The transcription factor GsWRKY23 gene from wild soybean confers enhanced salt tolerance by regulating GsPER3 expression via ROS homeostasis

The transcription factor WRKYs enable plants to initiate defense responses against multiple adverse conditions by regulating the expression of downstream target genes. The salt-tolerant wild soybean (Glycine soja) represents an important genetic resource for the molecular breeding and genetic improvement of salt-tolerant cultivated soybean (G. max). In this study, we identified GsPER3, a downstream target gene of GsWRKY23, using transcriptome sequencing (RNA-seq), combined with promoter cis-acting element analysis and GUS staining. The transcriptional regulation of GsWRKY23 on GsPER3 was further confirmed by yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), GUS activity, and dual-luciferase (DLR) assays. As a member of the peroxidase family, GsPER3 functions in scavenging reactive oxygen species (ROS). Using a soybean hairy-root transformation system, we investigated the mechanism by which GsWRKY23 enhances salt tolerance in wild soybean by regulating GsPER3. Results showed that GsWRKY23 specifically binds to the W-box element at position -486 in the GsPER3 promoter, thereby activating its expression. Under salt stress treatment, compared to the empty vector (EV) control, GsWRKY23-overexpressing (GsWRKY23-OE) plants exhibited upregulated root GsPER3 expression, and elevated peroxidase (POD) activity in both roots and leaves. In contrast, GsWRKY23-CRISPR/Cas9 (GsWRKY23-Cas9) plants showed suppressed GsPER3 expression and significantly decreased POD activity. Under salt stress, GsPER3-OE plants displayed enhanced salt tolerance, with superior performance in physiological parameters including plant fresh weight (FW), leaf relative water content (RWC), as well as lower relative electrolytic leakage (REL) level and malondialdehyde (MDA) content in roots and leaves compared to EV plants. Conversely, GsPER3-Cas9 plants showed the opposite trends. Moreover, GsPER3-OE plants exhibited lower ROS accumulation and higher antioxidant enzyme activities. Taken together, our findings demonstrate that GsWRKY23 enhances salt tolerance in wild soybean by activating GsPER3 expression, which in turn increases antioxidant enzyme activity and reduces ROS accumulation, thereby maintaining ROS homeostasis.