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Signalling drought in guard cells
S. Luan
Original ArticleBEES SGML
Plant, Cell and Environment (2002) 25, 229–237
Signalling drought in guard cells
S. LUAN
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
ABSTRACT
A number of environmental conditions including drought,
low humidity, cold and salinity subject plants to osmotic
stress. A rapid plant response to such stress conditions is
stomatal closure to reduce water loss from plants. From an
external stress signal to stomatal closure, many molecular
components constitute a signal transduction network that
couples the stimulus to the response. Numerous studies
have been directed to resolving the framework and molecular details of stress signalling pathways in plants. In guard
cells, studies focus on the regulation of ion channels by
abscisic acid (ABA), a chemical messenger for osmotic
stress. Calcium, protein kinases and phosphatases, and
membrane trafficking components have been shown to play
a role in ABA signalling process in guard cells. Studies also
implicate ABA-independent regulation of ion channels by
osmotic stress. In particular, a direct osmosensing pathway
for ion channel regulation in guard cells has been identified.
These pathways form a complex signalling web that monitors water status in the environment and initiates responses
in stomatal movements.
Key-words: Calcium; ion channel; osmosensing; protein
phosphorylation.
INTRODUCTION
Osmotic stress in plants can result from many environmental conditions, including drought, low temperature, saline
soil, pathogen attack and mechanical wounding. These
undesirable conditions interfere with plant growth and
development, leading to reduction in crop productivity.
During evolution, plant species that exist today have developed mechanisms to cope with stress conditions and to
eventually adapt to the changing environment. The capability of plants to survive stressful conditions is often
referred to as stress tolerance and it represents an important trait in agriculture. For most plants, stress tolerance is
a ‘developmental or physiological programme’ that
requires activation or induction by the stress signals. A
good example is stomatal closure induced by osmotic stress
(Zeiger, Farquhar & Cowan 1987; Mansfield, Hetherington
& Atkinson 1990; Assmann 1993). Under normal conditions, the opening and closing of the stomata (in most
plants) are geared to the light–dark cycle to maximize the
efficiency of light utilization for photosynthesis. Plants
Correspondence: Sheng Luan. E-mail: [email protected]
© 2002 Blackwell Science Ltd
respond to light by opening the stomata for CO2 uptake.
Upon darkness, stomata gradually close to prevent water
loss in the absence of light harvesting. However, if plants
experience water deficiency caused by low humidity, low
soil moisture or other conditions, stomatal closure is triggered, even during daytime. The reason for overriding CO2
uptake by water conservation is obvious: plants must prioritize survival over productivity (as do animals). Therefore, plants contain an ‘inducible programme’ that operates
stomatal closure upon exposure to water deficiency conditions. Similarly, plants also contain a number of other programmes that fine-tune physiological and developmental
processes of the whole plant under stress conditions. For
instance, a large array of genes is expressed differently
under normal or stressful conditions (Ingram & Bartel
1996; Bray 1997; Shinozaki & Yamaguchi-Shinozaki 1997).
Upon exposure to water stress, genes whose products are
involved in cell protection from osmotic stress are often
activated so that more osmolytes can be synthesized or
more water channels produced. When this inducible programme is understood at molecular, cellular and wholeplant levels, it will be possible to manipulate this programme to benefit agriculture. Recognizing the importance
of this research area, an increasing number of laboratories
have directed efforts to the study of stress tolerance mechanisms. A number of recent review articles are dedicated to
the studies on both stomatal regulation and gene expression by osmotic stress signals. I will focus only on the most
recent progress made on ion channel regulation in guard
cells in response to drought conditions.
STOMATAL MOVEMENTS AND ION
CHANNELS
Stomatal pores are responsible for gas exchange (CO2
uptake and water loss) between plants and the atmosphere.
The aperture of the stomatal pore must be finely tuned in
order to allow uptake of sufficient CO2 yet not to lose
excessive water to dessicate plants. This fine-tuning process
is controlled by a pair of guard cells that surround each stomatal pore. When guard cells swell due to increased turgor
pressure, the pore aperture enlarges. When guard cells lose
turgor pressure and shrink, stomatal pores become smaller.
The turgor pressure of guard cells is regulated by solute
concentration and water flow across cell membranes. Major
solutes in guard cells include K+ , Cl–, and malate. Transport
of these solutes is mainly achieved by active transporters
and ion channels in the cell membranes. Recent studies
have shown that ion channels play important roles in turgor
229
230 S. Luan
regulation in guard cells and these channels become critical
molecular targets of various signals including osmotic
stress. A number of review articles have provided detailed
information on ion channel function in stomatal movements (e.g. Assmann 1993; Hedrich 1994; Ward, Pei &
Schroeder 1995; Blatt & Grabov 1997; MacRobbie 1997).
ABA REGULATION OF ION CHANNELS IN
GUARD CELLS
A large body of literature addresses stomatal regulation by
the plant hormone abscisic acid (ABA). It has been well
established that ABA accumulates in the roots upon
drought conditions in the soil and moves upward to leaves
through the transpiration stream. Increase in ABA concentration around the guard cells causes stomatal closure.
ABA is therefore considered a long range chemical signal
for drought-induced stomatal closure. Several fundamental
questions limit the understanding of the mechanism of
ABA reception in guard cells. These include: ‘How is ABA
perceived by guard cells?’; ‘What are the targets for
ABA action?’; ‘What are the intermediate components for
ABA signalling in guard cells?’. Recent studies on ABA
action in guard cells have generated important insights concerning these questions.
Perception site of ABA
As a hormone, ABA requires a ‘receptor’ in order to function. Despite an extensive search during the past few
decades, the molecular identity of an ABA receptor
remains elusive. In fact, it remains open as to whether an
ABA receptor is localized extracellularly or intracellularly
or both (Anderson, Ward & Schroeder 1994; Allan et al.
1994; Schwartz et al. 1994). Clearly, it awaits molecular
characterization of ABA receptor(s) to resolve this issue.
ABA targets
As discussed earlier, ABA causes the closure of stomatal
pores that are held open by the turgor pressure of guard
cells. Ion channels are major players in regulation of guard
cell turgor. It is reasonable to make a tentative link between
ABA and ion channel regulation. Indeed, a number of studies indicate that ABA functions by regulating ion channel
activity in guard cells. Earlier studies on ABA regulation of
ionic fluxes in guard cells have been reviewed previously
(MacRobbie 1997). More recent studies using patch-clamp
techniques identify several types of ion channels as ABA
targets. These include inward (IKin) and outward (IKout) K+
channels and the slow anion channels in the plasma membrane (Blatt 1990; Lemtiri-Chlieh & MacRobbie 1994;
Schwartz et al. 1994; Pei et al. 1997). ABA inhibits the IKin
and activates the IKout, reducing influx and increasing efflux
of K+ in guard cells. This result is consistent with ABA function in the inhibition of stomatal opening and induction of
stomatal closure. The slow anion channel is responsible for
the efflux of anions such as Cl– (reviewed by Hedrich 1994).
Activation of anion channels by ABA has been suggested
to be a critical step in ABA-induced stomatal closure
because anion efflux triggers membrane depolarization
required for activation of IKout (Pei et al. 1997). Together,
these studies connect ion channel regulation to ABA
response in guard cells (Fig. 1). Between ABA and its
molecular targets lie many intermediate steps to execute
the signalling process. Using guard cells as a model system,
many laboratories have made important contributions to
the understanding of ABA signalling pathways in plants.
ABA signalling components
Calcium
Calcium is considered a second messenger for a number of
extracellular signals in plants (as well as in animals). Several
lines of evidence indicate that calcium may serve as a second
messenger for (part of) ABA action in guard cells. First, cellular Ca2+ level is up-regulated by ABA. This was described
in earlier studies using Ca2+ fluorescence dyes and electrophysiological techniques (McAinsh, Brownlee & Hetherington 1990; Schroeder & Hagiwara 1990; Gilroy et al. 1991;
Allan et al. 1994) followed by more recent confirmation
using transgenic expression of fluorescent protein indicators
(Allen et al. 1999). Secondly, elevation of Ca2+ in guard cells
is sufficient to induce part of ABA response. For example,
high levels of Ca2+ , such as ABA, initiate stomatal closure
and inhibit IKin in guard cells (Schroeder & Hagiwara 1989;
Blatt, Thiel & Trentham 1990; Gilroy et al. 1991; Luan et al.
1993) and activate anion channels (Ward et al. 1995).
Thirdly, buffering Ca2+ to a low level in guard cells blocks
part of the ABA response in guard cells (Lemtiri-Chlieh &
MacRobbie 1994; Leckie et al. 1998). Studies have also demonstrated clearly that some of the ABA signalling events in
guard cells are Ca2+-independent. These include activation
of IKout and regulation of stomatal aperture (Allan et al.
1994; Lemtiri-Chlieh & MacRobbie 1994) (Fig. 1).
Calcium channels
Recent studies provide a rather complex picture regarding
Ca2+ fluxes in guard cells. After ABA application, Ca2+
accumulates in the cytoplasm due to activation of several
possible pathways. One is the plasma membrane Ca2+ channels. A non-selective cation channel was first shown to parallel ABA-induced Ca2+ elevation in guard cells (Schroeder
& Hagiwara 1990). More recently, ABA-regulated Ca2+
channels have been identified in guard cells by patch-clamp
techniques (Hamilton et al. 2000; Pei et al. 2000). In the
study by Hamilton et al. (2000), a single channel current
was identified to conduct Ca2+ specifically across the plasma
membrane in Vicia guard cells. Activation of this current is
significantly altered by ABA application and by internal
Ca2+ concentration. While ABA increases the current,
internal Ca2+ inhibits it, suggesting a feedback control of
Ca2+ accumulation in guard cells. Pei et al. (2000) identified
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 229–237
Signalling drought in guard cells 231
Osmotic stress (Drought/Low humidity)
Sensor (?)
Osmotic shock
Induction of ABA
ABA receptor (?)
ABIs AAPK high pH NtSyr1
(H 2O2, S-1-P, IP3
IP6, cADPR)
Calcium
Anion channel
Inward K-channel
Outward K-channel
Turgor decrease
Open stomate
Cytoskeleton
Outward K-channel
Closed stomate
a Ca2+-permeable channel current in Arabidopsis guard
cells. The current is activated by both ABA and H2O2. ABA
induces production of H2O2. If H2O2 production is blocked
by an inhibitor, ABA induction of the Ca2+ channel or stomatal closure is also blocked, implicating H2O2 production
in the ABA signalling pathway between ABA and Ca2+ elevation. It is not known if the Ca2+ channels identified by
these two studies are the same type. The second source of
Ca2+ is from intracellular stores. Following earlier observations that IP3 triggers Ca2+ elevation in guard cells (Gilroy,
Read & Trewavas 1990), other ligands such as IP6, sphingosine-1-phosphate, and cyclic ADP-ribose have also been
shown to regulate Ca2+ signal when injected into the guard
cells (Leckie et al. 1998; Lemtiri-Chlieh, MacRobbie &
Brearley 2000; Ng et al.). In all cases, ABA induces accumulation of these compounds, which implicate IP6, sphingosine-1-phosphate, and cyclic ADP-ribose in ABAinduced calcium release. It is intriguing how ABA targets
different metabolic pathways that produce these signal
molecules. Further studies will resolve the molecular mechanism underlying ABA-induced production of these calcium-releasing molecules. Finally, Ca2+-induced Ca2+
release may be conducted by slow vacuolar channels in the
tonoplast (Ward & Schroeder 1994), counting for sustained
accumulation of calcium in the cytoplasm. Despite extensive electrophysiological analyses, none of the Ca2+ channels discussed above has been characterized at the protein
or gene level. An important direction for future research is
to identify the genes encoding Ca2+ channels in plant cells.
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 229–237
Figure 1. A simplified scheme of recent
progress on drought signal transduction in guard
cells. Arrows and bars indicate positive and
negative regulation, respectively. S-1-P,
sphingosine-1-phosphate. Other abbreviations are
identified in the text. All events lead to activation
of efflux channels and inhibition of influx channels,
which results in a decrease in guard cell turgor and
reduced stomatal aperture.
More precisely, connections must be built between the
genes and channel activities in vivo before further functional analyses can be conducted.
Calcium sensors
How does Ca2+ perform its function in guard cells? It is generally believed that Ca2+ transmits the signal downstream in
the pathway by interacting with protein sensors. These calcium sensors such as calmodulin (CaM), Ca2+-dependent
protein kinases (CDPK), bind Ca2+ and interact with and
regulate the activity of their target proteins (Roberts &
Harmon 1992; Zielinski 1998). Several studies point to the
importance of Ca2+-regulated protein kinases and phosphatases that may mediate Ca2+ function in guard cells and
in other plant cells. For example, two studies suggest that
calcineurin-like activity may mediate Ca2+ function in guard
cells (Luan et al. 1993; Allen & Sanders 1995). Calcineurin
is a Ca2+, calmodulin-regulated protein phosphatase identified in animals and fungi (Klee, Ren & Wang 1998). However, a functional homologue has not yet been identified in
plants. In a search for calcineurin-like proteins from plants,
Kudla et al. (1999) identified a family of calcium sensors
that are similar to both the regulatory B subunit of calcineurin and the neuronal Ca2+ sensor in animals (Olafsson,
Wang & Lu 1995; Klee et al. 1998). These unique plant Ca2+
sensors are referred to as calcineurin B-like (CBL) proteins
(Kudla et al. 1999). Using yeast two-hybrid screening, Shi
et al. (1999) identified a group of novel protein kinases
232 S. Luan
(CIPKs) as targets for CBL proteins, suggesting that the
CBL family of calcium sensors function by interacting with
a family of protein kinases. The specificity of interaction
between the CBL calcium sensors and their target kinases
may be important for their specific function in plants (Kim
et al. 2000). It is not known whether CBL-CIPK complexes
play a role in ABA signal transduction in guard cells.
Two studies suggest that Ca2+-dependent protein kinases
(CDPK) play a role in guard cell ion channel regulation
(Pei et al. 1996; Li, Lee & Assmann 1998). Pei et al. (1996)
micro-injected an active CDPK into the guard cells and
found activation of a novel Cl-channel in the tonoplast. Li
et al. (1998) revealed phosphorylation of KAT1, a putative
inward K+ channel in guard cells, by a CDPK-like activity.
There is still a gap between CDPK function and ABA signalling in guard cells.
SNARE
Another recent finding on ABA signalling in guard cells
was reported by Leyman et al. (1999). They identified a
putative membrane trafficking protein (SNARE) that is
involved in the signalling pathway for ABA regulation of
ion channels. Using an expression cloning strategy, Leyman
et al. isolated a syntaxin (t-SNARE) homologue from
tobacco (Nt-Syr1). When expressed in oocytes, Nt-Syr1
enhanced the ABA-dependent suppression of an endogenous Ca2+-regulated Cl-channel. This Cl-channel is strictly
activated by elevation of cellular Ca2+ and often used to
monitor the changes in the cellular Ca2+ level. Because
ABA elicits Ca2+ elevation in guard cells, it is speculated
that ABA may raise the Ca2+ level by a mechanism conserved among plants and animals. By injection of total
mRNA from tobacco, one would expect to clone the ABA
receptor that raises the cellular Ca2+ in an ABA-dependent
manner. Surprisingly, a syntaxin homologue was identified
that activates the Cl-channel in the oocyte upon exposure
to ABA. Moreover, manipulation of Nt-Syr1 also altered
ABA-dependent regulation of both K+ and anion channel
in guard cells (Leyman et al. 1999). Because syntaxin is a
member in the SNARE family, Nt-Syr1 may also play a role
in membrane trafficking in plants. This also implicates a
membrane fusion guidance molecule in ABA regulation of
ion channels. Studies in animal cells indicate that SNARE
proteins can interact with ion channels in the cell membrane (Sheng et al. 1994; Naren et al. 1998). Therefore, it is
conceivable that plant and animal SNAREs may have cellular functions other than membrane trafficking. More discussion on this topic can be found in a recent review (Blatt,
2000).
Ca2+-independent pathway and pH
ABA induces alkalization of guard cell cytosol, which
accompanies activation of outward K+ channel in the
plasma membrane (Blatt & Armstrong 1993). This study
suggests that pH may serve as a second messenger for ABA
in a calcium-independent pathway (MacRobbie 1997). In
contrast to IKout, IKin in guard cells is activated by acidifica-
tion of cytosol (Grabov & Blatt 1997), consistent with ABA
inhibition of IKin. More recent studies on cloned K+ channels provide a molecular basis for pH modulation of inward
K+ channels such as KAT1 and KST1 that are expressed in
guard cells (Hoth et al. 1997; Hoth & Hedrich (1999; Tang
et al. 2000). The fact that these channels are regulated in a
membrane-delimited manner indicates that pH may
directly modify the channel protein (Miedema & Assmann
1996; Hoth et al. 1997).
AAPK and ABIs
A function for protein kinases and phosphatases in ABA
response has been established. Earlier studies using kinase
and phosphatase inhibitors suggest that reversible phosphorylation modulates ion channel activity in guard cells
(Luan et al. 1993; Li et al. 1994; Thiel & Blatt 1994; Schmidt
et al. 1995). More recent studies combining both genetic
and molecular tools with patch-clamping techniques have
identified at least one protein kinase and two phosphatases
as important components in ABA regulation of ion channels in guard cells. Using a biochemical approach, two studies (Li & Assmann 1996; Mori & Muto 1997) identified an
ABA-activated protein kinase (AAPK) from guard cells of
Vicia faba. Cloning of this kinase and further genetic
manipulation in guard cells indicates that AAPK is
involved in the regulation of the slow anion channel by
ABA (Li et al. 2000). Because AAPK is not regulated by
Ca2+, AAPK regulation of the anion channel is located in
the Ca2+-independent branch of ABA signalling pathway.
Identification of AAPK raises a number of interesting questions. It provides a stepping stone for dissecting other intermediate components between ABA and AAPK and those
between AAPK and the channel. It will also be interesting
to find out what phosphatases counteract AAPK activity in
ion channel regulation.
At least two isoforms of protein phosphatase 2C are
involved in ABA signalling in guard cells. These phosphatases, ABI1 and ABI2, are identified as gene products
that render plants ABA-insensitive when mutated (Leung
et al. 1994; Meyer et al. 1994; Leung, Merlot & Giraudat
1997). Using abi mutant plants, Pei et al. (1997) examined
the ABA regulation of ion channels in guard cells. Consistent with the ABA-insensitive phenotype in the stomatal
regulation, anion channels in these mutants are not sensitive to ABA, resulting in constantly open stomata (Pei et al.
1997). When abi1 mutant protein is expressed in transgenic
tobacco, ABA regulation of inward and outward K+ channels, but not the anion channels, in guard cells was affected
(Armstrong et al. 1995). These results suggest that ABA signalling by ABI1 phosphatase may differ in different plant
species. Because the abi mutants are dominant, it is difficult
to predict the nature of the mutation in the proteins based
on initial genetic analysis. Further biochemical and genetic
studies have demonstrated that ABI phosphatases are negative regulators of ABA signalling (Sheen 1998; Gosti et al.
1999). As a result, ABI1 and ABI2 may negatively regulate
the anion channels and/or K+ channels. This is consistent
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 229–237
Signalling drought in guard cells 233
with the positive regulation of anion channels by AAPK (Li
et al. 2000). However, because studies on AAPK and ABIs
have been conducted in different plants and in a different
genetic context, it is premature to conclude whether AAPK
and ABIs serve as counteracting kinase and phosphatases
in anion channel regulation by ABA. Further work is
required to test this interesting possibility.
Protein farnesylation
Identification of era1 as an ABA-hypersensitive mutant in
Arabidopsis links protein farnesylation to ABA signalling
(Cutler et al. 1996; Pei et al. 1998). Mutation in a farnesyl
transferase beta subunit gene renders the mutant plants
more sensitive to ABA in comparison with the wild-type
plants, suggesting that protein farnesylation is required for
the function of a negative regulator of ABA signalling
pathway (Cutler et al. 1996). Guard cell anion channels and
stomatal behavior in era1 mutant plants display hypersensitivity to ABA (Pei et al. 1998). More recent studies indicate that protein farnesylation is also involved in many
developmental processes in Arabidopsis (Bonetta et al.
2000; Yalovsky et al. 2000; Ziegelhoffer et al. 2000). The
pleiotropic effect is consistent with the hypothesis that
covalent modification of proteins by farnesylation is
required for a number of cellular and developmental processes in plants as well as in other organisms. Finding the
specific target(s) for farnesylation in ABA signalling process will be an important goal for further studies.
OSMOSENSING OF ION CHANNELS:
AN ABA-INDEPENDENT PATHWAY
There are at least two pathways for drought signal transduction that target stomatal closure. One is transmitted
through production of ABA; the other is through direct
osmotic stress. Production of ABA often occurs under persistent drought conditions and can be considered as a
‘slower’ pathway for stomatal regulation under stress conditions. Direct hyperosmotic shocks cause rapid water loss
from guard cells (Raschke 1975). This osmotic stress may be
temporary and results from variations in air humidity, leading to rapid changes in stomatal aperture (Zeiger et al.
1987; Assmann 1993). A recent study has clearly demonstrated that stomatal regulation by air humidity is an ABAindependent process (Assmann, Snyder & Lee 2000).
Although extensive studies have been directed toward
understanding the ABA signalling pathway as discussed
above, much less is understood on how osmotic stress regulates stomatal aperture by the more direct, ABA-independent, osmosensing pathway.
Osmotic pressure generates a ‘secondary’ mechanical
signal because it can be translated into a membrane stretching force caused by ‘shrinking’ or ‘swelling’ of the cell. It has
long been recognized that membrane stretching activates a
class of mechanosensitive channels referred to as stretchactivated channels (SAC) (Kullberg 1987; Cosgrove &
Hedrich 1991; Sachs 1991; Pickard & Ding 1993; Ramaha© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 229–237
leo, Alexandre & Lassalles 1996). Opening of these channels is voltage-independent and operated simply by
pressure application. In order to regulate its osmotic status,
a cell must change the concentration of osmolytes in the cell
or in the medium according to the osmolarity of the
medium. It is apparently difficult for SACs to perform this
task.
More recently, several voltage-dependent ion channels
from mammalian cells have been shown to be regulated by
osmolarity in the extracellular medium (Morris 1990; Gosling, Smith & Poynder 1995; Schoenmakers, Vaudry &
Cazin 1995; Sachs 1997; Baraban et al. 1997). These osmosensitive, voltage-dependent channels play a critical role in
volume regulation of cells under osmotic stress (Moran
et al. 1996a; Cossins & Gibson 1997). For example, hypotonic swelling causes a concerted opening of K+ and Cl–
channels, leading to a net efflux of KCl and driving the loss
of cellular water to recover the cell volume. This process has
been found in many cell types in animals (Sachs 1997).
Stretch-activated channels have been identified in the
plasma membrane and tonoplast of guard cells (Alexandre
& Lassalles 1991; Cosgrove & Hedrich 1991). In a recent
report, Liu & Luan (1998) showed that both inward and
outward voltage-gated K+ channels are regulated by the
osmogradient across the plasma membrane of guard cells.
Hypotonic medium strongly activated IKin and suppressed
IKout whereas hypertonic medium abolished IKin and activated IKout. Organization of actin filaments, but not microtubules, may serve as a mediator for the osmoregulation of
IKin. These findings provide a mechanism for stomatal regulation under osmotic stress and a feedback mechanism for
accelerating stomatal movements.
In animals, cell volume regulation under osmotic stress is
crucial for the function and survival of many cell types. In
particular, hypotonic stress poses the danger of killing cells
due to swelling and bursting as most cells lack a strong
mechanical support such as the plant cell wall. During evolution, animal cells have acquired an extensive population
of membrane channels and pumps for osmoregulation. For
example, osmotic swelling would rapidly activate K+ and
Cl– efflux through voltage-dependent channels in intestine
cells that experience frequent changes in their environmental osmolarity. Loss of osmolytes reduces the osmogradient
across the cell membrane and prevents further swelling
(and bursting) and eventually restores cellular volume
(Tilly et al. 1996).
In plants, cells are confined in the cell wall that restricts
cell volume change caused by an osmogradient across the
cell membrane. Most plant cells swell and develop turgor
pressure against the cell wall because the extracellular
space in a plant is almost always filled with a ‘hypotonic’
solution. For most living plant cells, this turgor pressure can
be converted to cell volume change for cell growth and, in
cell types such as stomatal guard cells and Mimosa pulvini,
for cell ‘movement’ (Assmann 1993; Ward et al. 1995;
Moran, Yueh & Crain. 1996b). In guard cells, as in animal
cells, turgor is also regulated by the membrane channels
and pumps. Studies have shown that the inward (IKin) and
234 S. Luan
outward K+ channels (IKout) responsible for K+ fluxes across
the plasma membrane are among the most important channels in turgor regulation in guard cells (Assmann 1993;
Blatt & Grabov 1997; Maathuis et al. 1997). Moreover, as in
animal cells, osmotic stress can regulate the activity of these
membrane channels and thereby control the cell volume
(or turgor) (Liu & Luan 1998). However, in contrast to the
situation in animal cells where swelling activates outward
ion channels for loss of osmolytes, hypotonic swelling in
guard cells activated IKin, further increasing the cell turgor
and the volume. This distinction reflects the difference in
structural organization of animal and plant cells. More
importantly, ion channel regulation pattern seems to be
designed according to the physiological function of the cell.
Animal cells activate the ion efflux to recover the cell volume to the original level because they cannot afford further
swelling without a cell wall support (Cossins & Gibson
1997). Hypotonic swelling of guard cells is a way to open
the stomata, and activation of IKin is to speed up this process
under a water-sufficient condition (Liu & Luan 1998). Furthermore, cell wall provides the support that prevents cell
burst caused by the swelling process.
For guard cells, osmotic stress signalling may utilize both
ABA-dependent and -independent pathways, as both
induce stomatal closure by regulating activities of several
ion channels in guard cells (Assmann 1993; Blatt & Grabov
1997; MacRobbie 1997; Pei et al. 1997; Liu & Luan 1998).
Regarding the mechanism of osmosensing of ion channels
in guard cells, actin filament organization may play a vital
role. In animal cells, hypotonic swelling induces a reorganization of the actin filaments and activates outward channels
(Schwiebert, Mills & Stanton 1994; Moran et al. 1996a).
Although inward (instead of outward) K+ channel is activated by hypotonic swelling in guard cells, the mechanism
may be similar to that in animal systems. Indeed, osmotic
stress regulation of actin organization correlates well with
ion channel activity (Liu & Luan 1998). Swelling of guard
cells disrupts their actin filament network and causes activation of IKin. Under a hypertonic condition, actin filaments
in the protoplasts are organized into a network configuration that corresponds to a small IKin and a small stomatal
aperture. The correlation of actin filament organization in
guard cells and stomatal aperture under normal conditions
was described by Lee and colleagues (Kim et al. 1995;
Hwang et al. 1997) and is consistent with the osmosensing
mechanism observed by Liu & Luan (1998). Although actin
filament organization is linked to the ion channel regulation
in both animal and plant cells, little is understood on the
molecular mechanism underlying actin–ion channel interaction.
CONCLUDING REMARKS
Perhaps the best-defined mechanism in plant drought
response is stomatal regulation. Understanding the signalling network that links drought signal to guard cell turgor
has been an exciting research topic in plant biology for the
interest in both basic science and practical applications. As
a critical chemical messenger for drought, ABA has been
brought to the centre stage of stomatal regulation.
Although a number of components in ABA signalling have
been identified as briefly discussed above, it is not yet possible to build detailed molecular pathways. The most significant missing link is the ABA receptor or equivalent
component. Other important unknown players include
those between ABA and calcium. Despite the identification
of several molecules that regulate calcium levels in guard
cells, much less is known about the biochemical processes
that produce these signalling molecules. Although several
calcium channel activities have been detected by electrophysiological procedures, molecular characterization of calcium channels has yet to be carried out before a firm
conclusion can be drawn on the steps involved in ABAinduced calcium elevation in guard cells. Furthermore, the
protein components that translate calcium signal into ion
channel regulation are also missing from the picture. In the
direct, ABA-independent osmosensing pathway, ion channels are the molecular targets of osmotic regulation that
may involve cytoskeleton organization. However, none of
the osmosensitive ion channels has been identified at
molecular level. With these prominent questions, it is clear
that this field will remain one of the most exciting areas of
plant biology research in years to come.
ACKNOWLEDGMENTS
The author apologizes to many colleagues whose research
was not cited due to space constraints. Research in the
author’s laboratory was supported by National Institutes of
Health, The National Science Foundation, and the US
Department of Agriculture.
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Received 16 March 2001; received in revised form 17 June 2001;
accepted for publication 17 June 2001