the editor for their constructive feedback, as well as Prateep Nayak, Jennifer Silver, and Scott Slocombe for their feedback on earlier drafts. Kelompok Kerja Jejaring Kawasan Konservasi Perairan di Provinsi Bali. Available online at: http://nyegaragunung.net/wp-content/uploads/2013/11/ Keputusan-Gubernur-Bali-No.-1590-Tentang-Jejaring-KKP-Bali-low.pdf (Accessed August 15, 2015). 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 LT declares that, despite previously collaborating with the author DA as part of a large research project, their contributions to the research project were independent, the review process was handled objectively and no conflict of interest exists. Copyright © 2016 Berdej and Armitage. 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. # Factors Influencing Community Fishers' Leadership Engagement in International Small-Scale Fisheries Abigail M. Sutton<sup>1</sup> and Murray A. Rudd<sup>2</sup> \* <sup>1</sup> Environment Department, University of York, York, UK, <sup>2</sup> Department of Environment Sciences, Emory University, Atlanta, GA, USA #### Edited by: Annette Breckwoldt, Leibniz Center for Tropical Marine Ecology, Germany #### Reviewed by: Edward Jeremy Hind, Manchester Metropolitan University, UK Richard Nami Muallil, Mindanao State University - Tawi-Tawi College of Technology and Oceanography, Philippines > \Correspondence: Murray A. Rudd [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 27 February 2016 Accepted: 16 June 2016 Published: 28 June 2016 #### Citation: Sutton AM and Rudd MA (2016) Factors Influencing Community Fishers' Leadership Engagement in International Small-Scale Fisheries. Front. Mar. Sci. 3:116. doi: 10.3389/fmars.2016.00116 Local leadership is crucial to the functioning of local organizations in small-scale fishing (SSF) communities. By analyzing local leadership experiences of 54 international SSF researchers and practitioners, we aim in this paper to fill knowledge gaps that recent research has identified regarding our understanding of factors that influence the effectiveness of local leadership. Influencing factors are organized using modified versions of the Institutional Analysis and Development (IAD) framework, the Value-Belief-Norm (VBN) theory, and Schwartz's theory of cultural values. We identified factors that help shape leadership engagement and effectiveness at multiple levels, including: precursors to individual action that relate to potential SSF leaders' perceptions of threats and opportunities; institutional constraints at the individual level and community level; and high level governance issues. Precursors to individual action were numerous and multi-faceted, and individual behaviors were shaped by core values and attitudes, culture, experiences, and education. Motivation to participate in leadership can either be altruistic in nature or oriented toward self-enhancement. A lack of motivation for leadership could be attributed to the individualistic nature of many fishers. The availability of capital assets can facilitate or hinder participation in leadership. Individuals who may be willing to take on leadership roles were often hindered by lack of money and time, low educational attainment, or poor social cohesion among community members. The interactions between leaders and followers were crucial for effective leadership, especially a leader's perceived legitimacy and the ability of a community to groom appropriate successors. At the higher level, constant policy change and the resulting uncertainty were linked to decreasing motivation and apathy regarding SSF management at the local level, and disintegrating relationships between government level and local level actors. Our research highlights how local leadership and context are linked, and suggests potential researchable hypotheses that would in the future help further advance empirical and theoretical understanding of leadership influences in SSFs. Keywords: small-scale fisheries, leadership, institutional analysis and development framework, value-belief-norm theory, community-based fisheries management ## INTRODUCTION Uncertainty is pervasive in small-scale fisheries (SSFs) due to complex interactions within and between ecological and sociopolitical systems. SSFs are, as a result, often perceived to have low governability potential (Jentoft and Bavinck, 2014). This perception is exacerbated by a history of perceived failures by centralized, conventional fisheries management agencies (Imperial and Yandle, 2005; Pero and Smith, 2008). Consequently, decentralized or devolved fisheries management approaches (Rudd et al., 2003; Plummer and Fitzgibbon, 2004) have become increasingly popular since the 1980s (Jentoft, 1989; Pinkerton, 1989; Chuenpagdee et al., 2005). Decentralized governance systems transfer decision-making power to local government managers, while devolved governance involves the transfer of substantive decision-making power to local resource users (Rudd et al., 2003), often through community-based or co-management structures (Jentoft, 1989). If the devolution of SSFs is to be more than a way for governments to simply download their own management costs on communities (Wiber et al., 2010), engagement of community actors becomes central for success as they are tasked with performing key management functions (Rudd et al., 2003; Armitage, 2005). This is especially the case for the local leaders, who are crucial for successful community-based fisheries management (CBFM) (Muehlig-Hofmann, 2007; Bodin and Crona, 2008; Gutierrez et al., 2011; Sutton and Rudd, 2014, 2015; Al Mamun, 2015; Evans et al., 2015). While SSF leadership characteristics and functions have been examined at a relatively coarse scale (Sutton and Rudd, 2014), advances in other fields (e.g., Küpers and Weibler, 2008) suggested that detailed sharper focus on leadership concepts and methods could provide valuable insights regarding the role that leaders play in SSF management. In particular, there is a compelling need to also identify the social conditions that influence SSF leaders and leadership capabilities (Sutton and Rudd, 2014; Al Mamun, 2015), as those help shape ecological and socio-economic outcomes. Here we seek to strengthen our understanding about which conditions—at the level of individuals, communities, and higherlevels of governance—influence the capacity of local community members to successfully develop into leaders and engage in CBFM, thereby enhancing the delivery of positive ecological and socio-economic outcomes arising from the devolution of SSFs to their local communities. To do this, we conducted semi-structured interviews with 54 international SSF researchers and practitioners, focusing on the characteristics of leaders and the challenges that they face in SSF management. Our results thus provide broad insights into the influences and mechanisms affecting local leadership processes and outcomes in international SSFs. ## METHODS ## Theoretical Background Local leadership in SSF is influenced by numerous conditions across socio-political scales, at the level of the leader's own household, their community, and the political context within which their community is embedded. To help identify and organize our analysis, we drew on insights from the Institutional Analysis and Development (IAD) framework (Ostrom, 1990, 2005), Value-Belief-Norm (VBN) theory (Stern et al., 1999; Stern, 2000), and Schwartz's theory of cultural value (Schwartz, 1999, 2012). That combination helps to highlight conditions that influence the propensity of individuals to engage in SSF management leadership and to identify ways in which the broader social cultural and political environments might influence local leaders. #### Institutional Analysis Development (IAD) Framework The IAD framework is a universal policy analysis framework that helps organize and facilitate analyses of how institutions operate and change over time, allowing for greater understanding of the logic, design, and performance of institutional arrangements in a wide variety of settings and scales (Ostrom, 1990, 2005). We use it to organize our analysis and help identify key characteristics of leadership at the individual level and the institutions that catalyze or hinder the development of leaders. When viewed from an IAD perspective, community fisheries become a collection of social actors within an "action arena," the space where individuals interact, exchange ideas and services, and engage in contestation. The framework lays out how behavior is shaped by various sanctions and rewards associated with particular types of rules or social norms (i.e., about what, where, when, and how activities can be undertaken; by whom; and about permitted, required, or prohibited outputs and outcomes). In a capital asset-oriented IAD (Rudd, 2004, 2010), the state of the world is framed in terms of various capital assets (Figure 1), which can be accumulated or depleted. When valued assets and their resource flows are perceived to be threatened (hence linking to VBN theory, below), governments, communities, and leaders themselves have a range of options to alleviate adverse conditions that inhibit them achieving their objectives or adapting to changes in SSF context. Those investments can be in capital assets themselves (e.g., education and training to increase leadership capacity), in changing either the structure of the rules-in-use or their payoffs, and in implementing processoriented (rather than structural) changes in the governance system (i.e., designing participatory processes that enhance efficiency, equity, legitimacy, participation, accountability, fiscal equivalence, alignment with moral values, adaptability, resilience, robustness or sustainability—see McGinnis, 2011). Action arenas exist at multiple levels from a single household, to regional, national, or international governance organizations (Ostrom, 2005). The IAD framework can be used to structure the feedbacks between action arenas that are linked across different levels. Our primary focus is on the operational level, where individual SSF actors or organizations in their fishing communities make day-to-day decisions. However, outcomes from higher collective choice and political levels also affect them, creating facilitating or restrictive conditions that affect local leaders' capacity to engage and function in SSF leadership roles. When extending the IAD framework to multiple levels (Figure 2) in our SSF context, the lowest level (and that with the quickest cycle time) is that of the individual leader, who makes decisions that help him or her reach their personal objectives (e.g., earning a living and having enough money for educating children) or broader objectives regarding the state of capital assets in their community (e.g., infrastructure, social cohesion) or region (e.g., health of fish stocks). Individuals function within their community, and are influenced directly by actions of the community level (e.g., the aggregated outcomes of local fishers on fish stocks; social norms that influence where, when, and how an individual can fish). All actors at the operational level of households and communities are influenced by the actions and outcomes of higher level fisheries management and other organizations tasked with governing or supporting the operational level. For example, the formal rules that govern local fisheries are chosen at the higher level, as are choices about enforcement intensity and the allocation of resources to operational level activities like habitat restoration. At an even higher political level, activities and their outcomes shape general policy directions that reflect the desire of governments or other high-level organizations (e.g., donors). In our analysis, we found respondents who addressed issues at all levels and used the multilevel IAD framework to help organize and make sense of those comments. #### Value-Belief Norm (VBN) Theory The VBN theory (Stern et al., 1999; Stern, 2000) seeks to explain environmentally-significant behaviors. While fisheries leadership may not entirely be an environmental behavior per se, we believe that a modified VBN—used as a framework to organize comments about threat perceptions, actor objectives, and propensity to act in certain ways—is useful for framing thinking about SSF fisheries leadership. A key insight from VBN theory is that threat salience is influenced by a number of factors (i.e., cultural context, prior experiences, core values, access to information, and an actor's capabilities—Figure 3) that will affect the propensity of that actor to take action and influence the intensity of engagement, subject to institutional constraints. In theory, the more deeply rooted an individual's beliefs are, the more likely an individual is to be aware of the consequences of their behavior (López-Mosquera and Sánchez, 2012). Beyond environmental threat salience research, we believe that the theory can also be applied to perceptions of new opportunities that affect an individual's propensity to engage in behaviors that advance personal goals or become engaged with higher level entities or organizations that have goals reflecting the core values of that individual. For example, an individual fisher would be more likely to engage in a local SSF management if government organizations enforced rules against poaching by community outsiders. In the context of SSF leadership, individual leaders play a dual role: they act as individuals, making choices about personal actions that fulfill their objectives at the household level; and they also make decisions regarding community-level leadership actions. It is important to distinguish between the two because taking on a leadership role actually means that an individual also formally or informally fills a position at a level higher than the household level. Thus, attention needs to be paid to untangling the actions of individuals and to whether they are acting on behalf of their own household or as an actor with a particular SSF management role to fulfill. An individual's experience of working in a certain management or leadership context can shape their motivations to participate in future projects. Experiences with successful projects build reputation and credibility that can encourage future participation, while experiences with unsuccessful projects can discourage future participation. Social memory is the mechanism in which information regarding experiences is stored (Adger et al., 2005) and is embedded through community discussions and decision-making (McIntosh, 2000). #### Cultural Context Cultural values such as freedom, prosperity and security represent shared ideas about what is good, right and desirable in a society (Williams, 1970). Cultural values guide people to understand which behaviors are appropriate in various situations (Schwartz, 1999). Cultural values are numerous and can differ substantially between countries. Schwartz (2012) asserted that some values are congruent with each other while others conflict (Figure 4). With four quadrants, Schwartz (2012) defines the four major values types: openness to change; self-transcendence; conservation; and self-enhancement. The closer the values are, the more similar their underlying motivations, while the more distant they are, the more antagonist their underlying motivations (Schwartz, 2012). Therefore, conflicts can arise between individuals and groups that hold different values. The value of openness to change relative to the values of conservation captures the tension between independent thought and readiness to change, and values that encourage order, preservation of the past and resistance to change. Differences of values emphasizing self-enhancement relative to self-transcendence capture potential tensions between the concern for the interests of others (and the environment) and the pursuit of one's own interest. In synthesis, the IAD framework, and the VBN and cultural value theories facilitate the in-depth analysis of leadership. Individual-level factors we focus on include cultural values, prior experiences, and access to information, all of which influence an individual's propensity to engage in leadership roles. The link between individual-level factors and propensity to engage in leadership is based on the VBN theory (Figure 3). The intensity of engagement is constrained by capital assets (e.g., financial and social capital) and community-level activities (Figures 1, 2). Higher level factors at the political level directly and indirectly influence local-level leadership through policy direction and regulation setting. #### Empirical Implementation Interview Questions To collect contextual information on leadership we used semistructured interviews that offered participants the chance to explore issues they perceived as important (Longhurst, 2010). Interviews started with a general discussion on the fishery to obtain information about the fish stocks targeted, fishing methods used, perceived health of stocks and the environment, and governance arrangements. We then asked four theoreticallyguided questions (listed below) to help direct a conversation. Participants thus had the opportunity to develop arguments and engage in open discussions regarding key issues while minimizing interview time (Weiss, 1995). How do individuals come to be community leaders? The effectiveness of local leadership is related to the legitimacy or credibility of a leader. Theory assumes that individuals who have a connection to the community or who originate from the community are likely to be successful leaders (Ostrom, 2009). Legitimacy can also be enhanced through formal processes of elections and rotations (Hollander and Julian, 1970). In our interviews we sought to explicate the processes by which leaders most commonly emerges, and the conditions and factors that aided or hindered this emergence from an individual role as householder or small business person to an actor that took on a formal or informal leadership role at the community level. Why do people get involved with leadership roles? Motivations are an important precursor to the performance of certain behaviors (Giberson et al., 2005). The expression of inherent values is shown through motivations to act. Motivations can determine whether an individual will act in self-interest or for the interest of the wider community (Schwartz and Bilsky, 1987). Deciphering an individual's motivation for becoming involved with SSF leadership roles is therefore crucial. Are potential leaders prepared for leadership roles? Capacity building is often provided to local communities as part of CBFM projects (Pomeroy and Rivera-Guieb, 2005). Training programs are either directed at the wider community, specific key interest groups, or current leaders. Capacity building increases an individual's knowledge and skills, which can be then utilized in an action arena (Stern, 2000). Our question aimed to explore a range of tools and approaches used to enhance leaders' ability to function in SSF management. Do individuals receive external assistance to enhance their leadership capacity and meet their responsibilities as a leader? The introduction of CBFM structures often puts additional pressure on community resources. In many instances local organizations do not have the capacity to facilitate CBFM. For those communities, external assistance in terms of leadership, technical assistance, and the facilitation of access to resources is required (Pomeroy et al., 2001). Do you think there will be any challenges to leadership going on into the future? In addition to four theoretically guided questions, we included one final question that asked respondents to identify key future challenges regarding leadership in SSFs. The aim was to link leadership emergence to broader environmental, economic, political, and social landscapes. #### Sampling Method We selected cases deliberately to help ensure we covered as broad a range as possible of case study configurations, and to obtain opinions from individuals with diverse expertise. Four contextual variables that were potentially important for SSF success were used to broadly identify 16 general types of case study configurations: development status of the country where the fishery was located; whether fishers regularly participated in CBFM; fishery complexity, defined simply as single-species vs. multi-species fisheries; and management status (i.e., how established the SSF management arrangement was) (Table 1). Our aim was to include at least one case study from each of those possible combinations. Sampling was therefore theoreticallyinformed rather than random or representative. Once as many variable combinations as possible were covered with at least one interviewee, we added interviews opportunistically across case types until we reached our target of at least 50 interviews in total (a reasonable number for future Qualitative Comparative Analysis research—see Sutton and Rudd, 2015). Potential case studies were identified using academic journals, organization websites, project reports, and the Too Big to Ignore (TBTI) SSF database (toobigtoignore.net/issf/). After case studies were identified, potential interviewees were contacted via email. Our criterion for selecting interviewees was based on their involvement with the SSF. To be involved in this research, the individual had to either be a researcher of, or a practitioner within, a focused SSF. As such, our respondents included academic researchers, government scientists, representatives from NGOs and leaders in community-based organizations. This ensured we covered a range of insights and opinions on SSF leadership from individuals in different regions and with different backgrounds. Of 200 individuals contacted globally, interviews (via Skype or Google Hangouts) were conducted with 54 respondents between January and July 2015. Kingdon (2003) defined leadership as key individuals who by their skills, experience and personal characteristics are justified in being a central and influential role in social processes. Due to the complexity of leadership, the lack of a common definition for SSF leadership, and the difference in leadership structures between SSF communities, we decided not to have a fixed definition of leadership. Instead we left respondents to define leadership in a manner that was appropriate to their case study; for example, this included a single individual or a group of individuals, external or internal actors, and informal or formal leaders. As we took insights from both academics and practitioners, we had an even mix of respondents who were researchers or advisors to the SSF, and respondents who were themselves leaders. Interview questions were approved by the Department of Environment research ethics committee at the University of York in November 2014. Confidentiality agreements were signed by all interviewees and transcripts were stored on a private device. #### Data Analysis Interviews were transcribed and coded using NVivo software (www.qsrinternational.com). Theme identification is important to show recurrent unifying concepts or statements within data (Boyatzis, 1998). A priori themes were defined drawing on terminology likely to be important for theoretically-informed discussions of SSF leadership performance (i.e., terms relating to potential precursors to individual action; individual and community level action choices and constraints; interactions between various social groups; and higher level socio-political influences). As the interview transcripts were analyzed, themes and sub-themes were modified, refined and often combined to improve clarity. Further, theme structure evolved inductively with emergent themes reflecting representation of unanticipated interview responses (Bradley et al., 2007). ## RESULTS AND DISCUSSION #### Interview Results Our 54 interviews covered 52 case studies and 15 of 16 case study configurations (Table 1) from 34 countries (Figure 5). Conversations lasted between 30 and 120 min, resulting in over 46 h of interview recordings that were subsequently transcribed for textual analysis. In our subsequent reporting of results, we summarize the number of respondents who made reference to particular themes and provide selected interview excerpts. For confidentiality purposes, respondents are numbered R1, R2, etc. This research relied on the opinions and views expressed by our respondents. The potential for biases among our respondents was, we hope, minimized by collecting and reporting on information from a wide range of interviewees across diverse case configurations. #### Factors Affecting Individuals' Propensity to Engage in Leadership Cultural Background Individuals' perceived threats and propensity for taking action are influenced by shared culture and unique personal experiences. Culture influences an individual's behavior by shaping a repertoire of shared habits, skills, and values (Swidler, 1986). Cultural conditions can be either conducive for collective action or act as a barrier (Pomeroy et al., 2004; di Falco and Bulte, 2011), and either can influence leadership potential. We found Development status: using the Human Development Index (HDI), cases in very high and high HDI nations were ranked 1, and cases in medium and low HDI nations were ranked 0. Fisher participation: if fishers regularly participated in CBFM decision-making the case was ranked 1, and if not, the case was ranked 0. Fishery complexity: if the case SSF was mostly single-species in focus, the case was ranked 1 and if mostly multi-species focus, the case was ranked 0. Management arrangements: if SSF management techniques were fully established, the case study was ranked 1 and if new or unestablished, the case study was ranked 0. cases studies in this research that exhibited both possibilities, where cultural context was conducive to collective action and vice versa (Table 2). Seven of our cases studies highlighted cultural contexts that facilitated collective action. For small-scale aquaculture in northern Sri Lanka, collective action was traditionally practiced in cooperatives and associations. R1 emphasized that "if people are used to working collaboratively, its's easier." Fisheries and fish resources were an important part of the community's cultural identity in Velondraike, Madagascar. R2 stated that "it's completely intertwined with who they are as people", so that consequently community members actively participated in activities which focused on protecting those resources. Religion also influenced fishing activity and conservation measures. In Bangladesh, fishing activities ceased in line with Hindu and Muslim festivals. R3 noted that fishers have built a special connection to the fisheries, which has helped place a conservation value on fish stocks. The relative homogeneity of communities in the Khong District, Laos—in terms of ethnicity, language and culture—enabled effective information exchange between community members. R4 reported that this enabled individuals to easily evaluate the actions of others. For other contexts, collective action was hindered by cultural influences. In many SSFs, fishers had individualistic tendencies, which reduced the likelihood of collective action and of following a leader. R8 described the Bajau fishers of Wakatobi, Indonesia, as "rugged in their individualism" and questioned "why on earth would they accept someone being a leader, when they know everything they need to know." Similarly, fishers in Scotland preferred to act independently of regional grouping; that independent orientation, which was a valued trait among fishers in the region, hindered the potential of CBFM (R9). TABLE 2 \| Cultural values facilitate or restrict leadership and collective action in SSF management. In part, a fisher's individualism is attributed to the characteristics of the resource. Fisheries are a common pool resource, characterized by two defining features, excludability and subtractability. When fish stocks are declining, this can place fishers under pressure to participate in a race to fish (Ostrom, 1990). Independence and individualistic tendencies should not be regarded as undesirable characteristics, as they encourage the propensity to think and behave freely, facilitating the ability to make quick decisions (Poggie, 1980). However, in those cases, what is the likelihood of fishers working collectively, following a leader or becoming a leader themselves? Poggie (1980) recognized that CBFM needs to be compatible with the psycho-cultural characteristics of the fishing community: new management structures should encourage free thought in decision-making, independence, and the creation of community ownership whenever possible. #### Core Values Our respondents highlighted that individuals have different motivations for leadership (Table 3). The motivation of a leader influences his or her behavior and can consequently significantly TABLE 3 \| Core values are expressed in motivations for taking on leadership roles. influence the overall effectiveness of the organization (Giberson et al., 2005). We found that altruistic, self-enhancing, and environmental motivations all played motivating roles for individuals to engage as leaders in differing cases. Nine respondents attributed motivation for leadership to altruistic factors. In western Canada, R12 noted that older fishers believed that "it's time to give a little business back to the industry, the industry has been good to me and I'm going to put my time in." Similarly, older fishers in Bangladesh were found to be motivated to, "support their community and ensure the continued livelihoods for future generations" (R3). In Cambodia, R10 recognized that there will always be a member of the community who is committed to improving the life of community members. Many leaders were motivated for self-enhancement purposes. Simply getting paid was enough encouragement for poorer individuals in Malawi and Tanzania to take on leadership roles. Securing livelihood opportunities was particularly important in western Canada: "I think a lot of it is that this is their livelihood, this is how they and their families survive" (R12). The connections made with external, influential actors through leadership activities are a second motivating factor. One respondent (R17) stated that "individuals [in Argentina] are always trying to get help or trying to connect themselves to other levels, politically." R13 noted that leadership in Spain "brings all sorts of benefits, because you are the linking organization between all the fishers and the government; I think that's a big motivation." Social recognition was also a motivating factor according to two of our respondents. In Australia, R18 highlighted that fishers "are proud of the recognition they receive. . . they tend to be held in high regard by their communities and this social license is important to them and their families." In Laos, "leaders are people who were more interested in the prestige of the position, in the sense that they wanted to be known in their communities as important people" (R4). Environmental values were attributed as motivating factors by seven respondents. A member of a local environmental group in Taunton Bay, Maine had little confidence in the State government; his motivation for participating was to represent sound environmental policy (R23). In the Philippines, R29 highlighted that leaders "do not get paid for the work, it is purely a voluntary service, they believe in the cause of resource conservation and protection." Similarly, R30 commented that the leader of a marine protected area (MPA) in Spain was a local university professor; "he was on a mission for sustainability; he was really passionate about it." Our findings offer insight into the motivations of leaders in SSF and highlight different value structures. In line with the work of Schwartz (2012), it is possible to hypothesize that individuals with altruistic or biospheric tendencies are more likely to serve collective interests for the good of conservation, whilst those who express self-enhancement values are more likely to serve individual interests. However, individuals have multiple values which emerge at different times calling for a temporal component to future leadership research. #### Prior Life Experience—Early Education Our respondents identified education as a key factor that influenced fishers' behavior. The introduction or reestablishment of participatory approaches often included elements of education, training or capacity building. Education increases awareness and influences perceptions and beliefs that guide human behavior (Hungerford and Volk, 1990; Stern, 2000). Multiple educational approaches for increasing awareness were practiced in our case studies and targeted both children and adults. As early education is thought to influence threat salience and behavioral choice via its effect on worldviews (as opposed to skills- and awareness-building in adults, which can more directly and immediately affect perceptions regarding threat salience; Stern, 2000), we deal with each separately. Marine programs were developed for school children in seven countries including Tanzania and the Philippines. Increasing awareness from a young age embedded the importance of marine ecosystem sustainability (Table 4). R7 reported that after two decades of the marine program on Apo Island in the Philippines, local children had a strong sense of place and their marine environment was "sacred" to them. Similarly, an MPA organized by the Community of Arran Seabed Trust (COAST) in Scotland, UK, has received strong support from the local community. R19 attributed that level of support to "the continued presence of COAST at community events and awareness raising activities for children in local schools." #### Human Capital—Adult Education and Awareness of SSF Threats and Opportunities Human capital refers to the stock of knowledge that individuals possess in an action arena. The ability for individuals to adopt more profitable and secure livelihood strategies from SSF is in part dependent on education (Dercon and Krishnan, 1996). Adult members of the community benefited from awareness building opportunities that were created through the development of workshops, training programs, and community events (Table 5). R3 reported that programs in Bangladesh taught local fishers how to brand their fishery products and participate in micro-credit programs. The development of a co-management program in Spain increased local awareness of the importance of local fisheries resources to the local livelihoods. Consequently, R13 noted that fishermen were volunteering more of their time to participate in surveillance and monitoring. R30 reported that local ecological knowledge, a form of knowledge held by local resource users, was incorporated in Spanish MPA proposals, and that this "fostered a sense of ownership and that's what made it succeed." Many local fishers, however, have minimal formal education, and this can reduce their ability to participate in CBFM (Hollup, 2000; Vedeld, 2000; Glaser, 2003), a point that was reiterated by our respondents. In Sweden, R6 highlighted that language barriers hindered local fishers in their application for a Marine Stewardship Council (MSC) certification, which recognizes the sustainability of a fishery. Similarly, few community members had the level of education required for higher level positions of an MPA authority in Tanzania; R24 reported that "you have to be able to write on the computer and you have to be able to write in English, so that limits the number of people who can apply to the job." Many individuals simply do not have the capacity or disposition to be leaders. Respondents from the UK, Chile, Canada, and Ecuador highlighted that little or no capacity-building was targeted specifically at leadership. Lack of capacity-building for leadership was attributed to poor funding opportunities or leaders having too little time to attend workshops. Capacity building for leadership was provided for Beach Management Units (BMUs) around Lake Malawi and Lake Victoria in East Africa. However, R31 stated that local fisheries officers did not have the capacity to transfer knowledge on to their successors, and R22 added that training was one-off in nature, not followed by successive training that built skills over time. Several of our interviewees also reported that increased levels of awareness regarding other livelihood and investment opportunities, combined with the uncertain nature of fishing, could deter individuals from remaining in SSFs. In the Philippines, fishers were "less interested in managing the fishery because they don't depend on it anymore" (R32). In Argentina, "the sons and daughters of fishermen don't want to continue in fishing" (R17). Similarly, R31 emphasized that fishers around Lake Victoria were beginning to invest more in their children's education and that, as they did, their motivation to participate in SSF collective action, leadership and management was diminishing. TABLE 5 \| Human capital at the local level impacts an individual's ability to lead. #### Access to Resources #### Financial capital Many small-scale fishers are extremely poor and live well below the poverty line (Béné, 2003). Financial capital at the individual level is therefore often limited. Our respondents noted that fishers' poverty levels impacted on their ability to participate in CBFM in Tanzania, Bangladesh, Malawi, and Madagascar. In Vietnam, R15 stated that "the folks on board are also actively engaged in securing a livelihood, so there isn't a huge amount of time to spend doing project activities. This was reiterated by R37 who recognized that "people may be willing (to participate) but not able. . . an individual, whose livelihood relies on them being out in the industry—that is a constant problem. . . it's a catch 22." Timing issues were exacerbated by fishers working hours that are highly influenced by tides, and R23 reported, "no matter how carefully we planned, securing 100% attendance was impossible." Fishermen are increasingly being put under greater pressure due to dangerous working conditions, reduced stocks, and stricter regulations. It is inevitable that time will become even more restricted in the future (Salas et al., 2007). Therefore, the need to provide a secure income reduces the time fishers can devote to both leadership roles and collective action (Table 6). Manufactured capital such as fishing boats and technology are the stock of produced assets that people use over time (Rudd, 2004). The importance of manufactured capital was referred to by two of our respondents. Although this is a low level of coverage, we included it as a distinct category to emphasize the importance of further research on the influence of manufactured capital on leadership. In Bangladesh and Indonesia, a fisher's access to boats was the basis of their leadership. For the Bajau in Wakatobi, formal leadership among community members was an uncommon occurrence. However, R8 confirmed that "temporary leadership can emerge if an individual gets a bit more money, who maybe owns three boats and has a crew. . . this isn't policybased leadership, it's fisheries-based leadership but not because of the need to manage the fishery, it's just what you do to run your business." #### Social capital Social capital is an asset built on social networks (Rudd, 2000; Krishna, 2002). It facilitates the transmission of information and reputation, and is a key factor influencing the socioecological sustainability of CBFM (Rudd, 2004). While social capital by definition needs multiple actors to function, one can conceptualize that an individual's access to social capital their niche in the network—strongly affects their capacity to engage as an effective leader. Social capital is also an important TABLE 6 \| Financial capital influences leadership potential. resource from an organizational perspective at higher levels of management and political choice processes. Social capital was an important influencing factor in our case studies (Table 7). Trust and confidence between community members decreased the need for strict enforcement in the tilefish fishery in northeast USA (R42). Limited bonding social capital, or the bonds between likeminded people, was, however, also reported at the individual level. Poor social cohesion between fishers prevented collective action in the Galapagos Islands, Ecuador. R43 attributed this to the prevalence of fishers from mainland Ecuador who had stronger connections to their home communities. In Western Australia, bonding social capital was commonly weak among abalone fishers; R44 argued that this was due to "the historically fractious relationships between fishers." R5 recognized that social bonding between community members around the shore of Lake Malawi needed to be strengthened in order for shared objectives to be developed. A potential mechanism for increasing social capital was also highlighted. Experiences of working collectively are stored in the social memory of communities (Adger et al., 2005). Members of SSF organizations in Spain and Malawi who participated in prior CBFM projects had heightened confidence and trust in their collaborations with other fishers. In these communities, leaders used the experience of working collectively and the social memory of the fishing community to participate more effectively in subsequent projects. #### Community-Level Leadership Issues Leadership Legitimacy At the community level, individuals need to be considered in relation to the formal role that they play as leaders in fisheries management. Legitimacy is a psychological property of leadership that allows followers to perceive appropriate, proper, and just leadership (Tyler, 2005). Legitimacy is the common way of signaling acknowledgement of a leader (Hollander, 2012). By accepting a leader, followers influence the strength of a leader's influence and consequently the performance of the group. Over half of our respondents identified legitimacy as important and highlighted the numerous pathways individuals can become legitimate leaders (Table 8). Legitimacy can be achieved through formalized mechanisms of nominations, elections, and rotations, processes that define boundary rules and provide clarity regarding the leadership role within which individuals are placed and act. Elections also create a heightened psychological difference between followers and leaders (Hollander, 2012). To become a member of an Inshore Fisheries Group (IFGs) in Scotland, R9 reported that TABLE 7 \| Human capital at the local level impacts an individual's ability to lead. TABLE 8 \| Human capital at the local level impacts an individual's ability to lead. an individual had to meet certain criteria outlined by the organization's guidelines. In western Canada, to gain a place on the Board of Directors, prospective members were required to be nominated and elected by current members (R12). Individuals from regional groupings in New Zealand were nominated to become representatives on the New Zealand Rock Lobster Industry Council (NZRLIC) by other community members (R20). Elections increase legitimacy, but in some circumstances elections can also lead to unrealistic expectations of leaders and consequently they can become the subject of criticism (Hollander and Julian, 1970). Elections can, for instance, be corrupt (Hauck and Sowman, 2001) or poorly executed in the face of community members' low literacy rates (Xu and Ribot, 2004). Our case studies reiterated that the geographic origin of a leader can be important for leadership legitimacy. Local leaders who have a deep understanding of local processes and cultures are essential for collective action (Meaton and Low, 2003; Olsson et al., 2004; Beem, 2007; Bodin and Crona, 2008; Gutierrez et al., 2011). Calettas or fishing federations in Chile have strong social bonds, leading R33 to assert that when someone comes from another area, "he will always be an outsider." Leadership positions were maintained within family units in Quinta Roo, Mexico, and Apo Island (despite formal elections for barangay leadership in the Philippines). SSF leaders were also found to be traditional leaders in Malawi, Canada, Vietnam, Laos, the Philippines, and Malaysia, a factor that helped increase their legitimacy among community members. A leader's legitimacy can also be enhanced through his or her actions. In our case studies, a leaders' legitimacy was strengthened via their reputation, and the trust, accountability, and transparency that they engendered. In Madagascar, R34 noted that "community members have seen the benefit (of their leader), so trust had already been developed." Similarly, in the Philippines, R29 highlighted that "although leaders do not possess leadership skills at first, they evolve to be good leaders because of their first-hand knowledge. . . they gain the trust of the people in the community." The most important criteria of developing leadership in Jordan fisheries were transparency and openness (R25 and R26). #### Leaderful Issues at Community Level Creating "leaderful" organizations can be important for SSFs. A leaderful organization encourages each member of the community to gain experience of being a leader concurrently and collectively (Raelin, 2003). Due to the difficulties of leadership succession, it is important to expand the focus of leadership. The image of "successful leaders" has to shift from developing individual leaders to developing "leaderful organizations" of multiple leaders (Al Mamun, 2015), thereby increasing the pool of potential leaders. Succession is a social process determined by the interactions between leaders and their constituents, and the capabilities of local communities to produce new leaders (Hart, 1993). Our respondents identified several concerns about leadership succession (Table 9) and techniques to potentially facilitate more successful leadership succession planning. Motivation was found to be a limiting factor in leadership succession. R5 noted that local chiefs in Malawi had minimal motivation for leadership, as CBFM projects were implemented by the government. Reduced motivation among SSF leaders in Argentina was due to fluctuating support from governmental departments and poor success rates of prior CBFM projects; R17 reported that "the fishers started with a lot of motivation and strength, but the same people who are still in the fisheries are tired of continuing. . . it's really difficult to maintain the motivation." Similarly, R30 stated that due to reduced effectiveness of an MPA in Spain, the local leader is "totally deflated, he doesn't want to be involved anymore." Leadership succession was impacted by the lack of up-andcoming leaders. In northern Scotland, R50 reported that "we put an advert in the local press and invited applications from anybody who was interested. . .we didn't get many people who were interested." A limited pool of potential leaders was also experienced in Taunton Bay, Maine; R23 commented that the "area and the resource were just too small. . .we were a very limited number of people who were interested and that meant we were an inbred group by the end, we didn't get the fresh blood we were hoping for." An aging population of fishers contributed to concerns regarding leadership succession. Reporting from Spain, R13 noted that "many of the community leaders in the gooseneck barnacle industry are older, which could be problematic considering the dangerous nature of the fishery." R12 added that with the retirement of older fishers, years of cooperative expertise and local knowledge was likely to be lost. Despite concerns of an aging population, barriers to young, nascent leaders were also highlighted in some cases. In Tanzania, India, and Malaysia, older members of the community often discounted the authority of young members. R24 recognized that "you have an older guy and he doesn't want to listen to the younger guy who was supposed to be a leader, it's very difficult—it's definitely a cultural thing." To overcome concerns of leadership succession, new approaches should be developed to ensure the longevity of leadership. Capacity building was used in several of our case studies as a method to train individuals for leadership. A non-governmental organization (NGO) called Blue Ventures provided newly elected individuals in Bel Sur Mer, Madagascar, training in leadership and organization management skills (R2). R35 reported that in a regional project in the Caribbean, local fishers were given the opportunity to attend capacity building workshops and conferences on SSF. Similarly, R28 who worked for an NGO in Mexico, stated "over the last 3 years, we have worked quite heavily on leadership, working on administration and business training, because it's not something they are used to." Succession planning, the process which stabilizes the occupancy of key positions and consequently helps to ensure the #### TABLE 9 \| Succession is a beneficial attribute that helps the longevity of successful leadership. continued effective performance of an organization (Rothwell, 2010), is also explicitly needed. #### Vertical Collaborations between Communities and Agencies Nesting CBFM organizations in numerous institutional layers is crucial (Dietz et al., 2003). Community-based management has been reported to fail when communities lack linkages to higher levels of government (Lejano and Ingram, 2007; Cudney-Bueno and Basurto, 2009). Our cases studies reiterated the benefits of establishing and strengthening ties to different levels of SSF management organizations (Table 10). Linking social capital is important to this process and refers to the ability of groups to engage with external agencies to either influence policies or resource allocations (Rudd, 2000; Pretty, 2003). Several of the fishing organizations in our cases studies demonstrated effective linking roles. Fishing federations in Chile's co-management structure played important boundary spanning roles by communicating community issues to state agencies and vice versa (R36). The New Zealand Rock Lobster Industry Council (NZRLIC) provided a method of linking regional groups with the government in New Zealand. Our respondents also noted methods of enhancing linking social capital. In the Caribbean, R35 recommended the use of neutral platforms to facilitate the interaction of different actors including fishermen and government representatives. Similarly, in India, the Palk Bay Fisheries Management Platform was created to bring together key fishing stakeholders (R46). Local leaders are crucial in establishing and enhancing linking social capital. A key factor in the ability of communities to interact with higher levels of SSF management is the presence of educated, young individuals (Krishna, 2002). These individuals provide a mediating role by dealing with the complex procedures of a state and understanding complicated governmental language. The importance of an educated, younger generation was reiterated by our respondents. In Chile, some younger generations of fishers have been given the opportunity to study technical aspects of fishing and are thus more prepared and educated. R36 stated that these individuals "have a broader perspective on things." #### Elites and Power Traditional leaders have significant influence over community processes. Traditional leaders include religious or spiritual leaders, caste leaders, and local elites. The potential gains from TABLE 10 \| Interactions between different SSF organizations/agencies at different levels affect leadership. natural resources such as forestry and fishery products have often enticed local elites to act in self-interest. Consequently, the presence of local elites has been associated with embedded power inequalities and the ineffective use of community resources (Hauck and Sowman, 2001; Kull, 2002; Larson and Ribot, 2004; Njaya, 2007). Our respondents emphasized that local leadership is not immune from the abuse of elite capture (Table 11). R3 noted that formal positions in Bangladeshi co-management were often usurped by rich individuals, who were not members of the fishing community; consequently ethnic fishers (Jalyes) were unable to participate in decision-making. In Indonesia, R45 asserted that CBFM was not the best approach for fisheries management; collaborative or co-management should be implemented to allow for the careful monitoring of community elites by external actors. One respondent also noted that local elites also worked for the interest of the community. R5 commented on a village chief in Malawi who recognized the dangers of elite capture. The chief purposively did not sit on the Beach Village Committee (BVC) but instead orchestrated rotations when committee members became tired or unmotivated to perform leadership responsibilities. R5 referred to this individual as a "benevolent puppet master." Local elites have a strong influence on CBFM. As our case studies show, the activity of local elites can reduce the legitimacy of local leadership. In addition, the presence of local elites can lead to the dilution of wider community input, corruption, and improper use of community resources (Mahanty et al., 2006). However, elites can also help achieve successful SSF management, for example in Malawi and Mozambique, where traditional leaders have become advisors to SSF committees (Crona and Bodin, 2006). #### Interaction between Leadership Groups Implementing new management structures introduces new institutions, leadership, and potentially new power struggles into SSF communities. As Pinkerton (1989) recognized, key outputs of CBFM to consider are the new relationships that are created between different community organizations. It is especially important to consider how old and new institutions interact, and how power relationships play out (Amy, 1987). The interaction between old and new leadership proved to be an important influencing factor on the effectiveness of local leadership in our case studies (Table 12). Our case studies highlighted experiences where implementing agencies chose to create new leadership authorities within a community. The Galapagos National Park (GNP) was the #### TABLE 11 \| Elites have a profound influence on CBFM through their leadership. #### TABLE 12 \| Harmonious interactions between "old" and "new" leadership groups and elites. main administrator of the Galapagos Marine Reserve. In 2008, the Ecuadorian government approved a new constitution that created a new governing institution called the Galapagos Governing Council (GGC). R43 identified deep uncertainty about the function of the GNP and GGC since the new reforms were implemented in 2008. In Malawi, working relationships between the newly implemented and formalized BVCs and traditional village chiefs continued to influence CBFM effectiveness; R22 emphasized that there is "a blending of management systems where you have the chiefs and the villages on one hand and the government on the other; when there's transparency and accountability it's good and when there's not, it's bad." In the creation of the Gulf of Mannar's Bio-Reserve in India, managing authorities chose not to work through existing leaders but created parallel authorities, although R38 questioned "whether this was an entirely sensible decision." R3 reported that project officials in Bangladeshi co-management arrangements decided to hire new local leaders, as many community members were unhappy with the existing leadership. Limited research has been conducted on how existing leadership and newly implemented leadership can work together. Our case studies indicated that the transition is often complicated and characterized by uncertainty. Uncertainty is particularly evident in the responsibilities of each leadership group. Community members often questioned the legitimacy of their leaders, which reduced the overall effectiveness of leadership. It is important that agencies implementing CBFM consider the impact new leadership can have on exiting leadership and on the relationships leaders have with SSF communities. ## Interactions between Local Leaders and External Actors CBFM often requires external assistance from organizations such as NGOs, government agencies, and research organizations (Pomeroy et al., 2001). Depending on local leadership capabilities, external actors may need to perform leadership roles. Roles may include identifying management options, providing advice and expertise, and helping in community capacity building. Our respondents outlined a variety of experiences with external leadership (Table 13). #### TABLE 13 \| External assistance is important to the effectiveness of SSF leadership. Several respondents highlighted the positive experiences of working with external leaders. An external leader proved invaluable to local SSF in Argentina; R17 reported that "an outsider from Washington had a lot of experience and knew what was happening in other fisheries and how to manage resources. . . he organized and invited fishermen, students and researchers to visit communities in Chile, to learn of their experiences." Respondents from Vietnam and the Philippines recognized the work of system thinkers who could leverage important resources from international organizations and link them to communities who required extra help. Despite the importance of external leaders, barriers were also highlighted that restricted their effectiveness. Reflecting on the work of a governmental representative in Scotland, R50 commented that "does he add anything (to our community)? No, he's not as experienced in businesses as some of us are, he is not experienced in fisheries management, he's not nearly as knowledgeable about the fishery as our fishermen, so what does he add?" Concerns about the capacity of external leaders, in terms of resources and knowledge of local systems, were also highlighted by respondents from Malawi, Bangladesh, Madagascar, and the Solomon Islands. ## Higher Level Political Context #### Institutions and Management Institutional design—various management techniques, policy instruments, and other required, permitted, or prohibited activities and outputs—is used to influence SSF resource use at the local level (Ostrom, 1990; Rudd, 2004, 2010). Our case studies highlighted how rights-based approaches and direct payments provide economic incentives, which help shape fishers behavior (Table 14). If such approaches are designed properly, they provide incentives for fishers to balance resource stewardship, economic efficiency, and social welfare (Castrejón and Charles, 2013). Rights-based approaches used in our case studies included limited entry, individual transferable quotas (ITQs), individual fishery quota (IFQ) and territorial user rights in fisheries (TURFs). The implementation of rights-based approaches can be contentious due to the exclusion of some community members from the fishery (R12 and R42). R51 recognized that younger members of SSF communities found it difficult to obtain potentially expensive licenses. In northeast USA, a SSF management plan, which included a limited entry program and an IFQ, was initially met with resentment from excluded fishers. However, after concerns were addressed, R42 reported that the management plan now runs smoothly, has secured rights for local fishers, and has increased cooperation between community members. Similarly, the NZRLIC in New Zealand is made up of nine shares owned by regional groupings and incorporates separate TACs. Through the work of the NZRLIC and the use of TACs, R20 stated that fishers have heightened custodial attitudes resulting in higher levels of environmental stewardship. Economic incentives can be utilized to motivate fishermen to participate in and comply with CBFM. In a small Jordanian fisheries project, economic opportunities were created for local fishers by project officials who created partnerships with local businesses (R25 and R26). Similarly, in northeast USA, creative marketing ensured local fish was increasingly sold in local restaurants (R42). In Scotland, a major retailer invested in fisheries resources from a remote SSF; R50 noted "if fishermen can see quantifiable advantages of imposed management tools, those tools are more likely to be a hit with them." Payments to cover transport costs and a free lunch were given to participants of co-management projects in East Africa (R31). However, as R5 emphasized, "unfortunately, every time you pay someone for work that is in the collective interest, it reduces their incentive to contribute to anything else in the collective interest without being paid to do so." Economic incentives are powerful tools used to entice fishers to participate in SSF management. Increased motivation for participation and compliance with regulations was evident in our cases studies for those individuals who have access to rights and/or direct payouts. Those same individuals may be more inclined to follow a leader they perceive will maintain their access to economic incentives or even take on leadership roles themselves to maximize the outputs of their rights. However, as our results allude to, there are limitations to rights-based approaches and direct payouts. Reducing access to fisheries resources has social and economic costs to fishers and their families (Kitts et al., 2007). Poor fishers and younger members of the community are often unable to accces rights, which reduces the likelihood of their participation in CBFM and leadership activities. In addition, the longevity of direct pay-outs influences continued fishers' participation. #### Influences of Political Change An enabling political environment and government support is essential to sustain CBFM (Pomeroy and Berkes, 1997). Changes in government policies can cause knock-on impacts at all levels (Razzaque et al., 2000; Berkes, 2006). Ostrom (1996) found, frequent top-down changes of national, state, and local authority #### TABLE 14 \| Management techniques influence leadership potential at the local level. reduced the motivation of highly effective leaders and fishers to regularly participate in CBFM. Our results support the assertion that policy change creates uncertainty of the longevity of CBFM and is linked to changing attitudes among fishers at the local level (Table 15). Uncertainty about the longevity of CBFM organizations was evident in several of our case studies. In Argentina, the government went through several structural iterations for fisheries management and a recent change in the head of the Fisheries Department, which resulted in the decline of effective CBFM. R17 reported that "the State no longer supports local initiatives. . . the constant change and lack of support makes fisheries management difficult." The government of Tanzania leased an island off the coast of Zanzibar to a private company to run a no-take MPA. R24 suggested that the uncertainty surrounding lease renewal was a major concern for the longevity of the MPA. R52 expressed concerns about the uncertainty of continued funding to the English Inshore Fisheries and Conservation Authorities (IFCAs): "at the moment, we are fine; we are fine up until March 2016 when technically the money runs out. And, on paper, there's no more support funding from the government." Influences of policy uncertainty on individual behavior were reported by our respondents. In New Zealand, the rightsbased approach used in the NZRLIC was designed to engender a custodial attitude among fishers. However, R20 recognized that the government has "created so much uncertainty among the continued use of those rights that custodial attitudes and stewardship are being eroded." Reflecting on experiences of working with fishers in a Inshore Fisheries Group, R9 noted that "there's always a bit of suspicion from the fishermen, of anything to do with the government. . . if you have been in the fishing industry for 20 or 30 years, you will have seen a lot of changes. . . the fishermen are very wary." Activity at the government level is important to consider when researching SSF and leadership. Constant policy change and fluctuating government support creates uncertainty about the longevity of CBFM organizations and the flow of government resources available. Importantly, local leaders may be tied to the interests of particular politicians, which can compromise their ability to truly represent SSF communities (Scholtens, 2015). Our case studies reaffirm that uncertainty is linked to changing attitudes at the local level. Local leaders were found to lose motivation with CBFM in times of constant change due to limited support from government actors, and reduced credibility among community members. Fishers can also become apathetic to management processes, which influences the likelihood of participation. TABLE 15 \| Policy change affects local level leadership capacity/potential. ## CONCLUSIONS "Everything depends on leaders." (R16). Local leadership is crucial to CBFM and SSF success. Our research explored the factors that influenced the effectiveness of local leadership. Factors that helped shape leadership engagement and effectiveness were evident at multiple levels: the precursors to individual behavior relating to perceptions of threats and opportunities; institutional constraints on behavior at both the individual and community level; and higher level considerations. Interactions between the levels are intricate and complex, and contribute to uncertainty regarding potential leaders' willingness to engage in leadership roles, their balancing of personal vs. leadership goals, and the ultimate effectiveness of leadership. Thus, many factors either help or hinder leadership effectiveness, depending on the environmental, social, and political context within which SSFs operate. Precursors to individual action are numerous and multifaceted. Our research demonstrated that it can be useful to employ theoretically-derived frameworks to help clarify how individual behaviors are shaped by core values, culture, experiences, and education, and how resource limitations or institutions can constrain leadership engagement. Motivation to participate in leadership can be altruistic in nature (for environment or people) or more narrowly oriented toward self-enhancement. Future CBFM research on how and why individuals decide to become leaders could be useful to help guide interventions that might successfully increase engagement in SSF management. In addition, our respondents highlighted that fishers often display individualistic tendencies. Consideration needs to be given to how likely individuals are to participate in leadership roles or collective action. These fundamental individual characteristics of a SSF community have to be factored in when designing CBFM projects. Individuals and communities have a stock of capitals that they can use in SSF management. The availability of financial, human, and social capital can hinder or facilitate participation in leadership activities and collective action. At the individual level, we found that financial and human capital often restricted activity to such a point that SSF leadership potential was inhibited. Many fishers do not have the time or money available, or education level, needed to contribute effectively to SSF management leadership. The need for additional capacitybuilding aimed at local communities was frequently noted by our respondents. At the community level, the ability to work collectively and to follow a leader is particularly influenced by social capital. Although strong ties between community members were found in many SSF communities, historically fractious relationships between fishers, and between fishers and external actors can significantly reduce the likelihood of collective action. Interactions between leaders and followers are crucial to the effectiveness of leadership. Our findings suggest that local leadership is strongly influenced by perceptions of legitimacy among the local fishing community. Legitimacy may be achieved or enhanced through elections, by efforts to build reputation and trust, and via the geographic "credentials" of a leader. We also found, to a lesser degree, that external leaders could also be effective. However, external leaders often have to contend with a lack of trust from communities and limited resources beyond finances, and therefore have a more limited role to play in most SSFs. The ability of a community to produce appropriate leader successors was highlighted as a major concern by our respondents. They recommended developing more "leaderful organizations" to help facilitate long-term and effective leadership succession. Finally, our focus was primarily on factors that influenced leadership at the local level. Due to the political nature of leadership, it was also apparent that activities of higher-level actors considerably affected how local leaders could actively engage and be successful in their roles. Thus, there always needs to be consideration of the political environment within which SSFs operate. The uncertainty generated by policy change, in particular, can inhibit effective leadership due to fluctuating government support and access to resources. We found that constant policy change could also lead to the disintegration of relationships and trust between government departments and local actors, reduced motivation among fishers to engage in SSF leadership, and apathy toward SSF management initiatives. The management and governance of SSFs occurs in complex social environments. Local leadership is extremely important to the functioning of SSFs, and especially in contexts where communities and community organizations are tasked with key management roles in devolved CBFM. Our research outlines a variety of factors that influence the effectiveness of local leadership and that can help inform researchable future hypotheses, which will help further advance empirical and theoretical understanding of the role that local leadership plays in successful SSF management. Further research can build on this work to further decipher how different socialecological contexts influence the effectiveness of leadership engagement. ## AUTHOR CONTRIBUTIONS AS and MR conceived research, AS conducted and transcribed interviews, AS analyzed data; AS and MR wrote paper. ## FUNDING AS received funding from a University of York doctoral student scholarship. ## ACKNOWLEDGMENTS We thank all interviewees who contributed generously with their time. ## REFERENCES 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 © 2016 Sutton and Rudd. 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. # Deconstructing the Reality of Community-Based Management of Marine Resources in a Small Island Context in Indonesia #### Philipp Gorris \* Institute of Environmental Systems Research, Osnabrueck University, Osnabrueck, Germany This study offers a detailed analysis of community-based management (CBM) in a small island in Indonesia. In the study site, area-specific stewardship for a marine territory was informally institutionalized and, in addition to state rules, locally devised rules based on informal agreements have emerged. Using multiple methods for the analysis of the perceptions of the local community, this research examines the actual impact of the different rules on the fishing patterns in that sea territory, and illuminates the rationales of the local population to engage (or not) in the community-based approach to manage the marine resources. The study shows that the CBM initiative has to be seen as part of a convoluted regulatory system that impacts the fishing behavior in the sea territory. A lack of official authority to formally develop and especially to locally enforce rules represents a key challenges for the CBM initiative. This is further complicated by severe coordination problems between the local community and higher level state actors. The study further shows that the motivation of the community members to engage in the enforcement of the informal rules is strongly based on short-term economic considerations. For rules that are perceived to have a strong impact on the individual fishing yields, the fear of potential short-term economic losses constitutes a particular success factor of the local initiative since it motivates the members of the community to enforce local rules, especially when outside fishers break the rules. Yet, if rule-breaking is not perceived to decrease individual fishing yield, or if benefits of the generated yields are shared with the community as a compensation mechanism, the motivation of the community members to engage in rule enforcement ceases. Keywords: blast fishing, common-pool-resource regimes, fisheries management, Net-Map, perceptions, poison fishing, Spermonde Archipelago, territoriality ## INTRODUCTION Concerns about the world's oceans and coasts are rapidly growing (Rockström et al., 2009; Burke et al., 2011; Visbeck et al., 2013; Zondervan et al., 2013). One of the most severe threats for marine ecosystems and their associated natural resources emanates from the unregulated and uncontrolled resource use, i.e., an open access situation (The World Bank, 2006). Open access to marine resources is common all over the world since rules, regulations and management are often either lacking or not effectively enforced (Agardy et al., 2005). Such an open access situation is #### Edited by: Annette Breckwoldt, Leibniz Center for Tropical Marine Ecology Bremen, Germany #### Reviewed by: Louise Teh, University of British Columbia, Canada Lida Pet-Soede, PT Hatfield Indonesia, Indonesia > \Correspondence: Philipp Gorris [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 02 March 2016 Accepted: 22 June 2016 Published: 12 July 2016 #### Citation: Gorris P (2016) Deconstructing the Reality of Community-Based Management of Marine Resources in a Small Island Context in Indonesia. Front. Mar. Sci. 3:120. doi: 10.3389/fmars.2016.00120 Gorris CBM in an Indonesian Small Island Context widely assumed to lead to substantial sustainability deficits (Hardin, 1968; Ostrom, 1990; Agardy et al., 2005; The World Bank, 2006). The purpose of this article is to advance understanding of how to institute more effective communitybased marine resource management in a small island setting. Transforming an open access situation into any type of management regime requires the delineation of territory. While it appears to be more difficult to establish territoriality for marine areas than for terrestrial areas (The World Bank, 2006), research shows that it can be developed, legitimized and institutionalized (Kalikoski, 2007; Glaser et al., 2010). Marine territoriality implies area-specific stewardship coupled with legitimate rights to generate effective means based on formal and/or informal authority that steer human behavior in a specified sea area (Jones, 2014). In this regard, the concept of Common Pool Resource Regimes (CPRR) offers a useful point of departure (Ostrom, 1990; Young, 2006). The related literature holds that, apart from an open access situation, there are three proto-types of CPRR. These include the state CPRR, the private CPRR and the communal CPRR (Bromley and Cernea, 1989; Ostrom, 1990; Pomeroy and Berkes, 1997; Pomeroy and Rivera-Guib, 2006). In a "state CPRR," the state assumes control over the resources or specified territories. Individuals and groups can only use the resources with the consent of the state and must comply with the regulations made by government laws. The state can grant the right to exploit resources to individuals or groups, but control over the resources is exercised by the state (i.e., by government agencies; Bromley and Cernea, 1989). A "private CPRR" refers to the exclusive possession of an area or a set of resources by private entities. Such private entities may not necessarily be individuals, but private ownership can also be transferred to clearly defined groups (corporate private property; Bromley and Cernea, 1989). In a private CPRR, the control over a specified territory, its resources and its products is given to private entities (owner). They hereby gain the right to exercise their rewarded power to exclude others from the use of their terrain or prevent usage of their resources from non-owners. The third category is the "communal CPRR" which has attracted particular attention over the past decades (cf. Dearden et al., 2005; Berkes, 2007b). Such a community-based management (CBM) approach describes a management system of a clearly defined group of people for a set of natural resources or a particular area (Berkes, 2010). Since CBM encompasses many different management situations in which natural resources, whole ecosystems or territories are "owned" and managed by local groups, there is no general definition available. Yet, the quintessence of CBM is that management authority for a defined territory or set of resources is transferred to, or rests with, a clearly defined group at a local level, which shares certain common characteristics (e.g., ethnicity) or commonly resides in a geographical area (Armitage, 2005). Especially in tropical nations with weak state institutions, CBM has become a popular alternative approach for marine resource management. This is based on the notion that local actors are better suited to devise rules for addressing the roots of marine resource degradation (such as overfishing or the use of destructive fishing gears) than command-and-control approaches and other centrally organized solely government driven approaches (Ruddle, 1999; Ferse et al., 2010, 2014; Cinner et al., 2012). In fact, local communities all over the world have been involved in self-organized approaches to managing natural resources for centuries, and the idea of CBM originated from the acknowledgement of the effectiveness of such indigenous and traditional management systems for natural resources (Wade, 1988; Ostrom, 1990; Hidayat, 2005; Berkes, 2007a). A variety of management regimes for the sustainable use of natural resources has thus emerged based on local decision-making structures and formal or informal rules to secure the long-term socio-economic well-being of local populations (Borrini-Feyerabend et al., 2004, and references therein). The strength of such collaborative local endeavors is that communities can create solutions to local natural resource use problems, which are tailored to the particular local socio-cultural and environmental circumstances (Alcala, 1998; Armitage, 2005; Pomeroy and Rivera-Guib, 2006). Yet, it is also widely acknowledged today that CBM is not a one-size-fits-all solution to successful marine resource management (Berkes, 2004; Cudney-Bueno and Basurto, 2009; Cinner et al., 2012). Rather, CBM approaches for natural resources harbor a series of hazards and cannot be assumed to be a "panacea" or "blueprint" for successful natural resource management. Various studies have shown that their risk of failure is high (cf. Berkes, 2007b; Christie and White, 2007; Cudney-Bueno and Basurto, 2009; Cinner et al., 2012; Adhuri, 2013). Moreover, it cannot be simply assumed that if government actors endorse the development of CBM initiatives, new and effective rules will automatically emerge for successful CBM of natural resources (Schlager and Ostrom, 1992, 1999). Berkes (2004, p. 623) highlights in this regard that a "community" is a complex, elusive and multidimensional construct under constant change. Even small communities, therefore, cannot be seen as a unitary actor who per se acts toward the long-term benefit of the entire community. Rather, every community, whether small or large, is characterized by internal divergences of interests because any community is made up of various individuals and groups, which are embedded in larger systems and affected by influences from the outside (Berkes, 2004). Further empirical research is thus needed to better understand under which circumstances local initiatives can lead to improved sustainable marine resource use in a certain sea territory, and when CBM faces strong difficulties. Many studies have focused on the design of successful and persistent institutions in a self-organized CBM context that effect more sustainable resource use among the members of a community (cf. Wade, 1988; Ostrom, 1990; Cinner et al., 2012). Much less empirical research is available with regards to implementing CBM in the context of a regional resource use system, and in relation to CBM as part of a nested rule system to regulate resource use in a particular sea territory. In order to contribute to fill this gap, this study empirically investigates a CBM regime for the sea area surrounding Langkai Island, a small island located in the Indonesian Spermonde Archipelago off the coast of Makassar City. The objective of this research is two-fold: First, the study aims to provide a detailed analysis of what rules produced by which CPRR type actually have an impact on the fishing patterns in the sea territory as perceived by local resource users, and to illuminate potential challenges associated with implementing the rules generated by the different CPRR. The second objective of this article is to examine what motivates the local resource users to engage (or not) in the CBM of the marine resources. This article hereby complements previous more general work on CBM and informal rules in the Spermonde Archipelago by Deswandi (2012), Glaser et al. (2010, 2015) and Idrus (2009). The remainder of this article is structured as follows. The subsequent section provides an introduction of the study site, which outlines the particular fisheries related problems encountered and illuminates the presently implemented means to address them. Next, the methods applied in this research are described. The article then turns to the results. There, the article first focuses on understanding what rules actually affect the fishing patterns in the sea territory surrounding the study island based on the exploration of the perceptions of local fishers. The following section of the results then examines the rationales of the local fishers for engaging (or not) in the CBM initiative. The results are then discussed and put in a wider context. The article concludes with highlighting the main findings of the study and indicating further research needs to improve CBM initiatives for marine resource management. #### STUDY AREA: THE SPERMONDE ARCHIPELAGO, INDONESIA Indonesia is located within the Coral Triangle, one of the world's marine biodiversity hotspots (Burke et al., 2011). The country has about 81,000 km of coastline comprising about 4000 ha of mangrove forests and the national territory encompasses 5.8 million km<sup>2</sup> of sea area, of which ∼51,000 km<sup>2</sup> contain coral reefs (Syarif, 2009). The marine waters and its natural resources are of fundamental strategic, economic and environmental importance for Indonesia (Cribb and Ford, 2009). Yet, as a result of myriad anthropogenic pressures (Syarif, 2009), Indonesia is expected to experience the strongest decline in fisheries of any nation worldwide (Cheung et al., 2010). This is most severe for the people living in rural coastal areas and small islands, putting the livelihood security of millions of people at jeopardy (Ferrol-Schulte et al., 2013, 2015). In order to effect more sustainable resource use in Indonesia, a number of laws have been developed in an attempt to regulate the use of the country's fishery resources (cf. Syarif, 2009). These include for instance the ban of destructive fishing gears such as poison and blast fishing, and legislation to support the development of marine protected areas (Ferrol-Schulte et al., 2015). Yet, the different laws pertaining to the regulation of fisheries appear to only have little traction on the ground (Satria and Matsuda, 2004; Radjawali, 2012; Wever et al., 2012). Despite the existence of numerous Indonesian laws in the environmental realm, there have been only very few cases of effective enforcement through courts nationwide (Waddell, 2009). Especially in areas far away from larger towns and cities, the enforcement of government rules including the prohibition of blast and poison fishing by enforcement agencies is highly difficult. This study focuses on Langkai Island, a small island located at the outer margins of the Spermonde Archipelago, South Sulawesi (see Figure 1). The archipelago consists of ∼80–100 small islands inhabited by about 35,000 people (Sab and Katsuya, 2008). The islands greatly differ in terms of socio-economic characteristics (Glaeser and Glaser, 2010). The Spermonde Archipelago is home to one of the largest reef fisheries in Indonesia (Petsoede and Erdmann, 1998). Due to the physical characteristics of the islands, which hardly permit any land-based livelihood activities (Schwerdtner Máñez et al., 2012), fishery resources are of fundamental importance to provide the households in the archipelago with monetary and subsistence income (Pet-Soede et al., 2001; Glaser et al., 2015; Miñarro et al., 2016). Yet, similar to other areas in Indonesia and elsewhere in South-East Asia (Burke et al., 2011), the fisheries resources in the Spermonde Archipelago are increasingly depleted (Glaeser and Glaser, 2010; Glaser et al., 2010; Ferse et al., 2012) and the coral reef ecosystems are heavily degraded (Edinger et al., 1998; Plass-Johnson et al., 2015a,b, 2016). This jeopardizes the livelihoods of thousands of people as an ever growing number of fishers in the archipelago competes for increasingly scarce marine resources (Glaser et al., 2010; Deswandi, 2012; Miñarro et al., 2016). Moreover, unsustainable and destructive fishing practices including blast and poison fishing are used all over the archipelago and pose a major threat to the viability of marine resources and marine ecosystems (for more details on destructive fishing and its consequences on the marine ecosystems in the Spermonde Archipelago see esp. Pet-Soede et al., 1999; Chozin, 2008; Wilkinson, 2008; Idrus, 2009; Ferse et al., 2014; Pauwelussen, 2015). Effective means for more sustainable marine resource use are thus urgently needed to address this development. Unlike elsewhere in Indonesia, traditional customary fishery management systems such as the sasi laut in the Maluku Archipelago, for instance described in detail by Novaczek et al. (2001), are not found in the Spermonde Archipelago. Yet, in addition to official government laws, informal means to organize marine resource use have emerged in the Spermonde Archipelago. Today, local agreements between fishers (locally called kesepakatan) constitute informal rules, which have developed over time, and contribute to organizing the fishery in several areas in the Spermonde Archipelago (Glaser et al., 2010, 2015), including the sea territory around Langkai Island. #### METHODS The research applies a mixed-methods anthropological research approach to advance understanding of how to institute more effective community-based marine resource management in a small island setting. The study was conducted as part of the third phase of the joint German-Indonesian research program SPICE (Science for the Protection of Indonesian Coastal Marine Systems, 2012–2015) and builds upon the research conducted during the second phase of the SPICE program (2007–2010). Data for this study were collected over a 6 month field research period in the Spermonde Archipelago area from September 2012 to March 2013. Three visits of about 2 weeks each to Langkai Island were carried out. Further islands, including Lanyukang Island, Barrang Lompo Island, Lumu-Lumu Island, and Barrang Cadi Island, which are located in close vicinity of the study island (up to 2 h by boat), were visited for shorter time periods of about 2–5 days. In addition, a number of interviews with government officials on Sulawesi were conducted for the purpose of this study. Prior informed consent was obtained from all informants in this study. Moreover, the research was conducted in accordance with all ethical standards outlined in the Amended and Updated White Paper on Safeguarding Good Scientific Practice by the German Science Foundation [Deutsche Forschungsgemeinschaft (DFG), 2013]. The following section outlines the different methods used in this research (for more details on the methods see Bernard, 2006). #### Key Informant Interviews Using a semi-structured interview outline with open-ended questions, in-depth interviews were conducted with 69 informants on Langkai Island, on other small adjacent islands whose inhabitants frequently fish in the Langkai Island area, and government officials in Makassar City. The vast majority of respondents in all islands were fishers, but interviews were also conducted with traders and island officials with functions in the local administration structure<sup>1</sup> (for details see Table 1). In addition, interviews were carried out with government officials in Makassar City from the Water Police, BAPPEDA (Badan Perencana Pembangunan Daerah, responsible for marine spatial planning) and DKP (Dinas Kelautan dan Perikanaan, responsible for fisheries and marine conservation). Usually, an informant was not only interviewed once but visited several #### TABLE 1 \| Key informants. \*Some of the informants fall in multiple categories. times over the 6 months research period to inquire about different topics related to this study. Especially on Langkai Island, about eight informants served as central informants and conversations were held almost every day during the time spent on the island. In general, all interviews focused on understanding the development of the Langkai Island economy, changes of the social, economic and ecological circumstances, the different mechanisms in place that aim at organizing the appropriation of fishery resources in the sea territory surrounding Langkai Island, the impacts of these mechanisms on fishing behavior, and the reasons why some mechanisms work better than others. The particular topics covered in each interview were aligned to the expected knowledge of the informant, and sometimes adjusted to the actual knowledge of the interviewee. Except for the interviews with government officials, with whom more formal interviews were conducted, the interviews in the islands were not conducted as formal interviews since the topics covered highly sensitive matters such as the involvement in illegal fishing activities. Rather, after announcing the topic of this research, the intended use of the information, assuring anonymity to the individual respondents and obtaining informed consent from the informants, the interviews were carried out <sup>1</sup>Note that almost all of the positions in the administrative structure at the local level are voluntary and unsalaried, and the vast majority of the island officials relied more or less directly on fishery related livelihood activities such as fishing, trading, fishing boat construction etc. for their income. as informal conversations on the topic of the research to build as much trust as possible. Small groups of fishers, or individual fishers, were randomly approached at their homes or in public places in different areas of the small islands. Sometimes, upon recommendation by other island inhabitants, certain individuals were visited and asked to participate in the interviews because of their key role in CBM, or their anticipated in-depth knowledge of a particular aspect of the research. None of the conversations was recorded to further ensure an informal atmosphere and anonymity of the informant. Instead, particular effort was given to accurately document the content of the conversation in field notes during and after the conversations. All interviews were conducted by the author of this article with the help of a research assistant, who is a native speaker of the different local languages used in the area and has extended experience in working with the island communities on marine resource management in the Spermonde Archipelago and nearby areas. Where applicable, information received in one interview were triangulated in various interviews in the study island, on other islands, and on the Sulawesi "mainland" to verify data and cover a wider range of perspectives. ### Participatory Observation Participatory observation is a research method mainly used in cultural anthropology (Bernard, 2006). For this study, it was used to learn about social processes the interviewees may not be aware of, or are reluctant to talk about, and to further triangulate information obtained otherwise. The scope of participant observation in this study was limited, however, and only included attending relevant official meetings and informal gatherings, as well as observations of fishing behavior in the waters surrounding the island. #### Net-Map Interviews An adapted version of the participatory research method "Net-Map," described in detail by Schiffer and Hauck (2010), was used for this study. The method allows to visualize knowledge about the interplay of complex formal and informal social relations, the influence different actors exert on resource use patterns, and to unveil the social processes in natural resource management (cf. Gorris, 2015; Hauck et al., 2015). Two Net-Maps were developed in group interview sessions with fishers. The social relations that influence the fishing pattern in the Langkai Island waters, as perceived by the participants, were mapped. One group session was conducted with fishers from Langkai Island and the other group interview session was carried out with fishers from another nearby community in Barrang Lompo Island, who were wellknown for using illegal fishing methods. On Langkai Island, the Net-Map group was composed of eight participants. On Barrang Lompo Island, the Net-Map session consisted of six participants. It was not intended to ensure a representative sample of the respective island in these interviews, but rather to ensure that the interview participants had long-standing experience of fishing in the sea area around Langkai Island, and possessed in-depth knowledge on how the fishery in the area is organized. Hence, all interview participants in both Net-Map sessions were fishers who frequently fished in the sea territory surrounding Langkai Island and were thus equipped with in-depth knowledge of the organization of the fishery in the area. Moreover, it was sought to include representatives of the wide variety of different fishing gears used in the Langkai Island sea area. Potential candidates meeting these requirements were identified prior to the Net-Map session based on recommendations by key informants, or were key informants themselves as described above. Potential candidates were contacted at their residences, or their fishing boats after fishing trips. Yet, eventual participation in the Net-Map group interview depended on the availability and interest of the fisher. The Net-Maps were developed in a three-step process. A large sheet of paper was placed in front of the netmapping group. In a first step, the participants were invited to think of all actors that either are affected by, or themselves affect the management of natural resources in the waters surrounding the study island, i.e., who fishes in that area using what gear type, or who has an influence on the marine resource use patterns in the area. The identified actors were noted on cards and glued on the paper. In a second step, the netmapping group described who exercises influence affecting another actor. Influence of one actor toward another actor was indicated by an arrow on the paper. In a third step, the netmapping group participants were asked to judge how much influence they considered the different actors to have on the way marine resources are used in the area. A scale between one and four (four representing the highest possible influence) was used to determine the degree of influence of the respective actor. Discussions on the reasons for the thus constructed map followed. The netmapping approach, as adapted and used in this study, offers the opportunity to advance understanding of the de facto marine resource management through the visualization of social relations that affect marine resource use in the Langkai Island sea area. Data was digitalized and visualized using the social network analysis software Gephi. #### Survey The study was complemented by the results of a survey (for details see Supplementary Material in the online version of this article) with fishing households to provide socio-economic context data for Langkai Island (see Section Langkai Island: Fueling the Local Economy). The survey was conducted by a team of German and Indonesian researchers in several islands in the Spermonde Archipelago. This article only draws on the results of the survey interviews conducted on Langkai Island. A geographically stratified random sampling was used for selecting the respondents. Thirty-eight survey interviews were conducted representing about 20% of the island's fishing households. The survey participants were the household heads (all male). The households were selected in a lottery system from a list of fishers. Only descriptive statistics was used since the low absolute number of participants in the survey from Langkai Island does not allow for in-depth statistical analysis. ## RESULTS #### Langkai Island: Fueling the Local Economy At the end of the 1940s, only 10 people who were all fishers permanently lived on Langkai Island. During that time, the main fishing gear used by these fishers was hand-line and the most important targeted species was the Narrow-Barred Spanish Mackerel (Scomberomorus commerson, called Tenggiri in local language). Today, the island population has grown to 225 households of which 190 (∼84%) rely on fishing as their primary and mostly only source of income. This reflects the fundamental importance of marine resources to secure the local livelihoods. The results of the survey show that numerous fishing gears are used by Langkai islanders today (see Figure 2). Depending on the season, most fishers used different gear types during different times of the year. Yet, hand line still has remained the most commonly used fishing gear to target a variety of fishery resources (used by 41% of the fishers). The second most commonly used fishing gear is gillnet, used by 32% of Langkai Island's fishers. Further gears used include driftnet, fish trap, mobile lift net, and compressor diving, while some other gears were only used to a minor extent. Despite the introduction of new fishing gears over the past 50 years, which allowed the islanders to target a wider range of fishery resources, the hand-line fishery has remained particularly important for the local economy. The continuous importance of the hand-line fishery is due to the high abundance of economically valuable species in the area that can be caught by hand-line, and especially the occurrence of the Narrow-Barred Spanish Mackerel (which can be sold for ∼50–70,000 IDR<sup>2</sup> per kilo) in the sea area surrounding Langkai Island. Yet, the lucrative target fish, such as Mackerel, are unevenly distributed over the Spermonde Archipelago. Moreover, the increasingly depleted fish stocks and degrading fish habitats in the Spermonde Archipelago and the neighboring areas have motivated fishermen to search for fish in other areas than only the waters of their home islands. The sea territory surrounding Langkai Island has remained a particularly rich fishing ground where a wide range of valuable marine resources are still available. Hence, the area is not only subject to exploitation by local fishers from Langkai Island, but attracts many fishers from other islands and Sulawesi mainland fostering the competition for the valuable resources in the area. ## Organizing Marine Resource Use: Rules-in-Use in the Waters Surrounding Langkai Island Whilst not officially marked by flags or buoys, the "Langkai Island Waters" is a commonly acknowledged and relatively clearly defined marine territory surrounding the island. All interviewees from Langkai Island and from elsewhere, who frequently use the area for fishing, knew and acknowledged this. The interviewees were able to relatively precisely draw the borders of this area on a very large naval navigation map, and to describe the borders mainly based on aspects of the underwater topography and distinct features of the marine ecosystem. Since the area is perceived to belong to the island, the local community considers itself entitled to institute rules for the use of the area's fishery resources. Based on informal agreements, three locally devised rules were instituted for the use of marine resources in the Langkai Island Waters. These include the prohibition of (a) blast fishing, (b) poison fishing, and (c) the use of spear-guns for Mackerel fishing. The surveillance and enforcement of these local rules were carried out by the local resource users. In addition, an important role in the sustained implementation of these rules and for controlling what gear is used in the waters, so it was argued in the interviews, attributes to the elected island leader (Ketua Rukun Warga) to gain improved authority in rule enforcement. Yet, neither the local community in Langkai Island in general nor the island head in particular have a formal authority to develop and enforce such locally devised fisheries management rules for the Langkai Island Waters since the necessary official authority (by law) does not extent out to the sea territory, but only accrues to organizational matters on the community's land. As for the prohibition of blast and poison fishing, i.e., for the rules also found in national law, the enforcement of these rules for the Langkai Island Waters thus relies on the cooperation with the Water Police based in Makassar City. This is a difficult situation, so it was argued in the interviews, as the islanders do not hold official authority to detain rule-breakers until the police arrives. With regards to the spear-gun rule, there is no legal basis at all and the Water Police is not entitled to engage in enforcing this rule. Hence, the remaining option for the islanders to enforce all three rules at the local level is to apply alternative informal means for enforcement. Common practice is that, if somebody is spotted in the Langkai waters who uses or is suspected to use gears, which are prohibited by the local rules for the marine territory, fishers form a group, ideally with the island leader among them, and inform the respective fisher about the rules that apply to the Langkai Island Waters. Usually, according to the islanders, this is sufficient to scare the fishers away. If not, Langkai islanders may also throw stones at the rule-breakers. This common enforcement practice was also widely confirmed by fishers from other islands, who fish in the Langkai Island area, and whose inhabitants are particularly famous for fishing with bombs, poison and spear-guns. In fact, it was stated in the interviews in other islands that the interviewees heard that the fishers of Langkai Island would even confiscate the fishing gears, or set fishing boats on fire, which both would cause severe <sup>2</sup>At the time of this research, 1 Euro was equivalent to about 12,500 IDR (Indonesian Rupiah). economic loss for the fishers. While it was widely confirmed in the Langkai Island community and elsewhere that stones are used to scare rule-breakers away, the more drastic measures may also be a legend spread in the area. ## The Role of the Local Community in Marine Resource Management This section illuminates the perceived impact of the rules produced by the state CPRR and the CBM<sup>3</sup> on the fishing practice in the Langkai Island Waters based on the two Net-Map group interview sessions. Figure 3A shows the results of the group session with Barrang Lompo Island fishers, and Figure 3B shows the results of the session with Langkai Island fishers. Both groups identified fishers using different gear types in the Langkai Island Waters (for details see Figure 3). The fishers of Langkai Island created a more detailed picture of the fishing gears used in the area, which is certainly due to their more in-depth knowledge of the marine resources use patterns close to their island. With regards to who has an influence on marine resource use patterns in the area, both groups identified the Water Police (which is based in Makassar City) and the local community in Langkai Island. The notion of the Langkai islanders in both group sessions represents their influence on marine resource use patterns in the Langkai Island Waters. Government departments, and particularly the Department of Fishery and Marine Conservation (DKP), were only mentioned to have an impact by the Barrang Lompo Island group. The interview participants from Langkai Island did not see their direct influence on the resource use patterns in the Langkai Island Waters. This may be explained by the fact that Barrang Lompo <sup>3</sup>Note that the Fish Aggregation Devices (see below) are located outside the Langkai Island Waters and are thus not included in these interviews. Island is relatively close to Makassar City, where the government departments reside. Due to this proximity, and maybe also due to the fact that the Barrang Lompo Island residents are well-known throughout the Spermonde Archipelago for using destructive fishing, government programs such as awareness raising campaigns frequently target fishers from Barrang Lompo Island, whilst such activities occur very rarely on Langkai Island. Both groups argued in the interviews that the Water Police has a strong influence on poison and blast fishers (influence is marked by arrows in Figure 3), who attempt to fish in the area. The Langkai Island community was also found to affect these two types of fishing operations as a result of the informal agreements for this specific portion of marine territory. Moreover, the Langkai Islanders also influence the use of spear-gun fishers as, based on the local rules, they are not allowed to fish for Mackerel in that area. Hence, in the view of the both Net-Map groups, both government actors and the island community contribute to regulate marine resource use in the Langkai Island Waters. Participants of both sessions agreed that the Water Police has the maximum possible influence (indicated by the size of the dots in Figure 3) on the resources use patterns in the Langkai Island Waters due to their official power of apprehending fishers using illegal gears. Despite the fact that illegal fishers will most probably not be prosecuted in court, participants in both groups argued that, if caught by the Water Police, illegal fishers will still spend some days or even weeks in jail during which they cannot generate income for their family, and that they also have to spend a significant amount of money for their release. This means a substantial financial loss for these fishers. However, it was argued in both sessions that, while the Water Police generally exerts strong influence on the resource use patterns, patrolling only occurs rarely in the general Langkai Island area, as it is far from the police station in Makassar City, and patrolling the area of the actor on resource use in the area on a scale between 1 and 4 (the larger the dots, the higher is the perceived influence). requires high financial input in terms of gasoline. In addition, the large Water Police boats are visible from a long distance and, if they are in the area, fishers will not carry out any illegal fishing operations. Therefore, while the general influence of the Water Police in terms of deterring blast and poison fishing when in the area is considered high, their actual impact on avoiding illegal fishing operation in the Langkai Island Waters is limited due to their rare presence in the area. By the participants of the Barrang Lompo Island group, the other government actors were perceived to be less influential compared to the Water Police. It was argued that the influence of the other government actors stems from the awareness raising campaigns about the danger of blast and poison fishing, which led some of the respective fishers to reconsider their fishing practice. The perception on the influence of the local community on Langkai Island on the resources use patterns in the area slightly varies between the Net-Map sessions conducted in the two islands. The Barrang Lompo Island interview group saw less influence of the Langkai Island community compared to the Water Police. In contrast, the Langkai Island interview group also perceived the island community to have maximal influence. Participants in the Langkai Island group argued that they can develop rules for the area, which are complied with by the fishers from Langkai Island itself, and also by the majority of outsiders. Yet, the participants also highlighted that their means of actual enforcement is limited as they do not possess legal enforcement authority. The Langkai Island group reported that the cooperation with the police "is not always easy" as the police may be in other parts of the Spermonde Archipelago, or elsewhere, and may not come to Langkai Island, even upon request by the islanders. Therefore, Langkai Islanders usually rather tend to only scare rule-breakers away from the area instead of detaining them and cooperate with the police. The participants in the Barrang Lompo Island group argued along similar lines but especially highlighted that the islanders do not possess official authority to enforce rules in the Langkai Island Waters and, therefore, awarded the local community in Langkai Island with less than the maximum amount of "influence points." ## Community-Based Management of Marine Resources: Why Do Local Fishers Engage in Rule Enforcement? The support of local initiatives and the active engagement of a high share of the community in the enforcement of the related rules is a necessary precondition for a functioning rule system. This section illuminates the rationales behind the motivation of local fishers on Langkai Island to engage in the enforcement of the locally devised rules. Table 2 at the end of this section summarizes the fishers' rationales for engaging in the enforcement of the local rules. #### Blast Fishing Blast fishing is widely used in the Spermonde Archipelago. While there was also a more frequent use by fishers from Langkai Island up to the 1990s, today, only one fisher sometimes uses small bombs. The fishing practice by this fisher is despised by the other community members, but it was argued in the interviews that the other fishers cannot do much about it, except for trying to keep the fisher from operating the bombs in the Langkai Island Waters. Whilst a number of people on Langkai Island reported that they are also very strict on enforcing the blast fishing prohibition, in fact, other fishers reported that they tend to remain "inactive" in the enforcement of this rule and rather tolerate the use of blast fishing in the Langkai Island Waters for four main types of reasons. more general worry with regards to reciprocity was that the fishers using illegal fishing gears are believed to have very good relations with "important people" in Makassar City, which is why blast and poison fishers most probably will not be prosecuted for illegal fishing. Moreover, interviewees feared that they themselves would "get problems" if handing over illegal fishers to the police since it might be taken as an offense by "the important people in Makassar" to apprehend fishers who are under their protectorate. #### Poison Fishing The situation with poison fishing is different and at the time of this study there were no active poison fishers on Langkai Island. According to the informants, the prohibition of poison fishing was enforced much stricter locally than the prohibition of blast fishing. While the (1) issues in enforcement, and (2) reciprocity, as described in the previous section, remain the same in the given rationales for engaging in the enforcement of the poison fishing rule, in contrast, the (3) perceived strong negative impact on own fishery yield, and the (4) benefit-sharing differed for the case of poison fishing. #### Spear-Gun Fishing for Mackerels The third local rule-in-use relates to the prohibition of spear-gun fishing for Mackerel, one of the marine resource most valuable to the Langkai Island fishing community. This rule appeared to be at least as strictly enforced locally as the prohibition of poison fishing. The reasons behind (1) issues in enforcement, and (2) reciprocity, as already outlined before, also remain to some extent for this rule, but cooperation with state actors was not possible at all. Differences compared to the blast fishing rule again accrue to the (3) perceived strong negative impact on own fishery yield, and the (4) benefit-sharing. (3) Perceived strong negative impact on own fishery yield: The Mackerel fishery is vital for the local economy on Langkai Island. Hand-line and spear-gun are the two fishing gears most adequate to target Mackerel. According to the informants, the agreement to prohibit the use of spearguns for Mackerel fishing has two central reasons. First, as previously noted, the price for Mackerel caught by handline ranged between 50 and 70,000 IDR<sup>6</sup> per kilo at the time of this research. The kilo price for Mackerel caught by spear-gun was with 40–45,000 IDR much lower. The lower price results from the fact that the fish caught by speargun displays strong visible marks (i.e., the entry and exit injuries of the spear). To achieve the highest possible price for the amount of fish in the area, spear-guns are not used by the Langkai Island community, but only by outsiders. The spear-gun, however, is more effective than using hand-lines, and more fish can be caught in less time. If fishers from other areas use spear-guns, they have an advantage over the Langkai Island fishers and can catch a larger share of the total <sup>4</sup> It could not be revealed in further communication on the matter with marine biologists whether this perception holds true, or whether this is a misperception. <sup>5</sup> It could not be revealed in further communication on the matter with marine biologists whether this strong impact is true, or whether this is a misperception. <sup>6</sup>At the time of this research, 1 Euro was equivalent to about 12,500 IDR (Indonesian currency). fish in the area, but the overall yield will only be sold at a lower overall price. This would decrease the overall revenue that could be generated from the fish in the area. The second reason for the agreement is that the local Mackerel fishers perceived that, if Mackerel is caught by a spear-gun, the remaining fish will be scared away due to the fast movement of the spear and the blood spilled into the water. It was argued that, if only hand-lines are used to catch Mackerel, the "fellow fish" will not notice that "somebody" is missing and stay in the area while the use of spear-guns "scares them" away immediately. While fishers would prefer an overall legal prohibition of the use of spear-guns for Mackerel fishing in the entire archipelago, it was argued that the Langkai Island community can only influence what happens in the Langkai Island Waters. Both objectives of the rule thus relate to achieving the highest economic return from the overall abundance of the fish in the area. (4) Lack of benefit-sharing: The use of spear-guns for fishing Mackerel by outside fishers offers no economic incentives for the local community to tolerate it. #### Fish Aggregation Devices (FAD) In addition to the rules pertaining to the Langkai Island Waters, a further local informal agreement is found in the area. A Fish Aggregation Device (FAD, locally called rumpon) is a tool to attract fish and keep them nearby. It is an effective tool to concentrate fish in a certain area, which then can be easily harvested. Langkai Island fishers installed FAD westwards off the island, already outside of the area that is perceived to be the Langkai Island Waters. The general understanding among the fishers, not only in Langkai Island but also in other areas of the Spermonde Archipelago (cf. Chozin, 2008), is that who owns the FAD, and maintains it, also privately owns the fish that it aggregates, and that fishing around the FAD is prohibited, or requires the permission of the owner. The informal agreements regarding the FADs thus can be considered a private CPRR in which individuals own a set of marine resources in a defined marine area. For harvesting the fish around the FAD, some owners on Langkai Island collaborate with purse-seine fishers from other areas. The general agreement for the FAD is that if there is enough fish in the area, the purse-seine fishers will inform the owner that they now start to harvest. When harvesting a FAD, the catch will be shared and the total amount of harvested fish divided into four parts, of which one part goes to the FAD owner, whilst the other three parts go to the boat that harvests the fish<sup>7</sup> . If the FAD owner himself harvests the FAD, of course, he keeps the fish to himself. Since the rules associated with the FAD are no CBM rules, but the rules relate to a private CPRR, different issues arise compared to the CBM rules. The clearly economical nature underlying the motivation of the owner to engage in enforcement is obvious, and all owners reported that they try to enforce the rules as strictly as possible. <sup>7</sup>Note that there seem to be different agreements related to the FAD in the wider Spermonde Archipelago area. Chozin (2008) describes the FAD as a tool that is harvested by blast fishers using bombs. According to his detailed ethnographic description of another area in the Spermonde Archipelago, the sharing ratio is 2:3 in which the owner of the FAD gets two portions of the fish and the harvester gets three. As for the Langkai Island FAD, the FAD are harvested by Purse-Seine fishers, which also might explain the different share-ratio between the harvester and the FAD owner. "feel ashamed" and had to pay a high compensation fee. Outside fishers also reported that they feared hostility during Sawakung if they broke the rule, which would complicate their visits to the fishing grounds close Langkai Island. This shows that for the rules related to the FAD, the issues surrounding reciprocity support the compliance with the FAD rules due to the fear of social and economic sanction. ## DISCUSSION Effective means to address the unregulated and uncontrolled use of marine ecosystems and their associated natural resources are urgently needed (The World Bank, 2006; Young et al., 2007). While local approaches appear to be a promising means to achieve more successful natural resource management (Ruddle, 1999; Armitage, 2005; Ferse et al., 2010, 2014; Cinner et al., 2012), CBM harbors a series of hazards (Berkes, 2004; Cudney-Bueno and Basurto, 2009; Cinner et al., 2012). A better understanding of these hazards is needed to contribute to institute more successful CBM. In line with other observations from Indonesia and elsewhere, this study supports previous research that challenges the portrayal of CBM as isolated endeavors in which communities are buffered from the "outside" world (Agrawal and Gibson, 1999; Berkes, 2004, 2007b; Cudney-Bueno and Basurto, 2009; Seixas and Berkes, 2010; Adhuri, 2013; Pauwelussen, 2016). The results of this study show that particular problems emerge from "trans-local" variables, which hamper the effectiveness of the self-organized local endeavors. Moreover, the study illuminates that divergences in the economic rationales of the community members are an important factor which affect their motivations to engage (or not) in local approaches to managing marine resources. ## The Challenge of (Self-) Organizing Local Approaches to Managing Marine Resources in Context of an Entangled CPRR System The marine resource use patterns in the sea area around Langkai Island are impacted by a convoluted rule system generated by different types of CPRR. While the Indonesian state CPRR rules to ban highly destructive fishing are indeed found to have a perceived impact on the marine resource use in the waters surrounding Langkai Island, this study confirms wider observations that the enforcement of environmental law is fraught with difficulties (cf. Idrus, 2009; Glaser et al., 2010; Wever et al., 2012). Especially corruption, the long distance from the Water Police base to the case study area, and insufficient funds for adequate patrolling are central factors resulting in enforcement shortcomings of the rules produced by the state CPRR. This represents an eminent threat to the marine ecosystems and the abundance of fishery resources (Patlis et al., 2001; Dirhamsyah, 2006; Jones et al., 2011). Partly in response to the shortcomings of the state CPRR, local rules-in-use have emerged in the case study area despite the lack of legal authority to do so. Area-specific stewardship for a marine territory surrounding Langkai Island (CBM) and individual ownership (private CPRR) was informally institutionalized and locally devised rules based on informal agreements were instituted for a specified portion of the sea area surrounding Langkai Island. However, while the islanders' authority to devise rules for the Langkai Island Waters may be to some extent informally acknowledged by outside fishers, the selforganized local initiative lacks the official authority to formally develop and especially to locally enforce rules. As a result, close coordination between the local community and state actors is needed which represents a strong challenge, especially in context of a remote small island community. In consequence, the findings of this research further support the classical argument made for instance by Ostrom (1990, 2005) that, in order to contribute to increase effectiveness of self-organized local endeavors, and to reduce the challenge of coordination with higher level state actors for instituting and enforcing rules, a clear allocation of rights to the local level to devise rules, and the endowment of the community with appropriate legal means to enforce the rules, is essential. Moreover, Seixas and Berkes (2010), who explored success factors in multiple case studies on community-based enterprises in natural resource management, found in this regard that networks and partnerships which extend beyond the boundaries of a community are an important means to improve coordination in a nested rule system. Given the findings of this research together with the results of other studies from Indonesia and elsewhere (cf. Adhuri and Visser, 2006; Cudney-Bueno and Basurto, 2009; Gasalla, 2011), both aspects appear to be highly salient to effect more successful self-organized local natural resource management. #### CBM in a Regional Resource Use System The active engagement of the local population in the implementation of local regulations is a necessary precondition for a successful CBM initiative. In this respect, the analysis of local resource users' perceptions, which are socially constructed and informed by both personal experience and the information available (Clayton and Myers, 2009), are crucial to understand what motivates (or not) individuals to engage in CBM of marine resources (McClanahan et al., 2005; Walker-Springett et al., 2016). This study reveals how the divergences in the perceptions of the members in a community affect their motivation to engage in the CBM endeavor. Moreover, the findings particularly illustrate the challenges of dealing with factors that lie outside the influence sphere of a community. The vast majority of Langkai Island fishers cooperate and comply with "their" rules as produced by the CBM and the private CPRR. The fishers of the Langkai Island community neither use poison fishing, nor blast fishing, nor spear-guns in the Langkai Island Waters. Moreover, poaching at the FAD is perceived to be highly risky as it is difficult to conceal it in such a small island community. Thus, in fact, the rationales underlying the motivation of the islander to engage in rule enforcement, as reported in this study, mainly relate to rulebreaking of outsiders and, therefore, have to be understood in the context of defending the local resources against undesirable fishing behavior by non-community members. While research has shown that social sanctions can effectively induce intracommunity cooperation for collective action and compliance among community members (Ostrom, 1990, 1999, 2005), this study shows that this does not necessarily apply for non-community members. Rather, inter-community reciprocity concerns may arise when engaging in enforcing the local rules against outsiders, which can hamper the effective enforcement of the local rules. As a result, the reliance on social sanctions may be a pitfall in effecting rule compliance when the aim is to defend local resources against outsiders (see also, for instance, Cudney-Bueno and Basurto, 2009). The findings show that there are differences in the strictness of the enforcement of the local rules. These differences mainly stem from economic rationales of the community members. In fact, the motivation for the engagement in the enforcement of the local rules by the Langkai islanders are strongly based on shortterm economic considerations, i.e., on a "give-and-take" basis in the local context. If the fishing activities of rule-breakers are not perceived to strongly harm the fishing yield of individuals, and/or if benefits of the generated yields are shared with the Langkai Island community as a compensation mechanism for the environmental harm caused, the motivation of the affected community members to engage in rule enforcement seems to cease. As a result, the prohibition of the fishing activities, which are perceived to cause a stronger impact on the shortterm economic return of local fishers without compensation mechanisms are much stronger enforced. Ostrom (1990) raised concerns that the compliance of community members with self-organized rules that regulate the use of natural resources may be undermined, if resource users value the expected future opportunity of resource availability and possible future gains less than the value they can generate now or in the near future. While this may hold true for intra-community compliance to self-organized rules, the findings of this study indicate that the perceived danger of short-term economic losses of the local community members may be a particular success factor of a CBM initiative, if the aim of a CBM initiative involves to defend local resources against undesired use forms by outsiders. Despite the presence of diverse rules-in-use for organizing the marine resource use in the case study area, conservation thinking, i.e., the aim to preserve an intact local marine environment in the long run, played almost no role in the rationales given in all interviews. Rather, it appears that the locally devised rules in that area intend to ensure that the local community gets an adequate share of the diminishing local marine resources, which are exploited by a growing number of fishers from elsewhere. This also leads to concerns that the environmental conservation effects of the locally devised rules in the area may be limited and should be considered incidental. ## CONCLUDING REMARKS Especially in tropical nations with weak state institutions such as Indonesia, CBM has been widely advocated for its potential to achieve more effective natural resource management. However, detailed case study analyses of the challenges for implementing CBM in a particular sea territory remain very rare, but are particularly needed to understand the potential pitfalls for local approaches to marine resource management. This article provided a detailed analysis of a case study in Indonesia to contribute to fill this gap and help to institute more effective community-based marine resource management. The results of this study particularly emphasize the context dependence of the success of a CBM initiative for marine resources because a certain CBM initiative, even in what seems to be a small community in a remote island setting, is characterized by internal divergences, and by "trans-local" variables which create complex interdependencies. Especially divergences in the economic rationales of the community members are important factors which affect their motivation to engage (or not) in both the CBM and the private CPRR. While especially shortterm economic considerations appear to be a particular success factor in this study, such rationales underlying the motivation of community members to engage in CBM raise concerns about the sustainability orientation of the local measures. While the scope of this research with its narrow focus on a small sea territory appears limited, the study brings a suite of aspects to attention that are often overlooked, but are highly salient to understand the factors underlying successful CBM. Moreover, the situation in this island mirrors the situation of communities in other areas in Indonesia, and also in other countries with weak state institutions. Further research is especially needed on how to address the pitfalls of CBM that are induced by factors that lie beyond the reach of local communities, and on mechanisms for improved coordination between the different types of CPRR. This is particularly urgent for remote places such as a small island, where large portions of the population heavily depend on increasingly degraded resource systems to secure their livelihoods. Moreover, perception studies represent important means to assist marine planners, policy makers and natural resource managers to better understand the reality of CBM initiatives. ## AUTHOR CONTRIBUTIONS The author is responsible for data collection, analyses and writing of the manuscript. ## FUNDING I greatly appreciate the financial support from the SPICE III Project (grant number 03F0643A) funded by the German Ministry for Research and Education (BMBF) and the Leibniz Center for Tropical Marine Ecology (ZMT) in Bremen, Germany. Moreover, I acknowledge financial support from the Alexander-von-Humboldt (AvH) professorship for Environmental Economics of the University of Osnabrück (UOS). I also acknowledge support by Deutsche Forschungsgemeinschaft (DFG) and Open Access Publishing Fund of Osnabrück University. #### REFERENCES #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2016.00120 Archipelagic State, eds R. Cribb and M. Ford (Singapore: Institute of Southeast Asian Studies (ISEAS)), 1–27. Ostrom, E. (2005). Understanding Institutional Diversity. Oxford: Princeton. and Considerations, ed Food and Agriculture Organization (FAO) (Rome), 221–300. Zondervan, R., Gerhardinger, L. C., Torres de Noronha, I., Spalding, M. J., and Young, O. R. (2013). Ocean governance in the anthropocene. IGBP's Global Change Magazine 81, 24–27. Available online at: http:// www.igbp.net/news/features/features/oceangovernanceintheanthropocene.5. 64c294101429ba9184d483.html 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. The Guest Associate Editor, AB, declares that, despite having recently published with the author, PG, the review process was handled objectively and no conflict of interest exists. Copyright © 2016 Gorris. 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. # Analysis of Perceptions and Knowledge in Managing Coastal Resources: A Case Study in Fiji Jokim V. Kitolelei\* and Tetsu Sato\* Research Institute for Humanity and Nature, Kyoto, Japan Sustainable management of coastal resources depends on human knowledge and perceptions of natural resources and coastal environments. However, empirical evidence has been limited in order to understand linkages between knowledge, perceptions and collective actions to achieve sustainable resource management. This case study analyzed perceptions and knowledge among diverse stakeholders: villagers, government officials, scientists and staff of a non-governmental organization who are collaboratively working in a Fijian coastal community to manage the local coastal resources. Analyses were made using the integrated local environmental knowledge (ILEK) concept and frameworks of discourse analysis to clarify interlinkages between perceptions, knowledge and collective actions for a variety of examples. Research was conducted in Kumi village on the island of Viti Levu in Fiji, and the investigated projects included the management of a locally managed marine area, seaweed aquaculture, sea cucumber restoration and ginger plantations. These initiatives have shown that diverse knowledge on coastal resources and environments influence perceptions among people in a complex way, and transformation of perceptions produced new sets of knowledge through the generation of hypotheses regarding the management of coastal resources. Collective actions were promoted by the transformation of perceptions, and social learning processes were mobilized by these collective actions. Traditional institutions, cultures and leadership roles deeply embedded in the local communities had strong influences on shared perceptions among community members to provide foundations for collective actions. Dynamic transformations of perceptions promoted by integrated knowledge among community members were critical enablers of collective actions to achieve sustainable resource management. Keywords: coastal resource management, perceptions, integrated knowledge, stakeholders, collective actions #### INTRODUCTION Coastal communities across the Pacific Islands continue to face challenges in managing their marine and terrestrial resources effectively. Demand for seafood is growing (Delgado et al., 2003) and fisheries products are one of the highest traded food commodities worldwide (Asche et al., 2014). Pacific island developing countries experience slow economic growth and development, and almost 100% of the populations live within 100 km from the coast (Martinez et al., 2007). The fisheries resources are highly valued and provide 50–90% of animal protein in the Pacific islands #### Edited by: Annette Breckwoldt, Leibniz Center for Tropical Marine Ecology, Germany #### Reviewed by: Andrew M. Fischer, University of Tasmania, Australia Katrine Soma, Wageningen University and Research Centre, Netherlands #### \Correspondence: Jokim V. Kitolelei [email protected] Tetsu Sato [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 31 March 2016 Accepted: 15 September 2016 Published: 28 September 2016 #### Citation: Kitolelei JV and Sato T (2016) Analysis of Perceptions and Knowledge in Managing Coastal Resources: A Case Study in Fiji. Front. Mar. Sci. 3:189. doi: 10.3389/fmars.2016.00189 (Pacific Community, 2012). The life of people in these coastal areas depends on fisheries, tourism, agriculture, mining and small businesses, and they are vulnerable to various environmental problems in both terrestrial and marine areas (Kronen et al., 2009; Bidesi et al., 2011). Although Fijians are well renowned in adapting to their island environment (Veitayaki, 1995; Veitayaki et al., 2015), there have been continuously high levels of coastal fisheries exploitation in the past decade (Hand et al., 2005; Teh et al., 2009). As the threats of resource depletion, overfishing, consumerism, population growth and climate change have become profound, there is a growing need for research regarding the knowledge and perceptions of communities to promote collective actions that can ensure a sustainable use of natural resources. In Fiji, people have been managing their coastal resources through the introduction of new crops, implementation of locally managed and protected marine areas, and small-scale projects to restore depleted coastal resources (e.g., mangroves). These adaptive responses among people have been promoted through collective actions supported by their knowledge and perceptions on the surrounding coastal environment and resources. Also, traditional social systems and decision-making processes in Fijian communities are likely to play significant roles in these responses. In previous studies, perceptions have been recognized as a promoting factor of the process of changes in fisheries management (e.g., Cinner and Pollnac, 2004; Brewer, 2013; Bennett and Dearden, 2014; Barley-Kincaid and Rose, 2014). However, less attention was given to the transformation mechanisms of perceptions and the underlining knowledge systems that influence collective actions by local people with regard to the complex management of their multiple coastal resources and environments. In this paper, perceptions are defined as the cognitive framework of people to see the external world, to extract meanings and create collective actions with regard to the coastal resources they utilize in their daily lives. We assume that perceptions are dynamically transformed by the emergence of knowledge that is also dynamically produced and translated through livelihood and practices among people living in an ever-changing world. Locally-based scientists, who are embedded in communities, play an important role to integrate and systematize diverse knowledge. This could be either a residential type of living in the community as a member and stakeholder, or a visiting type, having the research bases in remote areas to "use" local communities as a field research site (Sato, 2014). We also recognize that there are various types of "bilateral knowledge translators," including residential and visiting researchers, government agencies, non-governmental or non-profit organizations (NGOs, NPOs) circulating and integrating transdisciplinary knowledge derived both from external scientists and from local communities (thus "bilateral") to create meanings of various knowledge components for local communities (Sato, 2014). In this paper, we analyze transformation mechanisms of knowledge and perceptions to promote collective actions and social learning among diverse stakeholders: villagers, government officials, scientists and NGO members, who are collaboratively working in a Fijian coastal community to manage the local coastal resources. The renewable resources discussed in this paper include coastal marine resources (seaweeds, Anadara clams and sea cucumbers) and a land resource (ginger). We analyzed the cases of collective actions among villagers to manage or restore these resources, and discuss (1) how knowledge production and translation contribute to the dynamic transformation of perceptions among stakeholders, (2) how collective actions can be promoted based on shared knowledge and perceptions, and (3) how social learning in collective actions influence perceptions and knowledge systems. We discuss the importance of knowledge translations to promote these processes, with special attention given to the function of knowledge and perceptions rooted in traditional institutions, rules and practices. Results of these analyses contribute to elucidate interlinkages between knowledge, perception and collective action to achieve effective community-based management of coastal resources in complex social-ecological systems. ## METHODOLOGICAL APPROACH #### Study Area The Republic of Fiji has 332 islands with a population of 909, 389 (CIA, 2015), and a total area of 18, 333 km<sup>2</sup> (Lane, 2008). There are more than 800 villages and settlements in Fiji, and these communities are dependent on both terrestrial and marine resources for survival and sustainable development (e.g., Kitolelei et al., 2011). Kumi village is located 17◦ South and 178◦ East on the eastern side of Viti Levu, the main island of Fiji (Figure 1). It has a population of 273 people with 84 households and is one of the seven villages in Verata District. The total area of the district is 235, 95 km<sup>2</sup> of which are coastal and marine and 140 km<sup>2</sup> terrestrial. The terrestrial ecosystems of Kumi village include secondary forests and grasslands, as well as farmlands and plantations. The marine ecosystems include coral reefs, seagrass beds, intertidal flats and mangrove forests. The diverse marine ecosystems are home to a variety of marine resources, which the villagers of Kumi harvest for subsistence and commercial uses. The marine resources that are sold in local markets include Anadara clams that are collected by women, and various finfishes. In Kumi and its district, the Anadara clams that can be found in shallow mudflats and seagrass beds, are a traditional totem that people respect and honor (Vunisea, 1996). Members of coastal communities in Fiji have traditional clan totems, including various marine species, that they revere and respect. The Anadara clams are totems and at the same time an important income source for Kumi villagers, and therefore enable community members to respect coastal management decisions that are relevant to protect their totems. Root crops such as cassava, taro and other vegetables are additional sources of income. Kumi villagers sell their marine and agricultural products in three main markets (Korovou, Nausori, and Suva), and at times products are also sold within the village itself. The average income of households in 2014 was \$79.2 FJD (∼38 US\$) per week as a result of the sale of fisheries catch. Kumi village has a community canteen and a truck that are managed by the community to provide services for its members. There are several other committees that are responsible for various community-related decision-making, and development projects of the village, including committees for: women's group, water supply, education, a shop cooperative, seaweed, sea cucumber, and ginger. Fijian coastal villages such as Kumi are characterized as closely-knit communities with strong traditional decision-making systems. For example, the traditional fishing boundaries designated to villages (the qoliqoli boundaries) are shared and followed amongst the clan members (Kitolelei et al., 2014). The tradition of "solesolevaki" is one such rule, implying the importance of working together as a group to achieve a given task for the community. The tabu tradition, in which fishing areas are closed for a certain period of time, is a tool that is still commonly practiced throughout Fijian communities (Johannes, 2002). Following such traditional rules and customs is constructive behavior in the communities, and breaking them can result in traditional punishment and social shaming. And while they are increasingly questioned and partly eroding (Vunisea, 2002), these strong and still widely-existing traditional institutions and rules remain one of the outstanding characteristics of Fijian community life. ## Field Research Methods This research focuses on perceptions of diverse stakeholders from within and outside Kumi village (see Table 1), who are collaborating in the coastal resource management of the village. Particular emphasis was placed on dynamically transforming perceptions among the villagers, who are the major actors and caretakers of these resources. The research used a transdisciplinary approach, including participatory observations, and individual as well as group interviews, which were conducted on three visits to the village and to different stakeholders' offices between October 2013 and June 2014. Twenty three stakeholders from a wide array of groups including government officials, university scientists, an officer of a conservation NGO, and Kumi villagers, were selected (Table 1). The rationale behind the selection was that they were actively involved in collective actions to manage Kumi's coastal resources, and built trust with the authors to collaborate throughout the research process. We could identify only a limited number of women who were actively taking responsibilities in decision-making of these collective actions, resulting in male dominance among interviewees from Kumi village. The exceptions were two elderly women who were respected among stakeholders and playing leading roles in these actions. Care was also taken to secure diversity among interviewees to avoid research bias (e.g., when several interviewees would belong to one clan) and to be able to triangulate and analyze the complex processes and interlinkages of knowledge production, transformation of perceptions, and individual as well as collective actions. Individual and group interviews were structured into two sets: the first one targeting Kumi villagers regarding ongoing collective actions, and the second one targeting external stakeholders regarding the roles and functions of "knowledge translators." All interviews and participatory observations were conducted by the first author, a graduate student of Kagoshima University at the time of this study. The university did not have a formal evaluation TABLE 1 \| Number of villagers and other external stakeholders involved in this study. committee for the ethical considerations related to social science field studies, which is why we strictly followed the Code of Ethics of the International Sociological Association (Section 2 Data Gathering, see International Sociological Association, 2011), including security, anonymity and privacy of the stakeholders involved in our study, and prior informed consent. Discourse analyses were conducted using a simplified form of qualitative sociological discourse analyses summarized by Ruiz (2009). The memos of interviews and group discussions as well as informally obtained narratives of these stakeholders in participatory observations were analyzed to extract their knowledge systems, perceptions and collective actions. In our preliminary field survey, we identified four ongoing collective actions in the village: a seaweed culture project, sea cucumber restoration, a ginger project, and the management of locally managed marine area (LLMA). Textual analyses were conducted with the accumulated discourses to extract sentences related to or containing words related to these collective actions. Contextual analyses were performed with these discourses to extract knowledge and perceptions behind these collective actions. In the analysis, knowledge was identified from discourses containing a set of information with regard to characteristics, status and modes of utilization of relevant coastal resources, and it was distinguished from those identifies as containing perceptions that represented the functional meanings of the available knowledge. The knowledge and perceptions thus extracted were then classified and mapped on the conceptual framework described below to visualize interlinkages between knowledge, perceptions and collective actions. All processes were interrelated so that, for example, the interpretations provided by categorization and mapping influenced the textual and contextual analyses. The processes were repeated until we reached an unambiguous interpretation as described in the results. ## Analytical Framing of Knowledge, Perceptions, and Collective Actions Over the years, various authors have documented diverse types of knowledge produced and shared in the Pacific Islands (Johannes, 1981; Thaman, 2002; Berkes, 2008; Campbell, 2009). The concept of integrated local environmental knowledge (ILEK) is introduced in this study as the key analytical element connecting knowledge, perception and collective action. The ILEK concept differs from previously introduced categories of local and empirical knowledge, such as traditional ecological knowledge (TEK; Berkes, 2008) or local ecological knowledge (LEK; Olsson and Folke, 2001), in its emphasis on dynamic and integrative views on knowledge (Sato, 2014). ILEK is generated by the interactions between diverse knowledge production processes in local communities, including scientific research, and integrates diverse types of knowledge produced and utilized by stakeholders (Figure 2). In this way, ILEK presents a range of solution-oriented knowledge systems in a transdisciplinary way, as it incorporates every relevant domain of science and technology as well as the empirical local knowledge and experiences required for the management of such complex social-ecological systems. ILEK is also characterized by its dynamic nature, constantly re-produced and transformed through interactions of the various involved stakeholders as knowledge producers. These stakeholders and "bilateral knowledge translators" play an important role in integrating and systematizing the diverse knowledge components that are used as bases for decision-making and collective actions. Such a dynamic and integrative view on knowledge with recognition of the diversities of knowledge producers and translators is essential in understanding its linkages with perceptions and collective actions in complex social-ecological systems. The analyses on interlinkages between knowledge, perceptions and collective actions were made based on our own observations and referring to previously accumulated information on diverse knowledge systems in Fijian communities (Sauni, 1999; Veitayaki, 2000; Lane, 2008; Govan, 2009; Teh et al., 2009) through the lens of ILEK. Figure 3 shows the conceptual framework of analysis for this study regarding knowledge (components of ILEK), perceptions, and individual as well as collective actions modified from Gregory (1997). There are many types of knowledge being continuously produced that become part of the available ILEK for stakeholders, which dynamically influences people's perceptions. Transformation of perceptions in turn influences ILEK by stimulating hypothesisgeneration and integration of new components of knowledge. Changes in a particular part of the perception systems influence behavioral patterns of each individual to create actions, which then sum up to collective actions to manage coastal resources and environments, especially when particular types of perceptions are shared among stakeholders. Individual and collective actions produce feedbacks to perceptions and knowledge by providing participants with opportunities of social learning. In other words, the perception arena in this framework is an agent connecting input (knowledge) and its outcome (actions). This analysis provided a set of snapshots of interlinkages between knowledge, perceptions and collective actions in the continuous processes of community-based coastal resource management, which extended beyond our study period. We aimed to extract important factors of collective actions by accumulating and analyzing these snapshots obtained in the limited study period. This conceptual framework assumes that collective actions can serve as a platform for social learning processes of all participating stakeholders, including residential and visiting researchers, to transform their knowledge systems (ILEK) and perceptions, thus resulting in adaptive improvements of the quality and impacts of actions. Transformed knowledge in this process may be disseminated to other villages in Fiji and potentially even beyond to be used for adaptive management of coastal resources in other regions. In this system, the transformation of perceptions by diverse stakeholders is assumed as a fundamental enabler of knowledge-based societal transformation toward sustainable futures of the coastal communities. ## RESULTS ## Diversities of Knowledge among Stakeholders The knowledge production processes and characteristics of produced knowledge varied among stakeholders with different interests and prioritized values. Table 2 represents diversities of prioritized values and framings of knowledge productions among major stakeholder groups working in Kumi village, extracted from the individual and group interviews. The gaps between the villagers and external stakeholder groups seem to be substantial, with little commonality and overlap expected for the knowledge produced from such framings and value systems. All these different knowledge components contribute to ILEK and are shared among villagers with different degrees of emphasis, which may result in individually isolated practices. Therefore, knowledge translation to extract and share new meanings of diverse knowledge sets is essential to create shared perceptions supporting collective actions. actions. TABLE 2 \| Prioritized values and framing of knowledge productions among diverse stakeholder groups working in Kumi village. In Fiji in general and also in Kumi village, sharing of knowledge among villagers is promoted through collaboration in activities such as fishing, farming, art and crafts, smallscale businesses and community functions. Fourteen among 15 interviewees in Kumi village stated that knowledge components created from community activities were shared in village meetings (5 interviewees) and through general customs and traditions of sharing knowledge (5), while others generally stated that it was shared (4). Younger generations were also mentioned to have an opportunity to share knowledge (1). A common practice in Fiji is producing and circulating knowledge during social functions where community members congregate during kava drinking sessions. During these events, local knowledge and experiences on coastal resources and their management are shared among members through informal conversation, and the members gain access to new knowledge sets when available. These social functions are basically open for the external stakeholders and therefore provide opportunities of interactions between different knowledge systems. Another common knowledge sharing process observed in this study was based on the communal way of life. Most houses are built at close proximity in the village and people are living in a closely-knit community resulting in information being easily spread from one household to the other. The term "coconut wireless" is given to this spreading of knowledge without any formal form of information-sharing. Various knowledge and skills derived from external stakeholders, including scientists, seemed to spread into the village through such processes, with translation of their meanings to fit to the villagers' context. On the other hand, opportunities for external stakeholders to learn and digest villagers' knowledge sets and perceptions seemed to be relatively limited except for the channels of kava ceremonies, resulting in mismatches between external interventions and villagers' perspectives and motivations. In this study, we found various types of knowledge translators contributing to mitigate these challenges, which will be described below. ## Interlinkages between Integrated Local Environmental Knowledge, Perceptions, and Collective Actions Figure 4 represents a snapshot of interlinkages between particular knowledge sets in ILEK, components of related perception systems, and relevant specific collective actions taken by the villagers during the study period. Through the discourse analyses, we identified seven categories of major knowledge sets, which were related to sea cucumber restoration, seaweed aquaculture, agriculture practices, and traditional resource management systems. These knowledge sets were classified into the basic knowledge often provided by external stakeholders such as government agencies and scientists (black), transformed knowledge sets translated from the basic knowledge by interactions among different stakeholders (blue), and traditional and empirical knowledge underlining almost all decision-making practices (green). Fourteen, seven and 11 villagers respectively referred to these three types of knowledge. Nine villagers mentioned that knowledge created and visualized shareable value in the community (indicating transformation of perceptions), and 13 stated that the knowledge motivated people to manage resources (mobilizing collective actions). These knowledge sets seemed to influence villagers' perceptions on the effectiveness of new and traditional resource management systems and techniques, the economic as well as other benefits related to resource management and their livelihoods, and on the potentials of knowledgebased consensus-building practices in the village. This transformation of perceptions in turn produced a series of collective actions. For example, influx of knowledge and changes of perceptions related to the effectiveness of new techniques of sea cucumber restoration and its potential economic benefits resulted in collective actions to restore sea cucumber resources in 2012. Knowledge related to seaweed aquaculture influenced perceptions on effective techniques and marketability, resulting in the implementation of a seaweed project in 2013. Various knowledge sets on farming techniques visualized the plausibility of producing new profitable products, and influenced the local perceptions on marketability of agricultural products, bringing about the implementation of a ginger project in 2013. On the other hand, we could not identify direct influences of particular knowledge sets on perceptions regarding implementation of the locally managed marine area (LMMA) in this study except for the indirect effects of knowledge and perceptions on traditional resource management systems and local leadership, which might be the prerequisites for the implementation of LMMA. This observation seemed to be reflected by the relatively longer time period after the initial launch of the LMMA in this village in 2007. The collective actions to manage the LMMA have been performed continuously to reach the stage maturity and stability of the management practices in this study period. Perceptions on the benefit of having a LMMA and its effective management systems might be less pronounced because they have already been shared and well-established among villagers. ## Social Learning and Hypothesis Generating Processes The original ideas and basic knowledge and skills for sea cucumber restoration and seaweed aquaculture were brought into Kumi village by Ministry of Fisheries and ginger farming by Ministry of Agriculture, while the LMMA system was disseminated by The University of the South Pacific scientists based on successful cases in other villages. However, these knowledge components and associated perceptions have been dynamically translated to allow new meanings through social learning in the processes of designing and conducting collective actions (Figure 4). Collective actions on sea cucumber restoration and seaweed aquaculture quickly transformed perceptions among participating community members on the effectiveness of materials and techniques of culture and restoration provided by the government agencies. People seemed to generate new perceptions on the value of more convenient, affordable and sustainable local materials for these practices, started testing these local materials based on the newly generated hypothesis, and brought about transformation of the knowledge system through social learning. Sea cucumber restoration also went through social learning processes regarding effective breeding of the species in small fenced enclosures in shallow waters. Villagers learned through their practices that sea cucumbers tended to congregate around the enclosure fence when sea cucumbers density in the enclosure was high. The knowledge derived from this observation transformed their perception on effective restoration techniques, generated a new hypothesis on density effects of sea cucumbers in the enclosure, and transformed their practices to induce possible spillover effects by breeding in the enclosure. On land, implementation of ginger planting supported by the government transformed their land use pattern for farming through learning by practice to utilize slopes on hills for ginger production, which had not been used for other crops so far. This collective action transformed their perceptions on improving livelihood and wellbeing by growing additional marketable products and produced new knowledge sets related to agriculture practices. The first LMMA in Kumi village was established to manage Anadara clams for the period from 2007 to 2009, and the success of this practice transformed perceptions of villagers with regard to potential impacts of LMMA upon their livelihood, wellbeing and sustainability of resources. Based on collaborations with The University of the South Pacific scientists, villagers also seemed to transform their perceptions on the values and impacts of their own management practices. This transformation of perceptions probably produced a new set of hypotheses regarding appropriate locations for LMMA setting and effects of shifting LMMA sites. The villagers had successively established and managed LMMAs from 2009 to 2011 and 2011 to 2016, but they had changed the LMMA site every time in between. This decision of selection and relocation of LMMA sites by villagers may be brought about by the transformed knowledge sets regarding appropriate environmental conditions of Anadara clam production, and impacts of shifting the LMMA location to improve environmental conditions. In all these examples, collective actions provided a platform of social learning among participants to transform their perceptions and generate new hypotheses with regard to the resources and their own management practices, producing new knowledge sets within their ILEK. ## Bilateral Knowledge Translators in the Community The dynamic transformations of ILEK and perceptions among villagers have been shared with other external stakeholders (i.e., knowledge producers) in various ways. Officials of the Ministry of Fisheries were frequently observed to visit Kumi to monitor the outcomes of the sea cucumber and seaweed projects. They collected data on the growth and quality of the products and observed locally-shaped restoration and aquaculture techniques, which were already disseminated to other villages. At the same time, they contributed new knowledge on technical developments in other villages to be shared with Kumi villagers. In the case of the LMMA, The University of the South Pacific scientists (including the lead author) played similar roles to promote knowledge circulation among villages that take collective action to establish and manage LMMAs. One community member of Kumi working in a company outside the village had also disseminated the success stories of LMMA in Kumi to other villages. All of these knowledge producers in and outside of the village can be regarded as "knowledge translators" (Crosby, 1997). In this study, they mobilized bilateral knowledge circulation by visualizing new meanings of locally developed knowledge and skills, to be shared with government, scientists and other communities in the area. Among these knowledge translators, all external stakeholders interviewed in this study recognized that the turaga ni koro played a significant role for Kumi as a link between the community and external translators (see Biturogoiwasa, 2001). The turaga ni koro is the headman of a Fijian village, chosen by the villagers and endorsed by the provincial government. He advises the traditional chief and other decision makers within the village regarding interventions from the external world. At the same time, we found that the turaga ni koro in Kumi advised external stakeholders including government agencies, NGO and university scientists with regard to conditions and needs of the village in general, especially with regard to resource managements (Figure 5). The village chief, elders, subclan chiefs, and religious leaders occasionally played a role of knowledge translators by traveling out of the community and attending meetings or visiting other communities within the province or region. By observing and learning from the marine resource management practices in other areas, they also shared their knowledge and influenced perceptions of the members of their own community. In addition, external translators from government, NGOs and The University of the South Pacific also directly visited the village to convey scientific knowledge. In all these processes, the turaga ni koro played a significant role as the gatekeeper of the community by controlling and promoting interactions between different knowledge systems and perceptions of diverse stakeholders, both within and outside of the village. He organized kava ceremonies in his house with the external stakeholders when they visited the village to promote knowledge exchange with the leader and decision-makers of the community. He guided these people around the village for interaction with other community members. Through these gatekeeping activities, the turaga ni koro seemed to translate the knowledge from both sides through the filters of his own perceptions, and blend external and local knowledge and skills to transform ILEK. ## Perceptions Rooted in Traditional Institutions In Kumi village, we found tabus in fishing practices, the sharing concept solesolevaki, clan totems, and traditional leadership as the fundamental institutions and decision-making systems of the community and its ILEK (Figure 4). Perceptions regarding the importance of these traditional aspects promoted collective actions based on consensus and collaboration among community members. A previous record found that the tabu tradition had been practiced in areas of Kumi fishing boundaries (Tawake et al., 2001). In this study, we found that knowledge on the tabu tradition was shared among Kumi villagers, and perceptions on its importance for the community served as the bases for collective actions of fisheries resource management (including the LMMA and sea cucumber restoration). The tradition of "solesolevaki" was shared and regularly practiced among Kumi villagers, including collective actions identified in this study. This practice seemed to provide behavioral foundations to integrate individual actions among different gender and age groups, to share responsibilities and collaborate in various labor-intensive community tasks. All interviewees in Kumi recognized that these collective actions were the result of community customs in the village. It was also likely that the cohesion among villagers to perform solesolevaki provided a platform for mutual support systems among group members, and mitigated potential conflicts of interest among participants of collective actions. The totem and related traditional knowledge on ecology and harvesting of the Anadara clams influenced the perceptions among Kumi villagers on the importance of managing the habitats of this important resource. This perception deeply rooted to their traditional culture provided a foundation to promote collective actions regarding the LMMA as well as the seaweed aquaculture on the mudflats, both of which were expected to contribute to improving the clam habitats. All traditional institutions and rules mentioned above were supported by the traditional village chief, elders, sub-clan chiefs and religion leaders and other important actors involved in decision-making on community level (Figure 4). The clan systems, centered around leadership of the chiefs with various traditional institutions and rules (such as tabus, solesolevaki, and totems), have been the oldest and most long-enduring institutions in Fiji, formed much before other institutions were brought into the communities by the colonial and current governments. We witnessed that these ancient institutions were still functioning well in Kumi village to date, to promote sharing of responsibility and collaboration among community members. ## DISCUSSION In this study, we found that dynamic production and circulation of ILEK contributed to transformations of perceptions regarding the status and values of coastal resources, the importance of locally developed techniques for resource restoration and management, the impacts of such new techniques on improving livelihood and well-being, and the significance of traditional institutions in achieving effective implementation of resource management projects. However, we also found that each component of knowledge in the ILEK such as knowledge on sea cucumber restorations or seaweed aquaculture did not directly correspond to particular sets of perceptions. Rather, the linkages between knowledge and perceptions seemed to be complicated in a way that each knowledge component influenced diverse sets of perceptions through different pathways of knowledge translations and meaning making. The resulting transformation of perceptions generated new hypotheses related to knowledge components which were often different from the original components. Our findings strongly suggest the importance of a complex systems approach to understand the interlinkages of knowledge and perceptions facilitated by knowledge translation and feedbacks through social learning and hypotheses generation. The transformation of perceptions had significant impacts on promoting various collective actions in this case study, supporting our initial theory of their fundamental function as an enabler of collective actions. However, ILEK and its constituent knowledge sets do not always produce collective actions toward sustainable directions. Collective actions are often influenced by the prioritized values among stakeholders and prospects of tangible outcomes of the actions. Previous case studies in communities of developing countries even showed mismatches between knowledge of stakeholders and actions taken that led local communities away from conservation practices (Bennett and Dearden, 2014). However, we found in this study that various knowledge sets introduced by external translators and digested by villagers via their own "knowledge translators" had transformed their perceptions to incorporate important aspects including more sustainable management techniques, and potentials to improve local livelihoods and well-being. The turaga ni koro, knowledge translator and gatekeeper of the village, seemed to play an essential role in this process. The turaga ni koro in Kumi village was likely to function as a residential researcher (i.e., knowledge producer) in the community as he integrated various types of knowledge to visualize resource values, effectiveness of techniques, and visions of management outcomes. Detailed comparative analyses of various types of knowledge producers and translators are needed to elucidate their core functions to transform perceptions to produce collective actions toward more effective coastal management measures. Collective actions among diverse stakeholders are essential for the success of community-based management of coastal resources, especially common property recourses including forestry and fisheries (Cox et al., 2010; Ratner et al., 2013). Collective actions are promoted by perceptions among involved stakeholders and underlying knowledge systems, while participating in collective actions again influences perceptions and knowledge systems of the participants through social learning processes (Shackleton et al., 2009). This interactive process is assumed to promote dynamic and adaptive transformations of local institutions by the relevant stakeholders to cope with complexities associated with coastal marine resource management. Collective actions observed in this study provided ample opportunities of social learning for both villagers and external stakeholders, including government officials and scientists, even though there were significant differences in prioritized values and the framing of knowledge. Collective actions apparently strengthened the perceptions of diverse stakeholders on the values of community practices and improved the local approaches to sustainable resource management. Continuous interaction between The University of the South Pacific scientists and villagers in the case of locally managed marine area was an essential factor to mobilize social learning processes of all parties involved. Monitoring activities by the Ministry of Fisheries and Agriculture officials were effective to promote social learning between these officials and members of the different communities they collaborated with. Understanding the functions of formal and informal mechanisms of knowledge translation, such as continuous networking and interaction of involved stakeholder groups, seems to be indispensable to support social learning. This study clearly showed the persisting importance of traditional institutions, rules and decision-making systems for producing collective actions that contribute to the sustainable management of coastal resources in Kumi village. The tabu traditions, solesolevaki practices, and the clan totems played indispensable functions to create respect for community decisions, unified actions toward common goals, and platforms for introducing sustainable management practices. These institutions were implemented and utilized in a consistent way under the strong leadership by the traditional chief of the village. Such strong traditional institutions and leadership foundation may be regarded as having limited potential to apply to societies in other parts of the world. However, if we take a closer look at the mechanisms that support these institutions, we can identify the fundamental parts played by shared respect of local rules and community decisions (tabu), recognition of importance of working together for common goals (solesolevaki), and understanding of linkages between cultural values and sustainable use of natural resources (totems). Societal mechanisms to maintain trusted leaderships were another fundamental factor to provide platforms for various collective actions in the community. All of these factors may in fact have a universal value, as important components of perceptions among community members toward their own community environments (natural, social, and cultural) and their own collaborative practices. The processes toward the creation of ILEK to enable the transformation of local perceptions incorporating these universal values, are essential to manage coastal common property resources, and are of interest to resource management researchers and practitioners in Fiji and beyond. This case study was conducted in a small coastal village with a relatively small sample size and gender imbalance as outlined above. The study period was limited to provide snapshots of a long and continuous process of resource management and community development practices in the village. Even though the research design had such drawbacks, it could reveal essential enablers of transformation of perceptions to promote various collective actions. In-depth interviews clearly focusing on specific collective actions combined with the qualitative analyses to extract knowledge and perceptions related to these ongoing actions seemed to be an appropriate approach to bring about core findings of this study that suggested broader applicability in research of coastal resource management. A more comprehensive research design to obtain more detailed discourses from a larger and balanced sample are expected to verify the effectiveness and limitations of the qualitative discourse analyses. Furthermore, an in-depth analysis of the inequality/gender imbalance in decision-making would be required to provide a better understanding of the power of transforming perceptions and their relationship to collective actions. ## CONCLUSION In conclusion, a dynamic production and circulation of ILEK in Kumi village contributed to the transformation of perceptions, promoting a series of collective actions for the sustainable management of the local coastal resources (marine and terrestrial). These collective actions provided ample opportunities of social learning for both villagers and external stakeholders, transforming their ILEK to generate new hypotheses and in turn influence their perceptions. Traditional institutions, rules and decision-making systems played essential roles in producing collective actions contributing to the sustainable management of various coastal resources, and these collective actions transformed and strengthened local perceptions on the universal values of traditional systems for their community. These observations were in good accordance with Ostrom's eight principles for managing common pool resources (Ostrom, 1990). The collective actions had a welldefined boundary and reflected both local needs and socialecological conditions. The rules and procedures of the actions were discussed and agreed among local stakeholders, and external stakeholders respected these decisions and drew lessons from them. The traditional rules and decision-making systems in place in Kumi village seemed to work well for preventing rule violation and solving conflicts. Probably the important remaining ## REFERENCES challenge is the sharing of responsibilities for sustainable coastal resource management with actors from a broader context, connected to both coastal resources and local livelihoods, such as seaweed and sea cucumber middlemen and traders, exporters of agricultural products, as well as policy makers and development agencies at national and international levels. To tackle this challenge, knowledge translators such as the ones identified in this study may play a significant role to promote collaborative interactions between the coastal communities and potential external stakeholders through knowledge integration and transformation of perceptions. ## AUTHOR CONTRIBUTIONS JK conducted all field research, wrote initial manuscript of the paper, and contributed to finalizing revised manuscript. TS provided theoretical background, analytical framework and conducted analyses of the data, and participated in field research. TS and JK jointly made blush up of the manuscript. ### ACKNOWLEDGMENTS This study was funded and supported by the international transdisciplinary research project at the Research Institute for Humanity and Nature (RIHN), The National Institute for Humanities, Japan, entitled "Creation and Sustainable Governance of New Commons through Formation of Integrated Local Environment Knowledge" (ILEK project, E-05-init) and the feasibility study funding from the Research Institute of Science and Technology for Society (RISTEX), Japan Science and Technology Agency (JST). We are grateful to the members of the ILEK project and RISTEX feasibility study, and colleagues in RIHN for their support for this research and stimulating discussions. We wish to express our sincere thanks to the people of Kumi Village and diverse stakeholders whom we worked together with during this research for their kind and productive collaboration. 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 © 2016 Kitolelei and Sato. 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. # Oceans of Discourses: Utilizing Q Methodology for Analyzing Perceptions on Marine Biodiversity Conservation in the Kogelberg Biosphere Reserve, South Africa Kristin Hagan<sup>1</sup> \* and Samantha Williams 2, 3 <sup>1</sup> Department of Human Geography, Planning and International Development, University of Amsterdam, Amsterdam, Netherlands, <sup>2</sup> Department of Environmental and Geographical Sciences, University of Cape Town, Cape Town, South Africa, <sup>3</sup> Department of Geography and Environmental Studies, Stellenbosch University, Stellenbosch, South Africa #### Edited by: Annette Breckwoldt, Leibniz Center for Tropical Marine Ecology, Germany #### Reviewed by: Fabio Badalamenti, National Research Council, Italy Elizabeth Ingrid Van Putten, CSIRO, Australia Mary Mackay, University of Tasmania, Australia > \Correspondence: Kristin Hagan [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 03 June 2016 Accepted: 15 September 2016 Published: 29 September 2016 #### Citation: Hagan K and Williams S (2016) Oceans of Discourses: Utilizing Q Methodology for Analyzing Perceptions on Marine Biodiversity Conservation in the Kogelberg Biosphere Reserve, South Africa. Front. Mar. Sci. 3:188. doi: 10.3389/fmars.2016.00188 This paper attempts to empirically investigate perceptions regarding marine biodiversity conservation among different stakeholders of the Kogelberg Biosphere Reserve, South Africa. The study's data was collected by following Q methodology in combination with semi-structured interviews and participant observation. Q methodology combines elements from quantitative and qualitative research traditions, providing researchers with a systematic and rigorous means to study human subjectivities. Primary data were gathered from stakeholders who either live, work, or have performed research in the Kogelberg Biosphere Reserve. A combination of interpretative discourse analysis and Q factor analysis was employed to identify perceptions. The results reveal that there are two operating discourses with clear stakeholder divisions. The science discourse is characterized by its scientific management-based ecological approach. On the other hand, the livelihoods discourse is primarily concerned about the social implications brought about by Kogelberg as a biosphere reserve. The paper goes on to argue that the meaning people attach to the concept of "marine biodiversity conservation" is relational as it is based on their lived experience. It further highlights the importance of performing context-specific social research of protected areas, as it is difficult for conservation projects to meet both ecological and social needs without understanding the viewpoints of engaged stakeholders and local communities. Keywords: marine biodiversity conservation, Kogelberg biosphere reserve, environmental discourses, environmental subjectivities, Q methodology, perceptions, nature conservation ## INTRODUCTION Loss of biodiversity is one of the most prominent aspects of the environmental crisis the world is facing. It is estimated that the earth is home to somewhere between 5 and 15 million species, of which only 1.8 million are known to science. While species dying out is a natural process, the current extinction rates are assumed to be 100 to 1000 times greater than the "normal" rate, which is largely due to human activities such as habitat destruction and fragmentation, overharvesting or pollution (Stoll-Kleemann and Bertzky, 2004: p. 1; Kearns, 2010: p. 7). Loss of biodiversity and rapid depletion of natural resources is present in all known ecosystems. While the ocean has been regarded as a source of infinite resources for a long time, it is estimated that 70% of the earth's commercially targeted fish species have been overfished to the point where their stocks are in grave danger of being depleted. On a global scale, some of the most threatened marine species include whales, dolphins, salmon, sea turtles, sharks, manatees and dugongs<sup>1</sup> . Biodiversity loss is therefore expressed as one of the main contemporary environmental concerns along with climate change and desertification (United Nations, 2002: p. 3). In response to reduce the loss of biodiversity worldwide, UNESCO has created 651 biosphere reserves (BR) in 120 countries worldwide as part of the Man and the Biosphere program (MAB). Biosphere reserves are experimental places, which see interdisciplinary approaches being tested to understand and manage changes and interactions between social and ecological systems, including conflict prevention and management of biodiversity. These reserves can be more closely described as areas comprising terrestrial, marine and coastal ecosystems, promoting biodiversity conservation of species as well as sustainable development of local human populations<sup>2</sup> . Such a reserve is an open area without any fences to "keep people out" and "nature in." Furthermore, BRs are governed in such a way that local communities, farmers, conservation agencies and local governments are committed to protect the landscape and its biodiversity together<sup>3</sup> . Participation of local communities in the management of biospheres is seen as crucial to make the project succeed, while at the same time aiming to include traditional ecological knowledge into ecosystem management. However, many attempts of conserving biodiversity have failed, and the BR concept as put forward by UNESCO is no exception (Stoll-Kleemann and Bertzky, 2004; Hyman, 2006). Many of the BRs neither have the resources nor the capacity to meet the global mandate put forward by UNESCO, a problem that is particularly evident in developing countries. Another important problem in a biosocio-economic system is that these systems are dynamic and complex, and consist of many interactions between humans and institutions. As a consequence, conflicts emerge on multiple levels. Stoll-Kleemann and Bertzky (2004: p. 2) note that biodiversity conflicts are often a result of the different preferences, values and objectives of different actors. Environmental arguments, such as protection and conservation of biodiversity might appear to be factual and scientific, but they are also meaningful, ethical and suggestive (Næss, 1974: p. xxiii), representing a certain discursive perception of an issue. However, the ways in which individuals think about and understand environmental problems such as biodiversity <sup>2</sup>UNESCO website, http://www.unesco.org/new/en/natural-sciences/ environment/ecological-sciences/biosphere-reserves/ Accessed 07.08.2015. conservation, is a vital issue in the study of environmental politics that often remains unexplored in the literature. Yet, this issue should be regarded as one of central importance because "until we know the 'discourses' people use about the environment, it will be very hard to judge what, and whether, environmental policies will be socially acceptable, and therefore capable of being implemented" (Barry and Proops, 1999: p. 338). Reality is socially constructed; therefore the analysis of meaning becomes central. In this way, it is not an environmental phenomenon in itself that is important, but the way in which society makes sense of this phenomenon. The meaning attributed to a concept such as "biodiversity conservation" affects the outcomes, institutions and laws, and further becomes the context, or discourse, in which environmental issues are talked about. These meanings do not appear out of nothing, but are the result of a particular set of operational routines and accepted norms and rules that give coherence to social life (Hajer and Versteeg, 2005: pp. 176–177). Understanding the local context and the local way of thinking about environmental issues is therefore crucial for creating development projects and plans that aim at protecting biodiversity. Furthermore, the study of environmental perceptions in particular is very important in creating an understanding of the social complexities embedded in the environmental crisis. This research has set out to contribute to the debate of biodiversity conservation and how differing discourses influence stakeholder perceptions and management of BRs. In order to investigate perceptions of marine biodiversity conservation, the study utilized a case study approach to understand how different stakeholders of a BR perceive biodiversity conservation of marine areas. The area of focus was the Kogelberg Biosphere Reserve located in South Africa. The study furthermore aimed to illustrate the utility of Q methodology for conducting perception-based research. The following sections will provide the background and context to the case study under investigation, before then turning to the actual process of applying Q methodology. ## CONSERVATION THROUGH THE ESTABLISHMENT OF BIOSPHERE RESERVES The Kogelberg Biosphere Reserve (KBR; Figure 1) was proclaimed as South Africa's first BR in 1998 (Turpie et al., 2009: p. 1). Some of the objectives highlighted in the establishment of BRs include the preservation and sustainable utilization of natural resources, as well as economic development that aims to be socially and environmentally just. It also includes education, monitoring and research as core and ongoing priorities (Tucker, 2013: p. 2). These areas are typically divided into core areas (where the highest level of protection is afforded and little or no consumptive uses occur), buffer zones (surround the core, and limited development and activities occur) and transitional zones where a range of activities and developments (i.e., including farming, residential or resort projects) take place<sup>4</sup> . Furthermore, <sup>1</sup>Marinebio website, http://marinebio.org/oceans/threatened-endangeredspecies/ Accessed 10.08.2015. <sup>3</sup>Kogelberg Biosphere Reserve website, http://www.kogelbergbiospherereserve.co. za/ Accessed 10.08.2015. <sup>4</sup>Kogelberg Biosphere Reserve Company website, http://www.kbrc.org.za/ Accessed 05.04.2016. BRs are managed by a range of organizations, including government departments, national parks authorities, provincial conservation bodies, local government departments, regional and municipal councils, non-governmental organizations (NGOs), community organizations, also with participation of researchers and universities. The KBR is located in an area known as the Cape Floral Kingdom, comprising approximately 100,000 hectares at land and at sea<sup>6</sup> . This area has approximately 5800 endemic plant species, which is more for its area than anywhere else in the world<sup>7</sup> . The KBR, also known as the "heart of the floral kingdom," contains rich wildlife with a variety of different bird, amphibian and mammal species, and boasts with South Africa's largest penguin colony. About 30% of the KBR consists of marine areas, which are particularly biodiversity rich. This is where the cold Atlantic currents meet the Indian Ocean's warm waters, creating a home for a variety of marine species. The reserve starts in the Atlantic Ocean, 7.5 km from land, and stretches two nautical miles out to sea<sup>8</sup> . Being located in the Overberg municipality, the area is surrounded by small villages that include Rooi- Els, Pringle Bay, Betty's Bay and Kleinmond (Turpie et al., 2009: p. 3). In terms of development and economic activity, the trade and services sectors make up almost half of the economic production, with tourism playing a vital role (Turpie et al., 2009: p. 9). Being in close proximity to the coast has meant that the inhabitants <sup>5</sup>Map courtesy of Johns and Johns (2001). Edited by Stephen Young. <sup>6</sup>KBRC Kogelberg Biosphere Reserve Company website, http://www.kbrc.org.za/ index.php?dirname=docs\_09about/history Accessed 20.06.2016. <sup>7</sup>Kogelberg Biosphere Reserve Company website, http://www.kbrc.org.za/index. php?dirname=docs\_03nature/flora Accessed 24.08.2016. <sup>8</sup>Kogelberg Biosphere Reserve Company website, http://www.kbrc.org.za/ Accessed 05.04.2016. of the surrounding villages have developed a dependency and relationship with the sea. Primary use of the coast and its resources include the harvesting of abalone (Haliotis midae), west coast rock lobster (Jasus lalandii), line fish and kelp. In the recent past, the harvesting of abalone has seen a moratorium being placed on the resource by the National department of forestry and fisheries as incidences of poaching and overharvesting are driving the species toward extinction. The tradition and history of fishing in one particular village, Kleinmond, has been well established and dates back many generations. However, the continued illegal harvesting and pressures for greater access to the coastal marine resources by local resource users (amongst others) of the KBR and elsewhere along the country's coast have been a subject of concern for the National department of forestry and fisheries (Turpie et al., 2009: pp. iv–vi; Sunde, 2014: p. 23). As a result strict controls have been implemented by resource managers, and various efforts directed to streamline conservation efforts and economic and livelihood considerations within the KBR. Management responsibility for the KBR is shared by a group of local and regional stakeholders. Its key management body is the Kogelberg Biosphere Reserve Company (KBRC), which works in collaboration with stakeholders from government, academia, business and NGOs<sup>9</sup> . In this management structure there are different subgroups or stakeholder working groups. The Kogelberg Marine Working Group (KMWG) is one such gathering of involved stakeholders. It was established in 2009, with the aim to contribute to the management of a no-take Marine Protected Area (MPA) in Betty's Bay (see Figure 1), which was established to facilitate the recovery of fish stocks and prevent marine species from being overharvested. The KMWG deals with marine and coastal environmental and social challenges (such as curbing poaching to protect the interest of the fishers) (Anchor Environmental, 2009: p. 4; (Hagan, 2016): pp. 15–18). The rationale for establishing a BR in the Kogelberg area was to ensure better biodiversity conservation through stakeholder involvement. It was envisaged that it would also address issues related to development pressures and poverty alleviation (Hyman, 2006: p. 23). However, the KBR has not achieved all of its desired successes, which has resulted in limited conservation and social developmental outcomes and stakeholders who struggle to cooperate (Hyman, 2006: p. 1; (Müller, 2010): p. 152; (Hagan, 2016): p. 56). While previous studies have pointed out that stakeholders in the KBR are facing management and cooperation difficulties as a result of divergent interests and perceptions (Hyman, 2006: p. 84–88; Müller, 2010: p. 152), this study has focused on stakeholders' perceptions of marine biodiversity conservation in more detail. The original study<sup>10</sup> looked at five key stakeholder groups of the KMWG: CapeNature, scientists, small-scale fishers, World Wide Fund for Nature (WWF) and Seawatch (Hagan, 2016: pp. 18–19). This article will not discuss the latter two, as only two individuals from each of these Non-Governmental Organizations (NGOs) were working directly on marine conservation in the KBR. CapeNature is a governmental institution that chairs the KMWG. They have the statutory responsibility for biodiversity conservation in the Western Cape as governed by the Western Cape Nature Conservation Board Act 15 of 1998<sup>11</sup> . Other stakeholders include both natural and social scientists, providing inputs in terms of management recommendations, monitoring and evaluation, as well as participation in stakeholder engagement. The fishers' group refers to men and women from the fishing villages of the KBR, whose livelihoods depend on small-scale fishing. #### MATERIALS AND METHODS In order to gain an understanding of the different ways marine biodiversity conservation in the KBR is perceived, data gathering was carried out using Q methodology in combination with semi-structured interviews and participant observation. The data from this process was analyzed using Q factor analysis and interpretative discourse analysis. A "discourse" is in this context understood as "a shared way of apprehending the world. Embedded in language, it enables those who subscribe to it to interpret bits of information and put them together into coherent stories or accounts. Discourses construct meanings and relationships, helping define common sense and legitimate knowledge. Each discourse rests on assumptions, judgments, and contentions that provide the basic terms for analysis, debates, agreements, and disagreements (Dryzek, 2013: pp. 9–10). #### Q Methodology In the 1930s, the psychologist Stephenson (1953) developed Q methodology as a means to systematically study human subjectivity. The methodology combines the strengths of both quantitative and qualitative research traditions, and is suitable to investigate questions about personal experience and matters regarding taste, values and beliefs (Baker, 2006: p. 2343). Q method is primarily used in psychology, but it has also been embraced by scientists as a means to investigate human subjectivity on a variety of issues, particularly in politics and health research (Eden et al., 2005: p. 414). In later years, Q method has also rapidly expanded to environmental studies (Dasgupta and Vira, 2005: p. 2; Eden et al., 2005: p. 414; Webler et al., 2009: p. 8). Previous publications in social environmental research have scrutinized a wide range of topics, including environmental policy (Addams and Proops, 2000), global environmental change (Niemeyer et al., 2005), environmental management (Bischof, 2010), successful biodiversity conservation (West et al., 2016), and animal rights (Kalof, 2000). The method has also been utilized in work on environmental policy and environmental discourses in order to gain a more thorough understanding of stakeholder perceptions (Dasgupta and Vira, 2005; Guimaraães, 2010; Pike et al., 2014). All Q studies are reconstructive and characterized by two key features. Firstly, the collection of data is done in form of Q sorts (Watts and Stenner, 2012: p. 178). This is typically <sup>9</sup>Kogelberg Biosphere Reserve Company website, http://www.kbrc.org.za/index. php?dirname=docs\_04projects/partners Accessed 12.05.2016. <sup>10</sup>This paper draws on research undertaken as part of the first author's master's dissertation. <sup>11</sup>CapeNature website, http://www.capenature.co.za/about-us/ Accessed 12.05.2016. (but not always) done by presenting people with a sample of statements about a topic, which is referred to as the Q-set. The selected respondents, called the P-set, are instructed to rank-order the statements from their personal point of view on a score sheet. Following this process, which is called the Q sort, people give their subjective meaning to the statements and thus reveal their subjective viewpoint (Van Exel and de Graaf, 2005: p. 1). Secondly, these Q sorts are factor-analyzed for establishing different patterns (Watts and Stenner, 2012: p. 178). Unlike standard survey analysis, Q methodology is not aimed at establishing patterns across individual characteristics such as age, gender and class. Instead it looks at patterns within and across individuals by focusing on their discursive understanding of a particular issue. It works on the assumption that there are a limited number of ordered patternings within a particular discursive realm, attempting to reveal those patterns in a structured and interpretable way (Barry and Proops, 1999: p. 339). The method is primarily explorative, for qualitative recognition of the mere existence of subjective views instead of measurement of pre-defined attitudes or perceptions. One of the main strengths of the method is that it provides statistically significant results from a reasonably low sample size (Brown, 1993: p. 94). Furthermore, it converts in-depth subjective information into quantifiable data in a way that traditional methods are not capable of Pike et al. (2014: p. 667). ## Administering the Q Sort Influenced by the steps created by Brown (1993) and later elaborated by Van Exel and de Graaf (2005), this Q study was conducted by following six steps; (1) defining the concourse; (2) developing the Q sample; (3) selecting the P-set; (4) Q sorting; (5) semi-structured interviews; and (6) analysis and interpretation. Employing Q methodology, the first and most important step is to identify all the possible statements the actors within the relevant domain could make about the subject matter (Van Exel and de Graaf, 2005 : p. 4), in this case marine biodiversity conservation. The concourse, or "the flow of communicability surrounding any topic" (Brown, 1993: p. 94) was collected through key informant interviews with two representatives from each stakeholder group, as well as interviews and informal conversations with other members of the identified stakeholders, living or working in the KBR. A snowball sampling method was employed after attending a KMWG meeting in order to meet and contact relevant respondents. A purposive sampling approach was also employed with people who had relevant views on the matter without being directly related to the KBR context. These included fishers from other parts of the coast (outside the KBR) as well as conservation biologists and politicians working with nature reserves in the Western Cape. This was done to triangulate the various ideas surrounding biodiversity conservation in general and of marine areas in particular. These interviews and conversations (about 40 in total) resulted in hundreds of statements, which were transcribed, coded and divided into categories. These categories emerged inductively from the coding process, focusing on the most re-occurring issues. For instance, issues related to the MPA came up frequently and were therefore included, while gender was only brought up once and thus excluded. The Q sample was selected by choosing a few statements from each category (Webler et al., 2009: p. 10). Particular emphasis was placed on interviews with people living and working in the KBR, minutes from meetings of the KMWG and scientific literature from the area. This resulted in 45 statements being collated. In this way, the selection procedure was based on field observation and interview data, in contrast to being based on pre-existing theory and categorizations. In addition to the Q sorting task, a key focus of the study was to emphasize the qualitative interview in combination with each Q sort. The Q sorts and interviews were set up to be no longer than 1 h, therefore the amount of statements had to be reduced accordingly. The selection procedure used experts (social scientists who had worked in the KBR during the last 2 years but were no longer actively involved) as a means of piloting the suitability of the Q sample. This resulted in a final Q set consisting of 23 statements (see Table 1 below). The next step was to develop the P-set, which is a "structured sample of respondents who are theoretically relevant to the problem under consideration. (...) The aim is to have four or five persons defining each anticipated viewpoint, which are often two to four, and rarely more than six" (Van Exel and de Graaf, 2005: p. 6). From the three stakeholder groups, eleven key informants were selected; four scientists (two social, two natural scientists), four fishers and the three CapeNature managers responsible for the KBR. As there is a limited amount of dedicated people who are engaged in the KMWG or the daily operations of the coastal areas of the KBR, the authors prioritized key informants with high levels of influence and engagement. The original study, which also involved Seawatch and WWF, contained 15 respondents for Q sorting. One of the benefits of Q methodology is that only few participants are needed to give statistical significant results. According to Barry and Proops (1999), as few as 12 participants can generate statistically meaningful results, because each participant's Q sort provides a vast amount of information (Barry and Proops, 1999: p. 334). The Q set was given to the respondent in form of a deck of randomly numbered cards. Each card contained one statement from the final Q sample. The respondent was first instructed to sort the deck into three piles; "agree," "neutral/undecided," and "disagree," depending on his/her personal point of view. Thereafter, the respondent was instructed to sort out the statements on a score sheet with a pyramidal, or "quasi-normal," sorting distribution, ranging from "strongly disagree" (−4) to "strongly agree" (4). The sorting distribution was pre-arranged; the whole Q set had to be allocated a ranking relative to one another within this distribution (see Figure 2). Each Q sorting was combined with an interview. During the sorting procedure, the respondent could choose whether to talk the researcher through each statement, or to sort first and do a follow-up interview afterwards. After each sorting, the respondent was asked to elaborate on his/her point of view, explain the most salient statements, and discuss whether there was any themes the respondent felt missing in the deck. The Q sorts were subject to Q factor analysis, which is the most quantitative part of Q. The factor analysis was carried out #### TABLE 1 \| Statements, with scores on the two extracted discourses, sorted from consensus to strongest deviation. with the help of PQMETHOD-2.3512, particularly designed for Q methodology. The package correlates every respondent's Q sort with every other Q sort. The resulting correlation matrix was then used for a centroid factor analysis (to define centers of gravity in the matrix and express these in specific terms; (Brown, 1993): p. 113). Varimax rotation was then used to rotate the remaining factors into a "simple structure" in order to extract factors that are significant according to the protocols of Q (Barry and Proops, 1999: p. 341). The package extracts all significant factors and conveys them as the "best estimate" of the sorts that represent them (Barry and Proops, 1999), capturing the common essence of the sorts. Based on their correlation to certain factors, the package provides a way of recognizing fundamentally different viewpoints and grouping respondents around these. The factors resulting from this analysis are not necessarily represented by any specific individual, but rather represent an "ideal type," which is a virtual respondent that is fully representing one of the distinguished viewpoints (Bischof, 2010: p. 605). Usually, each respondent has aspects of more than one "ideal" sort in their personal sort. Q sorts that come closest to this ideal are listed. The significance of a factor is determined statistically by its Eigenvalue (i.e., the sum of squares of the factor loadings). Eigenvalues higher than 1 are considered significant (Van Exel and de Graaf, 2005: p. 18). Another statistical criterion is the composite reliability<sup>13</sup> of a factor, which depends on how many respondents define it. The more respondents define a factor, the higher the reliability (Dasgupta and Vira, 2005: p. 14). A factor should be defined by at least five respondents. This will result in a factor reliability of 95%, which is sufficient to obtain a clear factor reading (du Plessis, 2005: p. 168). Correlation between an individual Q sort and shared factor was considered significant if a factor loading exceeded ±0.36 (West et al., 2016: p. 186). The "ideal" Q sorts resulting from this procedure were interpreted along with the interview data to gain a better understanding of the outcomes of the factor analysis. As most respondents expressed their view on each single statement in the Q deck and answered interview questions related to these themes, the interview data carried extensive amounts of information that could be directly attached to each quote. This data assisted in interpreting the meaning of each statement and understanding the rationale behind why statements were sorted in a particular order (Gallagher and Porock, 2010: p. 298). In addition to the factor analysis, interview data and observations from the field were subject to interpretative discourse analysis to triangulate the results. Interpretative discourse analysis is committed to gaining an in-depth understanding of the actors' frame of reference, and possesses a view of language as being constructive rather than merely representational. It emphasizes the social construction of meaning and the central role of language as a symbolic medium in constructing social reality (Heracleous, 2006: pp. 11–12). Prior to the Q factor analysis, all the interviews and accompanied field observations were coded and analyzed separately, focusing on elements such as language, content, meaning, knowledge system and worldview. Later the interviews were compared with each other and analyzed. Therefore, interview data and field observations were subjected to interpretative discourse analysis on its own, and assisted the Q factor analysis by including respondents' interpretations of statements when analyzing the factors. #### RESULTS The factor analysis revealed that there are two operating discourses in the KBR, and that these discourses have a clear stakeholder division. All respondents in the P-set loaded on a factor. The three CapeNature representatives loaded on factor 1, and the four fishers on factor 2. The natural and social scientists were split. The two natural scientists load on factor 1, while <sup>12</sup>Schmolck, P. (2015) The PQ Method Page, online: http://schmolck.userweb. mwn.de/qmethod/ Accessed 01.02.2016. <sup>13</sup>In PQMethod the formula Rxx = 0,80p / [1 + (p − 1), 080] is built into the program. 0,80 is the presumed average reliability of the Q sorts comprising the factor, while p is the number of those Q sorts. Rxx is the test-retest reliability coefficient. When p = 5 Q sorts the factor reliability is Rxx = 0,80(5) / [1 + (5 − 1),80] = 0,9524 (from du Plessis, 2005:169). the two social scientists load on factor 2. The discourse held by CapeNature and the natural scientists will in the following be referred to as the "scientific discourse," while the fishers and the social scientists adhere to the "livelihood discourse." The Eigen value of the scientific discourse is 5.3067, while it is 2.1598 for the livelihood discourse. The composite reliability is 97.3 and 96%, respectively. The two "ideal type" Q sorts for the scientific and the livelihood discourse are shown below in Table 1. In the table they are presented from statements of "strongest consensus" to statements of "strongest deviation." Statements of high consensus refer to statements that have been sorted similarly on the "ideal type" Q sorts of both discourses. It shows what aspects do not distinguish significantly between the two discourses. Consensus does not automatically mean that the statement has scores in the middle (near 0), it can also be non-neutral. Table 1 shows that, according to the factor analysis, statements #2, #4, #6, #7, #8, #10, #11, #17, #21, and #23 do not distinguish considerably between the different discourses but show common grounds between the two. The statements of strongest deviation show what issues differ most between the two discourses. Table 1 shows that the most important statements of distinction are statements #1, #3, #5, #9, #12, #13, #18, #19, and #20. These statements are statistically significant and therefore central when describing the discourses; they show the distinguishing issues and their relational importance. The interview data carried vital information explaining the respondents' thoughts on the issues highlighted in the statements, as well as the reasons why they sorted the way they did. Furthermore, the results of the interpretative discourse analysis have been triangulated with the Q factor analysis to validate the factor interpretation. The following section will present the scientific discourse and the livelihood discourse in closer detail, interpreting both data from the Q factor analysis and the interpretative discourse analysis. ## The Scientific Discourse What is distinctive about this account is its normative management-based ecological approach. This discourse displays a strong concern about the destructive impact human behavior has on the environment. It emphasizes the necessity of creating management plans and projects to reach conservation objectives, and that these projects need to involve the fishers to succeed optimally. Three statements are particularly important for this discourse, namely #13, #20, and #5. These three statements happen to be the statements of strongest agreement for this discourse, while also being among the most distinguishing ones to the livelihood discourse. The statements of strongest disagreement are #6, #8, and #22, however none of these statements are of great significance in defining this discourse in a comparative perspective to the other one, as all three are statements of consensus. There is support for the idea that all citizens should take responsibility for environmental problems, and that the South African government has a statutory duty to protect marine biodiversity. According to this view, the Kogelberg belongs to the South African state and therefore all South Africans, not only the locals who live in the reserve. As commented by a natural scientist: "Just because they happen to live there next to that piece of coast, I don't see that that necessarily means ownership or users rights. Theoretically, all of the resources belong to the state. That's what it says in the constitution. (...) People living outside the KBR have concerns and a right to know that that is being managed properly for the benefit of all South Africans, not just the people who happen to live in it." Marine conservation in this context involves regulating people's utilization of the sea's resources. As pointed out by another natural scientist: "I do think that it's never fully recognized that, if you look at our law, all the sea and it's resources are held in trust for all South Africans. Not just the people who happen to live by the sea." These statements therefore concur with dominant discourses which stress that MPA's are particularly important in this regard in order to keep marine areas healthy, which is necessary for protecting marine species. While a desire to strive toward "pristine" conservation ideals is present in the scientific discourse, regulation of human activities and more specifically the presence of people are cited as an important environmental and social challenge in achieving this state. In the interviews, this was demonstrated by the following response: "A MPA, which is less than 10% of an entire coastline, needs to be pristine. Not to do away with people's right to catch fish. But to have an area where we know what it used to be like. That is also an ideal place to monitor change. Climate change and change that doesn't come through human pressures. So it's got a very critical need." (CapeNature Official). This discourse expresses support for the viewpoint that local communities' use of marine resources is an important factor in species being threatened. In the interviews, both scientists and CapeNature representatives directly referred to the tragedy of the commons scenario when discussing the issue of fishers as protectors (statement #13). Following this view, the primary objective of the fishers is to optimize their daily economic return. According to one CapeNature official, "obviously fishermen are concerned about their resource, but the problem is the tragedy of the commons scenario. If you don't catch the fish, the next person is going to catch the fish." Having to act according to one's own self-interest instead of "the common good" is closely connected to the socio-economic reality of fishers' dependency on marine species to provide means of securing the necessities of life. According to this approach, conservation is also a matter of education, as locals harvesting marine resources may not always have sufficient knowledge of the biological repercussions. Central here is the assumption that protecting marine species will result in gains for the fishers in the long term. While holding positions for conservation with limited human interference to protect stocks, there is also recognition of the idea that it is important to include local fishers and other environmental users in conservation efforts, and that this is regarded a prerequisite for management projects to succeed. To this a scientist explained: "We must include fishers, otherwise we are doomed to fail. The more desperate and poor the fishers are, the more difficult it is. It's quite easy in America or Australia where you've got an educated fishing population. It's not a walk in the park, but it's a hell of a lot easier than when you've got a desperately hungry fishing population that has had the disservice of an apartheid education." #### The Livelihood Discourse What is distinct of this view is a strong concern about the social implications brought about by the Kogelberg as a BR, particularly injustice toward the fishers and the fishing communities. The key concern is that although nature needs to be sustained, conservation of marine areas cannot deprive people of their livelihoods. Importantly, this discourse also expresses a strong opposition to the Kogelberg MPA. During an interview this was passionately expressed by a fisher who thought that "The MPA is absolutely worthless. As far as the fishermen are concerned, it was stolen from them." The most influential statements of agreement in defining this discourse are #18 and #19, as well as #3 and #12. The most important statements of disagreement are #1 and #9. In the interviews, the fishers describe their respective villages and the ocean as something that is part of them, and something that belongs to them. With family bounds dating back generations, they believe that living from fishing is their inherited right and part of their identity. Conserving nature is talked about as a way of life where one coexists with other species, in contrast to creating and enforcing policies. The current arrangement with the MPA and fishing rights processes is considered as unfair and unnecessary. These sentiments, which were continuously expressed during interviews with the fishers, were related to their opinions on commercial fishing boats that catch vast amounts of fish. A fisher from Kleinmond explains: "I think the big boats are taking all our fish out of the water. Then when we go to sea there is nothing left for us. It's a major problem for us because they come very near to the shore." Another aspect linked to this is a feeling of inequality. Being among the poorest in the KBR, the fishers feel restricted by fishing rights and the fact that they are prohibited from fishing in the MPA as they have previously done. A fisher explained his position by adding: "People should be taken into consideration. I think it can't just be imposed without the public impact. They just took an area and declared it a protected area. No one could say anything at all. It was the fisher's favorite fishing spots. And now they are sentenced out of it. It's illegal to fish there, and that are the best places to fish. Then you get a clash of interests. The fishermen feel they are not acknowledged when it becomes illegal. The responsibility is certainly taken away from us." What also became apparent was that some fishers hold resentments toward white people of the area whom they believe are not penalized for "breaking the rules," e.g., when they are planting alien trees in their gardens or dislocating sand from the beaches. This can be understood as an expression of injustice on behalf of the poor fishing population, as they believe conservation restrictions are imposed on them alone. The livelihood discourse indicates that the fishers think and understand marine conservation differently than the other stakeholders. However, the social scientists have sorted their Q sorts similarly to the fishers and therefore loaded higher on factor 2, or the livelihood discourse. The interview data shows that although the fishers and the social scientists load on the same factor, there is an important difference between these two groups. While the fishers refer to their own personal experience and livelihood challenges, the social scientists who work in these communities emphasize that although they do not relate to the situation in the same way, they do understand and generally support the viewpoint of the fishers. To this a social scientists responded to statement #2 (living with the sea is my way of existing) by adding: "That's not relevant to me but I can see that it's relevant to a lot of people who live in the Kogelberg. So I would strongly agree with somebody who said that, of course. My job is linked to it, but I wouldn't say it's my way of existing personally." One of the biggest concerns for the fishers, and also recognized as important aspect by the social scientists, is that the current functioning of the KBR is depriving people of their livelihood. Here, a social scientist added: "The KBR is a particular concept. It's a foreign concept to most people out there. It was not very well brainstormed, not very well discussed, not very well implemented. So I can understand that most people don't really like what they see there because most of the projects run by the KBR have been very conservationist. There's very little livelihood development, or socio-economic benefits to the community living there." Therefore, the focus on social issues needs to be understood in the context of several social challenges, which include abalone poaching, violence, crime and drug abuse. Uncertain fishing rights and stricter conservation controls being exercised not only expose fishers and their livelihoods to vulnerabilities, but also exacerbate existing community challenges. ## Importance of Stakeholders' Lived Experience While the factor analysis demonstrates that there are two distinct discourses operating among the KBR stakeholders, certain viewpoints are shared between the two. "Consensus" is found among more "neutral" or less important statements, such as #10, #11, #23, but also among non-neutral statements such as #2 and #21. What is important to note is that although there is (dis- )agreement between the discourses, this (dis-)agreement is found on two different parts of the discourses. Although both groups disagree strongly with a statement, this disagreement is based on a different way of thinking about the subject matter. For instance, both the scientific and the livelihood discourse respondents strongly disagreed with statement #22 ("I need to be allowed to do what I need to do to make a living, although it means that some plant or animal species might go extinct."). Although there is consensus among the stakeholders that they disagree with this statement, the interview data show that three of the fishers had problems sorting this statement before it eventually ended up on strongly disagree. While one fisher said he would rather die hungry, the other fishers explained this as a difficult dilemma that is hard to answer. "I would rather die poor than to exploit that for my benefit (point at the sea). I would feel bad when I die if I plundered to get a nice car. That's not what I'm about. Maybe that's why I'm so poor (laughs)." Fisher, Kleinmond "When you got to eat, you got to eat. Either you go extinct or it goes extinct. It's a difficult one." Fisher, Pringle Bay The respondents falling under the scientific discourse recognized that there is a difference between not wanting to cause something to go extinct in theory and actually being in that situation. There was general agreement that letting a species go extinct is very selfish and morally wrong, however, because the respondents in this group have never been in that position they emphasized that it was hard to relate to it. Here a natural scientist added: "Me putting it in the 'I disagree with' is obviously indicative of my upbringing and social conditions in life and the fact that I haven't been put in the position where it's me or something else." This phenomenon was also found in other consensus quotes, such as statement #2 ("Living with the sea is my way of existing."), a non-neutral statement both discourses agree with. The sea provides a livelihood for all the respondents, although somewhat indirectly for some interviewed stakeholders. Nevertheless, respondents generally expressed a strong relationship with the sea, predominantly on different grounds. While recreational and job-related activities were crucial to both systems of belief, the supporters from the scientific discourse talked about ecosystem services, while the fishers brought up their direct dependency on consuming and selling marine species to sustain their livelihoods. What these examples indicate is the importance of how lived experience influences stakeholders' thoughts and ideas. People in the KBR experience and understand nature in different ways depending on how they live their lives. The concept of "biodiversity conservation" has different meanings to different stakeholders, and this meaning emerges in relation to practice. This research therefore supports the claim that our definition of "nature" is constructed by us giving it a certain meaning, as well as by discursive processes. Thus, what we understand as "natural" is also social and cultural (Escobar, 1999: p. 2). ## DISCUSSION #### The MPA As a Source of Dispute All the stakeholders who participated in this study emphasized the importance of conserving both natural resources and livelihoods, as the two are closely linked. In this regard, the primary concern of the scientific discourse was the natural environment, while the social issues related to the BR were of greatest importance to the respondents falling under the livelihood discourse. In contrast to the CapeNature representatives and the natural scientists who considered it a necessary conservation means, fishers perceived the imposition of a protected area as taking away their responsibility to act as custodians of "their" marine resources. This finding is not surprising, as other studies that included documenting and analyzing perceptions of biodiversity conservation in South Africa have found similar attitudes recorded from community members living adjacent or near protected areas (Sunde and Isaacs, 2008; Watts and Faasen, 2009; Williams, 2013). Research undertaken by Faasen (2006) and Watts and Faasen (2009) in the Tsitsikamma, South Africa, for instance investigated whether synergies existed between biodiversity conservation and sustainable rural development, and documented local community members' perceptions of the no-take policy of the MPA in the area. This work highlighted that local communities harbor discontent and opposing views toward the conservation mandate of the management authorities and that there was a need to foster better involvement and participation of community members in decision-making processes. Similarly, Williams' (2013) study in the same area highlighted that the local communities historically had access to various fishing sites until the proclamation of the national park and subsequently the establishment of a "no-take" MPA. It was found that community members and fishers alike did not regard the current management and status quo as legitimate, and continuously referred to historical and traditional fishing practices as evidence of their rights to access the current MPA and its fisheries resources (Williams, 2013: p. 13). While opposing views of what exactly constitutes conservation and how this may result in discontent especially from neighboring communities toward MPAs, Sunde and Isaacs (2008: pp. xiii, 19–22) noted that a key area of concern relates to the fact that communities perceive themselves as bearing the costs of marine conservation with limited benefits in return. What exacerbates the conflict is that in some cases current practices of protected areas were not perceived as a legitimate conservation approach among communities, especially where locals were not involved in the conceptualization or implementation of these areas. These examples show that conflict and disputes between stakeholder groups are already found on the discursive level, as stakeholders possess dissimilar systems of belief. These trends are disturbing, as creating and sustaining MPAs is a key conservation strategy for the South African government, which has expressed its commitment to meet international and related national obligations toward protecting its biodiversity. One of these is to ensure that local communities participate in conservation efforts, which could come up against several challenges if these efforts threaten to undermine local livelihoods (Sunde and Isaacs, 2008: p. xiv). #### Championing Conservation Cooperation Conflicts and disputes over natural resources are present across the world and therefore not unique to the South African context. What is significant in the South African experience is that the conservation approach was influenced by historical and political trends of the time. This meant that the country's conservation approaches were largely influenced by discriminative events and practices, and resulted in differing discourses in relation to environmental protection. These differences saw a conservation paradigm that was based on being exclusionary, riddled with conflict, and alienating the majority of the country's people to the objectives of conservation areas (Carruthers, 1989: p. 215). However, with the advent of democracy in the early 1990's, there was a need to address the deep inequalities and misconceptions that were woven into everyday discourses and the legal fabric of environmental legislation and management. Perceptions about the environment and the protectionist approaches demanded urgent attention in the government's postapartheid environmental planning (Wynberg, 2002: p. 234), and emphasis was placed on the need for meaningful engagement between stakeholders involved in conservation planning and management. South Africa has made significant progress in developing policies to address environmental priorities and social development. Yet the debilitating legacies of apartheid, coupled with contemporary politics, environmental concerns and pressures to ensure and promote sustainable use and access to natural resources, still present various challenges for achieving conservation goals. One such challenge is ensuring effective implementation and enforcement of policy and legislation, as well as monitoring policy outcomes<sup>14</sup> . This is related to the continued top-down decision-making processes in natural resource management, marginalization of local communities, and the dominant scientific narrative in conservation management, which have been well documented in earlier studies in the regional context (Sunde and Isaacs, 2008: p. 5; Müller, 2010: p. 153; Sowman, 2011: p. 299; Williams, 2013: p. 13; Sowman et al., 2014: p. 31). Many biodiversity-rich areas are subject to conservation strategies of some form and should include people as part of its biodiversity. In a developing country context, these people are typically among the economically poor who depend on natural resources to contribute to their livelihoods. While various conservation paradigms, such as the dominant discourse of sustainable development, recognize inclusive and participatory approaches, their implementation often fails. However, the ways in which people relate to nature, biodiversity, or species extinction varies greatly. Being rooted in different discourses, the ways in which environmental concepts are perceived depend on people's relational lived experience. Locals might not agree to the fundamental principles of conservation and, while being recognized as stakeholders, it has been difficult to integrate their system of belief into existing conservation approaches. Here, the importance of understanding these environmental discourses becomes apparent and highlights the need for more contextspecific research of BRs and protected areas, including the social environment that is part of these systems. A key issue that results in cooperation difficulties is related to discursive ideas of how biodiversity should be protected (Hyman, 2006: pp. 84–88). This was stressed by fishers who claimed that their interests and perceptions on marine biodiversity conservation, as well as their traditional way of life, were marginalized and not considered in conservation objectives. It is important to stress however that even if these conditions were met, this would not necessarily mean better cooperation between the stakeholders managing a protected area. What is key though is to acknowledge that communities and local stakeholders are part of the area that needs protection, and that their "buy-in" and inputs are necessary in order to collectively work toward meeting conservation objectives. Coupled to this ideal is that conservation practices should be viewed as socially just and should work toward "understanding how people perceive an issue [which] is essential to the whole process of 'problem identification,' both normatively and politically" (Barry and Proops, 1999:338). The KBR is a case in which different discursive ideas of how biodiversity should be protected result in cooperation difficulties among stakeholders and limited outcomes (Hyman, 2006: pp. 84–88). On these grounds, the authors would like to stress the importance and encourage more context-specific socioeconomic research regarding the establishment, implementation and maintenance of protected areas. Conservation efforts and projects will continue to fail if the belief systems, inputs and ways of life of local communities (and other relevant stakeholders) are not taken into account. In this regard, conservation should be a part of people's discourses, part of their livelihoods and not seen as a burden, exercise or an approach that instills fear or compromising on one's livelihood. While including people and incorporating their knowledge and way of life does not automatically mean that conservation will succeed, it is an important priority that cannot be overlooked and should be included when initiating and implementing conservation objectives. #### Evaluation of Q Methodology This study has demonstrated that Q methodology can provide a valuable tool for researching environmental subjectivities. Drawing on the strengths of both qualitative and quantitative research traditions, it offers a promising method for studying perception-based research and makes an important contribution to science as it is able to identify and analyze multiple discourses. The application of Q methodology in all its stages is an efficient, yet demanding task. Nevertheless, it provides a reliable and logical framework for studying perceptions with validated results. The statistical nature of the Q factor analysis provides outcomes and data interpretations that are less prone to researcher bias. By combining interpretative discourse analysis with Q in this study, it revealed that this combination worked well in verifying results and providing deeper meaning and insights to the data. The study undertaken here thus concurs with the findings of Wolsink (2004), who emphasized that Q is particularly suitable for research that combines it with other research methods (Wolsink, 2004: p. 2676), such as participant observation. #### Limitations One methodological limitation is related to the sample size; in this regard, a small P-sample size carries some limitations. Here, it implies a finite number of factors to reach the Eigenvalue level of 1.0. This is because the Eigenvalue ≤ 1.0 indicates that the unrotated factor explains less than the variance of one respondent, so with a small number of respondents this might happen sooner. Thus, it may be that a larger sample size, and particularly when recruited from other stakeholder groups, would have resulted in more discourses. Another methodological shortcoming is the double meaning as well as reasoning of some statements, which required careful qualitative analysis of the interview data and thereby put extra emphasis on the interpretative discourse analysis. This study has studied a very heterogeneous group of people with major socio-economic diversities. This represented a challenge particularly when selecting statements, as the stakeholder groups tended to use language quite differently. One can therefore not reject the possibility that sensitivity to formulations has had an impact on the Q sorting procedure. <sup>14</sup>South African National Biodiversity Institute website: http://www.sanbi. org/biodiversity-science/science-policyaction/biodiversity-policy Accessed 22.06.2016. Particularly consensus statements following the Q factor analysis might be a result of the statements being poorly formulated. The possibility therefore exists that some of the consensus statements could have been sorted differently if it was formulated in another way. It should also be noted that although there were clear stakeholder groupings in this study, other members of these groups might not share the same ideas, as these groups were not homogenous. The fishers as example might perceive marine conservation differently than the overall local community. Although all the fishers in this study shared similar ideas, a Q study that only focused on the fishers might have shown a wider spectrum of perceptions within the fishing community. Perceptions of marine biodiversity conservation are part of a larger environmental discourse, which is further related to people's wider ontological worldview and systems of knowledge. Additionally, it is not static and will change and develop over time. Therefore, this research can only provide a limited description of the prevailing discourses, at best highlighting the most prominent similarities and differences. The authors therefore note that the research is not directly transferable or intended to prescribe conservation management. The aim is rather to demonstrate that the use of Q methodology is relevant and can provide reliable analysis for scrutinizing perceptionbased research. Q methodology allows researchers to understand the perceptions and interests of people in their own terms and categories rather than making assumptions. Therefore, based on this study's outcomes, the authors would strongly encourage further application of Q methodology in other studies in order to increase the amount of research that aims to conceptualize and analyze context-specific environmental challenges. ## CONCLUSION Conserving biodiversity is an important endeavor and one of the greatest contemporary environmental challenges. The KBR is an exceptionally biodiversity-rich area, which should be conserved. In doing this, the need for stakeholder engagement and cooperation has been identified and established in the form of the KBRC and various working groups (Anchor Environmental, 2009: p. 4). Previous studies have shown that stakeholders in the KBR are facing management and cooperation difficulties by pointing at divergent interests and perceptions (Hyman, 2006: pp. 84–88; Müller, 2010: p. 152). This study has contributed to research on how different perceptions and understandings of conservation influence the conservation objectives and activities of a BR. In doing so, this study set out to investigate how marine biodiversity conservation is perceived in the KBR by examining different discursive realms, and how stakeholders adhere to these discourses. It targeted stakeholders of the #### REFERENCES Anchor Environmental (2009). Kogelberg Coast Integrated Management Plan: Report of the Organized Conservation Focus Group Meeting 13 October KMWG, who is tasked with promoting and ensuring coastal conservation and development. This study has found varying ideas of what constitutes biodiversity conservation and how it should be implemented. It further highlighted some of the difficulties and challenges for cooperation in this particular case. The case study has presented two different ways of perceiving marine biodiversity conservation among stakeholders of the KBR, which is grounded in different discourses. While these two differing discourses have highlighted specific positions, there was a common concern shared for the current environmental situation in the reserve. This was revealed in the importance and shared belief that protecting nature is of significant importance and to everyone's benefit. Another important finding was that there was a shared belief by stakeholders who all agreed that local communities and their livelihoods should be a key consideration in all conservation approaches. While researching perceptions is not an easy task, this study employed Q methodology to demonstrate how perception based research can be validated. This study provided insights into the discourses present at a particular time in the area. Taken the complexity of the issue and the shortcomings of doing this exploratory study, in many ways, this research begs a companion piece to describe the different discourses in closer detail. In conclusion, the authors would like to emphasize the importance of increasing the number of research projects that study environmental discourses, as it is crucial to understand the social context and implications for conservation initiatives. While there is a need to conserve biodiversity globally, it is important to bring people into the debate and how they think, talk about and see themselves in or as part of the natural environment. Understanding the ways in which people think about conservation in particular is key when considering that it is not only a scientific problem but also a societal problem. ## AUTHOR CONTRIBUTIONS KH conducted research as part of her masters course under the supervision of SW. For this publication KH and SW worked in co-operation to produce the manuscript submitted here. ## ACKNOWLEDGMENTS The authors would like to thank Prof. Maarten Wolsink for methodological and analytical guidance. We are also grateful to Dr. Yves Van Leynseele and Dr. Maarten Bavinck for comments, and Dr. Judy McKenzie for help with the analysis. We also want to thank Stephen Young for creative inputs, as well as three anonymous reviewers for their comments during the manuscript review phase. 2009. Available online at: http://www.anchorenvironmental.co.za/Documents/ Pdfs/Kogelberg%20CIMP/Minutes%20-%20Conservation%20AgenciesFocus %20meeting%20KCIMP.pdf (Accessed June 21, 2016). Baker, R. M. (2006). Economic rationality and health and lifestyle choices for people with diabetes. Soc. Sci. Med. 63, 2341–2353. doi: 10.1016/j.socscimed.2006.06.007 Addams, H., and Proops, J. (2000). Social Discourse and Environmental Policy: An Application of Q Methodology. Cheltenham: Edward Elgar. 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 © 2016 Hagan and Williams. 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. # Putting Lifeworlds at Sea: Studying Meaning-Making in Marine Research #### Rapti Siriwardane-de Zoysa<sup>1</sup> \* and Anna-Katharina Hornidge1, 2 <sup>1</sup> Department of Social Sciences, Leibniz-Center for Tropical Marine Ecology, Bremen, Germany, <sup>2</sup> Institute of Sociology, University of Bremen, Bremen, Germany An individual's "lifeworld" guides perceptions, the attachment of meaning and in sum, the interpretation of reality in everyday life. Yet the lifeworld (Ger. Lebenswelt) has been an undertheorized concept within interdisciplinary marine research. Through a two-stage analysis, we critically engage with the philosophical foundations, heuristic value and the methodological versatility that the interpretivist concept of the lifeworld stands to offer, drawing from contemporary marine scholarship. With two illustrative case studies exploring the lived realities of vastly different waterworlds in rural Uzbekistan and Sri Lanka, we further engage with the strengths and limitations of integrating a lifeworlds analysis into interdisciplinary work on localized perceptions. As a second step, we analyze the efficacy of adopting a phenomenological-lifeworlds approach in order to inductively explore diverse realities of coastal and sea-based peoples, while acknowledging the terrestrially-bound and anthropocentric genesis of the lifeworld as a concept. Therefore, in order to enliven hybrid thematic currents, conceptual debates and methodologies on "marine lifeworlds" on its own terms, we propose two thematic vantage points for interdisciplinary intervention by: (a) critically engaging with cognitive-material meanings and lived interpretations of "saltwater" realities; (b) tracing multiple modes of sociality and being with/in-the-world that go beyond human entanglements. In sum, we argue that while the lifeworlds concept affords spaces through which to study the complexities and ambivalences rife in surface-level perceptions, it promises the means with which to sidestep over-simplistic inferences to the vague and embattled notion of "culture," while widening horizons for reflective and experimental-experiential lines of inquiry. #### Keywords: lifeworlds, meaning making, applied phenomenology, marine epistemologies, seascapes ## INTRODUCTION "Only the magic and the dream are true—all the rest's a lie." –Rhys (1999) Jean Rhys' novel, set in nineteenth century plantation Jamaica, offers a postcolonial feminist retelling of the tragic tale of "Bertha" Mason, Edward Rochester's mad wife, who remains locked away in an unforgiving turret of Charlotte Bronte's Jane Eyre. In the latter Victorian novel she is dragged out, fighting tooth and claw, more harpy-like than animal, more mythological than misplaced. #### Edited by: Wen-Cheng Wang, National Taiwan Normal University, Taiwan #### Reviewed by: Edward Jeremy Hind, Manchester Metropolitan University, UK Jan Maarten Bavinck, University of Amsterdam, Netherlands \Correspondence: > Rapti Siriwardane-de Zoysa [email protected]* #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 10 May 2016 Accepted: 26 September 2016 Published: 08 November 2016 #### Citation: Siriwardane-de Zoysa R and Hornidge A-K (2016) Putting Lifeworlds at Sea: Studying Meaning-Making in Marine Research. Front. Mar. Sci. 3:197. doi: 10.3389/fmars.2016.00197 104 Yet in Rhys's (1999) Wide Sargasso Sea, Bertha—or Antoinette Mason—a fiercely intelligent and imaginative young Creole woman is presented in a very different lifeworld that is her own, on an island far removed from the cultivated frostiness of English country life among the landed gentry. It is this lifeworld that the stranger—Rochester—whom she weds, comes to fear and ultimately loathe, with its tropical "mountains and hills, rivers and the rains... its sunsets of whatever color... its beauty and its magic... its indifference and cruelty." The growing revulsion that he harbors toward his new wife lies inimically coupled to this antithetical world that he covertly delights in othering—replete with disease and rumor, obeah ritual and languid decadence. To Rochester, Granbois seems unreal and hallucinatory, while England appears surreal and dream-like to many of the young Anglophone women of the West Indies, yearning for "return" to an island they had never set foot upon. It is this slippage between embodied presence and absence, of wakefulness and the dream-like, in which the two lifeworlds in the Wide Sargasso Sea are so intimately interwoven. Yet, what is in a lifeworld and why should it matter in community-based research? Within ongoing debates on inter and transdisciplinary approaches in addressing "real life" problems, the inclusion of local lifeworlds in order to guide researcher reflexivity, in determining research processes and the interpretation of findings have repeatedly been stressed (Pohl and Hadorn, 2007). While the notion of "lifeworlds" (Ger. Lebenswelt) is often used metaphorically in order to place emphasis on the salience of local perceptions and worldviews, its conceptual and empirical uses and limitations remain under-researched across the coastal and marine social sciences. Since its formative stages of conceptual development the "lifeworld" has remained a relatively nebulous and opaque concept when perceived from outside the disciplinary frames of interpretive sociology and psychology. Furthermore, it is often perceived as being methodologically elusive and complex for practical application in empirical field-based contexts. While much ethnographic analyses bearing the term lifeworlds exist, they arguably pay scant lip-service in concretely conceptualizing what precisely is meant by the term. Furthermore, the concept offers little recourse to social scientists that remain wary (and weary) of wielding the hollow notion of "culture" as an explanatory force, given its dangerous ambiguity and inclination toward essentialist theorizing. Rather than attempting to tame and pin down a linear, all-encompassing definition of lifeworlds, this paper serves as an open invitation to socio-environmental scholars and policy analysts who are increasingly turning toward perceptions-based, interpretative and social constructivist thinking in order to invigorate community-based research. The second section provides a kaleidoscopic glimpse into the various threads of lifeworld theorizing, drawing attention to the often slippery axes between the following dualisms— (a) the individual and the collective, (b) the experiential and the ideational, (c) of appearance (exteriorized) and essence (interiorized) and (d) the subjective (first person) and the objective (third person). The third part of the paper proceeds to reflect on the flexible application of the lifeworld concept through the use of two empirical studies of fluid waterworlds (see Anderson and Peters, 2014), one implicating freshwater and the other saltwater. Following on from an empirically grounded discussion, the fourth section offers critical insights into whether the study of "marine lifeworlds" holds much conceptual purchase and empirical relevance at all, given the vast corpus of maritime and coastal-related social science research that has embraced an interpretive perspective, though not necessarily a phenomenological one. While briefly attending to some of the reasons for the apparent absence of marine lifeworlds-inspired research as opposed to more coastal-related foci, we provide critical points of departure and thematic interventions through which the study of marine epistemologies and ontologies (i.e., ways of knowing and being) may enliven existing interpretivist research endeavors. ## UNBRAIDING THE LIFEWORLD: THE ANATOMY OF A CONCEPT The understanding and study of social reality has been a core preoccupation across diverse sub-disciplines including social philosophy, existential anthropology, interpretivist sociology, and cognitive psychology. Since the early 1900s, the notion of the "lifeworld" has often appeared in the social sciences and the humanities, as an integrative concept with which to describe the particularities of an individual's lived experience in everyday life. However, before engaging with this comparatively hydra-headed term, the very philosophical and epistemological foundations of the lifeworld approach warrants further exploration. Contemporary approaches to lifeworlds thinking, emerged as the progeny of two vastly influential theoretical traditions in the late nineteenth and early twentieth century, particularly across the Austro-German and French philosophical milieu. At its broadest, phenomenology—as a philosophical tradition and movement—can be traced back to the Austrian School founded by Franz Brentano that built upon classical Hegelian notions of the experience of being in the world. Broadly framed, early phenomenological philosophers like Husserl (1970), Heidegger (1977), Mannheim (1929), Merleau-Ponty (1981), and Lyotard (1991) were concerned themselves with the structures of experience in everyday understanding, and in the interplay of common sense and how particular forms of "knowing" and "being" arise from experience (Roth, 2004; Bengtsson, 2013). These currents vastly influenced the work of humanist thinkers and writers like Jean-Paul Sartre, Albert Camus, and Simone de Beauvoir, as they engaged with broader interrogations of being and not-being, social alienation, otherness, and nihilism. Phenomenology is primarily concerned with the experience of "being there" (Ger. Dasein)—in all our humanness—that far transcend the surface meanings of ordinary perception and experience. Modes of existence were understood as being patterned by and grounded in very situated spatio-temporal, socio-relational and symbolic spheres of everyday life. In the contemporary context, phenomenological approaches still remain influential within fields such as area studies, urban and medical anthropology, peace and conflict studies, natural resource governance, educational psychology, social work, nursing practice, management research, and disability studies, incorporating diverse methods spanning the qualitativeinterpretive, quantitative and the poetic-reflective (see Nieuwenhuys, 1994; Ehrich, 2005; Jackson, 2005, 2012; Johansson et al., 2008; Oberkircher and Hornidge, 2011; Finlay, 2013; Siriwardane, 2015). Phenomenology can be therefore seen as a distinct epistemological philosophy—a style of thinking. Scholar-practitioners, particularly within the field of critical pedagogical research, often drew attention to its ontological, existential currents, treating it as a "way of being" or as a philosophy of life in itself (Hultgren, 1995, p. 379). This position bears far-reaching implications on how research practice and writing could be ethically and reflectively re-learned, an aspect that we will be exploring in greater depth. The second strand of lifeworlds thinking originated from the hermeneutic tradition, latterly branching into the sub-field of phenomenological hermeneutics. Hermeneutics can be broadly defined as the theory of interpretation (Dahlberg et al., 2008, p. 66), implicating scholars such as Gadamer (1975) and Ricoeur (1974), who were particularly engaged in exploring the gaps inherent between perception, language, embodied expression and action, together with the wider processes of storying human experience. Later work like that of Johansson and Emilson (2010) and Bengtsson (2013) grew out of the Gothenburg tradition with a focus interaction-based meaning-making, for example in the context of educational research on preschool children and their non-linguistic (yet highly expressive) routines of inter-subjective play-based worlds. While it is evident that there is no singular way of defining and doing phenomenological lifeworlds research, it must be borne in mind that phenomenology, and consequentially early theorizations of the lifeworld, did not explicitly engage with matters of empirical research. Indeed, early phenomenological thinking stood as a distinctly philosophical (and ethical) project. For thinkers like Edmund Husserl (1970), who first introduced the term "lifeworld" in a treatise written between the wartime years of 1916 and 1917, the notion arguably stood out as a dynamic counter-concept to the privileged role of scientific rationality and the crisis of the modern technological world. Phenomenology reflected the disenchantment of contemporary thinkers with the ideals of natural science and technology as "models for philosophical engagement" (Madsen, 2002, p. 10). The lifeworld then stood, not as a radical juxtaposition or dialectical Other, but as a world of commonly shared experience, encompassing worlds of belief (doxa), of preconceived notions of prejudice and illusion for example. In this light, the world of scientific rationality and knowledge production was but one lifeworld among others. While we have thus far explored the ontological and epistemological foundations of the lifeworld approach, how then can this multi-stranded approach be accessed with some degree of conceptual clarity? Husserl never quite as cogently defined what was meant as a lifeworld. Moreover, his work left to be asked why "worlding" metaphors mattered when exploring perceptions, attitudes and meaning-making processes. In other words, would not a singular focus on self-expressed perceptions, not seem as sufficient as empirical facts? These questions take us back to the German philosopher Heidegger (1977), who was arguably the first to integrate phenomenology with classical hermeneutics, borrowed from neo-Kantian, Kierkergaadian, and Weberian theoretical currents (Bengtsson, 2013). From a Heideggerian vantage point, perceptions were mere surface articulations. They were often conscious and could be rationalized logically, yet their salience, preconditioning and the more subtle tacit forms of knowing, thinking and feeling that still remained relatively less apparent. Perceptions then, at its simplest, were akin the proverbial tip of the iceberg. Often, the distance between perceptions and actions, believing and doing, could not simply be explained by chipping away at subjective attitudes or collectively recognized norms and mores. The lifeworld to Heidegger was about "being-in-the-world" (Ger. in der Welt sein). What this concretely translates to is the conceptualization of phenomena and experience that are lived and inter-subjectively experienced, yet could still remain tacit. It differed from Husserl's critique on the "natural attitude" (Ger. natürliche Einstellung) of phenomena as materially known and felt (Dahlberg et al., 2008, p. 33). Therefore, multiple lifeworlds as differently conceived and lived—could exist in a single material realm, for example a coastal stretch inhabited by seaweed farmers, dive operators, aquaculturalists, hoteliers and naval entities. However, Heidegger's conceptualization of the lifeworld concept sat within this wider philosophical project, and was a long distance away from being empirically translatable for research practice. For the French philosopher Merleau-Ponty (1981), the lifeworld approach adopted a more differentiated hue, which he conceptualized as "being-to-the-world" (Fr. entre au monde), in which the human body (and its embodied practices of everyday life) comprised the primary site of knowing, feeling and being. In his view, the "Eye" and the "Mind" (implying the Cartesian mind-body dualism) were not disconnected but mutually reinforcing, in which the world came to be interpreted and known through how it was materially, relationally and symbolically felt. In this light, it remains telling why interpretive scholarship within the fields of medical and educational psychology and disability studies for example, tend to be influenced by Merleau-Ponty's foundational work on embodiment. Moreover, there exists a recent and steadily growing body of marine/maritime scholarship that attends to the affective and multisensory meanings and subjectivities produced by dwelling with the sea—whether in terms of "finding one's sea legs" as an embodied experience of enskilment related to fishing and sea navigation (Pálsson, 2000), or through (masculinist) sensibilities of getting high on a "stoke" when surfing a wave (Evers, 2004). Ultimately however, it was the Austrian sociologist, Schütz (1932, 1960) who consciously attempted to extricate lifeworlds thinking as a purely theory-based endeavor, into a practical concept for empirical analysis. Schütz, like Merleau-Ponty, was influenced by early Husserlian currents, but his primary focus rested on locating patterned structures through which lifeworlds could be understood. For him, the very act of conceptualizing (and contextualizing) lifeworlds—both literally and metaphorically—as "worlds" (i.e., domains or realms of experience, knowing, doing and feeling), was paramount to the exercise of grounding the concept. As he posited, "in using the term 'world'...we mean only that different people are consociates, contemporaries, predecessors or successors to one another and that they accordingly experience one another and act upon one another in the different ways in question" (Schütz, 1960, p. 143). For Schütz, the lifeworld was bound through temporal and spatial dimensions comprising four interlocking socio-material worlds. The individual's immediate environment, the social world of contemporaries (Mitwelt), interlocks with the precedent world of predecessors (Vorwelt) and successors (Folgewelt). While the immediate environment (Umwelt) appears to be shaped by direct, close relationships to family members and friends, the surrounding world (Mitwelt) is characterized by the interaction with those actors and social structures potentially subject to the individual's personal experience. This experience stands in relation to the individual through typification—the process of conceptually identifying, differentiating, naming, sorting, and assigning symbolic meaning to perceived material and relational phenomena, that begins in infancy. As the Vorwelt is shaped by relationships to ancestors and interpretations of the past, the Folgewelt is shaped by relations and actions directed to/at the future (Schütz, 1932, p. 160). Together, these four worlds of the individual constitute the reality of everyday life, or the Schützian interpretation of the "lifeworld." It was for the American-Austrian-German sociologists Berger and Luckmann (1967), that the differentiation between objective and subjective lifeworlds appeared paramount in adding more nuance to the interpretive study of reality. The subjective lifeworld, formed via typifications, constitutes the researchers' own lifeworld including those that are encountered during the research process. On the other hand, the objective lifeworld however appears as the naturalized milieu, setting spatial and temporal boundaries that are concretely lived, and may not be apparent within collective consciousness. These boundaries however are not simply limiting; they are generative in the sense that the spatial-temporal scope of an individual's lifeworld directly depends on the zone of operation" (Wirkzone) characterized by the geographic, social, as well as the mental mobility of a person. Therefore, diverse practices of small-scale as well as industrial fishing are not merely treated as a livelihood, but as a way of being-with-the-world and as "a way of life" (Weeratunge, 2009). Yet, at this point it is must be noted that a Schützian reading of lifeworlds can be critiqued for its focus on the individual as a primary subject of analysis. Thus, collective lifeworlds, were somewhat simplistically interpreted as the additive stratification of individual experience, making for the interpretation of "shared reality" as merely the sum of its constituent parts. Indeed, the work of Berger and Luckmann (1967) proved influential in sociological institutionalist theory building, given its heuristic methodology in studying normative change, and the interplay of collective roles, norms, discourses and practices (see March and Olsen, 2005). Yet arguably, the analytical tools offered in tracing trajectories of institutional change remain relatively less defined. Meanwhile, two other influential German lifeworld theorists that warrant brief discussion: the Frankfurt School critical theorist Habermas (1955, 1984), and the phenomenological hermeneutic philosopher Gadamer (1975). In combining Chicago School pragmatism and early currents of structuralfunctionalist thinking, Habermas' view of the lifeworld stood in stark contradiction to what he defined as the "systems world" constituting the exteriorized rationalization of everyday action, as evident in modern bureaucracy for example. It was then the focus on the interaction between the two realms—in which the systems-world often "colonized" an individual's lifeworld, through tacit influences such as media practices that steer collective thinking and action. One of the more compelling tropes through which this tension is illustrated can be found within the substantial raft of fisheries-related governance literatures and environmental management practices that explores interactive encounters between bureaucratic, scientific and locally-situated knowledges, particularly within diverse co-management structures and other communicative contexts, whether more participatory or top-down (see Wilson and Jacobsen, 2013). The work of Gadamer (1975) on the other hand, took Husserl's conceptualizations further by integrating the notion of Vorurteile (preconceptions). His work contributed to reflexive praxis-oriented research that set the foundations for a practiced attitude of exposing and confronting prejudgements, particularly through intersubjective encounters. Thus, a Gadamerian reading potentially offers conceptual insights into questions of individual agency and resistance, regulatory and informal norm-based compliance, constituting interwoven thematic currents that are gaining increased traction within interdisciplinary fisheries research. In order to chart their diverse epistemological currents, Figure 1 depicts how the concept of the lifeworld evolved. Having explored the salience of understanding researcher lifeworlds, how does one set about reflecting upon, documenting and storying the lifeworlds of others? As the following section illustrates illustrates, since the concept entered the realm of social science discourse, much has been done in the way of creatively translating and operationalizing lifeworld approaches into research processes, while simultaneously guiding researcher ethics and reflexivity. Moreover, it critically examines the conceptual and empirical advantages and limitations of applying a lifeworlds approach to contemporary research on two different types of lived "waterworlds"—one in an arid, landlocked freshwater site in Central Asia, and the other constituting a saltwater milieu in a South Asian coastal setting. ## OPERATIONALIZING LIFEWORLDS IN EMPIRICAL RESEARCH The paradox of attending to and understanding the substance of individual lifeworlds, particularly if it is interpreted as constituting the implicitly lived or presupposed realm of everyday life, has been a central topic of debate within anthropological and sociological research. First how can the tacit and unexpressed (or inexpressible), emerge to the surface of consciousness? Second, how can a researcher—particularly one who is relatively distanced from the lifeworld of those she partners—explore at times unexpressed meanings? Thus, the attempt at bridging this epistemic distance, between the interiorized (implicit) and the exterior (the manifest), tends to emerge as a leap of faith. Furthermore, what questions of power and authorial voice materialize when claims are made about conclusively studying and writing about individual and collective lifeworlds? On one hand, lifeword theorizations have almost exclusively been individual-centered. On the other, the ability to write about lifeworlds, may run the risk of potentially strengthening truthclaims through textual preeminence. This double-bind, between the persuasive currency of being able to extract knowledge on lifeworlds, and the (em)powered researcher to grasp the otherwise implicit and tacitly known may potentially result in a dangerous cocktail of epistemic privilege and representational objectivity. Researcher positionality and reflexivity have therefore remained a central concern, particularly within the scope of fieldbased qualitative research. Ethnographers in particular have been doubly conscious of the early colonial origins of their enterprise, and continue to contribute to lively debates on the ability of fully grasping meanings and implications by one's socio-political positioning during fieldwork (see Lynch, 2000). In contemporary phenomenological lifeworld research, this is often achieved through a three-step iterative process in which experience is bracketed, otherwise known as "bridling." The first stage entails a systematic effort to suspend judgment, by stepping outside preconceived notions of how things are expected to seem and to work. The second stage requires the conscious effort of dwelling with and within the phenomena in question. Put differently, a researcher's attention may be re-focused to her content of what is experienced, and what makes experience possible in the first place. The process of cultivating generative openness to the first and second stage interpretations of meaning comes to bear, allowing one to consciously compare alternative templates and mental maps of how the same phenomena has been interpreted in the past. Taken together, these steps prevent a fieldbased researcher from acting upon pre-existing assumptions and interpreting lived realities too hastily. Moreover, it lays bare the fact that as researchers, "we can be self-reflective without being self-aware" Dahlberg et al. (2008, p. 165). This quotation takes into account the significance of experience that influences further actions, and the consequences that come about by reflecting with, rather than on others. Johansson et al. (2008, p. 2) see this as a concrete form of bridling, which does not make the pretense of abandoning all pre-assumptions, but instead embraces the possibility of "slowing down the process of understanding in order to see the phenomena in a new way," often integrating multisensory subjectivities and relationalities. For example Peters (2010) in problematizing scholarly representations of the sea as a mere metaphorical image of life on shore, draws attention to the very linear act of objectifying the sea through dynamics of voyaging, trade, empire-building and territorialization. Therefore, in (re)centering fluidities beyond spatially bounded terms, Anderson and Peters (2014, p. 5) calls for the imperative need to enliven scholarly engagements with diverse marine epistemologies that see (water)worlds as being in "flux, changeable, processual and in a constant state of becoming." As varied as the conceptual interpretations of watery lifewords has been, so have been the methodologies with which to research them. Oberkircher and Hornidge (2011) operationalized the phenomenological concept in the form of "water lifeworlds" through a contemporary Schützian interpretation by paying closer attention to how objective and subjective realities were coupled in Khorezm, Uzbekistan. Their methodological substance therefore entailed social facts and processes they themselves observed (e.g., everyday actions and decision-making trajectories) combined with the discursive-symbolic world of narrative reflections, mental maps and new or emergent concepts. Once again, the epistemological frame was driven by the particular problem-based research puzzle in question. They examined why perceivable forms of water saving were hardly present in an arid terrain such as Khorezm, and across a sociocultural milieu that seemingly articulated counter-rationalities on the sacredness of water and its centrality in establishing social status. By attempting to understand nuances inherent in the inbetweenness of these divergent rationalities, the temporal and spatial boundedness of the "objectively" perceived lifeworld was first explored. This entailed how farmers constructed their picture of time and space alongside diverse water users and managers. Furthermore, these lifeworlds comprised meanings about vegetation seasons, flows of freshwater, and how times of scarcity and abundance were cognitively labeled. As a second framing, typologies, values, and institutions (as rules, norms, and rationalities) were explored. In particular, they focused on how individuals compared, categorized and classified their diverse Khorezmian waterworlds. The authors did this by identifying several layers of typologies: (a) types of water (for example locally embedded meanings around hard water, muddy water, and freshwater, literally translated as being delicious); (b) types and roles of people (for example diverse groups of "upper people" in the social hierarchy), "water persons" linked to the ancient institution of mirabs in Central Asia, fermers or large farm operators etc. and (c) types of land. These boundaried pictures and typologies were compared with intersecting values and institutions—as discursively articulated, for example, through diverse state-led management principles, commonsensical logics and socio-religious rationalities on water provision and use. What the study drew attention to was why the rationality of water saving stood out as a "missing concept" in these diverse water lifeworlds, despite the ubiquitous Uzbek expression—suv hayot—or water is life (p. 406). By squarely drawing out and comparing rationales that prevent water saving as opposed to those that valorized the need to do so, the authors drew attention to the seemingly "messy" oscillation of lifeworld dynamics as they unfolded amid the complexities of everyday life. As we proceed to illustrate, this contemporary Schützian use of lifeworlds phenomenology, as a means to study problem-centered empirical questions, offers a number of conceptual and methodological advantages, as well as potential limitations and pitfalls. On the other hand, Siriwardane's (2015) island ethnography on fisher lifeworlds in postwar Sri Lanka, with a distinct focus on the interactions between geographically and ethnoreligiously diverse groups of migrants, settlers, and locals offers a different starting point. In asking why particular fisher collectives were othered as veritable "outsiders" or strangers, the study drew inspiration from existential anthropological readings on lifeworlds (see Jackson, 2005, 2012; Das, 2006). In this context, the "everyday" was taken as a realm that was not simply normed, routinized and rendered ordinary, but also as a site of active production, particularly in the way that power asymmetries come to be produced and contested. Moreover, in forewarning against the tendency of treating the concept the lifeworld as a "blanket term to encompass and 'explain away' every (ambiguous) facet of lived life" (Siriwardane, 2015, p. 96), the lifeworld concept was reshaped to suit everyday translocal and livelihood-based experiences and sensibilities. At first glance, local hostilities directed at bilingual fisher migrants who shared long biographical histories of seasonal mobility seemed intuitive. They often encamped near local settlements that practiced similar forms of craft-based fishing. Their apparent bilingualism also actively worked in the disfavor of other migrant groups, given the fact that many continued to mask outward expressions of hybrid ethno-linguistic belonging, particularly when interacting with locally embedded military institutions. Yet, upon further exploration, it could be argued that the very rationalities around belonging, place-based identities, (historic) social presence, and "home" did not always cohere. The normative underpinnings through which communal insiders and outsiders were differently framed lay in a host of interpretations entailing crisscrossing: (a) pioneer narratives (i.e., "Who fished here first," "Who cleared this land?"), (b) discourses on ancestral belonging and homeland (e.g., the primacy of having lived in the east coast, despite having been serially displaced over wartime), (c) biographical livelihood identities bound through "blood" and inter-generational enskilment, and (d) navigational imaginaries and historic legitimacies of mobility, through westeast coast sojourning. The institutionalized backdrop against which postwar militarized insider-outsider frames were being established was hardly ever articulated when exploring intergroup amity or hostility. For example, the vorwelt (pre-world) of bilingual fisher migration trajectories during wartime established encampments that were perceived by local residents as sites of exemption and rule breaking. This further exacerbated localized antagonistic perceptions, even between diverse migrant groups. In comparing different lifeworld conceptualizations between both Oberkircher and Hornidge (2011) and Siriwardane (2015), it can be ascertained that such phenomenological approaches enable researchers, often not trained in field-based qualitative ethnographic work, to explore local terms through meanings (as lived) that go far beyond their semantic definitions and terminology. For example, if the pervasively uttered phase suv hayot was taken literally, as a blanket cultural expression defining Uzbek life—the paradoxes around practices that otherwise imply that water is unproductively allocated and used across the Uzbek hydraulic bureaucracy, would have remained relatively underexplored. In a similar vein, homogenizing all migrant fishers as strangers and outsiders would have led to the problematic glossing over multiple modes of sociality and ways of relating-to/with-the world (vernacularly theorized as sambandam) along liminal coastlines. Thus, the close attention paid to what seemed at face value as contradicting realities, enabled both studies to elude the trap of over-simplified and essentialist readings of localized "culture." Furthermore, turning to the lifeworld as an empirically applied methodology enables researchers to interrogate their own lifeworlds and potentially, check biased western-centered rationales and framings of aspects such as time, space, notions of reciprocity, and systems of socio-economic exchange. While this opens up collaborative spaces for co-production and interpretation of data between researchers and those they partner, and for self-reflecting on epistemic power and privilege, the lifeworlds approach also enables us to acknowledge and capture nuance and ambivalence. The distance between "representation and practice" (Busby, 2000, p. 34), and what is discursively articulated and what is ultimately enacted, often appears as a central trope in localized fisheries research for example, taking contexts in which institutional norms are both sanctified as well as broken under specific circumstances (Siriwardane, 2015, p. 147). The lifeworlds approach therefore calls for a cultivated sense of epistemological un-knowing, embodied in the German phenomenological notion of Gelassenheit (of letting be or to dwell, Dahlberg et al., 2008, p. 81 and 100). The concept may also act as an epistemological starting point that can be used across diverse socio-cultural and regional contexts. While the concept may provide a heuristic vantage point through which a non-Eurocentric de-centered study could be envisaged, its claim toward epistemological universality may also act to its disadvantage. For example, phenomenology remains a deeply humanistic endeavor. Therefore, the lifeworld as a concept is inevitably an Anthropos-centric one, which encompasses more than just human interactions and engagements. While social meanings around inter-species relations (for example values toward non-human sentience for example), may visibly appear within lifeworlds writing, the means through which socio-nature can be seen as a subject possessing agency (and not passively objectivized as foreground that is acted upon) still remains fertile ground for further theoretical work (see Kohn, 2013; Viveiros de Castro, 2016). Meanwhile, a commonly articulated limitation of the lifeworlds approach can be found in its methodological individualism. The stratification of individual experience is seen as constituting collective or communal lifeworlds, a reading that has often been critiqued for its simplicity and inability to account for normative transformations. The old quandary of seeing and describing the world through the eyes of others remains a paradoxical task. Typologies and typifications therefore serve to essentialize and legitimate particular interpretations of reality, often in ways that may be complicit to existing power inequalities and forms of social injustice, for example politically legitimated ethno-racial, gendered and class-based classifications. Both Oberkircher and Hornidge (2011) and Siriwardane (2015) point toward the limitations inherent in typologizing "categories" of people as if social identities were container-like constructs, even if these labels were to an extent self-assigned. At the same time, their work allude to the difficulty in formulating alternative framings, which may well be far removed from daily discourse and practice. Therefore, by no means does this critical discussion stand to offer pat solutions to long-standing and debated questions on the preeminence of focusing on individuals as a unit of analysis, or on the other hand, on groups and collective framings. Moreover, the age-old philosophical agency-structure debate that our discussion forecloses further problematizes the dialectical relationship between individual capacities and freedom of choice, against the inherent constraints set by institutional rules and wider societal norms. As the following section proceeds to illustrate, the use of phenomenological lifeworlds has remained a marginal current, particularly across interdisciplinary coastal- and marine-related social science scholarship. However, as we proceed to argue, "marine lifeworlds"-inspired research (although not explicitly having drawn on phenomenological currents) have historically constituted a vast corpus of work, particularly in the fields of maritime and marine anthropology, together with coastal and cultural geography. While fisheries-related accounts of diverse "peoples of the sea" have often depicted an anthropos-centric bias, we further explore what inclusionary forms of more-thanhuman lifeworld research could be further pursued in ways that more expansively engage with the newly emergent sub-fields of multi- and interspecies ethnography. It is a conversation that draws interdisiciplinary marine researchers, particularly from the natural sciences, into a lateral dialogue with the social sciences and the environmental humanities on the practice of hybrid phenomenologies of the sea in order to push for more non-representional, de-centered and non-western centric explorations of oceanic relationalities and connections that prefigure a broader politics of life. ## COASTAL OR MARINE LIFEWORLDS? DE-TERRESTRIALIZING AND UN-HUMANIZING A CONCEPT The very notion of lifeworlds remains to be taken as an open-ended concept that is malleable enough to be creatively reworked and applied across multiple socio-environmental contexts. Yet phenomenologically-inspired lifeworlds research has traditionally privileged the study of terrestrially-bound themes. It can be argued that the problem lies with the humanistic social sciences that have been less forthcoming in putting lifeworlds out at sea. When the role of the sea in imperial and colonial expansion came to be understood beginning in the fifteenth century, the ocean was still overwhelmingly and paradoxically perceived in Enlightenment scholarship as "a quintessential wilderness" (Mack, 2011, p. 17), an atemporal place and as cultural tabula rasa. As Emile Cocco writes (Cocco, 2013, p. 6), "the sociological ignorance of the sea is quite striking against the major role played by the maritime environment in literature, religion or philosophical thought" despite critical interventions made by philosophers such as G.W.F. Hegel who "celebrated the sea for its uttermost importance in the development of state, economy and European identity." Over at least the last three decades, coastal and historic geography, maritime anthropology, sociology and cartography have made significant conceptual and epistemological inroads to grounding and understanding the diversity of marine spaces and "peoples of the sea," distinguished by everyday processes of sense-making and daily practices of cohabiting fluid waterworlds (see Acheson, 1981; Astuti, 1995; Steinberg, 2001; Cordell, 2007; van Ginkel, 2007; Peters, 2010). Seas and coastlines were therefore more than mere resource bases and sites of socioeconomic extraction, value and exchange. Seascapes have been perceived as spaces of enskillment and ancestral belonging, as dreamscapes of danger and presence, and as sites of desire and dwelling, while practices such as voyaging and coasting have historically been interpreted in relational terms, that connected expansive networks of social groups and distant spaces (see Firth, 1946; McWilliam, 2003; D'Arcy, 2006, 2013; McCormack, 2007; Hau'ofa, 2008; Cohen, 2010; Lehman, 2013). Mack (2011) argues that the majority of community-based research has been undertaken on coastal spaces, overwhelmingly focused on land-dwelling (and often gendered) social groups such as fisherfolk, traders, seamen, dockworkers, coastguards and surfers, for example (see Nieuwenhuys, 1994; Laderman, 2014). Yet a smaller corpus of research engages with liminal spaces, mariners and ship-based societies, from cruise liners to piracy networks and floating armories (see Rediker, 2004; Langewiesche, 2005; Gharibyan-Kefalloniti and Sims, 2012). Meanwhile the study of marine scientific research expeditions and commercial seabed mining ventures mark an exciting new turn in the study of floating societies and of underwater verticalities (see Helmreich, 2009; Steinberg and Peters, 2015). Recent strands of interpretative marine research, particularly across the fields of cultural geography, anthropology and sociology have predominantly been concerned with two key, interlocking questions. The first concerns interrogations of how traditionally earth-bound, "land-locked" disciplines such as human geography and sociology, together with their very "grounded" methodologies (evidenced in terms such as fieldwork) could be put out to sea. As an increasing number of cultural geographers argue, the mere thematic expansion on marine topics and the study of the sea as a "different" space barely answers this rallying call; indeed, conscious efforts to start thinking "from the water" is required in order to "chart new representations, understandings and experiences of the sea, plotting water worlds that are more than a "perfect and absolute blank" (Anderson and Peters, 2014, p. 4). Yet in actuality, the disciplinary boundaries through which these conceptual and epistemological modes of understanding (and practice) unfold remain relatively less permeable, especially when marinecentered and land-based social and natural scientists continue to talk away from one another. In part, these disciplinary gaps foreground the pressing need to "de-terrestrialize the Academy" (Hornidge, 2015). It draws attention to the urgency to foster deeper and more explorative efforts of putting into dialogue (as opposed to uncritically comparing) the diverse worlds of hinterland, coastal and marinebased societies, and their social-natural assemblages. Moreover, it seeks to question the very conceptual and methodological assumptions that have arguably favored terrestrially derived interpretations of reality. For example, as Mack (2011, p. 23) argues, much theory-work and empirical refocusing is needed in order to bring the study of seascapes to the same level of conceptual and methodological sophistication as the study of landscape geography or anthropology. The second overarching conundrum rests on how expanding the many ways in which de-centered non-human-centric vantage points in studying seas, oceans and their manifold connectivities could be better explored. It comes as little surprise that the overwhelmingly humanistic hue of lifeworld theorizing in the past—best illustrated through what Kirksey and Helmreich (2010, p. 546) refer to as the paradox of human exceptionalism—that placed the (thinking-being) Anthropos at the center of its empirical inquiry. Inevitably the lifeworld then constituted a humanized gaze of the world, as evidenced in the case of anthropomorphized writings for example. Arguably the mere presence of the so-called "non-human" both lively and inanimate, in an epistemological sense, unwittingly came to be patterned around the figure of the human, and its broader material and symbolic implications for sociopolitical and economic life—invariably recast as food stock, tradable commodities, and land/seascape backdrops among others. How then have more recent endeavors into delineating marine epistemologies taken shape? Moreover, what can be said of their inherent limitations, while reimagining more inclusive and hybrid templates implicating non-linear phenomenologies of the sea? Two distinct and inter-related thematic strands within inductive social science research stand to be taken as critical points of departure through which a lifeworlds approach could be potentially enlivened. The first entails a significant body of largely coastal ethnographic and historic research undertaken through the interpretive lens of "saltwater" realms, meanings and interactions (see Sharp, 2002; McNiven, 2004; Schneider, 2012). The second constitutes the lively and dynamically growing field of critical ocean geography that attempts to rupture, stimulate and experiment with novel ways of thinking and writing through/with (as opposed to on) "wet ontologies" (Steinberg and Peters, 2015), while weaving in both interspecies being and becomings, together with the material flows, processes and social lives of inanimate objects and previously understudied forms of lived dimensionality such as volume and marine verticality (see Anderson, 2012; Sammler and House-Peters, 2013; Muttenzer, 2015). We first turn to phenomenologically-inspired work on saltwater realities and processes of sense-making. In the history of science, saltwater has been both a powerful substance as well as a metaphor to think with/through. As Helmreich (2011, p. 133) reminds us, the very blueness of seawater became a "matter of cultural construal, rather than of sheer empiricity" when invoking the famous proclamation made by the German anthropologist Franz Boaz. In the contemporary context, marine realities referenced through saltwater networks and figurations—including people, places, the "non-human" (i.e., fish, waves, technologies) and their forms of interaction and movement, are seen through collectives such as the Australian indigenous Saltwater People Network (NAILSMA), and the Canadian grassroots fisher organization the Saltwater Network. Moreover, in scholarly writing, the inference to "saltwater people" (Sharp, 2002; McNiven, 2004) came to be synonymous with indigenity and aboriginal forms of sociospatial mobility, knowing, and interacting across localized seas that were at the same time spiritscapes, imbued with maritime rituals and complex historiographies of their own. Of late, varied sub-fields under the rubric of "salty geographies" have been gaining greater appeal among interpretative scholars particularly across Anglo-American and postcolonial contexts. In attending to translocal voices calling for the "historicization of the ocean"—not only does it aim to reflexively de-terrestrialise academic lenses through which multistanded histories and sociologies have been conventionally interpreted, but it also attempts to trouble the stability of geopolitical identities and the very temporalities under which they have been (re)made and naturalized. Once more, social-natural assemblages and meshworks have stood as dominant conceptual and epistemological frames with which to enliven hitherto understudied connectivities, agencies and socionatural-political dynamics not only between conventional outcasts, un/familiar figures and material spaces (e.g., buccaneers and wreckers, port harbors and littoral utopias), but also of questions around non-human presence, interaction and their transformations, a line of inquiry we will later revisit. However, two important methodological limitations present themselves when figuratively and empirically conceptualizing the material-symbolic substance of saltwater worlds. The first shortcoming entails the problematic conflation of "saltwater" realms with notions of indigeneity. While significant advances within this body of literature have predominantly focused on postcolonial and decolonial aboriginal histories and interpretive framings, the specificity of this term arguably runs the risk of uncritically accepting a sense of "authentically" dwelling with the sea. Its conceptual framing potentially forecloses "non-traditional" sensibilities and practices that entail entire coastal (and marine) lifeworlds in their own right. Second, while seascapes themselves can be theoretically imagined as "a cosmologically totalizing" realm rather like terrestrial desertscapes (Siriwardane, 2015, p. 158), there emerges the tendency of essentializing or "othering" the sea as a world that is entirely detached from land-based sensibilities. As postcolonial geographers such as Connery (2006) posit, the ontological distanciation between land and sea is strongly suggestive of a Eurocentric imaginary. Furthermore, complex land-sea interactions inevitably determine how life is experienced and lived, for as Ingold (2000, p. 167) asserts, everyday perception formations are never passive processes, and are structured against frames of socio-environmental meaning-making. However, the ways in which land-sea distinctions are typified, typologized and taken for granted as objective reality (in a Schützian sense) may remain intensely differentiated. Thus, what is considered to be typically "of the sea" or "of the land" may be separately interpreted and lived, however it is important to bear in mind that since Bronislaw Malinowski's Argonauts (Malinowski, 1922) among others, anthropological writing reveal that the absolute spatio-cognitive separation between watery realms and the terra firma are barely universal (see Anderson and Peters, 2014, p. 8). Another emergent field within interdisciplinary marine research is what could be broadly framed as "interspecies worldings," if we were to more meaningfully reuse the term borrowed from the environmental humanities (see DeLoughrey, 2015). While engaging more productively with broader questions around human hubris, anthropocentricity, and of racialized universality particularly evident within highly politicized debates on the Anthropocene, through which it troubles notions of "both indigeneity as well as interspecies ontologies in an era of sea level rise that is catalyzing new oceanic imaginaries" (p. 352). Having emerged at the crossroads of three interdisciplinary currents constituting environmental studies, animal studies, and Science and Technology Studies, multispecies ethnography (as a predecessor to interspecies theorizing) sought to bring a host of less visible and understudied organisms, from fungi to mollusks and oceanic microbes into anthropological conversations by virtue of acknowledging that they possessed "legibly biographical and politics lives of their own" Kirksey and Helmreich (2010, p. 545). Epistemologically, this conceptualization departed from conventional ways of thinking about the non-human as object, and rather as bodies and substances habiting and co-constituting shared human social worlds. In plainer Schützian terms, such worlds are reversely peopled by more-than-human forms of life and inanimate objects positioned across subjectivities, spatialities and temporalities of worlds that are pre/past, contemporary and future (also see Viveiros de Castro, 2016, pp. 156–157). However, the means with which to draw out this relational ontology without unduly falling into the trap of anthropomorphism has always remained a challenge. The "morethan-human" was conceptually privileged over more deficitcentered "non-human" subjectivities. Second, it strove to explore diverse, multi-stranded and power-laden networks, assemblages and meshworks implicating more-than-human entanglements everyday life, which enlist not only animals, plants, and microorganisms, but also objects, technologies, knowledge forms, minerals, air, water, and energy flows for example. For example, Probyn (2013) in tracing people following fish, stories a complex figuration of how pelagic herrings, anchovies, sardines, local corporates, and Japanese universities co-produce internationally tradable tuna that she termed as a "more-than-human fish," replete with its own individualized historic records that would put a contemporary biometric identity card to shame. While a more comprehensive description of the generative trajectory through which multi/interspecies epistemologies developed goes beyond the scope of this paper, it is worth noting that the earliest scholars (including Donna Haraway, Paul Rabinow, Eduardo Kohn, and Anna Tsing among others) who have written on these relationalities have argued that human nature and living by default encompasses pluri-worlds (see Kirksey and Helmreich, 2010, pp. 549–548). Indeed, as decolonial scholars often posit, post-Enlightenment rationalities and hierarchies privileging mastery over Nature and concomitant narratives of stewardship have in turn historically muted these existential states (see Belcourt, 2015). While the conceptual fault lines between multi- and inter-species ethnography remain blurred, arguably the latter focuses on communicative worlds comprising multiplications of associations shaped through networks, events, circulations and other forms of encounter. Lively vocabularies, particularly in the overlapping disciplines of cultural geography and anthropology follow these interspecies (life)worldings, comprising for example Ingold and Pálsson's (2013) understandings of "biosocial becomings" and Latimer's (2013) notion of "being alongside" as opposed to "being with." Lifeworld-inspired sensibilities also offer nuanced understandings of powerful yet invisible materialities (and their performativities), like in the case of Robertson's (2014) study on island groundwater networks and flows in Kiribati and their multiple enactments. For others like Peters and Steinberg (2015), multi-sensory, corporeal and affective engagements with the sea (for example, salt on skin, the performativity of a recreational beach) calls to attention values of not just "thinking from the sea, but how we can think with the sea" and what this means in widening explorative horizons for understanding multiple modes of marine sense-making. As a start then, it would seem prudent to acknowledge that what these fluid ontologies spell are arguably less visible and cognitively less graspable dimensions such as volume, liminality/mobilities, the unruliness of depth, and of vertical territorializations for example (see Steinberg and Peters, 2015). Yet these multisensory and embodied forms of knowing can be further enriched critically by hybridizing older lifeworld readings for example Merleau-Ponty's being-with-the-world. To take this concept further would mean to use it in prefiguring traditional meanings of spatial and temporal depth. It would also warrant critical reflections on the limits to knowing and feeling, contemplated through what Mazis (2010, p. 123) eloquently puts as "the further displacement of the human into the world's play of becoming." These conceptual and epistemological currents have further crisscrossed with the recent turn toward non-representational ethnography, particularly within the disciplines of human geography, anthropology and sociology (see Thrift, 2008). Not only does it emphasize the tracing of more-than-human relations, but pays attention to the very events, practices, socioand pre-cognitive structures of feelings, mobilities, including the extra-textual and "non-discursive dimensions of spatially and temporally complex lifeworlds" that have otherwise stood concealed by conventional ethnographic styles that have "been in the habit of uncovering meanings and values that apparently await our discovery" (Vannini, 2014, pp. 1–2). In this context, embodied actions and movements themselves speak and enact, rather like the surfed waves that people allude to as watery "places" that conjoin together (Anderson, 2012). At first glance, it may appear that conventional phenomenological-lifeworlds research has little to lend an open-ended and experimental epistemology, particularly one that has little to embellish in terms of drawing forth externalized meanings in order to render any objective explanation. Yet upon closer inspection, the very experientialist spirit that is warranted of immersive lifeworlds research (see Jackson, 2012) beckons what a non-representational ethnographic journey would entail, not in the least self-reflexivity. The experimental becomes the experiential and vice versa, making for a compelling case for critical conversations and border crossings between relational concepts cleaved within contemporary cultural geographies and anthropologies on the one hand, with neo-classical theorizings and operationalizations of the multi-stranded concept of the phenomenological-lifeworld. ## CONCLUSION We argue for a more conscious engagement with the concept and diverse epistemological foundations of lifeworlds (Ger. Lebenswelten) in interdisciplinary coastal and marine research. Our discussion serves as an open invitation for interdisciplinary scholars to more critically reflect the advantages together with the shortcomings of diverse lifeworlds conceptualizations. At the same time, we reiterate the double bind that contemporary phenomenological praxis finds itself in. On the one hand, the philosophical complexity and the diverse epistemological foundations of lifeworlds theorizing make its entry into presentday interdisciplinary research relatively more challenging. On the other hand, the apparent paucity of perceptions-based research on marine-centric/specific knowledges and the experience of everyday life makes for an urgent case for the integration of lifeworlds approaches. Eventually, it is the attempt to free the lifeworlds concept from a singularly land-based lens that makes further research into marine-based phenomenology far more appealing and pressing at the same time. How then, could the endeavor for embarking on lifeworlds research across multiple coastal and marine realms, possibly begin? Epistemologically, this multi-stranded concept opens up reflective spaces through which we, as interdisciplinary researchers, could unpack experiences and meanings around our own positionality. Methodological processes such as bridling offer practical techniques through which to consciously suspend judgment and explore biases and assumptions that are implicit to our own lifeworlds. Through two illustrative case studies on diverse waterworlds, we have shown how surface level perceptions-based research may still run the risk of perpetuating subjective assumptions often taken as constituting "objective" reality. Conceptually, we reveal how the integration of a specific lifeworlds approach within interdisciplinary work warrants active reflection, depending on the research puzzle or question that it seeks to understand. Empirical community-based fieldwork is hardly a process that entails passive encounters between "subjects," sets of data, and their forms of knowledge generation. While it offers little recourse to meta-level analysis, it provides the means to detangle fine-grained nuances across multiple and locally-situated realities that are often regarded as being "messy," encompassing values, norms, worldviews and actions that may often sit in contradiction to each other. While the concept affords the space through which to study the complexities, ambiguities and ambivalences inherent across both land- and seabased societies, it further promises the means through which to sidestep over-simplistic and essentialist inferences to the vague and embattled notion of "culture." Methodologically, while the concept favors the study of an individual's interpretation as the primary unit of analysis, it provides varied empirical layers through which implicit meanings could be drawn to the surface. Abstractions of course are never entirely static nor complete, in similar ways that knowledge(s) and forms of knowing are constantly in flux. Having problematized the endeavor of: (a) "reading the world" through categories of knowing (e.g., beliefs, mental maps), (b) of being and becoming (e.g., identities, material movements, flows); (c) of multiple socialities (e.g., more-than-human assemblages), and; (d) through experience (e.g., events, routinized social practices), the methodological foundations of the lifeworld enable us to work with concretely situated frames that people use to guide as well as to challenge perceptions and behavior. The lifeworld approach presents an empirical frame and an integrative research agenda through which diverse modes of dwelling with, and working the sea could be investigated, transcending a vast body of work related to coastal communities and spaces. Several thematic vantage points stand to be taken as points of departure in enlivening deeper forays into "marine lifeworlds." Rather than merely deliberating on surface-level perceptions, the lifeworld enables us to think #### REFERENCES beyond them. Novel and hybrid approaches to understanding marine epistemologies/forms of knowing would therefore require an ongoing engagement with how varied conceptual strands, methodological devices and thematic foci could be reworked in creative ways in order to consciously unhinge the concept from its terrestrially-bound roots, which at the same time naturalize the nature-cultural binary. Thus, thinking through place-based and materially interpreted realms such as saltwater-worlds and their manifold socialites and interactive entanglements which in turn solicit new ways of thinking, feeling and writing with/alongside oceans and seascapes (i.e., wet/fluid ontologies and interspecies worldings) are but an open-ended starting point. Attempts at integrating and tracing dynamic flows of lifeworld matter, relationships and symbolic meanings and events—from fish and oceanic currents to in/visible material flows and events that are constitutive of everyday life—opens up fertile ground and exciting imaginative possibilities with which to launch an inductively-shaped concept out to sea. ### AUTHOR CONTRIBUTIONS All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication. RS: Conception or design of the work; Manuscript drafting and critical revision of the article. AH: Critical input on the article. #### ACKNOWLEDGMENTS We thank Ryan McAndrews, Edward Jeremy Hind and Jan Maarten Bavinck for their insightful comments on previous drafts of this paper. Postcolonial Approaches, eds E. DeLoughrey, J. Didur, and A. Carrigan (New York, NY; Oxford: Routledge), 352–372. 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 © 2016 Siriwardane-de Zoysa and Hornidge. 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. # Fishers' Perceptions of the Recurrence of Dynamite-Fishing Practices on the Coast of Tanzania Robert E. Katikiro<sup>1</sup> \* and Jairos J. Mahenge<sup>2</sup> <sup>1</sup> Department of Agricultural Economics and Business, College of Agricultural Sciences and Fisheries Technology, University of Dar es Salaam, Dar es Salaam, Tanzania, <sup>2</sup> Dar es Salaam Marine Reserves System (DMRS), Marine Parks and Reserves Unit, Dar es Salaam, Tanzania Dynamite or blast fishing remains the most pervasive destructive fishing method in the coastal waters of Tanzania. It constitutes a major threat to small-scale fisheries through degradation of reefs and other critical habitats for fish. The Tanzanian Government has implemented several measures including a high rate of patrols and surveillance campaigns in the sea to try to deter the use of dynamite in fishing. However, most of these measures have failed to reduce its occurrence over the past years. Little is known on why existing management measures are failing to generate effective solutions to address dynamite and other forms of destructive fishing activities. This study was undertaken to examine perceptions of fishers on the persistent recurrence of dynamite-fishing activities within the present fisheries' management regulations. A random sample of 180 individual fishers was interviewed in two coastal districts using a household survey questionnaire. Key informants, semi-structured interviews and participant observations were used to collect additional data. Almost two thirds of survey respondents felt that an apparent recurrence of dynamite fishing is attributed to the inconsistencies of the fisheries management approaches deployed to thwart the use of dynamite. However, other factors such as absence of appropriate organization of the many involved actors, kinship ties, and migrant fishers played crucial role on the persistence of dynamite fishing. There was a common perception among all survey participants that lack of trust and transparency toward and within anti-dynamite initiatives negatively impacted the involvement of fishers in their efforts to reduce the use of dynamite. An improved situation where decision-making processes are coordinated among fishers, non-governmental, and governmental stakeholders is important to support ongoing management measures, in order to increase legitimacy, and long-term success of efforts to get rid of destructive fishing practices among small-scale fishers in Tanzania. Keywords: dynamite fishing, fishers' perceptions, destructive gears, interviews, coastal tanzania ## INTRODUCTION Humans have been the primary drivers of the changes in the coastal marine environment (Lotze et al., 2006; Crain et al., 2008; Sale et al., 2008; Waycott et al., 2009) and are expected further to exacerbate pressures on coastal ecosystems (Boldt et al., 2014; Jiao et al., 2015; Lucas and Smith, 2016). The increased human pressure on marine ecosystems has caused many environmental #### Edited by: Annette Breckwoldt, Leibniz Center for Tropical Marine Ecology (Leibniz-Gemeinschaft), Germany #### Reviewed by: Jon Olaf Olaussen, Trondheim Business School, Høgskolen i Sør-Trøndelag, Norway Jennifer O'Leary, California Polytechnic State University, USA > \Correspondence: Robert E. Katikiro [email protected] #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 22 April 2016 Accepted: 01 November 2016 Published: 16 November 2016 #### Citation: Katikiro RE and Mahenge JJ (2016) Fishers' Perceptions of the Recurrence of Dynamite-Fishing Practices on the Coast of Tanzania. Front. Mar. Sci. 3:233. doi: 10.3389/fmars.2016.00233 117* problems, particularly the loss of important ecosystem services (Halpern et al., 2012). Several studies (Costello et al., 2010, 2012; Coll et al., 2016) show that fishing presents one of the greatest human pressures on marine ecosystems, and one of its main threats is through destructive fishing techniques (Dayton et al., 1995; Watling and Norse, 1998; Wilkinson, 2004; Mak et al., 2005; Fox and Caldwell, 2006; Benn et al., 2010; Fenner, 2014; Petrossian, 2015). The use of destructive fishing techniques, including explosives and poisons, has been part of many small-scale fisheries for decades (Mak et al., 2005; Palma, 2010; Authman et al., 2013; Petrossian, 2015). The literature (Saila et al., 1993; Mazlan, 2005; Fox and Caldwell, 2006; Glaser et al., 2015) shows that explosive or blast fishing has been and still is occurring in many countries around the world. For example, explosives have been used in Hong Kong waters for at least a century, leading to the introduction of legislation to ban explosives in 1903 (Cornish and McKellar, 1998). A study on the status of destructive fishing activities in the Pacific Islands showed that by the early 1980s, nearly half of the cases on reef degradation were related to damage from explosives and poisons (Veitayaki et al., 1995). In the Western Indian Ocean (WIO) region, these methods, particularly dynamite or blast fishing, are still common, especially on the coast of Tanzania (Samoilys and Kayange, 2008; Wells, 2009; Braulik et al., 2015b; Slade and Kalangahe, 2015). For example, dynamite fishing has been experienced in Tanzania since the 1960s and although it was declared illegal in the 1970s it has continued largely unabated since that time (Guard and Masaiganah, 1997; Wells, 2009; Braulik et al., 2015b). Wagner (2004) asserted that in the 1980s and 1990s, the frequency of dynamite-fishing events along the coast of mainland Tanzania reached a peak. Some extreme cases of dynamite events were also recorded. For example, over 441 blasts were recorded from October to November 1996 in Mtwara, while in Songo Songo Archipelago, 30 blasts were heard every 3 h (Francis et al., 2002). Likewise, 100 blasts were recorded during one 6-h period at Mpori Reef in the same year (Francis et al., 2002). While destructive fishing methods are not a new phenomenon, they have posed seemingly intractable challenges to scholars and policy makers seeking to phase them out (Wells, 2009; Nurdin and Grydehøj, 2014; Heber Dunning, 2015; Petrossian, 2015; Slade and Kalangahe, 2015). The devastating impacts of destructive fishing techniques on marine ecosystems and human populations have received considerable attention in scientific studies (Jennings and Lock, 1996; McManus et al., 1997; Riegl and Luke, 1999), and the effects of dynamite fishing have also been investigated in detail (Saila et al., 1993; Guard and Masaiganah, 1997; Pet-Soede and Erdmann, 1998). Dynamite explosions are known to leave coral reefs in rubble and kill more fish than are harvested (Guard and Masaiganah, 1997; Mak et al., 2005; Fox and Caldwell, 2006). They are also potentially dangerous to the people who use them (Guard and Masaiganah, 1997). However, a lack of capacity to enforce fishing legislation, especially in the Pacific, Southeast Asia, and sub-Saharan Africa, coupled with the quest for high catch rates from dwindling fish stocks have resulted in an extreme increase of these methods in areas dominated largely by small-scale fishers (Kuperan and Sutinen, 1998; Sumaila et al., 2006). Different coastal states have already designed strategies and developed measures to curb increasing threats to marine fisheries from destructive fishing practices (Agnew et al., 2009; Munyi, 2009; Wells, 2009; FAO and UNEP, 2010). However, destructive fishing remains the stark reality of fishing activities in some regions, particularly in the above-mentioned regions (Mangi and Roberts, 2006; Braulik et al., 2015b; Giraldes et al., 2015; Sheppard, 2016). For example, the initiative by the Beach Management Units supported by the World Wide Fund for Nature (WWF), monitoring blast frequency at Songo Songo, located in the Lindi region in Tanzania, for 15 months up to late October 2014, recorded 8765 blasts, an average of 21 per day (Liganga, 2015). A large-scale vessel-based survey conducted over 2692 km of Tanzania's coast in 31 days recorded 318 blasts, where the highest intensity area for blasting was in the vicinity of Dar es Salaam City (Braulik et al., 2015b). However, Tanzania had previously reported an effective control of dynamite fishing in the late 1990s (Darwall and Guard, 2000). This happened when a massive crackdown on dynamite fishing was achieved under the joint operation (dubbed operation "pono") between the Tanzanian Navy and Marine Police with support from the local community members who had been sensitized about and mobilized to take actions against dynamite fishing. Nonetheless, numerous WIO nations have virtually no dynamite fishing (Braulik et al., 2015a,b). Despite many advanced efforts to phase down dynamite fishing, including the success registered during the Tanzania's 1990s anti-dynamite campaign, these practices persist along much of the Tanzanian coast, including Tanga, Pangani, Bagamoyo, Temeke, Mtwara, Rufiji, and Kilwa (SeaSense, 2010; Anderson, 2011; Slade and Kalangahe, 2014). Critics argue that Tanzanian government authorities are overlooking the fact that fishers have an important role and should be at the center of actions targeting a halt of dynamite fishing. Arguably, the active role and potential capability of fishers to stop dynamite fishing remains largely unutilized and their actions have not been scaled up by fishing authorities at local governments to meet targets to halt destructive fishing. More recently, there has been some backlash against anti-dynamite campaigns (Slade, 2011; Slade and Kalangahe, 2015), where even the strongest support to fight destructive fishing activities, offered by fishing communities as symbol of their cultural value to fisheries resources, have not prevented the use of dynamite. This has eventually resulted in a fisheries management stalemate, making the use of dynamite virtually impossible to alleviate (Braulik et al., 2015b). Government reports indicate that the most acute illegal fishing issue in Tanzania is dynamite fishing (United Republic of Tanzania (URT), 2016). A recent government report tabled in the parliament during the 2016/17 budget session shows that 35 detonators, 17 explosives, and 252 kg of urea—used for home-made blasts—were confiscated during the 2015 reporting period. Anecdotal evidence suggests that dynamite incidences in Tanzania are now probably more prevalent than they have ever been. Halting dynamite will require more comprehensive information, based on the perceptions and roles of fishers in these widespread dynamite-fishing activities, and support from government and non-governmental stakeholders for the fishing community to enhance their alternative livelihood strategies. This paper therefore attempts to investigate the perceptions of fishers on the recurrence of dynamite-fishing activities within the present fisheries legislation and regulations that call for fishers to collaborate in fishery management. Management of fisheries in mainland Tanzania is guided by the Fisheries Act of 2003 and is supported by the Fisheries Regulations of 2009 (Fisheries Division, 2014). The research was guided by the following questions: why do fishers still resort to dynamite? Are there any internal mechanisms coastal communities might employ to discourage the resumption of dynamite fishing? Furthermore, this study aimed to unravel what social interactions (e.g., cooperation among fishers, reciprocity, and trust), if any, can be used to persuade communities whose livelihood takes place in an environment of dynamite activities, to shoulder efforts with government and other fisheries stakeholders such as community-based fisheries organizations and local Non-Governmental Organisations (NGOs) to halt these activities. A successful strategy to understand perceptions of fishers on dynamite-fishing is critical for processes dedicated to reduce it substantially. Fishers perceptions should then be fed into projects and initiatives that seek to reduce destructive fishing practices (Foster and Vincent, 2010; Heyman and Granados-Dieseldorff, 2012). Understanding perceptions of fishers is crucial for local efforts to reduce, and optimally, prevent degradation of fisheries resources, and thus promote conservation efforts (Bacalso et al., 2013; Katikiro, 2014a). ## MATERIALS AND METHODS #### Study Sites Two coastal districts in Tanzania (Mtwara and Temeke) were chosen as case study sites for this research (Figure 1). Temeke district (municipality) was at the time of this research one of the three administrative districts within the Dar es Salaam region. Dar es Salaam is Tanzania's largest city and the most important one for both commercial and governmental activities. However, in 2015, two other districts were established within the Dar es Salaam region. One of this is Kigamboni, which covers part of the area previously under the jurisdiction of Temeke district. Temeke has the largest coastal stretch compared to Kinondoni and Ilala—two other districts that previously made up the Dar es Salaam region (National Bureau of Statistics (NBS), 2014). The 2012 population and housing census shows that Temeke district has 1,368,881 inhabitants, accounting for about 31% of Dar es Salaam's population United Republic of Tanzania (URT) (2013). Temeke's socio-economic profile indicates that there are 1450 registered fishers and about 2000 that are not registered (Temeke Municipal Council, 2010) Mtwara district is located on the southernmost region of Tanzania. Based on the population census of 2012, Mtwara district had a population of 336,302 people. The fishery in the district is quite extensive with over 4500 fishers (Everett et al., 2014). Its remoteness and proximate location to the northern side of Mozambique enable easy access to dynamite perpetrators and reduce effective control of destructive fishing activities attributed to the absence of effective border control of fishing activities. The majority of households in these two districts depend on fishing, crop farming, and small-scale business for their livelihoods (Market Axis, 2014; NBS RC's Office Dar es Salaam, 2014; Katikiro et al., 2015). The study districts were chosen for representing the ones possibly more directly affected by dynamite incidences in both rural and urban areas, which reflects retardation in actions to thwart destructive fishing activities. However, a recent study by Braulik et al. (2015b) using a combination of manual and semi-autonomous detection, which recorded a total of 318 blasts between March and early April 2015, revealed that 70% of the blasts came from Temeke district. Mtwara contains a substantial high number of recurrences of dynamiting events where in some areas more than two blasts per hour were heard (day and night). Both study districts have at least community-driven initiatives that patrol against illegal fishing and at that operate a database for collecting information on dynamite incidences. For example, SeaSense—a NGO that targets the conservation of flagship species such as sea turtles and marine mammals with support from local communities—recorded 1120 dynamite blasts in 2008 in Temeke district and some other parts of Dar es Salaam (SeaSense, 2012). Mtwara benefited from the NGO SHIRIKISHO from the late 1990s to the early 2000s where it led massive anti-dynamite campaigns (Katikiro, 2014b). The site selection was also based on: the predominance of migrant fishers who are often associated with destructive fishing methods, the significant large number of fish traders, which enables ready marketing for fishery products, and having a significant number of villages whose people have strong economic and cultural relations with marine fisheries. It was also assumed based on previous work on fisheries management in the area (Katikiro, pers. observation) that individuals taking part in this study would be aware of effects of dynamite fishing on fish stocks, the environment and human health. District fisheries officials assisted in the selection of study sites. Three villages were selected randomly in every district, making six villages altogether. The characteristics of each area to fit in the study was verified by a research team upon arrival in the district. This was made by crosschecking if the village had at least two of the criteria mentioned above. The population and sample sizes for the individual fishers surveyed are provided in Table 1. ### Data Collection Two months were spent in each district to collect data using a mixed-method approach with triangulation and the use of secondary sources including gray literature, reports, published materials and institutional documents on local fisheries and conservation activities. An overview of the methods is provided in Table 2. Semi-structured interviews were held with a purposive, snowballed sample of 24 community members of the six study villages. Participants for semi-structured interviews were selected because they were active in or associated with the fishery, were information-rich on challenges facing fisheries in their villages and would provide different viewpoints. This could either be TABLE 1 \| Villages and sample of individual fishers interviewed during household surveys. Source: Ward Executive Offices (2015). directly as a fisher (illegal or legal), fish processor, fish trader, or indirectly as member of a fishing household, key stakeholders such as NGOs focusing on fisheries issues, or those working as food vendors, net repairers and other fisheries-related jobs. A summary of the topics explored during these semi-structured interviews is shown in Table 3. Semi-structured interviews aimed at the perspective of local institutions such as village and traditional leaders, kinship relations and fishers' organizations on how the problem and effects of dynamite fishing are framed in their understanding of improving fisheries management. The interviews took up #### TABLE 2 \| Data collection methods used in this study. to 50 min each and were all held in Swahili. Questions were open and phrased to avoid the responses being prompted by the interviewer. The interviewer took notes for each semi-structured interview. No recording was done because of the sensitive nature of the topic and to guard the privacy of interviewees. A questionnaire survey among local fishing households was then conducted through face-to-face interviews with a random sample of 180 individual fishers. Only one individual fisher, in most cases the head of household, was interviewed (see Table 1). Surveys are frequently used to examine perceptions and attitudes (Blair et al., 2013). For this study, the survey was designed to provide quantitative information on dynamitefishing activities and fishers' perceptions of its reoccurrence. The structured survey questionnaires (Annex 1) comprised questions on specific details of the surveyed households, such #### TABLE 3 \| Summary of the main topic questions addressed in semi-structured interviews. as experience with destructive fishing methods; fishing patterns (e.g., seasons, operational details, species abundance, compliance to regulations, etc.); environmental and health risks of using dynamite; sources of dynamite; proportion of fishers who use dynamite; initiatives and NGOs in place to "combat" dynamite use; fishing license and knowledge on change in fishing activities. The survey also covered themes related to characteristics of dynamite fishers, attitudes of fishers toward dynamite, perceived negative impacts of dynamite fishing on their livelihoods and direct environment, and the level of consultation and involvement of local fishers in decision-making processes on dynamite and fishing in general. Survey questions were asked in Swahili. The questions were kept short and mainly closed with fixed alternative answers (mainly yes and no). Scale questions were also asked to allow respondents to agree or disagree with particular statements. In most cases, a five-point Likert scale was used for scale questions. In addition, 21 key informants were selected to provide a broad representation of environmental organizations, fish traders, government officials engaged in fisheries management and law enforcement, and local business leaders, particularly from the tourism and hotel sectors. Key informants were interviewed using open-ended questions. The interviews focused on their perceptions on the health of fisheries resources, the occurrence of dynamite blasts, the existing regulations and enforcement measures, recent reforms in the fisheries sector, village committees to monitor and control destructive fishing, and the future condition and direction of the fishery if dynamite use remains active. Key informants were chosen utilizing both opportunistic and snow-balling techniques. Interviews ranged from 30 to 70 min. Each interview was subsequently transcribed and analyzed for key words and statements. Because dynamite fishing is illegal and perpetrators will not want to be known to outsiders, precautions were taken to ensure that interviewees would not be influenced by wariness of and discomfort about being interviewed. This was done through protecting their identity and conducting interviews in a conversational and rather informal manner. Throughout the fieldwork, participant observation was undertaken in all six villages to make additional observations that capture the complexity of destructive fishing activities and validate the interviews and survey data during the analysis. Detailed field notes and photos were taken (where the situation allowed) to record observations and were used for triangulating interview data. ## Positionalities of the Researchers The first author was formerly employed by the Marine Parks and Reserves Unit (MPRU). The second author is still employed by the MPRU. The MPRU is a government institution established under the Marine Parks and Reserves Act 29 of 1994, and its roles among others is to oversee the management of marine protected areas in mainland Tanzania. Part of the study areas were within the marine protected area jurisdiction (Mnazi Bay-Ruvuma Estuary Marine Park located in the southernmost coast of Tanzania) while the other one was not. Based on the authors' professional duties, they may already have been known in the study areas. This relationship enabled networking and knowledge exchange, but clearly also created a by-product, i.e., to be considered as a "government marine conservation official." In the beginning of the research for this study, the authors were not sure if this connection would favor them or not. The authors tried to ensure that participants did not feel as mere research subjects. Obviously, some of the local community members still perceived the authors as "government officials" and not academic researchers, hence their radical criticisms about dynamite fishing, management strategies and the government's role in these. To address the issue of positionality explicitly, the authors were open about the limits of their research in effecting changes in the lives of individuals who agreed to be interviewed. They were also open in answering any questions people had about their research and clearly stated that they were trying to understand the persisting dynamite fishing activities from the participants' perspective. Given the fact that respondents for this study were drawn from local community members, government and NGOs, the relationships of power encountered were significantly different. In the interview context, it often became clear that the authors were expected to be the power holders because they were the ones asking the questions. However, the authors attempted to traverse the landscape of power relations during interviews by attempting to create momentary spaces where their positionalities and those of respondents complemented each other. The time spent in each study site also helped to build trust as the interviews were not conducted in the form of one-off encounters which often make people more suspicious. Undoubtedly, some respondents remained suspicious to this research work, but the authors tried to strike the balance by building mutual trust and rapport where they continued to present themselves as impartial, seeking information related to fisheries and livelihoods for academic purposes. #### Data Analysis Data from the household surveys were analyzed using descriptive statistics. After data were entered and confirmed in a spreadsheet, information was processed with the Statistical Package for the Social Sciences (SPSS 20). Qualitative information and extensive field notes were coded inductively, and themes and categories were identified accordingly (Grbich, 2007). Further analyses were then performed using content analysis (Patton, 2002). The data generated with all interview methods and secondary sources were then categorized and used in a complementary way for the presentation of the results. ## RESULTS #### Socio-Economic and Demographic Profile of Household Survey Respondents Table 4 presents the basic socio-demographic characteristics of the questionnaire survey sample. Most households surveyed had at least one person from their household employed full-time in fishing and were thus receiving over 50% of their income from fisheries. Most households surveyed showed negative perceptions regarding the government support for their fisheriesdependent livelihoods by reiterating that government support for fishing activities was not enough. Furthermore, most household interviewees stated that local fishing interests such as continued fishing in areas that are set aside as no-take areas, or their preference for certain gear types, were not being addressed by the current fisheries' management regimes, resulting in conflicts between fishers and government agencies responsible for fisheries. \The focus was to interview the household head; in their absence any other person aged above 18 was interviewed. ## Perceptions on Government-Led Enforcement Campaigns Questionnaire survey responses indicated that fishers and local people who are not engaging in fishing alike are knowledgeable about the importance of patrols in the sea to stop dynamite activities. Enforcement campaigns were identified by 68% of survey respondents as a contributory agent to ensure protection of fisheries resources. The majority of survey respondents (72%) indicated that although enforcement campaigns, which happen sporadic depending on availability of funds and human resources, provided useful practical means to deter dynamite activities, they do not offer incentives for a de-dynamite pathway. Some of the incentives referred to included modern fishing gears such as large nets, cooling devices for storing fish and engine powered boats. More than half of the survey respondents (55%) believed that preparations and eventually implementation of patrols at some instances led to dynamite suspects preparing strategies to avoid being arrested. Participant observation during fieldwork and further inquiry with key informants confirmed that often not every dynamite perpetrator encountered by patrols was arrested. Many survey respondents (47%) indicated that the government agenda to stop dynamite fishing is unclear and that little attention is paid to this problem at any given time as compared with other aspects related to the misuse and degradation of natural resources (such as illegal logging and poaching of wild animals). Almost 27% of survey respondents argued that dynamite practices were rarely resolved with law enforcement campaigns because of absence of targeted actions and ill-equipped patrolling protocols, in combination with a lack of dedicated efforts to eradicate the networks of dynamite suppliers. Instead, most dynamite perpetrators arrested by law-enforcing agents would usually end up in a situation where the suspects would retaliate by insulting or trying to harm informers. Further, it was mentioned by these respondents that the suspects may relocate into areas where enforcement is not yet existing. The majority of survey respondents (84%) cited lack of seriousness by responsible government agencies, incompatible models of resource management, bribery and unwillingness to enforce law, as factors for continued dynamite-fishing activities in their areas. When asked why the implemented enforcement measures were incapable of resolving the dynamite problem despite a high rate of patrols and surveillance campaigns, 82% of key informants argued that some of these strategies were flawed and corrupted because of different perspectives and weights given to the problem by people carrying out those campaigns. This in turn leads to leakages of information eventually reaching the potential offenders. Eventually, the potential culprits temporarily refrain from dynamite fishing or relocate to other fishing grounds during the campaigns. This was also confirmed by participant observation where the authors observed potential dynamite fishers postponing their fishing schedules after being tipped off on the forthcoming patrol missions. Over 70% of key informants remarked that enforcement campaigns also fail to succeed because of political interests, dictating what should be done. At times when election polling is nearing, they were asked to disengage their campaigning duties and respond to immediate pressures of politicians. But once they reduce their enforcement efforts, it becomes almost impossible to either operate campaigns or stop the spread of dynamite fishing. In the end, as explained by nearly 50% of key informants, halting dynamite through enforcement seems to be ineffective because the socio-political environment cannot credibly support such efforts. Despite these failures, a good proportion of survey respondents (48%) believed that enforcement activities usually end at least with some type of explicit reduction of dynamite supply and other illegal fishing activities. ## Community Awareness and Initiatives to Curb Dynamite Fishing Over 70% of survey respondents who began fishing longer than the past 20 years stated that dynamite was already used when they began their fishing career. The vast majority of survey respondents (90%), and almost all participants in the semistructured interviews, agreed having noticed a series of locally driven sensitisation and awareness-raising activities spearheaded by village leaders that aimed to reduce dynamite fishing over the last 10–20 years. However, only 18% of survey respondents reported having received support, information and training to stop dynamite (and other destructive) fishing activities. Almost all key informants agreed that all fishing activities should be non-destructive. When asked why they thought dynamite is reoccurring when every fisher was aware of its effects, these key informants said that it was only another malpractice in the society (like cattle robbery), which requires a case-by-case solution and should not be generalized to all fishers. Further probing of community awareness on the dynamite problem led to the responses summarized in Table 5. Information gathered from government and NGO reports, triangulated with data generated from participant observations, showed that significant efforts have been undertaken to inform communities about the effects of dynamite not only in the study sites but along the coastal districts of Tanzania. These included sponsored radio programmes to raise awareness among the local communities on the ecological and human health impacts of dynamite fishing, participatory videos (Slade, 2011) and dissemination of awareness materials. Awareness and capacity-building programmes, however, were cited by almost 45% of survey respondents to be confined to areas where the pioneer NGOs on anti-dynamite campaigns were based, and were thought to underrepresent opinions of fishers. These activities were reported to be more in Temeke than Mtwara because Temeke enjoyed relatively high coverage by the SeaSense organization. This organization however, as commented by key informants, has been somewhat dormant in recent years following lack of funding and aging of some of its founding members. The study revealed that local people are willing to report events of dynamite fishing, but credible and trustworthy government officials where they could report to are not organized to ensure a timely flow of information and subsequent actions. Several respondents (32%) of the semi-structured interviews TABLE 5 \| Responses (n = 180) to the yes/no questions on awareness of the dynamite fishing problem during the household survey. Several survey statements were asked to probe the respondents' view of dynamite fishing activities. All respondents were required to answer each statement as: 1, strongly agree; 2, somewhat agree; 3, undecided/unsure; 4, somewhat disagree; 5, strongly disagree. The answers across the five categories are provided in Table 6. observed that it was extremely difficult for local fishers to locate these agents. Furthermore, it emerged during household survey interviews that people who had voluntarily taken the task to monitor dynamite perpetrators and report them to the responsible authorities sometimes faced serious threats by the dynamite fishers. In some instances, dynamite detonators were placed at house compounds to intimidate the volunteers, especially in villages of Mtwara district, and there were reports of injuries after acid was splashed on their faces. Interview results indicated that while many fishers were aware of the effects of dynamite use, they find it difficult to report plans or people engaged in it because they feel they were not protected against these. Indeed, perpetrators could be close relatives, and in certain circumstances perpetrators could be linked to influential people in their communities who cannot easily be punished or fined through the current legal mechanisms and institutions. ## New Entrants into the Fishery and Actions against Dynamite Fishing While some projects by the government and NGOs were mentioned to have existed in the study sites—Temeke having more active organizations than Mtwara—over 55% of key informants and 70% of participants in the semi-structured interviews indicated that these projects often overlooked the component of empowering fishers for actions against destructive fishing. Almost all survey respondents (95%) considered that new groups joining fishing activities were not adequately introduced to options of less-destructive fishing. They suggested that these groups of individuals, which usually lack exposure to fishing or have little knowledge and experience in fishing, should receive adequate information about different gears, and the issuing of #### TABLE 6 \| Distribution of responses (n = 180) across the range of the five answer categories in a Likert scale. fishing licenses should also be done in a more meaningful and transparent way than is currently done. To date, anyone can gain access to fishing through the existing licensing system by paying around US \$10 annually. Respondents claimed that better inclusion of new entrants into fisheries management activities is crucial to accentuate stewardship toward the use of less harmful fishing gear. Most household survey respondents (80%) stated that because of lack of skills and adequate knowledge on fishing, new entrants are often persuaded easily to adopt destructive techniques in fishing as a way to meet their immediate income needs (Figure 2). ## Government Approaches to Reduce Dynamite Fishing Statements of nearly 40% of key informants indicated that local government authorities in the study districts did not have clear and consistent plans to protect fish stocks from dynamite activities. Most of these informants believed that poor progress in combating dynamite is partly attributed to lack of commitment by the relevant authorities where fisheries is given less priority compared to other sectors. However, nearly 70% of survey respondents clearly stated that they would value positively measures that allow fishers to share their knowledge and their greater involvement in decision making to curb dynamite Many key informants (48%) mentioned that the dynamite problem is often approached in a fragmented manner, with the population and the environment suffering in favor of economic and political considerations. Information from household surveys also showed that local authorities have often stigmatized fishers in general as criminals. The views of almost 60% of key informants made it clear that as long as fishers feel criminalized and left behind in management and development plans, any efforts to persuade them to support a reduction of dynamite fishing are likely to encounter significant opposition and little commitment from community members. Fifty six percent of survey respondents said that this is sometimes caused by unreliable information on whether certain individual fishers or fishing villages are producing or stockpiling materials used for dynamite fishing. To their knowledge, some of the information is baseless and diverting the truth from actual dynamite activities. According to the key informants, false and misleading information therefore often leads to reduced commitments to act against dynamite. #### Improving Actions against Dynamite Fishing Almost all participants from all interview methods used in this study, including informal conversations in the study sites, agreed that many fishers were willing to participate or invest in actions and initiatives that would result in the reduction of dynamite activities. They gave various recommendations for improving actions already undertaken, and designing of new activities to support anti-dynamite initiatives (Table 7). While the majority of survey respondents (85%) supported the idea of deploying the Tanzanian Navy to address the dynamite problem; it was, however, noted that this cannot be the final solution but just a quick remedy to the dynamite-fishing dilemma. According to the key informants, military actions as seen during the operation pono in 1998–1999, could positively address the dynamite-fishing crisis. However, lack of a clear long-term policy on how to integrate the military in antidynamite operations did reduce its mission. Arguably, as opined by some key informants, the inefficiency of deploying the military was demonstrated by continued dynamite activities when the operations were concluded, or by complaints about incidents of human-rights violations, as was the case in other natural resource cases like the operation Tokomeza Ujangili of 2013. Tokomeza Ujangili was a planned nationwide operation to combat poaching (Legal and Human Rights Centre (LHRC), 2015). It was ostensibly aimed at poachers but was terminated following widespread charges of human-rights abuses against local community members. It was observed during the fieldwork for this study that without efficient government support, the village environment management committees in the study sites have not been successful in stopping the use of dynamite and other destructive fishing activities. Over 50% of the key informants argued that despite being chosen among local community members, these committees seem to have no impact on fishers' decision to continue fishing with dynamite and violate the fishing regulations. There were also complaints among local communities that these committees have been part of the problem by allegedly being involved in assisting members of their families or clans and in-migrant fishers to circumvent enforcement of fisheries regulations. At the same time, while both fishers and members of village environment management committees agree that dynamite fishing has a profound impact, including the likelihood to harm the fishers, their perceptions on the destructive effects on the environment remain vague. Almost 60% of key informants said that planning for a national goal for reducing dynamite activities, i.e., hoping to cut the use of dynamite to a certain degree within a particular timeframe, would increase the effectiveness of antidynamite campaigns. They also mentioned that a significant loophole including absence of provisions in the current fisheries legislation that could impose heavy and deterrent penalties to suspects should be addressed, which allows suspected offenders to legally purchase explosives. Overall, 22% of semistructured interview respondents emphasized that there should be a background check on actual intention, occupation and criminal records before one is legally able to purchase explosives and detonation materials, which are commonly known to be destined for the mining sector. Furthermore, around 54% of the key informants and 20% of survey respondents mentioned that there have been various initiatives such as educational programmes and fishing grants for purchases of fishing equipment in place to combat dynamite use over the past decade, and new initiatives (both community-based and governmentdriven) could benefit from their lessons. The establishment of institutional arrangements needed to co-ordinate fishing activities and interactions between fishers and fisheries officials, and mainstream legal fishing activities, were also mentioned during the key informant interviews, especially by those who had knowledge of the recent government initiative for the formulation of a Multi-Agency Task Team (MATT). The MATT initiative was launched in 2015 by the Tanzanian Government to help find a lasting and effective solution to the widespread incidents of environmental crimes, including dynamite fishing. It was further said that such intervention efforts need to be focused to undermine and outsmart any efforts by dynamite fishers. For some key informants, the high level of interest demonstrated by a number of NGOs especially WWF in Tanzania should be integrated into government initiatives and actions. ## DISCUSSION The integration of fishers' perceptions may enhance their acceptance of and positive reactions toward curbing destructive fishing activities. The most noted barrier for fishers and local communities at large was the perceived low level of attention paid to their values, beliefs and livelihood challenges by policymakers and government authorities responsible for fisheries. The lack of trust and transparency toward and within anti-dynamite initiatives also seems to be a major factor to thwart destructive fishing. Participants therefore perceived that their daily needs were widely ignored, which contributed to reduced commitment to address the dynamite problem. Although there are cases from elsewhere in the world where fishers were reported to be active in anti-dynamite programmes (Murshed-e-Jahan et al., 2009; Heber Dunning, 2015), the findings of this study show that Tanzania still has a long way to go in this respect. The household survey respondents stated that if government agencies would accord opportunities, such as fishers having key leading roles in anti-dynamite activities and sufficient participation of local people during the design and implementation of anti-dynamite programmes, their own capacity and confidence to act against the offenders would increase significantly. Basing plans and actions on opinions of all fishers irrespective of their methods of fishing would mean that the majority of fishers would no longer feel carved out in core issues of their livelihood (Hauzer et al., 2013). Doing so could lead to more participatory formulations of antidynamite strategies, with well targeted actions such as reducing supply of dynamite for addressing destructive fishing activities. This however may not be a panacea to destructive activities when there is not much transparency and trust in decision-making processes. While the challenges facing small-scale fisheries in Tanzania cannot be overemphasized, anecdotal evidence suggests that some fishers use illegal and destructive fishing methods to secure control over resources. Despite the fact that initiatives by NGOs and government departments (to phase out dynamite use) have already provided some notable results (Slade and Kalangahe, 2015; United Republic of Tanzania (URT), 2016), there is little evidence to date that this has positively influenced attitudes and perceptions toward reducing dynamite practices by either minimizing frequency or occurrence of blasts. The majority of the household survey respondents opposed these initiatives, explaining that they were not consistent with the reality of core problems of their livelihoods and seemed to divide the community instead of prompting solutions in any perceptible way. This is a context-based concern and reduces the long-term options to act against dynamite and other destructive fishing techniques. The greatest opportunity to address the local needs is to give greater recognition to and actively seek to improve fishing-based livelihoods. This may involve development of sustainable schemes that provide multiple benefits to local fishers. Typical of these include conservation programmes in Bangladesh offering hilsa fishers economic incentives (Islam et al., 2016) and compensations to fishers impacted by marine renewable energy projects in the UK and Ireland (Reilly et al., 2016). The recurrences of dynamite blasts in many coastal areas suggest that measures already undertaken to combat destructive fishing activities in coastal waters of Tanzania have proven less successful (Sjöstedt and Sundström, 2015). While such programmes are often implemented jointly by various stakeholders, they still seem to lack detailed plans on how to integrate local community members and consider their livelihood needs. Additional steps need to be taken to deepen the co-operation between various stakeholders in combating this notorious way of catching fish. For example, Tanzania has since early 2015 started to develop a national task force and strategy that would guide activities, especially prohibition campaigns, against dynamite fishing. Although compliance and surveillance strategies are exclusively a government-led activity and not specifically aimed at putting fishers on eye-level to government officials, implementing anti-dynamite activities with fishers taken on board could help fishers feel the legitimacy of the processes and support it. While there would appear hierarchical differences because of power asymmetries between fishers and government authorities, a substantial number of key informants in this study noted that this would break down barriers that had previously prevented appropriate communication between groups with different interests. This would be in line, for example, with the case in Papua New Guinea (Rochers and Ame, 2005), where lack of credible and trustworthy communication channels between managers and fishers was the most often mentioned potential barrier to the reduction of illegal fishing activities. The uncertainty about effective communication pathways provides a reason for some individuals or groups of fishers to minimize their support for initiatives toward anti-dynamite cooperation and will have to be further investigated by future research. The strong perceptions of lacking consultation and participatory involvement in decision-making on dynamite fishing are not only relevant to Tanzanian fishers. A growing literature reported complaints by fishers (and those working in fisheries) about their lack of participation in management processes (Mikalsen and Jentoft, 2001, 2003; Symes and Phillipson, 2009; Trimble and Berkes, 2013; Holm et al., 2015). But the reasons behind this perceived or real lack of participation are always critical and challenging aspects. In the current study, it remained unclear how participation of fishers could in fact influence decision-making for sustainable fisheries, including the design of community-managed fishing areas. Participation is anticipated to include the ultimate users of resources as active participants in decision making and certainly allow their needs to be accommodated. Pita et al. (2010) elaborated that fishers in Scotland feel that the nature of their participation does not allow them to have a strong voice and stake on matters contested. Their study also suggested that presence of many representatives of fisheries management institutions lead to low and passive participation of fishers in the implementation of management actions. As a result, fishers were found to have a small role, which is consistent with what de Vivero et al. (2008) defined as the participation paradox. Fishers found themselves losing prominence and importance, fading into the spectrum of interests that compete with their own (Pomeroy et al., 2001; Wilson et al., 2003; Yandle, 2003; Gray and Hatchard, 2008). When the processes were open to a broader group of stakeholders, exchange of information and the management process could gradually become more open and transparent (Mikalsen and Jentoft, 2003, 2008; Trimble and Berkes, 2013). When many groups are involved, fishers may feel less stigmatized and the chances to consider their concerns may increase. Official approaches to tackle dynamite fishing should avoid direct accusations of fishers as "the sole perpetrators." Blaming, judging and eventually criminalizing fishers about reoccurring destructive fishing activities not only violates their basic rights and contributes to ignoring their voice, but also comes with severe social dynamic consequences. Norton (2015) found that for South Africa, the highly restrictive laws do not decrease incidents of degradation of natural resources without addressing the reasons that have created poor conditions for fishing communities in the first place. Instead, they lead to higher rates of illegal fishing activities (Norton, 2015). This may result in further consequences, including the relocation of destructive practices to other areas. For example, many survey respondents during this study said that some fishers who were unable to access the fish stocks because of poor fishing gears have been arrested for trying to purchase materials used for preparing dynamite bombs. Criminalizing fishers without first addressing their livelihood concerns is likely to have a disproportionate impact on more vulnerable groups of fishers, like those without efficient fishing gear, appropriate skills and capital. This study also highlighted the existence of low priority and coverage of the dynamite crisis in the Tanzanian media. Until recently, the manner of how NGOs and communitybased initiatives prioritized the anti-dynamite agenda remained vague. There are few cases of conservation volunteers, both local and foreign, who could act as champions to instill people transiting to legal and less destructive fishing practices. A lack of political will and strong socio-cultural patterns such as kinship and family relations in coastal areas seem to have influenced a high tolerance of destructive fishing activities and promoted low national attention. When the capacity of local institutions and actors on anti-dynamite campaigns could be improved by working closely with fishers, in an open and transparent way (Pet-Soede and Erdmann, 1998; Kokorsch et al., 2015), measures and strategies deployed to reduce dynamite fishing could be viewed as more legitimate. So far, most of these measures and strategies were viewed by fishers as illegitimate, and even village governments may seem to give a go ahead for dynamite fishing in their areas as opposition to what they perceive to threaten their access to resources. While some regions around the world have similar problems with compliance to fishing regulations like Tanzania, they are strictly enforced in other countries of the WIO region such as in Mozambique and Kenya. Despite the positive support some fishers in Tanzania have shown toward the implementation of fishing regulations, already designed measures to reduce the use of dynamite are not easy to implement and enforce on a long-term basis. Sometimes, corruption issues become vivid, especially when untrustworthy officials side with offenders and get away with destructive practices. The current fisheries legislation does not explicitly address dynamite issues, resulting in a lack of penalties attached to destructive actions. While a review of the current fisheries legislation is still underway, the process will also need to take in measures that would limit the supply of dynamite materials. Ideally, anti-dynamite campaigns, especially patrols and surveillance, should serve as a tool to identify areas of improvement to make sure that every fishing household has the opportunity to benefit from fish stocks and to identify persistent barriers for enhancement of livelihoods. But too often, as was the case in this study, they do not, and fishers have been dissatisfied with the current approach to curb not only dynamite but also other destructive fishing techniques. Given the extent to which the coastal marine environment is facing degradation from dynamite practices, especially in peri-urban fishing areas, the findings of this study show a need to address a critical set of fisheries' management issues. Despite the lack of data for site-specific management decisions to halt dynamite use, this article has pointed out some general interactions between fishers and fisheries officials that need to be improved through partnership between different fishers and the government. Those interactions combine with the good governance which takes in crucial issues for both parties to determine relevant information that could help curb dynamite use. Future studies should explore how transition to legal fishing techniques could deter dynamite-fishing activities at the local level and how they could be favored by individual fishers whose livelihoods so far depended directly or indirectly on dynamite fishing. ## CONCLUSION While Tanzania boasts of various initiatives already in place to combat dynamite fishing, the lack of a nationally unified fisheries management approach and of institutional arrangements needed to co-ordinate and mainstream legal fishing activities, contribute to the low success of these initiatives. Fishers interviewed generally felt ignored by fishing authorities, criminalized as employing destructive fishing methods, while they were given little chance to express their opinions, views and involvement in the use of dynamite. Fishers need to participate in meaningful ways for actions against dynamite use to be effective. Despite the fact that different fishers in the study sites were not completely certain of the potential of top-down measures such as patrols and surveillance campaigns, an overall negative attitude toward these initiatives prevails. This is one of the many barriers toward the success of limiting dynamite use over time. Consideration of fishers' perceptions and their heterogeneous behavior are prerequisites for the development of strategies to legitimize actions against dynamite use and other destructive fishing techniques, and will increase responsibilities and accountability of fishers at individual levels. If destructive and non-destructive fishers a like participate in the management process, there could be potential to change their destructive fishing behavior rather than feeling ignored by policy makers. There is not much scope for local fishers to play a crucial role in the success of management measures in situations where different stakeholders often manifest clearly divergent values and interests, as in the case of dynamite, where the "greedy" are likely to benefit the most. ## AUTHOR CONTRIBUTION RK conceived and designed the study. RK and JM conducted interviews for data collection. JM contributed secondary data. RK analyzed the data and wrote the paper. ## FUNDING This work was completed with financial support to the first author from Evangelisches Studienwerk e.V. Villigst (grant number 850661). ## ACKNOWLEDGMENTS We are grateful for the valuable contributions of the individuals and organizations we interviewed and for their participation in this study. We also thank fisheries officials and village government leaders in the study sites for supporting this research. We would like to thank the two reviewers and the editor for useful comments. ## SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2016.00233/full#supplementary-material ## REFERENCES Unregulated (IUU) Fishing on Marine Biodiversity and Habitats. Rome: FAO. Available online at: http://www.fao.org/docrep/012/i1490e/i1490e00.pdf threatens coastal ecosystems. Proc. Natl. Acad. Sci. U.S.A. 106, 12377–12381. doi: 10.1073/pnas.0905620106 Conflict of Interest Statement:* The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The handling Editor declared a past supervisory role with one of the authors, RK and states that the process nevertheless met the standards of a fair and objective review. Copyright © 2016 Katikiro and Mahenge. 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. # The Role of Perceptions for Community-Based Marine Resource Management #### Katharina Beyerl 1, 2 \, Oliver Putz <sup>1</sup> and Annette Breckwoldt <sup>3</sup> 1 Institute for Advanced Sustainability Studies, Potsdam, Germany, <sup>2</sup> Geography Department, Humboldt-Universität, Berlin, Germany, <sup>3</sup> Social Science Department, Leibnitz Center for Tropical Marine Ecology, Bremen, Germany Every community-based marine resource management (CBMRM) inherently takes place in a highly complex social–ecological environment, and stakeholder perceptions related to various aspects of the natural and social environment guide behavior in every stage of the management process. This paper provides an introduction to the psychology of perception with regard to marine resource management. In particular, it offers a typology of CBMRM relevant perceptions along with an analysis of psychological, societal, and physical factors that modulate them. Based on this analysis, we propose the introduction of specially trained local Perception Experts (PE's), whose role will be to recognize and reflect individual perceptions of involved stakeholders, and to communicate them at community meetings where decisions are made. This empirically testable addition to current CBMRM schemes could help to increase participation, develop management measures that fit the capacities of the involved stakeholders more accurately, and hence, contribute to a faster rehabilitation of marine resources. Keywords: perception, community-based resource management, psychology, participation, attitudes, norms, values ## INTRODUCTION In times of increasing socio–ecological pressures, sustainable resource management is more important than ever. Conservation and resource use behaviors are motivated by a variety of factors and understanding the psychological underpinnings may offer valuable insights for resource management approaches. Key here is stakeholder perceptions, which affect the management process from earliest conception to the actual implementation and monitoring. The centrality of the issue is increasingly acknowledged and recently there have been calls for perceptions to be considered as a part of natural resource management strategies (Jefferson et al., 2015; Bennett, 2016). Community-based marine resource management (CBMRM), where communities manage the marine resources upon which they depend for daily life, constitutes a common management scheme that makes apparent the essential role stakeholder perceptions play in such efforts. Coastal and island communities around the world have typically used and managed their crucial marine resources autonomously based on experience handed down from generation to generation (Zann and Vuki, 1998; McMillen et al., 2014). In today's context, these endeavors are often instigated or supported by outside partners [e.g., governmental agencies, non-governmental organizations (NGO's), academic research teams], who offer supplemental ecological analyses #### Edited by: E. Christien Michael Parsons, George Mason University, USA #### Reviewed by: Edward Jeremy Hind-Ozan, Manchester Metropolitan University, UK Emily Lux Cella, ICF, USA \Correspondence: Katharina Beyerl [email protected] #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 08 June 2016 Accepted: 02 November 2016 Published: 22 November 2016 #### Citation: Beyerl K, Putz O and Breckwoldt A (2016) The Role of Perceptions for Community-Based Marine Resource Management. Front. Mar. Sci. 3:238. doi: 10.3389/fmars.2016.00238 along with advice on contemporary management methods (Mühlig-Hofmann et al., 2006; Glaser et al., 2015). Whatever the exact circumstances, every CBMRM procedure inherently takes place in a highly complex social–ecological environment (Glaeser and Glaser, 2010, 2011). As such, it is influenced by external factors (e.g., environmental changes, market access, and demands) as well as internal community-specific conditions (e.g., inherited ownership structures, hierarchies, religious influences, or societal obligations). An individual's perception of either of these significantly shapes the dynamic of the entire project, including decisions on management measures and their execution. Community-based resource management frequently encounters problems due to stakeholder misunderstandings, lack of commitment, non-compliance, or conflicts (Bloomfield et al., 2012; Glaser et al., 2015). In our opinion, a commonly underestimated cause for this predicament is the differential perception of environmental changes, coping strategies, and social processes on part of individual stakeholders. For example, community members may evaluate ecological conditions quite differently, therefore reaching dissimilar, possibly even incompatible conclusions regarding management demands. Once in place, the specific responses to such given challenges might be considered efficacious by some, yet completely unsuccessful by others. Throughout this process, the perceptions that stakeholders have of each other can lead to further dissonance among them. We believe that insights from environmental psychology can prove essential for addressing these obstacles (see also Jefferson et al., 2015; Walker-Springett et al., 2016). The main objective of this paper, therefore, is to offer practitioners involved in CBMRM an introduction to the psychology of perception as it relates to resource management within local coastal communities. Here, we will assume a slightly unorthodox approach, where perception is defined and assessed through a carefully hewn phenomenological lens. Accordingly, the emphasis will lie on the structure of perception as the necessary condition of the possibility of experiencing the world in a meaningful way. Going a step further than merely acknowledging the importance of perceptions, we propose the introduction of specially trained Perception Experts (PE's) as a possible, empirically testable addition to community-based resource management approaches. ## ENVIRONMENTAL PSYCHOLOGY BACKGROUNDS ON PERCEPTIONS AND CBMRM-RELATED BEHAVIOR Environmental psychology "examines the influence of the environment on human experiences, behavior and well-being, as well as the influence of individuals on the environment, that is, factors influencing environmental behavior, and ways to encourage pro-environmental behavior" (Steg et al., 2013b, p. 2). In doing so, environmental psychology has generated and adopted a series of theories explaining behavior and the factors that shape it. Of these, some are particularly valuable for the CBMRM context, such as Ajzen's theory of planned behavior (1991), the norm activation model (Schwartz, 1977), the protection motivation theory (Rogers and Prentice-Dunn, 1997), and the transactional model of stress and coping (Lazarus and Folkman, 1984). More recent models include the integrative socio-cognitive model of private proactive adaptation to climate change (MPPACC), which focuses on adaptation to weather extremes (Grothmann and Patt, 2005), and Bamberg's stage model of self-regulated behavior change (2013). Space limitation and purpose of this article preclude us from examining every theory in detail, but suffice it to say aspects of each underlie the present discussion. In the following, we will describe what psychologists mean by perception, outline the role it plays in CBMRM, identify different stakeholders whose perceptions affect CBMRM, highlight what is being perceived, and summarize the main factors shaping CBMRM-relevant perceptions. ## Perception Defined Psychologists commonly envision perception as that process by which individuals organize sensory information and interpret it as "having been produced by properties of objects or events in the external, three-dimensional world" (Gerrig and Zimbardo, 2008). This definition is as oversimplified as it may be useful. It reduces perception to merely the act of sensing physical stimuli and to creating mental representations of environmental information. One could ask though, whether the activities in receptor cells and neurons in the brain, both clearly indispensable physiological aspects of perception, are by themselves sufficient to qualify as perception. Moreover, it is not clear whether the representational scenario, according to which we encounter objects as mental intermediaries, is truly the most adequate conception of how we perceive the world. Phenomenologists have long argued that perception is unmediated and confronts us not with mental images of objects, but with the objects themselves (Gallagher and Zahavi, 2012) 1 . Hence, defining perception as the operation of organizing sensory information into mental images seems overly reductionistic. A more comprehensive account of perception is used in studies assessing people's responses to unpredictable and potentially adverse challenges. Risk perception has been defined as an individual's "subjective judgment about the risk associated with some activity, event, or technology" (Böhm and Tanner, 2013, p. 24). This obviously involves not only the sensation of objects, but also higher cognitive processes such as reasoning. The perceiving individual logically discerns its future actions over and against the seeming facts with which it finds itself confronted. It does so with regard to held knowledge, previous experiences, and values. While clearly more refined, <sup>1</sup>The problem with representational theories is simply that it is unclear how the phenomenal subject would know that an intramental image represents a given extramental object. If indeed it is the resemblance to the extramental object that provides an intramental image with its representational quality, as representational theories would have it, a subject must have direct access to the extramental object to realize said quality. (For a detailed discussion see Gallagher and Zahavi, 2012). this definition overemphasizes reflective cognition and largely ignores the affective aspects of evaluating one's circumstances. In the present study, we use perception as the subjective way people experience, think about and understand someone or something. This involves conscious and unconscious processes of meaning making in a complex social and natural world, as well as affective states and reactions. The objects of perception can be quite concrete or abstract, animate or nonanimate entities, simple or complex, all depending on where the attention of the perceiving subject is focused. To put it into phenomenological terms, perception has an intentional structure and as such, is always about or of something (Gallagher and Zahavi, 2012). It is not merely the reception of information, but a process that involves the interpretation of phenomena within a given context. Rather than the external world impressing itself upon a passive subject, the subject's attention focuses perception onto a "perceptive field," thereby allowing the rest of the world to recede into the unconscious background. What directs the subject's attention is not only its physical surroundings, but also its interests and needs. In other words, whether a subject perceives objects while engaged in fishing is partially determined by the physical qualities of the objects sensed, yet largely also by his attention resulting from his desire to catch fish. One can say, then, that perception, further influenced by psychological and physiological factors, constitutes the background of experience and thus, guides a phenomenal subject's conscious acts (Merleau-Ponty, 1962). At first sight, this working definition may resemble the psychological concept of attitudes, but upon closer analysis it reveals an additional dimension that renders it more comprehensive. Attitudes are defined as the evaluation of an attitude object (Eagly and Chaiken, 1993; Haddock and Maio, 2012), whereas our use of perception refers to a more integral process involving the experience and interpretation of encountered reality. In this sense, it comes close to the common use of the term perception as "the way people think about or understand someone or something" (Merriam-Webster Online Dictionary, 2016). ## The Dynamics between Individual Perception and Group Behavior As mentioned above, CBMRM is always a social enterprise, insofar as it is a concerted, collaborative community effort involving specific actions toward a shared goal. In this sense, it is a form of group behavior. The group, here, is characterized not only by its common objective, but also by the interdependence of its members and their interactions (social structure), as well as a common social identity (Jonas et al., 2014). How individual stakeholders act depends to a great extent on the influence of the group, just as the group's overall actions are shaped by the individual behaviors of its members. In other words, CBMRM is invariably the result of a dynamic relationship between individual and group behaviors. This complex interrelation is commonly absent from portrayals of CBMRM, which remain on the meso-level of the group and depict the process as a rather straightforward progression of distinct steps from problem identification to implementation of management procedures (Figure 1). Accordingly, when the decline of marine resources is identified as a problem that cannot be addressed by the community alone, official partners are approached for assistance. Subsequently, strategies for marine resource management are developed and implemented. Depending on compliance and the evaluation of the process and results, the problem will be reassessed. Of course, this captures the process in theory, though at the expense of a more adequate analysis that takes into account the interaction of group and individual. In fact, the picture becomes even more intricate when one takes into consideration stakeholder perceptions that significantly shape individual behavior and, thus, CBMRM as group behavior. Simply put, whether and how community members act in terms of managing local marine resources depends on whether they perceive circumstances as requiring such actions. For instance, individual stakeholders might become aware of changes in environmental features or may simply develop an unreflected sense of change that raises concern. They may attribute reasons for why the observed changes exist and in turn infer the need for some kind of action to adjust the situation. Once coping strategies have been devised and implemented, individual stakeholders appraise them with regards to efficacy and associated costs. The behavioral outcomes and experiences provide feedback, which can result in a reappraisal of the situation, and of coping strategies. Thus, throughout the CBMRM process individual behavior presupposes perceptions, which in turn affect CBMRM as a group activity. ## Perceptions Influencing CBMRM Efforts Perceptions in the context of the CBMRM process are manifold. But before going into detail here, it is important to remember that much of environmental perception functions unconsciously. For instance, changing temperatures and precipitation rates, or dwindling fish stocks can be experienced as a new reality without immediate or continuous conscious reflection upon reasons and consequences. This poses two significant issues for CBMRM efforts. First, stakeholders may not be able to clearly voice their concerns during the planning phase. As a result, important aspects of the status quo of the resources to be managed may go unnoticed and the resulting management plans may not be entirely adequate for the given situation. Second, and closely related to the first problem, stakeholders may not agree with specific management proposals, but cannot articulate their reasons. Thus, CBMRM efforts may actually encounter serious challenges before they have really begun. Figure 2 gives a brief overview of three major areas environmental changes, coping strategies, and social processes whose contents overlap and are not independent of one another. For example, the perception of responsibilities for environmental changes and responsibilities for interventions are connected. The objective here is to offer a brief overview that helps identify the variety of possible perceptions relevant for CBMRM. Previous studies on perception in CBMRM have usually paid attention to specific segments of perceptions and provided very useful insights. Nevertheless, a more systematized approach, as we offer it here, might open up new perspectives for researchers, marine resource managers, and communities. #### Perceptions Related to Environmental Change At a very fundamental level, individual stakeholders may perceive physical changes in their environment, and based on it, assess the situation with regard to immediate or future intervention. Perceptions of impaired marine ecosystem health can include declining fish numbers and sizes, increases in algae density, the absence of known species and presence of novel species in familiar fishing grounds, altered coral colors along with increasingly fragile or broken calcareous structures, or changes in what is washed up on beaches. For the most part, these are readily sensed differences, but there are also other, more intricately perceived anomalies, such as declining catch over longer periods of time along with the associated impacts on income and food security, as well as the lack of specific species for traditional functions (Veitayaki et al., 2015). The perception of such changes can be interpreted not only as an unfortunate environmental deterioration but also as a risk to accustomed and valued lifestyles and personal well-being. Simultaneously to perceiving environmental changes, stakeholders may attempt explaining the situation as to gauge potential courses of action. Where responsibility is placed can make a significant difference regarding future behavior change and participation in any kind of CBMRM effort. For instance, if human behavior is perceived as a reason for the change, stakeholders will allocate culpability either to themselves or others and negotiate possible responses accordingly. However, if, for example, environmental deterioration is seen as divine punishment for human transgressions, stakeholders could FIGURE 2 \| Perceptions playing a role in the CBMRM process. consider morally offensive behaviors entirely unrelated to the treatment of natural resources and conclude remedies with no further ecological impact (Mortreux and Barnett, 2009; Kuruppu and Liverman, 2011). Whether the changes are perceived as due to human actions or due to a larger process, stakeholders are faced with the question of whether they are actually able to address the changes, and how. Their response to this shapes future decisions with respect to CBMRM. Thus far, there has been limited research focus on the perception of marine biodiversity change. Inquiries related to changes in marine biodiversity have largely been restricted to ecological and occasionally anthropological rather than more comprehensive studies encompassing psychological assessment (Mills et al., 2013; Young et al., 2016; Webster et al., 2017; Katikiro, 2014). One reason for this is clearly that here the detection of change is complex, involving a plethora of organisms, long timescales and various factors of change. Despite these undeniable difficulties, we nonetheless suggest intensifying a holistic strategy and encourage cooperation of marine biologists, ecologists, environmental psychologists, social scientists, governmental and non-governmental organizations, and communities. The benefits gained from including such a transdisciplinary approach for monitoring and evaluating environmental perceptions in CBMRM processes potentially far outweigh the methodological challenges associated with their implementation. #### Perceptions Related to Coping Strategies How stakeholders perceive coping strategies that have been devised and implemented in response to perceived environmental change is yet another area of inquiry for environmental psychologists (Grothmann and Patt, 2005). What makes this category interesting is that it reveals the importance of perceptions at every stage of the management effort. Already during the planning phase, stakeholders have a particular impression of a potential CBMRM strategy and will support the intended measures only insofar as they perceive them as adequate. As the strategy is implemented, they may experience the procedure quite differently, and it is at this point that some will withdraw their participation. When management measures have been in place for a while, stakeholders will most likely assess their success differently, once more potentially giving rise to conflict or disenchantment. In short, throughout the whole CBMRM process, stakeholder perceptions of the actual measures can decide the ultimate success of the entire enterprise. The perceptions related to CBMRM strategies can be divided into two broad categories: first, the perceived positive and negative effects of a measure and second, perceptions related to feasibility, including potential behavioral barriers and facilitators to engage in specific activities. Positive and negative effects of a measure do not only include its efficacy with regard to the restoration of the marine ecosystem, but also to the associated costs and benefits for individual stakeholders. These can be of a monetary nature, yet also related to the individual's invested time, energy, and social recognition or disapproval by others. No-take areas, for example, can be perceived as very effective to restore the marine ecosystem, but also as costly and consequently, undesirable. For instance, additional expenses for fuel to travel further to alternative fishing grounds and extra time spent on fishing trips may make it hard for some stakeholders living adjacent to the protected area to comply with such a measure. In addition to their perceived effects, single CBMRM measures will be judged by the involved individuals with regard to feasibility. Here, the perception of behavior facilitators and barriers is specifically relevant for stakeholder motivation. The extent to which people perceive themselves as able to exert an intended behavior depends on the perception of their individual skills, abilities and resources (Bandura, 1977). These assessments affect how actively stakeholders will engage in the community management effort. In addition, the sense of how circumstances allow stakeholders to bring to bear their abilities to partake in one or each decided measure plays a crucial role. Both aspects, the perception of individual abilities and enabling or hindering circumstances can be summarized as perceived behavioral control which is known as one important factor for motivating behavior (Ajzen, 2001, 2002). In Pacific small island contexts for example, where traditionally women fish in nearshore areas, establishing protected zones close to the beach would make it impossible for women to fish at all for lack of skills, abilities, and resources to go elsewhere (Mühlig-Hofmann, 2007). Finally, not only the perception of their own abilities, but also the perception of potential alternatives to make a living, will affect their motivation to engage in conservation measures that might impair their own subsistence. Taken together, these perceptions related to management strategies and measures can be key to resolving issues of stakeholder discontent with CBMRM and resulting lack of commitment. It is therefore absolutely crucial to reflect on them carefully throughout the entire process and particularly to anticipate them when precise management plans for a community are being conceived. #### Perceptions Related to Social Processes As outlined above, CBMRM is a group behavior involving various stakeholders and subgroups, which include, among others, the participating local communities of resource users, advising scientists, regulators, and government officials, as well as supporting NGO's. Social dynamics unfold both, within and between subgroups; individuals perceive and consequently interact with one another in their own group and also with individuals of other subgroups. These continuous perceptions of and experiences with one another constantly shape future expectations and behaviors. With respect to social perceptions, the perhaps most important subgroup of stakeholders are the resource users themselves, who in essence depend on the well-being of the local marine environment. As residents of the same village or region, these individuals likely engage with each other frequently and because of it stand in rather complex relationships with one another. Whether such a group can organize a facilitated effort to manage resources is dependent to a large extent on whether individual resource users perceive the activities of other group members as equitable, responsible, and just. Moral perceptions, as difficult as they are to diagnose, are absolutely key to the social dynamics of communities (Syme et al., 2000). They are therefore, also crucial for CBMRM. Concerns about justice come to bear already in the earliest stages of a CBMRM process, where negotiations of necessary management measures are strongly affected by who individual community members consider responsible for the state of the marine ecosystem (Montada and Kals, 2000; Fielding and Head, 2012; Kalamas et al., 2014). If others are seen as responsible for the observed environmental deterioration, stakeholders might not see any reason to act. Alternatively, group members could demand a greater contribution to the management efforts from those they deem accountable for the state of things. In any case, with the question of liability unresolved dissension within the community is almost inevitable. Hence, it is important, that the causes of marine environmental change are discussed transparently and a course of action is devised jointly along with a clear and accepted distribution of responsibilities. Once management measures have been established, stakeholder attention will shift somewhat from responsibility to equity, where individual contributions to the group effort are perceived in comparison with a stakeholder's own efforts (Van Lange, 1999). For instance, when others seemingly spend less labor, time, or money on altogether costly procedures, stakeholders might find their own involvement unduly taxing. Likewise, some community members may be perceived as disproportionately benefiting from the measures (e.g., if they live farther away from an established no-take area than the perceiving stakeholder or possess the means to travel further to fish when no-take areas were set up in their usual fishing spots). Perhaps the most damaging to stakeholder ambition would be if others are perceived as cheating. Fraudulent behavior undermines trust and therefore, the entire CBMRM effort, which as a group behavior is dependent on reliable stakeholder participation (Yandle et al., 2011; Van Lange et al., 2013). In turn, general participation according to the agreed-upon course of action can further a sense of community and ultimately increase motivation of individual stakeholders. Whatever the particular perceptions related to fairness, for the CBMRM process it is advisable to maintain a high degree of transparency at all times. In the best case, a social norm of fair co-operation would emerge. Although the perceptions of responsibilities and individual contributions are subjective and may not always be objectively verifiable, they will guide the CBMRM process and the motivation to engage in it. Aside from the actual resource users, other subgroups, such as government officials, NGO's, or research teams, play an integral part in a functional CBMRM process, and how they are seen by participating community members is crucial for overall success. When outsiders are perceived as competent, credible, perhaps even likeable, but most certainly culturally sensitive and consequently, as an acceptable authority, community members are far more likely to welcome advice and collaborate in a lengthy management approach (Fiske and Dupree, 2014). Of course, here past experiences with either of the participating outside subgroups decide current relationships: Some communities might have had extraordinarily positive experiences, whereas others could have gone through disappointments and may therefore be not as open and optimistic when it comes to new plans for co-operation. Although these factors are inherent to any human relationship and interaction, it might be helpful to be aware of their potential influence on CBMRM endeavors. Consequently, perceptions related to social processes are relevant in all stages of the CBMRM as it is by definition a group conduct extending over a longer period of time. #### Factors Modulating Individual Perceptions Perceptions are affected by a variety of individually and socially relevant psychological factors, of which Figure 3 offers a systematic overview. As we mentioned initially, perceptions are focused onto a perceptive field by physical, societal, or psychological circumstances, and their interactions. By way of illustration, how a community member perceives the state of the natural resources on which she relies is significantly affected by the environmental conditions she encounters, her relationships with others within the community, and her personal needs, interests, or psycho-physiological status. Thus, perceptions are not simply the result of a sensory input generating neural representations, but rather the product of a complex interaction between the perceiving individual and its FIGURE 3 \| Factors affecting perceptions and interpretations in the CBMRM context. surroundings. In view of such intricate reciprocity it does not surprise that even modulating influences of the most unlike kind can alter perceptions at each level (environmental change, coping strategies, or social processes). Analyzing single factors isolated from the rest is therefore prone to oversimplification. Nonetheless, in the following section we provide a brief overview with short introductions to the most relevant classes of perception modulating factors. the dialogue partners perceive each other as trustworthy and credible. Even more, successful communication may ride on whether stakeholders perceive their conversation partners as similar to themselves (Siegrist et al., 2000). How things are communicated and by whom, plays an important role in any group behavior. way as the concept of attitudes. In psychology, attitudes are defined as the evaluation of an attitude object which can be a person, place, thing, event, or action and include firstly a cognitive component referring to thoughts and beliefs about the attitude object, secondly an affective component which refers to emotions related to the attitude object, and thirdly a behavioral component relating to previous, current and anticipated behaviors related to the attitude object (Eagly and Chaiken, 1993; Ajzen and Fishbein, 2005; Haddock and Maio, 2012). Attitudes can vary in valence and intensity. They do not only affect the processing and interpretation of information but also bias attention. Although individuals might have positive attitudes toward environmental protection and sustainable resource use in general, their specific attitudes related to concrete CBMRM measures can vary greatly. Therefore, to elicit people's attitudes toward a certain measure, it is prudent not to rely on general statements, but to be as specific as possible with regard to the attitude object in question and about involved actions, contexts, and times. In that way, attitudes contribute to the explanation and prediction of behavior (Ajzen and Fishbein, 1977). perceives its contribution as hardly visible or unimportant (Latané et al., 1979; Kerr and Bruun, 1983; Williams and Karau, 1991; Stroebe et al., 1996; Kerr et al., 2007). Critical for the problem at hand is also the fact that stakeholders possess a social identity that "describes those aspects of a person's self-concept based upon their group memberships together with their emotional, evaluative and other psychological correlates" (Turner and Oakes, 1986, p. 240). As a member of a particular group, a stakeholder might seek to maximize the benefits of her own group over that of other stakeholder groups. Hence, some stakeholders will try to advance the interests of their own village, whereas others may act to achieve a common goal cooperatively with all subgroups and stakeholders (Tajfel and Turner, 1986; Turner et al., 1987; James and Greenberg, 1989; Lickel et al., 2000; Johnson et al., 2006). Being cognizant of social groups and addressing potentially existing prejudices or conflicts, which might hamper effective cooperation for CBMRM, would therefore be valuable (Nelson, 2009). (12) Cultural context. Zooming out from an individual to a societal perspective, it is clear that all the aforementioned factors need to be seen embedded in the cultural context. Cultural dimensions affect perceptions, group processes, and social practices (Hofstede, 2001; Triandis, 2001; House et al., 2004; Schwartz, 2006; Trompenaars and Hampden-Turner, 2012). Accordingly, if stakeholders from outside are involved in the CBMRM process, or if approaches developed elsewhere shall be applied, potential contradictions to the cultural context should be anticipated. Generally, it is important to bear in mind that the psychology on which the present discussion is based has its origin in Western thought. Applying its concepts and analyses elsewhere, as for example the Pacific Islands or African coastal regions, must be done cautiously and with the necessary consideration of cultural idiosyncrasies. ## Ramifications of the Psychological Insights on Perceptions This study set out to provide CBMRM practitioners with an introduction to the psychology of perception so that frequent problems of stakeholder misunderstandings, lack of commitment, non-compliance, or conflicts could be avoided. At this point, three conclusions of what has been said can be drawn: First, perceptions play a central role at every point of a CBMRM endeavor. As we have pointed out above, CBMRM is often oversimplified as the sequentially unfolding resource management process around the shared objectives of a homogenous community (Figure 1). However, this view neglects to a large part the heterogeneity of involved stakeholders as well as differences in their individual perceptions (Campbell and Vainio-Mattila, 2003). When stakeholder perceptions are taken into consideration, a far more complex picture begins to emerge (Figure 4). Perceptions affect every aspect of CBMRM, which is why an organized reflection on them throughout the process is necessary. Second, perceptions relevant to CBMRM can be grouped into three major areas with regards to (A) environmental changes, (B) chosen coping strategies, and (C) the involved social processes. These different types of perceptions act at various points of any CBMRM effort (Figure 4). Thus, perceptions of environmental changes are pivotal during the early stages of problem identification and the development of a joint strategy, but also during the evaluation of the implemented management schemes and subsequent adjustments to the strategy. Perceptions with respect to chosen coping strategies are of relevance during the development, implementation, and subsequent evaluation of the actual measures. Finally, social perceptions directly affect CBMRM efforts at every step of the way. With this in mind, practitioners can anticipate potential problems and their causes long before they occur. Moreover, they can respond more adequately to misunderstandings between stakeholders that may arise during the CBMRM process and which, if left unattended could hamper the management process. Third, perceptions in all three areas are shaped by a variety of psychological factors (1–12 in Figure 4). How stakeholders experience their situation and respond to it largely depends on their socio-demographic background, knowledge, attitudes, norms, or other kinds of psychological modulators. The ability of practitioners to identify any one of these factors can mark the difference between success and failure for a CBMRM effort. Taking them into consideration allows the development of more adaptive strategies tailored to the specific needs of a community in need of resource management. Equally important, it would help facilitate productive communication between stakeholders during the CMBRM process, especially once misunderstandings or conflict have ensued. Although the role of perceptions is increasingly acknowledged in the CBMRM literature (Pita et al., 2011; Jefferson et al., 2015), only little attention has been paid to psychological theories connecting perceptions to behavior (Bennett, 2016). As a result, perceptions that are crucial but not obvious are usually considered neither in CBMRM theory nor in CBMRM projects. Several studies focus on perceptions related to coping strategies, asking primarily about stakeholder perceptions of implemented fishing restrictions and their efficacy (e.g., McClanahan et al., 2005; Bloomfield et al., 2012; Cinner et al., 2014; Katikiro et al., 2015). Others address mainly environmental perceptions with regards to changes in catch size, fishery stock, condition of current fishing grounds, and the number and type of affected groups or species (Jefferson et al., 2014; Katikiro, 2014). A small minority of inquiries either combines the two, while even fewer include social perceptions (Gelcich et al., 2005; Abecasis et al., 2013; Deiye, 2015). Even though some of these studies control for socio-demographics and occasionally psychological factors such as personal values, virtually none of them have embedded their inquiry into psychological theory. For example, perceptions are almost never defined nor are different types of perceptions comprehensively distinguished. Moreover, how psychological factors shape perceptions and ultimately, behavior is a problem left unresolved. With the present summary of perceptions from the perspective of environmental psychology we hope to offer researchers and practitioners a theoretical foundation for more constructive management methods. Going beyond theoretical foundations, one question that might arise for practitioners is how to include the diverse range of perceptions in actual long-term CBMRM projects. Over the past decades, one strategy seeking to work with stakeholder perceptions is a participatory approach, where a special emphasis is placed upon developing natural resource management strategies jointly with every relevant stakeholder (DeCaro and Stokes, 2008; Ferse et al., 2010; Lin and Chang, 2011; Akbulut, 2012; Rabe and Saunders, 2013). Yet, against better intentions these approaches more often than not fail to consider the full breadth of relevant perceptions. As a result, they miss the inclusion of underprivileged members of society in decision-making processes over longer timescales and in effect, perpetuate existing power structures and inequalities (Akbulut, 2012; Rabe and Saunders, 2013). Although the idea of participatory, community-based management has valuable potential, the gap between expectations and reality is often undeniable. One reason for this might be that participatory projects are frequently facilitated by outside experts, who tend to face not only project-related constraints with regard to time and resources, but also lack local expertise, social embeddedness, and authority (Campbell and Vainio-Mattila, 2003; Akbulut, 2012). Furthermore, most resource management endeavors rely on the expertise of ecological experts rather than that of social and behavioral scientists (Campbell and Vainio-Mattila, 2003). It therefore is no exaggeration to say that approaches to community-based resource management considering stakeholder perceptions in their full complexity are still largely missing. In the following, we propose one potential solution to the challenge of working with perceptions in CBMRM that considers not only their diversity, but also addresses the need for continuity to work with stakeholder perceptions throughout the whole CBMRM process. ## PERCEPTION EXPERTS—A PRACTICAL PROPOSAL As clearly shown, perceptions of involved stakeholders affect every phase of the CBMRM process. Acknowledging the importance of perceptions for CBMRM contexts raises the question how psychological understandings of perceptions might enable community-based managers to detect possible inefficiencies and their causes early on to respond more flexibly, and how such a process could be anchored and unfold in a community-based context. As an empirically testable approach we propose that appointed individuals from the communities receive a tailored training program on psychological backgrounds. ## Core Tasks of Perception Experts and their Role in the Community When a CBMRM process is initiated and a management strategy is developed, usually certain tasks are defined and designated among the community members. These tasks (e.g., of fish-warden) focus mostly on environmental monitoring and watching over compliance with the decided rules and measures (Mühlig-Hofmann, 2007; Pomeroy et al., 2015). Already at that phase of the process, being aware of perceptions of involved stakeholders can offer insights in how management measures are assessed or where difficulties for compliance might arise. Therefore, from its earliest stages on, the CBMRM process would benefit from having trained individuals who work with perceptions of involved stakeholders to reach management approaches which fit stakeholders' needs and capabilities. The core task of such "perception experts" (PE's), would be mainly a reflective and communicative one. First, they are to reflect prevailing CBMRM-relevant perceptions together with involved community members and other stakeholders. Second, they should help to detect misunderstandings or biases which could then be clarified in communication within the community. Finally, by facilitating a transparent and respectful communication, the PE's will ensure that concerns, expectations, and needs of all CBMRM-stakeholders are taken seriously and will be articulated during regular CBMRM meetings. As modes of participation and decision-making can vary greatly across regional and cultural contexts, defining the specific role of the PE's would demand developing it together with local stakeholders, such as fishing communities and local institutions like universities or NGO's. Specifying the PE-role and agreeing on it within the community should vest some degree of authority and legitimization to the appointed individuals (Leaua and Ani¸tei, 2012). This should include, for example, being entitled to invite subgroup meetings, do interviews, and accompany stakeholders at their fishing- and CBMRM-relevant activities. Furthermore, the PE's role should allow them to speak and reflect on perceptions in regular CBMRM-related community meetings. As CBMRM is a long-term social–ecological process, perceptions and specific needs of involved people are likely to change over time (Roovers and van Buuren, 2016). PE's should therefore be in regular exchange with the stakeholders about perceptions of the status of the marine resources and management measures. Hence, the CBMRM strategies could be adapted dynamically depending on perceptions, ongoing environmental changes, and needs. Also, potentially needed support for individual stakeholders could be identified and provided more purposefully to help to reduce objective as well as psychological behavioral barriers. Perception experts would not only encourage community members to reflect their own perceptions and behaviors, but foster a participatory process in which stakeholders could shape the CBMRM process more actively. Decision makers, marine managers, as well as community members would engage in active feedback loops (Staats et al., 2000; Abrahamse et al., 2007). On the one hand, they would receive information on each other's perceptions of the environment, coping strategies and the process dynamics, and, on the other, get behavioral feedback on what CBMRM-measures proved useful for what reasons, or why single measures might suffer from a lack of acceptance. Since behavioral feedback is an essential factor for motivating behavior, experiencing that realistically negotiated goals can be achieved is likely to support people's motivation for remaining dedicated and committed over a longer timeframe (McCalley and Midden, 2002). In the case that additional necessity for conflict-mediation arises, which would need clarifying support going beyond the PE's competence, traditional and trusted conflict mediators could come into play (Alsop et al., 2006). Depending on the cultural context, these mediators could be, for instance, church members, village leaders, or others who usually engage in the role of traditional conflict mediators with the respective authority. ## Nomination of Perception Experts How PE's are selected will prove a complex issue, first and foremost because of the cultural and social diversity of communities engaging in CBMRM. As we have pointed out, the role of the PE's is primarily to listen to stakeholders, reflect upon their statements with regard to perceptions, and communicate their insights to the entire community during significant periods of the management process. Obviously, PE's must not only be respected and trustworthy authority figures within their community, they also need to possess some innate skills enabling them to engage stakeholders in open conversations and to create the safe spaces in which these exchanges can take place. Who qualifies as an authoritative voice within the community and how they are endowed with such a responsibility would clearly depend on the cultural background of the community in question. We cannot offer one general solution to the problem of PE choice, but a few general concerns regarding selection criteria can be outlined. First, as mentioned above, PE's need to be authority figures that are trusted and well-respected within the community. These may be individuals who already hold positions of authority and trust (e.g., religious figures, community elders, etc.) or persons who can fill such a position for the very first time. Either way, PE's will have to be self-confident, while at the same time humble enough not to overestimate their abilities. In other words, PE's need to be self-reflexive and aware of their own potential biases and prejudices. Second, PE's have to be competent communicators, which implies on the one hand an aptitude for listening and on the other hand, the ability to distill relevant information. Third, PE's need to be able networkers, who have proven their capacity for bringing together various community members from diverse backgrounds to address issues of communal import. Fourth, PE's will have to be empathic, as well as endowed with a certain non-verbal sensitivity. This may very well be the most important trait for a PE, simply because it underlies all previously mentioned abilities. Fifth, PE's will have to be creative, finding novel, and heretofore untested approaches to resolve issues such as potential conflicts or misunderstandings arising from stakeholder perceptions. In summary, one could say that PE's should be selected on their social standing within the community, their social competence, and their communicative skills. In addition, it would be important to recruit at least two PE's (or more, depending on the size of the community) to prevent loss of expertise should one PE be absent. Finding two individuals (or more) answering all of the demands listed above could be quite a challenge in itself, which is why it might be necessary to select two complementary individuals, who each possess some of the skills and together cover most or all of them. Of course, it would be necessary for both of them to be able to work together, which once more could be a function of the cultural context. For example, in strongly patriarchal societies with far-reaching gender separation, it may be both necessary as well as difficult to have a male and a female PE work together. Whatever the particular social structure of the community in question, it would be mandatory that the PE's will take into consideration perceptions of the greatest number of community members possible. ## Potential Training Contents PE's can be understood as "system experts" (Mieg, 2001, 2006) who have experience within the local human-environmental system and receive a science-based training to gain "interactional expertise" (Collins and Evans, 2007). Similar to training programs for fish-warden which have shown to benefit CBMRM efforts (Mühlig-Hofmann, 2007; Pomeroy et al., 2015), a specific training course could be developed for the PE's. Key partners for the development of such a training program could be psychology departments of local universities working in close cooperation with marine science departments, local governments, NGO's, and communities. Insights from areas of environmental, social, motivational, and communication psychology in combination with expertise from marine science and conservation studies could serve as the basis for creating training contents. To enhance a mutual learning process, the training should be interactive, based on a mix of contentual input by the trainers, interaction between the trainees, and practical exercises. Figure 5 illustrates a suggestion for potential contents of a three-step program consisting of first, a training session providing theoretical backgrounds and practical tools; second, a practice run using the learned skills; and third, a second training session where experiences from the practice run are reflected, and ideas and concepts laid out in the first training session are expanded. The first training session would focus on relevant perceptions in the CBMRM context and factors affecting them, as well as on mapping stakeholders that are involved in the whole process. Subsequently, ways how to elicit information about prevailing perceptions should be learnt and practiced. This could comprise interview techniques, group facilitation methods, and the use of qualitative and quantitative interview tools. Furthermore, ways of analyzing and integrating results to summarize and present them in community meetings should be introduced. Beyond that, prospective PE's should gain process-competence and get encouraged to reflect their own perceptions, assumptions, and role in the community. Between the first and the second session of the training course, the prospective PE's should get the opportunity to practice the approaches and tools they have acquired during the first training session at home in their communities. Exercises could encompass, for instance, getting an overview of the involved stakeholders in the local CBMRM-process, practicing observation skills, and doing test-interviews to compile a portfolio of factors that motivate or hinder relevant behaviors. Besides the value of practicing, the trainees could introduce and explain the role of the PE to the community and get first feedback and ideas to work with. Such exercises would also be important for the PE's to see if they feel comfortable with their new role and are willing to continue. For the second training session, all trainees should contribute updates from their communities that can be used to further analyze the role of stakeholder perceptions and ways to include them in CBRMR-related decision-making. Based on their observations and test-interviews, the training participants should use this session to compare the CBMRM-relevant perceptions and their main modulating factors that they could detect in their communities. Here, similarities and differences between single communities could be highlighted, and important factors that might have been overlooked so far could be added. In a next step, the analysis of local decision-making structures could serve as the foundation for the development of strategies to work with perceptions in the specific CBMRMprocesses. The second training session would also fulfill the purpose that the trainees could describe their experiences with the toolkit, potential difficulties they encountered, and where they see the need for additional support for their work in the communities. This support could include, for example, an association of local experts who could assist the PE's. One group of experts could be the PE-trainers at the university or NGO who could take a supervising role not only during the time of the training-course but also for later consultation. Supervision is an approach that has proved very useful in other fields of psychology (Davys and Beddoe, 2010). Furthermore, a network of local PE's could be established to work together, exchange, and support each other. Thereby, it would also be possible to co-create further strategies for the PEs. Last but not least, the competences of the PE's will not only be shaped by the initial training, but are likely to develop further over time and adapt to the needs and the composition of a community. If the PE-concept would prove valuable and should get established, experienced PE's could also train successors to ensure continuity. Thereby, the expertise could stay within the community. ## Potential Toolkit Contents To facilitate the work of the local PEs, a compilation of easy to use tools could be developed in addition to the training contents. These tools could comprise checklists and short summaries of facilitation and interview techniques (e.g., McFadzien et al., 2005, for a Pacific small island context example). There could be tools for quick-surveys with suggestions for open or closed questions regarding perceptions of marine resources, perceived reasons for environmental change, and perceptions of coping strategies. The tools could further focus on perceptions of responsibilities for environmental change and coping-responsibilities, as well as perceptions of the own role, abilities and perceived behavioral control. Additionally, the toolkit could include suggestions for community-specific stakeholder mapping, mapping of factors impeding and facilitating CBMRM-relevant behavior, and suggestions for summarizing the results in community-meetings. During the training course, the single tools could be introduced, tried out, and practiced between the first and the second training session. As part of a continuous co-creation-process, all tools could also be developed further and enhanced over time to incorporate the input from the communities. ## Putting the PE-Approach to the Test We are well aware of the fact that the proposed introduction of PE's to CBMRM raises a number of questions that are difficult to assess beforehand. As the introduction into the psychology of perceptions has made clear, perception and its modulators can be culturally determined, so that one has to wonder whether the training of PE's would have to be so culture-specific as to make it practically infeasible. How exactly can environmental psychologists and others preparing training material for PE's anticipate the particular cultural idiosyncrasies of individual coastal communities? Should PE's be community-members or external professionals? Would the comprehensive and time intensive task of a PE be manageable for a longer time on a voluntary level, and what kind of compensation would be necessary or advisable? And perhaps most importantly, would conflicts arising within communities during the CBMRM process exceed the competence of PE's and potentially put them into a vulnerable position within the community? Some of these questions can only be resolved when the proposal is put to the test. In order to gauge the efficacy of PE's in CBMRM it would be important to conduct pilot studies in a small number of communities, preferably from varying cultural backgrounds. To that end, it is imperative to carefully design both, the PE programs as well as the method by which they will be evaluated. Here, defining criteria for assessment is key. As we see it, possible indicators of success could be increased participation of various stakeholders in the negotiation process, a better understanding of ongoing environmental changes and management necessities, greater agreement among community members on adequacy of the chosen management measures, greater adherence to decided rules, as well as an overall reduction of conflict. Ultimately, however, the main criterion by which to establish the potential PE's may have for CBMRM would be a better and faster rehabilitation of marine resources. In all, the PE approach is just one proposal and other ways to work with perceptions are conceivable. Our overall hypothesis is that the explicit inclusion of perceptions would benefit CBMRM-processes. We therefore invite practitioners and scientists to develop and test ways to systematically incorporate environmental psychology expertise on perceptions and behavior to CBMRM endeavors. ## CONCLUSION Environmental psychology, as the science examining the relationship between human experience, behavior, and environment, provides theoretical and methodological expertise for understanding the role perceptions play for environmental behavior (Steg et al., 2013a). Therefore, the main objective of this paper has been to offer practitioners involved in CBMRM an introduction to the psychology of perception with regards to resource management within local marine communities. As has become apparent, perceptions are important and at work in every stage of the CBMRM process. They guide not only individual behavior, but also group conduct and, in the end, determine the welfare of the ecosystem in question. Stakeholder perceptions are nonetheless often disregarded in management planning, and usually receive attention only when obstacles are encountered. Given the importance perceptions have for resource management, it seems only prudent to make them a central part of the CBMRM process (Jefferson et al., 2015; Bennett, 2016). As one way to include perceptions in CBMRM endeavors we proposed the introduction of specially trained perception experts (PE's) recruited from the communities as a possible, empirically testable addition to community-based resource management approaches. PE's are to reflect CBMRM-relevant perceptions and related behaviors together with stakeholders, detect misunderstandings, and assure that stakeholders' concerns are being heard and taken seriously in CBMRM processes. Based on the systematic compilation of CBMRM-relevant perceptions, we suggested the development and empirical testing of a training course and a toolkit for local PE's through a cooperation of local universities, NGO's and communities. The described inclusion of perceptions in existing decisionmaking processes would build on traditional knowledge, beliefs, and norms, and acknowledge their importance. Decision-making procedures that have emerged over time within communities or cultures would be enriched without changing their fundamental structures. The implementation of PE's or similar approaches to ensure the inclusion of stakeholder perceptions could develop to be a "soft" way of participatory management and empowerment respecting existing and traditional decisionmaking structures of which Constantino et al. (2012) speak. Such an introduction of process advisors and trained local community facilitators has already proved valuable in other contexts of participatory decision-making like urban and regional planning and development projects (Bulkeley and Mol, 2003; Wongbusarakum et al., 2015). Going beyond CBMRM, developing a training program with focus on individual perceptions could contribute to local capacity building and is applicable to various contexts. The psychological concepts mentioned here are relevant to human behavior in general, also in the contexts of natural disasters, climate change related hazards, prevention, adaptation, rebuilding efforts, and even for health relevant behavior (Rogers and Prentice-Dunn, 1997; Milne et al., 2000; Grothmann and Patt, 2005; Steg et al., 2013a). Whenever human beings come together, they act based on their perceptions of the world. When conflicts arise in social situations, reflecting such perceptions, making at least some of them explicit, and taking them seriously can help to address concerns and misunderstandings in a respectful way. This may be a truism; yet, it still is overlooked time and time again during the conceiving and implementation of management plans. Paying greater attention to stakeholder perceptions would be a subtle, yet significant addition to current CBMRM practices and could help give rise to more sustainable futures not by relying solely on scientific data, but equally by emphasizing the way we experience ourselves within our natural and social contexts. ## AUTHOR CONTRIBUTIONS AB and KB co-developed the initial idea for the manuscript. KB and OP conceived the research, as well as the idea of Perception Experts, and wrote the main manuscript text. KB provided the psychological background of perception. OP supplied the background on phenomenology of perceptions. AB provided background on CBMRM and practical experience with fishing communities. All authors reviewed the manuscript. #### ACKNOWLEDGMENTS The authors thank Joeli Veitayaki, Ella Ritchie, Selina Stead, and Nick Polunin for their support during fieldwork, and Ilan Chabay, Gerhard Reese, Viliamu Iese, Harald A. Mieg, #### REFERENCES Christian Hoffmann, Akuila Cakacaka, Ina Richter, Tim Butler, and Andrew Parker for their highly valuable and much appreciated comments on earlier drafts of this article. Furthermore, we thank the Institute for Advanced Sustainability Studies. within the New Zealand Commercial Rock Lobster Fishery. Policy Stud. J. 39, 631–658. doi: 10.1111/j.1541-0072.2011.00425.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 © 2016 Beyerl, Putz and Breckwoldt. 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. # Multiple Drivers of Local (Non-) Compliance in Community-Based Marine Resource Management: Case Studies from the South Pacific #### Janne R. Rohe1, 2 \, Shankar Aswani <sup>3</sup> , Achim Schlüter 1, 2 and Sebastian C. A. Ferse<sup>4</sup> <sup>1</sup> Department of Social Sciences, Leibniz Centre for Tropical Marine Research, Bremen, Germany, <sup>2</sup> Department of Economics, Jacobs University, Bremen, Germany, <sup>3</sup> Departments of Anthropology and Ichthyology and Fisheries Science, Rhodes University, Grahamstown, South Africa, <sup>4</sup> Department of Ecology, Leibniz Centre for Tropical Marine Research, Bremen, Germany #### Edited by: Wen-Cheng Wang, National Taiwan Normal University, Taiwan #### Reviewed by: Michael Fabinyi, University of Technology Sydney, Australia Edward Jeremy Hind-Ozan, Cardiff University, United Kingdom > \Correspondence: Janne R. Rohe [email protected] #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 07 November 2016 Accepted: 18 May 2017 Published: 31 May 2017 #### Citation: Rohe JR, Aswani S, Schlüter A and Ferse SCA (2017) Multiple Drivers of Local (Non-) Compliance in Community-Based Marine Resource Management: Case Studies from the South Pacific. Front. Mar. Sci. 4:172. doi: 10.3389/fmars.2017.00172 The outcomes of marine conservation and related management interventions depend to a large extent on people's compliance with these rule systems. In the South Pacific, community-based marine resource management (CBMRM) has gained wide recognition as a strategy for the sustainable management of marine resources. In current practice, CBMRM initiatives often build upon customary forms of marine governance, integrating scientific advice and management principles in collaboration with external partners. However, diverse socio-economic developments as well as limited legal mandates can challenge these approaches. Compliance with and effective (legally-backed) enforcement of local management strategies constitute a growing challenge for communities—often resulting in considerable impact on the success or failure of CBMRM. Marine management arrangements are highly dynamic over time, and similarly compliance with rule systems tends to change depending on context. Understanding the factors contributing to (non-) compliance in a given setting is key to the design and function of adaptive management approaches. Yet, few empirical studies have looked in depth into the dynamics around local (non-) compliance with local marine tenure rules under the transforming management arrangements. Using two case studies from Solomon Islands and Fiji, we investigate what drives local (non-) compliance with CBMRM and what hinders or supports its effective enforcement. The case studies reveal that non-compliance is mainly driven by: (1) diminishing perceived legitimacy of local rules and rule-makers; (2) increased incentives to break rules due to market access and/ or lack of alternative income; and (3) relatively weak enforcement of local rules (i.e., low perceptions of risk from sanctions for rule-breaking). These drivers do not stand alone but can act together and add up to impair effective management. We further analyze how enforcement of CBMRM is challenged through a range of institutional; socio-cultural and technical/financial constraints, which are in parts a result of the dynamism and ongoing transformations of management arrangements. Our study underlines the importance of better understanding and contextualizing marine resource management processes under dynamic conditions for an improved understanding of compliance in a particular setting. Keywords: community-based marine resource management (CBMRM), compliance, enforcement, legitimacy, customary governance, transforming management, South Pacific ## INTRODUCTION The effect of formal and informal rule systems to manage natural resources largely depends on people's compliance behavior (Keane et al., 2008). In other words, rule compliance fundamentally influences the outcomes of conservation and related management interventions. In the marine realm, compliance has been linked to the ecological performance of marine protected areas (Pollnac et al., 2010; Campbell et al., 2012). In a broader sense, non-compliance with environmental regulations can threaten social and economic management objectives (Arias et al., 2015). Therefore, understanding drivers of (non-) compliance is crucial for the design and implementation of marine management efforts. Compliance can generally be defined as people's behavior that conforms to formal or informal rules which have emerged to influence actions (Tyler, 2006; Hauck, 2008). Within the fisheries context, the literature has highlighted different theoretical and empirical dimensions of compliance (Sutinen and Andersen, 1985; Kuperan and Sutinen, 1998; Hønneland, 1999; Sutinen and Kuperan, 1999; Hauck, 2008; Arias, 2015). Economic analyses of fisheries compliance have stressed that an individual's decision to comply or not with a rule is mainly based on a consideration of the potential economic costs (related to the certainty and severity of sanctions) and benefits of doing so (Sutinen and Andersen, 1985; Hatcher et al., 2000). More norm-based perspectives on compliance have emphasized internal and social incentives for (non-) compliance such as normative values, morality, perceptions of legitimacy and social justice (Hønneland, 1999; Jentoft, 2000; Raakjaer Nielsen, 2003; Hauck, 2008). The influence of the perceived legitimacy of rules on the rule acceptance by resource users has been widely stressed (Sutinen and Kuperan, 1999; Jentoft, 2000; Keane et al., 2008). Legitimacy aptly refers to the acceptance of decision-making and its outcomes by citizens (Van Tatenhove, 2013). It is related to the "perception that the actions and products of a certain entity are wished for and in accordance with a socially constructed set of norms, values, principles and definitions" (Van Tatenhove, 2011, p. 91). If resource users do perceive the rules and decisionmaking as legitimate, it is more likely that they choose to comply (De Vos and Van Tatenhove, 2011). The two perspectives, the more normative, i.e., norm-based, and the economic/rational choice view, on compliance are not mutually exclusive (Schlüter and Theesfeld, 2010). Furthermore, compliance is dynamic, changing in response to the local context. Thus, elements of both perspectives, as well as an analysis of how rules developed and what influenced them, are needed to gain a better understanding of compliance dynamics. Such analysis should therefore also question who defines rules and (non-) compliance as well as the power dynamics inherent in these processes (Hauck, 2008). Moreover, monitoring and enforcement is considered a key part of successful natural resource management (Ostrom, 1990; Gezelius, 2002; Keane et al., 2008) that can contribute to improved compliance behavior. It is often argued that the effectiveness of monitoring and enforcement influences how people evaluate the risks of rule-breaking (certainty and severity of sanctions) and thus determines the deterrent threat—which can influence people's consideration whether breaking a rule is worth the risk (Jackson et al., 2012). Marine governance and natural resource management systems are contextual, dynamic and continuously adapting to transforming social, political, economic and ecological conditions (Ostrom, 2007; Aswani and Ruddle, 2013). We argue that a better understanding of compliance dynamics, analyzing under what pressures and circumstances compliance can decrease or increase, is useful to gain improved insights into overall governance dynamics. This can be crucial to inform adaptive management of marine resources. In this article we conduct a study of two cases, located in Fiji and Solomon Islands, for an in-depth analysis of local compliance with local marine resource management. In both Melanesian countries inhabitants have long records of interaction with the marine environment. Customary tenure systems have been the prevailing management regime for inshore fisheries in the South Pacific for a long time (Johannes, 2002; Caillaud et al., 2004; Govan et al., 2009). However, these systems have not been static over time. In the late twentieth century customary marine tenure approaches seemed to be eroding due to various impacts of "westernization," e.g., the introduction of top-down management approaches and new fishing techniques as well as evolving market dynamics (Johannes, 1978). For the past two decades though, a reinvigoration of these initiatives has been ongoing, based upon communities' traditional knowledge and customary rights whilst integrating modern management principles and scientific advice (Johannes, 2002; Cinner and Aswani, 2007). Hence, in current practice, a hybrid that combines customary tenure systems and science-based conservation approaches is often promoted (Aswani and Ruddle, 2013). In many cases this happens in collaboration with nongovernmental organizations (NGOs) and other partner organizations, including from government, which brings in new actors and influences local management practices (Cohen and Steenbergen, 2015). These community-based management approaches have received wide recognition given their potential to promote local food security, sustainable fisheries management, and marine conservation (Govan, 2013; Weeks and Jupiter, 2013). Yet, customary governance and institutions—which are still at the core of these approaches—are being challenged by diverse socio-economic developments as well as cultural changes. This results in transformations, which have long been a feature of the Oceanic region (Aswani and Ruddle, 2013). Additionally, local leaders are constrained in their capacity to enforce local marine tenure rules owing to limited legal mandates. This is due to the fact that these rules, many of which are area-based (e.g., in the form of marine closures), are generally not legally gazetted under national law. In this study we will look at such marine closures, which we refer to as "managed areas." This study examines the emergent conditions that may challenge compliance with CBMRM—which can ultimately hinder it from achieving the above-mentioned social and ecological aims. We ask two questions: (1) what socio-cultural, economic and legal conditions drive local (non-) compliance within CBMRM? And (2) what challenges, and what supports, the effective enforcement of CBMRM? Previous studies in the region have examined local compliance with fishery rules and regulations (including national-legal; e.g., Pomeroy et al., 2015), where monitoring and enforcement mechanisms vary. Others (e.g., Jupiter et al., 2010) have examined compliance with CBMRM, including by "outsiders"—who do not have customary fishing rights to a given fishing ground. Overall, little attention has been given to a more in-depth assessment of compliance behavior of local villagers who have customary fishing rights within the respective managed area. Given locals' unique rights situation and the limited legal mandate for enforcing marine closures, it is important to elucidate different drivers of local (non-) compliance while scrutinizing the role of local social, political and economic contexts and their dynamism. This is the primary purpose of the present study. ## METHODS #### Study Sites Research was conducted at one site in each of Solomon Islands and Fiji (from now on referred to as SI and FJ, respectively). The case study sites were selected purposively, which allowed choosing cases that illustrate features or processes considered relevant for this study (Silverman, 2010). Research sites were selected to feature communities that: (1) directly use local marine resources, (2) have some form of management regime established for a considerable period of time, and (3) where management arrangements have been supported and accompanied by partner agencies as part of conservation and development initiatives. These three factors were considered because they are likely to influence (perceptions of) management and compliance dynamics. #### Fiji In Fiji, the Fisheries Act (Cap. 158) grants native Fijians customary fishing rights in their respective traditional fishing ground (qoliqoli) (Minter, 2008). Customary chiefs and clan heads can control access to fishing areas and make decisions regarding local marine tenure. Generally, chiefs and communities have decisive authority over local resource management (Veitayaki, 1998). Fishing areas are clearly demarcated and thus spatially define access rights (Weeks and Jupiter, 2013). Although the Fisheries Act remains the primary piece of legislation for inshore fisheries, customary right holders have unique and exclusive access rights to their traditional fishing ground. This creates a legal pluralist situation that can challenge local enforcement capacities in the face of current CBMRM practice. In Fiji a village on the island of Ovalau, Lomaiviti Province, served as local case study site (FJ, Figure 1). With its ∼350 inhabitants, FJ shares a traditional fishing ground with four other villages, although these have separate managed areas. In this village, marine resources and fishing have traditionally been an important part of peoples' lives. Yet, other livelihoods (mainly small-scale agriculture) are available and additional income-generating activities (mainly through employment in the nearby fish factory, especially for women) are practiced. The village set up a managed area in the form of a periodicallyharvested closure in front of the village about 10 years ago, with assistance of a regional network and other partners. The site has generally remained closed to any harvesting activities since its establishment. However, a small section of the managed area has been opened several times for a couple of days in cases of chiefly (or other important villagers') deaths. #### Solomon Islands In Solomon Islands the constitution and fisheries legislation also recognize customary rights. Diverse socio-cultural, historic and economic processes have created differential and contested territorial customary rights systems. As a result, customary marine tenure systems vary regionally and are generally more stratified, decentralized and politically eclectic than in Fiji (Aswani, 1997, 1999). FIGURE 1 \| Map of Fiji and research area. In Solomon Islands, our research was conducted in a village in Roviana Lagoon in the Western Province (SI, Figure 2). In this area, customary chiefs and elders control access to and use of marine resources. Villages in Roviana Lagoon are remote and only accessible by boat. Approximately 1,000 people live in SI. Households are highly dependent on marine resources. Fishing is the single or second most important livelihood, together with small-scale agriculture. Local marine resource management consists of a marine closure in front of the village that is permanently closed for all fishing and other marine harvesting activities. This site forms part of a marine conservation and development program initiated in the late 1990s that included 32 management sites across the Western Solomon Islands. The program was established with advice of foreign academic experts and was financially supported through external (international) donors (see Aswani et al., 2007). A community-based organization (CBO), founded in the course of the program, assisted with the management and implementation of the marine closure in collaboration with local leadership (customary and church leaders) and a local resource management committee that was created for this purpose. ## Qualitative Data Collection In this study we wanted to examine the complexities of locals' perceptions and behavior around compliance, while considering the local cultural context, including the meaning of norms and informal rules. Ethnographic data were collected using various qualitative methods (Corbin and Strauss, 2008; Silverman, 2010). We employed semi-structured and key-informant interviews (total n = 63; thereof 29 in Fiji and 34 in Solomon Islands), focus group discussions (total n = 5; thereof 3 in FJ and 2 in SI) and participant observations. Policy and legal documents (fisheries legislation) served as secondary data sources for triangulation. Data were collected over a 2 month period in each country, between April and June 2015 in Solomon Islands and between August and September 2015 in Fiji. A total of 99 persons participated in the study, 48 of which participated in Solomon Islands and 51 in Fiji. Interviews and focus groups involved respondents from different governance levels: village (n = 76), province (n = 4) and national (n = 19). At the national and provincial levels interviews were conducted in English. Interviews and focus groups with village respondents were held in the respective local language (Fijian and Roviana), with the help of local interpreters. Prior informed consent was sought orally from all research participants. Research was conducted in accordance with all ethical standards outlined in the White Paper on Safeguarding Good Scientific Practice by the German Research Foundation (DFG, 2013). An ethics approval was not required according to the DFG guidelines, as well as to our institutional guidelines and the regulations in the study locations. All required procedures for conducting research and obtaining research permits in the study locations were followed closely. #### Sampling At the village level, interviewees were selected purposively to cover a broad range of potentially relevant respondent characteristics and individual perceptions. The following contextual variables were developed for this sampling process: (1) marine resource users; (2) persons involved in local decisionmaking; (3) age; (4) gender; (5) church denomination (mainly for the Solomon Islands case study, where this emerged as a key aspect for people's role within and perceptions of the community); (6) involvement in other livelihoods apart from fishing (mainly for the Fijian case study, where this seemed to lower direct resource dependency and hence influence views on the marine closure and compliance issues). Interviews were used to get an overview of relevant themes and divergent views regarding local (non-) compliance. Selecting interviewees at the village level was done in an iterative process. Thus, initial analysis and results gained through the interviews were used to elaborate the above-mentioned contextual variables and to select further interviewees as well as key informants. Interviews at the village level were conducted until no new additional information or themes were emerging (i.e., until saturation was reached). Interviewees may not represent the view of everyone in the village due to the fact that we used non-random sampling. In order to minimize potential biases (e.g., due to personal networks and social relations) during the selection of research participants, we worked with interpreters who were not from the research sites. Focus groups built upon key themes that emerged from the interviews, allowing for data triangulation. Focus groups were comprised of seven to eight participants each. They were conducted separately for women, men, and in Fiji also for the youth, because there a stronger generational differentiation became apparent. The majority of the focus group discussants were involved in fishing. Some (especially older men) were or had also been involved in local decision-making, and some had a different income source (especially women in the Fijian case study through employment in the fish factory). Focus group participants were chosen purposefully in collaboration with local field assistants while seeking a good balance of potentially relevant characteristics of the participants (e.g., church denomination in the Solomon Islands case study). At the provincial and national levels, representatives from relevant ministries or departments (e.g., fisheries and environment), civil society (e.g., NGOs and international organizations) and academia were interviewed. Purposive and snowball sampling was used to identify respondents at these levels. Using a snowball sampling approach runs the risk that only certain opinions are heard due to path dependencies. In order to avoid this fallacy we sought specifically to interview actors with potentially opposing views. #### Interview Guidelines and Questioning For data collection at the village level we used indirect questioning, i.e., asking respondents about the compliance behavior of others and not (necessarily) their own. This was done in order to diminish potential response biases (people giving inaccurate information), as well as nonresponse biases (people refusing to give information) (Arias et al., 2015). Such biases were expected because questions around (non-) compliance touch upon sensitive issues, especially in a small village setting. The utility of such an assessment of what can be referred to as "perceived compliance" as a proxy for actual compliance has been demonstrated in other studies (Arias and Sutton, 2013). Yet, it should be taken into account that theories such as the social norms approach (Berkowitz, 2005) argue that people tend to overestimate the non-compliant and negative attitude of their peers beyond their actual behaviors. Nevertheless, it is also conceivable that a direct approach would not have revealed much non-compliance either, or that interviewees would have belittled their own infringements. Guidelines were used for the interviews and focus groups at the village level (see Supplementary Material). Through the interviews we first wanted to gain an understanding of the local marine resource management arrangements, changes regarding these arrangements, and peoples' perceptions of them. Therefore, interview questions firstly enquired about: existing and previous local marine resource management, perceptions of their purpose and benefits, issues regarding local decision-making, and peoples' participation in it. Secondly, questions were asked about: perceptions of local compliance with rules, enforcement, and local management of disputes. Similar questions and themes to the ones addressed in the interviews guided the focus groups, though in a more interactive and collective way (Mancini Billson, 2006). For example, participants were asked to discuss reasons for non-compliance and collectively identify and rank the most important drivers of non-compliant behavior, while being asked to explain for whom (which group) these drivers were important and why. Furthermore, participant observations (e.g., attending informal gatherings, a village market, and a church service; going to sea with fishers; accompanying locals on village walks) were used to further contextualize and triangulate information obtained otherwise. At the provincial and national levels interviews enquired about: respondents' perceptions of the potentials and challenges of local marine resource management in their country, their views on local compliance, enforcement, and the role of the national legislation (and, if applicable, of the respondents' agency) with regards to this. #### Data Analysis Data from all interviews and focus groups were transcribed and analyzed inductively through open, axial and selective coding (Corbin and Strauss, 2008). Qualitative data analysis software (MAXQDA) was used to identify and relate emerging themes and patterns from the transcripts and to link these to secondary data sources (e.g., legislations) as well as to the theoretical and empirical literature. Further information on the codes used for the analysis can be found in the Supplementary Material. ## RESULTS Data from our research reveal that in both study sites many locals—including some who were non-compliant—perceived local non-compliance with the marine closure as a problem with the potential to dismantle the social and ecological effects of these local management efforts. In SI, consensus was reached in the focus group discussions that people from different age groups, gender, and church denominations were inclined to disobey the managed area. In FJ, our data from interviews and focus groups reveal that mainly young male divers who lacked an alternative stable income tended to disobey the marine closure. In FJ, due to its relative closeness to the capital city, the managed area was challenged by external poachers (mostly commercial fishermen with fast boats) besides local non-compliance. In the following, we summarize the main aspects related to (non-) compliance that emerged from our two cases, before presenting individual results from each case study. In both case studies, results from the interviews and focus group discussions suggest that lowered perceived legitimacy of local rules (including the marine closures) and leadership was acting as a driver of non-compliance with the local marine resource management. Furthermore, economic incentives, e.g., related to market opportunities to sell fish, influenced noncompliance, too. Additionally, we find that the physicalgeographical conditions of the managed areas, mainly related to their size and location, constrained villagers in accessing their primary fishing grounds, and thus promoted non-compliant behavior. Finally, our study reveals different barriers for monitoring and enforcement in the context of the two case studies. In both countries (and both case study sites), managed areas were generally not legally gazetted, and thus enforceable, under national law. This means monitoring and enforcement of local marine management in FJ and SI relied on local villagers and customary governance systems. Yet, in both case study sites these were constrained in a number of ways. ## Fiji #### Lowered Legitimacy of Rules and Rule-Makers In FJ our study reveals two sets of aspects that partly constrained perceived legitimacy of the managed area, particularly for young fishermen. Firstly, data from interviews and focus groups reveal that a lack of participation and consultation regarding the local management, particularly about the size and location of the managed area, lowered perceptions of its legitimacy. Especially male youth, but partly also older women, voiced that they did not feel involved or consulted during the establishing process of the managed area—and thus perceived the managed area as illegitimately constraining their customary fishing rights. In Fiji, the Fisheries Act grants customary rights to fish inside a certain qoliqoli (traditional fishing ground) to every native Fijian whose mataqali (clan or other subdivision) has been registered by the Native Fisheries Commission. This basically applied to all inhabitants of FJ. Although older male informants expressed, and other sources confirmed, that a consultation process did take place, it might not have involved all actors within the village that currently impacted upon and were impacted by the managed area. The current youth were still too young to witness the establishment process of the managed area 10 years ago, and were hence not part of that consultation. Women did not participate actively during workshops for other reasons (e.g., due to traditional gender roles that constrain women from actively participating in such meetings, or time constraints related to family responsibilities, or work at the fish factory). Secondly, the current managed area varied from customary tenure arrangements practiced in earlier times with regards to its permanent nature as well as to its (perceived) main purpose. The first aspect relates to a change in the periods of closures. Data from our study reveal that the managed area was set up in a way that locals perceived as a permanent closure. Although customary leadership decided a few times to open one section of the managed area in the past in case of a chief's or another important villager's death, the "general status" of this part of the fishing ground was "closed." In previous times, the general status was "open," with temporal closures of a smaller section of the fishing ground to commemorate when a chief died. Therefore, villagers generally perceived the current managed area as permanent closure. This means the managed area was restricting fishing activities considerably more than previous practices used to. The second aspect relates to the perceived main purpose of the management interventions. Previously, customary approaches had served to impose periodic closures for special cultural and social events (commemoration of a chief's death). Indicated by consensus reached in a focus group discussion, these customary closures were thus perceived as mainly serving the (socio-cultural) purpose of having food for that special occasion and not for resource management. Focus group discussions disclosed that in current practice, locals perceived the aim of the managed area as being more focused on (longer-term) conservation objectives (notwithstanding that these conservation objectives are ostensibly linked to ensuring food security and thus allow for a continuation of the culture of fishing). Both factors, the lack of participation and consultation as well as perceptions of the altered nature and purpose of the management arrangement, were especially stressed by young fishermen. Among this constituency, they lowered feelings of ownership for the local management initiative and increased the perception that the restriction of their customary fishing rights by the managed area was illegitimate. This in turn then drove non-compliant behavior by this group. #### Market and Income Opportunities as Incentives There was consensus among respondents that in FJ especially young men resorted to poaching as (small-scale) income generating activity. They mainly did so when they did not have an alternative source of income, such as work in the near-by fish factory. Because the catch per unit effort (CPUE) was (perceived) higher inside the managed area, fishing there promised a fast catch and easy cash when selling the fish, which happened mainly on local markets. #### Physical-Geographical Conditions of Marine Closures In FJ the managed area was located right in front of the village, stretching from the shore to the outer reef slope. This area had been chosen because indigenous ecological knowledge indicated that it was especially ecologically relevant. Yet, its location clearly limited the accessibility of the (permitted) fishing grounds. It implied that local fishers either had to walk long distances during low tide or that they needed a boat—which were rare. There was only one bamboo raft and two fiberglass boats available in the village; the latter came with costs for the fuel. These limitations to access the fishing grounds outside the managed area, implying more time and money investments, were an impediment to compliance with the managed area. Respondents throughout the interviews agreed that this could further promote fishing inside the managed area. This was more likely when coupled with the above-mentioned economic incentives, and applied especially to young fishermen who lacked an alternative livelihood or income. #### Barriers for Monitoring and Enforcement Although most managed areas are not legally gazetted in Fiji, they do receive partial legal back-up under the Fisheries Act under two scenarios. Communities can "arrest" a poacher who is caught fishing inside the managed area and take this person to the police or closest fisheries office if: (1) it is a licensed fisher who is not respecting the managed area (more commonly applies to commercial fishers from outside the village who do not have customary fishing rights in this area), because license conditions prescribe that local (customary) management rules ought to be respected, or (2) someone is selling fish (no matter where the fish was caught) without having a license. Nonetheless, these scenarios have limited applicability to local non-compliance where people draw on customary fishing rights to engage in subsistence fishing. Under the current Fisheries Act small-scale sale of catch that exceeds subsistence needs has so far been tolerated for customary fishing right holders. This is why the second scenario does usually not apply to local poachers. Yet, this issue has been recognized as a gray area and will probably be addressed in the course of reviewing the fisheries legislation—a process that was initiated in 2006 and is still ongoing. #### Institutional Constraints In FJ it is common to announce local poachers in village meetings or during church services. Commonly the chief or village headman speaks to that person to issue an oral warning, too. Nevertheless, respondents agreed that the deterrent threat of these penalties was not very high, especially because there were no graduated sanctions (nothing more serious happens if that person poaches again). Further enforcement efforts of local marine resource management initiatives remained limited. In order to enforce managed areas under national law—in Fiji under one of the two scenarios described above—the police or another state authority would need to get involved. In Fiji local voluntary fish wardens are appointed to monitor local marine tenure rules (managed areas). At the same time these wardens are tasked with monitoring local compliance with national regulations, such as species restrictions and mesh size of nets, under the Fisheries Act. Fish wardens can be appointed by the Minister of Fisheries after receiving training by the Department of Fisheries. They are not paid nor do they receive any substantial financial support or equipment. Not all villages in Fiji have fish wardens—it rather depends on the initiative of the village itself and/or supporting partner organizations. Fish wardens or local leadership who want to report infringers to the police need to keep and demonstrate evidence of the infringement (e.g., confiscated catch, gear, boat). Yet, besides the training that fish wardens receive by the Department of Fisheries, they are not trained as prosecutors, and thus not familiar with the details of keeping evidence. Additionally, fish wardens often face police officers who are not aware of the fisheries legislation or who are reluctant to investigate and prosecute fisheries crimes. Furthermore, findings from FJ show that, in former times, the temporal closures on fishing areas declared by customary leaders were more strongly related to a socio-cultural purpose and tradition (chiefly deaths). As a result, these taboos were more respected than current managed areas because "it [was] part of tradition and [breaching it] would go directly against the chief," as expressed by one respondent in FJ. The current managed area had been supported by partner organizations that have more clearly brought in conservation objectives as part of a sustainability discourse. Consequently, locals perceived the managed area in FJ less as a strict taboo in the traditional sense. Thus, offenses were perceived to oppose rules that had been influenced by external actors, instead of directly opposing a chiefly decision. #### Socio-Cultural Constraints Our study reveals various socio-cultural constraints for local monitoring and enforcement in FJ. Firstly, fish wardens were constrained in reporting local infringers due to the strong network of clan and family relationships they were embedded in. This means fish wardens usually knew local infringers well and might therefore have been reluctant to report non-compliant behaviors, as this might have negatively impacted their own social relations. The same limitation applies to chiefs and clan heads, who would decide about consequences to take against local poachers if these were reported by the fish wardens or other community members. Secondly, the selection process for appointing fish wardens did not seem to be transparent and not everyone within the village was aware of the identity and the role of the wardens. This further impeded their work because some people might not have recognized their authority at all, or (again), perceived it as less legitimate. #### Technical and Financial Constraints In FJ villagers agreed that the area of the marine closure was too big to monitor from the shore, and monitoring would thus require patrols by boat. Yet, boats and/or money for fuel for the boats were lacking. Detection of infringers was further made difficult because poaching occurred at night most of the times. Additionally, respondents throughout interviews agreed that two fish wardens for the managed area were not sufficient, especially given that both of them were active fishermen, too. This means that many times they were involved in harvesting activities themselves while not being able to solely concentrate on monitoring the managed area. ## Solomon Islands #### Lowered Legitimacy of Rules and Rule-Makers In SI data from our study disclose that two developments generally lowered the perceived legitimacy of local leadership, which included chiefs and the council of elders who made decisions about marine tenure, persons who implemented these (e.g., rangers), and the church. One of these developments was associated with the (perceived) misuse of money; firstly in relation to logging activities that were ongoing on the main island opposite the village, but also with regards to the management of the managed area. Since inhabitants of the community owned the land where the logging company was operating, they had been receiving a substantial amount of "royalty payments" on a regular basis. This money was given to some of the leaders ("trustees") to share it among the community. A large proportion of interviewed villagers perceived that this did not happen in a transparent and equal way. This caused perceptions of unequal benefits and procedural unfairness in SI, and was given as a reason for non-compliant behavior as a payback strategy against the leaders. Similarly, many locals perceived that the money that was given by international donors to support the local CBO and resource management committee (including local rangers) was not being used transparently and in a way that was beneficial to everyone. Particularly some women expressed their growing mistrust toward the (predominantly male) leadership, highlighting that they no longer followed rules (i.e., the managed area) made by this leadership. As one woman pointed out when asked about conflicts with regards to the managed area: "Yes, there were also conflicts, especially with the money. These people [referring to the local leadership] are not reliable. [...] They are not transparent. [I] was one of these poachers [...]. [But] I was not poaching, [I] was showing that [I] was not happy about the decisions [the leadership was taking], so when they [rangers] came up, [I] would tell them 'you tell me where the money went, and then I stop fishing in this area."' In SI women actively participated in fishing and harvesting of other marine resources (mainly shells), thus providing an important food source for their families. Yet, they hardly took part in local decision-making processes, including with regards to the management of marine resources, and were underrepresented in leadership committees such as the council of elders. Over the last century chiefs were mostly male, too. The second development that led to diminished, and divergent, perceptions of the legitimacy of the council of elders and leadership in SI was related to the division of the predominant local church. In SI religious leaders played an important role within customary governance processes for a long time and were involved in ongoing conservation and management initiatives. The leader of the predominant local church gave his blessing for the managed area years back (in 2005), which was of great importance and meaning for locals in order to respect it as conservation area. Later, when this spiritual leader fell sick, discussions about his succession turned into a conflict between the two competing aspirants and their respective supporters. The church members were divided into two groups. The division affected the council of elders and other leadership meetings, too, because most of their members belonged to one or the other group. Since the death of the (former) spiritual leader in 2014, animosities between both groups increased further. The schism not only generated divergent views as to which spiritual leader to follow from now on, but also doubts about the acceptability of leadership, such as the council of elders, and their decisions. Data from interviews and focus groups reveal that it also generally deteriorated feelings of shared group membership and identity as well as trust within the community. This further diminished cooperative and prosocial behavior including with regards to the managed area, where this led to more non-compliance. A further aspect that is enhancing heterogeneity of the local population and adding to socio-cultural diversity is the fact that in SI, many people from other tribes and provinces were marrying into or out of the village. This was referred to as "intermarriage" and was a common theme coming up throughout interviews with diverse respondents. Although intermarriage does not necessarily lead to disunity, it was mostly raised as potential problem that had detrimental effects including on compliance with the managed area. An older fisherman in SI expressed: "[...] Those people who are married here, because they have different traditions and "kastoms" in their respective homes, they just don't tend to follow [the rules set by the local leaders, such as the managed area]." Our study shows that people's decisions to comply were influenced by the perceived compliance behavior of others in the community and perceived unequal distribution of benefits (from rule-breaking), as this statement by a male respondent in SI underlines: "I support not to fish in conservation areas. But if I keep on talk talk talk to members of the community not to go out [to fish inside the managed area], and people do not listen to me, do you think I will keep on talking talking talking? I will also want to have fish. In turn I will go and fish [inside the managed area], too." Finally, and similarly to findings from FJ, results from a focus group in SI reveal that locals perceived the current marine closure as an alteration of former customary practices and temporal closures. This was firstly due to its permanency, and secondly, due to a perceived shift from a socio-cultural purpose toward a stronger focus on conservation objectives. #### Market and Income Opportunities as Incentives In SI there was consensus throughout the focus group discussions that poaching within the managed area for an income intensified when new market access opportunities arose through middlemen and ships of the logging company operating nearby. The latter offered access to the market of the island's capital because villagers could put their coolers with fish for free on the logging ships when they made their way to the capital harbor, and sell it there. In SI alternative sources of cash income apart from fishing for sale were limited to marketing of local products, including copra, and operating small stores. Hence, for people wishing to earn some money (e.g., to pay school fees for their children or for church contributions) instead of living merely on a subsistence basis, poaching and selling the fish became more attractive. Again, there was consensus among respondents that the CPUE inside the managed area was higher (which is also related to the biophysical and ecological characteristics of that area), which further incentivized fishing inside the area for an "easy catch." #### Physical-Geographical Conditions of Marine Closures In SI the managed area was located directly in front of the village, too. The location of the marine closure had been decided based upon: indigenous ecological knowledge, scientific ecological surveys, and ease of monitoring (due to its proximity to the village), and the decision happened in consultation with the local leadership. Interview respondents similarly agreed that some (in this case especially older) people poached because they were "too tired to go far out to fish." Further, female respondents raised the complaint that the closure particularly constrained their fishing activities because it was set up in an area where mainly women used to fish. As one (female) respondent put it: "That is the number one place where [women] used to fish!" She further explained, and other respondents confirmed, that this was due to the area's closeness to the village. Women, due to their multiple responsibilities in the household and involvement with child care, could not afford to spend long hours fishing or go to more distant fishing grounds. #### Institutional Constraints for Monitoring and Enforcement In SI an interview respondent expressed that, apart from national laws that prohibited most customary penalties, the influence of missionaries and Christian imperatives weakened the effect of customary sanctions. This is related to the concept of "forgiveness" that was introduced by Christian missionaries in the early twentieth century. An old fisherman in SI explained: "Before missionaries came and before the church was here, our traditional governance [was] much more feared [...]. Because we only had one rule with[in] our traditions: you do something wrong which is against our traditions, we will kill you. [In the course of Christianization these penalty systems were weakened]: so when you break rule in our traditional way, the church comes in. That is forgiveness. When the church comes in, there is no longer value for our traditions." On the other hand, the involvement of the religious leaders in the marine conservation program in SI also served as a vehicle for reviving traditional systems and strengthened traditional leadership. Before the schisms of the local predominant church the fear of the spirit of the spiritual authority was very strong. But as the strength of this leadership weakened, so did the fear of retribution. In general, the perceived risk of sanctions for non-compliance with the managed area was very low in SI, and therewith the deterrence threat for rule-breaking. Previously, non-compliance with the managed area used to be addressed in village meetings and/or church gatherings to publicly announce and warn offenders. This became less common since the schism and the resulting division of the community, because generally less village and leadership meetings took place since then. Also, the effect of verbal warnings and public announcement recently decreased due to the community division and resulting divergent views on the legitimacy of leadership which made these announcements. #### Socio-Cultural Constraints In SI the church schism and related division of the community also affected monitoring activities of the local rangers who used to detect and "arrest" (stop/talk to) local infringers who fished inside the managed area. Back then, incidents were reported to the local resource management committee and/or to the council of elders who would then announce it in respective meetings. When data for this study were collected, these rangers were no longer active and no one else took on the responsibility of monitoring. This was partly due to the mentioned socioreligious division of the community, as well as to other disputes around (the perceived misuse of) money and logging, which significantly weakened the role of the supporting CBO and the rangers. #### Technical and Financial Constraints In SI villagers and rangers had access to wooden canoes that can be used for monitoring the managed area. However, lack of funding for the CBO that was previously supporting the management and equipping rangers, e.g., with torches for their night shifts, is another reason, besides the ones mentioned above, that was recently constraining monitoring activities. Rangers had previously complained about a lack of regular salaries. A self-sufficient and sustainable structure to finance monitoring activities was envisaged by the supporting external partners in SI but did not succeed. Still, other (mainly female) interview respondents conveyed their impression that rangers were receiving payments for their work in the past, which in their eyes increased (financial) inequalities in the village. #### DISCUSSION Natural resource governance and management systems adapt to and are conditioned by the larger social, political and economic realms they are embedded in Aswani and Ruddle (2013). Compliance with these systems is equally dynamic. Recognizing this dynamism, and analyzing under what pressures and circumstances compliance can decrease or increase, can help to understand how to tackle problems in adaptive management of marine resources. In order to do so, we have taken a snapshot of (non-) compliance in two case studies in Melanesia. Compliance has been recognized as fundamental for successful marine conservation (Keane et al., 2008; Arias et al., 2015; Cinner et al., 2016). Therefore, understanding drivers of (non-) compliance as well as barriers to enforcement is crucial in the assessment of marine management. In this study we find that people's compliance behavior is influenced by many factors. Non-compliance is partly driven by lowered perceived legitimacy of local decision-making and its outcomes. Furthermore, financial incentives and the physical-geographical conditions of the managed areas—constraining access to primary fishing grounds—can make fishing inside these areas more attractive or necessary. Finally, data from our study reveal that enforcement is impeded through various (institutional; socio-cultural; technical/financial) constraints, so that the deterrence threat for rule-breaking is rather low. These findings are consistent with other empirical studies and theoretical literature on compliance that have highlighted that both economic motivations as well as normative and social aspects—including around the process of how and by whom rules were set up—influence people's decision to comply (or not) (Gezelius, 2003; Hauck, 2008), and that monitoring and sanctioning is crucial, too (Ostrom, 1990; Gezelius, 2004; Keane et al., 2008). These drivers and factors can act together and add up to impair effective management. For example, in FJ we find that young fishermen were more likely to poach when they did not have an alternative livelihood or other income. They did so more readily: firstly, because they felt that the managed area was illegitimately constraining their customary fishing rights (partly because they did not participate in its establishment process), and secondly, because they knew that potential penalties were unlikely (since they were making use of their customary fishing rights and did not have to fear sanctions under national law). In SI more people were poaching, and increasingly did so to make money as a consequence of easier market access, because perceptions of the legitimacy of leadership diminished as a result of the schism and perceived money misuse. Also, the community division resulting from the schism had weakened the role and endeavors (as well as acceptance thereof) of leadership and rangers with regards to monitoring and enforcing the managed area. Nonetheless, in SI the leader of the predominant local church had previously been a vehicle of the conservation initiatives, which first increased their perceived legitimacy amongst villagers. This again points to the dynamism of governance and local leadership, as well as their potential vulnerabilities. It is conceivable that a revitalization of this leadership will result in the re-establishment of the managed area and its rules and monitoring. This highlights the need to develop "fallback systems," especially when conservation initiatives build upon such local leadership structures. #### Contextualizing Drivers of Non-compliance In FJ the fact that current young fishermen were too young to witness the establishment process of the managed area, and thus did not participate or feel involved in decision-making for local marine resource management, turned into a driver for noncompliance for this particular group. Women were generally less involved in the consultation about the managed area, too. But because many women were employed in the fish factory, they did not perceive this as negatively as young fishermen, who depended more directly on fishing for their livelihood. Also, young local fishermen had witnessed commercial fishermen from the capital city poaching in the managed area. This might have further encouraged young local fishermen to fish inside the managed area because in the face of such a race for fish locals may have wondered why they should obey the marine closure if others were reaping the benefits. Other studies have similarly revealed that young age, which often goes along with less participation in the process of developing the rules, can lower support for and compliance with such rules (e.g., Schlüter and Madrigal, 2012; Madrigal-Ballestero et al., 2013). This highlights the importance of participation with equal access for and representation of all groups affected to generate legitimacy and increase rule acceptance (Jentoft, 2000; Van Tatenhove, 2013). In the cases studied here it was obviously not practically feasible to involve future fishing generations in the participation process 10 years ago. Yet, it shows that participatory, outreach and consultative processes should take place continuously and repeatedly to renegotiate management arrangements if necessary. In order to do so, funding programs that support partner organizations engaged in CBMRM would need to consider longer-term funding cycles and perspectives. Van Tatenhove (2011, 2013) describes the process of increasing rule acceptance through participatory measures as "input-legitimacy," whereas "output-legitimacy" refers to whether decision-making succeeds in promoting common welfare for all people affected by these decisions. In SI the perceived misuse of money on behalf of the leadership and people involved in the management of the managed area led to perceptions of unequal benefits, lowering output-legitimacy. The experience of procedural unfairness through such unfair decision-making and/or outcomes thereof can erode "feelings of shared group membership with the authority concerned" as well as the identification with the rules that this authority establishes (Jackson et al., 2012, p. 1053). The previously-quoted statement by a fisherwoman in SI shows that this was the case, as she expressed that she perceived fishing inside the managed area no longer as poaching, but as a way to show her disagreement with the rules and with the unfair behavior of leadership. Generally, trust and cooperation among resource users are proven to be crucial for effective local governance of commonpool resources (CPRs) and make a sustainable use of CPRs more likely (McCay and Acheson, 1987; Gibson et al., 2000; Basurto et al., 2016). This elucidates why a decrease in trust, including toward leadership, and cooperative behavior within the community that followed from the schism, recently diminished compliance with the marine closure in SI. Intermarriages were adding to this because they can counteract feelings of shared group membership toward leadership and increase tenurial claims over fishing access. This intensifies fishing pressure and potential conflicts over (access to) resources. Also, people from outside of the community bring in their own tribal identity and traditions, which are very diverse across Solomon Islands (Aswani, 2002). Finally, an increasing number of people (temporarily) migrate out of the communities for marriage or employment, but they uphold their customary rights. This can result in increased non-awareness of "outside rights holders" with regards to local rules and management arrangements. All together, these factors might imply less respect for the local customary leaders and the decisions made by them. Although the schism and intermarriages revealed in SI can be seen as rather case specific phenomena, they also demonstrate the high degree of stratification and contestation of socio-political and tenurial systems in Solomon Islands (Aswani, 1999). Furthermore, the case demonstrates the dynamic and dual role that the church can play in local marine management, as pointed out before. In both case studies villagers perceived the marine closures as being more focused toward achieving conservation objectives, instead of serving primarily cultural and social purposes like former customary closures used to (Cinner and Aswani, 2007; Foale et al., 2011). Management had shifted from periodic closures to periodic openings/permanent closures, which limits the flexibility of these marine tenure systems (Hviding, 1998). Both management sites had been partly supported by and/or implemented in collaboration with external partners. They exemplify the hybrid approach that lays the basis for much of the current CBMRM practice that merges customary management and traditional ideas with conservation practice and sustainability discourses. Yet, locals distinguished between these practices and the underlying objectives, as other studies have found, too (Jupiter et al., 2014; Cohen and Steenbergen, 2015). Our study shows that this is likely to affect perceptions of legitimacy of these rule systems and thus local compliance with CBMRM. Also, violations of rules that were externally influenced were considered less severe than a breach of customary norms and taboos. These aspects should be considered by partner agencies that work with communities in the frame of CBMRM initiatives. Market access and the lack of alternative livelihoods were increasing non-compliance in both case studies. Numerous studies have highlighted market access and proximity, and the commercialization of marine resources, as key drivers for resource (over-) exploitation, with the potential to affect local management regimes (Aswani, 2002; Cinner and McClanahan, 2006; Brewer et al., 2009; Cinner et al., 2012, 2016). Similarly, the reliance on fishing as single livelihood has been proven to negatively affect local compliance with marine conservation areas (Arias et al., 2015). On the other hand, evidence also suggests that high dependence on marine resources at the community level can be a contributing factor for sustainable local marine resource management (Cinner et al., 2016). The apparent contradiction may be explained by the potentially contrasting effects of dependence on marine resources at the household and the community level. While communities with a high overall reliance on marine resources can be compelled into collective action by this dependency (Ostrom, 2009), individual households within a community might be more strongly compelled to break the rules if marine resources constitute their sole option to generate income and sustenance. The location and size of the managed areas restricted access to the most accessible (and productive) fishing grounds in front of the villages. In both cases location and size were decided based upon indigenous ecological knowledge. The locations of the closures were also selected due to their proximity to the villages and so to enhance feasibility of monitoring. Yet, because the managed areas constrained villagers in their ability to fish, this proximity partly turned into an additional driver for non-compliant behavior in both case studies. This illustrates how a feature that was initially seen as an advantage can turn into disadvantage when conditions and context change. Furthermore, it shows that there can be critical trade-offs between achieving ecological objectives and social acceptance (compliance) with regards to managed areas. Ecological requirements for conservation might require a certain spatial and geographical scale for management, which might not in all contexts be socially acceptable (Johannes, 2002; Foale and Manele, 2004; Mills et al., 2010), and hence less complied with. Besides, it points to the fact that the costs and benefits of conservation efforts might be unevenly affecting different social groups (see Eder, 2005 for a case study from Philippines). In SI especially older people and women were constrained by the managed area. In FJ particularly young fishermen who needed to fish because they were lacking an alternative income, but who at the same time did not have a boat to reach more distant fishing grounds, were bearing a higher burden. In this light the importance of continuous participative and communicative measures in order to increase ownership, legitimacy and support of management rules (Jentoft, 2000; Pomeroy et al., 2015) becomes even more evident. Such measures should take into account differentiated impacts of management efforts on diverse social groups (Gurney et al., 2015) and potentially adapt marine management to ensure more equitable arrangements. At the same time, the just-mentioned reasons for noncompliant behavior again highlight the importance of acknowledging the wider social and economic context of illegal fishing practices and non-compliance. For examples from Indonesia, where existing local elites and complex patronage (patron-client) networks have contributed to non-compliance with marine management rules, and thus hampered conservation efforts, see Lowe (2002), Ferse et al. (2012), and Kusumawati and Visser (2016). #### Addressing Barriers to Monitoring and Enforcement Customary governance systems are not static over time but have always operated within dynamic socio-cultural, political and economic contexts (Aswani and Ruddle, 2013). This has similarly affected legal and institutional aspects of monitoring and enforcement, such as penalty systems. Also, as the Solomon Islands case study demonstrates, some institutions that strengthened customary tenure systems in a certain setting can have a different effect when the context changes. At the same time, findings from FJ and SI reveal that diverse socio-cultural constraints can limit local monitoring and enforcement efforts, especially in a setting where sociocultural relationships between tribes, clans and families are highly complex and an important social capital, as well as a crucial aspect of local culture and identity. This shows how socio-cultural values that remain from customary systems and traditions can also potentially hinder the effective implementation of hybrid management arrangements if enforcement responsibilities solely rely on the local level. Customary fishing rights, which are recognized by national law as part of customary law, are at the core of the local marine resource management schemes studied here. Yet, the local management arrangements (marine closures) as such were not legally recognized by any national law. This has created a legal pluralist situation where different legal ideas and systems exist within a single setting (Scaglion, 2004; Hinz, 2008; Jentoft et al., 2009). It is crucial to evaluate how customary and state law interact or to which extent one of them is dominant (Jentoft, 2011). Generally, customary institutions and law remain the core means to resolve disputes in rural communities in Pacific societies until now (NZLC, 2006), including in the context of marine resources. Hence, they play a key role for the enforcement of marine tenure systems. In the past, customary penalties for breaking taboos included beatings, banishment or destruction of property. Breaching marine customary taboos, such as marine closures, was mainly punished by compensation payments in form of traditional money or livestock, social alienation or exclusion (Cinner and Aswani, 2007; Jupiter et al., 2010). Yet, the national legal systems have largely constrained the customary penalty systems given that nowadays most customary penalties are prohibited under national law. This effectively made the customary approaches less powerful. In the cases studied here, customary law was locally decisive for managing the use of and access to marine resources. Nevertheless, in the current settings the customary systems alone were no longer capable of enforcing these local rules and to sanction non-compliance. This was partly due to national laws that restricted the customary penalty systems, without (yet) providing an adequate substitute. This does not only show how modern legal systems can potentially lead to a (partial) erosion of customary management (Cinner and Aswani, 2007). It also highlights the need to clearly define and establish the roles and responsibilities of other (including government) actors involved in current CBMRM practice in order to ease their implementation and effective enforcement. In FJ and SI, sanctions that were practiced in cases of local non-compliance, such as oral warnings and public announcements, were showing limited success. Furthermore, those in charge of supervising compliance with local rules can be challenged by conflicting allegiances in pursuing their tasks, as is exemplified by the fish wardens in FJ. A similar situation was observed in an East African setting by de la Torre-Castro (2006), who cautioned that the local context, in particular the cultural setting, kinship and alliances, need to be carefully considered in the design of co-management institutions. Previous studies have noticed similar shortcomings of local enforcement in local marine resource management in the South Pacific, while also highlighting financial and technical constraints for local monitoring (Minter, 2008; Jupiter et al., 2010; Pomeroy et al., 2015). These studies have called for communicative measures, such as awareness programs and conflict resolution exercises, as well as improved administration under the fisheries legislation and increased law enforcement. The latter aspect might imply registering local management plans and penalty systems under national legislation. The new Fisheries Management Act of Solomon Islands (enacted in 2015, implementation is underway) offers communities the right to do so. It shall thus help to make local rules and penalties legally enforceable. This could contribute to restrengthening local enforcement capabilities while involving government actors more directly in enforcement efforts, too. Yet, the limited financial, personnel and time resources of the government to exercise these responsibilities and therewith reach out to the local level should be kept in mind. Also, such "legalization" should account for the flexibility of marine tenure systems in their continuously evolving forms and allow for adaptive management (Hviding, 1998). Although voluntary compliance is preferred and likely to increase through participatory and communicative measures that enhance legitimacy (Jentoft, 2000), a certain degree of enforcement is often necessary (Arias, 2015) to create or increase the deterrent threat for rule-breaking. Effective sanction mechanisms are also crucial to avoid "contingent compliance," because individuals base their decision (not) to follow rules on the (perceived) compliance of others, too (Pomeroy et al., 2015), as our study shows as well. Furthermore, the importance of having graduated sanctions for successful CPR management has been highlighted (Ostrom, 1990). Graduated sanctions are flexible to the seriousness and context of the offense (increasing with the frequency and severity of the infringement) and might hence be perceived as more legitimate. The existence of graduated sanctions has been positively related to resource users' compliance behavior (Cinner et al., 2012). However, in both case studies, graduated sanctions were not provided for in the current setting. Generally, sanctions could include social sanctions—which have been proven successful in inducing community cooperation and compliance (Ostrom, 1990). The design and perceived fairness of the enforcement system are again likely to influence perceptions of legitimacy (Pomeroy et al., 2015). Sanction mechanisms should thus also be formulated in a participatory manner, e.g., through consultations on which sanctions could be locally feasible and desirable. ## CONCLUDING REMARKS Our study reveals multiple drivers for local non-compliance with local marine resource management in two case studies in the South Pacific. In other words, this article outlines that locals fished inside the marine closures studied here for a number of different reasons and exposes how these reasons were influenced by dynamic social, political and economic contexts. Perceived legitimacy of decision-making and decision-makers was considerably influencing compliance behavior in our case studies. To address this driver of local (non-) compliance, broad participatory, transparent and communicative efforts are crucial (see also Ferse et al., 2010). These should involve women and youth—both groups are often less involved in local decisionmaking. Incentives to break rules due to market access and/or lack of alternative livelihoods can be difficult to tackle as they mostly lie beyond the local reach. Yet, it becomes clear that the governance system needs to react when such external factors change. This might imply the development of new rules. Our results show that multiple drivers of (non-) compliance interact. Hence, when legitimacy of local management rules and leadership is high/increased, it may more readily outweigh other incentives for rule-breaking. Vice versa, when the strength of local leadership is decreasing this might negatively affect compliance dynamics. Our study highlights that drivers of (non-) compliance are highly contextual. They react to and depend on the broader dynamics of marine governance systems. In other words, if the context of the governance system changes, this can affect rule compliance decisively. We therefore argue that it is important to build fallback mechanisms into governance arrangements that allow for adaptive management of marine resources. By showing that perceptions essentially shape people's compliance behavior, our study brings to attention the importance of assessing local perceptions of local rules, objectives and outcomes of resource management processes, as well as of people and actors involved in management. This also underlines the importance of qualitative research in the context of marine resource management (see also Barclay et al., 2017). In sum, while the scope of this research with its focus on two case studies appears limited, our study reveals under what pressures and circumstances compliance can decrease or increase. This understanding can inform future design and implementation of adaptive CBMRM and thus suggests applicability of the findings to the broader context of CBMRM in the region and beyond. ## AUTHOR CONTRIBUTIONS JR conducted the field research and data analysis and wrote the initial manuscript. All authors contributed substantially to revisions by providing theoretical, analytical, and empirical input. ## ACKNOWLEDGMENTS We sincerely thank all people who participated in this study as well as the local field assistants and interpreters who helped to facilitate research on the ground. We further wish to express our thanks to diverse stakeholders, including the Governments of Fiji and Solomon Islands, as well as community leaders, for their kind and productive collaboration. It helped immensely in facilitating this research project in both countries and in securing research permits. This work is part of the REPICORE project (grant 01LN1303A), funded by the German Ministry for Research and Education (BMBF) in the frame of the "Research for Sustainable Development Framework Program" (FONA), and contributes to the ICSU and UNESCO-sponsored "Program on Ecosystem Change and Society" (PECS). Finally, we thank the two reviewers for their insightful comments on a previous draft of this manuscript. #### REFERENCES #### SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2017.00172/full#supplementary-material in the Coral Triangle. Conserv. Lett. 3, 291–303. doi: 10.1111/j.1755-263X.2010. 00134.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 Rohe, Aswani, Schlüter and Ferse. 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. # Chinese Trader Perceptions on Sourcing and Consumption of Endangered Seafood Michael Fabinyi 1,2,3 \, Kate Barclay <sup>1</sup> and Hampus Eriksson3,4 <sup>1</sup> Faculty of Arts and Social Sciences, University of Technology Sydney, Sydney, NSW, Australia, <sup>2</sup> Australian Research Council Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD, Australia, <sup>3</sup> WorldFish, Penang, Malaysia, <sup>4</sup> Australian National Centre for Ocean Resources and Security, University of Wollongong, Wollongong, NSW, Australia Growing trade networks through globalization have expanded governance of local environments to encompass multiple scales. The governing role of market actors, such as traders and consumers in importing countries, has been recognized and embraced for sustainable seafood sourcing and trade. The perceptions that affect the conduct of these actors are a potential influence on governance of distal environments. In this paper we investigate the perceptions of sea cucumber traders in China. Sea cucumbers are an important global fishery commodity predominantly traded to China, the world's largest seafood market, and seven traded species are endangered globally. We examine what traders and consumers in China perceive as important issues in seafood markets, and where they perceive the responsibility for sustainable fisheries to lie, to interpret what scope there is for sustainability to become an important issue in China's seafood markets. We find that clusters of perceptions about cultural status, quality, health and food safety, and country of origin influence decisions that consumers make. These norms are rooted in sociocultural practice and drive current trade strategies. While traders do want to mitigate risks and secure supplies, food safety, product quality and country of origin are viewed as more important concerns than stock sustainability. Responsibility for sustainable fishing is perceived to be that of national governments in production countries. Trading practices and consumer perceptions together pose a serious challenge to sustainable seafood markets, further confounded by clandestine cross-border gray trade into China. Keywords: sea cucumber, China, seafood trade, sustainability, food safety, environmental governance, sustainable seafood movement ## INTRODUCTION Environmental governance is strongly affected by what actor groups perceive the important issues to be, and who they perceive as being responsible for addressing those issues (Robbins, 2004; Lakoff, 2010; Bennett, 2016; Beyerl et al., 2016; Cox and Pezzullo, 2016). How these perceptions influence governance has been explored in diverse fields. Work on "mental models," behavioral economics and psychology has focused on understanding individual representations of the external environment (Jones et al., 2011; Beyerl et al., 2016), while much work in social anthropology and political ecology has examined how broader socio-political contexts and relationships contribute #### Edited by: Annette Breckwoldt, Alfred-Wegener-Institut für Polar- und Meeresforschung, Germany #### Reviewed by: Lydia Chi Ling Teh, University of British Columbia, Canada Sebastian Christoph Alexander Ferse, Leibniz Centre for Tropical Marine Research (LG), Germany > \Correspondence: Michael Fabinyi [email protected] #### Specialty section: This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science Received: 07 December 2016 Accepted: 24 May 2017 Published: 15 June 2017 #### Citation: Fabinyi M, Barclay K and Eriksson H (2017) Chinese Trader Perceptions on Sourcing and Consumption of Endangered Seafood. Front. Mar. Sci. 4:181. doi: 10.3389/fmars.2017.00181 toward the perceptions that shape environmental governance (Li, 2007; West, 2016). In this context, "governance" is viewed not as the sole domain of governments, but more broadly to encompass multiple scales and actors. We use Kooiman et al.'s (2005, p. 17) definition of governance as "the whole of public as well as private interactions taken to solve societal problems and create societal opportunities. It includes the formulation and application of principles guiding those interactions and care for institutions that enable them." For example, small-scale fisheries governance is heavily influenced by diverse perceptions about the value of biodiversity and the environment (Foale and Macintyre, 2005), the state of the environment (Beyerl et al., 2016), the role of governance agencies (Jentoft, 2000), and social relations among stakeholders (Coulthard et al., 2011). With an increase in fisheries trade and the increasing role of private actors in certification and ecolabels, perceptions of consumers and traders can also have significant influence on environmental governance via international seafood markets. Consumer and retailer perceptions in international markets about the relative importance of sustainably caught fish can lead to the introduction of certification and ecolabels, which have influenced the development of fisheries management in source countries (Gutierrez et al., 2016). China is the world's largest seafood market, and the perceptions that influence decisions among traders in this market are important to understand. In this paper, we draw on interview data on sea cucumber trade in Hong Kong and mainland China to examine if and how consumer and trader perceptions about sea cucumbers and the sustainability of sea cucumber fisheries affect environmental governance. Conventionally, sustainable fishing has been perceived as the responsibility of governments and fisheries management institutions in producing countries. Contemporary globalization of social-ecological systems through markets, however, has caused vulnerabilities to people and environments that are difficult for national governments in producing countries to regulate (Berkes et al., 2006; Liu et al., 2013). For example, more than 50% of terrestrial and marine species threats in Malaysia and Papua New Guinea (PNG), two iconic global biodiversity hotspots, are linked to global trade (Lenzen et al., 2012). Seafood is the most highly-traded animal protein (Rabobank, 2015), so patterns of fisheries production and sustainability can thus be strongly influenced by the preferences and perceptions of actors (e.g., consumers and traders) in locations far from the original site of production. Modern market pressure is one of the central drivers of the status of biomass in marine environments and a central challenge to developing more sustainable production (Cinner et al., 2013; Kittinger et al., 2015). The sustainable seafood movement (SSM) has consequently developed as a way to effectively link market actors along the entire supply chain with the general discourse and specific aspects of sustainability, responding to the lack of effective measures related to the problem of declining fish stocks by governments (Gutiérrez and Morgan, 2015). It has expanded rapidly in recent years (Bush et al., 2013). The largest eco-label for seafood, the Marine Stewardship Council (MSC), now covers 306 certified fisheries in over 30 countries with a total of 9.5 million metric tons of seafood caught annually, representing approximately 10% of the global harvest (MSC, 2016a). The perception in some countries that market actors are part of environmental governance—a central idea underlying the SSM—has therefore had a significant impact on global fisheries. As the world's leading consumer of food fish—up to 38% of global food fish by 2030 according to one source (Kobayashi et al., 2015)—the Chinese market is of particular importance for marine resource governance. The amount of seafood consumed per capita in China has been rising steadily over the past several decades, especially since the early 1980s when China's economy began to boom: from less than 5 kg in 1980 to almost 35 kg today (FAOSTAT, 2016)<sup>1</sup> . China is therefore a hugely influential market in global seafood consumption. Yet the perceptions, priorities and assumptions that drive the Chinese market are quite different to those of more well-documented markets in the USA and Europe, and remain comparatively under-examined. In this paper we assess interviews with sea cucumber traders to examine how Chinese trader and consumer perceptions affect environmental governance in the frame of this international seafood trade. The sea cucumber trade is a particularly useful case to explore because the sourcing network connects Chinese consumers to global production in small-scale fisheries in poorer nations, as well as more developed industrial fisheries in developed countries, and high-technology aquaculture. Sea cucumbers have long been consumed in China as an item in banquets and as a health food. As the middle classes have expanded in China, sea cucumber consumption has also increased. Domestic production of sea cucumbers has escalated rapidly since the early 2000s (Fabinyi and Liu, 2014), while imports have also expanded. Between 1996 and 2011, the number of countries serving the Chinese sea cucumber market expanded from 35 to 83 and over 90% of the world's tropical coastline now lies within countries that export sea cucumbers to Hong Kong (Eriksson et al., 2015). This surge in consumption has had significant effects for countries that supply the market (Eriksson and Clarke, 2015), and seven traded species are endangered (Purcell et al., 2014b). Globally, at least 38% of sea cucumber fisheries are considered overfished and 24 countries have closed or attempted to close their sea cucumber fisheries due to overfishing (Purcell et al., 2013). Although the majority of the sea cucumber market in China is in dried form—bêchede-mer (BDM)—there is also a growing market for frozen and fresh (live) sea cucumbers (Purcell et al., 2014a). The traditional drying process is low-tech and enables stockpiling in production locations without refrigeration. This method has facilitated export from some of the least developed and most remote tropical islands of the world (Kinch et al., 2008). We recognize that the trade in sea cucumbers has highly significant effects on other societally important concerns, such as local economic development and poverty alleviation (Barclay et al., 2016), but the focus of this study is on the question of environmental sustainability within the international seafood trade sector. We do not adopt a formal definition of perceptions in this study, but use the term in a general sense to refer to "the way in which something is regarded, <sup>1</sup>FAOSTAT Database. Available online at: http://faostat3.fao.org/browse/FB/CL/E). understood or interpreted" (Oxford Dictionary, 2016). The paper examines perceptions about sea cucumber consumption, specifically relating to banqueting, food safety and health, and quality and country of origin. It then addresses perceptions about the governance of sea cucumber fisheries, including perceptions about environmental sustainability and trade regulation. We ask the following questions, which we return to in the Discussion section: ## METHODS In September 2015 in China, interviews were conducted at major wholesale markets for dried seafood, where sea cucumbers are sold: Sheung Wan in Hong Kong, Yidelu in Guangzhou, Jingshen in Beijing, and Tongchuan in Shanghai. Interviews were conducted together with a research assistant (one in Guangzhou and Hong Kong, one in Beijing, and one in Shanghai) in order to interpret from Cantonese when the interviewee did not speak Putonghua (sometimes called Mandarin), or to assist in the translation of some Putonghua when necessary. Interviews in Hong Kong and Guangzhou were conducted in a mixture of Cantonese and Putonghua, while interviews in Beijing and Shanghai were conducted in Putonghua. Some interviews extended up to an hour, while most lasted for approximately 30 min. Interviews were conducted until effective saturation of information took place, i.e., each new interview yielded little or no new data (Morse, 1995). In all, 30 traders or representatives of trade organizations were interviewed in detail (see Table 1 and Supplementary Material). More interviews were undertaken with traders in Hong Kong and Guangzhou compared to Beijing and Shanghai because of the disproportionately large number of traders who specialized in sea cucumbers in these locations, reflecting their importance as key trading nodes. Interviews were semi-structured (Bernard, 2006), and focused on a range of topics related to the trade, including trade structure, buyer preferences, pricing and market trends, and marine resource governance in China and in source countries. Observations were also conducted, as well as many brief informal conversations with traders and price checks at retail outlets. Most traders of sea cucumber trade with TABLE 1 \| Interviews in Hong Kong and mainland China. a broader portfolio of dried seafood products that includes abalone, fish maw, scallops, and shark fin. Traders specialize in one or more of these products to differing degrees; 10 of the traders we interviewed focused predominantly on sea cucumber. Our interviews focused on wholesale trading operations which specialized in sea cucumber, and which were of relatively larger scale, with an office in the market and several employees. Interviews were also carried out with three key informant researchers from Australia and Hong Kong, identified by the authors as experts with detailed knowledge of the sea cucumber trade in Hong Kong and China. The topics for these interviews included similar questions used for the traders in order to triangulate their responses, and questions on particular areas of their expertise (e.g., environmental regulations, trading practices between Hong Kong and China). We did not directly interview international consumers themselves but asked traders, as key informants, for their views on consumer demand. Because of the sensitive nature of some of the interview questions in relation to trade practices, they were not recorded, but detailed notes were taken. These notes were then qualitatively analyzed for patterns that emerged (Bernard, 2006). The paper also draws on some unpublished data from 20 earlier interviews with Beijing seafood restaurant operators in 2012, and two interviews with traders of dried seafood products in Beijing in 2014 (see Fabinyi and Liu, 2014, 2016, for full elaboration of the methods used in the earlier studies). We also draw on existing published research on sea cucumber consumption and trade, and other secondary data, such as technical reports. This study was carried out in accordance with the recommendations of the University of Technology Sydney Human Research Ethics Committee with informed consent from all subjects. All subjects gave verbal informed consent. The protocol was approved by the University of Technology Sydney Human Research Ethics Committee (reference number 2014000548). The Committee only required verbal consent, not written. ## RESULTS Table 2 provides a summary of perceptions about sea cucumber consumption and marine resource governance in China and their environmental and socio-economic implications. ## Perceptions about Sea Cucumber Consumption in China #### General Consumption Preferences Two major types of sea cucumbers are distinguished by Chinese traders, and consumption preferences differ depending on geographical location (Figure 1). Japanese spiky sea cucumbers, Apostichopus japonicus, are found in temperate waters in parts of China (especially Liaoning and Shandong provinces) and neighboring countries such as Korea, Russia, and Japan. While they are often captured in the wild, they are also cultured in large quantities (Chen, 2003; Han et al., 2016a). Commonly referred to as cishen, (spiky sea cucumber), the most highly TABLE 2 \| Summary of perceptions in China that influence BDM consumption and governance. valued individuals of these sea cucumbers reach the highest prices on the markets and are regarded as the best of all sea cucumbers (prices of USD316–1,892/kg). This is for several reasons: their spiky appearance is viewed as appealing (the longer the spikes, the better); they are of a suitable size to be served individually, whole; and they are regarded as having better nutrition and health benefits than other types of sea cucumbers. In much of northern China, these temperate sea cucumbers dominate the market. In contrast, tropical sea cucumbers include a far greater diversity of species and are sold in far greater quantities in southern Chinese markets. While A. japonicus is also the most highly-priced type of sea cucumber in southern markets, unlike in northern markets there are also significant numbers of tropical sea cucumbers. Their popularity in southern markets is likely due to their natural distribution in the south, as well as the historical linkages of southern Chinese communities with source locations of tropical sea cucumbers (e.g., in Southeast Asia) (Tagliacozzo and Chang, 2011). These larger tropical sea cucumbers are also sometimes served whole, but are more often sliced up. They can be served individually or with other ingredients. The prices of sea cucumbers vary significantly—from less than USD64/kg for dried, cheaper species of lower quality, to more than USD1,800/kg for high-value species of excellent quality (Table 3). A. japonicus was the most expensive type of sea cucumber in all locations, followed by sandfish (Holothuria scabra; H. lessoni) (price of USD64–359/kg) and teatfish (H. fuscogilva, H. nobilis, H. whitmaei) (price of USD96– 319/kg). Other types of tropical sea cucumbers commonly sold include prickly redfish (Thelenota ananas), South American sea cucumbers (Isostichopus fuscus, I. badionotus), and curryfish (A. herrmanni) (prices of USD96–294/kg). Purcell (2014) has conducted a more comprehensive survey of tropical sea cucumber prices in southern China. #### Banqueting Sea cucumbers were and are consumed for two major reasons: as a luxury status item, and as a health product. Records of sea cucumbers as part of royal cuisines have existed since the #### TABLE 3 \| Prices of selected sea cucumbers in USD/kg, September 2015. The column on conservation status was taken from the IUCN Red List (IUCN, 2016). For these species, high quality products are consumed in banquets and mid- to lower- quality products are consumed at home and for family occasions. Ming Dynasty (1368–1644), and they became more prominent during the Qing Dynasty (1644–1912) as one of the "eight great sea delicacies" (Yang and Bai, 2015, p. 9). In contemporary times, the most highly-priced sea cucumbers form part of luxury seafood banquets, served (often in a soup) together with other delicacies such as shark fin, bird's nest soup, reef fish, lobster and abalone. In China, such banquets are central for professional and social advancement. The social relationships and "connections" (guanxi) necessary to "get ahead" can only be formalized through the shared experience of eating together (Mason, 2013; Harmon, 2014). The emphasis in such banquets is to give "face" (mianzi) to guests, and a key way to give face is to offer "face dishes" (miancai). Expensive dishes such as those containing sea cucumber therefore serve the function of showing the guests that the host values and honors them. These banquets provide the social context for the consumption of high-value sea cucumbers. Recently, however, the luxury sea cucumber market has witnessed a downturn. First, the government's anti-corruption campaign (Jeffreys, 2016) is viewed by traders to have had a significant effect on the sales of dried seafood generally. Hong Kong traders of dried seafood widely reported reduced profits over the past 2–3 years. One Hong Kong trader advised that the price of most kinds of BDM had dropped by 30–50% in the past year or so; others did not give specific figures or estimates but noted that demand had dropped, especially from mainland China. Guangzhou-based traders similarly noted how Japanese spiky sea cucumber had dropped from USD1,892/kg to USD1,260/kg in the course of 2015. Beijing traders reported significant declines in sales over the past years (see also Fabinyi and Liu, 2016). One Beijing trader spoke of an 80% drop in sales; another described a 70–80% drop in sales. Several dried seafood traders had closed since 2013. The anti-corruption campaign began shortly after Xi Jinping's ascent to the leadership in late 2012, and has continued and even intensified since then. One of the specific targets of this campaign was government officials using public funds at banquets. While sea cucumbers are not exclusively eaten at such banquets, a considerable proportion of them are. Secondly, another factor relating to demand has been the slowdown in the Chinese economy. Due to government policies aiming to transition to a slower but more sustainable economic growth pattern, since 2011 the growth rate of the Chinese economy has been slowing. However, the market for many of the mid- and low value sea cucumbers will likely continue to expand, especially in new markets throughout China, largely due to their popularity as a health product. #### Health and Food Safety Sea cucumbers are also consumed for perceived health benefits, and they have been long documented in handbooks of Traditional Chinese Medicine (Yang et al., 2015). The name, haishen, literally means "sea ginseng," and sea cucumbers are regarded as being especially good for kidney function and against impotence. Chefs and traders that we interviewed cited the numerous benefits of eating sea cucumbers: "Sea cucumbers have the function of self-repair and regeneration. So eating them will have a positive effect on our health. And it is a zero cholesterol, low fat food," noted one. Another described how "if you eat it for a month, your immunity will be enhanced, and cancer cell growth will be restricted." Others spoke of the positive effects of sea cucumber consumption on skin, their anti-aging properties, and their high levels of vitamins. Sea cucumbers are often sold in pharmacies, next to other traditional Chinese medicines. They are also sometimes incorporated into different products such as soap, "Holothurian wine," and capsules (Figures 2A,B; see also Purcell et al., 2014a). Scientific research is ongoing to try to confirm the health benefits of sea cucumbers (Kiew and Don, 2012). The importance of health as a driver of sea cucumber consumption is linked to strong concerns about food safety in China. Consumers are very keen to eat food that is considered safe, because of the high prevalence of food safety crimes and scandals in recent years in China (Klein, 2013). An example of a prominent food safety concern in the marine sector is the use of antibiotics and other chemicals in aquaculture. This can be seen in product marketing: some farmed A. japonicus sea cucumbers, for example, are sold with the national Chinese organic certification in Beijing, which is administered by the China Organic Food Certification Center under the Ministry of Agriculture. Much of the frozen sea cucumbers and other types of seafood sold in Beijing supermarkets are sold with labels emphasizing "pollution-free," "natural" characteristics in Hong Kong (D). (Figure 2C). Some sea cucumbers have labels asserting they are "non-additive" and "chemical free" "to assure consumers that no additives have been used to artificially increase the reconstitution ratio of the product" (Purcell et al., 2014a, p. 49). One Beijingbased trader described the potential for "green" labeling that focused not on environmental sustainability of the production, but on food safety: "in the past nobody asked about these things, but more and more people do now." Because of these strong associations with health, many people and families in China also consume sea cucumbers in less formal restaurant settings, among family gatherings, or regularly at home. Traders also noted that unlike shark fin, sea cucumber could be easily prepared at home by people with no formal training. There is therefore also a very large market for lower-valued sea cucumber that is consumed by diverse groups of people. #### Quality and Place of Origin A central factor affecting the price of sea cucumbers is the quality of the processing (Purcell, 2014). Most traders stressed the importance of good processing, and many traders simply stated that they would only try and buy good-quality sea cucumbers, and avoid those that were poorly processed. Dryness was the most important factor mentioned by many traders; related to this was the expansion rate (i.e., how much they convert from dry to wet weight). Other characteristics looked for by traders included the way the sea cucumber was cut; the saltiness (the less salty the better); the size; the shape (straight and symmetrical, not curly); and the extent of damage. These characteristics determine the "grade" of the sea cucumber, and their eventual price. Linked to these perceptions about the quality of sea cucumbers are perceptions about country or region of origin. Products from PNG and some Southeast Asian countries, for example, had a poor reputation for processing. Most traders suggested that the quality was very low, due to poor processing (e.g., poor handling of catch, poor drying, poor cutting, bad shape, high salt content). One trader noted that because of the poor quality of PNG products his company had stopped buying from there. Only one trader suggested that the quality of PNG products was high, citing the high quality of the "seawater" in PNG, meaning that it was low in industrial pollution compared to many production locations around Asia. In Beijing and Shanghai, most traders had not heard of PNG, and did not know where it was geographically located. In contrast, place of origin branding is used for certain countries that are perceived to produce high quality seafood products. Different seafood products are associated with different countries. The best abalone and rock lobsters are perceived to come from Australia, for example, whereas the best salmon is perceived to be from Norway. For A. japonicus, Japanese sea cucumbers are regarded as the best quality, and within Japan, sea cucumbers from the northern region of Hokkaido are viewed as particularly high quality. Within China, cultured A. japonicus from Liaoning and Shandong provinces are viewed as the best sea cucumbers. For tropical species, Australian sea cucumbers are regarded as the best. Because of this reputation for quality based on place of origin, traders regularly advertise their tropical sea cucumbers as being Australian when there is no way of telling the actual origin of the product. Almost all of the sandfish (H. scabra) sold in Hong Kong, for example, is simply marketed as "Australian bald sea cucumber" (Figure 2D). These include undersized sea cucumbers that are unlikely to be from there because of the sizing requirements for Australian fisheries (e.g., 20 cm in Queensland; DEEDI, 2011). By contrast, despite the high numbers of these types of sea cucumber exported to Hong Kong from countries such as Philippines and Indonesia (Conand et al., 2014), no branding from these countries was observed. Such practices indicate that mislabeling is likely widespread, and that traders exploit consumer preferences for Australian products and the weak system for seafood traceability in China (Xiong et al., 2016). ## Perceptions about Governance of Sea Cucumber Trade #### Stock Sustainability Consumer preferences for food that is considered safe and healthy significantly overshadow any concerns about stock sustainability that may be present. A recent survey of 300 middleclass seafood consumers in Beijing and Shanghai, for example, found strong support (mean score 3.7 out of 5) for the statement that "Compared to sustainability, I am more concerned about food safety when consuming seafood products" (Fabinyi et al., 2016, p. 7). Many traders acknowledged problems of environmental sustainability, including with sea cucumbers—some noted, for example, that the supply of sea cucumbers from certain locations was becoming more difficult to source, and many Hong Kong and Guangzhou-based traders knew of management measures in other countries. One Hong Kong trader who had had long dealings with exporters in PNG, for example, was very supportive in principle of the need to manage PNG's sea cucumbers, saying that this was "good for the country" and "good for the livelihoods of villagers." Other traders agreed with the broad notion that countries should sustainably manage their fisheries, and saw advantages to their business because of this. Four traders, for example, suggested that it could help to stabilize prices, while two others suggested it could help to stabilize supply. As one noted, "if they are sustainably managed, of course this will stabilize the supply, and eventually the price." One Guangzhou-based trader suggested that sustainable management was a good idea "because sea cucumbers are slow to grow" and hence are vulnerable to overfishing. Overfishing was therefore recognized as a problem by some traders because of its impact on supply. Other traders were less concerned about environmental sustainability: "I don't care about these things; if there are no sea cucumbers left there [in PNG] I can just go and buy them from somewhere else" one Guangzhou trader stated. Another one stated that "it would be better to catch them all at one time; it's not my business if there are no sea cucumbers for harvesting anymore." One Guangzhou trader advised that "there are still lots of sea cucumbers in the wild, and sustainability is not a concern compared to shark fin. So sustainability wouldn't be helpful for me to improve the business." This last comment refers to the widespread perception in China that sharks are threatened, in part due to an intensive environmentalist campaign featuring celebrities (Fabinyi et al., 2016). Because there is no such widespread perception in China about the threatened status of many types of sea cucumbers, there is no perceived gain to marketing them as "environmentally sustainable" in the manner promoted by the SSM. Nearly none of the traders were willing to seriously engage with sustainability actions and initiatives, or said that certification schemes could improve their business. Only one Hong Kongbased trader suggested that eco-labeling may attract the interest of buyers. One other Guangzhou based trader noted that ecolabeling "may attract traders whose target customers are from high end. I think it may be good for branding, but it would need a long time to set the image." Most traders were instead very skeptical about the potential of eco-labels to improve their business, had not heard of the MSC, for example, and were not interested in MSC certification. As one trader stated, "I don't think it can improve my business and I think buyers and customers from Hong Kong and China don't care about this." Others expressed a high level of cynicism toward such schemes: one trader, for example, asserted that "everyone knows that these certifications are just bought by companies anyway, and aren't actually worth anything, so no-one will pay extra for them." Cynicism toward abstract institutions such as certification and indeed the food system more broadly is widespread in China (Hanser, 2010; Klein, 2013). As we discuss in the Discussion and Conclusion, however, the status of the MSC and the discussion of sustainability issues in China does have the potential to change. Others focused on the more general issue of responsibility for sustainability. One Guangzhou trader, for example, pointed out that private certification is unnecessary for seafood products, because "the Chinese government is already very strict with regulation."Another Guangzhou-based trader suggested that "most countries already have sustainable management in place, so these types of certification aren't necessary." As another Guangzhou-based trader put it: "Of course this [sustainability] is a very big problem. But I just sell these products. It's the responsibility of governments to regulate their fisheries properly." The implication of such comments is that the responsibility for sustainable management of fisheries is perceived to be the role of governments (Fabinyi et al., 2016). From this perspective, sustainability is associated with state regulatory frameworks and is not perceived as something markets should or even could deliver. #### Trading Perceptions about trading practices have a strong influence over the extent to which this trade can be effectively governed for sustainability (Clarke, 2004). The structure of the sea cucumber trade in Hong Kong and mainland China is complex, subject to change over time, and enmeshed in a broad range of other economic and social institutions. Considerable uncertainty remains about trade routes, which tend to be fluid, opaque and diverse. One key route is from source countries into Hong Kong, re-export to Guangzhou, and then throughout mainland China. This is because Hong Kong is a free port with no tariffs but sea cucumber imports to the rest of the country attract tariffs of up to 30% (depending on the trade relationship of the country of origin with China). Officially, seafood transferred from Hong Kong to the mainland is subject to the tariff. Sea cucumber, along with other forms of seafood, thus appears to be transported into China through clandestine channels to avoid the tariff. The majority of Hong Kong-based traders reported selling most of their products to mainland China. When selling to mainland Chinese buyers, Hong Kong-based traders advised that buyers from the mainland take responsibility for transportation to mainland China. As one trader described: "once we have sold the product to them, everything, including both the physical logistics of getting the product back to China, and the legal implications, is completely their responsibility. We don't ask too many questions. Every country has its own way of doing business, and that is how they request for us to do business." In this way, responsibility for trade legality is perceived to be that of the trader in China. Guangzhou is the major trade hub for sea cucumbers brought into mainland China from Hong Kong. There are approximately 1,000 members of the Guangzhou Dried Seafood and Nut Industry Association (GDSNIA), the primary trade association in the Yidelu market area. When asked about potential opportunities for exporters to export directly to Guangzhou (bypassing Hong Kong), Guangzhou-based traders simply advised that this would be a more expensive way for exporters to do business, and that it would be much cheaper for exporters simply to sell in Hong Kong in order to avoid the taxes. Some Guangzhou-based traders pointed out that they did not need to invest overseas and buy directly because it was far easier for them to just go to Hong Kong and inspect the goods there: "Why should we go overseas, with all of the risks, when we can just go to Hong Kong and check the product quality?" The GDSNIA explained that it advised all of its members to do business completely legally, but acknowledged that it did not control the way its members did their business. How the sea cucumbers are imported into Guangzhou, and the regulations that are supposed to govern the cross-border trade, was not something traders wanted to talk about in detail. This is understandable, given recent high-profile cases of prosecution against seafood traders in Guangdong province, and even more recent crackdowns by the central Chinese government against smuggling (Godfrey, 2014, 2015). Some traders advised that sea cucumbers were hidden within other cheaper types of seafood that would be subject to lower taxes. Others noted that vehicle transports into mainland China are only rarely inspected in any detail. There is also the possibility of "parallel goods trading," which involves the practice of transporting small quantities of goods across the border in very high frequencies (Chan, 2015). Vietnam is another possible gray trade route into China (To and Shea, 2012; Eriksson and Clarke, 2015). Some Guangzhou-based traders simply advised that these matters were all the responsibility of a "logistics company," and that they did not know about any of the customs regulations. Smuggling practices have also been documented at point of export from producing countries to evade regulation and trade tariffs (e.g., The Hindu, 2016). In such an environment, where formal governance institutions are weak and the trade is clandestine, perceptions about social relationships and informal institutions take on new significance (Nee and Opper, 2012). Of particular importance is the perception of trust among business partners, which is more important than obeying the formal regulations regarding tariffs. This trust is in many instances cultivated over long-term family relationships that are built up over time (Cheung and Chang, 2011). Most of the seafood traders in both Beijing and Shanghai, for example, are also of either Guangdong or Fujianese origin, and many have family or long-term links with traders in Guangzhou. The importance of these types of social relations in professional contexts has been discussed in a great deal of literature in Chinese anthropology and sociology (e.g., Fei, 1992 [1947]; Wank, 1999). Guanxi, or the establishment of social relationships via trading of gifts, favors and banquets, for example, remains an indispensable part of professional success in contemporary China (Yang, 1994). These perceptions about the nature of social relationships are therefore central not only in creating demand for sea cucumbers in banquets discussed in 3.1.2, but also as part of the social relations needed to successfully engage in the sea cucumber trade—including the illegal gray trade. The gray trade, founded on these social relationships, is a significant barrier to sustainability and improved environmental governance of the sea cucumber trade (Wu and Sadovy de Mitcheson, 2016). The importance of trust among business partners extends to source countries. In many cases, such exporters of seafood also tend to be of Chinese background (Tagliacozzo and Chang, 2011). Chinese traders who invested in overseas source countries for sea cucumbers emphasized the necessity of having a trusted local partner: "We have a trusted partner [in South Asia] who we buy from, and we finance him. He has a good relationship with the people on the ground, he knows the government, he knows the local people and the local traders. He is much better suited to working with these matters. But we have been working with him for years. If you were to come into my office and propose this sort of thing straightaway, that wouldn't be possible after just 5 minutes." Social norms and networks, and how they are perceived to operate, are therefore factors that strongly influence possibilities for governance of the trade. ## DISCUSSION AND CONCLUSION ## What Do Traders and Consumers in China Perceive as Important Issues in Sea Cucumber Markets? We asked three questions at the onset of our analyses. When answering the first question we note that there is a range of perceptions in China that contribute to the nature of the sea cucumber trade and demand for sea cucumber products. The diversity of consumers across Chinese provincial cuisine, social class and occasion, and a matching wide range of products on offer to cater for each of them, illustrates an immense complexity and challenge for understanding market drivers and the effects of sustainability initiatives. We also emphasize that these perceptions are not time-bound characteristics of a certain culture or society, and we do not intend to imply that the Chinese market cannot be concerned with environmental sustainability at all. Chinese consumer preferences for lower-trophic level freshwater fish such as carp, for example, is a perception that arguably has more positive effects for the sustainability of fisheries (Han et al., 2016b). We also note that while we did not directly interview consumers themselves in this study, our assertions about consumer preferences are based not only on responses from traders but also earlier research conducted with consumers and restaurant operators (Fabinyi and Liu, 2014; Fabinyi et al., 2016). Traders are in many cases also consumers of sea cucumbers. Trader perceptions and consumer preferences constitute a part of social practices that have significant influences on environmental governance (Table 2). In the Pacific, for example, discussions are taking place about whether it is possible to market and brand Pacific BDM to appeal to consumer preferences (IUCN, 2015). The social context of banqueting and giving gifts driven by dominant perceptions about how to achieve success in professional and societal contexts—has provided social pressure to consume high-value sea cucumbers. The increasing demand for healthy and safe food in China means that the market for midand low-valued types of sea cucumbers consumed in everyday meals outside of banquets—driven by a widespread perception in China that sea cucumbers are good for health—is rapidly expanding. The speed of modern expansion has caught new source countries unprepared, and many do not have the capacity to effectively manage their fisheries in the face of such pressure (Eriksson et al., 2015). Consumer preferences for sea cucumbers from certain countries seem to be driven by perceptions about the quality of seafood in these areas. However, mislabeling and a lack of traceability means that the stronger fisheries regulations in some desired source countries—such as Australia—do not necessarily lead to increased demand for actual Australian sea cucumbers that are more sustainably fished. When we summarize and interpret the consumer and trader perceptions from our study, we find that while markets are dynamic there is no clear indication that sustainability is going to become an important feature in the short- to medium- term. ## Where Do Traders and Consumers Perceive the Responsibility for Sustainable Sea Cucumber Fisheries to Lie? In China, the perception that consumers and retailers are active participants in and responsible for sustainable resource management is not widespread (Fabinyi et al., 2016). Instead, the responsibility for ensuring sustainability is seen to lie with the governments of fishing countries, and not the importing governments. Although China is a signatory to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), this is patchily enforced (Wu and Sadovy de Mitcheson, 2016) and there are no comparable regulations to those of the EU regarding Illegal, Unreported and Unregulated (IUU) fishing (Miller et al., 2014). Clandestine forms of trade to avoid tariffs are widely practiced, so that profits based on social networks and relationships are seen as preferable to following abstract state laws on tariffs that are poorly enforced. While these perceptions and practices about trade are not directly focused on environmental sustainability, they have the effect of seriously diminishing traceability and transparency, and hence the capacity to successfully govern trade. This means that even if the Chinese government were to start regulating more strongly for sustainability of imports (such as through improved enforcement of CITES regulations), the trade would first need to be brought into the legal sphere through enhanced regulation of the gray trade. Taken together with the perceptions on sea cucumber consumption discussed earlier, these perceptions about trading practices and governance have indirectly contributed to the decline and in some cases collapse of sea cucumber fisheries (Purcell et al., 2013). ## What Scope Is There for Sustainability to Become an Issue in China's Sea Cucumber Markets? Despite the fact that twenty-first century seafood consumers are increasingly materially connected to the distant environments from where products originate (Rabobank, 2015), complex multi-level supply systems tend to camouflage patterns of exploitation and sustainability that have the potential to influence consumer perceptions (Crona et al., 2016). Actors at the recipient end of supply chains are geographically removed from sourcing environments and activities, so their perceptions may not be influenced by direct experience and there is little consumer-facing traceability. Perceptions about environmental sustainability in China do not currently translate into market pressures toward sustainably caught seafood and its trade. While there is awareness of environmental problems, traders are not incentivized by consumer preferences to sell sustainably caught seafood as they are in some other countries and for other seafood. Consumers perceive environmental issues that affect them personally, such as pollution, as more important than issues of stock depletion in source countries. Improving the environmental sustainability of fisheries can improve the food safety and quality of products; however, our interviews with traders and previous research on consumer perceptions show that traders and consumers do not link these factors, but see food safety and quality as quite separate from environmentally sustainable fishing. In 2004, Clarke (2004) found that stock sustainability was not a key factor of consideration for sea cucumber traders and emphasized that it seemed unlikely that sustainability initiatives would come from the trade domain. The narratives by traders interviewed 11 years later in our study still center on the same issues as those in Clarke's study. The trade hence seems continuously driven along its current trajectory with social structures and norms that are essentially the same as they were in 2004. This raises a key question on how perceptions can be influenced and changed to better account for sustainability issues. There is currently a great deal of work undertaken to increase awareness and potentially change perceptions of market actors within China. Consumer awareness campaigns, for example, have worked with celebrities to spread the idea that sustainability is a problem in shark fisheries. Jeffreys (2016) argues that these campaigns have had minimal impact on consumption practices because they do not address the social norms, pressures and expectations associated with buying shark fin. However, while consumer awareness may not be sufficient on its own to introduce sustainability into seafood markets, it is likely to at least be part of a transition to sustainable seafood markets. In this regard, there may be considerable scope for campaigners to provide awareness about the endangered wild status of many high-profile types of seafood consumed in China, such as sea cucumbers. Similarly, given that food safety is a far more dominant concern in the Chinese market, expanding the notion of "environmental sustainability" to make linkages to food safety where possible may be another potential avenue to influence consumers. There are also many other activities taking place in China that work with additional actors other than consumers in the seafood market, such as restaurants, hotels, supermarkets and other retailers (e.g., Zhou, 2016). It will be important to observe how such interventions unfold in the near future. More broadly, this paper has contributed to the discussion concerning how to respond to the environmental sustainability challenges presented by globalization. Researchers from numerous fields have highlighted how what happens in one location can have environmental implications in another location (Berkes et al., 2006; Liu et al., 2013; Eriksson et al., 2015). The SSM was developed in part to respond to this challenge, linking actors across the length of the market chain through transparency institutions and infrastructure (Mol, 2015) embodied in certification. From this perspective, market actors around the world are viewed to be just as responsible for environmental governance through establishment of sustainable seafood markets as the government-based fisheries managers of countries where the seafood is produced (Oosterveer and Spaargaren, 2011). Producers of seafood invest in having their operations certified as sustainable, while wholesalers, retailers and consumers can exercise their market power to choose sustainably caught seafood. The rapid rise of this SSM has been affected by many geographically and historically specific factors (Gutiérrez and Morgan, 2015). Non-governmental environmental campaigns, media and scientific coverage has been focused in some areas and not others, and has changed over time. For example, awareness of the MSC label can vary widely globally, from 13% in Canada up to 71% in Switzerland (MSC, 2016b). More generally, the perception that market actors can be responsible for environmental governance through establishing and regulating sustainable seafood markets is also geographically and historically specific, and can change if broad cultural perceptions around sustainability and consumption shift, for example as a result of media campaigns. The perceptions among Chinese traders and consumers that we have described above pose a significant challenge to the SSM. The ways in which consumer and trader perceptions interact with influences on the trade highlight that on their own, market-based initiatives may currently struggle to provide the sorts of environmental governance impacts needed to ensure that sea cucumbers are harvested more sustainably (Jacquet et al., 2010). We suggest that this provides further evidence that the social context in which the SSM operates needs to be closely considered (Gutiérrez and Morgan, 2015; Adolf et al., 2016; Gutierrez et al., 2016). In particular, the ways in which market-based initiatives interact with the state, and the extent to which the perception that market actors can be responsible for market governance is shared across society, will be crucial for improved environmental governance through sustainable seafood markets. For China, we have argued that market-based initiatives have been hampered by limited government success in areas such as regulating the gray trade and improving traceability, and by a widespread perception that market actors are not primarily responsible for environmental governance. Ultimately, the success of the market-based SSM will be determined by the presence of regulating and enabling conditions in states and societies. ## AUTHOR CONTRIBUTIONS KB and MF conceived the research. MF conducted the fieldwork. MF, KB, and HE analyzed the data. MF, KB, and HE wrote the manuscript. ## FUNDING This research was funded by a grant from The David and Lucile Packard Foundation (KB, MF), by a Society in Science—Branco Weiss Fellowship (MF), and by an Australian Research Council grant (MF, DP140101055). ## ACKNOWLEDGMENTS Thank you to all of the traders in Hong Kong, Guangzhou, Beijing and Shanghai who spared their time to be interviewed. Thank you to Marielle Dumestre, Calton Law and Yvonne Sadovy from Hong Kong University, Hanlin Jia, Neng Liu, Yunzhun Lu, and Yining Zhang from Peking University, Lin Lin from the Jingshen Seafood Market, Wei Li from the Shanghai Administration Institute, Steve Purcell from Southern Cross University, Theo Simos from the University of Adelaide, and Allen To from WWF Hong Kong for assistance with fieldwork and/or helpful conversations that provided insight into the Chinese sea cucumber market. HE acknowledges the support by SwedBio at the Stockholm Resilience Centre. We thank the reviewers and the editor for their constructive comments that improved the paper. None of these individuals or organizations bear responsibility for the arguments presented in this paper. ## SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2017.00181/full#supplementary-material ### REFERENCES 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 Fabinyi, Barclay and Eriksson. 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	2025-04-07T03:56:59.438843	11-2-2021 14:45	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0094d209-0e07-4730-b988-02f5f7cca9c7", "url": "https://www.frontiersin.org/research-topics/4034/fishing-for-human-perceptions-in-coastal-and-island-marine-resource-use-systems", "author": "Hans von Storch\|\|Wen-Cheng Wang\|\|Beate M.W. Ratter\|\|Annette Breckwoldt", "title": "Fishing for Human Perceptions in Coastal and Island Marine Resource Use Systems", "publisher": "Frontiers Media SA", "isbn": "", "section_idx": 0 }
00a6a373-fd58-400b-bd1a-e87d014d5110.0	The Molecular and Cellular Basis of Retinal Diseases • Steven J. Pittler and Steven J. Fliesler	doab	2025-04-07T03:56:59.502214	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 0 }
00a6a373-fd58-400b-bd1a-e87d014d5110.1	The Molecular and Cellular Basis of Retinal Diseases Printed Edition of the Special Issue Published in Cells Steven J. Pittler and Steven J. Fliesler Edited by www.mdpi.com/journal/cells	doab	2025-04-07T03:56:59.502313	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 1 }
00a6a373-fd58-400b-bd1a-e87d014d5110.2	The Molecular and Cellular Basis of Retinal Diseases	doab	2025-04-07T03:56:59.502358	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 2 }
00a6a373-fd58-400b-bd1a-e87d014d5110.3	The Molecular and Cellular Basis of Retinal Diseases Editors Steven J. Pittler Steven J. Fliesler MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Steven J. Pittler Department of Optometry and Vision Science, Vision Science Research Center, University of Alabama at Birmingham USA Steven J. Fliesler Departments of Ophthalmology and Biochemistry and the Neuroscience Graduate Program, The State University of New York (SUNY)—University at Buffalo USA 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 Cells (ISSN 2073-4409) (available at: https://www.mdpi.com/journal/cells/special issues/basis retinal diseases). 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 978-3-03936-654-5 (Hbk) ISBN 978-3-03936-655-2 (PDF) Cover image courtesy of Steven Pittler. 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	2025-04-07T03:56:59.502382	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 3 }
00a6a373-fd58-400b-bd1a-e87d014d5110.5	About the Editors Steven J. Pittler is a professor at the Department of Optometry and Vision Science, a senior scientist and director of the Vision Science Research Center, with secondary appointments in the Departments of Ophthalmology and Biochemistry and Molecular Genetics. He received his undergraduate training in biochemistry from Michigan State University (1983). He completed his doctoral studies in Biochemistry at Michigan State University (1989), after which he was an NRSA Neurobiology Postdoctoral Fellow (Ocular Molecular Genetics) at Cullen Eye Institute, Baylor College of Medicine. In 1991, he was an assistant professor of Biochemistry and Molecular Biology at the University of South Alabama. In 1995, he became Director of the Center for Eye Research, at the College of Medicine, University of South Alabama. Moreover, in 1995, Dr. Pittler was the recipient of the Cogan Award for excellence in vision research from the Association for Research in Vision and Ophthalmology. He came to the University of Alabama in Birmingham in 1999, and was promoted to the rank of professor in 2000. In 2013, he received the National Eye Institute Audacious Idea Award. Steven J. Fliesler is a SUNY Distinguished Professor, UB Distinguished Professor, the Meyer H. Riwchun Endowed Chair Professor of Ophthalmology, and Vice-Chair/Director of Research in the Department of Ophthalmology, State University of New York (SUNY)—University at Buffalo (UB). He also holds concurrent appointments as a professor in the Department of Biochemistry and in the Neuroscience Graduate Program at UB, as well as being a Department of Veterans Affairs Research Career Scientist at the Buffalo VA Medical Center, VA Western NY Healthcare System. Dr. Fliesler obtained a PhD in biochemistry from Rice University, completed a postdoctoral fellowship at the Cullen Eye Institute/Baylor College of Medicine, and was previously on the faculties of Baylor College of Medicine, Bascom Palmer Eye Institute/University of the Miami School of Medicine, and the Saint Louis University School of Medicine, prior to joining the faculty of SUNY—University at Buffalo in 2008. His research is focused on inborn errors of cholesterol and isoprenoid metabolism and their impact on the development, structure and function of the retina, as well as on blast injury to the eye, using animal models. He has published more than 150 peer-reviewed journal articles, book chapters and review articles, and is the editor of two books. His research program has been funded continuously for more than 35 years by multiple grants from the NEI/NIH and private foundations, as well as, more recently, MERIT Awards from the U.S. Department of Veterans Affairs. Dr. Fliesler recently served on the Board of Trustees of the Association for Research in Vision and Ophthalmology (ARVO), representing the Retinal Cell Biology (RC) Section, as well as being President and immediate past President of ARVO. In 2009, he was inducted as a Silver-tier Fellow of ARVO (FARVO) and, in 2014, became a Gold-tier FARVO. In addition, he is a past councilor for North America, and Treasurer and President of the International Society for Eye Research (ISER). Dr. Fliesler is the Editor-in-Chief of Experimental Eye Research and serves on six other journal editorial boards, including Molecular Vision and the Journal of Lipid Research.	doab	2025-04-07T03:56:59.502497	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 5 }
00a6a373-fd58-400b-bd1a-e87d014d5110.6	Preface to "The Molecular and Cellular Basis of Retinal Diseases" Our goal with this series of articles was to bring together many prominent vision scientists to report on various aspects of ocular disease, with a focus on the use of animal models to elucidate the underlying mechanisms of pathobiology involved. Rather than limit the focus to only certain aspects of retinal research, we contacted a broad representation of vision scientists studying the retina, in the hopes of compiling a more comprehensive overview of the field. We believe that you will find, herein, a breadth of studies touching on many of the key areas of current retina research. > Steven J. Pittler, Steven J. Fliesler Editors	doab	2025-04-07T03:56:59.502760	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 6 }
00a6a373-fd58-400b-bd1a-e87d014d5110.7	Characterizing the Retinal Phenotype in the High-Fat Diet and Western Diet Mouse Models of Prediabetes Bright Asare-Bediako 1, Sunil K. Noothi 2, Sergio Li Calzi 2, Baskaran Athmanathan 3, Cristiano P. Vieira 2, Yvonne Adu-Agyeiwaah 1, Mariana Dupont 1, Bryce A. Jones 4, Xiaoxin X. Wang 5, Dibyendu Chakraborty 2, Moshe Levi 5, Prabhakara R. Nagareddy 3 and Maria B. Grant 2,\* Received: 9 January 2020; Accepted: 13 February 2020; Published: 18 February 2020 Abstract: We sought to delineate the retinal features associated with the high-fat diet (HFD) mouse, a widely used model of obesity. C57BL/6 mice were fed either a high-fat (60% fat; HFD) or low-fat (10% fat; LFD) diet for up to 12 months. The effect of HFD on body weight and insulin resistance were measured. The retina was assessed by electroretinogram (ERG), fundus photography, permeability studies, and trypsin digests for enumeration of acellular capillaries. The HFD cohort experienced hypercholesterolemia when compared to the LFD cohort, but not hyperglycemia. HFD mice developed a higher body weight (60.33 g vs. 30.17g, p < 0.0001) as well as a reduced insulin sensitivity index (9.418 vs. 62.01, p = 0.0002) compared to LFD controls. At 6 months, retinal functional testing demonstrated a reduction in a-wave and b-wave amplitudes. At 12 months, mice on HFD showed evidence of increased retinal nerve infarcts and vascular leakage, reduced vascular density, but no increase in number of acellular capillaries compared to LFD mice. In conclusion, the HFD mouse is a useful model for examining the effect of prediabetes and hypercholesterolemia on the retina. The HFD-induced changes appear to occur slower than those observed in type 2 diabetes (T2D) models but are consistent with other retinopathy models, showing neural damage prior to vascular changes. Keywords: retinal phenotype; neural infarcts; vascular leakage	doab	2025-04-07T03:56:59.502835	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 7 }
00a6a373-fd58-400b-bd1a-e87d014d5110.8	1. Introduction Diabetes is now considered a worldwide epidemic [1,2]. Recent reports indicate that over 90% of diabetic individuals have type 2 diabetes (T2D) [3,4]. The most common microvascular complication of diabetes is diabetic retinopathy (DR) [2]. Despite a growing number of different approaches to arrest DR, the incidence and prevalence of DR continues to rise [5]. The understanding of the pathogenesis of DR remains incomplete [4], and this is, in part, due to the lack of readily available models that completely recapitulate the metabolic phenotype [6]. The high-fat diet (HFD) mouse model has been described as a robust model for investigating obesity-associated T2D and its related metabolic complications [7]. Studies have shown that HFD-fed mice develop obesity, impaired glucose tolerance, and reduced insulin sensitivity [8,9] with systemic manifestations involving adipose tissue [10], liver [8], and kidneys [11]. However, the ocular changes associated with the HFD model have not been fully investigated. Moreover, the typical Western diet (WD; 40% fat) has also been given to rodents to recapitulate obesity-driven pathology. However, to mimic the features of T2D, the administration of low-dose Streptozotocin (STZ) is also given to the WD mice [12–14]. The retinal response to high fat exposure would likely involve local changes in the expression of lipid transport proteins, such as the liver X receptors (LXRs). The LXRs are the key transcription factors that regulate lipid and cholesterol metabolism [15]. While liver X receptor alpha (LXR α) is expressed only in some tissues, the expression of liver X receptor beta (LXRβ) is ubiquitous [12]. Previously we showed that whole body LXR α/β deficiency resulted in the generation of increased numbers of acellular capillaries, while LXR agonists improved DR in Streptozotocin (STZ)-induced diabetes [12] and in diabetic Leprdb/db (db/db) mice [16]; however, it is not known if the WD modulates the expression of LXR in the retina. Retinopathy is typically characterized by macroglia activation and gliosis identified by glial fibrillary acidic protein (GFAP) overexpression, which can be considered as a marker for retinal damage [17,18]. In the healthy mammalian retina, GFAP is expressed only in astrocytes and not in Muller cells. Following inherited or acquired retinal pathology, GFAP is expressed also in Muller cells [19,20]. GFAP expression in Muller cells has been widely used as a cellular marker for retinal pathology [21–25]. Hypoxia-inducible factor 1 alpha (HIF-1 α) is known to be a key regulator of a tissue's response to hypoxia [26] and plays a role in obesity-induced metabolic syndrome. It has been shown that HFD leads to gradual increase in HIF-1 α and associated pathological changes in the liver [27,28]. However, the role of HIF-1 α in the retina of WD-fed mice is not known. A better understanding of DR in obesity-driven models is needed and may facilitate the optimal choice of disease models for future investigations. Thus, in the present study, we hypothesized that HFD and WD feeding would result in a distinct retinal phenotype and a time course slower than that observed in models of T2D, such as the db/db mouse [29] or the high fructose and high fat fed mouse [30]. For this purpose, we characterized not only systemic endpoints of glucose and lipid metabolism but also the function of the retina and development of retinal pathology, including retinal vascular changes and changes in expression of the critical proteins LXRβ, HIF-1 α, and GFAP.	doab	2025-04-07T03:56:59.502986	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 8 }
00a6a373-fd58-400b-bd1a-e87d014d5110.10	2.1. Animals All animal experiments were approved by the University of Alabama at Birmingham (IACUC-20467, approved on 06/16/2016) and Georgetown University (animal project #2017-0059, approved on 10/27/2017), and followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals. Six to eight-week-old C57BL/6J mice were fed either a low-fat diet (LFD) (10%kcal fat, 70%kcal carbohydrate, 20% protein), a Western diet (40% kcal fat, 43% kcal carbohydrate, 17%kcal protein), or a HFD (60% kcal fat, 20% kcal carbohydrate, 20% kcal protein) for up to 12 months. Diets were purchased from Research Diets, Inc, New Brunswick, NJ, USA. Full details of the composition of each diet is given in Supplementary Table S1. ### 2.2. Body Composition, Glucose Tolerance, and Insulin Sensitivity Testing The fat mass, lean mass, and water content of the animals were measured by magnetic resonance imaging using EchoMRI (Echo Medical Systems, LLC, Houston, TX, USA). For glucose and insulin tolerance tests, mice were fasted for 5-6 h, injected intraperitoneally with D-glucose at 1.5 g/kg of lean mass and tail bled for glucose and insulin measurements. Blood glucose and insulin levels were measured 0, 15, 30, 45, 60, and 120 min after glucose administration. The insulin sensitivity index (ISI) was estimated using the Matsuda–Defronzo method [31].	doab	2025-04-07T03:56:59.503243	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 10 }
00a6a373-fd58-400b-bd1a-e87d014d5110.11	2.3. Electroretinogram (ERG) ERGs were performed using a LKC Bigshot ERG system. Briefly, mice were dark-adapted overnight. The animals were anesthetized with ketamine (80 mg/kg total body mass) and xylazine (15 mg/kg total body mass), then dilated with atropine/phenylephrine under dim red light. Once dilated, animals were exposed to 5 full-field white light flashes at 0.25 and 2.5 cd.s/m<sup>2</sup> under scotopic conditions. The animals were then light-adapted for 5 min and exposed to 10–15 full-field white light flashes at 10 and 25 cd.s/m<sup>2</sup> under photopic conditions. Responses were averaged and analyzed using the LKC EM software. ### 2.4. Fundus Photography and Fluorescein Angiography Fundus photography and fluorescein angiography were performed using the Phoenix Micron IV retinal imaging microscope (Phoenix Technology Group, Pleasanton, CA, USA). Briefly, mice were anesthetized with ketamine and xylazine, then dilated with atropine/phenlylephrine, as described above. Once dilated, the animals were placed on the instrument and fundus photographs were taken. Animals were then given intraperitoneal injection of fluorescein (AK-FLUOR 10%, Sigma Pharmaceuticals, North Liberty, IA, USA) and the retinal vasculature was imaged with blue light illumination after 5–8 min when all the vessels were filled.	doab	2025-04-07T03:56:59.503448	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 11 }
00a6a373-fd58-400b-bd1a-e87d014d5110.12	2.5. Acellular Capillaries Quantification Trypsin digestion of the retina was performed according to a previously published protocol [32,33]. Briefly, eyeballs were enucleated and incubated in 4% paraformaldehyde overnight. Retinas were isolated, washed, and digested in elastase solution (40 Units elastase/mL; Sigma-Aldrich, St. Louis, MO, USA) to remove the non-vascular tissue. The vascular beds were mounted on glass slides followed by staining with periodic acid–Schi ff's base and hematoxylin. About 5–6 fields from the central to mid-periphery were imaged and the number of acellular capillaries per square millimeter were quantified.	doab	2025-04-07T03:56:59.503552	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 12 }
00a6a373-fd58-400b-bd1a-e87d014d5110.13	2.6. Immunohistochemistry Immunohistochemical staining of mouse retinas was performed according to a previously published protocol [34]. Briefly, mice were euthanized and eyes were immediately enucleated and fixed in 4% paraformaldehyde (PFA) solution for 15 min. Cornea and lenses were carefully removed and posterior cups were incubated in 15% sucrose solution in phosphate-bu ffered saline (PBS) overnight at 4 ◦C after washing briefly in PBS. Posterior cups were transferred to 30% sucrose in PBS for 3–4 h, then embedded in optimal cutting temperature (O.C.T) medium and immediately frozen on dry ice. The frozen samples were stored at −80 ◦C until further processing. The sections were thawed at room temperature for 4 h, washed in PBS for 5 min, and permeabilized with 0.25% Triton-X in PBS for 5 min at room temperature. Sections were blocked with 10% horse serum in 1% bovine serum albumin (BSA) for 2 h then incubated with primary antibody diluted in blocking solution (1:100 dilution) overnight at 4 ◦C. The antibodies used were rabbit anti-GFAP (Abcam, MA, USA), mouse mAB HIF-1 α antibody (Novus Biologicals, CO, USA), LXR-β polyclonal antibody (Invitrogen, IL, USA), rabbit anti-Vimentin (Cell Signaling Technology, MA, USA), and isolectin GS-IB4 Alexa Fluor 568 (Life Technologies, OR, USA). Sections were then washed and incubated in fluorescent-labeled secondary antibodies (goat anti-rabbit IgG Alexa Fluor 488, Life Technologies, OR, USA) for 1 h at room temperature, followed by washing and incubation with <sup>4</sup>,6-diamidino-2-phenylindole, dihydrochloride (DAPI) solution (Life Technologies, OR, USA) for 5 min at room temperature. Finally, sections were washed and mounted with anti-fade mounting medium (Vector Laboratories, CA, USA) for imaging. Image analysis was completed in a masked fashion using four images taken at defined positions and quantified using ImageJ software. The analysis was performed in a masked fashion by three separate observers, then averaged. To achieve unbiased results, positive and negative controls were included alongside experimental test and control groups. Fluorescent microscopy was performed by trained masked operators. To address selection bias in immunofluorescence, the entire areas of retinal cross-sections were imaged.	doab	2025-04-07T03:56:59.503615	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 13 }
00a6a373-fd58-400b-bd1a-e87d014d5110.14	2.7. Statistics All experiments were repeated at least 3 times. All data were assessed using one-way ANOVA. When the results were significant, we determined which means differed from each other using Tukey's multiple-comparisons test. Results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism, with p < 0.05 considered statistically significant. Only significant comparisons are shown in the figures. All the examiners were blinded to the identities of the samples they were analyzing.	doab	2025-04-07T03:56:59.503779	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 14 }
00a6a373-fd58-400b-bd1a-e87d014d5110.15	3. Results ### 3.1. HFD Mice Have Normal Glucose Levels but Are Insulin-Resistant We first sought to validate our model by confirming in our cohort that HFD feeding led to similar degrees of body weight gain as reported in the literature [10,35]. Mice on HFD showed increased body weights by 4 weeks of feeding (p = 0.0020). This increase was sustained throughout the 12-month observation period (Figure 1A). At 12 months, HFD mice had moderately higher lean mass (difference of 5.580 ± 1.003g, p < 0.0001) and water content (difference of 4.517 ± 0.876, p = 0.0001) but a markedly increased fat mass (difference of 21.15 ± 2.362, p < 0.0001) compared to LFD controls (Figure 1B). Unexpectedly, chronic high-fat feeding did not cause hyperglycemia. Despite feeding mice with a HFD for 12 months, the HbA1c levels were not different between HFD and LFD mice (Figure 1C). At 12 months, there was no difference in fasting blood glucose levels (Figure 1D, basal) and intraperitoneal glucose tolerance test (IP-GTT) did not show any significant differences between LFD and HFD mice (Figure 1D). However, due to the very high levels of insulin in HFD mice (0.6 ng/mL for LFD and 3.5 ng/mL for HFD; Figure 1E, basal), the insulin sensitivity index demonstrated that HFD mice had much lower insulin sensitivity compared to LFD mice (Figure 1E,F). Also, plasma total cholesterol levels were higher in HFD mice compared to LFD (174.4 vs. 114.9, p = 0.0008) (Figure 1G) Figure 1. Cont. Figure 1. Body weight, glucose levels, and insulin sensitivity of high-fat diet (HFD) mice vs. low-fat diet (LFD) mice. (A) Body weights as measured for mice on LFD (green) and HFD (red) for 12 months. (\* p < 0.000001; n = 6). (B) Lean mass, fat mass and water content of LFD mice vs. HFD mice. (C) Glycated hemoglobin (HbA1c) levels measured for the mice after 6 months and 12 months. (D–G) Glucose curves (p > 0.46 for all time points), insulin curves (p < 0.0018 for all time points), insulin sensitivity index, and total cholesterol levels for LFD mice vs. HFD mice, respectively, following intraperitoneal glucose tolerance test (IP-GTT) after 12 months of feeding. ### 3.2. HFD Mice Have Functional Deficits in Their Retinas Full-field ERG under both scotopic and photopic conditions was performed at 6 months and 12 months of HFD feeding (Figure 2A–D). HFD mice at 6 months showed significantly reduced aand b-wave amplitudes under scotopic conditions (p = 0.00125 and p = 0.000002 for 0.25 cd.s/m<sup>2</sup> and 2.5 cd.s/m<sup>2</sup> stimulus luminance, respectively) but not photopic conditions when compared to LFD mice. After 12 months of feeding of the respective diets, the difference was not significant (p = 0.183 and p = 0.154 for 0.25 cd.s/m<sup>2</sup> and 2.5 cd.s/m<sup>2</sup> stimulus luminance respectively) (Figure 2C,D). Interestingly, when comparing 6 and 12 months of LFD feeding, the mice experienced marked reductions in both the a- and b- waves under both photopic and scotopic conditions at 12 months (Figure 2E,F), but no significant difference was noted in the HFD-fed mice (Figure 2G,H). Figure 2. Assessment of retinal function of LFD mice versus HFD mice by electroretinogram (ERG). The amplitudes of a-waves and b-waves were assessed under both scotopic and photopic conditions for LFD mice and HFD mice after 6 months (A,B) and 12 months (C,D). LFD mice showed a significant reduction in retinal response between 6 months and 12 months of feeding (E,F), but HFD mice did not (G,H); (n = 4 for both groups). ### 3.3. Fundus Photography shows Neural Retinal Lesions in HFD Mice In humans, DR is associated with retinal lesions such as hemorrhages, microaneurysms, exudates, and "cotton wool spots" [36]. Fundus photography using Micron IV demonstrated retinal pathology in the HFD mice. Though not statistically significant, HFD mice showed a trend of increased numbers of "lipid-laden-like" lesions (Figure 3A) after 6 months (p = 0.057). However, with 12 months of feeding, HFD mice showed significantly higher number of lesions in the retina (Figure 3B). Figure 3. Assessment of retinal lesions by fundus photography (A,B) and vascular leakage by fluorescein angiography (C,D). HFD mice developed more neural infarcts ((A,B), white arrows) than LFD mice. No infarct was observed for LFD after 6 months (A). However, vascular leakage was observed in HFD mice after 12 months of feeding ((D), white arrows). ### 3.4. Vascular Permeability Changes in HFD Mice A hallmark of DR in humans is increased vascular permeability, ultimately leading to diabetic macular edema in humans. To determine if HFD mice developed a breakdown in the blood–retinal barrier, we assessed vascular leakage by fluorescein angiography (FA). At 6 months of HFD feeding, FA did not show any evidence of retinal vascular leakage and were similar to FAs in LFD controls (Figure 3C). However, after 12 months of HFD feeding, increased leakage of fluorescein was observed in the retina compared to LFD control retinas (Figure 3D). ### 3.5. Acellular Capillary Formation in HFD Mice A well-established feature of diabetic microvascular dysfunction is an increase in the number of acellular capillaries in the retina, defined as basal membrane tubes lacking endothelial cells and pericyte nuclei. At 12 months of HFD feeding, there was no significant increase in acellular capillary numbers in the HFD mice (Figure 4B,C) compared to the LFD mice (Figure 4A,C). However, the HFD retinas showed lower vascular densities compared to LFD retinas (Figure 4D). Figure 4. Enumeration of acellular capillaries in LFD and HFD mice after 12 months of feeding. Red arrows indicate acellular capillaries in the retinas of LFD ( A) and HFD (B) mice. There was no significant di fference in the number of acellular capillaries between both groups ( C) (p = 0.086). However, HFD retinas showed lesser vascular densities compared to LFD retinas ( D). ### 3.6. Retinal Damage, Hypoxia, and Lipid Transport in WD Mice While the HFD represents a diet with 60% fat content that is used as a model of obesity and T2D, the WD with 40% fat content has garnered popularity as it represents a regimen closer to that actually ingested by humans. Since the WD diet has lower fat content and is not associated with hyperglycemia, we hypothesized that if retinal changes were present they would be subtle compared to those we observed with HFD feeding. To test the validity of our hypothesis, we performed IHC studies and first examined whether there was evidence of glial activation by examining expression of the glial marker GFAP after 6 months of WD feeding. Although there was no statistically significant di fference (p = 0.88) in the total expression of GFAP between retinas of WD and LFD mice (Figure 5A–C), increased expression of GFAP was observed in selected Vimentin-positive Muller cells in the WD mice (Figure 5G–I) compared to LFD (Figure 5D–F). Increased expression of GFAP in Muller cells is supportive of increased oxidative stress and inflammation in these cells, and suggests that the impact of WD is not experienced uniformly across all Muller cells [37,38]. To assess whether WD feeding induced retinal hypoxia, changes in HIF-1 α expression were examined by IHC. After 6 months of WD feeding, a significant increase (p = 0.025) in expression of HIF-1 α was seen in WD mice (Figure 6D) compared to LFD mice (Figure 6C). This was not observed after 3 months of WD feeding (Figure 6A,B). Quantitation of HIF-1 α expression is shown in Figure 6E, demonstrating that WD-fed mice exhibit higher levels than LFD-fed mice. Co-localization with isolectin, a known vascular endothelial cell marker, showed increased expression of HIF- 1 α in some endothelial cells in WD mice (I–K) but not in LFD mice (F–H). Higher magnification images from two di fferent WD samples are shown in Figure 6L,M. Retinal lipid content is regulated in part by liver X receptor beta (LXRβ) expression. We next examined changes in LXRβ expression in the two experimental cohorts. In control mice, LXRβ localized predominantly in the ganglion cell layer, as well as the inner nuclear layer (Figure 7A), which is the location of the bipolar cells, horizontal cells, and amacrine cells. There was a significant reduction in expression of LXRβ in WD only in the ganglion cell layer (p = 0.0079) after 3 months of feeding (Figure 7B). However, after 6 months of WD feeding, WD mice (Figure 7E) showed significantly reduced expression of LXRβ in the ganglion cell layer (p = 0.0374), inner nuclear layer (p < 0.0001), and outer nuclear layer, as well as in the photoreceptors of the outer nuclear layer (p = 0.0020). The expression of LXRβ was reduced after 6 months compared to 3 months of feeding in both LFD (p < 0.0001) and WD (p < 0.0001) in the nuclear and ganglion cell layers, suggesting an age-related loss in LXRβ. Figure 5. Cont. Figure 5. Retinal glial fibrillary acidic protein (GFAP) expression after 6 months of feeding. Some Muller cells in Western diet (WD) retinas express GFAP (A,C, white arrows), but not in LFD (A,B), indicating that the impact of WD is not uniform across all Muller cells. Co-localization with Vimentin, a known Mueller cell marker, showed increased expression of GFAP in some Mueller cells in WD mice (G–I) but not in LFD mice (D–F). Figure 6. Cont. Figure 6. Retinal hypoxia-inducible factor 1 alpha (HIF-1α) expression after 3 and 6 months of WD feeding. There was increased expression of HIF-1α in WD retinas (D, white arrows) compared to LFD retinas (C), as shown by quantification (E). Also, there was no significant difference in expression of HIF-1α after 3 months of feeding (A,B). Co-localization with isolectin, a known vascular endothelial cell marker, showed increased expression of HIF-1α in some endothelial cells in WD mice (I–K) but not in LFD mice (F–H). (L,M) Magnified merged images from two different WD samples. Figure 7. Retinal liver X receptor beta (LXRβ) expression after 3 and 6 months of feeding. After 3 months of either WD or LFD feeding, there was significant reduction in the expression of LXRβ in only the ganglion cell layer of WD mice (B) compared to LFD mice (A). However, after 6 months of feeding, there was reduced expression of LXRβ in the ganglion cell layer as well as inner and outer nuclear layers of WD mice (E, white arrows) compared to LFD mice (D). Quantification of LXR in the inner nuclear layer (INL) and outer nuclear layer (ONL) at 3 months shows reductions in the ganglion cell (GC) layer (C). At 6 months, reductions are seen in the INL, ONL, and ganglion cell (GC) layer of the WD-fed mice when compared to LFD mice.	doab	2025-04-07T03:56:59.503837	1-5-2021 17:32	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00a6a373-fd58-400b-bd1a-e87d014d5110", "url": "https://mdpi.com/books/pdfview/book/2654", "author": "", "title": "The Molecular and Cellular Basis of Retinal Diseases", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783039366545", "section_idx": 15 }

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