Eur Food Res Technol (2008) 227:1599–1611

DOI 10.1007/s00217-008-0884-0

ORIGINAL PAPER

EVects of a pretreatment with nitric oxide on peach (Prunus persica L.) storage at room temperature

Francisco B. Flores · Paloma Sánchez-Bel · Mónika Valdenegro · Félix Romojaro · María Concepción Martínez-Madrid · María Isabel Egea

Received: 17 March 2008 / Revised: 11 April 2008 / Accepted: 21 April 2008 / Published online: 10 May 2008© Springer-Verlag 2008

Abstract Peach is characterised by a rapid senescenceassociated with a high production of autocatalytic ethyleneat the beginning of ripening, a fact which reduces markedlyits postharvest shelf-life. The application of antisenescentcompounds after harvesting has been assayed to solve thisproblem. One of the newest and more promising com-pounds is the free radical gas nitric oxide (NO). In thiswork, peaches of cv. ‘Rojo Rito’ were treated with5 �L L¡1 of NO for 4 h, at 20 °C, and then stored at thesame temperature for 14 days. Untreated fruits stored underthe same conditions were used as control fruits in the exper-iment. Key physiological parameters of senescence (ethyl-ene production and respiratory rate) and quality parameters(Wrmness, titrable acidity, total soluble solids and colour)were analysed. A particular emphasis was placed on theanalysis of the oxidative status and the antioxidant capacityduring storage and as a response to the NO treatment. Theethylene production and respiratory rate of fruits treatedwith NO were lower than those of control fruits. Treatedfruits underwent a lesser loss of Wrmness during storage.The degree of disintegration of cell membranes, assessed asthe percentage of electrolyte leakage, was also lower in

fruits treated with NO. NO did not seem to aVect lipid per-oxidation or LOX activity, but it did aVect PPO activity.The treatment with NO stimulated POX activity and, espe-cially, SOD and CAT activities. It seems that total carote-noids and free ASC were not inXuenced by thepretreatment; however, the oxidised form of ASC, DHA,showed a slight increase. NO seemed to have a beneWcialeVect on the oxidation equilibrium and the antioxidantcapacity of peach fruit. A delay in the initiation of thesenescence of fruits treated with NO, that extended thepostharvest shelf-life, was observed.

Keywords Prunus persica · Senescence · Ethylene · Nitric oxide · Oxidative stress · Antioxidant capacity · Postharvest

Introduction

Peach is a climacteric fruit, the ripening of which is associ-ated with the autocatalytic production of ethylene [1, 2].The postharvest shelf-life of peach fruits is limited becauseof their rapid senescence, once ripening has been triggered.This implies a serious problem for the marketing of thisfruit [3, 4].

Disorders in cell membrane integrity, that cause the lossof semi-permeability, are one of the initial and main symp-toms of senescence. Although the role of ethylene as aninductor of the loss of this integrity and, therefore, of senes-cence, has not yet been proven, the hypothesis that ethylenecan accelerate the process, probably through the interven-tion of Ca2+ as a secondary messenger, has been proposedalready [5, 6].

During fruit ripening and senescence, cell homeostasisand a disproportionate increase in reactive oxygen species

F. B. Flores (&) · P. Sánchez-Bel · M. Valdenegro · F. Romojaro · M. I. EgeaDpto. de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, Apartado de Correos 164, 30100 Espinardo, Murcia, Spaine-mail: cta018@cebas.csic.es

M. C. Martínez-MadridDpto. de Agroquímica y Medio Ambiente, Escuela Politécnica Superior, Universidad Miguel Hernández, Ctra. Beniel Km. 3.2, 03312 Orihuela, Alicante, Spain

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(ROS) production, causing oxidative stress, take place [7].The signiWcance of this oxidative stress for the fruit qualityand postharvest shelf-life, and the inXuence of ripening ingeneral and of ethylene in particular, has been discussedwidely [8]. ROS are partially reduced species of molecularoxygen which can oxidise uncontrollably the diVerent cellcomponents and lead to the oxidative destruction of the cell[7, 9]. ROS cause the peroxidation of the membrane lipids,leading to cell membrane alterations [9]. The increase inROS production during ripening and senescence is causedby the loss of integrity of cell membranes, which seriouslyaVects two key cell organelles: the mitochondria and chlo-roplast.

The alteration of cell membrane integrity, the primaryresponse of senescence, disrupts the composition and func-tionality of mitochondria and chloroplast, altering the pro-tein complexes within the organelles’ membranes. Thisnegatively aVects the functionality of such complexes andstimulates an abnormal ROS production, causing an oxida-tive stress that can be considered the secondary response ofsenescence [7, 10]. This has been observed clearly in mel-ons, where ripening is characterised, Wrst, by the disintegra-tion of cell membranes [11] and, second, by an associatedoxidative stress [12].

In order to cope with this oxidative stress, plants havediVerent enzymatic and non-enzymatic antioxidant systems[13]. Some studies show that, due to the accumulation ofROS during ripening, these defence systems enter intoaction in order to neutralise them and, therefore, they play akey role in this physiological process [14, 15]. Among theenzymatic systems that remove ROS, the activities of cata-lases (CAT, E.C. 1.11.1.6), peroxidases (POX, E.C.1.11.1.7) and superoxide dismutases (SOD, E.C. 1.15.1.1)stand out. The non-enzymatic systems are usually smallmolecules that are able to reduce free radicals by oxidisingthemselves, the oxidised form still being stable. Amongthem, we can highlight ascorbic acid, carotenoids and phe-nolic compounds [16].

Oxidative stress is also induced by a rise in lipid peroxida-tion, a process in which ROS are generated and in whichlipoxygenase (LOX, E.C. 1.13.11.12) is the key pro-oxidisingenzyme [6, 17]. In apples, a positive correlation between LOXactivity and ethylene biosynthesis has been observed [18]. Intomato, the genetic expression of diVerent LOX isoformsseems to be regulated by ethylene, but also by developmentalfactors [19, 20]. Also, it has been observed that ethyleneinduces the activity of some antioxidant enzymes in tomato,like CAT and SOD [21]. This apparently paradoxical actionof ethylene can be interpreted as an attempt by the cells toconfront the loss of oxidative equilibrium they suVer as aresult of the increase in ROS during ripening and senescence[14]. The balance between ROS production and their removal

by the antioxidant defence systems determines the speed ofripening and senescence processes and, therefore, the exten-sion of the fruit shelf-life [22].

Another process that becomes apparent during peachsenescence is internal browning due to the action of theenzyme polyphenol oxidase (PPO, E.C. 1.14.18.1), whichcatalyses the oxidation of phenolic compounds [23]. Inexperiments to evaluate the storage of avocado treated withethylene and its antagonist 1-methylcyclopropene (1-MCP), the possibility that the ripening hormone couldinduce PPO activity has been considered [24, 25].

Considering all these facts, one strategy to try to delaythe senescence of climacteric fruits like peach during theirpostharvest life could be pretreatments with an antagonistof the biosynthesis or action of ethylene, which would playthe role of an antisenescent agent [26]. Treatments offruits and vegetables using diVerent compounds with theseproperties have been tested. One of the most recentlyassessed, with the most promising results in diVerent horti-cultural products, is the free radical gas nitric oxide (NO)[27]. NO takes part in the regulation of diVerent physio-logical processes in plants, from development and hor-mone signalling to regulation of stomatal closure [28–30].Treatment with NO of climacteric fruits, like kiwi, andnon-climacteric fruits sensitive to ethylene action, likestrawberry, considerably extends their postharvest life anddelays their senescence [31, 32]. In other plant organssuVering autocatalytic production of ethylene, like carna-tion Xowers, it has been observed that treatments with thissubstance, as a gas or by means of a compound thatreleases the active agent, cause an extension of the post-harvest life of the cut Xower [33]. Leshem [34] put for-ward the idea that NO inXuences the ripening andsenescence of diVerent plant products by inducing inhibi-tion of ethylene production. We have only found two pub-lications where pretreatments of peaches with NO havebeen described, both for variety Feichen. One focusedmainly on the eVects of this free radical gas on ethylenebiosynthesis [35] and the other on the composition of cellmembrane fatty acids [36]. We have not found an analysisof the possible eVects of exogenous NO on the oxidativeequilibrium and antioxidant capacity of fruits treated withthis compound.

Considering all this background, an experiment involv-ing the pretreatment of peach fruits with NO immediatelyafter harvest has been designed and carried out in order toassess whether this compound induces a delay of senes-cence and extends the postharvest life of the fruits duringstorage at room temperature (20 °C), without negativeeVects on the Wnal quality. A special emphasis was placedon the eVects of this antisenescent agent on the oxidationequilibrium and antioxidant capacity of peach.

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Materials and methods

Plant material and experimental design

Peaches (Prunus persica L.), variety “Rojo Rito”, from anorchard in Cieza (Murcia, Spain), were harvested at thecommercial ripening state and transported to the laboratory.They were separated into two batches of 96 fruits each(aprox. 15 kg), and a sample of 24 fruits was analysedimmediately and processed, in order to represent day 0 ofthe experiment.

One of the batches was stored in a thermostatised cham-ber at (20 § 1 °C), while the other was placed in a 32-L,airtight chamber with a gas inlet and outlet ducts, where thetreatment with NO, in the absence of oxygen at 20 °C, wascarried out. The need to apply the free radical gas com-pound in an anaerobic atmosphere is due to the rapid oxida-tion it suVers in the presence of oxygen [32]. The containerwith the fruits in it was sealed and N2 injected for 10 min,to sweep the air inside. Then, the gas outlet duct was closedand the container was Wlled with 5 �L L¡1 NO. The dosingwas carried out by introducing the NO from a bottle ofcompressed NO/N2 gas, having 5 �L L¡1 NO and beingequipped with a manometer and a Xowmeter. In order toguarantee the Wlling of the whole volume of the containerwith NO, gas from the bottle was injected for 7 min at aXow rate of 5 L min¡1. Fruits were kept in the airtight con-tainer with the NO for 4 h and, afterwards, they were trans-ferred to the chamber at 20 °C for their storage. At the endof the pretreatment and before opening the container toremove the fruits, the gas outlet duct was opened andanother injection with N2 was carried out, for 10 min, toremove every trace of NO.

The storage assay was carried out over 14 days (at20 § 1 °C), and samples were taken after 3, 6, 10 and14 days, apart from the sample of day 0. One sample, fromcontrol untreated fruits, from fruits treated with NO andfrom the fruits taken on day 0, included three subsamples of8 fruits each, that is, a total of 24 fruits. The analyticaldeterminations were carried out in these subsamples, induplicate for each sub-sample.

Except for the determinations of ethylene production,respiratory rate, electrolyte leakage and quality parameters,that were carried out using fresh material on the day ofsampling, the rest of the analytical determinations were car-ried out using frozen material cut into pieces, which wereimmediately frozen in liquid N2 and stored at ¡70 °C.

Analytical determinations

The physiological determinations performed were ethyleneproduction, by GC-FID, according to Martínez-Madridet al. [37], and respiratory rate measured as CO2 produc-

tion, by GC-TCD, according to Serrano et al. [38]. Thedeterminations of ethylene and CO2 production were car-ried out by placing each subsample of eight fruits in a her-metically sealed container, of a known volume andequipped with a silicone septum. After 1 h, 1 and 0.5 mL ofthe internal atmosphere of the container were extractedwith a syringe for the determination of ethylene and CO2,respectively. The ethylene was quantiWed in a Hewlett–Packard HP gas chromatograph (model 5890), Wtted with aXame ionisation detector and a stainless-steel column(3 m £ 3.2 mm) diameter, packed with 80/100 mesh acti-vated alumina. The Xow rates of carrier gas (nitrogen),hydrogen and air were 32, 26 and 400 mL min¡1, respec-tively, and the temperatures of the column, injector anddetector were 70, 150 and 175 °C, respectively. The quanti-Wcation was carried out by calibration, point-by-point, withan external standard, and the results were expressed asnL g¡1 h¡1. The CO2 was analysed in a Shimadzu GC-14Agas chromatograph Wtted with a thermal conductivity detec-tor. For the chromatographic separation, a TEKNOK-ROMA molecular-sieve column of 5 Å, 2-m length and3.2-mm diameter was used. The temperature of the columnwas 55 °C and that of the detector and injector was 115 °C.The pressure of the carrier gas (He) was 4 kg cm¡2. ThequantiWcation was carried out by calibration, point-by-point, with an external standard, and the results wereexpressed as mg CO2 kg¡1 h¡1.

The following quality parameters were determined: col-our, total soluble solids (TSS) content and titratable acidity,according to methods described by Martínez-Madrid et al.[39]. Results were expressed as parameter ‘a’ on the colourspace of the Commission Internationale de l’Eclairage CIELab (L*a*b*) system, ºBrix and g/100 g FW of malic acid,respectively. The ripening index was estimated as the quo-tient between TSS and titratable acidity. The pulp Wrmnesswas determined by penetrometry, according to the methoddescribed by Flores et al. [40], and the results wereexpressed in Newton (N).

In order to assess the oxidation status, cell membraneintegrity, lipid peroxidation index and activities of the pro-oxidising enzymes LOX and PPO were determined. Cellmembranes integrity was assessed by estimation of theirsemipermeability loss, measuring the percentage of elec-trolyte leakage according to the method described by Ben-Amor et al. [41]. The lipid peroxidation index was mea-sured as the production of malonyl-dialdehyde (MDA),using the thiobarbituric acid-reactive substances (TBARS)assay [42], with the modiWcations of Martínez-Solanoet al. [43], but introducing the following changes: 2.0 g offrozen material were taken, 4.0 mL of 0.1 g/100 mL TCAwere added for homogenisation and the Wrst centrifugationlasted 30 min. The results were expressed as nmol MDAg¡1 h¡1.

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The enzymatic extraction was carried out by homogenis-ing 3.0 g of frozen plant material in 6.0 mL of extractionbuVer, using a Polytron. The extraction buVer contained50 mmol L¡1 potassium phosphate pH 7.8, 1 mmol L¡1

ethylenediaminotetra-acetic acid (EDTA), 0.2 g/100 mLTriton X-100, 2 g/100 mL poly(vinylpyrrolidone) (PVP),5 mmol L¡1 L-cysteine and 1 mmol L¡1 phenylmethyl-sul-fonyl Xuoride (PMSF). The homogenisation was carried outin an ice-bath in order to maintain the temperature at 4 °C.The homogenate was centrifuged at 10,000 g for 15 min, at4 °C, and the supernatant was Wltered through nylon Wlters(Miracloth). This Wltered extract was used for the determi-nation of the LOX and PPO activities, and also of the activ-ities of the antioxidant enzymes CAT, POX and SOD, aswill be explained later. For the calculations of all speciWcenzymatic activities, protein concentration in the extractswas determined with the DC Protein kit (Bio-Rad), basedon the method of Lowry et al. [44] and using bovine serumalbumin as the standard.

LOX activity was determined as described in Martínez-Solano et al. [43], with the methodology of Minguez-Mos-quera et al. [45]. To a reaction mix containing 2.0 mL of200 mmol L¡1 potassium phosphate buVer (pH 6.5) and0.04 mL of enzymatic extract was added 0.04 mL of70 mmol L¡1 linoleic acid to initiate the reaction. This lastreagent was prepared according to Minguez-Mosqueraet al. [45]. LOX activity was determined by measuring theabsorbance at 234 nm wavelength, for 60 s, at 30 °C. TheLOX speciWc activity was expressed in SI units of catalyticactivity: nkat mg¡1 of protein. The molar extinction coeY-cient value (�M) employed in the calculation of this activitywas 25,000 M¡1 cm¡1, the �M of hydroperoxi-octadecadie-noic (HPOD) acid—the product of the peroxidation of lino-leic acid under these conditions.

PPO activity was determined according to Chazarraet al. [46]. To a reaction mix containing 2.1 mL of50 mmol L¡1 potassium phosphate buVer (pH 6.5) and0.1 mL of extract was added 0.3 mL of 50 mmol L¡1 chlor-ogenic acid to start the reaction. PPO activity was deter-mined by measuring the absorbance at 400 nm wavelength,for 60 s, at 25 °C. The speciWc enzyme activity wasexpressed in SI units of catalytic activity: pkat mg¡1 of pro-tein. The molar extinction coeYcient value (�M) employedin the calculation of this activity was 1,018 M¡1 cm¡1, the�M of the o-benzoquinone product of the oxidation of chlor-ogenic acid under these conditions.

The antioxidant capacity of the peaches was evaluatedby determination of the contents of the antioxidant com-pounds ascorbic (ASC) and dehydroascorbic (DHA) acids,the total carotenoids content and the activities of the anti-oxidant enzymes CAT, POX and SOD. ASC and DHAwere determined according to Wimalasiri and Wills [47]with the modiWcations described by Egea et al. [48], but

taking 3.0 g of frozen plant material for the extraction. Theresults were expressed as mg/100 g FW. Total carotenoidscontent was determined according to Romojaro et al. [49].Pigments extraction was carried out by homogenisation of5.0 g of frozen material with 25 mL acetone–methanol(1:1) solution. The homogenate was Wltrated through glasswool, followed by transfer to 50 mL ethyl ether and sapon-iWcation with 20 mL of a methanolic solution of KOH(20 mg L¡1), and 30 mL NaCl 10 g/100 mL were added to theextract in each one of the last two extraction steps, transferto ethyl ether and saponiWcation. The aqueous solution iseliminated in both the steps and the Wnal ethyl ether extractwas Wltrated through glass wool. Total carotenoids in theethyl ether extract were spectrophotometrically measuredat 450 nm and results were expressed as mg �-carotene/100 g FW.

The extraction for the determination of the antioxidantenzymes activities was carried out as previously described.CAT activity was analysed according to Aebi [50] in a mixcontaining 2.0 mL of 50 mmol L¡1 potassium phosphatebuVer (pH 7.0) and 0.1 mL of enzymatic extract. The reac-tion was initiated by adding 0.5 mL of 40 mmol L¡1 H2O2.The enzymatic activity was determined by measuring thedecrease in the absorbance at 240 nm wavelength, for 60 s at30 °C, due to the H2O2 consumption. Values of activity wereexpressed in SI units of catalytic activity: pkat mg¡1 of pro-tein. The molar extinction coeYcient value (�M) of H2O2

(39.58 mM¡1 cm¡1) was used to calculate this activity. POXactivity was analysed using guaiacol as substrate, accordingto the methodology of Field and Hall [51] but with modiWca-tions. The reaction mix contained 1.5 mL of 50 mmol L¡1

potassium phosphate buVer (pH 7.0) with 20 mmol L¡1 ofguaiacol and 0.1 mL of extract; 0.5 mL of 40 mmol L¡1

H2O2 was added to start the reaction. POX activity was deter-mined after an incubation period of 5 min, measuring theabsorbance increase at 470 nm wavelength, during 120 s at30 °C, due to the formation of a chromogen polymer that isthe product of the enzymatic reaction. The speciWc activitywas expressed in SI units of catalytic activity: pkat mg¡1 ofprotein. A molar extinction coeYcient (�M) of26.60 M¡1 cm¡1 was used, which corresponds to guaiacol(2-methoxyphenol), the proton donor for the reaction. SODactivity was analysed indirectly, as the inhibition by SOD ofcytochrome c reduction. This reduction was determinedspectrophotometrically at 550 nm using xanthine/xanthineoxidase as the source of superoxide radical [52]. The test wascarried out at 25 °C in a 3.0-mL cuvette containing50 mmol L¡1 potassium phosphate buVer (pH 7.8),0.1 mmol L¡1 EDTA, 1 mmol L¡1 cytochrome c,1 mmol L¡1 xanthine and an aliquot of enzymatic extract.The reaction was initiated by adding xanthine oxidase to themix. One SOD unit (U) was deWned as the quantity ofenzyme that produces a 50% inhibition of the reduction of

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cytochrome c. Values of the speciWc enzymatic activity wereexpressed in milliunits of SOD activity, mU mg¡1 of protein.

The experimental results are shown in graphs and tablesas the mean § standard error (SE) of determinations madefor each sample. A statistical analysis of the Student’s t testof comparison of means considering a conWdence intervalof 95 % (P < 0.05) was applied for the factor sample treat-ment, using the statistical package SPSS (version 14.0), toevaluate the signiWcance of the means obtained from thetwo kinds of samples: control peaches and NO-treatedpeaches.

Results

Physiological parameters: ethylene production and respiratory rate

For both the ethylene production and respiratory rate,peaches treated with NO showed signiWcantly lower levels(P < 0.05) than untreated control fruits, although the evolu-tion of both parameters, until day 10 for ethylene and day14 for respiration, was similar in treated and untreatedfruits (Fig. 1a, b). Ethylene production increased up to amaximum after 10 days at 20 °C (78.48 and67.8 nL g¡1 h¡1 for control and treated fruits, respectively)and then decreased in both the cases, although the decreasewas sharper in fruits treated with NO (72.42 and35.57 nL g¡1 h¡1 after 14 days for control and treatedfruits, respectively) (Fig. 1a).

Regarding the respiratory rate, there was a slightdecrease after 3 days of storage (from 96.76 mgCO2 kg¡1 h¡1 on day 0 to 86.72 and 80.72 mgCO2 kg¡1 h¡1 on day 3 for control and treated fruits,respectively); then, a slight increase in both cases wasobserved until day 10 and, Wnally, a sharp rise was detectedin the last sampling (107.04 and 105.42 mg CO2 kg¡1 h¡1

after 14 days for control and treated fruits, respectively).Between days 3 and 10 of storage, a lower respiratory ratewas observed in treated fruits and signiWcant diVerences(P < 0.05) existed in the samplings of days 3 and 6 of stor-age (Fig. 1b).

Quality parameters

Fruit softening was signiWcantly greater (P < 0.05) in con-trol fruits from day 6, at 20 °C, although the evolution wassimilar in NO-treated and untreated fruits. A decline duringthe whole period of storage was detected (from 40.67 N onday 0 to 10.01 and 13.67 N on day 14 for control and NO-treated fruits, respectively) (Fig. 2).

The NO treatment did not seem to aVect TSS content,titratable acidity or colour evolution, since no signiWcant

diVerences were observed between the control and treatedfruits except on day 3 of storage, when treated fruitsshowed signiWcantly (P < 0.05) higher values of TSS con-tent (Table 1). The TSS content gradually increased fromday 6 of storage, while titratable acidity graduallydecreased from the beginning of the experiment, showing a

Fig. 1 Evolution of ethylene production (a) and respiratory rate (b) inpeach fruits treated with 5 �L L¡1 NO (unWlled circle) and in untreatedcontrol fruits (Wlled circle) during storage at 20 °C. Data represent themeans § SE (vertical bars) of determinations made in duplicate foreach of three replicates of each sample. *SigniWcant diVerences existedbetween NO-treated and untreated fruits on the speciWc day of sam-pling (P < 0.05)

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sharper decrease at the end of storage, between days 10 and14 at 20 °C. No signiWcant diVerences existed regarding thematurity index of NO-treated and untreated fruits. A risewas observed, especially, at the end of the storage period(Table 1).

No signiWcant diVerences were found regarding colourevolution between the two kinds of fruit, control andtreated with NO. Parameters ‘L’ and ‘b’ remained practi-cally constant throughout the whole storage period, andonly parameter ‘a’ showed a slight increase both at thebeginning and at the end of the experiment, in both thesamples (Table 1).

Fruit oxidation status: electrolyte leakage, lipid peroxida-tion index, LOX and PPO activities

The percentage of electrolyte leakage, and, therefore, theloss of semipermeability of the cell membranes, increasedduring storage (from 63.8% on day 0 to 87.97 and 84.71%after 14 days for control and treated fruits, respectively),and it was higher for untreated fruits until the end of thestorage, when diVerences in % electrolyte leakage betweenthe two kinds of sample were no longer signiWcant(P < 0.05) (Fig. 3).

The evolution of the lipid peroxidation index, deter-mined as the rate of MDA production, was similar in boththe samples, treated and untreated fruits, and there were nosigniWcant diVerences between them. A gradual increase ofMDA production was observed from the beginning of stor-age until day 10, at 20 °C, and, later on, there was a sharpupsurge between days 10 and 14 of conservation (Table 2).

Regarding LOX activity, an increase from the beginningof the conservation until day 6 at 20 °C was observed(16.25 and 16.62 nkat mg¡1 protein, respectively, for con-trol and NO-treated fruits). Then, the activities were stabi-lised at around 14–15 nkat mg¡1 protein. No signiWcantdiVerences were found between the untreated and NO-treated fruits (Table 2).

PPO activity was slightly lower in the case of peachestreated with NO, for which it decreased from the beginningof storage, while in control fruits it remained steady. Sig-niWcant diVerences existed between NO-treated and controlfruits only at the end of the storage period, on days 10 and14 at 20 °C (P < 0.05) (Fig. 4).

Fruit antioxidant capacity: contents of ASC, DHA and total carotenoids, and activities of the antioxidant enzymes SOD, CAT and POX

No signiWcant diVerences were found between fruits treatedwith NO and control fruits regarding ASC content, which

Fig. 2 Evolution of fruit Wrmness for peach fruits treated with5 �L L¡1 NO (unWlled circle) and untreated control fruits (Wlled circle)during storage at 20 °C. Data represent the means § SE (vertical bars)of determinations made in duplicate for each of three replicates of eachsample. *SigniWcant diVerences existed between NO-treated and un-treated fruits on the speciWc day of sampling (P < 0.05)

Table 1 Evolution of titratable acidity, expressed as g/100 g of malic acid, total soluble solids content (TSS), expressed in ºBrix, maturity indexand colour, expressed as parameter ‘a’, of peach fruits treated with 5 �L L¡1 NO and untreated control fruits, during storage at 20 °C

*SigniWcant diVerences existed between NO-treated and untreated fruits on the speciWc day of sampling (P < 0.05). Values represent themeans § SE of determinations made in duplicate for three repetitions of each sample

Days at 20 °C

Titratable acidity in control fruits

Titratable acidity in treated fruits

TSS content in control fruits

TSS content in treated fruits

Ripening index in control fruits

Ripening index in treated fruits

Colour ‘a’ parameter in control fruits

Colour ‘a’ parameter in treated fruits

0 0.55 § 0.02 14.85 § 0.16 26.92 § 0.86 7.64 § 1.59

3 0.51 § 0.01 0.52 § 0.01 13.60 § 0.06* 14.10 § 0.40* 26.51 § 0.79 27.25 § 0.45 10.13 § 1.18 11.12 § 0.51

6 0.45 § 0.02 0.44 § 0.01 13.60 § 0.23 13.67 § 0.52 30.47 § 1.18 31.35 § 1.27 11.25 § 0.13 11.36 § 0.87

10 0.45 § 0.01 0.47 § 0.01 15.23 § 0.27 15.50 § 0.57 34.09 § 1.54 32.72 § 2.13 11.99 § 0.59 11.28 § 0.42

14 0.35 § 0.01 0.39 § 0.01 15.60 § 0.40 16.27 § 0.74 44.40 § 3.20 41.41 § 2.43 13.99 § 0.81 13.54 § 0.77

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showed a sharp reduction from 10.51 mg/100 g FW on day0 to 4–5 mg/100 g FW after 3 days of storage at 20 °C.Subsequently, levels remained constant around these lattervalues until the end of the conservation (Table 3).

However, diVerences occurred between the two kinds offruits regarding DHA content, which was higher in peachestreated with NO—signiWcantly so after three and 6 days ofstorage at 20 °C (P < 0.05). The content increased in bothsamples, but the rise was higher in NO-treated fruits,although by the end of the conservation it was similar inboth types of peach sample (Table 3).

Regarding the evolution of the total carotenoids content,no signiWcant diVerences were found between the two kinds

of sample; the levels of these pigments gradually increasedfrom the beginning of the storage (Table 3).

SOD activity increased gradually and similarly in bothcontrol and treated fruits until day 6 of storage at 20 °C.From this point, this activity showed a sharp increase, espe-cially in treated fruits, and the levels after 10 and 14 days ofstorage at room temperature in these samples were signiW-cantly higher than for control fruits (P < 0.05). At the endof the experiment, SOD activity of fruits treated with NOwas 36.27 mU mg¡1 protein and that of control fruits was25.42 mU mg¡1 protein (Fig. 5).

A very signiWcant increase (P < 0.05) of CAT activity inpeaches treated with NO was observed in comparison withcontrol fruits after 6 days of storage at 20 °C (Fig.6). Themost marked increase of this activity took place betweendays 3 and 6 of storage at 20 °C, when it increased from11.25 to 37.82 pkat mg¡1 protein in NO-treated fruits, andfrom 6.02 to 19.78 pkat mg¡1 protein in control, untreatedfruits (Fig. 6).

The increase in POX activity was gradual at the beginningof the experiment but it became intense at the end, when,from 34.24 pkat mg¡1 protein on day 10, it reached 50.03pkat·mg¡1 protein on day 14 in treated fruits, while increas-ing from 27.5 to 58.26 pkat mg¡1 protein for untreated fruitsover the same period of time (Fig. 7). POX activity washigher in fruits treated with NO except for the last samplingon day 14, and these diVerences were signiWcant only ondays 3 and 14 of storage at 20 °C (P < 0.05) (Fig. 7).

Discussion

A classical strategy for extending the postharvest shelf-lifeof fruit is refrigerated storage, but the application of lowtemperatures for peach is limited since it is a fruit sensitiveto chilling injury [53]. An alternative is to block the ethyl-ene synthesis and/or action, which could be fulWlled by theapplication of compounds that are antagonists of the bio-synthesis and/or action of this plant hormone involved inclimacteric fruit ripening and senescence [54, 55].

Table 2 Production of malonyl-dialdehyde (MDA) and LOX activity, expressed as nmol g¡1 h¡1 and nkat mg¡1 of protein, respectively, of peachfruits treated with 5 �L L¡1 NO and untreated control fruits, during storage at 20 °C

Values represent the means § SE of determinations made in duplicate for three repetitions of each sample

Days at 20 ºC

MDA production in control fruits

MDA production in treated fruits

LOX activity in control fruits

LOX activity in treated fruits

0 1647 § 0.41 11.82 § 0.25

3 18.61 § 0.30 19.09 § 0.33 13.89 § 0.16 13.89 § 0.27

6 23.77 § 0.84 22.59 § 0.51 16.25 § 0.12 16.62 § 0.18

10 21.52 § 0.81 21.61 § 0.36 14.57 § 0.40 14.68 § 0.72

14 30.32 § 0.79 30.61 § 0.59 15.41 § 0.36 15.63 § 0.16

Fig. 3 Evolution of electrolyte leakage in peach fruits treated with5 �L L¡1 NO (unWlled circle) and untreated control fruits (Wlled circle)during storage at 20 °C. Data represent the means § SE (vertical bars)of determinations made in duplicate for each of three replicates of eachsample. *SigniWcant diVerences existed between NO-treated and un-treated fruits on the speciWc day of sampling (P < 0.05)

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1606 Eur Food Res Technol (2008) 227:1599–1611

In the particular case of peaches, diVerent storage tech-nologies have been assayed, which imply pretreatmentswith ethylene antagonists that can be introduced in the cir-cuit of commercial handling of horticultural products, like1-MCP and acetaldehyde (AA); that is, products with anti-senescent properties [56, 57]. The free radical gas nitricoxide, NO, has antisenescent properties similar to those of1-MCP and AA, as has been observed in tests with diVerentfruits, vegetables and Xowers [31].

Pretreatment of peaches with 5 �L L¡1 NO for 4 h, at20 °C, immediately after harvesting induced signiWcantchanges in certain parameters characteristic of the physiol-ogy of ripening and senescence, like the decreases of respi-

ratory rate and ethylene production (Fig. 1a, b). Leshemet al. [31] suggested that the antisenescent action of NO onplant products takes place via the inhibition of ethylene bio-synthesis. NO is also produced endogenously by plants, andit has been shown that there is an inverse correlationbetween the production of this free radical and that of ethyl-ene during the development and ripening of strawberriesand avocados [34, 58]. The inhibitory eVect of NO on eth-ylene biosynthesis seems to occur through the inhibition ofACC oxidase (ACO, EC 1.14.17.4) activity and it does notseem to aVect ACC synthase (ACS, E.C. 4.4.1.14) activity,as has been observed in peach [35]. Tierney et al. [59],using spectroscopic techniques of electron nuclear doubleresonance and electronic paramagnetic resonance, observedthat ACC oxidase can bind NO and form a binary complexto which the natural substrate of the enzyme, the precursorof ethylene 1-aminocyclopropane-1-carboxylic acid(ACC), also binds to form a stable, ternary complex unableto oxidise ACC and yield ethylene.

The apparent antisenescent action of NO is also reXectedin the reduced respiratory rate of peaches treated with NOand stored for 3, 6 or 10 days at 20 °C. One of the classicalsymptoms of senescence is an increase in the respiration ofthe aVected tissue and organ, as we and other authors [60]have observed during the postharvest life of peaches atroom temperature. The results on the evolution of thisparameter seem to indicate that this antisenescent action islost as the storage period extends, as can be observed infruits after 10 and, especially, 14 days at 20 °C (Fig. 1b).

An essential quality parameter in peach ripening is Wrm-ness loss. In this matter, the action of NO seems to be bene-Wcial since the decrease of this parameter was somewhatreduced (Fig. 2). This eVect of NO has been observed alsoby Zhu et al. [35] and Zhu and Zhou [36] in peaches previ-ously treated with 5 or 10 �L L¡1 NO and then stored at 5or 25 °C, but not in those fruits treated with 15 �L L¡1—which suVered an even higher loss of Wrmness than controlfruits. The advantage of NO over the other, previouslymentioned ethylene antagonists, 1-MCP and AA, is theshorter period of treatment needed to achieve signiWcant

Fig. 4 Evolution of PPO activity in peach fruits treated with 5 �L L¡1

NO (unWlled circle) and untreated control fruits (Wlled circle) duringstorage at 20 °C. Data represent the means § SE (vertical bars) ofdeterminations made in duplicate for each of three replicates of eachsample. *SigniWcant diVerences existed between NO-treated and un-treated fruits on the speciWc day of sampling (P < 0.05)

Table 3 Evolution of the ASC and DHA contents, expressed as mg/100 g FW, and of the total carotenoids content, expressed as mg ß-carotene/100 g FW, for peach fruits treated with 5 �L L¡1 NO and untreated control fruits during storage at 20 °C

*SigniWcant diVerences existed between NO-treated and untreated fruits on the speciWc day of sampling (P < 0.05). Values represent themeans § SE of determinations made in duplicate for three repetitions of each sample

Days at 20 °C

ASC content in control fruits

ASC content in treated fruits

DHA content in control fruits

DHA content in treated fruits

Total carotenoids in control fruits

Total carotenoids in treated fruits

0 10.51 § 0.79 4.42 § 0.63 2.79 § 0.01

3 4.36 § 0.68 5.03 § 0.47 4.39 § 0.08* 6.77 § 0.39* 3.26 § 0.12 3.48 § 0.07

6 5.16 § 0.84 4.53 § 1.00 5.21 § 0.14* 6.62 § 0.13* 3.48 § 0.11 3.51 § 0.01

10 5.29 § 0.44 4.72 § 0.60 6.24 § 0.80 8.29 § 0.70 3.87 § 0.17 4.04 § 0.10

14 3.55 § 0.16 5.11 § 0.70 8.02 § 0.94 7.82 § 0.60 4.44 § 0.08 4.48 § 0.01

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eVects, like the reduction of Wrmness loss. In the case ofpeach, and with a dose of 5 �L L¡1 1-MCP, the optimumperiod of treatment for inhibition of fruit softening is 20 h[61]. Liu et al. [62] found that a 24-h treatment was neces-sary, although they showed that the use of doses higherthan 0.4 �L L¡1 did not aVect the texture loss. An inhibitoryeVect of AA on peach softening was found when a 24-htreatment was carried out with 2000–3000 �L L¡1 of thiscompound [63]. In our case, we have observed a signiWcantinhibition of the Wrmness loss of the fruits after 6 days ofstorage with a concentration of 5 �L L¡1 NO applied foronly 4 h (Fig. 2). Zhu et al. [35] and Zhu and Zhou [36]also observed inhibition of Wrmness loss in peaches treatedwith 5 or 10 �L L¡1 NO for only 3 h.

Liguori et al. [61] did not observe a correlation, withregard to the eVects of 1-MCP in peach, between Wrmnessloss and ethylene production, because the inhibition of fruitsoftening was higher at 20 °C than at 2 °C, and a reductionof ethylene production was only found when treated fruitswere stored at 2 °C, but not at 20 °C. This result is surpris-ing if we take into account that Wrmness loss in peachseems to be closely related to ethylene production [64]. Liuet al. [62] found a clear correlation between the maximumcontent of soluble pectin and the peak of ethylene produc-tion, both suVering the same delay in peaches treated with

Fig. 5 Evolution of SOD activity in peach fruits treated with 5 �L L¡1

NO (unWlled circle) and untreated control fruits (Wlled circle) duringstorage at 20 °C. Data represent the means § SE (vertical bars) ofdeterminations made in duplicate for each of three replicates of eachsample. *SigniWcant diVerences existed between NO-treated and un-treated fruits on the speciWc day of sampling (P < 0.05)

Fig. 6 Evolution of CAT activity in peach fruits treated with 5 �L L¡1

NO (unWlled circle) and untreated control fruits (Wlled circle) duringstorage at 20 °C. Data represent the means § SE (vertical bars) ofdeterminations made in duplicate for each of three replicates of eachsample. *SigniWcant diVerences existed between NO-treated and un-treated fruits on the speciWc day of sampling (P < 0.05)

Fig. 7 Evolution of POX activity in peach fruits treated with 5 �L L¡1

NO (unWlled circle) and untreated control fruits (Wlled circle) duringstorage at 20 °C. Data represent the means § SE (vertical bars) ofdeterminations made in duplicate for each of three replicates of eachsample. *SigniWcant diVerences existed between NO-treated and un-treated fruits on the speciWc day of sampling (P < 0.05)

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1608 Eur Food Res Technol (2008) 227:1599–1611

0.5 �L L¡1 1-MCP. It is already known that fruit softeningis due, to a great extent, to the degradation of protopectin.In this work, we have observed a correlation between ethyl-ene production and Wrmness loss throughout the time ofstorage, both for control fruits and for fruits treated withNO (R2 > 0.90). In the latter case, the signiWcant decreaseof ethylene production at the end of the storage has notbeen considered. Unlike peaches treated with 1-MCP [61],the respiration signiWcantly decreased in the Wrst 3 and6 days of storage in fruits treated with NO (Fig. 1b). In 24-hpretreatments of peaches with AA vapour, Lurie and Pesis[63] found that fruits which were later kept at 20 °C, or at2 °C and then transferred to 20 °C, suVered a lower Wrm-ness loss—that was related to a higher insoluble pectin con-tent and a lower increase of polygalacturonase activity.

Other quality parameters analysed like colour, titratableacidity, TSS content and maturity index were not aVectedby the NO pretreatment (Table 1). Zhu et al. [35] observeda lower increase of the TSS content in peaches during stor-age at 5 or 25 °C after treatment with 5 or 10 �L L¡1 NO,but the contrary was observed when they were treated with15 �L L¡1. Treatments of peaches with AA [63] do notshow signiWcant and clear diVerences regarding the evolu-tion of TSS content and titratable acidity. Liguori et al. [61]did not Wnd signiWcant diVerences in these quality parame-ters due to 1-MCP treatment, but Liu et al. [62] foundeVects of this ethylene antagonist on these parameters,which showed lesser values in treated fruits. These authorsconsider these changes to be within the general eVect of 1-MCP as a ripening inhibitor. The signiWcantly (P < 0.05)higher TSS content on day 3 of storage for fruits treatedwith NO could be due to the intrinsic variability of thefruits (Table 1).

The treatment with NO positively aVected the cell mem-brane integrity, as reXected in the lower increase of theelectrolyte leakage (Fig. 3). The loss of this integrity is oneof the main and primary symptoms of senescence [5, 6]and, therefore, it can be inferred that NO acts as an antise-nescent agent, as considered by Leshem and Wills [27].Zhu and Zhou [36] also observed a signiWcant decrease ofelectrolyte leakage in peaches treated with 5 or 10 �L L¡1

NO, when compared to untreated fruits or fruits treatedwith 15 �L L¡1.

No publications on the inXuence of 1-MCP or AA on theintegrity of the cell membranes in peaches or other fruits ofthe Prunus genus during their postharvest life at room tem-perature have been found, but treatment of watermelonswith 5 �L L¡1 1-MCP at 20 °C inhibited the increase ofelectrolyte leakage that this fruit suVered when stored in thepresence of ethylene at 20 °C [65].

But this better maintenance of cell membrane semiper-meability by fruits treated with NO is not reXected in alower lipid peroxidation index, measured as MDA produc-

tion, or in a lower LOX activity when compared with thecontrol fruits (Table 2), although both parameters increasedduring storage as the fruit senescence progressed. Zhu andZhou [36] considered that NO inXuences lipid peroxidationin peach, due to changes it seems to cause in the lipid com-position of the cell membrane, but they did not measure thelipid peroxidation index. NO possibly exerts its inhibitoryeVect on lipid peroxidation by reaction with the peroxylradicals generated from fatty acids, so that this process isinterrupted. However, although NO per se seems to act asan inhibitor of lipid peroxidation, the radical peroxynitriteformed from NO in the presence of the radical superoxidecan initiate and intensify this lipid peroxidation and, in thisaspect, NO does act as an inductor of this oxidative process[66]. Regarding LOX activity, Zhu et al. [35] observed aninhibitory eVect of NO in a similar experiment of treatmentof peaches with 5 or 10 �L L¡1 NO followed by storage at5 or 25 °C. These authors proposed that this eVect is due tothe binding of NO to the Fe3+ from the LOX active site,which is reduced to Fe2+, therefore inactivating the catalyticactivity of the enzyme [35, 67]. We have not observed thateVect of NO on LOX activity (Table 2).

As mentioned in the Introduction section, the keyenzyme in lipid peroxidation is LOX and, therefore, theactivity of this enzyme contributes to the development ofthe oxidative stress which occurs in fruits during ripeningand senescence [17, 68]. For this reason, a correlationbetween the LOX activity and lipid peroxidation index(MDA production) is to be expected, as has been observedin tomato [22] and apple [69] ripening and senescence. Wehave also found a correlation between these two parametersin peach until day 10 of conservation (R2 > 0.90). At theend of storage, a sharp increase of lipid peroxidation thatwas not reXected in a parallel sharp increase of LOX activ-ity was observed (Table 2). This increase of lipid peroxida-tion can be attributed to the non-enzymatic oxidationreactions caused by the presence of high levels of ROS[22]. This accumulation of ROS at the end of the storageperiod can be related to the increase in the respiratory rate(Fig. 1b). One of the main sources of ROS is respiration,where the oxygen free radicals are generated sequentially inthe respiratory chain in the mitochondria [7, 70].

DiVerences regarding the activity of PPO, the other pro-oxidising enzyme under analysis, occurred in the last stagesof storage, when it was signiWcantly (P < 0.05) lower infruits treated with NO (Fig. 4). PPO oxidises polyphenolsto quinones, which, in later reactions, give rise to mela-nines, the pigments responsible for the appearance ofbrown colour in pulp [71]. PPO is found in the thylakoidmembranes, while its natural substrates, the soluble pheno-lic compounds, are found mainly in the vacuole [72, 73].When the cell membranes disintegrate, as happens duringsenescence, the cell compartmentation is lost and the con-

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Eur Food Res Technol (2008) 227:1599–1611 1609

tact between enzyme and substrates unchains PPO activity[72]. The lesser alteration of the structure and compositionof cell membranes in fruits treated with NO could explainthe lower PPO activity observed in these samples (Figs. 3,4).

No diVerences existed between the treated and untreatedfruits regarding the evolution of free ASC content(Table 3). The sharp decrease of ASC levels during the Wrst3 days of storage could be explained by the high sensitivityof this antioxidant compound to postharvest conditions.ASC is one of the most eVective antioxidants and the Wrst tobe consumed in order to counteract oxidative stress inplants [74, 75]. In general, vitamin C decreases as ripeningprogresses, and one strategy to avoid this metabolic loss isto hasten ripening by ethylene treatment, but no clear andpositive results have been observed in this aspect [26]. Inany case, a relationship between ethylene and the evolutionof ASC content seems to exist, at least in peach, since fruitstreated with 0.5 �L L¡1 1-MCP before storage at 22 °C hada higher vitamin C content than control fruits after 10 daysof storage, and if fruits underwent a second treatment onday 5 of storage, the level of vitamin C was even higherafter 10 days at 22 °C [62].

Treatment with NO seemed to accelerate the conversionof ASC to DHA (Table 3). That is, the ASC, which is thereduced form and the one with the higher antioxidant activ-ity, seems to be oxidised to DHA, also with antioxidantactivity but to a lower degree. This conversion of ASC toDHA is reversible, but, on the contrary, the oxidation ofDHA to diketogluconic acid is not and the product of thisreaction does not have antioxidant capacity [48]. As hasbeen mentioned, respiration is one of the main biologicalsources of ROS, and the respiratory rate of fruits treatedwith NO was lower than that of control fruits, except at theend of the experiment (Fig. 1b). It can be considered thatfruits treated with NO must confront a lower concentrationof ROS due to their lower respiration and, therefore, theASC consumption needed for removal of these free radicalsis not so elevated and the degree of irreversible oxidation todiketogluconic acid is inferior, so DHA accumulates. Onthe contrary, in control fruits, DHA accumulation waslower (Table 3). At the end of storage, after 14 days at20 °C, a sharp increase of the respiratory rate was found inboth the treated and untreated fruits, and the aim of remov-ing the excess of ROS caused by the increase of aerobicmetabolism would have hampered the DHA accumulationin fruits treated with NO since this compound is consumedin this action (Table 4).

A decrease of the total carotenoids content could havebeen expected as the oxidative stress progressed in the fruit,but, on the contrary, it showed a gradual increase (Table 3).The explanation for this result could be found in the protec-tion provided by ASC to carotenoids, because the ROS

removal by the action of the former would prevent the con-sumption of the latter for that aim [74, 75]. The increase inthe content of these pigments is also due to the conversionof chloroplasts into chromoplasts, a characteristic processof ripening which continues after harvesting, as has beenobserved in apple [76]. Carotenoids are the main pigmentsresponsible for colour in ripe peaches, and the evolution oftheir content is closely correlated to the values of parameter‘a’ employed to measure fruit colour (R2 > 0.90).

The enzymatic antioxidant systems seem to enter intoaction, to counteract oxidative stress, at a more advancedstage of ripening. Thus, SOD activity only increased clearlyfrom day 10 of storage at 20 °C, and CAT activity after6 days (Figs. 5 and 6). That is, these sudden increases ofSOD and CAT activities occurred when the oxidative bal-ance had already been altered, as observed from theincreases of lipid peroxidation index and LOX activity(Table 3). Regarding the POX activity, the increase wasmore gradual, and it augmented sharply only at the end ofthe conservation period (Fig. 7). It must be highlighted thatthis activity seemed to be closely related to lipid peroxida-tion (R2 > 0.90). This correlation could be explained by therole of POX in the progress of this oxidative process [77].This delay in the increase of POX activity in comparisonwith CAT and SOD could be explained by the sequence ofantioxidant enzyme activities triggered to remove ROS,since POX was the last one to be stimulated and it is lessspeciWc than the other two. POX activity has been related toa large number of biochemical and physiological processeswhich can cause quantitative and qualitative changes dur-ing the development and ripening of fruit [78]. The signiW-cantly higher level of POX activity in control fruits at theend of storage could be related to the higher ROS produc-tion in these fruits, stimulating a higher activity of thisenzyme (Fig. 7).

NO treatment seemed to have a beneWcial eVect on theenzymatic antioxidant systems, especially on the SOD andCAT activities (Figs. 5 and 6) and, to a lower extent, onPOX (Fig. 7). The results obtained seem to indicate thatNO, as an antisenescent agent, aVects positively theresponse to the oxidative stress associated with fruit senes-cence. It has been observed that NO seems to have an anti-oxidant activity in the leaves of rice, where it delays theinitiation of senescence induced by the plant-growth regu-lator methyl jasmonate, probably due to its capacity forremoving ROS [79].

We have not found publications where the eVects of 1-MCP on the oxidation equilibrium and the antioxidantcapacity of peach are studied, but, in other fruits likeloquat, apple and pear, this type of study has been carriedout [80–82]. Loquat fruit treated with this ethylene antago-nist at a concentration of 5 �L L¡1, for 12 h at 20 °C,suVered, during their posterior storage, a lower accumula-

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1610 Eur Food Res Technol (2008) 227:1599–1611

tion of superoxide anion, a better maintenance of cell mem-brane integrity and lower LOX and PPO activities incomparison with untreated control fruits [82]. In the case ofpear, it has been observed that a treatment with 100 nL L¡1

1-MCP, at 0.5 °C for 23 h, resulted in a better preservationof the antioxidant capacity, in comparison with theuntreated control. This implied a lesser disintegration ofcell membranes, as assessed by electrolyte leakage, a lowercontent of hydrogen peroxide, a higher accumulation oftotal ASC and, Wnally, a higher activity of antioxidantenzymes (CAT, SOD, POX and ascorbate peroxidase) [80].Vilaplana et al. [81] also observed, in the apple cultivar‘Golden Smoothe’, a higher maintenance of the enzymaticantioxidant capacity, in terms of POX activity, combinedwith a lower oxidative damage, reXected in a lower lipidperoxidation index, during storage at 1 °C after a treatmentwith 625 nL L¡1 1-MCP at 0.5 °C. Larrigaudière et al. [80]and Vilaplana et al. [81] posed the hypothesis that 1-MCPcould have positive eVects on ripening which are not dueexclusively to its action as a competitive inhibitor of ethyl-ene. This hypothesis could be supported also by the resultsobtained in loquat, a non-climacteric fruit, treated with 1-MCP, regarding its oxidation status and antioxidant capac-ity [82]. However, this possibility must be considered care-fully since these last authors also observed that treatment ofloquat with 100 �L L¡1 ethylene, at 20 °C for 12 h, inducedhigher activities of the pro-oxidising enzymes LOX andPPO and a higher loss of the semipermeability of cell mem-branes, during postharvest at 20 °C. Moreover, we havementioned already, in the Introduction section, the possibleregulation by ethylene of LOX expression and activity andPPO, CAT and SOD activities, the Wrst two being pro-oxi-dising enzymes and the other two antioxidant ones [18, 20,21, 24].

In the case of AA application as an antisenescent agentin fruits, in order to prolong postharvest life by preservingfruit quality, we have not found any study of its eVects onthe oxidative stress and antioxidant capacity of treatedfruits. In principle, it does not seem to have a great inXu-ence, considering the results obtained regarding the evolu-tion of fermentation and the antioxidant capacity ofstrawberries stored in a modiWed atmosphere [83]. No sig-niWcant diVerences were found with regard to the activitiesof antioxidant enzymes in strawberries stored for 12 days at2 °C in an atmosphere of 20 g/100 g CO2, in comparisonwith the control fruits stored in air. This fact does not allowsupporting a relationship of the antioxidant enzymes’ activ-ities with the signiWcant rise of the AA level, main meta-bolic consequence of the induction of fermentation of fruitssubjected to high CO2 concentration.

From the results of this study of peach fruit storage atroom temperature, we can conclude that a pretreatmentwith 5 �L L¡1 of the free radical gas nitric oxide, for 4 h at

20 °C, eVectively extended the postharvest life of the fruitat room temperature. NO seems to act as an antisenescentagent which induces (1) a clear delay in the onset of senes-cence, reXected in a lower ethylene production, respiratoryrate and degree of cell membrane disintegration; (2) a bettermaintenance of fruit quality, in terms of Wrmness and (3) ahigher antioxidant capacity, manifested as the higher activi-ties of the antioxidant enzymes, especially CAT and SOD.Finally, although this aspect has not been studied in thiswork, it is worth mentioning the possible dependence of theNO eVects on the application dose of the gas. In the particu-lar case of peach, Zhu et al. [35] and Zhu and Zhou [36]observed positive eVects on the postharvest life when it wasapplied at 5 or 10 �L L¡1, but when the dose was 15 �L L 1,there were no eVects or they were contrary to thoseexpected. This dependence of the eVects of NO on the doseof application has been observed also in other horticulturalproducts like strawberry [32].

Acknowledgments We thank the agricultural cooperative “Finca LaCarrichosa”, from Cieza (Murcia, Spain), and Mr. J. Molina for provid-ing the fruits for this experiment. This work was funded by two CICYTresearch projects Wnanced by the Spanish Ministry of Education andScience (Refs. AGL2003-01457 and AGL2007-60447).

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