Changes in Polar Metabolites Content during Natural and Methyl-Jasmonate-Promoted Senescence of Ginkgo biloba Leaves.

The present study clarified changes in the contents of polar metabolites (amino acids, organic acids, saccharides, cyclitols, and phosphoric acid) in leaf senescence in Ginkgo biloba with or without the application of methyl jasmonate (JA-Me) in comparison with those in naturally senescent leaf blades and petioles. The contents of most amino acids and citric and malic acids were significantly higher in abaxially, and that of myo-inositol was lower in abaxially JA-Me-treated leaves than in adaxially JA-Me-treated and naturally senescent leaves. The levels of succinic and fumaric acids in leaves treated adaxially substantially high, but not in naturally senescent leaves. In contrast, sucrose, glucose, and fructose contents were much lower in leaf blades and petioles treated abaxially with JA-Me than those treated adaxially. The levels of these saccharides were also lower compared with those in naturally senescent leaves. Shikimic acid and quinic acid were present at high levels in leaf blades and petioles of G. biloba. In leaves naturally senescent, their levels were higher compared to green leaves. The shikimic acid content was also higher in the organs of naturally yellow leaves than in those treated with JA-Me. These results strongly suggest that JA-Me applied abaxially significantly enhanced processes of primary metabolism during senescence of G. biloba compared with those applied adaxially. The changes in polar metabolites in relation to natural senescence were also discussed.

Phenylalanine Increases the Production of Antioxidant Phenolic Acids in Ginkgo biloba Cell Cultures.

The aims of this study were to evaluate the antioxidant properties, to investigate the content of major secondary metabolites in Ginkgo biloba cell cultures, and to determine the change in the production of phenolic acids by adding phenylalanine to the culture medium. Three in vitro methods, which depend on different mechanisms, were used for assessing the antioxidant activity of the extract: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays. The extract showed moderate activity both in the DPPH and in the reducing power assays (IC50 = 1.966 ± 0.058 mg/mL; ASE/mL = 16.31 ± 1.20); instead, it was found to possess good chelating properties reaching approximately 70% activity at the highest tested dose. The total phenolic, total flavonoid, and condensed tannin content of G. biloba cell culture extract was spectrophotometrically determined. The phenolic acid content was investigated by RP-HPLC, and the major metabolites-protocatechuic and p-hydroxybenzoic acids-were isolated and investigated by 1H NMR. The results showed that phenylalanine added to G. biloba cell cultures at concentrations of 100, 150, and 200 mg/150 mL increased the production of phenolic acids. Cultures that were grown for 3 weeks and collected after 4 days of phenylalanine supplementation at high concentration showed maximal content of phenolic acids (73.76 mg/100 g DW).

Effect of Methyl Jasmonate on the Terpene Trilactones, Flavonoids, and Phenolic Acids in Ginkgo biloba L. Leaves: Relevance to Leaf Senescence.

The present study compared the effects of natural senescence and methyl jasmonate (JA-Me) treatment on the levels of terpene trilactones (TTLs; ginkgolides and bilobalide), phenolic acids, and flavonoids in the primary organs of Ginkgo biloba leaves, leaf blades, and petioles. Levels of the major TTLs, ginkgolides B and C, were significantly higher in the leaf blades of naturally senesced yellow leaves harvested on 20 October compared with green leaves harvested on 9 September. In petioles, a similar effect was found, although the levels of these compounds were almost half as high. These facts indicate the importance of the senescence process on TTL accumulation. Some flavonoids and phenolic acids also showed changes in content related to maturation or senescence. Generally, the application of JA-Me slightly but substantially increased the levels of TTLs in leaf blades irrespective of the difference in its application side on the leaves. Of the flavonoids analyzed, levels of quercetin, rutin, quercetin-4-glucoside, apigenin, and luteolin were dependent on the JA-Me application site, whereas levels of (+) catechin and (-) epicatechin were not. Application of JA-Me increased ferulic acid and p-coumaric acid esters in the petiole but decreased the levels of these compounds in the leaf blade. The content of p-coumaric acid glycosides and caffeic acid esters was only slightly modified by JA-Me. In general, JA-Me application affected leaf senescence by modifying the accumulation of ginkogolides, flavonoids, and phenolic acids. These effects were also found to be different in leaf blades and petioles. Based on JA-Me- and aging-related metabolic changes in endogenous levels of the secondary metabolites in G. biloba leaves, we discussed the results of study in the context of basic research and possible practical application.

Characterization, Function, and Transcriptional Profiling Analysis of 3-Hydroxy-3-methylglutaryl-CoA Synthase Gene (GbHMGS1) towards Stresses and Exogenous Hormone Treatments in Ginkgo biloba.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is one of the rate-limiting enzymes in the mevalonate pathway as it catalyzes the condensation of acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA. In this study, A HMGS gene (designated as GbHMGS1) was cloned from Ginkgo biloba for the first time. GbHMGS1 contained a 1422-bp open-reading frame encoding 474 amino acids. Comparative and bioinformatics analysis revealed that GbHMGS1 was extensively homologous to HMGSs from other plant species. Phylogenetic analysis indicated that the GbHMGS1 belonged to the plant HMGS superfamily, sharing a common evolutionary ancestor with other HMGSs, and had a further relationship with other gymnosperm species. The yeast complement assay of GbHMGS1 in HMGS-deficient Saccharomyces cerevisiae strain YSC6274 demonstrated that GbHMGS1 gene encodes a functional HMGS enzyme. The recombinant protein of GbHMGS1 was successfully expressed in E. coli. The in vitro enzyme activity assay showed that the kcat and Km values of GbHMGS1 were 195.4 min-1 and 689 µM, respectively. GbHMGS1 was constitutively expressed in all tested tissues, including the roots, stems, leaves, female flowers, male flowers and fruits. The transcript accumulation for GbHMGS1 was highest in the leaves. Expression profiling analyses revealed that GbHMGS1 expression was induced by abiotic stresses (ultraviolet B and cold) and hormone treatments (salicylic acid, methyl jasmonate, and ethephon) in G. biloba, indicating that GbHMGS1 gene was involved in the response to environmental stresses and plant hormones.

Molecular cloning of two novel peroxidases and their response to salt stress and salicylic acid in the living fossil Ginkgo biloba.

BACKGROUND AND AIMS: Peroxidase isoenzymes play diverse roles in plant physiology, such as lignification and defence against pathogens. The actions and regulation of many peroxidases are not known with much accuracy. A number of studies have reported direct involvement of peroxidase isoenzymes in the oxidation of monolignols, which constitutes the last step in the lignin biosynthesis pathway. However, most of the available data concern only peroxidases and lignins from angiosperms. This study describes the molecular cloning of two novel peroxidases from the 'living fossil' Ginkgo biloba and their regulation by salt stress and salicylic acid. METHODS: Suspension cell cultures were used to purify peroxidases and to obtain the cDNAs. Treatments with salicylic acid and sodium chloride were performed and peroxidase activity and gene expression were monitored. KEY RESULTS: A novel peroxidase was purified, which preferentially used p-hydroxycinnamyl alcohols as substrates and was able to form dehydrogenation polymers in vitro from coniferyl and sinapyl alcohols. Two peroxidase full-length cDNAs, GbPrx09 and GbPrx10, were cloned. Both peroxidases showed high similarity to other basic peroxidases with a putative role in cell wall lignification. Both GbPrx09 and GbPrx10 were expressed in leaves and stems of the plant. Sodium chloride enhanced the gene expression of GbPrx09 but repressed GbPrx10, whereas salicylic acid strongly repressed both GbPrx09 and GbPrx10. CONCLUSIONS: Taken together, the data suggest the participation of GbPrx09 and GbPrx10 in the developmental lignification programme of the cell wall. Both peroxidases possess the structural characteristics necessary for sinapyl alcohol oxidation. Moreover, GbPrx09 is also involved in lignification induced by salt stress, while salicylic acid-mediated lignification is not a result of GbPrx09 and GbPrx10 enzymatic activity.

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.

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.

Molecular cloning and expression analysis of a WRKY transcription factor gene, GbWRKY20, from Ginkgo biloba.

WRKY transcription factors are important regulators of diverse plant life processes. Our aim was to clone and characterize GbWRKY20, a WRKY gene of group IIc, derived from Ginkgo biloba. The cDNA sequence of GbWRKY20 was 818 bp long, encoding a 271-amino acid proteins and containing two introns and three exons. The proteinic molecular weight was 30.99 kDa, with a relevant theoretical isoelectric point of 8.15. Subcellular localization analysis confirmed that the GbWRKY20 protein localized to the nucleus. In total, 75 cis-regulatory elements of 19 different types were identified in the GbWRKY20 promoter sequence, including some elements involved in light responsiveness, anaerobic induction and circadian control, low-temperature responsiveness, as well as salicylic acid (SA) and auxin responsiveness. Expression pattern analysis of plant samples from different developmental stages and tissue types, revealed differential GbWRKY20 expression. The GbWRKY20 transcript was downregulated 12 h after heat treatment and at 4-12 h after drought treatment, but was upregulated 12 h after NaCl, cold and methyl jasmonate treatments. For abscisic acid and SA treatments, the GbWRKY20 transcript was upregulated at 24 h. In summary, GbWRKY20 encoded a newly cloned WRKY transcription factor of G. biloba that might be involved in plant growth and plant responses to abiotic stresses and hormones treatments.

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.

Physiological, Transcriptomic, and Metabolic Responses of Ginkgo biloba L. to Drought, Salt, and Heat Stresses.

Ginkgo biloba L. is highly adaptable and resistant to a range of abiotic stressors, allowing its growth in various environments. However, it is unclear how G. biloba responds to common environmental stresses. We explored the physiological, transcriptomic, and metabolic responses of G. biloba to short-term drought, salt, and heat stresses. Proline, H2O2, and ABA contents, along with CAT activity, increased under all three types of stress. SOD activity increased under salt and heat stresses, while soluble protein and IAA contents decreased under drought and salt stresses. With respect to metabolites, D-glyceric acid increased in response to drought and salt stresses, whereas isomaltose 1, oxalamide, and threonine 2 increased under drought. Piceatannol 2,4-hydroxybutyrate and 1,3-diaminopropane increased under salt stress, whereas 4-aminobutyric acid 1 and galactonic acid increased in response to heat stress. Genes regulating nitrogen assimilation were upregulated only under drought, while the GRAS gene was upregulated under all three types of stressors. ARF genes were downregulated under heat stress, whereas genes encoding HSF and SPL were upregulated. Additionally, we predicted that miR156, miR160, miR172, and their target genes participate in stress responses. Our study provides valuable data for studying the multilevel response to drought, salinity, and heat in G. biloba.

Cryopreservation of Ginkgo biloba cell culture: effect of pretreatment with sucrose and ABA.

The report describes the impact of preculture with sucrose and sucrose + ABA on desiccation and cryopreservation tolerance of cell cultures of Ginkgo biloba L., an important landscape and medicinal tree. Callus clumps were incubated on MS medium supplemented with high sucrose concentrations (up to 24 percent, w/v), employed alone or with ABA (2-10 mg per L) for various durations followed by desiccation for 0-240 min and cryopreservation. The beneficial effect of preculture on regrowth after desiccation without cryopreservation was only observed for the cells with water content of 20 percent FW and was not influenced by presence of ABA. However, preculture of calli in presence of ABA resulted in a lower desiccation rate as compared with untreated controls and calli pretreated with sucrose alone. In calli precultured with sucrose alone, post-thaw regrowth was occasional regardless of the sugar concentration in the medium, while pretreatment of calli with ABA and sucrose ensured stable regrowth after cryopreservation. The highest post-thaw regrowth of 54 percent was achieved for calli precultured on medium supplemented with 10 percent (w/v) sucrose and 2 mg per L ABA for 21 days followed by desiccation for 150 min. The different effects of preculture treatments on post-thaw regrowth were associated with significant changes in content and in composition of endogenous soluble sugars in calli. Sucrose and glucose accumulated preferentially in ABA-precultured calli, while the fructose content was higher in calli precultured in absence of ABA. The possible role of preculture on desiccation and cryopreservation tolerance of G. biloba cell cultures is discussed.

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.

Impact of elevated CO2 and O3 concentrations on biogenic volatile organic compounds emissions from Ginkgo biloba.

In natural environment with ambient air, ginkgo trees emitted volatile organic compounds 0.18 microg g(-1) h(-1) in July, and 0.92 microg g(-1) h(-1) in September. Isoprene and limonene were the most abundant detected compounds. In September, alpha-pinene accounted for 22.5% of the total. Elevated CO(2) concentration in OTCs increased isoprene emission significantly in July (p<0.05) and September (p<0.05), while the total monoterpenes emission was enhanced in July and decreased in September by elevated CO(2). Exposed to elevated O(3) increased the isoprene and monoterpenes emissions in July and September, and the total volatile organic compounds emission rates were 0.48 microg g(-1) h(-1) (in July) and 2.24 microg g(-1) h(-1) (in September), respectively. The combination of elevated CO(2) and O(3) did not have any effect on biogenic volatile organic compounds emissions, except increases of isoprene and Delta3-carene in September.

Changes of main secondary metabolites in leaves of Ginkgo biloba in response to ozone fumigation.

To investigate the effect of elevated O3 on the accumulation of main secondary metabolites in leaves of Ginkgo biloba L., four-year-old trees were exposed in open-top chambers with ambient air and the air with twice ambient O3 concentration in Shenyang in 2006. Elevated O3 increased the concentrations of terpenes, but decreased the concentrations of phenolics in G. biloba leaves. The results showed that secondary compounds from G. biloba leaves responded to the elevated O3 exposure in a different way when compared to previous studies which showed elevated O3 increased the concentrations of phenolics but had no effect on the terpenes in leaves of other deciduous trees. Furthermore, reduced synthesis of phenolics may decrease the resistance of G. biloba to O3 and other environmental factors. On the other hand, the induced synthesis of terpenes may enhance the antioxidant abilities in G. biloba leaves at the end of O3 fumigation.

[Exogenous nitric oxide accelerates soluble sugar, proline and secondary metabolite synthesis in Ginkgo biloba under drought stress].

The effects of exogenous nitric oxide donor sodium nitroprusside (SNP) on substance metabolism of Ginkgo biloba leaves under drought stress were studied. The results showed that 250 micromol/L SNP (Fig.2) treatment under 35% relative soil water content (RSWC) stress (Fig.1) raised remarkably soluble sugar content (Fig.3), proline content (Fig.4), phenylalanine ammonia lyase (PAL) activity (Fig.5), flavonoids (Fig.6) and ginkgolides content (Fig.7) of G. biloba leaves. Hemoglobin, used as NO scavenger, counteracted the effects of SNP in raising the soluble sugar (Fig.3), proline (Fig.4), flavonoid (Fig.6), ginkgolide content (Fig.7) and PAL activities (Fig.5), which indicates that the effects of sodium nitroprusside were through the nitric oxide released from sodium nitroprusside. We propose from these results that the roles of flavonoids and ginkgolides are the same as those of soluble sugars and proline under drought stress. NO may alleviate the damage caused by drought stress through raising soluble sugar, proline, flavonoid and ginkgolide content.

Effect of supplementing terpenoid biosynthetic precursors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures.

The effect of precursor feeding on the production of bilobalide and ginkgolides was studied with suspension cell cultures of Ginkgo biloba. The precursors greatly influenced the productivity of bilobalide and ginkgolides. Precursor supplementation increased the accumulation of both bilobalide and ginkgolides, and with positive effect on cell growth. The GA accumulation by cell cultures was influenced by precursors upstream in the metabolism, whereas the BB accumulation was under the influence of downstream precursors of the terpenoid biosynthetic pathway. Furthermore, precursor feeding modified the ratios of the BB, GA and GB in cells and cell cultures of G. biloba. The studies also aid in understanding effect of precursor feeding on the bilobalide and ginkgolides biosynthetic pathway.

Molecular cloning, characterization and expression of a novel trehalose-6-phosphate synthase homologue from Ginkgo biloba.

In many organisms, trehalose acts as protective metabolite against harsh environmental stresses, such as freezing, drought, nutrient starvation, heat and salt. Herein a cDNA (designated as GbTPS, GenBank Accession Number AY884150) encoding a trehalose-6-phosphate synthase homologue was isolated and characterized from the living fossil plant, Ginkgo biloba, which is highly tolerant to drought and cold. GbTPS encoded an 868-amino-acid polypeptide with a predicted isoelectric point of 5.83 and molecular mass of 97.9 kD. Amino acid sequence alignment revealed that GbTPS shared high identity with class II trehalose-6-phosphate synthase homologues (67% identical to AtTPS7), but had only 17% and 23% of identity with OstA from Escherichia coli and ScTPS1 from S. cerevisiae, respectively. DNA gel blot analysis indicated that GbTPS belonged to a small multi-gene family. The expression analysis by RT-PCR showed that GbTPS expressed in a tissue-specific manner in G. biloba and might involve in leaf development. GbTPS was also found to be induced by a variety of stresses including cold, salt, drought and mannitol.

[Pharmacological treatment of vascular cognitive impairment]. / Farmakoterapia zaburzen funkcji poznawczych uwarunkowanych czynnikami naczyniowymi.

Vascular dementia is second most common cause of dementia. The paper highlights the most important trends in pharmacological treatment of vascular dementia. Result of a clinical trial of some agents appears to be promising. Pentoxifyline appears to be useful in multi-infarct vascular dementia. Nimodipine produced improvement in subcortical dementia. Some other agents like ginkgo biloba, acetylocholinesterase inhibitors, memantine and other also have shown mild benefit or at least were associated with some stabilization of dementia.

Contribution of a phytotoxic compound to the allelopathy of Ginkgo biloba.

Ginkgo (Ginkgo biloba L.) has not changed over 121 million years. There may be unknown special strategy for the survival. Gingko litter inhibited the growth of weed species ryegrass (Lolium multiflorum L.). The inhibition was greater with the litter of the close position than that of the far position from the gingko tree. A phytotoxic substance, 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid (HHPEBA) was isolated in the litter. HHPEBA concentration was greater in the litter of the close position than that of the far position from the tree. HHPEBA inhibited the ryegrass growth at concentrations greater than 3 µM. HHPEBA was estimated to be able to cause 47-62% of the observed growth inhibition of ryegrass by the gingko litter. Therefore, HHPEBA may contribute to the inhibitory effect caused by ginkgo litter and may provide a competitive advantage for gingko to survive through the growth inhibition of the neighboring plants.

The behavior of pollination drop secretion in Ginkgo biloba L.

Pollination drop (PD) secretion plays a critical role in wind pollination in many gymnosperms. We conducted detailed investigations on PD secretion in Ginkgo biloba, and found that PDs could not form when the micropyle was removed, but were able to form after removal of the shoot, leaves, ovular stalk, or ovular collar. The duration and volume of the PD increased under high relative humidity, but addition of salt or sugar did not affect PD secretion, its size, or its duration. Morphological and anatomical observations showed that many secretion cells at the nucellus tip contributed to secreting the PD after the formation of pollen chamber. Under laboratory conditions, the PD persisted for approximately 10 d if not pollinated, and re-formed five times after it was removed, with the total volume of PDs reaching approximately 0.4 µL. These results suggested that PDs can be continuously secreted by the tip of the nucellus cells during the pollination stage to increase the chance of capturing pollen from the air. Importantly, PD secretion is an independent behavior of the ovule and PDs were produced apoplastically.