Drought Resistance Function And Regulation Mechanism Analysis Of Grapevine Transcription Factor VlbZIP30 Gene

Grapevine is one of the important fruit tree crops.Its fruits are mainly used for fresh food,wine making and drying.It can also be processed into fruit vinegar or transformed into health products that are beneficial to human health.Grapevine occupies a very important position in China's agricultural industry and regional economic development.Xinjiang,Ningxia,Gansu,and Shaanxi are the main grape producing areas,and their grape area and output account for more than 40% of China's total.However,there is a shortage of water resources in these areas,and most vineyards do not have irrigation conditions.The lack of water has a serious impact on the growth,development and yield of grapes.Water resources have become one of the main factors restricting the high-quality green development of grapes.Therefore,screening grape drought-resistant genes and studying its regulation mechanism are of great significance to the creation of new high-quality drought-resistant grape germplasm and the sustainable development of grape industry in China.Basic leucine zipper transcription factors are one of the most extensively and conserved transcription factors in the eukaryote proteins.bZIP transcriptional factors have been reported that involved in various biological functions such as the expression of LEA,plant development,light signaling,pathogen defence,responsive to abiotic stress and ABA signaling,and so on.They are particularly important in playing a key role in participating in plant drought resistance.In the early stage of the project,we had identified the grape bZIP transcription factor family and analyzed its expression under different stresses.The grape bZIP30 gene which specifically expressed under drought stress was selected.On the basis of this,we first isolated VlbZIP30 from Kyoho,and overexpression VlbZIP30 in the model plant A.thaliana and Thompson Seedless grape for further explore the function and the mechanism of VlbZIP30.The main results are as follows:1.Cloning of VlbZIP30 gene and analysis of its promoter activity.The complete ORF length of VlbZIP30 is 978 bp which encodes 325 amino acids.Amino acid sequence analysis showed that,VlbZIP30 contains a basic leucine zipper domain and 4 phosphorylation sites(C1,C2,C3,and C4).A phylogenetic analysis indicated that VlbZIP30 is most closely related to the group A ABF/DPBF.A 2000 bp promoter region was cloned from‘Kyoho'grape genome DNA,and a plant expression vector containing the GUS reporter gene was constructed.After transformation to A.thaliana,GUS activity was detected in seeds,seedlings,inflorescence stems,trichomes,flowers and siliques,and the GUS activity was induced by ABA or mannitol treatment.In addition,the expression of VlbZIP30 was induced in grape leaves under ABA and dehydration treatment.These results suggest that VlbZIP30 might be involved in the process of grapes responding to drought stress.2.VlbZIP30 gene may enhance the drought resistance of Arabidopsis thaliana by regulating the expression of drought stress-related genes.Overexpression of VlbZIP30 significantly enhanced Arabidopsis resistance to osmotic and drought stress during seedling and adult stages.Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage.We identified an A.thaliana G-box motif(CACGTG)and a potential grapevine G-box motif(MCACGTGK)in the promoters of the39 selected A.thaliana genes upregulated in the transgenic plants and in the 35 grapevine homologs,respectively.Subsequently,using two grapevine-related databases,we found that74%(23/31) and 84%(21/25) of the detected grapevine genes were significantly upregulated by ABA or drought stress,respectively,suggesting that these genes are involved in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine.3.VlbZIP30 improves drought resistance by activating drought-responsive genes and promoting lignin biosynthesis through the regulation of lignin biosynthetic genes.Transgenic grape plants overexpressing VlbZIP30 exhibited lignin deposition(mainly G and S monomers)in the stem secondary xylem under control conditions,which resulted from the up-regulated expression of VvPRX4 and VvPRX72.The overexpression of VlbZIP30 improves drought tolerance,characterized by a reduction in water loss rate,maintenance of an effective photosynthesis rate and increased lignin content(mainly G monomer)in leaves under drought conditions.EMSA,luciferase reporter and chromatin immunoprecipitation(ChIP)-q PCR assays indicated that VlbZIP30 directly binds to the G-box cis-element in the promoters of lignin biosynthetic(VvPRX N1)and drought-responsive(VvNAC17)genes,to regulate their expression.4.Using RNA-seq and ChIP-seq correlation analysis,we systematically analyzed the regulation mechanism of VlbZIP30 gene involved in regulating the drought resistance of grapes.Through chromatin immunoprecipitation(ChIP)-seq and RNA-seq analysis,the direct VlbZIP30 target genes were identified at a genome-wide scale.The ChIP-seq analysis determined that VlbZIP30 binds to DNA sequences containing an ACGTG core motif,termed a G-box.By combining the ChIP-seq and RNA-seq results,48 VlbZIP30-induced target genes were identified.Through ChIP-q PCR analysis confirmed that VlbZIP30 binds directly to the promoters of four of the target genes(VvNAC26,VvDHN1,VvGRAS17 and VvVQ6)containing a G-box motif.In addition,overexpression of VlbZIP30 led to less H₂O₂accumulation compared with the wild type under drought conditions in both Arabidopsis thaliana and grapevine.In summary,these results indicated that VlbZIP30 promotes the activity of ROS scavenging by regulating the expression of downstream target genes,thereby conferring drought resistance of grapevines.5.In summary,in this study we found that grapevine VlbZIP30 can activate their expression by binding to the G-box(ACGTG)element on the promoter of lignin biosynthesis genes(VvPRX4 and VvPRX72),thereby promoting lignin deposition on the stems of grapevines.In addition,under drought stress,the leaves of overexpressing VlbZIP30 transgenic plants increased the biosynthesis of lignin,maintained an effective photosynthetic rate,and enhanced the ROS scavenging activity to improve the drought resistance of the plants.Studies have shown that this is due to VlbZIP30 can directly and specifically bind to the G-box elements on the promoters of the lignin biosynthesis gene(VvPRX N1)and drought response genes(VvNAC17,VvNAC26,VvDHN1,VvGRAS17,and VvVQ6)to activate their expression,thereby improving the drought resistance of transgenic grapevines.