DHAR activity was assayed essentially as described (Hossain and Asada, 1984). Tobacco leaves were ground in extraction buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, and 1 mM MgCl₂), and soluble protein was obtained after a 5-min centrifugation at 13,000 rpm. DHAR was assayed from an equal amount of protein as described (Bradford, 1976) in 50 mM K₂HPO₄/KH₂PO₄, pH 6.5, 0.5 mM DHA, and 1 mM GSH and its activity followed by an increase in absorbance at 265 nm. MDHAR (10⁻⁸ mol NAPDH oxidized/min/mg protein), SOD (units to inhibit nitroblue tetrazolium photoreduction by 50%), CAT (10⁻⁶ mol H₂O₂ reduced/min/mg protein), GR (10⁻⁸ mol NAPDH oxidized/min/mg protein), and APX (10⁻⁸ mol Asc oxidized/min/mg protein) activities were determined as described (Gainnopolitis and Pies, 1977; Aebi, 1984; de Pinto et al., 2000). Ascorbate oxidase activity present in apoplastic wash fluid (obtained as described below) was determined from the decrease in A₂₆₅ (extinction coefficient of 14 mM⁻¹cm⁻¹) at 25°C in a reaction mixture containing 0.1 M sodium phosphate, pH 5.6, 0.5 mM EDTA, and 100 μM Asc as described (Pignocchi et al., 2003).