Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

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.     

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.     

Early events in division of the generative cell ofOrnithogalum virens

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

Microfilament bundles of F-actin inSpirogyra observed by fluorescence microscopy

Microfilament bundles (MFBs) of F-actin were observed by fluorescence microscopy in cells ofSpirogyra treated with rhodamine-phalloidin. Four types of MFBs could be recognized on the basis of locality and appearance: those dispersed in the cytoplasm near the cell surface; those beneath the plasma membrane running parallel to each other; those at the edges of the chloroplast; and those surrounding the nucleus. Each type exhibited a unique behavior during the cell cycle. Microfilament bundles dispersed in the cytoplasm came together at the middle of the cell to form a fibril ring at the mitotic prophase. The fibril ring decreased in diameter, causing the development of a furrow in the protoplast that progressed from the outside to the inside. After the completion of furrowing, the MFBs in the fibril ring dispersed beneath the plasma membrane. Microfilament bundles surrounding the nucleus formed a net-like cage which became invisible at the mitotic anaphase, while MFBs seen at the chloroplast edges persisted there during the cell cycle without changing their position. Parallel MFBs running perpendicular to the long axis of the cell were seen at all stages in the cell cycle.Abbreviations MF microfilament - MFB microfilament bundle - MT microtubule     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

Plastid differentiation during Cucurbita pepo (Cucurbitaceae) pollen grain development

The development of plastids in the pollen of Cucurbita pepo was followed from meiosis to pollen maturation by quantitative light and electron microscopy. Plastids are initially undifferentiated, then divide, and at late microspore stage differentiate into amyloplasts containing starch. Later the amyloplasts form lobes and divide. Amyloplasts containing a single starch grain are present from the early bicellular stage. Plastid development is considered in relation to such cytoembryological features as the pollen does not dehydrate at anthesis and germination begins 3 min after pollination.     

The coexistence of bicellular and tricellular pollen in Annona cherimola (Annonaceae): Implications for pollen evolution

Most angiosperms release bicellular pollen. However, in about one-third of extant angiosperms, the second pollen mitosis occurs before anthesis such that pollen is tricellular upon release. The shift from bicellular to tricellular development has occurred several times independently, but its causes are largely unknown. In this work, we observed the coexistence of both kinds of pollen at anther dehiscence in Annona cherimola, a species that belongs to the basal angiosperm family Annonaceae. Examination of pollen cell number during anther development showed that this coexistence was due to a late mitosis starting shortly before pollen shedding. Both types of pollen germinated equally well over the course of development. Because variable proportions of bicellular and tricellular pollen were observed at different sampling times, we tested the role of temperature by performing field and growth chamber experiments, which showed that higher temperatures near anthesis advanced the time of pollen mitosis II. The results show that selection could favor the production of tricellular pollen under certain environmental circumstances that prime rapid pollen germination and provide evidence of a system in which developmental variation persists, but that can be modified by external factors such as temperature.     

芒果花药发育的细胞化学研究

芒果花药发育中,花药药壁体细胞中淀粉粒多糖和脂滴类物质一直很少,仅药室内壁细胞中有零星淀粉粒分布.到二胞花粉早期,花粉营养细胞中的大液泡消失,开始积累淀粉粒.芒果成熟花粉中储存营养物质主要是淀粉粒,而脂类物质一直很少.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

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.     

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     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

Reorganization of the actin and microtubule cytoskeleton throughout blue-light-induced differentiation of characean protonemata into multicellular thalli

Summary The reorganization of the actin and microtubule (MT) cytoskeleton was immunocytochemically visualized by confocal laser scanning microscopy throughout the photomorphogenetic differentiation of tip-growing characean protonemata into multicellular green thalli. After irradiating dark-grown protonemata with blue or white light, decreasing rates of gravitropic tip-growth were accompanied by a series of events leading to the first cell division: the nucleus migrated towards the tip; MTs and plastids invaded the apical cytoplasm; the polar zonation of cytoplasmic organelles and the prominent actin patch at the cell tip disappeared and the tip-focused actin microfilaments (MFs) were reorganized into a homogeneous network. During prometaphase and metaphase, extranuclear spindle microtubules formed between the two spindle poles. Cytoplasmic MTs associated with the apical spindle pole decreased in number but did not disappear completely during mitosis. The basal cortical MTs represent a discrete MT population that is independent from the basal spindle poles and did not redistribute during mitosis and cytokinesis. Preprophase MT bands were never detected but cytokinesis was characterized by higher-plant-like phragmoplast MT arrays. Cytoplasmic actin MFs persisted as a dense network in the apical cytoplasm throughout the first cell division. They were not found in close contact with spindle MTs, but actin MFs were clearly coaligned along the MTs of the early phragmoplast. The later belt-like phragmoplast was completely depleted of MFs close to the time of cell plate fusion except for a few actin MF bundles that extended to the margin of the growing cell plate. The cell plate itself and young anticlinal cell walls showed strong actin immunofluorescence. After several anticlinal cell divisions, basal cells of the multicellular protonema produced nodal cell complexes by multiple periclinal divisions. The apical-dome cell of the new shoot which originated from a nodal cell becomes the meristem initial that regularly divides to produce a segment cell. The segment cell subsequently divides to produce a single file of alternating internodal cells and multicellular nodes which together form the complexly organized characean thallus. The actin and MT distribution of nodal cells resembles that of higherplant meristem cells, whereas the internodal cells exhibit a highly specialized cortical system of MTs and streaming-generating actin bundles, typical of highly vacuolated plant cells. The transformation from the asymmetric mitotic spindle of the polarized tip-growing protonema cell to the symmetric, higher-plant-like spindle of nodal thallus cells recapitulates the evolutionary steps from the more primitive organisms to higher plants.Abbreviations FITC fluorescein isothiocyanate - MF microfilament - MT microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline     

An analysis of the role of actin during pollen activation leading to germination in pear (Pyrus communis L.): treatment with cytochalasin D

Summary Major stages of actin organization during activation leading to germination of pear (Pyrus communis L.) pollen were disrupted by treatment with 5 g/ml cytochalasin D (CD), and the effects of the drug were monitored with rhodamine-phalloidin staining. CD induced the formation of granules or short rods in the place of the filamentous arrays that occur in normally developing pollen. Filamentous arrays, however, returned upon removal of CD. Pollen incubated directly in CD showed a gradual disappearance of circular actin profiles and their replacement by either granules or, less frequently, short rods. These granules and rods initially had a random distribution in the cell, but with time in CD they became localized at one of the three germination apertures. Pollen was also allowed to reach three stages of microfilament (MF) organization (initial fibrillar arrays, interapertural MFs, and MFs confined beneath a single aperture) prior to being continously exposed to CD. After CD treatment, germination was blocked and the number of cells containing short rods increased, but movement of actin to a single aperture continued. Finally, when pollen at different stages of MF organization was treated with a CD pulse and then transferred to drug-free medium, germination was delayed regardless of the stage of MF organization at the time of treatment. The results indicate that an uninterrupted progression of actin organization is essential for pollen germination, but that movement of actin in the cell is CD-insensitive.     

青葙花药发育的结构和组织化学观察

对苋科植物青葙Celosia argentea花药发育的结构和组织化学（多糖和脂滴）特征进行观察。青葙小孢子发生为同时型,四分体为四面体型。药壁为典型四层,绒毡层属于同型绒毡层。成熟花粉为二胞型。早期花药中的淀粉粒和脂滴均较少,绒毡层细胞至小孢子晚期退化为体积较大的脂块。二胞花粉时期的中层细胞退化为脂滴。早期二胞花粉中先出现多糖颗粒,晚期的成熟花粉中积累大量淀粉粒和较少的脂滴为营养储存物。