Some permeability properties of angiosperm pollen grains,pollen tubes and generative cells

Summary The permeability of pollen grains, pollen tubes and generative cells of Helleborus foetidus and Galanthus nivalis has been investigated using four probes spanning a wide range of molecular weights: 4,6-diamidino-2-phenyl indole (DAPI; mol.wt. 350). Evans blue (mol.wt. 960), FITC-dextran (average mol.wt. 19400) and FITC-albumin (average mol.wt. 67000). DAPI penetrated into the vegetative cells of desiccated and hydrated pollen, and also entered growing pollen tubes. In contrast, the generative cells of hydrated pollen and of pollen tubes were highly resistant to penetration, as they were when isolated in osmotically balancing medium. Evans blue failed to enter intact generative cells under any of the conditions tested. The dye ultimately entered the vegetative cells of some pollen grains, but these were non-germinable. Growing pollen tubes invariably resisted penetration. Neither of the high molecular weight conjugates entered germinable pollen grains or intact pollen tubes. The results suggest that it is highly unlikely that DNA fragments of high molecular weight can enter viable pollen, pollen tubes or generative cells under any normal conditions.     

Microtubular organization during asymmetrical division of the generative cell inGagea lutea

Video microscopy and conventional or Confocal Laser Scanning Microscopy after DAPI staining and anti-α-tubulin labelling were used to study the asymmetrical division of the generative cell (GC) inGagea lutea. Pollen was cultured for up to 8 hr in a medium containing 10% poly (ethylene glycol), 3.0% to 3.8% sucrose, 0.03% casein acid hydrolysate, 15 mM 2-(N-morpholinoethane)-sulphonic acid-KOH buffer (pH 5.9) and salts. In the pollen grain, the GC had a spherical or ovoid shape and contained a fine network of intermingled microtubules. As the GC entered into the pollen tube, it assumed a cylindrical shape with a length often exceeding 250 μm. A cage of microtubules then developed around the nucleus. The presence of dense and thick microtubular bundles in front of the generative nucleus within the GC coincided with the translocation of the nucleus to the leading end of the GC. No pre-prophase band was ever detected, but a distinct prophase spindle of microtubules was formed. In some GCs a tubulin-rich dot became visible at each pole of the spindle. After nuclear envelope breakdown, the bundles of microtubules spread between the chromosomes and became oriented into parallel arrays. The spindle became shorter at metaphase, and there was no tubulin labelling at the site of the metaphase plate. At anaphase, the microtubular apparatus lost its spindle-shape and a bridge of prominent bundles of microtubules connected the two daughter nuclei. At telophase, the site of the cell plate remained unstained by the anti-α-tubulin antibody, but a distinct phragmoplast of microtubules was formed more closely to the leading nucleus, resulting in the formation of unequal sperm cells (SCs). The leading SC was up to 2.5 times smaller than the following SC and it contained a smaller or equal number of nucleoli.     

Male cytoplasmic diminution and male germ unit in young and mature pollen of Cymbidium goeringii: a 3-dimensional and quantitative study

Summary Pollinia of Cymbidium goeringii (Orchidaceae) were examined at a young stage (approx. 2 weeks before anthesis) and at anthesis using transmission electron microscopy, computer-assisted serial reconstruction and quantitative cytology. At the young stage, the generative cell (GC) has a spherically shaped main body with a large nucleus and 1 to 4 cytoplasmic extensions (up to 10 m long) physically associated with a groove on one side of the vegetative nucleus (VN). By anthesis, some GCs lose their cytoplasmic extensions, becoming spherical. The GC surface becomes highly wrinkled, the distance between the GC and the VN increases and the two may separate completely. Enucleated cytoplasmic bodies (ECBs) are apparently generated by the GC, as evidenced by their similar ultrastructural appearance and distribution near the GC. ECBs are likely produced from two sites: the GC surface and the tips of cytoplasmic extensions of the GC. Vesicle-containing bodies (VCBs) may be generated from the lateral surface of the GC and may also represent a degeneration stage of ECBs, finally fusing with lipid bodies. Heritable organelles are rare and usually absent in the GC of Cymbidium: 0.176 mitochondria and 0.0042 plastids per GC were observed at the young stage and no mitochondria and 0.019 plastids at anthesis. Of 16 cellular parameters examined during GC development, all decreased with the exception of the surface area and volume of ECBs/ VCBs (which remained stable) and GC vacuoles (which increased). Significant decreases in 11 parameters presumably relate to dehydration of the pollen and male cytoplasmic diminution.Abbreviations 3-D three-dimensional - ECB enucleated cytoplasmic body - GC generative cell - MGU male germ unit - RER rough endoplasmic reticulum - VCB vesicle-containing body - VN vegetative nucleus     

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.     

Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

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

Summary 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.Abbreviations DAPI 4,6-diamino-2-phenylindole - MGU male germ unit - MT microtubule - SC 1 the leading sperm cell physically associated with the vegetative nucleus - SC 2 the trailing sperm cell     

Juxtaposition of the generative cell and vegetative nucleus in the mature pollen grain of amaryllis (Hippeastrum vitatum)

Summary Computer-generated, three-dimensional reconstructions from serial ultrathin sections were used to investigate the spatial organization and extent of association between the generative cell and vegetative nucleus within the mature pollen grain of amaryllis. In all cases examined, the highly lobed vegetative nucleus was found in close proximity and positioned laterally to the elongated, oval shaped generative cell. Numerous projections of the vegetative nucleus come to within 53 nm of the inner vegetative cell plasma membrane which surrounds the generative cell. These areas of close association may continue transversely around the generative cell for a distance of up to 4 m. Although an association exists between the generative cell and vegetative nucleus of the mature pollen grain, it is apparent that several changes must take place after pollination in order to achieve the high amount of close contact that occurs between the vegetative nucleus and the numerous terminal cell extensions of the leading sperm in the pollen tube of amaryllis (Mogensen 1986). Thus, this study demonstrates that the spatial organization among components of the male germ unit in the mature pollen grain does not necessarily reflect relationships that ultimately exist among these components within the pollen tube.     

Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

Summary The three-dimensional organization of microtubules in generative cells during their development in pollen grains of Hippeastrum vittatum and the dynamic changes that occur were studied by collecting large quantities of fixed and isolated generative cells for immunofluorescence microscopy. The framework configuration and the arrangement pattern of the microtubule organization was investigated. The microtubule framework changed in shape from being spherical at an early stage to being long spindle-shaped at maturity: various transitional forms were observed: ellipsoidal, pear-shaped and short spindle-shaped. The microtubule arrangement making up this framework changed correspondingly from the original network, which was random in distribution, to axially oriented long bundles via an intermediate pattern composed of a mixture of networks with long bundles. However, cells with the same framework configuration might be heterogeneous in microtubule arrangements.     

Striped projections of the outer membrane of the generative cell in Convallaria majalis pollen

In pollen grains of Convallaria majalis the outer membrane of the generative cell (GC) is the inner membrane of the vegetative cell (VC). Striped projections (SP) at the cytoplasmic face of the outer membrane of the GC were revealed by chemical fixation and also by a rapid freeze-fixation and freeze-substitution. The projections, located in groups on the protruding lobes of the GC, were arranged parallel to each other and were equally spaced (40 nm apart). The length of the SP, estimated from grazing sections of GC, was 400 nm. Each projection was composed of T-shaped elements, about 35 nm high, spaced at an average distance of 25 nm. SP were observed in mature, hydrated, activated and germinated pollen grains and seemed to be associated with microtubules and microfilaments of the VC. No evidence exists yet of SP on the sperm cell membrane. Immunogold labelling with anti-myosin antibodies showed many gold particles attached preferentially to the surface of the protruding lobes of the GC in the area of the projections. These results may suggest that the SP of Convallaria GC contain myosin-like protein and play an important role in the motility of the GC during pollen tube growth.     

Isolation of generative cells and their protoplasts from pollen ofLilium longiflorum

Summary Methods are described for the isolation of large quantities of generative cells and their protoplasts from the pollen ofLilium longiflorum. First, large numbers of pollen protoplasts were enzymatically isolated from immature pollen grains. When they were gently disrupted mechanically, the pollen contents including spindle-shaped generative cells were released. The generative cells were separated from other structures by Percoll density gradient centrifugation. They were nearly spherical, but had a callosic cell wall. The isolated generative cells were then re-treated in enzyme solution to yield authentic protoplasts. The generative cell protoplasts, gametoplasts, were uniform in size and contained a condensed haploid nucleus with relatively little cytoplasm.     

Isolation of generative cells from pollen of Phoenix dactylifera

Summary Generative cells in pollen of Phoenix dactylifera had a convoluted surface and a small cytoplasmic volume, and condensed chromatin in the nucleus. Treatment of hydrated pollen with pectinase followed by grinding in a hypotonic medium released the generative cells from pollen. Convolutions of the plasma-membrane surface in vivo were retained by generative cells in vitro. Generative cells had a much lower solute concentration than did vegetative cells, a property that explains why hypotonic shock was effective in releasing intact generative cells from pollen.     

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.     

Microtubule organization of in situ and isolated generative cells in Zephyranthes grandiflora Lindl

Summary Microtubule organization in the generative cells of Zephyranthes grandiflora was investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. The experimental materials used were generative cells located within pollen grains and tubes (i.e., in situ) as well as those artificially isolated after osmotic shock or grinding treatments of the pollen grains. Diverse microtubule organization patterns were revealed. In situ, the generative cells appeared spindle-shaped and contained mainly longitudinally oriented microtubule bundles, although other types were found as well. After isolation, as the alteration in microtubule patterns took place, the spindle-shaped generative cells became ellipsoidal and then spherical. In the ellipsoidal cells a transitional form consisting of a mixture of microtubule bundles and meshes could be found. In spherical cells the mesh structure appeared to be the predominant pattern. These results indicate that the microtubule cytoskeleton of the generative cells can change easily from one structural form to another in accordance with environmental conditions and may play an important role in determining the cell shape.     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Early events in division of the generative cell ofOrnithogalum virens

Summary Ornithogalum virens is a bicellular pollen species. In mature pollen, the generative nucleus is at advanced prophase. Mitosis of the generative cell is resumed just after pollen rehydration and prometaphase occurs within 10 min of germination. Prometaphase is manifested by nuclear envelope breakdown and the appearance of spindle microtubules in the nucleoplasm region. At this stage the number of cytoplasmic microtubules located in the generative cell periphery appears to decrease. Endoplasmic reticulum-like cisternae originating from the nuclear envelope tend to be spaced around the chromosomes, outside the area of the forming mitotic spindle. Some also begin to penetrate the spindle area. The results are discussed in terms of the generative cell cycle in bicellular pollen.     

Immunoelectron microscopy of myosin associated with the generative cells in pollen tubes ofNicotiana tabacum L.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.     

Sperm cells in pollen tubes ofNicotians tabacum L.: three-dimensional reconstruction,cytoplasmic diminution,and quantitative cytology

Summary The organization of the sperm cells and vegetative nucleus (male germ unit) ofNicotiana tabacum was examined 18 h after semivivo pollination using transmission electron microscopy, computerassisted serial section reconstruction and quantitative cytology. Based on a measurement of 11 cellular parameters in nine reconstructed sperm cell pairs, there are no statistically significant differences between the two cells. The S_vn is characterized by a strapshaped cytoplasmic extension that is physically associated with the surface of the vegetative nucleus. The nucleus is located adjacent to the sperm crosswall, with sperm organelles being distributed between the nucleus and the extension. The S_ua is a tapered cell with cytoplasmic areas at both poles and deep axial invaginations near the crosswall. This cell has a centrally-located nucleus and a largely polar distribution of organelles. Three mechanisms for cytoplasmic diminution were observed that appear to contribute actively to the loss of cytoplasmic volume and organelles: (1) enucleated cytoplasmic body production in the S_ua; (2) vesiculation at the tip of the cytoplasmic projection of the S_vn; and (3) vesicle-containing body accumulation in the periplasm of both the S_vn and S_ua.Abbreviations 3-D three-dimensional - ECB enucleated cytoplasmic body - MGU male germ unit - S_vn leading sperm cell - S_ua trailing sperm cell - TEM transmission electron microscopy - VCB vesicle-containing body     

The organization of the cytoskeleton in the generative cell and sperms ofHyacinthus orientalis

Summary The microtubular cytoskeleton of the generative cell (GC) ofHyacinthus orientalis has been studied until the formation of the sperm cells (SCs). Immunofluorescence procedures in combination with confocal laser scanning microscopy (CLSM) has enabled the visualization of the organization of the microtubular cytoskeleton. Chemical fixation and freeze-fixation electron microscopy have been used to investigate the cytoskeleton and the ultrastructural organization of the GC and SCs. During pollen activation the GC is spindle-shaped. Microtubules (MTs) are organized as bundles and distributed in proximity of the GC plasmamembrane, forming a basket-like structure. Following migration through the pollen tube, the basket-like structure becomes more intertwined. During the nuclear division the MTs are involved in the segregation of the chromosomes and kinetochores are clearly discernible. Association with organelles is also observed. The chromosomes of the GC remain condensed until they separate in two sperm nuclei. The pre-prophase band was never observed. At the end of the GC division the microtubular network reorganizes in the two SCs.Abbreviations CLSM confocal laser scanning microscopy - DAPI 46-diamidino-2-phenyl-indole - F-S freeze-substitution - GC generative cell - MT microtubule - PBS phosphate buffered saline - R-F rapid freeze-fixation - SC sperm cell - TBS tris buffered saline - VN vegetative nucleus