Abstract: INTRODUCTION: Heat stress severely impairs plant growth and crop productivity. WRINKLED1 (WRI1), an AP2/EREBP-class transcription factor in Arabidopsis thaliana, orchestrates carbon partitioning between glycolysis and fatty acid biosynthesis, playing pivotal roles in development and stress adaptation. Elucidating its molecular function under high-temperature stress is critical for improving thermotolerance in crops.  RATIONALE: While WRI1's metabolic regulatory function is established, its role in heat response remains unexplored. To decipher the molecular mechanism of WRI1-mediated thermotolerance, we integrated genetic approaches (wild-type, wri1-4, and WRI1-OE) with RT-qPCR and phenotyping under controlled heat stress.  RESULTS:In this study, histochemical GUS staining of pWRI1::GUS transgenic lines demonstrated constitutive WRI1 expression throughout Arabidopsis seedlings, with significantly enhanced transcription in cotyledons under heat stress (HS) (P<0.05). Prolonged HS induced gradual transcriptional attenuation, though levels remained elevated versus optimal temperature (22°C). RT-qPCR confirmed thermo-responsive WRI1 upregulation (peak:1h HS, 3-fold induction), followed by threshold-dependent decline, indicating acute early-phase responsiveness. Endogenous immunoassays revealed reduced WRI1 protein accumulation under HS, suggesting HS-impaired protein stability or post-translational regulatory mechanisms. Thermotolerance phenotyping of WT, wri1-4, and WRI1-OE lines showed superior HS survival in WRI1-OE, with acquired thermotolerance exceeding basal thermotolerance across genotypes, confirming WRI1-mediated positive thermoregulation. The survival rate of WRI1-OE overexpression seedlings reached approximately 75%-85%, whereas that of wild-type and complementary lines was less than 10%. WRI1-OE reduced reactive oxygen species (ROS) accumulation, while RT-qPCR excluded direct transcriptional regulation of HSF/HSP genes (e.g., HSFA2, HSP101). Differential gene expression across genotypes nevertheless indicated WRI1's auxiliary role in thermotolerance via ROS scavenging and indirect proteostasis maintenance.  CONCLUSION: This study establishes WRI1 as a master regulator of thermotolerance, functioning through synergistic activation of chaperone networks (HSFA2-HSPs) and reactive oxygen species (ROS) scavenging. The discovery of its cross-pathway coordination mechanism provides novel insights into plant thermal adaptation, while positioning WRI1 as an ideal target for breeding climate-resilient crops.

Key words: Arabidopsis, WRI1, heat stress, gene expression