Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

Abstract

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Keywords: Botrytis cinerea; cell wall acetylation; cuticles; epidermis; mRNA sequencing; peroxidase; trichomes.

Figure 1

Resistance to B. cinerea and toluidine blue staining of wild type and cell wall acetylation mutants (rwa2-3, tbl29, and axy4-3). (A) B. cinerea infection defined by lesion area measured. Relative mean values to the wild type are shown and the error bars represent standard deviations (N > 8). An asterisk indicates statistically significant difference from the wild type by Student's t-test (P < 0.05). (B) Toluidine blue staining pattern of 3-week old plants. The same results were obtained in three independent analyses. The arrowhead indicates the staining of a trichome.

Figure 2

Enhanced surface permeability in rwa2. (A) Water loss from detached leaves of wild type, rwa2-1 and rwa2-3 plants. Rosette leaves were detached from 4-weeks old plants and incubated on the lab bench. Water loss was determined as percentage weight loss compared to time zero. The data are average ± SD of four leaves per time point. (B) Water relations of wild type and rwa2-3 leaves measured by gas exchange analysis. Values are means ± SD of four leaves from individual plants. (C) The aperture of guard cells in response to exogenous ABA and drought. The apertures of guard cells were measured in epidermal peels from leaves treated with 10 μM ABA (left) or in epidermal peels from detached leaves that had been incubated on the bench for 1 h (right) (from plants incubated at high humidity over-night). The data is average of 40 guard cells ± SD. The experiment was repeated three times with similar result. An asterisk indicate a statistically significance difference from the wild type by Student's t-test (P < 0.05).

Figure 3

Trichome integrity is altered in rwa2. (A) Macroscopic appearance of 3-weeks old wild type, rwa2-1 and rwa2-3 grown in growth chamber with 12 h dark/light cycle. (B) Trichomes of the wild type (right) and rwa2-3 (left). The arrow indicates a collapsed trichome on rwa2-3. The scale bar equals 0.5 mm. (C) The percentage of “normal looking” trichomes [as depicted in (B left)] on the leaves of wild type, rwa2-1 and rwa2-3. Leaves were destained in ethanol followed by quantification of the total number of trichomes and the number of collapsed trichomes when viewed under the microscope. The data is the average of three independent experiments ± SD. The number of non-collapsed trichomes on rwa2-1 and rwa2-3 leaves are significant different from wild type as tested by Student's t-test (P < 0.05) as indicated by the asterisks. (D) Trichome birefringence of the wild type (left) and rwa2-3 (right) leaves. Scale bar equals 1 mm.

Figure 4

Epicuticular wax and cutin composition analysis of leaves of 4-weeks old wild type, rwa2-1 and rwa2-3 plants. (A) Composition of epicuticular waxes extracted by dipping in chloroform and derivatized with BSTFA. (B) Composition of depolymerized residual-bound lipids extracted extensively with methanol/chloroform followed by NaOMe-catalyzed acetylation. Values are means of at least five independent samples and the error bars represent the standard deviations. An asterisk indicate a statistically significance difference from the wild type by Student's t-test (P < 0.05).

Figure 5

Ultrastructural organization of the epidermis. Electron microscopic images of ultra-thin sections of the outer cell wall in the upper leaf epidermis were obtained for the wild type (A), rwa2-1 (B), and rwa2-3 (C). The cuticle layer is indicated by the white arrowheads. The electron dense deposits are indicated by black arrowheads. All scale bars equal 500 nm.

Figure 6

Ultrastructural organization of the trichomes. Transverse ultra-thin sections of the trichomes in the wild type (A), and rwa2-3 (B). Scale bars equal 50 μm. Lines across the cell wall are artifacts due to wrinkling of the ultra-thin sections. Scanning electron micrographic images of trichomes in the wild type (C) and the rwa2-3 (D). Scale bars equal 200 μm. Inserts (100 × 100 μm) represent close-up images of trichome branches. Close-up images of the trichome base in the wild type (E) and rwa2-3 (F). Scale bars equal 50 μm. All images were acquired by electron microscopy.

Figure 7

Global transcript analysis. (A) Shown is a principal component analysis of the entire dataset with the individual samples plotted. Wild type samples are shown as diamonds and rwa2 samples are shown as squares with the blue to orange transition showing the time course of the experiment. The first two PCA vectors are utilized that explain 86% of the total variance. (B) VENN diagrams showing the overlap of genes differentially expressed (left side: upregulated genes, right side repressed genes) in rwa2 control vs. wild type control, and wild type infected with B. cinerea vs. wild type mock across the time points. The genes that are differentially expressed in rwa2 control vs. the corresponding wild-type samples have a high overlap with the expression profile of genes in wild type infected with B. cinerea. The numbers in brackets represent the overlap expected by chance. The overlaps are highly significant both for induced (P < 0.001) and for repressed genes (P < 0.001) as determined by χ²-test.

Figure 8

Increased peroxidase activity and hydrogen peroxide accumulation in rwa2. (A) DAB staining for hydrogen peroxide in untreated (left) and in response to treatment with potato dextrose broth (PDB) (right). Five micro liters of 2% (v/v) PDB was placed on each side of the mid vain for 48 h followed by DAB staining. Chlorophylls were extracted in 96% (v/v) ethanol. (B) Peroxidase activities accumulate at trichome bases in uninfected rwa2 leaves. Leaves were stained with DAB in the presence of 0.1% (v/v) H₂O₂ for 1 h. Chlorophylls were extracted in 96% (v/v) ethanol. (C) Extracellular peroxidase activity in the wild type and rwa2-3 leaves. Detached leaves were treated with mock (PDB) or B. cinerea for 24 and 48 h. To measure peroxidase activity, the leaves were floated with the adaxial side downwards in TMB and H₂O₂. Peroxidase activity was measured as absorbance at 654 nm. The data is average of three samples ± SD. The asterisks indicate significant difference between wild type and rwa2-3 as determined by Two-Way ANOVA test (^*P < 0.05; ^**P < 0.01).

Figure 9

RWA2 is expressed abundantly in leaves, particularly in trichomes in Arabidopsis. RWA2 expression was analyzed by GUS staining in 3-weeks old Arabidopsis plants, 10 independent transformed lines were tested. (A) The negative control (promoter-less GUS line). (B) A representative RWA2 promoter:GUS fusion line. (C,D) Close-up images of leaves and trichomes in a representative RWA2 promoter:GUS fusion line.