Bot. Bull. Acad. Sin. (1998) 39: 161_165

Lin and Kao — Effect of exogenous H₂O₂ on leaf senescence

Effect of oxidative stress caused by hydrogen peroxide on

senescence of rice leaves

Jaw-Neng Lin and Ching Huei Kao¹

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

(Received September 25, 1997; Accepted February 26, 1998)

Abstract. Lipid peroxidation is considered to be an important mechanism of leaf senescence. The peroxidation of lipids can be initiated by free radicals. H₂O₂ itself is active oxygen species and can react with superoxide radicals to form more reactive hydroxyl radicals in the presence of trace amounts of Fe or Cu. Thus, it is of interest to investigate the relationship between lipid peroxidation and H₂O₂-promoted senescence in detached rice leaves. H₂O₂ effectively promoted senescence and increased malondialdehyde levels in detached rice leaves under both light and dark conditions. However, the promotion of senescence by the exogenous level of H₂O₂ is not associated with an increase in its endogenous level. Thiourea (a scavenger of hydroxyl radical) almost completely prevented H₂O₂-promoted senescence and the H₂O₂-induced decrease in superoxide dismutase and ascorbate peroxidase activities in light and darkness. It seems that promoted senescence by H₂O₂ in detached rice leaves is associated free radical-induced lipid peroxidation.

Keywords: Free radical scavengers; H₂O₂; Leaf senescence; Lipid peroxidation; Oryza sativa; Oxidative stress.

Abbreviations: APOD, ascorbate peroxidase; ASC, ascorbic acid; GR, glutathione reductase; GSH, reduced glutathione; MDA, malondialdehyde; SB, sodium benzoate; SOD, superoxide dismutase; TU, thiourea.

Introduction

Hydrogen peroxide (H₂O₂) is a constituent of oxidative plant metabolism. It is a product of peroxisomal and chloroplastic oxidative reactions (Del Rio et al., 1992). H₂O₂ itself is an active oxygen species. H₂O₂ can also react with superoxide radicals to form more reactive hydroxyl radicals in the presence of trace amounts of Fe or Cu (Thompson et al., 1987). The hydroxyl radicals initiate self-propagating reactions leading to peroxidation of membrane lipids and destruction of proteins (Asada and Takahashi, 1987; Bowler et al., 1992; Halliwell, 1987). It has been reported that H₂O₂ promotes senescence of detached leaves (Begam and Choudhuri, 1992; Mondal and Choudhuri, 1981; 1982; Parida et al., 1978; Sarkar and Choudhuri, 1981) and induction of senescence is accompanied by an increase in endogenous H₂O₂ level (Mondal and Choudhuri, 1981). Lipid peroxidation is considered to be an important mechanism of leaf senescence (Dhindsa et al., 1981; 1982; Kunnert and Ederer, 1985; Strother, 1988; Thompson et al., 1987). The peroxidation of lipids can be initiated by free radicals (Girotti, 1985; Thompson et al., 1987). Thus, accelerated senescence in leaves could be the result of an H₂O₂-mediated increase in rates of oxidative reactions. We thus examined the relationship between lipid peroxidation and H₂O₂-promoted senescence in detached rice leaves.

Materials and Methods

Rice (Oryza sativa L. cv. Taichung Native 1) was cultured as previously described (Kao, 1980). The apical 3-cm segments excised from the third leaves of 12-day-old seedlings were used. A group of 10 segments was floated in a Petri dish containing 10 mL of test solutions. Incubation was carried out at 27°C in light (40 µmol m^-2 s^-1) or in darkness.

For protein determination, 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 supernatant liquids were used for determination of protein by the method of Bradford (1976). Protein levels were expressed as mg g^-1 fresh weight. Malondialdehyde (MDA) was extracted with 5% (w/v) trichloroacetic acid and determined according to Heath and Packer (1968). MDA level is routinely used as an index of lipid peroxidation and was expressed as nmol g^-1 fresh weight.

The H₂O₂ level was colorimetrically measured as described by Jena and Choudhuri (1981). H₂O₂ was extracted by homogenizing 50 mg leaf tissue with 3 mL of phosphate buffer (50 mM, pH 6.5). The homogenate was centrifuged at 6,000 g for 25 min. To determine H₂O₂ level, 3 mL of extracted solution was mixed with 1 mL of 0.1% titanium sulphate in 20% H₂SO₄ (v/v), and the mixture was then centrifuged at 6,000 g for 15 min. The intensity of the yellow colour of the supernatant was measured at 410 nm. H₂O₂ level was calculated using the extinction coefficient (0.28 µmol^-1cm^-1).

¹Corresponding author. Fax: 886-2-23620879; E-mail: kaoch@cc.ntu.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

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 optical density 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 ans Asada (1981). The decrease in ascorbate concentration was followed as a 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 ascorbate. Glutathione reductase (GR) was determined by the method of Foster and Hess (1980). Enzyme activity was expressed on the basis of gram fresh weight.

All experiments were performed three times, and within each experiment, treatments were replicated 4 times. Similar results and identical trends were obtained each time. The data reported here are from a single experiment.

Results and Discussion

The senescence of detached rice leaves was followed by measuring the decrease of protein. Effects of H₂O₂ on the levels of protein, malondialdehyde (MDA), and endogenous H₂O₂, and the activity of catalase of detached rice leaves are shown in Figure 1. H₂O₂ was found to promote protein degradation under both light and dark conditions. The MDA level in H₂O₂-treated detached rice

leaves was higher than that in water-treated controls under both light and dark conditions. After incubation in darkness, protein levels in water- and H₂O₂-treated leaves were lower than those in the light. The MDA level in water- and H₂O₂-treated detached rice leaves incubated in darkness was higher than that incubated in light. This shows that H₂O₂-promoted senescence of detached rice leaves is linked to lipid peroxidation. Our results are consistent with those obtained by previous investigators (Mondal and Choudhuri, 1981; Parida et al., 1978; Sarkar and Choudhuri, 1981), who showed that H₂O₂-promoted the senescence of detached leaves.

The addition of H₂O₂ is expected to result in an accumulation of endogenous H₂O₂ in detached rice leaves. Contrary to our expectation, endogenous H₂O₂ did not accumulate in H₂O₂-treated detached rice leaves (Figure 1). H₂O₂ treatment resulted in a decrease in endogenous H₂O₂ level when compared with water-treated controls.

The fact that the addition of H₂O₂ did not result in an accumulation of endogenous H₂O₂ in detached rice leaves can be explained considering that H₂O₂ is being used in metal-catalyzed reactions such as lipid peroxidation. This suggestion is supported by the observation that light incubation resulted in a higher endogenous H₂O₂ level and lower lipid peroxidation level compared to dark (Figure 1).

Catalase converts H₂O₂ to form oxygen and water. H₂O₂ treatment increased in catalase activity (Figure 1). Endogenous H₂O₂ level was observed to be lower and catalase activity was higher in dark-treated detached rice leaves than in light-treated ones. Thus, the possibility that the increase in catalase activity is associated with the decrease in endogenous H₂O₂ level in H₂O₂-treated detached rice leaves cannot be excluded. The addition of exogenous H₂O₂ has been shown to stimulate the expression of catalase (Prasad et al., 1994). Recent work by Willekens et al. (1997) clearly demonstrated that catalase can effectively remove exgenous H₂O₂.

Since the promotion of senescence of detached rice leaves by exogenous H₂O₂ is not associated with an increase in endogenous H₂O₂, the possibility that a factor other than H₂O₂ per se may affect the senescence of detached rice leaves cannot be excluded. It has been shown that H₂O₂ can react with superoxide to form more reactive hydroxyl radicals (Thompson et al., 1987). Since lipid peroxidation is generally considered to be induced by free radicals (Girotti, 1985; Thompson et al., 1987), it is possible that the effects of exogenous H₂O₂ on the senescence of detached rice leaves is mediated through them, especially hydroxyl radicals. If this suggestion is correct, then the promotive effect of exogenous H₂O₂ on the senescence of detached rice leaves should be prevented by the addition of free radical scavengers such as ascorbic acid (ASC, destruction of active oxygen species, particuallary H₂O₂), reduced glutathione (GSH, scavenger of free radicals), and thiourea (TU, scavenger of hydroxyl radicals). This is essentially what we see in Figures 2 and 3. TU (10 mM) almost completely prevents H₂O₂-promoted senescence in

Figure 1. Effects of H₂O₂ on the levels of protein, MDA, and endogenous H₂O₂, and the activity of catalase of detached rice leaves under light and dark conditions. Protein, MDA, and endogenous H₂O₂ levels and catalase activity were determined at 4 d after treatment. Vertical bars represent standard errors (n=4). Only those standard errors larger than symbol size are shown.

Lin and Kao — Effect of exogenous H₂O₂ on leaf senescence

light and darkness, respectively. In darkness, TU and GSH have syngeristic effect on preventing H₂O₂-promoted senescence.

Superoxide dismutase (SOD), ascorbate peroxidase (APOD), and glutathione reductase (GR) have protective roles in scavenging free radicals. Treatment with H₂O₂ decreased SOD and APOD activities in detached rice leaves incubated in light and darkness (Table 1). H₂O₂ had no effect on GR activity in either light- or dark-incubated detached rice leaves. TU was observed to be able to prevent the decrease in SOD and APOD activities by H₂O₂.

In conclusion, the result of the present investigation suggest that H₂O₂-promoted senescence is correlated with free radical-induced lipid peroxidation.

Table 1. Effect of thiourea (TU) on superoxide dismutase (SOD), ascorbate peroxidase (APOD), and glutathione reductase (GR) activities in detached rice leaves treated with H₂O_2.

Enzymes^b

Treatment^a SOD APOD GR

Light

H₂O 40.6 ± 1.2 112.6 ± 1.0 8.4 ± 0.6

H₂O₂ 24.5 ± 0.6 90.7 ± 0.9 7.3 ± 0.4

H₂O₂ + TU 37.7 ± 1.0 98.2 ± 0.8 7.3 ± 0.6

Dark

H₂O 27.3 ± 1.6 65.8 ± 0.3 6.5 ± 0.5

H₂O₂ 17.3 ± 1.0 58.4 ± 0.7 6.0 ± 0.1

H₂O₂ + TU 22.0 ± 0.9 63.1 ± 0.6 6.5 ± 0.7

^aThe concentration of H₂O₂ and TU was 10 mM.

^bEnzyme activities were determined after 4 d in light or in darkness.

Figure 2. Effect of ascorbic acid (ASC), reduced glutathione (GSH), sodium benzoate (SB), and thiourea (TU) on protein levels of detached rice leaves treated with H₂O₂ (10 mM) in the light. Protein was determined at 4 d after treatment. Vertical bars represent standard errors (n=4). Only those standard errors larger than symbol size are shown.

Acknowledgements. This work was supported by the National Science Council of the Republic of China (NSC 86-2313-B002-044). This paper is part XXXX of the series Senescence of Rice Leaves.

Literature Cited

Asada, K. and M. Takahashi. 1987. Production and scavenging of active oxygen in photosynthesis. In D. J. Kyle, C. B. Osmond, and C. J. Arntzen (eds.), Photoinhibition, Elsevier, Amsterdam, pp. 227_287.

Begam, H.H. and M.A. Choudhuri. 1992. H₂O₂ metabolism during senescence of two submerged angiosperms Hydrilla and Ottelia: Changes in enzyme activities in light and darkness. Biochem. Physiol. Pflanzen 188: 105_115.

Bowler, C., M. Van Montagu, and D. Inze. 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 83_116.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248_254.

Del Rio, L.A., L.M. Saudalio, J.M. Palma, P. Bueno, and F.J. Corpas. 1992. Metabolism of oxygen radicals in peroxi

Figure 3. Effect of sodium benzoate (SB), reduced glutathione (GSH), and thiourea (TU) on protein levels of detached rice leaves treated with H₂O₂ (10 mM) in the dark. The concentration of SB, GSH, and TU was 0.5, 0.5 and 10 mM, respectively. Protein was determined at 4 d after treatment. Vertical bars represent standard errors (n=4). Only those standard errors larger than symbol size are shown.

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

somes and cellular implications. Free Rad. Biol. Med. 13: 557_580.

Dhindsa, R.S., P. Plumb-Dhindsa, and T.A. Thorpe. 1981. Leaf senescence: Correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32: 93_101.

Dhindsa, R.S., P. Plumb-Dhindsa, and D.M. Reid. 1982. Leaf senescence and lipid peroxidation: Effects of some phytohormones, and scavengers of free radicals and singlet oxygen. Physiol. Plant. 56: 453_457.

Foster, J.G. and J.L. Hess. 1980. Responses of superoxide dismutase and glutathione reductase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. Plant Physiol. 66: 482_487.

Girotti, A.W. 1985. Mechanism of lipid peroxidation. J. Free Rad. Biol. Med. 1: 87_95.

Halliwell, B. 1987. Oxidative damage, lipid peroxidation and antioxidant protection in chloroplasts. Chem. Phys. Lipids 44: 327_340.

Heath, R.L. and L. Packer. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125: 189_198.

Jena, S. and M.A. Choudhuri. 1981. Glycolate metabolism of three submerged aqutatic angiosperms during aging. Aquat. Bot. 12: 345_354.

Kao, C.H. 1980. Senescence of rice leaves IV. Influence of benzyladenine on chlorophyll degradation. Plant Cell Physiol. 21: 1255_1262.

Kato, M. and S. Shimizu. 1987. Chlorophyll metabolism in higher plants. VII. Chlorophyll degradation in senescing tobacco leaves: phenolic-dependent peroxidative degradation. Can. J. Bot. 65: 729_735.

Kunnert, K.J. and M. Ederer. 1985. Leaf aging and lipid peroxidation: the role of the antioxidant vitamin C and E.

Physiol. Plant. 65: 85_88.

Mondal, R. and M.A. Choudhuri. 1981. Role of hydrogen peroxide in senescence of excised leaves of rice and maize. Biochem. Physiol. Pflanzen 176: 700_709.

Mondal, R. and M.A. Choudhuri. 1982. Regulation of excised leaves of some C₃ and C₄ species by endogenous H₂O₂. Biochem. Physiol. Pflanzen 177: 403_417.

Nakano, Y. and K. Asada. 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22: 867_880.

Paoletti, F., D. Aldinucci, A. Mocali, and A. Capparini. 1986. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal. Biochem. 154: 536_541.

Parida, R.K., M. Kar, and D. Mishra. 1978. Enhancement of senescence in excised rice leaves by hydrogen peroxide. Can. J. Bot. 56: 2937_2941.

Prasad, T.K., M. Anderson, B.A. Martin, and C.R. Stewart. 1994. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6: 65_74.

Sarkar, U. and M.A. Choudhuri. 1981. Effect of some oxidants and antioxidants on senescence of isolated leaves of sunflower with special reference to glycolate content, glycolate oxidase, and catalase activities. Can. J. Bot. 59: 392_396.

Strother, S. 1988. The role of free radicals in leaf senescence. Gerontology 34: 151_156.

Thompson, J.E., R.L. Legge, and R.F. Barber. 1987. The role of free radicals in senescence and wounding. New Phytol. 105: 317_344.

Willekens, H., S. Chamnogpol, M. Davey, M. Schraudner, C. Langebartels, M. Von Montagu, D. Inze, and W. Van Camp. 1997. Catalase is a sink for H₂O₂ and is indispensable for stress defence in C₃ Plants. EMBO J. 16: 4806_4816.