Functional evolution and diversification of the CYP82D subfamily members shape 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 the 6/8-hydroxylated variants biosynthesized by the cytochrome P450 subfamily CYP82D. Our study analyzes metabolic differences between Scutellaria baicalensis and Scutellaria barbata, suggesting 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 rapid identified 261 flavonoids and delineated five clades associated with various catalytic functions of CYP82Ds. This approach uncovered a unique catalytic mode for 6/8-hydroxylated function under flavanone substrates and the first instance of 7-O-demethylation of flavonoid substrates catalyzed by cytochrome P450. Ancestral sequence reconstruction and functional validation demonstrated that gradual neofunctionalization of CYP82Ds has driven the chemical diversity of flavonoids in Scutellaria throughout its evolutionary history. Our study enhances the understanding of flavonoid diversity, elucidates the intricate roles of CYP82Ds in Scutellaria plants, and underscores the extensive catalytic versatility of cytochrome P450 members within plant taxa.