Ultrastructural characterization of the locomotory cytoskeletal system of the spermatozoid inGinkgo biloba

Summary The development of the locomotory cytoskeletal system of sperm is carefully coordinated with the development of the sperm inGinkgo biloba. Here we report further ultrastructural characterization of the locomotory cytoskeletal system in the developing spermatid and mature spermatozoid, particularly with respect to the initiation and early development of the flagellar apparatus. A multilayered structure (MLS) assembles from an electron-dense matrix that self-organizes after blepharoplast breakup and then further elongates. At the tail of the assembling MLS, the spline microtubules connect to an anterior beak of the nuclear envelope. Nuclear-pore complexes are found on the nuclear envelope close to this beak. The mitochondria which elongate and line up one behind the other are tightly associated with the MLS. The MLS ofG. biloba is composed of an upper layer of parallel spline microtubules and a lower layer consisting of a fibrous lamellar strip composed of paralled fibers about 9 nm in diameter. Higher-magnification images show that the fully assembled fibers of the lamellar strip consist of subunits which suggest that protofilaments are involved in the assembly processes. A unique cytoskeletal system of the spermatozoid inG. biloba is given by the anterior bundle of microtubules. This bundle, in which microtubules are arranged parallel to each other, forms between the plasmalemma and the MLS and is about 214–392 nm in cross section. These microtubules expand spirally along the MLS band. Other details of cellular fine structure of the mature spermatozoid are described.     

The origin and development of vascular cambium in girdled stems ofEucommia ulmoides Oliv.

We conducted anatomical studies of girdled stems ofEucommia ulmoides at various developmental stages to elucidate the origin and development of callus and the vascular cambium. In the transverse view, ray initial cells in the cambial zone began to divide both periclinally and anticlinally 2 d after girdling. Fusiform initial cells started to enlarge at 3 d, then gradually proliferated via periclinal divisions. Thus, the callus was derived from the ray initial cells of the cambial zone as well as from fusiform initial cells. In the tangential view, callus cells derived from ray initial cells were short while those from fusiform initial cells were long, thereby producing a heterogeneous structure. However, the fusiform initial cells underwent transverse divisions 10 d after girdling, which resulted in shorter cells and a homogeneous callus structure. Afterward, some short cells divided transversely while others elongated, so that a heterogeneous form was regained. Finally, the vascular meristem that was girdled early in its development redifferentiated from short and long cells in the callus. The long cells developed into fusiform initials, with the short ones becoming ray initials.     

Protective effects of ginkgo biloba against acetaminophen-induced toxicity in mice

Background: The analgesic acetaminophen (AAP) causes a potentially fatal, hepatic centrilobular necrosis when taken in overdose. It was reported that these toxic effects of AAP are due to oxidative reactions that take place during its metabolism. Objective: In this study, we aimed to investigate the possible beneficial effect of Ginkgo biloba (EGb), an antioxidant agent, against AAP toxicity in mice. Methods: Balb/c mice were injected i.p. with: (1) vehicle, control (C) group; (2) a single dose of 50 mg/kg Ginkgo biloba extract, EGb group; (3) a single dose of 900 mg/kg i.p. acetaminophen, AAP group, and (4) EGb, in a dose of 50 mg/kg after AAP injection, AAP + EGb group. Serum ALT, AST, and tumor necrosis factor-alpha (TNF-α) levels in blood and glutathione (GSH), malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and collagen contents in liver tissues were measured. Formation of reactive oxygen species in hepatic tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lusigenin probe. Tissues were also examined microscopically. Results: ALT, AST levels, and TNF-α were increased significantly (p < 0.001) after AAP treatment, and reduced with EGb. Acetaminophen caused a significant (p < 0.05–0.001) decrease in GSH levels while MDA levels and MPO activity were increased (p < 0.001) in liver tissues. These changes were reversed by EGb treatment. Furthermore, luminol and lusigenin CL levels in the AAP group increased dramatically compared to control and reduced by EGb treatment (p < 0.01). Conclusion: Our results implicate that AAP causes oxidative damage in hepatic tissues and Ginkgo biloba extract, by its antioxidant effects protects the tissues. Therefore, its therapeutic role as a “tissue injury-limiting agent” must be further elucidated in drug-induced oxidative damage.     

The anatomy of the chi-chi of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> suggests a mode of elongation growth that is an alternative to growth driven by an apical meristem

The chi-chi of Ginkgo biloba L. are cylindrical woody structures that grow downwards from the branches and trunks of old trees, eventually entering the soil where they give rise to adventitious shoots and roots. Examination of segments of young chi-chi taken from a mature ginkgo tree revealed an internal woody portion with irregular growth rings of tracheid-containing secondary xylem covered by a vascular cambium and bark. The cambium was composed of both fusiform cells and parenchymatous ray cells. Near the tip of the chi-chi, these two types of cambial cells had orientations ranging between axial, radial and circumferential with respect to the cylindrical form of the chi-chi. The xylem rays and tracheids that derived from the cambium showed correspondingly variable orientations. Towards the base of the chi-chi, the fusiform cells and young tracheids were aligned parallel to the axis, indicating that the orientation of the cambial cells in basal regions of the chi-chi gradually became normalised as the tip of the chi-chi extended forwards. Nevertheless, in such basal sites, tracheids near the centre of the chi-chi showed variable orientations in accordance with their mode of formation during the early stages of chi-chi development. The initiation of a chi-chi is proposed to derive from a localised hyperactivity of vascular cambial-cell production in the supporting stem. The chi-chi elongates by tip growth, but it does so in a manner different from organ growth driven by an apical meristem. It is suggested that the chi-chi of Ginkgo is an “evolutionary experiment” that makes use of the vascular cambium, not only for its widening growth but also for its elongation.     

Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees

The activity of a -(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000–100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min^–1 mg^–1 of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min^–1 cell^–1) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.     

The nuclear lamina in male generative cells ofGinkgo biloba

Using selective extraction and diethylene glycol distearate embedment and embedment-free electron microscopy, we demonstrated nuclear lamina-like structures in sperm cells ofGinkgo biloba. A well-organized nuclear matrix network was also observed. Further studies were undertaken to determine whether or not lamin-like components exist in the pollen and sperm cells. Immunofluorescence staining using monoclonal antibodies against different animal lamins revealed lamins localized in the nuclear compartment of the sperm cells. Western blotting showed that in pollen grains there are two positive crossreaction bands at 66 kDa and 86 kDa, recognized by antibodies specific to animal lamins; in sperm cells there was only one, at 66 kDa. These results indicate that nuclear lamina containing both A-type and B-type lamins was present in male generative cells ofG. biloba. The data imply that plant lamins share some homology with animal lamins and may be conserved during evolution.     

Somatic embryogenesis from immature zygotic embryos ofGinkgo biloba

Immature zygotic embryos ofG. biloba were taken, at various developmental stages, from ovules harvested in November 1993. Zygotic embryos showing the beginning of the cotyledonary development cultured on modified Murashige & Tücker (1969) media proliferated intensely. In fact, 98.5% of the immature zygotic embryos produced embryogenic and undifferentiated tissues (calluses), in proportions varying depending on the hormonal composition of the induction media. After two weeks of culture, direct embryogenesis was observed on the hypertrophic cotyledons when benzyladenine 10 M was used as the sole plant growth regulator in the induction media. The addition of different concentrations of NAA (5–10–20 M) and of BA (5 M) to the induction media led to an indirect embryogenesis after two months, when the calluses were transferred to the development media without auxin. The highest frequency of embryogenic tissues (90–95%) and the highest number of somatic embryos per explant (9.6) were obtained with benzyladenine (10 M) as the sole exogenous growth factor. Some embryos isolated mechanically or in situ on the callus developed as far as the later cotyledonary stage.Abbreviations AUX Auxin - BA Benzyladenine - CYT Cytokinin - IZE Immature zygotic embryo - MT Murashige & Tücker (1969) medium - NAA Naphtaleneacetic acid     

The secretory apparatus of Ginkgo biloba: structure,differentiation and analysis of the secretory product

Summary The differentiation of the secretory cavities of Ginkgo stem and the structural organization of the epithelial cells were followed by light and electron microscopy. The mode of formation of the cavities is schizo-lysigeneous. Functional complexes of leucoplasts and associated endoplasmic reticulum (ER) membranes are assumed to be the site of synthesis and translocation of the lipophilic secretory product. Most of the endoplasmic reticulum membranes are paired. The content of the cavities was directly collected and analysed by low- and high-resolution mass spectrometry. The cavities contain anacardic acids and cardanols, which are long-chain phenol lipids not characteristic of Ginkgo. The relationship between the plastid/ER complexes and the production of these secondary metabolites is discussed.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Embryogenesis from microspores of Ginkgo biloba L., a medicinal woody species

Summary The present work establishes that isolated microspores of Ginkgo biloba L. cultured at densities of 1.5 to 5·10⁴ per milliliter in Bourgin and Nitsch (1967) liquid medium are able to divide, both in the presence and in the absence of exogenous growth regulators, and to germinate by growing a pollen tube. In all experiments the microspores exhibited various modes of division leading to embryo formation in the liquid medium. Four weeks later, the microspores which had been previously submitted to various electrical stresses showed pro-embryo development earlier than those which had not. After ten weeks the number of embryos was found to be 300 to 5300 ml^–1 following the experiments. When the embryos exhibited a slower growth in liquid medium, they were transferred onto various solid media for maturation. Two months later, embryos had proliferated visibly.Abbreviations BN Bourgin and Nitsch (1967) medium - IAA Indole-3-acetic acid - KIN Kinetin - GS Growth substances     

Comparative anatomy and ultrastructure of active and dormant vascular cambium in rhizome ofBotrychium ternatum

Vascular cambium ofBotrychium ternatum rhizome varied according to age, position and season was studied by light and electron microscopy. Cambium at the 6th internode (6-year-old cambium) had the greatest number of active cambial cells in August and September, thus it was in the most active stage. The active cells were characterized by the presence of a large vacuole, few storage materials such as starch grains within plastids or lipid droplets, a thin tangential wall; and various cell organelles in the thin peripheral layer of cytoplasm. When the 6-year-old cambium reached its dormant season after November, the dormant cells were filled with numerous storage materials and had few cell organelles. Our observations suggested that the initiation and cessation of cambial activity may be correlated with the annual life cycle of this plant: the vegetative and reproductive leaves began to emerge in June and July, respectively, and the sporophyll withered in November after the spore dispersal. Most cambial cells at the 10th internode, which remained in a dormant state throughout the year, were filled with numerous storage materials. Our results indicated that the activity of vascular cambium in the 10th internode was determinate.     

Direct embryogenesis from female haploid protoplasts of Ginkgo biloba L., a medicinal woody species

Summary Haploid protoplasts isolated from prothallus (i.e. female gametophyte) of Ginkgo biloba, at densities ranging from 5×10⁴ to 10⁵ protoplasts per milliliter, were able to divide and form microclones which directly evolved into embryos, when they were cultured in two different liquid media. These were: the Murashige and Tucker medium (1969) modified by omitting ammonium ions and supplementing with glutamine, benzyladenine and various levels of naphthaleneacetic acid; or the Bourgin and Nitsch medium (1967) without growth regulators, supplemented with coconut milk. Three months later, the number of embryos ranged from 165 to 1900 embryos ml^–1 depending on the culture medium. After four months, embryos at whatever stage (globular, oblong or heart) exhibited a slow growth, which delayed the transfer onto solid media.Abbreviations BA 6-benzyladenine - BN Bourgin and Nitsch (1967) medium - MT Murashige and Tucker (1969) medium - NAA naphthaleneacetic acid     

Stem anatomy of Dolichos lablab Linn (Fabaceae): Origin of cambium and reverse orientation of vascular bundles

Secondary growth in the stem of Dolichos lablab is achieved by the formation of eccentric successive rings of vascular bundles. The stem is composed of parenchymatous ground tissue and xylem and phloem confined to portions of small cambial segments. However, development of new cambial segments can be observed from the obliterating ray parenchyma, the outermost phloem parenchyma and the secondary cortical parenchyma. Initially cambium develops as small segments, which latter become joined to form a complete cylinder of vascular cambium. Each cambial ring is functionally divided into two distinct regions. The one segment of cambium produces thick-walled lignified xylem derivatives in centripetal direction and phloem elements centrifugally. The other segment produces only thin-walled parenchyma on both xylem and phloem side. In mature stems, some of the axial parenchyma embedded deep inside the xylem acquires meristematic activity and leads to the formation of thick-walled xylem derivatives centrifugally and phloem elements centripetally. The secondary xylem comprises vessel elements, tracheids, fibres and axial parenchyma. Rays are uni-multiseriate in the region of cambium that produces xylem and phloem derivatives, while in some of the regions of cambium large multiseriate, compound, aggregate and polycentric rays can be noticed.     

Cytokinins in the leaves of Ginkgo biloba. II. Metabolism and transport of 8 (14C) zeatin applied to shoot explants

Irrespective of their age, leaves of Ginkgo biloba metabolised applied 8 (¹⁴C) zeatin to compounds of similar chromatographic properties. Glucosylation is apparently not a normal feature of cytokinin metabolism in immature leaves. However, the application of zeatin to these leaves did result in the formation of metabolites which co-chromatographed with glucosylated cytokinins. As far as cytokinin metabolism is concerned therefore, this application of excess zeatin allowed immature leaves to behave as mature or senescing leaves. Overall metabolism was fastest in immature leaves. From the metabolites formed it would appear as if oxidation, which resulted in the formation of a metabolite which co-eluted with N-(purin-6-yl)glycine, was also important in immature leaves. In senescing leaves glucosylation was the major form of metabolism. Extraction and re-application of the polar metabolites (formed from zeatin) to mature leaves resulted in the formation of compounds which co-chromatographed with zeatin. This suggests that these compounds could serve as precursors for zeatin or could be hydrolysed to form zeatin.Very little of the applied radioactivity was exported from the leaves irrespective of their physiological age. When the metabolites, obtained after zeatin application to mature leaves, were extracted and reapplied to the leaves, export of radioactive material was much improved. The results suggest that should cytokinins such as zeatin be translocated to mature leaves of this deciduous gymnosperm their export from the leaves would be unlikely unless first metabolised. In all probability the metabolites concerned are cytokinin glucosides.The financial support of the C.S.I.R., Pretoria, is gratefully acknowledged.     

Molecular cloning, characterization and expression of atpA and atpB genes from Ginkgo biloba

The chloroplast ATP synthase (ATPase) utilizes the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. The chloroplast ATPase α and β subunits are the essential components of multisubunit protein complex. In this paper, the full-length cDNA and genomic DNA of ATPase α (designated as GbatpA) and β (designated as GbatpB) subunit genes were isolated from Ginkgo biloba. The GbatpA and GbatpB genes were both intronless. The coding regions of GbatpA and GbatpB were 1530 bp and 1497 bp long, respectively, and their deduced amino acid sequences showed high degrees of identity to those of other plant ATPase α and β proteins, respectively. The expression analysis by RT-PCR revealed that GbatpA and GbatpB both expressed in tissue-specific manners in G. biloba and might involve in leaf development. The recombinant GbATPB protein was successfully expressed in E. coli strain using pET28a vector with ATPase activity as three times high as the control, and the results showed that the molecular weight of the recombinant protein was about 54 kDa, a size that was in agreement with that predicted by bioinformatics analysis. This study provides useful information for further studying on overall structure, function and regulation of the chloroplast ATPase in G. biloba, the so-called “living fossil” plant as one of the oldest gymnosperm species. These authors contributed equally to this work     

Structure and function of the tentpole in the reproductive process of Ginkgo biloba L.

The tentpole is a unique structure of the female gametophyte in Ginkgo biloba; however, its exact functions in the reproductive process are unclear. In the present study, we used semi-thin sectioning and electron microscopy to study the structure and function of the tentpole during fertilization in G. biloba. The tentpole was always initiated between two or more deeply immersed archegonia. Before fertilization, the tentpole had developed into a column-like structure, protruding toward the archegonial chamber; cells at the periphery of tentpole were loosely ranged, and abundant lipid droplets and starch grains were accumulated in the tentpole cells. After fertilization, the tentpole degenerated, and some membranous debris was overlaid on its surface. In addition, there were significant decreases in the lipids and starch grains. These results suggested that the tentpole led to the degeneration of the megaspore membrane and then supported the pliable apex of the nucellar tissues. Importantly, the tentpole also contributed to supplying nutrition for fertilization and embryo development.     

Deterrents extracted from the leaves of Ginkgo biloba: effects on feeding and contact chemoreceptors

Powdered dried ginkgo tree leaves were subjected to various chemical extractions and successive extracts were monitored for antifeedant activity against larvae of Pieris brassicae. Many fractions moderately inhibited food intake, and some were deterrent at levels as low as 25–50 ppm. Some behaviourally highly active fractions were tested electrophysiologically for neural responses in the maxillary taste sensilla. These extracts appeared to stimulate deterrent receptors. There were distinct differences in responses between Pieris brassicae and P. rapae. Ginkgolide A, B, and C each strongly stimulated deterrent receptors in P. rapae, which corresponds with the observation (Matsumoto & Sei, 1987) that these compounds are effective antifeedants for this species. No toxic effects were observed in insects after feeding for 24 h on diets containing ginkgo extracts.

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Résumé Des feuilles de Gingko séchées et broyées ont été soumises à différentes extractions. On a testé l'activité déterrante (antiapprétant) des extraits successifs sur les larves de Pieris brassicae. Un grand nombre de fractions inhibent modérément la consommation, et certaines sont déterrantes à des concentrations aussi faibles que 25–50 ppm. Par électrophysiologie, on a testé la réponse nerveuse des sensilles gustatives maxillaires à certaines fractions qui montraient une forte action sur le comportement. Il s'avère que ces extraits stimulent les récepteurs déterrants. Les réponses diffèrent pour P. brassicae et P. rapae. Les gingkolides A, B et C stimulent chacun fortement les récepteurs déterrants chez P. rapae, ce qui correspond aux observations (Matsumoto & Sei, 1987) selon lesquelles ces composés sont des antiappétants efficaces pour cette espèce. On n'a pas observé d'effet toxique sur les insectes nourris durant 24 heures par des diètes contenant des extraits de Gingko.

     

Cloning and Characterization of a Root-specific Expressing Gene Encoding 3-hydroxy-3-methylglutaryl Coenzyme a Reductase from Ginkgo biloba

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34) catalyzes the first committed step in mevalonic acid (MVA) pathway for biosynthesis of isoprenoids. The full-length cDNA encoding HMGR was isolated from Ginkgo biloba for the first time (designated as GbHMGR, GenBank accession number AY741133), which contained a 1713 bp ORF encoding 571 amino acids. The GbHMGR genomic DNA sequence was also obtained, revealing GbHMGR had four exons and three introns. The deduced GbHMGR protein showed high identity to other plant HMGRs and contained two trans-membrane domains and a catalytic domain. The three dimensional model of GbHMGR represented a typical spatial structure of HMGRs. The Southern blot and RT-PCR assay results indicated that GbHMGR belonged to a small gene family, and expressed in a tissue-specific manner with a low level expression being only found in root. The potential significance of GbHMGR gene was also discussed.