Wild soybean (Glycine soja) transcription factor GsWRKY40 plays positive roles in plant salt tolerance

Wild soybean (Glycine soja), a relative of cultivated soybean, shows high adaptability to adverse environmental conditions. We identified and characterized a wild soybean transcription factor gene, GsWRKY40, that promotes plant salt stress. GsWRKY40 was highly expressed in wild soybean roots and was up-regulated by salt treatment. GsWRKY40 was localized in nucleus and demonstrated DNA-binding activities but without transcriptional activation. Mutation and overexpression of GsWRKY40 altered salt tolerance of Arabidopsis plants. To understand the molecular mechanism of GsWRKY40 in regulating plant salt resistance, we screened a cDNA library and identified a GsWRKY40 interacting protein GsbHLH92 by using yeast two-hybrid approach. The physical interaction of GsWRKY40 and GsbHLH92 was confirmed by co-immunoprecipitation (co-IP), GST pull-down, and bimolecular fluorescence complementation (BiFC) techniques. Intriguingly, co-overexpression of GsWRKY40 and GsbHLH92 resulted in higher salt tolerance and lower ROS levels than overexpression of GsWRKY40 or GsbHLH92 in composite soybean plants, suggesting that GsWRKY40 and GsbHLH92 may synergistically regulate plant salt resistance through inhibiting ROS production. qRT-PCR data indicated that the expression level of GmSPOD1 gene encoding peroxidase was cooperatively regulated by GsWRKY40 and GsbHLH92, which was confirmed by using a dual luciferase report system and yeast one-hybrid experiment. Our study reveals a pathway that GsWRKY40 and GsbHLH92 collaboratively up-regulate plant salt resistance through impeding GmSPOD1 expression and reducing ROS levels, providing a novel perspective on the regulatory mechanisms underlying plant tolerance to abiotic stresses.