Hydrogen Sulfide Regulates Inward-Rectifying K+ Channels in Conjunction with Stomatal Closure1[OPEN]

Hydrogen sulfide affects inward-rectifying K+ channels in guard cells and implicates an additional, but as yet unidentified, signaling pathway in stomatal closure. Hydrogen sulfide (H2S) is the third biological gasotransmitter, and in animals, it affects many physiological processes by modulating ion channels. H2S has been reported to protect plants from oxidative stress in diverse physiological responses. H2S closes stomata, but the underlying mechanism remains elusive. Here, we report the selective inactivation of current carried by inward-rectifying K+ channels of tobacco (Nicotiana tabacum) guard cells and show its close parallel with stomatal closure evoked by submicromolar concentrations of H2S. Experiments to scavenge H2S suggested an effect that is separable from that of abscisic acid, which is associated with water stress. Thus, H2S seems to define a unique and unresolved signaling pathway that selectively targets inward-rectifying K+ channels.

Hydrogen sulfide (H 2 S) is a small bioactive gas that has been known for centuries as an environmental pollutant (Reiffenstein et al., 1992). H 2 S is soluble in both polar and, especially, nonpolar solvents (Wang, 2002), and has recently come to be recognized as the third member of a group of so-called biological gasotransmitters. Most importantly, H 2 S shows both physical and functional similarities to the other gasotransmitters nitric oxide (NO) and carbon monoxide (Wang, 2002), and it has been shown to participate in diverse physiological processes in animals, including cardioprotection, neuromodulation, inflammation, apoptosis, and gastrointestinal functions among others (Kabil et al., 2014). Less is known about H 2 S molecular targets and its modes of action. H 2 S can directly modify specific targets through protein sulfhydration (the addition of an -SH group to thiol moiety of proteins; Mustafa et al., 2009) or reaction with metal centers (Li and Lancaster, 2013). It can also act indirectly, reacting with NO to form nitrosothiols (Whiteman et al., 2006;Li and Lancaster, 2013). Among its molecular targets, H 2 S has been reported to regulate ATP-dependent K + channels (Yang et al., 2005), Ca 2+ -activated K + channels, T-and L-type Ca 2+ channels, and transient receptor potential channels (Tang et al., 2010;Peers et al., 2012), suggesting H 2 S as a key regulator of membrane ion transport.
ABA is an important player in plant physiology. Notably, upon water stress, ABA triggers a complex signaling network to restrict the loss of water through the transpiration stream, balancing these needs with those of CO 2 for carbon assimilation. In the guard cells that surround the stomatal pore, ABA induces an increase of cytosolic-free Ca 2+ concentration ([Ca 2+ ] cyt ), elevates cytosolic pH (pH i ), and activates the efflux of anions, mainly chloride, through S-and R-type anion channels. The increase in [Ca 2+ ] cyt inactivates inward-rectifying K + channels (I KIN ); anion efflux depolarizes the plasma membrane, and together with the rise in pH i , it activates K + efflux through outwardrectifying K + channels (I KOUT ; Blatt, 2000;Schroeder et al., 2001). These changes in ion flux, in turn, generate an osmotically driven reduction in turgor and volume and closure of the stomatal pore. All three gasotransmitters have been implicated in regulating the activity of guard cell ion channels, but direct evidence is available only for NO (Garcia-Mata et al., 2003;Sokolovski et al., 2005). Here, we have used two-electrode voltage clamp measurements to study the role of H 2 S in the regulation of the guard cell K + channels of tobacco (Nicotiana tabacum). Our results show that H 2 S selectively inactivates I KIN and that this action parallels that of stomatal closure. These results confirm H 2 S as a unique factor regulating guard cell ion transport and indicate that H 2 S acts in a manner separable from that of ABA.

RESULTS AND DISCUSSION
To address whether H 2 S-induced stomatal closure is mediated by changes in the activities of these channels, we recorded currents by two-electrode voltage clamp. I KIN and I KOUT currents were resolved on the basis of their voltage-dependent kinetics and challenging with H 2 S donor GYY4137 (for p-methoxyphenyl(morpholino) phosphinodithioic acid). Follow impalement currentvoltage recordings were carried out to confirm a stable baseline of channel activities for 5 to 10 min before the H 2 S donor GYY4137 was added. We observed a complete response to the H 2 S donor within 2 to 3 min of additions, indicating a halftime for response to the donor of less than 120 s. Most impalements could be held for 20 to 30 min only and therefore, did not allow us to assess current recovery after washout of the H 2 S donor. Figure 1A shows current traces and the mean steadystate currents as a function of voltage (I-V curves) from guard cells before and after 5 min of exposure to 10 mM GYY4137. The I-V curves show the characteristic voltage dependence of both I KOUT and I KIN as previously described (Blatt, 1992;Gradmann et al., 1993;Garcia-Mata et al., 2003). In 10 mM KCl, voltages positive of 240 mV yielded I KOUT that increased in amplitude with the voltage. Mean I KOUT at +30 mV was +120 6 28 and +91 6 30 mA cm 22 before and after exposure to GYY 4137, respectively, indicating a small but not very significant effect on the I KOUT . Voltages negative of 2100 mV were marked by a strong inward-directed current typical of I KIN , and the current amplitude increased with negative-going voltages. We found that I KIN at 2220 mV was reduced by roughly 90% by H 2 S donor treatments, yielding a mean I KIN of 221 6 8 mA cm 2 2 compared with 2169 6 12 mA cm 22 for the control. Exposure to the H 2 S donor also affected the halftimes for I KIN activation. Mean halftimes for I KIN activation at 2220 mV were 710 6 70 ms in the control and 1,230 6 230 ms after exposure to 10 mM GYY4137, indicating a significant change in gating of I KIN (Fig. 1B).
Steady-state current through any ion channel depends on the ensemble conductance (G), which is the product of the number of functional channels at the plasma membrane (N), the single-channel conductance for a given ion species (g X ), and the gating characteristics of the channel that describe the open probability of the channel (P o ). Plotting the conductance of I KIN before and after exposure to H 2 S as a function of voltage allowed a separation of the differences in the gating characteristics before and after H 2 S donor treatments (Fig. 1C). The G-V curves were jointly fitted to a modified Boltzmann function (Eq. 1) to determine the maximum Figure 1. H 2 S selectively affects I KIN . A, Currents through I KOUT and I KIN above and below the voltage axis, respectively, recorded under voltage clamp from tobacco guard cells. Voltages were clamped from a holding voltage of 2100 mV in 6-s steps between 2220 and +120 mV and 4-s steps between 280 and +40 mV. Data are means 6 SE of n = 5 guard cells bathed in 5 mM Ca 2+ -MES buffer (pH 6.1) with 10 mM KCl before (white circles) and 5 min after (black circles) adding 10 mM GYY4137. Insets, Current traces for I KOUT and I KIN for control and H 2 S treatment. Currents are cross referenced to the currentvoltage curves by symbols. Bars = 2 s (horizontal); 50 mA cm 22 (vertical). B, Mean activation halftimes as a function of voltage derived from current traces, including those in A, and cross referenced by symbols. C, Steady-state conductance as a function of voltage normalized to G max in the control (black line) and with 10 mM GYY4137 (dashed line). Inset, Steady-state conductance as a function of voltage. Curves were jointly fitted to Boltzmann function (Eq. 1) with gating charge-d held in common.
conductance (G max ) and the gating characteristics of I KIN (Table I). For joint fittings, d was held in common, and it yielded statistically and visually satisfactory fittings with a value of 21.66 6 0.04. As expected from the I-V data, the H 2 S donor suppressed the G max significantly by up to 90% relative to the control. We cannot distinguish from these data whether this effect was mediated through a change in the number of channels available for activation (N) or the single-channel conductance (g K ). Such detail would require single-channel analysis. However, we noted that the H 2 S donor displaced V 1/2 by approximately 212 mV ( Fig. 1C; Table I), indicating that the H 2 S not only resulted in a decrease of the maximum conductance but also affected the voltage dependence for gating of I KIN . An action on V 1/2 cannot be explained solely by an effect on N or g K . In short, H 2 S selectively inactivated I KIN .
The inactivation of the K + current is consistent with GYY4137 action in suppressing K + uptake and promoting stomatal closure, and it argues against earlier (and statistically undocumented) claims that H 2 S donors promote stomatal opening (Lisjak et al., 2010(Lisjak et al., , 2011. To assess the action of H 2 S effect on stomatal movement, we measured apertures from stomata treated with different concentrations of the H 2 S donor. Epidermal peels were placed in opening buffer under light of 150 mmol m 22 s 21 for 2 h to open the stomata before transfer to 5 mM Ca 2+ -MES (pH 6.1) with 60 mM KCl supplemented with 0, 0.1, 1, or 10 mM GYY4137 for 90 min. Apertures were recorded immediately before and after H 2 S treatments, and the data were normalized to the controls (Fig. 2). Exposure to the control buffer alone yielded stomatal apertures of 6.6 6 1.8 mm; treatments with H 2 S donor resulted in stomatal apertures ranging from 5.9 to 4.8 mm. Fitting the data to the hyperbolic decay function yielded an apparent K i of 160 6 40 nΜ for GYY4137. We carried out parallel measurements of I KIN to determine its dose dependence after treating guard cells in buffer supplemented with 0, 0.1, 1, or 10 mM GYY4137. Figure 2 also shows the mean values for I KIN again normalized to the control treatment. Increasing the concentration of H 2 S donor, indeed, enhanced I KIN inactivation. Fitting these data to hyperbolic decay function gave a K i of 120 6 70 nM for GYY4137, a value that did not significantly differ from that for stomatal closure compared with t test (P = 0.735). These results, thus, confirm the close kinetic relationship between I KIN inactivation and stomatal closure in H 2 S.
The similar effects of H 2 S and ABA on I KIN and stomatal aperture prompted us to explore the connection between H 2 S and ABA signaling, which was suggested by García-Mata and Lamattina (2010). A similar set of protocols was used as above. Epidermal peels were pretreated for 2 h with opening buffer and light for 90 min before treatments in 5 mM Ca 2+ -MES (pH 6.1) with 10 mM KCl with and without supplement of five distinct combinations of stomatal effectors: 10 mM GYY4137, 10 mM hypotaurine (HT), 10 mM GYY4137 + 10 mM HT, 20 mM ABA, and 20 mM ABA + 10 mM HT. HT interacts with free sulfide to form thiotaurine, effectively scavenging free H 2 S in solution (Ortega et al., 2008). Figure 3A shows the percentage of stomatal closure induced by each treatment relative to the control. Measurements were carried out separately at the Consejo Nacional de Investigaciones Científicas y Técnicas and yielded starting apertures (4.9 6 0.1 mm) that differed quantitatively from those recorded at the University of Glasgow. Qualitatively, however, the results were consistent between data sets. Exposure to 10 mM GYY4137 and 20 mM ABA resulted in 60% and 80% reductions in stomatal aperture, corresponding to apertures of 3.2 6 0.1 and 2.2 6 0.07 mm, respectively. Treatment with HT alone had no effect on stomatal aperture. When epidermal peels were treated with both 10 mM GYY4137 and HT, the effect of the H 2 S donor was alleviated, yielding apertures of 4.6 6 0.1 mm, similar to those of the control. Treatment of epidermal peels with 20 mM ABA + 10 mM HT partially suppressed the effect of ABA on aperture, resulting in a reduction to 70% pore width compared with control treatment.
Given the role of [Ca 2+ ] cyt in ABA signaling and control of I KIN (Blatt, 2000;Garcia-Mata et al., 2003), we sought to test whether the H 2 S-induced effect on I KIN might be mediated by the Ca 2+ intermediate. For this purpose, we loaded guard cells from the microelectrode with 50 mM EGTA, which chelates and buffers Ca 2+ to suppress its elevation (Grabov and Blatt, 1998;Chen et al., 2010;Wang et al., 2012). After being impaled, guard cells were held for a period of 5 min to ensure loading with EGTA. Thereafter, the guard cells were either maintained in 5 mM Ca 2+ -MES (pH 6.1) with 10 mM KCl or challenged with 10 mM GYY4137 for a period of 10 min. In the absence of H 2 S donor, we observed no substantive effect on I KIN . The mean amplitude at 2200 mV was 2217 6 29 mA cm 22 . In the presence of H 2 S donor, I KIN was suppressed, yielding a mean current of 261 6 11 mA cm 22 (Fig. 4A). EGTA did yield a small but not very significant recovery of I KIN in the presence of the H 2 S donor (Fig. 4B). These results indicate that H 2 S acts in a manner that is largely independent of [Ca 2+ ] cyt . We also investigated the effect of the above compounds and their combinations on I KIN , again following the same set of protocols. Figure 3B displays the mean percentage reduction of the I KIN amplitude at 2200 mV before and after the exposure to each of the treatments. H 2 S resulted in almost complete loss of I KIN , which is shown in Figure 1. ABA treatment reduced I KIN by 62%, resulting in a mean current of 2170 6 39 mA cm 22 at 2200 mV. Interestingly, exposure of guard cells to 10 mM HT yielded I KIN of 2292 6 64 mA cm 22 , marginally greater in amplitude compared with 2241 6 40 mA cm 22 for the control, although this difference was not very significant. Suppression of the current by H 2 S was blocked when guard cells were treated with the combination of H 2 S donor and scavenger, resulting in I KIN of similar amplitude as the control treatment. In contrast, the reduction of I KIN evoked by ABA was not prevented by adding HT, which yielded a mean I KIN of 2174 6 35 mA cm 22 . Altogether, these data indicate that H 2 S acts in a manner paralleling that either of ABA or upstream of the hormone.
Stomatal movement is a highly coordinated process that is generally recognized to engage several signaling networks leading to the regulation of K + channels, anion channels, and H + -ATPases at the plasma membrane as well as a complementary assembly of transporters at the tonoplast (Blatt, 2000). For ABA-evoked stomatal closure, this process includes inactivation of I KIN through changes in [Ca 2+ ] cyt and activation of the I KOUT mediated by a rise in pH i (Blatt, 1990;Blatt and Armstrong, 1993;Garcia-Mata et al., 2003;Siegel et al., 2009). Our findings that H 2 S differentially affects I KIN and I KOUT and that I KIN inactivation is dose dependent with an apparent K i in the low nanomolar range confirm a subcellular target for the action of this gasotransmitter. The timescale of the H 2 S-triggered changes in channel gating is entirely in keeping with posttranslational regulation, which is in contrast with the slower effects of ABA that, over timescales of many minutes or hours, clearly rely on the transcription regulation and trafficking of the channel proteins (Pilot et al., 2003;Sutter et al., 2007;Eisenach, et al., , 2014. These findings together with evidence that H 2 S mediates stomatal closure and that its scavenging partially suppresses closure in ABA suggest a connection with the hormone, albeit a loose one. Notably, H 2 S scavenging failed to reverse ABAevoked inactivation of I KIN (Fig. 3), and we found that  . H 2 S affects stomatal aperture and K + current in a dosedependent manner with similar concentration dependencies. Mean stomatal apertures 6 SE (n = 50) recorded from epidermal peels of tobacco (triangles) and mean I KIN 6 SE (n = 5) at 2200 mV (squares) normalized to the controls without GYY 4137 treatment. A hyperbolic decay function was fitted to each set of data, yielding a K i of 160 6 40 nM GYY4137 for the aperture response (solid line) and 120 6 70 nM GYY4137 for current inactivation (dashed line).
stomatal closure was enhanced when treated with ABA and the H 2 S scavenger compared with treatment with ABA alone. These observations are difficult to reconcile with a role for H 2 S as an intermediate in ABA signaling per se and instead, suggest a partial overlap in signaling pathways.
This interpretation is in agreement with recent studies showing an ABA dependency of H 2 S effect on stomatal movements (García-Mata and Lamattina, 2010;Scuffi et al., 2014). It also complements substantial evidence for a separate set of intermediates, especially ROS and NO, that trigger the elevation of [Ca 2+ ] cyt and are important for ABA-mediated stomatal closure (Pei et al., 2000;Lamattina, 2002, 2003), and it agrees with our finding that Ca 2+ buffering did not substantially rescue I KIN . Guard cells are thought to produce NO in response to ABA through the activity of nitrate reductases , and NO action is also dependent on the secondary messengers cGMP and cADPR . Garcia-Mata et al. (2003) showed that NO promoted the inhibition of I KIN and activated anion efflux through an enhanced sensitivity of internal Ca 2+ release to Ca 2+ influx across the plasma membrane. Notably, the effects of [Ca 2+ ] cyt on I KIN gating and especially, V 1/2 are much more pronounced than observed with H 2 S. Furthermore, NO is also able to modulate I KOUT by direct nitrosylation of the channel or an associated regulatory protein (Sokolovski and Blatt, 2004), but we observed little evidence of an effect on I KOUT current. Therefore, these observations implicate a parallel and as yet uncharacterized signaling pathway that acts on I KIN , thereby overlapping with the well-known pathways leading from ABA to I KIN and stomatal closure.
How might H 2 S act to modulate I KIN ? Nitrosylation of Cys sulfhydryl groups on either the channel itself or on closely associated regulatory proteins has been suggested to mediate the NO-induced block of I KOUT (Sokolovski and Blatt, 2004), and such modifications may be linked to ROS modification of residues within the voltage sensor domain . H 2 S is also capable of covalently modifying protein targets, and the mechanism is equally relevant to I KIN and the proteins that regulate these channels, including protein kinases and phosphatases (Thiel and Blatt, 1994;Li et al., 1998;Michard et al., 2005). In animals, for example, H 2 S activates ATP-dependent K + channels through the sulfhydration of a Cys residue of the sulfonurea SUR protein (Babenko et al., 2000). Addition of the SUR inhibitor glibenclamide antagonized the H 2 S response and prevented the hypotensive effect of H 2 S (Zhao et al., 2001). In other cases, sulfhydration is suppressed by the reducing agent dithiothreitol and mutants defective in H 2 S production (Mustafa et al., 2009). Of interest, glibenclamide has also been shown to abolish stomatal closure triggered by ABA and external Ca 2+ through the inhibition of anion and I KOUT (Leonhardt et al., 1999). More recent studies, however, have shown only partial suppression by glibenclamide of stomatal closure in ABA, whereas the response to H 2 S was completely abolished (García-Mata and Lamattina, 2010). These findings suggest that ABC proteins, a major target of glibenclamide, may contribute to channel regulation in guard cells upon H 2 S exposure. At present, however, there is not sufficient information from any system that would enable realistic predictions of the possible motifs for sulfhydration.
No doubt, future studies with transgenic Arabidopsis lines defective in H 2 S, NO production, and ABA sensitivity should help clarify the role of H 2 S in these processes (Hetherington and Woodward, 2003). What is clear, however, is that H 2 S is active in selectively regulating I KIN of guard cells over timescales consistent  Figure 1. Guard cells were bathed in 10 mM KCl (white circles) or 10 mM KCl supplemented with 10 mM GYY4137 (gray circles) and loaded from the microelectrode with 50 mM EGTA. The I-V curve for guard cells treated with only 10 mM GYY4137 (black circles) from Figure 1 is included for visual reference. Data are means 6 SE of n = 5 guard cells for each data set. Curves were jointly fitted to Boltzmann function (lines), with V 1/2 and gating charge-d held in common. Insets present current traces recorded under voltage clamp. Bars = 2 s (horizontal) and 200 mA cm 22 (vertical). B, Mean I KIN at 2205 mV from the current recordings for control (white bars) and 10 mM GYY4137 (black bars) treatments in the presence of EGTA and 10 mM GYY4137 without EGTA (gray bars). Lettering indicates statistical differences by ANOVA (P , 0.001) as determined by Student Newman-Keuls test.
with short-term posttranslational modification of specific target proteins. Furthermore, our evidence implicates H 2 S in a signaling pathway that is separable from that of ABA, although both ABA and H 2 S modulate stomatal behavior in parallel.

Plant Material, Chemicals, and Stomatal Assays
Tobacco (Nicotiana tabacum) plants were grown in Levington F2+S compost under long-day conditions (16-h-light/8-h-dark cycle; temperature approximately 26°C and 22°C for day and night, respectively; relative humidity of 60% and 70% for day and night, respectively) under 100 mmol m 22 s 21 of light.
Epidermal peels were obtained from the abaxial side of tobacco leaves and placed in opening buffer comprised of 10 mM Na + -MES (pH 6.1; 10 mM MES titrated to pH 6.1 with NaOH) with 60 mM KCl under light of 150 mmol m 22 s 21 for 2 h before treatment with the H 2 S donor GYY4137 (Sigma) in the same buffer. Stomata were imaged before and after 90 min of H 2 S treatment using an LD Achroplan 403 Objective and an Axio-Cam HRc Digital Camera (Zeiss). Apertures were tracked for individual stomata and quantified using IMAGEJ version 1.48 (image.nih.gov/ij/).

Guard Cell Electrophysiology
Currents were recorded under two-electrode voltage clamp using Henry's EP Software Suite (http://www.psrg.org.uk). Microelectrodes were constructed to give tip resistances of greater than 100 MV and filled with 200 mM K + -acetate (pH 7.5) to minimize interference arising from anion leakage from the microelectrode (Blatt and Slayman, 1983;Blatt, 1987;Wang and Blatt, 2011). Electrolyte filling solutions were equilibrated against the resin-bound Ca 2+ buffer BAPTA [for 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid; Ca 2+ sponge; Invitrogen] to prevent Ca 2+ loading of the cytosol from the microelectrodes. K + channel currents were recorded from guard cells bathed under continuous superfusion with 5 mM Ca 2+ -MES (pH 6.1; 5 mM MES titrated to pH 6.1 with Ca(OH) 2 ; [Ca 2+ ] = 1 mM) plus 10 mM KCl alone and supplemented with reagents as indicated. Recordings typically included a 2-s holding voltage at 2100 mV and two to six steps to voltages between 2220 and +40 mV. Surface areas of the impaled guard cells were calculated assuming a spheroid geometry .
Current voltage analysis and fittings were carried out using Henry's EP Software Suite and SigmaPlot 11 (SPSS; Systat Software). Conductance-voltage curves were fitted by joint nonlinear least squares and the Marquardt-Levenberg algorithm using a modified Boltzmann function of the form where G max is the maximum conductance, V is the membrane voltage, V 1/2 is the voltage at which half-maximum activation of channels occurs, d is the apparent gating charge, and F, R, and T have their usual meanings.

Statistical Analysis
Results are reported as means 6 SE of n observations, with significance determined using Student's t test and ANOVA at P , 0.05.