Mapping the dimerization specificity of bZIP transcription factors in bread wheat

Abstract Dimerization is pivotal in target-binding interactions among transcription factors (TFs), including bZIPs, bHLHs, and many zinc finger proteins. The bZIP family comprises an α-helix class of TFs that features a basic region binding to the major groove of double-stranded DNA (dsDNA), followed by a leucine zipper motif that facilitates dimerization through coiled-coil structures, forming homo- or heterodimers. In allohexaploid bread wheat ( Triticum aestivum cv.), bZIPs and their homeologs utilize dimerization motifs to create specific bZIP pairs with distinct regulatory functions. The dimerization pattern of bZIPs is essential for gene regulation. Following a genome-wide analysis, we identified 265 bZIP TFs in bread wheat. By applying criteria from previous studies to evaluate bZIP dimerization across humans, Drosophila, and Arabidopsis, we categorized bZIP TFs into different groups based on their predicted dimerization propensity. Amino acid sequence analysis of wheat bZIP TFs led to several conclusions: unlike Drosophila and humans, wheat bZIPs are longer, with some reaching lengths of up to 14 heptads. Based on the amino acids at the a, d, g, and e positions in each heptad, wheat bZIPs are predominantly predicted to form homodimers. However, the mechanisms underlying wheat bZIP dimerization specificity and stability, particularly in plants, remain poorly understood. To experimentally validate the predicted dimer assembly, eight key bZIP TFs containing the bZIP domain were cloned and expressed for structural analysis. Using purified bZIP proteins, thermal denaturation studies demonstrated a change in dimerization stability as the length of the leucine zipper varies. In summary, this study enhances our understanding of the role of leucine zippers in determining the specificity and stability of bZIP dimers, which subsequently influence DNA binding and gene regulation in plants.