Advertisement

Osmotic Treatment for Quantifying Cell Wall Elasticity in the Sepal of Arabidopsis thaliana

  • Aleksandra Sapala
  • Richard S. SmithEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2094)

Abstract

Elastic properties of the cell wall play a key role in regulating plant growth and morphogenesis; however, measuring them in vivo remains a challenge. Although several new methods have recently become available, they all have substantial drawbacks. Here we describe a detailed protocol for osmotic treatments, which is based on the idea of releasing the turgor pressure within the cell and measuring the resulting deformation. When placed in hyperosmotic solution, cells lose water via osmosis and shrink. Confocal images of the tissue, taken before and after this treatment, are quantified using high-resolution surface projections in MorphoGraphX. The cell shrinkage observed can then be used to estimate cell wall elasticity. This allows qualitative comparisons of cell wall properties within organs or between genotypes and can be combined with mechanical simulations to give quantitative estimates of the cells’ Young’s moduli. We use the abaxial sepal of Arabidopsis thaliana as an easily accessible model system to present our approach, but it can potentially be used on many other plant organs. The main challenges of this technique are choosing the optimal concentration of the hyperosmotic solution and producing high-quality confocal images (with cell walls visualized) good enough for segmentation in MorphoGraphX.

Key words

Plasmolysis Cell wall Shrinkage Biomechanics Sepal 

Notes

Acknowledgment

We thank Daniel Kierzkowski for guidance in tissue dissection and Gabriella Mosca and Mingyuan Zhou for comments on the manuscript.

References

  1. 1.
    Forouzesh E, Goel A, Mackenzie SA, Turner JA (2013) In vivo extraction of Arabidopsis cell turgor pressure using nanoindentation in conjunction with finite element modeling. Plant J 73:509–520CrossRefGoogle Scholar
  2. 2.
    Beauzamy L, Derr J, Boudaoud A (2015) Quantifying hydrostatic pressure in plant cells by using indentation with an Atomic Force Microscope. Biophys J 108:2448–2456CrossRefGoogle Scholar
  3. 3.
    Milani P, Gholamirad M, Traas J, Arnéodo A, Boudaoud A, Argoul F et al (2011) In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using Atomic Force Microscopy. Plant J 67:1116–1123CrossRefGoogle Scholar
  4. 4.
    Kierzkowski D, Nakayama N, Routier-Kierzkowska A-L, Weber A, Bayer E, Schorderet M et al (2012) Elastic domains regulate growth and organogenesis in the plant shoot apical meristem. Science 335:1096–1109CrossRefGoogle Scholar
  5. 5.
    Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6:850–861CrossRefGoogle Scholar
  6. 6.
    Hong L, Dumond M, Tsugawa S, Sapala A, Routier-Kierzkowska A-L, Zhou Y et al (2016) Variable cell growth yields reproducible organ development through spatiotemporal averaging. Dev Cell 38:15–32CrossRefGoogle Scholar
  7. 7.
    Elsayad K, Werner S, Gallemí M, Kong J, Guajardo ERS, Zhang L et al (2016) Mapping the subcellular mechanical properties of live cells in tissues with fluorescence emission—Brillouin imaging. Sci Signal 9:1–13CrossRefGoogle Scholar
  8. 8.
    Peaucelle A, Braybrook SA, LeGuillou L, Bron E, Kuhlemeier C, Höfte H (2011) Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis. Curr Biol 21:1720–1726CrossRefGoogle Scholar
  9. 9.
    Sampathkumar A, Krupinski P, Wightman R, Milani P, Berquand A, Boudaoud A et al (2014) Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells. elife 3:e01967.  https://doi.org/10.7554/eLife.01967CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Beauzamy L, Louveaux M, Hamant O, Boudaoud A (2015) Mechanically, the shoot apical meristem of Arabidopsis behaves like a shell inflated by a pressure of about 1 MPa. Front Plant Sci 6:1–10CrossRefGoogle Scholar
  11. 11.
    Majda M, Grones P, Sintorn IM, Vain T, Milani P, Krupinski P, Zagorska-Marek B, Viotti C, Jonsson H, Mellerowicz E, Hamant O, Robert S (2017) Mechanochemical polarization of contiguous cell walls shapes plant pavement cells. Dev Cell 43:290–304CrossRefGoogle Scholar
  12. 12.
    Routier-Kierzkowska A-L, Weber A, Kochova P, Felekis D, Nelson BJ, Kuhlemeier C et al (2012) Cellular force microscopy for in vivo measurements of plant tissue mechanics. Plant Physiol 158:1514–1522CrossRefGoogle Scholar
  13. 13.
    Hayot CM, Forouzesh E, Goel A, Avramova Z, Turner J (2012) Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation. J Exp Bot 63:2525–2540CrossRefGoogle Scholar
  14. 14.
    Bolduc J-E, Lewis LJ, Aubin C-E, Geitmann A (2006) Finite-element analysis of geometrical factors in micro-indentation of pollen tubes. Biomech Model Mechanobiol 5:227–236CrossRefGoogle Scholar
  15. 15.
    Wang L, Hukin D, Pritchard J, Thomas C (2006) Comparison of plant cell turgor pressure measurement by pressure probe and micromanipulation. Biotechnol Lett 28:1147–1150CrossRefGoogle Scholar
  16. 16.
    Park YB, Cosgrove DJ (2012) A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases. Plant Physiol 158:1933–1943CrossRefGoogle Scholar
  17. 17.
    Mosaliganti KR, Noche RR, Xiong F, Swinburne I, Megason SG (2012) ACME: Automated Cell Morphology Extractor for comprehensive reconstruction of cell membranes. PLoS Comput Biol 8(12):e1002780.  https://doi.org/10.1371/journal.pcbi.1002780CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Robinson S, Huflejt M, Barbier de Reuille P, Braybrook S, Schorderet M, Reinhardt D et al (2017) An automated confocal micro-extensometer enables in vivo quantification of mechanical properties with cellular resolution. Plant Cell 29:2959–2973CrossRefGoogle Scholar
  19. 19.
    Bringmann M, Bergmann DC (2017) Tissue-wide mechanical forces influence the polarity of stomatal stem cells in Arabidopsis. Curr Biol 27:877–883CrossRefGoogle Scholar
  20. 20.
    Weber A, Braybrook S, Huflejt M, Mosca G, Routier-Kierzkowska AL, Smith RS (2015) Measuring the mechanical properties of plant cells by combining micro-indentation with osmotic treatments. J Exp Bot 66:3229–3241CrossRefGoogle Scholar
  21. 21.
    Barbier de Reuille P, Routier-Kierzkowska A-L, Kierzkowski D, Bassel GW, Schüpbach T, Tauriello G et al (2015) MorphoGraphX: a platform for quantifying morphogenesis in 4D. elife 4:05864.  https://doi.org/10.7554/eLife.05864CrossRefPubMedGoogle Scholar
  22. 22.
    Oparka KJ (1994) Plasmolysis: new insights into an old process. New Phytol 67:571–591CrossRefGoogle Scholar
  23. 23.
    Mosca G, Sapala A, Strauss S, Routier-Kierzkowska AL, Smith RS (2017) On the micro-indentation of plant cells in a tissue context. Phys Biol 14:015003.  https://doi.org/10.1088/1478-3975/aa5698CrossRefPubMedGoogle Scholar
  24. 24.
    Roeder AHK, Chickarmane V, Cunha A, Obara B, Manjunath BS, Meyerowitz EM (2010) Variability in the control of cell division underlies sepal epidermal patterning in Arabidopsis thaliana. PLoS Biol 8:e1000367.  https://doi.org/10.1371/journal.pbio.1000367CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hervieux N, Dumond M, Sapala A, Routier-Kierzkowska AL, Kierzkowski D, Roeder AHK et al (2016) A mechanical feedback restricts sepal growth and shape in Arabidopsis. Curr Biol 26:1019–1028CrossRefGoogle Scholar
  26. 26.
    Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Biosystems Science and Engineering, ETH ZurichMattenstrasse, BaselSwitzerland
  2. 2.John Innes Centre, Computational and Systems Biology, Norwich Research ParkNorwichUK

Personalised recommendations