Volume 8, Issue 11, Pages 1665-1674 (November 2015) Cytosolic Ca2+ Signals Enhance the Vacuolar Ion Conductivity of Bulging Arabidopsis Root Hair Cells  Yi Wang, Julian Dindas, Florian Rienmüller, Melanie Krebs, Rainer Waadt, Karin Schumacher, Wei-Hua Wu, Rainer Hedrich, M. Rob G. Roelfsema  Molecular Plant  Volume 8, Issue 11, Pages 1665-1674 (November 2015) DOI: 10.1016/j.molp.2015.07.009 Copyright © 2015 The Author Terms and Conditions

Figure 1 Epidermal Root Cells, Studied with Triple-Barreled Microelectrodes Located in the Cytosol. (A) Fluorescence of Lucifer yellow injected into the cytosol of an epidermal root cell with a triple-barreled microelectrode. (B) Cartoon of a microelectrode positioned in the cytosol of an epidermal root cell. The microelectrode is measuring the plasma membrane potential (Epm), between the cytosol (cs) and apoplast (ap), which was on average −172 mV (SE = 3, n = 8). (C) Two barrels of the microelectrode were filled with 300 mM KCl and used to clamp the plasma membrane voltage, applying a bipolar staircase protocol with 20-mV increments as the input signal. Upper traces, output of the voltage-recording electrode. Note: due to the high conductance, a low voltage clamp accuracy was obtained and the measured voltage clamp steps were only 14 mV (SD = 2, n = 8). Lower traces, current applied via the current-injection electrode. (D) Current–voltage relations of eight epidermal root cells measured with triple-barreled electrodes located in the cytosol. The current measured at the start of the voltage pulse is shown as closed symbols and that at the end of the pulse by open symbols. The lines with large symbols represent the average conductance determined by linear regression. Molecular Plant 2015 8, 1665-1674DOI: (10.1016/j.molp.2015.07.009) Copyright © 2015 The Author Terms and Conditions

Figure 2 Epidermal Root Cells Examined with Triple-Barreled Microelectrodes Located in the Vacuole. (A) Fluorescence of Lucifer yellow injected into the vacuolar lumen of an epidermal root cell with a triple-barreled microelectrode. (B) Cartoon of a microelectrode impaled through the plasma membrane (pm) and vacuolar membrane (vm) into the lumen of a vacuole (vl). The microelectrode is measuring the electrical potential difference between the vacuolar lumen and cytosol (cs), as well as that between the cytosol and apoplast (ap). Note: the plasma membrane potential (Epm) and vacuolar membrane potential (Evm) are polarized in reverse directions relative to the bath electrode, so the electrical potential measured by the microelectrode is Et = Epm − Evm. On average, Et was −141 mV (SE = 4, n = 16). (C) Two barrels of a microelectrode were filled with 300 mM KCl and used to clamp the voltage, applying a bipolar staircase protocol with 20-mV increments as the input signal. Upper traces, output of the voltage-recording electrode; lower traces, current applied via the current-injection electrode. (D) Current–voltage relations of 16 epidermal root cell vacuoles measured with triple-barreled electrodes. The current measured at the start of the voltage pulse is shown as closed triangles and that at the end of the pulse by open triangles. The solid lines with large triangles represent the average conductance, determined by linear regression. For comparison, the average conductance measured by electrodes in the cytosol (as shown in Figure 1) is shown by the dashed lines with circles. Molecular Plant 2015 8, 1665-1674DOI: (10.1016/j.molp.2015.07.009) Copyright © 2015 The Author Terms and Conditions

Figure 3 Impact of Voltage Clamp Steps at the Vacuolar Membrane on the Plasma Membrane Potential in Root Hair Cells. (A) Cartoon of a root hair cell impaled with a double-barreled electrode in the vacuole and a single-barreled electrode in the cytosol. The symbols at the electrodes match those at current traces and voltage traces of B. (B) Upper traces, current injected through the first barrel of the double-barreled electrode positioned in the vacuole. The vacuolar membrane was clamped from the free running membrane potential, with a staircase protocol using 20-mV increments as input signals. Middle traces, voltage recorded by the second barrel of the double-barreled electrode. Lower traces, voltage recorded by the single-barreled electrode located in the cytosol. Inset, enlargement of voltage traces recorded by the single-barreled electrode; only traces of the two most extreme voltages are shown. Note: voltage clamp steps at the vacuolar membrane cause only small changes in the plasma membrane potential, which slightly increase in time at the most negative clamp potential. Symbols correspond to those in A. (C) Cartoon of a root hair cell impaled with a single- and double-barreled electrode, both located in the vacuole. The symbols at the electrodes match those at current traces and voltage traces of D. (D) Upper traces, current injected through the first barrel of a double-barreled electrode into vacuoles as in (B). Middle traces, voltage recorded by the second barrel of the double-barreled electrode. Lower traces, voltage recorded by the single-barreled electrode, also located in the vacuole. Symbols correspond to those in C. Molecular Plant 2015 8, 1665-1674DOI: (10.1016/j.molp.2015.07.009) Copyright © 2015 The Author Terms and Conditions

Figure 4 Transient Changes of the Cytosolic Ca2+ Level Evoked by Impalement of Microelectrodes, as well as Injection of Ca2+ or BAPTA. (A) Fluorescent signals from the root of an Arabidopsis seedling expressing R-GECO1, a Ca2+-sensitive red fluorescent protein. The color code on the left depicts the fluorescence intensity, normalized for the average signal obtained from the region of interest (indicated by the dashed border) at the start of the experiment (t = 1 min). Colored panels display the normalized R-GECO1 intensity before impalement (circle), just after impalement of two microelectrodes (square), and 2 min later (triangle). The corresponding transmission microscope image is shown on the right. (B) Average changes in normalized R-GECO1 signal caused by impalement of microelectrodes into bulging root hair cells (closed circles, n = 26 ± SE), or in control cells (closed triangles, n = 7 ± SE). The time point immediately after impalement (or an arbitrary time point for the control experiments) was set to t = 0, symbols correspond to those in the colored panels in (A). (C) Average changes in normalized R-GECO1 signal evoked by impaling a single-barreled electrode filled with 1 M CaCl2 (t = 0) and subsequent current injection of Ca2+ (Iinj = 1-5 nA) as indicated by the black bars below the trace (n = 10 ± SE). (D) Average changes in R-GECO1 fluorescence intensity evoked by impalement with a single-barreled electrode filled with 10 mM BAPTA (t = 0) and subsequent current injection of BAPTA (Iinj = 1–5 nA) as indicated by the black bars below the trace (n = 6 ± SE). In all graphs, the R-GECO1 fluorescence intensity was normalized to the mean value in the region of interest, at the start of the measurement (t = 1 min in B and t = 2 min in C and D). Molecular Plant 2015 8, 1665-1674DOI: (10.1016/j.molp.2015.07.009) Copyright © 2015 The Author Terms and Conditions

Figure 5 Stimulation of the Vacuolar Ion Conductance by an Increase of the Cytosolic Free Ca2+ Concentration in R-GECO1 Expressing Root Hair Cells. (A) Sudden increase of the cytosolic free Ca2+ concentration after injection of BAPTA into the cytosol of a root hair cell (colored panels). The color code depicted in the bar on the left indicates the R-GECO1 fluorescence intensity, normalized to the average value measured in the region of interest (as indicated by the dashed border) at the start of the experiment. The image on the left was obtained at the start of the experiment (circle), the second during a sudden change of the cytosolic free Ca2+ level (square), and the third 3 min later (triangle). The corresponding transmission microscope image is shown on the right. (B) Changes in R-GECO1 fluorescence intensity of a bulging root hair cell, plotted against time, which was injected with BAPTA as indicated by the black bars above the x-axis (data from the same cells as in A, with corresponding symbols). R-GECO1 fluorescence intensity was normalized to the average value in the region of interest at the start of the measurement. (C) Voltage protocol used to clamp the vacuolar membrane from the free running potential with 20-mV steps. (D) Current traces measured with a double-barreled electrode impaled into the vacuole of the same cell as depicted in (A), with corresponding symbols. Note the transient increase in current across the vacuolar membrane during elevation of the cytosolic Ca2+ concentration. (E) Changes in conductance of the vacuolar membrane (determined by linear regression of current–voltage graphs) plotted against the velocity of increase of the R-GECO1 signal intensity (in normalized units, nu). Cells were grouped into clusters with slow and small (gray symbols) and rapid and large (closed symbols) changes of the cytosolic Ca2+ levels. (F) Steady-state currents (ISS) measured at the end of 2-s voltage pulses as shown in (D), plotted against the change in membrane voltage. Data are shown for cells with rapid and large Ca2+ changes (black symbols in E), before (open circles), during (closed squares), and after (open triangles) Ca2+ elevations (n = 9 ± SE). Molecular Plant 2015 8, 1665-1674DOI: (10.1016/j.molp.2015.07.009) Copyright © 2015 The Author Terms and Conditions

Figure 6 Stimulation of the Vacuolar Ion Conductance by an Increase of the Cytosolic Free Ca2+ Concentration in FURA-2 Injected Root Hair Cells. (A) FURA-2 fluorescence ratio images of a root hair cell (colored panels) and the corresponding transmission microscope image. The bar on the left indicates the color code of the FURA-2 fluorescence ratio signal (F345 nm/F390 nm excitation wavelengths). Images were obtained before (white circle), during (black circle), and after (white triangle) a sudden increase of the cytosolic Ca2+ level. (B) Current traces of the vacuole in the same cell as depicted in (A), clamped from the free running membrane using a bipolar staircase protocol with 20-mV increments. The conductance of the vacuole was measured before (left, white circle), during (middle, black circle), and after (right, open triangle) the increase of the cytosolic free Ca2+ concentration. (C) Voltage clamp protocol used to clamp the vacuolar membrane potential. (D) Normalized current–voltage plots of vacuolar membranes before (open circles), during (closed circles), and after (open triangles) increases in the cytosolic free Ca2+. Data were normalized relative to the current measured at ΔV = 80 mV, before elevation of the cytosolic Ca2+ level (n = 5 ± SE). Molecular Plant 2015 8, 1665-1674DOI: (10.1016/j.molp.2015.07.009) Copyright © 2015 The Author Terms and Conditions