Duplicated CYP71D enzymes promote metabolic innovation through regiospecific oxidation at a sesquiterpenoid biosynthetic locus in Medicago truncatula.

Sesquiterpenoid chemical diversity is frequently generated through cytochrome P450 (CYP)-mediated oxidation of terpene scaffolds. In Medicago truncatula, the sesquiterpene alcohol himachalol, produced by the terpene synthase MtTPS10, has been implicated in pathogen defense, yet the enzymes responsible for its downstream modification remain undefined. Here, we characterize a compact locus containing two sesquiterpene synthases and three CYP71D genes. Using heterologous expression, we show that two duplicated CYP71D enzymes catalyze regiospecific oxidations of himachalol. CYP71D81 hydroxylates the C4 position to produce himachal-2-en-4α,7β-diol, whereas CYP71D82 oxidizes C15 to generate 7β-hydroxyhimachal-2-en-15-al. Phylogenetic analysis indicates that these enzymes arose through duplication followed by functional divergence. Consistent with a shared role in stress-responsive metabolism, MtTPS10, CYP71D81, and CYP71D82 exhibit coordinated induction following methyl jasmonate and salicylic acid treatment. Together, these findings identify oxidative tailoring enzymes associated with MtTPS10-derived metabolism and demonstrate how duplication within a TPS-CYP locus promotes metabolic innovation by generating structurally distinct sesquiterpenoids from a common precursor.