Calcium-regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts (original) (raw)
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The essential role of anionic transport in plant cells: the pollen tube as a case study
Journal of Experimental Botany, 2011
Plasma membrane anion transporters play fundamental roles in plant cell biology, especially in stomatal closure and nutrition. Notwithstanding, a lot is still unknown about the specific function of these transporters, their specific localization, or molecular nature. Here the fundamental roles of anionic transport in plant cells are reviewed. Special attention will be paid to them in the control of pollen tube growth. Pollen tubes are extreme examples of cellular polarity as they grow exclusively in their apical extremity. Their unique cell biology has been extensively exploited for fundamental understanding of cellular growth and morphogenesis. Non-invasive methods have demonstrated that tube growth is governed by different ion fluxes, with different properties and distribution. Not much is known about the nature of the membrane transporters responsible for anionic transport and their regulation in the pollen tube. Recent data indicate the importance of chloride (Cl -) transfer across the plasma membrane for pollen germination and pollen tube growth. A general overview is presented of the well-known accumulated data in terms of biophysical and functional characterization, transcriptomics, and genomic description of pollen ionic transport, and the various controversies around the role of anionic fluxes during pollen tube germination, growth, and development. It is concluded that, like all other plant cells so far analysed, pollen tubes depend on anion fluxes for a number of fundamental homeostatic properties.
Identification and Characterization of Stretch-Activated Ion Channels in Pollen Protoplasts
Plant Physiology, 2004
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 ...
Sexual Plant Reproduction, 2008
The presence of both calcium (Ca 2+ ) and proton (H + ) apical gradients is necessary for polarized cell elongation to occur in pollen tubes. So far, most of these studies have been carried out in lily pollen tubes, using chemical probes. Yet, lily is a refractory model for molecular genetics, with no easy protocol available for the construction of stable transgenic lines. Tobacco, however, is well suited for both transformation and cell biology, with sexual organs that are accessible, easy to handle and visualize. Pollen tubes are in an ideal size range for subcellular imaging analyses using modern microscopy techniques. Ion homeostasis in tobacco pollen tubes has not been precisely characterized so far. Here, we characterize the H + and Ca 2+ spatial and temporal patterns in tobacco pollen tubes by the use of two fluorescent genetic probes, pHluorin and the YC3.1 yellow CaMeleon, and direct measurement of extracellular flux by ion-sensitive vibrating probes. A distinct 0.4 pH unit acidic gradient was found to stretch from the tip up to 40 lm into the tube shank. This gradient intensity displayed 1-4 min period oscillations and is reduced in the non-growing phase of an oscillatory cycle. Furthermore, sub-membrane and sub-apical alkaline domains were detected. Extracellular H + fluxes oscillated between 10 and 40 pmol cm -2 s -1 . Fourier and continuous wavelet analyses showed tubes with one or two major oscillatory components in both extra and intracellular H + oscillations. Cytosolic Ca 2+ was imaged by confocal microscopy, showing a V-shaped 40 lm gradient extending from the tip, from 0.2 to 1.0 lM, which oscillates with a 1-4 min period, but with only one major oscillatory component. Extracellular Ca 2+ fluxes oscillate in most pollen tubes, between 2 and 50 pmol cm -2 min -1 and, like in H + , with one or two major oscillatory peaks. A combination of confocal and widefield microscopy showed that H + and Ca 2+ displayed different patterns and shapes inside the cell, sometimes suggesting a structurally complementary role for these 2 second messengers in the growth process. These data suggest that fluxes at the apex of the pollen tube are directly responsible for establishment and maintenance of the gradient.
Control of pollen tube growth: role of ion gradients and fluxes
New Phytologist, 2003
Introduction 540 II. Ion gradients and flux patterns 541 III. Oscillations 544 IV. The need for a Ca 2+ store 547 V. Intracellular targets for Ion activity 549 VI. Extracellular targets for ions: the cell wall 552 VII. Ions in navigation 554 VIII. Role of ions in self-incompatibility 555 IX. The plasma membrane; site of global coordination and control 556 X. A model for pollen tube growth 557 XI. Conclusions 558
Ions play a crucial role in the control of pollen tube growth. In this review we focus on four that seem especially important: calcium (Ca 2+ ), protons (H + ), potassium (K + ), and chloride (Cl -). Ca 2+ in the extracellular medium is essential for growth; it forms a steep intracellular tip-focused gradient, and exhibits a prominent extracellular tip-directed Ca 2+ influx. pH is also essential for growth. H + form an intracellular gradient consisting of a slightly acidic domain at the extreme apex and an alkaline band located along the clear zone. H + also exhibit an apical influx, but in contrast to Ca 2+ show an efflux along the clear zone, in the region occupied by the intracellular alkaline band. K + and anions (possibly Cl -) appear to participate in the growth process, as evidenced by the striking extracellular fluxes that are associated with tube elongation. K + exhibits an apical influx, while an anion displays an apical efflux. An exciting finding has been the discovery that pollen tube growth oscillates in rate, as do all the ionic expressions noted above. While the ionic activities and fluxes show the same period as growth, they usually do not show the same phase. The exploration of phase relationships, using cross-correlation analysis, reveals that most ion expressions lag growth. Thus, intracellular Ca 2+ activity follows growth rate by 1-4 s, whereas extracellular Ca 2+ influx follows growth rate by 12-15 s (130 • ). These observations suggest that Ca 2+ is a follower rather than a leader in growth. Despite the knowledge that has been gained, several aspects of ionic expression and function remain to be determined. Their elucidation will contribute greatly to our overall understanding of the control of pollen tube growth. 48 P.K. Hepler et al.
Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms
Journal of Experimental Botany, 2005
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 Ca 21 , 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 [Ca 21 ] i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,59 dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca 21 . The [Ca 21 ] i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca 21 concentration falls below 150 nM, again a large increase in apical [Ca 21 ] 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 [Ca 21 ] 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.
International Journal of Developmental Biology, 2009
In order to cope with reproduction in a dry environment without any sort of motility, plants have developed a very specialized and unique sexual system. Of special notice, the two sperm cells that will perform the double fertilization typical of higher plants are carried by one of the fastest growing cells in nature, the pollen tube. This tube develops from the vegetative cell of the pollen grain upon germination on the female tissues. While it cannot be considered as a canonical excitable cell, pollen tubes depend for most of their fundamental functional features on a close regulation of ion dynamics, namely in terms of polarization of extracellular fluxes and formation of standing cytosolic free ion gradients, namely of calcium (Ca 2+ ) and protons (H + ). In turn, these imply that plasma membrane transporters are polarized, or polarly regulated, and that internal signaling cascades transduce this spatial information into the basic features of growth and morphogenesis needed for pollen tubes to target correctly the ovules and discharge the sperm cells. Because of the singularity of this organization, and the ease with which pollen tubes can be experimentally handled, recent years have witnessed an accumulation of data at many levels, from basic biophysical and cell biology characterization, to gene assignment and transcriptomic description of pollen development. In this review we aim to organize this information in terms of the basic biophysical features of membrane function and integrate it into conceptual testable hypotheses on how the dynamics of ion regulation may underlie fundamental properties of cell development.
Mercury-sensitive water channels as possible sensors of water potentials in pollen
The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32 ± 0.31 × 10 -3 cm s -1 . Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed.
A Compartmental Model Analysis of Integrative and Self-Regulatory Ion Dynamics in Pollen Tube Growth
PLoS ONE, 2010
Sexual reproduction in higher plants relies upon the polarised growth of pollen tubes. The growth-site at the pollen tube tip responds to signalling processes to successfully steer the tube to an ovule. Essential features of pollen tube growth are polarisation of ion fluxes, intracellular ion gradients, and oscillating dynamics. However, little is known about how these features are generated and how they are causally related. We propose that ion dynamics in biological systems should be studied in an integrative and self-regulatory way. Here we have developed a two-compartment model by integrating major ion transporters at both the tip and shank of pollen tubes. We demonstrate that the physiological features of polarised growth in the pollen tube can be explained by the localised distribution of transporters at the tip and shank. Model analysis reveals that the tip and shank compartments integrate into a self-regulatory dynamic system, however the oscillatory dynamics at the tip do not play an important role in maintaining ion gradients. Furthermore, an electric current travelling along the pollen tube contributes to the regulation of ion dynamics. Two candidate mechanisms for growth-induced oscillations are proposed: the transition of tip membrane into shank membrane, and growth-induced changes in kinetic parameters of ion transporters. The methodology and principles developed here are applicable to the study of ion dynamics and their interactions with other functional modules in any plant cellular system.