Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II

Abstract

Background In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. Results We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. Conclusions The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I

“…An aliquot of 100 µL from the full reaction, representing 5 µg of the starting DP12-2AB acceptor, was removed and injected into a Superdex 75 10/300 GL column attached to an Agilent 1260 In nity II high pressure liquid chromatography system at a ow rate of 0.5 mL/min of 50 mM ammonium formate. RG-I products were detected by multi-angle light scattering coupled with size-exclusion chromatography (SEC-MALS) as described for determation of RG-II molecular mass 45 . Detection was performed using an Optilab t-rEX differential refractometer (Wyatt Technology) connected in series with a Dawn Heleos 8 MALS detector.…”

A putative rhamnogalacturonan-II CMP-β-Kdo transferase identified using CRISPR/Cas9 gene edited callus to circumvent embryo lethality

“…As described in 40 , the callus AIR (x g) were first resuspended in x mL of NaOAc (50 mM, pH 5.2), and then treated with 5 U/g endopolygalacturonase (EPG) at 30 °C for 24 hrs, shaking at 150 rpm. The EPG solublized AIR and the insoluble residue were separated by filtration through a nylon mesh (100 μm pre size).…”

“…To specifically detect Kdo and Dha, the glycosyl residue of RG-II were analyzed as their trimethylsilyl methyl glycodide (TMS) derivatives by Gas-liquid chromatography with electron impact mass spectrometry (GLC-EI-MS) as described in 40 . In general, RG-II (x μg) was suspended in x μL methanolic HCl (1 M) in screw-top glass tubes secured with Teflon-lined caps and heated at 80 °C for 18 hrs.…”

Insights into pectin O-acetylation in the plant cell wall: structure, synthesis, and modification

“…1 A representative model structure of pectin. The schematic shown here highlights the four main structural domains; homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) ( Caffall and Mohnen, 2009 ), rhamnogalacturonan-II (RG-II) ( Barnes et al, 2021 ), and xylogalacturonan (XGA) ( Jensen et al, 2008 ). The order and degree of side chain substitution is not known.…”