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

## TL;DR

This study shows that introducing a gene from soybean into a lupin plant improves its ability to tolerate alkaline soil by enhancing root growth, antioxidant activity, and beneficial soil microbes.

## Contribution

The novel contribution is demonstrating that heterologous expression of GsEXPA8 in Lupinus angustifolius enhances alkaline tolerance through multiple physiological and microbial mechanisms.

## Key findings

- GsEXPA8 overexpression in lupinus improves root biomass, leaf vigor, and antioxidant enzyme activity under alkaline stress.
- Transgenic lines show increased proline accumulation and reduced malondialdehyde levels, indicating better osmotic adjustment and stress mitigation.
- Rhizosphere microbial communities are enriched with beneficial taxa like Pseudomonas and Lysobacter in GsEXPA8-overexpressing plants.

## 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.