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

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 (Setaria italica), characterized by a compact genome, self-pollination, and a short growth cycle, has emerged as a C(4) model plant. Here, we revealed two SCR paralogs in foxtail millet-SiSCR1 and SiSCR2-which exhibit high sequence conservation with ZmSCR1/1h (Zea mays), OsSCR1/2 (Oryza sativa), and AtSCR (Arabidopsis thaliana), particularly within the C-terminal GRAS domain. Both SiSCR genes exhibited nearly identical secondary structures and physicochemical profiles, with promoter analyses revealing five conserved cis-regulatory elements. Robust phylogenetic reconstruction resolved SCR orthologs into monocot- and dicot-specific clades, with SiSCR genes forming a sister branch to SvSCR from its progenitor species Setaria viridis. Spatiotemporal expression profiling demonstrated ubiquitous SiSCR 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 SiSCR genes and other functional genes. Abscisic acid (ABA) treatment led to a significant downregulation of the expression level of SiSCR genes in Yugu1 roots, and the expression of the SiSCR genes in the roots of An04 is more sensitive to PEG6000 treatment. Drought treatment significantly upregulated SiSCR2 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 SiSCR genes were expressed in root cortical/endodermal initial cells, endodermal cells, cortical cells, and leaf stomatal complexes. Strikingly, ectopic expression of SiSCR genes in Arabidopsis 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 SiSCR genes, providing critical insights into their roles in root/shoot development and abiotic stress signaling in foxtail millet.