Pectin-like heteroxylans in the early-diverging charophyte Klebsormidium fluitans.

Background and aims: The cell walls of charophytic algae both resemble and differ from those of land plants. Cell walls in early-diverging charophytes (e.g. Klebsormidiophyceae) are particularly distinctive, in ways that may enable survival in environments that are incompatible with land-plant polymers. This study therefore investigates the structure of Klebsormidium polysaccharides.

Methods: The 'pectin' fraction (defined by extractability) of Klebsormidium fluitans, solubilised by various buffers from alcohol-insoluble residues (AIRs), was digested with several treatments that (partially) hydrolyse land-plant cell-wall polysaccharides. Products were analysed by gel-permeation and thin-layer chromatography.

Key results: The Klebsormidium pectic fraction made up ~30-50% of its AIR, was optimally solubilised at pH 3-4 at 100°C, and contained residues of xylose ≈ galactose > rhamnose > arabinose, fucose, mannose, glucose. Uronic acids were undetectable and the pectic fraction was more readily solubilised by formate than by oxalate, suggesting a lack of chelation. Some land-plant-targeting hydrolases degraded the Klebsormidium pectic fraction: digestion by α-l-arabinanase, endo-β-(1⟶4)-d-xylanase, and α-d-galactosidase suggests the presence of β-(1⟶4)-xylan with terminal α-l-arabinose, α-d-galactose and (unexpectedly) rhamnose. 'Driselase' released oligosaccharides of xylose and rhamnose (~1:1) and graded acid hydrolysis of these oligosaccharides indicated a 'rhamnoxylan' with rhamnose side-chains. Partial acid hydrolysis of Klebsormidium pectic fraction released rhamnose plus numerous oligosaccharides, one of which comprised xylose and galactose (~1:2 Gal/Xyl), suggesting a galactoxylan. Lichenase was ineffective, as were endo-β-(1⟶4)-d-galactanase, endo-β-(1⟶4)-d-mannanase, β-d-xylosidase and β-d-galactosidase.

Conclusions: Klebsormidium pectic fraction possesses many land-plant-like linkages but is unusual in lacking uronic acid residues and in containing rhamnoxylan and galactoxylan domains. Uronic acids allow land-plant and late-diverging charophyte pectins to form Ca2+-bridges, facilitating cell-wall polymer association; their absence from Klebsormidium suggests that neutral heteroxylans rely on alternative cross-linking mechanisms. This lack of dependency on Ca2+-bridges may confer Klebsormidium's ability to grow in the acidic, metal-rich environments which it tolerates.