The physiological and biochemical responses to freezing stress of olive plants treated with salicylic acid

ISSN 1021�4437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 4, pp. 443–450. © Pleiades Publishing, Ltd., 2014.

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1 INTRODUCTION

Freezing stress (<0°C) harmfully affects plantgrowth and development, limits their geographic dis�tribution, and significantly reduces agronomic pro�ductivity. The main target of freezing injury is cellmembranes, which are the primary cause of cellulardehydration in plants exposed to freezing stress [1].Like other abiotic stresses, exposure to freezing tem�perature leads to the accumulation of reactive oxygenspecies (ROS) in plant cells, followed by the increasein lipid peroxidation that are major causes of mem�brane damage [2]. To alleviate and protect from lowtemperature�induced oxidative injury, plants haveevolved mechanisms to scavenge these toxic and reac�tive species by antioxidant compounds and enzymaticantioxidant systems, such as superoxide dismutase(SOD), guaiacol peroxidase (POD), ascorbate perox�idase (APX), and catalase (CAT) [3]. SOD is a metal�binding enzyme that scavenges the toxic superoxideradicals and catalyzes the conversion of two superox�

1 This text was submitted by the authors in English.

ide anions into oxygen and H2O2. Then, POD andCAT convert H2O2 into H2O and O2, whereas APXdecomposes H2O2 by oxidation of co�substrates, suchas phenolic compounds and/or antioxidants [4]. Inaddition, polyphenol oxidase (PPO) catalyzes the oxi�dation of o�diphenols to o�diquinones, as well as thehydroxylation of monophenols [5]. PPO is also animportant enzyme in the response of plants againstfreezing stress, and it can help to avoid serious oxida�tive damage induced by freezing [5]. In order toaccommodate the oxidative stresses, it is crucial thatplants maintain the activities of these antioxidantenzymes. Under severe stress conditions, however, theantioxidant capacity may not be sufficient to minimizethe harmful effect of oxidative injury [6]. Therefore,the search for signal molecules that mediate the stresstolerance is an important step in our better under�standing how plants acclimate to the adverse environ�ment [6].

Salicylic acid (SA) is a hormone�like substancewith ubiquitous distribution among plants. Previousstudy indicated that SA is a natural signal molecule forthe activation of plant defenses [7]. Several studies alsosupported a major role of SA in modulating the plantresponse to several abiotic and biotic stresses, such asultraviolet light, drought, salt, chilling, and freezing[6, 8, 9]. SA pretreatment could directly or indirectly

The Physiological and Biochemical Responses to Freezing Stressof Olive Plants Treated with Salicylic Acid1

A. Hashempoura, M. Ghasemnezhada, R. Fotouhi Ghazvinia, and M. M. Sohanib

a Department of Horticultural Science, Faculty of Agriculture, University of Guilan, Rasht, Iran;fax: +98�131�669�0281; e�mail: [email protected]

b Department of Biotechnology, Faculty of Agriculture, University of Guilan, Rasht, IranReceived May 21, 2013

Abstract—One�year�old olive (Olea europaea L. cv. Zard) plants were treated with 0.5, 1, and 2 mM salicylicacid (SA) and then exposed to nonfreezing and freezing temperatures (–5, –10, and –20°C) for 10 h.Untreated plants served as a control. Exposure to freezing temperatures caused a considerable increase in ionleakage and lipid peroxidation in olive leaves. Treatment with suitable exogenous SA (1.0 mM) prevented theincrease in the ion leakage and lipid peroxidation caused by freezing temperatures, especially at –5 and –10°C.SA�induced freezing tolerance was accompanied by increased activities of antioxidant enzymes, such as gua�iacol peroxidase, catalase, ascorbate peroxidase, and polyphenol oxidase, as compared to control plants. Pro�line, total phenolic content, and antioxidant capacity of olive leaves were declined significantly after exposureto freezing temperature, and their content decreased with lowering of freezing temperatures, while treatmentwith 1 mM SA induced a significant increase in their content. As a summary of these results, suitable concen�tration of SA (1 mM) could enhance freezing tolerance of olive plant by increasing antioxidant enzyme activ�ities and decreasing MDA content through cell membrane integrity maintenance.

Keywords: Olea europaea, freezing stress, salicylic acid, antioxidant enzymes, ion leakage, lipid peroxidation

DOI: 10.1134/S1021443714040098

Abbreviations: APX—ascorbate peroxidase; CAT—catalase;DPPH—1,1�diphenyl�2�picrylhydrazyl; NBT—nitro blue tetra�zolium; POD—guaiacol peroxidase; PPO—polyphenol oxidase;PVPP—polyvinyl polypyrrolidone; SA—salicylic acid; SOD—superoxide�dismutase; TBA—thiobarbituric acid.

RESEARCH PAPERS

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alter freezing tolerance and antioxidant enzyme activ�ities during cold stress, which had a higher ability toendure chilling�induced injury [6]. The chilling injuryalleviated as electrolyte leakage in leaves was signifi�cantly reduced after the application of low SA concen�trations to maize, cucumber, and rice plants [9, 10].Ta gin et al. [6] reported that SA could increase freez�ing tolerance in winter wheat leaves by affecting anti�oxidant enzymes, such as POD, CAT, and PPO.Mora�Herrera et al. [8] showed that SA induced freez�ing tolerance in potato and inhibited APX and CATactivities. More recently, Yang et al. [9] reported thatthe suitable exogenous SA enhanced cold tolerance inwatermelon through the activation of antioxidantenzymes, such as POD, APX, CAT, and SOD.

The olive is an evergreen tree of economic valuedue to the quality of its oil. An important factor in olivegrowing is the minimum temperature in winter andearly spring. Below −12°C the trees suffer severe dam�age, while at −7°C to the aerial parts of the plant arealready damaged, which is accompanied by leaf dropand twig desiccation that can reduce the productivityand threaten plant survival [11]. These conditionsoccurs quite frequently in many places, where theolive is cultivated, such as in north and central parts ofIran [12], where freezing have proved to be lethal toolive plants on several occasions in the past decade.

However, the increasing demand for olive oil hasextended the cultivation of olive trees into regions withcolder climate than those of original Mediterraneanbasin. Moreover, better oil can be yielded from olivetrees growing in colder climates [11], and this has ledto olive trees being cultivated where there is a recurrentdanger of freezing. Concerning the physiological andbiochemical response mechanisms of olive trees dur�ing autumn and winter, some studies have reported asignificant increase in the activities of phenylalanine

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ammonia�lyase (PAL), PPO, APX, and CAT levels [5,13]. However, under severe freezing temperatures, theantioxidant system may not be sufficient to minimizethe harmful effects of oxidative injury and a necessityto use signal molecules, such as SA, that mediate thisharmful effects arises [6].

Up to now, there is hardly any report regarding theeffects of SA on improvement of olive freezing toler�ance. Based on works on other plants [6, 8–10], it washypothesized that freezing stress can lead to theincrease in ion leakage and lipid peroxidation in oliveleaves. These phenomena are due to the accumulationof ROS in plant cells, while SA�pretreatment can alle�viate freezing injury by enhancing antioxidant defenseactivities followed by decreasing in membrane oxida�tive damage due to the reduction of ion leakage andlipid peroxidation. In the present study, we investi�gated the effects of SA concentrations, freezing tem�peratures, and their combined effects on ion leakage,lipid peroxidation, proline content, total protein con�tent, total phenolic content, antioxidant capacity, andantioxidant enzyme (SOD, POD, CAT, AOX, andPPO) activities in one�year�old olive plants.

MATERIALS AND METHODS

One year�old frost sensitive olive (Olea europaea L.cv Zard) plants, which propagated by terminal semi�hardwood cuttings were used in this study. Plants weregrown in 1�L polyethylene bags containing a sandy�loam substrate: organic matter mixture (2 : 1, v/v).Before exposure to freezing temperatures, plants werekept outside during winter (until January 20) to inducenatural cold acclimation. Minimum and maximumdaily temperatures at experimental area (Rasht, Iran)were recorded during the experimental period and areshown in Fig. 1.

Salicylic acid pretreatment and freezing stress. Onthe fifth and sixth days before exposure to freezingtemperatures, plants were sprayed with 0.5, 1, or 2 mMsalicylic acid (SA, 15 mL/plant) solution, until it ranoff. Furthermore, at the same time, plants were irri�gated with 10 mL of SA solution. Distilled water was asa control. The bags were covered with a layer of glasswool to protect the roots from freezing damage. Theplants were divided into two groups. Some plants weretransferred to an open greenhouse and kept for 24 h asa nonfrozen control, and the second group was placedinto a programmable test chamber (KATO, Japan) forwhole plant freezing treatment. The chamber temper�ature was decreased stepwise by 1.5°C/h until −5°Cand thereafter 5°C/h until −20°C. Plants were exposed tofreezing temperatures (–5, –10, and –20°C) for 10 h.Relative humidity inside the chamber was kept at 45–50%, and darkness conditions were simulated. Afterexposing olive plants to each freezing temperature, thechamber temperature was increased stepwise up to4°C and held consistent overnight for slow thawing.

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THE PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO FREEZING STRESS 445

For freezing stress evaluation, the leaves of thethird node from the top were used. A part of leaf sam�ples were frozen in liquid nitrogen and kept at –80°Cuntil further biochemical analysis. The rest of the leafsamples were used to determine freezing injury damage.

Ion leakage. Ion leakage of five leaves per replica�tion was measured as described by Dexter et al. [14]with some modification. Samples were cut into equalpieces (10 mm in diameter), placed in the test�tubecontaining 10 mL of distilled water, and kept at 45°Cfor 30 min in a water bath. The initial conductivity ofthe solution was measured using a Mi 306 EC/TDSconductivity meter (Milwaukee Instruments, Hun�gary). The tubes were then kept in a boiling water bathfor 10 min, and their conductivity was measured onceagain after cooling to room temperature. Ion leakage (%)was calculated as initial EC/final EC × 100.

Lipid peroxidation (MDA content). The level ofmembrane damage was measured by the determina�tion of MDA as the end product of membrane lipidperoxidation [15]. Leaves were weighed and homoge�nized in the solution containing 10% TCA and thencentrifuged at 10000 g for 10 min. To 1.5 mL of thesupernatant aliquot, 1.5 mL of 20% (w/v) TCA con�taining 0.5% (w/v) TBA were added. The mixture washeated at 95°C for 60 min, cooled to room tempera�ture, and centrifuged at 10000 g for 10 min. The absor�bance of the supernatant was read at 532 and 600 nmagainst TCA solution as a reagent blank. The contentof MDA was determined using the extinction coeffi�cient of 1.55/(M cm) and expressed in nmol MDA perg fr wt.

Enzyme activities. Olive leaves (0.5 g) were homog�enized in 1 mL of 50 mM potassium phosphate buffer,pH 7.0, containing 1 mM of EDTA in the presence ofPVP. The homogenate was centrifuged at 15000 g for15 min at 4°C. The supernatant was used to measurethe activities of SOD, APX, POD, and PPO and todetermine total protein content. All assays were doneat 25°C using a T80 spectrophotometer (PG Instru�ment, England).

SOD (EC 1.15.1.1) activity was determined bymeasuring its ability to inhibit the photoreduction ofnitro blue tetrazolium (NBT) according to the meth�ods of Beauchamp and Fridovich [16]. The reactionmixture contained 50 mM phosphate buffer (pH 7.0),200 mM methionine, 1.125 mM NBT, 1.5 mMEDTA, 75 μM riboflavin, and 0–50 µL of the enzymeextract. Riboflavin was added as the last component.Reaction was carried out in test�tubes at 25°C underillumination supplied by two fluorescent lamps(20 W). The reaction was initiated by switching on thelight and allowed to run for 15 min, and light switchingoff stopped the reaction. The tubes were then immedi�ately covered with aluminum foil in order to stop thereaction, and absorbance of the mixture was then readat 560 nm. Identical tubes with complete reactionmixture containing no enzyme extract and developingmaximum color served as a control. A non�illumi�

nated complete reaction mixture with no color devel�opment served as a blank. Under experimental condi�tions, the initial rate of reaction, as measured by thedifference in the increase of absorbance at 560 nm inthe presence and absence of leaf extract was propor�tional to the amount of enzyme. One unit SOD activ�ity was defined as the amount of enzyme required toinhibit 50% of the rate of NBT reduction measured at560 nm. The SOD activity of the extract was expressedas activity unit/g fr wt.

POD (EC 1.11.1.7) activity in leaves was assayed bythe oxidation of guaiacol in the presence of H2O2. Theincrease in absorbance was recorded at 470 nm [17].The reaction mixture contained 100 μL of crudeenzyme extract, 500 μL of 5 mM H2O2, 500 μL of28 mM guaiacol, and 1900 μL of 50 mM potassiumphosphate buffer (pH 7.0). POD activity of the extractwas expressed as activity unit/(g fr wt min).

CAT (EC 1.11.1.6) activity was assayed accordingthe method of Beers and Sizer [18]. The decomposi�tion of H2O2 was monitored by the decrease in absor�bance at 240 nm. The assay mixture contained 2.6 mLof 50 mM potassium phosphate buffer (pH 7.0),400 μL of 15 mM H2O2, and 40 μL of enzyme extract.The CAT activity of the extract was expressed as activ�ity unit/(g fr wt min).

APX (EC 1.11.1.11) activity was measured accord�ing to Nakano and Asada [19]. The reaction mixturecontained 50 mM (pH 7.0) potassium phosphatebuffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbate,1.0 mM H2O2, and 100 μL of the enzymes extract.H2O2�dependent oxidation of ascorbate was followedby a decrease in the absorbance at 290 nm. The APXactivity of the extract was expressed as activity unit/(gfr wt min).

PPO (EC 1.10.3.1) activity was assayed with 4�methylcatechol as a substrate as described in [20] withsome modifications. The assay of the enzyme activitywas performed using 2 mL of 0.1 mM sodium phos�phate buffer (pH 6.8), 0.5 mL of 100 mM 4�methyl�catechol, and 0.5 mL of the enzyme solution. Theincrease in absorbance at 420 nm was recorded. ThePPO activity was expressed as activity unit/(100 g fr wtmin).

Proline content. Proline content was determinedspectrophotometrically by adopting the ninhydrinmethod of Bates et al. [21]. 300 mg of fresh leaf sam�ples were homogenized in sulfosalicylic acid; then2 mL of each acid ninhydrin and glacial acetic acidwere added. The samples were heated at 100°C for60 min. The mixture was extracted with toluene, free tol�uene was quantified at 520 nm using L�proline as a stan�dard, and its content was expressed as µmol/g fr wt.

Protein content. Protein content was determinedaccording to the method of Bradford [22] using BSAas a standard.

Total phenolic content and antioxidant capacity.Leaf sample (1 g) was extracted with 10 mL of metha�

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nol and then used for determination of total phenoliccontent and DPPH scavenging capacity. Total phenolswere determined with a spectrophotometer using themodified Folin–Ciocalteu colorimetric method [23].The methanolic extract (125 μL) was mixed with375 μL of distilled water in a test�tube followed by theaddition of 2.5 mL of 10% Folin–Ciocalteu reagentand allowed to stand for 6 min. Then, 2 mL of 7.5%Na2CO3 was added. Each sample was incubated for90 min at room temperature in darkness, and absor�bance at 760 nm was measured. Results were expressedas mg gallic acid (GAE)/g fr wt.

The antioxidant activity was evaluated by 1,1�diphe�nyl�2�picrylhydrazyl (DPPH) free radical scavengingmethod [24]. 50 μL of the leaf extract was added to950 μL of 0.1 mM DPPH, vortexed, and incubated atroom temperature in darkness. The absorbance of thesamples was measured at 517 nm using the spectro�photometer after 30 min.

Percentage of DPPH�scavenging activity was cal�culated as % inhibition of DPPH = (A517 control –A517 sample/A517 control) × 100.

Statistical analysis. The experiment was in com�pletely randomized factorial design. Temperaturetreatments orders were randomly assigned for thegrowth chamber. Then treated olive plants with differ�ent levels of SA were assigned in growth chamber, ran�domly. Statistical analysis was carried out using SASsoftware (v. 9.1, SAS Institute, United States). Theanalysis of variance (ANOVA) between treatmentmeans was carried out using Tukey’s test at p < 0.05.Values presented in the text indicate mean values ± SEof three replicates. The graphics were done using Excelsoftware.

RESULTS

Ion Leakage

Ion leakage reflects the level of cell membraneinjury as a result of oxidative damage. Both freezingstress and SA affected ion leakage percentage, and thesignificant difference in ion leakage was observedamong the treatments (Fig. 2a). Freezing stressinduced a significant increase in the ion leakage fromolive leaves, and the application of SA could signifi�cantly suppress the ion leakage at freezing stress con�ditions. The 1 mM SA�treated olive plants were shown tohave the lower leaf ion leakage at –5, –10, and −20°C,that were 36.8, 38.8, and 24%, respectively, as compareto the control plants (Fig. 2a).

Lipid Peroxidation

The production of MDA was used as an indicator ofmembrane lipid peroxidation, another indicator ofdamage done by stress in plant membranes. MDAcontent in olive plants significantly increased withlowering freezing temperature down to −10°C; there�after, it relatively decreased at −20°C. The percentageof increase was 75% in control plants at −10°C ascompared to nonfreezing temperature conditions. Adecline in the MDA content was observed in olivestreated by SA (Fig. 2b). Under freezing temperaturesof –10 and −20°C, the 1 mM SA�treated olives hadthe lower leaf MDA content, which reduced signifi�cantly from 37 to 26%, respectively, as compared tocontrol plants (Fig. 2b).

Enzyme Activities

In general, plants activate a number of antioxidantenzymes when exposed to low temperature in order toprotect them against potentially cytotoxic ROS. To

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Fig. 2. Ion leakage (a) and lipid peroxidation (MDA) (b) of olive leaves cv Zard pretreated with water (1, control), 0.5 (2), 1 (3),and 2 mM (4) SA under nonfreezing and freezing temperatures at –5, –10, and –20°C for 10 h. Vertical bars indicate standarderrors from means (n = 3). LSDs (P < 0.05) for ion leakage = 3.6, for lipid peroxidation = 1.8.



find out the relation between antioxidant enzymes andexogenous SA at freezing temperatures, the activitiesof antioxidant enzymes in olive plants treated withexogenous SA at freezing temperatures were assayed.

The activity of SOD significantly increased whenthe plants were exposure to freezing temperatures, andthe highest SOD activity was observed at −10°C, andthereafter its activity was decreased at −20°C (Fig. 3a).Untreated plants when exposed to −10°C had 21%more SOD activity than at nonfreezing temperature.

However, there is no significant difference betweenSA�treated and untreated plants regarding to theirSOD activity.

The highest POD activity was observed at −10°C,followed by a decrease at −20°C. Untreated controlplants when exposed to −10°C have showed 61% morePOD activity than at nonfreezing temperature. SAtreatment could significantly increase the activity ofPOD (Fig. 3b). The 1 mM SA�treated olives that wereexposed to −10°C had highest POD activity, which

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Fig. 3. Activity of SOD (a), POD (b), APX (c), CAT (d), PPO (e) and protein content (f) of olive cv. Zard leaves pretreated withwater (1, control), 0.5 (2), 1 (3), and 2 mM (4) SA under nonfreezing and freezing temperatures at –5, –10, and –20°C for 10 h.Vertical bars indicate standard errors from means (n = 3). LSDs (P < 0.05) for SOD = 44.65, POD = 3.26, APX = 77.13, CAT =30.92, PPO = 0.24, protein content = 9.07.

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increased significantly by 31%, compared to the oliveplants without SA (control) at the same temperature(Fig. 3b).

The data also showed that freezing temperaturessignificantly induced APX activity (Fig. 3c). The high�est APX activity was assayed at −10°C, and thereafterits activity decreased. The olive plants sprayed with1 mM SA showed significantly higher APX activity(Fig. 3c). The 1 mM SA�treated plants showed 29%more APX activity at −10°C than untreated plants atthis temperature

According to the results in Fig. 3d, freezing stresssignificantly reduced CAT activity. The plants treatedwith 1 mM SA maintained the higher CAT activityunder stress temperatures (Fig. 3d). However, withincreasing SA concentration CAT activity decreased.

Under freezing temperatures, the PPO activity inolive plants followed almost similar trend as that ofPOD. The percentage of increase in control plants was43% at −10°C as compared to nonfreezing tempera�ture. SA treatment at 1 mM level significantlyincreased the PPO activity (Fig. 3e). The 1 mM SA�treated olives exposed to −10°C had highest PPOactivity (by 23% higher than control at the same tem�perature).

Protein and Proline Contents

According to the results presented in Fig. 3f, theprotein content increased significantly at −10°C andthereafter decreased at −20°C. The 1 mM concentra�tion of SA increased protein content at –5 and −10°Cas compared to control plants (Fig. 3f). The increasewas 40% at −10°C, as compared to control plants atsame temperature.

Freezing stress significantly decreased prolinecontent in olive leaves (Fig. 4). The plant sprayedwith SA showed a significant increase in proline con�tent under freezing temperature, especially in plantsexposed to −5°C. The highest proline content(1.7 μmol/g fr wt) was observed in plants pretreatedwith 2 mM SA under −5°C (Fig. 4).

Total Phenolic Content and Antioxidant Capacity

Total phenolic content of freezing�treated plantswas significantly lower than that of the nonfreezingcontrol plants (Fig. 5a). In control plants, the percent�age of decrease was 31% at −20°C as compared toplants at nonfreezing temperature. The total phenoliccontent was significantly different among SA treat�ments, and the 1 mM SA�treated olives at −10°C hadthe highest content (16.6 mg GAE/g fr wt).

The results showed that antioxidant capacity ofolive plants increased after freezing treatment at −5°C,and then decreased at –10 and −20°C. Pretreatmentwith exogenous SA induced a significant increase inantioxidant capacity at both freezing and nonfreezingtemperatures (Fig. 5b). Under freezing stress temper�atures, the 1 mM SA�treated olives had the higherantioxidant capacity that increased significantly by 19and 26% under –10 and −20°C, respectively, as com�pared to the control plants.

DISCUSSION

There is hardly any report regarding the effects ofSA on the improvement of freezing tolerance in olive.In the present study, the freezing stress�induced oxida�tive damage to olive was evaluated and the protectiveeffect of suitable exogenous SA was measured. Expo�sure to freezing�temperatures (–5, –10, and −20°C)caused a significant increase in ion leakage (Fig. 2a)and MDA content (Fig. 2b) in olive leaves, indicatingthat freezing stress could cause damages to the integ�rity of the cellular membranes and to cellular compo�nents, such as lipids. The present results are in accor�dance with a previous report, which has shown thatpercentage of ionic leakage in olive cultivars subjectedto different freezing temperatures significantlyincreased while lowering freezing temperature downto −20°C [25].The plants treated with suitable exoge�nous SA (1.0 mM) showed a significantly lower ionleakage and MDA content, thereby alleviating thedamage normally caused by freezing stress in oliveleaves (Figs. 2a, 2b). This result is in agreement withthose of Kang and Saltveit [10] and Yang et al. [9], whohave found the ion leakage and lipid peroxidation con�tent were significantly reduced by the application ofthe lower concentrations of SA to maize, rice, cucum�ber, and watermelon. Also, Ta gin et al. [6] reportedthat exogenous SA decreased freezing injury (deter�mined by ion leakage) in winter wheat leaves exposedto different freezing temperatures.

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Fig. 4. Proline content in olive cv. Zard leaves pretreatedwith water (1, control), 0.5 (2), 1 (3), and 2 mM (4) SAunder nonfreezingand freezing temperatures at –5, –10,and –20°C for 10 h. Vertical bars indicate standard errorsfrom means (n = 3). LSDs (P < 0.05) for proline content =0.17.



The ability of SA�treated plants to protect cellularmembranes during freezing stress may be attributed, inpart, to the higher activities of antioxidant enzymes,such as SOD, POD, CAT, APX, and PPO, which areknown to be up�regulated by SA. The data about anti�oxidant enzyme activities support this suggestion, asshowed in Figs. 3a–3e. The results showed that as oliveplants were exposed to freezing temperatures, theactivities of antioxidant enzymes (SOD, POD, APX,and PPO) increased up to −10°C and thereafter, theiractivities were declined at −20°C, while CAT activitydecreased continuously down to −20°C. Under stressconditions, an enhanced activity of almost all theseenzymes has been reported [3]. Cansev et al. [13] andOrtega�García and Peragón [5] demonstrated thatAPX, CAT, PPO, NADPH oxidas, and phenylalanineammonia�lyase (PAL) activities increased during coldacclimatization in the olive tree. However, in ourexperiments, CAT activity decreased continuouslydown to −20°C. The application of exogenous SA canincrease freezing tolerance in olive plants by increas�ing activities of antioxidant enzymes, including POD,APX, CAT, and PPO (Figs. 3b–3e). However, SAapplication did not have a significant effect on theactivity of SOD (Fig. 3a). Our results are in agreementwith previous studies that showed a suitable exogenousSA increased freezing tolerance in winter wheat andenhanced cold tolerance in watermelon by the activa�tion of antioxidant enzymes, such as SOD, POD,APX, CAT, and PPO [6, 9]. Here, we also found thatthe application of 1 mM SA increased antioxidantenzyme activities; however, the higher concentrationof SA (2 mM) contrarily decreased oxidant enzymeactivities. Consequently, it reduced freezing toleranceof olive plants. The application of1 mM SA could sig�nificantly maintain the higher CAT activity as com�

pared to control at freezing temperatures. This resultwas consistent with findings of Yang et al. [9] whoreported an increase in the CAT activity after SA treat�ment of watermelon seedlings.

After exposure to freezing temperatures and recov�ery at 4°C, total protein content of olive plantsincreased during temperature lowering down to −10°Cand then markedly decreased at −20°C (Fig. 3f).Exogenous SA can have an important role in the regu�lation of proteins [6]. The application of 1 mM SAincreased total protein content in the olive plant whensubjected to freezing stress, especially at −10°C. Thisresult was supported by that of Ta gin et al. [6], whoreported that SA could increase freezing tolerance inwinter wheat leaves by increasing accumulation ofapoplastic proteins, including antifreeze proteins.

In the present study, freezing stress significantlydecreased proline content, whereas SA treatment sig�nificantly increased proline content (Fig. 4). Prolinemay inhibit membrane lipid peroxidation in plant tis�sue by acting as an antioxidant to neutralize the chill�ing�induced free radicals [26]. Heber et al. [27]showed that proline was able to inhibit freezing�induced inactivation of membrane activities.

The phenolic compounds have been proven to havethe ability to scavenge free radicals and inhibit mem�brane lipid peroxidation of seedlings [28]. Exposingolive plants to freezing temperatures reduced totalphenolic content, whereas antioxidant capacity wasincreased following freezing temperature treatment at−5°C and decreased thereafter (Figs. 5a, 5b). SuitableSA concentration (1 mM) could enhance antioxidantcapacity of olive plants by increasing enzymatic anti�oxidant activity and total phenolic content. The paral�lel increases in the total phenolic content and antioxi�dant capacity might be due to the increased PAL activ�

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Fig. 5. Total phenolic content (a) and antioxidant capacity (b) in the leaves of olive cv. Zard pretreated with water (1, control),0.5 (2), 1 (3), and 2 mM (4) SA under nonfreezing and freezing temperatures at –5, –10, and –20°C for 10 h. Vertical bars indi�cate standard errors from means (n = 3). LSDs (P < 0.05) for total phenolic content = 0.65, for antioxidant capacity = 0.06.

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ity induced by suitable SA treatments (1 mM). Ourresults are in accordance with a previous report thatthe high level of DPPH�radical scavenging has beencorrelated with increased chilling tolerance [10].

In present study, the effect of SA and freezing tem�peratures on antioxidant enzymes in olive plants wereinvestigated. Our results suggested that treatment with1 mM SA could reduce electrolyte leakage and MDAcontent in olive leaves by enhancing the antioxidantenzyme activities and protein content. Also, weobserved that −10°C was the best freezing temperaturefor the elevation of biochemical responses of oliveplants under artificially simulated freezing stress inlaboratory conditions.

REFERENCES

1. Levitt, J., Responses of Plants to Environmental Stress.Chilling, Freezing, and High Temperature Stresses, NewYork: Academic, 1980.

2. Kenadal, E.J. and McKersie, B.D., Free radical andfreezing injury to cell membranes of winter wheat,Physiol. Plant., 1989, vol. 76, pp. 86–94.

3. Lee, D.H. and Lee, C.B., Chilling stress�inducedchanges of antioxidant enzymes in the leaves of cucum�ber: in gel enzyme activity assays, Plant Sci., 2000,vol. 159, pp. 75–85.

4. Davis, D.G. and Swanson, H.R., Activity of stress�related enzymes in the perennial weed leafy spurge(Euphorbia esula L.), Environ. Exp. Bot., 2001, vol. 46,pp. 95–108.

5. Ortega�García, F. and Peragón, J., The response ofphenylalanine ammonia�lyase, polyphenol oxidase andphenols to cold stress in the olive tree (Olea europaea L.cv. Picual), J. Sci. Food Agric., 2009, vol. 89, pp. 1565–1573.

6. Ta gin, E., Atící, Ö., Nalbanto lu, B., and Popova, L.P.,Effects of salicylic acid and cold treatments on proteinlevels and on the activities of antioxidant enzymes inthe apoplast of winter wheat leaves, Phytochemistry,2006, vol. 67, pp. 710–715.

7. Klessig, D.F. and Malamy, J., The salicylic acid signalin plants, Plant Mol. Biol., 1994, vol. 26, pp. 1439–1458.

8. Mora�Herrera, M.E., López�Delago, H., Castillo�Morales, A., and Foyer, C.H., Salicylic acid and H2O2function by independent pathways in the induction offreezing tolerance in potato, Physiol. Plant., 2005,vol. 125, pp. 430–440.

9. Yang, J.H., Gao, Y., Li, Y.M., Qi, X.H., andZhang, M.F., Salicylic acid�induced enhancement ofcold tolerance through activation of antioxidativecapacity in watermelon, Sci. Hortic., 2008, vol. 118,pp. 200–205.

10. Kang, H.M. and Saltveit, M.E., Chilling tolerance ofmaize, cucumber and rice seedling leaves and roots aredifferentially affected by salicylic acid, Physiol. Plant.,2002, vol. 115, pp. 571–576.

11. Palliotti, A. and Bongi, G., Freezing injury in the oliveleaf and effects of mefluidide treatment, J. Hortic. Sci.,1996, vol. 71, pp. 57–63.

12. Fereydooni, H., Zeinanlo, A., Akhademi, M.,Tamaskani, A.A., and Bageri, M., Investigation on frostdamage and selection of olive tolerance in olive culti�vars, Proc. 6th Congr. Iranian Hort. Sci. (Iran, Rasht),2009, pp. 123–127.

13. Cansev, A., Gulen, H., and Eris, A., Cold�hardiness ofolive (Olea europaea L.) cultivars in cold�acclimatedand non�acclimated stages: seasonal alteration of anti�oxidative enzymes and dehydrin�like proteins, J. Agric.Sci., 2009, vol. 147, pp. 51–61.

14. Dexter, S.T., Tottingham, L.F., and Graber, L.F., Investi�gations of hardiness of plants by measurement of electricalconductivity, Plant Physiol., 1932, vol. 7, pp. 63–78.

15. Heath, R.L. and Parker, L., Photoperoxidation in iso�lated chloroplasts. I. Kinetics and stoichiometry of fattyacid peroxidation, Arch. Biochem. Biophys., 1968,vol. 125, pp. 189–198.

16. Beauchamp, C. and Fridovich, J., Superoxide dismu�tase: improved assays and an assay applicable to acryla�mide gels, Anal. Biochem., 1971, vol. 44, pp. 276–287.

17. Chance, B. and Maehly, S.K., Assay of catalase and per�oxidase, Methods Enzymol., 1955, vol. 2, pp. 764–775.

18. Beers, P.F. and Sizer, I.W., A spectrophotometric assaymeasuring the breakdown of hydrogen peroxide by cat�alase, J. Biol. Chem., 1952, vol. 195, pp. 133–138.

19. Nakano, Y. and Asada, K., H2O2 is scavenged by ascor�bate�specific peroxidase in spinach chloroplasts, PlantCell Physiol., 1981, vol. 22, pp. 867–880.

20. Luh, B.S. and Phithakpol, B., Characteristics ofpolyphenoloxidase related to browning in clingpeaches, J. Food Sci., 1972, vol. 37, pp. 264–268.

21. Bates, L.S., Waldren, R.P., and Teare, I.D., Rapiddetermination of free proline for water stress studies,Plant Soil, 1973, vol. 39, pp. 205–207.

22. Bradford, M.M., A rapid and sensitive method forquantitation of microgram quantities of protein utiliz�ing the principle of protein–dye binding, Anal. Bio�chem., 1976, vol. 72, pp. 248–252.

23. Singleton, V.L. and Rossi, J.A., Colorimetry of totalphenolics with phosphomolybdic�phosphotungsticacid reagents, Am. J. Enol. Vitic., 1965, vol. 16,pp. 144–158.

24. Blois, M.S., Antioxidant determinations by the use of astable free radical, Nature, 1958, vol. 181, pp. 1199–1200.

25. Bartolozzi, F. and Fontanazza, G., Assessment of frosttolerance in olive (Olea europaea L.), Sci. Hortic., 1999,vol. 81, pp. 309–319.

26. Xin, Z. and Li, P., Relationship between proline andabscisic acid in the induction of chilling tolerance inmaize suspension�cultured cells, Plant Physiol., 1993,vol. 103, pp. 607–613.

27. Heber, U., Tyankova, L., and Santarius, K.A., Effectsof freezing on biological membranes in vivo and in vitro,Biochim. Biophys. Acta, 1973, vol. 291, pp. 23–37.

28. Tusda, T., Shiga, K., Ohshima, K., Kawakishi, S., andOsawa, T., Inhibition of lipid peroxidation and theactive oxygen radical scavenging effect of anthocyaninpigments isolated from Phaseolus vulgaris L., Biochem.Pharmacol., 1996, vol. 52, pp. 1033–1039.

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The physiological and biochemical responses to freezing stress of olive plants treated with salicylic acid