ATBS1-INTERACTING FACTOR 2 Positively Regulates Freezing Tolerance via INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR-Induced Cold Acclimation Pathway.

The INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR (ICE1/CBF) pathway plays a crucial role in plant responses to cold stress, impacting growth and development. Here, we demonstrated that ATBS1-INTERACTING FACTOR 2 (AIF2), a non-DNA-binding basic helix-loop-helix transcription factor, positively regulates freezing tolerance through the ICE1/CBF-induced cold tolerance pathway in Arabidopsis. Cold stress transcriptionally upregulated AIF2 expression and induced AIF2 phosphorylation, thereby stabilizing the AIF2 protein during early stages of cold acclimation. The AIF2 loss-of-function mutant, aif2-1, exhibited heightened sensitivity to freezing before and after cold acclimation. In contrast, ectopic expression of AIF2, but not the C-terminal-deleted AIF2 variant, restored freezing tolerance. AIF2 enhanced ICE1 stability during cold acclimation and promoted the transcriptional expression of CBFs and downstream cold-responsive genes, ultimately enhancing plant tolerance to freezing stress. MITOGEN-ACTIVATED PROTEIN KINASES 3 and 6 (MPK3/6), known negative regulators of freezing tolerance, interacted with and phosphorylated AIF2, subjecting it to protein degradation. Furthermore, transient co-expression of MPK3/6 with AIF2 and ICE1 downregulated AIF2/ICE1-induced transactivation of CBF2 expression. AIF2 interacted preferentially with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) and MPK3/6 during the early and later stages of cold acclimation, respectively, thereby differentially regulating AIF2 activity in a cold acclimation time-dependent manner. Moreover, AIF2 acted additively in a gain-of-function mutant of BRASSINAZOLE-RESISTANT 1 (BZR1; bzr1-1D) and a triple knockout mutant of BIN2 and its homologs (bin2bil1bil2) to induce CBFs-mediated freezing tolerance. This suggests that cold-induced AIF2 coordinates freezing tolerance along with BZR1 and BIN2, key positive and negative components, respectively, of brassinosteroid signaling pathways.