The COBRA-Like9 from Glycine tabacina enhanced salt tolerance in cultivated soybean

Soybean is one of the major sources of high-quality protein and oil and plays a crucial role in ensuring nutritional and economic security. The rapidly increasing population, soybean supply-demand imbalance, and yield losses due to soil salinity collectively necessitate the development of salt-tolerant cultivars. Perennial wild Glycine species harbor-rich genetic diversity and constitute a reservoir of stress-tolerant genes. This study systematically characterized the COBL gene family in the salt-resilient Glycine tabacina (Labill.) Benth., a wild soybean that tolerates 500 mM NaCl with 100% survival compared with only 5% in cultivated soybean. We identified 44 GtCOBL genes nearly double the number in cultivated soybean driven by tetraploidization and tandem duplication events. We further identified a novel salt-induced gene, GtCOBRA-Like9 (GtCOBL9), from G. tabacina, which encodes a plasma membrane protein possessing a cellulose-binding motif and a GPI anchor, supporting its potential role in cell wall modification. Functional validation revealed that overexpression of GtCOBL9 in cultivated soybean-enhanced seedling salt tolerance. Elevated cellulose content in the roots of GtCOBL9-OE lines suggest that GtCOBL9 may function in cell wall modeling. Notably, we uncovered a novel interaction between GtCOBL9 and the ion-transport regulators GmAKT1 and GmCAX1. These genes were upregulated in GtCOBL9 overexpression lines and roots of GtCOBL9-OE-1 showed higher K⁺ retention and increased Na⁺ efflux while no significant difference in total ion content between GtCOBL9-OE lines and wildtype, suggesting that GtCOBL9 is associated with ion flux dynamics rather than bulk accumulation. Additionally, GtCOBL9-OE lines exhibited reduced reactive oxygen species accumulation, lower expression of GmRbohB2 and GmAOX1, higher catalase (CAT) activity, and increased proline content, suggesting a role in oxidative stress mitigation. Taken together, this study identifies a novel GtCOBL9 that may confers salt tolerance to cultivated soybean through cell wall modification, ionic and oxidative adjustments, however underlying molecular mechanisms require further investigations.