Methylation dynamics modified by GhDMT9, playing an vital role in drought response of cotton

Firstly and successfully created ghdmt9 mutant using CRISPR/Cas 9 system in cotton. Systematically and comprehensively interpreted DNA methylation alterations between wild type (WT) and ghdmt9 mutant. Transcription factor lsh was identified to interact with methyltransferase GhDMT9 by ChIP-seq method. DNA methylation is a stable epigenetic modification with essential roles in plant drought response. It is known that methyltransferase mutant is necessary for the regulation of methylation variations, but this epigenetic molecular mechanism based on methyltransferase mutant in responding to drought stress was still unclear in cotton. In this study, we aim to decipher the epigenetic code of drought response regulated by methyltransferase gene GhDMT9 in cotton, providing valuable information for the molecular research of drought resistance in cotton. We successfully created the first cotton methyltransferase mutant ghdmt9 using CRISPR/Cas9 method and performed methylation variations analysis with whole-genome bisulfite sequencing (WGBS) and transcriptome analysis based on ghdmt9 mutant. In addition, specific antibody of methyltransferase GhDMT9 was prepared and used for Chromatin Immunoprecipitation (ChIP-seq) analysis. The results indicated that ghdmt9 mutant interpreted approximately 2.06% methylation variations under drought stress. Demethylation variations, mainly derived from the CHG and CHH contexts, were closely correlated with drought response. Whether at normal growth stage or under drought stress, the number of up-regulated genes induced by demethylation variations was apparently higher than the number of down-regulated genes, especially genes regulating lipids and lipid-like molecules and hormone-related genes. In addition, fiber quality of ghdmt9 mutant was obviously better than that of wild type (WT). Interestingly, a transcription factor lsh (lysine-specific histone) was found to interact with methyltransferase gene GhDMT9 to activate its hyper-methylation function of target genomic regions by ChIP-seq analysis. Overall, our results extend our understanding of the epigenetic regulation of methyltransferase GhDMT9 in drought response and contribute to further investigations of the epigenetic mechanisms underlying abiotic stresses in cotton.