| Rice (Oryza sativa L.) is a major cereal crop that is feeding half of the world’s population. Not only rice but all other crops are also affording both biotic and abiotic environmental hazards. Here, we selected the rice plant for genome-wide study to analyze the impact of these hazards due to its facile genetics, small size and trim genome. It is already reported, to endure un-favourable environmental condition plant activates various molecular, physiological and biochemical processes. Cytosine (5mC)-DNA methylation is a stable epigenetic regulatory mechanism that is also playing a key role as an additive process for stresses regulation in plants and frequently associates with the silencing of genes and transposable elements (TEs). In fact, plants modulate their physiological and developmental machinery through genome-wide gene expression changes in response to these stresses but the actual mechanism is under explored. In order to find the role of DNA methylation for abiotic stresses like salt and osmotic stress adoptation in rice, we also created comprehensive genome-wide profiles of DNA methylation and differential genes expression (DGE) by using high-throughput techniques. As a result, we observed a variable level of methylation in stressed samples as compared to control. Further, it was analyzed hypo and hyper methylation of the individual genes caused their differential expression under two comparative conditions. The methods, research, results and novelties achieved in this study are as follow.First, we analyzed the genome-wide expression patterns of 10 genes(OsCMT1, OsCMT2, OsCMT3, OsDNMT2, OsMET1-1, OsMET1-2, OsDRM1a, OsDRM1b, OsDRM2, OsDRM3) of cytosine-DNA methyltransferases belonging to four subfamilies CMT (chromomethyltransferase), DNMT2 (DNA methyltransferase 2), MET1 (DNA methyltransferase 1) and DRM (domains rearranged methyltransferase) in rice. DNA methyltransferases (DNA-MTases) not only initiate (de novo) but also maintain the process of DNA methylation. Tissue-specific gene expression analysis showed that all family members varied widely in their expression and specificities and might be involved in some basic metabolic pathways. Similarly, the expression of all rice cytosine DNA MTase genes was not regulated by plant hormones except OsDRM1a and OsDRM1b, which were downregulated by jasmonic acid. The transcription level of 10 genes in rice shoots and roots was also measured under salt and osmotic stress. Meanwhile, quantitative polymerase chain reaction data of the japonica and indica rice cultivars revealed that there is large variation in the expression activities of all genes. The results provide a foundation to further explore the roles of DNA MTases and the epigenetic regulation of abiotic stress responses in rice.Secondly, first time Genome-wide comparative methylome analysis was performed through methylated DNA immunoprecipitation (MeDIP or mDIP) sequencing technique in japonica rice under conserved conditions of salt and osmotic stress. It was observed, control sample contained high level of methylation as compared to stresses. Methylation specific PCR (MSP), quantitative MSP and bisulfite sequencing (BS-seq) were used to validate MeDIP-sequencing results and we find a great consistency between them. The maximum unique reads 67,933,970 (69.35%) were measured under control (CK) sample as compared to salt and osmotic stress where, the unique mapped reads were only 64,858,665 (66.21%) and 65,173,609 (66.53%) respectively. The presence of maximum unique mapped reads in control predicts the high level of methylation as compared to stresses. Here, it can be proposed that with the passage of time under stressed condition maximum number of stress responsive genes get demethylation and highly expressed for the regulation of target stress. Methylome data also predicted that two different natures of stresses created different epigenetical changes for their regulation in the same genome. Similarly, we also observed the maximum number of peaks (76,858,704) under control condition as compared to both stresses where the maximum peaks were 75,321,706 under NaCl and 75,163,830 under PEG 6000 stress and overall the difference in the percentage of methylation level among two stresses was not so high. At gene level, the methylation quantity was measured in different regions like in CpG islands, upstream2k,5-UTR, CDS, intron,3-UTR and in downstream2k of multi samples. The distribution of reads in different gene regions represents the feature of genome-wide methylation patterns. Here, it was observed, Upstream2k (promoter) highly methylated (about 43.14% in CK,42.35% in NaCl and 43.81% in PEG) as compared to any other part and the second highly methylated (hypermethylated) regions were downstream2k (about 34.76% in CK,33.75% in NaCl and 34.75% in PEG) and coding sequences (CDS) (about 30.91% in CK,30.17% in NaCl and 33.10% in PEG) under three conditions. Previously it was observed, high level of methylation of promoter repressed the level of transcription highly and the situation was not diverse in our experiment. Finally, we found four methylation modified types in each sample regarding promoter and gene body as, only promoter was modified; only gene body was modified, both promoter and gene body were modified and neither of promoter and gene body was modified.Further, a systematic distribution of MeDIP-seq data and mRNA-seq data was done on the chromosomes to find a relationship between DNA methylation and gene expression. The unique hybridization (72.95% of the total hybridization) between two data’s resulted 33,009 individual genes that were differentially expressed under two comparative conditions. It was analyzed, a variety of different genes families and hormones are involved to support this process. We also succeeded to discover the important genes and transcription factors (TFs) of different abiotic stresses responsive protein families (ZFP, DUF, MYB, BURB, bZIP, F-Box, Coiled-coil domain, helix-loop-helix etc.) and most of the selected TFs are novel and not reported previously. Then quantitative PCR was applied in support of these important up and down regulated genes and results were analyzed for the validation of two purposes. First, we confirmed their expression fold (up and down regulation) under CK vs. NaCl or CK vs. PEG conditions. Secondly, we observed their expression patterns under salt and osmotic stresses in different intervals of time (0 h,3 h,6 h,12 h and 24 h) both in root and shoot of rice seedling. It confirmed a variation in their expression for target stress regulation.GO (Gene ontology) enrichment and KEGG pathway analysis was performed on the same calculation formula (Qvalue<0.05) and was explored, majority of biological process, cellular components and molecular functions were affected by both stresses on the base of DNA methylation.Finally, by focusing on zinc finger protein (ZFP) family, we succeeded to discover 14-novel transcription factors (TFs) belonging to different ZFP families (like, GRF zinc finger family, SOZ11-C2H2 zinc finger, DOF zinc finger, RING-type, SWIM zinc finger, LSD1, GATA zinc finger and C3HC4 zinc finger protein family). All the TFs were given the names (ZFP37, ZFP26, ZFP88, ZFP34, ZFP36, ZFP31, ZFP27, ZFP74, ZFP160, ZFP55, ZFP57, ZFP96, ZFP45, ZFP35) on the basis of molecular weight of their protein. Out of 14-genes,9 were up-regulated while,5 were down regulated under both stresses and are located on the chr3, chr4, chr5, chr6, chr7, chr8, chr10, chrl 1 and chr12. RT-PCR and qRT-PCR of the selected genes showed a clear difference in their expression patterns under control and stressed conditions that indicate their indispensable involvement in target stress regulation. In brief, this dissertation not only uncovers the fact that "cytosine DNA methylation is also a fundamental epigenetic regulatory process in plants under environmental hazards" but providing the basic raw material to laboratory to explore the importance of selected novel genes through further functional analysis. It also reveals that epigenetic process is playing a vital role through DNA methylation/demethylation to protect the rice crop against abiotic stresses like salinity and osmotic stress for better growth and yield. In general, hypo (decrease) and hyper (increase) level of methylation of the target genes cause their differential expression for stress regulation. |
PDF Full Text Request