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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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,

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....

Representative 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.......

Disclosures

The authors have no conflicts of interest.

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

NameCompanyCatalog NumberComments
Agarose, low melting pointHeliconB-5000-0.1for sample fixation
Brush--for section moving
CantileversNanoTools, GermanyNT_B150_v0020-5Model: Biosphere B150-FM
CantileversNT-MDT, RussiaFMG01/50Model: FMG01
Cyanoacrylate adhesive--for vibratomy
Glass slidesHeinz Herenz1042000for vibratomy and AFM calibration
ImageAnalysis P9 SoftwareNT-MDT, Russia-for data analysis
Leica DM1000 epifluorescence microscopeLeica Biosystems, Germany11591301for section check
NaOCl--for seed sterilization
Nova PX 3.4.1 SoftwareNT-MDT, Russia-for experiments conducting
NTEGRA Prima microscope with HD controllerNT-MDT, Russia-for AFM and data acquisition
Petri dish 35 mmThermo Fisher Scientific153066for sample fixation
Tip pipette 1000 µLThermo Fisher Scientific4642092-
Tip pipette 2-20 µLThermo Fisher Scientific4642062-
Ultrapure water---
Vibratome Leica VT 1000SLeica Biosystems, Germany1404723512for sample sectioning

References

  1. Zerzour, R., Kroeger, J., Geitmann, A. Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties. Developmental Biology. 334 (2), 437-446 (2009).
  2. Braybrook, S. A., Peaucelle, A.

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Plant Cell WallCell GrowthBiomechanical CharacterizationAtomic Force MicroscopyVibratome SectioningAgarosePlant OrganTissueNon fixedNon dehydratedCell Wall Properties

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