Dynamicity of histone H3K27ac and H3K27me3 modifications regulate the cold-responsive gene expression in Oryza sativa L. ssp. indica.

Cultivated in tropical and subtropical regions, Oryza sativa L. ssp. indica is largely affected by cold-stress, especially at the seedling stage. The present model of the stress-responsive regulatory network in plants entails the role of genetic and epigenetic factors in stress-responsive gene expression. Despite extensive transcriptomic studies, the regulation of various epigenetic factors in plants cold-stress response is less explored. The present study addresses the effect of genome-wide changes of H3K27 modifications on gene expression in IR64 rice, during cold-stress. Our results suggest a positive correlation between the changes in H3K27 modifications and stress-responsive gene activation in indica rice. Cold-induced enrichment of H3K27 acetylation promotes nucleosomal rearrangement, thereby facilitating the accessibility of the transcriptional machinery at the stress-responsive loci for transcription activation. Although H3K27ac exhibits uniform distribution throughout the loci of enriched genes; occupancy of H3K27me3 is biased to intergenic regions. Integration of the ChIP-seq data with transcriptome indicated that upregulation of stress-responsive TFs, photosynthesis-TCA-related, water-deficit genes, redox and JA signalling components, was associated with differential changes of H3K27ac and H3K27me3 levels. Furthermore, cold-induced upregulation of histone acetyltransferases and downregulation of DNA methyltransferases was noted through the antagonistic switch of H3K27ac and H3K27me3. Moreover, motif analysis of H3K27ac and H3K27me3 enriched regions are associated with putative stress responsive transcription factors binding sites, GAGA element and histone H3K27demethylase. Collectively our analysis suggests that differential expression of various chromatin and DNA modifiers coupled with increased H3K27ac and depleted H3K27me3 increases DNA accessibility, thereby promoting transcription of the cold-responsive genes in indica rice.

Understanding the early cold response mechanism in IR64 indica rice variety through comparative transcriptome analysis.

BACKGROUND: Cellular reprogramming in response to environmental stress involves alteration of gene expression, changes in the protein and metabolite profile for ensuring better stress management in plants. Similar to other plant species originating in tropical and sub-tropical areas, indica rice is highly sensitive to low temperature that adversely affects its growth and grain productivity. Substantial work has been done to understand cold induced changes in gene expression in rice plants. However, adequate information is not available for early gene expression, especially in indica variety. Therefore, a transcriptome profile was generated for cold shock treated seedlings of IR64 variety to identify early responsive genes. RESULTS: The functional annotation of early DEGs shows enrichment of genes involved in altered membrane rigidity and electrolytic leakage, the onset of calcium signaling, ROS generation and activation of stress responsive transcription factors in IR64. Gene regulatory network suggests that cold shock induced Ca2+ signaling activates DREB/CBF pathway and other groups of transcription factors such as MYB, NAC and ZFP; for activating various cold-responsive genes. The analysis also indicates that cold induced signaling proteins like RLKs, RLCKs, CDPKs and MAPKK and ROS signaling proteins. Further, several late-embryogenesis-abundant (LEA), dehydrins and low temperature-induced-genes were upregulated under early cold shock condition, indicating the onset of water-deficit conditions. Expression profiling in different high yielding cultivars shows high expression of cold-responsive genes in Heera and CB1 indica varieties. These varieties show low levels of cold induced ROS production, electrolytic leakage and high germination rate post-cold stress, compared to IR36 and IR64. Collectively, these results suggest that these varieties may have improved adaptability to cold stress. CONCLUSIONS: The results of this study provide insights about early responsive events in Oryza sativa l.ssp. indica cv IR64 in response to cold stress. Our data shows the onset of cold response is associated with upregulation of stress responsive TFs, hydrophilic proteins and signaling molecules, whereas, the genes coding for cellular biosynthetic enzymes, cell cycle control and growth-related TFs are downregulated. This study reports that the generation of ROS is integral to the early response to trigger the ROS mediated signaling events during later stages.

Analysis of DNA Methylation Profile in Plants by Chop-PCR.

Plants, when challenged with any unfavorable condition, such as biotic or abiotic stress, adapt to the stress via physiological or structural changes. DNA methylation, an important epigenetic factor, plays an integral role in determining chromatin dynamicity and in turn regulates the process of gene transcription in eukaryotes. DNA methylation resulting in 5-methylcytosine interferes with the transcription process by hindering accessibility of the transcriptional machinery. Transcriptionally active genes are predominantly hypomethylated, whereas repressed genes exhibit hypermethylation. It can thus be interpreted that the presence of methylation in the promoter and upstream regions of loci represses their transcription and vice versa. Chop-PCR is a targeted DNA methylation detection technique that uses partial digestion by methylation-sensitive restriction enzymes (MSREs) followed by PCR amplification. The presence of cytosine methylation at the cleavage sites of the MSREs protects the DNA against digestion and therefore can be amplified using PCR. Enzymatic cleavage occurs unhindered at unmethylated restriction sites and subsequent PCR amplification of the target sequence is not observed.

Comparative analysis of Histone modifications and DNA methylation at OsBZ8 locus under salinity stress in IR64 and Nonabokra rice varieties.

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region -2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.