Functional characterization of flavonoid/stilbene modifying enzymes in rice, medicago legumes and sorghum

Abstract

Flavone C-glycosides and tricin-type metabolites (both soluble and lignin-bound) are two predominant types of specialized metabolites that accumulate in vegetative tissues of cereal crops. In rice (Oryza sativa), two flavone synthases, OsF2H and OsFNSII, were reported to direct flavanones towards independent pathways that form flavone C-glycosides and tricin-type metabolites, respectively. It was also demonstrated that completion of tricin biosynthesis pathway requires a cytochrome-P450 enzyme, CYP75B4, as a chrysoeriol 5´-hydroxylase while such activity is absent in its homolog CYP75B3. Their precise roles in the biosynthesis of flavone C-glycosides and lignin-bound tricin have remained unknown. Examination of their expression pattern and biochemical analyses of rice mutants revealed that CYP75B3 is the sole F3´H in flavone C-glycosides biosynthesis whereas CYP75B4 alone provides sufficient 3´,5´-hydroxylation activities for tricin-lignin deposition. Thus, CYP75B3 and CYP75B4 represent two different pathway-specific enzymes recruited together with OsF2H and OsFNSII, respectively. Phylogenetic and in vitro analyses further suggested that these enzymes are highly conserved amongst grasses. Tricin is a predominant flavonoid amongst monocots but occurs largely in unrelated dicots. Although tricin biosynthesis has been intensively studied in Poaceae, it remained largely elusive in tricin-accumulating dicots. The roles of a subgroup of CYP75B subfamily flavonoid B-ring hydroxylases (FBHs) from two tricin-accumulating legumes, Medicago truncatula and alfalfa (M. sativa), were investigated to better understand how they recruited tricin biosynthesis. Five Medicago cytochrome P450 CYP75B FBHs were found to be phylogenetically distant from other legume CYP75B members. Among them, MtFBH-4, MsFBH-4 and MsFBH-10 are 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 5´-hydroxylation activities. M. truncatula mtfbh-4 mutants were tricin-deficient, indicating that MtFBH-4 is indispensable for tricin biosynthesis. This study revealed that these Medicago legumes had acquired the tricin pathway through molecular evolution of CYP75B FBHs after speciation from other non-tricin-accumulating legumes. Moreover, their evolution is independent from that of grass-specific CYP75B apigenin 3´-hydroxylases/chrysoeriol 5´-hydroxylases for tricin production and Asteraceae CYP75B flavonoid 3´,5´-hydroxylases catalyzing the production of delphinidin-based pigments. Finally, this study examined the role of an O-methyltransferase isolated from Sorghum bicolor in stilbene biosynthesis. While flavonoids are ubiquitous amongst terrestrial plants, stilbenes have only been identified in some distantly-related lineages like grapevines, peanut, and sorghum. Stilbene biosynthesis is thus likely a consequence of convergent evolution. Sorghum was previously demonstrated to accumulate resveratrol when challenged by fungal pathogens. This study further revealed that some sorghum cultivars accumulate two methoxylated analogs of resveratrol, namely pinostilbene and pterostilbene, when infected by a pathogenic fungus Colletotrichum sublineola. In vitro, in planta and proteomics analyses showed that a pathogen-inducible O-methyltransferase (SbSOMT) is co-expressed with stilbene synthase for pterostilbene biosynthesis in response to C. sublineola infection. Under in vitro conditions, spore germination and growth of C. sublineola were inhibited by pterostilbene in a dose-dependent manner. SbSOMT was found to be phylogenetically distant from stilbene O-methyltransferases previously identified in other stilbene-accumulating plants, strongly suggesting that sorghum independently recruited pterostilbene biosynthesis.