Study On The Function And Mechanism Of VviWRKY Transcription Factor Regulating Proanthocyanidin Metabolism In Grape

Grape leucoanthocyanidin reductase(VviLAR)is a key reductase that regulates the synthesis of grape proanthocyanidin monomers.Its reductive product(+)-catechin is an important starting unit of procyanidin synthesis and plays an important role in grape procyanidin synthesis.In order to explore the expression regulation of leucoanthocyanidin reductase in grape,we selected four methods to screen transcription factors that may bind to VviLARs promoter and the interaction between promoter and transcription factor was verified by yeast one-hybrid and double luciferase assay.Meanwhile,the mechanism and function of VviWRKYs transcription factors binding to VviLARs promoter were analyzed.The main findings are as follows:1 The transcription factors interacting with VviLARs promoters were screened.Twenty-one transcription factors were screened through four pathways,and the interaction between the screened transcription factors and the VviLARs promoter was verified by yeast one-hybrid and and the double-luciferase test was used to reverse the verification.The results showed that 4 Vvi MYBs transcription factors and 7 VviWRKYs transcription factors could bind to VviLARs promoter,among which VviWRKY6-2,Vvi MYB3R-5,Vvi MYBPA8 and Vvi MYB169 could bind and activate VviLAR1 promoter.However,VviWRKY57-2 and VviWRKY20-2 could bind and inhibit the VviLAR1 promoter.VviWRKY57-1,Vvi MYBPA8 and Vvi MYB169 could bind and activate the VviLAR2 promoter,while VviWRKY40-1,VviWRKY20-2,VviWRKY11-1,VviWRKY65-1,Vvi MYB172 and Vvi MYB3R-5 could bind and inhibit the VviLAR2 promoter.2 The binding of VviWRKY TFs interacting with VviLARs promoter to Vvi ANR1 promoter was verified.Yeast one-hybrid verified that VviWRKY6-2,VviWRKY11-1,VviWRKY40-1,VviWRKY65-1 and VviWRKY57-1 could also bind to the Vvi ANR1 promoter,while VviWRKY6-2 and VviWRKY20-2 didn’t bind to the Vvi ANR1 promoter.The effect of transcription factors on Vvi ANR1 promoter activity was tested by dual luciferase assay,the result showed that VviWRKY57-2 could up-regulate the promoter activity of Vvi ANR1,while VviWRKY40-1,VviWRKY11-1,VviWRKY57-1 and VviWRKY65-1 inhibited the promoter activity of Vvi ANR1.3 The interaction between VviWRKY TFs interacting with VviLARs promoters was analyzed.The formation of homodimers or heterodimers is a common interaction between WRKY TFs,which affects the binding of WRKY TFs to promoters.The interaction between seven VviWRKY TFs was verified by yeast two-hybrid assay and the bi-molecular fluorescence complementation test.The results showed that VviWRKY6-2,VviWRKY11-1and VviWRKY20-2 could form homodimers by themselves,while VviWRKY57-1 and VviWRKY20-2 could form heterodimers.Subcellular localization technique found that VviWRKY6-2,VviWRKY11-1,VviWRKY20-2,VviWRKY57-1 and VviWRKY20-2 were all localized in the nucleus.Dual-luciferase assay showed that the heterodimer formed by VviWRKY57-1 and VviWRKY20-2 could inhibit the promoter activity of VviLAR2.4 The effect of VviWRKYs interacting with VviLARs promoter on proanthocyanidin synthesis in plant was verified.By transiently transforming seven OE-VviWRKYs transcription factors in tobacco leaves,it was found that VviWRKY6-2 and VviWRKY57-1could significantly increase procyanidin content in tobacco leaves,while VviWRKY40-1,VviWRKY11-1,VviWRKY57-2,VviWRKY65-1 and VviWRKY20-2 inhibited the synthesis of proanthocyanidins in tobacco leaves.OE-VviWRKY20-2 and OE-VviWRKY57-1 were stably transformed into grape callus.The results showed that OE-VviWRKY20-2 could significantly inhibit proanthocyanidin synthesis in grape callus,while OE-VviWRKY57-1could significantly increase proanthocyanidin content in grape callus.