A Ca²⁺-mediated signaling pathway modulates ion homeostasis via HKT1;1 during Arabidopsis seed germination under salt stress

SUMMARY Soil salinization severely constrains seedling establishment by disrupting cellular Na⁺/K⁺ homeostasis. Calcium (Ca²⁺) signaling contributes to the re-establishment of ion balance during early growth, yet the mechanisms linking Ca²⁺ perception to ion transport regulation remain unclear. Here, we identify a Ca²⁺-responsive regulatory module in Arabidopsis thaliana comprising CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6 (CAMTA6), the TYPE 2C PROTEIN PHOSPHATASE PP2C49, and the HIGH-AFFINITY K⁺ TRANSPORTER HKT1;1, and define their coordinated roles during germination under salt stress. Spatial promoter analyses revealed that NaCl induces CAMTA6 expression at cotyledon margins, while CaCl₂ stimulates HKT1;1 transcription in the radicle, consistent with CAMTA6-mediated repression of HKT1;1 . In camta6 mutants, PP2C49 expression expanded beyond its normal radicle-restricted domain, indicating CAMTA6-dependent spatial control. Promoter activation assays in planta demonstrated CAMTA6-dependent transactivation of the HKT1;1 and PP2C49 promoters. Treatment with the PP2C inhibitor sanguinarine enhanced germination under salinity in the wild type, but not in hkt1 nor in the salt-tolerant camta6 and pp2c49 mutants. Sanguinarine restricted CAMTA6 promoter activity to cotyledon margins, suppressed PP2C49 expression, and enhanced HKT1;1 accumulation in the radicle, collectively supporting improved Na⁺/K⁺ balance. Transcriptome profiling further revealed additional Ca²⁺-responsive PP2C genes under CAMTA6-dependent regulation. Together, these findings establish a Ca²⁺-regulated transcriptional network coordinating ion homeostasis during germination and suggest strategies to support seedling performance in saline environments. Significance statement Salinity impairs germination largely by disrupting Na⁺/K⁺ homeostasis, yet the signaling pathways that protect seedlings at this stage remain poorly defined. We identify a calcium-responsive regulatory mechanism that spatially coordinates transcriptional control of genes involved in ion transport during early development, providing a mechanistic basis for improving seedling establishment in saline soils.