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.     

Ultrastructure and microtubule organization of isolated generative cells ofAllemanda neriifolia at interphase and prophase

Summary The ultrastructure of isolated generative cells ofAllemanda neriifolia at interphase and prophase was studied. The microtubule organization of the isolated cells was also investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. After the generative cells had been isolated from the growing pollen tubes by osmotic shock, most of the cells were at prophase and only a few were at interphase. The interphase cell is spindle shaped and contains an ellipsoidal nucleus. In addition to the usual organelles, the cytoplasm of the interphase cell contains numerous vesicles (each measuring 40–50 nm in diameter) and two sets of longitudinally oriented microtubule bundles — one in the cortical region and the other near the nucleus. Most of the prophase cells are spherical in shape. Based on the ultrastructure and the pattern of microtubule cytoskeleton organization three types of prophase cells can be recognized. (1) Early prophase cell, which contains the usual organelles, numerous vesicles, and a spherical nucleus with condensed chromosomes. Longitudinally oriented microtubule bundles can no longer be seen present in the early prophase cell. A new type of structure resembling a microtubule aggregate appears in the cytoplasm. (2) Mid prophase cell, which has a spherical nucleus containing chromosomes that appear more condensed than those seen in the early prophase cell. In addition to containing the usual organelles, the cytoplasm of this cell contains numerous apparently randomly oriented microtubules. Few vesicles are seen and microtubule aggregates are no longer present. (3) Late prophase cell, typified by the lack of a nuclear envelope. Consequently, the chromosomes become randomly scattered in the cytoplasm. Microtubules are still present and some become closely associated with the chromosomes. The changes in the ultrastructure and in the pattern of microtubule organization in the interphase and prophase cells are discussed in relation to the method of isolation of the generative cells.     

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.     

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The microtubule organizational changes in the isolated generative cells of Allemanda schottii were followed using immunofluorescence and confocal laser scanning microscopy. Due to the improved resolution and the lack of out-of-focus flares, the microtubule cytoskeleton of the generative cells could be visualized more clearly than using conventional epifluorescence systems. Immediately after isolation the microtubule cytoskeleton of the generative cells was cage-like composed of longitudinally oriented microtubule bundles. Later, some bundles began to depolymerize and at the same time some smaller bundles appearred. The smaller bundles unlike the longitudinal bundles crisscrossed throughout the cell. Later still, the cells became spherical. Both the longitudinal and the smaller bundles disappearred. At the same time some of the microtubules began to aggregate around the nucleus. These perinuclear microtubules were apparently not very stable, because soon afterwards,they started to disintegrate. By the time the cells became completely spherical,the cytoplasm became filled with diffuse fluorescence indicating that the tubulin was no longer existing in a polymerized form but in a monomeric form inside the cell. After the fuberlin had completely depolymerized the microtubules started to reform. The sequence of events leading to the reformation of the microtubule cytoskeleton in the spherical cells was as follow: A few nucleating centres began to form first. Then the nucleating centres gave rise to microtubule bundles. The bundles extended and aggregated to form a reticulate network. This cytoskeletal network appearred stable and well organized. It also had a lot of microtubule-bundle junctions. The network persisted after Triton X-l00 extraction.     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants

Large quantities of intact generative cells and their protoplasts were isolated from pollen protoplasts of four liliaceous plants, and their structural features were investigated. The generative cells, liberated from the vegetative cell cytoplasm of the pollen protoplasts, were initially spindle-shaped with two long, oppositely oriented extensions, and were surrounded by two cell membranes, one on each side of a wall of uniform thickness. The generative nuclei, stained with 4′,6-diamidino-2-phenylindole (DAPI), showed ellipsoidal and highly condensed chromatin, whereas the generative cell cytoplasm, whose quantity was widely different from species to species, showed no fluorescence, suggesting the absence of plastid and mitochondria! DNA, although many mitochondria were present. The isolated generative cells, which were spindle-shaped at first, became spherical in shape in vitro. Immunocytochemistry and transmission electron microscopy revealed that this change was associated with the depolymerization of an axial array of microtubules present in generative cells in situ. These results are discussed in relation to the function of the generative cell within the bicellular pollen of angiosperms.     

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.     

Identification of higher plant GlsA,a putative morphogenesis factor of gametic cells

GlsA has been identified in an asexual-reproductive-cell (gonidia)-deficient mutant of Volvox as a chaperone-like protein essential for gonidia production. In this study, we isolated an angiosperm glsA (LlglsA) gene expressed during Lilium longiflorum pollen development. Immunoblot analyses showed that the strong LlGlsA expression occurred in the generative cell and its pattern during pollen development corresponded to that of alpha-tubulin. Morphological analyses succeeded in visualizing the dispersion of the strong LlGlsA signal in developing generative cells. In addition, multiple-immunofluorescence staining of LlGlsA and alpha-tubulin revealed that some of the dot-like LlGlsA signals were co-localized with microtubule filaments. From those results, we suggest that angiosperm GlsA functions as a chaperone modifying various structures during male gametic cell formation.     

Generative cells ofLilium longiflorum possess translatable mRNA and functional protein synthesis machinery

Metabolic labelling with [³⁵S]-methionine demonstrated that generative cells ofLilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [³⁵S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

Unique actin and microtubule arrays co-ordinate the differentiation of microspores to mature pollen in Nicotiana tabacum

The complex cellular events that occur during development of the male gametophyte of higher plants suggest a role for the cytoskeleton. This investigation has revealed that unique microtubule arrays mediate events that occur during microspore development; both actin and microtubule arrays have important roles during the asymmetrical microspore mitosis and unique actin arrays mediate events that occur during early pollen development. Migration of the nucleus to the generative pole during cellular polarization of the microspore is mediated by a microtubule cage that encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it to the pole prior to the asymmetrical mitosis. During entry into mitosis, the microtubule cage becomes modified and transforms into the asymmetrical mitotic spindle. Actin is localized within the region of the mitotic spindle and in the phragmoplast. Following mitosis, actin networks enclose first the generative cell and then the vegetative nucleus. These actin networks function during migration of the generative cell and vegetative nucleus toward the centre of the pollen grain. Mature pollen contains a dense cortical actin meshwork and a disc-shaped microtubule array enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of unique actin and microtubule arrays is an essential determinant of microspore and pollen development.     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L．)、鸢尾(Iris tectorium Maxim．)和朱顶红(Hippeastrum vittatum Herb．)的生殖细胞质膜表面的凝集素受体进行标记。结果显示：在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布。这可能是导致部分精子表面不能被同种凝集素标记的重要原因。此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同。在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体。在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体。此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示。     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L.)、鸢尾(Iris tectorium Maxim.)和朱顶红(Hippeastrum vittatum Herb.)的生殖细胞质膜表面的凝集素受体进行标记.结果显示:在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布.这可能是导致部分精子表面不能被同种凝集素标记的重要原因.此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同.在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体.在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体.此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示.     

Fusion Experiments of Isolated Generative Cells in Several Angiosperm Species

The generative cells used for fusion experiments were isolated from pollen grains of Zephyranthes candida and Lycoris radiata by “2-step osmotic shock” and from those of Hippeastrum vittata, Hemerocallis minor and Iris tectorum by “weak enzyme treatment” as reported previously. Using PEG method, fusions have been successfully induced between generative cells of the same species mentioned above, between generative cells of Z. candida and L. radiata, between generative cells and petal protoplasts in L. radiata, and between generative cells of L. radiata and hypocotyl protoplasts of Brassica napus. In all cases either homokaryons or heterokaryons could be obtained. Fusion of nuclei was observed sometimes in homokaryons of generative cells in L. radiata. The generative nuclei in fusion products could be well identified by labelling the generative cells before fusion with DAPI. FDA test demonstrated that most of the fusion products were viable. Factors affecting fusion efficiency including cell density, PEG concentration, duration of PEG treatment and effect of calcium ions were studied in fusion of generative cells in Z. candida. Our experiments indicate that isolated generative cells are likely to be deprived of cell wails and may be regarded as a special kind of protoplasts for direct fusion experiments.     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

黄蝉花生殖细胞有丝分裂期间微管的动态

用微管免疫荧光方法观察了黄蝉花生殖细胞在花粉管中进行有丝分裂时的微管动态。微管在不同分裂期的分布情形很不一样。当生殖细胞由花粉进入花粉管后,细胞便立刻开始分裂进入早前期,在这阶段微管以一个紧密微管网笼子形式存在生殖细胞内。之后,细胞进入中前期,在此阶段细胞核扩大,染色体变粗,而存在细胞内的微管网逐渐变为疏松散漫状,跟着细胞进入晚前期,而微管笼子则由网状变为纵向排列状。分裂进入早中期微管变细并呈波浪状,微管由笼子结构过渡到纺锤体结构。进入中期,纺锤体全部形成,在纺锤体内可以清楚地看到两种不同类型的微管束,一种附着在染色体上,而另一种则从一极延伸至另一极。跟着细胞进入早后期,在这一阶段姊妹染色体分开并分别移向两极,在赤道板位置微管明显减少。之后,细胞进入晚后期,姊妹染色体集中在两极,极端有新微管出现。在两个染色体团之间又汇集了许多类似成膜体微管的微管。细胞进入分裂末期,存在赤道板位置的微管又再次减少,而在中央部位则新形成一“成膜体联接区”,把两个新形成的精子连接着。     

The male germ unit of Rhododendron: quantitative cytology,three-dimensional reconstruction,isolation and detection using fluorescent probes

Summary The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.     

Cortical microtubule organization in Vitis protoplasts as affected by concentration of enzyme isolation medium and duration of incubation

Protoplasts of Vitis rotundifolia Michx. cv. Summit were isolated from mesophyll of axenic shoot cultures under different enzyme concentrations and digestion times. Viability and plating efficiency were assessed and related to the cortical microtubule network, visualized using immunofluorescence. Higher concentrations of enzyme isolation medium significantly decreased protoplast viability and plating efficiency. However, the cortical microtubule network appeared stable, at all concentrations with dense, continuous microtubule strands in both random and parallel arrays. In contrast, longer vs shorter enzyme incubation duration resulted in significantly lower plating efficiency, which was correlated with changes in cortical microtubule organization. With longer incubation, the frequency of parallel microtubule strands decreased; microtubule organization showed increasing disruption, microtubule strands were shortened, fragmented and exhibited only a weak fluorescence labeling. Both high enzyme concentration and prolonged incubation periods negatively affected protoplast regenerability, but in different ways. Microtubule organization was sensitive to duration of incubation, but not to enzyme concentration. It is concluded that the presence of a well-developed cortical microtubule network does not gurantee regeneration. Other factors related to isolation appear to be involved.