Study Of Differential Expression Of Defense-related Genes And Epigenetic Mechanism Of Arabidopsis In Response To Sulfur Dioxide

Environmental stresses are severely affecting plant growth and productivity. Sulfur dioxide (SO2) is one of the most common and harmful air pollutants. In order to further understanding plant molecular response to SO2, we investigated transcriptional profiling, DNA methylation alterations and microRNA (miRNA) gene-expression pattern in Arabidopsis thaliana (4-week-old) exposed to30mg/m' SO2for72h.Transcriptional profiling using Affymetrix GeneChip technology identified494genes differentially expressed (≥2-fold change) between SO2fumigation group and the control, including220up-regulated genes and274down-regulated genes. According to Arabidopsis Gene Ontology resource, those genes were mainly involved in cellular metabolism, binding, transcriptional regulation, structural molecule, signal transduction, material transportation, and so on. A number of defense-related genes encoding antioxidant enzymes, glutathione S-transferase, cytochrome p450, and heat shock proteins were up-regulated. Moreover, numerous genes encoding pathogenesis-related proteins and enzymes required for the phenylpropanoid pathway and for cell wall modification were highly activated upon SO2exposure. We selected nine of the significantly differentially expressed defense-related genes for analysis using semi-quantitative RT-PCR and confirmed that their expression was up-regulated under SO2stress. The enhancement of reactive oxygen species (ROS) production and activation of genes involved in the signal transduction of ethylene and auxin suggest that ROS and phytohormone may play important roles in transcriptional regulation during plant adaptation to SO2stress.By using methylation sensitive amplification polymorphism (MSAP) technique,12primer-pair combinations were employed to generate703amplified bands. The fully methylated loci increased along with a decrease in unmethylated and hemimethylated loci. The MSAP ratios were37%, showing more hypermethylated sites and less hypomethylated sites in CCGG context in SO2-treated plants. These data showed that DNA methylation level of global genome was obviously promoted after SO2exposure, which resulted in genome stability and played an important role in adaptation to environmental stress. Bisulfite sequencing analysis of DNA methylation in NIT2and ACS6promoter regions showed that cytosine residues were hypomethylated in both CG and CHH (where H is C, A or T) contexts, and methylation levels of the promoters decreased in Arabidopsis plants exposed to SO2. DNA hypomethylation of NIT2and ACS6promoter regions is associated with high gene transcript levels in SO2-treated plants, suggesting that NIT2and ACS6expression was directly regulated by promoter DNA methylation. These data suggest that SO2-induced epigenetic change may be closely related to the gene activation in plant response to SO2stress. RT-PCR analysis results showed that SO2induced sequential expression of Arabidopsis miRNA. The expression of miR162a, miR167a, miR167c, miR319c was up-regulated, but miR398a and miR398c was decreased along with increase in transcript level of target gene CSDl and CSD2. The1000bp upstream sequence of SO2-induced miRNAs were further analyzed by using the PlantCARE database. A lot of stress and hormone response elements were identified, which play key roles in regulating miRNA expression.The results showed that SO2induce differential expression of Arabidopsis defense-related genes, and that epigenetic mechanism play important roles in transcriptional regulatory in plants adaptation to environmental stress.