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

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 α-(1→5)-l-[3H]arabino- or β-(1→4)-d-[3H]galacto-oligosaccharides as model acceptor substrates. The 3H-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 [3H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [3H]Xyloglucan–pectin complexes were detectable <4 min after [3H]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 [3H]xyloglucan that was anionic remained almost constant at ∼50% for ≥6 days, showing that the xyloglucan–pectin bond was stable in vivo. Some [3H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only ∼30% of the sloughed [3H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that ∼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.