Ion-transport proteins of guard cells have been studied in great detail because their activity is linked directly to stomatal movement. Guard cell ion transport differs from that in most other plant cells because ion transport in guard cells occurs in two directions (to enable stomatal opening as well as closure). By contrast, other plant cells (such as mesophyll- or epidermal pavement cells) grow irreversibly. It is therefore highly likely that the properties of ion transporters, and their regulation by various signals, differ between different cell types. Cell type-specificity of ion-transport genes has rarely been documented, but each cell seems to run a unique set of transport proteins. For instance, the expression rate of the K+-channel gene, GORK, is very high in guard cells, but it is virtually absent from mesophyll cells (Ache et al., 2000).
Figure 2. Overview of ion-transport proteins that have been identified or postulated in the plasma membrane (upper scheme) and vacuolar membrane (lower scheme) of guard cells. Transport proteins, for which one or more genes have been isolated, are shown in black, while those for which only physiological evidence exists are in grey. 1. Outward-rectifying K+ channel, GORK. 2. Inward-rectifying K+ channel, KAT1, KAT2, AKT1, AKT2/3 and AtKC. 3. Hyperpolarization-activated Ca2+ channel. 4. Ca2-ATPase, ACA. 5. R-type anion channel. 6. S-type anion channel. 7. H+-ATPase, AHA. 8. NO3− transporter, CHL1. 9. Cl− transporter. 10. Slow vacuolar (SV) channel; 11. Fast vacuolar (FV) channel. 12. Vacuolar K+-selective (VK) channel. 13. K+ transporter, NHX. 14. H+-pyrophosphatase. 15. V-type H+-ATPase, V1 and V0-subcomplexes. 16. Ca2+-dependent protein kinase (CDPK)-activated anion channel. 17. Hyperpolarization-activated anion channel. 18. Malate carrier, AttDT. 19. Ca2+-carrier, CAX; ACA. 20. Vacuolar ACA. 21. Voltage-gated Ca2+ channel. 22. Inositol triphosphate (IP3)- and inostitol hexakis-phosphate (IP6)-gated Ca2+ channels. 23. Cyclic ADP ribose-activated Ca2+ channel.
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Plasma membrane transport K+ transport The importance of K+ uptake during stomatal opening has been undisputed, ever since Fischer showed, in 1968, that stomatal movement in epidermal strips depends on the uptake of K+ (Fischer, 1968). However, the nature of the proteins facilitating K+ uptake remained unclear until Schroeder et al. (1984) showed the presence of K+-selective ion channels in the plasma membrane of V. faba guard cells. Another breakthrough came 8 yr later with the isolation of the first plant K+-channel genes (Anderson et al., 1992; Sentenac et al., 1992), which was followed by the guard cell-specific expression of the K+ channel encoded by KST1 and KAT1 (Müller-Röber et al., 1995; Nakamura et al., 1995).
Plant plasma membrane K+-channel types can be divided into three groups: outward-rectifying channels, which become more active at positive potentials (Fig. 3c); inward-rectifying channels, activating at more negative potentials (Fig. 3d); and a third type of channel that is largely voltage-independent. The first two types of K+ channels have been recorded in guard cells of various plant species, such as V. faba (Schroeder et al., 1987; Blatt, 1988) A. thaliana (Roelfsema & Prins, 1997; Pei et al., 1997), Nicotiana bethamiana and N. tabacum (Armstrong et al., 1995; Dietrich et al., 1998), Zea mays (Fairley-Grenot & Assmann, 1993) and Populus tremula ×P. tremuloides (Langer et al., 2004). Based on their voltage dependence, outward-rectifying K+ channels are, at physiological conditions, responsible for K+ extrusion, while inward-rectifying channels mediate K+ uptake. Genes encoding voltage-independent K+ channels are only expressed at low rates in guard cells and the activity of these channels has not, so far, been recognized for guard cells (Blatt et al., 1990; Blatt, 1992; Roelfsema & Prins, 1997, 1998).
Figure 3. K+ channel currents in guard cells of intact plants and through ‘Shaker like’ K+ channels expressed in Xenopus oocytes. (a) Plasma membrane current of an Arabidopsis thaliana var. Landsberg erecta guard cell on the abaxial side of a leaf. The plasma membrane was stepped from a holding potential of −80 mV, to test potentials ranging from −180 mV to 20 mV, at 20-mV increments. Note the activation of inward- and outward-rectifying K+ channels with increasing negative- and positive potentials, respectively. (b) Current–voltage relationship of steady-state currents from the same cell as in (a). Note the activation of inward- and outward-rectifying K+ channels at potentials negative of −120 mV and positive of −60 mV, respectively. (c) Whole-oocyte currents of GORK recorded in a bath solution containing 30 mm KCl, 1 mm CaCl2, 1 mm MgCl2 and 10 mm Tris/Mes, pH 7.5. Currents were elicited from a holding potential of −80 mV to test potentials ranging from −80 to 50 mV, at 10-mV increments. (d) Oocyte currents of KAT1 recorded in 30 mm KCl, 1 mm CaCl2, 1 mm MgCl2 and 10 mm Mes/Tris, pH 5.6. Currents were elicited with test potentials ranging from 20 to −150 mV, at 10-mV increments.
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The gating of outward- and inward-rectifying K+ channels differs in a crucial way: activation of outward K+ channels is dependent on the extracellular K+ concentration, while that of inward K+ channels is not. Outward-rectifying K+ channels respond to changes in the extracellular K+ concentration in such a way that the channel always activates at potentials slightly more positive than the Nernst potential for K+ (Blatt, 1988; Roelfsema & Prins, 1997; Gaymard et al., 1998; Ache et al., 2000). In contrast, the activation of inward channels is not affected by K+ (Blatt, 1992; Roelfsema & Prins, 1997), and these channels do not exclusively facilitate K+ uptake, but can mediate K+ extrusion at very low extracellular K+ concentrations (Véry et al., 1995; Brüggemann et al., 1999). In intact plants of V. faba, the K+ concentration in the guard cell wall has been estimated at between 3 and 40 mm (Felle et al., 2000; Roelfsema & Hedrich, 2002). The outward K+ channel of guard cells in intact V. faba becomes active at potentials close to −50 mV, while the inward rectifier activates at potentials negative of −100 mV (Roelfsema et al., 2001). In intact plants of A. thaliana (Fig. 3a,b) and Populus (Langer et al., 2004) the outward K+ channels also activate at −50 mV, but the threshold potential of inward K+ channels is more negative than −100 mV. A species-specific threshold potential for activation was not found by using patch-clamp recordings (Dietrich et al., 1998), indicating that the threshold potential is set by cytoplasmic factors that are lost during the whole-cell configuration.
The first two plant K+ channels were cloned from Arabidopsis, and denoted KAT1 (Anderson et al., 1992) and AKT1 (Sentenac et al., 1992); this was followed by the identification of the first KAT1 homolog, KST1, from potato (Müller-Röber et al., 1995). All these genes encode inward-rectifying K+ channels when expressed in Xenopus oocytes (Schachtman et al., 1992; Müller-Röber et al., 1995; Véry et al., 1995) or yeast cells (Bertl et al., 1995). These and later studies revealed that all members of the ‘Shaker like’ K+ channels are localized in the plasma membrane and function as K+-selective ion channels (Dietrich et al., 2001; Véry & Sentenac, 2002). KST1 and KAT1 are highly expressed in guard cells (Müller-Röber et al., 1995; Nakamura et al., 1995) and therefore soon became known as guard cell K+-uptake channels. Mutants of A. thaliana, with an En-transposon inserted in the open reading frame of KAT1, were therefore expected to lack inward K+ channels. However, recordings with intracellular microelectrodes revealed no difference between inward K+ currents of the KAT1 En-insertion mutant and wild type (Szyroki et al., 2001). Furthermore, these loss-of-function mutants displayed normal K+-uptake kinetics and stomatal opening. This led to a search for other K+-channel genes expressed in guard cells, which could back up for the loss of KAT1. Indeed, guard cells were found to express the K+-channel genes KAT2 (Pilot et al., 2001), AKT1, AKT2/3 and AtKC1, although at lower expression levels than that of KAT1 (Szyroki et al., 2001). Functional K+ channels probably assemble four subunits, encoded by one or more of these genes, to form a homomeric or a hetromeric complex (Dreyer et al., 1997). This might explain why the introduction of a nonfunctional KAT1 gene lowered the inward K+ conductance of guard cells by 70% (Kwak et al., 2001). Nonfunctional KAT1 subunits may form tetrameric channel proteins in combination with all five wild-type genes, thereby creating nonfunctional K+ channels.
The properties of inward K+ channels are thus not encoded by a single gene, but are probably the result of different properties encoded by at least five genes (Szyroki et al., 2001). The subunits KAT1, KAT2 and AKT1 all encode inward-rectifying K+ channels in heterologous expression systems. The fourth gene, AtKC1, probably does not encode a functional K+ channel itself, but its expression can alter the properties of K+ channels formed by the other subunits (Reintanz et al., 2002). The fifth gene expressed in guard cells, AKT2/3, encodes a largely voltage-independent channel when expressed in Xenopus oocytes (Marten et al., 1999; Lacombe et al., 2000). This channel probably plays an important role in the phloem of the shoot (Marten et al., 1999), but not in roots (Birnbaum et al., 2003). Voltage-independent K+ channels have not been recognized for guard cells, indicating that the properties the AKT2/3 subunits may change through interaction with other K+-channel subunits (Ivashikina et al., 2003), or as a result of phosphorylation (Chérel et al., 2002).
In contrast to the inward-rectifying K+ channels, outward-rectifying K+ channels in the guard cells of A. thaliana are encoded by a single gene only, named GORK (Ache et al., 2000). Disruption of the GORK gene causes a complete loss of the outward-rectifying K+ channels in the plasma membrane of guard cells (Hosy et al., 2003). The stomata of the GORK loss-of-function mutant close more slowly after treatment with ABA than do wild-type stomata (Hosy et al., 2003). However, the fact that stomata still close in response to ABA and darkness in gork-1, indicates that guard cells possess an alternative transport system capable of releasing K+ during stomatal closure.
Anion transport The efflux of K+ from guard cells during stomatal closure is electrically neutralized by a concomitant efflux of anions. Anions pass through the guard cell membrane via channels that conduct a range of small anions (Hedrich & Marten, 1993; Schmidt & Schroeder, 1994). Two types of channels conducting anions have been identified in the guard cell membrane: rapid (R)-type (Keller et al., 1989; Hedrich et al., 1990); and slow (S)-type (Schroeder & Hagiwara, 1989; Linder & Raschke, 1992) anion channels (Fig. 4). The most obvious difference between these channels is a strong decrease in the open probability of R-type channels when cells are clamped to potentials negative of −50 mV and a slow inactivation after returning to more positive potentials (Kolb et al., 1995), while S-type channels exhibit only a weak voltage-dependent deactivation (Linder & Raschke, 1992; Schroeder & Keller, 1992; Dietrich & Hedrich, 1994).
Figure 4. R- and S-type anion channels in the guard cell plasma membrane. (a) Current traces of R-type anion channels of a far depolarized guard cell in an intact Vicia faba plant (Roelfsema et al., 2001). The currents were elicited from a holding potential of −140 mV to test potentials ranging from −120 to 80 mV at 20-mV increments. (b) Current traces of S-type anion channels of a V. faba guard cell in an epidermal strip bathed in 5 mm CsCl, 1 mm CaCl2 and 1 mm Mes/BTP, pH 6.0. The currents were elicited from a holding potential of 20 mV to test potentials ranging from 0 to −180 mV, at −20-mV increments. Note the slow deactivation of S-type anion channels at the most negative membrane potentials. (c) Current–voltage relationship of the same cell as in (a) sampled during the last 100 ms of the test pulses. Note the maximum conductance at −80 mV. (d) Current–voltage relationship of the same cell as in (b) sampled during the last 500 ms of the test pulses. The reversal potential at 0 mV indicates an incomplete block of outward-rectifying K+ channels under the conditions applied.
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Guard cells in intact plants often maintain membrane potentials negative of −100 mV (Roelfsema et al., 2001; 2002) and the difference in gating between S-type and R-type anion channels may thus be of physiological importance. Anion-channel activation will normally depolarize the guard cell plasma membrane (see II. 3). However, in cells with a membrane potential negative of −120 mV, a depolarization cannot be initiated by R-type channels. Indeed, we have observed guard cells in intact plants with a negative membrane potential and a high activity of R-type channels (own unpublished results). In these cells a depolarization can be initiated only through changes in the voltage dependence of R-type channels or through the activation of S-type anion channels. A change in the voltage dependence of R-type anion channels can be caused by anions at the extracellular side of the channels. Organic anions, such as malate, acetate and proprionate, shift the half-maximal activation potential of R-type channels to more negative values (Hedrich & Marten, 1993; Dietrich & Hedrich, 1998) and thus increase the potency of the channel to initiate a depolarization. The effect of malate is probably the most significant, as this anion accumulates at high concentrations in guard cells during stomatal opening (Outlaw & Lowry, 1977; Raschke & Schnabl, 1978). Anion channels are permeable to malate (Hedrich et al., 1994; Schmidt & Schroeder, 1994) and thus will extrude this organic anion during stomatal closure. The apoplastic concentration of malate was found to rise in response to high CO2 concentrations (Hedrich et al., 1994) and during prolonged illumination (Lohaus et al., 2001).
Apart from a difference in gating characteristics, R- and S-type channels also differ in their sensitivity to anion-channel blockers. R-type channels are blocked by stilbene derivates, such as DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; Hedrich & Marten, 1993), while S-type channels are insensitive to DIDS (Schwartz et al., 1995, own unpublished results). S-type channels are blocked by NPPB (5-nitro-2-(3-phenylpropylamio)benzoic acid), 9-AC (anthracene-9-carboxylic acid) and niflumic acid (Schroeder et al., 1993; Schwartz et al., 1995), but these blockers are not specific because they also inhibit R-type anion channels (Marten et al., 1992) and outward-rectifying K+ channels (Garrill et al., 1996).
Apart from the difference in gating properties and their sensitivity to stilbene derivatives, R- and S-type anion channels have many features in common. Their single-channel conductance and ion selectivity are similar, which indicates that both channel types have a common pore structure or are just two gating modes of a single protein (Dietrich & Hedrich, 1994). Both channel types were also found in A. thaliana guard cells (Pei et al., 1997; Pei et al., 2000); however, the genes encoding these plasma-membrane anion channels are still unknown. Several genes homologous to animal ClC channels were found in the Arabidopsis genome, but these genes probably encode anion channels of intracellular membranes (Geelen et al., 2000; Lurin et al., 2000). In agreement with these results, ClC channels were not identified in purified plasma membranes of cell-suspension cultures of A. thaliana (Marmagne et al. 2004). Instead, voltage-dependent anion channel (VDAC) proteins were present in the plasma membrane, an anion channel normally associated with the mitochondria. Future experiments need to provide unequivocal evidence for VDAC proteins as genuine plasma-membrane anion channels that harbor the properties of R- and S-type anion channels.
Compared to the anion-release system of the guard cell membrane, little is known about the anion-uptake transporters. During stomatal opening guard cells accumulate anions, which electrically neutralize the uptake of K+. Anion uptake is a flexible process that depends on the extracellular presence of anions. As mentioned in II. 1, guard cells accumulate Cl− and malate in equal concentrations when stomata, in epidermal strips, are supplied with Cl− (Raschke & Schnabl, 1978). However, if no plasma membrane-permeable anions are applied, K+ uptake is completely balanced by malate synthesis (Raschke & Schnabl, 1978). Other anions, such as NO3− can also support stomatal opening (Guo et al., 2003). Arabidopsis mutants, with reduced levels of the NO3− transporter, CHL1, displayed smaller stomatal apertures than wild type, when NO3− was supplied to detached leaves (Guo et al., 2003). Stomatal opening, however, was similar to that of wild-type Arabidopsis in the presence of Cl−, indicating that the CHL1 transporter enables the uptake of NO3− into guard cells, but does not represent the Cl− uptake system. Most likely, guard cells also possess a carrier that couples the uptake of Cl− with that of H+, but this transporter still remains to be identified.
Ca2+ transport The interaction between extracellular K+ and Ca2+ on stomatal movement has been studied extensively (see reviews Raschke, 1975a; MacRobbie, 1987). In general, high extracellular K+ and Ca2+ concentrations act antagonistically, as stomatal opening is stimulated by K+ and inhibited by Ca2+. The sensitivity to extracellular Ca2+, however, differs between species. Whereas the presence of 0.25 mm extracellular Ca2+ reduced stomatal opening in C. communis by > 50% (DeSilva et al., 1985a), it reduced stomatal apertures to < 25% in V. faba (Fischer, 1972) and A. thaliana (Roelfsema & Prins, 1995). The latter data reflect an effect of Ca2+ at an extracellular K+ concentration of 50 mm; at lower K+ concentrations the Ca2+ effects become even more apparent (Fischer, 1972). Extracellular Ca2+ probably inhibits stomatal opening by evoking increases in the intracellular Ca2+ concentration (McAinsh et al., 1995). It has long been thought that high external Ca2+ concentrations would enhance leak Ca2+ currents across the plasma membrane. Such leak currents of Ca2+ would be supported by the high concentration gradient for Ca2+ across the plasma membrane. The apoplastic Ca2+ concentration is ≈ 100 µm (Felle et al., 2000; Roelfsema & Hedrich, 2002), while the cytoplasmic concentration ranges from 100 to 300 nm (Webb et al., 2001; Levchenko et al., 2005). Recently, it was shown that guard cells do not simply leak Ca2+ but instead possess a plasma-membrane Ca2+ sensor (CAS) (Han et al., 2003). The CAS sensor triggers a rise in the intracellular Ca2+ concentration, in response to an increase in the extracellular Ca2+ concentration. Guard cells expressing antisense constructs of CAS were no longer responsive to a rise in the extracellular Ca2+ concentration.
Calcium may enter guard cells via nonselective Ca2+ channels in the plasma membrane. These nonselective channels have been found in a large number of plant cells and are, in general, permeable to monovalent and divalent cations (Demidchik et al., 2002). In guard cell protoplasts, Ca2+-permeable channels were identified that are activated by stretch (Cosgrove & Hedrich, 1991) or ABA (Schroeder & Hagiwara, 1990). Nonselective cation channels that activate upon hyperpolarization were suggested by Lohse & Hedrich (1992) (Fig. 5b,c). The physiological relevance of these channels in guard cells became apparent after Grabov & Blatt (1998) showed hyperpolarization-induced rises in the cytoplasmic Ca2+ concentration. Subsequent studies showed that these channels are stimulated by 50 µm H2O2 in A. thaliana (Pei et al., 2000) and through phosphorylation and ABA in V. faba (Hamilton et al. 2000; Köhler & Blatt, 2002).
Figure 5. Electrical properties of Vicia faba guard cell protoplasts after elimination of the plasma membrane K+ and anion currents, redrawn after Lohse & Hedrich (1992). (a) Current–voltage relationship of the plasma membrane H+-ATPase measured in the whole-cell patch clamp mode. A voltage ramp was applied from −240 to 10 mV. (b) Voltage relationship of whole-cell currents, with ATP in the pipette and in the absence (white symbols) or presence (black symbols) of 5 mm LaCl3 in the bath solution. (c) Voltage relationship of whole-cell currents remaining after ATP depletion in the absence of LaCl3.
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The Ca2+ homeostasis in plant cells is maintained by Ca2+ transporters in the plasma membrane, vacuolar membrane and endoplasmic reticulum (ER) (Sanders et al., 2002). The plasma membrane harbors P-type ATPases (ACAs) that can transport Ca2+ against steep concentration gradients. These Ca2+ ATPases are characterized by an autoinhibitory domain at the N-terminus that binds Ca2+/calmodulin (Sze et al., 2000; Axelsen & Palmgren, 2001). At high cytoplasmic Ca2+ concentrations the N-terminus can be released from the core protein, thereby activating the Ca2+ pump. The latter mechanism provides a negative-feedback system that automatically counteracts a rise in cytoplasmic Ca2+. Ca2+ regulates the ACA pumps also via an additional pathway involving Ca2+-dependent protein kinases (CDPK) (Sze et al., 2000). Genes encoding Ca2+-permeable channels in the guard cell plasma membrane have not yet been cloned (Hetherington & Brownlee, 2004), but they may be homologous to the ionotropic glutamate receptors (Lacombe et al., 2001).
H+ transport The guard cell membrane is energized by a P-type ATPase that translocates H+ from the cytoplasm to the guard cell wall (Fig. 5a). This proton pump is the only primary active transporter in the plasma membrane, apart from a number of P-type ATPases that transport Ca2+ or heavy metals (Axelsen & Palmgren, 2001). The guard cell H+-ATPase has a low activity at pH 7.5 and becomes more active at acidic pH values (Becker et al., 1993). This pH dependence indicates a role for the proton pump in maintaining H+ homeostasis. Guard cells maintain a cytoplasmic pH of 7.5–7.8 (Blatt & Armstrong, 1993; Grabov & Blatt, 1997); a shift to more acidic values thus automatically activates the plasma membrane H+-ATPase. In addition, H+-ATPases are regulated through an autoinhibitory domain at the C-terminus. This domain is released from the catalytic site, after binding of a 14-3-3 protein, based on phosphorylation-dependent (Würtele et al., 2003), as well as phosphorylation-independent, mechanisms (Fuglsang et al., 2003). Binding of the phosphorylated C-terminus to 14-3-3 proteins is stabilized by fusicoccin, a fungal toxin that hyperstimulates stomatal opening (Braunsgaard et al., 1998; Kinoshita & Shimazaki, 1999).
Vacuolar membrane transport A proportion of the ions that accumulate in the guard cell during stomatal opening will remain in the cytoplasm, while the rest is sequestered in the vacuoles. Guard cells posses several small vacuoles that may be interconnected to form a reticulum (Palevitz et al., 1981; Faraday et al., 1982). Swollen guard cells contain only few but large vacuoles that shrink and become fragmented during stomatal closure (Diekmann et al., 1993). However, a detailed study of changes in vacuole size during stomatal movement has not been conducted. A vacuolar sequestration is of importance for ions, such as Ca2+, that also act as second messengers. Vacuoles are an important intracellular store of Ca2+ and are probably involved in the release of Ca2+ during signaling events in guard cells (MacRobbie, 1998; Sanders et al., 2002; Hetherington & Brownlee, 2004). Furthermore, sequestration of malate is important, as this anion negatively feeds back on carbon fixation, by inhibition of phospho(enol)pyruvate (PEP) carboxylase (Schnabl & Kottmeier, 1984b; Raschke et al., 1988). Indeed, most of the malate in guard cell protoplasts of V. faba is located in the vacuole (Schnabl & Kottmeier, 1984a).
Although a number of channels, carriers and pumps at the vacuolar membrane have been identified (Fig. 3), the regulation of vacuolar membrane transport is poorly understood. In general, transport activities were studied using isolated vacuoles, devoid of cytoplasmic components that regulate these transporters in intact cells. Recording techniques that measure the activity of ion transporters at more physiological conditions are technically demanding, but will be essential for a better understanding of ion transport regulation in the future. Likewise, little is known about the membrane potential of the guard cell vacuole. A potential difference of 20–50 mV, negative at the cytoplasmic site, is often assumed (MacRobbie, 1998), but no direct recordings of vacuoles in intact guard cells exist. Furthermore, the vacuolar membrane potential presumably changes during stomatal movements, but the extent to which changes in this potential contribute to ion release remains unknown.
K+ transport Three types of K+-permeable channels have been determined in the vacuolar membrane of guard cells (Allen et al., 1998). Based on differences in gating characteristics, these channels were classified as slow vacuolar (SV), fast vacuolar (FV) (Hedrich et al., 1986; Hedrich & Neher, 1987; Schulz-Lessdorf & Hedrich, 1995; Allen & Sanders, 1996) and vacuolar K+-selective (VK) channels (Ward & Schroeder, 1994; Allen & Sanders, 1996). SV channels represent the most prominent ion conductance in the vacuolar membrane and slowly activate at depolarizing potentials (more positive at the cytoplasmic side), conditions that allow K+ release from the vacuole. However, at large depolarization, the K+ flux reverses and K+ is transported into the vacuole via SV channels (Ivashikina & Hedrich, 2005). FV channels are similar in this respect, as they also activate with depolarization, but their activation is much faster. The Ca2+ dependence of FV and SV channels differs – FV are inhibited by Ca2+ concentrations of > 0.1 µm (Hedrich et al., 1986; Allen & Sanders, 1996), whereas SV channels are inactive at low concentrations of Ca2+, but are activated by Ca2+ concentrations of > 0.5 µm (Hedrich & Neher, 1987). Both channels are cation permeable and thus may enable K+ release, probably under different cytoplasmic conditions. VK channels represent the third type of K+-selective channel in the vacuole (Ward & Schroeder, 1994). These channels are nonrectifying and thus appear as K+-selective leaks that may enable K+ release or uptake at conditions that do not support the activation of SV- or FV channels (Allen et al., 1998).
Vacuolar K+ channels may be encoded by the KCO (TPK) genes, as all of these channels, apart from TPK4, are targeted to the vacuolar membrane (Czempinski et al., 2002; D. Becker et al., unpublished). The KCO channels have been renamed TPK (which stands for two pore domain K+ channels) (Becker et al., 2004), with the exception of KCO3, which has only one pore domain. The pore domains of the TPK channel and KCO3 contain the GYGD motif, suggesting that they encode ion channels with a K+-selective conductance (Czempinski et al., 1999). This was recently proven for TPK4, the sole Arabidopsis TPK channel located in the plasma membrane (Becker et al., 2004). Whether or not this is also the case for vacuolar TPK channels is currently under investigation.
K+ ions are taken up into vacuoles against the vacuolar membrane potential, the transport therefore is probably mediated by secondary active carriers. K+ uptake may be carried out by the Na+/H+ (NHX)-transporters, well known for their ability to transport Na+ (Gaxiola et al., 1999), but which also mediate H+-coupled K+ transport (Venema et al., 2003; Cellier et al., 2004). In addition, less well-characterized transporters are found in the Arabidopsis genome; these transporters have high sequence similarity to known H+/K+ carriers (Sze et al., 2004).
Anion transport The vacuolar membrane potential supports a flow of anions from the cytoplasm into the vacuole through anion channels. For guard cell vacuoles, only a single publication has reported on anion channels (Pei et al., 1996). This anion channel was reported to be activated by CDPK and to facilitate the flow of Cl− and malate2– into vacuoles. For other cell types, voltage-dependent anion channels have been described that open at negative membrane potentials, indicating that they indeed support anion sequestration into vacuoles (Pantoja & Smith, 2002; Hafke et al., 2003). This may be important for the sequestration of malate, as the cytoplasmic concentration of this anion has to be kept low in order to prevent the inhibition of PEPcarboxylase (Raschke et al., 1988; Schnabl & Kottmeier, 1984b). For malate, the uptake process into the vacuole is complex, as malate has two negative charges at neutral pH, but may lose one upon entry into the acidic vacuole. Monovalent malate anions could either be trapped in the vacuole, or flow back into the cytoplasm, depending on the selectivity of anion channels. Alternatively, malate may be transported into the vacuole by carriers, such as the malate carrier, AttDT (Emmerlich et al., 2003).
Anions can also be released via anion channels, but this requires depolarization of the vacuolar membrane. If the membrane potential would approximate 0 mV, anions would flow down their concentration gradient into the cytoplasm. Vacuolar anion channels may be encoded by genes homologous to the bacterial and animal ClC channels. Disruption of the AtClC-a gene resulted in plants that accumulate less NO3− when grown at high levels of NO3− (Geelen et al., 2000). However, the activity of ClC channels, in general, and their role in the vacuolar membrane, in particular, still await confirmation by direct measurements. Patch clamp studies, on isolated vacuoles of plants overexpressing a CLC channel or lacking one, may help to establish the function of these genes.
Ca2+ transport The large vacuole of guard cells plays an important role in Ca2+ homeostasis (Sanders et al., 2002). Although the Ca2+ concentration of guard cell vacuoles has not been studied in detail, it is generally assumed that it is in the mm range, as recorded for rhizoid cells of Riccia fluitants and root cells of Z. mays (Felle, 1988). Ca2+ accumulates in the vacuoles as a result of the activity of ATP-dependent ACA pumps (Sze et al., 2000; Axelsen & Palmgren, 2001) as well as of the CAX-encoded Ca2+/H+ antiporters (Hirschi et al., 1996). The ACA pumps are regulated by Ca2+/calmodulin, as are their counterparts at the plasma membrane, while the regulation of the CAX carriers seems to be more indirect and involves several CXIP proteins that closely interact with CAX1 and -4 (Cheng & Hirschi, 2003).
In contrast to the limited information available about Ca2+ sequestration into the vacuoles of guard cells, many reports have focused on Ca2+-release channels. Because of the steep concentration gradient, the opening of Ca2+-permeable channels in the vacuolar membrane results in a Ca2+ flux to the cytoplasm. This, in turn, leads to a rise in the cytoplasmic Ca2+ concentration, which may encode a signal for downstream responses (Evans & Hetherington, 2001; Sanders et al., 2002). Based on patch clamp recordings, plant vacuolar membranes seem to harbor a number of ligand-gated Ca2+-permeable channels. In beet roots, vacuolar membrane Ca2+ currents are induced by inositol triphosphate (IP3) and cyclic-ADP ribose (Allen et al., 1995), while guard cell vacuolar membranes have also been shown to be sensitive for cyclic-ADP ribose (Leckie et al., 1998) and inositol hexakis-phosphate (IP6) (Lemtiri-Chlieh et al., 2003). In addition to these ligand-gated Ca2+ channels, a voltage-gated Ca2+ channel (VVCa-Channel) has been described for the guard cell vacuolar membrane (Allen & Sanders, 1994).
The single-copy gene, AtTPC (Furuichi et al., 2001), is likely to encode one of the Ca2+ channels in the vacuolar membrane, as its gene product was localized to this membrane by using a proteomics approach (Carter et al., 2004). Genes homologous to the animal IP3- and ryanodine-receptors have not been identified in the Arabidopsis genome (Schwacke et al., 2003). Apparently, the genes encoding these receptors in plants have developed independently from the animal counterparts, or have diverged considerably from them during evolution.
H+ transport Two types of H+-translocating pumps – V-type H+-ATPases (V-ATPases) and pyrophosphatases (PPases) – co-reside in the same vacuolar membrane (Hedrich et al., 1986; 1989; Sze et al., 1999). The V-type ATPase is a protein complex similar to the F1/F0-ATPases and consists of a large number of different subunits (Sze et al., 2002; Kluge et al., 2003). The V1 subcomplex catalyzes ATP hydrolysis and rotates, during H+ translocation, with respect to the V0 subcomplex in the membrane. The stoichiometry of these pumps is variable: values were determined ranging from more than 3 (Davies et al., 1994) to 1 H+ per hydrolyzed ATP (Müller & Taiz, 2002). A low stoichiometry is probably important for very acidic vacuoles because a high proton motive force can inverse the proton flux through V-type ATPases (Gambale et al., 1994). For guard cell vacuoles the stoichiometry may be higher, as these compartments contain high concentrations of K+ and thus may not have such a low pH. V-type ATPases are probably regulated by the redox state as well as through changes in the cytoplasmic nucleotide concentration (Kluge et al., 2003), but no information is available about this type of regulation in guard cells. The importance of these transporters in guard cell physiology is evident from studies on the det3 mutant (Schumacher et al., 1999). These mutants are affected in subunit C of the V-ATPase and display only 70% of the V-ATPase activity compared to wild-type cells. Guard cells of det3 did not close in response to an increase in the extracellular Ca2+ concentration or to the application of H2O2 (Allen et al., 2000). The absence of these responses was therefore linked to an altered Ca2+ homeostasis in det3 guard cells.
In addition to the V-ATPases, PPases in Arabidopsis contribute to the generation of the electrochemical gradient across vacuolar membranes (Drozdowicz & Rea, 2001). These relatively simple proteins use the hydrolysis of PPi to pump H+ into the vacuolar lumen. The activity of these pumps is highest in young growing tissues, in which PPi is available as a by-product of biosynthesis (Maeshima, 2000). Because the synthetic rate of macromolecules is probably less pronounced in mature guard cells, it is unlikely that PPases play a major role in energizing the guard cell vacuolar membrane.