Effects of water stress on berry quality and anthocyanin metabolism in Marselan grape

Abstract：【Objective】The present experiment was undertaken to explore the appropriate water stress threshold for promoting grape quality and anthocyanin formation at the eastern foot of Helan Mountain, and also to provide a basis for water-saving irrigation.【Methods】The Marselan (Vitis vinifera L.) was used as the experimental material. In 20 days after anthesis (20 DAA), the berries were treated with no water stress (Control), mild water stress (T1) and moderate water stress (T2). The vertical and horizontal diameter, 100 grains weight, titratable acid and total soluble solid content of grape berries were determined. The contents of total anthocyanins were determined by pH differential method, the grape skin was ground into power in the liquid nitrogen and approximately 0.1 g powder was added into 1 mL methanol- 1% formic acid. The solution was digested for 10 min in the dark at constant temperature shaker. The mixture was then centrifuged for 5 min at 8000×g and 4 ℃ and the supernatant was collected for further analysis. The extraction procedure was repeated for three times and the collected superna-tants were pooled and evaporated at 37 ℃ in an evaporator. The residual material was re-suspended in 1.5 mL of methanol solution. The relative anthocyanin content was measured at 530 nm and 657 nm with an ultraviolet spectrophotometer. Individual anthocyanins were measured with a HPLC. The contents of individual sugar and organic acid were determined by HPLC according to a previously described method with minor modifications. The juice was contrifuged at 12 000×g for 10 min, and the supernatant was extracted and filtered through a 0.22 µm filter and used for subsequent analysis. The separating column was Hypersil GOLD^TM Amino (250 mm×4.6 mm, 5 µm) and C18 (250 mm×4.6 mm, 5 µm) column. Acetonitrile-water mixture (75∶25) and methanol-KH2PO4 mixture (3∶97) were used as the mobile phase at flow rate of 0.5 mL·min-1 and 0.8 mL·min^-1 . The column temperature of 25 ℃ and injection volumn of 10 uL were adopted. The analysis was carried out with three technical replicates and the corresponding sugar and acid contents were calculated using a standard curve. Quantitative realtime PCR (qRT-PCR) was used to detect the expression levels related to anthocyanin metabolism, total RNA was used as template for reverse transcription according to the PrimeScript^TM RT Reagent Kit with gDNA Eraser Reverse Transcription Kit instruction. VvActin was selected as the internal reference, and primers of VvPAL, VvF3'5'H, VvLAR and VvUFGT genes were designed with Primer 5.【Results】With the increase of water stress degree, berry vertical and horizontal diameter, 100 grains weight and total acid content gradually decreased, but total soluble solids content showed the opposite pattern. In each treatment, the contents of glucose, fructose and total anthocyanins in berries gradually increased, and their contents under mild water stress (T1) and moderate water stress (T2) were significantly higher than those of control. At 120 DAA, compared with the control, the glucose content increased by 16.36% and 15.36%, the fructose content increased by 7.10% and 8.19%, and the total anthocyanin content increased by 18.57% and 21.05% under T1 and T2 conditions, respectively. However, the contents of total phenols, tannins, tartaric acid and malic acid in berries showed opposite trend. At 120 DAA, compared with control, the contents of total phenols and tannins increased by 20.00% and 30.30%, 14.58% and 6.87% under T1 and T2 conditions. The contents of tartaric acid decreased by 17.35% and 6.91%, and the contents of malic acid decreased by 45.08% and 48.16% under T1 and T2 conditions. A total of 17 types of anthocyanins were detected in grape skins, and the content of dimethyl anthocyanin 3-O-glucoside was the highest in the mature stage. Compared with the control, which increased by 52.85% and 43.11% under T1 and T2 conditions at 110 DAA and 120 DAA, T1 and T2 treatments can increase the expression level of VvPAL, and T2 treatment can increase the expression profile of VvF3’5’H and VvUFGT in each period. At 60 DAA to 90 DAA, T2 treatment can increase the expression level of VvLAR.【Conclusion】The moderate water stress treatment could increase the contents of monosaccharides, anthocyanins, total phenols and tannins, reduce the contents of organic acids from berry set to maturity. Mild water stress significantly increased the contents of dimethyl anthocyanin-3-O-glucoside and dimethyl anthocyanin- 3-O- cafeylated glucoside. Moderate water stress treatment before the veraison stage could significantly increase the expression levels of VvF3’5’H and VvUFGT, and promote the biosynthesis of anthocyanins, which provided theoretical reference for the cultivation and efficient water saving management of Marselan grapevine in the eastern foot of Helan Mountain.