Gssg assay kit

The concentrations of MDA, GSH, GSSG, and nitric oxide (NO) were detected following the respective reagent instructions of the MDA, GSH, GSSG assay kit (Nanjing Jiancheng Bioengineering Institute), and NO assay kit (Beyotime). The optical density was determined by a microplate reader.

Glutathione a tripeptide compound having important functions in the antioxidant processes involved in the plant defense mechanism and also a metal chelator (Guo et al., 2012 (link)). The reduced glutathione (GSH) was determined using the total glutathione (T-GSH) and oxidized glutathione (GSSG) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The methods were principally based on the previously described enzymatic recycling method evolving cyclic reaction of DTNB (Rahman et al., 2006 (link)). Briefly, frozen leaves were homogenized in extraction buffer (1:4 ratio, wt/vol). Afterward, the homogenate was centrifuged at 3,500 rpm for 10 min at 4°C, the supernatant was collected, and stored at 4°C (−20°C if required to keep overnight) until further analysis according to the manufacturer’s protocol. The absorbance of the assay mixture was measured twice at 412 nm, first at 30 s and second at 10 min 30 s of the reaction initiation. T-GSH and GSSG content were determined using given formulas. The GSH content was calculated by subtracting the GSSG content from the T-GSH content according to the formula provided in the kit, expressed in micromoles per gram fresh leaf weight.

The cell sample collection and protein extraction and concentration evaluation were the same as above for the determination of LDH and MDA. Thereafter, the concentration of GSH and GSSG was determined using a GSH assay kit (A006-1-1) and GSSG assay kit (A061-2-1) (Jiancheng, Nanjing, China). Experiments were performed in triplicate.