title: Molecular cloning and characterization of three genes encoding dihydroflavonol-4-reductase from Ginkgo biloba in anthocyanin biosynthetic pathway.
abstract: Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.
authors.affiliation: Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratories, Hubei Huanggang, China ; College of Chemistry and Life Science, Huanggang Normal University, Huanggang, China ; College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Hua
authors.forename: Li
authors.initials: L
authors.lastname: Linling
authors.forename: Cheng
authors.initials: C
authors.lastname: Shuiyuan
authors.forename: Cao
authors.initials: C
authors.lastname: Fuliang
authors.forename: Xu
authors.initials: X
authors.lastname: Feng
authors.forename: Yuan
authors.initials: Y
authors.lastname: Honghui
authors.forename: Wu
authors.initials: W
authors.lastname: Conghua
mesh_terms: D000429:Alcohol Oxidoreductases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000096:biosynthesis*; D001483:Base Sequence; D055162:Biocatalysis; D053898:Biosynthetic Pathways / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D005419:Flavonoids / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000528:radiation effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000528:radiation effects; D020441:Ginkgo biloba / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D007527:Isoenzymes / Q000145:classification / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011794:Quercetin / Q000031:analogs & derivatives / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
chemical_list: D000872:Anthocyanins; D005419:Flavonoids; D007527:Isoenzymes; D010937:Plant Growth Regulators; D010940:Plant Proteins; C080220:aromadedrin; D011794:Quercetin; C003377:taxifolin; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; C001532:leucocyanidin