Panax notoginseng is an important medicinal plant, and saponins are the primary active components that are the key determinants of pharmaceutical quality. Research has indicated that methyl jasmonate (MeJA) enhances the saponins accumulation in P. notoginseng, but the specific MeJA-responsive transcription factors (TFs) that regulate this process remains unidentified. Given the critical role of MYB TFs in the regulation of plant secondary metabolism, this study aimed to elucidate the regulatory mechanisms of the MYB TF family in P. notoginseng under MeJA treatment. Genome-wide screening led to the identification 110 MYB genes, followed by a comprehensive analysis of their phylogenetic relationships, conserved motifs, gene structures, cis-acting elements, chromosomal localization, and collinearity. Integrated transcriptomic and metabolomic analyses showed that MeJA treatment significantly altered the expression patterns of 84 MYB genes while also promoting saponin accumulation in P. notoginseng leaves. Co-expression network analysis revealed a significant correlation between PnMYB38 and saponin metabolites, highlighting the pivotal regulatory function of this TF. Subcellular localization experiments confirmed nuclear localization of PnMYB38. Yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase assays demonstrated that PnMYB38 directly and specifically bound to the promoters of key saponin biosynthesis genes (PnSE and PnDS), thereby inducing their expression. This study comprehensively characterized the functional role of PnMYB38 in regulating MeJA-mediated saponin biosynthesis in P. notoginseng, proposed a ‘MeJA-PnMYB38-saponin biosynthesis’ regulatory network that provided novel insights into the transcriptional regulatory mechanism of saponin biosynthesis, and established a foundation for molecular breeding targeting MYB TFs and metabolic engineering.

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