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The Dof fa m ily of plant transcription factors

Shuichi Yanagisa w a
Dof proteins are m e mbers of a m ajor fa m ily of plant transcription factors. The proteins have si m ilar DNA-binding properties because of the highly conserved DNA-binding dom ain. How ever, recent studies are disclosing their diverse roles in gene expression when associated w ith plant-specific phenom ena including light, phytohormone and defense responses, seed developm ent and germ ination. Based on the structural diversity indicated by the complete catalog of Arabidopsis Dof proteins, Dof genes appear to have evolved multiple ti m es, preceding and paralleling the diversification of angiosperm s. Such gene multiplication m ight have led to the functional diversification of Dof proteins proceeding differently in distinct plant species.

Plant gene expression involves classes of transcription factors that have specifically evolved to regulate plant-specific genes and/or to mediate a variety of plant-specific signals. The Dof family is a typical example of such transcription factors. Many Dof proteins have already been discovered in both monocots and dicots. Currently, we know only a fraction of their biological roles. However, it is evident that Dof proteins play vital roles in plant gene expression. In the complete Arabidopsis genome sequence, 37 putative Dof genes are discernible [1]. The presence of so many Dof genes suggests that multiplication of Dof genes might be linked with the formation of sophisticated transcriptional control in higher plants.
What are Dof proteins?

acid sequences in the proteins are divergent, coinciding with their expected diverse functions. The inhibitory effects of the metal chelators and the absolute requirement of cysteine residues for putative zinc coordination were repeatedly demonstrated in studies of the DNA interaction of several Dof proteins [6,8,11,14]. Furthermore, two aromatic amino acid residues (Tyr and Trp) conserved among all Dof domains are necessary for sufficient DNA-binding of a pumpkin Dof protein [15]. However, whether the Dof domain includes a zinc finger has still to be determined. By contrast, the assumption that Dof proteins are unique to plants has been verified in a recent comparative study using the complete genome information from Arabidopsis, Drosophila, Caenorhabditis elegans and yeast [1].
Dof proteins as transcription factors

Shuichi Yanagisa w a Dep t of L i fe S ci en c es , Gradua t e S choo l of Art s and S ci en c es , The U n i vers it y of To k yo , Ko m aba , M eguro , To k yo 153-8902 , Japan . e-m a il: c syanag @ m a il . e cc . u-t o k yo . a c . j p

The first Dof protein (ZmDof1) was found as a DNA-binding protein of maize [2]. The DNA-binding domain in ZmDof1 includes a C2C2-type zinc-fingerlike motif, although the amino acid sequence of this domain is largely different from those of other zinc-finger domains (Fig. 1a). The divalent metal chelators, 1, 10-o-phenanthroline and EDTA, inhibited the DNA-binding of ZmDof1, and mutations on cysteine residues that had been expected to coordinate to a zinc ion also prevented the DNA-binding activity [3]. This suggested that the DNA binding domain contained a variant of zinc fingers, and it was therefore referred to as the Dof (DNA-binding with one finger) domain [3,4]. During recent years, >20 cDNAs encoding a Dof domain have been isolated from maize and other plant species, including Arabidopsis, tobacco, pumpkin, potato, barley, wheat and rice [3,513]. Dof proteins, which are typically composed of 200400 amino acids, are now defined as DNA-binding proteins that have a highly conserved Dof domain. In spite of intensive homology in the Dof domain, the rest of the amino
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Co-transfection experiments with expression vectors for Dof proteins and their putative target promoters fused to reporter genes have provided direct evidence that Dof proteins function as transcriptional activators or repressors through direct interaction with DNA. For instance, ZmDof1 enhanced reporter gene expression depending on the binding sites in the promoters, whereas co-expression of another maize protein (ZmDof2) suppressed the positive effect of ZmDof1 in maize mesophyll protoplasts [16]. A barley Dof protein (BPBF) also activated the transcription from a putative target promoter in particle-bombarded endosperm, but BPBF with mutations on the cysteine residues for putative zinc coordination did not [9]. In accordance with its activity as a transcription factor, nuclear localization of ZmDof1 was also shown [16,17]. Interestingly, the transcriptional activation domain of ZmDof1 can function not only in plant cells but also in animal cells and in yeast [17]. Like aromatic residues in the transcriptional activation domains of animal factors GATA-4 and VP16, a tryptophan residue in the transcriptional activation domain of ZmDof1 is of particular importance for the function [17]. In addition, the C-terminal part of BPBF also functions as a transcriptional activation domain in yeast [18]. Therefore, although Dof proteins are unique to plants, some Dof proteins might regulate transcription through a mechanism evolutionarily conserved among all eukaryotes.

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(a)
CPRCASRDTKFCYYNNYNTSQPRHFCKGCRRYWTKGGTLRNVPVGGGTRK

Table 1. A variety of put at ive roles f or Dof prot eins in plants

Dof protei n Z m Dof1 PBF BPBF WPBF O BP1 D A G1 D A G2 N t BBF1 A O BP OsDof3 S t Dof1 Species M a i ze M a i ze Bar l ey W hea t Arab i dopsi s Arab i dopsi s Arab i dopsi s Toba cc o Pu m p ki n R ic e Po t a t o Put ati ve role L i gh t response Endosperm spe ci f icit y Or t ho l ogs of PBF Or t ho l ogs of PBF Defense response Seed germ i na ti on Seed germ i na ti on A ux i n response A ux i n response G i bbere lli n response G uard c e ll spe ci f icit y Refs [16 ,21] [ 7] [ 9] [ 9] [5 , 14] [31] [13] [6 , 32] [ 8] [11] [12]

Dof 1 100 NLS

Activation 200 238 amino acids

(b)
450

Arabidopsis GST6 promoter

+1

TGACGACCTCCTCTACACTTTT

AAAAGGAAAG

Dual function of the Dof dom ain

OBF

OBP1

OBP1 OBP1

Maize 22-kDa zein promoter 250

+1

TGTCACATGTGTAAAGGTGAAGAGATCATGCATGTCATTCCACGTAG

O2

PBF

O2

O2

TRENDS in Plant Science

Fig. 1. (a) S che m e of Z m Dof 1 as an exa m p l e of t he Dof pro t e i n do m a i n stru ct ure . The Dof do ma i n , a nu cl ear l o c a li za ti on s i gna l (NLS) and t he t rans c r i p ti ona l a cti va ti on do m a i n are i nd ic a t ed i n ye ll o w , p i n k and purp l e , respe cti ve l y. A ser i ne-s t re tch i s i nd ic a t ed i n green . The c ys t e i ne res i dues for pu t a ti ve c oord i na ti on of z i n c are sho w n i n red l e tt ers i n t he Dof do m a i n a m i no a ci d sequen c e . (b) Exa m p l es of Dof-b i nd i ng s it es i n p l an t pro m o t ers . The b i nd i ng s it es for Dof pro t e i ns and bZ IP pro t e i ns are i nd ic a t ed i n red and b l ue , respe cti ve l y. Dof bZ IP i n t era cti ons and t rans c r i p ti on s t art s it es are i nd ic a t ed by b i-d i re cti ona l arro ws and ben t arro ws , respe cti ve l y.

Dom ain structure

Transcription factors sometimes contain multiple DNA-binding domains. For example, some plant MYB proteins have a single MYB DNA-binding domain, but others contain imperfect repeats of the MYB domain [19]. In addition, single MYB-domain proteins and two- or three-repeat MYB proteins have been suggested to bind DNA in different ways [19]. Similarly, plant-specific WRKY transcription factors possess different numbers of WRKY DNA-binding domains, which allows the proteins to be classified into subgroups [20]. However, in the case of Dof proteins, only a single copy of the Dof domain can always be found in their N-terminal regions. By contrast, the minimal transcriptional activation domain of ZmDof1 has been mapped in 44 amino acid residues of the C-termini [17]. The C-terminal regions of other Dof proteins (Arabidopsis OBP1, 2 and 3, and barley BPBF) also include transcriptional activation domains [10,18]. Taken together, Dof proteins usually appear to consist of two major domains: an N-terminal conserved DNA-binding domain and a C-terminal domain for transcriptional regulation (Fig. 1a). Serine-stretches that are frequently located immediately downstream of the Dof domains might be the molecular hinges linking the two domains.
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The strong similarity among Dof DNA-binding domains suggested that all Dof proteins display similar DNA-binding specificity. Indeed, an AAAG sequence or its reversibly orientated sequence, CTTT, is always found in the binding sequences of individual Dof proteins [2,57,912,14,16,21], except a pumpkin Dof protein (AOBP) that recognizes an AGTA motif [8]. Furthermore, binding- site selection experiments with four maize Dof proteins established an (A/T)AAAG sequence as the recognition core [22]. A different preference of each protein on the sequence flanking the core motif was also shown, but it affected the DNA recognition only to a limited extent [22]. Similar to the many cases of other transcription factors, precise selection of target promoters by each Dof protein in vivo might require other factors in addition to the DNA sequence. Dual activity of the Dof domain might be a clue to understanding why each Dof protein can act on correct target promoters in vivo. Like other zinc fingers [23], the Dof domain is known to be a bi-functional domain that mediates not only DNA-binding but also proteinprotein interactions. The first proteinprotein interaction was reported by Bei Zhang et al. [5]. An Arabidopsis Dof protein (OBP1), which was isolated as a protein specifically interacting with basic leucine zipper (bZIP) proteins (OBF4 and 5), promoted the binding of the bZIP proteins to DNA through its Dof domain [5,10]. An OBP1-binding site in close vicinity to an OBF-binding site was indeed found on a target promoter, Arabidopsis GST6 promoter (Fig. 1b) [14]. Another example of the DofbZIP interaction can be seen in the interaction of PBF (a maize endospermspecific Dof protein) with O2 (a maize bZIP protein involved in endosperm-specific gene expression) [7]. The physiological significance of the PBFO2 interaction is shown by the requirement of the PBF-binding site for O2-dependent activation [24,25] and conservation of the set of PBF- and O2-binding sites in many promoters of cereal storage-protein genes [26,27]. Furthermore, another target promoter of O2 in the endosperm (the maize cyPPDK promoter)

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Table 2. List of Arabidopsis Dof genes

Dof gene
a

G ene code A t1 A t1 A t1 A t1 A t1 A t1 A t1 A t1 A t1 A t1 A t2 A t2 A t2 A t2 A t2 A t3 A t3 A t3 A t3 A t3 A t3 A t3 A t4 A t4 A t4 A t4 A t4 A t4 A t4 A t5 A t5 A t5 A t5 A t5 A t5 A t5 A t5 g07640 g21340 g26790 g28310 g29160 g47650 g51700 g64620 g65935 g69570 g28510 g28810 g34140 g37590 g46590 g21270 g45610 g47500 g50410 g52440 g55370 g61850 g00940 g21030 g21040 g21050 g21080 g24060 g38000 g02460 g39660 g60200 g60850 g62430 g62940 g65590 g66940

Cl one F24B9 F24J8 T24P13 F3H9 F28N24 F16N3 F19C24 F1N19 F12P19 F10D13 T17D12 F8N16 T14G11 F13M22 F13A10 M XL8 F9K21 F1P2 F11C1 F22O6 T22E16 F21F14 T18A10 T13K14 T13K14 T13K14 F7J7 T19F6 F20D10 T22P11 M I J24 F15L12 M A E1 K19B1 M Q B2 K21L13 K8A10

EST

Synonymous

A t Dof1 . 1 A t Dof1 . 2 A t Dof1 . 3 A t Dof1 . 4 A t Dof1 . 5 d A t Dof1 . 6 A t Dof1 . 7 A t Dof1 . 8 A t Dof1 . 9 A t Dof1 . 10 A t Dof2 . 1 A t Dof2 . 2 A t Dof2 . 3 A t Dof2 . 4 A t Dof2 . 5 A t Dof3 . 1 A t Dof3 . 2 A t Dof3 . 3 A t Dof3 . 4 A t Dof3 . 5 A t Dof3 . 6 A t Dof3 . 7 A t Dof4 . 1 A t Dof4 . 2 A t Dof4 . 3 A t Dof4 . 4 A t Dof4 . 5 A t Dof4 . 6 A t Dof4 . 7 A t Dof5 . 1 A t Dof5 . 2 A t Dof5 . 3 A t Dof5 . 4 A t Dof5 . 5 A t Dof5 . 6 A t Dof5 . 7 A t Dof5 . 8
a b

A V540721 BE038318 T14116 A V564595 A I999028 A A395802 A I993066 BE038318 A V442796 A V559509 A W004398 T22381 A V543166 H77029 A V537390 A V531601 A V560727 T88559 N38019 A I995889 A V519437 T43764

O BP2 A D O F1 D A G2 A D O F2 O BP1 O BP3 D A G1 and BBFa O BP4

Combinatorial regulation by distinct classes of transcription factors is a persuasive explanation for seeming discrepancies between DNA-binding in vitro and the action in vivo of transcription factors [30]. It is likely that the dual function of the Dof domain contributes to correct choice and/or selective activation or repression of target genes in vivo. However, the exact meanings of these proteinprotein interactions in transcriptional control still remain to be evaluated in vivo. Also, it is still unknown what determines the specificity of these interactions in spite of the conserved amino acid sequence within the Dof domain. In a recent report, the DofMYB interaction was mediated through the C-terminal of BPBF but not through the Dof domain [18]. Analogous to the case of O2 protein, both the MYB-binding and the Dof binding sites were necessary for the MYB protein to activate the transcription from a target promoter [18].
Divergent roles of Dof proteins

N o m en cl a t ure g i v i ng f i rs t t he c hro m oso m e nu m bers and t hen orders on t he c hro m oso m e . G enBan k a cc ess i o n nu m bers . E ven i f m u lti p l e ESTs w ere found , on l y one i s sho w n . '' i nd ic a t es no G enBan k a cc ess i on nu m b er . c The or i g i na l na m es used i n pub lic a ti on or for reg i s t ra ti on i n a da t abase . '' i nd ic a t es no o t her na m e . d The open read i ng fra m e (ORF) for A t Dof1 . 6 w as es ti m a t e d us i ng a M e t c odon t h a t w as d i fferen t fro m t he pu t a ti ve t rans l a ti ona l s t ar t M e t c odon for t he ORF , A t 1 g47650 .

is also a target of ZmDof1 in leaf mesophyll cells, although no DofbZIP interaction associated with the activity of the cyPPDK promoter has been identified [21]. These examples reveal an interesting character of the Dof domains, but it still uncertain whether every Dof protein interacts with a bZIP protein. The Dof domain also mediates other types of proteinprotein interactions. The Dof domain showed a specific affinity to distinct Dof domains, although the biochemical and biological meanings of this affinity have not been clarified yet [28]. In addition, Dof domains interacted with maize HMG (chromatin-associated high mobility group) proteins and different HMG proteins enhanced DNA-binding of Dof proteins to different extents [28,29].
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Divergent physiological roles of Dof proteins have been suggested to date (Table 1). The suggested roles do not have anything in common apart from all being linked to plant-specific gene expression. Maize Dof1 was suggested to be a regulator for the light-responsive expression of multiple genes, including the C4 photosynthetic phosphoenolpyruvate carboxylase gene [16,21]. Maize PBF and its orthologs of wheat and barley appear to be transcription factors controlling endosperm-specific expression of storage proteins [7,9]. Arabidopsis OBP1 might regulate defense gene expression in response to salicylic acid and oxidative stress signals [5,14]. Arabidopsis DAG1 and DAG2 have been shown to act on a maternal switch controlling seed germination by isolating mutants with a T-DNA insertion in their respective genes [13,31]. Dof proteins are probably also involved in phytohormone-regulated expression. A tobacco Dof protein, NtBBF1, is a candidate for the activator of auxin-inducible expression of a plant oncogene rolB in apical meristems and vascular tissues [6,32]. Participation of Dof proteins in gene silencing has also been suggested from the binding of a pumpkin Dof protein (AOBP) to the silencer region of the auxin-inducible ascorbate-oxidase gene promoter [8]. Some gibberellin (GA) responses might involve Dof proteins as well. A rice Dof protein, OsDof3, might be a mediator for GA signaling during germination [11]. Recently, another function of Dof proteins was suggested in the regulation of guard cell-specific gene expression in potato [12]. Although every role has not been confirmed conclusively, Dof proteins apparently function in the regulation of various processes. Transcriptional and post-transcriptional regulations matching the suggested physiological functions have also been reported on expression of several Dof proteins. For example, expression of PBF gene is strictly restricted to certain developmental stages in the endosperm [7,9], and

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AtDof4.2 AtDof4.4 AtDof4.3 AtDof4.5 OsDof5 AtDof3.3 AtDof5.5 AtDof5.2 AtDof1.5 AtDof2.3 AtDof1.3 AtDof1.10 AOBP AtDof1.2 AtDof3.5 AtDof3.2 AtDof5.3 AtDof2.1 AtDof4.6 AtDof1.8 AtDof2.5 AtDof3.7 AtDof4.1 NtBBF1 NtBBF2a OsDof1 OsDof2 AtDof3.4 AtDof5.8 AtDof1.6 AtDof1.7 AtDof3.1 AtDof4.7 AtDof5.7 AtDof1.4 AtDof2.4 AtDof5.1 AtDof3.6 AtDof1.1 AtDof2.2 StDof1 ZmDof3 AtDof5.4 ZmDof1 ZmDof2 OsDof4 AtDof5.6 ZmPBF BPBF WPBF OsDof3

DNEMN--VMPPP--RV Dof DNQVNGLKRPPPS-RV Dof DNQVNDVKPPPPPPRV Dof DNQVNDVKPPPPPPRV Dof Dof KTLKKPTKILP Dof NK KTLKKPTKILP Dof NK LKKPDKILP Dof NK DLTAEKRPDKIIP Dof SK DLTAEKRPDKIIA Dof SK EKTTELKKPDKILP Dof NK EKSTALKKPDKLIP Dof NK LKKPDKILP Dof NK

QSSVSQMILAEIQQGNHQPF QSSISEMVSVENQPINHQSF QPSVAQMVSVGIQPGSHKPF QPSVARMVSVETQRGNNQPF

Group I

LSANPAALSR LCANPAALSR LQANPAALSR

Group II

LQANPAAMAR LQANPAAMSR LQANPAALSR IDLAAVFA LRTTPEP LRTTPEP IDLALVYA MELGLAYA IDLSLAFA IDLNLAFA QDLNLGFS KDLNLAFS QDLNLLSF KDLNLLSF NDLSLSFS HDLNLAFN QDLNLGFS QDLNLAFP QDLNLAFP

Dof RSR Dof SRR PEQSLR Dof HKR PPELALR Dof NKR LGGEAQN Dof NKR GGERKARP-EKDQAVN Dof NKR PVERKARP-EKDQALN Dof NKR VA ERKARP-Q-EK-LN Dof NKR MET RKARP-Q-EK-VN Dof NKR MTTMSTRP-Q-EP-RN Dof NKK PTERKPRP-QKEQAIN HIERKIRPHQKDQALN STERRARP-QKEKALN GTERRARP-QKEKALN Dof Dof Dof Dof NKR SKK NKR NKR

Group III

LPDLNP LPDLNP LPDLNP

MPSEFSESR MPSEPNQTR MQDLTSAA MQDP--AA

QEQLP Dof QEQLS Dof AEPLP Dof QEQLK Dof QEQLK Dof QPVLK Dof QQNLK Dof QPQLK Dof

PLQTTPVLFPQ PLQATPVLFPQ

Group IV

SSIESLSSFNQDLHQKLQQQR SSIETLSCLNQDLHWRLQQQR

RARQA-NVALPEAALK Dof RNRRTKS RARLA-NIPLPETALK Dof RNKRTKN RARIAKV-PLPEAALN Dof RNKRSKS RARQA-NIPPLAGPLK Dof RNNKKGK RARLAKNSQPPEGALK Dof RN-KKGK RARLAKV-PLPEAALN Dof RNKRSKS RARLAR-IPLPEPGLK Dof RHAKRAK Dof A KRS APAPAVGEGDP Dof APG-----GDP Dof Dof TKRS Dof GERKPRPQLPEALK- Dof AEKKSRPK-PEQKVE Dof PKRP AEKKPRPK-PEQKVE Dof PKRS Dof SKRS

QQSSM QQHQ QQAQQFPFL

VKVED Group V VKMEDSNNQLNLSR NLSR VKMEEQPNLANLSR VKMEDN-HLGNISR

QQMQSFPFL DQDQS--GLYLPstop DPDPACIFLNLPstop DTDPA-LFLNLPstop VPDPN---VYLPstop

VDHAYW PP--YW PPAAYW

Group VI

TMGLFPNVLPTLMPTGGG TGMNFANVLPTFMSGGFD TGMNFANVLPTFMSVGFE PCADFPNVLPTFVSTGFE

Group VII

TRENDS in Plant Science

DAG1 gene is expressed in flowers but not in embryos in accordance with its maternal role [13,31,33]. In addition, OBP1 mRNA is induced by plant defense
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signals [10,14]. OsDof3 expression is induced by GA, but an antagonistic phytohormone of GA, abscisic acid, prevents the GA effect on OsDof3 expression [11].

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Fig. 2. Dof pro t e i n re l a ti onsh i ps . The phy l ogene tic t ree was c rea t ed us i ng fu ll sequen c es of Arab i dops i s Dof pro t e i ns , us i ng t he C l us t a l W progra m . Dof pro t e i ns fro m o t her spe ci es (i nd ic a t ed i n red) are a l so cl ass i f i ed i n t o subgroups . The f i rs t one or t w o l e tt ers of t he na m es i nd ic a t e t he i n iti a l s of t he spe ci es (ex c ep t t ha t of pu m p ki n , w h ich i s A O BP): A t , Arab i dops i s t ha li ana ; B , bar l ey ; N t , N ic o ti ana t aba c u m ; Os , Oryza sa ti va ; S t , So l anu m t uberosu m ; W , w hea t , Z m , Zea m ays . The s i gna t ure m o ti fs c onserved a m ong m e m bers of t he sa m e group are sho w n i n b l ue . Exa m p l es of m o ti fs c onserved i n on l y so m e m e m bers are sho w n i n green . N on-ho m o l ogous sequen c es be t w een m o ti fs are i nd ic a t ed by bars . A lt hough no c onserved m o ti fs are found i n A t Dof5 . 6 or OsDof5 , t hey were sugges t ed t o form subgroups w it h o t hers on t he bas i s of t he c a lc u l a ti on us i ng t he C l us t a l W progra m .

A recent study using the Arabidopsis GeneChip system also showed that the expression of Dof genes were controlled differently in various environmental conditions [34]. However, the ZmDof1 activity appears to be modulated in a post-translational manner in response to light [16,17]. A protein kinase inhibitor was recently observed to activate ZmDof1, suggesting that protein phosphorylationdephosphorylation might be included in the regulation for Dof proteins (S. Yanagisawa, unpublished).
Evolutionary diversification

Acknow ledge m ents I w ou l d lik e t o t han k H i rosh i A k anu m a and Terunao Ta k ahara for t he i r c r itic a l read i ng of t he m anus c r i p t , and Cher i Chen for ass i s t an c e i n survey i ng Arab i dops i s Dof pro t e i ns . Resear ch support was prov i ded i n part by t he M i n i s t ry of Edu c a ti on , Cu lt ure , Sport s , S ci en c e and Te chno l ogy, Japan (no . 13780549 and no . 13039006).

The complete catalog of Dof proteins in a single plant species is useful for viewing the structural and functional diversity among Dof proteins as a whole. In a survey of the complete Arabidopsis genome sequence, 37 putative Dof genes have been found [1]. Because several Arabidopsis Dof proteins and their genes have been named in a non-systematic manner, they are listed in Table 2 in the order of their chromosomal positions. Although, at this stage, it is impossible to evaluate the expression of these genes exactly, ESTs corresponding to the genes in the database suggest that most of the genes are expressed, whereas the AtDof1.9 gene appears to be a pseudogene owing to a stop codon within the Dof domain. A phylogenetic tree categorizes Arabidopsis Dof proteins into seven subgroups (Fig. 2). Although the primary structures, except the Dof domain, are largely divergent among all Dof proteins, detailed comparison revealed small structural motifs that could become hallmarks, substantiating the validity of the classification. Amino acid substitutions within the Dof domain were also in accord with the classification. A recent genome-wide analysis suggested that the Arabidopsis genome had been generated by four different large-scale genome duplications 100 million to 200 million years ago (during the formative period in the diversification of angiosperms) [35]. Obviously, such genome duplications caused multiplication of Dof genes. For instance, generation of the AtDof3.2 and AtDof5.3 genes, which are located close to one another in the phylogenetic tree, is a result of genome duplication that was estimated to have occurred 100 million years ago. The same genome duplication also gave rise to the divergence of the AtDof2.5 and AtDof3.7 genes in Group III, and the divergence of the AtDof1.1 and AtDof2.2 genes in Group V. By contrast, other close relationships between the AtDof1.7 and
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AtDof3.1 genes and between the AtDof4.7 and AtDof5.7 genes reflect different genome duplications, which were estimated to have occurred 140 million years ago and 170 million years ago, respectively. Accordingly, the formation of subgroups appears to be originated from the multiplication of a Dof gene from a much older period, presumably before the divergence of monocots and dicots, which was estimated to have occurred between 180 million and 220 million years ago [36,37]. This is further supported because the same subgroups include both monocot and dicot Dof proteins. Thus, the complete catalog of Arabidopsis Dof proteins might disclose both the diversity common to all higher plants (represented by subgroups) and the diversity specific to dicots or species (represented by each member in the same subgroup). There is limited information about the relationship between functional diversification and gene multiplication. However, it is not unexpected that members in the same subgroup show redundant, overlapping or related functions. Indeed, OsDof3, which is specifically expressed in the scutellum and the endosperm of rice in response to GA during germination, forms a subgroup together with other Dof proteins associated with endosperm development (PBF, BPBF and WPBF). In addition, recent studies revealed that two closely related Arabidopsis Dof genes in Group III [DAG1 (AtDof3.7) and DAG2 (AtDof2.5)] are maternal genes involved in the control of seed germination, although their actions are opposite [13,31]. Furthermore, the phylogenetic tree of Dof proteins suggests that the branching of Dof genes after the divergence of monocots and dicots might produce further functional diversity in a dicot- or monocotspecific, or species-specific manner. For example, Group VI (Fig. 2) includes only a single Arabidopsis Dof protein but two maize Dof proteins (ZmDof1 and ZmDof2). These maize proteins display different expression patterns and related but different functions [16]. Many roles have already been suggested for Dof proteins. However, more roles should come to light in the future because the complete list of Arabidopsis Dof proteins suggests that most Dof proteins have yet to be characterized. Although further analyses should establish the function of each Dof protein, including gain-of-function and loss-of-function experiments, it is apparent that Dof proteins are involved in diverse plant physiology. This is not surprising because the structural and functional diversification of Dof proteins appears to have occurred at an early stage of higher plant evolution. Comparative studies with lower plants, such as mosses and algae, might reveal the origin and the primary role of Dof proteins, and also the evolutionary linkage between the functional diversification of Dof proteins and the emergence of higher plants in the world.

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