SummaryMalate is a characteristic metabolite in the photosynthesis of C4 and CAM plants. Furthermore, changes in the intracellular concentration of this organic acid provide part of the osmotic motor for guard cells. Since alterations in the malate concentration influence the photosynthetic capacity on one side and stometal action on the other, it was studied whether the extracellular malate level represents an indicator of changes in the ambient CO2 concentration and a key regulator of ion transport in guard cells.Here it is demonstrated that alterations in the ambient C02 level modify the extracellular malate concentration of Vicia faba leaves. Elevated external malete caused stometal closure in a Concentrationdependent manner (Kin real = 0.3 mM). Slight variations in the external malate concentration strongly regulate the voltsge-depandent properties of GCAC1, an anion-release channel in the plasma membrane of guard cells. Suparfusion of guard ceil protoplasts with malate levels in the physiological range (Kin ma~ = 0.4 mM) caused the voltage gate to shift towards the resting potential of the cell-activeting GCAC1. Single-channel conductance was dependent on the extracellular chloride concentration (Kin c= = 3 mM). In the absence of extraceliular chloride the plasma membrane lacked anion conductance until the addition of malate induced channel opening. Isophthalete was a powerful agonist in both malateinduced processes, channel regulation and stomatal closure, indicating that modulation of GCACl is a key step in stomatal action. It was thus concluded that feedback regulation of volume and turgor with respect to the ambient CO2 concentration via maletesensitive anion channels may provide a CO 2 sensor to guard cells.

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Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca(2+). Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H(+)-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H(+)-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H(+)-pump as carried by Ca(2+). Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H(+) current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5-3.8 μA · cm(-2)) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands.

Summary Ion channels in the plasma membrane of guard cells provide key mechanisms in signal perception and volume regulation during stomatal movement. Recent studies have suggested that the strongly voltage‐dependent, inactivating guard cell anion channel (GCAC1) acts as a sensor of the ambient extracellular CO2 concentration and as a target of modulation by nucleotides and Ca2+ ions. Applying the patch‐clamp technique it is demonstrated here that GCAC1 is activated by cytoplasmic ATP in a pH‐dependent manner. When the apoplastic pH was buffered to 5.6 and the cytosolic pH dropped step‐wise from 7.8 to 5.6, the single‐channel activity increased as a function of proton concentration. This pH‐sensitivity is characterized by a titratable site with an apparent pK value around 6.9. While the steepness and direction of the transmembrane pH gradient did not affect the kinetics of activation, deactivation and fast inactivation of the whole‐cell anion current, the kinetics of slow inactivation and reactivation were strongly influenced. When at a constant intracellular proton concentration of pH 7.2 the external pH decreased from 7.2 to 5.6 the time constants of slow inactivation and the half‐times of reactivation increased two‐ and sevenfold, respectively. The mechanism of nucleotide activation of GCAC1 was quantitatively analysed on the level of single‐channel events. Using inside‐out, cell‐free membrane patches, GCAC1 half‐activated around 0.4 mM ATP. The sigmoidal dose‐dependence of anion channel activation could be well fitted with an apparent Hill coefficient of 3.6. This behaviour might indicate that the activation process involves a strongly cooperative interaction of four ATP‐binding sites. Neither ATP nor its non‐hydrolysable analogue AMP‐PMP, which also activated GCAC1, altered the voltage‐dependent gating. AMP‐PMP stimulation and the insensitivity of GCAC1 towards the phosphatase inhibitor, okadaic acid, and the kinase inhibitors, staurosporine and H‐7, provided evidence that nucleotide binding rather than phosphorylation caused channel activation. Since the time‐ and voltage‐dependent activity of GCAC1 is strongly modulated by ATP and protons, this channel is capable of sensing changes in the energy status, acid metabolism and the H+ ATPase activity of guard cells.

The plasma-membrane H+-pump in guard cells generates the driving force for the rapid ion fluxes required for stomatal opening. Since our electrophysiological studies revealed a two fold higher pump-current density in guard cells than in mesophyll cells of Viciafaba 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.Abbreviations: PM = plasma membrane; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis