Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: An atomic force microscopy study

The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of “Index of Plasticity” ($\eta$), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus ($\frac{H}{E^{*}}$).

Figure 1

(a) Schematic structure of a root growth region (adapted from Ref. [4]). (b) The different designated zones in the root: (1) meristem ($\sim 350\mu \text{m}$); (2) accelerating elongation ($\sim 900\mu \text{m}$); (3) decelerating elongation ($\sim$1.2 mm); (4) mature (reference) ($\sim 500\mu \text{m}$); (5) rest of the root-lateral root emergence zone ($\sim$2.5 cm) [5].

Figure 2

An example of a single loading-unloading indentation curve from which the index of plasticity can be determined. This curve was generated at a distance of 0.5 mm from the root tip in water. ABC is area under the loading curve, and DBC is area under the unloading curve.

Figure 3

An example of force-versus-displacement curve on the sample surface. A is the loading curve, and B is the unloading curve.

Figure 4

(a) Confocal image of a root in experimental conditions (for AFM) on a glass substrate attached with a fixative. (b) Confocal image of a root in normal conditions (scale bar: 100 $\mu$m).

Figure 5

A selected range of force-indentation (F-I) curves along the length of the root. (a) F-I curves at 0.2 mm distance from the root tip (meristem zone). (b) F-I curves at 0.6 mm distance from the root tip (accelerating elongation zone). (c) F-I curves at 1 mm distance from the root tip (decelerating elongation zone). (d) F-I curves at 1.5 mm distance from the root tip (mature zone). In each panel a and b are loading and unloading curves in water, respectively, and c and d are loading and unloading curves in sorbitol, respectively.

Figure 6

The index of plasticity $\eta$ values for living root samples in water and sorbitol. The index of plasticity $\eta$ plots are observed to be in a similar reproducible range over a sample of individual sets within the remits of biological variability. (a) Index of plasticity $\eta$ values range in five different sets of root samples where experiments in A-water and B-sorbitol on each root were typically performed one after the other. (b) A single graph of the index of plasticity $\eta$ of root from the root tip in both water and sorbitol.

Figure 7

The ratio of hardness to reduced modulus $\frac{H}{E^{*}}$ values in five different sets of roots samples where experiments in (a) water and (b) sorbitol on each living root were performed one after the other.