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.     

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.     

The anti-microtubule drug carbetamide stopsNicotiana sylvestris pollen tube growth in the style

Summary Recently, we found that the anti-microtubule drugs colchicine and propham caused the absence of microtubules and thus loss of cytoplasmic zonation in in vitro growing pollen tubes ofNicotiana sylvestris, but did not seriously affect growth. In the present study we used the herbicide carbetamide as an anti-microtubule drug. It had the same effect as colchicine and propham: the cytoplasm, including the generative cell, was no longer concentrated in the tip but was distributed randomly. In addition, ultrastructural investigations have shown that even the vesicle zone, usually found at the very tip of pollen tubes, had disappeared in some tubes. Nonetheless, in vitro growth was not inhibited by more than 20% over a period of 22 h.In contrast, tube growth in plants ceased 1 cm down in the style when carbetamide was applied to the stigma before pollination. At the lowest concentration causing this effect, microtubules of the vegetative cell had disappeared and the cytoplasm was distributed randomly, as it was for in vitro grown tubes. It can be concluded that microtubules of the vegetative cell are essential for pollen tube growth in the style.Abbreviations DAPI 4,6-diamidmo-2-phenylindole - EGTA ethyleneglycerol-bis-(aminoethyl ether) tetraacetic acid - DIC differential interference contrast - GC generative cell - IC₅₀ inhibition concentration 50% - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-Test pollen tube growth test - SAM substrate adhesion molecule - VC vegetative cell     

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     

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     

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     

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     

Use of a nuclear-targeted β-glucuronidase fusion protein to demonstrate vegetative cell-specific gene expression in developing pollen

To examine the site of expression of the tomato anther-specific gene, LAT52, in the developing male gametophyte, the LAT52 gene promoter was fused to a nuclear-targeted version of the β-glucuronidase (GUS) gene and introduced into tobacco. Transformed plants expressing GUS activity showed nuclear localization of the GUS reaction product to the vegetative cell of the pollen grain. No staining or localization was detected in the generative cell, at pollen maturation or during pollen tube growth in vitro. These results clearly demonstrate differential gene expression within the male gametophyte, and highlight regulatory events which determine the differing fates of the vegetative and generative cells following microspore mitosis.     

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.     

Differentiation of generative and vegetative cells in angiosperm pollen

 Cellular differentiation of a generative and a vegetative cell is an important event during microspore and pollen development and is requisite for double fertilization in angiosperms. The generative cell produces two sperm cells, or male gametes, whereas the vegetative cell produces an elongated pollen tube, a gametophytic cell, to deliver the male gametes to the embryo sac. For typical differentiation of the gametic and gametophytic cells, cell polarity, including nuclear positioning, must be established prior to microspore mitosis and be maintained during mitosis. Microtubules are closely involved in the process of asymmetric cell division. On the other hand, alteration of the chromatin composition seems to be responsible for the differential gene expression between the generative and vegetative cells. Cytoplasmic regulatory molecules, which affect chromatin configuration, are postulated to be unequally distributed to the two cells at the asymmetric cell division. Thus, typical differentiation of the cells is accomplished by a cellular mechanism and a molecular mechanism, which might be independent of each other. These results are discussed in relation to one model that accounts for the different fates of generative and vegetative cells during sexual plant reproduction. Received: 3 September 1996 / Revision accepted: 23 September 1996     

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.     

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.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Association of the Generative Cell and Vegetative Nucleus in Pollen Tubes of Rhododendron

Mixed fluorescence/bright field microscopy of Rhododendron pollentubes in the first 72 h after germination reveals a lens-shapedgenerative cell which divides to give two associated spermswithin the original cell boundary. The generative cell is closelyassociated with the vegetative nucleus which precedes it in92 per cent of pollen tubes. Three-dimensional reconstruction from serial thin sections ofa pollen tube fixed 24 h after germination shows that the associationbetween the generative cell and vegetative nucleus is extremelycomplex. Elongated tails of the generative cell physically enfoldthe vegetative nucleus and penetrate into enclaves within it.The association has been clarified by use of the periodic acid-phosphotungsticacid-chromic acid technique to enhance electron contrast ofthe plasma membranes surrounding the generative cell. In thisbicellular system, the male germ unit association is apparentlyinitiated after pollen maturity but prior to generative celldivision. Pollen tube, generative cell, male germ unit, plasma membrane, vegetative nucleus, Rhododendron, Ericaceae     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata

Summary The ultrastructure of the cytoskeleton inNicotiana alata pollen tubes grownin vitro has been examined after rapid freeze fixation and freeze substitution (RF-FS). Whereas cytoplasmic microtubules (MTs) and especially microfilaments (MFs) are infrequently observed after conventional chemical fixation, they occur in all samples prepared by RF-FS. Cortical MTs are oriented parallel to the long axis of the pollen tube and usually appear evenly spaced around the circumference of the cell. They are always observed with other components in a structural complex that includes the following: 1. a system of MFs, in which individual elements are aligned along the sides of the MTs and crossbridged to them; 2. a system of cooriented tubular endoplasmic reticulum (ER) lying beneath the MTs, and 3. the plasma membrane (PM) to which the MTs appear to be extensively linked. The cortical cytoskeleton is thus structurally complex, and contains elements such as MFs and ER that must be considered together with the MTs in any attempt to elucidate cytoskeletal function. MTs are also observed within the vegetative cytoplasm either singly or in small groups. Observations reveal that some of these may be closely associated with the envelope of the vegetative nucleus. MTs of the generative cell, in contrast to those of the vegetative cytoplasm, occur tightly clustered in bundles and show extensive cross-bridging. These bundles, especially in the distal tail of the generative cell, are markedly undulated. MFs are observed commonly in the cytoplasm of the vegetative cell. They occur in bundles oriented predominantly parallel to the pollen tube axis. Although proof is not provided, we suggest that they are composed of actin and are responsible for generating the vigorous cytoplasmic streaming characteristic of living pollen tubes.Abbreviations EGTA ethylene glycol bis-(-aminoethyl ether), N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - MF microfilament - MT microtubule - PEG polyethylene glycol - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution     

Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains

We examined changes in the localization of cytoplasmic rRNA during pollen development inNicotiana tabacum SR-1. The rRNA was visualized byin situ hybridization, and the signal intensity of rRNA in microspore, vegetative and generative cell was quantified by microphotometry. The amount of rRNA per microspore or pollen section increased about 5 times from microspore to mature pollen grain and kept increasing even in the late stage of pollen development after PMI. The increase of rRNA occur in both vegetative and generative cells. The results suggest that synthesis of rRNA occur even after PM I in both vegetative and generative cells.     

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.     

Comparison of density of nuclear pores on vegetative and generative nuclei in pollen of Tradescantia

The freeze-etch technique has been used to expose surface views of vegetative and generative nuclear envelopes in both ungerminated and germinated pollen of Tradescantia paludosa. A comparison of the density and total numbers of nuclear pores on the two nuclei indicates that vegetative nuclei have at least twice as many pores as generative nuclei. These findings are compatible with the concept that the vegetative nucleus plays a more active role in pollen tube development than the generative nucleus.