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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Cold Transiently Activates Calcium-Permeable Channels in Arabidopsis Mesophyll Cells^1,[W]

Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute of Biosciences, Wurzburg University, 97082 Wurzburg, Germany (A.C., N.I., V.L., E.K., E.J., R.H.); Institute of Biophysics, National Research Council, 16149 Genova, Italy (A.C.); School of Biological Sciences, University of Wales, Bangor LL57 2UW, Wales, United Kingdom (N.I.); Department of Biophysics, Institute of Biology, Maria Curie-Sklodowska University, 20–033 Lublin, Poland (E.K.); and Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521 (J.-K.Z.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore >3.34 Å (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants.

The molecular mechanisms enabling plant cells to sense temperature are largely unknown. In animal cells, the sensory system is capable of detecting thermal stimuli over a broad temperature spectrum. Currently, the existence of various thermosensors is discussed. The latter belong to the transient receptor potential superfamily of cation channels. These channels function as detectors of chemical and physical stimuli, such as heat and cold, as well as mechanical forces (Clapham, 2003; Patapoutian et al., 2003; Calixto et al., 2005).

By applying the patch-clamp technique to Arabidopsis mesophyll cells, we were able to record transient cold-induced nonselective Ca²⁺-permeable cation channels in plants.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Repetitive CIPCs in Arabidopsis Mesophyll Cells

Rosette leaves of 8- to 10-week-old plants were mounted into the recording chamber and continuously perfused with standard bath solution (5 mM KCl, 1 mM CaCl₂, and 5 mM MES/BisTris-propane, pH 6.0). Mesophyll cells were impaled with a voltage-recording microelectrode, and temperature was monitored by a thermistor placed near the recording microelectrode. This arrangement allowed us to record temperature and free-running membrane potential simultaneously. To cold-stimulate the leaf, bath perfusion was switched from a reservoir at 26°C to a precooled one (1°C). Upon stepping the temperature from 26°C to 16°C, transient membrane potential changes could be elicited reproducibly and uniformly (Fig. 1A ). In line with the observation of Plieth et al. (1999), a drop in temperature to just 19°C did not elicit the full response. A full response was characterized by a rapid depolarization from a resting level of about –150 mV (in Fig. 1, A and B, resting potentials range from –162 to –152 mV) to –50 mV. In this initial phase, the velocity of cooling was maximal. From this peak depolarization, the membrane potential recovered to reach a plateau (around –100 mV in Fig. 1B). The second phase of the CIPC thus developed when the temperature reached the set value. In phase 3, when the temperature was shifted to 26°C again, the membrane potential repolarized to its prestimulus level. In similar experiments performed on apoaequorin-expressing Arabidopsis leaves (Knight et al., 1991), we measured both potential and cytosolic calcium changes (Supplemental Fig. S1), confirming previous findings that show that the initial phase of depolarization is associated with Ca²⁺ influx (Lewis and Spalding, 1998; Plieth et al., 1999; Knight, 2002).

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Temperature- and time-resolved membrane potential changes in response to cold stimulations of mesophyll cells of intact leaves from Arabidopsis plants. A, Time course of voltage changes (top) induced by the temperature profile (bottom trace). B, A single voltage transient (indicated by arrows in A) at higher temporal resolution (top) and corresponding temperature profile (bottom trace).

Previous studies documented that isolated cell wall-free mesophyll protoplasts still respond to external stimuli such as blue light (Stoelzle et al., 2003). But are turgor-free protoplasts still sensitive to cold? To test the cold response of mesophyll protoplasts, we again used Arabidopsis plants expressing the calcium reporter apoaequorin in the cytosol. Upon cold induction, cytosolic Ca²⁺ increased (Fig. 2 ) in a similar fashion as intact leaves or leaf discs (Supplemental Fig. S1). When perfusing noncooled media, only minor changes in cytosolic calcium were observed (Fig. 2). Thus, under these conditions, cell wall- and turgor-free mesophyll protoplasts operate a cold receptor rather than one responding to sheering stress.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Transient cold-induced increase in cytosolic Ca2+ in Arabidopsis mesophyll protoplasts. Relative luminescence of protoplasts with reconstituted aequorin was followed before and after application (indicated by the vertical arrow) of precooled solution (cold shock, n = 4) or room temperature bath solution (room temperature, n = 6). Data are means ± SE.

To gain new insights into the nature of CIPCs, we applied the patch-clamp technique to the isolated mesophyll protoplasts of Arabidopsis. The cytosolic (patch pipette) solution contained 150 mM CsCl, 1 mM MgCl₂, 10 mM EGTA, 1 mM MgATP, pH 7.4. The external solution contained 100 mM CaCl₂ and 10 mM CsCl, pH 5.6. Under these conditions, with K⁺-selective channels effectively blocked by Cs⁺ and Ca²⁺ (Hedrich et al., 1995; Becker et al., 2004), cold-induced inward currents could be recorded in the whole cell configuration (Fig. 3A ). To impose a steep temperature gradient, a bath perfusion system of high speed (approximately 2 mL/min) was used. Under these conditions, cold-induced inward currents showed the same temperature-dependence as the CIPCs of intact Arabidopsis leaves (compare with Fig. 1); currents activated at the onset of the temperature drop reached a peak value and inactivated to a new steady state when the temperature reached the intended value (Figs. 3A and 4A ). Upon steps to the prestimulus temperature, currents declined completely (Fig. 4A). Similarly to the calcium/aequorin measurements (Fig. 2), ionic currents were not induced by bath perfusion with room temperature-adapted solutions (Fig. 3B). To study the voltage dependence of cold-induced transient conductance (CITC), we clamped the membrane to –49 mV and applied the cold stimulus (Fig. 4B). During a step to +51 mV, the outward currents increased in amplitude (Fig. 4B); then a voltage ramp from +51 to –189 mV was applied. During the voltage ramp, currents reversed direction around –7 mV (Fig. 4C). At potentials negative of about –90 mV, currents slowly decayed (Fig. 4C).

View larger version (6K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Current transients in Arabidopsis mesophyll protoplasts elicited by a cold stimulus. A, Transient inward current recorded in mesophyll protoplasts in the whole-cell configuration of the patch-clamp technique. The arrow indicates the application of the cold stimulus (see "Materials and Methods"). The holding potential was –49 mV. B, Protoplast current response to perfusion with cold (15°C) and room temperature (26°C) solutions. Only protoplasts that show a measurable cold-induced current were challenged with room temperature bath solution. Bars represent the mean ± SE of nine protoplasts.

View larger version (7K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Kinetics and current-voltage relation of the cold-induced plasma membrane response in mesophyll protoplasts. A, The time course of cold-induced current transient was recorded in the whole-cell configuration of the patch-clamp technique. The upward and downward arrows indicate the start and the end of the cold stimulus, respectively. The holding potential was –39 mV. Spikes superimposing the current trace result from voltage ramps (+51 to –109 mV in 500 ms) similar to that shown in B, top. B, Ionic currents (bottom) elicited by the voltage ramp (top) during cold stimulation. Note that the removal of the cold stimulus (recovery) completely restored the initial resistance. C, Current-voltage relationship derived from voltage ramp stimulation as shown in B. Cold-induced currents were characterized by outward rectification and by a reversal voltage in this experiment of –7 mV. Inset: When 150 mM of Cl– in the pipette was substituted by 150 mM of gluconate–, the reversal voltage did not change significantly (–12 mV). c, Control; r, recovery.

What is the charge carrier of the CITC? When in the internal standard solution CsCl was replaced by cesium gluconate (150 mM), the signal was not altered significantly (Fig. 4C, inset), suggesting that the CITC represents a cation channel rather than anion channel. In external media containing 10 mM Cs⁺ and standard internal solution (CsCl = 150 mM) at a holding potential of –49 mV, transient outward currents were elicited by cold treatment (Fig. 5A , top). After reaching the peak of the transient current, a fast voltage ramp was applied (Fig. 5B, trace 1), and cold-induced currents reversed direction at –67 ± 4 mV (mean ± SD, n = 4), i.e. very close to the Nernst potential for Cs⁺ ([E₁(Cs⁺)] = –66.4 mV) and very far from the Nernst potential of Cl^– ([E₁(Cl^–)] = +67.0 mV). This strong experimental evidence again points to the cationic nature of the CITC. Outward current shown in Figure 5A (top trace) is mediated by Cs⁺ moving from the cytosol to the external solution.

View larger version (6K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Cation selectivity of the cold-activated current. A, Time-dependent current transients induced by cold stimulation in bath solutions containing 10 mM Cs+ (top trace) and 100 mM Ca2+ and 10 mM Cs+ (bottom trace). The holding potential was –49 mV. Note that in the absence of external Ca2+, current transients are outwardly directed. B, Current-voltage relationship of ionic currents following voltage ramps (which have been substituted in A by numbers for the sake of clarity) applied during cold stimulation in solutions containing 10 mM Cs+ (1) and 100 mM Ca2+ and 10 mM Cs+ (2). In this experiment, the reversal voltage of (1) and (2) were –65 mV and –5 mV, respectively.

To estimate the unitary conductance of the CITC, single channel fluctuations were measured in the whole-cell configuration. The trace shown in Figure 6 was obtained positioning the protoplast a few millimeters away from the tube releasing the cold solution. Perfusion of solution kept at room temperature did not elicit a measurable electrical response. Cold treatments, however, transiently activated macroscopic currents from which we could resolve single ion channel fluctuations (Fig. 6B) with an estimated chord conductance of 33 ± 7 pS (mean ± SD; Fig. 6B). The reversal voltage estimated from voltage ramps (which have been substituted in Fig. 6A by asterisks for the sake of clarity) was in agreement with the value obtained from macroscopic currents (like those of Fig. 4) recorded under similar conditions. Thus, the macroscopic cold-induced whole-cell currents shown in Figures 3 to 5 seem to be carried by the 33-pS single channel of Figure 6B.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Cold-induced single channel events. A, Transient inward current in mesophyll protoplasts in the whole-cell configuration elicited by cold application (indicated by the vertical arrow). The protoplast was placed slightly away (about 5 mm) from the tube releasing the cold solution. B, From the magnification of the trace shown in A, delimited by the arrows, single channel fluctuations became apparent. The trace was further filtered with a four-pole Bessel digital filter at 300 Hz. Single channel conductance was deduced from the correspondent histogram. C and Oi indicate the closed and open (conductive) states of the channels, respectively. The holding potential was –47 mV, and data were filtered at 500 Hz and acquired with a sampling time of 500 µs. Pipette solution: 150 mM cesium gluconate, 1 mM MgCl2, 10 mM EGTA, 1 mM MgATP, 10 mM HEPES/Tris, pH 7.4. Bath solution: 10 mM CsCl, 100 mM CaCl2, 10 mM MES/Tris, pH 5.6. Osmolarity of both pipette and bath solutions was adjusted to 400 mosmol kg–1 with D-sorbitol. The measured reversal voltage in this condition was –12 mV. Asterisks in A were inserted in the place of voltage ramps (omitted for the sake of clarity) from which Vrev was obtained.

CIPCs in Selected Arabidopsis Cold-Sensitive and Ion Channel Mutants

To find the gene corresponding to the CITC, we performed a screen on selected Arabidopsis mutants measuring CIPC in mesophyll cells of intact leaves.

The promoters of some stress-related genes, such as RD29, are very sensitive to cold, drought, and osmotic stress. In screens with a mutagenized Arabidopsis population expressing RD29 promoter::luciferase constructs, plants with low (LOS for low expression of osmotically responsive genes) and high (HOS for high expression of osmotically responsive genes) bioluminescence were isolated (Ishitani et al., 1998; Guo et al., 2001). Thus, los and hos mutants appear altered in the cold signaling pathway. When we challenged these temperature-sensitive mutants with a cold stimulus, however, they did not show an altered cold response (Supplemental Fig. S2). This indicates that the mutation in the cold pathway is downstream of the CITC. Some HOS genes, such as HOS9, mediate cold tolerance through a calcium-binding factor (CBF)-independent pathway (Zhu et al., 2004). The latter network consisting of 10 CBFs and 25 CBF-induced protein kinases (CIPKs), however, was associated with cold signaling as well (CBL1; Cheong et al., 2003). A key player in CBF-CIPK calcium-sensing network is CIPK1 (Ok et al., 2005). We thus included clb1 and cipk1 into our studies. Just like los and hos, cbl1 and cipk1 responded in a wild type-like manner (Supplemental Fig. S2). Although we have not tested all mutants within this pathway, our preliminary findings indicate that the CBF-CIPK network may respond to calcium entry via the CITC, but mutations in this system seem not to feedback on the CITC. Recently, Glu-like receptor channels and potentially CNG-gated channels have been discussed as Ca²⁺-entry pathways (Meyerhoff et al., 2005). The dnd1 mutant (Clough et al., 2000), lacking a CNG channel essential for pathogen defense, however, did not show differences in the cold response (Supplemental Fig. S2).

Because we could not exclude the possibility that cold stimuli activate ion channels already identified, we inspected the mesophyll plasma membrane cation channel composition. Previous studies have shown that in mesophyll cells, membrane hyperpolarization does not activate inward potassium currents, a fact in line with the lack of KAT1, KAT2, AKT1, and AKT2 potassium channel gene expression in this cell type (Ivashikina et al., 2003). Upon depolarization, however, a K⁺ outward rectifier activates. We have shown that GORK, the Shaker-like K⁺-selective outward rectifier expressed in the shoot and root of Arabidopsis (Ache et al., 2000), is transcriptionally activated by cold (Becker et al., 2003). In contrast to the CITC, GORK is not cold activated, but outward currents through this K⁺ channel drop upon reduction in temperature without a shift toward negative membrane potentials (A. Carpaneto, A. Naso, N. Ivashikina, H. Heber, F. Gambale, and R. Hedrich, unpublished data). Likewise, gork-1, a GORK loss-of-function mutant (Hosy et al., 2003), exhibited a wild-type cold response (Supplemental Fig. S2).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In this study, we focused on the early mesophyll plasma membrane signaling events following rapid cooling treatment. Applying the patch-clamp technique to isolated Arabidopsis mesophyll protoplasts, we were able to measure the predicted transient cold-activated ion channels. The fact that ionic currents activated at the onset of the temperature drop suggests that the CITC is the first step that leads the plant cell to sense the rapid cooling. As monovalent ion cesium, a well-known potassium channel blocker, was able to permeate the cold-induced channel, we can conclude that the diameter of the permeation pore is larger than 3.34 Å (the ionic diameter of Cs⁺). The suggested pore size and a unitary conductance of about 33 pS group CITC into the class of wide-pore ion channels (Hille, 1992). In line with previous data showing that rapid cooling application induces intracellular calcium rise (for review, see Knight 2002), we found that calcium can permeate through the CITC and that under our experimental condition, the permeability ratio P_Ca:P_Cs was 0.7.

The cold response of ion channels has been the subject of very few earlier studies (Ding and Pickard, 1993b; Ilan et al., 1995). In their studies on onion epidermis protoplasts, Ding and Pickard (1993b) could document the cold stimulation of a previously characterized channel (Ding and Pickard, 1993a) preactivated by membrane stretch. This behavior is different from that of the cold-induced channel investigated here. The latter is electrically silent in the absence of a cold stimulus but activates when the temperature drops from e.g. 25°C to 15°C (Fig. 3A). The temperature-sensitive channel in onion protoplasts, however, did not show a pronounced response in this temperature range. Moreover, the mechanosensitive channel did not inactivate (Ding and Pickard, 1993b), while the cold-induced conductance is clearly transient. As many temperature-sensitive channels present also modulation induced by mechanical stress (Kung, 2005; Voets et al., 2005), we tried to investigate if CITC is mechanosensitive. However, in cell-free excised patches, we could not record any cold-activated channels, indicating that an important cytosolic factor is lost upon patch excision, or, alternatively, that the channel density is not enough high to allow single channel measurements in excised patch. This prevented our ability to clearly determine or refuse the mechanosensitive nature of CITC.

Working with V. faba guard cells, Ilan et al. (1995) explored the temperature sensitivity of voltage-activated inward and outward K⁺ rectifiers. While the whole-cell current of the inward rectifier dropped with the decrease in temperature from about 30°C to 25°C and finally 13°C, the outward rectifier was characterized by a peak at 20°C. Both primarily voltage-dependent channels, however, did not require a cold stimulus to activate.

The CITC in Arabidopsis mesophyll protoplasts represents a nonselective cation channel. Up to now, several cation channels have been described. Demidchik et al. (2002), Davenport and Tester (2000), and Tyerman et al. (1997) identified cation channels when searching for channels involved in the response to salt stress. This channel type was found permeable to sodium ions but blocked by Ca²⁺. Another cation channel type was recognized when studying the electrical properties of the plasma membrane to hydrogen peroxide or blue light (Stoelzle et al., 2003); however, the latter conditions activated a hyperpolarization-dependent cation channel. In guard cells and their subsidiary cells, a depolarization-activated cation channel was identified recently (Buchsenschutz et al., 2005). Future studies will thus have to prove whether or not this channel is expressed in mesophyll cells and responds to cold in a CITC-like manner. So far, our cold-induced channel does not share the properties with Arabidopsis cation channels described before.

In the search for the CITC gene, we tested several Arabidopsis mutants (see "Results"). However, we could not find significant differences in CIPC between these mutants and wild-type plants. Ongoing screens for cold mutants in the future will identify CITC genes. This possibly will help to gain insight into the molecular basis of temperature sensing in plants and to develop cold-tolerant crops.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Membrane Potential Measurements

Mesophyll preparations were obtained from the rosette leaves of 8- to 10-week-old Arabidopsis (Arabidopsis thaliana) plants, and the leaf sections were fixed to the experimental chamber by adhesive tape and continuously perfused with the standard bath solution (5 mM KCl, 1 mM CaCl₂, and 5 mM MES/BisTris-propane, pH 6.0). Temperature was controlled by a homemade Peltier device (Becker et al., 2004) and directly monitored using a thermistor placed in the bath solution near the recording microelectrode. Electrodes were pulled from borosilicate-glass capillaries (Hilgenberg; ø outside 1.0 mm, ø inside 0.58 mm), filled with 300 mM KCl, and had a tip resistance ranging from 50 to 100 M. Cells were impaled using a micromanipulator (type 5171; Eppendorf) combined with a piezo translator (P-280.30; Physik Instumente). The electrodes were connected via an Ag/AgCl half-cell to a microelectrode amplifier (VF-102; Bio-Logic) connected to a head stage with an input impedance of 10¹¹ . The free running membrane potential of mesophyll cells was contemporarily recorded on a chart recorder and digitalized.

Simultaneous Membrane Potential and Ca²⁺ Recordings in Leaf Segments of Arabidopsis

C24 plants expressing apoaequorin were preincubated for 6 h under complete darkness in solution containing 0.1 mM KCl, 0.1 mM CaCl₂, 5 mM MES/BisTris-propane, pH 6.0, along with 5 µM coelenterazine (for reconstitution of active aequorin). Prior to measurements, the segments were fixed to the experimental chamber by adhesive tape and continuously perfused with standard solution (5 mM KCl, 1 mM CaCl₂, and 5 mM MES/BisTris-propane, pH 6.0). Cells were impaled using borosilicate glass electrodes of a 50- to 100-M tip resistance filled with 300 mM KCl. The electrodes were connected via an Ag/AgCl half-cell to a head stage (1 G; HS-2A; Axon Instruments) and an Axoclamp-2B amplifier (Axon Instruments). The free running membrane potential was digitalized (ME-RedLab) and recorded on hard disc. Cold-induced [Ca²⁺]_cyt increases were measured using a CCD camera Visiluxx Imager (Visitron Systems GMBH).

Isolation of Mesophyll Protoplasts

Epidermal-free fully developed Arabidopsis rosette leaves were incubated for 30 min in enzyme solutions containing 0.8% (w/v) cellulase (Onozuka R-10), 0.1% pectolyase (Sigma), 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1 mM CaCl₂, and 10 mM MES/Tris, pH 5.6. Osmolarity of the enzyme solution was adjusted to 400 mosmol kg^–1 with D-sorbitol. Released protoplasts were filtered through a 100-µm nylon mesh and washed twice in 1 mM CaCl₂ buffer (osmolarity of 400 mosmol kg^–1, pH 5.6).

Bioluminescence Measurements with Protoplasts

Stimulus-induced cytosolic Ca²⁺ signals were measured in mesophyll cells expressing cytosolic apoaequorin (Knight et al., 1996; Baum et al., 1999). For reconstitution, the mesophyll protoplasts were floated on 1 mM KCl, 0.1 mM CaCl₂, 4 mM MES/Tris, pH 5.6, osmolarity of 420 mosmol kg^–1 with mannitol in the presence of 2.5 µM coelenterazine (Nanolight Technologies). Bioluminescence of the preparation was determined using a cooled photomultiplier-based chemoluminometer (model 9829A; Thorn EMI Electron Tubes). Protoplasts were placed into a 3.5-mL cuvette (Sarstedt) in the following solution: 1 mM KCl, 1 mM CaCl₂, 4 mM MES/Tris, pH 5.6, osmolarity of 420 mosmol kg^–1 with mannitol. Cold stimulus was applied via a precooled syringe through a luminometer port. To determine the relative luminescence L/L_max, after each experiment the aequorin was discharged by adding 1 M CaCl₂, 10% ethanol solution. The relative luminescence was determined from the ratio of the actual luminescence and the total luminescence emitted from the probe and plotted as a function of time.

Patch-Clamp Recordings

Patch-clamp recordings were performed in the whole-cell mode using an EPC-7 amplifier (List-Medical-Electronic). Data were digitized by ITC-16 interface (Instrutech) and analyzed using software Pulse and PulseFit (HEKA Elektronik) and IGORPro (Wave Metrics). Patch pipettes were prepared from Kimax-51 glass capillaries (Kimble Products) and coated with silicone (Sylgard 184 silicone elastomer kit; Dow Corning GmbH). The standard pipette solution (cytoplasmic side) contained 150 mM CsCl, 1 mM MgCl₂, 10 mM EGTA, 1 mM MgATP, and 10 mM HEPES/Tris, pH 7.4. The standard external solution contained 100 mM CaCl₂, 10 mM CsCl, and 10 mM MES/Tris, pH 5.6. For the selectivity experiments, the pipette solution was replaced by 150 mM cesium gluconate, 1 mM MgCl₂, 10 mM EGTA, 1 mM MgATP, 10 mM HEPES/Tris, pH 7.4, and the external solution by 10 mM CsCl, 10 mM MES/Tris, pH 5.6. Osmolarity of all solutions was adjusted to 400 mosmol kg^–1 with D-sorbitol. The command voltages were corrected off-line for liquid junction potential (Neher, 1992). For the standard internal and external solutions, liquid junction potential was –9 mV; for the cesium gluconate internal solution and the external standard solution, it was –17 mV. Temperature was controlled by a homemade Peltier device (Becker et al., 2004). At the end of each experimental session, the temperature profile was measured by a 1.5-mm diameter thermistor (GM103 Thermometrics) placed in front of the perfusion tube. The measured temperature dropped from 26°C to 15°C within 10 s. The high speed of the flux (approximately 2 mL/min), together with the critical positioning of the tube in front of the protoplast, resulted in a high probability (>50%) to lose the cell, making these experiments demanding. The total number of successful protoplasts investigated by the patch-clamp technique was 39. Chemicals were obtained from Sigma-Aldrich. The permeability ratio between calcium and cesium was calculated using the following equation (Lewis, 1979): P_Ca/P_Cs = 1/4 ([Cs]_in exp[FV/RT] – [Cs]_out)/[Ca]_out (1 + exp[FV/RT]), where R, T, F, and V have the usual meaning, and [Cs]_in, [Cs]_out, and [Ca]_out are the activities of internal cesium and external cesium and calcium, respectively. The activity coefficients (Robinson and Stokes, 1959) given to internal cesium and external cesium and calcium were 0.724, 0.693, and 0.518, respectively.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Cold-induced changes in membrane potential and cytosolic Ca²⁺.

Supplemental Figure S2. Cold-induced potential changes in mesophyll cells from selected Arabidopsis cold-sensitive and ion channel mutants.

Note Added in Proof

We recently detected a similar CITC in Arabidopsis guard cell protoplasts in agreement with recent calcium measurements performed by Dodd et al. (Dodd AN, Jakobsen MK, Baker AJ, Telzerow A, Hou S-W, Laplaze L, Barrot L, Poethig RS, Haseloff J, Webb AAR [2006] Time of day modulates low-temperature Ca²⁺ signals in Arabidopsis. Plant J 48: 962–973).

	ACKNOWLEDGMENTS

 
We thank Hervé Sentenac (Institut National de la Recherche Agronomique, Montpellier) for gork-1, Petra Dietrich (University of Erlangen-Nürnberg) for dnd-1, and Jörg Kudla (University of Münster) for cbl1 and cipk1 and Arabidopsis mutants. We are grateful to Marc and Heather Knight (Oxford University, UK) and Franco Gambale, Michael Pusch, and Joachim Scholz-Starke (IBF-CNR, Italy) for comments and suggestions on the manuscript.

Received October 9, 2006; accepted November 7, 2006; published November 17, 2006.

	FOOTNOTES

 
¹ This work was supported by Deutsche Forschungsgemeinschaft (grants to R.H.), by SFB 576 (short-term stipend to E.K.), and by Alexander von Humboldt (stipend to A.C.).

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: Rainer Hedrich (hedrich{at}botanik.uni-wuerzburg.de).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.090928

^* Corresponding author; e-mail hedrich{at}botanik.uni-wuerzburg.de; fax 49–931–8886157.

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