Nitrogen-mediated regulation of the biosynthesis of furanocoumarin and functional characterization of AdEH genes in Angelica dahurica var. formosana

Nitrogen is a key environmental factor shaping the development and accumulation of bioactive compounds in medicinal plants. In Angelica dahurica var. formosana , furanocoumarins (FCs) are the principal active constituents, yet epoxide hydrolases (EHs) genes in their biosynthetic pathway remain uncharacterized，and the molecular mechanism by which nitrogen controls this pathway is also unclear. In this study, seedlings of A. dahurica var. formosana were subjected to five nitrogen levels. Analyses of phenotypes and the content of furanocoumarin showed that while phenotypic indices displayed an initial increase followed by a decrease, the total furanocoumarin content consistently declined with higher nitrogen. Additionally, the responses varied among different furanocoumarin derivatives. Transcriptome data showed that low-nitrogen stress substantially upregulated genes involved in phenylpropanoid and furanocoumarin biosynthesis, whereas medium-to-high nitrogen suppressed these pathways and induced the expression of genes involved in nitrogen assimilation. WGCNA analysis identified a Brown module that was significantly and positively correlated with the accumulation of multiple furanocoumarins, and two AdEHs candidate genes were selected from this module. Functional verification confirmed that both proteins catalyze the hydrolysis of oxypeucedanin and biacangelicol. Among them, AdEH1 demonstrates a higher affinity for biacangelicol, whereas AdEH2 exhibited a greater affinity for oxypeucedanin. Both AdEHs localize to the cytoplasm. qRT-PCR verified that low-nitrogen significantly induces the expression of both AdEHs . In summary, nitrogen deficiency redirects metabolic resources toward furanocoumarin synthesis in A. dahurica var. formosana is, underscoring the importance of rational nitrogen fertilizer management. This work also first identifies and functionally characterizes two EHs in the FCs biosynthetic pathway.