Systematic investigation and expression profiles of the GbR2R3-MYB transcription factor family in ginkgo (Ginkgo biloba L.).

As one of the largest families of transcription factors, the R2R3-MYB family plays a significant role in plant growth, development, and response to hormone and environmental stress. To explore its evolutionary mechanism and potential function in Ginkgo biloba, a gymnosperm of great economic and ecological value, we presented a comprehensive analysis of the R2R3-MYB genes in ginkgo. Sixty-nine GbR2R3-MYB genes were identified and these genes could be classified into 33 groups based on the characteristics of the amino acid sequence of the R2R3-MYB domain and gene structure. Syntenic analyses indicated that few tandem and segmental duplications possibly resulted in the contraction of the GbR2R3-MYB gene family. Based on the transcriptome data, expression profiles of eight different tissues and different developmental stages of leaf and kernel showed that GbR2R3-MYB genes had distinct temporal and spatial expression characteristics. Specific expression patterns of the sixteen GbR2R3-MYB genes were also identified in response to different abiotic stresses and hormonal exposures. Further investigation revealed that GbR2R3-MYB19 was located in the nucleus and possessed transcriptional activity, implying its potential roles in the regulation of multiple biological processes. Our findings provide a robust basis for future comprehensive evolutionary and functional analyses of GbR2R3-MYB genes in ginkgo.

Rapid Authentication of Ginkgo biloba Herbal Products Using the Recombinase Polymerase Amplification Assay.

Species adulteration in herbal products (HPs) exposes consumers to health risks. Chemical and morphological methods have their own deficiencies when dealing with the detection of species containing the same active compounds in HPs. In this study, we developed a rapid identification method using the recombinase polymerase amplification (RPA) assay to detect two species, Ginkgo biloba and Sophora japonica (as adulteration), in Ginkgo biloba HPs. Among 36 Ginkgo biloba HP samples, 34 were found to have Ginkgo biloba sequences, and 9 were found to have Sophora japonica sequences. During the authentication process, the RPA-LFS assay showed a higher specificity, sensitivity and efficiency than PCR-based methods. We initially applied the RPA-LSF technique to detect plant species in HPs, demonstrating that this assay can be developed into an efficient tool for the rapid on-site authentication of plant species in Ginkgo biloba HPs.

The presumed ginkgophyte Umaltolepis has seed-bearing structures resembling those of Peltaspermales and Umkomasiales.

The origins of the five groups of living seed plants, including the single relictual species Ginkgo biloba, are poorly understood, in large part because of very imperfect knowledge of extinct seed plant diversity. Here we describe well-preserved material from the Early Cretaceous of Mongolia of the previously enigmatic Mesozoic seed plant reproductive structure Umaltolepis, which has been presumed to be a ginkgophyte. Abundant new material shows that Umaltolepis is a seed-bearing cupule that was borne on a stalk at the tip of a short shoot. Each cupule is umbrella-like with a central column that bears a thick, resinous, four-lobed outer covering, which opens from below. Four, pendulous, winged seeds are attached to the upper part of the column and are enclosed by the cupule. Evidence from morphology, anatomy, and field association suggests that the short shoots bore simple, elongate Pseudotorellia leaves that have similar venation and resin ducts to leaves of living GinkgoUmaltolepis seed-bearing structures are very different from those of Ginkgo but very similar to fossils described previously as Vladimaria. Umaltolepis and Vladimaria do not closely resemble the seed-bearing structures of any living or extinct plant, but are comparable in some respects to those of certain Peltaspermales and Umkomasiales (corystosperms). Vegetative similarities of the Umaltolepis plant to Ginkgo, and reproductive similarities to extinct peltasperms and corystosperms, support previous ideas that Ginkgo may be the last survivor of a once highly diverse group of extinct plants, several of which exhibited various degrees of ovule enclosure.

Authentication of Ginkgo biloba herbal dietary supplements using DNA barcoding.

Ginkgo biloba L. (known as ginkgo or maidenhair tree) is a phylogenetically isolated, charismatic, gymnosperm tree. Herbal dietary supplements, prepared from G. biloba leaves, are consumed to boost cognitive capacity via improved blood perfusion and mitochondrial function. A novel DNA mini-barcode assay was designed and validated for the authentication of G. biloba in herbal dietary supplements (n = 22; sensitivity = 1.00, 95% CI = 0.59-1.00; specificity = 1.00, 95% CI = 0.64-1.00). This assay was further used to estimate the frequency of mislabeled ginkgo herbal dietary supplements on the market in the United States of America: DNA amenable to PCR could not be extracted from three (7.5%) of the 40 supplements sampled, 31 of 37 (83.8%) assayable supplements contained identifiable G. biloba DNA, and six supplements (16.2%) contained fillers without any detectable G. biloba DNA. It is hoped that this assay will be used by supplement manufacturers to ensure that their supplements contain G. biloba.

Chloroplast phylogenomics indicates that Ginkgo biloba is sister to cycads.

Molecular phylogenetic studies have not yet reached a consensus on the placement of Ginkgoales, which is represented by the only living species, Ginkgo biloba (common name: ginkgo). At least six discrepant placements of ginkgo have been proposed. This study aimed to use the chloroplast phylogenomic approach to examine possible factors that lead to such disagreeing placements. We found the sequence types used in the analyses as the most critical factor in the conflicting placements of ginkgo. In addition, the placement of ginkgo varied in the trees inferred from nucleotide (NU) sequences, which notably depended on breadth of taxon sampling, tree-building methods, codon positions, positions of Gnetopsida (common name: gnetophytes), and including or excluding gnetophytes in data sets. In contrast, the trees inferred from amino acid (AA) sequences congruently supported the monophyly of a ginkgo and Cycadales (common name: cycads) clade, regardless of which factors were examined. Our site-stripping analysis further revealed that the high substitution saturation of NU sequences mainly derived from the third codon positions and contributed to the variable placements of ginkgo. In summary, the factors we surveyed did not affect results inferred from analyses of AA sequences. Congruent topologies in our AA trees give more confidence in supporting the ginkgo-cycad sister-group hypothesis.

Expression patterns of a cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis and environmental stress in Ginkgo biloba.

The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. A cDNA sequence encoding the CAD gene was isolated from the leaves of Ginkgo biloba L, designated as GbCAD1. The full-length cDNA of GbCAD1 was 1,494 bp containing a 1,074 bp open reading frame encoding a polypeptide of 357 amino acids with a calculated molecular mass of 38.7 kDa and an isoelectric point of 5.74. Comparative and bioinformatic analyses revealed that GbCAD1 showed extensive homology with CADs from other gymnosperm species. Southern blot analysis indicated that GbCAD1 belonged to a multi-gene family. Phylogenetic tree analysis revealed that GbCAD1 shared the same ancestor in evolution with other CADs and had a further relationship with other gymnosperm species. GbCAD1 was an enzyme being pH-dependent and temperature-sensitive, and showing a selected catalyzing. Tissue expression pattern analysis showed that GbCAD1 was constitutively expressed in stems and roots, especially in the parts of the pest and disease infection, with the lower expression being found in two- to four-year-old stem. Further analysis showed the change in lignin content had some linear correlation with the expression level of GbCAD1 mRNA in different tissues. The increased expression of GbCAD1 was detected when the seedling were treated with exogenous abscisic acid, salicylic acid, ethephon, ultraviolet and wounding. These results indicate that the GbCAD1 gene may play a role in the resistance mechanism to biotic and abiotic stresses as well as in tissue-specific developmental lignification.

Ginkgo biloba extract (EGb 761) attenuates oxidative stress induction in erythrocytes of sickle cell disease patients

Sickle cell disease promotes hemolytic anemia and occlusion of small blood vessels due to the presence of high concentrations of hemoglobin S, resulting in increased production of reactive oxygen species and decreased antioxidant defense capacity. The aim of this study was to evaluate the protective action of a standardized extract of Ginkgo biloba (EGb 761), selected due to its high content of flavonoids and terpenoids, in erythrocytes of patients with sickle cell anemia (HbSS, SS erythrocytes) subjected to oxidative stress using tert-butylhydroperoxide or 2,2-azobis-(amidinepropane)-dihydrochloride, in vitro. Hemolysis indexes, reduced glutathione, methemoglobin concentrations, lipid peroxidation, and intracellular reactive oxygen species were determined. SS erythrocytes displayed increased rates of oxidation of hemoglobin and membrane lipid peroxidation compared to normal erythrocytes (HbAA, AA erythrocytes), and the concentration of EGb 761 necessary to achieve the same antioxidant effect in SS erythrocytes was at least two times higher than in normal ones, inhibiting the formation of intracellular reactive oxygen species (IC50 of 13.6 µg/mL), partially preventing lipid peroxidation (IC50 of 242.5 µg/mL) and preventing hemolysis (IC50 of 10.5 µg/mL). Thus, EGb 761 has a beneficial effect on the oxidative status of SS erythrocytes. Moreover, EGb 761 failed to prevent oxidation of hemoglobin and reduced glutathione at the concentrations examined.

A doença falciforme promove anemia hemolítica e oclusão dos pequenos vasos, causados pela presença de altas concentrações de hemoglobina S, cujas consequências incluem a produção aumentada de espécies reativas de oxigênio e diminuição da capacidade de defesa antioxidante. O objetivo desse estudo foi avaliar a ação protetora de um extrato padronizado de Ginkgo biloba (EGb 761), selecionado devido ao seu alto conteúdo de flavonóides e terpenóides, em eritrócitos de pacientes com anemia falciforme (HbSS, eritrócitos SS) submetidos ao estresse oxidativo usando terc-butil-hidroperóxido e 2,2-azobis-(amidinopropano)-diidrocloreto, in vitro. Índices de hemólise, glutationa reduzida, concentração de metemoglobina, peroxidação lipídica e espécies reativas de oxigênio foram determinados. Eritrócitos de pacientes com anemia falciforme apresentaram taxas aumentadas de oxidação da hemoglobina e peroxidação lipídica e a concentração de EGb 761 necessária para atingir o mesmo efeito antioxidante foi pelo menos duas vezes maior em relação aos eritrócitos normais (HbAA, eritrócitos AA), inibindo a formação de espécies reativas de oxigênio (IC50 de 13.6 µg/mL), prevenindo parcialmente a peroxidação lipídica (IC50 de 242.5 µg/mL) e prevenindo a hemólise (IC50 de 10.5 µg/mL). Portanto, EGb 761 possui um efeito benéfico no estado oxidativo dos eritrócitos SS. Entretanto, o EGb 761 não preveniu a oxidação da hemoglobina e da glutationa reduzida, nas concentrações examinadas.

Biology and chemistry of Ginkgo biloba.

Ginkgo biloba has been existing on earth since 200 million years and is considered as a "living fossil". It is among the most sold medicinal plants in the world. A number of secondary metabolites representing terpenoids, polyphenols, allyl phenols, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids have been isolated from the plant. However, the main bioactive constituents are terpene trilactones and flavonoid glycosides which are considered responsible for the pharmacological activities of its standardized leaf extract. Scattered information is available on the extraction and analysis of these pharmacologically important constituents which have been compiled in the present review.

Genetic relationships of ornamental cultivars of Ginkgo biloba analyzed by AFLP techniques.

Eight primer combinations that produced clear and a large number of polymorphic bands were screened from 64 EcoR I/Mse I primer combinations (Mse I fluorescent labeled). The genetic relationships of 21 ornamental cultivars of Ginkgo biloba L. from the United States of America, Holland, Japan, France, and China were analyzed. These primer combinations produced a total of 1 119 bands, 229 specific loci (including 54 absent bands, and 175 monomorphic bands). Among them, 983 polymorphic bands (PPB), accounting for 88%, were detected. The percentage of identification per primer combination was as high as 100%. The average PPB of 14 foreign cultivars was 35.86% and the average PPB of seven domestic cultivars was 31.51%. Genetic similarity coefficient (SC) among all cultivars varied from 0.4899 to 0.8499, and all cultivars were divided into the four clusters when SC was set at 0.7300. The cultivars from the same origin did not fall into the same group. The cultivars from France and China were classified into three groups. According to the comprehensive analyses based on specific loci, similarity coefficient, and clustering results, eight cultivars 'Fastigiata', 'Tit', 'Tubifolia', 'Daeryinxing', 'Variegata', 'Horizontalis, 'Pendula', and 'Yiyuanyeziyinxing' were considered to be important germplasms of ornamental cultivars of Ginkgo biloba.

[Origin of Ginkgo biloba L. Phylogenetic approach]. / Origine du Ginko biloba L. Approche phylogénétique.

A cladistic analysis on fossil and modern Gymnosperms (20 taxa) is presented and discussed with particular mention of Ginkgo biloba L. origin. The consensus tree obtained from 68 characters (59 informative characters) shows a monophyletic clade containing all plants bearing micropylate ovules ('Micropylophytes'). Medullosales appear at the base of this clade. Ginkgo forms the sister group of the Dicranophyllales + Coniferales. The obtained phylogeny implies that the Ginkgoales ancestor is to be found during the Upper Carboniferous.