Malondialdehyde (MDA) content measurement (TBARS assay)

The MDA content was measured through a reaction with thiobarbituric acid (TBA) (Heath and Packer 1968). Five hundred mg of plant tissue was homogenized in cold ethanol (80% v/v). Homogenized samples were centrifuged at 11,200×g for 15 min. The supernatant was mixed with 0.5% (w/v) of TBA in 20% (w/v) trichloroacetic acid (TCA) and the mixture was placed in a water bath at 90 °C for 1 h. Then samples were cooled in an ice bath to stop the reaction and centrifuged at 11,200×g for 5 min. The absorbance of the mixture was measured at 532 and 600 nm by spectrophotometer (Ultraspec 2000 UV Pharmacia, Biotech, Sweden). MDA concentration was measured according to the absorbance of the MDA–TBA complex. This absorbance was estimated by subtracting the non-specific absorption at 600 nm from the absorption at 532 nm. The results of the TBARS assay were normalized according to the concentration of total protein and reported as the nmol of MDA/mg protein. The measurements were performed in triplicates for each group on days 1, 7, 14, and 21.