Cadmium stress on antioxidant activity of two Alternanthera sp.

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1 Journal 55 of Scientific & Industrial Research J SCI IND RES VOL 7 SEPT - OCT 13 Vol. 7, September - October 13, pp Cadmium stress on antioxidant activity of two Alternanthera sp. M Devi Chinmayee, Princymol Stephan, M S Anu, A Mary Sheeba, I Mini and T S Swapna* Department of Botany, University College, Thiruvananthapuram, Kerala 95 3, India Received 19 March 13; revised May 13; accepted May 13 This study presents antioxidant response of two species of Alternanthera (A. sessilis & A. tenella) under cadmium (Cd) stress conditions. Plants were grown in soil treated with different concentrations of Cd depending upon threshold level, and enzymatic and non-enzymatic antioxidants were analysed after one month. Activity of enzymatic antioxidants [superoxide dismutase (SOD; E.C ), catalase (CAT: EC ), polyphenol oxidase (PPO; EC ), peroxidase (POX; E.C ), ascorbate peroxidase (APX: EC ) and glutathione reductase (GR: EC 1...)] were estimated. Cd induced oxidative stress enhanced antioxidant enzyme activities and nonenzymatic antioxidant concentrations in both species of Alternanthera. With Cd concentration, proline and lycopene increased, fluctuation was noticed in phenol concentration and carotenoids and chlorophyll content reduced. Both species of Alternanthera showed enhancement in antioxidant action and tolerance against Cd induced stress and thus can act as a suitable candidate for phytoremediation of Cd. Keywords: Antioxidant enzymes, Cadmium, Heavy metal stress, Non-enzymatic antioxidants Introduction Cadmium (Cd), one of the most toxic pollutants found in air, water and soil, interacts with photosynthetic, respiratory and nitrogen metabolism of plants resulting in growth inhibition, low biomass accumulation, reduction of root length, leaf area and even plant death, although mechanisms involved in its toxicity are still not completely understood 1,. Cd is a widespread heavy metal in environment with long half-life and high toxicity 3. It can easily be taken up by plants and then enters food chain, resulting in a serious health issue for animals and humans. Therefore, evaluation of effect of heavy metals on higher plants and their responses to excessive metal concentrations as stressors 5 is important. To overcome heavy metal toxicity in soil, plant cells are equipped with enzymatic mechanisms to eliminate or reduce their damaging effects. Non enzymatic compounds like phenolics have free radical scavenging activity that protect membrane lipids from oxidation 1,. Most of the plant species including crops and weeds cannot survive on polluted sites due to toxic effects of heavy metals. Thus, it is urgent to remediate heavy metal contaminated soils 7. Available remediation methods (soil dressing, soil washing and replacement of polluted soils) are too expensive, time consuming and also require huge amount *Author for correspondence swapnats@yahoo.com of water. Therefore, phytoremediation has attracted great attention as a new and inexpensive technology 9. This study evaluates physiological responses of two species of Alternanthera (A. sessilis & A. tenella; Family: Amaranthaceae) to various Cd concentrations, with reference to production of enzymatic and nonenzymatic antioxidants. Response of antioxidant enzymes [superoxide dismutase (SOD; E.C ), catalase (CAT: EC ), polyphenol oxidase (PPO; EC ), peroxidase (POX; E.C ), ascorbate peroxidase (APX: EC ) and glutathione reductase (GR: EC 1...)] involved in stress tolerance was also investigated. Experimental Section Air dried garden soil (bulk density, 1.3 g/cm³; specific gravity,.7; moisture content,.71%; ph., and soil carbon, 7.75 mg -1 was used as control. Plants were raised in garden soil artificially polluted with different concentration of cadmium sulphate (CdSO ) after analyzing threshold level of plants (, 3 and 5 mg/kg) against control. Plants were harvested after one month, washed with double distilled water, blotted leaves and roots were separated. Enzymatic Antioxidants SOD activity was assayed spectrophotometrically by measuring its ability to inhibit photochemical reduction

2 CHINMAYEE et al: Cd STRESS ON ANTIOXIDANT ACTIVITY OF TWO ALTERNANTHERA Sp. 559 a) Superoxide dismutase activity in two species of b) Peroxidase activity in two species of Alternanthera under Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg c) Polyphenol oxidase activity in two species of Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg 1 d) Ascorbate Peroxidase activity in two species of Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg e) Catalase activity in two species of Alternanthera 1 1 Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg 1 1 f) Glutathione Reductase activity in two species of Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg Fig. 1 Effect of on following enzymatic activities in two species of Alternanthera: a) SOD; b) POX; c) PPO; d) APX; e) CAT; and f) GR of Nitro blue Tetrazoilum (NBT) 1. One unit of SOD is the amount of extracts that gives 5% inhibition in the rate of NBT reduction. CAT was determined by consumption of hydrogen peroxide (H O ) 11 and was monitored spectrophotometrically at nm for 3 min. PPO activity was expressed as changes in absorbance at 95 nm min -1 g -1 fresh weight of tissue 1. Standard method was adopted for POX and increase in absorbance due to oxidation of guaiacol [extinction coefficient (EC). mm -1 cm -1 ] was monitored at 7 nm 13. APX activity was determined by decrease in absorbance of ascorbate at 9 nm of 1 ml reaction mixture containing.1mm EDTA and enzyme extract 1 and activity was calculated by using EC. mm -1 cm -1. GR activity was determined spectrophotometrically by monitoring glutathionedependant oxidation of NADPH at 3 nm, and calculated by using EC. mm -1 cm -1. Non-enzymatic Antioxidants Proline was analysed spectrophotometerically at 5 nm using toluene for a blank as per Bates et al 15 Chlorophyll content was measured by Arnon s method 1, total carotenoids and lycopene as per reported 17 procedure, and total phenols by Folin-Ciocalteau method 1. Results and Discussion SOD activity in A. tenella leaves showed significant increase at 3 mg kg -1 Cd (Fig. 1a). SOD activity in stem and root recorded increase in mg kg -1 Cd, but in roots

3 5 J SCI IND RES VOL 7 SEPT - OCT 13 a) Phenol content in two species of Alternanthera Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg b) Proline content in two species of Alternanthera under Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg c) Chlorophyll content in two species of Alternanthera under the Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg d) Carotenoid content in two species of Alternanthera Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg e) Lycopene content in two species of Alternanthera under Cadmium mg/kg Cadmium 3 mg/kg Cadmium 5 mg/kg Fig. Effect of on following non-enzymatic antioxidants in two species of Alternanthera: a) phenol; b) proline; c) chlorophyll; d) carotenoid; and e) lycopene it was increased 3 mg kg -1 of Cd. SOD is an essential component of antioxidative stress defense system in plants, catalyzing dismutation of superoxide into oxygen and H O 19. POX activity (Fig. 1b) increased with increase in Cd concentration in both species except A. tenella leaves, where it was elevated only in 3 mg kg -1 of Cd. It is reported that POX activity response to excess Cd can vary among plant species and among different tissue. Increased POX activity in two species of Alternanthera noticed in present study might be sufficient to protect proteins, chlorophyll and lipids of some parts of plants against ROS (reactive oxygen species). PPO activity was induced in response to oxidative stress in test plants (Fig. 1c). APX activity (Fig. 1d) registered enhancement with increase in Cd concentration in all parts of two species of Alternanthera. In both plants, CAT activity increased with increasing Cd concentration (Fig. 1e), demonstrating that any H O formed as a result of SOD activity was consumed by CAT and or POX. CAT is one of the most important plant enzymes catalyzing dismutation of H O into oxygen and water 19. CAT activity varies during seed maturation and germination 1, seedling development and leaf maturation. GR activity increased significantly with increase in Cd concentration (Fig. 1f), may be because synthesis of glutathione (GSH) 3. GR contains a highly conserved disulphide bridge between 7 th Cys and 1 st Cys, which may undergo cleavage by heavy metals at toxic concentrations. GR catalyzes reduction of oxidized glutathione (GSSG) in an NADPH-dependent reaction. GR, therefore, plays an essential role in protection of chloroplasts against oxidative damage by maintaining a

4 CHINMAYEE et al: Cd STRESS ON ANTIOXIDANT ACTIVITY OF TWO ALTERNANTHERA Sp. 51 high GSH/GSSG ratio. Results obtained in present study are in agreement with reports in A. philoxeroides under zinc stress, which considerably changed activities of SOD, CAT and APX, and also reduced GSH and ascorbic acid (AsA). Therefore, zinc induced oxidative stress in A. philoxeroides lea ves show enhancement of antioxidant enzyme activities and antioxidant concentrations 5. Phenolic compounds are secondary metabolites and play many roles including synthesis of lignin during stress condition. A. tenella showed high phenolic concentration in root as compared to leaves and stem, under high concentration of Cd (Fig. a). Phenolic compounds in A. sessilis fluctuated according to Cd concentration and recorded high value in stem under 5 mg kg -1 Cd treatment. Cd treatment induced increase in proline content on leaf and stem of A. tenella, but proline content was elevated in stem and root of A. sessilis. In root of A. tenella and leaves of A. sessilis, proline content did not show variations under stress (Fig. b). Accumulation of free proline in response to heavy metal exposure is widespread among plants. Exposure to Cd 7 disturb plant water balance. Proline-mediated alleviation of water deficit stress could substantially contribute to Cd tolerance 7 through mechanisms as osmoregulation, protection of enzymes against denaturation, and stabilization of protein synthesis. Chlorophyll and carotenoid content seemed to be reduced with increase in Cd concentration in both leaves and stem of two species (Fig. c & d). Lycopene content increased in proportion with Cd concentration in all three plant parts of both species (Fig. e). Decreased chlorophyll content associated with heavy metal stress may be the result of inhibition of enzymes responsible for chlorophyll biosynthesis. Cd is reported to affect chlorophyll biosynthesis and inhibit protochlorophyll reductase and aminolevulinic acid (ALA) synthesis 9. Similar results are obtained in Brassica juncea and plant exhibited decline in growth, chlorophyll content and carotenoids with Cd and lead (Pb) but Cd was found to be more detrimental than Pb treatment 3. Conclusions Two species of Alteranthera had the ability to cope with metal stress using effective oxidative stress defense mechanisms. Changes in SOD, CAT, POX, PPO, APX and GR showed a clear correlation with Cd concentrations and thus Cd caused oxidative stress as evidenced by decrease in chlorophyll and carotenoid content. A significant increment was demonstrated in activities of major antioxidant enzymes (CAT, GR, PO, SOD, APX and POX), which are involved in detoxification of ROS. However, magnitude of increase in activity depends on both the enzyme and plant parts (leaves, stem or root). 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