title: Molecular and Biochemical Analysis of Two Rice Flavonoid 3'-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain.
abstract: Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3',4'-dihydroxylated leucocyanidin. 3'-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3'H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3'H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.
authors.affiliation: National Institute of Agricultural Science, Rural Development Administration, JeonJu 54874, Korea. psk2779@korea.kr.
authors.forename: Sangkyu
authors.initials: S
authors.lastname: Park
authors.affiliation: National Institute of Agricultural Science, Rural Development Administration, JeonJu 54874, Korea. nancmj90@naver.com.
authors.forename: Min Ji
authors.initials: MJ
authors.lastname: Choi
authors.affiliation: National Institute of Agricultural Science, Rural Development Administration, JeonJu 54874, Korea. jy0820@korea.kr.
authors.forename: Jong Yeol
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Korea. kjkpj@inu.ac.kr.
authors.forename: Jae Kwang
authors.initials: JK
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Korea. sunhwa@khu.ac.kr.
authors.forename: Sun-Hwa
authors.initials: SH
authors.lastname: Ha
authors.affiliation: National Institute of Agricultural Science, Rural Development Administration, JeonJu 54874, Korea. limsh2@korea.kr.
authors.forename: Sun-Hyung
authors.initials: SH
authors.lastname: Lim
mesh_terms: D019943:Amino Acid Substitution; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D005419:Flavonoids / Q000096:biosynthesis*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006900:Hydroxylation; D012275:Oryza / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000254:growth & development; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D005419:Flavonoids; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C038888:flavonoid 3'-hydroxylase
keywords: anthocyanins; flavonoid 3′-hydroxylase; flavonoids; leucocyanidins; pigmented rice; proanthocyanidins