The co-chaperone AtDJB3 regulates HSC70-1-mediated expression of heat shock genes and thermotolerance in Arabidopsis.

Heat stress disrupts protein homeostasis, triggering the heat shock response (HSR) to maintain cellular proteostasis. A key aspect of this response is the release of heat shock factors (HSFs) from HSP70-mediated attenuation under heat shock conditions, making HSP70 a central regulator of HSR. However, the role of HSP70 co-chaperones in this process remains largely unexplored in plants. Our study identifies AtDJB3, a heat-inducible class II J-domain protein (JDP), as a critical modulator of HSR. Microscopy and cell fractionation show that loss of AtDJB3 impairs HSC70-1 recruitment to heat-induced protein aggregates, maintaining HSFA1d bound to HSC70-1 in the cytoplasm. Chromatin immunoprecipitation (ChIP) and promoter luciferase assays revealed that AtDJB3 is required for HSFA1d enrichment at promoters of key heat-inducible genes, linking AtDJB3 to transcriptional activation of genes, including HSC70-1, HSP90, HSP17.6, FES1A, and HOP3. Consistent with this, atdjb3 mutants displayed compromised thermotolerance, evidenced by their inability to survive prolonged heat stress of 37°C. Conversely, overexpression of AtDJB3 conferred enhanced thermotolerance, supporting its positive regulatory role in HSR. We propose that AtDJB3 not only contributes to the solubilization of heat-induced protein aggregates but also promotes HSFA1d activation by diverting HSC70-1 to aggregates, thereby releasing HSFA1d to drive the transcriptional heat shock response.