Ginkgo biloba is a singular and relict gymnosperm indigenous to China. Its distinctive fleshy episperm is rich in unique metabolites, ginkgolic acids, which protect the developing seed from biotic stresses. The unique nature of the tissue and its metabolites has made it highly challenging to elucidate the molecular and cellular mechanisms governing ginkgolic acid biosynthesis and regulation. In this study, we performed the mass spectrometry imaging of G. biloba seed, revealing that ginkgolic acids primarily accumulate in the secretory cavities of the episperm. We constructed a single-cell expression atlas of the G. biloba episperm and identified seven cellular types: meristem cells, subepidermal cells, lignified cells, trancheid cells, parenchymal cells, secretory cavity cells, and epidermis cells. Based on the analysis of upregulated gene expression in secretory cavity cells, pseudotime analysis of cell differentiation, and gene expression trajectory analysis, we precisely identified the key enzyme-encoding genes highly associated with ginkgolic acid biosynthesis. This approach elucidated the cellular and molecular mechanisms underlying secretory cell differentiation, secretory cavity formation, and ginkgolic acid biosynthesis and accumulation in response to exogenous jasmonic acid induction. By constructing a molecular interaction network, it was determined that the GbWRKY35, encoded by Gb_25334, is the core transcription factor. We further identified the signaling proteins that interact with GbWRKY35, confirming its central positive regulatory role in ginkgolic acid biosynthesis. As a core transcription factor, GbWRKY35 regulates ginkgolic acid biosynthesis through stimulating the expression of GbAAE16. This study provides the first spatially resolved investigation into the molecular and cellular regulatory mechanisms of ginkgolic acid biosynthesis in the episperm under jasmonic acid induction.

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