Nitrated Cyclic GMP Modulates Guard Cell Signaling in Arabidopsis
Takahiro Joudoi, Yudai Shichiri, Nobuto Kamizono, Takaaki Akaike, Tomohiro Sawa, Jun Yoshitake,
Naotaka Yamada and Sumio Iwai
Plant Cell; originally published online February 8, 2013;
DOI 10.1105/tpc.112.105049
This information is current as of September 26, 2013
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© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY
This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
Nitrated Cyclic GMP Modulates Guard Cell Signaling
in Arabidopsis
W
Takahiro Joudoi,a Yudai Shichiri,a Nobuto Kamizono,a Takaaki Akaike,b Tomohiro Sawa,b Jun Yoshitake,b
Naotaka Yamada,c and Sumio Iwaia,1
a Department
of Horticultural Science, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan
of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan
c Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan
b Department
Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence
and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this
pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the
nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical
analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive
oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 39,59-cyclic monophosphate (8-bromocGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening
but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to
stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-59diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates
that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard
cells.
INTRODUCTION
Stomata are regulated pores on the surface of aerial plant
organs; the opening and closing of these pores controls the
diffusion of gasses into and out of air spaces in the plant tissues.
Stomata are formed by pairs of guard cells, which sense and
rapidly respond to environmental signals such as light, humidity,
carbon dioxide, and pathogens, and also respond to hormones
including abscisic acid (ABA), auxin, and ethylene (Schroeder
et al., 2001a; Melotto et al., 2006; Shimazaki et al., 2007; Shope
et al., 2008). Guard cells provide a convenient model system for
studying signal transduction (Schroeder et al., 2001b; Assmann
and Wang, 2001). Numerous signaling components act in the
induction of stomatal closure, including the important signaling
compound nitric oxide (NO) (Besson-Bard et al., 2008; Neill
et al., 2008). NO is a reactive free radical that can diffuse across
biological membranes; it is a second messenger in animals and
also regulates various processes in plants, including root growth,
cell wall lignification, senescence, chlorophyll biosynthesis, and
stomatal closure (Lamattina et al., 2003).
In animals, NO enhances the activity of a soluble guanylate
cyclase, which stimulates the production of cyclic GMP (cGMP),
a crucial signaling molecule involved in smooth muscle relaxation,
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Takaaki Akaike (takakaik@
gpo.kumamoto-u.ac.jp).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.105049
in inhibition of platelet aggregation, and in sensing systems,
including vision and olfaction (McDonald and Murad, 1995). In
plants, NO-induced increases in cGMP levels have been reported in plants such as spruce (Picea abies), tobacco (Nicotiana
tabacum), soybean (Glycine max), and Arabidopsis thaliana
(Pfeiffer et al., 1994; Durner et al., 1998; Suita et al., 2009;
Dubovskaya et al., 2011). In plants, inhibitors of animal guanylate
cyclase suppressed the expression of genes encoding Phe
ammonia-lyase and PATHOGEN-RELATED1 protein (Durner et al.,
1998). These inhibitors also prevented NO-induced plant cell death
(Durner et al., 1998) and NO-induced stomatal closure (Neill et al.,
2002). The cell-permeating analog of cGMP, 8-bromoguanosine
39,59-cyclic monophosphate (8-bromo-cGMP), induced expression of genes encoding PATHOGEN-RELATED1 protein, Phe
ammonia-lyase, cation transporters, and chalcone synthase
(Bowler et al., 1994; Durner et al., 1998; Maathuis, 2006; Suita
et al., 2009). Recent work identified a novel plant NO-sensitive
guanylate cyclase (Mulaudzi et al., 2011). Thus, a NO/cGMP
signaling cascade resembling that in animal cells also appears
to operate in plants.
The presence of cGMP has been demonstrated in guard cells,
a finding that agrees with the idea of a specific role for cGMP in
stomatal function (Pharmawati et al., 2001). In addition, NO was
shown to induce stomatal closure (García-Mata and Lamattina,
2001) and to inhibit stomatal opening (Yan et al., 2007; Zhang
et al., 2007). Inhibitors of animal guanylate cyclase also prevented NO-induced stomatal closure in plants (Neill et al., 2002).
These results suggested that the NO/cGMP signaling cascade
functions in stomatal closure. However, 8-bromo-cGMP did not
induce stomatal closure in pea (Pisum sativum; Neill et al., 2002).
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
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By contrast, it did induce stomatal opening in Commelina
communis, Tradescantia albiflora, fava bean (Vicia faba), and
Arabidopsis (Cousson and Vavasseur, 1998; Pharmawati et al.,
1998, 2001; Cousson, 2003). Thus, the issue of whether the NO/
cGMP cascade operates in guard cell signaling is still controversial.
Work in animals has provided evidence that 8-nitro-cGMP is
present and physiologically relevant in vivo. Sawa et al. (2007)
used mammalian cells to demonstrate that NO-dependent
guanine nitration of cGMP does occur and that the nitrated
derivative cGMP, 8-nitro-cGMP, is produced under physiological and pathophysiological conditions. This nitrated derivative
activated cGMP-dependent protein kinases and induced vasodilatation in a manner similar to that of native cGMP. In addition,
8-nitro-cGMP possesses unique electrophilic properties, as it
triggered the production of reactive oxygen species (ROS) and
modified thiol residues of proteins. This novel 8-nitro-cGMP–
mediated posttranslational modification, named S-guanylation,
was studied using the redox sensor protein Keap1. S-Guanylated Keap1 induced the expression of heme oxygenase 1,
which elicits cytoprotection during Salmonella infection (Zaki
et al., 2009). This finding raised the question of whether nitrated
cGMP functions as a guard cell signaling molecule in plants.
Here, we examined the possibility that nitrated cGMP is
present in plants and participates in guard cell signaling. We
found that ABA, NO, and ROS induced 8-nitro-cGMP synthesis
in guard cells and that 8-nitro-cGMP triggered stomatal closure.
The effects of cGMP and 8-nitro-cGMP differed considerably:
cGMP caused stomata to open in the dark, whereas 8-nitro-cGMP
caused stomata to close in the light. Thus, a NO/nitrated
cGMP signaling cascade that is distinct from the classical
NO/cGMP cascade operates in guard cells.
RESULTS
Nitrated cGMP Acts as a Signaling Molecule in Guard Cells
To test whether guanylate cyclase is involved in the signaling
pathways that lead to stomatal closure or opening, we treated
Arabidopsis epidermal cells with two guanylate cyclase inhibitors,
1-H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and
6-anilino-5,8-quinolinequinone (LY83583). Although these reagents
alone had no effects on stomatal aperture (see Supplemental
Figure 1 online), they inhibited ABA- and 1-hydroxy-2-oxo-3-(3aminopropyl)-3-isopropyl-1-triazene (NOC5; NO donor)–induced
stomatal closure (Figures 1A and 1C; see Supplemental Figure 2
online), which suggests that guanylate cyclase is involved in
ABA- and NO-induced stomatal closure. We applied cGMP (the
product of guanylate cyclase) and 8-bromo-cGMP (a membranepermeating analog of cGMP) to epidermal strips, but these molecules did not induce stomatal closure (Figure 1B), consistent with
previous observations in pea and Arabidopsis (Neill et al., 2002;
Dubovskaya et al., 2011). However, cGMP or 8-bromo-cGMP
Figure 1. Guanylate Cyclase Inhibitors Prevented ABA- and NO-Induced Stomatal Closure.
Change in stomatal aperture (mm) in response to various treatments. The stomatal aperture in untreated cells measured 3.44 6 0.05 mm, 3.45 6 0.03
mm, and 3.48 6 0.03 mm for (A), (B), and (C), respectively. Error bars represent SE (n = 100). The double asterisks indicate a significant difference (P <
0.01) compared with values for untreated cells.
(A) Effect of guanylate cyclase inhibitors (ODQ and LY83583) on ABA-induced stomatal closure.
(B) Effect of cGMP on ABA-induced stomatal closure.
(C) Effect of cGMP and ODQ on NOC5-induced stomatal closure.
Nitrated Cyclic GMP–Guard Cell Signaling
applied in combination with ABA and ODQ did induce stomatal
closure (Figure 1B). This effect was abolished by the addition of
the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Furthermore, when NOC5 was applied with cGMP and ODQ, stomata closed (Figure 1C). These data
indicate that cGMP is required, but not sufficient, for stomatal
closure and that its effect occurs only in the presence of NO.
Next, we examined a T-DNA knockout mutant of NOdependent guanylate cyclase, nogc1. In this mutant, ABA and
NOC5 did not induce stomatal closure (Figure 2A), indicating
that NOGC1 is involved in ABA- and NO-induced stomatal closure. We also applied cGMP and 8-bromo-cGMP to epidermal
strips from the nogc1 mutant, but these molecules did not induce
stomatal closure (see Supplemental Figure 3 online), consistent
with the observation in Figure 1B. When ABA and NOC5 were
applied with cGMP (Figure 2B), the stomata closed, confirming
that NO and cGMP are required for stomatal closure.
Mass Spectrometric Identification of 8-Nitro-cGMP
To assess the possibility that cGMP nitration occurs in plant
cells, we first performed mass spectrometry (MS) by liquid
chromatography–ion trap–time-of-flight. MS analysis of 8-nitrocGMP showed the deprotonated molecule ion mass-to-charge
ratio (m/z) 389.03 [M-H2] (calculated C10H10N6O9P2, 389.0247)
with a retention time of 10.01 min (Figure 3A). The plant cell
extracts also showed a deprotonated molecule ion m/z 389.03
at the same retention time. The precursor ion (m/z 389.03) of
8-nitro-cGMP was fragmented by collision-induced dissociation
to produce product ions at m/z 178.00, 195.03, 273.04, and
291.04 (Figure 3B). The plant cell extracts showed an identical
precursor ion at m/z 389.03 and the same product ions,
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confirming that 8-nitro-cGMP synthesis did occur in the plant
cells (Figure 3C).
Detection of 8-Nitro-cGMP by Immunocytochemistry
To further examine 8-nitro-cGMP formation in guard cells, we
conducted immunocytochemical analyses using a monoclonal
antibody against 8-nitro-cGMP and a fluorescent secondary
antibody (Figures 4 and 5). Weak fluorescent background signals were detected in untreated guard cells. Epidermal strips
treated with 8-nitro-cGMP showed strong fluorescence, but
strips treated with 8-bromo-cGMP or cGMP did not differ from
untreated strips (see Supplemental Figure 4 online). These results confirmed that the antibody specifically recognized 8-nitrocGMP and that 8-nitro-cGMP permeated the guard cell membrane.
We detected strong fluorescence in guard cells treated with
ABA, NOC5, and S-nitroso-N-acetyl-DL-penicillamine (SNAP;
NO donor) but not in epidermal pavement cells (Figure 4),
showing that most of the 8-nitro-cGMP in epidermal tissues was
found in the guard cells. A NO synthase inhibitor, N-nitro-L-Arg
methyl ester (L-NAME), reduced the ABA-induced immunofluorescence, and the NO scavenger cPTIO decreased the immunofluorescence induced by ABA and NOC5 (Figure 4). These results
indicated that 8-nitro-cGMP forms in a NO-dependent manner.
Addition of ODQ, a guanylate cyclase inhibitor, also caused
a decrease in ABA- and NOC5-induced immunofluorescence
(Figure 5). When cGMP was applied to epidermal strips together
with NOC5 and ODQ, guard cells showed strong fluorescence
that resembled the fluorescence observed in NOC5-treated
guard cells. These results indicate that cGMP and NO were
required for 8-nitro-cGMP formation in guard cells, which is consistent with our findings from the stomatal aperture assays (Figure 1).
Figure 2. ABA - and NO-Induced Stomatal Closure Requires NO-Dependent Guanylate Cyclase.
(A) Effect of ABA, NOC5, and 8-nitro-cGMP (NcGMP) on stomatal aperture in the nogc1 mutant. The stomatal aperture in untreated cells measured
3.17 6 0.05 mm.
(B) Effect of cGMP on ABA- and NOC5-induced stomatal closure in the nogc1 mutant.
Gray bars, treated with 30 mM ABA; black bars, treated with 100 mM NOC5. 2cGMP, treated with ABA or NOC5 without cGMP; +cGMP, treated
with ABA or NOC5 with 100 mM cGMP. The stomatal aperture in untreated cells measured 3.38 6 0.10 mm for the ABA series and 3.42 6 0.16 mm
for the NOC5 series. Error bars represent SE (n = 80). The double asterisks indicate a significant difference (P < 0.01) compared with values for
untreated cells.
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Figure 3. Identification of 8-Nitro-cGMP in Plant Cell Extracts by MS.
(A) LC-MS/MS chromatogram showing detection of the molecular ion (m/z 389.03 [M-H2]). Authentic 8-nitro-cGMP (top panel) and plant cell extracts
(bottom panel).
(B) Negative ion MS spectra and tandem MS product ions spectra of 8-nitro-cGMP. MS full scan of authentic 8-nitro-GMP (top panel) and product ion
scan of m/z 389.03 (bottom panel).
(C) Negative ion MS spectra and tandem MS product ions spectra of plant cell extract. MS full scan of plant cell extract (top panel) and product ion scan
of m/z 389.03 (bottom panel). The inserted diagram shows the fragmentation pattern.
Quantitative Analyses of 8-Nitro-cGMP by Liquid
Chromatography–Tandem MS
Quantitative analyses of 8-nitro-cGMP were performed by liquid
chromatography–tandem MS (LC-MS/MS) in multiple reaction
monitoring (MRM) mode. The intensities of MS fragments specific to 8-nitro-cGMP in epidermal cells treated with 30 mM ABA
or 100 mM NOC 5 were approximately six- to sevenfold higher
than in untreated epidermal cells (Figure 6A). 8-Nitro-cGMP
synthesis was stimulated even at 1 mM ABA. By contrast, ABAinduced 8-nitro-cGMP synthesis was impaired in the mutant of
the early ABA signaling component protein phosphatase 2C
ABA-INSENSITIVE1 (ABI1, abi1-1; see Supplemental Figure 5
online). These results indicate that ABA and NO induced 8-nitro-
cGMP. We then examined the time course of 8-nitro-cGMP
synthesis (Figure 6B). ABA did not induce 8-nitro-cGMP within 3
min, but within 5 min, ABA induced an increase of ;260% in
8-nitro-cGMP concentrations, which peaked at 15 min.
8-Nitro-cGMP Synthesis Depends on ROS
We used 2,7-dichlorofluorescin diacetate (H2DCF-DA) and diaminofluorescein–2 diacetate (DAF-2 DA) to monitor ROS and
NO generation in guard cells treated with ABA. ABA induced a
250% increase in ROS within 3 min, and ROS concentrations
peaked at 3 to 5 min and then gradually decreased (Figure 7A).
ABA also increased NO production in guard cells; there was
a 200% increase in NO within 3 min after ABA treatment, and
Nitrated Cyclic GMP–Guard Cell Signaling
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Figure 4. ABA- and NO-Induced Synthesis of 8-Nitro-cGMP in Guard Cells.
(A) Fluorescence micrographs of epidermal tissues showing fluorescent signals from antibody against 8-nitro-cGMP. Bars = 20 mm.
(B) Fluorescence intensity per guard cell expressed as the ratio of treated cells to untreated cells. Error bars represent SE (n = 3). The asterisks indicate
a significant difference (P < 0.05) compared with values for untreated cells.
NO continuously increased to 370% in 15 min (Figure 7A). The
generation of NO and ROS occurred simultaneously and preceded 8-nitro-cGMP synthesis (Figure 6B).
To test whether ABA-induced ROS are involved in 8-nitro-cGMP
formation, we used DTT (a reducing agent), 1,2-dihydroxy-3,5benzenedisulfonic acid (Tiron; a superoxide scavenger), and
catalase (a hydrogen peroxide [H2O2] scavenger). These reagents
suppressed ROS generation induced by ABA (Figure 7B) and reduced 8-nitro-cGMP formation induced by ABA (Figures 8A and
8B). In the abi1-1 mutant, where ABA-induced ROS generation did
not occur (see Supplemental Figure 6 online), ABA did not induce
8-nitro-cGMP synthesis (see Supplemental Figure 5 online). Furthermore, when epidermal tissues were treated with H2O2, 8-nitrocGMP increased fivefold (Figure 8C). These results showed that
ROS were required for ABA-induced 8-nitro-cGMP formation.
Roles of cGMP and 8-Nitro-cGMP in Guard Cell Signaling
To examine the responses to the different cGMP derivatives, we
applied each compound exogenously to epidermal strips and
Figure 5. 8-Nitro-cGMP Synthesis Induced by ABA and NO Depended on cGMP.
(A) Fluorescence micrographs of epidermal tissues showing fluorescent signals from antibody against 8-nitro-cGMP. Bars = 20 mm.
(B) Fluorescence intensity per guard cell expressed as the ratio of treated cells to untreated cells. Error bars represent SE (n = 5). The asterisks indicate
a significant difference (P < 0.05) compared with values for untreated cells.
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Figure 6. Quantitative Analyses of 8-Nitro-cGMP by LC-MS/MS.
(A) Levels of 8-nitro-cGMP in epidermal tissues treated with ABA and NOC5. Epidermal tissues were treated with ABA (1, 30 mM) or NOC5 (100 mM) for
30 min. The double asterisks indicate a significant difference (P < 0.01) compared with the control value.
(B) Time course of 8-nitro-cGMP synthesis in epidermal tissues treated with ABA. Epidermal tissues were treated with ABA (30 mM) for the indicated period.
Error bars represent SE (n = 3). fr wt, fresh weight.
examined the change in stomatal aperture. In the light, 8-nitrocGMP induced stomatal closure in a dose-dependent manner
(Figure 9A). Under identical conditions, 8-bromo-cGMP did not
induce stomatal closure. These results indicate that the NO
signaling molecule responsible for the closure of stomata is not
cGMP but is rather 8-nitro-cGMP. When epidermal strips with
closed stomata were exposed to 1 to 100 mM 8-bromo-cGMP in
the dark, stomata opened (Figure 9B), which agrees with previously reported results (Cousson, 2003). At the same concentrations, 8-nitro-cGMP was ineffective, which indicates that the
signaling molecule responsible for stomatal opening was not 8nitro-cGMP but was instead cGMP.
Figure 7. ROS and NO Generation in Guard Cells Treated with ABA.
(A) Time course of ROS and NO generation in guard cells treated with ABA. Epidermal tissues were treated with ABA (30 mM) for the indicated period.
Circles: Fluorescence intensity from guard cells loaded with H2DCF-DA, which detects ROS. Triangles: Fluorescence intensity from guard cells loaded
with DAF-2 DA, which detects NO. Fluorescence intensity per guard cell was expressed as the ratio of treated cells to untreated cells. Error bars
represent SE (n = 30).
(B) Effects of DTT, Tiron, and catalase on ABA-induced ROS generation in guard cells. Epidermal tissues were treated with ABA (30 mM) in the absence
or presence of DTT (2 mM), Tiron (100 mM), or catalase (CAT; 1000 units) for 5 min. Fluorescence intensity per guard cell was expressed as the ratio of treated
cells to untreated cells. Error bars represent SE (n = 30). The double asterisks indicate significant differences (P < 0.01) compared with ABA-treated cells.
Nitrated Cyclic GMP–Guard Cell Signaling
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Figure 8. 8-Nitro-cGMP Synthesis Is Dependent on ROS.
(A) Fluorescence micrographs of epidermal tissues showing fluorescent signals from antibody against 8-nitro-cGMP. Epidermal tissues were treated
with ABA (30 mM) in the absence or presence of DTT (2 mM), Tiron (100 mM), or catalase (CAT; 1000 units) for 30 min. Bars = 20 mm.
(B) Fluorescence intensity per guard cell expressed as the ratio of treated cells to untreated cells. Error bars represent SE (n = 3). The asterisk indicates
a significant difference (P < 0.05) compared with the control value.
(C) H2O2-induced 8-nitro-cGMP synthesis. Epidermal tissues were treated with 100 mM H2O2 for 30 min. 8-Nitro-cGMP levels in epidermal tissues were
determined by means of LC-MS/MS. Error bars represent SE (n = 3). The double asterisk indicates a significant difference (P < 0.01) compared with the
untreated control. fr wt, fresh weight.
Components of the 8-Nitro-cGMP Signaling Pathway
in Guard Cells
Calcium acts as a signaling component in ROS and NO signaling pathways in guard cells. To determine whether Ca2+
mediates 8-nitro-cGMP signaling in guard cells, we used the
cell-permeating Ca2+-chelator bis-(o-aminophenoxy) ethane-N,
N,N’,N’- tetra-acetic acid acetoxymethyl ester (BAPTA-AM). This reagent inhibited stomatal closure induced by NOC5 and 8-nitro-cGMP
(see Supplemental Figure 7 online; Figure 10A) and. Cyclic
adenosine-59-diphosphate-ribose (cADPR), a second messenger modulating intracellular calcium levels (Guse, 1999), has
been shown to serve as a downstream messenger of NO in pea
and fava bean (Neill et al., 2002; Garcia-Mata et al., 2003). To
determine whether cADPR mediates 8-nitro-cGMP signaling in
guard cells, we used two antagonists of cADPR production,
nicotinamide and 8-bromo-cADPR. Although the two inhibitors
themselves had no effect on stomatal aperture (see Supplemental
Figure 1 online), both inhibited NO- and 8-nitro-cGMP–dependent
stomatal closure (see Supplemental Figure 8 online; Figure 10B).
Figure 9. Stomatal Response to 8-Nitro-cGMP and 8-Bromo-cGMP.
(A) Stomatal closure in the light. The stomatal aperture in untreated cells measured 3.87 6 0.05 mm (for the 8-Br-cGMP series) and 3.74 6 0.05 mm (for
the 8-nitro-cGMP series).
(B) Stomatal opening in the dark. The stomatal aperture in untreated cells measured 2.60 6 0.06 mm (for the Br-cGMP series) and 2.55 6 0.04 mm (for the
8-nitro-cGMP series). Error bars represent SE (n = 100). The double asterisks indicate a significant difference (P < 0.01) compared with values for untreated cells.
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These results suggested that Ca2+ acts through ADP-ribosyl cyclase in 8-nitro-cGMP signaling.
SLOW ANION CHANNEL1 (SLAC1) is an essential protein
functioning downstream of ABA, ROS, NO, and Ca2+ in mediating stomatal closure (Negi et al., 2008; Vahisalu et al., 2008;
Geiger et al., 2010). In the slac1-2 mutant, stomatal closure induced by 8-nitro-cGMP or ABA was impaired, indicating that
SLAC1 acts downstream of 8-nitro-cGMP (Figure 10C). ABI1
acts upstream of 8-nitro-cGMP as described above. Nonetheless, there are reports showing that it functions at other steps
of ABA guard cell signaling. For example, it acts downstream
of NO (Desikan et al., 2002) and cADPR (Wu et al., 2003), which
act as signaling molecules in 8-nitro-cGMP signaling. To test
whether ABI1 acts in 8-nitro-cGMP signaling, we examined the
effects of abi1-1 on stomatal closure. We found that NOC5 and
8-nitro-cGMP did not trigger stomatal closure in abi1-1 mutant
plants (see Supplemental Figure 9 online). In the nogc1 mutant,
ABA and NO did not induce stomatal closure, but 8-nitro-cGMP
could induce stomatal closure (Figure 2A), indicating that NOGC1
acts upstream of 8-nitro-cGMP.
DISCUSSION
8-Nitro-cGMP Is a Novel Signaling Molecule in Plants
Neill et al. (2002) previously demonstrated that NO is a downstream component of ABA guard cell signaling and that ABA- and
NO-induced stomatal closure was inhibited by ODQ, a guanylate
cyclase inhibitor, which suggests that an NO/cGMP signaling
cascade operates in guard cells. The reaction product of guanylate cyclase is cGMP, which would be expected to induce
stomatal closing, but its membrane-permeating analog, 8bromo-cGMP, did not induce this response. However, when 8bromo-cGMP was applied together with ABA and ODQ, stomata
closed. Based on these results, Neill et al. (2002, 2008) suggested that cGMP is required but not sufficient for stomatal
closure and that other ABA-induced factors might be required.
We obtained similar results in Arabidopsis. Two guanylate cyclase inhibitors, ODQ and LY83583, prevented ABA- and NOinduced stomatal closure. The T-DNA knockout mutant of
NO-dependent guanylate cyclase was impaired in ABA- and
NO-induced stomatal closing. When cGMP was exogenously
applied together with ODQ and ABA, stomata closed, which
suggests that cGMP-induced stomatal closure depends on an
ABA-induced factor (or factors). Our experiments using nogc1 mutants confirm these results: cGMP-induced stomatal closure occurred in the presence of ABA. NO is thought to be an ABA-induced
factor because NO scavengers eliminated the effect of ABA and
because cGMP-induced stomatal closure requires the presence
of an NO donor. Thus, ABA-induced NO interacted with cGMP
to elicit stomatal closure.
Sawa et al. (2007) reported that NO-dependent nitration of
cGMP, producing 8-nitro-cGMP, occurs in mammalian cells
under physiological and pathological conditions. This nitrated
derivative seems to act as an NO signaling molecule in mammalian cells. We hypothesized that NO-dependent guanine nitration
Figure 10. Downstream Components in 8-Nitro-cGMP Signaling.
(A) Effect of BAPTA-AM on 8-nitro-cGMP–induced stomatal closure in the wild type. The stomatal aperture in untreated cells measured 3.43 6 0.15 mm.
NcGMP, 8-nitro-cGMP.
(B) Effect of antagonists of cADPR on 8-nitro-cGMP–induced stomatal closure in the wild type. The stomatal aperture in untreated cells measured
3.70 6 0.03 mm. Br-cADPR, 8-bromo-cADPR.
(C) 8-Nitro-cGMP–induced stomatal closure was impaired in the slac1-2 mutant. The stomatal aperture in untreated cells measured 2.93 6 0.10 mm. Error
bars represent SE (n = 100). The double asterisks indicate a significant difference (P < 0.01) compared with values for cells treated with 8-nitro-cGMP.
Nitrated Cyclic GMP–Guard Cell Signaling
of cGMP may also occur in plants and that the resulting 8-nitrocGMP acts as a signaling molecule in guard cells. To test this
hypothesis, we performed MS and immunocytochemical analyses with monoclonal antibodies against 8-nitro-GMP. These
analyses demonstrated that NO and cGMP induced the synthesis of 8-nitro-cGMP in guard cells. Furthermore, 8-nitrocGMP induced stomatal closure in a dose-dependent manner.
We concluded that NO induces the production of 8-nitro-GMP
as a signaling molecule that regulates stomatal aperture. A NO/
8-nitro-cGMP signaling cascade therefore operates in guard cells.
A role for cGMP in kinetin- and natriuretic peptide–induced
stomatal opening has been described in T. albiflora (Pharmawati
et al., 1998). In C. communis and Arabidopsis, cGMP mediated
auxin-induced stomatal opening (Cousson and Vavasseur,
1998; Cousson, 2003). We also tested the possibility that
8-nitro-cGMP functions in stomatal opening in Arabidopsis.
8-Bromo-cGMP caused stomata to open, as previously reported (Cousson, 2003), but 8-nitro-cGMP did not. We conclude
that cGMP and its nitrated derivative play different roles in guard
cell signaling: cGMP promotes stomatal opening in the dark,
whereas 8-nitro-cGMP promotes stomatal closure in the light.
ROSs Are Essential for 8-Nitro-cGMP Synthesis
ROS and NO are key signaling molecules that mediate many
developmental and physiological processes. Pathogens elicited
rapid NO and H2O2 production, which triggers plant defense
signaling, including expression of defense-related genes, hypersensitive responses, and stomatal closure (Delledonne et al.,
1998, 2001; Durner et al., 1998; Lee et al., 1999; de Pinto et al.,
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2002; Melotto et al., 2006). In addition, NO and H2O2 are important signaling molecules involved in other processes such as
root development, root gravitropism, and seed germination (Joo
et al., 2001; Pagnussat et al., 2002; Bailly, 2004; Bethke et al.,
2004; Hu et al., 2005; Li et al., 2007). ROS and NO have been
identified as essential components of the signaling network inducing stomatal closure in response to ABA, jasmonate, darkness, UV, and high CO2 concentrations (Zhang et al., 2001; Neill
et al., 2002; Desikan et al., 2004; She et al., 2004; He et al., 2005;
Liu et al., 2005; Kolla and Raghavendra, 2007; Kolla et al., 2007).
In response to stressors, guard cells generate ROS and NO simultaneously; this indicates a close functional relationship between ROS and NO in guard cell signaling (Bright et al., 2006).
Several research groups have suggested that ROS is an upstream component in the NO signaling network involved in
stomatal closure in response to UV-B, ABA, chitosan, and extracellular calmodulin (He et al., 2005; Bright et al., 2006; Li et al.,
2009; Srivastava et al., 2009). In ABA inhibition of stomatal
opening, ROS also acts upstream of NO (Yan et al., 2007).
In this article, we showed that ROS and NO were required for
ABA-induced 8-nitro-cGMP formation, which indicates that guanine nitration of cGMP occurred in the presence of both NO and
ROS. NO and ROS act synergistically to mediate stomatal closure
through guanine nitration of cGMP. In other words, the NO and
ROS signaling pathways converge at guanine nitration of cGMP.
The Role of ABI1 in 8-Nitro-cGMP Signaling
ABI1 acts at a very early step of ABA signaling. ABA binding to
the ABA receptor (RCAR/PYR/PYL) leads to inactivation of ABI1,
Figure 11. Proposed Model for 8-Nitro-cGMP Signaling in Guard Cells.
Stimuli, including ABA, jasmonate (JA), UV, high concentrations of CO2, and pathogen attacks, induce increased NO synthesis that results in cGMP
production. The same stimuli also induce ROS, which react with NO to form RNSs. RNSs react with cGMP to form 8-nitro-cGMP. 8-Nitro-cGMP
triggers cytoplasmic [Ca2+] elevation and then activates slow anion channels, inducing stomatal closure. In the dark, other plant hormones, including
cytokinin, auxin, and natriuretic peptide, induce decreases in both NO and ROS and increases in cGMP, with the result being stomatal opening.
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The Plant Cell
which triggers ABA signaling (Ma et al., 2009; Park et al., 2009).
This led to our expectation that ABI1 functions upstream of 8nitro-cGMP. In this article, we showed that ABA did not induce
8-nitro-cGMP synthesis and ROS generation in abi1-1 mutants,
indicating that ABI1 acts upstream of 8-nitro-cGMP. We also
found that NO and 8-nitro-cGMP did not elicit stomatal closure
in abi1-1 mutants. Two possible explanations may account for
these results. First, ABI1 may act downstream of 8-nitro-cGMP.
ABI1 regulates SLAC1 (Geiger et al., 2009; Brandt et al., 2012)
and KAT1 potassium channels (Sato et al., 2009), which play
central roles in guard cell signaling. 8-Nitro-cGMP directly or
indirectly regulates ABI1 activity, leading to stomatal closure.
In the second possible explanation, ABI1 functions in parallel
with 8-nitro-cGMP. ABI1 activates or induces the synthesis of a
component that functions downstream of 8-nitro-cGMP. This
component is not activated or not synthesized in the abi1-1
mutant, which thus does not respond to 8-nitro-cGMP. The role
of ABI1 in 8-nitro-cGMP signaling will require further study.
NO/8-Nitro-cGMP Signaling Cascade
On the basis of the results presented here, and in view of the
data published by other groups, we propose a model of NO
signaling in guard cells (Figure 11). Biotic and abiotic stressors,
such as pathogen attack, ABA, UV, jasmonate, and high CO2
concentrations, induce increased NO synthesis (Delledonne
et al., 1998; Durner et al., 1998; Neill et al., 2002; He et al., 2005;
Liu et al., 2005; Kolla and Raghavendra, 2007), which causes
higher cGMP levels through NOGC1. The same stressors also
induce the formation of ROSs (Delledonne et al., 2001; Zhang
et al., 2001; Desikan et al., 2004; She et al., 2004; Suhita et al.,
2004; He et al., 2005; Kolla et al., 2007), which react with NO or
nitrite to form reactive nitrogen oxide species (RNS) (van der
Vliet et al., 1997). RNS may react with cGMP to produce 8-nitrocGMP (Yermilov et al., 1995; Sawa et al., 2007). 8-Nitro-cGMP
triggers cytoplasmic [Ca2+] elevation and then activates slow
anion channel1, which induces stomatal closure. In the dark,
other plant hormones, such as cytokinin, auxin, and natriuretic
peptides, induce decreases in NO and ROS (She and Song,
2006; Song et al., 2006) and activate membrane-bound (NOindependent) guanylate cyclase to form cGMP (Pharmawati
et al., 2001). In this case, cGMP nitration does not occur; thus,
the native cGMP induces stomatal opening. Guanine nitration of
cGMP is therefore considered to be a critical factor for switching
the guard cell signaling pathway from stomatal opening to stomatal closure. We conclude that 8-nitro-cGMP is an important
signaling molecule that mediates a dynamic signaling cascade
involving NO/cGMP- and ROS-mediated pathways in guard
cells.
METHODS
Plant Materials
Seeds of the Arabidopsis thaliana mutant slac1-2 were kindly provided by
Koh Iba (Kyushu University, Japan). Seeds of the Arabidopsis ecotype
Columbia-0 and mutant lines nogc1 (CS820406) and abi1-1 were obtained from the Arabidopsis Biological Research Center. Seedlings were
grown in soil in a growth chamber for 1 to 2 months at 20 to 25°C under an
11-h-light/13-h-dark cycle (50 mmol m22 s21).
Stomatal Aperture Assay
Epidermal strips were peeled from the abaxial surface of young but fully
expanded leaves. To study stomatal closing, strips were kept in opening
medium (10 mM MES-KOH, pH 6.15, 50 mM KCl, and 0.1 mM CaCl2) for
3 h in the light (50 mmol m22 s21) before transfer to opening medium
containing one or more of various reagents (30 mM ABA, 100 mM cPTIO,
100 mM NOC5, 2 mM ODQ, 2 mM LY83583, 100 mM cGMP, 1 to 100 mM
8-bromo-cGMP, 1 to 100 mM 8-nitro-cGMP, 10 mM 8-bromo-cADPR, 250
mM BAPTA-AM, and 5 mM nicotinamide). 8-Nitro-GMP was synthesized
as described (Sawa et al., 2007). NOC5 and cPTIO were from Dojindo.
ODQ was from Sigma-Aldrich. LY83583 was from Merck. Other reagents
were from Wako Pure Chemical Industries. The strips were incubated in
this medium for 2 h in the light. To investigate stomatal opening, epidermal
strips were floated in opening medium for 2 h in the dark and were then
incubated in opening medium containing 8-bromo-cGMP or 8-nitrocGMP for 3 h in the dark. During treatments, epidermal strips were kept at
23°C. After treatment, stomata were photographed with a charge-coupled
device camera (DS-Fi1; Nikon) mounted on a microscope (Eclipse E600;
Nikon). Stomatal apertures (inner diameters of the stomatal pores) were
measured using a digital microanalyzer (Japan Polaroid Digital Products).
At least four strips, with 20 stomata in each strip, were evaluated for each
treatment. cPTIO, NOC5, cGMP, 8-bromo-cGMP, and 8-nitro-cGMP
were dissolved in water. ABA, LY83583, and ODQ were dissolved in 0.1 to
0.01% DMSO, and 8-bromo-cADPR was dissolved in 0.01% ethanol. At
these concentrations, DMSO and ethanol did not affect stomatal aperture
in our experimental conditions. Experiments were repeated at least four
times, and representative results are shown.
Identification of 8-Nitro-cGMP by MS
About 5 g leaves treated with 30 mM ABA for 2 h were ground in liquid
nitrogen with a mortar and pestle and resuspended in extraction medium
(2% acetic acid/80% ethanol). The mixture was incubated for 30 min at
4°C and centrifuged at 15,000g for 20 min. The supernatants were removed and the pellets were rehomogenized two times with extraction
medium. The supernatants were combined and applied to OASIS WAXSPE columns (Waters) preconditioned with 1 mL methanol and 1 mL
water. The columns were rinsed with 2 mL 2% (v/v) acetic acid/water and
2 mL methanol and then eluted with 2 mL 5% (v/v) NH4OH/methanol. The
eluates were dried under vacuum and resuspended in 0.6 mL 1% (v/v)
formic acid/water (Sigma-Aldrich) and then were separated by HPLC
fractionation on a Mightysil RP-18 column (250 3 4.6-mm i.d.; Kanto
Chemical) with a binary solvent system comprising 0.1% (v/v) formic acid/
water (A) and 0.1% (v/v) formic acid/methanol (B) at 40°C with flow rate of
1.0 mL/min. The linear solvent gradient program applied was: 0 to 3 min B,
5%; 3 to 9 min B, 5 to 100%; and 9 to 20 min B, 100%. The injection
volume was 20 mL each time. All 8-nitro-cGMP fractions (at 11.2 to 12.0
min) were concentrated under vacuum and reconstituted with 200 mL
0.1% formic acid.
Detection and identification of 8-nitro-cGMP were performed with a
liquid chromatography–ion trap–time-of-flight mass spectrometer (Shimadzu).
The fractionated samples were eluted from reverse-phase HPLC on
a CAPCELL PAK C18 column (150 3 3.0-mm i.d.; Shiseido Fine
Chemicals), with a binary solvent system comprising 0.1% (v/v) formic
acid/water (A) and 0.1% (v/v) formic acid/methanol (B) at room temperature (;20°C) with a flow rate of 0.20 mL/min. The linear solvent gradient
program applied was as follows: 0 to 1 min, 5% solvent B; 1 to 6 min, 5 to
100% solvent B; and 6 to 20 min, hold at 100% solvent B. The injector
volume selected was 10 mL. The MS was operated with a probe voltage of
Nitrated Cyclic GMP–Guard Cell Signaling
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23.5 kV, collision-induced dissociation temperature of 200°C, block
heater temperature of 200°C, nebulizer gas (nitrogen) flow of 1.5 L/min,
ion accumulation time of 50 ms, tolerance of 0.05 m/z, MS range of m/z
100 to 500, tandem MS range of m/z 100 to 500, and collision-induced
dissociation parameters as follows: energy, 125 mV; collision gas, 100%.
The precursor ion of 8-nitro-cGMP was m/z 389.03 (calculated
C10H10N6O9P2, 389.0247). LCMS solution version 3.50 software (Shimadzu)
was used for data acquisition and processing.
immunostain solution A) was added, and the samples were incubated for
60 min at room temperature. After the slides were washed, they were
mounted with Vectashield (Vector Laboratories) and observed with
a fluorescence microscope (Eclipse E600; Nikon) equipped with a chargecoupled device camera (DS-Fi1; Nikon). Fluorescence intensities were
measured using EZ-C1 FreeViewer version 3.70 software (Nikon). At least
100 guard cells were evaluated. The data are average values from at least
three independent experiments.
Quantitative Analyses of 8-Nitro-cGMP by LC-MS/MS
ROS Detection in Guard Cells
Leaves were homogenized (30 s 3 3) in ice-cold 10 mM MES-KOH, pH
6.15, containing 2 mM DTT using a homogenizer (AM-5; Nihonseiki
Kaisha). Epidermal tissues were collected on 40-mm nylon mesh and kept
in opening medium for 3 h in the light before incubation in opening
medium containing ABA, NOC5, and hydrogen peroxide for the indicated
times. The treated epidermal tissues (100 to 200 mg) were ground with
a microtube and pestle and resuspended in the extraction medium. The
mixture was incubated for 30 min at 4°C and centrifuged at 15,000g for 20
min. The supernatants were removed and the pellets were rehomogenized
two times with the extraction medium. The supernatants were combined
and separated on OASIS WAX-SPE columns as described above. The
eluates were dried and then dissolved in 0.1 mL water, followed by
analysis of 8-nitro-cGMP contents by LC-MS/MS in MRM mode. 15Nlabeled 8-nitro-cGMP (8-15NO2-cGMP) was synthesized as described
(Fujii et al., 2010) and was used as an internal standard. LC-MS/MS was
performed with a 3200 QTRAP LC-MS/MS system (AB SCIEX) after reverse-phase HPLC on a Mightysil RP-18 column (150 3 2.0-mm i.d.) with
a binary solvent system comprising 0.1% (v/v) formic acid/water (A) and
methanol (B). The linear solvent gradient program applied was as follows:
0 to 7 min, 0 to 100% solvent B; 7 to 20 min, hold at 100% solvent B. The
total flow rate was 0.20 mL/min, and the injection volume was 10 mL.
Ionization was achieved using electrospray in the negative mode with the
spray voltage set at 24.5 kV. Nitrogen was used as the nebulizer gas and
nebulizer pressure was set at 50 p.s.i. The source temperature, the declustering potential, and collision energy were set to 350°C, 50 V, and
35 V, respectively. Quantification was performed using the MRM mode at
m/z 389 to 195 for 8-nitro-cGMP and m/z at 390 to 196 for 8-15NO2-cGMP.
Analyst 1 software (AB SCIEX) was used for data acquisition and processing. The data are average values from at least three independent
experiments.
Epidermal tissues were incubated in opening medium for 3 h and were
then transferred to opening medium containing 30 mM ABA and incubated
for 3 to 15 min in the light. The treated epidermal tissues were collected on
40-mm nylon mesh, floated in opening medium containing 10 mM H2DCF-DA
(Wako Pure Chemical Industry) for 20 min in the dark, and then collected
on 40-mm nylon mesh. The dye-loaded epidermal tissues were washed
with opening medium and floated in opening medium in the dark for
10 min. Control experiments (with only 0.01% DMSO added) showed no
stimulation of ROS production. Fluorescence intensities were measured
as described above, and data shown were obtained by subtracting the
control values (without ABA) from the values for treated samples. At least
30 guard cells were evaluated. The data are average values from at least
three independent experiments.
NO Detection in Guard Cells
Epidermal tissues were incubated in opening medium for 3 h, transferred
to opening medium containing 10 mM DAF-2 DA (Sekisui Medical), and
incubated for 20 min in the dark. The tissues were washed with opening
medium and floated in opening medium in the dark for 10 min. The dyeloaded epidermal tissues were transferred to opening medium containing
30 mM ABA and incubated for 3 to 15 min in the light. Fluorescence
intensities were measured for at least 30 guard cells, and the data shown
were obtained by subtracting the control values (without ABA) from the
values for the treated samples. The data are average values from at least
three independent experiments.
Statistical Analysis
All data are presented as means 6 SE. The statistical significance of
differences between two groups was determined by Student’s t test.
Immunocytochemical Analyses
Epidermal tissues were incubated in opening medium for 3 h and were
then transferred to opening medium containing one or more of various
reagents (30 mM ABA, 25 mM L-NAME, 100 mM cGMP, 100 mM NOC5,
200 mM SNAP, 100 mM cPTIO, 2 mM ODQ, 100 mM Tiron, 2 mM DTT, and
1000 units of catalase). L-NAME, NOC5, cPTIO, and SNAP were from
Dojindo. Other reagents were from Wako Pure Chemical Industries. After
1 h, the tissues were fixed with Zamboni solution (4% paraformaldehyde,
10 mM picric acid, and 0.1 M phosphate buffer, pH 7.4) at 4°C for 4 h. After
the epidermal strips were washed three times with PBS, fixed strips were
placed on silanized glass slides (Dako). Preparations were digested enzymatically (20 mM MES-KOH, pH 5.5, 0.7% cellulysin, and 0.2 M
mannitol) at 25°C for 15 min, washed with PBS, and treated with 0.5%
Triton X-100 at room temperature for 15 min. After the preparations were
washed again with PBS, they were blocked with 3% BSA in PBS at 4°C
overnight and then washed with PBS. The slides were then incubated with
antibodies against 8-nitro-cGMP (1G6; Sawa et al. 2007) in Can Get
Signal immunostain solution A (Toyobo) for 2 h at room temperature. The
slides were washed in PBST (PBS and 0.05% Tween 20) four times for 5
min each. The fluorescent secondary antibody (2 mg/mL Alexa 488–
conjugated goat anti-mouse IgG [Invitrogen] dissolved in Can Get Signal
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: NOGC1 (At1g62580), ABI1 (At4g26080), and SLAC1
(At1g12480).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Effect of Inhibitors on Stomatal Aperture.
Supplemental Figure 2. Inhibitors of Guanylate Cyclase Reduced
NOC5-Induced Stomatal Closure.
Supplemental Figure 3. Effect of cGMP and 8-Bromo-cGMP on
Stomatal Aperture in the nogc1 Mutant.
Supplemental Figure 4. Fluorescence Micrographs of Epidermal
Strips Showing Fluorescent Signals from Anti-8-Nitro-cGMP.
Supplemental Figure 5. ABA-Induced 8-Nitro-cGMP Synthesis in the
abi1-1 Mutant.
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The Plant Cell
Supplemental Figure 6. ABA-Induced ROS Generation Did Not Occur
in the abi1-1 Mutant Guard Cells.
Supplemental Figure 7. Effect of BAPTA-AM on NOC5-Induced
Stomatal Closure.
Supplemental Figure 8. Effect of Antagonist of Cyclic ADPR on
NOC5-Induced Stomatal Closure.
Supplemental Figure 9. Effect of 8-Nitro-cGMP on Stomatal Aperture
in the abi1-1 Mutant.
ACKNOWLEDGMENTS
We thank Koh Iba (Kyushu University, Japan) for kindly providing slac1-2.
We thank members of the Vegetable Science Laboratory at Kagoshima
University for help and suggestions. This work was supported by Grantsin-Aid for Scientific Research (B: 21390097) and Scientific Research
on Innovative Areas (Research in a Proposed Research Area, 20117001
and 20117005) from the Ministry of Education, Sciences, Sports, and
Technology, Japan.
AUTHOR CONTRIBUTIONS
S.I., T.A., and T.S. designed the research and analyzed data. T.J., Y.S.,
N.K., J.Y., N.Y., and S.I. performed research. S.I., T.A., T.S., and N.Y.
wrote the article.
Received September 11, 2012; revised December 26, 2012; accepted
January 16, 2013; published February 8, 2013.
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