Figure 4.

Hydrogen peroxide (H₂O₂) production in Arabidopsis plants with reduced cell wall peroxidase and NADPH oxidase activity. The accumulation of H₂O₂ was detected in wild‐type (Col‐0), prx33 and rbohd T‐DNA insertion lines and in TRV‐GFP and TRV‐PRX gene‐silenced Arabidopsis plants by 3,3′‐diaminobenzidine (DAB) (A–C) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) (D, E) staining methods. Five‐ to six‐week‐old Arabidopsis plants (whole rosettes) were spray inoculated with Alternaria brassicicola conidial suspension at a concentration of 5 × 10⁵ conidia/mL distilled water. The stainings were carried out with mock‐inoculated (A) and Alternaria brassicicola‐infected (B–E) plants (2 days after inoculation). Bars, 50 µm. The intensity of the fluorescence signal emitted by DCFH‐DA‐stained leaves (D) was quantified under UV light using an AlphaImager Mini gel documentation system (E). Results are presented as an average pixel fluorescence intensity and represent the means of two experiments (n = 30 leaves in the middle position for each genotype/treatment) ± standard error (SE). Statistical analysis was performed using one‐way analysis of variance (ANOVA) and Tukey’s post hoc test. Different letters indicate statistically significant results. prx33 and TRV‐PRX plants accumulate less H₂O₂ than the corresponding control plants. Microscopic observation of the patterns of H₂O₂ accumulation reveals that prx33 and TRV‐PRX plants show apoplastic H₂O₂ accumulation in a reduced number of cells in comparison with the corresponding control plants after inoculation with A. brassicicola. Leaves of the rbohd knockout line lacking apoplastic H₂O₂ accumulation after A. brassicicola infection are shown as a reference. Decreased H₂O₂ production in rbohd plants was also confirmed by the DCFH‐DA staining method. The mean level of base fluorescence for mock‐inoculated Col‐0 plants was 1240 ± 34.