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.
Highlights
Flavonoids are a large family of plant secondary metabolites, that exist widely throughout the plant kingdom
Characterization of G. biloba DFR cDNA Clones By searching the blastx results from expressed sequence tags generated from a G. biloba cDNA library, we identified three different cDNA clones with high similarity to other DFRs, designated GbDFR1, GbDFR2 and GbDFR3
To examine if the GbDFRs belongs to a multigene family, aliquots of 20 mg genomic DNA were digested with Sma I, EcoR V, Sac I for GbDFR1, Kpn I, Pvu II, Xma I for GbDFR2 and DraI, Cla I and Hind III for GbDFR3
Summary
Flavonoids are a large family of plant secondary metabolites, that exist widely throughout the plant kingdom. Flavonoids are divided into several structural classes, including flavanones, isoflavonoids, flavonols, anthocyanins, catechins, and condensed tannins, and are abundant in fruits, leaves and flowers [1]. Over 9000 different flavonoid compounds have been identified [2], many of which are involved in biological processes such as pigmentation of flowers, auxin transport regulation, seed development, protection against UV-B damage, defense against pathogens and pests, and pollen viability [3,4,5]. Several flavonoids are active ingredients in herbal medicines and appear to confer health benefits to humans when consumed regularly [6]. Particular attention has been placed on the anthocyanins, catechins, and proanthocyanidins because of their antioxidant activities and their interactions with human health [6,7]. The biosynthetic pathway of flavonoids are well established in plants. Many flavonoid biosynthetic genes are induced under stress conditions and, flavonoid levels increase during exposure to biotic and abiotic stresses, such as wounding, drought, metal toxicity and nutrient deprivation [8,9]
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