The polysaccharide-rich plant cell wall provides mechanical strength to plant cells and is loosened during plant growth. Structural investigations of the wall polymers at the molecular level are important for understanding these diverse functions of the wall. We have developed several solid-state NMR techniques to determine the polysaccharide conformations and intermolecular interactions in intact cell walls. A T1 relaxation-compensated proton-driven 13C spin diffusion experiment enables us to suppress intramolecular cross peaks in 2D spectra and selectively detect only long-range cross peaks. This allowed us to determine unambiguous spatial contacts between cellulose and pectins in primary plant cell walls. We have adopted a CHHC technique to measure the H4-H6 distances in glucose, which allows us to restrain the hydroxymethyl conformations of surface and interior cellulose in the cell walls of model plants. To determine how wall-loosening proteins bind to wall polysaccharides, we developed two methods. Under dynamic nuclear polarization to enhance the NMR sensitivity by 30 fold, we can selectively excite and transfer protein 13C magnetization to polysaccharides, thus determining the binding target of the protein. Using this approach, we showed that bacterial expansin binds xyloglucan-enriched regions of the cellulose microfibrils in Arabidopsis primary cell walls to loosen the wall. For proteins that cannot be produced recombinantly, we developed a paramagnetic relaxation enhancement NMR technique whereby the naturally extracted protein is tagged by a paramagnetic ion to speed up nuclear spin relaxation to polysaccharides in close proximity to the protein. Using this method, we showed that β-expansin binds the non-covalent junctions between highly substituted and lowly substituted glucuronoarabinoxylan to loosen the cell wall. These novel solid-state NMR techniques enlarge the toolbox for molecular-structure characterization of complex polysaccharides in cell walls, and provide unique insights into the functionally relevant three-dimensional structure of the cell wall.

Solid-state NMR methods to determine glycan structure, intermolecular interactions and protein binding in plant cell walls

253rd National Meeting (2017)

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