Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes

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BIOLOGIA PLANTARUM 51 (1): 98-103, 2007 98 Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes in two sesame cultivars

F. FAZELI*1, M. GHORBANLI* and V. NIKNAM** Department of Biology, Faculty of Science, Teacher Training University, No. 49, Mofateh Ave., Tehran 15614, I.R.. Iran* School of Biology, University College of Science, University of Tehran, Tehran 14155, I.R. Iran**

Abstract The effects of drought on growth, protein content, lipid peroxidation, superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and polyphenol oxidase (PPO) were studied in leaves and roots of Sesamum indicum L. cvs. Darab 14 and Yekta. Four weeks after sowing, plants were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity for next four weeks. Fresh and dry masses, and total protein content in leaves and roots decreased obviously under drought. However, several new proteins appeared and content of some proteins was affected. Measurement of malondialdehyde content in leaves and roots showed that lipid peroxidation was lower in Yekta than in Darab 14. Severe stress increased SOD, POX, CAT and PPO activities in leaves and roots, especially in Yekta. According to the present study Yekta is more resistant to drought than Darab 14.

Additional key words: catalase, malondialdehyde, peroxidase, polyphenol oxidase, Sesamum indicum, superoxide dismutase.

Introduction Water stress, induces numerous biochemical and physiological responses in plants (e.g. Pattangual and Madore 1999). A common effect of drought stress, similarly as of other environmental stresses, is to cause oxidative damage (Smirnoff 1998). Generation of reactive oxygen species (ROS) lead to lipid peroxidation (Chen et al. 2000, Sreenivasulu et al. 1999) protein degradation (Jiang and Zhang 2001) and nucleic acid damages (Hagar et al. 1996). To prevent or alleviate injuries from ROS, plants have evolved an antioxidant defence system that includes non-enzymic compounds like ascorbate, gluthatione, tocopherol, carotenoids, flavonoids and enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase

(POX), ascorbate peroxidase (APX), gluthatione reductase (GR) and polyphenol oxidase (PPO) (Agarwal and Pandey 2004, Núñez et al. 2003/4, Sergi and Alegre 2003). Co-operation among these enzymes is essential for the effective protection from ROS (Scebba et al. 1998). Sesame is considered a drought-resistant crop (Weiss 2000), and its cultivation is extended beyond the tropic and subtropic zones to the temperate and sub-temperate zones of the world (Ali et al. 2000). The objective of this work was to investigate the effect of drought stress on biomass, protein content, lipid peroxidation and activities of antioxidant enzymes in leaves and roots of two sesame cultivars.

Materials and methods Seeds of two sesame (Sesamum indicum L.) cultivars (Darab 14 and Yekta) were sown in plastic pots filled with 3 kg of sandy-loam soil maintained to field capacity (FC). Pots of each cultivar were devided into four groups (four replicates each) and then were watered for four weeks to 100, 75, 50 and 25 % FC. The water supplies

⎯⎯⎯⎯ Received 2 June 2005, accepted 22 January 2006. Abbreviations: CAT - catalase; FC - field capacity; L-DOPA - L-3,4-dihydroxyphenylalanine, MDA - malondialdehyde; NBT - nitroblue tetrazolium; POX - peroxidase; PPO - polyphenol oxidase; ROS - reactive oxygen species; SDS-PAGE - sodium dodecyl-sulphate polyacrylamide gel electrophoresis; SOD - superoxide dismutase; TBA - thiobarbituric acid; TCA - trichloroacetic acid. Acknowledgements: This work was supported by Teacher Training University of Tehran. The authors also acknowledge Biology Department of Teacher training University and Laboratory of Plant Physiology of University of Tehran. 1 Corresponding author; fax: (+98) 21 88848940, e-mail: fazeli69@yahoo.com DROUGHT AND ANTIOXIDANT ENZYMES 99 were maintained by weighing the pots. Plant growth was carried out, in a controlled condition: 16-h photoperiod, irradiance of 250 μmol m-2 s-1, 32/22 °C day/night temperature, 40 - 45 % air humidity. Root and third leaves of each plant were sampled and assayed in the experiments. Dry mass of leaves and roots was determined after drying the samples at 60 °C for 48 h to constant mass. Malondialdehyde (MDA) was determined according to Heath and Packer (1968). Fresh plant material (0.2 g) was homogenized with 5 cm3 0.1 % (m/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10 000 g for 5 min at 25 °C. 4 cm3 of TCA 20 % containing 0.5 % (m/v) thiobarbituric acid (TBA) was added to 1 cm3 of supernatant. The mixture was heated at 95 °C for 30 min and then quickly cooled on ice. The content was centri-fuged at 10 000 g for 10 min at 25 °C. The absorbance of supernatant was measured at 532 nm. The value for non-specific absorption at 600 nm was subtraced. The amount of MDA-TBA complex was calculated from the coefficient of absorbance 155 mM-1cm-1. For determination of total protein content and enzyme activity, 1 g of plant material was homogenized in 5 cm3 of 1 M Tris-HCl buffer (pH 6.8). The homogenate was centrifuged in a refrigerated centrifuge at 13 000 g for 20 min, and the supernatant obtained was used for protein determination and enzyme assay. All the steps were carried out at 4 °C. Protein content of the extracts was determined according to the method of Bradford (1976), using bovine serum albumin as the standard. Total superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) as described by Beauchamp and Fridovich (1971). The reaction mixture consisted of 0.1 cm3 enzyme extract, 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 0.1 mM EDTA, and 2 μM riboflavin. Test tubes were shaken and placed 30 cm from three 30 W fluorescent lamps. The reduction in NBT was measured by reading absorbance at 560 nm. Blanks and controls were run in the same manner but without illumination and enzyme, respectively. One unit of SOD was defined as the amount of enzyme that produced a 50 % inhibition of NBT reduction under the assay conditions (Giannopolitis and Ries 1977). Peroxidase (POX; EC 1.11.1.7) activity was assayed as described by Abeles and Biles (1991). The reaction mixture consisted of 4 cm3 of 0.2 M acetate buffer (pH 4.8), 0.4 cm3 H2O2 (3 %), 0.2 cm3 of 20 mM benzidine and 0.05 cm3 enzyme extract. The rate of benzidine oxidation was measured at 530 nm. Catalase (CAT; EC 1.11.1.6) activity was estimated by the decrease in absorbance of H2O2 at 240 nm as a consequence of H2O2 consumption (Aebi 1974). The