1706 enzima: catalasa

Postharvest Biology and Technology 20 (2000) 81–89

Catalase enzyme activity is related to tolerance of mandarinfruits to chilling

Jose M. Sala *, Marıa T. LafuenteInstituto de Agroquımica y Tecnologıa de Alimentos (IATA), Consejo Superior de In6estigaciones Cientıficas (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-conditioningtreatment 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 hightemperature to cold storage a rapid decline in CAT activity was associated with increased chilling injury. Greaterchilling 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 storageperiod 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 thecultivars 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 maybe a major antioxidant enzyme involved in the defence mechanism of mandarin fruits against chilling stress. Ourresults also suggest that the different effectiveness of the heat-conditioning treatments in increasing chilling toleranceof ‘Fortune’ mandarins may be related to induction of CAT activity during heating and on its persistence during coldstorage. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Acclimation; Catalase; Citrus fruit; Cold-stress; High-temperature conditioning; Hot-water dips; Oxidative stress

www.elsevier.com/locate/postharvbio

1. Introduction

Exposure to low temperature causes chillinginjury (CI) expressed as rind staining and peel

pitting in ‘Fortune’ mandarin fruits, whereas thecultivars ‘Clementine’ and ‘Clemenules’, are resis-tant to CI. The CI-resistant cultivars can bestored at very low temperatures to extend themarket period, withstand long-distant transportor undergo quarantine treatments to control Med-iterranean fruit fly (Martınez-Javega and Cu-querella, 1984; Sala, 1998). High-temperature

* Corresponding author. Tel.: +34-96-3900022; fax: +34-96-3636301.

E-mail address: [email protected] (J.M. Sala).

0925-5214/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

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J.M. Sala, M.T. Lafuente / Posthar6est Biology and Technology 20 (2000) 81–8982

conditioning has been shown to increase chillingtolerance in different crops (Lurie, 1998).Postharvest hot-water treatment (HWT) over50°C for 1–3 min (Wild 1990; Rodov et al.,1995; Schirra and Mulas, 1995) or hot air treat-ment (HAT) at 35–37°C at high humidity for1–3 days (Ben-Yehoshua et al., 1987; Lafuenteet al., 1997) improve citrus fruit resistance to CI.Both heat pretreatments reduce CI in ‘Fortune’mandarins (Mulas et al., 1995; Gonzalez-Aguilaret al., 1997; Lafuente et al., 1997). However,HWT treatments were not able to maintain theheat-induced resistance to cold stress after pro-longed storage in ‘Fortune’ mandarins, whereasthe HAT for 3 days at 37°C did so (Mulas et al.,1995).

Chilling temperatures may induce oxidativestress in plant tissues (Purvis and Shewfelt,1993), including fruits (Hariyadi and Parkin,1991; Sala, 1998). Sala (1998) found that themain difference between chilling-sensitive andchilling-tolerant cultivars is in the higher abilityof the chilling-tolerant to break down H2O2 byCAT activity and co-operation of APX and GRactivities. Subsequently, it was reported thatheating ‘Fortune’ mandarins at 37°C for 3 daysinduced 2.5-, 1.4-, and 1.2-fold increases in theactivities of catalase (CAT), superoxide dismu-tase (SOD) and ascorbate peroxidase (APX), re-spectively, and that the differences in theactivities produced by the heat treatment weremaintained during cold storage (Sala and La-fuente, 1999). In mustard seedlings, however, aheat acclimation treatment resulted in decreasedCAT activity during the induced thermoprotec-tion period (Dat et al., 1998). Aminotriazole (3-amino-1,2,4-triazole) (AT) is an inhibitor of CATactivity in the presence of hydrogen peroxide,and has been used to investigate the role of CATin animals and plants exposed to different stressconditions (Halliwell and Gutteridge, 1993;Prasad, 1997).

We have tested the importance of CAT, APX,GR and SOD on the tolerance of mandarinfruits to low-temperature stress, and the effect ofAT on response of chilling-tolerant ‘Clementine’and ‘Clemenules’ mandarins and on the heat-in-duced tolerance to cold stress in ‘Fortune’ man-

darins. The effects of short HWT on CATactivity and protection of fruit against CI underprolonged cold storage in relation to the mainte-nance of the heat-induced CAT activity has alsobeen 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 atrandom from 20-year-old trees grafted onto ‘Sat-suma’ mandarin (Citrus unshiu Marc.) and sourorange (Citrus aurantium L.) rootstock andgrown at Sagunto, Valencia, Spain. ‘Clementine’(Citrus reticulata Blanco, cv. ‘Fina’) and‘Clemenules’ (Citrus reticulata Blanco, cv.‘Nules’) fruits were obtained from a packinghouse in Almenara, Castellon, Spain.

‘Fortune’ mandarin fruits were randomly di-vided into three lots containing three replicatesof 20 fruits to estimate chilling damage, and often fruits per storage period to analyse CATactivity: (1) stored immediately for up to 28 daysat 2°C and 80–85% RH; (2) subjected to a 3-dayheat-conditioning treatment at 90–95% RH and37°C (HAT) and then stored under the sameconditions as the fruits of the first lot; (3) sub-merged for 3 min in a recirculating hot waterbath at 5390.3°C (HWT) consisting of a stain-less steel chamber (volume 120 l) and a Honey-well water bath controller temperature unit(90.1°C; model Versapak 84, UK). After HWTall fruit were air-dried and stored as the non-treated fruit. The RH was measured using anelectronic probe (Eliwell, EWS28).

Two additional experiments were conductedusing AT to inhibit CAT activity. In the firstexperiment, ‘Fortune’ mandarin fruits were se-lected and randomly divided into two lots. Thefirst lot was treated by dipping the fruits twice inan aqueous solution of 150 mM AT (Sigma) for15 s, conditioned at 37°C and 90–95% RH for 3days and then subdivided into two groups, whichwere stored at 2 or 12°C at 80–85% RH for up

J.M. Sala, M.T. Lafuente / Posthar6est Biology and Technology 20 (2000) 81–89 83

to 8 weeks. The second lot (controls), not treatedwith AT, was heat-conditioned for 3 days at 37°Cand subdivided into two groups, which werestored under the same conditions as the first lot.In the second experiment, ‘Clementine’ and‘Clemenules’ fruits were divided into two lots, onewas used as a control and the other was treatedby dipping the fruits twice in a 30-mM ATaqueous solution for 15 s. AT-treated and non-treated fruits were stored at 2 or 12°C and 80–85% RH for up to 6 weeks.

In each group, fruits of the three cultivarsstudied were randomly divided into three repli-cates of 27 fruits and peel damage was evaluatedweekly. Three replicate samples of four fruits ofeach group stored at 2°C were sampled after 2, 4,6 and 8 weeks storage in ‘Fortune’ mandarins andafter 2 and 6 weeks in ‘Clementine’ and‘Clemenules’ fruits for assessment in enzyme ac-tivities. The coloured outer layer of skin (flavedotissue) was separated from the whole fruit, cutinto 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 sever-ity of peel damage was evaluated by the methodpreviously described by Lafuente et al. (1997). Arating scale based on surface necrosis and inten-sity 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 offrozen flavedo tissue ground in 10 ml of 100 mMpotassium phosphate, pH 6.8 at 4°C and thencentrifuged twice at 27 000×g for 15 min at 4°C.The supernatant was used to determine CAT ac-tivity by the method of Kar and Mishra (1976) ina final volume of 5 ml, which contained 1 ml ofenzyme extract (400–800 mg protein). The unit ofCAT activity was defined as the amount of en-zyme, which decomposes 1 mmol H2O2 per minuteat 25°C.

APX was extracted from 1 g of frozen flavedotissue ground in 10 ml of 50 mM potassiumphosphate buffer, pH 7.0, containing 0.1 mMethylenediamine tetraacetic acid (EDTA), 1 mMascorbic acid and 1% polyvinyl-polypyrrolidone(PVPP) at 4°C. The homogenate was centrifu-gated at 27 000×g for 15 min at 4°C twice andthe supernatant used to determine the APX activ-ity by the method of Asada (1984) in a finalvolume of 3 ml, which contained 100–300 ml ofenzyme extract (40–240 mg protein). The unit ofAPX was defined as the amount of enzyme thatoxidised 1 mmol of ascorbate per minute at 25°C.

GR was extracted from 1 g of frozen flavedotissue ground in 10 ml of 100 mM potassiumphosphate buffer, pH 7.5, containing 0.5 mMEDTA at 4°C. The homogenate was centrifugedtwice at 27 000×g for 15 min at 4°C. The super-natant was used to determine GR activity by themethod of Smith et al. (1988) in a final volume of3 ml, which contained 100 ml of enzyme extract(40–80 mg protein). The unit of GR was definedas the amount of enzyme that catalysed the oxida-tion of 1 mmol of NADPH per minute. Theactivity of the GR solution used for the standardcurve was determined by the method of Carlbergand Mannervik (1985).

SOD was extracted from 1 g of frozen flavedotissue ground in 10 ml of 50 mM potassiumphosphate buffer, pH 7.8, containing 1.33 mMdiethylenetriamine pentaacetic acid at 4°C andthen centrifuged twice at 27 000×g for 15 min at4°C. The supernatant was used to determine SODactivity by the method of Oberley and Spitz(1986) in a final volume of 3 ml, which contained60–70 ml of enzyme extract (24–56 mg protein).The unit of SOD was defined as the amount ofenzyme which gave half-maximal inhibition.

Activities of all enzymes were expressed as spe-cific activities (units per mg protein fresh weight).Protein was determined by the method of Brad-ford (1976), using bovine serum albumin (BSA) asa standard.

2.4. Statistical design

Experimental data are the mean9S.E. of threereplicates of the determinations for each sample.


3. Results

3.1. Effect of heat-conditioning treatments onchilling injury and catalase acti6ity of ‘Fortune’mandarins

Chilling symptoms appeared by 2 weeks at 2°Cstorage in the non-conditioned ‘Fortune’ fruits(Fig. 1(A)). The CI index of these fruits increasedcontinuously for up to 4 weeks. The susceptibilityof the ‘Fortune’ mandarins decreased consider-

ably in HAT fruit. The CI index of fruits exposedto this treatment was about 0.3 after 4 weeksstorage, whereas that of the non-conditioned fruitwas 1.8. Dipping the fruits for 3 min at 53°C alsoincreased the tolerance of ‘Fortune’ mandarins tochilling, although this treatment was less effective.The dip treatment delayed cold-induced peel dam-age but chilling symptoms were apparent by 3weeks at 2°C. After 4 weeks storage, the CI indexof dipped fruits was about 61% that of non-condi-tioned fruits.

CAT activity in the flavedo of ‘Fortune’ man-darins quickly increased after the HWD treatmentat 53°C but this increase was lower than thatinduced by heating the fruits for 3 days at 37°C(Fig. 1(B)). In general, the decline in CAT activityin heat-conditioned and in non-conditioned fruitsafter cold storage followed a different pattern.After 3 weeks, fruit dipped for 3 min at 53°Cshowed similar CAT activity to that of non-condi-tioned fruits. However, in the 3-day conditionedfruits the activity of the enzyme remained consid-erably higher than in non-conditioned fruitsthroughout the 4 weeks of exposure to lowtemperature.

3.2. Effect of AT on heat-induced tolerance tocold stress and on the acti6ity of the enzymes ofthe antioxidant system in ‘Fortune’ mandarins

The effectiveness of the HAT increasing chillingtolerance of ‘Fortune’ mandarins was reducedwhen CAT activity was inhibited by AT. After 1week of storage at 2°C the CI index of these fruitswas even higher than that of the non-treatedfruits, which showed slight peel damage after 14days at 2°C (Sala and Lafuente, 1999). Peel dam-age was, however, negligible in conditioned fruitstreated with AT for up to 8 weeks storage at anon-chilling temperature (12°C; Fig. 2).

AT treatment barely affected the activities ofAPX, GR and SOD in this citrus cultivar (Fig.3B, C and D). However, the activity of the en-zyme CAT was reduced to about 25–33% by ATover the storage period studied in the HAT fruit(Fig. 3A). The CAT activity in the HAT fruitstreated with AT ranged from 8.1 to 6.5 units mgprotein−1 (Fig. 3A) after 2 weeks storage at 2°C;

Fig. 1. Effect of a hot-water dip treatment at 53°C for 3 minand 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 themean of three replicate samples9S.E.


Fig. 2. CI index of ‘Fortune’ mandarin fruits treated with ATand conditioned for 3 days at 37°C and 90–95% RH and storedfor up to 8 weeks at 2°C. Fruits stored at 2°C () and 12°C(). Values are the mean of three replicate samples9S.E.

Fig. 3. Effect of AT on CAT, APX, GR and SOD of ‘Fortune’mandarin fruits conditioned for 3 days at 37°C and stored forup to 8 weeks at 2°C. Data are expressed as relative activity(histograms) calculated as the ratio of the activity of the enzymesin the flavedo of the AT-treated fruits with respect to thenon-treated ones (%) and as absolute enzyme activity (closedcircle) in fruits treated with AT. Each value is the mean of threereplicate samples9S.E.

whereas it ranged between 33.1 and 19.9 units mgprotein−1 in the non-AT treated fruits (Sala andLafuente, 1999).

3.3. Effect of AT on peel damage and on theacti6ity of the enzymes of the antioxidant systemin ‘Clementine’ and ‘Clemenules’ mandarins

The fruits of ‘Clementine’ and ‘Clemenules’ cul-tivars were chilling-tolerant. These cultivars didnot show peel damage for up to 8 weeks at 2°Cbut peel damage appeared after 2 weeks storage at2°C when fruits were treated with AT (Fig. 4). Nopeel damage occurred during storage of the AT-treated and the non-treated fruits at 12°C (datanot shown).

The effect of AT treatment on the pattern ofchanges in the activities of the enzymes of theantioxidative system in non-conditioned ‘Clemen-tine’ and ‘Clemenules’ fruits is shown in Fig. 5. Inboth cultivars, the activity of CAT was reduced toabout the 40–50% and remained lower in AT-treated fruits during storage at low temperature(Fig. 5(A, B)). The activities of APX (Fig. 5(C,


Fig. 4. Peel damage of ‘Clementine’ and ‘Clemenules’ man-darin fruits stored for up to 6 weeks at 2°C. The extent ofinjury was evaluated in fruits non-treated ( ) and treated withAT (). Values are the mean of three replicate samples9S.E.

Fig. 5. Effect of AT on CAT, APX, GR and SOD activity of‘Clementine’ (A, C, E and G) and ‘Clemenules’ (B, D, F andH) mandarin fruits non-treated ( ) and treated (b) with ATand stored for up to 6 weeks at 2°C. Each value is the meanof three replicate samples9S.E.

D)), GR (Fig. 5(E, F)) and SOD (Fig. 5(G, H))showed, in general, little change when the fruitswere treated with AT.

4. Discussion

Exposure to non-lethal high-temperature condi-tioning treatments protected ‘Fortune’ fruitagainst cold stress. Cold stress-induced injury inplants may be related to toxic oxygen forms


(Purvis and Shewfelt, 1993). It has been alsoreported that heat shock can result in an oxidativestress, which induces genes involved in the oxida-tive stress defence (Storozhenko et al., 1998). Adecline in CAT activity during cold stress hasbeen described in different plants (Omran, 1980;MacRae and Ferguson, 1985). Ferguson andDunning (1986) found in cell cultures that evenwith substantial inhibition of CAT by AT, therewas little effect on H2O2 levels in the cells proba-bly because of the participation of other enzymesto keep the peroxide levels down. Conflicting re-sults concerning the effect of temperature precon-ditioning on CAT activity in plants have beenreported. A beneficial temperature pretreatmentdid not induce an increase in CAT activity insquash but reduced the decline after transfer offruit to cold temperature (Wang, 1995). CATactivity decreased in mustard seedlings (Dat et al.,1998) and increased in maize seedlings (Prasad,1997) during heat acclimation. In previous paperswe have reported that the antioxidant enzymesystem appears to be involved in the tolerance ofmandarin fruits to chilling (Sala, 1998) and in theheat-induced chilling tolerance of ‘Fortune’ man-darin fruits (Sala and Lafuente, 1999).

Our results indicate that the persistence of heat-induced CAT activity during cold storage affectsthe effectiveness of the heat conditioning treat-ment. The HWT increased chilling tolerance andCAT activity in the fruits, but after removing thefruits from high temperature to cold storage, CATactivity declined at the same time as resistance tochilling. However, fruits protected from develop-ing chilling symptoms by HAT had higher CATactivity than the dipped and the non-conditionedfruits over the storage period (Fig. 1(B)). Thedifference in the effectiveness of the two high-tem-perature conditioning treatments may therefore berelated to the different ability they confer on‘Fortune’ fruits to maintain CAT activity andmetabolise hydrogen peroxide. This oxygen formhas no unpaired electron and can therefore passthrough biological membranes. If hydrogen per-oxide reaches the plant nucleus in sufficient con-centration and reacts with intracellular metal ions,it will result in hydroxyl free radical activity andcell damage to the plant (Halliwell and Gutte-ridge, 1993).

To further elucidate the importance of CAT,‘Fortune’ fruits were treated with AT before heat-ing them. AT inhibits CAT activity without inter-fering with CAT synthesis (Prasad, 1997) andallows plant tissues to accumulate hydrogen per-oxide during exposure to cold temperatures. ATmarkedly inhibited CAT activity of HAT ‘For-tune’ mandarin fruits over the storage period (Fig.3A), and reduced the effectiveness of the treat-ment. It is interesting to note that conditionedfruits treated with AT showed considerable CIafter 7 days storage at 2°C whereas, as we haveshown in the present and previous papers, CIoccurs after 14 days in non-conditioned ‘Fortune’fruits (Lafuente et al., 1997; Sala and Lafuente,1999). Our results also demonstrate that the chill-ing-tolerant ‘Clementine’ and ‘Clemenules’ culti-vars show peel damage after storage at 2°C butnot at 12°C when were treated with AT, and thatAT barely influences APX, GR and SOD activi-ties in the mandarin cultivars used in this study,which further reinforces the idea that CAT playsa special role in acclimation to chilling in man-darin fruits. Our results in mandarins agree, there-fore, with those found by Prasad (1997) in maizeseedlings.

The involvement of different biochemical andphysiological mechanisms in high temperature-in-duced tolerance to chilling has been investigated(Lurie, 1998). Some of these heat-induced re-sponses, such as the increase in polyamine levels,are not maintained after transferring citrus fruitsto cold storage (Gonzalez-Aguilar et al., 2000),whereas avocado and tomato fruits tolerance tochilling temperatures correlated with the contin-ued presence of HSPs at low temperature (Woolfet al., 1995; Sabehat et al., 1996). To our knowl-edge, the present study is the first suggesting thatthe maintenance of heat-induced CAT activity inflavedo cells during the chilling period affects theability of heat shock to increase chilling tolerance.The maintenance of increased CAT activity bypaclobutrazol has also been related to the resis-tance of wheat seedlings to heat and paraquatinjury (Kraus and Fletcher, 1994).

In conclusion: (1) the effectiveness of the HATincreasing chilling tolerance was considerably re-duced when the induction of CAT activity in


response to high-temperature conditioning wasinhibited by AT; (2) peel damage occurred inchilling-tolerant citrus cultivars stored at 2°C butnot at 12°C when fruit were treated with AT; and(3) the maintenance of the heat-induced CATactivity appear to be important to maintain thebeneficial effect of heat conditioning. Our datasuggest that CAT operates in the defence mecha-nism of mandarin fruit against low temperaturestress and that the different effectiveness of theheat-conditioning treatments in increasing chillingtolerance of ‘Fortune’ mandarins may be relatedto induction of CAT activity during heating andon its persistence during cold storage.

Acknowledgements

This work was supported by research grantsALI-96-0506-CO1 from the Comision Interminis-terial de Ciencia y Tecnologıa (CICYT), Spain,and FAIR-CT98-4096 from the European Union.The statistical advice of Mr Lopez-Santovena, thetechnical assistance of M.J. Pascual and the provi-sion of fruits by the growers, Messrs. Orts andLlusar, are also gratefully acknowledged.

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1706 enzima: catalasa