Spatiotemporal Analysis Reveals Mechanisms Controlling Reactive Oxygen Species and Calcium Interplay Following Root Compression

Abstract

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.