Xyloglucan−pectin linkages are formed intra-protoplasmically, contribute to wall-assembly, and remain stable in the cell wall

Zoë A. Popper, Stephen C. Fry

We tested two hypotheses for the mechanism by which xyloglucan-pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using alpha-(1-->5)-L-[(3)H]arabino- or beta-(1-->4)-D-[(3)H]galacto-oligosaccharides as model acceptor substrates. The (3)H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [(3)H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [(3)H]Xyloglucan-pectin complexes were detectable <4 min after [(3)H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan-pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [(3)H]xyloglucan that was anionic remained almost constant at approximately 50% for > or =6 days, showing that the xyloglucan-pectin bond was stable in vivo. Some [(3)H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only approximately 30% of the sloughed [(3)H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that approximately 50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan-pectin bonds are stable and the pectic moiety aids wall-assembly.