Molecular Mechanism Of VvERF95 In Regulating Grape Rachis Degreening During Storage

Grape is one of the important fruit trees cultivated worldwide.Due to its thin and juicy skin and rich nutrition,the grape skin and seeds are rich in proanthocyanidins and resveratrol and other functional nutrients,which are deeply appreciated by consumers.Grape is a non-climacteric type of fruit,and there is no obvious peak of ethylene release.However,some studies have shown that the respiration intensity of the rachis is more than10 times higher than that of fruit.The increase in ethylene release accelerates the aging process,resulting in rachis browning which casued rachis severely reduces the quality of the grapes during post-harvest storage.In this study,it was found that 1-MCP treatment could delay grape rachis browning,and ethylene release was reduced and chlorophyll content was higher in rachis after treatment.According to literature reports and the results of this study,it was speculated that ethylene regulation of chlorophyll degradation played an important role in browning process of rachis.However,the molecular mechanism by which ethylene regulates chlorophyll degradation in grape is rarely reported.In this study,’Shine Muscat’grapes were used as materials to study the delayed browning of the rachis by 1-MCP during postharvest storage,and to clarify the regulation of ethylene response factor ERF on grape rachis degreening and chlorophyll degradation genes during storage.The main findings are as follows:1.VvERF95 was cloned from ’Shine Muscat’ grape,encoding 130 amino acids,containing an AP2/ERF conservative domain,belonging to IXb subgroup of ERF transcription factor family.The expression of VvERF95 is regulated by ethylene;VvERF95 is a nuclear membrane co-localization protein with transcriptional autoactivation.2.The mature ’Shine Muscat’ grape was stored for 4 weeks and treated with CK and1-MCP.The ethylene release peak appeared in CK treated grape rachis after 3 days of storage,the browning was serious after 2 weeks of storage,the water loss rate was as high as60 % after storage,and the chlorophyll content decreased to 0.055 mg/g;The 1-MCP-treated grape rachis showed a peak of ethylene release after 1 weeks storage,severe browning after3 weeks storage,water loss rate was less than 50 % after storage,and chlorophyll content decreased to 0.07 mg/g.3.Quantitative analysis showed that compared with CK,the expression of VvERF95 in grape rachis treated with 1-MCP was significantly decreased.RNA was extracted from grape rachis treated with CK and 1-MCP,and the expression levels of chlorophyll degradation pathway genes were analyzed.The results showed that the expression patterns of VvERF95 and chlorophyll degradation related genes showed the same or opposite trend in CK and 1-MCP treatments.4.To explore whether VvERF95 regulates the expression of chlorophyll degradation-related genes,cis-element in the promoters of chlorophyll degradation-related genes were analyzed and verified by yeast one-hybrid,LUC and EMSA assays.The results of promoter analysis showed that the promoters of chlorophyll degradation-related genes all contained the binding elements of ERF transcription factors;yeast one-hybrid and LUC assays showed that VvERF95 could bind to the promoter of VvPAO1 and positively regulate the promoter activity of VvPAO1;The promoter was segmented and yeast one-hybrid was performed again to determine that the bound cis-acting element was CAGCC,that is,VvERF95 could bind to the DRE cis-acting element CAGCC of the VvPAO1 promoter to positively regulate the expression of VvPAO1.EMSA test confirmed these results.5.VvERF95 overexpression vector with 35 S promoter was constructed,and VvERF95 function was verified by meristem callus of stably transformed grapes.Three overexpressed grape transgenic lines OE-6,OE-7 and OE-10 were obtained.Phenotypic observation,chlorophyll content determination and quantitative analysis of chlorophyll degradation pathway related genes were carried out in these three lines.The results showed that the leaves of the overexpression lines were yellower than the control,the measured chlorophyll content was significantly lower than that of the control,and the expression levels of VvERF95 and chlorophyll degradation-related genes were significantly increased,indicating that overexpression of VvERF95 in grapes can promote chlorophyll.The expression of degradation-related genes increased,which promoted the degradation of chlorophyll.