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.     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 1. Descriptive cytology and three-dimensional organization

The generative cell is initiated as a small, lenticular, unpolarized cell with a cell wall traceable to two origins: the external segment originates as intine, while an inner callose positive cell wall forms de novo. As the lenticular generative cell begins its migration into the pollen cytoplasm, the generative cell becomes polarized both externally and internally, displaying a characteristic shape and patterns of organelle distribution oriented with respect to the vegetative nucleus and independent of pollen aperture location. Separation of the generative cell from the pollen wall begins at the end opposite the vegetative nucleus and results in an elongating protuberance at the opposite end of the generative cell; this becomes associated with a preformed groove located on the surface of the vegetative nucleus. The generative cell subsequently separates from the intine near the vegetative nucleus and moves progressively toward the opposite end of the cell; during this separation, the edge of the wall facing the intine becomes callose-positive and remains so until separating from the intine. The generative cell becomes a free cell within the pollen, which is in physical association with the vegetative nucleus. Generative cell organization and organelle content become increasingly polarized during maturation, with microtubules evident both in the elongating protuberance of the generative cell and in association with organelles. The generative nucleus migrates away from the vegetative nucleus and toward the plastid-rich end of the generative cell, whereas mitochondria are more generally distributed within the cell. Generative cell polarization is made permanent during mitotic division and cytokinesis, i.e., two sperm cells differing in morphology are formed: the larger cell associated with the vegetative nucleus (S_vn) contains a majority of the mitochondria, and the smaller, unassociated sperm cell (S_ua) receives the plastids.     

杜仲花粉不对称分裂的超微结构观察

运用透射电镜对杜仲花粉发育进程进行了观察研究。结果显示,杜仲小孢子的第一次分裂为不等分裂,形成小的生殖细胞和大的营养细胞。分裂开始前小孢子的营养极形成许多小液泡,建立细胞极性;然后随着核膜的解体核周围的细胞器逐渐向纺锤体区靠近,围绕在纺锤体周围。花粉第一次有丝分裂完成后,生殖细胞所获得的细胞器开始分布在细胞的两侧,后来移向生殖细胞的营养极,而紧贴花粉壁的生殖极无细胞器分布。这种生殖细胞早期的细胞极性,可能为进一步分裂形成精细胞奠定基础。     

Pollen mitosis in the slipper orchid Cypripedium fasciculatum

Pollen mitosis in the slipper orchid Cypripedium fasciculatum was studied using correlated methods of immunofluorescence and transmission electron microscopy. Unlike the more highly evolved orchids, the cypripedioid orchids shed pollen as monosulcate monads. Prior to pollen mitosis, the microspore nucleus migrates to a proximal position opposite the aperture, as is typical of monocotyledons. There is no distinct generative pole microtubule system (GPMS) like that recently reported in development of pollen polarity in the vandoid moth orchid Phalaenopsis. Instead, microtubules in early prophase are concentrated around the nucleus and extend into the cytoplasm toward the future generative pole. Once the nucleus has migrated to the continuous surface opposite the aperture, microtubules surround the nucleus evenly and show no tendency to be more concentrated in the generative domain. The mitotic spindle, which develops from the perinuclear microtubules, is asymmetrically placed in the microspore and is cone-shaped. The generative pole is broad and closely appressed to the continuous spore surface, while the vegetative pole is pointed and located in the interior of the microspore. As the chromosomes move poleward, microtubules proliferate in the interzone and a phragmoplast develops. The phragmoplast expands in a hemispherical path beyond the interzone following an array of microtubules that radiates from the generative nucleus. Data from this study indicate that evolution of pollen in orchids includes a shift in location of the generative cell from proximal to distal and the evolution of a GPMS, in addition in the well-known trend toward increased pollen aggregation and loss of exine.     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.     

DEVELOPMENT OF OMNIAPERTURATE POLLEN IN TRILLIUM KAMTSCHATICUM (LILIACEAE)

Ultrastructural changes during omniaperturate pollen development in Trillium kamtschaticum Pall, was examined using transmission electron microscopy. The pollen mother cells are not enveloped within a thick callosic wall. The microspores resulting from successive meiosis are divided by scanty deposition of callosic wall in the tetrad. A primexine/exine template is not recognizable within the tetrad during formation of exinous components. Preexinous globules, originating from vesicles in the callosic wall, accumulate electron-dense materials and develop into exinous globules. The preexinous globules have ca 10 nm wide contacts with tilted and invaginated plasma membrane of the microspore within the callosic wall. After dissolution of the callosic wall, the microspores separate and mitosis subsequently leads to the formation of a generative cell and vegetative cell encased in a loose aggregation of developing exinous globules. When the generative cell is at the pollen grain surface, the channeled zone is initiated at the opposite side of the microspore on the surface of the vegetative cell. Just before pollen maturity, a new layer develops under the channeled zone. Thus, development of the omniaperturate pollen grains of T. kamtschaticum involves some processes that are distinct from those of Canna and Heliconia and some that are similar.     

Pollen Tetrads of Hedycarya arborea J. R. et G. Forst. (Monimiaceae)

Hedycarya has pollen in permanent tetrads. H. arborea, the New Zealand species, differs from others studied, in having a cap of more or less imperforate tectum at the distal pole of each grain. This polar region is not an aperture and the pollen tube emerges through a papillose part of the external wall of each grain. Transmission electron microscope studies of immature and mature tetrads reveal a most unusual exine structure. "Radial processes" develop by accumulation of sporopollenin around unit membranes of similar dimensions to the plasmalemma, and extend from just beyond the intine to the tectal region. The entire exine is considered ectexinous. During development, members of a tetrad are interconnected by cytoplasmic channels and the synchronous division into generative and vegetative nuclei within each tetrad is attributed to their presence. The channels become closed by the deposition of intine. Comparisons are made with exine structure in some other members of the woody Ranales and with some other plants with tetrad pollen.     

莴苣花粉发育过程中ATP酶的分布

对莴苣花粉发育过程中ATPase的分布特征做了研究。四分体早期的小孢子细胞质中开始出现ATPase反应颗粒。之后,小孢子在发育过程中,花粉内壁聚集大量体积较大的ATPase反应颗粒,并一直保持到花粉即将成熟。在小孢子发育晚期,在花粉萌发孔处和小孢子大液泡中也特异性地聚集了较多ATPase颗粒。二胞花粉刚形成的生殖细胞表面呈现大量的ATPase反应颗粒,当生殖细胞脱离花粉内壁移入营养细胞,ATPase反应颗粒基本消失。生殖细胞分化过程中生殖细胞的ATPase反应颗粒逐渐低于营养细胞中的。在成熟花粉中,精细胞中的ATPase反应颗粒比营养细胞中的少,且主要集中在细胞核中。结果显示花粉发育过程中ATPase的特异分布与花粉发育的一些生物学事件密切相关。     

Mitosis and Amitosis of Generative Cell Nuclei in Artificially Germinated Pollen Tubes in Zephyranthes candida(Lindl.)Herb.

This paper deals with the comportmem of the vegetative nucleus and its spatial association with the generative cell and sperm cells in the artificially germinated pollen tubes of Zephyranthes candida (Lindl.) Herb. before and after generative cell mitosis with the use of DNA-specific fluochrome 4′,6-diamidino-2-phenylindole (DAPI). The induction of amitosis and abnormal mitosis of generative cell nuclei by cold-pretreatment of the pollen prior to germination was studied in particular. In normal case, the generative cell, after appressing to the vegetative nucleus for certain time, underwent mitosis to form two sperms, while the vegetative nucleus became markedly elongated, diffused, and exhibited blurring of its fluorescence. After division, a pair of sperms remained shortly in close connexion with the vegetative nucleus. Then the vegetative nucleus returned to its original state. In the pollen tubes germinated from cold-pretreated pollen, amitosis of some generative cell nuclei were frequently observed. Amitosis took place via either equal or unequal division with a mode of constriction. During amitosis, the dynamic change of vegetative nucleus and its intimate association with generative cell afore described did not occur. Sperm nuclei produced from amitosis could farther undergo amitisis resulting in micronnclei. Factors affecting the amitosic rate of generative cells, such as pollen developmental stage, temperature and duration of cold-pretreatment, were studied. Besides amitosis, cold-pretreatment also induced some abnormal mitotic behavior leading to the formation of micronuclei. Based on our observations and previously reported facts in other plant materials, it is inferred that the vegetative nucleus plays an important role in normal mitosis of generative cell and development of sperms.     

Generative cell division and sperm cell formation in barley

Summary Shortly before and during division, the generative cell of barley (Hordeum vulgare L.) is located near the vegetative nucleus, in the peripheral layer of the highly vacuolated vegetative cell at the aperture pole. This position is also characteristic of the two resulting sperm cells. Conventional mitosis of the generative cell is followed by cytokinesis through cell plate formation. Just after division, the two sperm cells are enclosed together within a common inner vegetative cell plasma membrane, and they gradually separate from each other only during pollen maturation. The space between the generative or sperm cell plasma membrane and the vegetative cell plasma membrane is very thin and appears to be devoid of a cell wall. Both the generative cell and the young sperm cells contain a normal set of organelles; plastids devoid of starch are only sporadically observed. Our data indicate that in Hordeum vulgare the generative cell divides after migrating inside the pollen grain. This follows the pattern of development well established for several species with tricellular pollen.     

The male germ unit in the pollen and pollen tubes ofPetunia hybrida: Ultrastructural,quantitative and three-dimensional features

Summary The male germ unit ofPetunia hybrida was examined quantitatively and qualitatively at the ultrastructutral level. Three-dimensional reconstructions, the determination of nuclear and cytoplasmic volumes and surface areas, and organelle counts were obtained from serial ultrathin sections and computer analysis. In the mature pollen grain, an elongated generative cell is found in direct physical association with and partially surrounded by the vegetative nucleus. The mature generative cell lacks plastids and has mitochondria equally distributed at both of its tapering ends. In the pollen tube, the sperm cells are physically associated by cytoplasmic connections to each other and to the surrounding vegetative cell membrane. At full style length, the lobed vegetative nucleus and sperm pair are found in close association near the end of the pollen tube. The two sperms of a pair are not strongly dimorphic.     

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.     

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.     

土麦冬离体萌发花粉管中生殖细胞与营养核的动态变化

主要报道了土麦冬人工培养萌发花粉管中生殖细胞与营养核的动态变化。多数花粉管中,生殖细胞与营养核贴合后,开始进行有丝分裂,贴合时,营养核略呈弥散状态。在分裂早中期,生殖细胞与营养核分开,从贴合到分开大约经历３－５ｈ,精子形成后,不与营养核连接。ＤＡＰＩ对生殖细胞的有丝分裂有抑制作用。少数花粉管中,生殖细胞核进行无丝分裂,有缢裂和劈裂两种方式。生殖细胞核发生缢裂的花粉管中,未观察到生殖细胞与营养核的贴     

Microsporogenesis,Megasporogenesis and the Formation of Male and Female Gametophyte in Coix lacryma-jobi L.

The development of the anther wall follows the monocotyledonous type. During meiotic stages of prophase I, some cytoplasmic channels are observed on the walls between meiotic cells. which divide synchronously. Cytokinesis in the microspore mother cell is of the successive type and gives rise to iscbilateral tetrad. The cell wall between the generative cell and the vegetative cell in early stage shows PAS positive reaction. The mature pollen grain is of 3-celled type. The development of the female gametophyte follows the polygonum-type; the antipodal cells proliferate to form a multicellular tissue mass. Many starch grains are present in the central cell. The nucleus of the mature egg cell is located at the micropylar end; a great deal of starch grains are in the cytoplasm surrounding the nucleus, while vacuoles of various size distribute throughout its cytoplasm but are more and larger at the chalaza,1 end. The nucleus of the synergid cell is located at the micropytar end where a filiform apparatus is formed and many small vacuoles are present at the chalazal part.     

The spermatogenous body cell of the coniferPicea abies (Norway spruce) contains actin microfilaments

Summary In conifer pollen, the generative cell divides into a sterile stalk cell and a body cell, which subsequently divides to produce two sperm. InPicea abies (Norway spruce, Pinaceae) this spermatogenous body cell contains actin microfilaments. Microfilament bundles follow the spherical contour of the body cell within the cell cortex, and also traverse the cytoplasm and enmesh amyloplasts and other organelles. In addition, microfilaments are associated with the surface of the body cell nucleus. The sterile stalk cell also contains microfilament bundles in the cytoplasm, around organelles, and along the nuclear surface. Within the pollen grain, microfilament bundles traverse the vegetative-cell cytoplasm and are enriched in a webbed cage which surrounds the body cell. Microfilaments were identified with rhodamine-phalloidin and with indirect immunofluo-rescence using a monoclonal antibody to actin. The majority of evidence in literature suggests that the spermatogenous generative cell in angiosperms does not contain actin microfilaments, so the presence of microfilaments within the spermatogenous body cell inP. abies appears to be a fundamental difference in sexual reproduction between conifers and angiosperms.     

Cytological Observations of Microsporogenesis and Pollen Development in Wheat in Vivo

Cytological observations of microsporogenesis and pollen development in vivo in wheat were carried on by means of phase contrast optics, which could avoid the distortions resulted from materials difficult to be fixed. The cytological changes were observed as follows: 1. Just before first mitosis of pollen, many strands of cytoplasm arose from one side of the nucleus facing the aperture, and moved swingingly toward the aperture. And then these strands of cytoplasm combined into one mass and protruded in the large vacuole. 2. There was a alteration in the direction of the spindle axis from obliquity to parallelism in anaphase. 3. Forming course of the wall between generative and vegetative cell. 4. Dynamic course of the disintegration of wall between vegetative and generative cell as well as that the generative cell came into the vetetafive cell.     

Anther culture of Sorghum bicolor (L.) Moench

The sequence of pollen development from the tetrad stage to the mature tricellular grain was studied in freshly harvested anthers of Sorghum bicolor. This pattern of development was then compared with that occurring during panicle pretreatment and subsequent anther incubation in vitro. It was found that during pretreatment at 7° C mitoses of the vegetative cell were induced in up to 30% of the pollen. During anther incubation procallus development was highly polarised with contributions from both the generative and vegetative cells. After pretreatment at 14 or 20° C the generative cell became detached from the pollen wall and it was not possible to determine whether subsequent development involved only the vegetative cell or both the vegetative and generative cells.Although retarded pollen grains were observed both in vivo and in vitro, and were occasionally seen to divide in culture, they did not appear to be the source of the procalluses produced.     

洋葱花粉发育过程中ATP酶的分布特征(简报)

在真核细胞中,除了线粒体和叶绿体ATPase的功能是合成ATP外,其余部位ATPase是水解ATP以获取生物能量的代谢酶,在生物体细胞内广泛存在。探索ATPase在细胞中的分布状态是研究细胞生理状态的一种重要手段。ATPase在细胞中的多少可反映出细胞当时的生活状态,这一特征已被初步用于探索小麦和水稻雄性不育的细胞生物学研究中,希望通过比较可育花药和不育花药中ATPase的分布差异寻找雄性不育的机理,发现