title: Cloning and Characterization of a Flavonoid 3'-Hydroxylase Gene from Tea Plant (Camellia sinensis).
abstract: Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.
authors.affiliation: Horticulture, Northwest A&amp;F University, 3 Taicheng Road, Yangling 712100, China. zhoutianshan@nwsuaf.edu.cn.
authors.forename: Tian-Shan
authors.initials: TS
authors.lastname: Zhou
authors.affiliation: Conagen Inc., 15 DeAngelo Dr., Bedford, MA 01730, USA. rui.zhou@conagen-inc.com.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhou
authors.affiliation: Horticulture, Northwest A&amp;F University, 3 Taicheng Road, Yangling 712100, China. yyben@163.com.
authors.forename: You-Ben
authors.initials: YB
authors.lastname: Yu
authors.affiliation: Horticulture, Northwest A&amp;F University, 3 Taicheng Road, Yangling 712100, China. xiaoyao1583@gmail.com.
authors.forename: Yao
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Horticulture, Northwest A&amp;F University, 3 Taicheng Road, Yangling 712100, China. lidhua29@163.com.
authors.forename: Dong-Hua
authors.initials: DH
authors.lastname: Li
authors.affiliation: Horticulture, Northwest A&amp;F University, 3 Taicheng Road, Yangling 712100, China. xiaobin@nwsuaf.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Xiao
authors.affiliation: Conagen Inc., 15 DeAngelo Dr., Bedford, MA 01730, USA. Oliver.yu@conagen-inc.com.
authors.forename: Oliver
authors.initials: O
authors.lastname: Yu
authors.affiliation: Tea Research Institute, Chinese Academy of Agricultural Sciences, 9 Meiling South Road, Hangzhou 310008, China. yjyang@mail.tricaas.com.
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Yang
mesh_terms: D028241:Camellia sinensis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular / Q000379:methods*; D019295:Computational Biology / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis; D018525:Germination; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018520:Plant Shoots / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D012689:Sequence Homology, Nucleic Acid
chemical_list: D005419:Flavonoids; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C038888:flavonoid 3'-hydroxylase
keywords: Camellia sinensis; flavan 3-ols; flavonoid 3′-hydroxylase; heterologous expression; nitrogen depletion