Synergistic effect of light and fusicoccin on stomatal opening : epidermal peel and patch clamp experiments

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K⁺ channel blocker, tetraethyl-ammonium⁺ (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (−60 millivolts) at which K⁺ channels are inactivated. These results are indicative of FC activation of a H⁺ pump. FC effects on the magnitude of inward and outward K⁺ currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H⁺ ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.     

Light-dependent Influx and Efflux of Potassium of Guard Cells during Stomatal Opening and Closing

Stomata in epidermal strips of Vicia faba opened in light and closed in darkness when floated on dilute K⁺ solutions. Opening and closing, respectively, paralleled the fluxes of labeled K⁺ into and out of the strips. The gain and loss of K⁺ by the strips were shown by colbaltinitrite stain to be centered at guard cells. Intact epidermal cells, however, appeared to take up K⁺, complicating interpretation of the data.     

Relationship of Temperature to Stomatal Aperture and Potassium Accumulation in Guard Cells of Vicia faba

Epidermal strips of Vicia faba were floated on 10 millimolar KCl at various temperatures and for several time periods. The diameter of the stomatal aperture was determined microscopically and K⁺ content was estimated and expressed as the per cent of the guard cell stained. Stomatal opening was associated with increased K⁺ in guard cells, but the quantitative association was modified both by time and temperature. At low temperatures (0-20 C) there was a prolonged Spannungsphase while at higher temperatures (30-45 C) motorphase was exhibited. During the motorphase there was a rapid opening of the stomates which was highly correlated with K⁺ influx. At treatment periods of 360 minutes and temperatures higher than 25 C there appeared to be a maintenance phase during which K⁺ concentration of the guard cells decreased without an equivalent decrease in aperture.     

Effects of Cations and Abscisic Acid on Chlorophyll a Fluorescence in Guard Cells of Vicia faba

The effects of cations and abscisic acid on chloroplast activity in guard cells of Vicia faba were investigated by analysis of the transient of chlorophyll a fluorescence. When epidermal strips containing guard cells as the only living cells were incubated in water and illuminated with strong light, chlorophyll a fluorescence rose rapidly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K⁺ or Na⁺, and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca²⁺ suppressed the enhancement by Na⁺ and, to a lesser extent, that by K⁺. Abscisic acid also suppressed the enhancement by K⁺ and Na⁺. Since the fluorescence decline reflects the increase of intrathylakoid H⁺ concentration necessary for photophosphorylation, the acceleration of the decline by K⁺ (or Na⁺ in the absence of Ca²⁺) implicates chloroplast activity in ion accumulation by guard cells in the light. The differential effects of phosphate and chloride suggest that chloroplast activity may be involved in malate formation in guard cells in the light.     

Presence of Chloride Reduces Malate Production in Epidermis during Stomatal Opening

When stomata of isolated epidermis of Vicia faba are allowed to open in the presence of K⁺ and iminodiacetate (an impermeant zwitterion), malate is formed in the epidermis; the increases in malate content follow a nearly linear relationship with stomatal aperture. Stomata of leaf sections of V. faba floated on water during opening also exhibit this relationship. When isolated epidermis is offered KCI, this relationship is not observed and less malate is detected at comparable stomatal apertures. The data indicate that Cl⁻, if present at concentrations ≥ 10⁻⁵ eq liter⁻¹, can partially satisfy the anion requirement of guard cells of V. faba during stomatal opening. Discrepancies between earlier reports on the relative roles Cl⁻ and malate play as counterions for K⁺ in guard cells of V. faba could now be explained as resulting from variations in the availability of Cl⁻ to guard cells.     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.     

The involvement of a P38-like MAP kinase in ABA-induced and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

SB203580 is a specific inhibitor of p38 mitogen-activated protein (MAP) kinase and has been widely used to investigate the physiological roles of p38 in animal and yeast cells. Here by using an epidermal strip bioassay, laser-scanning confocal microscopy and whole-cell patch clamp analysis, we assess the effects of pyridinyl imidazoles-like SB203580 on the H₂O₂ signaling in guard cells of Vicia faba L. The results indicated that SB203580 blocks H₂O₂- or ABA-induced stomatal closure, ABA-induced H₂O₂ generation, and decrease in K⁺ fluxing across plasma membrane of Vicia guard cells by application of ABA and H₂O₂, whereas its analog SB202474 had no effect on these events. Thus, these results suggest that activation of p38-like MAP kinase modulates guard cell ROS signaling in response to stress.     

Isotachophoretic Analysis of Ions in Guard Cells of Vicia faba

Isotachophoretic analysis of ions was performed on guard cells of Vicia faba cv. Ryosai Issun with either open or closed stomata. In guard cells of open stomata, K⁺ and malic acid concentrations were 5–7 and 5–10 times higher, respectively, than in guard cells of closed stomata. The content of citric acid (plus isocitric acid) also increased during stomatal opening, but the increment was smaller than that of malic acid. Sodium ions, phosphoric and glyceric acids were present in low concentrations but did not increase during the opening. Other cations and anions could not be measured because of low concentrations. Malic acid provided 68–79% of the counter anions for the potassium taken up by guard cells during stomatal opening.     

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.     

Stomatal opening quantitatively related to potassium transport: evidence from electron probe analysis

When stomata of Vicia faba opened (from a stomatal aperture of about 2 micrometers to one of 12 micrometers) the solute content of the guard cells increased by 4.8 × 10⁻¹² osmoles per stoma. During the same time an average of 4.0 × 10⁻¹² gram equivalents of K⁺ were transported into each pair of guard cells. This amount of K⁺, if associated with dibasic anions, is sufficient to produce the changes in guard cell volume and osmotic pressure associated with stomatal opening. Analysis of Cl, P, and S showed that these elements were not transported in significant amounts during stomatal opening. This finding suggests that the anions balancing K⁺ were predominantly organic. K⁺ was specifically required because no other elements, likely to be present as cations, were found to accumulate in appreciable quantities in guard cells of open stomata.     

The kinetics of stomatal responses to VPD in Vicia faba: electrophysiological and water relations models

Abstract. An Ohm's law analogy is frequently employed to calculate parameters of leaf gas exchange. For example, resistance to water vapour loss is calculated as the quotient of vapour pressure difference (VPD) and vapour loss by transpiration. In the present research, this electrical analogy was extended. Steady-state transpiration as a function of VPD, assayed in leaflets of Vicia faba using gas exchange techniques, was compared with steady-state K⁺ current magnitude as a function of voltage in isolated guard cell protoplasts of Vicia faba, assayed using the patch clamping technique in the whole cell configuration. An electrophysiological model originally developed to explain the kinetics of current changes following step changes in voltage across a cell membrane was used to fit the kinetics of transpiration changes following step changes in VPD applied to leaflets of Vicia faba. Following step increases in VPD, transpiration exhibited an initial increase, reflecting the increased driving force for water loss and, for large step increases in VPD, a transient decrease in stomatal resistance. Transpiration subsequently declined, reflecting stomatal closure. By analogy to electrophysiological responses, it is hypothesized that the humidity parameter that is sensed by guard cells is VPD. Two models based on epidermal water relations were also applied to transpiration kinetics. In the first model, the transient increase in transpiration following a step increase in VPD was attributed partially to an increase in the Physical driving force (VPD) and partially to a transient decrease in stomatal resistance resulting from reduced epidermal backpressure. In the second model, the transient decrease in stomatal resistance was attributed to a direct response of the guard cells to VPD. Both models based on water relations gave good fits of the data, emphasizing the need for further study regarding the metabolic nature of the guard cell response to humidity.     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.     

Availability of Chloride Affects the Balance between Potassium Chloride and Potassium Malate in Guard Cells of Vicia faba L

Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0.1 equivalent of K⁺ per liter in the absence or presence of Cl⁻. In the absence of Cl⁻, iminodiacetate, a presumed impermeant zwitterion, served as anion. With no Cl⁻ in the medium, 91% of the K⁺ imported into the guard cells during stomatal opening was neutralized by malate production; import of Cl⁻ (presumably from the rest of the epidermal tissue) contributed 6%. In the presence of Cl⁻, 50% of the necessary negative charges were provided by malate synthesis, 45% by Cl⁻ import. Stomatal opening was not obviously affected by the chloride concentration in the incubation medium, but malate production declined roughly linearly with the logarithm of [Cl⁻] between 10⁻⁵ and 10⁻¹ equivalent per liter.     

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)     

A Membrane-delimited Effect of Internal pH on the K+ Outward Rectifier of Vicia Faba Guard Cells

ABA stimulation of outward K⁺ current (I _K,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pH _i ). However, the underlying mechanism by which I _K,out is affected by pH _i has remained unknown. Here, we demonstrate that pH _i regulates outward K⁺ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pH _i was voltage insensitive and independent of the two free calcium levels tested, 50 nm and 1 μm. The single-channel conductance was only slightly affected by pH _i . Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pH _i enhances the magnitude of I _K,out by increasing the number of channels available for activation. The fact that the pH _i effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of I _K,out by pH _i , and by implication, components of hormonal signal transduction pathways that are downstream of pH _i , are membrane-delimited. Received: 5 June 1996/Revised: 1 August 1996     

Identification and biochemical characterization of the plasma-membrane H+-ATPase in guard cells of Vicia faba L.

The plasma-membrane H⁺-pump in guard cells generates the driving force for the rapid ion fluxes required for stomatal opening. Since our electrophysio-logical studies revealed a two fold higher pump-current density in guard cells than in mesophyll cells of Vicia faba L. we elucidated the biochemical properties of this proton-translocating ATPase in plasma-membrane vesicles isolated from both cell types. The capability of the H⁺ —ATPase to create an H⁺ gradient is maintained in plasma-membrane vesicles derived from purified guard cells via blender maceration, high-pressure homogenization and polymer separation. The H⁺-pumping activity of these vesicles coincides with the presence of two polypeptides of approx. 100 and 92 kDa which are recognized by a monoclonal antibody raised against the plasma-membrane H⁺-ATPase from Zea mays L. coleoptiles. Comparison of H⁺-pumping activities of isolated membranes revealed an approximately two fold higher activity in guard cells than in mesophyll cells with respect to the total membrane protein content. Furthermore, we demonstrated by western blotting that the difference in pump activities resulted from a higher abundance of the electroenzyme per unit membrane protein in guard-cell plasma membranes. We suggest that the high H⁺-pump capacity is necessary to enable guard cells to respond to sudden changes in the environment by a change in stomatal aperture.     

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.     

Effect of CO2 on Volume Change of Guard Cell Protoplast from Vicia faba L

Guard cell protoplasts (GCP) were isolated from epidermal stripsof Vicia faba L. by enzymatic digestion. The presence of non-osmoticvolume in the protoplast was suggested by the relationship betweenprotoplast volume and the mannitol concentration of the suspendingmedium. Light illumination caused swelling of GCP only whenKCl was present in the suspending medium. Dark treatment causedshrinking of GCP irrespective of the presence of 10 mM KCl.In the presence of 10 µM abscisic acid (ABA), GCP shrank.Light-induced swelling was suppressed at concentrations of ambientCO₂ higher than that in normal air. Promotion of swelling wasnot always observed at lower CO₂ concentration. These volumechange responses to light, ABA and CO₂ suggest that GCP retainsits physiological activity as a guard cell. The osmotic contributionof K⁺ to volume increase was lower than expected. Ambient CO₂seems to have some effect on the contribution of K⁺ to osmoregulationof GCP. (Received January 30, 1982; Accepted June 25, 1982)     

K+ channels of stomatal guard cells: Abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH

The activation by abscisic acid (ABA) of current through outward-rectifying K⁺ channels and its dependence on cytoplasmic pH (pH_i) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H⁺-selective microelectrodes to record membrane potentials and pH_i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH_i. Following impalements, stable pH_i values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pH_i rose over periods of 5–8 min to values 0.27±0.03 pH units above the pH_i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K⁺ channel current (I_{K, out}) and, once evoked, both pH_i and I_{K, out} responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in I_{K, out}. Butyrate concentrations of 10 and 30 mM (pH₀ 6.1) caused pH_i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH_i and concomitant swell in the steady-state conductance of I_{K, out}. The rise in pH_i and i_{K, out} in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K⁺ channels (I_{K, in}). The pH_i dependence of I_{K, out} was consistent with a cooperative binding of at least 2H⁺ (apparent pK_a = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of I_{K, out} in ABA and, thus, affirm a role for H⁺ in signalling and transport control in plants distinct from its function as a substrate in H⁺-coupled transport. Additional evidence implicates a coordinate control of I_{K, in} by cytoplasmic-free [Ca²⁺] and pH_i.Abbreviations ABA abscisic acid - [Ca²⁺]_i cytoplasmic free [Ca²⁺]_i - E_K K⁺ equilibrium potential - I_{K, out}, I_{K, in} outward-, inward-rectifying K⁺ channel (current) - I-V current-voltage (relation) - Mes 2-(N-morpholino)ethanesulfonic acid - pH_i cytoplasmic pH - Tes 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid - V_m membrane potential We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship.     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996