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

Abstract

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 μm) was incubated in living Rosa cell cultures and cell‐free media with and without 1.2 mm H3BO3 and cationic chaperones (Ca2+, Pb2+, 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. Pb2+ (20–2500 μm) was highly effective at pH 1.75–4.0, but not 4.75–7.0. Cationic peptides were effective at approximately 1–30 μm; 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 mm) 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.

Arabinogalactan-Proteins as Boron-Acting Enzymes, Cross-Linking the Rhamnogalacturonan-II Domains of Pectin

“…Although the above reaction (Figure 3) occurs only very slowly in vitro at all pH values tested (~1.75-7.0) [23], it can be greatly expedited by the addition of traces of certain 'nonbiological' divalent metal cations, especially those with an ionic radius of >0.11 nm, e.g., Pb 2+ , Sr 2+ and Ba 2+ [7,31,32,53,54]. Pb 2+ was highly effective at pH ~2-4, but not at higher pH values (Figure 5e) [23].…”

“…The negative charges on RG-II can be essentially abolished if the pH of the reaction mixture is reduced to about 2 (a pH at which B bridges are stable) or below. However, even at pH 2, B(OH) 3 alone does not dimerise RG-II in vitro [23].…”

“…With silver-staining on PAGE, in contrast, the dimer is detected with much greater sensitivity than an equal mass of monomer [5,22]. As will be discussed later, recent evidence suggests that B may also form weak bridges via the side chain B of RG-II [23]; their weakness is of particular interest as this type of B bridging would be reversible in vivo, unlike the stable bridging via side chain A. The plastic-backed TLC plate was Merck silica gel.…”

“…The evidence for this is that even though RG-II is highly water-soluble, aqueous buffers fail to extract any 5-or 10-kDa RG-II from the cell wall. It might be argued that any free RG-II would have been lost during cell wall isolation; however, free RG-II is also absent in the spent media of cell-suspension cultures [23], where any naturally free RG-II would have accumulated. The only known way of obtaining free RG-II is artificially-to digest the cell wall's pectin enzymically.…”

Linking the key physiological functions of essential micronutrients to their deficiency symptoms in plants

“…Boron‐driven dimerization of RG‐II occurs predominantly inside the cell, in the Golgi apparatus, before its release into the apoplast (Begum & Fry, 2022). Furthermore, the B‐bridged RG‐II complex appears to be extremely stable and resistant to degradation, hence it is unlikely that B‐deficient plants can access already‐formed RG‐II‐B complexes for new growth (Begum et al ., 2023).…”