High resolution imaging of temporal and spatial changes of subcellular ascorbate, glutathione and H₂O₂ distribution during Botrytis cinerea infection in Arabidopsis.

In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H₂O₂) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H₂O₂ accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H₂O₂ in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size), chlorosis and necrosis of the leaves

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

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 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

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

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

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