Identification and functional analysis of DNA methylation-related enzyme gene family in Eleutherococcus senticosus

Eleutherococcus senticosus, a medicinal and edible plant recognized historically in East Asia, is characterized by a rich composition of saponins and other biologically active ingredients. In this study, 11 cytosine-5 DNA methyltransferases (C5-MTase) and 6 demethylases (dMTase) genes were identified within the genome of E. senticosus. Specifically, two major EsC5-MTases (EsMET1a and EsCMT3b) and two EsdMTases (EsROS1a and EsDME1) were screened through correlation analysis, examining the relationship between their gene expression levels and DNA methylation ratio within E. senticosus genome. Promoter function analysis revealed that the expression of these four prominent genes was regulated by abiotic stresses such as drought and light. In vitro catalytic validation demonstrated the functionality of EsMET1a, EsCMT3b, EsROS1a, and EsDME1 proteins in modifying DNA methylation patterns of key enzyme genes associated with saponin synthesis in E. senticosus. Additionally, transient in vivo overexpression of the four central genes could alter the saponin content by modulating the DNA methylation levels and secondary metabolic processes in E. senticosus. Molecular docking studies further elucidated the possible mechanism of DNA methylation and demethylation catalyzed by these four primary proteins.