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

SUMMARY

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 characterized the making and breaking of boron bridges in vivo and in vitro at ‘apoplastic’ pH. RG‐II (13–26 μ m) was incubated in living Rosa cell cultures and cell‐free media with and without 1.2 m m H ₃BO ₃ and cationic chaperones (Ca ²⁺, Pb ²⁺, polyhistidine, or arabinogalactan‐protein oligopeptides). The cross‐linking status of RG‐II was monitored electrophoretically. Dimeric RG‐II was stable at pH 2.0–7.0 in vivo and in vitro. In‐vitro dimerization required a ‘catalytic’ cation at all pHs tested (1.75–7.0); thus, merely neutralizing the negative charge of RG‐II (at pH 1.75) does not enable boron bridging. Pb ²⁺ (20–2500 μ m) was highly effective at pH 1.75–4.0, but not 4.75–7.0. Cationic peptides were effective at approximately 1–30 μ m; higher concentrations caused less dimerization, 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 (>40 m m) blocked RG‐II dimerization in vitro, but did not cleave existing boron bridges. Rosa cells did not take up d‐[U‐ ¹⁴C]apiose; therefore, exogenous apiose would block only apoplastic RG‐II dimerization 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 monomerization of RG‐II. Divalent metals and cationic peptides catalyse RG‐II dimerization via co‐ordinate and ionic bonding respectively (possible and impossible, respectively, at pH 1.75). Exogenous apiose may be useful to distinguish intra‐ and extra‐protoplasmic dimerization.

Significance Statement

This study demonstrates the nature of the interaction between the cell‐wall pectic domain rhamnogalacturonan‐II (RG‐II) and cationic chaperones necessary for efficient RG‐II dimerization via borate diester bonds at acidic pH, resembling the acid growth conditions in the cell wall. Boron bridging is irreversible at apoplastic pH, so boron‐starved cells cannot salvage boron from RG‐II, and wall loosening in ‘acid growth’ is not achieved by pH‐dependent modification of RG‐II cross‐linking.