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Catalase enzyme activity is related to tolerance of mandarin fruits to chilling
Postharvest Biology and Technology 20 (2000) 81 – 89 www.elsevier.com/locate/postharvbio
Catalase enzyme activity is related to tolerance of mandarin fruits to chilling Jose M. Sala *, Marı´a T. Lafuente Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA), Consejo Superior de In6estigaciones Cientı´ficas (CSIC), Apartado de Correos 73, Burjassot 46100, Valencia, Spain Received 17 December 1999; accepted 4 May 2000
Abstract The effect of a postharvest hot-water dip treatment (HWT) at 53°C for 3 min and a 3-day heat-conditioning treatment at 37°C with air (HAT) at 90–95% RH on chilling tolerance and catalase (CAT) activity was compared in ‘Fortune’ mandarins. The HWT treatment increased CAT activity in the fruit, but after they were removed from high temperature to cold storage a rapid decline in CAT activity was associated with increased chilling injury. Greater chilling tolerance and CAT activity was induced when fruits were conditioned for 3 days at 37°C and 90 – 95% RH. The CAT activity in fruits exposed to HAT was higher than in the dipped and the non-heated fruits over the storage period at 2°C. An inhibitor of CAT activity, 3-amino-1,2,4-triazole (AT), caused peel damage in HAT ‘Fortune’ mandarins and in the chilling-tolerant ‘Clementine’ and ‘Clemenules’ cultivars stored at 2°C but not at 12°C (non-chilling temperature). CAT activity was reduced about two to three times by AT upon cold storage in the cultivars studied. Little difference was found in the activity of ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) between AT-treated and non-treated fruits. The data indicate that CAT may be a major antioxidant enzyme involved in the defence mechanism of mandarin fruits against chilling stress. Our results also suggest that the different effectiveness of the heat-conditioning treatments in increasing chilling tolerance of ‘Fortune’ mandarins may be related to induction of CAT activity during heating and on its persistence during cold storage. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Acclimation; Catalase; Citrus fruit; Cold-stress; High-temperature conditioning; Hot-water dips; Oxidative stress
1. Introduction Exposure to low temperature causes chilling injury (CI) expressed as rind staining and peel * Corresponding author. Tel.: +34-96-3900022; fax: + 3496-3636301. E-mail address: [email protected] (J.M. Sala).
pitting in ‘Fortune’ mandarin fruits, whereas the cultivars ‘Clementine’ and ‘Clemenules’, are resistant to CI. The CI-resistant cultivars can be stored at very low temperatures to extend the market period, withstand long-distant transport or undergo quarantine treatments to control Mediterranean fruit fly (Martı´nez-Ja´vega and Cuquerella, 1984; Sala, 1998). High-temperature
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conditioning has been shown to increase chilling tolerance in different crops (Lurie, 1998). Postharvest hot-water treatment (HWT) over 50°C for 1–3 min (Wild 1990; Rodov et al., 1995; Schirra and Mulas, 1995) or hot air treatment (HAT) at 35 – 37°C at high humidity for 1 – 3 days (Ben-Yehoshua et al., 1987; Lafuente et al., 1997) improve citrus fruit resistance to CI. Both heat pretreatments reduce CI in ‘Fortune’ mandarins (Mulas et al., 1995; Gonzalez-Aguilar et al., 1997; Lafuente et al., 1997). However, HWT treatments were not able to maintain the heat-induced resistance to cold stress after prolonged storage in ‘Fortune’ mandarins, whereas the HAT for 3 days at 37°C did so (Mulas et al., 1995). Chilling temperatures may induce oxidative stress in plant tissues (Purvis and Shewfelt, 1993), including fruits (Hariyadi and Parkin, 1991; Sala, 1998). Sala (1998) found that the main difference between chilling-sensitive and chilling-tolerant cultivars is in the higher ability of the chilling-tolerant to break down H2O2 by CAT activity and co-operation of APX and GR activities. Subsequently, it was reported that heating ‘Fortune’ mandarins at 37°C for 3 days induced 2.5-, 1.4-, and 1.2-fold increases in the activities of catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), respectively, and that the differences in the activities produced by the heat treatment were maintained during cold storage (Sala and Lafuente, 1999). In mustard seedlings, however, a heat acclimation treatment resulted in decreased CAT activity during the induced thermoprotection period (Dat et al., 1998). Aminotriazole (3amino-1,2,4-triazole) (AT) is an inhibitor of CAT activity in the presence of hydrogen peroxide, and has been used to investigate the role of CAT in animals and plants exposed to different stress conditions (Halliwell and Gutteridge, 1993; Prasad, 1997). We have tested the importance of CAT, APX, GR and SOD on the tolerance of mandarin fruits to low-temperature stress, and the effect of AT on response of chilling-tolerant ‘Clementine’ and ‘Clemenules’ mandarins and on the heat-induced tolerance to cold stress in ‘Fortune’ man-
darins. The effects of short HWT on CAT activity and protection of fruit against CI under prolonged cold storage in relation to the maintenance of the heat-induced CAT activity has also been investigated.
2. Material and methods
2.1. Plant material, storage and treatments Fruits of three mandarin cultivars were used. ‘Fortune’ (Citrus clementina Hort. ex Tanaka× Citrus reticulata Blanco) fruit were harvested at random from 20-year-old trees grafted onto ‘Satsuma’ mandarin (Citrus unshiu Marc.) and sour orange (Citrus aurantium L.) rootstock and grown at Sagunto, Valencia, Spain. ‘Clementine’ (Citrus reticulata Blanco, cv. ‘Fina’) and ‘Clemenules’ (Citrus reticulata Blanco, cv. ‘Nules’) fruits were obtained from a packing house in Almenara, Castello´n, Spain. ‘Fortune’ mandarin fruits were randomly divided into three lots containing three replicates of 20 fruits to estimate chilling damage, and of ten fruits per storage period to analyse CAT activity: (1) stored immediately for up to 28 days at 2°C and 80–85% RH; (2) subjected to a 3-day heat-conditioning treatment at 90–95% RH and 37°C (HAT) and then stored under the same conditions as the fruits of the first lot; (3) submerged for 3 min in a recirculating hot water bath at 539 0.3°C (HWT) consisting of a stainless steel chamber (volume 120 l) and a Honeywell water bath controller temperature unit (9 0.1°C; model Versapak 84, UK). After HWT all fruit were air-dried and stored as the nontreated fruit. The RH was measured using an electronic probe (Eliwell, EWS28). Two additional experiments were conducted using AT to inhibit CAT activity. In the first experiment, ‘Fortune’ mandarin fruits were selected and randomly divided into two lots. The first lot was treated by dipping the fruits twice in an aqueous solution of 150 mM AT (Sigma) for 15 s, conditioned at 37°C and 90–95% RH for 3 days and then subdivided into two groups, which were stored at 2 or 12°C at 80–85% RH for up
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to 8 weeks. The second lot (controls), not treated with AT, was heat-conditioned for 3 days at 37°C and subdivided into two groups, which were stored under the same conditions as the first lot. In the second experiment, ‘Clementine’ and ‘Clemenules’ fruits were divided into two lots, one was used as a control and the other was treated by dipping the fruits twice in a 30-mM AT aqueous solution for 15 s. AT-treated and nontreated fruits were stored at 2 or 12°C and 80 – 85% RH for up to 6 weeks. In each group, fruits of the three cultivars studied were randomly divided into three replicates of 27 fruits and peel damage was evaluated weekly. Three replicate samples of four fruits of each group stored at 2°C were sampled after 2, 4, 6 and 8 weeks storage in ‘Fortune’ mandarins and after 2 and 6 weeks in ‘Clementine’ and ‘Clemenules’ fruits for assessment in enzyme activities. The coloured outer layer of skin (flavedo tissue) was separated from the whole fruit, cut into small pieces, frozen in liquid N2 and stored at −70°C for enzyme assays.
2.2. CI index CI symptoms in ‘Fortune’ mandarin are small, brown, pit-like depressions on the peel. The severity of peel damage was evaluated by the method previously described by Lafuente et al. (1997). A rating scale based on surface necrosis and intensity of browning was used: 0 =no pitting; 1= slight; 2=medium; 3= severe pitting.
2.3. Enzyme assays CAT was extracted from 1 g fresh weight of frozen flavedo tissue ground in 10 ml of 100 mM potassium phosphate, pH 6.8 at 4°C and then centrifuged twice at 27 000× g for 15 min at 4°C. The supernatant was used to determine CAT activity by the method of Kar and Mishra (1976) in a final volume of 5 ml, which contained 1 ml of enzyme extract (400 – 800 mg protein). The unit of CAT activity was defined as the amount of enzyme, which decomposes 1 mmol H2O2 per minute at 25°C.
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APX was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ascorbic acid and 1% polyvinyl-polypyrrolidone (PVPP) at 4°C. The homogenate was centrifugated at 27 000 × g for 15 min at 4°C twice and the supernatant used to determine the APX activity by the method of Asada (1984) in a final volume of 3 ml, which contained 100–300 ml of enzyme extract (40–240 mg protein). The unit of APX was defined as the amount of enzyme that oxidised 1 mmol of ascorbate per minute at 25°C. GR was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 0.5 mM EDTA at 4°C. The homogenate was centrifuged twice at 27 000× g for 15 min at 4°C. The supernatant was used to determine GR activity by the method of Smith et al. (1988) in a final volume of 3 ml, which contained 100 ml of enzyme extract (40 – 80 mg protein). The unit of GR was defined as the amount of enzyme that catalysed the oxidation of 1 mmol of NADPH per minute. The activity of the GR solution used for the standard curve was determined by the method of Carlberg and Mannervik (1985). SOD was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 50 mM potassium phosphate buffer, pH 7.8, containing 1.33 mM diethylenetriamine pentaacetic acid at 4°C and then centrifuged twice at 27 000× g for 15 min at 4°C. The supernatant was used to determine SOD activity by the method of Oberley and Spitz (1986) in a final volume of 3 ml, which contained 60 – 70 ml of enzyme extract (24–56 mg protein). The unit of SOD was defined as the amount of enzyme which gave half-maximal inhibition. Activities of all enzymes were expressed as specific activities (units per mg protein fresh weight). Protein was determined by the method of Bradford (1976), using bovine serum albumin (BSA) as a standard.
2.4. Statistical design Experimental data are the mean9 S.E. of three replicates of the determinations for each sample.
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3. Results
3.1. Effect of heat-conditioning treatments on chilling injury and catalase acti6ity of ‘Fortune’ mandarins Chilling symptoms appeared by 2 weeks at 2°C storage in the non-conditioned ‘Fortune’ fruits (Fig. 1(A)). The CI index of these fruits increased continuously for up to 4 weeks. The susceptibility of the ‘Fortune’ mandarins decreased consider-
ably in HAT fruit. The CI index of fruits exposed to this treatment was about 0.3 after 4 weeks storage, whereas that of the non-conditioned fruit was 1.8. Dipping the fruits for 3 min at 53°C also increased the tolerance of ‘Fortune’ mandarins to chilling, although this treatment was less effective. The dip treatment delayed cold-induced peel damage but chilling symptoms were apparent by 3 weeks at 2°C. After 4 weeks storage, the CI index of dipped fruits was about 61% that of non-conditioned fruits. CAT activity in the flavedo of ‘Fortune’ mandarins quickly increased after the HWD treatment at 53°C but this increase was lower than that induced by heating the fruits for 3 days at 37°C (Fig. 1(B)). In general, the decline in CAT activity in heat-conditioned and in non-conditioned fruits after cold storage followed a different pattern. After 3 weeks, fruit dipped for 3 min at 53°C showed similar CAT activity to that of non-conditioned fruits. However, in the 3-day conditioned fruits the activity of the enzyme remained considerably higher than in non-conditioned fruits throughout the 4 weeks of exposure to low temperature.
3.2. Effect of AT on heat-induced tolerance to cold stress and on the acti6ity of the enzymes of the antioxidant system in ‘Fortune’ mandarins
Fig. 1. Effect of a hot-water dip treatment at 53°C for 3 min and a 3-day heat-conditioning treatment at 37°C and 90–95% RH on CI index (A) and CAT activity (B) of ‘Fortune’ mandarins stored for up to 4 weeks at 2°C. Each value is the mean of three replicate samples 9S.E.
The effectiveness of the HAT increasing chilling tolerance of ‘Fortune’ mandarins was reduced when CAT activity was inhibited by AT. After 1 week of storage at 2°C the CI index of these fruits was even higher than that of the non-treated fruits, which showed slight peel damage after 14 days at 2°C (Sala and Lafuente, 1999). Peel damage was, however, negligible in conditioned fruits treated with AT for up to 8 weeks storage at a non-chilling temperature (12°C; Fig. 2). AT treatment barely affected the activities of APX, GR and SOD in this citrus cultivar (Fig. 3B, C and D). However, the activity of the enzyme CAT was reduced to about 25–33% by AT over the storage period studied in the HAT fruit (Fig. 3A). The CAT activity in the HAT fruits treated with AT ranged from 8.1 to 6.5 units mg protein − 1 (Fig. 3A) after 2 weeks storage at 2°C;
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Fig. 2. CI index of ‘Fortune’ mandarin fruits treated with AT and conditioned for 3 days at 37°C and 90–95% RH and stored for up to 8 weeks at 2°C. Fruits stored at 2°C ( ) and 12°C ( ). Values are the mean of three replicate samples 9S.E.
whereas it ranged between 33.1 and 19.9 units mg protein − 1 in the non-AT treated fruits (Sala and Lafuente, 1999).
3.3. Effect of AT on peel damage and on the acti6ity of the enzymes of the antioxidant system in ‘Clementine’ and ‘Clemenules’ mandarins The fruits of ‘Clementine’ and ‘Clemenules’ cultivars were chilling-tolerant. These cultivars did not show peel damage for up to 8 weeks at 2°C but peel damage appeared after 2 weeks storage at 2°C when fruits were treated with AT (Fig. 4). No peel damage occurred during storage of the ATtreated and the non-treated fruits at 12°C (data not shown). The effect of AT treatment on the pattern of changes in the activities of the enzymes of the antioxidative system in non-conditioned ‘Clementine’ and ‘Clemenules’ fruits is shown in Fig. 5. In both cultivars, the activity of CAT was reduced to about the 40–50% and remained lower in ATtreated fruits during storage at low temperature (Fig. 5(A, B)). The activities of APX (Fig. 5(C,
Fig. 3. Effect of AT on CAT, APX, GR and SOD of ‘Fortune’ mandarin fruits conditioned for 3 days at 37°C and stored for up to 8 weeks at 2°C. Data are expressed as relative activity (histograms) calculated as the ratio of the activity of the enzymes in the flavedo of the AT-treated fruits with respect to the non-treated ones (%) and as absolute enzyme activity (closed circle) in fruits treated with AT. Each value is the mean of three replicate samples 9S.E.
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Fig. 4. Peel damage of ‘Clementine’ and ‘Clemenules’ mandarin fruits stored for up to 6 weeks at 2°C. The extent of injury was evaluated in fruits non-treated ( ) and treated with AT ( ). Values are the mean of three replicate samples 9 S.E.
D)), GR (Fig. 5(E, F)) and SOD (Fig. 5(G, H)) showed, in general, little change when the fruits were treated with AT.
4. Discussion Exposure to non-lethal high-temperature conditioning treatments protected ‘Fortune’ fruit against cold stress. Cold stress-induced injury in plants may be related to toxic oxygen forms
Fig. 5. Effect of AT on CAT, APX, GR and SOD activity of ‘Clementine’ (A, C, E and G) and ‘Clemenules’ (B, D, F and H) mandarin fruits non-treated ( ) and treated (b) with AT and stored for up to 6 weeks at 2°C. Each value is the mean of three replicate samples 9S.E.
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(Purvis and Shewfelt, 1993). It has been also reported that heat shock can result in an oxidative stress, which induces genes involved in the oxidative stress defence (Storozhenko et al., 1998). A decline in CAT activity during cold stress has been described in different plants (Omran, 1980; MacRae and Ferguson, 1985). Ferguson and Dunning (1986) found in cell cultures that even with substantial inhibition of CAT by AT, there was little effect on H2O2 levels in the cells probably because of the participation of other enzymes to keep the peroxide levels down. Conflicting results concerning the effect of temperature preconditioning on CAT activity in plants have been reported. A beneficial temperature pretreatment did not induce an increase in CAT activity in squash but reduced the decline after transfer of fruit to cold temperature (Wang, 1995). CAT activity decreased in mustard seedlings (Dat et al., 1998) and increased in maize seedlings (Prasad, 1997) during heat acclimation. In previous papers we have reported that the antioxidant enzyme system appears to be involved in the tolerance of mandarin fruits to chilling (Sala, 1998) and in the heat-induced chilling tolerance of ‘Fortune’ mandarin fruits (Sala and Lafuente, 1999). Our results indicate that the persistence of heatinduced CAT activity during cold storage affects the effectiveness of the heat conditioning treatment. The HWT increased chilling tolerance and CAT activity in the fruits, but after removing the fruits from high temperature to cold storage, CAT activity declined at the same time as resistance to chilling. However, fruits protected from developing chilling symptoms by HAT had higher CAT activity than the dipped and the non-conditioned fruits over the storage period (Fig. 1(B)). The difference in the effectiveness of the two high-temperature conditioning treatments may therefore be related to the different ability they confer on ‘Fortune’ fruits to maintain CAT activity and metabolise hydrogen peroxide. This oxygen form has no unpaired electron and can therefore pass through biological membranes. If hydrogen peroxide reaches the plant nucleus in sufficient concentration and reacts with intracellular metal ions, it will result in hydroxyl free radical activity and cell damage to the plant (Halliwell and Gutteridge, 1993).
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To further elucidate the importance of CAT, ‘Fortune’ fruits were treated with AT before heating them. AT inhibits CAT activity without interfering with CAT synthesis (Prasad, 1997) and allows plant tissues to accumulate hydrogen peroxide during exposure to cold temperatures. AT markedly inhibited CAT activity of HAT ‘Fortune’ mandarin fruits over the storage period (Fig. 3A), and reduced the effectiveness of the treatment. It is interesting to note that conditioned fruits treated with AT showed considerable CI after 7 days storage at 2°C whereas, as we have shown in the present and previous papers, CI occurs after 14 days in non-conditioned ‘Fortune’ fruits (Lafuente et al., 1997; Sala and Lafuente, 1999). Our results also demonstrate that the chilling-tolerant ‘Clementine’ and ‘Clemenules’ cultivars show peel damage after storage at 2°C but not at 12°C when were treated with AT, and that AT barely influences APX, GR and SOD activities in the mandarin cultivars used in this study, which further reinforces the idea that CAT plays a special role in acclimation to chilling in mandarin fruits. Our results in mandarins agree, therefore, with those found by Prasad (1997) in maize seedlings. The involvement of different biochemical and physiological mechanisms in high temperature-induced tolerance to chilling has been investigated (Lurie, 1998). Some of these heat-induced responses, such as the increase in polyamine levels, are not maintained after transferring citrus fruits to cold storage (Gonzalez-Aguilar et al., 2000), whereas avocado and tomato fruits tolerance to chilling temperatures correlated with the continued presence of HSPs at low temperature (Woolf et al., 1995; Sabehat et al., 1996). To our knowledge, the present study is the first suggesting that the maintenance of heat-induced CAT activity in flavedo cells during the chilling period affects the ability of heat shock to increase chilling tolerance. The maintenance of increased CAT activity by paclobutrazol has also been related to the resistance of wheat seedlings to heat and paraquat injury (Kraus and Fletcher, 1994). In conclusion: (1) the effectiveness of the HAT increasing chilling tolerance was considerably reduced when the induction of CAT activity in
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response to high-temperature conditioning was inhibited by AT; (2) peel damage occurred in chilling-tolerant citrus cultivars stored at 2°C but not at 12°C when fruit were treated with AT; and (3) the maintenance of the heat-induced CAT activity appear to be important to maintain the beneficial effect of heat conditioning. Our data suggest that CAT operates in the defence mechanism of mandarin fruit against low temperature stress and that the different effectiveness of the heat-conditioning treatments in increasing chilling tolerance of ‘Fortune’ mandarins may be related to induction of CAT activity during heating and on its persistence during cold storage.
Acknowledgements This work was supported by research grants ALI-96-0506-CO1 from the Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT), Spain, and FAIR-CT98-4096 from the European Union. The statistical advice of Mr Lo´pez-Santoven˜a, the technical assistance of M.J. Pascual and the provision of fruits by the growers, Messrs. Orts and Llusar, are also gratefully acknowledged.
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Catalase enzyme activity is related to tolerance of mandarin fruits to chilling Jose M. Sala *, Marı´a T. Lafuente Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA), Consejo Superior de In6estigaciones Cientı´ficas (CSIC), Apartado de Correos 73, Burjassot 46100, Valencia, Spain Received 17 December 1999; accepted 4 May 2000
Abstract The effect of a postharvest hot-water dip treatment (HWT) at 53°C for 3 min and a 3-day heat-conditioning treatment at 37°C with air (HAT) at 90–95% RH on chilling tolerance and catalase (CAT) activity was compared in ‘Fortune’ mandarins. The HWT treatment increased CAT activity in the fruit, but after they were removed from high temperature to cold storage a rapid decline in CAT activity was associated with increased chilling injury. Greater chilling tolerance and CAT activity was induced when fruits were conditioned for 3 days at 37°C and 90 – 95% RH. The CAT activity in fruits exposed to HAT was higher than in the dipped and the non-heated fruits over the storage period at 2°C. An inhibitor of CAT activity, 3-amino-1,2,4-triazole (AT), caused peel damage in HAT ‘Fortune’ mandarins and in the chilling-tolerant ‘Clementine’ and ‘Clemenules’ cultivars stored at 2°C but not at 12°C (non-chilling temperature). CAT activity was reduced about two to three times by AT upon cold storage in the cultivars studied. Little difference was found in the activity of ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) between AT-treated and non-treated fruits. The data indicate that CAT may be a major antioxidant enzyme involved in the defence mechanism of mandarin fruits against chilling stress. Our results also suggest that the different effectiveness of the heat-conditioning treatments in increasing chilling tolerance of ‘Fortune’ mandarins may be related to induction of CAT activity during heating and on its persistence during cold storage. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Acclimation; Catalase; Citrus fruit; Cold-stress; High-temperature conditioning; Hot-water dips; Oxidative stress
1. Introduction Exposure to low temperature causes chilling injury (CI) expressed as rind staining and peel * Corresponding author. Tel.: +34-96-3900022; fax: + 3496-3636301. E-mail address: [email protected] (J.M. Sala).
pitting in ‘Fortune’ mandarin fruits, whereas the cultivars ‘Clementine’ and ‘Clemenules’, are resistant to CI. The CI-resistant cultivars can be stored at very low temperatures to extend the market period, withstand long-distant transport or undergo quarantine treatments to control Mediterranean fruit fly (Martı´nez-Ja´vega and Cuquerella, 1984; Sala, 1998). High-temperature
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conditioning has been shown to increase chilling tolerance in different crops (Lurie, 1998). Postharvest hot-water treatment (HWT) over 50°C for 1–3 min (Wild 1990; Rodov et al., 1995; Schirra and Mulas, 1995) or hot air treatment (HAT) at 35 – 37°C at high humidity for 1 – 3 days (Ben-Yehoshua et al., 1987; Lafuente et al., 1997) improve citrus fruit resistance to CI. Both heat pretreatments reduce CI in ‘Fortune’ mandarins (Mulas et al., 1995; Gonzalez-Aguilar et al., 1997; Lafuente et al., 1997). However, HWT treatments were not able to maintain the heat-induced resistance to cold stress after prolonged storage in ‘Fortune’ mandarins, whereas the HAT for 3 days at 37°C did so (Mulas et al., 1995). Chilling temperatures may induce oxidative stress in plant tissues (Purvis and Shewfelt, 1993), including fruits (Hariyadi and Parkin, 1991; Sala, 1998). Sala (1998) found that the main difference between chilling-sensitive and chilling-tolerant cultivars is in the higher ability of the chilling-tolerant to break down H2O2 by CAT activity and co-operation of APX and GR activities. Subsequently, it was reported that heating ‘Fortune’ mandarins at 37°C for 3 days induced 2.5-, 1.4-, and 1.2-fold increases in the activities of catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), respectively, and that the differences in the activities produced by the heat treatment were maintained during cold storage (Sala and Lafuente, 1999). In mustard seedlings, however, a heat acclimation treatment resulted in decreased CAT activity during the induced thermoprotection period (Dat et al., 1998). Aminotriazole (3amino-1,2,4-triazole) (AT) is an inhibitor of CAT activity in the presence of hydrogen peroxide, and has been used to investigate the role of CAT in animals and plants exposed to different stress conditions (Halliwell and Gutteridge, 1993; Prasad, 1997). We have tested the importance of CAT, APX, GR and SOD on the tolerance of mandarin fruits to low-temperature stress, and the effect of AT on response of chilling-tolerant ‘Clementine’ and ‘Clemenules’ mandarins and on the heat-induced tolerance to cold stress in ‘Fortune’ man-
darins. The effects of short HWT on CAT activity and protection of fruit against CI under prolonged cold storage in relation to the maintenance of the heat-induced CAT activity has also been investigated.
2. Material and methods
2.1. Plant material, storage and treatments Fruits of three mandarin cultivars were used. ‘Fortune’ (Citrus clementina Hort. ex Tanaka× Citrus reticulata Blanco) fruit were harvested at random from 20-year-old trees grafted onto ‘Satsuma’ mandarin (Citrus unshiu Marc.) and sour orange (Citrus aurantium L.) rootstock and grown at Sagunto, Valencia, Spain. ‘Clementine’ (Citrus reticulata Blanco, cv. ‘Fina’) and ‘Clemenules’ (Citrus reticulata Blanco, cv. ‘Nules’) fruits were obtained from a packing house in Almenara, Castello´n, Spain. ‘Fortune’ mandarin fruits were randomly divided into three lots containing three replicates of 20 fruits to estimate chilling damage, and of ten fruits per storage period to analyse CAT activity: (1) stored immediately for up to 28 days at 2°C and 80–85% RH; (2) subjected to a 3-day heat-conditioning treatment at 90–95% RH and 37°C (HAT) and then stored under the same conditions as the fruits of the first lot; (3) submerged for 3 min in a recirculating hot water bath at 539 0.3°C (HWT) consisting of a stainless steel chamber (volume 120 l) and a Honeywell water bath controller temperature unit (9 0.1°C; model Versapak 84, UK). After HWT all fruit were air-dried and stored as the nontreated fruit. The RH was measured using an electronic probe (Eliwell, EWS28). Two additional experiments were conducted using AT to inhibit CAT activity. In the first experiment, ‘Fortune’ mandarin fruits were selected and randomly divided into two lots. The first lot was treated by dipping the fruits twice in an aqueous solution of 150 mM AT (Sigma) for 15 s, conditioned at 37°C and 90–95% RH for 3 days and then subdivided into two groups, which were stored at 2 or 12°C at 80–85% RH for up
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to 8 weeks. The second lot (controls), not treated with AT, was heat-conditioned for 3 days at 37°C and subdivided into two groups, which were stored under the same conditions as the first lot. In the second experiment, ‘Clementine’ and ‘Clemenules’ fruits were divided into two lots, one was used as a control and the other was treated by dipping the fruits twice in a 30-mM AT aqueous solution for 15 s. AT-treated and nontreated fruits were stored at 2 or 12°C and 80 – 85% RH for up to 6 weeks. In each group, fruits of the three cultivars studied were randomly divided into three replicates of 27 fruits and peel damage was evaluated weekly. Three replicate samples of four fruits of each group stored at 2°C were sampled after 2, 4, 6 and 8 weeks storage in ‘Fortune’ mandarins and after 2 and 6 weeks in ‘Clementine’ and ‘Clemenules’ fruits for assessment in enzyme activities. The coloured outer layer of skin (flavedo tissue) was separated from the whole fruit, cut into small pieces, frozen in liquid N2 and stored at −70°C for enzyme assays.
2.2. CI index CI symptoms in ‘Fortune’ mandarin are small, brown, pit-like depressions on the peel. The severity of peel damage was evaluated by the method previously described by Lafuente et al. (1997). A rating scale based on surface necrosis and intensity of browning was used: 0 =no pitting; 1= slight; 2=medium; 3= severe pitting.
2.3. Enzyme assays CAT was extracted from 1 g fresh weight of frozen flavedo tissue ground in 10 ml of 100 mM potassium phosphate, pH 6.8 at 4°C and then centrifuged twice at 27 000× g for 15 min at 4°C. The supernatant was used to determine CAT activity by the method of Kar and Mishra (1976) in a final volume of 5 ml, which contained 1 ml of enzyme extract (400 – 800 mg protein). The unit of CAT activity was defined as the amount of enzyme, which decomposes 1 mmol H2O2 per minute at 25°C.
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APX was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 50 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ascorbic acid and 1% polyvinyl-polypyrrolidone (PVPP) at 4°C. The homogenate was centrifugated at 27 000 × g for 15 min at 4°C twice and the supernatant used to determine the APX activity by the method of Asada (1984) in a final volume of 3 ml, which contained 100–300 ml of enzyme extract (40–240 mg protein). The unit of APX was defined as the amount of enzyme that oxidised 1 mmol of ascorbate per minute at 25°C. GR was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 0.5 mM EDTA at 4°C. The homogenate was centrifuged twice at 27 000× g for 15 min at 4°C. The supernatant was used to determine GR activity by the method of Smith et al. (1988) in a final volume of 3 ml, which contained 100 ml of enzyme extract (40 – 80 mg protein). The unit of GR was defined as the amount of enzyme that catalysed the oxidation of 1 mmol of NADPH per minute. The activity of the GR solution used for the standard curve was determined by the method of Carlberg and Mannervik (1985). SOD was extracted from 1 g of frozen flavedo tissue ground in 10 ml of 50 mM potassium phosphate buffer, pH 7.8, containing 1.33 mM diethylenetriamine pentaacetic acid at 4°C and then centrifuged twice at 27 000× g for 15 min at 4°C. The supernatant was used to determine SOD activity by the method of Oberley and Spitz (1986) in a final volume of 3 ml, which contained 60 – 70 ml of enzyme extract (24–56 mg protein). The unit of SOD was defined as the amount of enzyme which gave half-maximal inhibition. Activities of all enzymes were expressed as specific activities (units per mg protein fresh weight). Protein was determined by the method of Bradford (1976), using bovine serum albumin (BSA) as a standard.
2.4. Statistical design Experimental data are the mean9 S.E. of three replicates of the determinations for each sample.
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3. Results
3.1. Effect of heat-conditioning treatments on chilling injury and catalase acti6ity of ‘Fortune’ mandarins Chilling symptoms appeared by 2 weeks at 2°C storage in the non-conditioned ‘Fortune’ fruits (Fig. 1(A)). The CI index of these fruits increased continuously for up to 4 weeks. The susceptibility of the ‘Fortune’ mandarins decreased consider-
ably in HAT fruit. The CI index of fruits exposed to this treatment was about 0.3 after 4 weeks storage, whereas that of the non-conditioned fruit was 1.8. Dipping the fruits for 3 min at 53°C also increased the tolerance of ‘Fortune’ mandarins to chilling, although this treatment was less effective. The dip treatment delayed cold-induced peel damage but chilling symptoms were apparent by 3 weeks at 2°C. After 4 weeks storage, the CI index of dipped fruits was about 61% that of non-conditioned fruits. CAT activity in the flavedo of ‘Fortune’ mandarins quickly increased after the HWD treatment at 53°C but this increase was lower than that induced by heating the fruits for 3 days at 37°C (Fig. 1(B)). In general, the decline in CAT activity in heat-conditioned and in non-conditioned fruits after cold storage followed a different pattern. After 3 weeks, fruit dipped for 3 min at 53°C showed similar CAT activity to that of non-conditioned fruits. However, in the 3-day conditioned fruits the activity of the enzyme remained considerably higher than in non-conditioned fruits throughout the 4 weeks of exposure to low temperature.
3.2. Effect of AT on heat-induced tolerance to cold stress and on the acti6ity of the enzymes of the antioxidant system in ‘Fortune’ mandarins
Fig. 1. Effect of a hot-water dip treatment at 53°C for 3 min and a 3-day heat-conditioning treatment at 37°C and 90–95% RH on CI index (A) and CAT activity (B) of ‘Fortune’ mandarins stored for up to 4 weeks at 2°C. Each value is the mean of three replicate samples 9S.E.
The effectiveness of the HAT increasing chilling tolerance of ‘Fortune’ mandarins was reduced when CAT activity was inhibited by AT. After 1 week of storage at 2°C the CI index of these fruits was even higher than that of the non-treated fruits, which showed slight peel damage after 14 days at 2°C (Sala and Lafuente, 1999). Peel damage was, however, negligible in conditioned fruits treated with AT for up to 8 weeks storage at a non-chilling temperature (12°C; Fig. 2). AT treatment barely affected the activities of APX, GR and SOD in this citrus cultivar (Fig. 3B, C and D). However, the activity of the enzyme CAT was reduced to about 25–33% by AT over the storage period studied in the HAT fruit (Fig. 3A). The CAT activity in the HAT fruits treated with AT ranged from 8.1 to 6.5 units mg protein − 1 (Fig. 3A) after 2 weeks storage at 2°C;
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Fig. 2. CI index of ‘Fortune’ mandarin fruits treated with AT and conditioned for 3 days at 37°C and 90–95% RH and stored for up to 8 weeks at 2°C. Fruits stored at 2°C ( ) and 12°C ( ). Values are the mean of three replicate samples 9S.E.
whereas it ranged between 33.1 and 19.9 units mg protein − 1 in the non-AT treated fruits (Sala and Lafuente, 1999).
3.3. Effect of AT on peel damage and on the acti6ity of the enzymes of the antioxidant system in ‘Clementine’ and ‘Clemenules’ mandarins The fruits of ‘Clementine’ and ‘Clemenules’ cultivars were chilling-tolerant. These cultivars did not show peel damage for up to 8 weeks at 2°C but peel damage appeared after 2 weeks storage at 2°C when fruits were treated with AT (Fig. 4). No peel damage occurred during storage of the ATtreated and the non-treated fruits at 12°C (data not shown). The effect of AT treatment on the pattern of changes in the activities of the enzymes of the antioxidative system in non-conditioned ‘Clementine’ and ‘Clemenules’ fruits is shown in Fig. 5. In both cultivars, the activity of CAT was reduced to about the 40–50% and remained lower in ATtreated fruits during storage at low temperature (Fig. 5(A, B)). The activities of APX (Fig. 5(C,
Fig. 3. Effect of AT on CAT, APX, GR and SOD of ‘Fortune’ mandarin fruits conditioned for 3 days at 37°C and stored for up to 8 weeks at 2°C. Data are expressed as relative activity (histograms) calculated as the ratio of the activity of the enzymes in the flavedo of the AT-treated fruits with respect to the non-treated ones (%) and as absolute enzyme activity (closed circle) in fruits treated with AT. Each value is the mean of three replicate samples 9S.E.
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Fig. 4. Peel damage of ‘Clementine’ and ‘Clemenules’ mandarin fruits stored for up to 6 weeks at 2°C. The extent of injury was evaluated in fruits non-treated ( ) and treated with AT ( ). Values are the mean of three replicate samples 9 S.E.
D)), GR (Fig. 5(E, F)) and SOD (Fig. 5(G, H)) showed, in general, little change when the fruits were treated with AT.
4. Discussion Exposure to non-lethal high-temperature conditioning treatments protected ‘Fortune’ fruit against cold stress. Cold stress-induced injury in plants may be related to toxic oxygen forms
Fig. 5. Effect of AT on CAT, APX, GR and SOD activity of ‘Clementine’ (A, C, E and G) and ‘Clemenules’ (B, D, F and H) mandarin fruits non-treated ( ) and treated (b) with AT and stored for up to 6 weeks at 2°C. Each value is the mean of three replicate samples 9S.E.
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(Purvis and Shewfelt, 1993). It has been also reported that heat shock can result in an oxidative stress, which induces genes involved in the oxidative stress defence (Storozhenko et al., 1998). A decline in CAT activity during cold stress has been described in different plants (Omran, 1980; MacRae and Ferguson, 1985). Ferguson and Dunning (1986) found in cell cultures that even with substantial inhibition of CAT by AT, there was little effect on H2O2 levels in the cells probably because of the participation of other enzymes to keep the peroxide levels down. Conflicting results concerning the effect of temperature preconditioning on CAT activity in plants have been reported. A beneficial temperature pretreatment did not induce an increase in CAT activity in squash but reduced the decline after transfer of fruit to cold temperature (Wang, 1995). CAT activity decreased in mustard seedlings (Dat et al., 1998) and increased in maize seedlings (Prasad, 1997) during heat acclimation. In previous papers we have reported that the antioxidant enzyme system appears to be involved in the tolerance of mandarin fruits to chilling (Sala, 1998) and in the heat-induced chilling tolerance of ‘Fortune’ mandarin fruits (Sala and Lafuente, 1999). Our results indicate that the persistence of heatinduced CAT activity during cold storage affects the effectiveness of the heat conditioning treatment. The HWT increased chilling tolerance and CAT activity in the fruits, but after removing the fruits from high temperature to cold storage, CAT activity declined at the same time as resistance to chilling. However, fruits protected from developing chilling symptoms by HAT had higher CAT activity than the dipped and the non-conditioned fruits over the storage period (Fig. 1(B)). The difference in the effectiveness of the two high-temperature conditioning treatments may therefore be related to the different ability they confer on ‘Fortune’ fruits to maintain CAT activity and metabolise hydrogen peroxide. This oxygen form has no unpaired electron and can therefore pass through biological membranes. If hydrogen peroxide reaches the plant nucleus in sufficient concentration and reacts with intracellular metal ions, it will result in hydroxyl free radical activity and cell damage to the plant (Halliwell and Gutteridge, 1993).
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To further elucidate the importance of CAT, ‘Fortune’ fruits were treated with AT before heating them. AT inhibits CAT activity without interfering with CAT synthesis (Prasad, 1997) and allows plant tissues to accumulate hydrogen peroxide during exposure to cold temperatures. AT markedly inhibited CAT activity of HAT ‘Fortune’ mandarin fruits over the storage period (Fig. 3A), and reduced the effectiveness of the treatment. It is interesting to note that conditioned fruits treated with AT showed considerable CI after 7 days storage at 2°C whereas, as we have shown in the present and previous papers, CI occurs after 14 days in non-conditioned ‘Fortune’ fruits (Lafuente et al., 1997; Sala and Lafuente, 1999). Our results also demonstrate that the chilling-tolerant ‘Clementine’ and ‘Clemenules’ cultivars show peel damage after storage at 2°C but not at 12°C when were treated with AT, and that AT barely influences APX, GR and SOD activities in the mandarin cultivars used in this study, which further reinforces the idea that CAT plays a special role in acclimation to chilling in mandarin fruits. Our results in mandarins agree, therefore, with those found by Prasad (1997) in maize seedlings. The involvement of different biochemical and physiological mechanisms in high temperature-induced tolerance to chilling has been investigated (Lurie, 1998). Some of these heat-induced responses, such as the increase in polyamine levels, are not maintained after transferring citrus fruits to cold storage (Gonzalez-Aguilar et al., 2000), whereas avocado and tomato fruits tolerance to chilling temperatures correlated with the continued presence of HSPs at low temperature (Woolf et al., 1995; Sabehat et al., 1996). To our knowledge, the present study is the first suggesting that the maintenance of heat-induced CAT activity in flavedo cells during the chilling period affects the ability of heat shock to increase chilling tolerance. The maintenance of increased CAT activity by paclobutrazol has also been related to the resistance of wheat seedlings to heat and paraquat injury (Kraus and Fletcher, 1994). In conclusion: (1) the effectiveness of the HAT increasing chilling tolerance was considerably reduced when the induction of CAT activity in
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response to high-temperature conditioning was inhibited by AT; (2) peel damage occurred in chilling-tolerant citrus cultivars stored at 2°C but not at 12°C when fruit were treated with AT; and (3) the maintenance of the heat-induced CAT activity appear to be important to maintain the beneficial effect of heat conditioning. Our data suggest that CAT operates in the defence mechanism of mandarin fruit against low temperature stress and that the different effectiveness of the heat-conditioning treatments in increasing chilling tolerance of ‘Fortune’ mandarins may be related to induction of CAT activity during heating and on its persistence during cold storage.
Acknowledgements This work was supported by research grants ALI-96-0506-CO1 from the Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT), Spain, and FAIR-CT98-4096 from the European Union. The statistical advice of Mr Lo´pez-Santoven˜a, the technical assistance of M.J. Pascual and the provision of fruits by the growers, Messrs. Orts and Llusar, are also gratefully acknowledged.
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