Plant growth and development are tightly regulated by environmental conditions, and high-temperature stress has become a major threat to vegetative productivity. Tree peony (Paeonia ostii) is a highly valued ornamental plant widely cultivated across Asia and Europe. Although heat stress is known to severely impair its normal development, the underlying molecular mechanisms remain largely elusive. Here, we investigated the cellular, molecular and genetic basis of thermotolerance in P. ostii. Physiological analyses revealed that high temperature markedly suppressed growth by altering key leaf physiological parameters. Through transcriptome sequencing and expression profiling, we identified PoDHN1, a dehydrin-encoding gene markedly induced by heat stress. Silencing PoDHN1 significantly compromised thermotolerance. We further demonstrated that PoDREB2A, a DREB transcription factor, bound to a DRE motif within the PoDHN1 promoter. In addition, PoDREB2A acted as an upstream activator of PoDHN1 by activating the intact PoDHN1 promoter, thereby enhancing thermotolerance in tree peony. Importantly, we found that the stress associated protein PoSAP8 physically interacts with PoDREB2A and enhances its activation of PoDHN1 promoter. Collectively, our findings establish the PoSAP8/PoDREB2A-PoDHN1 regulatory module, which is activated by high temperature and functions as a positive regulator of thermotolerance in tree peony. This study advances our understanding of heat stress responses in woody plants and provides potential genetic targets for breeding heat-tolerant tree peony cultivars.
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