Abstract

Soil alkalinity and heavy metal toxicity are major abiotic stressors that severely limit plant growth and crop productivity. Wild soybean ( Glycine soja ) exhibits strong alkaline tolerance, making it a valuable genetic resource for improving cultivated soybean. Previous studies identified Gshdz4 and GsNAC019 as key alkaline-tolerant transcription factors, and GsEXPA8 as an alkaline-tolerant expansin protein. This study establishes a hierarchical "nucleus-nucleus-membrane" regulatory model in wild soybean, wherein the nuclear master transcription factor Gshdz4 transcriptionally upregulates GsNAC019 , which in turn activates the expression of the plasma membrane-localized expansin GsEXPA8 , collectively enhancing alkaline tolerance. We further investigated the role of Gshdz4 in conferring resistance to combined sodium bicarbonate and cadmium chloride stress in soybean. Through integrated RNA-seq and CUT&Tag-seq analyses, we identified Gshdz4 as a direct binder and regulator of diverse stress-responsive genes-including GmDEAH5 , GmHSP22.3 , GmACR4 , and GmATG1c . Under alkaline stress, Gshdz4 modulates GmSLX8, GmSF3, and GmPP4 ; under cadmium stress, it regulates GmGMFL01 and GmUNC , establishing a broad-spectrum defense mechanism. Additional targets encompass splicing factors, heat shock proteins, ABA signaling components, and ethylene-responsive factors. GO and KEGG enrichment analyses confirmed that Gshdz4 participates in multiple hormonal pathways (ABA, IAA, ET, JA) and stress response signaling. Our findings highlight Gshdz4 as a master transcriptional regulator under multiple stresses and provide a theoretical foundation for molecular breeding strategies to enhance soybean resilience.