Molecular Mechanisms Of Arabidopsis MAPKKKx Participating In ABA Response And MAPKKKy Regulating ROS Accumulation In Oilseed Rape

Mitogen-activated protein kinases(MAPKs/MPKs)cascade pathway is a highly conserved signal module in plants that plays a very important role in response to a wide range of environmental stimuli.The function of MAPKKK members as the upstream regulator of the MAPK cascade,has not been fully reported due to its large number and possible redundant functions.In this study,on the basis of previously screened two MAPKKKs,one is Arabidopsis MAPKKKx mediating ABA induced leaf senescence and the other is an oilseed rape MAPKKKy gene in regulating reactive oxygen species(ROS)accumulation.Here,we present new evidence in the molecular mechanism of MAPKKKx involved in ABA response and the ROS accumulation of MAPKKKy by employing various experimental methodologies such as real-time quantitative RT-PCR(q RT-PCR),Dual Luciferase Reporter Assay(Dual-LUC),Electrophoretic mobility shift assay(EMSA)etc.The main findings are as follows:1.Arabidopsis MAPKKKx gene mediated dark and age-induced leaf senescence.q RT-PCR results show upregulation of the gene expression level in senescencent tissues.A delayed senescence phenotype was observed in mapkkkx compared with WT.Dark induced phenotypic analysis and chlorophyll content comparison results showed that overexpression of MAPKKKx gene could promote leaf senescence,but the mapkkkx mutant did not delay senescence significantly in dark conditions,which might be caused by redundancy between genes.2.The MAPKKKx gene of Arabidopsis acts downstream of the ABA core signal pathway and is regulated by ABF3 and ABF4.The results of q RT-PCR showed that the transcription level of MAPKKKx gene in abf3 and abf4 mutants was significantly decreased.We speculated that these two transcription factors were regulators of MAPKKKx.Further we used a Dual-LUC assay to confirm that ABF3 and ABF4 could transcribe the expression of fluorescent enzyme gene driven by MAPKKKx promoter.EMSA results further validated that ABF3 and ABF4 could bind directly to ABA responsive element(AREB)in the MAPKKKx promoter.Therefore,we conclude that ABF3 and ABF4 transcription factors are the direct upstream regulators of MAPKKKx gene,and can regulate plant leaf senescence by regulating the expression of MAPKKKx gene.3.We dentified MPK3/MPK6 as a downstream target of Bna MAPKKKy.By using MPK3/MPK6 specific antibody and Western blotting confirmed that the phosphorylation of MPK3/MPK6 in Bna MAPKKKy overexpression lines was significantly increased,while the phosphorylation of MPK3/MPK6 in atmapkkky was significantly decreased.4.Previous experiments confirmed that oilseed rape Bna MAPKKKy could induce reactive oxygen species(ROS)production and cell death.We further investigated the role homologous Arabidopsis gene At MAPKKKy and found that the seeds of knockout mutant atmapkkky were larger.The innovation point of this project is that the transcription factors ABF3 and ABF4 regulate Arabidopsis MAPKKKx.Further we also demonstrated that rapeseed Bna MAPKKKy could phosphorylate MPK3 and MPK6,and its homologue At MAPKKKy in Arabidopsis negatively regulates seed size.