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

Abstract

Most pectic rhamnogalacturonan-II (RG-II) domains in plant cell walls are borate-bridged dimers. However, the sub-cellular locations, pH dependence, reversibility and biocatalyst involvement in borate bridging remain uncertain. Experiments discussed here explored these questions, utilising suspension-cultured plant cells. In-vivo pulse radiolabelling showed that most RG-II domains dimerise extremely quickly (<4 min after biosynthesis, thus while still intraprotoplasmic). This tallies with the finding that boron withdrawal causes cell wall weakening within 10–20 min, and supports a previously proposed biological role for boron/RG-II complexes specifically at the wall/membrane interface. We also discuss RG-II monomer ↔ dimer interconversion as monitored in vitro using gel electrophoresis and a novel thin-layer chromatography method to resolve monomers and dimers. Physiologically relevant acidity did not monomerise dimers, thus boron bridge breaking cannot be a wall-loosening mechanism in ‘acid growth’; nevertheless, recently discovered RG-II trimers and tetramers are unstable and may thus underpin reversible wall loosening. Dimerising monomers in vitro by B(OH)3 required the simultaneous presence of RG-II-binding ‘chaperones’: co-ordinately binding metals and/or ionically binding cationic peptides. Natural chaperones of the latter type include highly basic arabinogalactan protein fragments, e.g., KHKRKHKHKRHHH, which catalyse a reaction [2 RG-II + B(OH)3 → RG-II–B–RG-II], suggesting that plants can ‘enzymically’ metabolise boron.

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“…The three major domains are HG, RG‐I and RG‐II, which can readily be distinguished after digestion of whole cell walls with endo‐polygalacturonase (EPG). This enzyme cleaves the HG domains (after pre‐treatment with mild alkali to remove the methyl and acetyl ester groups) to yield GalA 1 , 2 and 3 , and in the process also releases intact RG‐I (M r ~ 50–500 kDa; Kaczmarska et al, 2022 ) and RG‐II (M r ~ 5 kDa; Kobayashi et al, 1999 ; Begum & Fry, 2023 ). It is suspected that land‐plant pectin comprises a large polysaccharide in which HG, RG‐I and RG‐II are glycosidically linked like beads on a string.…”

“…and 3 , and in the process also releases intact RG-I (M r $ 50-500 kDa;Kaczmarska et al, 2022) and RG-II (M r $ 5 kDa;Kobayashi et al, 1999;Begum & Fry, 2023). It is suspected that land-plant pectin comprises a large polysaccharide in which HG, RG-I and RG-II are glycosidically linked like beads on a string.…”