VOZ1, a transcriptional repressor of DREB2C, mediates heat stress responses in Arabidopsis

Main conclusion Under normal growth conditions, Arabidopsis VOZ1 interacts with DREB2C and acts as a transcriptional repressor by reducing DNA binding of DREB2C. Under heat stress conditions, VOZ1 is degraded by ubiquitination, and DREB2C, which is freed from VOZ1, functions as a transcription activator. To investigate the mechanism by which the DEHYDRATION-RESPONSIVE ELEMENT-BINDING FACTOR 2C (DREB2C)-dependent signaling cascade regulates heat stress (HS) responses, we performed a yeast two-hybrid screening using the DREB2C APETALA2 (AP2) DNA-binding domain as the bait against a cDNA library derived from Arabidopsis. We identified VASCULAR PLANT ONE-ZINC-FINGER 1 (VOZ1) and further verified positive VOZ1 colonies by repeating the X-alpha-Gal second screening and pull-down assay in vitro. Deletion analysis of VOZ1 demonstrated that the amino acid residues in its transcriptional regulatory, zinc finger and NAC domains are essential for the DREB2C-AP2 interaction. Although the HsfA3 promoter was strongly transactivated by DREB2C in Arabidopsis protoplasts, transient co-expression of VOZ1 (35S:VOZ1) with DREB2C (35S:DREB2C) in Arabidopsis protoplasts resulted in a significant decrease in the activity of GUS fused to the HsfA3 promoter (Prom (HsfA3) :GUS), indicating that VOZ1 acts as a repressor of DREB2C. In electrophoretic mobility shift assays (EMSAs), the signal generated by binding of DREB2C to DRE gradually decreased with increasing VOZ1 level, providing evidence that the interaction of the DREB2C AP2 DNA-binding domain with DRE is blocked by VOZ1. Additionally, a voz1 voz2-2 double knockout mutant exhibited increased HS tolerance, likely due to the suppressive function of VOZs. Taken together, these results demonstrate that VOZ1 functions as a negative regulator of HS-inducible DREB2C signaling by blocking access to the AP2 DNA-binding domain of DREB2C.