Epigenetic Mechanism Of Gene Methylation-mediated Phenotypic Plasticity Of Arabidopsis Thaliana

The ability of plants to exhibit diversed phenotypes in order to adapt to the environment is called phenotypic plasticity.Epigenetic variations,in particular,DNA methylations are believed to play a major role in phenotypic plasticity.However,because of the relatively few empiric cases,the internal mechanism of the epigenetic methylation regulation is largely unknown.Therefore,at present study,the relationship between DNA methylation and phenotypic plasticity was studied to reveal the internal molecular mechanism of phenotypic plasticity.DNA methylation-mediated phenotypic plasticity of Arabidopsis thaliana 27 populations of Arabidopsis thaliana were selected and comparatively observed on their cotyledon length,leaf length,leaf area,flowering time,plant height,leaf number,rosette diameter,number of silique,silique size,mature time,seed size,disease spot,trichome number and branch number before and after demethylation agent 5-azacytidine(5-azaC)treatment.The results showed great phenotypic diversity were found among the 27 A.thaliana genotypes.After treated with 5-azaC,the phenotypic diversity of the A.thaliana genotypes decreased significantly.For example,the height of higher height A.thaliana genotypes decreased after 5-azaC treatment while lower height populations tend to increase.These results indicated that the phenotypic plasticity of A.thaliana was determined by the level of methylation of the relevant trait pathway.Epigenetic mechanism of phenotypic diversity of leaf length in Arabidopsis thaliana The leaf length diversity of the 27 Arabidopsis thaliana genotypes was observed and analyzed with its correlation of DNA methylation.The results showed that a great leaf length diversity existed among the 27 different A.thaliana genotypes.After treated with 5-azaC,leaf length diversity of A.thaliana genotypes decreased significantly,with shortening in the long-leaved genotypes while elongating in short-leaved ones.Leaf length of the 27 different A.thaliana genotypes are significantly correlated with DNA methylation of four genes(SWP,ARF2,GRF1,EBP1)involved in A.thaliana leaf development.Three A.thaliana genotypes with different leaf lengths were selected and used for gene expression analysis.The expressions of these four genes are positively correlated with its DNA methylation level,respectively.However,relative expression level differences among these three genotypes decreased significantly after 5-azaC treatment.These results indicate that the decreased difference of gene expression levels lead to the smaller leaf length diversity.In summary,the leaf length diversity is regulated by methylated-mediated gene expressions of the leaf development pathway.ICE1 methylation variation primarily determines phenotypic polymorphisms of freezing tolerance in Arabidopsis thaliana Cold stress is a major abiotic factor encountered by plants during their life cycle.Plants often demonstrate phenotypic polymorphisms for cold tolerance,but the mechanisms remain unclear.In the present study,A.thaliana accessions collected from 28 sites had 50%lethal temperature(LT50)values ranging from-4.9? to-11.3?,which closely correlated with the cold climate factors of the collection sites.As the upstream regulator of CBF pathway,ICE I coding regions and its promoter regions from the tested accessions demonstrated the greatest variability in the methylation levels among all members of CBF pathway,with 5 to 122 methylated cytosines,which is the only gene significantly negatively correlated with freezing tolerance.Demethylation by 5-azacytidine,as well as cmtl and drm mutants resulted in 30.0-78.3%and 10.0-48.3%increases in the freezing tolerance,respectively,with approximate decreases of 1.9-3.60C and 0.7-3.6? in the LT50,upon upregulation of members of the CBF pathway,with the exception of AtCBF2.These data indicate that ICE1-methylation-regulated transcription of the CBF pathway is responsible for phenotypic polymorphisms of freezing tolerance in A.thaliana.In order to further verify the important regulation of ICE1 methylation on its cold tolerance,we conducted a transgenic test.The A.thaliana transgenic plants 35S:AaICE1(DC),which had only 13 methylation sites,had higher EaICE1-DC gene expression than A.thaliana 35S:AaICE1(HGG1)transferred to 52 methylation sites.The effect of exogenous ICE1 gene on the expression pattern of downstream CBF pathway in A.thaliana had a similar pattern.AtCBF3 and AtCOR47 transcription factors weren't expressed under warm conditions.However,AtCBF3 reached a maximum expression at 4h of cold treatment,while AtCOR47 gene expression increased gradually with the treatment time.Meanwhile,the expression of AtCBF3 and AtCOR47 in 35S:AaICE1(DC)plants were significantly higher than that of 35S:AaICE1(HGG1)plants and the expression of Col-0 was the lowest.In conclusion,the results demonstrated that the apparent genetic variation of ICE]and DNA methylation may play an important role in plant cold tolerance phenotypic diversity.FVE methylation variation primarily determines phenotypic polymorphisms of flowering time in Arabidopsis thaliana Flowering time is one of the important phenotypic features of plants.However,it still remains unclear how epigenetic variation regulates plant phenotypic diversity in flowering time.In this study,the flowering time of 27 Arabidopsis thaliana populations with different ecological genotypes demonstrated abundant phenotypic diversity,which the flowering time ranged from 19d to 55d.The flowering time of late-flowering A.thaliana genotypes decreased after 5-azaC treatment while early-flowering ones tended to increase.Meanwhile,the flowering time variation among the 27 different ofgenotypes decreased significantly.Flowering time of different A.thaliana genotypes are significantly correlated with DNA methylation of thirteen genes(COP1?GI?TOE2?FVE?FLD?FLM?FY?LHPI?CSTF64?PRC2?TPS1?PHYA?PHYB)involved in A.thaliana flower development.Three A.thaliana genotypes with different flowering time were selected and used for gene expression analysis.It showed that expressions of these thirteen genes are negatively correlated with its DNA methylation level,respectively.Furthermore,a cytosine-substituted FVE fve-3+35S::FVE(CS)was constructed and transferred into a FVE mutant A.thaliana.The results showed that the flowering time of fve-3+35S::FVE(CS)and fve-3+35S::FVE were significantly ahead than the receiptor,and fve-3+35S::FVE(CS)flowered earlier than fve-3+35S::FVE.The results were consistent with the relative expression of the genes,and correspond to the methylation sites and level of FVE gene.The results provided an empiric evidence that the variation of the FVE methylation regulated the flowering time.Therefore,this indicates that the gene expression of a single gene can be regulated by methylation,confering to phenotypic plasticity ofA.thaliana.