Making and breaking of boron bridges in the pectic domain rhamnogalacturonan-II at apoplastic pH in vivo and in vitro.

Cross-linking of the cell-wall pectin domain rhamnogalacturonan-II (RG-II) via boron-bridges between apiose residues is essential for normal plant growth and development, but little is known about its mechanism or reversibility. We characterised the making and breaking of boron-bridges in vivo and in vitro at 'apoplastic' pH. RG-II (13-26μM) was incubated in living Rosa cell-cultures and cell-free media with and without 1.2mM H 3 BO 3 and cationic chaperones (Ca 2+ , Pb 2+ , polyhistidine, or arabinogalactan-protein oligopeptides). RG-II's cross-linking status was monitored electrophoretically. Dimeric RG-II was stable at pH 2.0-7.0 in vivo and in vitro. In-vitro dimerisation required a 'catalytic' cation at all pHs tested (1.75-7.0); thus, merely neutralising RG-II's negative charge (at pH 1.75) does not enable boron-bridging. Pb 2+ (20-2500μM) was highly effective at pH 1.75-4.0, but not 4.75-7.0. Cationic peptides were effective at ~1-30μM; higher concentrations caused less dimerisation, probably because two RG-IIs then rarely bonded to the same peptide molecule. Peptides were ineffective at pH 1.75, their pH optimum being 2.5-4.75. d-Apiose (>40mM) blocked RG-II dimerisation in vitro, but did not cleave existing boron-bridges. Rosa cells did not take up d-[U- 14 C]apiose; therefore, exogenous apiose would block only apoplastic RG-II dimerisation in vivo. In conclusion, apoplastic pH neither broke boron-bridges nor prevented their formation. Thus boron-starved cells cannot salvage boron from RG-II, and 'acid growth' is not achieved by pH-dependent monomerisation of RG-II. Divalent metals and cationic peptides catalyse RG-II dimerisation via co-ordinate and ionic bonding respectively (possible and impossible, respectively, at pH1.75). Exogenous apiose may be useful to distinguish intra- and extra-protoplasmic dimerisation.