Boosting transcriptional activities by employing repeated activation domains in transcription factors

Enhancing the transcriptional activation activity of transcription factors (TFs) has multiple applications in organism improvement, metabolic engineering, and other aspects of plant science, but the approaches remain unclear. Here, we used gene activation assays and genetic transformation to investigate the transcriptional activities of two MYB TFs, PRODUCTION OF ANTHOCYANIN PIGMENT 1 (AtPAP1) from Arabidopsis (Arabidopsis thaliana) and EsMYBA1 from Epimedium (Epimedium sagittatum), and their synthetic variants in a range of plant species from several families. Using anthocyanin biosynthesis as a convenient readout, we discovered that homologous naturally occurring TFs showed differences in the transcriptional activation ability and that similar TFs induced large changes in the genetic program when heterologously expressed in different species. In some cases, shuffling the DNA-binding domains and transcriptional activation domains (ADs) between homologous TFs led to synthetic TFs that had stronger activation potency than the original TFs. More importantly, synthetic TFs derived from MYB, NAC, bHLH, and ethylene-insensitive3-like (EIL) family members containing tandemly repeated ADs had greatly enhanced activity compared to their natural counterparts. These findings enhance our understanding of TF activity and demonstrate that employing tandemly repeated ADs from natural TFs is a simple and widely applicable strategy to enhance the activation potency of synthetic TFs.