The phytohormone abscisic acid (ABA) reports on the water status of the plant and induces stomatal closure. Guard cell anion channels play a central role in this response, because they mediate anion efflux, and in turn, cause a depolarization-induced K ؉ release. We recorded early steps in ABA signaling, introducing multibarreled microelectrodes in guard cells of intact plants. Upon external ABA treatment, anion channels transiently activated after a lag phase of Ϸ2 min. As expected for a cytosolic ABA receptor, iontophoretic ABA loading into the cytoplasm initiated a rise in anion current without delay. These ABA responses could be elicited repetitively at resting and at largely depolarized potentials (e.g., 0 mV), ruling out signal transduction by means of hyperpolarizationactivated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of Ϸ100 nM background Ca 2؉ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca 2؉ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetate. The chain of events appears very direct, because none of the tested putative ABA-signaling intermediates (inositol 1,4,5 trisphosphate, inositol hexakisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose), could mimic ABA as anion channel activator. In patchclamp experiments, cytosolic ABA also evoked anion current transients carried by R-and S-type anion channels. The response was dose-dependent with half-maximum activation at 2.6 M ABA. Our studies point to an ABA pathway initiated by ABA binding to a cytosolic receptor that within seconds activates anion channels, and in turn, leads to depolarization of the plasma membrane. stomatal closure T he plant hormone abscisic acid (ABA) provides a developmental signal serving as a chemical switch between, for instance, dormancy and growth of seeds and buds (1). Furthermore, this sesquiterpene is involved in the transmission of environmental changes like drought, saline, and cold periods into stress-adaptation processes (2). Based on the time scale of the individual ABA responses, they can be subdivided into fast (seconds up to minutes) and slow (hours up to days, or even months) signaling processes. Stomatal closure represents the fastest ABA response known so far, characterized by a half-time of Ϸ5 min, and presumably does not involve gene activation. Fast stomatal closure is accomplished by the release of potassium and chloride via voltage-dependent ion channels and by metabolic degradation of the major organic anion malate (3, 4).In search for ABA signaling intermediates, guard cells have been challenged with well characterized modulators operating in signal transduction pathways of animal cells (5-8). To a large extent, the respective substances were injected into guard cells of excised epidermal peels, and the corresponding responses were monitored (9-12). After microinjection, photolysis of caged inositol 1,4,...

Summary During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double‐barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (ΔG < 5 nS), ABA predominantly activated voltage‐independent (slow (S)‐type) anion channels. During strong responses (ΔG > 5 nS), however, ABA activated voltage‐dependent (rapid (R)‐type) in addition to S‐type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl− from guard cells and a rapid but confined reduction in stomatal aperture.

Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore .3.34 Å (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants.