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Journal of Experimental Botany, Vol. 49, Special Issue, pp. 351–360, March 1998 Co-ordination of signalling elements in guard cell ion channel control A. Grabov1 and M.R. Blatt Laboratory of Plant Physiology and Biophysics, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK Received 3 November 1997; Accepted 10 November 1997 Abstract Fine regulation of solutes transport across the guard cell plasma membrane for osmotic modulation is essential for the maintenance of the proper stomatal aperture in response to environmental stimuli. The major osmotica, K+, Cl− and malate are transported through selective ion channels in the plasma membrane and tonoplast of guard cells. To date, a number of ion channels have been shown to operate in the guard cell plasma membrane: outwardly- and inwardlyrectifying K+ channels (I and I ), slowly- and K,in K,out rapid-activating anion channels, and stretch-activated non-selective channels. Slow and fast vacuolar channels (SV and FV) and voltage-independent K+-selective (VK) channels have been found at the guard cell tonoplast. On the molecular level, the regulation of the stomatal aperture is achieved by precise spatial and temporal co-ordination of channel activities through a network of signalling cascades that can be triggered, among others, by plant hormones. ABA and auxin as regulators of stomatal aperture have received most attention to date. It is clear now that the effect of these hormones on ion channels is mediated by second messengers pH and [Ca2+] . The effect of ABA is geni i erally associated with increases in pH , while auxin i acidifies the cytosol. Both of these hormones may induce elevation in [Ca2+] . Phosphorylation is another i important factor in cellular signalling. The ABI1 gene encoding a 2C-type protein phosphatase has been shown to be a key element of ABA-dependent cascades. Plasma membrane voltage is also an important component of signalling and channel control, and has recently been shown to influence cytosolic-free [Ca2+]. Thus, transduction of these, and associated cytoplasmic signals is clearly non-linear, and is prob1 To whom correspondence should be addressed. Fax: +44 1233 813 140. © Oxford University Press 1998 ably important for providing a plasticity of cellular response to external and environmental stimuli. Understanding the interdependence and hierarchy of signalling elements now presents a major challenge for research in plant biology. Key words: Ion channels, stomatal aperture, signalling pathway, second messengers, guard cells. Introduction Guard cell control of stomatal aperture is crucial to balancing transpiration stream and gas exchange for photosynthesis. As transpiration and CO exchange can 2 not be regulated independently, the demand of photosynthetic machinery can lead to excessive water evaporation under adverse environmental conditions. Thus, the size of the stomatal aperture optimizes gas exchange for overall plant performance. This balance of requirements is achieved by the precise tuning of guard cell turgor pressure and on the molecular level is accomplished by means of fine temporal and spatial co-ordination of different solute transporters. It is well known that K+, Cl−, malate, and some other organic anions are the major contributors to the guard cell osmolarity ( Willmer and Fricker, 1996). Guard cell response to the variety of the hormonal and environmental stimuli is due to the rapid transport of these ions across the plasma membrane and tonoplast ( Willmer and Fricker, 1996; Blatt and Grabov, 1997a, b). The variety of environmental and hormonal stimuli affecting stomatal aperture is enormous and includes CO , 2 light, temperature, gaseous pollutants, as well as all of the major plant hormones. Remarkably, on the cellular level these stimuli appear to converge on a few cytoplasmic signals, known as second messengers, and these 352 Grabov and Blatt in turn regulate a handful of transport proteins. Second messengers like cytosolic-free H+ (pH ) and Ca2+ i ([Ca2+] ) are key components of signal transduction i chains in stomata aperture regulation (Blatt and Thiel, 1993; Assmann, 1993; Bush, 1995; McAinsh et al., 1997; Blatt and Grabov, 1997b). Phosphorylation of some signal cascade and target elements also contributes to these events (Pei et al., 1996, 1997; Blatt and Grabov, 1997a; Grabov et al., 1997). Finaly, membrane voltage is an essential in controlling transport proteins in the membrane, the activities of which are voltage sensitive, and in second messenger generation (Thiel et al., 1992). Guard cell ion channels Plasma membrane channels There are at least three different types of ion channels in the guard cell plasma membrane implicated for the regulation of stomatal aperture (Blatt, 1991; Blatt and Grabov, 1997a): outwardly rectifying K+ channels (I ), K,out inwardly rectifying K+ channels (I ), and anion chanK,in nels. Experimental factors that control the activities of these channels have given us much insight into possible regulatory mechanisms even without reference to any external stimuli. Inwardly rectifying K+ channels are involved in K+ uptake during stomatal opening (Schroeder, 1988; Blatt, 1988, 1991; Grabov and Blatt, 1997). I is only moderK,in ately sensitive to membrane voltage showing an apparent gating charge of 1.3–1.7 (Blatt, 1992; Grabov and Blatt, 1997). However, I is strongly dependent upon apoplastic K,in pH. Increasing [H+] outside promotes the K+ current in a voltage-dependent maner shifting V by 22 mV per pH 1/2 decade and accelerating activation kinetics (Blatt, 1992). At neutral external pH these channels are activated by membrane voltages more negative than −100 mV with V close to −200 mV, but at pH 6.0 I is gated at 1/2 K,in more positive voltages (V #−180 mV ). The current is 1/2 also sensitive to cytosolic pH. Lowering pH from pH 7.6 i to 6.9 stimulates I by a factor of up to 6. (Grabov and K,in Blatt, 1997). The channel activity is also reduced at elevated [Ca2+] (Schroeder and Hagiwara, 1989; Lemtiri-Chlieh i and MacRobbie, 1994; Grabov and Blatt, 1997). By contrast to the situation with pH no kinetic detail is available for I activity and its gating characteristics as K,in a function of [Ca2+] . i The outwardly rectifying K+ channel is activated by depolarization and has been demonstrated to contribute to K+ efflux during stomatal closure. This channel is up-regulated by pH increases and is virtually insensitive i to [Ca2+] (Hosoi et al., 1988; Blatt and Armstrong, 1993; i Lemtiri-Chlieh and MacRobbie, 1994; Grabov and Blatt, 1997). I voltage sensitivity and kinetics are dependent K,out upon the K+ gradient across the plasma membrane (Blatt, 1988; Blatt and Thiel, 1993). Activation and deactivation kinetics of I in Vicia faba guard cells slow with K,out elevation of external K+ and voltage dependent opening follows E ensuring that I operates as one-way valve K K,out to prevent reflux of K+ into the guard cell. Recent studies have shown that I activation is dependent on the K,out co-operative interaction of 2 K+ ions with the channel, but at a site (or sites) distinct from the channel pore. The apparent K for interaction was strongly voltage1/2 dependent, accounting for the equivalence of (negative) membrane voltage and [ K+] in regulating channel activity (Blatt and Gradmann, 1997). A positive shift in the plasma membrane potential is essential for I activation when the membrane voltage K,out is situated negative of E . Plasma membrane depolarizaK tion under these conditions is evoked by an inward current (Thiel et al., 1992; Ward et al., 1995; Blatt and Grabov, 1997b). At least one component of this current has been suggested to be mediated by anion channels (Hedrich et al., 1990; Thiel et al., 1992; Schroeder and Keller, 1992). Two types of anion current with markedly different kinetics have been identified in the guard cell plasma membrane. One of these currents demonstrates rapid activation on membrane depolarization (t = 1/2 50 ms) and inactivates with t ~10 s at voltages near to 1/2 and positive of −100 mV ( Keller et al., 1989; Hedrich et al., 1990). The second anion current has been shown to activate and deactivate slowly (t =5–30 s) and shows 1/2 no time-dependent inactivation (Schroeder and Keller, 1992; Linder and Raschke, 1992). Of these two anion currents, the slow current is implicated for sustained long-term depolarization (Schroeder and Hagiwara, 1989; Schroeder and Keller, 1992; Linder and Raschke, 1992; Grabov et al., 1997). Both types of anion currents are activated with increasing [Ca2+] and i exhibit roughly similar single channel conductance. In fact it has been suggested that the same channel protein may account for both gating modes (Blatt and Thiel, 1993). Indeed, changes in anion current kinetics are known to occur in response to ATP depletion ( Thomine et al., 1995), ABA stimulation or phosphorylation (Grabov et al., 1997). Schulz-Lessdorf et al. (1996) have argued that fast activating anion channels recognize the H+ gradient across the plasma membrane. Regardless of the relationships between the two anion currents, the fast activating form can be ruled out as a major pathway for anion efflux during stomatal closure. Its narrow voltage range for activation and the fact that the current inactivates within seconds cannot be reconciled with the requirement for sustained channel functioning over periods of 20–30 min or more during closure (Blatt and Grabov, 1997a; Blatt and Thiel, 1993). Tonoplast channels As the vacuole occupied of approximately 80–90% of the cell volume, this organelle dominates guard cell osmoreg- Co-ordination of ion channels in guard cells 353 ulation. Much of the solute lost during stomatal closure originates from the vacuole. Equally, osmotic accumulation on stomatal opening is associated predominantly with this storage organelle. The importance of vacuole in this function implies a high degree of transport co-ordination at the tonoplast and between the two membranes (MacRobbie, 1995a, b). Current knowledge of tonoplast ion transport relevant to stomata movement remains relatively poor. To date, patch-clamp measurements have uncovered three different types of cation-selective channels operating at the tonoplast. One channel recently identified appears to be voltage-independent and selective for K+ and has been designated the VK channel ( Ward and Schroeder, 1994; Allen and Sanders, 1996). This channel is activated by elevated [Ca2+] within physiological range (Allen and i Sanders, 1996). Two other channels originally identified at the tonoplast have been designated as SV and FV for Slow and Fast Vacuolar, respectively. The SV channel is voltage-, H+- and Ca2+-dependent and is permeable to Ca2+ and K+ ( Ward and Schroeder, 1994; SchulzLessdorf and Hedrich, 1995; Allen and Sanders, 1996). Estimates of the permeability of this channel are highly variable ( Ward and Schroeder, 1994; Schulz-Lessdorf and Hedrich, 1995; Allen and Sanders, 1996). The FV channel has also been shown to be voltage-dependent, to be inhibited by high [Ca2+] , and to activate and deactivate i rapidly (Hedrich and Neher, 1987). Recent findings have been estimated K+/Cl− selectivity of this channel in barley mesophyll to be as high as 3051, indicating the importance of this channel for tonoplast electrogenesis and K+ transport ( Tikhonova et al., 1997). It now appears that all three cation channels co-exist in the guard cell vacuole (Allen and Sanders, 1996), raising the possibility that their interaction may be important for guard cell function. Finally an anion channel with high selectivity for Cl− over K+ has recently been found at the tonoplast of Vicia faba guard cells. The activity of this channel is dependent on protein phosphorylation (Pei et al., 1996). This channel could function in anion uptake during stomatal opening, however, no direct evidence is currently available. Ion channel control In parallel with this growing catalogue of ion channels, our understanding of mechanisms by which these channels are controlled has expanded dramatically over the past decade. Most important for this development, the link between stomatal movement and the hormone abscisic acid has provided a key physiological basis from which to explore ion channel regulation. Abscisic acid acts as a universal stress signal in plants. It accumulates in the leaf tissues under adverse environmental conditions including drought and high salinity and promotes stomatal closure preventing transpirational water loss ( Willmer and Fricker, 1996). H+ signalling in guard cell cytoplasm The protonic messenger was first proposed to be major signalling element in the guard cell when it was recognized that ABA action on stomata is strictly associated with cytoplasm alkalinizations of 0.2–0.4 pH units (Gehring et al., 1990; Irving et al., 1992; Blatt and Armstrong, 1993; Blatt and Thiel, 1993). The key role of H+ messenger for stomatal function has been confirmed in the experiments in which ABA-induced stomatal closure was blocked when pH was clamped at a constant low value i (Blatt and Armstrong, 1993). On the molecular level, ABA induced stomatal closure is accomplished by activation of I (Blatt and Armstrong, 1993) and anion K,out channels (Grabov et al., 1997), providing electrically neutral solute efflux from the guard cells. I was found K,out to be strongly stimulated by increasing pH (Blatt, 1992; i Blatt and Armstrong, 1993; Miedema and Assmann, 1996; Grabov and Blatt, 1997), suggesting it as the major target of H+ signalling. Like the effect on stomatal closure, suppressing changes in pH effectively blocked i the I response to ABA (Blatt and Armstrong, 1993). K,out The effects of ABA mediated through pH are also i evident in the characteristics of the other major K+ channel at the plasma membrane. I was found to K,in exhibit a sensitivity to pH inverse to that of I (Blatt, i K,out 1992; Blatt and Armstrong, 1993; Grabov and Blatt, 1997), and in line with the cytosolic alkalinization imposed by ABA. pH , however, accounted only in part i for this effect (Blatt and Armstrong, 1993), suggesting an intervention of another cytosolic factor, very likely [Ca2+] i (Schroeder and Hagiwara, 1989; Blatt and Thiel, 1993; Lemtiri-Chlieh and MacRobbie, 1994; Grabov and Blatt, 1997). It is worth noting that work with another plant hormone auxin has also implicated a role for pH signalling. i Auxin at low concentration is known to open stomata (Snaith and Mansfield, 1982; Marten et al., 1991) and has been shown to reduce cytosolic pH (Irving et al., 1992). Mobilization of the protonic messenger after treatment with low concentration of auxin activates I K,in providing pathway for K+ influx (Blatt, 1992; Blatt and Thiel, 1994; Grabov and Blatt, 1997). K+ uptake is driven by the H+ pump, that, probably, also is enhanced by [ H+] elevation (Blatt, 1987; Kurkdjian and Guern, i 1989; Felle, 1989). The effect of auxin on stomatal movement, however, shows two distinct phases. While stomatal opening is promoted at low micromolar concentration, the effect of auxin at high concentrations (100 mM and above) is to close stomata (Marten et al., 1991; Lohse and Hedrich, 1992). I inhibition by high auxin concenK,in trations parallels this behaviour (Blatt and Thiel, 1994). 354 Grabov and Blatt The crucial role of pH in auxin signalling has been i elucidated in experiments where the H+ signal was disrupted by pH buffering (Blatt and Thiel, 1994). No effect i of auxin on I was observed under these experimental K,in conditions. Furthermore, peptide homologous to the C-terminus of the maize auxin binding protein mimicked high auxin concentrations and imposed a rise in cytosolic pH. The effect again was to reduce I activity and K,in cytosolic buffering abolished the peptides effect on I K,in ( Thiel et al., 1993). Experimentally, cytosolic acidification can be achieved by an addition of weak acids to the bath where protonated and dissociated forms of acid come to equilibrium (Blatt, 1992; Blatt and Armstrong, 1993; Grabov and Blatt, 1997). Provided the protonated form easily crosses the plasma membrane it will dissociate in the cytoplasm as pH is normally higher than the weak acid pK . This i a approach has been used to clamp pH imposing acid loads i on the cell and hence to titrate guard cell K+ channels against pH as the pH shift is strictly dependent on the i i weak acid concentration in the bath (Grabov and Blatt, 1997). On the basis of such experiments, low pH was found i to stimulate I while inactivating I (Blatt, 1992; K,in K,out Blatt and Armstrong, 1993; Grabov and Blatt, 1997). The precise mechanism of this interaction remains obscure. However, some conclusions may be drawn from these experiments. Voltage-dependent gating of both I K,in and I is practically unaffected by the cytosolic pH. K,out Thus, in each case the maximal channel conductivity (g ) was affected without a change in V . For I , Kmax 1/2 K,in acid pH stimulated the current, while g of outward Kmax rectifier was down-regulated under the same conditions (Grabov and Blatt, 1997). Elevated levels of H+ in the cytosol could screen membrane surface charge and, hence, distort the total electrical field encountered by the channel ( Hille, 1992). This mechanism of H+ action on the guard cell K+ channels can be ruled out because the effect of low pH on both I and I was practically voltagei K,in K,out independent at least over pH range from 6.9 to 8.0 (Blatt, i 1992; Blatt and Armstrong, 1993; Miedema and Assmann, 1996; Grabov and Blatt, 1997). It appears more likely that pH affects the number of i channels (N ) that can be gated by transmembrane voltage. Maximal conductivity of the channel ensemble is the product of N and the single channel conductivity (c ). K Thus, H+ binding could affect either c or N. For I K K,in competition between H+ and K+ for a common binding site within the channel pore (Hille, 1992) can also be ruled out. Increasing H+ concentration stimulated the current whereas competition would be expected to block it. Such a mechanism could explain a decrease in I . K,out However H+ competition is known to lead to flickery block that can appear as a reduction in single-channel conductance. These effects were not detected in patch clamp studies of the outward rectifier (Miedema and Assmann, 1996). In short, it is most likely that I is K,out regulated by pH through allosteric interactions that result i in an increase of the pool of active channels (Blatt and Armstrong, 1993; Miedema and Assmann, 1996; Grabov and Blatt, 1997). The effect of pH on the inward rectifier has yet to be i studied at the single channel level. However, the Hill model was well fitted to the pH dependence of g also Kmax in the case of I As fittings were based essentially on K,in. the assumption that H+ affects the availability of the channels for voltage-dependent gating, it is suggested that the mechanism of I regulation by cytosolic H+ is K,in similar but antiparallel to I (Grabov and Blatt, 1997). K,out Titration of g against pH indicated that I has one Kmax i K,in proton binding site with pK =6.3 suggesting the involvea ment of histidine in H+ binding. Titration of an I K,out yielded a Hill coefficient of 2 with an apparent pK of 7.4 a that was rather close to the resting pH =7.6. i The events immediately upstream of H+ signalling remains to be studied. Buffering capacity of the guard cell is enormous and corresponds to an apparent cytosolic buffer concentration of 275 mM with pK =6.9. These a calculations indicate that approximately 30 mM of H+ are bound (or released) by the cytosolic H+ binding sites when pH is modified by 0.2 pH units (Grabov and Blatt, i 1997). Where this huge H+ flux originates from is unclear. It is very likely that the vacuole is the source of protons when the guard cell is stimulated by auxin, as this plant hormone failed to acidify the cytoplasm in the vacuolefree protoplasts (Frohnmeyer et al., 1997). Ca2+ as second messenger In animal tissues Ca2+ is well known as a second messenger controlling many cellular processes including fertilization, cell growth, secretion, sensory perception, and neuronal signalling (Berridge, 1993; Bootman and Berridge, 1996). The evidence accumulated in the last decade points to a similar role for Ca2+ in the signal transduction cascades in plants. In guard cells, environmental stress, pathogens and plant hormones have been shown to evoke increases in [Ca2+] . Schroeder and i Hagiwara, 1990; McAinsh et al., 1990; Knight et al., 1991; Irving et al., 1992; Bush, 1993; McAinsh et al., 1997). ABA-induced stomatal closure may be associated with increased [Ca2+] as well. However, the physiological i significance of ABA-dependent Ca2+ signalling is still questionable, as stomata may close in ABA without any visible [Ca2+] elevation (Gilroy et al., 1991; McAinsh i et al., 1992; Allan et al., 1994). It is very likely therefore that both Ca2+-dependent and Ca2+-independent signalling cascades transduce the ABA signal in the guard cell (MacRobbie, 1992). The switch between these two signalling pathways may be triggered by ambient temperature (Allan et al., 1994). Co-ordination of ion channels in guard cells 355 Another source of concern about the Ca2+ second messenger has been its widespread occurrence and ability to mediate sometimes opposing responses. [Ca2+] i increases may lead to such different events as stomatal opening after stimulation by auxin or stomatal closure after stimulation by ABA (Bush, 1993; McAinsh et al., 1997). It is not yet known how the Ca2+ signal is targeted. However, the kinetics of [Ca2+] elevation (the ‘Ca2+ i signature’) is probably important for the encoding of information contained in the cytoplasmic signal. [Ca2+] i oscillations are well known in animal cells, where [Ca2+] i waves can be shown to propagate within the cytoplasm or between adjacent cells (Boitano et al., 1994; Hajnóczky and Thomas, 1997). The information carried by the [Ca2+] signal can be encoded by the frequency and i magnitude of such oscillations (Dolmetsch et al., 1997). Similar [Ca2+] transients and oscillations have frequently i been observed in plant tissues including guard cells in response to environmental or hormonal stimuli (Schroeder and Hagiwara, 1990; Knight et al., 1991; McAinsh et al., 1995; Johnson et al., 1995). The mechanism of [Ca2+] fluctuations in the plant cell i cytoplasm is not yet understood. Recent findings, however, indicate that elevation of [Ca2+] may be triggered i by the plasma membrane hyperpolarization. Work from this laboratory (Grabov and Blatt, 1998) has shown that [Ca2+] increases can be driven by membrane voltage i transitions between voltages near −40 mV and −200 mV ( Fig. 1). Oscillations in free-running voltage can be recorded at the guard cell plasma membrane (Gradmann et al., 1993; Blatt and Thiel, 1994). These oscillations are similar to cardiac action potentials, but occur with periods of s or even min. Co-existence of voltage-dependent [Ca2+] increases ( Fig. 1) and action potential-like voltage i oscillations may explain the [Ca2+] transients observed i Fig. 1. Hyperpolarization of the plasma membrane evokes [Ca2+] i increases. Measurement from a single Vicia guard cell in an epidermal strip bathed in 5 mM Ca2+–MES, pH 6.1 with 10 mM KCl. The guard cell was loaded with fura-2 iontophoretically. [Ca2+] was recorded as i fura-2 fluorescence ratio (F /F , low panel ). Plasma membrane was 340 390 hyperpolarized under 2-electrode voltage clamp by a 20 s voltage pulse from −40 to −200 mV (upper panel ). in the guard cells (McAinsh et al., 1997), since repetitive [Ca2+] elevation may be triggered by the hyperpolarizi ation phase of membrane voltage oscillations. Potentially, there are two pathways for elevating [Ca2+] : (1) Ca2+ influx across the plasma membrane and i (2) Ca2+ release from internal stores. Both mechanisms function in animal tissues, including cardiac muscles and neurons, and give rise to increases in [Ca2+] through i processes of Ca2+-induced Ca2+ release (CICR; (Berridge, 1993)). In these tissues, small [Ca2+] increases i triggered by Ca2+ flux through plasma membrane Ca2+ channels induce explosive Ca2+ release from internal stores (Berridge, 1993; McPherson and Campbell, 1993; Ehrlich et al., 1994; Sitsapesan et al., 1995). Similar mechanisms of CICR may operate in plant cells. At least some elements that could contribute to CICR are known to occur in plants ( Ward and Schroeder, 1994; Sanders et al., 1995). There are a few direct indications that Ca2+ channels operate in the plant cell plasma membrane. Ca2+-permeable channels activated by depolarization were found at the carrot cell plasma membrane ( Thuleau et al., 1994b). These channels also appear to be recruited at depolarized voltages into an active pool, as could be determined from prolonged prepulses ( Thuleau et al., 1994a). Piñeros and Tester found Ca2+ channels in cells of wheat roots (Piñeros and Tester, 1995). These channels were activated at the voltages more positive than −130 mV and were proposed to function as mediators of nutrient uptake. There has also been evidence of [Ca2+] i transients in guard cell protoplasts triggered by ABA. Schroeder and Hagiwara (1990) proposed that these transients were associated with the activation at the plasma membrane of Ca2+-permeable channels. Recently, Ca2+ channels activated by hyperpolarization have been found in tomato cells (Gelli and Blumwald, 1997). Tension-activated Ca2+-selective channels, have been indicated to function in onion epidermal cells (Ding and Pickard, 1993; Pickard and Ding, 1993). At the guard cell plasma membrane, there is evidence of Ca2+permeable channels that may be activated by mechanical stress (Cosgrove and Hedrich, 1991). As the regulation of osmolarity is central for stomata physiology, these channels could be important for feedback control of solute flux during stomatal movements. Somewhat more information is to hand about mechanisms of Ca2+ release within guard cells. In animal cells two major families of Ca2+ channels, identified as IP 3 and ryanodine receptors, share remarkable structural similarity and mediate Ca2+ release from internal stores (Berridge, 1993). It appears that both of these Ca2+release mechanisms operate in plants. The rise of [Ca2+] i triggered by IP (and the consequent inactivation of I ) 3 K,in was directly demonstrated by photolysis of caged IP 3 injected into guard cells (Blatt et al., 1990; Gilroy et al., 356 Grabov and Blatt 1990). Ryanodine receptors of animal cells (RyR) are usualy localized on a modified part of the ER (Berridge, 1993). Although, there is an indication for Ca2+ release channels in the ER of higher-plant mechanoreceptor organs ( Klüsener et al., 1995), no channels with sensitivity to ryanodine or cADPR (a cytoplasmic ligand for RyR; Sitsapesan et al., 1995) have been found in the plant cell ER to date. It is most probable that plant RyR-like channels are located at tonoplast, hence the release of Ca2+ from vacoular-enriched microsomes of beet was triggered by cADPR (Allen et al., 1995; Muir and Sanders, 1996). A special mechanism of CICR has been suggested to function in Ca2+ mobilization in plant cells in which vacuolar SV channels mediate Ca2+ release and act in concert with K+-selective VK channels ( Ward et al., 1995). The proposal is consistent with the co-existence of these channels in guard cell vacuoles (Allen and Sanders, 1996). However, the contribution of SV channels to CICR has been questioned, because the channel open probability is extremely low at the physiologically relevant tonoplast voltages, vacuolar and cytosolic [Ca2+] (Pottosin et al., 1997). Elevation of [Ca2+] has no effect on I (Schroeder i K,out and Hagiwara, 1989; Blatt and Thiel, 1993; LemtiriChlieh and MacRobbie, 1994), while [Ca2+] has been i demonstrated to be a very potent regulator of I and K,in the anion current at the guard cell plasma membrane (Schroeder and Hagiwara, 1989; Hedrich et al., 1990; Blatt and Thiel, 1993; Lemtiri-Chlieh and MacRobbie, 1994; Kelly et al., 1995). Elevating [Ca2+] from 0.1 to i above 1 mM has been shown to activate the anion current and to inactivate I almost completely (Schroeder and K,in Hagiwara, 1989; Kelly et al., 1995). Details of the mechanisms by which [Ca2+] acts on these channels is less i clear. Increasing [Ca2+] affects the voltage-dependence of i gating rather than single channel conductance giving rise to I inactivation (Schroeder and Hagiwara, 1989, K,in Grabov and Blatt, 1997). It is interesting that I was largely unaffected by high K,in [Ca2+] when EGTA was used as the Ca2+ buffer in patchi clamp experiments ( Kelly et al., 1995). Kelly et al. (1995) suggested that this dependence on the buffer was related to the dynamics of Ca2+ binding with the buffer. However, as it is not clear how the kinetic properties of the Ca2+ buffer affect steady-state [Ca2+] , it is difficult i to explain the difference in I characteristics between K,in the two buffers. Whatever the explanation for their observations, the data make it clear that estimating the Ca2+sensitivity of I in vivo is not straightforward in patch K,in clamp experiments. Roles for protein phosphorylation in guard cell signalling The phosphorylation state of key regulatory (or target) proteins may drastically affect ion transport. At the guard cell plasma membrane these effects are seen in H+ pumping (Shimazaki et al., 1992; Li and Assmann, 1996), K+ channels (Luan et al., 1993; Li and Assmann, 1996; Thiel and Blatt, 1997) and slow anion channels (Schmidt et al., 1995; Grabov et al., 1997). At the tonoplast SV channels have been shown to be regulated by calcineurin (protein phosphatase 2B) (Allen and Sanders, 1995). The recognition of phosphatase-kinase activities in guard cells signalling was boosted in recent years by the cloning of the ABI1 gene, the mutation of which leads to a loss of sensitivity to ABA in Arabidopsis ( Koornneef et al., 1984). Sequence and biochemical analysis of the gene product has confirmed that it functions as a 2C-type protein phosphatase (Leung et al., 1994; Meyer et al., 1994; Bertauche et al., 1996). Thus, the abi1 mutant provides an invaluable tool for the study of ABA signalling. Armstrong et al. (1995) have since linked the mutant phenotype with abberant control of the guard cell K+ channels using transgenic N. benthamiana plants carrying the mutant abi1-1 gene. These studies showed that the abi1–1 gene not only reduced I in the absence of K,out ABA, but eliminated the response of the current in its presence. The transgene was also found to interfere with ABA-evoked control of I and to block stomatal closK,in ure. Furthermore, the background of I activity, as K,out well as I and I responses to ABA and stomatal K,in K,out closure could be ‘rescued’ by treatments with protein kinase antagonists. So, how can abi1 gene action be understood? The simplest explanation is that the (dominant) mutant protein phosphatase interferes with a wildtype homologue in tobacco preventing protein dephosphorylation. The kinase antagonists, then, redress the consequent imbalance in phosphorylation of the target protein(s) to reinstate K+ channel sensitivity to ABA. This interpretation is entirely consistent with the phosphatase activities shown by the wild-type and mutant gene products (Bertauche et al., 1996). Anion channel function is also known to respond to protein (de-)phosphorylation. Evidence from pharmacological studies have shown that protein phosphatase antagonists such as okadaic acid and calyculin A (both protein phosphatase 1/2A antagonists) alter anion channel characteristics ( Thiel and Blatt, 1994) and rundown in patch electrode recordings of guard cell protoplasts (Schmidt et al., 1995). Recently, slow anion channels were found to be activated by ABA in guard cells of N. benthamiana (Grabov et al., 1997), and Arabidopsis (Pei et al., 1997). Again the activation was dependent on the phosphorylation state of the proteins involved in the signal transduction cascade. Pei et al. (1997) compared anion currents measured either in the presence or absence of ABA and/or with additions of okadaic acid. Statistical comparisons indicated that the current was activated by ABA and that this activation could be suppressed by Co-ordination of ion channels in guard cells 357 okadaic acid. The effect appeared to be a purely scalar increase in the number of channels in the active pool and was not observed in abi1 mutant plants. Grabov et al. (1997), by contrast, made use of intracellular microelectrode methods, recording anion currents from individual guard cells before and throughout treatments with ABA and calyculin A. They found that ABA reversibly stimulated the anion current and that this stimulation was associated with alterations in the kinetics of voltagedependent activation that favoured the active state of the channels. Grabov et al. (1997) also observed that calyculin A acted synergistically with ABA in promoting the anion current, and that the effects in every case were independent of the abi1 transgene. How can these two sets of data be reconciled? Pei et al. (1997) based their assessment on a contentious comparison of the Arabidopsis anion current in the presence of ABA with the N. benthamiana current (Grabor et al., 1997) recorded in the absence of ABA. In fact, resting activities of the anion channels were roughly equivalent, despite the obvious difference of species and very different methods used to record these currents. It is likely that subtle differences in kinase/phosphatase cascades mean that Arabidopsis and N. benthamiana respond differently to protein phosphatase antagonists (compare also Schmidt et al., 1995), and this factor might well explain the discrepancy in action of the abi1 gene product. Equally important, technical differences—including exchange of the cytosol with patch electrode filling solutions (Pei et al., 1997)—raises the possibility that different parts of the same kinase/phosphatase cascade could be favoured in each case. Regardless of these complications, the fact that both sets of experiments demonstrate an action of ABA on the anion current is salutary. Interaction between signalling elements and its significance for stomata regulation It comes as no surprise that signalling pathways should interact, even when evoked by a single stimulus such as ABA ( Trewavas, 1992). Sufficient evidence is now to hand to demonstrate interactions between signalling elements of protein (de-)phosphorylation, [Ca2+] and pH . i i What is less clear at present are the points of interaction and the extent to which the various signals are interdependent. These issues are particularly relevant both to determining the hierarchy of events behind stimulusresponse coupling per se, and to establishing the role(s) of each element, whether primary to signal transmission or secondary in adaptive conditioning of the response. Some progress is now being made through epistatic and equivalent physiological analyses. However, these problems will continue to pose major challenges to understanding guard cell transport control, as it does to plant cell signalling generally. A case in point can be drawn from work on pH and i [Ca2+] signals evoked by ABA. From analysis of the i current kinetics and voltage-dependence, it is evident that pH and [Ca2+] controls of I are fundamentally differi i K,in ent (Schroeder and Hagiwara, 1989; Blatt and Armstrong, 1993; Grabov and Blatt, 1997). Nonetheless, these two signalling elements almost certainly interact as well in guard cells. Grabov and Blatt (1997) observed that experimentally imposing a decrease in pH below about 7.0 i resulted in the rise of [Ca2+] in approx. 50% of the cells i examined. The precise mechanism of this pH-induced [Ca2+] rise is unknown. One possible explanation lies in i the pH-sensitivity of Ca2+ release mechanisms within the cell, for example the binding of IP to its receptor that 3 leads to cytosolic Ca2+ release (Busa, 1986), although it is normally alkaline rather than acid pH favours IP i 3 mediated Ca2+ release (Taylor and Richardson, 1991). In the guard cells pH is known to affect the gating of i vacuolar ion channels that may contribute to [Ca2+] i changes either directly or indirectly ( Ward and Schroeder, 1994; Schulz-Lessdorf and Hedrich, 1995). It is also possible that pH could affect Ca2+ influx across the i plasma membrane. Elevation of [Ca2+] in turn potentially may affect i phosphorylation cascades. Protein kinases and phosphatases dependent on Ca2+ are ubiquitous in animal cells and serve as one point of convergence for Ca2+ signalling and protein phosphorylation cascades. In plant cells there is now considerable evidence for Ca2+-, Ca2+/calmodulinand Ca2+/phospholipid-dependent kinases (Nickel et al., 1991; Shimazaki et al., 1992; Watillon et al., 1993) and phophatases (Luan et al., 1993; Allen and Sanders, 1995). Calcium-dependent protein kinase activity is known to activate an ABA-responsive promoter in Arabidopsis leaf protoplasts (Sheen, 1997) and a Cl− channel in the guard cell tonoplast (Pei et al., 1996). It is interesting that the ABI1 gene product, although it contains a putative EF-hand Ca2+-binding motif and was expected to be Ca2+-regulated, has proven to be insensitive to [Ca2+] i (Bertauche et al., 1996). Finally, protein phosphorylation also affects pH signal i transduction. Blatt and Grabov (1997b) have reported that the abi1 transgene in N. benthamiana drastically affects K+ channel sensitivity to experimentally-imposed changes in pH . They found that reducing pH with 3 and i i 10 mM butyrate, equivalent to reductions of 0.3–0.5 pH i units, had only marginal effects on the K+ currents in the transgenic plants compared with the wild type. The observations are significant, because Armstrong et al. (1995) noted that the mutant transgene affected the K+ channel response to ABA but had no effect on the rise in pH evoked by the hormone. Thus, these two sets of data i indicate that events downstream of pH are dependent on i the phosphorylation state of one or more target proteins. In conclusion, there can be little doubt now about the 358 Grabov and Blatt complexity of signalling events that underlie stomatal control. The emergence of pH as a second messenger, i along with [Ca2+] and protein phosphorylation events, i emphasizes our relative ignorance about the origins and interactions of each of these components, quite apart from the mechanism(s) by which each targets downstream elements. 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