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Keywords:

  • Calcium;
  • ion channel;
  • osmosensing;
  • protein phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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 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 whole-plant 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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.

image

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.

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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 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 ABA-induced 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. 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 (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 acidification 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 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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 ABA-independent 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 stretch-activated channels (SAC) (Kullberg 1987; Cosgrove & Hedrich 1991; Sachs 1991; Pickard & Ding 1993; Ramahaleo, 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 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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 ABA-induced 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Stomatal movements and ion channels
  5. Aba regulation of ion channels in guard cells
  6. Osmosensing of ion channels: an aba-independent pathway
  7. Concluding remarks
  8. Acknowledgments
  9. REFERENCES

Received 16 March 2001; received in revised form 17 June 2001; accepted for publication 17 June 2001