The plant cell walls are made of a complex network of interacting polymers including polysaccharides and lignin, which is a complex polyphenolic network. They can be classified into primary and secondary cell walls depending on their composition and physical characteristics. Primary cell walls found in all growing cells are thin, extensible, and are mainly composed of cellulose, hemicellulose, and pectin, whereas secondary cell walls, found only in some mature cell types, are thick, less flexible and are composed mainly of cellulose, heteroxylan, mannan, and lignin (1). Secondary cell walls are the main constituents of plant lignocellulosic biomass from major feedstocks used in biofuels industry (2, 3). Cellulose alone represents the most abundant renewable source available worldwide (about 1.5x1012 tons of the total annual biomass) produced via photosynthesis. However, due to lignin, the accessibility to this renewable source is reduced, which negatively impacts biofuels industry (4, 5).
Despite technological advances allowing the isolation and structural analysis of individual polysaccharides from cell walls of various plant tissues, our understanding about how these polysaccharides are organized into specific molecular three-dimensional (3D) architectures is very limited (6, 7). Elucidating this 3D organization of plant cell walls is a prerequisite for the full understanding of how plants can adapt to environmental and growth conditions specific to cell types. For structural analysis, the individual polysaccharides are first extracted from cell walls by treatments with various chemicals. However, the 3D structure that these polymers adopt within the cell wall is lost and can only be predicted through molecular computer modeling. X-ray diffraction and magic-angle spinning solid-state nuclear magnetic resonance (ssNMR), along with better computer modeling allowed the determination of isolated semi-crystalline structure of cellulose microfibrils (8–12). These studies showed that the b(1–4)-d-glucan chains of cellulose microfibrils can fold in a two-fold helical screw conformation (one 360° twist per two glycosidic bonds) forming stiffened sheets in parallel arrangement via inter and intra chains hydrogen bonds and sheets are held together through weak van-der waals forces to form microfibrils (13). However, the dynamic of the interactions of these cellulose microfibrils with other cell wall polymers (in particular hemicellulose and lignin) is poorly characterized (14–16).
The major hemicellulose in the primary cell walls of dicots is xyloglucan (XyG, which consists of a backbone of b(1, 4)-linked glucosyl chains), whereas heteroxylan (HX), which consists of a backbone of b(1, 4)-linked xylosyl chains, is the main hemicellulosic polymer in primary and secondary cell walls of monocots. These polymers were thought to cross-link cellulose microfibrils within cell walls. However, recent study using ssNMR have shown that while only a minor portion of XyG interacts with cellulose microfibrils (17), the majority of HX binds to cellulose microfibrils in a flattened two-fold helical screw fold, which has similar rigidity to the cellulose microfibrils (18). Despite these advances, we are still lacking a clear model for the 3D network formed by the interactions of these polysaccharides. Multidimensional ssNMR, which relies on the resolution of 13C chemical shifts of cell wall polysaccharides in 2D and 3D 13C-13C correlation NMR spectra, has been instrumental in probing these interactions in native cell wall from never-dried plant tissues (19, 20). However, the low natural abundance of 13C isotope in plant material (1.1%) results in limited NMR sensitivity and makes it challenging to apply multidimensional (2D/3D) correlation experimental schemes to achieve high resolution (15). In addition, the cost of 13C-labeled CO2 needed to grow plants in standard growth chambers further limited accessibility of researchers to ssNMR analysis (17).
In this work, we describe a simple and cost-effective protocol for 13C-labelling of plant materials using 13C-labeled precursors (e.g., glucose, sucrose, CO2) to facilitate accessibility to multidimensional ssNMR analysis. This protocol allows more than 60% 13C-labelling of plant cell wall and can be adapted to any plant species. This important enabling technique allows high-resolution solid-state NMR analysis to be more accessible to our research community, helping to address many unsolved questions related to biomass structure.