Proline Content and Antioxidant Enzymes Activities of Flowers

Harvested flowers (four replicates/treatment) were homogenized in a 50 mM potassium-phosphate ice-cold extraction buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v) polyvinylpyrrolidone (PVPP), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.05% Triton X-100 (pH = 7.0). Protein content was determined using bovine serum albumin as described previously (Chrysargyris et al., 2017).

Proline was determined according to acid-ninhydrin and toluene method at 520 nm (Khedr et al., 2003). Results were expressed in micrograms of proline per gram of fresh weight. Catalase (CAT) and superoxide dismutase (SOD) activity were assayed according to Jiang and Zhang (2002). CAT was assayed in a reaction mixture (1.5 mL) containing 50 mM K-phosphate buffer (pH 7.0), 10 mM H₂O₂ and an enzyme aliquot. The reduction of H₂O₂ was measured at 240 nm. The results were expressed in CAT units/mg of protein (1 unit = 1 mM of H₂O₂ reduction per min). SOD was assayed using a photochemical method; a reaction mixture (1.5 mL) containing 50 mM K-phosphate buffer (pH 7.5), 13 mM methionine, 75 μM nitro blue tetrazolium (NBT), 0.1 mM EDTA, 2 μM riboflavin and an enzyme aliquot. The reaction began by exposing the mixture to a light source of two 15 watt fluorescent lamps for 15 min and was stopped by placing the tubes in the dark. Absorbance was determined at 560 nm and activity was expressed in units/mg of protein. Peroxidase activity (POD) was determined as described by Tarchoune et al. (2012) following the increase in absorbance at 430 nm. Calculations were performed using the coefficient of extinction of 2.47 mM/cm. One POD unit was defined as the amount of enzyme to decompose 1 μmol of H₂O₂ per minute. Results were expressed in units/mg of protein. The activity of APX was determined according to Zhu et al. (2004) by the decrease in the absorbance of ascorbate at 290 nm. Results were expressed in units APX/mg of protein.