BnaGRP3 mediates salt tolerance via Na+/K+ homeostasis and BnaPIPs interactions in Brassica napus.

Rapeseed (Brassica napus L.) is a major oilseed crop with considerable potential for improving saline-alkali soils, yet the molecular mechanisms underlying its salt tolerance remain unclear. This study investigates the functional roles and regulatory mechanisms of glycine-rich protein 3 (BnaGRP3) in rapeseed under salt stress. We employed molecular genetics, phenotypic and biochemical evaluation of transgenic rapeseed and Arabidopsis, transcriptome sequencing, protein interaction assays including immunoprecipitation-mass spectrometry (IP-MS), yeast two-hybrid (Y2H), luciferase complementation (LCA), and bimolecular fluorescence complementation (BiFC) assays, gene expression analysis by RT-qPCR, and hydrogen peroxide (H2O2) permeability assays conducted in yeast. BnaGRP3 was induced by salt stress and enhanced salt tolerance. Transcriptome analysis revealed that BnaGRP3 modulated expression of ion transporters under salt stress, especially NHX1 and SKOR. BnaGRP3 physically interacted with four plasma membrane intrinsic proteins (BnaPIPs). Overexpression of these BnaPIPs improved salt tolerance in Arabidopsis and increased H2O2 tolerance when expressed in yeast. In addition, these BnaPIPs formed both homomeric and heteromeric complexes, suggesting they may facilitate H2O2 permeability. BnaGRP3 enhances salt tolerance by maintaining Na+/K+ homeostasis and, through its interactions with BnaPIPs, may participate in the regulation of H2O2 balance·H2O2 potentially serves as a bridge linking BnaGRP3-mediated ion homeostasis and redox regulation. The previously uncharacterized BnaGRP3-BnaPIP module broadens the mechanistic framework of GRP-mediated salt stress responses, thereby expanding our understanding of salt tolerance mechanisms in Brassica napus.