Hemicellulose-remodelling transglycanase activities from charophytes: towards the evolution of the land-plant cell wall

Transglycanases remodel cell-wall polymers, having a critical impact on many physiological processes. Unlike xyloglucan endotransglucosylase (XET) activity, widely studied in land plants, very little is known about charophyte wall-modifying enzymes - information that would promote our understanding of the 'primordial' wall, revealing how the wall matrix is remodelled in the closest living algal relatives of land plants, and what changed during terrestrialisation. We conducted various in-vitro assays for wall-remodelling transglycosylases, monitoring either (a) polysaccharide-to-[H-3]oligosaccharide transglycosylation or (b) non-radioactive oligosaccharide-to-oligosaccharide transglycosylation. We screened a wide collection of enzyme extracts from charophytes (and early-diverging land plants for comparison) and discovered several homo- and hetero-transglycanase activities. In contrast to most land plants, charophytes possess high trans-beta-1,4-mannanase activity, suggesting that land plants' algal ancestors prioritised mannan remodelling. Trans-beta-1,4-xylanase activity was also found, most abundantly in Chara, Nitella and Klebsormidium. Exo-acting transglycosidase activities (trans-beta-1,4-xylosidase and trans-beta-1,4-mannosidase) were also detected. In addition, charophytes exhibited homo- and hetero-trans-beta-glucanase activities (XET, mixed-linkage glucan [MLG]:xyloglucan endotransglucosylase and cellulose:xyloglucan endotransglucosylase) despite the paucity or lack of land-plant-like xyloglucan and MLG as potential donor substrates in their cell walls. However, trans-alpha-xylosidase activity (which remodels xyloglucan in angiosperms) was absent in charophytes and early-diverging land plants. Transglycanase action was also found in situ, acting on endogenous algal polysaccharides as donor substrates and fluorescent xyloglucan oligosaccharides as acceptor substrates. We conclude that trans-beta-mannanase and trans-beta-xylanase activities are present and thus may play key roles in charophyte walls (most of which possess little or no xyloglucan and MLG, but often contain abundant beta-mannans and beta-xylans), comparable to the roles of XET in xyloglucan-rich land plants.

Automated summary

Transglycanases play a crucial role in remodeling cell-wall polymers, with charophytes showing high mannanase and xylanase activities compared to land plants. Various homo- and hetero-transglycanase activities were identified in charophytes, providing insights into the evolution of cell walls in land plants. This study highlights the importance of transglycanases in charophyte cell-wall remodeling and suggests potential differences in enzyme activities between charophytes and early-diverging land plants.