The Molecular Mechanism Of VvERFs And VvEIN3/EILs In Synergistic Regulation Of Grape Rachis Browning

Grape(Vitis vinifera L.)is one of the main economic fruit trees in China,its fruit is delicious and nutritious,but the rachis browning is easy to occur in postharvest storage,which seriously shortens the storage life and shelf life.In the previous study,ethylene was the key factor to affect the senescence and browning of rachis,and the accumulation of carotenoids was closely related to ethylene.However,the mechanism of how they jointly regulate the browning of grape rachis after harvest has not been reported.In this study,by analyzing the physiological changes and transcriptome data of rachis treated with 1-MCP during postharvest storage,the effects of ethylene and carotenoids on grape rachis browning were determined,and the key differential genes of ethylene pathway VvEIN3/EILs and VvERFs,were screened by genetic transformation,yeast one-hybrid system and dual-luciferase test to explore the molecular mechanism of VvEIN3/EILs and VvERFs involved in ethylene synthesis and carotenoid synthesis during grape postharvest rachis browning.The main results are as follows:1.Compared with CK,1-MCP significantly inhibited ethylene release and carotenoid accumulation in rachis of Shine-Muscat grape during postharvest storage.2.By analyzing the transcriptome data processed by CK and 1-MCP,the key differential genes were screened in the ethylene biosynthesis and signal transduction pathway and carotenoid biosynthesis pathway.In the 1-MCP treatment group(1-MCP2)compared with the control group(CK2),the expression of VvEIL2 in the ethylene signal transduction pathway was up-regulated by 2.3-fold,the expression of VvEIL4 gene was down-regulated by 4.3-fold,and the expression levels of VvERF75,VvERF95,VvERF98 and VvERF111 genes were down-regulated.In the carotenoid biosynthesis pathway,the expression levels of synthesis-related genes VvPDS1(100246195)and VvCRTISO(100263250)were up-regulated by 2.1-fold and 3.3-fold,respectively.Moreover,the expression levels of degradation-related genes VvZEP(100263360)and VvZEP(100232944)were up-regulated by 2.7-fold and 3.1-fold,respectively.3.In exploring the regulation mechanism of VvEIL4 on ethylene synthesis and signal transduction,the function of VvEIL4 was verified by genetic transformation of Arabidopsis,and there was a significant increase in ethylene production in transgenic Arabidopsis lines with over-expressed VvEIL4 gene.The hypocotyls and roots length of etiolated seedlings were significantly shorter,which were 33%and 55%of the control,respectively.The yeast one-hybrid system and LUC activity analysis were used to further verify the regulation mechanism of VvEILs involved in ethylene synthesis and transduction.The results showed that VvEIL4 interacts with VvERF95 and VvERF98 promoters in the regulation of ethylene synthesis and transduction pathways,and VvEIL4 positively regulates the activity of VvERF95 promoter and negatively regulates the activity of VvERF98 promoter.At the same time,the feedback regulation mechanism of VvEIL2、VvEIL4、VvERF75、VvERF95、VvERF98 and VvERF111 has been confirmed,that is,VvEIL2 and VvEIL4 negatively regulate the activity of the VvACO2 promoter,and VvERF75,VvERF95,VvERF98,VvERF111 positively regulate VvACS5 expression by binding to the DRE element in the VvACS5 promoter.In addition,transient transformation grape plantlets leaves to verify the function of VvERF75 and VvERF95,the results showed that the ethylene production of the leaves with over-expressed VvERF75 and VvERF95 was significantly increased,which was increased by 1.38-fold and 1.86-fold,respectively.At the same time,the expression level of VvACSs,the genes related to ethylene synthesis also increased significantly.4.In exploring the mechanism that VvEIL2 affects grape rachis browning through regulation of carotenoid biosynthesis,the function of VvEIL2 was verified by genetic transformation of Arabidopsis,and it was found that the amount of carotenoids was significantly reduced in Arabidopsis with over-expressed VvEIL2,which was 72%of the empty vector control.Using yeast one-hybrid system and LUC activity analysis to verify the regulation mechanism of VvEIL2 involved in carotenoid synthesis and metabolism,the results indicated that VvEIL2 directly binds to the promoter of VvCRTISO to negatively regulates its expression.In addition,VvEIL2 can be combined with the promoter of VvERF95 to positively regulate its expression,and with the promoter of VvERF98 to negatively regulate its expression.