Abstract
High-temperature (HT) stress during flowering causes male sterility and yield loss in soybean. MYB transcription factors are key regulators under abiotic stress, yet their function and mechanism in regulating male fertility under HT stress in soybean are not fully understood. In this study, a MYB transcription factor GmMYB21a in soybean was identified. GmMYB21a was induced by HT stress in soybean restorer line and was specifically expressed in pollen. Through overexpression and knockout experiments, we demonstrated that GmMYB21a positively regulated pollen viability and germination under HT stress. Overexpression of GmMYB21a significantly enhanced these traits in restorer line, whereas knockout plants exhibited the opposite effect. Transcriptome sequencing revealed that GmMYB21a overexpression upregulated numerous stress-responsive genes, particularly those involved in flavonoid biosynthesis and sugar metabolism. In addition, molecular experiments confirmed that GmMYB21a bound to the promoter of flavonoid synthesis gene GmCHI2-A and promoted its expression. In summary, our research indicated GmMYB21a enhanced the HT-tolerance of male fertility in soybean restorer line through reactive oxygen species scavenging and flavonoid synthesis. This study aims to elucidate the thermotolerance mechanism in soybean male fertility and identify genetic resources for breeding HT-tolerant restorer lines.