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At least 73 records · Page 4Linked to original sources
PubMed2005

Actin polymerization promotes the reversal of streaming in the apex of pollen tubes.

Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.

Actin Cytoskeleton↗
PubMed2005

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms.

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.

Caffeine↗
PubMed2005

Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana.

The expression of "classical" arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana was characterized by RT-PCR. Transcripts of Agp6 and Agp11 were consistently found to be more abundant in pollen tubes and seem to be pollen-specific. Transcripts of other AGP genes were also detected in pollen but in lesser amounts and in a non-specific fashion. Two reference genes, ubiquitin-conjugating enzyme 9 and tubulin beta-4 chain, were evaluated and selected for gene expression studies in pollen. Expression characterization was complemented with immunolocalization studies using monoclonal antibodies specific to several glycosidic epitopes of AGPs. These studies were performed on in vitro germinated pollen tubes with the antibodies MAC207 and LM2. MAC207 produced labelling at the tip of the pollen tube, while LM2 produced a ring-like fluorescence around the emerging region of the tube, suggesting a role of AGPs during Arabidopsis pollen tube germination. To our knowledge, this is the first report establishing the presence of AGPs on Arabidopsis pollen tubes.

Antibodies, Monoclonal↗
PubMed1991

The pollen tube cytoskeleton.

In the last few years the role of pollen and the pollen tube in the fertilization process in higher plants has received considerable attention. By ultrastructural, biochemical and immunofluorescent investigations it has been shown that a cytoskeletal apparatus plays a central role in pollen tube growth. Microfilaments and microtubules, in which main components are, respectively, actin and tubulin, represent the most investigated cytoskeletal components. New information has been recently provided by the identification of myosin and also of a kinesin-like protein. The pollen tube cytoskeleton consists of two different cytoskeletal systems: the vegetative cell cytoskeleton, namely the cytoskeleton of the pollen grain and pollen tube, and the gamete cytoskeleton (generative cell and sperm cell cytoskeleton). The vegetative cell cytoskeleton plays a fundamental role in assuring the cytoplasmic movement of organelles, vesicles and gametes from the pollen grain to the pollen tube apex and consists mainly of microtubules and microfilaments. Also myosin and the kinesin-like protein are involved in the process of organelle and vesicle movement. The gamete cytoskeleton has a central role in sperm cell formation and in the reshaping process during gamete movement inside the pollen tube. It consists mostly of microtubules and partially characterized microtubule-associated structures. Actin filaments have recently also been identified.

Animals↗
PubMed2006

Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization.

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the beta-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.

Amino Acid Sequence↗
PubMed1998

Guidance in vitro of the pollen tube to the naked embryo sac of torenia fournieri

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.

Journal Article↗
PubMed2006

How pollen tubes grow.

Sexual reproduction of flowering plants depends on delivery of the sperm to the egg, which occurs through a long, polarized projection of a pollen cell, called the pollen tube. The pollen tube grows exclusively at its tip, and this growth is distinguished by very fast rates and reaches extended lengths. Thus, one of the most fascinating aspects of pollen biology is the question of how enough cell wall material is produced to accommodate such rapid extension of pollen tube, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth. This review discusses recent advances in our understanding of the mechanism of pollen tube growth, focusing on such basic cellular processes as control of cell shape and growth by a network of cell wall-modifying enzymes, molecular motor-mediated vesicular transport, and intracellular signaling by localized gradients of second messengers.

Arabidopsis↗
PubMed2006

Membrane trafficking and polar growth in root hairs and pollen tubes.

Root hairs and pollen tubes extend by rapid elongation that occurs exclusively at the tip. Fundamental for such local, tip-focused growth (so-called 'tip growth') is the polarization of the cytoplasm that directs secretory events to the tip, and the presence of internal gradients and transmembrane flux of ions, notably Ca2+, H+, K+, and Cl-. Electrophysiological and imaging studies using fluorescent markers have sought to link ion gradients with growth and membrane trafficking. Current models recognize membrane trafficking as fundamental to tip growth, notably its role in supplying lipid and protein to the new plasma membrane and cell wall that extend the apex of the cell, and a complementary role for endocytosis in retrieving excess membrane and in recycling various protein fractions. The current state of knowledge is reviewed here in order to highlight the major gaps in the present understanding of trafficking as it contributes to polar growth in these cells and recent results, that suggest a role for membrane trafficking in the active regulation of ion channel turnover and activity during polar tip growth, are discussed.

Animals↗
PubMed2003

Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2+ and GTPases.

Pollen tube growth relies on an extremely fast delivery of new membrane and wall material to the apical region where growth takes place. Despite the obvious meaning of this fact, the mechanisms that control this process remain very much unknown. It has previously been shown that apical growth is regulated by cytosolic free calcium ([Ca(2+)](c)) so it was decided to test how changes in [Ca(2+)](c) affect endo/exocytosis in pollen tube growth and reorientation. The endo/exocytosis was assayed in living cells using confocal imaging of FM 1-43. It was found that growing pollen tubes exhibited a higher endo/exocytosis activity in the apical region whereas in non-growing cells FM 1-43 is uniformly distributed. During pollen tube reorientation, a spatial redistribution of exocytotic activity was observed with the highest fluorescence in the side to which the cell will bend. Localized increases in [Ca(2+)](c) induced by photolysis of caged Ca(2+) increased exocytosis. In order to find if [Ca(2+)](c) changes were modulating endo/exocytosis directly or through a signalling cascade, tests were conducted to find how changes in GTP levels and GTPase activity (primary regulators of the secretory pathway) affect the apical [Ca(2+)](c) gradient and endo/exocytosis. It was found that increases in GTP levels could promote exocytosis (and growth). Interestingly, the increase in [GTP] did not significantly affect [Ca(2+)](c) distribution, thus suggesting that the apical endo/exocytosis is regulated in a concerted but differentiated manner by the Ca(2+) gradient and the activity of GTPases. Rop GTPases are likely candidates to mediate the Ca(2+)/GTP cross-talk as shown by knock-down experiments in growing pollen tubes.

Amino Acid Sequence↗
PubMed1976

Studies on the physiology of pollen and pollen tube growth. 1. pinus roxburghii.

Pollen grains and pollen tubes of Pinus roxburghii were subjected to histochemical technique with a view to determining the distributional pattern of reserve substances (ascorbic acid, proteins starch, polysaccharides, nucleic acids) and some enzymes (acid phosphatase, glucose-6-phosphatase, peroxidase, esterase, cytochrome oxidase, succinate dehydrogenase). An attempt is made to correlate the activity of different enzymes in relation to pollen tube growth.

Enzymes↗
PubMed1994

Uridine Diphosphate Glucose Metabolism and Callose Synthesis in Cultured Pollen Tubes of Nicotiana alata Link et Otto.

Membrane preparations from cultured pollen tubes of Nicotiana alata Link et Otto contain a Ca2+ -independent (1-3)-[beta]-D-glucan (callose) synthase activity that has a low affinity for UDP-glucose, even when activated by treatment with trypsin (H. Schlupmann, A. Basic, S.M. Read [1993] Planta 191: 470-481). Therefore, we investigated whether UDP-glucose was a likely substrate for callose synthesis in actively growing pollen tubes. Deposition of (1-3)-[beta]-glucan occurred at a constant rate, 1.4 to 1.7 nmol glucose min-1, in tubes from 1 mg of pollen from 3 h after germination; however, the rate of incorporation of radioactivity from exogenous [14C]-sucrose into wall polymers was not constant, but increased until at least 8 h after germination, probably due to decreasing use of internal reserves. UDP-glucose was a prominent ultraviolet-absorbing metabolite in pollen-tube extracts, with 1.6 nmol present in tubes from 1 mg of pollen, giving a calculated cytoplasmic concentration of approximately 3.5 mM. Radioactivity from [14C]-sucrose was rapidly incorporated into sugar monophosphates and UDP-glucose by the growing tubes, consistent with a turnover time for UDP-glucose of less than 1 min; the specific radioactivity of extracted UDP-[14C]glucose was equal to that calculated from the rate of incorporation of [14C]sucrose into wall glucans. Large amounts of less metabolically active neutral sugars were also present. The rate of synthesis of (1-3)-[beta]-glucan by nontrypsin-treated pollen-tube membrane preparations incubated with 3.5 mM UDP-glucose and a [beta]-glucoside activator was slightly greater than the rate of deposition of (1-3)-[beta]-glucan by intact pollen tubes. These data are used to assess the physiological significance of proteolytic activation of pollen-tube callose synthase.

Journal Article↗
PubMed2000

Integrin-like proteins in the pollen tube: detection, localization and function.

The distribution of integrin-like proteins in the pollen tube was examined by immunofluorescent labeling and western blotting techniques using antibodies against human placenta integrin vitronectin receptor (VnR), and alpha(v), beta3 and beta1 integrin subunits. Pseudocolor-coded confocal images showed intense immunostaining within 10 and 5 microm of the tip of the pollen tube in Lilium davidii and Nicotiana tabacum respectively. In both segments the site near the plasma membrane was labeled. Western blotting analyses revealed cross-reaction of anti-beta3, anti-alpha(v) and anti-VnR with the proteins in the plasma membrane preparation of L. davidii and Hemerocallis citrina pollen tube. These studies provide evidence for the first time that the integrin-like protein is present in pollen tubes, and it may be mainly composed of alpha(v) and beta3 subunits in lily pollen tubes. In a functional assay, neither anti-VnR antibody nor the Arg-Gly-Asp-Ser tetrapeptide inhibited pollen tube growth of N. tabacum in vitro, but both of them depressed tube growth on the stigma and in style under quasi in vivo culture conditions. The integrin-like proteins localized in the tip and periphery of the pollen tube appeared to play roles in growth of the pollen tube tip and interaction with the extracellular matrix of the style.

Antibodies, Monoclonal↗
PubMed2006

Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes.

Rac/Rop-type Rho-family small GTPases accumulate at the plasma membrane in the tip of pollen tubes and control the polar growth of these cells. Nt-RhoGDI2, a homolog of guanine nucleotide dissociation inhibitors (GDIs) regulating Rho signaling in animals and yeast, is co-expressed with the Rac/Rop GTPase Nt-Rac5 specifically in tobacco (Nicotiana tabacum) pollen tubes. The two proteins interact with each other in yeast two-hybrid assays, preferentially when Nt-Rac5 is prenylated. Transient over-expression of Nt-Rac5 and Nt-RhoGDI2 depolarized or inhibited tobacco pollen tube growth, respectively. Interestingly, pollen tubes over-expressing both proteins grew normally, demonstrating that the two proteins functionally interact in vivo. Nt-RhoGDI2 was localized to the pollen tube cytoplasm and effectively transferred co-over-expressed YFP-Nt-Rac5 fusion proteins from the plasma membrane to this compartment. A single amino acid exchange (R69A), which abolished binding to Nt-RhoGDI2, caused Nt-Rac5 to be mis-localized to the flanks of pollen tubes and strongly compromised its ability to depolarize pollen tube growth upon over-expression. Based on these observations, we propose that Nt-RhoGDI2-mediated recycling of Nt-Rac5 from the flanks of the tip to the apex has an essential function in the maintenance of polarized Rac/Rop signaling and cell expansion in pollen tubes. Similar mechanisms may generally play a role in the polarized accumulation of Rho GTPases in specific membrane domains, an important process whose regulation has not been well characterized in any cell type to date.

Cell Division↗
PubMed2001

Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii.

The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.

Actins↗
PubMed1999

[Immunofluorescence studies on the localization of integrin-like protein in plant pollen tubes].

The Strong fluorescence signals were obtained in pollen tube of Lilium davidii Duch with labeled anti-VnR integrin serum, and anti-beta 3, alpha v integrin subunit cytoplasmic domain serum separately. The highest density of immunolabel was in the tip of pollen tube. There was little or no immunolabel in control experiment using non-immune serum, second antibody alone and anti-FnR, LnR integrin serum separately. In pollen of Prunus persica f. rubro-piena Schneid, fluoresence signals were also obtained in tube using labeled anti-beta 1, beta 3 integrin subunit cytoplasmic domain serum separately and in apertures using anti-beta 1 serum. Preliminary results show that during the germination of pollen and the growth of pollen tube, there may be integrin-like proteins in pollen tube, consisting of alpha v and beta s integria subunits in the pollen tube of Lilium davidii Duch, which is the receptor of vitronectin-like protein.

Extracellular Matrix↗
PubMed1999

Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx.

We have shown that Rop1At, a pollen-specific Rop GTPase that is a member of the Rho family of small GTP binding proteins, acts as a key molecular switch controlling tip growth in Arabidopsis pollen tubes. Pollen-specific expression of constitutively active rop1at mutants induced isotropic growth of pollen tubes. Overexpression of wild-type Arabidopsis Rop1At led to ectopic accumulation of Rop1At in the plasma membrane at the tip and caused depolarization of pollen tube growth, which was less severe than that induced by the constitutively active rop1at. These results indicate that both Rop1At signaling and polar localization are critical for controlling the site of tip growth. Dominant negative rop1at mutants or antisense rop1at RNA inhibited tube growth at 0.5 mM extracellular Ca(2+), but growth inhibition was reversed by higher extracellular Ca(2+). Injection of anti-Rop antibodies disrupted the tip-focused intracellular Ca(2+) gradient known to be crucial for tip growth. These studies provide strong evidence for a Rop GTPase-dependent tip growth pathway that couples the control of growth sites with the rate of tip growth through the regulation of tip-localized extracellular Ca(2+) influxes and formation of the tip-high intracellular Ca(2+) gradient in pollen tubes.

Arabidopsis↗
PubMed2005

Pectin methylesterase, a regulator of pollen tube growth.

The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.

Arabidopsis↗
PubMed2006

Species preferentiality of the pollen tube attractant derived from the synergid cell of Torenia fournieri.

The synergid cell of Torenia fournieri attracts pollen tubes by a diffusible but yet unknown chemical attractant. Here we investigated the species difference of the attractant using five closely related species in two genera, namely T. fournieri, Torenia baillonii, Torenia concolor, Lindernia (Vandellia) crustacea, and Lindernia micrantha. These five species have an exserted embryo sac, and ablation experiments confirmed that their synergid cells attracted the pollen tube. When ovules of T. fournieri and one of the other species were cultivated together with pollen tubes of each species, pollen tubes were significantly more attracted to synergid cells of the corresponding species. The attraction was not affected by the close proximity of embryo sacs of different species. This suggests that the attractant is a species-preferential molecule that is likely synthesized in the synergid cell. The calcium ion, long considered a potential attractant, could not serve as the sole attractant in these species, because elevation of the calcium ion concentration did not affect the observed attraction. In vivo crossing experiments also showed that the attraction of the pollen tube to the embryo sac was impaired when pollen tubes of different species arrived around the embryo sac, suggesting that the species preferentiality of the attractant may serve as a reproductive barrier in the final step of directional control of the pollen tube.

Biological Factors↗