EsWRKY regulate saponin biosynthesis via binding, DNA methylation, and protein interactions in Eleutherococcus senticosus

WRKY transcription factors (TFs) are essential in regulating plant secondary metabolism, yet their role in the saponin biosynthesis of Eleutherococcus senticosus remains unclear. This study aimed to identify and characterize EsWRKY TFs associated with saponin biosynthesis. Bioinformatics analyses and correlation studies indicated the strongest correlation of EsWRKY7 , EsWRKY35 , EsWRKY40 , and EsWRKY88 with saponin production. Subcellular localization confirmed their nuclear distribution; regulatory activity was influenced by light and DNA methylation, with Es WRKY7, Es WRKY35, and Es WRKY40 possessing transcriptional autoactivation ability. Overexpression experiments revealed that Es WRKY7 and Es WRKY88 negatively regulated saponin biosynthesis genes, reducing saponin content, while Es WRKY35 and Es WRKY40 acted as positive regulators, promoting saponin accumulation. Through the application of Y2H and BiFC assays, a yeast library screening uncovered several proteins that interact with Es WRKYs. Furthermore, three regulatory mechanisms mediated by Es WRKY in saponin biosynthesis were elucidated: direct promoter binding, transcriptional regulation via DNA methylation-sensitive elements, and regulation through protein complex formation. Molecular dynamics simulations provided insights into the variations in the regulatory function of Es WRKYs upon protein interaction. These findings unveil the multifaceted modulatory network of saponin biosynthesis in E. senticosus and lay a foundation for enhancing saponin accumulation through metabolic engineering. • Four core EsWRKYs transcription factors were identified to oppositely regulate saponin biosynthesis. • Es WRKYs directly bind promoters of saponin synthases ( EsFPS / EsSS / EsSE ) to control their expression. • A novel bidirectional interplay between Es WRKYs and DNA methylation fine-tunes promoter binding. • Protein interactions modulate Es WRKYs' DNA-binding stability, thereby affecting transcriptional activity. • A triple-mechanism framework (direct binding, methylation sensing, protein interactome) is proposed.