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,round plastic planter 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 biosynthetic 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. Exogenous application of S-ABA improves the color of table grapes by stimulating the anthocyanin synthesis and accumulation in the grape skin . Our results are similar to those reported previously for “Flame Seedless” grapes, in which applications of 300 mg/L ABA during or after véraison were more effective at increasing anthocyanin concentrations than application before véraison . In ‘Crimson Seedless’ grapes, anthocyanin concentrations increased with application of 400 mg/L S-ABA at 17 DAV, but the response varied widely between seasons depending on S-ABA dosage and time of application .

Exogenous S-ABA application is thought to simulate plant stress responses and accelerate ripening processes . High ABA concentrations are believed to be perceived by grapevines as a drought stress signal . Subsequently, water stress leads to changes in grape secondary metabolism, significantly increasing flavonoid levels and, especially, anthocyanin biosynthesis . In our study, in addition to increasing anthocyanin concentration, exogenous S-ABA improved both berry color intensity and uniformity. This is important because the visual assessment of berry color characteristics determine, in part, the commercial value of table grapes . Grape clusters with more intense and uniform berry color have higher consumer acceptance. Improved color characteristics such as increased color coverage of grape berries, and uniformity of berry color within a cluster, were also observed in “Benitaka” and “Rubi” table grapes following application of 400 mg/L S-ABA at 7 DAV and 15 days before harvest . In our experiments, berries from the seedless grape Selection 21 treated with one or two applications of 400 mg/L S-ABA presented higher CIRG values than the controls. Besides, improving cluster color and attractiveness, exogenous S-ABA can potentially decrease the time to harvest, a feature that is very advantageous for grape commercialization . The observed increase in anthocyanin concentration resulting from S-ABA application does not necessarily result in an increase in total polyphenol content; polyphenols include phenolic acids, stilbenes, coumarins, tannins, and flavonoids , as reported in “Alachua” muscadine grapes and in “Isabel” grapes . Environmental factors such as temperature, rainfall, and altitude could also influence berry polyphenol concentrations. Inaddition, the berry ripening stage is directly correlated to the concentration and proportion of several polyphenols that impact the organoleptic properties, nutritional value, and antioxidant capacities of the grapes .

S-ABA application can negatively affect berry firmness, an important characteristic for the successful postharvest handling of grapes for the fresh fruit market because it influences transportability and shelf life . ABA application is known to cause loosening and decreased rigidity of the cell wall, resulting in fruit softening and a higher probability of berry cracking . Treatment of grapes with exogenous S-ABA can result in changes in the regulation of proline-rich cell wall proteins and in the induction of cell wall degrading genes, such as polygalacturonases that promote pectin solubilization and depolymerization . The effect of S-ABA on berry firmness was also observed in “Flame Seedless” grapes, where it caused softening similar to that caused by ethephon application , as well as in ‘Crimson Seedless’ and “Red Globe” grapes . Therefore, it is still necessary to evaluate if the benefits of applying exogenous ABA to improve berry color can outweigh a potential reduction in the shelf life of treated grapes. Multiple applications of exogenous ABA can promote anthocyanin accumulation for longer periods of time . It is possible that more than one ABA application could induce a milder response at later grape phenological stages or that the effects of a second application could take more time to be evident. In this study, the second application of 400 mg/L S-ABA significantly increased the total anthocyanin content at the time of harvest, which supported the latter hypothesis and confirmed that two S-ABA applications had a more pronounced effect than only one application. The higher total anthocyanin concentration observed with S-ABA application appeared to result from a transient effect of S-ABA, because the anthocyanin concentration of grapes that received only one S-ABA application remained essentially constant between 28 and 35 DAV. It may, therefore, be inferred that the action of S-ABA decreases over time and that its levels increase with a second application, allowing maintenance of its activity. Three applications of 400 mg/L S-ABA at 1-week intervals prior to véraison resulted in an earlier accumulation of anthocyanin in “Cabernet Sauvignon” grapes, but no differences in anthocyanin concentration at harvest were observed in grapes that received different treatments . The increase in endogenous ABA concentration in grape berries occurs at the beginning of véraison and extends until the establishment of maturation when endogenous ABA concentrations peak. ABA concentration then decreases until harvest, the period over which the decrease occurs ranges from 13 to 20 days depending on the cultivar . Application of exogenous S-ABA close to véraison,round plastic plant pot when ABA naturally reaches its highest concentration in berries, was shown to be more effective in increasing anthocyanin accumulation than application at other times . S-ABA application significantly increased endogenous ABA levels 7 days after application in “Carménère” grapes, 40 days later, the ABA levels in the treated berries remained higher than those from control . Berry color is affected both by total anthocyanin concentration and by anthocyanin composition.

The five main grape anthocyanins differ from each other in the number and position of the hydroxyl and methyl groups on the B-ring. Cyanidin and peonidin are dihydroxylated precursors of red anthocyanins in grape skin, whereas delphinidin, petunidin, and malvidin are trihydroxylated precursors of blue and purple anthocyanins . Accumulation of individual anthocyanins in grapes may be induced by S-ABA application and varies with the cultivar. In “Noble” and “Alachua” muscadine grapes, application of S-ABA during véraison and again at 8 DAV for “Noble” or again at 13 DAV for “Alachua” resulted in higher levels of accumulation of all evaluated anthocyanins in “Noble” grapes but not in “Alachua” grapes, which only presented higher accumulation of peonidin-3-diglucoside compared to the control . Changes in the proportions of individual anthocyanins resulting from S-ABA application were also observed in “Cabernet Sauvignon” grapes, both in berries and in wine . In “Isabel” grapes, application of S-ABA increased the accumulation of individual anthocyanins both in must and in processed whole juice . Similar results were observed in wines prepared from “Merlot” grapes treated with a racemic mixture of ABA . This treatment resulted in changes in the proportions of anthocyanins, increased total phenol and flavonol content, and increased antioxidant activity . However, it should be considered that application of racemic mixtures of enantiomers may result in a range of plant responses because R-ABA is not found in plants and is less active and less effective than S-ABA. The two enantiomeric forms may have different effects on gene expression and on physiological responses . Anthocyanin accumulation in grape berries during véraison is probably triggered by increased sugar and ABA concentrations in the berry skin, which activate the expression of genes involved in anthocyanin biosynthesis . The activation threshold for genes involved in anthocyanin production was reported to be between 9 and 10◦Bx . S-ABA application at 7 DAV, when anthocyanin biosynthetic genes are normally induced, followed by a second application at 21 DAV, when endogenous ABA concentrations are close to maximal or are beginning to decrease, can upregulate their expression even further or maintain them at a constant level for a longer period of time. Anthocyanins are produced through multiple pathways that are controlled by MYB transcription factors. These transcription factors are responsive to ABA and are associated with the regulation of the biosynthetic genes CHI, F3H, DFR, LDOX, and UFGT . The transcription factors VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevines and are not functional in white grape cultivars . Transcription factors affect the ratio of tri-/dihydroxylated anthocyanins through trans-regulation of flavonoid 3-hydroxylase and flavonoid 30 5 0 -hydroxylase gene expression . During anthocyanin biosynthesis, F3H is responsible for the hydroxylation of naringenin at position 30 , generating dihydrokaempferol, a dihydroflavonol that can be hydrolyzed at position 30 or 50 of the B-ring by the enzymes F30 H or F30 ,50 H, which are responsible for the hydroxylation of the B-ring of flavonoids. F30 H activity promotes accumulationof the cyanidin and peonidin anthocyanin groups, whereas F30 ,50 H activity results in the production of delphinidin and its derivatives petunidin and malvidin. These two enzymes compete in controlling di- and trihydroxylated anthocyanin synthesis . In our study, treatment of hybrid grapes with two applications of 400 mg/L S-ABA primarily favored the accumulation of delphinidin-3-glucoside and malvidin-3-glucoside ; therefore, such treatment decreased the difference between the concentrations of diand trihydroxylated anthocyanins in the grapes. This is consistent with previous results obtained for “Aki Queen” grapes , in which the application of S-ABA stimulated the gene expression of F30 ,50 H relative to F30 H. In addition, the concentrations of petunidin and malvidin increased in the berries, thereby increasing the proportion of trihydroxylated anthocyanins and decreasing the proportions of cyanidin and peonidin anthocyanins relative to the total anthocyanins . In this study, the expression of the main enzymes leading to anthocyanin biosynthesis were analyzed. Future experiments to study changes in expression of F30 H and F30 ,50 H encoding genes are still required to gain a better insight into the impact of exogenous ABA applications on the differential accumulation of specific anthocyanins. Our results indicate that application of S-ABA increased the expression of the UFGT gene and the transcription factors at 28 DAV, but this was not observed for the treatment with only one S-ABA application. Anthocyanin accumulation begins when all genes involved in the biosynthetic pathway are induced, especially UFGT . Anthocyanidins are unstable and are easily degraded to colorless compounds; therefore, before anthocyanins are transported, they must be stabilized by the addition of a glucose residue at position 3 of the C-ring .