Essay 14.1 Calcium Gradients and Oscillations in the Growing Pollen Tube
Peter Hepler and Alenka Lovy-Wheeler, University of Massachusetts, Amherst, Massachusetts, USA
Pollen tube growth delivers the two sperm cells to the embryo sac, and thus is essential for sexual reproduction in higher plants [see Malhó, R. (Ed.) (2006) Plant Cell Monographs (Vol. 3)]. The process has important and unique features: It is extremely fast, reaching rates of up to 1 cm per hour in corn; it is highly polarized, with growth being confined to the tip ("tip-growth"); and it possesses a guidance mechanism that determines the direction of growth.
Several aspects of tip-growth have been deciphered; for example, the Golgi apparatus produces vesicles containing cell wall precursor material, and through cytoplasmic streaming these vesicles flow to the apex of the pollen tube, where they fuse with the plasma membrane and secrete their contents into the cell wall. It is unclear how these events are orchestrated to achieve the high degree of polarity evident during pollen tube growth, although recent work indicates that different ions, including notably calcium and protons, play an important role (Hepler et al. 2006).
It has been known for many years that both calcium ions and protons are essential for growth (Hepler et al. 2006). Calcium ions must be present in the growth medium at a concentration above 10 μM, but below 10 mM. A high proton concentration or low pH (e.g., 4.5 to 6.5) also facilitates both pollen germination and tube growth. Considerable work on the intracellular and extracellular expression on these ions has revealed unique features that are crucial for the regulation of pollen tube growth (Holdaway-Clarke and Hepler 2003). For example, ion imaging reveals that the growing pollen tube possesses a "tip-focused" gradient of free calcium at its extreme apex (Figure 1, top images), in which the concentration extends from 3000 nM or higher (e.g., 10,000 nM; Messerli et al. 2000) at the plasma membrane to a basal level of 200 nM or less within 20 μm from the tip.
Figure 1 Growing pollen tubes exhibit a pronounced "tip-focused" calcium gradient (top, left and right). The gradient oscillates between high (left) and low (right) levels. The pollen tube was injected with the calcium sensitive dye, fura-2-dextran, and photographed using ratio-metric ion imaging. Growing pollen tubes also exhibit a pH gradient in which the tip is slightly acidic (bottom, left and right). Back from the tip is a prominent alkaline band. This band oscillates between high (left) and low (right) pH. The pollen tube was injected with the pH sensitive dye, BCECF-dextran, and subjected to ratio-metric ion imaging. Bar = 10 μm. (From Hepler et al. 2006.)
Protons or pH also exhibit marked gradients; studies on oscillating lily pollen tubes show that the apex contains an acidic region with an alkaline domain or band towards the base of the clear zone (Feijó et al. 2001; Lovy-Wheeler et al. 2006). The gradient is large and extends from pH 6.8 at the tube apex to pH 7.5 in the alkaline band, which is 10–30 μm from the tip (Figure 1, bottom images).
Studies on the extracellular status of calcium and protons have relied on the ion-selective vibrating electrode, a device that rapidly measures the local ion concentration in the medium at two different points—one close to the pollen tube and another 10 μm away. With knowledge of these concentrations and their difference, it is possible, using Fick′s law, to calculate the flux or movement of calcium ions or protons relative to the growing pollen tube (Holdaway-Clarke and Hepler 2003).
Results from studies using the extracellular ion-selective electrodes show that growing pollen tubes exhibit a tip-directed influx of calcium in which the point of ion entry coincides with the location of the intracellular gradient and also with the position of maximal cell elongation (Hepler et al. 2006). Extracellular protons also show an influx at the apex of the growing pollen tube. However, in contrast to calcium ions, protons display a marked efflux along the side of the tube, close to the base of the clear zone, in a position that corresponds to the location of the intracellular alkaline band (Feijó et al. 2001). Extracellular protons thus constitute a current loop, which may be driven by a proton ATPase located on the plasma membrane.
The results above become even more interesting when it is realized that the magnitude of the intracellular calcium and proton gradients and the extracellular fluxes of these ions, oscillate (Messerli et al. 2000; Feijó et al. 2001; Holdaway-Clarke and Hepler 2003). Moreover, these oscillations display a period (i.e., 15–50 s) that is identical to the oscillation in the rate of pollen tube growth. For intracellular calcium, the concentration at the tip will periodically change (oscillate) from 3–4 μM to less than 1 μM (Figure 2), while the extracellular calcium influx varies between 0 and 17 pmol cm-2 sec-1 (Feijó et al. 2001; Holdaway-Clarke and Hepler 2003) (Figure 3).
Figure 2 Oscillation of intracellular calcium plotted against growth rate. Note that both oscillations show the same period. Cross-correlation analysis reveals that the calcium peak follows the peak on growth rate by 1–4 sec. (From Cárdenas and Hepler, unpublished results.)
Figure 3 Oscillation of extracellular calcium plotted against growth rate. The extracellular calcium peak follows the growth rate peak by 11–15 sec. and is thus out of phase with intracellular calcium. [From Holdaway-Clarke et al. (1997) Plant Cell 9: 1999–2010.]
Determination of the phase relationship between intracellular calcium and growth rate, using cross-correlation analysis, reveals that the high point of the gradient actually lags behind the growth rate peak by 1–4 sec (Messerli et al. 2000). Changes in intracellular calcium thus follow growth and presumably cannot serve as a prime regulator because it is the preceding growth event that defines the changes in calcium, rather than calcium defining growth. A further puzzling issue is the result showing that the oscillation of extracellular calcium influx is even further delayed (11–15 sec), being out of phase with growth and with the oscillation of the intracellular gradient (compare Figures 2 and 3).
The delay indicates that calcium ions do not immediately enter the cytoplasmic pool; if they did, they would be detected by the intracellular calcium indicator. A possible explanation is that the extracellular influx of calcium is governed by changes in the ion-binding properties within the cell wall rather than movement across the plasma membrane (Holdaway-Clarke and Hepler 2003). In pollen tubes, the apical cell wall is composed largely of pectin, which is secreted in its methyl ester form. De-esterification of the methyl groups by pectin methyl esterase yields carboxyl residues, which bind calcium and form calcium-pectate cross-bridges. Thus, calcium binding to the carboxyl residues may account for the bulk of the observed extracellular current.
Protons or pH changes also exhibit pronounced oscillations during pollen tube growth (Feijó et al. 2001; Lovy-Wheeler et al. 2006) (Figure 4). Recent studies reveal that the pH in the alkaline band increases 11 sec before the next growth cycle, whereas a pH decrease in the acidic tip follows the previous growth cycle by 8 sec (Lovy-Wheeler et al. 2006). As shown in Figure 4, the pH rises quickly and reaches a plateau before the marked increase in growth. However, following the peak in growth rate there is a sharp decrease in the pH (Messerli and Robinson 1998; Lovy-Wheeler et al. 2006). These recent findings are particularly exciting because they provide the first evidence of an ionic change—namely, the increase in pH in the alkaline band, that precedes growth. As a leading event, the rise in pH, which is presumably controlled by a proton ATPase on the apical plasma membrane, emerges as a potential prime regulator of pollen tube growth.
Figure 4 Oscillation in pH plotted against growth rate. The alkaline band rises 11 sec. in anticipation of the increase in growth rate. Following the peak in growth by 8 sec, there is an influx of protons, which is indicated by a sharp decline in pH. (From Lovy-Wheeler et al. 2006.)
Progress is also being made on the mechanisms by which calcium and protons enter and exit the pollen tube. It has seemed plausible that the process of cell elongation would deform the apical plasma membrane and open stretch- activated channels, which would allow entry of calcium ions. Recently such a channel has been detected in the apex of lily pollen tubes (Dutta and Robinson 2004). Given that proton influx shows a similar phase delay from growth as calcium, it is possible that these ions enter the same, or similar channel. Work in progress is drawing attention to other channels that could regulate ion influx, including notably a cyclic nucleotide-gated channel (Sze et al. 2006).
The efflux and/or accumulation of calcium and protons requires energy because these ions are expelled against a substantial gradient. We have already referred to a proton ATPase on the plasma membrane, but proton pumps driven by ATP or pyrophosphate—which are associated with internal membrane organelles, such as the ER and vacuoles—may also contribute to pollen development and tube growth (Sze et al. 2006). Calcium ions are also either expelled to the outside or sequestered into organelles, such as the ER, mitochondria, and vacuoles. Recent work provides evidence for an autoinhibited Ca2+-ATPase that is located on the plasma membrane; furthermore, mutant studies reveal that this enzyme is essential for normal pollen tube growth (Sze et al. 2006).
Figure 5 Lily pollen tube exhibits a prominent cortical actin fringe in the apical domain (a). The fringe can start 1–5 μm behind the apex and extend for an additional 5–10 μm. In the shank of the tube, the actin microfilaments are evenly dispersed throughout the thickness of the tube. This pollen tube was prepared by rapid-freeze fixation and freeze substitution. The pollen tube was then rehydrated and probed with anti-actin antibodies, and imaged by confocal laser scanning microscopy (b). Lily pollen tube prepared as above but stained with an antibody to lily ADF (actin depolymerizing factor). Note that ADF colocalizes with the cortical actin fringe. Bar = 10 μm. (From Lovy-Wheeler et al. 2005, 2006.)
Calcium ions and protons clearly play a major role in pollen tube growth, but what are they doing? A considerable amount of work makes it evident that these ions—especially calcium—regulate cytoplasmic streaming, facilitate membrane trafficking and the process of secretion, control cell wall assembly, participate in self-incompatibility, and influence tube guidance (Malhó 2006). Of the several potential targets for calcium and protons, here we briefly focus on the cytoskeleton, which appears to participate in many of the processes mentioned above. Firstly, it is important to note, based on recent work, that the actin cytoskeleton displays a unique structure in the pollen tube apex (Lovy-Wheeler et al. 2005). In lily pollen tubes, this consists of a cortical fringe of F-actin that starts 1–5 μm behind the tip and extends basally for another 5–10 μm (see Figure 5a). It is additionally important to note that this cortical actin fringe occupies the same domain as the alkaline band. Distal to the fringe in the pollen tube shank, the actin filaments are longitudinally oriented but dispersed evenly throughout the thickness of the tube (see Figure 5a).
Calcium ions and protons can control the structure and activity of the actin cytoskeleton through the modulation of different actin-binding proteins. For example, at basal levels of calcium villin, an actin-binding protein found in pollen tubes will bundle actin microfilaments. However, at elevated concentrations, such as are found in the tip-focused gradient at the apex, calcium together with calmodulin causes villin to unbundle actin. The closely related protein gelsolin, in the presence of elevated calcium, will in addition fragment existing filaments, explaining why there is very little F-actin in the extreme apex of the tube. Profilin, which binds G-actin, will retard polymerization in high levels of calcium. Finally, myosin, the motor protein for streaming, is inhibited in high levels of calcium explaining why directed motion is not observed in the extreme apex of the pollen tube.
Protons, or the lack thereof, can also affect the actin cytoskeleton. The high pH in the alkaline band would be expected to activate ADF/cofilin—another actin-binding protein that occurs commonly in pollen tubes—and stimulate the remodeling of the existing actin cytoskeleton. Recent work reveals that ADF colocalizes with the cortical-actin fringe and thus occurs specifically in a region where the pH of the alkaline band oscillates (Lovy-Wheeler et al. 2006) (see Figure 5b). When the pH rises above 7.2, ADF will facilitate the fragmentation of existing actin microfilaments, and stimulate new polymerization. Thus both the apical calcium gradient and the alkaline band could participate in the process of actin turnover and remodeling, and thereby affect the structure and organization of elements thought to be important for pollen tube cell polarity.
Calcium ions and protons may also have a pronounced effect on membrane trafficking. It is a paradigm in plant and animal cells that elevated calcium levels facilitate secretion. Thus given the elevated level of calcium in the tip-focused gradient, it can be appreciated that secretion of vesicles that contribute to the growth of the pollen tube will be locally enhanced. Indeed, it is reasonable to imagine that the intracellular calcium gradient, by directing the location of secretion, defines the direction of pollen tube growth.
Finally, we draw attention to the effect of calcium and protons on the structure and yielding properties of the cell wall. Increased levels of calcium will stabilize and strengthen the wall through the formation of pectate bridges. On the other hand, increased levels of protons will weaken the wall. Here, the lower pH can either inhibit certain isoforms of pectin methylesterase and thus reduce the number of carboxyl groups, or it can enhance pectin hydrolyases and degrade pectin cross-links in the cell wall.
In summary, calcium ions and protons play crucial roles in the control of pollen tube growth (Figure 6). Intracellular calcium and protons could facilitate vesicle fusion and thus control the amount and location of secretion. They may also control the obligatory remodeling of the actin cytoskeleton that is essential for cell elongation. The strict spatial location of the intracellular ion activity is important in both instances, because it will dictate where vesicle fusion or actin polymerization can occur and thus determine the overall growth polarity of the pollen tube. In the extracellular space, calcium and protons are also major contributors to the structure and yielding properties of the wall. The continued elucidation of the interaction of these ion-dependent processes may bring us closer to an understanding of the central regulator or pacemaker that controls oscillatory pollen tube growth.
Figure 6 A summary diagram showing the high calcium and pH domains together with other structural features of the growing pollen tube. The diagram emphasizes the many processes that calcium ions and protons can regulate. (From Hepler et al. 2006.)
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