Characterization and molecular cloning of sos3: A gene important for salt tolerance and potassium nutrition in higher plants

Abstract

The major goal of my dissertation research was to use genetic approaches to identify and characterize the components (genes) that are important for salt tolerance in Arabidopsis. Identification and characterization of such genes might provide insights into why these genes are important, and how these genes function in salt response and salt tolerance in higher plants. During my dissertation research, the sos3-1 mutant was isolated and characterized. The mutant plants are hypersensitive to Na⁺ and unable to grow with low K⁺. Increased Ca²⁺ levels can partially suppress the growth defect of the mutant plants under salt stress and fully restore their growth under low K⁺. These results suggest that SOS3 may be a Ca²⁺-mediated regulator that controls K⁺ and Na+ homeostasis in Arabidopsis. The SOS3 gene was cloned by map-based cloning techniques. SOS3 encodes a protein sharing significant sequence similarity with the B subunit of calcineurin from yeast and neuronal calcium sensor from animals. SOS3 contains three putative EF-hand calcium binding domains and a putative myristoylation motif at its NH₂-terminus. SOS3 binds calcium and is myristoylated in vitro. A mutation in SOS3 that destroys the conserved myristoylation motif abolishes SOS3 myristoylation, but not its calcium binding in vitro. Furthermore, the defect in Ca²⁺ binding of the sos3 does not affect its myristoylation. These results indicate the independence of calcium binding and myristoylation of SOS3. Mutant sos3-1 has a nine-base-pair deletion in the second conserved EF-hand Ca²⁺ binding domain, which leads to misfunction. of sos3 in vivo. To determine if myristoylation is also important for SOS3, the wild-type SOS3 cDNA and the SOS3 cDNA with a disrupted conserved myristoylation sequence were tested for their capability to complement the sos3-1. It was found that an intact conserved myristoylation sequence is essential for SOS3 function. These results indicate that both calcium binding and myristoylation are essential for the function of SOS3.