Targeted manipulation of bZIP53 DNA-binding properties influences Arabidopsis metabolism and growth

: bZIP transcription factors regulate diverse processes in eukaryotic cells. Arabidopsis bZIP members of the C and S1 groups form heterodimers and synergistically control metabolic reprogramming during stress responses. However, their functional characterization is complicated due to an overlapping heterodimerization network and high redundancy. In this study, we develop a simple but powerful approach for generating dominant negative mutants of bZIP factors with high specificity. By applying in vitro DNA-binding, reporter gene and protoplast two-hybrid assays, and plant mutant analysis, we show that phosphorylation-mimicking substitution of conserved serines in the DNA-binding domain of bZIP monomeric subunits suffices for the disruption of the interaction of both bZIP homo- and heterodimers with cognate DNA. This results in the transcriptional inactivation of target genes. The dominant-negative effect is achieved by the unaltered function of the intrinsic nuclear localization signal and dimerization properties of the mutated bZIP protein. Our findings not only reveal an additional regulatory mechanism of bZIP10 intracellular localization, but also provide evidence of the involvement of bZIP53 in the diurnal adjustments of amino acid metabolism. Our data demonstrate the advantages and the suitability of this new approach for the artificial inactivation of bZIP transcription factors in plants, and it may also be of use for other organisms.