Rhamnogalacturonan I (RG-I) is a structurally complex pectic polysaccharide found in the cell walls of all vascular plants. Due to its complexity, there is limited knowledge about RG-I’s structural variability and sequence pattern across different cells, tissues, and species, and how these patterns influence interactions with cellulose and other glycopolymers and regulate plant cell wall properties and functions. Developing new methods to assess RG-I’s fine structure is crucial for understanding these structure-function relationships. 

RG-I Structure in Arabidopsis: A schematic of RG-I structures in Arabidopsis reveals that arabinan side chains are substituted with single arabinofuranose (Araf) residues in 2-, 3-, and 2,3-positions. Arabinans are directly attached to the backbone, but the type of linkage is unknown. Both β1-4 and β1-6 linked galactan side chains are observed, with β1-6 linked galactans substituted with α1-3-linked Araf residues. Some galactan side chains are terminated by glucuronic acid (GlcA) residues carrying methyl-ether at the O-4 position. Arabinogalactans are observed, but their attachment to the backbone—whether via galactose (Gal) or arabinose (Ara) residues—is unclear. The linkage to the RG-I backbone is resolved by NMR only for the single-Gal side chain (β1-4). Acetylation on rhamnose (Rha) of the backbone is the major acetyl modification found, with mono- and di-acetylated galacturonic acid (GalA) residues also observed. Terminal β-linked Araf residues are observed, but their linkage is unknown.The || symbol indicates that the pictured structure is a composite of individual backbone disaccharide fragments with an attached branch but that the connection between these building blocks has not been established. In other words, the sequence and distribution of side chains is unknown. 

Enzymatic Toolbox for Linkage-Specific Deconstruction of RG-I. Lead Researcher: Breeanna Urbanowicz 

We are identifying new enzymes from gut bacteria capable of breaking down different portions of the RG-I molecule. These enzymes help obtain simpler fragments that can be studied using analytical methods to understand side chain structure and patterning. 

Development of Chemical and Spectroscopic Methods for Structural Characterization of RG-I. Lead Researchers: Christian Heiss, Li Tan, Parastoo Azadi 

Plant cell wall polysaccharides vary in solubility, and even a single type can differ in solubility based on its association with other cell wall components. We are developing new methods to solubilize all cell wall polysaccharides together for analysis by nuclear magnetic resonance spectroscopy and mass spectrometry providing a comprehensive picture of polysaccharide composition and structure. 

Lead Researcher: Debra Mohnen 

To further understand RG-I biosynthesis and function, we use the robust HEK293 cell heterologous protein expression system developed at the CCRC. This system allows us to express functional plant cell wall glycosyltransferases (GTs) and GT complexes to study RG-I synthesis initiation, backbone elongation mechanisms, and the identity and characteristics of enzymes that add glycosyl and non-glycosyl substituents to the backbone.