Functional Analysis Of VaWRKY55 In Cold And Drought Stresses Responses In Vitis Amurensis

Abiotic stresses such as cold and drought causes substantial yield losses,limit crop growth and distribution across diverse environments.Grape is an important fruit crop in the China.However,the commonly grown varieties are susceptible to cold and drought stresses.Previous studies in our laboratory indicated that WRKY transcription factors may participate in the cold and drought stresses response in grapes.But limited information is available about its regulation mechanism.In this study,V.amurensis,a wild Vitis species with excellent cold and drought stress resistance,was used as parent material and cold stress-related transcription factor VaWRKY55 was cloned.V.amurensis was subjected to cold and drought stress to understand the expression pattern of VaWRKY55.The VaWRKY55 was transformed through overexpression into Arabidopsis(Col)to evaluate its function under cold and drought stress,and uncover the mechanism of enhanced drought stress tolerance in the transgenic Arabidopsis.RNA-seq analysis was conducted to unravel the signal pathways through which drought tolerance was induced.The main results obtained were as follows;1.Based on the published genome sequence from 'Pinot Noir',primers were designed to amplify the coding sequences of VaWRKY55 in V.amurensis.The VaWRKY55 protein sequence had 99%similarity with VvWRKY55 from 'Pinot Noir' genomes.The expression pattern of VaWRKY55 under cold and drought stress was evaluated using real time PCR(qRT-PCR).The VaWRKY55 showed up-regulated expression patterns in cold temperature(4 ?)and drought stress,which indicate its involvement in the cold and drought stresses responses in V.amurensis.2.A vector was generated by using 35S as promoter to overexpress VaWRKY55 in Arabidopsis.Three positively transformed fourth generation transgenic lines were selected and used to evaluate the phenotypes under the abiotic stresses.The phenotypic analysis showed slightly increased survival rates,but only significant in one transgenic line compared to wild type under cold stress.When the Arabidopsis plants were subjected to drought stress all the three transgenic lines showed significantly higher survival rates compared to the wild type.These results clearly indicate that VaWRKY55 involved in the drought stress related biological processes and enhanced drought stress tolerance in the transgenic Arabidopsis.3.The biochemical and physiological analysis showed that there was elevated levels of soluble sugar and proline contents and,reduced quantities of hydrogen peroxide and electrolyte leakage in the transgenic Arabidopsis compared to the wild type under drought conditions.The transgenic lines also had lower stomata density,which may explain the lower water loss and higher relative water content in the leaves of transgenic Arabidopsis plants compared to the wild type.The results indicate that the overexpression of VaWRKY55 could enhance the drought stress tolerance by altering the physiology and leaf structure of the transgenic Arabidopsis.4.To understand the mechanism of drought tolerance at the molecular level due to the overexpression of VaWRKY55,RNA-seq was performed.When compared to the wild type after drought treatment,the transgenic lines showed upregulated expression of jasmonic acid biosynthesis and downstream signaling related genes which included AtAOC1,AtAOC2,AtLOX2,AtAOS,AtPR1,At3G05727,AtPDF1.2 and AtPR4.These results suggest that overexpression of VaWRKY55 enhanced drought tolerance in Arabidopsis through jasmonic acid related signaling pathway.