Elisabeth S. Pierson

Cytoskeleton and Cytoplasmic Organization of Pollen and Pollen Tubes

Publisher Summary The pollen grain and the pollen tube fulfill important functions in the sexual reproduction of higher plants. The pollen grain is concerned with conveying the two sperm cells or their progenitor, the generative cell, to the female gametophyte. The pollen tube serves as a guide and a pathway for the sperm cells on their course to the embryo sac so that they can complete double fertilization. This chapter focuses on the cytoskeleton of the vegetative cytoplasm of pollen grains and pollen tubes. The cytoskeleton is involved in numerous intracellular processes, such as organelle movement, organization of the cytoplasm, endocytosis and exocytosis, cytomorphogenesis, meiotic and mitotic division, and cell wall deposition. The distribution of calcium in pollen tubes is discussed in relationship to pollen tube growth, intracellular movement, and the activity of the cytoskeleton. The movements of the organelles and nuclei inside the pollen grain and the pollen tube are also described.

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection.

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.

Isolation of male and female gametes in higher plants.

SummaryThe availability of generative cells, sperm cells, embryo sacs and egg cells from angiosperm plants in isolated conditions has opened up many prospects: study of the mechanism of recognition and fusion between gametes of opposite sex and detailed observation of the process of fertilization, biochemical and genetic analysis of gamete-specific components and genetic engineering in combination with in vitro fertilization. This review provides a list of about ninety publications, in which the isolation of male or female angiosperm gametes and the closely related generative cells and embryo sacs is reported. The species used are summarized in two tables. A description is given of the diverse isolation techniques, which consist of enzymatic digestion, bursting of pollen by osmotic shock, squashing, grinding and micro-dissection. Viability of isolated cells and yield, two important aspects of biotechnological manipulation, are emphasized. A critical evaluation of the most significant results obtained so far with isolated material is presented together with notes on prospects for the future.

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation.

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50–100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.

Periodic deposition of arabinogalactan epitopes in the cell wall of pollen tubes of Nicotiana tabacum L.

The monoclonal antibodies MAC 207 and JIM 8, recognizing arabinogalactan epitopes, were used to localize the corresponding antigenic sites in pollen tubes of Nicotiana tabacum L. grown in vitro or semi in vivo. The analysis of the immunofluorescence labelling was performed by means of confocal laser scanning microscopy. Most pollen tubes were labelled along their length, with the exception of the tip region, in a ring-like pattern with remarkable periodicity. The diameter of the rings was approx. 12 μm and the distance between two rings was about 6 μm. No labelling of the cytoplasm, the vegetative nucleus or the generative cell was observed. The presence of labelling in the non-apical tube wall after pectinase and cellulase digestion indicates that the epitopes for MAC 207 and JIM 8 are located in the inner callosic sheath of the pollen-tube wall.

Enforced growth-rate fluctuation causes pectin ring formation in the cell wall of Lilium longiflorum pollen tubes

Normally growing lily (Lilium longiflorum Thunb.) pollen tubes cultured in standard sucrose medium display a relatively steady tip-growth pattern and a rather even pectin sheath in the cell wall. In an attempt to better understand pulsatory growth, observed in some species, e.g., Petunia, and its possible role in causing the formation of thickened cell wall rings, we have imposed marked fluctuations in the growth-rate of lily pollen tubes. The appropriate growth-perturbing conditions were achieved by modulating the medium osmolarity or by applying caffeine, a non-turgor inhibitor, in a specially designed incubation chamber with a controlled medium flow. The relatively non-esterified pectin deposition in the wall of the growth-interrupted pollen tubes was detected by immunofluorescence microscopy using a monoclonal antibody, JIM 5. The observations show that the periods of slow or inhibited growth correspond to the times when the thickened walls are deposited. Since the growth fluctuations were induced by both turgor- and non-turgor-related means, the proposed endogenous regulatory role of turgor pressure is questioned. Other factors, such as the tip-focused Ca2+ gradient which was demonstrated by ratiometric ion imaging, and the alteration in the extensibility of the cell wall, which correlated with pectin esterification/de-esterification, emerge as candidates for the regulation of growth fluctuations.

Ultrastructural changes of Arabidopsis thaliana pollen during final maturation and rehydration.

Several ultrastructural changes occur during dehydration and subsequent rehydration of Arabidopsis thaliana pollen. The cytoplasmic channels, present in the outer part of the intine of the mature, dehydrating pollen grain, degenerate and develop into electron-dense inclusions. At the same time a large quantity of electron-dense material is deposited in the cavities of the exine. A large number of vesicles is produced in the vegetative cell, and they become predominantly located in the peripheral region near the intine. Starch of amyloplasts is consumed and the lipid bodies which originally surround the sperm cells become randomly distributed. In addition, the individual lipid bodies become enveloped by single rough endoplasmic reticulum cisterns.

Video microscopic observations of living, isolated embryo sacs of Nicotiana and their component cells

SummaryLiving embryo sacs and megagametophytic cells of Nicotiana alata and Nicotiana tabacum were obtained using enzymatic maceration and microdissection. The yields of isolated embryo sacs, egg apparatus and central cells were up to 35%, 40% and 35%, respectively. Vectorial movement of organelles and undulations of tubular structures, presumably endoplasmic reticulum, were observed in eggs, synergids and central cells using video-enhanced microscopy. Despite evident viability using the fluorochromatic reaction, the egg displays much less organelle movement and therefore appears to be quiescent. The large vacuole of the central cell is traversed by mobile strands of cytoplasm through which organelles migrate. A polygonal network is located at the periphery of the central cell, which may contribute to anchorage of the cell with the embryo-sac wall. The observation of organelle movement provides direct evidence of the condition of the cell and may be a useful approach for assessing cell vigor.

The microtubule cytoskeleton and the rounding of isolated generative cells of Nicotiana tabacum

SummaryUpon squashing of the pollen grain, the isolated generative cell ofNicotiana tabacum looses its spindle shape to become spherical; this phenomenon is independent of the sucrose concentration used. The time necessary for this change can vary from 1 min (0% sucrose) to 20 min (30% sucrose). The microtubular cytoskeleton was studied by means of immunofluorescence and electron microscopy. Just after isolation, 5 to 15 clearly visible bundles in microtubules organized in a basket-like structure are present. After 15 min in medium with 15% sucrose, the microtubular cytoskeleton disappears, and a diffusely spread tubulin can be observed. Neither the addition of 10–20 μM taxol to the medium, nor the omission of Ca2+ to the medium has any effect on the changes in cell shape and loss of microtubular bundles after isolation.