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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.

Key Message: Our results showed that GbIRL1 belongs to the PCBER protein family. Besides, IRL1 gene was a novel gene regulating lignin change and also effecting the accumulation of flavonoids in Ginkgo. A cDNA encoding the IFR-like protein was isolated from the leaves of Ginkgo biloba L., designated as GbIRL1 (Accession no. KC244282). The cDNA of GbIRL1 was 1,203 bp containing a 921 bp open reading frame encoding a polypeptide of 306 amino acids. Comparative and bioinformatic analyses revealed that GbIRL1 showed extensive homology with IFLs from other gymnosperm species. Phylogenetic tree analysis revealed that GbIRL1 shared the same ancestor in evolution with other PCBERs protein and had a further relationship with other gymnosperm species. The recombinant protein was successfully expressed in E. coli strain with pET-28a vector. The vitro enzyme activity assay by HPLC indicated that recombinant GbIRL1 protein could catalyze the formation the TDDC, IDDDC from DDDC, DDC. Tissue expression pattern analysis showed that GbIRL1 was constitutively expressed in stem and roots, especially in the parts of the pest and fungal infection, with the lower expression being found in 1- or 2-year old stem. The increased expression of GbIRL1 was detected when the seedlings were treated with Ultraviole-B, ALA, wounding and ethephon, abscisic acid, salicylic acid. Correlation analysis between GbIRL1 activity and flavonoid accumulation during Ginkgo leaf growth indicated that GbIRL1 might be the rate-limiting enzyme in the biosynthesis pathway of flavonoids in Ginkgo leaves. Results of RT-PCR analysis showed that the transcription level of change in GbIRL1 power correlated with flavonoid contents, suggesting IRL1 gene as a novel gene regulating lignin change and also effecting the accumulation of flavonoids in Ginkgo.