Advanced NMR analysis of the physical interactions leading to biochemical cohesiveness and enzymatic recalcitrance of grass cell

This is a proposal to use advanced solid-state NMR methods to elucidate the architecture and arrangement of cell walls of plants, grass species in particular. It was conceived within and will be carried out with strong participation from the DOE-funded Energy Frontier Research Center (EFRC) on Lignocellulose Structure and Formation (CLSF), directed by the PI of this proposal. The work will make use of state-of-the-art multi-dimensional solid-state NMR and 13C-enriched cell walls from the grass species Brachypodium distachyon to address several open questions about the architecture of grass cell walls, including: What proportion of the cellulose microfibril surface interacts with arabinoxylan, the dominant hemicellulose of grass cell walls? Do pectins interact extensively with cellulose surfaces, as seen in recent NMR studies of dicot cell walls? What fraction of arabinoxylan is mobile, what fraction rigid (thus presumably bound to cellulose surfaces)? Does dehydration irreversibly alter polysaccharide interactions within the cell wall? The answers to these and related questions will shed light on the important physical interactions that contributes to cell wall cohesiveness and recalcitrance to enzymatic deconstruction. This knowledge is relevant to the EMSL science theme entitled Biological Interactions and Dynamics because it characterizes biological polymers of relevance to biofuel or biofuel precursor production from plant biomass. For initial work we will prepare 13C-labeled cell walls from Brachypodium callus cultures grown on medium containing 13C-glucose. For some experiments we will selectively remove specific components with enzymes or chemical extractants/solvents. The specific aims of this proposal will be addressed by first identifying 13C chemical shifts for the major components of the grass cell wall (building on the published literature) and then assessing hemicellulose-cellulose cross-peaks in two- and three-dimensional NMR spectra, as described in the proposal. These methods have recently been worked out successfully for Arabidopsis cell walls by Mei Hong and collaborators, but many of these results are not applicable to grass cell walls which replace most of the pectin and much of the xyloglucan with arabinoxylan. Thus work is particularly relevant to DOE because most cellulosic feedstocks for biofuels are expected to come from sugarcane, switchgrass, Miscanthus, corn stover and other species in the grass family (Poaceae). We will make use of Brachypodium because its genome has been sequenced by JGI and it is being developed as a major model system for grass genetics. Our results will enhance these efforts as well as elucidate one of the key mechanisms of cell wall recalcitrance and set the stage for future work to combine the power of Brachypodium genetics with the advanced instrumentation capability of EMSL. The NMR capabilities and expertise at EMSL are critical for this proposal: the resolution and sensitivity enhancements obtained from running experiments at 20.0 T will provide unprecedented molecular-scale details for both interfacial components in this important system.