Multiscale investigation from subcellular to tissue scale of onion epidermal plant cell wall mechanical properties

Open Access
Zamil, Mohammad S
Graduate Program:
Agricultural and Biological Engineering
Doctor of Philosophy
Document Type:
Date of Defense:
December 04, 2014
Committee Members:
  • Virendra Puri, Dissertation Advisor
  • Virendra Puri, Committee Chair
  • Nicole Robitaille Brown, Committee Member
  • Jeffrey M Catchmark, Committee Member
  • Md Amanul Haque, Committee Member
  • Ho Jae Yi, Special Member
  • Plant cell walls
  • Mechanical characterization
  • Tensile loading
  • MEMS
  • Biomaterials
  • Bridging subcellular and tissue scales
The physical and mechanical properties of cell walls, their shape, how they are arranged and interact with each other determine the architecture of plant organs and how they mechanically respond to different environmental and loading conditions. Due to the distinctive hierarchy from subcellular to tissue scale, plant materials can exhibit remarkably different mechanical properties. To date, how the subcellular scale arrangement and the mechanical properties of plant cell wall structural constituents give rise to macro or tissue scale mechanical responses is not yet well understood. Although the tissue scale plant cell wall samples are easy to prepare and put to different types of mechanical tests, the hierarchical features that emerge when moving towards a higher scale make it complicated to link the macro scale results to micro or subcellular scale structural components. On the other hand, the microscale size of cell brings formidable challenges to prepare and grip samples and carry mechanical tests under tensile loading at subcellular scale. This study attempted to develop a set of test protocols based on microelectromechanical system (MEMS) tensile testing devices for characterizing plant cell wall materials at different length scales. For the ease of sample preparation and well established database of the composition and conformation of its structural constituents, onion epidermal cell wall profile was chosen as the study material. Based on the results and findings of multiscale mechanical characterization, a framework of architecture-based finite element method (FEM) computational model was developed. The computational model laid the foundation of bridging the subcellular or microscale to the tissue or macroscale mechanical properties. Being microscale in size, the subcellular scale mechanical characterization of plant cell walls presents four major formidable challenges to address, namely: (1) excise or cut the sample from a cell, (2) pick and place the sample, (3) grip the sample, and (4) carry test with tensile testing device having force and displacement resolution suitable for submicron scale biological soft materials. By fusing focused ion beam (FIB) based sample preparation and MEMS-based mechanical characterization technique, a novel test protocol was developed, which was able to address the aforementioned four major hurdles. The MEMS-based tensile testing technique was designed, developed, and fabricated to accommodate soft and fragile sample such as a cell wall. Using FIB-based sample preparation and MEMS-based sample characterization technique, 15x5 µm samples were excised from onion outer epidermal peel and characterized under tensile loading both in major and minor growth directions of a cell. The measured mean modulus, fracture strength, and fracture strain in the major growth direction were 3.7 ± 0.8 GPa, 95.5 ± 24.1 MPa, and 3.0 ± 0.5%, respectively. The corresponding properties along the minor growth direction were 4.9 ± 1.2 GPa, 159 ± 48.4 MPa, and 3.8 ± 0.5%, respectively. The fracture strength and fracture strain were significantly different (p<0.05), whereas modulus of elasticity values were not significantly different (p>0.05), along the major and minor growth directions. The wall fragment level modulus of elasticity anisotropy for a dehydrated cell wall was 1.23, suggesting a limited anisotropy of the cell wall itself compared with tissue-scale results. However, this technique requires the use of a scanning electron microscope, which exposes the sample to vacuum that dries up the sample. As water content has significant impact on the mechanical response of cell wall, to accommodate cell wall samples with water, another novel test protocol was developed and supported by a new MEMS tensile testing device. For preparing never dried cell wall samples, a cryotome based technique was developed. The new MEMS device uses a separate 3D force sensor having very low stiffness with large sample placing area; this allowed the test to be conducted under optical microscope. Using cryotome based technique, subcellular scale cell wall samples were tested for two different water state conditions; which allowed the investigation of the quantitative role of water to wall mechanics. Like tissue sample, the cell wall at subcellular scale showed biphasic material behavior. However, instead of a transition zone between linear elastic or viscoelastic and linear plastic zones, the subcellular scale samples showed plateau-like trend exhibiting sharp drop in modulus value. The critical ranges of stress (20-40 MPa) and strain (5-12%) of the plateau zone were identified. The strain energy of 1.3 MJ m-3 calculated from the critical stress-strain range was in accordance with the previously estimated hydrogen bond energy in cell wall. It was also observed that subcellular scale sample shows very large lateral/axial deformation (0.8±0.13) at fracture, from which an estimate of the free space in the wall network was made. In addition, by investigating the wall mechanical properties at three different water states, it was suggested that the contribution of water is critical for plastic flow behavior of matrix polymers. The cells in plant tissue are joined together by a distinct layer called the middle lamella (ML), which bridges the gap between the subcellular- and tissue-scale mechanical properties. However, the nanoscale size of the ML presents formidable challenges to its characterization as a separate layer. Consequently, the mechanical properties of the ML under tensile loading are as yet unknown. The ML samples were prepared the same way as mentioned in the earlier two different techniques: FIB based and cryotome based. The only difference was the samples were cut including ML contact area between the two adjacent cells. Our test results showed that even at a subcellular scale, the ML appears to be stronger than the wall fragments. There was also evidence that the ML attached at the corner of cells more strongly attached than at the rest of the contact area. The contribution of the additional ML contact area was estimated to be 40.6 MPa. Wall fragment samples containing an ML layer were also significantly stronger (p < 0.05) than the wall fragments without an ML layer. Apart from direct excision of cell wall samples within the boundary of single cell, another technique was developed to characterize cell wall at subcellular scale while the sample is tested at tissue scale. The same MEMS-based tensile testing device was used as mentioned in the cryotome based technique. The tissue samples were stained by fluorescent polystyrene beads (500 nm in diameter), which can be tracked by a digital image correlation based image analysis technique. Thus, it was possible to investigate the strain field of wall samples at subcellular scale sample by stretching the sample at tissue scale. The Young's modulus values of individual cell walls of dehydrated and rehydrated samples were 3.0±1.0 GPa and 0.4±0.2 GPa, respectively, and are different from the Young's modulus values of the global tissue-scale dehydrated and rehydrated samples, which were 1.9±0.3 GPa and 0.08±0.02 GPa, respectively. The Poisson's ratio showed more than a three-fold increase due to hydration. The insights gained from experimentations of cell wall at different length scales and the intercellular adhesion were used to link the microscale (subcellular) to macroscale (tissue) mechanical properties. To scale up, a structure-based multiscale finite element method (FEM) computational model was developed, which takes the subcellular scale mechanical properties and the extracellular parameters and properties of the ML as inputs. To bridge micro and macroscales, a 3D repetitive volume element (RVE), which includes both subcellular and extracellular parameters, was built with ABAQUS® (a finite element modeling software). The two dimensionally arranged RVEs makes up a tissue patch of multiple cells. In a single RVE, wall fragments from four adjacent cells are attached by surface cohesive contact to define the ML interaction. By changing the shape parameters of a cell, four different RVEs were constructed, which allowed the quantitative investigation of cell shape to tissue scale mechanical responses. The RVE finite element simulations were run within 1% strain to ensure elastic only deformation. It was observed that change in the ML contact stiffness has little to no impact on tissue level mechanical responses. However, anisotropy in modulus values was observed for all levels of ML contact stiffness examined. For the range of shape factor values considered, E2 (modulus in transverse direction) and ν21 remain almost unchanged. However, E1 (modulus in longitudinal direction), ν12, and E2/E1 showed substantial change in values when the width to length ratio (WL) value is 1. The G12 (shear modulus in 12 direction, i.e., 1=longitudinal plane orientation direction and 2=transverse direction) values were different for all WL ratios. The experimental results showed good agreement in the anisotropy of modulus value (1.25) observed in the computational model (1.29). A framework is presented to scale up subcellular scale mechanical properties to tissue scale. Lastly, this study suggests that there are important insights of cell wall mechanics and structural features that can only be investigated by carrying tensile characterization of samples not confounded by extracellular parameters. To the best of our knowledge, the plant cell wall at subcellular scale was never characterized under tensile loading. By coupling the structure based multiscale modeling and mechanical characterizations at different length scales, an attempt was made to provide novel insights towards understanding the mechanics and architecture of cell wall. This study also suggests that a multiscale investigation is essential for garnering fundamental insights into the hierarchical deformation of biological systems.