Different antioxidant responses to salt stress in two different

ORIGINAL PAPERDifferent antioxidant responses to salt stress in two different provenances of Carthamus tinctorius L.Najoua Karray-Bouraoui •Faten Harbaoui •Mokded Rabhi •Ine`s Jallali •Riadh Ksouri •Houneida Attia •Najoua Msilini •Mokhtar Lachaaˆl Received:29September 2010/Revised:22November 2010/Accepted:13December 2010ÓFranciszek Go´rski Institute of Plant Physiology,Polish Academy of Sciences,Krako ´w 2010Abstract Seedlings of two Tunisian Carthamus tinctori-us L.provenances (Kairouan and Tazarka)differing insalt sensitivity were hydroponically grown at 0and 50mM NaCl over 21days.Leaves of Kairouan (salt-sensitive)showed a 48%restriction in their growth at 50mM NaCl although they accumulated less sodium than those of Tazarka (less salt-sensitive)that maintained an unchanged growth.Salt treatment induced oxidative stress in C.tinctorius and the effect was more pronounced in the leaves of the more salt sensitive provenance,Kairouan.Both provenances exhibited a stimulation of antioxidant enzyme activities with higher catalase (CAT)and superoxide dis-mutase (SOD)activities in Tazarka and higher peroxidase (POD)activity in Kairouan.But,it seems that antioxidant activities were more correlated with polyphenol content.Actually,leaves of Tazarka experienced higher polyphenol and antioxidant activity than Kairouan at 50mM NaCl.Hence,moderate salinity (3g NaCl L -1)enhanced bioac-tive molecule yield in the less salt sensitive provenance,Tazarka.In addition, C.tinctorius was found rich inascorbic acid,but the moderate salt stress enhanced its production only in the sensitive provenance.Keywords Antioxidant activity ÁAntioxidant enzymes ÁAscorbic acid ÁFree radical-scavenging ÁPolyphenol concentrationIntroductionAromatic and medicinal plants (AMPs)exhibit from the antiquity,a considerable source of bioactive compounds and flavoring substances that can be used in food,per-fumery,and pharmaceutical industries.AMPs secondary metabolites are commonly responsible for their pre-servative effects,which is correlated with the presence of high amounts of antioxidative and antimicrobial constitu-ents in their tissues (Hirasa and Takemasa 1998).In arid and semi-arid regions,AMPs are often subjected to severe environmental conditions (salinity,drought,light stress,extreme temperatures,etc.)and their productivity depends on several environmental factors (Belaqziz et al.2009).Hence,an optimization of their culture conditions is nee-ded to improve their yields.Such an optimization should take into account two major parameters:biomass produc-tion and secondary metabolite yield.In Tunisia,arid and semi-arid regions cover about two-third of the total area where salinity is a major factor affecting plant growth and yield (Hachicha and Mtimet 1994).Currently,salt-affected soils in Tunisia cover 10%of the total area of the country (Hachicha 2007).In these regions,AMPs are often irrigated with relatively saline waters.Salinity induces a wide variety of plant responses ranging from altered gene expression and cellular metabolism to changes in growth rates and crop yieldsCommunicated by R.Aroca.N.Karray-Bouraoui and F.Harbaoui equally contributed to the elaboration of this work.N.Karray-Bouraoui (&)ÁF.Harbaoui ÁH.Attia ÁN.Msilini Áchaaˆl Unite´de Physiologie et de Biochimie de la Tole ´rance au Sel des Plantes,Faculte´des Sciences de Tunis,Campus Universitaire,2092Tunis El Manar,Tunisiae-mail:najouakarraybouraoui@yahoo.fr M.Rabhi ÁI.Jallali ÁR.KsouriLaboratory of Extremophile Plants (LPE),Biotechnology Centre of Borj Cedria (CBBC),P.O.Box 901,2050Hammam-Lif,TunisiaActa Physiol PlantDOI 10.1007/s11738-010-0679-3(Munns and Tester2008).Currently,NaCl seemed to be the most abundant salt found in salty environments because of its ability to compete with various nutrients, especially with K?,resulting in nutrient deficiency and specific toxicity(Tester and Davenport2003).One of the major traits of salt tolerance at the cell level is sodium sequestration in vacuoles,preventing in this way meta-bolic activities from its harmful effects.To maintain equal osmotic potentials in vacuoles and cytoplasm,solutes that do not disturb cell metabolism are accumulated in cyto-plasm(Tester and Davenport2003;Munns and Tester 2008).If this sodium sub-cellular compartmentalization is not sufficient,oxidative stress can occur due to an enhanced production of reactive oxygen species(ROS) (Ashraf2009).Levels of ROS are regulated by their rate of generation,their rate of reaction with target substances (such as proteins,lipids,and/or nucleic acids),their potential rate of degradation,and their rate of scavenging/ neutralizing by enzymatic and/or non-enzymatic antioxi-dants(Hodges2003).Their accumulation leads to lipid peroxidation and a loss of membrane integrity(Sairam and Srivastava2002).Hence,to survive and protect their tissues from toxic effects of these potentially cytotoxic species of activated oxygen,plants evolved different adaptive mechanisms to reduce salt-induced oxidative damages.This involves a cascade of antioxidants stopping the propagation of oxidative chain reactions(Hajlaoui et al.2009).These antioxidants are usually grouped into enzymatic and non-enzymatic systems.The former include superoxide dismutases(SOD),peroxidases (POD),catalase(CAT),and ascorbate–glutathione cycle enzymes(Noctor and Foyer1998;Smirnoff2005).The latter commonly include glutathione(GSH),ascorbic acid,carotenoids,tocopherols,essential oils,and poly-phenols.Phenolic compounds and ascorbic acid constitute the main powerful natural antioxidants(Balasundram et al.2006;Herna´ndez et al.2010).They are also highly applied in food industry,cosmetic,pharmaceutical,and medicinal materials(Maisuthisakul et al.2007).They retard or inhibit lipid autoxidation by acting as radical scavengers(Namiki1990)and,consequently,are essen-tial antioxidants and protect cells against oxidative chain propagation.In plants,polyphenol synthesis and accu-mulation is generally stimulated in response to biotic and abiotic stresses(Naczk and Shahidi2004),such as salinity (Navarro et al.2006).Enhanced synthesis of secondary metabolites under stressful conditions is believed to pro-tect cells from oxidative effects(Abdul-Jaleel et al.2007). The presence of these natural antioxidants gives plants a high capacity to tolerate salinity.This work was under-taken to study physiological behaviors and leaf antioxi-dant responses to salinity in two Tunisian provenances of Carthamus tinctorius,one of the most important species of the family Asteraceae and one of the most widespread spices in the world.MethodsPlant materialC.tinctorius seeds were collected in two Tunisian sites: Tazarka and Kairouan.In both sites,safflower plants are irrigated with relatively saline water containing about4g NaCl L-1.Culture conditionsSeeds were sterilized in a diluted sodium hypochlorite solution,washed for15min,imbibed for1day in distilled water,and then sown in Petri dishes with wetfilter paper for germination in the dark in a growth room at25°C. Seven days after germination,seedlings were transferred into individual pots containing1L aerated,fourfold diluted nutrient solution of Hoagland and Arnon(1950).Photo-period was16h with150l mol m-2s-1PAR at the plant level.Day/night temperature and relative humidity regimes were22/18°C and60/80%,respectively.Three weeks after germination,an initial harvest was performed(7plants per provenance).Then,two treatments were started.In thefirst, seedlings were cultivated in the same nutrient solution and considered as control.In the second treatment,50mM NaCl was added to the medium.Thefinal harvest was performed after21days of treatment and leaves were sampled,then oven-dried at60°C(for3days)for the determination of dry weights and sodium(Na?)concen-trations(seven replicates per treatment).Besides,fresh leaf samples(4th pair from the apex)from each plant were immediately frozen in liquid nitrogen and stored at-80°C until performing biochemical analysis.Determination of Na?concentrationFrom each of the oven-dried leaf samples,0.2g was digested with25mL nitric acid(HNO30.5%).Na?con-centrations in the digests were determined with aflame emission photometry(Jenway PFP7).Measurement of electrolyte leakageElectrolyte leakage was determined as described by Dionisio-Sese and Tobita(1998).Leaf samples(approxi-mately200mg fresh weight)were submerged into10mL distilled water and kept at32°C over2h.Then,the initial electrical conductivity of the medium(EC1)was measured. After that,the leaf tissues were killed by autoclaving atActa Physiol Plant121°C for20min to release all electrolytes,cooled to 25°C,and then thefinal electrical conductivity(EC2)was measured.The electrolyte leakage(EL)was calculated as EL=1009(EC1/EC2).Determination of lipid peroxidationSalt-induced oxidative damage(membrane lipid peroxi-dation)was assayed by measuring the amount of mal-onyldialdehyde in0.5g leaf fresh weight according to Buege and Aust(1972).For MDA extraction,0.5g from each leaf sample was homogenized with5mL TCA(tri-chloroacetic acid0.1%).The homogenate was centrifuged for5min at10,000g.For each milliliter of the aliquot, 4mL TCA(20%)containing TBA(thiobarbituric acid 0.5%)was added.The mixture was heated at95°C for 30min and then cooled quickly on an ice bath.Afterwards, the mixture was centrifuged for10min at10,000g and the absorbance of the supernatant was measured at532nm. Measurements were corrected for unspecific turbidity by subtracting the absorbance at600nm.The concentration of MDA was calculated using an extinction coefficient of 155mM-1cm-1.Protein extraction and enzyme assaysAliquots of frozen leaf material were ground to afine powder with liquid nitrogen and soluble proteins were extracted with50mM potassium phosphate buffer(pH7), 1mM EDTA,1mM dithiothreitol,5%polyvinylpirroli-done,and5%glycerol.The homogenate was centrifuged at 13,000g for10min and supernatant was used for the determination of soluble protein concentration and anti-oxidative enzyme activities.Protein concentration was determined according to Bradford(1976)using bovine serum albumin as a standard.Catalase(CAT)activity was measured spectrophoto-metrically according to the method of Aebi(1984)by monitoring the decline in the absorbance at240nm,as H2O2 was consumed.Thefinal volume(3mL)of the reaction mixture contained50mM sodium phosphate buffer(pH 7.0),to which30%H2O2was added(OD=0.52–0.55at 240nm).The reaction was activated by adding100mL of the leaf crude extract to this solution.CAT activity was expressed as units(l mol of H2O2decomposed per minute) per milligram of protein.Staining for CAT was performed using the method of Woodburry et al.(1971).The gel was soaked in5mM H2O2for10min.After a brief rinse,CAT isozymes were detected by incubating the gels in1%(w/v) ferric chloride and1%(w/v)potassium ferricyanide until achromatic bands appeared.For the measurement of guaiacol peroxidase(POD) activity,the reaction mixture contained25mM phosphate buffer(pH7.0),0.05%guaiacol,10mM H2O2,and enzyme.POD activity was determined by the increase in absorbance at470nm due to guaiacol oxidation(Nakano and Asada1981).POD isoforms were visualized on gels according to Vallejos(1983).The gel wasfirst incubated for30min in a0.1M sodium acetate buffer(pH4.0) containing1%(v/v)guaiacol.Then,the revelation was achieved in a solution containing afinal concentration of 4.7mM3-amino-9-ethylcarbazole,38mM N,N-dimethyl formamide,0.1M sodium acetate buffer(pH5.0),CaCl2 (0.1M),and H2O230%.SOD activity assays were performed by running45l g protein aliquots by12%acrylamide native PAGE gels at 4°C.Staining was carried out as described by Beauchamp and Fridovich(1971).The gels werefirst soaked in50mM sodium phosphate(pH7.5)containing 4.8mM3-(4,5-dimethylthiazol-2-4)-2,5-diphenyl tetrazolium bromide (MTT)in the dark for20min then soaked in50mM sodium phosphate(pH7.5)containing0.4%(v/v) N,N,N0,N0-tetramethylethylenediamine(TEMED),and26l M riboflavin then subsequently illuminated for10min.The three SOD isozymes,Fe-SOD,Mn-SOD,and Cu/Zn-SOD were identified using inhibitors.Mn-SOD was visualized by its insensitivity to5mM H2O2and2mM KCN.Cu–Zn SOD was sensitive to2mM KCN.Fe-SOD was inhibited by5mM H2O2(Navari-Izzo et al.1998).Polyphenol extractionHarvested leaves were dried at room temperature for 1week.Leaf extracts were obtained by magnetic stirring for30min of2.5g of dry powder with25mL acetone (80%).The extracts were then kept for24h at4°C,filtered through a Wattman no.4filter paper,evaporated under vacuum to dryness,and stored at4°C until analyzed.Total phenolic concentrationTotal phenolics were assayed using the Folin–Ciocalteu reagent,following the method of Singleton and Rosi(1965) (based on the reduction of a phosphowolframate–phos-phomolybdate complex by phenolics to blue reaction products)slightly modified by Dewanto et al.(2002).An aliquot of125l L tenth-diluted sample extract was added to0.5mL distilled water and0.125mL Folin-Ciocalteu reagent(Fluka,47641).The mixture was shaken and after 6min,it was added with 1.25mL Na2CO3(7%).The solution was then adjusted with distilled water to afinal volume of3mL and thoroughly mixed.After incubation in the dark,its absorbance was read at760nm versus the prepared blank.Leaf total phenolic concentration was expressed as milligrams gallic acid equivalents per gram dry weight(mg GAE g-1DW)through the calibrationActa Physiol Plantcurve with gallic acid.All samples were analyzed in three replicates.Antioxidant assaysEvaluation of total antioxidant capacityThe assay is based on the reduction of Mo(VI)to Mo(V)by the extract and subsequent formation of a green phosphate/ Mo(V)complex at acid pH(Prieto et al.1999).An aliquot (0.1mL)of leaf acetonic extract(described in polyphenol extraction paragraph)was added to1mL reagent solution (0.6M sulfuric acid,28mM sodium phosphate,and4mM ammonium molybdate).Tubes were incubated in a thermal block at95°C for90min.Thereafter,the mixture was kept at ambient temperature;the absorbance of each solution was measured at695nm(Anthelie Advanced2,SECOMAN) against a blank.The antioxidant capacity was expressed as milligram gallic acid equivalent per gram dry weight (mg GAE g-1DW).The calibration curve range was 0–500l g mL-1.All samples were analyzed in triplicate. Scavenging ability on DPPH radicalDPPH-quenching ability of C.tinctorius extracts was measured according to Hatano et al.(1988).One milliliter of the extract at known concentrations was added to0.5mL DPPH methanolic solution(0.2mM).The mixture was vigorously shaken and left standing at room temperature in the dark for30min.The absorbance was then measured at 517nm and corresponds to the extract ability to reduce the radical DPPH to the yellow-colored diphenylpicrylhydr-azine.The antiradical activity was expressed as IC50 (l g mL-1)that stands for the antiradical dose required to cause50%inhibition.The ability to scavenge the DPPH radical was calculated using the following equation:DPPH scavenging effect(%)=[(A0-A1)/A0]9100where A0 and A1stand,respectively,for the absorbance of the control and the sample both at30min.All samples were analyzed in triplicate.A lower IC50value corresponds to a higher anti-oxidant activity of the plant extract.Extraction and determination of ascorbic acid concentrationThe assay is based on the reduction of Fe3?to Fe2?by ascorbic acid in acid solution(Okamura1980).Then Fe2? forms complexes with bipyridyl,producing a pink color that absorbs at525nm.Aliquots of leaf frozen tissues were ground in a cold mortar using liquid nitrogen.Then,they were deproteinized in ice-cold6%(w/v)trichloroacetic acid,using a cold mortar and a pestle.The mixture was rapidly transferred on cold ice until TCA crystallization.After vigorous shaking,the mixture was centrifuged at 15,000g for10min at4°C.Total ascorbate and dehydro-ascorbate concentrations were determined according to the method of Kampfenkel et al.(1995).An aliquot of0.2mL supernatant was added with0.4mL phosphate buffer (0.2mM,pH7.4),vigorously shaken then incubated for 15min at42°C.Thereafter,1mL TCA(10%),0.8mL orthophosphoric acid(H3PO442%),0.8mL2,20-pipyridyl (4%),and0.4mL FeCl3(3%)were added and well mixed. The reaction mixture(4.0mLfinal volume)was incubated for40min at42°C before measuring the absorbance at 525nm at25°C.After that,DHA was reduced to AsA by pre-incubating each sample with0.2mL dithiothreitol (DTT10mM).Excess DTT was removed with0.2mL N-ethylmaleimide(NEM0.5%)for total AsA determina-tion.DHA concentration was calculated as the difference between those of total ascorbate and AsA.Statistical analysisData were subjected to a one-way ANOVA test using SPSS 11.0for Windows and means were compared according to Duncan’s multiple-range test at5%level of significance. ResultsLeaf growth,water content,and sodium concentrationUnder non-saline conditions,Kairouan provenance expe-rienced significantly higher leaf dry weight than Tazarka provenance(Table1).Under saline conditions,leaf bio-mass production of Tazarka was not affected by salinity, whereas that of Kairouan was reduced by48%.These results were concomitant with a higher leaf Na?concen-tration in Tazarka than in Kairouan.However,leaf tis-sue hydration was not affected by salt stress in both provenances(Table1).Lipid peroxidation and electrolyte leakageThe extent of oxidative damage was estimated as the con-centration of malondialdehyde(MDA),a product of lipid peroxidation.Under non-saline conditions,leaf MDA concentration was5.52and5.97mmol g-1FW,respectively, in Tazarka and Kairouan(Fig.1a).Under saline conditions, both provenances exhibited a significant increase in lipid peroxidation,but this salt-induced effect was more pro-nounced in Kairouan(MDA:9.20mmol g-1FW)than in Tazarka(MDA:6.73mmol g-1FW).These results were concomitant with a significant increase in electrolyte leakage (EL)under salinity in Kairouan as well as in Tazarka.But, huge differences between the two provenances were noticed.Acta Physiol PlantActually,while EL increased by156%in Kairouan,it was increased by only49%in Tazarka.Total protein concentrationThree weeks after salt treatment,leaf protein concentration in Kairouan was reduced by24.2%(Fig.1b).In Tazakara, however,it was not reduced.Leaf antioxidant enzyme activitiesUsing different specific inhibitors(KCN for Cu/Zn-SOD and H2O2for both Cu/Zn-SOD and Fe-SOD),only two SOD isoforms(Cu/Zn-SOD1and Cu/Zn-SOD2)were detected in leaf soluble proteins of the two safflower provenances.In Tazarka,salt treatment did not affect SOD profile,whereas in Kairouan,it led to a noticeable decrease in band intensity of both SOD isoforms(Fig.2a).Kairouan and Tazarka POD activities were significantly enhanced under salt treatment,but NaCl effect was much higher in the former(4times that of the control)than in thelatter(1.4time that of the control;Table2).Similar to these results,the electrophoretic analysis showed that regardless of the treatment,POD activity was relatively high in both provenances.Moreover,this activity signifi-cantly increased in Kairouan under salinity;however in Tazarka,it seems to remain unchanged(Fig.2b).Catalase(CAT)is involved in scavenging hydrogen peroxide formed by the dismutation of superoxide anions catalyzed by SOD.Its activity under non-saline conditions was79and33unit mg-1protein,respectively,in Kairouan and Tazarka.Under salinity,it was decreased by50%in the former and increased by91%in the latter.These resultsTable1Leaf dry weight(DW,mg plant-1),water content(mL g-1DW),and Na?concentration(mmol g-1DW)in two Tunisian C.tinctorius provenances(Kairouan and Tazarka)grown at0and50mM NaCl over3weeksParameters Kairouan Tazarka0mM NaCl50mM NaCl0mM NaCl50mM NaClLeaf dry weight740.5±57.2c383.8±51.2a558.5±51.4b546.5±69.4b Water content 6.94±0.57ab 6.24±0.78a7.42±0.41b 6.72±0.82ab Na?concentration0.04±0.01a 1.74±0.10b0.05±0.01a 2.10±0.15cValues are mean of seven replicates.In each line,values followed by different letters are significantly different according to Duncan’s test at p B0.05050050BActa Physiol Plantwere supported by the electrophoretic analysis that revealed higher CAT intensity in Tazarka and drastically declined one in Kairouan in response to salt stress (Fig.2c).Leaf polyphenol concentration and total antioxidant capacityThe total phenolic assay was performed on dry weight basis(Fig.3a).Leaf concentrations of these compounds ranged from6to7mg GAE g-1DW in the control.In salt-treated plants,they were enhanced by200%in Tazarka and only90%in Karouan.Total antioxidant capacity of C.tinctorius leaf extracts was expressed as gallic acid equivalents per gram dry weight(Fig.3b).Our results revealed a similar behavior in both provenances,with a significant increase in antioxidant capacity under salt stress.However,this increase was more pronounced in Tazarka than in Kairouan.Ascorbic acid concentrationAscorbic acid concentrations indicate a noticeable richness of this species in this natural antioxidant (264–280mg100g-1FW;Fig.4a).Salt treatment induced a high increase in ascorbic acid concentration in Kairouan (403mg100g-1FW)and a slight but significant decrease in Tazarka(222mg100g-1FW).The dehydroascorbate fraction was not affected by salinity in Tazarka(about50% in both treatments),whereas in Kairouan,it was reduced from61%in the control to36%in salt treatment(Fig.4a).DPPH radical-scavenging activityThe antiradical potential of leaf acetonic extracts to scav-enge the synthetic radical(DPPH)is shown in Fig.4b.As evidence by thisfigure,Kairouan extracts exhibited higher activity to quench DPPH,in control medium as well as in saline medium.Salt treatment led to considerably increased IC50values in both provenances.These data suggest that the photosynthetic organs(especially those of Tazarka) lacked efficient antiradical activity.DiscussionBassil and Kaffka(2002)found that safflower response to increasing salinity included reduced plant height,biomass, leaf area,capitula number and order,and earlier matura-tion.The two provenances studied in the present work showed different leaf dry weights.The higher values observed in Kairouan were due to a higher leaf growth rate that could be genetically controlled.In addition,Kairouan was shown to be more sensitive to salt stress than TazarkaTable2Leaf total protein concentration(mg g-1FW)and CAT and POD activities(unit mg-1protein)in two C.tinctorius Tunisian prov-enances(Kairouan and Tazarka)grown at0and50mM NaCl over3weeksParameters Kairouan Tazarka0mM NaCl50mM NaCl0mM NaCl50mM NaClProtein concentration8.76±0.30c 6.64±0.35b 5.06±0.44a 4.90±0.13a POD activity67.4±2.04a275.7±6.9d127.8±5.16b182.4±2.4c CAT activity78.9±8.0c39.0±2.6a32.8±1.6a62.5±4.4bValues are mean of three replicates.In each line,values followed by different letters are significantly different according to Duncan’s test at p B0.05Acta Physiol Plantin terms of leaf biomass production(Table1).Several authors reported that the salt-induced growth reduction can be related to salt osmotic effects,which affect cell turgor and expansion(Rozema and Van Diggelen1991).In our case,the salt sensitivity of Kairouan was not due to an osmotic effect since leaf water content was not signifi-cantly decreased under saline conditions(Table1).These results suggest that sodium ions were absorbed,transported and compartimentalized in vacuoles thus contributing to cell osmotic adjustment(Hasegawa et al.2000).Such sodium partitioning was more efficient in Tazarka as its leaf Na?content was much higher than that of Kairouan. Indeed,sodium sequestration in vacuoles is positively correlated with salt tolerance in plants(Tester and Davenport2003).Salt stress is known to result in extensive lipid peroxi-dation,which has often been used as indicator of salt-induced oxidative damage in membranes(Herna´ndez and Almansa2002).Moreover,cell membrane stability has been largely used as a criterion to distinguish stress tolerant and sensitive cultivars(Premchandra et al.1992).It is presumed that the lowest values of EL and MDA concen-tration obtained in Tazarka leaves could account for less-affected membrane permeability and better protection against oxidative stress under salt stress.Low levels of EL and lipid peroxidation may be considered as one of the reasons for the observed lower sensitivity of Tazarka leaves exposed to50mM NaCl.Similar results were reported by Dionisio-Sese and Tobita(1998)who found that the susceptible variety of rice presented a high level of MDA and higher solute leakage.Demiral and Tu¨rkan (2005)observed in two rice cultivars that MDA level increased under salt stress in the roots of IR28(sensitive cultivar)but showed no change in Pokkali(salt-resistant cultivar).Masood et al.(2006)observed that the low values of EL and MDA concentration obtained with Azolla pin-nata may account for lower lipid peroxidation levels and less-affected membrane permeability.The low values of EL and MDA may explain the observed lower sensitivity of Tazarka leaves treated with50mM NaCl.Salt-treated plants responded to the salt-induced oxida-tive stress by a biosynthesis stimulation of both enzymatic (SOD,POD,and CAT)and non-enzymatic molecules (polyphenols and ascorbic acid).While SOD activity decreased in saline growth medium in Kairouan leaves, SOD activity did not change in salt-stressed leaves of Tazarka.Since SOD are multimeric metalloproteins,they have different isoforms,based on the metals present at their active sites.The most common SOD isoforms in litera-ture are copper/zinc-containing superoxide dismutase (Cu/Zn-SOD),manganese-containing(Mn-SOD),iron-containing(Fe-SOD),and nickel-containing(Ni-SOD) (Ashraf2009).Our results suggest that quenching of superoxide radicals was found to be more important in the chloroplastic compartment,than in the mitochondrial and cytosolic compartments,due to the presence of two Cu/Zn-SOD isoforms.Elkahoui et al.(2005)also postu-lated that after native polyacrylamide gel electrophoresis (PAGE)analysis,five SOD isoenzymes were detected and the increase in SOD activity observed in salt-treated cells of Catharanthus roseus seemed to be mainly due to iso-enzymes SOD1,2,and3.Herna´ndez et al.(2001)also reported that the induction of chloroplastic Cu/Zn-SOD activity in pea plants can constitute a protective role against the production of O2-under salt stress.Recent works showed that salt tolerance is closely related to the efficiency of antioxidant enzymes(Ben Amor et al.2005;Herna´ndez et al.2001).Other authors showed that several enzymes are involved in detoxifying ROS and reducing their damaging effects on plants(Xiong and Zhu0 50 Acta Physiol Plant2002).Superoxide dismutases(SOD)react with superoxide radicals to produce H2O2(Bowler et al.1992),which is converted on water by peroxidases(POD)and eliminated by catalase(CAT).Hence,it has been observed that CAT and POD have a significant role in plant defense against oxidative stress.In C.tinctorius,we found that in Kairouan such a role was especially due to POD,whereas in Tazarka, it was mainly due to CAT(Table2;Fig.2).Similar salt-induced reductions in CAT activity were reported in sev-eral glycophytes and halophytes(Lee et al.2001;Parida et al.2004;Ben Amor et al.2006).Actually,H2O2seems to be an important signal molecule since it acts as a direct link between environmental stresses and cell adaptive responses(Foyer and Noctor2003).Peroxidases are involved not only in scavenging H2O2produced in chlo-roplasts but also in growth and developmental processes (Dionisio-Sese and Tobita1998).Mittal and Dubey(1991) compared two sets of rice cultivars differing in salt toler-ance to determine a possible correlation between peroxi-dase activity and salt tolerance.Our results agree with those of Mittal and Dubey(1991)since we noticed an increase in POD activity in leaves of both provenances with the highest one in the salt-sensitive one(Kairouan). Taken together,the results of growth,lipid peroxidation, and electrolyte leakage show that antioxidant enzyme activities(SOD,CAT,and POD)in the two provenances of C.tinctorius did not award their leaves a complete pro-tection against the deleterious effects of ROS.In addition to the antioxidant enzymes,C.tinctorius response to oxidative stress also involved non-enzymatic molecules such as ascorbic acid and phenolic compounds. The more salt-sensitive provenance showed higher ascorbic acid concentrations under saline conditions than the less sensitive one.This suggests that the indirect involvement of vitamin C in Kairouan was predominant,which explains the high POD activity in this provenance.Actually, ascorbate acts in coordination with glutathione and anti-oxidant enzymes in the ascorbate–glutathione cycle to control hydrogen peroxide concentration within the cell (Asensi-Fabado and Munne´-Bosch2010).But despite these differences,vitamin C concentrations in C.Tinctorius are higher than those found in numerous conventional species announced as part of the alimentary diet,such as carrot (6mg100g-1FW),banana(10–30mg100g-1FW), citrus(40–50mg100g-1FW),spinach(51mg100g-1 FW),and tomato(20–25mg100g-1FW)(Davey et al. 2000).Thus,elevated levels of endogenous ascorbic acid in plants are necessary to offset oxidative stress in addition to the regulation of other plant metabolic processes(Smirnoff 2005).Ascorbate levels and its regenerative enzymes depend on the environmental factors(Davey et al.2000). Subsequently,the high concentrations of this natural anti-oxidant in the leaves of both provenances may strengthen the pool of antioxidants in plants and ensure the protection of their photosynthetic machinery.Total polyphenol concentrations in safflower leaves were higher in Tazarka leaves as compared to those of Kairouan,in control as well as in saline medium.These concentrations were higher than those of several species cited in literature.Similar results were found in Cynara cardunculus(Falleh et al.2008).This abundance of phe-nolic compounds is characteristic of the Asteraceae family as reported by Djeridane et al.(2006).The values found in both provenances of C.tinctorius are equal or slightly exceed those found in Cynara cardunculus and those of other medicinal plants such as Ruta montana,Teucrium polium,and Thymelea hirsuta in which the levels of polyphenols were,respectively,3.13,4.92,and6.81mg GAE g-1DW(Djeridane et al.2006).Phenolic compounds also show antioxidant activities against a variety of oxidisable compounds.They widely occur in the plant kingdom and exhibit a wide range of physiological properties (Falleh et al.2008).In the halophyte Cakile maritima, Ksouri et al.(2007)found that the less tolerant accession, Tabarka,accumulated less polyphenols and showed lower antioxidant activity and higher MDA concentration at 100mM NaCl than the more tolerant accession,Jerba. They hypothesized that the difference in polyphenol accumulation capacity participated in the difference in salt tolerance of the two accessions.Other studies confirmed theirfindings and demonstrated that the salt stimulating effect on total polyphenols in a species depends on its salt tolerance or sensitivity(Falleh et al.2008;Hajlaoui et al. 2009).Nevertheless,their roles in various plant species under saline conditions need more investigations to be understood(Wahid and Ghazanfar2006).Salt treatment improved antioxidant activity in the leaves of both provenances.However,Tazarka,the less sensitive one,exhibited higher antioxidant activity under saline as well as under non-saline conditions.In salt-treated plants,this activity showed a positive correlation with polyphenol concentrations,with higher R2values in Taz-arka(Table3).Similar results were found in Mentha pulegium by Karray-Bouraoui et al.(2010)who suggested Table3Determination coefficient(R2)between leaf total polyphe-nol content(mg GAE g-1DW)and its whole antioxidant activity(mg GAE g-1DW)in two Tunisian C.tinctorius provenances(Kairouan and Tazarka)grown at0and50mM NaCl over3weeks Parameters Kairouan Tazarka0mMNaCl50mMNaCl0mMNaCl50mMNaCl R20.1970.8320.1800.711Acta Physiol Plant。