The Mechanisms Of MAPK6 Regulating The Involvement Of Transcription Factors MYB39 And ERF109 In Salt Acclimation In Arabidopsis

Mitogen-activated protein kinase(MAPK)cascades are common signaling patterns in eukaryotes that deliver stimuli signal in cells and elicit cellular responses.The mitogen-activated protein kinase(MAPK)module consists of three kinases,which are MAP3 K,MAP2K,and MAPK.These kinases in turn activate each other through phosphorylation,thereby transferring signals from the receptor to the corresponding downstream effector factors.The protein phosphorylation cascade links extracellular stimulation with a wide range of cellular responses.However,the number of MAPK substrates found in plants to date is limited.MAPK6,as an important component of MAPK kinase signaling pathway,participates in biological responses to biotic and abiotic stress.Previous studies have found that MAPK6 is involved in the salt acclimation process,but the molecular mechanism is unknown.Our previous research results showed that MAPK6 participated in salt acclimation regulation,but its control mechanism underlying the regulation processes is unknown.In order to clarify the molecular mechanism of MAPK6 regulating salt acclimation,the transcriptome and quantitative phosphorylation proteome analysis were carried out using wild-type Arabidopsis and mapk6 mutants as materials under salt acclimation treatment.Therefore,MYB39 and ERF109 were preliminarily identified as the transcription factors that may be regulated by MAPK6 in salt acclimation.The research of this paper is mainly from the following aspects:(1)Bioinformatic analysis including the phylogenetic tree analysis,protein conserved domain(PCD)and sequence characteristics of transcription factor binding target gene promoters were performed in MYB39 or ERF109 transcription factor,providing references for further understanding of the role of transcription factors in the salt acclimation pathway.(2)The previous results of yeast two-hybrid analysis in the laboratory have showed that there was the interaction between MAPK6 and MYB39 or ERF109.In this study,the interaction between MAPK6 and MYB39 was verified by the LUC complementation imaging assay.Therefore,we speculate that MAPK6 may interact with MYB39.In order to verify this hypothesis,further phosphorylation assay in vitro and mass spectrometry analysis will be needed to determine phosphorylation sites of MYB39 or ERF109 as MAPK6 substrates.(3)In order to detect whether the interaction between MAPK6 and MYB109 affects the biological function of At MYB39,mapk6/myb39 double mutants were obtained by traditional hybridization on the basis of single mutants.The phenotypes of mapk6,myb39 and mapk6/myb39 double mutants in salt acclimation were analyzed.The results showed that mapk6 single mutant and mapk6/myb39 double mutant displayed the phenotype of salt acclimation defect,whereas that of myb39 single mutant was not obvious.We speculated this result is due to that At MYB39 in myb39T-DNA insertion mutant is knockdown,rather than knockout.Then the knockout mutant of MYB39 gene was obtained by CRISPR/Cas9 technology,and the phenotype of myb39 in salt acclimation was analyzed.It was found that the albinism of mutant leaf was earlier than that of wild type,implying that At MYB39 plays a crucial role in the process of salt acclimation.The work of obtaining mapk6/myb39 double mutants by CRISPR/Cas9 is in progress.(4)In order to detect whether the interaction between MAPK6 and ERF109 affects the biological function of At ERF109,mapk6/erf109 double mutants were obtained by traditional hybridization on the basis of single mutants.The phenotypes of mapk6,erf109 single mutant and mapk6/erf109 double mutants in salt acclimation were analyzed and we found that either single mutants or double mutant had the similar salt acclimation deficiency phenotype,indicating that MAPK6 and ERF109 might belong to the same signal pathway and interact genetically.(5)Col-0,mapk6,erf109,and mapk6/erf109 were subjected to salt acclimation,and transcriptome sequencing was performed after the material was collected.The differentially expressed genes regulated by At MAPK6 and At ERF109 during salt acclimation were analyzed for further elaboration of mechanism of At MAPK6 regulating At ERF109 transcription factors involved in salt acclimation.Transcriptome analysis showed that the gene expression of phenylpropanol synthesis and metabolism was significantly changed after salt domestication,in contrast with none salt domestication.Previous studies indicate that phenylpropanol is closely related to secondary metabolites,which may enhance plant stress resistance.Compared with salt acclimated salt stress and salt acclimation,differentially expressed genes are mainly related to the oxidation-reduction process,and changes in the expression of oxidation-reduction-related genes will cause changes in the content of reactive oxygen species(ROS)in plants.The differentially expressed genes of salt acclimated salt stress and non-acclimated salt stress are mainly related to organic cyclic compound metabolic process.As a central molecular switch controlling the lifespan of transcription factors,organic cyclic compound is involved in the development of cell apoptosis and cell transformation.According to transcriptome results,we speculate that the acclimation conditions activate the gene expression of the secondary metabolic pathway,enhance the stress resistance of plants,and thus enable them to cope with the high-salt stress environment.