Saline–alkali environments restrict soybean production in China. Wild soybean genes can be used to improve the alkaline tolerance of cultivated soybean in molecular breeding. The expansin protein family promotes cell wall expansion. In this study, the relative expression levels of expansin family genes in wild soybean treated with 50 mM NaHCO₃ were measured at 0, 3, 6, and 12 h, and the relative expression of GsEXPA8 was found to be higher at 12 h. Wild soybean was treated with abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA), and jasmonic acid (JA), and GsEXPA8 was found to respond to ABA and IAA signals. Sequence analysis shows that GsEXPA8 has DPBB_EXPA and expansin domains. Subcellular localization analysis shows that GsEXPA8 was localized in the cytoplasm in protoplasts and the cell membrane or wall in tobacco, indicating that it has nuclear membrane localization signals. GsEXPA8 overexpression reduced the malondialdehyde content in transgenic plants treated with NaHCO₃ and increased peroxidase activity before treatment. After the transformation of soybean roots from hair roots, GsEXPA8 was found to be expressed in the outer root cells and promote the development of thicker, shorter roots, thereby improving the plant’s alkaline tolerance. Stable GsEXPA8 transformation improved saline alkaline tolerance via the regulation of the alkali stress-related genes GmKIN1, GmRD22, GmDnaJA6, GmNFYC1, and GmMYB14. These findings provide support for further research on alkali-tolerance regulation pathways and molecular breeding for alkali tolerance.

Soil alkalinity severely restricts the cultivation of Lupinus angustifolius, a valuable legume. Wild soybean (Glycine soja) is a leguminous plant with extremely strong alkaline resistance (pH 8.5). Transferring the alkali-tolerant genes from wild soybeans into lupinus can effectively enhance the alkali tolerance. In this study, we combined transcriptome profiling and genetic transformation to elucidate the molecular basis of alkaline stress response in lupinus. RNA-seq analysis of root tips under acid (HCl, pH 4.0) and alkali (NaHCO₃, pH 8.5) stress revealed 104,353 annotated unigenes, with differential expression patterns highlighting enrichment in cellular component, binding, and catalytic activity categories. KEGG pathway analysis indicated that early responses involved ribosome-related pathways, while later stages activated plant hormone signaling and MAPK pathways. Notably, no homeodomain-leucine zipper (HD-Zip) family genes were identified in the lupinus genome. Therefore, we transferred GsHZ4, an alkali-resistant HD-Zip transcription factor from wild soybean into lupinus hairy roots via Agrobacterium rhizogenes-mediated transformation. Overexpression of GsHZ4 significantly enhanced antioxidant enzyme activities (CAT, POD, and SOD) and reduced malondialdehyde content under NaHCO₃ stress. Furthermore, the promoter of GsHZ4 expression was strongly induced by indole-3-acetic acid (IAA). Key alkali-responsive genes (LaKIN, LaMYB34, LaDnaJ1, LaDnaJ20, LaNAC22, and LaNAC35) were upregulated in transgenic lines, suggesting that GsHZ4 integrates into the endogenous stress-regulation network. Our findings demonstrate that heterologous expression of GsHZ4 can enhance alkaline tolerance of lupinus, providing a novel strategy for breeding stress-resistant varieties and expanding lupinus cultivation in saline–alkali soils.

Soil alkalinity and heavy metal toxicity are major abiotic stresses 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 revealed Gshdz4 as a master transcriptional regulator under multiple stresses and provide a theoretical foundation for molecular breeding strategies to enhance soybean resilience.

Lupinus angustifolius is an important leguminous ornamental species, but its productivity is often compromised by alkaline soil stress. GsEXPA8, an expansin gene identified in wild soybean (Glycine soja), has been implicated in alkali stress tolerance. In this study, we examined how heterologous expression of GsEXPA8 in lupinus affects its biochemical, molecular, and rhizospheric responses to alkali stress. Under NaHCO₃-induced alkaline conditions, transgenic lines overexpressing GsEXPA8 displayed improved leaf vigor, greater root biomass and length, elevated activities of antioxidant enzymes (CAT and POD), increased proline accumulation, and reduced malondialdehyde levels compared to the wild type. Expression analysis revealed time-dependent up-regulation of several alkali-responsive genes (LaSOS1, LaNCED3, LaMYB39, LaNAC56, LaNHX6, and LaP5CS). Moreover, the rhizosphere microbial community was significantly restructured, with a marked increase in beneficial microbial taxa such as Pseudomonas and Lysobacter. We also found that the endogenous lupinus homolog LaEXPA8 is alkali-inducible. Overexpression of LaEXPA8 similarly enhanced alkaline tolerance, whereas CRISPR/Cas9 knockout lines showed no clear phenotypic alteration, suggesting potential functional redundancy within the expansin family. Notably, LaEXPA8 and GsEXPA8 differed in their temporal regulation of downstream genes, indicating both conserved and distinct regulatory roles. Our results demonstrate that GsEXPA8 improves alkali tolerance in lupinus through integrated mechanisms: promoting root growth, enhancing antioxidant and osmotic adjustment capacity, dynamically modulating stress-related gene expression, and enriching beneficial rhizosphere microbiota. This work provides the critical report of modifying alkali tolerance by manipulating an expansin gene alongside the associated rhizosphere microbiome, offering a combined strategy for breeding stress-resistant ornamentals.