Early sex determination of Ginkgo biloba based on the differences in the electrocatalytic performance of extracted peroxidase.

Ginkgo biloba is a dioecious plant. Male ginkgoes are mainly used in landscaping, while females are mainly used for fruit production. However, sex identification of ginkgo is a difficult task, especially at the seedling stage. In this work, we present for the first time the use of electrochemical techniques for the identification of ginkgo sex based on the differences in peroxides within male and female ginkgos. Graphene was used to concentrate peroxides in ginkgo extract, thereby improving electrochemical signal sensitivity. The electrochemical reduction of hydrogen peroxide catalyzed by peroxidase was used as a prob for sex determination in ginkgo. This electrochemical identification technique can be used not only for the analysis of adult ginkgo, but also successfully for the analysis of tissue culture seedlings and live seedlings. This electrochemical sensor has excellent discrimination ability due to the difference in peroxidase content in the leaves and petiole of ginkgo of different sexes. This electrochemical sensor allows for a rapid identification of the sex of ginkgo and has a very strong potential for field analysis.

Characterization of a novel levopimaradiene synthase gene responsible for the biosynthesis of terpene trilactones in Ginkgo biloba.

Terpene trilactones (TTLs) are the main medicinal compounds of Ginkgo biloba. Levopimaradiene synthase (LPS) is the crucial enzyme that catalyzes TTLs biosynthesis in G. biloba. In this study, a novel LPS gene (designated as GbLPS2) was cloned from G. biloba leaves. The open reading frame of GbLPS2 gene was 2520 bp in length, encoding a predicted polypeptide of 840 amino acids. Phylogenetic analysis revealed that the GbLPS2 was highly homologous with reported LPS proteins in other plants. On the basis of the genomic DNA (gDNA) template, a 4308 bp gDNA sequence of GbLPS2 and a 913 bp promoter sequence were amplified. Cis-acting elements in promoter analysis indicated that GbLPS2 could be regulated by methyl jasmonate (MeJA) and abscisic acid (ABA). Tissue-specific expression analysis revealed that GbLPS2 was mainly expressed in roots and ovulate strobilus. MeJA treatment could significantly induce the expression level of GbLPS2 and increase the content of TTLs. This study illustrates the structure and the tissue-specific expression pattern of GbLPS2 and demonstrates that exogenous hormones regulated the expression of GbLPS2 and TTL content in G. biloba. Our results provide a target gene for the enhancement of TTL content in G. biloba via genetic engineering.

Efficient removal of ginkgotoxin from Ginkgo biloba seed powder by combining endogenous enzymatic hydrolysis with resin adsorption.

BACKGROUND: Ginkgotoxin including 4'-O-methylpyridoxine (MPN) and MPN-5'-glucoside (MPNG) is responsible for Ginkgo seed food poisoning. The purpose of the work reported was to prepare detoxified Ginkgo seed powder and at the same time to retain the nutritional and functional components of Ginkgo seed powder to the maximum extent. RESULTS: Resin adsorption technology was firstly employed to remove ginkgotoxin from water extract of Ginkgo seed powder. Under optimal conditions, the adsorption efficiency of the optimal resin for MPN could reach 100%, and that for MPNG could only reach 85.4 ± 0.93%. Resin adsorption alone could not effectively remove MPN and MPNG simultaneously. Endogenous enzymatic hydrolysis was next attempted to transform MPNG to MPN. MPNG could be completely hydrolyzed to MPN by endogenous enzyme(s) at 40 °C and pH 5.0 in 180 min. Ginkgotoxin only in the form of MPN in the enzymatic hydrolysate was then adsorbed with resin and the conditions were statistically optimized. The adsorption efficiency of MPN reached 98.89 ± 0.99% under the optimized conditions. CONCLUSIONS: Removal of ginkgotoxin by combining endogenous enzymatic hydrolysis with resin adsorption could preserve the main nutritional and functional components of Ginkgo seed powder to the most extent, and did not change its main characteristics. The ginkgotoxin removal method developed in this work is a relatively simple and efficient approach. © 2020 Society of Chemical Industry.

Effect of Bacillus natto solid-state fermentation on the functional constituents and properties of Ginkgo seeds.

In the present investigation, fibrinolytic Ginkgo seeds were produced by solid-state fermentation (SSF) with Bacillus natto strains, and some parameters of the fermentation processes were investigated. Under optimal fermentation conditions, the fibrinolytic activity of Ginkgo seeds reached 3,682 ± 43 IU/g with the fermentation parameters of relative humidity 80%, initial water content 73%, fermentation temperature 38°C, inoculation volume 18%, and fermentation time 38 hr, respectively. The fermented Ginkgo seeds possessed a superior potential for the production of Nattokinase. What's more, the fermented Ginkgo seeds possessed higher total flavonoid and lower ginkgolic acids contents, which could enhance bioactivity and guarantee food safety. Sensory evaluations indicated that Ginkgo seeds produced by SSF could also be consumed as a kind of popular food. PRACTICAL APPLICATIONS: Fermented food is popular in countries. It can not only improve the sensory properties of the products, reduce undesirable constituents, and make nutrients easily absorbable, but also improve the nutritional properties. Ginkgo biloba L is one of the oldest species that has existed on earth for more than 200 million years. However, the application of Ginkgo seeds has been limited because of the ginkgolic acids. In a previous study, immobilized Bacillus natto acted upon Ginkgo seeds to enhance the bioactivity and safety of fermented Ginkgo seeds. However, separating the fermented Ginkgo seeds from the liquid needs a large amount of energy. The solid-state fermentation of Ginkgo seeds is a good choice to produce functional Ginkgo seed products.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and ß-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 ß-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/ß-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities.

Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

ß-Galactosidase from Ginkgo biloba seeds active against ß-galactose-containing N-glycans: purification and characterization.

In this study, we purified an acidic ß-galactosidase to homogeneity from Ginkgo biloba seeds (ß-Gal'ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified ß-Gal'ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, ß-Gal'ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that ß-Gal'ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing ß-galactosyl residues were used as substrates, ß-Gal'ase Gb-1 showed substantial activity for ß1-4 galactosyl residue and modest activity for ß1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of ß-Gal'ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.

Purification and substrate specificity of a Ginkgo biloba glycosidase active in ß-1,2-xylosidic linkage in plant complex type N-glycans.

The ß-xylosidase, which is active against plant complex type N-glycans, was purified to homogeneity from Ginkgo biloba seeds. The N-terminal amino acid sequence, G-S-A-A-G-N-R-, of the Ginkgo ß-xylosidase (ß-Xyl'ase Gb) was consistent with the deduced internal amino acid sequence of an Arabidopsis ß-xylosidase (AtBXL1). ß-Xyl'ase Gb hydrolyzed the ß1-2 xylosyl residue from Xylß1-2Manß1-4GlcNAcß1-4GlcNAc-PA and Xylß1-2Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA, but not that from Manα1-6(Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA.

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.

Expression patterns of an isoflavone reductase-like gene and its possible roles in secondary metabolism in Ginkgo biloba.

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.

Distinct expression patterns of two Ginkgo biloba 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase/isopentenyl diphospahte synthase (HDR/IDS) promoters in Arabidopsis model.

1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) or isopentenyl diphosphate synthase (IDS) is an enzyme at the final step of the MEP pathway. The multi-copy nature of IDS gene in a gymnosperm Ginkgo biloba is known. To evaluate the function of each isogene, the roles of the promoters were examined in Arabidopsis model. Among the promoters of GbIDS series, about 1.3 kb of GbIDS1pro and 1.5 kb of GbIDS2pro were cloned and fused with GUS. The GbIDS1pro::GUS was introduced into Arabidopsis to show GUS expression in most organs except for roots, petals, and stamina, whereas the GbIDS2pro::GUS was expressed only in the young leaves, internodes where the flower and shoot branched, and notably in primary root junction. This pattern of GUS expression correlated with high transcript level of GbIDS2 compared to that of GbIDS1 in Ginkgo roots. Methyl jasmonate (MeJA) treatment resulted in down-regulated GbIDS1pro activity in Arabidopsis leaves and upregulated GbIDS2pro activity in roots. The same pattern of gene regulation in roots was also seen upon treatments of gibberellins, abscisic acid, and indole butyric acid.

Developmental pattern of Ginkgo biloba levopimaradiene synthase (GbLPS) as probed by promoter analysis in Arabidopsis thaliana.

Levopimaradiene synthase (GbLPS) of Ginkgo biloba catalyzes the first committed step in ginkgolide biosynthesis by converting geranylgeranyl diphosphate into levopimaradiene, which subsequently undergoes complex oxidation step and rearrangement of carbon skeleton, leading to formation of ginkgolides. To assess the organ-specificity and developmental characteristics of GbLPS expression, the GbLPS promoter-driven GUS expression in transgenic Arabidopsis was studied. Histological analysis of the transgenic Arabidopsis plant showed that the GUS accumulation was mainly localized in the epidermis of leaves, phloem of the shoots, ovaries and stamens of flowers, and vasculature of roots. These observations correlate with the occurrence of LPS transcripts in roots and male strobili of G. biloba. Treatment of methyl jasmonate on the transformant exhibited significant upregulation of the reporter gene in the roots with little change in leaves and flowers. The present findings support biosynthesis of ginkgolide in the roots of Ginkgo plant and suggest translocation occurs through the phloem.

Isolation, characterization, and function analysis of a flavonol synthase gene from Ginkgo biloba.

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.

Season- and age-associated telomerase activity in Ginkgo biloba L.

Telomeres have lately received considerable attention in the development of broad-leaved tree species. In order to determine tissue-, sex-, season- and age-specific changes in telomerase activity in ginkgo trees, analyses of the telomerase repeat amplification protocol were carried out. In all of the tissues detected (embryonal callus, microspore tissues and leaves) telomerase activity was found, with differences between these activities statistically significant (P < 0.05). The highest telomerase activity was found in embryonal callus, suggesting that ginkgo trees have tissue-specific telomerase activity. Tissues containing high levels of dividing cells also have high levels of telomerase activity. No significant difference of telomerase activity was found between male and female trees (P > 0.05). In the annual development cycle, the highest telomerase activity was found in April and a decreasing trend over time in the four age groups studied: 10, 20, 70 and 700 year. The most obvious decline appeared in trees of the 700 year old group, suggesting that ginkgo trees have season-specific telomerase activities and trees of various ages react differently to seasonal changes. The mean annual telomerase activity showed a regular decreasing trend in all leaf samples analyzed from 10 to 700 year old ginkgo trees. We conclude that maintenance of telomere length depends on season- and age- associated telomerase activity. An optimal telomere length is regulated and maintained by telomerase in Ginkgo biloba L.

Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba.

Chalcone isomerase (CHI, EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthesis pathway catalyzing the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones. In this investigation, both the cDNA sequence and the genomic sequence encoding the chalcone isomerase from Ginkgo biloba L. (designated as GbCHI) were isolated from the leaves. The GbCHI gene contained two introns and three extrons and encoded a peptide of 244 amino acids with a predicted molecular mass of 26.29 kDa and a pI of 7.76. RQPCR showed that GbCHI was expressed in a tissue-specific manner in G. biloba. Expression of GbCHI was also up-regulated by UV-B irradiation or treatment with 5-aminolevulinic acid or three plant growth regulator-ethylene, abscisic acid, and chlormequat-and these effects were consistent with analysis of the GbCHI promoter region. The recombinant protein was successfully expressed in an E.coli strain with the pET-28a vector. In vitro enzyme activity, assayed by HPLC, indicated that recombinant GbCHI protein could catalyze the formation of naringenin from 6'-hydroxychalcone. RQPCR analysis showed that CHI activity correlated with changes in transcription level of the CHI gene, GbCHI activity was also positively correlated with total flavonoid levels in ginkgo leaves, suggesting CHI as a key gene regulating flavonoid accumulation in ginkgo leaves.

Effects of elevated O3 and/or elevated CO2 on lipid peroxidation and antioxidant systems in Ginkgo biloba leaves.

Four-year-old seedlings of Ginkgo biloba were exposed to elevated O(3), elevated CO(2) and elevated O(3) plus elevated CO(2) in open-top chambers (OTCs) to study the responses of antioxidant system in Ginkgo biloba leaves. No significant changes in reactive oxygen production and scavenging systems were detected in seedlings exposed to high CO(2). Significant increase in H(2)O(2) and MDA content were induced by elevated O(3). The ascorbate content and antioxidative enzymes activity were increased significantly by exposure to high O(3) as well. But the promoted ability in scavenging did not prevent the increase in H(2)O(2) content and cell membrane lipid peroxidation. The increase was mitigated by high CO(2) in the combined exposure, but the effect was hardly significant.

The presence of sinapyl lignin in Ginkgo biloba cell cultures changes our views of the evolution of lignin biosynthesis.

Suspension cell cultures (SCCs) from one of the oldest seed plants, Ginkgo biloba, show unpredictable alterations in the nature of the lignins, such as is the recruitment of sinapyl alcohol for lignin biosynthesis, compared with the woody tissues of the same species, which lack syringyl (S) lignins. These results show that, in this gymnosperm, the genes involved in sinapyl alcohol biosynthesis are latent and that their regulatory regions respond, by initiating gene expression, to the developmental signals and the environmental clues, which condition its in vitro culture. G. biloba SCCs not only synthesize S lignins but also their extracellular proteome contains both class III peroxidases capable of oxidizing sinapyl alcohol and enzymes involved in H2O2 production, observation which suggests that the peroxidase branch for the oxidative coupling of sinapyl alcohol units into lignins is operative. The incomplete knowledge of the G. biloba peroxidase-encoding genes led us to purify, characterize and partially sequence the peroxidase responsible for monolignol oxidation. When the major peroxidase from G. biloba SCCs (GbPrx) was purified to homogeneity, it showed absorption maxima in the visible region at 414 (Soret band), and at 543 and 570 nm, which calls to mind those shown by low-spin ferric peroxidases. However, the results also showed that the paraperoxidase-like character of GbPrx is not an obstacle for oxidizing the three monolignols compared with high-spin ferric peroxidases. Taken together, these results mean that the time at which the evolutionary gain of the segment of the route that leads to the biosynthesis of S lignins took place in seed plants needs to be revised.

Molecular cloning and function analysis of an anthocyanidin synthase gene from Ginkgo biloba, and its expression in abiotic stress responses.

Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA and genomic DNA sequences of ANS gene (designated as GbANS) were isolated from Ginkgo biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA analysis showed that GbANS gene had three exons and two introns. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbANS at the similar positions like other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The recombinant protein was successfully expressed in E. coli strain with pET28a vector. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.

The Pdx1 family is structurally and functionally conserved between Arabidopsis thaliana and Ginkgo biloba.

Vitamin B6 is one of the most important compounds in living organisms, and its biosynthesis has only recently been understood. Because it is required for more than 100 biochemical reactions, lack of the vitamin is fatal. This is of special importance to mammals and humans, which cannot biosynthesize the vitamin and thus depend on its external uptake. Here we describe the cloning of a vitamin B6 biosynthetic gene GbPDX1 from Ginkgo biloba. The gene is expressed in seeds, leaf and trunk tissue. Using yeast 2-hybrid and pull-down assays, we show that the protein can interact with itself and with members of Arabidopsis thaliana AtPDX1 and AtPDX2 families. Furthermore, we prove the function of GbPDX1 in vitamin B6 biosynthesis by complementation of an Arabidopsis AtPDX1.3 mutant rsr4-1, at the phenotypical level and increasing vitamin B6 levels caused by ectopic GbPDX1 expression in the mutant background. Overall, this study provides a first description of Ginkgo vitamin B6 metabolism, and demonstrates a high degree of conservation between Ginkgo and Arabidopsis.