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

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,3Hxyloglucan and 12C,1Hxyloglucan 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, 13Cglucose-grown Rosa cells were transferred into 12Cglucose medium 6 h after a approximately 2 h pulse of l-1-3Harabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound 3Hxyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized 12C,1Hxyloglucan, it became covalently attached to previously wall-bound 13C, 3Hxyloglucan. In protocol B, 12Cglycerol- or 12Cglucose-grown Rosa cells were transferred into 13Cglucose medium, 20 or 60 min before a approximately 2 h pulse of 3Harabinose. The buoyant density of the earliest wall-bound 3Hxyloglucan showed that it had a 12C/13C ratio of approximately 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized 13C, 3Hxyloglucan became covalently attached to previously synthesized 12Cxyloglucan. During the following 7 days in culture, the mean buoyant density of the 3Hxyloglucan increased, showing that later-synthesized 13C,1Hxyloglucan can be covalently attached to previously wall-bound 12C,13C,3Hxyloglucan. 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.