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 primordium2,3. 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 slower4,5. In the maize root, the transition of cells from division to elongation was accompanied by a decrease in elastic moduli in all tissues of the root. The moduli remained low in the elongation zone and increased in the late elongation zone6.
Despite the availability of various methods, the large arrays of biochemical and genetic information on cell wall biology obtained annually are rarely compared with the mechanical properties of cell walls. For example, mutants on cell wall-related genes often have altered growth and development4,7,8, but are rarely described in terms of biomechanics. One of the reasons for this is the difficulty of conducting measurements at the cellular and subcellular levels. Atomic force microscopy (AFM) is currently the primary approach for such analyses9.
In recent years, numerous AFM-based studies on plant cell wall biomechanics have been carried out. The mechanical properties of cell walls of the outer tissues of Arabidopsis2,3,4,5,10,11 and onion12, as well as of cultured cells13,14,15, have been investigated. However, the superficial cells of a plant may have cell walls whose mechanical properties differ from those of the inner tissues6. In addition, plant cells are pressurized by turgor which makes them stiffer. To get rid of the influence of turgor pressure, researchers have to use plasmolyzing solutions2,3,4,5,10,11 or decompose the values obtained into turgor and cell wall contributions12. The first approach leads to sample dehydration and changes the thickness and properties of the cell wall16, while the second approach requires additional measurements and complicated mathematics, and applies only to cells of relatively simple shape12. The cell wall properties of internal tissues can be evaluated on cryosections17 or sections of plant material impregnated with resin8. However, both methods involve dehydration and/or impregnation of samples, which inevitably leads to changes in properties. The properties of isolated or cultured cells are difficult to relate to the physiology of the whole plant. Both cultivation and isolation of plant cells can affect the mechanical properties of their cell walls.
The method presented here complements the aforementioned approaches. Using it, the primary cell walls of any tissue and at any stage of plant development can be examined. Sectioning and AFM observations were performed in liquid which avoids sample dehydration. The problem of turgor was solved as the cells are cut. The protocol describes work with maize and rye roots, but any other sample can be examined if it is suitable for vibratome sectioning.
The AFM studies described here were performed using the force-volume technique. Different instruments use different names for this method. However, the basic principle is the same; a force-volume map of the sample is obtained by a sinusoidal or triangular motion of the cantilever (or sample) to achieve a certain loading force at each analyzed point, while recording the cantilever deflection18. The result combines a topographic image of the surface and the array of force-distance curves. Each curve is used to calculate the deformation, stiffness, Young's modulus, adhesion, and energy dissipation at a specific point. Similar data can be obtained by point-by-point force-spectroscopy after scanning in contact mode19, although it is more time-consuming.
Protocol
Representative Results
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge Dr. Dmitry Suslov (Saint Petersburg State University, Saint Petersburg, Russia) and Prof. Mira Ponomareva (Tatar Scientific Research Institute of Agriculture, FRC KazSC RAS, Kazan, Russia) for providing maize and rye seeds, respectively. The presented method was developed within the framework of the Russian Science Foundation Project No. 18-14-00168 awarded to LK. The part of the work (obtaining of the results presented) was performed by AP with the financial support of the government assignment for the FRC Kazan Scientific Center of RAS.
Materials
| 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 |
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