Expression dynamics of DNA methylation machinery during ethephon-induced sex-conversion in Cannabis sativa: an in silico genome-wide analysis

DNA methylation is an important epigenetic mechanism involved in plant growth, development, and responses to environmental cues. Although Cannabis sativa exhibits notable floral sex plasticity and ethylene-induced feminization has significant agricultural importance, the DNA methylation machinery associated with this process has not been systematically investigated. An in-silico genome-wide analysis identified ten DNA methylation-related genes in the C. sativa genome, comprising six cytosine-5 DNA methyltransferases (C5-MTases) and four DNA demethylases (dMTases). Phylogenetic, gene structure, conserved domain, and motif analyses demonstrated that these gene families are highly conserved and share structural features with homologues from other plant species. Promoter analysis revealed numerous cis-regulatory elements associated with light, phytohormone, and stress responses, including ethylene-responsive motifs. Functional validation by quantitative expression profiling revealed distinct transcriptional differences between male and female flowers and dynamic changes in the expression of several methyltransferase and demethylase genes during ethephon-induced feminization. These transcriptional changes were accompanied by altered expression of flowering- and sex-associated genes, including AGL11 , SOC1 , SQUAMOSA1 , LFY , AP3 , Men8 , SHT , and bHLH91 , throughout floral development. This study provides the first comprehensive characterization of DNA methyltransferase and DNA demethylase gene families in C. sativa . The expression patterns observed during ethephon-induced feminization identified candidate genes for future investigations into DNA methylation-associated regulatory pathways causing floral development and sex plasticity in C. sativa.