Received: 26 February, 2009. Accepted: 9 December, 2009. Original Research Paper Plant Stress ©2009 Global Science Books Uptake of Heavy Metals, and Antioxidative Enzymes in Brassica juncea L. Seedlings as Affected by Zn in Binary Combinations with Other Heavy Metals Rupinder Kaur • Renu Bhardwaj • Ashwani K. Thukral * Department of Botanical and Environmental Sciences, Guru Nanak Dev University Amritsar, 143005, India Corresponding author: * akthukral@rediffmail.com ABSTRACT The present study attempts to understand the uptake of heavy metals and stress tolerance in Brassica juncea L. seedlings under the effect of Zn in binary combinations with Cr, Ni, Co and Cu through the production of antioxidative enzymes - superoxide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). It was observed that the order of uptake of heavy metals by the seedling in single metal solutions was Zn > Cu > Co > Cr > Ni. Zn in binary combination with other heavy metals, mutually decreased the uptake of each other, the maximum decrease being 66.1% in the uptake of Cr in (Cr100+Zn100) binary solution. All the metals, whether applied singly or in combinations, significantly increased the activities of antioxidative enzymes, except for CAT. Zn was the most effective metal in increasing the activities of antioxidative enzymes. At 100 mg l -1 , it increased the activities of GR, GPX and APX by 101%, 64%, and 42% respectively, whereas maximum SOD activity (16 mM UA mg -1 protein) was induced by 100 mg l -1 Cr. Of all the binary combinations, Zn+Co and Zn+Ni were most effective in increasing the activities of GPX and GR, respectively, whereas Zn+Cu and Zn+Cr increased the activities of APX and SOD, respectively. Binary interaction models revealed that Cr, Ni, Co and Cu act antagonistic to Zn to increase the activity of GR, whereas for GPX, APX and SOD, these metals in binary combinations with Zn, were mutually antagonistic, thereby causing a negative interactive effect. Cr, Co and Cu were mutually antagonistic to Zn for catalase activity, whereas interaction between Ni and Zn was synergistic for this enzyme. _____________________________________________________________________________________________________________ Keywords: chromium, cobalt, copper, metal interactions, nickel Abbreviations: ANOVA, analysis of variance; APX, ascorbate peroxidase; CAT, catalase; EDTA, ethylenediaminetetraacetic acid; GDHP, guaiacol dehydrogenation product; GPX, guaiacol peroxidase; GR, glutathione reductase; GSSG, glutathione disulphide; NADPH, nicotinamide adenine dinucleotoide phosphate; NBT, nitroblue tetrazolium chloride; ROS, reactive oxygen species; SOD, superoxide dismutase INTRODUCTION The advent of technology in the late 20 th century ushered in an era of industrial revolution with the accompanying unde- sirable generation of waste products on a vast scale. Indus- trial wastes impregnated with heavy metals, besides being detrimental to the health of humans and animals, exten- sively damage living and natural resources in the environ- ment. Heavy metals are nondegradable pollutants that undergo bioaccumulation along higher trophic levels of the food chain, thereby accentuating their toxic effects. Phyto- remediation is a technology for the abatement of pollution that embraces unique property of plants for the uptake and accumulation of heavy metals to decontaminate polluted soils and waters. The hypertolerance of plants to metals is a key characteristic required for hyperaccumulation of metals, and the tolerance capacity of plants depends on an inter- related network of physiological and molecular mechanisms (Khan et al. 2009). Plants exposed to heavy metals evoke a vast array of defense mechanisms, such as immobilization, exclusion, chelation and compartmentalization of heavy metal ions, expression of stress proteins and activation of ethylene response to stress (Cobbett 2000). Much of the injury to the plants caused by stress exposure is associated with oxidative damage at the cellular level. The generation of reactive oxygen species (ROS) is implicated as a stress response (Dietz et al. 1999; Mollar et al. 2007). Heavy metal stress in plants results in the production of ROS such as O 2 , H 2 O 2 and - OH. ROS are also generated in plant cells during normal metabolic processes, where their production is well regulated, but metal toxicity enhances the formation of ROS up to 30-fold (Mittler 2004), leading to distur- bances in the metabolic pathway and macromolecule dam- age (Hegedus et al. 2001). However, plants are surfeited with various mechanisms to combat excessive ROS forma- tion. The deleterious effects resulting from cellular oxida- tive stress may be alleviated by the antioxidative defence machinery of the plant (Halliwell 1987) comprising of en- zymatic and non-enzymatic free radical scavengers. Enzy- matic scavengers include SOD, APX, GPX, GR, dehydro- ascorbate reductase and monodehydroascorbate reductase. The non-enzymatic antioxidants include ascorbic acid, - tocopherol, glutathione, carotenoids and polyamines, etc. (Wang et al. 2004; Narang at el. 2008a). The antioxidative enzyme defence layer is largely provided by specific en- zymes including SODs as a family of metalloenzymes, cata- lyzing the dismutation of the superoxide anion to H 2 O 2 and molecular oxygen (Alsher et al. 2002). A fine regulation of H 2 O 2 is achieved by the enzymes and metabolites of the ascorbate glutathione cycle that are crucial for determining the steady state levels of O 2 and H 2 O 2 (Narang et al. 2008b). Hyperaccumulator plants used for phytoremedation have acclimated themselves well against homeostatic dis- turbances and cellular damages by evoking antioxidative enzyme induction as a general adaptive response to toxic effects of heavy metals (Van and Clijsters 1998). The extent of such tolerance and the degree of adaptation is highly variable in which the efficiency and capacity of the detoxi- ®