Functional characterization of the transcription factors Gm/GsERF160 from G.max and G.soja in conferring salt...

Soil salinization is a major challenge for the sustainable development of global agriculture. Cl⁻-dominated anion toxicity is less studied than Na⁺ because its uptake and transport are more complex. Thus, it has become a key issue for understanding salt tolerance in soybean and other plants. Our previous studies attribute the superior salt tolerance of Glycine soja over Glycine max to greater root Cl⁻ retention, reduced shoot transport, and stronger root GsCLC-c2 expression under salt stress. However, the upstream transcriptional regulators governing CLC-c2 gene expression in G. max and G. soja remain unclear. Using G. max (cv. N23674) and G. soja (acc. BB52), we combined yeast one-hybrid (Y1H), subcellular localization, hairy-root transformation, and transgenic Arabidopsis assays to reveal how the ERF transcription factors Gm/GsERF160 regulate CLC-c2 expression to enhance salt tolerance. Phylogenetic analysis indicates that GsERF160 has five extra amino acids than GmERF160, with only one difference in the conserved domain and nearly identical three-dimensional structures. Both the GmERF160 and GsERF160 genes exhibited markedly salt stress-inducible expression. GmERF160 and GsERF160, which are localized in the nucleus, can bind to the promoter region of GmCLC-c2 and subsequently activate its expression. Compared with GmERF160-Cas9 or WT plants, overexpression of Gm/GsERF160 in soybean hairy-root composite plants and Arabidopsis seedlings under salt stress alleviated salt damage. This was reflected by increased chlorophyll and relative water content (RWC) in leaves or shoots, as well as reduced malondialdehyde (MDA), relative electrolyte leakage (REL), and Cl⁻/NO₃⁻ and Na⁺/K⁺ ratios in both roots and shoots. Together, these results demonstrate that the conserved, salt-inducible Gm/GsERF160-CLC-c2 module positively regulate GmCLC-c2 expression, thereby mediating root Cl⁻ uptake and shoot transport to maintain shoot Cl⁻ homeostasis and confer salt tolerance in soybean. These findings provide valuable regulatory targets and candidate genes for breeding of salt-tolerant soybean cultivars.