Phosphatidylinositol 4,5-bisphosphate is important for ...
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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
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1.4
PLCδ1PH MT2a PLCδ1PH
Fluo
resc
ence
inte
nsity
at
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/ cy
toso
l InitialAfter 3 h
Light Darkness
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resc
ence
inte
nsity
at
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/ cy
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l
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
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
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
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120
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140
150
Prot
opla
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olum
e (%
of
initi
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Darkness
PI4P ControlControl
Light
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atal
ape
rtur
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m)
ControlU-73122U-73343
0.60.81.01.21.41.61.8
U-73122U-73343
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:PL
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/ cy
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atal
ape
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m) Control
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atal
ape
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m) Control
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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.
806 Yuree Lee et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
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
)
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–120
–80
–40
0
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a
c
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l (22
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0)
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2(28
)
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DAG (11)
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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).
Roles of PtdIns(4,5)P2 in stomatal opening 807
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
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
808 Yuree Lee et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
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.
Roles of PtdIns(4,5)P2 in stomatal opening 809
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
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.
810 Yuree Lee et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
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
Roles of PtdIns(4,5)P2 in stomatal opening 811
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 803–816
(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.
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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/
Roles of PtdIns(4,5)P2 in stomatal opening 813
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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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