Figure 1.
H2O2 production at the level of stomata.
Epidermal peels from Arabidopsis leaves were first loaded with the fluorescent probe H2DCF-DA and then treated either on the cuticle side (A and B) or on the underside (C) with 150 µM CP to analyse the origin of the ROS signalling. (A) Single guard cells photographed at 2-minute intervals after treatment with CP (same peel). Bar = 30 µm. (B) Photographs of representative regions of three different peels treated with CP for 5, 10 or 30 min respectively, or 30 min with MES buffer (control). Fluorescence microscopy (H2DCF-DA), light microscopy (bright field) and merged pictures (merge) are shown in order to facilitate the localisation of stomata. The bar is 40 µm and applies to all photographs. (C) Peels treated on the underside (the side devoid of cuticle) with CP. Bars = 100 µm (5 min) and 30 µm (10 and 30 min).
Figure 2.
Stomatal closure induced by CP.
(A) Open and closed stomata in Arabidopsis epidermal peels. Bar = 10 µm. (B) Measurement of stomatal aperture after 30 min, 1 and 2 h of treatment with 150 µM CP or MES buffer (control). Results are the mean of 100 measurements ± SEM. Statistical analysis was performed by unpaired t-test (treated vs. control). Asterisks indicate statistically significant difference at P<0.05. The experiment was repeated with similar results.
Figure 3.
Phosphorylation of Arabidopsis MPK6 and MPK3 after treatment with 150 µM CP.
(A) Analysis carried out with human phospho-p44/42 antibodies (pERK1/2) on protein extracts obtained after 0, 5, 15, 30 and 60 min of treatment. pMPK6 and pMPK3 indicate phosphorylation. (B) Image analysis showing the phosphorylation level normalized to the respective protein amount. Error bars indicate SD of three biological replicates. Statistical analysis was performed by unpaired t-test by comparing the phosphorylation level to the appropriate background level (0 h). Asterisks indicate statistically significant difference at P≤0.05.
Figure 4.
Expression analysis of genes related to defence-signalling pathways, camalexin synthesis and ROS production.
Significant variations (P≤0.05) in the level of expression are reported in the figure with a colour scale (up-regulated genes = yellow; down-regulated genes = blue). Arabidopsis leaves were treated on the lower surface with 150 µM CP (treated sample) or sterile distilled water (calibrator sample) for 1, 3, 6, 12 or 24 h. Actin-2 was used as the endogenous reference gene. Relative gene expression values (2−ΔΔCt or fold change values) from three biological replicates and two technical replicates are shown. Statistical analysis was performed by unpaired t-test (treated sample vs. calibrator sample per each time point). The qPCR results and the locus ID of each gene can be seen in Table S1.
Figure 5.
Phytoalexin production by Arabidopsis leaves treated with CP.
(A) Leaves treated on the lower surface with 150 µM CP or sterile distilled water (control). The drops were collected after 15 and 30 min, 1, 3, 6, 9, 12 or 24 h of treatment and the phytoalexin release measured by fluorescence analysis (λex = 320 nm, λem = 386 nm). The fluorescence value was normalized to the number of droplets analysed and expressed as relative fluorescence units. Error bars indicate SD of three biological replicates. Statistical analysis was performed by unpaired t-test (treated vs. control). Asterisks indicate statistically significant difference at P<0.05. (B) Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) of camalexin extracted from the leaves treated for 12 and 24 h. The retention time (Rt) of the peak indicated by the arrow is the same as the camalexin standard, as experimentally determined. (C) Fluorescence emission spectrum of the peaks eluted at 11.2 min compared to a camalexin standard.
Figure 6.
Resistance induction assays against Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst).
Arabidopsis detached leaves were treated on the lower surface with 150 µM CP, sterile distilled water (control) or 0.1% chitosan for 24 h before pathogen inoculation. (A) B. cinerea strain PM10 was inoculated by placing a single 10-µl drop of a suspension 2×105 conidia ml−1 in 1% Sabouraud Maltose Broth on a side of the middle vein. Infected leaves were incubated for 3 days at 22°C before taking photos and measuring the lesion size. (B) Pst strain DC3000 was inoculated by placing a single 10-µl drop of a suspension 108 colony-forming units (CFU) ml−1 (OD600 = 0.2) in sterile distilled water containing 0.02% Silwet L-77 on a side of the middle vein. Infected leaves were incubated for 3 days at 28°C before taking photos and determining bacterial titer. (C) Measurement of the lesion diameter (mm) ± SD after 3 days of incubation with B. cinerea (n = 8). (D) Counting of CFU mg−1±SD after 3 days of incubation with Pst DC3000 (n = 8). Statistically significant differences among treatments are indicated at P≤0.05.