Cloning and Functional Analysis of the SiMAPKKK17 Gene in Foxtail Millet (Setaria italica)

Mitogen-activated protein kinase kinase kinases (MAPKKKs) play important roles in plant responses to abiotic stresses; however, the function of SiMAPKKK17 in mediating drought tolerance in foxtail millet remains unclear. In this study, the expression pattern, subcellular localization, and biological function of SiMAPKKK17 were investigated to clarify its role in the drought stress response. Tissue expression analysis showed that SiMAPKKK17 was expressed across developmental stages and in multiple organs, with the highest transcript level observed at the booting stage and comparatively higher expression in vegetative tissues, including roots, stems, and leaves. Subcellular localization analysis demonstrated that SiMAPKKK17 was localized to both the plasma membrane and the nucleus, suggesting potential involvement in membrane-associated signal transduction and nuclear regulatory processes. To evaluate its function, foxtail millet lines overexpressing SiMAPKKK17 were generated and subjected to drought stress. Compared with wild-type plants, the overexpression lines exhibited enhanced drought tolerance, as indicated by greener and more upright upper leaves, higher aboveground fresh weight, greater plant height, and larger leaf area under drought conditions. Transcriptome analysis of OE4 and WT plants under drought stress identified 3919 upregulated genes and 2965 downregulated genes in OE4 compared with WT. These differentially expressed genes were mainly enriched in chloroplast-related cellular components, as well as biological processes and metabolic pathways related to cellular amide metabolism, ion transport, carbon metabolism, photosynthesis, carbon fixation, purine metabolism, and amino acid biosynthesis. Taken together, these results indicate that SiMAPKKK17 acts as a positive regulator of drought tolerance in foxtail millet, potentially through modulation of photosynthesis- and metabolism-related pathways. This study provides evidence for the molecular mechanisms underlying drought tolerance in foxtail millet and identifies SiMAPKKK17 as a promising candidate gene for the development of drought-resistant cultivars.