Abstract
Curcuma wenyujin (C. wenyujin) 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 C. wenyujin. However, its DNA methylation patterns remain uncharacterized. In this study, we identified five CwC5-MTases and three CwdMTases based on the transcriptome of C. wenyujin. 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 C. wenyujin 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 CwDXS. 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 C. wenyujin 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.