Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth

Abstract

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Figure 1

(A) Schematic representation of stiffness-tomography imaging by AFM. The tip of the cantilever is pushed into the sample under controlled conditions, thereby causing its indentation. The resulting deformation of the cantilever is monitored. This information is used to construct a stiffness profile of the sample as a function of penetration depth (z-position). i, Position of the tip according to the sample; ii, Profile of the indented spot of the sample (stiffness is coded by different color/gray values); iii, FD curve displaying the deformation of the cantilever as a function of the position of the tip (dashed line represents segments fitted with the Hertz model); iv, Calculated Young’s modulus of the indented spot; v, reconstructed 3D stiffness tomography matrix of the sample. (B) Optical image of a typical A. thaliana cell. The shadow of the AFM cantilever is visible on the right-hand side of the image. (C) A typical FD curve recorded on a medium-sized Arabidopsis cell.

Figure 2

Growth curve of a suspension culture of A. thaliana, monitored by temporal changes in optical density at a wavelength of 646 nm (OD₆₄₆) and pH-monitored with the passage of time in culture. The error bars represent standard errors of the repeated optical absorbance measurements for each sample.

Figure 3

Cell-wall stiffness profiles of suspension cultures of A. thaliana down to an indentation depth of 10 nm after 4, 7, 10, 13, 17, and 20 days of cultivation. The histograms are constructed by pooling stiffness values obtained on three cell categories (six cells in total). Each graph corresponds to 6 × 1024 FD curves obtained on the three cell categories, with each cell sampled at two different locations. The insets correspond to a single 2 μm × 2 μm stiffness map recorded on a medium-sized cell, arbitrarily chosen for illustration among the recorded data set.

Figure 4

Average stiffness of the cell wall (down to an indentation depth of 80 nm) as a function of cultivation time.

Figure 5

Changes in average cell-wall stiffness (± SE) as a function of indentation depth, on the 4th, 7th, 10th, 13th, 17th, and 20th days of culture. The averaged values for the three size categories (small, medium, and large) are presented. The dots indicating experimental points are connected with lines for visual clarification.

Figure 6

(A) FTIR spectra of the cell walls of A. thaliana, isolated from suspension cultures on the indicated days of cultivation. (B) The data from the subfigure A are shown in the form of a heat map. The absorbance value for each wavenumber divided by the sum of the column of all values for that wavenumber is shown using the color code shown at the right side of the figure. An average spectrum is shown at the top of the figure.