SOS2-FREE1 regulates SOS1 tonoplast sorting to promote Na⁺ compartmentalization in vacuole during salt stress response

Soil salinity significantly affects plant survival and limits crop productivity. Under salt stress, plants can transport sodium ions (Na⁺) out of cells and sequester them into vacuoles for detoxification. The salt excretion process is governed by the SALT OVERLY SENSITIVE (SOS) pathway, which involves the calcium sensors SOS3 and SOS3-LIKE CALCIUM BINDING PROTEIN 8, the protein kinase SOS2, and the plasma membrane Na⁺/H⁺ antiporter SOS1. While previous studies have provided insights into Na⁺ transport through the SOS system, the role of this pathway in Na⁺ compartmentalization within vacuoles remains poorly understood. In this study, we demonstrate that SOS1 partially internalizes to the tonoplast under salt stress, which is crucial for Na⁺ compartmentalization in vacuoles in Arabidopsis (Arabidopsis thaliana). We show that SOS2 phosphorylates the endosomal sorting complex required for transport-I (ESCRT-I) component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which disrupts its interaction with VPS23A, an ESCRT-I component. This phosphorylation event inhibits the formation of intraluminal vesicles (ILVs) in prevacuolar compartments and multivesicular bodies (PVCs/MVBs), thereby remodeling endosomal sorting during salt stress. Additionally, our previous research indicated that SOS2-mediated phosphorylation of FREE1 leads to vacuole fragmentation by altering endomembrane fusion, thereby regulating intracellular Na⁺ homeostasis. Taken together, our findings reveal how the SOS2-FREE1 module orchestrates both endomembrane fusion and endosome sorting processes to enhance plant salt tolerance, providing novel insights into the cellular mechanisms underlying salt stress adaptation.