Li LinLing

Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

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.

Characterization of a cinnamoyl-CoA reductase gene in Ginkgo biloba : Effects on lignification and environmental stresses

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyzes key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. The full-length cDNA of GbCCR is 1178 bp and contains a 972 bp open reading frame (ORF) encoding a 323 amino acid protein. The deduced GbCCR protein showed high identities with other plant CCRs, and had closer relationship with Picea abies , sharing 56.3% homology. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Phylogenetic tree analysis revealed that GbCCR shared the same ancestor with other CCRs, but the divergence time is early. Southern blot analysis indicated that GbCCR belonged to a multi-gene family. The expression analysis by quantitative real-time polymerase chain reaction (QRT-PCR showed that GbCCR was seen in a tissue specific manner in Ginkgo biloba ; it had the highest expression in injured stems, and a high expression in four years old stems, while it had the lowest in endosperm. GbCCR was also found to be significantly up-regulated by gibberellin (GA), but the expression was weakly induced by Agrobacterium treatment. QRT-PCR analysis showed that GbCCR activity correlated with changes in transcription level of the GbCCR gene, and GbCCR activity was also positively correlated with total lignin accumulation in developments of Ginkgo stem. In light of these properties and expression pattern, we suggested that the corresponding enzyme is probably involved in constitutive lignification and defense. Key words : Ginkgo biloba L., GbCCR, gene expression, lignification, defense.