Download Co-ordination of signalling elements in guard cell ion

Journal of Experimental Botany, Vol. 49, Special Issue, pp. 351–360, March 1998
Co-ordination of signalling elements in guard cell ion
channel control
A. Grabov1 and M.R. Blatt
Laboratory of Plant Physiology and Biophysics, Wye College, University of London, Wye, Ashford,
Kent TN25 5AH, UK
Received 3 November 1997; Accepted 10 November 1997
Abstract
Fine regulation of solutes transport across the guard
cell plasma membrane for osmotic modulation is
essential for the maintenance of the proper stomatal
aperture in response to environmental stimuli. The
major osmotica, K+, Cl− and malate are transported
through selective ion channels in the plasma membrane and tonoplast of guard cells. To date, a number
of ion channels have been shown to operate in the
guard cell plasma membrane: outwardly- and inwardlyrectifying K+ channels (I
and I
), slowly- and
K,in
K,out
rapid-activating anion channels, and stretch-activated
non-selective channels. Slow and fast vacuolar channels (SV and FV) and voltage-independent K+-selective
(VK) channels have been found at the guard cell tonoplast. On the molecular level, the regulation of the
stomatal aperture is achieved by precise spatial and
temporal co-ordination of channel activities through a
network of signalling cascades that can be triggered,
among others, by plant hormones. ABA and auxin as
regulators of stomatal aperture have received most
attention to date. It is clear now that the effect of
these hormones on ion channels is mediated by second
messengers pH and [Ca2+] . The effect of ABA is geni
i
erally associated with increases in pH , while auxin
i
acidifies the cytosol. Both of these hormones may
induce elevation in [Ca2+] . Phosphorylation is another
i
important factor in cellular signalling. The ABI1 gene
encoding a 2C-type protein phosphatase has been
shown to be a key element of ABA-dependent cascades. Plasma membrane voltage is also an important
component of signalling and channel control, and has
recently been shown to influence cytosolic-free
[Ca2+]. Thus, transduction of these, and associated
cytoplasmic signals is clearly non-linear, and is prob1 To whom correspondence should be addressed. Fax: +44 1233 813 140.
© Oxford University Press 1998
ably important for providing a plasticity of cellular
response to external and environmental stimuli.
Understanding the interdependence and hierarchy of
signalling elements now presents a major challenge
for research in plant biology.
Key words: Ion channels, stomatal aperture, signalling
pathway, second messengers, guard cells.
Introduction
Guard cell control of stomatal aperture is crucial to
balancing transpiration stream and gas exchange for
photosynthesis. As transpiration and CO exchange can
2
not be regulated independently, the demand of photosynthetic machinery can lead to excessive water evaporation
under adverse environmental conditions. Thus, the size
of the stomatal aperture optimizes gas exchange for
overall plant performance. This balance of requirements
is achieved by the precise tuning of guard cell turgor
pressure and on the molecular level is accomplished by
means of fine temporal and spatial co-ordination of
different solute transporters. It is well known that K+,
Cl−, malate, and some other organic anions are the major
contributors to the guard cell osmolarity ( Willmer and
Fricker, 1996). Guard cell response to the variety of the
hormonal and environmental stimuli is due to the rapid
transport of these ions across the plasma membrane and
tonoplast ( Willmer and Fricker, 1996; Blatt and Grabov,
1997a, b).
The variety of environmental and hormonal stimuli
affecting stomatal aperture is enormous and includes CO ,
2
light, temperature, gaseous pollutants, as well as all of
the major plant hormones. Remarkably, on the cellular
level these stimuli appear to converge on a few cytoplasmic signals, known as second messengers, and these
352
Grabov and Blatt
in turn regulate a handful of transport proteins. Second
messengers like cytosolic-free H+ (pH ) and Ca2+
i
([Ca2+] ) are key components of signal transduction
i
chains in stomata aperture regulation (Blatt and Thiel,
1993; Assmann, 1993; Bush, 1995; McAinsh et al., 1997;
Blatt and Grabov, 1997b). Phosphorylation of some
signal cascade and target elements also contributes to
these events (Pei et al., 1996, 1997; Blatt and Grabov,
1997a; Grabov et al., 1997). Finaly, membrane voltage is
an essential in controlling transport proteins in the membrane, the activities of which are voltage sensitive, and in
second messenger generation (Thiel et al., 1992).
Guard cell ion channels
Plasma membrane channels
There are at least three different types of ion channels in
the guard cell plasma membrane implicated for the regulation of stomatal aperture (Blatt, 1991; Blatt and Grabov,
1997a): outwardly rectifying K+ channels (I
),
K,out
inwardly rectifying K+ channels (I ), and anion chanK,in
nels. Experimental factors that control the activities of
these channels have given us much insight into possible
regulatory mechanisms even without reference to any
external stimuli.
Inwardly rectifying K+ channels are involved in K+
uptake during stomatal opening (Schroeder, 1988; Blatt,
1988, 1991; Grabov and Blatt, 1997). I
is only moderK,in
ately sensitive to membrane voltage showing an apparent
gating charge of 1.3–1.7 (Blatt, 1992; Grabov and Blatt,
1997). However, I is strongly dependent upon apoplastic
K,in
pH. Increasing [H+] outside promotes the K+ current in
a voltage-dependent maner shifting V by 22 mV per pH
1/2
decade and accelerating activation kinetics (Blatt, 1992).
At neutral external pH these channels are activated by
membrane voltages more negative than −100 mV with
V close to −200 mV, but at pH 6.0 I
is gated at
1/2
K,in
more positive voltages (V #−180 mV ). The current is
1/2
also sensitive to cytosolic pH. Lowering pH from pH 7.6
i
to 6.9 stimulates I
by a factor of up to 6. (Grabov and
K,in
Blatt, 1997). The channel activity is also reduced at elevated
[Ca2+] (Schroeder and Hagiwara, 1989; Lemtiri-Chlieh
i
and MacRobbie, 1994; Grabov and Blatt, 1997). By
contrast to the situation with pH no kinetic detail is
available for I
activity and its gating characteristics as
K,in
a function of [Ca2+] .
i
The outwardly rectifying K+ channel is activated by
depolarization and has been demonstrated to contribute
to K+ efflux during stomatal closure. This channel is
up-regulated by pH increases and is virtually insensitive
i
to [Ca2+] (Hosoi et al., 1988; Blatt and Armstrong, 1993;
i
Lemtiri-Chlieh and MacRobbie, 1994; Grabov and Blatt,
1997). I
voltage sensitivity and kinetics are dependent
K,out
upon the K+ gradient across the plasma membrane (Blatt,
1988; Blatt and Thiel, 1993). Activation and deactivation
kinetics of I
in Vicia faba guard cells slow with
K,out
elevation of external K+ and voltage dependent opening
follows E ensuring that I
operates as one-way valve
K
K,out
to prevent reflux of K+ into the guard cell. Recent studies
have shown that I
activation is dependent on the
K,out
co-operative interaction of 2 K+ ions with the channel,
but at a site (or sites) distinct from the channel pore. The
apparent K
for interaction was strongly voltage1/2
dependent, accounting for the equivalence of (negative)
membrane voltage and [ K+] in regulating channel activity
(Blatt and Gradmann, 1997).
A positive shift in the plasma membrane potential is
essential for I
activation when the membrane voltage
K,out
is situated negative of E . Plasma membrane depolarizaK
tion under these conditions is evoked by an inward
current (Thiel et al., 1992; Ward et al., 1995; Blatt and
Grabov, 1997b). At least one component of this current
has been suggested to be mediated by anion channels
(Hedrich et al., 1990; Thiel et al., 1992; Schroeder and
Keller, 1992). Two types of anion current with markedly
different kinetics have been identified in the guard cell
plasma membrane. One of these currents demonstrates
rapid activation on membrane depolarization (t =
1/2
50 ms) and inactivates with t ~10 s at voltages near to
1/2
and positive of −100 mV ( Keller et al., 1989; Hedrich
et al., 1990). The second anion current has been shown
to activate and deactivate slowly (t =5–30 s) and shows
1/2
no time-dependent inactivation (Schroeder and Keller,
1992; Linder and Raschke, 1992).
Of these two anion currents, the slow current is implicated for sustained long-term depolarization (Schroeder
and Hagiwara, 1989; Schroeder and Keller, 1992; Linder
and Raschke, 1992; Grabov et al., 1997). Both types of
anion currents are activated with increasing [Ca2+] and
i
exhibit roughly similar single channel conductance. In
fact it has been suggested that the same channel protein
may account for both gating modes (Blatt and Thiel,
1993). Indeed, changes in anion current kinetics are
known to occur in response to ATP depletion ( Thomine
et al., 1995), ABA stimulation or phosphorylation
(Grabov et al., 1997). Schulz-Lessdorf et al. (1996) have
argued that fast activating anion channels recognize the
H+ gradient across the plasma membrane. Regardless of
the relationships between the two anion currents, the fast
activating form can be ruled out as a major pathway for
anion efflux during stomatal closure. Its narrow voltage
range for activation and the fact that the current inactivates within seconds cannot be reconciled with the requirement for sustained channel functioning over periods of
20–30 min or more during closure (Blatt and Grabov,
1997a; Blatt and Thiel, 1993).
Tonoplast channels
As the vacuole occupied of approximately 80–90% of the
cell volume, this organelle dominates guard cell osmoreg-
Co-ordination of ion channels in guard cells 353
ulation. Much of the solute lost during stomatal closure
originates from the vacuole. Equally, osmotic accumulation on stomatal opening is associated predominantly
with this storage organelle. The importance of vacuole in
this function implies a high degree of transport
co-ordination at the tonoplast and between the two
membranes (MacRobbie, 1995a, b).
Current knowledge of tonoplast ion transport relevant
to stomata movement remains relatively poor. To date,
patch-clamp measurements have uncovered three different
types of cation-selective channels operating at the tonoplast. One channel recently identified appears to be voltage-independent and selective for K+ and has been
designated the VK channel ( Ward and Schroeder, 1994;
Allen and Sanders, 1996). This channel is activated by
elevated [Ca2+] within physiological range (Allen and
i
Sanders, 1996). Two other channels originally identified
at the tonoplast have been designated as SV and FV for
Slow and Fast Vacuolar, respectively. The SV channel is
voltage-, H+- and Ca2+-dependent and is permeable to
Ca2+ and K+ ( Ward and Schroeder, 1994; SchulzLessdorf and Hedrich, 1995; Allen and Sanders, 1996).
Estimates of the permeability of this channel are highly
variable ( Ward and Schroeder, 1994; Schulz-Lessdorf and
Hedrich, 1995; Allen and Sanders, 1996). The FV channel
has also been shown to be voltage-dependent, to be
inhibited by high [Ca2+] , and to activate and deactivate
i
rapidly (Hedrich and Neher, 1987). Recent findings have
been estimated K+/Cl− selectivity of this channel in
barley mesophyll to be as high as 3051, indicating the
importance of this channel for tonoplast electrogenesis
and K+ transport ( Tikhonova et al., 1997). It now
appears that all three cation channels co-exist in the
guard cell vacuole (Allen and Sanders, 1996), raising the
possibility that their interaction may be important for
guard cell function. Finally an anion channel with high
selectivity for Cl− over K+ has recently been found at
the tonoplast of Vicia faba guard cells. The activity of
this channel is dependent on protein phosphorylation (Pei
et al., 1996). This channel could function in anion uptake
during stomatal opening, however, no direct evidence is
currently available.
Ion channel control
In parallel with this growing catalogue of ion channels,
our understanding of mechanisms by which these channels
are controlled has expanded dramatically over the past
decade. Most important for this development, the link
between stomatal movement and the hormone abscisic
acid has provided a key physiological basis from which
to explore ion channel regulation. Abscisic acid acts as a
universal stress signal in plants. It accumulates in the leaf
tissues under adverse environmental conditions including
drought and high salinity and promotes stomatal closure
preventing transpirational water loss ( Willmer and
Fricker, 1996).
H+ signalling in guard cell cytoplasm
The protonic messenger was first proposed to be major
signalling element in the guard cell when it was recognized
that ABA action on stomata is strictly associated with
cytoplasm alkalinizations of 0.2–0.4 pH units (Gehring
et al., 1990; Irving et al., 1992; Blatt and Armstrong,
1993; Blatt and Thiel, 1993). The key role of H+ messenger for stomatal function has been confirmed in the
experiments in which ABA-induced stomatal closure was
blocked when pH was clamped at a constant low value
i
(Blatt and Armstrong, 1993). On the molecular level,
ABA induced stomatal closure is accomplished by activation of I
(Blatt and Armstrong, 1993) and anion
K,out
channels (Grabov et al., 1997), providing electrically
neutral solute efflux from the guard cells. I
was found
K,out
to be strongly stimulated by increasing pH (Blatt, 1992;
i
Blatt and Armstrong, 1993; Miedema and Assmann,
1996; Grabov and Blatt, 1997), suggesting it as the major
target of H+ signalling. Like the effect on stomatal
closure, suppressing changes in pH effectively blocked
i
the I
response to ABA (Blatt and Armstrong, 1993).
K,out
The effects of ABA mediated through pH are also
i
evident in the characteristics of the other major K+
channel at the plasma membrane. I
was found to
K,in
exhibit a sensitivity to pH inverse to that of I
(Blatt,
i
K,out
1992; Blatt and Armstrong, 1993; Grabov and Blatt,
1997), and in line with the cytosolic alkalinization
imposed by ABA. pH , however, accounted only in part
i
for this effect (Blatt and Armstrong, 1993), suggesting an
intervention of another cytosolic factor, very likely [Ca2+]
i
(Schroeder and Hagiwara, 1989; Blatt and Thiel, 1993;
Lemtiri-Chlieh and MacRobbie, 1994; Grabov and
Blatt, 1997).
It is worth noting that work with another plant hormone auxin has also implicated a role for pH signalling.
i
Auxin at low concentration is known to open stomata
(Snaith and Mansfield, 1982; Marten et al., 1991) and
has been shown to reduce cytosolic pH (Irving et al.,
1992). Mobilization of the protonic messenger after treatment with low concentration of auxin activates I
K,in
providing pathway for K+ influx (Blatt, 1992; Blatt and
Thiel, 1994; Grabov and Blatt, 1997). K+ uptake is
driven by the H+ pump, that, probably, also is enhanced
by [ H+] elevation (Blatt, 1987; Kurkdjian and Guern,
i
1989; Felle, 1989). The effect of auxin on stomatal
movement, however, shows two distinct phases. While
stomatal opening is promoted at low micromolar concentration, the effect of auxin at high concentrations (100 mM
and above) is to close stomata (Marten et al., 1991; Lohse
and Hedrich, 1992). I
inhibition by high auxin concenK,in
trations parallels this behaviour (Blatt and Thiel, 1994).
354
Grabov and Blatt
The crucial role of pH in auxin signalling has been
i
elucidated in experiments where the H+ signal was disrupted by pH buffering (Blatt and Thiel, 1994). No effect
i
of auxin on I
was observed under these experimental
K,in
conditions. Furthermore, peptide homologous to the
C-terminus of the maize auxin binding protein mimicked
high auxin concentrations and imposed a rise in cytosolic
pH. The effect again was to reduce I
activity and
K,in
cytosolic buffering abolished the peptides effect on I
K,in
( Thiel et al., 1993).
Experimentally, cytosolic acidification can be achieved
by an addition of weak acids to the bath where protonated
and dissociated forms of acid come to equilibrium (Blatt,
1992; Blatt and Armstrong, 1993; Grabov and Blatt,
1997). Provided the protonated form easily crosses the
plasma membrane it will dissociate in the cytoplasm as
pH is normally higher than the weak acid pK . This
i
a
approach has been used to clamp pH imposing acid loads
i
on the cell and hence to titrate guard cell K+ channels
against pH as the pH shift is strictly dependent on the
i
i
weak acid concentration in the bath (Grabov and Blatt,
1997).
On the basis of such experiments, low pH was found
i
to stimulate I
while inactivating I
(Blatt, 1992;
K,in
K,out
Blatt and Armstrong, 1993; Grabov and Blatt, 1997).
The precise mechanism of this interaction remains
obscure. However, some conclusions may be drawn from
these experiments. Voltage-dependent gating of both I
K,in
and I
is practically unaffected by the cytosolic pH.
K,out
Thus, in each case the maximal channel conductivity
(g
) was affected without a change in V . For I ,
Kmax
1/2
K,in
acid pH stimulated the current, while g
of outward
Kmax
rectifier was down-regulated under the same conditions
(Grabov and Blatt, 1997). Elevated levels of H+ in the
cytosol could screen membrane surface charge and, hence,
distort the total electrical field encountered by the channel
( Hille, 1992). This mechanism of H+ action on the guard
cell K+ channels can be ruled out because the effect of
low pH on both I
and I
was practically voltagei
K,in
K,out
independent at least over pH range from 6.9 to 8.0 (Blatt,
i
1992; Blatt and Armstrong, 1993; Miedema and Assmann,
1996; Grabov and Blatt, 1997).
It appears more likely that pH affects the number of
i
channels (N ) that can be gated by transmembrane voltage.
Maximal conductivity of the channel ensemble is the
product of N and the single channel conductivity (c ).
K
Thus, H+ binding could affect either c or N. For I
K
K,in
competition between H+ and K+ for a common binding
site within the channel pore (Hille, 1992) can also be
ruled out. Increasing H+ concentration stimulated the
current whereas competition would be expected to block
it. Such a mechanism could explain a decrease in I
.
K,out
However H+ competition is known to lead to flickery
block that can appear as a reduction in single-channel
conductance. These effects were not detected in patch
clamp studies of the outward rectifier (Miedema and
Assmann, 1996). In short, it is most likely that I
is
K,out
regulated by pH through allosteric interactions that result
i
in an increase of the pool of active channels (Blatt and
Armstrong, 1993; Miedema and Assmann, 1996; Grabov
and Blatt, 1997).
The effect of pH on the inward rectifier has yet to be
i
studied at the single channel level. However, the Hill
model was well fitted to the pH dependence of g
also
Kmax
in the case of I
As fittings were based essentially on
K,in.
the assumption that H+ affects the availability of the
channels for voltage-dependent gating, it is suggested that
the mechanism of I
regulation by cytosolic H+ is
K,in
similar but antiparallel to I
(Grabov and Blatt, 1997).
K,out
Titration of g
against pH indicated that I
has one
Kmax
i
K,in
proton binding site with pK =6.3 suggesting the involvea
ment of histidine in H+ binding. Titration of an I
K,out
yielded a Hill coefficient of 2 with an apparent pK of 7.4
a
that was rather close to the resting pH =7.6.
i
The events immediately upstream of H+ signalling
remains to be studied. Buffering capacity of the guard
cell is enormous and corresponds to an apparent cytosolic
buffer concentration of 275 mM with pK =6.9. These
a
calculations indicate that approximately 30 mM of H+
are bound (or released) by the cytosolic H+ binding sites
when pH is modified by 0.2 pH units (Grabov and Blatt,
i
1997). Where this huge H+ flux originates from is unclear.
It is very likely that the vacuole is the source of protons
when the guard cell is stimulated by auxin, as this plant
hormone failed to acidify the cytoplasm in the vacuolefree protoplasts (Frohnmeyer et al., 1997).
Ca2+ as second messenger
In animal tissues Ca2+ is well known as a second messenger controlling many cellular processes including fertilization, cell growth, secretion, sensory perception, and
neuronal signalling (Berridge, 1993; Bootman and
Berridge, 1996). The evidence accumulated in the last
decade points to a similar role for Ca2+ in the signal
transduction cascades in plants. In guard cells, environmental stress, pathogens and plant hormones have been
shown to evoke increases in [Ca2+] . Schroeder and
i
Hagiwara, 1990; McAinsh et al., 1990; Knight et al.,
1991; Irving et al., 1992; Bush, 1993; McAinsh et al.,
1997). ABA-induced stomatal closure may be associated
with increased [Ca2+] as well. However, the physiological
i
significance of ABA-dependent Ca2+ signalling is still
questionable, as stomata may close in ABA without any
visible [Ca2+] elevation (Gilroy et al., 1991; McAinsh
i
et al., 1992; Allan et al., 1994). It is very likely therefore
that both Ca2+-dependent and Ca2+-independent signalling cascades transduce the ABA signal in the guard cell
(MacRobbie, 1992). The switch between these two signalling pathways may be triggered by ambient temperature
(Allan et al., 1994).
Co-ordination of ion channels in guard cells 355
Another source of concern about the Ca2+ second
messenger has been its widespread occurrence and ability
to mediate sometimes opposing responses. [Ca2+]
i
increases may lead to such different events as stomatal
opening after stimulation by auxin or stomatal closure
after stimulation by ABA (Bush, 1993; McAinsh et al.,
1997). It is not yet known how the Ca2+ signal is targeted.
However, the kinetics of [Ca2+] elevation (the ‘Ca2+
i
signature’) is probably important for the encoding of
information contained in the cytoplasmic signal. [Ca2+]
i
oscillations are well known in animal cells, where [Ca2+]
i
waves can be shown to propagate within the cytoplasm
or between adjacent cells (Boitano et al., 1994; Hajnóczky
and Thomas, 1997). The information carried by the
[Ca2+] signal can be encoded by the frequency and
i
magnitude of such oscillations (Dolmetsch et al., 1997).
Similar [Ca2+] transients and oscillations have frequently
i
been observed in plant tissues including guard cells in
response to environmental or hormonal stimuli
(Schroeder and Hagiwara, 1990; Knight et al., 1991;
McAinsh et al., 1995; Johnson et al., 1995).
The mechanism of [Ca2+] fluctuations in the plant cell
i
cytoplasm is not yet understood. Recent findings, however, indicate that elevation of [Ca2+] may be triggered
i
by the plasma membrane hyperpolarization. Work from
this laboratory (Grabov and Blatt, 1998) has shown that
[Ca2+] increases can be driven by membrane voltage
i
transitions between voltages near −40 mV and −200
mV ( Fig. 1). Oscillations in free-running voltage can be
recorded at the guard cell plasma membrane (Gradmann
et al., 1993; Blatt and Thiel, 1994). These oscillations are
similar to cardiac action potentials, but occur with periods
of s or even min. Co-existence of voltage-dependent
[Ca2+] increases ( Fig. 1) and action potential-like voltage
i
oscillations may explain the [Ca2+] transients observed
i
Fig. 1. Hyperpolarization of the plasma membrane evokes [Ca2+]
i
increases. Measurement from a single Vicia guard cell in an epidermal
strip bathed in 5 mM Ca2+–MES, pH 6.1 with 10 mM KCl. The guard
cell was loaded with fura-2 iontophoretically. [Ca2+] was recorded as
i
fura-2 fluorescence ratio (F /F , low panel ). Plasma membrane was
340 390
hyperpolarized under 2-electrode voltage clamp by a 20 s voltage pulse
from −40 to −200 mV (upper panel ).
in the guard cells (McAinsh et al., 1997), since repetitive
[Ca2+] elevation may be triggered by the hyperpolarizi
ation phase of membrane voltage oscillations.
Potentially, there are two pathways for elevating
[Ca2+] : (1) Ca2+ influx across the plasma membrane and
i
(2) Ca2+ release from internal stores. Both mechanisms
function in animal tissues, including cardiac muscles and
neurons, and give rise to increases in [Ca2+] through
i
processes of Ca2+-induced Ca2+ release (CICR;
(Berridge, 1993)). In these tissues, small [Ca2+] increases
i
triggered by Ca2+ flux through plasma membrane Ca2+
channels induce explosive Ca2+ release from internal
stores (Berridge, 1993; McPherson and Campbell, 1993;
Ehrlich et al., 1994; Sitsapesan et al., 1995). Similar
mechanisms of CICR may operate in plant cells. At least
some elements that could contribute to CICR are known
to occur in plants ( Ward and Schroeder, 1994; Sanders
et al., 1995).
There are a few direct indications that Ca2+ channels
operate in the plant cell plasma membrane. Ca2+-permeable channels activated by depolarization were found
at the carrot cell plasma membrane ( Thuleau et al.,
1994b). These channels also appear to be recruited at
depolarized voltages into an active pool, as could be
determined from prolonged prepulses ( Thuleau et al.,
1994a). Piñeros and Tester found Ca2+ channels in cells
of wheat roots (Piñeros and Tester, 1995). These channels
were activated at the voltages more positive than −130
mV and were proposed to function as mediators of
nutrient uptake. There has also been evidence of [Ca2+]
i
transients in guard cell protoplasts triggered by ABA.
Schroeder and Hagiwara (1990) proposed that these
transients were associated with the activation at the
plasma membrane of Ca2+-permeable channels. Recently,
Ca2+ channels activated by hyperpolarization have been
found in tomato cells (Gelli and Blumwald, 1997).
Tension-activated Ca2+-selective channels, have been
indicated to function in onion epidermal cells (Ding and
Pickard, 1993; Pickard and Ding, 1993). At the guard
cell plasma membrane, there is evidence of Ca2+permeable channels that may be activated by mechanical
stress (Cosgrove and Hedrich, 1991). As the regulation
of osmolarity is central for stomata physiology, these
channels could be important for feedback control of
solute flux during stomatal movements.
Somewhat more information is to hand about mechanisms of Ca2+ release within guard cells. In animal cells
two major families of Ca2+ channels, identified as IP
3
and ryanodine receptors, share remarkable structural
similarity and mediate Ca2+ release from internal stores
(Berridge, 1993). It appears that both of these Ca2+release mechanisms operate in plants. The rise of [Ca2+]
i
triggered by IP (and the consequent inactivation of I )
3
K,in
was directly demonstrated by photolysis of caged IP
3
injected into guard cells (Blatt et al., 1990; Gilroy et al.,
356
Grabov and Blatt
1990). Ryanodine receptors of animal cells (RyR) are
usualy localized on a modified part of the ER (Berridge,
1993). Although, there is an indication for Ca2+ release
channels in the ER of higher-plant mechanoreceptor
organs ( Klüsener et al., 1995), no channels with sensitivity
to ryanodine or cADPR (a cytoplasmic ligand for RyR;
Sitsapesan et al., 1995) have been found in the plant cell
ER to date. It is most probable that plant RyR-like
channels are located at tonoplast, hence the release of
Ca2+ from vacoular-enriched microsomes of beet was
triggered by cADPR (Allen et al., 1995; Muir and
Sanders, 1996).
A special mechanism of CICR has been suggested to
function in Ca2+ mobilization in plant cells in which
vacuolar SV channels mediate Ca2+ release and act in
concert with K+-selective VK channels ( Ward et al.,
1995). The proposal is consistent with the co-existence of
these channels in guard cell vacuoles (Allen and Sanders,
1996). However, the contribution of SV channels to
CICR has been questioned, because the channel open
probability is extremely low at the physiologically relevant
tonoplast voltages, vacuolar and cytosolic [Ca2+]
(Pottosin et al., 1997).
Elevation of [Ca2+] has no effect on I
(Schroeder
i
K,out
and Hagiwara, 1989; Blatt and Thiel, 1993; LemtiriChlieh and MacRobbie, 1994), while [Ca2+] has been
i
demonstrated to be a very potent regulator of I
and
K,in
the anion current at the guard cell plasma membrane
(Schroeder and Hagiwara, 1989; Hedrich et al., 1990;
Blatt and Thiel, 1993; Lemtiri-Chlieh and MacRobbie,
1994; Kelly et al., 1995). Elevating [Ca2+] from 0.1 to
i
above 1 mM has been shown to activate the anion current
and to inactivate I
almost completely (Schroeder and
K,in
Hagiwara, 1989; Kelly et al., 1995). Details of the mechanisms by which [Ca2+] acts on these channels is less
i
clear. Increasing [Ca2+] affects the voltage-dependence of
i
gating rather than single channel conductance giving rise
to I
inactivation (Schroeder and Hagiwara, 1989,
K,in
Grabov and Blatt, 1997).
It is interesting that I
was largely unaffected by high
K,in
[Ca2+] when EGTA was used as the Ca2+ buffer in patchi
clamp experiments ( Kelly et al., 1995). Kelly et al. (1995)
suggested that this dependence on the buffer was related
to the dynamics of Ca2+ binding with the buffer.
However, as it is not clear how the kinetic properties of
the Ca2+ buffer affect steady-state [Ca2+] , it is difficult
i
to explain the difference in I
characteristics between
K,in
the two buffers. Whatever the explanation for their observations, the data make it clear that estimating the Ca2+sensitivity of I
in vivo is not straightforward in patch
K,in
clamp experiments.
Roles for protein phosphorylation in guard cell signalling
The phosphorylation state of key regulatory (or target)
proteins may drastically affect ion transport. At the guard
cell plasma membrane these effects are seen in H+ pumping (Shimazaki et al., 1992; Li and Assmann, 1996), K+
channels (Luan et al., 1993; Li and Assmann, 1996; Thiel
and Blatt, 1997) and slow anion channels (Schmidt et al.,
1995; Grabov et al., 1997). At the tonoplast SV channels
have been shown to be regulated by calcineurin (protein
phosphatase 2B) (Allen and Sanders, 1995). The recognition of phosphatase-kinase activities in guard cells
signalling was boosted in recent years by the cloning of
the ABI1 gene, the mutation of which leads to a loss of
sensitivity to ABA in Arabidopsis ( Koornneef et al.,
1984). Sequence and biochemical analysis of the gene
product has confirmed that it functions as a 2C-type
protein phosphatase (Leung et al., 1994; Meyer et al.,
1994; Bertauche et al., 1996). Thus, the abi1 mutant
provides an invaluable tool for the study of ABA
signalling.
Armstrong et al. (1995) have since linked the mutant
phenotype with abberant control of the guard cell K+
channels using transgenic N. benthamiana plants carrying
the mutant abi1-1 gene. These studies showed that the
abi1–1 gene not only reduced I
in the absence of
K,out
ABA, but eliminated the response of the current in its
presence. The transgene was also found to interfere with
ABA-evoked control of I
and to block stomatal closK,in
ure. Furthermore, the background of I
activity, as
K,out
well as I
and I
responses to ABA and stomatal
K,in
K,out
closure could be ‘rescued’ by treatments with protein
kinase antagonists. So, how can abi1 gene action be
understood? The simplest explanation is that the (dominant) mutant protein phosphatase interferes with a wildtype homologue in tobacco preventing protein dephosphorylation. The kinase antagonists, then, redress the
consequent imbalance in phosphorylation of the target
protein(s) to reinstate K+ channel sensitivity to ABA.
This interpretation is entirely consistent with the phosphatase activities shown by the wild-type and mutant
gene products (Bertauche et al., 1996).
Anion channel function is also known to respond to
protein (de-)phosphorylation. Evidence from pharmacological studies have shown that protein phosphatase antagonists such as okadaic acid and calyculin A (both
protein phosphatase 1/2A antagonists) alter anion channel
characteristics ( Thiel and Blatt, 1994) and rundown in
patch electrode recordings of guard cell protoplasts
(Schmidt et al., 1995). Recently, slow anion channels
were found to be activated by ABA in guard cells of N.
benthamiana (Grabov et al., 1997), and Arabidopsis (Pei
et al., 1997). Again the activation was dependent on the
phosphorylation state of the proteins involved in the
signal transduction cascade. Pei et al. (1997) compared
anion currents measured either in the presence or absence
of ABA and/or with additions of okadaic acid. Statistical
comparisons indicated that the current was activated by
ABA and that this activation could be suppressed by
Co-ordination of ion channels in guard cells 357
okadaic acid. The effect appeared to be a purely scalar
increase in the number of channels in the active pool and
was not observed in abi1 mutant plants. Grabov et al.
(1997), by contrast, made use of intracellular microelectrode methods, recording anion currents from individual
guard cells before and throughout treatments with ABA
and calyculin A. They found that ABA reversibly stimulated the anion current and that this stimulation was
associated with alterations in the kinetics of voltagedependent activation that favoured the active state of the
channels. Grabov et al. (1997) also observed that calyculin A acted synergistically with ABA in promoting the
anion current, and that the effects in every case were
independent of the abi1 transgene.
How can these two sets of data be reconciled? Pei et al.
(1997) based their assessment on a contentious comparison of the Arabidopsis anion current in the presence of
ABA with the N. benthamiana current (Grabor et al.,
1997) recorded in the absence of ABA. In fact, resting
activities of the anion channels were roughly equivalent,
despite the obvious difference of species and very different
methods used to record these currents. It is likely that
subtle differences in kinase/phosphatase cascades mean
that Arabidopsis and N. benthamiana respond differently
to protein phosphatase antagonists (compare also
Schmidt et al., 1995), and this factor might well explain
the discrepancy in action of the abi1 gene product. Equally
important, technical differences—including exchange of
the cytosol with patch electrode filling solutions (Pei
et al., 1997)—raises the possibility that different parts of
the same kinase/phosphatase cascade could be favoured
in each case. Regardless of these complications, the fact
that both sets of experiments demonstrate an action of
ABA on the anion current is salutary.
Interaction between signalling elements and its significance
for stomata regulation
It comes as no surprise that signalling pathways should
interact, even when evoked by a single stimulus such as
ABA ( Trewavas, 1992). Sufficient evidence is now to
hand to demonstrate interactions between signalling elements of protein (de-)phosphorylation, [Ca2+] and pH .
i
i
What is less clear at present are the points of interaction
and the extent to which the various signals are interdependent. These issues are particularly relevant both to
determining the hierarchy of events behind stimulusresponse coupling per se, and to establishing the role(s)
of each element, whether primary to signal transmission
or secondary in adaptive conditioning of the response.
Some progress is now being made through epistatic and
equivalent physiological analyses. However, these problems will continue to pose major challenges to understanding guard cell transport control, as it does to plant cell
signalling generally.
A case in point can be drawn from work on pH and
i
[Ca2+] signals evoked by ABA. From analysis of the
i
current kinetics and voltage-dependence, it is evident that
pH and [Ca2+] controls of I
are fundamentally differi
i
K,in
ent (Schroeder and Hagiwara, 1989; Blatt and Armstrong,
1993; Grabov and Blatt, 1997). Nonetheless, these two
signalling elements almost certainly interact as well in
guard cells. Grabov and Blatt (1997) observed that experimentally imposing a decrease in pH below about 7.0
i
resulted in the rise of [Ca2+] in approx. 50% of the cells
i
examined. The precise mechanism of this pH-induced
[Ca2+] rise is unknown. One possible explanation lies in
i
the pH-sensitivity of Ca2+ release mechanisms within the
cell, for example the binding of IP to its receptor that
3
leads to cytosolic Ca2+ release (Busa, 1986), although it
is normally alkaline rather than acid pH favours IP i
3
mediated Ca2+ release (Taylor and Richardson, 1991).
In the guard cells pH is known to affect the gating of
i
vacuolar ion channels that may contribute to [Ca2+]
i
changes either directly or indirectly ( Ward and Schroeder,
1994; Schulz-Lessdorf and Hedrich, 1995). It is also
possible that pH could affect Ca2+ influx across the
i
plasma membrane.
Elevation of [Ca2+] in turn potentially may affect
i
phosphorylation cascades. Protein kinases and phosphatases dependent on Ca2+ are ubiquitous in animal cells
and serve as one point of convergence for Ca2+ signalling
and protein phosphorylation cascades. In plant cells there
is now considerable evidence for Ca2+-, Ca2+/calmodulinand Ca2+/phospholipid-dependent kinases (Nickel et al.,
1991; Shimazaki et al., 1992; Watillon et al., 1993) and
phophatases (Luan et al., 1993; Allen and Sanders, 1995).
Calcium-dependent protein kinase activity is known to
activate an ABA-responsive promoter in Arabidopsis leaf
protoplasts (Sheen, 1997) and a Cl− channel in the guard
cell tonoplast (Pei et al., 1996). It is interesting that the
ABI1 gene product, although it contains a putative
EF-hand Ca2+-binding motif and was expected to be
Ca2+-regulated, has proven to be insensitive to [Ca2+]
i
(Bertauche et al., 1996).
Finally, protein phosphorylation also affects pH signal
i
transduction. Blatt and Grabov (1997b) have reported
that the abi1 transgene in N. benthamiana drastically
affects K+ channel sensitivity to experimentally-imposed
changes in pH . They found that reducing pH with 3 and
i
i
10 mM butyrate, equivalent to reductions of 0.3–0.5 pH
i
units, had only marginal effects on the K+ currents in
the transgenic plants compared with the wild type. The
observations are significant, because Armstrong et al.
(1995) noted that the mutant transgene affected the K+
channel response to ABA but had no effect on the rise in
pH evoked by the hormone. Thus, these two sets of data
i
indicate that events downstream of pH are dependent on
i
the phosphorylation state of one or more target proteins.
In conclusion, there can be little doubt now about the
358
Grabov and Blatt
complexity of signalling events that underlie stomatal
control. The emergence of pH as a second messenger,
i
along with [Ca2+] and protein phosphorylation events,
i
emphasizes our relative ignorance about the origins and
interactions of each of these components, quite apart
from the mechanism(s) by which each targets downstream
elements. Indeed, any current perspective of this, now
expanding network of controls will almost certainly
appear naive before the close of this decade. Thus, while
the present task remains to characterize these and related
signalling events, both upstream and downstream in each
case, equally it must be to understand how these events
are integrated in vivo.
Acknowledgements
This work was supported by the Gatsby Charitable Foundation
and the Biotechnology and Biological Sciences Research
Council.
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