Developmental synchronization of male and female gametophytes in Ginkgo biloba and its neck mother cell division prior to fertilization

This study investigated male and female gametophytes in Ginkgo biloba, while a droplet of fluid was present in the fertilization chamber and found that the central cell, the generative cell and the neck mother cell divided simultaneously prior to fertilization. In male gametophytes, the generative cell divided to yield two sperm cells. Concomitantly, the two neck mother cells of the archegonium increased in size then divided asymmetrically resulting in two big cover cells and two small base cells. Each cell had a fixed end in direct contact with an adjacent jacket cell and a free end overlapping its counterpart. This unique arrangement could allow for their free ends to swing into the fertilization chamber as a result of the force from the interior of the archegonium where a polar periclinal division had occurred to produce a canal cell and an egg. The subsequent withdrawal of the content of the archegonium may facilitate the entry of sperm into the archegonium. The neck apparatus closed after the fertilization occurred. The concurrence of the above divisions and the delicate structure of neck apparatus suggest that the gametophytes undergo a synchronization process to become receptive at the time of fertilization. However, the formation of neck cells and the opening time of neck apparatus of the archegonia within the same ovule were slightly different, which could lead to the formation of zygotes at a temporally distinct interval. The earlier formed zygote may progress as the only mature embryo in the ovule.     

地钱颈卵器发育和卵发生的显微观察及细胞化学研究

对地钱（Marchantia polymorpha）颈卵器发育和卵发生过程进行了显微观察和细胞化学的研究,颈卵器起始于原始细胞,该细胞呈乳突状,经横分裂产生基细胞和顶细胞,顶细胞经3次纵斜向分裂和1次横分裂产生初生细胞,初生细胞是颈卵器内的第一个细胞,经横分裂产生中央细胞和颈沟母细胞,前者产生1个腹沟细胞和1个卵细胞,后者最终产生4个颈沟细胞。颈卵器的成熟表现为颈部显著伸长和腹部膨大,卵细胞成熟时具有不规则的核,细胞质内含有丰富的囊泡和颗粒物,卵细胞周围充满粘性物质,细胞化学研究表明,该粘性物质为多糖,卵细胞质中深染色的颗粒可能为脂类物质,腹沟细胞自产生后就逐渐退化,颈沟细胞的退化迟于腹沟细胞,其数量通常为4个,偶尔可见5个颈沟细胞或具有双核的现象。     

Ultrastructure of vascular cambial cell cytokinesis in pine seedlings preserved by cryofixation and substitution

Summary.  Trees depend on the secondary vascular cambium to produce cells for new xylem and phloem. The fusiform cells of this lateral meristem are long and narrow, presenting special challenges for arranging the mitotic spindle and phragmoplast. Fusiform cambial cells of Pinus ponderosa and Pinus contorta were studied by cryofixation and cryosubstitution which preserved ultrastructure and phases of cytokinesis with a resolution not previously attained. Membranous structures including the plasma membrane, tonoplast, and those of other organelles were smooth and unbroken, indicating that they were preserved while the protoplasm was in a fully turgid state. Mitotic spindles separated daughter chromosomes diagonally across the radial width of the cells. The cell plate was initiated at an angle to the cell axis between the anaphase chromosomes by a microtubule array which organized vesicles at the phragmoplast midline. Within the phragmoplast, vesicles initially joined across thin tubular projections and then amalgamated into a tubulo-vesicular network. Axial expansion of the cell plate generated two opposing phragmoplasts connected by a thin, extended bridge of cell plate and cytoplasm that was oriented along the cell axis. In the cytoplasmic bridge trailing each phragmoplast, the callose-rich tubular network gradually consolidated into a fenestrated plate and then a complete cell wall. Where new membrane merged with old, the parent plasmalemma appeared to be loosened from the cell wall and the membranes joined via a short tubulo-vesicular network. These results have not been previously reported in cambial tissue, but the same phases of cytokinesis have been observed in cryofixed root tips and suspension-cultured cells of tobacco. Received February 11, 2002; accepted May 31, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada. Abbreviations: CFS cryofixation and cryosubstitution; ER endoplasmic reticulum; HPF high-pressure freezing; PPB preprophase band.     

阔鳞瘤蕨颈卵器形成与卵发生的初步研究

运用光学显微镜与透射电镜对阔鳞瘤蕨（Phymatosorus hainanensis(Noot.) S.G.Lu）颈卵器形成和卵发生进行了研究。阔鳞瘤蕨颈卵器产生于雌配子体生长点下方分枝毛状体内侧。切片观察表明颈卵器起源于配子体表面的原始细胞,该细胞经两次不等分裂形成3个细胞,上下两个细胞分别发育为颈卵器的颈部与底部壁细胞,中间的细胞为初生细胞,含有较丰富的细胞器。初生细胞进行两次不等分裂产生颈沟细胞、腹沟细胞与卵细胞。成熟颈卵器内颈沟细胞和腹沟细胞退化,卵细胞上表面产生受精孔。本研究阐述了阔鳞瘤蕨颈卵器形成和卵发生的细胞学过程,对阐明蕨类植物雌性生殖器官的发育特征有一定的科学意义。     

乌蕨配子体发育及卵发生的显微观察

采用光学显微镜对乌蕨的配子体发育及卵发生的过程进行了研究,以阐明蕨类植物颈卵器发育特征,为揭示蕨类植物有性生殖机制以及鳞始蕨科的演化提供依据。结果表明:(1)乌蕨孢子黄褐色,具单裂缝,表面平滑或呈疣状纹饰;孢子接种12d萌发,萌发类型为书带蕨型,原叶体发育类型为铁线蕨型。(2)半薄切片观察表明,乌蕨颈卵器产生于原叶体生长点下方的表面细胞,即颈卵器原始细胞,该细胞经过两次分裂形成纵向3层细胞,最上层细胞发育为颈卵器的颈部壁细胞,中间层细胞即初生细胞再经过两次不等分裂产生颈沟细胞、腹沟细胞和卵细胞,此三细胞最初紧密贴合,随着颈卵器的发育,卵细胞与腹沟细胞间从两侧向中间产生分离腔,且腹沟细胞与颈沟细胞开始退化;分离腔逐渐向中间扩大,直至出现孔状结构,即受精孔;颈卵器发育后期,在卵细胞上表面形成染色较深的卵膜,颈沟细胞与腹沟细胞退化成絮状物。     

Cytokinesis in Coleochaete orbicularis (Charophyceae): an ancestral mechanism inherited by plants

Recently, highly vacuolate cells of Arabidopsis were shown to exhibit "polarized" cytokinesis, in which the phragmoplast and cell plate contact the mother cell wall and then progress from one side of the cell to the other, rather than forming uniformly outward from the cell center (Cutler and Ehrhardt, 2002, Proceedings of the National Academy of Sciences, USA 99: 2812-2817). It was not known if such a mechanism was unique to flowering plants or whether it occurred more broadly in the plant clade. To determine if a polar mechanism of cell division might have been characteristic of the first plants, differential interference contrast optics were used to examine living cells of the charophycean green alga Coleochaete orbicularis, a close relative of plants, with cytokinesis involving a phragmoplast. By recording images in different focal planes over time, such "polarized" cytokinesis was found in cells dividing either parallel or perpendicular to the edge of this radially symmetrical organism. Previously reported differences between these two types of division in Coleochaete were clarified. Polarized cytokinesis appears to be an ancestral mechanism of plant cell division inherited from the highly vacuolate cells of the charophycean algal ancestors of plants.     

SCANNING ELECTRON MICROSCOPY OF ANTHERIDIA AND ARCHEGONIA OF ANEMIA MEXICANA KLOTZSCH

Antheridia and archegonia of the fern Anemia mexicana were viewed with scanning electron microscopy (SEM). The mature antheridium is composed of a cap, ring, and basal cell with spermatozoids inside. The archegonium neck is composed of a neck canal cell surrounded by four rows of neck cells. The ventral canal cell and egg were not observed. The neck bends toward the notched meristem. The neck cells usually are uniform in shape and arrangement, but in some archegonia, shape and arrangement of neck cells was irregular. The apex of these archegonia often appeared swollen because of the random cell arrangement. In the presence of water, the antheridium cap is partially detached and the spermatozoids emerge. At this time, the neck cells open at the end of the archegonium in preparation for fertilization. The basic morphology of the antheridia and archegonia is similar to previous reports, although SEM provides more structural detail and a better three-dimensional visualization of these reproductive structures.     

水蕨颈卵器的形成与发育

主要运用电子透射显微镜技术对水蕨（Ceratopteris thalictroides（L．）Brongn）颈卵器形成与发育进行了研究。结果表明：水蕨颈卵器是由原叶体分生组织内颈卵器原始细胞形成的。该原始细胞经2次分裂形成3层细胞,上下两层发育成颈卵器颈部与底部的壁细胞,中层为初生细胞。初生细胞是颈卵器内雌配子发生的第一个细胞,该细胞经2次不等分裂形成1个卵细胞,1个腹沟细胞、1个双核的颈沟细胞。本研究首次阐明了水蕨颈卵器内细胞的发育顺序和特征。     

水蕨颈卵器的形成与发育

主要运用电子透射显微镜技术对水蕨(Ceratopteris thalictroides(L.)Brongn)颈卵器形成与发育进行了研究。结果表明:水蕨颈卵器是由原叶体分生组织内颈卵器原始细胞形成的。该原始细胞经2次分裂形成3层细胞,上下两层发育成颈卵器颈部与底部的壁细胞,中层为初生细胞。初生细胞是颈卵器内雌配子发生的第一个细胞,该细胞经2次不等分裂形成1个卵细胞,1个腹沟细胞、1个双核的颈沟细胞。本研究首次阐明了水蕨颈卵器内细胞的发育顺序和特征。     

Postmeiotic cytokinesis and pollen aperture number determination in eudicots: effect of the cleavage wall number

Summary.  In eudicot postmeiotic tetrads, apertures are usually joined in pairs in highly conserved areas. These appear to be located at the last points of contact persisting at the end of cytokinesis between the cytoplasm of the future microspores. In order to investigate the relationship between cytokinesis and aperture formation, aperture distribution within postmeiotic tetrads and the progression of meiosis were studied in Nicotiana tabacum cv. Ambalema. This variety (inbred line) produces about 85% tricolporate pollen and 15% tetracolporate pollen grains. In addition, about 7% of tetrads are composed of four equal-sized microspores and a supernumerary pseudomicrospore of small size and an equal proportion of tetrads exhibit unpaired apertures (these apertures are not joined in pairs within tetrads). Observation of cytokinesis indicates that both unpaired apertures and pseudomicrospores could result from the persistence of late communications between microsporocytes. Observations of tetrads indicate that an increase in the number of elements that are separated during cytokinesis is correlated with an increase in microspore aperture number. All data converge to support the hypothesis that aperture site determination is partly controlled by the number of walls formed to separate the different elements of the tetrad. Received May 22, 2002; accepted October 29, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Laboratoire de Ecologie, Systematique et Evolution, Batiment 362, Université Paris Sud, 91405 Orsay cedex, France.     

Accessory phragmoplast corrects abnormal cytokinesis in wheat x rye hybrids

The compensation for phragmoplast dysfunction in the male meiosis of F1 wheat × rye hybrids was described. In pollen mother cells (PMCs), he transition from central spindle fibers (forming a solid bundle) to phragmoplast (hollow cylinder) was blocked. This blockage suppresses the centrifugal movement of the phragmoplast and cell-plate formation. As a result, cells become binucleate. Sometimes, two nuclei fuse and form one restitution nucleus. In PMCs of the wheat × rye F1 hybrid D-144 gp 06 year (T. aestivum n. 93-60 t 9 × S. cereale n. Saratovskaya 7) with this phenotype, an additional phragmoplast is formed at the late telophase. This occurs by a common mechanism for the development of the immobile phragmoplast in the meiosis in bicotyledons; new phragmoplasts arise as a result of microtubule polymerization starting from the spindle poles. The accessory phragmoplast facilitates a new cell plate assembly and achievement of cytokinesis.     

Dynamics of microtubular cytoskeleton in higher plant meiosis. VI. Mechanisms of the successive cytokinesis

Intracellular morphological processes of successive cytokinesis in cereal pollen mother cells during normal and abnormal meiosis were studied. It was shown that the central spindle fiber system transforms into a phragmoplast at telophase. A model of centrifugal movement of the phragmoplast as a modification of B-anaphase has been proposed.     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.     

峨眉凤丫蕨配子体发育及卵发生的研究

用显微观察及透射电镜技术对峨眉凤丫蕨的配子体发育及卵发生过程进行了观察研究,以探讨其卵发生细胞学机制及蕨类植物演化关系。结果表明:(1)峨眉凤丫蕨孢子接种7～9d萌发,经丝状体和片状体阶段发育为心形原叶体,成熟原叶体雌雄同株,在原叶体基部产生精子器,在原叶体生长点下方产生颈卵器。(2)卵发生研究表明,峨眉凤丫蕨颈卵器产生于生长点下方的表面细胞,该细胞经2次分裂形成3层细胞,中间者为初生细胞,它经2次不等分裂产生卵细胞、腹沟细胞和颈沟细胞;新产生的卵与腹沟细胞间连接紧密,有发达的胞间连丝,随着发育,卵细胞与腹沟细胞之间产生分离腔,而腹沟细胞与卵细胞始终通过孔区相连;发育中期,卵核形成大量核外突;发育后期,在卵细胞外侧形成卵膜,孔区演变为受精孔,核外突数量减少。     

Altering of phragmoplast orientation as a result of excessive centrifugal movement in the absence of cell plate

The inability of phragmoplast to stop its centrifugal movement after reaching the mother cell membrane is described in abnormal meiosis with the arrest of cell plate formation. The excess of phragmoplast expansion leads to rotation of the whole telophase figure (phragmoplast with daughter nuclei) within the cell through 90 degrees. It has been suggested that this phenomenon may occur because of a the lack of signal stopping cytokinesis. Such a signal arises due to formation of daughter cell membranes.     

Feed-back regulation of successive meiotic cytokinesis

The paper considers a number of abnormal phenotypes with impaired temporal regulation of cytokinesis during the meiotic division of pollen mother cells. The phenomenon of “non-stop” cytokinesis with blocked arrest of the phragmoplast centrifugal motion and cell plate growth as well as incomplete and premature cytokinesis are described. The obtained data suggested a model for regulation of the processes involved in the arrest of the main cytokinesis processes during its completion in the plant meiosis.     

The cytoskeleton in the unique cell reproduction by conidiogenesis of the long-neck yeast Fellomyces (Sterigmatomyces) fuzhouensis

Summary. The morphology of conidiogenesis and associated changes in microtubules, actin distribution and ultrastructure were studied in the basidiomycetous yeast Fellomyces fuzhouensis by phase-contrast, fluorescence, and electron microscopy. The interphase cell showed a central nucleus with randomly distributed bundles of microtubules and actin, and actin patches in the cortex. The conidiogenous mother cell developed a slender projection, or stalk, that contained cytoplasmic microtubules and actin cables stretched parallel to the longitudinal axis and actin patches accumulated in the tip. The conidium was produced on this stalk. It contained dispersed cytoplasmic microtubules, actin cables, and patches concentrated in the cortex. Before mitosis, the nucleus migrated through the stalk into the conidium and cytoplasmic microtubules were replaced by a spindle. Mitosis started in the conidium, and one daughter nucleus then returned to the mother via an eccentrically elongated spindle. The cytoplasmic microtubules reappeared after mitosis. A strong fluorescence indicating accumulated actin appeared at the base of the conidium, where the cytoplasm cleaved eccentrically. Actin patches then moved from the stalk together with the retracting cytoplasm to the mother and conidium. No septum was detected in the long neck by electron microscopy, only a small amount of fine “wall material” between the conidium and mother cell. Both cells developed a new wall layer, separating them from the empty neck. The mature conidium disconnected from the empty neck at the end-break, which remained on the mother as a tubular outgrowth. Asexual reproduction by conidiogenesis in the long-neck yeast F. fuzhouensis has unique features distinguishing it from known asexual forms of reproduction in the budding and fission yeasts. Fellomyces fuzhouensis develops a unique long and narrow neck during conidiogenesis, through which the nucleus must migrate into the conidium for eccentric mitosis. This is followed by eccentric cytokinesis. We found neither an actin cytokinetic ring nor a septum in the long neck, from which cytoplasm retracted back to mother cell after cytokinesis. Both the conidium and mother were separated from the empty neck by the development of a new lateral wall (initiated as a wall plug). The cytoskeleton is clearly involved in all these processes. Correspondence and reprints: Department of Biology, Faculty of Medicine, Masaryk University, Tomešova 12, 602 00 Brno, Czech Republic.     

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis

The phragmoplast, a structure crucial for the completion of cytokinesis in plant cells, is composed of antiparallel microtubules (MTs) and actin filaments (AFs). However, how the parallel structure of phragmoplast MTs and AFs is maintained, especially during centrifugal phragmoplast expansion, remains elusive. Here, we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein (AtMAC). When AtMAC was deleted, the phragmoplast showed disintegrity during centrifugal expansion, and the resulting phragmoplast fragmentation led to incomplete cell plates. Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis. Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro, and the truncated AtMAC protein, N-CC1, was the key domain controlling the ability of AtMAC. Further analysis showed that N-CC1(51–154) is the key domain for binding MTs, and N-CC1(51–125) for binding AFs. In conclusion, AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion, which is required for complete cytokinesis.     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 g of cytochalasin B per ml but inhibited by 10 M oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday