Superoxide anion radical (O2·−) and hydrogen peroxide (H2O2) content

Content of O₂^·− was assayed spectrophotometrically according to the previously described procedures^49,115,116. Discs of approximately 0.8 cm in diameter were cut out from the second fully expanded leaves and incubated for 1 h in the dark with 3 ml of mixture pH 7.8 (0.05 M KH₂PO₄/K₂HPO₄, 0.1 mM EDTA, 10 mM NaN₃ and 0.05% NBT). Nitroblue tetrazolium (NBT) undergoes reduction by O₂^·− and forms colorful diformazan. After the incubation, the samples were heated 15 min at 85 °C. After the cooling down, the absorbance was measured at 580 nm. The amount of O₂^·− was presented as the absorbance level per 1 g of leaf fresh weight (FW).

H₂O₂ content was evaluated by the titanium (Ti⁴⁺) method^49,117,118. The amount of 0.4 g of plant leaf tissue in 3 biological replicates was homogenized in TissueLyser (Qiagen) with cooled 1.5 ml of 0.1 M potassium-phosphate buffer (pH 7.8) and subsequently centrifuged at 14,000×g at 4 °C by 25 min to collect supernatant. A reaction mixture contained 400 µl of tissue extract, 600 µl of potassium-phosphate buffer and 500 µl of titanium reagent (0.6 mM PRL and 0.6 mM PTO in a ratio 1:1) in 2 technical replicates per sample was incubated 10 min at room temperature. After the incubation, the absorbance was measured at 508 nm using Ultrospec 1100 pro spectrophotometer (Amersham). The level of H₂O₂ was calculated according to the standard curve and expressed as µmol H₂O₂ per 1 g of FW.