Analysis of endogenous ABA concentration Measurement of stomatal
Samples (0.1 g dry weight: DW) were homogenized in 20 mL 80% ethanol and filtered. The O2− radical activity was analyzed by the nitric method (Sankat and Maharaj, 1997). The reaction mixture was composed of 50 μL extracted sample, 100 μL of a mixture of 65 mM potassium phosphate – 35 mM borax – 0.5 mM EDTA buffer (pH 8.2), 100 μL 0.5 mM hypoxanthine, 50 μL 10 mM hydroxylammonium chloride with 1 mg mL−1 hydroxylamine-O-sulphonic acid, and 100 μL distilled water. To this PMSF mixture, 100 μL of 5 mU mL−1 xanthine oxidase was added for a total volume of 500 μL. The resultant mixture was incubated at 37 °C for 30 min; 1 mL of a mixture of 30 μM N-(1-naphthyl) ethylenediamine dihydrochloride, 3 mM sulphanilic acid, and 4.2 M glacial acetic acid was added to induce diazo dye formation at 25 °C for 30 min. Distilled water was added to the blank instead of xanthine oxidase, and 80% (v/v) ethanol instead of the extracted sample was used as the control. The absorbance at 550 nm was determined using a spectrophotometer (model: U-2910, Hitachi, Tokyo, Japan). The antioxidant activity (EC50) was determined at the mid-point (50%) between zero and full inhibition of diazo dye formation.