The Arabidopsis thaliana NAC transcription factor family: structure-function relationships and determinants of ANAC019 stress signaling

Abstract

Abstract Transcription factors (TFs) are modular proteins minimally containing a DNA-binding domain (DBD) and a transcription regulatory domain (TRD). NAC proteins comprise one of the largest plant TF-families. They are key regulators of stress perception and developmental programs, and most share an N-terminal NAC domain. Based on analyses of gene expression data and phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and divergent C-termini. Nine of ten NAC domains analyzed bind a previously identified NAC DNA-target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analyzed is dependent on the C-terminal region for transactivational activity. In silico analyses show that NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of abscisic acid (ABA)-signaling, conferring ABA-hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of ANAC019 DBD or TRD alone also resulted in ABA-hypersensitivity. Expression of stress-responsive marker genes (COR47, RD29b, ERD11) was also induced by full-length and truncated ANAC019. Domain-swapping was used to analyze the specificity of this function. Replacement of the NAC domain of ANAC019 with analogous regions of other NAC TFs resulted in chimeric proteins, which also have the ability to regulate ABA-signaling positively. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA-hypersensitivity. In conclusion, our results demonstrate that biochemical and functional specificity is associated with both DBDs and TRDs in NAC TFs.