The ontogeny of the vascular cambium inGinkgo biloba roots

Observation was made on early ontogeny of vascular cambium in the developing root ofGinkgo biloba L. After completion of root elongation, the vascular meristem gradually acquires cambial characteristics. Strips of the periclinal division of cells in transverse section are observed on the inner side of phloem when the primary xylem and phloem in the stele have been established. The strips are united into a continuous layer between phloem and xylem. In tangenital section, the procambium shows a homogeneous structure, which is initially composed of short cells with transverse end walls and subsequently, of long cells with tapering ends. Then, the procambium is organized into two systems of cells; axial strands of short cells with transverse end walls resulting from the sporadic transverse divisions of long cells, and long cells with tapering ends. Still later, the short cells are divided frequently in a trasverse plane exhibiting one or a few cells in width and several decades of cells in height, while the long cells are elongated. The frequency of transverse divisions of the short cells decreases in subsequent stages. Eventually, the short cells in axial strands are vertically separated from one another by the elongation of neighboring long cells and by the decrease in the frequency of transverse divisions of short cells themselves. Cambial initials occur in two forms; ray initials a few cells in height and one cell in width derived from the short cells, and fusiform initials with tapering ends derived from the long cells.     

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.     

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.     

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     

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     

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     

Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

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     

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.     

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.     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

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.     

Reconstruction of the flagellar apparatus and microtubular cytoskeleton inPyramimonas gelidicola (Prasinophyceae,Chlorophyta)

Summary The absolute configuration of the flagellar apparatus inPyramimonas gelidicola McFadden et al. has been determined and shows identity withP. obovata, indicating that they are closely related. Comparison with the flagellar apparatus of quadriflagellate zoospores from the more advancedChlorophyceae suggest thatPyramimonas may be a primitive ancestral form. The microtubular cytoskeleton has been examined in detail and is shown to be unusual in that it does not attach to the flagellar apparatus. Cytoskeletal microtubules are nucleated individually, and this is interpreted as an adaptation to the methods of mitosis and scale deployment. In view of the primitive nature of these processes, it is proposed that this type of cytoskeletal organization may represent a less advanced condition than that of the flagellar root MTOCs (microtubule organizing centers) observed in theChlorophyceae.     

A cytoskeletal system predicts division plane in meiosis ofSelaginella

Summary An ultrastructural investigation of the monoplastidic microsporocytes ofSelaginella arenicola revealed a unique cytoskeletal array that predicts the future division plane before nuclear division takes place. By midprophase of the first meiotic division, the single plastid has divided once and the two plastids lie on opposite sides of the nucleus which is elongated in the plane of the incipient metaphase I spindle. A cytoplasmic structure, the procytokinetic plate (PCP), predicts the division plane of of both plastid and cytoplasm. The PCP consists of a distinct concentration of vesicles lying in the future division plane and an elaborate system of microtubules aligned parallel to the long axis of plastids and nucleus. Microtubules of the axially aligned system appear to terminate in clusters of vesicles in the central zone of the PCP. The PCP with axially aligned microtubules is as predictive of the division plane in these meiotic cells as is the girdling preprophase band of microtubules in mitotic cells.     

Microtubules and the flagellar apparatus in zoospores and cysts of the fungusPhytophthora cinnamomi

Summary A correlated immunofluorescence and ultrastructural study of the microtubular cytoskeleton has been made in zoospores and young cysts ofPhytophthora cinnamomi. Labelling of microtubules using antibodies directed towards tubulin has revealed new details of the arrangement of the flagellar rootlets in these cells, and of the variability that occurs from cell to cell. Most of the variation exists at the distal ends of the rootlets, and may be correlated with differences in cell shape in these regions. The rootlets have the same right and left configuration in all zoospores. The arrangement of the rootlet microtubules at the anterior end of the zoospores raises the possibility that the microtubules on the left hand side of the groove may not comprise an independent rootlet which arises at the basal bodies.The absolute configuration of the flagellar apparatus has been determined from ultrastructural observations of serial sections. In the vicinity of the basal bodies, there is little, if any, variation between individuals, and the structure of the flagellar apparatus is similar to that described for related species of fungi. Two ribbon-like coils surround the central pair of microtubules at the distal tip of the whiplash flagellum, and clusters of intramembranous particles, similar to ciliary plaques, have been found at the bases of both flagella. There are two arrays of microtubules associated with the nucleus in the zoospores. One array lies next to the outer surface of the nuclear envelope, and probably functions in the shaping and positioning of the apex of the nucleus. The nuclear pores in this region are aligned in rows alongside these microtubules. The second array is formed by kinetochore microtubules which extend into a collar-like arrangement of chromatin material around the narrow end of the (interphase) nucleus. During encystment, all flagellar rootlets are internalized when the flagella are detached at the terminal plate. The rootlets arrays are no longer recognizable 5–10 minutes after the commencement of encystment.     

Monoclonal antibodies to surface and cytoskeletal components of the spermatozoid ofPteridium aquilinum

Summary Detergent extracted spermatozoids of the fernPteridium aquilinum were used as mixed antigen preparations for raising monoclonal antibodies in order to obtain reagents for detecting as yet uncharacterized components of the plant cytoskeleton. Selected antibodies were studied by immunofluorescence microscopy of developing spermatids and mature spermatozoids. Some reacted directly with fixed cells, others required permeabilization treatments with cold methanol or Triton X-100. AntibodiesPas2D9 andPas6D7 bind to glycoprotein antigenic determinants that are exposed on the surface of the plasma membrane. Several antibodies interact with cytoskeletal components.Pas1D3,Pas5D8 andPas5F4 bind to the cytoskeleton of permeabilized cells including the flagella. Three react specifically with the flagellar band or associated components:Pas2G6 reacts with the whole flagellar band but shows a prominent binding to basal bodies,Pas5E2 binds exclusively to basal bodies, andPas5E7 detects mitochondria associated with the flagellar band. Cross-reactions to wheat root tip cells at different stages of the cell cycle are described inMarc andGunning (1988).Abbreviations MLS multilayered structure - MT microtubule - MAb monoclonal antibody - MAP microtubule associated protein     

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.

---

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.