**2.2 Thiobarbituric acid reactive substances (TBARS) determination**

Lipid peroxidation was measured as the amount of TBARS determined by the thiobarbituric acid (TBA) reaction as described by Heath and Packer (1968). Fresh control and treated roots (0.3 g) were homogenized in 3ml of 20% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 3,500 x g for 20 min. To 1 ml of the aliquot of the supernatant, 1ml of 20% TCA containing 0.5% (w/v) TBA and 100 ml 4% butylated hydroxytoluene (BHT) in ethanol were added. The mixture was heated at 95ºC for 30min and then quickly cooled on ice. The contents were centrifuged at 10,000 x g for 15 min and the absorbance was measured at 532 nm. Value for non-specific absorption at 600 nm was substracted. The concentration of TBARS was calculated using an extinction coefficient of 155 mM-1cm-1

## **2.3 Heme oxygenase preparation and assay**

Roots (0.3 g) were homogenized in a Potter-Elvehejm homogenizer using 4 vol. of ice-cold 0.25M sucrose solution containing 1mM phenylmethyl sulfonyl fluoride, 0.2mM EDTA and 50mM potassium phosphate buffer (pH 7.4). Homogenates were centrifuged at 20,000 x g for 20min and supernatant fractions were used for activity determination. Heme oxygenase activity was determined as previously described with minor modifications (Muramoto et al. 2002). The standard incubation mixture in a final volume of 500ml contained 10mmol potassium phosphate buffer (pH 7.4), 60 nmol NADPH, 250ml HO (0.5mg protein), and 200 nmol hemin. Incubations were carried out at 378ºC during 60min. Activity was determined by measuring biliverdin formation, which was calculated using the absorbance change at 650 nm employing an 1 value of 6.25mM-1cm-1 (vismax 650 nm)

### **2.4 Glutathione determination**

Non-protein thiols were extracted by homogenizing 0.3 g of roots in 3.0 ml of 0.1 N HCl (pH 2.0), and 1 g PVP. After centrifugation at 10,000 x g for 30 min at 4°C, the supernatants were used for analysis. Total glutathione (GSH plus GSSG) was determined in the homogenates spectrophotometrically at 412 nm, after precipitation with 0.1 N HCl, using yeastglutathione reductase, 5,5' dithio-bis-(2-nitrobenzoic acid) (DTNB) and NADPH. GSSG was determined by the same method in the presence of 2-vinylpyridine and GSH content was calculated from the difference between total glutathione and GSSG (Anderson, 1985).
