Arabidopsis PUB49 nuclear E3 ligase promotes HIT4-dependent heat-induced chromocenter decondensation and thermotolerance

In Arabidopsis thaliana, HEAT-INTOLERANT 4 (HIT4) mediates heat-induced chromocenter decondensation, a process essential for plant thermotolerance; however, its molecular regulation remains unclear. Using TurboID-based proximity labeling, bimolecular fluorescence complementation, and pull-down assays, we observed that PUB49 directly interacts with HIT4. PUB49 is a nuclear protein possessing both U-box E3 ubiquitin ligase and peptidyl-prolyl isomerase domains. CRISPR/Cas9-generated pub49 knockout mutants exhibited heat-sensitive phenotypes like those of the hit4-1 missense mutant. Subnuclear localization analysis revealed that the PUB49-HIT4 complex localizes to chromocenters under normal conditions and relocates to the nucleolus before chromocenter decondensation in response to heat stress. In contrast, the PUB49-HIT4S227Y complex formed numerous granules throughout the nucleus, independent of temperature. These results, suggest that functional HIT4 is required for temperature-dependent subnuclear trafficking of PUB49. Furthermore, chromocenter decondensation was incomplete in pub49 mutants under heat stress. Complementation analysis demonstrated that only the U-box domain, rather than peptidyl-prolyl isomerase, was sufficient to restore full chromocenter decondensation and thermotolerance. These findings demonstrate that nuclear U-box E3 ubiquitin ligase is involved in heat-responsive chromatin remodeling and HIT4-mediated thermotolerance.