Assembly of a simple scalable device for micromechanical testing of plant tissues.

Tensile testing is widely used to evaluate the mechanical properties of biological materials including soft primary plant tissues. Commercially available platforms for tensile testing are often expensive and limited in customizability. In this chapter, we provide a guide for the assembly and use of a simple and low-cost micromechanical testing apparatus suitable for research and educational purposes. The build of the setup is presented with scalability and universality in mind and is based on a do-it-yourself mind frame towards mechanical tests on plant organs and tissues. We discuss hardware and software requirements with practical details on required components, device calibration and a script to run the device. Further, we provide an example in which the device was used for the uniaxial tensile test of onion epidermis.

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity in vitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.

Multiscale stress-strain characterization of onion outer epidermal tissue in wet and dry states.

UNLABELLED: • PREMISE OF THE STUDY: Quantitative measurements of water's effects on the tension response of plant tissue will assist in understanding the regulatory mechanism underlying expansive growth. Such measurements should be multiscale in nature to account for plants' hierarchical structure.• METHODS: Outer onion epidermal tissues were cut and bonded to uniaxial displacement-controlled mechanical loading devices to apply and measure the force on the sample. Fluorescent polystyrene beads (500 nm in diameter) were dispersed on the sample surface under various levels of tensile load conditions to obtain displacement maps with a confocal fluorescent microscope. The resulting strain was measured using a digital image correlation technique by tracking individual bead displacements. The applied forces were obtained by measuring the displacement of the calibrated force-sensing device. Tissue- and cell-scale mechanical properties were quantified by calculating the applied stress and the corresponding global and local strains.• KEY RESULTS: 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. Poisson's ratio increased more than 3-fold due to hydration.• CONCLUSION: The results on global, cell-to-cell, and point-to-point mechanical property variations suggest the importance of the mechanical contribution of extracellular features including the middle lamella, cell shape, and dimension. This study shows that a multiscale investigation is essential for fundamental insights into the hierarchical deformation of biological systems.

Mechanical characterization of outer epidermal middle lamella of onion under tensile loading.

UNLABELLED: • PREMISE OF THE STUDY: The cells in plant tissue are joined together by a distinct layer called the middle lamella (ML). Understanding the mechanical properties of the ML is crucial in studying how tissue-level mechanical properties emerge from the subcellular-level 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.• METHODS: Here, we characterize the ML from a subcellular sample excised from two adjacent cells and composed of two wall fragments and a single line of ML in between. Two techniques, cryotome sectioning and milling with a focused ion beam, were used to prepare ML samples, and tensile experiments were performed using microelectromechanical system (MEMS) tensile testing devices.• KEY RESULTS: 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 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.• CONCLUSIONS: The tensile properties of the ML might not have a major impact on the tissue-scale mechanical properties. This conclusion calls for further study of the ML, including characterization under shear loading conditions and elucidation of the contributions of other extracellular parameters, such as cell size and shape, to the overall tissue-level mechanical response.