Identification of Eleutherococcus senticosus NAC transcription factors and their mechanisms in mediating DNA methylation of EsFPS, EsSS, and EsSE promoters to regulate saponin synthesis.

ABSTRACT

BACKGROUND: The formation of pharmacologically active components in medicinal plants is significantly impacted by DNA methylation. However, the exact mechanisms through which DNA methylation regulates secondary metabolism remain incompletely understood. Research in model species has demonstrated that DNA methylation at the transcription factor binding site within functional gene promoters can impact the binding of transcription factors to target DNA, subsequently influencing gene expression. These findings suggest that the interaction between transcription factors and target DNA could be a significant mechanism through which DNA methylation regulates secondary metabolism in medicinal plants. RESULTS: This research conducted a comprehensive analysis of the NAC family in E. senticosus, encompassing genome-wide characterization and functional analysis. A total of 117 EsNAC genes were identified and phylogenetically divided into 15 subfamilies. Tandem duplications and chromosome segment duplications were found to be the primary replication modes of these genes. Motif 2 was identified as the core conserved motif of the genes, and the cis-acting elements, gene structures, and expression patterns of each EsNAC gene were different. EsJUB1, EsNAC047, EsNAC098, and EsNAC005 were significantly associated with the DNA methylation ratio in E. senticosus. These four genes were located in the nucleus or cytoplasm and exhibited transcriptional self-activation activity. DNA methylation in EsFPS, EsSS, and EsSE promoters significantly reduced their activity. The methyl groups added to cytosine directly hindered the binding of the promoters to EsJUB1, EsNAC047, EsNAC098, and EsNAC005 and altered the expression of EsFPS, EsSS, and EsSE genes, eventually leading to changes in saponin synthesis in E. senticosus. CONCLUSIONS: NAC transcription factors that are hindered from binding by methylated DNA are found in E. senticosus. The incapacity of these NACs to bind to the promoter of the methylated saponin synthase gene leads to subsequent alterations in gene expression and saponin synthesis. This research is the initial evidence showcasing the involvement of EsNAC in governing the impact of DNA methylation on saponin production in E. senticosus.