Role Of H₂O₂and NO In SO₂-Induced Alteration Of DNA Methylation In Arabidopsis

DNA methylation is one of the most important heritable epigenetic modifications of the genome and plays an important role in maintaining the DNA stability and normal development plant. Adverse environments such as drought, salt, heavy metals can induce to changes of DNA methylation level in plants, which may have important consequences for plant adaptations. Current results have shown that SO²stress can cause the alteration of genome DNA methylation in Arabidopsis thaliana; SO²can cause reactive oxygen species （ROS） increasing in lower epidermis cells in Arabidopsis; however, in the ground organization of SO²-fumigated Arabidopsis plants the expression of nitrate reductase（NR） and nitrite NO reductase （NIR） will increase, which suggests that at this time NO content in the cell increased. We speculate that in alteration of SO²-induced DNA methylation modify in SO²-fumigated plants, increased ROS and NO play an important role. Therefore we find out the role of H²O²and SNP in alteration of SO²-induced DNA methylation in H²O²and exogenous NO donor（SNP） treated plants.Transcription of methytranferase was determinated by RT-PCR in SO², AsA+SO², c-PTIO+N^aN³+SO²-treated Arabidopsis plants. The experimental results showed that transcription of MET1, CMT3, DRM1improved in SO²-treated Arabidopsis plants respetively; transcription of MET1, CMT3in AsA+SO², c-PTIO+N^aN³+SO²-treated Arabidopsis plants diminished. The clean of ROS and NO will block DNA methylaion of plants. The present results indicated that ROS and NO play important role in alteration of DNA methylation.H²O²-induced DNA methylation alteration of a gene encoding nitrilase2（NIT2） protein in Arabidopsis shoots was investigated by using bisulfite sequencing, meanwhile ROS level and NIT2gene expression were also detected. The experimental results showed that the total methylation level in promoter region of NIT2gene did not change much after plants exposed to100μmolL^-1 H²O²for3h, but for different DNA contexts, cytosine residues could have different methylaiton status in H²O²-exposed samples. In control plants, the methylation levels of CHG （where H is C, A or T） and CG contexts were35%and93.1%respectively, while in H²O²-exposed samples, the methylation levels of CHG and CG contexts were50%and96.7%respectively. For CHH context, cytosine residues could be hypomethylated, hypermethylated, or demethylated. Exposure to H²O²increased the intracellular ROS level in guard cells of Arabidopsis leaf, and also induced NIT2transcript level increasing in plants exposed to100μmolL^-1 H²O²for3h. The present results indicated that H²O²can induce NIT2gene expression and cytosine methylation changes, which can affect plant physiology contributing to plant adaptation to environmental stress. Our results also suggest that increased ROS may play a key role in triggering cytosine methylation and gene expression.Transcription of methytranferase was determinated by RT-PCR in0.2mmol’L^-1 SNP-treated plants, mRNA contents of nitrate reductase and nitrite reductases increased significantly conparaed with control.Transcription levels of METl, MET2, CMT3, DRM1, DRM2changed, expression of antioxidant enzymes genes POD, GPX7up-regulat significantly, but the same condition, the heat shock transcription factor （HsfA2） heat shock protein Hsp17.7and Hsp17.6b transcription level there is no obvious change. The results showed that SNP caused the context of NO and induced expression of methytranferase in arabidopsis plants.Therefor, NO involves the processof SO²inducing alteration of DNA methylation modify.