Nanoscale regulation of ROS signaling at the plasma membrane tunes the plant response to osmotic stress

The spatiotemporal organization of proteins and lipids within membranes is crucial for ensuring proper cellular signaling. While the segregation of proteins and lipids into membrane nanodomains is well established, it remains unclear whether nanodomains can generate gradients of small diffusible molecules. In plants, reactive oxygen species (ROS), especially hydrogen peroxide (H ₂ O ₂ ), act as key signaling molecules in response to environmental stimuli such as osmotic stress. However, how extracellular H ₂ O ₂ affects intracellular signaling has remained unknown. Here, we show that osmotic stimulation induces the formation of localized, H ₂ O ₂ -rich nanoenvironments at the cytoplasmic face of the plasma membrane (PM) in Arabidopsis root cells. Using a PM-tethered H ₂ O ₂ biosensor, we found that these oxidized nanodomains arise from the clustering of RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs) and RHO OF PLANTS 6 (ROP6), in coordination with aquaporin-mediated H ₂ O ₂ transport via the PLASMA MEMBRANE INTRINSIC PROTEIN2;7 (PIP2;7). These local redox hotspots at the PM create a feedforward loop in which H ₂ O ₂ enhances ROP6 nanoclustering, thereby amplifying ROS signaling. Disruption of H ₂ O ₂ production or transport dampens both ROP6 clustering and anisotropic cell expansion, indicating a crucial role for spatially confined redox signaling in regulating plant growth under osmotic stress. Our findings propose a model in which ROP6/RBOHD-F/PIP2;7 nanodomains function as discrete redox signaling units, redefining ROS signaling at the PM as a structured, signal-specific, and compartmentalized process.