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Environmental and Experimental Botany 74 (2011) 162– 170

Contents lists available at ScienceDirect

Environmental and Experimental Botany

j o ur nal homep age : www.elsev ier .com/ locate /envexpbot

igh temperature positively modulates oxidative protection in salt-stressedashew plants

érgio Luiz Ferreira-Silvaa, Eduardo Luiz Voigtb, Evandro Nascimento Silvaa,osemir Moura Maiab, Adilton de Vasconcelos Fontenelea, Joaquim Albenisio Gomes Silveiraa,∗

Departamento de Bioquímica e Biologia Molecular/Instituto Nacional de Ciência e Tecnologia em Salinidade (INCTsal/CNPq), Universidade Federal do Ceará, CP 6033,EP 60451-970 Fortaleza, Ceará, BrazilLaboratório de Estudos em Biotecnologia Vegetal, Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte,ampus Universitário Lagoa Nova, Caixa Postal 1648, CEP 59078-970 Natal, RN, Brazil

r t i c l e i n f o

rticle history:eceived 17 August 2010eceived in revised form 4 April 2011ccepted 23 May 2011

eywords:eat stressalt stressombined stressesntioxidant enzymes

a b s t r a c t

This work evaluated the oxidative protection mechanisms triggered by high temperatures in salt-stressedcashew (Anacardium occidentale) plants. In the first experiment, cashew plants in a greenhouse were sub-jected to a wide range of NaCl concentrations under natural conditions involving high temperatures. Inthe second experiment, the plants were exposed to 100 mM NaCl alone, heat alone (42 ◦C) or a combi-nation of both heat and NaCl. Data analysis from the two experiments revealed that salt-stressed plantswere favored by high temperatures in terms of oxidative protection, as indicated by a decrease in lipidperoxidation and H2O2 concentration. The H2O2 concentration and lipid peroxidation results were cor-roborated in long-term salt exposure in a greenhouse; however, greenhouse plants that were subjectedto high salinity exhibited mild protein oxidation. High temperature positively modulated protein content

onenzymatic antioxidantnacardium occidentale

and the activities of catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) in salt-stressed plants, but salinity exerted a negative effect on APX activity. The changes in ascorbate redox statewere favorable for cashew protection under high salinity combined with heat. The data demonstrate thathigh temperature is essential for the oxidative protection of salt-stressed cashew plants, which displayan efficient protection mechanism represented by the activities of CAT, SOD and APX as well as favorablechanges in the ascorbate redox state under acute salt stress.

. Introduction

Plant species cultivated in tropical semi-arid regions face a com-lex combination of abiotic stress factors, such as drought, salinity,igh temperatures and high radiation (Cavalcanti et al., 2004;ittler, 2006). Some species adapted for those regions, such as

ashew plants (Anacardium occidentale L.), have developed genetic,olecular and physiological mechanisms to overcome adverse

onditions (Silveira et al., 2003). Indeed, cashew plants displayood growth performance when cultivated in dry, hot and salineites in Brazil (Ferreira-Silva et al., 2008). Recent studies have

emonstrated that plants exhibit complex responses when theyre simultaneously subjected to combined abiotic stresses (Wangt al., 2003; Mittler, 2006).

Abbreviations: APX, ascorbate peroxidase; AsA, ascorbic acid; CAT, catalase;HA, dehydroascorbate acid; ROS, reactive oxygen species; SOD, superoxide dis-utase.∗ Corresponding author. Tel.: +55 85 3366 9821; fax: +55 85 3366 9821.

E-mail address: silveira@ufc.br (J.A.G. Silveira).

098-8472/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rioi:10.1016/j.envexpbot.2011.05.015

Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

The molecular, biochemical and physiological mechanismsinvolved in plant response to combined stresses are very complex(Kreps et al., 2002; Rizhsky et al., 2002, 2004; Mittler, 2006; Kotaket al., 2007; Kant et al., 2008). Oxidative metabolism is currently themost promising area of plant biology for investigating the complexmetabolic networks involved in environmental stress responses(Mittler et al., 2004; Gechev and Hille, 2005; Møller et al., 2007;Slesak et al., 2007). Although oxidative metabolism represents acommon route for signaling and gene expression involved in abioticstress response and tolerance (Neill et al., 2002), the understandingof this process is incipient and fragmentary (Mittler, 2002; Mittleret al., 2004).

High temperatures, whether isolated or in combination withother stresses, are capable of inducing dramatic changes in plantmetabolism (Penfield, 2008; Baisakh and Subudhi, 2009; Ruellandand Zachowski, 2010; Silva et al., 2010). Heat stress is often accom-panied by water deficiency and stomatal closure (Wahid et al.,

2007), which reduces CO2 availability and may decrease the CO2/O2ratio in chloroplasts (Foyer and Noctor, 2000). These changes canstrongly affect photosynthetic efficiency, a process considered to bemore sensitive to heat (Wise et al., 2004). In addition, heat stress

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an trigger expression of several genes and proteins, such as heathock proteins, which are involved in cell protection (Howarth andugham, 1993; Chen et al., 2002; Iba, 2002; Kotak et al., 2007).

Salt stress induces stomatal closure causing a decrease in theO2/O2 ratio in chloroplasts, which restricts CO2 assimilation byhe Calvin cycle (Andersson, 2008) and increases RuBisCO oxyge-ase activity, stimulating the photorespiratory pathway (Zelitcht al., 2009; Foyer et al., 2009). Under these conditions, theADP+/NADPH ratio is diminished and the photosynthetic electron

ransport chain becomes over-reduced by energy excess (Foyer andoctor, 2000). These disturbances can cause excessive productionf reactive oxygen species (ROS), which can induce oxidative stressMøller et al., 2007). Thus, the interaction between salinity and highemperature can generate oxidative stress in plants and is therefore

potential danger (Mittler, 2006).Plants have developed several antioxidative defenses against

xcessive ROS production generated by salinity (Miller et al.,010) or high temperature (Silva et al., 2010). This defense sys-em involves several enzymes, such as superoxide dismutase (SOD),scorbate peroxidase (APX) and catalase (CAT), together with theain non-enzymatic antioxidants, ascorbate (AsA) and glutathione

GSH) (Smirnoff, 2000; Shigeoka et al., 2002). SOD and APX repre-ent the first line of defense against ROS produced in chloroplastsAsada, 2006), while CAT is the main scavenger of H2O2 gener-ted by photorespiration (Del Río et al., 2006; Foyer et al., 2009).lutathione and ascorbate constitute essential components of thelutathione–ascorbate cycle, which is the main route for ROS scav-nging, especially in chloroplasts (Kocsy et al., 2002; Mittler, 2002).

In this work, antioxidative protective mechanisms were evalu-ted in leaves of cashew plants exposed to long-term exposure toncreasing NaCl concentrations and high temperatures. Next, thelants were subjected to both combined and isolated conditionsf high temperature and salinity. The cashew was chosen as anxperimental model because it is a well-adapted, semi-arid speciesften exposed to salt, drought and hot environments (Silveira et al.,003), and because it displays an effective photochemical protec-ion mechanism under adverse environmental conditions (Souzat al., 2005).

We tested the hypothesis that high temperature is favorableor oxidative protection in cashew leaves under conditions of highalinity. The data demonstrate that cashew leaves have an efficientntioxidant enzymatic protection system especially when in pres-nce of high temperature. This system comprises CAT, SOD, andPX activities together with non-enzymatic ascorbate buffer com-onent. The involvement of the combination of high temperaturend salt stress in oxidative response is discussed.

. Materials and methods

.1. Plant material and initial growth conditions

Cashew nuts (A. occidentale L.), CCP 06 genotype, were providedy EMBRAPA, Brazil. The nuts were surface-disinfected with 5%w/v) sodium hypochlorite, washed in distilled water and sown inermiculite (medium texture and density, 0.12 g/cm3) in 2 L plasticots. The substrate humidity was maintained at 70% of the ver-iculite holding capacity by irrigation with one-fourth strengthoagland and Arnon (1950) nutrient solution. Thirty-five days after

owing, plants were utilized in the Experiments I and II. Plants werenitially grown in a greenhouse at high temperatures, means rang-

ng from 27 ◦C to 39 ◦C (day) and 23 ◦C to 25 ◦C (night). The meanelative humidity varied from 62% (day) to 82% (night) under natu-al sun light with an average maximum photosynthetic photon fluxensity (PPFD) of 850 �mol m−2 s−1 and a photoperiod of 12 h.

xperimental Botany 74 (2011) 162– 170 163

2.2. Experiment I – effects of NaCl-salinity levels on physiologicaland oxidative responses in a greenhouse under high temperatureconditions

In this experiment, cashew plants were exposed to increasingNaCl levels in a greenhouse under natural environmental condi-tions with high temperatures, as described above. The 35-day-oldplants with eight fully expanded leaves were exposed to increasingNaCl concentrations (50, 100, 150 and 200 mM) dissolved in one-fourth strength Hoagland and Arnon (1950) nutrient solution. Thesalt treatments were applied gradually, increasing the NaCl con-centration by 50 mM per day to avoid osmotic shock. Plants grownwithout NaCl served as a control. The nutrient solution containingsalt treatments was applied every 2 days until 70% of the vermi-culite holding capacity was reached. NaCl was applied for 15 daysafter the first irrigation with 50 mM in each treatment. On the dayof harvest, the plants treated with 200 mM NaCl had a transpirationrate of 15% of the control plants. The leaf discs were immediatelyharvested for the determination of membrane damage (K+ leakage)and relative water content. Then fully expanded leaf samples werefrozen in liquid N2 and stored at −80 ◦C.

2.3. Experiment II – isolated and combined effects of NaCl-salinityand high temperature on the oxidative response in cashew leaves

In order to evaluate the isolated and combined effects of hightemperature and salinity in the oxidative response, a second exper-iment in a growth chamber was performed. The experiment wascarried out under controlled conditions with high relative humid-ity (70%) and low PPFD (100 �mol m−2 s−1). This low light intensitywas chosen in order to avoid photo-oxidative damage induced bylight excess combined with heat or salt. Thirty-five-day-old cashewplants were previously grown in the absence (control) or pres-ence of 100 mM NaCl (salt stress) for 15 days in greenhouse, asdescribed in Experiment I. Then the plants were subjected to thefollowing treatments in a growth chamber: control (control plantsexposed to 27 ◦C for 12 h), salt alone (plants previously cultivatedin 100 mM NaCl were exposed to 27 ◦C for 12 h), heat alone (controlplants exposed to 42 ◦C for 12 h), and NaCl and heat (plants previ-ously cultivated in 100 mM NaCl exposed to 42 ◦C for 12 h). The hightemperature treatments were performed by increasing the temper-ature to 42 ◦C at a rate of 5 ◦C per hour. The leaf temperatures weremeasured with an IRGA (infrared gas analyzer, LI-COR, mod. 6400XT, USA), and they reached values of 27 ± 1.8 ◦C and 42 ± 1.5 ◦C,corresponding to the treatments of 27 ◦C and 42 ◦C, respectively.

2.4. Na+ content, relative water content, membrane damage (K+

leakage) and stomatal conductance

The Na+ concentration in leaf tissues was measured by flamephotometry, leaf membrane damage (MD) was estimated on thebasis of leaf K+ leakage, and the leaf relative water content (RWC)was measured by successive weighing of leaf discs (fresh, fullysaturated and dry), as previously described (Cavalcanti et al.,2004). Stomatal conductance was performed daily using a portableporometer (LI-COR 1600, USA). All measurements were madebetween 09:30 and 10:00 a.m., when environmental conditionsshowed low variation. A similar mature leaf from the middle ofthe shoot was chosen for each measurement.

2.5. Hydrogen peroxide, lipid peroxidation and proteincarbonylation

Hydrogen peroxide was determined according to the method ofCheeseman (2006). Samples of fresh leaves (0.1 g) were powderedin liquid N2 and extracted in 100 mM potassium phosphate buffer

1 and Experimental Botany 74 (2011) 162– 170

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Table 1Dry weight (DW), relative water content (RWC), stomatal conductance (gs) and Na+

content in the leaves of cashew plants exposed to increasing NaCl concentrationsover 15 days in a greenhouse under high temperatures. The data represent the meanof four replicates. In each column, the values followed by the same letter did notdiffer at the 0.05 probability level (Tukey’s test).

NaCl (mM) Leaf DW(g plant−1)

RWC (%) gs

(mmol m−2 s−1)Na+

(mmol kg−1 DW)

0 4.34a 92a 106a 121c50 3.44b 92a 51b 495b

64 S.L. Ferreira-Silva et al. / Environmental

pH 6.4) containing 5 mM KCN. The reaction was performed at5 ◦C for 30 min, and the absorbance was read at 560 nm. The H2O2oncentration was expressed as �mol g−1 FW. Lipid peroxidationas determined by measuring the thiobarbituric acid-reactive sub-

tances (TBARS) according to Heath and Packer (1968). The TBARSoncentration was calculated using of the molar extinction coeffi-ient (155 mM−1 cm−1) and expressed as nmol g−1 FW. Protein car-onylation was determined after derivatization of carbonyl groupsith dinitrophenylhydrazine (DNPH), according to Levine et al.

1994). The carbonyl content was calculated using a molar absorp-ion coefficient for aliphatic hydrazones of 22 mM−1 cm−1, and theesults were expressed as �mol carbonyl group mg−1 protein.

.6. Ascorbate redox state

The content of total ascorbate (AsA + DHA) was determinedy adding the leaf extract to a mixture of potassium phosphateuffer (pH 7.4) containing 2 mM DTT, 0.5% (w/v) N-ethylmaleimide,0% (w/v) TCA, 45% (w/v) H2PO4, 4% (w/v) bipyridyl, and 5%w/v) FeCl3. The reaction was performed at 40 ◦C for 30 min, andhe absorbance was read at 525 nm. The content of AsA + DHAnd AsA was estimated using l-ascorbate as a standard andxpressed as �mol g−1 FW. The oxidized ascorbate (DHA) contentas obtained by subtracting the reduced fraction from the total

ontent (Kampfenkel et al., 1995).

.7. Enzyme assays

Protein extraction for enzymatic assays was performed accord-ng to Zimmermann et al. (2006). The activity of superoxideismutase (SOD; EC: 1.15.1.1) was determined by adding leafxtract to a mixture containing 50 mM potassium phosphate bufferpH 7.8), 0.1 mM EDTA, 13 mM l-methionine, 2 �M riboflavin,nd 75 �M p-nitro blue tetrazolium chloride (NBT) in the dark.he reaction was performed under illumination (30 W fluorescentamp) at 25 ◦C for 6 min. The absorbance was measured at 540 nmGiannopolotis and Ries, 1977). One SOD activity unit (AU) wasefined as the amount of enzyme required to inhibit NBT photore-uction by 50% (Beauchamp and Fridovich, 1971), and the activity

s expressed as AU g−1 FW min−1. The ascorbate peroxidases activ-ty (APX; EC: 1.11.1.1) was assayed after the reaction of the extractn the presence of 50 mM potassium phosphate buffer (pH 6.0) and.5 mM ascorbic acid. The reaction started when 0.1 mL of 30 mM2O2 was added, and the decreasing absorbance at 290 nm wasonitored over 300 s (Nakano and Asada, 1981). The APX activ-

ty was estimated by utilizing the molar extinction coefficient ofscorbate (2.8 mM−1 cm−1) expressed as �mol AsA g−1 FW min−1.he catalase activity (CAT; EC: 1.11.1.6) was determined after theeaction of the extract in the presence of 50 mM potassium phos-hate buffer (pH 7.0) containing 20 mM H2O2. The reaction tooklace at 30 ◦C, with monitoring of the absorbance at 240 nm over00 s (Havir and Mchale, 1987). CAT activity was calculated accord-

ng to the molar extinction coefficient of H2O2 (36 mM−1 cm−1) ands expressed as �mol H2O2 g−1 FW min−1.

.8. Catalase and superoxide dismutase zymograms

Native electrophoresis was performed at 4 ◦C on a 1.5 mm poly-crilamide mini-slab gel in standard tris-glycine buffer (pH 8.3).amples of 10 �L (20 �g protein per lane) for SOD and 20 �L (40 �grotein per lane) for CAT were loaded into each lane and thenlectrophoresed at 80 V through the stacking gel for 15 min and

t 120 V through the separating gel for 60 min. In-gel detection ofOD isoenzymes was done via nitroblue tetrazolium (NBT) reduc-ion by photochemically generated superoxide radicals as originallyescribed by Beauchamp and Fridovich (1971). The identification

100 3.32b 92a 50b 482b150 2.98c 89a 23c 666a200 2.48d 92a 12d 626a

of SOD isoforms was performed by the inhibition-specific assaysutilizing H2O2 and KCN according to Martinez et al. (2001). For CATactivity, the gels were stained according to Chandlee and Scandalios(1984) as follows: the gels were soaked in 0.01% H2O2 solution for5 min, washed twice in water and incubated for 5 min in 1% of bothFeCl3 and K3[Fe(CN)6].

2.9. Statistical analysis

The data were analyzed by a completely randomized design withfive treatments (Experiment I) and a factorial design 2 × 2 (Experi-ment II) with four replicates per treatment in each experiment. Thereplicates were represented by an individual pot containing a sin-gle plant. The data were analyzed by ANOVA and the means werecompared by the Tukey test at a 0.05 probability level.

3. Results

3.1. Physiological and oxidative stress indicators in plantssubjected to salinity and heat

Cashew plants were subjected to increasing NaCl concentrationsin greenhouse conditions under high temperatures (Experiment I).Leaf dry weight (DW) was reduced by 24% in plants exposed to100 mM NaCl for 15 days in a greenhouse, compared with the con-trol. In Experiment II, the heat treatment, alone or in combinationwith NaCl 100 mM, did not change the biomass of the salt pre-treated plants (data not shown). The leaf relative water content wasnot altered by the salt treatments, but the stomatal conductancewas strongly reduced by salinity (Table 1). The leaf Na+ contentincreased 300% in plants subjected to 100 mM NaCl.

The leaf K+ leakage increased approximately 50% in plantstreated with 100, 150 and 200 mM NaCl, compared with controls(Fig. 1A). The protein carbonylation (an oxidative stress indica-tor) increased 25% at all NaCl concentrations higher than 100 mM(Fig. 1B), but the leaf H2O2 content was not affected by salttreatments (Fig. 1C). Unlike what was observed in ExperimentI, salt-treated plants under controlled conditions (27 ◦C) showedincreased H2O2 (37%) and TBARS (38%) content, compared with thatof controls (Fig. 2A and B). Heat alone increased H2O2 and TBARScontent by 37% and 30%, respectively, whereas the combination of100 mM NaCl with heat did not affect H2O2 and TBARS levels.

3.2. High temperature favored the activities of SOD, APX, CAT andprotein expression in salt-stressed plants

In Experiment I, SOD activity was not changed by salttreatments, and zymogram analysis revealed five isoforms, char-acterized as two Fe–SODs and three Cu–Zn–SODs (Fig. 3A). The

activities of all five isoforms were unchanged by salt treatment.In Experiment II, SOD activity was not changed by isolated salttreatment (27 ◦C), but isolated heat and the combination of NaCland heat significantly increased SOD activity compared to controls

S.L. Ferreira-Silva et al. / Environmental and Experimental Botany 74 (2011) 162– 170 165

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Fig. 1. Electrolyte leakage (A), content of protein carbonyl groups (B) and hydrogenperoxide (C) in leaves of cashew plants exposed to increasing NaCl concentrationsover 15 days in a greenhouse under high temperatures. The bars represent the mean,at

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Fig. 2. Content of hydrogen peroxide (A) and TBARS (B) in leaves of cashew plants

200 mM NaCl treatment (Fig. 6A). The ascorbate (AsA/AsA + DHA)

nd the error bars show the standard deviation of four replicates. Bars marked withhe same letter did not differ at the 0.05 probability level (Tukey’s test).

Fig. 4A). These results indicate that heat-induced upregulation ofOD activity might have contributed to the prevention of lipid per-xidation.

In Experiment I (greenhouse), the APX activity was not alteredy 50 mM NaCl, but it decreased progressively as the NaCl level

ncreased, reaching a 50% decrease in 200 mM NaCl compared to

he control (Fig. 3B). In contrast, in Experiment II, APX activity wastrongly stimulated by heat (70%). In plants exposed to the com-ination of NaCl and heat, it was upregulated by (44%), but NaCl

exposed to isolate and combined stresses with NaCl and heat in a chamber growth.The data represent the mean of four replicates. In each column, the values followedby the same letter did not differ at the 0.05 probability level (Tukey’s test).

alone caused a 21% decrease compared to the control (Fig. 4B). TheCAT activity was markedly stimulated by NaCl-salinity in the green-house, reaching a 470% increase in the 100 mM treatment group(Fig. 3C). At NaCl levels higher than 100 mM, the activity decreasedprogressively but still reached a 33% increase in 200 mM NaCl, com-pared with the control.

The CAT zymogram showed results consistent with total activitymeasurements, but it was not possible to separate CAT isoenzymes(Fig. 3C). In Experiment II, CAT activity was increased (40%) by heatalone and the combination of heat and NaCl, but its activity wasnot changed in salt treatment relative to the control (Fig. 4C). Theheat-induced upregulation of SOD, APX and CAT protein levels wasconsistent with the strong increase in the protein profiles revealedby SDS-PAGE (Fig. 5).

3.3. High temperature and salinity induced changes in theascorbate redox state

The content of total ascorbate (AsA + DHA) was not altered bysalinity, but the content of reduced ascorbate (AsA) decreased (30%)while the oxidized ascorbate (DHA) increased (45%) under the

redox state was about 34% lower in the plants exposed to 200 mMNaCl in relation to the control (Fig. 6B). In cashew plants kept undercontrolled conditions, the data obtained were consistent with those

166 S.L. Ferreira-Silva et al. / Environmental and Experimental Botany 74 (2011) 162– 170

Fig. 3. Total activity and zymogram of SOD (A), total APX activity (B), and totalaNbn

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iapa(rtc

Fig. 4. Total activity and zymogram of SOD (A), total APX activity (B), and totalactivity and zymogram of CAT (C) in leaves of cashew plants exposed to isolated

ctivity and zymogram of CAT (C) in leaves of cashew plants exposed to increasingaCl concentrations over 15 days in a greenhouse under high temperatures. Thears represent the mean of the four replicates. Bars marked with the same letter didot differ at the 0.05 probability level (Tukey’s test).

btained in the greenhouse, especially the salt-induced changes inhe ascorbate redox state. The total ascorbate content was highestn the combined NaCl and heat treatment followed by isolated heat,

hereas in the salt alone treatment it was decreased (Fig. 6C).The content of reduced ascorbate (AsA) significantly decreased

n all treatments, and dehydroascorbate (DHA) increased in heatnd combined NaCl and heat treatments compared to controllants. Ascorbate redox state (AsA/AsA + DHA) decreased in NaCllone and showed further reductions in combined heat and NaCl

Fig. 6D). Under acute salt stress (150 and 200 mM of NaCl), theesults suggest a coordinated action of CAT and APX together withhe ascorbate redox balance to remove H2O2 excess with a directhemical reaction between ascorbate and H2O2.

and combined stresses with NaCl and heat in a chamber growth. The data representthe mean of four replicates. In each column, the values followed by the same letterdid not differ at the 0.05 probability level (Tukey’s test).

4. Discussion

4.1. High temperature favors oxidative protection in salt-stressedcashew plants

The results obtained in this study strongly suggest that cashew

is a species singularly adapted to high temperature. This adaptationis clearly associated with up-regulation of proteins that could con-tribute to increased activity of protective enzymes. Heat stimulated

S.L. Ferreira-Silva et al. / Environmental and E

Fig. 5. Soluble protein SDS-PAGE of cashew leaves subjected to the isolated andcw2

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ombined stresses with NaCl and heat in a chamber growth. The protein separationas performed in 12.5% polyacrylamide gel containing 0.1% SDS. Each well contained

0 �L of protein extract.

he activities of SOD, APX and CAT (under controlled conditions),ontributing to better oxidative protection under high salinity con-itions. High temperature also favored the AsA redox buffer, eitherlone or in combination with salt stress. Thus, our data show aomplex synergistic and beneficial interaction between high tem-erature and salinity in terms of oxidative metabolism.

The combination of salinity with heat favored oxidative pro-ection (indicated by the decrease in the H2O2 and TBARS levels),ompared with plants subjected to heat or salt alone. The interpre-ation of this interaction is complex because a possible beneficialole of the salinity on oxidative protection in those conditions can-ot be excluded. On the other hand, the role of heat was apparentlyost important in the upregulation of antioxidative enzymes com-

ared to salinity because salt alone did not induce increases in theAT, SOD and APX activities. Recent studies have demonstrated thatlants exhibit complex responses when they are simultaneouslyubjected to combined abiotic stresses (Rizhsky et al., 2002, 2004;

ang et al., 2003; Mittler, 2006).The data reported here suggest that cashew plants are unusual

nd that their responses are unexpected because exposure to tem-eratures as high as 42 ◦C for 12 h is usually deleterious to corelant functions (Rizhsky et al., 2004; Mittler, 2006; Wahid et al.,007; Silva et al., 2010). Furthermore, a beneficial effect triggeredy high temperature in improving oxidative protection in plantsubjected to high salinity has not been previously reported. Cashews a species adapted to and widely cultivated in semi-arid regions ofrazil and Asia, where is frequently subjected to hot, dry and salineonditions (Silveira et al., 2003; Abreu et al., 2008; Ferreira-Silvat al., 2010).

The upregulation triggered by heat, alone or in combination

ith salinity, stimulated the antioxidative defense enzymes rep-

esented by increases of SOD, APX and CAT activities. Althoughalinity caused moderate increases in protein carbonylation underreenhouse condition, the H2O2 concentration and lipid peroxi-

xperimental Botany 74 (2011) 162– 170 167

dation remained unchanged under salinity combined with hightemperature. Thus, the low level of oxidative stress observed insalt-stressed cashew leaves was associated with a more effectiveantioxidant system triggered by heat. It has been widely reportedthat the SOD, APX, and CAT enzymes play an essential role in thedefense against oxidative stress generated by salinity (AzevedoNeto et al., 2006; Ashraf and Ali, 2008), and the activity of theseenzymes is upregulated by heat in other plant species (Ali et al.,2005; Guo et al., 2006; Silva et al., 2010). However, the biochemi-cal mechanisms that control the interactions between salinity andhigh temperature in terms of oxidative metabolism are virtuallyunknown (Mittler, 2006).

The salinity and heat (isolated or in combination) can triggerdistinct networks involving perception, signal transduction cas-cades and gene expression (Mittler, 2002; Wang et al., 2003; Kantet al., 2008). The oxidative metabolic network plays a central role inthese stress conditions, but the biochemical, molecular and phys-iological mechanisms in plants grown under those conditions arepoorly understood (Foyer and Noctor, 2005; Mittler, 2006; Silvaet al., 2010). Several works have reported that heat treatment trig-gers several metabolic changes, increasing the expression of severalgenes, transcription factors and genes associated with protectionproteins, such as heat shock proteins (Rizhsky et al., 2004; Kotaket al., 2007; Tian et al., 2009). Heat stress induces expressive alter-ations in oxidative metabolism, which activates gene expressionand increases antioxidative enzyme activities (Mittler et al., 2004;Ma et al., 2008).

The data obtained under controlled conditions indicate thatthe hydrogen peroxide concentrations showed similar trend in alltreatments when compared to those found for lipid peroxidation.In salt-stressed plants grown under high temperature, the H2O2and TBARS content were unchanged. These results demonstratethat salt stressed plants, when in the presence of heat, display amore efficient mechanism against oxidative damage compared tocontrols. These two stressing factors could induce a unique and pos-itive interaction (synergism) compatible with an effective oxidativeprotection. These results reinforce the hypothesis that the sum ofthe response of two individual abiotic stresses is frequently differ-ent from that triggered by the combination of both (Rizhsky et al.,2002, 2004; Mittler, 2006; Silva et al., 2010).

In general, significant H2O2 accumulation in plant tissue mayoccur under salt stress, indicating excessive ROS accumulation bymetabolic disturbances (Huang et al., 2005; Zhu et al., 2007). Hydro-gen peroxide can be scavenged by catalase and other peroxidases(Miller et al., 2010). The maintenance of hydrogen peroxide con-centrations at unchanged levels in salt stressed cashew leavesunder high temperatures and salinity (greenhouse) could be aconsequence of an efficient balance between ROS production andscavenging. Thus, salt stressed cashew plants under high tempera-ture were able to avoid substantial accumulation of H2O2.

4.2. High temperature upregulates the SOD, CAT and APXactivities and alters the AsA redox state which mitigates thesalt-induced oxidative damage

In the greenhouse (high temperature), SOD activity did notchange as the salinity levels increased, probably because the salt-untreated plants (control) were grown under high heat conditions.In this situation, heat may have induced upregulation of SOD activ-ity. Increase in SOD activity in plants previously treated with saltfollowed by heat exposure was associated with unaltered changesin lipid peroxidation. Thus, SOD activity seems to play a central role

in oxidative protection of cashew plants under these conditions.Some reports have shown that high temperatures upregulate SODactivity and gene expression (Gupta et al., 1993; Guo et al., 2006;Tang et al., 2006). SOD is critical for scavenging the O2

•− generated

168 S.L. Ferreira-Silva et al. / Environmental and Experimental Botany 74 (2011) 162– 170

Treatment

NaCl+HeatHeatNaClControl

Asc

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te c

onte

nt( μμ μμ

mol

. g-1

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0

3

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ASA DHA

Asc

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te c

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nt( μμ μμ

mol

. g-1

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0

2

4

6

8

10

ASADHA

Treatment

NaCl+HeatHeatNaClControl

Redox state (%

)

[AsA

/(AsA

+DH

A)]

0

18

36

54

72

Redox state (%

)

[AsA

/(AsA

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A)]

0

14

28

42

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70(A)

(C) (D)

(B)

aa a

a a

aa aa b

b bb a a

a

b

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aa

b

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ca bc

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Fig. 6. The content of total (AsA + DHA), reduced (AsA) and oxidized (DHA) ascorbate, and ascorbate AsA/(AsA + DHA) redox state in the leaves of cashew plants exposed toi res (AD lumn(

b(

wit2eg2ci2ud

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ncreasing NaCl concentrations over 15 days in a greenhouse under high temperatu) in a growth chamber. The data represent the mean of four replicates. In each co

Tukey’s test).

y the electron transport chain in chloroplasts and mitochondriaAlscher et al., 2002; Miller et al., 2010).

The increase in SOD activity by heat alone or in combinationith salt occurred in parallel to upregulation of three Cu–Zn–SOD

soforms (Fig. 4A). These SOD isoforms are essential for the elimina-ion of superoxide radical in chloroplast and cytosol (Alscher et al.,002; Asada, 2006). This strategy is essential in leaf tissues that arexposed to abiotic stresses, such as salt and heat, which are able toenerate superoxide radicals (Song et al., 2005; Munns and Tester,008). Several works have reported that SOD activity is crucial tohloroplast protection against oxidative stress under high salin-ty conditions (Bor et al., 2003; Gomez et al., 2004; Elkahoui et al.,005). Thus, the increase in the activities of these SODs isoformsnder salt stress is essential to cell protection against oxidativeamage.

The coordination among SOD and APX activities is crucial tocavenging ROS generated under salt and heat conditions (Songt al., 2005; Asada, 2006). The H2O2 produced by the SOD activ-ty in the water–water cycle is eliminated by chloroplastic APXShigeoka et al., 2002; Asada, 2006). Heat positively modulated APXctivity but salt alone or in combination with high temperatureecreased the enzymatic activity in cashew. Apparently, salt stressxerts negative effects on expression, synthesis and/or activity ofPX isoforms. This enzyme has several isoforms that are widelyistributed in plant cell (Shigeoka et al., 2002). Some works haveeported that that salinity may decrease the APX activity in some

lant species (Gomez et al., 2004; Vital et al., 2008; Xie et al., 2008).

The thylakoid and stoma APX isoforms are crucial for scav-nging excess H2O2 in chloroplasts (Shigeoka et al., 2002). Recenttudies have reported that these two isoforms operate in concert

and B) and subjected to isolated and combined stresses with NaCl and heat (C and, the values followed by the same letter did not differ at the 0.05 probability level

with the cytosolic-APX 1 isoform (Davletova et al., 2005; Marutaet al., 2010). Cytosolic APX isoforms contribute markedly to oxida-tive homeostasis, signalization and chloroplast protection againstoxidative damage generated by several abiotic stresses (Song et al.,2005). Recent efforts highlight that APX is the main cytosolicH2O2-scavenging enzyme involved with H2O2-signaling and thatcrosstalk between APX and CAT plays a role in the control of theintracellular H2O2 levels (Mittler, 2002; Palatnik et al., 2002).

Catalase activity was strongly stimulated by salinity in thegreenhouse (high temperature) and in the combination of NaCland heat (chamber experiment). These results suggest that heatis the main factor responsible for CAT upregulation, demonstrat-ing again that oxidative protection in this species is dependenton high temperature. Catalase is important for the elimination ofH2O2, especially when it accumulates to high concentrations dur-ing photorespiration (Foyer and Noctor, 2000; Foyer et al., 2009).Catalase activity might also suppress excess H2O2 in other cell com-partments due to H2O2 leakage across the peroxisome membrane(Mittler, 2002). Several studies have reported that heat and salinitymay induce increases in CAT activity (Guo et al., 2006; Ali et al.,2005; Demiral and Türkan, 2005; Sekmen et al., 2007).

The salt-induced upregulation of CAT activity from 50 mM to150 mM NaCl was followed by a significant decrease under a veryhigh salinity level (200 mM NaCl). At this salt level, APX activity wasalso decreased. Therefore, at least theoretically, the role of thesetwo enzymes in the removal of the H2O2 could have been compro-

mised. However, the plant cells have several redundant pathwaysinvolved with H2O2-scavenging, represented by several peroxi-dases and non-enzymatic compounds such as ascorbate (Palatniket al., 2002). Indeed, the maintenance of unaltered levels of H2O2 in

and E

cpb2

aa(haahidircyba

stshsspr

rjmar

A

emf

R

A

A

A

A

A

A

A

B

B

S.L. Ferreira-Silva et al. / Environmental

ashew treated with very high salinity could have involved othereroxidases as well as a direct chemical reaction between ascor-ate and H2O2 involving AsA redox buffering power (Del Río et al.,006).

Ascorbate peroxidase catalyzes H2O2 removal, utilizing reducedscorbate (AsA) as the electron-specific donor, and its increasedctivity may change the AsA/DHA ratio towards the oxidized formNoctor and Foyer, 1998). In this study, the combination of NaCl andeat decreased AsA content and increased DHA content, indicating

possible utilization of AsA as a direct scavenger of H2O2, as APXctivity was decreased (Del Río et al., 2006). This mechanism mayave contributed to suppression of H2O2 accumulation, lipid perox-

dation and consequent high oxidative damage under conditions ofecreased APX and CAT activities. Ascorbate is widely distributed

n plant cells, especially in the chloroplasts where they act as aedox buffer system (Smirnoff, 2000). In the ascorbate–glutathioneycle, H2O2 removal by APX consumes reduced ascorbate (AsA) andields its oxidized form (DHA), which is reduced by dehydroascor-ate reductase (DHAR) using glutathione (GSH) as substrate (Foyernd Noctor, 2000).

The highlight of this study is that the combination of twotressful factors with potential to trigger an antagonistic interac-ion caused a synergism in terms of oxidative protection for thealt stressed plants. The adaptive response to the combination ofeat and salinity activated by cashew apparently involved exclu-ive adaptive responses which are not triggered by either of thesetresses alone. These unique responses may be typical of adaptedlants to extreme environments, such as those of arid and semi-aridegions.

The results allow us to conclude that high temperature isequired to upregulate oxidative defense in cashew leaves sub-ected to salt stress. This protection mechanism involves the

odulation of the activities of catalase, superoxide dismutase andscorbate peroxidase as well as favorable changes in the ascorbateedox state under acute salt stress condition.

cknowledgements

We thank the Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq), Fundac ão Cearense de Apoio ao Desenvolvi-ento Científico e Tecnológico (FUNCAP) and INCT-sal CNPq/MCT

or financial support. J.A.G.S. is an honored researcher of CNPq.

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