Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties

Cellular morphogenesis involves changes to cellular size and shape which in the case of walled cells implies the mechanical deformation of the extracellular matrix. So far, technical challenges have made quantitative mechanical measurements of this process at subcellular scale impossible. We used micro-indentation to investigate the dynamic changes in the cellular mechanical properties during the onset of spatially confined growth activities in plant cells. Pollen tubes are cellular protuberances that have a strictly unidirectional growth pattern. Micro-indentation of these cells revealed that the initial formation of a cylindrical protuberance is preceded by a local reduction in cellular stiffness. Similar cellular softening was observed before the onset of a rapid growth phase in cells with oscillating growth pattern. These findings provide the first quantitative cytomechanical data that confirm the important role of the mechanical properties of the cell wall for local cellular growth processes. They are consistent with a conceptual model that explains pollen tube oscillatory growth based on the relationship between turgor pressure and tensile resistance in the apical cell wall. To further confirm the significance of cell mechanics, we artificially manipulated the mechanical cell wall properties as well as the turgor pressure. We observed that these changes affected the oscillation profile and were able to induce oscillatory behavior in steadily growing tubes.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

Model for calcium dependent oscillatory growth in pollen tubes

Experiments have shown that pollen tubes grow in an oscillatory mode, the mechanism of which is poorly understood. We propose a theoretical growth model of pollen tubes exhibiting such oscillatory behaviour. The pollen tube and the surrounding medium are represented by two immiscible fluids separated by an interface. The physical variables are pressure, surface tension, density and viscosity, which depend on relevant biological quantities, namely calcium concentration and thickness of the cell wall. The essential features generally believed to control oscillating growth are included in the model, namely a turgor pressure, a viscous cell wall which yields under pressure, stretch-activated calcium channels which transport calcium ions into the cytoplasm and an exocytosis rate dependent on the cytosolic calcium concentration in the apex of the cell. We find that a calcium dependent vesicle recycling mechanism is necessary to obtain an oscillating growth rate in our model. We study the variation in the frequency of the growth rate by changing the extracellular calcium concentration and the density of ion channels in the membrane. We compare the predictions of our model with experimental data on the frequency of oscillation versus growth speed, calcium concentration and density of calcium channels.     

The role of pectin in plant morphogenesis

The presence of a polysaccharidic cell wall distinguishes plant cells from animal cells and is responsible for fundamental mechanistic differences in organ development between the two kingdoms. Due to the presence of this wall, plant cells are unable to crawl and contract. On the other hand, plant cell size can increase by several orders of magnitude and cell shape can change from a simple polyhedron or cube to extremely intricate. This expansive cellular growth is regulated by the interaction between the cell wall and the intracellular turgor pressure. One of the principal cell wall components involved in temporal and spatial regulation of the growth process is pectin. Through biochemical changes to pectin composition and biochemical configuration, the properties of this material can be altered to trigger specific developmental processes. Here, the roles of pectin in three systems displaying rapid growth - the elongation zone of the root, the tip region of the pollen tube, and organ primordia formation at the shoot apical meristem - are reviewed.     

Understanding pollen tube growth: the hydrodynamic model versus the cell wall model

Scientific progress stimulates the evolution of models used to understand and conceptualize biological behaviors. The widely accepted cell wall model of pollen tube growth explains stochastic growth of the apical pectin wall, but fails to explain the mechanism driving oscillations in growth and cell signaling. Recent advances led to the formulation of a new hydrodynamic model that explains the mechanism that drives both stochastic and oscillatory growth, as well as oscillations in cell signaling and ion fluxes. A critical analysis of evidence that has been used to challenge the validity of the hydrodynamic model yields new information on turgor pressure, cell mechanical properties and nonlinear dynamics in pollen tube growth. These results may have broader significance for plant cell growth.     

The pollen tube: a soft shell with a hard core

Plant cell expansion is controlled by a fine‐tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in‐depth knowledge of cell wall mechanics. Pollen tubes are tip‐growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20–90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM‐based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.     

Pectin and the role of the physical properties of the cell wall in pollen tube growth of<Emphasis Type="Italic"> Solanum chacoense</Emphasis>

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.     

Dynamic,high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation

The pollen tube is the most rapidly growing cell in the plant kingdom and has the function to deliver the sperm cells for fertilization. The growing tip region of the cell behaves in a chemotropic manner to respond to the guidance cues emitted by the pistil and the female gametophyte, but how it perceives and responds to these directional triggers is virtually unknown. Quantitative assessment of chemotropic behavior can greatly be enhanced by the administration of pharmacological or other biologically active agents at subcellular precision, which is a technical challenge when the target area moves as it grows. We developed a laminar flow based microfluidic device that allows for continuous administration of two different solutions with a movable interface that permits the dynamic targeting of the growing pollen tube apex over prolonged periods of time. Asymmetric administration of calcium revealed that rather than following the highest calcium concentration as would be expected with simple chemotropic behavior, the pollen tube of Camellia targets an optimal concentration suggesting the presence of two superimposed mechanisms. Subcellular application of pectin methyl esterase (PME), an enzyme that modifies the growth behavior by rigidifying the pollen tube cell wall, caused the tube to turn away from the agent – providing important evidence for a previously proposed conceptual model of the growth mechanism.     

Regulator or Driving Force? The Role of Turgor Pressure in Oscillatory Plant Cell Growth

Turgor generates the stress that leads to the expansion of plant cell walls during cellular growth. This has been formalized by the Lockhart equation, which can be derived from the physical laws of the deformation of viscoelastic materials. However, the experimental evidence for such a direct correlation between growth rate and turgor is inconclusive. This has led to challenges of the Lockhart model. We model the oscillatory growth of pollen tubes to investigate this relationship. We couple the Lockhart equation to the dynamical equations for the change in material properties. We find that the correct implementation of the Lockhart equation within a feedback loop leading to low amplitude oscillatory growth predicts that in this system changes in the global turgor do not influence the average growth rate in a linear manner, consistent with experimental observations. An analytic analysis of our model demonstrates in which regime the average growth rate becomes uncorrelated from the turgor pressure.     

Chemically mediated mechanical expansion of the pollen tube cell wall

Morphogenesis of plant cells is tantamount to the shaping of the stiff cell wall that surrounds them. To this end, these cells integrate two concomitant processes: 1), deposition of new material into the existing wall, and 2), mechanical deformation of this material by the turgor pressure. However, due to uncertainty regarding the mechanisms that coordinate these processes, existing models typically adopt a limiting case in which either one or the other dictates morphogenesis. In this report, we formulate a simple mechanism in pollen tubes by which deposition causes turnover of cell wall cross-links, thereby facilitating mechanical deformation. Accordingly, deposition and mechanics are coupled and are both integral aspects of the morphogenetic process. Among the key experimental qualifications of this model are: its ability to precisely reproduce the morphologies of pollen tubes; its prediction of the growth oscillations exhibited by rapidly growing pollen tubes; and its prediction of the observed phase relationships between variables such as wall thickness, cell morphology, and growth rate within oscillatory cells. In short, the model captures the rich phenomenology of pollen tube morphogenesis and has implications for other plant cell types.     

The Role of the Cytoskeleton and Dictyosome Activity in the Pulsatory Growth of Nicotiana tabacum and Petunia hybrida Pollen Tubes

Pollen tubes of Nicotiana tabacum and Petunia hybrida show pulsatory growth. Phases of slow growth lasting minutes are interrupted by pulse-like elongations lasting 10–20 seconds involving an increase of growth rate by up to 24-fold. Inhibition of dictyosome activity with brefeldin A or monensin did not result in an inhibition of pulsatory growth but eventually stopped pollen tube elongation. In contrast to this the inhibition of the cytoskeletal elements with cytochalasin D and colchicine caused the pollen tubes to abandon the pulse-like elongations. It was concluded that the activity of the dictyosomes does not have a controlling function in the mechanism of pulsatory growth, even though it is necessary for pollen tube elongation, since cell wall material is provided by secretory vesicles deriving from the Golgi apparatus. In contrast the cytoskeletal elements, actin and microtubules, seem to play an important regulatory role in the pulse-like elongations. In addition, it was observed that during the experiments several pollen tubes burst upon the completion of a pulse-like expansion, indicating on the one hand that the internal turgor is the driving force of the pulse-like expansions. On the other hand, the bursting shows that the pollen tube cell wall is rather weak at the end of a pulse, indicating that at this point of time it is either thinner or less stable than during the slow growth phase or at the beginning of a pulse.     

Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.     

Cellular oscillations and the regulation of growth: the pollen tube paradigm

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86-94, 2001.     

Pressure–Induced Cell Wall Instability and Growth Oscillations in Pollen Tubes

In the seed plants, the pollen tube is a cellular extension that serves as a conduit through which male gametes are transported to complete fertilization of the egg cell. It consists of a single elongated cell which exhibits characteristic oscillations in growth rate until it finally bursts, completing its function. The mechanism behind the periodic character of the growth has not been fully understood. In this paper we show that the mechanism of pressure – induced symmetry frustration occurring in the wall at the transition-perimeter between the cylindrical and approximately hemispherical parts of the growing pollen tube, together with the addition of cell wall material, is sufficient to release and sustain mechanical self-oscillations and cell extension. At the transition zone, where symmetry frustration occurs and one cannot distinguish either of the involved symmetries, a kind of ‘superposition state’ appears where either single or both symmetry(ies) can be realized by the system. We anticipate that testifiable predictions made by the model () may deliver, after calibration, a new tool to estimate turgor pressure from oscillation frequency of the periodically growing cell. Since the mechanical principles apply to all turgor regulated walled cells including those of plant, fungal and bacterial origin, the relevance of this work is not limited to the case of the pollen tube.     

How to shape a cylinder: pollen tube as a model system for the generation of complex cellular geometry

Expansive growth in plant cells is a formidable problem for biophysical studies, and the mechanical principles governing the generation of complex cellular geometries are still poorly understood. Pollen, the male gametophyte stage of the flowering plants, is an excellent model system for the investigation of the mechanics of complex growth processes. The initiation of pollen tube growth requires first of all, the spatially confined formation of a protuberance. This process must be controlled by the mechanical properties of the cell wall, since turgor is a non-vectorial force. In the elongating tube, cell wall expansion is confined to the apex of the cell, requiring the tubular region to be stabilized against turgor-induced tensile stress. Tip focused surface expansion must be coordinated with the supply of cell wall material to this region requiring the precise, logistical control of intracellular transport processes. The advantage of such a demanding mechanism is the high efficiency it confers on the pollen tube in leading an invasive way of life.     

A model for predicting growth responses in plants to changes in external water potential: Zea mays primary roots

In response to osmotic step changes, three distinct phases have been noted in the growth response of Zea mays primary roots. They are cessation or slowing of growth over a period of 15–20 minutes, tissue contraction, and a damped oscillatory return to nearly normal growth rate, all within a period of about one hour. A system model of the tissue response is presented to explain such behavior and to serve in a predictive capacity to govern future experiments.It is supposed that for turgor pressure in excess of a cell wall yield threshold, plastic flow is the major component of wall deformation, and that when turgor falls below yield threshold, elastic deformation is dominant. The equations of the model describe growth rate as a function of time in terms of the following properties; plastic flow, elastic deformation, permeability to water, and solute uptake. They are derived from basic equations of feedback interactions between internal osmotic pressure and growth rate, and between wall softening, turgor and growth rate.The model predicts oscillatory growth rate regulation, and phase and amplitude relationships between turgor pressure and growth rate. The simplest model which accounts for all observations is that of biphasic deformation, two modes of wall softening, and a dual feedback system involving osmotic and yield threshold control of growth rate.It should be noted that to predict the time course of turgor pressure, osmotic pressure, yield pressure, and growth rate, two initial conditions and six system parameter values are sufficient. So far only the initial values of growth rate and its derivative can be obtained for Zea mays primary roots. However, values for wall softening and hardening coefficients (including the strain and turgor independent component), plastic extensibility, water permeability and dilution rate coefficients have not been obtained as yet for Zea roots. Values for some of these parameters have been obtained for other roots, coleoptiles, and giant algal cells.Lest the reader despair, it should be pointed out that experimental observations coupled with simulation studies will help establish restricted ranges of values that the system parameters might assume. These can then be compared with known values in the literature and values experimentally obtained in the future.     

Rheological analysis of viscoelastic cell wall changes in maize coleoptiles as affected by auxin and osmotic stress

The effects of auxin and osmotic stress on elongation growth of maize (Zea mays L.) coleoptile segments are accompanied by characteristic changes in the extensibility of the growth-limiting cell walls. At full turgor auxin causes growth by an increase in wall extensibility (wall looseining). Growth can be stopped by an osmotically produced step-down in turgor of 0.45 MPa. Under these conditions auxin causes the accumulation of a potential for future wall extension which is released after restoration of full turgor. Turgor reduction causes a reversible decrease in wall extensibility (wall stiffening) both in the presence and absence of auxin. These changes in vivo are correlated with corresponding changes in the rheological properties of the cell walls in vitro which can be traced back to specific modifications in the shape of the hysteretic stress-strain relationship. The longitudinally load-bearing walls of the coleoptile demonstrate almost perfect viscoelasticity as documented by a nearly closed hysteresis loop. Auxin-mediated wall loosening causes an increase of loop width and thus affects primarily the amount of hysteresis in the isolated wall. In contrast, turgor reduction by osmotic stress reduces loop length and thus affects primarily the amount of viscoelastic wall extensibility. Pretreatment of segments with anoxia and H₂O₂ modify the hysteresis loop in agreement with the conclusion that the wall-stiffening reaction visualized under osmotic stress in vivo is an O₂-dependent process in which O₂ can be substituted by H₂O₂. Cycloheximide specifically inhibits auxin-mediated wall loosening without affecting wall stiffening, and this is mirrored in specific changes of the hysteresis loop. Corroborating a previous in vivo study (Hohl et al. 1995, Physiol. Plant. 94: 491–498) these results show that cell wall stiffening in vivo can also be demonstrated by Theological measurements with the isolated cell wall and that this process can be separated from cell wall loosening by specific changes in the shape of the hysteresis loop.     

Replicase proteins as determinants of phloem-dependent long-distance movement of tobamoviruses in tobacco

Summary The turgor pressure of growing pollen tubes of the lily (Lilium longiflorum Thunb.) has been recorded using a turgor pressure probe. Insertion of the probe's micropipette was routinely accomplished, providing recording periods of 20 to 30 min. Probe insertion did not affect tube growth. The stable turgor values ranged between 0.1 and 0.4 MPa, the mean value being 0.209 ± 0.064 MPa (n=106). A brief increase in turgor, generated by injection of oil through the pressure probe, caused the tube to burst at its tip. Burst pressures ranged between 0.19 and 0.58 MPa, that is, individual lily pollen tubes do not withstand turgor pressure approaching twice their regular turgor pressure. In contrast, parallel experiments using the incipient plasmolysis technique yielded a mean putative turgor pressure of 0.79 MPa either using sucrose (n=24) or mannitol (n=25). Surprisingly, turgor pressure was not significantly correlated with tube growth rate which ranged from zero to 13 m/min. Nor did it correlate with tube length over the tested range of 100 to 1600 m. In addition the influence of the medium's osmolality was surprisingly low: raising the external osmotic pressure from 0.36 to 1.08 MPa, with sucrose or mannitol, only caused mean turgor pressure to decline from 0.27 to 0.18 MPa. We conclude that growing lily pollen regulates its turgor pressure remarkably well despite substantial variation in tube growth rate, tube length, and osmotic milieu.     

Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate

Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.     

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.