TCP genes: a family snapshot ten years later

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1 Review TCP genes: a family snapshot ten years later Mar Martín-Trillo 1 and Pilar Cubas 2 1 Facultad de Ciencias del Medio Ambiente, Campus Tecnológico de la Fábrica de Armas, Avda. Carlos III, s/n E-45071, Toledo, Spain 2 Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología/CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain TCP genes encode plant-specific transcription factors with a bhlh motif that allows DNA binding and protein protein interactions. The TCP gene family has five members in the lycophytes and >20 members in the eudicots. Gene duplication and diversification has generated two clades (class I and II) with slightly different TCP domains. Here, we summarize our current knowledge of the evolution of this family, their regulation, the biochemical activity of their proteins and the biological function of some members, in particular, in the control of cell proliferation in developing tissues. Increasing knowledge of the functions of TCP genes should enable their use as tools to modulate plant growth patterns and to generate novel morphologies in species of agronomical interest. A gene family is born The TCP gene family was first described in 1999, as a small group of plant genes encoding proteins sharing the socalled TCP domain, a 59-amino acid basic helix loop helix (bhlh) motif that allows DNA binding and protein protein interactions [1,2]. This domain was initially identified in four proteins encoded by apparently unrelated genes, from which the name TCP was derived: teosinte branched1 (tb1) from maize (Zea mays) [3], CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus) [4], and the PROLIFERATING CELL FACTORS 1 and 2 (PCF1 and PCF2) from rice (Oryza sativa) [1]. The tb1 gene is a major determinant of strong apical dominance in domesticated maize [3]. CYC is involved in the control of floral bilateral symmetry in Antirrhinum [4]. PCF1 and PCF2 are factors that bind to the promoter of the rice PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) gene [1], which encodes a protein involved in DNA replication and repair, maintenance of chromatin structure, chromosome segregation and cell-cycle progression. A decade later, a snapshot of TCP genes needs a wide angle lens to encompass this ever-growing family. TCP genes have been found in various plant species, and new roles in plant development have been elucidated. These discoveries emphasize the importance of this plant-specific gene family in the evolution and developmental control of plant form. Genome and transcriptome sequencing in non-model plant species is leading to the discovery of even greater numbers of TCP genes of Corresponding author: Cubas, P. (pcubas@cnb.csic.es). unknown functions, whose roles will need to be determined partly with the help of information available in genetic model systems. Here, we summarize current knowledge of this family, its evolutionary history, the biochemical function of its proteins and the known roles of some members in the control of plant morphology and evolution. Evolution of the TCP gene family The TCP transcription factors are ancient proteins. Although they do not appear to be present in the unicellular algae Chlamydomonas, they have been found in pluricellular green algae, such as Cosmarium and Chara, in the moss Physcomitrella patens, in ferns and in the lycophyte Selaginella [5,6]. In these species, TCP genes form small families of five to six members. During plant evolution, duplication and diversification has generated larger families comprising tens of members in gymnosperms [5] and angiosperms [7 16]. More than 20 members have been identified in whole-genome searches carried out in Arabidopsis thaliana [7,8], rice [5,14,15] (see Table 1 for complete list), poplar (Populus) [5] and grapevine (Vitis vinifera) (D. Lijavetzky and J.M. Martínez Zapater, personal communication). Two types of TCP protein can be distinguished, based on differences within their TCP domains: class I [17] (also known as PCF class [7] or TCP-P class [5]) and class II (also known as TCP-C class) [5]. Class I contains rice PCF1 and PCF2, and class II includes CYC and tb1 (Figures 1 and 2; Online Supplementary Material Table S1). The most striking difference between these two classes is a four-amino acid deletion in the basic domain of class I relative to class II proteins. Other class-diagnostic residues for each class are also found within the helices and the loop of the TCP domain (Figure 2). Outside this domain, other motifs, specific to subclades, are conserved [2,5,11,14,18]. For instance, an residue arginine-rich motif (the R domain, Figure 1) [2] and a glutamic acid cysteine glutamic acid stretch (the ECE motif) [11], both of unknown function, are only present in a subset of class II proteins. So far, it has not been possible to determine which class is more related to the ancestral TCP proteins, because even green algae have members belonging to these two distinct groups. In angiosperms, class I is formed by a group of relatively closely related proteins, whereas class II can be further subdivided into two clades also based in differences within the TCP domain (Figures 1 and 2). The CIN clade /$ see front matter ß 2009 Elsevier Ltd. All rights reserved. doi: /j.tplants Available online 4 December

2 Table 1. Arabidopsis and rice TCP gene nomenclature Identifier (Arabidopsis) Name a Alternative name Refs (alternative name) Type Identifier (rice) Name b Alternative name Refs (alternative name) At1g67260 AtTCP1 CYC/TB1 Os04g11830 PCF1 OsTCP15 [5] PCF At4g18390 AtTCP2 CIN Os08g43160 PCF2 OsTCP23 [5] PCF At1g53230 AtTCP3 CIN Os11g07460 PCF3 OsTCP19, [5,14] PCF OsTCP26 At3g15030 AtTCP4 MEE35 [83] CIN Os01g11550 PCF5 OsTCP1 [5,14] CIN At5g60970 AtTCP5 CIN Os03g57190 PCF6 OsTCP8, [5,14] CIN OsTCP14 At5g41030 AtTCP6 PCF Os01g55100 PCF7 OsTCP4 [5] CIN At5g23280 AtTCP7 PCF Os12g42190 PCF8 OsTCP22, [5,14] CIN OsTCP29 At1g58100 AtTCP8 PCF Os03g49880 OsTB1 OsTCP13 [5] CYC/TB1 At2g45680 AtTCP9 PCF Os01g41130 OsTCP3 PCF At2g31070 AtTCP10 CIN Os01g55750 OsTCP5 OsTCP2 [14] CIN At2g37000 AtTCP11 PCF Os01g69980 OsTCP6 OsTCP3 [14] PCF At1g68800 AtTCP12 BRC2 [32] CYC/TB1 Os02g42380 OsTCP7 OsTCP4 [14] PCF At3g02150 AtTCP13 PTF1, [67,69] CIN Os02g51280 OsTCP9 OsTCP5 [14] PCF TCP10 At3g47620 AtTCP14 PCF Os02g51310 OsTCP10 OsTCP6 [14] CIN At1g69690 AtTCP15 PCF Os02g58180 OsTCP11 PCF At3g45150 AtTCP16 PCF Os03g30880 OsTCP12 PCF At5g08070 AtTCP17 CIN Os04g44440 OsTCP17 OsTCP10 [14] PCF At3g18550 AtTCP18 BRC1, [32,33] CYC/TB1 Os05g43760 OsTCP18 OsTCP11 [14] CIN TBL1 At5g51910 AtTCP19 PCF Os06g12230 OsTCP19 OsTCP12 [14] PCF At3g27010 AtTCP20 PCF Os07g04510 OsTCP20 OsTCP13 [14] CIN At5g08330 AtTCP21 CHE [64] PCF Os07g05720 OsTCP21 OsTCP14 [14] CIN At1g72010 AtTCP22 PCF Os08g33530 OsTCP22 OsTCP15 [14] CYC/TB1 At1g35560 AtTCP23 PCF Os09g24480 OsTCP24 OsTCP17, [14,30] CYC/TB1 REP1 At1g30210 AtTCP24 CIN Os09g34950 OsTCP25 OsTCP18 [14] PCF Os12g02090 OsTCP27 OsTCP20 [14] CIN Os12g07480 OsTCP28 OsTCP21 [14] PCF a See Ref. [7] for names. b See Ref. [17] and [5] for names Type exemplified by CINCINNATA (CIN) of Antirrhinum, contains genes involved in lateral organ development [18 24] and the CYC/TB1 clade (or ECE clade) [11] includes genes mainly involved in the development of axillary meristems giving rise to either flowers or lateral shoots. The CIN clade could be more ancient than the CYC/TB1 clade because, for example, in Selaginella and Physcomitrella, which have neither flowers nor branches, all class II members are CINtype [6]. The CYC/TB1 clade probably evolved later within angiosperms. In monocots, the tb1 gene and its orthologues control the growth and development of both reproductive and vegetative axillary structures (inflorescences, flowers and lateral shoots) [3,25 30]. Phylogenetic analyses indicate that the CYC/TB1 clade experienced two duplications at the base of core eudicots, giving rise to three types of genes: CYC1, CYC2 and CYC3 [11]. Genetic studies in Arabidopsis suggest that the CYC1 subclade has retained the tb1 role related to the control of shoot branching [31 33]. CYC2 includes CYCLOIDEA-like (CYC-like) genes, which have had a key role in the evolution of floral dorsoventral asymmetry (zygomorphy) [34 36]. This clade has expanded by duplication in groups where this key innovation evolved, such as the Leguminosae [12,37 40], Asteraceae [41 43] and Lamiales [4,13,44 59]. The CYC3 subclade contains genes such as Arabidopsis BRANCHED2 (BRC2), expressed both in branch and flower primordia which appears to have a minor role in the control of shoot branching, and with unclear functions during flower development [32,33]. Biochemical function of TCP proteins Biochemical characterisation of TCP factors has been carried out mainly in rice [1,17]. PCF1 and PCF2 were identified as rice DNA-binding proteins that recognized the PCNA promoter [1]. The basic region of the TCP domain, highly conserved in all family members, is necessary (but not sufficient) for DNA binding because deletion of this region completely abolished DNA binding activity in yeast one-hybrid assays [1]. Moreover, rice TCP proteins can recognize specific DNA motifs. Random binding-site selection experiments and electrophoretic mobility shift assays (EMSAs) revealed different consensus binding sites for class I and class II factors. The consensus for class I is GGNCCCAC [17]. This same motif [or shorter versions, (T)TGGGCC, GCCCR, GG(A/T)CCC] has been subsequently found in Arabidopsis gene promoters putatively controlled by class I factors [60 64]. Interestingly, the consensus of class II binding sites was found to be distinct but overlapping with that of class I sites: G(T/C)GGNCCC [17,65]. A complementary motif, gggaccac, has been identified, by in vitro selection, as a binding site for class II AtTCP4 [21] and its core, GGACCA, is over-represented in AtTCP4- regulated genes [21]. This suggested that sequences containing both sites, G(T/C)GGNCCCAC, could be recognized 32

3 Figure 1. The TCP gene family. Unrooted phylogenetic tree showing relationships among predicted TCP proteins of the eudicot Arabidopsis thaliana (At), monocot Oryza sativa (Os) and representative members of Antirrhinum majus (Am), Lotus japonicus (Lj), Solanum lycopersicum (Sl), Gerbera hybrida (Gh), Solanum tuberosum (St), Pisum sativum (Ps) and Zea mays (Zm). Class I is highlighted in blue, and class II is highlighted in yellow. Blue, green and red lines indicate the PCF, CYC/TB1 and CIN clades, respectively. On the right is the protein structure indicating conserved motifs: TCP domain (blue), R domain (red). The position of the microrna mir319 recognition sequence in the mrna is indicated in green (not to scale). The R domain is absent in all class I proteins and present in all CYC/TB1 proteins (with some exceptions; i.e. Os08g33530 and OsREP1; [5,14]). Ancestral class II CIN proteins probably had an R domain because a derivative of this motif has been identified in both Selaginella and Physcomitrella class II CIN-like proteins [6]. This motif has been lost in most angiosperm CIN-like proteins (with some exceptions, i.e. AtTCP2, AtTCP24 [7], Os02g51310, Os07g04510 and Os07g05720 [5,14]). The mir319 site is only present in a subset of the CIN-like genes [5,6,18]. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 alrt) and the reliability for internal branching was assessed using the bootstrap method [91,92]. Only the TCP domain was used for the analysis. The scale bar represents amino acid length. Genes encoding an R domain and/or containing a microrna mir319 target site are indicated in bold. Arabidopsis genes are named after Refs [7,32,64,69] (Table 1, main text). Rice gene names are either TIGR identifiers (Table 1, main text) or named according to Refs [17,29,30,39,40]. by both types of protein, which could coordinately or competitively regulate transcription through the binding of TCP sites [7,17,60]. More recently, the class II factor CYC has been shown to bind class I sites [66], indicating that this sequence specificity is not absolute, and that it allows for even more competition possibilities among TCP factors. TCP proteins form homo- and heterodimers (so far only described among members of the same class) [17]. DNA binding appears to require dimer formation because protein deletions (outside the TCP domain) preventing dimerisation result in the loss of the DNA-binding ability [17]. Moreover, heterodimers bind DNA more efficiently than do homodimers [17]. Therefore, it is possible that different heteromeric combinations bind to slightly divergent cis-regulatory elements, recognize target genes with different affinity, or modulate each other s activity [7]. This would add another level of complexity to the regulatory networks controlled by these factors. 33

4 Figure 2. Alignment of the predicted amino acid sequence from selected members of the TCP family. Amino acids are expressed in the standard single letter code. Black boxes highlight residues conserved in both TCP classes; yellow, residues conserved in class I; grey, conserved in class II; light-green, conserved in CYC/TB1 proteins; darkgreen, conserved in CIN-like proteins; pink, conserved in CYC-like proteins; blue, conserved in TB1/BRC1/BRC2-like proteins. Amino acids with the same hydrophobicity or charge are considered to be synonymous. As DNA-binding proteins, the TCP factors are expected to be targeted to the nuclei. Indeed, bipartite or monopartite nuclear localization signals (NLS) have been identified in many of them, and nuclear localisation has been confirmed for a few TCP members by immunoprecipitation of nuclear extracts or GFP-protein fusions [1,17,32,64,67,68]. By contrast, GFP fusions of PTF1/ TCP13 have been described as being targeted to either nuclei [67] or chloroplasts [69]. These apparently contradictory results might suggest that PTF1/TCP13 could be targeted to both compartments. A few additional TCP factors (AtTCP11, AtTCP15, AtTCP17, AtTCP22 and AtTCP23) have been predicted to contain chloroplast-targeting peptides (ctp) [5,70], raising the possibility that some of these factors might control the transcription of chloroplast genes [69]. TCP proteins bind to functional cis-acting elements at the promoter of several genes [1,17,21,60 65,69,71]. However, the molecular mechanisms by which TCP proteins control transcription are still poorly understood. For instance, can TCP factors activate transcription by themselves? A few TCPs have been reported as self-activating when used as bait in yeast two-hybrid assays (M. Martín, F. Chevalier and P. Cubas, unpublished) [17,24] but none of the class I rice proteins tested so far can trans-activate reporter genes driven by class I sites, in cell culture cotransfection assays [17]. Therefore, it seems likely that at least some TCP factors are not transcriptional activators per se, but require interaction with other proteins to control transcription. TCP binding sites are often associated with other cisacting elements, indicating that TCPs could function as part of multimeric regulatory modules [63,72]. For example, in the promoter of the Arabidopsis CYCLINB1;1 gene (AtCYCB1;1), TCP-binding sites cooperate with the M-specific activator (MSA) element (which controls the G2/ M phase-specific timing of expression) [73] to promote high levels of transcription [60]. TCP sites have been found in association with the cis-element telo box at the promoter of AtPCNA2, AtCYTOCHROME C-1 and 2 and a large number of ribosomal protein genes [61,72,74,75], and they act in synergy with the telo box in artificial promoters driving expression of reporter genes [61]. Putative TCP protein partners could participate in those transcriptional complexes. For instance, a telo box-binding factor, AtPURa interacts in yeast-two hybrid assays with AtTCP20, suggesting that these two proteins could act together at the promoter of ribosomal protein genes [61]. Another TCP factor, CCA1 HIKING EXPEDITION (CHE), which binds to the CCA1 promoter and causes down-regulation of the gene [64], interacts with TIMING OF CAB EXPRESSION1 (TOC1), a transcriptional activator of CCA1 [76]. It has been proposed that CHE sequesters TOC1, preventing its interaction with other transcription factors at the CCA1 promoter [64]. AtTCP24 negatively interacts with Armadillo BTB Arabidopsis Protein (ABAP1) to regulate the transcription of AtCDT1a and AtCDT1b [65]. Finally, Antirrhinum the TCP-Interacting with CUP (TIC) protein binds CUPULIFORMIS (CUP), a NAC-domain transcription factor that regulates organ boundaries [77] but their transcriptional role has not been tested yet. TCP proteins can act as transcriptional activators or repressors but this does not seem to be determined by their type of TCP domain: both classes include transcriptional activators (i.e. class-i AtTCP20 enhances transcription of CYCB1;1 [60], class-ii PTF1/AtTCP13 promotes transcription of PSBD [69]) and repressors (i.e. class-i CHE [64] and class-ii AtTCP24 [65], see above). It is even possible that some TCP proteins function either as activators or repressors depending on their interactions with other proteins [63]. Moreover, some TCP protein interactions could be unrelated to transcriptional control. PTF1/AtTCP13, which promotes transcription of PSBD in chloroplasts [69], has been independently identified in yeast two-hybrid assays as an interactor with histidine-containing phosphotransmitters (AHPs), which mediate His-to-Asp phosphor- 34

5 Review elay signal transduction, such as that of the cytokinin signalling pathway [67]. This raises the possibility that this factor could also act by modulating cytokinin signal transduction. Protein protein interactions could be mediated partly through the conserved amphipathic helices of the TCP domain [2], one of which contains the signature LxxLL (or the closely related fxxll, where L is leucine, x is any amino acid and f is a hydrophobic amino acid), which has been shown to mediate binding of transcriptional co-activators to ligand nuclear receptors in animals [78]. However, other parts of the proteins could, in some cases, enhance their specificity: ABAP1 interacts with AtTCP24 but not with the closely related AtTCP3, AtTCP5, PTF1/ TCP13, or AtTCP17 factors, which contain almost identical TCP domains (Figure 2) [65].This suggests that the highly divergent, fast evolving sequences outside the TCP domain are, in some cases, essential for their functional specificity. By contrast, Antirrhinum CUP can bind both TIC and CYC, two divergent TCP proteins belonging to different classes (Figure 1) [77]. All these interactions, so far demonstrated in vitro, need to be confirmed in vivo during plant development. Role of TCP genes during development It has long been suggested that class I genes promote plant growth and proliferation, based mainly on the expression of rice PCF1/PCF2 and AtTCP20 in meristematic tissues, and on the proposed role for these genes as transcriptional Trends in Plant Science Vol.15 No.1 activators of PCNA and CYCB1;1, respectively [1,60]. However, direct evidence for such a role is still missing. Most class I single mutants that have been analysed have mild or no phenotypic defects, probably as a result of genetic redundancy [60,63,79,80]. Whereas in Arabidopsis, ectopic expression of AtTCP20 fused to the EAR repressor domain (which probably inactivates several class I genes) leads to severe phenotypes, suggesting complex but as yet unclear roles in the regulation of cell division, expansion and differentiation [63]. By contrast, the proposed role of class II genes in preventing growth and proliferation is based directly on phenotypes observed in single and multiple mutants. cyc-type mutants have reduced floral bilateral symmetry (in species with zygomorphic flowers, Figure 3a) [4,30,39,41,43,54, 56,81], cin-type mutants have defects in lateral organs (Figure 3b d) [18 24] and tb1/brc1-type mutants have excessive shoot branching (Figure 3e, f) [3,25,27 29,32, 33]. These apparently unrelated phenotypes are all due to changes in the proliferation patterns of the tissues in which the corresponding genes are expressed. CIN-type genes limit cell proliferation at the margins of the developing leaf primordia in Antirrhinum, Arabidopsis and tomato (Solanum lycopersicum). In Antirrhinum and Arabidopsis mutants, leaf cells keep on dividing for a longer period of time compared with wild-type plants, thus generating larger leaves, with altered shape and crinkled surface [18,20,21,22,65]. In tomato, mutant compound leaves have larger leaflets with continuous growth of leaf margins Figure 3. Developmental defects caused by class II TCP gene loss-of-function in different plant species. In each panel, wild-type (left) and mutant (right) phenotypes are shown. (a) Double cyc dich Antirrhinum mutant flower showing loss of dorsoventral asymmetry [4]. (b) cin Antirrhinum mutant showing crinkly leaves caused by negative leaf curvature [20]. (c) jaw-d Arabidopsis mutant with high levels of microrna mir319, and reduced expression of AtTCP2, 3, 4, 10 and 24. These plants have defects in leaf shape and curvature similar to those seen in Antirrhinum cin mutants [18]. (d) Ectopic expression of mir319 with the FILAMENTOUS FLOWER promoter in tomato leads to downregulation of several LANCEOLATE-like genes and generates plants with larger leaflets and continuous growth of leaf margins [23]. (e) tb1 maize mutant showing loss of apical dominance and increased number of branches [3]. (f) brc1 Arabidopsis mutant with more rosette branches than wild-type plants, owing to an increased frequency of bud outgrowth [32]. (a) Reproduced with permission from John Innes Institute; (b,c) reproduced, with permission, from Refs [20] and [18], respectively; (d,e) kindly provided by N. Ori and Y. Eshed, and by J. Doebley, respectively; (f) reproduced, with permission, from Ref. [32]. 35

6 (Figure 3d) [23]. Antirrhinum CYC reduces cell proliferation at the dorsal region of young floral meristems, thus limiting the number of dorsal floral organs formed in this region. Later, it causes the abortion of the most dorsal stamen to form a staminode [4,56]. Maize tb1 and AtBRC1 promote a reversible growth arrest in axillary buds that, upon downregulation of the genes, enables buds to resume normal growth to generate a branch (Figure 3e, f) [3,25,32,33]. What is the relationship between class II genes and the control of cell proliferation and growth? It has been reported that there is an inverse correlation between class II TCPs mrna levels and the expression of some cell cycle markers (i.e. HISTONE H4, CYCLIND3b) [20,81,82], but this could simply reflect an arrest of cell division in the regions where these genes are expressed. So far, there is no evidence that TCPs directly repress HISTONE or CYCLIND3 genes transcription. Although few TCP targets are known [1,21,60,64 66,69], some are directly involved in cell-cycle progression. For instance, class II AtTCP24 negatively regulates the transcription of the pre-replication control (pre-rc) factor genes CDT1a and CDT1b, which are required for licensing cells into the S phase and DNA replication [65]. Moreover, class II proteins could locally antagonise the above-mentioned activity of class I factors in the transcription of PCNA [1], or CYCB1;1 [60]. Other class II factors could affect proliferation more indirectly: for instance, PTF1/TCP13 might modulate the response to the cytokinin signalling pathway [67], thus affecting cell division. A recent study using leaf maturation markers proposes that, rather than retarding proliferation, CINlike genes act as heterochronic regulators that promote differentiation [22]. In addition, genome-wide analyses indicate that class I binding sites are over-represented in the promoters of genes that have several key cellular activities, such as protein synthesis, mitochondrial oxidative phosphorylation and transcription of chloroplast genes, indicating that class I genes could be controlling some of these processes [60 62,69,75]. Interference with these cellular activities through class II proteins could have dramatic effects on the proliferation and growth rates of cells and tissues. Another exciting field of research suggests that NACdomain transcription factors have a close connection with TCPs, both as interaction partners and as downstream targets. The Antirrhinum NAC-domain protein CUP interacts with TIC, a class I TCP factor, and suggests a model for the establishment of organ boundaries based on the localized expression of NAC and TCP factors [77]. More recently, it has been shown that transgenic Arabidopsis plants expressing chimeric repressors of AtTCP3, AtTCP2, AtTCP4, AtTCP5, AtTCP10, PTF1/TCP13, AtTCP17, or AtTCP24 induce ectopic expression of the NAC-domain containing CUP-SHAPED COTYLEDON (CUC) genes [24]. By contrast, gain of function of AtTCP3 suppresses the expression of CUC genes and results in cotyledon fusion [24], a phenotype previously observed in AtTCP2 and AtTCP4 over-expressing plants [18]. In addition, in plants expressing dominant negative versions of TCP3, there is downregulation of mir164, which targets the cleavage of CUC gene transcripts. Therefore, CIN-like genes could modulate the expression of CUC genes through the transcriptional regulation of mir164 [24]. In this context, other TCP genes could be promoting organ fusion during normal development. In the Asteraceae Gerbera hybrida, GhCYC2 expression correlates with petal fusion in ray florets. Moreover, reduced GhCYC2 function leads to frequent splitting of trans flower petals and ectopic GhCYC2 expression causes petal fusion and tube-shaped disk florets [41]. Other roles for TCP genes are beginning to emerge in relation to the control of cell elongation [18,21,24,41,63,66], male and female gametophyte development [80,83], embryogenesis [84], seed germination [79], jasmonic acid synthesis and leaf senescence [21], and photomorphogenesis [85]. Finally, the identification of CHE as a new component of the circadian clock [64] further reveals the multiple roles that these genes have in plants. Transcriptional and post-transcriptional control of TCP genes Transcriptomic data indicate that class I genes are widely expressed during plant development, and post-transcriptional control of class I TCP genes has not yet been reported. By contrast, the function of class II TCP genes seems to be tightly controlled at multiple levels. This is not surprising given their strong effects on proliferation and growth, which need to be limited to the developmental stages and tissues where their functions are required. Class II genes show dynamic and spatially restricted patterns of mrna distribution (e.g. Refs [4,32,20]) that are generated through not only transcriptional control, but also strictly regulated mrna stability. A subset of CIN-type genes are microrna mir319 targets in all angiosperms that have been analysed to date (Figure 1) [18,21,22 24]. This pathway evolved late because, although mir319 has been detected in Physcomitrella, CIN-like genes do not have the target sequence in this species [6,86]. Expression of mir319-resistant CIN-like genes, ectopically or under their own promoter, leads to strong reductions in growth, fusion of cotyledons, lack of shoot apical meristem development and lethality, supporting the importance of this level of control for plant viability [18,23,24]. Introns might also have a regulatory role in these genes. Many tb1-like and CYC-like genes have introns, in some cases located in their 3 0 UTRs [4,42]. UTRs modulate mrna stability, localization and translational efficiency, and UTR introns, which are relatively rare, have been reported to influence expression levels (e.g. Refs [87,88]). Putative regulation through alternative splicing has also been observed. For instance, BRC1 cdnas have been isolated from tissues other than axillary buds, which retain introns leading to truncated proteins lacking the R domain (C. Poza and P. Cubas, unpublished). Finally, although it remains to be demonstrated, it is likely that the highly conserved phosphorylatable residues of the TCP domain are involved in the modulation of DNA binding, subcellular localization, protein interactions or protein degradation. Concluding remarks and future research The plant-specific TCP gene family is emerging as a group of genes encoding transcriptional regulators that have central roles during plant development. Some of these 36

7 genes, mainly from class II, affect local patterns of cell proliferation and control morphological traits determinant of evolutionary success, such as flower shape, leaf form and shoot branching. TCP genes also participate in other key biological processes, such as hormone synthesis and circadian rhythm regulation. Over the past ten years, some knowledge has been gained about the evolution of this family and its molecular functions, but these areas of research are still in the early stages. It is unclear how the emergence of novel TCPs and new cis-regulatory elements are associated with the generation of new morphological traits, such as floral zygomorphy or optional suppression of lateral shoot outgrowth. Phylogenomic comparison and RNAi functional analyses of TCP genes in relevant species throughout the plant kingdom should help address these questions. From a mechanistic point of view, it remains to be demonstrated that class I and class II factors act antagonistically by competing for common targets or partners. If this scenario is true, TCP factors, acting in partially overlapping expression domains, could establish complex combinatorial networks of transcription factor activity, which need to be elucidated. Biologically significant TCP homoand heterodimers and additional TCP protein partners are likely to be characterised in the near future using proteomic approaches, and target genes controlled by these transcriptional complexes should be identified by in vivo high-throughput techniques, such as chromatin immunoprecipitation combined with massive parallel DNA sequencing (ChIP-Seq) [89]. The isolation of TCP protein partners and direct downstream genes might help researchers to understand the genetic pathways through which TCP genes affect the proliferation chromatin immunoprecipitation combined with massive parallel DNA sequencing (ChIP-Seq) versus differentiation status of cells. Finally, because of the probable functional redundancy of class I members, analysis of this class might need to be determined by RNA-induced gene silencing and chimeric repressor silencing techniques [24,63,90]. Future work should reveal new functions for this family of plant-specific genes. Furthermore, our increasing knowledge of TCP genes function and regulation should enable researchers to use them as tools to modulate plant growth patterns and to generate novel morphologies in species of agronomical interest. Acknowledgements We thank members of our lab (M.L. Rodriguez, F. Chevalier, E. Gonzalez and S. Otero), J.M. Martínez-Zapater, S. Prat and D. Bradley for comments on the article. 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