In Arabidopsis thaliana， the factor HEAT-INTOLERANT 4 (HIT4) orchestrates chromocenter relaxation， a process critical for heat stress tolerance， yet the precise molecular pathway underlying this process is yet enigmatic. Through TurboID-mediated proximity labeling in combination with bimolecular fluorescence complementation and pull-down assays， we identified a direct interaction between Plant U-box 49 (PUB49) and HIT4. PUB49 is a nuclear protein that simultaneously functions as a U-box E3 ubiquitin ligase and peptidyl-prolyl cis-trans isomerase. pub49 null mutants generated via CRISPR/Cas9 displayed thermosensitive phenotypes similar to those observed in the hit4-1 missense allele. Subnuclear imaging demonstrated that the PUB49–HIT4 association resides in chromocenters at 23oC but moves to the nucleolus prior to chromocenter relaxation during heat stress at 37oC. Conversely， the PUB49– HIT4S227Y variant formed numerous nuclear granules irrespective of temperature. Our findings indicate that wild-type HIT4 is required to enable PUB49 trafficking in response to heat stress. Moreover， chromocenter relaxation was not fully achieved in pub49 mutants under sustained heat stress at 37oC. In addition， complementation assays showed that the U-box E3 motif alone， but not the peptidyl-prolyl isomerase domain of PUB49， is responsible for heat-induced chromocenter relaxation and plant thermotolerance. Collectively， these results establish nuclear U-box E3 ubiquitin ligase as a key determinant in heat-induced chromatin remodeling and HIT4-mediated tolerance to thermal stress.
