Figure 2.

Accumulation of H₂O₂ (A) and Superoxide (B) in Col-0 Leaves Grown under SD or LD Conditions.

Accumulation of H₂O₂ and superoxide was detected using DAB (diaminobenzidine; Sigma-Aldrich) and NBT (nitroblue tetrazolium; Sigma-Aldrich) substrates, respectively. Rosettes were excised at the end of the light period, and incubated on Petri dishes containing 0.1 mg ml⁻¹ solution of DAB (pH 3.8) or a 5 mg ml⁻¹ solution of NBT overnight in darkness. In the subsequent morning, the dishes were transferred to growth light (130 μmol photons m⁻² s⁻¹ at 20°C) for 1 h and, thereafter, the rosettes were incubated in ethanol until chlorophyll was bleached.