Isofraxidin Biosynthesis in <em>Chloranthus</em>: 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 dihydroxycoumarin in this clade, exhibits significant pharmaceutical potential. However, its biosynthetic basis remains uncharacterized. Here, we assembled a high-quality triploid genome of Chloranthus erectus (8.57 Gb, contig N50=8.76 Mb) to explore the evolution of defensive metabolism. Genomic analysis revealed an ancient WGD event and expanded gene families associated with pathogen resistance. Metabolomic analysis identified at least 49 coumarin compounds in Chloranthus plants, significantly exceeding previous records. Integrated omics revealed 267 candidate biosynthetic genes across 9 enzyme families governing isofraxidin biosynthesis. PAL-4CL established the phenylpropanoid backbone, amplified COSY genes correlated with umbelliferone accumulation, and CYP71/OMT catalyzed terminal modifications. This study deciphers the chemical defense evolution in early angiosperms and enables engineering of plant-derived antimicrobials.