phosphatidylinositol 4,5-bisphosphate is important for

Phosphatidylinositol 4,5-bisphosphate is important forstomatal opening

Yuree Lee1, Yong-Woo Kim2, Byeong Wook Jeon1, Ki-Youb Park1, Su Jeoung Suh3, Jiyoung Seo1, June M. Kwak4,

Enrico Martinoia1,3, Inhwan Hwang2 and Youngsook Lee1,*1POSTECH-UZH Global Research Lab., Division of Molecular Life Sciences, POSTECH, Pohang, 790-784, Korea,2Center for Plant Intracellular Trafficking, POSTECH, Pohang, 790-784, Korea,3Institut fur Pflanzenbiologie, Universitat Zurich, 8008 Zurich, Switzerland, and4Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA

Received 20 June 2007; revised 21 July 2007; accepted 25 July 2007.

*For correspondence (fax +82 54 279 2199; e-mail [email protected]).

Correction added after online publication, 31 October 2007: correction to author’s name.

Summary

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]

inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a

reduction in free PtdIns(4,5)P2, decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by

phospholipases C and D. However, it is less clear how PtdIns(4,5)P2 modulates stomatal opening. We found

that in response to white light irradiation, the PtdIns(4,5)P2-binding domain GFP:PLCd1PH translocated from

the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P2 increases at the plasma

membrane upon illumination. Exogenously administered PtdIns(4,5)P2 substituted for light stimuli, inducing

stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P2 targets we performed patch-

clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P2. Genetic analyses

using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P2 in stomatal opening. The

reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was

countered by exogenous application of PtdIns(4,5)P2. The phenotype of reduced stomatal opening in the

pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest

that PIP5K4 produces PtdIns(4,5)P2 in irradiated guard cells, inhibiting anion channels to allow full stomatal

opening.

Keywords: PtdIns(4,5)P2, anion channel, PIP kinase, phospholipase C, stomatal opening, guard cells.

Introduction

Guard cells sense environmental and physiological stimuli,

and tightly regulate the stomatal aperture by responding

sensitively to a wide variety of exogenous and internal

stimuli such as light, temperature, internal CO2 concentra-

tion and ABA. ABA-induced stomatal closure (Hetherington,

2001; Schroeder et al., 2001; Fan et al., 2004) involves

changes in reactive oxygen species (Pei et al., 2000; Zhang

et al., 2001), phosphatidylinositol 3-kinase activity

(Park et al., 2003), calcium oscillations (McAinsh et al., 1990;

Allen et al., 2000) and actin organization (Eun and Lee, 1997).

Phospholipases C (PLC) and D (PLD) participate in the ABA-

induced stomatal closure response by producing the

calcium-mobilizing secondary messenger inositol 1,4,5-

trisphosphate [Ins(1,4,5)P3; Hunt et al., 2003] and phospha-

tidic acid (PA). Phosphatidic acid binds to ABI1, a negative

regulator of ABA responses (Leung et al., 1997), decreasing

its PP2C-type phosphatase activity (Zhang et al., 2004;

Mishra et al., 2006). The ultimate targets of many signal

mediators are ion channels and pumps, which are respon-

sible for ion influx and efflux and the resulting changes in

osmotic potential that lead to stomatal opening and closure.

There has been far less investigation into the stomatal

opening process than into stomatal closure (Dietrich et al.,

2001). Light, which is a potent stimulus for inducing stoma-

tal opening, activates the plasma membrane H+-ATPase by

phosphorylation of its C-terminus (Kinoshita and Shimazaki,

1999), allowing binding of a 14-3-3 protein and activation of

the proton pump (Emi et al., 2001; Kinoshita and Shimazaki,

ª 2007 The Authors 803Journal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 52, 803–816 doi: 10.1111/j.1365-313X.2007.03277.x

2002). Activation of the plasma membrane H+-ATPase is a

prerequisite for stomatal opening as it leads to hyperpolar-

ization of the membrane potential, which catalyzes opening

of inward-rectifying K+ channels (Schroeder et al., 1987) and

provides the driving force for K+ influx into guard cells. The

positive charges of K+ ions are counterbalanced by malate

synthesis within the guard cells, and by Cl– ions which enter

by proton co-transport (Roelfsema and Hedrich, 2005).

Although the role of anion channels in ABA-induced stoma-

tal closure is better known, they may also be involved in the

regulation of stomatal opening. Slow anion channels are

activated by depolarization and increasing cytosolic Ca2+

levels, releasing Cl– and other anions (Hedrich et al., 1990;

Schroeder and Keller, 1992). Together with the outward-

rectifying K+ channels, which also open in response to

depolarization of the membrane potential (Schroeder et al.,

1987), anion channel opening results in a decline in osmotic

potential, with consequent water efflux and stomatal clo-

sure. As various anion channel inhibitors induce stomatal

opening, it was suggested that these channels also play a

role in the opening process (Schroeder et al., 1993; Schwartz

et al., 1995; Leonhardt et al., 1999). The slow anion channels

remain activated at hyperpolarized membrane potentials,

often as negative as )200 mV (Linder and Raschke, 1992),

and supply a background flux of anions that generate a small

shunt-like pathway, controlling against further hyperpolar-

ization and over-opening of the stomata.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is

an important signal molecule that is involved in various

processes such as pollen tube growth (Kost et al., 1999;

Monteiro et al., 2005), salt and osmotic stress (DeWald et al.,

2001), vesicle trafficking (Martin, 2001), actin organization

(Janmey, 1994; Caroni, 2001), modulation of the plasma

membrane vanadate-sensitive H+-ATPase (Memon and

Boss, 1990), ion channel activity (Hilgemann et al., 2001;

Liu et al., 2005) and guard cell movements (Jung et al.,

2002). Guard cells have been shown to contain PtdIns(4,5)P2

(Parmar and Brearley, 1993) and in Vicia faba guard cells,

PtdIns(4,5)P2 levels transiently decrease following applica-

tion of ABA, suggesting a role in the ABA signaling cascade

for stomatal closure (Lee et al., 1996). Furthermore, the PLC

inhibitor 1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl)a-

mino]hexyl]-1H-pyrrole-2,5-dione (U-73122) inhibited ABA-

induced calcium oscillations in guard cells and stomatal

closure, providing supporting evidence for the importance

of PtdIns(4,5)P2 hydrolysis by PLC in the ABA-induced

stomatal closure process (Staxen et al., 1999). In addition,

PtdIns(4,5)P2 activates PLD (Qin et al., 1997), and following

ABA application the transient increase in PLD activity

releases PA, which has an inhibitory effect on the inward

K+ channel (Jacob et al., 1999). However, PtdIns(4,5)P2 also

appears to be involved in stomatal opening. This was

demonstrated using the PtdIns(4,5)P2-binding protein

GFP:PLCd1PH, which inhibited not only ABA-induced

stomatal closure, but also light-induced stomatal opening

when expressed in guard cells (Jung et al., 2002).

Phosphatidylinositol 4,5-bisphosphate is generated from

phosphatidylinositol 4-phosphate (PtdIns(4)P) or phospha-

tidylinositol 5-phosphate (PtdIns(5)P) by phosphatidylinosi-

tol phosphate kinase (PIP kinase). In Arabidopsis, although

there are 11 type I/II PIP kinases that are predicted to produce

PtdIns(4,5)P2 from either PtdIns(4)P or PtdIns(5)P (Mueller-

Roeber and Pical, 2002), this activity has only been con-

firmed for PIP5K1 and PIP5K10 (Mikami et al., 1998; Perera

et al., 2005). The PIP kinase PIP5K1 belongs to the B

subfamily, which contains putative membrane occupation

and recognition nexus (MORN) repeats, and it is expressed

strongly in procambial cells (Elge et al., 2001). In Arabidop-

sis, PIP5K1 expression is induced rapidly by drought, salt

and ABA (Mikami et al., 1998) and is regulated by a soluble

protein kinase (Westergren et al., 2001). The PIP kinase

PIP5K10 belongs to the A subfamily, which lacks MORN

repeats, and is most abundant in inflorescence stalks and

flowers; its Vmax is 10-fold lower than PIP5K1 (Perera et al.,

2005). Although the presence and absence of MORN repeats

suggests membrane and non-membrane localizations for

PIP5K1 and PIP5K10, respectively, their cellular localizations

and physiological functions remain undetermined.

In this paper we confirm that PtdIns(4,5)P2 promotes

stomatal opening and identify a mechanism of its action: it

inhibits anion current activation. Moreover, we describe a

gene encoding a PIP5K that is expressed in guard cells, and

show that this lipid kinase generates PtdIns(4,5)P2 in vitro.

We present a number of lines of evidence that support a role

for this gene in light-induced stomatal opening.

Results

PtdIns(4,5)P2 binding domain GFP:PLCd1PH translocates to

the plasma membrane in response to white light irradiation

GFP:PLCd1PH (phospholipase Cd1 pleckstrin homology do-

main) binds PtdIns(4,5)P2 and is widely used as a specific

biosensor for the lipid (Stauffer et al., 1998). It can be used to

visualize the minute amounts of this lipid that exist in plant

cells (Stauffer et al., 1998; Kost et al., 1999). Previously, we

reported that overexpression of GFP:PLCd1PH in guard cells

inhibited light-induced stomatal opening, probably by

interfering with the normal interactions between

PtdIns(4,5)P2 and other molecules (Jung et al., 2002).

Therefore, this result suggests that PtdIns(4,5)P2 is impor-

tant for light-induced stomatal opening. To test whether

illumination leads to increased PtdIns(4,5)P2 content, we

overexpressed GFP:PLCd1PH (Figure 1a) in V. faba guard

cells and observed the localization of fluorescence before

and after 3 h of irradiation with 170 lmol m)2 sec)1 white

light (Figure 1b). Translocation was quantified by measuring

the green fluorescence intensity of GFP from microscopic

804 Yuree Lee et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816

images. Fluorescence images of guard cells were scanned

along two lines drawn at right angles to the long axis of the

cells, at about 25% of the distance from both ends (Figure 1c,

left). From the resulting intensity profiles (Figure 1c, right),

the average peak pixel intensities of the cell boundary

(which should include the plasma membrane) and the cell

interior were obtained. The ratios of the two values were

compared before and after irradiation.

Initially, the intensity of fluorescence at the cell bound-

ary was similar to that of the cytosol (mean

SE = 1.09 � 0.02%, P > 0.05; Figure 1d, the first white

bar). However, following 3 h of irradiation with white

light, the fluorescence intensity was higher at the cell

boundary than in the cytosol (1.37 � 0.03%, P < 0.001),

indicating translocation of GFP:PLCd1PH from the cytosol

to the plasma membrane. Although GFP:PLCd1PH can

bind Ins(1,4,5)P3 as well, it is unlikely that the increase in

the fluorescence ratio was caused by a decrease in the

Ins(1,4,5)P3 level in the cytosol, as GFP:PLCd1PH was

expressed at a high level in the cytosol using the 35S

promoter, and its fluorescence is independent of whether

it is in the bound or free state.

In order to control for circadian clock-dependent trans-

location during the 3-h experiment, we also measured the

fluorescence changes in darkness. We observed that fluo-

rescence at the cell boundary increased slightly during the

experimental period (1.16 � 0.02%, P < 0.05) compared with

that of the cytosol. However, under light irradiation, the

extent of increase in fluorescence at the cell boundary was

significantly higher than that in the dark (P < 0.001).

During stomatal opening the vacuole swells. As a result,

the cytosol moves close to the nuclear area or to the

periphery of the cell, a process that may resemble translo-

cation of the protein to the nucleus or plasma membrane. To

assess the extent of this effect, we constructed a fusion of

GFP and the cytosolic Arabidopsis protein metallothionein

2a (MT2a; Lee et al., 2004) as a negative control for trans-

location (Figure 1b,d). Initially, the fluorescence intensity of

GFP:MT2a at the cell boundary was 1.06 � 0.03% of that in

the cytosol (P > 0.1). However, after 3 h of irradiation with

35S PLCδ1PH NOS

35S MT2a NOS

GFP:PLCδ1PH

GFP : MT2a

Dark Light 3 h

PM regionCytosol

1.0

1.1

1.2

1.3

1.4

PLCδ1PH MT2a PLCδ1PH

Fluo

resc

ence

inte

nsity

at

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/ cy

toso

l InitialAfter 3 h

Light Darkness

0.9

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0Time (min)

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inte

nsity

at

PM

/ cy

toso

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30 60 90 120 150

(a)

(b)

(c)

(d) (e)

GFP

GFP

Figure 1. GFP:PLCd1PH translocates from cyto-

sol to plasma membrane in response to illumi-

nation in Vicia faba guard cells.

(a) Diagrams showing the GFP:PLCd1PH and

GFP:MT2a fusion constructs in 326GFP-3 G vec-

tor. NOS; terminator derived from the nopaline

synthase.

(b) Fluorescence images of guard cells express-

ing GFP:PLCd1PH or GFP:MT2a in darkness or

after 3 h illumination. Bars = 10 lm.

(c) Measurement of fluorescence intensity in the

plasma membrane and cytosol. Guard cell fluo-

rescence images were scanned along two lines

(white bar) drawn at right angles to the long axis

of the cells, at about 25% of the distance from

both ends (left). From the resulting intensity

profiles (right), the average peak pixel intensities

of the cell boundary (black bar) and the cell

interior (white bar) were obtained.

(d) Relative pixel intensity of plasma membranes

from guard cells transformed with GFP:MT2a

and GFP:PLCd1PH in darkness or after 3 h of

illumination. Means � SE from 60–100 cells are

shown.

(e) Light- and dark-induced changes in the fluo-

rescence ratio of GFP:PLCd1PH at the plasma

membrane (PM) versus GFP:PLCd1PH in the

cytosol. GFP:PLCd1PH fluorescence was visual-

ized using time-lapse confocal microscopy for

1 h each of light and dark conditions as indicated

by white and black bars at the bottom. Mean-

s � SE from 17 cells are shown.

Roles of PtdIns(4,5)P2 in stomatal opening 805


white light, this had increased to 1.15 � 0.02% (P < 0.05),

relative to the cytosol. This value was similar to that

observed for GFP:PLCd1PH after 3 h in the dark (P > 0.1),

but different from that following 3 h of irradiation (P < 0.001,

Figure 1d). Therefore, we conclude that light induces trans-

location of GFP:PLCd1PH from the cytosol to the plasma

membrane independently of the circadian clock. The trans-

location of GFP:PLCd1PH was partially reversed upon trans-

fer of the cells to darkness after the light treatment

(Figure 1e, n = 17), further supporting the light dependency

of the process. The plasma membrane is a major target in

the guard cell signaling cascade, and the light-dependent

translocation of GFP:PLCd1PH to this membrane suggests a

function for PtdIns(4,5)P2 in the cellular light signaling

process.

Stomatal opening is induced by PtdIns(4,5)P2

The results described above suggest that PtdIns(4,5)P2 is a

factor that mediates stomatal opening. Therefore, we tested

whether or not application of exogenous PtdIns(4,5)P2 can

induce stomatal opening. Vicia faba guard cells were incu-

bated in a medium containing PtdIns(4,5)P2 mixed with

shuttle carriers (Ozaki et al., 2000) that assist in intracellular

delivery of PtdIns(4,5)P2, after which their stomatal aper-

tures were measured. Under darkness, treatment of epider-

mal tissues with 10 lM PtdIns(4,5)P2 significantly enhanced

circadian clock-dependent stomatal opening (P < 0.01). In

contrast, when PtdIns(4,5)P2 was replaced by PtdIns(4)P, no

significant difference could be observed between the

experimental and control stomata (P > 0.1, Figure 2a). The

specificity of PtdIns(4,5)P2-induced stomatal movement was

further tested using other phosphoinositides, including

PtdIns(3)P, PtdIns(5)P, PtdIns(3,4)P2 and PtdIns(3,5)P2. Only

PtdIns(3,4)P2 slightly increased the stomatal aperture. None

of the other lipids tested showed a significant effect (P > 0.1,

Figure 2a,b). The effect of PtdIns(4,5)P2 on stomatal opening

was concentration dependent between 1 and 30 lM (Fig-

ure 2c). In Commelina communis, a similar and statistically

significant effect was observed on stomatal opening fol-

lowing a 2-h application of PtdIns(4,5)P2 (P < 0.01, data not

shown). We speculated that if exogenously applied

PtdIns(4,5)P2 induced stomatal opening by increasing

PtdIns(4,5)P2 levels at the plasma membrane, then it

should also have induced translocation of GFP:PLCd1PH to

the plasma membrane. Indeed, a significant increase in

GFP:PLCd1PH fluorescence at the cell boundary was

observed at 60 min after application of PtdIns(4,5)P2

(P < 0.01, Figure 2d and Supplementary Figure S1a; n = 13),

whereas no such translocation was observed after applica-

tion of PtdIns(4)P (P > 0.1, Figure 2d and Supplementary

Figure S1b; n = 9).

As PtdIns(4,5)P2 is cleaved by PLC, it is possible that

PLC inhibition may represent a mechanism for increasing

PtdIns(4,5)P2 levels, and consequently stomatal opening.

This hypothesis was tested by investigating the effect of

U-73122 (a specific inhibitor of PLC in guard cells, as reported

by Staxen et al., 1999) on stomatal opening. The guard cells

2

3

4

5

6

7

8

Stom

atal

ape

rtur

e (μ

m) Control

1 µM PIP2

10 µM PIP2

20 µM PIP2

30 µM PIP2

90

100

110

120

130

140

150

Prot

opla

st v

olum

e (%

of

initi

al)

Darkness

PI4P ControlControl

Light

0.6

0.8

1.0

1.2

1.4

1.6

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GFP

:PL

Cδ1

PH

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/ cy

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atal

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m)

ControlU-73122U-73343

0.60.81.01.21.41.61.8

U-73122U-73343

GFP

:PL

Cδ1

PH

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m) Control

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atal

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20 40 60 80 100 120

0Time (min)

20 40 60 80 100 120

U73122 PI45P2

(a) (b)

(c) (d)

(e) (g)

(f)

Figure 2. Phosphatidylinositol 4,5 bis-phosphate [PtdIns(4,5)P2] enhances

stomatal opening in darkness and induces swelling of guard cell protoplasts

of Vicia faba.

(a, b) Stomatal aperture of guard cells treated with 10 lM of various kinds of

phosphoinositides, including PtdIns(4,5)P2 and phosphatidylinositol 4-phos-

phate [PtdIns(4)P]. The epidermal peels were maintained in darkness for the

entire experiment, which began 0.5 h prior to the photoperiod and ended at

2.5 h. During this time the stomata exhibited circadian clock-driven opening

movements. Values represent the means � SE from (a) 113–187 and (b) 116–

195 stomata.

(c) Stomatal apertures of guard cells treated with various concentrations of

PtdIns(4,5)P2 in darkness. Values represent the means � SE of 100–161

stomata.

(d) PtdIns(4,5)P2-induced changes in the localization of GFP:PLCd1PH fluo-

rescence of guard cells in darkness. Before and after treatment with 20 lM

PtdIns(4,5)P2 or PtdIns(4)P, GFP:PLCd1PH fluorescence was analyzed follow-

ing the protocol described in Figure 1. n = 13 for PtdIns(4,5)P2 and n = 9 for

PtdIns(4)P.

(e) Stomatal aperture of guard cells treated with 0.1 lM 1-[6-[((17b)-3-methoxy-

estra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) or its

inactiveanalog1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-

2,5-pyrrolidinedione (U-73343) in the dark. Values represent the means � SE of

153–213 stomata.

(f)U-73122-inducedchanges in the localizationofGFP:PLCd1PHfluorescenceof

guard cells in darkness. Before and after treatment with 0.5 lM U-73122 or

U-73343,GFP:PLCd1PHfluorescencewasanalyzed.n = 17forU-73122andn = 6

for U-73343.

(g) Effect of PtdIns(4,5)P2 or U-73122 on the volume of guard cell protoplasts.

Values represent the means � SE from 225–275 protoplasts.



treated with U-73122 showed statistically significant

increases in stomatal opening compared with the control

(P < 0.001), whereas those treated with its inactive analog,

1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl) amino]hex-

yl]-2,5-pyrrolidinedione (U-73343), did not (P > 0.1, Fig-

ure 2e). After exposure to U-73122 the stomatal apertures

reached the maximum after 2 h and remained in that state for

5 h (data not shown). This effect of U-73122 on stomatal

opening can be attributed to increased levels of PtdIns(4,5)P2

at the plasma membrane, as evidenced by the translocation

of GFP:PLCd1PH fluorescence to the plasma membrane

60 min after U-73122 treatment (P < 0.01, Figure 2f and

Supplementary Figure S1c; n = 17). Guard cells treated with

inactive U-73343 did not show any noticeable translocation

of GFP:PLCd1PH fluorescence (P > 0.1, Figure 2f and Sup-

plementary Figure S1d; n = 6).

To confirm the role played by PtdIns(4,5)P2 in stomatal

opening, we tested whether PtdIns(4,5)P2 could substitute

for light in inducing protoplast swelling via an increase in

osmotic pressure (Zeiger and Hepler, 1977; Amodeo et al.,

1992). We observed a similar degree of swelling in guard cell

protoplasts that were treated with either 10 lM PtdIns(4,5)P2

or irradiated with white light for 20 min (P > 0.05, Figure 2g).

There was no significant change in the volume of protop-

lasts incubated in darkness without PtdIns(4,5)P2 or in the

presence of PtdIns(4)P (P > 0.05, Figure 2g). In addition, the

volume of guard cell protoplasts treated with U-73122

increased more than that of the controls (P < 0.01, Fig-

ure 2g). These results provide additional support for the

suggestion that PtdIns(4,5)P2 can substitute for light in

inducing stomatal opening.

Slow anion current is inhibited by PtdIns(4,5)P2

Stomatal opening requires the coordinated and balanced

activities of many ion channels and transporters. To exam-

ine whether or not PtdIns(4,5)P2 induces stomatal opening

via alteration of ion channel activities we performed whole-

cell patch clamping of V. faba guard cell protoplasts and

analyzed K+ and anion channel activities before and after

+30 mV–120 mV

+30 mV

40

Cur

rent

(pA

)

0

–120

–80

–40

0

Time (sec)

a

c

b

a: Initialb: NPPBc: Washout

–400

–300

–200

–100

0

100

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rent

(pA

)

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IT10

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rent

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100

150

200

Time

Contro

l (22

)

PI4P (2

0)

PI45P

2(28

)

PI34P

2(7)

DAG (11)

Δ I/I

T0

(%)

*

20 40 60 80 100 0Time (sec)

20 40 60 80 100

0Time (sec)

20 40 60 80 100

(a) (b)

(c) (d)

Figure 3. Phosphatidylinositol 4,5 bis-phosphate [PtdIns(4,5)P2] inhibits the slow anion current activated by a depolarizing voltage stimulus applied to Vicia faba

guard cell protoplasts.

(a) Identification of S-type anion currents. a, Whole-cell patch-clamp recordings showing typical slow anion currents. The membrane potential was held at +30 mV to

activate the S-type channel, then hyperpolarized to )120 mV for 60 sec. b, External application of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) resulted in

inhibition of slow anion currents within 5 min. c, Following removal of the inhibitor by perfusion with control bath medium, the slow anion current recovered within

10 min.

(b) Slow anion current increases with time when the membrane potential is kept depolarized at +30 mV. After establishing the whole cell configuration, the

membrane potential was held at +30 mV for 3 min, after which the voltage was stepped to )120 mV (IT0) for 60 sec. The membrane potential was held at +30 mV for

the next 10 min, after which the same voltage step to )120 mV was repeated (IT10).

(c) Slow anion current of guard cell protoplasts treated with PtdIns(4,5)P2. Starting 5 min after the first recordings (IT0), 10 lM PtdIns(4,5)P2 was applied to

protoplasts for 5 min, after which the second recordings (IT10) were made.

(d) The effect of phosphoinositides on the time-dependent (10 min at +30 mV) increase in anion current. PtdIns(4,5)P2 inhibited the time-dependent anion current

increase, whereas the control and phosphatidylinositol 4-phosphate [PtdIns(4)P] did not (numbers in the parenthesis indicate the number of cells tested). The star

indicates a significant difference in the I/IT0 values of protoplasts treated with 10 lM PtdIns(4,5)P2, compared with the non-treated time control (P < 0.05).



application of PtdIns(4,5)P2. Inward (n = 14) and outward

(n = 14) K+ channel activities were unaltered by 10 lM

PtdIns(4,5)P2 (data not shown).

As anion channels inhibit stomatal opening (Schwartz

et al., 1995; Leonhardt et al., 1999), inhibition of their

activities may represent a mechanism for enhancing this

process. In order to measure anion currents, we used a

pipette solution containing 0.3 lM free Ca2+ and 200 lM

guanosine 5¢-triphosphate (GTP), which have been shown

to enhance anion currents across the plasma membrane of

guard cells (Hedrich et al., 1990). S-type anion currents were

identified by their typical time dependence and sensitivity to

50 lM 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB;

Figure 3a). The current magnitude measured after a 10-min

exposure to +30 mV (IT10) increased in comparison to initial

currents (IT0), a result that was expected as depolarization

activates anion channels (Figure 3b; Schroeder and Keller,

1992). Treatment with PtdIns(4,5)P2 inhibited this time-

dependent increase in anion currents (Figure 3c). To quan-

tify these effects and to test whether or not 10 lM

PtdIns(4,5)P2 specifically inhibits the current, we compared

the magnitude of steady-state anion currents at the end of

60 sec hyperpolarizing voltage steps applied before and

after treatment with various lipids. The magnitude of current

change relative to initial current (DI/IT0) (%) = [(IT10 – IT0)/IT0] ·100 (relative current increase) was about 160 � 56% in

untreated control cells. Protoplasts treated with 10 lM

PtdIns(4)P showed a magnitude of DI/IT0 similar to the time

control (164 � 50%). In contrast, anion currents from pro-

toplasts treated with 10 lM PtdIns(4,5)P2 showed DI/IT0 of

40 � 20%, significantly lower than the time control or

PtdIns(4)P (P < 0.05, Figure 3d). We tested the effects of

phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and

1-palmitoyl-2-oleoyl-sn-glycerol (DAG) on anion current,

as PtdIns(3,4)P2 has been reported in the guard cells of

C. communis (Parmar and Brearley, 1993), and DAG is a

product of PtdIns(4,5)P2 hydrolysis, as well as an inducer of

stomatal opening (Lee and Assmann, 1991). Protoplasts

treated with 10 lM PtdIns(3,4)P2 and DAG exhibited slightly

reduced DI/IT0 values, but these effects were not statistically

significant (PtdIns(3,4)P2, 92 � 52%; DAG, 78 � 20%). There-

fore, we conclude that of the lipids tested, PtdIns(4,5)P2 was

the most effective at inhibiting development of an anion

current.

Arabidopsis PIP5K4 mutants exhibit reduced

stomatal opening

To test whether PtdIns(4,5)P2 is important for stomatal

opening in vivo we used a genetic approach. The enzyme

that produces PtdIns(4,5)P2 is PIP kinase (PI4P5K and

PI5P4K), and 11 different PIP kinases have been identified in

Arabidopsis (Mueller-Roeber and Pical, 2002). We obtained

Arabidopsis mutants from the SALK T-DNA insertion

populations deficient for these genes and tested their sto-

matal opening. We observed altered stomatal opening in a

mutant deficient in PIP5K4 (At3g56960); the T-DNA insertion

in pip5k4 was confirmed by polymerase chain reaction (PCR)

of genomic DNA. The T-DNA was inserted into the first exon

of PIP5K4, 1192 nucleotides downstream of the initiation

codon (Figure 4a). To confirm that the pip5k4 mutant does

not generate a PIP5K4 transcript, reverse transcriptase (RT)-

PCR was performed using total RNA. As expected, the

PIP5K4 transcript was not amplified from pip5k4, whereas it

was amplified from wild-type (WT) Arabidopsis (Figure 4b).

Under natural light, the pip5k4 mutant exhibited delayed

stomatal opening (data not shown), and this phenotype was

confirmed by performing a stomatal opening test in the dark

or under white light irradiation (Figure 4c). At the beginning

of the photoperiod (T = 0 h), the apertures of pip5k4 stomata

did not differ significantly from WT (P > 0.05), whereas after

3 h of illumination with 170 lmol m)2 sec)1 white light, the

mean aperture size of pip5k4 stomata (2.78 � 0.03 lm) was

significantly smaller than that of WT (4.20 � 0.03 lm,

P < 0.001).

If the reduced stomatal opening in pip5k4 was due to

decreased production of PtdIns(4,5)P2, replenishment of

PtdIns(4,5)P2 should enable recovery of normal movement.

We tested this idea by treating epidermal strips of pip5k4

plants with exogenous PtdIns(4,5)P2. These strips were

incubated in medium containing 10 lM PtdIns(4,5)P2 and

irradiated with white light, after which stomatal apertures

were measured (Figure 4d). We observed a reduction in

the light-induced opening of peeled epidermis compared

with that in detached whole leaves. Stomatal apertures

reached maximal opening after 4 h of illumination. The

stomatal apertures of PtdIns(4,5)P2-treated pip5k4 (2.94 �0.05 lm) were similar to those of WT (3.02 � 0.06 lm,

P > 0.5), and significantly larger than those of pip5k4

without treatment (2.32 � 0.06 lm, P < 0.01; Figure 4d).

The stomatal apertures of PtdIns(4)P- and PtdIns(3,4)P2-

treated pip5k4 plants were not significantly different from

those of untreated pip5k4 plants (Figure 4e). These results

indicate that the reduced stomatal opening observed for

pip5k4 mutants is most likely due to a reduced level of

PtdIns(4,5)P2.

We tested whether U-73122 differentially affects stomatal

responses in WT and pip5k4 mutant plants. Epidermal layers

of WT and pip5k4 leaves were peeled off and incubated in a

solution containing 0.1 lM U-73122 or U-73343 under dark-

ness. In WT plants, the guard cells treated with U-73122

showed statistically significant increases in stomatal open-

ing compared with the control (P < 0.001), whereas those

treated with its inactive analog U-73343 did not (P > 0.1).

Similar responses to the two drugs were observed in the

pip5k4 plants (Figure 4f).

To ensure that the reduced stomatal opening in pip5k4

was indeed due to the deficiency in PtdIns(4,5)P2 caused by



mutation of PIP5K4, we transformed pip5k4 plants with a

construct expressing the full-length cDNA of PIP5K4 driven

by its own promoter. The complemented lines expressing

PIP5K4 exhibited similar stomatal opening to WT (Fig-

ure 5b). Thus, the phenotype of reduced stomatal opening

in the pip5k4 mutant was recovered in complemented lines

(Figure 5b).

PIP5K4 is expressed in guard cells and localized

to the plasma membrane

To verify that PIP5K4 is expressed in guard cells, we per-

formed RT-PCR using the same total RNA preparations

of Arabidopsis guard cell and mesophyll cell protoplasts as

those described by Mori et al. (2006). The guard cell prepa-

ration showed little contamination with mesophyll cells

(Figure 1a of Mori et al., 2006). We determined that PIP5K4

was expressed in both cell types (Figure 6a). If PIP5K4 is

important for light-induced stomatal opening and is

responsible for light-dependent production of PtdIns(4,5)P2

at the plasma membrane (as suggested by the results shown

in Figure 1), it should localize to the plasma membrane. We

investigated the localization of PIP5K4 using V. faba guard

cells that had been transformed by biolistic bombardment

with vector expressing GFP:PIP5K4. The fluorescence was

localized to the plasma membrane (Figure 6b and Supple-

mentary Figure S2b), and this localization did not alter in a

light-dependent manner (data not shown). Free GFP was

localized to the cytosol regardless of the light condition

(Figure 6b and Supplementary Figure S2a).

wt pip5k4

PIP5K4

Tubulin +1192 ATG

pROK2

wt - light pip5k4 - light

pip5k4 - dark wt - dark

0

1

2

3

4

5

0 1 2 3

0 1 2 3 4 5

0 1 2 3

Time (h)

Stom

atal

ape

rtur

e (µ

m)

1.0

1.5

2.0

2.5

3.0

3.5

Time (h)

Stom

atal

ape

rtur

e (µ

m) wt

pip5k4 pip5k4 + PI45P 2

1.0

1.5

2.0

2.5

Stom

atal

ape

rtur

e (µ

m)

WT-control pipk4 -control

WT-U73122 pipk4 -U73122WT-U73343 pipk4 -U73343

0.5

1.0

1.5

2.0

2.5

3.0

Time (h) 0 1 2 3 4 5

Time (h)

Stom

atal

ape

rtur

e ( μ

m)

pipk4 pipk4 + PI34P 2

pipk4 + PI45P 2 pipk4 + PI4P

(a) (b)

(c) (d)

(e) (f)

Figure 4. Mutation of Arabidopsis PIP5K4 (At3g56960) results in delayed stomatal opening.

(a) Genetic structure of PIP5K4 and site of the T-DNA insertion. Boxes represent exons. ROK2, T-DNA present in the SALK Arabidopsis mutants.

(b) reverse transcriptase-polymerase chain reaction amplification of PIP5K4 mRNA. PIP5K4 transcript was amplified from wild-type (WT), but not PIP5K4 knockout

plants (pip5k4). TUBULIN was amplified as a positive control.

(c) Stomatal apertures of WT and pip5k4 plants. Results shown are from four independent experiments (mean � SE). n (dark) = 80–97, n (light) = 428–790.

(d) Effects of Phosphatidylinositol 4,5 bis-phosphate [PtdIns(4,5)P2] on stomatal apertures of WT and pip5k4 plants. Epidermal strips from wild-type and pip5k4

leaves were peeled and incubated on medium with or without 10 lM PtdIns(4,5)P2 under 170 lmol m–2 sec–1 white light. Values represent the means � SE from

140–240 stomata.

(e) No effect of phosphatidylinositol 4-phosphate [PtdIns(4)P] or phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] on stomatal opening movement in pip5k4

plants under 170 lmol m)2 sec)1 white light. Values represent the means � SE from 83–147 stomata.

(f) Stomatal aperture of guard cells treated with 0.1 lM of 1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) or its

inactive analog 1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione (U-73343). Epidermal strips from wild type and pip5k4 leaves

were peeled and incubated on medium with 0.1 lM U-73122 or U-73343 in the dark. Values represent the means � SE of 50–148 stomata.



PIP5K4 has PIP kinase activity

PIP5K4 comprises a conserved PIP kinase catalytic domain, a

dimerization domain and the repeated MORN motif

(Mueller-Roeber and Pical, 2002). To test whether PIP5K4

exhibits PIP kinase activity, we purified the entire kinase

protein or the catalytic domain of PIP5K4 without MORN

repeats (D1–388) fused to glutathione-S-transferase (GST).

Full-length proteins were less stable than the catalytic

domain lacking the MORN repeats; thus we added twice as

much of the full-length protein (full-length protein, 10 lg;

catalytic domain, 5 lg) to the kinase assay. We determined

the kinase activity of the purified fusion proteins and GST

alone (as a negative control) using exogenous PtdIns(4)P as

the substrate. Both GST–PIP5K4 fusion proteins (with or

without MORN repeats) exhibited PIP kinase activity when

supplied with PtdIns(4)P, whereas GST alone did not (Fig-

ure 6c). GST–PIP5K4 did not show phosphatidylinositol (PI)

kinase activity when supplied with PtdIns as a substrate

(Figure 6c). These results indicate that PIP5K4 encodes an

active PIP kinase, which can take PtdIns(4)P, the major

PtdInsP in the cell, as a substrate and produce PtdIns(4,5)P2.

Discussion

In this paper we provide evidence that PtdIns(4,5)P2 is an

important signal mediator for stomatal opening, and we

identify PIP5K4 as an enzyme that synthesizes PtdIns(4,5)P2.

We demonstrate that the fluorescence intensity of a

PtdIns(4,5)P2-binding peptide (GFP:PLCd1PH; Stauffer et al.,

1998) is stronger at the plasma membrane than in the cyto-

sol of guard cells irradiated with white light, but not in those

in darkness (Figure 1b), suggesting a light-dependent

increase in PtdIns(4,5)P2 levels at the plasma membrane of

guard cells. The increase in PtdIns(4,5)P2 levels could be due

to a light-induced increase in synthesis and/or a decrease in

hydrolysis of PtdIns(4,5)P2. Regardless of this, the light-

dependent appearance of PtdIns(4,5)P2 at the plasma

membrane is consistent with the suggestion that it plays a

wt pip5k4

PIP5K4Tubulin

PIP5K4–1

PIP5K4–2

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 1 2 3

Time (h)

Stom

atal

ape

rtur

e (µ

m)

wtpip5k4PIP5K4-1PIP5K4-2

(a)

(b)

Figure 5. The reduced stomatal opening of pip5k4 is recovered by comple-

mentation in pip5k4 lines expressing PIP5K4 from its own promoter.

(a) reverse transcriptase-polymerase chain reaction amplification of PIP5K4

mRNA. PIP5K4 transcript was amplified from wild-type (WT) and comple-

mented lines, but not from pip5k4. Tubulin was amplified as a positive

control.

(b) Stomatal apertures of WT, pip5k4 and complemented pip5k4 lines 1 and 2

(PIP5K4-1 and PIP5K4-2, respectively) under 170 lmol m)2 sec)1 white light.

The complemented lines were produced by transforming pip5k4 plants with a

construct expressing the full-length cDNA of PIP5K4 under its own promoter.

Results shown are from three independent experiments (mean � SE,

n = 149–188).

GC MC

PIP5K4

UBQ

Origin

PI45P2

Protein

SubstratePI4P − PI PI4P − PI PI4P

GST GST-PIP5K4Δ1-388

GST-PIP5K4

GFP alone GFP:PIP5K4

(a)

(b)

(c)

Figure 6. Characterization of PIP5K4.

(a) Detection of PIP5K4 mRNA expression in guard cell (GC) and mesophyll

(MC) protoplasts using reverse transcriptase-polymerase chain reaction.

UBQ, encoding the ubiquitin-conjugating enzyme E2, was amplified as a

positive control.

(b) Fluorescence (upper panels) and corresponding bright-field images

(bottom panels) of guard cells expressing GFP alone or GFP:PIP5K4.

Bars = 10 lm.

(c) Phosphatidylinositol phosphate (PIP) kinase activity of bacterially

expressed glutathione-S-transferase (GST)-PIP5K4 in vitro. The purified

GST-PIP5K4 fusion proteins without membrane occupation and recognition

nexus repeats (D1–388) or the full-length enzyme were assayed for PIP kinase

activity, as described in Experimental procedures. The whole plate is shown in

a box on the left. The bottom region of the same plate is enlarged on the right.

Correction added after online publication, 31 October 2007: GC label in (a)

corrected.



role in light signal transduction. Further support for this idea

comes from the observation that exogenous application of

PtdIns(4,5)P2 induced stomatal opening and swelling of

guard cell protoplast in darkness (Figure 2). Phosphatidyl-

inositol 4-phosphate, a metabolite and precursor of

PtdIns(4,5)P2, is not responsible for the stomatal opening

effect of PtdIns(4,5)P2, as shown in Figure 2. This result is

consistent with the previous observation that PtdIns(4)P-

binding protein, which presumably reduces the free

PtdIns(4)P levels, exerts an effect opposite to that of

PtdIns(4,5)P2-binding protein in stomatal opening move-

ment (Jung et al., 2002). The result is also consistent with

the recent observation that PtdIns(4,5)P2 synthesis, but not

PtdIns(4)P synthesis, is the rate-limiting step in the plant

phosphoinositides pathway (Im et al., 2007). In addition,

anion channel activity was altered by PtdIns(4,5)P2, but

not by PtdIns(4)P (Figure 3), which may explain, at least

partly, why they have different effects on stomatal opening

movement. Another hydrolysis product of PtdIns(4,5)P2,

Ins(1,4,5)P3, is well known for its effect on stomatal closing

(Blatt et al., 1990; Gilroy et al., 1990), which would appear to

preclude an effect on stomatal opening. Moreover, U-73122,

which inhibits the production of Ins(1,4,5)P3, showed effects

similar to that of PtdIns(4,5)P2.

Possible targets for the action of PtdIns(4,5)P2 at the guard

cell plasma membrane include ion pumps and channels.

Recent studies in animals have shown that PtdIns(4,5)P2

regulates a variety of ion transporters and channels, activat-

ing Na+/Ca+ exchangers (Hilgemann and Ball, 1996),

inwardly rectifying potassium channels (Huang et al.,

1998) and the epithelial sodium channel (Yue et al., 2002).

In addition, PtdIns(4,5)P2 regulates the cystic fibrosis

transmembrane conductance regulator (CFTR), which func-

tions as an anion channel enabling the passage of chloride

or other anions across an electrochemical gradient (Himmel

and Nagel, 2004). Among the many potential target trans-

porters in guard cells, we tested anion channel activities, and

observed that those were inhibited by PtdIns(4,5)P2 (Fig-

ure 3). The slow anion channel can play a role as a negative

regulator of stomatal opening. An anion channel, when

activated, releases anions, and thus depolarizes membrane

potential in plant cells. Interestingly, it retains a significant

opening at strongly hyperpolarized potentials, as low as

)200 mV (Linder and Raschke, 1992; Schroeder and Keller,

1992; Schroeder et al., 1993), acting as a leak pathway that

inhibits further hyperpolarization of membrane potential.

Therefore, activation of an anion channel inhibits over-

activation of inward K+ channels that are responsible for the

K+ uptake necessary for stomatal opening. Supporting the

idea of anion channels as negative regulators of stomatal

opening, various studies have demonstrated that anion

channel inhibitors enhance opening (Schroeder et al., 1993;

Schwartz et al., 1995; Leonhardt et al., 1999). It is noteworthy

that the anion channel CFTR responds differently to

PtdIns(4,5)P2 depending on its phosphorylation status:

application of PtdIns(4,5)P2 to non-phosphorylated CFTR

activates a chloride current, whereas phosphorylated CFTR

is inhibited. In most cases, PtdIns(4,5)P2 regulates channel

activity via direct binding. It would be interesting to elucidate

the mechanism by which PtdIns(4,5)P2 modulates anion

channel activity in guard cells. However, this awaits molec-

ular identification of the anion channels at the plasma

membrane of these cells.

In addition to the slow anion channel, PtdIns(4,5)P2 can

modulate other channels or pumps that are important for

stomatal opening, and a candidate might be the inward K+

channel, which plays an important role in stomatal opening.

However, we could not find any effect of PtdIns(4,5)P2 on K+

channel activity. In contrast to our result, recently published

data have demonstrated that PtdIns(4,5)P2 restores activity

of shaker-type K+ channels run down following patch

excision (Liu et al., 2005). Although these different results

may be due to cell type, they are most likely a PtdIns(4,5)P2

concentration effect. We used 10 lM PtdIns(4,5)P2, whereas

Liu et al. (2005) used concentrations up to 500 lM, which are

unlikely to represent true physiological values. At relatively

low concentrations of PtdIns(4,5)P2 (20 lM), they were

unable to detect any significant change in K+ current from

the giant patch, a result that is consistent with our data. In

addition, while we used V. faba guard cells, they used oocyte

cells expressing the gene encoding the K+ channel.

The increase in fluorescence of the PtdIns(4,5)P2 indicator

at the plasma membrane of irradiated cells is indirect

evidence for de novo synthesis of PtdIns(4,5)P2 at this site.

Recently, PtdIns(4,5)P2 synthesis, but not PtdIns(4)P synthe-

sis, was shown to be the rate-limiting step in the plant

phosphoinositides pathway. In these experiments, the ratio

of PtdIns(4)P to PtdIns(4,5)P2 in WT tobacco cells was found

to be ‡10:1, whereas in tobacco cells expressing human

PIPKIa, a 100-fold increase in plasma membrane

PtdIns(4,5)P2 was observed without any change in the

PtdIns(4)P level (Im et al., 2007). To investigate possible

changes in PtdIns(4,5)P2 synthesis in response to light, we

measured PIP kinase activity in guard cell extracts. However,

we were unable to obtain consistent results, most likely

because of a very low level of enzyme activity in this cell

type. As an alternative approach to test the importance of

PtdIns(4,5)P2 synthesis in light signal transduction leading to

stomatal opening, we screened PIP kinase knockout mutants

for altered stomatal opening. Among the two knockout

mutants tested, pip5k4, which contains a mutation in PIP5K4

(At3g56960), exhibited a smaller stomatal aperture under

light (Figure 4c), while pip5k3, which contains a mutation in

PIP5K3 (At3g56960), did not differ from WT with respect to

stomatal movement (data not shown). Normal stomatal

opening movements were recovered in the pip5k4 mutant

by application of PtdIns(4,5)P2 (Figure 4d), and complemen-

tation using lines expressing PIP5K4 under its own promoter



(Figure 5b). These results suggest that for normal stomatal

opening sufficient PtdIns(4,5)P2 must be present in the

plasma membrane and that PIP5K4 contributes to the

synthesis of PtdIns(4,5)P2. The possibility that PIP5K4 affects

stomatal movement via some of its other functions is

remote, although it still remains to be shown that an inactive

kinase mutant of PIP5K4 cannot complement stomatal

movement. Consistent with this explanation, we observed

localization of this enzyme at the plasma membrane (Fig-

ure 6b). Other PIP5Ks may also participate in this pathway, as

light-induced stomatal opening was not completely inhibited

in pip5k4 plants (Figure 4c), and most PIP5Ks except for

PIP5K3, 6, and 10 are present in guard cells, although the

expression of no single gene predominates in this cell type

(Leonhardt et al., 2004). Under darkness, the PtdIns(4,5)P2

level may not differ much between WT and knock-out guard

cells, as the PLC inhibitor enhanced stomatal opening to

similar extents in the two genotypes of plants under

darkness. It is possible that PIP5K4 is mainly responsible

for the light-dependent increase of PtdIns(4,5)P2 production

and that other PIP5Ks produce PtdIns(4,5)P2 in the dark.

How is PIP5K activity regulated in guard cells? With the

exception of one report, which showed that its activity is

reduced by phosphorylation (Westergren et al., 2001), little is

known about the regulation of PIP5K activity in plants. In

animal cells, the activity of PIP5K I isoforms is often regulated

by small Rho GTP-binding proteins such as RhoA, Rac1 and

Cdc42 (Chong et al., 1994; Weernink et al., 2004). Plants

contain a unique subfamily of Rho GTPases, the Rop GTPases

(for Rho-related proteins from plants; Li et al., 1998; Yang,

2002) that are most similar to the mammalian RAC GTPases.

Rop GTPases play roles in guard cell signaling (Lemichez

et al., 2001; B.W. Jeon, J.-U. Hwang, J.M. Kwak, Z. Yang and

Y. Lee, our unpublished results), as well as in many other

processes including pollen tube growth and actin organiza-

tion, in which PtdIns(4,5)P2 was also found to be important

(Lin and Yang, 1997; Kost et al., 1999; Fu et al., 2002). Further

investigation will be required to determine whether or not

Rop GTPases act as upstream regulators of PIP5Ks.

Light-dependent increases in PtdIns(4,5)P2 levels at the

plasma membrane can be caused not only by increased

synthesis but also by a reduction in PtdIns(4,5)P2 hydrolysis

by PLC. The Arabidopsis genome contains nine putative

phosphatidylinositol-specific phospholipase C (PI-PLC) iso-

forms (Mueller-Roeber and Pical, 2002; Hunt et al., 2004), of

which only PLC1 and PLC2 have been characterized (Hiray-

ama et al., 1995, 1997). Expression of PLC1 is induced under

environmental stress (Hirayama et al., 1995) and decreased

expression using antisense PLC1 reduces the inhibitory

effect of ABA on germination and downregulates the

expression of many drought/cold-inducible genes (Sanchez

and Chua, 2001). Previous physiological experiments have

suggested that PI-PLCs are also important for ABA signal

transduction in guard cells (Staxen et al., 1999; Hunt et al.,

2003; Mills et al., 2004). At concentrations that also inhibit

recombinant PI-PLC activity, the PLC inhibitor U-73122

inhibits stomatal guard cell responses to ABA and cytosolic

Ca2+ oscillations (Staxen et al., 1999). In addition, it has been

observed that reducing the level of PI-PLC in tobacco guard

cells partially interferes with ABA inhibition of stomatal

opening (Hunt et al., 2003; Mills et al., 2004). However, little

is known about the function of PI-PLCs in light-induced

stomatal opening. We investigated the involvement of PLC

on stomatal opening using U-73122. The specificity of

U-73122 as an inhibitor of PLC in guard cells was rigorously

shown by Staxen et al. (1999), who showed that U-73122,

but not its inactive analog U-73343, reduced activity in a

recombinant plant PI-PLC, stomatal closing movement and

Ca2+ oscillation. We observed that guard cells treated with

U-73122 had an accelerated circadian clock-induced stoma-

tal opening in darkness. Guard cells treated with the inactive

analog, U-73343, were no different from control cells with

respect to stomatal opening (Figure 2e). Treatment with

U-73122 also induced swelling of guard cell protoplast in the

dark (Figure 2g). Therefore, it is possible that PLCs are also

involved in the regulation of light-induced stomatal open-

ing. An interesting question that remains to be answered is

whether or not the PLCs involved in light signaling are the

same as those in ABA signaling.

In summary, our results demonstrate that PtdIns(4,5)P2 is

an important factor for light-induced stomatal opening and

that PIP5K4 is at least partially responsible for the production

of PtdIns(4,5)P2 in guard cells. For a better understanding of

the stomatal opening process, we will need to elucidate how

PIP5K is regulated, what other enzymes are able to synthe-

size PtdIns(4,5)P2 in guard cells, and how PtdIns(4,5)P2

regulates the anion channel activity.

Experimental procedures

Plant materials and chemicals

Vicia faba, C. communis and A. thaliana plants were grown for 3, 5and 5 weeks, respectively, in a greenhouse at 22 � 2�C with light/dark cycles of 16/8 h. For the delivery of bisphosphorylated phos-phoinositides into the cells, Shuttle PIPTM Carrier-1, histone H1(Molecular Probes, http://probes.invitrogen.com. was used, andPtdIns(4,5)P2 delivery was confirmed using BODIPY tetramethyl-rhodamine-X C6-PtdIns(4,5)P2 (Molecular Probes). The fluorescencewas evenly distributed at the plasma membrane and displayed apunctuate staining pattern inside the cell. For stomatal movementassays, synthetic PtdIns(4,5)P2 (L-a-phosphatidylinositol 4,5-diphosphate), PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(3)P, PtdIns(4)P,and PtdIns(5)P with dioctanoyl acyl chains were used (Sigma-Aldrich, http://www.sigmaaldrich.com/). For electrophysiologicalrecordings, sodium salts of PtdIns(4)P and PtdIns(4,5)P2 were pur-chased from Sigma-Aldrich, and 1-palmitoyl-2-oleoyl-sn-glycerol(DAG) from Avanti Polar Lipids (http://www.avantilipids.com/), andthe lipid solutions were sonicated immediately before treatment.U-73122 and U-73343 were purchased from Sigma-Aldrich anddissolved in dimethyl sulfoxide.



Measurement of stomatal apertures

Abaxial epidermal layers of V. faba or Arabidopsis leaves werefirst peeled and then incubated in a solution containing10 mM KCl and 10 mM 2-(N-morpholine)-ethanesulfonic acid(MES)-KOH (pH 6.1) with or without drugs or phosphoinositides.In order to supply phosphoinositides, Shuttle PIPTM Carrier-1,histone H1 (Molecular Probes) and phosphoinositides weremixed immediately before the experiments, allowed to equili-brate for 5 min, and added to the buffer solution to generate aworking concentration of phosphoinositides. A control solutionwas prepared from an equivalent amount of Shuttle Carrier-1without phosphoinositides. In some Arabidopsis experimentsthat did not necessitate treatment with drugs or lipids, wholeleaves were incubated in buffer solution, and then the epidermallayer was peeled off just before observation. The samples wereincubated in darkness 0.5 h prior to the beginning of the pho-toperiod, observed at 1 h intervals with bright field microscopy,Axioskop 2 (Carl Zeiss, http://www.zeiss.com/) and photographedusing a CCD camera, Axio Cam (Carl Zeiss). Aperture size wasmeasured from photographs using the Interactive Measurementsoftware package, AXIOVISION 3.0.6 (Carl Zeiss). In all experimentstreatment with light or lipids began immediately after the initialmeasurement.

Measurement of the volume of guard cell protoplasts

Guard cell protoplasts of V. faba were isolated following a proceduremodified from Kruse et al. (1989). The youngest fully expandedleaves of 3- to 5-week-old plants were homogenized in a Waringblender 7010 (http://www.waringproducts.com) for 40–50 sec at10000 g to remove epidermal and mesophyll cells. After washingwith tap water, the epidermal fragments were collected on a 220 lmnylon mesh (Small Parts, http://www.smallparts.com/), then incu-bated in enzyme solution for 30 min at 21�C, with rotation at 100 rpm.The enzyme solution comprised 4.5 parts distilled water and 5.5 partsbasic solution (0.5 mM CaCl2, 0.5 mM MgCl2, 10 lM KH2PO4, 5 mM K+-MES [pH 5.5] and 0.45 M D-mannitol) containing 1% (w/v) cellulysin(Calbiochem, http://www.calbiochem.com), 0.3% (w/v) BSA, 0.1%(w/v) polyvinylpyrrolidone (PVP) and 1 mM ascorbic acid. The par-tially digested epidermal fragments were then incubated in basicsolution containing 1.5% (w/v) cellulase RS (Yakult Honsha, http://www.yakult.co.jp), 0.3% (w/v) BSA, 0.02% pectolyase Y-23 (YakultHonsha) and 1 mM ascorbic acid (pH 5.5), at 21�C, with rotation at60 rpm. After 40 to 50 min, protoplasts were collected and resus-pended in a solution containing 0.35 M D-mannitol, 1 mM CaCl2,

10 mM KCl, 1 mM MES (pH 6.2) and 1 mM ascorbic acid.To measure change in protoplast volume, the protoplasts were

incubated in darkness for 30 min in media with or without lipidsand then photographed using bright field microscopy (Axioskop2, Carl Zeiss) with a CCD camera (Axio Cam, Carl Zeiss).Diameters of protoplasts were measured from photographsusing the Interactive Measurement software package AXIOVISION

3.0.6 (Carl Zeiss) and the volumes were calculated by theequation 4/3*p*r.

Electrophysiological recordings

Patch electrodes were pulled from glass capillaries (Kimax-51,Kimble, http://www.kimble.com) using a two-stage puller (PP-83,Narishige, http://www.narishige.co.jp/) and filled with an intra-cellular solution comprising 150 mM tetraethylammonium chlo-ride (TEA-Cl), 10 mM HEPES, 2 mM MgCl2, 4 mM MgATP, 200 lM

Na2GTP, 6.7 mM EGTA, 3.35 mM CaCl2, and adjusted to pH 7.2with 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)–HCl andto 400 mmol kg)1 with D-mannitol. The free Ca2+ concentration inthe pipette solution was about 0.3 lM (Schroeder and Keller,1992). The bath solution contained 40 mM CaCl2, 2 mM MgCl2,10 mM MES (adjusted to pH 5.6 with TRIS-HCl and to400 mmol kg)1 with D-mannitol). The pipette resistance was»10 MW and current data were obtained using an Axon Instru-ments Axopatch 200A amplifier (Axon Instruments, http://www.axon.com/). PCLAMP software (Axon Instruments) was usedfor voltage pulse stimulation, online data acquisition and dataanalyses. The current response of the protoplast in the whole-cellconfiguration was recorded after the membrane potential hadbeen held at +30 mV for 3 min to activate the anion channels(Schroeder et al., 1993). Current amplitudes were compared forthe same cell before and at 5 min following drug treatments.Only cells that maintained >1 GW whole seal resistancethroughout the experiment were included in the analyses.

For application of lipid to the patch-clamped cells, the lipid wasN2-dried, then added to the bath solution. The mixture wassonicated for 2.5–3 min immediately prior to application to thewhole-cell patches and released from a micropipette positionedabout 150 lm from the target cell. The inner diameter of the drugpipette was nearly double that of the protoplast and the solutionflow rate was kept extremely low to reduce perturbation of thepatched cell.

Biolistic gene bombardment into V. faba guard cells

Vectors expressing GFP and GFP:PLCd1PH were introduced intoV. faba guard cells using a bombardment technique (ParticleDelivery System-1000/He; Bio-Rad, http://www.bio-rad.com/) des-cribed previously (Park et al., 2003). The bombarded leaves werethen placed onto a Petri dish lined with wet filter paper and keptin the dark at 22 � 2�C. Between 15 and 20 h after bombardmentthe leaves were transferred to a solution containing 10 mM KCland 10 mM MES-KOH (pH 6.1), then either exposed to white light(0.2 mmol m)2 sec)1) or kept in the dark. Epidermal peels wereremoved from the leaves and observed using an Axioskop 2fluorescence microscope (Carl Zeiss). For time-lapse recording ofsingle cells, a confocal microscope, FLUOVIEW FV1000 (Olympus,http://www.olympus-global.com/) was used.

Assay for translocation of GFP:PLCd1PH between

the plasma membrane and cytoplasm

Vector expressing GFP:PLCd1PH was introduced into guard cells bybiolistic bombardment. Using the image edit tool of KS Lite version2.0 (Kontron, http://www.kontron.com/), fluorescence images ofguard cells were scanned along two lines drawn at right angles tothe long axis of the cells, at about a 25% distance from both ends.The lines seldom crossed the nucleus, which is usually located atthe center of guard cells. From the resulting intensity profiles, theaverage peak pixel intensities of the cell boundary and interior wereobtained. The ratios of these two values were compared before andafter irradiation.

Verification of AtPIP5K4 knockout plants and

generation of complemented lines of pip5k4

A T-DNA insertion line of PIP5K4 was obtained from the SalkInstitute Genomic Analysis Laboratory (http://signal.salk.edu/



cgi-bin/tdnaexpress, stock number: SALK_001138). Homozygotepip5k4 plants were selected by PCR using the genomic DNA as thetemplate using the T-DNA specific primer 5¢-GCGTGGACCGCTT-GCTGCAACT-3¢ (LBb1 primer, see http://signal.salk.edu/tdna_FAQs.html) and the PIP5K4 specific primers 5¢-GACGGGAGTCCTGAAT-GGGAT-3¢ and 5¢-GCAGCTACATATTTTTCATCTTGTC-3¢. To confirmthat the pip5k4 mutant does not generate a PIP5K4 transcript,RT-PCR was performed. Total RNA was extracted from whole leavesand RT-PCR was performed using primers 5¢-GAAATGATGAGA-CTAGAGGCTGAAGGGTTC-3¢ and 5¢-GAGACTTGTTTCAATTATC-CTCAGTGAAGAC-3¢.

To generate complemented lines of pip5k4, pip5K4 plants weretransformed with a pCAMBIA1302 (BIOS) vector containing a 1.3 kbfragment upstream of the 5¢ of the PIP5K4 coding sequence fused tothe PIP5 K4 cDNA. The cording sequence of PIP5K4 was amplifiedfrom cDNA generated from total RNA by PCR using primerscontaining SphI and PmlI restriction sites (5¢-GCATGCATCAG-CAAGGAAACAAAGCTGTGTTC-3¢ and 5¢-CACGTGTCAATTATCCT-CAGTGAAGACCTTG-3¢) and the 1.3 kb promoter region wasamplified from genomic DNA using a forward primer containingXmaI (5¢-CCCGGGTTTTCGATTCCAACGATGAGAACCAA-3¢) and areverse primer containing SphI (5¢-GCATGCCTTCTTAAACTAAT-AAAACTTTTCTCTAAGATACC-3¢).

PIP kinase activity assay

Escherichia coli strain Rosetta (DE3) was used for the expression ofrecombinant PIP5K4 proteins fused to GST. An overnight culture ofRosetta expressing GST-PIP5K4 was diluted 1:100 with fresh culturemedium and grown at 37�C with shaking at 200 rpm until an OD600

of 0.6–0.8 was reached, at which point isopropyl-D-thiogalactoside(IPTG) was added to a final concentration of 30 lM. Cells wereincubated for an additional 12 h at 16�C and collected by centrifu-gation and kept frozen at )70�C until required for the purification ofthe GST-PIP5K4 protein.

The kinase activity of GST-PIP5K4 was measured as described byLee et al. (1996). Phosphorylation of PtdInsP was undertaken atroom temperature for 10 min in a 50 ll mixture containing 50 mM

HEPES-KOH (pH 7.4), 3 mM MgCl2, 10 mM 2-glycerophosphate,2 mM dithiothreitol, 240 mM NaCl, 10 lg PI or PtdIns(4)P (Sigma-Aldrich) and 10 lCi of c32P-ATP (Amersham-Pharmacia Biotech,http://www5.amershambiosciences.com/) and the purified GST-PIP5K4 fusion protein. For control experiments, purified GST wasused in place of the fusion protein. For lipid extraction, 300 ll ofchloroform:methanol:0.7 M HCL (8:4:3, v/v) was added to thesample and vortexed for 20 sec. Phase separation was facilitatedby centrifugation at 1600 g for 1 min in a tabletop centrifuge. Theupper phase was removed and the lower chloroform phase waswashed once more with a fresh upper phase. Divalent cations,which bind to PtdInsP and retard its mobility during TLC, wereremoved by vigorous mixing with 150 ll of chloroform and 150 ll ofan aqueous solution containing 2 M KC1 and 2 mM EDTA. Theaqueous phase was discarded, and the solvent was evaporatedunder a stream of N2 and dissolved in 30 ll of chloroform. Lipidswere spotted onto silica gel 60 thin layer chromatography (TLC)plates (Merck, http://www.merck.com/) impregnated with 1%potassium oxalate and 2 mM EGTA, and separated usingchloroform:methanol:4 N ammoniumhydroxide (90:70:20, v/v).Plates were autoradiographed using a phosphorimager, FLA-2000R(Fujifilm, http://www.fujifilm.com/). To assist identification ofthe PtdIns(4,5)P2 band, cold phospholipid standards were runin parallel lanes on the same TLC plate and visualized by exposingthe plate to iodine vapor in a sealed tank.

RT-PCR analyses of guard cell and mesophyll

cell protoplasts

We used the same total RNA preparations from highly pure guardcell and mesophyll cell protoplasts as those reported previously(Mori et al., 2006). Equal amounts of each cDNA were used for a25 ll PCR reaction containing 400 nM primers, 1· reaction buffer,400 lM each nucleotide (dNTP), 2.5 U Ex Taq polymerase (Takara,http://www.takara-bio.com/) and 1 ll each cDNA. The PCR mixtureswere denatured at 94�C for 2.5 min, followed by 37 (PIP5K4) or 35(UBQ, ubiquitin-conjugating enzyme E2, At5 g25760) cycles ofamplification (94�C, 30 sec; 54�C for PIP5 K4 or 60�C for UBQ, for30 sec; 72�C for 1 min 10 sec). Each PCR reaction was repeatedtwice. We used the following primers for the PCR reactions: PIPK-F,5¢-TAAAGTGCTTCTGAGGATGCTTGCAGC-3¢; PIPK-R, 5¢-GAAATC-ACGGAAACGTCTCGAGTACAG-3¢; UBQ-F, 5¢-TAGAGATGCAG-GCATCAAGAGCGCGACT-3¢; and UBQ-R, 5¢-GCGGCGGAGGCGTGTATACATTTGTGCCA-3¢.

Acknowledgements

We thank Dr Nava Moran for a critical reading of the manuscript.This work was supported by grants awarded to YL from the CropFunctional Genomics Center of Korea (grant no. CG1-1-23),POSTECH Core Research Program (grant no. 2.0005412.01) andGlobal Research Program of the Ministry of Science and Technol-ogy (grant no. 40001795.01) to IH from Biogreen 21 (Korea)program, to EM from Swiss National Foundation, and to JMK fromNSF (grant no. MCB-0614203).

Supplementary Materials

The following supplementary material is available for this articleonline:Figure S1. Individual values for GFP:PLCd1PH fluorescence intensityat the plasma membrane and in the cytosol of guard cells treatedwith phosphoinositides or drugs. (a,b) GFP:PLCd1PH fluorescenceintensity at the plasma membrane and cytosol 60 min after appli-cation of (a) PtdIns(4,5)P2 or (b) PtdIns(4)P. The cells shown here arethe same as those shown in Figure 2d. (c,d) GFP:PLCd1PH fluores-cence intensity at the plasma membrane and cytosol 90 min afterapplication of (c) U-73122 or (d) U-73343. The cells shown here arethe same as those shown in Figure 2f.Figure S2. Optically sectioned fluorescence images of guard cellsexpressing (a) GFP alone or (b) GFP:PIP5K4. Pictures were takenevery 1 lm from the bottom of the cell to the top. Bars = 10 lm.Note that GFP:PIP5K4 is mostly at the plasma membranethroughout the entire cell surface, whereas free GFP is localizedin cytoplasm.This material is available as part of the online article from http://www.blackwell-synergy.com

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phosphatidylinositol 4,5-bisphosphate is important for

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