Control of Ion Fluxes in Stomatal Guard Cells

Opening and closing of the stomatal pore is associated with very large changes in K-salt accumulation in stomatal guard cells. This review discusses the ionic relations of guard cells in relation to the general pattern of transport processes in plant cells, in plasmalemma and tonoplast, involving primary active transport of protons, proton-linked secondary active transport, and a number of gated ion channels. The evidence available suggests that the initiation of stomatal opening is regulated through the uptake mechanisms, whereas initiation of stomatal closing is regulated by control of ion efflux at the plasmalemma, and of fluxes to and from the vacuole. In response to a closing signal there are large transient increases in efflux of both Cl^? (or Br^?) and Rb⁺ (K⁺) at the plasmalemma, with also a probable increase in anion flux from vacuole to cytoplasm and decrease in anion flux from cytoplasm to vacuole. A speculative hypothetical sequence of events is discussed, by which the primary response to a closing signal is an increase in Ca²⁺ influx at the plasmalemma, producing depolarisation and increase in cytoplasmic Ca²⁺. The consequent opening of Ca²⁺-sensitive Cl^? channels, and voltage-sensitive K⁺ channels (also Ca²⁺-sensitive?) in the plasmalemma, and of a Ca²⁺-sensitive nonspecific channel in the tonoplast, could produce the flux effects identified by the tracer work; this speculation is also consistent with the Ca²⁺-sensitivity of the response to closing signals and with evidence from patch clamping that such channels exist in at least some plant cells, though not yet all shown in guard cells.     

Early ABA Signaling Events in Guard Cells

The plant hormone abscisic acid (ABA) regulates a wide variety of plant physiological and developmental processes, particularly responses to environmental stress, such as drought. In response to water deficiency, plants redistribute foliar ABA and/or upregulate ABA synthesis in roots, leading to roughly a 30-fold increase in ABA concentration in the apoplast of stomatal guard cells. The elevated ABA triggers a chain of events in guard cells, causing stomatal closure and thus preventing water loss. Although the molecular nature of ABA receptor(s) remains unknown, considerable progress in the identification and characterization of its downstream signaling elements has been made by using combined physiological, biochemical, biophysical, molecular, and genetic approaches. The measurable events associated with ABA-induced stomatal closure in guard cells include, sequentially, the production of reactive oxygen species (ROS), increases in cytosolic free Ca²⁺ levels ([Ca²⁺]_i), activation of anion channels, membrane potential depolarization, cytosolic alkalinization, inhibition of K⁺ influx channels, and promotion of K⁺ efflux channels. This review provides an overview of the cellular and molecular mechanisms underlying these ABA-evoked signaling events, with particular emphasis on how ABA triggers an “electronic circuitry” involving these ionic components.     

The chemiosmotic model of stomatal opening revisited

Stomata are light‐activated biological valves in the otherwise gas‐impermeable epidermis of aerial organs of higher plants. Stomata often regulate rates of photosynthesis and transpiration in ways that optimize whole‐plant carbon gain against water loss. Each stoma is flanked by a pair of opposing guard cells. Stomatal opening occurs by light‐activated increases in the turgor pressure of guard cells, which causes them to change shape so that the stomatal pore between them widens. These increases in turgor pressure oppose increases in cellular osmotic pressure that result from uptake of K⁺. K⁺ uptake occurs by a chemiosmotic mechanism in response to light‐activated extrusion of H⁺ outward across the plasma membrane of the guard cell. The initial changes in cellular membrane potential lead to the opening of inward‐rectifying K⁺ channels, after which K⁺ is taken up along its electrochemical gradient. Changes in membrane potential resulting from K⁺ uptake may be balanced by accumulation of Cl^?ions by guard cells and/or by synthesis of malic acid within each cell. Malic acid also acts to buffer increases in cytosolic pH caused by H⁺ extrusion. This review describes how the application of patch‐clamp technology to guard cell protoplasts has enabled investigators to elucidate the mechanisms by which H⁺ is extruded from guard cells, the types of ion channels present in the guard cell plasma membrane, how those ion channels are regulated, and the signal transduction processes that trigger stomatal opening and closing.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

The mechanism of stomatal closing by salicylic acid inCommelina communis L.

The mechanism of stomatal closing by salicylic acid (SA) has been investigated. The addition of 1 mM SA to fully opened stomata resulted in a significant reduction of 75% in stomatal aperture. Stomata in the treatment of SA with EGTA closed as observed in the treatment of SA. However, the addition of catalase with SA completely inhibited stomatal closing. Stomatal closing induced by SA was also reduced by Ca²⁺. To understand the relation bewteen stomatal closing by SA and catalase activity, the effect of SA on catalse activity and the effect of AT (catalase inhibitor) on stomatal closing was investigated. SA inhibited 32% of catalase activity. Stomata in isolated epidermis floated on an incubation solution containing 0.1 mM AT closed from 9.6 μm to 3.2 μm after 1 hour. SA stimulated K⁺ efflux as much as the twice of the control in isolated strips. SA inhibited 53% of photosynthetic activity at the light intensity of 1000 μmole m² s¹ on SA infiltrated leaves. A similar result was found on stomatal conductance in SA infiltrated leaves. These results indicate that SA inhibit catalase activity and increase the concentration of H₂O₂ in guard cell cytoplasm. H₂O₂ oxidize the plasma membrane and increase the membrane permeability of K⁺. The mass efflux of K⁺ induce the loss of turgor pressure and lead to stomatal closing. The inhibition of photosynthetic activity by SA suggests that stomatal closing by SA is also related with the decrease of photosynthetic activity.     

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

AtALMT12 represents an R‐type anion channel required for stomatal movement in Arabidopsis guard cells

Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S‐type) and rapid (R‐type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage‐independent anion channel component in guard cells. In contrast, the R‐type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R‐type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark‐ and CO₂‐induced stomatal closure, as well as in response to the drought‐stress hormone abscisic acid. Patch‐clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R‐type currents compared with wild‐type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage‐dependent anion currents reminiscent to R‐type channels could be activated. In line with the features of the R‐type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R‐Type channels, like voltage‐dependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long‐term anion and K⁺ efflux via SLAC1 and GORK, respectively.     

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Guard cells undergo constitutive and pressure-driven membrane turnover

Summary. During stomatal movement, guard cells undergo large and reversible changes in cell volume and consequently surface area. These alterations in surface area require addition and removal of plasma membrane material. How this is achieved is largely unknown. Here we summarize recent studies of membrane turnover in guard cells using electrophysiology and fluorescent imaging techniques. The results implicate that membrane turnover in guard cells and most likely in plant cells in general is sensitive to changes in membrane tension. We suggest that this provides a mechanism for the adaptation of surface area of guard cells to osmotically driven changes in cell volume. In addition, guard cells also exhibit constitutive membrane turnover. Constitutive and pressure-driven membrane turnover were found to be associated with addition and removal of K⁺ channels. This implies that some of the exo- and endocytic vesicles carry K⁺ channels. Together the results demonstrate that exo- and endocytosis is an essential process in guard cell functioning. Correspondence and reprints: Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt, Federal Republic of Germany.     

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Regulating role of acetylcholine and its antagonists in inward rectified K+ channels from guard cell protoplasts ofVicia faba

The inward rectified potassium current ofVicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K⁺ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K⁺ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K⁺ current is inhibited by 60% –75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K⁺ channels has no effect on the inward K⁺ current regulated by ACh, suggesting that there are inward K⁺ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Roles of heterotrimeric G proteins in guard cell ion channel regulation

Stomata are formed by pairs of surrounding guard cells and perform important roles in photosynthesis, transpiration and innate immunity of terrestrial plants. Ionic solutes in the cytosol of guard cells are important for cell turgor and volume change. Consequently, trans-membrane flux of ions such as K⁺, Cl⁻, and malate2⁻ through K⁺ channels and anion channels of guard cells are a direct driving force for turgor change, while the opening of calcium permeable channels can serve as a trigger of cytosolic free calcium concentration elevations or oscillations, which play second messenger roles. In plants, heterotrimeric G proteins have fewer members than in animals, but they are well investigated and found to regulate these channels and to play fundamental roles in guard cell function. This mini-review focuses on the recent understanding of G-protein regulation of ion channels on the plasma membrane of guard cells and their participation in stomatal movements.Key words: guard cell, heterotrimeric G protein, ion channel, arabidopsis thaliana, stomata, plasma membrane, patch clampHeterotrimeric G proteins, composed of Gα, Gβ and Gγ subunits, are key elements of cellular signal transduction networks. In plant species, fewer members of G proteins are present than in animals. For example, only one Gα subunit (GPA1), one Gβ subunit (AGB1) and two Gγ subunits (AGG1 and AGG2) are reported in Arabidopsis while 23 Gα, 5 Gβ and 12 Gγ subunits have been identified in human.¹ All three kinds of subunits are expressed in guard cells. Ubiquitous expression of GPA1 throughout plant was ascertained by northern and promoter::GUS analyses and RT-PCR results also indicate guard cell expression.^2–4 AGB1 is ubiquitously expressed throughout the plant and its promoter::GUS transgenic lines show strong expression in guard cells.^5–7 For Gγ subunits, RNA blots show AGG1 and AGG2 expression throughout the plant, however, reporter gene analysis shows guard cell expression of AGG2 but not AGG1.^7–9 The guard cell expression of G protein subunits implies the function of G protein in guard cell signaling and stomatal movement regulation.Stomata are microscopic pores in the epidermis of terrestrial plants, which serve as the mouths of plants for gas change since through them CO₂ enters leaves for photosynthesis and water vapor is lost as transpiration.^10–13 In addition, stomatal movements induced by pathogen and pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) are a component of the plant innate immunity system.^14–16 Biotic and abiotic stresses (e.g. water deficiency, cold, pathogens) and their induced phytohormone changes (e.g. abscisic acid [ABA], ethylene) have been widely investigated in stomatal movement regulation, and stomatal apertures are directly regulated by volume change of the surrounding guard cell pairs. The accumulation/release of ionic solutes through ion channels on the guard-cell plasma membrane together with malate production/metabolism induces water influx/efflux driving increase/decrease of cell turgor and volume which co-operates with the radial reinforcement of the guard cell walls to widen/shrink stomatal aperture.^10,17 Given that mature guard cells lack plasmodesmata with neighboring cells, all ion uptake and efflux must pass through ion channels and ion transporters on the plasma membrane.In Arabidopsis guard cells, the model cell type for cell signaling of the model plant species, all three kinds of ion channels (K⁺ channels, anion channels and Ca²⁺-permeable channels) have been investigated and found to be regulated by heterotrimeric G proteins.^10,17 Their ion channel activities can be measured in intact guard cells, guard cell protoplasts, or cell membrane patches using the patch clamp technique.^15,18,19 Patch clamping can be used to measure ion fluxes in whole cells or even through a single ion channel.^20,21 The patch clamp technique under the whole-cell recording configuration can measure the currents through hyperpolarization-activated inward K⁺ channels which account for K⁺ accumulation during stomatal opening, and the depolarizationactivated outward K⁺ channels which, together with R-type and S-type anion channels, mediate solute removal during stomatal closure. Besides these ionic fluxes which directly elicit changes in turgor, Ca²⁺-permeable channels which participate in Ca²⁺ signaling are also regulated by G proteins. For better visualization of the currents through K⁺, anion and Ca²⁺permeable channels, real current traces and their idealized current/voltage relationships are indicated in Figure 1. The G-protein regulation of inward and outward K⁺ channels, S-type anion channels, and Ca²⁺-permeable channels and their significance for stomatal movements will be discussed below, and the genes encoding them which have been explored up to now also will be discussed.Open in a separate windowFigure 1Current traces and idealized current/voltage relationships of wild type guard cell plasma membrane ion channels involved in G-protein regulation (A–C), ABA inhibition of whole-cell inward K⁺ currents. (A) indicates inward K⁺ currents of wild type guard cell protoplasts in response to hyperpolarizing voltages under control conditions [Scale bar is shown in (B)]; (B) indicates inward K+ currents of wild type guard cell protoplasts with ABA treatment; (C) indicates the idealized current/voltage relationship of inward K⁺ currents for control (gray) and ABA treatments (black). (D–F), ABA activation of slow anion currents. (D) indicates anion currents of wild type under control condition and (E) shows current after ABA treatment; (F) indicates the idealized current/voltage relationship of anion currents for control (gray) and ABA treatments (black). (G–I), ABA activation of currents through Ca²⁺-permeable channels. (G) indicates currents through Ca²⁺-permeable channels of wild type under control condition and (H) shows current after ABA treatments; (I) indicates the idealized current/voltage relationship of currents through Ca²⁺-permeable channels for control (gray) and ABA treatments (black).     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

The mechanism of stomatal movement in Allium cepa L.

In the guard cells of Allium cepa leaves, no starch was found either when the stomata were open or closed. The lack of other soluble polysaccharides that could be hydrolyzed during the opening reaction of the stomata (Schnabl, Planta 1977, in press) leads to the question, how is the osmotic effect, which is the basis of the stomatal movement, achieved in Allium? It is shown in this paper, by histochemical and microprobe analyses, that in Allium — as in other plant species—the K⁺ concentration of the guard cells increases during stomatal opening. The charges of the K⁺ ions in the guard cells seem to be fully compensated by imported Cl^- ions. This could mean that if starch is present in the guard cells, as in the majority of plant species, its major role in the mechanism of stomatal movement is to deliver the cuunteranions for the imported K⁺ ions.     

K+ channels of stomatal guard cells: bimodal control of the K+ inward-rectifier evoked by auxin

The influence of the auxins indole-3-acetic acid (IAA) and 1-napthylene acetic acid (NAA) on K⁺ channels and their control was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes to record membrane potentials and to monitor K⁺ channel currents under voltage clamp during exposures to 0.1–100 µM IAA and NAA. Following impalements, challenge with either IAA or NAA in the presence of 10 mM KCl resulted in the concerted modulation of at least four different currents with distinct kinetic characteristics and concentration dependencies. Equivalent concentrations of benzoic acid were wholly without effect. Most striking, current carried by inward-rectifying K⁺ channels (I_K,in) exhibited a bimodal response to both IAA and NAA which was reversed on washing the auxins from the bathing medium. The steady-state current was augmented 1.3- to 2-fold at concentrations between 0.1 and 10 µM and antagonized at concentrations near 30 µM and above. Auxin agonism of I_K,in was time- and voltage-independent. By contrast, I_K,in inactivation at the higher auxin concentrations was marked by a voltage-dependence and slowing of the kinetics for current activation. Inactivation of I_K,in by the auxins was relieved when cytoplasmic pH (pH_i) was clamped near 7.0 in the presence of 30 mM Na⁺-butyrate. In addition to the control of I_K,in, current carried by a second class of (outward-rectifying) K⁺ channels rose in a monotonic and largely voltage-independent manner with auxin concentrations about 10 µM and above, and IAA and NAA also activated an inward-going current with a voltage dependence characteristic of guard cell anion channels. Further changes in background current were consistent with a limited activation of the H⁺-ATPase. Over the concentration range examined, the auxins evoked membrane hyperpolarizations and depolarizations of up to ±12–19 mV, depending on the free-running membrane potential prevailing before auxin additions. Prolonging exposures to 100 µM auxin beyond 3–5 min frequently elicited rapid transitions to voltages near E_K as well as regenerative action potentials. However, in every case the voltage response was a predictable consequence of auxin action on the K⁺ channels and, at 100 µM auxin, on the anion current. These results demonstrate a control of K⁺ channel activity by auxin, consistent with the roles of these channels in mediating K⁺ flux for stomatal movements; the data associate a bimodal characteristic with the activity of I_K,in, implicating pH_i as a putative intermediate in its control, and offer strong evidence for a multiplicity of signal cascades evoked by auxin; finally, they highlight a coordinate modulation of transport activities by auxin, thereby drawing a close analogy to the pattern of stimulus-response coupling in abscisic acid.