Phosphatidylinositol 3-phosphate is required for abscisic acid-induced hydrogen peroxide production in rice leaves

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Phosphatidylinositol 3-phosphate is required for abscisic acid-induced

hydrogen peroxide production in rice leaves

Kuo Tung Hung and Ching Huei Kao

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China; *Author for correspondence (e-mail: [email protected])

Received 9 August 2004; accepted in revised form 28 January 2005

Key words: Abscisic acid, Hydrogen peroxide, NADPH oxidase, Oryza sativa, Phosphatidylinositol 3-kinas, Phosphatidylinositol 3-phosphate

Abstract

Rice leaves produce H2O2in response to abscisic acid (ABA), which results in induction of senescence and

accumulation of NH4+. The upstream steps of the ABA-induced H2O2production pathway in rice leaves

remain largely unclear. In animal cells, H2O2production in neutrophils is activated by phosphatidylinositol

3-phosphate (PI3P), a product of phosphatidylinositol 3-knase (PI3K). In the present study, we examined whether PI3P plays a role in H2O2 production in rice leaves exposed to ABA. We found that PI3K

inhibitors LY 294002 (LY) or wortmannin (WM) inhibited ABA-induced H2O2production, senescence and

NH4+accumulation. Hydrogen peroxide almost completely rescued the inhibitory effect of LY or WM. It

appears that PI3P plays a role in ABA-induced H2O2production, senescence, and NH4+accumulation in

rice leaves.

Abbreviations:ABA – abscisic acid; AOS – active oxygen species; DMSO – dimethyl sulfoxide; FW – fresh weight; GS – glutamine synthetase; LY – LY 294002; MDA – malondialdehyde; PAL – phenylalanine ammonia-lyase; PI3K – phosphatidylinositol 3-kinase; PI3P – phosphatidylinositol 3-phosphate; WM – wortmannin

Introduction

Recently, many researchers have focused on the functional aspect of H2O2. H2O2is a constituent of

oxidative metabolism and is itself an active oxygen species (AOS). Because H2O2is a small, diffusible,

and ubiquitous molecule that can be synthesized, as well as destroyed, rapidly in response to exter-nal stimuli, it fulfills the important criteria for an intracellular messenger.

AOS, originating from the plasma-membrane NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2to form O2 Æ, followed

by dismutation of O2 Æ to H2O2, has been a recent

focus in AOS signaling. The neutrophil NADPH oxidase consists of two plasma-membrane pro-teins, gp91phox and p22phox, which together form heterodimeric flavocytochrome b 558, and three

cytosolic regulatory proteins, p40phox, p47phox, and p67phox, which translocate to the plasma-membrane after stimulation to form the active complex (Segal and Abo 1993; Henderson and Chappell 1996). Plant homologues of the animal NADPH oxidase protein subunits have been identified, and some of their genes have been sequenced (Xing et al. 1997; Keller et al. 1998; Torres et al. 1998; Sagi and Fluhr 2001). These observations have demonstrated the presence of a plant NADPH oxidase resembling

the neutrophil NADPH oxidase. In fact, in several plants systems investigated, the oxidative burst and the accumulation of H2O2 are mediated by the

activation of plasma-membrane NADPH oxidase (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; Hung and Kao 2004). Since the discovery in the early to mid-1980s, the enzyme responsible for D-3 phosphorylation of phosphoinositides, that is the phosphatidylinositol 3-kinase (PI3K), has been the subject of intensive study (Charpenter and Cantley 1996). There are three types of PI3K in animal cells (Toker and Cantly 1997). In plants, only type III PI3K has been reported (Hong and Verma 1994; Welters et al. 1994; Bunney et al. 2000). It has been shown that NADPH oxidase is activated by binding phosphatidylinositol 3-phosphate (PI3P), a prod-uct of PI3K, to the PX domain of p40phox(Ellson et al. 2001). Jung et al. (2002) demonstrated that guard cells overexpressing PI3P-binding protein resulted in decreased stomatal closure in response to ABA. They also showed the similar effects in guard cells treated with PI3K inhibitors wort-mannin (WM) or LY 294002 (LY) and concluded that PI3P is required for ABA-induced stomatal closure (Jung et al. 2002). This conclusion is sup-ported further by the recent observation that H2O2

partially reversed the effect of WM or LY on ABA-induced stomatal closure (Park et al. 2003).

We have previously shown that ABA not only increases the content of H2O2, but also promotes

senescence (protein loss) and induces lipid peroxi-dation, as judged by the content of malondialde-hyde (MDA), in rice leaves (Hung and Kao 2003). Recently, we also found that NADPH oxidase is an enzyme responsible for H2O2generation in

ABA-treated rice leaves and H2O2is involved in

ABA-induced senescence of rice leaves (Hung and Kao 2004). In this paper, we investigated the possibility that PI3P, as found in animal cells and guard cells, activates H2O2 generation in ABA-treated rice

leaves by using PI3K inhibitors WM and LY.

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 wa-ter. These seeds were then germinated in Petri dishes with wetted filter paper at 37C under dark conditions. After 48 h incubation, uniformly ger-minated 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 nat-ural sunlight 30C day/25 C night and 90% rel-ative humidity. The apical 3 cm of the third leaf was used in all experiments. A group of ten seg-ments was floated in a Petri dish containing 10 ml of test solution. Incubation was carried out at 27C in the dark.

Test solutions included ABA (45 lM), H2O2

(1 mM), and PI3K inhibitors, LY (100 lM) and WM (1 lM). All chemicals were purchased from Sigma Co. (St. Louis, MO, USA). LY and WM stock solutions were prepared in 100% dimethyl sulfoxide (DMSO).

Determinations of protein, H2O2, lipid peroxidation,

and NH4+

For protein extraction, leaf segments were

homogenized in 50 mM sodium phosphate buffer (pH 6.8). The extracts were centrifuged at 17,600 · g for 20 min, and the supernatants were used for determination of protein by the method of Bradford (1976). The H2O2 content was

mea-sured colorimetrically as described by Jana and

Choudhuri (1981). H2O2 was extracted by

homogenizing leaf tissue with phosphate buffer (50 mM, pH 6.5) containing 1 mM

hydroxyl-amine. The homogenate was centrifuged at

6000· g for 25 min. To determine H2O2content,

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

mixture was then centrifuged at 6000· g for 25 min. The absorbance was measured at 410 nm. The H2O2 content was calculated using the

extinction coefficient 0.28 lmol 1cm 1. MDA, routinely used as an indicator of lipid peroxida-tion, was extracted with 5% (w/v) trichloroacetic acid and determined according to Heath and Packer (1968). NH4+ was extracted and its

con-centration determined as described previously (Chien and Kao 2000).

Enzyme assays

For extraction of glutamine synthetase (GS), leaf samples were homogenized with 10 mM Tris-HCl buffer (pH 7.6, containing 1 mM MgCl2, 1 mM

EDTA and 1 mM 2-mercaptolethanol) using a chilled pestle and mortar. The homogenate was centrifuged at 15,000· g for 30 min and the resulting supernatant was used for determination of GS activity. The whole extraction procedure was carried out at 4C. GS was assayed by the method of Oak et al. (1980). The reaction mix-ture contained in a final volume of 1 ml was 80 lmol Tris–HCl buffer, 40 lmol L-glutamic

acid, 8 lmol ATP, 24 lmol MgSO4, and 16 lmol

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 C, was stop-ped 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 lmol L-glutamate

c-monohydroxa-mate formed per minute. Phenylalanine ammo-nia-lyase (PAL) was extracted and determined according to Hyodo and Fujinami (1989). The calculation was based on the extinction coeffi-cient (9500 M 1cm 1) for trans-cinnamic acid. One unit of activity for PAL was defined as the amounts of enzyme which caused the formation of 1 nmol trans-cinnamic acid per hour. For protease extraction, leaf samples were homoge-nized in prechilled pestle and mortar with 10 mM Tris–HCl buffer (pH 7.4) containing 10 mM 2-mercaptoethanol at 4C. The homogenate was centrifuged at 15,000· g for 30 min and the resulting supernatant was used for protease as-say. 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 A280 per

hour.

Statistical analysis

Statistical differences between measurements (n = 4) on different treatments or on different times were analyzed following Duncan’s multiple range test.

Results

The most obvious character of leaf senescence is yellowing. Chlorophyll loss has long been consid-ered the principal criterion of senescence. The protein breakdown that occurs during leaf senes-cence has been recognized since the earliest studies performed. We have shown that protein break-down precedes chlorophyll loss during rice leaf senescence (Kao 1980). Thus, senescence of rice leaves in the present investigation was followed by measuring the decrease of protein.

LY and WM have been shown to be inhibitors for PI3K in animal cells (Vlahos et al. 1994; Ui et al. 1995). These inhibitors also affected pro-duction of PI3P in Arabidopsis protoplasts (Kim et al. 2001). Figure 1 shows that the effect of LY or WM concentration on protein content of rice leaves in the presence or absence of 45 lM ABA. ABA alone decreased protein content in rice leaves, indicating that ABA is effective in pro-moting senescence of rice leaves. LY or WM alone had no effect on protein content. When applied together with ABA, LY concentrations between 10 and 100 lM produced a clear protection against protein loss. The optimal concentration of LY in inhibiting ABA-promoted senescence of rice leaves was observed to be 100 lM. WM at 1 lM was observed to be equally effective as 10 lM in inhibiting ABA-promoted protein loss in rice leaves. Visual observation indicated that appear-ance of rice leaves treated with ABA + LY or ABA + WM is basically similar to that of control leaves, indicating that the effect of LY or WM on ABA-induced senescence is biologically significant. MDA is routinely used as an indicator of lipid peroxidation. It is known that ABA is effective in causing lipid peroxidation in rice leaves (Hung and Kao 2003). Recently, we demonstrated that H2O2

is involved in ABA-induced lipid peroxidation of rice leaves (Hung and Kao 2004). Thus, we investi-gated the effect of LY or WM on ABA-induced

MDA and H2O2 production in rice leaves.

Detached rice leaves were treated with 45 lM ABA, 100 lM LY, 1 lM WM or 0.2% (w/v) DMSO (solvent control) and the contents of MDA and H2O2were determined 2 days after treatment.

LY or WM alone had no effect on MDA and H2O2 contents in rice leaves (Figures 2a, b).

ABA-induced MDA and H2O2production in rice

leaves (Figures 2a, b).

NH4+ is a central intermediate of nitrogen

metabolism (Miflin and Lea 1976). GS is the key enzyme in NH4+ assimilation and catalyzes that

ATP-dependent condensation of NH4+ with

glu-tamate to produce glutamine (Miflin and Lea 1976). GS in plants has been reported to be par-ticularly prone to proteolysis under oxidation stress conditions (Ortega et al. 1999; Palatnik et al. 1999; Ishida et al. 2002). PAL catalyzes the elimination of NH4+from phenylalanine and the

production of trans-cinnamate (Hahlbrock and

Grisebech 1979). NH4+ released from PAL

reaction is known to be trapped in glutamine molecule by the action of GS (Sakurai et al.

2001). Our recent work indicated that

ABA-induced NH4

+

accumulation is attributable to the ABA-induced increase in the specific activity of PAL and protease and decrease in the activity of GS in rice leaves, and meanwhile, H2O2 has been shown to be required for the

ABA-induced NH4+ accumulation in rice leaves

(Hung and Kao 2005). It seems that in rice leaves one of the earliest events following ABA treat-ment is the generation of H2O2, which then causes

changes in PAL, GS and protease and subsequent accumulation of NH4+(Figure 3). If PI3P indeed

plays a role in the ABA-induced H2O2generation

in rice leaves, then NH4+accumulation caused by

ABA are expected to be inhibited by LY or WM. As shown in Figure 4d, it is indeed the case. LY or WM treatment was also observed to be effec-tive in inhibiting the decrease in the activity of GS (Figure 4a) and the increase in the specific activ-ities of protease (Figure 4b) and PAL (Figure 4c) in rice leaves caused by ABA.

If H2O2 generation is the primary function of

PI3P in the ABA-induced senescence and NH4+

accumulation, then H2O2should be able to bypass

Figure 2. Effect of LY or WM on the contents of MDA (a) and H2O2 (b) in ABA-treated rice leaves in the presence or

absence of H2O2. The concentrations of ABA, LY, WM, and

H2O2 were 45 lM, 100 lM, 1 lM, and 1 mM, respectively.

All measurements were determined 2 days after treatment in the dark. Values with the same letter are not significantly different at p<0.05 level, according to Duncan’s multiple range test.

Figure 1. Effect of LY or WM concentration on protein content in rice leaves treated with or without ABA. Protein content was determined 2 days after treatment in the dark. Values with the same letter are not significantly different at p<0.05 level, according to Duncan’s multiple range test.

a deficiency of PI3K activity and induce senescence and NH4+ accumulation. Indeed, H2O2 (1 mM)

almost completely reverses the effect of LY or WM on the ABA-induced decrease in protein content (data not shown) and increase in NH4+ content

(Figure 4d). We also observed that H2O2reversed

the effect of LY or WM on ABA-induced increase in MDA content (Figure 2a), decrease in the activity of GS (Figure 4a), and increase in the specific activities of protease (Figure 4b) and PAL (Figure 4c) in rice leaves.

Discussion

In recent years there has been a growing interest in the functional aspects of H2O2. H2O2is a

constit-uent of oxidative metabolism and is itself an AOS. In addition, in view of characteristics of H2O2,

which can be generated in normal cells, and is rel-atively stable and diffusible through cell mem-branes (Foyer et al. 1997; del Rı´o et al. 1998), it is suitable to act as a signal molecule in cell function. It has been shown that H2O2 per se promotes

senescence and NH4+accumulation in rice leaves

(Lin and Kao 1998; Hung and Kao 2005). There are reports indicating that H2O2is required for

ABA-induced senescence and NH4+accumulation in rice

leaves (Hung and Kao 2004, 2005). Induction of senescence and NH4+ accumulation caused by

ABA was found to be preceded by an increase in endogenous H2O2content in rice leaves (Hung and

Kao 2003, 2005). Recently, we were able to show that NADPH oxidase in rice leaves is involved

Figure 4. Effect of LY or WM 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 H2O2. The concentrations of ABA, LY, WM, and H2O2were 45 lM,

100 lM, 1 lM, and 1 mM, respectively. All measurements were determined 2 days after treatment in the dark. Values with the same letter are not significantly different at p<0.05 level, according to Duncan’s multiple range test.

Figure 3. Regulation of NH4+accumulation in ABA-treated

rice leaves. fi Stimulation of the reaction;  inhibition of the reaction.

ABA-induced H2O2 production (Hung and Kao

2004). Inhibitors of NADPH oxidase such as di-phenyleneiodonium chloride (DPI) and imidazole (IMD) prevented ABA-induced H2O2production

and inhibited promoted senescence, ABA-induced accumulation of NH4+, and ABA-caused

changes in enzymes (PAL, protease, and GS) responsible for NH4+accumulation in rice leaves

(Hung and Kao 2004; 2005).

In the present study, we investigated the role of PI3P in ABA-induced H2O2 production in rice

leaves using PI3K inhibitors. It appears that PI3P is important in ABA-induced H2O2 production

based on two lines of evidence. First, two PI3K inhibitors, LY or WM, commonly inhibited the ABA-induced H2O2 production (Figure 2b).

Sec-ond, H2O2 reversed the inhibitory effect of the

PI3K inhibitors on the ABA-induced senescence (data not shown), lipid peroxidation (Figure 2a), NH4+ accumulation (Figure 4d), increase in the

specific activities of protease (Figure 4b) and PAL (Figure 4c), and decrease in the activity of GS (Figure 4a). PI3P has also been shown to be important in the ABA-induced H2O2generation in

guard cells (Jung et al. 2002; Park et al. 2003). It appears that a role of PI3P in the ABA-induced H2O2generation is not confined to guard cells.

NADPH oxidase is known to be H2O2-generating

enzyme in the ABA-treated rice leaves (Hung and Kao 2004). In neutrophils, PI3P regulates H2O2

production by binding to the noncatalytic compo-nent p40phoxof the NADPH oxidase (Ellson et al. 2001). However, a rice homolog of p40phoxhas not been reported. Therefore, the detailed mechanism of the action of PI3P during H2O2production in

rice leaves await further investigation.

The fact that PI3K inhibitors, which reduced the ABA-induced H2O2production (Figure 2b), were

able to prevent the ABA-induced senescence (Figure 1) and NH4+accumulation (Figure 4d) of

rice leaves strengthens further our previous con-clusion that H2O2 is necessary for ABA-induced

senescence and NH4+accumulation of rice leaves

(Hung and Kao 2004, 2005). Acknowledgements

This work was supported the National Science Council of the Republic of China (NSC93-2313-B-002-062).

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