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

Abstract

TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the 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 ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.

Subgroup-specific intrinsic disorder profiles of arabidopsis NAC transcription factors: Identification of functional hotspots

“…1B). 9 However, conserved ID patterns were reveled for several NAC subgroups ( Fig. 2), in accordance with the suggestion that disorder patterns are constrained compared to sequences.…”

“…1) with interspersed conserved sequence motifs. 9 Our recent work 10 characterizing NAC ID suggested that the large disordered TRD of ANAC0046 forms an entropic chain which is well-suited for molecular fishing of e.g., RCD1 (Fig. 1A).…”

“…Two of these regions coincide with previously defined sequence motifs, the LP and WQ motifs. 9,15 The WQ motif is necessary for transcriptional activity of SND, 1,15 whereas the function of the LP motif remains elusive. However, conservation of this motif and coincidence with a MoRF strongly suggests functional significance.…”

“…(Left) Phylogentic NAC subgroups derived from a tree generated using MEGA4 software based on a multiple alignment of complete Arabidopsis NAC proteins. 9 The color underlining the protein names corresponds to the color of the graphs shown at right. (Right) Predicted disorder as a function of alignment position for the transcription regulatory domains of NAC proteins from the subgroups shown.…”

“…Due to the lack of sequence similarity of the TRDs previous phylogenetic NAC analysis was based on only the NAC domain. 9 In this study, complete NAC sequences were used to derive dendograms for all Arabidopsis NAC proteins. ID predictions using metaPrDOS 11 confirmed that the ID patterns are not conserved in the NAC family (Fig.…”