Cold Transiently Activates Calcium-Permeable Channels
in Arabidopsis Mesophyll Cells1[W]
Armando Carpaneto, Natalya Ivashikina, Victor Levchenko, Elzbieta Krol, Elena Jeworutzki,
Jian-Kang Zhu, and Rainer Hedrich*
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.)
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.
Plants recognize daily and annual temperature
changes and integrate them into their developmental
program. Cold stimulation of plant cells result in
membrane depolarization, increase in cytoplasmic calcium, and transcription of cold- and touch-responsive
genes (Thomashow, 2001; Knight, 2002; Braam, 2005).
Early studies identified cold-induced potential
changes (CIPCs) in Cucumis, Beta, and Glycine roots,
Avena and Hordeum coleoptiles, Lonicera and Hedera
leaves, Limnobium root hairs, and Allium epidermal
cells (Minorsky, 1989). Later, a large and transient
depolarization was recorded in the moss Conocephalum
conicum (Krol et al., 2003) and in mesophyll cells of
Arabidopsis (Arabidopsis thaliana), Helianthus annuus,
and Vicia faba (Krol et al., 2004). Recently, in Arabidopsis plants expressing the calcium reporter apoaequorin in the cytoplasm (Knight et al., 1996; Plieth
et al., 1999; Knight, 2002), it was found that a temperature drop of several degrees caused an immediate
and transient rise in cytosolic calcium. Temperature
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.).
* Corresponding author; e-mail [email protected].
de; fax 49–931–8886157.
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 ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.106.090928
sensing in Arabidopsis depends on both the cooling
rate (Plieth et al., 1999) and the final temperature to
which cooling occurs (Knight, 2002). Previous findings
showed that rapid cooling (i.e. cooling rates greater
than 1°C–10°C/min) acts on a variety of physiological
processes in plants, such as protoplasmic streaming,
plant growth, phloem translocation, cell motility, and
water absorption, in a different way from gradual
changes in external temperature of the same amplitude (for review, see Minorsky, 1989).
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 Ca21-permeable cation
channels in plants.
RESULTS
Repetitive CIPCs in Arabidopsis Mesophyll Cells
Rosette leaves of 8- to 10-week-old plants were
mounted into the recording chamber and continuously
Plant Physiology, January 2007, Vol. 143, pp. 487–494, www.plantphysiol.org Ó 2006 American Society of Plant Biologists
487
Carpaneto et al.
Fig. S1), confirming previous findings that show that
the initial phase of depolarization is associated with
Ca21 influx (Lewis and Spalding, 1998; Plieth et al.,
1999; Knight, 2002).
Cold Induces Calcium Signals in Cell Wall-Free
Mesophyll Protoplasts
Previous studies documented that isolated cell wallfree 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 Ca21 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 turgorfree mesophyll protoplasts operate a cold receptor
rather than one responding to sheering stress.
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).
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 2150 mV (in Fig. 1, A
and B, resting potentials range from 2162 to 2152 mV)
to 250 mV. In this initial phase, the velocity of cooling was maximal. From this peak depolarization, the
membrane potential recovered to reach a plateau
(around 2100 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
488
Mesophyll Membrane Harbors a Cold-Sensitive
Ionic Conductance
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 K1-selective
channels effectively blocked by Cs1 and Ca21 (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, coldinduced inward currents showed the same temperaturedependence 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 249 mV and
applied the cold stimulus (Fig. 4B). During a step to
151 mV, the outward currents increased in amplitude
(Fig. 4B); then a voltage ramp from 151 to 2189 mV
was applied. During the voltage ramp, currents reversed direction around 27 mV (Fig. 4C). At potentials
negative of about 290 mV, currents slowly decayed
(Fig. 4C).
Plant Physiol. Vol. 143, 2007
Transient Cold-Induced Ion Channel
CITC Represents a Calcium-Permeable Cation Channel
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 6 7 pS (mean 6 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.
Because one cannot exclude the possibility that
CITC may be able to sense changes in membrane tension due to temperature-dependent membrane lipid
rearrangements, we tried to record the cold-induced
channel in cell-free excised patches. In eight different
protoplasts, we could not record any cold-activated
currents both in inside-out (n 5 2) and outside-out
patches (n 5 6). In this context, it should be mentioned
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 Cs1 and standard
internal solution (CsCl 5 150 mM) at a holding potential of 249 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 267 6 4 mV (mean 6 SD,
n 5 4), i.e. very close to the Nernst potential for Cs1
([E1(Cs1)] 5 266.4 mV) and very far from the Nernst
potential of Cl2 ([E1(Cl2)] 5 167.0 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 Cs1 moving from the
cytosol to the external solution.
To study the calcium permeability of the CITC, we
added 100 mM Ca21 to the external solution containing
10 mM Cs1; at a holding potential of 249 mV, transient
inward currents were elicited by cold treatments
(Fig. 5A, bottom trace). The cold-induced currents
reversed direction at 25 6 2 mV (mean 6 SD, n 5 6;
Fig. 5B, trace 2; see also Fig. 4C). This value is neither
close to the Nernst potential for Ca21 (,200 mV) nor
for Cs1, indicating that cold-induced channels are
cation nonselective with PCa/PCs 5 0.7 6 0.1. Note that
the inward current displayed in Figure 5A (bottom
trace) results from movement of Ca21 from the external medium to the cytosol.
To estimate the unitary conductance of the CITC,
single channel fluctuations were measured in the
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 patchclamp technique. The arrow indicates the application of the cold
stimulus (see ‘‘Materials and Methods’’). The holding potential was
249 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 6 SE of nine protoplasts.
Figure 2. Transient cold-induced increase in cytosolic Ca21 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 5
4) or room temperature bath solution (room temperature, n 5 6). Data
are means 6 SE.
Plant Physiol. Vol. 143, 2007
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Carpaneto et al.
that Ding and Pickard (1993a, 1993b) recorded a
mechanosensitive channel in onion (Allium cepa) epidermis protoplasts that had a peak of activity at 6°C.
To test our Arabidopsis mesophyll protoplast system
accordingly, we applied mechanical stretch to the
excised patches, but upon rapid cooling we could,
however, not detect any cold-activated stretch channels. But, in line with the experiments of Qi et al.
(2004), we identified a stretch-activated anion channel
at room temperature too (data not shown). The latter
channel was characterized by a reversal potential
following the Nernst potential for Cl2 and a single
channel conductance of approximately 15 pS. These
properties clearly distinguish the CITC from mechanosensitive channels.
CIPCs in Selected Arabidopsis Cold-Sensitive and Ion
Channel Mutants
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 239 mV. Spikes superimposing
the current trace result from voltage ramps (151 to 2109 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
490
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 promoterTluciferase 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 Ca21-entry pathways
(Meyerhoff et al., 2005). The dnd1 mutant (Clough et al.,
as shown in B. Cold-induced currents were characterized by outward
rectification and by a reversal voltage in this experiment of 27 mV.
Inset: When 150 mM of Cl2 in the pipette was substituted by 150 mM of
gluconate2, the reversal voltage did not change significantly (212 mV).
c, Control; r, recovery.
Plant Physiol. Vol. 143, 2007
Transient Cold-Induced Ion Channel
GORK is not cold activated, but outward currents
through this K1 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
Figure 5. Cation selectivity of the cold-activated current. A, Timedependent current transients induced by cold stimulation in bath solutions containing 10 mM Cs1 (top trace) and 100 mM Ca21 and 10 mM Cs1
(bottom trace). The holding potential was 249 mV. Note that in the
absence of external Ca21, 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 Cs1 (1)
and 100 mM Ca21 and 10 mM Cs1 (2). In this experiment, the reversal
voltage of (1) and (2) were 265 mV and 25 mV, respectively.
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 K1 outward rectifier activates. We have shown
that GORK, the Shaker-like K1-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,
Plant Physiol. Vol. 143, 2007
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 coldactivated 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 Cs1). 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 PCa:PCs
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 temperaturesensitive 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 coldinduced 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.
491
Carpaneto et al.
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 247 mV, and data were filtered at 500 Hz and acquired with a sampling
time of 500 ms. 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 kg21 with D-sorbitol. The measured reversal voltage in this condition was 212 mV. Asterisks in A were inserted in
the place of voltage ramps (omitted for the sake of clarity) from which Vrev was obtained.
Working with V. faba guard cells, Ilan et al. (1995)
explored the temperature sensitivity of voltageactivated inward and outward K1 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 voltagedependent 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 Ca21. Another
cation channel type was recognized when studying the electrical properties of the plasma membrane
492
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 depolarizationactivated 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.
Plant Physiol. Vol. 143, 2007
Transient Cold-Induced Ion Channel
MATERIALS AND METHODS
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 MV. 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 V. The free running membrane potential of mesophyll cells
was contemporarily recorded on a chart recorder and digitalized.
Simultaneous Membrane Potential and Ca21 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 mM 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-MV tip resistance filled with 300 mM KCl. The electrodes were connected
via an Ag/AgCl half-cell to a head stage (1 GV; 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 [Ca21]cyt increases were measured using a CCD camera Visiluxx
Imager (Visitron Systems GMBH).
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 kg21 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 29 mV; for the cesium gluconate internal solution and the external standard
solution, it was 217 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): PCa/PCs 5 1/4
([Cs]in exp[FV/RT] 2 [Cs]out)/[Ca]out (1 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 Ca21.
Supplemental Figure S2. Cold-induced potential changes in mesophyll
cells from selected Arabidopsis cold-sensitive and ion channel mutants.
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 kg21 with D-sorbitol. Released
protoplasts were filtered through a 100-mm nylon mesh and washed twice in
1 mM CaCl2 buffer (osmolarity of 400 mosmol kg21, pH 5.6).
Bioluminescence Measurements with Protoplasts
Stimulus-induced cytosolic Ca21 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 kg21 with mannitol in
the presence of 2.5 mM 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 kg21 with
mannitol. Cold stimulus was applied via a precooled syringe through a
luminometer port. To determine the relative luminescence L/Lmax, 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
Plant Physiol. Vol. 143, 2007
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 Ca21 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.
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