Membrane-Delimited Phosphorylation Enables the Activation of the Outward-Rectifying K Channels in Motor Cell Protoplasts of Samanea saman

Outward-rectifying K channels activated by membrane depolarization (Kout or KD channels) control K+ efflux from plant cells. To find out to what extent phosphorylation is required for the activity of these channels, the patch-clamp method was applied to protoplasts from the legume Samanea saman in both whole-cell and isolated-patch configurations. In the absence of either Mg2+ or ATP in the "cytosolic" solution, the KD channel activity declined completely within 15 min. This decline could be reversed in excised, inside-out patches by restoring MgATP (1 mM) to the cytoplasmic side of the membrane. Mg2+ (1 mM) plus 5[prime]-adenylylimidodiphosphate (1 mM), a nonhydrolyzable ATP analog, did not substitute for ATP. Mg2+ (1 mM) plus adenosine 5[prime]-O-(3-thiotriphosphate) (25 to <100 [mu]M), an irreversibly thiophosphorylating ATP analog, sustained channel activity irreversibly. 1-(5-IsoquinolinesulphonyI)-2- methylpiperazine (100 [mu]M), a broad-range kinase inhibitor, blocked the activity of KD channels in the presence of MgATP. These results strongly suggest that the activation of the outward-rectifying K channels by depolarization depends critically on phosphorylation by a kinase tightly associated with the KD channel.     

Calcium-Activated K+ Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatal Closure

Stomatal closing requires the efflux of K+ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K+ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca2+ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K+-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca2+ concentration. These vacuolar K+ (VK) channels had a single channel conductance of 70 pS with 100 mM KCI on both sides of the membrane and were highly selective for K+ over NH4+ and Rb+. Na+, Li+, and Cs+ were not measurably permeant. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K+ channels in the initiation and control of K+ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K+-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca2+ over K+ selectivity (permeability ratio for Ca2+ to K+ of ~3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca2+-induced Ca2+ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K+ and Ca2+ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca2+-induced Ca2+ release from higher plant vacuoles is discussed.     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Stomatal Movement and Sucrose Uptake by Guard Cell Protoplasts of Commelina benghalensis L.

Sucrose concentration in guard cells of epidermal strips ofCommelina benghalensis increased with stomatal opening. Sucroseuptake patterns were investigated using guard cell protoplastsof C. benghalensis. Sucrose (0.5 mM) uptake into these protoplastswas sensitive to pH, with an optimum at pH 6. Uptake of sucroseinto guard cell protoplasts was inhibited by 2,4-dinitrophenol(DNP), diethylstilbestrol (DES) and (ptrifluoromethoxy)carbonylcyanide phenylhydrozone (FCCP), while DCMU and o-phenanthrolinehad no effect on the uptake of sucrose. Fusicoccin (FC) stimulatedsucrose influx. The influence of pH and the effect of the metabolicinhibitors on the sucrose uptake into the guard cell protoplastsare consistent with an energy dependent membrane-function. (Received July 7, 1986; Accepted September 26, 1986)     

The Investigation of Stomatal Ionic Relations using Guard Cell Protoplasts: I. METHODOLOGY

Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. 1. Methodology.—J exp. Bot.36: 1726–1738. A study was made of the methodology for the production and useof guard cell protoplasts in ion transport studies, with particularemphasis placed on the effects of the composition of the externalmedium on protoplast survival and performance. Addition of externalKCl to media during the production of guard cell protoplastsfrom Commelina communis L. was found to improve viability andto increase K⁺ content and physiological competence of the isolatedprotoplasts. Addition of low levels (20 x 10^–3 mol m^–3)CaCl₂ increased protoplast yield and the maintenance of viabilityin long-term incubation. Ambiguities and uncertainties werefound in the application of methods commonly used for the assessmentof viability of isolated protoplasts. Poor yields (despite highpercentage recoveries) together with difficulties in the assessmentof viability were considered to pose major potential problemsin the use of guard cell protoplasts in ion transport studies. Key words: Guard cell protoplasts, ion transport, Commelina communis     

Adenine and Pyridine Nucleotide Status of Isolated Vicia Guard Cell Protoplasts During K+-Induced Swelling

The levels of ATP, ADP, and reduced and oxidized pyridine nucleotideswere determined in guard cell protoplasts of Vicia faba duringtheir swelling in 10 mu potassium iminodiacetate. Upon additionof K⁺, the ATP/ADP ratio dropped from 9 to about 6 (0 to 1 minof incubation), followed by a steep increase up to 15 between1 and 3 min. After 3 min, the protoplasts began to increasein diameter from about 15 µm to nearly 17 µm after5 min of incubation, accompanied by a rapid decrease of theATP/ADP ratio to about 7. During this time, NAD and NADPH showedoppositely directed oscillations, while the pools of NADH andNADP stayed rather constant. As the sum of all pyridine nucleotidesdid not change, an interconversion of NAD and NADPH must haveoccurred during the swelling. The levels.of glucose-6-P dehydrogenaseactivity, which were assayed under the same conditions, showedoscillations parallel to those of the ATP/ADP ratios and oppositeto those of the NADPH/NAD ratios. The results are discussedwith respect to the regulatory mechanisms during guard cellswelling. (Received April 21, 1984; Accepted August 1, 1984)     

Ferricyanide Reduction by Guard Cell Protoplasts

Guard cell protoplasts of Commelina communis L. reduced exogenousferricyanide at pH values lower than 5?0; upon addition of NADH,reduction of ferricyanide by guard cell protoplasts was stimulatedover the pH range 4?0 to 9?0 with two peaks of activity at pH5?0 and between pH 8?0 and pH 9?0. Calcium chloride (1?0 molm^–3) and MgCl2 (1?0 mol m^–3) increased the NADH-stimulatedreduction of ferricyanide. Superoxide dismutase and cyanidehad little effect on the NADH-stimulated reduction of ferricyanideby guard cell protoplasts, but, salicylhydroxamic acid completelyinhibited this activity. The NADH-stimulated reduction of ferricyanidealso occurred in the cell-free supernatant. Horseradish peroxidasedid not reduce ferricyanide in the absence of NADH over a broadrange of pH (4?0 to 9?0). However, in the presence of NADH,horseradish peroxidase reduced ferricyanide over the pH range5?0 to 9?0 with maximal activity at pH 8?0. The NADH-stimulatedreduction of ferricyanide by horseradish peroxidase showed similarproperties to those observed with guard cell protoplasts. Mannitol,superoxide dismutase, and cyanide did not inhibit the NADH-stimulatedreduction of ferricyanide by horseradish peroxidase; SHAM, however,completely inhibited the reduction of ferricyanide by horseradishperoxidase. Catalase inhibited the NADH-stimulated reductionof ferricyanide by horseradish peroxidase by 20%, while absenceof oxygen in the assay medium stimulated this activity over60%. We propose that the reduction of ferricyanide in the presenceof NADH by guard cell protoplasts, can be explained in termsof peroxidase activity associated with the plasma membrane andsecreted to the extracellular medium. However, the capacityof guard cell protoplasts to reduce ferricyanide at acid pHvalues where little peroxidase activity occurs may indicatethe presence of a plasma membrane redox system in guard cellsof C. communis. Key words: Commelina, guard cell protoplasts, ferricyanide reduction, peroxidase, redox system     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

过氧化氢对蚕豆气孔运动和质膜K+通道的影响

不同浓度H2 O2 可使蚕豆 (ViciafabaL .)叶片气孔关闭 ,抑制气孔张开 ,10mmol/L的H2 O2 最有效 ,10 μmol/L的H2 O2 仍明显使气孔关闭。且 10 μmol/L的H2 O2 抑制气孔张开作用能被EGTA所消除 ,表明Ca2 参与低浓度H2 O2 使气孔关闭的过程。 2mmol/L的H2 O2 可使质膜内向K 通道电流明显减小 ,而外向K 通道电流显著增加。因此 ,H2 O2 促进蚕豆气孔关闭主要是通过抑制K 通过保卫细胞质膜内向流入 ,或加强K 外向流出实现的     

Hydrogen Sulfide Regulates Inward-Rectifying K+ Channels in Conjunction with Stomatal Closure

Hydrogen sulfide (H₂S) is the third biological gasotransmitter, and in animals, it affects many physiological processes by modulating ion channels. H₂S has been reported to protect plants from oxidative stress in diverse physiological responses. H₂S closes stomata, but the underlying mechanism remains elusive. Here, we report the selective inactivation of current carried by inward-rectifying K⁺ channels of tobacco (Nicotiana tabacum) guard cells and show its close parallel with stomatal closure evoked by submicromolar concentrations of H₂S. Experiments to scavenge H₂S suggested an effect that is separable from that of abscisic acid, which is associated with water stress. Thus, H₂S seems to define a unique and unresolved signaling pathway that selectively targets inward-rectifying K⁺ channels.Hydrogen sulfide (H₂S) is a small bioactive gas that has been known for centuries as an environmental pollutant (Reiffenstein et al., 1992). H₂S is soluble in both polar and, especially, nonpolar solvents (Wang, 2002), and has recently come to be recognized as the third member of a group of so-called biological gasotransmitters. Most importantly, H₂S shows both physical and functional similarities to the other gasotransmitters nitric oxide (NO) and carbon monoxide (Wang, 2002), and it has been shown to participate in diverse physiological processes in animals, including cardioprotection, neuromodulation, inflammation, apoptosis, and gastrointestinal functions among others (Kabil et al., 2014). Less is known about H₂S molecular targets and its modes of action. H₂S can directly modify specific targets through protein sulfhydration (the addition of an -SH group to thiol moiety of proteins; Mustafa et al., 2009) or reaction with metal centers (Li and Lancaster, 2013). It can also act indirectly, reacting with NO to form nitrosothiols (Whiteman et al., 2006; Li and Lancaster, 2013). Among its molecular targets, H₂S has been reported to regulate ATP-dependent K⁺ channels (Yang et al., 2005), Ca²⁺-activated K⁺ channels, T- and L-type Ca²⁺ channels, and transient receptor potential channels (Tang et al., 2010; Peers et al., 2012), suggesting H₂S as a key regulator of membrane ion transport.In plants, H₂S is produced enzymatically by the desulfhydration of l-Cys to form H₂S, pyruvate, and ammonia in a reaction catalyzed by the enzyme l-Cys desulfhydrase (Riemenschneider et al., 2005a, 2005b), DES1, that has been characterized in Arabidopsis (Arabidopsis thaliana; Alvarez et al., 2010). Alternatively, H₂S can be produced from d-Cys by d-Cys desulfhydrase (Riemenschneider et al., 2005a, 2005b) and in cyanide metabolism by β-cyano-Ala synthase (García et al., 2010). H₂S action was originally related to pathogenesis resistance (Bloem et al., 2004), but in the last decade it has been proven to have an active role in signaling, participating in key physiological processes, such as germination and root organogenesis (Zhang et al., 2008, 2009a), heat stress (Li et al., 2013a, 2013b), osmotic stress (Zhang et al., 2009b), and stomatal movement (García-Mata and Lamattina, 2010; Lisjak et al., 2010, 2011; Jin et al., 2013). Moreover, H₂S was reported to participate in the signaling of plant hormones, including abscisic acid (ABA; García-Mata and Lamattina, 2010; Lisjak et al., 2010; Jin et al., 2013; Scuffi et al., 2014), ethylene (Hou et al., 2013), and auxin (Zhang et al., 2009a).ABA is an important player in plant physiology. Notably, upon water stress, ABA triggers a complex signaling network to restrict the loss of water through the transpiration stream, balancing these needs with those of CO₂ for carbon assimilation. In the guard cells that surround the stomatal pore, ABA induces an increase of cytosolic-free Ca²⁺ concentration ([Ca2+]_cyt), elevates cytosolic pH (pH_i), and activates the efflux of anions, mainly chloride, through S- and R-type anion channels. The increase in [Ca2+]_cyt inactivates inward-rectifying K⁺ channels (I_KIN); anion efflux depolarizes the plasma membrane, and together with the rise in pH_i, it activates K⁺ efflux through outward-rectifying K⁺ channels (I_KOUT; Blatt, 2000; Schroeder et al., 2001). These changes in ion flux, in turn, generate an osmotically driven reduction in turgor and volume and closure of the stomatal pore. All three gasotransmitters have been implicated in regulating the activity of guard cell ion channels, but direct evidence is available only for NO (Garcia-Mata et al., 2003; Sokolovski et al., 2005). Here, we have used two-electrode voltage clamp measurements to study the role of H₂S in the regulation of the guard cell K⁺ channels of tobacco (Nicotiana tabacum). Our results show that H₂S selectively inactivates I_KIN and that this action parallels that of stomatal closure. These results confirm H₂S as a unique factor regulating guard cell ion transport and indicate that H₂S acts in a manner separable from that of ABA.     

Properties of Two Outward-Rectifying Channels in Root Xylem Parenchyma Cells Suggest a Role in K+ Homeostasis and Long-Distance Signaling

Xylem parenchyma cells (XPCs) control the composition of the transpiration stream in plants and are thought to play a role in long-distance signaling as well. We addressed the regulation, selectivity, and dependence on the apoplastic ion concentrations of two types of outward rectifiers in the plasma membrane of XPCs, to assess the physiological role of these conductances. In whole-cell recordings, the membrane conductance at depolarization was under the control of cytosolic Ca2+: at physiological Ca2+ levels (150 nM) the K+ outward-rectifying conductance (KORC) predominated, whereas at elevated Ca2+ levels (5 [mu]M), only the nonselective outward-rectifying conductance (NORC) was active. No such regulatory effect of Ca2+ was observed in inside-out experiments. The voltage dependence of whole-cell KORC currents strongly depended on apoplastic K+ concentration: an increase in apoplastic K+ resulted in a positive shift of the current-voltage curve, roughly following the shift in Nernst potential of K+. KORC is impermeable to Na+, but does translocate Ca2+ in addition to K+. In contrast to KORC, NORC selected poorly among monovalent cations and anions, the relative permeability PC+/PA- being about 1.9. Gating of NORC was largely unaffected by the level of K+ in the bath. Under all ionic conditions tested, NORC tail currents or single-channel currents reversed close to 0 mV. Using an in vivo xylem-perfusion technique, tetraethylammonium (an inhibitor of KORC) was shown to block K+ transport to the shoot. These data support the hypothesis that release of K+ to the xylem sap is mediated by KORC. The molecular properties of these two conductances are discussed in the light of the distinct physiological role of XPCs.     

Guard Cell Pressures and Wall Properties during Stomatal Opening

Pressures required to produce stomatal apertures of differentwidths have been measured in guard cells of T. virginiana andC. communis. These pressures are lower than those that coulddevelop in guard cells at full turgor on account of their osmoticproperties. During the Spannungsphase the metabolism of guardcells seems to contribute towards a reduction of the elasticmodulus of their walls, whereas during the motorphase the volumetricmodulus of the cell as a whole, apparently increases gentlyat first and then more markedly as maximum apertures are approached.     

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba

Guard cell protoplasts of Vicia faba treated with 10 [mu]M (+)abscisic acid (ABA) in the light exhibited a 20% decrease in diameter within 1.5 h, from 24.1 to 19.6 [mu]m. Within 10 s of administration of ABA, a 90% increase in levels of inositol 1,4,5-trisphosphate was observed, provided that cells were treated with Li+, an inhibitor of inositol phosphatase activity, prior to incubation. Concomitantly, levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased 20% compared to levels in control cells; levels of label in the membrane lipids phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol did not change significantly in response to ABA treatment. These results show that phosphoinositide turnover is activated in response to ABA in guard cells. We conclude that phosphoinositide signaling is likely to be a step in the biochemical cascade that couples ABA to guard cell shrinking and stomatal closure.     

Light-Induced De-Epoxidation of Violaxanthin in Guard Cell Protoplasts of Vicia faba

Photosynthetic pigments of Vicia guard cell protoplasts (GCPs)from abaxial epidermis were analyzed by reverse-phase HPLC.Violaxanthin decreased and zeaxanthin increased in GCPs afterlight illumination. The epoxidation state of GCPs decreasedfrom 0.82 (dark) to 0.37 (light), suggesting operation of thexanthophyll cycle in GCPs of Vicia faba. (Received March 15, 1993; Accepted May 10, 1993)     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

保卫细胞的光合作用在光调节的气孔运动中的功能

本文介绍植物叶片上保卫细胞中叶绿体在光诱导气孔开放过程中的作用等研究进展,并对叶肉细胞中的光合作用与气孔运动之间的关系也作简要分析和讨论。     

Nitrate-Sensitive ATPase Activity and Proton Pumping in Guard Cell Protoplasts of Commelina

ATPase activity was measured in crude homogenates of guard cellprotoplasts of Commelina communis L. using a linked enzyme assay.A low level of azide-sensitive ATPase activity was detectedwith a pH optimum of 6.8. This activity was stimulated by 0.01%(v/v) Triton X-100, and the pH optimum shifted to pH 7.4. Nitrate-sensitiveATPase activity was measured in the presence of azide and showeda pH optimum around pH 8.0. Proton pumping activity in a mixedpopulation of vesicles from GCP was monitored using fluorescencequenching of quinacrine. Mg-ATP dependent proton pumping wasobserved at pH 8.0, but not at pH 6.6. The activity at pH 8.0was inhibited by nitrate and DCCD but not vanadate. These dataindicate that activity of the tonoplast proton pump was beingmeasured. There was, however, no evidence for a tonoplast cation(K⁺)/proton antiporter under these assay conditions as potassiumdid not reduce the initial rate of pH gradient formation orincrease the rate of collapse of a pre-formed gradient afterinhibition of the pump. Key words: Tonoplast ATPase, proton pump, guard cell protoplasts, Commelina     

Vanadate Sensitive ATPase and Phosphatase Activity in Guard Cell Protoplasts of Commelina

Flicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPaseand phosphatase activity in guard cell protoplasts of Commelina.—J.exp. Bot. 38: 642–648. Phosphatase activity was measured in extracts of guard cellprotoplasts of Commelina communis L. using the artificial substratep-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.Ammonium molybdate (Ol mol m^–3) and sodium vanadate (1–0mol m^–3) gave almost complete inhibition of phosphataseactivity at pH 60. ATPase assays were, therefore, conductedin the presence of 0–2 mol m ^–3 molybdate and vanadatewas used as a specific inhibitor of plasmamembrane ATPase activity.Vanadate sensitive ATPase activity showed a pH optimum of 6.6and activity was stimulated by KC1. These properties are characteristicof plasmamembrane proton pumping ATPases in other systems andsuggest that proton extrusion in guard cells could be mediatedby a similar enzyme. The maximum ATPase activity is sufficientto account for all the proton flux observed during the stomatalopening response. Key words: ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate     

Photosynthesis-Dependent Volume Regulation in Guard Cell Protoplasts from Vicia faba L.

A rapid and convenient procedure was developed for isolatingguard cell protoplasts (GCPs) from epidermal strips of Viciafaba L. The mean rates of O₂ uptake in the dark and evolutionin light of the isolated GCPs were 200 and 290 µmol O₂mg^–1 Chl h^–1, respectively, showing net O₂ evolutionin light. Photosynthetic O₂ evolution was suppressed completelyby 5 µM DCMU. Addition of 5 µM DCMU to the incubationmedium after 30 min of light exposure also suppressed the light-inducedswelling of GCP, indicating possible participation of PS IIin volume regulation in GCP. ⁴Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Yatabe machi, Tsukuba,Ibaraki 305, Japan. (Received December 17, 1983; Accepted March 21, 1984)     

Abscisic Acid Activates a 48-Kilodalton Protein Kinase in Guard Cell Protoplasts

A 49- and a 46-kD Ca2+-independent protein kinase and a 53-kD Ca2+-dependent protein kinase were detected in Vicia faba guard cell protoplasts (GCPs) by an in-gel protein kinase assay using myelin basic protein as a substrate. A 48-kD protein kinase designated as abscisic acid (ABA)-responsive protein kinase (ABR kinase) appeared when GCPs were treated with ABA. The activation of ABR kinase was suppressed by the protein kinase inhibitor staurosporine, indicating that a putative activator protein kinase phosphorylates and activates ABR kinase. The treatment of GCPs with 1,2-bis(o-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid, a calcium chelator, suppressed the activation of ABR kinase, suggesting that an influx of extracellular Ca2+ is required for the activation. Staurosporine and K-252a inhibited both the activity of ABR kinase and the stomatal closure induced by ABA treatment of V. faba epidermal peels. These results suggest that ABR kinase and its activator kinase may consist of a protein kinase cascade in a signal transduction pathway linking ABA perception to stomatal closure. The mobility of the 53-kD Ca2+-dependent protein kinase in sodium dodecyl sulfate-polyacrylamide gel was shifted upon Ca2+ binding to the enzyme, thus exhibiting the characteristics of a Ca2+-dependent or calmodulin-like domain protein kinase. This kinase may be the activator of ABR kinase.