Abscisic acid deficiency causes changes in cuticle permeability and pectin composition that influence tomato resistance to Botrytis cinerea

Abstract

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.

Figure 1.

Pectin degradation during B. cinerea infection in sitiens and wild-type tomato. Degradation of pectin at 32 h after B. cinerea inoculation (A) or mock inoculation (B) was detected with monoclonal antibody JIM7 and secondary labeling with fluorescein isothiocyanate. Some sites with cell wall degradation are indicated with arrows. Bars = 50 μm. At least 10 samples from different plants were examined for the wild type and sitiens, and representative images are shown.

Figure 2.

Transmission electron micrographs of wild-type and sitiens transverse leaf sections. Micrographs show leaf epidermal cell walls of the wild type (A), sitiens (B), sitiens + 100 μm ABA in 0.05% ethanol (C), and sitiens + 0.05% ethanol (D). The cuticle (cu) is visible as the dark (electron-dense) apposition on the cell wall (cw). E, Examples of cell wall abnormalities found in cell wall and cuticle of sitiens leaf. Similar observations were made in sections of three different wild-type and sitiens plants. The experiment was repeated with similar results.

Figure 3.

Leaf surface hydrophobicity and trichome density. A, Images of droplets on wild-type and sitiens leaf surfaces illustrating the differences in surface tension. B, Hydrophobicity determined by measuring the contact angle of a 10-μL droplet of distilled water on the leaf surface by the sessile drop method. Mean contact angles were averages of at least 10 measurements. Fourth leaves of 5-week-old plants (seventh leaf stage) were used. Error bars indicate se. A Student’s t test indicated that differences between the wild type and sitiens were statistically significant, indicated by the star (P < 0.001). C, Number of trichomes per 10 mm². Error bars indicate se. A Mann-Whitney test revealed a significant difference between the wild type and sitiens, indicated by the star (P < 0.001). D, Representative photographs of leaf sections illustrating differences in trichome density between the wild type (top) and sitiens (bottom). [See online article for color version of this figure.]

Figure 4.

Cuticle and cell wall permeability of sitiens and the wild type. A, Extraction of chlorophyll from equal amounts of wild-type and sitiens intact leaves for 0.5 to 4 h in 80% ethanol at room temperature. B, Total chlorophyll extracted from leaves of the wild type, sitiens, and sitiens complemented with ABA by spraying 100 μm ABA in 0.05% ethanol until runoff twice per week during their development at different time intervals with sd. C, Leaf discs excised from the fourth leaf of 5-week-old wild-type and sitiens plants floating on their adaxial side on a 0.05% toluidine blue solution in water for 1 h. sitiens plants were complemented with ABA by spraying 100 μm ABA in 0.05% ethanol until runoff twice per week during development. As a control, a mock solution containing 0.05% ethanol was used. Biological replicates of the experiments produced similar results.

Figure 5.

Correlation between leaf surface permeability and resistance against B. cinerea. A, Schematic representation of a 5-week-old tomato plant (seventh leaf stage) with leaf numbers indicated. B, Permeability index and disease index calculated on four leaf discs of each leaf of 5-week-old (seventh leaf stage) wild-type and sitiens plants. Disease index was evaluated using four scoring categories (0, resistant; 1, slightly spreading lesion; 2, moderately spreading lesion; 3, severely spreading lesion), and permeability index was calculated based on the surface area stained with toluidine blue using four categories (0, 0%–25%; 1, 25%–50%; 2, 50%–75%; 3, 75%–100%). Data represent means ± se of three different plants with four discs per leaf and per plant (n = 12). A significant correlation was observed between permeability and resistance in sitiens, as indicated by the Spearman’s rank correlation coefficient (ρ = −0.770, P < 0.01), but not for the wild type (ρ = −0.330).

Figure 6.

Pectinase treatments of wild-type and sitiens leaf discs. A, Leaf discs excised from fifth leaves of wild-type and sitiens plants floating at room temperature on 1 mL of pectinase (Pectinex Ultra SPL; Sigma-Aldrich) solutions at different enzyme concentrations (units mL⁻¹). Photographs were taken after 3 d. B, Quantification of the leaf disc surface area with the image-analysis software APS Assess 2.0. Two biological replicates gave similar results. WT, Wild type.

Figure 7.

Cell wall composition of wild-type (WT) and sitiens plants. Monosaccharide composition of the cell walls of fifth leaves of 5-week-old wild-type and sitiens plants. Results are expressed as a percentage of cell wall dry weight. Bars indicate se (n = 4; n = 2 for Gal [gal], GalA [galA], and methylesterified GalA [galAmet]).

Figure 8.

Electropherograms obtained from capillary electrophoresis of wild-type and sitiens oligosaccharides released into B. cinerea inoculation droplets. Oligosaccharides released into B. cinerea inoculation droplets on fifth leaves of 5-week-old plants were sampled at 0, 6, 12, and 48 hpi and labeled with 8-amino-1,3,6-pyrenetrisulfonic acid. Signal intensity as relative fluorescence units is given in the ordinate, and scan numbers are given in the abscissa. For each sample, three biological replicates were analyzed, of which one is represented here.

Figure 9.

Schematic representation of the putative oligosaccharide signaling in the wild type and sitiens. In sitiens, the perception by the plant of a defective cuticle might lead to the constitutive expression of chitinases and glucan endo-1,3-β-glucosidases (1), releasing elicitor-active molecules from the fungal cell wall (2). The higher degree of methylesterification in the sitiens cell wall delivers more active host elicitors. Both types of elicitors can have a synergistic effect or even interact with each other to form even more elicitor-active complexes (3). Together with the higher permeability of the sitiens cuticle, the signaling would be faster and more effective with defense gene expression and the resistant phenotype that typifies the mutant as a consequence. In the wild type, basal expression of chitinases and glucan endo-1,3-β-glucosidases is not high enough to release elicitors from the germinating B. cinerea, and expression is induced only upon infection. The cuticle is not permeable, which delays the diffusion of signaling molecules/complexes into the cytosol of epidermal cells. Because of the absence of early signaling reactions, defense is delayed, resulting in cell wall breakdown, cell death, and fungal spreading. co, Conidium; cu, cuticle; ep, epidermal cell layer; me, mesophyll cell layer; pe, point of penetration.