The Gshdz4-GsNAC019-GsEXPA8 Multi-Component Module Enhances Alkaline Stress Tolerance in Lupinus angustifolius

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.