A comparative study of the postharvest performance of an ABA-deficient mutant of oranges: II. Antioxidant enzymatic system and phenylalanine ammonia-lyase in non-chilling and chilling peel disorders of citrus fruit

A comparative study of the postharvest performance of an ABA-deficient mutant of oranges: II. Antioxidant enzymatic system and phenylalanine ammonia-lyase in non-chilling and chilling peel disorders of citrus fruit
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  Postharvest Biology and Technology 37 (2005) 232–240 A comparative study of the postharvest performance of anABA-deficient mutant of orangesII. Antioxidant enzymatic system and phenylalanineammonia-lyase in non-chilling and chilling peeldisorders of citrus fruit Jos´e M. Sala a , Mar´ıa T. Sanchez-Ballesta a , Fernando Alf ´erez a , Maurizio Mulas b ,Lorenzo Zacarias a , Mar´ıa T. Lafuente a , ∗ a  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 b  Dipartimento di Economia e Sistemi Arborei dell’Universit`a di Sassari, Via E. Nicola 1, 07100 Sassari, Italy Received 22 January 2005; accepted 16 May 2005 Abstract Postharvest rind disorders of citrus fruit may be caused by chilling but also by other stress conditions at non-chilling temper-atures. The mechanisms involved in the tolerance of citrus fruit to these postharvest physiological disorders and how they arerelated to each other are not well understood. In the present work, we have examined changes in the activities of the antioxidantenzymes, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), and of phenylalanine ammonia-lyase (PAL), the initial rate-controlling enzyme in phenolic synthesis, in ‘Navelate’ oranges and inits yellow abscisic acid-deficient mutant ‘Pinalate’ in relation to their susceptibility to chilling injury (CI) and to non-chillingpeel pitting. ‘Navelate’ oranges developed CI during storage at 2 ◦ C and very slight postharvest non-chilling peel pitting at12 ◦ C. By contrast, ‘Pinalate’ fruit did not show CI and were more susceptible to non-chilling peel pitting than ‘Navelate’ fruit.No important differences in the activities of the enzymes GR and APX were found in fruit of both phenotypes when storedat 2 ◦ C or 12 ◦ C. By contrast, ‘Pinalate’ fruit, the chilling-tolerant cultivar, had higher CAT activity than ‘Navelate’ orangesduring storage at 2 ◦ C, while it was lower at the temperature causing non-chilling peel pitting. PAL activity barely changed inresponse to cold stress in the mutant but increased in the chilling-susceptible ‘Navelate’ fruit. On the other hand, the increase inPAL occurring in ‘Pinalate’ fruit at 12 ◦ C might be associated with their greater development of non-chilling peel pitting. Theoverall results indicate that the enzyme PAL might be a good biochemical marker for both chilling-induced superficial scald and ∗ Corresponding author. Tel.: +34 963900022; fax: +34 963636301.  E-mail address: (M.T. Lafuente).0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.postharvbio.2005.05.006   J.M. Sala et al. / Postharvest Biology and Technology 37 (2005) 232–240  233 non-chilling peel pitting and that CAT may play a role protecting ‘Navelate’ and ‘Pinalate’ fruit from conditions favoring bothphysiologicaldisorders.Inaddition,SODcouldbeinvolvedinthehighertoleranceof‘Navelate’orangestodevelopnon-chillingpeel pitting.© 2005 Elsevier B.V. All rights reserved. Keywords:  Ascorbate peroxidase; Abscisic acid; Catalase; Chilling injury; Ethylene; Glutathione reductase; Navelate; Non-chilling peel pitting;Pinalate; Phenylalanine ammonia-lyase; Superoxide dismutase 1. Introduction Citrus peel disorders may be induced by differentabioticfactorsandpresentdifferentsymptomology,buthow they are related to each other is not understood(Grierson, 1986). It is well known that resistance of  plants to stress conditions causing damage depends onthe species and genotype. As shown in the first paperof this series, ‘Navelate’ ( Citrus sinensis  L. Osbeck)fruit is prone to develop CI, manifested as superficialscald, while its yellow-abscisic acid-deficient mutant‘Pinalate’ is tolerant to cold stress. In addition, theABA-deficient mutant, which is unable to accumulateABA in response to conditions favouring water lossand very prone to dehydration, is much more suscep-tible to develop non-chilling peel pitting than ‘Nave-late’ fruit (Alf ´erez et al., 2005). Therefore, this mutantmaybeespeciallyusefultounderstandthemechanismsunderlying chilling-induced superficial scald and non-chilling peel pitting disorders.Oxidative stress has been associated with damageof stressed plants (Dat et al., 2000). Superoxide anion,hydrogen peroxide and the hydroxyl radical are reac-tive oxygen species (ROS) that can result in oxidiza-tion of proteins, unsaturated fatty acids and DNA,causing cellular damage and eventually cell death(Mittler, 2002). Plants have evolved efficient antiox- idantsystemstoscavengeROS.Amongthesesystems,antioxidant enzymes, including superoxide dismutase(SOD; EC, catalase (CAT; EC,and the enzymes ascorbate peroxidase (APX; EC1.11.1.11) and glutathione reductase (GR; EC the Halliwell-Asada cycle are the most efficient pro-tective mechanisms against oxidative stress (Mittler,2002).Oxidative stress has been proposed as one of thecauses of cold-temperature damage in mandarin fruit(Sala, 1998) and of damage caused by water stress(Senaratnaetal.,1985).Previousstudiessuggestedtheparticipation of the antioxidant enzymatic system inthe tolerance of ‘Fortune’ mandarins to chilling con-ditions causing peel pitting (Sala and Lafuente, 1999)and in the peel pitting process occurring in ‘Navelina’fruit at non-chilling temperatures (Sala and Lafuente,2004),butfurtherinvestigationisneededtounderstandwhethertheantioxidantsystemmayplayaroleprotect-ing citrus fruit against this non-chilling rind disorder.Additional research is also necessary to elucidate itsparticipation in the susceptibility of different ‘Navel’orangecultivarstodevelopbronzenon-depressedareasat low temperature.The inhibition of phenylpropanoid biosynthesisby co-suppression of phenylalanine ammonia-lyase(PAL; EC may lead to precocious celldeath (Maher et al., 1994). The participation of  this enzyme in the reduction of the developmentof peel pitting chilling symptoms in ‘Fortune’ man-darins has been demonstrated (Lafuente et al., 2001). However, the involvement of PAL in the develop-ment of chilling symptoms different to pitting, assuperficial scald occurring in ‘Navelate’ oranges, orin non-chilling-related citrus peel disorders remainsunknown.Theaimofthisworkhasbeentoinvestigatewhetherthe capacity of the enzymes of the antioxidant path-ways to respond to storage conditions favouring CIand non-chilling peel pitting is modified in fruit of themutant ‘Pinalate’ with respect to its parental ‘Nave-late’. In addition, we have investigated, using fruitof both phenotypes, the involvement of the enzymePAL in the development of chilling-induced brownnon-depressed areas in the flavedo of ‘Navel’ oranges,which differs from the chilling-induced peel pittingsymptoms described in other citrus cultivars, and inthe development of the non-chilling-related citrus peeldisorder.  234  J.M. Sala et al. / Postharvest Biology and Technology 37 (2005) 232–240 2. Materials and methods 2.1. Fruit samples and storage conditions ‘Navelate’ ( Citrus sinensis  L. Osbeck) orange fruitand fruit of its ABA-deficient mutant ‘Pinalate’ wereharvested at commercial maturity and stored at 2 and12 ◦ C under air at 85–90% RH for up to 8 weeks asdescribed in the first paper of this series (Alf ´erez etal., 2005). Fruit samples used throughout this studywere from the same harvest and batches of fruit usedin that paper. Three replicates of 10 fruit per tempera-ture and storage period were used to analyze changesin the activities of the enzymes SOD, CAT, APX, GRand PAL. Flavedo tissue, the coloured outer layer of skin, was separated from the whole fruit, frozen inliquid N 2 , homogenized, and stored at  − 70 ◦ C forenzyme assays. Antioxidant enzymes were extractedfromrepresentative1gfreshweight(FW)samplesandPAL from 0.4g acetone powder prepared as describedbelow.Resultsaregivenasthemeansofthreereplicatesamples ± S.E.M. 2.2. SOD, CAT, GR and APX assays The activities of the enzymes SOD, CAT, APXand GR were analyzed in the flavedo of ‘Navelate’and ‘Pinalate’ oranges as previously described by Sala(1998). Each value is the mean of three replicate sam-ples containing 10 different fruit ± S.E.M.SOD was extracted from 1g FW of frozen flavedotissue with 10ml of cold 50mM potassium phosphatebuffer,pH7.8,containing1.33mMdiethylenetriaminepentaacetic acid (DETAPAC) in a mortar and pestle at4 ◦ C. The homogenate was centrifuged twice at thistemperature for 15min at 27,000 × g  and the super-natant used to analyze SOD spectrophotometrically bythe method of  Oberley and Spitz (1986). The super- oxide radicals were generated by xanthine–xanthineoxidase and nitro blue tetrazolium (NBT) was used asindicator of superoxide radical production. One unit of SOD was defined as the amount of enzyme that gavehalf-maximal inhibition.Frozen flavedo tissue (1g FW) was pulverized in amortar and pestle with 10ml of cold 100mM potas-sium phosphate buffer, pH 6.8 at 4 ◦ C to extract CAT.Theextractwascentrifugedasdescribedaboveandthesupernatant used to determine the activity of CAT at25 ◦ C following the method of  Kar and Mishra (1976).OneunitofCATwasdefinedastheamountofenzyme,which decomposes 1  mol H 2 O 2  per minute at 25 ◦ C.APX was extracted from 1g of FW flavedo tissuewith 10ml of cold 50mM potassium phosphate buffer,pH7.0,containing0.1mMethylenediaminetetraaceticacid (EDTA), 1mM ascorbic acid and 1% polyvinyl-polypyrrolidone (PVPP) at 4 ◦ C. The homogenate wascentrifuged twice as described above and the super-natant used to determine APX activity as described byAsada (1984). One unit of APX was defined as theamount of enzyme that oxidized 1  mol of ascorbateper minute.GRwasextractedfrom1gofFWflavedotissuewith10ml of cold 100mM potassium phosphate buffer, pH7.5,containing0.5mMEDTAat4 ◦ C.Thehomogenatewas centrifuged twice at 4 ◦ C for 15min at 27,000 × g andthesupernatantusedtoassayGRspectrophotomet-rically by the method of  Smith et al. (1988). The activ- ities of the GR solutions used for the standard curveweredeterminedaccordingtoCarlbergandMannervik (1985). One unit of GR was defined as the amountof enzyme that catalyzed the oxidation of 1  mol of NADPH per minute. 2.3. PAL activity determination PAL (EC activity was determined in threereplicatesamplesfromflavedoacetonepowderaccord-ing to Mart´ınez-T´ellez and Lafuente (1997). Flavedosamples were collected from the total surface of fruit stored at 2 and 12 ◦ C at the times indicated.Flavedo tissue was ground with 10ml of acetone, pre-viously chilled to  − 20 ◦ C, per gram of flavedo. Thehomogenate was filtered through a Buchner funnel,the residue washed twice with chilled acetone and theresulting powder dried at room temperature. PAL wasextracted from 0.4g of acetone powder with 15mlof 0.1M sodium borate buffer, pH 8.8, containing0.02M   -mercaptoethanol. The extract was purifiedby salting out proteins with ammonium sulphate toa final saturation of 46% supernatant. The precipi-tated PAL enzyme was dissolved in 4.5ml of 0.1Mammonium acetate buffer, pH 7.7, containing 0.02M  -mercaptoethanol and the PAL activity measured bydeterminingtheabsorbanceofcinnamicacidat290nmoveraperiodof2hat40 ◦ C.Thereactionmixturecon-tained 2ml of the purified enzyme extract and 0.6ml   J.M. Sala et al. / Postharvest Biology and Technology 37 (2005) 232–240  235 l -phenylalanine 0.1M in a total volume of 6ml. PALactivityisexpressedonadry-matterbasisasnanomolesof cinnamic acid per gram of acetone powder flavedotissue per hour. The results are the mean of three repli-cate samples ± S.E.M. 3. Results and discussion The involvement of the antioxidant enzymatic sys-tem in the different susceptibility of ‘Navelate’ and‘Pinalate’ fruit to chilling-induced superficial scaldand non-chilling peel pitting and how PAL activity isinvolved in these processes has been examined. 3.1. Antioxidant enzymatic system in non-chillingand chilling peel disorders on fruit of ‘Navelate’and of its ABA-deficient mutant ‘Pinalate’ SODisthefirstlineofdefensefromdamagescausedby oxygen radicals (Mittler, 2002). The activity of this enzyme in freshly harvested ‘Navelate’ and ‘Pinalate’fruit was similar and declined during storage at 2 ◦ Cin fruit of both cultivars (Fig. 1). At this temperature,SODactivityin‘Pinalate’waslowerthanin‘Navelate’fruit. Therefore, this enzyme appears not to play a rolein the higher tolerance of ‘Pinalate’ fruit to chilling.However, we cannot rule out its participation in thelower susceptibility of ‘Navelate’ fruit to develop non-chillingpeelpittingat12 ◦ C,asSODactivitywaslowerin fruit of the mutant held at this temperature (Fig. 1).In addition, SOD activity of ‘Pinalate’ fruit stored forprolongedperiodsat12 ◦ Cwassimilartothatoffreshlyharvestedfruit,whileitincreasedatthistemperaturein‘Navelate’ oranges.CAT activity of freshly harvested fruit of both phe-notypes was also similar. However, the activity of thisenzyme was lower in ‘Pinalate’ than in ‘Navelate’ fruitheld at the temperature (12 ◦ C) causing non-chillingpeel pitting (Fig. 2B). As fruit of the mutant are much more susceptible to this physiological disorder, thatresult might indicate the involvement of CAT in thedefense of ‘Navel’ oranges against this rind disorder.In addition, our results show that ‘Pinalate’ fruit, thechilling tolerant cultivar, presented higher CAT activ-ity than ‘Navelate’ oranges after 2 weeks storage at2 ◦ C and remained higher for the whole storage periodat this temperature (Fig. 2A). Whether the different Fig. 1. Changes in SOD activity in ‘Navelate’ and ‘Pinalate’ fruitstored at 2 and 12 ◦ C. Black symbols correspond to ‘Pinalate’ andwhitesymbolsto‘Navelate’fruit.Resultsarethemeanof3replicatesamples ± S.E.M. CAT behaviour found in both cultivars kept at 2 and12 ◦ C is related to different CAT isoenzymes operat-ing at these temperatures in ‘Pinalate’ and ‘Navelate’orangesdeservesfurtherconsideration.However,fromthese data, it appears that oxidative stress may partic-ipate in both citrus fruit physiological disorders andthat their occurrence may depend on the ability of harvested fruit to maintain high CAT activity duringstorage at different temperatures. Previous investiga-tions suggested that CAT may be a major antioxi-dant enzyme participating in the defence mechanismof mandarin fruit against chilling conditions causingflavedo pit-like depressions (Sala and Lafuente, 2000).‘Navelate’orangespresentedadifferentchillingsymp-  236  J.M. Sala et al. / Postharvest Biology and Technology 37 (2005) 232–240 Fig. 2. Changes in CAT (A and B), APX (C and D) and GR (F and G) activities in ‘Navelate’ and ‘Pinalate’ fruits stored at 2 and 12 ◦ C. Black symbols correspond to ‘Pinalate’ and white symbols to ‘Navelate’ fruit. Results are the mean of 3 replicate samples ± S.E.M. tomology. Thus, the results obtained here may indicatethat ROS may also participate in the development of chilling-induced flavedo superficial scalded areas andfurthersuggestthatCATmightprotect‘Navel’orangesagainst low temperature stress conditions causing thischilling-associated peel disorder. Little is known aboutthe participation of this enzyme in the development of non-chilling peel pitting. Recently, it has been shownthat CAT activity decreased in ‘Navelina’ oranges keptunder temperature conditions (20 ◦ C) favouring non-chilling peel pitting and that such a decrease was lowerin fruit exposed to RH conditions reducing its inci-dence (Sala and Lafuente, 2004). Considering theseresults, it was suggested that CAT might play a role inthe tolerance of citrus fruit to this disorder. The resultsof the present work further support this idea since thedecline in CAT activity occurring in ‘Pinalate’ fruit at12 ◦ C was higher than that of ‘Navelate’ oranges and
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