The Transcriptional Response To Salt Stress Directed By MdWRKY9 In Malus Domestica Borkh.

Plant response and adaptation to salt stress is controlled by multiple genes via a complex mechanism.The transcription factors play a central role in salt stress response after receiving the upstream signaling via regulating many downstream genes to cooperate for the salt tolerance.WRKY is a superfamily of transcription factors.A lot of researches reported that WRKY take part in the the biotic stress response,but recent reports found that WRKY were also involved in abiotic stress response.We also found that WRKYs change their expressions under salt stress in apple rootstock.But the role of WRKYs in salt response remained unclear.In order to uncover the role of MdWRKY9 in salt response in apple plants,functional research of MdWRKY9 was carried out to reveal the mechanism.Our research will contribute to the clarification of the molecular mechanism for salt response in Malus genus.The results are as follows:1.The ORF(Open Reading Frame)of salt responsive MdWRKY9 gene was cloned.The qPCR result indicated that MdWRKY9 was induced early by salt stress in both leaves and roots.The bioinformatics analysis showed that MdWRKY9 has the conserved domain of WRKYGQK,and is a WRKY ?transcription factor.MdWRKY9 was located in nucleus.It didn't exhibited transcriptional activities in the yeast system,but showed transcription inhibition in protoplast system,inferring it is a possible transcriptional inhibitor.2.The transgenic M26 plants and calli overexpressing of MdWRKY9 were obtained by Agrobacterium-mediated transformation.The transgenic M26 apple plants and calli overexpressing of MdWRKY9 exhibited significantly increased salt tolerance than the control.The results of ChIP-qPCR,EMSA,and qPCR confirmed that MdWRKY9 could directly bind to the promoter of MdNHX4 and repress its expression,which leads to the decreased compartmentation of K+to the vacuole and enhanced the salt tolerance of transgenic M26 apple plant and calli.3.The treatment of immunoprecipitated MdWRKY9 protein by phosphatase confirmed that MdWRKY9 could be phosphorylated in vivo.The in vivo(BiFC and Co-IP)and in vitro(pull-down)results found that MdWRKY9 interacts with MdMAPK6 and MdMAPK6 interacts with MdMAPKK9.The in vitro phosphorylation confirmed that MdWRKY9 could be phosphorylated by MdMAPKK9/MdMAPK6 cascade.The results of ChIP-qPCR and the GUS activity detection in transient transgenic apple calli confirmed that the phosphorylation of MdWRKY9 by MdMAPK6 enhances the binding of MdWRKY9 to the promoter of MdNHX4,and further inhibited its expression.4.Under normal condition,the transgenic M26 apple plants overexpressing of MdWRKY9 exhibited the dwarfing with shorter internodal distance and also lower rooting rate,longer but fewer roots than control M26 plants.The brassinosteroid(BR)and castasterone(CS)contents were significantly decreased in transgenic M26 apple plants overexpressing of MdWRKY9 and the dwarfing phenotype of the transgenic M26 apple plants could be partially reversed via exogenous BL treatment.5.The results of ChIP-qPCR,EMSA,and qPCR confirmed that MdWRKY9 directly binds to the promoter of MdDWF4,the BR rate-limiting synthetase,and represses the expression of MdDWF4,which leads to the decrease of BR level in transgenic apple.RNA interfered MdWRKY9 in transiently transformed apple calli,led to significant increase of MdDWF4,suggesting MdWRKY9 plays a critical role in regulating the expression of MdDWF4.Taken together,MdWRKY9 regulates the salt response of apple via controlling its ion homoeostasis and positively slowing down the plant growth to increase its salt tolerance.