AtDjB3 regulates Hsc70-1-mediated expression of the 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. We present microscopy and cell fractionation-based evidence to demonstrate that the loss of AtDjB3 impairs Hsc70-1 recruitment to heat-induced protein aggregates, thereby maintaining HsfA1d bound to Hsc70-1 in the cytoplasm. This inhibited the expression of many heat-inducible genes, including HSP70s , HSP90 , HSP18.2 , FES1A , HSFA2 , and HSFA7A . Consistent with this, AtDJB3 mutants displayed compromised thermotolerance, as evidenced by their inability to survive a prolonged heat stress of 37°C. Conversely, overexpression of AtDJB3 conferred enhanced thermotolerance, further supporting its positive regulatory role in the HSR. We propose that AtDjB3 not only contributes to the solubilization of heat-induced protein aggregates but also promotes Hsf activation by diverting Hsc70-1 to these aggregates, thereby releasing Hsfs to drive the transcriptional heat shock response.