Cloning And Functional Characterization Of HvXTH1 In Drought Tolerance In Tibetan Wild Barley

Drought is a major abiotic stress that negatively regulates plant growth and development.It results in a decline in crop production.Identification of drought-tolerant germplasm,related genes and tolerance mechanism is of great importance to improve crop drought tolerance/resistance.The Tibetan wild barley provides natural sources for plant biotic and abiotic stress tolerance due to its wide genetic diversity.In this study,genome-wide identification,characterization and expression analysis of xyloglucan endotransglucosylase/hydrolase gene family in barley?Hordeum vulgare?were performed.HvXTH1,one of the HvXTH gene family members,was cloned and analyzed.Silencing and overexpression of HvXTH1 were conducted for function characterization.Digital gene expression profile and metabolites analysis were performed to illustrate the drought tolerance mechanism of HvXTH1.The main results are as follows:1.A total of 24 HvXTH genes?named as HvXTH1-HvXTH24?was identified with the recently completed genome of barley?H.vulgare?.Phylogenetic analysis showed that 24 HvXTH genes could be classified into three phylogenetic groups: Group I/II,III-A and III-B and HvXTH15 was in the ancestral group.All HvXTH protein members,except HvXTH15,had a conserved N-glycosylation site.The genomic location of HvXTHs on barley chromosomes showed that the 24 genes are unevenly distributed on 7 chromosomes,with 10 of them specifically located on chromosome 7H.Structure-based sequence alignment demonstrates that each XTH possesses a highly conserved domain?ExDxE?responsible for catalytic activity.Expression profiles based on the barley genome database showed that HvXTH family members display different expression patterns in different tissues and at different stages.2.HvXTH1 was cloned from two Tibetan wild barley genotypes?drought-tolerant XZ5 and drought-sensitive XZ54?and a drought-tolerant cv.Tadmor.We compared the bioinformatics of HvXTH1 among two Tibetan wild barley genotypes and cv.Tadmor.Two cis-acting regulatory elements were found in the promoter region of HvXTH1 in cv.Tadmor,but not in two Tibetan wild barley genotypes.Amino acid sequence of HvXTH1 showed two functional domains?Glyco_hydro₁6 and XET_C?in the three genotypes.Subcellular localization analysis showed that HvXTH1 is a plasma membrane protein.The expression level of HvXTH1 in XZ5,XZ54,Tadmor and Golden Promises changed differently in response to different plant growth regulators,where higher upregulation was found under 6-Benzylaminopurine?6-BA?and gibberellin acid 3?GA3?in XZ5;2,4-Dichlorophenoxyacetic acid?2,4-D?,6-BA,and brassinosteroid?BR?treatments induced the expression of HvXTH1 in Tadmor.Under different levels of PEG-induced drought stress,HvXTH1 was more sensitive to 5% PEG 6000 induced drought stress.3.Virus-induced gene silencing of HvXTH1 in XZ5 was performed.The result showed that BSMV:HvXTH1 plants showed an increase in shoot fresh weight and a reduction in water loss compared with BSMV:? plants under drought stress,while HvXTH1-overexpressing lines exhibited a decrease in shoot fresh weight,a faster water loss and a larger stomatal aperture than that of the wild type Golden Promise in response to drought.4.The digital gene expression profile of shoot and metabolites of leaves from Golden Promise and HvXTH1-overexpressing?HvXTH1-OX?plants were conducted for further analysis of drought tolerance mechanism via HvXTH1.Digital gene expression profile and metabolites analysis demonstrated that the expression level of ABA-associated genes such as WRKY,LHCB,PIF reduced in HvXTH1-overexpressing plants when compared with Golden Promise in response to drought,and metabolites related to phenylalanine and tyrosine biosynthesis and metabolism such as tyrosine and ferulic acid decreased.Based on these DEGs and metabolites,we elucidated the role of HvXTH1 played in response to drought in barley.