Gshdz4-GmU2AFb-GmCML27 Regulatory Pathway Reshapes Root System Architecture and Enhances Alkaline Tolerance in...

Alkaline soil limits soybean production. This study elucidates the molecular mechanism by which the Gshdz4-GmU2AFb-GmCML27 module regulates soybean alkaline tolerance. Transcriptome analysis, yeast one-hybrid and dual-luciferase assays confirm that the HD-Zip transcription factor Gshdz4 binds to the CAATAA motif in the GmU2AFb promoter and activates its transcription. Subcellular localization verifies the nuclear distribution of GmU2AFb. Overexpression of GmU2AFb improves alkaline tolerance by increasing antioxidant enzyme activities and proline levels, reducing MDA accumulation, facilitating root development, and upregulating alkaline-responsive genes, including GmSOD1, while gene knockout impairs stress resistance. Combined Y2H, BiFC and LCI assays validate the nuclear protein interaction between GmU2AFb and the calcium-binding protein GmCML27, and overexpression of GmCML27 also enhances antioxidant capacity and root growth in soybean. Co-overexpression of the two genes generates obvious synergistic effects; compared with single-gene overexpression lines, co-transgenic plants possess higher antioxidant levels and elevated transcription of downstream alkaline-tolerant genes, accompanied by alleviated growth inhibition under alkaline stress. In summary, Gshdz4 transcriptionally activates GmU2AFb, and the interaction between GmU2AFb and GmCML27 connects RNA splicing with calcium signaling pathways to synergistically trigger downstream defense responses and promote root development, thereby enhancing soybean alkaline tolerance at multiple layers. This work provides candidate genes for molecular breeding of alkali-resistant soybean.