Integrated multi-omics demonstrates DNA demethylation-driven activation of the RgMYB2-RgG10H4 axis enhancing iridoid...

Rehmannia glutinosa roots produce a group of lipophilic bioactive components known as iridoid glycosides. However, the molecular mechanisms by which DNA methylation regulates the biosynthesis of iridoid glycosides in R. glutinosa remain unknown. Herein, the development of R. glutinosa roots and the content of iridoid glycosides in the Wenxian region were significantly higher than those in Xinxiang. Low methylation level contributed to the accumulation of iridoid glycosides and the expression of related enzyme genes. Demethylation promoted both roots growth and development, as well as the accumulation of iridoid glycosides. Up-regulated genes including aldehyde dehydrogenase (RgALDH13), 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (RgHDR1), geraniol 10-hydroxylases (RgG10H3 and RgG10H4), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (RgDXR1), uroporphyrinogen decarboxylase (RgUPD1), and various transcription factors, collectively form the regulatory network for iridoid glycoside biosynthesis. Furthermore, the primary active region of the RgG10H4 promoter is located in the -164 bp region, where the RgMYB2 protein specifically binds to the TAACCA motif in the RgG10H4 promoter. Collectively, low DNA methylation enhances core gene expression, promoting iridoid glycoside accumulation, with RgMYB2-RgG10H4 positively regulating this process. The study provides new insights into the regulation of iridoid glycosides biosynthesis in plants by DNA methylation.