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The primary plant cell wall is a complex matrix composed of interconnected polysaccharides including cellulose, hemicellulose, and pectin. Changes of this dynamic polysaccharide system play a critical role during plant cell development and differentiation. A better understanding of cell wall architectures can provide insight into the plant cell development. In this study, a Raman spectroscopic imaging approach was developed to visualize the distribution of plant cell wall polysaccharides. In this approach, Surface-enhanced Raman scattering (SERS through self-assembled silver nanoparticles) was combined with Raman labels (4-Aminothiophenol. 4ATP) and targeted enzymatic hydrolysis to improve the sensitivity, specificity, and throughput of the Raman imaging technique, and to reveal the distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell (OEC) wall. This technique significantly decreased the required spectral acquisition time. The resulted Raman spectra showed a high Raman signal. The resulted Raman images successfully revealed and characterized the pectin distribution and its co-localization pattern with xyloglucan in OEC wall., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

The plant's recalcitrant cell wall is composed of numerous polysaccharides, including cellulose, hemicellulose, and pectin. The most abundant hemicellulose in dicot cell walls is xyloglucan, which consists of a β-(1- > 4) glucan backbone with α-(1- > 6) xylosylation producing an XXGG or XXXG pattern. Xylose residues of xyloglucan are branched further with different patterns of arabinose, fucose, galactose, and acetylation that varies between species. Although xyloglucan research in other species lag behind Arabidopsis thaliana , significant advances have been made into the agriculturally relevant species Oryza sativa and Solanum lycopersicum , which can be considered model organisms for XXGG type xyloglucan. In this review, we will present what is currently known about xyloglucan biosynthesis in A. thaliana , O. sativa , and S. lycopersicum and discuss the recent advances in the characterization of the glycosyltransferases involved in this complex process and their organization in the Golgi. [ABSTRACT FROM AUTHOR]

The plant cell wall is a complex network of polysaccharides. A better understanding of the plant cell wall polysaccharide content, distribution, and interactions among them could be instrumental for our further understanding of plant cell wall in general. Confocal Raman microscopy (CRM) is a powerful tool that could reveal details about chemical landscape of the plant cell wall with micrometer resolution. However, the low signal‐to‐noise (S/N) ratio of Raman spectral signal led to low throughput of Raman imaging, which limited its application in plant cell wall study. In this study, the interaction between the cell wall polysaccharides and the pectin enzyme endo‐polygalacturonase (EPG) was characterized by analyzing Raman images collected before and after EPG treatment at high scanning speed. The obtained low S/N ratio Raman spectra were processed with principal component analysis (PCA) and hierarchical clustering analysis (HCA) to recover information‐rich signature changes in Raman signal dataset that reflected the changes in the polysaccharides caused specifically by the enzymatic hydrolysis. The PCA reconstruction technique significantly improved the S/N ratio of the spectra dataset while kept the Raman signal intact. Further analysis of principal components (PCs) revealed the pectin distribution and its interaction with the enzyme, which provided organizational details of pectin inside the onion plant cell wall. The technique could be used to reveal polysaccharide organization and distribution inside plant cell walls. [ABSTRACT FROM AUTHOR]

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 two-dimensional (2D) and three-dimensional high-resolution magic-angle-spinning 13C solid-state nuclear magnetic resonance spectra of the 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 semiquantitative 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 cellulose-surface cellulose cross-peaks, indicating that pectins come into direct contact with the cellulose microfibrils. Approximately 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 types of polysaccharides in the primary wall of Arabidopsis form a single cohesive network, while structural proteins form a relatively separate domain. [ABSTRACT FROM AUTHOR]

Pectin is a critical component of the plant cell wall, supporting wall biomechanics and contributing to cell wall signaling in response to stress. The plant cell carefully regulates pectin methylesterification with endogenous pectin methylesterases (PMEs) and their inhibitors (PMEIs) to promote growth and protect against pathogens. We expressed Aspergillus nidulans pectin methylesterase (AnPME) in Arabidopsis thaliana plants to determine the impacts of methylesterification status on pectin function. Plants expressing AnPME had a roughly 50% reduction in methylester content compared with control plants. AnPME plants displayed a severe dwarf phenotype, including small, bushy rosettes and shorter roots. This phenotype was caused by a reduction in cell elongation. Cell wall composition was altered in AnPME plants, with significantly more arabinose and significantly less galacturonic acid, suggesting that plants actively monitor and compensate for altered pectin content. Cell walls of AnPME plants were more readily degraded by polygalacturonase (PG) alone but were less susceptible to treatment with a mixture of PG and PME. AnPME plants were insensitive to osmotic stress, and their susceptibility to Botrytis cinerea was comparable to wild type plants despite their compromised cell walls. This is likely due to upregulated expression of defense response genes observed in AnPME plants. These results demonstrate the importance of pectin in both normal growth and development, and in response to biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

The polysaccharide structure and dynamics in the primary cell wall of the model grass Brachypodium distachyon are investigated for the first time using solid-state nuclear magnetic resonance (NMR). While both grass and non-grass cell walls contain cellulose as the main structural scaffold, the former contains xylan with arabinose and glucuronic acid substitutions as the main hemicellulose, with a small amount of xyloglucan (XyG) and pectins, while the latter contains XyG as the main hemicellulose and significant amounts of pectins. We labeled the Brachypodium cell wall with 13C to allow two-dimensional (2D) 13C correlation NMR experiments under magic-angle spinning. Well-resolved 2D spectra are obtained in which the 13C signals of cellulose, glucuronoarabinoxylan (GAX), and other matrix polysaccharides can be assigned. The assigned C chemical shifts indicate that there are a large number of arabinose and xylose linkages in the wall, and GAX is significantly branched at the developmental stage of 2 weeks. 2D 13C-13C correlation spectra measured with long spin diifusion mixing times indicate that the branched GAX approaches cellulose microfibrils on the nanometer scale, contrary to the conventional model in which only unbranched GAX can bind cellulose. The GAX chains are highly dynamic, with average order parameters of ∼0.4. Biexponential 13C T1 and 1H T1ρ, relaxation indicates that there are two dynamically distinct domains in GAX: the more rigid domain may be responsible for cross-linking cellulose microfibrils, while the more mobile domain may fill the interfibrillar space. This dynamic heterogeneity is more pronounced than that of the non-grass hemicellulose, XyG, suggesting that GAX adopts the mixed characteristics of XyG and pectins. Moderate differences in cellulose rigidity are observed between the Brachypodium and Arabidopsis cell walls, suggesting different effects of the matrix polysaccharides on cellulose. These data provide the first molecular-level structural information about the three-dimensional organization of the polysaccharides in the grass primary wall. [ABSTRACT FROM AUTHOR]

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 13C-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 13C T1 and ±H T1ρ 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. [ABSTRACT FROM AUTHOR]