Convergent recruitment of 5′-hydroxylase activities by CYP75B flavonoid B-ring hydroxylases for tricin biosynthesis in Medicago legumes

• Tricin (3′,5′-dimethoxylated flavone) is a predominant flavonoid amongst monocots but occurs only in isolated and unrelated dicot lineages. Although tricin biosynthesis has been intensively studied in monocots, it has remained largely elusive in tricin-accumulating dicots. • We investigated a subgroup of cytochrome P450 (CYP) 75B subfamily flavonoid B-ring hydroxylases (FBHs) from two tricin-accumulating legumes, Medicago truncatula and alfalfa (Medicago sativa), by phylogenetic, molecular, biochemical and mutant analyses. • Five Medicago cytochrome P450 CYP75B FBHs are phylogenetically distant from other legume CYP75B members. Among them, MtFBH-4, MsFBH-4 and MsFBH-10 were expressed in tricin-accumulating vegetative tissues. In vitro and in planta analyses demonstrated that these proteins catalyze 3′- and 5′-hydroxylations critical to tricin biosynthesis. A key amino acid polymorphism, T492G, at their substrate recognition site 6 domain is required for the novel 50-hydroxylation activities. Medicago truncatula mtfbh-4 mutants were tricindeficient, indicating that MtFBH-4 is indispensable for tricin biosynthesis. • Our results revealed that these Medicago legumes had acquired the tricin pathway through molecular evolution of CYP75B FBHs subsequent to speciation from other nontricin-accumulating legumes. Moreover, their evolution is independent of that of grass-specific CYP75B apigenin 3′-hydroxylases/chrysoeriol 5′-hydroxylases dedicated to tricin production and Asteraceae CYP75B flavonoid 3′,5′-hydroxylases catalyzing the production of delphinidinbased pigments.