Functional Interaction of the SNARE Protein NtSyp121 in Ca2+ Channel Gating, Ca2+ Transients and ABA Signalling of Stomatal Guard Cells
a Laboratory of Plant Physiology and Biophysics, IBLS—Plant Sciences, University of Glasgow, Glasgow G12 8QQ, UK
b Current address: Blackfriars, 64 St Giles, Oxford OX1 3LY, UK
1 To whom correspondence should be addressed. E-mail m.blatt{at}bio.gla.ac.uk, fax (+44) 0141 330 4447, tel. (+44) 0141 330 4771.
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Abstract |
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There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K+ and Cl– channel responses to the stress-related hormone abscisic acid (ABA), but left open a question about functional impacts on signal intermediates, especially on Ca2+-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca2+ channel gating and its consequent effects on cytosolic-free [Ca2+] ([Ca2+]i) elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca2+]i elevation independently of the plasma membrane Ca2+ channels. Consistent with these observations, Ca2+ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca2+]i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca2+]i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca2+ channels at the plant cell plasma membrane and, because [Ca2+]i plays a key role in the control of K+ and Cl– channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.
Key Words: Ca2+ channel, hyperpolarization-activated • abscisic acid • membrane vesicle traffic • cytosolic-free [Ca2+] elevation • Nicotiana • plant pathogen defense
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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Cytosolic-free Ca2+ concentration ([Ca2+]i) plays important roles in many signalling cascades of plants cells (Hepler, 2005; Hetherington and Brownlee, 2004). Changes in [Ca2+]i often depend on the activation of Ca2+ channels at the plasma membrane and Ca2+ influx that triggers Ca2+ release from intracellular stores (Hetherington and Brownlee, 2004), much as has been described for Ca2+-induced Ca2+ release (CICR) in animal cells (Berridge, 1998). Such [Ca2+]i transients engage in a wide range of physiological responses, including root hair, pollen growth, and fertilization (Foreman et al., 2003; Franklin-Tong et al., 2002; Holdaway-Clarke and Hepler, 2003), plant defense (Durner et al., 1998; Klusener et al., 2002; Lamotte et al., 2004), and nodulation (Ehrhardt et al., 1996; Shaw and Long, 2003), as well as osmotic solute loss from guard cells and stomatal closure (Blatt, 2000; Hetherington and Brownlee, 2004).
In guard cells, Ca2+ entry through plasma membrane Ca2+ channels has been shown to trigger Ca2+ release from intracellular Ca2+ stores, raising [Ca2+]i (Garcia-Mata et al., 2003; Grabov and Blatt, 1998; Hamilton et al., 2000; Staxen et al., 1999). The plasma membrane Ca2+ channels are activated by abscisic acid (ABA) (Hamilton et al., 2000) as well as reactive oxygen species (ROS) (Kwak et al., 2003; Pei et al., 2000) and are subject to control by protein (de)phosphorylation (Köhler and Blatt, 2002; Sokolovski et al., 2005). These increases in [Ca2+]i serve to inactivate inward-rectifying K+ channels (Grabov and Blatt, 1999; Schroeder and Hagiwara, 1989) and activate Cl– channels at the plasma membrane, which, together with outward-rectifying K+ channels, facilitates osmotic solute loss and stomatal closure. [Ca2+]i transients are coupled to membrane voltage (Grabov and Blatt, 1998) and probably serve as part of a homeostatic feedback loop to regulate the K+ and Cl– channels (Blatt et al., 2007). Additionally, oscillations in [Ca2+]i have been associated with a stress ‘memory’ of guard cells—so-called ‘programmed’ closure (Allen et al., 2001)— that is thought to suppress stomatal re-opening after drought and ABA challenge.
Parallel to their functions in controlling ion transport, Ca2+ signals also play an important role in the traffic of membrane proteins and vesicles within plant cells. At least for traffic to and from the plasma membrane, where physical access is possible in vivo, kinetic and physiological studies have identified changes in capacitance that accompany the increase or decrease in membrane surface area during vesicle membrane fusion and removal, respectively (Blatt and Thiel, 2003; Pratelli et al., 2004; Thiel and Battey, 1998). These capacitance changes are consistent with the predicted vesicle dimensions derived from ultrastructural studies (Phillips et al., 1988; Picton and Steer, 1983) and from imaging studies using fluorescent styryl dyes to label internalized membrane (Meckel et al., 2004). [Ca2+]i and guanosine nucleotides (Carroll et al., 1998; Homann and Tester, 1997) are known to affect a number of these exo- and endocytotic events, although evidence for exocytotic pathways both dependent and independent of [Ca2+]i elevations underscores the complexity of secretory processing (Homann and Tester, 1997; Sutter et al., 2000). Significant, too, are observations of a selective endocytosis of K+ channel protein and its recycling that is triggered by ABA (Sutter et al., 2007).
Remarkably, one further parallel can be drawn juxtaposing ABA- and [Ca2+]i-mediated regulation of K+ channel gating with that of channel traffic in guard cells. Previously, this laboratory identified two Q-SNAREs, NtSyp121 (= NtSyr1) from tobacco and AtSyp121 from Arabidopsis, associated with ABA and drought stress (Leyman et al., 1999). Both NtSyp121 and AtSyp121 are syntaxin-like SNAREs with close homologues in mammals and yeast (Leyman et al., 1999). Both proteins localize to the plasma membrane (Leyman et al., 1999, 2000), and we now know that both contribute to secretory vesicle traffic (Geelen et al., 2002; Sutter et al., 2006b). SNAREs (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors) comprise a family of membrane trafficking proteins that are conserved among all eukaryotes and are essential for vesicle fusion and membrane traffic. They sustain neurotransmitter release in nerves, cell wall delivery and budding in yeast, and the growth and development of plant cells (Jahn et al., 2003; Surpin and Raikhel, 2004; Sutter et al., 2006a). Complementary SNAREs are localized to different membrane compartments and selectively interact to draw vesicle and target membrane surfaces together for fusion.
We found that disrupting NtSyp121 function, by expressing a dominant-negative, (so-called) Sp2 fragment in vivo, had severe effects on growth, tissue development and on traffic to the plasma membrane (Geelen et al., 2002). The Sp2 fragment corresponds to the cytosolic domain of NtSyp121 and its effects in trafficking assays was understood to arise from competition for SNARE partners. Significantly, ABA-mediated control of the K+ and Cl– channels was lost when guard cells were injected either with the same Sp2 fragment or with Clostridium neurotoxin BotN/C that cleaves the Q-SNARE and yields a product similar to the Sp2 fragment (Leyman et al., 1999). We have since observed that the Sp2 fragments of NtSyp121 and AtSyp121 affect traffic as well as anchoring of KAT1—the inward-rectifier K+ channel of Arabidopsis guard cells—at the plasma membrane (Sutter et al., 2006b). These observations indicated a role for the Q-SNARE in determining the local distribution of the K+ channel within the plasma membrane, and they raise questions about additional functions in regulating ion channel activities and the integration of SNARE- and Ca2+-mediated channel control.
To explore these questions, we have examined the actions of the Sp2 fragment of NtSyp121 on the ABA- and voltage-activated Ca2+ channels from tobacco guard cells, and we have employed complementary strategies to characterize the functional interaction of the SNARE fragment with evoked [Ca2+]i transients in vivo. Our results show that the Sp2 fragment of NtSyp121 prevents stomatal closure and selectively suppresses evoked [Ca2+]i transients by altering Ca2+ channel gating and reducing Ca2+ entry across the plasma membrane. These observations offer primary evidence for an action of the Q-SNARE on Ca2+ channels at the guard cell plasma membrane, and they indicate [Ca2+]i homeostasis as one target for SNARE function in vivo.
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RESULTS |
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Expressing NtSyp121-Sp2 Blocks Stomatal Closure in ABA
Previous studies showed that the sensitivity to ABA of all three of the predominant ion channels at the guard cell plasma membrane could be suppressed by injecting the Sp2 fragment of NtSyp121 directly into tobacco and Vicia guard cells (Leyman et al., 1999). Although it was therefore anticipated that Sp2 action should suppress stomatal closure in ABA, the means to testing this prediction were not to hand at the time. To this end, we took advantage of Sp2 expression in Sp2-14 transgenic tobacco under the control of the mammalian corticosteroid-inducible promoter that is activated to give maximal Sp2 expression within 12 h treatment with 1–10 µM dexamethasone (Geelen et al., 2002). Figure 1A shows the effect of Sp2 fragment expression on stomatal closure evoked by ABA and recorded using epidermal peels from the same tobacco plants before and 24 h after treatments with 10 µM dexamethasone, and from wild-type tobacco. In the absence of dexamethasone, the stomata of both wild-type and Sp2-14 tobacco achieved maximum closure within 60 min of superfusion with 20 µM ABA. However, 24 h after dexamethasone treatments, stomata from the same Sp2-14 tobacco plants showed a complete loss of sensitivity to ABA additions and an increase, albeit not very significant, in the steady-state mean of apertures.
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Because the currents carried by two of the channels responsive to ABA—the inward-rectifying K+ channel (IK,in) and the Cl– (anion) channel (ICl)— are regulated early on by elevated [Ca2+]i (Blatt, 2000; Hetherington and Brownlee, 2004), we explored the effects of the nitric oxide (NO) donor S-nitroso-N-acetyl-penacillamine (SNAP) as a means to elevating [Ca2+]i (Garcia-Mata et al., 2003). This approach avoided manipulating extracellular [K+] and [Ca2+] (Allen et al., 2001) and the additional effects such changes might have on stomatal aperture (Willmer and Fricker, 1996). Figure 1B shows the effect of Sp2 fragment expression on stomatal closure evoked by SNAP, again recorded using epidermal peels from the same tobacco plants before and 24 h after treatments with 10 µM dexamethasone, and from wild-type tobacco. As with ABA, in the absence of dexamethasone, the stomata of both wild-type and Sp2-14 tobacco closed in response to the NO donor, although the effect was appreciably slower than observed with ABA. After dexamethasone treatments, stomata from the same Sp2-14 tobacco plants showed a significant reduction in the amplitude of closure over a similar time period. However, a complete loss of sensitivity similar to that in ABA was not observed.
One intriguing feature of interference with membrane secretory traffic, at least in yeast, is an increase in the specific gravity of the cells (Novick et al., 1980), possibly associated with an elevation in osmotic solutes. Geelen et al. (2002) noted previously that expressing the Sp2 fragment of NtSyp121 led to abberant cell structures, hypertrophy and turgidity of the leaves. Although these observations were made over periods of many days, they do suggest a build-up of osmotica within the cells. To test whether the effect on stomatal behaviour of Sp2 expression might be related to an effect on osmotic solute content, we first measured the total osmotic content of tobacco leaves by freezing-point depression osmometry. Figure 2 summarizes the results of five independent experiments. Comparison of total osmotic content showed no significant difference between leaf tissues of wild-type and Sp2-14 transgenic tobacco in the absence of dexamethasone, but yielded an appreciable increase in the Sp2-14 transgenic plants 84 h after treatment with 10 µM dexamethasone (Figure 2A). As a second test, we examined the time course for this increase in osmotic content. Figure 2B incorporates the results from six experiments (six plants) and indicates a significant increase only after a lag time of the first 24 h following induction of Sp2-14 expression with dexamethasone. However, the osmotic content rose steadily thereafter, leading to roughly a doubling within the first week of dexamethasone treatment. Thus, although expression of the Sp2 fragment does affect the osmotic solute content of the leaf—and therefore must also influence cellular turgor—the response appeared too slow to account for a general loss of ABA sensitivity of guard cells within the first 24 h of transgene induction.
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NtSyp121-Sp2 Expression Affects Voltage-Evoked [Ca2+]i Elevation
One of the principal actions of NO in guard cells is mediated through guanylate cyclase and its stimulation of cyclic-ADP-ribose-mediated Ca2+ release, and its effect is to elevate [Ca2+]i, both at rest and in response to membrane voltage (Garcia-Mata et al., 2003; Garcia-Mata and Lamattina, 2003). Because these same [Ca2+]i signals are essential for ABA-mediated control of IK,in and ICl (Sokolovski et al., 2005) and might account for the loss of sensitivity to ABA and (partially) to NO (Figure 1), we explored the effect of the Sp2 fragment on evoked [Ca2+]i transients. For this purpose, measurements were carried out on intact guard cells in epidermal peels from wild-type and Sp2-14 transgenic tobacco, both before and 24 h after inducing Sp2 fragment expression with 10 µM dexamethasone. The guard cells were impaled with three-barrelled microelectrodes, the membrane clamped to –50 mV and the cells loaded by iontophoresis with the Ca2+-sensitive fluorescent dye Fura2. Increases in [Ca2+]i were evoked by 20-s clamp voltage steps and slow (100-s) voltage ramps between –50 and –220 mV. The ratio of Fura2 fluorescence averaged from the entire cell and from a 2-µm peripheral band around the cell was used to quantify the overall maxima, rise to peak and recovery. Fluorescence recorded from the peripheral band was used to determine the voltage threshold initiating [Ca2+]i increases by comparison with the resting [Ca2+]i before the start of each ramp (Garcia-Mata et al., 2003).
Figure 3 shows [Ca2+]i measurements from representative guard cells of the same Sp2-14 transgenic tobacco plant before (above) and 24 h after (below) treatment with dexamethasone. In each case, the guard cells were subjected to voltage ramps and Fura2 fluorescence was recorded at 1-s intervals throughout. Individual ratio images are shown on the right (a–d) corresponding to time points indicated along the voltage time-lines (centre). The kymographs (left) shows the kinetics of the [Ca2+]i rise and its recovery for bands stretching from the cell periphery (P) to the perinuclear (PN) region (tagged line in frames a and c, upper right). In the absence of Sp2 expression, voltage negative of approximately –130 mV (red arrow, upper centre) led to a rise in [Ca2+]i, first at the cell periphery and, within 5–10 s, also centrally within the cell. The [Ca2+]i rise was most pronounced near the cell periphery and perinuclear region, in part reflecting the high density of endoplasmic reticulum (see also Grabov and Blatt, 1998). Sp2 fragment expression had no significant effect on the resting [Ca2+]i at –50 mV (see Table 1). However, the same voltage ramp protocol uncovered a pronounced shift in sensitivity, with voltages near –200 mV only evoking a [Ca2+]i rise (red arrow, lower centre) and a much reduced [Ca2+]i transient. No significant difference was observed in the rate of [Ca2+]i rise once evoked, nor in its recovery when the voltage was returned to –50 mV. Similar results were obtained in a further six independent experiments (compare also Figure 5 of Garcia-Mata et al. (2003)) and are summarized in Table 1 together with data for [Ca2+]i transient maxima recorded from the guard cells in response to a standard 20-s voltage step to –220 mV. Brief inspection shows that Sp2 fragment expression led to a mean shift of greater than (–)45 mV in the voltage threshold for [Ca2+]i transients, and roughly a 50% reduction in the peak amplitude of transients evoked by voltage steps to –220 mV. Considering that change in [Ca2+] of one order of magnitude nominally equates to a 29-mV shift in the equilibrium voltage, ECa, for Ca2+ transport, the effects of Sp2 expression appeared most evident in a change in voltage threshold needed to evoke a [Ca2+]i rise.
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2 µm). Thresholds (red arrows, center): –Dex, –131 mV and +Dex, –184 mV for the data shown. The mean change in threshold, 
Vthresh, was +46 ± 5 mV (n = 7).NtSyp121-Sp2 Suppresses Ca2+ Channel Gating at the Plasma Membrane
Previous studies (Hamilton et al., 2000) have shown that voltages negative of approximately –100 mV activate a 12-pS Ca2+ channel with bursting characteristics and permeable to Ba2+ at the guard cell plasma membrane, and that its activation triggers intracellular Ca2+ release and a [Ca2+]i rise. Significantly, the voltage-dependence of this channel—and, hence, of evoked [Ca2+]i transients—is subject to ABA (Hamilton et al., 2000) and reactive oxygen species (Kohler et al., 2003; Pei et al., 2000). Thus, the Ca2+ channel is thought to play a major role in feedback coupling of K+ and Cl– efflux with membrane voltage (Blatt, 2000; Blatt et al., 2007). Unlike intracellular Ca2+ release, the plasma membrane Ca2+ channel is not appreciably responsive to NO (Garcia-Mata et al., 2003). We reasoned therefore that if the Sp2 fragment targeted the plasma membrane Ca2+ channel, but had little influence on intracellular Ca2+ release, this fact might explain the relatively weak suppression of NO-evoked stomatal closure as well as the shift in the voltage threshold evoking a [Ca2+]i rise (Figures 1 and 3): in effect, NO should promote intracellular Ca2+ release and could be expected to short-circuit a primary influence of the SNARE fragment at the plasma membrane.
To test this idea, we recorded Ca2+ channel currents under patch clamp from guard cell protoplasts, in the absence of K+ and Cl– and with 1 mM Ba2+ inside and 30 mM Ba2+ outside to eliminate K+ and Cl– currents (Hamilton et al., 2000). Ca2+ currents were recorded from inside-out macro-patches of wild-type tobacco with and without added Sp2 and from Sp2-14 transgenic tobacco with and without prior treatment with dexamethasone. As before (Köhler and Blatt, 2002; Sokolovski et al., 2005), we found that recordings could be maintained for 20 min or more with ATP present, both in excised macro- and micro-patch recordings. Figure 4 shows results from one of five experiments. In each case, Ca2+ currents from wild-type and un-induced Sp2-14 transgenic plants gave a characteristic inward current that showed significant activation near –100 mV under voltage clamp during slow (10-s) voltage ramps; however, the same experimental protocols yielded appreciable current activation only negative of –150 mV from guard cell protoplasts harvested 24 h after inducing Sp2 fragment expression and from macro-patches of wild-type protoplasts with added Sp2 fragment. Fitting current–voltage curves jointly to a Boltzmann function (see Figure 4 legend, and Hamilton et al. (2000)) yielded voltages for half-maximal activation (V1/2) of –110 ± 9 from wild-type and Sp2-14 transgenic plants before induction, and –181 ± 7 from Sp2-14 transgenic plants 24 h after induction with dexamethasone. Significantly, best fittings were obtained with the maximum conductance held in common—without change ±Sp2—indicating a principle effect on the voltage-dependence of channel gating rather than on the number of active channels.
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the voltage sensitivity coefficient, V1/2 the voltage giving half-maximal current activation, and z, F, R and T have their usual meanings. For the fitting shown, parameters gmax and
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We also recorded single-channel currents from wild-type tobacco guard cells using the inside-out configuration to enable Sp2 fragment additions directly to the cytoplasmic face of the membrane. Again, measurements were carried out in the absence of K+ and Cl– and with 1 mM Ba2+ inside and 30 mM Ba2+ outside to eliminate K+ and Cl– currents (Hamilton et al., 2000). Figure 5 summarizes the results from one of four independent experiments. Shown are segments of a continuous trace from one experiment and the corresponding open-channel point distribution histograms (right of each segment) and analysis of mean open probability (Po, below each segment) carried out on overlapping 1-s intervals and corrected for the total number of channels estimated in the patch. No activity was observed with the membrane clamped to –50 mV, but, on stepping to –180 mV, four open channel levels were observed in the cell-attached configuration and at least seven channels were recovered after excising the inside-out patch in the presence of 1 mM ATP in the bath (cytosolic side of the membrane). In each case, an appreciable flickering channel activity was recorded over periods of tens of seconds with the voltage held at –180 mV. However, this activity was rapidly lost when 7 µM of the Sp2 fragment of NtSyp121 was introduced into the bath (cytoplasmic) solution. Analysis of the channel current yielded amplitudes for openings in each case of 2.7 ± 0.2 pA at –180 mV, as expected for a 12-pS conductance, and open probabilities (Po) from all four experiments yielded a 6 ± 1-fold reduction, consistent with the whole-cell recordings at this voltage (Figure 4). Separate experiments using a similar concentration of bovine serum albumin (Figure 5, bottom) failed to influence the Ca2+ channel characteristics, indicating that the effects of the Sp2 fragment on channel gating were not a general phenomenon associated with the presence of a soluble protein or charge masking (Hille, 2001). We return to these observations below.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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Membrane vesicle traffic and ion transport are closely coordinated in eukaryotes and contribute to cellular homeostasis, signalling and development. In addition to their roles in membrane vesicle delivery and fusion, several membrane trafficking proteins interact with ion channels, notably to facilitate neurotransmitter release and neuronal signalling (Kang et al., 2006; Leung et al., 2005). There is now growing evidence in plants that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling. Work from this laboratory (Leyman et al., 1999) first identified a role for the SNARE proteins NtSyp121 in stomatal response to ABA, demonstrating that a soluble, inhibitory (dominant-negative) fragment of this SNARE—a so-called Sp2 fragment—blocked K+ and Cl– channel responses to ABA. Subsequent work showed that ABA-evoked endocytosis in guard cells of the Ca2+-sensitive K+ channel, KAT1, is too slow to account for the rapid response of the K+ current to ABA (Sutter et al., 2007) and that Sp2 fragments of NtSyp121 and its Arabidopsis homologue, AtSyp121, affect ion channel distribution within microdomains and mobility within the plane of the plasma membrane (Sutter et al., 2006b). In toto, these observations have implicated the SNAREs in signal coupling through the physical localization of the channel protein, but they have left open a question about functional impacts on signal intermediates, especially of [Ca2+]i.
The studies outlined above now demonstrate one mode of action for the plasma membrane SNARE that is mediated directly through alterations Ca2+ channel gating and its consequent effects on [Ca2+]i elevation through Ca2+-induced Ca2+ release. We show that expressing the Sp2 fragment of NtSyp121 affects three, causally related characteristics of guard cells: (1) Sp2 expression blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor SNAP which promotes [Ca2+]i elevation independent of the plasma membrane Ca2+ channels (Garcia-Mata et al., 2003). (2) Ca2+ channel gating at the plasma membrane is altered by the Sp2 fragment of NtSyp121 in a manner effective in reducing the potential for triggering a rise in [Ca2+]i. Finally, (3) we show directly that Sp2 expression in vivo leads to a pronounced suppression of [Ca2+]i elevation through a displacement in its sensitivity to membrane hyperpolarization. In addition, measurements of tobacco leaf tissues after inducing Sp2 expression showed that increases in cellular osmotic strength were not temporally correlated with the loss in sensitivity of the guard cells to ABA (Figures 1 and 2). These observations offer primary evidence for the functional coupling of the SNAREs with Ca2+ channels at the plant cell plasma membrane and, because [Ca2+]i plays a key role in the control of K+ and Cl– channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure and ABA signalling.
Ca2+ Channels as a Functional Target for SNARE Action
A key observation was our finding that the Sp2 fragment of NtSyp121 displaced the voltage-sensitivity of the Ca2+ channels to more negative voltages (Figure 4). Ensemble channel current is determined by the product of the number of channels, the single-channel conductance and the open probability (Po) of the channel. Changes in channel number and single-channel conductance are nominally scalars that do not affect the voltage-sensitivity of the current (Hille, 2001). So the effect on the voltage-sensitivity for [Ca2+]i elevation (Figure 3) implied a significant alteration in the voltage-sensitivity of the Ca2+ current and in Ca2+ channel open probability. These expectations were borne out in voltage clamp recordings both in tobacco expressing the Sp2 fragment (Figure 4) and in excised patches on addition of the fragment (Figure 5). A detailed analysis showed that the primary effect was on steady-state Po rather than on the single channel current amplitude—and, hence, conductance—and, because Po was low in the presence of the Sp2 fragment, this inactivation probably also accounts for the apparent loss of channel number (see also Köhler et al. (2002)). By contrast, no significant effect was observed with additions of another soluble protein—bovine serum albumin—at similar concentrations. The difference argues strongly against effects on gating of surface charge masking (Hille, 2001) and suggests a functional association with the plasma membrane SNARE (Leyman et al., 1999; Sutter et al., 2006b).
Significantly, expression of the Sp2 fragment of NtSyp121 also affected voltage-evoked [Ca2+]i transients, suppressing [Ca2+]i elevation by driving the voltage threshold for a [Ca2+]i rise some 40 mV negative from the control (Figure 3), but without an appreciable effect on the kinetics for [Ca2+]i elevation or its recovery (Table 1). Because intracellular Ca2+ release is normally triggered by Ca2+ entry through the plasma membrane Ca2+ channels (Grabov and Blatt, 1998, 1999; Hamilton et al., 2000), the shift in threshold points to a direct causal link between Sp2 action on these Ca2+ channels and their efficacy in evoking intracellular Ca2+ release. Indeed, this pattern mirrors, in reverse, the effect of ABA on Ca2+ channel gating and its displacement of the voltage threshold for [Ca2+]i elevations (Grabov and Blatt, 1998; Hamilton et al., 2000). By contrast, the general absence of an effect on the kinetics for [Ca2+]i elevation, once triggered, suggests that subsequent Ca2+ release from intracellular stores was unaffected by Sp2 expression. This observation also explains the correspondingly weak suppression of stomatal closure by NO, even after expressing the Sp2 fragment in vivo (Figure 1): NO acts primarily on intracellular Ca2+ release and it elevates [Ca2+]i, even at resting membrane voltages independently of the Ca2+ channel gating at the plasma membrane (Garcia-Mata et al., 2003; Blatt et al., 2007).
A Mechanism for Ion Channel Regulation
It is of interest that interactions are well documented between various SNARE proteins and mammalian Ca2+ channels, notably N-type Ca2+ channels of synaptic junctions, and some parallels may be drawn to the action of the SNARE fragment in the guard cells. NtSyp121 and other syntaxin-like Q-SNAREs (Jahn et al., 2003; Sutter et al., 2006a) comprise a single, C-terminal membrane-spanning domain, an adjacent cytosolic protein-interaction (H3) domain, and three N-terminal regulatory (HA/HB/HC) coils. Co-expression of N-type Ca2+ channels with the Q-SNARE syntaxin 1A that normally reside together at the presynaptic membrane is known to suppress Ca2+ channel gating by stabilizing an inactive state of the channel (Arien et al., 2003; Bezprozvanny et al., 2000; Degtiar et al., 2000). SNARE and Ca2+ channel interactions in this case are dictated by the so-called ‘synprint’ site of the Ca2+ channel, identified with two adjacent sequences in the cytosolic loop between domains II and III of the Ca2+ channel 
subunit (Rettig et al., 1996)—although additional interactions have been implicated (Bezprozvanny et al., 2000)—and by the H3 helix (Bezprozvanny et al., 2000) as well as N-terminal sequences (Jarvis et al., 2002) of the SNARE. The molecular identity of the guard cell Ca2+ channels remains unknown. However, it is significant that the Sp2 fragment of NtSyp121 incorporates the analogous SNARE domains (= HA/HB/HC + H3) and that this same SNARE fragment is sufficient to block ABA-mediated control of the guard cell K+ and Cl– channels (Leyman et al., 1999) as well as channel anchoring at the plasma membrane (Sutter et al., 2006b). Thus, one plausible explanation for Sp2 action, at least on the Ca2+-sensitive Cl– channels and inward-rectifying K+ channels, lies in a physical interaction with the Sp2 fragment that suppresses the plasma membrane Ca2+ channel and its ability to evoke a rise in [Ca2+]i. This conclusion also accords with recent evidence that ABA-evoked traffic of the inward-rectifying K+ channel, KAT1, is too slow to account for its rapid inactivation by the hormone (Sutter et al., 2007). Nonetheless, a direct test of this interpretation must await the molecular identity of the Ca2+ channels.
Its interaction in Ca2+ signalling aside, we note that NtSyp121 must target other ion channel functions as well. The action of the Sp2 fragment on the outward-rectifying K+ channels is a case in point. This K+ current is Ca2+-independent in guard cells, but is nonetheless activated in the presence of ABA (Blatt, 2000; Hetherington and Brownlee, 2004; Blatt et al., 2007) and its response to ABA is suppressed by the Sp2 fragment (Leyman et al., 1999). Indeed, our observations outlined above do not rule out additional and direct interactions of the SNARE with either of the Ca2+-dependent ion channels to affect their gating and currents. Examples of such interactions include the interactions of Syntaxin 1A with K+ channels that co-localize within microdomains of mammalian cell plasma membranes (Bravo-Zehnder et al., 2000; Martens et al., 2004), and interactions that affect subtly the gating of several mammalian Kv-type K+ channels (Kang et al., 2006; Leung et al., 2003, 2005).
Physiological Implications of SNARE Action in Ca2+ Signalling
The impact of the Sp2 fragment—and, by inference, of NtSyp121—on Ca2+ channel gating and [Ca2+]i transients carries a number of implications, both for membrane vesicle traffic and for Ca2+ signalling generally. At the synapse, Ca2+ plays a fundamental role in regulating the tethering and subsequent interactions of SNARE proteins leading to vesicle fusion (Jahn et al., 2003), with associated C2 domain (Ca2+- and lipid-binding) proteins, so-called synaptotagmins, facilitating late steps in fusion (Davis et al., 1999; Garcia et al., 2000; Mackler et al., 2002). Although synaptotagmin-like proteins occur in plants, including three in Arabidopsis (www.tair.org), little is known of their function(s). However, elevated [Ca2+]i is known to promote exocytosis in vivo in barley (Homann and Tester, 1997; Zorec and Tester, 1992) and maize root cap cells (Carroll et al., 1998), and has been associated with exocytosis at the tips of pollen and root hairs (cf. Roy et al. (1999), also Campanoni and Blatt (2006) for review]. Furthermore, [Ca2+]i acts at two discrete steps during vesicle exocytosis and recycling, at least in maize coleoptile protoplasts (Sutter et al., 2000). The onset of ABA-evoked endocytosis is also temporally correlated with elevated [Ca2+]i in guard cells and may play a role in longer-term adaptive control of ion channel activities (Sutter et al., 2007). Thus, the NtSyp121-Ca2+ channel association itself may impart a degree of control on vesicle traffic mediated by the SNARE much as has been proposed for homotypic fusion in yeast (Seeley et al., 2002; Takita et al., 2001).
There are recent indications, too, for coordinated SNARE activity and Ca2+ signalling in at least two other physiological responses in plants: gravitropism and pathogen defence. Gravitropism is associated with statoliths—dense, membrane-delimited storage organelles also known as amyloplasts—that ‘sediment’ and deform the plasma membrane and tonoplast in both roots and in the endodermis of shoots to affect cell expansion and growth (Masson et al., 2002). Significantly, mutations in the genes for two vacuole-associated SNAREs, AtVti11 (= ZIG) and AtSyp22 (= SRG3), suppress gravitropism and exhibit an abnormal distribution of endodermal statoliths and vacuole fragmentation (Kato et al., 2002; Morita et al., 2002; Surpin et al., 2003; Yano et al., 2003). Although these phenotypes have been interpreted in the context of vacuole ‘plasticity’ (Yano et al., 2003), there are indications that statolith position also affects local changes in [Ca2+]i as an early step in signal transduction (Edwards and Pickard, 1987; Gehring et al., 1990). Finally, both the Arabidopsis and barley homologues of NtSyp121, AtSyp121 (= PEN1) and ROR2, as well as a close homologue AtSyp122, are known to contribute to pathogen defence responses and, thus, are potentially linked with the associated Ca2+-dependent signalling events (Dangl and Jones, 2001). Mutants of AtSyp121 and ROR2 suppress penetration resistance to fungal pathogens (Collins et al., 2003), while AtSyp122 has been implicated in plant responses to bacterial pathogen attack (Nuhse et al., 2003). The close parallels of NtSyp121 and AtSyp121 functions in ABA signalling and ion channel control (Leyman et al., 1999; Sutter et al., 2006b, 2007) now provide a concrete framework with which to interpret the SNARE action on Ca2+ signalling during pathogen defense.
In conclusion, we find that the Sp2 fragment of NtSyp121 prevents stomatal closure and selectively suppresses evoked [Ca2+]i transients by affecting the voltage-dependence for Ca2+ channel gating at the plasma membrane. These observations offer primary evidence for the action of a SNARE on Ca2+ channels at the plant plasma membrane, and they point to its close interaction in [Ca2+]i homeostasis in vivo.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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Plant Material
Both Nicotiana tabacum L. and the transgenic tobacco line Sp2-14 that expresses the Sp2 fragment under induction by dexamethasone (Leyman et al., 1999) were used in this study. Dexamethasone treatments were carried out as before on the intact plants (Geelen et al., 2002). Epidermal peels were prepared as before (Grabov and Blatt, 1998) and protoplasts isolated enzymatically in a modification of the methods described previously (Hamilton et al., 2000). In the latter case, digestions were in 100 mM glycine, 2% Cellulase Onozuka RS (Yakult Honsan, Tokyo, Japan), 1.5% Cellulysin (Calbiochem, USA), 0.4% Pectolyase Y-23 (Kikkoman, Tokyo, Japan), 10 mM CaCl2, 1 mM MgCl2, 5 mM MES-NaOH, pH 5.7, adjusted to 300 m Osm with D-sorbitol. Protoplasts were collected after 30 min incubation at 28°C on a rotary shaker (10 rpm), washed twice with the same solution minus digestive enzymes and kept on ice until use. For impalement of intact guard cells, epidermal strips were mounted as described before (Hamilton et al., 2000; Sokolovski et al., 2005). All operations were carried out on a Zeiss Axiovert microscope with 40x and 63x LWD D.I.C. optics (Zeiss, Oberkochen) at 20–22°C. Solutions were added (20 chamber volumes min–1) by gravity feed and removed by aspiration.
Apertures and Osmometry
Stomatal apertures were recorded in epidermal peels mounted as described above, and measurements were carried out under continuous superfusion. Aperture dimensions were determined using a calibrated eyepiece micrometer. Osmotic contents of leaves were determined by freezing-point depression osmometry using a Wescor 5520 osmometer (Logan, USA). Leaves were harvested, mid-veins removed, and fresh weight recorded before extraction of soluble matter using a potter with distilled H2O added at a 1:1 w/v ratio to the tissue. Particulate matter was removed by centrifugation at 10 000 g for 10 min at 4°C, and the osmotic contents assayed according to the manufacturer's instructions.
Electrophysiology
For whole-cell patch and single-channel recording, pipettes were pulled using a Narashige PP-81 puller (Narashige, Tokyo) in two stages (input resistances, 10–30 M
) and were coated with Sigmacote (Sigma) and wax to reduce capacitance. Connections to amplifier and bath were via a 0.2-M KCl|Ag–AgCl liquid junctions, and junction potentials were taken into account (Barry and Lynch, 1991). Single-channel currents were recorded using an Axopatch 200B patch amplifier (Axon Instruments, Redwood City) and analyzed offline after filtering at 1 kHz. Voltages quoted are referenced to the physiological orientation of the membrane, the voltage on the cytosolic side relative to the extracellular side. Voltage clamp recordings and Fura2 injections of intact guard cells were carried out as before (Grabov and Blatt, 1998). Whole-cell currents were recorded under voltage clamp driven using Heka Pusle+PulseFit v8.53 software (Heka Electronik, Göttingen, Germany). Normally, a holding voltage of –50 mV was used with voltage steps chosen between +80 and –180 mV or voltage ramps run between 0 and –200 mV. All recordings were analyzed and leak currents subtracted using standard methods (Leyman et al., 1999; Sutter et al., 2006b) with Henry III software (Y-Science, University of Glasgow, Glasgow, UK).
[Ca2+]i Measurements
[Ca2+]i was determined by Fura2 fluorescence ratio photometry from fluorescence images gathered at 0.5- to 10-s intervals using cooled Pentamax-512 CCD camera and GenIV intensifier (Princeton Instruments, Princeton NJ) as described previously (Garcia-Mata et al., 2003; Hamilton et al., 2000). Dye was injected into intact guard cells by iontophoresis using three-barrelled microelectrodes, with barrels filled with 200 mM K+-acetate or 0.1 mM Fura2, while clamping the membrane voltage to –50 mV. Measurements were corrected for background before dye loading and analyzed using MetaFluor and MetaMorph software (Universal Imaging, West Chester, PA). Fura2 fluorescence was calibrated in vitro and in vivo after permeabilization (Grabov and Blatt, 1998). Estimates of dye loading indicated final Fura2 concentrations <10 µM (Grabov and Blatt, 1998).
Numerical Analysis
Channel amplitudes were calculated from point-amplitude histograms estimated from open events of >5-ms duration beyond closed levels determined from periods of no channel activity (Colquhoun and Sigworth, 1995) and were fitted to sums of Gaussian distributions using a Hooke-Jeeves algorithm (Hooke and Jeeves, 1961). Estimates of closed-amplitude distributions were obtained in Henry III software by similar fittings using single half-amplitude distributions for the most positive currents. Channel numbers were estimated from the maximum number of concurrent openings and from binomial distributions of open events (Horn, 1991). Channel openings and open probabilities were determined as before (Hamilton et al., 2000, 2001).
Chemicals and Solutions
We use the terms ‘inside’ and ‘outside’ with reference to the physiological sidedness of the membrane. Protoplasts were bathed in Ba2+-HEPES, pH 7.5 (HEPES buffer titrated to its pKa with Ba(OH)2) adjusted to 300 m OsM with sorbitol, and pipettes were filled with similar solutions. For cell-attached recording, pipette and bath contained 30 mM Ba2+; for whole-cell recordings, pipettes contained 1 mM Ba2+ and the bath contained 30 mM Ba2+; for excised, inside-out patches, pipettes contained 30 mM Ba2+ and the bath contained 1 mM Ba2+; 1 mM (Mg2+)2ATP was included on the cytosolic side of the membrane in each case. For measurements from intact guard cells, bath solutions comprised 5 mM Ca2+-MES, pH 6.1 (MES buffer titrated to its pKa with Ca(OH)2), with additions of 0.1 or 10 mM KCl. The Sp2 fragment of NtSyp121 was obtained by expression in E. coli and purified by NTA affinity chromatography as described previously (Leyman et al., 1999). All other chemicals and compounds were from Sigma (Poole, UK).
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Acknowledgements |
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This work was supported by BBSRC grants P09640 [GenBank] , BB/D/001528/1, and BB/D500595/1, by Leverhulme Trust grant F00179 [GenBank] /T, and by a John Simon Guggenheim Fellowship to MRB. No conflict of interest declared.
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