Effect of chilling on activated oxygen-scavenging enzymes in low temperature-sensitive and -tolerant cultivars of rice (Oryza sativa L.)

iENCE ELSEVIER

Plant Science 109 (1995) 105-113

Effect of chilling on activated oxygen-scavenging enzymes in low temperature-sensitive and -tolerant cultivars of rice (Oryza sativa L.) Haruo Saruyama *, Masatoshi Tanida Hokkaiab Green-Bio Institute, Higashi 5 Kita 15. Naganma,

Yubari-gun, Hokkaido 069-13, Japan

Received 15 February 1995; revision received 26 April 1995; accepted 15 May 1995

when seedlings of the rice (Oryza sativa L.) cultivar K-sen 4 were exposed, at the germination and the leaf stages, to 5°C for 7 days (chilling), they withered after incubation at 25°C (rewarming). In contrast, the cultivar Dunghan Shah showed chilling tolerance and successful growth after the rewarming. We tried to find whether the difference in cold sensitivity correlates with superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) (activated oxygen-scavenging system), NADP-dependent isocitrate dehydrogenase (NADP-ICDH) (TCA cycle), and glucose-bphospahte dehydrogenase (G6P-DH) (pentose phosphate cycle) activities. CAT activities in both cultivars were drastically decreased by the chilling. In D. Shah, these activities were recovered and stimulated by the rewarming. However, in K-sen 4, the rewarming decreased the activities in the embryo and root at the leaf stage. For APX at the germination stage, chilling resistance was found with both cultivars, but the rewarming enhanced the activities effectively in D. Shah while not as effectively in K-sen 4. APX activities at the leaf stage in both cultivars were not affected very much by the chilling. However, only 10% of the initial activity was detected in K-sen 4 root after the rewarming. The other enzymes, SOD, GR, ICDH and G6P-DH, displayed no significant differences in cold sensitivities between the 2 cultivars. We concluded that the tolerance of rice cultivars to chilling injury is closely linked to the cold stability of CAT and APX.

Keywords: Rice (Oryza sativa L.); Chilling; Activated oxygen; Catalase; Ascorbate peroxidase

1. Introduction In plants, activated oxygen is produced as superoxide radicals CO;‘), hydrogen peroxide (H202) and hydroxyl radicals (OH’) during meta* Corresponding author, Tel.: +0081 123892046, Fax: +0081 123892641.

bolic processes. The electron transport system in mitochondria and the photosystems in chloroplasts are well known as processes where such toxic oxygen species are formed [l-4]. These activated oxygens injure. the cellular components of proteins, membrane lipids and nucleic acids 151.In order to escape from these oxidative injuries, plants have developed enzymatic systems for

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scavenging these highly active forms of oxygen. Superoxide dismutase (SOD) (EC 1.15.1.1) reacts with superoxide radicals and converts them to O2 and hydrogen peroxide. Hydrogen peroxide is then detoxified to water and 0, by catalase (CAT) (EC I. II. 1A) and/or ascorbate peroxidase (APX) (EC 1.11.1.11) [1,6]. The ascorbate-dependent H,Ozscavenging system needs monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) in addition to APX [2,7,8]. Thus, functional co-operation among these enzymes is very important for the effective scavenging of these harmful activated oxygens. Activated oxygens are suggested to be responsible for cold injury in plants [9- 121. Since the level of activated oxygens in vivo depends upon the balance between the production of the active oxygens and the capacity of scavenging for them, the cold stability of these enzymes and/or the effective response of the enzyme-synthesizing system to low temperatures stress can be expected to be critically linked to plant growth. In fact, we have found that there is a correlation between the CAT activity in rice seed embryos and germination ability at low temperatures (Tanida, to be published elsewhere). In this study, we examined the role of activated oxygen-scavenging enzymes, especially SOD, CAT, APX, and GR, in the tolerance against low temperatures. In addition, the effects of chilling on NADPdependent isocitrate dehydrogenase (NADP-ICDH) (EC 1.1.1.42) and glucose-6phospahte dehydrogenase (G6P-DH) (EC 1.1.1.49) were also examined, because NADPH is needed as a cofactor in the regeneration process of GSH (reduced glutathione) by GR [2,7]. 2. Materials and methoda 2.1. Plant materials Two cultivars of rice (Oryza sativa L.) were used. At IO”C, Dunghan Shali can germinate, while K-sen 4 can scarcely germinate. 2.2. Chilling treatment Plants were germinated and grown at 25°C on moist paper in a tray with constant light (approx. 3000 lx). After incubation at 25OC for 2-3 days

(denoted as the germination stage) or for 4-6 days (denoted as the leaf stage, where the first and second leaf appeared), seedlings were exposed at 5°C for 7 days (chilling). Then they were transferred to 25°C and incubated at 25°C again (rewarming). The effect of the chilling on the plants was examined by measuring the length of the buds or leaves of seedlings. 2.3. Preparation of enzyme fraction The embryos and buds at the germination stage, and embryos, leaves and roots at the leaf stage were homogenized at 4°C in the presence of protein extraction buffer (50 mM potassium phosphate buffer, pH 7.0, 1% Triton X-100 and 7 mM 2-mercaptoethanol) with mortar and pestle. The homogenate was then centrifuged at 14 000 rev./mm for 20 min twice and the supematant was used as the crude extract for the enzyme assay. The crude extract of each enzyme after the chilling (7 days) and after rewarming (2 days) was also prepared in this manner. 2.4. Enzyme assays All enzyme activities were measured at 25°C using a Shimadzu UV-2100 spectrophotometer. SOD activity was measured by the NBT method according to Beyer and Fridovich [ 131 using the SOD test Wako kit (Wake Chemical Co., Japan). The activity is shown as % inhibition of xanthine oxidase reaction. CAT activity was assayed according to Aebi [14] in a reaction mixture (0.9 ml) containing 50 mM phosphate buffer, pH 7.0, 45 mM Hz02 and the enzyme fraction. The reaction was started by adding Hz02 solution and the activity was determined by monitoring the decrease of absorbance at 240 nm, as a result of the Hz02 consumption. APX activity was determined according to Chen and Asada [ 151with minor modification. The reaction mixture (1.0 ml) was composed of 50 mM potassium phosphate (pH 7.0) containing NaNs, 0.5 mM ascorbate, 1.54 mM H202 and the enzyme fraction. The oxidation of ascorbate was started by H202 and the decrease in the absorbance at 290 nm was monitored. GR activity was measured according to Klapheck et al. [16] with some modifications. The reaction mixture (1 .O ml) contained 0.15 mM

H. Saruyama, M. Tank&a/PlantSrjence 109 (1995) 105-113

Tris-HCl,

pH 8.0, 1 mM EDTA,

0.1 mM

NADPH, 0.5 mM GSSG (oxidizedgiutathione) and the enzyme fraction. The reaction was initiated by addition of GSSG solution and the decrease in absorbance at 340 nm, due to oxidation of NADPH, was monitored. NADP-ICDH activity was measured by monitoring the increase of absorbance at 340 nm according to Ni et al. [17] with some modifications. The reaction mixture (1.0 ml) contained 0.15 mM Tris-HCl, pH 8.0, 4 mM MgC12, 0.26 mM NADP+, 2.5 mM DL-&citrate and the enzyme fraction. The reaction was initiated by addition of DL-isocitrate.

107

G6P-DH was assayed according to Muto and

Uritani [ 181.Activitywascalculatedby the difference of NADP+ reduction measured at 340 nm between 2 assays; one assay was carried out in a mixture containing 50 mM Tris-HCl, pH 7.7, 1.7 mM ~-glucose 6-phospahte (G6P), I.7 mM 6phospho-Dgluconate (6PG), 1.0 mM NADP+ and the enzyme fraction, and the other containing all these materials except G6P.

2.5. Protein assay Protein concentration was determined according to the method of Bradford [19] using the Bio-

(B) K-sen 4

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Fis. 1. Effectof chilling on growth of rice cukivars (A) Dunghan Shali and (B) K-sen 4. D. Shali and K-sen 4 were incubated at 25°C (control, 60 ). Arrows A and B indicate when seedlings at the germination stage and the leaf stage, mwly, were transferredfrom 25°C to SOCand incubated for 7 days (chilling, - ). Arrows C and D indicate the time at which chilled seedlings were transferred from SC to 25°C and then incubated at 25oC again (regrowth of seedlings at the @nation stage, +-+; regrowth of leaf stage seedlings, Lm ).

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108

Rad protein CA., USA).

assay kit (Bio-Rad

Laboratories,

ined by measuring the capability of regrowth at 25°C after chilling (5OCfor 7 days). D. Shali showed good recovery after chilling at both the germination and leaf stages (Fig. 1A). However, in the case of K-sen 4, chilling had a lethal effect (Fig. 1B). Chilled K-sen 4 seedlings at the early germination stage could restart growth for 6 days at 25°C but eventually stopped growing with withering of the seedlings (Fig. 1B). The effect of chilling at the leaf stage was severe with no growth occurring after transfer of the chilled seedlings to 25°C (Fig. 1B).

2.6. Materials NADP+ (potassium salt), G6P, 6PG, GSH and GSSG were obtained from Boehringer Mannheim (Germany). DL-Isocitrate was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3. Results 3.1. Effect of chilling on seedling growth The cold sensitivity of rice cultivars was exam-

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Fig. 2. Effect of chilling on activities of SOD, CAT, APX, and GR in D. Shali (A) and K-WI 4 (B) at the germination stage. Seedlings grown at 25’C for l-2 days (control, o), were exposed to 4°C for 7 days (chilling, q ), then transferredto 25°C and incubated for 2 days (rewarming, m). Activities of SOD (% inhibition /mg protein), CAT (~oles/mg protein/min), APX @noles/mp protein/mm), and GR (nmolcs/mg protein/mm) in embryo and bud were measured as described in the Materials and methods. Control activities of D. Shah: SOD, 475 f 69 (embryo), 1080 f 117 (bud); CAT, 27.0 + 4.7 (embryo), 32.2 * 5.9 (bud); APX, 0.49 * 0.02 (embryo), 0.58 f 0.08 (bud); GR, 43.4 * 0.92 (embryo), 36.0 f 10.2 (bud). K-sen 4: SOD, 354 f 124 (embryo), 598 l 89 (bud); CAT, 48.3 f 1.8 (embryo), 54.6 t 19.7 (bud); APX, 0.86 f 0.32 (embryo), 0.93 f 0.39 (bud); GR, 44.4 + 2.2 (embryo), 42.3 f 2.3 (bud). Each value is the mean of resultsfrom at least 3 separate experiments * S.D.

H. Saruyama,M. TaniablPlantScience109 (1995) 105-113

3.2. Effect of chilling on activated oxygenscavenging enzymes at the germination stage The effects of chilling on SOD, CAT, APX and GR at the germination stage were examined, and the results are summarized in Fig. 2. In the case of SOD, both chilled cultivars of D. Shah and K-sen 4 showed almost the same activities as those of the control ones. However, interestingly, after 2-day incubation of the chilled seedlings at 25”C, the SOD activities were increased in both cultivars (Fig. 2a,e).

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With CAT, activities decreased drastically after the chilling in both D. Shah and K-sen 4 (Fig. 2b,f). D. Shali exhibited 48% and 28% of the control activities in embryo and bud, respectively, while in K-sen 4, they were only 31% and 23%, respectively. In D. Shah, these activities were recovered and stimulated by 243% and 161% in embryo and bud, respectively, on incubation of the chilled seedlings at 25’C for 2 days. No such stimulation occurred with K-sen 4 (Fig. 2f). Chilling treatment did not induce. the decrease

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Fig. 3. Effect of chilling on activities of SOD, CAT, APX, and GR in D. Shali (A) and K-sen 4 (B) at the leaf stage. Seedlings were grown at 25’C for 4-5 days (control, Q, chilled at 4’C for 7 days (chilling, El ), then transferred to 25°C and incubated for 2 days {rewarming, n ). Activities of SOD (% inhibitiotimg protein), CAT @moles/mg protein/min), APX (pmoles/mg protein/mitt), and OR (mnoledmg protein/mitt) in embryo, leaf and root were measured as described in Materials and methods. Control activities of D. Shah SOD, 821 f 164 (embryo), 838 f 185 (leaf), 749 f 49 (root); CAT, %.3 f 1.7 (embryo), 50.2 f 6.2 (leaf), 52.6 * 12.3 (root); APX, 2.20 + 0.20 (embryo), 1.17 f 0.12 (leaf), 2.48 f 0.31 (root); GR, 50.8 f 4.0 (embryo), 46.6 f 4.8 (leaf), 82.6 * 8.3 (root). ILsen 4: SOD, 768 f 100 (embryo), 587 * 108 (leaf), 654 f 34 (root); CAT, 98.7 f 2.0 (embryo), 52.6 * 6.2 (leaf), 109.9 f 4.4 (root); APX, 2.56 f 0.30 (embryo), 0.61 l 0.24 (leaf), 2.33 * 0.50 (root); GR, 50.8 f 5.0 (embryo), 54.0 * 4.5 (leaf), 81.0 f 11.2 (root). Each value is the mean of results from at least 3 separate experiments f S.D.

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of APX activity, except in the case of K-sen 4 embryo - which showed about a 40% decrease (Fig. 2c,g). After transfer of the chilled plants to 25°C remarkable increases of 361% and 244% of the activity were observed in D. shali embryo and bud, respectively. In K-sen 4, 191% increase in the activity was only induced in embryos. GR in both cultivars was stable to low temperature stress (Fig. 2d,h).

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Fig. 5. Effect of chilling on activities of ICDH and G6P-DH in D. Shah (A) and K-sen 4 (B) at the leaf stage. The seedlings were grown at 25OCfor 4-5 days (control, Ii3), chilled at 4°C for 7 days (chilling, a), then transferred to 25°C and incubated for 2 days (rewarming, n ). Activities of ICDH (nmoles/mg protein/mm) and G6P-DH (nmolcs/mg protein/mitt) in embryo, leaf and root were measured as described in Materials and methods. Control activities of D. Shah: ICDH, 106.4 f 15.7 (embryo), 32.3 * 6.0 (leaf), 119.1 * 11.2 (root); G6P-DH, 82.5 & 7.0 (embryo), 30.2 f 1.2 (leaf), 81.9 f 8.1 (root). Ksen 4: ICDH, 98.4 l 4.0 (embryo),34.9 f 0.1 (leaf), 108.7 f 3.3 (root); G6P-DH, 73.0 * 10.0 (embryo), 19.0 f 1.0 (leaf), 50.0 f 12.3 (root). Each value is the mean of results from at least 3 separate experiments f S.D. Fig. 4. Effect

of chilling on activities of ICDH and G6P-DH in Shah (A) and K-sen 4 (B) at the germination stage. The seedlings were grown at 2YC for l-2 days (control, I3), chilled at 4°C for 7 days (chilling, q ), then transferred to 25% and incubated for 2 days (rewarming, n ). Activities of ICDH (nmoles/mg protein/mitt) and G6P-DH (nmoles/mg protein/mitt) in embryo and bud were measured as described in Materials and methods. Control activities of D. Shah: ICDH, 50.8 zt 5.5 (embryo), 50.3 & 5.1 (bud); G6P-DH, 50.0 zt 3.4 (embryo), 49.2 f 2.3 (bud). K-sen 4: 44.4 f 4.5 (embryo), 50.8 f 13.4 (bud); G6P-DH, 54.0 f 4.2 (embryo), 52.4 k 4.2 (bud). Each value is the mean of results from at least 3 separate experiments * SD. D.

3.3. Effect of chilling on activated scavenging enzymes at the leaf stage

oxygen-

The effects of the chilling on SOD, CAT, APX and GR activities at the leaf stage were examined, and the results are shown in Fig. 3. SOD activities did not change very much with the chilling, as was the case in the germination stage. When the chilled seedlings were returned to 25°C 1.5 to 2.5-fold increases of activities were observed (Fig. 3a,e).

I-i. Saruyama. M. Tank&z/Plant Science 109 (1995) 105-113

CAT activity in all tissues examined decreased with the chilling (Fig. 3b,f). In D. Shah, all of these decreased activities increased on incubation at 25°C for 2 days, except for leaf activity. In contrast, the decreased activities of embryo and root in K-sen 4 were not recovered by the re-incubation at 25OC, and the activities decreased further from 60 to 33% and 18% to 16%, respectively. Next, we examined the cold sensitivity of APX. As shown in Fig. 3c,g, no drastic change of activity after chilling was observed, except for the case of K-sen 4 roots in which activity was about 60% of the control level. A very clear difference in cold sensitivity of APX was observed in the roots between these 2 cultivars after transfer of the chilled seedlings to 25°C. Activity in roots of D. Shah was the same as the control activity, but only 10% of the activity remained in K-sen 4 (Fig. 3g). As for as GR activities, no significant effect of the chilling was observed in either cultivar (Fig. 3d,h). 3.4. Effect of chilling on NADP-ICDH

and G’6P-

DH

We also examined the cold sensitivity of NADPICDH and G6P-DH. At the germination and the leaf stage (Figs. 4 and 5, respectively), NADPICDH activities did not decrease much with the chilling. Two-day incubation at 25°C of the chilled plants showed rather increased activities. In the case of G6P-DH, chilling treatment did not induce a drastic decrease of the activities. After transfer of the chilled plant to 25”C, the activities increased in embryos, buds and leaves in both cultivars. 4. D&Eluuion The content of unsaturated fatty acids in membrane lipids is strongly suggested to be directly linked to the chilling tolerance in plants [20-221. However, unsaturated fatty acids are easily peroxidized by hydroxyl radicals that are converted from 0;’ [2,23,24]. Thus, dismutation of 0; by SOD might be the primary step for the defense against chilling injury. In this research, total SOD activities were not changed by the chilling and we did not find any significant difference in cold sensitivity in SOD between cold-sensitive and

111

-tolerant rice cultivars (Figs. 2 and 3). From these results, SOD itself seems to be cold-tolerant. However, it cannot be ruled out that activities of some SOD isozymes may vary by the chilling, since isoxymes of SOD exhibit different characteristics and intercellular localization [a]. In addition, the SOD activities in all tissues examined increased after transfer of the chilled plants from 5°C to 25°C (Figs. 2 and 3). Increases in SOD mRNA levels after the low temperature stress were also observed in Nicotiana phmbaginifolia [25]. Thus, raising the temperature from 5°C to 25°C might stimulate the production of 0; that triggers SOD synthesis. The increase of SOD activity by raising the temperature from 5°C to 25°C suggested an increased production of HzOz. An abrupt increase in Hz02 levels with cold treatment was also reported in wheat seedlings [ 111. Hz02 is detoxified by CAT, APX and glutathione peroxidase (GPX), but no significant GPX activity was detected in rice cultivars (data not shown). A drastic decrease of CAT activities after the chilling was observed in these cultivars in the germination and the leaf stages (Figs. 2 and 3). Thus, rice catalase is coldlabile similar to pea, cucumber, maize, rye and wheat [26-291. Furthermore, CAT at the leaf stage in K-sen 4 was irreversibly damaged, since recovery of the decreased activities by the incubation at 25OC was very low in embryos (33%) and roots (14%). This sharply contrasted with the case of D. Shali, where there was effective recovery of CAT activity (Fig. 3b,f). The cessation of growth of chilled seedlings of K-sen 4 at 25°C seems to be attributable to the cold sensitivity of CAT and/or the gene expression of CAT. The cold lability of CAT suggested the significance of the co-operative function of APX in compensating for the loss of CAT activities. The important role of APX in relation to the increase of oxidative tolerance has been reported for many plants [18,26,30]. Specific activities of APX compared to those of CAT were very low (see legends of Figs. 2 and 3). However, the higher affinity for HzOz [ 15,31,32] suggested that APX can work at low temperatures and compensate for the loss of CAT activities. In fact, APX activity in D. Shah was almost the same as the control, or rather increased by the chilling (Figs. 2c and 3~). In con-

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trast, K-sen 4 showed a decrease in activities by chilling of the embryo at the germination stage (Fig. 2g) and of the roots at the leaf stage (Fig. 3g). Therefore, D. Shah seems to make up for the decrease of CAT activity caused by the chilling, while K-sen 4 could not. Furthermore, the rewarming enhanced APX activity in D. Shali by 361% and 244%, in embryo and bud, respectively (Fig. 2~). However, in K-sen 4, the increase of activity was very low (Fig. 2g). Thus, the decrease of activity in the embryo and lower stimulation of APX activities does not seem to be sufftcient for H202 detoxification, since chilled K-sen 4 could continue growth at 25°C for a while, before finally withering (Fig. 1B). On the other hand, at the leaf stage, APX activities in D. Shali were not changed much by the chilling and the rewarming treatment. In contrast, the activity in K-sen 4 root was decreased by the chilling and almost lost after the rewarming at 25°C (Fig. 3~). Therefore, irreversible inactivation of APX in addition to CAT in root (Fig. 3b) seems to severely reduce H202scavenging capacity, which leads to quick cessation of growth after rewarming of K-sen 4 (Fig. 1B). GR is necessary for the regeneration of ascorbate which is required for the activity of APX. In addition, the importance of GR in relation to oxidative stress has been reported for many plants [33-381. GR requires NADPH as a cofactor [3,8]. NADP-linked dehydrogenases such as ICDH and G6P-DH are important for the production of a reduced form of NADP during the imbibition and the germination process [39]. Therefore, we examined the effect of chilling on these enzymes. In the case of rice cultivars, GR activities did not change very much by the chilling as shown in Figs. 2d,h and 3d,h. Similarly, as shown in Figs. 4 and 5, neither ICDH nor G6P-DH was inhibited by the chilling and the rewarming experiments. These results indicated that GR, ICDH and G6P-DH were cold-stable and not responsible for the loss of APX activity in K-sen 4 by the chilling. We found clear differences in the cold sensitivity of CAT and APX between the cold-sensitive and tolerant rice cultivars. Our results strongly suggest that functional co-operation between CAT and APX is important for the defense against low temperature injury. The mechanism by which these

activated oxygen-scavenging enzymes can work co-operatively, in response to low temperature stress, is not yet known. Further analysis of the regulational gene expression in these enzymes should elucidate the mechanism of cold tolerance in rice. References 111K. Asada, Production and scavenging of active oxygen in chloroplasts, in: J.G. Scandalios (Ed.), Current Communications 5, In Cell and Molecular Biology, Molecular Biology of Free Radical Scavenging Systems. Cold Spring Harbor Laboratory Press, New York, 1992, pp. 173-192. 121C. Bowler, M. Van Montagu and D. Inzd, Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol., 43 (1992) 83-116. [31 S. Puntarulo, R.A. Sanchez and A. Boveris, Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination. Plant Physiol., 86 (1988) 626-630. 141 S. Puntaruio, M. Galleano, R.A. Sanchez and A. Boveris, Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination. Biochim. Biophys. Acta, 1074 (1991) 277-283. IS1 C.A. Rice-Evans, A.T. Diplock and M.C.R. Symons, Mechanisms of radical production, in: R.H. Burdon, P.H. van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology. Vol. 22, Techniques in Free Radical Research. Elsevier, Amsterdam, 1991, pp. 19-50. (61 J.G. Scandalios, Response of plant antioxidant defense genes to environmental stress, in: J.G. Scandahos and T.R.F. Wright (Eds.), Advances in Genetics. Vol. 28, Genomic Responses to Environmental Stress. Academic Press, San Diego, 1990, pp. I-41. Y. Nakano and K. Asada, [71 M.A. Hossain, Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol., 25 (1984) 385-395. 181 A. Polle, K. Chakrabarti, W. Schiirmann and H. Rennenberg, Composition and properties of hydrogen peroxide decomposing systems in extracellular and total extracts from needles of Norway spruce (Picea abies L., Karst.). Plant Physiol., 94 (1990) 312-319. 191 R.R. Wise and A.W. Naylor, Chilling-enhanced photooxidation. Evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. Plant Physiol., 83 (1987) 278-282. [lOI R.A.J. Hodgson and J.K. Raison, Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition. Planta, 183 (1991) 222-228. 1111 T. Okuda, Y. Matsuda, A. Yamanaka and S. Sagisaka, Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment. Plant Physiol., 97 (1991) 1265-1267.

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