Compartment-specific accumulation of H₂O₂.

<p>The subcellular accumulation of H₂O₂ was quantified on transmission electron micrographs by determining the area of CeCl₃ staining in the different cell compartments. Measurements were performed 12, 24, 48 and 96 hpi at the IS and the AIS. Graphs show means with standard errors which document the percentage of areas covered by CeCl₃ precipitates in the individual cell compartments. n>40 for the individual cell compartments. Different lowercase letters indicate significant differences (p<0.05) between the individual cell compartments whereas uppercase letters indicate significant differences between the total amount of CeCl₃ staining for all analyzed cell compartments taken together. Data were analyzed with the Kruskal-Wallis test followed by post-hoc comparison according to Conover. nd = not detected.</p

Compartment-specific changes in glutathione labeling.

<p>Labeling was evaluated within leaf mesophyll cells of <i>Arabidopsis thaliana</i> Col-0 inoculated with <i>Botrytis cinerea</i> and compared to CL. Measurements were performed 0 h ( = CL, represented by the dotted line), 12, 24, 48 and 96 hpi at the IS and the AIS. Shown are means with standard errors and changes in the amount of gold particles bound to glutathione per µm² in the respective cell compartment. n>20 for peroxisomes and vacuoles and n>60 for other cell structures. Significant differences were calculated using the Mann-Whitney U-test; *, **, and ***, respectively, indicate significance at the 0.05, 0.01 and 0.001 levels of confidence. Square = AIS; triangle = IS.</p

Changes in the relative number of chloroplasts.

<p>Number of chloroplasts was evaluated per cell on longitudinal semithin-sections of the palisade cell layer and the spongy parenchyma in <i>Arabidopsis thaliana</i> Col-0 leaves inoculated with <i>Botrytis cinerea</i> and compared to CL. Measurements were performed 0 h ( = CL, represented by the dotted line), 12, 24, 48 and 96 hpi at the IS and the AIS. Shown are means with standard errors which document changes in the number of chloroplasts. n>100 chloroplasts per treatment. Significant differences were calculated using the Mann-Whitney U-test; *, **, and ***, respectively, indicate significance at the 0.05, 0.01 and 0.001 levels of confidence. Square = AIS; triangle = IS.</p

Visualization of H₂O₂ by CeCl₃ staining.

<p>No staining was observed in cells of mock-inoculated controls (A: 96 hpi). At the AIS H₂O₂ accumulation (arrows) was detected within cell walls 48 hpi (B) and inside mitochondria (M) and plastids (P) 96 hpi (inset in C). Note that plastoglobuli (white asterisks) within chloroplasts (C) and plastids (P) increased in size with advancing infection (best seen in B and E when compared to A). At the IS small dark precipitates of H₂O₂ staining (arrows) were observed 24 hpi (D) in cell walls (arrows) but not in the other cell compartments and 48 hpi (E) in cell walls, in the matrix of mitochondria (M, inset upper right corner) and stroma of chloroplasts (inset lower right corner). Dark precipitates (arrows) were observed at the IS throughout the cytoplasm of leaf cells 96 hpi (F). At the IS 96 hpi only remnants of organelles such as plastids (P) and nucleus (N) could be found in the condensed cytoplasm. H = hyphae, ICS = intercellular spaces, Px = peroxisomes, St = starch, V = vacuoles. Bars in inset of A, C, and E = 0.25 µm, D = 0.5 µm. Bars in all other images 1 µm.</p

Symptom development on <i>Arabidopsis thaliana</i> Col-0 leaves after the inoculation with <i>Botrytis cinerea</i>.

<p>No symptoms were observed on mock-inoculated controls after 0 h (A), 24 h (B), 48 h (C), and 96 h (D). On infected leaves, visible symptoms remained absent 12 hpi (E) and 24 hpi (F), but developed 48 hpi when one large necrotic area appeared at the IS (arrow in G) and chlorosis developed around this area. Strong symptoms could be observed 96 hpi when the necrotic spot expanded in size and chlorosis appeared on the whole leaf (H). Black frames indicate the areas that were harvested for analysis of ultrastructure and ascorbate or glutathione levels. Square = AIS, circle = IS.</p

Changes in the size and fine structure of chloroplasts.

<p>Size and fine structures of chloroplasts was evaluated by TEM on longitudinal ultrathin sections within the mesophyll of leaves from <i>Arabidopsis thaliana</i> Col-0 leaves inoculated with <i>Botrytis cinerea</i> and compared to CL. Measurements were performed 0 h ( = CL, represented by the dotted line), 12, 24, 48 and 96 hpi at the IS and the AIS. Shown are means with standard errors. n>100 chloroplasts per treatment. Significant differences were calculated using the Mann-Whitney U-test; *, **, and ***, respectively, indicate significance at the 0.05, 0.01 and 0.001 levels of confidence. Square = AIS; triangle = IS.</p

Compartment-specific changes in ascorbate labeling.

<p>Labeling was evaluated within leaf mesophyll cells of <i>Arabidopsis thaliana</i> Col-0 inoculated with <i>Botrytis cinerea</i> and compared to CL. Measurements were performed 0 h ( = CL, represented by the dotted line), 12, 24, 48 and 96 hpi at the IS and the AIS. Shown are means with standard errors and changes in the amount of gold particles bound to ascorbate per µm² in the respective cell compartment. n>20 for peroxisomes and vacuoles and n>60 for other cell structures. Significant differences were calculated using the Mann-Whitney U-test; *, ** and ***, respectively, indicate significance at the 0.05, 0.01 and 0.001 levels of confidence. Square = AIS; triangle = IS.</p