Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.     

Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis

Ca2+ dynamics in the growing pollen tube have been well documented in vitro using germination assays and Ca2+ imaging techniques. However, very few in vivo studies of Ca2+ in the pollen grain and papilla cell during pollination have been performed. We expressed yellow cameleon, a Ca2+ indicator based on green fluorescent protein, in the pollen grains and papilla cells of Arabidopsis (Arabidopsis thaliana) and monitored Ca2+ dynamics during pollination. In the pollen grain, [Ca2+]cyt increased at the potential germination site soon after hydration and remained augmented until germination. As in previous in vitro germination studies, [Ca2+]cyt oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca2+]cyt was higher than had been observed in vitro. In the pollinated papilla cell, remarkable increases in [Ca2+]cyt occurred three times in succession, just under the site of pollen-grain attachment. [Ca2+]cyt increased first soon after pollen hydration, with a second increase occurring after pollen protrusion. The third and most remarkable [Ca2+]cyt increase took place when the pollen tube penetrated into the papilla cell wall.     

水稻非花粉型细胞质雄性不育系及其保持系花药发育过程中Ca2+的分布变化

钙在高等植物中被称为第二信使,与植物的有性生殖有关。为了研究水稻（Oryza sativa L.）花药中钙的定位与花粉败育的关系,利用焦锑酸钾沉淀法研究了非花粉型细胞质雄性不育系G37A及其保持系G37B花药的发育过程及其细胞中Ca^2＋ 的分布变化。研究发现,在2个材料间花药中钙的分布存在大量差异。G37B的可育花药在花粉母细胞时期及二分体时期,很少看到有Ca^2＋的沉积;而在单核花粉时期,Ca^2＋沉积急速地增加,主要定位在绒毡层细胞、花粉外壁外层及乌氏体的表面;随后花药壁上沉积的Ca^2＋减少而花粉的外壁外层仍然有很多Ca^2＋沉积物。相反,G37A的不育花药在花粉母细胞时期和二分体时期有大量的Ca^2＋沉积在小孢子母细胞和花药壁,中间层和绒毡层特别多。在二分体时期之后,不育花药的Ca^2＋沉积减少,特别是绒毡层内切向质膜附近的Ca^2＋几乎消失。但是同时期的可育花药中,有大量的Ca^2＋沉积在绒毡层。不育花药的Ca^2＋沉积在开花几天后消失。根据研究结果推测在不育花药发育早期中更多的钙离子与花粉败育有一定的关系。     

Involvement of extracellular calcium influx in the self-incompatibility response of Papaver rhoeas

We have previously demonstrated that increases in cytosolic free Ca2+ are triggered by the self-incompatibility (SI) response in incompatible Papaver rhoeas (the field poppy) pollen. However, one key question that has not been answered is whether extracellular Ca2+ may be involved. To address this question, we have used an ion-selective vibrating probe to measure changes in extracellular Ca2+ fluxes around poppy pollen tubes. Our data reveal several findings. First, we confirm that there is an oscillating Ca2+ influx directed at the apex of the pollen tube; we also provide evidence that Ca2+ influx also occurs at the shanks of pollen tubes. Second, upon challenge with self-incompatibility (S) proteins, there is a stimulation of Ca2+ influx along the shank of incompatible pollen tubes, approximately 50 microm behind the pollen tube tip. This demonstration of SI-induced Ca2+ influx suggests a role for influx of extracellular Ca2+ in the SI response.     

Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes.

The distribution of intracellular free calcium ions ([Ca2+]i) was measured in pollen tubes of Lilium longiflorum using video imaging microscopy and the calcium sensitive indicators fura-2 and quin-2. The mean [Ca2+]i in growing pollen tubes measured with fura-2 shows a maximum of 1.7 to 2.6 microM in the tube tip and decreases almost exponentially to 60 to 100 nM at 100 microns behind the tip. Using quin-2, the maximum [Ca2+]i was also found in the tube tip but with a lower Ca2+ concentration, namely 1 microM. Addition of the calcium channel blocker La3+ caused a decrease of the [Ca2+]i maximum in the tube tip, indicating a heterogeneous distribution of Ca2+ channels along the plasma membrane of pollen tubes. The [Ca2+]i increased after addition of vanadate or compound 48/80. This suggests an involvement of a calmodulin-dependent Ca2+ pump in generation of the Ca2+ gradient in lily pollen tubes. The high [Ca2+]i found in the tube tip with fura-2 seems to indicate the real Ca2+ concentration and is probably responsible for vesicle fusion, fragmentation of actin filaments, and inhibition of cytoplasmic streaming.     

Characterization of a birch pollen allergen, Bet v III, representing a novel class of Ca2+ binding proteins: specific expression in mature pollen and dependence of patients' IgE binding on protein-bound Ca2+.

A cDNA coding for a birch pollen allergen, Bet v III, with significant sequence homology to Ca2+ binding proteins was isolated from an expression cDNA library using serum IgE from a patient who was allergic to pollen. The deduced amino acid sequence of the pollen allergen contained three typical Ca2+ binding sites. Peptides mimicking the Ca2+ binding sites of Bet v III were synthesized and shown to bind 45Ca in blot overlays. The binding of patients' IgE to the recombinant allergen depended on the native protein conformation and protein-bound Ca2+. Depletion of Ca2+ led to a reversible loss of the IgE binding thus representing a conformational IgE epitope adopted by a polypeptide upon Ca2+ binding. By RNA hybridization it was demonstrated that Bet v III is expressed preferentially in mature pollen. Bet v III therefore represents a pollen allergen which because of its unique structural features also belongs to a novel class of Ca2+ binding proteins.     

Identification and characterization of stretch-activated ion channels in pollen protoplasts

Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.     

水稻胞质雄性不育系珍汕97A及其保持系珍汕97B绒毡层发育过程中的Ca2+分布变化

利用焦锑酸钾沉淀法研究了野败不育系珍汕97A及其保持系珍汕97B绒毡层细胞的发育过程及其细胞中Ca2 的分布变化。研究发现保持系绒毡层细胞在单核花粉晚期才开始迅速解体,而不育系绒毡层细胞在花粉母细胞时期就开始出现核膜、细胞膜解体,此过程持续到二核花粉时期。珍汕97A绒毡层细胞从花粉母细胞时期开始,细胞质内有少量颗粒状的Ca2 沉淀;减数分裂时期,绒毡层细胞的内切向壁表面有大量大颗粒的Ca2 沉淀;单核花粉时期绒毡层细胞周围集聚一层Ca2 沉淀。而保持系绒毡层细胞遮花粉母细胞时期和减数分裂时期细胞内没有Ca2 沉淀;单核花粉时期绒毡层细胞内的Ca2 沉淀主要分布在解体的细胞质内。推测绒毡层细胞结构发育的异常和Ca2 的异常分布可能与花粉的败育有关。     

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.     

Calcium Channel Activity during Pollen Tube Growth and Reorientation

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.     

Effect of extracellular calcium,pH and borate on growth oscillations in Lilium formosanum pollen tubes

Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.     

Ca2+ influx into lily pollen grains through a hyperpolarization-activated Ca2+-permeable channel which can be regulated by extracellular CaM

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.     

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.     

Pollen Tube Growth and the Intracellular Cytosolic Calcium Gradient Oscillate in Phase while Extracellular Calcium Influx Is Delayed

Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3- to 5-sec intervals showed that the tip-focused [Ca2+]i gradient oscillates with the same period as growth. Similarly, measurement of the extracellular inward current, using a noninvasive ion-selective vibrating probe, indicated that the tip-directed extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+]i gradient oscillates in phase with growth, the influx of Ca2+o lags by ~11 sec. Ion influx thus appears to follow growth, with the effect that the rate of growth at a given point determines the magnitude of the ion influx ~11 sec later. To explain the phase delay in the extracellular inward current, there must be a storage of Ca2+ for which we consider two possibilities: either the inward current represents the refilling of intracellular stores (capacitative calcium entry), or it represents the binding of the ion within the cell wall domain.     

Localization of pectins and Ca2+ ions in unpollinated and pollinated wet (Petunia hybrida Hort.) and dry (Haemanthus albiflos L.) stigma

The subcellular localization of Ca2+ ions as well as esterified and deesterified pectins in unpollinated and pollinated wet (Petunia hybrida) and dry (Haemanthus albiflos) stigma was analyzed. Stigmas with different surfaces were found to differ in Ca2+ and pectin localization. In a wet Petunia hybrida stigma, Ca2+ ions were present in the exudate occurring in the intercellular spaces of secretory tissue before pollination. The exudate of an unpollinated stigma was the site of the localization of large amounts of deesterified pectins. Stigma penetration by pollen tubes induced the lysis of this category of pectins. The epidermal cells walls of the dry Haemanthus albiflos stigma before pollination lacked free and loosely bound Ca2+ ions. Pollination induced an accumulation of these ions in the apoplast of the stigma epidermal cells. In cells walls of an unpollinated stigma, mainly esterified pectins were present. Their deesterification took place after pollination at the site of pollen grain adhesion and then at the site of pollen tube growth. These results have shown that wet and dry stigmas differ in pectin metabolism and in the mechanism of forming a calcium environment at the site of pollen grain germination.     

甘蓝型油菜花粉离体培养

通过对甘蓝型油菜花粉发育阶段和活力的检测确定花粉发育的时期,分离出单核晚期花粉进行离体培养.结果表明,(1)筛选出适合油菜小孢子花粉离体培养的液体培养基为T_1+怀特维生素(White's vitamins)+2%椰子汁+0.5 mol/L麦芽糖,在此培养基上花粉的成熟率可达25.1%,萌发率达6.3%.(2)筛选出适合成熟花粉离体萌发液体培养基为0.6 mol/L麦芽糖+1.6 mmol/L硼酸+2.9 mmol/L硝酸钙+29.6 μmol/L VB_1,在此培养基上,自然成熟花粉的萌发率可达75.2%.将离体培养成熟的花粉培养在萌发培养基,萌发的花粉占成熟花粉的66.3%.     

Exogenous Ca2+ Regulation of Pollen Tube Growth and Division of Generative Nucleus in Nicotiana tabacum

Cytological and statistical studies on the effects of exogenous Ca2 + on in vitro pollen tube growth and generative nucleus (GN) division of tobacco (Nicotiana tabacum L. ) were conducted in an artificial experimental system. Under normal cultured conditions, the rate of GN division increased logarithmically in general, and reaches the climax at about 10 - 18 h. Among the treatments with various Ca2 + concentrations, 10- 3 mol/L was the optimal concentration for pollen tube growth, whereas other Ca2+ concentrations showed increasing inhibitory effect with the time of culture. Generally, Ca2 + concentrations at 10-2 and 10-3 mol/L favored GN division more than the others. Compared with 10-3 mol/L Ca2 + concentration at 10-2 mol/L benefitiated GN division at earlier stage of the treatment, but afterwards showed inhibitory effect gradually. Besides, the authors designed another series of experiments, in which 10-2, 10-1 mol/L Ca2+ (final concentrations) or 2,10 mmol/L EG-TA was respectively added to the medium containing 10-3 mol/L Ca2+ at 10 h of culture. Pollen tube growth was inhibited by the high Ca2+ treatments, especially being severely effected by 10-l mol/L Ca2 + from which wall, thickening of the tube tip, amitotic division of GN leading to micronucleus formation occurred. 10-2 mol/L Ca2 + treatment, however, promoted GN division at the earlier stage of treatment ( 10 - 12 h). EGTA treatments inhibited both pollen tube growth and GN division.     

Aluminium Effects on Pollen Germination and Tube Growth of Chamelaucium uncinatum. A Comparison with Other Ca2+Antagonists

An interaction between aluminium (Al) and calcium (Ca) may bea cause of Al toxicity in plants. The pollen tube is a suitablesystem to test the interaction between Al and Ca since Ca ionsplay a pivotal role in pollen germination and tube growth. Weinvestigated how Al and other known blockers of Ca²⁺-permeablechannels (trivalent cations, ruthenium red, verapamil and nifedipine)influence pollen of an Australian native species Geraldton waxflower(Chamelaucium uncinatum). Pollen germination was inhibited bymicromolar concentrations of trivalent cations (La³⁺>Al³⁺>Gd³⁺)and ruthenium red, but it was relatively insensitive to a micromolarconcentration of verapamil. Exposure of the growing pollen tubesto micromolar concentrations of Al³⁺and La³⁺, and a millimolarconcentration of Ca²⁺chelator ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA) led to rapid tip bursting.In contrast, exposure to Gd³⁺, nifedipine, ruthenium red, verapamiland the organic trivalent cation tris (ethylenediamine)cobalt(TEC³⁺) caused only inhibition of pollen tube growth. The Al³⁺-relatedpollen tube bursting was reduced significantly by increasingeither solution pH from 4.5 to 6 or activity of Ca²⁺from 0.25to 5 m M. In contrast, La³⁺-related pollen tube bursting wasinsensitive to changes in Ca²⁺activity. The results are discussedin terms of Al interactions with cell wall Ca²⁺and the plasmamembrane Ca²⁺-permeable channels. Copyright 1999 Annals of BotanyCompany Aluminium toxicity, Ca²⁺-channel blockers, cell wall, Chamelaucium uncinatum, pollen germination, pollen tube growth.     

Localized Apical Increases of Cytosolic Free Calcium Control Pollen Tube Orientation

To reach the ovule, pollen tubes must undergo many changes in growth direction. We have shown in previous work that elevation of cytosolic free calcium ([Ca2+]c) can manipulate orientation in growing pollen tubes, but our results suggested that [Ca2+]c changes either in the tip or in more distal regions might regulate the critical orienting mechanism. To identify the spatial location of the orienting motor, we combined the techniques of ion imaging with confocal microscopy and localized photoactivation of loaded caged Ca2+ (nitr-5) and diazo-2 (a caged Ca2+ chelator) to manipulate [Ca2+]c in different pollen tube domains. We found that increasing [Ca2+]c on one side of the pollen tube apex induced reorientation of the growth axis toward that side. Similarly, a decrease in [Ca2+]c promoted bending toward the opposite side. These effects could be mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca2+ channel blocker (GdCl3); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl3. Manipulation of [Ca2+]c in regions farther back from the apical zone also induced changes in growth direction, but the new orientation was at random. We observed communication of these distal events to the tip through a slow-moving [Ca2+]c wave. These data show that localized changes of [Ca2+]c in the tip, which could result from asymmetric channel activity, control the direction of pollen tube growth.