Identification of DNA Methyltransferase/Demethylase Genes and 5-Azacytidine’s Impact on β-Elemene and Methylation in Curcuma wenyujin

<i>Curcuma wenyujin</i> (<i>C. wenyujin</i>) is a Dao-di geoherb. It depends on specific ecological conditions. DNA methylation (5mC) mediates environmental stress responses, regulating both growth and bioactive compound synthesis. This implies epigenetic control of secondary metabolism in <i>C. wenyujin</i>. However, its DNA methylation patterns remain uncharacterized. In this study, we identified five CwC5-MTases and three CwdMTases based on the transcriptome of <i>C. wenyujin</i>. They were responsible for DNA methylation and demethylation, respectively. Structural and integrated phylogenetic analysis classified the five CwC5-MTases into four subfamilies: CwMET, CwCMT, CwDRM, CwDNMT. The three CwdMTases were grouped into the ROS subfamily. Both CwC5-MTases and CwdMTases exhibited the closest evolutionary relationship to their homologs in monocots. Treatment of <i>C. wenyujin</i> seedlings with the DNA methyltransferase inhibitor 5-azacytidine (5-Az) enhanced terpenoid biosynthesis. QPCR analysis demonstrated that this treatment significantly upregulated key biosynthetic genes, with the exception of <i>CwDXS</i>. Subsequent GC detection further revealed a concomitant increase in the accumulation of β-elemene. Furthermore, Methylation-Sensitive Amplification Polymorphism (MSAP) analysis revealed that 5-Az altered global DNA methylation patterns. It primarily induced demethylation events. Finally, we explored the nature of these MSAP bands with altered methylation patterns. Gene identification and the effects of 5-Az on terpenoid biosynthesis and methylation not only elucidate the potential role of DNA methylation in secondary metabolism in <i>C. wenyujin</i> but also provide novel insights into the molecular mechanisms underlying its geoherbalism. This research opens a new avenue for breeding high-yield and stress-tolerant cultivars.