Botanical Studies (2008) 49: 323-334.

*

Corresponding author: E-mail: shexiaoping530@163.com

or shexiaoping@snnu.edu.cn; Tel: +86-0-29-85310265;

Fax: +86-0-29-85303736.

INTRODUCTION

Stomata are the main routes for leaf gas exchange,

controlling CO

2

uptake and transpiration. Stomatal move-

ments are regulated by both internal and external factors.

The opening of stomata is stimulated by low CO

2

concen-

trations, a range of natural and synthetic cytokinins, and

blue light and other photosynthetically active wavelengths.

Stomatal closure occurs in response to environmental cues

like low air humidity and high temperature (Jewer and

Incoll, 1980; Assmann, 1993; Willmer and Fricker, 1996;

Liang et al., 2002; Hung et al., 2005). Light and dark are

the most important environmental factors affecting stoma-

tal movement (Zeiger, 1983; Cousson et al., 1995), which

is also regulated by the redox active molecule hydrogen

peroxide (H

2

O

2

) (Neill et al., 2002; Laloi et al., 2004).

Previous evidence showed that H

2

O

2

functions as an en-

dogenous signalling molecule mediating responses to vari-

ous stresses and stimuli (Finkel, 2000; Neill et al., 2002).

There is now compelling evidence that H

2

O

2

is involved in

abscisic acid (ABA)-induced stomatal closure (Pei et al.,

2000; Zhang et al., 2001a, b; Meihard et al., 2002). Recent

research provides exciting evidence that H

2

O

2

is a key sig-

naling molecule mediating dark-induced stomatal closure

(Desikan et al., 2004; She et al., 2004).

A growing body of evidence has shown that numerous

protein kinases with close sequence similarity to mam-

malian mitogen-activated protein kinases (MAPKs) have

been identified in plants (Hirt, 1997; Mizoguchi et al.,

1997; Zhang and Klessig, 2001; Ichimura et al., 2002).

Increasing evidence has shown that MAPKs play an im-

portant role in plant signal transduction related to biotic

and abiotic stresses. Activation of MAPKs has been ob-

served in plants exposed to pathogens (He et al., 1999),

cold (Jonak et al., 1996), salinity (Mikolajczyk et al.,

2000), drought (Jonak et al., 1996), and wounding (Usami

MAPK kinase and CDP kinase modulate hydrogen

peroxide levels during dark-induced stomatal closure in

guard cells of Vicia faba

Xi-Gui SONG

1,2

, Xiao-Ping SHE

1,

*, Lin-Ying GUO

1

, Zhao-Ni MENG

1

, and Ai-Xia HUANG

1

1

School of Life Sciences, Shaanxi Normal University, Xi¡¦an 710062, People¡¦s Republic of China

2

Middle School Attached to Shaanxi Normal University, Xi¡¦an 710062, People¡¦s Republic of China

(Received January 26, 2007; Accepted April 10, 2008)

ABSTRACT.

We used 2¡¦-amino-3¡¦-methoxyflavone (PD98059) (an inhibitor of mitogen-activated protein

kinase kinase, MEK) and Trifluoperazine (TFP) (a specific inhibitor of calcium-dependent protein kinase,

CDPK) to investigate the role of MEK/CDPK and its effects on H

2

O

2

levels of guard cells in the dark-induced

stomatal closure in Vicia faba. We provide evidence that both PD98059 and TFP reduced H

2

O

2

levels in guard

cells and promoted stomatal opening significantly in the dark, implying that MEK/CDPK mediated dark-

induced stomatal closure by influencing H

2

O

2

levels of guard cells. In addition, like ascorbic acid (ASA), an

important reducing substrate for H

2

O

2

removal, but unlike diphenylene iodonium (DPI), an inhibitor of the

H

2

O

2

-generating enzyme NADPH oxidase, PD98059 and TFP not only reduced exogenous H

2

O

2

levels in

guard cells in light, but also eliminated the H

2

O

2

that had been generated during a dark period and promoted

stomatal opening. The results suggest MEK and CDPK are probably involved in restraining the H

2

O

2

scavenging enzyme and elevating H

2

O

2

levels in guard cells during dark-induced stomatal closure. Of course,

the probability of MEK and CDPK acting as the target downstream of H

2

O

2

in the signaling transduction

chain is not excluded.

Keywords: Dark; Hydrogen peroxide; MAPK kinase and CDP kinase; Stomatal closure; Vicia faba.

Abbreviation: ASA, ascorbic acid; CDPK, calcium-dependent protein kinase; DCF, dichlorofluorescein;

DMSO, dimethyl sulfoxide; DPI, diphenylene iodonium; H

2

DCF-DA, 2, 7-dichlorofluorescein diacetate;

MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; H

2

O

2

, hydrogen peroxide; PD98059, 2¡¦

-amino-3¡¦-methoxyflavone; ROS, reactive oxygen species; TFP, Trifluoperazine.

PhySIOlOgy

324

Botanical Studies, Vol. 49, 2008

et al., 1995; He et al., 1999). Plant MAPKs also can be ac-

tivated by ABA (Knetsch et al., 1996; Burnett et al., 2000;

Heimovaara-Dijkstra et al., 2000). Most physiological re-

sponses to activation of protein kinases in stimulus factors

like light, hormones, stress, and pathogen attack regulated

stomatal aperture (Sopory and Munshi, 1998). Some evi-

dence suggests that the guard cell-specific protein kinase,

ABA-activated protein kinase (AAPK), is essential for

ABA-induced stomatal closing (Li et al., 2000). Burnett et

al. (2000) reported that ABA activation of additional types

of protein kinases had been found in pea epidermal peels.

Jiang et al. (2003) reported that MEK is an important

regulator of stomatal movement, which is believed to me-

diate the H

2

O

2

generation induced by ABA in guard cells

of Vicia faba. In addition, plant cells contain a group of

kinases, designated as calcium-dependent protein kinases

(CDPKs), which are dependent only on calcium and do not

require CaM for activation (Harmon et al., 1987). Wang

and Wu (1999) reported that CDPKs might be involved in

the ABA-mediated signal transduction cascades of stoma-

tal movement.

Recently, elegant work from Gomi et al. (2005)

showed that the involvement of a specific MAPK has

been suggested in jasmonic acid signaling and MAPK-

silenced plants showed misregulation of stomatal aperture.

In addition, a potential crosstalk between a CDPK and a

MAPK signaling pathway mediated by ethylene during

stress responses in guard cells has also been reported

(Ludwig et al., 2005), as have the effects of CDPKs CPK6

and CPK3 on stomatal aperture and ion channel activity

in guard cells (Mori et al., 2006). Up to now, the involve-

ment of MAPKs and CDPKs in darkness-mediated sto-

matal closure has not been addressed, and the effect of

MEK and CDPK on H

2

O

2

levels in the process needs to be

demonstrated. In the present study, we seek for evidence

by means of stomatal bioassay and laser-scanning confocal

microscopy that MEK and CDPK mediate dark-induced

stomatal closure based on 2¡¦, 7¡¦,-dichlorodihydrofluo-

rescein diacetate (H

2

DCF-DA). The effect of MEK and

CDPK on H

2

O

2

levels in dark-induced stomatal closure in

Vicia faba is also studied.

MATERIAlS AND METhODS

Chemicals

Molecular probes 2¡¦, 7¡¦-dichlorodihydrofluorescein di-

acetate (H

2

DCF-DA, from Biotium, Hayward, California)

was dissolved in dimethyl sulfoxide (DMSO) to produce

a 10 mM stock solution. Diphenylene iodonium (DPI),

Trifluoperazine (TFP), DMSO and 2-(N-morpholino)

ethanesulfonic acid (MES) were obtained from Sigma-

Aldrich (St. Louis, MO). 2¡¦-amino-3¡¦-methoxyflavone

(PD98059) were purchased from Calbiochem (an affiliate

of Merck KGaA, Darmstadt, Germany). Unless stated

otherwise, the remaining chemicals were of the highest

analytical grade available and were sourced from various

Chinese suppliers.

H

2

DCF-DA, PD98059 and DPI were

dissolved in

DMSO. The final concentration of the

solvent was 0.5%

(v/v), which did not induce any significant change

in guard

cell viability or stomatal aperture.

Plant materials

Broad bean (Vicia faba L.) was grown in controlled-

environment plant growth chamber with a humidity of

80%, a photo flux density of 300 £gmolm

-2

s

-1

PAR gener-

ated by cool white fluorescent tubes (Philips, New York,

NY), and an ambient temperature 25¡Ó2¢XC with a 14-h light

and 10-h dark cycle. The epidermis was peeled carefully

from the abaxial surface of the youngest, fully expanded

leaves of 4-week-old seedlings and cut into pieces about 5

mm wide and 5 mm long.

Stomatal bioassay

Stomatal opening and closing were monitored using the

method of McAinsh et al. (1996) with slight modifications.

To study the role of MEK and CDPK in dark-induced sto-

matal movement, freshly prepared abaxial epidermis was

first incubated in CO

2

-free MES/KCl (10 mM MES/KOH,

50 mM KCl, 100 £gM CaCl

2

, pH 6.15) buffer, which in-

cluded various treating reagents (PD98059, TFP, ASA or

DPI), in the dark for 3 h at 25¢XC. Final stomatal apertures

were recorded with a light microscope and an eyepiece

graticule previously calibrated with a stage micrometer.

To study the effects of PD98059 and TFP on stomatal

closure caused by exogenous H

2

O

2

, epidermal strips were

incubated in MES/KCl buffer with H

2

O

2

alone, or in buf-

fer containing PD98059, TFP, or other compounds for 3

h under light (300 £gmol m

-2

s

-1

) conditions at 25¢XC, and

then the apertures were recorded. To study the effects of

MEK and CDPK on stomata that had closed in the dark,

strips were incubated in MES/KCl buffer for 3 h in dark

at 25¢XC, and then were treated with fresh buffer alone, or

buffer containing PD98059, TFP, or other compounds for

another 3 h in dark at 25¢XC. Final stomatal apertures were

recorded.

To avoid any potential rhythmic effects on stomatal

aperture, experiments were always started at the same

time each day. In each treatment, we scored 30 randomly

selected apertures per replicate, and treatments were

repeated thrice. The data presented are the means of 90

measurements¡Ós.e.

Dye loading of h2DCF-DA

H

2

O

2

measurement was performed as the method of

Allan and Fluhr (1997) with some modifications. To

study the effects of MEK and CDPK on H

2

O

2

levels in

guard cells caused by darkness and exogenous H

2

O

2

,

the epidermal strips were treated for 3 h as described

for stomatal bioassay and were immediately placed into

loading Tris-KCl buffer (Tris 10 mM and KCl 50 mM,

pH 7.2) containing 50 £gM of H

2

DCF-DA for 10 min in

darkness at 25¡Ó2¢XC. To study the effects of MEK and

CDPK on H

2

O

2

levels generated in guard cells held in the

SONG et al. ¡X MEK and CDPK in dark responses

325

dark, strips were incubated in MES/KCl buffer for 3 h in

darkness at 25¢XC, and then in fresh MES/KCl containing

PD98059, TFP, or other reagents for another 3 h. After

these steps, H

2

DCF-DA was loaded in Tris-KCl buffer.

laser-scanning confocal microscopy

After excess dye was washed off with fresh Tris-KCl

loading buffer in darkness, the epidermal strips were im-

mediately examinated by TCS SP5 laser-scanning confo-

cal microscopy (Leica Lasertechnik Gmbh, Heidelberg,

Germany) with the following settings: excitation 488 nm,

emission 530 nm, power 10%, PMT 959, zoom about 4,

normal scanning speed, frame 512¡Ñ512 pixel. Images

acquired from the confocal microscope were analyzed

using Leica image software, Time-Course, and Photoshop.

In the time-course plot experiments for the changes

in DCF fluorescence intensity, epidermal strips were

incubated in MES/KCl buffer for 3 h in darkness at 25

¢XC, and then H

2

DCF-DA was loaded for 10 min. After

these steps, PD98059, TFP, or other reagents were added

directly to Tris-KCl buffer. The change in intensity of

dichlorofluoroscein (DCF) fluorescence was recorded at

about 800 s, and guard cell images were taken at 0, 100,

300, 500, 700 s.

To enable the comparison of changes in signal intensity,

confocal images were taken under identical conditions (in

manual setup) for all samples, and in each treatment we

measured three epidermal strips, and the treatment was

repeated at least thrice. The selected confocal images rep-

resented the same results from three replications.

Monitor of the fluorescence spectrum of h

2

DCF-

DA in vitro

The fluorescence spectrum of H

2

DCF-DA was mea-

sured on a Hitachi F-2500 fluorescence spectrophotometer

(Ltd., Tokyo, Japan), using Sarstedt REF67.754 cuvettes.

Apparatus settings: excitation at 488 nm, emission at 530

nm, PMT voltage 950 V. To study the effects of MEK and

CDPK inhibitors on the fluorescence spectrum of H

2

DCF-

D A in vitro at 25¡Ó2¢XC, the treatments were as follows:

Tris-KCl buffer including 50 £gM of H

2

DCF-DA only,

H

2

DCF-DA + 10 £gM H

2

O

2

, H

2

DCF-DA + H

2

O

2

+ 10 £gM

PD98059, H

2

DCF-DA + H

2

O

2

+ 10 £gM TFP, H

2

DCF-DA

+ H

2

O

2

+ 100 £gM ASA, and H

2

DCF-DA + H

2

O

2

+ 10 £gM

DPI. The fluorescence intensity of the above treatments

was determined respectively. Each treatment was per-

formed at least thrice. The data from three replications

were the same.

RESUlTS

Effects of PD98059 and TFP on dark-induced

stomatal closure

PD98059 is a potent and selective cell permeable in-

hibitor of MAPK kinase (MEK) and thus an invaluable aid

in elucidating the role of MEK in a variety of biological

systems (Alessi et al., 1995). Previous studies suggested

that PD98059 abolished the ABA-inducted stomatal clo-

sure in Pisum stativum, implying that ABA effects in pea

epidermal peels require MAPK activation (Burnett et al.,

2000), and MEK specifically mediates the ABA-induced

H

2

O

2

generation in guard cells in Vicia faba (Jiang et al.,

2003). Additionally, Wang and Wu (1999) reported that

CDPK might also be involved in ABA-mediated signal

transduction cascades in regulation of stomatal movement.

Previous research demonstrates that trifluoperazine (TFP)

inhibits plant CDPKs (Polya and Micucci, 1985; Zhou and

Zhang, 2004). Recent studies have shown that H

2

O

2

is an

essential signaling molecule involving in dark-induced

stomatal closure (Desikan et al., 2004; She et al., 2004).

Therefore, we expected that PD98059 and TFP could af-

fect dark-induced H

2

O

2

levels and stomatal closure in Vi -

cia faba.

After examining the effects of PD98059 or TFP

on dark-induced stomatal closure, we found that both

PD98059 and TFP at a concentration of 10 £gM significant-

ly reversed darkness-induced stomatal closure (P<0.01)

(Figure 1A and B). So we suppose that darkness induces

stomatal closure via a pathway involving MEK and

CDPK.

Dark-induced stomatal closure is related to endogenous

H

2

O

2

(Desikan et al., 2004; She et al., 2004). To investigate

the relation between the mediating of MEK/CDPK in

dark-induced stomatal closure and the change of H

2

O

2

levels in guard cells, epidermal strips were treated with

ASA and DPI. The former is the most important reducing

substrate for H

2

O

2

removal (Noctor and Foyer, 1998), and

the latter is an inhibitor of the H

2

O

2

-generating enzyme,

NADPH oxidase (Lee et al., 1999). The results show that

both ASA and DPI induced stomatal opening in darkness

in a dose-dependent manaer (Figure 1C, D). The effects of

ASA and DPI on stomatal aperture were signicant (P<0.01)

at 100 and 10 £gM, respectively. These results suggest that,

probably like ASA and DPI, MEK/CDPK modulates H

2

O

2

levels during dark-induced stomatal closure in guard cells.

Both PD98059 and TFP affect the dark-induced

h

2

O

2

levels of guard cells

Having established that both MEK and CDPK mediate

dark-induced stomatal closure (Figure 1A, B), we used

H

2

DCF-DA, a specific probe for intracellular H

2

O

2

(Al-

lan and Fluhr, 1997), to measure H

2

O

2

levels directly in

guard cells. Upon entering the cell, the nonpolar H

2

DCF-

DA is hydrolyzed to the oxidatively sensitive, more polar,

nonfluorescent compound fluorophore dichlorofluorescein

(H

2

DCF). H

2

DCF is rapidly oxidized to the highly fluores-

cent DCF by intracellular H

2

O

2

(Allan and Fluhr, 1997).

H

2

DCF-DA loads readily into guard cells, and its optical

properties make it amenable to analysis using laser-scan-

ning confocal microscopy.

As shown in Figure 2B, darkness could induce an

intense DCF fluorescence in guard cells over the light

treatment (Figure 2A), which is consistent with previous

326

Botanical Studies, Vol. 49, 2008

Figure 1. Effects of protein kinase inhibitors on dark-induced

stomatal closure. Isolated epidermal strips were incubated at 25

¢XC in CO

2

-free MES[2-(N-morpholino) ethanesulfonic acid]/KCl

containing different concentrations of (A) PD98059 (0, 5, 10,

50 £gM), (B) TFP (0, 1, 10, 20 £gM), (C) ASA (0, 10, 100, 1000,

10000 £gM) and (D) DPI (0, 0.1, 1, 10, 100 £gM) for 3 h in dark-

ness. Stomatal apertures were determined. Values are the means

of 90 measurements¡Ós .e. forming three independent experi-

ments.

Figure 2. Effects of protein kinase inhibitors on the dark-induced H

2

O

2

levels of guard cells. Guard cells of V. faba: (A) treated with

MES/KCl only in light for 3 h; (B) in darkness alone for 3 h; (C) in darkness with 10 £gM PD98059; (D) in darkness with 10 £gM

TFP; (E) in darkness with 100 £gM ASA; (F) in darkness with 10 £gM DPI, for 3 h. Above treated strips were immediately loaded with

H

2

DCF-DA in Tris¡VKCl buffer for 10 min in darkness. Then excess dye was removed, and strips were examined using laser-scanning

confocal microscopy; (G) shows the average fluorescent intensity of guard cells in images from (A) to (F). Data are the means¡Ós.e.

Guard cells shown in image (a`) to (f`) represent guard cells shown in images (A) to (F). The insets show the bright-field images

corresponding to the fluorescence images (a`) through (f`). The length of scale bar in image (F) and (f`) represents 40 £gm and 16 £gm

for image (A) to (F) and (a`) to (f`), respectively. The bar in inset of image (f`) represents 8 £gm for all the insets. Each experiment was

performed at least thrice, and the selected confocal image represented the same results from about nine time measurements.

reports (Desikan et al., 2004; She et al., 2004). However,

darkness-induced DCF fluorescence in guard cells was

largely prevented by PD98059 and TFP (Figure 2C, D).

Similarly, ASA and DPI also substantially suppressed

dark-induced DCF fluorescence (Figure 2E, F). Taking

these results from Figures 1 and 2 together, we suggest

that MEK and CDPK may be the upstream signal mol-

ecule mediating H

2

O

2

levels in the dark-induced stomatal

closure of Vicia faba, and they can elevate the levels of en-

dogenous H

2

O

2

and promote stomatal closure in darkness.

Effects of PD98059/TFP on stomatal closure

and DCF fluorescence in guard cells induced by

exogenous h

2

O

2

Having established that MEK and CDPK mediate dark-

induced stomatal closure and modulate the H

2

O

2

levels of

guard cells in Vicia faba, we wanted further insight into

how MEK and CDPK affect H

2

O

2

levels in guard cells.

Epidermal strips were incubated in MES/KCl with H

2

O

2

alone or H

2

O

2

with PD98059, TFP and other compounds

for 3 h in light. As shown in Figure 3, exogenous ap-

plication of H

2

O

2

promoted stomatal closure in light,

which is consistent with the previous data (Zhang et al.,

2001b; Desikan et al., 2004; She et al., 2004). PD98059,

SONG et al. ¡X MEK and CDPK in dark responses

327

TFP, ASA or DPI did not cause any changes in stomatal

aperture in light. However, similar to ASA (an important

reducing substrate for H

2

O

2

removal), but not to DPI

(an inhibitor of the H

2

O

2

-generating enzyme, NADPH

oxidase), PD98059 and TFP prevented the stomatal

closure induced by exogenous H

2

O

2

in light. The effects

were significant (P<0.01; Figure 3).

To further clarify whether or not MEK/CDPK can

affect exogenous H

2

O

2

-induced DCF fluorescence, the

epidermal strips were treated with H

2

O

2

in the presence of

PD98059, TFP, ASA or DPI for 3 h in light, and then H

2

O

2

levels were measured. As shown in Figure 4, a striking

DCF fluorescence in guard cells was observed after

treatment with 10 £gM exogenous H

2

O

2

in light (Figure 4B)

compared with the control (Figure 4A). However, H

2

O

2

-

induced DCF fluorescence in guard cells was abolished

by PD98059, TFP (Figure 4C, D) and ASA (an important

reducing substrate for H

2

O

2

removal) (Figure 4E), but

not by DPI (an inhibitor of the H

2

O

2

-generating enzyme,

NADPH oxidase) (Figure 4F). From these results we know

that, like ASA, both PD98059 and TFP not only prevented

stomatal closure by exogenous H

2

O

2

, but also reduced ex-

ogenous H

2

O

2

levels in guard cells in light.

The closed stomata caused by dark can be

reopened by PD98059 and TFP

To confirm the effects of MEK and CDPK on H

2

O

2

guard cell levels in response to darkness, epidermal strips

were incubated in MES/KCl for 3 h in darkness and then

Figure 3. Stomatal closure induced by exogenous H

2

O

2

can be

prevented by PD98059 and TFP. Isolated epidermal strips of V.

faba were incubated at 25¢XC in CO

2

-free MES/KCl alone, or

MES/KCl containing 10 £gM H

2

O

2

, 10 £gM PD98059, 10 £gM

TFP, 100 £gM ASA, 10 £gM DPI, 10 £gM H

2

O

2

+10 £gM PD98059

(H+P), 10 £gM H

2

O

2

+10 £gM TFP (H+T), 10 £gM H

2

O

2

+100 £gM

ASA (H+A), 10 £gM H

2

O

2

+10 £gM DPI (H+D) for 3 h under

light (300 £gmol m

-2

s

-1

). Stomatal apertures were determined

after 3 h incubation. Values are the means of 90 measurements¡Ó

s.e of three independent experiments.

Figure 4. Exogenous H

2

O

2

-induced DCF fluorescence in guard cells is reduced by protein kinase inhibitors. Guard cells shown in

image (A) were treated in light for 3 h with buffer only, and those in image (B) were treated with 10 £gM H

2

O

2

; (C) 10 £gM H

2

O

2

+10

£gM PD98059; (D) 10 £gM H

2

O

2

+10 £gM TFP; (E) 10 £gM H

2

O

2

+100 £gM ASA; (F) 10 £gM H

2

O

2

+ 10 £gM DPI, in light for 3 h. Above

treated strips were loaded with H

2

DCF-DA for 10 min in darkness. Then excess dye was removed, and the strips were examined by

laser-scanning confocal microscopy. (G) shows the average fluorescent intensity of guard cells in images from (A) to (F). Data are the

means¡Ós.e. Other explanations are the same as in Figure 2.

328

Botanical Studies, Vol. 49, 2008

ferent cells (Cohen, 1997; Hirt, 1997). MAPKs are them-

selves activated via dual phosphorylation on threonine

and tyrosine residues by MAPK kinases (MEKs), which

in turn are activated via phosphorylation by MAPKK ki-

nases (MAPKKKs). MAPK-based signalling cascades are

ubiquitous components of all eukaryotic cells (Hirt, 1997;

Mizoguchi et al., 1997). A large number of these enzymes

have already been identified in plants, and it seems very

likely that they transduce responses to many external sig-

nals and plant hormones (Hirt, 1997; Mizoguchi et al.,

1997). CDPKs are another class of serine/threonine protein

kinases unique to plants and some protists. A large fam-

ily of CDPKs has been identified recently in higher plants

(Roberts and Harmon, 1992). CDPKs are dependent only

on calcium and do not require CaM for activation (Har-

mon et al., 1987). Among the cells in epidermis CDPKs

are only expressed in the stomatal guard cells (Hong et al.,

1996). Previous research shows that both MAPKs and CD-

PKs might be involved in the ABA-mediated signal trans-

duction cascades that regulate stomata movement (Wang

and Wu, 1999; Burnett et al., 2000; Jiang et al., 2003). A

crosstalk between the MAPK and CDPK pathways was

mediated by ethylene and involved the generation of reac-

tive oxygen species in guard cells (Ludwig et al., 2005).

This is the first study, to our knowledge, to show the role

of MEK and CDPK in dark-induced stomatal closure in

Vicia faba.

Previous studies have suggested that PD98059 and TFP

are inhibitors of MEK and CDPK activity, respectively

(Polya and Micucci, 1985; Alessi et al., 1995; Burnett et

al., 2000; Jiang et al., 2003; Zhou and Zhang, 2004). A

report from Lu et al. (2002) showed that the inhibition of

MAPKK activity by PD98059 could interfere with the

ability of ABA to trigger postgermination growth arrest

treated with fresh buffer alone or buffer containing vari-

ous reagents for another 3 h in dark. As shown in Figure

5, like ASA, the most important reducing substrate for

H

2

O

2

removal, both PD98059 and TFP promoted the

reopening of stomata that had closed in the dark, but DPI

(an inhibitor of the H

2

O

2

-generating enzyme, NADPH

oxidase) did not (Figure 5). The results suggest that H

2

O

2

is necessary to maintaining stomatal closure in darkness,

once the stomata are closed, continued H

2

O

2

production

in dark is neither required (at least by a DPI-sensitive en-

zyme) nor significant.

PD98059 and TFP reduce levels of h

2

O

2

generated by darkness

The effects of PD98059 and TFP on the levels of H

2

O

2

generated in guard cells held in the dark were also mea-

sured. After an incubation of 3 h in darkness, epidermal

strips were loaded with H

2

DCF-DA, washed, and exam-

ined by laser-scanning confocal microscopy. During the

examination of DCF fluorescence, PD98059, TFP, ASA, or

DPI was added to the buffer. As shown in Figure 6A, the

fluorescence intensity of controls showed no change within

800 s. PD98059 and TFP reduced the DCF fluorescence

intensity compared with the control (Figure 6B and C),

as did ASA (Figure 6D). However, DPI did not reduce it

(Figure 6E).

Images of guard cells treated with PD98059, TFP,

ASA, and DPI at 0 and 3 h were also obtained. As shown

in Figure 7, the DCF fluorescence of guard cells treated

with PD98059, TFP, and ASA for 3 h in dark (Figure 7C,

D, E) was less than that of the control (Figure 7B) and 0

h treatment (Figure 7A). However, the DCF fluorescence

of guard cells treated with DPI for 3 h (Figure 7F) was

unchanged. The results suggest that both PD98059 and

TFP reduce endogenous H

2

O

2

levels that have been

generated by darkness in guard cells.

The effects of PD98059/TFP on the fluorescence

spectrum of h

2

DCF-DA in vitro

As shown in Figure 8, in vitro, the fluorescence

intensity of H

2

DCF-DA only was 103, but in the presence

of H

2

O

2

, the value increased. The inhibitors of MEK/

CDPK, PD98059, and TFP had no effect on the fluores-

cence intensity of H

2

DCF-DA, but ASA significantly

reduced it (P<0.01). In addition, the fluorescence intensity

of H

2

DCF-DA was also reduced by DPI to some extent.

DISCUSSION

Protein phosphorylation/dephosphorylation is now

known to play a very important role in response to exog-

enous factors like light, hormones, stress, and pathogen

attack in plants (Stone and Walker, 1995; Sopory and

Munshi, 1998; Schenk and Snaar-Jagalska, 1999). MAPKs

are serine/threonine protein kinases that phosphorylate a

range of substrates to activate various cellular responses,

including gene expression and membrane transport, in dif-

Figure 5. The clos ed s tomata during dark expos ure can be

reopened by protein kinase inhibitors. Strips of V. faba incubated

at 25¢XC in CO

2

-free MES/KCl for 3 h in darkness were treated

with fresh buffer alone or buffer containing 10 £gM PD98059, 10

£gM TFP, 100 ASA, or 10 £gM DPI for another 3 h in dark, and

then stomatal apertures were determined. Values are the means

of 90 measurements¡Ós.e. of three independent experiments.

SONG et al. ¡X MEK and CDPK in dark responses

329

Figure 6. Time-course plots of changes in intensity of DCF fluorescence. Epidermal strips of V. faba, incubated in CO

2

-free MES/KCl

for 3 h in the dark were loaded with H

2

DCF-DA for 10 min in the dark, washed, and examined by laser-scanning confocal microscopy.

During image acquisition, Tris-KCl buffer only (A, control), 10 £gM PD98059 (B), 10 £gM TFP (C), 100 £gM ASA (D), and 10 £gM

DPI (E) were added directly to the buffer. (A-E) Time-course plots of changes in the DCF fluorescence intensity of guard cells; higher

intensity stands for higher H

2

O

2

concentration. Arrow in image (A) to (E) indicates the addition of reagents. Fluorescence images of

stomata were taken at 0, 100, 300, 500 and 700 s after the addition of reagents. The scale bar in the stoma of image (E) is 20 £gm for all

the images.

330

Botanical Studies, Vol. 49, 2008

tion in ABA-regulated stomatal movement in Arabidopsis

(Mustilli et al., 2002), Jiang et al. (2003) reported MAPK

could specifically regulate and amplify the ABA-induced

H

2

O

2

generation in guard cells of Vicia faba. Using laser-

scanning confocal microscopy, we found that like ASA,

an important reducing substrate for H

2

O

2

removal, and

DPI, an inhibitor of the H

2

O

2

-generating enzyme NADPH

oxidase (Figure 2E, F), both PD98059 and TFP prevented

darkness-induced H

2

O

2

levels (Figure 2C, D), suggesting

that MEK and CDPK may be an upstream signal mol-

ecule regulating H

2

O

2

levels in darkness-induced stomatal

closure in Vicia faba.

Given that MEK and CDPK mediated in the dark-

induced change of H

2

O

2

levels in Vicia faba guard cells,

we wanted to further explore how MEK and CDPK

affected the levels of H

2

O

2

in dark-induced stomatal

closure. We found that, like ASA, but unlike DPI (Figure

4E, F; Figure 7E, F), both PD98059 and TFP not only

eliminated the H

2

O

2

-induced DCF fluorescence of

guard cells in light (Figure 4C, D), but also removed

by in-gel kinase assays. Desikan et al. (2001) provided

evidence that PD98059 could inhibit the activity of a

MAPK-like enzymew AtMPK4 in cell suspension cultures

of Arabidopsis var. Landsberg erecta. Jiang et al. (2003)

reported that PD98059 reversed ABA-induced stomatal

closure and H

2

O

2

generation. In addition, CDPK exhibits

a Ca

2+

-induced electrophoretic mobility shift, and its Ca

2+

-

dependent catalytic activity can be inhibited by TFP in

Vicia faba (Li et al., 1998) and in Arabidopsis thaliana

(Hong et al., 1996). Wang and Wu (1999) reported that

addition of TFP significantly reversed the inhibitory

effect of ABA on stomatal opening, suggesting CDPKs

are involved in ABA-regulated stomatal closure. The

results of the present study showed that PD98059 and

TFP stopped darkness-induced stomatal closure (Figure

1). From these results we presume that dark-induced sto-

matal closure occurs via a pathway involving MEK and

CDPK and that the two protein kinases are key signaling

components of this closure. Previous research shows that

OST1, one of the AAPK, acts upstream of H

2

O

2

produc-

Figure 7. Protein kinase inhibitors reduce the H

2

O

2

levels generated by darkness. Epidermal strips of V. faba, were incubated in CO

2

-

free MES/KCl buffer for 3 h in darkness, and then in fresh MES/KCl containing PD98059, TFP, or other reagents for another 3 h.

After this step, H

2

DCF-DA was loaded in Tris-KCl buffer for 10 min in darkness and washed. (In Figure 7A at 0 h in the dark, strips in

MES/KCl for 3 h in darkness, and then H

2

DCF-DA was loaded). At 0 h in dark (A) and 3 h, the strips were examined by laser-scanning

confocal microscopy. Image of guard cells held in dark for another 3 h in the presence of buffer only (B), and buffer and (C) 10 £gM

PD98059, (D) 10 £gM TFP, (E) 100 £gM ASA, (F) 10 £gM DPI. (G) shows the average fluorescence intensity of guard cells in images

from (A) to (F). Data are the means¡Ós.e. Other explanations are the same as in Figure 2.

SONG et al. ¡X MEK and CDPK in dark responses

331

H

2

O

2

generated by the dark (Figure 7C, D). The DCF

fluorescence intensity changes in the time-course plots

showed that the DCF fluorescence caused by darkness

was eliminated by PD98059/TFP and ASA (Figure 6B,

C, D), but not by DPI (Figure 6E). In vitro, as shown in

Figure 8, the fluorescence intensity of H

2

DCF-DA was not

reduced by the inhibitors of MEK/CDPK PD98059 and

TFP, however, the intensity could be reduced by ASA. In

addition, DPI also reduced the fluorescence intensity of

H

2

DCF-DA to some extent, but DPI had no effect on the

H

2

O

2

-induced DCF fluorescence of guard cells (Figure

4F), and the reason should be studied in the future. These

results were consistent with the observation that PD98059/

TFP reversed exogenous H

2

O

2

-induced stomatal closure

(Figure 3) and promoted the reopening of stomata that had

closed in the dark (Figure 5).

ASA is the major antioxidant that scavenges H

2

O

2

, and

the balance between H

2

O

2

production and the ASA redox

state establishes whether the H

2

O

2

concentration rises to a

level that can trigger stomatal closure. Dehydroascorbate

reductase (DHAR) catalyzes the reduction of dehydro-

ascorbate (oxidized ascorbate) to ASA and thus contrib-

utes to the regulation of the ASA redox state (Chen and

Gallie, 2004). Previous research provided evidence that

stomatal pores in many species open in the morning but

close in the afternoon to limit water loss (Assmann, 1993;

Assmann and Wang, 2001). The level of H

2

O

2

increases

during the afternoon while the Asc redox state in guard

cells decreases. Plants with an increased guard cell Asc re-

dox state were generated by increasing DHAR expression,

and these exhibited a reduction in the level of guard cell

H

2

O

2

(Chen and Gallie, 2004). Here, our data imply that,

in the darkness, MEK and CDPK are probably involved

in restraining the H

2

O

2

scavenging enzyme DHAR to el-

evate the H

2

O

2

levels of guard cells in the darkness. When

PD98059/TFP inhibits MEK/CDPK, DHAR expression

increases. H

2

O

2

scavenging is no longer restrained, and

H

2

O

2

levels decline. Of course, further experiments should

be performed to confirm the effects of PD98059/TFP on

antioxidant activities.

By stomatal bioassay both PD98059 and TFP not only

prevented stomatal closure by exogenous H

2

O

2

in light

(Figure 3), but also promoted reopening of stoma induced

to close in the dark (Figure 5), implying that MEK/CDPK

may be an upstream or downstream signal molecule

of H

2

O

2

in guard cells. Using confocal microscopy we

provide evidence that both PD98059 and TFP not only

abolished H

2

O

2

-induced DCF fluorescence of guard cells

in light (Figure 4 C, D), but also removed H

2

O

2

that had

been generated by darkness (Figure 6B, C; Figure 7C,

D). MEK and CDPK are suggested to be involved in

restraining the H

2

O

2

scavenging enzyme to elevate H

2

O

2

levels. Of course, the probability of MEK and CDPK

acting as the target downstream of H

2

O

2

in the signaling

transduction chain is not excluded. It is well known that

H

2

O

2

induces the activation of a MAPK in Arabidopsis

(Desikan et al., 1999; Grant et al., 2000; Desikan et al.,

2001). Mizoguchi et al. (1998) have shown that H

2

O

2

-

induced MAPK cascade induces specific stress-responsive

gene expression. H

2

O

2

also activates AtMPK6 (MEK-

like) in Arabidopsis leaf protoplasts (Kovtun et al., 2000).

Therefore, H

2

O

2

may have an autocatalytic function to

facilitate its own generation, e.g. once an H

2

O

2

-generation

system was triggered, a small amount of H

2

O

2

could

accelerate MEK or CDPK activation due to the formation

of an H

2

O

2

feedback-loop. This crosstalk of H

2

O

2

and

MEK or CDPK may lead to the formation of a self-ampli-

fication loop. Once the inhibition of H

2

O

2

-induced closure

by PD98059/TFP was achieved by reducing the activities

of MEK/CDPK, the signal transduction pathway in guard

cells was completely blocked.

In summary, the roles and functions of MAPK cascades

and CDPKs in plants have been widely reported, but the

whole picture is still fragmented. Furthermore, the role

of MAPK cascades and CDPKs in stomatal responses to

environmental stresses is not fully understood. Here, we

suggest that MEK/CDPK mediates dark-induced stoma-

tal closure by influencing H

2

O

2

levels of guard cells in

Vicia faba. Furthermore, our data show that MEK/CDPK

modulation of H

2

O

2

levels in guard cells is probably relat-

ed to its restraint of the H

2

O

2

scavenging enzyme system

during darkness-induced stomatal closure. These results

will no doubt help us to gain further insight into the roles

Figure 8. Monitoring of the fluorescence spectrum of H

2

DCF-

DA in vitro. The treatments, to seek for the effects of MEK and

CDPK inhibitors on the fluorescence spectrum of H

2

DCF-DA

in vitro, were as follows: Tris-KCl buffer including 50 £gM of

H

2

DCF-DA only (dye), H

2

DCF-DA + 10 £gM H

2

O

2

(dye + H

2

O

2

),

H

2

DCF-DA + H

2

O

2

+ 10 £gM PD98059

(dye + H

2

O

2

+PD), H

2

D-

CF-DA + H

2

O

2

+ 10 £gM TFP

(dye + H

2

O

2

+ TFP), H

2

DCF-DA

+ H

2

O

2

+ 100 £gM ASA (dye + H

2

O

2

+ ASA), and H

2

DCF-DA +

H

2

O

2

+ 10 £gM DPI (dye + H

2

O

2

+ DPI). The fluorescence spec-

trum of H

2

DCF-DA was measured on a Hitachi F-2500 fluores-

cence spectrophotometer (Hitachi Ltd., Tokyo, Japan). Apparatus

settings: excitation at 488 nm, emission at 530 nm, PMT voltage

950 V. The fluorescence intensity of the above treatments was

determined. Each treatment was performed at least thrice. The

data from three replications were the same.

332

Botanical Studies, Vol. 49, 2008

of MEK and CDPK and the relationship between MEK/

CDPK and the change to H

2

O

2

levels in guard cell signal

transduction. However, little is known about the com-

plex molecular network operating during the guard cell

stomatal movement triggered by darkness, and whether or

not MEK/CDPK acts as the target downstream of H

2

O

2

in

guard cells in the darkness in Vicia faba. These problems

should be further studied in future. Schematic representa-

tion of the relationship among darkness, MEK/CDPK,

and H

2

O

2

signalling in stomatal guard cells was shown as

follows by our results.

Acknowledgments. The work was financially supported

by the Natural Science Research Plan of Shaanxi Province

of People¡¦s Republic of China (2005C112).

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