Function And Molecular Mechanism Of Grapevine Ubiquitin Ligase Gene VviPUB19 In Response To Abiotic Stress

Grapevine is an important economic fruit tree,and European grape(Vitis vinifera L.)has been widely used in fresh food,drying,winemaking and grape seed extract.However,most European grapes have low resistance to low temperature,drought and salt stress,and these abiotic stresses will limit the growth and development of grapes,thereby affecting the grape quality and yield.In this study,the U-box ubiquitin ligase gene VviPUB19 and its upstream promoter were cloned from Vitis vinifera L.cv.Thompson Seedless,and the gene expression pattern and promoter activity of VviPUB19 under abiotic stresses were analyzed.The proteins interacting with VviPUB19 were screened,and the function of VviPUB19 and its interacting proteins against abiotic stress was verified,and the molecular mechanism of its resistance to abiotic stress was studied.The main results of this study are as follows:1.Analysis of gene expression and screening of interacting proteins of ubiquitin ligase VviPUB19 in grapevine.The CDS region of VviPUB19 was obtained by PCR amplification using the c DNA of ’Thompson Seedless’.The N-terminus of VviPUB19 contains an UND domain and a U-box domain,and the C-terminus contains four repeated ARM motifs.RTq PCR analysis showed that VviPUB19 was expressed in different tissues,and there were significant differences;And the expression was significantly induced by low temperature,drought,salt and exogenous hormones.Meanwhile,the VviPUB19 promoter was cloned,it was showed that the VviPUB19 promoter contained cis-acting elements related to light,ABA,SA,Me JA and low temperature response.GUS activity analysis showed that light,low temperature and exogenous hormone treatments activated VviPUB19 promoter activity,while dark treatment inhibited its activity.By Pull down tandem mass spectrometry assay,48 candidate proteins were screened for interaction with VviPUB19,including proteins related to biotic and abiotic stress.2.VviPUB19 reduces the tolerance of grapevine to low temperature stress by degrading VviICE1 and VviCBF1.The expression of PUB19 gene was induced by cold treatment in both’Beibinghong’ and ’Thompson Seedless’,and reaching a peak at 12 h;the induced fold was higher in ’Thompson Seedless’.It was showed that overexpression of VviPUB19 reduced the cold tolerance of grape and Arabidopsis,and the resistance phenotype of Arabidopsis atpub19 mutants could be restored by VviPUB19.Further RT-q PCR analysis showed that overexpression of VviPUB19 reduced the expression of CBF and COR genes.Pull down tandem mass spectrometry assay screened a protein VviICE1 interacting with VviPUB19.VviICE1 protein is a key transcription factor of the ICE-CBF-COR cold response pathway.Then the interaction of VviPUB19 with the transcription factors VviICE1,VviICE2,VviICE3,VviCBF1 and VviCBF2 were verified by yeast two-hybrid and bimolecular fluorescence complementation(Bi FC)assays.Protein degradation assays showed that VviICE1 and VviCBF1 proteins were both degraded by VviPUB19 at 22°C and 4°C,and inhibited by proteasome inhibitor MG132.Yeast one-hybrid and dual-luciferase assays demonstrated that VviICE1,VviICE2 and VviICE3 interacted with the VviPUB19 promoter and significantly enhanced the VviPUB19 promoter activity.At the same time,it was found that VviPUB19 interacted with cold-related ubiquitin ligases VviHOS1 and VviPUB24,and these three E3 proteins had specific degradation relationships at 22℃ and 4℃.3.Functional analysis of the interaction between VviPUB19 and VviTPS10 under low temperature stress.Co-expression analysis showed that the trehalose 6-phosphate synthases gene VviTPS9 and VviTPS10 were co-expressed with VviPUB19.RT-q PCR analysis showed that the gene expression of VviTPS9 and VviTPS10 was induced by low temperature treatment.Yeast two-hybrid assay showed that VviPUB19 interacted with VviTPS9 and VviTPS10,and the UND and ARM domains of VviPUB19 and the TPP domain of VviTPS9 and VviTPS10 were involved in their interaction.Protein degradation assay showed that VviTPS10 protein was degraded by VviPUB19 at both 22℃ and 4℃,and could be inhibited by MG132.Transcription factors VviICE1,VviICE3 and VviCBF1 significantly enhanced promoter activity by interacting with VviTPS10 promoter.The heterologous transformation of Arabidopsis showed that overexpression of VviTPS10 significantly improved the cold tolerance of plants.4.VviPUB19 interacts with VviExo70 B to regulate drought and salt stress responses.Overexpression of VviPUB19 reduced the sensitivity of grape callus and Arabidopsis to ABA and tolerance to mannitol and Na Cl,and decreased the drought and salt tolerance of Arabidopsis.Yeast two-hybrid and Bi FC assays indicated that VviPUB19 and its conserved domain ARM interacted with VviExo70 B.VviExo70 B is a subunit of exocyst in exocytosis.The ubiquitinated protein degradation assay showed that the presence of VviPUB19 accelerated the degradation of VviExo70 B protein,and aggravated the degree of polyubiquitination of VviExo70 B,which was inhibited by MG132.Overexpression of VviExo70 B increased the sensitivity of grape callus and Arabidopsis to ABA and tolerance to mannitol and Na Cl,and enhanced the drought and salt tolerance of Arabidopsis.5.Functional analysis of interaction between VviPUB19 and transcription factor VviERF105 e in abiotic stresses.Co-expression analysis showed that VviERF105 s and VviPUB19 were co-expressed.The results of yeast two-hybrid showed that VviPUB19 interacted with VviERF105 a,VviERF105 c and VviERF105 e.RT-q PCR results showed that the expression of VviERF105 e was significantly induced by abiotic stresses.The UND domain of VviPUB19 was further demonstrated to be involved in the interaction with VviERF105 e by yeast two-hybrid and Bi FC assays.The protein degradation assay showed that the degradation of VviERF105 e by VviPUB19 was inhibited by MG132.Overexpression of VviERF105 e in grape callus and Arabidopsis increased plant tolerance to cold,drought and salt stresses.In conclusion,the ubiquitin ligase VviPUB19 plays a negative role in plant resistance to abiotic stresses by regulating the stability of related proteins in ICE-CBF-COR cold response pathway,trehalose metabolism pathway,exocytosis and ERF transcription factor.