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Pollen grains are the carriers of male gametes in flowering plants. After landing on the stigma, they germinate and develop a cytoplasmic extension – the pollen tube – that grows up to the ovule, where the sperm cells are released. This event is a key point in plant sexual reproduction. The pollen tubes are an extreme example of cellular polarity and exclusive apical growth, and have been extensively used for basic cell biology studies involving cellular growth and morphogenesis (Boavida et al., 2005; Michard et al., 2009).
The use of noninvasive methods has demonstrated a close link between intracellular ion fluctuations and ionic fluxes across the plasma membrane, and the cellular phenomena that occur during the formation and elongation of the pollen tube (Mascarenhas, 1993; Feijóet al., 1995, 2001, 2004; Cheung, 1996; Hepler et al., 2001). For example, oscillatory H+ gradients have been shown to exist and to be intimately associated with polarized pollen tube growth. A constitutive alkaline band in the clear zone and a growth-dependent acidic tip were later shown to be based molecularly on the differential distribution of a P-H+-ATPase (Feijóet al., 1999; Certal et al., 2008).
Several groups have detected the presence of [Ca2+] tip focused gradients (e.g. Holdaway-Clarke et al., 1997; Feijóet al., 2001; Holdaway-Clarke & Hepler, 2003; Michard et al., 2009). Additionally, ion-specific self-referencing electrodes have been used to study ionic fluxes during pollen tube growth, demonstrating an uneven distribution of the different fluxes. Non-oscillatory ionic fluxes of typical permeable ions in cells, such as H+, Ca2+, K+ and Cl−, have been observed from the grain and along the tube shank, while the tip zone displays distinctive oscillatory fluxes for the same ions, and usually in the reverse direction to that on the shank (Feijóet al., 1999; Moreno et al., 2007). These oscillatory fluxes have been shown to be essential to tube growth and show a similar period of oscillation to that of growth, although with distinct phase delays (Holdaway-Clarke et al., 1997; Messerli et al., 1999; Feijóet al., 2001; Zonia et al., 2002; Holdaway-Clarke & Hepler, 2003). Early studies by Weisenseel & Jaffe (1976) demonstrated that pollen tube germination and growth have an absolute requirement for extracellular K+, Ca2+, a trace of boron, and a somewhat acidic pH. Conversely, substitution experiments with nonpermeable anions (cellobinate or methylsulfonate) showed that inorganic anions were not required for the growth of the pollen tubes; but it should be noted that these authors never estimated the chloride concentration in the germination medium, and therefore were not able to determine the contributions of reagent contamination or pollen release. Nevertheless, there are several indications of the presence of anionic fluxes from the pollen tube. Trofimova & Molotkovskii (1993) reported that the activation of Nicotiana tabacum pollen grains was accompanied by Cl− efflux. Zonia et al. (2001, 2002) have confirmed the occurrence of large Cl− fluxes from the tip of the growing pollen tube of Lilium longiflorum and of Nicotiana tabacum, leading to the accumulation of anions in the extracellular medium. In particular, Cl− fluxes exceeded by a factor of 10 other previously reported H+ and Ca2+ fluxes in the same species. Furthermore, substitution of nitrate by chloride in the germination medium was the only treatment that induced a preference of growth reorientation under an electric field towards the cathode (Malhóet al., 1992). Recent studies by Breygina et al. (2009a,b), measuring chloride concentration and plasma membrane voltage polarization, showed that cytoplasmic Cl− is involved in the polarization of the lily pollen tube during germination. This group showed that the anion channel inhibitor 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) inhibited Cl− release from the grain (during hydration) and from the tube tip, and stopped cell growth. These effects were also achieved by the extracellular addition of high [Cl−], possibly by equilibrating the intracellular [Cl−]. This study also demonstrated an NPPB-induced depolarization of the membrane, with additional disruption of the functional compartmentalization of the cytoplasm necessary for polar growth.
Doubts about the nature of the Cl− currents measured by Zonia et al. (2002) were raised by Messerli et al. (2004), who suggested that the ion-selective Cl− probes are sensitive to the concentration of buffer and indirectly detect changes in H+ gradients in the bathing solution, and therefore the Cl− fluxes measured were contaminated by H+ fluxes. It should be noted, however, that Messerli et al. (2004)did not reproduce the exact measurement conditions used by Zonia et al. (2002), and did not take fully into account the pharmacological evidence. In addition, transcriptomic studies have demonstrated a number of anion/chloride membrane transporters and channels to be specifically and highly expressed in the pollen of Arabidopsis (Pina et al., 2005). The promoter of a cation-chloride co-transporter (CCC) was also found to be highly expressed in pollen (Colmenero-Flores et al., 2007). The contradictory interpretations of chloride fluxes in pollen call for a direct analysis of plasma membrane chloride channel activity. Arguments have also been made elsewhere that the issues of the existence and relevance of chloride currents in pollen tubes have not yet been resolved (Hepler et al., 2006; Kunkel et al., 2006; Moreno et al., 2007).
There have been great efforts to identify the putative channels underlying the detected macroscopic fluxes in pollen. Most of the information gathered relates to the identification of passive K+ currents with different characteristics by means of patch-clamp techniques. Three types of K+ channel in lily have been reported by Obermeyer & Kolb (1993) and suggested to be involved in inward influx during pollen tube growth. Two outward channels have also been reported (Obermeyer & Kolb, 1993; Obermeyer & Blatt, 1995). Griessner & Obermeyer (2003) detected, for the first time, both inward and outward K+ currents in the plasma membrane of protoplasts from L. longiflorum pollen tubes. The inward currents showed different activation kinetics from, and higher current density than, those detected in the grain. K+ channels have also been found in pollen protoplasts from Arabidopsis (Fan et al., 2001) and Brassica chinensis, which are distinctly regulated by pH and/or Ca2+ (Fan et al., 1999, 2003; Fan & Wu, 2000). Mouline et al. (2002) cloned, for the first time, a pollen-specific K+ channel of the Shaker family (SPIK) from Arabidopsis and showed that disruption of the SPIK coding sequence strongly affected inwardly rectifying K+ channel activity in the pollen plasma membrane, resulting in impaired pollen tube growth. Dutta & Robinson (2004) detected K+ and Ca2+ single-channel currents from outside-out patches from L. longiflorum pollen grain and tube tip protoplasts, which were attributed to putative stretch-activated channels. A hyperpolarization-activated inward Ca2+ channel regulated by actin filaments was identified in Arabidopsis (Wang et al., 2004).
Here we used the patch-clamp technique to investigate Cl− currents across the plasma membrane of pollen protoplasts from L. longiflorum. In addition, we studied the possible regulation of the currents by [Ca2+]in, a cation known to form a constitutive elevated domain at the tip which is essential for directed apical growth (Pierson et al., 1994, 1996; Holdaway-Clarke et al., 1997; Messerli & Robinson, 1997). The whole-cell currents can be split into three anion conductances which cross the membrane of the pollen protoplasts through putative anionic channels and which show distinct regulation by [Ca2+]in. Outside-out isolated patches displayed isolated single-channel anionic currents with at least three conductive states, which could underlie the macroscopic whole-cell currents. The currents measured may account for the extracellular fluxes measured by Zonia et al. (2002) and thus their possible physiological role is discussed.
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By employing the patch-clamp technique in the whole-cell configuration, we identified three distinct anionic current populations regulated by [Ca2+]in (ICl1, ICl2 and ICl3) sharing strong outward rectification. Throughout this work we have treated ICl1, ICl2 and ICl3 as individual currents. Each of these currents comprises instantaneous and slow activation components (Iinst, I1 and I2) undergoing similar magnitudes of rundown and inhibition. Similarly, Marten et al. (1999) detected whole-cell K+ currents resulting from the heterologous expression of AKT3 (Arabidopsis Potassium Transporter 3, a potassium channel from vascular tissue of Arabidopsis) in Xenopus oocytes which presented instantaneous and time-dependent components. These authors also described similar inhibition profiles and rundown behaviour for the two current components through the potassium channel. In addition, the heterologous expression of the Ca2+-activated channel mTMEM16A in HEK293 cells produced whole-cell currents with a similar profile to those detected here, with faster and slower activation components (Schroeder et al., 2008). Under control conditions (P1/B1), ICl1 was identified as the current lost by rundown, possibly being modulated by an unknown intracellular regulator that was diluted in the pipette solution replacing the intracellular ionic contents. ICl3 was identified as the current that was inhibited by NPPB, and ICl2 was the current that remained after the loss of ICl1 and that was insensitive to NPPB. We do not exclude the possibility that ICl1 could also be inhibited by NPPB. Nevertheless, the attempts to seal in the presence of NPPB proved unsuccessful. All I/V curves showed Vrev close to the expected ECl− and the positive currents diminished with low [Cl−]out, with a corresponding shift of Vrev towards positive potentials, strong indications that this anion is the major contributor to these currents. Tail analysis confirmed the anionic nature of the currents, with the I/V curves being linear, reversing at the equilibrium potential expected for Cl− and NO3−, undergoing rundown and being partially inhibited by NPPB.
Nevertheless, the three current populations displayed distinctive features, with ICl1 showing features that were clearly distinct from those of the other two. ICl1 increased with higher [Ca2+]in, displaying maximum currents in the presence of 0.5 mM [Ca2+]in, while ICl2 and ICl3 had higher values for μM [Ca2+]in. As the time constants that characterized the rise of the currents did not vary with the tested [Ca2+]in, it is likely that Ca2+ regulates the number of open channels contributing to the current at a given moment. The G/V curve for ICl1 was different from those of ICl2 and ICl3, with different potential values for the half-maximum chord conductance (Vh) and the slope factor (Vs). The currents measured with symmetrical NO3− showed a shift of the midpoint potential (Vh) by c. +30 mV, also indicating a different sensitivity of the channels to the potential in the presence of this anion. The average inhibition caused by 500 μM NPPB for ICl3 (measured with nM [Ca2+]in; +160 mV) was significantly different from the inhibition values obtained for the test groups (measured with μM and mM [Ca2+]in).
To control the [Ca2+]in tested, we used a very low [Ca2+]out (3 mM) and an additional general blocker for Ca2+ currents (Gd3+; Wang et al., 2004) to prevent the entrance of Ca2+ into the cell. The presence of Gd3+ also facilitated giga-seal formation (Dunina-Barkovskaya et al., 2004). Under these conditions, the success of sealing and the maintenance of stable giga-seals was low (10%), but once a high resistance seal was established it remained stable for several hours. While not physiological, in this study we used a broad range of potential values for studying the currents, a common procedure in this kind of patch-clamp approaches. The extreme positive values were necessary to attain linearity of the currents with the applied potentials. These values could then be used for the determination of the conductance ratios and the respective Boltzmann parameters that were used for comparison purposes between experimental groups. Although we had no indications of seal destabilization in the transition to whole cell, because of the strong outward rectification observed, we had to consider the possibility of the negative currents being attributable to the passage of ions directly between the pipette and the bathing solutions. Attempts to subtract the possible leak current led to an overestimation of the current values, which became positive for negative potentials. Additional experimental evidence showed that the negative currents measured were certainly passing through channels in the membrane: they underwent rundown and were partially inhibited by NPPB, just as observed for the positive currents. Therefore, the anionic channels described here displayed the ability to conduct ions both inwardly and outwardly, according to the anionic electrochemical gradient found under natural conditions.
We consistently observed that the currents suffered rundown over time (c. 90 min). Binder et al. (2003) observed a similar behaviour over time while studying the Cl− currents in protoplasts from the marine alga Valonia utricularis and attributed it to a retarded equilibration of the cytosol with the pipette solution, as indicated by alterations of the Vrev of the detected currents until it stabilized near the predicted ECl−. In this study, we observed no drifts of the reversal potential of the measured currents from ECl−, which may indicate that the equilibration is faster in these protoplasts. This also applied to the currents measured with symmetrical NO3−. Alternatively, the natural intracellular anion concentration may be similar to those in the pipette solution. We observed that the currents from occasional outside-out patches had the same characteristics as, although smaller amplitudes than, those measured from full protoplasts. A previous study by Dutta & Robinson (2004) showed no Cl− currents in outside-out patches from lily pollen protoplasts, although the same group could measure unitary current events attributable to K+ and Ca2+. As we were able to measure whole-cell currents of very small amplitude in outside-out patches, it is possible that these authors may have misinterpreted the macroscopic even current levels with the absence of channel activity (Fig. 8a). Although uncommon, macroscopic currents have been observed from outside-out patches (e.g. Whitehead et al., 1998; Artigas & Gadsby, 2002). Here, we obtained values for whole-cell currents measured from protoplasts of between 1.3 and 15 nA, in response to a voltage command of +160 mV. The currents measured under similar conditions from outside-out patches were smaller, as expected, although in some cases they reached c. 1 nA. Another experimental difference that could explain the absence of Cl− channels was the use of extremely high [Ca2+]out (40 mM) by Dutta & Robinson (2004). There is evidence of inward K+ currents in Arabidopsis pollen grain protoplasts regulated by [Ca2+]out (Fan et al., 2001).
Whole-cell data do not allow us to determine if these currents cross the membranes of these cells through one or more than one type of channel. Additionally, each of these three isolated populations may represent different channels sharing similar conductance and/or pharmacological characteristics. The identification of single-channel events from isolated outside-out patches provided some hints about the nature of the outward rectifying whole-cell currents. This experimental configuration has been successfully used to identify unitary currents through plant channels (Hedrich & Jeromin, 1992; Piñeros & Kochian, 2003; Wang et al., 2004; Wu et al., 2010). As we detected macroscopic currents from spontaneous outside-out vesicles that formed during whole-cell measurements with 8–10 MΩ pipettes, our strategy was to use pipettes with a smaller aperture than those used to study the whole-cell currents (25 MΩ), in order to obtain smaller patches of membrane that could, eventually, show single-channel events. Our results demonstrated the presence of single-channel anion currents across the plasma membrane of lily pollen protoplasts displaying at least three conductive substates. The detected single event currents ranged from a few pA up to 30 pA, for currents measured at ± 100 mV. These events superimpose a baseline which probably represents the summation of currents from a number of channels. The direction and amplitude of the currents followed what was expected according to the electrochemical gradient for the anions in solution (with Cl− as the major permeant anion). The effect of the inhibitor NPPB confirmed the anionic nature of the currents. The results suggest the presence of open channels at all times for the different potentials, in agreement with the measured whole-cell currents. Moreover, the measurements were made with the same intra- and extracellular solutions used for studying the macroscopic anion currents, and therefore it is likely that they contribute to these currents.
The whole-cell current ICl1 showed Boltzmann parameters that were clearly distinct from those of the other two current populations for all the experimental conditions tested. Moreover, ICl2 and ICl3 had similar dependences of the conductance on the applied Vm values (Fig. 3, Table 2). The most straightforward interpretation of these results is that there are two channel types in the membrane of lily pollen protoplasts with different sensitivities to the membrane potential. The single-channel currents depicted in Fig. 10 seem to result from the activity of one type of channel, the electrical behaviour of which is characterized by at least three conductive states. Although it is unlikely, these states could also derive from the activity of two or more channels. Only the positive molecular identification of the channel (or channels) responsible for the observed currents will provide, hopefully in the near future, a definitive answer to these questions.
Several anion channels have been identified in the plasma membrane of plant cells. These have been associated with various processes in plants, such as stomatal closure, hormone signalling, membrane excitability, cellular osmoregulation, growth regulation and anionic nutrition (reviewed in Barbier-Brygoo et al., 2000; Roberts, 2006; Angeli et al., 2007). The regulation of stomatal opening and closure in Vicia faba has been extensively studied and involves the coordination of a variety of ionic fluxes. Namely, the S-type anionic channel is responsible for the entrance of anions during stomatal closure. This channel is highly regulated by [ATP]in (phosphorylation), [Ca2+]in, and ABA. Other anion channels have been identified in the plasma membrane of these cells. The R-type, involved in membrane excitability and auxin sensing, is also activated by [ATP]in (nucleotide binding) and [Ca2+]in, and regulated by intracellular pH and extracellular anions. Both show a higher permeability to NO3− than to Cl−. Most of the described anion channels are inward rectifiers (channels through which the exit of anions from the cell is greater than their entrance). Some outward rectifiers have been detected in root cells and have been suggested to be involved in repolarization of the membrane potential after sodium uptake by roots in saline soils (Skerret & Tyerman, 1994). The anionic selectivity of the channels to NO3− and Cl−, as well as their pharmacology and regulation, depends on species and cell type.
The majority of the currents reported here were measured from protoplasts of hydrated pollen grains, before the onset of germination. These results, together with the single-channel currents measured from the same cellular preparation, demonstrate beyond doubt the existence of anionic fluxes across channels in the plasma membrane of ungerminated grains. Moreover, we report anionic currents from germinated pollen which probably result from the same kind of putative channels responsible for the currents across the plasma membrane of the hydrated grain. It has been established that pollen tube germination does not require extracellular anions (Weisenseel & Jaffe, 1976). Nevertheless, our results, combined with those of Zonia et al. (2002) and Breygina et al. (2009a), suggest that pollen grains may have considerable anionic reserves, which are readily available for transport across the membrane. If this is the case, the channels responsible for the currents we observed could be involved in the exit of anions during grain hydration and tube growth processes. Although displaying the same general characteristics (e.g. strong outward rectification, deactivation transient currents), our results suggest that the relative contributions of the three current populations to the total amount of anionic current are different before and after tube germination. The existence of Ca2+ domains has been demonstrated by means of imaging techniques, with intracellular nM values for [Ca2+]in in most of the tube, and an increasing concentration gradient towards the tip up to μM concentrations (Pierson et al., 1994; Holdaway-Clarke et al., 1997; Messerli & Robinson, 1997; Messerli et al., 2000). There are suggestions that [Ca2+]in may reach mM domains close to the plasma membrane at the tip. On the basis of our results, one can speculate that the channels responsible for ICl1 may be involved in the anionic apical effluxes detected at the tip of the growing tube (Zonia et al., 2002) where [Ca2+]in reaches its maximum. We do not exclude, however, the possibility that the anionic currents from lily protoplasts may additionally be distinctly regulated by other intracellular or extracellular effectors, reflecting the different stages of pollen physiology. For instance, it has been shown that inositol 3,4,5,6-tetrakisphosphate modulates the oscillatory Cl− efflux at the pollen tube apex, thereby playing a possible role in tube growth and volume regulation (Zonia et al., 2002). A recent study (Chen et al., 2010), in which concurrent measurements of anionic currents and [Ca2+]inin vivo were performed, suggests the existence of three modes by which anionic S-type currents may be regulated, one of which is Ca2+-dependent. Additionally, both Ca2+-dependent and Ca2+-independent currents showed an extra regulation involving protein dephosphorylation, investigated with okadaic acid, a protein phosphatase antagonist.
We speculate that many of the channels already present in the plasma membrane of the grain protoplasts may suffer a spatial redistribution to specific regions of the membrane during pollen tube germination and growth or that they undergo recycling. Previous studies in the growing pollen tube showed proton fluxes (per area unit) similar to those detected in the hydrated grain, probably attributable to relocation/recycling of the transporters, or de novo synthesis, balancing transporter concentrations (Feijóet al., 1999; Certal et al., 2008). The results presented here seem to confirm that the same putative channels could be responsible for the currents measured from hydrated pollen and during pollen tube growth, but responding to the natural spatial and temporal variation of [Ca2+]in (and other factors). At this point we cannot discriminate between a redistribution of the channels initially present in the membrane and the de novo synthesis of these membrane proteins. We observed a great variation in current density between cells, probably reflecting differences in the number of active channels in the membrane. The error associated with the mean I/V curves obtained with the measured currents normalized to their respective capacity values was greater than the error associated with the nonnormalized currents, particularly in ICl1. Therefore, we chose to use the original current values. For comparison purposes, the normalized values are shown in Table 3. Under physiological conditions it is expected that the membrane potential of the hydrated grain and that of the pollen tube are intrinsically negative (Feijóet al., 1995; Breygina et al., 2009a,b). We have estimated the equivalent ionic fluxes to the Cl− currents measured with nM Ca2+, by assuming a spherical shape for the membrane surface and calculating the corresponding current density (pA cm−2). The values were transformed to fluxes by dividing them by the product of the unitary charge for Cl− and the Faraday constant. For the negative membrane potential values (−160 to −60 mV), we obtained values for the fluxes ranging on average from 300 to 4000 pmol cm−2 s−1, according to a given radius (10 to 3 μm, respectively). These values were estimated from Iinitial and are comparable to those reported by Zonia et al. (2002) which were measured with vibrating probes and which showed an oscillatory efflux that ranged from 50 to 8000 pmol cm−2 s−1, under a low extracellular Cl− concentration (c. 1.1 mM). We followed the same methodology to estimate the fluxes for the germinated pollen, assuming a tube diameter of 15 μm with a length of 200 μm. The values ranged from 90 to 167 pmol cm−2 s−1, the upper limit being much lower than the peak value reported by Zonia et al. (2002). This could reflect the different anionic electrochemical gradients. Alternatively, one must take into account that the net fluxes obtained with the vibrating probes were not measured under voltage control and reflected the sum of both electrogenic and nonelectrogenic transport systems. The whole-cell currents measured from the germinated pollen tube were greater than those measured from the grain (c. 3 times) but the estimated fluxes for the germinated pollen tube were smaller (3.3 times for −60 mV and 23 times for −160 mV). These observations further support a possible relocation of the channels throughout a greater membrane surface, or a preferential migration towards the tube resulting in the clustering of channels. This would also explain the macroscopic currents measured from outside-out patches. Here we were unable to address this point further without establishing the actual localization of the channel(s).
The currents described here share similarities with the few outwardly rectifying depolarization-activated anion channels (OR-DAACs) already described in plants. Nevertheless, the kinetic properties of the currents studied here are distinctive from the former. An anion channel that only allows outward current flow has been identified in protoplasts derived from wheat (Triticum aestivum) roots. It activates in < 100 ms and is positively regulated by nM [Ca2+]in (Skerret & Tyerman, 1994). Outward rectifier channels in roots of L. albus display a slower kinetics (seconds) which is dependent on the holding potential (Zhang et al., 2004). The currents measured here from lily protoplasts were not dependent on the holding potential (−100, 0 and +100 mV; data not shown). The channels described are involved in stabilization of the plasma membrane voltage in different ionic environments. The T. aestivum OR-DAAC is probably involved in Cl− uptake in high-salinity conditions, to maintain a negative membrane potential after the influx of Na+; the L. albus OR-DAAC is involved in the adaptation to phosphorus deficiency. We suggest that the putative anionic channels from the plasma membrane of lily pollen protoplasts may be specific to these cells and have a unique function during hydration and tube growth, possibly keeping the membrane potential within negative physiological values, while counterbalancing the cationic movements across the membrane, and maintaining the electroneutrality of the medium. By following these equilibration movements, it is expected that chloride also plays a role in the transport and equilibration of water, as previously demonstrated (Zonia et al., 2002).