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.

Lupinus angustifolius is an important ornamental plant; however, its poor tolerance to alkaline soils limits its cultivation and production. Based on the alkaline-tolerance-related Gshdz4-GsNAC019-GsEXPA8 regulatory module previously screened and identified in soybean, we used Agrobacterium rhizogenes-mediated transformation to overexpress in lupine roots the combinations Gshdz4-GsNAC019-GsEXPA8 (HNE), Gshdz4-GsNAC019 (HN), and GsNAC019-GsEXPA8 (NE) to investigate their effects on root development and alkaline tolerance. RT-PCR confirmed the successful generation of all overexpression lines. Under 100 mM NaHCO₃ stress, all overexpression lines exhibited less wilting and longer survival than the wild type (WT), with the HNE line showing the best phenotype. Physiological measurements showed that the overexpression lines had significantly higher proline content, antioxidant enzyme (SOD, CAT, POD) activities, and root activity, as well as lower malondialdehyde content. DAB and NBT staining of leaves indicated reduced accumulation of O₂⁻ and H₂O₂, suggesting enhanced antioxidant capacity. Root architecture analysis revealed that root length, surface area, volume, tip number, and fork number were significantly increased in HNE, HN, and NE lines compared with WT, with the most pronounced effect observed in HNE. Bioinformatics analysis and qPCR confirmed that Gshdz4 binds to and activates the promoter of the endogenous LaNAC072 (the lupine homolog of GsNAC019), while GsNAC019 binds to and activates the promoter of the endogenous LaEXPA8 (the lupine homolog of GsEXPA8), thereby triggering the endogenous alkaline tolerance regulatory mechanism. Furthermore, the overexpression combinations significantly upregulated the expression of alkaline stress-responsive genes, including LaSOS1, LaNHX6, LaP5CS, LaMYB39, and LaDnaJ1. This study provides theoretical support for molecular breeding of alkaline-tolerant lupine.

Alkaline soil limits soybean production. This study elucidates the molecular mechanism by which the Gshdz4-GmU2AFb-GmCML27 module regulates soybean alkaline tolerance. Transcriptome analysis, yeast one-hybrid and dual-luciferase assays confirm that the HD-Zip transcription factor Gshdz4 binds to the CAATAA motif in the GmU2AFb promoter and activates its transcription. Subcellular localization verifies the nuclear distribution of GmU2AFb. Overexpression of GmU2AFb improves alkaline tolerance by increasing antioxidant enzyme activities and proline levels, reducing MDA accumulation, facilitating root development, and upregulating alkaline-responsive genes, including GmSOD1, while gene knockout impairs stress resistance. Combined Y2H, BiFC and LCI assays validate the nuclear protein interaction between GmU2AFb and the calcium-binding protein GmCML27, and overexpression of GmCML27 also enhances antioxidant capacity and root growth in soybean. Co-overexpression of the two genes generates obvious synergistic effects; compared with single-gene overexpression lines, co-transgenic plants possess higher antioxidant levels and elevated transcription of downstream alkaline-tolerant genes, accompanied by alleviated growth inhibition under alkaline stress. In summary, Gshdz4 transcriptionally activates GmU2AFb, and the interaction between GmU2AFb and GmCML27 connects RNA splicing with calcium signaling pathways to synergistically trigger downstream defense responses and promote root development, thereby enhancing soybean alkaline tolerance at multiple layers. This work provides candidate genes for molecular breeding of alkali-resistant soybean.

Soil salinization severely constrains plant growth and agricultural productivity. Identifying key genes conferring alkali tolerance and elucidating their regulatory mechanisms is of great significance for breeding salt–alkali-tolerant leguminous crops. In this study, we investigated the soybean genes GmCML27 and its potential interacting partner GmU2AFb, and explored their functions in response to alkali stress in narrow-leaf lupine (Lupinus angustifolius) through bioinformatics analyses. Protein interaction prediction and experimental validation further supported a potential association between GmCML27 and GmU2AFb. Using an Agrobacterium rhizogenes (A. rhizogenes)-mediated hairy root transformation system, composite plants carrying empty vector (CK), GmCML27-overexpression construct (GC), GmU2AFb-overexpression construct (GU), or both GmCML27 and GmU2AFb overexpression constructs (GCU) were generated and evaluated under NaHCO₃-induced alkali stress. The results showed that all transformed composite plants exhibited enhanced alkali tolerance, with the GCU plants displaying the strongest phenotype. Compared with CK plants, transformed composite plants maintained higher catalase (CAT) activity, proline (Pro) content, and root vitality, while accumulating lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and retaining more intact root architecture. Furthermore, the expression levels of alkali tolerance-related genes, including LaMYB39, LaSOS1, LaNHX6, LaKIN, LaDnaJ1, and LaP5CS, were significantly upregulated in transgenic lines. Collectively, our findings demonstrate that both GmCML27 and GmU2AFb positively regulate alkali tolerance in lupine, and their co-expression confers enhanced alkali tolerance compared with single-gene overexpression. These two genes may jointly contribute to alkali stress adaptation through regulation of antioxidant capacity and stress-responsive pathways. This study highlights the potential roles of GmCML27 and GmU2AFb in lupine responses to alkali stress and provides candidate gene resources for future improvement of lupine alkali tolerance.

Splicing factors, as core determinants of splice-site selection and dynamic spliceosome assembly, play pivotal roles in stress responses. This review systematically categorizes splicing factors involved in plant abiotic stress responses according to their functions as major spliceosomal components, dividing them into small nuclear ribonucleoproteins (snRNPs) and associated components, spliceosome assembly and disassembly factors, splicing regulatory factors, and proteins related to non-canonical RNA splicing. On this basis, we summarize their regulatory mechanisms of these factors under salt, drought, abscisic acid (ABA) signaling, temperature, and oxidative stresses. Through analyses across multiple species—including Arabidopsis thaliana, rice, maize, soybean, and wheat—we reveal both the evolutionary conservation and species-specific divergence of splicing-factor-mediated regulation. Currently, a large amount of research is still mainly at the transcriptome analysis or single phenotype validation stages, lacking in-depth analysis of direct targets, splicing isomer functions, and molecular mechanisms. Furthermore, current research is heavily concentrated on Arabidopsis, with relatively insufficient functional validation and breeding applications in crops such as maize and wheat. Despite substantial progress, several bottlenecks remain for translational applications in breeding, such as functional redundancy among splicing factor family members, growth penalties associated with overexpression, and tissue-specific and developmental-stage-dependent effects. To address these challenges, we discuss promising strategies, including CRISPR/Cas9-mediated splice-site editing, the use of inducible or tissue-specific promoters, and targeted modulation of upstream kinases, although extensive field trials and rigorous evaluations remain necessary. Collectively, this review provides a theoretical framework for understanding the roles of splicing factors in RNA-level regulation of plant stress adaptation and highlights their potential for breeding improvement.