The polysaccharide-rich cell walls (CWs) of plants perform essential functions such as maintaining tensile strength and allowing plant growth. Using two- and three-dimensional magic-angle-spinning (MAS) solid-state NMR and uniformly (13)C-labeled Arabidopsis thaliana, we have assigned the resonances of the major polysaccharides in the intact and insoluble primary CW and determined the intermolecular contacts and dynamics of cellulose, hemicelluloses, and pectins. Cellulose microfibrils showed extensive interactions with pectins, while the main hemicellulose, xyloglucan, exhibited few cellulose cross-peaks, suggesting limited entrapment in the microfibrils rather than extensive surface coating. Site-resolved (13)C T(1) and (1)H T(1ρ) relaxation times indicate that the entrapped xyloglucan has motional properties that are intermediate between the rigid cellulose and the dynamic pectins. Xyloglucan absence in a triple knockout mutant caused the polysaccharides to undergo much faster motions than in the wild-type CW. These results suggest that load bearing in plant CWs is accomplished by a single network of all three types of polysaccharides instead of a cellulose-xyloglucan network, thus revising the existing paradigm of CW structure. The extensive pectin-cellulose interaction suggests a central role for pectins in maintaining the structure and function of plant CWs. This study demonstrates the power of multidimensional MAS NMR for molecular level investigation of the structure and dynamics of complex and energy-rich plant materials.

Plant cell wall (CW) polysaccharides are responsible for the mechanical strength and growth of plant cells; however, the high-resolution structure and dynamics of the CW polysaccharides are still poorly understood because of the insoluble nature of these molecules. Here, we use 2D and 3D magic-angle-spinning (MAS) solid-state NMR (SSNMR) to investigate the structural role of pectins in the plant CW. Intact and partially depectinated primary CWs of Arabidopsis thaliana were uniformly labeled with (13)C and their NMR spectra were compared. Recent (13)C resonance assignment of the major polysaccharides in Arabidopsis thaliana CWs allowed us to determine the effects of depectination on the intermolecular packing and dynamics of the remaining wall polysaccharides. 2D and 3D correlation spectra show the suppression of pectin signals, confirming partial pectin removal by chelating agents and sodium carbonate. Importantly, higher cross peaks are observed in 2D and 3D (13)C spectra of the depectinated CW, suggesting higher rigidity and denser packing of the remaining wall polysaccharides compared with the intact CW. (13)C spin-lattice relaxation times and (1)H rotating-frame spin-lattice relaxation times indicate that the polysaccharides are more rigid on both the nanosecond and microsecond timescales in the depectinated CW. Taken together, these results indicate that pectic polysaccharides are highly dynamic and endow the polysaccharide network of the primary CW with mobility and flexibility, which may be important for pectin functions. This study demonstrates the capability of multidimensional SSNMR to determine the intermolecular interactions and dynamic structures of complex plant materials under near-native conditions.

A newly created object-oriented program for automating the process of fitting molecular-mechanics parameters to ab initio data, termed ParFit, is presented. ParFit uses a hybrid of deterministic and stochastic genetic algorithms. ParFit can simultaneously handle several molecular-mechanics parameters in multiple molecules and can also apply symmetric and antisymmetric constraints on the optimized parameters. The simultaneous handling of several molecules enhances the transferability of the fitted parameters. ParFit is written in Python, uses a rich set of standard and nonstandard Python libraries, and can be run in parallel on multicore computer systems. As an example, a series of phosphine oxides, important for metal extraction chemistry, are parametrized using ParFit. ParFit is in an open source program available for free on GitHub ( https://github.com/fzahari/ParFit ).