Antioxidant enzymes activities and rindstaining in ‘Navelina’ oranges as affected by storage relative humidity and ethylene conditioning

Postharvest Biology and Technology 31 (2004) 277–285

Antioxidant enzymes activities and rindstaining in ‘Navelina’ oranges as affected by storage relative humidity and ethylene conditioning José M. Sala, Mar´ıa T. Lafuente∗ Instituto de Agroqu´ımica y Tecnolog´ıa de Alimentos (IATA), Consejo Superior de Investigaciones Cient´ıficas (CSIC), Apartado de Correos 73, Burjassot 46100, Valencia, Spain Received 22 May 2003; accepted 9 October 2003

Abstract The involvement of active oxygen detoxifying enzymes on postharvest rindstaining occurring in citrus fruit at non-chilling temperature has been investigated. Changes in the activities of superoxide reductase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) in the flavedo of ‘Navelina’ (Citrus sinensis L. Osbeck) oranges stored at 22 ◦ C under high (85–90%) and low (55–60%) relative humidity (RH) have been examined. In addition, the effect of conditioning the fruit for 4 days with 10 ␮l l−1 ethylene at 22 ◦ C and 85–90% RH before being transferred to air at the same temperature and 65–70% RH was studied. The SOD activity increased during storage of fruit at 22 ◦ C under low (55–60% RH) or high (85–90% RH) humidity, being the rate of increase higher in fruit kept under low humidity. By contrast GR decreased and a slower decline rate was observed in fruit stored at low RH. A significant reduction in the activities of CAT and APX also occurred in ‘Navelina’ oranges maintained at 85–90% RH, but not at 55–60% RH. Thus, fruit kept under high RH, which showed higher rindstaining, presented lower SOD, CAT, GR and APX activities than fruit stored under low RH. The incidence of this physiological disorder was reduced by the ethylene pre-treatment. No significant differences in the activities of these enzymes were found in fruit examined after 4 days of ethylene or air treatment. GR activity of fruit pre-treated with ethylene and then held under air was significantly higher than that of their respective control air-treated fruit. Conversely, CAT was higher in fruit continuously held under air and no significant differences in APX and SOD were found. These results indicate that SOD, CAT, GR and APX may play a role in the lower rindstaining incidence observed in ‘Navelina’ fruit continuously exposed to low RH, as compared with fruit held under high RH, while GR may be involved in the beneficial effect of ethylene reducing this non-chilling physiological disorder. © 2003 Elsevier B.V. All rights reserved. Keywords: Abscisic acid; Ascorbate peroxidase; Catalase; Citrus; Ethylene; Glutathione reductase; Non-chilling postharvest physiological disorder; Superoxide reductase; Water stress

1. Introduction ∗ Corresponding author. Tel.: +34-963-900022; fax: +34-963-636301. E-mail address: [email protected] (M.T. Lafuente).

The damage of plant tissues has been associated with active oxygen species (AOS) arising under stress conditions such as exposure to ozone (Reich and

0925-5214/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2003.10.002

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Amundson, 1985), chilling (Wise and Naylor, 1987), UV-light (Boldt and Scandalios, 1997) and water stress (Senaratna et al., 1985; Sgherri et al., 1994). The level of AOS in vivo depends upon the balance between their generation and the capacity to remove them. Major AOS-scavenging mechanisms of plants include superoxide reductase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2). The balance between SOD, APX, GR and CAT activities in cells is crucial for determining the steady-state level of superoxide radicals and hydrogen peroxide. It has been reported that APX might be responsible for the fine modulation of AOS for signalling, whereas CAT might be responsible for the removal of excess AOS during stress (Mittlet, 2002). The protective function of those enzymes against different stress conditions has been reported in plants (MacRae and Ferguson, 1985; Jiang and Zhang, 2001; Rubio et al., 2002) but also in horticultural crops, including fruit such as melons and mandarins (Sala, 1998; Ben-Amor et al., 1999). The involvement of plant hormones in the stress-induced antioxidant system has been also reported (Ben-Amor et al., 1999; Jiang and Zhang, 2001; Michaeli et al., 2001; Arora et al., 2002). Contrasting results have been found on the relationship between ethylene and the AOS detoxifying enzymes. Thus, the deleterious effect of ethylene favouring chilling in cantaloupe melons was associated with a reduction in the activities of CAT and SOD (Ben-Amor et al., 1999), while CAT activity increased with ethylene during cold storage of apples and CAT and SOD have been closely related to the onset of apple ripening, signalled by an ethylene burst (Masia, 1998). On the other hand, exogenous ethylene during storage of spinach leaves caused a decrease in levels of CAT, APX and GR (Hodges and Forney, 2000), while the application of aminoethoxyvinylglicine reduced the nitrate-induced ethylene production and the activation of the enzymes CAT, APX and GR in chickpea nodules (Mann et al., 2002). The active oxygen detoxifying enzymes have been shown to participate in the beneficial effect of postharvest heat conditioning treatments protecting citrus fruit against chilling (Sala and Lafuente, 1999), but their involvement in the susceptibility of citrus fruit to other non-chilling postharvest physiological disorders is still unknown. ‘Navelina’ oranges (Citrus sinensis L. Os-

beck) are prone to develop postharvest rindstaining during storage at non-chilling temperature. This disorder, manifested as extensive collapsed and dry areas of the flavedo (outer coloured part of the peel) and part of the albedo (inner part of the peel) that becomes dark with time, has been shown to be influenced by storage RH and ethylene (Lafuente and Sala, 2002; Alférez et al., 2003). The incidence of rindstaining in this citrus cultivar has been shown to be favoured by increasing environmental RH during storage (Lafuente and Sala, 2002). However, different postharvest treatments which raised the humidity to over 96% RH reduced ‘noxan’, a similar physiological blemish occurring in Shamouti oranges (Ben Yehoshua et al., 2001). These different effects of the RH environment could be due to the different peel water status at harvest since rindstaining is aggravated by fruit exposure to changes in RH (Agust´ı et al., 2001; Alférez et al., 2003), and low differences in the water regimen during fruit storage may lead to a marked difference in the induction of this type of blemish (Ben Yehoshua et al., 2001). These and previous results (Casas and Garc´ıa-Bataller, 1986; Sala et al., 1992) suggest the involvement of water stress in the development of this non-chilling physiological disorder. AOS have been shown to be associated with plant tissue damage caused by water stress (Senaratna et al., 1985) and also to be produced under physiological conditions altering ethylene production (Nandwal et al., 2000; Mann et al., 2002). However, how postharvest ethylene and RH conditions may affect the AOS detoxifying enzymes SOD, CAT, GR and APX in citrus fruit has not been reported. The aim of this study was to investigate the effect of storage relative humidity and of an ethylene pre-treatment on changes in the activities of these antioxidant enzymes in the flavedo of ‘Navelina’ oranges stored at a non-chilling temperature. Our results revealed that those enzymes are good candidates to be involved in the mechanisms underlying tolerance of citrus fruit to non-chilling rindstaining.

2. Materials and methods 2.1. Fruit and storage conditions Mature ‘Navelina’ orange fruit were harvested at random from 12 trees grafted onto Troyer citrange

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rootstock (Citrus Sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf), which were grown in a commercial orchard in Lliria (Valencia, Spain). All the fruit were harvested after colour break (orange colour) and transferred immediately to the laboratory, where fruit free of visual defects and of uniform size were selected. In a first experiment, fruit were divided into two groups, one group stored at 22 ◦ C under air at 85–90% RH for up to 28 days, and the other at the same temperature under air at 55–60% RH for the same period of time. Three replicates of 25 fruit were included in each group to estimate the rindstaining incidence, and of five fruit per storage period to analyse changes in the activities of the enzymes SOD, CAT, APX and GR. In a second experiment, we studied the effect of conditioning the fruit with ethylene before being transferred to air at a different RH on the development of rindstaining and on the activities of the AOS detoxifying enzymes. To that end, fruit were divided into two groups, which contained the same replicates and number of fruit as in the first experiment. Fruit from the first group, control group, were held under air for 4 days at 22 ◦ C and 85–90% RH, while fruit included in the other group were treated for the same period of time with 10 ␮l l−1 ethylene at 85–90% RH and 22 ◦ C in experimental chambers. Thereafter, fruit from both groups were transferred to air at the same temperature and 65–70% RH (simulation of environmental RH). Flavedo tissue, the coloured outer layer of skin, was separated from the whole fruit, frozen in liquid N2, homogenised, and stored at −70 ◦ C for enzyme assays. Enzymes were extracted from representative 1 g fresh weight (FW) samples and the results are given as the means of three replicate samples ± S.E.M. 2.2. Determination of rindstaining incidence Symptoms of rindstaining are manifested as collapsed and drying areas of the flavedo (the outer coloured part of the peel) and part of the albedo (the inner part of the peel) that become dark brown with time. To estimate the incidence of this physiological disorder, fruit were visually inspected and the percentage of fruit showing rindstaining calculated. The results are the means of three replicate samples of 25 fruit ± S.E.M.

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2.3. SOD, CAT, GR and APX assays The activities of the enzymes SOD, CAT, APX and GR were analysed in the flavedo of ‘Navelina’ oranges as previously described by Sala (1998). Each value is the mean of three replicate samples containing five different fruit ± S.E.M. SOD was extracted at 4 ◦ C from 1 g FW of frozen flavedo tissue with 10 ml of cold 50 mM potassium phosphate buffer, pH 7.8, containing 1.33 mM diethylenetriamine pentaacetic acid (DETAPAC) in a mortar and pestle. The homogenate was centrifuged twice at 4 ◦ C for 15 min at 27,000 × g and the supernatant used to assay SOD spectrophotometrically by the method of Oberley and Spitz (1986). The superoxide radicals were generated by xanthine–xanthine oxidase and nitro blue tetrazolium (NBT) was used as indicator of superoxide radical production. One unit of SOD was defined as the amount of enzyme that gave half-maximal inhibition. One gram FW of frozen flavedo tissue was pulverised in a mortar and pestle with 10 ml of cold 100 mM potassium phosphate buffer, pH 6.8, at 4 ◦ C to extract CAT. The homogenate was centrifuged as described above and the supernatant used to determine the activity of CAT at 25 ◦ C following the method of Kar and Mishra (1976). One unit of CAT was defined as the amount of enzyme, which decomposes 1 ␮mol H2 O2 per minute at 25 ◦ C. APX was extracted from 1 g of FW flavedo tissue with 10 ml of cold 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM ethylene diaminetetraacetic acid (EDTA), 1 mM ascorbic acid and 1% polyvinyl-polypyrrolidone (PVPP) at 4 ◦ C. The homogenate was centrifuged twice as described above and the supernatant used to determine APX activity as described by Asada (1984). One unit of APX was defined as the amount of enzyme that oxidised 1 ␮mol of ascorbate per minute. GR was extracted from 1 g of FW flavedo tissue with 10 ml of cold 100 mM potassium phosphate buffer, pH 7.5, containing 0.5 mM EDTA at 4 ◦ C. The homogenate was centrifuged twice at 4 ◦ C for 15 min at 27,000 × g and the supernatant used to assay GR spectrophotometrically by the method of Smith et al. (1988). The activities of the GR solutions used for the standard curve was determined according to Carlberg and Mannervik (1985). One unit of GR was defined

Experimental data are the mean ± S.E.M. of three replicates of the determinations for each sample. ANOVA analysis were performed by means of SAS/STAT GLM procedure, using the orthogonal contrast test for means comparison (P ≤ 0.05).

3. Results 3.1. Effect of storage relative humidity on fruit rindstaining and on SOD, CAT, APX and GR activities The incidence of rindstaining was lower in ‘Navelina’ fruit stored under low RH (Table 1). No postharvest rind disorder was developed in fruit held at high (85–90% RH) and low RH (55–60% RH) for up to 4 days storage at 22 ◦ C. After 13 days, 77.7% of fruit stored under high RH showed rindstaining, while the percentage of damaged fruit at low RH was 54.7%. The incidence of rindstaining increased thereafter, with a faster increase in fruit stored at 85–90% RH. Thus, by 28 days, 64.0% and 90.3% of rindstained fruit were found at 55–60% and 85–90% RH, respectively. Changes in the activities of the enzymes SOD, CAT, APX and GR in ‘Navelina’ fruit stored at 22 ◦ C depended on the environmental RH selected. The pattern of changes in the activities of these enzymes Table 1 Incidence of rindstaining in ‘Navelina’ fruit stored for up to 28 days at 22 ◦ C under air at 55–60 and 85–90% RH Days at 22 ◦ C

Rindstaining (%) 55–60% RH

4 13 21 28

0.0 54.7 58.3 64.0

± ± ± ±

0.0a 5.5a 8.3a 4.0a

85–90% RH 0.0 77.7 89.0 90.3

± ± ± ±

0.0a 1.2b 4.2b 9.7b

Values represent means ± S.E.M. For the same storage period, values labelled with the same letter are not different at the 5% significance level.

(A)

1800 1600 1400 1200 1000

CAT activity (u g FW-1)

2.4. Statistical design

2000

100

APX activity (u gFW -1)

as the amount of enzyme that catalysed the oxidation of 1 ␮mol of NADPH per minute.

SOD activity (u gFW -1)

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10

GR activity (u gFW -1)

280

(B)

80

60

40

(C)

9 8 7 6 5

(D) 1.6 1.4 1.2 1.0 0.8 0.6 0

5

10

15

20

Days at 22˚C Fig. 1. Changes in SOD (A), CAT (B), APX (C), and GR (D) activities in the flavedo of ‘Navelina’ orange fruit stored for up to 20 days at 22 ◦ C under air at 55–60% RH (䊐) and at 85–90% RH (䊏). Values are the mean of three replicate samples ± S.E.M.

differed during holding of the fruit at this temperature but, with one exception (13 days storage APX activity, Fig. 1C), fruit kept under low RH presented significantly higher activities than their corresponding

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control fruit held at high RH. The SOD activity continuously increase during storage of fruit at 22 ◦ C either at 55–60% RH or 85–90% RH (Fig. 1A). However, the rate of increase was higher in ‘Navelina’ oranges kept under low RH. Thus, by the 13th day, when differences in rindstaining incidence of fruit began to be apparent, a 1.2-fold increase in SOD activity occurred in fruit held at high RH, while a 1.7-fold increase was observed in those held at low RH. This difference persisted in fruit stored for 20 days, which showed noticeable differences in the severity of rindstaining. A reduction in CAT (Fig. 1B) and APX (Fig. 1C) activities was found in ‘Navelina’ oranges kept at 85–90% RH. Such reduction mainly occurred at the beginning of the storage period (4 days). By contrast, CAT activity showed little change during fruit storage at low RH, while APX slightly increased for up to 4 days and then remained nearly constant. Therefore, the activities of both enzymes were, in general, significantly higher (P ≤ 0.05) in fruit held under low RH. The pattern of changes in GR activity was opposite to that observed in SOD activity. A sharp decrease in GR was induced by holding the fruit at 22 ◦ C and 85–90% RH (Fig. 1D). GR activity also decreased in oranges stored at low RH, but the rate of decrease was much lower. Thus, a 2-fold decrease in GR was found in fruit kept for 20 days at high humidity, while a 1.4-fold decrease occurred in those stored at 55–60% RH. 3.2. Effect of ethylene conditioning on fruit rindstaining and on SOD, CAT, APX and GR activities To study how AOS detoxifying enzyme activities are related to the effect of ethylene conditioning on the susceptibility of ‘Navelina’ oranges to rindstaining, a group of fruit was treated for 4 days with 10 ␮l l−1 ethylene at 22 ◦ C and 85–90% RH, while control fruit were treated with air under the same conditions, before being transferred to air at a different RH (65–70% RH). Fruit used in this second experiment were harvested 1 month later than those used in the first experiment from the same orchard. Conditioning the fruit for 4 days with 10 ␮l l−1 ethylene significantly reduced the incidence of rindstaining in ‘Navelina’ fruit (Table 2). Little damage was observed in fruit held for 4 days under air or ethylene. A sharp increase in the per-

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Table 2 Incidence of rindstaining in ‘Navelina’ fruit pre-treated for 4 days with air (air-pre-treated) or with ethylene (ethylene-pre-treated) at 85–90% RH and 22 ◦ C (4 days) and thereafter transferred to air at 65–70% RH at the same temperature (7–28 days) Day

RH (%)

Rindstaining (%) Air-pre-treated

4 7 17 24 28

85–90 65–70 65–70 65–70 65–70

4.0 72.3 90.7 90.3 92.0

± ± ± ± ±

2.3a 4.3a 5.3a 4.0a 4.0a

Ethylene-pre-treated 6.1 42.3 49.3 59.7 60.3

± ± ± ± ±

1.3a 2.2b 4.7b 4.8b 4.2b

Values represent means ± S.E.M. For the same storage period, values labelled with the same letter are not different at the 5% significance level.

centage of rindstained fruit was observed thereafter, when control air pre-treated fruit and those pre-treated with ethylene were transferred from 85–90% RH to air at 65–70% RH (4–7 days, Table 2). However, such increase was much lower in ethylene-treated fruit. The percentage of damaged oranges slightly increased thereafter in both groups and remained significantly lower in the ethylene conditioned fruit throughout the whole storage period. Ethylene by itself did not induce changes in the activities of the AOS detoxifying enzymes as no significant differences (P ≤ 0.05) in these activities were found between fruit treated for 4 days at 22 ◦ C and 85–90% RH with air or ethylene (Fig. 2). APX activity appeared to be higher in fruit treated for 4 days with ethylene (Fig. 2C) but the differences found between air- and ethylene- treated fruit were only significant at P = 0.06. The activities of the enzymes SOD (Fig. 2A), APX (Fig. 2C) and GR (Fig. 2D) increased by transferring fruit treated with air for 4 days at 85–90% RH to air at 65–70% RH (21 days), while CAT (Fig. 2B) decreased. Conditioning the fruit with ethylene favoured the rise in the activity of the enzyme GR and the decline in CAT. Thus, significant differences (P ≤ 0.05) in the activities of both enzymes were found between fruit conditioned with air and ethylene by 21 days storage at 22 ◦ C. The increase in the APX activity occurring in ‘Navelina’ fruit from 4 to 21 days storage was similar in air- and ethylene-treated fruit and, by 21 days, no significant differences were found in fruit

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) --1 SOD activity (u gFW

4. Discussion

(A) 1000 800 600 400 200

CATactivity (ugFW

-1

)

(B) 60

40

20

APXactivity (ugFW

-1

)

(C) 10 8 6 4 2

GRactivity(u gFW

-1

)

(D) 0.4

0.3

0.2

4

21

Days at 22 ˚C Fig. 2. Activities of the enzymes SOD (A), CAT (B), APX (C) and GR (D) in the flavedo of ‘Navelina’ oranges treated for 4 days with air (black bars) or 10 ␮l−1 ethylene (grey bars) at 22 ◦ C and 85–90% RH and then exposed to air at 22 ◦ C and 65–70% RH for an additional period of 17 days storage (21 days). Values are the mean of three replicate samples ± S.E.M.

previously exposed to both treatments (P > 0.05). No significant difference was either found in SOD activities of fruit pre-treated with air and ethylene after this storage period (Fig. 2A).

It has been reported that the tolerance of plants to stress conditions originating damage may be associated with their higher ability to remove AOS (Senaratna et al., 1985; Ben-Amor et al., 1999) and that AOS detoxifying enzymes, SOD, CAT, GR and APX, may play a role protecting citrus fruit from chilling-induced damage (Sala, 1998; Sala and Lafuente, 1999). In the present work we have focused our attention on evaluating the effect of storage RH and of an ethylene pre-treatment, affecting the development of a postharvest non-chilling (22 ◦ C) physiological disorder (rindstaining) in ‘Navelina’ oranges, on the activities of these enzymes. As shown in Fig. 1, the ability of ‘Navelina’ fruit kept continuously at low RH to metabolise oxygen toxic forms was higher than that of fruit held under high RH, as the activities of the AOS detoxifying enzymes examined here were significantly higher in fruit stored at 55–60% RH. Thus, SOD activity might play a role in the dismutation of superoxide radicals, whereas CAT, APX and GR activities would contribute, at least to some extent, to the elimination of hydrogen peroxide. From these results and the fact that fruit kept continuously under high humidity showed a higher susceptibility to develop rindstaining, it cannot be ruled out that the tolerance of ‘Navelina’ fruit to this physiological disorder may be associated with their ability to remove AOS. Previous findings showed that mild water stress can result in the increased generation of AOS and activate the antioxidant enzyme system in plants (Senaratna et al., 1985; Jiang and Zhang, 2002a) and documented that these water stress-related events may be dependent on the stress-induced abscisic acid (ABA) (Jiang and Zhang, 2002b). Interestingly, we have recently shown that the rise in ABA occurring during storage of ‘Navelina’ oranges under 55–60% RH was higher (1.7-fold increase after 4 days) than that occurring in fruit stored at 85–90% RH (Lafuente and Sala, 2002), while results from the present work demonstrate that fruit kept under low RH presented higher SOD, CAT, GR and APX activities. Therefore, ABA could contribute in part to maintain higher the activities of the oxygen detoxifying enzymes in ‘Navelina’ fruit held under low RH, which presented lower rindstaining (Table 1).

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Besides studying the effect of keeping ‘Navelina’ fruit at a constant high or low storage RH on the activities of the antioxidant enzymes, we have examined in the present paper how those enzymes were influenced by conditioning the fruit with ethylene at high RH (85–90% RH) before exposing them to a different RH (65–70% RH, simulation of environmental conditions that may occur during commercial conditions), which has been previously shown to provoke rindstaining in ‘Navelina’ fruit (Lafuente and Sala, 2002). Ethylene has been shown to provoke or enhance the induction of brown necrotic tissue areas in some commercial horticultural crops (Hyodo et al., 1978; Ben-Amor et al., 1999), but also may participate in the defensive mechanism of plants against stress (Yang and Hoffman, 1984). Previous results in citrus fruit demonstrate that ethylene induction during fruit cold storage plays a role in reducing the development of chilling-induced peel damage, although applying ethylene at a non-chilling temperature did not reduce the incidence of this disorder when fruit were subsequently held at a chilling temperature (Lafuente et al., 2001). By contrast, we have found that the incidence of rindstaining occurring in ‘Navelina’ oranges at 22 ◦ C when were transferred from 85–90 to 65–70% RH may be reduced by applying ethylene immediately after fruit harvest (Table 2), while a marked and transient increase in ethylene production has been associated with rindstaining occurring in navel oranges exposed to changes in storage RH (Alférez et al., 2003). Ethylene may be involved in different mechanisms which protect plants against stresses, but its role on the process of rindstaining is not understood. It has been well documented that the activity of some AOS detoxifying enzymes increase, while others decrease in response to ethylene, with the specific profiles dependent upon the species examined and whether ethylene was applied or stress-induced (Masia, 1998; Ben-Amor et al., 1999; Hodges and Forney, 2000; Mann et al., 2002). To our knowledge, the present study is the first reporting the effect of ethylene on the activities of the AOS scavenging enzymes in citrus fruit. We have found that a 4 days treatment with 10 ␮l l−1 ethylene at 22 ◦ C did not significantly alter the activities of the AOS detoxifying enzymes in ‘Navelina’ fruit, as compared to the data found in control fruit treated for 4 days with air (Fig. 2). In addition, our data

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show that after transferring ‘Navelina’ fruit pre-treated with air or ethylene at 85–90% RH to air at 65–70% RH, CAT activity was lower in ethylene pre-treated fruit and no difference was observed in SOD, indicating that the beneficial effect of ethylene on reducing postharvest citrus rindstaining would not be related to the removal of the excess of AOS by increasing CAT or SOD. In melons it has been suggested, however, that ethylene might be responsible, in association with low temperature, for a reduced capacity of fruit to remove AOS through lowered activity of CAT and SOD and of favouring the chilling-induced necrosis (Ben-Amor et al., 1999). It is also interesting that storage of spinach leaves in ambient air + ethylene enhanced the decrease in levels of AOS detoxifying enzymes and promoted senescence, a genetically regulated process which leads to cell death (Hodges and Forney, 2000). Our data also show that APX activity of ‘Navelina’ fruit conditioned with ethylene at 22 ◦ C and 85–90% RH and then transferred to air at 65–70% RH was not significantly higher than that of their control air-treated fruit (see 21 days, Fig. 2). By contrast, although this hormone did not induce an increase in GR activity (4 days, Fig. 2), it enhanced its induction after subsequent transference of ‘Navelina’ fruit to air at 65–70% RH (see 21 days, Fig. 2). This suggests that GR may be a good candidate to be involved in the ethylene-induced rindstaining tolerance and, therefore, we cannot rule out the participation of the Halliwell–Asada cycle in the beneficial effect of ethylene on reducing ‘Navelina’ rindstaining. In this cycle, the efficacy of the enzyme APX to remove hydrogen peroxide depends on the tissues ability to regenerate ascorbic acid and this is mediated by reduced glutathione, which in turn depends on the GR activity and NADPH. Thus, the higher is the GR activity the higher would be the APX efficiency removing hydrogen peroxide. Furthermore, glutathione peroxidase is an important enzyme participating in the removal of hydrogen peroxide that requires an glutathione regenerating cycle and this depends on GR and NADPH (Mittlet, 2002). The activities of the enzymes SOD, APX and GR increased and that of CAT declined when air-conditioned ‘Navelina’ fruit were transferred from 85–90 to 65–70% RH in concordance with the sharply increased development of rindstaining. It could be thought that the decline in CAT activity occurring in

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the air-treated fruit under the experimental conditions assayed (4–21 days) was associated with the development of rindstaining but the ethylene-pre-treatment favoured the decline in CAT (Fig. 2B) and reduced rindstaining (Table 2). The induction of the other enzymes could be ascribed to an adaptive fruit response to cope with environmental conditions originating rindstaining. Nevertheless, such increases were not sufficient to avoid ‘Navelina’ fruit damage. Avoiding AOS production might be as important as activating scavenging of AOS. It should be considered that mechanisms that might reduce AOS production during stress include anatomical and physiological adaptations and it has been shown that ethylene application may led to morphological and physiological changes which could increase plant resistance to a subsequent stress-induced tissue damage (Liptay et al., 1982). We cannot rule out the involvement of ethylene-induced resistance mechanisms different of those underlying oxidative stress which would reduce the incidence of rindstaining occurring after exposing the fruit to changes RH, but our results pointed out that the beneficial effect of ethylene reducing rindstaining may involve changes in GR. Our results showing that the more mature fruit harvested in the second experiment had lower SOD, CAT and GR activities and were more susceptible to develop rind damage than fruit from the first experiment may further indicate that the maturity stage and the antioxidant enzymatic system might participate in the ‘Navelina’ rindstaining development. Nevertheless, it should be considered that rindstaining is aggravated by fruit exposure to changes in RH (Alférez et al., 2003) and fruit used in the second experiment were subjected to storage RH changes, while the RH was maintained constant in the first experiment. On the other hand, as the different peel water status at harvest might also influence the susceptibility of ‘Navelina’ fruit to develop rindstaining, it is difficult to ascertain from the present results the influence of fruit maturity on the incidence of RS found here. It is also important to note that the response of plant antioxidant system to water stress may depend on the severity of the imposed stress and on the plant developmental stage (Baisak et al., 1994; Synková and Valcke, 2001). Based on the assumption that the activities of the antioxidant enzymes reflect the need for detoxification of the respective AOS, and considering the global results

of the present paper illustrating that the AOS detoxifying enzymes may be operating in the rindstaining process, further research should be conducted to characterise the involvement of different AOS in the development of this non-chilling physiological disorder in citrus fruit. In summary, our results indicate that: (1) SOD, CAT, APX and GR enzymes may participate in the lower incidence of rindstaining found in ‘Navelina’ fruit continuously stored under low than under high RH; (2) ethylene (4 days 10 ␮l l−1 ) by itself did not significantly alter the activities of the AOS detoxifying enzymes in ‘Navelina’ oranges but conditioning the fruit with this ethylene treatment enhanced their GR activity and reduced postharvest rindstaining.

Acknowledgements This work was supported by research grants ALI-93-117 and ALI-96-0506-CO3 from the Comisión Interministerial de Ciencia y Tecnolog´ıa (CICYT), Spain. We gratefully acknowledge the statistical advice of Mr. Lopez-Santoveña and the technical assistance of Mrs. D. Arocas and M.J. Pascual. Special thanks are due to Mr. J. Llacer and V. Ortell from Brogdex C. Iberica S.A. for their help and for allowing us to use their facilities. The provision of fruit by Mr. M. Sabater is also gratefully acknowledged.

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