Transcriptional redirection of quercetin-oriented flavonoid biosynthesis underpins ecotype differentiation in Tetrastigma hemsleyanum

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 ecotype divergence. This axis may contribute to the enrichment of quercetin-derived flavonoids and is linked to the enhanced antioxidant and anti-inflammatory potential of SE materials. These findings provide potential targets for quality evaluation and breeding.