Lipid peroxidation and antioxidative response in Arabidopsis thaliana exposed to cadmium and

ORIGINAL PAPERLipid peroxidation and antioxidative response in Arabidopsis thaliana exposed to cadmium and copperEwa Sko ´rzyn ´ska-Polit ÆMaria Dra ˛_zkiewicz ÆZbigniew KrupaReceived:26January 2009/Revised:30June 2009/Accepted:27August 2009/Published online:15September 2009ÓFranciszek Go´rski Institute of Plant Physiology,Polish Academy of Sciences,Krako ´w 2009Abstract Formation of lipid hydroperoxides,malondial-dehyde (MDA)and hydroxyalkenals (HAEs),membranedamages and antioxidative response of plants expressed as changes in glutathione S -transferase activity (GST)and anthocyanin accumulation were studied in Arabidopsis thaliana (L.)Heynh cv.Columbia plants treated for 7days with various concentrations:5,25,50,100l M Cd and Cu.Increased lipid hydroperoxide content was metal concen-tration-dependent.The level of MDA ?HAE was elevated in Cd-and Cu-treated plants,but it was metal concentra-tion-dependent under Cu stress.Electrolyte leakage measurements showed a larger membrane damage under Cu-than Cd-treatment.In Cu-stressed plants,GST activity was always enhanced in comparison with control,while in plants exposed to Cd it dropped slightly at lower metal concentrations;but at 100l M Cd it was even higher than in plants treated with the same Cu concentration.Antho-cyanin accumulation was considerably higher under Cu than Cd stress.Both lipid peroxidation and antioxidative response was stronger in Cu-than Cd-treated Arabidopsis thaliana plants.Various mechanisms of defense against the lipid peroxidation products,depending on the metal type,are discussed.Keywords Anthocyanin ÁArabidopsis thaliana ÁCadmium ÁCopper ÁGlutathione-S -transferase ÁLipid peroxidationAbbreviations BHT 2(6)-Di-tert -butyl-p -cresol CDNB 1-Chloro-2,4-dinitrobenzene GST Glutathione S -transferase HAEs Hydroxyalkenals MDA Malondialdehyde LOX LipoxygenaseIntroductionHeavy metals are environmental contaminants emanating from industries,anthropogenic activities and use of agro-chemicals.Among them,Cd is considered biologically as a non-essential element,although at low concentration it was found to have stimulatory effect on root growth of Allium sativum (Liu et al.2003),and in marine diatoms T.weissflogii was able to replace the Zn in the specific carbonic anhydrase (CA)(Lane and Morel 2000).A few terrestrial plant species,e.g.Thlaspi sp.(Lombi et al.2000)and Arabidopsis halleri (Bert et al.2002)were reported to accumulate high concentration of Cd without any symp-toms of its toxicity.However,at high concentration it is extremely toxic to most of plants as well as animals.As a consequence Cd exposed plants show various symptoms of injury such as chlorosis,growth inhibition,reduction in water and nutrient uptake (Das et al.1997;Sanita di Toppi and Gabbrielli 1999;Siedlecka et al.2001),and changes in photosynthetic activity (Krupa 1999),induction/inhibitionCommunicated by M.N.V.Prasad.Present Address:E.Sko´rzyn ´ska-Polit (&)Department of Plant Physiology and Biotechnology,The John Paul II Catholic University of Lublin,Konstantynow1H,20-708Lublin,Poland e-mail:eskorzynska@kul.lublin.pl M.Dra ˛_zkiewicz ÁZ.Krupa Department of Plant Physiology,Maria Curie-Skłodowska University,Akademicka 19,20-033Lublin,PolandActa Physiol Plant (2010)32:169–175DOI 10.1007/s11738-009-0393-1of enzymes(Siedlecka et al.2001),damage to proteins (Romero-Puertas et al.2002)and lipid matrix(Shah et al. 2001).Another heavy metal Cu,which is an essential micronutrient for plants,can also be a toxic element when applied in amounts higher than its optimal level.Its excess induced alterations in the photosynthetic and respiratory processes,enzyme activity,DNA,and membrane integrity resulting in growth inhibition or in plant senescence or even in plant death(Maksymiec1997;Dra˛_z kiewicz et al. 2004;Demirevska-Kepova et al.2004).Among plants’response to heavy metals the changes in LOX activity were found(Gallego et al.1996;Sko´rzyn´ska-Polit and Krupa2003;Djebali et al.2005).Lipid peroxi-dation expressed as MDA level was shown to increase under heavy metal stress(Gallego et al.1996;Cho and Park2000;Shah et al.2001).Although lipid peroxidation is a natural metabolic process in the cell,it is still an unclear phenomenon occurring in plants and animals.It is suggested to be responsible for numerous effects observed in the biological systems(see Spiteller1996;2003).The reaction can occur both in non-enzymatic and enzymatic way,and lipid hydroperoxides(LOOH)are formed as by-products.Lipoxygenases(LOXs)are engaged in enzymatic lipid peroxidation.They transform polyun-saturated fatty acids to LOOH that are converted to dif-ferent secondary compounds.In non-enzymatic processes, reactive oxygen species(ROS)formed in different cell compartments(especially superoxide radical O2•)and transition metal ions are engaged in the initiation of lipid peroxidation(Kappus1985;Shewfelt and Purvis1995). Non-enzymatic lipid peroxidation starts after hydrogen abstraction from an unsaturated fatty acid and a lipid rad-ical is formed on adding molecular oxygen,and after that radical chain reaction can occur(Kappus1985).During enzymatic lipid peroxidation,the peroxyl radicals(LOO•) are transformed within the enzyme complex to corre-sponding anions(LOO-)and therefore lose their reactivity, whereas peroxyl radicals generated in non-enzymatic reactions are not deactivated(Spiteller2003).They react with molecules in their close vicinity causing changes in their molecular structure resulting in cell death.However,there are defense mechanisms in the cells protecting them against the lipid peroxidation.Low molecular mass antioxidant like ascorbate(AA),glutathi-one(GSH),tocopherols and carotenoids are especially important,because they directly scavenge free radicals (Cadenas1989;Shewfelt and Purvis1995;Bartosz1997). As long as reducing agents,like glutathione,or compounds with free phenolic groups are available,the amount of LOOHs is low(Spiteller2003).Flavonoids have phenolic groups which make them perfect scavengers and quenchers of ROS and/or lipid radicals.On the other hand,flavonoids can also chelate metals like Fe2?/3?and Cu?/2?(Rice-Evans et al.1997;Dixon and Steele1999;Duthie and Crozier2000).Tsuda et al.(1996)found that some anthocyanins from Phaseolus vulgaris seed coat had a strong antioxidative activity in the liposomal system and reduced the formation of malondialdehyde by UVB irra-diation.Recently,many reports have focused on glutathi-one S-transferases(GST).These enzymes are suspected to have an important protective function.Plant GSTs attach glutathione to electrophilic compounds(like xenobiotics or flavonoids)which tags them for vacuolar sequestration. Some studies show that GSTs catalyze glutathione-depen-dent isomerization and the reduction of toxic organic hydroperoxides(Edwards et al.2000).We have found an increase in LOX activity in Arabid-opsis thaliana under heavy metal stress(Sko´rzyn´ska-Polit and Krupa2003).Activity of this enzyme was especially high,when linolenic acid was used as substrate(Sko´rzyn´ska-Polit et al.2006).The high activity of the enzyme may result in enhanced non-enzymatic lipid peroxidation;therefore,we decided to widen our studies and check whether and how the level of non-enzymatic products of lipid peroxidation is changed under heavy metal stress in this plant species.We were interested in GST activity and anthocyanin content which were almost not taken into consideration in relation to lipid peroxidation during studies of plants.Such examina-tions were mainly carried out in vitro and in animals.We hypothesized that GST activity and anthocyanin level play a role in scavenging of lipid peroxidation in plants under heavy metal stress.We also expected the different antioxi-dative response in plants exposed to Cu,which is transition metal,than in plants exposed to Cd.Materials and methodsPlant growth conditionsPlants of Arabidopsis thaliana(L.)Heynh cv.Columbia were cultivated as described earlier(Dra˛_z kiewicz et al. 2003),and they were treated with different concentrations of Cd or Cu:5,25,50and100l M in the form CdSO4ÁH2O or CuSO4Á5H2O,respectively.After7days of heavy metal treatment,leaves of at leastfive plants(for individual metal concentration and control)were cut,weighed,frozen in liquid nitrogen and analyzed.Sample preparation for measurement of lipid peroxidationOne gram of fresh leaves was ground in3mL of20mM phosphate buffer,pH7.4.To1mL of tissue homogenate, 10l l of0.5M BHT in acetonitrile were added to prevent sample oxidation.Determination of lipid hydroperoxidesQuantitative assay for lipid hydroperoxides was carried out using the BIOXYTECH LPO-560test(Oxis,USA).This assay is based on the oxidation of ferrous ions to ferric ions by hydroperoxides under acidic conditions.The ferric ion binds with the indicator dye,xylenol orange to form a stable colored complex.Absorbance of the complex was measured at560nm.To eliminate H2O2interference from the LOOH measurement,samples were pretreated with catalase to decompose the existing H2O2. Determination of MDA?HAE(methanesulfonic acid solvent procedure)Determination of malondialdehyde(MDA)and hydrox-yalkenals(HAE)in leaf extract was carried out using the Oxis Research LPO-586test(Oxis,USA).The application of the hydrochloric acid solvent procedure to estimate only MDA level was inappropriate because of interference with anthocyanins;therefore,methanesulfonic acid solvent procedure was chosen by us.This assay is based on the reaction of the chromogenic reagent N-methyl-2-phenyl-indole with MDA and hydroxyalkenals at45°C.One molecule of either MDA or hydroxyalkenal reacts with two molecules of the reagent to yield a stable chromophore with maximal absorbance at586nm.Electrolyte leakage measurementsLeaf disks(0.2g)(6mm diameter each)from fresh leaves of control and metal-treated plants were washed with 40mL of ultrapure water(MilliQ,Millipore,USA)for 3min to remove surface adhered electrolytes and dried withfilter paper.Samples were placed in test tubes con-taining10mL of ultrapure water and were gently shaken for3h at25°C.Conductivity(L1)was measured using MPC227meter(Mettler Toledo,Switzerland).Then,the samples were heated in a thermoblock at95°C for20min, and thefinal conductivity(L2)was measured.Leakage electrolyte(EL)was defined as EL(%)=(L1/L2)9100. Estimation of GST activityThe plant material was powdered in liquid nitrogen and homogenized in extraction buffer(50mM phosphate buffer, pH7.0)according to Milosevic and Slusarenko(1996).The activity of glutathione S-transferase(GST)[E.C.2.5.1.18] was measured according to Mannervik and Guthenberg (1981).The enzyme activity was determined spectrophoto-metrically at340nm by measuring the formation of the conjugate of glutathione(GSH)and1-chloro-2,4-dinitrobenzene(CDNB)at30°C for3min.The activity of the enzyme was expressed as D A340min-1mg-1protein. Determination of anthocyanin contentAnthocyanin extraction was performed according to Lange et al.(1971)and Bette and Kutschera(1996)in our mod-ification.Anthocyanins were extracted from1g of leaves with20mL of mixture:n-propanol:HCl:H2O(18:1:81, v/v/v).The samples were heated in a boiling water bath for 10min,and then they were incubated for24h in the dark at4°C.Extracts werefiltered,and absorbance was measured at535and650nm.Anthocyanin content was expressed as A535per gram fresh weight after correction for chlorophyll:A535=A535-0.229A650.Statistical analysisThe presented values are the means of the samples from three independent experiments,each in three repli-cates±SE.Kruskal–Wallis one-way analysis of variance on ranks was performed using SigmaStat3.0software for calculations.ResultsThe content of lipid hydroperoxides was elevated in Cd-treated Arabidopsis thaliana plants(Fig.1);their increase was metal concentration-dependent.In plants growing in the nutrient medium with Cu excess,the lowest level of lipid hydroperoxides was observed at25l M Cu;but it was still higher than in control.In these plants enzymatic lipid peroxidation could be switched into non-enzymatic one.At the highest Cu excess in the nutrient solution a very high content(almost22times higher than control)of lipid hydroperoxides was measured(P B0.001).In Cd-treated Arabidopsis thaliana plants,elevated level of MDA?HAE was measured(Fig.2).It was very sim-ilar at individual Cd concentrations(P B0.05).In leaves of the plants growing in the medium with Cu excess,metal concentration-dependent increase in MDA?HAE content was found(Fig.2),and at100l M Cu the level increased to178%of control(P B0.001).Leakage of electrolytes from leaves of Arabidopsis thaliana increased under heavy metal treatment,and it was higher in the plants exposed to Cu than Cd(Fig.3).The highest electrolyte leakage(238%of control)was found in the plants grown at50l M Cu(P B0.001).At5and25l M Cd in the nutrient solution GST activity reached approx.95%of control(Fig.4).At50l M Cd it was close to control,but at100l M Cd in the nutrient solution GST activity increased to151%of control(P B0.001).When Arabidopsis thaliana plants were grown at Cu excess in the nutrient medium GST activity was almost the same as in control (Fig.4),except of plants exposed to 100l M Cu.In these ones the enzyme activity increased to 124%of control (P B 0.01),but it was lower than at 100l M Cd.Similarly,the content of anthocyanins both in Cd-and Cu-treated plants was higher in comparison with control (Fig.5).The level of anthocyanins was exceptionally high (5.5times higher than control)in plants growing for 7days at 25l M Cu in the nutrient medium (P B 0.001).DiscussionThe lipid peroxidation under heavy metal stress as a marker of oxidative stress is a well-known phenomenon (Gallegoet al.1996;Mazhoudi et al.1997;Lozano-Rodriguez et al. 1997;Verma and Dubey2003;Cho and Park2000;Dixit et al.2001;Metwally et al.2005),but these studies were limited to determination of MDA content.In our study both enzymatic and non-enzymatic lipid peroxidation was taken into consideration.During enzymatic lipid peroxidation, where LOX is engaged,the peroxyl radical(LOO•)is not able to escape from the enzyme complex,then peroxyl anion(LOO-)is formed which combines with the proton to lipid hydroperoxide(LOOH)(see Spiteller2003).LOX activity increased in Arabidopsis thaliana plants under Cd-and Cu-stress(Sko´rzyn´ska-Polit and Krupa2003).In the present study,we have found an elevated level of lipid hydroperoxides(which is related to enhanced LOX activ-ity)in the metal-stressed Arabidopsis thaliana plants increased significantly with increasing Cd or Cu concen-tration(Fig.1).Lipid peroxides formed in non-enzymatic reaction are unstable and decompose to form a complex series of compounds,including reactive carbonyl compounds. Polyunsaturated fatty acid peroxides generate malondial-dehyde(MDA)and hydroxyalkenals(HAEs)(Kappus 1985;Uchida2003).HAE is known to be a major aldehyde produced during peroxidation of linoleic acid and arachi-donic acid(Uchida2003).In Cd-treated Arabidopsis thaliana plants,the level of MDA?HAE was elevated.It was independent of the metal concentration but correlated with electrolyte leakage (r=0.8492,P\0.1).Thus,MDA?HAE could be responsible for membrane damage in these plants.How-ever,under Cu excess the level of MDA?HAE increased significantly with increasing the metal concentration in the nutrient medium.The difference in the level of MDA?HAE as well as lipid hydroperoxides in Cd-treated plants in comparison with plants exposed to Cu excess resulted from chemical properties of the metals.Copper as a transition metal can catalyze Fenton type reaction resulting in increased MDA?HAE level in Arabidopsis thaliana leaves.When the supply of substrate exceeds a certain limit,enzymatic lipid peroxidation switches to non-enzymatic lipid peroxi-dation.Then,iron ion liberated from LOX reacts with LOOH molecules to produce alkoxy radical(LO•)in a Fenton type reaction(Halliwell and Gutteridge1990). From this moment,non-enzymatic lipid peroxidation begins.The higher level of hydroperoxides and MDA?HAE in Cu-treated plants as a result of enhanced lipid peroxidation led to disorder of the plasmatic mem-branes,andfinally to the electrolyte leakage which was higher than in Cd-stressed plants.There are no data about relationship between the activity of antioxidative system(which includes GST activity, anthocyanin and GSH content)and lipid peroxidation products in plants under Cd and Cu stress.This part of the antioxidative system is also responsible for scavenging free radicals.The content of GSH decreased in Cu-treated Arabidopsis thaliana(Dra˛_z kiewicz et al.2003),but it increased in Cd-treated plants(Sko´rzyn´ska-Polit et al. 2003)in comparison with control.GSH plays an important role in detoxification of lipid peroxyl radicals.It can directly scavenge the radicals or it participates in the reaction of nucleophilic attack of GSH on hydroperoxides catalyzed by GST(Edwards et al.2000;Schro¨der2001). Thus,the enzyme can catalyze the reduction of lipid hydroperoxides to the less-toxic monohydroxy alcohols (A˚lin et al.1985;Edwards et al.2000).GST activity increased in plants exposed to Cd(Dixit et al.2001; Iannelli et al.2002)and in Scenedesmus bijugatus under Cu stress(Nagalakshmi and Prasad2001).In Cu-treated Arabidopsis thaliana plants GST activity was higher than in control,and its dependency on the metal concentration (despite small differences between GST activity in plants exposed up to50l M Cu and control)resembled the dependency between lipid hydroperoxides generation and metal concentration,because significant correlation was found between GST activity and lipid hydroperoxide level (r=0.9416,P\0.05).Thus,lipid hydroperoxides could activate GST in A.thaliana exposed to Cu.However,both in Cd-and Cu-treated Arabidopsis thali-ana plants activity of GST seems to be not related to anthocyanin level,although anthocyanins have been found to be a substrate for some members of multi-gene family of plant GSTs(Alfenito et al.1998).On the other hand,the transport of anthocyanins in the form of GS-conjugates into the vacuole remains to be elucidated(Schro¨der2001).In Arabidopsis thaliana plants under Cd stress elevated level of anthocyanins was observed,but under Cu excess it strongly increased especially at25l M Cu.Production of anthocyanins increased under heavy metals stress in Zea mays(Rangel and Kordan1987).Krupa et al.(1996) observed various responses of winter rye plants to various metals,from a mild or more pronounced inhibition of anthocyanins accumulation(Pb and Ni),through its strong stimulation(Zn)to without any effect(Cd).The pigments are non-enzymatic antioxidants what was shown by Tsuda et al.(1996)in liposomal system in vitro.Like other flavonoids,they can act as scavengers and quenchers,or they can chelate metals(Rice-Evans et al.1997;Dixon and Steele1999;Duthie and Crozier2000).In in vitro exper-iment,Kondo et al.(1992)obtained Cd-commelinin in which the complexation metal Mg2?was replaced with Cd2?.Conversely to Tsuda et al.(1996),we did notfind an inhibitory effect of anthocyanins on MDA formation.It was particularly visible at25l M Cu,where a high content of anthocyanins was not accompanied by decreased level of MDA?HAE,but it cannot be excluded that thepigment chelated Cu ions.However,a high,although not quite significant correlation(r=0.8098,P\0.1)occur-red between anthocyanins and lipid hydroperoxide levels, when Arabidopsis thaliana were exposed to Cd.Moreover, in these plants the increase in hydroperoxide level was related to LOX activity(Sko´rzyn´ska-Polit and Krupa 2003),and a higher content of MDA?HAE to the ele-vated level of free radicals(O2•and OH•)(Sko´rzyn´ska-Polit et al.2003).The elevated level of MDA?HAE under Cd stress allows us to suggest that liberation of iron from LOX,resulting from a high level substrate free fatty acids (FFAs),did not occur.Thus,in Cd-treated plants enzymatic lipid peroxidation did not switch to non-enzymatic one. The activity of GST was related neither to HAE nor anthocyanin level,but the pigments could be responsible for antioxidative protection against MDA?HAE but without GST participation.Under Cu stress the generation both of lipid hydroper-oxides and MDA?HAE depended on the metal concen-tration.Lipid hydroperoxides seem to be formed in the enzymatic pathway,but additionally switching enzymatic into non-enzymatic peroxidation can take place.In Cu-treated Arabidopsis thaliana GST activity was signifi-cantly correlated with hydroperoxides level.Detoxification of lipid hydroperoxides was postulated as possible function of plant GST(Schro¨der2001and refs therein),and our results confirm it.In Cu-treated Arabidopsis thaliana plants both lipid peroxidation and antioxidative response were stronger than under Cd stress.Summing up,in A.thaliana exposed to Cd or Cu the level of lipid hydroperoxides was significantly dependent on metal concentration.Moreover,MDA?HAE level was also Cu-concentration 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