Quantifying hydrostatic pressure in plant cells by using indentation with an atomic force microscope

Abstract

Plant cell growth depends on a delicate balance between an inner drive-the hydrostatic pressure known as turgor-and an outer restraint-the polymeric wall that surrounds a cell. The classical technique to measure turgor in a single cell, the pressure probe, is intrusive and cannot be applied to small cells. In order to overcome these limitations, we developed a method that combines quantification of topography, nanoindentation force measurements, and an interpretation using a published mechanical model for the pointlike loading of thin elastic shells. We used atomic force microscopy to estimate the elastic properties of the cell wall and turgor pressure from a single force-depth curve. We applied this method to onion epidermal peels and quantified the response to changes in osmolality of the bathing solution. Overall our approach is accessible and enables a straightforward estimation of the hydrostatic pressure inside a walled cell.

Figure 1

Atomic force microscopy on onion epidermal peels. (a) Schematic of the setup (not to scale). (b) Top view of the onion epidermis and the AFM cantilever; cells are typically elongated (here their axes are roughly parallel to the cantilever). (c) Example of topographic (height) image (150 × 150 μm scan size); each of the two crests corresponds to the long axis of a cell (approximate height = 10 μm). (d) Typical force-depth curves—extension (blue) and retraction (red)—obtained on a cell. To see this figure in color, go online.

Figure 2

Schematics of the methodology to infer pressure: The topographic (height) image yields the local mean, κ_M, and Gaussian, κ_G, curvatures at the indentation point; the cell wall modulus, E, and the cell stiffness, k, are deduced from force-depth curves. Together with an independent measurement of wall thickness, t, these data enable the estimation of cell hydrostatic pressure, P. To see this figure in color, go online.

Figure 3

Extraction of mechanical and geometrical parameters from AFM data. (a) Fits of one typical AFM curve (blue) using a linear fit on the upper part of the curve (red line, 75–100% of maximal force) and a Hertz fit on the bottom part of the curve (green curve, corresponding indentation depth ≤300 nm). (b) Typical surface height measured by AFM (yellow) and its corresponding polynomial fit (blue), which is used to compute curvatures. To see this figure in color, go online.

Figure 4

Estimation of turgor pressure. (a) Example of AFM extension force-depth curves obtained on a cell, when turgid (blue) and when deflated (red) after puncture. (b) Pressure values obtained in 41 specific cells (blue crosses) from seven different peels. (Red) Mean value of each peel and its associated standard deviation. To see this figure in color, go online.

Figure 5

Measurements in NaCl solutions. Ten cells were followed as the bath osmotic pressure Π was increased. (a) AFM force-depth extension curves on one cell. (b) Apparent cell stiffness, k. (c) Young’s modulus values E_e deduced from the extension curves. (d) Estimated pressure, P. To see this figure in color, go online.

Figure 6

Measurements in sorbitol solutions. Ten cells were followed as the bath osmotic pressure Π was increased up to 1.47 MPa, then kept overnight at 0 MPa (after which the cells were measured) and then increased back to 1.47 MPa. (a) AFM force-depth extension curves on one cell. (b) Apparent cell stiffness, k. (c) Young’s modulus values E_e deduced from the extension curves. (d) Estimated pressure, P. To see this figure in color, go online.

Figure 7

Summary of results with osmotic treatments (using data from Figs. 5 and 6). (a) Changes in deduced Young’s modulus E with turgor pressure, P. (Red) NaCl solutions, modulus averaged over 10 cells. (Dark blue) Sorbitol solutions, modulus averaged over nine cells (all cells from Fig. 6 except No. 4). (Light blue) Sorbitol experiment, cell No. 4. (Circles) Values obtained on the second day with sorbitol. (b) Changes in estimated turgor pressure, P, with bath osmotic pressure Π. Same colors and symbols as in (a). To see this figure in color, go online.