Functional evolution and diversification of CYP82D subfamily members have shaped flavonoid diversification in the genus Scutellaria.

Flavonoids, the largest class of polyphenols, exhibit substantial structural and functional diversity, yet their evolutionary diversification and specialized functions remain largely unexplored. The genus Scutellaria is notable for its rich flavonoid diversity, particularly of 6/8-hydroxylated variants biosynthesized by the cytochrome P450 subfamily CYP82D. Our study analyzes metabolic differences between Scutellaria baicalensis and Scutellaria barbata, and the results suggest that CYP82Ds have acquired a broad range of catalytic functions over their evolution. By integrating analyses of metabolic networks and gene evolution across 22 Scutellaria species, we rapidly identified 261 flavonoids and delineated five clades of CYP82Ds associated with various catalytic functions. This approach revealed a unique catalytic mode for 6/8-hydroxylation of flavanone substrates and the first instance of 7-O-demethylation of flavonoid substrates catalyzed by a cytochrome P450. Ancestral sequence reconstruction and functional validation demonstrated that gradual neofunctionalization of CYP82Ds has driven the chemical diversity of flavonoids in the genus Scutellaria throughout its evolutionary history. These findings enhance our understanding of flavonoid diversity, reveal the intricate roles of CYP82Ds in Scutellaria species, and highlight the extensive catalytic versatility of cytochrome P450 members within plant taxa.