Download Anion-Channel Blockers Inhibit S-Type Anion

Plant Physiol. (1995) 109: 651-658
Anion-Channel Blockers Inhibit S-Type Anion Channels and
Abscisic Acid Responses in Guard Cells'
Amnon Schwartz, Nitza Ilan, Martin Schwarz, jennifer Scheaffer, Sarah M. Assmann*, and Julian I . Schroeder
Department of Agricultura1 Botany, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76-1 00,
Israel (A.S., N.I.); Department of Biology and Center for Molecular Genetics, University of California at San
Diego, La Jolla, California 92093 (M.S., J.S., 1.13.); and Department of Biology, Pennsylvania State University,
University Park, Pennsylvania 16802 (S.M.A.)
l h e effects of anion-channel blockers on light-mediated stomatal
opening, on the potassium dependence of stomatal opening, on
stomatal responses to abscisic acid (ABA), and on current through
slow anion channels in the plasma membrane of guard cells were
investigated. The anion-channel blockers anthracene-9-carboxylic
acid (9-AC) and niflumic acid blocked current through slow anion
channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid
reversed ABA inhibition of stomatal opening in V. faba L. and
Commelina communis 1. The anion-channel blocker probenecid
also abolished ABA inhibition of stomatal opening in both species.
Additional tests of 9-AC effects on stomatal aperture in Commelina
revealed that application of this anion-channel blocker allowed
wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100
mM) KCI conditions. These results indicate that anion channels can
function as a negative regulator of stomatal opening, presumably by
allowing anion efflux and depolarization, which prohibits ion uptake in guard cells. Furthermore, 9-AC prevented ABA induction of
stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced
stomatal closure and inhibition of stomatal opening is discussed.
Ion transport across the plasma membrane of guard cells
via ion channels, pumps, and carriers is thought to be
integral to the regulation of stomatal aperture (Schroeder
and Hedrich, 1989; Assmann, 1993). During stomatal opening, membrane hyperpolarization by an H+-ATPase (Assmann et al., 1985; Serrano et al., 1988; Lohse and Hedrich,
1992) creates an electrical gradient for Kt uptake via inward-rectifying K+ channels (Schroeder et al., 1987). Estimates of the transmembrane electrochemical gradient for
C1- indicate that C1- uptake is active (Assmann and
Zeiger, 1987); it is hypothesized that C1- enters via
Cl-/H+ symport or Cl-/OH- antiport (Zeiger et al., 1978).
Uptake of ions and production of osmotically active organic solutes (Ogawa et al., 1978; Poffenroth et al., 1992)
This research was supported by Bi-national Science Foundation grant No. 89-00235 to A.S. and S.M.A., by U.S. Department of
Agriculture (USDA) grant No. 92-37100-7537/92-37100-8333
to
S.M.A., and by USDA grant No. 92-37304-7757 and National
Science Foundation grant MCB 9004977 to J.I.S.
* Corresponding author; e-mail sma39psuvm.psu.edu; fax
1-814-865-9131.
drive water influx, resulting in guard cell swelling and
stomatal opening.
Stomatal closure requires that an electrochemical driving
force be created for ion efflux that drives water loss and
guard cell shrinkage. A mechanism is required that can
strongly and continuously depolarize the membrane potential, thus creating a gradient for K+ efflux through
outward-rectifying K+ channels (Schroeder et al., 1987;
Blatt, 1990). Anion efflux resulting from the opening of
anion channels in the plasma membrane of guard cells
provides a mechanism that can produce prolonged depolarization positive of the Kf equilibrium potential (Keller
et al., 1989; Schroeder and Hagiwara, 1989).
At least three types of currents with permeability to
anions are present in the guard cell membrane (Keller et al.,
1989; Schroeder and Hagiwara, 1989; Hedrich et al., 1990;
Cosgrove and Hedrich, 1991; Schroeder and Keller, 1992).
One is a stretch-activated anion channel (Schroeder and
Hedrich, 1989; Cosgrove and Hedrich, 1991). Better studied
are slow (S-type) (Schroeder and Hagiwara, 1989) and
rapid (R-type) (Keller et al., 1989) anion channels. R-type
anion channels show a strong voltage dependence, with a
threshold for activation positive of approximately - 100
mV, maximal activation at -30 to -70 mV, and decreasing
activity at more positive membrane potentials (Keller et al.,
1989). R-type anion channels activate and deactivate relatively rapidly, thereby giving rise to transient depolarization (Hedrich et al., 1990; Schroeder and Keller, 1992).
S-type anion channels are voltage regulated and allow
large and sustained anion efflux in the entire physiological
range of membrane potentials (Schroeder and Hagiwara,
1989; Schroeder and Keller, 1992). Slow anion channels
remain activated at voltages as negative as -200 mV
(Linder and Raschke, 1992).S-type channels activate slowly
and in a sustained manner in response to depolarization
(Schroeder and Keller, 1992). The sustained activation of
slow anion channels suggests that these anion channels can
promote long-term ion efflux during stomatal closing
(Schroeder and Hagiwara, 1989; Schroeder and Keller,
1992). Furthermore, the activation of slow anion channels
over the entire physiological membrane potential range
Abbreviations: 9-AC, anthracene-9-carboxylic acid; DIDS, 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid; NPPB, 5-nitro2,3-phenylpropylaminobenzoicacid.
651
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652
Schwartz et al.
suggests that anion-channel regulation is required during
stomatal opening and closing. Elevated cytosolic Ca2+ has
been shown to enhance slow anion currents, providing a
mechanism that can contribute to ABA-mediated Ca2+dependent stomatal closure (Schroeder and Hagiwara,
1989).
Given current understanding of stomatal mechanisms, it
would be predicted that opening of anion channels would
prevent stomatal opening and promote stomatal closure by
favoring loss of osmotically active anions and, simultaneously, promoting membrane depolarization (Schroeder
and Hedrich, 1989). This has led to the hypothesis that
anion-channel blockers might enhance stomatal opening
and inhibit stomatal closure. Severa1 anion-channel blockers have been shown to block activity of R-type channels
(Marten et al., 1992, 1993) and S-type channels (Schroeder
et al., 1993). Detailed investigations of the effects of these
chemicals on physiologically regulated stomatal apertures
can be used to determine the functions of guard cell anion
channels. An initial anion-channel-blocker study has provided support for the hypothesis that anion channels represent an essential mechanism for regulation of stomatal
closing (Schroeder et al., 1993). Stomatal closing, produced
by simultaneous application of ABA and malate to guard
cells, was inhibited by the anion-channel blocker NPPB,
and anion channel blockers led to increased stomatal opening in the light (Schroeder et al., 1993).In the present study
effects of anion channel blockers on ABA signaling as the
sole stimulus were analyzed. To investigate the role of
anion channels in the control of stomatal opening as well as
stomatal closure by ABA, detailed studies of the effects of
anion-channel blockers on slow anion channels and on the
time course and K+ dependence of stomatal movements
were performed.
MATERIALS A N D M E T H O D S
Plant Material
Plants of Commelina communis L. and Vicia faba L. were
grown in Rehovot, Israel, as described by Karlsson and
Schwartz (1988) and in Cambridge, MA, as described by
Fairley-Grenot and Assmann (1991). Plants of V . faba L.
were grown in La Jolla, CA, as described previously
(Schroeder and Fang, 1991). Fully expanded young leaves
from plants 3 to 4 weeks old were used in a11 experiments.
Stomatal Movement Assays
Closed stomata for experiments in which stomatal opening was assayed were obtained as follows. Detached leaves
were floated on tap water in a Petri dish in darkness for at
least 1 h. Abaxial epidermes were then removed, floated on
a solution containing 10 mM KC1, 10 mM Mes, pH 6.1 (4.8
mM KOH), and kept in darkness at 27°C for approximately
30 min. This procedure resulted in apertures of O to 2 pm.
Peels were then transferred to treatment solutions and
incubated under 0.160 mmol m-’s-’ white light (daylight
fluorescent bulbs, 40 W, Sylvania, GTE) at 27°C. Treatment
solutions for experiments in which stomatal opening was
Plant Physiol. Vol. 109, .I995
assayed contained 10 mM Mes, pH 6.1 (4.8 mM KOH), and,
unless otherwise noted, 100 mM KCl for experiments
with Commelina and 30 mM KC1 for experiments with Vicia.
Peels were incubated for 3 h (Fig. l), during which measurements of apertures were taken with an ocular
micrometer.
To obtain open stomata for experiments in which stomatal closure was assayed, intact detached leaves were submerged in tap water and illuminated with 0.300 mmol ni-’
S-’ white light for approximately 2 h. Abaxial epi’dermes
were then peeled and floated on a solution containing 100
mM KCl, 10 mM Mes, pH 6.1 (4.8 mM KOH), while baseline
measurements of stomatal apertures were taken. Initial
apertures were 15 to 17 pm. Peels were then transferred to
50 mM KC1, 10 mM Mes, pH 6.1 (4.8 mM KOH), and
incubated under 0.160 mmol m-’s-’ white light at 27°C
for 30 min before aperture measurement.
Values presented are the averages of 60 to 120 stomatal
apertures, measured in at least three different experiments.
SES were calculated and are presented, except in cases in
which error bars are smaller than the data points. I11 the
niflumic acid experiments, approximately 50% of guard
cells appeared damaged, despite the use of low concentrations of that anion-channel blocker. Stomata with damaged
guard cells were not measured.
Patch-Clamp Solutions and Experiments
The patch-clamp pipette solution that equilibrates with
the cytosol of guard cells contained 136 or 150 mM (ZsC1,
3.35 mM CaCI,, 7.6 mM EGTA-Tris, 10 mM Hepes, 1.8 mM
MgCl,, 200 p~ guanosine Y-[y-thioltriphosphate, 4 mM
MgATP, 4 mM Tris, pH 7.10 (free Ca2+ was approximately
0.2 p ~ )The
. extracellular solution bathing guard cells contained 30 mM CsC1,2 mM MgCl,, 1 mM CaCl,, 10 mM Mes,
pH 5.6, unless otherwise noted. The osmolalities of the
pipette and bath solutions were adjusted to 480 mosmol
kg-’ by the addition of D-sorbitol.
Patch-clamp studies of slow anion channels in guard
cells were performed as described previously (Schroeder
and Hagiwara, 1989; Schroeder and Keller, 1992). Patchclamp recordings were obtained using an Axopatch 1D
amplifier (Axon Instruments, Foster City, CA). On-line
data acquisition, voltage pulse protocols, and data analysis
were executed with a TL-125 interface (Axon Instruments)
and a 25-MHz 386-based microcomputer. For quantification of 9-AC block of slow anion channels, the membrane
potential was held continuously at +40 mV. Under our
experimental conditions, this procedure activates slow anion channels (Schroeder and Keller, 1992) while inactivating rapid anion channels. To confirm that rapid anion
channels were inactivated during analysis, repetitive linear
changes in membrane potential of 7 s duration (voltage
ramps) from -100 to +40 mV were applied to guard cells
(Keller et al., 1989; Schroeder and Keller, 1992).
Chemicals
9-AC was obtained from Aldrich. Probenecid and niflumic acid were from Sigma. Anion-channel blockers were
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Anion-Channel
Blockers and ABA Response in G u a r d Cells
dissolved in 100% ethanol or in 0.1 M NaOH. For the
patch-clamp experiments with niflumic acid, a 100 mM
stock solution of niflumic acid was prepared in 0.2 M Tris
and 0.2 M KOH and subsequently brought to pH 9.5 with
HCI. The final concentration of ethanol in the treatment
solutions was not more than 0.1%. The final concentration
of NaOH, Tris, or HC1 was not more than 100 p ~and
, the
pH of the treatment solutions was adjusted as necessary.
RESULTS
The effect of the anion-channel blocker 9-AC on lightstimulated stomatal opening was analyzed as a function of
time and as a function of the extracellular K+ concentration. Figure 1A shows that the rate of stomatal opening in
Commelina was accelerated by the presence of 300 p~ 9-AC
and final stomatal apertures were increased. It is well
documented that high concentrations of approximately 50
mM Kt are required to produce wide stomatal opening in
epidermal peels of C. communis (Schwartz et al., 1988).
Interestingly, Figure 1B shows that 300 p~ 9-AC produced
a striking enhancement of light-induced stomatal opening,
allowing stomata to open even with low (1 mM) KCl concentrations in the incubation medium. In the absence of
9-AC, opening of Commelina stomata in epidermal peels
did not occur under such conditions (Fig. 1B). In 1 mM KC1,
average apertures were less than 1 pm in the absence of
9-AC but were approximately 11 pm in the presence of 300
/.LM 9-AC (Fig. 1B). In the absence of 9-AC, 100 mM extracellular KC1 was required to achieve similar stomatal apertures of 11 pm.
In further experiments the effects of anion-channel blockers on ABA regulation of stomatal opening and stomatal
closing were studied. Surprisingly, the inhibition of stomatal opening by ABA was alleviated when 9-AC was added
to Commelina epidermal peels (Fig. 2). 9-AC abolished the
inhibitory effect of 10 p~ ABA on stomatal opening in the
presence of 100 mM KC1 (Fig. 2A) and partially reversed the
ABA effect in 1 mM KCl (Fig. 2B). These data suggest that
anion-channel activation, in addition to proton pump and
inward KC channel inhibition, contributes to ABA-medi-
653
ated inhibition of stomatal opening (see ”Discussion”). Figure 3 shows that 9-AC not only reversed ABA inhibition of
stomatal opening but also abolished ABA promotion of
stomatal closure at similar effective concentrations. In both
cases, ABA effectiveness decreased as 9-AC concentrations
increased from O to 300 p ~ with
,
saturation occurring at
approximately 300 p~ 9-AC.
As illustrated in Figure 4A, the anion-channel blockers
probenecid and niflumic acid (Greger, 1991) also prevented
ABA inhibition of stomatal opening in Commelina. The Ki
for 50% reversal of ABA inhibition of stomatal opening is
approximately 120 p~ for 9-AC (Fig. 3), 400 p~ for probenecid, and only 18 p~ for niflumic acid (data not shown).
When used at concentrations that are effective for Commelina, a11 three of these anion-channel blockers also reversed ABA inhibition of stomatal opening in V. faba
(Fig. 4B) and also produced increased stomatal opening
(Fig. 4B).
The effect of 9-AC on S-type anion channels was tested in
patch-clamp experiments on guard cell protoplasts to determine whether the 9-AC effects on stomatal apertures
described above correlate with inhibition of slow anion
channels. V. faba was chosen for these experiments because
of the significantly greater ease of obtaining high resistance
seals in this species versus Commelina and because patchclamp data could be compared with epidermal peel data
from the same species. Large, deactivating ionic currents
were recorded by stepping the membrane potential of
guard cell protoplasts from +40 to -90 mV (Fig. 5A). These
deactivating currents have been demonstrated to be carried
by selective efflux of anions via slow anion channels in V .
faba guard cells (Schroeder and Keller, 1992; Schmidt and
Schroeder, 1994). Extracellular perfusion of guard cells
with 300 p~ 9-AC inhibited on average 84% ( n = 7) of the
slow anion current, as shown in Figure 5B. Remova1 of
extracellular 9-AC by bath perfusion resulted in recovery
of slow anion currents, suggesting that 9-AC blocks slow
anion channels from the extracellular side of the membrane
( n = 5, data not shown). The block of slow anion currents
by 9-AC was determined at 9-AC concentrations ranging
from 20 to 500 p~ in 12 guard cells. The dose dependence
Figure 1. The anion-channel blocker 9-AC increases the rate and extent of light-induced stomatal opening in C. communis. A, Time course
of stomatal opening in epidermal peels incubated in 1O 0 mM KCI, 1O mM Mes, 4.8 mM KOH
(pH 6.1) under 0.1 60 mmol m-‘s-’ white light,
without (A) or with (O) 300 p~ 9-AC. B, The
effect of 300 p~ 9-AC on steady-state stomatal
aperture of C. communis epidermal peels measured in different KCI concentrations (all with 1 O
mM Mes, 4.8 mM KOH, pH 6.1) under 0.160
mmol m-’ s-’ white light. Apertures in each
treatment were measured every hour until
steady-state was reached. Steady-state with 1
mM KCI was reached after 5 to 6 h.
O
30
60
90
120
Time, min.
150
180 O
20
40
60
80
100 120
KCl, mM
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Schwartz et al.
654
Figure 2. 9-AC can reverse ABA inhibition of
stomatal opening in C. communis. Time courses
of stomatal opening are shown in the presence
of 300 ~ L M
9-AC (A),
in the presence of 10 ~ L M
ABA (V),and in the presence of 1O p~ ABA plus
300 ~ L M9-AC (A).A, The incubation solution
contained 1O0 mM KCI, 1O mM Mes (pH 6.1). B,
The incubation solution contained 1 mM KCI, 10
mM Mes (pH 6.1).
Plant Physiol. Vol.
18
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15
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12
3
Li
109, 1995
B
3
Y
Li
a,
a
cd
3
9
cd
6
8O
3
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Y
v1
O
2
O
6 0
4
2
4
6
Time, h.
of anion current block (Fig. 6) agrees well with the dose
dependence for 9-AC inhibition of ABA-induced stomatal
movements (Figs. 3 and 4).
In further experiments, inhibition of slow anion-channel
currents in guard cells by niflumic acid was tested as
illustrated in Figure 7. Typical large, slow anion-channel
currents were observed in the entire physiological voltage
range (Fig. 5, A and C). Exposure of guard cells to 50 WM
niflumic acid greatly inhibited slow anion channels at a11
membrane potentials analyzed ( n = 5; Fig. 5, B and C).
Interestingly, in contrast to 9-AC (Figs. 5 and 6), NPPB, and
IAA-94 (Schroeder et al., 1993), niflumic acid block of slow
anion channels was not reversible, even when guard cells
were perfused with the control bath solution for durations
of up to 30 min (data not shown).
D I SC USSI ON
Blockers of Cuard Cell Anion Channels
9-AC and niflumic acid block anion channels in the
plasma membrane of guard cells (Figs. 5-7). 9-AC and the
anion-channel blockers niflumic acid and probenecid promote stomatal opening, even under conditions in which
opening is usually inhibited (Figs. 1 4 ) . These results and
findings showing anion-channel block by NPPB and
IAA-94 (Marten et al., 1992; Schroeder et al., 1993) indicate
that anion-channel blockers developed for animal systems
(Greger, 1991) are also effective in blocking plant anion
channels. The anion-channel blockers tested here also block
R-type anion channels at similar concentrations and rela-
E
3
n
8
c
s
c,
c
8
2
H
cd
c
cd
O
100
200
300
400 O
100
200
300
400
9-AC, ,u,M
Figure 3. The effect of 9-AC on ABA (10 p ~regulation
)
of stomatal opening (A) and stomatal closing (B) in C. communis.
A, To asses the effects of 9-AC on ABA inhibition of stomatal opening, epidermal peels with initially closed stomata (see
“Materials and Methods”) were incubated in 50 mM KCI, 10 p~ ABA, 10 mM Mes (pH 6.1) and in the presence of the
indicated 9-AC concentrations under 0.1 60 mmol m-’s-’ white light for 3 h before aperture measurement. B, To asses the
effects of 9-AC on ABA promotion of stomatal closure, stomata were initially opened and subsequent incubation conditions
were 1O p~ ABA, 50 mM KCI, 10 mM Mes, pH 6.1, and the indicated 9-AC concentrations. Peels were illuminated with 0.1 60
mmol m-* s-’ white light for 30 min and apertures were then measured. Apertures at the start of closing experiments
averaged 15.3 p m .
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Anion-Channel Blockers and ABA Response in Cuard Cells
18
I
I
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15
12
9
B
z
2
6
3
D
a
m
18
*
n
-
M
*
8
;j
O
o -1
*-
B. 300 p M 9AC
-
r--
15
-200
12
O
10
20
30
40
50
60
9
Time, sec.
6
3
n
Control
ABA
ABA
9-AC
ABA
ABA
Proben. Nif. acid
Figure 4. The effects of the anion-channel blockers 9-AC, probenecid (Proben.), and niflumic acid (Nif. acid) on stomatal opening in the
presence of ABA in C. communis (A) and V. faba (6). For experiments
with Commelina, peels were incubated in 50 mM KCI, 10 y~ ABA,
10 mM Mes (pH 6.1), under 0.160 mmol m-2 s-’ white light for 3 h
before aperture measurement. Experimental conditions were identical for experiments with Vicia, except that the incubation medium
contained 30 mM KCI. The anion-channel blockers were 9-AC (300
p ~ )probenecid
,
(500 p ~ )and
,
niflumic acid (30 and 2 0 ~ L Mfor
Commelina and Vicia, respectively). Control indicates the degree
of stomatal opening without addition of ABA and anion-channel
blockers.
tive effectiveness (Marten et al., 1992), supporting indications that there is a structural relation between S-type and
R-type anion channels (Schroeder et al., 1993; Schmidt and
Schroeder, 1994; Zimmermann et al., 1994). However, Rtype channels exhibit little activity under the hyperpolarized membrane potentials associated with stomatal opening (Hedrich et al., 1990). Furthermore, R-type anion
channels are completely blocked by DIDS (Ki = 0.6 p ~ ;
Marten et al., 1993), whereas S-type anion currents are not
effectively blocked by DIDS at concentrations of 100 ~ L M
(Schroeder et al., 1993). The finding that DIDS has little or
no effect on stomatal opening in response to light
(Schwartz et al., 1991; Schroeder et al., 19931, as well as the
finding that S-type but not R-type channels are active at
membrane potentials negative of -100 mV (Schroeder and
Keller, 1992), suggests that the effects of anion-channel
blockers on stomatal opening reported here are likely to
predominantly reflect the effects of these blockers on Stype anion channels. The observed effects of anion-channel
blockers on stomatal closure (Fig. 3; Schroeder et al., 1993)
also suggest a predominant contribution of S-type anion
channels. Although R-type anion channels are likely to also
participate in this response, DIDS alone does not inhibit
stomatal closing (Schroeder et al., 1993), in contrast to
abolishment of ABA-induced stomatal closure by 9-AC
(Fig. 38) and abolishment of stomatal closure in response to
Figure 5. Block of slow anion currents in V. faba guard cells by
9-AC. Top inset, The membrane potential was hyperpolarized from
+40 to -90 mV. A, Hyperpolarization to -90 mV produced a large
anion efflux current that showed a slow relaxation (deactivation). 6,
Exposure of the same guard cell to extracellular 9-AC (300 p ~ )
resulted in inhibition of slow anion channels.
co-application of ABA and malate by NPPB (Schroeder et
al., 1993).
Effects of 9-AC on Light-lnduced Stomatal Opening
The enhanced light-induced stomatal opening observed
under low KCl conditions in the presence of 9-AC (Fig. lB),
the greater rate of stomatal opening in the presence of this
anion-channel blocker (Fig. 1, A and B), and the increased
stomatal apertures observed when anion channels are
blocked (Fig. 4; Schroeder et al., 1993) indicate a central role
of anion channels in the regulation of light-mediated stomata1 opening, in addition to their initially suggested role
in stomatal closure (Keller et al., 1989; Schroeder and Hagiwara, 1989). When anion channels are blocked, such that
they cannot contribute to anion efflux and membrane depolarization, Commelina stomata in epidermal peels can
e,
&
2
o
60
40
O
.3
4
20
o
1
10
100
1000
9-AC, ,UM
Figure 6. Concentration dependence of inhibition of slow anion
channels in Vicia guard cells by 9-AC. Large steady-state slow anion
currents ranging in magnitude from approximately -1 50 to -600 pA
at -90 mV were recorded, and the relative block of slow anion
currents in each cell was determined as previously described (Schroeder et al., 1993). Data from 12 guard cells are shown.
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Schwartz et al.
656
indicating that downregulation of slow anion channels
would be required during stomatal opening.
+35 mV
+30 mV
Plant Physiol. Vol. 109, 1995
Effects of Anion-Channel Blockers on ABA-lnduced
lnhibition of Stomatal Opening
500
-1000
-50:
-
A. Control
T F
-
L
250
O
-250
-500
,
,
,
-150 -100
I
-50
,
II
O
,
,
50
Membrane potential, pA
Figure 7. Block of slow anion channels in Vicia guard cells by
niflumic acid (Nif. acid). A, Large, slow anion currents were recorded
in response to voltage pulses ranging from +35 to -145 mV (voltage
protocol is illustrated in the top inset). B, Extracellular perfusion of
guard cells with 50 p~ niflumic acid inhibited slow anion channels
at membrane potentials ranging from +35 to -145 mV. C, Steadystate current voltage curves of whole-cell currents illustrated in A and
B. T h e bath solution contained 39 mM CaCI, (Marten et al., 1992)
instead of 30 mM CsCl (see “Materials and Methods”)in niflumic acid
block experiments.
open in response to light in solutions with KC1 concentrations as low as 1 mM. In the absence of 9-AC, Commelina
stomata require 100 mM KC1 to achieve the same extent of
opening (Fig. 1). Enhancement of stomatal opening by
9-AC suggests that in addition to H+ pump activation
downregulation of anion channels may be an important
component in the mechanism that allows membrane hyperpolarization in response to light-mediated stomatal
opening. The voltage dependence of slow anion channels
in guard cells shows that these anion channels are open at
membrane potentials in the range of -100 to -200 mV
(Linder and Raschke, 1992; Schroeder and Keller, 1992),
In this study we have also analyzed inhibition of stoinatal opening by the phytohormone ABA. Surprisingly,
9-AC, niflumic acid, and probenecid counteract ABA inhibition of stomatal opening in both Commelina and Vicia
(Figs. 2 4 ) . 9-AC reverses the inhibitory effect of ABA on
stomatal opening completely under high KC1 conditions
and partially under low (1 mM) KC1 conditions (Fig. 2, A
and B). ABA and elevation in cytosolic Ca2+ inhibit inward
KC channels (Schroeder and Hagiwara, 1989; Blatt, 1990;
Lemtiri-Chlieh and MacRobbie, 1994; Schwartz et al., 1994),
an effect not reversed by 9-AC and one that would slow K+
uptake and thus stomatal opening, particularly under low,
externa1 K+ concentrations. Incomplete reversal by 9-AC of
the ABA effect under low KC1 conditions (Fig. 2B) i,,e consistent with inhibition of proton pumps and inward KC
channels by ABA (Blatt, 1990; Lemtiri-Chlieh and MacRobbie, 1994; Schwartz et al., 1994). However, the finding that
anion-channel blockers can completely or partially reverse
ABA inhibition of stomatal opening (Figs. 2 4 ) demonstrates that proton pumps and inward K+ channels are not
completely inhibited by ABA. The presence of residual
inward K+ channel currents after ABA-induced reduction
in inward K+ currents supports this suggestion (Blatt, 1990;
Lemtiri-Chlieh and MacRobbie, 1994; Schwartz et al., 1994).
The reversal of ABA inhibition of stomatal opening by
anion-channel blockers and the activation of S-type anion
channels at strongly hyperpolarized potentials suggest that
anion-channel activation contributes significantly to ABA
inhibition of stomatal opening.
Effects of 9-AC on ABA-Mediated Stomatal Closure
The mechanisms of stomatal opening and stomatal closure are not simply the reverse of one another (Schroeder
and Hedrich, 1989; Kearns and Assmann, 1993).Therefore,
it was also of interest to investigate the effect of 9-AC on
ABA promotion of stomatal closure. Stomatal closing induced by ABA has been suggested to require slow anionchannel activation (Schroeder and Hagiwara, 1989; Schroeder et al., 1993). Consistent with this hypothesis, block of
anion channels completely abolishes stomatal closing triggered by combined exposure of guard cells to ABA and 40
mM malate (Schroeder et al., 1993).In the present study we
analyzed anion-channel blocker effects under more physiological conditions, with ABA as the sole stimulus for
stomatal closing. 9-AC abolishes ABA stimulation of stomata1 closure (Fig. 38). The demonstration that the anionchannel blocker 9-AC inhibits ABA effects on stomatal
closure implies that, in addition to previously documented
regulation of K+ channels by ABA (Blatt, 1990; LemtiriChlieh and MacRobbie, 1994; Schwartz et al., 1994), anionchannel activation is necessary in response to ABA. Enhancement of outward Kt channel currents and inhibition
of proton pumps alone would not suffice to stimulate K+
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Anion-Channel Blockers and ABA Response in Guard Cells
efflux, since the membrane potential would be polarized to
the K+ equilibrium potential, which prevents net K+ release required for stomatal closing. The presented effects of
anion-channel blockers therefore suggest that anion-channel-mediated anion efflux is central to ABA-induced stomatal closure (Keller et al., 1989; Schroeder and Hagiwara,
1989; Schroeder et al., 1993).Furthermore, the weak voltage
dependence of slow anion channels (Fig. 7) indicates the
need for signal-dependent modulation of these anion channels according to current models of stomatal regulation
(Schroeder and Hagiwara, 1989; Schroeder and Hedrich,
1989; Kearns and Assmann, 1993).ABA activation of anion
channels is likely to function as a rate-limiting mechanism
necessary for stomatal closure (Keller et al., 1989; Schroeder
and Hagiwara, 1989). Consistent with such a rate-limiting
mechanism, the concentration dependence for S-type anion-channel inhibition by 9-AC (Fig. 6) is similar to the
9-AC concentration dependence for relief of ABA effects on
both stomatal opening and closing (Fig. 3). Note that, in
contrast to anion-channel block, 70% block of K+ channels
by barium (Schroeder et al., 1987) alone does not reduce stomatal responses significantly (H.H. Fang and J.I.
Schroeder, unpublished data). Anion-channel modulation
in conjunction with K+ channel and H+ pump modulation
could provide a basis for tight control of stomatal
movements.
Possible ABA Regulation Mechanisms
Under some conditions, particularly when plants are
grown at higher temperatures, ABA elevates cytosolic Ca2+
levels (McAinsh et al., 1990,1992; Schroeder and Hagiwara,
1990; Allan et al., 1994). Since the activities of both S-type
and R-type anion channels are enhanced by increased Ca2+
concentrations (Schroeder and Hagiwara, 1989; Hedrich et
al., 1990), one transduction mechanism could be that ABA
activates these anion channels via elevation of cytosolic
Ca2+ levels. The Ca2+ activation of S-type anion channels
has been suggested to occur indirectly, implicating additional unknown intermediate mechanisms in S-type anionchannel activation (Schroeder and Hagiwara, 1989). In addition to Ca2+-induced stomatal closure, Ca2+independent mechanisms have been proposed to play
central roles during ABA-induced stomatal regulation (for
review see, MacRobbie, 1992; Allan et al., 1994). Note that
effects of Ca2+ channel blockers on ABA-mediated stomatal regulation (DeSilva et al., 1985; Schwartz, 1985) are less
pronounced than the marked effects of anion-channel
blockers on stomatal regulation (Figs. 14; Schroeder et al.,
1993). These data are consistent with an essential control
function of anion channels in stomatal regulation in response to either Ca2+ signals or Ca2+-independent mechanisms of ABA action. Further studies will be required to
directly analyze whether and by which intermediate mechanisms ABA activates anion channels in guard cells.
ACKNOWLEDCMENTS
The assistance of Mrs. Aviva Ben-David and Mr. Yosi Shamia
(Hebrew University) in performing some of the epidermal peel
657
experiments is acknowledged. We thank Mrs. Judie Murray (University of California, San Diego) for support in preparation of the
manuscript and Dr. John Ward for comments concerning the
manuscript.
Received May 31, 1995; accepted July 6, 1995.
Copyright Clearance Center: 0032-0889/95/ 109/0651/OS.
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