Cladode chlorenchyma tissues (0.5 g) were collected at 12:00, frozen in liquid nitrogen, and immediately ground with a mortar and pestle. They were ground again with 20 mg of Polyclar AT in 2 ml of grinding medium, which contained 50 mM Tris·HCl (pH 7.5), 5 mM dithiothreitol, 0.2 mM disodium EDTA, 0.5% (w/v) polyvinylpyrrolidone, and 1% (v/v) Triton X-100. The homogenate was centrifuged at 10 000 × g for 10 min at 4 °C, and the supernatant was concentrated about tenfold in Amicon Ultra-2 centrifugal filter devices (Merck Millipore Ltd., MA, USA). We used a SOD Assay Kit-WST (Dojindo Co., Kumamoto, Japan), in which the superoxide anion reduces WST-1 ((2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2 4-disulphophenyl)-2H-tetrazolium, monosodium salt) to yellow formazan, which can be quantified from absorbance at 450 nm. Antioxidants inhibit yellow WST-1 formation. In brief, the supernatants were mixed with WST-1 and then with the enzyme solution and incubated at 37 °C for 20 min. Absorbance at 450 nm was measured in a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that caused a 50% decrease in formazan formation. The activity was expressed as units per milligram of protein, and the relative value (%) was based on the value in the control at the start of treatment. Protein contents were determined with the Bradford reagent (Sigma Aldrich Co., St. Louis, MO, USA) according to Bradford (1976 (link)). The activity was calculated as the mean of 3 measurements (three plants per treatment).
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