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. 1998 Apr 14;95(8):4778-83.
doi: 10.1073/pnas.95.8.4778.

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

A Grabov  1 , M R Blatt
Affiliations

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

A Grabov et al. Proc Natl Acad Sci U S A. .

Abstract

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near -50 mV and -200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 microM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5-10 s and relaxed over 40-60 s thereafter. The [Ca2+]i increases showed a voltage threshold near -120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in "conditioning" [Ca2+]i signals that regulate ion channels for stomatal function.

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Figures

Figure 1
Figure 1
Membrane voltage drives oscillations in cytosolic free [Ca2+] ([Ca2+]i). Concurrent records of voltage, [Ca2+]i, and clamp current (B and C) from three Vicia guard cells bathed in 5 mM Ca2+-Mes, pH 6.1, with 10 mM KCl. Time scales, 1 min. (A) Rise in [Ca2+]i and [Ca2+]i oscillations (lower trace) coincident with spontaneous membrane hyperpolarizations and oscillations in free-running voltage (upper trace). Data from one cell. (B) Cyclic increases in [Ca2+]i (lower trace) evoked under voltage clamp by 20-s steps from −50 to −200 mV (top trace). Clamp current (middle trace) shows inactivation of the inward-rectifying K+ channel current (IK,in) with each [Ca2+]i rise. Note the initial IK,in activation during the first 1–2 s of each step, and the early inactivation of IK,in during the later voltage steps to −200 mV before full [Ca2+]i recovery. (C) [Ca2+]i increase (Left, lower trace) evoked by transfer to 0.1 mM KCl in 5 mM Ca2+-Mes, pH 6.1, to hyperpolarize the free-running voltage (Left, upper trace). No change in [Ca2+]i was observed when membrane voltage was clamped to −75 mV during exposure to 0.1 mM KCl (Right, upper and lower traces). Clamp current (Right, middle trace) was dominated by outward-rectifying K+ channels (not shown; see ref. 20). Open bars (above traces) indicate periods of exposure to 0.1 mM KCl.
Figure 2
Figure 2
Increases in [Ca2+]i are strongly dependent on membrane voltage. (A) Clamp steps of 20 s from −50 mV to voltages between −80 and −200 mV (top trace), clamp current (middle trace), and [Ca2+]i record (bottom) show a small increase in [Ca2+]i at voltages near −150 mV with a pronounced rise and IK,in inactivation at −200 mV. Time scale, 1 min. Data from one guard cell in 5 mM Ca2+-Mes, pH 6.1, with 10 mM KCl. (B) Peak [Ca2+]i at the end of 20-s voltage steps and rate of rise in [Ca2+]i (d[Ca2+]i/dt as linear fittings ± SE, Inset) during the first 5 s of the voltage steps in A plotted against clamp voltage. Histograms are means ± SE for peak [Ca2+]i at −50 mV and for 20-s voltage steps at −190 (n = 16).
Figure 3
Figure 3
Increases in [Ca2+]i are dependent on the extracellular Ca2+ concentration and triggered by Ca2+ influx. Data from one Vicia guard cell in 5 mM K+-Mes, pH 6.1, and KCl (= 10 mM K+), with 0.02–20 mM CaCl2 (A–C) and from a second cell with 2 mM MnCl2 instead of CaCl2 (D). (A) [Ca2+]i record during exposures to 0.02, 0.2, and 2 mM CaCl2 (bars above). The cell was clamped to −50 mV throughout with 4-s steps to −200 mV at times indicated (⊔). Time scale, 2 min. (B) Mean rate of rise in [Ca2+]i (d[Ca2+]i/dt) during voltage steps plotted as a function of extracellular Ca2+ concentration shows roughly a 10-fold increase with each decade change in [Ca2+]o. (C) [Ca2+]i rise evoked on stepping to −200 mV continues to increase after returning to −50 mV. Data from the A in 2 mM [Ca2+]o replotted on an expanded time scale. Period of clamp step to −200 mV indicated by vertical hairlines. Time scale, 10 s. (D) Fura2 fluorescence (Upper) recorded on excitation with 340 nm (f340, fine line), 360 nm (f360, heavy line), and 390 nm (f390, dotted line) light, and the corresponding [Ca2+]i signal (Lower). Time scale, 20 s. Note the approximate 20% quench of fluorescence excited at all three wavelengths during the 20-s step to −200 mV (⊔, Upper) and the absence of any change in [Ca2+]i. (Inset) Fura2 fluorescence excited at 340, 360, and 390 nm during the first 20-s voltage step (⊔) in Fig. 1B for comparison.
Figure 4
Figure 4
Membrane hyperpolarization evokes a wave of high [Ca2+]i that moves centripetally. Fura2 fluorescence ratio images (Upper, a–e) from one Vicia guard cell bathed in 5 mM Ca2+-Mes, pH 6.1, and 10 mM KCl. Pseudo-color scale, 0–2 μM [Ca2+]i. Microelectrode position (masked from images) indicated in schematic (Lower). Corresponding [Ca2+]i traces (Lower, cross-referenced to images by letter) determined as pixel-sums from the peripheral 3- μm (red trace) and central regions (blue trace) of the cell (see schematic). Membrane voltage was stepped from −50 mV to −200 mV for 20 s at the time indicated above trace (⊔). Time scale, 30 s. Ratio images and trace obtained from measurements time-averaged over 400-ms intervals. Image time points were 2 s before (a), then 2 s (b), 5 s (c), and 15 s (d) after the start, and finally 48 s (e) after the end of the voltage step. (Lower Inset) [Ca2+]i traces replotted on expanded time scale. Vertical dotted lines indicate time points at which traces first rose beyond 3 SD from the means of each trace determined from the 10-s period preceding the voltage step. Time scale, 5 s.
Figure 5
Figure 5
Membrane voltage and abscisic acid (ABA) interact to facilitate [Ca2+]i increases. Data from Vicia guard cells bathed in 5 mM Ca2+-Mes, pH 6.1, and 10 mM KCl before and after adding 20 μM ABA to the bath. (A) ABA displaces the threshold for [Ca2+]i rise to more positive voltages. Fura2 fluorescence ratios recorded while driving membrane voltage in 1-min ramps from −50 to −200 mV. Data from one Vicia guard cell before (lower trace) and 4 min after (upper trace) adding ABA. [Ca2+]i signals (points) smoothed (solid lines) by empirical, least-squares fitting of data points to a sigmoid function (27). Thresholds (arrows) taken as time points at which the fitted curves first exceeded 10 nM above the means determined from the 10-s period preceding the voltage ramps (fine horizontal lines). Scale: horizontal, 5 s; vertical, 40 nM. (B) [Ca2+]i increases are augmented and prolonged by ABA, but are potentiated only on membrane hyperpolarization. Data from one Vicia guard cell before and during ABA exposure (diagonal-filled bar). Fura2 fluorescence ratios recorded while driving membrane voltage in 20-s steps from −50 to −180 mV at times indicated above trace (⊔). Time scale (bar), 1 min.
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