The miR167-ARF8 Module Integrates HSFA1-HSFA2 and RVE4/8-ERF53/54 Transcriptional Cascades to Enhance Plant Thermotolerance

Heat stress (HS) is a major environmental factor limiting plant survival and crop productivity, prompting the evolution of adaptive responses driven by transcriptional reprogramming coordinated by Class A1 HS TRANSCRIPTION FACTORS (HSFA1s). Here, we demonstrate that the miR167–AUXIN RESPONSE FACTOR 8 (ARF8) module functions as a central regulatory hub, enhancing thermotolerance by integrating both HSFA1-dependent and HSFA1-independent signaling cascades. Under HS, the expression of ARF6 and ARF8 is repressed, while miR167 is induced. Disruption of ARF6/ARF8 or overexpression of miR167 significantly improves heat tolerance, whereas ARF8 overexpression increases HS sensitivity. ARF8 directly binds to the promoters of HSFA1 genes and heat shock protein (HSP) genes, repressing their transcription and thereby impairing the heat-responsive gene network. In addition, ARF8 physically interacts with HSFA1 proteins, further inhibiting their transactivation activity post-translationally. Notably, ARF8 also regulates HSFA2, a major amplifier of the HS response, through both HSFA1-dependent and HSFA1-independent mechanisms. Genetic evidence supports a model in which the HSFA1–HSFA2 axis functions downstream of the miR167–ARF8 module, but contributes only partially to the overall thermotolerance phenotype. Beyond the HSFA1–HSFA2 pathway, ARF8 also suppresses the RVE4/8–ERF53/54 transcriptional cascade, a circadian-regulated, HSFA1-independent module that promotes thermotolerance. This dual regulation allows ARF8 to fine-tune HS adaptation through integration of both canonical and noncanonical signaling routes. Together, these findings establish the miR167–ARF8 module as a central integrator of HS response pathways. This work not only reveals new mechanistic complexity in plant heat response networks but also identifies a conserved regulatory node with promising potential for genetic improvement of heat-resilient crops.