Co-Ordination of Signalling Elements in Guard Cell Ion Channel Control

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 ably important for providing a plasticity of cellular response to external and environmental stimuli. Fine regulation of solutes transport across the guard Understanding the interdependence and hierarchy of cell plasma membrane for osmotic modulation is signalling elements now presents a major challenge essential for the maintenance of the proper stomatal for research in plant biology. aperture in response to environmental stimuli. The major osmotica, K+,Cl− and malate are transported Key words: Ion channels, stomatal aperture, signalling through selective ion channels in the plasma mem- pathway, second messengers, guard cells. brane 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 inwardly- Introduction + rectifying K channels (IK,in and IK,out), slowly- and Guard cell control of stomatal aperture is crucial to rapid-activating anion channels, and stretch-activated balancing transpiration stream and gas exchange for non-selective channels. Slow and fast vacuolar chan- photosynthesis. As transpiration and CO2 exchange can nels (SV and FV) and voltage-independent K+-selective not be regulated independently, the demand of photosyn- (VK) channels have been found at the guard cell tono- thetic machinery can lead to excessive water evaporation plast. On the molecular level, the regulation of the under adverse environmental conditions. Thus, the size stomatal aperture is achieved by precise spatial and of the stomatal aperture optimizes gas exchange for temporal co-ordination of channel activities through a overall plant performance. This balance of requirements network of signalling cascades that can be triggered, is achieved by the precise tuning of guard cell turgor among others, by plant hormones. ABA and auxin as pressure and on the molecular level is accomplished by regulators of stomatal aperture have received most means of fine temporal and spatial co-ordination of attention to date. It is clear now that the effect of different solute transporters. It is well known that K+, these hormones on ion channels is mediated by second Cl−, malate, and some other organic anions are the major 2+ messengers pHi and [Ca ]i. The effect of ABA is gen- contributors to the guard cell osmolarity (Willmer and erally associated with increases in pHi, while auxin Fricker, 1996). Guard cell response to the variety of the acidifies the cytosol. Both of these hormones may hormonal and environmental stimuli is due to the rapid 2+ induce elevation in [Ca ]i. Phosphorylation is another transport of these ions across the plasma membrane and important factor in cellular signalling. The ABI1 gene tonoplast (Willmer and Fricker, 1996; Blatt and Grabov, encoding a 2C-type protein phosphatase has been 1997a, b). shown to be a key element of ABA-dependent cas- The variety of environmental and hormonal stimuli ff cades. Plasma membrane voltage is also an important a ecting stomatal aperture is enormous and includes CO2, component of signalling and channel control, and has light, temperature, gaseous pollutants, as well as all of recently been shown to influence cytosolic-free the major plant hormones. Remarkably, on the cellular [Ca2+]. Thus, transduction of these, and associated level these stimuli appear to converge on a few cyto- cytoplasmic signals is clearly non-linear, and is prob- plasmic signals, known as second messengers, and these

1 To whom correspondence should be addressed. Fax: +44 1233 813 140.

© Oxford University Press 1998 352 Grabov and Blatt in turn regulate a handful of transport proteins. Second 1988; Blatt and Thiel, 1993). Activation and deactivation + 2+ messengers like cytosolic-free H (pHi) and Ca kinetics of IK,out in Vicia faba guard cells slow with 2+ + ([Ca ]i) are key components of signal transduction elevation of external K and voltage dependent opening chains in stomata aperture regulation (Blatt and Thiel, follows EK ensuring that IK,out operates as one-way valve 1993; Assmann, 1993; Bush, 1995; McAinsh et al., 1997; to prevent reflux of K+ into the guard cell. Recent studies Blatt and Grabov, 1997b). Phosphorylation of some have shown that IK,out activation is dependent on the signal cascade and target elements also contributes to co-operative interaction of 2 K+ ions with the channel, these events (Pei et al., 1996, 1997; Blatt and Grabov, but at a site (or sites) distinct from the channel pore. The 1997a; Grabov et al., 1997). Finaly, membrane voltage is apparent K1/2 for interaction was strongly voltage- an essential in controlling transport proteins in the mem- dependent, accounting for the equivalence of (negative) brane, the activities of which are voltage sensitive, and in membrane voltage and [K+] in regulating channel activity second messenger generation (Thiel et al., 1992). (Blatt and Gradmann, 1997). A positive shift in the plasma membrane potential is essential for IK,out activation when the membrane voltage Guard cell ion channels is situated negative of EK. Plasma membrane depolarization under these conditions is evoked by an inward Plasma membrane channels current (Thiel et al., 1992; Ward et al., 1995; Blatt and There are at least three different types of ion channels in Grabov, 1997b). At least one component of this current the guard cell plasma membrane implicated for the regula- has been suggested to be mediated by anion channels tion of stomatal aperture (Blatt, 1991; Blatt and Grabov, (Hedrich et al., 1990; Thiel et al., 1992; Schroeder and Keller, 1992). Two types of anion current with markedly 1997a): outwardly rectifying K+ channels (I ), K,out different kinetics have been identified in the guard cell inwardly rectifying K+ channels (I ), and anion chan- K,in plasma membrane. One of these currents demonstrates nels. Experimental factors that control the activities of rapid activation on membrane depolarization (t = these channels have given us much insight into possible 1/2 50 ms) and inactivates with t ~10 s at voltages near to regulatory mechanisms even without reference to any 1/2 and positive of −100 mV ( Keller et al., 1989; Hedrich external stimuli. et al., 1990). The second anion current has been shown Inwardly rectifying K+ channels are involved in K+ to activate and deactivate slowly (t =5–30 s) and shows uptake during stomatal opening (Schroeder, 1988; Blatt, 1/2 no time-dependent inactivation (Schroeder and Keller, 1988, 1991; Grabov and Blatt, 1997). IK,in is only moder- 1992; Linder and Raschke, 1992). ately sensitive to membrane voltage showing an apparent Of these two anion currents, the slow current is implic- gating charge of 1.3–1.7 (Blatt, 1992; Grabov and Blatt, ated for sustained long-term depolarization (Schroeder 1997). However, IK,in is strongly dependent upon apoplastic and Hagiwara, 1989; Schroeder and Keller, 1992; Linder + + pH. Increasing [H ] outside promotes the K current in and Raschke, 1992; Grabov et al., 1997). Both types of a voltage-dependent maner shifting V by 22 mV per pH 2+ 1/2 anion currents are activated with increasing [Ca ]i and decade and accelerating activation kinetics (Blatt, 1992). exhibit roughly similar single channel conductance. In At neutral external pH these channels are activated by fact it has been suggested that the same channel protein − membrane voltages more negative than 100 mV with may account for both gating modes (Blatt and Thiel, − V1/2 close to 200 mV, but at pH 6.0 IK,in is gated at 1993). Indeed, changes in anion current kinetics are − more positive voltages (V1/2 # 180mV).Thecurrentis known to occur in response to ATP depletion (Thomine also sensitive to cytosolic pH. Lowering pHi from pH 7.6 et al., 1995), ABA stimulation or phosphorylation to 6.9 stimulates IK,in by a factor of up to 6. (Grabov and (Grabov et al., 1997). Schulz-Lessdorf et al. (1996) have Blatt, 1997). The channel activity is also reduced at elevated argued that fast activating anion channels recognize the 2+ [Ca ]i (Schroeder and Hagiwara, 1989; Lemtiri-Chlieh H+ gradient across the plasma membrane. Regardless of and MacRobbie, 1994; Grabov and Blatt, 1997). By the relationships between the two anion currents, the fast contrast to the situation with pH no kinetic detail is activating form can be ruled out as a major pathway for ffl available for IK,in activity and its gating characteristics as anion e ux during stomatal closure. Its narrow voltage 2+ a function of [Ca ]i. range for activation and the fact that the current inactiv- The outwardly rectifying K+ channel is activated by ates within seconds cannot be reconciled with the require- depolarization and has been demonstrated to contribute ment for sustained channel functioning over periods of to K+ efflux during stomatal closure. This channel is 20–30 min or more during closure (Blatt and Grabov, up-regulated by pHi increases and is virtually insensitive 1997a; Blatt and Thiel, 1993). 2+ to [Ca ]i (Hosoi et al., 1988; Blatt and Armstrong, 1993; Lemtiri-Chlieh and MacRobbie, 1994; Grabov and Blatt, Tonoplast channels 1997). IK,out voltage sensitivity and kinetics are dependent As the vacuole occupied of approximately 80–90% of the upon the K+ gradient across the plasma membrane (Blatt, 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 preventing transpirational water loss (Willmer and originates from the vacuole. Equally, osmotic accumula- Fricker, 1996). tion on stomatal opening is associated predominantly with this storage organelle. The importance of vacuole in H+ signalling in guard cell cytoplasm this function implies a high degree of transport co-ordination at the tonoplast and between the two The protonic messenger was first proposed to be major membranes (MacRobbie, 1995a, b). signalling element in the guard cell when it was recognized Current knowledge of tonoplast ion transport relevant that ABA action on stomata is strictly associated with to stomata movement remains relatively poor. To date, cytoplasm alkalinizations of 0.2–0.4 pH units (Gehring patch-clamp measurements have uncovered three different et al., 1990; Irving et al., 1992; Blatt and Armstrong, types of cation-selective channels operating at the tono- 1993; Blatt and Thiel, 1993). The key role of H+ messen- plast. One channel recently identified appears to be volt- ger for stomatal function has been confirmed in the age-independent and selective for K+ and has been experiments in which ABA-induced stomatal closure was designated the VK channel (Ward and Schroeder, 1994; blocked when pHi was clamped at a constant low value Allen and Sanders, 1996). This channel is activated by (Blatt and Armstrong, 1993). On the molecular level, 2+ ABA induced stomatal closure is accomplished by activa- elevated [Ca ]i within physiological range (Allen and Sanders, 1996). Two other channels originally identified tion of IK,out (Blatt and Armstrong, 1993) and anion at the tonoplast have been designated as SV and FV for channels (Grabov et al., 1997), providing electrically ffl Slow and Fast Vacuolar, respectively. The SV channel is neutral solute e ux from the guard cells. IK,out was found voltage-, H+- and Ca2+-dependent and is permeable to to be strongly stimulated by increasing pHi (Blatt, 1992; Ca2+ and K+ (Ward and Schroeder, 1994; Schulz- Blatt and Armstrong, 1993; Miedema and Assmann, Lessdorf and Hedrich, 1995; Allen and Sanders, 1996). 1996; Grabov and Blatt, 1997), suggesting it as the major Estimates of the permeability of this channel are highly target of H+ signalling. Like the effect on stomatal ff variable (Ward and Schroeder, 1994; Schulz-Lessdorf and closure, suppressing changes in pHi e ectively blocked Hedrich, 1995; Allen and Sanders, 1996). The FV channel the IK,out response to ABA (Blatt and Armstrong, 1993). ff has also been shown to be voltage-dependent, to be The e ects of ABA mediated through pHi are also 2+ + inhibited by high [Ca ]i, and to activate and deactivate evident in the characteristics of the other major K rapidly (Hedrich and Neher, 1987). Recent findings have channel at the plasma membrane. IK,in was found to been estimated K+/Cl− selectivity of this channel in exhibit a sensitivity to pHi inverse to that of IK,out (Blatt, barley mesophyll to be as high as 3051, indicating the 1992; Blatt and Armstrong, 1993; Grabov and Blatt, importance of this channel for tonoplast electrogenesis 1997), and in line with the cytosolic alkalinization and K+ transport (Tikhonova et al., 1997). It now imposed by ABA. pHi, however, accounted only in part appears that all three cation channels co-exist in the for this effect (Blatt and Armstrong, 1993), suggesting an 2+ guard cell vacuole (Allen and Sanders, 1996), raising the intervention of another cytosolic factor, very likely [Ca ]i possibility that their interaction may be important for (Schroeder and Hagiwara, 1989; Blatt and Thiel, 1993; guard cell function. Finally an anion channel with high Lemtiri-Chlieh and MacRobbie, 1994; Grabov and selectivity for Cl− over K+ has recently been found at Blatt, 1997). the tonoplast of Vicia faba guard cells. The activity of It is worth noting that work with another plant hor- this channel is dependent on protein phosphorylation (Pei mone auxin has also implicated a role for pHi signalling. et al., 1996). This channel could function in anion uptake Auxin at low concentration is known to open stomata during stomatal opening, however, no direct evidence is (Snaith and Mansfield, 1982; Marten et al., 1991) and currently available. has been shown to reduce cytosolic pH (Irving et al., 1992). Mobilization of the protonic messenger after treat- ment with low concentration of auxin activates I Ion channel control K,in providing pathway for K+ influx (Blatt, 1992; Blatt and In parallel with this growing catalogue of ion channels, Thiel, 1994; Grabov and Blatt, 1997). K+ uptake is our understanding of mechanisms by which these channels driven by the H+ pump, that, probably, also is enhanced + are controlled has expanded dramatically over the past by [H ]i elevation (Blatt, 1987; Kurkdjian and Guern, decade. Most important for this development, the link 1989; Felle, 1989). The effect of auxin on stomatal between stomatal movement and the hormone abscisic movement, however, shows two distinct phases. While acid has provided a key physiological basis from which stomatal opening is promoted at low micromolar concen- to explore ion channel regulation. Abscisic acid acts as a tration, the effect of auxin at high concentrations (100 mM universal stress signal in plants. It accumulates in the leaf and above) is to close stomata (Marten et al., 1991; Lohse tissues under adverse environmental conditions including and Hedrich, 1992). IK,in inhibition by high auxin concen- drought and high salinity and promotes stomatal closure trations parallels this behaviour (Blatt and Thiel, 1994). 354 Grabov and Blatt

The crucial role of pHi in auxin signalling has been clamp studies of the outward rectifier (Miedema and + elucidated in experiments where the H signal was dis- Assmann, 1996). In short, it is most likely that IK,out is ff ff rupted by pHi bu ering (Blatt and Thiel, 1994). No e ect regulated by pHi through allosteric interactions that result of auxin on IK,in was observed under these experimental in an increase of the pool of active channels (Blatt and conditions. Furthermore, peptide homologous to the Armstrong, 1993; Miedema and Assmann, 1996; Grabov C-terminus of the maize auxin binding protein mimicked and Blatt, 1997). ff high auxin concentrations and imposed a rise in cytosolic The e ect of pHi on the inward rectifier has yet to be ff pH. The e ect again was to reduce IK,in activity and studied at the single channel level. However, the Hill ff ff cytosolic bu ering abolished the peptides e ect on IK,in model was well fitted to the pH dependence of gKmax also (Thiel et al., 1993). in the case of IK,in. As fittings were based essentially on Experimentally, cytosolic acidification can be achieved the assumption that H+ affects the availability of the by an addition of weak acids to the bath where protonated channels for voltage-dependent gating, it is suggested that + and dissociated forms of acid come to equilibrium (Blatt, the mechanism of IK,in regulation by cytosolic H is 1992; Blatt and Armstrong, 1993; Grabov and Blatt, similar but antiparallel to IK,out (Grabov and Blatt, 1997). 1997). Provided the protonated form easily crosses the Titration of gKmax against pHi indicated that IK,in has one = plasma membrane it will dissociate in the cytoplasm as proton binding site with pKa 6.3 suggesting the involve- + pHi is normally higher than the weak acid pKa. This ment of histidine in H binding. Titration of an IK,out ffi approach has been used to clamp pHi imposing acid loads yielded a Hill coe cient of 2 with an apparent pKa of 7.4 = on the cell and hence to titrate guard cell K+ channels that was rather close to the resting pHi 7.6. + against pHi as the pHi shift is strictly dependent on the The events immediately upstream of H signalling weak acid concentration in the bath (Grabov and Blatt, remains to be studied. Buffering capacity of the guard 1997). cell is enormous and corresponds to an apparent cytosolic ff = On the basis of such experiments, low pH was found bu er concentration of 275 mM with pKa 6.9. These i + to stimulate IK,in while inactivating IK,out (Blatt, 1992; calculations indicate that approximately 30 mM of H Blatt and Armstrong, 1993; Grabov and Blatt, 1997). are bound (or released) by the cytosolic H+ binding sites The precise mechanism of this interaction remains when pHi is modified by 0.2 pH units (Grabov and Blatt, obscure. However, some conclusions may be drawn from 1997). Where this huge H+ flux originates from is unclear. these experiments. Voltage-dependent gating of both IK,in It is very likely that the vacuole is the source of protons ff when the guard cell is stimulated by auxin, as this plant and IK,out is practically una ected by the cytosolic pH. Thus, in each case the maximal channel conductivity hormone failed to acidify the cytoplasm in the vacuole- ff free protoplasts (Frohnmeyer et al., 1997). (gKmax) was a ected without a change in V1/2. For IK,in, acid pH stimulated the current, while g of outward Kmax Ca2+ as second messenger rectifier was down-regulated under the same conditions (Grabov and Blatt, 1997). Elevated levels of H+ in the In animal tissues Ca2+ is well known as a second messen- cytosol could screen membrane surface charge and, hence, ger controlling many cellular processes including fertiliza- distort the total electrical field encountered by the channel tion, cell growth, secretion, sensory perception, and (Hille, 1992). This mechanism of H+ action on the guard neuronal signalling (Berridge, 1993; Bootman and cell K+ channels can be ruled out because the effect of Berridge, 1996). The evidence accumulated in the last low pHi on both IK,in and IK,out was practically voltage- decade points to a similar role for Ca2+ in the signal independent at least over pHi range from 6.9 to 8.0 (Blatt, transduction cascades in plants. In guard cells, environ- 1992; Blatt and Armstrong, 1993; Miedema and Assmann, mental stress, pathogens and plant hormones have been 2+ 1996; Grabov and Blatt, 1997). shown to evoke increases in [Ca ]i. Schroeder and ff It appears more likely that pHi a ects the number of Hagiwara, 1990; McAinsh et al., 1990; Knight et al., channels (N) that can be gated by transmembrane voltage. 1991; Irving et al., 1992; Bush, 1993; McAinsh et al., Maximal conductivity of the channel ensemble is the 1997). ABA-induced stomatal closure may be associated 2+ product of N and the single channel conductivity (cK). with increased [Ca ]i as well. However, the physiological + ff 2+ Thus, H binding could a ect either cK or N. For IK,in significance of ABA-dependent Ca signalling is still competition between H+ and K+ for a common binding questionable, as stomata may close in ABA without any 2+ site within the channel pore (Hille, 1992) can also be visible [Ca ]i elevation (Gilroy et al., 1991; McAinsh ruled out. Increasing H+ concentration stimulated the et al., 1992; Allan et al., 1994). It is very likely therefore current whereas competition would be expected to block that both Ca2+-dependent and Ca2+-independent signal- it. Such a mechanism could explain a decrease in IK,out. ling cascades transduce the ABA signal in the guard cell However H+ competition is known to lead to flickery (MacRobbie, 1992). The switch between these two signal- block that can appear as a reduction in single-channel ling pathways may be triggered by ambient temperature conductance. These effects were not detected in patch (Allan et al., 1994). Co-ordination of ion channels in guard cells 355 Another source of concern about the Ca2+ second in the guard cells (McAinsh et al., 1997), since repetitive 2+ messenger has been its widespread occurrence and ability [Ca ]i elevation may be triggered by the hyperpolariz- 2+ to mediate sometimes opposing responses. [Ca ]i ation phase of membrane voltage oscillations. increases may lead to such different events as stomatal Potentially, there are two pathways for elevating 2+ 2+ opening after stimulation by auxin or stomatal closure [Ca ]i:(1)Ca influx across the plasma membrane and after stimulation by ABA (Bush, 1993; McAinsh et al., (2) Ca2+ release from internal stores. Both mechanisms 1997). It is not yet known how the Ca2+ signal is targeted. function in animal tissues, including cardiac muscles and 2+ 2+ 2+ However, the kinetics of [Ca ]i elevation (the ‘Ca neurons, and give rise to increases in [Ca ]i through signature’) is probably important for the encoding of processes of Ca2+-induced Ca2+ release (CICR; 2+ 2+ information contained in the cytoplasmic signal. [Ca ]i (Berridge, 1993)). In these tissues, small [Ca ]i increases 2+ 2+ 2+ oscillations are well known in animal cells, where [Ca ]i triggered by Ca flux through plasma membrane Ca waves can be shown to propagate within the cytoplasm channels induce explosive Ca2+ release from internal or between adjacent cells (Boitano et al., 1994; Hajnoćzky stores (Berridge, 1993; McPherson and Campbell, 1993; and Thomas, 1997). The information carried by the Ehrlich et al., 1994; Sitsapesan et al., 1995). Similar 2+ [Ca ]i signal can be encoded by the frequency and mechanisms of CICR may operate in plant cells. At least magnitude of such oscillations (Dolmetsch et al., 1997). some elements that could contribute to CICR are known 2+ Similar [Ca ]i transients and oscillations have frequently to occur in plants (Ward and Schroeder, 1994; Sanders been observed in plant tissues including guard cells in et al., 1995). response to environmental or hormonal stimuli There are a few direct indications that Ca2+ channels (Schroeder and Hagiwara, 1990; Knight et al., 1991; operate in the plant cell plasma membrane. Ca2+-per- McAinsh et al., 1995; Johnson et al., 1995). meable channels activated by depolarization were found 2+ The mechanism of [Ca ]i fluctuations in the plant cell at the carrot cell plasma membrane (Thuleau et al., cytoplasm is not yet understood. Recent findings, how- 1994b). These channels also appear to be recruited at 2+ ever, indicate that elevation of [Ca ]i may be triggered depolarized voltages into an active pool, as could be by the plasma membrane hyperpolarization. Work from determined from prolonged prepulses (Thuleau et al., this laboratory (Grabov and Blatt, 1998) has shown that 1994a). Pin˜eros and Tester found Ca2+ channels in cells 2+ [Ca ]i increases can be driven by membrane voltage of wheat roots (Pin˜eros and Tester, 1995). These channels transitions between voltages near −40 mV and −200 were activated at the voltages more positive than −130 mV (Fig. 1). Oscillations in free-running voltage can be mV and were proposed to function as mediators of 2+ recorded at the guard cell plasma membrane (Gradmann nutrient uptake. There has also been evidence of [Ca ]i et al., 1993; Blatt and Thiel, 1994). These oscillations are transients in guard cell protoplasts triggered by ABA. similar to cardiac action potentials, but occur with periods Schroeder and Hagiwara (1990) proposed that these of s or even min. Co-existence of voltage-dependent transients were associated with the activation at the 2+ 2+ [Ca ]i increases (Fig. 1) and action potential-like voltage plasma membrane of Ca -permeable channels. Recently, 2+ 2+ oscillations may explain the [Ca ]i transients observed Ca 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 2+ two major families of Ca channels, identified as IP3 and ryanodine receptors, share remarkable structural 2+ 2+ similarity and mediate Ca release from internal stores Fig. 1. Hyperpolarization of the plasma membrane evokes [Ca ]i 2+ increases. Measurement from a single Vicia guard cell in an epidermal (Berridge, 1993). It appears that both of these Ca - 2+ strip bathed in 5 mM Ca2+–MES, pH 6.1 with 10 mM KCl. The guard release mechanisms operate in plants. The rise of [Ca ]i cell was loaded with fura-2 iontophoretically. [Ca2+] was recorded as i triggered by IP3 (and the consequent inactivation of IK,in) fura-2 fluorescence ratio (F340/F390, low panel ). Plasma membrane was hyperpolarized under 2-electrode voltage clamp by a 20 s voltage pulse was directly demonstrated by photolysis of caged IP3 from −40 to −200 mV (upper panel ). injected into guard cells (Blatt et al., 1990; Gilroy et al., 356 Grabov and Blatt 1990). Ryanodine receptors of animal cells (RyR) are cell plasma membrane these effects are seen in H+ pump- usualy localized on a modified part of the ER (Berridge, ing (Shimazaki et al., 1992; Li and Assmann, 1996), K+ 1993). Although, there is an indication for Ca2+ release channels (Luan et al., 1993; Li and Assmann, 1996; Thiel channels in the ER of higher-plant mechanoreceptor and Blatt, 1997) and slow anion channels (Schmidt et al., organs (Klu¨sener et al., 1995), no channels with sensitivity 1995; Grabov et al., 1997). At the tonoplast SV channels to ryanodine or cADPR (a cytoplasmic ligand for RyR; have been shown to be regulated by calcineurin (protein Sitsapesan et al., 1995) have been found in the plant cell phosphatase 2B) (Allen and Sanders, 1995). The recog- ER to date. It is most probable that plant RyR-like nition of phosphatase-kinase activities in guard cells channels are located at tonoplast, hence the release of signalling was boosted in recent years by the cloning of Ca2+ from vacoular-enriched microsomes of beet was the ABI1 gene, the mutation of which leads to a loss of triggered by cADPR (Allen et al., 1995; Muir and sensitivity to ABA in Arabidopsis (Koornneef et al., Sanders, 1996). 1984). Sequence and biochemical analysis of the gene A special mechanism of CICR has been suggested to product has confirmed that it functions as a 2C-type function in Ca2+ mobilization in plant cells in which protein phosphatase (Leung et al., 1994; Meyer et al., vacuolar SV channels mediate Ca2+ release and act in 1994; Bertauche et al., 1996). Thus, the abi1 mutant concert with K+-selective VK channels (Ward et al., provides an invaluable tool for the study of ABA 1995). The proposal is consistent with the co-existence of signalling. these channels in guard cell vacuoles (Allen and Sanders, Armstrong et al. (1995) have since linked the mutant 1996). However, the contribution of SV channels to phenotype with abberant control of the guard cell K+ CICR has been questioned, because the channel open channels using transgenic N. benthamiana plants carrying probability is extremely low at the physiologically relevant the mutant abi1-1 gene. These studies showed that the 2+ tonoplast voltages, vacuolar and cytosolic [Ca ] abi1–1 gene not only reduced IK,out in the absence of (Pottosin et al., 1997). ABA, but eliminated the response of the current in its 2+ ff Elevation of [Ca ]i has no e ect on IK,out (Schroeder presence. The transgene was also found to interfere with and Hagiwara, 1989; Blatt and Thiel, 1993; Lemtiri- ABA-evoked control of IK,in and to block stomatal clos- 2+ Chlieh and MacRobbie, 1994), while [Ca ]i has been ure. Furthermore, the background of IK,out activity, as demonstrated to be a very potent regulator of IK,in and well as IK,in and IK,out responses to ABA and stomatal the anion current at the guard cell plasma membrane closure could be ‘rescued’ by treatments with protein (Schroeder and Hagiwara, 1989; Hedrich et al., 1990; kinase antagonists. So, how can abi1 gene action be Blatt and Thiel, 1993; Lemtiri-Chlieh and MacRobbie, understood? The simplest explanation is that the (domin- 2+ 1994; Kelly et al., 1995). Elevating [Ca ]i from 0.1 to ant) mutant protein phosphatase interferes with a wild- above 1 mM has been shown to activate the anion current type homologue in tobacco preventing protein dephos- and to inactivate IK,in almost completely (Schroeder and phorylation. The kinase antagonists, then, redress the Hagiwara, 1989; Kelly et al., 1995). Details of the mech- consequent imbalance in phosphorylation of the target 2+ + anisms by which [Ca ]i acts on these channels is less protein(s) to reinstate K channel sensitivity to ABA. 2+ ff clear. Increasing [Ca ]i a ects the voltage-dependence of This interpretation is entirely consistent with the phos- gating rather than single channel conductance giving rise phatase activities shown by the wild-type and mutant to IK,in inactivation (Schroeder and Hagiwara, 1989, gene products (Bertauche et al., 1996). Grabov and Blatt, 1997). Anion channel function is also known to respond to ff It is interesting that IK,in was largely una ected by high protein (de-)phosphorylation. Evidence from pharmaco- 2+ 2+ ff [Ca ]i when EGTA was used as the Ca bu er in patch- logical studies have shown that protein phosphatase ant- clamp experiments (Kelly et al., 1995). Kelly et al. (1995) agonists such as okadaic acid and calyculin A (both suggested that this dependence on the buffer was related protein phosphatase 1/2A antagonists) alter anion channel to the dynamics of Ca2+ binding with the buffer. characteristics (Thiel and Blatt, 1994) and rundown in However, as it is not clear how the kinetic properties of patch electrode recordings of guard cell protoplasts 2+ ff ff 2+ ffi the Ca bu er a ect steady-state [Ca ]i,itisdi cult (Schmidt et al., 1995). Recently, slow anion channels ff to explain the di erence in IK,in characteristics between were found to be activated by ABA in guard cells of N. the two buffers. Whatever the explanation for their obser- benthamiana (Grabov et al., 1997), and Arabidopsis (Pei vations, the data make it clear that estimating the Ca2+- et al., 1997). Again the activation was dependent on the sensitivity of IK,in in vivo is not straightforward in patch phosphorylation state of the proteins involved in the clamp experiments. signal transduction cascade. Pei et al. (1997) compared anion currents measured either in the presence or absence Roles for protein phosphorylation in guard cell signalling of ABA and/or with additions of okadaic acid. Statistical The phosphorylation state of key regulatory (or target) comparisons indicated that the current was activated by proteins may drastically affect ion transport. At the guard ABA and that this activation could be suppressed by Co-ordination of ion channels in guard cells 357 ff okadaic acid. The e ect appeared to be a purely scalar A case in point can be drawn from work on pHi and 2+ increase in the number of channels in the active pool and [Ca ]i signals evoked by ABA. From analysis of the was not observed in abi1 mutant plants. Grabov et al. current kinetics and voltage-dependence, it is evident that 2+ ff (1997), by contrast, made use of intracellular microelec- pHi and [Ca ]i controls of IK,in are fundamentally di er- trode methods, recording anion currents from individual ent (Schroeder and Hagiwara, 1989; Blatt and Armstrong, guard cells before and throughout treatments with ABA 1993; Grabov and Blatt, 1997). Nonetheless, these two and calyculin A. They found that ABA reversibly stimu- signalling elements almost certainly interact as well in lated the anion current and that this stimulation was guard cells. Grabov and Blatt (1997) observed that experi- associated with alterations in the kinetics of voltage- mentally imposing a decrease in pHi below about 7.0 2+ dependent activation that favoured the active state of the resulted in the rise of [Ca ]i in approx. 50% of the cells channels. Grabov et al. (1997) also observed that calycu- examined. The precise mechanism of this pH-induced 2+ lin A acted synergistically with ABA in promoting the [Ca ]i rise is unknown. One possible explanation lies in anion current, and that the effects in every case were the pH-sensitivity of Ca2+ release mechanisms within the independent of the abi1 transgene. cell, for example the binding of IP3 to its receptor that How can these two sets of data be reconciled? Pei et al. leads to cytosolic Ca2+ release (Busa, 1986), although it (1997) based their assessment on a contentious compar- is normally alkaline rather than acid pHi favours IP3- ison of the Arabidopsis anion current in the presence of mediated Ca2+ release (Taylor and Richardson, 1991). ff ABA with the N. benthamiana current (Grabor et al., In the guard cells pHi is known to a ect the gating of 2+ 1997) recorded in the absence of ABA. In fact, resting vacuolar ion channels that may contribute to [Ca ]i activities of the anion channels were roughly equivalent, changes either directly or indirectly (Ward and Schroeder, despite the obvious difference of species and very different 1994; Schulz-Lessdorf and Hedrich, 1995). It is also ff 2+ methods used to record these currents. It is likely that possible that pHi could a ect Ca influx across the subtle differences in kinase/phosphatase cascades mean plasma membrane. ff 2+ ff that Arabidopsis and N. benthamiana respond di erently Elevation of [Ca ]i in turn potentially may a ect to protein phosphatase antagonists (compare also phosphorylation cascades. Protein kinases and phosphat- Schmidt et al., 1995), and this factor might well explain ases dependent on Ca2+ are ubiquitous in animal cells the discrepancy in action of the abi1 gene product. Equally and serve as one point of convergence for Ca2+ signalling important, technical differences—including exchange of and protein phosphorylation cascades. In plant cells there the cytosol with patch electrode filling solutions (Pei is now considerable evidence for Ca2+-, Ca2+/calmodulin- et al., 1997)—raises the possibility that different parts of and Ca2+/phospholipid-dependent kinases (Nickel et al., the same kinase/phosphatase cascade could be favoured 1991; Shimazaki et al., 1992; Watillon et al., 1993) and in each case. Regardless of these complications, the fact phophatases (Luan et al., 1993; Allen and Sanders, 1995). that both sets of experiments demonstrate an action of Calcium-dependent protein kinase activity is known to ABA on the anion current is salutary. activate an ABA-responsive promoter in Arabidopsis leaf protoplasts (Sheen, 1997) and a Cl− channel in the guard Interaction between signalling elements and its significance cell tonoplast (Pei et al., 1996). It is interesting that the for stomata regulation ABI1 gene product, although it contains a putative EF-hand Ca2+-binding motif and was expected to be 2+ 2+ It comes as no surprise that signalling pathways should Ca -regulated, has proven to be insensitive to [Ca ]i interact, even when evoked by a single stimulus such as (Bertauche et al., 1996). ffi ff ABA (Trewavas, 1992). Su cient evidence is now to Finally, protein phosphorylation also a ects pHi signal hand to demonstrate interactions between signalling ele- transduction. Blatt and Grabov (1997b) have reported 2+ ments of protein (de-)phosphorylation, [Ca ]i and pHi. that the abi1 transgene in N. benthamiana drastically What is less clear at present are the points of interaction affects K+ channel sensitivity to experimentally-imposed and the extent to which the various signals are interde- changes in pHi. They found that reducing pHi with 3 and pendent. These issues are particularly relevant both to 10 mM butyrate, equivalent to reductions of 0.3–0.5 pHi determining the hierarchy of events behind stimulus- units, had only marginal effects on the K+ currents in response coupling per se, and to establishing the role(s) the transgenic plants compared with the wild type. The of each element, whether primary to signal transmission observations are significant, because Armstrong et al. or secondary in adaptive conditioning of the response. (1995) noted that the mutant transgene affected the K+ Some progress is now being made through epistatic and channel response to ABA but had no effect on the rise in equivalent physiological analyses. However, these prob- pHi evoked by the hormone. Thus, these two sets of data lems will continue to pose major challenges to understand- indicate that events downstream of pHi are dependent on ing guard cell transport control, as it does to plant cell the phosphorylation state of one or more target proteins. signalling generally. In conclusion, there can be little doubt now about the 358 Grabov and Blatt complexity of signalling events that underlie stomatal Blatt MR, Armstrong F. 1993. K+ channels of stomatal guard cells: abscisic acid-evoked control of the outward rectifier control. The emergence of pHi as a second messenger, 2+ mediated by cytoplasmic pH. Planta 191, 330–41. along with [Ca ]i and protein phosphorylation events, Blatt MR, Grabov A. 1997a. Signal rebundancy, gates and emphasizes our relative ignorance about the origins and integration in the control of ion channels for stomatal interactions of each of these components, quite apart movement. 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