Bot. Bull. Acad. Sin. (2005) 46: 21-28

HUNG and KAO — Nitric oxide counteracts H₂O₂ action

Nitric oxide counteracts the senescence of rice leaves induced by hydrogen peroxide

Kuo Tung HUNG and Ching Huei KAO*

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China

(Received February 17, 2004; Accepted July 26, 2004)

Abstract. In the present study, we evaluate the protective effect of nitric oxide (NO) against the senescence of rice leaves promoted by hydrogen peroxide (H₂O₂). Senescence of rice leaves was determined by decreases in protein content. H₂O₂ treatment resulted in (1) increases in leaf H₂O₂ content, (2) induction of leaf senescence, (3) increases in lipid peroxidation, (4) decreases in ascorbic acid (AsA) and reduced form glutathione (GSH) contents, and (5) increases in antioxidative enzyme activities (ascorbate peroxidase, glutathione reductase, and peroxidase). NO donors [N-tert-butyl-a-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA + NaNO₂] were effective in reducing H₂O₂-induced leaf senescence. PBN prevented H₂O₂-increased H₂O₂ content, H₂O₂-induced lipid peroxidation, H₂O₂-decreased AsA and GSH contents, and H₂O₂-increased antioxidative enzyme activities. The protective effect of PBN on H₂O₂-promoted senescence, H₂O₂-increased H₂O₂ content and lipid peroxidation, H₂O₂-decreased AsA and GSH contents, and H₂O₂-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to the NO released. Reduction of H₂O₂-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge H₂O₂.

Keywords: Hydrogen peroxide; Lipid peroxidation; Nitric oxide; Oryza sativa.

Introduction

Hydrogen peroxide (H₂O₂) is a constituent of oxidative plant metabolism and is itself an active oxygen species (AOS). H₂O₂ can also react with superoxide radicals to form more active hydroxyl radicals in the presence of trace amounts of Fe or Cu (Van Breusegem et al., 2001). The hydroxyl radicals initiate self-propagating reactions leading to peroxidation of membrane lipids and destruction of proteins (Halliwell and Gutteridge, 1989). H₂O₂ has been shown to promote leaf senescence (Parida et al., 1978; Mondal and Choudhuri, 1981; Sarkar 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). Lipid peroxidation is considered an important mechanism of leaf senescence (Thompson et al., 1987; Strother, 1988). The peroxidation of lipids can be initiated by active oxygen species (Thompson et al., 1987; Halliwell and Gutteridge, 1989). Thus, H₂O₂-induced leaf senescence is mediated, at least in part, through lipid peroxidation.

In mammalian systems, nitric oxide (NO), a bioactive molecule, is produced mainly from L-arginine by NO synthase (NOS). However, in plants neither the protein nor the appropriate gene for NOS activity have been detected (Lamattina et al., 2003). Recent work demonstrates that

the mammalian NOS antibodies recognize many NOS-unrelated plant proteins (Butt et al., 2003), suggesting that inferring the presence of plant NOS using the immunological technique may be inappropriate. These results led to speculation that the plant NOS-like enzyme could be structurally different from the mammalian NOS. Recently, a pathogen-inducible plant NOS has been identified as a variant P protein of the mitochondrial glycine decarboxylase complex (Chandok et al., 2003; Wendehenne et al., 2003). Additionally, Guo et al. (2003) reported the presence of an NOS gene (AtNOS1) in Arabidopsis. AtNOS1 turned out to be a protein very similar to a group of bacterial proteins with putative GTP-binding or GTPase domains. Neither the variant P protein nor the purified AtNOS1 protein had sequence similarities to any mammalian NOS. It is increasingly evident that plant nitrate reductase also catalyzes a NAD(P)H-dependent reduction of nitrite to NO (Yamasaki et al., 1999; Rockel et al., 2002, Sakihama et al., 2002; Lamattina et al., 2003). Evidence is mounting that NO acts as an important messenger in plant physiological processes, including growth, development, and defense responses (Noritake et al., 1996; Gouvea et al., 1997; Dangl, 1998; Beligni and Lamattina, 2000, 2001; Neill et al., 2002; Pagnussat et al., 2002; Jih et al., 2003; Zhao et al., 2004). NO has been shown capable of counteracting the toxicity of paraquat and diquat, which are known to generate superoxide radicals, in potato leaves (Beligni and Lamattina, 1999a,b; 2002) and block H₂O₂ production induced by jasmonic acid in tomato leaves (Orozco-Cárdenas and Ryan, 2002). NO seems to be a potent antioxidant, and its

*Corresponding author. Fax: +886-2-23620879; E-mail: kaoch@ntu.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

centrifuged at 6,000 g for 25 min. The absorbance was measured at 410 nm. The H₂O₂ content was calculated using the extinction coefficient 0.28 µmol^-1 cm^-1. Lipid peroxidation in leaf tissue was determined by measuring thiobarbituric acid reactive substances (TBARS). The leaf tissue was extracted with 5% (w/v) trichloroacetic acid and determined according Heath and Packer (1968). GSH in 5% (w/v) 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

Peroxidase (POD) activity was measured using a modification of the procedure of MacAdam et al. (1992). Activity was calculated using the extinction coefficient (26.6 mM^-1 cm^-1 at 470 nm) for tetraguaiacol. Catalase (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 mM^-1 cm^-1 at 240 nm) for H₂O₂ (Kato and Shimizu, 1987). Superoxide dismutase (SOD) was determined according to Paoletti et al. (1986). Ascorbate peroxidase (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 mM^-1 cm^-1 at 290 nm) for AsA. Glutathione reductase (GR) was determined by the method of Foster and Hess (1980). One unit of activity for CAT, POD, SOD, APOD, and GR was defined as the amount of enzyme which degraded 1 µmol H₂O₂ per min, caused the formation of 1 µmol tetraguaiacol per min, inhibited 50% the rate of NADH oxidation observed in control, degraded 1 µmol of AsA per min, and decreased 1 A₃₄₀ per min, respectively.

Statistical Analysis

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

Results and Discussion

The most obvious character of leaf senescence is yellowing. Chlorophyll loss has long been considered the principal criterion of senescence. The protein breakdown that occurs during leaf senescence has been recognized since the earliest studies performed. We have shown that protein breakdown 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. The changes in protein and TBARS content in detached rice leaves treated with 10 mM H₂O₂ in the dark are shown in Figures 1A and 1B. The decease in protein and increase in TBARS were evident at 2 days after H₂O₂ treatment. Clearly, H₂O₂ is effective in promoting the senescence of rice leaves. To be sure that the described effect of H₂O₂ on leaf senescence

ability to directly scavenge AOS may be behind its action (Beligni and Lamattina, 2002).

In rice leaves, we have shown that NO counteracts oxidative stress induced by paraquat, dehydration, and polyethylene glycol (Cheng et al., 2002; Hung et al., 2002). More recently, we have shown that the promotion of rice leaf senescence by abscisic acid and methyl jasmonate, both of which induce H₂O₂ production and lipid peroxidation, can be counteracted by NO donors (Hung and Kao, 2003, 2004). In the present investigation, we examined the effect of NO on the H₂O₂-induced senescence of 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°C under dark conditions. After a 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 (Agriculture Experimental Station, National Taiwan University, Taipei, Taiwan) with natural light 30°C day (12 h)/25°C 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°C in the dark.

Test solutions included H₂O₂, NO donors, and a NO scavenger [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, c-PTIO]. N-tert-butyl-a-phenylnitrone (PBN), 3-morpholino-sydonimine (SIN-1), and sodium nitroprusside (SNP) were used as NO donors. We also used a solution containing ascorbic acid (AsA) and NaNO₂ as another NO donor. All chemicals were purchased from Sigma Co. (St. Louis, MO, USA).

Determinations of Protein, H₂O₂, Lipid peroxidation, GSH, and AsA

Chlorophyll was determined according to Wintermans and De Mots (1965) after extraction in 96% (v/v) ethanol. 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) and antioxidant enzyme activities. The H₂O₂ content was measured 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 hydroxylamine. The homogenate was centrifuged at 6,000 g for 25 min. To determine H₂O₂ content, extracted solution was mixed with 0.1% (v/v) titanium chloride in 20% (v/v) H₂SO₄. The mixture was then

HUNG and KAO — Nitric oxide counteracts H₂O₂ action

was related to an increase in the leaf H₂O₂ content, H₂O₂ concentration was determined in detached rice leaves treated with either water or 10 mM H₂O₂ (Figure 1C). H₂O₂ content in control leaves remained unchanged during 3 days of incubation in the dark. The increase in H₂O₂ content in H₂O₂-treated detached rice leaves was clearly evident at 2 days after treatment.

H₂O₂ treatment resulted in a marked increase in TBARS content, indicating that H₂O₂ brings about lipid peroxidation (Figure 1B). In previous work, when free radical scavengers such as AsA, GSH, sodium benzoate, and thiourea were used together with H₂O₂, it was found that

they prevented H₂O₂-promoted senescence and lipid peroxidation (Lin and Kao, 1998). All these results suggest that H₂O₂ treatment caused an oxidative stress.

Plant cells are equipped with several AOS detoxifying enzymes and antioxidants to protect them against oxidative damage. Antioxidant enzymes include SOD, APOD, GR, CAT, and POD (Foyer et al., 1997). AsA and GSH are two main water-soluble antioxidants (Foyer et al., 1997). The striking increase in lipid peroxidation seen in rice leaves treated with H₂O₂ (Figure 1B) may reflect the change in the activities of antioxidative enzymes and in the contents of antioxidants. It was observed that, H₂O₂-treated rice leaves had higher activities of APOD (Figure 2B), GR (Figure 2C), and POD (Figure 2E) than the controls at 2 days after dark incubation. Increased CAT activity by H₂O₂ was observed at 3 days after treatment (Figure 2D). However, H₂O₂ had no effect on SOD activity in rice leaves (Figure 2A). Treatment with H₂O₂ significantly decreased AsA and GSH contents compared with the control leaves, with the increase occurring 2 day after treatment (Figures 3A and 3B). The increased activities of APOD, GR, and POD and the decreased contents of AsA and GSH in rice leaves in response to H₂O₂ further suggest a strong induction of oxidative stress.

Figure 1B and 1C show that no changes in TBARS or H₂O₂ contents were observed in the control leaves. Lipid peroxidation and dark-induced senescence of rice leaves appear to have no direct relationship under dark

Figure 1. Changes in the contents of protein (A), MDA (B), and H₂O₂ (C) in rice leaves treated with H₂O₂. Detached rice leaves were treated with either water or 10 mM H₂O₂ in the dark. Values with the same letter are not significantly different at P < 0.05, according to Duncan's multiple range test.

Figure 2. Changes in the activities of SOD (A), APOD (B), GR (C), CAT (D) and POD (E) in rice leaves treated with either water or 10 mM H₂O₂ in the dark. Values with the same letter are not significantly different at P < 0.05, according to Duncan's multiple range test.

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

conditions. The changes in the activities of SOD (Figure 2A), APOD (Figure 2B), GR (Figure 2C), CAT (Figure 2D), and POD (Figure 2E) and the contents of AsA (Figure 3A) and GSH (Figure 3B) which occurred in detached rice leaves after incubation in water may be a manifestation of changes in metabolism associated with excision and consequent senescence.

In the present investigation, we show that the promotion of senescence in detached rice leaves by H₂O₂ is associated with lipid peroxidation or oxidative stress. NO is known to counteract oxidative stress in plants (Beligni and Lamattina, 1999a,b; 2002; Beligni et al., 2002; Cheng et al., 2002; Hung et al., 2002; Hung and Kao, 2003, 2004). Thus, it is of great interest to know whether the protective power of NO is also active in the H₂O₂-promoted senescence of rice leaves.

To study the role of NO, various donors were employed, with the assumption that they release NO. Typically, NO is applied to plant tissues via a NO-donor, that is, a molecule that will generate NO, sometimes after passage into

cells. Chamulitrat et al. (1993) postulated that PBN can undergo oxidative decomposition to release NO. SIN-1 is known to generate both superoxide anion and nitric oxide which spontaneously form peroxynitrite (Spiecker et al., 1993). Peroxynitrite has been shown to react with H₂O₂ to yield nitrite ion and oxygen (Martinez et al., 2000). This reaction has been suggested to be the mechanism of NO cytoprotective actions in animals (Wink et al., 1993). NO is released from SNP mainly by decomposition of pure solutions due to photochemical reactions or by the various reducing metabolites, including thiols, in biological organelles (Rochelle et al., 1994; Rao and Cederbaum, 1995; Ioannidis et al., 1996). As shown in Table 1, all NO donors, such as PBN, SIN-1, SNP, and a mixture of AsA and NaNO₂, are effective at inhibiting H₂O₂-promoted senescence of rice leaves.

To investigate whether the protective effect induced by PBN treatment was the result of the production of NO, 100 µM c-PTIO, a NO-specific scavenger, was applied along with 100 µM PBN. The effect of PBN on H₂O₂-promoted protein loss and H₂O₂-induced increase in lipid peroxidation and H₂O₂ content could be reversed by c-PTIO (Table 2). We also observed that PBN counteracted H₂O₂-induced increases in antioxidant enzyme activities, and c-PTIO reversed the effect of PBN-decreased enzyme activities (Table 3). Furthermore, the effect of PBN on H₂O₂-decreased AsA and GSH contents could be reversed by c-PTIO (Table 4). Clearly, the effect of NO donor PBN would then be attributable to NO released.

It has been shown that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge AOS, H₂O₂ and O₂^•- (Beligni and Lamattina, 2002). If NO acts as an antioxidant, it may reduce H₂O₂ content in H₂O₂-treated rice leaves. Since NO reduces H₂O₂-increased H₂O₂ content (Table 2), it appears that it indeed has the ability to scavenge AOS. Orozco-Cárdenas and Ryan (2002) also reported that NO blocked the H₂O₂ production that was induced by jasmonic acid. We also observed that NO donors blocked abscisic acid- and methyl jasmonate-induced H₂O₂ production in rice leaves (Hung and Kao, 2003, 2004).

Figure 3. Changes in the contents of AsA (A) and GSH (B) in rice leaves treated with either water or 10 mM H₂O₂ in the dark. Values with the same letter are not significantly different at P < 0.05, according to Duncan's multiple range test.

HUNG and KAO — Nitric oxide counteracts H₂O₂ action

NO has been shown to act as iron ligand in haemoproteins (Stamler et al., 1992). Ferrer and Barceló (1999) found that the NO donor SNP (5 mM) and NO (55 µM) itself were able to inhibit POD activity in the xylem of Zinnia elegans. Clark et al. (2000) also demonstrated that NO donor (0.8 mM) inhibited the activities of tobacco CAT and APOD, heme-containing enzymes. However, our results show that PBN alone had no effect on the lipid peroxidation and activities of POD and APOD in rice leaves (data not shown). Thus, the reduction of H₂O₂-induced increase in antioxidant enzyme activities by NO is not likely due to a direct NO-mediated inhibition of the enzymes. Why is PBN able to counteract H₂O₂-induced senescence, but not able to inhibit the activities of POD, CAT, and APOD in rice leaves? In the present investigation, we used a µM concentration range of PBN and did not measure the concentration of NO released by PBN in rice leaves. It is possible that the concentration of NO released by PBN was high enough to scavenge H₂O₂, but not sufficient to inhibit the activities of heme-containing enzymes.

In conclusion, the results presented in this paper provide evidence that NO acts as an antioxidant in inhibiting H₂O₂-promoted rice leaf senescence.

Acknowledgements. This work was supported by Grant NSC 91-2313-B-002-364 from the National Science Council of the Republic of China.

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In the present investigation, we found that NO reduced H₂O₂-increased lipid peroxidation (Table 2) and antioxidant enzyme activities (Table 3) in rice leaves. These results are in agreement with our previous work, in which we demonstrated that NO counteracted paraquat-increased lipid peroxidation and antioxidant enzyme activities (Hung et al., 2002). Because lipid peroxidation and the increase in antioxidant enzyme activities are the consequence of AOS (Thompson et al., 1987) and NO acts as an AOS scavenger, the reduction of lipid peroxidation and antioxidant enzyme activities could be a result of low levels of H₂O₂ in rice leaves treated with NO and H₂O₂ (Table 1). The fact that NO counteracts H₂O₂-decreased AsA and GSH (Figures 3A and 3B) should result in an increased capacity to scavenge H₂O₂ in rice leaves treated with NO and H₂O₂ compared to rice leaves treated with H₂O₂ alone and might account in part for the lower contents of H₂O₂ observed in rice leaves treated with both NO and H₂O₂ (Table 2).

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

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Botanical Bulletin of Academia Sinica, Vol. 46, 2005