全 文 ：Received 5 Apr. 2004 Accepted 6 Jul. 2004
Supported by the Natural Science Research Plan of Shaanxi Provine (99SM20, 2003C101).
* Author for correspondence. Tel: +86 (0)29 85308451; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1292-1300
Role and Relationship of Nitric Oxide and Hydrogen Peroxide in
Light/Dark-regulated Stomatal Movement in Vicia faba
SHE Xiao-Ping*, SONG Xi-Gui, HE Jun-Min
(College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China)
Abstract: Role and relationship of NO and H2O2 in light/dark-regulated stomatal movement in Vicia faba
L. were investigated by epidermal strip bioassay and laser-scanning confocal microscopy. Results showed
that the effects of exogenous sodium nitroprusside (SNP, NO-releasing compound) and H2O2 on stomatal
closure were more significant in light than those in the dark. Dark-induced closure of stomata was largely
prevented not only by 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific
NO scavenger and NG-nitro-L-arg-methyl eater (L-NAME), an inhibitor of NO synthase (NOS) in mammalian
cells that also inhibits plant NOS, but also by addition of ascorbic acid (Vc) and exogenous catalase (CAT),
which are an important reducing substrate for H2O2 removal and an H2O2 scavenger, respectively. Experi-
ments based on fluorescent probe DAF-2 DA and H2DCF-DA showed that the level of endogenous NO and
H2O2 in guard cells was greater in the dark than that in light. These results prove that light/dark regulates
stomatal movement via influencing NO and H2O2 production. In addition, H2O2-induced NO production and
stomatal closure in light were abolished partly by cPTIO and L-NAME. Interestingly, SNP-induced H2O2
accumulation and stomatal closure were reversed by Vc and CAT in light. These show that NO and H2O2
cross talk in light/dark-regulated stomatal movement. Furthermore, L-NAME could reverse stomatal
closure and NO generation induced by darkness and H2O2 in light, we presume that the NO generation in
guard cells of Vicia faba is likely related to NOS-like enzyme.
Key words: nitric oxide; hydrogen peroxide; light/dark; stomatal movement; Vicia faba
There is now compelling evidence that nitric oxide (NO)
and hydrogen peroxide (H2O2) function as signalling mol-
ecules in the world of plants (Durner and Klessing, 1999;
Neill et al., 1999). Both biotic and abiotic stresses can in-
duce NO and H2O2 synthesis, such as extremes of
temperatures, UV irradiation, excess excitation energy,
ozone exposure, phytohormones, dehydration, wounding,
elicitor and pathogen challenge and so on. Establishing
contact with the interactional manner in many cases, NO
and H2O2 participate in and regulate together many impor-
tant developmental processes. A great of evidence has been
accumulated suggesting that H2O2 and NO function as sig-
naling molecules in plants mediating a range of responses
to environmental stress (Foyer et al., 1997; Bolwell, 1999;
Dat et al., 2000). NO was shown to act synergistically with
reactive oxygen species (ROS) to increase host cell death
of soybean suspension cells (Delledonne et al., 1998). Upon
exposure to pathogens, plants rapidly produce reactive oxy-
gen intermediates such as superoxide (O2
-. ) and H2O2 (the
so-called “oxidative burst”), leading to the hypersensitive
response (HR) (Wojtaszek, 1997; Alvarez et al., 1998), and
NO also mediates plant defense responses against patho-
gens (Dangl, 1998). Other studies showed that H2O2 and
NO production after cryptogein activation were almost
identical, with the earliest responses detectable after less
than 1 min and full activation between 6 and 12 min (Foissner
et al., 2000). One noteworthy point is that NO has been
reported to inhibit the activity of tobacco catalase (CAT)
and ascorbate peroxidase (APX) and subsequently in-
creased the intracellular H2O2 concentration in plant (Clark
et al., 2000). It is clear that both NO and H2O2 can mediate
the transcription of specific genes. NO induced phenylala-
nine ammonia lyase (PAL) expression via a salicylic acid
(SA) independent pathway (Durner et al., 1998), while the
PAL induction by H2O2 required SA (Chamnongpol et al.,
1998). These show that NO and H2O2 occur complementary
action in the activation of defense responses (Camp et al.,
1998).
Stomatal closure is induced by many abiotic and biotic
factors, such as osmotic stress, darkness, high CO2
concentrations, and some mechanical stresses (Kearns and
Assmann, 1993). Light/dark is one of the most important
factors of environment to regulate stomatal movement
(Zeiger, 1983). The change of level of ABA in guard cell is
SHE Xiao-Ping et al.: Role and Relationship of Nitric Oxide and Hydrogen Peroxide in Light/Dark-regulated Stomatal Move
ment in Vicia faba 1293
connected with stomatal movement regulated by light/dark
(Michael and William, 1991; Wang et al., 1997). As we know,
ABA is one of the most studied phytohormones due to its
key participation in stomatal movement. Previous studies
suggest that both NO and H2O2 can affect stomatal
movement: NO induces stomatal closure and enhances the
plant adaptive responses against drought stress (Mata
et al., 2001), and NO synthesis in guard cells is required for
ABA-induced stomatal closure (Mata and Lamattina, 2002;
Neill et al., 2002). The new research shows that NO is in-
volved in salicylic acid (SA) regulating stomatal movement
(Liu et al., 2003a). Resemblingly, H2O2 is also involved in
SA (Dong et al., 2001) and ABA (Allen et al., 2000; Pei
et al., 2000) regulating stomatal movement. Recently, Liu
et al. (2003b) proposed that NO and H2O2 were involved
mutually and dependently in regulating of stomatal
movement. However, to our knowledge, the role of NO and
H2O2 in light/dark-regulated stomatal movement has not
been investigated, and the interrelationship between NO
and H2O2 in this process was unknown. Here the work
provides evidence that H2O2 and NO in guard cells of Vicia
faba are involved in light/dark-regulated stomatal movement,
and guard cells in epidermal peels of V. faba generate H2O2
and NO in response to darkness, which synthesis is essen-
tial for stomatal closure. Additionally, the interrelationship
of H2O2 and NO in light/dark regulated stomatal movement
was studied by means of epidermal strip bioassay and la-
ser-scanning confocal microscopy based on DAF-2 DA
and H2DCF-DA.
1 Materials and Methods
1.1 Plant materials
Broad bean (Vicia faba L.) was grown in greenhouse
with a humidity of about 80%, a photon flux density of 300
µmol.m-2.s-1, and an ambient temperature (25 ± 2) °C with
a 14-h light and 10-h dark cycle. Fully expanded leaves of 3-
week-old seedlings were used in the experiment.
1.2 Chemicals
Molecular probes including 4,5-diaminofluorescein
diacetate (DAF-2 DA, from Sigma) and dichlorofluorescein
diacetate (H2DCF-DA, from Biotium) were dissolved in dim-
ethyl sulfoxide (DMSO, from Amresco.) to produce a stock
solution, which was aliquoted. Sodium nitroprusside (SNP),
2, 4- carboxyphenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-
3-oxide (cPTIO), NG-nitro-L-arg-methyl eater (L-NAME), 2-
(N-morpholino) ethanesulfonic acid (MES) and catalase
(CAT, from bovine liver) were obtained from Sigma. Unless
stated otherwise, the remaining chemicals were of analyti-
cal grade from Chinese companies.
1.3 Stomatal bioassays
Stomatal bioassay was performed as described by
McAinsh et al. (1996) with slight modifications. Immedi-
ately prior to each experiment, the epidermis was peeled
carefully from the abaxial surface of the youngest, fully
expanded leaves of 3-week-old plants. To study the role
and interaction of NO and H2O2 in stomatal movement in-
fluenced by light/dark, freshly prepared abaxial epidermis
was incubated in CO2-free MES/KCl buffer (10 mmol/L MES/
KOH, 50 mmol/L KCl, 100 µmol/L CaCl2, pH 6.15), which
included various compounds, in light (a photon flux den-
sity of 300 µmol.m-2.s-1) and dark with the same tempera-
ture of 25 °C for 3 h. Stomatal apertures were measured
under a light microscope with a calibrated micrometer scale.
Data are presented as the mean of three independent
experiments.
1.4 Measurement of endogenous NO and H2O2 by confo-
cal laser scanning microscopy
NO and H2O2 measurement was performed by using
their fluorescent indicator dye DAF-2 DA and H2DCF-DA
as described previously (Allan and Fluhr, 1997; Kojima et
al., 1998) with slight modifications. The epidermis was
peeled carefully from leaves and cut into 5-mm length. Epi-
dermal strips were incubated in MES/KCl buffer including
various compounds for 3 h in light and in the dark,
respectively. After this step, the strips were placed into
Tris/KCl buffer (Tris 10 mmol/L and KCl 50 mmol/L, pH 7.2)
containing DAF-2 DA at a final concentration of 10 µmol/L
for 30 min, or H2DCF-DA at 50 µmol/L for 10 min, in the dark
at 26 oC. After washed off excess dye with fresh Tris/KCl
buffer apart from light, examination of peels was performed
using confocal microscopy (Leica, TCS SP2) with the fol-
lowing settings: excitation 488 nm, emission 505–530 nm,
normal scanning speed and frame 512× 512. Images ac-
quired from the confocal microscope were analyzed by us-
ing LEICA IMAGE software and treated by photoshop soft-
ware (Dong et al., 2001; Zhang et al., 2001; Neill et al.,
2002). The experiments were repeated at least three times in
each treatment, and obtained the same results.
2 Results
2.1 Effects of exogenous SNP and H2O2 on stomatal
behavior
NO is a short-lived free radical gas, and it is typically
applied to plants via the administration of NO-releasing
compounds such as SNP (sodium nitroprusside). V. faba
epidermal strips were treated with increasing concentra-
tions SNP and H2O2. Both SNP (Fig.1A) and H2O2 (Fig.1B)
induced stomatal closure in light, and the effects were in a
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041294
dose-dependent manner. In darkness, as shown in Fig.1,
SNP or H2O2 treatment promoted stomatal closure slightly
over the control. The results imply that the endogenous
NO and H2O2 level of guard cells in the dark is higher than
that in light, so effects of exogenous SNP and H2O2 on
stomatal aperture in light are more significant than those in
the dark.
2.2 Effects of cPTIO, L-NAME, CAT and Vc on stomatal
aperture
To determine whether endogenous NO and H2O2 are
involved in the stomatal movement regulated by light/dark,
the epidermal strips were treated with cPTIO (the specific
NO scavenger), L-NAME (an inhibitor of NO synthase),
CAT (the H2O2 scavenger) and Vc (one of the anti-oxidant
to eliminate ROS) in light and dark. Figure 2 shows that
cPTIO and L-NAME did not cause any changes of sto-
matal aperture in light. However, dark-induced stomatal clo-
sure was readily reversed by cPTIO or by L-NAME.
Similarly, both CAT and Vc could reverse partly the sto-
matal closure induced by darkness, but in light, treatments
with CAT or Vc had less effect. These data indicate that the
level of endogenous NO and H2O2 of guard cells in the dark
is greater than that in light, so effects of cPTIO, L-NAME,
CAT and Vc on stomatal aperture in darkness are more
significant than that in light. Furthermore, reversibility of
dark-induced stomatal closure by L-NAME shows that V.
faba guard cells are likely to possess a NOS-like enzyme,
and guard cells generate NO in response to darkness via
the NOS-like enzyme.
2.3 Reversibility of H2O2-induced stomatal closure by
cPTIO and L-NAME
As preceding data, we know that H2O2 can induce sto-
matal closure (Fig.1B). To determine whether NO is involved
in this process, the epidermal strips were treated with H2O2
in the presence of either cPTIO or L-NAME, respectively.
Figure 3 shows that both cPTIO and L-NAME reversed
Fig.1. Effects of sodium nitroprusside (SNP) and H2O2 on
stomatal behavior. The epidermal strips were incubated in CO2-
free MES-KCl containing different concentrations of SNP (A),
and H2O2 (B) for 3 h under light (300 µmol.m-2.s-1) and dark
conditions. Stomatal apertures were determined. Values are the
means of 90 measurements ± SE of three independent experiments.
Error bars are indicated.
Fig.2. Effects of cPTIO, NG-nitro-L-arg-methyl eater (L-
NAME), CAT and Vc on stomatal aperture. Isolated epidermal
strips were incubated in CO2-free MES-KCl, containing various
compounds (cPTIO 200 µmol/L, L-NAME 25 µmol/L, Vc 100
µmol/L and CAT 100 units/mL) for 3 h under light and dark
conditions, and then stomatal apertures were determined. Values
are the means of 90 measurements ± SE of three independent
experiments. Error bars are indicated.
Fig.3. Reversibility of H2O2-induced stomatal closure by cPTIO
and L-NAME. The epidermal strips were treated with H2O2 100
µmol/L, cPTIO 200 µmol/L and L-NAME 25 µmol/L alone, or
with H2O2 at 100 µmol/L in the presence of cPTIO 200 µmol/L
(H+P) or L-NAME 25 µmol/L (H+N) for 3 h under light and dark
conditions, and then stomatal apertures were determined. Values
are the means of 90 measurements ± SE of three independent
experiments. Error bars are indicated.
SHE Xiao-Ping et al.: Role and Relationship of Nitric Oxide and Hydrogen Peroxide in Light/Dark-regulated Stomatal Move
ment in Vicia faba 1295
largely the H2O2-induced stomatal closure in light and in
the dark, indicating that NO production is required for sto-
matal closure induced by H2O2, and H2O2 induces NO syn-
thesis via the action of a NOS-like enzyme.
2.4 Effects of CAT and Vc on SNP-induced stomatal
closure
To determine whether H2O2 is also involved in the sto-
matal closure induced by NO, the epidermal strips were
treated with SNP in the presence of either CAT or Vc,
respectively. Figure 4 shows that CAT and Vc could effec-
tively abolish stomatal closure induced by SNP whether in
light or in the dark. The results show that H2O2 is required
for the stomatal closure induced by NO.
2.5 Endogenous NO production in response to exog-
enous H2O2 and light/dark in guard cells
To determine the variations of endogenous NO during
light/dark-regulated stomatal movement, epidermal strips
were loaded with the cell-permeable NO sensitive
fluorophore DAF-2 DA, which allows the detection of NO
presence in both animal and plant cells (Kojima et al., 1998).
Slight auto fluorescence observed and produced by DAF-
2 DA was associated with the inner walls of the guard cells
in the situation of light treatment (Fig.5A), but after dark-
ness treatment, there was a striking green fluorescence in
guard cells (Fig.5B). Moreover, fluorescence not only was
near the stomatal pore, but also spread out apparently in
the cytosol. Additionally, dark-induced DAF-2 DA fluo-
rescence in guard cells was largely prevented by cPTIO
(Fig.5C). Similarly, treatment with L-NAME also substan-
tially suppressed dark-induced DAF-2 DA fluorescence (Fig.
5D). These results prove that NO production of V. faba
guard cells is regulated by light and dark, and NO genera-
tion induced by darkness is likely related to NOS-like
enzyme, which are consistent with the preceding conclu-
sion from stomatal bioassays (Fig.2).
The possibility of an increase in NO production induced
by H2O2 was also examined using DAF-2 DA. As shown in
Fig.5E, exogenous application of H2O2 enhanced the rela-
tive fluorescence intensity of DAF-2DA in guard cells un-
der the light comparing with treatments of light alone
(Fig.5A). H2O2-induced DAF-2 DA fluorescence in guard
cells was substantially prevented by cPTIO (Fig.5F) or L-
NAME (Fig.5G), suggesting that H2O2 substantially induces
NO synthesis, and the NO synthesis is via the action of a
NOS-like enzyme in V. faba guard cells.
2.6 Endogenous H2O2 production in response to exog-
enous NO (SNP) and light/dark in guard cells
Experimentally, we used H2DCF-DA, a specific probe
for intracellular H2O2 (Allan and Fluhr, 1997), to measure
H2O2 production in light/dark-regulated stomatal movement.
Figure 5B showed that the increase of fluorescence in guard
cells treated with darkness was observed over the light
treatment (Fig.5A). Moreover, darkness-induced H2DCF-
DA fluorescence in guard cells was largely prevented by
CAT (Fig.5C). Pretreatment with Vc also substantially sup-
pressed darkness-induced H2DCF-DA fluorescence
(Fig.5D). These results suggest that H2O2 production of V.
faba guard cells is regulated by light and dark, which are in
accordance with the preceding conclusion from stomatal
bioassays (Fig.2).
Effects of CAT and Vc on SNP-induced stomatal clo-
sure (Fig.4) let us know whether endogenous H2O2 change
is involved in stomatal closure induced by SNP. As showed
in Fig.5E, in light, exogenous application of SNP enhanced
stupendously the H2DCF-DA fluorescence in guard cells,
but not in the subsidiary cells. Figure 5F and G show that
SNP did not induce H2DCF-DA fluorescence increase in
the presence of CAT or Vc. These results further indicated
that NO induced H2O2 accumulation in guard cells.
3 Discussion
In this work, we demonstrate that H2O2 and NO are in-
volved in light/dark-regulated stomatal movement in V. faba.
SNP and H2O2, when applied exogenously in light, induce
stomatal closure, and the effects are in a dose-dependent
manner (Fig.1), which is the same as the previous results
(Liu et al., 2003b). In darkness, the effects of SNP and H2O2
on stomatal closure are very unconspicuous. However, the
dark-induced stomata closure can be reversed by cPTIO,
L-NAME, CAT and Vc, which have no significant affect on
Fig.4. Effects of CAT and Vc on SNP-induced stomatal closure.
The epidermal strips were treated with SNP 100 µmol/L, CAT
100 units/mL and Vc 100 µmol/L alone, or with SNP 100 µmol/L
in the presence of CAT 100 units/mL or Vc 100 µmol/L for 3 h
under light and dark conditions, and then stomatal apertures were
determined. Values are the means of 90 measurements ± SE of
three independent experiments. Error bars are indicated.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041296
SHE Xiao-Ping et al.: Role and Relationship of Nitric Oxide and Hydrogen Peroxide in Light/Dark-regulated Stomatal Move
ment in Vicia faba 1297
stomatal aperture in light (Fig.2). So we speculate that the
levels of endogenous NO and H2O2 in light are different
from that in darkness. Concretely, the levels of NO and
H2O2 in light are low, but in dark that is high. So, exog-
enous SNP and H2O2 can induce effectively stomatal clo-
sure in light but have less effect in darkness. Certainly,
their scavengers or inhibitors can abolish stomatal closure
induced by darkness but have little effect on stomatal aper-
ture in light, indicating intensively that comparative high
levels of endogenous NO and H2O2 are required for full
stomatal closure induced by darkness.
We used the molecular probes DAF-2 DA and H2DCF-
DA to directly measure the change of NO and H2O2 within
guard cells, respectively. The two compounds are converted
to the membrane-impermeant DAF-2 and H2DCF by es-
terases when they are taken up by the cell. The triazole-like
substance generated by DAF-2 and NO can emit strongly a
kind of green fluorescence (Kojima et al., 1998). Similarly,
H2DCF is rapidly oxidized to the highly green fluorescent
DCF by intracellular H2O2 (Allan and Fluhr, 1997). By laser-
scanning confocal microscopy, we provide evidence that
in the dark the endogenous NO fluorescence is very strik-
ing (Fig.5B) as well as H2O2 fluorescence (Fig.5B) over the
light (Fig.5A, A), respectively. Both cPTIO and L-NAME
can prevent dark-induced DAF-2 DA fluorescence (Fig.5C,
D). Analogously, both CAT and Vc can suppress dark-
induced H2DCF-DA fluorescence (Fig.5C, D). These re-
sults support clearly that darkness can induce NO and H2O2
generation, and the levels of NO and H2O2 in darkness is
affirmatively higher than those in light, which are consis-
tent with the results of stomatal bioassays (Figs.1, 2).
Both NO and H2O2 are freely diffusible and membrane-
permeable. From being molecules of somewhat novelty
interest, in the last few years NO and H2O2 have emerged
to be crucial players in the plant cell signalling, particularly
under various stressful situations (Potikha et al., 1999; Dat
et al., 2000; Pedroso et al., 2000; Pei et al., 2000). Previous
studies in plant immune response suggest that NO and
H2O2 act in parallel pathways to activate a cascade of down-
stream signals, such as the expression of pathogenesis-
related 1 (PR-1) and phenylalanine ammonia lyase (PAL)
(Delledonne et al., 1998; Beligni and Lamattina, 1999). Lum
et al. (2002) have also provided evidence that besides act-
ing as two independent signals in plant-immune response,
NO and H2O2 are interrelated and may cooperate to pro-
duce a rapid oxidative burst during the hypersensitive
response, the produced H2O2, NO as well as other free
radical species may interact together in the herpersensitivity
response during pathogen infections. Wounding is a com-
mon consequence of pathogen challenge of plants, during
which the generation and increased accumulation of NO
and H2O2 are frequently observed (Delledonne et al., 1998).
A correlation between H2O2, NO and antioxidant levels has
also been demonstrated recently by de Pinto et al. (2002),
they propose that both H2O2 and NO regulate cellular anti-
oxidant levels to affect programmed cell death（PCD）,
at least in some systems. Here, by epidermal strip bioassay
and laser-scanning confocal microscopy, we have investi-
gated the relationship of H2O2 and NO in light/dark-regu-
lated stomatal movement. Guard cells generated NO in re-
sponse to H2O2 in light, exogenous H2O2 increased the
level of NO in guard cells (Fig.5E), and such NO production
was required for full stomatal closure in response to H2O2
(Fig.3). Both cPTIO and L-NAME reversed largely the H2O2-
induced stomatal closure in light (Fig.3), and H2O2-induced
DAF-2 DA fluorescence in guard cells was substantially
prevented by cPTIO or L-NAME (Fig.5F,G). Interestingly,
SNP could also induce H2O2 accumulation in guard cells
under the light (Fig.5E). CAT and Vc abolished stomatal
closure induced by SNP (Fig.4), and SNP did not induce
H2O2 fluorescence increase in the presence of CAT and Vc
(Fig.5F,G). The results of our present study clearly indi-
cate a causal and interdependent relationship between NO
and H2O2 in light/dark-regulated stomatal movement of V.
faba guard cells, and in the process, this cross talk of NO
and H2O2 may lead to the information of a self-amplifica-
Fig.5. Endogenous NO and H2O2 production in response to light/dark, exogenous H2O2 (100 µmol/L) or sodium nitroprusside (SNP)
(100 µmol/L) in guard cells. Guard cells shown in image A and A treated in light alone. B and B in the darkness alone. C and D in the
darkness including cPTIO(200 µmol/L) and L-NAME (25 µmol/L), respectively. C and D in the darkness including CAT (100 units/
mL) and Vc (100 µmol/L), respectively. E, F, and G in light including H2O2 (100 µmol/L), H2O2 (100 µmol/L)+cPTIO (200 µmol/L) and
H2O2 (100 µmol/L)+L-NAME (25 µmol/L), respectively. E, F, and G in light including SNP (100 µmol/L), SNP (100 µmol/L)+CAT
(100 units/mL) and SNP (100 µmol/L)+Vc (100 µmol/L), respectively. Epidermal strips were loaded with DAF-2 DA from A to G or
H2DCF-DA from A to G apart from light, then washed off excess dye and examined by laser scanning confocal microscopy. The guard
cell in image A is representative of guard cells in image a. The inset shows the bright-field image corresponding to the fluorescence image
A. The image B-G and A-G are the same. Each experiment was repeated at least 3 times and obtained the same results. The length of
the bar in image (g) =40 µm for a-g and a-g; the bar in image (G) =16 µm for A-G and A-G; the bar in inset of image (G) = 8 µm for
all the insets.
←
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041298
tion loop about them.
Although NO is well recognized as an important signal-
ing molecule, its source has always been controversial. Pre-
vious researches have suggested three possible sources
of NO in plants: (a) synthesized through chemical reactions,
e.g. oxidation and reduction of different nitrogenous com-
pounds by various carotenoids (Cooney et al., 1994); (b)
enzymatically produced from nitrite (NO2-) by the action of
plant nitrate reductase (NR) (Yamasaki, 2000; Yamasaki and
Sakihama, 2000; Desikan et al., 2002); and (c) via the action
of a NOS-like enzyme using arginine as substrate (Sen and
Cheema, 1995; Cueto et al., 1996). NOS is a family of well
characterized enzymes in mammalian cells that catalyze the
conversion of L-arginine to L-citrulline and NO. The pres-
ence of NOS-like enzyme and its activity are well docu-
mented in many publications (Sen and Cheema, 1995;
Wojtaszek, 1997; Delledonne et al., 1998; Wojtaszek, 2000).
A NOS enzyme activity has been purified from pea peroxi-
somes (Barroso et al., 1999), and it is particularly exciting
that a partial cDNA clone for pea NOS has been obtained
(Corpas et al., 2001). In the study, stomatal closure induced
by whether darkness or H2O2 in light was readily reversed
by L-NAME (Figs.2, 3), which is an arginine analogous
NOS inhibitor. Similarly, by means of laser-scanning con-
focal microscopy based on fluorescence probe DAF-2 DA,
we found NO generation induced by whether darkness or
H2O2 in light was largely abolished by L-NAME (Fig.5D,
G). Our studies support that both darkness and H2O2 in-
duce NO production mediated probably by the action of
NOS-like enzyme in V. faba guard cells.
Acknowledgements: The authors wish to thank Prof.
HUANG Chen and Mr. LUO Yu for their help in this study.
References:
Allan A C, Fluhr R. 1997. Two distinct source of elicited reactive
oxygen species in tobacco epidermal cells. Plant Cell, 9: 1559-
1572.
Allen G J, Chu S P, Schumacher K, Shinmazaki C T, Vafeados D,
Kemper A, Hawke S D, Tallman G, Tsien R Y, Harper J F,
Chory J, Schroeder J I. 2000. Alteration of stimulus-specific
guard cell calcium oscillations and stomatal closing in
Arabidopsis det3 mutant. Science, 289: 2338-2342.
Alvarez M E, Pennell R I, Meijer P J, Ishikawa A, Dixon R A,
Lamb C. 1998. Reactive oxygen intermediates mediate a sys-
temic signal network in the establishment of plant immunity.
Cell, 92: 773-784.
Barroso J B, Corpas F J, Carreras A, Sandalio L M, Valderrama R,
Palma J M, Luupianez J A, del Rio L A. 1999. Localization of
nitric oxide synthase in plant peroisomes. J Biol Chem, 274:
36729-36733.
Beligni M V, Lamattina L. 1999. Is nitric oxide toxic or protective?
Trends Plant Sci, 4: 299-300.
Bolwell G P. 1999. Role of active oxygen species and NO in plant
defense. Curr Opin Plant Biol, 2: 287-294.
Camp W V, Montagu M V.1998. H2O2 and NO: redox signals in
disease resistance. Trends Plant Sci, 3: 330-334.
Chamnongpol S, Willekens H, Moeder W, Langebartels C,
Sandermann H, Jr van M M, Inze D, van C W. 1998. Defense
activation and enhanced pathogen tolerance induced by H2O2
in transgenic tobacco. Proc Natl Acad Sci USA, 95: 5818-
5823.
Clark D, Durner J, Navarre D A, Klessig D F. 2000. Nitric oxide
inhibition of tobacco catalase and ascorbate peroxidase. Mol
Plant Microbe In, 13: 1380-1384.
Cooney R V, Harwood R J, Custer L J, Franke A A. 1994. Light-
mediated conversion of nitrogen dioxide to nitric oxide by
carotenoids. Environ Health Perspect, 102: 460-462.
Corpas F J, Barroso J B, del Rio L A. 2001. Peroxisomes as a
source of reactive oxygen species and nitric oxide signal mol-
ecules in plant. Trends Plant Sci, 6: 145-150.
Cueto M, Hernandez-Perera O, Martin R, Bentura M L, Rodrigo
J, Lamas S, Golvano M P. 1996. Presence of nitric oxide
synthase activity in roots and nodules of Lupinus albus. FEBS
Lett, 398: 159-164.
Dat J, Vandenbeele S, Vranova E, van Montagu M, Inzé D, van
Breusegem F. 2000. Dual action of the active oxygen species
during plant stress response. Cell Mol Life Sci, 57: 779-795.
Dangl J. 1998. Innate immunity. Plants just say NO to pathogens.
Nature, 394: 525-527.
Delledonne M, Xia Y, Dixon R A, Lamb C. 1998. Nitric oxide
signal functions in plant disease resistance. Nature, 394: 585-
588.
de Pinto M C, Tomassi F, de Gara L. 2002. Changes in the anti-
oxidant systems as part of the signaling pathway responsible
for the programmed cell death activated by nitric oxide and
reactive oxygen species in tobacco bright-yellow 2 cells. Plant
Physiol, 130: 698–708.
Desikan R, Griffiths R, Hancock J, Neill S J. 2002. A new role for
an old enzyme: nitrate reductase-mediated nitric oxide genera-
tion is required for abscisic acid-induced stomatal closure in
Arabidopsis thaliana. Proc Natl Acad Sci USA, 99: 16314-
16318.
Dong F-C, Wang P-T, Zhang L , Song C-P. 2001. The role of
SHE Xiao-Ping et al.: Role and Relationship of Nitric Oxide and Hydrogen Peroxide in Light/Dark-regulated Stomatal Move
ment in Vicia faba 1299
hydrogen peroxide in salicylic acid-induced stomatal closure in
Vicia faba guard cells. Acta Phytophysiol Sin , 27: 296-302.
Durner J, Klessig D F. 1999. Nitric oxide as a signal in plants.
Curr Opin Plant Biol, 2: 369-374.
Durner J, Wendehenne D, Klessig D F. 1998. Defense gene induc-
tion in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-
ribose. Proc Natl Acad Sci USA, 95: 10328-10333.
Foissner I, Wendehenne D, Langebartels C, Durner J. 2000. In
vivo imaging of an elicitor-induced nitric oxide burst in tobacco.
Plant J, 23: 817–824.
Foyer C H, Lopez-Delgado H, Dat J F, Scott I M. 1997. Hydro-
gen peroxide and glutathione-associated mechanisms of
acclamatory stress tolerance and signaling. Physiol Plant, 100:
241-254.
Kearns E V, Assmann S M. 1993. The guard cell-environmental
connection. Plant Physiol, 102: 711-715.
Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y,
Nagoshi H, Hirata Y, Nagano T. 1998. Detection and imaging
of nitric oxide with novel fluorescent indicators:
diaminofluoresceins. Anal Chem, 70: 2446-2453.
Liu X, Zhang S Q, Lou C H. 2003a. Involvement of nitric oxide in
the signal transduction of salicylic acid regulating stomatal
movement. Chin Sci Bull, 48: 449-452.
Liu X , Zhang S-Q , Lou C-H. 2003b. Cross talk between hydro-
gen peroxide and nitric oxide in regulation of stomatal movement.
Prog Nat Sci, 13: 355-358. (in Chinese)
Lum H K, Butt Y K C, Lo S C L. 2002. Hydrogen peroxide in-
duces a rapid production of nitric oxide in Mung bean (Phaseolus
aureus). Biol Chem, 6: 205-213.
Mata C G, Lamattina L. 2001. Nitric oxide induces stomatal clo-
sure and enhances the adaptive plant responses against drought
stress. Plant Physiol, 126: 1196-1204.
Mata C G, Lamattina L. 2002. Nitric oxide and abscisic acid cross
talk in guard cells. Plant Physiol, 128: 790-792.
McAinsh M R, Clayton H, Mansfield T A, Hetherington A M.
1996. Changes in stomatal behavior and guard cell cytosolic
free calcium in response to oxidative stress. Plant Physiol,
111: 1031-1042.
Michael J H, William H O. 1991. Rapid adjustment of guard cell
abscisic acid levels to current leaf-water status. Plant Physiol,
95: 171-173.
Neill S J, Desikan R, Clarke A, Hancock J T. 1999. H2O2 Signaling
in Plant Cells: Plant Response to Environment Stress. UK:
BIOS Scientific Publishers. 59-63.
Neill S J, Desikan R, Clarke A, Hancock J T. 2002. Nitric oxide is
a novel component of abscisic acid signaling in stomatal guard
cells. Plant Physiol, 128: 13-16.
Pedroso M C, Magalhaes J R, Durzan D. 2000. A nitric oxide
burst precedes apoptosis in angiosperm and gymnosperm
callus cells and foliar tissues. J Exp Bot, 51: 1027-1036.
Pei Z M, Murata Y, Benning G, Thomine S, Klusener B, Allen G
J, Grill E, Schroeder J L. 2000. Calcium channels activated by
hydrogen peroxide mediate abscisic acid signaling in guard
cells. Nature, 406: 731-734.
Potikha T S, Collins C C, Johnson D I, Delmer D P, Levine A.
1999. The involvement of hydrogen peroxide in the differen-
tiation of secondary walls in cotton fibers. Plant Physiol, 119:
849-858.
Sen S, Cheema I R. 1995. Nitric oxide synthase and calmodulin
immunoreactivity in plant embryonic tissue. Biochem Arch,
11: 221-227.
Wang H-B, Wang X-C, Chen J , Cao M, Li Y-Y . 1997. Relation-
ship of stomata sensitivity to ABA and ABA binding proteins on
the plasmalemma of guard cells in Vicia faba. Acta Bot Sin ,
39: 126-129. (in Chinese with English abstract)
Wojtaszek P. 1997. Oxidative burst: an early plant response to patho-
gen infection. Biochem J, 322: 681-692.
Wojtaszek P. 2000. Nitric oxide in plants to NO or not to NO.
Phytochemistry, 54: 1-4.
Yamasaki H. 2000. Nitrite-dependent nitric oxide production
pathway: implications for involvement of active nitrogen spe-
cies in photoinhibition in vivo. Philos Trans R Soc Lond B
Biol Sci, 355: 1477-1488.
Yamasaki H, Sakihama Y. 2000. Simultaneous production of ni-
tric oxide and peroxynitrite by plant nitrate reductase: in vitro
evidence for the NR-dependent formation of active nitrogen
species. FEBS Lett, 468: 89-92.
Zhang X, Zhang L, Dong F C, Gao J F, Galbraith D W, Song C P.
2001. Hydrogen peroxide is involved in abscisic acid induced
stomatal closure in Vicia faba. Plant Physiol, 126: 1438-1448.
Zeiger E. 1983. The biology of stomatal guard cells. Annu Rev
Plant Physiol, 34: 441-475.
(Managing editor: HE Ping)