Mapping the dimerization specificity of bZIP transcription factors in bread wheat

Dimerization plays a pivotal role in target-binding interactions among various transcription factors (TFs), including bZIPs, bHLHs, MYBs, and zinc finger TFs. The bZIP family is defined by an -helix class of TFs containing a basic region that binds to the major groove of double-stranded DNA (dsDNA), followed by a leucine zipper motif that mediates dimerization through coiled-coil structures, forming homo- or heterodimers. In polyploid bread wheat (Triticum aestivum cv.), bZIPs and their homeologs interact through the dimerization motifs to form specific bZIP pairs with distinct regulatory functions. The dimerization pattern of bZIPs is critical for gene regulation, however, the mechanisms underlying their specificity remain poorly understood. Here, we focus on the dimerization specificity of coiled-coil - helical bZIP proteins in bread wheat, highlighting the leucine zipper region as a key dimerization motif. We identified 265 bZIPs containing the bZIP motif, following dimerization criteria established in humans, Drosophila, and Arabidopsis based on the presence of specific amino acids at the a, d, e, and g positions in heptads. Eight key bZIP TFs were cloned, expressed, and purified for the structural analysis. This study not only provides the dimerization trend exhibited by bZIPs during thermal stability analysis but also provides the reversibility pattern followed by the thermal denaturation exposure at 85{degrees}C. Our study also points towards that how amino acid substitutions and the length of the leucine zipper influence the formation of coiled-coil structures among bZIP homeologs of polyploid wheat. Overall, the study advances our understanding of the role of leucine zippers, in determining the specificity and stability of bZIP dimers, which in turn governs DNA binding and gene regulation in plants.