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.

Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids

“…Before catalytic assay, recombinant proteins were prepared and purified. Subsequently, the purified proteins were subjected to the biochemical analysis using DHK, DHQ, or DHM as substrate in the presence of NADPH, respectively, and the reaction products were analyzed by HPLC in comparison to authentic standards, relative retention time and UV spectra (Cheng et al, 2013). As shown in Figure 6 , formation of leucodelphinidin (LEUD) was observed when using DHM as substrate in the in vitro reaction system with FhDFR1, FhDFR2, or FhDFR3 proteins, whereas only FhDFR2 could convert DHQ to LEUC because of relative lower catalytic efficiencies.…”

“…Substrate specificities of FhDFR proteins were carried out as described by Cheng et al (2013). Shortly, DHK, DHM, and DHQ bought from Sigma were dissolved in methanol at 10 mg/mL.…”

“…Mutations in DFR may explain the color transition in some species such as Andean genus Iochroma (Des Marais and Rausher, 2008; Smith and Rausher, 2011; Smith et al, 2013). To our knowledge, various DFR genes have so far been isolated from a wide range of plant species, such as Lotus japonicus (Shimada et al, 2005), Camellia sinensis (Singh et al, 2009), Medicago truncatula (Xie et al, 2004), Malus domestica (Fischer et al, 2003), Pyrus communis (Fischer et al, 2003), Citrus sinensis (Piero et al, 2006), Ipomoea batatas Lam (Wang et al, 2013), Ginkgo biloba (Cheng et al, 2013), and Populus trichocarpa (Huang et al, 2012). However, most of the genes were isolated from dicot plants and few studies have focused on the functionally divergence of the members in the DFR gene family, especially in monocot species.…”

Comparative Metabolomic Analysis Reveals Distinct Flavonoid Biosynthesis Regulation for Leaf Color Development of Cymbidium sinense ‘Red Sun’

“…The enzyme activity of DFR decreased at first and then increased, while the activity of ANS increased at first and then decreased, but the coding genes expression level of the two enzymes was significantly down-regulated. DFR from different plants has specific substrates biases for DHK, DHQ and DHM [6,31], and the downstream DFRs and ANSs is necessary for large sum of anthocyanin accumulation in Phalaenopsis [32,33]. These unstable anthocyanins eventually went through the action of UFGT to form stable anthocyanins [34].…”

Association of New Mutant Allele, w4‐nw, at W4 Locus with Near‐White Flower Color in Soybean

“…GenBank accession numbers of amino acid sequences of DFRs are as follows: Arabidopsis thaliana , BAA85261; Clitoria ternatea , BAF49294; Crataegus monogyna , AAX16491; Fagopyrum esculentum , ACZ48698; Glycine max , ABM64803; Lotus japonicus , BAE19949; Medicago truncatula , XP_013466134; Prunus avium , AJO67977; and Vitis vinifera , NP_001268144. Boxed region denotes a putative NADPH‐binding region in the DFR proteins (Hua et al, 2013). Thick black boxes indicate an additional 18 amino acid residues and deletion of 11 amino acid residues in w4‐nw‐a and w4‐nw‐b , respectively.…”