Improvement of color and increase in anthocyanin content of ‘ Niagara Rosada ’ grapes with application of abscisic acid

This study aimed to assess the application effect of abscisic acid (S-ABA) on color, and total anthocyanin and phenolic compound contents of the ‘Niagara Rosada’ grapes. The experiment was carried out in a commercial vineyard located in Jales, SP, Brazil, during 2014/2015. The randomized block design was used with four treatments and five replications. The treatments consisted of applying different concentrations of S-ABA: 0 mg L -1 (control); 400 mg L -1 of S-ABA at the beginning of maturation (BM); 400 mg L -1 of S-ABA in the BM + 200 mg L -1 25 days after the first application (25 DAFA) and 400 mg L -1 of S-ABA in IM + 400 mg L -1 at 25 DAFA. At harvest, the following variables were evaluated: luminosity (L*), saturation (C*), hue (h°) and color index (CIRG). The total anthocyanin content, as well the total phenolic compounds of berries were also determined. The application of 400 mg L -1 of S-ABA in the BM + 200 mg L -1 to 25 DAFA significantly increased the total anthocyanin content of the ‘Niagara Rosada’ grapes, improving the color of the berries as well.

This study aimed to assess the application effect of abscisic acid (S-ABA) on color, and total anthocyanin and phenolic compound contents of the 'Niagara Rosada' grapes.The experiment was carried out in a commercial vineyard located in Jales, SP, Brazil, during 2014/2015.The randomized block design was used with four treatments and five replications.The treatments consisted of applying different concentrations of S-ABA: 0 mg L -1 (control); 400 mg L -1 of S-ABA at the beginning of maturation (BM); 400 mg L -1 of S-ABA in the BM + 200 mg L -1 25 days after the first application (25 DAFA) and 400 mg L -1 of S-ABA in IM + 400 mg L -1 at 25 DAFA.At harvest, the following variables were evaluated: luminosity (L*), saturation (C*), hue (h°) and color index (CIRG).The total anthocyanin content, as well the total phenolic compounds of berries were also determined.The application of 400 mg L -1 of S-ABA in the BM + 200 mg L -1 to 25 DAFA significantly increased the total anthocyanin content of the 'Niagara Rosada' grapes, improving the color of the berries as well.

INTRODUCTION
The State of São Paulo is the second largest producer of table grapes in Brazil (Agrianual, 2015), with emphasis on the cultivation of common or hybrids grapes, such as 'Niagara Rosada'.This cultivar is a colored mutation of 'Niagara' (Vitis labrusca L. x Vitis vinifera L.), and it has a foxy flavor and scent, and still presents satisfactory sugars level that attracts consumers.'Niagara Rosada' is cultivated mainly in the eastern region of São Paulo, and there are, however, other winegrowing regions, such as the northwest and southwest of the State of São Paulo, represented respectively by the cities of Jales and São Miguel Arcanjo.
Traditionally, the Northwest region of the São Paulo State stands out in the cultivation of table grapes (V.vinifera), however, a decrease in the production of these grapes has been observed in the last years and the increase in the production of common grapes (V.labrusca L. x V. vinifera L.), predominantly the 'Niagara Rosada', which, during the last two years had an increase in production of 17.4% (IEA, 2017).
The climate conditions of the northwest region allow the harvesting of the 'Niagara Rosada' grapes from July to October, the offspring of the eastern and southwestern regions of the state, traditional growing regions where the harvesting of the summer crops is carried out from December to February, and the winter crops from March to June.Associated with the best price obtained in the off-season, there is also a significant reduction of labor and agricultural pesticides in the growing of 'Niagara Rosada' when compared with fine table grapes.However, the warm climate and the low thermal amplitude during the maturation stage of the grapes in the Northwest region make them not to reach optimal color levels (Gardin et al., 2012) due to the reduction of anthocyanin synthesis (Sozim, 2011).Thus, it is necessary to apply plant regulators that aid in these compounds synthesis.
Historically, the acquisition value of ABA used to be high, however, as time went by, a cheaper production method was developed, designated as S-ABA, which is more accessible to grape growers (Cantín et al., 2007).Several studies suggest that exogenous applications of ABA increase the anthocyanin content in the skin of colored grapes, surpassing even the ethephon, playing a vital role in the activation of flavonoids (Cantín et al., 2007;Peppi et al., 2007;Sandhu et al., 2011;Roberto et al., 2013;Koyama et al., 2014a;Domingues Neto et al., 2017).
Color is one of the decisive attributes when marketing table grapes, since those with better color uniformity receive better values because color plays a direct role in attracting consumers, therefore, it is one of the most important ampelographic features, as well as being one of the main characteristics to be considered to evaluate the quality by phenotyping of a potential genotype (Rustioni et al., 2013).Anthocyanins, as an example of some phytochemicals of the polyphenol class, are responsible for the color on red grape berries and their accumulation seem to be regulated, at least in part, by abscisic acid -ABA (Lacampagne et al., 2010) and these pigments are considered important for the commercial value of the grapes (Rustioni et al., 2015), as well as act as antioxidants in the human body, preventing several degenerative diseases (Nixdorf and Hermósín-Gutiérrez, 2010).The expression of anthocyanins depends on internal factors, such as abscisic acid, which induces the transmission factor, MYB1A, a protein responsible for regulating the transcription of genes that make up the biosynthetic route of the anthocyanins of the colored grapes (Jeong et al., 2004).
Considering the increase of 'Niagara Rosada' grape in São Paulo State and the lack of studies involving the application of plant regulators to improve grape quality, this study had as objective to evaluate the effect of S- Tecchio et al. 1401 ABA application in order to increase color, total anthocyanins content and the total phenolic compounds of 'Niagara Rosada' grape.

MATERIALS AND METHODS
The experiment was carried out in a commercial 6-year old vineyard of 'Niagara Rosada' (V.labrusca x V. vinifera) grapevine, grafted on the IAC 572 'Jales' (Vitis caribaea x 101-14 Mgt) rootstock, located in Jales, SP (20º 16 'S, 50º 33' W and average altitude of 480 m), during 2014/2015 season.Vines were trained in an overhead trellis system, spaced at 2.50 x 2.00 m distance and irrigated by micro sprinklers.
The climate of the region according to Köppen is Aw (tropical climate with dry winter season), with annual average rainfall of 1221.6 mm.The annual average temperature is of 24.4°C, with a minimum average of 14.0°C and maximum of 33.0°C (CEPAGRI, 2016).
The randomized block design was used with four treatments and five replications, and each plot was composed by one vine.The treatments consisted of the application of different concentrations of the isomer S-ABA (100 g L -1 of active ingredient, Valent BioSciences Co.): 0 (control); 400 mg L -1 of S-ABA at the beginning of maturation (BM); 400 mg L -1 of S-ABA in BM + 200 mg L -1 at 25 days after the first application (25 DAFA) and 400 mg L -1 of S-ABA in BM + 400 mg L -1 at 25 DAFA.When preparing the solution for all treatments, BreakThru ® , a non-ionic spreader (0.3 mL L -1 ) was added.The beginning of maturation of the berries was when they started softening and color change was considered, with soluble solids of 10 °Brix and titratable acidity of 1.02% of tartaric acid.During all applications, the clusters were pulverized in the morning using a backpack sprayer under 40 kgf cm 2 of pressure with JA1 hollow cone jet nozzles until the berries were completely and even covered, the syrup volume employed was of 800 L ha -1 .
At harvest, 10 representative clusters per plot were selected, from which 100 berries were collected (10 berries per bunch), which were collected from the lower, middle and upper parts of the cluster (3:4:3) in order to determine color parameters, and total anthocyanin and total phenolic compounds content.
For total anthocyanin assessments, berries were cut in half and the seeds were removed, and then, they were pulverized with liquid nitrogen, according to Lee and Francis (1972).The readings were performed on a Genesys 10S Spectrophotometer, UV-VIS at 535 nm.In order to calculate the total anthocyanins concentration, the formula proposed by Teixeira et al. (2008) was used.Then, the quantification of total phenolic compounds was carried out using the Folin-Ciocalteau method, according to Bucic-Kojic et al. (2007), using 50% ethanol as an extractor.The readings were performed on a Genesys 10S Spectrophotometer, UV-VIS at 765 nm.The data obtained were submitted to analysis of variance and the means were compared by Tukey test, at 5% probability.

RESULTS AND DISCUSSION
It was noticed that two applications of S-ABA, one performed at the beginning of maturation and the other 25 days after the first application (400 + 200 mg L -1 or 400 + 400 mg L -1 ), provided lower values of luminosity Means followed by the same letter in the column do not show any significant difference by the Tukey's test at 5% probability.BM = Beginning of maturation; 25 DAFA = 25 days after the first application (DAFA).
(L*) of berries (29.88 and 28.60, respectively), on a scale from 0 to 100 and from black to white, respectively (Table 1).There was no significant difference for L* between control and 400 mg L -1 of S-ABA applied at the beginning of maturation, with means of 32.98 and 32.62, respectively.
Lower L* indicate darker color, and it means that two applications of S-ABA provided an increase in the 'Niagara Rosada' grape color, which may reflect directly on its commercialization; once consumers are attracted to, among other factors, the color of berries, a factor that might favor its commercial value (Roberto et al., 2012;Domingues Neto et al., 2017).
There was no significant difference between the different S-ABA concentrations regarding chroma (C*), which are responsible for color saturation; hue angle (h°) and color index (CIRG), which ranged from 9.71 to 10.31 for C*, from 31.72 to 35.45 for h° and from 3.42 to 3, 81 for the CIRG.These results corroborate with those found by Roberto et al. (2012), that obtained for 'Benitaka' table grape, with two applications of S-ABA 200 + 200 mg L -1 and 400 + 400 mg L -1 , both at 7 days after maturation and 15 days before harvest, a decrease of L*, presenting no changes in the C* and h°.However, the CIRG found by these authors in the control treatment was lower than all treatments that underwent the application of S-ABA, which was not observed in this study.The results of the present study are similar to those found in studies, which also evaluated S-ABA, with 'Redglobe' (Peppi et al., 2007), 'Flame Seedless' (Peppi and Fidelibus, 2008) and 'Crimson Seedless' grapes.
In experiments with 'Isabel' grape (Koyama et al., 2014b), 'Benitaka' (Roberto et al., 2012) and 'Flame Seedless' (Peppi and Fidelibus, 2008) grapes, the S-ABA application decreased L* and C* values.For 'Rubi' grape, Roberto et al. (2013) found differences in the variable hº, but the S-ABA provided lower values than those of the control.Several factors can interfere with the results of these variables, such as the grape maturation, cultivar and cultural practices, among others.
The 'Niagara Rosada' grapes which received two S-ABA applications, regardless of their concentrations, presented total anthocyanin contents twice as high as compared to the control treatment, with average values of 0.80 and 0.40 mg 100 g -1 , respectively.The synthesis of anthocyanins can occur, constantly, from the beginning of maturation to harvest, as well as during the first half of maturation (Mullins et al., 1992), which justifies the positive effect of S-ABA application on two grape ripening stages.Several studies have demonstrated that the exogenous application of S-ABA increases the anthocyanin content in grapes (Ban et al., 2003;Yamamoto et al., 2015, Domingues Neto et al., 2017).It is possible to state that, with the results obtained in this study and those already available in the literature, that the S-ABA triggers the secondary metabolism of 'Niagara Rosada' grapes and other grape cultivars, specifically the anthocyanin biosynthesis, which is the pigment that imparts a pink/purple color on fruits, flowers and vegetables.
Despite providing a greater accumulation of total anthocyanins, the application of S-ABA presented no effect on total phenolic compounds content of 'Niagara Rosada' grapes (Table 2), with average values varying from 33.02 to 39.85 mg 100 g -1 . Similar results were found in 'Isabel' grapes, in which applications of S-ABA 400 + 400 mg L -1 (7 days after maturation and 10 days before harvest) provided an accumulation of total anthocyanins, however, with no difference in total phenolic compounds content (Koyama et al., 2014b;Yamamoto et al., 2015).
The S-ABA had an effect on the enhancement of color characteristics, as well as on the synthesis and accumulation of total anthocyanins of 'Niagara Rosada' grapes, especially when it was applied twice.Thus, the use of S-ABA is a promising alternative for viticulture, once it makes possible, addition of more value to the fruits, since the grapes with a better color uniformity get better values in the market, and 'Niagara Rosada' grapes, have in their color, one of the decisive parameters to obtain better prices.
Since there was no significant difference in the treatments with two S-ABA applications, 400 mg L -1 of S-ABA at the beginning of maturation + 200 mg L -1 25 days after the first application can be used for color improvement, considering the use of a lower concentration during the second application, aiming to reduce the cost of the process.Means followed by the same letter in the column do not show any significant difference by the Tukey's test at 5 % probability.BM = Beginning of maturation; 25 DAFA = 25 days after the first application (DAFA).

Conclusions
Two applications of S-ABA (400 mg L -1 of S-ABA at the beginning of maturation + 200 mg L -1 25 days after the first application) are efficient in improving the color of berries, as well the anthocyanins accumulation of 'Niagara Rosada' grapes.