In situ detection of superoxide anion radical was performed
using the nitroblue tetrazolium(NBT) staining method as described
by Kawai-Yamada et al. [29]. Detached leaves were vacuum-infiltrated
with 10 mM NaN3 in 10 mM potassium phosphate buffer
(pH 7.8) for 1 min, and incubated in 1 mg ml1 nitroblue tetrazolium
(in 10 mM potassium phosphate buffer, pH 7.8) for 20 min
in the dark at room temperature. In situ detection of hydrogen peroxide
was performed using the 3,30 0-diaminobenzidine (DAB) staining
method as described by Thordal-Christensen et al. [30].
Detached leaves were vacuum-infiltrated with 1 mg ml1 DAB
solution (pH 3.8) for 1 min and incubated in the dark at room temperature
for 6 h. Stained leaves were cleared by boiling in acetic
acid/glycerol/ethanol (1:1:3 (v/v/v)) solution before photographs
were taken.