AtHAP5A modulates freezing stress resistance in Arabidopsis through binding to CCAAT motif of AtXTH21.

Several eukaryotic Heme-associated proteins (HAPs) have been reported to bind specifically to DNA fragments containing CCAAT-box; however, the physiological functions and direct targets of these HAP proteins in plants remain unclear. In this study, we showed that AtHAP5A as a transcription factor interacted with CCAAT motif in vivo, and AtXTH21, one direct target of AtHAP5A, was involved in freezing stress resistance. The AtHAP5A overexpressing plants were more tolerant, whereas the loss-of-function mutant of AtHAP5A was more sensitive to freezing stress than wild-type plants. Chromatin immunoprecipitation (ChIP) assay demonstrated that AtHAP5A could bind to five fragments that contained CCAAT motifs in the AtXTH21 promoter. Similarly, the AtXTH21 overexpressing plants exhibited improved freezing resistance, while xth21 knockdown mutants displayed decreased freezing resistance. Notably, the modulated freezing resistance of AtHAP5A overexpressing plants and knockout mutant could be reversed by the xth21 mutant and AtXTH21 overexpressing plants, respectively, indicating that AtHAP5A might act upstream of AtXTH21 in freezing stress. Additionally, modulation of AtHAP5A and AtXTH21 expression had the same effects on abscisic acid (ABA) sensitivity and reactive oxygen species (ROS) metabolism. Taken together, these results demonstrated that AtHAP5A modulates freezing stress resistance in Arabidopsis through binding to the CCAAT motif of AtXTH21.