Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots

Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.The walls of growing plant cells must fulfill two simultaneous and seemingly contradictory requirements. First, they must expand to accommodate cell growth, which is anisotropic in many tissues and determines organ morphology. Second, they must maintain their structural integrity, both to constrain the turgor pressure that drives cell growth and to provide structural rigidity to the plant. These requirements are met by constructing primary cell walls that can expand along with growing cells, whereas secondary cell walls are deposited after cell growth has ceased and serve the latter function.One of the major constituents of both types of cell walls is cellulose, which exists as microfibrils composed of parallel β-1,4-linked glucan chains that are held together laterally by hydrogen bonds (Somerville, 2006). Microfibrils are 2 to 5 nm in diameter, can extend to several micrometers in length, and exhibit high tensile strength that allows cell walls to withstand turgor pressures of up to 1 MPa (Franks, 2003). In vascular plants, cellulose is synthesized by a multimeric cellulose synthase (CESA) complex composed of at least three types of glycosyl transferases arranged into a hexameric rosette (Somerville, 2006). After delivery to the plasma membrane, CESA initially moves in alignment with cortical microtubules (Paredez et al., 2006), but its trajectory can be maintained independently of microtubule orientation. For example, in older epidermal cells of the root elongation zone in Arabidopsis (Arabidopsis thaliana), cellulose microfibrils at the inner wall face are oriented transversely despite the fact that microtubules reorient from transverse to longitudinal along the elongation zone (Sugimoto et al., 2000), suggesting that microtubule orientation and cellulose deposition are independent in at least some cases.Depending on species, cell type, and developmental stage, cellulose microfibrils may be surrounded by additional networks of polymers, including hemicelluloses, pectins, lignin, and arabinogalactan proteins (Somerville et al., 2004). Hemicelluloses are composed of β-1,4-linked carbohydrate backbones with side branches and include xyloglucans, mannans, and arabinoxylans. Xyloglucan is thought to interact with the surface of cellulose and form cross-links between adjacent microfibrils (Vissenberg et al., 2005). In some cell types, pectin or lignin may also participate in cross-linking or entrapment of other cell wall polymers. It is unclear how the associations between networks of different cell wall components are relaxed to allow for cell wall expansion during growth.Several models have been proposed for the behavior of cell wall components during wall expansion. The passive reorientation hypothesis (also called the multinet growth hypothesis; Preston, 1982) postulates that in longitudinally expanding cells, cellulose microfibrils are synthesized in a transverse pattern and are then reoriented toward the longitudinal axis due to the strain generated by turgor pressure (Green, 1960). This phenomenon has been observed in the multicellular alga Nitella (Taiz, 1984). In higher plants, there is less direct evidence for passive reorientation, and another hypothesis holds that wall expansion involves active, local, and controlled remodeling of cellulose microfibrils along a diversity of orientations (Baskin, 2005). Such remodeling could be achieved by proteins such as xyloglucan endotransglycosylases (XETs), which break and rejoin xyloglucan chains, and expansins, which loosen cell walls in vitro in a pH-dependent manner (Cosgrove, 2005). Marga et al. measured cellulose microfibril orientation at the innermost layer of the cell wall before and after in vitro extension and did not observe reorientation (Marga et al., 2005). This suggests that processes other than microfibril reorientation might be involved in wall expansion, at least under certain circumstances or in some wall layers. Thus, the degree to which cellulose microfibrils are reoriented after their synthesis during wall expansion has remained unclear.One difficulty in resolving this problem has been the inability to directly image cellulose microfibrils in the growing cell wall. Existing methods to assess cellulose structure and orientation in plant cell walls are limited by the low contrast of cellulose in transmission electron microscopy, the ability to image only the surface of the wall using field emission scanning electron microscopy, and the use of polarized light microscopy in combination with dyes such as Congo red to measure only the bulk orientation of cellulose microfibrils (Baskin et al., 1999; Sugimoto et al., 2000; Verbelen and Kerstens, 2000; MacKinnon et al., 2006). In addition, the sample manipulation required for the former two methods has the potential to introduce artifacts (Marga et al., 2005). Although cellulose microfibril orientation differs at the inner and outer surfaces of the cell wall (Sugimoto et al., 2000) and presumably changes over time, the dynamics of cellulose reorientation during cell wall expansion have not been observed to date.In this study, we tested fluorescent dyes for their potential to allow imaging of cellulose distribution in the walls of Arabidopsis seedlings by confocal microscopy. We used one of these dyes to characterize the distribution of cellulose in wild-type root cells and in mutants with reduced cellulose or xyloglucan. By directly observing the fine structure of cellulose over time in growing wild-type root cells, we concluded that cellulose microfibrils in these cells reorient in a transverse to longitudinal direction as predicted by the passive reorientation hypothesis.