Tobacco pollen tubes as cellular models for ion dynamics: improved spatial and temporal resolution of extracellular flux and free cytosolic concentration of calcium and protons using pHluorin and YC3.1 CaMeleon (original) (raw)
Related papers
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.
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
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.
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.
2017
Pollen tubes (PTs) are one of the best characterized plant cell types in many respects. The identification of key players involved in tube growth offers the perspective of an integrative understanding of cell morphogenesis processes. One outstanding feature of PTs is their prominent dependence on ion dynamics to promote and regulate growth. Many reports have identified and characterized membrane transport proteins, such as channels, transporters, and pumps, as well as their regulatory mechanisms, some of which themselves are dependent on ions such as Ca and H. The signaling network that governs growth is based on a strict spatial distribution of signaling molecules, including apical gradients of Ca, H, and reactive oxygen species. A central role for ion homeostasis, and more generally membrane transport systems, is proposed to underlie the spatiotemporal establishment of the signaling network that controls the PT self-organization and morphogenesis. Here, we review the latest progre...
Pollen Tube Growth and the lntracellular Cytosolic Calcium Gradient Oscillate in Phase
Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3to 5-sec intervals showed that the tip-focused [Ca2+Ii 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 tipdirected extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+Ii gradient oscillates in phase with growth, the influx of Ca2+o lags by -11 sec. lon 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.
Ion dynamics and hydrodynamics in the regulation of pollen tube growth
Sexual Plant Reproduction, 2001
A coherent picture of pollen tube growth is beginning to emerge that couples ion dynamics with biochemical, biophysical and cytological processes in ordered and controlled feedback circuits that define the nature of polarized apical growth. It is a paradox, however, that complete understanding of the mechanical forces that drive cell elongation in this system still remains to be fully achieved.
2007
Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3to 5-sec intervals showed that the tip-focused [Ca2+Ii 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 tipdirected extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+Ii gradient oscillates in phase with growth, the influx of Ca2+o lags by -11 sec. lon 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 repre...
Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2 Ј ,7 Ј -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H ϩ ) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 M or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.