Premature cytokinesis in pollen mother cells of transgenic tobacco plants (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.)

For the majority of dicotyledonous plants, cytokinesis in PMC is staged only once, i.e., after the completion of two cycles of caryokinesis. In the article, a cytological picture and the frequency characteristics of anomalies are shown, in which the cytokinesis in the PMCs of transgenic tobacco plants was already initiated after the first meiotic division. It is shown that, in such cells, the basic processes of cytoskeletal reorganization, which is typical for the simultaneous type of cytokinesis, remained unmodified. However, in most cases, premature division of cytoplasm took place with abnormalities, e.g., with the formation of a membranous “tunnel” or “gash.” It has been detected that the initialization of an additional round of cytokinesis is not an obstacle to the performance of the nuclear cycle or cytokinesis after the second meiotic division. Thus, in the presence of this anomaly, there is a change in the type of cytoplasm division, i.e., of simultaneous to successive.     

Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants

Large quantities of intact generative cells and their protoplasts were isolated from pollen protoplasts of four liliaceous plants, and their structural features were investigated. The generative cells, liberated from the vegetative cell cytoplasm of the pollen protoplasts, were initially spindle-shaped with two long, oppositely oriented extensions, and were surrounded by two cell membranes, one on each side of a wall of uniform thickness. The generative nuclei, stained with 4′,6-diamidino-2-phenylindole (DAPI), showed ellipsoidal and highly condensed chromatin, whereas the generative cell cytoplasm, whose quantity was widely different from species to species, showed no fluorescence, suggesting the absence of plastid and mitochondria! DNA, although many mitochondria were present. The isolated generative cells, which were spindle-shaped at first, became spherical in shape in vitro. Immunocytochemistry and transmission electron microscopy revealed that this change was associated with the depolymerization of an axial array of microtubules present in generative cells in situ. These results are discussed in relation to the function of the generative cell within the bicellular pollen of angiosperms.     

Recent progress on sperm characterization in flowering plants

The organization, isolation and physiology of the angiosperm male gamete has recently emerged as an area of special interest. Detailed in vivo studies have revealed: (1) that the functional unit of male reproduction is a'male germ unit,'composed of two sperm cells and a physically associated vegetative nucleus; (2) that the two sperm cells are often cytoplasmically dimorphic, and (3) that some sperm cells appear to undergo preferential fertilization, preferentially fusing with either the egg or the proendospermaric central cell. Male gamete isolation has provided enrichments of up to 10⁷ cells ml^–1, with sperm integrity and viability demonstrated by the flunrochromatic reaction and ATP levels. Although results from biochemical and hybridoma antibody characterization are still preliminary, they indicate that sperm cells possess polypeptide expression patterns that are different from those of surrounding cells. These findings suggest the existence of an independent developmental program in the angiosperm male gamete.     

Ultrastructural Observations of Papaver rhoeas Mature Pollen Grains

The mature pollen grain of Papaver rhoeas is bicellular. The vegetative cell contains numerous mitochondria; endoplasmic reticulum is not very extensive and there are few ribosomes and plastids. Golgi bodies are in a very active state. The generative cell is lobed and spindle-shaped. The cytoplasm contains many, generally longitudinally arranged, bundles of microtubules. Other organelles are few in number, and include mitochondria, Golgi bodies and short cisternae of endoplasmic reticulum.     

ISOLATION OF A NUCLEUS-ASSOCIATED MICROTUBULE SCAFFOLD FROM SPIROGYRA (ZYGNEMATALES, CHAROPHYCEAE)

A phragmosome-like perinuclear scaffold attaches to helical chloroplast bands in the peripheral cytoplasm of Spirogyra crassa Kützing by rigid, stalk-like cytoplasmic strands. Balanced integration of forces within this scaffold by microfilament-based tension conveyed along stabilized microtubules (MTs) within the stalks controls transverse centering of the lenticular nucleus in the cylindrical cell prior to mitosis. Nuclei with their associated scaffolds were released from rapidly frozen, shattered cells upon thawing in a MT-stabilizing buffer and could be purified by a procedure combining filtration, differential centrifugation, and isopyknic centrifugation in a linear density gradient. In the osmotically indifferent gradient medium Nycodenz™, nuclei with adhering scaffolds attained a buoyant density of 1.26–1.27 g·cm⁻³. The nuclei maintained their energetically unfavorable, lenticular shape in vitro and appeared structurally preserved as judged by video-enhanced differential interference contrast and by fluorescent staining of DNA with 4'-6'-diamidino-2-phenylindole. The stalks of the scaffold, though still radiating from the nuclear rim, lost their straightness and were often severed during the isolation process. The distal ends of many unsevered stalks and of stalk fragments were still associated with chloroplast fragments. Persistent flexural rigidity of the scaffold stalks and indirect immunofluorescence indicated preservation of MTs. In addition to tubulin, both actin and an intermediate filament antigen of M _r ≈ 68 kDa were detected in the isolated nuclei with associated scaffolds by immunoblot with monoclonal antibodies.     

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF. Mol. Reprod. Dev. 50:79–85, 1998. © 1998 Wiley-Liss, Inc.     

The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments

The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed.     

Critical contact residues that mediate polymerization of nematode major sperm protein

The polymerization of protein filaments provides the motive force in a variety of cellular processes involving cell motility and intracellular transport. Regulated assembly and disassembly of the major sperm protein (MSP) underlies amoeboid movement in nematode sperm, and offers an attractive model system for characterizing the biomechanical properties of filament formation and force generation. To that end, structure-function studies of MSP from the nematode Caenorhabditis elegans have been performed. Recombinant MSP was purified from Escherichia coli using a novel affinity chromatography technique, and filament assembly was assessed by in vitro polymerization in the presence of polyethylene glycol. Prior molecular studies and structure from X-ray crystallography have implicated specific residues in protein-protein interactions necessary for filament assembly. Purified MSP containing substitutions in these residues fails to form filaments in vitro. Short peptides based on predicted sites of interaction also effectively disrupt MSP polymerization. These results confirm the structural determination of intermolecular contacts and demonstrate the importance of these residues in MSP assembly.     

被子植物生殖细胞和精细胞

随着被子植物精细胞分离技术的突破和细胞生物学以及分子生物学技术的发展,对被子植物精细胞的研究不断深入。在以前细胞生物学研究的基础上结合近年来的分子生物学研究结果对被子植物雄性生殖细胞的产生、精细胞的形成和发育以及有关精细胞的表面蛋白质、精细胞的特异启动子、精细胞cDNA文库的构建等分子生物学研究进展和今后的发展趋势进行了综述。     

Molecular aspects of microtubule dynamics in plants

Microtubules are highly dynamic structures that play a major role in a wide range of processes, including cell morphogenesis, cell division, intracellular transport and signaling. The recent identification in plants of proteins involved in microtubule organization has begun to reveal how cytoskeleton dynamics are controlled.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻。在其活体花粉的4个发育阶段(Ⅰ．小孢子晚期;Ⅱ．二细胞早期;Ⅲ．二细胞晚期;Ⅳ．三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化。发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移。而这些位移与微丝骨架的结构变化和运动有密切关系。在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移。在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架。这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据。     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 1. Descriptive cytology and three-dimensional organization

The generative cell is initiated as a small, lenticular, unpolarized cell with a cell wall traceable to two origins: the external segment originates as intine, while an inner callose positive cell wall forms de novo. As the lenticular generative cell begins its migration into the pollen cytoplasm, the generative cell becomes polarized both externally and internally, displaying a characteristic shape and patterns of organelle distribution oriented with respect to the vegetative nucleus and independent of pollen aperture location. Separation of the generative cell from the pollen wall begins at the end opposite the vegetative nucleus and results in an elongating protuberance at the opposite end of the generative cell; this becomes associated with a preformed groove located on the surface of the vegetative nucleus. The generative cell subsequently separates from the intine near the vegetative nucleus and moves progressively toward the opposite end of the cell; during this separation, the edge of the wall facing the intine becomes callose-positive and remains so until separating from the intine. The generative cell becomes a free cell within the pollen, which is in physical association with the vegetative nucleus. Generative cell organization and organelle content become increasingly polarized during maturation, with microtubules evident both in the elongating protuberance of the generative cell and in association with organelles. The generative nucleus migrates away from the vegetative nucleus and toward the plastid-rich end of the generative cell, whereas mitochondria are more generally distributed within the cell. Generative cell polarization is made permanent during mitotic division and cytokinesis, i.e., two sperm cells differing in morphology are formed: the larger cell associated with the vegetative nucleus (S_vn) contains a majority of the mitochondria, and the smaller, unassociated sperm cell (S_ua) receives the plastids.     

Plant microtubules reorganization under the indirect UV-B exposure and during UV-B-induced programmed cell death

The role of microtubules in cellular pathways of UV-B signaling in plants as well as in related structural cell response become into focus of few last publications. As microtubules in plant cell reorient/reorganize (become randomized, fragmented or depolymerized) in a response to direct UV-B exposure, these cytoskeletal components could be involved into UV-B signaling pathways as highly responsive players. In the current addendum, indirect UV-B-induced microtubules reorganization in cells of shielded Arabidopsis thaliana (GFP-MAP4) primary roots and the correspondence of microtubules depolymerization with the typical hallmarks of the programmed cell death in Nicotiana tabacum BY-2 (GFP-MBD) cells are discussed.     

Flowering plant sperm cells: Isolation from pollen of Gerbera jamesonii (asteraceae)

Sperm cells were isolated from pollen of Gerbera jamesonii by gentle grinding in a medium containing buffer, osmoticum, protectants and ions. Sperm nuclei were identified by means of the DNA fluorochrome DAPI. The sperm cells were observed by Nomarski optics and by scanning electron microscopy. Isolated sperm were osmotically sensitive and excluded Evans Blue. The structure of the isolated sperm cells was similar in shape and size to that of sperm cells extruded from fresh pollen and in fixed pollen.     

Lectin-mediated sperm agglutination of bufo arenarum and leptodactylus chaquensis spermatozoa

Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination.     

Optimization of culture conditions for the formation of sperm cells in pollen tubes of tradescantia

A culture medium and culture conditions are described that enable generative cell division and sperm formation to occur in a large proportion (greater than 70%) of the pollen tubes of Tradescantia paludosa within six to eight hours of culture of pollen. The nature of the nitrogen source, speed of shaking, and ratio of pollen to medium are important parameters in determining the extent of sperm formation. Addition of the plant hormones indole acetic acid, gibberellic acid, and kinetin to the growth medium does not influence generative cell division.     

Microtubule Associated Proteins in Plants and the Processes They Manage

Microtubule associated proteins （MAPs） are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band, and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification, transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis. This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.     

Tissue- and development-specific distributions of cytoskeletal elements in growing cells of the maize root apex

ABSTRACT

Indirect immunofluorescence performed using sections of actively growing maize root apices fixed and then embedded in low-melting-point Steedman's wax has proved efficient in revealing the arrangements and reorganizations of motility-related cytoskeletal elements which are associated with root cell development and tissue differentiation. This powerful, yet relatively simple, technique shows that specific rearrangements of both microtubular (MT) and actin microfilament (MF) arrays occur in cells as they leave the meristem and traverse the transitional region interpolated between meristem and elongation region. Cytoskeletal and growth analyses have identified the transition zone as critical for both cell and root development; it is in this zone that cell growth is channelled, by the cytoskeleton, into a strictly polarized mode which enables root tips to extend rapidly through the soil in search of water and nutrients. An integrated cytoskeletal network is crucial for both the cytomorphogenesis of individual cells and the overall morphogenesis of the plant body. The latter process can be viewed as a reflection of the tight control which cytoskeletal networks exert not only over cell division planes in the cells within meristematic apices but also over the orientation of cell growth in the meristem and elsewhere. Endoplasmic MTs interconnecting the plasma membrane with the nucleus are suggested to be involved in cell division control; they may also act as a two-way cytoskeletal communication channel for signals passing to and fro between the extracellular environment and the genome. Moreover, the dynamism of endoplasmic MTs exerts direct effects on chromatin structure and the accompanying nuclear architecture and hence can help exert a cellular level of control over cell growth and cell cycle progression. Because the inherent dynamic instability of MTs depends on the concentration of tubulin dimers within the cytoplasm, we propose that when asymmetric cell division occurs, it will result in two daughter cells which differ in the turnover rates of their MTs. This phenomenon could be responsible for different cell fates of daughter plant cells produced by such cell divisions.