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First published online January 26, 2007; 10.1104/pp.106.091405 Plant Physiology 143:1140-1151 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Osmo-Sensitive and Stretch-Activated Calcium-Permeable Channels in Vicia faba Guard Cells Are Regulated by Actin Dynamics1,[OA]State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China
In responses to a number of environmental stimuli, changes of cytoplasmic [Ca2+]cyt in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca2+ channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca2+ channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca2+ channel inhibitor Gd3+. Disruption of actin filaments activated SA Ca2+ channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca2+]cyt imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca2+ elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca2+ channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca2+]cyt and consequently inhibits overswelling of guard cells. This SA Ca2+ channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.
Stomata form pores on leaf surfaces that facilitate CO2 uptake for photosynthesis and regulate transpirational water vapor loss. A number of stimuli, such as light, CO2, drought, humidity, and the phytohormone abscisic acid (ABA), regulate the aperture of the stomata by controlling the turgor of the two guard cells that surround each stomatal pore (for review, see Mansfield et al., 1990  ; Assmann, 1993). Turgor changes are driven by fluxes of K+ and anions through ion channels in the plasma and vacuolar membranes, Suc accumulation/removal, and metabolism between starch and malate (for review, see Assmann, 1993; MacRobbie, 1998). An increase of [Ca2+]cyt has been shown to be a common and key intermediate, both inactivating inward K+ channels and activating slow anion channels (Schroeder and Hagiwara, 1989), which leads to stomatal closure (for review, see Blatt, 2000; McAinsh et al., 2000). More recent studies demonstrated that [Ca2+]cyt oscillations in guard cells are important for stomatal closure movements (Allen et al., 2000, 2001). The changes of [Ca2+]cyt result from both Ca2+ release from and sequestration to intracellular stores (such as tonoplasts) and Ca2+ entry and efflux across the plasma membrane (PM; Köhler et al., 2003; for review, see MacRobbie, 1998; Hetherington and Brownlee, 2004). Ca2+ efflux from tonoplasts through ion channels has been studied extensively. Two tonoplast Ca2+-permeable ion channels, the fast- and slow-vacuole channels, exist in stomatal guard cells and are regulated by inositol 1,4,5-triphosphate (Allen et al., 1995), calcineurin (Allen and Sanders, 1995), and cyclic ADP-ribose (Leckie et al., 1998). Compared to the studies on tonoplast Ca2+-permeable ion channels and the substantial body of evidence on regulation of [Ca2+]cyt in guard cells (Ward et al., 1995; Ng et al., 2001; for review, see Assmann, 1993; MacRobbie, 1998; Schroeder et al., 2001), little is known about the regulatory mechanisms for Ca2+ channels in the PM of guard cells (Hamilton et al., 2000; Pei et al., 2000; Köhler et al., 2003). Hydrogen peroxide (H2O2) has been demonstrated to mediate ABA signaling to increase [Ca2+]cyt in guard cells by activating Ca2+ channels in the PM (Pei et al., 2000; Klüsener et al., 2002), whereas there is also a line of evidence suggesting that the ABA and H2O2 pathways diverge further downstream in their actions on the outward K+ channels, questioning the role of H2O2 as a critical second messenger regulating guard cell ion channels in response to ABA (Köhler et al., 2003).
Stomatal response to osmotic stress is regulated via a feedback mechanism (Liu and Luan, 1998
Actin microfilaments are dynamic cellular components and they can be assembled or disassembled into actin monomers at spatially defined sites of a living cell during physiological processes. It is established that in animal cells, as a signal transducer, dynamic structural changes in actin microfilaments contribute to several signaling processes involving regulation of ion channel activities (Schwiebert et al., 1994
Identification of the Osmo-Sensitive and Voltage-Dependent Ca2+-Permeable Channels in Vicia Guard Cells
Considering that Ba2+ has been commonly used for Ca2+ channel identification (Hamilton et al., 2000
To further confirm the nature of the recorded inward currents, Gd3+, an inorganic blocker of Ca2+-permeable channels, was applied. Lemtiri-Chlieh et al. (2003) reported that the 100 µM Gd3+ dramatically blocks PM Ca2+-permeable channels in V. faba guard cell protoplasts but with no effect on the inward K+ channel currents. Ding and Pickard (1993) showed that Gd3+ blocks mechanosensory (SA) Ca2+-selective channels in epidermal cells. In this study, addition of 100 µM Gd3+ to the bath solutions dramatically inhibited the activation of the currents by the hypotonic treatment (Fig. 1, trace 1, n = 5; compared to trace 5). The reversal potential of the inward currents activated by hypotonic bath solution was approximately +12 mV, which is close to the theoretical equilibrium potential for Ba2+ (EBa = 14.9 mV) and far from that for Cl– under the given conditions (ECl = –34.6 mV). These results demonstrate that the recorded osmo-regulated inward currents are predominantly carried by an influx of Ba2+ or Ca2+ through Ca2+-permeable channels in the PM of Vicia guard cell protoplasts. These osmo-sensitive Ca2+ channels in guard cells are inhibited under hypertonic conditions and activated under hypotonic conditions.
Actin dynamics has been demonstrated to regulate various types of ion channels in animal and plant cells as discussed in the introduction. To investigate if actin dynamics are involved in the osmo regulation of Ca2+-permeable channels in guard cells, the actin polymerization inhibitor CD and the actin filament stabilizer phalloidin were applied to test their effects on activity of the osmo-regulated Ca2+-permeable channels. Compared to the control (the isotonic bath solutions; Fig. 2A , trace 2, n = 8), the hypertonic treatment exerted a slight inhibition of the whole-cell inward Ba2+ currents (Fig. 2A, trace 1, n = 8). Addition of 20 µM CD to the hypertonic bath solutions dramatically increased the inward Ba2+ currents (Fig. 2A, trace 3, n = 8). Conversely, application of 100 µM phalloidin to the pipette solutions had no effect on the inward currents under the isotonic conditions (the osmolality of both bath and pipette solutions at 500 mosmol; Fig. 2B, trace 2 versus trace 1, n = 6). Furthermore, in the presence of phalloidin, activation of the inward currents by the hypotonic treatment was abolished (Fig. 2B, trace 3 versus trace 1, n = 6). These results demonstrate that the effect of osmo gradients on the inward currents was mediated by actin dynamics. The blockage in depolymerization of actin filaments abolishes activation of Ca2+-permeable channels by hypotonic treatment, which suggests that the hypotonic treatment may first induce depolymerization of actin filaments and consequently cause activation of the Ca2+-permeable channels in guard cells.
Characterization of the SA Ca2+ Channels in the PM of Guard Cells Under the control conditions, the inward channel activity in the isolated outside-out membrane patches was not observed at any voltage tested (from 60 mV to –120 mV) without application of a pressure-mediated stretch to the membrane (the top trace in Fig. 3A shows the recording at –60 mV without application of a stretch). However, the inward currents were elicited at –60 mV after application of a positive pressure to the outside-out membrane patch (blowing into the interior of the glass pipette) and the channel activity was increased along with the increase of the applied pressure from 3 to 15 kPa (Fig. 3A). For example, the open probability (NPo, as defined in "Materials and Methods") was increased by nearly 200% (from 0.31–0.91) when the applied pressure was increased from 9 to 15 kPa (Fig. 3, B and C). This pressure- or stretch-induced activation of the channels was reversible. The channel activity disappeared once the pressure was released (back to 0 kPa; see bottom trace of Fig. 3A).
As shown in Figure 4 , the current amplitude (Fig. 4, A and B) and the NPo (Fig. 4, A and C) of the SA channels are both voltage dependent when a 9-kPa positive pressure was applied to an outside-out membrane patch via the pipette. The derived single-channel conductance was 19.7 pS and the reversal potential was near 15 mV (Fig. 4B, n = 8). The value of reversal potential is close to the theoretical EBa under the given conditions (EBa = 14.9 mV, ECl = –34.6 mV), indicating that the single-channel currents can be ascribed to an influx of Ba2+ through the Ca2+-permeable channels. This notion was further supported by the result that the single-channel conductance increased (Fig. 5A ) and the reversal potential shifted to more positive values (Fig. 5B) along with the increase of Ba2+ concentration in the bath solutions. The relationship for single-channel conductance versus Ba2+ concentration was well fitted by the Michaelis-Menten kinetic equation with a Km at approximately 1.98 mM (Fig. 5A), which is similar to that reported previously (Hamilton et al., 2000 ). The results presented in Figure 5B also show that the measured reversal potentials merged well with the calculated Nernst potentials.
When Ba2+ in the bath solution was substituted with Ca2+, similar voltage-dependent SA currents were observed with application of a 9-kPa positive pressure to an outside-out membrane patch (Fig. 4D). Compared to the results presented in Figure 4, A to C, the Ca2+ current amplitude and the NPo of the channels were similarly voltage dependent (Fig. 4, D–F). The single-channel conductance and the reversal potential of the inward Ca2+ currents were 11.8 pS and 23 mV, respectively (Fig. 4E, n = 4). The relative permeabilities of Ba2+ and Ca2+ were calculated by the simplified Goldman-Hodgkin-Katz equation (Hille, 1993 ) and the derived ratio of PCa2+/PBa2+ was 1.91. Figure 6 shows reversible inhibitory effects of Gd3+, a Ca2+ channel blocker, on the activity of the identified SA channels. The NPo of the channels was significantly reduced in an inhibitor concentration-dependent manner. Under the application of a 9-kPa positive pressure to the membrane patch at –60 mV, the NPo of the SA channels was decreased by nearly 60% or 95% after the addition of 10 µM (Fig. 6, B and F) or 100 µM Gd3+ (Fig. 6, C and G) compared to the control (Fig. 6, A and E), respectively. The removal of Gd3+ from the bath solution resulted in restoration of the channel activity (Fig. 6, D and H).
SA of the Ca2+-Permeable Channels Are Regulated by Actin Dynamics As described above, the SA Ca2+ channels recorded at the single-channel levels shared similar electrophysiological properties with the osmo-sensitive whole-cell inward currents. Figure 7 presents results showing that the SA Ca2+ channels are similarly regulated by actin dynamics compared to the whole-cell recording results as shown in Figure 2. Under the application of a 9-kPa positive pressure to the outside-out membrane patch at –60 mV, addition of 20 µM CD dramatically increased the activity of the SA Ca2+ channels (Fig. 7, B and G versus A and F; n = 7), suggesting that CD-induced depolymerization of actin filaments enhanced the sensitivity of the channels to the stretch. Not surprisingly, the presence of 100 µM phalloidin in the pipette solutions significantly impaired the CD's stimulatory effects on channel activation (Fig. 7, C–E, H, and J).
Osmo Regulation of the SA Ca2+ Channels under the Cell-Attached Configuration Osmo regulation of the SA Ca2+ channels was also investigated under the cell-attached configuration. As shown in Figure 8 , channel activity was activated by suction applied to the cell membrane (Fig. 8, B and G versus A and F), and the activation was significantly reduced when the osmolality of the bath solutions was increased from 500 to 600 mosmol (Fig. 8, C and H versus B and G). Channel activity was greatly enhanced when the osmolality of the bath solutions was decreased from 500 to 400 mosmol (Fig. 8, D and I versus B and G). SA channels were still activated when a hypotonic (400 mosmol) treatment was applied in the absence of suction (Figs. 8, E and J). These results further support the notion that SA Ca2+ channels in the PM of guard cells account for the osmo-sensitive whole-cell currents.
Actin Dynamics Regulates [Ca2+]cyt in Guard Cell Protoplasts Fluo 3-AM, a fluorescent calcium indicator, was employed to test if actin dynamics or osmotic change would regulate the [Ca2+]cyt in guard cells. Most of the tested protoplasts (approximately 80%) were loaded successfully with Fluo 3-AM and the ones with relative stronger fluorescence were chosen for further experiments. Under control conditions (incubation solutions containing 0.1% dimethyl sulfoxide as solvent for CD), the fluorescence of the protoplasts remained at a stable level during a 60-min observation (Fig. 9A ). Addition of 20 µM CD in the incubation solution significantly increased the fluorescence intensity after 20 min (Fig. 9B), while addition of 100 µM Gd3+ impaired the CD-induced increase of fluorescence (Fig. 9C). The results indicate that CD-induced depolymerization of actin filaments may elevate [Ca2+]cyt by stimulating Ca2+ influx through Ca2+ channels in the PM. Similarly, hypotonic treatment (400 mosmol compared to 500 mosmol as the control) increased the fluorescence intensity in guard cell cytoplasm (Fig. 9D), while this hypotonicity-induced increase of fluorescence intensity was blocked by 100 µM Gd3+ (Fig. 9E). In addition, a hypertonic treatment (600 mosmol compared to 500 mosmol as the control) did not significantly affect the fluorescence intensity (Fig. 9F), while subsequent hypotonic treatment significantly increased the fluorescence intensity (Fig. 9F). The addition of 20 µM CD significantly increased the fluorescence intensity even under the hypertonic condition (Fig. 9G). Figure 9H shows the time kinetics of the changes in the relative fluorescence intensity in the guard cell protoplasts under the various treatments, as indicated in the legends of Figure 9. The results of [Ca2+]cyt imaging presented in Figure 9 correlate well with those from the patch-clamping experiments, and both results lead to the same conclusion that actin dynamics mediates osmotic regulation of Ca2+ channel-facilitated Ca2+ influx across the PM of guard cells.
Stomatal movements are controlled by turgor-driven guard cell volume changes. Voltage-dependent K+ channels have been implicated critically in these processes by serving as osmotic-sensing targets in guard cells for a positive feedback loop (Liu and Luan, 1998 ). In this study, we report SA Ca2+ channels in the PM of V. faba guard cells, which are activated by mechanical stretch applied to the PM either by changing osmotic concentration of the bath solutions or by directly exerting a pressure to the PM via micropipettes. More importantly, the observed osmo regulation of the channels is mediated by actin dynamics. Depolymerization of actin filaments results in activation of the channels, whereas the inhibition of actin depolymerization blocked the activation of Ca2+-permeable channels.
Compared to the Ca2+-permeable channels in V. faba guard cells reported by Hamilton et al. (2000)
SA channels or mechano-sensitive channels have been reported in a wide variety of species from bacteria and yeasts to animals and plants (Morris, 1990
A K+ channel-based positive feedback model has been proposed to explain the osmo-regulation mechanisms for stomatal movements (Liu and Luan, 1998
It is known that cytoplasmic Ca2+ elevation may inhibit IKin and stimulate slow anion channels (such as Cl– channels), thus contributing to regulation of stomatal movement (for review, see Schroeder et al., 2001
Liu and Luan (1998)
As discussed by Liu and Luan (1998)
Guard Cell Protoplast Preparation
Vicia faba plants were grown in soil mixture in a growth chamber under a 12-h light (100 µmol m–2 s–1)/12-h dark cycle and temperatures of 22°C and 15°C for daylight and night, respectively. Plants were watered twice a week with tap water and relative humidity was kept at approximately 50%. Guard cell protoplasts were prepared by an enzymatic method as described previously (Wang et al., 1998
Standard whole-cell and single-channel recording techniques were applied in this study. All experiments were conducted at room temperature (approximately 22°C) under dim light. Glass micropipettes were made by using a two-step puller (model PP-83, Narishige) and fire polished by a microforge (model MF-83, Narishige). For the control conditions of both whole-cell and outside-out single-channel recordings, the bath solutions contained 50 mM BaCl2, 0.1 mM dithiothreitol, 10 mM MES/Tris, pH 5.6, and the osmolality was adjusted to 500 mosmol with sorbitol. The pipette solutions were composed of 10 mM BaCl2, 0.1 mM dithiothreitol, 2 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and 10 mM HEPES/Tris, pH 7.1, and the osmolality was adjusted to 500 mosmol with sorbitol. For some specific treatments, BaCl2 was replaced with CaCl2, or some reagents (such as GdCl3, CD, phalloidin, etc.) were added into the solutions, or the osmolality of the solutions was adjusted. All changes made with solution composition or osmolality are indicated in the text or in figure legends.
For the whole-cell recordings, the seal resistance between the membrane and the micropipette was greater than 2 G Patch-clamp recordings were performed using an Axopatch-200B amplifier (Axon Instruments) connected to a microcomputer via an interface (TL-1 DMA Interface, Axon Instruments). pCLAMP software (Version 6.0.4, Axon Instruments) was used to acquire and analyze the whole-cell and single-channel currents. SigmaPlot software was used to draw I-V plots and data analysis.
A Ca2+ fluorescent dye, Fluo 3-AM, was used to monitor changes in relative [Ca2+]cyt in guard cell protoplasts. Protoplasts were isolated as described above and then incubated in a solution containing 10 µM Fluo 3-AM, 0.2% (v/v) pluronic F-127 (freshly added from 1 mM stock solution), 5 mM MES, pH 5.0 (adjusted with Tris), with osmolality adjusted to 500 mosmol with sorbitol, and incubated in the dark for 1.5 h at 4°C. This incubation resulted in 80% of the protoplasts being successfully loaded with Fluo3. The preincubated protoplasts were washed with and kept in a solution containing 10 mM MES, pH 6.0, 50 mM KCl, 1 mM CaCl2, with osmolality adjusted to 500 mosmol with sorbitol. Fluo3 fluorescence was imaged under a laser scanning confocal microscope (Bio-Rad, MRC-1024, equipped with Krypton/Argon laser light). The wavelengths of excitation and emission light were 488 nm and 515 nm, respectively. Three-dimensional scanning was applied with 1-µm Z-series project steps in 2-min cycles, and the three-dimensional reconstruction was used to display the variations of cytosolic calcium, as shown in Figure 9.
All chemicals were obtained from Sigma unless otherwise indicated in the text. Received October 21, 2006; accepted January 12, 2007; published January 26, 2007.
1 This work was supported by the National Science Foundation of China (a competitive fund for Creative Research Groups; grant no. 30421002) and by the Chinese National Key Basic Research Project (grant no. 2006CB100100 to W.H.W.). 
2 Present address: Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, State Key Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wei-Hua Wu (wuwh{at}public3.bta.net.cn).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.091405 * Corresponding author; e-mail wuwh{at}public3.bta.net.cn; fax 8610–6273–4640.
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ABSTRACT
RESULTS
ka, 1999
, control; 
, 20 µM CD; 
, 20 µM CD + Gd3+; 
, hypotonicity; 
, hypotonicity + Gd3+; 
, osmoregulation; 
, hypertonicity + 20 µM CD). The fluorescent intensity at the beginning of each treatment was taken as 100% in H. All data points shown in H are presented as mean ± SE (n = 5).
in all experiments. Membrane potential was clamped to 0 mV, and current data were acquired when the voltages were clamped from –100 mV to 40 mV using a voltage-ramp method at a speed of 0.014 mV/ms or 1 mV/ms, as indicated in figure legends, at 5 min after obtaining whole-cell configuration. Data were filtered at 1 kHz (1 ms/sample) before storage onto the disc of the computer. For the single-channel recordings from the outside-out membrane patches, the bath and pipette solutions were the same as those for the whole-cell recording experiments. For the cell-attached recordings, the pipettes were filled with the bath solution used for the whole-cell recording experiments. The seal resistance was no less than 10 G