Isofraxidin biosynthesis in Chloranthus: genomic insights into metabolic evolution of an early angiosperm phytoalexin

As an early-diverging angiosperm lineage, Chloranthaceae produces specialized coumarins with documented antimicrobial and anti-inflammatory activities, which contribute to its ecological success. Isofraxidin, the most representative simple coumarin in this clade, exhibits significant pharmaceutical potential. However, its biosynthetic basis remains uncharacterized. Here, we assembled a high-quality triploid genome of <i>Chloranthus</i> sp<i>icatus</i> (8.57 Gb, contig N50 = 8.76 Mb) to explore the evolution of defensive metabolism. Genomic analysis revealed an ancient whole-genome duplication event and expanded gene families associated with pathogen resistance. Metabolomic analysis identified at least 49 coumarin compounds in <i>Chloranthus</i> plants, significantly exceeding previous records. Integrated omics revealed 267 candidate biosynthetic genes across 9 enzyme families governing isofraxidin biosynthesis. Building on the upstream synthesis of the phenylpropanoid backbone, this study identifies amplified coumarin synthase (COSY) genes linked to umbelliferone accumulation, and specific CYP450s and O-methyltransferases catalyzing final structural modifications. This work elucidates the evolution of chemical defenses in early angiosperms and enables the engineering of plant-derived antimicrobials.