| The jasmonic acid（JA）signal pathway has been shown to play an important role in Arabidopsis immunity.Many genes are identified to take part in the JA signal pathway.Therefore,revealing the regulation of the expression of these genes will provide insights into the mechanisms of the signal pathway.DNA methylation is a stable epigenetic phenomenon and has been shown to play a key role in the regulation of the expression of some genes.In this study,using the methylation sequencing data and transcriptome sequencing data from Arabidopsis wild-type,methylase and demethylase mutants in public databases,we analyzed the methylation and expression of the genes involved in the JA signal pathway in the mutants defective in methylases and demethylases.The study will uncover if DNA methylation is implicated in the regulation of expression of JA signal pathway genes in Arabidopsis.Further,the expression of some genes was detected in rdd and drm1/2 mutants at four-week-old seedlings,and some genes in the mutant drm1/2 were detected after inoculation with Xanthomonas campestris pv.campestris（Xcc）.Below are the main results.1.Effects of DNA methylases on the DNA methylation and transcriptional activity of JA signal pathway-related genes in ArabidopsisBy text mining,at least 84 genes were found to be involved in the JA signal pathway in Arabidopsis.On the whole,these genes can be divided into four categories:genes in JA and its derivatives synthesis（27）,core signaling genes（19）,genes for transcription factor（33）and defense response genes（PDF1.2,PR4,THI2.1,VSP1 and VSP2）（5）.DNA methylation within the upstream regions of genes AtPLAI,AOC2,OPR3,ACX1,JOX3,ASK2,JAZ7,JAZ11,MYC5/NIG1,ERF1 and ATAF2/ANAC081 was found to be significantly affected by DNA（de）methylases.The methylation in the upstream region of AtPLAI was mainly distributed in the 200bp upstream region,and the methylation levels of CG,CHG and CHH were 84.68%,48.98%and 44.74%,respectively.The DNA methylation in the upstream region of ATAF2/ANAC081 was also mainly happening in the 200bp upstream region,and the CG and CHH methylations were 21.95%and 88.56%,respectively.The DNA methylation in the upstream regions of AOC2,OPR3,JOX3,ASK2,JAZ7 and MYC5/NIG1 was mainly occurring in the upstrteam regions from 200 to 500 bp.After the mutation of the methyltransferase MET1,the CG methylation levels in the gene body regions of 25 genes were significantly decreased by more than 10%and decreased to less than 1%,indicating that MET1 plays a pivotal role in maintaining their CG methylation.Mutations in the methylases CMT3,DRM1/2,and DDM1 resulted in changes in the CG methylation levels within the gene bodies of 7,8,and 15 genes,respectively.Among them,the CG methylation of AtPLAI,OPR3,ASK2,JAZ7 and MYC5 was regulated by four methylases.After MET1 mutation,the transcriptional activity of 22 genes was enhanced and that of five was attenuated.Mutations in CMT3 resulted in the increased transcriptional activity of 15 genes,with only the activity of ACX3 being decreased.Most transcription factors in the JA signaling have enhanced transcriptional activity after CMT3 mutation.The transcription level of VSP1 in cmt3 is more than 14 times higher than that in wild type.In drm1/2,the transcriptional activity of 16 genes was enhanced and that of 11 were decreased.Among them,the transcription level of VSP1 in drm1/2 was 23.88 times higher than that in wild type.LOX2 and AOS,two synthetase-encoding genes,also showed higher transcriptional activities in drm1/2,which were 2.43 times and 1.59 times that of the wild type,respectively.Mutations in DDM1 enhanced the expression of 21 genes and weakened the expression of 5 genes.In ddm1,the transcription level of PDF1.2 was 11.73 times higher than that in wild type,and the transcription level of THI2.1 was 12.87 times higher than that in wild type.Among the four methylase mutants,the transcriptional activity of ACX6,JOX2,JAZ8,WRKY18,ANAC055,ATAF2 and MED25 was increased.The transcriptional levels of AOS,AOC2,and VSP1 were affected by the methylases CMT3,DRMl/2,and DDM1,and their transcriptional activities were significantly up-regulated in all the three mutants.2.Effects of DNA demethylases on the DNA methylation and transcriptional activity of JA signaling-related genes in ArabidopsisMethylation within the gene body of LOX2 is regulated by both methylases and demethylases.Mutation of ROS1 resulted in markedly increased CG and CHG methylations within the gene body of LOX2.In rdd,CG methylation in the gene body regions of KAT2,JOX1,JAZ2,JAZ3 and MYC4 was increased by more than 10%,and CG and CHG methylations in the gene body regions of LOX2 were increased by 31.08%and 21.75%,respectively.Mutation of ROS1 caused an increase in CG methylation within the 500 bp upstream regions of ASK2,JAZ1,JAZ11 and ERF1 by 44.25%,60.16%,84.37%and 70.18%,respectively;while in rdd,the CG methylation within the 500 bp upstream regions of JAZ11,MYC5 and ERF1 was increased by 77.25%,37.34%and 31.23%,respectively.After ROS1 mutation,the transcript levels of OPR3,WRKY70 and ERF1 were increased by 1.56-fold,1.48-fold and 2.37-fold,respectively.In rdd,the transcriptional activities of AOS,ACX5,JOX2 and JOX4,which are involved in JA synthesis,were all increased,while the transcription level of PDF1.2 was decreased to only 19%of that of wild type,and the transcript levels of WRKY60 and WRKY70 were only 34%and 24%of that of wild type,respectively.3.qRT-PCR analyses of some JA signal pathway genes in rdd and drm1/2 mutantsThe results of qRT-PCR revealed that the expression levels of COI1,MYC2,ORA59,PDF1.2 and VSP1 were extremely or significantly up-regulated,and the expression levels of EFR1 and ANAC019 were significantly down-regulated in rdd.However,there was no significant change in the expression level of MED25 between wild type and rdd according to the results of qRT-PCR.In drm1/2,the qRT-PCR results showed that the expression levels of LOX2,AOS,COI1,JAZ1,JAZ3,JAZ9,ERF1 and MYC2 were significantly down-regulated,the expression level of ORA59 was not significantly altered,while those of ATAF2,PR1,PDF1.2 and VSP1 were significantly up-regulated.In drm1/2 infected by Xcc,the qRT-PCR results demonstrated that the expressions of AOS,COI1,MYC2 and PDF1.2 were significantly activated,while the expressions of LOX2,ERF1,ORA59,PR1 and VSP1 were repressed.It is speculated that Xcc suppresses the SA signal pathway,but activates the JA signal pathway in drm1/2. |
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