2.9 In situ histochemical localization of reactive oxygen species (ROS)
To detect reactive oxygen species (ROS), histochemical staining with nitroblue tetrazolium (NBT) was performed following Dong et al. (2009) with minor modifications. Detached leaves were first vacuum-infiltrated in their appropriate solution (with or without NBT). For superoxide free radical (O-2. ) characterization, leaf samples were soaked in 6 mM NBT solution containing 50 mM sodium phosphate (pH 7.5) for 12 h under darkness. To detect hydrogen peroxide (H2O2), detached leaves were immersed in 5 mM of 3, 3’ diaminobenzidine (DAB) solution containing 10 mM MES (pH 3.8) for 12 h under darkness. After that, the adaxial surface of the leaf was exposed to moderately high light (500 μmol m-2 s-1) for 1h. The dark-blue spots reveal the interaction between NBT and the generated O-2.; however, the brown spots on the leaf reflect the interaction between DAB and formed hydrogen peroxide (H2O2) at the presence of peroxidase. Both reactions (DAB and NBT) were stopped by soaking the leaves with lacto-glycerol-ethanol (1:1:4 by vol). Chl was removed from the leaves prior to imaging by boiling leaves in their respective solutions (NBT or DAB) for 2 min then the solutions were discarded these solutions and leaves were re-boiled in water for 2 to 3 times (1 min each). Then leaves were incubated in alcohol (99.5%) as described by Zulfugarov et al. (2014) till complete removal of Chl. Afterwards, leaves devoid of Chl were preserved in 50% ethanol till photographed.