Angiosperm species that produce sperm cell pairs or generative cells with polarized distribution of DNA-containing organelles

We examined the distribution of DNA-containing organelles (plastids or mitochondria) in pollen from 115 angiosperm species (104 genera, 56 families) to determine whether there was polarized distribution of sperm cells (SC) and generative cells (GC). Using an improved method of fixation and squashing, we found polarization in the GC or SC pairs in mature pollen (obtained from the flower at anthesis) from five species (Abelia spathulata, Campsis grandiflora, Erythrina variegata, Limonium sinuatum, and Tecoma capensis). Including Plumbago, polarization is now known to occur in six genera representing four unrelated families. The results show that the production of pairs of dimorphic SC occurs in species other than those in the genus Plumbago, in which this phenomenon has already been well characterized. These results suggest that differentiation of SC and preferential double fertilization are not restricted to Plumbago and may occur in a variety of angiosperms. In species with significant polarization in GC or SC pairs, polarization of GC occurs at an especially early stage when GC are freed and become spherical, or just after microspore mitosis. This suggests that the distribution of DNA-containing organelles is strictly controlled during the early development of the GC.     

Development of male gametes in flowering plants

The male gametes of angiosperms consist of two sperm cells within a pollen grain or a pollen tube. They are derived from a single generative cell, which is formed as the smaller cell by unequal cell division in the microspore after meiosis. Limited information is available about these male gametic cells, beyond observations by electron microscopy, because each is surrounded by the cytoplasm of a larger vegetative cell. Recently, large quantities of generative cells and sperm cells have been isolated from pollen grains or pollen tubes of various plant species, and their physiological, biochemical and molecular characterization is now possible. Although almost all the available results are still preliminary, it is evident that the male gametic cells are peculiar in terms both of cell structure and composition. For example, they are rich in axial microtubules which maintain the spindle-like shape of each cell. However, they lack plastids which are DNA-containing cytoplasmic organelles. Biochemical characterization of their proteins indicates the presence of male gamete-specific polypeptides. These findings suggest, not unexpectedly, the possibility of male gamete-specific gene expression and of a strict genetic mechanism that controls the formation of male gametes.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

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.     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 2. Quantitative cell and organelle dynamics of the male reproductive cell lineage

Quantitative cell and organelle dynamics of the male gamete-producing lineage of Plumbago zeylanica were examined using serial transmission electron microscopic reconstruction at five stages of development from generative cell inception to sperm cell maturity. The founder population of generative cell organelles includes an average of 3.88 plastids, 54.9 mitochondria, and 3.7 vacuoles. During development the volume of the pollen grain increases from 6,200 μm³ in early microspores to 115,000 μm³ at anthesis, cell volume of the male germ lineage decreases more than 67% from 362.3 μm³ to 118.4 μm³. By the time the generative cell separates from the intine, plastid numbers increase by >600%, mitochondria by 250%, and vesicles by 43 times. A cellular projection elongates toward and establishes an association with the vegetative nucleus; this leading edge contains plastids and numerous mitochondria. When the generative cell completes its separation from the intine, organellar polarity is reversed and plastids migrate to the opposite pole of the cell. Cytoplasmic microtubules are common in association with cellular organelles. Plastids accumulate at the distal end of the cell as a linked mass, apparently adhered by lateral electron dense regions. Before division of the highly polarized generative cell, plastids decrease in number by 16%, whereas mitochondria increase by ∼90% and vacuoles increase by ∼140% from the prior stage. After mitosis, the resultant sperm cells differ in size and organelle content. The sperm cell associated with the vegetative nucleus (S_vn) contains 62.7% of the cytoplasm volume, 87% of the mitochondria, 280.4 vesicles (79% of those in the generative cell), and 0.6% of the plastids. At maturity, the S_vn mitochondria increase by 31% and the cell contains an average of 0.4 plastids, 158.9 vesicles, and 0.36 microbodies. The mature unassociated sperm (S_ua) contains 39.8 mitochondria (up 3.3%), 24.3 plastids (down 31%), 91.1 vesicles (up 54.9%), and 3.18 microbodies. The small number of organelles initially in the generative cell, followed by their rapid multiplication in a shrinking cytoplasm suggests a highly competitive cytoplasmic environment that would tend to eliminate residual organellar heterogeneity. Cell and cytoplasmic volumes vary as a consequence of fluctuations in the number and size of large vesicles or vacuoles, as well as loss of cytoplasmic volume by (1) formation of “false cells” involving amitotic cytokinesis, (2) “pinching off” of cytoplasm, and (3) dehydration of pollen contents prior to anthesis.     

Ultrastructure of Male Gametophyte in wheat I. The Formation of Generative and Vegetative Cells

The present study of the formation of the generative and vegetative cells in wheat has demonstrated some cytological details at the ultrastructural level. The phragmoplast formed in telophase of the first microsporic mitosis extended centrifugally until it connected with the intine of the pollen grain. A new cell wall was then formed to separate the generative and the vegetative cells. By unequal cytokinesis the former is small and the latter large. In early developmental stage of male gametophyte, the organelles in the cytoplasm of the generaVive cell and the vegetative cells are similar, including mitochondria, dictyosomes, rough endoplasmic retieulum, free and clustered ribosomes and plastids, but microtubules were observed only in the early cytokinesis stage. In the further developmental stage of the male gemetophyte, the generative cell gradually detached from the intine of pollen grain and grew inward to the cytoplasm of the vegetation cell. When the generative cell became round and free in the cytoplasm of the vegetative cell, the wall materials between plasma membranes of the cytoplasm of the generative and the vegetative cells disappeared completely, so that it was a naked cell with a double-layer membrane at this time. The heterogeneity between both cells was then very conspiceous. The organelles in the cytoplasm of the generative cell have hardly any changed besides the degeneration of plastids, but in vegetative cytoplasm the mitochondria and plastids increased dramatically both in number and size. The rapid deposition of starch in the plastids of the cytoplasm of the vegetative cell made the most conspicuous feature of the vegetative cell in mature pollen grain. The significance of the presence of a temporary cell wall in generative cell and heterogeneity between generative and vegetative cells are discussed.     

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.     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

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.     

The pollen-specific gene Ntp303 encodes a 69-kDa glycoprotein associated with the vegetative membranes and the cell wall

The pollen-specific gene Ntp303 belongs to the class of late pollen specific genes. It is first transcribed directly after pollen mitosis. Biochemical properties, appearance and precise location of the NTP303 protein during pollen development and pollen tube growth were studied by amino-acid micro-sequencing, protein gel blotting and immuno-localization. Antisera were raised against recombinant proteins, encoded by sequences of the pollen-specific Ntp303 gene. The antibodies specifically recognized a 69-kDa glycoprotein. Electron-microscopic immuno-localization of the protein revealed the presence of high concentrations of the NTP303 protein at the vegetative plasma membranes that surround the vegetative cell, the generative cell and the sperm cells of pollen and pollen tubes. The generative plasma membranes of the generative cell and the sperm cells were negative. NTP303 protein was also present in the cell walls and in callose plugs. With this method it was shown that the NTP303 protein was already present in mid-bicellular pollen, after the first, asymmetrical pollen mitosis. Possible functions for the NTP303 protein are discussed in relation to its properties and its association with the vegetative plasma membranes. Received: 9 September 1999 / Revision accepted: 4 November 1999     

Populations of plastids and mitochondria during male reproductive cell maturation in Nicotiana tabacum L.: A cytological basis for occasional biparental inheritance

The dynamics of plastid and mitochondrial populations in male reproductive cells of tobacco (Nicotiana tabacum L.) were examined during development using serial ultrathin sections and transmission electron microscopy to reconstruct 58 generative cells and 31 sperm cells at selected stages of maturation from generative cell formation through gametic fusion. The first haploid mitosis resulted in incomplete exclusion of plastids providing an average of 2.81 plastids and 82.7 mitochondria for each newly formed generative cell. During generative-cell maturation, plastid content decreased to an average of 0.48 plastids/generative cell at anthesis owing to autophagy of organelles. Plastids were present in low frequency within generative and sperm cells in the pollen tube and appeared to be transmitted, according to observations immediately prior to fertilization. This forms a cytological basis for genetic reports of occasional biparental plastid inheritance. In contrast, mitochondria were transmitted in larger numbers, and approximately 80 mitochondria per generative cell or sperm cell pair were retained throughout development. This provides a potentially stable source for the transmission of male mitochondrial DNA, if present at fertilization.Abbreviations GC generative cell - SC sperm cell We thank Dr. Frank J. Sonleitner, for helpful suggestions on the statistical calculations and Dr. Bing-Quan Huang for technical assistance in the preparation of embryo sacs during fertilization. This research was supported in part by U.S. Department of Agriculture grant 91-37304-6471. We gratefully acknowledge use of the Samuel Roberts Noble Electron Microscopy Laboratory of the University of Oklahoma.     

The male germ unit in the pollen and pollen tubes ofPetunia hybrida: Ultrastructural,quantitative and three-dimensional features

Summary The male germ unit ofPetunia hybrida was examined quantitatively and qualitatively at the ultrastructutral level. Three-dimensional reconstructions, the determination of nuclear and cytoplasmic volumes and surface areas, and organelle counts were obtained from serial ultrathin sections and computer analysis. In the mature pollen grain, an elongated generative cell is found in direct physical association with and partially surrounded by the vegetative nucleus. The mature generative cell lacks plastids and has mitochondria equally distributed at both of its tapering ends. In the pollen tube, the sperm cells are physically associated by cytoplasmic connections to each other and to the surrounding vegetative cell membrane. At full style length, the lobed vegetative nucleus and sperm pair are found in close association near the end of the pollen tube. The two sperms of a pair are not strongly dimorphic.     

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     

Unequal distribution of ubiquitinated proteins during Pinus bungeana pollen development

The production of gametogenesis is a charming and complicated event in higher plants, during that stage the protein population undergoes substantial alterations. But few attentions have been paid to the possible roles of the UPP in gymnosperm gametogenesis. In the present study, DNA-specific probe 4′,6-dimidino-phenylindole was employed to assess Pinus bungeana pollen developmental stage. It was revealed that the division of pollen mother cell occurred in late April. The uninucleate microspore then underwent three asymmetric divisions, forming a mature pollen grain including a tube cell and a generative cell together with two degenerated prothallial cells in early May. Immunofluorescence labeling of ubiquitinated proteins (UbPs) with an anti-ubiquitin antibody indicated that fluorescence signal was detected in both cytosol and nuclear of the microspore at the uninucleate stage. In the two-cell pollen grain, a brighter fluorescence was always detected in the first prothallial when compared with that in central cell. Similarly, unequal distribution of UbPs was observed again during the division of the central cell into the antheridial initial and the second prothallial cell. The high intensity of the fluorescence in the two degenerated prothallial cells remained in the mature pollen grain, but only a faint signal could be detected in the tube cell or the generative cell deriving from the division of the antheridial initial. The unequal distribution of UbPs was further unveiled by immunogold labeling among prothallial cells, generative cells and tube cells in mature pollen grains. Besides, Coomassie brilliant blue cytochemistry was also performed to illustrate the general subcellular distribution of total proteins in the two-cell and matured pollen grains. All these results indicated that the prothallial cells have high ratio of UbPs, and that the ubiquitin-mediated proteolysis might have an important role during pine pollen development.     

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana

Summary Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultra-structure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyo-somes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.     

Ultrastructural features of Aloe ciliaris pollen

Summary Both the internal anatomy and the external morphology of the mature pollen grain of Aloe ciliaris have been studied, together with the cytological changes occurring during pollen activation. In mature pollen, the generative cell (GC) and the vegetative nucleus (VN) are closely associated with each other, and both can be found in the central part of the grain. In the generative cytoplasm, some organelles and microtubular bundles are present. In the vegetative cell, dictyosomes, stacks of rough endoplasmic reticulum, mitochondria, plastids, vacuoles, ribosomes, and masses of fibrillar material have been described. During pollen activation, important changes occur in both the generative and vegetative cells (VC). In the GC, the microtubular bundles become clearly visible, and the GC and VC gradually move towards the germ pore. The RER cisterns become free from the stacks, and organelles, such as dictyosomes, become very active. The fibrillar masses gradually decrease in number, and the individual fibrils become more evident and clearer in resolution.This research was carried out in the framework of contract no. BAP-0204-I of the Biotechnology Action Programme of the Commission of the European Communities     

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.     

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.     

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.