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ABSTRACT: The primary cell wall of higher plants consists of a mixture of polysaccharides whose spatial proximities and interactions with each other are not well understood. We recently obtained the first 2D and 3D high-resolution magic-angle-spinning (MAS) 13C solid-state NMR spectra of uniformly 13C-labeled primary cell wall of Arabidopsis thaliana, which allowed us to assign the majority of 13C resonances of the three major classes of polysaccharides: cellulose, hemicellulose, and pectins. In this work, we measured the intensity buildup of 13C-13C cross peaks in a series of 2D 13C correlation spectra, to obtain semi-quantitative information about the spatial proximities between different polysaccharides. Comparison of 2D spectra measured at different spin diffusion mixing times identified intermolecular pectin - cellulose cross peaks as well as interior cellulose - surface cellulose cross peaks. The intensity buildup time constants are only modestly longer for cellulose-pectin cross peaks than for interior-surface cellulose cross peaks, indicating that pectins come into direct contact with the cellulose microfibrils. About 25-50% of the cellulose chains exhibit close contact with pectins. The 13C magnetization of the wall polysaccharides is not fully equilibrated by 1.5 s, indicating that pectins and cellulose are not homogeneously mixed on the molecular level. We also assigned the 13C signals of cell-wall proteins, identifying common residues such as Pro, Hyp, Tyr and Ala. The chemical shifts indicate significant coil and sheet conformations in these structural proteins. Interestingly, few cross peaks were observed between the proteins and the polysaccharides. Taken together, these data indicate that the three major polysaccharides in the primary wall of Arabidopsis form a single cohesive network, while structural proteins form a relatively separate domain.
Biochemistry 11/2012; · 3.42 Impact Factor
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ABSTRACT: 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.
Magnetic Resonance in Chemistry 07/2012; 50(8):539-50. · 1.44 Impact Factor
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ABSTRACT: 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.
Biochemistry 02/2011; 50(6):989-1000. · 3.42 Impact Factor
