@article {161131, title = {Targeted Bisulfite Sequencing and DNA Methylation Analysis in Rice Endosperm and Other Tissues.}, journal = {Methods Mol Biol}, volume = {2988}, year = {2026}, month = {2026}, pages = {71-82}, abstract = {

DNA methylation is a major epigenetic mark on chromatin. Tissue-specific DNA methylation in the regulatory regions of key genes modulates their tissue-specific expression. To study DNA methylation levels at specific sequence contexts across tissues or during various environmental conditions, whole-genome DNA methylation analysis is widely used. However, whole-genome methylation analysis is an expensive, time-consuming technique and requires specific skills. To understand the role of DNA methylation in a small number of candidate loci, targeted bisulfite (BS) PCR-sequencing analysis is an attractive option. Here, we describe a detailed method to understand DNA methylation by targeted-BS PCR analysis using rice endosperm tissues. This method can be readily extended to multiple monocots, across various tissues and various stress conditions.

}, keywords = {DNA Methylation, DNA, Plant, Endosperm, Epigenesis, Genetic, Genome, Plant, High-Throughput Nucleotide Sequencing, Oryza, Polymerase Chain Reaction, Sequence Analysis, DNA, Sulfites}, issn = {1940-6029}, doi = {10.1007/978-1-0716-4981-7_7}, author = {Pal, Avik Kumar and Gandhivel, Vivek Hari-Sundar and Shivaprasad, P V} } @article {162685, title = {Loss of function of chromatin remodeler OsCLSY4 leads to RdDM-mediated mis-expression of endosperm-specific genes affecting grain qualities.}, journal = {PLoS Genet}, volume = {21}, year = {2025}, month = {2025 Dec}, pages = {e1011956}, abstract = {

In plants, RNA-directed DNA methylation (RdDM) sequence-specifically targets transposable elements (TEs) and repeats, often in a tissue-specific manner. In triploid endosperm tissue, RdDM also acts as a parental dosage regulator, mediating spatio-temporal expression of genes required for its development. It is unclear how RdDM is initiated and established in endosperm. Rice endosperm-specific imprinted chromatin remodeler OsCLSY3 recruits RNA polymerase IV to specific genomic sites for silencing and optimal gene expression. Here we show that, in addition to OsCLSY3, ubiquitously expressed OsCLSY4 is also crucial for proper reproductive growth and endosperm development. Loss of function of OsCLSY4 led to reproductive and nutrient-filling defects in endosperm. Using genetic and molecular analysis, we show that both OsCLSY3 and OsCLSY4 play overlapping and unique silencing roles in rice endosperm, by targeting specific and shared genomic regions such as TEs, repeats and genic regions. These results indicate the importance of optimal expression of two OsCLSYs in regulating endosperm-specific gene expression, genomic imprinting and suppression of specific TEs. Results presented here provide new insights into the functions of rice CLSYs as upstream RdDM regulators in rice endosperm development, and we propose that functions of their homologs might be conserved across monocots.

}, keywords = {Chromatin Assembly and Disassembly, DNA Methylation, DNA Transposable Elements, Edible Grain, Endosperm, Gene Expression Regulation, Plant, Genomic Imprinting, Oryza, Plant Proteins}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1011956}, author = {Pal, Avik Kumar and Rana, Saumyapriya and Dey, Riju and Shivaprasad, Padubidri V} } @article {156893, title = {Transcriptional and epigenomic changes in response to PEG-triggered osmotic stress in rapeseed (Brassica napus L.).}, journal = {J Exp Bot}, year = {2025}, month = {2025 Apr 03}, abstract = {

Drought hinders growth, development, and productivity of higher plants. While physiological and molecular background of plant responses to drought has been extensively studied, the role of post-translational modifications of histones or DNA methylation in response to dehydration remains largely elusive. In this study, we deciphered genome-wide changes in transcriptome and histones modifications in response to dehydration in rapeseed (Brassica napus L.). High-throughput transcript profiling (RNA-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) of PEG-treated rapeseed plants revealed genome-scale changes in transcription and histone methylation patterns, specifically in histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 tri-methylated lysine 27 (H3K27me3) sites. We have identified gene sets with altered transcript profiles as well as histone methylation marks in response to osmotic stress. Several proline biosynthesis regulatory genes coding for Delta 1-Pyrroline-5-Carboxylate Synthetases (P5CS) displayed changes in H3K4me3 and/or H3K36me3 enrichment post PEG treatment. Targeted bisulphite sequencing further identified stress-dependent gene body DNA methylation in one of the BnP5CSA gene copies that correlates with its stress-induced activation. By integrating physiological, transcriptional and epigenomic data, our study contributes to a better understanding of the drought response control in crop plants.

}, keywords = {ChIP; DNA methylation; Drought responsive genes; H3K27me3; H3K4me3; Histone methylation; P5CS genes; Proline; Rapeseed; Transcriptome.}, issn = {1460-2431}, doi = {10.1093/jxb/eraf123}, url = {https://pubmed.ncbi.nlm.nih.gov/40178034/}, author = {Prasad, Melvin and Shetty, Prateek and Pal, Avik Kumar and Rig{\'o}, G{\'a}bor and Kant, Kamal and Zsigmond, Laura and Nagy, Istv{\'a}n and Shivaprasad, P V and Szabados, L{\'a}szl{\'o}} } @article {149375, title = {Upstream regulator of genomic imprinting in rice endosperm is a small RNA-associated chromatin remodeler.}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Sep 06}, pages = {7807}, abstract = {

Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small RNAs (sRNAs) mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in the sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its likely paternal imprinted nature. These important features are modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines, we observe defects in endosperm development and a loss of around 70\% of all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms are also\ dependent on OsCLSY3. We observed that many imprinted genes and seed development-associated genes are\ under the control of OsCLSY3. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

}, keywords = {Chromatin Assembly and Disassembly, DNA Methylation, DNA Transposable Elements, Endosperm, Gene Expression Regulation, Plant, Genomic Imprinting, Oryza, Plant Proteins, Plants, Genetically Modified, RNA, Plant, RNA, Small Untranslated}, issn = {2041-1723}, doi = {10.1038/s41467-024-52239-z}, author = {Pal, Avik Kumar and Gandhivel, Vivek Hari-Sundar and Nambiar, Amruta B and Shivaprasad, P V} }