**2.5 Classical antioxidant enzymes**

Extracts for determination of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities were prepared from 0.3 g of roots homogenized under ice-cold conditions in 3 ml of extraction buffer, containing 50 mM phosphate buffer (pH 7.4), 1 mM EDTA, 1 g PVP, and 0.5% (v/v) Triton X-100 at 4 °C. The homogenates were centrifuged at 10,000 × g for 20 min and the supernatant fraction was used for the assays.

CAT activity was determined in the homogenates by measuring the decrease in absorption at 240 nm in a reaction medium containing 50 mM potassium phosphate buffer (pH 7.2) and 2 mM H2O2. The pseudo-first order reaction constant (k′ = k[CAT]) of the decrease in H2O2

absorption was determined and the catalase content in pmol mg−1 protein was calculated using k = 4.7 × 107 M−1s−1.

APX activity was measured immediately in fresh extracts and was assayed as described by Nakano and Asada (1981), using a reaction mixture (1 ml) containing 50 mM K-phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM Na-Ascorbate and 0.1 mM EDTA. The hydrogen peroxide-dependent oxidation of Ascorbate was followed by a decrease in the absorbance at 290 nm (ε: 2.8 mM –1 cm–1). One unit of APX forms 1 µmol of ascorbate oxidized per minute under the assay conditions.

GR activity was measured by following the decrease in absorbance at 340 nm due to NADPH oxidation. The reaction mixture contained tissue extract, 1 mM EDTA,0.5 mM GSSG, 0.15 mM NADPH and 50 mM Tris–HCl buffer (pH 7.5) and 3 mM MgCl2 (Schaedle and Bassham 1977).

#### **2.6 Histochemical analysis**

In order to analyze H2O2 generation roots were excised and immersed in a 1% solution of 3,3'-Diaminobenzidine (DAB) in Tris-HCl buffer (pH 6.5), vacuum-infiltrated for 5 min and then incubated at room temperature for 16 h in the absence of light. Roots were illuminated until appearance of brown colors characteristic of the reaction of DAB with H2O2.

In the same way to show O2 .- production roots were excised and immersed in a 0.1% solution of NBT in K-phosphate buffer (pH 6.4), containing 10 mM Na-azide, and were vacuum-infiltrated for 5 min and illuminated until appearance of dark spots, characteristic of blue formazan precipitate.

#### **2.7 Isolation of RNA and RT-PCR analysis**

Total RNA was extracted from soybean roots by using the Trizol reagent (Gibco BRL). Four micrograms of total RNA were treated with RNase-free DNase I (Promega, CA, USA) and then 1.0 µg was reversed transcribed into cDNA using random hexamers and M-MLV Superscript II RT (Invitrogen, CA, USA). PCR reactions were carried out using *Glycine max* HO-1 and 18S specific primers, as previously described (Yannarelli and others, 2006). The PCR profile was set at 94°C for 1 min and then 29 cycles at 94°C for 0.5 min, 54°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 7 min. Each primer set was amplified using an optimized number of PCR cycles to ensure the linearity requirement for semi-quantitative RT-PCR analysis. The amplified transcripts were visualized on 1.5% agarose gels with the use of ethidium bromide. Gels were then scanned (Fotodyne Incorporated, WI, USA) and analyzed using Gel-Pro Analyzer 3.1 software (Media Cybernetics, MD, USA).

#### **2.8 Protein determination**

Protein concentration was evaluated by the method of Bradford (1976), using bovine serum albumin as a standard.

#### **2.9 Statistics**

Values in the text, figures and tables indicate mean values ± SEM. Differences among treatments were analyzed by one-way ANOVA, taking p<0,05 as significant according to Tukey's multiple range test.
