FIGURE 2.

Mechanical wounding-induced the accumulation of NO, H₂O₂, and ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{–•}}$ in local leaf and systemic leaf. The local (fourth) leaf was pre-treated with distilled water, 200 μM L-NAME, 200 μM tungstate, 200 μM c-PTIO, 5 mM DMTU, or 100 μM DPI prior to wounding. The local leaves were harvested 3 h after wounding and the in situ NO was determined using DAF-2DA and detected by a confocal laser scanning microscope system (CLSM). The green fluoresce intensity of DAF-2DA indicates the degree of NO accumulation. Bar, 100 μm. H₂O₂ and ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{–•}}$ were determined in local leaf by incubating in DAB or NBT and photographed with a microscopy. Images of DAB and NBT stained leaves showing localized H₂O₂ and ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{–•}}$ accumulation as dark-brown and dark-blue spots, respectively. Bars, 2 mm. At least five leaves were imagined for each replicate and one representative is shown (D). The other batch of local and systemic (fifth) leaves was also collected and NO concentration (A), H₂O₂ content (B) as well as ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{–•}}$ production rate (C) were determined. Means denoted by the same letter did not significantly differ at P < 0.05 for each treatment according to Tukey’s test.