Role of OsCZMT1 in Na⁺ and Mg²⁺ transport and salinity insensitivity

Salinity stress is one of the most important abiotic stress factors affecting rice production worldwide. Using a forward genetics approach, a salt-insensitive TILLING line 1 (sitl1) rice mutant was isolated from a TILLING population. The sitl1 mutant exhibited reduced root growth and leaf chlorophyll content when grown under normal conditions, owing to a reduced ability to take up Mg²⁺; however, the defective phenotype could be restored by Mg²⁺ supplementation. Ionomic analysis revealed that the salinity insensitivity of sitl1 was the result of significantly reduced Mg²⁺ and Na⁺, and whole genome and RNA sequencing revealed that the sitl1 mutant caused a frameshift in the OsCZMT1 protein's CorA-like ZntB cation transfer domain. Transient expression of an OsCZMT1-sGFP fusion protein revealed that OsCZMT1 was localized to the plasma membrane, and comprehensive expression analysis revealed that OsCZMT1 was mainly expressed in the roots and leaves of seedlings and highly upregulated in response to salinity stress. Complementation assays in yeast and CRISPR/Cas9-mediated knock-out mutants reveal that OsCZMT1 possesses both Mg²⁺ and Na⁺ transport activity. Taken together, these findings suggest that the identified OsCZMT1 frameshift mutation is responsible for the reduced Na⁺ and Mg²⁺ transport abilities, and that those reduced transport abilities confer salinity insensitivity. Data availability statement: The study's raw whole-genome sequencing and RNA-sequencing data have been accessioned in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under BioProject Accession Number PRJNA648465 (SRR12334077 – SRR12334084).