The Plant Journal (2004) 37, 578±588
doi: 10.1111/j.1365-313X.2003.01985.x
ABA depolarizes guard cells in intact plants, through a
transient activation of R- and S-type anion channels
M. Rob G. Roelfsemay, Victor Levchenkoy and Rainer Hedrich
Julius-von-Sachs-Institut fuÈr Biowissenschaften, Lehrstuhl fuÈr Molekulare P¯anzenphysiologie und Biophysik,
UniversitaÈt WuÈrzburg, Julius-von-Sachs-Platz 2, D-97082, Germany
Received 15 September 2003; revised 4 November 2003; accepted 11 November 2003.
For correspondence (fax ‡49 931 8886157; e-mail [email protected]).
y
Both authors contributed equally to this study.
Summary
During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces
transpiration. Stomatal closure is accompanied by large ion ¯uxes across the plasma membrane, carried by
K‡ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard
cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled
electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient
depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the
presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of
ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied
from one hormone application to the other. The transient depolarization correlated with the activation of
anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate
increase in plasma membrane conductance (DG < 5 nS), ABA predominantly activated voltageindependent (slow (S)-type) anion channels. During strong responses (DG > 5 nS), however, ABA
activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the
combined activation of these two channel types leads to the transient depolarization of guard cells. The
nature of this ABA response correlates with the transient extrusion of Cl from guard cells and a rapid but
con®ned reduction in stomatal aperture.
Keywords: abscisic acid (ABA), membrane potential, transient depolarization, guard cell, R-type and S-type
anion channel, intact plant.
Introduction
During periods of limiting water supply, plants reduce their
stomatal conductance, to decrease transpiration (Assmann
and Shimazaki, 1999; Schroeder et al., 2001). Stomatal
closure, however, also reduces CO2 uptake and thereby
limits carbon assimilation. To optimize the uptake of CO2
and the evaporation of H2O, plants have developed signalling pathways, which tightly control stomatal opening.
These pathways regulate the activity of ion transport proteins at the plasma membrane and tonoplast (Assmann and
Shimazaki, 1999; MacRobbie, 1998; Schroeder et al., 2001).
Changes in the activity of these transporters alter the ionic
composition of guard cells and thereby provide the driving
force for stomatal movement.
Recently, we developed a method to study plasma membrane transport of single guard cells in the intact plant
578
(Roelfsema et al., 2001). These cells responded to light
and CO2, with large membrane potential changes that
altered the direction of the K‡ ¯ux across the plasma
membrane (Roelfsema et al., 2001, 2002). Membrane
potential changes of this magnitude, induced by light
and CO2, had not been recorded with guard cells in epidermal strips or protoplasts thereof. Probably, the mechanical stress during isolation of epidermal strips or
protoplasts, in combination with loss of the natural environment, alters the responsiveness of guard cells to these
signals (Roelfsema and Hedrich, 2002).
In addition to the light- and CO2-signalling pathways
previously studied, plants are equipped with a droughtsensing system. Here, the phytohormone abscisic acid
(ABA) plays a central role (Assmann and Shimazaki,
ß 2004 Blackwell Publishing Ltd
ABA-activation of R- and S-type anion channels
1999; Schroeder et al., 2001), as mutants insensitive to ABA
(abi) tend to wilt (Koornneef et al., 1984; Roelfsema and
Prins, 1995). These abi mutants lack ABA-induced plasma
membrane responses recognized for wild-type guard cells
(Armstrong et al., 1995; Pei et al., 1997). In wild-type guard
cells, an effect of ABA on at least three types of ion channels
has been reported. ABA enhances the activity of outward K‡
channels (Blatt and Armstrong, 1993) and slow (S)-type
anion channels (Grabov et al., 1997; Pei et al., 1997), while
that of inward K‡ channels is reduced (Blatt and Armstrong,
1993; Lemtiri-Chlieh and MacRobbie, 1994; Schwartz et al.,
1994).
The sensitivity of stomata to ABA depends on other
signals, such as CO2 and indole-3-acetic acid (IAA)
(Raschke, 1987). This explains, to some extent, why all
guard cells do not display the same response to the stress
hormone. In guard cells, ABA can induce rises in cytoplasmic Ca2‡, but only a limited number of the cells display this
response (Allen et al., 1999; Gilroy et al., 1991; McAinsh
et al., 1990). In some experiments, ABA was even found to
turn spontaneous Ca2‡ oscillations off (KluÈsener et al.,
2002).
Although targets of ABA regulation have been recognized
for guard cells, the sequence of events leading to stomatal
closure still awaits a detailed analysis. Recordings with ionselective miniature electrodes at the guard cell wall showed
an increase of Cl extrusion starting a few minutes after
ABA application (Felle et al., 2000). The Cl concentration
peaks 15 min after stimulus onset and returns to prestimulus values within an hour. Tracer ¯ux experiments
with epidermal peels also showed that ABA temporarily
increases the ef¯ux of anions (MacRobbie, 1987).
Altogether, these data point to an ABA-induced transient
activation of anion channels in guard cells. So far, however,
such a transient response has not been measured for guard
cells in epidermal strips or protoplasts thereof.
Here, we attempt to bridge the gap between data from
transpiration measurements, ion ¯ux recordings and
changes in ion channel activity, by monitoring the ABA
response of single guard cells in their natural environment:
the intact plant. In line with ion ¯ux measurements, guard
cells transiently depolarized in response to ABA, because of
the activation of both rapid (R)-type and S-type anion
channels.
Results
ABA-induced stomatal closure and membrane
depolarization
Guard cells surround the stomatal pore and therefore can
be easily ABA stimulated via the leaf surface. The responsiveness of guard cells to ABA, applied via leaf surface
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
579
Figure 1. Effect of 10 mM ABA, applied via leaf cuticle perfusion, on stomatal
opening.
(a) Abaxial side of a V. faba leaf, an experimental solution (5 mM KCl, 5 mM
potassium citrate (pH 5.0), 0.1 mM CaCl2 and 0.1 mM MgCl2), ran between
the objective and the leaf surface (volume 0.3 ml) at ¯ow rate of
1.5 ml min 1. The leaf was illuminated with white light at a photon ¯ux
density of 500 mmol m 2 sec 1, projected from the adaxial side at an area of
20 mm diameter. At t ˆ 10 min (left ®gure), the experimental solution was
changed to one containing 10 mM ABA. Stomatal closure occurred during
the following 20 min (right ®gure) for the upper stoma, but not for the lower
stoma.
(b) Changes in stomatal aperture plotted against time, from the same
stomata as in (a) (*, upper stoma; , lower stoma). At t ˆ 10 min, the
solution on the cuticle was changed for one containing 10 mM ABA.
perfusion, was tested microscopically. Figure 1(a) depicts
the abaxial epidermis of a Vicia faba leaf, with an ABAresponsive stoma adjacent to an ABA-insensitive one. The
upper, ABA-responsive stoma started to close 8 min after
the introduction of the stress hormone and reached its
maximal closure within 20 min (Figure 1a,b). Out of 37
stomata tested, 14 closed upon exposure to 10 mM ABA.
The other 23 stomata did not respond to ABA, just as the
lower stoma in Figure 1(a). Based on this heterogeneity, it
can be assumed that at least 30% of the guard cells will
show an ABA-induced change in the electrical properties of
the plasma membrane.
Plasma membrane responses to ABA were monitored for
guard cells that were impaled with double-barrelled electrodes. Upon impalement, the membrane potential of these
580 M. Rob G. Roelfsema et al.
cells transiently depolarizes and reaches a new stable value
in 2±4 min (Roelfsema et al., 2001). After reaching a stable
membrane potential, 71 out of 85 cells could be classi®ed as
`depolarized'; these cells had an average membrane potential of 74 mV (SD 10). The other 14 cells were classi®ed as
`hyperpolarized' and had an average membrane potential
of 112 mV (SD 16). Note, however, that the recorded
membrane potentials may be affected by electrical leaks
caused by the impalement. These potential leaks are small
for hyperpolarized cells (see Experimental procedures),
but may have an impact on the membrane potential of
depolarized cells.
In line with the observation that some stomata do not
close in response to ABA, an ABA-induced change in
the membrane potential could not be observed in 40 cells,
2 of which were hyperpolarized before ABA application
(Figure 2a). In the other 45 cells, ABA depolarized the
Figure 2. ABA-induced changes in the electrical properties of the plasma
membrane of guard cells in intact V. faba plants.
(a±d) Guard cell membrane potentials before, during and after the application of 10 mM ABA, as indicated in the bar below the graphs. Four types of
responses could be distinguished: (a) Cells not responding to ABA; (b)
depolarized cells, further depolarizing after application of ABA; (c) depolarized cells, further depolarizing with ABA and transiently hyperpolarizing
after washing out ABA; and (d) hyperpolarized cells becoming depolarized
in the presence of ABA.
(e) Plasma membrane current of a guard cell clamped continuously at
100 mV. The guard cell was exposed to 10 mM ABA, as indicated by the
bar below the graph. ABA induced ®rst a transient increase of inward
current, followed by a steady increase of inward current at lower amplitude.
plasma membrane (Figure 2b±d). The amplitude of the
ABA-induced depolarization was variable and depended
on the membrane potential before application of the stress
hormone. Cells that could be classi®ed as `depolarized',
further depolarized upon exposure to ABA (Figure 2b,c).
The depolarization reached its maximal value (DEm ˆ
22 mV, SD 10) 5 min (SD 1) after application of the stimulus.
Following this transient, the membrane potential partially
recovered to a value 12 mV (SD 8) more positive than that
before application of ABA. A subsequent removal of ABA
from the perfusion solution caused a recovery of the membrane potential to a pre-stimulus value. Before reaching
the pre-stimulus value, however, a large percentage of
the `depolarized' cells showed an overshoot response
(Figure 2c). Guard cells that could be classi®ed as `hyperpolarized' (Roelfsema et al., 2001) had an average membrane potential of
112 mV (SD 16) and strongly
depolarized upon exposure to ABA (DEm ˆ 57 mV, SD 19,
Figure 2d). Again, the cells spontaneously re-polarized in
the presence of the hormone; however, a complete recovery of the membrane potential occurred only after the
removal of ABA. The plasma membrane responses thus
split into four groups: (i) depolarized or hyperpolarized nonresponsive cells; (ii) depolarized cells further depolarizing
with ABA; (iii) depolarized cells with an overshoot after
removal of ABA; and (iv) hyperpolarized cells with a strong
ABA-induced depolarization.
All ABA-responsive cells shared an initial transient depolarization. The most positive potential reached during this
depolarization was independent of the membrane potential
before hormone application (Figure 2b±d). The most depolarized potential was 52 mV (SD 12) and 55 mV (SD 11)
for initially depolarized or hyperpolarized cells, respectively. The underlying change in ion channel activity was
explored through voltage clamp studies. Guard cells were
exposed to the hormone, while their plasma membrane
was constantly clamped at 100 mV. At this potential, the
plasma membrane ion conductance was small, as voltagedependent K‡ channels were not active (Roelfsema et al.,
2001). ABA triggered a transient increase in inward current,
which reached a peak value approximately 5 min after
introduction of the hormone (Figure 2e). During prolonged
ABA stimulation, the inward current dropped again, but
remained larger than that before stimulus onset. This
response could be elicited by the stress hormone at concentrations as low as 1 mM (data not shown).
Transient changes in the activity of plasma membrane
ion channels
To study the ionic basis of the ABA-induced increase in
plasma membrane conductance, the membrane potential
of guard cells was clamped at regular intervals to a range of
test potentials (diamonds in Figure 3a). Before exposure to
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
ABA-activation of R- and S-type anion channels
581
Figure 4. ABA-responsive guard cell, in which the conductance of outward
rectifying channels remains unchanged.
(a) Membrane potential trace of a guard cell exposed to 10 mM ABA (dark
area of bar below the graph). Diamonds indicate the time points at which
voltage clamp protocols were applied.
(b) Current traces from the same cell as in (a), symbols correlate. Arrows
indicate the 0-nA level. Cells were clamped from a holding potential of
100 mV to potentials ranging from 180 to 0 mV with 20-mV increments.
Note that ABA does not affect the time-dependent outward currents. ABA
transiently increases instantaneous inward currents ( ) that slowly deactivate at potentials from 120 to 160 mV. The increase in instantaneous
current levels off (&) after prolonged exposure to ABA.
Figure 3. ABA induced changes in plasma membrane conductance.
(a) Membrane potential trace of a guard cell exposed to 10 mM ABA (dark
area of bar below the graph). Diamonds indicate the time points at which
voltage clamp protocols were applied.
(b) Current traces from the same cell as in (a), symbols correlate. Arrows
indicate the 0-nA level. Cells were clamped from a holding potential of
100 mV to potentials ranging from 180 to 0 mV with 20-mV increments.
Note that the introduction of ABA ( ) caused a dramatic increase of currents
measured directly after the capacity compensation peak; concurrently, timeactivated outward currents are reduced. The currents virtually recovered to
pre-stimulus values after a prolonged exposure to ABA (&).
(c) Instantaneous currents, sampled directly after termination of the capacity
compensation peak, plotted against the clamp voltage (symbols correspond
to (a)). Note that ABA triggered an increase of inward current at test
potentials negative of 40 mV ( ). The effect of ABA was reversed after
prolonged exposure (&). The removal of ABA resulted in a further decrease
of inward currents, at potentials negative of 60 mV (&).
(d) Steady-state currents, sampled at the end of the 2-sec test pulses, plotted
against the clamp voltage (symbols correspond to (a)). Note that ABA
caused a large shift of the zero current potential to more positive values.
ABA, the guard cell membrane conductance was dominated by inward and outward rectifying K‡-selective channels (Figure 3b, open circle; Roelfsema et al., 2001). In
contact with ABA, a dramatic change in the plasma membrane conductance properties was observed (Figure 3b,
closed circle). In this cell, ABA-stimulated ion channels that
activate instantaneously (Figure 3b,c) and inhibited timedependent outward rectifying K‡ channels (Figure 3b,d).
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
Upon prolonged hormone application, these conductance
changes were reversed (Figure 3b±d, closed squares), in
line with the transient depolarization (Figure 3a). After the
removal of ABA, the cell became transiently hyperpolarized
(Figure 3a), which correlated with an increase of outward
current recorded at membrane potentials negative of
60 mV (Figure 3c, open squares).
The effect of ABA on outward rectifying K‡ channels
varied; in 14 out of 27 cells, the stress hormone decreased
the conductance of these channels, as in Figure 3. However,
in 10 other cells depolarizing in response to ABA
(Figure 4a), a less than 10% change in current, mediated
by outward K‡ channels, was found (Figure 4b). In contrast
to the variable effect of ABA on outward K‡ channels, the
stress hormone stimulated instantaneously activating
channels in all transiently depolarizing cells (Figures 3b,c
and 4b). In Figure 4(b), these channels slowly deactivate at
potentials ranging from 120 to 160 mV, a feature reminiscent of S-type anion channels (Linder and Raschke, 1992;
Schroeder and Hagiwara, 1989).
Based on the voltage-dependent activation and deactivation, the inward current triggered by ABA is most likely
conducted by anion channels. However, non-selective
cation channels (Demidchik et al., 2002) or voltage-independent K‡ channels (Marten et al., 1999) could also contribute to the inward conductance. To exclude the latter
582 M. Rob G. Roelfsema et al.
40 mV (Figure 5b). This behaviour, together with the
Nernst potential of K‡ 73 mV, indicates that the inward
current is not carried by K‡-selective channels. The current
may thus be conducted by anion- or non-selective channels, which are not affected by Cs‡ and Ba2‡.
The reversal potential of ABA-stimulated channels was
determined using electrodes ®lled with 300 mM CsCl and a
perfusion solution with KCl. Although a low activity of
inward K‡ channels was still recorded at these conditions
(data not shown), it ensured that K‡ is the main cation in the
guard cell wall (Roelfsema and Hedrich, 2002). At a holding
potential of 100 mV, ABA supply induced a transient
inward current (Figure 5c). Before and after ABA application, the reversal potential was determined with fast (2 sec)
voltage ramps from 180 to 60 mV (Figure 5d). During the
ABA response, the reversal potential shifted from 30 to
20 mV, the latter potential being close to the Nernst potential of Cl . If the ABA-induced current was carried by nonselective K‡-conducting (Demidchik et al., 2002) cation
channels, a shift of the reversal potential to values negative
of 30 mV would have been expected. Instead, the reversal
potential shifted to more positive values, showing that the
ABA-induced current is predominantly carried by anion
channels.
ABA activates both R- and S-type anion channels
Figure 5. Ionic nature of ABA-induced inward current.
(a) Current traces of a guard cell impaled with a double-barrelled electrode
containing 300 mM CsCl and exposed to Ba2‡ via the perfusion solution
¯owing over the leaf. The guard cell was clamped stepwise from a holding
potential of 100 mV to test potentials ranging from 180 to 0 mV with 20mV increments. Arrow indicates the 0-nA level. Note that the time-dependent outward as well as the inward rectifying K‡ channels are blocked.
(b) Current traces of the same guard cell as in (a), exposed to a short pulse
(40 sec) of 10 mM ABA as indicated by the dark area in the bar below the
graphs. ABA induced a transient inward current at a holding potential of
100 mV (left graph) as well as at a holding potential of 40 mV (right
graph).
(c) Current trace of a guard cell impaled with an electrode containing
300 mM CsCl, exposed to a perfusion solution with 5 mM KCl and clamped
to 100 mV. In contact with 10 mM ABA (dark area in bar below the graph),
the inward current across the plasma membrane transiently increased.
(d) Current±voltage relations of the same guard cells as in (c), determined
with a ramp of 2 sec, from 180 to 60 mV, before and during the ABA
response (Symbols correlate to (c)). The clamp protocol was applied from a
holding potential of 100 mV. Note that during the ABA response the
reversal potential shifts from 30 to 20 mV.
possibility, we ®lled electrodes with 300 mM CsCl and used
a perfusion solution containing BaCl2. The presence of
these K‡-channel blockers eliminated currents carried by
inward and outward rectifying K‡ channels (Figure 5a).
Under these conditions, ABA still elicited a transient inward
current at a holding potential of 100 mV as well as at
ABA activation of anion channels was further studied, using
a BaCl2-containing perfusion solution and KCl in the microelectrode. Ba2‡ blocked the inward rectifying K‡ channels
and reduced the current carried by outward rectifying K‡
channels (Figure 6b, closed circle; Schroeder et al., 1987).
Ba2‡ did not affect the time course of anion channel activation, as ABA stimulation peaked 5 min (SD 1, n ˆ 21) after
stimulus onset, just as in the absence of Ba2‡. Under both
conditions, guard cell anion channels could be repetitively
activated by ABA, each exposure resulting in an increase of
inward current (Figure 6a). Although ABA repetitively activated inward currents with a similar time course, the amplitude levelled off with each exposure. The increase of inward
current was accompanied by an increase in conductance of
instantaneously activating channels (Figure 6b, closed circle and square). The properties of these channels differed
from one ABA exposure to another. During the ®rst exposure, the instantaneous activating channels had a peak
conductance at 100 mV (Figure 6c), a feature characteristic for R-type anion channels (Dietrich and Hedrich, 1998;
Hedrich et al., 1990; Keller et al., 1989; Kolb et al., 1995;
Schroeder and Keller, 1992). During the second (data not
shown) and third (Figure 6d) exposure to ABA, the peak
conductance at 100 mV decreased. The remaining current
was reminiscent of S-type anion channels (Linder and
Raschke, 1992; Schroeder and Hagiwara, 1989; Schroeder
and Keller, 1992), as it displayed a more linear current
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
ABA-activation of R- and S-type anion channels
Figure 6. Repetitive activation of anion channels by ABA.
(a) Plasma membrane current of a guard cell clamped at 100 mV and
exposed three times to 10 mM ABA (dark areas in the bar below the graph).
The membrane was clamped to a range of de®ned test potentials at regular
intervals, indicated by diamonds. The recording was carried out with an
experimental solution containing 5 mM BaCl2 to inhibit outward and inward
rectifying K‡ channels.
(b) Current traces of the same cell as in (a), symbols correlate. Arrows
indicate the 0-nA level. The plasma membrane was clamped from 100 mV
to test potentials ranging from 180 to 0 mV with 20-mV increments; for
clarity, only traces of 180, 120, 60 and 0 mV are shown. Note that the
presence of Ba2‡ reduced the currents carried by inward and outward
rectifying K‡ channels (*). The introduction of ABA ( and &) caused an
increase of instantaneously activating currents.
(c) Instantaneous currents, sampled after termination of the capacity compensation peak, plotted against the clamp voltage. Currents were measured
before (*) and during ( ) the ®rst application of ABA.
(d) Same as in (c), but now for current sampled before (&) and during (&)
the third ABA application. Note that the inward current has a peak value
at 100 mV during the ®rst ABA application, which was absent during the
third application.
voltage relation and slowly deactivates at 180 mV
(Figure 6b, open square).
The relative contribution of R- and S-type anion channels
to the ABA-induced increase in conductance was estimated. Instantaneous current±voltage relations were ®tted
with an equation (see Experimental procedures), which
sums the current carried by R-type and S-type anion channels. This analysis revealed that in 6 out of 13 responses, Rtype anion channels dominated the anion conductance. In
the latter experiments, the entrance of Ba2‡, via Ca2‡- and
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
583
Ba2‡-permeable channels (Demidchik et al., 2002; Hamilton
et al., 2000; Pei et al., 2000) cannot be excluded, and thus
Ba2‡ may have altered the activation state of the two anion
channel types (Hedrich et al., 1990; Schroeder and
Hagiwara, 1989). The ABA effect was therefore analysed
in the absence of Ba2‡ as well. Under these conditions, the
distinct properties of both anion channel types were apparent in response to voltage steps from 100 to 180 mV
(Figure 7a,c). In cells dominated by S-type anion channels,
the current instantaneously increased at
180 mV
(Figure 7a), while an instantaneous decrease was observed
for cells with R-type anion channels (Figure 7c). The latter
current change is because of a rapid deactivation of anion
channels (within 40 msec) (Kolb et al., 1995), after stepping
to 180 mV. Because of the distinct gating properties of
both channel types, cells dominated by S-type anion channels displayed a linear instantaneous current±voltage relation (Figure 7b), while cells dominated by R-type anion
channels exhibited a pronounced peak current (Figure 7d).
The relative contribution of both channel types to the conductance increase induced by ABA was resolved using
Equation 1 (see Experimental procedures). The anion channel type dominating the ABA-induced conductance
increase depended on the magnitude of the response,
which was de®ned as the total increase in anion conductance. In cells with a small response (DGinst < 2.5 nS),
S-type channels formed the dominant anion conductance
(Figure 7e). At intermediate responses (2.5 nS < DGinst
< 5 nS), S-type channels were dominant in the majority
of cells, but in other cells, R-type channels formed the
dominant anion conductance (Figure 7e). Finally, an equal
number of cells with R- or S-type anion channels were
found for large ABA responses (DG > 5 nS, Figure 7e).
Guard cell responses to short of ABA pulses
To test if the activation of anion channels required the
continuous presence of ABA, we applied pulses of the
phytohormone shorter than 150 sec. Figure 8(a) depicts
the current trace of a cell clamped at 100 mV and exposed
to 50 mM ABA for 60 sec. Following the ABA pulse, the
inward current transiently increased, reaching a peak value
approximately 4.5 min after the onset of hormone application. The same time course was determined when
responses to short ABA pulses were averaged (Figure 8b).
The chain of events triggered by ABA pulses thus showed
kinetics similar to those induced by continuous ABA supply
(Figure 2e). Upon application of an ABA pulse, however, a
small outward current followed the initial transient in
inward current (Figure 8b), while an inward current
remained with prolonged ABA supply (Figure 2e).
Finally, we examined if successive ABA pulses could
reproducibly induce current transients in guard cells. The
cell displayed in Figure 8(c) was exposed to 50 mM ABA for
584 M. Rob G. Roelfsema et al.
Figure 8. Guard cells responses to short pulses of ABA.
(a) Plasma membrane current of a guard cell constantly clamped to
100 mV in a perfusion solution containing BaCl2; 50 mM ABA was applied
for 60 sec, as indicated by the bar below the trace. The pulse of ABA induced
a transient increase in inward current peaking 4.5 min after start of the ABA
application.
(b) Average change in the plasma membrane current of guard cells exposed
to short (30±150 sec) pulses of ABA (10±50 mM). The average was calculated
for eight experiments, in which ABA was applied at t ˆ 0; error bars
represent SE. The membrane current was normalized for the peak current
(Im, peak ˆ 1). ABA induced a transient change in current, which peaked
between 4 and 5 min after start of hormone application. Note that average
current change becomes positive relative to the pre-stimulus value 10 min
after the ABA application.
(c) Plasma membrane current of a single guard cell (different from the cell
shown in (a)) exposed two times to 50 mM ABA for 30 sec, as indicated by the
bars below the trace. The current trace is interrupted by 16 min. Note that
both ABA pulses triggered current changes with a similar time coarse, but
with an increased amplitude during the second application.
Figure 7. Contribution of R- and S-type anion channels to the ABA-induced
increase in plasma membrane conductance.
(a) Current trace of guard cell exposed to ABA in an experimental solution
containing 5 mM KCl. The plasma membrane was clamped from a holding
potential of 100 mV to a test potential of 180 mV, which caused an
instantaneous increase of inward current, followed by a time-dependent
activation of inward rectifying K‡ channels.
(b) ABA-induced change in plasma membrane current, plotted against the
clamp voltage for the same cell as in (a). The linear current±voltage relation
indicates that the instantaneous current is carried by S-type anion channels.
(c) As in (a), but now the inward current instantaneously decreased at
180 mV followed by a slow activation of inward rectifying K‡ channels.
(d) ABA-induced change in plasma membrane current, plotted against the
clamp voltage for the same cell as in (c). Inward currents peak around
120 mV, indicating that the current in this cell was mainly carried by R-type
anion channels. The instantaneous current±voltage plots of (b) and (d) were
®tted with the following equation: Im ˆ …Vm Vrev † …Gslow ‡ Grapid =
…1 ‡ e…dF …Vhalf Vm †=RT † †† , where Gslow and Grapid represent the conductivity
of S- and R-type anion channels, Vhalf and d are the half maximal activation
potential and the gating charge of rapid anion channels, and F, R and T have
their usual meaning.
(e) Number of cells, which had either S-type (Gslow) or R-type anion channels
(Grapid) as the dominant anion channel, binned for ®ve classes of ABAinduced increases in plasma membrane conductance (Gtotal). The values of
Gslow and Grapid were determined from 34 experiments of which the instantaneous current±voltage plots were ®tted with the equation above. Note that
S-type anion channels are dominant in cells with a small response to ABA,
while cells with a larger ABA response also can have R-type anion channels
as the dominant anion conductance.
30 sec; this pulsed ABA treatment was repeated after
30 min. Both ABA applications resulted in current transients with a similar time course; however, the amplitude
increased during the second ABA application (Figure 8c).
The `run up' in the latter experiment, together with the `run
down' in Figure 6, shows that the responsiveness of a
single guard cell can change in time.
Discussion
Heterogeneity in guard cell responsiveness to ABA
Exogenously applied ABA induced stomatal closure, but
stomata within a leaf did not respond uniformly. Even
neighbouring stomata, sometimes, were found to differ
in their sensitivity towards the stress hormone (Figure 1).
A variable ABA sensitivity has already been reported for
stomata in epidermal strips (Raschke, 1987). In our studies,
the variation in responsiveness did not correlate with the
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
ABA-activation of R- and S-type anion channels
localization of the stomata within the leaf, indicating that
this feature is not strictly coupled to the well-documented
patchiness in stomatal opening (Mott and Buckley, 2000).
Mutant analysis showed that the sensitivity to ABA can
change more than 10-fold with a single gene mutation
(Hugouvieux et al., 2001; Klein et al., 2003; Pei et al.,
1998; Roelfsema and Prins, 1995). Natural variation in the
transcription rate of such genes or in the phosphorylation
state of the encoded proteins may underlie variations in
guard cell sensitivity to ABA.
In agreement with the variations in hormone sensitivity
of stomata, some guard cells depolarized upon exposure to
ABA, while the membrane potential of others remained
unchanged. Similar results have been obtained for ABAinduced Ca2‡ signals in guard cells (Allen et al., 1999; Gilroy
et al., 1991; McAinsh et al., 1990). A variable hormone
sensitivity of guard cells could be bene®cial for plants, as
it provides a mechanism for ®ne-tuning the ABA-mediated
drought response. At a moderate increase in the ABA level,
only a small population of stomata will close, while higher
hormone concentrations sequentially will close other populations. This all-or-none response may be bene®cial for the
plant, as it could prevent oscillations in stomatal aperture,
which hamper an optimal CO2 supply for photosynthesis.
Such oscillations occur at small stomatal apertures (Kaiser
and Kappen, 2001) and thus would be inevitable if all
stomata reduce their aperture in response to ABA. When
instead some stomata close while others remain open,
oscillations will be prevented and stomata can still respond
to CO2 even at high concentrations of ABA (Leymarie et al.,
1998).
Activation of R- and S-type anion channels
In all ABA-responsive cells, the stress hormone increased
the activity of ion channels that activated instantaneously.
Based on the changes of the instantaneous current±voltage
relation, the channels activated by ABA were identi®ed as
R- and S-type anion channels. Both channel types facilitate
the ef¯ux of anions and depolarize the plasma membrane
(Keller et al., 1989; Schroeder and Hagiwara, 1989). Previous reports on guard cells in isolated epidermal strips and
protoplasts thereof revealed that ABA increases the conductance of S-type anion channels in guard cells of
Arabidopsis thaliana (Pei et al., 1997) and Nicotiana
benthamiana (Grabov et al., 1997). An effect of ABA on
R-type anion channels, however, had not been recognized
before.
R- and S-type anion channels have been described for
guard cells of V. faba (Keller et al., 1989; Schroeder and
Hagiwara, 1989; Schroeder and Keller, 1992), as well as for
A. thaliana (Pei et al., 1997, 2000), indicating that both
channel types are conserved within the plant kingdom. In
V. faba, the properties of R- and S-type anion channels are
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
585
similar with respect to their selectivity and single-channel
conductance, which led to the hypothesis that both channels represent different gating modes of a single protein
(Dietrich and Hedrich, 1994). Both anion channel types
however display an obvious difference in gating characteristics and voltage dependence (Linder and Raschke, 1992;
Schroeder and Keller, 1992). The open probability of R-type
anion channels strongly decreases at membrane potentials
negative of 100 mV, a feature not found for S-type channels. Guard cells in intact plants can have membrane
potentials more negative than 100 mV (Roelfsema et al.,
2001). In the absence of gating modi®ers, such as extracellular anions (Dietrich and Hedrich, 1998), R-type channels, therefore, are not capable of inducing an initial
depolarization. However, once a guard cell becomes depolarized, the activation of R-type channels can boost stomatal closure. We found that guard cells displaying a
moderate response to ABA predominantly activated S-type
anion channels, while cells with a large response showed
the activity of R-type anion channels, too. This suggests a
role for R-type anion channels in fast ABA-induced stomatal
closure.
Regulation of voltage-dependent K‡ channels
Guard cells in intact plants apparently respond to ABA with
the transient activation of two types of anion channels. As a
result, the plasma membrane depolarizes and the activity of
K‡ channels is altered through voltage-dependent regulation. In addition, ABA can alter the maximum conductance
of voltage-dependent K‡ channels. Previously, ABA has
been shown to increase the activity of outward rectifying
K‡ channels (Blatt and Armstrong, 1993; Lemtiri-Chlieh and
MacRobbie, 1994), but this response was not observed in
patch clamp experiments by others (Schwartz et al., 1994).
In intact plants, only few cells showed a conductance
increase of outward K‡ channels, while in the majority of
cells, the conductance remained unchanged or decreased.
The ABA stimulation of outward K‡ channels in epidermal
strips depends on an alkalinization of the cytoplasm (Blatt
and Armstrong, 1993). In intact plants, such an ABA-dependent cytoplasmic pH-change may thus not occur.
ABA also inhibits inward rectifying K‡ channels of guard
cells in epidermal strips (Blatt and Armstrong, 1993) and
protoplasts thereof (Lemtiri-Chlieh and MacRobbie, 1994;
Schwartz et al., 1994). For guard cells in intact plants, the
effect of ABA on these channels could not be precisely
determined because of overlap with currents carried by
S-type anion channels. However, if ABA would have inhibited inward K‡ channels, hyperpolarized cells would have
become more hyperpolarized (Roelfsema and Prins, 1998).
For guard cells in intact plants (Figure 2d), this was not
observed; instead, ABA depolarized guard cells to potentials where inward K‡ channels are inactive.
586 M. Rob G. Roelfsema et al.
Mutants of A. thaliana lacking the voltage-dependent
inward K‡ channel Arabidopsis thaliana 1 (KAT1; Szyroki
et al., 2001), or the outward K‡ channel guard cell outward
rectifying K‡ channel (GORK; Hosy et al., 2003) display
stomatal movements similar to those of wild-type plants.
The mutants lacking GORK are completely devoid of timeactivated outward K‡ channels, but still close in response to
darkness and ABA, although the latter response is slowed
down to some extent (Hosy et al., 2003). Apparently,
changes in the conductance of voltage-dependent K‡ channels only have a small effect on stomatal movement. The
activation of anion channels however depolarizes guard
cells and can alter the direction of K‡ transport across
the plasma membrane. This leads to the conclusion that
anion channels represent a prime target for ABA action in
guard cells.
Transient efflux of Cl
The transient ABA activation of anion channels is well in
agreement with ion-selective electrode recordings of the
apoplastic Cl concentration in intact leaves (Felle et al.,
2000). Furthermore, a temporarily increase of the guard cell
Cl conductance was already predicted based on tracer ¯ux
experiments with epidermal strips (MacRobbie, 1981).
Apparently, the stress hormone causes a large, but transient, ef¯ux of anions followed by a small but steady extrusion. This will cause rapid reduction in the stomatal
aperture (as shown in Figure 1b), but does not cause complete stomatal closure. The following low, but persisting,
activation of anion channels may prevent re-opening of the
stomata. An incomplete stomatal closure allows a rapid reopening of the stomata when the ABA level in the leaf drops
again (Cummins et al., 1971). In contrast, a large and steady
activation of anion channels would have resulted in a
complete loss of guard cell turgor and in turn complete
stomatal closure. In the latter situation, re-opening of stomata would be a tardy process, as the stomata would have
to overcome the `Spannungsphase' before they start opening again (Sharpe et al., 1987).
Experimental procedures
Guard cell measurements on intact plants
Broad bean (V. faba L. cv. GruÈnkernige Hangdown, Gebag, Hannover, Germany) plants were grown in a green house. A leaf of a 4±
6-week-old plant was mounted with the adaxial side to a Plexiglas
holder in the focal plane of an upright microscope (Axioskop 2FS,
Carl Zeiss, GoÈttingen, Germany). The cells were visualized with a
water immersion objective (Achroplan 40/0.80 W, Carl Zeiss).
The solution between the objective and the leaf surface (volume
0.3 ml) was constantly exchanged at ¯ow rate of 1.5 ml min 1. The
standard experimental solution contained 5 mM KCl, 5 mM potassium citrate (pH 5.0), 0.1 mM CaCl2 and 0.1 mM MgCl2; where
indicated 5 mM BaCl2 was added instead of 5 mM KCl, () ABA
was obtained from Lancaster (Newgate, UK) and added at a concentrations ranging from 1 to 50 mM. The leaves were illuminated,
at an area with a diameter of 20 mm, by the microscope lamp
(HAL 12 V/100 W, Carl Zeiss) at a photon ¯ux density of
500 mmol m 2 sec 1 at the adaxial side, which corresponds to a
density of 20 mmol m 2 sec 1 at the abaxial side of the leaf.
Guard cells were impaled with double-barreled electrodes
pulled from borosilicate glass capillaries and ®lled with 300 mM
KCl or 300 mM CsC1, as described previously by Roelfsema et al.
(2001). The reference electrode (300 mM KCl agarose bridge) was
placed in the solution on the leaf surface. A potential difference
may exist between the guard cell wall and perfusion solution; this
potential was recorded with blunt electrodes brought in contact
with the guard cell wall (Roelfsema et al., 2001). An average surface potential of 4 mV was measured; this potential was not
affected by ABA. Both barrels of the intracellular electrode were
connected via Ag/AgCl half cells to a microelectrode ampli®er (VF102, Bio-Logic, Claix, France); the membrane potential was
clamped to test voltages using a differential ampli®er (CA-100,
Bio-Logic). The data were ®ltered at 300 Hz and sampled at 1 kHz
using the same system and software as described earlier by
Roelfsema et al. (2001).
Although the approach used here is far less invasive than the
measurements carried out previously with epidermal strips or
turgor-free protoplasts, the impalement of a single cell introduces
electrical leaks that may affect the membrane potential. First,
microelectrode ampli®ers only have a con®ned resistance and
conduct a leak current. The ampli®ers used here have a resistance
of 100 GV and will cause a leak current of 2 pA at 100 mV. The
depolarization resulting from this current will depend on the
resistance of the plasma membrane. For V. faba guard cells in
intact plants, the highest resistance is found in the voltage range
from 100 to 70 mV; here, a value of approximately 5 GV was
determined (Roelfsema et al., 2001). A leak current of 2 pA
through the ampli®ers, thus, will cause a maximum depolarization
of 10 mV. Second, a leak current may be conducted because of an
imperfect connection between glass electrode and the plasma
membrane. Such a leak conductance will be non-selective and
conduct current over the whole voltage range. It, therefore, cannot
be distinguished from voltage-independent ion channels and will
depolarize the membrane. Note, however, that a large percentage
of cells became hyperpolarized after the removal of ABA
(Figure 2c,d). The conductance of the voltage-independent
channels and leaks is small in hyperpolarized cells (approximately
200 pS, Roelfsema et al., 2001), we therefore conclude that, in
these cells, the connection between the electrode and plasma
membrane was tight. However, a number of cells remained
depolarized after ABA washout; for the latter cells, the possibility
of an impalement-induced leak conductance cannot be excluded.
Numerical analysis
Instantaneous current±voltage relations were ®tted with SIGMA2000 (SPSS Science, Chicago, IL, USA) using the following
equation:
PLOT
Im ˆ …Vm
Vrev † …Gslow ‡ Grapid =…1 ‡ e…dF …Vhalf
Vm †=RT †
††
…1†
where Im is the membrane current, Vm is the membrane potential,
Vrev is the reversal potential, Gslow is the voltage-independent
conductance, Grapid is the voltage-dependent conductance, d is
the gating charge, F is the Faraday constant, Vhalf is the half
maximal activation potential, R is the gas constant and T is the
temperature. Current±voltage relations were ®tted, assuming that
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
ABA-activation of R- and S-type anion channels
Erev ˆ 0 mV and using the following constraints: Gslow > 0;
Grapid > 0; 150 mV < Vhalf < 50 mV and 1 < d < 5. Only instantaneous current±voltage relations, which could be ®tted with a
regression coef®cient larger than 0.90, were used for further
analysis.
Acknowledgements
We thank R. Steinmeyer (University of WuÈrzburg) for help with the
analysis, P. Dietrich (University of WuÈrzburg) and D. Sanders
(University of York) for their helpful discussions. This research
was supported by grants of the Deutsche Forschungsgemeinschaft
and the KoÈrber award to R.H.
Notes added in proof
During the reviewing process, a paper has appeared, which con®rms part of our observations: Raschke, K., Shabahang, M. and
Wolf, R. (2003) The slow and the quick anion conductance in whole
guard cells: their voltage-dependent alternation, and the modulation of their activities by abscisic acid and CO2. Planta, 217,
639±650.
This paper is dedicated to Hidde B.A. Prins.
References
Allen, G.J., Kuchitsu, K., Chu, S.P., Murata, Y. and Schroeder, J.I.
(1999) Arabidopsis abi1-1 and abi2-1 phosphatase mutations
reduce abscisic acid-induced cytoplasmic calcium rises in guard
cells. Plant Cell, 11, 1785±1798.
Armstrong, F., Leung, J., Grabov, A., Brearley, J., Giraudat, J. and
Blatt, M.R. (1995) Sensitivity to abscisic acid of guard-cell K‡
channels is suppressed by abi1-1, a mutant Arabidopsis gene
encoding a putative protein phosphatase. Proc. Natl. Acad. Sci.
USA, 92, 9520±9524.
Assmann, S.M. and Shimazaki, K.I. (1999) The multisensory guard
cell. Stomatal responses to blue light and abscisic acid. Plant
Physiol. 119, 809±815.
Blatt, M.R. and Armstrong, F. (1993) Potassium channels of stomatal guard cells: abscisic acid-evoked control of the outward
recti®er mediated by cytoplasmic pH. Planta, 191, 330±341.
Cummins, W.R., Kende, H. and Raschke, K. (1971) Speci®city and
reversibility of the rapid stomatal response to abscisic acid.
Planta, 99, 347±351.
Demidchik, V., Davenport, R.J. and Tester, M. (2002) Nonselective
cation channels in plants. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 53, 67±107.
Dietrich, P. and Hedrich, R. (1994) Interconversion of fast and slow
gating modes of GCAC1, a guard cell anion channel. Planta, 195,
301±304.
Dietrich, P. and Hedrich, R. (1998) Anions permeate and gate
GCAC1, a voltage-dependent guard cell anion channel. Plant
J. 15, 479±487.
Felle, H.H., Hanstein, S., Steinmeyer, R. and Hedrich, R. (2000)
Dynamics of ionic activities in the apoplast of the sub-stomatal
cavity of intact Vicia faba leaves during stomatal closure evoked
by ABA and darkness. Plant J. 24, 297±304.
Gilroy, S., Fricker, M.D., Read, N.D. and Trewavas, A.J. (1991) Role
of calcium in signal transduction of Commelina guard cells.
Plant Cell, 3, 333±344.
Grabov, A., Leung, J., Giraudat, J. and Blatt, M.R. (1997) Alteration
of anion channel kinetics in wild-type and abi1-1 transgenic
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588
587
Nicotiana benthamiana guard cells by abscisic acid. Plant J.
12, 203±213.
Hamilton, D.W., Hills, A., KoÈhler, B. and Blatt, M.R. (2000) Ca2‡
channels at the plasma membrane of stomatal guard cells are
activated by hyperpolarization and abscisic acid. Proc. Natl.
Acad. Sci. USA, 97, 4967±4972.
Hedrich, R., Busch, H. and Raschke, K. (1990) Calcium ion and
nucleotide dependent regulation of voltage dependent anion
channels in the plasma membrane of guard cells. EMBO J. 9,
3889±3892.
Hosy, E., Vavasseur, A., Mouline, K. et al. (2003) The Arabidopsis
outward K‡ channel GORK is involved in regulation of stomatal
movements and plant transpiration. Proc. Natl. Acad. Sci. USA,
100, 5549±5554.
Hugouvieux, V., Kwak, J.M. and Schroeder, J.I. (2001) An mRNA
cap binding protein, ABH1, modulates early abscisic acid signal
transduction in Arabidopsis. Cell, 106, 477±487.
Kaiser, H. and Kappen, L. (2001) Stomatal oscillations at small
apertures: indications for a fundamental insuf®ciency of stomatal feedback-control inherent in the stomatal turgor mechanism.
J. Exp. Bot. 52, 1303±1313.
Keller, B.U., Hedrich, R. and Raschke, K. (1989) Voltage-dependent
anion channels in the plasma membrane of guard cells. Nature,
341, 450±453.
KluÈsener, B., Young, J.J., Murata, Y., Allen, G.J., Mori, I.C., Hugouvieux, V. and Schroeder, J.I. (2002) Convergence of calcium
signalling pathways of pathogenic elicitors and abscisic acid
in Arabidopsis guard cells. Plant Physiol. 130, 2152±2163.
Klein, M., Perfus-Barbeoch, L., Frelet, A., Geadeke, N., Reinhardt,
D., Mueller-Roeber, B., Martinoia, E. and Forestier, C. (2003) The
plant multidrug resistance ABC transporter At MRP5 is involved
in guard cell hormone signalling and water use. Plant J. 33,
119±129.
Kolb, H.A., Marten, I. and Hedrich, R. (1995) Hodgkin-Huxley analysis of a GCAC1 anion channel in the plasma membrane of
guard cells. J. Membr. Biol. 146, 273±282.
Koornneef, M., Reuling, G. and Karssen, C.M. (1984) The isolation
and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana. Physiol. Plant. 61, 377±383.
Lemtiri-Chlieh, F. and MacRobbie, E.A.C. (1994) Role of calcium in
the modulation of Vicia guard cell potassium channels by abscisic acid: a patch-clamp study. J. Membr. Biol. 137, 99±107.
Leymarie, J., LasceÁve, G. and Vavasseur, A. (1998) Interaction of
stomatal responses to ABA and CO2 in Arabidopsis thaliana.
Aust. J. Plant Physiol. 25, 785±791.
Linder, B. and Raschke, K. (1992) A slow anion channel in guard
cells, activating at large hyperpolarzation, may be principal for
stomatal closing. FEBS Lett. 313, 27±30.
MacRobbie, E.A.C. (1981) Effects of ABA in `isolated' guard cells of
Commelina communis L. J. Exp. Bot. 32, 563±572.
MacRobbie, E.A.C. (1987) Ionic relations of guard cells. In Stomatal
Function (Zeiger, E., Cowan, G.D. and Farquhar, I.R., eds). Stanford, CA: Stanford University Press, pp. 125±162.
MacRobbie, E.A.C. (1998) Signal transduction and ion channels in
guard cells. Philos. Trans. R. Soc. Lond. B, 353, 1475±1488.
Marten, I., Hoth, S., Deeken, R., Ache, P., Ketchum, K.A., Hoshi, T.
and Hedrich, R. (1999) AKT3, a phloem-localized K‡ channel, is
blocked by protons. Proc. Natl. Acad. Sci. USA, 96, 7581±7586.
McAinsh, M.R., Brownlee, C. and Hetherington, A.M. (1990) Abscisic acid-induced elevation of guard cell cytosolic Ca2‡ precedes
stomatal closure. Nature, 343, 186±188.
Mott, K.A. and Buckley, T.N. (2000) Patchy stomatal conductance:
emergent collective behaviour of stomata. Trends Plant Sci. 5,
258±262.
588 M. Rob G. Roelfsema et al.
Pei, Z.M., Kuchitsu, K., Ward, J.M., Schwarz, M. and Schroeder, J.I.
(1997) Differential abscisic acid regulation of guard cell slow
anion channels in Arabidopsis wild-type and abi1 and abi2
mutants. Plant Cell, 9, 409±423.
Pei, Z.M., Ghassemian, M., Kwak, C.M., McCourt, P. and Schroeder,
J.I. (1998) Role of farnesyltransferase in ABA regulation of guard
cell anion channels and plant water loss. Science, 282, 287±290.
Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen,
G.J., Grill, E. and Schroeder, J.I. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in
guard cells. Nature, 406, 731±734.
Raschke, K. (1987) Action of abscisic acid on guard cells. In Stomatal Function (Zeiger, E., Farquhar, G.D. and Cowan, I.R., eds).
Stanford, CA: Stanford University Press, pp. 253±279.
Roelfsema, M.R.G. and Hedrich, R. (2002) Studying guard cells in
the intact plant: modulation of stomatal movement by apoplastic factors. New Phytol. 153, 425±431.
Roelfsema, M.R.G. and Prins, H.B.A. (1995) Effect of abscisic acid
on stomatal opening in isolated epidermal strips of abi mutants
of Arabidopsis thaliana. Physiol. Plant. 95, 373±378.
Roelfsema, M.R.G. and Prins, H.B.A. (1998) The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced
by apoplastic acidi®cation. Planta, 205, 100±112.
Roelfsema, M.R.G., Steinmeyer, R., Staal, M. and Hedrich, R.
(2001) Single guard cell recordings in intact plants: light-induced
hyperpolarization of the plasma membrane. Plant J. 26, 1±13.
Roelfsema, M.R.G., Hanstein, S., Felle, H.H. and Hedrich, R. (2002)
CO2 provides an intermediate link in the red light response of
guard cells. Plant J. 32, 65±75.
Schroeder, J.I. and Hagiwara, S. (1989) Cytosolic calcium regulates
ion channels in the plasma membrane of Vicia faba guard cells.
Nature, 338, 427±430.
Schroeder, J.I. and Keller, B.U. (1992) Two types of anion channel
currents in guard cells with distinct voltage regulation. Proc.
Natl. Acad. Sci. USA, 89, 5025±5029.
Schroeder, J.I., Raschke, K. and Neher, E. (1987) Voltage dependence of K‡ channels in guard-cell protoplasts. Proc. Natl. Acad.
Sci. USA, 84, 4108±4112.
Schroeder, J.I., Allen, G.J., Hugouvieux, V., Kwak, J.M. and Waren,
D. (2001) Guard cell signal transduction. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 627±658.
Schwartz, A., Wu, W.H., Tucker, E.B. and Assmann, S.M. (1994)
Inhibition of inward K‡ channels and stomatal response by
abscisic acid: an intracellular locus of phytohormone action.
Proc. Natl. Acad. Sci. USA, 91, 4019±4023.
Sharpe, P.J.H., Wu, H. and Spence, R.D. (1987) Stomatal
mechanics. In Somatal Function (Zeiger, E., Farquhar, G.D.
and Cowan, I.R., eds). Stanford, CA: Stanford University Press,
pp. 91±114.
Szyroki, A., Ivashikina, N., Dietrich, P. et al. (2001) KAT1 is not
essential for stomatal opening. Proc. Natl. Acad. Sci. USA, 98,
2917±2921.
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 578±588