Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea

Abstract

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.

Figure 1.

Phylogenetic analysis of Cas1p from C. neoformans and RWA proteins from selected plant species. Alignment was generated by ClustalX (2.0) using full-length sequence and the phylogeny calculated with Phylip. Proteins used for analysis were AtRWA1 (NP_568662.1), AtRWA2 (NP_001118592.1), AtRWA3 (NP_180988.3), and AtRWA4 (NP_001031111.1) of Arabidopsis, CnCas1 (XP_568628.1) of C. neoformans, Os1g631100 (NP_001043644.1), Os5g582100 (NP_001056436.1), and Os3g314200 (NP_001049932.1) of rice (Oryza sativa), SmRWA (XP_002969943.1) of S. moellendorfii (lycophyte), Pp226366 (XP_001783265.1), Pp211822 (XP_001765248.1), and Pp65220 (XP_001752616.1) of P. patens (moss), and Pt2298475 (XP_002298511.1), Pt2317264 (XP_002317300.1), Pt2314931 (XP_002314967.1), and Pt2312277 (XP_002312313.1) of poplar (Populus trichocarpa). The numbers at the nodes indicate bootstrap values in percentage of 1,000 trials.

Figure 2.

Identification of rwa loss-of-function mutants. A, Gene structures are shown with black boxes indicating exons and shaded areas indicating introns. Triangles indicate T-DNA insertions. Small arrows directly above the genes indicate forward and reverse primers used in genotyping and RT-PCR. B, Transcripts of mutated RWA genes could not be detected except in rwa2-2 and rwa2-3, where a truncated transcript was present. WT, Wild type.

Figure 3.

Cell wall acetylation levels in rwa mutants. Acetic acid was determined after saponification of AIR derived from mature leaves. Rosette leaves of eight 6-week-old short-day-grown plants were pooled for AIR preparation, and three pools per genotype were analyzed. The qrt1 mutant was added to the analysis as a parental line (rwa1-1 and rwa4-1 mutants are homozygous for the qrt1 mutation; Preuss et al., 1994). rwa2-1 displayed a significant reduction of about 20% in acetic acid release compared with the Col-0 wild type (WT; P < 0.05, t test), whereas none of the other samples were significantly different from each other. Values shown are means ± se (n = 3).

Figure 4.

Expression profiles of the four RWA genes. RNA was isolated from different tissues and quantified by real-time PCR. The expression levels are shown relative to expression in leaves. Three reference genes were used for the analysis, ACT2, UBC, and PP2AA3, and ΔC_T values were calculated using the average C_T values for the three reference genes.

Figure 5.

rwa2-1 and rwa2-3 mutants are allelic. Acetic acid release was determined on saponified cell wall material derived from mature leaves. rwa2-1, rwa2-3, and F1 generation (rwa2-1 × rwa2-3) all displayed a significant approximately 10% reduction in acetic acid release compared with the wild type (P < 0.01, pair-wise t test). Values shown are means ± se (n = 6). WT, Wild type.

Figure 6.

Indirect immunofluorescence detection of cell wall epitopes at the epidermis of equivalent transverse sections of wild-type (WT) and rwa2 stems. For the right panels, the LM23 epitope was rarely detected at the epidermal cell corners of stem sections pretreated with phosphate buffer (pH 7.0; A) but the epitope was unmasked after pretreatment with sodium carbonate (pH 12.4; C and D) and to a lower extent when the RWA2 gene was disrupted (B). For the left panels, regardless of the pH pretreatment, the LM10 xylan epitope was not detected at the epidermis of wild-type or rwa2 plants, suggesting that the polysaccharide recognized by LM23 at the epidermis is xylogalacturonan and not xylan. Double-headed arrows show the occurrence of the LM23 epitope at the epidermis. Arrowheads indicate the outer surface of the epidermis. Bars = 20 μm.

Figure 7.

RWA2 plays a key role in acetylation of both pectic and nonpectic polysaccharides. Cell wall extracts were treated with EPG and PME for 24 h. Acetic acid release upon saponification was determined for supernatant and residue as well as for the starting material. The sugar content in cell wall material (CWM) and supernatant was calculated per g dry weight of cell wall material. For the residue, the sugars are calculated per g dry weight of residue. Values shown are means ± se (n = 5). WT, Wild type.

Figure 8.

O-Acetylation of xyloglucan is affected in rwa2. Destarched walls were treated with a xyloglucan-specific endoglucanase and their xyloglucan oligosaccharide composition was profiled by matrix-assisted laser-desorption ionization time of flight mass spectrometry (OLIMP). For one-letter xyloglucan nomenclature, see Fry et al. (1993). Values shown are means ± se (n = 3 biological replicates, each with 8–10 technical replicates). WT, Wild type.

Figure 9.

RWA2 localizes to the ER. GFP-tagged RWA2 protein was transiently expressed under the control of the 35S promoter in N. benthamiana leaves. Constructs for expressing mCherry-tagged markers for Golgi (A–D) and ER (E and F) were coinfiltrated with the RWA2 constructs. The RWA2 protein colocalizes with the ER-specific marker protein and loosely with the Golgi-specific marker protein. A, RWA2 with N-terminal GFP fusion. B, Golgi marker tagged with mCherry. C, Merged image of A and B. E, RWA2 with C-terminal GFP fusion. F, ER marker tagged with mCherry. G, Merged image of E and F. D and H, Four-times magnified images of C and G, respectively. GFP and mCherry signals are shown as green and magenta, respectively. Overlapping signals in the merged images are shown as white.

Figure 10.

rwa2 mutants displayed increased resistance to B. cinerea. A, Photographs of leaves 3 d after inoculation with B. cinerea. B, Average lesion size 3 d after inoculation with spores of B. cinerea. Values are means ± se (n ≥ 13). Asterisks indicate a significant difference between genotypes (P < 0.001, Mann-Whitney test). The experiment was repeated three times with similar results. WT, Wild type.