Proc. Nati. Acad. Sci. USAVol. 87, pp. 9305-9309, December
1990Botany
Repetitive increases in cytosolic Ca2+ of guard cells by
abscisic acidactivation of nonselective Ca2+ permeable channels
(stomata/ion channel/receptor-operated Ca2+ channel/fura-2)
JULIAN I. SCHROEDER* AND SUSUMU HAGIWARAtDepartment of
Physiology, Jerry Lewis Neuromuscular Research Center, University
of California at Los Angeles School of Medicine, Los Angeles, CA
90024
Communicated by Bernard 0. Phinney, August 29, 1990 (received
for review June 28, 1990)
ABSTRACT Many signal-transduction processes in higherplant cells
have been suggested to be triggered by signal-induced opening of
Ca21 channels in' the plasma membrane.However, direct evidence for
activation of plasma-membraneCa21 channels by physiological signals
in higher plants has notyet been obtained. In this context, several
lines of evidencesuggest that Ca2+ flux into the cytosol of guard
cells is a majorfactor in the induction of stomatal closing by
abscisic acid(ABA). ABA closes stomatal pores, thereby reducing
transpi-rational loss of water by plants under drought conditions.
Todirectly investigate initial events in ABA-induced signal
trans-duction in guard cells, we devised an experimental
approachthat allows simultaneous photometric measurements of
cyto-solic Ca2+ and patch-clamp recordings of ion currents
acrossthe plasma membrane of single Vicia faba guard cells.
Usingthis approach, we found that the resting cytosolic Ca21
con-centration was 0.19 ± 0.09 FM (n = 19). In responsive
guardcells, external exposure to ABA produced transient
repetitiveincreases in the cytosolic free Ca2+ concentration. These
Ca21transients were accompanied by concomitantly occurring
in-creases in an inward-directed ion current. Depolarization of
themembrane terminated both repetitive elevations in cytosolicCa2+
and inward-directed ion currents, suggesting that ABA-mediated Ca2+
transients were produced by passive influx ofCa2+ from the
extracellular space through Ca2+-permeablechannels. Detailed
voltage-clamp measurements revealed thatABA-activated ion currents
could be reversed by depolariza-tions more positive than -10 mV.
Interestingly, reversalpotentials of ABA-induced currents show that
these currentsare not highly Ca2+-selective, thereby permitting
permeation ofboth Ca2 and K+. These results provide direct evidence
forABA activation of Ca2+-permeable ion channels in the
plasmamembrane of guard cells. ABA-activated ion channels
allowrepetitive elevations in the cytosolic Ca2+ concentration,
which,in turn, can modulate cellular responses promoting
stomatalclosure.
Elucidation of the molecular mechanisms responsible
forstimulus-dependent activation of Ca2l fluxes is central
tounderstanding the initial events in signal transduction inhigher
plant cells (1). Control of stomatal pore movements bythe
physiological growth regulator abscisic acid (ABA) pro-vides an
opportune system for the investigation of mecha-nisms underlying
Ca2"-dependent signal transduction (2-6).
Stomatal pores permit diffusion of CO2 into leaves
forphotosynthetic carbon fixation and the transpiration of
watervapor to the atmosphere. This exchange of gases is regulatedby
movements ofguard cell pairs that surround each stomatalpore. When
water stress develops, the plant growth regulatorABA triggers
stomatal closing synergistically with Ca2+ (2,3). Recent detailed
investigations of several plant species
have shown that stomatal closing in response to ABA followsa
nonuniform behavior such that stomatal pores in distinctsmall
patches of the leaf surface area- close in response toABA, whereas
stomata in other small areas of the leafepidermis remain open
(7-11). This nonuniform ABA re-sponse has been suggested to play a
key role in ABA-dependent regulation of carbon fixation (7-11).
Stomatal movements are mediated by changes in the ioncontent of
guard cells, which depend on large ion fluxesacross guard-cell
membranes (5, 6). Stomatal opening re-quires organic-anion
synthesis and K+ uptake (5). Inward-conducting Ca2l-regulated K+
channels represent a majorpathway for K+ uptake (4, 12). Stomatal
closing is producedby release of K+ and anions from guard cells
(13, 14).ABA-mediated closing of stomata proceeds in a
Ca2+-dependent manner (2, 3). The modulation of voltage-dependent
anion channels (4, 15) by cytosolic Ca2+ (4) andthe resulting
activation of outward-conducting K+ channels(12) have been
suggested to provide a molecular basis for theCa2' dependence of
ABA-induced stomatal closure (4).Recent research has indicated that
ABA can elevate the
cytosolic free Ca2+ concentration ([Ca2+]cyt) in guard cells
ofCommelina communis (16). The mechanisms by which[Ca2+]cyt is
elevated remain unknown. In the present studywe have attempted to
determine the effects of ABA on[Ca2+]cyt in Vicia faba guard cells
by adopting a method ofinvestigation that allows the simultaneous
monitoring of[Ca2 ]cyt and ion currents across the plasma
membrane(plasmalemma) ofguard cells. This approach permits
distinc-tion between signal-dependent activation of Ca2+ channels
inthe plasma membrane and release of Ca2+ from
intracellularorganelles.
METHODSCell Isolation. V. faba plants were grown in a
controlled
environment growth chamber (Conviron E15) at 20°C with a12-hr
light, 12-hr dark day/night cycle and the use of fluo-rescent and
incandescent illumination at a photon fluencerate of 150 ,uE m 2 s-
(where E = 1 mol of photons). Guardcell protoplasts were isolated
from 2- to 3-week-old V. fabaplants by 60- to 75-min incubation in
1.7% Cellulase OnozukaRS (Yakult Honsha, Tokyo), 1.7% Cellulysin
(CalBiochem),0.026% Pectolyase Y-23 (Seishin Pharmaceutical,
Tokyo)following a described procedure (17).
Patch Clamp and Solutions. The tight-seal whole-cell
con-figuration of the patch-clamp technique (18) was applied
toisolated guard-cell protoplasts as described (12, 17).
Duringrecordings, cells were bathed in solutions that contained
2
Abbreviations: ABA, abscisic acid; [Ca2+]cyt, cytosolic free
Ca2+concentration.*To whom reprint requests should be sent at
present address:Department of Biology C-016, University of
California at SanDiego, La Jolla, CA 92093.tDeceased April 1,
1989.
9305
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 87 (1990) 9307
[Ca2+]
1 pM
0
2 min ABA
FIG. 2. ABA-induced Ca2+ transient in a V. faba guard cell.
Theguard cell was externally exposed to 1 ,uM ABA. Membrane
potentialof the cell was held at -40 mV. Intracellular solution
contained 100mM potassium glutamate, and extracellular solution
included 10 mMpotassium glutamate.
Using whole-cell patch-clamp techniques in conjunctionwith
measurements of [Ca2+],y, allows the simultaneousmonitoring of ABA
effects on both transmembrane ion cur-rents and [Ca2+]cyt. This
approach permits direct identifica-tion of the mechanisms by which
[Ca2+]cyt elevations aretriggered. Ca2+ release from intracellular
stores may increase[Ca2+]cyt without a concomitantly occurring
plasma-membrane Ca2+ current, whereas activation of plasma-membrane
Ca2+ channels would increase both [Ca2+]cyt andinward Ca2+
currents.
In the present study, the effects of ABA on [Ca2+J]cytmembrane
current, or membrane potential were recorded in37% of the cells
studied (n = 62). When studying stomatalmovements in epidermal
strips, ABA treatment was ob-served to close approximately
one-third of the stomata (J.I. S.and P. Zanirato, unpublished
results). In the present studyresponsive guard cells showed
characteristic effects as de-scribed below.When guard cells were
externally perfused with 1 ,uM
ABA, a rapid and transient increase in [Ca2+]cyt was
recorded(Fig. 2). Peak amplitudes of the initial Ca2+ transient
ob-served after ABA application ranged from 0.5 to 5
AM.Simultaneous Effects of ABA on Transmembrane Ion Cur-
rents and [Ca2+Jcyt. To study the mechanisms by whichtransient
rises in [Ca2+]cyt were mediated, ion currents acrossthe plasma
membrane and [Ca2+Jcyt were simultaneouslymeasured. When guard
cells were continuously exposed toABA by bath perfusion and the
membrane potential (V.m) washeld at -54 mV, repetitive rises in
[Ca2+]cyt were observed(Fig. 3). With each rise in [Ca2+Jcyt (Fig.
3; lower trace), aconcomitant rise in inward ion current across the
guard-cellplasma membrane was seen (Fig. 3, upper trace). These
data
suggest that Ca2l influx through ABA-activated ion channelsmay
be responsible for ABA-mediated increases in [Ca2+]cyt.To test the
suggestion that rises in [Ca2J]cyt were mediated
by passive ion-channel-mediated flux into the cell, the
mem-brane was depolarized to 0 mV (Fig. 3). Depolarizations to 0mV
eliminated transient elevations in [Ca2+]cy, and inducedsmall
outward currents (Fig. 3). After depolarization to 0 mVfor several
minutes, the membrane was repolarized to -54mV. Upon repolarization
to -54 mV immediate continuationof the occurrence of transients in
[Ca2+]cyt was observed.Each transient Ca2+ elevation was
accompanied by a tran-sient increase in an inward ion current (Fig.
3).Subsequently the membrane was polarized for periods
lasting from 1.5 min to 30 sec to +5 mV, followed by -54 mV,+5
mV, and -54 mV (Fig. 3). Each depolarization to +5 mVled to
termination of transient elevations in [Ca2+]cy, as wellas
cessation of inward currents, whereas each hyperpolar-ization
resulted in transient [Ca2+]cyt elevations accompaniedby
concomitantly occurring inward currents showing thatCa2+ influx
across the plasma membrane contributed toABA-induced Ca2+
transients (Fig. 3).
Nonselective Current Activation by ABA. The Ca2+ equi-librium
potential with 1 mM Ca2+ in the external medium and
+20 current
pA 1 IV KJ-20 -
ii
2+i /12 MM [Ca0 ---------
---,)-----------------------------------------------------------------
fABA 1 5 min
-54
membrane potential |Vm=54 V OmV -54mV +5 I-54mV
FIG. 3. Simultaneous recordings ofABA-induced [Ca2+]cyt
elevations (lowertrace) and concomitantly occurring inwardion
currents (downward deflections in up-per trace) in a V.faba guard
cell. The guardcell was exposed continuously to 5 ,AMABA by bath
perfusion. ABA-inducedrises in [Ca2+]cyt and inward ion
currentsdepended on the imposed membrane po-tential (VIm, indicated
at bottom) as de-scribed in text. Internal solution included100 mM
potassium glutamate, and externalsolution included 10 mM
potassiumglutamate.
Botany: Schroeder and Hagiwara
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9308 Botany: Schroeder and Hagiwara
0.5 pM ABA20 PA- losI
-3 mvFIG. 4. ABA-activated ion currents re-
corded in whole-cell mode of patch-clamptechnique. Downward
deflections corre-spond to ABA-activated inward ion cur-rents, and
upward deflections are ABA-induced outward currents. ABA was
ap-plied for several seconds as indicated bythe bar on the top,
from a perfusion pipettelocated in the vicinity of the guard
cell.Membrane potentials are indicated at theupper left of current
traces. Internal solu-tion included 100 AtM EGTA, 80 mM po-tassium
glutamate, and 20 mM KCI; ex-ternal solution included 10 mM
KCL.
+50 mV) and the K+ equilibrium potential (EK+ = -52
mV).Furthermore, the reversal potential of ABA-activated cur-rents
was more negative than equilibrium potentials of allother ions in
the pipette and bath solutions (See Methods;EfreeMg2+ = -1 mV; Ecl-
= +10 mV; EH+ = +61 mV).Equilibrium potentials were calculated
after corrections forionic activities (27).
DISCUSSION
The reversal potential ofABA-activated currents (Figs. 4 and5)
and current-mediated Ca2' elevations (Fig. 3) showed thatCa2+ ions
as well as K+ ions were permeable to ABA-activated channels.
Similar reversal potentials of ABA-activated currents were found
when using other recordingsolutions, confirming the conclusion that
ABA-induced ele-vations in [Ca2+]cy were mediated by Ca2+ influx
throughnonselective ion channels.
Ca2+-channel activation by physiological stimuli such asplant
hormones, light, and fungal elicitors has been suggestedto play a
primary role in the initiation of signal-transductionprocesses of
higher plant cells (for review, see ref. 1). Thefinding that ABA
activates Ca2+-permeable currents in guardcells provides direct
evidence for signal-activated, Ca2+-permeable channels in the
plasma membrane ofa higher plantcell.Mechanism of ABA-Induced
Channel Activation. ABA-
dependent Ca2+-permeable channels were activated in a
20-
0.
c O
C)m
c -20(D
A
A
-50 -40 -30
A
-20 -10 0 10 20 30 40Membrane potential, mV
FIG. 5. Peak amplitudes of ABA-activated ion currents
recordedfrom the guard cell of Fig. 4 as function of membrane
potential; thereversal potential was interpolated to -11 mV.
repetitive manner during continuous ABA applications (Fig.3).
This repetitive activation pattern during continuous ABAapplication
is unlike the activation of transmitter-receptorion channels, when
the receptor and the ion channel consti-tute one molecular entity.
In general, such receptor-ionchannel complexes in animal cells show
activation followedby desensitization at high ligand
concentrations. However,recovery of receptor-ion channel proteins
from desensitiza-tion typically requires removal ofthe ligand from
the externalmedium (28). Therefore, the repetitive activation
pattern ofABA-activated currents during continuous ABA
applicationsuggests that intermediate signaling events may be
requiredbetween ABA exposure at the plasma membrane and ion-channel
opening.
In animal cells, receptor-operated Ca2l channels havebeen
postulated to explain Ca2' influx into a variety of cellsthat do
not possess voltage-gated ion channels for electricalexcitability,
as well as into electrically excitable cells (29).However, direct
evidence for the existence of receptor-operated Ca2l channels
remains sparse to date (29). In onedetailed study of peritoneal
mast cells, second-messenger-dependent Ca2' influx was found in 26%
of the investigatedcells, indicating that receptor-activated Ca2+
influx mayunderlie complex activation and Ca2+-translocation
mecha-nisms (30).Although elevations in [Ca2+]cyt of guard cells
correlate
with ABA-activated inward currents, the possibility cannotbe
excluded that release of Ca2+ from intracellular organellesprovides
an additional contribution to the observed rises in[Ca2+],yt.
During prolonged depolarizations, no increases in[Ca21]cyt were
observed (Fig. 3), suggesting that Ca2' releasefrom intracellular
organelles may require hyperpolarizationand/or Ca2' influx across
the plasma membrane. Ca2+-induced Ca2' release from intracellular
organelles, as foundin animal cells (31), could lead to additional
elevation in[Ca2+]cy1 under these conditions. Our results deviate
from thehypothesis that Ca2+ release from intracellular organelles
isthe initial mechanism of ABA-induced stomatal closing,which was
derived from injection of synthetic caged inositolphosphates into
guard cells (32, 33). Further investigationswill be needed to
assess the possible contribution of Ca2+release from intracellular
organelles to ABA-mediated risesin [Ca2]+cyt.ABA Responses of Guard
Cells. Our data correlate with
recent findings by McAinsh et al. (16), which showed ABA-
-23 mV
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Proc. Natl. Acad. Sci. USA 87 (1990) 9309
dependent elevation of [Ca2"],y, in guard cells from C.communes.
The more repetitive nature of Ca2" elevationsobserved in the
present study as well as subsequent extrusionof Ca2" may account
for the difficulty in resolving ABA-induced changes in 45Ca2"
fluxes in leaf epidermi (for detaileddiscussion, see ref. 34).
Small 45Ca2" flux changes in ABA-treated epidermi (34) may also be
attributed to the recentfindings of several groups that show that
stomata respond toABA treatment by closing in small domains and by
remainingopen in other small domains of the leaf epidermis (7-11).
Thisdifferential ABA response has been suggested as the basis
forthe physiologically observed regulation of photosyntheticcarbon
fixation by ABA (7-11). The nonuniform occurrenceof ABA-induced
stomatal closure in leaves (7-11) may also beresponsible for our
findings that approximately one-third ofthe stomata in the
epidermis of V. faba leaves closed afterexposure to ABA and 37% of
the guard cells studied by patchclamping responded to ABA.
Physiological modification ofthe primary ABA signaling mechanisms
may account for theobservation of nonuniform closure of stomata by
ABA. Thecorrelation between physiological ABA responses in
leaves(7-11) and results on ABA-activated Ca2"-permeable chan-nels
from the present study calls for further investigation todetermine
whether modification of initial events in ABA-mediated signal
transduction contribute to physiological ef-fects of ABA on
photosynthetic carbon fixation in leaves(7-11).
Effects of [Ca2J1cyt on Guard-Cell Ion Transport. Increasesin
[Ca2J]cy, have been suggested to play a key role in
inducingstomatal closing by ABA (refs. 2-4 and 16; for reviews,
seerefs. 5, 6, and 35). ABA-induced stomatal closing is mediatedby
release of K+ and anions from guard cells (13, 14).Increases in
[Ca2+]CYJ to the micromolar level have beenshown to inhibit inward
rectifying K+ channels and to acti-vate voltage-dependent anion
channels (4). The resultingefflux of anions through anion-selective
channels (15) could,in turn, depolarize guard cells sufficiently
(4) to activateoutward rectifying K+ channels (12, 17). In addition
ABA-dependent enhancement of K+ currents has been reported(36).
Simultaneous activation of anion channels and K+channels would
permit ion efflux across the plasma mem-brane required for stomatal
closing (4-6).
Equivocal comparison of steady-state anion currents (15)with
non-steady-state anion currents (4) has led to the sug-gestion that
two types of anion channels prevail in the plasmamembrane of guard
cells with different voltage dependencies(15). However, direct
comparison of steady-state currentsshows that the voltage
dependencies of Ca2'-activated anioncurrents (4) and single-anion
channel currents (15) are similar(37). In the present study
ABA-activated Ca2'-permeablecurrents could be investigated in the
absence of anion chan-nels due to the inactivation and wash-out of
these voltage-dependent anion channels (4, 37).
Previous results have suggested that other second messen-gers,
in addition to [Ca2+]CYt, may be important for
regulatingvoltage-dependent anion channels (4, 37). Additional
second-messenger processes may be triggered by ABA, as the
ABAactivation of Ca2+-permeable channels appears to occur bymeans
of intermediate coupling mechanisms (see above).Whole-cell
patch-clamp experiments executed in conjunctionwith [Ca2+]cyt
measurements, as used here, may allow furtherinsight into initial
signal-transduction events of ABA-mediated stomatal closure.
We thank H. Fang for careful handling of protoplast
preparationsand Dr. J. Kourie for comments on the manuscript. We
thank Drs.R. Penner and J. Vergara for help and advice during
initial Ca2+
measurements and Dr. E. Tobin for the use of growth chambers.
Thisresearch was supported by National Institutes of Health
GrantNS09012-21 to the late S. Hagiwara and by Alexander von
Humboldtand National Institutes of Health Fellowships to J.1.S.
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