The mechanical properties of cell walls play a vital role in plant development. Atomic-force microscopy (AFM) is widely used for characterization of these properties. However, only surface or isolated plant cells have been used for such investigations, at least as non-embedded samples. Theories that claim a restrictive role of a particular tissue in plant growth cannot be confirmed without direct measurement of the mechanical properties of internal tissue cell walls. Here we report an approach of assessing the nanomechanical properties of primary cell walls in the inner tissues of growing plant organs. The procedure does not include fixation, resin-embedding or drying of plant material. Vibratome-derived longitudinal and transverse sections of maize root were investigated by AFM in a liquid cell to track the changes of cell wall stiffness and elasticity accompanying elongation growth. Apparent Young’s modulus values and stiffness of stele periclinal cell walls in the elongation zone of maize root were lower than in the meristem, i.e., cell walls became more elastic and less resistant to an applied force during their elongation. The trend was confirmed using either a sharp or spherical probe. The availability of such a method may promote our understanding of individual tissue roles in the plant growth processes.

The cellulose-enriched tertiary cell walls present in many plant fibers have specific composition, architecture, machinery of formation, and function. To better understand the mechanisms underlying their mode of action and to reveal the peculiarities of fibers from different plant species, it is necessary to more deeply characterize the major components. Next to overwhelming cellulose, rhamnogalacturonan I (RG-I) is considered to be the key polymer of the tertiary cell wall; however, it has been isolated and biochemically characterized in very few plant species. Here, we add RG-I to the list from the phloem fibers of the Phaseolus vulgaris stem that was isolated and analyzed by nuclear magnetic resonance (NMR), dynamic light scattering, and immunolabeling, both within tissue and as an isolated polymer. Additionally, fibers with tertiary cell walls from nine species of dicotyledonous plants from the orders Malphigiales, Fabales, and Rosales were labeled with RG-I-related antibodies to check the presence of the polymer and compare the in situ presentation of its backbone and side chains. The obtained results confirm that RG-I is an obligatory polymer of the tertiary cell wall. However, there are differences in the structure of this polymer from various plant sources, and these peculiarities may be taxonomically related.

Biomechanical parameters of plant tissues and organs are increasingly recognized as key factors in plant development and application, increasing the demand for convenient devices for their study. The paper presents an original device for performing a three-point bending test using the inverse method, which is a modification of the classical (straight) three-point test. The designed device was tested in experiments to determine the modulus of elasticity of flax plant stems, and the results were compared with data obtained using the vibration method and the straight three-point bending test on a commercial instrument. Due to the high sensitivity associated with its design features, the device for the inverse three-point bending test is characterized by being able to adequately measure elastic moduli in plant stems over a wide range of values, from tens of MPa to tens of GPa. It also allows checking the effect of humidity, temperature, and water content on the mechanical properties of samples and is equipped with an automation system. The proposed device is quite affordable and can be effectively used both for young stem parts, whose mechanical properties are based on a hydroskeleton, and for mature, poorly hydrated parts with cell walls highly developed in sclerenchymatous tissues.

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).