Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

Summary

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

Abstract

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Introduction

Plant cells are surrounded by a cell wall that is a complex structure composed of interacting networks of polysaccharides, proteins, metabolites, and water that varies in thickness from 0.1 to several µm depending on the cell type and the phase of growth^1,2. Cell wall mechanical properties play an essential role in the growth of plants. Low stiffness values of the cell wall have been proposed as a precondition for cell growth and cell-wall expansion, and there is increasing evidence that all cells sense mechanical forces to perform their functions. However, it is still debated whether changes in the physi....

Protocol

1. Preparation of the plant material and growth conditions

1. To generate the needed plant material, sterilize the seeds of Arabidopsis wild type and mutant lines of interest.
   NOTE: In this protocol, we used the following: ttl1: T-DNA insertion lines Salk_063943 (for TTL1; AT1G53300) - Columbia-0 (Col-0) wild-type; the Procuste1 (prc1-1) mutant, which consists of a knock-out mutation (Q720stop) in the CESA6 gene (AT5G64740), as previously described in^10,11; and ttl1 x prc1-1 double mutant.
   1. Put an approximate volu....

Results

Force-Indentation experiments
The following text presents some results expected when a force-indentation experiment is conducted to show the typical output to expect when the protocol is well executed.

Force-displacement curves
Representative force indentation plots that were obtained indenting live samples at a position placed in the center of the cell of the root elongation zone are presented in Figure 2. When the AF.......

Discussion

Cell and cell-wall mechanics are increasingly becoming relevant to gain insight into how mechanics affects growth processes. As physical forces propagate over considerable distances in solid tissues, the study of changes in the physical properties of the cell wall and how they are sensed, controlled, tuned, and impact the plant's growth are becoming an important field of study^2,3,8.

A method is pr.......

Materials

Name	Company	Catalog Number	Comments
1 x Phosphate-Buffered Saline (PBS)			Include sodium chloride and phosphate buffer and is formulated to prevent osmotic shock and maintain water balance in living cells.
AFM software	Bruker, Billerica, MA, USA
Atomic force microscopy (AFM)	BioScope Catalyst, Bruker, Billerica, MA, USA
Catalyst Probe holder-fluid	Bruker, Billerica, MA, USA	CAT-FCH	A probe holder for the Bioscope Catalyst, designed for fluid operation in contact or Tapping Mode.  Also compatible with air operation.
Cryoscopic osmometer; model OSMOMAT 030	Gonotech, Berlin, Germany
Murashige & Skoog Medium	Duchess Biochemie	M0221	Original concentration, (1962)
Optical inverted microscope coupled to the AFM	Olympus IX81, Miami, FL, USA
PEGAMIL	ANAEROBICOS S.R.L., Buenos Aires, Argentina	100429	Neutral, non acidic silicone glue
Petri dishes	Deltalab	200201.B	Polystyrene, 55 x 14 mm, radiation sterile.
Propidium iodide	Sigma	P4170	For root viability test.
Silicon nitride probe, DNP-10, cantilever A	Bruker, Billerica, MA, USA	DNP-10/A	For force modulation microscopy in liquid operation. Probe tip radius of 20-60 nm. 175-μm-long triangular cantilever,  with a spring constant of 0.35 N/m.
Tweezers	Sigma	T4537