Extraction and Measurement of Ascorbate and Glutathione

The leaves of rapeseed plant (0.5 g) had been homogenized in 5% meta-phosphoric acid containing 1 mM EDTA (centrifuged at 11,500 × g; 15 min at 4°C). Supernatant was collected for the assay of AsA and GSH pool. To determine total ascorbate, the oxidized fraction was reduced by adding 0.1 M dithiothreitol for 1 h at room temperature and then read at 265 nm using 1.0 unit AO. Oxidized ascorbate (DHA) content had been assayed by subtracting reduced AsA from total AsA (Hasanuzzaman et al., 2011; Nahar et al., 2016b). The glutathione pool had been determined according to previously described methods (Yu et al., 2003; Hasanuzzaman et al., 2011). Standard curves with known concentrations of GSH and GSSG had been used to calculate the unknown GSH and GSSG pool of plant sample. The content of reduced GSH had been calculated by subtracting GSSG from total GSH.