Redox Metabolite Quantification

Redox metabolite (Asc, DHA, GSH, GSSG) levels were measured based on an enzymatic cycling method (Quevel and Noctor, 2007; Noctor et al., 2016). Carefully weighed frozen leaf material (approximately 100 mg) was finely ground in liquid nitrogen and extracted in cold 0.2 M HCl. To remove cellular debris, samples were centrifuged at 16,000 g for 10 min, 4°C. Phosphate buffer (0.2 M, pH 5.6, 0.5 ml) was added and the extracts brought to pH 4–5 by carefully adding small volumes of 0.2 N NaOH with vortexing. The added volume was noted and taken into account in the final calculations.

To determine the total glutathione pool, in triplicate in a 96-well plate, 10 μl of the neutralized extract was added to a reaction mixture where the final concentration was 0.1 M phosphate buffer containing 5 mM EDTA, pH 7.5, 0.5 mM NADPH and 0.6 mM 5,5 dithiobis 2-nitro-benzoic acid, pH 7.5. After shaking, the reaction was initiated by the addition of glutathione reductase (final concentration 0.01 U). The reaction was monitored at 412 nm for 2 min at 5 s intervals and the slope of the line from the first 90 s was used to determine total glutathione concentration, using a standard curve of free GSH (ranging from 0 to 250 pmol). To measure GSSG, the neutralized plant extracts as well as a concentration range of GSSG standards (0–100 pmol) were incubated with 2-vinylpyridine, which forms an insoluble precipitate with GSH. After centrifugation at 16,000 g for 10 min, room temperature, GSSG concentration was determined by the assay as described above. Reduced GSH was calculated by subtracting 2 × GSSG from the total GSH.

To determine reduced ascorbate (Asc) levels, in triplicate in a 96-well plate, 40 μl of the neutralized extract was added to a reaction mixture of 0.1 M phosphate buffer, pH 5.6 (final concentration). After shaking, the absorbance was read at 265 nm. The reaction initiated by the addition of 0.2 U of ascorbate oxidase, prepared in 0.2 M phosphate buffer, and incubated with gentle shaking for 8 min at room temperature. Again, the absorbance was read at 265 nm and the difference used to calculate Asc levels based on an Asc standard curve (ranging from 40 to 240 μM) that was included on each plate. In parallel, 100 μl of extract was added to a reaction mixture with a final concentration of 0.1 M phosphate buffer, pH 5.6 and 1.25 mM dithiothreitol, that reduces dehydroascorbate to Asc. After incubation for 30 min at room temperature, total ascorbate levels were measured.