In fact, PEG and SA changed components of ascorbate pool (ASA and DHA) not the total ascorbate, resulted in change of redox ratios (ASC/DHA and GSH/GSSG).
Our results demonstrated that, the total ascorbate was stable under PEG stress and SA treatment.
To estimate the total ascorbate, 200 μl of 150 mM Na-phosphate buffer (pH 7.4) and 200 μl of 10 mM DTT were added to 200 μl of the supernatant.
Similarly, the total ascorbate (AsA + DHA) content suffered a decline, but of a lesser magnitude, in all treatments and at all time points.
The values of total ascorbate weren't affected by PEG-simulated drought stress and SA pretreatment.
The concentrations of total ascorbate and ASC were calculated using a standard curve with known amount of ASC and expressed in μmol g−1 FW.
There were no significant differences in the contents of total ascorbate (Fig. 8A and 8F), total glutathione (Fig. 8C and 8H) and GSH (Fig. 8D and 8I) between Mn-excess roots and controls, while the content of ASC (Fig. 8B and 8G) and the ratios of ASC/(ASC + DHA) (Fig. 8E) and GSH/(GSH + GSSG) (Fig. 8J) were significantly lower in Mn-excess roots than in controls.
The ratio of GSH to total glutathione did not significantly differ between Mn-excess leaves and controls (Fig. 7J), while the ratio of ASC to total ascorbate was slightly lower in the former (Fig. 7E).
Redox state of ascorbate was expressed as the ratio of ASC to total ascorbate (ASC/ASC + DHA).
Estimation of the ascorbate (ASC), dehydroascorbate (DHA) and total ascorbate was performed by method of Law et al. (1983).
