title: Completion of Tricin Biosynthesis Pathway in Rice: Cytochrome P450 75B4 Is a Unique Chrysoeriol 5'-Hydroxylase.
abstract: Flavones are ubiquitously accumulated in land plants, but their biosynthesis in monocots remained largely elusive until recent years. Recently, we demonstrated that the rice (Oryza sativa) cytochrome P450 enzymes CYP93G1 and CYP93G2 channel flavanones en route to flavone O-linked conjugates and C-glycosides, respectively. In tricin, the 3',5'-dimethoxyflavone nucleus is formed before O-linked conjugations. Previously, flavonoid 3',5'-hydroxylases belonging to the CYP75A subfamily were believed to generate tricetin from apigenin for 3',5'-O-methylation to form tricin. However, we report here that CYP75B4 a unique flavonoid B-ring hydroxylase indispensable for tricin formation in rice. A CYP75B4 knockout mutant is tricin deficient, with unusual accumulation of chrysoeriol (a 3'-methoxylated flavone). CYP75B4 functions as a bona fide flavonoid 3'-hydroxylase by restoring the accumulation of 3'-hydroxylated flavonoids in Arabidopsis (Arabidopsis thaliana) transparent testa7 mutants and catalyzing in vitro 3'-hydroxylation of different flavonoids. In addition, overexpression of both CYP75B4 and CYP93G1 (a flavone synthase II) in Arabidopsis resulted in tricin accumulation. Specific 5'-hydroxylation of chrysoeriol to selgin by CYP75B4 was further demonstrated in vitro. The reaction steps leading to tricin biosynthesis are then reconstructed as naringenin → apigenin → luteolin → chrysoeriol → selgin → tricin. Hence, chrysoeriol, instead of tricetin, is an intermediate in tricin biosynthesis. CYP75B4 homologous sequences are highly conserved in Poaceae, and they are phylogenetically distinct from the canonical CYP75B flavonoid 3'-hydroxylase sequences. Recruitment of chrysoeriol-specific 5'-hydroxylase activity by an ancestral CYP75B sequence may represent a key event leading to the prevalence of tricin-derived metabolites in grasses and other monocots today.
authors.affiliation: School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, China (P.Y.L., C.L.); andInstitute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China (H.L.).
authors.forename: Pui Ying
authors.identifier: http://orcid.org/0000-0001-5025-1308
authors.initials: PY
authors.lastname: Lam
authors.affiliation: School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, China (P.Y.L., C.L.); andInstitute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China (H.L.).
authors.forename: Hongjia
authors.identifier: http://orcid.org/0000-0003-3102-0082
authors.initials: H
authors.lastname: Liu
authors.affiliation: School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, China (P.Y.L., C.L.); andInstitute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China (H.L.) clivelo@hku.hk.
authors.forename: Clive
authors.identifier: http://orcid.org/0000-0002-1995-3843
authors.initials: C
authors.lastname: Lo
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D047309:Flavones / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis*; D055786:Gene Knockout Techniques; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D047309:Flavones; D005419:Flavonoids; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C017769:tricin; D006899:Mixed Function Oxygenases; C007054:chrysoeriol