Systematic Characterization of the OSCA Family Members in Soybean and Validation of Their Functions in Osmotic Stress

Since we discovered OSCA1, a hyperosmolarity-gated calcium-permeable channel that acted as an osmosensor in Arabidopsis, the OSCA family has been identified genome-wide in several crops, but only a few OSCA members’ functions have been experimentally demonstrated. Osmotic stress seriously restricts the yield and quality of soybean. Therefore, it is essential to decipher the molecular mechanism of how soybean responds to osmotic stress. Here, we first systematically studied and experimentally demonstrated the role of OSCA family members in the osmotic sensing of soybean. Phylogenetic relationships, gene structures, protein domains and structures analysis revealed that 20 GmOSCA members were divided into four clades, of which members in the same cluster may have more similar functions. In addition, GmOSCA members in clusters III and IV may be functionally redundant and diverged from those in clusters I and II. Based on the spatiotemporal expression patterns, GmOSCA1.6, GmOSCA2.1, GmOSCA2.6, and GmOSCA4.1 were extremely low expressed or possible pseudogenes. The remaining 16 GmOSCA genes were heterologously overexpressed in an Arabidopsis osca1 mutant, to explore their functions. Subcellular localization showed that most GmOSCA members could localize to the plasma membrane (PM). Among 16 GmOSCA genes, only overexpressing GmOSCA1.1, GmOSCA1.2, GmOSCA1.3, GmOSCA1.4, and GmOSCA1.5 in cluster I could fully complement the reduced hyperosmolality-induced [Ca(2+)](i) increase (OICI) in osca1. The expression profiles of GmOSCA genes against osmotic stress demonstrated that most GmOSCA genes, especially GmOSCA1.1, GmOSCA1.2, GmOSCA1.3, GmOSCA1.4, GmOSCA1.5, GmOSCA3.1, and GmOSCA3.2, strongly responded to osmotic stress. Moreover, overexpression of GmOSCA1.1, GmOSCA1.2, GmOSCA1.3, GmOSCA1.4, GmOSCA1.5, GmOSCA3.1, and GmOSCA3.2 rescued the drought-hypersensitive phenotype of osca1. Our findings provide important clues for further studies of GmOSCA-mediated calcium signaling in the osmotic sensing of soybean and contribute to improving soybean drought tolerance through genetic engineering and molecular breeding.