Boron cross-linking of rhamnogalacturonan-II in vivo and in vitro: effects of pH and cationic chaperones.
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Date
07/12/2021Author
Begum, Rifat Ara
Metadata
Abstract
Rhamnogalacturonan-II (RG-II) is a ~5 kDa pectic polysaccharide domain, which typically
represents 1–4% of the total polysaccharide of the primary cell wall in dicots. It has a complex
structure with a backbone of 8-10 galacturonic acid (GalA) residues, to which are attached 6
different sidechains (A–F). In vivo, RG-II is prevalent in its dimeric form, which is produced
by borate-diester bridge formation between two sidechain A apiose residues from two RG-II
molecules. RG-II dimerisation is important for maintaining cell wall porosity, thickness, and
biophysical properties for cell growth. But dimerisation is not inevitable even in the presence
of boric acid; it also requires the presence of a cationic ‘chaperone’.
In this project, the effect of acidic pH on RG-II was studied in vitro, to understand the role of
RG-II and its dimerisation during auxin induced acid growth of cells. The effect of alkaline pH
on RG-II was investigated to understand the importance of ester groups affecting the
charge:mass ratio of RG-II. In-vivo 14C-radiolabelling using Paul’s scarlet Rosa and
Arabidopsis cultures was conducted to understand the kinetics of RG-II dimerisation. RG-II
monomers and dimers were separated and observed using polyacrylamide gel electrophoresis
and silver nitrate staining, and newly synthesised RG-II domains were traced in vivo by pulse-labelling with [14C]glucose.
In-vitro study on the effect of acidic pH showed that RG-II dimerisation is favoured at
physiologically acidic pH (~4.0–5.0, mimicking the apoplastic pH during auxin-induced
growth) in presence of boron and cationic chaperones. Alkaline pH affected the esterification
status of RG-II, hence controlling the sites accessible to pectolytic enzymes and affecting the
charge:mass ratio of RG-II. The in-vivo 14C-radiolabelling kinetic study on living Rosa and
Arabidopsis cells showed that [14C]RG-II is synthesised and dimerised intra-cellularly, rather
than after secretion. Another study supported this intracellular localisation of dimerisation by
showing the inability of added apoplastic apiose (which cannot enter the symplast) to prevent
RG-II dimerisation. A study conducted on glycosylinositol phosphorylceramide (GIPCs) gave
inconclusive results regarding its hypothesized role as a boron-donor.
Alongside these studies, it was found that the three classical lysine-rich arabinogalactan
proteins of A. thaliana (AtAGP17, AtAGP18 and AtAGP19) are potential biological chaperones
for promoting RG-II dimerisation: specifically, certain highly basic peptide fragments (DP 10–
13) of these three AGPs promoted RG-II dimerisation in vitro. Overall, these studies open the
possibility of conducting future studies for revealing the detailed biochemical character and
biological roles of RG-II and the proposed AGPs as cationic chaperones for RG-II dimerisation
in vivo.