Determining how long grape berries are competent to induce the expression of anthocyanin biosynthetic genes may help determine the optimal time, number, and frequency of S-ABA applications. Currently, little is known about the potential benefits of multiple applications, which may be desirable if a single application results in an insufficient response. The aim of the present study was to determine the effects of S-ABA applications at different concentrations and times on the quality and biochemical properties of berries from the seedless grape Selection 21 hybrid during three growing seasons in the region of Paraná, Brazil. We evaluated a variety of parameters including: grape color development, berry phenolic profiles, and gene expression of transcriptional regulators and biosynthetic enzymes of the anthocyanin pathway after treatments with S-ABA. The results of this report indicate that two S-ABA applications during and after véraison extend the competency of grape berries to respond to ABA and induce the accumulation of anthocyanins, resulting in higher grape berry coloration. The study was conducted during three consecutive seasons in a commercial vineyard located in Marialva, state of Paraná , Brazil , using 4-year-old vines of hybrid seedless grape Selection 21 grafted onto IAC 766 Campinas rootstock. According to the Köppen classification, growing bags the climate of the region is Cfa , with an average temperature below 18◦C in the coldest month and above 22◦C in the hottest month and an average annual rainfall of 1,596 mm. The region’s soil is classified as dystroferric red latosol .
The vines were trained using a bilateral overhead trellis system, where vines were spaced at 2.5 m × 2.5 m , and each vine had 6.25 m2 total canopy area. Cane pruning was performed during the 2013, 2014, and 2015 seasons and was followed by application of 3% hydrogenated cyanamide to the two apical buds to induce and standardize sprouting. The number of canes per vine was evenly adjusted to 40 and the number of shoots per vine was also established to 40 . Considering that a grape bunch of the Selection 21 weighs on average 460 g, the load per vine is 18.40 kg, which represents an estimated yield of 29.44 tons/ha. Furthermore, to avoid drifting, a non-treated vine was left as side border between two treated vines, which almost duplicated the experimental area. In each plot, all grape bunches were treated , and the bunch samples were collected from random positions at each side of the canopy to account for intracanopy variability. Control plants were not subjected to any treatment, instead, they were sprayedwith water at the same time and following the same procedures as the S-ABA treatments.The effects of applying S-ABA isomer at different concentrations and times were evaluated in terms of berry quality traits. ProTone R, the commercial growth regulator used in this study, has an active ingredient concentration of 100 g/L S-ABA. As shown in Figure 1, the initial treatments tested in the 2013 and 2014 seasons corresponded to: control or water spray, 200 mg/L S-ABA application at 7 days after véraison , 400 mg/L S-ABA application at 7 DAV, 200 mg/L S-ABA application at 7 DAV plus an additional application at 21 DAV, and 400 mg/L S-ABA at 7 DAV plus an additional application at 21 DAV. In the 2015 season, only the control and treatments of 400 mg/L S-ABA with one or two applications were performed and analyzed.
Berry samples from the 2015 season were collected from each treatment at four different times: 7 DAV , 14 DAV, 28 DAV, and 35 DAV for further targeted analyses . For all seasons, a randomized complete block experimental design was used, with five treatments and three to four replicates, and with each plot consisting of one vine . Véraison was determined by measuring soluble solid content and firmness of grape berries randomly sampled in the experimental vineyard. At véraison, the mean grape SSC concentration was 9◦Bx, and 20% of the berries in more than 50% of the grape clusters presented softening . The berries presented a mean of 11◦Bx at 7 DAV, the time of the first S-ABA application, and a mean of 13◦Bx at 21 DAV, the time of the second S-ABA application. For treatment applications, grape clusters were sprayed in the morning using a knapsack sprayer at a pressure of 568.93 psi with JA1 hollow cone nozzle tips at a volume of 800 L/ha to provide complete and uniform coverage. In addition, 0.3 mL/L of Break-Thru R a non-ionic surfactant was added to all treatments. During the trials, the standard regional cultivation practices with regard to nutrition, weed control, and pest and disease management were used.Clusters of each plot were manually harvested when SSC stabilized . The clusters were cleaned, and damaged berries were discarded. Color coverage of the bunches was determined using 10 grape clusters per plot by visually rating the clusters on a scale of 1–5 using the following scale: 0–20%, 21–40%, 41–60%, 61–80%, and 81–100% coverage .
The same grape clusters used for evaluating color coverage measurements were used for berry sampling. For physicochemical analyses, two berries were collected from the upper, middle, and lower portion of each grape bunch, yielding a total of 70 berries per plot. Total anthocyanins and color index of red grapes were determined in berry samples from all seasons. The following variables were analyzed only for the 2013 and 2014 seasons: color coverage, total polyphenols, and berry firmness. All physiological analyzes were performed in the Laboratory of Fruit Analysis of the Agricultural Research Center, Londrina State University, Brazil. The total anthocyanin concentration of the berries was determined using 30 berries per plot, which were frozen and stored at −20◦C. The berry skins were removed using tweezers, taking care to remove only the skin, without pulp. The skins were washed once with water, followed by washing in deionized water and drying with absorbent paper. A 5-g skin sample was then placed in a polystyrene tube containing 50 mL of acidified methanol and stored in the dark for 48 h at room temperature. The tubes were then removed from the dark and stirred manually for 5 s. Absorbance was determined using a Genesys 10S spectrophotometer at 520 nm with the solvent as blank. The results were expressed in milligram malvidin-3-glucoside per gram of skin . The CIRG was determined using 10 berries per plot with a CR-10 colorimeter , using the CIELAB color system. The following variables were determined for the berry equatorial section: lightness , saturation , and hue . CIRG was then determined using the following equation: CIRG = / . Total polyphenol determination was performed using 30 berries per plot based on a modified Folin–Ciocalteu method. In summary, the absorbance of each sample was measured after 15 min at 765 nm using a Genesys 10S spectrophotometer against a blank sample prepared with water instead of the extract. Determination of total polyphenol was calculated from the calibration curve obtained with gallic acid. The results were expressed in milligram total polyphenols per 100 g of sample . The berry firmness was performed with a TA.XT2i Texture Analyzer , at 25 ± 1 ◦C, nursery grow bag analyzing the equatorial position of 10 berries with pedicels per plot. Each berry was placed on the base of the equipment and compressed using a cylindrical probe with a diameter of 35 mm parallel to the base. A constant force of 0.05 N at a speed of 1.0 mm/s was applied to promote the cracking of the sample. The berry firmness was then determined .Application of abscisic acid increased the total anthocyanin concentration in berry skins of the seedless grape Selection 21 during the 2013 and 2014 seasons, regardless of the S-ABA concentration and time of application . However, berries that received 400 mg/L of S-ABA at 7 and 21 DAV had significantly higher, almost two to three times more, anthocyanin concentrations than other treatments. According to the CIRG, berries from control treatments had a green to a yellow color in both seasons . In 2013, berries treated with one or two applications of 200 mg/L S-ABA or one application of 400 mg/L S-ABA at 7 DAV, and those in the 2014 season that were treated with one application of 200 mg/L S-ABA developed a pink color . Remarkably, berries of the 2013 season treated with two applications of 400 mg/L S-ABA and berries of the 2014 season treated with one or two 400 mg/L S-ABA applications, developed a stronger red color .
For both the 2013 and 2014 seasons, color coverage was lowest in control grapes and highest in grapes treated with two applications of 400 mg/L S-ABA. Increase in total polyphenols was evident in grapes subjected to two 400 mg/L S-ABA applications during the 2013 and 2014 seasons. These berries also presented the lowest mean berry firmness . Importantly, the increased softening due to S-ABA application did not result in higher frequency of cracked berries in any of the studied seasons. Qualitative assessment of berry cracking was performed visually. Further analyses of the effect of 400 mg/L S-ABA treatments on CIRG, total and individual anthocyanins concentrations, and gene expression of transcription factors and biosynthetic enzymes were performed with grape berries collected from the 2015 trial. Measurements of CIRG confirmed previous results obtained during the 2013 and 2014 seasons, at the time of harvest , grapes treated with two S-ABA applications had the highest CIRG values . Grape bunches from the control treatment presented pink berries , whereas those treated with one or two applications of S-ABA had red berries . As determined in the previous seasons, berries treated with 400 mg/L S-ABA also presented higher total anthocyanin content than the control at 14 and 28 DAV . At 28 DAV, grapes treated with one or two applications of 400 mg/L S-ABA presented total anthocyanin concentrations almost three times higher than the control. Even 3 weeks after the first application , berries treated with only one S-ABA application showed a total anthocyanin content similar to those treated with two S-ABA applications. Nonetheless, the second application of 400 mg/L S-ABA significantly affected the total anthocyanin accumulation at the time of harvest . S-ABA altered the concentrations and proportions of individual anthocyanins in berries from the seedless grape Selection 21 . With the exception of petunidin- 3-glucoside, S-ABA application significantly improved the concentrations of all the measured anthocyanins. Cyanidin-3- glucoside and peonidin-3-glucoside levels increased at 14 DAV, 1 week after the first S-ABA application. The second S-ABA application stimulated the accumulation of the anthocyanin delphinidin-3-glucoside at 28 DAV, yielding differences relative to both the control and to the samples treated with only one S-ABA application. At 28 DAV, the concentrations of peonidin-3-glucoside and malvidin-3-glucoside increased after exogenous S-ABA application but were not further increased by the second application. At the time of harvest , peonidin-3-glucoside and cyanidin-3-glucoside were the dominant pigments present after all treatments. Delphinidin- 3-glucoside, cyanidin-3-glucoside, and peonidin-3-glucoside presented higher accumulation following the second application of 400 mg/L S-ABA, but the number of applications did not affect the accumulation of malvidin-3-glucoside. As presented in Figure 4, treatment with 400 mg/L S-ABA significantly increased the expression of the transcription factors VvMYBA1 and VvMYBA2 and the expression of the bio-synthetic genes CHI, F3H, DFR, and UFGT 1 week after the first application . Three weeks after the first S-ABA application , expression of CHI, F3H, and DFR genes remained high, but this was not observed for the transcription factors VvMYBA1 and VvMYBA2 or the UFGT gene. Four weeks after the first S-ABA application , no significant differences were observed in the expression of genes or transcription factors between berries that received one S-ABA application and those that received the control treatment. The two applications of 400 mg/L S-ABA induced expression of the genes CHI, F3H, DFR, and UFGT and the transcription factors VvMYBA1 and VvMYBA2 at 14 and 28 DAV . F3H expression was the most affected by S-ABA application, displaying higher levels than the control until the final stages of berry maturation at 35 DAV,whereas the remaining genes presented no differences from the control at harvest. Overall, the gene expression results indicate that a second S-ABA application contributed to the maintenance of the expression of the transcription factors VvMYBA1 and VvMYBA2 and the genes F3H and UFGT at higher levels than in the control for an extended period of time.