The Function Analysis Of RNA-directed DNA Methylation In Regulation Of Salt Stress Response In The Promoter Region Of Arabidopsis Thaliana

Genome expression is mainly influenced by the chromatin structure, which is governedby processes often associated with epigenetic regulation, including histone post-translationalmodification and DNA methylation. DNA methylation is an important epigenetic mark forthe regulation of gene expression in eukaryotes. RNA-directed DNA methylation (RdDM),which was first discovered in viroid-infected tobacco, is an important regulatory phenomenoninvolved in repressive epigenetic modifications that can trigger transcriptional gene silencing(TGS) under abiotic stress in plants. The gain or loss of DNA methylation is correlated with aconsiderable decrease or increase in the corresponding amount of mRNA abundance and withthe presence or absence of24-nt siRNAs at each silenced epiallele. Approximately one-thirdof methylated DNA loci in Arabidopsis is rich in siRNAs, implying an primary determinantof siRNAs in DNA methylation. Increasing evidence has shown that the activity of siRNAscould be triggered by various environmental stimuli to affect the targeting chromatinstructure.Although the ABA-dependent salt stress signal transduction pathway has beenintensively studied, whether or not DNA methylation/demethylation is involved in thispathway remains unknown. The mechanism of how the siRNA mediates DNA methylation byRdDM to respond to abiotic stress also needs to be elucidated. Our findings provide a novelABA-dependent salt stress regulatory signal pathway and allows for a better understanding ofthe role of siRNAs in controlling the RdDM pathway to regulate gene expression in responseto abiotic stress. The main results and conclusions presented in this thesis are as follows:(1) AtMYB74encodes a R2R3-MYB transcription factor and differentially expresses invarious tissues.By analyzing the database of GENEVESTIGATOR and Arabidopsis eFP Browser withsalt stress treatment,1286genes were found. Promoter analysis of the selected genes was then carried out in Arabidopsis epigenome maps to identify hypermethylated regions. Theresults showed that gene candidates with promoter hypermethylation in response to salt stresswere identified. AtMYB74was chosen for our further study.The full-length cDNA corresponding to the AtMYB74mRNA is975bp and encodes aputative protein of324amino acids. The R2and R3MYB DNA binding repeats (amino acids13to65and66to117, respectively) of AtMYB74are highly conserved with all other MYBproteins in Arabidopsis and in other plant species. AtMYB74is constitutively expressed invarious tissues at low abundance by qRT-PCR analysis and GUS staining detection. Inaddition, onion epidermal cells transformed with an expression plasmid for theAtMYB74-GFP fusion protein exhibited GFP fluorescence in the nucleus.(2) Transgenic plants overexpressing AtMYB74show hypersensitivity to ABA and saltstress during seed germination.Overexpression of AtMYB74also induced the expression of salt stress responsive genes,such as AtRD29B, AtRAB18, and AtRD20. However, under NaCl or ABA treatment, thegermination of OE transgenic seeds was inhibited more severely than that of WT plants andthe germination rates of RNAi transgenic seeds were similar to those of WT plants. Moreover,application of ABA biosynthesis inhibitors (tungstate) in the medium suppressed thehypersensitivity of OE transgenic seeds to NaCl. The reduced germination rates of the OElines suggest that AtMYB74is involved in the response to salt stress during seed germinationin an ABA-dependent manner in plants.(3) Dynamic DNA methylation results in AtMYB74activation in response to ABA andsalt stress.The level of AtMYB74transcripts increased significantly by both NaCl and ABAtreatments in WT plants, indicating that AtMYB74responds to salt stress and ABA signals atthe transcriptional level. In addition, in the RdDM mutants, the NaCl and ABA treatmentsdemonstrate much less changes in the accumulation of AtMYB74mRNA than that in WT,suggesting the involvement of RdDM in the transcriptional regulation of AtMYB74.Meanwhile, the deficiency of active DNA demethylation in ROS1-RNAi and ZDP-RNAitransgenic plants resulted in a slight increment after treatment. The200bp promoter regionapproximately500bp upstream of the transcription initiation site of AtMYB74was analyzed by bisulfite sequencing. WT plants treated with NaCl and ABA exhibited the a visiblereduction in total5-meC contents compared with the control. Interestingly, the percentage ofCpHpH methylation was nearly halved in the treated plants, and only around10%reductionin CpG contexts was detected. And the RNAi transgenic plants showed only a slightreduction in all context of DNA methylation.Time-course analysis revealed that the mRNA level under salt stress showed a increasefrom0.5h, with the highest levels occurring at3h, accompanied by the lowest percentage ofCpHpH methylation. After6h, AtMYB74expression under salt stress was appearently higherin treated plants than that in the control; a lower percentage of CpHpH methylation was alsodemonstrated in WT plants. Similar results were obtained in ABA-treated plants. Theseresults indicate that stress increases the expression of AtMYB74by reducing dynamic DNAmethylation in the CpHpH context.(4) DNA methylation is controlled by the accumulation of24-nt siRNAs targetingAtMYB74promoter.Five24-nt siRNAs (ASRP215119, ASRP41948, ASRP27256, ASRP13208, andASRP2423) located in a cluster were identified to target at a narrow region (-603bp to-477bp) of the2.9kb promoter of AtMYB74. Moreover, bisulfite sequencing analysis of individualclones showed that DNA methylation considerably changes in or near the siRNA targetregion. However, no obvious changes were detected in the5-azaC-treated WT, ddc, dcl3, rdr2,ROS1-RNAi, and ZDP-RNAi transgenic plants, indicating that CpHpH hypomethylation isdependent on RdDM. The accumulation of five24-nt siRNAs was substantially reducedunder the salt stress and ABA in WT plants compared with those in dcl3plants by siRNAqRT-PCR and Northern Blot analysis, suggesting that decreases in DNA methylation causedby the reductions in24-nt siRNA accumulation lead to the activation of AtMYB74under saltstress.(5) Exogenous24-nt siRNAs direct RdDM in the AtMYB74promoter in Nicotianabenthamiana leaves.Considering that the siRNAs diced from the hairpin RNA could induce transcriptionalsilencing of the target genes, two types of expression cassettes were constructed to generatesiRNAs and express reporter gene (R1and R2). In the reporter gene cassettes of pBI121, gusA was driven by the35S promoter (P1), AtMYB74promoter (P2), and mutated AtMYB74promoter with the deletion of a200bp region where these siRNAs are targeted (P3). GUSactivities between groups P1and P1-R1, P2and P2-R2, and P2and P3-R1did not showobvious changes after transient cotransformation. The GUS activity of group P2-R1wasobviously lower than that of group P2. In addition, bisulfite sequencing analysis revealed thatthe DNA methylation in the200bp promoter region increased43%due to the siRNAsyielded by the hairpin RNA. The constructs P1-R1, P2-R1, and P3-R1could efficientlygenerate siRNAs in the transformed tobacco. Overall, results demonstrate that the ectopicexpression of artificial siRNAs targeting the AtMYB74promoter also can regulate GUSexpression through RdDM.(6) Both RdDM and ABA-responsive cis-elements (ABREs) contribute to the regulationof AtMYB74under salt stress in the ABA-dependent pathway.Four putative ABREs were identified by bioinformatics analysis in the AtMYB74promoter. ChIP-PCR detection found that ABI5occupancy on the AtMYB74promoter viaABREs. Although the transcripts of AtMYB74in abi5increased under stress, they remainedlower than those in WT plants, indicating that ABREs also participate in the regulation ofAtMYB74. GUS staining showed that stress-induced accumulation of AtMYB74transcriptscould be detected in P3transgenic seedlings, suggesting that increasing levels of GUS maybe caused by ABREs’ regulation. Furthermore, in abi5, both the CpHpH DNA methylation ofAtMYB74and the accumulation of five24-nt siRNAs remarkably decreased. Theseobservations reveal that both ABREs and RdDM participate in the regulation of the AtMYB74promoter activity to respond to salt stress.