ANTIOXIDATIVE ENZYME RESPONSE OF HEAVY METAL ...

ANTIOXIDATIVE ENZYME RESPONSE OF HEAVY METALHYPERACCUMULATOR ALYSSUM MURALE TO Ni +2 STRESSSelcen BABAOĞLU 1 , Şebnem KUŞVURAN 2 , Şebnem ELLİALTIOĞLU 3 ,Leyla AÇIK 1 , Nezaket ADIGÜZEL 11 Gazi University, Faculty of Science and Art, Department of Biology2 Çukurova University, Faculty of Agriculture, Department of Horticulture3 Ankara University, Faculty of Agriculture, Department of HorticultureABSTRACTSoil contamination with heavy metals has become a worldwide problem, leading to losses inagricultural yield and hazardous health effects as they enter the food chain. Several techniques forremoving heavy metal contamination from soil, water and sediment have been developed and manyfactors limit the applicability of existing techniques. Another promising environmental technology stillin its infancy is phytoremediation, whereby living plants are applied to clean up soils or waterways.This approach exploits the ability of various plant species to thrive in high metal environments whileaccumulating large amounts of toxic elements. Advantages compared with existing remediationmethods include minimal site destruction and destabilisation, low environmental impact and favourableaesthetics. Alyssum (Brassicaceae) genus includes 48 Ni hyperaccumulator taxa in total 170. Plantsposess a number of antioxidant molecules and enzymes that protect them against oxidative damage. Inthis study A. murale’s antioxidative enzyme responses to Ni +2 differed; elevated concentrations of Ni +2resulted in increase in SOD and catalase activity but decrease in glutathione activity.INTRODUCTIONMan’s energy and chemical consumption is the main cause of trace element pollution in the biosphere.Non- ferrous metal industry, mining, waste disposal, pesticides, fertilizers or metal-contaminatedsludge are important sources of metal dispersion in terrestrial and aquatic environment (Lepp, 1981).Soil contamination with heavy metals has become a worldwide problem, leading to losses inagricultural yield and hazardous health effects as they enter the food chain (Schickler and Caspi, 1999).Several techniques for removing heavy metal contamination from soil, water and sediment have beendeveloped, including precipitation, ion exchange, field bioremediation using bacteria and fungi andsome high impact technologies. Many factors limit the applicability of existing techniques. Anotherpromising environmental technology still in its infancy is phytoremediation, whereby living plants areapplied to clean up soils or waterways. This approach exploits the ability of various plant species tothrive in high metal environments while accumulating large amounts of toxic elements. Advantagescompared with existing remediation methods include minimal site destruction and destabilisation, lowenvironmental impact and favourable aesthetics (Nedelkoska and Doran, 2000). Hyperaccumulatorplants are found in Brassicaceae, Euphorbiaceae, Asteraceae, Lamiaceae or Scrophulariaceae plantfamilies (Macnair, 1993). Alyssum (Brassicaceae) genus includes 48 Ni hyperaccumulator taxa in total170 (Kramer et al., 1996).Uptake of phytotoxic amounts of metal by higher plants or algae can result in inhibition of severalenzymes, and in increase in activity (=induction of others). Two mechanisms of enzyme inhibitionpredominate: (1) binding of the metal to sulphydryl groups, involved in the catalytic action orstructural integrity of enzymes, and (2) deficiency of an essential metal in metalloproteins or metalproteincomplexes, eventually combined with substitution of the toxic metal for the deficient element.

The induction of some enzymes is considered to play a significant role in the stress metabolism,induced by the metal toxicity (Van Assche, 1989). One possible mechanism via which elevatedconcentrations of heavy metals may demage plant tissues is the stimulation of free radical production,by imposing oxidative stress (Foyer et al., 1997). Plants posess a number of antioxidant molecules andenzymes that protect them against oxidative damage. In the study described herein, we axamine theresponse of antioxidative enzymes such as superoxide dismutase (SOD), glutathione and catalaseimproved against Ni +2 stress in heavy metal hyperaccumulator Alyssum murale.MATERIALS AND METHODSSeeds were collected from Alyssum murale populations growing in metalliferous regions of EasternTurkey. Seed germination and plant growth took place in growth chamber. Seeds were germinated invermiculite and seedlings were transferred to hydroponic culture containing Hoagland's solution(Hoagland, 1938) . Ni +2 was supplied as NiSO 4 .6H 2 O at two levels (0,1 and 1 mmol/L) on thetwentieth day and treated plants were harvested after four days. Plant leaves were ground with a mortarand pestle in liquid nitrogen and homogenized in phosphate buffer. The homogenate was centrifugedand the supernatant was used for enzyme assays. SOD, catalase and glutathione enzyme levels weremeasured (Çakmak, 1994).RESULTS AND DISCUSSIONMany Alyssum taxa are known as Ni +2 hyperaccumulators and A. murale is the most common speciesknown as a candidate for phytoremediation studies. In our study A.murale’s enzyme responses to Ni +2differed; elevated concentrations of Ni +2 resulted in increase in SOD and catalase activity but decreasein glutathione activity.SOD aktivitesi70060050040030020010001 2 3SOD aktivitesiFigure 1. SOD activity in the leaves of the Ni treated Alyssum murale (U/m/mg T.A)1)Control group, 2) 0,1 mM nickel group, 3) 1 mM nickel groupKatalaz aktivitesi1,41,210,80,60,40,201 2 3Katalaz aktivitesiFigure 2. Catalase activity in the leaves of the Ni treated Alyssum murale (μmol/ dak/mg T.A)1) Control group, 2) 0,1 mM nickel group, 3) 1 mM nickel group

Glutatyon aktivitesi1,4001,2001,0000,8000,6000,4000,2000,0001 2 3Figure 3. Glutathione activity in the leaves of the Ni treated Alyssum murale (μmol/ m/mg T.A)1-Control group, 2) 0,1 mM nickel group, 3) 1 mM nickel groupThe significant induction of SOD activity, increase of catalase activity and the significant reduction inGR activity display a typical antioxidative enzyme response pattern in Ni treated Alyssum murale. Thisactivity patterns show that Ni +2 stress induces the antioxidant enzyme system.REFERENCESÇakmak, I., 1994, Activity as ascorbate-dependent H 2 O 2 scavenging enzymes and leaf chlorosis areenhanced in magnesium and potassium deficient leaves but not in phosphorus deficientleaves. J.Exp. Bot., 45;1259-1266Foyer CH, Lopez-Delgado H, Dat JF, Scott IM, 1997, Hydrogen peroxide and glutathione associatedmechanisms of acclimatory stress tolerance and signaling, Physiol Plant, 100:241-254Hoagland, D.R., and Arnon, D.I., 1938, The water culture method for growing plants without soil.Circ. Calif. Agr. Exp. Sta., 347-461.Krämer,U., Charnack, J.M., Baker, A.J.M, 1996, Free histidine as a metal chelator in plants thataccumulate nickel, Nature, 379, 635-638Lepp, N.W. (ed), 1981, Effect of heavy metal pollution on plants, Vol.2. Applied Science Publishers,London.Macnair, M,R.,1993, Tansley Review No.49 The genetics of metal tolerance in vascular plants, NewPhytol, 124, 541-559Nedelkoska, T.V., Doran, P.M., 2000, Characteristics of heavy metal uptake by plant species withpotential for phytoremediation and phytomining, Minerals Engineering, 13, 549-561Schickler, H., Caspi H.,1999, Response of antioxidative enzymes to nickel and cadmium stress inhyperaccumulator plants of the genus Alyssum, Physiologia Plantarum, 105(1): 39-44 Jan.Van Assche, F., Clijsters, H.,1990, Effects of metals on enzyme activity in plants, Plant, Cell andEnvironment, 13, 195-206