The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998     

Origin of sperm cell association in the “male germ unit” ofBrassica pollen

The association of the two sperm cells inBrassica napus pollen following the generative cell division was investigated. The generative cell during division is located in the center of the pollen grain, within the vegetative cell. The space present between the two cells is slightly irregular as seen following standard glutaraldehyde fixation. After completion of mitosis vesicles appear in the equatorial plane, coalescing centripetally to form a cell plate which fuses with the membrane of the generative cell, dividing it in two sperm cells. They are isolated from the vegetative cell by the space between the two cell membranes and are separated from each other by a similar space resulting from the cell plate formed during cytokinesis.     

Generative cell division and sperm cell formation in barley

Summary Shortly before and during division, the generative cell of barley (Hordeum vulgare L.) is located near the vegetative nucleus, in the peripheral layer of the highly vacuolated vegetative cell at the aperture pole. This position is also characteristic of the two resulting sperm cells. Conventional mitosis of the generative cell is followed by cytokinesis through cell plate formation. Just after division, the two sperm cells are enclosed together within a common inner vegetative cell plasma membrane, and they gradually separate from each other only during pollen maturation. The space between the generative or sperm cell plasma membrane and the vegetative cell plasma membrane is very thin and appears to be devoid of a cell wall. Both the generative cell and the young sperm cells contain a normal set of organelles; plastids devoid of starch are only sporadically observed. Our data indicate that in Hordeum vulgare the generative cell divides after migrating inside the pollen grain. This follows the pattern of development well established for several species with tricellular pollen.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

Fine structure of sperm cells in pollen grains ofBeta

Summary The structure of sperm cells in mature trinucleate pollen grains ofBeta vulgaris L. was studied with the electron microscope. The ellipsoidal sperm cell nuclei and cytoplasm are products of mitosis and cytokinesis of the ellipsoidal generative cell. Each sperm cell is separated from the vegetative cytoplasm by two contiguous membranes which enclose its cytoplasm and nucleus. Microtubules present in the sperm cell cytoplasm may be responsible for sperm cell motility.Approved as Journal paper Nr. 846, Utah Agricultural Experiment Station, Logan, Utah.     

Ultrastructure of Male Gametophyte in Wheat II. Formation and Development of Sperm Cell

Ultrastructural events in wheat sperm cell development were examined from the division of generative cell stage to the maturation of sperm cell in pollen grains. The results are smnmarized as follows: 1. The generative cell in forming microspore by mitosis goes through a series of changes including tile displacement and transformation. It finally becomes a spindle-shaped cell getting ready for another mitosis. The generative cell at this stage is naked. it is only surrounded by both membranes of its own and vegetative cell Most part of the generative cell is occupied by the conspicuous elliptical nucleus with highly condensed chromatin. With the exception of ribosomes, the organelles in the thin layer of generativc cell cytoplasm are obviously fewer and smaller than those in the vegetative cytoplasm. The mierotubules may also be seen in the cytoplasm of spindle-shaped generative cell parallel to the long axis of the cell. There is no amyloplast in generative cell. 2. When the generative cell has moved to the position close to the vegetative nucleus again, it begins to divide. The formation of sperm cells as the result of mitosis of generative cell, and the development of sperm cell involves the following main changes. The shape of the sperm cell tranforms from spherical to elliptical, finally it forms an elongated cell with a tail-like structure. At the sametime, the distribution of cytoplasm gradually concentrated at one end of the sperm cell to form the cytoplasmic extension, so that the so called "tail" of the sperm cell is formed. There are more organelles, especially the mitochondria, assembling in this part. The sperm cell just formed after mitosis is naked and the enclosed plasma membrane is discontinuous. The sperm cell membrane is enclosed by vegetative cell membrane, and the double membranes may be completed at a later stage. It is considered that the period which follows is very short, the deposition of wall material, the callose, occurs to fill up continuously the space between two membranes, but soon after this period the cell wall becomes discontinuous and the wall material is obviously decreased. The significance of the position of the generative cell before its mitosis and the morphological changes during the development of the sperm cell are discussed in this paper.     

Male reproductive cell development in Nicotiana tabacum: male germ unit associations and quantitative cytology during sperm maturation

Generative and sperm cells were examined at four stages of development from generative cell formation to sperm cell maturation using serial transmission electron microscopy. The generative cell and vegetative nucleus are associated in a male germ unit association during pollen maturation and tube elongation, except for generative cell mitosis. At late stages of prophase, this association loosens; the generative cell separates from the vegetative nucleus at metaphase. Slender, unbranched, or occasionally branched projections may be found at one or both ends of the generative cell, or they may be single, blunt, and short. Slender projections are rare during anaphase and telophase. The vegetative nucleus moves back into apposition with one sperm cell at the end of mitosis. During the re-establishment of the association, the vegetative nucleus first touches the end of the leading sperm cell and then moves next to the middle of the sperm nucleus. As the sperm cells enter interphase, a conventional association is re-established between one cell and the vegetative nucleus through one or more long and slender cytoplasmic extensions; these associations are maintained throughout later passage in the pollen tube. During maturation, a significant increase occurs in the surface area of the sperm cells (particularly in the sperm cell in association with the vegetative nucleus), and a lesser increase in nuclear volume and surface area. Other sperm cell parameters, including those of heritable organelles, remain unchanged during sperm cell maturation.     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.     

A novel class of MYB factors controls sperm-cell formation in plants

In contrast to animals, the plant male germline is established after meiosis in distinctive haploid structures, termed pollen grains. The germline arises by a distinct asymmetric division of the meiotic products . The fates of the resulting vegetative and generative cells are distinct. In contrast to the larger vegetative cell, arrested in the G1 phase of the cell cycle, the smaller generative cell divides once to produce the two male gametes or sperm cells. Sperm cells are delivered to the female gametes by the pollen tube, which develops from the vegetative cell. In spite of recent efforts to understand pollen development , the molecular pathway controlling sperm-cell ontogenesis is unknown. Here, we present the isolation of DUO1, a novel R2R3 MYB gene of Arabidopsis, as the first gene shown to control male gamete formation in plants. DUO1 is specifically expressed in the male germline, and DUO1 protein accumulates in sperm-cell nuclei. Mutations in DUO1 produce a single larger diploid sperm cell unable to perform fertilization. DUO1 appears to be evolutionarily conserved in several plant species and defines a new subfamily of pollen-specific MYB genes.     

Generative Cell Division and Sperm Cell Association in the Pollen Grain of Sambucus nigra

Generative cell division in tricellular pollen grains of Sambucusnigra L. (Caprifoliaceae) has been examined with light and electronmicroscopy. During division the generative cell is located inthe centre of the pollen grain, near to the nucleus of a surroundingvegetative cell. Conventional mitosis of the generative cellis followed by cytokinesis through centrifugal cell plate formation.Two sister sperm cells remain in spatial contact with each otherand are surrounded, as formerly their progenitor cell was, bythe vegetative cell. From the changes of shape of the generativecell during division and of the sperm cells it may be assumedthat the space between these cells and the vegetative one containsa labile, non-rigid, wall material. No plastids have been observedin the generative cell and its mitochondria appear to be unequallydistributed between the two future sperm cells during division. Sambucus nigra L., generative cell division, pollen, sperm cell association     

Preliminary observations on the formation of the male germ unit in pollen tubes ofCyphomandra betacea sendt.

Summary The structure of the generative cell and its association with the vegetative nucleus in the pollen tube ofCyphomandra betacea Sendt. were observed with the electron microscope. The generative cell, bounded by its own plasma membrane and the inner plasma membrane of the vegetative cell, possesses the cytoplasmic extension which lies within the embayments of a vegetative nucleus. The generative cell contains the normal complement of organelles and, especially, microtubules which cluster into several groups adjacent to the plasma membrane, oriented along the longitudinal axis of the cell. In the pollen tube reaching the lower end of the style aftersemivivo pollination, both of the sperm cells are elongated and polyribosomes and microtubules are the outstanding feature in the cytoplasm. The two sperm cells are connected by a common transverse cell wall, while cytoplasmic channels exist in both the periplasm of the two sperm cells and the transverse wall. The leading sperm cell (S_vn) is closely associated with the vegetative nucleus. Thus the present study demonstrates the existence of the male germ unit in the pollen tube ofC. betacea. The possible cytoplasmic continuity between the sperm cells and between the gametes and vegetative cell is considered.Abbreviations S_vn sperm cell physically associated with the vegetative nucleus - S_ua sperm cell unassociated with the vegetative nucleus - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Pollen vegetative cell-specific expression of Bra r 1: useful tool for observation of the vegetative nucleus and identification of transgenic pollen by nuclear-targeted GFP

Bra r 1 encodes a novel Ca²⁺-binding protein specifically expressed in pollen and is localized in cytoplasm of pollen and pollen tubes. In this study, we demonstrated the expression of green fluorescent protein (GFP) with a nuclear localization signal under the control of Bra r 1 promoter in tobacco pollen. A fluorescent signal was detected in the vegetative nucleus (VN) but not in generative and sperm cell nuclei, indicating pollen vegetative cell-specific expression of Bra r 1. The fluorescent signal in elongating pollen tubes was stronger than that in mature pollen, indicating that the expression of Bra r 1 was more activated during pollen tube growth. This result suggests that Bra r 1 protein might be necessary for pollen tube growth. The pattern of green fluorescence in the VN revealed that VN chromatin is dispersed during the mid-bicellular pollen stage and condensed at the mature stage. This suggests that the level of chromatin condensation might be linked with gene expression in pollen vegetative cells. We also found that the expression of GFP and its targeting of the VN have no detrimental effect on pollen maturation and pollen tube growth. Expression of GFP in pollen thus makes rapid non-destructive monitoring of transgenic pollen and pollen tubes possible. The GFP which moved into the VN was found to be a convenient tool for observation of the VN and could be useful as a selectable marker of transgenic pollen for the analysis of pollen-specific genes. Received: 6 December 2000 / Revision accepted: 20 March 2001     

Unequal distribution of DNA-containing organelles in generative and sperm cells of Erythrina crista-galli (Fabaceae)

The generative cell at anthesis in the mature pollen grain of Erythrina crista-galli (Fabaceae) was examined by 4,6-diamidino-2-phenylindole(DAPI)-fluorescence microscopy using the squash method. An unequal, polarized distribution of DNA-containing organelles (plastids and/or mitochondria) within the generative cell was observed in every mature pollen grain examined. Polarization of DNA-containing organelles is obvious when generative cells are freed and assume a spherical shape soon after microspore mitosis, as revealed by fluorescence-microscopic observations of specimens embedded in Technovit 7100 resin and thin-sectioned at different developmental stages. Early establishment of polarized localization of organelles in young generative cells of E. crista-galli and maintenance of this unequal distribution until pollen maturation strongly suggests that the organelles may still be clustered at pollen mitosis. Production of a dimorphic pair of sperm cells, as has been reported in Plumbago zeylanica, was observed in some pollen tubes germinated in vitro. The differentiation of the two sperm cells is discussed in relation to possible preferential double fertilization in angiosperms. Received: 28 July 1999 / Revision accepted: 8 November 1999     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

被子植物生殖细胞与精细胞的分离方法

开花植物精细胞的发育经历一个独特的后减数分裂过程,在此过程中每个花粉母细胞减数分裂的产物——小孢子经不对称有丝分裂产生1个大的营养细胞和1个小的生殖细胞,随后生殖细胞经过正常的有丝分裂产生2个精细胞。近几年,随着高通量组学技术的不断完善,利用组学技术比较分析生殖细胞和精细胞的分子特征、揭示决定精细胞命运与功能以及受精识别的重要分子已成为植物生殖生物学备受关注的课题。开展此项研究的关键是建立能获得大量高纯度的生殖细胞与精细胞分离纯化技术。该文综述了被子植物生殖细胞和精细胞分离方法的主要研究进展,分析了关键方法的特点和要点以及不同方法之间的差异和共性,以期为相关领域的研究人员提供借鉴。     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.