Download Ca2+ signalling and control of guard-cell volume in stomatal

196
Ca2+ signalling and control of guard-cell volume in stomatal
movements
Michael R Blatt
Stomatal guard cells are unique as a plant cell model and,
because of the depth of knowledge now to hand on ion
transport and its regulation, serve as an excellent model for the
analysis of stimulus-response coupling in higher plants. Parallel
controls – mediated by Ca2+, H+ protein kinases and
phosphatases — regulate the gating of the K+ and Cl– channels
that facilitate solute flux for stomatal movements. A growing
body of evidence now indicates that oscillations in the cytosolic
free concentration of Ca2+ contribute to a ‘signalling cassette’,
which is integrated within these events through an unusual
coupling with membrane voltage. Additional developments
during the past two years point to events in membrane traffic
that play complementary roles in stomatal control. Research in
these areas, especially, is now adding entirely new dimensions to
our understanding of guard cell signalling.
Addresses
Laboratory of Plant Physiology and Biophysics, Imperial College of
Science, Technology and Medicine at Wye, Wye, Kent TN25 5AH, UK;
e-mail: [email protected]
Current Opinion in Plant Biology 2000, 3:196–204
1369-5266/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
ABA
abscisic acid
ARF
ADP-ribosylation factor G-protein
concentration of cytosolic free calcium
[Ca2+]i
cADPR
cyclic ADP–ribose
EK
K+ equilibrium voltage
GEF
guanine-nucleotide exchange factor
ICl
non-inactivating Cl– current
IK,in
inward-rectifying K+ current
IK,out
outward-rectifying K+ current
IP3
inositol-1,4,5-trisphosphate
Nt-Syr1
Nicotiana syntaxin-related protein 1
PLD
phospholipase D
SNARE
soluble N-ethylmaleimide-sensitive factor attachment
protein receptor
Introduction
Stomata are pores that form between pairs of specialised
cells, called guard cells, and that are found on the epidermis
of all aerial parts of most higher plants. Guard cells open and
close the stoma to regulate gas exchange between the intercellular spaces within the plant tissue and the surrounding
environment, thereby influencing two of the processes that
are most important to the vegetative plant, photosynthesis
and transpiration. Thus, the control of stomatal aperture has
long been recognised as directly influencing vegetative
yields, as well as water resource and arable land management. Guard cells have also become a focus of attention in
fundamental research as a ‘model’ for higher-plant cells.
Their ability to integrate environmental and endogenous
signals, and their position within the leaf tissue has enabled
explorations of the signal cascades that link guard-cell
membrane transport to stomatal control. Of these, the signalling mechanisms evoked by the water-stress hormone
abscisic acid (ABA) have received the greatest attention and,
in the past four years, have yielded some of the most exciting new findings in plant cell signalling.
Stomatal movements are achieved through changes in
guard-cell volume, which, in turn, are driven by the accumulation (during opening) and loss (during closing) of
osmotically active solutes (i.e. KCl and/or other K+ salts).
Flux of these inorganic solutes must take place across the
plasma membrane because mature guard cells lack functional plasmodesmata [1,2]. K+ transport is dominated by
two classes of K+ channels. The first of these permits K+
movement into, but not out of, the cell and thus gives rise
to an inward-rectifying current (IK,in); the second facilitates
K+ flux out of, but not into, the cell and therefore appears
as an outward-rectifying current (IK,out). These two currents contribute to K+ flux during stomatal opening and
closing, respectively, and can be clearly distinguished by
their whole-cell, single-channel and pharmacological properties [2–4]. The movement of Cl– through so-called ‘slow’
or ‘non-inactivating’ Cl– channels (ICl) accounts for much
of the charge balancing of the fluxes of K+, especially during stomatal closure when it is essential that the voltage
across the guard-cell plasma membrane is brought positive
of the K+ equilibium voltage (EK) for the loss of K+
through IK,out [4].
Clearly, effective control of stomatal aperture, and hence
of guard-cell volume, requires the coordinated regulation
of both K+ and Cl- fluxes, and implies a high degree of
integration between the signalling pathways that determine the activities of IK,in, IK,out and ICl. We know now
(see Figure 1) that ABA first, evokes an inward-directed
current, mediated at least in part by ICl [5,6]; second, inactivates IK,in, which normally mediates K+ uptake [7]; and
third, activates the current through IK,out, which together
with ICl facilitates a net loss of KCl. At least two parallel
signalling pathways that engender changes in cytosolicfree calcium concentration ([Ca2+]i) and pH (pHi)
underpin these events [2–4]. Nonetheless, major gaps
remain in our understanding of these signalling pathways.
Even the function and processing of the [Ca2+]i signal,
itself, is proving surprisingly complex. Furthermore, key
developments during the past two years point to related
events in membrane traffic that play equally important
roles in stomatal control. Research in these areas, especially, is adding entirely new dimensions to our
understanding of guard-cell signalling and of plant cell
biology in general.
Ca2+ signalling and control of guard-cell volume in stomatal movements Blatt
197
Figure 1
ABA leads to a concerted modulation (⊕,
activation; O, inactivation) of three plasma
membrane ion channels: the two dominant K+
currents (a) IK,in, (b) IK,out, and (c) the slowactivating anion current ICl (and, probably also
(d) the H+-ATPase). ABA binds to an as yet
unidentified receptor (X), but that is
postulated to be on the plasma membrane.
Binding may activate a G-protein (Gα) and
trigger activation of phospholipase C (PLC)mediated hydrolysis of phosphoinositol
bisphosphate to IP3, thereby releasing Ca2+
from intracellular stores and the vacuole. (e)
ABA also affects Ca2+ entry across the
plasma membrane and its interaction with
intracellular Ca2+-release pathways. High-gain
switch-like Ca2+-induced and (f) cADPRmediated Ca2+ release from intracellular
stores affects IK,in, ICl and the H+-ATPase.
Parallel but independent increases in pHi act
on IK,in, IK,out, ICl, and deplete the substrate for
the H+-ATPase. Additional targets and
interactions with the abi1 protein
phosphatase (abi1) and other protein kinase
(PK)/protein phosphatase (PP) activities (P*)
are discussed in several recent reviews
[8,10,83].
(b)
Outside
+
Cytoplasm
P*
K+
abi1
(d)
H+
abi1
P*
(a)
Vacuole
[H + ] i
abi1
K+
-
K+
(c)
+
?
Cl + P*
? P* +
+
(e) Ca 2+
H+
[Ca 2+ ] i
?
ABA
Cl -
X
Gα
IP3 ?
?
Ca2+
? +
(f) cADPR
P*
Ca2+
PLC
IP3 ?
+
PK / PP
P*
Current Opinion in Plant Biology
[Ca2+]i increases and Ca2+ channels
Early debates centred around the origins of the [Ca2+]i signal, whether it is internal or external to the guard cell.
Considerable evidence now indicates that increases in
[Ca2+]i arise from both Ca2+ entry across the plasma membrane and release from intracellular stores [8–10].
Furthermore, the vacuole is certainly not the only source of
Ca2+ within the cell [8]. Some uncertainty remains about
the relative contributions of various pathways, which may
reflect an inherent redundancy (and, hence, plasticity)
within the Ca2+ cascade. Nonetheless, we can expect that
the specialisation, and localisation to selected
membranes within the cell, of [Ca2+]i-signalling elements
is also important for signal encoding [11,12].
Within the guard cell, Ca2+ release and Ca2+-mediated suppression of IK,in are mediated by inositol-1,4,5-trisphosphate
(IP3) [13,14], which is produced rapidly in response to ABA
[15,16]. This lipid metabolite probably interacts with IP3-sensitive Ca2+ channels within the cell to potentiate a rise in
[Ca2+]i [17]. Several studies have, however, described a second class of endomembrane Ca2+-release channels in higher
plants [17,18]. These channels are sensitive to the alkaloid
ryanodine and are activated by binding cyclic ADP–ribose
(cADPR), a metabolite of nicotinamide adenine dinucleotide.
A statistical analysis of cADPR-injected mesophyll had implicated a role for this agonist in r29A and kin2 gene expression
evoked by ABA [19]. More recently, Leckie et al. [20••] have
provided direct evidence of cADPR-sensitive, Ca2+-permeable channels in guard-cell vacuoles and suggested that they
function as a Ca2+-release pathway during ABA stimulation.
In support of this hypothesis, they observed increases in
[Ca2+]i following injection of cADPR into guard cells, and a
slowing of stomatal closure in response to ABA when guard
cells were preloaded with the cADPR antagonist 8NH2cADPR. Grabov and Blatt [21•] offer parallel evidence that
voltage-evoked increases in [Ca2+]i are suppressed by ryanodine in intact guard cells. The latter study is of particular
interest because it offers the first quantitative analysis of the
[Ca2+]i dependence of IK,in. Grabov and Blatt [22••] had previously found evidence of a hyperpolarisation-activated Ca2+
channel at the plasma membranne, implicated by the sensitivity of [Ca2+]i increases to the membrane voltage. In the
more recent study [21•], they made use of changes in clamp
voltage to elicit changes in [Ca2+]i, monitoring IK,in under
voltage clamp. They found a high (‘switch-like’) sensitivity of
the current to [Ca2+]i, with an apparent K1/2 (~330 µM) only
marginally above the mean resting [Ca2+]i. Whether ABA
affects the [Ca2+]i dependence of IK,in is not known; however, ABA does strongly influence the voltage sensitivity of
198
Physiology and metabolism
[Ca2+]i increases, as well as affecting the kinetics of the [Ca2+]i
rise and its return to resting concentrations [22••]. So, we can
anticipate a compound action of ABA both on Ca2+ entry
across the plasma membrane and on its release from cADPRdependent or other Ca2+ stores.
The spatial distribution of internal Ca2+-release pathways
is also likely to affect the kinetics of [Ca2+]i changes and
Ca2+-signal encoding. The vacuole is an obvious source of
Ca2+ and the tonoplast contains slow vacuolar (SV)-type
channels that may be related to animal ryanodine-receptor
Ca2+ channels [23•], and contribute to Ca2+ release [24].
Nonetheless, Ca2+ channels have also been identified in
the endoplasmic reticulum (ER) of mechanosensitive
organs in Bryonia [25] and probably the ER of Lepidium
roots [26]. In Brassica florets, Ca2+ release appears to be
triggered by IP3 in vesicular compartments that are distinct
from the vacuole [27]. Furthermore, in the giant alga
Chara, the action potential (which is initiated by Ca2+mediated activation of Cl- channels [28,29•,30]) draws on
IP3-dependent Ca2+ release from a non-vacuolar compartment close to the plasma membrane. In a particularly
elegant study, Plieth et al. [31••] preloaded Chara with
Mn2+, targeting either all intracellular stores (by incubation
in Mn2+ medium) or only the central vacuole (by injection
with Mn2+). They found that Mn2+ quenches the fluorescence of the Ca2+-sensitive dye Fura2 and, because it also
permeates many Ca2+ channels, it provides an assay for
divalent release. When they evoked action potentials,
Plieth et al. [31••] observed a quenching of Fura2 fluorescence in cells that were uniformly loaded with Mn2+, but
not in cells in which Mn2+ was loaded only into the vacuole. Whether spatial differences in Ca2+ release
underpins [Ca2+]i signalling in guard cells has yet to be
determined, but there is certainly evidence of local differences in [Ca2+]i dynamics between the peripheral cytosol
and regions of the cell close to the nucleus [22••]. To summarise, both the pharmacological and kinetic
characteristics, as well as the spatial distribution of Ca2+release pathways within the guard cell, can be expected to
play important roles in defining the nature of the [Ca2+]i
response to ABA.
[Ca2+]i oscillations
Ca2+ mediates a diversity of responses, even within a single cell. The issue of how a single second messenger could
give rise to such varied responses was resolved only when
it was recognised that the frequency and location of a
sequence of repetitive increases in [Ca2+]i within the cell
might determine the nature of the response(s) [12,32,33].
Frequency modulation of [Ca2+]i increases or ‘spikes’ has
been shown to give rise to ‘Ca2+ signatures’ that encode
specific responses, including gene expression [34,35] and
calmodulin-dependent protein kinase activity [36•]. In animals, [Ca2+]i oscillations initiate Ca2+ entry across the
plasma membrane; local [Ca2+]i elevation then triggers further Ca2+ release from intracellular stores (i.e.
Ca2+-induced Ca2+ release [CICR]) before the Ca2+ is
eliminated from the cell or sequestered within organelles.
The result is repetitive [Ca2+]i spikes, each with a duration
of a few seconds or less.
Interest in [Ca2+]i signalling in plants has been heightened
by observations that [Ca2+]i may oscillate in response to
external stimuli [37–40]. In guard cells, however, these
oscillations often take place over periods of more than
10 min, with discrete [Ca2+]i maxima lasting for 2–4 min or
more when evoked by a rise in the extracellular Ca2+ concentration or CO2 [40,41]. A similar oscillation pattern has
now been observed in response to ABA. Staxen et al. [42•]
reported [Ca2+]i increases from approximately 200 nM at
rest to 400–600 nM within 15–30 min of exposure to 10 nM
ABA. Significantly, treatments with higher concentrations
of ABA that resulted in a pronounced decrease in stomatal
aperture also produced a reduced amplitude in the [Ca2+]i
oscillations and a longer duration of the [Ca2+]i elevation. A
role for IP3-mediated Ca2+ release was suggested because
the ABA-evoked oscillations were partially suppressed by
treatment with the phospholipase C antagonist U73122.
This pattern of slow and irregular oscillations in [Ca2+]i
raises questions about the origins and role of this signal in
the guard cell. One explanation relates [Ca2+]i oscillations
to the voltage status of the cell. Guard cells, like many
higher-plant cells, have two states of membrane voltage:
one state close to EK, and the other largely K+-insensitive
and typified by voltages well negative of EK [43,44].
Transitions between these states take place in response to
stimuli, including ABA, that can give rise to oscillations in
voltage with periods of between 10 s and many minutes
[43,45]. Because membrane hyperpolarisation also evokes
a Ca2+ influx and a consequent rise in [Ca2+]i, it is most
likely that the [Ca2+]i oscillations mirror an underlying
oscillation in plasma membrane voltage [22••]. These characteristics — and the effect of ABA in altering the voltage
threshold for the [Ca2+]i rise [22••] — suggest a role for
[Ca2+]i elevation in a feedback mechanism that ‘tunes’ the
[Ca2+]i-dependent currents IK,in and ICl to the prevailing
transport status of the guard cell. Thus, [Ca2+]i feedback
creates a response ‘cassette’ for osmotic balance (Figure 2),
switching the membrane between states in which net
uptake and net loss of K+ and Cl– take place [8,44].
Membrane traffic and cell volume
The activation of Ca2+ channels may also play a role in
events one step removed from the regulation of K+ and Cl–
fluxes. Between the open and closed state of the stomatal
pore, the volumes of the guard cells often change by a factor of two or more and extensive reorganisation of the
vacuolar membrane is known to occur [2,46]. Such volume
changes cannot be accommodated by lateral expansion and
compression of the bilayer [47,48] and must, therefore, be
accompanied by substantial changes in total membrane
matter. A coordinate regulation of vesicular traffic to and
from both the plasma membrane and the tonoplast is
therefore implied. MacRobbie [49] began to explore some
Ca2+ signalling and control of guard-cell volume in stomatal movements Blatt
Figure 2 Legend
The voltage–[Ca2+]i response ‘cassette’ cycle. (a) Negative membrane
voltage (downward arrow indicates amplitude) triggers Ca2+ influx
across the plasma membrane that (b) stimulates intracellular Ca2+
release, activates Cl– channels for Cl– efflux and inactivates IK,in. (c)
The rise in [Ca2+]i, bias to Cl– efflux and additional changes in
membrane conductance (not shown) lead to membrane depolarisation,
inactivate the Ca2+ influx and engage Ca2+ extrusion processes. (d)
Finally, with [Ca2+]i reduced, the Cl– flux inactivates, IK,in re-activates
and the membrane voltage hyperpolarises. Black and grey arrows
represent active and inactive channels, respectively.
Figure 2
(a) Outside
Cytoplasm
Ca2+
Intracellular
compartment
K+
Cl−
In the first of these, Homann [50•] and Kubitscheck et al.
[51••] used in vivo capacitance recording and membranesurface labelling with styryl dyes to quantify membrane
exocytosis and endocytosis in guard-cell protoplasts during
osmotically driven volume changes. Capacitance recording
makes use of sine-wave retardation to determine membrane capacitance [52–54], which is directly proportional to
the membrane surface area. An increase in capacitance
represents the sum of all exocytotic events less the sum of
all endocytotic events. Endocytotic events can subsequently be isolated by simultaneously monitoring
internalisation of fluorescent styryl dyes, such as FM1-43
[55]. Homann [50•] found that the membrane surface area
of Vicia guard-cell protoplasts increased under hypo-osmotic conditions and decreased after hypertonic treatment,
and that the rate of change in each case was graded in
response to the magnitude of the osmotic potential difference. Kubitscheck et al. [51••] subsequently combined
these techniques with FM1-43 dye measurements. Their
results demonstrate a rapid internalisation of membrane
during hyperosmotic stimulation that is quantitatively consistent with the decrease in plasma membrane surface area
(and also capacitance). Intriguingly, the dye also accumulated gradually within the cell in a ring 1–2 µm below the
plasma membrane, even in the absence of an osmotic step.
This steady internalisation suggests that constitutive
endocytosis continues under constant osmotic pressure,
resulting in the accumulation of a pool of sub-plasma
membrane vesicles. Whether these vesicles are available
for re-incorporation into the plasma membrane remains to
be determined. The role of ABA and of Ca2+ in these
events can also be expected to draw attention in the near
future. So far, all of the evidence indicates that Ca2+ does
not have a direct impact on the membrane trafficking that
is evoked by osmotic gradients [50•]. It may be that osmotic potential- and ABA-sensitive trafficking controls overlap
only in part. Whether or not this is the case, experimental
results point to a highly dynamic process of membrane
cycling that is responsive to membrane tension.
Ca2+
IP3
Ca2+
of these issues in Chara more than two decades ago. Yet,
despite their implied function in stomatal movements, the
dynamics of membrane trafficking within the guard cell
were largely ignored until recently. Two recent developments have no placed membrane trafficking in the
limelight of stomatal physiology and ABA signalling.
199
cADPR
+
(b) Outside
Ca2+
Ca2+
∆ψ
Cytoplasm
Ca2+
Intracellular
compartment
K+
Cl−
Ca2+
IP3
Ca2+
cADPR
+
(c) Outside
Ca2+
Ca2+
∆ψ
Cytoplasm
Ca2+
Intracellular
compartment
K+
Cl−
Ca2+
Ca2+
Ca2+
Ca2+
+
(d) Outside
∆ψ
Cytoplasm
Ca2+
Intracellular
compartment
K+
Cl−
Ca2+
Ca2+
Ca2+
Ca2+
+
∆ψ
Ca2+
channel
Cl–
channel
K+
channel
Current Opinion in Plant Biology
200
Physiology and metabolism
Figure 3
(a) SNARE as a scaffolding/actuator element
(b) SNARE as a receptor/signal
intermediate coupling element
ABA
Net endocytosis
Ca2+
pHi
Ion channel control
Net endocytosis
Ca2+
pHi
Ion channel control
Current Opinion in Plant Biology
Two models for the dual functioning of Nt-Syr1 (and other SNARE
proteins) in controlling ion channels and vesicle trafficking. Soluble
ABA (orange) binding to its receptor (blue) alters both net endocytosis
and the permeation of ions (yellow) through ion channels (red).
SNARE elements (green) play a role in secretion (exo/endocytosis)
and function either (a) as scaffolding elements or (b) as receptorcoupling elements that interact directly with ion channels. The
receptor-associated coupling element is coloured pink.
The second development has come from the identification of the Nicotiana syntaxin homologue Nt-Syr1.
Syntaxins belong to a group of integral membrane proteins that form the core of the molecular machinery for
vesicle trafficking and membrane fusion (see below).
Surprisingly, Leyman et al. [56••] isolated the Nt-Syr1
gene by expression cloning in a heterologous screen
designed to identify an ABA receptor. Their studies
demonstrate not only that ABA enhances Nt-Syr1 transcript and Nt-Syr1 protein levels in the plant but also
that disrupting Nt-Syr1 function, with either the
Clostridial neurotoxin BotN/C (an endopeptidase that
specifically cleaves the syntaxin) or by loading guard
cells with the soluble (carboxy-truncated) portion of
Nt-Syr1, prevents both K+ and Cl– channel responses to
ABA in vivo. These results, and observations of a high
molecular weight band recognised by Nt-Syr1 antibodies, suggest that Nt-Syr1 functions in a complex that is
competitively blocked through substitution with the carboxy-truncated protein. They also indicate that Nt-Syr1
functions within or very close to the early steps of the
ABA signal cascade, either as a scaffolding protein, a second messenger or a modulator of the activity/availability
of one or more signalling elements.
Further evidence of membrane trafficking in
stomatal control?
Two additional lines of evidence hint at roles for membrane trafficking in guard-cell volume control and ABA
signalling. First, in screening for ABA-hypersensitive
mutants of Arabidopsis, Cutler et al. [57] identified Era1,
the gene that encodes a protein farnesyl-transferase.
Farnesyl- and geranylgeranyl-transferases covalently add
lipid moieties to small GTPases, anchoring the latter at the
membrane surface [58] to target GTPase activity in a variety of cellular functions. Although the substrate(s) for Era1
is not known, Rac- and Rho-type GTPases, which are
important for cytoskeletal function [59], are commonly farnesylated. In fact, a Rho-like GTPase, Rop1, that is
localised to the apex of Pisum pollen tubes [60] has been
implicated in microfilament-based intracellular transport
of vesicles. Intriguingly, actin organisation has also been
suggested to regulate guard-cell ion channels and stomatal
movements, although the function of actin in this case has
been interpreted in the context of control by mechanical
stress rather than membrane trafficking [61–63].
The Era1 farnesyl-transferase also contributes to ABA signalling. Pei et al. [64•] have reported an enhanced
Ca2+ signalling and control of guard-cell volume in stomatal movements Blatt
sensitivity of guard-cell Cl– channels to ABA in the era1
mutant and in wild-type Arabidopsis plants after treatment
with broad-range farnesyl-transferase antagonists. The
era1 phenotype is dominant and persists throughout the
life cycle of the plant. So, although these results do not distinguish between possible roles for Era1 as an
ABA-signalling element per se and as a secondary element
necessary to maintain the signalling machinery, they do
implicate a role for small GTPases and potentially for
membrane trafficking in guard-cell function.
Second, Jacob et al. [65•] have recently reported a transient rise in the phospholipase D (PLD) activity of
guard-cell protoplasts following stimulation with ABA.
They also observed a reduction in the activity of IK,in
that complemented the action of the cADPR antagonist
nicotinamide when the protoplasts were treated with the
PLD by-product phosphatidic acid. It is worth noting
that both PLD and phosphatidic acid have been implicated in secretion by the barley aleurone [66].
Phosphatidic acid contributes to the regulation of vesicle
trafficking at the Golgi apparatus [67], and PLD activity
has been associated with the functioning of ARF- and
Rho-GTPases in the secretory processes of mammals
and yeast [68,69]. Once again, the data suggest a link
between membrane trafficking and ABA.
Coordinating membrane traffic and ion
channel control
How might membrane trafficking contribute to ion-channel control and ABA signalling? Membrane cycling and
fusion between the membranes of eukaryotic cells are
facilitated by a family of SNARE (i.e. soluble N-ethylmaleimide-sensitive factor attachment protein receptor)
proteins that are associated with each membrane [70–72].
During synaptic transmission, which depends on vesicle
fusion and release of a neurotransmitter, the target
(t-SNARE) proteins syntaxin and SNAP-25 form a stable
ternary complex with the vesicle (v-SNARE) protein
synaptobrevin that serves to bring the two membrane
bilayers into close proximity for fusion [73]. In yeast and
plants, SNARE proteins are thought to be essential for the
related functions of interorganelle transport, growth and
cell division [72,74]. Thus, SNAREs have been recognised
as contributing to response mechanisms and not as components of signal cascades per se.
Even so, SNAREs are probably important for signal
transduction, its plasticity and adaptation. SNAREs may
contribute to ion-transport regulation simply by mediating the selective addition and removal of transport
proteins from the membrane. These events can take
place with remarkable rapidity. A recent study of the
Arabidopsis GNOM gene, which encodes a GTP–GDP
exchange factor (GEF) for an ADP-ribosylation factor Gprotein (ARF) GTPase, is a case in point. Steinmann
et al. [75••] report that treatments with the ARF–GEF
antagonist brefeldin A result in dramatic alterations in
201
protein localisation within 15–30 min, including a loss of
the polar distribution of the putative auxin efflux carrier
protein Pin1. Auxin efflux, itself, is altered within 10 min
of brefeldin A treatments [76], suggesting that substantial changes to the make-up of the membrane transport
proteins take place within the same time period. The
data certainly highlight the dynamic flux of membrane
and protein within the plant cell.
The role(s) carried out by SNAREs may prove to be much
more subtle still. In neurons, SNAREs, including syntaxins, bind to and regulate Ca2+ channels [77,78] as well as
CFTR Cl- channels [79]. Syntaxins interact with proteins
that may not be related to secretion processes directly,
including the orphan G-protein-coupled receptor CIRL
[80] and tomosyn, a microfilament-associated protein [81].
Furthermore, SNARE proteins have been associated with
responses to membrane tension and osmotic stress [82]. So,
one possibility (Figure 3) is that SNARE proteins such as
Nt-Syr1 [56••] contribute to the scaffolding of an ABAreceptor complex and, therefore, serve dual roles by
marrying the functions of a signalling element and of the
machinery for membrane trafficking.
Conclusions
Research into the cellular and molecular mechanisms of
stomatal control has begun to take on new dimensions in
relating signalling pathways with response ‘cassettes’ that
comprise several ion transporters and their control mechanisms. Understanding how these cassettes are assembled
will present a major challenge in the coming years.
Defining the interactions within these cassettes is already
providing a clearer picture of the functions of Ca2+ as a second messenger. At the same time, attention to the related
processes of membrane dynamics and trafficking promises
to add to our understanding of both cellular transport and
volume control, as well as to the guard-cell system as a
higher-plant cell model.
Update
Following up the evidence of a hyperpolarisation-activated
rise in [Ca2+]i, Hamilton et al. [84••] have now identified a
small-conductance Ca2+ channel at the plasma membrane
by patch-clamp of Vicia guard-cell protoplasts. The channel shows characteristics that are entirely consistent with
those expected from the previous studies, including a
dependence on negative voltage and block by Gd3+ and
La3+ [21•,22••]. Furthermore, ABA evokes a large increase
in channel opening, even in isolated membrane patches.
These results suggest that the ABA receptor is physically
close to the Ca2+ channel and localised to the inner surface
of the plasma membrane.
Acknowledgements
I am grateful to Ulrike Homann and Gerhard Thiel for comments on the
manuscript. Work from the author’s laboratory was supported by
Biotechnology and Biological Sciences Research Council grants C10234,
C09640 and P09561, European Union-Biotech grant CT96-0062, and the
British Council.
202
Physiology and metabolism
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
macological data presented are consistent with a ryanodine-sensitive
release pathway.
22. Grabov A, Blatt MR: Membrane voltage initiates Ca2+ waves and
•• potentiates Ca2+ increases with abscisic acid in stomatal guard
cells. Proc Natl Acad Sci USA 1998, 95:4778-4783.
This is a seminal work that links plasma-membrane voltage to oscillations of
[Ca2+]i in guard cells. The data implicate the function of a plasma-membrane
Ca2+-channel that is activated at negative voltages and demonstrate an action
of ABA in altering the kinetics and voltage-dependence of these evoked
[Ca2+]i increases. A report of an ABA-sensitive, hyperpolarisation-activated
Ca2+ channel at the guard-cell plasma membrane will follow shortly.
1.
Wille A, Lucas W: Ultrastructural and histochemical studies on
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See ‘Update’.