Critical Review NAC Transcription Factor Gene Regulatory and Protein–Protein Interaction Networks in Plant Stress Responses and Senescence

Michael K. Jensen Karen Skriver*

Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, Copenhagen, Denmark

Abstract Plant-specific NAM/ATAF/CUC (NAC) transcription factors (TFs) have recently received considerable attention due to their significant roles in plant development and stress signaling. Here, we summarize progress in understanding NAC TFs in stress responses and senescence. We focus on interactions between the DNA-binding NAC domain and target genes, and between the large, mostly disordered transcription regulatory domain

of NAC TFs and protein interaction partners. Recent studies have identified both up-stream regulators of NAC genes and down-stream NAC target genes, outlining regulatory networks associated with NAC–protein interactions. This connects molecular interactions and signal pathway intersections with C 2014 biological functions with promising use in agriculture. V IUBMB Life, 00(00):000–000, 2014

Keywords: NAC transcription factors; stress response; senescence; gene regulatory networks; interactomes

Introduction Some transcription factor (TF) families are highly expanded, while others have restricted evolutionary proliferation. The NAM/ATAF/CUC (NAC) TFs have only been identified in plants—from mosses to angiosperms—with approximately 110 genes in the model Arabidopsis thaliana (1) and 150 genes in rice (2). Although NAC proteins were initially associated with development (3), they are increasingly appreciated for their roles in stress responses and senescence (4, 5). The most well-characterized, stress-associated NAC TFs belong to sub-group III-3 (1), also known as stress-responsive NAC (SNAC) (2). Three closely related members of this sub-

C International Union of Biochemistry and Molecular Biology V

Volume 00, Number 00, Month 2014, Pages 00–00 *Address correspondence to: Karen Skriver, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen, Denmark. Tel: 144-20642133; Fax: 145-35321567. E-mail: [email protected] Present address of Michael K. Jensen: Novo Nordisk Foundation Center  6, for Biosustainability, Technical University of Denmark, Kogle Alle DK-2970 Hïrsholm, Denmark. Received 6 February 2014; Accepted 28 February 2014 DOI 10.1002/iub.1256 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)

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group, ANA019, ANAC055, and ANAC072 (RD26), are induced by drought, high salinity, and the hormones abscisic acid (ABA) and jasmonic acid (JA). ABA controls many physiological processes in plants and is recognized for its role in abiotic stress responses (6), whereas JA is implicated in regulation of wounding and biotic stress responses (6). Overexpression of each of the three NAC TFs results in up-regulation of several stress-inducible genes and improved drought tolerance (7), and ANAC019 and ANAC072 function as positive regulators of ABA signaling (1, 8). In addition, ANA019 and ANAC055 are implicated in JA-signaling following pathogen infection (9, 10). Other sub-group III-3 NAC TFs are also implicated in abiotic and biotic stress responses (11). The most successful application of NAC TFs in crop engineering so far also involves a rice sub-group III-3 NAC TF, SNAC1, whose over-expression enhances drought resistance and salt tolerance in transgenic rice plants. Importantly, SNAC1 transgenics have higher seed set in the field than the negative control under severe drought stress conditions (12). Recently ANAC096 of sub-group IV-1 (1) was also shown to mediate ABA-dependent adaptation to dehydration and osmotic stresses in Arabidopsis (13). Thus, NAC TFs regulate abiotic stress tolerance and hold the potential for improving stress tolerance in transgenic plants. Uauy et al. (14) showed in 2006 that replacement of a non-functional NAC allele in modern wheat with a functional

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FIG 1

Structure of NAC proteins. (a) X-ray structure of the NAC019 NAC DBD in complex with the ANAC019 binding site 50 TCAGTCTTGCGTGTTGGAACACGCAACAGGGA-30 (PDB accession code 3SWP). Specific phosphates of the NACBS protected from cleavage in uranyl photo-footprinting in the presence of ANAC019 are shown by bold lines and the NAC binding core is underlined (24). (b) Schematic structure of an intrinsically disordered NAC C-terminus. Sub-group specific sequence motifs, representing putative protein interaction determinants, some of which may represent regions with local structure, for example, preformed structural elements or molecular recognition features, are shown. (c) ID and interactions of NAC TFs. ID was predicted using PONDR-FIT (25) with a threshold value of 0.5. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ancestral counterpart results in increased wheat protein, zinc, and iron content. This initiated great interest in the role of NAC TFs in senescence. Leaf senescence is a key process in plants and involves the action of a large number of TFs among which NAC TFs are prominent (15). ORESARA1 (ORE1)/ ANAC092/NAC2 and ORESARA1 SISTER1 (ORS1) of sub-group II-3 and AtNAP of sub-group III-2 are positive regulators of senescence (16–18), whereas JUNGBRUNNEN1 (JUB1) of subgroup V-1 is a negative regulator (19). For example, overexpression of ORE1 in transgenic plants triggers early senescence, and ORE1 functions by controlling the expression of various senescence-associated genes (SAGs) (16). ORE1 is one of several senescence-associated NAC TFs which is also implicated in abiotic stress responses (20, 21). In this review, we relate insights on NAC structure to specific interactions with target genes and other proteins in gene regulatory networks (GRNs) and protein–protein interaction networks relevant to stress responses and senescence. The focus is on selected Arabidopsis NAC TFs for which detailed molecular interactions have been characterized.

scription regulatory domains (TRDs) (Fig. 1). The crystal structure of the 168 amino acid residues NAC domain from Arabidopsis ANAC019 revealed a novel dimeric TF fold consisting mainly of b-sheets (22). Recently, interactions of the NAC domain with a palindrome of the seven base pair ANAC019 binding site (BS) (23) were probed both by crystallography and in solution (24). The NAC domain inserts an outer b strand with the highly conserved WKATQTD sequence into the major groove of DNA (Fig. 1a) in a manner similar to that of the plant-specific WRKY TF family and the mammalian family of glia cell missing (GCM) TFs. This mechanistic similarity may be explained either by a common evolutionary origin or mechanistic convergence of these TFs. Several conserved basic amino acid residues are within DNA-binding distance. However, changing of only Arg-88 into alanine resulted in significant impairment of DNA binding, suggesting that this residue is most import for DNA binding (24). This NAC–DNA model provides a platform for investigating NAC–DNA interactions and for engineering—possibly to redirect NAC TFs in GRNs.

NAC Structure as an Interaction Platform

Several recent studies have addressed NAC binding to synthetic oligonucleotides and target promoters using different methods (Table 1). Binding of ANAC019 has been investigated in several independent studies. The minimal sequence required for ERD1 promoter binding by ANAC019 contains the NAC core motif, as in the case of binding by ANAC055 and

The NAC Domain NAC TFs contain an N-terminal dimerization and DNA-binding domain (DBD), the NAC domain, and diverse C-terminal tran-

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What is the NAC Binding Site?

NAC Transcription Factor Gene Regulatory and Protein–Protein Interaction Networks

TABLE 1 NAC

DNA-binding by stress and senescence associated NAC proteins

Method

ANAC019 (055/072) Y1H

Synthetic DNA/target promoter (name)

Ref.

50 -ACATGCGTGTNNNNNNNGA-30 (ERD1)a

(7)

0

0

a

ANAC019

EMSA

5 -TGCGTGGACATGCGTGGACATGCGTGGACATGCGTGGACATGCG-3 (VSP1)

(9)

ANAC019

SELEX, EMSA

50 -WKNCGTR-30

(23)

ANAC019

X-ray, footprint 50 -TCAGTCTTGCGTGTTGGAACACGCAACAGGGA-30 0

0

(24)

ANAC019

EMSA

5 -CAGTCTTGCGTGTTGGAACACGCAACAGTC-3

(1)

ATAF1

PBM

50 -TVCGTR-30 and 50 -TTVCGT-30

(27)

0

0

ATAF1

ChIP

5 -. . .TTGCGTA. . .-3 (131 bp of NCED3)

(27)

ATAF2

EMSA

50 -AAATAAGAAGGCAAATATAATTTGATAACAACTATT-30 (35 bp of NIT2)

(28)

ORE1

SELEX, EMSA

50 -TWRCGTRDW-30

(23)

0

0

ORE1

EMSA

5 -CAGTCTTGCGTGTTGGAACACGCAACAGTC-3

(1)

ORE1

MUCb

50 -VMGTRNNNNN(N)YACR-30

(29)

0

0

ORE1

EMSA/CHiP

5 . . .-ACGTANNNNNCGTG-. . .3 (40/141 bp of BNF1)

(29)

ORS1

SELEX

50 -RCGTRNNNN(N)RYACGCAA-30

(17)

JUB1

SEELX

50 -TGCCGTNNNNNNNACG-30 50 -TGCCGTNNNNNNNCCGC-30 0

0

(19)

JUB1

EMSA/ChIP

5 -. . .GATGCCGTTAGAGACACG. . .-3 (40 bp of DREB2A)

(19)

AtNAP

Y1H

50 -. . .CACGTAAGT. . .-30 (52 bp of SAG113)

(30)

0

0

AtNAP

EMSA

5 -GCACGTAAGTGT-3

NTL6

ChiP

50 -TGGACCATGTATTTACAAAAACGTGAGATC-30

NTL4

ChIP, 50 -. . .TTATGTCGTACGAAA. . .-30 (AtrbohC) 50 -. . .TACGTGGCGTAATCC. . .-30 (AtrbohE) Activation as.

(32)

ANA096

EMSA

50 -. . .CACGTATGAAGCGTCTGTTTTCGTGGT. . .-30 (49 bp of RD29A)

(13)

CBNAC

EMSA/ChIP

50 -TAATAATGCTTAGTTATAAATTACT-30 (239 bp of PR1)

a

(30) (31)

(33, 34)

NAC core motif is underlined. Methylumbelliferyl b-D-cellobioside (MUC) substrate (29). W 5 AT; K 5 G, T; R 5 A, G; V 5 ACG; M 5 A, C; D 5 T, G, A; Y, C, T.

b

ANAC072 (7). This is in accordance with the sequence needed for binding of ANAC019 and ANAC055 to the VEGETATIVE STORAGE PROTEIN1 (VSP1) promoter (9). In addition, the NAC binding site (BS) TTNCGTA was obtained by systematic evolution of ligands by exponential enrichment (SELEX) (23). In all cases, the BSs contain the CGT[AG] core motif. However, biochemical analysis also shows decreased susceptibility of specific phosphates outside the core of the palindromic NAC BS to cleavage in uranyl photo-footprinting in the presence of ANAC019. This demonstrates that bases flanking the core NAC BS are also involved in binding (24) (Fig. 1a). A sequence similar to the ANAC019 BS was recently identified for ATAF1 using newer technology based on protein binding microarrays

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(PBMs), which represents an unbiased and sensitive strategy for searching for consensus BSs (26). This identified T[A,C,G]CGT[AG] and TT[A,C,G]CGTC as the most significant descriptors for ATAF1 binding. TTGCGTA is also present in the 131 bp promoter fragment shown by chromatin immunoprecipitation (ChIP) to be responsible for the binding of ATAF1 to its target gene 9-cis-epoxycarotenoid dioxygenase-3 (NCED3) (27). Interestingly, ATAF2, a close paralog of ATAF1 (1), binds an unrelated sequence in the nitrilase 2 (NIT2) gene (28). Originally T[TA][GA]CGT[GA][TCA][TA], also containing the NAC core motif, was selected as the preferred BS for ORE1 (23). A bipartite BS was later defined for ORS1 (17) and ORE1, and one of the sites of the bipartite BS is conserved in the

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ORE1 target promoter of BIFUNCTIONAL NUCELASE1 (BFN1) (29). The BS of JUB1 in the promoter of DEHYDRATIONRESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) (19), VND INTERACTING2 (VNI2) in the promoters of COLD-REGULATED (COR) and RESPONSIVE TO DEHYDRATION (RD) (20), and AtNAP in the promoter of SAG113 (30) also contain the NAC core motif. Interestingly, the relatively distant, membrane anchored NAC TFs NTL6 of sub-group I-1 and NTL4 of subgroup I-2 and ANAC096 also bind to promoter regions of the Pathogenesis-related1 (PR1) (31) gene, the Atrboh A, C, and E genes encoding ROS biosynthetic proteins (32), and the RD29A gene (13), respectively, which all contain the NAC core motif. In contrast, calmodulin (CaM)-binding (CB) NAC recognizes an unrelated site in the PR1 promoter (33, 34). In conclusion, NAC TFs involved in stress responses and senescence bind to a promoter region with the NAC core CGT[AG] motif. The different flanking sequences are also in contact with the NAC domain (24) and are likely determinants of both affinity and specificity of target promoter binding. ATAF2, which is also stress inducible (35), represents an exception and binds to an unrelated sequence in the NIT2 promoter encoding an auxin hormone synthesis gene (28). Based on NAC domain sequence characteristics (1), ATAF2 is also likely to be able to bind to the NAC core motif. However, sub-specificities may be used by the NAC TFs in specialized cases such as regulation of the auxin pathway. NAC BSs with less stringent or without a NAC core motif have also been identified for NAC TFs which are not primarily associated with stress responses and senescence (4, 33). Unraveling the GRNs controlled by TFs benefits from the identification of cis-regulatory elements to which they can bind. Thus, although the NAC BSs shown in Table 1 have not been identified in a systematic analysis for NAC BSs, the information may prove useful for identification of direct NAC target genes from accumulating data for NAC regulons.

The C-Terminal Transcription Regulatory Domains Although NAC TFs are grouped according to their N-terminal DBD structures, they also contain a TRD. Several reports have shown that the NAC C-termini function either as transcriptional activators (1, 7, 36) or repressors (33, 37). In a systematic analysis of the NAC TF family, 10 NAC proteins, representing functionally important and phylogenetically different clades and including ANAC019, ORE1, ATAF1, and NTL6, were analyzed for their ability to activate transcription in yeast. The transactivational activity of nine of these NAC proteins depends on their C-terminal regions (1). Truncation of the C-terminal regions showed that the activity depends on short, group-specific sequence motifs such as the WQ and LP motifs (36, 38). Jensen et al. (1) further dissected structure– function aspects of NAC TF modularity. Interestingly, not only full-length ANAC019 but also the ANAC019 DBD or TRD alone confers ABA hypersensitivity when ectopically expressed in plants, demonstrating the in vivo functional power of both of these domains. Domain-swapping experiments showed that chimeric proteins in which the ANAC019 NAC domain is

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replaced with the analogous domain from other NAC TFs also functions as positive regulators of ABA signaling. In contrast, replacing the ANAC019 TRD with other NAC TRDs abolished ANAC019-mediated ABA hypersensitivity, demonstrating in vivo functional specificity of a NAC TRD (1). However, Taoka et al. (36) in 2004 showed that fusing TRDs from three different NAC TFs to the NAC domain of CUC2 maintained the shoot-inducing functionality of native CUC2. This suggests that domain-specific features associated with NAC TF functional specificity are difficult to predict. In contrast to the structurally well-defined DBDs of TFs, the TRDs have a propensity for flexible protein segments that do not form fixed tertiary structures. This feature is known as protein intrinsic disorder (ID) (39). Systematic in silico analysis of 10 NAC TFs suggested that their TRDs are mostly disordered (Fig. 1b) and contain short conserved subgroup-specific sequence motifs (1). The motifs are often located in a short structured region within a large disordered region or at the border between ordered and disordered regions (1). Such motifs may represent sites for protein interactions in the form of molecular recognition features (40) with a propensity for target-induced folding or pre formed structural elements (41). Experimental analysis of ID in plant proteins is still limited (42) and has only been experimentally addressed for two NAC TFs, barley senescence-associated HvNAC005 and HvNAC013. These TFs were shown by biophysical analysis to be mostly disordered (38). Furthermore, HvNAC013 interacts with barley RADICAL-INDUCED CELL DEATH1 (RCD1) via the very Cterminal part of its TRD. Surprisingly, a more extended conformation may form in the NAC TRD upon interaction with RCD1 (38). Thus, fuzziness, defined as disorder in the partner bound state (43), may characterize the interaction of the HvNAC013 TRD with RCD1. Clearly, a goal is to improve structural information on the NAC TRDs to develop knowledge for future engineering of these malleable and powerful NAC TRDs. Whereas knowledge about NAC gene regulatory networks has increased dramatically in recent years (4), a similar gain of knowledge has not been obtained for NAC–protein interactions. Many interactions can be expected for the large disordered NAC TRDs (Figs. 1b and 1c) which, due to their flexibility, may be able to adapt to several structurally diverse interaction partners (42). The low number of interaction partners identified so far may reflect the challenges of identifying interaction partners of self-activating protein regions using yeast two hybrid screenings, and of regulatory proteins participating in transient interactions using protein purification methods.

NAC Gene Regulatory and Protein Networks in Stress Responses and Senescence Identification of Specific NAC Target Genes Several approaches have been used for identification of NAC downstream regulons. Microarray analyses have addressed

NAC Transcription Factor Gene Regulatory and Protein–Protein Interaction Networks

FIG 2

Strategies for identification of direct NAC target genes. (a) Identification of ATAF1 target genes combining PBM-based identification of ATAF1 binding sites and ATAF1 co-expression analysis under perturbation. ChIP verified the suggestion from initial analyses that NCED3 is a direct target gene of ATAF1 (27). (b) Identification of ERD1 as a direct target of ANAC019/055/072. Identification of ERD1 promoter binding TFs exploited the ability of fusions between yeast GAL4 AD and transcription factors encoded by an Arabidopsis expression library to activate expression of the marker genes HIS3 and lacZ by binding to copies of an ERD1 promoter fragment fused to the HIS3 and lacZ genes (7). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

putative downstream NAC targets (7, 16). However, within the last few years many direct NAC targets genes have also been identified using different approaches. The approach used for identification of the ATAF1 target gene NCED3 was based on the ATAF1 BS determined by PBM analysis (27) in combination with co-expression analysis (Fig. 2a). The relevance of coexpression analysis is supported by the observations that coexpression occurs among TFs and their target genes and that co-expression clusters can be enriched for common TF binding sites (15). The analysis resulted in 25 top-ranking genes coexpressed with ATAF1 showing strong induction by ABA, drought, osmotic, and salt stresses. These genes were analyzed for the PBM-derived ATAF1 BS in their promoters. This identified the key regulatory ABA biosynthetic gene NCED3 as a likely ATAF1 target. Furthermore, an ATAF1 BS was identified in the NCED3 promoter region that was enriched in ChIPs from ATAF1 overexpressing plants (27). A different strategy using yeast one-hybrid screening also proved useful for identification of direct NAC target genes, as demonstrated by the identification of ANAC019, ANAC055, and ANAC072 as ERD1 binding and regulating TFs (7). In this system, identification of ERD1 promoter binding TFs was dependent on the ability of fusions between the yeast GAL4 activation domain (AD) and a protein from an Arabidopsis expression library to activate expression of the marker genes HIS3 and lacZ (7) (Fig. 2b).

Sub-Group III Networks: From Stress Responses to Senescence The first indications of a NAC-centered GRN came from the identification of ANAC019, ANAC055, and ANAC072 as direct regulators of ERD1, which encodes a Clp protease regulatory subunit (Fig. 3d) (44). Overexpression of one of the three NAC genes is not sufficient for up regulation of ERD1 (7), but depends on co-expression with the zinc finger homeodomain TF ZFHD1.

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ZFHD1 also recognizes a cis-acting element in the ERD1 promoter and is up-regulated by drought, high salt, and ABA (45). This suggests cooperative regulation of stress responses by different TF families in drought tolerance (Fig. 3a) through physical interaction between the NAC and the ZFHD1 TFs (45). Microarray analysis of transgenic plants over expressing one of the three NAC TFs revealed up-regulation of the expression of several stress and ABA-inducible genes including RD29B, COR47, ERD11, and FER1 genes (1, 7) further supporting the role of these TFs in abiotic stress signaling. The first direct up-stream regulators of these three NAC TFs were recently identified in a yeast one-hybrid screen (21). The key ABA-regulating bZIP TFs, ABA RESPONSIVE ELEMENT BINDING FACTOR3 (ABF3), and ABF4 (46), were identified by their ability to bind to NAC promoter fragments, in accordance with the ABA-association of the three NAC TFs (1, 7, 47). ANAC019 and ANAC055 also interact with another key mediator of ABA-signaling, ABI4, an AP2 family TF (48). Five members of the MYB TF family, MYB2, MYB21, MYB108, MYB112, and MYB116 also bound to the promoter fragments. In contrast, the four AP2 TFs, C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT BINDING FACTOR1 (CBF1), CBF2, CBF3, and CBF4 bound to only the ANAC072 promoter. Modeling of expression and mutant analysis suggested further differential contribution to the regulation of the expression of the three NAC genes by potential regulators in stress responses and senescence. For example, of the MYB TFs which bind to the promoters of all three NAC TFs, only MYB2 and MYB108 are implicated in senescence, and although all four CBF TFs can regulate ANAC072, they may play different roles in binding under cold, osmotic and salt stresses, senescence, and during Botrytis cinerea infection (21). Biotic stress GRNs are also appearing for the three NAC TFs. ANAC019 and ANAC055 are implicated in JA-dependent

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FIG 3

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Gene regulatory and protein–protein interaction networks of NAC TFs in stress responses and senescence. Direct interactions between the NAC TFs and DNA are shown by arrows (positive effect on expression) or blocked lines (negative effect on expression). Protein–protein interactions are shown by straight lines, and agonistic effects of these interactions are shown by arrows, whereas antagonistic effects are shown by blocked lines. Dashed arrows and lines refer to effects which may involve additional components. (a) Sub-group III-3 ANAC019, ANAC055, and ANAC072 GRN main associations are: MYC2 with JA-signaling and pathogen defense; MYBs, ABFs, and ABI with ABA-signaling and abiotic stress responses, CBF1, 2, and 3 with cold responses, and CBF4 also with ABA. Up-regulation of sub-group III-II ANAC019, ANAC055, and ANAC072 improves drought tolerance either directly or through interactions with ZFHD1. (b) Sub-group III-3 ATAF1 GRN. (c) Sub-group III-3 ATF2 GRN. (d) Sub-group III-2 AtNAP GRN. (e) Sub-group III-2 VNI1 GRN. (f) Sub-group V-1 JUB1 GRN. (g) Sub-group II-3 ORE1 GRN. (h) Sub-group IV-1 ANAC096 GRN. (i) Sub-group I-1 NTL6 GRN. (j) Sub-group I-2 NTL4 GRN. (k) Sub-group I-1 CBNAC GRN.

NAC Transcription Factor Gene Regulatory and Protein–Protein Interaction Networks

defense responses with their MeJA-induced gene expression depending on the JA-signaling components CORONTATINE INSENSITIVE1 (COI1) and the basic helix–loop–helix TF AtMYC2. In addition, over expression of ANA019 partially rescues the phenotype of the atmyc2-2 mutant (9). The phenotype of the anac019anac055 double mutant, which exhibits decreased expression of the JA marker genes LIPOXYGENASE 2 (LOX2) and VSP1 (a direct ANAC019 target), also shows increased resistance to the necrotrophic pathogen B. cinerea (9). Recently, ChIP was used to show that ANAC019, ANAC055, and ANAC072 are direct in vivo targets of MYC2 (49). Coronatine, a toxin produced by the pathogenic bacterium Pseudonomas syringae and mimicking JA isoleucine, activates the three NAC genes. The NAC TFs mediate coronatine-induced stomatal reopening and bacterial propagation by inhibiting accumulation of the immune signaling hormone salicylic acid (SA), thereby promoting pathogen virulence. The NAC TFs exert this inhibitory effect by binding (at least in the case of ANAC019) to and repressing ISOCHORISMATE SYNTHASE1 (ICS1) and by activating S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE (BSMT1) involved in SA biosynthesis and metabolism, respectively. Together these studies elucidate the complexity and intersections of the GRNs of ANAC019, ANAC055, and ANAC072 involving ABA- and JA-signaling and enhancing and repressing adaptive abiotic and biotic stress responses as well as developmental senescence signaling (Fig. 3a). ANAC019 was originally identified as an interaction partner of the small RING-H2 protein RHA2a (47), a positive regulator of ABA-signaling, and a functional E3 ubiquitin ligase (50). Additional RING-H2 proteins also interact with ANAC019 in yeast two-hybrid assays (Fig. 3a) (47). However, it remains to be determined if these proteins regulate ANAC019 by ubiquitin-dependent degradation. A putative function of the interaction between ANAC019 and CTD phosphate-like1 (CPL1), which is essential for regulation of stress-responsive gene expression, also remains elusive (51). Thus, a functional role has so far only been revealed for the interaction with ZFHD1. The NAC DBD has been shown to mediate the interaction with RHA2a and ZFHD1 (Fig. 1c), demonstrating the ability of this domain to interact with molecules other than DNA. Clearly, additional interaction partners, also targeting the disordered TRDs, can be expected. ATAF1 targets the ABA-biosynthesis gene NCED3 in vivo, and ATAF1-mediated induction of NCED3 expression correlates with increased ABA levels (Fig. 3b) (27). Plants overexpressing ATAF1 have reduced stomatal aperture (11), and transient expression of NCED3 in guard cells also decreases stomatal aperture (52) suggestive of overlapping physiological functions of ATAF1 and NCED3. Thus, ATAF1 may regulate ABA biosynthesis in a positive feed-back loop by binding to and activating the expression of NCED3. Plants overexpressing ATAF1 display stunted growth and delayed flowering, which is also the case for ABA-hypersensitive plants overexpressing the ATAF1 interaction partner kinase SnRK1.1/

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AKIN10 (53, 54). This interaction is likely to involve the relatively disordered TRD of ATAF1 (53) (Fig. 1c). Future studies should address the functional and physiological consequences of this interaction in planta. However, ATAF1 seems largely dispensable for SnRK1 signaling as ataf1 mutant plants display wild-type phenotypes (27). In addition to NCED3, several other putative ATAF1 targets show ATAF1-dependent expression perturbations. This is the case for ABI2, which as a typical clade A type 2C protein phosphate (PP2C) functions as a negative regulator of ABA signaling by dephosphorylating ABAactivated kinases (55). Although speculative, the positive correlation between ATAF1 and ABI2 may reflect a negative feedback loop to decrease endogenous ABA levels associated with ATAF1 induction to limit accelerated ABA signaling and thereby physiological defects (27). ATAF2, closely related to ATAF1, is also induced by stresses (Fig. 3c) (35). Recently, ATAF2 was shown to be involved in auxin hormone biosynthesis via regulation of NIT2 (28) encoding an enzyme converting indole-3-3acetonitril (IAN) into auxin in a pathway that can be activated by wounding and pathogens (56). Expression of both ATAF2 and NIT2 was induced by IAN treatment, and ATAF2 activates the promoter of the NIT2 genes by direct binding. Further studies are needed to clarify how ATAF2 contributes to tryptophandependent auxin biosynthesis, which can be activated by wounding and pathogens, and if this is related to plant immunity (28). The involvement of group III NAC TFs in senescence is clear from analysis of AtNAP (Fig. 3d). A T-DNA insertion knockout of AtNAP displays a delay in leaf senescence, and overexpression of AtNAP in young leaves causes precocious senescence suggesting that AtNAP is a positive regulator of senescence (18). SAG113 is a direct target of AtNAP (30), and both AtNAP and SAG113 are induced by leaf senescence and ABA. SAG113, which encodes a Golgi-localized protein phosphatase 2C family protein phosphatase, mediates inhibition of stomatal closure resulting in water loss, and triggering senescence in leaves (30, 57). The expression of VNI2 is induced by senescence and by salt in an ABA-dependent manner, and overexpression of VNI2 enhances resistance to salt stress and prolongs leaf longevity, similarly to the direct target genes COR15A, COR15B, RD29A, and RD29B (Fig. 1e) (20). Thus, VNI2 integrates ABA-mediated abiotic stress signals and ageing. VNI2 is also an interaction partner of VASCULAR-RELATED NAC-DOMAIN7 (VND7) associated with programmed cell death (PCD) (37), suggesting that abiotic stress adaptation, senescence, and PCD are integrated via a VIN2 centered GRN. In conclusion, several group III NAC TFs are convergence points for different signaling pathways related to stress and senescence, and regulated by several different phytohormones.

JUB1: A Negative Regulator of Senescence JUB1, a reactive oxygen species (ROS)-responsive NAC TF, delays senescence (Fig. 3f) (19). The TF decreases intracellular

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H2O2 levels and activates the AP2 TF gene DREB2A which is important in abiotic stress responses (58). DREB2A targets the RD29A (58) and HsfA3 (59) genes, and HsfA3 in turn regulates the expression of genes encoding HEAT SHOCK PROTEINS (HSPs) and H2O2 scavenging enzymes, and has been suggested to be part of a positive feedback loop together with HsfA1e and HsfA2 (60). A model was proposed in which JUB1 acts through the HsfA3-associated feedback loop in lowering the intracellular H2O2 level, resulting in prolonged longevity and increased stress tolerance (19).

ORE1 Networks As a key senescence-control TF ORE1 directly targets the BFN1, SAG29/SWEET15, and SINA1 genes (29), relating ORE1 to different physiological functions (Fig. 3g). ORE1 and BFN1 have overlapping expression patterns, and ANAC092 and BFN1 were suggested to constitute a regulatory cascade resulting in nucleic acid degradation during senescence and PCD (29). SAG29, encoding a putative sugar transporter, accelerates senescence when overexpressed (61), possibly by altering sugar transport, and SINA1, encoding a putative E3 ubiquitin ligase, may mediate protein ubiquitination and degradation. ORE1 itself is controlled by mRNA degradation by the transacting micro(mi)RNA164 (62). miR164 in turn is negatively regulated both by ETHYLENE INSENSITIVE 2 (EIN2), central to ethylene signaling (62), and by its downstream target gene EIN3, encoding a key TF in ethylene signaling which directly represses miR164 accumulation (63). This again allows ORE1 mRNA accumulation (63). The Golden2-like TFs, GLK1 and GLK2, were recently shown to interact with the ORE1 TRD (64) (Figs. 1c and 3f). GLK1 and GLK2 are important for chloroplast development and maintenance and thereby for leaf maintenance (65). Whereas the interaction does not affect the transactivating ability of ORE1, it antagonizes GLK transcriptional activity. A model was proposed in which ORE1 accumulation and interaction with the GLKs at late stages of senescence result in ORE1GLK heterodimers incapable of activating photosynthetic GLK target genes (64). This regulatory interaction reveals a novel mechanism for fine tuning senescence and demonstrates the functional power of the ORE1 TRD in protein–protein interactions.

ANAC096: A Novel Player in Intersecting Abiotic Stress GRNs The involvement of ANAC096 in ABA-mediated stress responses was suggested from a screen for mutants with ABAhyposensitive phenotypes (Fig. 3h) (13). ANAC096 functions as a positive regulator of ABA-responsive signaling under dehydration and osmotic stress, and a major proportion of ABAresponsive genes are transcriptionally regulated by ANAC096. It directly interacts with ABF2 and ABF4 (Fig. 1c), and ANAC096 and ABF2 synergistically activate RD29A transcription. Furthermore, the anac096 abf2 abf4 triple mutant is more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant.

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ANAC096 was therefore suggested to synergistically activate, with the ABFs, ABA-inducible genes in response to dehydration and osmotic stress (13). This is one of several examples of NAC TF interplay with other types of TFs which may be essential for rapid and specific regulation of gene expression upon stress exposure. In addition to enhancing specificity, such mechanisms allow synergistic effects from different pathways involved in different functions. Interestingly, ANAC096 interacts with ABF2 and ABF4 but not with ABF3 (13). The explanation for this differentiation of specificity remains elusive. The fact that ANAC096 has an agonistic, and ANAC019 an antagonistic relationship with ABF2 with respect to RD29A expression, also raises a biochemical question. As the NAC domains are highly conserved (1), ANAC096 and ANAC019 may use their mostly disordered and variable TRDs for the interaction with the ABFs (13). This region of ANAC096 has also been suggested to be responsible for the interaction with the corepressor protein TOPLESS (TPL) (13).

Networks of Membrane-Anchored NAC TFs The plasma membrane-anchored NTL6 of sub-group I-1 is proteolytically processed upon exposure to cold and migrates to the nucleus, where it induces the expression of the direct target genes PR1, PR2, and PR5, indicating that NTL6 mediates cold-induced pathogenesis (Fig. 3i) (31). In addition, ABA promotes NLT6 processing, and transgenic plants overexpressing processed NTL6 are hypersensitive to ABA and high salinity (66). Taken together, these observations indicate that NTL6 integrates plant responses to both biotic and abiotic stress conditions (66). It was recently shown that Snf1-related protein kinase (SnRK) 2.8-mediated phosphorylation of NTL6 is necessary for nuclear import of NTL6 (67). Overexpression of NTL6 results in enhanced drought resistance, as does overexpression of SnRK2.8, and SnRK2.8 knock-out compromises the effect of NTL6 overexpression. This suggests that the SnRK2.8-mediated phosphorylation of NTL6 is important for the NTL6mediated induction of drought-resistance (67). The ABA, drought, and heat inducible NTL4 gene encodes a sub-group I-2 membrane-anchored NAC TF which promotes ROS production during drought-induced senescence in Arabidopsis (Fig. 3j). NTL4 directly up-regulates the Atrboh (A, C, and E) genes encoding ROS biosynthetic proteins (32). Leaf senescence is accelerated in transgenic plants overexpressing proteolytically processed NTL4 under drought conditions, and these transgenics are hypersensitive to drought and ROS accumulates in their leaves. These observations indicate that NTL4 regulates ROS generation as part of processes controlling drought-induced leaf senescence in Arabidopsis (32). In conclusion, membrane-anchored NTL TFs are nodes connecting different signaling pathways of abiotic and biotic stress responses and of abiotic stress responses and senescence. An interesting mechanism of suppression of basal pathogen resistance has been reported for CBNAC/NTL9 whose membrane anchoring is debated (Fig. 3k) (33, 68). cbnac1

NAC Transcription Factor Gene Regulatory and Protein–Protein Interaction Networks

mutants display enhanced resistance to the bacterial pathogen P. syringae (Pst) through relief of PR1 repression. SUPPRESSOR OF NONEXPRESSOR OF PR GENES1, INDUCIBLE1 (SNI1), also a negative regulator of PR1 expression, interacts with CBNAC and enhances binding of CBNAC to the PR1 promoter. Thus, CBNAC and SNI1 could function synergistically as negative regulators of PR1 expression and disease resistance to prevent detrimental effects of constitutive activation of defense on plant growth (34), or they could be Pst effector targets guarded by specific Arabidopsis R genes. The C-terminal of the NAC TF is also used for their interaction in this case (Fig. 1c).

Conclusion Recent knowledge of NAC-TF centered gene regulatory and protein–protein interaction networks in stress responses and senescence has revealed novel aspects of the biological roles and molecular mechanisms of these powerful TFs. These regulators are themselves regulated transcriptionally and posttranscriptionally at the RNA level, as well as through protein– protein interactions, post translational modifications, and possibly regulated protein turn-over. Structure analysis has revealed how the NAC domain targets both bases from the NAC core BS and from flanking regions present in target genes mediating diverse processes important to plant growth and production. It has also recently become clear from studies of HvNAC013, ORE1, ATAF1, and CBNAC how the mostly disordered NAC TRDs possess enormous potential for regulatory interactions. In particular, the interactions of NAC TFs with unrelated TFs raise the question of how and why plants apply such interactions in stress responses. Under stress, plants experience dramatic transcriptional reprogramming to improve survival. Here, it may be a clear advantage to use multiple pathways. In addition, the physiologically important cooperative interactions between NAC TFs and unrelated TFs may affect both DNA binding affinity and specificity through interactions with several cis-acting promoter elements and activity of the interacting TFs. Future research should illuminate the structural basis of these interactions. In general, the NAC-centered networks reveal intersections between pathways involved in abiotic stresses, biotic stresses, senescence, and PCD. Near future research in the NAC field should further address NAC-centered GRNs using systems biology and modeling strategies and structure-defined yeast 2-hybrid or in planta screenings. Detailed understanding of these molecular networks is a prerequisite for engineering of NAC TFs which represent an enormous future breeding potential for agriculture.

Acknowledgements This work was supported by the Danish Agency for Science Technology and Innovation grant numbers 274-07-0173 (to K.S.)

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