Cytosolic abscisic acid activates guard cell anion channels without preceding Ca2+ signals

Abstract

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 approximately 2 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 hyperpolarization-activated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of approximately 100 nM background Ca2+ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca2+ 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 patch-clamp 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 microM 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.

Fig. 1.

Plasma membrane responses of V. faba guard cells in intact plants to ABA and light. (A) Reversible changes in the free-running membrane potential of a guard cell exposed to ABA and darkness. Note that K⁺ conductances in A and B were eliminated with Ba²⁺ in the external solution and Cs⁺ in the pipette. (B) Dependence of ABA-induced anion channel activation on the plasma membrane potential. ABA (20 μM) was applied with repetitive pulses of 40 s (black squares above the trace) to a single guard cell at three holding potentials (lower trace).

Fig. 2.

Simultaneous recordings of the cytoplasmic free [Ca²⁺] and ABA-induced anion currents. (A) ABA responses of a V. faba guard cell in an intact plant, recorded at a holding potential of –100 mV before and after injection of FURA-2. (Left) Response to ABA before loading. (Right) ABA response after loading of FURA-2. The upper trace shows the FURA-2 F₃₄₅/F₃₉₀ fluorescent ratio. (B) Guard cell stimulated with voltage pulses and ABA. A FURA-2 loaded guard cell was clamped to a holding potential of –100 mV and to test potentials of 0 and –180 mV (lower trace). Hyperpolarization, but not depolarization, evoked an increase in cytoplasmic free [Ca²⁺] (upper trace), followed by an increase in inward current (arrow, middle trace). Subsequent application of ABA (black bar) induced an inward current without a change in cytoplasmic free [Ca²⁺]. (C) Average change in cytoplasmic free [Ca²⁺] (upper trace) of six guard cells displaying typical ABA-induced anion currents (lower trace). Note that ABA did not affect the cytoplasmic free [Ca²⁺]. Error bars represent SE. (D) Average change in cytoplasmic free [Ca²⁺] (upper trace) of 11 guard cells clamped to –275 mV for 1 s, starting after 20 s, from a holding potential of –100 mV. Note that strong hyperpolarizations triggered large changes in the cytoplasmic free [Ca²⁺] and were followed by a transient increase in inward current. Error bars represent SE.

Fig. 3.

Cytoplasmic application of BAPTA, ABA, and the putative second messenger InsP₃.(A) Repression of the ABA-induced anion current transient by cytoplasmic BAPTA. (Left) Response to external ABA before loading BAPTA. (Right) Strong inhibition of the ABA response after simultaneous loading of BAPTA and FURA-2. The cell was loaded by means of the third barrel of the microelectrode filled with 2 mM FURA-2 and 50 mM BAPTA. Upper trace shows the FURA-2 F₃₄₅/F₃₉₀ fluorescent ratio. (B) Guard cell response to externally and cytoplasmically applied ABA. Anion current transients were triggered with ABA externally (black bar) and subsequently through a 30-s loading pulse of ABA by means of the third barrel of the microelectrode filled with 100 μM ABA (arrows). Note that during injection the membrane potential remained clamped at –100 mV, the inward loading current by means of the third barrel, therefore, is compensated by an outward current. (C) Current response to the putative signaling intermediate InsP₃. Before and after InsP₃ injection, anion current transients were elicited with extracellular ABA (black bars) at a holding potential of –100 mV.

Fig. 4.

Kinetics of ABA-induced activation of anion channels in guard cell protoplasts. (A) Current–voltage relations in the presence of 10 μM cytosolic ABA, (open symbols) and without ABA (filled symbols), at time points indicated. Voltage ramps were applied from –158 to + 82 mV in 1,500 ms, and the holding potential was –158 mV. Note the activities of R- and S-type channels. (B) Time courses of peak currents from voltage ramps as shown in Upper, recorded after whole-cell access. Time courses are shown for representative experiments in the absence (filled symbols, n = 26) and presence (open symbols, n = 24) of 10 μM ABA in the pipette solution. (C) Peak currents of R- and S-type channels were plotted as a function of time after gaining whole-cell access. Currents were normalized to their maxima. S-type current amplitudes were determined from linear interpolation between the current at –98 and 2 mV during voltage ramps as shown in A. R-type current amplitudes were expressed as the difference between peak and S-type currents. (D) Single-channel fluctuations of an outside-out patch, excised from a whole cell at the peak of ABA-induced anion currents (≈2 min). The recordings were conducted at several time points, indicated as the time after gaining whole-cell access.

Fig. 5.

Activation of anion currents in guard cell protoplasts by extracellular and intracellular ABA. (A) Voltage ramps in the presence of different ABA concentrations in the pipette solution were applied and peak currents were determined, as in Fig. 4. The maximum currents during the ABA responses were normalized to the cell capacitance and plotted as a function of the ABA concentration (error bars represent SE, n indicates number of cells). Two cells with unusually high ABA responses were not included in the analysis. Filled symbols represent data obtained with a pipette solution containing 50 mM BAPTA instead of 10 mM EGTA. (B) Time courses of peak currents from voltage ramps as shown in Fig. 4A Upper, recorded after whole-cell access. Time courses are shown for representative experiments in which 20 μM ABA was given at the extracellular face (filled symbols) or 10 μM ABA was applied by means of the pipette solution (open symbols). (Inset) Average peak currents after intracellular (open symbols), or extracellular (filled symbols) ABA application, or without ABA (contr.). Error bars represent SE, n indicates number of cells.