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Effect of Salicylic acid on the antioxidant system


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Effect of salicylic acid on the antioxidant sys

tem in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures
Ren-Hua Huang a,c , Ji-Hong Liu b , Yun-Mei Lu a,c , Ren-Xue Xia a,?
College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, PR China b National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, PR China c College of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621002, PR China Received 27 January 2007; accepted 25 June 2007
a

Abstract Effects of salicylic acid (SA) on active oxygen metabolism and the antioxidant system in the pulp of ‘Cara cara’ navel orange (C. sinensis L. Osbeck) fruit stored at 6 ? C and 20 ? C were investigated through analysis of the contents of malondialdehyde and hydrogen peroxide, the activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione reductase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and ascorbate peroxidase (AsA-POD, EC 1.11.1.11), and non-enzyme components such as ascorbate (AsA), dehydroascorbate (DHAsA), glutathione (GSH) and oxidized glutathione (GSSG) in the AsA-GSH cycle. The results showed that the control fruit (dipped in water) had lower contents of malondialdehyde and activities of antioxidant enzymes at 6 ? C, and lower contents of ascorbate and glutathione at 20 ? C. During storage, the contents of hydrogen peroxide and malondialdehyde together with activities of SOD and CAT gradually increased at both storage temperatures. SA-pretreatment accelerated hydrogen peroxide accumulation and the increase in SOD, but it signi?cantly slowed down malondialdehyde and CAT rate of increase during storage compared to controls under these two temperatures. At the end of storage, malondialdehyde contents in SA-pretreated fruit were 12.6 and 27.6% lower than those in control fruit. The activities of GR and DHAR and the contents of AsA and GSH during fruit storage declined but the SA-pretreatment reduced the rate of this decline. The SA-pretreated fruit had higher values of AsA/DHAsA than those in control fruit at the same temperature, and these ratios were highest at 6 ? C. These results indicate that low storage temperature and exogenous SA can reduce lipid peroxidation by regulating the antioxidant system, and suggest that pretreatment with SA combined with lower storage temperature might provide a useful means of maintaining bene?cial antioxidant activity during storage of navel orange. ? 2007 Elsevier B.V. All rights reserved.
Keywords: Salicylic acid; Antioxidant system; Citrus sinensis; Temperature; Storage

1. Introduction Active oxygen species (AOS) are generated enzymatically as by-products of normal metabolism through mitochondrial respiration and the cytochrome P450 system, and are stimulated by enzyme activity and as a result of non-enzymatic reactions of oxygen with organic compounds (Slater, 1972). Levels of AOS in vivo depend upon the balance between their generation and the capacity to remove them. Major AOS-scavenging activity of

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Corresponding author. Tel.: +86 27 87284181. E-mail address: renxuexia@mail.hzau.edu.cn (R.-X. Xia).

plants includes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (AsA-POD, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2). The balance of these enzyme activities in cells is crucial for determining the steady-state levels of superoxide radicals (O2 ?? ) and hydrogen peroxide (H2 O2 ). SOD converts O2 ?? to H2 O2 , which is then decomposed by CAT, a process widely present in both plants and animals (Fridovich, 1986). In addition, AOS can also be removed through a series of oxidation–reductions involving ascorbate (AsA) and glutathione (GSH) (Dalton et al., 1991). AsA is regenerated in a reduced GSH-dependent reaction catalyzed by DHAR and is then utilized by plants through

0925-5214/$ – see front matter ? 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.06.018

Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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an AsA-POD reaction and converted to dehydroascorbate (DHAsA). The AsA regeneration and utilization recycling system protects plants from possible injury from AOS. The generation of AOS and the cellular antioxidant defense systems in fresh fruit has been widely studied (e.g. Wang and Jiao, 2000; Wang and Li, 2006). The antioxidant enzyme activities and rindstaining in ‘Navelina’ navel oranges has been previously studied and it has been shown that SOD activities increase and CAT activity decreases during storage (Sala and Lafuente, 2004). Recently ‘Cara cara’ navel orange (C. sinensis L. Osbeck) had become an important citrus cultivar, as a result of its special features of nutritional value, red pigmentation and antioxidant compounds. It is desirable that nutritional levels in the pulp can be maintained as long as possible after harvest. However, dehydration occurs at room temperature (20 ? C), leading to ?esh woodiness. It has been well documented that antioxidant enzymes and non-enzyme components play an important role in protecting the plant cell against different stress conditions (MacRae and Ferguson, 1985; Jiang and Zhang, 2001; Rubio et al., 2002), including fruit crops such as mandarin (Sala, 1998). Salicylic acid (SA) is also involved in activation of the stressinduced antioxidant system when plants are exposed to stress (Borsani et al., 2001; Senaratna et al., 2004), and is now considered to be a hormonal substance that plays a key part in regulating plant growth and development. However, it is still unclear if SA treatment and various temperatures during postharvest storage affect the AOS-scavenging system in citrus fruit. To gain insight into this issue, the current study was carried out to investigate effects of SA on changes in the antioxidant system in the pulp of ‘Cara cara’ navel oranges at different storage temperatures. 2. Materials and methods 2.1. Fruit materials and pretreatments Fruit of ‘Cara cara’ navel orange (C. sinensis L. Osbeck) was harvested at a commercially mature stage (20th December) from Zigui county, Hubei province (China), and uniform (based on size and colour) and healthy fruit were selected for use. They were sterilized with 2% (v/v) sodium hypochlorite for 2 min, after which they were washed with tap water and air-dried, then treated with 2.0 mM SA for about 30 min (SApretreated fruit, SPTF), or with water (control fruit, CF). Each treatment was composed of three replicates of 30 fruit each and the entire experiment was repeated three times. The fruit were packed in perforated, low-density polyethylene bags and placed at 6 or 20 ? C in a temperature-controlled storeroom under air at 75–80% relative humidity. The equatorial pulp tissue was separated from the whole fruit, frozen in liquid nitrogen, and ground into ?ne powder and stored at ?70 ? C until use. 2.2. Measurement of malondialdehyde (MDA) and H2 O2 contents Finely ground powder of 1 g fresh weight (FW) was homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid, followed

by centrifugation at 10,000 × g for 5 min. The resulting supernatant was used for MDA and H2 O2 analysis based on the thiobarbituric acid reaction and titanium reaction, as respectively described by Peever and Higgins (1989) and Wang and Jiao (2000). 2.3. Extraction and analysis of SOD and CAT SOD was extracted at 4 ? C from 1 g FW ?nely ground powder in 10 mL of a cold solution of 1.33 mM diethylenetriamine pentaacetic acid in 50 mM potassium phosphate buffer (pH 7.8). After the homogenate was centrifuged twice at 4 ? C for 15 min at 27,000 × g, the supernatant was kept for the SOD assay by a spectrometric method (Oberley and Spitz, 1984). The superoxide radicals were generated by xanthine–xanthine oxidase and nitro-blue tetrazolium was used as an indicator of superoxide radical production. One unit of SOD was de?ned as the amount of enzyme (after 20 min) with gave half-maximal inhibition. CAT was extracted from 1 g of ?nely ground powder in 10 mL of cold 100 mM potassium phosphate buffer (pH 6.8) at 4 ? C. The homogenate was centrifuged as described above and the supernatant was used to determine the activity of CAT at 25 ? C following the method of Kar and Mishra (1976). One unit of CAT was de?ned as the amount of enzyme decomposing 1 mol H2 O2 per min at 25 ? C. 2.4. Measurement of enzymes in the AsA-GSH cycle Five grams of fruit tissue (FW) was homogenized with a solution of 2.0 mM ethylenediaminetetracetic acid, disodium salt and 2.0 mM dithiothreitol in 5.0 mL of 0.1 M Tris–HCl buffer (pH 7.8). The homogenate was centrifuged for 30 min at 20,000 × g (4 ? C), and the supernatant was used for the GR assay using a method modi?ed from Smith et al. (1988). 100 L of crude enzyme extract was added to a solution containing 0.05 M Tris–HCl buffer (pH 7.5), 3.0 mM MgCl2 , 0.5 mM GSSG, 2.0 mM ethylenediaminetetracetic acid, 0.15 mM NADPH to a total volume of 1.0 mL. The activity of GR, expressed as nanomoles of NADPH oxidized per mg of protein per min, was determined by monitoring the glutathione-dependent oxidation of NADPH at 340 nm with a spectrophotometer (Shimadzu UV-2450, Japan). Five g of fruit tissue (FW) was homogenized in a cold mortar with 5 mL of potassium phosphate buffer (0.1 M, pH 7.3) containing 1.0 mM ethylenediaminetetracetic acid and 2.0 mM dithiothreitol, followed by centrifugation for10 min at 12,000 × g (4 ? C) before AsA-POD and DHAR assays were carried out. AsA-POD activity, expressed as nanomoles of ascorbate oxidized per mg of protein per min, was detected according to Amako et al. (1994). The ?nal sample volume was made up to 1.0 mL composed of 0.05 M potassium phosphate (pH 7.0), 0.1 mM ethylenediaminetetracetic acid, 0.5 mM AsA, 0.5 mM H2 O2 , and 100 l of enzyme extract. DHAR activity, expressed as nanomoles of NADPH oxidized per mg of protein per min, was assayed by measuring the rate of NADPH oxidation at 340 nm (Shigeoka et al., 1980) in

Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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a reaction mixture of 1.0 mL containing 0.05 M potassium phosphate (pH 6.1), 0.2 mM NADPH, 2.5 mM DHAsA, 2.5 mM GSH, 0.6 unit of GR (from Sigma) and 100 l of crude enzyme extract. The reaction was started by adding DHAsA. 2.5. Determination of non-enzyme components of the AsA-GSH cycle Five grams of fruit tissue (FW) was homogenized in 5 mL of 5% (w/v) trichloroacetic acid. The homogenized tissue was ?ltered through four layers of Miracloth and centrifuged for 10 min at 16,000 × g (4 ? C) before the supernatant was used for assay of AsA and DHAsA (Chen and Wang, 2002). The mixture for the AsA assay, composed of 1.0 mL of the supernatant, 0.5 mL 100 mM phosphate buffer (pH 7.7), 1.0 mL 10% trichloroacetic acid, 1.0 mL 44% H3 PO4 , 1.0 mL 4% 2,2 -bipyridyl and 0.5 mL 3% FeCl3 , was incubated for 60 min at 37 ? C and then cooled down to room temperature. Absorbance of the colored solution was recorded at 525 nm. The assay buffer for total AsA, including 1.0 mL supernatant, 0.25 mL 100 mM phosphate buffer (pH 7.7) and 0.25 mL 0.2 mM dithiothreitol, was kept for 10 min at room temperature, followed by addition of 1.0 mL trichloroacetic acid (10%), 1.0 mL H3 PO4 (44%), 1.0 mL 2,2 -bipyridyl (4%) and 0.5 mL FeCl3 (3%). The ?nal mixture was incubated for 60 min at 37 ? C before absorbance at 525 nm was recorded. DHAsA concentration was calculated by subtracting AsA from total AsA. Five grams of fruit tissue (FW) was homogenized in 5.0 mL of ice-cold 60.0 mM phosphate solution with N2 . The homogenate was centrifuged for 15 min at 20,000 × g (4 ? C) and the supernatant was subjected to GSH and GSSG measurement as described by Castillo and Greppin (1988). Total GSH equivalents were determined by 0.1 mL of extract reacting with 60 mM KH2 PO4 -2.5 mM ethylenediaminetetracetic acid buffer (PH 7.5), 0.6 mM 5,5 -dithiobis (2-nitrobenzoic acid) in 200 mM Tris–HCl (pH 8.0), 1 unit of GR (from Sigma), and 0.2 mM NADPH. The reaction was followed as the rate of change in absorbance at 412 nm with a spectrophotometer (Shimadzu UV-2450, Japan), and the total glutathione content was calculated based on a standard curve. GSH was determined from the reaction mixture by mixing 0.1 mL of extract with 60 mM KH2 PO4 -2.5 mM ethylenediaminetetracetic acid buffer (pH 7.5), and 0.6 mM 5,5 -dithiobis (2-nitrobenzoic acid) in 200 mM Tris–HCl (pH 8.0). The mixture was incubated at 30 ? C for 10 min, and the reaction was followed as the rate of change in absorbance at 412 nm. GSSG was determined by subtraction of GSH from total glutathione. 2.6. Statistical analysis All data presented were means of three replicates along with standard errors of means. Data were further subjected to analysis of variance, and means were compared using least signi?cance difference (LSD) test (SAS Insti-

tute, Cary, USA). Differences at p < 0.05 were considered signi?cant. 3. Results 3.1. Effect of SA and storage temperature on H2 O2 and MDA production Minor changes in H2 O2 content were observed when control fruit were stored at either 6 or 20 ? C in the ?rst 30 days, but contents increased markedly at 45 days, and continuously increased to the end of storage of the fruit. H2 O2 content in the SA-pretreated fruit showed nearly the same trend as that of the control fruit, although contents in the former were significantly (p < 0.05) higher than in the latter at the same storage temperature (Fig. 1A). Lipid peroxidation, measured on the basis of MDA content, was investigated during storage (Fig. 1B). The MDA content in all fruit showed a notable increase during storage at both temperatures, and was higher in the fruit stored at 20 ? C than at 6 ? C. However, MDA content in SA-pretreated fruit was signi?cantly (p < 0.05) lower than that in the controls at the same storage temperature. At the end of storage, MDA content in pretreated fruit was 12.6 and 27.6% lower than that in control fruit at 20 and 6 ? C, respectively.

Fig. 1. The contents of H2 O2 (A) and MDA (B) in the pulp of ‘Cara cara’ navel orange affected by salicylic acid pretreated during different storage temperatures. CF + 6 ? C (— —), SPTF + 6 ? C (— —), CF + 20 ? C (— —), SPTF + 20 ? C (— —). The data are displayed with mean ± standard deviation (bars) of three replications. CF and SPTF stand for control fruit and SA-pretreated fruit, respectively.

Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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Fig. 2. The activities of SOD (A) and CAT (B) in the pulp of ‘Cara cara’ navel orange affected by salicylic acid pretreated during different storage temperatures. CF + 6 ? C (— —), SPTF + 6 ? C (— —), CF + 20 ? C (— —), SPTF + 20 ? C (— —). The data are displayed with mean ± standard deviation (bars) of three replications. CF and SPTF stand for control fruit and SA-pretreated fruit, respectively.

3.2. Effect of SA and storage temperature on CAT and SOD activity SOD activities in all fruit increased during storage with the maximal increase occurring at 15 days after storage (Fig. 2A). Fruit stored at 6 ? C had lower SOD activity. Treatment with SA led to an increase in SOD activity at both temperatures, except the last day of fruit stored at 20 ? C. CAT activity gradually increased in control fruit, and in the fruit stored at 20 ? C was slightly higher than in those at 6 ? C. After SA-pretreatment, CAT activities were signi?cantly (p < 0.05) reduced compared with those in fruit without SA-pretreatment at both temperatures (Fig. 2B). CAT activity decreased within the ?rst 30 days of storage at both temperatures, and remained constant thereafter. 3.3. Effect of SA and storage temperature on enzymes in the AsA-GSH cycle In?uence of SA and storage temperature on activities of enzymes involved in the H2 O2 -scavenging system in the ascorbate-glutathione cycle was investigated (Fig. 3). In CF, GR activity slightly declined within the ?rst 30 days, followed by a remarkable decrease from 45 to 105 days. No difference in GR

Fig. 3. The activities of GR (A), AsA-POD (B) and DHAR (C) in the pulp of ‘Cara cara’ navel orange affected by salicylic acid pretreated during different storage temperatures. CF + 6 ? C (— —), SPTF + 6 ? C (— —), CF + 20 ? C (— —), SPTF + 20 ? C (— —). The data are displayed with mean ± standard deviation (bars) of three replications. CF and SPTF stand for control fruit and SA-pretreated fruit, respectively.

activities between SA-pretreated and control fruit at 20 ? C was observed during the storage, whereas GR activity in pretreated fruit was signi?cantly (p < 0.05) higher than that in the controls at 6 ? C (Fig. 3A). During storage, the activity of AsA-POD in control fruit was higher at 20 ? C than that at 6 ? C (Fig. 3B), while there was no signi?cant difference between that in pretreated and control fruit at 20 ? C. In addition, AsA-POD activity of the untreated fruit at 6 ? C was signi?cantly decreased at 15 and 60 days, but the fruit stored at 20 ? C exhibited an increase in AsA-POD activity at the same time point. Furthermore, the activity in SApretreated fruit was signi?cantly (p < 0.05) higher than that in control fruit at 6 ? C, with the exception of 30 days and 75 days,

Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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3.4. Effect of SA and storage temperature on non-enzyme components in the AsA-GSH cycle Non-enzyme components, such as AsA and DHAsA, are important for reducing toxicity of AOS during storage. Fig. 4 showed the results during storage of pretreated and control fruit and at different temperatures. AsA in all fruit declined at both

Fig. 4. The values of AsA (A), DHAsA (B) and AsA/DHAsA (C) in the pulp of ‘Cara cara’ navel orange affected by salicylic acid pretreated during different storage temperatures. CF + 6 ? C (— —), SPTF + 6 ? C (— —), CF + 20 ? C (— —), SPTF + 20 ? C (— —). The data are displayed with mean ± standard deviation (bars) of three replications. CF and SPTF stand for control fruit and SA-pretreated fruit, respectively.

while no marked difference was detected at 20 ? C irrespective of treatment. DHAR activity in pulp of control fruit exhibited a gradual decrease during storage at 6 ? C, while a signi?cant increase was detected in controls at 20 ? C at 30 days. It is noted that DHAR activity in CF at 20 ? C was higher than that at 6 ? C at any time point. The activity of DHAR in SA-pretreated fruit showed a decrease during storage, but the decrease was mild compared with that in controls under the same storage temperature (Fig. 3C).

Fig. 5. The values of GSH (A), GSSG (B) and GSH/GSSG (C) in the pulp of ‘Cara cara’ navel orange affected by salicylic acid pretreated during different storage temperatures. CF + 6 ? C (— —), SPTF + 6 ? C (— —), CF + 20 ? C (— —), SPTF + 20 ? C (— —). The data are displayed with mean ± standard deviation (bars) of three replications. CF and SPTF stand for control fruit and SA-pretreated fruit, respectively.

Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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temperatures during storage. In the controls, the rate of reduction was faster at 6 ? C than at 20 ? C during the ?rst 30 days, however, the results were opposite after 45 days. In addition, the reduction of the AsA contents in pretreated fruit was less than that in the controls at the same temperature (Fig. 4A). DHAsA in all fruit remained constant within the ?rst 30 days, then rapidly increased from 45 days to 105 days. After 45 days, DHAsA contents in pretreated fruit were signi?cantly (p < 0.05) lower than those in the controls (Fig. 4B). Changes in ratios of AsA to DHAsA presented a similar trend to that of AsA with prolonged storage. However, the values of AsA/DHAsA in SA-pretreated fruit were signi?cantly (p < 0.05) higher than those in the controls at the same temperature (Fig. 4C). As shown in Fig. 5A, GSH contents in all fruit decreased during storage at both temperatures, but the decrease rate in the controls was faster. The result showed that at the end of storage under the treatments of CF + 20 ? C, SPTF + 20 ? C, CF + 6 ? C, and SPTF + 6 ? C, the GSSG contents of the fruit dropped to 66.9, 70.2, 72.8 and 72.2% of that at the onset of storage (Fig. 5B). The ratios of GSH to GSSG gradually decreased in all fruit, but pretreated fruit consistently exhibited higher values than controls at both temperatures (Fig. 5C). 4. Discussion Fruit development is a complex process involving regulation of a number of biochemical and developmental processes that lead to changes in oxidative metabolism (Brennan and Frenkel, 1977). Oxidative processes during fruit senescence were measured based on content of H2 O2 and lipid peroxidation. It is worth mentioning that the method to analyse H2 O2 is based on the oxidation of titanium and not speci?c. Therefore, the data for H2 O2 may exceed the actual value in the pulp. In this study, less accumulation of H2 O2 and MDA in control fruit was observed at 6 ? C than at 20 ? C suggesting that metabolism in ‘Cara cara’ navel orange pulp was signi?cantly slower at lower temperatures. Bowler and Fluhr (2000) reported that H2 O2 produced in response to a variety of stimuli acts as signaling molecule/second messenger and contributes to the phenomenon of cross-tolerance. On the other hand, pretreatment with SA signi?cantly increased the H2 O2 contents whatever storage temperature (Fig. 1A), as has been reported in tomato and cucumber (Sanchez-Casas and Klessig, 1994), thaliana (Rao et al., 1997) and rice (Vasudha et al., 2001). The mechanism underlying this increase remained to be clari?ed. One possibility is that SA facilitated an increase in SOD activity conversion of O2 ?? into H2 O2 . Another possibility is that exogenous SA penetrated into the pulp (free salicylic acid content was 3315–3567 ng g?1 FW in CF and 3743–3805 ng g?1 FW in SPTF) and combined with iron ions, leading to activation of the Haber-Weiss system, which negatively affected decomposition of H2 O2 . It has been suggested that an SA-induced increase in H2 O2 was mediated by an inhibition of CAT and AsA-POD (Dat et al., 2000; Landberg and Greger, 2002). However, our results suggested that accumulation of H2 O2 was independent of inhibition of AsA-POD (Fig. 3B). It is known that free radicals produced during fruit ripening, if not inactivated, can induce lipid peroxidation (Dhindsa et al., 1982),

leading to deteriorative changes associated with senescence (Du and Bramlage, 1994). Involvement of lipid peroxidation and H2 O2 in tissue senescence of pear fruit has been demonstrated (Brennan and Frenkel, 1977). However, in our work, lipid peroxidation in the pulp of SA-pretreated fruit, revealed by MDA contents, was signi?cantly lower than that in controls, although H2 O2 was higher in the former (Fig. 1), which may be due to inhibition of other AOS by SA, especially the ? OH radical, one of the major and direct factors associated with biosynthesis of MDA. As shown in Fig. 2B, activity of CAT in controls at both temperatures increased continuously during storage, and was higher at 20 ? C than at 6 ? C. This suggests that this enzyme plays a role in metabolic responses of orange pulp to stress factors (low temperature). It is well documented that CAT is an important enzyme catalyzing H2 O2 into H2 O and O2 , functioning in removal of excess AOS during stress. However, the content of H2 O2 in control fruit also increased during storage, especially in the middle stage (30–60 days), which might be ascribed to the fact that production of H2 O2 predominated over CAT scavenging activity. On the other hand, CAT activities in pretreated fruit were signi?cantly lower than those in controls at both temperatures (Fig. 2B). Our results were consistent with previous reports on maize (Janda et al., 1999), rice, wheat, and cucumber (Shim et al., 2003). In addition, it is important to mention that low activity of CAT in the pulp of SPTF was concurrent with a rise in activities of other components of the antioxidant system. SOD has been implicated as an essential component for defense against the potential toxicity of oxygen (McCord, 1979) and this defensive action was affected by temperature. Lower SOD activity was measured in the pulp sampled from control fruit stored at 6 ? C in comparison with fruit at 20 ? C, in line with results from strawberry (Vicente et al., 2006). This phenomenon may probably be due to the lower production of O2 ?? in cells at low temperature, or slow metabolism at the temperature. On the other hand, SA-pretreatment signi?cantly increased the activity of SOD whatever storage temperature (Fig. 2A), which is in agreement with the work on Agrostis stolonifera by Larkindale and Huang (2004). The increase might be related to promotion of activity of SOD with different isoforms such as Fe-SOD and Mn-SOD. It has been reported that stress tolerance of plants may be associated with their ability to remove AOS (Senaratna et al., 1985; Ben-Amor et al., 1999), in which enzymes responsible for the ascorbate-glutathione cycle, such as AsA-POD, DHAR, and GR, may play key roles (Sala, 1998; Sala and Lafuente, 1999). In the presence of NADPH, GR contributes to the regeneration of ascorbate (Saruyama and Tanida, 1995), which, along with AsA-POD, decomposes H2 O2 . In addition, GR catalyzes the NADPH-dependent reduction of GSSG to GSH, keeping the ratio of GSH/GSSG high (Gamble and Burke, 1984). During storage at 6 ? C, activities of GR, AsA-POD, and DHAR of control fruit maintained relatively lower levels (Fig. 3). GR and DHAR activities showed a decrease when the storage period was extended, suggesting that they may be ascribed to an adaptive response to cope with environmental conditions and are important in regulating fruit senescence. In addition, treatment

Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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with SA led to higher activities of GR, AsA-POD, and DHAR than control fruit at 6 ? C, although such differences did not hold true for fruit stored at 20 ? C. This suggested that the in?uence of SA on the antioxidative enzyme system may be related to temperature. Janda et al. (1999) also showed that pretreatment with SA for 1 day provided protection in maize plants against low temperature stress and induced increased antioxidant activity. AsA, essential metabolites and powerful regulators for regulating cell functions, play a pivotal role in antioxidant defense (Smimoff, 1995). In our work, AsA content in control fruit stored at 6 ? C were lower than that at 20 ? C from 0 to 30 days, which was totally opposite during the rest period. AsA functions as a co-substrate of plant POD, for production of DHAsA (Halliwell, 1982). On the other hand, there was no any in?uence of temperature on DHAsA, and DHAsA increased remarkably after 45 days. This demonstrated that both storage temperatures and duration were instrumental in mobilizing the antioxidant defense of AsA in ‘Cara cara’ navel orange fruit tissue. AsA content in the pulp of SA-pretreated fruit was higher than that in the controls at both temperatures (Fig. 4A), while the opposite trend was observed concerning DHAsA, especially after 45 days (Fig. 4B). High AsA contents in the pulp of pretreated fruit may result from an acceleration of biosynthetic pathways or a decrease in catabolism through an accumulation of DHAsA. Accumulation of DHAsA suggests that catabolism may be an important reason for this. Both changes lead to a shift from the reduced form to the oxidized form, and as observed, to a decrease in the AsA/DHAsA ratios (Fig. 4C). Like AsA, glutathione is a low-molecular-mass compound with recognized antioxidant functions. In exists in cells with two forms, reduced (GSH) and oxidized forms (glutathione disul?de, GSSG), the former dominating. In this study, GSH content was higher in control fruit stored at 6 ? C than at 20 ? C, while negligible differences were observed in GSSG between the two temperatures. Similarly, the relatively lower GR and DHAR activities were also observed in the pulp of controls at 6 ? C. These results may be relative to changes of GR and DHAR. On the other hand, GSH content in pulp of pretreated fruit was higher than that in controls whatever storage temperature (Fig. 4A), whereas pretreatment with SA did not affect GSSG content notably. This result was consistent with those of Wang et al. (2006). Recently, Wang and Li (2006) proposed that SA induced Ca2+ movement from vacuoles and intercellular spaces to the cytoplasm which could induce the AsA-GSH cycle, leading to a GSH increase. Apart from GSH, the ratio of reduced-to-oxidized glutathione (GSH/GSSG) has to be taken into consideration. It has been suggested that a high GSH/GSSG ratio is necessary for several physiological functions including activation and inactivation of redox-dependent enzyme systems (Ziegler, 1985). Kocsy et al. (2001) also suggested that a change in GSH/GSSG was more important than GSH content in cell resistance to oxidative stress. The present study showed that SA pretreated fruit had higher GSH/GSSG than controls during storage at both temperatures, in agreement with Kn¨ rzera o et al. (1999) who reported that SA increased cold tolerance of soybean by promoting the ascorbate-glutathione cycle.

In conclusion, the work presented here showed that the antioxidant system (SOD, CAT, GR, AsA-POD, DHAR, AsA and GSH) may participate in less lipid peroxidation in the pulp of ‘Cara cara’ fruit stored at 6 ? C than that at 20 ? C, and that application of SA could increase antioxidant enzyme activity and thus delay membrane lipid peroxidation (with the exception of CAT), and improve levels of antioxidant components (AsA and GSH) as well. Therefore, we suggest that pretreatment with SA in combination with low storage temperature may be a useful strategy for prolonging orange postharvest life and maintaining nutritional conditions during storage. Acknowledgements This work was supported by project from Migrant, Ministry of Science and Technology (2003EP090018; 2004EP090019;) and NSFC (30671435), PR China. References
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Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018

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Please cite this article in press as: Huang, R.-H., et al., Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures, Postharvest Biol. Technol. (2007), doi:10.1016/j.postharvbio.2007.06.018


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