Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes

The germination and growth of pollen grains of Nicotiana tabacum and N. alata with the anti-microtubule drug oryzalin retarded significantly the movement of the vegetative nucleus (VN) and the generative cell (GC) from the grain to the tube apex but had no effect on pollen tube elongation. In N. tabacum, only 11% and 48% of the pollen tubes treated with oryzalin for 6 h and 12 h, respectively, had the VN and GC in the tube mainly in its middle part. In corresponding control materials, 79% and 99% of pollen tubes contained the VN and GC close to the apex. Indirect immunofluorescence microscopy and related studies of the tubes grown in the presence of oryzalin revealed complete absence of microtubules (MTs) but apparently intact microfilaments (MFs). These results suggested that the movement of VN and GC from the grain into the tube is possible when no MTs but only MFs are present, but the movement is then slow. In control tubes, the parallel orientation of MT bundles and extensions of VN were interpreted to represent the structural organization needed for the MT-dependent movement of VN.     

Differential mechanisms of movement between a generative cell and a vegetative nucleus in pollen tubes of Nicotiana tabacum as revealed by additions of colchicine and nonanoic acid

The growth of pollen tubes of Nicotiana tabacum cultured in a petri dish was divided into five stages by the behaviors of pollen tubes, vegetative nuclei (VNs), and generative cells (GCs). Pollen tubes continued to elongate during every stage. Colchicine, at a concentration of 1 mM, preferentially inhibited the movement of the VNs but not that of GCs. Nonanoic acid preferentially inhibited the movement of GCs. These results suggest that different mechanisms of movement exist between VNs and GCs.     

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.     

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

Summary 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.     

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     

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     

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.     

The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance

Organellar DNA in mature pollen grains of eight angiosperm species (Actinidia deliciosa Lindl., Antirrhinum majus L., Arabidopsis thaliana (L.) Heynh., Medicago sativa L., Musa acuminata Colla, Pelargonium zonale (L.) L'Hér, Petunia hybrida Vilm. and Rhododendron mucronatum (Blume) G. Don, in which the modes of organellar inheritance have been determined genetically, was observed by fluorescence microscopy using Technovit 7100 resin sections double-stained with 4′,6-diamidino-2-phenylindole (DAPI) and 3,3′-dihexyloxacarbocyanine iodide (DiOC₆). The eight species were classified into four types, based on the presence or absence of organellar DNA in mature generative cells: namely (1) type “m+p+”, which has both mitochondrial and plastid DNA (P. zonale), (2) type “m+p–”, which only has mitochondrial DNA (M. acuminata), (3) type “m−p+”, which only has plastid DNA (A. deliciosa, M. sativa, R. mucronatum), and (4) type “m−p−”, which has neither mitochondrial nor plastid DNA (A. majus, A. thaliana, P. hybrida). This classification corresponded to the mode of organellar inheritance determined by genetic analysis. The presence or absence of mitochondrial and plastid DNA corresponded to paternal/biparental inheritance or maternal inheritance of the respective organelle, respectively. When organellar DNA was present in mature generative cells (m+ or p+), the DNA content of the organelles in the generative cells started to increase immediately after pollen mitosis one (PMI). In contrast, the DNA content of organelles in generative cells decreased rapidly after PMI when organellar DNA was absent from mature generative cells (m− or p−). These results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI. Received: 22 December 1998 / Accepted: 8 February 1999     

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.     

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     

Differential localization of the LIM domain protein PLIM-1 in microspores and mature pollen grains from sunflower

 PLIM-1 is a LIM domain protein specifically expressed in pollen grains. Using two PLIM-1-specific monoclonal antibodies we studied its expression and intracellular location at various developmental stages of sunflower (Helianthus annuus L.) pollen. Our studies show that the protein appears at the microspore stage in a limited number of cytoplasmic bodies, becomes undetectable in bicellular pollen, and reappears in tricellular pollen grains in cortical patches particularly concentrated in the F-actin-enriched germination cones of the vegetative cell. The developmental stage-dependent, different location of the protein suggests a dual function during pollen development. While this function in microspore development remains obscure, the high concentration of PLIM-1 in the germination cones of mature pollen suggests that it participates in the germination process as well as in pollen tube growth. Received: 11 August 1998 / Revision accepted: 15 December 1998     

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.     

Biogenesis and function of the lipidic structures of pollen grains

 Pollen grains contain several lipidic structures, which play a key role in their development as male gametophytes. The elaborate extracellular pollen wall, the exine, is largely formed from acyl lipid and phenylpropanoid precursors, which together form the exceptionally stable biopolymer sporopollenin. An additional extracellular lipidic matrix, the pollen coat, which is particularly prominent in entomophilous plants, covers the interstices of the exine and has many important functions in pollen dispersal and pollen-stigma recognition. The sporopollenin and pollen coat precursors are both synthesised in the tapetum under the control of the sporophytic genome, but at different stages of development. Pollen grains also contain two major intracellular lipidic structures, namely storage oil bodies and an extensive membrane network. These intracellular lipids are synthesised in the vegetative cell of the pollen grain under the control of the gametophytic genome. Over the past few years there has been significant progress in elucidating the composition, biogenesis and function of these important pollen structures. The purpose of this review is to describe these recent advances within the historical context of research into pollen development. Received: 1 November 1997 / Revision accepted: 3 February 1998     

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.     

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

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

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997     

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