Erdei, Sára

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Erdei Sára

University of Szeged

Volume 46(3-4):89-90, 2002Acta Biologica Szegediensishttp://www.sci.u-szeged.hu/ABS1Department of Molecular Plant Biology, Faculty of Horticultural Science, Szent István University, Budapest, Hungary,2Plant Physiology Research Group, Hungarian Academy of Sciences, Budapest, HungarySára Erdei1*, Attila Hegedûs1,2, Gábor Hauptmann1,2, József Szalai1,2, Gábor Horváth1,2*Corresponding author. E-mail: serdei@omega.kee.hu89Heavy metal induced physiological changes in the antioxidativeresponse systemProceedings of the 7th Hungarian Congress on Plant Physiology, 2002S2-P07Heavy metal stress induced alterations in the activities ofseveral representatives of the enzymatic antioxidant defensesystem such as guiacol peroxidase (POD), catalase (CAT)and ascorbate peroxidase (APX) were comparatively studiedin barley seedlings treated by Zn and Cd. Although rootswere the main sites of metal accumulation, the oxidativedamage was detrimental mainly in the leaves. Our exper-iments clearly show that the investigated heavy metals havequite different effects on plant metabolism and induce theprotective enzymatic system in different ways.It is inevitable that we should cope with the environ-mental damages induced by increased concentration of heavymetals. One of the common characteristics of these events isthe oxidative stress generating active oxygen species (AOS)(Salin 1987).Plants have evolved various protective mechanisms toeliminate or reduce AOS species. One of them is the enzym-atic antioxidant system, including SOD, POD, CAT andAPX. Each of these enzymes has physiological functionunder non-stressed conditions, but their activity/or quantityis increased under oxidative stress.The comparison of the the antioxidative enzymaticsystem in seedlings exposed to two essentially differentheavy metals, namely Zn and Cd should reveal essentialelements of this defense system. While Zn is an essentialmicroelement that is indispensable for normal plant growthat low concentration and is toxic only at high concentration,Cd has no vital function in plants developing under “natural”conditions.Materials and MethodsPlant materialBarley seedlings (Hordeum vulgare L. cv. Triangle) weregrown in hydroponics of a half strength Hoagland solutionin a growth chamber as described (Hegedüs et al. 2001). Theseven-day-old seedlings were transferred into fresh mediumsupplemented with different concentrations of CdCl2 orZnSO4.Determination of Cd and Zn content contentThe metal content was determined by atomic absorptionspectroscopy (Horváth et al. 1996).Chlorophyll determinationThe chlorophyll content was determined in 80 % acetoneextract of 0.1 g leaf as described by Arnon (1949).Preparation for enzyme activitiesOne g of plant tissue was homogenized with three fold excessof buffer containing 0.05 M Tris (pH 8.0), 0.004 M citricacid, 0.008 M cysteine, 0.005 M ascorbic acid, 0.01 M MgCl2and sucrose (Arulsekar and Parfitt 1986). The tissue extractwas centrifuged at 10000xg for 30 min. The supernatant wasused for further analyses.POD and CAT activities were determined using guaiacoland H2O2 substrates, respectively as described by Chance andMaehly (1955), while the APX activity was measured ac-cording to Nakano and Asada (1981). The activities wereexpressed in mkat/g.fr.w.Results and DiscussionBoth heavy metals have been accumulated in the plantsproportionally with the progress of incubation time and metalconcentration. The primary site of the metal accumulationwas the root.The increasing Cd accumulation resulted in the reductionof the chlorophyll content of leaves but there was no sub-stantial, visible sign of the Zn treatment. The highest (1mM)Zn concentration and the longest (7day) treatment had noeffect on the chlorophyll content of leaves, while 0,3-1mMCd concentrations resulted in 60 % reduction after a 4-daytreatment. The latter observation is not surprising, as Cd hasbeen proved to be an aggressive, oxidative damage inducingagent and an effective competitor for essential metal co-factors participating in the chlorophyll biosynthesis (Sanitádi Toppi and Gabbrielli 1999). Zn-ions at high concentrationalso induce oxidative damage (Chaoui et al. 1997), but at thesame time it is an essential cofactor of chlorophyll-synthe-tizing d-aminolevulinic acid dehidratase. Our observation canbe explained at least in two different ways. For one theintracellular localisation of Zn is different from that of Cd,for the other the antioxidative protective system can over-come the detoriate effect of Zn.To answer these questions we have determined theactivity of characteristic antioxidative enzymes.The POD activity was determined in Zn- and Cd-treatedseedlings, and the data are presented in Figure 1. The PODactivity did not change in roots after heavy metal treatment.90On the contrary, enhanced POD activity was detected inleaves, although the extent of increase was different in Zn andCd treated seedlings. Cd-treatment even at the lowest concen-tration resulted in a substantial increase of enzyme activity,while Zn provoked considerable enzyme activity increaseonly at the highest concentration and aft er longer incubationtime (7 day).Catalase which is located mostly in peroxysomes andparticipates in the breakdown of the photorespiratory H2O2(Foyer et al. 1994) could not be detected in roots and showedno significant alterations in neither experimental system.TheAPX is one of the most important enzymes playing crucialrole in eliminating poisonous H2O2 from plant cells (Foyeret al. 1994). In Cd-treated seedlings the APX activity of rootswas proportionally increased with higher Cd concentrationand further enhanced during further incubation. It should beemphasized that at all Cd concentrations the APX activitywas dramatically reduced after the third day of treatment. Inleaves the APX activity exhibited similar changes as thosefound in roots but no inhibition could be detected at thehighest, 1 mM Cd concentration. Zn-treatment did not casuedsignificant changes in APX activity at any concentrations andafter any days of treatment neither in roots nor in leaves. Thisfact is an additional proof emphasizing the essentiallydifferent effects of the heavy metal Zn and Cd on plantmetabolism.We conclude, that there is a fundamental difference in themetabolic changes induced by the heavy metal Zn and Cd.Cadmium induces drastic changes that are reflected in thedecrease of chlorophyll content and the radical changes in theactivity of the antioxidative enzyme system. Another heavymetal, zinc induces much milder changes in the plant meta-bolism that is reflected in the constant chlorophyll content,the CAT and APX activity. The only parameter indicating thetoxicity of Zn ions at high concentration was the increase ofPOD activity in leaves. We conclude, that the reason of lesstoxicity of Zn should be either in the different localizationof Zn or the most effective phytochelation of the heavy metal.On the basis of our experiments we suggest a simple,cheap method for the early detection of heavy metal damageon plants; namely the determination of POD activity ofdifferent plant tissues. By means of this test we can make adistinction between the consequences of a detrimental (i.e.Cd) and a less harmful (i.e. Zn) heavy metal exposure of theplants.AcknowledgmentsThe authors are grateful to late Mrs. Nóra Koch for herskillful technical assistance. This work was partly supportedby the Hungarian National Science and Research Foundation(OTKA 026078).ReferencesArnon DJ (1949) Copper enzymes in isolated chloroplasts.Polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1-15.Arulsekar S, Parfitt DE (1986) Isozyme analysis. Procedures for stonefruits, almond, grape, walnut, pistachio and fig. Hort Sci 21:928-933.Chance B, Maehly AC (1955) Assay of catalases and peroxidases. MethodsEnzymol 2:764-817.Chaoui A, Mazhoudi S, Ghorbal MH, El Ferjani E (1997) Cadmium andzinc induction of lipid peroxidation and effects on antioxidant enzymeactivities in bean (Phaseolus vulgaris L.). Plant Sci. 127:139-147.Elstner EF (1982) Oxygen activation and oxygen toxicity. Annu. Rev. PlantPhysiol 33:73-96.Foyer CH, Lelandais M, Kunert JK (1994) Photooxidative stress in plants.Physiol. Plant 92:696-717.Hegedûs A, Erdei S, Horváth G (2001) Comparative studies of H2O2detoxifying enzymes in green and greening barley seedlings undercadmium stress. Plant Sci 160:1085-1093.Horváth G, Droppa M, Oravecz Á, Raskin VI, Marder JB (1996) Formationof the photosynthetic apparatus during greening of cadmium-poisonedbarley leaves. Planta 199:238-243.Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867-880.Salin ML (1987) Toxic oxygen species and protective systems of thechloroplast. Physiol. Plant 72:681-689.Sanitá di Toppi L, Gabbrielli R (1999) Response to cadmium in higherplants. Environ. Exp. Bot 41:105-130.Van Assche F, Clijsters H (1990) Effects of metals on enzyme activity inplants. Plant Cell Env 13:195-206.Figure 1. POD activity after Cd (A) and Zn (B) treatment in the leaves of barley seedlings. (®) control; (l) 0,1 mM; (n) 0,3 mM; (s) 1 mM Zn andCd, respectively.A B

Heavy metal induced physiological changes in the antioxidative response system

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Heavy metal induced physiological changes in the antioxidative response system

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