Roles Of RDM1,AGO4,NRPD1 And ROS1 In The Establishment Of CHG Methylation And Growth As Well As Development Of Edm2 Mutant

IBM1(INCREASE IN BONSAI METHYLATION 1)is a histone H3K9 demethylase,which can erase the histone H3K9 methylation occurring on the active transcribed gene as the result of Pol ? transcription,resulting in the inability to form CHG methylation on such genes.Two anti-silencing factors ASI1(ANTI-SILENCING 1)and EDM2(ENHANCED DOWNY MILDEW 2)in Arabidopsis thaliana perform important functions in maintaining IBM1 expression by forming protein complexes through the bridge protein ASI1immunoprecipitation protein 1,AIPP1(ASI1-IMMUNOPRECIPITATED PROTEIN 1)EDM2 is found to play an important role in maintaining the normal expression of IBM1;the expression level of IBM1 in the edm2 mutant is significantly reduced,which leads to a visible developmental phenotypes similar to ibml mutant plants,such as shorter plant height,increased branching,and smaller as well as dark green leaves,partially abnormally developed floral organs,reduced seed setting,and defective seeds.In order to investigate the causes of the abnormal phenotype of edm2,and to explore whether the increase in CHG methylation caused by the ibm1 mutation is affected by mutations of the RNA-directed DNA methylation(RdDM)pathway genes,we crossed edm2 with RdDM pathway mutants as well as ros1 mutant(ROS1 is a DNA demethylase),and obtained a series of double mutants,namely edm2 rdm1,edm2 ago4,edm2 ros1 and edm2 nrpd1.The results from phenotypic observations showed that edm2 rdm1,edm2 ago4,and edm2 ros1 can restore,or at least in part,the developmental defects of edm2 mutant seedlings,but edm2 nrpdl exacerbated the developmental defects of mutant seedlings.We performed genome-wide DNA methylation and transcriptome sequencing for these mutants,and analyzed the resulting data by means of bioinformatics approaches,and found that CHG methylation on the active genes in these double mutants is changed,and the genes involved in biosynthesis and signaling of IAA as well as GA and defense responses has also been altered,and,in effect,their combined actions cause the developmental phenotype of edm2 mutants.The results we have obtained are as follows:1.Previous studies suggested that the developmentally defective phenotype of edm2 was caused by a decrease in the expression of IBM1,which caused hypermethylation of the BNS gene,thereby reducing its gene expression.The results of this study showed that the BNS gene expression is upregulated in edm2 rdm1,edm2 ago4 and edm2 ros1,but the expression of BNS gene in edm2 nrpd1 was relatively low.However,analysis of methylation sequencing data revealed that,compared with the edm2 mutant,only the edm2 rdm1 double mutant showed a decrease in the methylation level of the BNS gene;instead,the methylation of BNS gene in the edm2 ros1 double mutant is aggravated.These results suggest that BNS gene expression is not only controlled by DNA methylation.2.Whole genome DNA methylation analysis of double mutants found that the introduction of rdm1 or ago4 mutation into edm2 mutants can significantly reduce CHG hypermethylation of some genes in edm2 mutants.Therefore,it is believed that the RdDM pathway may help establish and stabilize CHG methylation caused by edm2 mutation in certain genomic regions.In addition,the introduction of ros1 mutants in the edm2 background can significantly increase CHG methylation at many loci.Therefore,we postulate that CHG methylation on a collection of actively transcribed genes caused by edm2 mutation may be impaired by ROS1.However,the introduction of the nrpd1 mutation into the edm2 mutant can either increase CHG methylation on particular genes or reduce CHG methylation on other genes,and the number of genes with exacerbated CHG methylation is larger than that of the genes with impaired CHG methylation.As is the case in edm2 rdml and edm2 ago4,we speculate that,in edm2 nrpd1,NRPD1 may have the effects on inhibition of CHG methylation on some genes,but may play roles in facilitating the establishment of CHG methylation,or affecting CHG methylation through the RdDM pathway.Another possibility is that a large number of these CHG highly methylated genes in edm2 nrpd1 are caused by the decreased expression of ROS1.Therefore,on the basis of the above-mentioned results,we propose that CHG methylation in the edm2/ibm1 mutant is modulated by the RdDM pathway as well as the ROS1-mediated DNA demethylation pathway.3.mRNA sequencing results showed that rdm1,ago4,rosl and nrpd1 mutations each affected the expression of tens of genes participating in IAAand GA biosynthesis and signaling,thus leading to the developmental defects of edm2.In edm2 mutant plants,the expression of genes involved in biosynthesis of and signal transduction of auxin were suppressed,resulting in a dwarfism phenotype of the plants.However,double mutation impaired or aggravated the dwarfism phenotype of such plants by raising or lowering the expression of these genes;the expression of several genes associated with GA-mediated signal transduction pathways in edm2 mutants were significantly reduced,while edm2 rdml and edm2 ago4 mutant plants has reverted to the wild-type expression levels of the aforementioned genes,indicating that the edm2 mutant may weaken the GA signaling pathway and lead to the shorter plant height and other developmental phenotypes,and rdml and ago4 restored the GA signaling transduction through an unknown mechanism.4.The mRNA sequencing results also showed that a large number of genes in the edm2 mutant plants related to the plant defense responses were significantly increased,such as the responses to cold,salinity stress,damage,UV light,abscisic acid,and so on.However,the expression of these genes was restored or partially restored in edm2 rdm1,edm2 ago4 and edm2 ros1,but was further increased in edm2 nrpd1.The edm2 mutant plants had a higher ABA content than Col-0 plants did and shows enhanced sensitivity to ABA.These results collectively suggest that the abnormal developmental phenotypes of the edm2 mutant are probably caused by the overexpression of defense-related genes.5.In addition,compared with edm2,the pro35S::EDM2 overexpression transgenic lines restored the expression levels of genes involved in auxin responses,GA responses and defense responses,which implies that the restoration of expression of these genes in the above-mentioned double mutants does not necessarily depend on the EDM2.So,it is speculated that the double mutant may affect the expression of target genes downstream of EDM2.In summary,this study found that loss of function of RDM1,AGO4,ROS1,or NRPD1 attenuated or enhanced developmental defects of edm2 by changing the expression of BNS genes,genes implicated in auxin,GA,and ABA biosynthesis and signaling,and genes associated with defenses,which implicate that the establishment of CHG methylation on the active transcribed gene in the edm2/ibm1 mutant is modulated by the RdDM pathway and ROS1.Based on these findings,we speculate that ceertain downstream target genes of EDM2/IBM1 are also the targets of action of RdDM pathway or ROS1.