Chemical and enzymatic fractionation of cell walls from Fucales: insights into the structure of the extracellular matrix of brown algae

Abstract

Background and aims: Brown algae are photosynthetic multicellular marine organisms evolutionarily distant from land plants, with a distinctive cell wall. They feature carbohydrates shared with plants (cellulose), animals (fucose-containing sulfated polysaccharides, FCSPs) or bacteria (alginates). How these components are organized into a three-dimensional extracellular matrix (ECM) still remains unclear. Recent molecular analysis of the corresponding biosynthetic routes points toward a complex evolutionary history that shaped the ECM structure in brown algae.

Methods: Exhaustive sequential extractions and composition analyses of cell wall material from various brown algae of the order Fucales were performed. Dedicated enzymatic degradations were used to release and identify cell wall partners. This approach was complemented by systematic chromatographic analysis to study polymer interlinks further. An additional structural assessment of the sulfated fucan extracted from Himanthalia elongata was made.

Key results: The data indicate that FCSPs are tightly associated with proteins and cellulose within the walls. Alginates are associated with most phenolic compounds. The sulfated fucans from H. elongata were shown to have a regular α-(1→3) backbone structure, while an alternating α-(1→3), (1→4) structure has been described in some brown algae from the order Fucales.

Conclusions: The data provide a global snapshot of the cell wall architecture in brown algae, and contribute to the understanding of the structure-function relationships of the main cell wall components. Enzymatic cross-linking of alginates by phenols may regulate the strengthening of the wall, and sulfated polysaccharides may play a key role in the adaptation to osmotic stress. The emergence and evolution of ECM components is further discussed in relation to the evolution of multicellularity in brown algae.

Keywords: Brown algae; ECM; FCSP; Fucales; Himanthalia elongata; cell wall architecture; extracellular matrix; fucose-containing sulfated polysacharides; plant cell wall evolution; sequential extractions; sulfated fucan.

Fig. 1.

Extraction and fractionation of the cell wall content from algal biomass. (A) Chemical sequential extraction procedure. The final extracts were obtained after elimination of the alginate content from the solubilized materials by a calcium chloride precipitation. (B) Enzymatic sequential extraction procedure. An alcohol-insoluble residue is used as a starting material. (C) Partial hydrolytic extraction procedure used to study fucan structures from Himanthalia elongata. The alginate content is removed by a calcium chloride precipitation.

Fig. 2.

Composition of sequential extracts. (A) Chemical sequential extraction procedure. (B) Enzymatic sequential extraction procedure. The tables refer to the composition of the extracts (nd, not determined). The graphs refer to the composition of the extracts with respect to the respective extraction yields as shown in Table 1. The empty squares in the graphs indicate that values were not determined.

Fig. 3.

Heatmap showing the distribution of polymers in extracts according to their fractionation patterns by anion-exchange chromatography (A) from the chemical sequential extraction procedure and (B) from the enzymatic sequential extraction procedure. For each polymer within an extract, a total value of 100 % was assigned and the corresponding signals in fractions were adjusted accordingly. Colour intensity was applied to match values. PC, P. canaliculata; AN, A. nodosum; HE, H. elongata; FV, F. vesiculosus; FS, F. serratus; nd, not determined.

Fig. 4.

Size exclusion chromatography profiles of the extracts: (A) from the chemical sequential extraction procedure and (B) from the enzymatic sequential extraction procedure.

Fig. 5.

Profiles of the fucan fractions from H. elongata after autohydrolysis (AmbW, AmbS) and subsequent acid hydrolysis (AmbWH, AmbSH): (A) Size exclusion chromatography profiles and (B) C-PAGE profiles.

Fig. 6.

¹H-NMR spectra of the fucan fractions from H. elongata after autohydrolysis and acid hydrolysis.

Fig. 7.

Cell wall model for brown algae from the order Fucales. Cellulose microfibrils are sparse and with a ribbon shape. Alginates and fucose-containing sulfated polysaccharides (FCSPs; including sulfated fucans) form the greater part of cell wall polymers, the latter acting as cross-linkers between cellulose microfibrils. Considering their respective charge densities, putative short-chained hemicellulose molecules might act as intermediates between the cellulose microfibrils and the FCSPs. Phenols are likely to be associated with alginates and proteins. Large amounts of iodide are also found in the wall, but its association with other polymers remains elusive.

Fig. 8.

Proposed scenario for the evolution of anatomical organizations in relation to the evolution of the composition and the structure of the ECM within Stramenopiles.