Exogenous Vitamin D Mitigates Salt Stress in Arabidopsis thaliana via Ion Homeostasis and Calcium‐Mediated Signaling

Understanding the physiological, biochemical, and molecular responses of Arabidopsis thaliana to salt stress and the potential of exogenous vitamin D application to alleviate these responses is critical for developing effective biostimulant strategies under increasing soil salinization. However, knowledge about how vitamin D2 and vitamin D3 mediate salt stress responses at the molecular level remains limited. The physiological, biochemical, and molecular responses of A. thaliana Col‐0 seedlings treated with exogenous vitamin D2 (50 ppm) and vitamin D3 (75 ppm) under 100 mM NaCl salt stress were investigated. Treatment significantly influenced all measured parameters. Both vitamin D forms consistently alleviated salt‐induced damage, with combination treatments maintaining higher photosynthetic pigment content, better growth performance, and stronger suppression of oxidative stress markers (reduced MDA, H 2 O 2 , and superoxide anion) compared with salt stress alone. Antioxidant enzyme activities (CAT, POX, APX, GR) were reduced under vitamin D treatment. Ion analysis revealed partial Na + exclusion and elevated Ca 2+ content under nonsaline vitamin D application, whereas RT‐qPCR analysis demonstrated coordinated upregulation of SOS1 and NHX1 under vitamin D3 combination treatment, alongside modulation of CNGC10, GORK, and SIP3 expression. It is concluded that exogenous vitamin D enhanced salt tolerance in A. thaliana through calcium‐mediated activation of Na + exclusion and vacuolar sequestration pathways, with vitamin D3 appearing more effective for growth recovery and ionic regulation at the concentrations tested. The mechanistic insights provided by correlation and clustering analyses suggest that vitamin D operates as a genuine upstream regulator of salt stress signaling rather than a compensatory antioxidant supplement, offering a promising foundation for vitamin D‐based biostimulant applications in saline agriculture. Relative to salt stress alone, the combination treatments lowered MDA by up to about 37% and hydrogen peroxide by roughly 83%, restored chlorophyll a to more than four times the salt‐stressed value, and increased seedling dry weight by approximately 150%.