Hydrogen peroxide is required for abscisic
acid-induced NH
4
+
accumulation in rice leaves
Kuo Tung Hung, Ching Huei Kao
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China Received 1 September 2004; accepted 15 November 2004
Summary
The role of H2O2in abscisic acid (ABA)-induced NH4
+
accumulation in rice leaves was
investigated. ABA treatment resulted in an accumulation of NH4+in rice leaves, which
was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of
NH4+in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H2O2,
was observed to be effective in inhibiting ABA-induced accumulation of NH4+in rice
leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-a-phenylnitrone, PBN), which
have previously been shown to prevent ABA-induced increase in H2O2contents in rice
leaves, inhibited ABA-induced increase in the content of NH4+. Similarly, the changes of
enzymes responsible for NH4+ accumulation induced by ABA were observed to be
inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H2O2was found to
increase NH4+ content, decrease GS activity, and increase protease and PAL-specific
activities in rice leaves. Our results suggest that H2O2is involved in ABA-induced NH4+
accumulation in rice leaves.
&2005 Elsevier GmbH. All rights reserved.
Introduction
NH4+ is a central intermediate of nitrogen
metabolism (Miflin and Lea, 1976). Glutamine
synthetase (GS) is the key enzyme in NH4+
assimila-tion and catalyzes the ATP-dependent condensaassimila-tion of NH4+with glutamate to produce glutamine (Miflin
and Lea, 1976). Phenylalanine ammonia-lyase (PAL) catalyzes the elimination of NH4+ from
phenylala-nine and produces trans-cinnamate (Hahlbrock and
www.elsevier.de/jplph KEYWORDS Abscisic acid; H2O2; NH4+; Oryza sativa
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.11.007
Abbreviations: ABA, abscisic acid; AOS, active oxygen species; c-PTIO, 2-(4-carboxy-2-phenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide; DMTU, dimethylthiourea; DPI, diphenyleneiodonium chloride; FW, fresh weight; GS, glutamine synthetase; IMD, imidazole; NO, nitric oxide; PAL, phenylalanine ammonia-lyase; PBN, N-tert-butyl-a-phenylnitrone
Corresponding author. Fax: +886 2 23620879. E-mail address: [email protected] (C.H. Kao).
Grisebach, 1979). NH4+, released from PAL reaction,
is known to be trapped in the glutamine molecule by the action of GS (Razel et al., 1996;van Heerden et al., 1996). Sakurai et al. (2001) provided evidence to show that GS is partly coupled to the reaction of PAL in developing rice leaves. Cd-induced NH4+ accumulation in rice leaves has been
proved to be associated with the decrease in GS activity and the increase in PAL-specific activity (Hsu and Kao, 2004).
It is generally believed that GS activity in plants is regulated at the transcriptional level (Hirel et al., 1987;Forde et al., 1989;Edwards et al., 1990;
Roche et al., 1993; Sukanya et al., 1994). Beside transcriptional regulation, GS activity in plants might also be regulated at the level of turnover. Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria (Levine, 1983; Rivett and Levine, 1990). Stieger and Feller (1997)have shown that GS degradation in illuminated chloroplasts requires the function of the photosynthetic electron transport chain. Chlor-oplastic GS of wheat seedlings has been reported to be particularly prone to degradation under oxida-tive stress conditions (Palatnik et al., 1999). By incubating soybean root extracts enriched in GS in a metal-catalyzed oxidation system to produce the hydroxyl radical, Ortega et al. (1999)have shown that GS is oxidized and that the oxidized GS is inactive and more susceptible to proteolysis than nonoxidized GS. It is clear that GS degradation requires the production of active oxygen species (AOS). We also demonstrated that paraquat, which is known to produce AOS, decreased GS activity and increased NH4+ content in rice leaves in the light
(Chien et al., 2002). It has been shown that the specific activity of protease (or proteolysis) in-creased under photooxidative environmental con-ditions and treatment with a hydroxyl radical-generating system or H2O2 (Casano and Trippi,
1992; Casano et al., 1990, 1994). Kumar and Knowles (2003) demonstrated that the specific activity of PAL induced by wounding in potato tuber is related to the ability to produce superoxide radicals.
Recently, many investigators have focused on the functional aspects of H2O2. H2O2is a constituent of
oxidative metabolism and is itself an AOS. Because H2O2 is relatively stable and diffusible through
membrane, it is generally thought to serve as a signal molecule under various abiotic stresses (Neill et al., 2002), in acclimation to photooxidative stress (Karpinski et al., 1999), in plant–pathogen interactions (Levine et al., 1994), and in abscisic acid (ABA)-induced stomatal closure (Zhang et al., 2001).
We have previously shown that NH4+accumulation
is associated with ABA-promoted senescence of rice
leaves (Chen et al., 1997). Evidence was also
presented to show that ABA-induced NH4+ in rice
leaves is attributed to a decrease in GS activity (Chen et al., 1997). In recent studies, we found that ABA increases the content of H2O2 in rice
leaves (Hung and Kao, 2003). Here we have
examined the possible involvement of H2O2 in
ABA-induced NH4+accumulation in rice leaves.
Materials and methods
Plant materials
Rice (Oryza sativa L., cv. Taichung Native 1) seeds were sterilized with 2.5% sodium hypochlor-ite 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 in 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 sunlight at 30 1C day/25 1C night and 90% relative humidity. The apical 3 cm of the third leaf was used in all experiments. A group of 10 segments was floated in a Petri dish containing 10 mL of test solution. Incubation was carried out at 27 1C in the dark.
Determination of NH
4+NH4+ was extracted by homogenizing leaf
seg-ments with a pestle and mortar using 0.3 mmol L1 sulphuric acid (pH 3.5). The homogenate was
centrifuged for 10 min at 39,000gn. The
super-natant was used to determine NH4+content by the
method of Weatherburn (1967). NH4+ content was
calculated using an extinction coefficient of
3.9982 mmol1cm1 and expressed as mmol g1
fresh weight (FW).
Enzyme assays
For extraction of GS, leaf samples were homo-genized with 10 mmol L1 Tris–HCl buffer (pH 7.6, containing 1 mmol L1 MgCl2, 1 mmol L1 EDTA and
1 mmol L1 2-mercaptoethanol) using a chilled
mortar and pestle. The homogenate was
centri-fuged at 15,000gn for 30 min and the resulting
activity. The whole extraction procedure was carried out at 4 1C. GS was assayed by the method of Oak et al. (1980). The reaction mixture contained in a final volume of 1 mL was 80 mmol Tris–HCl buffer, 40 mmol L-glutamic acid, 8 mmol ATP, 24 mmol MgSO4, and 16 mmol NH2OH, the final
pH was 8.0. The reaction was started with the addition of the enzyme extract and, after incuba-tion for 30 min at 30 1C, was stopped by adding 2 mL 2.5% (w/v) FeCl3and 5% (w/v) trichloroacetic acid
in 1.5 N HCL. After centrifugation, the absorbance of the supernatant was read at 540 nm. One unit of
GS activity is defined as 1 mmol L-glutamate
g-monohydroxamate formed per min. PAL was
ex-tracted and determined according to Hyodo and
Fujinami (1989). The calculation was based on the extinction coefficient [9500 (mmol L1)1cm1] for trans-cinnamic acid. One unit of activity for PAL was defined as the amount of enzyme which caused the formation of 1 nmol trans-cinnamic acid h1. For protease extraction, leaf samples were homo-genized in prechilled mortar and pestle with
10 mmol L1 Tris–HCl buffer (pH 7.4) containing
10 mmol L12-mercaptoethanol at 4 1C. The homo-genate was centrifuged at 15,000gnfor 30 min and
the resulting supernatant was used for protease assay. Protease was assayed according to the
method described by Sheoran and Garg (1978).
One unit of protease activity was defined as the amount of enzyme which increased 0.01 A280h1.
The method of Bradford (1976) was used to
determine protein content in enzyme extracts.
Statistical analysis
Statistical differences between measurements (n ¼ 4) on different treatments or on different times were analyzed following the Duncan’s multi-ple range test or Student’s t-test.
Results and discussion
NH4+ content in the control leaves remained
unchanged during the first 24 h of incubation in the dark and increased subsequently (Fig. 1). It is clear that ABA-treated rice leaves had higher NH4 +
content than the control leaves at 48 and 72 h after treatment (Fig. 1).
GS is the primary enzyme responsible for NH4+
assimilation in plants (Miflin and Lea, 1976). We observed that GS activity in the control leaves remained unchanged during 48 h of incubation and the decrease in GS activity in ABA-treated rice leaves was evident 36 h after treatment (Fig. 2A).
However, ABA had no effect on the specific activity of GS in rice leaves (Fig. 2D). Since NH4+is known to
be released through the action of PAL, the first
enzyme in the phenylpropanoids (Hahlbrock and
Grisebach, 1979), it is possible that ABA-induced NH4+accumulation is associated with the increase in
the activity or the specific activity of PAL in rice
NH 4 + ( µ mol g -1 FW) 0 0 5 10 15 12 24 36 48 60 72 20 ABA H2O2 * * Time (h)
Figure 1. Changes in NH4+contents in rice leaves treated
with either water or 45 mmol L1ABA in the dark. Vertical
bars represent standard errors (n ¼ 4). Asterisk
repre-sents value that is significant at Po0:05 level by
Student’s t-test when compared to water control.
GS (units g -1 FW ) 0 2 4 6 8 PA L (unit s m g -1 pro tein ) 0.0 0.5 1.0 1.5 2.0 ABA H2O Time (h) 0 12 24 36 48 Pr otease ( u nits m g -1 pr otein ) 0.0 0.5 1.0 1.5 2.0 2.5 GS (units m g -1 pro tein ) 0.00 0.05 0.10 0.15 0.20 PAL (units g -1 FW ) 0 1 2 3 Time (h) 0 12 24 36 48 Pro tea se (uni ts g -1 FW) 0 20 40 60 80 A D B E C F * * * * * *
Figure 2. Changes in the activities and the specific activities of GS (A,D), PAL (B,E) and protease (C,F) in rice
leaves treated with either water or 45 mmol L1 ABA in
the dark. Vertical bars represent standard errors (n ¼ 4).
Asterisk represents values that are significant at Po0:05
level by Student’s t-test when compared to water control.
leaves. It was found that NH4+accumulation caused
by ABA is associated with the specific activity of PAL (Fig. 2E), but not the activity of PAL (Fig. 2B). GS activity in plants might be regulated at the level of turnover (Stieger and Feller, 1997; Ortega et al., 1999; Palatnik et al., 1999). The decrease in GS activity in ABA-treated rice leaves is most likely related to the increase in the activity or the specific activity of protease. As shown in Figs. 2C and F, ABA was observed to be effective in increasing the specific activity, rather than activity of protease. ABA-induced decrease in the activity of GS and increase in the specific activities of PAL and protease in rice leaves (which occurred 36 h after treatment) are prior to ABA-induced
accumu-lation of NH4+ (which was observed 48 h after
treatment) (Figs. 1 and 2). It appears that GS, PAL, and protease are the enzymes responsible for
ABA-induced NH4
+
accumulation.
NH4+is a central intermediate in the metabolism
of nitrogen in plants. NH4+ can also be produced
during nitrate assimilation and photorespiration (Miflin and Lea, 1976). Since our experiments were conducted in the dark, NH4+ accumulation induced
by ABA is unlikely to have been produced from photorespiration. Previously, we have shown that NH4+ accumulation in rice leaves by methyl
jasmo-nate is attributable to an increase in reduction of nitrate (Chen and Kao, 1998). ABA is biologically
similar to methyl jasmonate (Weidhase et al.,
1987). It appears that an increase in nitrate
reduction induced by ABA may also contribute NH4
+
accumulation in ABA-treated rice leaves. Further experiments are required to clarify this possibility.
We have previously shown that ABA-induced H2O2
production in rice leaves is evident 24 h after treatment (Hung and Kao, 2004). In several plant
systems, H2O2 has been shown to function as a
signal molecule (Levine et al., 1994; Rao et al., 1997; Karpinski et al., 1999; Casano et al., 2001;
Zhang et al., 2001;Neill et al., 2002). It seems that the accumulation of H2O2in rice leaves induced by
ABA may play an important role in regulating the increase in NH4+content in rice leaves. To test this
hypothesis, dimethylthiourea (DMTU), a chemical trap for H2O2(Levine et al., 1994;Rao et al., 1997;
Casano et al., 2001), was used. As indicated in
Fig. 3D, ABA-induced NH4+ accumulation was
sig-nificantly reduced by DMTU. DMTU treatment was also observed to be effective in inhibiting the decrease in the activity of GS and the increase in the specific activities of protease and PAL in rice leaves caused by ABA [Figs. 3(A)–(C)].
AOS, originating from the plasma-membrane NADPH oxidase, which transfers electrons from
cytoplasmic NADPH to O2to form O2
, followed by dismutation of O2
to H2O2, has been a recent
focus in AOS signaling. There are reports indicating that oxidative burst and the accumulation of H2O2
are mediated by the activation of plasma-mem-brane 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 H2O2 in plants (Levine et al., 1994; Auh and
Murphy, 1995;Bestwick et al., 1997;Alvarez et al., 1998; Orozco-Ca´rdenas and Ryan, 1999; Jiang and Zhang, 2002). Previously, we also demonstrated that ABA-induced H2O2accumulation in rice leaves
can be inhibited by low-concentration (25 mmol L1)
DPI and 0.1 mmol L1 IMD, indicating that
ABA-dependent H2O2 generation originated, at least in
part, from plasma-membrane NADPH oxidase
(Hung and Kao, 2005). As shown in Fig. 4D, when rice leaves were treated with DPI and IMD, ABA-induced accumulation of NH4+ in rice leaves was
reduced. DPI and IMD also inhibited ABA-induced changes in the activities or specific activities of
the enzymes responsible for NH4+ accumulation
[Figs. 4(A)–(C)]. GS (uni ts g -1 FW) 0 2 4 6 8 PAL ( unit s m g -1 protein) 0.0 0.5 1.0 1.5 2.0 Protease (units mg -1 protein) 0 1 2 3 H2O ABA ABA+DMTU NH 4 + ( µ mol g -1 FW) 0 2 4 6 8 10 H2 O ABA ABA +D MTU A B D C a b c a b b b b a a b c
Figure 3. Effect of DMTU on the activities of GS (A), the specific activities of protease (B) and PAL (C), and the
content of NH4+(D) in rice leaves treated with ABA. The
concentrations of ABA and DMTU were 45 mmol L1 and
5 mmol L1, respectively. All measurements were
deter-mined 2 days after treatment in the dark. Vertical bars represent standard errors (n ¼ 4). Values with the same
letter are not significantly different at Po0:05 level,
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 phosphatidylinositol 3-phosphate to one of the components (Ellson et al., 2001). Phosphatidylino-sitol 3-phosphate is a product of phosphatidylino-sitol 3-kinase. Recently,Jung et al. (2002)andPark et al. (2003) demonstrated that wortmannin or LY 294002, inhibitors of phosphatidylinositol 3-kinase, inhibited ABA-induced H2O2production and
stoma-tal closing and H2O2 partially reversed the effects
of wortmannin or LY 294002 on ABA-induced stomatal closing. They suggested that phosphati-dylinositol 3-phosphate is important in NADPH
oxidase-mediated H2O2 production during
ABA-induced stomatal closing. We have preliminary data indicating that wortmannin or LY 294002 prevented ABA-induced H2O2 production and
ABA-induced NH4+accumulation in rice leaves (Hung and
Kao, unpublished observations). Work in this direc-tion is presently under further investigadirec-tion.
Nitric oxide (NO) is a bioactive free radical implicated in a number of physiological processes in plants, including growth, development, and defense responses (Lamattina et al., 2003). It has been shown that NO is able to counteract the
toxicity of paraquat and diquat, which is known to generate superoxide radicals, in potato and rice leaves (Beligni and Lamattina, 1999; Hung et al.,
2002). More recently, we have shown that
ABA-induced H2O2 production in rice leaves can be
reduced by NO donor N-tert-butyl-a-phenylnitrone
(PBN) (Hung and Kao, 2003). Here, we show that
PBN is effective in reducing ABA-induced accumu-lation of NH4+ (Fig. 5D), decrease in the activity of
GS (Fig. 5A), and increase in the specific activities of protease and PAL (Figs. 5B and C) in rice leaves. Meanwhile, these PBN effects can be reversed by 2- (4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazo-line-1-oxyl-3-oxide (c-PTIO), a NO-specific scaven-ger (Fig. 5), suggesting that the PBN effects are attributable to NO released.
If H2O2 indeed plays an important role in
ABA-induced accumulation of NH4+ in rice leaves,
exogenous H2O2is expected to increase the content
of NH4+ and influence the activities or specific
activities of enzymes related to NH4+accumulation
in rice leaves. As shown inFig. 6, it is indeed the case. Meanwhile, PBN counteracted the effect of H2O2and the protective effect of PBN was reversed
by c-PTIO (Fig. 6).
We have previously shown that paraquat, an AOS-generating chemical, decreases GS activity and
GS (uni ts g -1 FW) 0 2 4 6 8 PAL ( unit s m g -1 protein) 0.0 0.5 1.0 1.5 2.0 Protease (units mg -1 protein) 0 1 2 3 H2O ABA ABA +PB N ABA +PBN +c-P TIO NH 4 + ( µ mol g -1 FW) 0 2 4 6 8 10 H2O ABA AB A+P BN AB A+P BN+c-PTIO A B C D a b c c c c a a b c a b b a c c
Figure 5. Effect of PBN on the activities of GS (A), the specific activities of protease (B) and PAL (C), and the
content of NH4+ (D) in ABA-treated rice leaves in the
presence or absence of c-PTIO. The concentrations of
ABA, PBN, and c-PTIO were 45 and 100, and 100 mmol L1,
respectively. All measurements were determined 2 days after treatment in the dark. Vertical bars represent standard errors (n ¼ 4). Values with the same letter are not significantly different at Po0.05 level, according to Duncan’s multiple range test.
GS (units g -1 FW) 0 2 4 6 8 PAL ( unit s m g -1 protein) 0.0 0.5 1.0 1.5 2.0 Protease (units mg -1 protein) 0 1 2 3 H2O ABA ABA+DPI NH 4 + ( µ mol g -1 FW) 0 2 4 6 8 10 ABA+ IMD H2O ABA ABA+DPIABA+ IMD A B C D a b c d c b c a c b b a c a b b
Figure 4. Effect of DPI and IMD on the activities of GS (A), the specific activities of protease (B) and PAL (C),
and the content of NH4+ (D) in rice leaves treated with
ABA. The concentrations of ABA, DPI, and IMD were 45
and 25 mmol L1
, and 0.1 mmol L1, respectively. All
measurements were determined 2 days after treatment in the dark. Vertical bars represent standard errors (n ¼ 4). Values with the same letter are not significantly
different at Po0:05 level, according to Duncan’s multiple
increases NH4+ accumulation in rice leaves in the
light (Chien et al., 2002). It has been demonstrated that GS in plants is particularly prone to proteolysis under oxidative stress conditions (Ortega et al., 1999; Palatnik et al., 1999; Chien et al., 2002;
Ishida et al., 2002), and the specific protease activity increases under photooxidative environ-mental conditions and treatment with hydroxyl radical-generating system or H2O2 (Casano and
Trippi, 1992;Casano et al., 1990, 1994). It appears that the decrease in GS activity in ABA-treated rice leaves (Fig. 2A) may be, at least in part, attribu-table to the increase in specific protease activity (Fig. 2F).
Kumar and Knowles (2003) demonstrated that specific activity of PAL induced by wounding in potato tuber was related to the ability to produce superoxide radicals. Here, we provided evidence to show that H2O2is involved in ABA-induced specific
PAL activity in rice leaves. It is not known whether PAL in plants is prone to proteolysis under oxidative stress conditions. The fact that ABA treatment resulted in an increase in specific activities of both PAL and protease (Fig. 2E and F), suggests that PAL is resistant to proteolysis in rice leaves.
Our results indicated that H2O2 production
precedes the changes of enzymes associated with NH4
+
accumulation, and NH4
+
accumulation in ABA-treated rice leaves. Clearly, the links between ABA treatment, H2O2 production, enzymes responsible
for NH4+ accumulation, and NH4+ accumulation are
well established. The results reported here also suggest that the changes in enzyme activities or specific activities related to NH4+accumulation, and
the increase in NH4+ content demonstrated in rice
leaves are a consequence of H2O2 production
caused by ABA.
Acknowledgements
This study has been financially supported by 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 C-K, Murphy TM. Plasma membrane redox enzyme is
involved in the synthesis of O2 and H2O2 by
Phyto-phothora elicitor-stimulated rose cells. Plant Physiol 1995;107:1241–7.
Beligni MV, Lamattina L. Nitric oxide protects against cellular damage produced by methyl violgen herbi-cides in potato plants. Nitric Oxide Biol Chem 1999;3: 199–208.
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 1997;9:209–21.
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.
Casano LM, Trippi VS. The effect of oxygen radicals on proteolysis in isolated oat chloroplasts. Plant Cell Physiol 1992;33:329–32.
Casano LM, Go´mez LD, Trippi VS. Oxygen- and
light-induced proteolysis in isolated oat chloroplasts. Plant Cell Physiol 1990;31:377–82.
Casano LM, Lascano HR, Trippi VS. Hydroxyl radicals and a thylakoid-bound endopeptidase are involved in light-and oxygen-induced proteolysis in oat chloroplasts. Plant Cell Physiol 1994;35:145–52.
Casano LM, Martin M, Sabater B. Hydrogen peroxide mediates the chloroplastic Ndh complex under photo-oxidative stress in barley. Plant Physiol 2001;125: 1450–8. GS (units g -1 FW ) 0 2 4 6 8 PAL (units mg -1 pr ote in) 0.0 0.5 1.0 1.5 2.0 H2O P rotease (uni ts mg -1 p rot ein ) 0.0 0.5 1.0 1.5 2.0 2.5 NH 4 + ( µ mol g -1 FW) 0 2 4 6 8 10 H2O 2 H2O 2+P BN H2O 2+PBN+c-PTIO H2O H2O 2 H2O 2 +PB N H2O 2+P BN +c-P TIO A B C D a b c c c a b a a a a a b b c c
Figure 6. Effect of PBN on the activities of GS (A), the specific activities of protease (B) and PAL (C), and the
content of NH4+ (D) in H2O2-treated rice leaves in the
presence or absence of c-PTIO. The concentrations of
H2O2, PBN, and c-PTIO were 10 mmol L1 and 100 and
100 mmol L1, respectively. All measurements were
de-termined 2 days after treatment in the dark. Vertical bars represent standard errors (n ¼ 4). Values with the same
letter are not significantly different at Po0:05 level,
Chen SJ, Kao CH. Methyl jasmonate, ammonium, and leaf senescence in rice. J Plant Physiol 1998;152:353–7. Chen SJ, Hung KT, Kao CH. Ammonium accumulation is
associated with senescence of rice leaves. Plant Growth Regul 1997;21:195–201.
Chien H-F, Lin CC, Wang J-W, Chen CT, Kao CH. Changes in ammonium ion content and glutamine synthetase activity in rice leaves caused by excess cadmium are a consequence of oxidative damage. Plant Growth Regul 2002;36:41–7.
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.
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.
Edwards JW, Walker EL, Coruzzi GM. Cell-specific expression in transgenic plants reveals nonoverlapping roles for chloroplast and cytosolic glutamine synthe-tase. Proc Natl Acad Sci USA 1990;87:3459–63. 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. Nature Cell Biol 2001;
3:679–82.
Forde BG, Day HM, Turton JF, Shen W-J, Cullimore JV, Oliver JE. Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmen-tal and spatial patterns of expression in transgenic Lotus corniculatus plants. Plant Cell 1989;1:391–401. Hahlbrock R, Grisebach H. Enzymic controls in the biosynthesis of lignin and flavonoids. Annu Rev Plant Physiol 1979;30:105–30.
Hirel B, Bouet C, King B, Layzell D, Jacobs F, Verma DPS. Glutamine synthetase genes are regulated by ammo-nia provided externally or by symbiotic nitrogen fixation. EMBO J 1987;6:1167–71.
Hsu YT, Kao CH. Cadmium toxicity is reduced by nitric oxide in rice leaves. Plant Growth Regul 2004;42: 227–38.
Hung KT, Kao CH. Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid. J Plant Physiol 2003;160:871–9.
Hung KT, Kao CH. Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves. J Plant Physiol 2004;161:1347–57.
Hung KT, Chang CJ, Kao CH. Paraquat toxicity is reduced by nitric oxide in rice leaves. J Plant Physiol 2002; 159:159–66.
Hyodo H, Fujinami H. The effects of 2,5-norbornadiene on the induction of the activity of 1-aminocyclopro-pane-1-carboxylate synthase and of phenylalanine ammonia-lyase in wounded mesocarp tissue of Cucur-bita maxima. Plant Cell Physiol 1989;30:857–60. Ishida H, Anzawa D, Kokubun N, Makino A, Mae T. Direct
evidence for non-enzymatic fragmentation of
chlor-oplastic glutamine synthetase by a reactive oxygen species. Plant Cell Environ 2002;25:625–31.
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.
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;284:654–7.
Kumar GNM, Knowles NR. Wound-induced superoxide production and PAL activity decline with potato tuber
age and wound healing ability. Physiol Plant
2003;117:108–17.
Lamattina L, Garcı´a-Mata C, Graziano M, Pagnussat G. Nitric oxide: the versatility of an extensive signal molecule. Annu Rev Plant Biol 2003;54:109–36. Levine RL. Oxidative modification of glutamine
synthe-tase. I. Inactivation is due to loss of one histidine residue. J Biol Chem 1983;258:11823–7.
Levine A, Tenhaken R, Dixon RA, Lamb CJ. H2O2from the
oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 1994;79:583–93. Miflin BJ, Lea PJ. The pathway of nitrogen assimilation in
plants. Phytochemistry 1976;15:873–85.
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. Oak A, Stulen I, Jones K, Winspear MJ, Boesel IL. Enzymes
of nitrogen assimilation in maize roots. Planta 1980;148:477–84.
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 ML, 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 ML, Narva´ez-Va´squez J, Ryan CA.
Hydrogen 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.
Ortega JL, Roche D, Sengupta-Gopalan C. Oxidative turnover of soybean root glutamine synthetase. In vitro and in vivo studies. Plant Physiol 1999;119:1483–95. Palatnik JF, Carrillo N, Valle EM. The role of
photosyn-thetic electron transport in the oxidative degradation of chloroplastic glutamine synthetase. Plant Physiol 1999;121:471–8.
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.
Rao MV, Paliyath G, Ormrod DP, Murr DP, Watkins CB.
Influence of salicylic acid on H2O2production,
oxida-tive stress, and H2O2-metabolizing enzymes. Salicylic
acid-mediated oxidative damage requires H2O2. Plant
Physiol 1997;115:137–49.
Razel RA, Ellis S, Singh S, Lewis NG, Towers GHN. Nitrogen recycling in phenylpropanoid metabolism. Phytochemistry 1996;41:31–5.
Rivett AJ, Levine RL. Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes. Arch Biochem Biophys 1990;278:26–34.
Roche D, Temple SJ, Sengupta-Gopalan C. Two classes of differentially regulated glutamine synthetase genes are expressed in the soybean nodule: a nodule-specific class and a constitutively expressed class. Plant Mol Biol 1993;22:971–83.
Sakurai N, Katayama Y, Yamaya T. Overlapping expression of cytosolic glutamine synthetase and phenylalanine
ammonia-lyase in immature leaf blades of rice. Physiol Plant 2001;113:400–8.
Sheoran IS, Garg OP. Effect of salinity on the activities of RNase, DNase and protease during germination and early seedling growth of mung bean. Physiol Plant 1978;44:171–4.
Stieger PA, Feller U. Requirements for the light-stimu-lated degradation of stromal proteins in isolight-stimu-lated pea (Pisum sativum L.) chloroplasts. J Exp Bot 1997;48: 1639–45.
Sukanya R, Li MG, Snustad DP. Root- and shoot-specific responses of individual glutamine synthetase genes of maize to nitrate and ammonium. Plant Mol Biol 1994;26:1935–46.
van Heerden PS, Towers GHN, Lewis NG. Nitrogen metabolism in lignifying Pinus taeda cell cultures. J Biol Chem 1996;271:12350–5.
Weatherburn MW. Phenol-hypochlorite reaction for de-termination of ammonia. Anal Chem 1967;39:971–4. Weidhase RA, Kramell HM, Lehmann J, Liebisch HW, Lerbs
W, Parthier B. Methyl jasmonate induced changes in the polypeptide pattern of senescing barley leaf segments. Plant Sci 1987;51:177–86.
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 2001;126: 1438–48.
