Screening and identification of miRNAs related to sexual differentiation of strobili in Ginkgo biloba by integration analysis of small RNA, RNA, and degradome sequencing.

BACKGROUND: Ginkgo biloba, a typical dioecious plant, is a traditional medicinal plant widely planted. However, it has a long juvenile period, which severely affected the breeding and cultivation of superior ginkgo varieties. RESULTS: In order to clarify the complex mechanism of sexual differentiation in G. biloba strobili. Here, a total of 3293 miRNAs were identified in buds and strobili of G. biloba, including 1085 known miRNAs and 2208 novel miRNAs using the three sequencing approaches of transcriptome, small RNA, and degradome. Comparative transcriptome analysis screened 4346 and 7087 differentially expressed genes (DEGs) in male buds (MB) _vs_ female buds (FB) and microstrobilus (MS) _vs_ ovulate strobilus (OS), respectively. A total of 6032 target genes were predicted for differentially expressed miRNA. The combined analysis of both small RNA and transcriptome datasets identified 51 miRNA-mRNA interaction pairs that may be involved in the process of G. biloba strobili sexual differentiation, of which 15 pairs were verified in the analysis of degradome sequencing. CONCLUSIONS: The comprehensive analysis of the small RNA, RNA and degradome sequencing data in this study provided candidate genes and clarified the regulatory mechanism of sexual differentiation of G. biloba strobili from multiple perspectives.

Ginkgo agroforestry practices alter the fungal community structures at different soil depths in Eastern China.

Agroforestry practices aim to achieve environmentally friendly land use. Fungi play a primarily role in soil organic carbon and nutrient maintenance, while the response of the soil fungi community to land use changes is little explored. Here, a high-throughput sequencing method was applied to understand the fungal community structure distinction in ginkgo agroforestry systems and adjacent croplands and nurseries. Our results showed that the agroforestry systems achieved better soil fertility and carbon contents. The agroforestry practices significantly altered the composition of soil fungal communities comparing with pure gingko plantation, adjacent cropland, and nursery. The dominant fungal phyla were always Ascomycota and Basidiomycota. The relative abundance of Ascomycota was correlated with the TN and AP, while the abundance of Basidiomycota was negatively correlated with the TN and NN. The soil organic carbon, total nitrogen, and nitrate nitrogen explained 59.80% and 63.36% of the total variance in the fungal community composition in the topsoil and subsoil, and the available phosphorus also played a key role in the topsoil. Considering soil fertility maintenance and fungal community survival and stability, the agroforestry systems achieved better results, and the ginkgo and wheat system was the best among the five planting systems we studied. In the ginkgo and wheat system, applying readily available mineral nitrogen fertilizer either alone or in combination with organic amendments will improve the soil quality and fertility.

Determination and Comparison of 4'- O-Methylpyridoxine Analogues in Ginkgo biloba Seeds at Different Growth Stages.

The antivitamin B6, 4'- O-methylpyridoxine (MPN); its glucoside, 4'- O-methylpyridoxine-5'-glucoside (MPNG); and vitamin B6 compounds, including pyridoxal (PL), pyridoxamine, pyridoxine, pyridoxal-5'-phosphate (PLP), and pyridoxamine-5'-phosphate, exist in Ginkgo biloba seeds, which are widely used as food and medicine. This work aimed to determine the MPN analogues in G. biloba seeds at different growth stages in terms of cultivars and ages of trees. The highest total MPN contents of 249.30, 295.62, and 267.85 µg/g were obtained in the mature stages of three selected G. biloba samples. The total contents of vitamin B6 compounds decreased significantly in the entire growth period of the three samples. Principal-component analysis revealed that MPN and MPNG were important contributors in the MPN-analogue metabolism of G. biloba seeds. The influence of the cultivar on the content and composition of MPN analogues was greater than that of the age of the G. biloba tree.

Direct assessment of phytochemicals inherent in plant tissues using extractive electrospray ionization mass spectrometry.

An ambient pressure ionization mass spectrometric strategy called internal extractive electrospray ionization mass spectrometry (iEESI-MS) has been developed and applied for direct profiling of labile phytochemicals inherent in various native plant tissues, including leaves, roots, and fruits. By passing the electrospray solvent through the plant tissue, a variety of phytochemicals, such as amino acids, sugars (e.g., glucose, sucrose, polysaccharides, etc.), and alkaloids, were continuously extracted from the sample interior, driven toward the natural/cut electro-spraying tip, and vaporized into gaseous ions for mass spectrometric interrogation. Phytochemical patterns obtained by iEESI-MS permit a rapid differentiation between various species of ginkgo plant and strawberry maturity stages, as well as characterization of physiological/pathologic conditions of chlorophytum comosum. Our experimental results further demonstrate that the established iEESI-MS approach is potentially useful for direct phytochemomics studies with minimal biodegradation, allowing elucidation of plant metabolism with high speed, specificity, and simplicity of analysis.

Elemental analysis of Ginkgo biloba leaf samples collected during one vegetation period.

The object of our work was the identification and quantification of inorganic elements in Ginkgo biloba L. leaves (Ginkgonis folium, Ginkgoaceae) by X-ray fluorescence analysis. The plant material was obtained from a 50-years-old female tree at the Comenius University Botanical Garden (Bratislava, Slovakia). Leaves were collected from early May to late September, with the last sample consisting of fallen leaves. The elements analyzed were: phosphorus, sulfur, potassium, calcium, scandium, iron, zinc, yttrium, molybdenum, tellurium, samarium, gadolinium, dysprosium, iridium, thallium and lead. The amounts of the monitored heavy metals were below the limits specified in Ph. Eur. 7 and PhS 1.

Improving flavonoid extraction from Ginkgo biloba leaves by prefermentation processing.

This paper presents a prefermentation treatment method involving fungi to improve flavonoid extraction from the leaves of Ginkgo biloba . The fungi employed for this treatment were screened from the soil present under an ancient ginkgo tree. Seventy-six strains belonging to 23 genera were isolated and identified by a molecular identification method employing 18S rDNA sequences. Thirty-three strains grew well using ginkgo leaves as the growth medium. One strain, Gyx086, with higher extracted yield of flavonoids and more similar to the control, was finally selected for prefermentation processing. The major fermentation factors were optimized by response surface methodology. The optimal conditions for the highest total falvonoid yield were 27.8 °C for temperature, 64.2% for moisture content, and 61 h for fermentation time. Under the optimal condition, a actual total flavonoid yield of 27.59 ± 0.52 mg/g dry weight culture sample was obtained, which was about 70% higher than that of unfermented gingko leaf samples.

Estaquia de Ginkgo biloba L. utilizando três substratos / Ginkgo biloba L. cutting using three substrates

Ginkgo biloba é arbórea, decídua, cuja folhagem se torna amarelada no outono antes da queda das folhas, o que a torna valorizada em jardinagem. A estaquia é um método de propagação vegetativa baseado na capacidade das células em retomarem o processo de divisão celular, formando raízes em estacas destacadas de ramos provenientes de plantas matrizes. O presente trabalho teve como objetivos verificar a influência de diferentes substratos, assim como, a aplicação da auxina sintética o ácido indol butírico (AIB) no enraizamento de estacas de Ginkgo biloba. No inverno de 2005, ramos foram coletados e transportados até o Laboratório de Macropropagação, onde foram confeccionadas estacas sem folhas, com 10-12 cm de comprimento. Os tratamentos com regulador vegetal (T) foram T1- 0 mg L-1 AIB em solução; T2- 4000 mg L-1 AIB em solução; T3- 8000 mg L-1 AIB em solução; T4- 0 mg kg-1 AIB em talco; T5- 4000 mg kg-1 AIB em talco e T6- 8000 mg kg-1 AIB em talco. Para cada tratamento foram utilizados três diferentes substratos (S), S1- areia, S2- fibra de casca de coco (coxim) e S3- casca de arroz carbonizada. Após 120 dias da instalação, foram avaliadas as porcentagens de estacas enraizadas, vivas, com calos e mortas; o número de raízes por estaca e o comprimento das três maiores raízes por estaca. Os melhores resultados no enraizamento foram obtidos com estacas tratadas com 4000 e 8000 mg kg-1 AIB em talco, utilizando o coxim como substrato (45,00 e 46,25 por cento de enraizamento, respectivamente).

Ginkgo biloba is an arboreal and deciduous species, the foliage of which becomes yellowish in the autumn, before leaf drop, increasing its value for gardening. Cutting is a method of vegetative propagation based on the capacity of cells to recover the cell division process, originating roots in cuttings detached from branches of stock plants. This study aimed to verify the influence of different substrates, as well as the application of the synthetic auxin indole-3-butyric acid (IBA) in Ginkgo biloba cutting rooting. In the winter of 2005, branches were collected and sent to the Macropropagation Lab, where cuttings of 10-12cm length were made without leaves. The treatments with plant growth regulator (T) were T1- 0 mg L-1 IBA solution, T2- 4000 mg L-1 IBA solution, T3- 8000 mg L-1 IBA solution, T4- 0 mg kg-1 IBA in talc, T5- 4000 mg kg-1 IBA in talc, T6- 8000 mg kg-1 IBA in talc. Each treatment was planted in three substrates (S), S1- sand, S2- coir and S3- carbonized rice hull. After 120 days, the percentages of cuttings that were rooted, alive, with callus and dead were evaluated, besides the number of roots per cutting and the length of the three highest roots per cutting. The best results regarding rooting were obtained for cuttings treated with 4000 and 8000 mg kg-1 IBA in talc, by using coir as substrate (45.00 and 46.25 percent rooting, respectively).

[Studies on dynamic change of total ginkgolic acids in Ginkgo biloba leaves of different aged trees and different collecting seasons].

OBJECTIVE: To study dynamic change of total ginkgolic acids in Ginkgo biloba leaves of the different aged trees and different collecting seasons. METHOD: The content of total ginkgolic acids in G. biloba leaves was determined by HPLC. A Alltima C18 (4.6 mm x 250 mm, 5 microm) and the mobile phase of methanol and 1% acetic acid (90:10) were used, the flow rate was 1.0 mL x min(-1), and the wavelength was 310 nm. The content were calculated with external standard method. RESULT: The content of total ginkgolic acids in G. biloba leaves was in the range of 0.48% to 2.51% in different collecting seasons. The content reached maximum at the end of May and the beginning of June, and then declined gradually. In different aged trees, the content in the older ages was lower than that in the younger ages. CONCLUSION: The results provide scientific basis for the collecting season of G. biloba leaves.

[Study on dynamical changes of Ginkgo biloba resources in Pizhou city of Jiangsu Province base on RS and GIS].

OBJECTIVE: To analyse the dynamical changes of the Ginkgo biloba's resources from 2001 to 2006, in Pizhou city, Jiangshu province by useing spatial analytical function of GIS and RS technology. METHOD: Use the GIS and RS technology, extracted the information of G. biloba by scientific investigation, researched the spatial distribution and dynamical changes of G. biloba based on landsat 5 TM: the Apr. 3rd, 2001; Jan. 16th, 2005; July 30th, 2006. RESULT: Ginkgo biloba's resource was 1.61 x 10(5) hm2 in 2001, 1.84 x 10(5) hm2 in 2005, 1.88 x 10(5) hm2 in 2006. CONCLUSION: Ginkgo biloba's resource rised from 1.61 x 10(5) hm2 to 1.88 x 10(5) hm2 from 2001 to 2006, showed the gradually rise.

Formation of archegonium chamber is associated with nucellar-cell programmed cell death in Ginkgo biloba.

The archegonium chamber in Ginkgo biloba L. is a pathway for spermatozoids swimming towards the archegonium for fertilization. The objective of this study was to elucidate the mechanism of archegonium chamber formation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and DNA ladder demonstrated that the nucellar cell death, coordinated with the archegonium chamber formation, was a process of programmed cell death. Cytochemical localization of Ca(2+) in these nucellar cells was determined by means of in situ precipitation with potassium pyroantimonate and electron microscopic visualization, in order to study the relation between Ca(2+) and programmed cell death. The results showed an early uptake of the mitochondrial calcium particles in the nucellar cells undergoing programmed cell death. Together with other dynamic changes in Ca(2+) subcellular distribution, this indicates that Ca(2+) may play a role in the regulation of mitochondria-mediated programmed events in the nucellar cells.

[Study on pharmacognosy of Ginkgo leaf].

The primary study of Ginkgo leaf such as crude drug macroscopic and powder characteristics were carried out, and the flavonoids content in the leaf of Ginkgo in different areas of Gansu province was determined by HPLC, in order to provide scientific references for the exploitation of Ginkgo in Gansu province.

[Study on the comparison of polysaccharides in Ginkgo biloba leaves].

OBJECTIVE: To determine the suitable tree's age, tree's sex and gathering seasons for Ginkgo biloba leaves. METHODS: The twelve different polysaccharides were obtained by extracting and precipitating from Ginkgo biloba leaves and to see if there were differences among them. The concentration of Ginkgo biloba leaf polysaccharides with the highest gain ratio will be determined by HPLC. RESULT: The average gain ratio of Ginkgo biloba leaf polysaccharides was 4.29%, among them the gain ratio of 10-years old female Ginkgo biloba leaf collected in the last ten days of September was the highests, its polysaccharides concentration was 61.5% with RSD = 2.5% (n = 6). CONCLUSION: The gain ratios were different in different Ginkgo biloba leaves and the changing rules provide scientific basis for the GAP of medicinal plants.

A 300-million-year record of atmospheric carbon dioxide from fossil plant cuticles.

To understand better the link between atmospheric CO2 concentrations and climate over geological time, records of past CO2 are reconstructed from geochemical proxies. Although these records have provided us with a broad picture of CO2 variation throughout the Phanerozoic eon (the past 544 Myr), inconsistencies and gaps remain that still need to be resolved. Here I present a continuous 300-Myr record of stomatal abundance from fossil leaves of four genera of plants that are closely related to the present-day Ginkgo tree. Using the known relationship between leaf stomatal abundance and growing season CO2 concentrations, I reconstruct past atmospheric CO2 concentrations. For the past 300 Myr, only two intervals of low CO2 (<1,000 p.p.m.v.) are inferred, both of which coincide with known ice ages in Neogene (1-8 Myr) and early Permian (275-290 Myr) times. But for most of the Mesozoic era (65-250 Myr), CO2 levels were high (1,000-2,000 p.p.m.v.), with transient excursions to even higher CO2 (>2,000 p.p.m.v.) concentrations. These results are consistent with some reconstructions of past CO2 (refs 1, 2) and palaeotemperature records, but suggest that CO2 reconstructions based on carbon isotope proxies may be compromised by episodic outbursts of isotopically light methane. These results support the role of water vapour, methane and CO2 in greenhouse climate warming over the past 300 Myr.

Long-term growth of Ginkgo with CO(2) enrichment increases leaf ice nucleation temperatures and limits recovery of the photosynthetic system from freezing.

The importance of subzero temperature interactions with elevated CO(2) on plant carbon metabolism has received rather little attention, despite their likely role in influencing future vegetation productivity and dynamics. Here we focused on the critical issues of CO(2)-enrichment effects on leaf-freezing temperatures, subsequent membrane damage, and recovery of the photosynthetic system. We show that growth in elevated CO(2) (70 Pa) results in a substantial and significant (P<0.01) increase (up to 4 degrees C) in the ice nucleation temperature of leaves of Maidenhair tree (Ginkgo biloba), which was observed consistently throughout the 1999 growing season relative to their ambient CO(2) (35 Pa) counterparts. We suggest that increased sensitivity of leaves to ice damage after growth in elevated CO(2) provides an explanation for increased photoinhibition observed in the field early and late in the growing season when low nighttime temperatures are experienced. This new mechanism is proposed in addition to the earlier postulated explanation for this phenomenon involving a reduction in the rate of triose-P utilization owing to a decrease in the rate of carbohydrate export from the leaf.

Changes in N-linked oligosaccharides during seed development of Ginkgo biloba.

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-beta-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans. The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc2 approximately 0Man3Xyl1Fuc1 approximately 0GlcNAc2) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc2Man3Xyl1Fuc1GlcNAc2 and GlcNAc1Man3Xyl1Fuc1GlcNAc2 decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages. Concerning the endogenous substrates for plant endo-beta-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.

[Habitats and present situations of 13 rare precious medicinal materials in Funiu mountains].

The living resources are very abound in the Funiu mountains area because of its special geographical position and climate, and there are also many rare precious living beings and Chinese medicinal herbs, for example, Taxus mairei, Ginkgo biloba, Gastrodia elata, Dendrobium huashanense, Physochlaina infunbularis, Cypripedium japonicum, Dysosma versipellis, Moschus moschiferus, Megalobatrachus davidanus, Succinum, etc, and conscious protection and rational development is an urgent task.

Studies on the callus cultures of Ginkgo biloba and its metabolites-ginkgolides.

The production of ginkgolides in callus culture of Ginkgo biloba was reported. The affection of some physical factors and chemical substances on the induction and growth of calli was also investigated. A biologically quantitative method (platelet aggregation induced by PAF) and HPLC were successfully used for the determination of Ginkgolides A and B in all kinds of callus cultures. The result showed that the content of Ginkgolides B in the callus cultures varies from 0.005% to 0.01%, which is one of the best results for the callus culture of G.biloba in the world.