Characterization of the Arabidopsis thaliana CBF1 transcription factor: Functional role of two evolutionarily conserved signature sequences

Over the course of evolution plants have adapted to the environment by developing ways to cope with different biotic and abiotic stresses. Among the abiotic stresses, low temperatures represent a major limiting factor for the growth and development of plants. The CRT/DRE-Binding Factors (CBFs) are transcriptional activators that are rapidly activated in response to low temperature and in turn induce the expression of a battery of cold-regulated (COR) genes to increase plant freezing tolerance. Arabidopsis plants overexpressing CBF1, CBF2 or CBF3 are constitutively freezing tolerant, indicating that these regulators are master regulators of cold adaptation. CBF proteins belong to the APETALA2/Ethylene-Response Binding Protein (AP2/EREBP) family, which includes 145 members in Arabidopsis. Proteins in this family share high similarity within their AP2/EREBP DNA-binding domain. A unique feature of the CBF proteins is that they contain two conserved sequences flanking the DNA-binding domain. These sequences, represented by the consensus motifs PKK/RPAGRxKFxETRHP and DSAWR, are also found in CBF-like proteins from evolutionarily diverse plant species, suggesting that these "signature" sequences play a role in CBF activity. Overexpression of wild type CBF1 in Arabidopsis results in constitutive COR gene expression. Transgenic lines overexpressing CBF1 carrying alanine substitutions in the signature sequences showed reduced or no COR gene expression, indicating that these sequences play an important role in CBF1 activity. Analysis of protein levels revealed that alanine mutations throughout the DSAWR motif affect protein accumulation in planta, and could explain the lower COR gene accumulation in those plants. On the contrary, mutations in the PKKPAGR motif did not affect protein steady state levels; instead they impaired the ability of CBF1 to bind its cognate CRT/DRE element from the COR gene promoters. The most pronounced effect was observed when two conserved Arg and Phe residues were substituted with Lys and Ala, respectively; these substitutions were sufficient to abrogate DNA binding, indicating an essential role of those residues and potentially a base-specific recognition. Altogether these results indicated that DNA binding activity in the AP2/EREBP family extends beyond the canonical DNA-binding domain previously described to include the N-flanking PKKPAGR region. Based on these observations and secondary structure prediction studies, we developed a computational model describing CBF1 bound to the DNA. According to this model, CBF1 binds the DNA major groove through a three-stranded beta sheet, as described for other AP2/EREBP proteins. In addition, a helical stretch within the PKKPAGR motif makes essential interactions with the DNA minor groove in close proximity to a conserved thymine that is a specificity determinant in the CRT/DRE element bound by the CBF proteins. Additional investigations will elucidate whether residues within the PKKPAGR motif represent specificity switches for the recognition of the CRT/DRE promoter element by CBF proteins and whether a similar mechanism has been conserved in other protein of the AP2/EREBP family.