Osmotic stress activates two reactive oxygen species pathways with distinct effects on protein nanodomains and diffusion.

Physiological acclimation of plants to an ever-changing environment is governed by complex combinatorial signaling networks that perceive and transduce various abiotic and biotic stimuli. Reactive oxygen species (ROS) serve as one of the second messengers in plant responses to hyperosmotic stress. The molecular bases of ROS production and the primary cellular processes that they target were investigated in the Arabidopsis (Arabidopsis thaliana) root. Combined pharmacological and genetic approaches showed that RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) pathway and an additional pathway involving apoplastic ascorbate and iron can account for ROS production upon hyperosmotic stimulation. The two pathways determine synergistically the rate of membrane internalization, within minutes following activation. Live super-resolution microscopy revealed at single-molecule scale how ROS control specific diffusion and nano-organization of membrane cargo proteins. In particular, ROS generated by RBOHs initiated clustering of the PIP2;1 aquaporin and its removal from the plasma membrane. This process is contributed to by clathrin-mediated endocytosis, with a positive role of RBOH-dependent ROS, specifically under hyperosmotic stress.