Straining the root on and off triggers local calcium signalling.

A fundamental function of an organ is the ability to perceive mechanical cues. Yet, how this is accomplished is not fully understood, particularly in plant roots. In plants, the majority of studies dealing with the effects of mechanical stress have investigated the aerial parts. However, in natural conditions roots are also subjected to mechanical cues, for example when the root encounters a hard obstacle during its growth or when the soil settles. To investigate root cellular responses to root compression, we developed a microfluidic system associated with a microvalve allowing the delivery of controlled and reproducible mechanical stimulations to the root. In this study, examining plants expressing the R-GECO1-mTurquoise calcium reporter, we addressed the root cell deformation and calcium increase induced by the mechanical stimulation. Lateral pressure applied on the root induced a moderate elastic deformation of root cortical cells and elicited a multicomponent calcium signal at the onset of the pressure pulse, followed by a second one at the release of the pressure. This indicates that straining rather than stressing of tissues is relevant to trigger the calcium signal. Although the intensity of the calcium response increases with the pressure applied, successive pressure stimuli led to a remarkable attenuation of the calcium signal. The calcium elevation was restricted to the tissue under pressure and did not propagate. Strain sensing, spatial restriction and habituation to repetitive stimulation represent the fundamental properties of root signalling in response to local mechanical stimulation. These data linking mechanical properties of root cells to calcium elevation contribute to elucidating the pathway allowing the root to adapt to the mechanical cues generated by the soil.

Single-Cell Confinement Methods to Study Plant Cytoskeleton.

Progress in cytoskeletal research in animal systems has been accompanied by the development of single-cell systems (e.g., fibroblasts in culture). Single-cell systems exist for plant research, but the presence of a cell wall hinders the possibility to relate cytoskeleton dynamics to changes in cell shape or in mechanical stress pattern. Here we present two protocols to confine wall-less plant protoplasts in microwells with defined geometries. Either protocol might be more or less adapted to the question at hand. For instance, when using microwells made of agarose, the composition of the well can be easily modified to analyze the impact of biochemical cues. When using microwells in a stiff polymer (NOA73), protoplasts can be pressurized, and the wall of the well can be coated with cell wall components. Using both protocols, we could analyze microtubule and actin dynamics in vivo while also revealing the relative contribution of geometry and stress in their self-organization.