Estimation of lipid peroxides:

The level of lipid peroxidation products was estimated following the method of Heath and Packer . Fresh root/shoot samples (200 mg) were ground in 0.25% thiobarbituric acid (TBA) in 10% TCA using mortar and pestle. The mixture was heated at 95 8C for 30 min and then quickly cooled in an ice bath and centrifuged at 10 000_/g for 10 min. The absorbance of the supernatant was read at 532 nm and correction for unspecific turbidity was done by subtracting the absorbance of the same at 600 nm. A total of 0.25% TBA in 10% TCA served as blank. The concentration of lipid peroxides together with the oxidatively modified proteins of plants were quantified and expressed as total TBARS in terms of nmol g_1 fresh weight using an extinction coefficient of 155 mM_1 cm_1. TBARS are an index of lipid peroxidation.

Superoxide dismutase assay:

About 200 mg fresh tissues were homogenized in 5 ml of 100 mM Kphosphate buffer (pH 7.8) containing 0.1 mM EDTA, 0.1% (v/v) Triton X-100 and 2% (w/v) polyvinyl pyrrolidone (PVP). The extract was filtered through muslin cloth and centrifuged at 22 000_/g for 10 min at 4 8C. Supernatant was dialyzed in cellophane membrane tubings against the cold extraction buffer for 4 h with 3_/4 changes of the buffer and then used for the assay. The assay mixture in a total volume of 3 ml contained 50 mM sodium carbonate_/bicarbonate buffer (pH 9.8), 0.1 mM EDTA, 0.6 mM epinephrine and enzyme. Epinephrine was the last component to be added. The adrenochrome formation in the next 4 min was recorded at 475 nm in a UV-Vis spectrophotometer. One unit of SOD activity is expressed as the amount of enzyme required to cause 50% inhibition of epinephrine oxidation under the experimental conditions.

Catalase assay:
The activity of catalase was assayed according to Beers and Sizer [24]. Fresh samples (200 mg) were homogenized in 5 ml of 50 mM Tris_/NaOH buffer (pH 8.0) containing 0.5 mM EDTA, 2% (w/v) PVP and 0.5% (v/v) Triton X-100. The homogenate was centrifuged at 22 000_/g for 10 min at 4 8C and after dialysis supernatant was used for enzyme assay. Assay mixture in a total volume of 1.5 ml contained 1000 ml of 100 mM KH2PO4 buffer (pH 7.0), 400 ml of 200 mM H2O2 and 100 ml enzyme. The decomposition of H2O2 was followed at 240 nm (extinction coefficient of 0.036

mM_1 cm_1) by decrease in absorbance. Enzyme specific activity is expressed as mmol of H2O2 oxidized min-1 (mg protein)-1.

Guaiacol peroxidase assay:

Guaiacol peroxidase (EC 1.11.1.7) was assayed according to Egley et al. [25]. Fresh root/shoot samples weighing 200 mg were homogenized in 5 ml of cold 50 mM Na-phosphate buffer (pH 7.0). The homogenates were centrifuged at 22 000_/g for 10 min and the dialyzed enzyme extracts were used for the assay. Assay mixture in a total volume of 5 ml contained 40 mM Naphosphate buffer (pH 6.1), 2 mM H2O2, 9 mM guaiacol and 50 ml enzyme. Increase in absorbance was measured at 420 nm (extinction coefficient of 26.6 mM_1 cm_1) at 30 s intervals up to 2 min, using a Bausch and Lomb Spectronic-20 spectrophotometer (USA). Enzyme specific activity is expressed as mmol of H2O2 reduced min_1 (mg protein)_1.

Ascorbate peroxidase assay:

About 200 mg root/shoot samples were homogenized in 5 ml of 50 mM K-phosphate buffer (pH 7.8) containing 1% PVP, 1 mM ascorbic acid and 1 mM PMSF. After centrifugation at 22 000_/g for 10 min at 4 8C, the supernatant was dialyzed against the same extraction buffer and it served as enzyme. Ascorbate peroxidase was assayed according to Nakano and Asada. Reaction mixture in a total volume of 1 ml contained 50 mM K-phosphate buffer (pH 7.0), 0.2 mM ascorbic acid, 0.2 mM EDTA, 20 mM H2O2 and enzyme. H2O2 was the last component to be added and the decrease in absorbance was recorded at 290 nm (extinction coefficient of 2.8 mM_1 cm_1) using a UV-Vis spectrophotometer (ELICO, India) at 30 s intervals up to 7 min. Correction was made for the low, non enzymic oxidation of ascorbic acid by H2O2. The specific activity of enzyme is expressed as mmol ascorbate oxidized min-1(mg protein)-1.

Glutathione reductase assay:

Glutathione reductase was assayed according to Fresh root/shoot samples weighing 200 mg were homogenized using chilled mortar and pestle in 5 ml of 50 mM Tris_/HCl buffer (pH 7.6). The homogenate was centrifuged at 22 000_/g for 30 min at 4 8C and the supernatant after dialysis was used for enzyme assay. The reaction mixture in a total volume of 1 ml contained 50 mM Tris_/HCl buffer (pH 7.6), 0.15 mM NADPH, 1 mM GSSG, 3 mM MgCl2 and 200 ml enzyme extract. The reaction was monitored by decrease in absorbance of NADPH at 340 nm. The specific activity of enzyme is expressed as mmol NADPH oxidized min_1 (mg protein)-1.

Isoenzyme profile of catalase:

To determine the influence of Pb2+ toxicity in situ on changes in isoforms of catalase in growing rice seedlings, rice cv. Jaya was grown for 15 days under increasing concentrations of Pb(NO3)2 in the growth medium. Catalase was extracted from roots and shoots and polyacrylamide gel electrophoresis was performed in vertical slab gel following the method of Davis at 4oC Tris_/glycine (pH 8.3) was used as electrode buffer and 7.5% running and 3.5% stacking gels were used. Enzyme samples corresponding to 30 mg protein mixed with glycerol were layered on top of the stacking gel and electrophoretic run was completed using a current of 25 mA per slab. For detection of catalase isoforms, gels were soaked in 5 mM K-phosphate buffer (pH 7.0) and then transferred to a 5 mM H2O2 solution in the same buffer. After 10-min incubation, gels were rinsed with water and stained in a reaction mixture containing 2% (w/v) potassium ferricyanide and 2% (w/v) ferric chloride. The isozymes appeared as colourless bands on a deep blue background. Protein determination In all the enzyme preparations protein was determined by the method of Lowry et al. [30] using bovineserum albumin (BSA, Sigma) as standard. Determination of Free Amino Acids: Frozen plant samples were homogenised in a blender with 80% ethanol. Plant tissues were removed by centrifugation at 4000 rpm for 15min and the clear extract was mixed with chloroform (1:3 v/v). The upper phase was vacuum dried and stored at -25oC (Raggi, 1994). Residues were dissolved in 1 ml of buffer solution containing sodium acetate (8.2 g/L), methanol (7.5 %), formic acid (0.3 %), acetic acid (1.5 %) and octanoic acid (0.001%), membrane filtered and analyzed by LC3000 Amino Acid Analyser (Eppendorf, Biotronic, Maintal, Germany) equipped with a 75 x 6.0 mm BTC guard column and a 145 x 3.2 mm BTC 2140 main column. The running conditions used a flow rate of 0.2 ml/min., pressure of buffer from 0.0 to 50 bar, pressure of reagent from 0.0 to 150 bar, and reaction temperature of 123 oC. Amino acids derived from the column with ninhydrine were quantified at 440 nm for primary amino acids and at 570 nm for proline.