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.
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).
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.
1. Sample preparation for AFM measurements
2. AFM preparation and calibration
NOTE: The force-volume method of AFM generates a spatially resolved array of force-distance curves obtained at each point of the area studied. Obtain all parameters for the force-volume mode (cantilever stiffness, IOS, etc.) in contact mode. Similar procedures for instruments from other manufacturers have been described previously10,20.
3. Data acquisition
4. Data evaluation and post-processing
NOTE: Do not rely on elastic modulus values calculated automatically. Since the surface varies greatly in height, many artifact curves should be expelled.
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. This image is not convenient to be used as an external map for a further selection of satisfactory force curves. The DFL signal map (deflection/error map) that is presented on the right (Figure 2A) is better suited for this purpose.
The modulus maps of transverse and longitudinal sections of the maize root are shown in Figure 2B,C. The scanner that has been fully extended while trying to reach the bottom of the sample can result in erroneous measurements and even interrupted scans (top part of Figure 2C). Sometimes, after getting a good scan in contact mode, the instrument suddenly contracts the scanner completely, alarming that it has reached the maximum z-direction limit when it switches to the force-volume mode. It means that the sample was not immobilized properly and floated up. A new sample should be prepared.
The agarose contamination may also be a reason to prepare a new sample (Figure 2D,E). The agarose presence may be checked while obtaining the first scan in the contact mode (step 3.5). Ideally, the cell walls and some cell bottoms should be visible on such scans (Figure 2D); however, in case of inaccurate immobilization, the surface may be covered with agarose (Figure 2E), which masks the sample topography.
Figure 2F shows four different force curves recorded at different points of the same cell wall. The curve recorded at Point 1 shows no baseline. It means there was no separation of the cantilever tip from the cell wall. The modulus calculated from this curve was overestimated. The curve recorded at Point 3 shows a shoulder on the approaching part. This artifact may indicate that the cell wall was bent. Both curves recorded at Points 1 and 3 should be expelled. Points 2 and 4 show satisfactory force curves with similar values of moduli. The following criteria can be used to distinguish proper curves: (i) the baseline level should be evident on both approaching and retracting parts of the curve; (ii) there should be no irregularities such as shoulders or failures (appears as constant force values and a sudden decrease of force values in the middle of the slope, respectively); (iii) the selected model should fit the curve well. For all selected curves, the maximum applied force values should be similar. A maximum applied force that is significantly higher or lower than the expected value can also be used as a criterion for curve expelling. Using neural networks to discard poor-quality curves19 can speed up the processing of results.
Figure 1: Critical steps in sample preparation. (A) Root fragment embedded in a block of agarose and mounted on the vibratome stage. (B) Sample section placed on top of a layer of 1% agarose and immobilized with additional agarose in a Petri dish cap. Please click here to view a larger version of this figure.
Figure 2: Typical AFM images and measurements on primary roots. (A) Elasticity modulus and DFL maps of a transverse section of the rye root. Fully recorded (B) and interrupted (C) maps of elasticity modulus obtained on longitudinal and transverse sections of maize root. 3D view of clean (D) and agarose-covered (E) sections of maize roots. (F) A DFL map of a transverse section of maize root. Points 1-4 on the cell wall show the positions where the force curves presented on the right side of the image were recorded. The red line is for approaching, the blue line is for retraction, and the black line shows the contact mechanics model fit. The curves recorded at points 1 and 3 have artifacts and cannot be used for calculations. Rye roots were examined with sharp tips, and maize roots were examined with spherical tips. Abbreviations: Rhiz = rhizodermis, Exo = exodermis, OC = outer cortex, IC = inner cortex, End = endodermis, Per = pericycle, and VP = vascular parenchyma. Scale bars = 10 µm. Please click here to view a larger version of this figure.
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, and a mathematical model was constructed based on these properties6. The simulated root responded to the application of pressure imitating turgor by changes in linear size. These changes were in the same range and in the opposite direction as those exhibited by a freshly excised root in plasmolyzing medium6.
The sample preparation is critical for this protocol. The drying of the sample should be avoided during the entire preparation. Correct placement of the plant material to be sectioned is an important issue; the cell walls must be oriented strictly perpendicular to the future scanning plane. Otherwise, unsatisfactory force-deformation curves will be obtained because the cell wall has been bent or buckled. The agarose-embedded section should be tightly fixed with additional agarose (Figure 2B). The latter should not get onto the sample, otherwise, the agarose properties will be measured. The most important part of this protocol is to filter the resulting force curves. Even studying model objects, such as AFM calibration grids, may result in a significant proportion of erroneous force-deformation curves22. Direct use of the calculated modulus values without analyzing the underlying force-deformation curves can lead to incorrect conclusions (Figure 2F).
The technique is not limited by the localization of the cell or the size of plant organs. However, objects that are too small are difficult to cut, orient, and immobilize. Cells with a large lumen are difficult to work with since there is a high probability of scan interruption (Figure 2C).
With all the benefits of using non-fixed plant sections prepared with a vibratome, there are at least two crucial issues: the possibility of developing a stress reaction after cutting plant material, and the possibility of partial solubilization of material from the cut cell walls. Both can lead to changes in the composition and properties of cell walls. The presence of immediate stress response on the cut was tested on Arabidopsis apices10. No significant effect on the observed properties was reported. Experiments were conducted to test changes in the properties of cut cell walls during their incubation in water19. There was no stable trend in apparent Young's modulus values measured on the same wall of maize root for at least a half-hour period19. However, it is better to maintain the time of experiments as short as possible and check each specimen for such changes during incubation of the sample in water.
AFM is a powerful technique for studying plant cell walls. However, it is obvious that calculations of elasticity modulus, stiffness, and adhesion are based on several assumptions that are not fulfilled by plant cell walls. All models of contact mechanics used by AFM consider the investigated specimen as an infinite half-space of isotropic material, which is not true for plant cell walls. Simultaneously, it is assumed that the geometry and properties of the indenter are accurately measured and constant over the cantilever lifespan, which may also be erroneous. In addition, plant cell walls exhibit complex mechanical behavior involving elastic, viscoelastic, and plastic components, and usually only one of these can be measured in a single experiment. Nevertheless, the data obtained using AFM-based methods reliably correlate with the plant cell growth and mechanical performance in various organs and species3,4,5,6. All this means that both the technique itself and the understanding of the data obtained with it can be improved.
The authors have nothing to disclose.
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.
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 |