Jean-Marie Frachisse

Summary: Plants respond to mechanical stimuli by a rapid increase in cytosolic calcium. The intensity and kinetics of the calcium changes define calcium signatures important for biological responses. In this study, we determine the properties of a calcium‐permeable force‐gated channel localized at the plasma membrane called Rapid Mechanically Activated (RMA). Using patch clamp and pressure clamp, we characterized the kinetics of the Arabidopsis thaliana RMA channel upon stimulation by pressure pulses applied onto the plasma membrane. Combining pressure pulse protocols at different frequencies with modeling, we investigated the channel's capacity to transduce high frequency mechanical stimuli. The RMA channel rapidly activates in response to membrane tension, then it inactivates during prolonged stimulation. Upon repeated stimulations, the RMA current amplitude decreases irreversibly indicating that it undergoes attenuation. The channel kinetics were modeled with four chemical states and the model predicts that it behaves as a pass band filter in the 10 Hz–1 kHz range. In conclusion, due to its activation/inactivation, the RMA channel is a candidate for mediating cytosolic calcium signaling in response to mechanostimulation. Its attenuation and filtering properties suggest its involvement in the transduction of high frequency mechanical stimulation, such as those produced by insects' vibrations.

Mechanical stimulation of the root triggers signal transduction involving Reactive Oxygen Species (ROS) and calcium, but their relationships are unclear. This study aims to clarify the temporal and spatial interrelations between calcium and ROS following a localized lateral compression of the root. We combined a microfluidic valve rootchip to apply controlled compression, with fluorescent probes and wide-field or confocal microscopy to monitor H2O2 and calcium dynamics in root tissues simultaneously. Pharmacological inhibitors were used to investigate the causal links between H2O2 and calcium responses. In response to compression, we observed transient H2O2 accumulation, with characteristics similar to the calcium response observed previously in the same microfluidic system. H2O2 and calcium response occurred in 3 kinetic phases: a fast calcium increase relying on mechanosensitive channels and external calcium entry, followed by a long-lasting H2O2 accumulation and a slow calcium increase depending on NADPH oxidase activity. H2O2 accumulated in all root tissues while calcium increases were confined to the root center. These results suggest that two mechanotransduction mechanisms are involved in root response to compression. One mechanism relies on plasma membrane mechanosensitive channels, triggering a fast calcium increase. Another independent mechanism, relying on FERONIA, induces H2O2 accumulation, which drives the slower secondary calcium increase.

Summary: Plants respond to mechanical stimuli by a rapid increase in cytosolic calcium. The intensity and kinetics of the calcium changes define calcium signatures important for biological responses . In this study, we determine the properties of a calcium permeable force-gated channel localized at the plasma membrane called Rapid Mechanically Activated (RMA). Using patch-clamp and pressure-clamp, we characterized the kinetics of activation and inactivation of RMA channel upon stimulation by pulses of pressure applied onto the plasma membrane. Combining repetitive pressure pulse protocols at different frequencies with modeling, we investigated the channel’s capacity to transduce high frequency mechanical stimuli. RMA channel rapidly activates in response to membrane tension, then it inactivates during prolonged stimulation. Upon repeated stimulations, RMA current amplitude decreases irreversibly indicating that undergoes adaptation. The channel kinetics may be modeled with four chemical states and the model predicts that it behaves as a pass band filter in the 10 Hz - 1 kHz range. In conclusion, due to its activation/inactivation characteristics, RMA channel is a candidate to mediate cytosolic calcium signaling in response to mechano-stimulation. Its adaptation and filtering properties suggest its involvement in the transduction of high frequency mechanical stimulation such as those produced by insects’ vibrations.

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.

Throughout their life, plant root are submitted to mechanical stresses due to pressure exerted by the soil. So far, few studies addressed root cell deformation and calcium signaling elicited by soil compression. In this study, we designed a microchip inspired by pneumatic microvalve concept in order to deliver a lateral pressure to the root of a plant expressing the RGECO1-mTurquoise calcium reporter. 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. The calcium elevation was restricted to the tissue under pressure and did not propagate. Additionally, the calcium signals exhibited a remarkable attenuation upon repetitive stimulations. Significance statement In natural conditions, roots are subjected to diurnal hydraulic pressure variations producing a periodic root diameter fluctuation moreover during its progression, the lateral confinement of the root increases, generating additional lateral forces. Thus it is crucial for the root to be able to perceive and transduce these stimulations in order to progress in the heterogeneous soil network. Our microfluidic set-up, mimicking lateral soil pressure, revealed new features of the early root cellular responses. A cytosolic calcium elevation is observed upon increase but also upon release of the pressure, corresponding to variations in the shape of the cells of the cortex. Although the intensity of the calcium response increases with the pressure applied, successive pressure stimuli lead to attenuation of the calcium signal. Strain sensing and habituation to repetitive stimulation represent the fundamental properties of the “sensing system” allowing the root to adapt to the stresses generated by the soil.

Plants spend most of their life oscillating around 1–3 Hz due to the effect of the wind. Therefore, stems and foliage experience repetitive mechanical stresses through these passive movements. However, the mechanism of the cellular perception and transduction of such recurring mechanical signals remains an open question. Multimeric protein complexes forming mechanosensitive (MS) channels embedded in the membrane provide an efficient system to rapidly convert mechanical tension into an electrical signal. So far, studies have mostly focused on nonoscillatory stretching of these channels. Here, we show that the plasma-membrane MS channel MscS-LIKE 10 (MSL10) from the model plant Arabidopsis thaliana responds to pulsed membrane stretching with rapid activation and relaxation kinetics in the range of 1 s. Under sinusoidal membrane stretching MSL10 presents a greater activity than under static stimulation. We observed this amplification mostly in the range of 0.3–3 Hz. Above these frequencies the channel activity is very close to that under static conditions. With a localization in aerial organs naturally submitted to wind-driven oscillations, our results suggest that the MS channel MSL10, and by extension MS channels sharing similar properties, represents a molecular component allowing the perception of oscillatory mechanical stimulations by plants. Significance Mechanosensitive (MS) channels embedded in the membrane provide an efficient system to rapidly convert mechanical tension into a biological signal. Here we show that the plasma-membrane mechanosensitive channel MscS-LIKE 10 (MSL10) responds to pulsed membrane stretching with rapid activation and relaxation kinetics allowing fast adjustment of its activity to fluctuations in membrane tension. Under sinusoidal membrane mechanical stimulation, we found that MSL10 amplifies oscillatory signals near 1 Hz which corresponds to the frequencies of plant motions in the wind. Together with its localization in aerial organs, our results indicate that the MSL10 represents a molecular component for the perception of oscillations triggered by the wind in plants.

Throughout their life, plants are submitted to recurrent cyclic mechanical loading due to wind. The resulting passive oscillation movements of stem and foliage is an important phenomenon for biological and ecological issues such as photosynthesis optimization ^1–3 and thermal exchange ⁴ . The induced motions at plant scale are well described and analyzed, with oscillations at typically 1 to 3 Hz in trees ^5–10 . However, the cellular perception and transduction of such recurring mechanical signals remains an open question. Multimeric protein complexes forming mechanosensitive (MS) channels embedded in the membrane provide an efficient system to rapidly convert mechanical tension into electrical signal ¹¹ . Here we show that the plasma membrane mechanosensitive channel MscS-LIKE 10 (MSL10) from the model plant Arabidopsis thaliana responds to pulsed membrane stretching with rapid activation and relaxation kinetics in the range of one second. Under sinusoidal membrane stretching MSL10 presents a greater activity than under static stimulation and behaves as a large bandpass oscillation “follower” without filtering the signal in the range of 0.3 to 3 Hz. With a localization in aerial organs naturally submitted to oscillations, our results suggest that the mechanosensitive channel MSL10 represents a molecular component of a universal system of oscillatory perception in plants.

The root is an important organ for water and nutrient uptake, and soil anchorage. It is equipped with root hairs (RHs) which are elongated structures increasing the exchange surface with the soil. RHs are also studied as a model for plant cellular development, as they represent a single cell with specific and highly regulated polarized elongation. For these reasons, it is useful to be able to accurately quantify RH length employing standardized procedures. Methods commonly employed rely on manual steps and are therefore time consuming and prone to errors, restricting analysis to a short segment of the root tip. Few partially automated methods have been reported to increase measurement efficiency. However, none of the reported methods allow an accurate and standardized definition of the position along the root for RH length measurement, making data comparison difficult. We developed an image analysis algorithm that semi-automatically detects RHs and measures their length along the whole differentiation zone of roots. This method, implemented as a simple automated script in ImageJ/Fiji software that we termed Root Hair Sizer, slides a rectangular window along a binarized and straightened image of root tips to estimate the maximal RH length in a given measuring interval. This measure is not affected by heavily bent RHs and any bald spots. RH length data along the root are then modelled with a sigmoidal curve, generating several biologically significant parameters such as RH length, positioning of the root differentiation zone and, under certain conditions, RH growth rate. Image analysis with Root Hair Sizer and subsequent sigmoidal modelling of RH length data provide a simple and efficient way to characterize RH growth in different conditions, equally suitable to small and large scale phenotyping experiments.