Insoluble (1 → 3), (1 → 4)-β-D-glucan is a component of cell walls in brown algae (Phaeophyceae) and is masked by alginates in tissues

Abstract

Brown algae are photosynthetic multicellular marine organisms. They belong to the phylum of Stramenopiles, which are not closely related to land plants and green algae. Brown algae share common evolutionary features with other photosynthetic and multicellular organisms, including a carbohydrate-rich cell-wall. Brown algal cell walls are composed predominantly of the polyanionic polysaccharides alginates and fucose-containing sulfated polysaccharides. These polymers are prevalent over neutral and crystalline components, which are believed to be mostly, if not exclusively, cellulose. In an attempt to better understand brown algal cell walls, we performed an extensive glycan array analysis of a wide range of brown algal species. Here we provide the first demonstration that mixed-linkage (1 → 3), (1 → 4)-β-D-glucan (MLG) is common in brown algal cell walls. Ultra-Performance Liquid Chromatography analyses indicate that MLG in brown algae solely consists of trisaccharide units of contiguous (1 → 4)-β-linked glucose residues joined by (1 → 3)-β-linkages. This regular conformation may allow long stretches of the molecule to align and to form well-structured microfibrils. At the tissue level, immunofluorescence studies indicate that MLG epitopes in brown algae are unmasked by a pre-treatment with alginate lyases to remove alginates. These findings are further discussed in terms of the origin and evolution of MLG in the Stramenopile lineage.

Figure 1

Detection of MLG epitopes in brown algal extracts by glycan array analyses. The tree shows the schematic phylogenetic relationships of the 34 species of brown algae used in the study: they belong to 6 main orders of brown algae from the early diverging clade Dictyotales to the more recently diversified clades of Laminariales, Fucales and Ectocarpales. Cell walls were sequentially extracted and probed with the anti-mixed linkage glucan (MLG) and anti-alginates (LM7) antibodies. The heatmap represents the binding profile obtained from the glycan array analyses. The colour scale in relation to absorbance values is shown. For each antibody the highest mean signal value in the entire data set was set to 100 and all other signals adjusted accordingly. Values <5 were considered as background and discarded. Stars by algal names refer to the corresponding pictures shown on the side.

Figure 2

Deletion of MLG epitopes from glycan arrays by enzymatic treatment. Glycan arrays were treated with buffer only or with either a lichenase (10 µg/ml) or a laminarinase (7.4 µg/ml) for 5 min before probing with the anti-mixed linkage glucan (MLG) and anti-alginates (LM7) antibodies. Brachypodium dystachion is included as a positive control, while Arabidopsis thaliana is included as negative control.

Figure 3

UPLC analysis of lichenase oligomers. Ultra-Performance Liquid Chromatography (UPLC) analysis of the oligosaccharide products released by the lichenase digestion of cell wall extracts obtained from 6 brown algae belonging to 3 distinct orders (Ectocarpales, Fucales, Laminariales). Brachypodium dystachion is included as a positive control, while Arabidopsis thaliana and a commercial laminarin sample are included as negative controls. The chromatogram shows the relative abundance of the tri- (G4G3G) and tetra-saccharides (G4G4G3G). Note that the algal MLG has a very regular structure with trisaccharides only, in contrast to B. dystachion which has both tri- and tetrasaccharide units.

Figure 4

Detection of MLG in stipes from Laminariales by tissue printing. The tissue prints of stipes from Laminariales were probed by the anti-mixed linkage glucan (MLG) and anti-alginates (LM7) antibodies. The samples being naturally pigmented, negative controls are included to appreciate the signal due to the antibody binding. Corresponding cross sections of the stipes are shown on the side. A representative picture of a whole plant of Saccharina latissima is indicated for size indication. The LM7 epitopes are abundant in all tissues. MLG is of a more restricted occurrence and shows distinct spatial localization depending on the alga. It is mostly detected in the medulla for Laminaria hyperborea, while it is more largely distributed in the meristoderm and the cortex in S. latissima. m, meristoderm; co, cortex; md, medulla. The dot lines broadly indicate the different regions in tissues as observed by light microscopy.

Figure 5

Unmasking MLG in stipes from Laminariales by immunofluorescence imaging. (a) Overview of MLG detection in cross-sections of stipes from Laminaria hyperborea and Saccharina latissima. The diagram illustrates the distinct tissues under study. The micrographs show the indirect immunofluorescence detection of MLG epitopes for some of the tissues. The Calcofluor White (calc.) stains all cell walls in sections. MLG is not or only weakly detected. The strongest binding is obtained in the cortical cell walls from Laminaria hyperborea. (b) Indirect immunofluorescence detection in cortical cell walls from Saccharina latissima. MLG epitopes are strongly detected in all cell walls after a pre-treatment with alginate lyases. Equivalent sections are labelled with LM7 and indicates the loss of alginate detection after the enzymatic treatment. Equivalent sections further treated with a lichenase indicate a decrease in MLG detection. (c) Same sections observed at higher magnification indicate that the MLG epitopes are strongly detected after the enzymatic treatment in the most inner and outer parts of the cell walls, both in cortical and medulla cells. m, meristoderm; co, cortex; md, medulla. All scale bars = 50 μm.