2.2 Histochemical detection of the content of O2•-, H2O2 and the status of cell damage
The histological detection of O2•-and H2O2 in cotton leaves by using NBT (Nitroblue tetrazolium chloride, Beijing Boaotuo Technology Co., LTD) and DAB (DAB 4HCL, Beijing Boaotuo Technology Co., LTD) as the chromogenic substrate as described by Lee et al. (2023) , respectively. Well-grown leaves of CK and UV-B treated cotton seedlings were selected, and leaf discs of 1 cm diameter were punched out with a hole puncher. These leaf discs were then immersed with 5 ml NBT solution (0.5 mg/ml) or DAB solution (1 mg/ml), wrapped with foil and incubated for 12 h at room temperature. After incubation, the stained leaf discs were bleached in 90% ethanol at 70℃, and then stored in 50% glycerol until photographic documentation was completed. In leaves, NBT reacts with O2•- to form dark blue insoluble formazan compound, while DAB is oxidized by H2O2 in the presence of peroxidases to produce reddish brown precipitate. In addition, the content of O2•- in leaves was determined using the method of Elstner and Heupel (1976). A hydrogen peroxide kit (Cat. H2O2-1-Y, Suzhou Comin Biotechnology Co. Led, Jiangsu, China) was used to detect the content of H2O2.
Cell damage is characterized by membrane disruption, and the Evan’s blue stain can penetrate through the destabilized membrane and stain the cells (You et al., 2021). Here, Evan’s blue stain was used to evaluate the membrane damage of upland cotton seedlings under UV-B stress. Leaf discs were stained with Evans Blue solution (0.5%) (Shanghai Maokang Biotechnology Co., Led, China) for 12 h in the dark, then washed three times with distilled water and immersed in boiling anhydrous ethanol-glycerol (9:1 v/v) solution for 30 min to remove chlorophyll.