Plant CellPhysiol. 34(8): 1321-1327 (1993)
JSPP © 1993

Properties of the Signal Transduction Pathway in the Blue Light Responseof Stomatal Guard Cells of Vicia faba and Commelina benghalensis
Ken-ichiro Shimazaki1, Kenji Omasa2, Toshinori Kinoshita3
and Mitsuo Nishimura3
1

Biological Laboratory, College of General Education, Kyushu University,
Ropponmatsu, Fukuoka, 810 Japan
2
Environmental Biology Division, The National Institute for Environmental Studies,
Onogawa, Tsukuba, 305 Japan3
Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka, 812 Japan

Blue light-dependent proton extrusion in guard cell protoplasts from Vicia faba and lightdependent stomatal opening in the epidermis of Commelina benghalensis are inhibited by thecalmodulin (CaM) antagonist, A/-(6-aminohexyl)-5-chloro-l-naphthalenesulfononamide (W-7)
and the myosin light chain kinase (MLCK) inhibitor, l-(5-iodonaphthalene-l-sulfonyl)-l//hexahydro-l,4-diazepine (ML-7) [Shimazaki, K., Kinoshita, T. and Nishimura, M. (1992) Plant
Physiol. 99: 1416]. We now suggest that the inhibition occurs in the blue light signaling pathway
without affecting the proton pump. Addition of fusicoccin (FC), an activator of H + -ATPase, tothe protoplasts and the epidermis whose blue light-dependent proton extrusion and light-dependent stomatal opening had been inhibited by W-7 and ML-7, induced both proton extrusion andstomatal opening, respectively. Blue light-dependent proton extrusion was inhibited by K-252a, awide-range inhibitor of protein kinases, and KT5926, a selective inhibitor of MLCK. FC induced
proton extrusion in the presence of K-252a and KT5926. In contrast, phenylmercuric acetate(PMA), carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and MJV-dicyclohexylcarbodiimide (DCCD) inhibited both the proton extrusion and stomatal opening, but FC did not induce
the responses. These results suggest that W-7, ML-7, K-252a and KT5926 inhibit the signaltransduction process by which the perception of blue light is transduced into activation of the proton pump in guard cells, and that MLCK or MLCK-like protein is involved in the blue light response of stomata. The possibility that calcium-dependent, calmodulin independent protein
kinase [Harper, J.F. et al. (1991) Science 252: 951] functions rather than MLCK in the blue light
response of stomata should be noted, however.
Key words: Blue light response — Fusicoccin — Guard cells — H + -ATPase — Proton pump —Signal transduction.

Stomata on leaves open in response to blue light, andthe opening is mediated by an accumulation of potassium
salt in guard cells that surround the stomatal pore (Ogawa
et al. 1978, Zeiger 1983, lino et al. 1985, Sharkey andRaschke 1981, Zeiger et al. 1987). Blue light acts as an actiAbbreviations: CaM, calmodulin; CCCP, carbonyl cyanidem-chlorophenylhydrazone; DCCD, TV.yV-dicyclohexylcarbodiimide; DMSO, dimethylsulfoxide; FC, fusicoccin; ML-7, l-(5iodonaphthalene-l-sulfonyl)-l//-hexahydro-l,4-diazepine; MLCK,
myosin light chain kinase; PMA, phenylmercuric acetate; W-7, N(6-aminohexyl)5-chloro-l-naphthalenesulfononamide.

vator of the proton pump in the plasma membrane ofguard cells, and induces proton extrusion, thereby creating
an inside-negative electrochemical proton gradient acrossthe plasma membrane (Assmann et al. 1985, Shimazaki etal. 1986). The hyperpolarized membrane potential induces
the opening of voltage-gated potassium channels in the plasma membrane of guard cells, and potassium ions are taken
up electrophoretically into the cells (Assmann et al. 1985,
Schroeder et al. 1987). However, the molecular mechanism
by which the perception of blue light is transduced into activation of the proton pump remains largely unknown.
It is postulated that blue light-dependent proton extru-

1321

1322

K. Shimazaki et al.

sion, a driving force for potassium uptake, is mediated by
H + -ATPase in the plasma membrane of guard cells. Its activity is lost when cytoplasmic ATP is removed from thecells (Assmann et al. 1985). Blue light-stimulated swellingof Vicia guard cell protoplasts, which corresponds tostomatal opening, and light-induced stomatal opening in
Commelina and Vicia are inhibited by vanadate, an inhibitor of plasma membrane-type H + -ATPase (Gepstein etal. 1982, Vara and Serrano 1982, Schwartz et al. 1991,
Amodeo et al. 1992). Furthermore, immunocytochemical
evidence indicates that H + -ATPase is most abundant in theplasma membrane of Zea mays guard cells (Villalba et al.
1991).
Recent investigations have shown that blue light-dependent proton extrusion in Vicia guard cell protoplasts
and light-dependent stomatal opening in the epidermis ofCommelina benghalensis are inhibited by CaM antagonists(e.g. W-7) and MLCK inhibitors (e.g. ML-7), which suggests that CaM and MLCK may be involved in the signaltransduction process of the stomatal blue light response(Shimazaki et al. 1992).
In the present investigation, we show that FC, an activator of H + -ATPase in the plasma membrane, induces proton extrusion and stomatal opening in the presence of W-7
or ML-7. Specific inhibition of the light-signaling pathway
by K-252a and KT5926, inhibitors of MLCK, is also
reported.

(Sylvania EFR 150W) by passing the light through a blue
glass filter (Corning 5-60). Red light was provided by passing the light from a halogen lamp (Sylvania 82V, 300W)
through a red glass filter (Corning 2-61). Light intensity
was 20/imol m~2 s" 1 for the blue light and 600 fimol m~2
s" 1 for the red light. The bathing solution (2.5 ml) of the epidermis contained 10 mM MES-KOH (pH 6.1), 30 mM KC1
and 0.1 mM CaCl2.
Protein determination—Protein was determined by
the method of Bradford (1976).
Chemicals—ML-7, W-7 were purchased from Seikagaku Kogyo (Tokyo). K-252a and KT5926 were from
Kyowa Medex (Tokyo).
Results

Effect ofFC on proton extrusion in the presence of W7 and ML-7—When guard cell protoplasts from Vicia faba
were illuminated with a pulse of blue light, superimposedon the background red light, sustained proton extrusion
was induced (Fig. la). The magnitude of proton extrusion
was 0.510 nmol H + per pulse per fig of protein, with a maximum rate of 3.63 nmol H + h~' per fig of protein around
1.5 min after the pulse. The proton extrusion ceased 12 to
16 min after the pulse. Fusicoccin (10 ^M), an activator of
H + -ATPase in the plasma membrane, elicited sustained
proton extrusion without blue light (Fig. la). Proton extrusion reached a maximum rate of 2.38 nmol H + h~' per fig
of protein within 15 min, and showed a constant value forMaterials and Methods
at least 20 min. Blue light-dependent proton extrusion was
Plant materials—Plants of Vicia faba L. (Ryosai inhibited strongly by W-7 (200//M) and ML-7 (100 ^M)+
Issun) and those of Commelina benghalensis ssp. were cul- (Fig. lb, c), with a magnitude of 0.026 and 0.035 nmol H
tured in a greenhouse, and guard cell protoplasts from per pulse per fig of protein, respectively. The maximum+
Vicia were isolated enzymatically as described previously rates of proton extrusion were 0.17 and 0.34 nmol H h~'
per fig of protein with W-7 and ML-7, respectively. How(Shimazaki et al. 1992).
Measurements of blue light-dependent proton ex- ever, after the application of blue light in the presence of intrusion—Blue light-dependent proton extrusion by Vicia hibitors, further addition of FC (lOftM) to the protoplast
guard cell protoplasts was measured with a glass pH elec- suspension induced the proton extrusion (Fig. lb, c). The
trode (Beckman 39532) using a dual-beam protocol as de- rates of FC-induced proton extrusion were 1.50 and 2.39+
1
scribed previously (Shimazaki et al. 1992). Light intensity nmol H h" per fig of protein with W-7 and ML-7, respec2
was 100/imolm" s~' for the blue light pulse (30 s) and tively.
600/miol m~ 2 s~' for background red light, respectively.
Effect of FC on stomatal opening in the presence of
The reaction mixture (1.0 ml) consisted of 0.125 mM MESW-7 and ML-7—Support for the above observations was
NaOH (pH 6.0), 1 mM CaCl 2 ,0.4 M mannitol, 10 mM KC1 obtained from the effect of inhibitors on stomatal reand guard cell protoplasts (20-60 fig of soluble protein).
sponses in Commelina benghalensis epidermis. Stomata
Measurement of light-induced stomatal opening in epi- opened in response to light, and their apertures ranged
dermis—The stomatal aperture of the epidermis in Com- from 8 to \2fim after illumination for 2 h (Fig. 2, 5). Lightmelina benghalensis was determined using a calibrated dependent stomatal opening was suppressed completely byocular micrometer as described previously (Shimazaki et W-7 and ML-7; however, the stomata opened after furtheral. 1992), but with a slight modification. Apertures of 80 addition of FC in the presence of the inhibitor. Stomatal
stomata were measured microscopically within 6 min. The apertures induced by FC were larger than those induced by
epidermis was peeled from the abaxial side of the leaf with light, both in the presence and absence of inhibitor (Fig. 2).
forceps according to the method of Weyers and Travis
Inhibition of blue light-dependent proton extrusion by(1981). Blue light was obtained from a halogen lamp protein kinase inhibitors—The effects of K-252a, a wide-Blue light response of stomatal guard cells

a)

BL

FC

b)

c)

1323

BL
FC

-exti

FC

\

10 nmol H+

\
\

A\

V\

x.

1
\

\

V

i

f5 min\\Fig. 1 Blue light-dependent and FC-induced H + extrusion in guard cell protoplasts from Viciafaba L.
Blue light at 100//mol m 22s~' was given for 30 s, superimposed on the background red light at 600 /anol m~ s~'. Final concentration of FC was 10 fiM. (a) FC was
added to the protoplast suspension immediately after the blue light-dependent proton extrusion had ceased. Maximum rate of blue lightdependent proton extrusion was 3.68 nmol H + h" 1 per//g of protein, whereas the maximum rate of FC-induced H + extrusion was 2.38
nmol H + h~' per n% of protein, (b) ML-7 was added to the protoplast suspension at 100/iM, 15 min before the illumination, (c) W-7
was added to the protoplast suspension at 200 fiM, 5 min before the illumination with blue light.

15

b)

a)

I
10

range inhibitor of protein kinases isolated from Nocardiopsis sp., and of KT5926, a selective inhibitor of MLCK
which is a derivative of K-252a (Kase et al. 1987, Nakanishi
et al. 1988), on blue light-dependent proton extrusion were
examined. K-252a inhibited proton extrusion, and the inhibition was concentration-dependent (Table 1). It inhibitedblue light-dependent proton extrusion by about 75% at 1
^M, with values of 0.449 and 0.116 nmol H + per pulse per
fig protein in the control and in the presence of K-252a,
respectively (Table 1). KT5926 inhibited the blue light-dependent proton extrusion at the same range of concentration as that for K-252a (Table 1). It inhibited proton extru-

Q.

4

Table 1 Effects of K-252a and KT5926 on blue light-dependent proton extrusion in Vicia guard cell protoplasts

(0

S 5

Inhibitor
K-252a
o
z

u o 0
o

(MM)

0
1
10

1 it

Fig. 2 Induction of stomatal opening in the Commelina epidermis by FC in the presence of W-7 or ML-7.
W-7 and ML-7
were added to the bathing solution (2.5 ml) 5 min before illumination or addition of FC. Final concentrations of W-7, ML-7 andFC were 200, 100 and 10/<M, respectively. Illumination was performed for 2 h except for "dark" samples.

Concentration

KT59260
1

10Magnitude of H
extrusion(nmol H + pulse"1(jug protein)" 1 )
0.449
0.116
0.047
0.386
0.078
0.036

K-252a and KT5926 were added to the protoplast suspension
about 40 min before illumination with blue light. K-252a andKT5926 were dissolved in DMSO. Final concentration of DMSO
in the reaction mixture was 0.5%.

1324

K. Shimazaki et al.

sion by about 80% at 1 fiM, with magnitudes of 0.386 and0.078 nmol H + per pulse per fig protein in the control andin the presence of KT5926, respectively. Addition of FC tothe protoplast suspensions in the presence of these inhibitors induced rates of proton extrusion similar to those in
the controls (Fig. 3). These results suggest that K-252a andKT5926 inhibit the signal transduction pathway in the blue
light response of stomata without affecting the proton
pump.
Effect of FC on proton extrusion and stomatal open-

a)
PMA
o
(0
3

b)

CCCP

8L
I
^
—"\__^-^

FC

70 nmol H

BL

FC

X

01

v

X
15 min

1

J

FC

DCCD

BL

-J/

Fig. 4 Inhibition of blue light-dependent proton extrusion in
guard cell protoplasts by PMA, CCCP and DCCD.
PMA,
CCCP and DCCD were added to the suspension 15 min before
blue light illumination at final concetrations of 2.5, 5 and 200//M,
respectively. Magnitudes of blue light-dependent proton extrusion
were 0.465, 0.016, 0.032 and 0 nmol H + per pulse per n% of protein, for control, PMA, CCCP and DCCD, respectively. Maximum rates of proton extrusion induced by FC were 2.63, 0.56 and0.28 nmol H + h" 1 per /ig of protein, for control, PMA and
CCCP, respectively. Other experimental conditions were the sameas in Fig. 1.

ing in the presence of PMA, CCCP and
DCCD—Bluelight-dependent proton extrusion in guard cell protoplasts

15
a)

b)
FC

Light

I
| 10

d>
QL

<

Fig. 3 Inhibition of blue light-dependent proton extrusion in
Vicia guard cell protoplasts by K-252a and KT5926.
(a) Without inhibitor (b) 10 /iM K-252a (c) Without inhibitor (d) 10 ^M
KT5926. K-252a and KT5926 dissolved in DMSO were added at
10 fiM, 40min before blue light illumination. FC dissolved in
DMSO was added at 10//M after the blue light-dependent proton
extrusion had ceased. Magnitudes of the blue light-dependent proton extrusion were 0.486, 0.064, 0.349 and 0.031 nmol H + per
pulse per^g of protein, for (a), (b), (c) and (d), respectively. Maximal rates of FC-induced proton extrusion were 4.96, 5.12, 1.82
and 1.77 nmol H + h" 1 per /ig of protein, for (a), (b). (c) and (d),respectively. The reaction mixture (1 ml) contained 23.5 and 41.5//g of protein as guard cell protoplasts for (a), (b) and for (c), (d),respectively. Final concentration of DMSO was \% in the presence of FC. Other experimental conditions were the same as inFig. 1.

E

S 5

c/l

I
a < a a JC

£ S o <-> isI
P: Q

Fig. 5 Blocking of FC-induced stomatal opening in Commelina
epidermis by PMA, CCCP and DCCD.
PMA, CCCP and
DCCD were added at 2.5, 5 and 200//M, respectively, to thebathing solution 5 min before illumination or addition of FC.
Other experimental conditions were the same as in Fig. 2.

Blue light response of stomatal guard cells

1325

KT5926 has much larger Ks values; the values for protein kinase C, cAMP-dependent protein kinase and cGMP-dependent protein kinase were found to be 0.723, 1.20 and
0.158 fiM, respectively (Nakanishi et al. 1990). Thus, thesimilar concentration dependency for inhibition by K-252a
and KT5926 strongly suggests the involvement of MLCK in
the blue light response of stomata, although a permeabilityof the inhibitors across the plasma membrane of guard
cells should be considered. This agrees well with our previous results (Shimazaki et al. 1992). It should be noted, however, that the concentration required for 50% inhibition(IC50) was remarkably affected by ATP concentration in
the reaction system. For example, the IC50 values ofKT5926 were 0.072 and 0.39 fiM for 0.2 and 1.6 mM ATP,
respectively, in the phosphorylation reaction of myosin
light chain by chicken gizzard MLCK (Nakanishi 1990).
Since ATP concentration in the cytoplasm of guard cells ismost likely to be an order of mM, fiM range of the kinase
Discussion
inhibitor will be required to inhibit MLCK or MLCK-likeBlue light perceived by photoreceptors leads to activa- proteins in guard cells. Furthermore, the inhibitory action
tion of the proton pump in the plasma membrane of guard of K-252a and KT5926 seems to be specific to the lightcells through an unknown signal transduction process. The signaling pathway without affecting the proton pump, beblue light-sensitive pump, a terminal target of the blue light cause FC induced the same rate of proton extrusion in thesignal, has been suggested to be a H + -ATPase in the plas- presence of these inhibitors as those in the controls (Fig. 3).
ma membrane (Assmann et al. 1985, Schwartz et al. 1991,
Specific inhibition of the light signaling pathway by
Amodeo et al. 1992), although a redox process in the plas- W-7, ML-7, K-252a and KT5926 was supported by the conma membrane of guard cells cannot not be excluded trasting inhibitory effects of PMA, CCCP and DCCD.
(Raghavendra 1990, Pantoja and Willmer 1991, Gautier These chemical agents suppressed both proton extrusion
et al. 1992). A recent study has revealed that blue light- and the stomatal opening; however, FC failed to restore
dependent proton extrusion of Vicia guard cell proto- stomatal responses in the presence of these inhibitors.
plasts and light-dependent stomatal opening of Commelina These inhibitors affected the activity of the proton pump in
benghalensis are inhibited by W-7, a CaM antagonist, and guard cells. PMA markedly reduces the level of ATP, a
ML-7, a CaM-dependent MLCK inhibitor, suggesting the fuel for the pump, in guard cell protoplasts (Shimazaki etinvolvement of CaM and MLCK in the blue light response al. 1983). CCCP acts as a protonophore, which dissipatesof stomata (Shimazaki et al. 1992). In the present study, we the proton gradient across the membrane and reduces thefound that FC, an activator of H + -ATPase, induced both ATP level. DCCD acts as an inhibitor of H + -ATPases inproton extrusion and stomatal opening that had been in- the plasma membrane as well as that of the F-type ATPase
hibited by W-7 or ML-7. This suggests that W-7 and ML-7 of chloroplasts and mitochondria (Sze 1985).
inhibit the pathway of light signaling in the processes ofIn animal cells, MLCK phosphorylates myosin light
blue light response of stomata.
chain and this phosphorylation is essential for the contFrom these results, the inhibition of blue light-depend- raction of smooth muscle cells (Adelstein and Eisenberg
1980, Ito et al. 1989). MLCK-catalyzed phosphorylation ofent proton extrusion by K-252a and KT5926 will be expected. K-252a inhibits MLCK (Nakanishi et al. 1988), and myosin light chain is also required for the activation of
KT5926 selectively inhibits MLCK (Nakanishi et al. 1990). platelets (Saitoh et al. 1986). There are, however, few
As shown in Table 1, both K-252a and KT5926 at 1 fiM sup- reports on the functional role of MLCK in plant cells. In
pressed the blue light-dependent proton extrusion by about Chara cells, it has been suggested that the phosphorylation
80% in Vicia guard cell protoplasts, and these inhibitors of myosin causes the cessation of cytoplasmic streaming
2+
showed a similar concentration dependency. K-252a and (Tominaga et al. 1987). Recently, Ca -dependent protein
KT5926 inhibit MLCK in a competitive manner in respect kinase, which has a regulatory domain similar to CaM
with ATP, and have similar inhibition constants (Kj); the (Harper et al. 1991), has been proposed to inhibit the cytovalues were 0.020//M and 0.018 ^M for K-252a and plasmic streaming in Chara cells, and has been shown tophosphorylate a putative myosin light chain (McCurdy andKT5926, respectively. K-252a has similar K; values (around0.02 fiM) for protein kinase C, cAMP-dependent protein ki- Harmon 1992). The enzyme is sensitive to W-7 and K-252a,nase and cGMP-dependent protein kinase. In contrast, also (Harmon et al. 1987, Li et al. 1991). Thus, the involve-

was inhibited by PMA (2.5 ^M), CCCP (5 fiM) and DCCD
(200/iM) (Fig. 4a, b, c). The values for proton extrusion
were 0.016, 0.032 and 0 nmol H + per pulse per fig of protein, with PMA, CCCP and DCCD, respectively. Without
inhibitor the magnitude was 0.465 nmol H + per pulse per
fig of protein. In contrast to the inhibitory effects of W-7,
ML-7, K-252a and KT5926 (Fig. 1, 3), FC failed to induce
proton extrusion with these inhibitors. Light-dependent
stomatal opening was inhibited by PMA, CCCP and
DCCD (Fig. 5). FC did not induce stomatal opening in thepresence of these inhibitors.
These results suggest that W-7, ML-7, K-252a andKT5926 affected neither the energetic status nor the proton
pump in guard cells, but inhibited the process(es) by whichlight signal was transduced into activation of the proton
pump in the plasma membrane of the cells.

K. Shimazaki et al.

1326ment of Ca2+-dependent, calmodulin-independent protein
kinase in the blue light response of stomata could not be excluded.
FC activates H+-ATPase in the plasma membrane ofmost higher plant cells as well as that of guard cells, and induces the proton extrusion (Marre 1979, Shimazaki et al.
1986, Rasi-Caldogno et al. 1986, Assmann and Schwartz
1992, Johansson et al. 1993). The activation process is initiated by the attachment of FC to the FC-binding proteinin the plasma membrane (Aducci et al. 1988). Most recent
studies indicate that the signal of FC is transmitted to the
C-terminal inhibitory domain of the molecule of H+-ATPase via this binding protein (Johansson et al. 1993), thereby
increasing the activity of H+-ATPase. Since FC elicited proton extrusion in the presence of W-7, ML-7, K-252a andKT5926, the activation signal of FC could be transmitted
to the H+-ATPase molecule in the presence of these inhibitors. This suggests that the FC-binding protein acts on thesite closer to the molecule of H+-ATPase than the sites ofinhibition by antagonist and kinase inhibitors, or acts independently from the signaling pathway of blue light.
Finally, all of the results obtained in the present investigation are consistent with that MLCK or MLCK-likeprotein is involved in the blue light response of stomata.
However, more direct evidence will be required to show
this, because the data were solely provided from the use ofpharmacological tools that are usually used in animal cells.
This work was supported in part by grants from the Toray
Science Foundation (No. 89-3005) and from the Ministry of
Education, Science and Culture of Japan (Nos. 03640570,
03304006).

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(Received July 23, 1993; Accepted September 30, 1993)