title: Xyloglucan-pectin linkages are formed intra-protoplasmically, contribute to wall-assembly, and remain stable in the cell wall.
abstract: 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.
authors.affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK.
authors.forename: Zoë A
authors.initials: ZA
authors.lastname: Popper
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Fry
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D005936:Glucans / Q000737:chemistry / Q000378:metabolism*; D010368:Pectins / Q000737:chemistry / Q000378:metabolism*; D014990:Xylans / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005936:Glucans; D014990:Xylans; C042491:rhamnogalacturonan I; C029353:xyloglucan; D010368:Pectins