Postmeiotic cytokinesis and pollen aperture number determination in eudicots: effect of the cleavage wall number

Summary.  In eudicot postmeiotic tetrads, apertures are usually joined in pairs in highly conserved areas. These appear to be located at the last points of contact persisting at the end of cytokinesis between the cytoplasm of the future microspores. In order to investigate the relationship between cytokinesis and aperture formation, aperture distribution within postmeiotic tetrads and the progression of meiosis were studied in Nicotiana tabacum cv. Ambalema. This variety (inbred line) produces about 85% tricolporate pollen and 15% tetracolporate pollen grains. In addition, about 7% of tetrads are composed of four equal-sized microspores and a supernumerary pseudomicrospore of small size and an equal proportion of tetrads exhibit unpaired apertures (these apertures are not joined in pairs within tetrads). Observation of cytokinesis indicates that both unpaired apertures and pseudomicrospores could result from the persistence of late communications between microsporocytes. Observations of tetrads indicate that an increase in the number of elements that are separated during cytokinesis is correlated with an increase in microspore aperture number. All data converge to support the hypothesis that aperture site determination is partly controlled by the number of walls formed to separate the different elements of the tetrad. Received May 22, 2002; accepted October 29, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Laboratoire de Ecologie, Systematique et Evolution, Batiment 362, Université Paris Sud, 91405 Orsay cedex, France.     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto

The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g · ml⁻¹, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets. Received: 24 September 1997 / Accepted: 12 November 1997     

Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h⁻¹ in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location. Received: 1 December 1998 / Accepted: 21 January 1999     

Nuclear cytoplasmic domains,microtubules and organelles in microsporocytes of the slipper orchid Cypripedium californicum A. Gray dividing by simultaneous cytokinesis

Microsporocytes of the slipper orchidCypripedium californicum A. Gray divide simultaneously after second meiosis. The organization and apportionment of the cytoplasm throughout meiosis are functions of nuclear-based radial microtubule systems (RMSs) that define domains of cytoplasm - a single sporocyte domain before meiosis, dyad domains within the undivided cytoplasm after first meiosis, and four spore domains after second meiosis. Organelles migrate to the interface of dyad domains in the undivided cytoplasm after first meiotic division, and second meiotic division takes place simultaneously on both sides of the equatorial organelle band. Microtubules emanating from the telophase II nuclei interact to form columnar arrrays that interconnect all four nuclei, non-sister as well as sister. Cell plates are initiated in these columns of microtubules and expand centrifugally along the interface of opposing RMSs, coalescing in the center of the sporocyte and joining with the original sporocyte wall at the periphery to form the tetrad of microspores. Organelles are distributed into the spore domains in conjunction with RMSs. These data, demonstrating that cytokinesis in microsporogenesis can occur in the absence of both components of the typical cytokinetic apparatus (the preprophase band of microtubules which predicts the division site and the phragmoplast which controls cell-plate deposition), suggest that plant nuclei have an inherent ability to establish a domain of cytoplasm via radial microtubule systems and to regulate wall deposition independently of the more complex cytokinetic apparatus of vegetative cells.     

Successive microsporogenesis affects pollen aperture pattern in the tam mutant of Arabidopsis thaliana

Background and Aims

The tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which exhibits a modified cytokinesis with a switch from simultaneous to successive cytokinesis, was used to perform a direct test of the implication of cytokinesis in aperture-pattern ontogeny of angiosperm pollen grains. The aperture pattern corresponds to the number and arrangement of apertures (areas of the pollen wall permitting pollen tube germination) on the surface of the pollen grain.

Methods

A comparative analysis of meiosis and aperture distribution was performed in two mutant strains of arabidopsis: quartet and quartet-tam.

Key Results

While the number of apertures is not affected in the quartet-tam mutant, the arrangement of the three apertures is modified compared with the quartet, resulting in a different aperture pattern.

Conclusions

These results directly demonstrate the relationship between the type of sporocytic cytokinesis and pollen aperture-pattern ontogeny.     

Study of male sterility in Taiwania cryptomerioides Hayata (Taxodiaceae)

Summary. A study of male sterility over a period of three consecutive years on a conifer species endemic to Taiwan, Taiwania cryptomerioides Hayata (Taxodiaceae), was done for this article. With the aids of fluorescence and electron microscopic observations, the ontogenic processes in the fertile and sterile microsporangia are compared, using samples collected from Chitou Experimental Forest and Yeou-Shoei-Keng Clonal Orchard of the National Taiwan University, Nantou, Taiwan. The development of male strobili occurred from August to the end of March. Microsporogenesis starts with the formation of the archesporium and ends with the maturation of 2-celled pollen grains within the dehiscing microsporangium. Before meiosis, there was no significant difference in ultrastructure between the fertile and sterile microsporangia. Asynchronous pollen development with various tetrad forms may occur in the same microsporangium of either fertile or sterile strobili. However, a callose wall was observable in the fertile dyad and tetrad, but not in the sterile one. After dissolution of the callose wall, the fertile microspores were released into the locule, while some sterile microspores still retained as tetrads or dyads with intertwining of exine walls in the proximal faces. As a result, there was no well developed lamellated endexine and no granulate ectexine or intine in the sterile microspores. Eventually, the intracellular structures in sterile microspores were dramatically collapsed before anthesis. The present study shows that the abortion in pollen development is possibly attributed to the absence of the callose wall. The importance of this structure to the male sterility of T. cryptomerioides is discussed. Correspondence and reprints: Department of Life Science, National Taiwan University, 106 Taipei, Taiwan.     

Post-meiotic cytokinesis and pollen aperture pattern ontogeny: comparison of development in four species differing in aperture pattern

Pollen aperture patterns vary widely in angiosperms. An increasing number of studies indicate that aperture pattern ontogeny is correlated with the way in which cytokinesis that follows male meiosis is completed. The formation of the intersporal callose walls that isolate the microspores after meiosis was studied in four species with different aperture patterns (two monocots, Phormium tenax and Asphodelus albus, and two eudicots, Helleborus foetidus and Protea lepidocarpodendron). The way in which post-meiotic cytokinesis is performed differs between all four species, and variation in callose deposition appears to be linked to aperture pattern definition.     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

Changes in calcium and calmodulin levels during microsporogenesis,pollen development and germination in Gasteria verrucosa (Mill.) H. Duval

Summary The distribution of membrane calcium and calmodulin (CaM) has been fluorimetrically determined in the anther of Gasteria verrucosa with particular attention to sporogenous cells, meiocytes, microspores, pollen and stages of pollen germination and tube growth using chlortetracycline (CTC) and fluphenazine (FPZ). CTC and FPZ fluorescence in sporogenous cells is relatively higher than in the adjacent tapetal cells, indicating higher membrane calcium and CaM levels in the former cell type. However, during meiosis there is a significant increase in membrane calcium and CaM levels in the meiocytes compared to that found in the young microspores. CTC and FPZ fluorescence in the sporogenous cells, meiocytes and young microspores is punctate and slightly diffused throughout the cytoplasm. In the microspores of the tetrad and the young released microspores CTC fluorescence (CTCf) is polarized and mainly associated with the area opposite the future colporal region. FPZ fluorescence (FPZf) becomes polarized in the young microspore. Subsequently, there is a shift in the polarity, and most of the CTCf and FPZf in the old microspores and pollen is regionalized towards the colporal region, and the fluorescence is more diffused, indicating a change in the organellar-bound calcium and CaM. This final graded distribution of CTCf is maintained during pollen germination in that the growing pollen tubes invariably show a tip to base membrane-calcium gradient. In the tapetal cells a high level of Ca²⁺ is present during the microspore stage. During the preparation for anthesis the endothecium differentiation is marked by the presence of Ca²⁺. Post-treatment of labelled cells with a Ca²⁺ chelator such as EGTA resulted in a substantial decrease in diffuse and punctate CTCf. Alternatively, treatment of cells with non-ionic detergent Nonidet P-40 resulted in the total elimination of CTCf, suggesting that the observed CTC fluorescence was due to membrane-associated calcium. The cytological specification of CTC as a probe for calcium is discussed. From cytofluorometric measurements and atomic absorption, it became clear that the level of Ca²⁺ in the anther is high during the sporogenous and meiotic phases. An increase in CTCf and FPZf occurred after microspore mitosis. An interaction of Ca²⁺ transport from tapetum to the young pollen is postulated. These findings suggest that the level of Ca²⁺ in the anther during meiosis is generally relatively higher than at the sporogenous or young microspore stage. These findings are discussed in the light of available information on the role of Ca²⁺ and CaM-mediated processes such as cell division, callose synthesis and pollen-tube tip growth.     

A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3

Adenine phosphoribosyltransferase (APT) catalyzes the conversion of adenine and cytokinin bases to the corresponding nucleotides. An Arabidopsis thaliana mutant lacking the major APT isoform, APT1, is male sterile due to defects soon after meiosis. We have now used electron microscopy to define the effects of APT1 deficiency on pollen development to determine whether the changes might be attributed to adenine or cytokinin metabolism. Changes were observed in mutant anthers in both tapetal and pollen mother cells prior to meiosis with additional defects found at later stages, in both compartments. Principal changes include altered lipid accumulation in the tapetal cells, changes in pollen cell wall development, and a loss of synchrony in the development of the tapetum and microspores. Taken together our results suggest that APT1 deficiency causes a general metabolic decrease in energy metabolism, due to the lack of adenine recycling into adenylate nucleotides, which ultimately leads to pollen abortion. The early onset of meiosis in the mutant may be associated with altered cytokinin metabolism.     

The influence of tetrad shape and intersporal callose wall formation on pollen aperture pattern ontogeny in two eudicot species

Background and Aims

In flowering plants, microsporogenesis is accompanied by various types of cytoplasmic partitioning (cytokinesis). Patterns of male cytokinesis are suspected to play a role in the diversity of aperture patterns found in pollen grains of angiosperms. The relationships between intersporal wall formation, tetrad shape and pollen aperture pattern ontogeny are studied.

Methods

A comparative analysis of meiosis and aperture distribution was performed within tetrads in two triporate eudicot species with contrasting aperture arrangements within their tetrads [Epilobium roseum (Onagraceae) and Paranomus reflexus (Proteaceae)].

Key Results and Conclusions

Intersporal wall formation is a two-step process in both species. Cytokinesis is first achieved by the formation of naked centripetal cell plates. These naked cell plates are then covered by additional thick, localized callose deposits that differ in location between the two species. Apertures are finally formed in areas in which additional callose is deposited on the cell plates. The recorded variation in tetrad shape is correlated with variations in aperture pattern, demonstrating the role of cell partitioning in aperture pattern ontogeny.     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.     

Interaction between Cytokinesis-Related Callose and Cortical Microtubules in Dividing Cells of the Liverwort Riella helicophylla

Abstract: In juvenile walls of dividing cells of the liverwort Riella helicophylla the nitroso-derivative of photolysed Nifedipine (a calcium antagonist) stimulates the deposition of callose. This enhanced biosynthesis of β-1,3-glucan can only be observed in the cell plate, the juvenile cell walls and the walls of adjacent cells. An immunocytological analysis of this effect revealed that no cortical microtubules occurred at the sites of callose deposition. The cells of the control displayed a normal distribution of cortical microtubules at the plasma membrane as long as no callose was deposited along the corresponding walls. In a second set of experiments, inhibitors of microtubule polymerization and depolymerization (amiprophosmethyl and taxol, respectively) were used. At low concentrations, these substances also caused a significant stimulation of callose deposition in the plane of cell division. Based on these findings, we propose a regulatory model of callose and cellulose biosynthesis that depends on the binding of the cellulose/callose synthase complex to cortical microtubules that may be mediated by unknown binding protein(s).     

Links between morphology and function of the pollen wall: an experimental approach

The wall of pollen grains exhibits morphological variation in many features including apertures, ornamentation and thickness, but the function of these characters remains to be clarified. It has been suggested that they are involved in the accommodation of volume changes (harmomegathy). To investigate this further, we developed a protocol that induces a controlled hydration of the pollen without affecting its metabolism and we applied it to six species differing in their pollen wall morphology. The entry of water caused pollen swelling and volume increase leading to breakage of the wall and/or of the plasma membrane, such that the per cent of intact grains was negatively correlated with the level of hydration. Qualitative and quantitative differences were observed between the species. Breakage of the exine was observed only in pollen lacking apertures and with thin exine. Variation in the exine ornamentation and thickness could explain the interspecific differences observed for the rates of breakage of the plasma membrane. Our results suggest that pollen wall morphology matters for survival and maintenance of pollen integrity further to volume increase due to hydration. We propose a rationale for future studies that should allow disentangling the contribution of different pollen morphological and physiological features to harmomegathy.     

Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen

Summary. Mitochondrial DNA in the male reproductive cells of rapeseed (Brassica napus L.) was monitored by fluorescence microscopy of Technovit 7100 resin sections double-stained with 4,6-diamidino-2-phenylindole and 3,3-dihexyloxacarbocyanine iodide. Mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen. This result corresponds well with the maternal inheritance of mitochondria in rapeseed determined by previous genetic analyses. To better characterize the mode of inheritance of the mitochondrial linear plasmid in rapeseed, which is transmitted through pollen, we analyzed by indirect immunofluorescence microscopy the expression and localization of ORF6 protein, a putative RNA polymerase encoded by the plasmid. ORF6 protein was expressed in mature pollen and specifically localized in the cytoplasm of sperm cells in the mature pollen. This suggests that the genes encoded by the plasmid DNA are transcribed in the mature pollen by its own RNA polymerase (ORF6 protein) and that the gene expression in the generative cells may be needed for transmission of plasmid DNA through the pollen.Present address: Laboratory of Plant Molecular Biology, Nara Institute of Science and Technology, Ikoma, Nara, Japan.Correspondence and reprints: Department of Plant Biotechnology, National Institute of Agrobiological Sciences, 2-1-2 Kan-non-dai, Tsukuba 305-8602, Japan.     

Germination and early tube development in vitro of Lycopersicum peruvianum pollen: Ultrastructural features

Morphologic changes occurring during pollen grain activation and ultrastructural features of Lycopersicum peruvianum Mill. pollen tube during the first stages of growth in vitro have been studied. The more evident morphologic changes during activation, in comparison to those already described for mature inactive pollen, concern dictyosomes, rough endoplasmic reticulum (RER), and ribosomes. The dictyosomes are very abundant and produce large and small vesicles. Near the germinative pores both types of vesicles are present, while all along the remaining cell wall only the large type is observed. These latter react weakly to Thiéry's test and probably contain a callose precursor necessary for the deposition of a callosic layer lining at first only the inner side of the functioning pore and occasionally the other two pores, and subsequently the entire inner surface of the cell wall. The small vesicles, highly positive to Thiéry's test, are present only near the pores and could be involved in the formation of the pectocellulosic layer of the tube wall. The setting free of RER cisterns, which in the mature inactive pollen were aggregated in stacks, coinciding with polysome formation and resumption of protein synthesis, is in accord with the hypothesized role of RER cistern stacks as a reserve of synthesizing machinery. The pollen tube reaches a definitive spatial arrangement soon after the generative cell and vegetative nucleus have moved into it. At this stage four different zones that reflect a functional specialization are present. In the apical and subapical zone two types of dictysosome-originated vesicles, similar to those found in the activated pollen grain, are present. Their role in the formation of the callosic and pectocellulosic wall layers seems to be the same as in the activated pollen grain.Abbreviations ER endoplasmic reticulum - RER rough endoplasmic reticulum Research performed under CNR program Biology of Reproduction     

The relationship between airborne pollen distribution and the frequency of specific pollen sensitization at two climatically different locations in Switzerland

Pollen data reported from the two monitoring stations of Locarno-Monti, on the southern slopes of the Alps, and Zurich on the northern slopes of the Alps, for the years 1989–1993 were compared with meteorological data from the corresponding locations and with the prevalence of pollinosis and frequency of specific pollen sensitization in patients with pollinosis. It was so discovered that the recorded quantity of allergenic pollen types (Alnus, Corylus, Betula, Fraxinus, Poaceae, Castaneae,Olea andArtemisia) south of the Alps is higher by a factor of 2.9 than that north of the Alps. These differences are due to the distinctly milder climate in the canton of Ticino (south of the Alps) showing higher annual mean temperatures, more sunshine duration and less precipitation days with higher amounts of precipitation. The allergological data show additionally that the summer pollinosis, caused byCastanea andOlea, is probably responsible for the differing prevalence of pollinosis at the two sites. Finally, we can summarize that in Switzerland pollen from Poaceae, Betulaceae and Oleacea are the most important for pollinosis.