Dissecting The Molecular Mechanisms Of Two WRKY Transcription Factor Genes Regulating Leaf Senescence And Stress Tolerance In Arabidopsis Thaliana

WRKY transcription factors(TFs)are one of the largest families of transcriptional regulators and contain 72 members in Arabidopsis thaliana.WRKY TFs have diverse functions and are involved in regulating plant growth and development as well as responses to biotic,abiotic stress and development.So far,there are lots of reports on the functions of WRKY transcriptional factor genes,but the functions and regulatory mechanisms of some WRKY genes are still unknown.In this study,we aim to dissect the molecular mechanisms of two WRKY transcription factor genes,WRKYx and WRKYy,in regulating leaf senescence,salt stress and low potassium tolerance in Arabidopsis thaliana.First,we obtained the T-DNA insertion mutants and constitutive over-expression(OE)lines of WRKYx.The age-dependent senescence assay showed that the overexpression of WRKYx accelerated the progress of senescence while the mutation delayed the senescence.We identified some senescence-associated genes(SAGs)are up-regulated in WRKYx over-expression lines through RNA-Seq analysis.We further analyzed the transcriptional changes of putative target genes,SAG15,SAG29 and SAG113,through q RT-PCR.Dual-LUC and EMSA proved the above three genes were targets of WRKYx.Meanwhile,the interaction between RCD1 and WRKYx was identified and confirmed while the senescence phenotype was opposite.Dual-LUC demonstrated that RCD1 repressed the transcriptional activity of WRKYx.Besides,WRKYx is involved in regulating the response to low potassium,and OE lines of WRKYx is insensitive to low potassium,but no differences between mutant and wild-type was observed likely because of function redundancy with other homologous WRKY genes.Further AKT1 and KC1 were identified to be target genes of WRKYx through Dual-LUC and EMSA.We also found that WRKYy is responsive to salt stress.The germination and post-germination assays showed that the wrkyy mutant is sensitive and OE lines are tolerant to salt stress.WRKYy is not involved in the SOS(Salt Overly Sensitive)signaling pathway directly through the dual-LUC experiment.The phenotypic analysis showed that wrkyy mutant was sensitive to ABA treatment.Thus,WRKYx-mediated response to salt stress may be dependent on ABA,which needs to be further explored.In this study,we identified the functions and regulatory mechanisms of two Arabidopsis WRKY genes,which provided theoretical value in understanding the biological functions of WRKY gene family members in Arabidopsis and guiding the breeding of stress tolerant crops.