Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

Pollen tubes are one of the fastest growing eukaryotic cells.Rapid anisotropic growth is supported by highly active exocytosisand endocytosis at the plasma membrane, but the subcellularlocalization of these sites is unknown. To understand molecularprocesses involved in pollen tube growth, it is crucial to identifythe sites of vesicle localization and trafficking. This reportpresents novel strategies to identify exocytic and endocyticvesicles and to visualize vesicle trafficking dynamics, usingpulse-chase labelling with styryl FM dyes and refraction-freehigh-resolution time-lapse differential interference contrastmicroscopy. These experiments reveal that the apex is the siteof endocytosis and membrane retrieval, while exocytosis occursin the zone adjacent to the apical dome. Larger vesicles areinternalized along the distal pollen tube. Discretely sizedvesicles that differentially incorporate FM dyes accumulatein the apical, subapical, and distal regions. Previous workestablished that pollen tube growth is strongly correlated withhydrodynamic flux and cell volume status. In this report, itis shown that hydrodynamic flux can selectively increase exocytosisor endocytosis. Hypotonic treatment and cell swelling stimulatedexocytosis and attenuated endocytosis, while hypertonic treatmentand cell shrinking stimulated endocytosis and inhibited exocytosis.Manipulation of pollen tube apical volume and membrane remodellingenabled fine-mapping of plasma membrane dynamics and definedthe boundary of the growth zone, which results from the orchestratedaction of endocytosis at the apex and along the distal tubeand exocytosis in the subapical region. This report providescrucial spatial and temporal details of vesicle traffickingand anisotropic growth. Key words: Endocytosis; exocytosis, hydrodynamics, lipophilic FM dyes, pollen tube growth, vesicle trafficking Received 14 September 2007; Revised 23 November 2007 Accepted 7 January 2008     

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.     

Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4‐64 and charged nanogold. Time‐lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co‐localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin‐dependent and actin‐indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin‐dependent secretory activity occurs in the shank of pollen tubes.     

Pollen Tube Growth: a Delicate Equilibrium Between Secretory and Endocytic Pathways

Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo .     

A model of plasma membrane flow and cytosis regulation in growing pollen tubes

A model of cytosis regulation in growing pollen tubes is developed and simulations presented. The authors address the question on the minimal assumptions needed to describe the pattern of exocytosis and endocytosis reported recently by experimental biologists. Biological implications of the model are also treated. Concepts of flow and conservation of membrane material are used to pose an equation system, which describes the movement of plasma membrane in the tip of growing pollen tubes. After obtaining the central equations, relations describing the rates of endocytosis and exocytosis are proposed. Two cytosis receptors (for exocytosis and endocytosis), which have different recycling rates and activation times, suffice to describe a stable growing tube. Simulations show a very good spatial separation between endocytosis and exocytosis, in which separation is shown to depend strongly on exocytic vesicle delivery. In accordance to measurements, most vesicles in the clear zone are predicted to be endocytic. Membrane flow is essential to maintain cell polarity, and bi-directional flow seems to be a natural consequence of the proposed mechanism. For the first time, a model addressing plasma membrane flow and cytosis regulation were posed. Therefore, it represents a missing piece in an integrative model of pollen tube growth, in which cell wall mechanics, hydrodynamic fluxes and regulation mechanisms are combined.     

Not-so-tip-growth

In tip-growing plant cells such as pollen tubes and root hairs, surface expansion is confined to the cell apex. Vesicles containing pectic cell wall material are delivered to this apical region to provide the material necessarily to build the expanding cell wall. Quantification of wall expansion reveals that the surface expansion rates are not highest at the pole but instead in an annular region around the pole. These findings raise the question of the precise localization of exocytosis events in these cells. Recently, we used spatio-temporal image correlation spectroscopy (STICS) in combination with high temporal resolution confocal imaging to characterize the intracellular movement of vesicles in growing pollen tubes. These observations, together with the analysis of FRAP (fluorescence recovery after photobleaching) experiments, indicate that exocytosis is likely to occur predominantly in the same annular region where wall expansion rates are greatest. Therefore, tip growth in plant cells does not seem to happen exactly at the tip.Key words: tip growth, pollen tube, exocytosis, cell wall, expansion, root hair, plant cell growth, allometric growth, cytomechanics, cell mechanics, vesicle transport     

Antisense Oligodeoxynucleotide Inhibition as an Alternative and Convenient Method for Gene Function Analysis in Pollen Tubes

Antisense oligodeoxynucleotide (A-ODN) inhibition works well in animal cells. However, there have been few successful examples to date of its application in plants, and more specifically whether the technique can be used in pollen tubes as a model of plant cell growth. NtGNL1 plays an important role in pollen tube development and was thus selected as an indicator to assess the biological effects of A-ODN. An A-ODN inhibition technique was used to down-regulate NtGNL1 expression in tobacco pollen tubes and showed that A-ODNs could quickly enter pollen tubes through the thick wall and cell membrane and effectively block NtGNL1 expression. Phenotype analysis revealed that the down-regulation of NtGNL1 by A-ODNs resulted in abnormalities in endocytosis and subsequent vesicle trafficking, similar to the phenotypes of pollen tubes treated with NtGNL1 RNAi. This investigation confirmed that A-ODNs could specifically inhibit target gene expression, and furthermore demonstrated that A-ODN functioned in a concentration- and duration-dependent manner, because A-ODNs could be degraded when incubated with pollen tubes. Thus, the A-ODN technique was successfully used for gene function analysis in pollen tubes and appears to be an alternative and convenient technique when the in vitro pollen tube is used as the study model. This technique will greatly facilitate investigations on the molecular mechanism(s) underlying pollen tube growth.     

Growing Pollen Tubes Possess a Constitutive Alkaline Band in the Clear Zone and a Growth-dependent Acidic Tip

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H⁺) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 μM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.     

Effects of brefeldin A on pollen germination and tube growth. Antagonistic effects on endocytosis and secretion

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 microg mL(-1)) treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased, together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the retarded growth of pollen tubes induced by BFA.     

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.     

Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments

The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca²⁺, in vitro. Analysis of a mutant that bears a point mutation at the Ca²⁺ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca²⁺ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.     

Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside

Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth. They are secreted at pollen tube tips in Lilium longiflorum. Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species. In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L. longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum. With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips. Only A. cherimola showed evidence of AGPs at the pollen tube tip as does lily. The Yariv reagent causes arrest of tube growth in both A. cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species. We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion. Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium. After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site. During this recovery, esterified pectins colocalized with AGPs. Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.     

FIMBRIN1 Is Involved in Lily Pollen Tube Growth by Stabilizing the Actin Fringe

An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.     

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.     

Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth

Vacuolar sorting receptors (VSRs) are type‐I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type‐IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans‐Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP‐VSR/GFP‐LIVSR is located throughout the pollen tubes, excepting the apical clear‐zone region, whereas GFP‐LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.     

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca²⁺ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca²⁺ gradient.Abbreviations ER endoplasmic reticulum - MES 2-[N-morpholino] ethanesulfonic acid - MFs microfilaments     

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.     

Imaging elongating pollen tubes by green fluorescent protein

Pollen tube elongation is a dynamic process in which pollen tubes navigate and respond to female tissues to accomplish their mission of delivering the sperm cells for fertilization. The tube growth process itself is driven by regulated intracellular conditions that maintain the appropriate ionic environment, actin dynamics and a balance level of exocytosis and endocytosis to support growth at the tube apex. However, the interactive process within the pistil has not rendered itself accessible for direct observation. The contribution by individual cytosolic constituents of the pollen tube growth machinery remains to be determined. With the development of the green fluorescent protein reporter system, many of these questions can be addressed in live pollen tubes that elongate within the pistil and inchemically defined media. Analyses of the mechanisms that underlie pollen tube growth will be significantly facilitated. Received: 15 March 2001 / Accepted: 30 May 2001     

Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco

Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein-tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. RNA interference-induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P(2) in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P(2), but also by that of PI4Kβ1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P(2) is required for clathrin-dependent endocytosis in the tip of pollen tubes.