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

Shile Sun, Bingjun Yu, Jiaxin Yang, Jifeng Jiang, Dan Liang

Plant Physiology and Biochemistry; Volume 231, February 2026, 111035

Abstract

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.

See https://www.sciencedirect.com/science/article/pii/S0981942826000215

Fig. 3. The transcription factor GsWRKY23 binds to the W-box cis-acting element at position −486 of the promoter region of GsPER3 gene and activates its expression. (A) Y1H assay demonstrating the binding of GsWRKY23 to the GsPER3 promoter, Y187 yeast strain cotransformed by GAL4-AD and GsPER3pro:HIS as negative control, (SD-Leu) represents a selective medium deficient in leucine, and (SD-Leu-His) represents a selective medium deficient in both leucine and histidine. (B) The pattern diagram of the GsPER3 promoter shows that the sequence between −507 bp and −468 bp is a DNA probe containing a W-box cis-acting element. (C) EMSA experiment confirmed that the GsWRKY23 protein binds to the W-box element in the GsPER3 promoter region in vitro, with “+” indicating the addition of the corresponding protein or probe, and “-” indicating its absence. (D) Verification of GsWRKY23 binding to the GsPER3 promoter to activate a reporter gene by transient expression in tobacco leaves. (E) Determination of GUS activity. (F) Utilization of a plant live-imaging system to observe the transcriptional activation effect of GsWRKY23 on the GsPER3pro in tobacco leaves, where a stronger LUC fluorescence signal indicates a stronger activation effect, and (G) Quantitative analysis of the ratio of LUC to REN activities to determine the binding capacity of GsWRKY23 to the GsPER3pro sequence.