| Abstract: | In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.Growth and form in plants are controlled by the precisely oriented expansion of the walls of individual cells. The driving force for cell expansion is osmotic, but the rate and direction of expansion are controlled by the mechanical properties of the cell wall (Szymanski and Cosgrove, 2009). Expanding, primary cell walls are nanocomposite materials in which long microfibrils of cellulose, a few nanometers in diameter, run through a hydrated matrix of xyloglucans, pectins, and other polymers (Knox, 2008; Mohnen, 2008; Szymanski and Cosgrove, 2009; Scheller and Ulvskov, 2010). Native cellulose microfibrils are partially crystalline (Nishiyama, 2009; Fernandes et al., 2011). Formerly, primary wall cellulose was thought to have a unique crystal structure called cellulose IV1 (Dinand et al., 1996), but NMR evidence suggests the presence of forms similar to the better characterized cellulose Iα and Iβ crystalline forms together with large quantities of less ordered cellulose (Wickholm et al., 1998; Sturcová et al., 2004; Wada et al., 2004). Nevertheless, cellulose is much more ordered than any other component of the primary cell wall (Bootten et al., 2004), in keeping with its key role of providing strength and controlling growth.The stiffness of the cell wall is greatest in the direction of the cellulose microfibrils, where growth is directional and the predominant microfibril orientation is usually transverse to the growth direction (Green, 1999; MacKinnon et al., 2006; Szymanski and Cosgrove, 2009). Expansion of the cell wall then requires either widening of the spacing between microfibrils (Marga et al., 2005) or slippage between them (Cosgrove, 2005), or both, and the microfibrils reorient toward the direction of growth (Anderson et al., 2010). Polymer cross bridges between microfibrils (McCann et al., 1990) are thought to resist these deformations of the cell wall nanostructure and, thus, to control the rate of growth. Until recently, most attention was focused on bridging xyloglucans, hydrogen bonded to microfibril surfaces (Scheller and Ulvskov, 2010). However, there is evidence that not all xyloglucans are appropriately positioned (Fujino et al., 2000; Park and Cosgrove, 2012a) and that other bridging polymers may be involved (Zykwinska et al., 2007). It has also been suggested that bundles of aggregated microfibrils, not single microfibrils, might be the key structural units in primary cell walls (Anderson et al., 2010), as in wood (Fahlén and Salmén, 2005; Fernandes et al., 2011). If so, single microfibrils could bridge between microfibril bundles. In summary, the growth of plant cells is not well understood, and we need more information on how cellulose orientation is controlled and on the nature of the bridging polymers, the cellulose surfaces to which these polymers bind, and the cohesion between microfibril surfaces that might mediate aggregation.Cellulose microfibrils are synthesized at the cell surface by large enzyme complexes having hexagonal symmetry, sometimes called “rosettes” (Somerville, 2006). Each complex contains multiple cellulose synthases that differ between primary cell walls and wood, although the appearance of the complexes is similar (Somerville, 2006; Atanassov et al., 2009). The simultaneous synthesis, from the same end, of all the chains in a native cellulose microfibril is why they are parallel (Nishiyama et al., 2002, 2003), in contrast to the entropically favored antiparallel structure found in man-made celluloses like rayon (Langan et al., 2001). The number of chains in a microfibril and the number of cellulose synthases in the synthetic complex are evidently related. It is commonly assumed that the number of chains is divisible by six, matching the hexagonal rosette symmetry, and 36-chain models (Himmel et al., 2007) bounded by the hydrophilic [110] and [1-10] crystal faces, as in algal celluloses (Bergenstråhle et al., 2008), have been widely adopted. The assembly and orientation of cellulose are connected, as several cellulose synthase mutants have phenotypes defective in cellulose orientation and plant form as well as depleted in cellulose content (Paredez et al., 2008). In certain other mutant lines, the crystallinity of the microfibrils appears to be affected (Fujita et al., 2011; Harris et al., 2012; Sánchez-Rodríguez et al., 2012).Therefore, a detailed understanding of the structure of primary wall cellulose microfibrils would help us to understand cellulose synthesis as well as the growth and structural mechanics of living plants (Burgert and Fratzl, 2009). Primary cell walls and their cellulose skeletons also affect food quality characteristics like the crispness of salad vegetables and apples (Malus domestica; Jarvis, 2011). When biofuels are produced from lignocellulosic biomass, lignification leads to recalcitrance (Himmel et al., 2007), but some of the cell types in Miscanthus spp., switchgrass (Panicum virgatum), and arable crop residues have only primary walls with no lignin, and recalcitrance then depends on the nature of the cellulose microfibrils (Beckham et al., 2011).A relatively detailed structure has recently been proposed for the microfibrils of spruce (Picea spp.) wood (Fernandes et al., 2011), which are 3.0 nm in diameter, allowing space for only about 24 cellulose chains. Evidence from x-ray diffraction supported a “rectangular” shape (Matthews et al., 2006) bounded by the [010] and [200] faces. There was considerable disorder increasing toward the surface, and the microfibrils were aggregated into bundles about 15 to 20 nm across, with some, but not all, of the lateral interfaces being resistant to water (Fernandes et al., 2011). Disordered domains are a feature of other strong biological materials such as spider silk (van Beek et al., 2002).Therefore, it is of interest whether any of these features of wood cellulose might also be found in the cellulose microfibrils of primary (growing) cell walls. It would be particularly useful to characterize the disorder known to be present in primary wall microfibrils, that is, to define how cellulose that is not measured as “crystalline” differs from crystalline cellulose. Many of the experiments leading toward a structure for wood cellulose were dependent on exceptionally uniform orientation of the cellulose microfibrils (Sturcová et al., 2004; Fernandes et al., 2011). However, in growing cell walls, the microfibrils are not uniformly oriented. When microfibrils are first laid down at the inner face of the primary cell wall, their orientation is normally transverse to the direction of growth, but as the cell wall expands, the microfibrils reorient so that the orientation distribution, integrated across the thickness of the expanded cell wall, becomes progressively closer to random (Cosgrove, 2005; MacKinnon et al., 2006).This technical problem does not apply to the cell walls of celery (Apium graveolens) collenchyma, which are similar in composition to other primary cell walls but have their microfibrils oriented relatively uniformly along the cell axis (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b). Some structural information on celery collenchyma cellulose has already been derived from spectroscopic and scattering experiments (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b), confirming the disorder expected in a primary wall cellulose. Some of these experiments were analogous to what has been done on spruce cellulose (Fernandes et al., 2011), but insufficient data are available to specify the number of chains in each primary wall microfibril, the nature and location of the disorder, and the presence or absence of direct contact between microfibrils. Here, we report x-ray and neutron scattering and spectroscopic experiments addressing these questions and leading to a proposed structure for primary wall cellulose microfibrils. Characterizing a structure containing so much disorder presented unusual challenges, but disorder appears to be central to the enigmatic capacity of primary wall cellulose to provide high strength and yet to permit and control growth. |
