Genome-wide characterization of the HSF gene family in Setaria italica (foxtail millet) and functional elucidation of SiHSF3 in abiotic stress tolerance

Heat shock transcription factors (HSFs) are central regulators of plant stress signaling networks, orchestrating transcriptional responses to diverse environmental cues. In this study, we performed a comprehensive genome-wide identification and evolutionary analysis of the HSF gene family in Setaria italica (foxtail millet), an emerging model for C₄ cereal crops. Twenty-Five SiHSF genes were identified and mapped across six chromosomes, displaying uneven chromosomal distribution. Phylogenetic and duplication analyses revealed that segmental duplication was the primary force driving the expansion of the SiHSF family, whereas Ka/Ks ratios indicated strong purifying selection during evolution. Promoter inspection revealed a rich repertoire of cis-elements associated with light and phytohormone signaling, particularly in response to jasmonic acid and abscisic acid. Transcriptomic profiling highlighted diverse spatiotemporal expression patterns and a notable induction of SiHSF3 under salt and drought stress. qRT-PCR assays further validated the upregulation of SiHSF3 in response to salinity, dehydration, and heat treatments. Functional characterization demonstrated that SiHSF3 possesses transcriptional self-activation activity and significantly enhances yeast tolerance to osmotic and high-temperature stress. Collectively, these findings provide novel insights into the evolutionary diversification and functional specialization of HSF genes in foxtail millet and establish SiHSF3 as a promising candidate for engineering improved abiotic stress resilience in cereal crops.