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Osmo-Sensitive and Stretch-Activated Calcium-Permeable Channels in Vicia faba Guard Cells Are Regulated by Actin Dynamics1[OA] Wei Zhang, Liu-Min Fan2, and Wei-Hua Wu* 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 [Ca21]cyt in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca21 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 Ca21 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 Ca21 channel inhibitor Gd31. Disruption of actin filaments activated SA Ca21 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, [Ca21]cyt imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca21 elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca21 channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca21]cyt and consequently inhibits overswelling of guard cells. This SA Ca21 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 K1 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 [Ca21]cyt has been shown to be a common and key intermediate, both 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. * Corresponding author; e-mail [email protected]; fax 8610–6273–4640. 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 ([email protected]). [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.091405 1140 inactivating inward K1 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 [Ca21]cyt oscillations in guard cells are important for stomatal closure movements (Allen et al., 2000, 2001). The changes of [Ca21]cyt result from both Ca21 release from and sequestration to intracellular stores (such as tonoplasts) and Ca21 entry and efflux across the plasma membrane (PM; Köhler et al., 2003; for review, see MacRobbie, 1998; Hetherington and Brownlee, 2004). Ca21 efflux from tonoplasts through ion channels has been studied extensively. Two tonoplast Ca21-permeable ion channels, the fast- and slowvacuole 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 Ca21-permeable ion channels and the substantial body of evidence on regulation of [Ca21]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 Ca21 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 [Ca21]cyt in guard cells by activating Ca21 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 Plant Physiology, March 2007, Vol. 143, pp. 1140–1151, www.plantphysiol.org 2007 American Society of Plant Biologists Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Ca21 Channels in Vicia faba Guard Cell Protoplasts downstream in their actions on the outward K1 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). It has been hypothesized that some components, such as stretch-activated (SA) ion channels in the PM of guard cells could sense the osmotic change-induced cell turgor changes or membrane stretch and further transduce osmotic signals (MacRobbie, 1995). It is also suggested that the ABA-dependent and ABA-independent pathways might work together to regulate guard cell turgor under osmotic stress conditions (Liu and Luan, 1998). Voltage-dependent K1 channels and slow anion channels have been shown to be regulated by water stress-induced ABA signal, which contributes to regulation of guard cell turgor and stomatal aperture (Assmann and Wu, 1994; Schwartz et al., 1994; Pei et al., 1997, 1998). On the other hand, osmosensitive voltage-dependent K1 channels, regulated by the osmo gradient across the PM of guard cells, have been considered to be a positive feedback loop in an ABA-independent osmo-sensing pathway that accelerates stomatal movements (Liu and Luan, 1998). Both osmotic stress and physiological cell swelling during stomatal opening may cause stretch forces imposed on the PM of guard cells. SA Ca21-permeable channels (SA Ca21 channels) have been suggested to contribute to regulation of turgor pressure in Vicia guard cells (Cosgrove and Hedrich, 1991). However, regulatory mechanisms of SA Ca21 channels in the PM of guard cells and their roles in signal remain to be elucidated. 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; Cantiello, 1997; for review, see Janmey, 1998). In plants, it has become evident that actin microfilaments also function in signal transduction networks (for review, see Volkmann and Baluška, 1999). Actin dynamics has been proposed to be involved in regulation of Ca21 oscillations and the establishment of [Ca21]cyt gradients in pollen tubes (Fu et al., 2001; Wang et al., 2004). In stomatal guard cells, actin microfilaments respond to physiological stimuli, including light and ABA (Eun and Lee, 1997; Lemichez et al., 2001), and an increase in [Ca21]cyt mediates ABA-induced disruption of actin filaments (Hwang and Lee, 2001). Furthermore, disruption of actin filaments by cytochalasin D (CD), an inhibitor of actin cytoskeleton polymerization, has been shown to up-regulate the osmo-sensitive inward K1 channels and enhance stomatal opening (Kim et al., 1995; Eun and Lee, 1997; Liu and Luan, 1998). The actin cytoskeleton is suggested to serve as an osmo sensor, targeting K1 channels for turgor regulation in a positive feedback loop in guard cells (Liu and Luan, 1998). Similar to the observation in animal cells (Glogauer et al., 1995), actin dynamics in stomatal guard cells may play an important role in regulating SA Ca21 channels and thus modulating stomatal movements. In this study, we report SA Ca21permeable channels in the PM of Vicia faba guard cell, which are sensitive to osmotic changes or stretch forces and are regulated by actin dynamics. These SA Ca21-permeable channels may, at least in part, account for the osmo-sensitive macroscopic Ca21 currents across the PM of guard cells and serve as an osmotic-sensing target in a negative feedback loop. This negative feedback loop, together with the positive feedback loop proposed previously (Liu and Luan, 1998), may explain the mechanisms for osmo regulation of stomatal movements and oscillation (Cowan et al., 1997). RESULTS Identification of the Osmo-Sensitive and Voltage-Dependent Ca21-Permeable Channels in Vicia Guard Cells Considering that Ba21 has been commonly used for Ca21 channel identification (Hamilton et al., 2000; Pei et al., 2000), Ba21 was used in our patch-clamping experiments as the major charge-carrying ion to identify Ca21 channels, unless otherwise indicated. Under the control conditions with an osmolality at 500 mosmol for both bath and pipette solutions, inward currents with small amplitude were observed (Fig. 1, trace 3, n 5 12). When the bath isotonic solutions (osmolality at 500 mosmol) were changed to hypertonic solutions with an osmolality at 600 mosmol, the currents were inhibited by approximately 30% (Fig. 1, trace 2, n 5 12). In contrast, the inward currents were dramatically increased when the bath isotonic solutions (osmolality at 500 mosmol) were replaced with hypotonic bath solutions (osmolality at 400 mosmol; Fig. 1, trace 5, n 5 12). Both the inhibition and activation of the inward currents were reversible when the bath solutions were changed between the hypertonic and the isotonic (control) solutions or between the hypotonic and the isotonic solutions. Furthermore, when Ba21 was substituted by Ca21 in the hypotonic bath solution, the observed currents appeared similar to the currents when Ba21 were used under the same conditions (Fig. 1, trace 4, n 5 4; compared to trace 5). The results indicate that these channels are Ca21 permeable and activated or inhibited under the hypotonic or the hypertonic conditions, respectively. To further confirm the nature of the recorded inward currents, Gd31, an inorganic blocker of Ca21-permeable channels, was applied. Lemtiri-Chlieh et al. (2003) reported that the 100 mM Gd31 dramatically blocks PM Plant Physiol. Vol. 143, 2007 1141 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Zhang et al. 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 Ca21-permeable channels. Compared to the control (the isotonic bath solutions; Fig. 2A, trace 2, n 5 8), the hypertonic treatment exerted a slight inhibition of the whole-cell inward Ba21 currents (Fig. 2A, trace 1, n 5 8). Addition of 20 mM CD to the hypertonic bath solutions dramatically increased the inward Ba21 currents (Fig. 2A, trace 3, n 5 8). Conversely, application of 100 mM phalloidin to Figure 1. Osmo gradients regulate inward whole-cell Ba21 or Ca21currents in Vicia guard cell protoplasts. Whole-cell recordings of Ba21 (traces 1–3, and 5) or Ca21 (trace 4) currents in Vicia guard cell protoplasts challenged with osmotic and/or Gd31 treatments. Wholecell currents were recorded under application of ramp-mode voltage protocols from 2100 to 40 mV with increments at 0.014 mV/ms. Traces 1 to 5 represent whole-cell recordings subjected to the different treatments. The osmolality of the bath solutions for each treatment is indicated as follows, while the osmolality of the pipette solutions maintained unchanged for the different treatments. Trace 1, 400 mosmol 1 100 mM Gd31. Trace 2 (the hypertonic treatment), 600 mosmol. Trace 3 (control), 500 mosmol. Trace 4, 400 mosmol (BaCl2 was replaced by CaCl2). Trace 5 (the hypotonic treatment), 400 mosmol. Each trace (1–5) was plotted with the mean value of 12 recordings from 12 different cells, respectively. Ca21-permeable channels in V. faba guard cell protoplasts but with no effect on the inward K1 channel currents. Ding and Pickard (1993) showed that Gd31 blocks mechanosensory (SA) Ca21-selective channels in epidermal cells. In this study, addition of 100 mM Gd31 to the bath solutions dramatically inhibited the activation of the currents by the hypotonic treatment (Fig. 1, trace 1, n 5 5; compared to trace 5). The reversal potential of the inward currents activated by hypotonic bath solution was approximately 112 mV, which is close to the theoretical equilibrium potential for Ba21 (EBa 5 14.9 mV) and far from that for Cl2 under the given conditions (ECl 5 234.6 mV). These results demonstrate that the recorded osmoregulated inward currents are predominantly carried by an influx of Ba21 or Ca21 through Ca21-permeable channels in the PM of Vicia guard cell protoplasts. These osmo-sensitive Ca21 channels in guard cells are inhibited under hypertonic conditions and activated under hypotonic conditions. Actin Dynamics Modulates the Osmo Regulation of the Whole-Cell Ba21 Channel Activity 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 Ca21- Figure 2. Regulation of osmo-sensitive whole-cell Ba21 currents by actin dynamics. Whole-cell currents were recorded under application of ramp-mode voltage protocols from 2100 to 40 mV with increments at 0.014 mV/ms. A, Averaged (n 5 8) whole-cell Ba21 currents recorded under various conditions. Traces 1 to 3 were recorded under hypertonic conditions (bath solution osmolality at 600 mosmol, trace 1), isotonic conditions (both the bath and pipette solution osmolalilty at 500 mosmol, trace 2), and hypertonic conditions (the bath solution osmolality at 600 mosmol) in the presence of 20 mM CD in the bath (trace 3), respectively. B, Effects of phalloidin on the whole-cell Ba21 currents. The recording traces (1–3) are presented as the averaged whole-cell currents (n 5 8). Traces 1 to 3 were recorded under hypertonic treatment (600 mosmol, trace 1), isotonic conditions (trace 2), and hypotonic conditions (400 mosmol, trace 3), respectively, all in the presence of 100 mM phalloidin in the pipette solutions. Other components of the solutions used in the experiments were the same to the solutions for the control conditions as described in ‘‘Materials and Methods.’’ 1142 Plant Physiol. Vol. 143, 2007 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Ca21 Channels in Vicia faba Guard Cell Protoplasts 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 5 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 5 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 Ca21-permeable channels by hypotonic treatment, which suggests that the hypotonic treatment may first induce depolymerization of actin filaments and consequently cause activation of the Ca21-permeable channels in guard cells. Characterization of the SA Ca21 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 2120 mV) without application of a pressuremediated stretch to the membrane (the top trace in Fig. 3A shows the recording at 260 mV without application of a stretch). However, the inward currents were elicited at 260 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 5 8). The value of reversal potential is close to the theoretical EBa under the given conditions (EBa 5 14.9 mV, ECl 5 234.6 mV), indicating that the singlechannel currents can be ascribed to an influx of Ba21 through the Ca21-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 Ba21 concentration in the bath solutions. The relationship for single-channel conductance versus Ba21 concentration was well fitted by the MichaelisMenten 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 Ba21 in the bath solution was substituted with Ca21, 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 Ca21 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 Ca21 currents were 11.8 pS and 23 mV, respectively (Fig. 4E, n 5 4). The relative permeabilities of Ba21 and Ca21 were calculated by the simplified Goldman-Hodgkin-Katz equation (Hille, 1993) and the derived ratio of PCa21/PBa21 was 1.91. Figure 3. Analysis of strength-dependent SA channel activity in an isolated outside-out membrane patch. A, Current traces were recorded from the same outside-out membrane patch at 260 mV under different stretches between 0 and 15 kPa, as indicated. Stretch was applied to the membrane patch by exerting a positive pressure to the interior of the glass pipette. The bath and pipette solutions for the control conditions were used. Dotted lines indicate the state of the channel, and the letters c or o stand for the closed or open state, respectively. B and C, NPo chart derived from the data recorded at 9 and 15 kPa, respectively. The NPo values were the averaged results of the recordings from six membrane patches. Plant Physiol. Vol. 143, 2007 1143 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Zhang et al. Figure 4. Voltage dependence of the SA channel-mediated Ba21 (A–C) and Ca21 (D–F) currents. A and D, Current traces recorded from outside-out membrane patches at different voltages with application of 9-kPa positive pressure to the pipette interior. The solutions for the control conditions as described in ‘‘Materials and Methods’’ were used for the recordings, except that the Ba21-containing solution was used for A and the Ca21-containing solution was used for D. The dotted lines indicate the states of the channel and the letters c or o stand for the closed or open state, respectively. B and E, The I-V curves of the averaged (n 5 8 for B, n 5 4 for E) SA currents under the control conditions. The calculated single-channel conductance and the measured reversal potential are indicated. C and F, The voltage dependence of NPo of the SA channels. The data are presented as mean 6 SE (n 5 8 for C, n 5 4 for F) and fitted by application of Boltzmann equation to the relationship of NPo versus voltage. Figure 6 shows reversible inhibitory effects of Gd31, a Ca21 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 260 mV, the NPo of the SA channels was decreased by nearly 60% or 95% after the addition of 10 mM (Fig. 6, B and F) or 100 mM Gd31 (Fig. 6, C and G) compared to the control (Fig. 6, A and E), respectively. The removal of Gd31 from the bath solution resulted in restoration of the channel activity (Fig. 6, D and H). SA of the Ca21-Permeable Channels Are Regulated by Actin Dynamics As described above, the SA Ca21 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 Ca21 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 260 mV, addition of 20 mM CD dramatically increased the activity of the SA Ca21 channels (Fig. 7, B and G versus A and F; n 5 7), suggesting that CD-induced depolymerization of actin filaments enhanced the sensitivity of the channels to the stretch. Not surprisingly, the presence of 100 mM 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 Ca21 Channels under the Cell-Attached Configuration Osmo regulation of the SA Ca21 channels was also investigated under the cell-attached configuration. As shown in Figure 8, channel activity was activated by 1144 Plant Physiol. Vol. 143, 2007 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Ca21 Channels in Vicia faba Guard Cell Protoplasts Figure 5. Single-channel conductance and reversal potential of the SA channels are dependent on extracellular Ba21 concentration. Control solutions were used except that various concentrations of Ba21 were added to the bath solutions. Data in both A and B are presented as mean 6 SE (n 5 8). A, The relationship of single-channel conductance versus extracellular Ba21 concentration derived from the singlechannel recordings at 260 mV and 9 kPa positive pressure applied to the outside-out membrane patches. The curve is well fitted by the Michaelis-Menten equation. B, Comparison of the measured reversal potentials (black circles) with the theoretical equilibrium potentials (white circles) calculated by Nernst equation. 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 Ca21 channels in the PM of guard cells account for the osmo-sensitive whole-cell currents. Actin Dynamics Regulates [Ca21]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 [Ca21]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 mM CD in the incubation solution significantly increased the fluorescence intensity after 20 min (Fig. 9B), while addition of 100 mM Gd31 impaired the CD-induced increase of fluorescence (Fig. 9C). The results indicate that CD-induced depolymerization of actin filaments may elevate [Ca21]cyt by stimulating Ca21 influx through Ca21 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 mM Gd31 (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 mM 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 [Ca21]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 Ca21 channelfacilitated Ca21 influx across the PM of guard cells. DISCUSSION Stomatal movements are controlled by turgordriven guard cell volume changes. Voltage-dependent K1 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 Ca21 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 Ca21permeable channels. Compared to the Ca21-permeable channels in V. faba guard cells reported by Hamilton et al. (2000), the channels shown in this study may share similar characteristics, although it can be only clarified by further Plant Physiol. Vol. 143, 2007 1145 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Zhang et al. Figure 6. Reversible inhibition of the SA channel activity by Ca21 channel blocker Gd31. A to D, Current traces recorded from the same outside-out membrane patch in the presence of a 9-kPa positive pressure at 260 mV with addition of different concentrations of Gd31 in the bath solutions as indicated. Different concentrations of Gd31 were added to (B and C) or washed out (D) from the control bath solutions. Dotted lines indicate the state of the channels and the letters c or o stand for the closed or open state, respectively. E to H, NPo charts derived from the corresponding recordings of the SA Ca21 currents as shown in A to D, respectively. channel molecular identification, given that the experimental conditions were similar in two independent studies in spite of some minor differences. First, the channels in both studies are hyperpolarization activated. Second, although Hamilton et al. (2000) did not mention osmo regulation of the channels, there was an osmolality difference (about 40 mosmol) across the membrane (200 mosmol and 240 mosmol for the bath and pipette solutions, respectively) and both bath and pipette solutions had rather low osmolality in their experiments. Thus, it may be hypothesized that the channel activity shown in their study might be also osmo regulated. Third, the current amplitudes of the channels in the study by Hamilton et al. (2000) are much greater than that we show in this study, possibly due to different osmolality of solutions and other recording conditions. SA Ca21 Channels May Function as an Osmotic Signal Transducer in Stomatal Guard Cells in Vivo 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; Garrill et al., 1996; Ramahaleo et al., 1996; Sachs, 1997; Hamill and Martinac, 2001). SA Ca21 channels, as signal transducers, activated by mechanical stimuli, including stretch or osmotic swelling imposed on the PM, have been implicated in a number of physiological processes, such as cell movement, cell volume and turgor regulation (Pickard and Ding, 1993), and gravitropism (White and Broadley, 2003). The voltage-dependent Ca21-permeable channels in the PM of V. faba guard cells identified in this study are activated by stretch forces applied to the PM of guard cells. The pressures applied to the isolated membrane patches in our experiments may be much lower than the turgor pressure present in stomatal guard cells in vivo. A turgor pressure between 3 and 4 MPa has been suggested to be common for guard cells surrounding an open stoma (Franks, 2003). As demonstrated in yeast (Saccharomyces cerevisiae) and animal cells, SA channels are membrane tension dependent rather than pressure dependent (Gustin et al., 1988). According to Laplace’s law, T 5 Pd/4 (where T is tension on the membrane, P is applied pressure, and d is cell diameter), the larger the cell, the greater the sensitivity to osmotic pressure changes (Gustin et al., 1988). In most of our patchclamping experiments (Figs. 4–6 and 7), a 9-kPa positive pressure was applied to the membrane patches to activate SA Ca21 channels. Such a pressure gives a tension force at 7.5 relative units on the membrane patches, assuming that the membrane bleb formed at the tip of a micropipette averages approximately 2 mm 1146 Plant Physiol. Vol. 143, 2007 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Ca21 Channels in Vicia faba Guard Cell Protoplasts Figure 7. Actin dynamics regulate the SAchannel activity. A to E, Current traces recorded from the outside-out membrane patches in the presence of a 9-kPa positive pressure at 260 mV. The current traces shown in A and B were recorded from the same membrane patch, and the current traces shown in C to E were recorded from another membrane patch. Addition of CD (in the bath solution) or phalloidin (in the pipette solution) is as indicated; otherwise, control solutions were used. Dotted lines indicate the state of the channels and the letters c or o stand for the closed or open state, respectively. F to J, NPo charts derived from the corresponding recordings of SA Ca21 currents as shown in A to E, respectively. in diameter. Given that an average diameter of Vicia guard cell protoplasts is approximately 15 mm, 4 MPa turgor pressure in a guard cell is equivalent to a tension of 10,000 units on the PM of the guard cell according to Laplace’s law. Although most of the turgor pressuregenerated tension force is borne by the cell wall (Cosgrove and Hedrich, 1991) and the actual membrane tension in guard cells is much lower than this, it is reasonable to believe that such turgor pressureinduced membrane tension is sufficient to activate SA Ca21 channels in the PM of guard cells in vivo. Potential Roles of the Osmo-Regulated Ca21 Channels in Regulation of Stomatal Movements A K1 channel-based positive feedback model has been proposed to explain the osmo-regulation mechanisms for stomatal movements (Liu and Luan, 1998). In this model, during the initial opening of illuminated stomata, the H1 pump in the PM of guard cells is activated, resulting in a more negative membrane potential that activates the inward K1 currents (IKin), and K1 influx takes place accompanied by water influx, making guard cell swell. Cell swelling further activates IKin and therefore accelerates K1 and water influxes and consequently stimulates stomatal opening (Liu and Luan, 1998). One may question for this osmoregulation model what would stop continuous swelling of guard cells. The results presented in this study suggest that the osmo-sensitive inward Ca21 channels may mediate Ca21 influx and subsequent [Ca21]cyt elevation in response to turgor pressure-induced membrane tension and consequently inhibit further swelling of guard cells by inhibiting K1 influx (Assmann, 1993; Ward et al., 1995) and activating Cl2 efflux (Schroeder et al., 2001). Thus, this model provides further explanation for how the stomatal movements are finely controlled by the internal mechanisms. It is known that cytoplasmic Ca21 elevation may inhibit IKin and stimulate slow anion channels (such as Plant Physiol. Vol. 143, 2007 1147 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Zhang et al. Figure 8. Changes in osmo gradients across cell-attached membrane patches mimic the effect on the SA-channel currents of stretch applied to the membrane by suction. Control solutions were used except that osmolarities of the pipette solutions were adjusted to 500 mosmol (A and B), 600 mosmol (C), or 400 mosmol (D and E), respectively. A 2-kPa positive pressure was applied to the cell-attached membrane patches by suction, as indicated (B–D). Dotted lines indicate the state of the channels and the letters c or o stand for the closed or open state, respectively. F to J, NPo charts to the corresponding recordings as shown in A to E, respectively. Cl2 channels), thus contributing to regulation of stomatal movement (for review, see Schroeder et al., 2001). Therefore, the transporters for controlling [Ca21]cyt may play important roles during stomatal movement. SA Ca21 channels have been reported previously in the PM of V. faba guard cells (Cosgrove and Hedrich, 1991), although their role had not been well defined at that time. As discussed earlier, these SA Ca21 channels may operate in vivo and thus play roles in regulation of stomatal movement. In this study, we present an osmosensing negative feedback mechanism mediated by [Ca21]cyt. During the initial stage of stomatal opening, the SA Ca21 channels are activated by cell swelling that is caused by the influxes of K1 and water. As a result, [Ca21]cyt tends to be elevated, thereby inhibiting the H1 pump (Kinoshita et al., 1995) and IKin, which in turn decreases cell turgor. On the other hand, elevated [Ca21]cyt disrupts the actin filaments (Hwang and Lee, 2001), thus in turn enhancing the sensitivity of the SA Ca21 channels to swelling (Glogauer et al., 1995; this study) and accelerating [Ca21]cyt elevation. The interaction between disruption of actin filaments and elevation of [Ca21]cyt may form a negative loop to limit the sustained activation of IKin and cell swelling. During the initiation of stomatal closure induced by darkness, SA Ca21 channels may be activated by the existing cell turgor, contributing to regulation of increase or oscillation of [Ca21]cyt, which initiates the signaling events for stomatal closure (McAinsh et al., 1995; Allen et al., 2000, 2001). The activation of slow anion channels and inactivation of IKin by the increase of [Ca21]cyt, together with depolarization-driven K1 efflux, cause cell shrinking, which in turn inactivates SA Ca21 channels. Therefore, SA Ca21 channels may be also actively functioning during stomatal closure. The possible complex interaction between the positive and negative feedback mechanisms by which a number of ion transporters are regulated may explain stomatal oscillation under variable environmental conditions (Cowan et al., 1997). Liu and Luan (1998) reported that hypotonic or hypertonic condition caused gradual swelling or 1148 Plant Physiol. Vol. 143, 2007 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Ca21 Channels in Vicia faba Guard Cell Protoplasts Figure 9. Imaging of the [Ca21]cyt in Vicia guard cell protoplasts under various conditions. A to G, Confocal images of [Ca21]cyt changes in guard cell protoplasts. The intensities of Fluo 3-AM fluorescence are displayed as pseudocolors, representing the relative [Ca21]cyt, as illustrated by the pseudocolor bar. Each specific treatment is indicated at the top of each section. The osmolality of the bath solutions for the control, hypotonic, or hypertonic treatments was 500, 400, or 600 mosmol, respectively. The arrows in F and G or 0 time points in A to H indicate the beginning of each corresponding treatment. H, Time kinetics of changes in the fluorescent intensity subject to the different treatments, as shown in A to G, respectively (d, control; s, 20 mM CD; ,, 20 mM CD 1 Gd31; ;, hypotonicity; n, hypotonicity 1 Gd31; h, osmoregulation; ¤, hypertonicity 1 20 mM 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 6 SE (n 5 5). shrinking of Vicia guard cell protoplasts, respectively, but both swelling and shrinking of the cells saturated during the given time period. According to the K1-channel-based positive feedback model (Liu and Luan, 1998), the protoplasts will continuously swell at hypotonic condition or shrink at hypertonic condition. The swelling or shrinking of stomatal guard cells may be compromised by increased or decreased [Ca21]cyt as explained by the osmo-sensing negative feedback mechanism mediated by [Ca21]cyt presented in this study. Actin Cytoskeleton May Serve as an Osmo Sensor for Regulation of Ca21 Channels in Guard Cells As discussed by Liu and Luan (1998), the actin cytoskeleton may serve as an osmo sensor and target osmo-sensitive K1 channels in guard cells. This study shows that the actin cytoskeleton may also transduce osmotic signals to SA Ca21 channels under hypotonic conditions (induced, e.g., by high humidity or high water potential in vivo). On the other hand, we also show that the actin cytoskeleton may act as a stress sensor for regulation of SA Ca21 channels in the PM under hypertonic stress that may occur under drought or low-humidity conditions (for review, see Raschke, 1975; Zeiger, 1983; Grantz, 1990). Water stress-induced ABA triggers an elevation or oscillation in [Ca21]cyt in guard cells that is critical for maintenance of stomatal closure (for review, see Schroeder et al., 2001). Ca21 influx through Ca21 channels across the PM has been proposed to be involved in water stress- and ABAinduced signaling of stomatal closure (Hamilton et al., 2000; Pei et al., 2000). H2O2 and protein phosphorylation were implicated to mediate ABA signaling in regulating Ca21 channels in the PM (Hamilton et al., 2000; Pei et al., 2000; Köhler and Blatt, 2002). External ABA or Ca21 induces a disintegration of actin filaments in guard cells (Eun and Lee, 1997; Eun et al., 2001; Hwang and Lee, 2001; Lemichez et al., 2001). ABA-induced Plant Physiol. Vol. 143, 2007 1149 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2007 American Society of Plant Biologists. All rights reserved. Zhang et al. actin disruption in guard cells was demonstrated to be mediated by [Ca21]cyt and by protein phosphorylation and dephosphorylation (Hwang and Lee, 2001). In pollen tubes, tip F-actin and tip-focused calcium gradients oscillate in the opposite phase (Fu et al., 2001), indicating that high [Ca21]cyt might play a vital role in regulating actin dynamics and vice versa. Moreover, a study has shown that actin disruption stimulates Ca21 channels in fibroblasts (Glogauer et al., 1995). In this study, we demonstrate that actin dynamics regulates [Ca21]cyt by modulating SA Ca21 channels in the PM of guard cells. Therefore, the interplay between the dynamics of these two vital cellular components may be an important regulatory mechanism by which guard cells sense environmental changes and regulate stomatal movement. MATERIALS AND METHODS Guard Cell Protoplast Preparation Vicia faba plants were grown in soil mixture in a growth chamber under a 12-h light (100 mmol m22 s21)/12-h dark cycle and temperatures of 22C and 15C 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), with slight modification. Briefly, abaxial epidermises were peeled from fully expanded leaves of 3- to 4-week-old plants. Mesophyll cells were brushed off from the epidermal strips in distilled water, then the epidermis was transferred to enzyme solution I containing 0.7% (w/v) cellulysin (Calbiochem), 0.1% (w/v) polyvinylpyrrolidone-40, 0.3% (w/v) bovine serum albumin, and 0.5 mM ascorbic acid (freshly added from stock solution) dissolved in 45% (v/v) distilled water and 55% (v/v) basic solution (0.45 M sorbitol, 0.5 mM CaCl2, 0.5 mM MgCl2, 10 mM KH2PO4, 5 mM MES/Tris, pH at 5.5). The enzymatic mixture was incubated in a rotary water bath at 25C with rotation speed at 160 rpm for 30 min. The partially digested epidermal strips were thoroughly washed with the basic solution on 220-mm nylon mesh and transferred to the enzyme solution II (basic solution plus 1.5% [w/v] Cellulase RS [Yakult]), 0.3% (w/v) bovine serum albumin, 0.2% (w/v) Pectolyase Y-23 (Seishin Pharmaceutical), and 0.5 mM ascorbic acid, pH 5.5) and incubated in a rotary water bath at 21C with rotation speed at 60 rpm for 40 min. Released protoplasts were collected and washed twice by filtration and centrifugation at 800 rpm for 5 min. The isolated protoplasts were resuspended in the basic solution and kept on ice in the dark for at least 1 h before use for patch-clamping and [Ca21]cyt measurements. Patch-Clamping Recordings and Data Analysis Standard whole-cell and single-channel recording techniques were applied in this study. All experiments were conducted at room temperature (approximately 22C) under dim light. Glass micropipettes were made by using a twostep puller (model PP-83, Narishige) and fire polished by a microforge (model MF-83, Narishige). For the control conditions of both whole-cell and outsideout 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 GV in all experiments. Membrane potential was clamped to 0 mV, and current data were acquired when the voltages were clamped from 2100 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 singlechannel 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 GV for all excised-patch and cell-attached recordings. In the recordings from the outside-out membrane patches, the SA Ca21 channels were activated by a positive pressure applied to the interior of a glass pipette. For the cell-attached recordings, SA Ca21 channels were activated by a negative pressure (suction) applied to the interior of the glass pipettes. The strength of blow or suction was monitored by a barometer connected to the micropipette buffered via a water column. The data of single-channel recordings were analyzed with Fetchan software, and then, Simplex-LSQ fitting method and Gaussian fitting included in Pstat software were applied. After being fitted with graphic seeds, the area of the column was calculated for further calculation of channel NPo. Because most of the tested membrane patches showed multiple channels, NPo were expressed as NPo, where N represents the number of channels existing in the membrane patches and Po represents the open probability of a single channel. The NPo values were calculated using the equation (NPo 5 [A1 1 2A2 1 3A3 1.nAn]/[A0 1 A1 1 A2 1.An]), as described previously (López-López et al., 1998). 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 singlechannel currents. SigmaPlot software was used to draw I-V plots and data analysis. Cytosolic Calcium Measurements A Ca21 fluorescent dye, Fluo 3-AM, was used to monitor changes in relative [Ca21]cyt in guard cell protoplasts. Protoplasts were isolated as described above and then incubated in a solution containing 10 mM 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 4C. 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-mm 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. Chemicals All chemicals were obtained from Sigma unless otherwise indicated in the text. Received October 21, 2006; accepted January 12, 2007; published January 26, 2007. 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