Enzymatic innovations in <i>Angelica pubescens</i> reveal dual coumarin biosynthetic pathways driving metabolic diversification

Coumarins are structurally diverse phenylpropanoid derivatives with ecological and pharmacological significance, yet the biosynthetic logic underlying their diversification remains incompletely understood in non-model medicinal plants. Angelica pubescens (Apiaceae), widely used in traditional Chinese medicine, accumulates a rich repertoire of furanocoumarins and dihydrofuranocoumarins, making it an ideal system to investigate this metabolic complexity. Here, we combined chromosome-level genome assembly, transcriptome and metabolite profiling, phylogenetics, and heterologous expression assays to dissect coumarin biosynthesis in A. pubescens. We identified two functionally specialized O-methyltransferases, ApOMT1 and ApOMT2, which catalyze regioselective methylation of xanthotoxol and bergaptol to yield the furanocoumarins xanthotoxin and bergapten. We also characterized ApCYP736A121, a cytochrome P450 enzyme that converts osthenol to the dihydrofuranocoumarin columbianetin via a previously unknown mechanism. Gene expression and metabolite accumulation patterns across tissues and developmental stages revealed functional partitioning among pathway branches. Phylogenetic and syntenic analyses indicated that ApOMT1 and ApOMT2 arose through subfunctionalization following gene duplication, whereas ApCYP736A121 evolved via neofunctionalization from a distantly related CYP736 ancestor. Together, our findings uncover dual biosynthetic routes to structurally distinct coumarins in A. pubescens and provide insights into the evolutionary mechanisms contributing to metabolic innovation in Apiaceae. This work lays a foundation for future efforts to engineer coumarin pathways and understand their ecological functions in medicinal plants.