Abstract
SUMMARYCertain transglucanases can covalently graft cellulose and mixed‐linkage β‐glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero‐transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero‐transglucosylation, we studied Equisetum fluviatile hetero‐trans‐β‐glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo‐transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia‐produced EfHTG was up to approximately 300% increased by addition of methanol‐boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol‐stable factor is proposed to be expansin‐like, a suggestion supported by observations of pH dependence. Screening numerous low‐molecular‐weight compounds for hetero‐transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero‐transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero‐transglucosylation in planta by identifying factors that govern this reaction.
Highlights
Plant cells are surrounded by complex polysaccharide-rich cell walls, which determine plant morphology and play a crucial role during development (Popper et al, 2011)
We found that Equisetum contains extractable factors that strongly boost cellulose:xyloglucan endotransglucosylase (CXE) but not XyG endotransglucosylase (XET) or MLG:xyloglucan endotransglucosylase (MXE) activity
Appreciable activities at typical apoplastic pH values suggest that Equisetum fluviatile hetero-trans-b-glucanase (EfHTG) can act in plant cell walls
Summary
Plant cells are surrounded by complex polysaccharide-rich cell walls, which determine plant morphology and play a crucial role during development (Popper et al, 2011). Major land-plant hemicelluloses consist of a b-(1?4)-linked glucose and/or mannose or xylose backbone and some possess side chains: for example, xyloglucan (XyG), the most abundant hemicellulose in most land-plant primary walls (Popper et al, 2011), carries a-(1?6)-linked xylose side chains with or without attached galactose and fucose. These three polysaccharide classes are considered to form dense networks (Park and Cosgrove, 2012)
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