The participation of hydrogen peroxide in methyl jasmonate-induced NH4+ accumulation in rice leaves

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The participation of hydrogen peroxide in methyl

jasmonate-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 22 August 2006; received in revised form 14 October 2006; accepted 17 October 2006

KEYWORDS H2O2; Methyl jasmonate; NH4+; Oryza sativa

Summary

Ammonium is a central intermediate in the nitrogen metabolism of plants. We have previously shown that methyl jasmonate (MJ) not only increases the content of H2O2,

but also causes NH4 +

accumulation in rice leaves. More recently, H2O2is thought to

constitute a general signal molecule participating in the recognition of and the response to stress factors. In this study, we examined the role of H2O2 as a link

between MJ and subsequent NH4+ accumulation in detached rice leaves. MJ

treatment resulted in an accumulation of NH4+in detached 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 appear to be the enzymes responsible for the accumulation of NH4 +

in MJ-treated detached rice leaves. Dimethylthiourea (DMTU), a chemical trap for H2O2, was observed to be effective in inhibiting MJ-induced NH4

+

accumulation in detached rice leaves. Scavengers of free radicals (sodium benzoate, SB, and glutathione, GSH), nitric oxide donor (N-tert-butyl-a-phenylnitrone, PBN), the inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI, and imidazole, IMD), and inhibitors of phosphatidylinositol 3-kinase (wortmannin, WM, and LY 294002, LY), which have previously been shown to prevent MJ-induced H2O2

production in detached rice leaves, inhibited MJ-induced NH4+ accumulation.

Similarly, changes in enzymes responsible for NH4 +

accumulation induced by MJ were observed to be inhibited by DMTU, SB, GSH, PBN DPI, IMD, WM, or LY. Seedlings of rice cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)-sensitive and those of cultivar Tainung 67 (TNG67) are JA-insensitive. On treatment with JA, H2O2

accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ- and abscisic

www.elsevier.de/jplph

Abbreviations: ABA, abscisic acid; DMTU, dimethylthiourea; DPI, diphenyleneiodonium chloride; FW, initial fresh weight; GS, glutamine synthetase; IMD, imidazole; JA, jasmonic acid; LY, LY 294002; MJ, methyl jasmonate; PAL, phenylalanine ammonia-lyase; PI3K, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol 3-phosphate; ROS, reactive oxygen species; STS, silver thiosulfate; TN1, Taichung Native 1; TNG67, Tainung 67; WM, wortmannin

Corresponding author. Fax: +886 2 23620879. E-mail address:[email protected] (C.H. Kao).

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acid-induced accumulation of NH4+ and changes in enzymes responsible for NH4+

accumulation in detached rice leaves, suggesting that the action of MJ and ABA is ethylene dependent.

Introduction

Ammonium is a central intermediate in the nitrogen metabolism of plants. Glutamine synthe-tase (GS) is a key enzyme in NH4+assimilation and

catalyzes the ATP-dependent condensation of NH4+

with glutamate to produce glutamine (Miflin and

Lea, 1976). Phenylalanine ammonia-lyase (PAL)

catalyzes the elimination of NH4+ from

phenylala-nine producing trans-cinnamic acid (Hahlbrock and Grisebach, 1979). NH4+, released from the PAL

reaction, is known to be incorporated into gluta-mine 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 is

asso-ciated with decreases in GS activity and increases in PAL specific activity (Hsu and Kao, 2004).

GS activity in plants is known to be regulated at the levels of transcription and 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 photosynthetic electron transport chain. Chloroplastic GS of wheat seed-lings has been reported to be particularly prone to degradation under oxidative stress conditions ( Pa-latnik 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 participa-tion of reactive oxygen species (ROS). We also demonstrated that paraquat, which is known to produce ROS, decreased GS activity and increased NH4+content in rice leaves in the light (Chien et al.,

2002). It has been shown that protease specific activity (or proteolysis) increased under photoox-idative environmental conditions 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 PAL specific activity induced by wounding in potato tubers is related to the ability to produce superoxide radicals.

Recently, researchers have focused on the func-tional aspects of H2O2. H2O2 is a constituent of

oxidative metabolism and is itself a ROS. Because H2O2 is a small, diffusible, and ubiquitous molecule

that can be synthesized, as a stimulus, it fulfills the important criteria for an intracellular messenger (Neill et al., 2002;Foyer and Noctor, 2005). Thermoprotec-tion obtained by spraying salicylic acid or by heat acclimation was suggested to be achieved by a common signal transduction pathway involving very early increases in H2O2content (Dat et al., 1998). In

tomato plants, H2O2has been shown to act as a second

messenger for induction of defense genes in response to wounding and systemin (Orozco-Ca´rdenas et al., 2001). It has been demonstrated that H2O2is required

for the induction of cytosolic ascorbate peroxidase mRNA by oxidative stress (Morita et al., 1999). H2O2

has now also been shown to be a critical component of abscisic acid (ABA)-induced stomatal closure (Pei et al., 2000; Zhang et al., 2001; Kwak et al., 2003) and ABA-induced rice leaf senescence (Hung and Kao, 2004b), ABA-induced activities of ascorbate perox-idase and glutathione reductase in rice roots (Tsai and Kao, 2004), and gibberellic acid-induced programed cell death in barley aleurone cells (Fath et al., 2001). Methyl jasmonate (MJ) was first considered to be secondary metabolite with a possible application in the perfume industry (Demole et al., 1962). It is now evident that jasmonates are a class of plant hormones, which mediate various aspects of devel-opmental and stress responses (Creelman and Mullet, 1997). MJ has been shown to cause H2O2production

in parsley suspension-cultured cells (Kauss et al., 1994) and to act as a signal molecule for the induction of defense genes in tomato plants (Orozco-Ca´rdenas et al., 2001). We have previously shown that MJ not only increases the content of H2O2(Hung and Kao,

2004a), but also causes NH4 +

accumulation (Chen and Kao, 1998) in rice leaves. In this paper, we have examined the possible involvement of H2O2 in

MJ-induced NH4+accumulation in rice leaves.

Materials and methods

Plant materials and treatments

Rice (Oryza sativa L., cv. Taichung Native 1, TN1, or Tainung 67, TNG 67) seeds were sterilized with 2.5%

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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 of incubation, uniformly germinated seeds were selected and cultivated in a 500 ml beaker containing half-strength Kimura B solution as described previously (Hsu and Kao, 2005). The hydroponically cultivated seedlings were grown for 12 days in a phytotron (College of Agriculture, National Taiwan University, Taipei Taiwan) with natural sunlight at 3071 1C (day)/2571 1C (night) and 90% relative humid-ity. The apical 3 cm of the third leaf of TN1 was used in 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. In experiments with intact leaves of TN1 and TNG67 seedlings, jasmonic acid (JA) was added directly to half-strength Kimura B solution at the time when the third leaf was fully expanded. Determination of NH4+and H2O2

NH4+was extracted by homogenizing leaf segments with

a pestle and mortar using 0.3 mM sulfuric acid (pH 3.5). The homogenate was centrifuged for 10 min at 39,000gn.

The supernatant was used to determine NH4 +

content by the method of Weatherburn (1967). NH4+ content was

calculated using an extinction coefficient of 3.9 mmol1cm1 and expressed as mmol g1 initial fresh weight (FW). H2O2 content was measured

colorimetri-cally as described by Jana and Choudhuri (1982). H2O2

was extracted by homogenizing leaf samples with phosphate buffer (50 mmol L1_{, pH 6.5) containing}

1 mmol L1 hydroxylamine. The homogenate was centri-fuged at 6,000gnfor 24 min. To determine H2O2content,

the extracted solution was mixed with 0.1% titanium chloride in 20% (v/v) H2SO4. The mixture was then

centrifuged at 6000gn for 25 min. The absorbance was

measured at 410 nm. Using this method, we obtained that absorbance increased linearly with the amount of H2O2

and addition of H2O2to extracts resulted in the predicted

increase of absorbance, i.e. added H2O2 was fully

recovered (data not shown). The H2O2 content in leaf

extracts was calculated using an extinction coefficient of 0.28 mmol1_cm1_.

Enzyme assays

For extraction of GS, leaf samples were homogenized with 10 mmol L1 Tris–HCl buffer (pH 7.6, containing 1 mmol L1 MgCl2, 1 mmol L

1

EDTA and 1 mmol L1 2-mercaptoethanol) using a chilled pestle and mortar. The homogenate was centrifuged at 15,000gn for 30 min and

the resulting supernatant was used for determination of GS activity. The whole extraction procedure was carried out at 4 1C. GS was assayed by the method ofOaks 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 by addition of the enzyme extract and, after incubation for 30 min at

30 1C, was stopped by adding 2 ml 2.5% (w/v) FeCl3 and

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 mmolL-glutamate g-monohydroxamate formed per min.

PAL was extracted and determined according toHyodo 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 mmol trans-cinnamic acid per h. For protease extraction, leaf samples were homogenized in prechilled mortar and pestle with 10 mmol L1 Tris–HCl buffer (pH 7.4) containing 10 mmol L1

2-mercaptoetha-nol at 4 1C. The homogenate was centrifuged at 15,000gn

for 30 min and the resulting supernatant was used for protease assay. Protease was assayed according to the method described bySheoran and Garg (1978). One unit of protease activity was defined as the amount of enzyme which increased 0.01 A280per h. The method ofBradford

(1976)was used to determine protein content in enzyme extracts.

Preparation of silver thiosulfate (STS)

A stock of STS was prepared by mixing equal volumes of 0.01 mol L1AgNO3and 0.04 mol

1

Na2S2O03(Liu et al.,

1990).

Statistical analysis

Statistical differences between measurements (n ¼ 4) on different treatments or at different times were analyzed following the Duncan’s multiple range test or Student’s t-test.

Results and discussion

MJ increases NH

4+

and H

2

O

2

Effect of MJ on the enzymes related to NH

4+

accumulation

GS is the primary enzyme responsible for NH4+

assimilation in plants (Miflin and Lea, 1976). It is clear that the decrease in GS activity (expressed on the basis of g FW), rather than GS specific activity (expressed on the basis of mg protein), induced by MJ is closely associated with NH4+ accumulation

(Fig. 2A and D). Since NH4+is known to be released

through the action of PAL, the first enzyme in the pathway of phenylpropanoid biosynthesis ( Hahl-brock and Grisebach, 1979), it is likely that MJ-induced NH4

+

accumulation is associated with an

increase in the activity or the specific activity of PAL in rice leaves. NH4+ accumulation caused by

MJ was 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 MJ-treated rice leaves is most likely related to the increase in the activity or the specific activity of protease. As shown in Fig. 2C and F, MJ was observed to be effective in increasing the specific activity, rather than activity of pro-tease. MJ-induced decrease in the activity of GS and increases in the specific activities of PAL and protease in rice leaves (which occurred 24 h after treatment) occur prior to MJ-induced accumulation of NH4+ (which occurred 36 h after treatment) and

after the onset of H2O2production (which occurred

12 h after treatment) (Figs. 1 and 2). It appears that GS, PAL, and protease are the enzymes responsible for MJ-induced NH4+accumulation. The

fact that MJ treatment resulted in an increase in the specific activities of both protease and PAL (Fig. 2E and F), suggests that PAL is resistant to proteolysis in detached rice leaves.

There is a dramatic increase in NH4 +

content between 36 and 48 h for MJ treatment (Fig 1A), which does not correspond to the kinetic of GS activity and PAL specific activity (Fig. 2A and E), suggesting that other factors may also contribute to the increase in NH4+ content. NH4+ is known to be

produced during nitrate assimilation and photo-respiration (Miflin and Lea, 1976). Previously, we have shown that NH4+ accumulation in MJ-treated

detached rice leaves is attributable to an increase in reduction of nitrate (Chen and Kao, 1998). Since our experiments were conducted in the dark, NH4+

accumulation induced by MJ is unlikely to have been produced from photorespiration.

Effect of sodium benzoate (SB), reduce

glutathione (GSH), and nitric oxide (NO)

donor

Previously, we have shown that free radical scavengers, such as SB and GSH, partially inhibited the increase in H2O2content in rice leaves caused

by MJ (Hung and Kao, 2004a). NO is a bioactive free radical implicated in a number of physiological processes in plants (Lamattina et al., 2003). We have shown that MJ-induced H2O2 production in

rice leaves can be reduced by the NO donor N-tert-butyl-a- phenylinitrone (PBN) (Hung and Kao, 2004a). Here, we show that SB, GSH, and PBN are effective in reducing MJ-induced accumulation

NH 4 + (µ mol g -1 FW) 0 20 40 60 Time (h) 0 12 24 36 48 H2 O2 (µ mol g -1 FW) 0 10 20 30 40 50 MJ H2O * * * * * * A B Figure 1. Changes in NH4 +

(A) and H2O2 (B) contents in

detached rice leaves treated with either water or 45 mmol L1 MJ in the dark. Vertical bars represent standard errors (n ¼ 4). Asterisks indicate values that are significantly different between H2O and MJ

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of NH4+ (Fig. 3D), decreasing the activity of GS

(Fig. 3A), and increasing the specific activities of protease and PAL (Fig. 3B and C) in rice leaves. Meanwhile, the PBN effects can be reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimida-zoline-1-oxyl-3-oxide (c-PTIO), a NO-specific sca-venger (Fig. 3), suggesting that the PBN effects are attributable to NO release.

Effect of dimethylthiourea (DMTU), a

chemical trap for H

2

O

2

and NADPH oxidase

inhibitors

To demonstrate the involvement of H2O2 on the

effects induced by MJ in detached rice leaves, namely the increase in NH4+ content and the

changes in enzymes responsible for NH4+

accumula-tion, DMTU, a chemical trap for H2O2 (de Agazio

and Zacchini, 2001), was used. Detached rice

leaves were incubated in a solution containing 45 mmol L1 _{MJ with or without 5 mmol L}1 _DMTU.

The increase in the content of NH4 +

, the decrease in

the activity of GS, and the increase in the specific activities of PAL and protease caused by MJ was observed to be reduced by DMTU (Fig. 4).

ROS, originating from the plasma-membrane NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2to form O

d

2 , followed by

dismutation of O₂d to H2O2, has been a recent

focus in ROS signaling research (Neill et al., 2002). Diphenyleneiodonium chloride (DPI) and imidazole (IMD) are known to be inhibitors of NADPH oxidase (Orozco-Ca´rdenas et al., 2001). Recently, we also demonstrated that MJ-induced H2O2 accumulation

in detached rice leaves can be inhibited by low concentrations of DPI (1 mmol L1) and IMD (100 mmol L1) (Hung et al., 2006), indicating that MJ-dependent H2O2 generation is originates, in

part, from plasma-membrane NADPH oxidase. When detached rice leaves were treated with DPI and IMD, MJ-induced NH4+ accumulation in rice

leaves was reduced (Fig. 4D). DPI and IMD also inhibited the decrease in the activity of GS and the increase in the specific activities of PAL and protease caused by MJ (Fig. 4A, B and C).

GS (units g -1 FW) 0 2 4 6 8 PAL (units mg -1 protein) Time (h) 0 12 24 36 48 Protease (units mg -1 protein) 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 2.5 GS (units mg -1 protein) 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 Protease (units g -1 FW) 0 20 40 60 80 MJ H2O * * * * * * * * * * * * * * A B C F E D

Figure 2. Changes in the activities and the specific activities of GS (A, D), PAL (B, E), and protease (C, F) in detached rice leaves treated with either water or 45 mmol L1 MJ in the dark. Vertical bars represent standard errors (n ¼ 4). Asterisks indicate values that are significantly different between H2O and MJ treatments at Po0.05 by Student’s t-test.

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GS (units g -1 FW) 0 2 4 6 8 PAL (units mg -1 protein) 0.0 0.5 1.0 1.5 2.0 2.5 Protease (units mg -1 protein) 0 1 2 3 H2O MJ MJ+SB MJ+GSHMJ+PBN MJ+PBN+c-PTIO H2O MJ MJ+SB MJ+GSHMJ+PBN MJ+PBN+c -PTIO NH 4 + ( µ mol g -1 FW) 0 20 40 60 a a a a b c c d e b c c _c d b c c d e b c c c d A B C D

Figure 3. Effect of SB, GSH, and PBN on the activities of GS (A), the specific activities of protease (B) and PAL (C), and the content of NH4+(D) in MJ-treated detached rice leaves in the presence or absence of c-PTIO. The concentrations of

MJ, SB, GSH, PBN, and c-PTIO were 45 mmol L1_{, 10 mmol L}1_{, 30 mmol L}1_{, 100 mmol L}1_{, and 100 mmol L}1_,

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, according to Duncan’s multiple range test. PAL (units mg -1 protein) 0.0 0.5 1.0 1.5 2.0 2.5 GS (units g -1 FW) 0 2 4 6 8 Protease (units mg -1 FW) 0 1 2 3 NH 4 + ( µ mol g -1 FW) 0 20 40 60 A B C D a b b d c a b c c d a c b c d c a b b d H2O MJ MJ+DMTUMJ+DPIMJ+IM D H2O MJ MJ+DMTUMJ+DPIMJ+IM D

Figure 4. Effect of DMTU, 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 detached rice leaves treated with MJ. The concentrations of MJ, DMTU, DPI, and IMD were

45 mmol L1, 5 mmol L1, 1 mmol L1, 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, according to Duncan’s multiple range test.

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Collectively, these results appear to suggest that the changes in enzyme activities or specific activities related to NH4+ accumulation, and the

increase in NH4+content in detached rice leaves are

a consequence of H2O2 production caused by MJ.

This suggestion is supported further by the ob-servations that exogenous application of H2O2 is

able to increase NH4+content, decrease GS activity,

and increase protease and PAL specific activities in detached rice leaves (Hung and Kao, 2005).

Effect of phosphatidylinositol 3-kinase (PI3K)

inhibitors

The neutrophil NADPH oxidase consists of two plasma-membrane proteins, gp91phox and p22phox, which together form hetrerodimeric flavocyto-chrome b558, and three cytosolic regulatory

proteins, p40phox, p47phox, and p67phox, which translocate to the plasma-membrane after stimula-tion to form the active complex (Sagel and Abo, 1993; Henderson and Chappell, 1996). In neutro-phils, NADPH oxidase is activated by binding phosphatidylinositol 3-phosphate (PI3P), a product of PI3K, to the PX domain of p40phox(Ellson et al., 2001). It has been shown that PI3P is important in

NADPH oxidase-mediated H2O2 production during

ABA-induced stomatal closure (Jung et al., 2002;

Park et al., 2003), ABA-promoted leaf senescence (Hung and Kao, 2004b), MJ-promoted leaf senes-cence (Hung et al., 2006), and during auxin-induced root gravitropic responses (Joo et al., 2005). LY 294002 (LY) and wortmannin (WM) are inhibitors of PI3K, a product of which is PI3P. Recently, we also demonstrated that LY (100 mmol L1) or WM (1 mmol L1) inhibited MJ-induced H2O2 production in detached rice leaves

(Hung et al., 2006). When detached rice leaves were treated with a solution of LY or WM, MJ-induced accumulation of NH4+ in detached rice

leaves was reduced (Fig. 5D). LY or WM also inhibited the decrease in the activity of GS (Fig. 5A) and the increase in the specific activities of PAL and protease (Fig. 5B and C) in rice leaves caused by MJ. Exogenous H2O2 (1 mmol L1) was

observed to reverse the inhibitory effect of LY or WM on MJ-induced accumulation of NH4+ and

changes of enzymes responsible for the accumula-tion of NH4+(Fig. 5).

The fact that PI3K inhibitors, which reduced the MJ-induced H2O2 production (Hung et al., 2006),

were able to prevent the MJ-induced NH4 +

accumu-lation and changes in enzymes related to NH4 + GS (units g -1 FW) 0 2 4 6 8 PAL (units mg -1 protein) Protease (units mg -1 protein) 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 NH 4 + ( µ mol g -1 FW) 0 20 40 60 a b b c c d d d a a a b c d e f a b b c c d d d a b _b c c d d d A B C D MJ LY WM H₂O₂

Figure 5. Effect of LY and WM on the activities of GS (A), the specific activities of protease (B) and PAL (C), and the content of NH4+(D) in MJ-treated detached rice leaves in the presence or absence of H2O2. The concentrations of LY, WM,

MJ, and H2O2 were 100 mmol L 1

, 1 mmol L1 , 45 mmol L1and 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, according to Duncan’s multiple range test.

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accumulation in detached rice leaves (Fig. 5), strengthen further our conclusion that H2O2 is

necessary for MJ-induced NH4+ accumulation and

changes in enzymes related to NH4+accumulation.

JA induces H

2

O

2

and NH

4+

accumulation in

the leaves of cultivar TN1 seedlings but not

in cultivar TNG67

Fig. 6 shows the effect of JA concentrations, in the range 5–40 mmol L1, applied over a period of 3 days, on the contents of NH4+and H2O2in the second

leaves of rice seedlings. It is clear that increasing JA concentration progressively increases NH4+

con-tent in leaves of TN1 seedlings but not in leaves of TNG67 (Fig. 6B and D). It appears that, in terms of NH4

+

accumulation, TNG67 is a JA-insensitive culti-var and TN1 is a JA-sensitive. If H2O2is important in

regulating NH4+accumulation, then H2O2content is

expected to be increased in JA-treated seedlings of TN1 but not in TNG67. As indicated inFig. 6A and C, this is the case.

An increase in ethylene sensitivity is

associated with MJ- and ABA-induced NH

4+

accumulation in detached rice leaves

In our recent work, we reported that H2O2 is

necessary for ABA-induced NH4+ accumulation in

detached rice leaves (Hung and Kao, 2005). Since endogenous ABA content decreased in MJ-treated rice leaves (Wang and Kao, 1999), it is unlikely that the effect of MJ on NH4+ accumulation in detached

rice leaves is mediated through ABA. Previously, we demonstrated that MJ- and ABA-induced H2O2

production in detached rice leaves was inhibited by STS, an inhibitor of ethylene action, and concluded that one of the earliest events following MJ or ABA treatment is modulating ethylene sensitivity, which then causes the production of H2O2 (Hung et al., 2006). Here, we also observed

that treatment of rice leaves with STS inhibited the accumulation of NH4+, the decrease in GS activity,

and the increase in protease and PAL specific activities caused by MJ and ABA (Fig. 7). Results suggest that ABA- or MJ-induced NH4+accumulation

is ethylene dependent. JA (µM) JA (µM) 0 10 20 30 40 0 10 20 30 40 NH 4 + ( µ mol g -1 FW) 0 5 10 15 20 25 a _a a a a a b c c d TN1 H2 O2 (µ mol g -1 FW) 0 10 20 30 40 TNG67 a a a a a a b b b c A B C D

Figure 6. Effect of JA on the contents of H2O2 (A, C), and NH4 +

(B, D) in the second leaves of rice seedlings. Rice seedlings were cultivated in Kimura B solution in a Phytron with natural sunlight at 30 1C (day)/25 1C (night) at 90% relative humidity. JA was added to the Kimura B solution when the third leaves were fully expanded. H2O2 and NH4+

contents were determined 3 days after adding JA. Vertical bars represent standard errors (n ¼ 4). Values with the same letter are not significantly different at Po0.05, according to Duncan’s multiple range test.

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Conclusion

Our results indicated that H2O2 production

preceded the changes in enzyme activity asso-ciated with NH4+ accumulation in MJ-treated

de-tached rice leaves. In terms of NH4+, it was observed

that rice seedlings of TN1 are JA-sensitive and those of TNG67 are JA-insensitive. On treatment with JA, H2O2 accumulated in the leaves of TN1

seedlings but not in the leaves of TNG67. This work establishes the links between MJ (or JA) treatment, H2O2, enzymes responsible for NH4+ accumulation,

and NH4+accumulation.

Acknowledgment

This research was supported by the National Science Council of the Republic of China (NSC 94-2313-B-002-052).

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GS (units g -1 FW) 0 2 4 6 8 10 Protease (units mg -1 protein) 0 1 2 3 4 H2O _{STS ABA} ABA+STS MJ MJ+STS H2O _{STS ABA} ABA+STS MJ MJ+STS PAL (units mg -1 protein) 0.0 0.5 1.0 1.5 2.0 2.5 NH 4 + ( µ mol g -1 FW) 0 20 40 60 a a a b b c d d a b b c c d b _a a a a a b c d e A B C D

Figure 7. Effect of STS on the activities of GS (A), the specific activities of protease (B) and PAL (C), and the content of NH4+(D) in detached rice leaves in the presence or absence of ABA and MJ. The concentrations of STS, ABA and MJ were

200, 45, and 45 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, according to Duncan’s multiple range test.

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