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.