Still life: Pollen tube growth observed in millisecond resolution

Our recent work used novel methods to localize and track discrete vesicle populations in pollen tubes undergoing oscillatory growth. The results show that clathrin-dependent endocytosis occurs along the shank of the pollen tube, smooth vesicle endocytosis occurs at the tip, and exocytosis occurs in the subapical region. Here, growth of tobacco and lily pollen tubes is examined in greater temporal resolution using refraction-free high-resolution time-lapse differential interference contrast microscopy. Images were collected at 0.21 s intervals for 10 min, sequentially examined for millisecond details, compressed into video format and then examined for details of growth dynamics. The subapical growth zone is structurally fluid, with vesicle insertion into the plasma membrane, construction of new cell surface and cellular expansion. Incorporation of new membrane and wall materials causes localized disruption at the cell surface that precedes the start of the growth cycle by 3.44 ± 0.39 s in tobacco, and 1.02 ± 0.01 s in lily pollen tubes. Vesicle deposition increases after the start of the growth cycle and supports expansion of the growth zone. Growth reorientation involves a shift in the position and angle of the growth zone. In summary, these results support a new model of pollen tube growth.Key words: growth zone, oscillation, exocytosis, growth reorientation, differential interference contrast microscopy, refraction-free     

Propidium Iodide Competes with Ca2+ to Label Pectin in Pollen Tubes and Arabidopsis Root Hairs

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca²⁺ binding. We first show that Ca²⁺ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca²⁺ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca²⁺-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca²⁺ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca²⁺ in tip growth.The apical wall of tip-growing cells participates directly in the process of growth regulation (McKenna et al., 2009; Winship et al., 2010), yet few methods permit monitoring the wall properties of living cells. Despite this, several recent studies have enhanced our understanding of the apical cell wall. Chemical analyses of isolated pollen tube wall material have revealed a complex mixture of pectic polysaccharides with regions comprising long sequences of polygalacturonic acid. Important patterns of pectin methoxylation have been detected using immunocytochemical approaches, but these are limited to fixed cells (Dardelle et al., 2010). In a recent study, Parre and Geitmann (2005) used microindentation to show significant correlations between wall strength and growth rate. None of these techniques allow for easy investigation of the cell wall during growth.In an earlier study, we found that propidium iodide (PI) vitally stains pollen tubes of lily (Lilium formosanum) and tobacco (Nicotiana tabacum) and in particular reveals with great clarity the thickened apical cell wall (Fig. 1; McKenna et al., 2009). In addition, the apical PI fluorescence oscillates and in lily pollen tubes correlates tightly with oscillations in wall thickness measured by differential interference contrast (DIC) optics. Finally, these studies indicated that the PI fluorescence predicted cell growth rates with high confidence, suggesting that PI binding may provide useful information about the physical and chemical properties of the cell wall.Open in a separate windowFigure 1.PI fluorescence and growth rate oscillate in lily pollen tubes (A and B), Arabidopsis root hairs (C–E), and Arabidopsis pollen tubes (F and G). A, The top panel shows a DIC image of a lily pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 10 μm. B, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. C, DIC image (top panel) and PI fluorescence image (bottom panel) of an Arabidopsis root hair. Bar = 10 μm. D, Two PI fluorescence images of the same root hair focused on the apex representing peak (top) and trough (bottom) PI signals. Bar = 5 μm. E, A line graph showing the growth rate (blue) and peak PI fluorescence at the apex (red) for the same root hair shown in C and D. F, The top panel shows a DIC image of an Arabidopsis pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 5 μm. G, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. The growth rate between individual 3-s frames was smaller than the pixel size for our optics in both Arabidopsis cell types; to remove the noise this generated, a four-image (pollen) or five-image (root hair) running average is shown. A.U., Arbitrary units.PI is commonly used to visualize plant cell walls by wide-field fluorescence and confocal microscopy (Fiers et al., 2005; Tian et al., 2006; Estevez et al., 2008) and to select viable cells during cell sorting (Deitch et al., 1982; Jones and Senft, 1985). A positively charged phenanthridine derivative, the propidium ion stains cell walls but does not pass through the intact cell membranes of living cells. It readily diffuses into dead cells and forms highly fluorescent complexes by intercalation between base pairs of double-stranded nucleic acids, thus acting as an excellent indicator for cell vitality (Hudson et al., 1969). Binding to cell walls presumably occurs by a different mechanism, since it is not accompanied by the dramatic increase in fluorescence and shift in absorption and emission maxima observed when PI binds to nucleic acids. The mechanism of PI binding needs further exploration, as does the potential for broader use in other tip-growing plant cells.In this report, we test two hypotheses: first, that PI stains other tip-growing cells with pectin-containing cell walls; and second, that PI and Ca²⁺ bind to the same sites in these walls. This binding would occur through the interaction of partial positive charges caused by localized deficits in π-orbital electrons associated with three of the four nitrogen atoms of PI (Luedtke et al., 2005) coordinating with negatively charged carboxyl and hydroxyl groups on homogalacturonans (HGs), as has been suggested in Oedogonium bharuchae (Estevez et al., 2008).Our findings indicate that both hypotheses are satisfied. Notably, oscillatory changes in apical PI fluorescence occur and are observed to anticipate oscillations in growth rate in Arabidopsis (Arabidopsis thaliana) root hairs and Arabidopsis pollen tubes. In addition, competition studies indicate that PI and Ca²⁺ bind to the same sites in cell walls. Supporting these studies, we demonstrate that pectin methyl esterase (PME) creates more sites for PI binding, presumably by demethoxylating HGs as they are secreted, and that pectinase reduces PI fluorescence dramatically. However, unlike other pectin-binding dyes, PI does not block Ca²⁺ channels at the concentration used in live cell studies, nor does it alter oscillatory growth characteristics. Our findings provide evidence that PI may be employed as a quantitative measure of Ca²⁺-binding sites and thus may have use as an indicator of the degree of cross-linking of HGs and of cell wall extensibility.     

Localization of arabidopsis SYP125 syntaxin in the plasma membrane sub-apical and distal zones of growing pollen tubes

Tip growth in pollen tubes occurs by continuous vesicle secretion and delivery of new wall material, but the exact sub-cellular location of endocytic and exocytic domains remains unclear. Here we studied the localization of the Arabidopsis thaliana pollen specific syntaxin SYP125 using GFP-fusion constructs expressed in Nicotiana tobaccum pollen tubes. In agreement with the predicted role for syntaxins, SYP125 was found to be associated with the plasma membrane and apical vesicles in growing cells. At the plasma membrane, SYP125 was asymmetrically localized with a higher labeling 20–35 µm behind the apex, a distribution which is distinct from SYP124, another pollen-specific syntaxin. Competition with a related dominant negative mutant affected the specific distribution of SYP125 but not tip growth. Co-expression of the phosphatidylinositol-4-monophosphate-5-kinase 4 (PIP5K4) or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane.Taken together, our results corroborates previous observations that in normal growing pollen tubes, the asymmetric distribution of syntaxins helps to define exocytic sub-domains but requires the involvement of additional signaling and functional mechanisms, namely phosphoinositides and small GTPases. The localization of syntaxins at different membrane domains likely depends on the interaction with specific partners not yet identified.Key words: [Ca²⁺]_c, endocytosis, exocytosis, secretion, syntaxins, tip growth     

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.     

Pollen sporoplasts: dissolution of pollen walls

4-Methylmorpholine N-oxide monohydrate (MMNO·H₂O), a potent solvent for polysaccharides, is an effective vehicle for release of membrane-enclosed male gametophytes (sporoplasts) from spore walls. This release occurs in minutes when pollen (Lilium longiflorum Thunb.) is suspended in a melt of MMNO·H₂O at 75°C. Continued heating at 75°C leads to distintegration of the exine `shell' which coalesces into immiscible globules in the MMNO melt. These observations provide a general procedure for preparation of pollen sporoplasts and sporoplast outer membranes, and offer a new method for dissolving the sporopollenin component of the spore wall.     

Invertases of Lilium Pollen : Characterization and Activity during In Vitro Germination

Two different forms of invertase are found in pollen of lily (Lilium auratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partially purified and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K_m of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are β-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germination. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations.     

High temperature-induced thermotolerance in pollen tubes of tradescantia and heat-shock proteins

Growing pollen tubes of Tradescantia paludosa are protected from inhibition of growth at 41°C by a prior exposure to gradually increasing temperatures. Heat shock proteins (hsps) are not synthesized by pollen tubes as determined by labeling with [³⁵S]methionine and two-dimensional gel electrophoresis, during either a heat shock at 41°C or a gradual temperature increase to 41°C. A comparison after two-dimensional electrophoresis of silver-stained spots and radioactive spots after autoradiography of an extract of ungerminated pollen mixed with a trace amount of [³⁵S]methionine-labeled vegetative tissue heat shocked at 41°C to act as a hsps marker, indicates that the majority, if not all, of the major hsps are not present in the pollen grain at anthesis. The type of thermotolerance seen with pollen tubes can thus be achieved without the presence or the new synthesis of the hsps.     

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

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

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.     

Apical F‐actin‐regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall

In tip‐confined growing pollen tubes, delivery of newly synthesized cell wall materials to the rapidly expanding apical surface requires spatial organization and temporal regulation of the apical F‐actin filament and exocytosis. In this study, we demonstrate that apical F‐actin is essential for the rigidity and construction of the pollen tube cell wall by regulating exocytosis of Nicotiana tabacum pectin methylesterase (NtPPME1). Wortmannin disrupts the spatial organization of apical F‐actin in the pollen tube tip and inhibits polar targeting of NtPPME1, which subsequently alters the rigidity and pectic composition of the pollen tube cell wall, finally causing growth arrest of the pollen tube. In addition to mechanistically linking cell wall construction and apical F‐actin, wortmannin can be used as a useful tool for studying endomembrane trafficking and cytoskeletal organization in pollen tubes.     

beta-1, 3-Glucan Synthase from Lilium longiflorum Pollen

A particulate fraction from pollen tubes and ungerminated pollen of Lilium longiflorum incorporated ¹⁴C-glucose from UDP-glucose-¹⁴C into a lipid fraction and into β-1, 3-glucan. Partial hydrolysis of the glucan yielded laminaribiose as the only radioactive disaccharide. The preferred substrate was UDP-glucose, and enzyme activity was stimulated by glucose and by β-linked di- and trisaccharides. Enzyme from growing pollen tubes synthesized β-1, 3-glucan more rapidly and produced a higher proportion of alkali-insoluble glucan than did enzyme from ungerminated pollen. The onset of pollen tube growth may be dependent on altered activity of β-1, 3-glucan synthase.     

Nitric Oxide Participates in Cold-Inhibited Camellia sinensis Pollen Germination and Tube Growth Partly via cGMP In Vitro

Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.     

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.     

Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.     

Characterization and function of wall-bound exo-β-glucanases of Lilium longiflorum pollen tubes

Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward 1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases, as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important role in the regulation of lily pollen tube growth. Received: 3 January 2000 / Revision accepted: 8 March 2000     

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.     

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     

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.