Abstract
Heat stress significantly impacts rice productivity, making understanding of molecular mechanisms crucial for crop improvement. Here, we report a regulatory pathway in rice that integrates a membrane-localized E3 ubiquitin ligase (OsTT3.1), glycogen synthase kinase 2 (OsGSK2), and the heat shock factor B2c (OsHsfB2c) to modulate thermotolerance. We demonstrate that OsGSK2 directly interacts with and phosphorylates OsHsfB2c both in vitro and in vivo, a modification that promotes its nuclear translocation. Functional analyses revealed that mutations in both
OsGSK2
and
OsHsfB2c
enhance heat tolerance, while their overexpression reduces thermotolerance. Furthermore, we identified OsTT3.1 as an OsGSK2-interacting protein that promotes OsGSK2 degradation through ubiquitin-mediated proteolysis. Double mutant analysis (
ostt3.1
/
osgsk2
) showed increased heat tolerance compared to
ostt3.1
single mutants, confirming that OsTT3.1 acts upstream of OsGSK2. Transcriptomic profiling of
oshsfb2c
mutants under normal and heat stress conditions uncovers widespread dysregulation of genes involved in primary metabolism and stress defense, indicating that OsHsfB2c functions as a transcriptional repressor of heat-responsive pathways. Our findings establish a sophisticated regulatory cascade where OsTT3.1-mediated degradation of OsGSK2 relieves the phosphorylation of OsHsfB2c, thereby derepressing heat stress responses and enhancing thermotolerance in rice. This “brake-release” mechanism serves as a critical buffering system that allows rapid attenuation of the OsGSK2–OsHsfB2c repressive module under heat stress, ensuring timely and robust activation of stress responses and optimal metabolic reprogramming.