Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II

Malcolm O'Neill, Ian Black, Breeanna Urbanowicz, Vivek Bharadwaj, Mike Crowley, Sabina Koj, Maria Pena

Research output: Contribution to journalArticlepeer-review

17 Scopus Citations


Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD4)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD4 reduction to identify 3-, 4-, and 3,4-O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD4-reduced counterparts, suggest that methyl etherification of the β-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

Original languageAmerican English
Pages (from-to)329-344
Number of pages16
JournalSLAS Technology
Issue number4
StatePublished - 1 Aug 2020

Bibliographical note

Publisher Copyright:
© 2020 Society for Laboratory Automation and Screening.

NREL Publication Number

  • NREL/JA-2800-77252


  • methyl ester
  • methyl ether
  • pectic polysaccharides
  • plant cell wall
  • rhamnogalacturonan II (RG-II)


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