4.5. Biomarkers of Oxidative Stress

The levels of O₂^•− were quantified according to the method described by Gajewska and Skłodowska [68], using fresh plant material of roots and shoots (200 mg). After a 1 h reaction at dark conditions in a reaction mixture (2 mL), containing nitroblue tetrazolium (NBT) and sodium azide (NaN₃), an incubation period of 15 min at 85 °C was followed. At the end, the absorbance (Abs) of the obtained solution was recorded at 580 nm and O₂^•− levels were expressed in Abs_580nm h⁻¹ g⁻¹ fresh mass (f.m.). The quantification of H₂O₂ levels was performed in frozen samples, following the spectrophotometric assay of Alexieva et al. [69], which is based on the reaction between H₂O₂ and potassium iodide, forming a yellowish complex, measurable at 390 nm. Its content was determined through a standard curve, using known concentrations of H₂O₂ and later expressed in nmol g⁻¹ f.m.

LP was estimated by the evaluation of malondialdehyde (MDA) content, via spectrophotometry, following the procedure described by Heath and Packer [70]. Abs was recorded at 532 and 600 nm. The difference between Abs₅₃₂ and Abs₆₀₀ was calculated to eliminate non-specific turbidity. Considering the ε of 155 mM⁻¹ cm⁻¹, MDA content was determined and expressed in nmol MDA g⁻¹ f.m.