Abstract

Tetrastigma hemsleyanum exhibits pronounced ecotype divergence in tuberous root morphology, phytochemical composition, and bioactivity, yet the molecular basis of this differentiation remains unclear. By integrating market surveys, common-garden experiments, metabolomics, transcriptomics, and functional assays, we show that the southeastern (SE) ecotype consistently produces smaller tuberous roots but accumulates higher total flavonoids than the southwestern (SW) ecotype, indicating a genetically encoded metabolic preference. This chemical advantage translates into superior antioxidant capacity and anti-inflammatory efficacy, as validated by radical-scavenging assays and an LPS-induced zebrafish inflammation model. Untargeted metabolomics reveals a quercetin-centered flavonoid signature in SE materials, with SE-enriched discriminants dominated by quercetin-derived compounds (e.g., quercitrin, isorhamnetin, and eupafolin) that are strongly associated with bioactivity. Integrative transcriptomics and metabolomics revealed that SE accessions upregulated core pathway nodes, particularly ThCHIs and ThF3′H , which channel flux toward 3′-hydroxylated products and their derivatives. Functional characterization confirmed that ThF3′H is an endoplasmic reticulum-localized cytochrome P450 catalyzing the conversion of naringenin and kaempferol to eriodictyol and quercetin and promoting quercetin-derived metabolite accumulation. Mechanistically, ThWRKY75 directly bound the ThF3′H promoter and repressed its activity, and its higher expression in SW provides a regulatory basis for reduced quercetin-oriented flux. Moreover, an MYB-centered regulatory hub upstream of the ThCHI → ThF3H → ThFLS → ThF3′H branch further reinforced quercetin accumulation in SE through ecotype-biased expression of activators (e.g., ThMYB1 ) and repressors (e.g., ThMYB4 ). In summary, our study suggests that differential expression and regulation of the ThCHI→ThF3′H axis may be associated with