The functional divergence of plant ESCRT components TOL3, SNF7.1, and VPS4 during salt stress response

Salinity, a major abiotic stressor, impacts protein trafficking and alters transport routes. The endosomal sorting complex required for transport (ESCRT)is a key player for membrane protein sorting and is increasingly recognized as pivotal element in abiotic stress responses. However, the functional role of ESCRT proteins in cereal crops under salt stress remains unexplored. Using the as near as possible to nature DRD-BIBLOX (Dark-Root device Brick Black Box), barley ( Hordeum vulgare , L.), although relatively salt-tolerant, exhibits impaired germination, seedling growth, and an altered root system architecture (RSA) under high salinity. Shotgun proteomic analysis revealed spatio-temporal regulation of ESCRT proteins in shoots and roots during seedling development. We identified HvSNF7.1 and HvVPS4 as critical, but differently acting modulators of salt stress responses in barley tissues. In this study we further reveal novel functions of ESCRT-0-like TOM-1-like proteins (TOLs): Previously described as gatekeepers of degradative protein sorting and as regulators of abscisic acid signaling, growth, and heat stress responses in Arabidopsis ( Arabidopsis thaliana ), HvTOLs had not been characterized for their role in salinity adaptation during plant development. Functional analyses demonstrated lethality in Hv toldouble and Hv tolquadruple mutants under standard growth conditions. In Arabidopsis, loss of At TOL3 alone impaired germination and root growth under salt stress, underscoring the essential role of individual TOL proteins in the spatio-temporal regulation of abiotic stress adaptation. These findings highlight ESCRT-mediated trafficking as a key determinant of cereal resilience under saline conditions and provide a molecular framework for improving crop stress tolerance.