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

TLDR: In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder, and demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.

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.

Figures

Figure 6. ANAC019 is a positive regulator of ABA signaling. Transgenic plants overexpressing full-length ANAC019 (1-317), ANAC019(1-168) and ANAC019(169-317) are more sensitive to ABA than the Col-0 wild-type and anac019 mutant. (A) Germination efficiency of Col-0, anac019 mutant, 35S:ANAC019(1-317),

Full text

HAL Id: hal-00479244
https://hal.archives-ouvertes.fr/hal-00479244
Submitted on 30 Apr 2010
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entic research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diusion de documents
scientiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
The Arabidopsis thaliana NAC transcription factor
family: structure-function relationships and
determinants of ANAC019 stress signaling
Michael K Jensen, Trine Kjaersgaard, Michael M Nielsen, Pernille Galberg,
Klaus Petersen, Charlotte O’Shea, Karen Skriver
To cite this version:
Michael K Jensen, Trine Kjaersgaard, Michael M Nielsen, Pernille Galberg, Klaus Petersen, et al..
The Arabidopsis thaliana NAC transcription factor family: structure-function relationships and deter-
minants of ANAC019 stress signaling. Biochemical Journal, Portland Press, 2010, 426 (2), pp.183-196.
�10.1042/BJ20091234�. �hal-00479244�

1
The Arabidopsis thaliana NAC Transcription Factor Family: Structure-
Function Relationships and Determinants of ANAC019 Stress Signaling
Michael K. JENSEN, Trine KJAERSGAARD, Michael M. NIELSEN, Pernille GALBERG, Klaus
PETERSEN, Charlotte O’SHEA, and Karen SKRIVER*
Department of Biology, University of Copenhagen, Ole Maaloesvej 5, DK-2200 Copenhagen N, Denmark
*To whom correspondence should be addressed: Karen Skriver, KSkriver@bio.ku.dk, Phone: +45
35321712
SYNOPSIS
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.
Key words: NAC transcription factor, DNA-binding, Intrinsic disorder, Domain-swap,
Abscisic acid (ABA), Arabidopsis
Short title: Structural Determinants of Arabidopsis ANAC019 Function
Biochemical Journal Immediate Publication. Published on 08 Dec 2009 as manuscript BJ20091234
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20091234
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2009 The Authors Journal compilation © 2009 Portland Press Limited

2
ABBREVIATIONS
ABA Abscisic acid
ABRC Arabidopsis biological resource center
AGI Arabidopsis genome initiative
CaMV Cauliflower mosaic virus
CASTing Cyclic amplification and selection of targets
Col-0 Columbia ecotype 0
CUC CUP-SHAPED COTYLEDON (CUC)
DBD DNA-binding domain
EMBL European molecular biology laboratory
EMSA Electrophoretic Mobility Shift Assays
GST Glutathione-S-Transferase
ID Intrinsic disorder
NAC N
AM/ATAF1,2/CUC2
MEME Multiple EM for motif elicitation
MAST Multiple alignment and search tools
MORF Molecular Recognition Features
NACBS NAC-binding site
palNACBS Palindromic NACBS
TRD Transcription regulatory domain
TF Transcription factor
Rap1p Repressor activator protein1p
TAIR The Arabidopsis Information Resource
Biochemical Journal Immediate Publication. Published on 08 Dec 2009 as manuscript BJ20091234
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20091234
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2009 The Authors Journal compilation © 2009 Portland Press Limited

3
INTRODUCTION
Gene specific transcription factors (TFs) are DNA-binding regulatory proteins which
activate or repress the basal transcription apparatus at target gene promoters. TFs are
grouped into families based on sequence similarities, most often in the DNA-binding
domain (DBD)[1]. TFs from the same family often bind DNA in a sequence-specific
manner thereby only targeting promoters with a given consensus sequence [1]. Apart
from the DBD, TFs are characterized by having a transcription regulatory domain (TRD).
In contrast to the sequence similarities and well-defined structures of DBDs in members
of a specific TF family, TRDs have traditionally been classified according to their amino
acid profile as acidic, glutamine-, proline-, or serine/threonine-rich. Moreover, TRDs
often have a high degree of low-complexity sequences and propensity for flexible protein
segments that fail to self-fold into an ordered three-dimensional structure, commonly
referred to as intrinsic disorder (ID) [2].
Ongoing efforts to understand the structural and functional modularity of TFs have
traditionally been based on examination of the activity of truncated versions of the
proteins. Early in the dissection of TFs, it was found that the DBD could be separated
from the TRD with no loss of function of either module, as illustrated by the yeast GAL4
TF; a paradigm for eukaryotic transcriptional regulation studies [3]. Additionally,
Johnston et al. [3] showed that expression of a cytoplasmicly located version of GAL4,
without the N-terminal DBD, in a wild-type background enabled induction of the
expression of reporter genes to the same extent as expression of full-length GAL4. This
implied that GAL4 consists of separate domains conferring nuclear localization, protein-
protein interactions, DNA-binding, and transcriptional activation [3]. This seemingly
independent modularity has proved general to many TFs [4;5], and has led to the question
as to how functional specificity is substantiated.
Though a wealth of studies has aimed to characterize TF functionality, a few of them
should be highlighted as important attempts to shed light on functional specificity from
analysis of independent TF modules. In yeast, this question has been addressed by
deciphering regions within the transcriptional activator Rap1p (repressor activator
protein1p), showing that the C-terminal TRD has only a minor effect on cell growth
modulated by Rap1p [6]. Furthermore, domain swap experiments with combinations of
elements from DBDs of Rap1p homologues from different yeasts revealed that major
changes can be made to the amino acid sequence of this region without affecting Rap1p
function in telomere binding [6]. In another study, using a series of domain-swap
chimeres in which different parts of Drosophila melanogastor T-domain TFs, encoded by
the genes optomotor-blind (omb) and optomotor-blind related-1 (org-1), were mutually
exchanged, Porsch and co-workers investigated the relevance of individual domains for
proper eye development [7]. Their findings suggested that both transcriptional activation
and repression properties as well as DNA-binding specificity can contribute to the
functional characteristics of T-domain factors [7], highlighting the complexity of parsing
the functional specificity of seemingly independent TF domains.
Biochemical Journal Immediate Publication. Published on 08 Dec 2009 as manuscript BJ20091234
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20091234
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2009 The Authors Journal compilation © 2009 Portland Press Limited

4
In plants, the NAC (N
AM/ATAF1,2/CUC2) family constitutes a prominent group of TFs
[8]. The genomes of Arabidopsis thaliana (thale cress), tobacco and rice all contain more
than 100 genes encoding NAC TFs, making it one of the largest gene families in plants
[9;10]. Genes encoding NAC TFs were originally identified from forward genetic screens
as key regulators of developmental processes [8]. More recently, NAC TFs have also
been shown to be involved in regulation of stress responses in both model plants and
agronomically important crops [11;12].
We have previously identified the NAC protein ANAC019 as an interactor of the RING
(Really Interesting New Gene)-H2-type E3 ubiquitin-protein ligase, RHA2a, and solved
the crystal structure of the ANAC019 NAC domain [13;14]. This revealed a novel
protein fold consisting of a twisted E-sheet packing against an D-helix on both sides.
Moreover, using ANAC019 as a reference we have elucidated residues important for both
NAC homo- and heterodimerization and DNA-binding to the core consensus element
CGT[GA] [15]. Studies of ANAC019 gene expression have implicated a role in different
types of stress responses [14;16] and over-expression of ANAC019 caused increased
drought tolerance [16]. Moreover, it has been shown that ANAC019 regulates defense
responses upon fungal attack through a signal pathway related to the plant hormone
jasmonic acid [17]. Finally, Bu et al. [18], more recently verified our result on the
ANAC019-RHA2a interaction and coined RHA2a as a new positive regulator of abscisic
acid (ABA) phytohormone signaling [18].
With respect to parsing functional determinants of NAC DBDs and TRDs, Taoka et al.
[19], have shown that for members of the CUP-SHAPED COTYLEDON (CUC) sub-
family of NAC TFs the DBD confers functional specificity in terms of inducing
adventitious shoot formation on calli [19;20]. They showed that TRDs of three different
NAC TF origins could be fused to the NAC domain of CUC2 and still induce shoot
formations. Contrastingly, substituting CUC NAC domains with a NAC domain from a
member of the ATAF subfamily of NAC TFs did not induce shoot formation. However,
the NAC domain of the CUC subfamily member, CUC1, did not retain shoot formation
ability when fused to the potent TRD of the herpes simplex virus VP16 protein. This
suggested that certain structural features of the TRD which are difficult to predict from
the amino acid sequence were necessary for the function of CUC [19].
In this study, we have used a genome-wide approach taking advantage of the functional
diversity of the NAC proteins to dissect structure-function aspects of NAC TF
modularity. Our analyses showed that phylogenetic relationships based on NAC domain
sequences generally are in accordance with C-terminal sequence motifs of intrinsically
disordered TRDs and global NAC gene expression clusters. Additionally, our study
includes examination of truncated versions of ten phylogenetically distinct NAC proteins
to characterize the structural specificity of NAC DBDs and TRDs, and a chimeric-
strategy based on our identification of ANAC019 as a novel ABA regulator, to
investigate structure-function relationship of ANAC019 DBD and TRD. The findings
suggested multi-specificity of NAC TFs in terms of DNA-binding sites of target genes
and provide evidence for positive ABA-regulatory functionality to be associated with
both the ANAC019 TRD and DBD.
Biochemical Journal Immediate Publication. Published on 08 Dec 2009 as manuscript BJ20091234
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20091234
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "The arabidopsis thaliana nac transcription factor family: structure-function relationships and determinants of anac019 stress signaling" ?

Jensen et al. this paper investigated the functional specificity of gene specific transcription factors ( TFs ) and found that the TFs from the same family often bind DNA in a sequence-specific manner thereby only targeting promoters with a given consensus sequence. 

Q2. What future works have the authors mentioned in the paper "The arabidopsis thaliana nac transcription factor family: structure-function relationships and determinants of anac019 stress signaling" ?

Future systems-oriented temporal studies of selected tissues in response to environmental stresses are needed for an improved understanding of overlapping NAC gene expression patterns versus potential functional redundancy. Future studies using site-directed mutagenesis of SOG1, NTL8 and NAC2/ORE1 are needed to clarify this. All together, their results have laid out future directions for engineering of NAC TFs for an improved understanding and use of NAC networks and for investigating their role in plant stress perception. This suggested that powerful effects may result from engineering NAC TFs. 

Q3. What was used to search for motifs in the NAC proteins?

MEME (Multiple EM for motif Elicitation) was used to search for statistically significant motifs in the NAC proteins using either all NAC sequences or group and sub-group specific sequences as data set. 

Q4. What is the significance of the presence of NACBS in the promoter of all four genes?

The presence of NACBS in the 1kb promoter of all four genes (data not shown) highlights ANAC019 as a possible direct positive upstream regulator of ABA signaling genes. 

Q5. What are the main motifs of the CUC-like proteins?

Three motifs; LP (also named L; [19]), V and W, dominate the C-termini of the CUC-like proteins, which includes NAC2/ORE1 [34], of sub-group II-3. 

Q6. How many l of each dilution were spotted onto the plates?

5 l of each dilution was spotted onto SD/-Trp and SD/-Trp-His-Ade plates, which were then incubated at 30 °C for another seven days. 

Q7. What software was used for generating the alignments?

Multiple alignments were generated using ClustalW or ClustalX [21] followed by BoxShade (http://www.ch.embnetorg/software/BOX_form.html) for producing graphical presentations of the alignments and manual adjustments. 

Q8. What was the significance of the motifs identified?

In addition, the relevance of the motifs identified was evaluated by analysis of its occurrence in control groups, e.g. the NAC domain served as a control group for motifs identified in the C-terminal regions and vice versa. 

Q9. How long did the yeast cultures remain in the plates?

The transformed yeast cultures were plated onto SD/-Trp and SD/-Trp-His-Ade plates for seven days at 30 ºC before inspection of transactivation properties of the reporter constructs. 

Q10. What is the main reason for the lack of modularity of TFs?

Ongoing efforts to understand the structural and functional modularity of TFs have traditionally been based on examination of the activity of truncated versions of the proteins. 

Q11. What is the role of the ANAC019 DBD in ABA signaling?

The ability of the ANAC019 DBD to positively regulate ABA-signaling may be explained by its ability to dimerize with endogenous NAC domains and thereby enhance promoter binding [15] and subsequent activation of ABA-signaling genes. 

Q12. What is the role of ANAC019 in seedling development?

In summary, ANAC019(1-317) and truncated versions of ANAC019 displayed ABA-hypersensitivity during germination and early seedling development, suggesting that ANAC019 is a positive regulator of ABA-signaling. 

Q13. What is the effect of a substitution of ANAC019 on ABA signaling?

substituting ANAC019 TRD with SOG1 TRD could have a lethal effect on plants or hinder T-DNA insertion events, as no transformants were recovered from several attempts to ectopically express the ANAC019-SOG1 chimere (Figure 7B). 

Q14. What is the effect of ectopic expression of ANAC019 on root length?

In addition to confer reduced root length in plants expressing ANAC019, this ABA concentration has also been shown to compromise root length of other NAC members [28]. 

Q15. What are the tertiary structure information of the NAC proteins?

The divergent C-terminal regions of NAC proteins, for which no tertiary structure information is available, were also examined for sequence motifs (Figure 2B; Supplementary Table S2). 

Q16. What was used for the generation of chimeric constructs?

Vector pCAMBIA3300 and the primers and restriction enzymes listed in Supplementary Table S1 were used for generation of chimeric constructs for in planta expression. 

Q17. What is the significance of the ANAC003 and SOG1 sequences?

So far, ANAC003 and SOG1, or members of their clades, have not been characterized with respect to biochemical properties, and their sequences show a relatively low degree of similarity to the typical NAC domains. 

Q18. What was the binding pattern of GST-VOZ2(205-420) to palNA?

GST-VOZ2(205-420) also bound palNACBS, although with lower affinity than GST-ANAC019(1-168), which was also the case for both GSTNTL8(1-157) and GST-NTL6(1-168). 

Q19. What is the phylogenetic analysis of the NAC domains?

From the phylogenetic analysis of the NAC domains major sub-groups correlate with conserved motifs in the C-terminal TRDs of NAC proteins (Figure 2B and Supplementary Table 2). 

Q20. How did Johnston et al. study the ANAC019 TRD?

Though knowledge of how the isolated ANAC019 TRD exerts its function in vivo remains elusive, it is intriguing to acknowledge research performed more than two decades ago on the GAL4 transcription factor [3].