NAC transcription factors structurally distinct, functionally diverse

Transcriptional networks in plants

NAC transcription factors:structurally distinct,functionally diverse

Addie Nina Olsen 1,Heidi A.Ernst 2,Leila Lo Leggio 2and Karen Skriver 1

Institute of Molecular Biology,University of Copenhagen,?ster Farimagsgade 2A,DK-1353Copenhagen K,Denmark 2Department of Chemistry,Centre for Crystallographic Studies,University of Copenhagen,Universitetsparken 5,DK-2100Copenhagen ?,Denmark

NAC proteins constitute one of the largest families of plant-speci?c transcription factors,and the family is present in a wide range of land plants.Here,we sum-marize the biological and molecular functions of the NAC family,paying particular attention to the intricate regulation of NAC protein level and localization,and to the ?rst indications of NAC participation in transcription factor networks.The recent determination of the DNA and protein binding NAC domain structure offers insight into the molecular functions of the protein family.Research into NAC transcription factors has demon-strated the importance of this protein family in the biology of plants and the need for further studies.

Introduction

The investigation of transcription factor families is an important area of postgenomic research.NAC (NAM,ATAF1,2,CUC2)transcription factors (see Glossary)were ?rst described less than a decade ago.The NAC genes are speci?c to plants,in which they are abundant,with more than a hundred genes in Arabidopsis [1].Only a pro-portion of the NAC proteins have been studied to date and yet the family has been implicated in diverse processes,including developmental programmes [2–8],defence [9–12]and abiotic stress responses [11–14].The complex regu-lation of NAC transcription factors includes microRNA (miRNA)-mediated cleavage of mRNAs and ubiquitin-dependent proteolysis [15,16].Moreover,the structure of the NAC domain was recently determined,revealing a unique transcription factor fold [17].Here,we provide an overview of the current status of NAC research.

Biological functions of NAC proteins

Embryonic,?oral and vegetative development

The striking appearance of mutant phenotypes ?rst indicated the importance of the NAC gene family in plant biology.Most petunia (Petunia !hybrida )nam (no apical meristem )mutants lack the shoot apical meristem (SAM)and die at the seedling stage [2].Cotyledon fusions occur in these mutant seedlings,and plants developed from occasional escape shoots display aberrant ?oral development.NAM was the ?rst NAC

gene

Corresponding author:Skriver,K.(ks@apk.molbio.ku.dk).Available online 6January 2005

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to be characterized but was soon followed by the charac-terization of the Arabidopsis CUC2(CUP-SHAPED COTYLEDON2)gene[3].When combined,mutations in the CUC1and CUC2genes cause defects similar to the nam phenotype[3].The cuc1cuc2double mutants have fused cotyledons and the embryonic SAM is absent.When shoots are induced by regeneration from mutant calli, abnormal?owers are formed.

The mutant phenotypes and the expression patterns of NAM,CUC1and CUC2suggested a function for the gene products in boundary speci?cation and SAM formation [2,3].Thorough studies of the Arabidopsis CUC genes have since provided further information about the roles of NAC proteins in development[6,7,18–22].CUC1was found to encode a NAC-domain protein with high sequence similarity to CUC2[6].Functional redundancy was further demonstrated by the recent discovery that a third Arabidopsis NAC gene,CUC3,is involved in the establishment of the cotyledon boundary and the shoot meristem[7].

The cuc mutant phenotype prompted an investigation of the interaction between the CUC1and CUC2genes and the STM(SHOOT MERISTEMLESS)gene[6,18,21,22]. STM is a KNOTTED1-like homeobox(KNOX)gene involved in SAM formation and maintenance as well as in cotyledon separation[23,24].CUC1and CUC2were found to be required for STM expression during embryonic SAM formation[18](Figure1c).Furthermore,overexpres-sion of CUC1was shown to induce adventitious shoots on cotyledons through STM expression[6,22].Ken-ichiro Hibara et al.[22]examined the genetic interaction between CUC1and the AS1(ASYMMETRIC1)and AS2 genes,which are also important in SAM formation[25]. Their results indicated that CUC1also promotes SAM formation through an STM-independent pathway that is negatively regulated by AS1and AS2(Figure1d).AS1 encodes a MYB(myeloblastosis)domain transcription factor[26],and it is thus apparent from this single example of NAC gene function that research into the roles of NAC proteins in plant biology will contribute to an unravelling of transcription factor networks.Indeed,a recent study of cup(cupuliformis)mutants in Antirrhinum (snapdragon)showed that the CUP protein is involved in the establishment of aboveground organ boundaries and that it interacts with a TCP(TB1,CYC,PCF)domain transcription factor[8](Figure1b).Members of the TCP family of transcription factors are involved in the regu-lation of plant growth and development[27].Further-more,expression of an Arabidopsis NAC gene called NAP (NAC-like,activated by APETALA3/PISTILLATA)has been shown to be directly activated by a heterodimer of the APETALA3and PISTILLATA proteins,both of which are MADS(MCM1,AGAMOUS,DEFICIENS and SRF) box transcription factors essential for the speci?cation of ?oral organ identities[4].Other regulators of NAC gene expression have been identi?ed without evidence of immediate regulation.CUC1and CUC2spatial expression is affected by mutations in STM[6,18]and in PIN1(PIN-FORMED1),PID(PINOID)and MP (MONOPTEROS),genes involved in auxin signalling [20,28].Moreover,NAC1transcription is activated by NAC1[5]and CUC3transcription is stimulated by CUC1 and CUC2[7](Figure1a).

Lateral root formation and auxin signalling

A role in a different developmental program,the formation of lateral roots,has been demonstrated for NAC1.NAC1 was initially examined because of its predominant expres-sion in the root tip and in lateral root initiation points[5]. NAC1expression was shown to be induced by the hormone auxin[5],which is involved in lateral root production[29]. Over-and underexpression of NAC1increased or reduced lateral root formation,respectively.In addition,the auxin-responsive genes AIR3(AUXIN-INDUCED IN ROOT CULTURES3)and DBP(DNA-BINDING PROTEIN) were identi?ed in a screen for downstream targets of NAC1[5](Figure1c).DBP encodes a DNA-binding protein [30]and AIR3encodes a subtilisin-like protease that might weaken cell-to-cell connections to facilitate lateral root emergence[31].Detailed studies have demonstrated a speci?c activation of the AIR3promoter by NAC1[5]. Defence and abiotic stress

Several NAC proteins have been identi?ed because they interact with other proteins of biological importance.The wheat(Triticum sp.)geminivirus RepA-binding(GRAB) proteins GRAB1and GRAB2were identi?ed because of their ability to interact with the wheat dwarf geminivirus RepA protein[9],and Arabidopsis turnip crinkle virus (TCV)-interacting protein(TIP)was identi?ed because of its binding to the TCV capsid protein(CP)[10].TCV induces a hypersensitive response and systemic resistance in TCV-resistant Arabidopsis[32].The ability of TCV to induce resistance was dependent on interaction between TCV CP and TIP,suggesting that TIP is essential for the TCV resistance response pathway[10].

A function of NAC proteins in biotic stress responses has also been suggested by the induction of the potato (Solanum tuberosum)StNAC gene by Phytophthora infestans infection[11]and induction of several Brassica napus(rape)NAC genes by insect herbivory and fungal infection[12].The expression of several of these genes was also induced by abiotic stress such as wounding,cold shock and dehydration[11,12].Recently,it was reported that transgenic plants overexpressing three different Arabidopsis NAC genes(ANAC019,ANAC055and ANAC072)showed signi?cantly increased drought toler-ance[13].Furthermore,ANAC072{referred to as RD26 (RESPONSIVE TO DESICCATION26)in Ref.[14]}was shown to function in a novel abscisic acid(ABA)-dependent stress-signalling pathway[14].The ANAC genes belong to a subgroup of NAC genes de?ned by the wound-inducible ATAF1and ATAF2genes[11–14,33],and were upregulated by dehydration,high salinity and ABA, and some also by methyl jasmonate[13,14].In addition, the expression of ANAC072was shown to be induced by reactive oxygen species[14].Overexpression of the ANAC genes in transgenic plants revealed potential,stress-related target genes[13,14](Figure1c).ANAC072 transactivated the promoter of one of these genes, encoding a glyoxalase I family protein[14].These studies

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suggest that NAC proteins can be of importance for cross-talk between different pathways.

Expression analysis and NAC function

Expression analyses have substantiated the involvement of NAC genes in?ower development and reproduction [34–36]as well as in responses to hormones[37,38]and biotic[39]and abiotic[40–42]stress.NAC genes have also been implicated in light responses[43–46],programmed cell death[46,47]and senescence[48,49].

Regulation of NAC level and activity

Transcriptional regulation of NAC genes

Proper control of the level and the activity of transcription factors are essential,and the mechanisms regulating NAC activity are becoming apparent(Figure2).Knowledge of

Figure1.NAC transcription factors as regulators and regulatees.(a)Regulation of NAC gene expression.CUC1and CUC2gene expression is affected by mutations in the STM [6,18],PIN1,PID and MP genes[20,28].CUC3transcription is stimulated by CUC1and CUC2[7].The expression of several genes involved in shoot meristem formation affects CUC3spatial expression[7](not shown).TIR1and NAC1stimulate NAC1expression[5].NAP is a target of the AP3/PI MADS box heterodimer[4].A broken arrow indicates an effect on gene expression,whereas a solid arrow indicates that the gene is an immediate downstream target.Abbreviation:ARF,AUXIN RESPONSE FACTOR.

(b)Transcription factor interactions involving NAC proteins.Experimental data have demonstrated interactions between the NAC protein CUP and a TCP-domain transcription factor[8],homodimerization of NAC1[5]and the NAC domain of ANAC019[17,68],and heterodimerization of the Brassica napus NAC protein BnNAC14with BnNAC5-8,BnNAC485,BnNAC5-11and BnNAC3[12].BnNAC14also interacts slightly with BnNAC1-1,BnNAC18and the NAC domain of BnNAC5-7[12](not shown).(c)NAC target genes.CUC2and CUC1regulate the expression of the STM gene[18],and NAC1regulates the expression of the DBP and AIR3genes[5].ANAC072(RD26) transactivates expression of the GLY gene(encoding a glyoxalase I family protein)in vivo and microarray analysis identi?ed several genes regulated by ANAC072 overexpression or repression[14].Overexpression of ANAC019,ANAC055and ANAC072upregulated expression of partially overlapping groups of genes[13].(d)CUC1and shoot apical meristem(SAM)formation.CUC1promotes SAM formation both through an STM-dependent pathway and through an STM-independent pathway that is negatively regulated by AS1and AS2[22].

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transcription factors acting upstream of NAC genes is still limited (Figure 1a)but recent reports have supplied interesting examples of regulation at the transcriptional level.Studies of the transition from leaf cells to proto-plasts have suggested that the acquisition of pluripoten-tiality involves the activation of several silent NAC genes [50,51].A role in dedifferentiation is in accordance with NAC gene function in meristem development.Another example is the maize (Zea mays )endosperm NAC gene nrp1(NAM-related protein 1),which is regulated by gene-speci?c imprinting.Thus,paternally transmitted alleles are silenced,allowing maternal control of endosperm development [52].

Post-transcriptional control

miRNAs are small regulatory RNAs that pair with target mRNAs,thereby providing post-transcriptional repres-sion of the targets [53].Informatic analyses suggested that transcription factors involved in cell-fate determination are the predominant targets of miRNAs in plants [54].A subset of Arabidopsis NAC mRNAs,including CUC1,CUC2,NAC1,At5g07680and At5g61430,was initially predicted to be targeted by members of the miR164gene family [54],and CUC1and CUC2mRNAs were shown to be cleaved within their miR164complementary site [55].In addition,expression of miR164-resistant versions of CUC1and CUC2mRNAs,and overexpression of miR164proved that miR164is necessary for proper regulation of CUC1and CUC2[15,56].miR164-directed cleavage of NAC1,At5g07680and At5g61430was also detected,

further demonstrating the importance of post-transcrip-tional control of speci?c NAC genes [15,56](Figure 2).Phloem long-distance transport of the NAC mRNA CmNACP in pumpkin (Cucurbita maxima )represents another regulatory mechanism working at the RNA level [57].CmNACP mRNA is transported from the body of the plant to the shoot apex,a mechanism that might integrate physiological processes taking place in distant organs with developmental processes occurring in meri-stematic tissues [57](Figure 2).

Post-translational control

The repertoire of methods used to control NAC activity also includes post-translational regulation by ubiquitin-mediated protein degradation [16].Polyubiqui-tination of proteins through the sequential action of E1,E2and E3proteins targets them for degradation [58].A yeast two-hybrid screen identi?ed SINAT5(SINA of Arabidopsis thaliana 5)as an interaction partner of NAC1[16].SINAT5was shown to function as an E3ubiquitin–protein ligase and to target NAC1for proteasomal degradation,thereby attenuating auxin signalling (Figure 2).

TIR1(TRANSPORT INHIBITOR RESPONSE 1),which functions upstream of NAC1to regulate the NAC1transcript level [5](Figure 1a),also mediates ubiquitin-dependent protein degradation as a subunit of the SCF TIR1(Skp1,Cullin,F-box complex containing TIR1)E3com-plex.Auxin-promoted interactions between TIR1and auxin/indole-3-acetic acid (AUX/IAA)repressor proteins target the AUX/IAA proteins for degradation,allowing

Figure 2.Regulation of NAC protein level and localization.The regulatory mechanisms shown or hypothesized (indicated by a question mark)to affect NAC protein level or localization are listed on the right with arrows indicating the level at which the regulation occurs (DNA,mRNA or protein).Parentheses contain examples of NAC genes or proteins shown to be regulated by a particular mechanism as well as references to relevant articles,Figure 1,and Box 1.Abbreviation:HSINAC,HvSPY-interacting NAC;NES,nuclear export signal;NLS,nuclear localization signal.

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expression of auxin target genes[59,60].This shows that ubiquitin-dependent proteolysis can both promote and attenuate the auxin signal in lateral root formation.

Ubiquitin-dependent protein degradation might regu-late other NAC proteins.The Arabidopsis ANAC protein [61](renamed ANAC019[33])can interact with several different RING(really interesting new gene)domain E3s [61],and the expression of several NAC and RING genes is ABA inducible[38].

A barley(Hordeum vulgare)NAC protein was recently shown to interact with the SPINDLY(HvSPY)protein, a negative regulator of gibberellin responses.Based on sequence similarity,HvSPY is likely to be a protein N-acetylglucosaminyltransferase.This suggests that NAC proteins might also be regulated via modi?cation with N-acetylglucosamine[62](Figure2).NAC protein structure

Domains and molecular function

Database searches with the sequences of the cloned NAM and CUC2genes?rst revealed the presence of a conserved N-terminal region in the encoded proteins[2,3](Figure3). This family-de?ning domain was named the NAC domain [3].The C-terminal parts of NAC proteins are highly diverse and do not contain any known protein domains [33].However,sequence analysis does reveal?ve Arabidopsis NAC genes that appear to encode two NAC domains in tandem(At1g60280,At1g60300,At1g60340, At1g60350and At1g60380)as well as sequences predicted to contain transmembrane regions or nuclear import and/or export signals(Box1).

Elucidation of the molecular functions of NAC proteins began with the report of two NAC proteins being able to

Figure3.NAC domain sequences.Sequence alignment of the NAC domain of NAC proteins from a wide range of plant species.Red residues are common to at least half of the sequences,whereas blue residues are chemically similar in more than half of the sequences or similar to the dominating residue.OsNAC6(NAC gene from Oryza sativa6): Accession no.XP_463543,Oryza sativa(rice).StNAC:Accession no.CAC42087,Solanum tuberosum(potato).BnNAC14:Accession no.AAP35055,Brassica napus(rape). GRAB1:Accession no.CAA09371,Triticum sp.(wheat).ANAC019:Accession no.NP_175697,Arabidopsis(thale cress).CjAU084485:translation of expressed sequence tag (EST)sequence Accession no.AU084485,Cryptomeria japonica(Japanese cedar).PpBJ175589:translation of EST sequence Accession no.BJ175589,Physcomitrella patens subsp.patens(a moss).NAC1:Accession no.NP_175997,Arabidopsis(thale cress).PtCF663726:translation of EST sequence Accession no.CF663726,Pinus taeda(loblolly pine).NAM:Accession no.CAA63102,Petunia!hybrida(garden petunia).An arrow shows the position of the predicted two-residue insertion in the dysfunctional NAM protein,and asterisks mark the positions of the substitutions in the dysfunctional CUC1proteins.

Box1.Regulation of NAC localization

A range of molecular mechanisms regulates the localization of eukaryotic transcription factors,one of which is cytoplasmic seques-tration caused by the presence of a membrane anchor[73].Upon speci?c cues,membrane-bound precursors are proteolytically cleaved,allowing nuclear localization of the transcription factor.Two mechanisms of precursor cleavage have been described:regulated intramembrane proteolysis and regulated ubiquitin–proteasome-dependent processing[74].

When NAC proteins are analysed for the presence of transmem-brane regions using multiple prediction servers,several sequences are predicted to contain a transmembrane segment at their extreme C-termini.The membrane protein database ARAMEMNON(http:// aramemnon.botanik.uni-koeln.de/)provides the ability to compare the predictions of11different transmembrane span computation pro-grams for proteins from Arabidopsis and rice[75].The database contains19NAC proteins,14from Arabidopsis and?ve from rice (Oryza sativa).A subset of these database entries has a high degree of sequence similarity to turnip crinkle virus(TCV)-interacting protein (TIP),the Arabidopsis NAC protein required for the TCV resistance response[10].Some transmembrane prediction programs also predict that TIP contains a transmembrane region.It will therefore be interesting to determine the biological relevance of these in silico data. Nucleocytoplasmic shuttling guided by nuclear localization signals (NLSs)[76]and nuclear export signals(NESs)[77]can also regulate the activity of transcription factors.Five NAC proteins have been shown to localize to the nucleus[5,14,57,61,62],but the targeting signals responsible remain to be determined.Several the NAC sequences are predicted to contain an NLS when analysed by PredictNLS[78]and PSORT[79].However,no single conserved NLS site is discernable with these prediction tools.

Using the NES prediction tool NetNES[80],several NAC sequences are predicted to contain an NES in a region of the NAC domain rich in conserved hydrophobic amino acids,for example residues43–51in ANAC019.The properties of this region in the ANAC019NAC domain structure agrees well with the observations made in a structural comparison of known NESs[80].Experimental analysis of this and other possible localization signals is necessary for understanding the regulation of NAC localization.

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activate a cauli?ower mosaic virus(CaMV)35S promoter construct in yeast[2].Since then,three NAC proteins from Arabidopsis(NAC1,AtNAM and ANAC019)and several from Brassica napus have been shown to bind the CaMV 35S promoter[5,12,17,63].Recently,three ANAC proteins were shown to bind a fragment of the ERD1(EARLY RESPONSIVE TO DEHYDRATION STRESS1)pro-moter.Substitution analysis identi?ed the sequence CACG as the core DNA motif recognized by the ANAC proteins in the promoter fragment[13].The DNA-binding ability has been assigned to the NAC domain[5,17,63],but the mode of DNA recognition by the NAC domain is still unknown.

Assays in both yeast and plant cells have demon-strated that the C-terminal regions of several NAC proteins function as transcriptional activation domains [5,10,12–14,62,63].A common feature of NAC protein C-terminal regions is the frequent occurrence of simple amino acid repeats and regions rich in serine and threonine,proline and glutamine,or acidic residues [2,3,9,12,63,64].This feature and the sequence divergence are common characteristics of plant activation domains[65].

NAC phylogeny

The study of NAC proteins has so far focused on angiosperms.A phylogenetic analysis of105NAC proteins from Arabidopsis and75NAC proteins from rice(Oryza sativa)classi?ed the NAC domains into two groups,each containing sequences from both species,and several subgroups[33].Differences were observed between the two groups in NAC domain conservation and all the characterized NAC proteins belong to one group.Exami-nation of the C-terminal regions revealed short subgroup-speci?c motifs[33].The functional relevance of these conserved features is exempli?ed by the fact that one of the discovered motifs corresponds to miR164com-plementary sites.

The NAC gene family is not limited to monocots and dicots.Expressed sequence tag database searches reveal many NAC genes in conifers and,interestingly,in the moss Physcomitrella patens(Figure3).Recently,phylo-genetic pro?ling of the Arabidopsis proteome was used to identify proteins found solely within the plant lineage [66].A large proportion of the plant-speci?c proteins are transcription factors,indicating the importance of these proteins for the evolution of the plant lineage.The NAC proteins constitute one of the largest plant-speci?c families of transcription factors[1,66].Considering the presence of NAC genes in land plants ranging from mosses to eudicots,it will be of great interest to study the NAC gene family with regard to the evolution of plant development.

X-ray crystal structure

Recently,the structure of the NAC domain of ANAC019 was determined by X-ray crystallography[17].The NAC domain consists of a twisted antiparallel b-sheet that packs against an N-terminal a-helix on one side and a short helix on the other side(Figure4,centre).Tools for protein structure comparison have not revealed close structural homologues[17].The NAC domain does not possess any known DNA-binding motif but one face of the NAC domain is rich in positive charges and is probably involved in DNA binding[17](Figure4,bottom right).The central part of the NAC domain does share some struc-tural similarity with the large subdomain of the GCM (glial cells missing)DNA-binding domain,which is present in metazoans but not plants[67].Although the overall folds for the two proteins are different,it is possible that the two classes of transcription factors share some features of DNA recognition.

NAC proteins have been shown to homo-and hetero-dimerize,and the dimerization ability has been localized to the NAC domain[5,68]but can be in?uenced by the C-terminal regions[12].The NAC domain of ANAC019 preferentially forms a dimer in solution[68],and this is in accordance with the dimer seen in the crystalline state [17].The dimer contacts involve residues at the highly conserved N-terminal end of the NAC domain and consist of hydrophobic interactions,a short antiparallel b-sheet and two prominent salt bridges(Figure4,centre).The salt bridges are formed by the conserved arginine-19and glutamate-26.When these two residues are changed to alanine,the ANAC019NAC domain is found in its mono-meric form(A.N.Olsen et al.,unpublished).

Analysis of the genomic organization of the Arabidopsis NAC genes revealed conserved positioning of two introns in the NAC-domain-encoding sequence[63].The?rst exon encodes the sequence forming the dimerization interface and the a-helical segments of the domain,and the second exon encodes the b-strand core except for one strand, which is encoded by the50end of the third exon.It would be interesting to study intron positions of NAC genes from a range of land plants to gain insight into the evolutionary relevance of a possible correlation between exons and subdomains.

Structural interpretation of mutational changes

The mutational changes reported for dysfunctional NAC proteins[2,6]can now be evaluated based on structural information(Figures3and4).Thus,a recessive mutation in the NAM gene resulting from excision of the dTph1 transposable element is due to the addition of6bp to the gene[2].This inserts two extra amino acids into an N-terminal a-helix and abolishes NAM protein function, owing either to disruption of the dimer or to disruption of the interactions between the N-terminal helical region and the central b-sheet(Figure4,green).Mutant CUC alleles encoding single residue substitutions might also result in dysfunctional NAC proteins[6].The CUC1 mutant allele cuc1-1contains a mis-sense mutation that alters lysine-123to a threonine.This substitution might affect nuclear localization,DNA binding or the structural integrity of the NAC domain by abolishing a conserved salt bridge(Figure4,purple).The cuc1-4allele changes a leucine to a phenylalanine,a substitution that is likely to result in conformational stress and instability of the NAC domain by inserting a bulkier residue in a tight hydro-phobic pocket(Figure4,pink).CUC1mRNA was detected at normal levels in the cuc1-1mutant,suggesting that the mis-sense mutation is responsible for the mutant

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Lys35Glu48

Lys115

Glu136Leu59

N-terminal

C-terminal

Dimer interface

DNA

Figure 4.The NAC domain structure.The ANAC019NAC domain dimer (PDB code 1UT7)[17].Arginine-19and glutamate-26,which are involved in salt bridges at the dimer interface,are shown in ball-and-stick fashion.One monomer in the dimer is colour coded according to secondary structure:the two main helices in red,the main antiparallel b -sheet in blue and short extra helices and strands in yellow and cyan,respectively.The other monomer (in light grey)illustrates the positions of the amino acid residues corresponding to likely disruptive mutations in nam [2](green),in allele cuc1-1[6](purple)and in allele cuc1-4[6](pink).The coloured panels focus on the areas corresponding to the disruptive mutations,according to the colour code.(Green,top right)Lysine-35is the position preceded by the two-residue insertion in the predicted dysfunctional NAM protein.The orientation of the monomer in light grey is similar to the model in the centre,with the other monomer shown in black.The insertion could disrupt NAC protein function by disrupting dimerization or the important interaction between lysine-35in the helix (red)and glutamate-48in the central b -sheet.(Purple,top left)The view is rotated in the direction shown by the purple arrow compared with the orientation of the centre view.The mutation in allele cuc1-1corresponds to a substitution of lysine-115with threonine in ANAC019.The solvent exposure in this region is consistent with a role of lysine-115in nuclear localization or DNA binding.A water-mediated interaction with highly conserved glutamate-136might be important for the structural integrity of the monomer.(Pink,bottom left)This view is similar in orientation to the view in the centre.The mutation in allele cuc1-4corresponds to a substitution of leucine-59with phenylalanine in ANAC019.Because of the tight packing of

atoms around leucine-59,illustrated here by representation of residues within a 5A

?radius as van der Waals spheres,substitution with a bulkier aromatic residue is likely to cause signi?cant conformational stress in the structure.(Bottom right)The positively charged face of the NAC domain is likely to be involved in DNA binding (adapted from Ref.[17]).The NAC domain is shown in surface representation,in a similar orientation to the centre view,with positively charged residues mapped to the surface in blue and negatively charged residues mapped to the surface in red.Figure made with the programs Chimera [81](https://www.360docs.net/doc/9218198935.html,/chimera/)and Grasp [82].

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phenotype[6].However,expression of the mutant NAC proteins should be ascertained to con?rm the importance of the observed mutations.

Prospects for future research

In the few years since the discovery of the NAC tran-scription factor family,considerable knowledge has been gained about the physiological and molecular functions of NAC proteins.Nevertheless,this area of research is still in its infancy.The presence of large NAC gene families in a wide range of land plants makes the determination of NAC functions a daunting task.Future NAC research will therefore undoubtedly bene?t from large-scale approaches to functional analysis.

Identifying NAC target genes is of great interest. Overexpression of NAC genes combined with microarray analysis is one approach.Another is the identi?cation of transcription factor binding sites in genomic DNA using microarrays combined with chromatin immunopreci-pitation[69].Owing to functional redundancy,few knock-out mutants are likely to have informative phenotypes. As alternatives,RNA-induced gene silencing[70]and the chimeric repressor silencing technique[14,71] could be used.

In addition to functional genomics methods,improved understanding of NAC protein function will entail further studies of the molecular functions of these transcription factors.This will require detailed examination of the interactions of individual NAC proteins with DNA and with other proteins.Studying protein–protein inter-actions including viral proteins,RING proteins and TCP transcription factors could afford important information about plant defence processes,regulated protein degra-dation and plant development.Furthermore,the inter-action of NAC protein activation domains with the transcriptional machinery deserves attention.

The ability of NAC proteins to homo-and hetero-dimerize,and their interaction with TCP transcription factors suggest combinatorial regulation of transcription factor activity[72].Moreover,it is evident that NAC proteins carry out their function both downstream and upstream of other transcription factors.Hence,an essential goal of NAC protein research is to illuminate the complexities of transcription factor networks. Acknowledgements

Our work was supported by grants from the Danish Research Council and the Danish National Research Foundation(K.S.,L.L.L.and H.A.E.)and a PhD stipend from University of Copenhagen(A.N.O.).We thank John Mundy and Stanley Brown for reading the manuscript,as well as Anne M?lgaard and Michael K.Jensen for fruitful discussions.

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