Abstract

Jasmonic acid (JA) signaling is a key regulator of plant secondary metabolism, yet how it balances rapid activation with timely repression of ginsenoside biosynthesis remains unclear in Gynostemma pentaphyllum. Here, we investigated the transcriptional regulation mechanism of ginsenoside biosynthesis by JA-responsive basic helix-loop-helix (bHLH) transcription factors. By integrating transcriptome and phylogenetic analyses with yeast one-hybrid, electrophoretic mobility shift assays, dual-luciferase assays, transgenic hairy root overexpression, and RNA interference, together with metabolite profiling, we identified key regulators and defined their roles in the ginsenoside biosynthesis pathway of G. pentaphyllum. We characterized an antagonistic bHLH pair, GpGAB (g36559, Activator), and GpGIB (g21771, Inhibitor). GpGAB activated ginsenoside biosynthetic genes GpDS, GpPPDS, and GpUGT94AT1, whereas GpGIB repressed the expression of GpDS and GpPPDS. GpGAB also upregulated the expression of GpGIB, with GpGAB responding earlier than GpGIB to JA treatment. Taken together, our results support a model in which GpGAB and GpGIB form a time-delayed negative feedback loop that fine-tunes ginsenoside biosynthesis after JA induction, providing a mechanistic framework for dynamic transcriptional regulation of ginsenoside in G. pentaphyllum.