水分胁迫对马瑟兰葡萄果实品质及花色苷合成代谢的影响

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

【目的】探讨贺兰山东麓促进葡萄品质及花色苷形成的适宜水分胁迫阈值,为节水灌溉提供依据。【方法】以马瑟兰葡萄为试材,在花后20d,开始进行无胁迫(对照)、轻度(T1)和中度(T2)水分胁迫处理,测定了葡萄果实纵横径、百粒质量、可滴定酸含量、可溶性固形物含量,采用pH示差法测定总花色苷含量,采用高效液相色谱法(HPLC,High performanceliquidchromatography)测定果实糖酸、单体花色苷含量,采用实时荧光定量PCR法(qRT-PCR,Quantita-tivereal-timePCR)检测花色苷代谢途径相关基因的表达量。【结果】随水分胁迫程度的增加,果实纵横径、百粒质量和可滴定酸(TA,Titratableacidity)含量均下降,可溶性固形物(TSS,Totalsolublesolid)含量提高,在花后120d时,与对照相比,T1和T2的葡萄糖含量分别提高16.36%和15.36%、果糖含量分别提高7.10%和8.19%、总花色苷含量提高18.57%和21.05%,总酚和单宁含量分别提高20.00%和30.30%、14.58%和6.87%,T1和T2的酒石酸和苹果酸含量分别降低17.35%和6.91%、45.08%和48.16%;葡萄果皮中共检测出17种单体花色苷,在成熟期二甲花翠素3-O-葡萄糖苷含量最高,与对照相比,T1和T2分别提高52.85%和43.11%;在花后110d和120d时,T1和T2处理均可提高VvPAL的基因表达量,T2处理下VvF3'5'H、VvUFGT的基因表达量在各时期均有所上升,在花后60d至90d时,T2处理可提高Vv-LAR的基因表达量。【结论】葡萄在坐果期至成熟期采用中度水分胁迫处理可提高单糖、花色苷、总酚和单宁含量,降低有机酸含量,研究结果为贺兰山东麓马瑟兰葡萄栽培和高效节水提供理论参考。

【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 horizon-tal diameter,100 grains weight,titratable acid and total soluble solid content of grape berries were deter-mined.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 collect-ed 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 con-tents of individual sugar and organic acid were determined by HPLC according to a previously de-scribed method with minor modifications.The juice was contrifuged at 12000×g for 10 min,and the su-pernatant was extracted and filtered through a 0.22µm filter and used for subsequent analysis.The sepa-rating column was Hypersil GOLDTM Amino(250 mm×4.6 mm,5µm)and C18(250 mm×4.6 mm,5µm)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 real-time PCR(qRT-PCR)was used to detect the expression levels related to anthocyanin metabolism,total RNAwas used as template for reverse transcription according to the PrimeScriptTM 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 in-creased by 18.57%and 21.05%under T1 and T2 conditions,respectively.However,the contents of to-tal phenols,tannins,tartaric acid and malic acid in berries showed opposite trend.At 120 DAA,com-pared 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-glu-coside 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 Vv-LAR.【Conclusion】The moderate water stress treatment could increase the contents of monosaccha-rides,anthocyanins,total phenols and tannins,reduce the contents of organic acids from berry set to ma-turity.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 bio-synthesis of anthocyanins,which provided theoretical reference for the cultivation and efficient water saving management of Marselan grapevine in the eastern foot of Helan Mountain.