a genomic and expression compendium of thea genomic and expression compendium of the expanded pebp...

A Genomic and Expression Compendium of theExpanded PEBP Gene Family from Maize[W][OA]

Olga N. Danilevskaya*, Xin Meng, Zhenglin Hou, Evgueni V. Ananiev, and Carl R. Simmons

Pioneer Hi-Bred International Inc., a DuPont business, Johnston, Iowa 50131

The phosphatidylethanolamine-binding proteins (PEBPs) represent an ancient protein family found across the biosphere. Inanimals they are known to act as kinase and serine protease inhibitors controlling cell growth and differentiation. In plants themost extensively studied PEBP genes, the Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) and TERMINALFLOWER1 (TFL1) genes, function, respectively, as a promoter and a repressor of the floral transition. Twenty-five maize (Zea mays)genes that encode PEBP-like proteins, likely the entire gene family, were identified and named Zea mays CENTRORADIALIS(ZCN), after the first described plant PEBP gene from Antirrhinum. The maize family is expanded relative to eudicots (typicallysix to eight genes) and rice (Oryza sativa; 19 genes). Genomic structures, map locations, and syntenous relationships with ricewere determined for 24 of the maize ZCN genes. Phylogenetic analysis assigned the maize ZCN proteins to three major sub-families: TFL1-like (six members), MOTHER OF FT AND TFL1-like (three), and FT-like (15). Expression analysis demonstratedtranscription for at least 21 ZCN genes, many with developmentally specific patterns and some having alternatively splicedtranscripts. Expression patterns and protein structural analysis identified maize candidates likely having conserved gene func-tion of TFL1. Expression patterns and interaction of the ZCN8 protein with the floral activator DLF1 in the yeast (Saccharomycescerevisiae) two-hybrid assay strongly supports that ZCN8 plays an orthologous FT function in maize. The expression of otherZCN genes in roots, kernels, and flowers implies their involvement in diverse developmental processes.

The phosphatidylethanolamine-binding protein(PEBP) genes are found in all three major phylogeneticdivisions of prokaryotes, archaea, and eukaryotes(Banfield et al., 1998; Hengst et al., 2001; Chautardet al., 2004). Their conserved sequences indicate anancient common origin of a basic protein functionalunit. The seminal PEBP described was isolated frombovine brain as a soluble 23-kD basic cytosolic protein(Bernier and Jolles, 1984). Animal PEBP proteins weresubsequently shown to act as Raf kinase inhibitors(Yeung et al., 1999; Lorenz et al., 2003; Odabaei et al.,2004) in signaling cascades that control cell growth(Keller et al., 2004). Mouse PEBP exerts activity as a Serprotease inhibitor (Hengst et al., 2001). Overall, how-ever, mammalian PEBPs are multifunctional proteinswhose physiological roles are just beginning to beunderstood.

Plant PEBP-related genes were originally clonedfrom mutants with altered inflorescence architecture.These include Antirrhinum CENTRORADIALIS (CEN;Bradley et al., 1996), Arabidopsis (Arabidopsis thaliana)TERMINAL FLOWER1 (TFL1; Bradley et al., 1997), andtomato (Solanum lycopersicum) SELF PRUNING (SP;

Pnueli et al., 1998). The Antirrhinum cen and Arabi-dopsis tfl1 mutations cause normally indeterminateinflorescences to terminate in a flower (Shannon andMeeks-Wagner, 1991; Bradley et al., 1996, 1997). Thetomato sp mutation changes the indeterminate growthhabit to determinate, resulting in limited shoot growthand a bushy compact habit (Pnueli et al., 1998). Thesemutant phenotypes suggested these genes maintainthe indeterminate state of the inflorescence meristem.CEN is activated in the inflorescence apex a few daysafter the floral transition, whereas TFL1 and SP areexpressed in vegetative and inflorescence apices fromvery early stages where they delay the commitment toreproductive development (Bradley et al., 1997; Pnueliet al., 1998). The TFL1 and SP genes control bothvegetative and reproductive phase durations by main-taining both apical and inflorescence meristem inde-terminacy (Pnueli et al., 1998; Ratcliffe et al., 1998),while CEN controls only the inflorescence meristem(Bradley et al., 1996). Two distinct TFL1 homologs con-trol pea (Pisum sativum) vegetative and reproductivephase duration: The LATE FLOWERING gene main-tains shoot apical meristem indeterminacy, and DE-TERMINATE (DET) maintains inflorescence meristemindeterminacy (Foucher et al., 2003). TFL1 homologswith conserved function have now been identified inmany eudicotyledonous (Amaya et al., 1999; Pillitteriet al., 2004; Sreekantan et al., 2004; Kotoda and Wada,2005; Boss et al., 2006; Kim et al., 2006) and monocot-yledonous plants (Jensen et al., 2001; Nakagawa et al.,2002; Zhang et al., 2005). The Arabidopsis TFL1 pro-tein has recently been shown to be a mobile signal thatmoves from its localized transcriptional domain in the

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Olga Danilevskaya ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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central region of the inflorescence meristem to theentire meristem, where it exerts its activity. The TFL1protein is excluded from the floral meristem, allowingfloral primordia to develop into a flower (Conti andBradley, 2007).

The Arabidopsis FLOWERING LOCUS T (FT) geneis a member of the PEBP gene family, but it hasan opposing function to TFL1: It promotes the transi-tion from the vegetative to the reproductive phase(Kardailsky et al., 1999; Kobayashi et al., 1999). Criticalamino acid residues differentiate FT and TFL1 proteinsand dictate whether the protein acts as a floral activa-tor or repressor. These residues include Tyr (Y85) in FTand His (H88) in TFL1 (Hanzawa et al., 2005), togetherwith a stretch of 14 amino acid residues in exon 4 (Ahnet al., 2006). FT is a key activator of flowering, medi-ating both photoperiod and vernalization regulation(Baurle and Dean, 2006; Jaeger et al., 2006). The FTgene is transcribed in the leaf phloem (Takada andGoto, 2003), but the FT protein moves via the phloemto the shoot apex where it acts as a floral stimulus. Thisprotein provides a molecular explanation for the longhypothesized mobile flowering signal, known as theflorigen (Corbesier et al., 2007; Jaeger and Wigge, 2007;Mathieu et al., 2007). At the shoot apex, the FT proteininteracts with the bZIP transcription factor FLOWER-ING LOCUS D (FD), and together they activate themeristem identity gene APETALA1, a MADS-box pro-tein, triggering flower development (Abe et al., 2005;Wigge et al., 2005).

The function of FT is highly conserved in plants. Therice (Oryza sativa) FT homolog encoded by Headingdate3a (Hd3a; also known as OsFTL2) migrates fromleaves to the shoot apical meristem to induce thefloral transition (Tamaki et al., 2007). The tomato FThomolog SINGLE FLOWER TRUSS also regulatesflowering time and shoot architecture by generating agraft-transmissible signal (Lifschitz and Eshed, 2006;Lifschitz et al., 2006). In rice and perennial ryegrass(Lolium perenne), heading date quantitative trait loci(QTL) are associated with FT homologs (Kojima et al.,2002; Monna et al., 2002; Armstead et al., 2004). Inwheat (Triticum aestivum) and barley (Hordeum vulgare)the VERNALIZATION LOCUS3 (VRN3) gene corre-sponds to an FT function (Yan et al., 2006; Faure et al.,2007). FT homologous genes accelerate floral develop-ment in transgenic woody plants, such as citrus(Poncirus trifoliata L. Raf.; Endo et al., 2005) and poplar(Populus spp.; Bohlenius et al., 2006), a quality thatmay speed tree breeding programs. In addition toregulating flowering time, the poplar FT homologcontrols short-day fall-induced growth cessation andbud set (Bohlenius et al., 2006). In Norway spruce(Picea abies L. Karst.), an FT homolog mediates bud setand bud burst, indicating conservation of FT functionin gymnosperms (Gyllenstrand et al., 2007).

Four additional PEBP genes were found in theArabidopsis genome by homology: TWIN SISTER OFFT (TSF), BROTHER OF FT AND TFL1 (BFT), ARABI-DOPSIS THALIANA CENTRORADIALIS (ACT), and

MOTHER OF FT AND TFL1 (MFT). TSF and MFT seemto act as floral integrators redundantly with FT (Yooet al., 2004; Yamaguchi et al., 2005). The function ofACT and BFT remains unclear (Mimida et al., 2001).Small families of about six to eight genes are typicalwithin eudicots such as Arabidopsis, tomato, poplar,and grape (Vitis vinifera; Mimida et al., 2001; Carmel-Goren et al., 2003; Kotoda and Wada, 2005; Carmonaet al., 2007). Larger gene families were found in mono-cots such as rice (19 members), wheat (19), and maize(Zea mays; approximately 30; Chardon and Damerval,2005). In this article we identified the likely completeset of maize PEBP-related genes, which we named Zeamays CENTRORADIALIS (ZCN) after CEN from An-tirrhinum, the first PEBP-related plant gene described(Bradley et al., 1996). A combination of genomic,syntenic, phylogenetic, protein structural, and expres-sion analyses revealed an expanded, diverse genefamily. Included in this family are candidates for theprobable functional orthologs of TFL1 and FT govern-ing the floral transition, and also other members thatmay control developmental processes in roots, kernels,and flowers.

RESULTS

Identification and Characterization of the ZCNGene Family

Extensive searches of public and proprietary tran-script and genomic databases with six ArabidopsisPEBP proteins as BLAST queries (TFL1, FT, ATC, BFT,TSF, and MFT) identified 25 ZCN genes that werecompleted sequences from the appropriate bacterialartificial chromosome (BAC) clones (Table I). One ofthese, ZCN23, appears to be a pseudogene because onlyexon 4 was found in the 200-kb sequence of the cor-responding BAC. All complete 25 maize genes includedDNA fragments previously identified by Chardon andDamerval (2005). However, the final number of ZCNgenes will be established upon completion of themaize genome sequencing project.

The nearest genetic markers for each ZCN gene weredetermined from BAC contigs and used to position theZCN genes on the maize genetic map (Table I; Fig. 1).In addition to in silico mapping, the chromosome lo-cation of each ZCN gene was confirmed by PCR usingthe oat (Avena sativa)-maize addition lines (Supple-mental Fig. S1; Supplemental Table S1), which are a setof 10 oat lines each carrying a single maize chromo-some addition (Kynast et al., 2001). ZCN2, ZNC13,ZCN20, ZCN21, and ZCN26 mapped near centromeres(Fig. 1).

All the maize ZCN gene family members appearedto have generally low expression judging by the smallnumber of ESTs in all the databases queried. cDNAswere obtained by reverse transcription (RT)-PCR us-ing pairs of gene-specific primers designed close to thestart and stop codons (Supplemental Table S2). NocDNAs were obtained for ZCN13, ZCN21, and ZCN24,

PEBP Gene Family in Maize

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evidently because no expressing tissues were found(Table I). The intron/exon structures of the ZCN geneswere determined from the alignments of the cDNAand genomic sequences (Fig. 2). If a cDNA was notobtained, the genomic sequence was used to deter-mine the probable gene structure by alignment to theconserved coding regions and intron positions exhib-ited by other gene family members. Of the ZCN genesfound, 22 of 24 have a conserved genomic structureconsisting of four exons. The exceptions, ZCN2 andZCN20, have three exons that apparently resultedfrom fusion of exons 1 and 2. Most ZCN genes havea compact gene structure in the range of 1 to 3 kb. Thesizes of exon 2 and exon 3 are generally 63 and 42 bp,respectively, which are also the conserved exon sizesamong other plant PEBP-related genes (Carmel-Gorenet al., 2003; Zhang et al., 2005; Carmona et al., 2007).ZCN14, ZCN18, ZCN21, and ZCN24 are the largestgenes, with sizes between 5 to 9 kb, as a result ofexpanded introns. The complete amino acid sequencefor each gene was deduced from its cDNA sequenceand in a few cases its genomic sequence (Table I).

Protein Phylogenetic Analysis and Rice Synteny

Phylogenetic analysis was used to infer the evolu-tionary relationship between the 24 maize and 19 ricePEBP proteins. The six Arabidopsis PEBP proteins and

the wheat and barley FT functionally homologousproteins corresponding to VRN3 (Yan et al., 2006), wereincluded as phylogenetic references. Protein alignments(ClustalW algorithm) revealed dispersed protein con-servation, but with higher conservation over the ligand-binding site (Banfield and Brady, 2000). The mostconserved protein segments beginning with EDL/DPLand ending with RRR/GGR residues (PEPB domainpfam01161; Supplemental Table S3) were used for thephylogenetic analysis (Kumar et al., 2004). The PEBPproteins form three well-marked subfamilies, whichwe designate after the three Arabidopsis reference genes,TFL1-like, MFT-like, and FT-like (Fig. 3). The dendo-gram topology of the plant PEBP proteins is consistentwith a previous report (Chardon and Damerval, 2005).

The rice proteins, with the exception of OsTFL8,formed monophyletic subgroups with one or two maizeproteins. The previously reported rice-specific OsFTL5and OsFTL11 (Chardon and Damerval, 2005) are nowassociated with ZCN16 and ZCN17, respectively. Amongthe 24 maize ZCN proteins, 14 ZCN proteins appearedas seven paired monophyletic subgroups: ZCN1-ZCN3,ZCN4-ZCN5, ZCN7-ZCN8, ZCN9-ZCN10, ZCN13-ZCN21, ZCN18-ZCN24, and ZCN19-ZCN25. Thesegene pairs likely represent paralogs, a product of thetetraploid ancestry of maize (Gaut, 2001; Bruggmannet al., 2006).

Table I. ZCN gene family in maize

*, Genes with no RT-PCR detectable expression; **, a pseudogene containing only exon 4.

Gene Name Proteina cDNAGenBank Accessions,

cDNA

GenBank Accessions,

Genomics

Nearest

MarkerbChr.

Locusc

ZCN1 173 EST EU241917 EU241892 csu728a 3.09ZCN2 173 EST EU241918 EU241893 bngl1755 4.05ZCN3 173 EST EU241919 EU241894 umc1576 10.1ZCN4 176 EST EU241920 EU241895 umc1235 2.04ZCN5 173 EST EU241921 EU241896 umc1677 10.04ZCN6 178 RT-PCR EU241922 EU241897 csu166a 4.8ZCN7 192 RT-PCR EU241923 EU241898 umc1520 6.06ZCN8 175 EST EU241924 EU241899 umc2075 8.03ZCN9 172 EST EU241925 EU241900 umc1483 8.02ZCN10 172 EST EU241926 EU241901 umc1717 3.04ZCN11 180 EST EU241927 EU241902 umc21 6.04ZCN12 177 EST EU241928 EU241903 umc2272 3.07ZCN13* 185 No EU241904 bngl2323cent 5.04ZCN14 173 EST EU241929 EU241905 umc2147 8.03ZCN15 177 EST EU241930 EU241906 umc2074 6.01ZCN16 174 RT-PCR EU241931 EU241907 csu173 5.05ZCN17 179 RT-PCR EU241932 EU241908 AW681281 2.05ZCN18 173 RT-PCR EU241933 EU241909 umc1890 2.07ZCN19 175 RT-PCR EU241934 EU241910 umc2003 10.4ZCN20 175 EST EU241935 EU241911 est492090x 10.03ZCN21* 187 No EU241912 cent 2ZCN23** No 5#end No EU241913 csu230 3.02ZCN24* 173 No EU241914 umc1333 7.03ZCN25 174 RT-PCR EU241936 EU241915 umc1326 2.04ZCN26 187 RT-PCR EU241937 EU241916 est324143x 9.03

aThe number of amino acids in the deduced polypeptides. Corresponding full-length cDNAs were ESTsor generated in RT-PCR. bThe nearest chromosomal marker to the ZCN genes. cLocalization of theZCN genes to the chromosome bins.

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The TFL1-like subfamily is composed of six maizeand four rice proteins. Maize ZCN1-ZCN3-ZCN6 pro-teins group with rice proteins RCN1 and RCN3. ZCN1and ZCN3 genes seem to be a recent duplication,because they share 89.3% nucleotide identity withinexons and 79.4% nucleotide identity within introns(Supplemental Fig. S2). The other monophyletic groupis composed of maize proteins ZCN2-ZCN4-ZCN5and rice RCN2-RCN4, the latter thought to represent aduplicated rice chromosomal segment (Chardon andDamerval, 2005). The ZCN4-ZCN5 gene pair does notshare much intron homology, suggesting a less recentduplication. The MFT-like subfamily is the smallest,composed of three maize and two rice genes: ZCN9-ZCN10 grouped with OsMFT2, and ZCN11 groupedwith OsMFT1.

The FT-like subfamily is the largest, composed of 15maize and 13 rice genes. This subfamily forms twolarge monophyletic groups that we named FT-like Iand FT-like II. The FT-like I group is of particularinterest because it includes the key floral activatorsfrom Arabidopsis, FT, and TSF (Huang et al., 2005;Yamaguchi et al., 2005). The FT-like I group is dividedinto two main subgroups. One subgroup is composedof the Arabidopsis FT and TSF plus seven maize andfive rice proteins, including maize gene pairs ZCN18-ZCN24 and ZCN19-ZCN25 and rice OsFTL4 andOsFTL6. The other subgroup contains maize ZCN14and ZCN15 within the well-supported monophyleticgrouping of known monocot floral activators, includ-ing rice OsFTL2 (Hd3a) and OsFTL3 (Hd3b; Kojima

et al., 2002), wheat and barley VRN3, which are namedTaFT and HvFT (Yan et al., 2006). The FT-like II groupis composed of six maize and five rice proteins, but noArabidopsis proteins, which suggests diversificationand expansion of this subgroup after the monocot-eudicot radiation. The ZCN7-ZCN8 pair forms agrouping with ZCN12 and OsFTL9. ZCN13-ZCN21and ZCN26 are similar to OsFTL13 and OsFTL12,respectively. Only OsFTL8 does not directly groupwith any of the maize proteins.

Synteny analysis was performed to assess the corre-spondence between the phylogenetic groupings of thePEBP proteins and the prevailing rice-maize synteniccontext for their gene locations, recognizing that syntenyor departures therefrom can exist between these com-plex cereal genomes. The maize gene map positions andtheir syntenic rice counterpart(s) are summarized inFigure 1. In cases of a single maize-to-rice gene phylo-genetic correspondence, the synteny was present andunambiguous. These examples are ZCN11-OsMFT1,ZCN2-RCN2, ZCN16-OsFTL5, ZCN17-OsFTL11, ZCN20-OsFTL7, ZCN14-OsFTL1, ZCN12-OsFTL9, and ZCN26-OsFTL12. In cases where duplicated maize genescorresponded to a single rice gene, both maize duplicatedsegments might be considered syntenic to a single ricelocus. These examples are ZCN9/ZCN10-OsMFT2,ZCN13/ZCN21-OsFTL13, ZCN18/ZCN24-OsFTL4, andZCN19/ZCN25-OsFTL6. In one case, a single maizegene (ZCN15) corresponded to tandemly duplicatedrice genes (Hd3a/Hd3b), and the syntenic context wasapparent. More challenging for syntenic interpretation

Figure 1. ZCN gene positions on maize IBM2genetic map and syntenic rice genes. ZCNpositions are inferred from the nearest markersof the corresponding BAC fingerprinting contigs(Table I). Centromeric regions are shown asblack rectangles. Pseudogene, ZCN23, ismarked byan asterisk (*). Seven duplicated pairsof genes are marked by various colors: ZCN1/ZCN3, blue; ZCN4/ZCN5-ZCN19/ZCN25,red; ZCN9/ZNC10, burgundy; ZCN18/ZCN24,orange; ZCN7/ZCN8, green; ZCN13/ZCN21,framed red over the centromeres. Rice genes(their chromosomes in parentheses) with thehighest probability of synteny are shown onthe left side of the maize chromosome.





are genes involved in apparently ancient segmental du-plications prior to the rice-maize divergence. The largeduplicated segments of rice chromosomes 2 and 4,carrying RCN2-OsFTL5 and RCN4-OsFTL6 genes, re-spectively, appeared to be syntenic to maize genesZCN4-ZCN21 and ZCN5-ZCN19 on chromosomes 2and 10, but this synteny remains somewhat ambiguous.A similar situation exists for the rice RCN1-RCN3 genesthat reside in duplicated segments of rice chromosomes11 and 12. The closely related maize genes ZCN1, ZCN3,and ZCN6 mapped to duplicated segments of chromo-somes 3, 10, and 4, and synteny was difficult to assign.We were also not able to determine clear rice syntenywith the maize pair ZCN7/ZCN8. The complete maizegenome sequence may help clarify these and otherremaining inconsistencies.

Maize ZCN Protein Structural Analysis

All PEBP proteins share a common structure, with adominant feature being a central antiparallel b-sheetflanked by a small b-sheet on one side and two a-heliceson the other. All family members also possess a highlyconserved putative anion-binding site located at oneend of the central b-sheet, supporting the idea thatthe proteins are functioning in part through formingcomplexes with a phosphorylated ligand (Banfieldet al., 1998; Serre et al., 1998; Banfield and Brady, 2000;Ahn et al., 2006). The three-dimensional (3-D) struc-ture of the CEN proteins suggested a role as a kinaseregulator (Banfield and Brady, 2000), hinting that plantPEBP proteins may function as modulators of signal-ing cascades controlling plant growth and develop-

ment. The critical structural variations among distinctmembers occur at the loops connecting these second-ary structure elements and at the C-terminal regions.

To build 3-D models for the maize ZCN proteins,we used homology modeling with the ArabidopsisTFL1 (Protein Data Bank PDB:1wko A chain) and FT(PDB:1wkp A chain) proteins as structural templates.Out of 25 maize ZCN proteins, we focused on tworepresentative candidates (ZCN2 and ZCN14) basedon the highest sequence similarity to their Arabidopsis

Figure 3. Phylogenetic analysis of the maize, rice, and ArabidopsisPEBP proteins. The protein sequences of 51 plant PEBP gene productsincluding 45 Poaceae monocot (black) and six Arabidopsis (red) werelimited to the conserved EDL/DPL...RRR/GGR region encompassingmost of the complete proteins. The phylogenetic test used was theBootstrap test by minimum evolution method, as implemented byMEGA version 3.1 (Kumar et al., 2004). Dendogram branches arelabeled with percentage of 1,000 iterations supporting each branch.The scale bar at bottom reflects the frequency of amino acid substitu-tions between sequences as determined by the Poisson correctivedistance model.

Figure 2. ZCN exon/intron structures. Genomic structure of the ZCNgenes is represented by black boxes as exons and spaces between theblack boxes corresponds to introns. The sizes of the exons and intronscan be estimated using the vertical lines. Genes are arranged intophylogenetic subfamilies. The major subfamilies are denoted on the right.

Danilevskaya et al.




counterparts. ZCN2 has 58% amino acid sequenceidentity to TFL1, and ZCN14 has 72% sequence identityto FT. The models were constructed with MODELER,and refined further by guidance from stereochemistryviolation penalty rules. The overall refined modelsabided to their Arabidopsis templates with Ca RMSDs(root mean square deviations of Ca atomic coor-dinates) less than 1 A (Fig. 4, A and B). Comparisonbetween ZCN2 and TFL1 at the putative phosphate-binding site revealed that the key residues were 100%conserved, including Glu-109 (ZCN2)/Glu-112 (TFL1),His-87/90, Asp-71/74, Val-74/77, Leu-82/85, His-85/88, Asp-140/144, His-118/121, and Phe-120/123(Fig. 4A). Similarly, ZCN14 and FT were also highlyconserved in the binding site, again including Glu-107(ZCN14)/Glu-109 (FT), His-85/87, Asp-69/71, Ala-72/Val-74, Leu-80/82, Tyr-83/85, Gln-138/140, His-116/118, and Val-118/120. In addition, the previously iden-tified structural features for distinguishing TFL1 andFT specificity, including the interacting triads, Glu-112-His-88-Asp-144 of TFL1 corresponding to Glu-109-His-85-Asp-140 of ZCN2 and Glu-109-Tyr-85-Gln-140 of FTcorresponding to Glu-107-Tyr-83-Gln-138 of ZCN14 atthe binding site entrance, were all well preserved be-tween maize and Arabidopsis (Fig. 4, A and B). To in-vestigate whether the binding site could accommodatea phosphorylated ligand, the bovine PEBP structure(PDB:1b7a) was superimposed, and then its cocrystal-lized ligand phosphoric acid mono-(2-amino-ethyl)ester (OPE in PDB structure) was transferred into theZCN2 model. Further docking analysis regardinggeometric complementarity and binding energy sug-gested that the OPE could fit well into the cavity with-out serious stereo conflict (Supplemental Fig. S3).

Conservation of ZCN protein primary structures wasevaluated from the alignment of the ligand-bindingmotif and the external loop (Fig. 4C). All maize proteinspossess invariable residues in the critical positionsforming the ligand-binding pocket corresponding toArabidopsis TFL1/FT residues Asp-74/71, Asp-75/73,Pro-78/75, His-90/87, and His-121/118 (Fig. 4C). Thecritical His-88 in TFL1 that defines the opposing re-pressor/activator activities of TFL1 and FT, respec-tively, is conserved in all six maize TFL1-like proteins.All other maize ZCN proteins have in this position Tyr,corresponding to Tyr-85 in Arabidopsis FT, with theexception of ZCN11 (MFT-like) and ZCN17 (FT-like I),that have Leu and Asn, respectively. The distinct fea-ture of MFT-like proteins is substitution of conservedGlu-112/109 for Met in ZCN9 and ZCN10 or for Val inZCN11. All the other maize proteins have Glu in thisposition.

The external loop encoded by exon 4 is importantfor the opposing activities of Arabidopsis TFL1/FTproteins (Ahn et al., 2006). The external loop is clearlydefined in the alignment due to its variation in sizebetween subfamilies (Fig. 4C). The loops of TFL1-likeproteins are variable in size (14–17 residues) and haveonly two conserved residues despite the high aminoacid conservation over the remainder of the proteins

(100 invariant out of 180 total residues). The externalloop of the MFT clade is also variable, ranging from 14to 20 residues and having three invariant amino acids.The ZCN11 protein is the most diverged from theconsensus with the longest (20 residues) external loop.The external loop of the FT-like subfamily has auniform size of 14 amino acids with significant con-servation of amino acids between members of thegroup. For instance, nine out of 14 residues are invari-ant in the FT-like I group, and eight out of 14/15 in theFT-like II group. This is consistent with the previousobservation for the FT-like subfamily in other species(Ahn et al., 2006). In conclusion, the conservation ofprimary, secondary, and tertiary protein structures ofmaize ZCN proteins suggest their biochemical func-tions might also be preserved.

Expression Patterns of the ZCN Genes

As a step toward elucidating maize ZCN genefunction, we surveyed ZCN transcript accumulationacross a wide range of organs and tissues using themassively parallel signature sequence (MPSS) RNAprofiling database (Fig. 5). MPSS technology is anopen-ended platform that quantifies the level of tran-script accumulation of virtually all genes in a sampleby counting the number of individual mRNA mole-cules produced from each gene in the form of a 17-mertag signature sequence (Brenner et al., 2000). The 17-mer tag distributions confirmed the low level of ZCNgene expression across all tissues, as predicted fromtheir low EST abundance. ZCN14 and ZCN11 ap-peared to be the only highly expressed genes in thebroad set of tissues queried. The MFT-like subfamilyshowed preferred expression in kernel tissues. The FT-like II group was expressed mainly in leaves. To refineour analysis of gene expression patterns depicted byMPSS, we performed RT-PCR in nine different tissues:root, stem, immature leaf, mature leaf blade, shootapex, immature tassel, immature ear, embryo, andendosperm. For selected ZCN genes, a more detaileddevelopmental analysis was performed. Gene expres-sion was detected using RT-PCR with pairs of gene-specific primers corresponding to the first and lastexon of each gene (Supplemental Table S2). Thus, thisprimer design allowed us to detect alternative tran-script splicing patterns.

The maize TFL1-like subfamiliy showed similartissue expression patterns with expression in roots,young stems, immature ear, and tassel (Fig. 6A).Because the Arabidopsis TFL1 gene maintains shootapical and inflorescence meristem indeterminacy, wefocused on the expression of TFL1-like genes in de-veloping shoot apices, tassels, and ears (Fig. 6B). Theclosely related ZCN1 and ZCN3 genes are expressed inshoot apices during vegetative development and inboth tassel and ear primordia during reproductivedevelopment (Fig. 6B). ZCN1 and ZCN3 accumulatedsignificant amounts of unspliced pre-mRNA at early





stages. ZCN3-spliced mRNA appeared in significantproportions after the floral transition in the V9 stagetassel and ear primordia, whereas ZCN1 mRNA re-mained mostly unspliced. None of the other TFL1-likegenes accumulated unspliced transcript.

ZCN6 mRNA was detected at lower levels in shootapices and tassel primordia, but its expression is up-regulated in developing ears. ZCN2 and ZCN6 wereexpressed before and after the floral transition likeZCN1 and ZCN3, but they show a distinct pattern ofaccumulation (Fig. 6B). ZCN2 mRNA accumulationgradually increased in shoot apices around the floraltransition, peaking in V7 and V8 tassels and decreas-ing in later stages. ZCN6 had a complementary pat-tern, showing low transcript accumulation in V3 to V8

stage apices and a higher accumulation at the V9 stage.In developing ears, ZCN2 was active at all stagestested whereas ZCN6 was expressed at the V7 to V9stages and decreased later. ZCN4 and ZCN5 tran-scripts accumulated after the floral transition in bothtassel and ear primordia (Fig. 6B), suggesting they actspecifically in the developing inflorescence after thefloral transition, similar to snapdragon (Antirrhinummajus) CEN (Bradley et al., 1996) and pea DET (Foucheret al., 2003). Five out of the six TFL1-like maize geneswere expressed in roots (Fig. 6A).

MFT-like ZCN transcripts accumulated predomi-nantly in kernels with the exception of ZCN11, whichaccumulated in both kernel and seedling tissues (Fig.6C). Developmental profiling of the MFT-like genes

Figure 4. Structural models of twoZCN proteins and alignment ofmaize and Arabidopsis PEBP pro-teins. Protein structure ribbon pre-sentation of ZCN2 (A) and ZCN14(B). The secondary structures areassigned by Pymol and colored asorange. The external loop is high-lighted as magenta. The conservedresidue side chains in the bindingpocket are shown as sticks and thetriad residue side chains are repre-sented as stick and balls with atomscolor coded: green for carbon, bluefor nitrogen, and red for oxygen. C,Multiple sequence alignment of theconserved ligand-binding motifand the external loop of maizeZCN and the Arabidopsis TFL1,MFT, FT, and TSF proteins. Theexternal loop is boxed. Invariantamino acids are highlighted yellow,similar amino acids are highlightedgreen or blue. Intron positions aremarked by arrows above the align-ment. Asterisks (*) indicate invari-able and pound signs (#) variable,but critical amino acids forming theligand-binding pocket. Invariableresidues correspond to the follow-ing position in TFL1/FT proteins:Asp-74/71, Asp-75/73, Pro-78/75,His-90/87, His-121/118. VariableHis-88/Tyr-85 residues define theopposing activity of TFL1 and FT,respectively. Ampersand (&) signsmark Arg-133/130 and Arg-143/139 in the external loop that isconserved in most proteins, withthe exceptions of ZCN2, ZCN19,and ZCN25.

Danilevskaya et al.




was performed on whole kernels starting from 2 to 8 dafter pollination (DAP) and on dissected embryo andendosperm tissues from kernels at 10 to 26 DAP. ZCN9mRNA was detected in the embryo after 10 DAP andfaint expression was found in the endosperm after 14DAP. ZCN10 mRNA was detected only in the embryoafter 14 DAP with a significant proportion of unsplicedtranscript. ZCN11 transcript accumulated in ovulesbefore pollination and continued to accumulate inboth embryo and endosperm at all postpollinationstages (Fig. 6D).

The FT-like subfamily is a complex group composedof 15 maize ZCN genes in two groups named FT-like Iand FT-like II. The FT-like I group is composed ofseven maize genes that are further divided into twosubgroups, one with Arabidopsis FT-TSF, and theother formed by the FT functionally conserved mono-cot homologs (Fig. 3). The duplicated ZCN19-ZCN25pair of genes was expressed in roots. ZCN16 andZCN20 produced unspliced mRNA in many tissues.ZCN17 is actively transcribed in roots and stems.ZCN18 is expressed in the stem and leaves producinga mixture of spliced and unspliced transcript. ZCN24mRNA was not detected in this set of tissues (Fig. 6E).ZCN14 and ZCN15 are grouped tightly with FT ho-mologous monocot floral activators (Fig. 3), thus beingthe most favorable candidates for possessing FT func-tion. However, their expression patterns do not sup-port their function as floral activators. In the broad setof tissues surveyed, ZCN14 mRNA was detected in thetassel and ear primordia and the endosperm, whereasZCN15 expression was not detected in these samples(Fig. 6E). The original ZCN15 EST was found in apedicel cDNA library. In developing kernels ZCN15

mRNA was detected in whole kernels between 4 and 8DAP and in the dissected pedicels from later stagekernels (Fig. 6F). Dissected pedicels often contain thebasal endosperm. Therefore, ZCN15 transcript accu-mulates in kernels after fertilization, most likely in thebasal layer of the endosperm. This expression patternis inconsistent with ZNC15 as a floral activator. ZCN14transcript was detected in ovules and early developingkernels, but its level gradually decreased after 12 DAP(Fig. 6F). Because ZCN14 is the only FT-like gene whosetranscript accumulates in the ear and tassel primordia,we investigated its expression in these tissues as well(Fig. 6F). In shoot apices, weak transcript accumulationis detected at the V5 stage, coinciding with the floraltransition. ZCN14 mRNA levels increased in develop-ing tassels at the V7 stage and remained constant up tothe V9 stage. ZCN14 transcript accumulated in V7 stageear buds and later developing ear stages. The ZCN14expression pattern is quite different from ArabidopsisFT and rice Hd3a, which are transcribed exclusively inleaf blades (Corbesier et al., 2007; Jaeger and Wigge,2007; Mathieu et al., 2007; Tamaki et al., 2007). ThusZCN14 is unlikely a floral activator in maize.

The FT-like II group is composed of six maize genes.Three maize genes of this group, ZCN8, ZCN12, andZCN26, are expressed predominantly in leaf blades(Fig. 6G). Three other genes seem to be nonfunctionalor with a limited function because they produce eitherunspliced mRNA (ZCN7) or their expression was notdetected in any tissues tested (ZCN13 and ZCN21). Weinvestigated further the developmental expressionpattern of ZCN8, ZCN12, and ZCN26 from FT-like IIand ZCN14 and ZCN18 from FT-like I in stems, im-mature leaves, and leaf blades at the V2 to V9 stages

Figure 5. Maize ZCN family transcriptsurvey across various tissues usingMPSS technology. Relative ZCN genetranscript abundance measured asmean frequency of 17-mer tags in partper million in varies tissues. ZCN13was omitted from the survey due tolack of a gene-specific tag. ZCN2,ZCN20, and ZCN21 do not produceexpressed 17-mer tags.





Figure 6. ZCN gene expression patterns revealed by RT-PCR. RNA expression patterns of TFL1-like genes in a broad set of tissues(A), and in developing tassels and ears (B). Expression patterns of MFT-like genes in a broad set of tissues (C) and in developingkernels (D). Expression patterns of FT-like I genes in a broad set of tissues (E). ZCN14 and ZCN15 expression in developing

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(Fig. 6H). We also included ZCN7 to test mRNAsplicing during development; however, only un-spliced ZCN7 RNA was detected in stems and imma-ture leaves and no RNA was found in leaf blades atvarious developmental stages. Its paralog ZCN8 alsoproduced unspliced transcript in stems and immatureleaves, but in expanded leaf blades, ZCN8 mRNA wascompletely spliced (Fig. 6H). This finding indicatesthat ZCN8 activity may be regulated by mRNA splic-ing. Importantly, the spliced ZCN8 transcript was de-tected in leaf blades before the floral transition at theV3 stage. Its level increased at the V4 stages just beforethe floral transition and stayed high afterward. Thisexpression pattern makes ZCN8 an attractive candi-date as a floral activator. Another leaf-specific gene,ZCN12, was found to be activated in leaf blades at theV7 stage after the floral transition and continued to beexpressed at later stages (Fig. 6H). ZCN26 is expressedpredominantly in leaf blades and produced a signifi-cant amount of unspliced pre-mRNA. ZCN18 was ex-pressed mainly in the stem with weaker expression inleaf blades. ZCN14 mRNA was barely detected by RT-PCR in immature leaves and in leaf blades at vegeta-tive V4 and V5 stages, but after the floral transition, theZCN14 mRNA level increased and was reliably de-tected in leaf blades (Fig. 6H).

Yeast Two-Hybrid Interactions between DLF1 and Maize

FT-Like Proteins

In Arabidopsis the floral activator FT interacts withthe bZIP transcription factor FD to activate the floralidentity genes inducing flower development (Abe et al.,2005). FTand FD protein interaction was demonstratedgenetically and in the yeast (Saccharomyces cerevisiae)two-hybrid system (Abe et al., 2005; Wigge et al., 2005).Of the maize FT-like ZCN genes the mostly likelycandidate for FT function is ZCN8. Its expression pat-tern is consistent with a role as the floral activator. Inmaize the bZIP transcription factor DLF1 is an activatorof the floral transition and has function similar to theArabidopsis FD (Muszynski et al., 2006). We testedinteractions between DLF1 and ZCN8, ZCN12 andZNC14 proteins in the yeast two-hybrid system (TableII). The DLF1 protein shows self interaction that istypical for bZIP transcription factors that form dimersthrough Leu zippers. None of the ZCN proteins showedself interaction. Of three ZCN proteins tested, onlyZCN8 interacts with DLF1 in both combinations as bait

and prey (Table II; Supplemental Fig. S4). ZCN14interacts with DLF1 as bait but not as prey, indicatinga weaker or ambiguous interaction. This result stronglysupports the hypothesis that the ZCN8 gene functionsas a floral activator in maize similar to the ArabidopsisFT and rice Hd3a.

DISCUSSION

Maize Contains an Expanded PEBP Gene Family

Growing evidence indicates that PEBP-related pro-teins are potent and sometimes mobile regulators ofplant architecture, development, and seasonal growthadaptation (Bohlenius et al., 2006; Yan et al., 2006; Contiand Bradley, 2007; Corbesier et al., 2007; Tamaki et al.,2007). A survey study on this important gene family incereals was published earlier, but maize genes wererepresented largely by raw sequences available frompublic ESTand GSS (genome sample survey) databases(Chardon and Damerval, 2005). In this article we re-dress this gap by identifying and curating the entiremaize PEBP gene family, including complete gene andprotein structures, evolutionary relationships with riceand Arabidopsis, and spatiotemporal specific geneexpression patterns.

We identified 25 ZCN genes, although one is likely apseudogene. Eudicot genomes such as Arabidopsis andpoplar have about a half-dozen PEBP genes (Mimidaet al., 2001; Carmel-Goren et al., 2003; Carmona et al.,2007). Monocots are trending higher, with at least19 known in rice (Chardon and Damerval, 2005). Thephylogenetic analysis revealed three major ZCN sub-families, the TFL1-like, MFT-like, and FT-like, as in otherplant genomes, reinforcing the idea that the monocot-eudicot last common ancestor had at least three PEBPmembers (Chardon and Damerval, 2005). Gene dis-tribution between these rice subfamilies is 4:2:13(TFL1-like:MFT-like:FT-like). The ZCN subfamily dis-tribution is 6:3:15, which is nearly double the numberrelative to the presumed monocot ancestor. The largerFT-like subfamily fell into two groups with the FT-likeII group remaining monocot specific.

The maize PEBP gene family expansion (to 25 mem-bers) compared to rice (19 members) could be ex-plained by the ancient tetraploid ancestry of maize, inwhich a genome duplication occurred after divergencefrom the rice and maize common ancestor, followed bysubsequent diploidization en route to modern maize

Figure 6. (Continued.)kernels, pedicel (ZCN15), and in developing tassels and ears (ZCN14; F). Expression patterns of FT-like II genes in a broad set oftissues (G) and in developing stems and leaves (H). Horizontal arrows indicate spliced mRNA. White stars indicate the floraltransition. Asterisks (*) mark genes with no detectable expression. V3 to V10 indicates the vegetative stages of plant developmentdefined according to the appearance of the leaf collar of the uppermost leaf. The superscript numbers 1 and 2, mark the early andlater stages of V4 and V7, respectively, as judged by the shorter internode length at the early stages. Beginning of the floraltransition was determined by the elongation of the shoot apical meristem followed by the appearance of the spikelet pairmeristem that typically takes 1 to 2 d. Tissues are abbreviated as: EM, Embryo; EN, endosperm; IL, immature leaves; IT, immaturetassel; IE, immature ear; LB, leaf blade; SAM, shoot apical meristem; R, root; S, stem.





(Paterson et al., 2004; Swigonova et al., 2004; Bruggmannet al., 2006). This diploidization process is thought tohave shed at least 50% of the duplicated genes (Laiet al., 2004). The seven duplicated pairs of ZCN genesmay have survived this diploidization contraction,contributing to the higher overall family gene count.Syntenic relationships between rice and maize PEBPgenes were sometimes confusing due to gene dupli-cations and deletions during evolution of each species;however, for 13 rice genes their syntenic maize ZCNcounterparts were found (Fig. 1).

Most of the ZCN Genes Have DistinctExpression Patterns

Most of the ZCN genes appear to be functional,because all but one gene predicts a credible completeopen reading frame, and all but three genes (ZCN13,ZCN21, and ZCN24) have transcripts detected in atleast one tissue of 13 tested by RT-PCR. Although thethree poorly expressed genes may be heading towardevolutionary degeneracy, their open reading framesare complete, and important cryptic expression cannotbe ruled out. Further, the duplicated ZCN13 andZCN21 genes are located near centromeres 5 and 2,where expression may be suppressed due to epige-netic silencing spreading from the heterochromatin.ZCN20 is also centromeric and shows very low ex-pression.

The presence of duplicated genes raises the questionabout their functional redundancy. According to evo-lutionary models, duplicated genes may undergo dif-ferent selection processes: nonfunctionalization whereone copy loses function, hypofunctionalization whereone copy decreases in expression or function, neo-functionalization where one copy gains a novel func-tion, or subfunctionalization where the two copiespartition or specialize into distinct functions (Lynchand Conery, 2000; Otto and Yong, 2002; Duarte et al.,2006). These evolutionary fates may be indicated withdivergence in expression patterns or protein structure.We compared the expression patterns of the sevenduplicate ZCN gene pairs that were not duplicated inthe rice genome. Three maize gene pairs from the

FT-like subfamily suggest non/hypofunctionalization.These dominant/submissive pairs are ZCN19/ZCN25,ZCN18/ZCN24, and ZCN7/ZCN8. Both ZCN19/ZCN25 are expressed in roots, but ZCN19 has a rela-tively high level whereas ZCN25 transcript is barelydetected. ZCN18 is expressed in stems and leaves, buta ZCN24 transcript was not found in any tissue tested.The ZCN8/ZCN7 pair could be nonfunctionalizationinvolving differential splicing. Both ZCN8 and ZCN7are transcribed, but ZCN7 transcript remains unsplicedin all tissues tested, whereas ZCN8 mRNA is com-pletely spliced in the leaf blade where function isanticipated.

Possible subfunctionalization trends are inferred byexpression patterns for gene pairs ZCN1/ZCN3,ZCN4/ZCN5, and ZCN9/ZCN10, which showed clearexpression pattern shifts. ZCN1 and ZCN3 are bothexpressed in the shoot apices before the floral transitionand in the ear and tassel primordia after the floraltransition, but they produce different levels of fullyspliced mRNA. ZCN1 transcripts are mostly unspliced,whereas ZCN3 produces mostly spliced mRNA, andaccumulates this fully spliced mRNA in ears andtassels at later developmental stages. ZCN4 and ZCN5are expressed in developing ears and tassels after thefloral transition, but their expression is activated atsomewhat different stages of development. The dupli-cated genes ZCN9 and ZCN10 are expressed in kernels,but ZCN9 is expressed in the embryo and endosperm,whereas ZCN10 is embryo specific. The other dupli-cated pair ZCN13/ZCN21 may have undergone bilat-eral nonfunctionalization, as apparently neither gene isexpressed.

Nine ZCN Genes Display Regulated RNA Processing

In plants regulated RNA processing and alternativesplicing are common mechanisms of the posttran-scriptional regulation of gene expression (Reddy, 2007).Nine ZCN genes show distinct patterns of transcriptprocessing that appeared to be development and tis-sue dependent. The first pattern is displayed byZCN16 and ZCN20 that produce low levels of un-spliced pre-mRNA in many tissues accompanied byproperly spliced transcript in one tissue, leaf forZCN16, and tassel for ZCN20. The second pattern isdisplayed by ZCN10, ZCN18, and ZCN26 that areexpressed at relatively high levels predominantly inone tissue type, embryo, stem, and leaf blade, respec-tively. They produced unspliced pre-mRNAs and twoto three alternatively spliced transcripts per gene. Thethird pattern is displayed by the ZCN1/ZCN3 genesthat showed developmentally regulated alternativesplicing. ZCN1 pre-mRNA is mostly unspliced in theshoot apices before the floral transition and at earlystages of ear and tassel development, whereas ZCN3produces proportionally more fully spliced mRNA inthese tissues, and fully spliced mRNA in ear and tasselat later stages that suggests a more prominent role forZCN3 relative to ZCN1. The fourth and most intrigu-

Table II. Yeast two-hybrid interactions between DLF1 and FT-likeZCN8, ZCN12, and ZCN14 proteins

Interactions were assessed by transformed yeast colony growth onthe SD/-Leu/-Trp/-His dropout media (Supplemental Fig. S4) using thereciprocal combinations of the bait pGBKT7 vector and the preypGADT7 vector with insertions of the tested cDNAs. 1, Interaction; 2,no interaction; 6, a weak interaction only in one vector combination.

Proteins DLF1 ZCN8 ZCN12 ZCN14

DLF1 1

ZCN8 1 2

ZCN12 2 2

ZCN14 6 2

Danilevskaya et al.




ing pattern is displayed by ZCN8, which apparentlydemonstrates tissue-specific RNA maturation. A lowlevel of unspliced ZCN8 pre-mRNA is detected instem and immature leaves at V3 to V9 stage seedlings,but fully spliced ZCN8 transcripts capable of produc-ing a functional protein appeared in expanded leafblades at the same seedling stages. Its paralog, ZCN7,produced unspliced pre-mRNA in stems and imma-ture leaves, but no detectable transcripts in leaf blades.This indicates that ZCN8 may play a larger rolerelative to its duplicate ZCN7.

ZCN8 Is a Candidate Gene for the Floral Activator

FT in Maize

The Arabidopsis FT protein and its orthologs in otherplant species serve as long-range developmental sig-nals in activation of the floral transition (Lifschitz andEshed, 2006; Lifschitz et al., 2006; Corbesier et al., 2007;Jaeger and Wigge, 2007; Lin et al., 2007; Mathieu et al.,2007; Tamaki et al., 2007). Which maize ZCN gene(s)plays a similar floral activator function? Of 15 ZCNgenes in the FT-like subfamily, only ZCN8 and ZCN26are expressed in leaf blades before and after the floraltransition, and neither is expressed in the shoot apicalmeristem as would be expected for FT function (Fig. 6,G and H). ZCN8 is favored because it produces fullyspliced mRNA in leaves, whereas ZCN26 producesseveral splicing variants. However, there are inconsis-tencies that warrant discussion. First, the ZCN8 locushas no clear syntenic counterpart in rice. Second, ZCN8protein is not phylogenetically subgrouped with the FThomologous proteins that are floral activators in rice,barley, and wheat (Fig. 3). Third, ZCN8 has two sub-stitutions in the putative ligand-binding domain com-pared to Arabidopsis FT: Val-120(FT)/Leu-118(ZCN8)and Gln-140(FT)/His-138(ZCN8). Based on structuralanalysis, it is unlikely that these two substitutionsdramatically alter the ZCN8’s binding capability. Val-120 of FT is at the bottom of the binding site and its sidechain is approximately 6 A away from the modeledOPE according to the bovine protein (PDB: 1b7a), thusleaving ample space to accommodate Leu-118 in ZCN8without blocking the ligand binding. Gln-140 of FT is atthe binding cavity rim and fully exposed to solvent(PDB: 1wkp). The change to His-138 in ZCN8 seem-ingly maintains the Gln’s hydrophilic property, andmight even preserve the potential capability to hydro-gen bond to the incoming ligand through its nitrogenatom on the imidazolium ring, ND1 or NE2.

However, the yeast two-hybrid assay providesstrong evidence that ZCN8 likely functions as the FTfloral activator because only the ZCN8 protein interactswith the DLF1 bZIP transcription factor similar tointeraction of the FT and the bZIP transcription factorFD in Arabidopsis (Abe et al., 2005). Moreover, ZCN8is linked to marker umc2075 on chromosome 8 thatmaps in the vicinity of the flowering QTL vegetative-to-generative transition2 (Vladutu et al., 1999; Chardon et al.,2004), one of the strongest maize QTLs for flowering

time. Further functional analysis of ZCN8 as the maizefloral activator will require a genetic study of mutantsand/or transgenic plants. It is also important to point outthat none of the FT-like ZCN genes were expressed inimmature leaves, the tissue where the major floral reg-ulator indeterminate1 is expressed (Colasanti et al., 1998).

Maize Candidate Genes for the Floral Repressor TFL1

The Arabidopsis TFL1 gene plays an opposing role tothe FT gene, being a repressor of the floral transition(Kardailsky et al., 1999; Kobayashi et al., 1999). Thefunction of TFL1 in meristem indeterminacy mainte-nance is conserved in diverse species (Pnueli et al.,1998; Amaya et al., 1999; Nakagawa et al., 2002; Foucheret al., 2003; Pillitteri et al., 2004; Kotoda and Wada, 2005;Zhang et al., 2005; Boss et al., 2006). TFL1 seems to playa key role in the evolution of inflorescence architecture(Prusinkiewicz et al., 2007).

We identified six maize genes whose proteinsformed a well-supported monophyletic subfamilywith Arabidopsis TFL1. The important TFL1 proteinstructures are all preserved in the six maize homolo-gous proteins including His-88, which is critical forrepressing flowering (Hanzawa et al., 2005), and thedivergent external loop (Ahn et al., 2006). Modeling3-D structure of maize ZCN2 protein revealed that itsoverall fold structure, putative phosphate-binding site,and features distinguishing its floral repressive activ-ities are all preserved in the maize TFL1-like proteins.Thus, all six maize homologous proteins may poten-tially function similar to TFL1. The expression patternsof all six genes are also consistent with their putativerole in meristem maintenance, as all are expressed inthe shoot apices or the developing inflorescence. Ac-cording to their expression patterns, TFL1-like ZCNgenes can be divided into two groups. One group ofgenes (ZCN1, ZCN3, ZCN4, and ZCN6) is expressedbefore and after the floral transition, suggesting func-tions in both vegetative and reproductive meristems.The second group, ZCN4 and ZCN5, is expressed onlyin the developing inflorescence after the floral transi-tion, suggesting functions in maintenance of the inflo-rescence meristem. This suggestive partitioning offunction between several genes for the independentmaintenance of the vegetative and inflorescence mer-istem has been shown in Antirrhinum (Bradley et al.,1996), tobacco (Nicotiana tabacum; Amaya et al., 1999),and pea (Foucher et al., 2003).

Potential ZCN Gene Function in Root, Seed, andFlower Development

Despite their significant protein similarity, each ZCNfamily member may have partially overlapping or evendistinct biological functions based on their develop-mental expression differences. Among the maize ZCNgenes, we have ascribed eight with putative functionsrelating to floral transition and meristem determinacy,





guided by the examples of the Arabidopsis FT/TSF andTFL1 families, respectively (Kardailsky et al., 1999;Kobayashi et al., 1999; Yamaguchi et al., 2005).

The expression patterns of maize ZCN genes hint atroles in root development. The TFL1-like maize genesZCN1, ZCN3, ZCN2, and ZCN5 are expressed in theroot tips as fully spliced mRNA, suggesting a possiblefunction in the root apical meristem. In addition, amongthe FT-like subfamily, ZCN19, ZCN25, and ZCN17 areexpressed mainly in the roots. These could also be in-volved in root development perhaps as a mobile sig-nal akin to FT.

Another likely role for some of the ZCN genes isin kernel development. The seed-specific expressionof the maize MFT-like genes ZCN9 and ZCN10, andbroader kernel plus other tissues expression of ZCN11,suggests these MFT-like genes function during seeddevelopment. Indeed, the Arabidopsis MFT gene ishighly expressed in early developing seeds (Supple-mental Table S4). This finding supports the hypothesisthat Arabidopsis MFT may act during seed develop-ment in addition to its role as a redundant floral inducer(Yoo et al., 2004). Moreover, rice OsMFT1 and OsMFT2display high level expression (by MPSS) in developingand germinating seeds (Supplemental Table S5). TheMFT-like genes’ EST distributions from barley, rice, andwheat are apparently derived from spike or kernelcDNA libraries (Chardon and Damerval, 2005), sup-porting the hypothesis of a kernel-preferred functionfor the MFT-like genes in the Poaceae. Two genes fromthe FT-like subfamily, ZCN14 and ZCN15, are alsoexpressed in kernels. The highly restrictive expressionof ZCN15 in the basal layer of the endosperm afterfertilization indicates a possible novel function in devel-oping endosperm. In short, genes from both MFT-likeand FT-like subfamilies are expressed in developingkernels and may function in distinct aspects of kerneldevelopment.

ZCN14 expression in various tissues suggests itsinvolvement in other developmental processes beyondflowering time. ZCN14 is activated after the floraltransition in the tassel and ear primordia and expressedduring flower development and later in ovules andkernels. To date, ZCN14 is the only FT-like gene withapparent expression in the inflorescence meristem. RiceOsFTL1 syntenic to ZCN14 has a similar pattern ofexpression in spikes and kernels (Izawa et al., 2002;Chardon and Damerval, 2005). Barley HvFT2 and aputative wheat ortholog appear to have similar expres-sion in spikes and kernels (Chardon and Damerval,2005; Faure et al., 2007). This raises the possibility of arole for ZCN14 and related orthologs in plant seed andflower development well after the floral transition.

In conclusion, this study of the maize PEBP genefamily affirms the conserved protein structures andfunction in flowering time, but the gene expressionresults, including alternative splicing, point to likelyadaptation and specialization of physiological roles,further investigation of which this study may helpguide.

MATERIALS AND METHODS

Maize PEBP Gene Identification

A local implementation of the National Center for Biotechnology Infor-

mation BLASTX version 2.0 was used for sequence searching. The initial

protein queries used the six publicly known Arabidopsis (Arabidopsis thaliana)

founders of this gene family, AtTFL1 (At5g03840), AtCEN (At2g27550), AtBFT

(At5g62040), AtFT (At1g65480), AtTSF (At4g20370), and AtMFT (At1g18100).

Maize (Zea mays) sequence subject sources were proprietary ESTs and their

assemblies, publicly available ESTs, CDS, GSS, BACs, and The Institute for

Genomic Research genomic GSS assemblies AZM_4, AZM_5 (http://maize.tigr.

org/), and MAGI_4 (http://www.plantgenomics.iastate.edu/maize/). All po-

tential hits to conserved regions of the PEBP gene family were assembled and

curated, and additional rounds of searching were performed to extend the

genomic and/or transcript sequences across the most complete possible tran-

script region. The 33 unique sequences obtained originally by this survey were

used to design overgo probes for screening genomic BAC libraries. Selected

BACs were sequenced and assembled. The exon/intron structures of the ZCN

genes were deduced from alignments of cDNA and BAC genomic sequences

using the program Sequencher 4.7 (Gene Codes Corporation).

ZCN Gene Mapping and Synteny Analysis

The BAC physical map as fingerprinting contigs (http://www.genome.

arizona.edu/fpc/maize/WebAGCoL/WebFPC/) was used to find the nearest

available markers to position ZCN genes on the genetic IBM2 map (http://

www.maizegdb.org). For synteny comparisons, The Institute for Genomic

Research Rice Annotation Release 5 gene calls, rice (Oryza sativa)-maize

syntenic blocks (http://www.tigr.org/tdb/synteny/maize_IBMn/figureview_

desc.shtml), and a locally developed gene-centric synteny analysis between rice

and maize were used.

Tissue Preparations

Maize plants (genotype B73) were grown in the field. Vegetative growth

stages (V1–V9) were defined according to the appearance of the leaf collar of

the uppermost leaf (Muszynski et al., 2006). Shoot apices with one or two leaf

primordia attached were dissected from seedlings at V3 to V9 stages under the

dissecting microscope. Immature leaf blades were sampled from the plant

whorls. Fully expanded green leaves were sampled as leaf blade. Lateral ear

buds and ear primordia were sampled at the V6 to V10 stages. Kernels were

collected after pollination at 2 d intervals. After 10 DAP the embryo and the

endosperm tissues were dissected from the kernels.

RNA Isolation, RT-PCR, and cDNA Cloning

Small tissues such as shoot apices and ear buds were homogenized in 300

mL of TRIzol Reagent (Roche Diagnostics Corporation) using a 1.5 pestle

(VWR KT479521-1590). Immature leaves and leaf blades were ground with a

mortar and pestle in liquid nitrogen. Ground tissue (50 mg) was treated with

300 mL of TRIzol. Total RNA was isolated with TRIzol Reagent in combination

with Phase Lock Gel (Brinkmann Instruments Inc.) according to the manu-

facturer’s instructions. Complementary DNA synthesis was performed with

Superscript First-Strand Synthesis system (Invitrogen). RT-PCR amplification

was performed using Expand Long Template DNA polymerase (Roche).

Primers were designed according to the predicted gene structure deduced

from BAC sequences. Primers are shown in Supplemental Table S1. Two

microliters of the cDNA reaction was used for PCR amplification in a 50 mL

volume. The PCR conditions were 95�C for 2 min followed by 35 cycles at 94�C

for 45 s, 58�C for 45 s, 72�C for 1 min, and a final extension of 72�C for 10 min.

PCR products were cloned in pCR4-TOPO (Invitrogen) and sequenced.

Yeast Two-Hybrid Assay

The commercial kit MATCHMAKER GAL4 Two-Hybrid system 3 (Clon-

tech) was used according the manufacturer’s protocol. cDNAs of interacting

genes were cloned into pGBKT7 (the Bait vector) and pGADT7 (the Prey

vector) in reciprocal combinations. Both plasmids were transformed into the

AH109 yeast (Saccharomyces cerevisiae) strain and plated on the dropout media

SD/-Leu/-Trp. Individual colonies were then replated in parallel on two

plates with dropout media SD/-Leu/-Trp and SD/-Leu/-Trp/-His. Colony

growth on the His-minus media was indicative of protein interactions.

Danilevskaya et al.




MPSS Analysis

The DuPont MPSS (Solexa) 17-mer expression libraries isolated from over

200 diverse maize tissues and developmental stages was queried with ZCN

gene sequences. The MPSS samples were curated and grouped by tissue-

developmental criteria, and from these groupings the mean parts per million

for each MPSS 17-mer tag was calculated for 13 tissues, and then used to

represent that ZCN gene’s transcript abundance. Arabidopsis MPSS can be

found at http://mpss.udel.edu/at/?/.

Protein Structural 3-D Modeling

The starting coordinates for molecular dynamics simulations and homol-

ogous modeling were taken from the x-ray structures PDB:1wko A chain at

1.80 A resolution and PDB:1wpb A chain at 2.60 A resolution. The initial

structural coordinates of ZCN1 and ZCN14 were constructed using InsightII’s

MODELER module with its autoenergy minimization procedure (Accelrys).

Before further analysis and molecular dynamics, all the structures were

energy minimized under various constraints to relax the structure gradually,

first in virtual vacuum with the crystal waters if applicable, and then

subsequently in solvent water boxes.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers EU241892 to EU241916 and EU241917 to

EU241937.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Mapping of ZCN genes on oat-maize addition

lines.

Supplemental Figure S2. Alignment of ZCN1 and ZCN3 genes across

exon/intron sequences.

Supplemental Figure S3. Anion-binding site charge distribution in ZCN2

protein model structure.

Supplemental Figure S4. The yeast two-hybrid assay for interaction of the

DLF1 and the FT-like proteins ZCN8, ZCN12, and ZCN14.

Supplemental Table S1. A list of primers for ZCN gene mapping on the

oat-maize addition lines.

Supplemental Table S2. A list of primers for RT-PCR.

Supplemental Table S3. Multiple rice, maize, and Arabidopsis PEBP

protein alignment used for phylogenetic analysis.

Supplemental Table S4. MPSS expression analysis of Arabidopsis PEBP

genes.

Supplemental Table S5. MPSS expression analysis of rice MFT-like genes.

ACKNOWLEDGMENTS

The authors thank Jeff Habben for general support, Mike Muszynski and

Nic Bate for valuable comments, Laura Appenzeller for proofreading of the

manuscript, Sergei Svitashev and Stephane Deschamps for BAC library

screening and sequence assembly, Norbert Brugiere for pedicel RNA sam-

ples, and Pooja Patel for help with yeast two-hybrid vector construction.

Received September 21, 2007; accepted November 3, 2007; published Novem-

ber 9, 2007.

LITERATURE CITED

Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y,

Ichinoki H, Notaguchi M, Goto K, Araki T (2005) FD, a bZIP protein

mediating signals from the floral pathway integrator FT at the shoot

apex. Science 309: 1052–1056

Ahn JH, Miller D, Winter VJ, Banfield MJ, Lee JH, Yoo SY, Henz SR,

Brady RL, Weigel D (2006) A divergent external loop confers antago-

nistic activity on floral regulators FT and TFL1. EMBO J 25: 605–614

Amaya I, Ratcliffe OJ, Bradley DJ (1999) Expression of CENTRORADIALIS

(CEN) and CEN-like genes in tobacco reveals a conserved mechanism

controlling phase change in diverse species. Plant Cell 11: 1405–1418

Armstead IP, Turner LB, Farrell M, Skot L, Gomez P, Montoya T,

Donnison IS, King IP, Humphreys MO (2004) Synteny between a

major heading-date QTL in perennial ryegrass (Lolium perenne L.) and

the Hd3 heading-date locus in rice. Theor Appl Genet 108: 822–828

Banfield MJ, Barker JJ, Perry AC, Brady RL (1998) Function from structure?

The crystal structure of human phosphatidylethanolamine-binding protein

suggests a role in membrane signal transduction. Structure 6: 1245–1254

Banfield MJ, Brady RL (2000) The structure of Antirrhinum centroradialis

protein (CEN) suggests a role as a kinase regulator. J Mol Biol 297: 1159–1170

Baurle I, Dean C (2006) The timing of developmental transitions in plants.

Cell 125: 655–664

Bernier I, Jolles P (1984) Purification and characterization of a basic 23 kDa

cytosolic protein from bovine brain. Biochim Biophys Acta 790: 174–181

Bohlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S,

Strauss SH, Nilsson O (2006) CO/FT regulatory module controls timing

of flowering and seasonal growth cessation in trees. Science 312: 1040–1043

Boss PK, Sreekantan L, Thomas MR (2006) A grapevine TFL1 homologue

can delay flowering and alter floral development when overexpressed

in heterologous species. Funct Plant Biol 33: 31–41

Bradley D, Carpenter R, Copsey L, Vincent C, Rothstein S, Coen E (1996)

Control of inflorescence architecture in Antirrhinum. Nature 379: 791–797

Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E (1997) Inflores-

cence commitment and architecture in Arabidopsis. Science 275: 80–83

Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo

S, McCurdy S, Foy M, Ewan M, et al (2000) Gene expression analysis by

massively parallel signature sequencing (MPSS) on microbead arrays.

Nat Biotechnol 18: 630–634

Bruggmann R, Bharti AK, Gundlach H, Lai J, Young S, Pontaroli AC, Wei

F, Haberer G, Fuks G, Du C, et al (2006) Uneven chromosome contrac-

tion and expansion in the maize genome. Genome Res 16: 1241–1251

Carmel-Goren L, Liu YS, Lifschitz E, Zamir D (2003) The SELF-PRUNING

gene family in tomato. Plant Mol Biol 52: 1215–1222

Carmona MJ, Calonje M, Martınez-Zapater JM (2007) The FT/TFL1 gene

family in grapevine. Plant Mol Biol 63: 637–650

Chardon F, Damerval C (2005) Phylogenomic analysis of the PEBP gene

family in cereals. J Mol Evol 61: 579–590

Chardon F, Virlon B, Moreau L, Falque M, Joets J, Decousset L, Murigneux A,

Charcosset A (2004) Genetic architecture of flowering time in maize as

inferred from quantitative trait loci meta-analysis and synteny conservation

with the rice genome. Genetics 168: 2169–2185

Chautard H, Jacquet M, Schoentgen F, Bureaud N, Benedetti H (2004)

Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p

Ras GTPase-activating protein in Saccharomyces cerevisiae. Eukaryot

Cell 3: 459–470

Colasanti J, Yuan Z, Sundaresan V (1998) The indeterminate gene encodes

a zinc finger protein and regulates a leaf-generated signal required for

the transition to flowering in maize. Cell 93: 593–603

Conti L, Bradley D (2007) TERMINAL FLOWER1 is a mobile signal

controlling Arabidopsis architecture. Plant Cell 19: 767–778

Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A,

Farrona S, Gissot L, Turnbull C, et al (2007) FT protein movement

contributes to long-distance signaling in floral induction of Arabidopsis.

Science 316: 1030–1033

Duarte JM, Cui L, Wall PK, Zhang Q, Zhang X, Leebens-Mack J, Ma H,

Altman N, dePamphilis CW (2006) Expression pattern shifts following

duplication indicative of subfunctionalization and neofunctionalization

in regulatory genes of Arabidopsis. Mol Biol Evol 23: 469–478

Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M (2005) Ectopic

expression of an FT homolog from citrus confers an early flowering pheno-

type on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Res 14:

703–712

Faure S, Higgins J, Turner AS, Laurie DA (2007) The FLOWERING LOCUS

T-like gene family in barley (Hordeum vulgare). Genetics 176: 599–609

Foucher F, Morin J, Courtiade J, Cadioux S, Ellis N, Banfield MJ, Rameau

C (2003) DETERMINATE and LATE FLOWERING are two TERMINAL

FLOWER1/CENTRORADIALIS homologs that control two distinct

phases of flowering initiation and development in pea. Plant Cell 15:

2742–2754





Gaut BS (2001) Patterns of chromosomal duplication in maize and their

implications for comparative maps of the grasses. Genome Res 11: 55–66

Gyllenstrand N, Clapham D, Kallman T, Lagercrantz U (2007) A Picea

abies FT homolog is implicated in control of growth rhythm in conifers.

Plant Physiol 144: 248–257

Hanzawa Y, Money T, Bradley D (2005) A single amino acid converts a

repressor to an activator of flowering. Proc Natl Acad Sci USA 102: 7748–7753

Hengst U, Albrecht H, Hess D, Monard D (2001) The phosphatidyletha-

nolamine-binding protein is the prototype of a novel family of serine

protease inhibitors. J Biol Chem 276: 535–540

Huang T, Bohlenius H, Eriksson S, Parcy F, Nilsson O (2005) The mRNA

of the Arabidopsis gene FT moves from leaf to shoot apex and induces

flowering. Science 309: 1694–1696

Izawa T, Oikawa T, Sugiyama N, Tanisaka T, Yano M, Shimamoto K

(2002) Phytochrome mediates the external light signal to repress FT

orthologs in photoperiodic flowering of rice. Genes Dev 16: 2006–2020

Jaeger KE, Graf A, Wigge PA (2006) The control of flowering in time and

space. J Exp Bot 57: 3415–3418

Jaeger KE, Wigge PA (2007) FT protein acts as a long-range signal in

Arabidopsis. Curr Biol 17: 1050–1054

Jensen CS, Salchert K, Nielsen KK (2001) A TERMINAL FLOWER1-like

gene from perennial ryegrass involved in floral transition and axillary

meristem identity. Plant Physiol 125: 1517–1528

Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK, Nguyen

JT, Chory J, Harrison MJ, Weigel D (1999) Activation tagging of the

floral inducer FT. Science 286: 1962–1965

Keller ET, Fu Z, Brennan M (2004) The role of Raf kinase inhibitor protein

(RKIP) in health and disease. Biochem Pharmacol 68: 1049–1053

Kim DH, Han MS, Cho HW, Jo YD, Cho MC, Kim BD (2006) Molecular

cloning of a pepper gene that is homologous to SELF-PRUNING. Mol

Cells 22: 89–96

Kobayashi Y, Kaya H, Goto K, Iwabuchi M, Araki T (1999) A pair of

related genes with antagonistic roles in mediating flowering signals.

Science 286: 1960–1962

Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T, Yano M

(2002) Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes

transition to flowering downstream of Hd1 under short-day conditions.

Plant Cell Physiol 43: 1096–1105

Kotoda N, Wada M (2005) MdTFL1, a TFL1-like gene of apple, retards the

transition from the vegetative to reproductive phase in transgenic

Arabidopsis. Plant Sci 168: 95–104

Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for

molecular evolutionary genetics analysis and sequence alignment. Brief

Bioinform 5: 150–163

Kynast RG, Riera-Lizarazu O, Vales MI, Okagaki RJ, Maquieira SB, Chen

G, Ananiev EV, Odland WE, Russell CD, Stec AO, et al (2001) A

complete set of maize individual chromosome additions to the oat

genome. Plant Physiol 125: 1216–1227

Lai J, Ma J, Swigonova Z, Ramakrishna W, Linton E, Llaca V, Tanyolac B,

Park YJ, Jeong OY, Bennetzen JL, Messing J (2004) Gene loss and

movement in the maize genome. Genome Res 14: 1924–1931

Lifschitz E, Eshed Y (2006) Universal florigenic signals triggered by FT

homologues regulate growth and flowering cycles in perennial day-

neutral tomato. J Exp Bot 57: 3405–3414

Lifschitz E, Eviatar T, Rozman A, Shalit A, Goldshmidt A, Amsellem Z,

Alvarez JP, Eshed Y (2006) The tomato FT ortholog triggers systemic

signals that regulate growth and flowering and substitute for diverse

environmental stimuli. Proc Natl Acad Sci USA 103: 6398–6403

Lin MK, Belanger H, Lee YJ, Varkonyi-Gasic E, Taoka K, Miura E,

Xoconostle-Cazares B, Gendler K, Jorgensen RA, Phinney B, et al

(2007) FLOWERING LOCUS T protein may act as the long-distance

florigenic signal in the cucurbits. Plant Cell 19: 1488–1506

Lorenz K, Lohse MJ, Quitterer U (2003) Protein kinase C switches the Raf

kinase inhibitor from Raf-1 to GRK-2. Nature 426: 574–579

Lynch M, Conery JS (2000) The evolutionary fate and consequences of

duplicate genes. Science 290: 1151–1155

Mathieu J, Warthmann N, Kuttner F, Schmid M (2007) Export of FT protein

from phloem companion cells is sufficient for floral induction in

Arabidopsis. Curr Biol 17: 1055–1060

Mimida N, Goto K, Kobayashi Y, Araki T, Ahn JH, Weigel D, Murata M,

Motoyoshi F, Sakamoto W (2001) Functional divergence of the TFL1-

like gene family in Arabidopsis revealed by characterization of a novel

homologue. Genes Cells 6: 327–336

Monna L, Lin X, Kojima S, Sasaki T, Yano M (2002) Genetic dissection of a

genomic region for a quantitative trait locus, Hd3, into two loci, Hd3a and

Hd3b, controlling heading date in rice. Theor Appl Genet 104: 772–778

Muszynski MG, Dam T, Li B, Shirbroun DM, Hou Z, Bruggemann E,

Archibald R, Ananiev EV, Danilevskaya ON (2006) delayed flowering1

encodes a basic leucine zipper protein that mediates floral inductive

signals at the shoot apex in maize. Plant Physiol 142: 1523–1536

Nakagawa M, Shimamoto K, Kyozuka J (2002) Overexpression of RCN1

and RCN2, rice TERMINAL FLOWER 1/CENTRORADIALIS homo-

logs, confers delay of phase transition and altered panicle morphology

in rice. Plant J 29: 743–750

Odabaei G, Chatterjee D, Jazirehi AR, Goodglick L, Yeung K, Bonavida B

(2004) Raf-1 kinase inhibitor protein: structure, function, regulation of

cell signaling, and pivotal role in apoptosis. Adv Cancer Res 91: 169–200

Otto SP, Yong P (2002) The evolution of gene duplicates. Adv Genet 46:

451–483

Paterson AH, Bowers JE, Chapman BA (2004) Ancient polyploidization

predating divergence of the cereals, and its consequences for compar-

ative genomics. Proc Natl Acad Sci USA 101: 9903–9908

Pillitteri LJ, Lovatt CJ, Walling LL (2004) Isolation and characterization of

a TERMINAL FLOWER homolog and its correlation with juvenility in

citrus. Plant Physiol 135: 1540–1551

Pnueli L, Carmel-Goren L, Hareven D, Gutfinger T, Alvarez J, Ganal M,

Zamir D, Lifschitz E (1998) The SELF-PRUNING gene of tomato

regulates vegetative to reproductive switching of sympodial meristems

and is the ortholog of CEN and TFL1. Development 125: 1979–1989

Prusinkiewicz P, Erasmus Y, Lane B, Harder LD, Coen E (2007) Evolution

and development of inflorescence architectures. Science 316: 1452–1456

Ratcliffe OJ, Amaya I, Vincent CA, Rothstein S, Carpenter R, Coen ES,

Bradley DJ (1998) A common mechanism controls the life cycle and

architecture of plants. Development 125: 1609–1615

Reddy AS (2007) Alternative splicing of pre-messenger RNAs in plants in

the genomic era. Annu Rev Plant Biol 58: 267–294

Serre L, Vallee B, Bureaud N, Schoentgen F, Zelwer C (1998) Crystal

structure of the phosphatidylethanolamine-binding protein from bo-

vine brain: a novel structural class of phospholipid-binding proteins.

Structure 6: 1255–1265

Shannon S, Meeks-Wagner DR (1991) A mutation in the Arabidopsis TFL1

gene affects inflorescence meristem development. Plant Cell 3: 877–892

Sreekantan L, Clemens J, McKenzie MJ, Lenton JR, Croker SJ, Jameson

PE (2004) Flowering genes in Metrosideros fit a broad herbaceous model

encompassing Arabidopsis and Antirrhinum. Physiol Plant 121: 163–173

Swigonova Z, Lai J, Ma J, Ramakrishna W, Llaca V, Bennetzen JL,

Messing J (2004) Close split of sorghum and maize genome progenitors.

Genome Res 14: 1916–1923

Takada S, Goto K (2003) TERMINAL FLOWER2, an Arabidopsis homolog

of HETEROCHROMATIN PROTEIN1, counteracts the activation of

FLOWERING LOCUS T by CONSTANS in the vascular tissues of leaves

to regulate flowering time. Plant Cell 15: 2856–2865

Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K (2007) Hd3a

protein is a mobile flowering signal in rice. Science 316: 1033–1036

Vladutu C, McLaughlin J, Phillips RL (1999) Fine mapping and charac-

terization of linked quantitative trait loci involved in the transition of

the maize apical meristem from vegetative to generative structures.

Genetics 153: 993–1007

Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU, Weigel

D (2005) Integration of spatial and temporal information during floral

induction in Arabidopsis. Science 309: 1056–1059

Yamaguchi A, Kobayashi Y, Goto K, Abe M, Araki T (2005) TWIN SISTER

OF FT (TSF) acts as a floral pathway integrator redundantly with FT.

Plant Cell Physiol 46: 1175–1189

Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M, Sanchez A, Valarik

M, Yasuda S, Dubcovsky J (2006) The wheat and barley vernalization

gene VRN3 is an orthologue of FT. Proc Natl Acad Sci USA 103: 19581–19586

Yeung K, Seitz T, Li S, Janosch P, McFerran B, Kaiser C, Fee F, Katsanakis KD,

Rose DW, Mischak H, et al (1999) Suppression of Raf-1 kinase activity

and MAP kinase signalling by RKIP. Nature 401: 173–177

Yoo SY, Kardailsky I, Lee JS, Weigel D, Ahn JH (2004) Acceleration of

flowering by overexpression of MFT (MOTHER OF FT AND TFL1). Mol

Cells 17: 95–101

Zhang S, Hu W, Wang L, Lin C, Cong B, Sun C, Luo D (2005) TFL1/CEN-like

genes control intercalary meristem activity and phase transition in rice. Plant

Sci 168: 1393–1408

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