Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate

Abstract

Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a ≈ 2 h pulse of L-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C,3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a ≈ 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of ~ 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C,3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation accompanies, and probably causes, the integration of newly secreted xyloglucan into the cell-wall architecture.

Suppression and acceleration of cell elongation by integration of xyloglucans in pea stem segments

“…This integration is in agreement with the finding in cells of Rosa that XET-catalyzed interpolymeric transglycosylation integrates newly synthesized [ 13 C]xyloglucan into the cell wall xyloglucan (27,28). Large amounts of XET may exist in cell walls; XET constitutes 1.2% of total apoplastic proteins in Azuki hypocotyls (6).…”

In Vivo Colocalization of Xyloglucan Endotransglycosylase Activity and Its Donor Substrate in the Elongation Zone of Arabidopsis Roots

“…Such integration is another necessary element for continued cell expansion. A role for XET in xyloglucan integration has been supported by the demonstration that newly secreted xyloglucan chains undergo interpolymeric transglycosylation at the time of their binding to the cell wall (Thompson et al, 1998).Roles other than the two mentioned above can also be suggested. An enzyme with XET and xyloglucan endohydrolase activity is involved in the postgerminative mobilization of xyloglucan storage reserves in nasturtium ( Tropaeolum majus ) cotyledons (Farkaš et al, 1992; Fanutti et al, 1993).…”

Mixed‐linkage β‐glucan : xyloglucan endotransglucosylase, a novel wall‐remodelling enzyme from Equisetum (horsetails) and charophytic algae

“…It may also point to a role for MXE in wall strengthening during maturation, rather than in wall loosening during cell expansion. XTHs, in contrast, usually peak in young, rapidly growing tissue, where they may play a role in wall assembly (Ito and Nishitani, 1999;Thompson et al, 1997) and/or the restructuring of existing wall material to enable reversible wall loosening (Thompson and Fry, 2001). However, some XTHs may contribute to wall strengthening and contraction (e.g.…”

“…with both the donor and the acceptor-substrate of high M r ) has been demonstrated in vivo, establishing enzyme action as distinct from in-vitro activity (Fry, 2004;Thompson and Fry, 2001; Thompson et al, 1997). Xyloglucan remodelling by XET action could (i) restructure existing wall-bound xyloglucan molecules, thereby contributing to the reversible wall loosening required for cell expansion (Fry et al, 1992;Nishitani, 1997;Nishitani and Tominaga, 1992;Thompson and Fry, 2001;Van Sandt et al, 2007b), (ii) reconnect xyloglucan chains to enable the shrinkage of tension wood fibres during gravitropism in trees (Nishikubo et al, 2007), and (iii) help to integrate newly secreted xyloglucan chains into the cell-wall matrix (Ito and Nishitani, 1999;Thompson et al, 1997).…”