Effects of exogenous 6-BA on anthocyanin synthesis in ‘Merlot’ grape

Abstract：【Objective】Anthocyanin, a water-soluble pigment with antioxidant activity, is one of the crucial components for the red wine and table grape. It has a role in adaptation to adverse conditions and resistance to diseases. The climatic conditions have important impact on grape quality. In recent years, the wine grape industry has developed rapidly in Ningxia with favorable climate condition and geographical resources suitable for producing high-quality grapes. However, there is a problem of insufficient accumulation of anthocyanins and their derivatives due to the climate reasons in some regions. Plant grows regulators are applied to improve grape quality. The purpose of this study was to analyze the mechanism of the effect of 6-benzyladenine(6-BA), a synthetic cytokinin, on anthocyanin accumulation.【Methods】The experiment was conducted with seven- year- old vines of Vitis vinifera‘Merlot’ from 2016 to 2017 in Yuquanying Farm, Ningxia, where the soil in this region is typically sandy and the vineyard was planted in east-west lines. The plant spacing was 0.5 m×0.3 m and drip irrigation was adopted. The test set three 6-BA treatments with different concentrations: 100 mg·L^-1 , 200 mg·L^-1 and 400 mg·L^-1 , and spraying distilled water served as the control. The sprays were made on the 50th day after flowering on the clusters until dripoff. Berries were sampled five times from veraison to harvest. The samples were quickly frozen with liquid nitrogen and stored in a -80 ℃ freezer. Liquid chromatography was used separate the components. Chromatography is used to analyze the content of endogenous hormone ABA, which plays a critical role in the process of plant physiology and biochemistry. Anthocyanin quantity and composition were detected by spectroscopy and HPLC. Chemical analysis, HPLC analysis and transcriptional analysis were used to study the effect of 6-BA on the anthocyanin biosyn-thesis. qRT-PCR was carried out to quantify the expression levels of structural genes in the anthocyanin biosynthesis pathway including CHS1, F3’H, F3’5’H and UFGT.【Results】The results showed that different concentrations of 6-BA had an effect on the synthesis of anthocyanins. The treatment of 200 mg·L^-1 6-BA promoted fruit coloration and increased endogenous ABA content. The anthocyanin content in the treatment at 100 mg ·L^-1 (5.21 mg · g^-1 ), was higher than the control at 110 days after flowering. The results indicated that 6-BA 200 mg · L^-1 treatment significantly increased the cyanidin-3-acelylglucoside content at mature stage, which was 4.82 times that of the control. Low concentration of 6-BA showed effect on the expression of anthocyanin synthesis related genes at version and the initial maturation stages. Among them, the expression levels of CHS1, F3’H and F3’5’H genes were increased mainly at veraison. Compared with the control, the 100 mg·L^-1 treatment significantly increased the relative expression of CHS1 at 90 days after flowering. The changes in the ratio of delphinidin-3-acelylglucoside to cyanidin-3-acelylglucoside were associated with the ratio of the expression of F3’5’H gene to that of F3’H gene expression. The expression of both F3’H and F3’5’H gene was up-regulated at 70 days after flowering in the 100 mg ·L^-1 treatment, and in the 200 mg ·L^-1 treatment, the relative expression of F3’5’H gene was 2.34 times that of the control. The expression level of the UFGT gene was increased in the initial maturation period by 6-BA treatments at the two concentrations (17.29 and 5.87 times that of the control, respectively). However, anthocyanin content in the 400 mg · L^-1 treatment was lower than the control throughout fruit ripening. Besides, this treatment not only delayed coloration, but also inhibited the expression of genes related to anthocyanin synthesis. Furthermore, the correlation analysis displayed a significant positive correlation between sucrose and anthocyanin content in 200 mg · L^-1 treatment. 【Conclusion】At the late stage of veraison, low concentration of 6-BA promoted the accumulation of anthocyanins by increasing endogenous ABA, total soluble sugar content and the expression of related genes. Thus, ABA, sugars and the relevant genes play important roles in grape quality formation and low concentrations of 6-BA (100-200 mg ·L^-1 ) has an effect in the improvement of fruit quality and the accumulation of anthocyanins in‘Merlot’grape.