Cecilia Del Casino

Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.

Sucrose Synthase Is Associated with the Cell Wall of Tobacco Pollen Tubes

Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for defining the relationships between nutrients, growth, and cell morphogenesis. We used the pollen tube of tobacco (Nicotiana tabacum) as a cell model to characterize the main features of Sus with regard to cell growth and cell wall synthesis. Apart from its role during sexual reproduction, the pollen tube is a typical tip-growing cell, and the proper construction of its cell wall is essential for correct shaping and direction of growth. The outer cell wall layer of pollen tubes consists of pectins, but the inner layer is composed of cellulose and callose; both polymers require metabolic precursors in the form of UDP-glucose, which is synthesized by Sus. We identified an 88-kD polypeptide in the soluble, plasma membrane and Golgi fraction of pollen tubes. The protein was also found in association with the cell wall. After purification, the protein showed an enzyme activity similar to that of maize (Zea mays) Sus. Distribution of Sus was affected by brefeldin A and depended on the nutrition status of the pollen tube, because an absence of metabolic sugars in the growth medium caused Sus to distribute differently during tube elongation. Analysis by bidimensional electrophoresis indicated that Sus exists as two isoforms, one of which is phosphorylated and more abundant in the cytoplasm and cell wall and the other of which is not phosphorylated and is specific to the plasma membrane. Results indicate that the protein has a role in the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes.

DISTRIBUTION OF MICROTUBULES DURING THE GROWTH OF TOBACCO POLLEN TUBES

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α‐, β‐ and tyrosinated α‐tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer‐assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L.

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

An Ankyrin Repeat-Containing Protein, Characterized as a Ubiquitin Ligase, Is Closely Associated with Membrane-Enclosed Organelles and Required for Pollen Germination and Pollen Tube Growth in Lily

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

The organization of the cytoskeleton in the generative cell and sperms ofHyacinthus orientalis

SummaryThe microtubular cytoskeleton of the generative cell (GC) ofHyacinthus orientalis has been studied until the formation of the sperm cells (SCs). Immunofluorescence procedures in combination with confocal laser scanning microscopy (CLSM) has enabled the visualization of the organization of the microtubular cytoskeleton. Chemical fixation and freeze-fixation electron microscopy have been used to investigate the cytoskeleton and the ultrastructural organization of the GC and SCs. During pollen activation the GC is spindle-shaped. Microtubules (MTs) are organized as bundles and distributed in proximity of the GC plasmamembrane, forming a basket-like structure. Following migration through the pollen tube, the basket-like structure becomes more intertwined. During the nuclear division the MTs are involved in the segregation of the chromosomes and kinetochores are clearly discernible. Association with organelles is also observed. The chromosomes of the GC remain condensed until they separate in two sperm nuclei. The pre-prophase band was never observed. At the end of the GC division the microtubular network reorganizes in the two SCs.

The Microtubular Cytoskeleton during Pollen Development and Pollen Tube Growth in Nicotiana tabacum

The role of the male partner in the fertilization process in higher plants has been defined from long time (Stanley and Linskens, 1972). As shown by optical and ultrastructural investigations, some closely related steps such as pollen development, pollen release from the anther, pollen-stigma interaction, pollen tube emission and growth occur and each step is characterized by own morphological and physiological states.

Microtubular Motors in the Pollen Tube of Nicotiana tabacum

The Angiosperm pollen tube Is specialized in delivering the sperm cells during the process of fertilization (Cresti and Tiezzi, 1990). Microtubules and actin filaments are the main components of the vegetative cell cytoskeleton, which study has been extensively carried out mainly by electron and fluorescence microscopy (reviewed by Tiezzi, 1991). Many types of motility characterize the cytoplasm of the pollen tube, including the movement of generative and sperm cells and the translocation of organelles and vesicles (Pierson et al., 1990). The dependence of this movement on the cytoskeletal elements is not well understood, even though some recent observations seem to support the actin filament system as an active component (Tang et al., 1989). On the other hand the role of microtubules in the cytoplasmic movement and consequently in the tube growth is not yet clear. Some recent remarks (Astrom et al., 1991) have suggested possible correlations between the microtubule cytoskeleton and the movement of generative cell and vegetative nucleus.

The organization of microtubules during generative-cell division inConvallaria majalis

SummaryThe 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.

Graphene oxide impairs the pollen performance of Nicotiana tabacum and Corylus avellana suggesting potential negative effects on the sexual reproduction of seed plants

The production of graphene based materials (GBMs) is steadily increasing but the effects of the possible release of GBMs in the environment are far from being understood. Graphene oxide (GO) is among the most active GBMs and it causes widely varying effects on the vegetative body of seed plants. However, nothing is known yet about its potential effects on the reproductive process. This study addresses the effects of GO on pollen germination and pollen tube elongation in the model species Nicotiana tabacum and in the non-model species Corylus avellana. In vitro germination experiments were conducted without or with GO (control and treated samples, respectively) at concentrations of 25, 50 and 100 μg mL−1. Pollen germination and tube elongation were affected at GO concentrations ≥50 μg mL−1, decreasing by 20% and 19% in N. tabacum and by 68% and 58% in C. avellana, respectively. GO did not affect the viability of N. tabacum pollen, but doubled the frequency of bent tubes. Microscopy observations of pollen tubes exposed to a cellpermeant, dual-excitation ratiometric pH indicator revealed that GO affected the intracellular pH homeostasis. Further germination experiments on C. avellana conducted by inverting the pH conditions of the control and treated (100 μg GO mL−1) samples demonstrated that the main factor influencing the pollen performance is the acidic properties of GO. This might affect the reproductive process of numerous seed plants thus being relevant from an environmental point of view.