Flavonols are produced by the desaturation of dihydroflavanols, which is catalyzed by flavonol synthase (FLS). FLS belongs to the 2-oxoglutarate iron-dependent oxygenase family. The full-length cDNA and genomic DNA sequences of the FLS gene (designated as GbFLS) were isolated from Ginkgo biloba. The full-length cDNA of GbFLS contained a 1023-bp open reading frame encoding a 340-amino-acid protein. The GbFLS genomic DNA had three exons and two introns. The deduced GbFLS protein showed high identities with other plant FLSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbFLS at similar positions like other FLSs. GbFLS was found to be expressed in all tested tissues including roots, stems, leaves, and fruits. Expression profiling analyses revealed that GbFLS expression was induced by all of the six tested abiotic stresses, namely, UV-B, abscisic acid, cold, sucrose, salicylic acid, and ethephon, consistent with the in silico analysis results of the promoter region. The recombinant protein was successfully expressed in the E. coli strain BL21 (DE3) with a pET-28a vector. The in vitro enzyme activity assay by high performance liquid chromatography indicated that recombinant GbFLS protein could catalyze the formation of dihydrokaempferol to kaempferol and the conversion of kaempferol from naringenin, suggesting that GbFLS is a bifunctional enzyme within the flavonol biosynthetic pathway.

Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

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Flavonoids are secondary metabolites that contribute substantially to the quality of Ginkgo biloba. Plant flavonoid accumulation is controlled by transcriptional regulation of the genes that encode the biosynthetic enzymes, in which the R2R3-MYB transcription factor is a key factor. In this study, we describe the cloning and functional characterization of a R2R3-MYB transcription factor gene, GbMYBF2, isolated from G. biloba. GbMYBF2 encodes a protein belonging to a small subfamily of R2R3-MYB transcription factors. Comparative and bioinformatics analyses showed that GbMYBF2 is more closely related to the repressor R2R3-MYB subfamily involved in flavonoid biosynthesis. Tissue expression pattern analysis showed that GbMYBF2 was constitutively expressed in leaves, fruits, stems, and roots, wherein the level of transcription in the roots is significantly higher than that in the stems, leaves, and fruits. During G. biloba leaf growth, the transcription of GbMYBF2 is negatively correlated with the flavonoid content, suggesting that the GbMYBF2 gene is responsible for the repressed flavonoid biosynthesis. Transgenic Arabidopsis plants that overexpress GbMYBF2 exhibit an inhibition of flavonoid and anthocyanin biosynthesis compared with the untransformed Arabidopsis plants. In addition, the overexpression of GbMYBF2 in Arabidopsis clearly downregulates the expression of the structural genes that control the synthesis of flavonoids and anthocyanins. These findings suggest that GbMYBF2 may have a key role in repressing transcription in regulating the biosynthesis of flavonoids in G. biloba.