The Co-Expression of GmCML27 and GmU2AFb Enhances Tolerance to Alkaline Stress in Lupinus angustifolius.

Soil salinization severely constrains plant growth and agricultural productivity. Identifying key genes conferring alkali tolerance and elucidating their regulatory mechanisms is of great significance for breeding salt-alkali-tolerant leguminous crops. In this study, we investigated the soybean genes GmCML27 and its potential interacting partner GmU2AFb, and explored their functions in response to alkali stress in narrow-leaf lupine (Lupinus angustifolius) through bioinformatics analyses. Protein interaction prediction and experimental validation further supported a potential association between GmCML27 and GmU2AFb. Using an Agrobacterium rhizogenes (A. rhizogenes)-mediated hairy root transformation system, composite plants carrying empty vector (CK), GmCML27-overexpression construct (GC), GmU2AFb-overexpression construct (GU), or both GmCML27 and GmU2AFb overexpression constructs (GCU) were generated and evaluated under NaHCO3-induced alkali stress. The results showed that all transformed composite plants exhibited enhanced alkali tolerance, with the GCU plants displaying the strongest phenotype. Compared with CK plants, transformed composite plants maintained higher catalase (CAT) activity, proline (Pro) content, and root vitality, while accumulating lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and retaining more intact root architecture. Furthermore, the expression levels of alkali tolerance-related genes, including LaMYB39, LaSOS1, LaNHX6, LaKIN, LaDnaJ1, and LaP5CS, were significantly upregulated in transgenic lines. Collectively, our findings demonstrate that both GmCML27 and GmU2AFb positively regulate alkali tolerance in lupine, and their co-expression confers enhanced alkali tolerance compared with single-gene overexpression. These two genes may jointly contribute to alkali stress adaptation through regulation of antioxidant capacity and stress-responsive pathways. This study highlights the potential roles of GmCML27 and GmU2AFb in lupine responses to alkali stress and provides candidate gene resources for future improvement of lupine alkali tolerance.