1 : red light and pm h 2 3 corresponding author · 13/08/2018 · 3 36 abstract 37 stomatal...

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Short title: Red light and PM H+-ATPase in guard cells 1

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Corresponding author: 3

Toshinori Kinoshita, Ph.D 4

Professor 5

Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, 6

Nagoya 464-8602, Japan 7

Tel/Fax: +81-52-789-4778 8

e-mail: [email protected] 9

Plant Physiology Preview. Published on August 13, 2018, as DOI:10.1104/pp.18.00544

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Red light-induced phosphorylation of plasma membrane H+-ATPase in stomatal guard 10

cells 11

12

Eigo Andoa and Toshinori Kinoshitaa,b,* 13

14 aDivision of Biological Science, Graduate School of Science, Nagoya University, Chikusa, 15

Nagoya 464-8602, Japan; bInstitute of Transformative Bio-Molecules (WPI-ITbM), Nagoya 16

University, Chikusa, Nagoya 464-8602, Japan 17

*Address correspondence to [email protected] 18

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Short title: Red light and PM H+-ATPase in guard cells 20

21

One sentence summary: 22

Red light induces photosynthesis-dependent phosphorylation of plasma membrane 23

H+-ATPase in guard cells to promote stomatal opening in whole leaves. 24

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Author contributions: 26

E.A. and T.K conceived research plans; T.K. supervised the experiments; E.A. performed 27

most of the experiments; E.A. and T.K. designed the experiments and analyzed the data; E.A. 28

and T.K wrote the article. 29

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Funding 31

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of 32

Education, Culture, Sports, Science, and Technology, Japan (grant nos. 15H05956 and 33

15H04386 to T.K.) and by Grant-in-Aid for Japan Society for the Promotion of Science 34

Research Fellow (grant no. 26303 to E.A.). 35



3

ABSTRACT 36

Stomatal opening is stimulated by red and blue light. Blue light activates plasma membrane 37

(PM) H+-ATPase by phosphorylating its penultimate residue, threonine, via a blue light 38

photoreceptor phototropin-mediated signaling pathway in guard cells. Blue light-activated 39

PM H+-ATPase promotes the accumulation of osmolytes and thus osmotic influx of water 40

into guard cells, driving stomatal opening. Red light-induced stomatal opening is thought to 41

be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, 42

how red light induces stomatal opening and whether PM H+-ATPase is involved in this 43

process have remained unclear. In this study, we established an immunohistochemical 44

technique to detect the phosphorylation level of PM H+-ATPase in guard cells using whole 45

leaves of Arabidopsis (Arabidopsis thaliana) and unexpectedly found that red light induces 46

PM H+-ATPase phosphorylation in whole leaves. Red light-induced PM H+-ATPase 47

phosphorylation in whole leaves was correlated with stomatal opening under red light and 48

was inhibited by the plant hormone abscisic acid. In aha1-9, a knockout mutant of one of the 49

major isoforms of PM H+-ATPase in guard cells, red light-dependent stomatal opening was 50

delayed in whole leaves. Furthermore, the photosynthetic electron transport inhibitor, DCMU, 51

inhibited red light-induced PM H+-ATPase phosphorylation, as well as red light-induced 52

stomatal opening in whole leaves. Our results indicate that red light-induced PM H+-ATPase 53

phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight 54

into the photosynthetic regulation of stomatal opening. 55



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INTRODUCTION 56

Stomata are composed of pairs of guard cells in the plant epidermis and serve as the gate 57

for gas exchange, controlling CO2 uptake for photosynthesis as well as water loss by 58

transpiration. Plants respond to various environmental stimuli with changes in guard cell 59

turgor and volume, which leads to stomatal opening or closure (Inoue and Kinoshita, 2017; 60

Jezek and Blatt, 2017). The accumulation of K+ and/or sugars in guard cells drives osmotic 61

influx of water and thus stomatal opening. K+ accumulates through voltage-gated 62

inward-rectifying K+ channels in response to hyperpolarization of the plasma membrane 63

(PM) in guard cells (Inoue and Kinoshita, 2017; Jezek and Blatt, 2017). Sugars accumulate 64

via photosynthesis and starch degradation (Daloso et al., 2016). 65

Stomatal opening is stimulated by light, including blue and red light (Shimazaki et al., 66

2007; Inoue and Kinoshita, 2017). Blue light-induced stomatal opening is mediated by 67

blue-light photoreceptor phototropins (phot1 and phot2; Kinoshita et al., 2001; Doi et al., 68

2004; Ando et al., 2013). Blue light activates PM H+-ATPase via phosphorylation of the 69

penultimate C-terminal residue, threonine, even in isolated guard cell protoplasts (GCPs) or 70

epidermal tissue (Kinoshita and Shimazaki, 1999; Kinoshita and Shimazaki, 2002; Ueno et 71

al., 2005; Hayashi et al., 2011), which indicates that blue light-induced stomatal opening is a 72

guard cell-autonomous reaction. In addition, blue light induces starch degradation, resulting 73

in the accumulation of sugars in guard cells (Poffenroth et al., 1992; Talbott and Zeiger, 74

1993). A recent study suggested that blue light-activated PM H+-ATPase is required for this 75

degradation (Horrer et al., 2016; reviewed by Santelia and Lunn, 2017). In Arabidopsis 76

(Arabidopsis thaliana), all isogenes of PM H+-ATPase (AHA1–AHA11) are expressed in 77

guard cells (Ueno et al., 2005). Among them, AHA1 is the major isoform and is responsible 78

for blue light-induced stomatal opening (Yamauchi et al., 2016). 79

Red light-induced stomatal opening is inhibited by a photosynthetic electron transport 80

inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); thus, it is considered to depend 81

on photosynthesis (Sharkey and Raschke, 1981; Doi and Shimazaki, 2008; Wang et al., 2011; 82

Suetsugu et al., 2014). Red light leads to the accumulation of sugars in guard cells via 83

photosynthesis (Poffenroth et al., 1992; Talbott and Zeiger, 1993) and starch degradation 84

(Olsen et al., 2002). In addition, guard cells import sucrose synthesized in mesophyll 85



5

photosynthesis from apoplasts via the activity of H+/sucrose symporters in these cells (Lu et 86

al., 1997; Daloso et al., 2016). Red light also leads to the accumulation of K+ in guard cells 87

and PM H+-ATPase activity in guard cells is inferred to be required for this uptake (Hsiao et 88

al., 1973; Olsen et al., 2002); however, it is controversial whether red light activates PM 89

H+-ATPase in guard cells. Although there is a report describing red light-induced activation 90

of PM H+-ATPase in GCPs (Serrano et al., 1988), this finding has not been reproduced 91

(Roelfsema et al., 2001; Taylor and Assmann, 2001). If this does occur, it remains to be 92

revealed how red light activates PM H+-ATPase in guard cells because PM H+-ATPase 93

activity is regulated not only by phosphorylation of the C-terminal penultimate residue, 94

threonine, but also by modifications at other residues in the protein (Falhof et al., 2016). 95

Guard cells respond to intercellular CO2 concentration (Ci) rather than ambient CO2 (Mott, 96

1988). Illumination of guard cells with a small beam of red light does not induce a change in 97

PM potential (Roelfsema et al., 2001). Such localized light illumination is insufficient for 98

stomatal opening (Mott et al., 2008). A high concentration of CO2 activates anion channels 99

and depolarizes the PM of guard cells, while illumination of a large area of a leaf with red 100

light decreases substomatal CO2 concentration (Roelfsema et al., 2002). Elevated CO2 also 101

inactivates and activates inward- and outward-rectifying K+ currents, respectively (Brearley 102

et al., 1997). These reports suggest that red light-induced stomatal opening may be an 103

indirect response to the reduction of Ci by photosynthesis. In fact, a low concentration of CO2 104

also induces stomatal opening mediated by low CO2 signaling components, HIGH LEAF 105

TEMPERATURE1 (HT1; Hashimoto et al., 2006) and its downstream targets 106

CONVERGENCE OF BLUE LIGHT AND CO2 1 and 2 (CBC1 and CBC2; Hiyama et al., 107

2017). Low CO2 signaling most likely inhibits S-type anion channels (Xue et al., 2011; Hõrak 108

et al., 2016; Hiyama et al., 2017), which are critical channels not only for anion efflux during 109

stomatal closure (Vahisalu et al., 2008; Geiger et al., 2011), but also for indirect homeostatic 110

regulation of cytosolic pH and Ca2+, which in turn affects K+ uptake and thus stomatal 111

opening (Wang et al., 2012). The ht1 and cbc1 cbc2 mutants do not exhibit clear increase in 112

stomatal conductance in response to red light, as well as low CO2 levels (Matrosova et al., 113

2015; Hiyama et al., 2017). Moreover, it is noteworthy that a high concentration of CO2 114

inhibits PM H+-ATPase (Edwards and Bowling, 1985), giving rise to the hypothesis that the 115



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reduction of Ci may release PM H+-ATPase from the inactivating effects of high Ci. 116

Evidence has been presented that red light induces stomatal opening even under constant 117

Ci (Messinger et al., 2006; Lawson et al., 2008), which suggests that a Ci-independent 118

mechanism is also involved in red light-induced stomatal opening. Guard cells in most plant 119

species contain chloroplasts (Lawson, 2009), with the exception of orchids of the genus 120

Paphiopedilum (Zeiger, 1983), and photosynthesis in guard cell chloroplasts provides the 121

ATP required for H+-pumping by activated PM H+-ATPase (Tominaga et al., 2001). 122

Mesophyll photosynthesis may also promote the accumulation of ATP in guard cells during 123

light-induced stomatal opening (Wang et al., 2014a). In addition, recent studies have 124

suggested that sugars accumulate not only as osmolytes but also as metabolic substrates for 125

the production of ATP and/or malate2–, a counterion of K+, via glycolysis and the 126

tricarboxylic acid cycle in guard cells (Daloso et al., 2016). Furthermore, red/far-red light 127

photoreceptor phytochrome B (phyB) regulates a transcript of MYB60, a transcription factor 128

that affects stomatal apertures (Cominelli et al., 2005; Wang et al., 2010). 129

Previously, we established an immunohistochemical technique to visualize the 130

phosphorylation level of PM H+-ATPase in guard cells using isolated epidermis (Hayashi et 131

al., 2011). Conventional analyses using GCPs require large-scale cultivation of plants and 132

usually take a long time to prepare the protoplasts. By contrast, the immunohistochemical 133

technique using isolated epidermis enables us to analyze PM H+-ATPase more easily. 134

However, despite the great usefulness of the previously developed immunohistochemical 135

technique, it still requires isolation of the epidermis, so its range of application is restricted by 136

the availability of plant epidermis. To overcome this issue, we established an 137

immunohistochemical technique using whole leaves instead of isolated epidermis and 138

unexpectedly found that red light induces phosphorylation of the penultimate C-terminal 139

residue, threonine, of PM H+-ATPase in guard cells in whole leaves. We further characterized 140

this red light-induced phosphorylation of PM H+-ATPase and investigated its correlation with 141

stomatal opening in whole leaves. Our findings indicate that red light induces 142

photosynthesis-dependent phosphorylation of PM H+-ATPase in guard cells to promote 143

stomatal opening in whole leaves. 144



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145



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RESULTS 146

An immunohistochemical technique using whole leaves and detection of red 147

light-induced phosphorylation of PM H+-ATPase in guard cells 148

Previous success with immunohistochemical analyses using isolated epidermis enabled us 149

to analyze the phosphorylation status of PM H+-ATPase in guard cells more easily than with 150

conventional analyses using GCPs (Hayashi et al., 2011). However, that technique still 151

requires isolation of the epidermis, so it is impractical for studying plants from which 152

sufficient epidermis cannot be isolated. To overcome this weakness, we conducted 153

immunohistochemical analyses using whole leaves. To this end, a syringe was used to 154

infiltrate the fixative quickly into the leaves and then excessive leaf tissue, except the 155

epidermis, was removed enzymatically on microscope slides during tissue permeabilization 156

(see also Materials and Methods). To test whether the method would allow us to detect PM 157

H+-ATPase protein and phosphorylation of the penultimate residue, threonine, in guard cells, 158

we investigated phototropin (phot1 and phot2)-mediated blue light-induced phosphorylation 159

of PM H+-ATPase using anti-pThr and anti-H+-ATPase antisera. As expected, the 160

phosphorylation level of PM H+-ATPase in guard cells was statistically significantly higher 161

with weak blue light (5 μmol m–2 s–1) superimposed on background red light (600 μmol m–2 162

s–1) than with red light alone in grabra1 (gl1; Fig. 1A), the control plant of phot1 phot2 163

(Kinoshita et al., 2001). By contrast, blue light-induced phosphorylation was completely 164

abolished in phot1 phot2 (Fig. 1A). The amount of PM H+-ATPase did not differ depending 165

on the light conditions or genotype, as described previously (Fig. 1B; Hayashi et al., 2011). 166

These results verified that our new method works. 167

Interestingly, we found that the phosphorylation level of whole gl1 leaves illuminated 168

with red light alone was statistically significantly higher, by about 90%, than that of gl1 169

leaves kept in the dark (Fig. 1A). Consistent with this, red light also increased the 170

phosphorylation level by about 82% in phot1 phot2 (Fig. 1A). Red light-induced 171

phosphorylation of PM H+-ATPase in guard cells was confirmed in whole leaves from wild 172

type Col-0 and isolated epidermis immediately prepared from illuminated Col-0 leaves 173

(Supplemental Fig. S1), which suggests that it is not caused by mutations in GL1 or some 174

artifact inherent to the technique. In addition, we confirmed that the same fluence rate of red 175



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light used to illuminate whole leaves did not induce phosphorylation in isolated epidermis 176



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(Supplemental Fig. S2A). These results clearly indicate that the phosphorylation status of PM 177

H+-ATPase in guard cells is regulated by red light in whole leaves of Arabidopsis. 178

179

Red light-induced phosphorylation of PM H+-ATPase in guard cells correlates with 180

stomatal opening in whole leaves 181

The activation of PM H+-ATPase via phosphorylation of the penultimate residue, 182

threonine, of PM H+-ATPase is important in blue light-induced stomatal opening (Kinoshita 183

and Shimazaki, 1999; Kinoshita and Shimazaki, 2002). Thus, we investigated the relationship 184

between this phosphorylation and red light-induced stomatal opening in whole leaves. In 185

accordance with the phosphorylation status of PM H+-ATPase shown in Figure 1A and 186

Supplemental Figure S1A, red light statistically significantly induced stomatal opening in 187

whole leaves (Fig. 2). The size of the stomatal aperture in red light-illuminated leaves was 188

intermediate between that of leaves illuminated with both red and blue light, and dark-kept 189

leaves in both Col-0 (Fig. 2A) and gl1 (Fig. 2B). Red light-induced stomatal opening in 190

whole leaves was saturated for at least 60 min (Fig. 2A) and detectable even in phot1 phot2 191

(Fig. 2B). These results are consistent with previous gas-exchange experiments showing a red 192

light-dependent increase in stomatal conductance (Doi et al., 2004; Shimazaki et al., 2007; 193

Suetsugu et al., 2014; Yamauchi et al. 2016). In isolated epidermis, red light did not induce 194

stomatal opening within the measurement period used here (up to 180 min), and only blue 195

light superimposed on red light induced stomatal opening (Supplemental Fig. S2B). 196

Next, we performed a time-course experiment within a period of 60 min to characterize 197

the initial red-light responses using whole leaves. As shown in Figure 3A, the 198

phosphorylation level of PM H+-ATPase was upregulated within 10 min of the start of 199

illumination, and saturated around 30 min. Red light induced stomatal opening within at least 200

15 min after the start of illumination and the size of the aperture reached a stable level within 201

60 min (Fig. 3B). These results indicate that red light-induced phosphorylation of PM 202

H+-ATPase in guard cells correlates with red light-induced stomatal opening. 203

204



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Exogenous abscisic acid suppresses red light-induced phosphorylation of PM 205

H+-ATPase in guard cells and stomatal opening 206



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Abscisic acid (ABA) inhibits blue light-induced phosphorylation of PM H+-ATPase in 207

guard cells and stomatal opening (Zhang et al., 2004; Hayashi et al., 2011). Thus, we 208

investigated the effects of ABA on red light-induced phosphorylation of PM H+-ATPase in 209

guard cells and stomatal opening in whole leaves. As shown in Figure 4A, 20 µM ABA 210

completely suppressed red light-induced phosphorylation of PM H+-ATPase and partially 211

suppressed blue light-induced phosphorylation. The net blue light-induced phosphorylation 212



13

decreased by approximately 41% compared to that of untreated leaves (Supplemental Fig. 213

S3). Moreover, stomata in ABA-treated leaves did not open after exposure to red or blue light 214

(Fig. 4B). These results indicate that exogenous ABA decreases the apparent phosphorylation 215

level of PM H+-ATPase under any light quality in whole leaves. 216

217

AHA1, a major isoform of PM H+-ATPase in guard cells, is responsible for red 218



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light-induced stomatal opening 219

A recent study suggested that AHA1 constitutes the majority of PM H+-ATPase in guard 220



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cells, probably due to some posttranscriptional regulation, as the transcript level of AHA1 in 221

guard cells is not the highest among those of the isogenes; AHA1 is responsible for blue 222

light-induced stomatal opening, and the red light-dependent increase in stomatal conductance 223

is statistically significantly slower in aha1 plants than in the wild type (Yamauchi et al., 224

2016). To confirm the need for PM H+-ATPase for red light-induced stomatal opening 225

genetically, we analyzed stomatal apertures in whole leaves from PM H+-ATPase mutant 226

plants. As shown in Figure 5A, stomata in aha1-9, a null allele of AHA1 leading to 65% 227

reduction in the amount of PM H+-ATPase in guard cells (Yamauchi et al., 2016), were found 228

to have smaller apertures than in the wild type until 45 min after the start of red light 229

illumination. The aperture reached a level comparable to that in the wild type within 60 min, 230

in agreement with previous gas-exchange measurements (Yamauchi et al., 2016). By contrast, 231

the stomatal opening in aha2-5, a knock-out allele of AHA2 (Hayashi et al., 2011), was 232



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indistinguishable from that of the wild type (Fig. 5B). These results indicate that AHA1 is 233

responsible for stomatal opening in response to red light as well as blue light. The delayed 234

red light-induced stomatal opening in aha1-9 indicates that PM H+-ATPase is necessary to 235

accelerate red light-induced stomatal opening in whole leaves. 236

237

Red light-induced phosphorylation of PM H+-ATPase in guard cells depends on 238

photosynthesis 239

Red light-induced stomatal opening depends on photosynthesis (Sharkey and Raschke, 240

1981; Doi and Shimazaki, 2008; Wang et al., 2011; Suetsugu et al., 2014). Thus, we 241

investigated the effects of DCMU, an inhibitor of photosynthetic electron transport, on the 242

stomatal responses to red light in whole leaves. As shown in Figure 6A, red light-induced 243

phosphorylation of PM H+-ATPase was completely inhibited by 10 µM DCMU. Accordingly, 244

stomata in DCMU-treated leaves showed no significant increase in the size of apertures in 245

response to red light (Fig. 6B). The inhibition of red light-induced stomatal opening is in 246

agreement with the previous gas-exchange experiment showing that DCMU diminished the 247

red light-dependent increase in stomatal conductance (Suetsugu et al., 2014). Additional blue 248

light induced phosphorylation even with DCMU, as described previously (Suetsugu et al., 249

2014); however, the phosphorylation level appeared to be lower than that of untreated leaves 250

(Fig. 6A; Supplemental Fig. S3). Blue light-induced stomatal opening was also suppressed by 251

DCMU (Fig. 6B). Next, we investigated red light-induced phosphorylation of PM H+-ATPase 252

in guard cells in phyA phyB, a mutant of phytochromes. The results showed that red light 253

induced phosphorylation in phyA phyB without affecting the amount of PM H+-ATPase 254

(Supplemental Fig. S4). These results indicate that red light-induced phosphorylation of PM 255

H+-ATPase is regulated by photosynthesis not by phytochrome-mediated red light signaling. 256

257

Effects of sucrose or CO2 on phosphorylation of PM H+-ATPase in guard cells 258

To clarify the signaling components mediating photosynthesis and red light-induced 259

phosphorylation of PM H+-ATPase in guard cells, we first assumed that photosynthetic 260

products might be the responsible signal. Sucrose is the primary photosynthetic product and 261

accumulates in guard cells partly by photosynthesis in guard cell chloroplasts and partly by 262



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import from mesophyll cells via apoplasts (Poffenroth et al., 1992; Talbott and Zeiger, 1993; 263

Lu et al., 1997). In whole leaves, sucrose content increases beginning approximately 5 min 264



18

after the start of illumination (Okumura et al., 2016); in line with this, we found that red 265

light-induced phosphorylation of PM H+-ATPase in guard cells required more than 5 min of 266

illumination (Fig. 3A). Thus, we investigated the effects of sucrose, or the same concentration 267

of mannitol as an osmotic control, on the phosphorylation status of PM H+-ATPase in the 268

guard cells of isolated epidermis samples in the dark or under red light. As shown in Figure 269

7A, the application of sucrose to isolated epidermis did not induce phosphorylation of PM 270

H+-ATPase in guard cells in the dark. In addition, simultaneous application of sucrose and red 271



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light did not induce phosphorylation of PM H+-ATPase in isolated epidermis. 272

Red light causes the reduction of Ci via photosynthesis. A high concentration of CO2 may 273

inhibit PM H+-ATPase (Edwards and Bowling, 1985), raising the hypothesis that high Ci 274

inactivates PM H+-ATPase. Therefore, we investigated the effects of reduced Ci on the 275

phosphorylation status of PM H+-ATPase. In this experiment, isolated epidermis samples 276

were incubated with aeration by diffused ambient or soda lime-passed low CO2 air for 30 min 277

in the dark or under red light (Supplemental Fig. S5; see also Materials and Methods). As 278

shown in Figure 7B, low CO2 conditions neither induced phosphorylation of PM H+-ATPase 279

in the dark nor provoked red light-induced phosphorylation of PM H+-ATPase in isolated 280

epidermis. 281



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282



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DISCUSSION 283

New immunohistochemical technique for guard cells using whole leaves 284

Immunohistochemical staining using isolated epidermis enables one to determine the 285

amount of PM H+-ATPase using anti-H+-ATPase antibodies and the phosphorylation status of 286

PM H+-ATPase using anti-pThr antibodies in stomatal guard cells (Hayashi et al. 2011). 287

However, when using the method, it is necessary to prepare enough isolated epidermis for 288

experiments. Therefore, it is not practical for studying plants with small and/or few leaves, 289

such as dwarf or early-flowering mutants. In addition, the method cannot be used for analysis 290

of individual plants, such as genetic screening, in which only single and often small leaves 291

are used. Overcoming these issues, the newly developed immunohistochemical technique 292

using whole leaves enables various applications, regardless of the availability of isolated 293

epidermis. 294

Using this method, we found that red light induces phosphorylation of PM H+-ATPase in 295

guard cells of whole leaves (Figs. 1A and 3A). According to our results, in isolated epidermis, 296

red light did not induce phosphorylation of PM H+-ATPase in guard cells or stomatal opening 297

(Supplemental Fig. S2; Hayashi et al., 2011; Suetsugu et al., 2014). Hence, an additional 298

advantage of our modified technique is that stomatal responses in whole leaves can be 299

analyzed. 300

301

Red light induces photosynthesis-dependent phosphorylation of PM H+-ATPase in 302

guard cells and stomatal opening in whole leaves 303

Activation of PM H+-ATPase induces PM hyperpolarization and drives K+ uptake through 304

voltage-gated inward-rectifying K+ channels (Inoue and Kinoshita, 2017; Jezek and Blatt, 305

2017). Blue light activates PM H+-ATPase via phosphorylation of the penultimate C-terminal 306

residue of PM H+-ATPase, threonine (Kinoshita and Shimazaki, 1999; Kinoshita and 307

Shimazaki, 2002). Red light also leads to the accumulation of K+ in guard cells, which may 308

require PM H+-ATPase activity (Hsiao et al., 1973; Olsen et al., 2002). However, whether 309

and how red light activates PM H+-ATPase has remained unclear. We found that red light 310

induces DCMU-sensitive phosphorylation of the threonine of PM H+-ATPase in guard cells, 311

which correlates with red light-induced stomatal opening in whole leaves (Figs. 1–3, 6). PM 312



22

H+-ATPase was genetically confirmed to be necessary to accelerate red light-induced 313

stomatal opening (Fig. 5). The eventual stomatal opening in aha1-9 (Fig. 5A) suggests that 314

other PM H+-ATPase isoforms make minor contributions to red light-induced stomatal 315

opening. This idea is supported by the previous observation that activation of PM H+-ATPase 316

by fusicoccin ultimately induces full stomatal opening in aha1 (Kinoshita and Shimazaki, 317

2001; Yamauchi et al., 2016). These results strongly suggest that red light induces stomatal 318

opening through the activation of PM H+-ATPase by photosynthesis-dependent 319

phosphorylation in whole leaves and provide insight into the photosynthetic regulation of 320

stomatal opening. 321

The absence of red-light responses in isolated epidermis (Supplemental Fig. S2) suggests 322

that mesophyll photosynthesis is most likely to be required for red light-induced 323

phosphorylation of PM H+-ATPase and thus stomatal opening. Indeed, enhancement of red 324

light-induced stomatal opening by mesophyll cells was also reported in other species. In Vicia 325

faba, red light induced stomatal opening even in isolated epidermis (Schwartz and Zeiger, 326

1984), however, the amplitude of the response was estimated to be much smaller than that in 327

leaf discs (Hsiao et al., 1973; reviewed by Mott, 2009). In Chlorophytum comosum, red 328

light-dependent increase in stomatal conductance was abolished in albino leaf patches, in 329

which guard cell chloroplasts were indicated to be functional (Roelfsema et al., 2006). Such 330

enhancement could be explained, at least in part, by the hypothesis that mesophyll 331

photosynthesis primarily induces phsphorylation of PM H+-ATPase in guard cells. However, 332

measurable red light-induced stomatal opening in isolated epidermis in Vicia faba (Schwartz 333

and Zeiger, 1984) suggests that guard cell chloroplasts in this species partially contribute to 334

red light-induced stomatal opening. Even with the same incubation solution wherein red light 335

induced stomatal opening in Vicia isolated epidermis (Schwartz and Zeiger, 1984), red light is 336

not likely to induce phosphorylation of PM H+-ATPase in Arabidopsis isolated epidermis 337

(Supplemental Fig. S6). Nevertheless, guard cell chloroplasts in certain species might have 338

the potential to induce partial phosphorylation of PM H+-ATPase, which could cause the 339

interspecific difference in the red-light sensitivity of guard cells in isolated epidermis. 340

Analyses of red light-induced phosphorylation of PM H+-ATPase in whole leaves from 341

mutant or transgenic plants with a defect in photosynthetic activity, either in mesophyll cells 342



23

or in guard cells, would provide further insight into the issue regarding whether mesophyll 343

cells or guard cell chloroplasts contribute to this response. In addition, application of our 344

immunohistochemical technique to other species could be considered for future study. 345

346

Effects of ABA on red light-induced stomatal opening in whole leaves 347

The present study indicates that ABA suppresses red light-induced phosphorylation of PM 348

H+-ATPase in guard cells and stomatal opening (Fig. 4). ABA was suggested to have no 349

direct effect on photosynthesis (Kriedemann et al., 1975; Mawson et al., 1981; Lauer and 350

Boyer, 1992). Therefore, the inhibition of red light-induced stomatal opening by ABA may be 351

achieved by suppressing phosphorylation of PM H+-ATPase rather than by a direct limitation 352

of photosynthesis and may also be enhanced by ABA-induced stomatal closure via anion and 353

cation efflux from guard cells (Jezek and Blatt, 2017). In addition, the inactivation of PM 354

H+-ATPase in guard cells may also reduce photosynthesis-dependent sucrose accumulation as 355

osmolytes through H+/sucrose symporters in these cells, as previously predicted in silico (Sun 356

et al., 2014). A previous study indicated that ABA suppresses blue light-induced 357

phosphorylation of PM H+-ATPase via early ABA signaling components including ABI1, 358

ABI2, and OST1 (Hayashi et al., 2011). Further genetic elucidation is required to reveal 359

whether these ABA signaling components are involved in suppression of red light-induced 360

phosphorylation of PM H+-ATPase. 361

362

What mediates photosynthesis-dependent phosphorylation of PM H+-ATPase in guard 363

cells? 364

Neither sucrose as a major photosynthetic product, nor low CO2 conditions, which 365

mimics the reduced Ci caused by photosynthesis, induced phosphorylation of PM H+-ATPase 366

even with red light illumination (Fig. 7). These results suggest that simultaneous application 367

of sucrose and red light, or low CO2 conditions and red light, are not sufficient for 368

phosphorylation of PM H+-ATPase in guard cells. Taken together with a previous report 369

suggesting that high Ci inhibits PM H+-ATPase (Edwards and Bowling, 1985), reduced Ci 370

may be necessary but not sufficient for the activation of PM H+-ATPase. Thus, a 371

Ci-independent mechanism may also be involved in phosphorylation of PM H+-ATPase, 372



24

consistent with the Ci-independence of red light-induced stomatal opening (Messinger et al., 373

2006; Lawson et al., 2008). In summary, identification of the signal(s) mediating 374

photosynthesis-dependent phosphorylation of PM H+-ATPase in guard cells will be a critical 375

issue for future work and the combination of low CO2 and other putative signal(s) should be a 376

focus of further studies. 377

Ci is also suggested to affect transport of anions and cations across the PM of guard cells 378

(Brearley et al., 1997; Roelfsema et al., 2002). Reduced Ci is likely to inhibit S-type anion 379

channels via low CO2 signaling mediated by HT1, CBC1, and CBC2 (Hiyama et al., 2017). 380

Guard cells from cbc1 cbc2 showed reduced voltage-dependent inward-rectifying K+ current, 381

although the reduction of the K+ current did not limit the rate of stomatal opening induced by 382

fusicoccin (Hiyama et al., 2017). Red light-induced stomatal opening is delayed in aha1 (Fig. 383

5A; Yamauchi et al., 2016) and suppressed in ht1 and cbc1 cbc2 (Matrosova et al., 2015; 384

Hiyama et al., 2017; Supplemental Fig. S7). A possible explanation for these distinct 385

phenotypes is that proper regulation of anion and cation transporters at the PM of guard cells 386

is a prerequisite for the acceleration of red light-induced stomatal opening by activated PM 387

H+-ATPase. Alternatively, phosphorylation of PM H+-ATPase might be affected in ht1 and 388

cbc1 cbc2 regardless of Ci. Further analyses are required to test these hypotheses. 389

390

Light-induced stomatal opening in whole leaves mediated by PM H+-ATPase in guard 391

cells 392

Based on the present results, we propose a possible model for light-induced stomatal 393

opening in whole leaves mediated by PM H+-ATPase. As shown in Supplemental Figure S8, 394

blue light and red light induce phosphorylation of PM H+-ATPase via phototropin-mediated 395

signaling and photosynthesis, respectively. In addition, red light seems to enhance blue 396

light-induced phosphorylation of PM H+-ATPase in a photosynthesis-independent manner 397

(Suetsugu et al., 2014). Thus, photosynthesis at least provides the ATP required for 398

H+-pumping by activated PM H+-ATPase to promote stomatal opening (Tominaga et al., 399

2001; Suetsugu et al., 2014; Wang et al., 2014a). Our results suggest that blue light signaling 400

and photosynthetic status are integrated into the activation of PM H+-ATPase. Considering 401

that stomatal apertures are a limiting factor in photosynthesis (Wang et al., 2014b), this 402



25

mechanism may enable plants to promote stomatal opening and CO2 uptake in response not 403

only to the light environment but also to the actual CO2 demand caused by photosynthesis in 404

whole leaves. Further investigation of the phosphorylation level of PM H+-ATPase in whole 405

leaves under various light conditions should provide a better understanding of light-induced 406

stomatal opening mediated by PM H+-ATPase in whole leaves. 407



26

MATERIALS AND METHODS 408

Plant materials and growth conditions 409

Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) plants were used as the wild type in 410

the physiological experiments, unless otherwise stated. Col-0 was also used as a control plant 411

for aha1-9 (SAIL_1285_D12; Yamauchi et al., 2016), aha2-5 (SALK_022010; Haruta et al., 412

2010), ht1-9 (Hiyama et al., 2017), and cbc1 cbc2 (SALK_005187 for cbc1; SAIL_740_G01 413

for cbc2; Hiyama et al., 2017). glabra1 (gl1) and Landsberg erecta were used as control 414

plants for phot1-5 phot2-1 (phot1 phot2; Kinoshita et al., 2001) and phyA-201 phyB-1 (phyA 415

phyB; Mazzella et al., 1997), respectively. All plants were grown on soil under white 416

fluorescent lamps (approximately 50 µmol m–2 s–1) with a 16:8 h light/dark cycle in a growth 417

room. Growth temperature and relative humidity were approximately 20–24°C and 40–60%, 418

respectively. Three- to five-week-old plants were used for the experiments. 419

420

Light source 421

Red and blue light were produced from light-emitting photodiodes (ISL-150X150-H4RR 422

for red light only and ISL-150X150-H4RHB for both red and blue light; CCS) with a 423

manufacturer-provided power supply (ISC-201-2; CCS). Photon flux densities were 424

confirmed using a quantum meter (Li-250; Li-Cor). 425

426

Light illumination, chemical treatment, and aeration 427

Epidermal fragments were obtained by blending leaves for <10 s (<5 s twice) in Milli-Q 428

water (Millipore) with a Waring blender equipped with an MC1 mini container (Waring 429

Commercial) at full speed and collected in a 58 µm nylon mesh. Isolated epidermis or whole 430

leaves harvested from dark-adapted plants were incubated in or put on the basal buffer (5 mM 431

MES-1,3-bis[tris(hydroxymethyl)methylamino] propane [BTP; pH 6.5], 50 mM KCl, 0.1 mM 432

CaCl2) and illuminated with red light (600 µmol m–2 s–1) or kept in the dark. Blue light (5 433

µmol m–2 s–1) was superimposed on the background red light. For pretreatment of leaves with 434

ABA or DCMU, these compounds were dissolved in dimethyl sulfoxide (DMSO). The 435

harvested leaves were kept in the dark for 30 min on the basal buffer containing 20 µM ABA 436

or 10 µM DCMU, or DMSO alone, and then transferred to the indicated light conditions. The 437



27

final concentration of DMSO was 0.1% (v/v). To examine the effects of sucrose, isolated 438

epidermis was kept in the basal buffer containing 30 mM sucrose or the same concentration 439

of mannitol as an osmotic control for 30 min in the dark or under red light. The epidermis 440

was also illuminated or kept in the dark as described above without sugars. To examine the 441

effects of low CO2 conditions, isolated epidermis was incubated in a glass dish (internal 442

diameter: approximately 21 mm × 10 mm height) containing 1.5 mL basal buffer aerated with 443

ambient or soda lime-passed air (CO2: 30–40 ppm) at a flow rate of approximately 50 cm3 444

min–1 and illuminated or kept in the dark as described above. The basal buffer was aerated 445

from at least 30 min before and throughout the incubation. A schematic diagram of the 446

experimental set-up is shown in Supplemental Figure S5. 447

448

Antisera 449

Previously described antiserum against the catalytic domain of PM H+-ATPase or its 450

penultimate phosphorylated residue, threonine, designated as anti-H+-ATPase or anti-pThr, 451

respectively (Hayashi et al., 2010), were used to estimate the amount of the protein and its 452

phosphorylation level, respectively. 453

454

Immunohistochemical detection of PM H+-ATPase in guard cells using whole leaves 455

We modified a previously described whole-mount immunohistochemical technique 456

(Sauer et al., 2006) as described below to detect PM H+-ATPase in guard cells in whole 457

leaves. The pH of phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 458

mM KCl, 1.47 mM KH2PO4) was adjusted to 6.5 by HCl for sample rehydration and enzyme 459

treatment, whereas unadjusted PBS was used in all other experimental steps. To fix leaves, 460

fixative composed of 4% (w/v) formaldehyde with 0.3% (w/w) glutaraldehyde in 461

microtubule-stabilizing buffer (MTSB: 50 mM PIPES-NaOH [pH 7.0], 5 mM MgSO4, and 5 462

mM EGTA) was freshly prepared from paraformaldehyde (Wako) and 25% (w/w) 463

glutaraldehyde stock solution (Nacalai Tesque). After light treatment, the fixative was 464

immediately infiltrated into the leaves as follows: Leaves and the fixative were put into a 465

syringe with the top sealed by a gloved finger, into which a plunger was then inserted. The 466

syringe was inverted and excess air was pushed out of it. The top of the syringe was again 467



28

sealed by a gloved finger, after which negative pressure was applied several times. The 468

infiltration usually took less than 20 s. The infiltrated leaves were immersed in the fixative 469

for 1 h in the dark at room temperature. After washing with PBS, leaves were incubated three 470

to four times for 20 min each with pure methanol at 37°C to remove chlorophyll. Then, 471

central areas of the leaves without the midrib were cut out and incubated with xylene for 2 472

min at 37°C, pure ethanol for 5 min at room temperature, and 50% (v/v; in PBS) ethanol for 5 473

min at room temperature and washed with Milli-Q water twice. Materials were mounted on 474

MAS-coated microscope slides (Matsunami) in an orientation in which the abaxial side of the 475

leaf was attached to the slide. Rehydrated samples were digested with 1% (w/v) Cellulase 476

Onozuka R-10 (Yakult) with 0.5% (w/v) Macerozyme R-10 (Yakult) in PBS for 1 h at 37°C. 477

After digestion, all of the leaf tissue except for the abaxial epidermis attached to the slide was 478

removed stereomicroscopically in PBS, and the epidermal tissue left on the slide was washed 479

four times for 5 min each with PBS. The epidermis was permeabilized with 3% (v/v) 480

IGEPAL CA-630 (MP Biomedicals) with 10% (v/v) DMSO in PBS for 30 min at room 481

temperature. After washing, samples were incubated with blocking solution (3% [w/v] bovine 482

serum albumin Fraction V [Gibco] in PBS) for 1 h at room temperature. The primary 483

antiserum (anti-pThr or anti H+-ATPase) at a dilution of 1:3,000 in the blocking solution was 484

applied overnight at 4°C. Secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit 485

IgG [Invitrogen; A11034]) at a dilution of 1:500 in the blocking solution was applied for 3 h 486

at 37°C. The specimens were covered by a cover glass with 50% (v/v) glycerol. 487

488

Immunohistochemical detection of PM H+-ATPase in guard cells using isolated 489

epidermal tissue 490

We performed immunohistochemical analyses (Hayashi et al., 2011) with slight 491

modifications as described below. Epidermal tissue was fixed using 4% (w/v) formaldehyde 492

with 0.1% (w/w) glutaraldehyde in MTSB for 2 h at 4°C in the dark. After washing, 493

chlorophyll was removed by incubation with pure methanol for 10 min at 37°C. The 494

epidermal tissue was digested with supernatant of 3% (w/v) Driselase (Sigma Aldrich) and 495

0.5% (w/v) Macerozyme R-10 in PBS for 45 min at 37°C. Tissue permeabilization was 496

performed with 3% (w/w) Triton X-100 in PBS. Primary antiserum was applied overnight at 497



29

4°C. Other conditions (i.e., blocking and secondary antibody) were the same as described 498

above. 499

500

Estimation of phosphorylation level or amount of PM H+-ATPase in guard cells 501

Phosphorylation level or amount of PM H+-ATPase in guard cells was estimated based on 502

Alexa Fluor 488 fluorescence intensity. Fluorescent images were obtained as described 503

previously at the same exposure time within independent measurements (Hayashi et al., 504

2011). The fluorescence intensity of a pair of guard cells was measured using ImageJ 505

(National Institute of Health; Schneider et al., 2012) as described below. All images were 506

split into red, green, and blue channels using the “RGB stack” command. The fluorescence 507

intensity of each pair of guard cells was defined as the difference between the mean gray 508

value of the guard cells on the green channel and that of the neighboring area without guard 509

cells. The representative value of each specimen in an independent measurement was 510

calculated as the geometric mean of more than 30 pairs of guard cells from at least two leaves. 511

In each measurement, an additional specimen, in which normal serum was used instead of 512

primary antiserum, was simultaneously examined. The net fluorescence intensity was defined 513

as the difference between the representative values obtained for each antiserum and normal 514

serum and is expressed relative to the corresponding control. Data represent arithmetic means 515

of the relative values from at least three independent measurements with SDs. Red 516

light-induced phosphorylation of PM H+-ATPase in guard cells was confirmed by blind test 517

(Supplemental Fig. S9A). 518

519

Measurement of stomatal aperture 520

Isolated epidermal tissue was collected from a 58 µm nylon mesh and observed 521

microscopically. To measure the stomatal aperture in whole leaves, the leaves were collected 522

after the treatments and blended as described above, with the exception of ht1-9 and cbc1 523

cbc2; then, isolated epidermis samples were collected and observed immediately. Because a 524

high concentration of extracellular K+ keeps stomata open in isolated epidermis samples of 525

cbc1 cbc2 (Hiyama et al., 2017), leaves from cbc1 cbc2, ht1-9, a mutant of the component 526

upstream of CBC1 and CBC2, and control Col-0 plants were blended in potassium buffer (5 527



30

mM MES-BTP [pH 6.5], 50 mM KCl) to prevent possible stomatal closure, and isolated 528

epidermis samples were observed immediately with the same solution, as shown in 529

Supplemental Figure S7. The representative value of each specimen in an independent 530

measurement was calculated as the arithmetic mean of 30 stomatal apertures on the abaxial 531

side (five stomata per epidermis) from at least two leaves. Data represent arithmetic means of 532

the absolute mean values from at least three independent measurements with SDs. Red 533

light-induced stomatal opening was confirmed by blind test (Supplemental Fig. S9B). 534

535

Statistical analyses 536

For means expressed as relative values, one-tailed or two-tailed one-sample t tests were 537

carried out to evaluate whether the mean was statistically significantly different from a 538

corresponding control set to 1.0. Whether there was a difference between two independent 539

means was assessed using one-tailed or two-tailed Student’s t test. For means expressed as 540

absolute values, statistically significant increases in multiple means compared to a single 541

control were assessed using a one-tailed Dunnett’s test. Statistically significant differences 542

among all means were analyzed using a Tukey’s test. P values were calculated using R (Ver. 543

3.4.4; R Core Team, 2018) with the package “multcomp” (Ver. 1.4.8; Hothorn et al., 2008), 544

and P < 0.05 were considered statistically significant. 545

546

Accession numbers 547

Sequence data from this article can be found in the GenBank/EMBL data libraries under 548

the following accession numbers: GL1 (At3g27920), PHOT1 (At3g45780), PHOT2 549

(At5g58140), AHA1 (At2g18960), AHA2 (At4g30190), PHYA (At1g09570), PHYB 550

(At2g18790), HT1 (At1g62400), CBC1 (At3g01490), and CBC2 (At5g50000). 551

552

Supplemental Data 553

Supplemental Figure S1. Validation of red light-induced phosphorylation of PM H+-ATPase 554

in guard cells in whole leaves. 555

Supplemental Figure S2. Light-induced phosphorylation of PM H+-ATPase in guard cells 556

and stomatal opening in isolated epidermis. 557



31

Supplemental Figure S3. Inhibition of blue light-induced phosphorylation of PM H+-ATPase 558

in guard cells in whole leaves by ABA but not DCMU. 559

Supplemental Figure S4. Immunohistochemical detection of PM H+-ATPase in guard cells 560

in whole leaves from Landsberg erecta (Ler) and phyA phyB. 561

Supplemental Figure S5. Schematic diagram of the set-up for the aeration experiment. 562

Supplemental Figure S6. Effects of incubation solution on red light-induced 563

phosphorylation of PM H+-ATPase in guard cells in isolated epidermis. 564

Supplemental Figure S7. Light-induced stomatal opening in whole leaves from Col-0, ht1-9, 565

and cbc1 cbc2. 566

Supplemental Figure S8. Schematic diagram of the possible model for light-induced 567

stomatal opening in whole leaves mediated by PM H+-ATPase in guard cells. 568

Supplemental Figure S9. Blind tests of red-light responses. 569

570

ACKNOWLEDGEMENTS 571

We thank Yukari Kamiya and Dr. Maki Hayashi for technical assistance and advice in the 572

establishment of the immunohistochemical technique using whole leaves, Dr. Koji Takahashi 573

for participation in the blind test of the immunohistochemical analysis of the phosphorylation 574

level of PM H+-ATPase and stomatal aperture measurement, and Drs. Ken-ichiro Shimazaki 575

and Shin-ichiro Inoue for critical advice on this study. 576

577

FIGURE LEGENDS 578

Figure 1. Establishment of an immunohistochemical technique using whole leaves to detect 579

PM H+-ATPase in guard cells. Phosphorylation (A) and amount of PM H+-ATPase (B) in 580

guard cells in whole leaves from gl1 and phot1 phot2. Phosphorylation (A) or amount (B) of 581

the protein was detected using anti-pThr or anti-H+-ATPase antiserum, respectively. Mature 582

leaves harvested from dark-adapted plants were illuminated with red light (600 µmol m–2 s–1) 583

for 30 min (R30) followed by blue light (5 µmol m–2 s–1) superimposed on red light for 2.5 584

min (R + B2.5) or kept in the dark (D30). Typical fluorescent and corresponding bright field 585

images (upper panel) and quantification of the fluorescence intensities (lower panel) are 586

shown. Arrowheads indicate guard cells. Scale bars represent 50 µm. Data represent means of 587



32

relative values from three independent measurements with standard deviations (SDs). (A) 588

Daggers denote that the mean is statistically significantly higher than D30 of gl1 set to 1.0. 589

N.S., not significant (one-tailed one-sample t test; †P < 0.05, ††P < 0.01, †††P < 0.005, N.S., P 590

= 0.516). Asterisk indicates that the mean of R + B2.5 is statistically significantly higher than 591

that of R30 in gl1 (one-tailed Student’s test; *P < 0.005). (B) The means were not statistically 592

significantly different from D30 of gl1 set to 1.0 (two-tailed one-sample t test; N.S., P > 593

0.45). 594

595

Figure 2. Light-induced stomatal opening in whole leaves. (A) Mature leaves harvested from 596

dark-adapted Col-0 plants (0 min) were illuminated with red light (RL), both red and blue 597

light (RB), or kept in the dark (Dk) for 60, 120, or 180 min. Light intensities were 600 µmol 598

m–2 s–1 for red light and 5 µmol m–2 s–1 for blue light. Data represent means of representative 599

values from three independent measurements with SDs. Asterisks denote a statistically 600

significant increase in the mean compared to that of 0 min (one-tailed Dunnett’s test; *P < 601

0.05, **P < 0.01, ***P < 0.001). Hash signs indicate that the mean of RB is statistically 602

significantly higher than that of RL at each time point (one-tailed Student’s t test; #P < 0.05). 603

(B) Light-induced stomatal opening in whole leaves from gl1 and phot1 phot2. Mature leaves 604

harvested from dark-adapted plants were illuminated with red light (R60), both red and blue 605

light (RB60), or kept in the dark (D60) for 60 min. Light intensities were the same as in A. 606

Data represent means of representative values from four independent measurements with SDs. 607

Different letters indicate statistically significant differences among means (Tukey’s test, P < 608

0.05). 609

610

Figure 3. Initial responses to red light in guard cells in whole leaves. Mature leaves were 611

harvested from dark-adapted plants (0 min) and kept in the dark (Dk) for 60 min or 612

illuminated with red light (RL; 600 µmol m–2 s–1) for the indicated time. (A) 613

Immunohistochemical detection of phosphorylation of PM H+-ATPase in guard cells using 614

anti-pThr antiserum. Leaves were illuminated for 5, 10, 30, or 60 min. Typical fluorescent 615

and bright-field images (upper panel) and quantification of the fluorescence intensities (lower 616

panel) are shown. Arrowheads indicate guard cells. Scale bar represents 50 µm. Data 617



33

represent means of relative values from three independent measurements with SDs. Daggers 618

denote that the mean is statistically significantly higher than at 0 min set to 1.0. N.S., not 619

significant (one-tailed one-sample t test; †P < 0.05, ††P < 0.005, N.S., P > 0.07). (B) 620

Measurement of red light-induced stomatal opening. Leaves were illuminated for 15, 30, 45, 621

or 60 min. Data represent means of representative values from three independent 622

measurements with SDs. Asterisks denote statistically significant increases of the mean 623

compared to that of 0 min (one-tailed Dunnett’s test; *P < 0.05, **P < 0.01, ***P < 0.005; N.S., 624

P = 0.717). 625

626

Figure 4. Inhibition of light-induced phosphorylation of PM H+-ATPase in guard cells and 627

stomatal opening in whole leaves by the phytohormone ABA. Mature leaves harvested from 628

dark-adapted plants were pretreated with 20 µM ABA dissolved in 0.1% (v/v) DMSO 629

(+ABA) or 0.1% (v/v) DMSO alone (–ABA) for 30 min in the dark before light illumination. 630

(A) Immunohistochemical detection of phosphorylation of PM H+-ATPase in guard cells 631

using anti-pThr antiserum. Pretreated leaves were illuminated with red light (600 µmol m–2 s–632 1) for 30 min (R30) followed by blue light (5 µmol m–2 s–1) superimposed on red light for 2.5 633

min (R + B2.5) or kept in the dark (D30). Typical fluorescent and bright-field images (upper 634

panel) and quantification of the fluorescence intensities (lower panel) are shown. Scale bar 635

represents 50 µm. Data represent means of relative values from four independent 636

measurements with SDs. Daggers denote that the mean is statistically significantly higher 637

than D30 of –ABA set to 1.0. N.S., not significant (one-tailed one-sample t test; †P < 0.01, 638

N.S., P > 0.18). Asterisks indicate that the mean of R + B2.5 is statistically significantly 639

higher than that of R30 within each treatment and that the mean of R + B2.5 of +ABA is 640

statistically significantly lower than that of –ABA (one-tailed Student’s t test; *P < 0.01, **P < 641

0.005). (B) Measurement of light-induced stomatal opening under ABA treatment. Pretreated 642

leaves were illuminated with red light (R60), both red and blue light (RB60), or kept in the 643

dark (D60) for 60 min. Light intensities were the same as in A. Data represent means of 644

representative values from four independent measurements with SDs. Different letters 645

indicate statistically significant differences among means (Tukey’s test, P < 0.05). 646

647



34

Figure 5. Red light-induced stomatal opening in whole leaves from PM H+-ATPase mutants. 648

Mature leaves from aha1-9 (A) or aha2-5 (B) and Col-0 were harvested from dark-adapted 649

plants. The harvested leaves were kept in the dark (Dk) for 60 min or illuminated with red 650

light (RL; 600 µmol m–2 s–1) for 15, 30, 45, or 60 min. Data represent means of representative 651

values from three independent measurements with SDs. (A) Asterisks denote statistically 652

significant decrements of the mean of aha1-9 compared to that of Col-0 at each time point. 653

N.S., not significant (one-tailed Student’s t test; *P < 0.05, **P < 0.001, N.S., P > 0.07). (B) 654

The mean of aha2-5 was not statistically significantly lower than that of Col-0 at each time 655

point (one-tailed Student’s t test; N.S., P > 0.6). 656

657

Figure 6. The effects of the photosynthesis inhibitor DCMU on light responses of guard cells 658

in whole leaves. Mature leaves harvested from dark-adapted plants were pretreated with 10 659

µM DCMU dissolved in 0.1% (v/v) DMSO (+DCMU) or 0.1% (v/v) DMSO alone (–DCMU) 660

for 30 min in the dark before light illumination. (A) Immunohistochemical detection of 661

phosphorylation of PM H+-ATPase in guard cells using anti-pThr antiserum. Pretreated leaves 662

were illuminated with red light (600 µmol m–2 s–1) for 30 min (R30) followed by blue light (5 663

µmol m–2 s–1) superimposed on red light for 2.5 min (R + B2.5) or kept in the dark (D30). 664

Typical fluorescent and bright-field images (upper panel) and quantification of the 665

fluorescence intensities (lower panel) are shown. Arrowheads indicate guard cells. Scale bars 666

represent 50 µm. Data represent means of relative values from five independent 667

measurements with SDs. Daggers indicate that the mean is statistically significantly higher 668

than D30 of –DCMU set to 1.0. N.S., not significant (one-tailed one-sample t test; †P < 0.05, 669 ††P < 0.001, N.S., P > 0.58). Asterisks indicate that the mean of R + B2.5 is statistically 670

significantly higher than that of R30 within each treatment and that the mean of R + B2.5 of 671

+DCMU is statistically significantly lower than that of –DCMU (one-tailed Student’s t test; 672 *P < 0.05, **P < 0.01, ***P < 0.005). (B) Measurement of light-induced stomatal opening 673

under DCMU treatment. Pretreated leaves were illuminated with red light (R60), both red and 674

blue light (RB60), or kept in the dark (D60) for 60 min. Light intensities were the same as in 675

A. Data represent means of representative values from four independent measurements with 676

SDs. Different letters indicate statistically significant differences among means (Tukey’s test, 677



35

P < 0.05). 678

679

Figure 7. Effect of sucrose or CO2 concentration on phosphorylation of PM H+-ATPase in 680

guard cells in isolated epidermis. (A and B) Immunohistochemical detection of 681

phosphorylation of PM H+-ATPase in stomatal guard cells using anti-pThr antiserum. 682

Epidermal tissue was illuminated with red light (600 µmol m–2 s–1) for 30 min (R30) followed 683

by blue light (5 µmol m–2 s–1) superimposed on red light for 2.5 min (R + B2.5) or kept in the 684

dark (D30) with the indicated conditions. (A) Epidermis was incubated without (–) or with 30 685

mM mannitol (Man) or sucrose (Suc). (B) Experimental basal buffer was aerated with 686

ambient or soda lime-passed low CO2 air (CO2 = 30–40 ppm). Quantification of fluorescence 687

intensities is shown. Data represent means of relative values from independent measurements 688

(A: n = 4, B: n = 5) with SDs. Daggers denote that the mean is statistically significantly 689

higher than D30 of –sugar (A) or that of ambient (B) set to 1.0. N.S., not significant 690

(one-tailed one-sample t test; †P < 0.05, A: N.S., P > 0.08, B: N.S., P > 0.10). Asterisks 691

indicate that the mean of R + B2.5 is statistically significantly higher than that of R30 with a 692

corresponding control condition (one-tailed Student’s t test; *P < 0.01). 693



36

694



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Hsiao TC, Allaway WG, Evans LT (1973) Action spectra for guard cell Rb+ uptake and stomatal opening in Vicia faba. Plant Physiol. 51: www.plantphysiol.orgon October 23, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

http://www.ncbi.nlm.nih.gov/pubmed?term=Ando E%2C Ohnishi M%2C Wang Y%2C Matsushita T%2C Watanabe A%2C Hayashi Y%2C Fujii M%2C Ma JF%2C Inoue S%2C Kinoshita T %282013%29 TWIN SISTER OF FT%2C GIGANTEA%2C and CONSTANS have a positive but indirect effect on blue light%2Dinduced stomatal opening in Arabidopsis%2E Plant Physiol%2E 162%3A 1529%26%23x2013%3B1538&dopt=abstract

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ando&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=TWIN SISTER OF FT, GIGANTEA, and CONSTANS have a positive but indirect effect on blue light-induced stomatal opening in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ando&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

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1 : red light and pm h 2 3 corresponding author · 13/08/2018 · 3 36 abstract 37 stomatal...

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