Boron bridging of rhamnogalacturonan-II in Rosa and arabidopsis cell cultures occurs mainly in the endo-membrane system and continues at a reduced rate after secretion.

Abstract

Rhamnogalacturonan-II (RG-II) is a domain of primary cell-wall pectin. Pairs of RG-II domains are covalently cross-linked via borate diester bridges, necessary for normal cell growth. Interpreting the precise mechanism and roles of boron bridging is difficult because there are conflicting hypotheses as to whether bridging occurs mainly within the Golgi system, concurrently with secretion or within the cell wall. We therefore explored the kinetics of RG-II bridging. Cell-suspension cultures of Rosa and arabidopsis were pulse-radiolabelled with [14C]glucose, then the boron bridging status of newly synthesized [14C]RG-II domains was tracked by polyacrylamide gel electrophoresis of endo-polygalacturonase digests. Optimal culture ages for 14C-labelling were ~5 and ~1 d in Rosa and arabidopsis respectively. De-novo [14C]polysaccharide production occurred for the first ~90 min; thereafter the radiolabelled molecules were tracked as they 'aged' in the wall. Monomeric and (boron-bridged) dimeric [14C]RG-II domains appeared simultaneously, both being detectable within 4 min of [14C]glucose feeding, i.e. well before the secretion of newly synthesized [14C]polysaccharides into the apoplast at ~15-20 min. The [14C]dimer : [14C]monomer ratio of RG-II remained approximately constant from 4 to 120 min, indicating that boron bridging was occurring within the Golgi system during polysaccharide biosynthesis. However, [14C]dimers increased slightly over the following 15 h, indicating that limited boron bridging was continuing after secretion. The results show where in the cell (and thus when in the 'career' of an RG-II domain) boron bridging occurs, helping to define the possible biological roles of RG-II dimerization and the probable localization of boron-donating glycoproteins or glycolipids.