Hydrogen peroxide is necessary for abscisic
acid-induced senescence of rice leaves
Kuo Tung Hung, Ching Huei Kao
Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC Received 16 March 2004; accepted 12 May 2004
Summary
The role of H2O2 in abscisic acid (ABA)-induced rice leaf senescence is investigated.
ABA treatment resulted in H2O2 production in rice leaves, which preceded the
occurrence of leaf senescence. Dimethylthiourea, a chemical trap for H2O2, was
observed to be effective in inhibiting ABA-induced senescence, ABA-increased malondialdehyde (MDA) content, ABA-increased antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase), and ABA-decreased antioxidant contents (ascorbic acid and reduced glutathione) in rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of
NADPH oxidase, and KCN and NaN3, inhibitors of peroxidase, prevented ABA-induced
H2O2 production, suggesting NADPH oxidase and peroxidase are H2O2-generating
enzymes in ABA-treated rice leaves. DPI, IMD, KCN, and NaN3 also inhibited
ABA-promoted senescence, ABA-increased MDA contents, ABA-increased antioxidative enzyme activities, and ABA-decreased antioxidants in rice leaves. These results
suggest that H2O2is involved in ABA-induced senescence of rice leaves.
r2004 Elsevier GmbH. All rights reserved.
Introduction
The plant hormone abscisic acid (ABA) is a
sesquiterpenoid synthesized from xanthophylls
(Creelman, 1989; Taylor et al., 2000; Seo and Koshiba, 2002) and appears to influence several
physiological and developmental events (Creelman,
1989; Kende and Zeevaart, 1997). It has been suggested that ABA is one of the most effective plant hormones in terms of promoting leaf
senes-cence (Nooden, 1988). Applied ABA has been found
to promote leaf senescence in a wide range of plant www.elsevier.de/jplph KEYWORDS: Abscisic acid; H2O2; Leaf senescence; Lipid peroxidation; Oryza sativa
0176-1617/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.05.011
Abbreviations: AOS, Active oxygen species; APOD, Ascorbate peroxidase; AsA, Ascorbic acid; CAT, Catalase; DMTU, Dimethylthiour-ea; DPI, Diphenyleneiodonium chloride; FW, Fresh weight; GR, Glutathione reductase; GSH, Reduced glutathione; MDA,
Malondialdehyde; SOD, Superoxide dismutase Corresponding author. Fax: +886-2-23620879
species (Nooden, 1988; Creelman, 1989). An in-crease in endogenous ABA has been shown to
coincide with senescence of leaves (Gepstein and
Thimann, 1980;Yang et al., 2002).
Lipid peroxidation is considered to be an
im-portant mechanism of leaf senescence (Thompson
et al., 1987). Active oxygen species (AOS) can
initiate lipid peroxidation (Kellogg and Fridovich,
1975). It has been shown that ABA causes
genera-tion of AOS including H2O2(Guan et al., 2000;Pei et
al., 2000; Jiang and Zhang, 2001; Hung and Kao,
2003) and lipid peroxidation expressed as
malon-dialdehyde (MDA) production in plant cells (Bueno
et al., 1998). Thus, ABA leads to oxidative stress in plant cells.
Recently, many researchers have focused on the
functional aspects of H2O2. H2O2is a constituent of
oxidative metabolism and is itself an AOS. It has
been shown that H2O2 promotes leaf senescence
(Parida et al., 1978; Mondal and Choudhuri, 1981; Begam and Choudhuri, 1992;Lin and Kao, 1998) and induction of senescence is accompanied by an
increase in endogenous H2O2 content (Mondal and
Choudhuri, 1981; Hung and Kao, 2003). Because
H2O2 is relatively stable and diffusible through
membrane, it is generally thought to serve as a signal molecule under various abiotic stresses (Chamnongpol et al., 1998; Neill et al., 2002), in
acclimation to photooxidative stress (Karpinski et
al., 1999), in plant–pathogen interactions (Levine et al., 1994), and in ABA-induced stomatal closure (Zhang et al., 2001).
We have previously shown that ABA not only
increases the content of H2O2and the activities of
superoxide dismutase (SOD), ascorbate peroxidase (APOD), glutathione reductase (GR), and catalase (CAT), but also causes a decrease in ascorbic acid (AsA) and glutathione (GSH) contents in rice leaves (Hung and Kao, 2003). Meanwhile, protein loss (senescence) and lipid peroxidation were observed
in ABA-treated rice leaves (Hung and Kao, 2003).
All these results suggest that ABA causes oxidative stress and ABA-promoted senescence of rice leaves is mediated through oxidative stress. Here, we
have examined the role of H2O2 as a connection
between ABA and subsequent antioxidant defense and senescence in rice leaves.
Materials and methods
Plant material and chemicals
Rice (Oryza sativa L., cv. Taichung Native 1) was sterilized with 2.5% sodium hypochlorite for 15 min
and washed extensively with distilled water. These seeds were then germinated in Petri dishes with wetted filter paper at 37 1C under dark conditions. After 48 h incubation, uniformly germinated seeds were selected and cultivated in a 500 ml beaker containing half-strength Kimura B solution as
described previously (Chu and Lee, 1989). The
hydroponically cultivated seedlings were grown for 12 days in a Phytotron with natural light 30 1C day (12 h)/25 1C night (12 h) and 90% relative humidity. The apical 3 cm of the third leaf was used in all experiments. A group of ten segments was floated in a Petri dish containing 10 ml of test solution. Incubation was carried out at 27 1C in the dark.
Determinations of protein, H
2O
2, lipid
peroxidation, GSH, and AsA
The senescence of detached rice leaves was followed by measuring the decrease of protein content. For protein extraction, leaf segments
were homogenized in 50 mmol l1 sodium
phos-phate buffer (pH 6.8). The extracts were
centri-fuged at 17,600gnfor 20 min, and the supernatants
were used for determination of protein by the
method of Bradford (1976)and enzyme activities.
The H2O2content was measured colorimetrically as
described byJana and Choudhuri (1981). H2O2was
extracted by homogenizing leaf tissue with
phos-phate buffer (50 mmol l1, pH 6.5) containing
1 mmol l1 hydroxylamine. The homogenate was
centrifuged at 6000gn for 25 min. To determine
H2O2 content, the extracted solution was mixed
with 0.1% titanium sulphate in 20% (v/v) H2SO4. The
mixture was then centrifuged at 6000gnfor 25 min.
The absorbance was measured at 410 nm. The H2O2
content was calculated using the extinction
coeffi-cient 0.28 mmol1cm1. MDA, routinely used as an
indicator of lipid peroxidation, was extracted with 5% (w/v) trichloroacetic acid and determined
according to Heath and Packer (1968). GSH in 3%
sulfosalicylic acid extract and AsA in 5% (w/v) trichloroacetic acid extract were determined as
described by Smith (1985) and Laws et al. (1983),
respectively.
Enzyme assays
The enzyme assays in detail have been described
previously (Hurng and Kao, 1994). CAT activity
was assayed by measuring the initial rate of
disappearance of H2O2 (Kato and Shimizu, 1987).
The decrease in H2O2 was followed as the
decline in absorbance at 240 nm, and activity was calculated using the extinction coefficient
[40 (mmol l1)1cm1 at 240 nm] for H2O2 (Kato
and Shimizu, 1987). SOD was determined according to Paoletti et al. (1986). APOD was determined
according to Nakano and Asada (1981). The
decrease in AsA concentration was followed as the decline in optical density at 290 nm and activity was calculated using the extinction coefficient
[2.8 (mmol l1)1cm1at 290 nm] for AsA. GR was
determined by the method of Foster and Hess
(1980). One unit of activity for CAT, SOD, APOD, and GR was defined as the amount of enzyme which
degraded 1 mmol H2O2 per min, inhibited 50% the
rate of NADH oxidation observed in control, degraded 1 mmol of AsA per min, and decreased 1
A340per min, respectively.
Statistical analysis
The results presented were the mean of four replicates. Means were compared by either Stu-dent’s t-test or Duncan’s multiple range test at 5% level of significance.
Results
Yellowing is an obvious expression of leaf senes-cence and chlorophyll loss is often viewed as the principal criterion of senescence. The protein degradation during leaf senescence has been realized from earliest studies. We have shown that protein degradation precedes chlorophyll loss
dur-ing rice leaf senescence (Kao, 1980). Thus,
senes-cence of rice leaves in the present investigation was followed by measuring the decrease of protein. MDA is routinely used as an indicator of lipid peroxidation. The changes in protein and MDA contents in detached rice leaves treated with
45 mmol l1 ABA in the dark are shown in Figs. 1A
and B. The decrease in protein and increase in MDA was evident at 36 h after ABA treatment. Clearly, ABA is effective in promoting senescence of rice leaves. ABA treatment resulted in an increase in MDA, indicating that ABA brings about lipid perox-idation. Lipid peroxidation is caused by AOS (Kellogg and Fridovich, 1975; Thompson et al.,
1987). ABA treatment also caused an increase in
H2O2 content (Fig. 1C). The increase in H2O2 was
evident at 24 h after treatment of ABA, which preceded the decrease in protein and increase in
MDA. These results suggest that H2O2may play an
important role in regulating the senescence of rice leaves induced by ABA.
To demonstrate the involvement of H2O2 in the
effects induced by ABA in rice leaves, namely the
decrease in protein content and the increase in MDA content, dimethylthiourea (DMTU), a chemical
trap for H2O2(de Agazio and Zacchini, 2001), was
used. Detached rice leaves were incubated in a
solution containing 45 mmol l1ABA with or without
5 mmol l1DMTU. As indicated inFigs. 2A and B, the
decrease in protein and the increase in MDA in rice leaves caused by ABA were reduced by DMTU. Previously, we have shown that ABA increased the
ABA
H
2O
Protein (mg g -1 FW) 0 20 40 60 MDA (nmol g -1 FW) 0 10 20 30 40 Time (h) 0 12 24 36 48 H2 O2 ( µ mol g -1 FW) 0 10 20 30 40 ABA H2O*
*
*
*
*
*
*
(A) (B) (C)Figure 1. Changes in the contents of protein (A), MDA
(B), and H2O2(C) in rice leaves treated with either water
or 45 mmol l1 ABA in the dark. Values are means7SE
(n ¼ 4). Asterisks represent values that are significant at
Po0.05 level by Student’s t-test when compared to
activities of SOD, APOD, GR, and CAT and decreased
the contents of AsA and GSH in rice leaves (Hung
and Kao, 2003). DMTU was also observed to be effective in inhibiting ABA-increased activities of
SOD (Fig. 2C), APOD (Fig. 2D), GR (Fig. 2E), and CAT
(Fig. 2F) and ABA-decreased contents of AsA (Fig.
3A) and GSH (Fig. 3B) in rice leaves.
AOS, originating from the plasma-membrane NADPH oxidase, which transfers electrons from
cytoplasmic NADPH to O2to form O
d
2 , followed by
dismutation of Od
2 to H2O2, has been a recent
focus in AOS signaling. In several model systems investigated in plants, the oxidative burst and the
accumulation of H2O2appear to be mediated by the
activation of plasma-membrane NADPH oxidase
complex (Ogawa et al., 1997; del Rı´o et al., 1998;
Potikha et al., 1999; Pei et al., 2000; Orozco-Ca´rdenas et al., 2001; Jiang and Zhang, 2002).
Some chemical inhibitors of the NADPH oxidase complex found in mammalian neutrophils, such as diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibit the pathogen-, elicitor-, wound-, and
ABA-induced accumulation of H2O2in plants (Levine
et al., 1994;Auh and Murphy, 1995;Bestwick et al., 1977; Alvarez et al., 1998; Orozco-Ca´rdenas and Ryan, 1999; Jiang and Zhang, 2002). As shown in Fig. 4, when detached rice leaves were treated
with a solution of DPI (25 mmol l1) and IMD
(0.1 mmol l1), ABA-induced accumulation of H2O2
in rice leaves was reduced (Figs. 4C and F). DPI and
IMD also inhibited ABA-promoted leaf senescence (Figs. 4A and D), ABA-increased contents of MDA (Figs. 4B and E) and activities of SOD (Figs. 5A and 6A), APOD (Figs. 5B and 6B), GR (Figs. 5C and
6C), and CAT (Figs. 5D and 6D), and ABA-decreased
contents of AsA (Fig. 3A) and GSH (Fig. 3B).
Pr ot ein (m g -1 g FW) 0 10 20 30 40 MDA (n mo l g -1 FW) 0 10 20 30 40 S O D ( u nit s m g -1 prot ei n) 0.0 0.5 1.0 1.5 2.0 2.5 H2O DMTU AB A+D MTU ABA APO D (u nit s m g -1 prot ein ) 0 1 2 3 4 GR ( uni ts mg -1 pr ot e in ) 0.00 0.05 0.10 0.15 0.20 0.25 CAT ( uni ts m g -1 prote in) 0.0 0.1 0.2 0.3 0.4 H2O DMTU ABA +DM TU ABA (A) (B) (C) (D) (E) (F) a b d c a d c b c c a b b b a b c c a b c c a b
Figure 2. Effect of DMTU on the contents of protein (A), and MDA (B), and the activities of SOD (C), APOD (D), GR (E),
and CAT (F) in rice leaves treated with ABA. The concentrations of ABA and DMTU were 45 mmol l1 and 5 mmol l1,
respectively. All measurements were determined 2 days after treatment in the dark. Values are means7SE (n ¼ 4).
Another potential enzymatic source of H2O2
production is cell wall peroxidase (Papadakis and
Roubelakis-Angelakis, 1999; Blee et al., 2001). To test the possible involvement of cell wall
perox-idase, peroxidase inhibitors such as KCN and NaN3
were used. As is well known, KCN and NaN3are also
inhibitors of mitochondria respiration. To
distin-guish the effect of KCN and NaN3on respiration and
peroxidase activity, the concentrations of KCN and
NaN3 of 10 mmol l1 and 1 mmol l1, respectively,
were used, since these concentrations did not inhibit the respiration rate of detached rice leaves
(unpublished).Figure 7Cshows that the addition of
KCN and NaN3to detached rice leaves restored the
ABA-induced H2O2production to control levels. KCN
and NaN3also inhibited ABA-promoted senescence
(Fig. 7A), ABA-increased content of MDA (Fig. 7B)
and activities of SOD (Fig. 8A), APOD (Fig. 8B), GR
(Fig. 8C), and CAT (Fig. 8D), and ABA-decreased
contents of AsA (Fig. 8E) and GSH (Fig. 8F).
Discussion
Pei et al. (2000)were the first to demonstrate the
generation of H2O2 and its effects in guard cells
caused by ABA. In subsequent work, ABA-induced
increase in H2O2 has been reported for maize
seedlings, rice roots, and rice leaves (Jiang and
Zhang, 2001; Lin and Kao, 2001; Hung and Kao, 2003, Fig. 1C). On the other hand, ABA decreased
the release of H2O2 from germinating radish seeds
(Schopfer et al., 2001). It seems that H2O2
generation is not a common response to ABA and this response is not confined to guard cells.
In guard cells, ABA-induced H2O2 generation is
regulated by plasma-membrane NADPH oxidase (Pei
et al., 2000; Zhang et al., 2001). Recently, Jiang and Zhang (2002) also reported that plasma-membrane NADPH oxidase is involved in ABA- and water stress-induced antioxidant defense in leaves of maize seedlings. Here, we show that DPI and
IMD, inhibitors of NADPH oxidase (Levine et al.,
1994;Auh and Murphy, 1995;Bestwick et al., 1977; Alvarez et al., 1998; Orozco-Ca´rdenas and Ryan, 1999; Pei et al., 2000; Orozco-Ca´rdenas et al., 2001; Jiang and Zhang, 2002), reduced
ABA-induced H2O2production (Figs. 4C and F) and lipid
peroxidation (Figs. 4B and E), ABA-promoted
senescence (Figs. 4A and D), ABA-increased
anti-oxidative enzyme activities (Figs. 5 and 6), and
ABA-decreased antioxidants (Fig. 3) in rice leaves.
Similar results were obtained by using DMTU, a
chemical trap for H2O2 (Figs. 2 and 3).
Further-more, the increase in H2O2 content by ABA was
observed to be preceded the occurrence of leaf
senescence and the increase in MDA content (Figs.
1A and C). It appears that H2O2is involved in
ABA-induced senescence of rice leaves and that NADPH oxidase in rice leaf cells is involved in ABA-induced
H2O2production.
It has been shown that a high concentration of DPI can affect other enzymes potentially involved in the generation of AOS, including cell wall
peroxidase and nitric oxide synthase (Bolwell et
al., 1998; Orozco-Ca´rdenas et al., 2001; Schopfer et al., 2001). The fact that ABA-induced H2O2
accumulation in rice leaves can be inhibited by low
concentration (25 mmol l1) DPI, and can be
inhib-ited by both DPI and IMD strongly suggest that
ABA-dependent H2O2 generation originated, at least in
part, from plasma membrane NADPH oxidase.
0 2 4 6 8 10 H2O GSH (nmol g -1 FW) AsA ( µ mol g -1 FW) 0.0 0.5 1.0 1.5 2.0 (A) (B) ABA
ABA+DMTUABA+DPIABA+IMD
b a b b c a b b b c
Figure 3. Effect of DMTU, DPI, and IMD on the contents of AsA (A) and GSH (B) in rice leaves treated with ABA. The concentrations of ABA, DMTU, DPI, and IMD were
45 mmol l1, 5 mmol l1, 25 mmol l1, and 0.1 mmol l1,
respectively. All measurements were determined 2 days after treatment in the dark. Values are means7SE (n ¼ 4). Value with the same letter are not significantly
different at Po0.05 level, according to Duncan’s
Recently, a cell wall peroxidase has been
identified in French bean (Bolwell et al., 1998;
Blee et al., 2001), and a potentially
peroxidase-mediated H2O2production has been demonstrated
in Arabidopsis cultures challenged with a fungal
elicitor (Bolwell et al., 2002). Arabidopsis plants
transformed with an antisense bean peroxidase construct are hypersensitive with a fungal elicitor (Bolwell et al., 2002). Here, we show that KCN and
NaN3, inhibitors of peroxidase, prevented
ABA-induced H2O2 production (Fig. 7C) and lipid
peroxidation (Fig. 7B), ABA-promoted senescence
(Fig. 7A), ABA-increased antioxidative enzyme
activities (Figs. 8(A)–(D)), and ABA-decreased
anti-oxidants in rice leaves (Figs. 8E and F). It appears
that peroxidase is another H2O2-generating enzyme
in ABA-treated rice leaves. However, the endogen-ous rice peroxidase has yet to be identified.
Plasma-membrane NADPH oxidase transfers
elec-trons from cytoplasmic NADPH to O2to form O
d
2 ,
which is then dismutated to H2O2 by the action of
apoplastic SOD. It has been shown that NaN3
inhibits apoplastic Cu-Zn SOD (Ogawa et al.,
1997). Furthermore, KCN has the ability to
sca-venge H2O2 (Baker et al., 1998). It appears that
KCN and NaN3 are not fully specific to peroxidase,
which would explain the results that the addition of
KCN and NaN3to detached rice leaves restored the
ABA-induced H2O2production to control levels (Fig.
7C).
In plants, polyamines are thought to play an important role in growth, development and stress
P ro tein (m g g -1 FW) 0 10 20 30 40 50 MD A (n m o l g -1 FW) 0 10 20 30 40 H2 O2 ( µ mo l g -1 FW) 0 10 20 30 40 H2O DPI ABA +DPI ABA H2 O IMD ABA +IM D ABA a a c b a a c b c c a b b b a b c b a b c bc a b (A) (D) (B) (C) (E) (F)
Figure 4. Effect of DPI and IMD on the contents of protein (A, D), and MDA (B, E), and H2O2(C, F) in rice leaves treated
with ABA. The concentrations of ABA, DPI, and IMD were 45, 25 mmol l1, and 0.1 mmol l1, respectively. All
measurements were determined 2 days after treatment in the dark. Values are means7SE (n ¼ 4). Value with the same letter are not significantly different at Po0.05 level, according to Duncan’s multiple range test.
SOD (units mg -1 p rot ei n) 0 1 2 3 AP OD (units mg -1 pr ote in ) 0 1 2 3 4 GR (u nits mg -1 pr otei n ) 0.00 0.05 0.10 0.15 0.20 C A T (u nits mg -1 pr otei n ) 0.0 0.1 0.2 0.3 0.4 c c a b b b a b c c a b c c a b H2O DPI ABA+ DPI ABA H2O DPI ABA+DPI ABA (A) (B) (C) (D)
Figure 5. Effect of DPI on the activies of SOD (A), APOD (B), GR (C), and CAT (D) in rice leaves treated with ABA. The
concentrations of ABA and DPI were 45 and 25 mmol l1, respectively. All measurements were determined 2 days after
treatment in the dark. Values are means7SE (n ¼ 4). Value with the same letter are not significantly different at
Po0.05 level, according to Duncan’s multiple range test.
SOD (units m g -1 p rot ei n) 0 1 2 3 AP OD (u nits m g -1 pr ote in ) 0 1 2 3 4 GR (u nits mg -1 pr otei n) 0.00 0.05 0.10 0.15 0.20 CA T (u nits mg -1 pr otei n) 0.0 0.1 0.2 0.3 0.4 bc c a b c c a b b b a b c c a b H2O IMD ABA+ IMD ABA H2O IMD ABA+I MD ABA (A) (B) (C) (D)
Figure 6. Effect of IMD on the activies of SOD (A), APOD (B), GR (C), and CAT (D) in rice leaves treated with ABA. The
concentrations of ABA and IMD were 45 mmol l1and 0.1 mmol l1, respectively. All measurements were determined 2
days after treatment in the dark. Values are means7SE (n ¼ 4). Value with the same letter are not significantly
response (Walden et al., 1997). It has been shown
that H2O2 produced by diamine or polyamine
oxidase induced hypersensitive cell death in plants (Yoda et al., 2003). Previously, we have shown that ABA treatment had no effect on polyamine content
in rice leaves (Chen and Kao, 1991). Thus, diamine
or polyamine oxidase is unlikely to be affected by
ABA in rice leaves. An alternative source for H2O2
includes oxalate oxidase, an enzyme that degrades
oxalate to CO2 and H2O2 (Dumas et al., 1995).
Oxalate oxidase gene expression is induced by salt
stress, salicylate, and methyl jasmonate (Hurkman
and Tanaka, 1996). It is not known whether ABA will
activate oxalate oxidase in rice leaves. Further work is necessary to clarify this possibility.
Results observed in the present study suggest that NADPH oxidase, which shows sensitivity to DPI and IMD, and peroxidase, which is sensitive to KCN
and NaN3, are operating in ABA-treated rice leaves.
These two H2O2-generating enzymes were also
observed in tobacco protoplasts (Papadakis and
Roubelakis-Angelakis, 1999). It appears that when
rice leaves are treated with ABA, H2O2is generated
in the apoplast. Because apoplast has only a small
proportion of the cell’s antioxidant capacity, H2O2
will rapidly move into the cell to exert its effect on senescence. It has been suggested that peroxipor-ins or water channels (aquaporperoxipor-ins) may serve as
conduits for trans-membrane H2O2 transport (Neill
et al., 2002). Thus, H2O2can function as a mobile
signal in ABA-treated rice leaves, but whether H2O2
is the sole signal remains to be determined. When detached rice leaves are used to study senescence, wounding is always a problem. How-ever, in the present study, each long and narrow rice leaf was cut transversely, thus the area of
wounding was very small. Therefore, H2O2
genera-tion and senescence of detached leaves induced by ABA are unlikely to be complicated by the wound-ing effect. Since ABA is known to inhibit ethylene
production in detached rice leaves (Kao and Yang,
1983), ABA-induced H2O2 production and
senes-cence do not seem to be mediated through ethylene production.
The mechanism of AOS production and the molecules involved have been well investigated in animal cells, particularly in neutrophils. The NADPH oxidase complex, which consists of many components, is responsible for AOS production in neutrophil cells, and is activated by the binding of posphatidylinositol 3-phosphate to one of the
components (Ellson et al., 2001).
Phosphatidylino-sitol 3-phosphate is a product of phosphatidylino-sitol 3-kinase, which phosphorylates the D-3
position of phosphoinositides. Recently, Jung et
al. (2002)andPark et al. (2003)demonstrated that wortmannin or LY 294002, inhibitors of
phosphati-dylinositol 3-kinase, inhibited ABA-induced H2O2
production and stomatal closing and H2O2partially
reversed the effects of wortmannin or LY 294002 on ABA-induced stomatal closing. They suggested that phosphatidylinositol 3-phosphate is important in
NADPH oxidase-mediated H2O2 production during
ABA-induced stomatal closing. We have preliminary data indicating that wortmannin and LY 294002
prevented ABA-induced H2O2 production and
ABA-promoted senescence in rice leaves (Hung and Kao, unpublished observation). Work in this direction is presently under further investigation.
Prote in ( m g g -1 FW) 0 10 20 30 40 50 MDA (n mo l g -1 FW ) 0 10 20 30 40 H2O H2 O2 ( µmo l g -1 FW) 0 10 20 30 40 KCN NaN 3 ABA ABA +KCN ABA +NaN 3 (A) (B) (C) a a a b b c c c c a b c b b b a b b
Figure 7. Effect of KCN and NaN3 on the contents of
protein (A), and MDA (B), and H2O2 (C) in rice leaves
treated with ABA. The concentrations of ABA, KCN, and
NaN3were 45, 10 mmol l1, and 1 mmol l1, respectively.
All measurements were determined 2 days after treat-ment in the dark. Values are means7SE (n ¼ 4). Value with the same letter are not significantly different at
Acknowledgements
This work was supported by grant NSC 90-2313-B-002-267 from the National Science Council of the Republic of China.
References
Alvarez ME, Penell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C. Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 1998;92:773–84.
Auh CK, Murphy TM. Plasma membrane redox enzyme is
involved in the synthesis of O2and H2O2 by
Phy-tophthora elicitor-stimulated rose cells. Plant Physiol 1995;107:1241–7.
Baker CJ, Deahl K, Domek JM, Orlandi EW. Oxygen metabolism in plant/bacteria interactions: effect of DPI on the pseudo-NAD(P)H oxidase activity of
peroxidase. Biochem Biophys Res Commun
1998;252:461–4.
Begam HH, Choudhuri MA. H2O2 metabolism during
senescence of two submerged angiosperm Hydrilla and Ottelia: changes in enzyme activities in light and darkness. Biochem Physiol Pflanzen 1992;188:105–15. Bestwick CS, Brown IR, Bennett MHR, Mansfield JW. Localization of hydrogen peroxide accumulation dur-ing the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. Plant Cell 1977;9:209–21.
Blee KA, Jupe SC, Richard G, Bolwell GP. Molecular identification and expression of the peroxidase re-sponsible for the oxidative burst in French bean (Phaseolus vulgaris L) and related members of the gene family. Plant Mol Biol 2001;47:607–20.
Bolwell GP, Davies DR, Gerrish C, Auh CK, Murphy TM.
Comparative biochemistry of the oxidative
burst produced by rose and French bean cells reveals two distinct mechanisms. Plant Physiol 1998;116: 1374–85. S O D ( u n its mg -1 pr ot ein) 0.0 0.5 1.0 1.5 2.0 2.5 APO D (uni ts m g -1 pr ot e in ) 0.0 0.5 1.0 1.5 2.0 2.5 G R (uni ts m g -1 prote in) 0.00 0.05 0.10 0.15 0.20 CAT ( uni ts m g -1 p ro tei n) 0.0 0.1 0.2 0.3 0.4 As A ( µmo l g -1 FW ) 0 3 6 9 12 GS H (n mo l g -1 FW) 0.0 0.5 1.0 1.5 2.0 2.5 a a a b b c a a a a a b a b b c c c cd a b c d d a b b b b b H2O KCN NaN 3 AB A AB A+K CN ABA+ NaN 3 H2O KC N NaN 3 ABA AB A+KCN ABA+ NaN 3 c a b c c c (A) (D) (B) (C) (E) (F)
Figure 8. Effect of KCN and NaN3on the activies of SOD (A), APOD (B), GR (C), and CAT (D) and the contents of AsA (E)
and GSH (F) in rice leaves treated with ABA. The concentrations of ABA, KCN, and NaN3 were 45, 10 mmol l1, and
1 mmol l1, respectively. All measurements were determined 2 days after treatment in the dark. Values are means7SE
(n ¼ 4). Value with the same letter are not significantly different at Po0.05 level, according to Duncan’s multiple range test.
Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Genrrish C, Minibayeva F. The apoplastic oxidative burst in response to biotic stress in
plants: a three-component system. J Exp Bot
2002;53:1367–76.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.
Bueno P, Piqueras A, Kurepa J, Savoure´ A, Verbruggen N,
Van Montagu M, Inze´ D. Expression of antioxidant
enzymes in response to abscisic acid and high osmoticum in tobacco BY-2 cell cultures. Plant Sci 1998;138:27–34.
Chamnongpol S, Willekens H, Moeder W, Langebartels C,
Sandermann H, Van Montagu M, Inze´ D, Van Camp W.
Defense activation and enhanced pathogen tolerance
induced by H2O2in transgenic tobacco. Proc Natl Acad
Sci USA 1998;95:5818–23.
Chen CT, Kao CH. Senescence of rice leaves XXX. Levels of endogenous polyamines and dark-induced senescence of rice leaves. Plant Cell Physiol 1991;32:935–41. Chu C, Lee TM. The relationship between ethylene
biosynthesis and chilling tolerance in seedlings of rice (Oryza sativa). Bot Bull Acad Sin 1989;30:263–73. Creelman RA. Abscisic acid physiology and biosynthesis in
higher plants. Physiol Plant 1989;75:131–6.
de Agazio M, Zacchini M. Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in spermidine-treated maize roots. Plant Cell Environ 2001;24:237–44. del Rı´o LA, Pastori GM, Palma JM, Sandalio LM, Sevilla F,
Corpas FJ, Jime´nez A, Lo´pez-Huartas E, Herna´ndez JA.
The activated oxygen role of peroxisomes in senes-cence. Plant Physiol 1998;116:1195–200.
Dumas B, Freyssinet G, Pallett KE. Tissue-specific expression of germin-like oxalate oxidase during development and fungal infection of barley seedlings. Plant Physiol 1995;107:1091–6.
Ellson CD, Gobert-Gosse S, Anderson KE, Davidson K, Erdjument-Bromage H, Tempst P, Thuring JW, Cooper MA, Lim Z-Y, Holmes AB, Gaffney PRJ, Coadwell J, Chilvers ER, Hawkins PT, Stephens LR. PtdIns(3)P regulates the neutrophil oxidase complex by binding
to the PX domain of p40phox. Nat Cell Biol
2001;3:679–82.
Foster JG, Hess JL. Responses of superoxide dismutase and glutathione reductase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. Plant Physiol 1980;66:482–7.
Gepstein S, Thimann KV. Changes in the abscisic acid content of oat leaves during senescence. Proc Natl Acad Sci USA 1980;77:2050–3.
Guan L, Zhao J, Scandalios JG. Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic
stress: H2O2 is the likely intermediary signaling
molecule for the response. Plant J 2000;22:87–95. Heath RL, Packer L. Photoperoxidation in isolated
chloroplasts. I. Kinetics and stoichiometry of fatty
acid peroxidation. Arch Biochem Biophys
1968;125:189–98.
Hung KT, Kao CH. Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid. J Plant Physiol 2003;160:871–9.
Hurkman WJ, Tanaka CK. Effect of salt stress on germin
gene expression in barley roots. Plant Physiol
1996;110:971–7.
Hurng WP, Kao CH. Effect of flooding on the activities of some enzymes of activated oxygen metabolism, the levels of antioxidants, and lipid peroxidation in
senescing tobacco leaves. Plant Growth Regul
1994;14:37–44.
Jana S, Choudhuri MA. Glycolate metabolism of three submerged aquatic angiosperm during aging. Aquat Bot 1981;12:345–54.
Jiang M, Zhang J. Effect of abscisic acid on active oxygen species, antioxidative defense system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 2001;42:1265–73.
Jiang M, Zhang J. Involvement of plasma membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 2002;215:1022–30.
Jung J-Y, Kim Y-W, Kwak JM, Hwang J-U, Young J, Schroeder JI, Hwang I, Lee Y. Phosphatidylinositol 3-and 4-phosphate are required for normal stomatal movements. Plant Cell 2002;14:2397–412.
Kao CH. Senescence of rice leaves IV Influence of benzyladenine on chlorophyll degradation. Plant Cell Physiol 1980;21:1255–62.
Kao CH, Yang SF. Role of ethylene in the senescence of detached rice leaves. Plant Physiol 1983;73:881–5. Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen
G, Mullineaux P. Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 1999;84:654–7.
Kato M, Shimizu S. Chlorophyll metabolism in higher plants. VII. Chlorophyll degradation in senescing tobacco leaves: phenolic-dependent peroxidative de-gradation. Can J Bot 1987;65:729–35.
Kellogg EW, Fridovich I. Superoxide, hydrogen
peroxide, and singlet oxygen in lipid peroxidation by xanthine oxidase system. J Biol Chem 1975;250: 8812–7.
Kende H, Zeevaart JAD. The five classical plant hor-mones. Plant Cell 1997;9:1197–210.
Laws MY, Charles SA, Halliwell B. Glutathione and ascorbic acid in spinach chloroplasts: the effect of hydrogen peroxide and of paraquat. Biochem J 1983;210:899–903.
Levine A, Tenhaken R, Dixon RA, Lamb CJ. H2O2from the
oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 1994;79:583–93. Lin JN, Kao CH. Effect of oxidative stress by hydrogen
peroxide on senescence of rice leaves. Bot Bull Acad Sin 1998;39:161–5.
Lin CC, Kao CH. Abscisic acid induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings. Plant Sci 2001;160:323–9.
Mondal R, Choudhuri MA. Role of hydrogen peroxide in senescence of excised leaves of rice and maize. Biochem Physiol Pflanzen 1981;176:700–9.
Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 1981;22:867–80.
Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT. Hydrogen peroxide and nitric oxide as signalling molecules in plants. J Exp Bot 2002;53:1237–47. Nooden LD. Abscisic acid, auxin and other regulators of
senescence. in: Nooden LD, editor. Senescence and aging in plants. San Diego: Academic Press Inc.; 1988. p. 329–68.
Ogawa K, Kanematsu S, Asada K. Generation of super-oxide anion and localization of CuZn-supersuper-oxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification. Plant Cell Physiol 1997;38:1118–26.
Orozco-Ca´rdenas M, Ryan CA. Hydrogen peroxide is
generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc Natl Acad Sci USA 1999;96:6553–7.
Orozco-Ca´rdenas M, Narva´ez-Va´squez J, Ryan CA.
Hydro-gen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmo-nate. Plant Cell 2001;13:179–91.
Paoletti F, Aldinucci D, Mocali A, Capparini A. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal Biochem 1986;154:536–41.
Papadakis AK, Roubelakis-Angelakis KA. The generation of active oxygen species differs in tobacco and
grapevine mesophyll protoplasts. Plant Physiol
1999;121:197–205.
Parida RK, Kat M, Mishra D. Enhancement of senescence in excised rice leaves by hydrogen peroxide. Can J Bot 1978;56:2937–41.
Park KY, Jung J-Y, Park J, Hwang J-U, Kim Y-W, Hwang I, Lee Y. A role for phosphatidylinositol 3-phosphate in abscisic acid-induced reactive oxygen species genera-tion in guard cells. Plant Physiol 2003;132:92–8.
Pei ZM, Murata N, Benning G, Thomine S, Klu¨sener B,
Allen GJ, Grill E, Schroeder JI. Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in guard cells. Nature 2000;406:731–4. Potikha TS, Collins CC, Johnson DI, Delmer DP, Levine A.
The involvement of hydrogen peroxide in the differ-entiation of secondary walls in cotton fibers. Plant Physiol 1999;119:849–58.
Schopfer P, Plachy C, Frahry G. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light,
gibber-ellin, and abscisic acid. Plant Physiol
2001;125:1591–602.
Seo M, Koshiba T. Complex regulation of ABA biosynthesis. Trends Plant Sci 2002;7:41–8.
Smith IK. Stimulation of glutathione synthesis in photo-respiring plants by catalase inhibitors. Plant Physiol 1985;79:1044–7.
Taylor LB, Burbridge A, Thompson AJ. Control of abscisic acid synthesis. J Exp Bot 2000;21:1563–74.
Thompson JE, Legge RL, Barber RF. The role of free radical in senescence and wounding. New Phytol 1987;105:317–44.
Walden R, Cordeiro A, Tiburcio AF. Polyamines: small molecules triggering pathways in plant growth and development. Plant Physiol 1997;113:1009–13. Yang J, Zhang J, Wang Z, Zhu Q, Liu L. Abscisic acid
and cytokinins in the root exudates and leaves and their relationship to senescence and
remobiliza-tion of carbon reserves in rice subjected to
water stress during grain filling. Planta 2002;215: 645–52.
Yoda H, Yamaguchi Y, Sano H. Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants. Plant Physiol 2003;132:1973–81.
Zhang X, Zhang L, Dong F, Gao J, Galbraite DW, Song C-P. Hydrogen peroxide is involved in abscisic acid-induced
stomatal closure in Vicia faba. Plant Physiol
