Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca2+ Signals  Lena J. Voss, Rainer Hedrich, M. Rob G. Roelfsema  Molecular Plant  Volume 9, Issue 3, Pages 471-480 (March 2016) DOI: 10.1016/j.molp.2016.02.004 Copyright © 2016 The Author Terms and Conditions

Figure 1 Confocal Spinning Disc Images of Fluorescent Dyes, Injected into Tobacco Guard Cells with Multi-Barreled Microelectrodes. (A) Time course of images obtained during injection of Oregon Green BAPTA into a tobacco guard cell in an intact plant. See also Supplemental Movie 1. (B) Z stack of images of a tobacco guard cell loaded with fluorescent dye from an electrode located in the cytosol. Images are presented from the plane close to the mesophyll on the left to that covered by the cuticle on the right. See also Supplemental Movie 2. Note the bright fluorescent area associated with the nucleus in the middle of the cell, circular structures surrounding the chloroplasts in the plane close to the mesophyll, and a plane mostly occupied by the vacuole below the cuticle. The distance between the planes was approximately 1.6 μm. (C) Z stack of images of a tobacco guard cell loaded with fluorescent dye from an electrode located in the vacuole. See also Supplemental Movie 3. Note that the vacuole is a single compartment that surrounds the nucleus in the middle plane of the guard cell. The distance between the planes was approximately 2 μm. Scale bars represent 10 μm. Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions

Figure 2 Changes of Lucifer Yellow Fluorescence Intensity during Voltage Clamp of a Tobacco Guard Cell from −100 mV to −200 mV or −220 mV for 10 s. (A) False colored images of a guard cell in an intact tobacco plant loaded with Lucifer Yellow. See also Supplemental Movie 4. The cell was clamped from the holding potential of −100 mV (t = 10 and 60 s) to −200 mV from 20 to 30 s. Areas with high or low fluorescence intensity are indicated by color code as shown in the bar on the right. Scale bar represents 10 μm. (B) Changes in the fluorescence intensity of Lucifer Yellow shown relative to the values measured at t = 0 s, as indicated by the scale bar on the right. See also Supplemental Movie 5. Data are from the same cell as in (A). Note that the voltage pulse causes an increase in fluorescence signal in the region around the nucleus, while the signal decreases within the nucleus (t = 30 s). Scale bar represents 10 μm. (C) Average changes in fluorescence intensity of guard cells loaded with Lucifer Yellow, plotted against time. The cells were stimulated with voltage pulses from −100 mV to −200 mV (5 cells) or −220 mV (1 cell), as indicated by the black area in the bar above the x axis. Fluorescence values were normalized to their values at the start of the experiments. Signals were averaged for the region around the nucleus (filled circles), within the nucleus (filled triangles), and for the whole cell (open triangles). Error bars represent SE, n = 6. (D) Lucifer Yellow fluorescence intensity plotted for each pixel of the line shown in (A), t = 0. Fluorescence curves were obtained before (t = 0), during (t = 30), and after (t = 60) clamping the cell to −200 mV for 10 s. Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions

Figure 3 Changes in Fluorescence Intensity of the Fluorescent Protein Venus in Arabidopsis Guard Cells During Hyperpolarizing Pulses. (A) False colored images of an Arabidopsis guard cell in an epidermal strip that expresses Venus and was stimulated with a 10-s voltage pulse, starting at t = 20 s, from −100 mV to −180 mV. Images were obtained before (t = 10, left panel), during (t = 30, middle panel), and after (t = 60, right panel) the pulse. See also Supplemental Movie 6. The scale bar represents 10 μm. (B) Average changes in Venus fluorescence intensity of guard cells stimulated with pulses to −160 mV (2 cells), −180 mV (3 cells), or −200 mV (1 cell), as indicated by the black area in the bar above the x axis. Fluorescence signals were normalized to their values at the start of the experiments. Signals were averaged for the region around the nucleus (filled circles), within the nucleus (filled triangles), and the whole cell (open triangles). Error bars represent SE, n = 6. (C) Venus fluorescence intensity plotted for each pixel of the line shown in (A), left panel. Fluorescence curves were obtained before (t = 10), during (t = 30), and after (t = 60) a 10-s hyperpolarizing pulse to −180 mV. Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions

Figure 4 Changes in Cytosolic Volume during Voltage Clamp Pulses Are Blocked by Extracellular Cs+. (A) Voltage protocol (upper traces) and current response (lower traces) of a tobacco guard cell in an epidermal strip loaded with Lucifer Yellow. Current traces are given for a bath solution with 50 mM K+ (open symbol) as well as 50 mM Cs+ (closed symbol). In the presence of K+, the voltage pulse to −200 mV triggers a time-dependent activation of K+ uptake channels, followed by a transient inward current carried by S-type anion channels (Stange et al., 2010). Both the currents through inward K+ channels and S-type anion channels are absent in the presence of Cs+. (B and C) False colored images of the same guard cell as in (A) loaded with Lucifer Yellow and perfused with a bath solution containing either 50 mM KCl (B, see also Supplemental Movie 7), or 50 mM CsCl (C, see also Supplemental Movie 8). Changes in fluorescence intensity are given relative to the values at the start of the experiment, as indicated by the scale bar on the right. The cell was stimulated with a 10-s voltage pulse from −100 mV (t = 10 and t = 70) to −200 mV (t = 30). Note that Cs+ in the bath solution prevents voltage-induced changes in the Lucifer Yellow signal. Scale bars represent 10 μm. (D and E) Average changes in fluorescence intensity given relative to that at the start of the experiments plotted against time. Experiments were conducted with cells in a bath solution with 50 mM KCl (D) or 50 mM CsCl (E). Cells were stimulated with a 10-s voltage pulse from −100 mV to −180, or −200 mV, as indicated by the bar above the x axis. Fluorescence signals were averaged for regions surrounding the nucleus (closed circles), the area in the nucleus (closed triangles), and the whole cell (open triangles). Error bars represent SE, n = 11 (D), n = 7 (E). (F) Changes in the fluorescence intensity plotted against the change in current required to clamp the plasma membrane to −180 or −200 mV. Data obtained in bath solution with 50 mM KCl are given as open symbols, while closed symbols represent experiments with 50 mM CsCl in the bath solution. The line was obtained by linear regression of all data points. Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions

Figure 5 Current Injection with Gluconate Causes Reduced but Irreversible Changes in Cytosolic Volume. (A and B) False colored images that indicate the fluorescence intensity of Lucifer Yellow in tobacco guard cells stimulated with a 50-s voltage pulse starting at t = 20 s, from −100 mV to −180 mV. Images were obtained before (t = 10 s), during (t = 40 s and 70 s), and after (t = 100 s and 130 s) the voltage pulse. Guard cells were impaled with an electrode with the current injection barrel filled with 300 mM KCl (A, see also Supplemental Movie 9) or 300 mM K-gluconate in the tip (B, see also Supplemental Movie 10). Arrowheads indicate sites of cytosolic expansion. The bar on the right links the color code to the fluorescence intensity; the bar in the image on the left represents 10 μm. (C and E) Average changes in fluorescence intensity triggered by voltage pulses from −100 mV to −180, or −200 mV, as indicated by the black bar above the x axis. Average values are presented for the region around the nucleus (closed circles), in the nucleus (closed triangles), and the whole cell (open triangles). Guard cells were impaled with electrodes with 300 mM KCl (C) or 300 mM K-gluconate in the tip (E) in the current injection barrel of the triple-barreled microelectrode. Note that the changes in fluorescence intensity become smaller and irreversible with gluconate in the current injection barrel. Error bars represent SE, n = 7. (D and F) Cartoon depicting the sequence of events caused by hyperpolarization of the guard cell plasma membrane. (1) Hyperpolarization activates K+-selective channels that facilitate the K+ uptake. (2) The K+ current across the plasma membrane is compensated by an equal flux of K+ and Cl− through the electrode if the current injection barrel is filled with KCl (D) and predominantly by K+ in case of K-gluconate in the current injection barrel (F). Due to the accumulation of KCl (D) or K-gluconate (F), water from the vacuole is taken up into the cytosol, which causes the vacuole to shrink (3). Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions

Figure 6 Voltage-Induced Ca2+ Elevation Is Inhibited by Replacing Extracellular K+ with Cs+. (A) Changes in Oregon Green BAPTA fluorescence signal provoked by a voltage pulse of 10 s from the holding potential of −100 mV (t = 10 s, 40 s, 50 s, and 60 s) to −200 mV (t = 28 s). Oregon Green BAPTA was loaded into a guard cell in an epidermal strip that was kept in a bath solution with 50 mM CsCl (upper panel, see also Supplemental Movie 11) or in a bath solution with KCl (lower panel, see also Supplemental Movie 12). Average changes in fluorescence intensity relative to that at the start of the experiment are represented in false color, as indicated by the bar on the right. The bars in the left images represent 10 μm. (B) Changes in Oregon Green BAPTA signal provoked with voltage pulses to −180, or −200 mV, as in (A), averaged for whole guard cells obtained with epidermal strips kept in a bath solution with 50 mM KCl (open triangles) or 50 mM CsCl (filled triangles). Note that elevation of the Oregon Green BAPTA fluorescence is reduced in the presence of Cs+. Error bars represent SE, n = 9 (KCl), n = 10 (CsCl). Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions

Figure 7 Cytosolic Volume Changes Evoke Ca2+ Signals in Tobacco Guard Cells. (A) FURA-2 F345/F390 ratio of a tobacco guard cell plotted against time. The epidermal strip with the guard cell was incubated in a bath solution with 50 mM CsCl or 50 mM KCl and supplemented with 1 mM LaCl3, as indicated by the bars above the graph. The cell was stimulated with 10-s voltage pulses from a holding potential of −100 mV to −180 mV as indicated by the gray bars. Note that changes in the FURA-2 ratio triggered by the voltage pulses are blocked by the combination of Cs+ and La3+. (B and C) FURA-2 ratio (upper graphs) and current traces (lower graphs) of the same guard cell as in (A) stimulated with a voltage pulse from −100 mV to −180 mV (indicated by the gray bar). The cell was tested with a bath solution containing either 50 mM KCl (open symbols) or 50 mM CsCl (closed symbols) and in the absence (B) or presence (C) of 1 mM LaCl3. Note that Cs+ blocks the inward currents triggered during and after the voltage pulse. (D and E) Average changes in FURA-2 ratio triggered with voltage pulses from −100 mV to −180 mV. Experiments were conducted with bath solutions containing either 50 mM KCl or 50 mM CsCl as indicated below the x axis and without (D) or with (E) 1 mM LaCl3 in the bath solution. Error bars represent SE, n = 5. Molecular Plant 2016 9, 471-480DOI: (10.1016/j.molp.2016.02.004) Copyright © 2016 The Author Terms and Conditions