Tissue-Specific Expression Analysis and Functional Validation of Genes in Foxtail Millet () Under Hormone and Drought Stresses, and Heterologous Expression in .

The SCARECROW (SCR) transcription factor governs cell-type patterning in plant roots and Kranz anatomy of leaves, serving as a master regulator of root and shoot morphogenesis. Foxtail millet (), characterized by a compact genome, self-pollination, and a short growth cycle, has emerged as a C model plant. Here, we revealed two paralogs in foxtail millet- and -which exhibit high sequence conservation with (), (), and (), particularly within the C-terminal GRAS domain. Both genes exhibited nearly identical secondary structures and physicochemical profiles, with promoter analyses revealing five conserved -regulatory elements. Robust phylogenetic reconstruction resolved orthologs into monocot- and dicot-specific clades, with genes forming a sister branch to from its progenitor species . Spatiotemporal expression profiling demonstrated ubiquitous gene transcription across developmental stages, with notable enrichment in germinated seeds, plants at the one-tip-two-leaf stage, leaf 1 (two days after heading), and roots during the seedling stage. Co-expression network analysis revealed that there is a correlation between genes and other functional genes. Abscisic acid (ABA) treatment led to a significant downregulation of the expression level of genes in Yugu1 roots, and the expression of the genes in the roots of An04 is more sensitive to PEG6000 treatment. Drought treatment significantly upregulated expression in leaves, demonstrating its pivotal role in plant adaptation to abiotic stress. Analysis of heterologous expression under the control of the 35S promoter revealed that genes were expressed in root cortical/endodermal initial cells, endodermal cells, cortical cells, and leaf stomatal complexes. Strikingly, ectopic expression of genes in led to hypersensitivity to ABA, and ABA treatment resulted in a significant reduction in the length of the meristematic zone. These data delineate the functional divergence and evolutionary conservation of genes, providing critical insights into their roles in root/shoot development and abiotic stress signaling in foxtail millet.