Effects Of DNA Demethylases On DNA Methylation And Transcriptional Activities Of Disease Resistance-related Genes In Arabidopsis Thaliana

Plant disease resistance-related genes play a major role in disease resistance,therefore the regulation of their expressions has been paid attention by researchers.In recent years,DNA methylation modification has been proven to regulate the expression of eukaryotic genes.Plant DNA demethylases are responsible for inhibiting excessive methylation of endogenous genes to maintain their normal transcriptional activity.ROS1,DME,DML2 and DML3 in Arabidopsis are the proteins that have DNA demethylase activities.Using the high-throughput sequencing data of Arabidopsis wild-type plants and the above DNA demethylase mutants in the public database,i.e.,genome-wide DNA methylation sequencing data and transcriptome sequencing data,this study systematically analyzed the DNA methylation levels and transcriptional levels of NB-LRR(NUCLEOTIDE-BINDING,LEUCINE-RICH REPEAT)genes,LAC(LACCASE)genes and genes involved in the signaling pathways mediated by salicylic acid(SA),jasmonic acid(JA)and ethylene(ET)in Arabidopsis DNA demethylase mutants.The following are the main findings.1.There are 13 NB-LRR genes in Arabidopsis thaliana of which DNA methylation levels in the ros1,rdd and dme mutants were significantly different from those in the wild-type plants,that is,the DNA demethylases played roles in the regulation of DNA methylation of some NB-LRR genes in Arabidopsis thaliana.It was observed that the transcriptional levels of 22 Arabidopsis NB-LRR genes increased or decreased by at least 10%in the rosl,rdd and dme mutants compared to the wild-type plants.A significant difference(Padj?0.05)was observed in the transcriptional activities of AT4GI9520(in rosl)and AT5G45510(in rdd)compared with those in the wild-type plants.In dme mutants,50 NB-LRR genes were observed with significant differences(Padj?0.05)in the transcriptional activity compared with those of the wild-type plants.Thus,the transcriptional activities of some NB-LRR genes in Arabidopsis were regulated by different DNA demethylases.2.In the wild-type plants and different DNA demethylase mutants,some Arabidopsis LAC genes were observed to have high methylation levels in the upstream and transcribed regions.Arabidopsis LAC genes were found to have generally low transcriptional levels in the wild-type plants and different DNA demethylase mutants.AT5G48100/LAC15 was found to be strongly expressed in Arabidopsis endosperm cells,and the expression of AT5G48100/LAC15 appears to be particularly sensitive to methylation modification.3.The analysis revealed that the DNA methylation levels of some genes,which are related to the signaling pathway mediated by SA in Arabidopsis thaliana,increased or decreased by at least 5%in the mutants compared with those in the wild-type plants.The AT5G04230/PAL3 gene is found to be most sensitive to the mutation of genes encoding DNA demethylases.The analysis showed that the ROS1 mutation resulted in a significant change(Padj<0.001)in the transcriptional level of the AT5G04230/PAL3 gene.In the rdd mutants,the transcriptional activities of five genes related to the SA signaling pathway were significantly inhibited.While the mutation of DME caused significant changes in the transcriptional levels of 14 genes in this pathway,and most of the genes showed extremely significant differences in the transcriptional levels.In the dme mutants,there are more genes with significantly higher levels of transcription than those with significantly lower levels.4.The analysis showed that the DNA methylation levels within some genes,which are related to the signaling pathway activated by JA in Arabidopsis thaliana,increased or decreased by more than 5%compared with the wild-type plants.While the CG methylation levels within individual gene increased even by 70.82%,and the CHG methylation levels in the regions also increased significantly.It was observed that the transcriptional activities of six genes in the JA signaling pathway in two of the three DNA demethylase mutants differed by at least 10%from the wild-type.5.This study revealed that the DNA methylation levels within some genes involved in the disease resistance signaling pathway mediated by ET in Arabidopsis thaliana,were significantly regulated by different DNA demethylases.This study also showed that the transcriptional activities of nine of the genes,which involved in ET-mediated disease resistance signaling pathway,differed by at least 10%in both of the three mutants compared with the wild-type plants.The above results revealed the effects of DNA demethylases on the DNA methylation modification and transcriptional activities of Arabidopsis disease resistance-related genes,and deepened the understanding of the regulation of expressions of disease resistance-related genes in Arabidopsis thaliana.