Abstract
Alternative oxidase (AOX) serves as a critical terminal oxidase within the plant respiratory pathway, playing a significant role in cellular responses to various stresses. Foxtail millet (Setaria italica), a crop extensively cultivated across Asia, is renowned for its remarkable tolerance to abiotic stresses and minimal requirement for fertilizer. In this study, we conducted a comprehensive genome-wide identification of AOX genes in foxtail millet genome, discovering a total of five SiAOX genes. Phylogenetic analysis categorized these SiAOX members into two subgroups. Prediction of cis-elements within the promoter regions, coupled with co-expression network analysis, intimated that SiAOX proteins are likely involved in the plant's adaptive response to abiotic stresses. Employing RNA sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR), we scrutinized the expression patterns of the SiAOX genes across a variety of tissues and under multiple abiotic stress conditions. Specifically, our analysis uncovered that SiAOX1, SiAOX2, SiAOX4, and SiAOX5 display distinct tissue-specific expression profiles. Furthermore, SiAOX2, SiAOX3, SiAOX4, and SiAOX5 exhibit responsive expression patterns under abiotic stress conditions, with significant differences in expression levels observed between the shoot and root tissues of foxtail millet seedlings. Haplotype analysis of SiAOX4 and SiAOX5 revealed that these genes are in linkage disequilibrium, with Hap_2 being the superior haplotype for both, potentially conferring enhanced cold stress tolerance in the cultivar group. These findings suggest that both SiAOX4 and SiAOX5 may be targeted for selection in future breeding programs aimed at improving foxtail millet's resilience to cold stress.