Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

Summary

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Abstract

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young's modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Introduction

The mechanical properties of the plant cell wall determine the shape of the cell and its ability to grow. For example, the growing tip of the pollen tube is softer than the non-growing parts of the same tube¹. The primordia formation on Arabidopsis meristem is preceded by a local decrease in cell wall stiffness at the site of the future primordium^2,3. The cell walls of Arabidopsis hypocotyl, which are parallel to the main growth axis and grow faster, are softer than those that are perpendicular to this axis and grow slower^4,

Protocol

1. Sample preparation for AFM measurements

1. Plant material: Sterilize the seeds of maize (Zea mays L.) and rye (Secale cereale L.) with a 0.35% NaOCl solution for 10 min, wash 3x with distilled water, and then grow hydroponically in the dark at 27 °C for 4 days and 2 days, respectively. Primary roots were used for the experiment.
2. Preparation of solutions and sample for vibratome sectioning
   1. Prepare agarose solution for root embedding by dissolving 3% (w/w) low-melting-point agarose in water using a microwave oven.
      NOTE: All experiments were performed in water. If buffer or any other type o....

Results

Typical elastic modulus and DFL maps, as well as force curves obtained on rye and maize roots by the method described, are presented in Figure 2. Figure 2A shows elastic modulus and DFL maps obtained on the transverse section of rye primary root. The white areas in the modulus map (Figure 2A, left) correspond to an erroneous overestimation of Young's modulus due to the scanner reaching its limit in the z-direction.......

Discussion

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth, and therefore the future size and shape of the plant. The AFM-based method presented here complements existing techniques which are used to study the properties of plant cell walls. It allows the elasticity of cell walls, which belong to the inner tissues of the plant, to be investigated. Using the presented method, the mechanical properties of cell walls in different tissues of the growing maize root were mapped, a.......

Materials

Name	Company	Catalog Number	Comments
Agarose, low melting point	Helicon	B-5000-0.1	for sample fixation
Brush	-	-	for section moving
Cantilevers	NanoTools, Germany	NT_B150_v0020-5	Model: Biosphere B150-FM
Cantilevers	NT-MDT, Russia	FMG01/50	Model: FMG01
Cyanoacrylate adhesive	-	-	for vibratomy
Glass slides	Heinz Herenz	1042000	for vibratomy and AFM calibration
ImageAnalysis P9 Software	NT-MDT, Russia	-	for data analysis
Leica DM1000 epifluorescence microscope	Leica Biosystems, Germany	11591301	for section check
NaOCl	-	-	for seed sterilization
Nova PX 3.4.1 Software	NT-MDT, Russia	-	for experiments conducting
NTEGRA Prima microscope with HD controller	NT-MDT, Russia	-	for AFM and data acquisition
Petri dish 35 mm	Thermo Fisher Scientific	153066	for sample fixation
Tip pipette 1000 µL	Thermo Fisher Scientific	4642092	-
Tip pipette 2-20 µL	Thermo Fisher Scientific	4642062	-
Ultrapure water	-	-	-
Vibratome Leica VT 1000S	Leica Biosystems, Germany	1404723512	for sample sectioning