Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.Li Wang and Yi-Min Liu are considered joint first authors     

Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining

Summary Standard methods for visualising microfilament (MF) arrays in pollen tubes using rhodamine phalloidin (RP) involve treatment of living tubes with a variety of stabilising, permeabilising, and fixation agents. Video differential interference contrast (DIC) microscopy has been used to investigate the effect of these agents on normalNicotiana pollen tube structure and activity. Most of these agents were found to induce extensive axial translocations generally starting with tipward contractions. These movements were less extensive in the apex compared to more distal regions, however, tips often suffered swelling damage. RP staining patterns of the actin cytoskeleton were highly variable within fixation treatments. In addition to investigating agents used by other authors on pollen tubes, we investigated the efficacy of pretreatment withm-maleimido-benzyol N-hydroxysuccinimide ester (MBS). This resulted in less disruption to the pollen tubes, especially when used alone in growth medium. It also gave better HP-labelling than that achieved in standard aldehyde-fixed specimens. We conclude that standard preparation methods do not faithfully preserve in vivo cytoplasmic integrity in pollen tubes so that subsequent images of MF distribution may be misleading.Abbreviations GM growth medium - RP rhodamine phalloidin - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - DIC differential interference contrast - MF microfilament - PIPES piperazine-N, N-bis-2-ethanesulphonic acid - PME 50 mM PIPES buffer, pH 6.8, amended with 2.0 mM EGTA and 1 mM MgSO₄ - EB extraction buffer Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

In vitro germination and growth of lily pollen tubes is affected by calcium inhibitor with reference to calcium distribution

The calcium inhibitors A23187, EGTA and La³⁺ inhibit pollen grain germination and growth of pollen tubes of Lilium davidii var. unicolor at different concentrations. Treatment with 10⁻⁴ or 10⁻⁵ M ionophores A23187 reduced germination rate and resulted in distortion of pollen tube. Addition of 2 or 10 mM of the chelator EGTA disturbed the direction of pollen tube growth and extended the diameter of pollen tube as observed by light and confocal microscopy. The Ca²⁺-channel blocker lanthanum chloride (La³⁺) restrained germination or markedly caused transformation of pollen tube. Furthermore, all treatments led to disappearance of any calcium gradient. Calcium distribution in pollen grain and pollen tube was altered as shown by confocal microscopy for each treatment. This indicates that the inhibitors influence pollen development by affecting the calcium gradient which may play a critical role in germination and tube growth. Fourier transform infrared (FTIR) spectra indicated slight increases in contents of amide I and a substantial decrease in the content of aliphatic esters and saturated esters in treated pollen tubes compared with normal pollen tubes. The FTIR analysis confirmed that EGTA and La³⁺ weakened the accumulation of ester in pollen tubes, which may be associated with an increased content of amide I.     

The anti-microtubule drug carbetamide stopsNicotiana sylvestris pollen tube growth in the style

Summary Recently, we found that the anti-microtubule drugs colchicine and propham caused the absence of microtubules and thus loss of cytoplasmic zonation in in vitro growing pollen tubes ofNicotiana sylvestris, but did not seriously affect growth. In the present study we used the herbicide carbetamide as an anti-microtubule drug. It had the same effect as colchicine and propham: the cytoplasm, including the generative cell, was no longer concentrated in the tip but was distributed randomly. In addition, ultrastructural investigations have shown that even the vesicle zone, usually found at the very tip of pollen tubes, had disappeared in some tubes. Nonetheless, in vitro growth was not inhibited by more than 20% over a period of 22 h.In contrast, tube growth in plants ceased 1 cm down in the style when carbetamide was applied to the stigma before pollination. At the lowest concentration causing this effect, microtubules of the vegetative cell had disappeared and the cytoplasm was distributed randomly, as it was for in vitro grown tubes. It can be concluded that microtubules of the vegetative cell are essential for pollen tube growth in the style.Abbreviations DAPI 4,6-diamidmo-2-phenylindole - EGTA ethyleneglycerol-bis-(aminoethyl ether) tetraacetic acid - DIC differential interference contrast - GC generative cell - IC₅₀ inhibition concentration 50% - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-Test pollen tube growth test - SAM substrate adhesion molecule - VC vegetative cell     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

Microtubules are involved in maintaining the cellular polarity in pollen tubes ofNicotiana sylvestris

Summary The polarity of a growing pollen tube is clearly reflected by a distinct zonation of the cytoplasmic content. The vegetative nucleus and the generative cell (GC) are located in the tip region of the tube, and the basal cytoplasmic portion is highly vacuolated. Using pollen tubes ofNicotiana sylvestris Spegazz. & Comes grown in vitro, we examined the effects of varying concentrations of the microtubule inhibitors colchicine and propham. The depolymerization of the cortical microtubules by 25 M colchicine led to a disorganization of the cytoplasm, i.e., vacuolization of the tip region, and to a deranged position of both the vegetative nucleus and the generative cell. The same concentration of colchicine inhibited tube growth by 10–20% of the control. Mitosis of the GC was not affected. Only from concentrations of 200 M the configuration of the GC's microtubules was altered and an inhibition of mitosis was observed. At this concentration the disorganization of the cytoplasm was always reversible, but neither inhibition of mitosis nor derangement of the nuclear positioning was. At 1,800 M colchicine, pollen tube growth was inhibited by 50% of the control. Using propham, the same three steps of action were observed, although propham proved to be about a hundred times more effective than colchicine. We conclude that the cortical microtubules of the pollen tube are involved in maintaining cellular polarity, probably as a part of a heterogeneous cytoskeletal network including also microfilaments and membranous elements. Nuclear positioning seems to be dependent on both, the tube's cortical and the GC's microtubules. A possible involvement of the extracellular matrix in maintaining intracytoplasmic polarity is suggested.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethyleneglycol-bis-(aminoethyl ether) tetraacetic acid - GC generative cell - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES, 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-test pollen tube growth test - VN vegetative nucleus Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1⁻¹. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.     

Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin microfilaments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes (primarily lily) are frozen in liquid propane at –180°C, substituted at –80°C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of microfilaments starting about 1–5 m behind the extreme apex and extending basally for an additional 5–10 m. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal filaments in the shank are consistent features of the actin cytoskeleton.     

Distribution of elements in the lily pollen tube tip,determined with the Oxford scanning proton microprobe

Summary Pollen tubes ofLilium longiflorum were fixed with glutaraldehyde and investigated unsectioned with the Oxford scanning proton microprobe (SPM). Two-dimensional maps which show the distribution and concentration of phosphorus, sulphur, chlorine, potassium, calcium, iron, copper, zinc and arsenic are presented. The maps show that, within the pollen tube tip region, calcium and zinc exhibit relatively steep longitudinal concentration gradients compared to the more flat distributions of phosphorus and sulphur. Chlorine, potassium, iron and copper appear equally distributed along the tube. All elements with the expception of arsenic show the highest concentration within the cell protoplasm and not in the cell wall. Additional signals of arsenic, chlorine and potassium originate from the remaining fixative dried around the tube, containing also the free ions of the cell. The arsenic signals originate exclusively from the buffer used during fixation. The different maps are compared and discussed in relation to their significance to the pollen tubes.Abbreviations EDAX energy dispersive analysis of X-rays - EIXE electron induced X-ray emission - PIXE proton induced X-ray emission - SPM scanning proton microprobe     

Evaluation of fixative solutions for ultrastructural analysis of brown spider Loxosceles intermedia (Araneae: Sicariidae) tissues.

In view of the widely varying compositions of fixative solutions used for studying spiders, five different fixative formulas were tested for fixing male brown-spider (Loxosceles intermedia) gonad tissues. The brown spider represents a public health problem in Curitiba (Paraná State, Brazil). Morphological study of its gonads may aid in understanding the reproductive strategies of this species, and possibly in developing a reproduction control program. The fixatives tested contained glutaraldehyde alone or combined with paraformaldehyde, and the buffers cacodylate or phosphate, with or without the addition of sucrose or sodium chloride as osmolytes. Those containing 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM phosphate buffer with 200 mM sucrose, or in 200 mM sodium cacodylate, satisfactorily preserved mitochondria, the Golgi apparatus, and the membranes in general. These formulas were nearly isosmotic (439 mOsm/kg H2O and 455 mOsm/kg H2O respectively) to brown spider hemolymph (478 mOsm/kg H2O). With respective to the fixative agents, a glutaraldehyde-paraformaldehyde combination resulted in optimal fixation of Loxosceles intermedia cells. For other species of spiders, hemolymph osmolality should be considered, but the fixative formulas cited above would also probably yield good results.     

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.     

Localization of a 170 kDa myosin heavy chain in plant cells

Summary A polyclonal antibody directed against a 170 kDa myosin heavy chain from lily pollen tubes was employed to (a) assess the cellular distribution of the polypeptide using immunofluorescence methods, and (b) ascertain if similar polypeptides are present in pollen tubes and somatic cells of other species. Fluorescence is associated with particles of various size as well as an amorphous component, and is concentrated in the apical cytoplasm of lily and tobacco pollen tubes. Apical fluorescence is more extensive in lily than in tobacco, which may be related to different streaming patterns and apical zonation seen at the ultrastructural level. In suspension cells of tobacco andArabidopsis, fluorescence is concentrated around the nuclei. Dual localizations indicate that anti-myosin fluorescence may be associated with the presence of actin. Little or no staining was seen in controls consisting of either pre-immune serum or mono-specific IgG that had been preadsorbed with the 170 kDa polypeptide. Immunoblots show that a 170 kDa immunoreactive polypeptide is present in pollen tubes of tobacco andTradescantia virginiana in addition to lily, and in suspension culture cells of tobacco andArabidopsis and extracts of wholeArabidopsis seedlings. Our results show that a conserved 170 kDa myosin heavy chain is present in a variety of monocot and dicot cells. They are also consistent with the presence of multiple myosins in plants in general and pollen tubes in particular.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - Mf microfilament - Mt microtubule - PBS phosphate-buffered saline - PME 50 mM Pipes, 5mM EGTA - 2mM MgSO₄, pH6.9.     

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.     

A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes.

We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth.     

TipChip: a modular,MEMS‐based platform for experimentation and phenotyping of tip‐growing cells

Large‐scale phenotyping of tip‐growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high‐throughput execution of more sophisticated assays, an experimental platform, the TipChip, was developed based on microfluidic and microelectromechanical systems (MEMS) technology. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels equipped with microscopic experimental set‐ups. The tip‐growing cells (pollen tubes, filamentous yeast or fungal hyphae) may be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform, we were able to answer several outstanding questions on pollen tube growth. We established that, unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air, raising the question of how and where water is taken up by the cell. The platform opens new avenues for more efficient experimentation and large‐scale phenotyping of tip‐growing cells under precisely controlled, reproducible conditions.     

The Arabidopsis CrRLK1L protein kinases BUPS1 and BUPS2 are required for normal growth of pollen tubes in the pistil

In flowering plants, the interaction of pollen tubes with female tissues is important for the accomplishment of double fertilization. Little information is known about the mechanisms that underlie signalling between pollen tubes and female tissues. In this study, two Arabidopsis pollen tube‐expressed CrRLK1L protein kinases, Buddha's Paper Seal 1 (BUPS1) and BUPS2, were identified as being required for normal tip growth of pollen tubes in the pistil. They are expressed prolifically in pollen and pollen tubes and are localized on the plasma membrane of the pollen tube tip region. Mutations in BUPS1 drastically reduced seed set. Most of the bups1 mutant pollen tubes growing in the pistil exhibited a swollen pollen tube tip, leading to failure of fertilization. The bups2 pollen tubes had a slightly abnormal morphology but could still accomplish double fertilization. The bups1 bups2 double mutant exhibited a slightly enhanced phenotype compared to the single bups1 mutants. The BUPS1 proteins could form homomers and heteromers with BUPS2, whereas BUPS2 could only form heteromers with BUPS1. The BUPS proteins could interact with the Arabidopsis pollen‐expressed RopGEFs in the yeast two‐hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. The results indicated that the BUPSs may mediate normal polar growth of pollen tubes in the pistil.     

Effects of Nifedipine on Pollen Germination,Pollen Tube Growth and Division of Generative Nucleus in Nicotiana tabacum

The deals with the effects of nifedipine (Nif), a Ca2+ channel blocker of rather high specificity, on pollen germination, pollen tube growth and division of generative nucleus (GN) in experimentlly germinated pollen tubes of Nicotiana tabacurn L. Pollen germination was inhibited by the addition of 10-4 mol/L Nif whereas no significant inhibition by 10-7~10-5 mol/L Nif was observed. The effects of Nif on pollen tube growth were related to its concentration and duration of treatment. At the earlier stage, tube growth was promoted at the lower concentrations (10-7~10-5 mol/L), but was significantly inhibited at a concentration of 10 4 mol/L Nif. With increasing time of culture, even the lower concentrations also became harmful; the stronger the concentration, the earlier the transition from promotion to inhibition. Generally, inhibition of tube growth occurred within 24 hours of culture with different extent in various concentrations. Moreover, higher concentrations also tended to disturb tube morphology and cytoplasmic streaming. Nif was observed to perturb GN division at various concentrations, either blocked it completely at 10-4 mol/L, or only delayed it at 10-7~10-5 mol/L. The dynamics of membrane-associated calcium in pollen tubes was tested with chlorotetracycline (CTC). With increasing time of culture and escalating Nif concentration, CTC fluorescence weakened gradually, indicating that the physiological effects of Nif is mediated by its in hibition on Ca2+ channel activities.     

Effect of cadmium on pollen germination and tube growth in Lilium longiflorum and Nicotiana tabacum

Cadmium had a highly toxic effect on pollen germination and tube growth, which were greatly inhibited as metal concentrations increased. Cadmium concentrations up to 10(-2) M completely stopped pollen germination and pollen showed an increasing tendency to burst within 1 h. At low concentrations, the metal caused a slight stimulation of pollen germination, growth rate and tube elongation at the initial stages of tube development. Comparing the two plants studied, cadmium was more toxic for Nicotiana tabacum than for Lilium longiflorum pollen. Pollen tubes showed a range of strong morphological abnormalities, characterized by uneven or aberrant growth, including apical branching or swelling at the tip of the pollen tube. Cell wall intrusions at or near the tip were evident on the inner side, whereas a loose network formed from fibrillar material was observed on the outer layers. After prolonged cadmium exposure, round (ball-like) aggregates were embedded in a fine fibrillar network. Increased cadmium concentrations (10(-3)-10(-2) M) decreased or completely paralyzed cytoplasmic streaming. No typical cytoplasmic zonation existed, while cell organelles (plastids, lipid droplets) were relocated toward the tip. The vesicular apical zone was drastically reduced, with vesicles dispersed into the subapical region. Mitochondria were distributed throughout the subapical region and among the vesicles of the tube apex. Visible ultrastructural changes in cell organelles were not observed.     

Growth and cellular organization of Arabidopsis pollen tubes in vitro

Tricellular pollen tubes of Arabidopsis thaliana were cultured in vitro on solid media and studied with respect to growth, cellular organization and ultrastructure, cytoskeletal organization, organelle movement, deposition and structure of the wall and the occurrence of coated pits, all elements assumed to be relevant for tip growth. For our ultrastructural studies we used freeze fixation and freeze substitution. Although Arabidopsis pollen tubes are broadly similar to those of bicellular species such as Nicotiana tabacum and Lilium spec. and in vivo grown pollen tubes of Arabidopsis, some differences occurred. The density of the equally distributed, relatively small (85 nm) secretory vesicles (SV) in the tip is low (five/µm ²). In between the SV of the tip, membranous material, possibly smooth endoplasmic reticulum, fragments of rough endoplasmic reticulum and loose ribosomes are present. The wall in the tip is not amorphous but layered and a secondary wall is formed already in the flanks of the tip. The general pattern of organelle motion is reverse fountain-like, but individual organelles move in distinct lanes at speeds of up to 2 µm/s, and about half of the organelle population shows a moderate velocity or Brownian movement. These properties are discussed in relation to the low growth rate (10 µm/h) of Arabidopsis pollen grown in vitro. The two similar sperm cells are closely attached and are always found near the vegetative nucleus. No surrounding wall and no cytoskeletal elements were obvious in the sperm cells. The preferential location of the mitochondria at the wall and the large (up to 400 nm) coated pits are unique for angiosperm pollen tubes. The size of the coated pits may allow not only membrane retrieval but also pinocytosis.     

The regulation of actin organization by actin-depolymerizing factor in elongating pollen tubes

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)-tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1-expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H(+) concentrations in the apical region of elongating pollen tubes.