hot and chilling injury 01-main

Postharvest Biology and Technology 99(2015)114–119Contents lists available at ScienceDirectPostharvest Biology andTechnologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p o s t h a r v b ioChilling injury incidence and antioxidant enzyme activities of Carica papaya L.‘Frangi’as influenced by postharvest hot water treatment and storage temperatureN.Shadmani a ,S.H.Ahmad a ,∗,N.Saari b ,P.Ding a ,N.E.Tajidin aa Department of Crop Science,Faculty of Agriculture,University Putra Malaysia,Serdang,43400Selangor,D.E.,MalaysiabDepartment of Food Science,Faculty of Food Science and Technology,University Putra Malaysia,Serdang,43400Selangor,D.E.,Malaysiaa r t i c l ei n f oArticle history:Received 21February 2014Accepted 5August 2014Available online 18September 2014Keywords:Chilling injury CatalaseAscorbate peroxidaseSuperoxide dismutase activitya b s t r a c tThe effect of double-dip hot water treatment (42◦C for 30min and 49◦C for 20min)with two different storage temperatures (6and 12◦C,85–90%RH)and storage durations (0–3weeks)on peel and pulp chilling injury incidence and antioxidant enzyme (catalase,ascorbate peroxidase,superoxide dismutase)activities of Frangi papayas,were investigated,with untreated fruit as controls.Peel and pulp chilling injury (CI)incidence was reduced in the treated fruit,especially in fruit stored at 6◦C,which was accompa-nied by increased ascorbate peroxidase activity.Thus,hot water dips followed by low storage temperature could prolong the storage life and reduce CI incidence of Frangi papaya.©2014Elsevier B.V.All rights reserved.1.IntroductionThe export potential of Frangi papaya is restricted by its limited storage life due to its highly perishable nature,susceptibility to chilling injury (CI),and deterioration of postharvest quality during transportation and storage.Long-term storage at low temperature may cause physiological injuries (Maharaj and Sankat,1990),and reducing the transport temperature slows the ripening process.Postharvest hot water dip (HWD)treatments can be applied to overcome the risk of CI and the decay of quarantined fruit (Schirra et al.,2004).Heating “Eksotika”papaya at 38◦C for 6–24h before storage at low temperature has been reported to reduce chilling injury (Ali et al.,2000).Changes in fruit membranes and structure are the first response of CI at the cellular level,and this affects membrane permeability.A secondary reaction would appear with symptoms such as elec-trolyte leakage,reduction in metabolic energy and cell lysis (Raison and Orr,1990).Oxidative damage is considered to be a response of tissues that are susceptible to CI.Fruit subjected to abiotic stress,such as low or high temperatures (Sairam et al.,2000;Sala and Lafuente,2004),have been shown to produce elevated levels of the reactive oxygen species (ROS)that cause oxidative damage∗Corresponding author.Tel.:+60389474837;fax:+60389408445.E-mail addresses:hajar@.my ,hajar@.my ,sitihajarahmad@ (S.H.Ahmad).(Shigeoka et al.,2002).When plants are exposed to moderate heat treatments,weak oxidative stresses are induced,and this stress modulates antioxidant levels and causes tolerance to subsequent severe stress (Li,2003).The activity levels of antioxidant enzymes such as catalase,ascorbate peroxidase and superoxide dismu-tase have been related to tolerance to low temperatures and CI prevention (Imahori et al.,2008).The goal of a higher export rate of papaya with desirable qual-ities and minimised CI has yet to be achieved.Thus,it is critical to evaluate the CI incidence of papaya after hot water treatment and during storage.Heat treatments will provide more commercial benefits for local and regional producers and comply with quar-antine protocols from importing countries.Presently,there are no reports concerning the influence of postharvest hot water treat-ment and storage temperature on the chilling injury incidence of ‘Frangi’papaya.The objective of this study was to investigate the effect of hot water treatment and two different storage temper-atures (6and 12◦C)on chilling injury and antioxidant enzymes activities of ‘Frangi’papaya during a three week storage period.2.Materials and methods2.1.Sample preparation and heat treatmentPapaya (Carica papaya cv.Frangi)fruit at maturity stage 2,with less than 10%yellow skin,were harvested from a commercial farm in Lanchang,Pahang,Malaysia.The fruit were transported to/10.1016/j.postharvbio.2014.08.0040925-5214/©2014Elsevier B.V.All rights reserved.N.Shadmani et al./Postharvest Biology and Technology99(2015)114–119115the Postharvest Laboratory,Faculty of Agriculture,Universiti Putra Malaysia within3h of harvest.Fruit of uniform size(450–550g) and appearance,that were free from blemishes and defects,were selected.A double-dip hot water treatment was applied to selected fruit byfirst immersing fruit in hot water at42◦C for30min and then immediately immersing fruit in hot water at49◦C for20min (Alvarez and Nishijima,1987).The hot water dip was performed in a water bath,and the required temperature was kept constant with a thermostat.During the treatment,the internal fruit temper-ature was determined by inserting a thermocouple into the pulp of a few fruit samples.During thefirst dip,the internal tempera-ture was44.7◦C;during the second dip,the internal temperature increased to46.3◦C.The hot water dipped and control(without hot water dip)fruit were dipped in Folicur®(Tebuconazole fungicide, Bayer Advanced TM)for60s and then cooled at ambient tempera-ture(25±2◦C)for20min.Chilling injury incidence and antioxidant enzymes activities were evaluated each week for three weeks of storage at6and12◦C with85–90%RH.The samples were assessed on day0of storage,and this measurement was considered to be week0.2.2.Internal and external chilling injury assessmentFruit were removed from storage on weeks0–3,and then incu-bated at ambient temperature(25±2◦C)for2days.Then the fruit were assessed visually for external injuries such as the occur-rence of skin pitting,scald(from dark olive to light brown spots), shrivelling and external water soaking.Pulp water soaking,hard lumps(around the fruit seed cavity or throughout the pulp),and internal mesocarp regions that failed to soften,were evaluated as internal injuries.These areas,which failed to ripen,appeared slightly lighter orange in colour than normal tissue.The assessment was performed according to the following hedonic scale:1=no abnormality,2=trace symptoms,small pits(1–15%),3=moderate symptoms,small to medium pits,blotchy appearance(16–30%), 4=moderate to severe symptoms(31–50%),5=severe symptoms (more than50%affected)(Proulx et al.,2005).The CI incidence was calculated by logarithmic transformation of the data.2.3.Peel electrolyte leakage determinationPapaya peel electrolyte leakage percentage was measured according to previous methods,Woolf and Lay-Yee(1997)with some modifications.The papaya peel was removed with a blade, and the exposed pulp was cut into10disks of2g each with a10-mm cork borer.Ten disks from each treatment and storage tempera-ture werefloated in separate conicalflasks.Eachflask contained 10ml of0.4M mannitol and was incubated for3h in a water bath (903,Protech,Malaysia)at30◦C under continuous shaking,and initial conductivity was measured by a conductivity metre(Acc-umet AB30,Fisher Scientific,Singapore).Total conductivity was measured when conicalflasks withfloated peel disks were auto-claved at120◦C for20min and cooled at ambient temperature. The electrolyte leakage percentage was calculated by dividing the initial conductivity by the total conductivity.2.4.Enzyme analysis2.4.1.Catalase(CAT)extraction and assayFor the enzyme extraction,fruit samples removed from storage were transferred immediately to-80◦C until measurements were made.One gram of papaya pulp(2-cm cubes from the fruit’s equatorial region)was taken and ground in liquid nitrogen with a mortar and pestle.The CAT activity was measured according to previously described methods,Blume and McClure(1980)with some modifications.The extraction buffer contained5mL of 50mM sodium phosphate buffer(pH7.0).The reaction mixture (1mL)contained500␮L of100mM sodium phosphate buffer(pH 7.8),150␮L of enzyme extract,250␮L distilled water and100␮L of50mM H2O2.The homogenate was centrifuged at10,000×g for 15min at4◦C.The reaction was begun by adding100␮L of50mM H2O2.The absorbance was measured at240nm using a spectropho-tometer(UV-Visible Double Beam,U-2800,Hitachi,Japan).One unit of enzyme activity was defined as one micromole of hydrogen peroxide oxidised mL−1min−1at25◦C.The results were expressed as enzyme units per gram fresh weight(U/g FW).The CAT activity was calculated using an extinction coefficient of39.4mM cm−1. 2.4.2.Ascorbate peroxidase(APX)extraction and assayRemaining samples from those described in section2.5.1were extracted with5mL extraction buffer.The extraction buffer con-tained the following:250␮L of50mM sodium phosphate buffer (pH7.0),50␮L of1mM l-ascorbic acid,5␮L of0.1M ethylene-diaminetetraacetic acid(EDTA)and1%of polyvinylpyrrolidone (PVP).The homogenate was centrifuged at10,000×g for15min at4◦C.APX activity was assayed according to previously published methods,Nakano and Asada(1981)with some modifications.The reaction mixture contained500␮L of100mM sodium phosphate buffer(pH7.0),100␮L of5mM l-ascorbic acid,250␮L of distilled water,50␮L of enzyme extract and100␮L of1mM H2O2.The final reaction volume was adjusted to1mL and APX activity was measured by the oxidation of ascorbate at290nm.The reaction was started by adding1mM H2O2.One unit of enzyme activity was defined as one micromole of ascorbate oxidised per millil-itre per minute at25◦C.The results were expressed as U/g FW. The APX activity was calculated using an extinction coefficient of 2.8mM cm−1.2.4.3.Superoxide dismutase(SOD)extraction and assayRemaining samples were extracted with5mL extraction buffer. The extraction buffer contained5mL of50mM sodium phosphate buffer(pH7.8),and the homogenate was centrifuged at10,000×g for15min at4◦C.The assay was run using the SOD kit(Cell Tech-nology Inc.,USA)to draw the SOD standard curve.The absorbance was recorded at450nm using a microplate reader(Labomed,UVD-2950,Power Wave X340,Biotek Instruments Inc.,USA).Under these assay conditions,one unit of SOD activity inhibits the rate of increase in the absorbance(inhibition in reduction of WST-1 to a water-soluble formazan dye)at440nm by50%.The per-cent inhibition of the test sample correlated with SOD activity using a SOD standard curve.The results were expressed as U/g FW.2.5.Experimental design and data analysisThe experiments were conducted using a randomised complete block design in a factorial arrangement of treatments(two heat treatment levels×two storage temperatures×four storage dura-tions),with three replications.Three fruit per replicate,with three replicates,were assessed for each treatment,including the control. The experiment was repeated a total of three times,once each in June,July,and August2011.Thus,the data comprised3subsam-ples of fruit per replicate(pooled before statistical analysis)×3 replicates per season(block)×3seasons(blocks).The data were analysed using the analysis of variance in SAS,version9.13(SAS, Institute Inc.,Cary,NC,USA),and the means were separated by least significant difference(LSD)tests at P≤0.05.The regression analysis was conducted when the interaction effects between two factors were significant.116N.Shadmani et al./Postharvest Biology and Technology 99(2015)114–1190.050.10.150.20.250.30.35P e e l c h i l l i n g i n j u r y (l o g t r a n s f o r m e d )Storage tempera ture (°C)trol Fig.1.Effect of storage temperature on chilling injury to peels of control and hot water dipped ‘Frangi’papaya.Data shown are the means of three replications.Means followed by same letters are not significantly different according to the LSD test,P ≤0.05.n =9.3.Results and discussion3.1.Peel and pulp chilling injury incidenceThere was a significant difference in the CI development between hot water dipped and control fruit stored at 6◦C,and it could be concluded that the application of hot water as a pre-conditioning treatment induced tolerance of chilling (Fig.1).Similarly,hot water treated bananas had lower CI after 5days of storage at 7◦C (Wang et al.,2012).Chen et al.(2008)reported that heat pretreatment of bananas before storage at 8◦C delayed the induction of CI symptoms by 3days.Cold conditioning along with a hot water dip produced a decline in susceptible fruit CI.The results shown in Fig.1were similar to those for the reduc-tion of CI in heat-treated grapefruit stored at 2◦C (Sapitnitskaya et al.,2006),heat-treated pomegranates stored at 2◦C (Mirdehghan et al.,2007)and heat-treated ‘Flavorcrest’peaches stored at 0±0.5◦C (Murray et al.,2007).The tolerance induced by modulat-ing antioxidant systems could inhibit AOS accumulation (Sala and Lafuente,2000).Fig.2shows that as the storage period progressed,peel CI in control fruit increased remarkably.During weeks 1and 2,control fruit showed relatively constant rates of CI incidence.After week 2,control fruit showed increasing decay and areas of pitting on the fruit surfaces.Peel CI of treated fruit increased from week 0to week 2,but CI incidence was noticeably reduced compared to the control until week 3,and this might be attributed to the reduction of ethylene production after week 2of storage.However,the CI reduction of hot water dipped papaya after week 2may be attributable to the alterations in the surface gloss of the fruit.The possible modifications to the light reflectance characteristics could be induced by a loss of cuticular waxand/or00.10.20.30.40.50.60.7P e e l c h i l l in g i n j u r y(l o g t r a n s f o r m e d )Storage duration (week)Fig. 2.Relationships between peel chilling injury and storage duration of ‘Frangi’papaya in control ()and hot-water-dipped ()fruit.y (con-trol)=0.395x −0.22x 2+3.05x 3(R 2=0.76)and y(hot water dip)=−0.001+0.34x −0.09x 2(R 2=0.56).Solid lines indicate significant quadratic and cubic relationships.n =9.0.050.10.150.20.250.3P u l p c h i l l i n g i n j u r y (l o g t r a n s f o r m e d )Storage temperature (°C)Con trol Hot water dipFig.3.Effect of storage temperature on pulp chilling injury of control and hot-water-dipped ‘Frangi’papaya.Data shown are the means of three replications.Meansfollowed by the same letters are not significantly different by the LSD test,P ≤0.05.n =9.ultrastructural changes in the crystalline surface wax (Charles et al.,2008).Any modification in the skin could decrease the adhesion of pathogens,thus reducing colonisation.Hot water dipped fruit stored at 6◦C showed lower incidence of CI than heat-treated fruit stored at 12◦C (Fig.3).In addition,control fruit stored at 6◦C had significantly higher CI incidence than control fruit stored at 12◦C.Fig.3shows that the rate of chilling injury development increased in hot water dipped fruit when they were stored at 12◦C.The increase of CI incidence in hot water dipped fruit at 12◦C stor-age might be related to the low persistence of CAT activity after hot water treatment,as mentioned earlier with heat-treated ‘For-tune’mandarins (Sala and Lafuente,2000).CAT was correlated to the enhanced CI in hot water treated mandarins.CI symptoms were revealed at relatively high temperatures (e.g.,12◦C),and the symp-toms were exacerbated by reductions in temperature (Chen et al.,2008).Many CI symptoms may occur along with ripening progression and can be distinguished from senescence (Saltveit and Morris,1990).The results obtained for heat-treated bananas were differ-ent from those in this experiment.Pulp firmness in banana during storage both at ambient temperature for 10days and at low tem-perature (14◦C)for 8days did not show any heat damage in the treated fruit (Ummarat et al.,2011).Internal injury was lower in hot water dipped fruit stored at 6◦C compared with controlled fruit stored at the same temperature.The response of treated fruit to storage at lower temperatures with lower incidence of CI may be related to the antioxidant defence mechanism or to heat-shock treatment (Luengwilai et al.,2012).APX enzyme activity was sig-nificantly higher in hot-water-dipped fruit than in the control fruit when stored at 6◦C (Fig.6).These antioxidant enzymes may help control the oxidative responses that result from free radical species such as ROS (Wahid et al.,2007).3.2.Peel electrolyte leakageThe results shown in Table 1demonstrate that PEL was signif-icantly influenced by heat treatment and that heat-treated fruit had lower peel electrolyte levels than control fruit,by 24%.PEL was also significantly affected by storage duration,with a con-sistent increase (55%)until the end of the storage period.The increase of ion leakage during storage may be due to the respi-ration rate and is dependent on the integrity of the fruit tissue.The increases in respiration rate and ion leakage parameters are presumed to occur at the end of fruit ripening or when the fruit are exposed to rigorous stress conditions such as processing,cut-ting and/or exposure to high or low temperatures (Nyanjage et al.,1999).PEL was higher in control papaya than in heat-treated fruit (Table 1).The same useful effects of heat treatments have been mentioned earlier for other tropical and subtropical fruit,includingN.Shadmani et al./Postharvest Biology and Technology99(2015)114–119117 Table1Main and interaction effects of heat treatment,storage temperature and storage duration on peel and pulp chilling injury incidence and peel electrolyte leakage of‘Frangi’papaya.Factor Peel chilling injury(log y)Pulp chilling injury(log)Peel electrolyte leakage(%) Heat treatment(HT)No hot water dip(control)0.25a z0.18a21.36aHot water dip0.22a0.21a16.04bStorage temperature(ST)(◦C)60.26a0.20a19.34a120.22b0.18a18.06aStorage duration(SD)(weeks)00c0c10.81c10.24b0.17b19.75b20.34b0.23b20.10b30.39a0.40a24.15aHT×ST**nsHT×SD*ns nsSD×ST ns ns nsHT×ST×SD**nsns,not significant.y Data were log transformed.n=9.z Means within a column and factor followed by the same letter are not significantly different by the LSD test at P≤0.05.*Significant at P≤0.05.Table2Main and interaction effects of heat treatment,storage temperature and storage duration on catalase,ascorbate peroxidase and superoxide dismutase activity of‘Frangi’papaya.Factor Catalase(U/g FW)Ascorbate peroxidase(U/g FW)Superoxide dismutase(U/g FW)Heat treatment(HT)No hot water dip(control) 2.22a z41.13b85.84aHot water dip 1.77b48.07a84.40aStorage temperature(ST)(◦C)6 1.74b35.75b79.89b12 2.25a53.45a90.35aStorage duration(SD)(weeks)0 1.84b74.66b34.59c1 1.77b43.79b96.00a2 2.41a54.18a73.13b3 1.96b45.84b96.69aHT×ST ns ns*HT×SD*ns nsSD×ST ns**HT×ST×SD ns**ns,not significant.z Means within a column followed by the same letter were not significantly different by LSD test at P≤0.05.*Significant at P≤0.05.n=9.pepper,tomato,cucumber and citrus(Lurie,1998;Sanchez-Ballesta et al.,2000;Saltveit,2001).A protective effect of heat treatment was observed in tomato,with less ion movement across the membranes and enhanced resistance against chilling-induced damage(Saltveit, 2005).Relative conductivity of grape tissue was increased by12.1% after3h chilling at−2◦C.Ultrastructural alterations related to CI have often appeared in membrane systems such as chloroplasts, mitochondria,nuclei,plasma membranes and tonoplasts(Kratsch and Wise,2000).3.3.Catalase,ascorbate peroxidase and superoxide dismutase activityCAT is the enzyme that preserves cells against AOS;it cataly-ses the decomposition of hydrogen peroxide to oxygen and water (Ghasemnezhad et al.,2008).The CAT activity of control fruit was reduced from week0to week1but then increased consistently until week2.CAT activity decreased markedly until week3(Fig.4). The increase of CAT activity in fruit stored at12◦C(Table2)may be due to the decomposition of hydrogen peroxide,which would cause the formation of hydroxyl radicals and the alleviation of chilling-induced peel injury(Table1).Differences in CAT activity between storage temperatures reflect the physiological differences related to the function of the antioxidant system in fruit.ROS accumulation is one of the mechanisms that contribute to the loss of membrane integrity and membrane-bound enzyme activities.The increase of H2O2and lipid hydro-peroxides has been reported earlier during fruit ripening and senescence(Lacan and Baccou,1998).LowerCAT 11.522.533.540123Catalase(U/gFW)Storage duration (weeks)Fig.4.Relationships between storage duration and catalase activity(U/g FW) of‘Frangi’papaya in control()and hot-water-dipped ()fruit.y(con-trol)=2.12−2.92x+3.14x2−0.72x3(R2=0.50).Solid line indicates a significant cubic relationship.n=9.118N.Shadmani et al./Postharvest Biology and Technology 99(2015)114–11920304050607080900123A s c o r b a t e p e r o x i d a s e (U /g F W )Storage duration (weeks)Fig.5.Relationship between ascorbate peroxidase activity (U/g FW)and stor-age duration of ‘Frangi’papaya stored at 6◦C()and 12◦C ().y (12◦C)=39.21−4.37x +17.98x 2−4.68x 3(R 2=0.50).Solid line indicates a significant cubic relationship.n =9.expression was found in hot-water-treated bananas than in the control fruit during cold storage (Wang et al.,2012).The fluctuating trend of CAT activity in control fruit during stor-age could be the result of increasing H 2O 2content during storage,as proposed by Huang et al.(2008)on ‘Cara Cara’navel oranges.They noted that the H 2O 2content in control fruit increased,especially in the middle stage of fruit ripening (week 2,as shown for papaya;Fig.4).The constant trend of peel CI incidence in control fruit dur-ing weeks 1and 2could be due to the increase in CAT activity.The increase of CI incidence in control fruit during week 3was similar to the reduction of CAT activity during the same week (Fig.2,Fig.4).The findings for ‘Navel’and ‘Valencia’oranges were not consistent with the results of this experiment.Hot water treatment signifi-cantly increased CAT activity in both peel and juice during storage at 1◦C for 20days.In addition,the reduction of CI was paralleled by higher CAT activity (Bassal and El-Hamahmy,2011).According to the results on papaya in this study,it did not seem that higher CAT activity correlated with resistance to CI in hot water treated papaya.There was a significant cubic relationship between APX activity and storage duration in papaya fruit stored at 12◦C (Fig.5).Enzyme activity increased from week 0to week 2of storage and slightly decreased thereafter.The R 2=0.50indicated that 50%of the variability in APX activity at 12◦C was due to the storage duration.These cellular changes will result in oxidative stress injuries and increase postharvest senescence.The results obtained from storage at 6◦C may indicate that the ascorbate-glutathione cycle in papaya did not provide antioxidant protection during 3weeks of cold storage as mentioned above for melon and tomato fruit (Barreiro et al.,2001;Mondal et al.,2006).The decreasing trend of the APX activity observed from week 1to week 3in papaya fruit stored at 6◦C was similar with mume fruit stored for 16days at 6◦C (Imahori et al.,2008).In this experiment,the high-est APX activity at 12◦C storage was observed at week 3(Fig.5),and consequently the reduction of peel CI was observed at week 3(Fig.2).It is possible that in fruit stored at 12◦C,the APX isoforms were synthesised,reducing the concentration of hydrogen perox-ide and consequently protecting the papaya from oxidative stress,as previously described by Ghasemnezhad et al.(2008)for Sat-suma mandarins.Fig.6indicates that APX activity was significantly higher in hot water dipped fruit stored at 6◦C.The increased activ-ity of antioxidant enzymes (APX)induced by heat treatments can protect against the membrane damage produced by chilling stress,consequently enhancing chilling tolerance (Zhang et al.,2005).Con-trol fruit stored at 12◦C showed significantly higher APX activity than control fruit stored at 6◦C (Fig.6).It is known that free radi-cals are produced during fruit ripening at optimum temperature that cause deteriorative changes related with senescence,but stor-age at chilling temperatures retards the normal ripening process and inactivates the production of free radicals (Huang et al.,2008).102030405060612A s c o r b a t e p e r o x i d a s e (U /g F W )Storage tempera ture ( °C )trol water dipFig.6.Effects of storage temperature and hot water dip on ascorbate peroxidase activity (U/g FW)of control and hot-water-dipped ‘Frangi’papaya.Data shown are the means of three replications.Means followed by the same letters were not significantly different by the LSD test,P ≤0.05.n =9.The high level of APX induced in cold storage may play a signifi-cant role in the alleviation of cold damage (Wang et al.,2012).The results obtained were consistent with Fig.1,which showed peel CI reduction of hot water dipped fruit stored at 6◦C.During the first week of storage,SOD activity of fruit stored at 12◦C increased and then decreased markedly until week 2of storage.SOD activity increased from week 2to week 3of storage (Fig.7).SOD activity in mangoes stored at 12◦C was higher than that of those stored at 6◦C.These results suggest a link between SOD activity and (O 2•−)scavenging activity at 12◦C (Kondo et al.,2005).CI occurrence in papaya fruit was not totally correlated with SOD production at 6and 12◦C,but the decreasing trend of peel CI (Fig.2)from week 2to week 3in fruit stored at 12◦C was similar to the increase of SOD at 12◦C (Fig.7).The enhanced SOD activity after week 2at 12◦C may inhibit the accumulation of superoxide radicals dur-ing storage and consequently reduce tissue damage (Vicente et al.,2006).The effects of chilling on SOD activity could be described by the different durations of cold exposure,and its activity clearly depended on the temperature regime during chilling (Sala and Lafuente,2004).In grape berries,CAT and SOD activities were steadily reduced under chilling stress.The increase of SOD activity could improve the fruit’s ability to dismutate superoxide radicals.In addition,the increased CAT and APX activities may be related to the elimination of hydrogen peroxide.Injury to plant tissues has been correlated with AOS increases with stresses such as expo-sure to chilling temperatures.Oxidative damage is regarded as an early reaction of the sensitive tissues to CI (Imahori et al.,2008).Protection of the plant cell from oxidative damage during stress is the primary mechanism by which plants resist stress (Kraus and Fletcher,1994).4060801001201400123S u p e r o x i d e d i s m u t a s e (U /g F W )Storage duration (week)Fig.7.Relationship between storage duration and storage temperature on super-oxide dismutase activity (U/g FW)of ‘Frangi’papaya stored at 6◦C ()and 12◦C(),y (12◦C)=66.73+155x −142.5x 2−32.22x 3(R 2=0.58).Solid line indicates a significant cubic relationship.n =9.N.Shadmani et al./Postharvest Biology and Technology99(2015)114–1191194.ConclusionsHot water treated fruit stored at6◦C showed significantly lower CI incidence and higher ascorbate peroxidise activity.These results demonstrated that SOD,CAT and APX activities may contribute to the alleviation of CI in fruit stored at6◦C.The integrated action of SOD,CAT and APX,might efficiently remove the AOS.The bal-ance among SOD,CAT,and APX activities is essential to cell survival during cold storage(Sala and Lafuente,2004).Plant injury caused by different biotic and abiotic stresses often involves an imbalance between the production and removal of AOS(Wise,1995).AcknowledgementThis work was funded by the Faculty of Agriculture and Faculty of Food Science and Technology,Universiti Putra Malaysia,Serdang, Selangor,Malaysia.ReferencesAli,Z.M.,Ng,C.H.,Chua,S.T.,Othman,R.,Lazan,H.,2000.Heat treatment alleviates chilling injury symptoms,retards texture changes and maintains the capacity to produce ethylene in papaya fruits.In:Artcs,F.,Gil,M.I.,Conesa,M.A.(Eds.), Improving Postharvest Technologies of Fruits,Vegetables and Ornamentals.International Institute of Refrigeration,Paris,pp.519–524.Alvarez,A.M.,Nishijima,W.T.,1987.Postharvest diseases of Papaya.Plant Dis.71, 681–686.Barreiro,M.G.,Lidon,F.C.,Pinto,M.,2001.Physicochemical characterisation of the postharvest senescence of the winter melon‘Tendral’.Fruits56,51–58. Bassal,M.,El-Hamahmy,M.,2011.Hot water dip and preconditioning treatments to reduce chilling injury and maintain postharvest quality of‘Navel’and‘Valencia’oranges during cold quarantine.Postharvest Biol.Technol.60,186–191. Blume,D.E.,McClure,J.,1980.Developmental effects of Sandz6706oil of enzymes of phenolic and general metabolism in barely shoots grown in the dark or under low of high intensity light.Plant Physiol.65,234–238.Charles,M.T.,Benhamou,N.,Arul,J.,2008.Physiological basis of UV-C induced resis-tance to Botrytis cinerea in tomato fruit.III.Ultrastructural modifications and their impact on fungal colonization.Postharvest Biol.Technol.47,27–40. Chen,J.y.,He,L.h.,Jiang,Y.m.,Wang,Y.,Joyce,D.C.,Ji,Z.l.,Lu,W.j.,2008.Role of phenylalanine ammonia-lyase in heat pretreatment-induced chilling tolerance in banana fruit.Physiol.Plant.132,318–328.Ghasemnezhad,M.,Marsh,K.,Shilton,R.,Babalar,M.,Woolf,A.,2008.Effect of hot water treatments on chilling injury and heat damage in‘Satsuma’mandarins: antioxidant enzymes and vacuolar ATPase,and pyrophosphatase.Postharvest Biol.Technol.48,364–371.Huang,R.H.,Liu,J.H.,Lu,Y.M.,Xia,R.X.,2008.Effect of salicylic acid on the antioxi-dant system in the pulp of‘Cara cara’navel orange(Citrus sinensis L.Osbeck)at different storage temperatures.Postharvest Biol.Technol.47,168–175. Imahori,Y.,Takemura,M.,Bai,J.,2008.Chilling-induced oxidative stress and antiox-idant responses in Mume(Prunus mume)fruit during low temperature storage.Postharvest Biol.Technol.49,54–60.Kondo,S.,Kittikorn,M.,Kanlayanarat,S.,2005.Preharvest antioxidant activities of tropical fruit and the effect of low temperature storage on antioxidants and jasmonates.Postharvest Biol.Technol.36,309–318.Kratsch,H.A.,Wise,R.R.,2000.The ultrastructure of chilling stress.Plant Cell Environ.23,337–350.Kraus,T.E.,Fletcher,R.A.,1994.Paclobutrazol protects wheat seedlings from heat and paraquat injury.Is detoxification of active oxygen involved?Plant Cell Physiol.35,45–52.Lacan,D.,Baccou,J.C.,1998.High levels of antioxidant enzymes correlate with delayed senescence in nonnetted muskmelon fruits.Planta204,377–382.Li,M.H.,2003.Peroxidase and superoxide dismutase activities infig leaves in response to ambient air pollution in a subtropical city.Arch.Environ.Contam.Toxicol.45,168–176.Luengwilai,K.,Beckles,D.M.,Saltveit,M.E.,2012.Chilling injury of harvested tomato (Solanum lycopersicum L.)cv.Micro-Tom fruit is reduced by temperature pre-treatments.Postharvest Biol.Technol.63,123–128.Lurie,S.,1998.Postharvest heat treatments.Postharvest Biol.Technol.14,257–269. Maharaj,R.,Sankat,C.K.,1990.Storability of papayas under refrigerated and control atmosphere.Acta Hortic.269,375–386.Mirdehghan,S.H.,Rahemi,M.,Martínez-Romero,D.,Guillén,F.,Valverde,J.M.,Zap-ata,P.J.,Serrano,M.,Valero,D.,2007.Reduction of pomegranate chilling injury during storage after heat treatment:role of polyamines.Postharvest Biol.Tech-nol.44,19–25.Mondal,K.,Sharma,N.S.,Malhotra,S.P.,Kamal,D.,Singh,R.O.,2006.Oxidative stress and antioxidant systems in tomato fruits stored under normal and hypoxic conditions.Physiol.Mol.Biol.Plants12,145–150.Murray,R.,Lucangeli,C.,Polenta,G.,Budde,C.,bined pre-storage heat treatment and controlled atmosphere storage reduced internal breakdown of ‘Flavorcrest’peach.Postharvest Biol.Technol.44,116–121.Nakano,Y.,Asada,K.,1981.Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts.Plant Cell Physiol.22,867–880. Nyanjage,M.O.,Wainwright,H.,Bishop,C.F.H.,1999.Effects of hot-water treatment and storage temperature on electrolyte leakage of mangoes(Mangifera indica Linn.).J.Hortic.Sci.Biotechnol.74,566–572.Proulx,E.,Cecilia,M.,Nunes,N.,Emond,J.P.,Brecht,J.K.,2005.Quality attributes limiting papaya postharvest life at chilling and non-chilling temperatures.Proc.Fla.State Hortic.Soc.118,389–395.Raison,J.K.,Orr,G.R.,1990.Proposals for a better understanding of the molecular basis of chilling injury.In:Wang,C.(Ed.),Chilling Injury of Horticultural Crops.CRC Press,Boca Raton,FL,pp.145–164.Sairam,R.K.,Srivastava,G.C.,Saxena,D.C.,2000.Increased antioxidant activity under elevated temperatures:a mechanism of heat stress tolerance in wheat geno-types.Biol.Plant.43,245–251.Sala,J.M.,Lafuente,M.a.T.,2000.Catalase enzyme activity is related to tolerance of mandarin fruits to chilling.Postharvest Biol.Technol.20,81–89.Sala,J.M.,Lafuente,M.a.T.,2004.Antioxidant enzymes activities and rindstaining in ‘Navelina’oranges as affected by storage relative humidity and ethylene condi-tioning.Postharvest Biol.Technol.31,277–285.Saltveit,M.E.,2001.Chilling injury is reduced in cucumber and rice seedlings and in tomato pericarp discs by heat-shocks applied after chilling.Postharvest Biol.Technol.21,169–177.Saltveit,M.E.,2005.Influence of heat shocks on the kinetics of chilling-induced ion leakage from tomato pericarp discs.Postharvest Biol.Technol.36,87–92. Saltveit,M.E.,Morris,L.L.,1990.Overview on chilling injury of horticultural crops.In:Wang,C.Y.(Ed.),Chilling Injury of Horticultural Crops.CRC Press,Boca Raton, pp.3–15.Sanchez-Ballesta,M.T.,Zacarias,L.,Granell,A.,Lafuente,M.T.,2000.Accumulation of PAL transcript and PAL activity as affected by heat-conditioning and low-temperature storage and its relation to chilling sensitivity in mandarin fruits.J.Agric.Food Chem.48,2726–2731.Sapitnitskaya,M.,Maul,P.,McCollum,G.T.,Guy,C.L.,Weiss,B.,Samach,A.,Porat,R., 2006.Postharvest heat and conditioning treatments activate different molec-ular responses and reduce chilling injuries in grapefruit.J.Exp.Bot.57, 2943–2953.Schirra,M.,Mulas,M.,Fadda,A.,Cauli,E.,2004.Cold quarantine responses of blood oranges to postharvest hot water and hot air treatments.Postharvest Biol.Tech-nol.31,191–200.Shigeoka,S.,Ishikawa,T.,Tamoi,M.,Miyagawa,Y.,Takeda,T.,Yabuta,Y.,Yoshimura, K.,2002.Regulation and function of ascorbate peroxidase isoenzymes.J.Exp.Bot.53,1305–1319.Ummarat,N.,Matsumoto,T.K.,Wall,M.M.,Seraypheap,K.,2011.Changes in antiox-idants and fruit quality in hot water-treated‘Hom Thong’banana fruit during storage.Sci.Hortic.130,801–807.Vicente,A.R.,Martínez,G.A.,Chaves,A.R.,Civello,P.M.,2006.Effect of heat treat-ment on strawberry fruit damage and oxidative metabolism during storage.Postharvest Biol.Technol.40,116–122.Wahid,A.,Gelani,S.,Ashraf,M.,Foolad,M.R.,2007.Heat tolerance in plants.An overview.Environ.Exp.Bot.61,199–223.Wang,H.,Zhang,Z.,Xu,L.,Huang,X.,Pang,X.,2012.The effect of delay between heat treatment and cold storage on alleviation of chilling injury in banana fruit.J.Sci.Food Agric.92,2624–2629.Wise,R.R.,1995.Chilling-enhanced photooxidation:the production,action and study of reactive oxygen species produced during chilling in the light.Photo-synth.Res.45,79–97.Woolf,A.B.,Lay-Yee,M.,1997.Pretreatments at38◦C of‘Hass’avocado confer ther-motolerance to50◦C hot water treatments.Hortic.Sci.32,705–708.Zhang,J.,Huang,W.,Pan,Q.,Liu,Y.,2005.Improvement of chilling tolerance and accumulation of heat shock proteins in grape berries(Vitis vinifera cv.Jingxiu) by heat pretreatment.Postharvest Biol.Technol.38,80–90.。