Osmotic stress-induced CsRCI2E endosomal trafficking modulates CsPIP2 aquaporins at the plasma membrane in Camelina sativa

Rare Cold Inducible 2s (RCI2s) are membrane-associated proteolipids dynamically trafficking between the plasma membrane (PM) and the endomembrane system. Their expression is upregulated in response to abiotic stresses, including cold, heat, drought, and salinity, contributing to plant stress tolerance. CsRCI2E interacts with the water transport protein CsPIP2; 1, reducing its abundance at the PM under NaCl-induced stress. Consequently, CsRCI2E is considered a potential regulator of CsPIP2 endocytosis involved in maintaining cellular homeostasis. However, its precise role in membrane trafficking remains unclear. Therefore, this study aims to investigate the rapid internalization of CsRCI2E and CsPIP2 under mannitol-induced and NaCl-induced osmotic stress using a sucrose density gradient. CsRCI2E transcription levels increased significantly 3 h posttreatment with mannitol or NaCl. CsRCI2E overexpression enhanced stress tolerance and reduced reactive oxygen species accumulation-induced cellular damage during Camelina germination. Despite no concurrent change in CsRCI2E gene expression, the subcellular distribution of CsRCI2E and CsPIP2s (CsPIP2; 1 and CsPIP2; 2) shifted rapidly from the PM to the endomembrane within 0.5 h following osmotic stress. Additionally, CsRCI2E overexpression induced internalization and subcellular redistribution of CsRCI2E and CsPIP2s under osmotic stress and non-stress conditions. These findings suggest that CsRCI2E internalization functions as a sensing mechanism during the initial phase of osmotic shocks. Furthermore, elevated CsRCI2E levels promote CsPIP2s membrane trafficking from the PM to the endomembrane system, supporting water homeostasis in Camelina.