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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Extracellular Calmodulin-Induced Stomatal Closure Is Mediated by Heterotrimeric G Protein and H₂O₂¹

National Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China (Y.-L.C., Y.-M.X., J.C., X.-C.W.); Biotechnology Research Institute, National Grand Scientific Engineering of Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China (R.H.); and College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China (Y.-L.C., P.L.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Extracellular calmodulin (ExtCaM) exerts multiple functions in animals and plants, but the mode of ExtCaM action is not well understood. In this paper, we provide evidence that ExtCaM stimulates a cascade of intracellular signaling events to regulate stomatal movement. Analysis of the changes of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and H₂O₂ in Vicia faba guard cells combined with epidermal strip bioassay suggests that ExtCaM induces an increase in both H₂O₂ levels and [Ca²⁺]_cyt, leading to a reduction in stomatal aperture. Pharmacological studies implicate heterotrimeric G protein in transmitting the ExtCaM signal, acting upstream of [Ca²⁺]_cyt elevation, and generating H₂O₂ in guard cell responses. To further test the role of heterotrimeric G protein in ExtCaM signaling in stomatal closure, we checked guard cell responses in the Arabidopsis (Arabidopsis thaliana) G-subunit-null gpa1 mutants and cG overexpression lines. We found that gpa1 mutants were insensitive to ExtCaM stimulation of stomatal closure, whereas cG overexpression enhanced the guard cell response to ExtCaM. Furthermore, gpa1 mutants are impaired in ExtCaM induction of H₂O₂ generation in guard cells. Taken together, our results strongly suggest that ExtCaM activates an intracellular signaling pathway involving activation of a heterotrimeric G protein, H₂O₂ generation, and changes in [Ca²⁺]_cyt in the regulation of stomatal movements.

Heterotrimeric G proteins composed of -, -, and -subunits are a key intracellular signaling molecule in eukaryotic cells. The activation by G-protein-coupled receptor results in conformation change of the G protein due to GTP binding and the separation of G from the G dimer. GTP hydrolysis by GTPase activity of G results in the reassociation of G with G (Jones and Assmann, 2004). In plants, G protein has been found to be involved in ion-channel regulation (Aharon et al., 1998; Wang et al., 2001), control of seed germination (Ullah et al., 2002), pollen tube elongation (Ma et al., 1999), and responses to ABA (Wang et al., 2001). Genome sequencing revealed the existence of only one prototypical G (GPA1) in Arabidopsis (Arabidopsis thaliana; Ma et al., 1990). It was reported that G-subunit-null mutants, gpa1-1 and gpa1-2, were insensitive to ABA inhibition of whole-cell inward K⁺ currents and pH-independent ABA-activation of anion channels (Wang et al., 2001), suggesting G is a key component in ABA signaling. It is unknown whether G-subunit participates in calcium signaling in ABA regulation of guard cell responses.

Recently, reactive oxygen species (ROS) has been shown to be an important second messenger in signaling to developmental processes, such as polar growth of Fucus rhizoid cells (Coelho et al., 2002) and cell elongation in root growth (Demidchik et al., 2003), responses to environmental stresses (Baxter-Burrell et al., 2002), and guard cell movement (Pei et al., 2000). Evidence indicates that homeostasis of ROS depends on the activity of several enzymes involved in ROS generation as well as the activity of ROS scavenging enzymes (for review, see Mittler, 2002). Recently, guard cell-specific NADPH oxidases AtrbohD (Arabidopsis respiratory burst oxidase homologs D) and AtrbohF have been identified, and the double mutants of atrbohD/F are impaired in ABA-induced ROS generation, [Ca²⁺]_cyt increases, and stomatal closing (Kwak et al., 2003), suggesting that AtrbohD and AtrbohF NADPH oxidases and ROS play an important role in ABA signal transduction in guard cells. The evidence that Ca²⁺-sensing receptor (CAS) in Arabidopsis plasma membrane, which mediates extracellular Ca²⁺ induced cytosolic Ca²⁺ increase in guard cells (Han et al., 2003) suggests that CAS may regulate [Ca²⁺]_cyt status through functioning together with the regulation of Ca²⁺ influx and release from intracellular Ca²⁺ stores, Ca²⁺-ATPase, and Ca²⁺/H⁺ antiporter. However, it is unclear whether ROS also acts as a mediator in extracellular Ca²⁺ receptor mediated stomatal movement.

Calmodulin (CaM), a ubiquitous and abundant intracellular Ca²⁺ receptor, exists in all eukaryotic cells (for review, see Vetter and Leclerc, 2003). Recently, it has been found that CaM exists extracellularly to exert many functions in both animals and plants. In animals, extracellular CaM (ExtCaM) is present in body fluids, saliva, urine, and milk (Houston et al., 1997), stimulating the proliferation of cultured hepatocytes, melanoma cells, and fibroblasts (Goberdhan et al., 1993). In addition, ExtCaM inhibits tumor necrosis factor- release and augments neutrophil elastase release, preventing further cytotoxicity (Houston et al., 1997). In plants, extracellular peptides, such as systemin, CLAVATA3, and ENOD40, may act as intercellular signals, regulating some important processes, e.g. wound defense reaction, maintenance of shoot apical meristem, and nodule formation (Pearce et al., 1993; Yang et al., 1993; van de Sande et al., 1996; Trotochaud et al., 2000). ExtCaM has been found in many plant species. For example, ExtCaM was detected in the medium of suspension-cultured cells from Angelica dahurica, carrot, and tobacco (Sun et al., 1994, 1995). The existence of ExtCaM in plants suggests that it may have important functions. Indeed, ExtCaM stimulates proliferation of suspension-cultured cells of A. dahurica, Fenistum typhoides, and Sataria italica by enhancing cell wall regeneration and protoplast division (Sun et al., 1994) and accelerates pollen germination and tube growth (Ma and Sun, 1997; Ma et al., 1999; Shang et al., 2001). Pharmacological studies have implicated several signaling molecules, including heterotrimeric G protein (Ma et al., 1999), phosphoinositide, and cytosolic Ca²⁺ (Shang et al., 2001) in the signal transduction pathway of ExtCaM-enhanced pollen germination.

Stomatal movements regulate the loss of water to the atmosphere and the entry of CO₂ into the plants for photosynthetic carbon fixation. Many factors, such as ABA, CO₂, light/darkness, and temperature, are known to modulate stomatal movements (Schroeder et al., 2001). The involvement of intracellular CaM in stomatal movements has also been studied (Cottele et al., 1996). However, the effects of ExtCaM on stomatal movements are not well addressed. In our previous report, we demonstrated that ExtCaM existed in the walls of guard cells and that its exogenous application promoted stomatal closure (Chen et al., 2003). In this report, we investigate the intracellular signaling mechanism by which ExtCaM mediates stomatal movement using combined pharmacological, physiological, and genetic approaches. We have provided convincing evidence that ExtCaM triggers a cascade of intercellular signaling events involving heterotrimeric G protein, H₂O₂, and Ca²⁺in the regulation of stomatal closure.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

ExtCaM Induces [Ca²⁺]_cyt Increase in Guard Cells

Since [Ca²⁺]_cyt levels and oscillation have been shown to be a key mediator of guard cell movement (Allen et al., 1999, 2001), we were interested in whether ExtCaM has an effect on the dynamic changes of [Ca²⁺]_cyt in guard cells. Vicia faba guard cells have been a favorite model for the study of guard cell movement (Assmann, 1993). In this study, we applied 10^–8 M CaM to induce stomata closure (Chen et al., 2003) and used confocal laser scanning microscopy (CLSM) to visualize the fluorescence of fluo-3, which was loaded into guard cells. Among 27 V. faba guard cells, 63% of the cells showed the typical [Ca²⁺]_cyt changes responsive to ExtCaM (Fig. 1A). ExtCaM-induced dramatic elevation in [Ca²⁺]_cyt was found after 280 s incubation of CaM (Fig. 1D), while the control treatment (10^–8 M bovine serum albumin) did not cause obvious fluorescent changes in guard cells (data not shown).

View larger version (50K): [in this window] [in a new window]   Figure 1. Application of ExtCaM causes the changes of [Ca2+]cyt through the function of G protein in V. faba guard cells. A to C, Fluorescent changes in guard cells preloaded with 10 µM fluo-3 AM and (D–F) quantitative curve of [Ca2+]cyt responsive to the induction of 10–8 M CaM (A and D), pretreatment of 400 ng/mL PTX plus CaM (B and E), and 400 ng/mL CTX (C and F), respectively. [Ca2+]cyt was quantified based the relative fluorescent intensity referred to the standard serial calcium concentrations as described in "Material and Methods." All the start times in the following figures of this paper reorganize the time point of chemicals/protein treatment as zero time. Bar = 10 µm.

Heterotrimeric G Protein Might Be Involved in ExtCaM Promotion of Stomatal Closure

Having observed ExtCaM induction of [Ca²⁺]_cyt elevation and stomatal closure, we next sought to investigate whether other important signaling components might transduce this signal input. Heterotrimeric G proteins have been shown to regulate [Ca²⁺]_cyt by modulating Ca²⁺ channels in the plasma membrane of animal cells. It was reported that heterotrimeric G protein mediated ExtCaM stimulation of pollen germination (Ma et al., 1999). In guard cells, G protein activators such as cholera toxin (CTX; van Corven et al., 1993) and GTPS inhibited the influx of K⁺, and the effect of GTPS is prevented by buffering cytosolic Ca²⁺ to a low level, suggesting that activated G proteins may inhibit inward K⁺ channels via elevation of [Ca²⁺]_cyt (Fairley-Grenot and Assmann, 1991; Wu and Assmann, 1994). Using genetic approaches, G has been shown to mediate ABA signaling in regulating inward K⁺ channels and slow anion channels and stomatal movements in Arabidopsis (Wang et al., 2001; Coursol et al., 2003). Thus, we speculated that heterotrimeric G proteins may also mediate ExtCaM signaling in guard cells. In this study, we used pertussis toxin (PTX), an inhibitor of heterotrimeric G protein -subunit (Kuryshev et al., 1993), and CTX, an activator of heterotrimeric G protein -subunit (van Corven et al., 1993) to assess whether heterotrimeric G proteins act as a mediator in ExtCaM promotion of [Ca²⁺]_cyt elevation. As shown in Figure 2, CaM promotion of V. faba stomatal closure was greatly impaired when leaf epidermal strips were pretreated with PTX. In parallel with this effect, when V. faba guard cells were pretreated with PTX, 21 guard cells (n = 30) failed to trigger increase of [Ca²⁺]_cyt responsive to ExtCaM (Fig. 1, B and E). Meanwhile, CTX, an activator of heterotrimeric G protein, induced both stomatal closure (Fig. 2) and [Ca²⁺]_cyt increase in 16 of 22 guard cells during 480-s CTX treatment (Fig. 1, C and F). The effect of CTX on stomatal apertures and elevation in [Ca²⁺]_cyt resembled that of ExtCaM, further supporting the hypothesis that ExtCaM acts through [Ca²⁺]_cyt to regulate stomatal movement. Taken together, these results indicate that heterotrimeric G protein may mediate ExtCaM induction of V. faba stomatal closure by tuning [Ca²⁺]_cyt in guard cells.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of PTX or CTX on V. faba stomatal closure. Control, Open stomata were kept in MES buffer but minus CaM for 2 h, then the stomatal aperture was treated as 100%; CaM, open stomata were treated with 10–8 M CaM solution for 2 h; PTX, open stomata were treated with 400 ng/mL PTX solution for 2 h; PTX + CaM, open stomata were pretreated with 400 ng/mL PTX for 30 min, washed with MES buffer, then kept in CaM solution for 2 h; CTX, open stomata were treated with 400 ng/mL CTX for 2 h.

View larger version (17K): [in this window] [in a new window]   Figure 3. Application of ExtCaM causes stomatal closure through the function of G protein in Arabidopsis guard cells. A, Mutants gpa1-1 and gpa1-2 impaired stomatal closure induced by 10–8 M CaM, but not in wild type. Control, Epidermal strips of Ws plants were kept in MES buffer; WT (wild type) + CaM, epidermal strips of Ws plants were treated with 10–8 M CaM solution; gpa1-1 + CaM, epidermal strips of gpa1-1 plants were treated with CaM solution; gpa1-2 + CaM, epidermal strips of gpa1-2 plants were kept in 10–8 M CaM solution. B, cG constitutively overexpressing heterotrimeric G protein -subunit AtGPA1 stimulated stomatal closure induced by 10–8 M CaM. Control, Epidermal strips of Ws plants were kept in MES buffer; WT + CaM, epidermal strips of Ws plants were treated with 10–8 M CaM solution; cG1 + CaM, epidermal strips of cG1 plants were treated with 10–8 M CaM solution; cG2 + CaM, epidermal strips of cG2 plants were kept in 10–8 M CaM solution. Each assay was repeated three times. The data were presented as mean ± SE (n = 150).

Involvement of H₂O₂ in ExtCaM-Induced Stomatal Closure

It has been reported that ROS is a key regulator of stomatal movements (Purohit et al., 1994). For instance, H₂O₂ caused an increase in guard cell [Ca²⁺]_cyt, which was abolished in the presence of EGTA (McAinsh et al., 1996). H₂O₂ has been shown to be a signal molecule in ABA induction of stomatal closure. In this process, ABA induces H₂O₂ production in guard cells by activating NADPH oxidases, and then H₂O₂ causes a [Ca²⁺]_cyt increase by activating Ca²⁺ channels in the plasma membrane (Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003). In this study, we observed that H₂O₂ promoted stomatal closure in V. faba (Fig. 4A), which is consistent with the previous reports in Arabidopsis (Pei et al., 2000). To investigate whether H₂O₂ is involved in ExtCaM-induced stomatal closure, V. faba epidermal strips were incubated in ExtCaM solution containing diphenylene iodonium (DPI) or catalase (CAT), which was either an inhibitor of NADPH oxidases, the key enzyme in the production of H₂O₂, or the scavenger of H₂O₂ (Zhang et al., 2001; Qin et al., 2004). Under these conditions, both DPI and CAT abolished the stimulation of stomatal closure by ExtCaM (Fig. 4B), suggesting that stomatal closure induced by ExtCaM requires the production of H₂O₂, and NADPH oxidases might be involved in the generation of H₂O₂.

View larger version (15K): [in this window] [in a new window]   Figure 4. H2O2 mediates ExtCaM-induced V. faba stomatal closure. A, Effect of 5 x 10–5 M H2O2 in MES buffer on stomatal closure within 2 h. Control indicates no addition of H2O2 except MES buffer. B, Effects of DPI or CAT on stomatal closure-induced by CaM for 2 h. Control, Open stomata were kept in MES buffer under light for 2 h, then the stomatal aperture was treated as 100%; CaM, open stomata were treated with 10–8 M CaM solution; CaM + CAT, open stomata were treated with 100 units/mL CAT plus 10–8 M CaM; CaM + DPI, open stomata were kept in 10 µM DPI plus 10–8 M CaM. Each assay was repeated three times. The data were presented as mean ± SE (n = 150).

View larger version (42K): [in this window] [in a new window]   Figure 5. ExtCaM promotes production of H 2O2 that further induces changes of [Ca2+]cyt in V. faba guard cells. A, Fluorescence and (C) quantitative curve of H2O2 changes reflected with 50 µM H2DCF-DA after addition of 10–8 M CaM. B, Fluorescence and (D) quantitative curve of [Ca2+]cyt in V. faba guard cells preloaded with 10 µM fluo-3 AM responsive to the induction of 5 x 10–5 M H2O2. Control indicates no chemical addition in the buffer solution. Bar = 10 µm.

Heterotrimeric G Protein Mediates ExtCaM-Induced H₂O₂ Increase in Guard Cells

To investigate the relationship among heterotrimeric G protein, H₂O₂, and Ca²⁺, we performed the following experiments. First we tested this pathway in V. faba epidermal strips. As shown in Figure 6, A and B, 17 guard cells (n = 25) displayed an increase of H₂O₂ production induced by CTX. Similarly, CTX induction of stomatal closure was also blocked by either DPI or CAT (Fig. 7A), suggesting that heterotrimeric G protein may act upstream of H₂O₂ production of the regulation of stomatal closure. Once extracellular Ca²⁺ was chelated by EGTA, CTX failed to induce H₂O₂ increase in guard cells (data not shown), suggesting a likely requirement for Ca²⁺ in the H₂O₂ production induced by G protein.

View larger version (31K): [in this window] [in a new window]   Figure 6. Heterotrimeric G protein activator CTX enhanced H2O2 production in V. faba guard cells. A, Fluorescent changes and (B) quantitative curve of H2O2 reflected with 50 µM H2DCF-DA after addition of 400 ng/mL CTX. Control indicates no chemical addition in the buffer solution. Bar = 10 µm.

View larger version (28K): [in this window] [in a new window]   Figure 7. Analysis of functional relationship among heterotrimeric G protein, H2O2, and ExtCaM in guard cells. A, Effects of activator of G protein (400 ng/mL CTX) and inhibitor of NADPH oxidase (10 µM DPI) or scavenger of H2O2 (100 units/mL CAT) on V. faba stomatal closure. Control, Open stomata were kept in MES buffer for 2 h under light, then the stomatal aperture was treated as 100%; CTX, open stomata were treated with 10–8 M CTX solution; CTX + DPI, open stomata were treated with 400 ng/mL CTX plus 10 µM DPI; CTX + CAT, open stomata were pretreated with 400 ng/mL CTX plus 100 units/mL CAT. Each assay was repeated three times. The data were presented as mean ± SE (n = 150). B, Fluorescent changes and (C) quantitative curve of H2O2 reflected with 50 µM H2DCF-DA after addition CaM in WT and gap1. Bar = 40 µm.

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
In this study, we have provided strong evidence that ExtCaM stimulates stomatal closure through the activation of heterotrimeric G protein and subsequent promotion of H₂O₂ production and [Ca²⁺]_cyt elevation. Both genetic and pharmacological studies consistently support the hypothesis that ExtCaM mediating G protein activates the production of H₂O₂. Pharmacological data also support G protein regulation of ExtCaM-dependent [Ca²⁺]_cyt elevation; this remains to be confirmed by genetic studies. In ABA promotion of stomatal closure, it has been shown that ABA activates the production of H₂O₂, which in turn activates plasma membrane-localized calcium channels, leading to [Ca²⁺]_cyt elevation (Pei et al., 2000). Given that ExtCaM and G protein activate both H₂O₂ production and [Ca²⁺]_cyt elevation, it is tempting to propose that in ExtCaM mediated stomatal closure, H₂O₂ too acts as a second messenger to activate plasma membrane-localized calcium channels and [Ca²⁺]_cyt elevation. However, it is also possible that Ca²⁺ might also regulate H₂O₂ production. It has been reported that there are Ca²⁺ binding sites in NADPH oxidases and that this enzyme may be regulated by heterotrimeric G protein (Keller et al., 1998). The induction of ROS generated by oligogalacturonic acid involves a series of processes including receptor binding (Horn et al., 1989), activation of a heterotrimeric G protein (Legendre et al., 1992), influx of Ca²⁺ (Chandra et al., 1997), stimulation of phospholipase C (Legendre et al., 1993), and induction of a number of kinases (Chandra and Low, 1995). Nonetheless, future studies should be directed at understanding the functional relationship among G protein, H₂O₂, and calcium in ExtCaM-mediated stomatal movement.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials

Vicia faba plants were grown in potting mix in a growth chamber under a 12-h-light and 12-h-dark cycle, with a photon flux density of 0.30 mmol m^–2 s^–1, and day/night temperature cycle of 25°C ± 2°C and 20°C ± 2°C, respectively. Lower epidermis of fully expanded leaves from 3- to 4-week-old V. faba seedlings was used for bioassay and the measurements of cytosolic calcium and ROS. Arabidopsis (Arabidopsis thaliana) plants of cG overexpressing constitutively active form mutant of heterotrimeric G protein -subunit AtGPA1, which were obtained from Dr. L.G. Ma (Yale University), were grown in the presence of 70 nM dexamethasone (DEX; Sigma, St. Louis) according to the methods described by Okamoto et al. (2001). T-DNA insertion mutants gpa1-1 and gpa1-2 (from Nottingham Arabidopsis Stock Center) that lack function of G-protein -subunit (Ullah et al., 2001), and wild-type Ws were cultured as described by Wang et al. (2001). Fully expanded leaves of 4- to 6-week-old Arabidopsis plants were used for epidermal strip bioassay and ROS measurement.

Ca²⁺-Sensitive Fluorescent Dye Loading

The abaxial epidermal strips from V. faba were peeled gently and incubated in 10 µM 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, pentaacetoxymethyl ester (fluo-3 AM) loading buffer (10 mM MES-Tris, pH 6.1) at 4°C for 2 h in darkness. Because the activities of esterases at 4°C were low, fluo-3 AM permeated through the membranes without being hydrolyzed by esterases in cell walls. After washed three times with MES buffer, strips were kept at room temperature for 1 h. During this time, fluo-3 AM inside the cell was hydrolyzed by intracellular esterases and the hydrolyzed form of fluo-3 AM bound to free Ca²⁺ to indicate dynamic Ca²⁺ changes in guard cells (Shang et al., 2001).

H₂O₂-Sensitive Fluorescent Dye Loading

The abaxial epidermal strips from V. faba or Arabidopsis were peeled gently and incubated in 50 µM H₂DCF-DA buffer (10 mM MES-Tris, pH 6.1) for 15 min at room temperature and then washed three times before measurement.

CLSM Microscopy

The fluorescence in guard cells was measured using CLSM (Bio-Rad CLSM 1024, Hercules, CA) with the following settings: excitation = 488 nm, emission = 535 nm, frame 512 x 512. Images were recorded every 10 s. When the fluorescence stabilized around 100 s after scanning, the reagents were added directly to the buffer in which the strips were placed, and we treated this agent addition point as zero time in all assays, and fluorescence changes were recorded and the calcium or ROS relative fluorescence intensity was figured by subtracting the basal signal at different time points indicated in figure legends. Using pixel intensity standard curves created by calcium calibration kit (Molecular Probes, Eugene, OR), the calcium concentrations in cells was quantified (Shang et al., 2001). The experiments were repeated at least three times with 7 to 10 cells each time, and one time data were presented to illustrate the changes of fluorescence intensity.

Epidermal Strip Bioassay

After incubated in MES buffer (10 mM MES-Tris, pH 6.1, containing 30 mM KCl and 0.1 mM CaCl₂) for 90 min under light to open the stomata, the strips from V. faba or Arabidopsis were treated with the following procedures. For studying the effects of ExtCaM, CTX, or H₂O₂ on stomatal closure, the strips with open stomata were transferred to the above buffer containing 10^–8 M CaM, 400 ng/mL CTX, or 5 x 10^–5 M H₂O₂ solution, separately. For studying the effects of PTX, DPI, or CAT on CaM induction of stomatal closure, the strips with open stomata were either pretreated with 400 ng/mL PTX solution for 30 min and the strips transferred to and incubated in 10^–8 M CaM solution for 2 h, or the strips were transferred to and incubated in 10^–8 M CaM solution plus10 µM DPI or plus 100 units/mL CAT for 2 h, respectively. For investigating the effect of G protein on the stimulation of H₂O₂ production by ExtCaM, the strips with open stomata were transferred to and incubated in 400 ng/mL CTX solution plus 10 µM DPI or plus 100 units/mL CAT, respectively. Stomatal apertures were measured under microscope at indicated times with 50 randomly selected stomata. Each assay was repeated three times. The data were presented as mean ± SE (n = 150).

	ACKNOWLEDGMENTS

 
The authors thank Drs. L.M. Fan and L. Miao for their helpful discussion and critical reading of the manuscript, Nottingham Arabidopsis Stock Center, and Dr. L.G. Ma for providing Arabidopsis seeds.

Received June 8, 2004; returned for revision September 20, 2004; accepted September 27, 2004.

	FOOTNOTES

 
¹ This work was supported by the Major State Basic Research Program of China (grant no. G1999011704) and by the National Science Foundation of China (grant no. 30370129).

² These authors contributed equally to the paper.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.047837.

^* Corresponding author; e-mail xcwang{at}cau.edu.cn; fax 86–10–62733450.

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