Abstract
Continuous perception and rapid reaction to their environment are crucial features of the plants in the course of their growth and development. To maintain the water status and acclimate to environmental constraints plants have developed short-term (fast regulation of stomatal aperture and tissue hydraulics) and long-term (alteration of root system architecture and leaf abscission) responses. Despite their central role, the early cellular events that lead to these adaptive responses are largely unknown. Whereas the molecular bases of plant osmotic perception are not fully characterized, reactive oxygen species (ROS) constitute first cellular response and they are crucial secondary messengers during osmotic signaling. Thus, the main question of this Ph.D. thesis is to dissect the molecular transduction and, ultimately, the perception of the osmotic stimuli. The cell plasma membrane is the major coordinator of many environmental signaling complexes. In the context of hyperosmotic constraint, the cell responds by accumulation Reactive Oxygen Species (ROS), produced enzymatically by Respiratory Burst Oxidase Homolog (Rboh), and additional pathway involving apoplastic ascorbate and iron (Martiniere et al., 2019). By combining gene candidate approach and varieties of microscopy technics we demonstrated that Arabidopsis Rho GTPase, Rho of Plants (ROP6) is the upstream regulator hub of the two ROS producing agents and it’s necessary to some downstream plant responses to osmotic stress. Furthermore, we demonstrated the mechanism by which ROP6 can directly regulate ROS production. ROP6 can recruit its effectors, RbohD and RbohF, in “osmotic specific” nanodomains on the PM. Indeed, it is known that ROP6 participates also in auxin signaling in cells. But, whereas this stimulus induces ROP6 nanodomain formation, the Rbohs effector proteins are absent of these nanodomains. Consequently, auxin does not induce ROS accumulation in cells. This shows that nano-organization of a single ROP isoform in the membrane can encode for signal specificity. This work also demonstrated a crucial role of one of the activators of the ROPs in the GTP-ase cycle, guanine nucleotide exchange factor (GEF) GEF14. It appears that GEF14 could be the specific activator of ROP6 in osmotic stressed environment. Small GTPase are classically acting downstream of membrane receptors. In plants, Catharanthus roseus Receptor-like kinases (CrRLKs) are known to participate in the perception of cell wall environment that is strongly impacted during hyperosmotic stress. By using live imaging methods, we demonstrated that Feronia, a typical member of CrRLK, is necessary but not sufficient for osmotic signaling by acting indirectly on ROP6 signalization by regulating its diffusion. Further studies revealed that anionic lipid phosphatidylserine (PS), which has been described to fine-tune the spatiotemporal dynamics of small GTPases (Platre et al., 2019), can be regulated by Feronia. Our observation suggests that Feronia is able to acts quantitatively on ROP signaling both in roots and shoot through the regulation of PS amount and clustering at the PM, therefore serving as a rheostat for the small GTPase signaling.In summary, this work shed some light on part of the molecular machinery of the osmotic signaling cascade in plants, where the small GTPase and cell wall sensing machinery refine the regulation of protein localization and dynamics within the membrane.