Abstract
Hexokinases (HXKs) in plants are multifunctional enzymes that not only phosphorylate hexose but also function as glucose sensors, integrating nutrient, light, and hormone signaling networks to regulate cell metabolism and signaling pathways, thereby controlling growth and development in response to environmental changes. To date, limited information is available regarding the HXKs of foxtail millet (Setaria italica L.). In this study, six HXK genes were identified and characterized in foxtail millet. Phylogenetic analysis revealed that the foxtail millet hexokinases were classified into three subfamilies, corresponding to the two types (B-type and C-type) of hexokinases in plants. Gene structure and conserved motif analysis showed that the SiHXKs exhibited varying numbers of introns and exons, with proteins in each subfamily showing similar motif organization. Evolutionary divergence analysis indicated that the foxtail millet HXK and green foxtail HXK genes families underwent both positive and negative selection and experienced a large-scale duplication event approximately 1.18-154.84 million years ago. Expression analysis revealed that these genes are widely expressed in roots, stems, leaves, panicles, anthers, and seeds, with most genes showing significantly increased expression in roots under abiotic stress conditions, including 20% PEG 6000 (drought stress), 200 μmol/L NaCl (salt stress), and 1 μmol/L BR (brassinosteroid-mediated stress response). These results suggest that these genes may play a pivotal role in enhancing stress tolerance. Subcellular localization assay showed that SiHXK5 and SiHXK6 were predominantly localized in mitochondria. Haplotype analysis revealed that SiHXK3-H1 was associated with higher plant height and grain yield. These findings provide valuable insights into the functional characteristics of HXK genes, especially in the context of marker-assisted selection and the pyramiding of advantageous haplotypes in foxtail millet breeding programs.