Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al.

The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old Arabidopsis thaliana wild-type and almt1-mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force-distance curve measurements using the trimechanic-3PCS framework. The presence of single metal species Fe2+ or Al3+ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe2+ or Al3+ to 20 µM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root-extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe2+ and Al3+ with exuded malate is crucial for both CW stiffening and root-extension arrest. However, stiffness increase induced by single Fe2+ or Al3+ is not sufficient for arresting root growth in our experimental conditions.

Measuring external primary cell wall elasticity of seedling roots using atomic force microscopy.

Stiffness plays a central action in plant cell extension. Here, we present a protocol to detect changes in stiffness on the external epidermal cell wall of living plant roots using atomic force microscopy (AFM). We provide generalized instructions for collecting force-distance curves and analysis of stiffness using contact-based mechanical model. With this protocol, and some initial training in AFM, a user is able to perform indentation experiments on 4- and 5-day-old Arabidopsis thaliana and determine stiffness properties. For complete details on the use and execution of this protocol, please refer to Godon et al.1.

Nano-structural stiffness measure for soft biomaterials of heterogeneous elasticity.

Measuring the structural stiffness aims to reveal the impact of nanostructured components or various physiological circumstances on the elastic response of material to an external indentation. With a pyramidal tip at a nano-scale, we employed the atomic force microscopy (AFM) to indent the surfaces of two compositions of polyacrylamide gels with different softness and seedling roots of Arabidopsis thaliana. We found that the stiffness-depth curve derived from the measured force exhibits a heterogeneous character in elasticity. According to the tendency of stiffness-depth curve, we decomposed the responding force into depth-impact (FC), Hookean (FH) and tip-shape (FS) components, called trimechanic, where FS and its gradient should be offset at the surface or subsurfaces of the indented material. Thereby, trimechnic theory allows us to observe how the three restoring nanomechanics change with varied depth. Their strengths are represented by the respective spring constants (kC, kH, kS) of three parallel-connected spring (3PCS) analogs to differentiate restoring nanomechansims of indented materials. The effective Young's modulus Ê and the total stiffness kT (= kH + kS) globally unambiguously distinguish the softness between the two gel categories. Data fluctuations were observed in the elasticity parameters of individual samples, reflecting nanostructural variations in the gel matrix. Similar tendencies were found in the results from growing plant roots, though the data fluctuations are expectedly much more dramatic. The zone-wise representation of stiffness by the trimechanic-3PCS framework demonstrates a stiffness measure that reflects beneath nanostructures encountered by deepened depth. The trimechanic-3PCS framework can apply any mechanical model of power-law based force-depth relationship and is compatible with thin layer corrections. It provides a new paradigm for analyzing restoring nanomechanics of soft biomaterials in response to indenting forces.