GmRWP-RK1 Enhances Salt Tolerance by Modulating Antioxidant Defense, Ion Homeostasis and Stress-Responsive Pathways...

Soil salinity is rapidly spreading across agricultural regions and has become one of the most critical constraints on soybean growth, yield, and sustainable production. Despite the central role of transcription factors (TFs) in coordinating plant responses to abiotic stresses, the molecular mechanisms by which RWP-RK domain-containing TFs regulate salt-tolerant responses in soybean remain poorly understood. Our previous genome-wide characterization identified 28 RWP-RK TFs in soybean exhibiting abiotic stress-responsive expression, yet their biological functions under salt stress have not been experimentally validated. Here, we investigated a 981-bp GmRWP-RK1 encoding region and demonstrated its regulatory role in enhancing salt tolerance by activating antioxidant defence, Na⁺/K⁺ homeostasis, and transcriptional control of salt-responsive genes using a cross-species overexpression approach. The two Arabidopsis lines (OE1 & OE4) overexpressing GmRWP-RK1 demonstrated significantly improved salt tolerance, as evidenced by ~18% greater survival and enhanced germination compared to non-transgenic plants under salinity stress. This phenotype was supported by stronger antioxidant protection, as indicated by elevated proline levels, reduced MDA accumulation, and increased SOD and POD activities. At the molecular level, the transgenic lines also showed up-regulated expression of key stress-responsive genes (AtACS10, AtSUMO1, AtGBF1), confirming the regulatory influence of GmRWP-RK1 on salt-adaptation pathways. Consistent with the Arabidopsis results, GmRWP-RK1 overexpression in soybean hairy roots also led to improved salt-stress tolerance by accumulating significantly reduced ROS contents (27.38% lower H₂O₂ and 33.98% lower O₂^-), and maintained a balanced Na⁺/K⁺ ratio compared to that of non-transgenic hairy roots under salinity. Furthermore, GmRWP-RK1-overexpressing transgenic soybean hairy roots showed increased expression of stress-responsive genes, especially GmATG-5, GmOLP-1, and GmOLP-2. Overall, our results support a possible role of GmRWP-RK1 in soybean salt tolerance and provide a foundation for future functional and breeding-oriented studies.