Multi-omic analysis highlights new candidate genes involved in triterpenoid and sesquiterpenoid biosynthesis in Codonopsis pilosula (Franch.) Nannf

Codonopsis pilosula root is a notable resource for bioactive sesquiterpenoids and triterpenoids. Despite this, the regulatory networks governing sesquiterpenoid and triterpenoid biosynthesis within C. pilosula, remain predominantly uncharted. In this study, we performed extensive metabolome and transcriptome investigations to reveal the gene networks, pinpoint crucial candidate genes, and transcription factors that govern the production of triterpenoids and sesquiterpenoids in C. pilosula. Our research characterized 15 distinct triterpenoids and sesquiterpenoids, with 10 showing differential accumulation based on their origins. Utilizing Mfuzz clustering and weighted gene co-expression network analysis, we isolated metabolite-specific modules/clusters. A total of 46 genes and some transcription factors potentially involved in triterpenoid and sesquiterpenoid production were identified, comprising 20 genes linked to terpenoid skeleton synthesis and 26 genes tied to the generation and modulation of triterpenoids and sesquiterpenoids. Further assays, including yeast one-hybrid and electrophoretic mobility shift, indicated that CpbHLH2, CpbHLH3, and CpAP2/ERF2 may significantly influence triterpenoid and sesquiterpenoid biosynthesis by directly regulating the transcription of CpLUP4-14, CpSQLE-2, and CpFDFT1-8. In conclusion, our results provide valuable insights into the molecular mechanisms controlling triterpenoid and sesquiterpenoid biosynthesis in C. pilosula, enhancing genetic resources and guiding future research on these compounds' accumulation in C. pilosula root.