Vegetative Nucleus Research Articles

Organization of the early post-fertilization megagametophyte ofPopulus deltoides

Gynoecia ofPopulus deltoides, 48 to 60 h after pollination, were examined using transmission electron microscopy. The mature megagametophyte contains two synergids, an egg cell, a central cell with two unfused polar nuclei and three antipodals. Pollen tube passage between cells in the styloid, on the placenta and through the micropyle does not induce degeneration of surrounding cells. Pollen tube entry into the megagametophyte occurs through the degenerated synergid immediately adjacent to the filiform apparatus. The tube grows the length of the filiform apparatus 20–30 μm into the synergid, and then discharges its contents-two sperm cells, the vegetative nucleus, and pollen tube cytoplasm. Sperm cells appear progressively hypertrophied during their passage in the pollen tube. In the degenerate synergid, the sperm nucleus is located at one end of the cell and the remaining cytoplasm is diffuse, containing a large vacuole. Following transmission of sperm nuclei into the egg and central cell, one male nucleus fuses with the egg and the other fuses with the micropylar polar nucleus. Enucleate cytoplasmic bodies of apparent sperm origin were observed within the degenerated synergid. The gametic fusion mechanism in this plant appears to involve: (1) eccentric positioning of the nucleus within the sperm cell, (2) separation of cytoplasmic and nuclear portions of the sperm cell, and (3) fusion of the nuclear portion with the egg cell. This represents a previously undescribed mechanism by which male cytoplasmic organelles are eliminated from sperm cells prior to fusion.

Developmental Staging of Maize Microspores Reveals a Transition in Developing Microspore Proteins

A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady-state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period.

Nuclear DNA content and separation of Nicotiana sylvestris vegetative and generative nuclei at various stages of male gametogenesis

At all stages of male gametogenesis, generative and vegetative pollen nuclei of Nicotiana sylvestris can be distinguished without ambiguity after Feulgen or ethidium bromide staining. They differ by their morphology and their apparent DNA content, always lower in vegetative nuclei. These differences provide a basis for their separation by sedimentation and fluorometry. After elimination of the another somatic cells and after crushing the pollen, vegetative and generative nuclei are separated by two successive Percoll gradients (purity 80–90%). Analysis of the gradient fractions and final purification can be done with a cell sorter. DNAs of both types are isolated by a cetyltrimethylammonium method, followed by a RNase treatment. Yields are lower for vegetative than for generative nuclei, and decrease with the age of pollen. Molecular weights and digestibility by restriction enzymes are compatible with molecular analyses.

Fusion studies of pollen protoplasts and generative cell protoplasts in Lilium longiflorum

Cell fusion of pollen protoplasts and generative cell protoplasts, which were isolated from angiosperm pollen grains, was studied for the first time in Lilium longiflorum. Using an electric pulse, a high frequency of cell fusion was induced between pollen protoplasts, between generative cell protoplasts, and between pollen protoplasts and generative cell photoplasts. Each fusion product could be cultured for a prolonged period. In the cell resulting from fusion of two pollen protoplasts, the two generative cells divided after 8–10 days of culture resulting in the formation of four sperm cells, or male gametes. The cell resulting from fusion of two generative cell protoplasts survived for about 10 days, but no nuclear fusions or divisions were observed during the culture period. When a pollen protoplast and a generative cell protoplast were fused, the original generative cell from the pollen protoplast divided into two sperm cells within 10 days, as when two pollen protoplasts were fused. However, the generative nucleus transferred into the vegetative cell cytoplasm of the pollen protoplast by the fusion never divided and the chromatin became dispersed as in the vegetative nucleus. These results are discussed in relation to pollen ontogeny, in particular to the differentiation of generative and vegetative nuclei, which must be regulated by the cytoplasm of each cell after unequal microspore cell division.

Structure and Behaviour of Organelles in Living Pollen Tubes ofLilium longiflorum

The morphology and the movement of organelles, in particular of the endoplasmic reticulum (ER), have been studied using video-enhanced microscopy in living pollen tubes of Lilium longiflorum Thunb. The patterns of the cytoplasmic streaming in the pollen grains and tubes generally correspond with the descriptions by Iwanami (1956). We found, however, that the organelles move as single and independent elements reflecting highly individual characteristics. The apical zone of the pollen tube, the so-called 'clear' zone, is populated by numerous intensively vibrating organelles of small size, most probably Golgi vesicles. The migration of vesicles to the apical plasma membrane of the growing pollen tube is not a smooth flow of particles, but a turbulent process. Spherical bodies with a high contrast, probably polysaccharide (p-) particles and/or lipid drops, and some mitochondria are also visible in the extreme tip. Their penetration into the apex seems to be inhibited by the dense mass of agitated vesicles. The small vacuoles located in the central zone are very motile; they change their shape continuously. The tonoplast, therefore, must be very plastic and, to a certain extent, mechanically resistant. The vegetative nucleus and the generative cell maintained a stable shape despite constant collisions with the organelles of the vigorously streaming cytoplasm. The thin cytoplasmic layer in the vacuolate zone contains tubular ER (diameter about 0-3 /urn) arranged in complex closed networks. The tubules are permanently moving. Gentle undulations alternated with sudden dramatic distortions. Rapid fusions or divisions of tubules are common. This dynamic behaviour is taken as proof of the plasticity of the ER tubules and the high fluidity of the membranes of the ER. Organelles preferentially move along the ER tubules in a saltatory manner. It is assumed that the ER tubules are associated with cytoskeletal elements.

Microspore development in the anther culture of wheat(Triticum aestivum L.).

A cultivar (Chris) of hard red spring wheat (Triticum aestivum L.) and a one-step haploid generation medium were used to study microspore development and callus formation by using an anther culture technique. Cytological observations on microspore development were made 7, 10, 13 and 16 days after the anther was placed on the medium. The first mitotic division of the uninucleate microspore formed two nuclei, which were similar or different in size. Further divisions of the vegetative nucleus produced multicellular pollen grains that continued to grow and then were released from the exine. This extensive division of the vegetative nucleus could be the source of embryoid or callus generation.

RNA and Protein Synthesis in Germinating Pine Pollen

The results of autoradiographic experiments demonstrate that, as with the pollen of most other species, both the generative and vegetative nuclei of Loblolly Pine (Pinus taeda) actively engage in RNA synthesis from the very early stages of pollen germination. Unlike most other species, however, this newly synthesized RNA includes rRNA. Evidence is provided for the importance of this newly synthesized RNA in the process of continued pollen tube growth. One and two-dimensional gel electrophoretic analysis reveals a number of both qualitative and quantitative differences among the proteins synthesized during the early stages of germination and the later stages of pollen tube growth. One of the most notable of these is a 36 kD protein, the synthesis of which predominates during the later stages of pollen germination. A similar pattern of 36 kD protein synthesis is observed when mRNA extracted from pollen at each of these stages is translated in vitro.

Actomyosin and movement in the angiosperm pollen tube: an interpretation of some recent results

Recent confirmations of the presence of myosin in angiosperm pollen tubes indicate that an energy-transducing actomyosin system is involved in the motility system of the vegetative cells. Myosin has been localised by immunofluorescence on the surfaces of vegetative nuclei and generative cells. It has been shown to be associated with individual amyloplasts in grass pollen, and there are indications that it is present on other particulate bodies in the cytoplasm. The organelles in the leading part of the tube move along separate traffic lanes of acropetal and basipetal polarity, known from electron microscopy and phalloidin labelling to contain numbers of fibrils containing aggregates of actin microfilaments; in older segments the movement can be related to single, uniformly polarised, fibrils. Circulatory flow is maintained at the proximal end by the looping of the fibrils in the grain or at callose plugs. Such loops do not occur at the apex, where entering organelles undergo random movement before becoming associated with basipetal streams. Vegetative nuclei and generative cells interact with several fibrils, and it is suggested that they are held in the leading part of the protoplast in unstable equilibrium between acropetal and basipetal forces. Constantly changing form, especially of the vegetative nucleus, is one consequence of these varying stresses. Possible analogies with the intracellular motility system of the giant cells of the Characeae are noted, and it is suggested that lipid globuli and other nonorganellar bodies may be transported in the pollen tube by association with myosin-bearing membranes similar to those involved in endoplasm movement in the characean cells.

Myosin associated with the surfaces of organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes

ABSTRACT Myosin, detected by immunofluorescence using an antibody to bovine skeletal and smooth muscle myosin, has been localised on individual identifiable organelles from the grasses Alopecurus praten-sis and Secale cereale, and on the surfaces of vegetative nuclei and generative cells from pollen and pollen tubes of Hyacinthus orientalis and Hel-leborus foetidus. Taken in conjunction with recent evidence showing that the growing pollen tube contains an actin cytoskeleton consisting of numerous mainly longitudinally oriented microfilament bundles, and that isolated pollen-tube organelles show ATP-dependent movement along the actin bundles of the giant cells of the characeous algae, this finding suggests that an actomyosin motility system is present in pollen tubes, and indicates that the movements of the different classes of inclusions are driven by interaction of the surface myosin with the actin fibrils at the zones of contact.

Androgenetic vs. Gynogenetic Doubled Haploids of Tobacco

Cultivar development by doubled haploid (DH) methods employing in vitro anther culture has not been highly successful in tobacco, Nicotiana tabacum L., because of the low yielding ability of DH lines. The objective of this study was to compare DH lines developed from androgenetic and gynogenetic haploid sporophytes from the same parental plants. Single plants from ‘Ky10’, ‘Ky15’, and ‘Kyl7’ were self‐pollinated, crossed with pollen of N. africana Merx. and Buttler, and immature buds were anther cultured. Haploid sporophytes arising from embryo sac nuclei and vegetative nuclei of pollen were chromosome doubled and self‐pollinated. Ten random androgenetic DH lines (ADHs), 10 random gynogenetic DH lines (MDHs), and selfed progenies of the parental plant were assigned to a set, or cultivar family, and grown in three replications in each set in two environments. Data were collected for eight characters. Sixteen second cycle MDH lines were produced from one random first cycle line, Ky 10 MDH 1, and compared with the original Ky 10 parent, a first cycle MDH line composite, and Ky 10 MDH 1. The ADH and MDH lines were significantly different for all characters measured. The MDH lines were agronomically superior to ADH lines and more closely resembled the cultivars. The ADHs and MDHs yielded 79.9% and 91.5%, respectively, of the cultivars. A second cycle of MDH line production resulted in no further yield reduction. A model of heterozygosity and inbreeding depression in the parental cultivars could explain the results for yield of the MDH lines; it would not account for differences between ADH and MDH lines.

The ultrastructure of polymorphic pollen grains of Canna indica L.

Our investigations on Canna indica L. indicate that the pollen of this species is polymorphic: there are two types of pollen — a larger type and a comparatively smaller type. Transmission electron microscopy (TEM) revealed the presence of small vacuoles containing tannic substances in the generative cell (GC) of the larger grains: the GC of the mature grain contained a higher quantity of tannins than the GC of the immature grain. Mitochondria, lipid bodies, rough endoplasmic reticulum (RER) and microtubular bundles were present in the cytoplasm of the GC. Numerous mitochondria, lipid bodies and plastids were also present in the vegetative cell (VC), with the mitochondria clustered around the vegetative nucleus. The plastids were observed to be associated with the RER cisterns. During the maturation process, the number of starch grains contained in the plastids decreased.

The Male Gametophyte of Flowering Plants.

Angiosperm plants produce flowers, very beautiful and intricate structures, within which their reproductive devel? opment takes place. In flowering plants, as in other groups of plants, a diploid, spore-producing generation (sporo? phyte) altemates with a haploid, gamete-producing gen? eration (gametophyte). Unlike some of the evolutionarily more primitive plants, the male and female gametophytes of angiosperms are reduced to microscopic structures that are dependent on the tissues of the sporophyte for their development. The flower contains specialized structures, the anthers and the pistil or gynoecium, in which the male and female gametophytes, respectively, are formed. The functions of the gametophytes are the production of the sperm cells and the female cells, and their union in fertilization. In flowering plants, the pollen grain is the male gametophyte and the embryo sac is the female gametophyte. The male gametophyte completes its early development within the anther. The sequential stages of pollen devel? opment are shown in Figure 1. Microsporocytes or pollen mother cells (Figures 1A and 1K) are produced in the sporogenous tissue within the anther. The two divisions of meiosis transform these cells into haploid microspores, each pollen mother cell producing first a dyad (Figures 1B and 1L) and, after the second meiotic division, a tetrad of microspores (Figures 1C and 1M). The tetrad and each microspore within the tetrad are surrounded by a callose (1,3-?-glucan) wall (Figure 1M). Upon release from the tetrad, the microspores increase rapidly in volume and undergo a change in shape (Figures 1D, 1E, 1N, and 10). This is followed by a period of slower growth until the maximum volume of the pollen grains is reached before anthesis. Following release of microspores from the te? trads, there is an extended interphase period that terminates with a very unequal division of the microspore (mi? crospore mitosis), forming a vegetative cell and a generative cell, both of which are included within the confines of the cell wall of the original microspore. The vegetative cell constitutes the bulk of the young pollen grain, while the generative cell, which inherits a very small amount of the microspore cytoplasm, lies within the vegetative cell (Fig? ures 1F and 1G). In several plants, such as corn, the generative cell undergoes a mitotic division within the pollen grain, forming two sperm cells (Figures 1H and 1R). In most pollens, however, the generative cell completes its division during the growth of the pollen tube in the style. At ma urity, the male gametophyte consists of three cells, the vegetative cell and the two sperm cells, which lie within the cytoplasm of the vegetative cell (Figures 11 and 1S). For a short while following anthesis (the rupture of the anther and release of pollen), the mature pollen grain exists as a free organism until it is transported by wind, insects, or other agents to the stigma of an appropriate pistil. It then begins another phase of its life and development. Each pollen grain germinates by the extrusion of a tube through a germ pore in the pollen wall (Figures 1J and 1T). The tube then grows down into the style and the vegetative nucleus, and, depending on the plant species, the gener? ative cell or sperm cells move out of the pollen grain and i to the tube. Germination and pollen tube growth are relatively rapid events in most plants, the period from pollination to fertilization ranging from 1 hr to around 48 hr. The rate of pollen tube growth varies in different plant species; extremely rapid rates as high as 35 mm/hr have been reported for some plants. The pollen tube grows through the transmitting tissue of the style, enters the micropyle of the ovule, and reaches the embryo sac. It penetrates one of the synergids of the embryo sac (nor? mally the one that has begun to degenerate), the tube rup ures, and the sperm cells together with some of the other tube contents are discharged into the synergid. The two sperm move by a presently unknown mechanism, one fusing with the egg cell to form the diploid zygote. The other sperm fuses with the normally diploid central cell, giving rise to the primary endosperm nucleus. This com? pletes the process of double fertilization. A major recent advance is the observation that the two sperm cells in a pollen grain are often morphologically different (Russell,

Conformation and movement of the vegetative nucleus of the angiosperm pollen tube: association with the actin cytoskeleton

ABSTRACT Actin is present in the cytoplasm of the vegetative cell of angiosperm pollens in numerous fusiform, spiculate or toroidal bodies, and also as a sheath enveloping the vegetative nucleus. During activation following hydration, the compact cytoplasmic bodies are translated into skeins of extended fibrils, and circulatory movements begin in the cytoplasm. Throughout this period the vegetative nucleus, with fibrillar actin now associated with the surface, undergoes a continuous change of shape. In the extending tube following germination the actin cytoskeleton consists of numerous mainly longitudinally oriented fibrils. After entry into the tube the vegetative nucleus remains associated with the fibrils, usually extending greatly in length and developing attenuated, often pointed extensions. The observed conformations, which change continuously, suggest that varying local tensions are applied to the vegetative nucleus during passage through the tube. Cytochalasin D breaks up the actin fibril system and brings about a rapid contraction of the nucleus, at the same time eliminating the elastic extensions of the nuclear envelope. Nuclei isolated physically from unfixed tubes also contract in length as the fibrillar components of the cytoskeleton are detached. These findings indicate that the movement of the vegetative nucleus depends on local associations of the nuclear envelope with the actin cytoskeleton of the vegetative cell.

Three-dimensional observations on freeze-fractured frozen hydratedPapaver dubiumpollen with cryo-scanning electron microscopy

SUMMARY The ultrastructure of generative cells and vegetative cells of Papaver dubium L. pollen was studied with low-temperature scanning electron microscopy (LTSEM). Liquid propane freezing resulted in a very fast fixation. The on-line fracturing and observation of the pollen grains was performed at − 130°C. The specimens were fully hydrated and did not undergo morphological changes during the procedure. Unfractured and cross fractured organelles, generative cells, vegetative nuclei and membranes are visible in three-dimensional images. The LTSEM micrographs show a lobed vegetative nucleus with many nuclear pores and a wrinkled generative cell in longitudinal direction. The vegetative nucleus and the generative cell are situated close to each other but structural connections are not observed.

Cytochalasin effects on structure and movement in the pollen tube of Iris

Cytochalasins B and D in the concentration range 0.5–10 μg ml−1 produced similar effects on growth, movement and cytoplasmic structure in the pollen tubes of Iris spp. cultured in vitro. Continuous video recording showed that at 5 μg ml−1, CB was capable of stopping organelle circulation in as short a period as 20 s. The usually elongated vegetative nuclei were also arrested, and subsequently contracted irregularly. Generative cells were not radically changed in shape, but occasionally moved erratically before being halted. Detailed examination of CD- and CB-treated tubes regarded as being capable of recovering growth upon transfer to normal medium revealed several characteristic effects on cytoplasmic structure. Fibrils presumed to consist of, or contain, microfilament bundles are readily visible in the older parts of the living tube where they form the pathways of organelle movement; these were either condensed into amorphous columns or fragmented by treatment. In the distal parts of the tube, the cytoplasm had contracted into amorphous masses which continued to show very slow shape changes. With the arrest of extension growth, pectin accumulated over the tube tip and in patches along the flanks. In a medium containing 1 mM ATP, recovery from treatment was achieved in some instances within l min. Organelle circulation in the younger tubes was resumed, and fresh adventive tube tips were formed. The fibrillar system of the older tubes was not restored, however; instead, the cytoplasm in these zones formed aggregates which underwent continuous amoeboid movement, the organelles within moving rapidly in irregular trajectories with no indication of the resumption of the original long-range cyclotic flux. Some possible implications of the results are briefly discussed.

Ultrastructure of sperm cells and the male germ unit in pollen tubes ofNicotiana tabacum

The structure of sperm cells and their association with the vegetative nucleus in pollen tubes ofNicotiana tabacum grown in styles were observed with the electron microscope, demonstrating the existence of a male germ unit. The two sperm cells are arranged in tandem and are closely associated with the vegetative nucleus, which always takes the lead. The leading sperm cell (SC 1) has a long and narrow cytoplasmic projection which lies within the enclaves of the much lobed vegetative nucleus, thus forming a physical association. The trailing sperm cell (SC 2) and the SC 1 are not only joined by a common transverse cell wall but also are surrounded by a periplasm bounded by the plasma membrane of the sperm cells and that of the vegetative cell, thus forming a structural connection. The sperm cells are elongated, with cytoplasmic projections at the anterior end of the SC 1 and at both ends of the SC 2. The cytoplasm of both sperm cells includes mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, small vacuoles and axially oriented microtubules. No plastids were observed.

Identification of 2n-pollen producing interspecific hybrids of Lilium using flow cytometry.

DNA-content of nuclei from pollen was determined using flow cytometry. The pollen from diploid genotypes gave rise to a 1C- and a 2C-peak of identical size, representing the vegetative and the generative nuclei. Pollen of the interspecific hybrid (L. auratum×L. henryi) presented only 2C- and 4C-peaks, while in the interspecific hybrids (L.בEnchantment’×L.pumilum) 1C-, 2C- and 4C-peaks were distinguished. The different C-levels, representing the DNA-contents of the generative and vegetative nuclei in G1 and G2 phase respectively, can be related to haploid and diploid pollen. Results of a meiotic analysis, a number of crossing experiments and pollen measurements agreed with the outcome of the flow cytometric determinations. The flow cytometry method enables the large scale screening of collections of genotypes for their potential of 2n-pollen production.

Some ultrastructural data on the spore development in aHenneguya sp. parasite of the gill of a Brazilian fish

A myxosporean Henneguya sp. was found in the gill filaments of a freshwater fish, Hoplosternum littorale, from the lower Amazon river near Belém, Brazil. Plasmodia were represented by numerous vegetative nuclei, generative cells and sporogony stages. The spore body was ellipsoidal and each valve presented a tapering tail approximately 45.2 microns in length. The spores differed from those of previously described species in their shape and size. The developmental stages are described and discussed. This histozoic species is reported to be pathogenic, inducing great mortality in the host.

A Simple Method for Staining Nuclei of Mature and Germinated Maize Pollen

A sugar acetocarmine staining technique has been developed for staining the sperm and vegetative nucleus of mature and germinated maize pollen grains. This procedure is simple, stable and highly repeatable. The physiological properties of the mature maize pollen grains are first adjusted by using an in vitro germinating culture solution. This solution is 15% sucrose and contains 360 ppm calcium chloride dihydrate, and 120 ppm boric acid. One part fresh pollen grains is uniformly mixed with nine parts of the solution and left at room temperature for at least 5 hr. One part of this solution is then mixed with two parts of regular acetocarmine stain and left overnight. The color of this mixture is pinkish red or raspberry. The sugar in the mixture helps to increase color contrast between the pollen cytoplasm (light pink) and the nuclei (reddish purple), decreases the frequency of burst pollen, increases pollen expansion, stabilizes pollen figures and automatically seals the coverglass.

Generative cell division and sperm cell formation in barley

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.