Molecular identification of flavanone 3-hydroxylase and flavonol synthase from Cudrania tricuspidata

Cudrania tricuspidata (Moraceae) serves as a viable feed source for oligophagous silkworms in silk production. This plant also exhibits a wide range of biological activities and accumulates a rich diversity of flavonoids, including flavonols. Flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS) are key rate-limiting enzymes in flavonol biosynthesis. F3H catalyzes the conversion of flavanones into dihydroflavonols, and FLS subsequently converts dihydroflavonols into flavonols. However, F3H and FLS have not yet been identified in C. tricuspidata. In this study, we identified two F3H enzymes (CtrF3H1 and CtrF3H2) and one FLS enzyme (CtrFLS) from C. tricuspidata and functionally characterized them using in vitro and in vivo assays. Enzymatic assays demonstrated that CtrF3H1 and CtrF3H2 exhibit flavanone 3-hydroxylase activity toward flavanones, with CtrF3H2 displaying broader substrate-catalyzed activity and is capable of catalyzing five flavanones (naringenin, eriodictyol, hesperetin, liquiritigenin, and pinocembrin). Enzymatic kinetic analysis revealed that CtrF3H1 exhibits higher catalytic efficiency toward naringenin. Additionally, site-directed mutagenesis experiments have elucidated the molecular mechanisms underlying the differences in substrate selectivity between CtrF3H1 and CtrF3H2. Moreover, CtrF3H1 can increase the content of flavones and flavonols in Arabidopsis thaliana CtrF3H1/tt6, indicating that CtrF3H1 exhibits a degree of functional diversity in plants. CtrFLS can convert dihydroflavonol to flavonol in vitro, and CtrFLS overexpression in the fls mutant leads to increased levels of the flavonols kaempferol and quercetin. The discovery of CtrF3H and CtrFLS enhances our understanding of flavonoids biosynthesis and diversity in C. tricuspidata.