Heterologous Co-Expression of Gshdz4 and GmRSZ22 Modulates Alkali Stress Responses in Lupinus angustifolius

Abstract

Lupinus angustifolius is an ornamental species with high horticultural value, but its poor alkaline tolerance limits broader cultivation. Wild soybean (Glycine soja) is a legume plant with strong alkali tolerance. Gshdz4-GmRSZ22 was identified as a potential regulatory module that contributes to alkali stress resistance. SR proteins are essential splicing factors that play important roles in abiotic stress responses. Firstly, the serine/arginine-rich (SR) gene family in lupin was characterized and compared with that of wild soybean to reveal evolutionary differences. Using Agrobacterium rhizogenes transformation, we generated lupin roots overexpressing either GmRSZ22 alone (ROE) or the Gshdz4-GmRSZ22 combination (HROE), and assessed their effects on root development and alkali tolerance under 100 mM NaHCO3 stress (for phenotypic, physiological, histochemical, and gene expression analyses) or 25 mM NaHCO3 over a two-week period (for root architecture analysis). Compared to wild-type (WT), both overexpression lines showed reduced wilting, prolonged survival, increased proline content, elevated superoxide dismutase (SOD) activity, enhanced root activity, and lower malondialdehyde (MDA) levels. Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining indicated decreased reactive oxygen species (O2− and H2O2) in leaves, confirming improved antioxidant capacity. Root architecture analysis revealed that both ROE and HROE significantly enhanced root length, surface area, and root tip number, with HROE showing a more pronounced effect on branching. Promoter and RT-qPCR analyses suggested that Gshdz4 may be involved in the upregulation of the endogenous LaRSZ22 transcript levels, potentially contributing to a broader alkali-responsive transcriptional program. Furthermore, both ROE and HROE upregulated key stress-responsive genes (LaSOS1, LaNHX6, LaP5CS, LaKIN, and LaDnaJ1). Overall, the HROE line demonstrated the most robust alkali tolerance across phenotypic, physiological, root architectural, and gene expression responses. Our findings offer a valuable foundation for breeding programs aimed at improving lupin adaptation to alkaline soils.