Terpene trilactones (TTLs) have important medicinal value, but their low content in Ginkgo biloba leaves makes their exploitation extremely costly, thereby limiting the development of TTL-related industries. It was found that exogenous methyl jasmonate (MeJA) treatment increased the accumulation of TTLs, but the molecular mechanism is still unclear. Here, we identified two bHLH transcription factors in G. biloba, with the protein subcellular localizations in the nucleus. Expression of GbMYC2s was strongly induced by MeJA treatment, and the interactions between GbJAZs and GbMYC2s were demonstrated by yeast two-hybrid and bimolecular fluorescence complementation experiments. Overexpression of GbMYC2_4 and GbMYC2_5 enhanced Arabidopsis root sensitivity and significantly increased TTL content. In addition, GbGGPPS was found to be a common target of GbMYC2_4 and GbMYC2_5 by yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter assays and DAP-seq, and they achieved regulation of GbGGPPS by binding to the G-box. Further findings revealed that GbMYC2_4 and GbMYC2_5 bind the G-box not universally but selectively. Our study revealed that jasmonic acid signaling mediates TTL biosynthesis through the GbJAZ–GbMYC2–GbGGPPS module, which enriches the terpenoid biosynthesis regulatory networks and provides a research basis and target genes for enhancing TTL content through genetic engineering.

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