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

<i>Lupinus angustifolius</i> is an important leguminous ornamental species, but its productivity is often compromised by alkaline soil stress. <i>GsEXPA8</i>, an expansin gene identified in wild soybean (<i>Glycine soja</i>), has been implicated in alkali stress tolerance. In this study, we examined how heterologous expression of <i>GsEXPA8</i> in lupinus affects its biochemical, molecular, and rhizospheric responses to alkali stress. Under NaHCO₃-induced alkaline conditions, transgenic lines overexpressing <i>GsEXPA8</i> 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 (<i>LaSOS1</i>, <i>LaNCED3</i>, <i>LaMYB39</i>, <i>LaNAC56</i>, <i>LaNHX6</i>, and <i>LaP5CS</i>). 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 <i>LaEXPA8</i> is alkali-inducible. Overexpression of <i>LaEXPA8</i> similarly enhanced alkaline tolerance, whereas CRISPR/Cas9 knockout lines showed no clear phenotypic alteration, suggesting potential functional redundancy within the expansin family. Notably, <i>LaEXPA8</i> and <i>GsEXPA8</i> differed in their temporal regulation of downstream genes, indicating both conserved and distinct regulatory roles. Our results demonstrate that <i>GsEXPA8</i> 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.