GsEXPA8 Improves Alkaline Tolerance in Lupinus angustifolius by Modulating Root Architecture, Stress-Responsive Gene Expression, and Rhizosphere Microbiome

Abstract

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.

1. Introduction

Soil salinization–alkalization is one of the primary abiotic stresses constraining global agricultural sustainability [1]. Alkaline stress, characterized by high pH, elevated Na⁺ levels, and high concentrations of alkaline salts such as NaHCO₃, leads to reduced nutrient availability, ionic imbalance, and physiological drought, severely impairing root development and plant growth [2]. Root systems are particularly vulnerable under alkaline conditions, making root-related adaptive mechanisms central to plant tolerance.

Narrow-leaf lupinus (Lupinus angustifolius) is a leguminous plant valued as forage, green manure, and an ornamental. Its root system possesses biological nitrogen-fixation capacity, playing a significant role in improving soil structure and reducing dependence on nitrogen fertilizers. For instance, yellow lupinus (Lupinus luteus L.), with its high protein content, holds notable economic and ecological value [3]. It fixes atmospheric nitrogen through symbiosis with rhizobia, reducing fertilizer inputs and serving as a protein-rich feed source [3]. However, the growth and yield of lupinus are also significantly inhibited by alkaline soils. In response to alkaline stress, plants adapt not only through their intrinsic physiological and molecular regulatory networks, but also by actively modifying the rhizosphere microenvironment through changes in root morphology and exudate composition [4]. Current research on lupinus has primarily focused on its nutritional properties [5], mechanisms of organic acid secretion [6], drought stress [3], and heat stress [7], while studies on the alkali-tolerance mechanisms of narrow-leaf lupinus remain insufficient.

Expansins constitute a multigene family, encompassing α-expansins (EXPA), β-expansins (EXPB), expansin-like α, and expansin-like β subfamilies, which play crucial roles in plant growth and development, primarily through promoting root system expansion [8]. Recent studies indicate that cell wall remodeling-associated proteins are key players in plant responses to environmental stresses. For instance, overexpression of ZmEXPA3 in maize enhances growth under both normal and saline conditions via modifications to cell wall architecture [9]. As cell wall-loosening proteins, expansins are instrumental not only in regulating growth and development but also in mediating plant adaptation to diverse environmental challenges. The structural features, regulatory mechanisms, and functional roles of expansins exhibit considerable diversity across plant species. They modulate plant growth, organogenesis, and adaptation to stresses such as drought [10], heat [11], and plant diseases [12]. Wild soybean (Glycine soja) represents a valuable genetic reservoir harboring numerous genes conferring tolerance to saline-alkaline stress. GsEXPA8, a member of the expansin family in wild soybean belonging to the EXPA subfamily, has been demonstrated to enhance alkaline stress tolerance and participate in regulating root architecture in soybean seedlings [13]. However, the function of LaEXPA8, the homologous gene of GsEXPA8 in narrow-leaf lupinus (Lupinus angustifolius), remains unexplored.

Plant adaptation to alkaline stress involves not only intrinsic physiological and molecular regulation but also active modification of the rhizosphere environment through changes in root architecture and exudate composition. The rhizosphere microbial community, often regarded as the plant’s ‘second genome’, plays a crucial role in assisting nutrient acquisition, enhancing disease resistance, and improving abiotic stress tolerance [14].The introduction of exogenous genes into plants can alter soil microbial composition, though the underlying mechanisms remain incompletely understood [15,16,17]. 16S rRNA gene sequencing, a high-throughput metagenomic approach, enables the analysis of microbial community diversity, taxonomic composition, and ecological interactions in the rhizosphere soil of herbaceous plants [18], and further facilitates the isolation of beneficial strains [19]. Under stress conditions, plants often modulate root exudation to recruit beneficial microorganisms that aid in stress adaptation. For example, lotus expansin EXPA1 is recruited during intracellular and intercellular colonization by rhizobia [20]. Most leguminous plants establish symbiotic relationships with rhizobia, a group of nitrogen-fixing bacteria. Expansins, which promote cell enlargement by loosening the cell wall, hold considerable breeding potential for improving biological nitrogen fixation in legumes. GmEXPA11, regulated by GmPTF1, promotes soybean nodule enlargement and nitrogen fixation via interaction with GmNOD20 [21]. Therefore, elucidating how overexpression of the key gene GsEXPA8 drives the reassembly of the rhizosphere microbial community through modified root phenotypes and physiological metabolism in other legumes is essential for a comprehensive understanding of the mechanisms underlying plant alkali tolerance, stress-induced changes in root development, and the regulatory influence of transgenic root systems on the rhizosphere microbiome.

Based on previous findings regarding the potential role of GsEXPA8 in plant alkali tolerance [13], we propose that GsEXPA8 overexpression enhances adaptation of narrow-leaf lupinus to alkaline stress by promoting root development and physiological resilience, potentially accompanied by changes in rhizosphere microbial community structure. To test this hypothesis, we generated transgenic lupinus lines overexpressing GsEXPA8 and systematically assessed their phenotypic, biochemical analysis, and molecular responses under alkaline conditions. Furthermore, using 16S rDNA high-throughput sequencing, we compared the compositional changes in the rhizosphere microbial communities among transgenic soybean, transgenic lupinus, and their corresponding wild-type plants. From a novel ‘plant–microbe’ interaction perspective, this study aims to elucidate the integrated mechanism through which GsEXPA8 mediates rhizosphere microbial community dynamics to regulate alkali tolerance in lupinus. Additionally, through overexpression, CRISPR/Cas9-based knockout, and hairy root induction assays, we functionally characterized the homologous gene LaEXPA8 in its native host. These findings are expected to provide a new theoretical foundation and genetic resources for synergistically improving crop stress resistance through genetic modification and rhizosphere microbiome engineering.

2. Results

2.1. GsEXPA8 Positively Regulates Alkaline Tolerance in Lupinus

Previous studies have shown that overexpression of alkali-tolerance genes from wild soybean, such as GsHZ4, in the root system of narrow-leaf lupinus (Lupinus angustifolius) can enhance plant alkaline tolerance [4]. Heterologous expression of the wild soybean GsEXPA8 gene in soybean also improves alkaline stress resistance. To investigate the function of GsEXPA8 in the ornamental plant lupinus, this study generated transgenic lupinus lines overexpressing GsEXPA8 in roots. Positive transgenic plants were identified by RT-PCR (Figure S1). RT-PCR analysis demonstrated differential expression levels among the independent OE lines. OE1 showed the lowest expression level and correspondingly exhibited the smallest plant size under control conditions, suggesting a potential positive association between transgene expression and basal growth performance. Both wild-type (WT) and overexpression (OE) lines were treated with 50 mM NaHCO₃ for 12 days, and phenotypic changes were analyzed. Under alkaline stress, WT plants exhibited pronounced leaf wilting, whereas OE lines retained visibly greener leaves (Figure 1A). The fresh weight and root length of OE lines were significantly greater than those of WT plants after treatment (Figure 1B,C). Biochemical analysis revealed that CAT activity was significantly higher in OE plants compared to WT following stress treatment (Figure 1D). In contrast, MDA content was markedly lower in OE lines (Figure 1E). POD activity was significantly elevated in OE plants under both control and stress conditions relative to WT (Figure 1F). Pro content was also higher in OE lines after alkali treatment (Figure 1G). These results indicate that GsEXPA8 overexpression enhances the core antioxidant defense system in roots under stress, effectively protecting cellular membrane integrity and activating osmotic adjustment. Phenotypic observation under non-stress conditions indicated that the shoot parts of OE plants were substantially larger than those of WT, suggesting a growth-promoting effect of the transgene. Root architecture was further analyzed using a root scanning system in untreated plants (Figure 2A). OE lines developed more robust and extensive root systems compared to WT. Quantitative analysis showed that transgenic lines exhibited significantly greater root surface area, total root length, nodule number, root tip count, and node number (Figure 2B–F). Overall, transgenic plants displayed taller and more vigorous growth. These findings demonstrate that GsEXPA8 overexpression promotes root growth, branching, and the formation of root tip tissues in lupinus, which may enhance nutrient and water acquisition. These results are consistent with the effects of GsEXPA8 overexpression previously reported in soybean.

2.2. GsEXPA8 Upregulates the Expression of Alkaline Stress-Related Genes

To investigate the regulatory network through which GsEXPA8 confers alkali tolerance in lupinus, WT and root-specific GsEXPA8-overexpressing (OE) plants were treated with 100 mM NaHCO₃. The relative expression levels of key alkali-responsive genes—LaSOS1, LaNAC56, LaNHX6, LaP5CS, LaMYB39, and LaNCED3—were analyzed at 0, 3, 6, and 12 h after treatment (Figure 3A–F). Compared with WT, the expression of LaSOS1, LaNCED3, LaNAC56, and LaMYB39 was significantly upregulated in OE plants at 12 h post-treatment. LaNHX6 showed marked induction at earlier time points (3 h and 6 h). In contrast, LaP5CS transcript levels were already higher in OE plants before stress and gradually declined with prolonged treatment. These results indicate that GsEXPA8 enhances alkali tolerance by differentially regulating a suite of stress-related genes at distinct stages of the stress response.

2.3. Heterologous Expression of GsEXPA8 Enriches Distinct Microbial Communities in Different Plant Hosts

Following expression of GsEXPA8 in soybean roots, the protein was primarily localized in the intercellular space between the cell membrane and wall within the outermost and sub-outermost layers of root tips, suggesting a potential role in modulating root–microbe interactions [13]. To investigate the impact of GsEXPA8 transformation on the plant-associated microbial environment, we analyzed the rhizosphere soil microbiota of both transgenic soybean and lupinus. In soybean, 1756 Amplicon Sequence Variants (ASVs) were shared between the control and overexpression groups, while 1837 ASVs were unique to the control and 1728 ASVs were unique to the overexpression group (Figure 4A). In lupinus, 1993 ASVs were common to both groups, with 3111 ASVs unique to the wild-type (WT) and 2039 ASVs unique to the transgenic lines (Figure 4B). Venn diagram analysis further revealed that 118 microbial genotypes increased, and 94 decreased, in the rhizosphere of both species. Conversely, 107 genotypes increased in soybean but decreased in lupinus, while 151 increased in lupinus but decreased in soybean (Figure 4C). Heatmap analysis indicated a marked enrichment of beneficial rhizobacteria such as Pseudomonas (associated with growth promotion and disease suppression) and Pseudolabrys (with potential for environmental remediation). In contrast, taxa including the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group (closely related to rhizobia) and Bdellovibrio (a predator of various pathogens) were reduced (Figure 4D,E). This shift may reflect functional compensation by GsEXPA8 for certain rhizobial activities. In transgenic lupinus, we observed an increase in genera such as Lysobacter (a prolific producer of antimicrobial compounds and a resource for biopesticides), Phenylobacterium (capable of degrading recalcitrant aromatic pollutants), and Ramlibacter (involved in mineralization of organic phosphorus). Conversely, genera including Pseudoxanthomonas (a degrader of persistent pollutants), Nocardioides (a decomposer of chitin and organic matter), and the potential phytopathogen Cellulosimicrobium showed decreased abundance. Comparative analysis highlighted three genera—Sphingomonas (an efficient degrader and potential plant probiotic), Pseudolabrys (a common soil inhabitant with unclear plant effects), and Pseudarthrobacter (some strains of which promote plant growth or degrade pollutants)—that exhibited consistent directional changes in both soybean and lupinus rhizospheres (marked within red boxes in Figure 4D,E). Sphingomonas and Pseudolabrys increased in both systems, whereas Pseudarthrobacter decreased. Collectively, these results demonstrate that GsEXPA8 expression reshapes the rhizosphere microbiome, with both common and species-specific effects across different leguminous hosts. The potential benefits and risks associated with such transgenic-driven microbial shifts warrant further evaluation.

2.4. The Exogenous GsEXPA8 and the Endogenous LaEXPA8 Share High Homology

While investigating the enhanced alkaline tolerance conferred by the exogenous GsEXPA8, we also examined its endogenous homolog in narrow-leaf lupinus, LaEXPA8. Sequence analysis revealed that both proteins possess an identical DPBB_1 structural domain (Figure S2), suggesting potential functional conservation. Analysis of transcriptomic data under NaHCO₃ treatment (pH 8.5) showed that, among the expansin gene family, LaEXPA8 expression was significantly upregulated at 0, 3, and 6 h post-treatment, with a particularly pronounced increase observed at the 6-h time point (Figure 5A). This induction was confirmed by examining the FPKM values of LaEXPA8 across different time points. Subsequent qRT-PCR validation using roots treated with 0 or 100 mM NaHCO₃ for 6 h demonstrated a significant increase in LaEXPA8 transcript levels in wild-type plants (Figure 5C). In contrast, the expression of other expansin family members, including LaEXPA4 (Figure 5D), LaEXPA13 (Figure 5E), LaEXPA16 (Figure 5F), and LaEXPA18 (Figure 5G), was significantly downregulated under the same conditions. LaEXPB3 expression also showed a decreasing, though not statistically significant, trend (Figure 5H). These results indicate that not all expansin family members are upregulated under alkaline stress. The specific and significant induction of LaEXPA8 suggests its potential involvement in the alkaline stress response mechanism in lupinus.

2.5. Functional Validation of LaEXPA8 in Positively Regulating Alkaline Tolerance

To determine whether the endogenous LaEXPA8 gene enhances alkaline tolerance in lupinus, overexpression and CRISPR-Cas9 knockout constructs were generated (Figure S3). Wild-type (WT), overexpression (OE), and CRISPR mutant (CR) plants were treated with 100 mM NaHCO₃. Under non-stress conditions, OE plants exhibited larger and taller growth phenotypes compared to WT, while WT and CR lines showed no significant difference. After three days of treatment, CR plants began to show wilting symptoms. By 6 days, WT plants started to wilt, whereas CR plants were completely necrotic (Figure 6A; a top-view comparison is shown in Figure 6B). Biochemical analysis assays under alkaline stress revealed that, relative to WT, OE plants displayed significantly higher activities of POD and CAT, increased proline (Pro) content (Figure 6C–E), and a marked reduction in malondialdehyde (MDA) levels (Figure 6F). In contrast, CR plants showed no significant differences from WT in phenotypic appearance or any measured biochemical parameters under either control or stress conditions. These results indicate that LaEXPA8 overexpression enhances alkaline tolerance, while its knockout does not confer a negative phenotype, suggesting possible functional redundancy within the expansin gene family in lupinus. Root architecture analysis further demonstrated that OE lines developed more robust root systems (Figure 7A). Quantitative parameters, including total root length (Figure 7B), number of healthy roots (Figure 7C), total surface area (Figure 7D), lateral root count (Figure 7E), surface area (Figure 7F), and total root volume (Figure 7G), were all significantly greater in OE plants than in WT, whereas CR and WT lines did not differ significantly. Histochemical staining with DAB (Figure 8A) and NBT (Figure 8B) on leaves from treated and untreated plants showed that CR leaves consistently accumulated more reactive oxygen species (ROS), indicative of greater oxidative damage, under both conditions. Under non-stress conditions, WT and OE leaves showed minimal staining. After alkaline treatment, OE leaves exhibited only slight staining, whereas WT leaves displayed more severe ROS accumulation. In summary, similar to GsEXPA8 overexpression, ectopic expression of LaEXPA8 promotes root growth, branching, and root tissue proliferation, thereby enhancing alkaline tolerance and shoot vitality in lupinus. However, knockout of LaEXPA8 did not impair root development or alkaline stress tolerance, likely due to genetic redundancy within the expansin family. Since overexpression of both GsEXPA8 and LaEXPA8 improved plant biomass, we hypothesize that these genes may also enhance resistance to other abiotic stresses and ornamental traits, although further validation is required.

2.6. LaEXPA8 Upregulates the Expression of Alkaline Stress-Responsive Genes

To validate the relative expression levels of key alkaline stress-related genes and elucidate the regulatory mechanism of LaEXPA8, wild-type (WT) and overexpression (OE) plants were treated with 100 mM NaHCO₃. The transcript levels of LaSOS1 (Figure 8C), LaP5CS (Figure 8D), and LaNHX6 (Figure 8E) were analyzed at 0, 3, 6, and 12 h post-treatment. The results showed that in OE plants, LaSOS1 was significantly upregulated at 3 h and 6 h, LaP5CS was markedly induced at 6 h and 12 h, and LaNHX6 expression was significantly elevated at 3 h. These findings indicate that, similar to GsEXPA8, LaEXPA8 enhances plant alkaline tolerance by upregulating stress-responsive genes. However, the timing of induction differed between the two genes: LaSOS1 upregulation occurred earlier, while LaP5CS induction was delayed compared to the pattern observed with GsEXPA8. This suggests that LaEXPA8 and GsEXPA8 may function within distinct regulatory networks despite their conserved role in promoting stress tolerance.

3. Discussion

3.1. GsEXPA8 Enhances Alkaline Tolerance in Lupinus Through Coordinated Biochemical and Molecular Mechanisms

As previously established, the root system is a multifunctional organ critical for water and nutrient uptake, metabolite storage, anchorage, mechanical support, and interaction with the soil environment [8]. This study demonstrates that heterologous overexpression of the wild soybean-derived GsEXPA8 gene in lupinus systemically improves plant tolerance to alkaline stress. The enhanced tolerance was first evident at the phenotypic level. The observed variation in plant size among independent OE lines under control conditions may be associated with differences in transgene expression levels. The positive trend between expression level and plant size suggests a dosage-dependent effect of GsEXPA8 on plant growth. However, further quantitative correlation analysis would be required to confirm this relationship. Under alkaline stress, transgenic overexpression (OE) plants maintained higher biomass and developed more extensive root architecture compared to wild-type (WT) plants. This improvement directly enhances the capacity for water and nutrient acquisition, forming the foundational basis for stress resilience. At the biochemical level, OE plants exhibited a more active antioxidant defense system, characterized by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content, effectively mitigating oxidative damage. Concurrently, elevated proline (Pro) content indicated enhanced osmotic adjustment, alleviating osmotic stress induced by alkalinity. Further analysis showed that the endogenous homolog LaEXPA8 in Lupinus angustifolius is induced under alkaline stress and may participate in the stress response. However, phenotypic comparisons between wild-type and GsEXPA8-overexpressing lines under alkaline treatment indicated that the enhanced tolerance observed in the OE plants is primarily associated with ectopic expression of GsEXPA8. Meanwhile, the endogenous LaEXPA8 gene is induced under alkaline stress, and its potential contribution to stress tolerance cannot be completely excluded. Molecular investigations revealed that GsEXPA8 overexpression positively regulated a suite of key alkaline stress-responsive genes in lupinus. For instance: LaNHX6, a homolog of GmNHX6, is involved in cellular Na⁺/K⁺ and pH homeostasis, crucial for salt-alkali tolerance. Heterologous expression of GmNHX6 in Arabidopsis was shown to enhance alkaline tolerance by maintaining high K⁺ levels and a low Na⁺/K⁺ ratio [22]. LaP5CS, homologous to GmP5CS, encodes a bifunctional enzyme catalyzing the rate-limiting step in proline biosynthesis, linking its upregulation to the observed osmotic adjustment [23,24]. LaNCED3 (a homolog of AtNCED3) is a key enzyme in abscisic acid (ABA) biosynthesis. Studies indicate that NCED3 can be strongly induced by NaCl via an ABA-independent pathway, highlighting its role in stress signaling [25,26]. LaSOS1 encodes a Na⁺/H⁺ antiporter that contributes to cellular ion homeostasis by facilitating Na⁺ efflux, which is vital for salt tolerance [27]. LaNAC56 and LaMYB39 are transcription factors implicated in stress response regulation. NAC56 in rapeseed acts as a transcriptional activator involved in regulating reactive oxygen species (ROS) accumulation and cell death under stress [28,29], while MYB39 in maize enhances seedling drought tolerance [30]. Their upregulation suggests involvement in transcriptional reprogramming. These genes participate in critical pathways governing ion homeostasis, transcriptional regulation, and hormone synthesis. Therefore, GsEXPA8 does not function solely through its canonical cell wall-loosening activity. Instead, it acts as a key regulatory node, coordinating a multi-layered defense system. This system integrates the promotion of root growth, enhancement of biochemical resilience, and activation of core stress-signaling networks, collectively conferring strong alkaline tolerance to lupinus.

3.2. Heterologous Expression of GsEXPA8 Specifically Reshapes the Rhizosphere Microbial Community

This study further examined the potential impact of GsEXPA8 expression on the rhizosphere microbiome of host plants. Our analysis revealed significant, yet host-specific, restructuring of the rhizobacterial community in both transgenic soybean and lupinus hairy root systems. A common trend was the enrichment of certain plant-beneficial genera, such as Sphingomonas [31,32] and Pseudolabrys [33], in the rhizosphere of both species. However, distinct differences were observed: in soybean, a relative decrease occurred in rhizobial taxa closely associated with symbiotic nitrogen fixation, whereas in lupinus, increases were noted in genera involved in pollutant degradation (e.g., Phenylobacterium [34]) and nutrient mobilization (e.g., Ramlibacter [35,36]), alongside a reduction in potential pathogenic bacteria. These host-dependent shifts likely stem from inherent differences in root exudate profiles between plant species, compounded by specific alterations in exudate composition induced by the transgene in each host. For instance, Sphingomonas species possess diverse functionalities, including environmental remediation and production of high-value phytohormones, and have been implicated in the degradation of organometallic compounds [31] and in mitigating pesticide stress by colonizing roots and degrading chlorpyrifos while releasing beneficial metabolites [37]. The enrichment of Ramlibacter, which includes alkali-tolerant species such as R. alkalitolerans isolated from ginseng soil [38], may be particularly relevant under alkaline conditions. These findings suggest that the enhanced alkaline tolerance mediated by GsEXPA8 is not solely attributable to intrinsic biochemical adjustments within the plant. It may also involve a ‘plant–microbe’ interaction mechanism, whereby the gene facilitates the recruitment and enrichment of a beneficial rhizosphere microbiome that indirectly bolsters stress adaptation. Plant growth-promoting rhizobacteria (PGPR) can colonize the rhizosphere and enhance stress tolerance through various mechanisms, such as producing exopolysaccharides (EPS), phytohormones, ACC deaminase, and volatile compounds, inducing the accumulation of osmolytes and antioxidants, modulating stress-responsive gene expression, and altering root morphology [39]. Examples include Bacillus megaterium strains improving drought tolerance in rice [40,41] and various Bacillus species alleviating salt stress damage in maize [42,43,44,45]. Similarly, specific Pseudomonas strains have been shown to modulate mercury uptake in lupinus, underscoring the potential of soil–plant–microbe synergy. Collectively, our results indicate that GsEXPA8-mediated rhizosphere microbiome remodeling represents a contributory, host-specific layer to the overall alkaline tolerance phenotype, warranting deeper investigation into this synergistic tripartite interaction.

3.3. Structural Homology Between GsEXPA8 and LaEXPA8 Suggests Functional Conservation

Sequence analysis revealed a high degree of homology between the exogenous GsEXPA8 and the endogenous LaEXPA8, with both genes containing the canonical DPBB_1 domain characteristic of functional expansins. This structural conservation provides a critical molecular basis for the successful functionality of GsEXPA8 in lupinus, indicating that these homologous genes, despite their different species origins, likely encode proteins with similar biochemical activities, namely, cell wall loosening via disruption of hydrogen bonds between wall polysaccharides to promote cell elongation. Both GsEXPA8 and the endogenous LaEXPA8 can increase plant root length, which may be related to the enhanced activity of cell wall modifying proteins (such as expansin), and this needs to be directly verified by subsequent cell microscopy studies. Such functional conservation explains how an expansin gene from soybean can be effectively integrated into the growth and stress-response programs of lupinus.

This aligns with reports of expansins playing conserved roles across diverse species: for instance, EXPB2 is associated with cell wall loosening and fruit softening in strawberry [46]; certain expansins positively regulate drought tolerance in wheat [47]; and OsEXPB2, a root-predominant gene in rice, plays a key role in root hair formation and holds potential for transgenic root breeding to enhance abiotic stress tolerance [48]. Similarly, transcription factors such as NAC2 have been shown to improve growth and drought/heat tolerance in transgenic cowpea [49], and have been linked to freezing tolerance in Aegilops tauschii [50] and enhanced stress tolerance in Picea wilsonii [51]. Furthermore, this homology implies that the core cell wall-modifying function of expansins and their role in stress responses may be evolutionarily conserved across leguminous and broader plant lineages. However, comparative expression analysis revealed distinct kinetic profiles in the regulation of downstream stress-responsive genes. In LaEXPA8-overexpressing plants under alkaline stress, LaSOS1 was significantly upregulated at 3 and 6 h, whereas in GsEXPA8-overexpressing plants, its upregulation was pronounced at 12 h. LaNHX6 showed marked induction at 3 h in LaEXPA8 OE lines but exhibited a sustained increase from 3 to 6 h in GsEXPA8 OE lines. For LaP5CS, transcript levels peaked at 6 and 12 h in LaEXPA8 OE plants, while in GsEXPA8 OE plants, they were already elevated at 0 h and declined significantly by 6 h. These temporally distinct expression patterns suggest that while GsEXPA8 and LaEXPA8 share conserved biochemical functions, they may operate within nuanced, gene-specific regulatory networks, fine-tuning the stress response dynamics in lupinus.

3.4. LaEXPA8 Is a Potential Endogenous Regulator of Alkaline Tolerance

A notable finding of this study is the specific induction of the endogenous LaEXPA8 gene under alkaline stress, contrasting with the suppressed expression of other members within the same expansin family. This distinct expression pattern strongly suggests that LaEXPA8 is not merely a conventional expansin but may serve as a key endogenous regulator in the lupinus alkali-stress response. Given that the heterologous expression of GsEXPA8 significantly enhanced alkaline tolerance, it is plausible to speculate that the upregulation of endogenous LaEXPA8 represents an adaptive strategy employed by lupinus to counteract alkaline stress. Its function likely parallels that of GsEXPA8, potentially enhancing tolerance by promoting adaptive root growth under stress and modulating downstream stress-responsive genes. This insight shifts the perspective from the application of exogenous genes towards understanding intrinsic plant mechanisms. Direct functional validation via CRISPR/Cas9-mediated knockout of LaEXPA8 resulted in a significant reduction in root size, underscoring its essential role. This finding is crucial for elucidating the natural genetic basis of alkali tolerance in lupinus and may provide a novel target for precision molecular breeding utilizing the plant’s own homologous genes. Furthermore, LaEXPA8 may be functionally linked to critical regulatory pathways, such as ion homeostasis and abscisic acid biosynthesis.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The experiment was conducted at the Horticultural Experiment Center of Northeast Agricultural University. Lupinus angustifolius seeds used in the research were sourced from the commercial brand ‘Hua You Xiu’ [4]. The lupinus was cultured in pots containing a 1:1 mixture of nutrient soil and vermiculite. Plants were maintained under controlled conditions at 22–25 °C with a 16-h light/8-h dark photoperiod until the third pair of palmate compound leaves were fully developed. The plant was carefully removed from the pot, and the roots were gently rinsed with water to remove impurities. Subsequently, sterile surgical blades were used to separate the plant into aboveground and belowground parts. The fresh weight was measured using an electronic balance, and the length of the main root was determined by direct measurement. To evaluate the phenotypic differences under alkaline stress, a pot irrigation method was employed, and wild-type and overexpressing plants were treated with 0 mM (control) and 100 mM NaHCO₃ solutions, respectively.

Cultivated soybean were planted in the Experimental Practice and Demonstration Center of Northeast Agricultural University, Harbin, China.

4.2. Vector Construction

For generating overexpression lines, the full-length cDNA of GsEXPA8 was inserted into the BamH I and EcoR I sites of the pBI121 vector, while the full-length cDNA of LaEXPA8 was cloned into the Nco I site of pCAMBIA1302. For generating knockout lines, a CRISPR/Cas9 system was employed using the pHK2-Cas9-U6 vector (Biorun, Wuhan, China), modified from pCAMBIA1300 for dicot gene editing. A dual-target strategy was used for guide RNA design. The recombinant constructs were amplified in Escherichia coli DH5α cells. Purified plasmids were subsequently introduced into Agrobacterium rhizogenes strain K599 (Weidi, Shanghai, China) via the freeze–thaw method. Transgenic lupinus plants (overexpression and knockout lines) were generated through root transformation using A. rhizogenes infection, as previously described. WT soybean seeds were cultured until germination, portions of the hypocotyls were cut off, the seed coats were peeled, and the two cotyledons were separated and clipped with the nodes. Two true leaves were removed, and a knife tip was used to cut to growth point 1–2 to create explants. The recombinant GsEXP8 plasmid was introduced into Agrobacterium K599, followed by propagation. [52,53].

4.3. RNA Extraction and Reverse Transcription

Twenty-five-day-old wild-type (WT), overexpression (OE), and CRISPR knockout (CR) lupinus plants were treated with 100 mM NaHCO₃. Root samples were collected at 0, 3, 6, and 12 h post-treatment, with three biological replicates per time point. Total RNA was extracted using the TransZol Up Kit (ET111-01-V2; TransGen Biotech, Beijing, China). First-strand cDNA was synthesized from total RNA using the SPARKscript II RT Plus Kit (with gDNA Eraser) (AG0304-B, Sparkjade, Jinan, China). Gene-specific primers were designed for qRT-PCR analysis, which was performed using SYBR Green dye (AH0104-B, Sparkjade, Jinan, China) on a real-time PCR system. The relative expression levels of target genes (LaSOS1, LaNAC56, LaNHX6, LaP5CS, LaMYB39, and LaNCED3) were calculated using the 2^−ΔΔCT method [54], with Ubiquitin serving as the internal reference gene for normalization. All primer sequences are listed in Supplementary Table S1.

4.4. RNA-Seq Analysis

Lupinus plants were grown in Hoagland solution at pH 5.5 as the control group (Group A/a). For treatment groups, plants were exposed to Hoagland solution supplemented with NaHCO₃ (pH 8.5) for 3 h (Group D/d) or 6 h (Group E/e). Root tips were immediately frozen in liquid nitrogen and stored at –80 °C. Each group included three biological replicates, resulting in a total of 15 samples. These samples were subsequently submitted to BGI for transcriptome sequencing. The raw sequencing data have been deposited in the NCBI SRA database under accession number SUB14514279.

4.5. Root Scanning Assay

Wild-type (WT), overexpression (OE), and CRISPR knockout (CR) lupinus plants with uniform growth were selected, with 15 biological replicates per genotype. Root systems were carefully washed clean and scanned using a root scanner (LA-S; Wanshen, Hangzhou, China) according to the manufacturer’s instructions. Key morphological parameters were measured from the scanned images [55].

4.6. Microbiome Analysis

Rhizosphere soil samples (5 g each) were collected from 25-day-old untreated transgenic and non-transgenic lupinus and soybean plants, with 6 replicates per group, resulting in a total of 24 samples. Microbial community analysis was performed via 16S rDNA amplicon sequencing to assess composition, diversity, and structure. Sequencing was conducted on the DNBSEQ-G99 platform by LC-Bio Technologies (Hangzhou) Co., Ltd (Hangzhou, China) [56].

4.7. Determination of Biochemical Parameters

Wild-type (WT), overexpression (OE), and CRISPR knockout (CR) lupinus plants were treated with 0, 100 mM NaHCO₃ to assess biochemical responses under alkaline stress. Roots were harvested for analysis, with each sample consisting of a pool of five roots and three biological replicates per line. The following commercial assay kits were used according to the manufacturers’ protocols: peroxidase (POD) activity kit (No. M0105; Mengxi Biomed, Suzhou, China), malondialdehyde (MDA) content kit (No. G0109W; Geruisi Biotechnology, Suzhou, China), catalase (CAT) activity kit (No. G0105W; Geruisi Biotechnology, Suzhou, China), and proline (Pro) content kit (No. G1101W; Geruisi Biotechnology, Suzhou, China).

4.8. Diaminobenzidine (DAB) Staining

DAB staining solution was prepared using DAB powder (Biotopped, Beijing, China). Briefly, 0.1 g of DAB powder was dissolved in a brown bottle containing 45 mL of distilled water. The pH was adjusted to 5.7, and the volume was brought to 50 mL with distilled water. Leaves were vacuum-infiltrated with the DAB solution and incubated for 18 h in the dark. Subsequently, chlorophyll was removed by boiling the samples in absolute ethanol. After complete decolorization, the leaves were mounted on glass slides with glycerol and imaged using a digital camera [57].

4.9. Nitroblue Tetrazolium (NBT) Staining

The staining solution was prepared by dissolving 0.1 g of NBT powder (Biotopped, Beijing, China) in 50 mL of phosphate buffer (pH 7.5). The phosphate buffer was prepared by mixing 1.6 mL of NaH₂PO₄ buffer with 8.4 mL of Na₂HPO₄ buffer and diluting to a final volume of 200 mL with distilled water. Leaves were immersed in the NBT solution and incubated in the dark for 18 h. Decolorization, mounting, and imaging were performed following the same procedures described for DAB staining [57].

4.10. Statistical Analysis

Data are presented as the mean ± standard deviation (SD). Differences between two groups were evaluated using a two-tailed Student’s t-test. For multiple comparisons, statistical significance was assessed by either one-way or two-way analysis of variance (ANOVA) using GraphPad Prism 9.5.1 software.

5. Conclusions

This study demonstrates that heterologous overexpression of the GsEXPA8 gene from wild soybean significantly enhances alkaline tolerance in narrow-leaf lupinus (Lupinus angustifolius). GsEXPA8 promotes root elongation and branching under stress, establishing a more developed root architecture that may improve water and nutrient uptake. Concurrently, it mitigates oxidative damage and osmotic stress induced by alkalinity by elevating antioxidant enzyme (e.g., CAT) activity, reducing membrane lipid peroxidation (MDA), and increasing the accumulation of osmoregulatory compounds such as proline. Functioning as a regulatory node, GsEXPA8 is associated with the upregulation of multiple key stress-responsive genes involved in ion transport (LaSOS1), transcriptional regulation (LaNAC56, LaMYB39), and hormone synthesis (LaNCED3), thereby systemically reinforcing the plant’s stress-adaptation network. Notably, this study reveals for the first time that GsEXPA8 overexpression specifically reshapes the rhizosphere microbial community, enriching beneficial bacterial genera with plant growth-promoting, antimicrobial, and nutrient-mobilizing functions in lupinus. This indicates that the enhanced alkaline tolerance is partly derived from the optimization of beneficial plant-microbe interactions. Furthermore, the specific induction of the endogenous homologous gene LaEXPA8 under alkaline stress suggests a conserved functional role for this expansin family in alkali tolerance. In summary, GsEXPA8 enhances alkaline tolerance in lupinus through an integrated strategy involving the promotion of root development, enhancement of biochemical resilience, activation of molecular networks, and recruitment of a beneficial rhizosphere microbiome. These findings provide new insights into utilizing expansin genes for the genetic improvement of crop stress resistance and enhancing crop adaptability through rhizosphere microbiome engineering.