Heat shock transcription factor ZmHsfA5 coordinates with ZmHsp9 to enhance thermotolerance in maize

Heat shock transcription factors (HSFs) have roles in plant thermotolerance. However, the functional characterization and regulatory analyses of HSFs in maize remain limited. In this study, we cloned and characterized the previously uncharacterized maize HSF gene ZmHsfA5. Expression of ZmHsfA5 was significantly up-regulated under heat stress (HS). Overexpression of ZmHsfA5 enhanced thermotolerance in both maize seedlings and developing pollen by promoting proline accumulation and enhancing scavenging of reactive oxygen species. Conversely, ZmHsfA5 knockout mutants exhibited decreased thermotolerance. ZmHsfA5 physically interacted with the heat shock protein ZmHsp9. ZmHsp9 was significantly induced by HS, and transgenic experiments revealed that ZmHsp9 positively regulated thermotolerance, similarly to ZmHsfA5. Integrated analyses using CUT&Tag, RNA-seq, EMSA, and luciferase reporter assays demonstrated that ZmHsfA5 activated expression of oxidative stress-related genes by direct binding to heat stress elements in their promoters. Furthermore, ZmHsp9 stabilized the ZmHsfA5 protein under HS, thereby cooperatively enhancing transcriptional activation of the downstream target gene. These findings elucidate novel roles of the ZmHsfA5-ZmHsp9 module in maize thermotolerance and highlight its potential for enhancing adaptation to high-temperature environments.