Disorganization of Cortical Microtubules Stimulates Tangential Expansion and Reduces the Uniformity of Cellulose Microfibril Alignment among Cells in the Root of Arabidopsis

Abstract

To test the role of cortical microtubules in aligning cellulose microfibrils and controlling anisotropic expansion, we exposed Arabidopsis thaliana roots to moderate levels of the microtubule inhibitor, oryzalin. After 2 d of treatment, roots grow at approximately steady state. At that time, the spatial profiles of relative expansion rate in length and diameter were quantified, and roots were cryofixed, freeze-substituted, embedded in plastic, and sectioned. The angular distribution of microtubules as a function of distance from the tip was quantified from antitubulin immunofluorescence images. In alternate sections, the overall amount of alignment among microfibrils and their mean orientation as a function of position was quantified with polarized-light microscopy. The spatial profiles of relative expansion show that the drug affects relative elongation and tangential expansion rates independently. The microtubule distributions averaged to transverse in the growth zone for all treatments, but on oryzalin the distributions became broad, indicating poorly organized arrays. At a subcellular scale, cellulose microfibrils in oryzalin-treated roots were as well aligned as in controls; however, the mean alignment direction, while consistently transverse in the controls, was increasingly variable with oryzalin concentration, meaning that microfibril orientation in one location tended to differ from that of a neighboring location. This conclusion was confirmed by direct observations of microfibrils with field-emission scanning electron microscopy. Taken together, these results suggest that cortical microtubules ensure microfibrils are aligned consistently across the organ, thereby endowing the organ with a uniform mechanical structure.How do plants build organs with specific and heritable shapes? A part of the answer to this question lies in the control of growth. It is not growth rate per se that is crucial for morphogenesis but the directionality of growth. If growth rate were the same in all directions, i.e. isotropic, plant organs would be spherical; organs attain shapes other than spherical because their component cells grow at different rates in different directions, i.e. anisotropically. Understanding how cells control the anisotropy of their expansion is essential for understanding morphogenesis.Expansion anisotropy is characterized by the direction in which the maximal growth rate occurs and by the degree to which the maximum differs from the minimum. The direction of maximal expansion rate is known to be controlled by the direction of net alignment of cellulose microfibrils. Within a growing cell wall, microfibrils are aligned, on average, perpendicularly to the direction of maximal expansion rate, and the aligned cellulose microfibrils confer a mechanical anisotropy on the cell wall, which translates into expansion anisotropy (Green, 1980;Taiz, 1984). The wall can be considered a composite material with strong, rod-shaped particles (microfibrils) embedded in a compliant matrix. Such materials deform perpendic...

Genetic Evidence That Cellulose Synthase Activity Influences Microtubule Cortical Array Organization

“…Addition of the cellulase resulted in an 11.7% (n 5 28 control; n 5 34 cellulase treated) increase in cell width compared with mature cells of the same cell file. In contrast, the cells at this same position in untreated roots were 3.9% thinner, which is in agreement with the negative tangential expansion of this region reported by Baskin et al (2004). Therefore, the actual amount of swelling induced by the cellulase treatment was around 15% assuming that the more mature cells were resistant to the cellulase (P , 0.001 by equal variance t test comparing the ratios of cellulase-treated cell widths to untreated controls).…”

“…Arrays are transversely orientated where epidermal cells emerge from under the root cap marking the beginning of the elongation zone. The cortical arrays of cells remain transverse as cells move through the elongation zone, with some cells displaying obliquely oriented cortical arrays just before root hair emergence, which marks the point where elongation slows (Dolan et al, 1994;Sugimoto et al, 2000;Baskin et al, 2004). Shortly after root hair emergence, cell elongation ceases and the microtubules become longitudinally oriented.…”

Identification of a cellulose synthase-associated protein required for cellulose biosynthesis

“…To determine whether there was a quantitative difference in microfibril abundance or orientation, we quantified the retardance of the cell walls and the azimuth of the crystalline elements. The average retardance was about 1 nm, similar to that reported previously for Arabidopsis cell walls (27), and there was no significant difference between the genotypes in the three delineated zones of the root (Fig. 5C).…”

Update: Plant Cortical Microtubule Arrays

“…Since that time, several, but not all, examinations of microtubules and cellulose microfibrils support the hypothesis that microtubules guide the deposition of cellulose microfibrils, resulting in effects on cell shape (Baskin, 2001). Temperature-sensitive mutants and microtubule-related drug treatments have identified patchwork-like patterns of locally coaligned cellulose microfibrils that are variably coaligned relative to each other on an individual cell face in the absence of cortical microtubules, suggesting some level of cellulose microfibril self-assembly that requires microtubules for global microfibril coalignment (Sugimoto et al, 2003;Baskin et al, 2004). The relationship between cellulose deposition and the time and extent to which the cellulose pattern impacts cell shape are still very much in debate.…”

“…1) for the deposition of cellulose (Paredez et al, 2006), the main load-bearing polymer in the wall (McFarlane et al, 2014). Elimination or disorganization of microtubule arrays with inhibitors causes plant cells to expand more isotropically (Baskin et al, 1994), which correlates with increasing variability in the orientation of cellulose microfibrils (Baskin et al, 2004). Direct demonstrations, through imaging, that the cellulose synthase complexes are inserted into the plasma membrane at sites determined by microtubules (Crowell et al, 2009;Gutierrez et al, 2009), coupled with observations of mobile synthase complexes tracking along microtubules, have bolstered early hypotheses for a functional relationship between the microtubule array pattern and cellulose deposition.…”