Complex coumarins (CCs) represent characteristic metabolites found in Apiaceae plants, possessing significant medical value. Their essential functional role is likely as protectants against pathogens and regulators responding to environmental stimuli. Utilizing genomes and transcriptomes from 34 Apiaceae plants, including our recently sequenced Peucedanum praeruptorum, we conduct comprehensive phylogenetic analyses to reconstruct the detailed evolutionary process of the CC biosynthetic pathway in Apiaceae. Our results show that three key enzymes – p-coumaroyl CoA 2’-hydroxylase (C2’H), C-prenyltransferase (C-PT), and cyclase – originated successively at different evolutionary nodes within Apiaceae through various means of gene duplications: ectopic and tandem duplications. Neofunctionalization endows these enzymes with novel functions necessary for CC biosynthesis, thus completing the pathway. Candidate genes are cloned for heterologous expression and subjected to in vitro enzymatic assays to test our hypothesis regarding the origins of the key enzymes, and the results precisely validate our evolutionary inferences. Among the three enzymes, C-PTs are likely the primary determinant of the structural diversity of CCs (linear/angular), due to divergent activities evolved to target different positions (C-6 or C-8) of umbelliferone. A key amino acid variation (Ala161/Thr161) is identified and proven to play a crucial role in the alteration of enzymatic activity, possibly resulting in distinct binding forms between enzymes and substrates, thereby leading to different products. In conclusion, this study provides a detailed trajectory for the establishment and evolution of the CC biosynthetic pathway in Apiaceae. It explains why only a portion, not all, of Apiaceae plants can produce CCs and reveals the mechanisms of CC structural diversity among different Apiaceae plants.

Corydalis yanhusuoW.T. Wang is a classic herb that is frequently used in traditional Chinese medicine and is efficacious in promoting blood circulation, enhancing energy, and relieving pain. Benzylisoquinoline alkaloids (BIAs) are the main bioactive ingredients inCorydalis yanhusuo. However, few studies have investigated the BIA biosynthetic pathway inC. yanhusuo, and the biosynthetic pathway of species-specific chemicals such as tetrahydropalmatine remains unclear. We performed full-length transcriptomic and metabolomic analyses to identify candidate genes that might be involved in BIA biosynthesis and identified a total of 101 full-length transcripts and 19 metabolites involved in the BIA biosynthetic pathway. Moreover, the contents of 19 representative BIAs inC. yanhusuowere quantified by classical targeted metabolomic approaches. Their accumulation in the tuber was consistent with the expression patterns of identified BIA biosynthetic genes in tubers and leaves, which reinforces the validity and reliability of the analyses. Full-length genes with similar expression or enrichment patterns were identified, and a complete BIA biosynthesis pathway inC. yanhusuowas constructed according to these findings. Phylogenetic analysis revealed a total of ten enzymes that may possess columbamine-O-methyltransferase activity, which is the final step for tetrahydropalmatine synthesis. Our results span the whole BIA biosynthetic pathway inC. yanhusuo. Our full-length transcriptomic data will enable further molecular cloning of enzymes and activity validation studies.