Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum

Abstract

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca(2+) concentration. In Vicia faba, however, guard cell anion channels activate in a Ca(2+)-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca(2+) concentration. ABA triggered transient rises in cytoplasmic Ca(2+) in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca(2+) closely matched the activation of anion channels, while the Ca(2+) rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca(2+) level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca(2+) concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca(2+) in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca(2+)-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca(2+)-independent pathway.

Figure 1.

Guard cells in intact N. tabacum plants impaled with triple-barreled electrodes. A, Close up of an N. tabacum leaf impaled with triple-barreled electrode (left), guard cells in the abaxial epidermis were visualized with a water emersion objective (middle). Experimental solutions were perfused through a hypodermic needle (left back) over the cuticle to a suction pipette (right). B, Overview of the experimental setup with the upright microscope (middle), microelectrode amplifiers (left), and the N. tabacum plant (right). C, N. tabacum guard cell impaled with a triple-barreled electrode, through which FURA2 was injected, located with the tip in the cytoplasm. D, Guard cell injected with FURA2 through a triple-barreled electrode with the tip in the vacuole. E, Guard cell injected with FURA2 in the cytoplasm, but in which the FURA slowly appeared in the vacuole. F, Same guard cell as in E but 15 min later.

Figure 2.

ABA-induced changes in the plasma membrane conductance of an N. tabacum guard cell. A, Current trace of a guard cell clamped to a holding potential of −100 mV and exposed to 50 μm ABA (as indicated by the bar below the graph). Symbols indicate the time points at which voltage clamp step protocols were applied. B, Current traces from test pulses of the same cell as in A, symbols correlate. Arrows indicate the 0 pA level. Cells were clamped from a holding potential of −100 mV to potentials ranging from −180 to 0 mV with 20-mV increments. Note that the introduction of ABA (•) caused a dramatic increase of currents measured directly after the capacity compensation peak, while ABA had little effect on time-dependent outward currents. The currents virtually recovered to prestimulus values after a washout of ABA (□). C, Steady-state currents, sampled at the end of the 2-s test pulses, plotted against the clamp voltage (symbols correspond to A). Note that ABA increased inward current ranging from −40 to −140 mV and caused a large shift of the zero current potential to more positive values. D, Instantaneous currents, sampled directly after termination of the capacity compensation peak, plotted against the clamp voltage (symbols correspond to A). Note that ABA activated a channel with a linear instantaneous current-voltage relation and a reversal potential at −17 mV.

Figure 3.

N. tabacum guard cell in intact plant, with an ABA-induced rise of the cytoplasmic free Ca²⁺ concentration, matching an increase of anion currents. A, ABA responses of a guard cell clamped to −100 mV, loaded with FURA2, and exposed to 50 μm ABA as indicated by the bar below the graphs. Top trace, Cytoplasmic free Ca²⁺ concentration as calculated from the FURA2 F₃₄₅:F₃₉₀ fluorescent ratio. Note that ABA induces a transient rise in the free Ca²⁺ concentration. Lower trace, current trace at −100 mV. Note that ABA induces an inward current that matches the rise in the cytoplasmic free Ca²⁺ concentration. B, False color cytoplasmic free Ca²⁺ images of the same guard cell as in A, before, during, and after the response to ABA (symbols correspond to A). Color codes are linked to free Ca²⁺ concentration in the bar on the left. Note that ABA triggers a rise in the cytoplasmic free Ca²⁺ concentration throughout the cell, which is most obvious in the area surrounding the nucleus.

Figure 4.

N. tabacum guard cells in intact plants, in which ABA induced an increase in anion channel activity, accompanied by a delayed rise in the cytoplasmic free Ca²⁺ concentration (A) or without a change of the Ca²⁺ concentration (B). A and B, ABA responses of guard cells clamped to −100 mV, loaded with FURA2, and exposed to 50 μm ABA as indicated by the bar below the graphs. Top traces, cytoplasmic free Ca²⁺ concentration as calculated from the FURA2 F₃₄₅:F₃₉₀ fluorescent ratio. Note that ABA induces a transient rise in the free Ca²⁺ concentration that is delayed compared to the increase in current in A but no change in the Ca²⁺ concentration in B. Bottom traces, current trace at −100 mV. Note that ABA induces transient increases in inward current in both cells.

Figure 5.

ABA-induced change in free-running membrane potential and cytoplasmic free Ca²⁺ concentration of a N. tabacum guard cell in an intact plant. A, ABA response of a guard cell at its free-running membrane potential, loaded with FURA2, and exposed to 50 μm ABA as indicated by the bar below the graphs. Top traces, cytoplasmic free Ca²⁺ concentration as calculated from the FURA2 F₃₄₅:F₃₉₀ fluorescent ratio. Note that ABA induces a transient rise in the free Ca²⁺ concentration that matches the change in free-running membrane potential. Bottom traces, free-running membrane potential of the same cell as in the top trace. B, Average free Ca²⁺ concentration of N. tabacum guard cells as calculated from the FURA2 F₃₄₅:F₃₉₀ fluorescent ratio before (black bar) or at the peak of the ABA response (gray bar); cells not showing an ABA-induced change in cytoplasmic free Ca²⁺ were not included. Guard cells were clamped at a holding potential of −100 mV (left bars) or at their free-running membrane potential (average E_m = −55 mV, se = 5, n = 6, right bars). Error bars represent se, n is indicated below the bars. Note that the average free Ca²⁺ concentration is lower in cell at their free-running membrane potential and is raised by ABA irrespective of current- or voltage-clamp conditions.

Figure 6.

Ca²⁺-dependent activation of plasma membrane anion channels in guard cell protoplasts of N. tabacum. A, Cl⁻-induced shift in reversal potential of anion channels. The current-voltage relation was obtained with voltage clamp step protocols from a holding potential of −158 mV to a preconditioning voltage of +62 mV and test potentials ranging from +82 to −218 mV. Measurements were carried out with a pipette solution containing 10 mm ATP, 110 nm free Ca²⁺, and 150 mm Cl⁻. A change of the extracellular Cl⁻ concentration from 80 to 8 mm shifted the reversal potential from 7.5 mV (se = 0.2, n = 4) to 41.3 mV (se = 3.5, n = 4). B and C, Kinetics of activation of anion channels in guard cell protoplasts after establishing the whole cell configuration (t = 0) with 10 mm ATP (B) or 1 mm ATP (C) in the pipette solution. The plasma membrane was clamped to −158 mV and Ca²⁺ was given via the patch pipette either at a free Ca²⁺ concentration of 0 (○), 110 (□), or 380 (▵) nm. Error bars represent se, n = 6 to 12. Note that at 10 mm ATP, 380 nm Ca²⁺ activates anion channels during the first 30 s, but the current decays to the same value as with 0 or 110 nm during prolonged measurements. At 1 mm ATP, 110 and 380 nm Ca²⁺ activate anion channels during the first 30 s and inhibit slow inactivation.

Figure 7.

Kinetics of ABA-induced activation of anion channels in guard cell protoplasts from N. tabacum. A, Average plasma membrane currents at −158 mV of N. tabacum guard cell protoplasts, patched with pipettes containing 1 mm ATP and 0 mm free Ca²⁺. ABA was omitted (○) or applied through the patch pipette at a concentration of 10 μm (•). Note that ABA inhibits the time-dependent deactivation of anion channels. B, Current traces obtained with voltage clamp step protocols from a holding potential of −158 mV to a preconditioning voltage of +60 mV and test potentials ranging from +82 to −218 mV. For clarity only current traces of every second voltage step (ΔV = −40 mV) are shown. C, Current-voltage relation of representative protoplasts recorded 4.5 min after establishing the whole cell configurations in the presence or absence of 10 μm ABA in the pipette. The protoplasts were challenged with fast voltage clamp ramps from a holding potential of −158 mV to +82 mV in 1,500 ms.