Calcium ionophore A 23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum

The effects of the calcium inonophore A 23187 on growing pollen tubes of Lilium longiflorum Thunb. cv. Ace were investigated with the light and electron microscope. Tip growth is slowed down and stopped within 20 min after application of 5x10^-5 M ionophore A 23187. The main effects are the disappearance of the clear zone at the pollen tube tip and a thickening of the cell wall at the tip and at the pollen tube flanks. This effect on cell wall formation is confirmed under the electron microscope: The vesicular zone in treated pollen tubes is reduced, numerous vesicular contents are irregularly integrated in the pollen tube wall not only in the tip, but over a long distance of the pollen tube wall. In addition, effects on mitochondria and dictyosomes are observed. These results are interpreted as a disorientation of the Ca²⁺-based orientation mechanism of exocytosis after equilibration of the Ca²⁺-gradient     

Calcium accumulations within the growing tips of pollen tubes

Pollen of L. longiflorum was grown in 45Ca-labeled medium and washed with nonradioactive medium. Whole, labeled pollen was then frozen and autoradiographed at -78 degrees C. The autoradiographs show striking accumulations of 45Ca in the growing tips of the pollen tubes. This result is obtained when the pollen is labeled for times as short as 1 min, or as long as 5 h. In most cases, the tip concentration is about two to four times greater than that in the bulk of the pollen tube, and extends for a length of about 20 mum. In autoradiographs of tubes longer than 1 mm, a small fraction of cells show a distinctly larger 45Ca accumulation, the tip containing more than 100 times that in the rest of the cell. The 1- to 5-h labeling experiments show that calcium is relatively concentrated within the cytoplasm of the growing tip. The 1- to 3-min labeling experiments suggest that calcium may enter the tip faster than it enters other regions. These patterns of calcium accumulation and flux may be related to the localized secretion of vesicles at the grow;ng tip.     

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.     

Pectin methylesterase, a regulator of pollen tube growth

The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.     

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.     

Targeting and Regulation of Cell Wall Synthesis During Tip Growth in Plants

Root hairs and pollen tubes are formed through tip growth, a process requiring synthesis of new cell wall material and the precise targeting and integration of these components to a selected apical plasma membrane domain in the growing tips of these cells. Presence of a tip-focused calcium gradient, control of actin cytoskeleton dynamics, and formation and targeting of secretory vesicles are essential to tip growth. Similar to cells undergoing diffuse growth, cellulose, hemicelluloses, and pectins are also deposited in the growing apices of tip-growing cells. However, differences in the manner in which these cell wall components are targeted and inserted in the expanding portion of tip-growing cells is reflected by the identification of elements of the plant cell wall synthesis machinery which have been shown to play unique roles in tip-growing cells. In this review, we summarize our current understanding of the tip growth process, with a particular focus on the subcellular targeting of newly synthesized cell wall components, and their roles in this form of plant cell expansion.     

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.     

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.     

A model for the mechanism of tip extension in pollen tubes

Three main mechanisms are proposed to account for the tip growth of pollen tubes. (1) The tip region is supported against the internal osmotic pressure of the cell by a fibrillar network, composed mainly of microfilaments, that is stabilized by calcium ions. Tip extension is promoted by a lowering of the local cytoplasmic calcium ion concentration, through uptake by the mitochondria and/or endoplasmic reticulum, which leads to a weakening of the fibrillar network. (2) Vesicles, derived from dictyosomes in the main body of the tube, fuse with the apical plasma membrane, providing new membrane and further carbohydrate for the wall. The rate of fusion is proportional to the rate of diffusion of calcium ion across the plasma membrane at the tip. (3) The callose lining present in the pollen tube wall, except at the tip, renders the wall impermeable and restricts entry of calcium ions to the apical plasma membrane. This restriction limits the rate of vesicle fusion, and tube growth, to the tip.This model is discussed in the light of previous observations on the growth and structure of pollen tubes under normal and experimental conditions.     

External application of gametophyte-specific ZmPMEI1 induces pollen tube burst in maize

Regulated demethylesterification of homogalacturonan, a major component of plant cell walls, by the activity of pectin methylesterases (PMEs), plays a critical role for cell wall stability and integrity. Especially fast growing plant cells such as pollen tubes secrete large amounts of PMEs toward their apoplasmic space. PME activity itself is tightly regulated by its inhibitor named as PME inhibitor and is thought to be required especially at the very pollen tube tip. We report here the identification and functional characterization of PMEI1 from maize (ZmPMEI1). We could show that the protein acts as an inhibitor of PME but not of invertases and found that its gene is strongly expressed in both gametophytes (pollen grain and embryo sac). Promoter reporter studies showed gene activity also during pollen tube growth toward and inside the transmitting tract. All embryo sac cells except the central cell displayed strong expression. Weaker signals were visible at sporophytic cells of the micropylar region. ZmPMEI1–EGFP fusion protein is transported within granules inside the tube and accumulates at the pollen tube tip as well as at sites where pollen tubes bend and/or change growth directions. The female gametophyte putatively influences pollen tube growth behavior by exposing it to ZmPMEI1. We therefore simulated this effect by applying recombinant protein at different concentrations on growing pollen tubes. ZmPMEI1 did not arrest growth, but destabilized the cell wall inducing burst. Compared with female gametophyte secreted defensin-like ZmES4, which induces burst at the very pollen tube tip, ZmPMEI1-induced burst occurs at the subapical region. These findings indicate that ZmPMEI1 secreted by the embryo sac likely destabilizes the pollen tube wall during perception and together with other proteins such as ZmES4 leads to burst and thus sperm release.     

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.     

The block of intracellular calcium release affects the pollen tube development of Picea wilsonii by changing the deposition of cell wall components

Two potent drugs, neomycin and TMB-8, which can block intracellular calcium release, were used to investigate their influence on pollen tube growth and cell wall deposition in Picea wilsonii. Apart from inhibiting pollen germination and pollen tube growth, the two drugs largely influenced tube morphology. The drugs not only obviously disturbed the generation and maintenance of the tip-localized Ca(2+) gradient but also led to a heavy accumulation of callose at the tip region of P. wilsonii pollen tubes. Fourier transform infrared (FTIR) spectroscopy analysis showed that the deposition of cell wall components, such as carboxylic acid, pectins, and other polysaccharides, in pollen tubes was changed by the two drugs. The results obtained from immunolabeling with different pectin and arabinogalactan protein antibodies agreed well with the FTIR results and further demonstrated that the generation and maintenance of the gradient of cross-linked pectins, as well as the proportional distribution of arabinogalactan proteins in tube cell walls, are essential for pollen tube growth. These results strongly suggest that intracellular calcium release mediates the processes of pollen germination and pollen tube growth in P. wilsonii and its inhibition can lead to abnormal growth by disturbing the deposition of cell wall components in pollen tube tips.     

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.     

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.     

Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins

In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.     

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     

The local cytomechanical properties of growing pollen tubes correspond to the axial distribution of structural cellular elements

Morphological studies of pollen tubes have shown that the configuration of structural cellular elements differs between the growing apex and the distal part of the cell. This polarized cellular organization reflects the highly anisotropic growth behavior of this tip growing cell. Accordingly, it has frequently been postulated that physical properties of pollen tubes such as cell wall plasticity should show anisotropic distribution, but no experimental evidence for this has been published hitherto. Using micro-indentation techniques, we quantify pollen tube resistance to lateral deformation forces and analyze its visco-elasticity as a function of distance from the growing apex. Our studies reveal that cellular stiffness is significantly higher at the distal portion of the cell. This part of the cell is also completely elastic, whereas the apex shows a visco-elastic component upon deformation. To relate these data to the architecture of the particular pollen tube investigated in this study, Papaver rhoeas, we analyzed the distribution of cell wall components such as pectin, callose, and cellulose as well as the actin cytoskeleton in this cell using fluorescence label. Our data revealed that, in particular, the degree of pectin methyl esterification and the configuration of the actin cytoskeleton correlate well with the distribution of the physical properties on the longitudinal axis of the cell. This suggests a role for these cellular components in the determination of the cytomechanics of pollen tubes.     

Expression of a polygalacturonase enzyme in germinating pollen of <Emphasis Type="Italic">Brassica napus</Emphasis>

Penetration of pollen tubes through stigmatic tissues in Brassica napus L. may involve the release of cell wall modifying enzymes from the pollen tube tip. We examined the expression of a pectin-degrading polygalacturonase (PG) enzyme in unpollinated and early and late pollinated stigmas via immunoblotting and immuno-light microscopy using a PG polyclonal antibody. Immunoblotting analysis indicated that PG enzyme was present at low levels in unpollinated stigmas and at high levels in pollinated stigmas. The level of PG did not detectably increase between early and late pollinated stigmas. Immuno-light microscopy demonstrated that PG enzyme was present in ungerminated pollen grains, stigmatic papillae and in the tip of pollen tubes growing into the papillar wall. This latter evidence suggests that PG enzyme may play an important role in papillar cell wall penetration during pollination although other interpretations of the role of pollen PG should not be discounted. Received: 9 November 2000 / Accepted: 7 December 2000     

Quin-2 fluorescence in lily pollen tubes: Distribution of free cytoplasmic calcium

Summary Using the fluorescent calcium probe Quin-2, we could demonstrate a tip-to-base gradient of free calcium within growing pollen tubes ofLilium longiflorum. This result shows that it is possible to visualize calcium ions using Quin-2 in plant cells which are surrounded by a cell wall and that the calcium gradients demonstrated by CTC fluorescence (indicating membrane-bound calcium) and PIXE (showing total calcium) is paralleled by a gradient of free calcium in pollen tubes.     

Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Despite the vital role that flavonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the flavonol-deficient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of flavonols in this process, wild-type and flavonol-deficient pollen tubes were subjected to cytological and ultrastructural analyses and screened for differences. The results showed that before disruption of the flavonol-deficient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural differences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any significant difference. However, for the first time, obvious morphological differences were observed in the wall of the flavonol-deficient pollen tubes. We conclude that flavonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins. Received: 23 February 1998 / Accepted: 13 August 1998