An ABF5b-HsfA2h/HsfC2a-NCED2b/POD4/HSP26 module integrates multiple signaling pathway to modulate heat stress tolerance in wheat.

Heat stress caused by increasing global temperature has become a major factor limiting yield in wheat. Heat shock transcription factors (Hsfs), as the primary regulators in plant responses to heat stress, play essential roles in modulating both basal and acquired thermotolerance in plants. However, the underlying molecular mechanisms remain to be elucidated. By analysing the wheat transcriptome after subjecting wheat to heat treatments for different time intervals, we identified gene TaHsfA2h that showed a significant positive regulatory response to heat stress. Heat stress tolerance was enhanced by overexpression of TaHsfA2h and constrained by its RNA interference. RNA-seq analysis demonstrated that the overexpression of TaHsfA2h significantly enhanced the expression levels of genes involved in ABA and ROS signalling pathways. Additionally, we identified TaABF5b, a critical regulatory factor in the ABA signalling pathway, as being capable of modulating the expression of TaHsfA2h. Notably, TaHsfA2h interacted with TaHsfC2a both in vivo and in vitro. Similarly, overexpression of TaHsfC2a significantly enhanced heat stress tolerance, whereas knockout dramatically reduced tolerance. The presence of TaHsfC2a significantly enhanced the regulatory activity of TaHsfA2h. TaHsfA2h and TaHsfC2a can co-regulate the expression levels of heat stress tolerance-related genes, including TaNCED2B, TaPOD4 and TaHSP26, thereby enhancing wheat's tolerance to heat stress. Overall, our findings revealed a positive regulatory function of the ABF5b-HsfA2h/HsfC2a-NCED2b/POD4/HSP26 module on wheat heat stress tolerance. This discovery further expanded the functionality of a plant heat stress response model, providing a theoretical foundation for the development of heat-tolerant wheat varieties.