A Genomic and Expression Compendium of the
Expanded 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. In
animals they are known to act as kinase and serine protease inhibitors controlling cell growth and differentiation. In plants the
most extensively studied PEBP genes, the Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) and TERMINAL
FLOWER1 (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 (typically
six to eight genes) and rice (Oryza sativa; 19 genes). Genomic structures, map locations, and syntenous relationships with rice
were determined for 24 of the maize ZCN genes. Phylogenetic analysis assigned the maize ZCN proteins to three major subfamilies: TFL1-like (six members), MOTHER OF FT AND TFL1-like (three), and FT-like (15). Expression analysis demonstrated
transcription for at least 21 ZCN genes, many with developmentally specific patterns and some having alternatively spliced
transcripts. Expression patterns and protein structural analysis identified maize candidates likely having conserved gene function of TFL1. Expression patterns and interaction of the ZCN8 protein with the floral activator DLF1 in the yeast (Saccharomyces
cerevisiae) two-hybrid assay strongly supports that ZCN8 plays an orthologous FT function in maize. The expression of other
ZCN 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 phylogenetic
divisions of prokaryotes, archaea, and eukaryotes
(Banfield et al., 1998; Hengst et al., 2001; Chautard
et al., 2004). Their conserved sequences indicate an
ancient common origin of a basic protein functional
unit. The seminal PEBP described was isolated from
bovine brain as a soluble 23-kD basic cytosolic protein
(Bernier and Jolles, 1984). Animal PEBP proteins were
subsequently 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 Ser
protease inhibitor (Hengst et al., 2001). Overall, however, mammalian PEBPs are multifunctional proteins
whose physiological roles are just beginning to be
understood.
Plant PEBP-related genes were originally cloned
from mutants with altered inflorescence architecture.
These include Antirrhinum CENTRORADIALIS (CEN;
Bradley et al., 1996), Arabidopsis (Arabidopsis thaliana)
TERMINAL FLOWER1 (TFL1; Bradley et al., 1997), and
tomato (Solanum lycopersicum) SELF PRUNING (SP;
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described 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 subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.109538
250
Pnueli et al., 1998). The Antirrhinum cen and Arabidopsis tfl1 mutations cause normally indeterminate
inflorescences to terminate in a flower (Shannon and
Meeks-Wagner, 1991; Bradley et al., 1996, 1997). The
tomato sp mutation changes the indeterminate growth
habit to determinate, resulting in limited shoot growth
and a bushy compact habit (Pnueli et al., 1998). These
mutant phenotypes suggested these genes maintain
the indeterminate state of the inflorescence meristem.
CEN is activated in the inflorescence apex a few days
after the floral transition, whereas TFL1 and SP are
expressed in vegetative and inflorescence apices from
very early stages where they delay the commitment to
reproductive development (Bradley et al., 1997; Pnueli
et al., 1998). The TFL1 and SP genes control both
vegetative and reproductive phase durations by maintaining both apical and inflorescence meristem indeterminacy (Pnueli et al., 1998; Ratcliffe et al., 1998),
while CEN controls only the inflorescence meristem
(Bradley et al., 1996). Two distinct TFL1 homologs control pea (Pisum sativum) vegetative and reproductive
phase duration: The LATE FLOWERING gene maintains shoot apical meristem indeterminacy, and DETERMINATE (DET) maintains inflorescence meristem
indeterminacy (Foucher et al., 2003). TFL1 homologs
with conserved function have now been identified in
many eudicotyledonous (Amaya et al., 1999; Pillitteri
et al., 2004; Sreekantan et al., 2004; Kotoda and Wada,
2005; Boss et al., 2006; Kim et al., 2006) and monocotyledonous plants (Jensen et al., 2001; Nakagawa et al.,
2002; Zhang et al., 2005). The Arabidopsis TFL1 protein has recently been shown to be a mobile signal that
moves from its localized transcriptional domain in the
Plant Physiology, January 2008, Vol. 146, pp. 250–264, www.plantphysiol.org Ó 2007 American Society of Plant Biologists
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PEBP Gene Family in Maize
central region of the inflorescence meristem to the
entire meristem, where it exerts its activity. The TFL1
protein is excluded from the floral meristem, allowing
floral primordia to develop into a flower (Conti and
Bradley, 2007).
The Arabidopsis FLOWERING LOCUS T (FT) gene
is a member of the PEBP gene family, but it has
an opposing function to TFL1: It promotes the transition from the vegetative to the reproductive phase
(Kardailsky et al., 1999; Kobayashi et al., 1999). Critical
amino acid residues differentiate FT and TFL1 proteins
and dictate whether the protein acts as a floral activator or repressor. These residues include Tyr (Y85) in FT
and His (H88) in TFL1 (Hanzawa et al., 2005), together
with a stretch of 14 amino acid residues in exon 4 (Ahn
et al., 2006). FT is a key activator of flowering, mediating both photoperiod and vernalization regulation
(Baurle and Dean, 2006; Jaeger et al., 2006). The FT
gene is transcribed in the leaf phloem (Takada and
Goto, 2003), but the FT protein moves via the phloem
to the shoot apex where it acts as a floral stimulus. This
protein provides a molecular explanation for the long
hypothesized mobile flowering signal, known as the
florigen (Corbesier et al., 2007; Jaeger and Wigge, 2007;
Mathieu et al., 2007). At the shoot apex, the FT protein
interacts with the bZIP transcription factor FLOWERING LOCUS D (FD), and together they activate the
meristem identity gene APETALA1, a MADS-box protein, triggering flower development (Abe et al., 2005;
Wigge et al., 2005).
The function of FT is highly conserved in plants. The
rice (Oryza sativa) FT homolog encoded by Heading
date3a (Hd3a; also known as OsFTL2) migrates from
leaves to the shoot apical meristem to induce the
floral transition (Tamaki et al., 2007). The tomato FT
homolog SINGLE FLOWER TRUSS also regulates
flowering time and shoot architecture by generating a
graft-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). In
wheat (Triticum aestivum) and barley (Hordeum vulgare)
the VERNALIZATION LOCUS3 (VRN3) gene corresponds to an FT function (Yan et al., 2006; Faure et al.,
2007). FT homologous genes accelerate floral development 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 that
may speed tree breeding programs. In addition to
regulating flowering time, the poplar FT homolog
controls short-day fall-induced growth cessation and
bud set (Bohlenius et al., 2006). In Norway spruce
(Picea abies L. Karst.), an FT homolog mediates bud set
and bud burst, indicating conservation of FT function
in gymnosperms (Gyllenstrand et al., 2007).
Four additional PEBP genes were found in the
Arabidopsis genome by homology: TWIN SISTER OF
FT (TSF), BROTHER OF FT AND TFL1 (BFT), ARABIDOPSIS THALIANA CENTRORADIALIS (ACT), and
MOTHER OF FT AND TFL1 (MFT). TSF and MFT seem
to act as floral integrators redundantly with FT (Yoo
et al., 2004; Yamaguchi et al., 2005). The function of
ACT and BFT remains unclear (Mimida et al., 2001).
Small families of about six to eight genes are typical
within eudicots such as Arabidopsis, tomato, poplar,
and grape (Vitis vinifera; Mimida et al., 2001; CarmelGoren et al., 2003; Kotoda and Wada, 2005; Carmona
et al., 2007). Larger gene families were found in monocots such as rice (19 members), wheat (19), and maize
(Zea mays; approximately 30; Chardon and Damerval,
2005). In this article we identified the likely complete
set of maize PEBP-related genes, which we named Zea
mays CENTRORADIALIS (ZCN) after CEN from Antirrhinum, the first PEBP-related plant gene described
(Bradley et al., 1996). A combination of genomic,
syntenic, phylogenetic, protein structural, and expression analyses revealed an expanded, diverse gene
family. Included in this family are candidates for the
probable functional orthologs of TFL1 and FT governing the floral transition, and also other members that
may control developmental processes in roots, kernels,
and flowers.
RESULTS
Identification and Characterization of the ZCN
Gene Family
Extensive searches of public and proprietary transcript and genomic databases with six Arabidopsis
PEBP proteins as BLAST queries (TFL1, FT, ATC, BFT,
TSF, and MFT) identified 25 ZCN genes that were
completed sequences from the appropriate bacterial
artificial chromosome (BAC) clones (Table I). One of
these, ZCN23, appears to be a pseudogene because only
exon 4 was found in the 200-kb sequence of the corresponding BAC. All complete 25 maize genes included
DNA fragments previously identified by Chardon and
Damerval (2005). However, the final number of ZCN
genes will be established upon completion of the
maize genome sequencing project.
The nearest genetic markers for each ZCN gene were
determined from BAC contigs and used to position the
ZCN genes on the maize genetic map (Table I; Fig. 1).
In addition to in silico mapping, the chromosome location of each ZCN gene was confirmed by PCR using
the oat (Avena sativa)-maize addition lines (Supplemental Fig. S1; Supplemental Table S1), which are a set
of 10 oat lines each carrying a single maize chromosome addition (Kynast et al., 2001). ZCN2, ZNC13,
ZCN20, ZCN21, and ZCN26 mapped near centromeres
(Fig. 1).
All the maize ZCN gene family members appeared
to have generally low expression judging by the small
number of ESTs in all the databases queried. cDNAs
were obtained by reverse transcription (RT)-PCR using pairs of gene-specific primers designed close to the
start and stop codons (Supplemental Table S2). No
cDNAs were obtained for ZCN13, ZCN21, and ZCN24,
Plant Physiol. Vol. 146, 2008
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Danilevskaya et al.
Table I. ZCN gene family in maize
*, Genes with no RT-PCR detectable expression; **, a pseudogene containing only exon 4.
Gene Name
Proteina
cDNA
ZCN1
ZCN2
ZCN3
ZCN4
ZCN5
ZCN6
ZCN7
ZCN8
ZCN9
ZCN10
ZCN11
ZCN12
ZCN13*
ZCN14
ZCN15
ZCN16
ZCN17
ZCN18
ZCN19
ZCN20
ZCN21*
ZCN23**
ZCN24*
ZCN25
ZCN26
173
173
173
176
173
178
192
175
172
172
180
177
185
173
177
174
179
173
175
175
187
No 5#end
173
174
187
EST
EST
EST
EST
EST
RT-PCR
RT-PCR
EST
EST
EST
EST
EST
No
EST
EST
RT-PCR
RT-PCR
RT-PCR
RT-PCR
EST
No
No
No
RT-PCR
RT-PCR
GenBank Accessions, GenBank Accessions,
cDNA
Genomics
EU241917
EU241918
EU241919
EU241920
EU241921
EU241922
EU241923
EU241924
EU241925
EU241926
EU241927
EU241928
EU241929
EU241930
EU241931
EU241932
EU241933
EU241934
EU241935
EU241936
EU241937
EU241892
EU241893
EU241894
EU241895
EU241896
EU241897
EU241898
EU241899
EU241900
EU241901
EU241902
EU241903
EU241904
EU241905
EU241906
EU241907
EU241908
EU241909
EU241910
EU241911
EU241912
EU241913
EU241914
EU241915
EU241916
Nearest
Markerb
Chr.
Locusc
csu728a
bngl1755
umc1576
umc1235
umc1677
csu166a
umc1520
umc2075
umc1483
umc1717
umc21
umc2272
bngl2323cent
umc2147
umc2074
csu173
AW681281
umc1890
umc2003
est492090x
cent
csu230
umc1333
umc1326
est324143x
3.09
4.05
10.1
2.04
10.04
4.8
6.06
8.03
8.02
3.04
6.04
3.07
5.04
8.03
6.01
5.05
2.05
2.07
10.4
10.03
2
3.02
7.03
2.04
9.03
a
The number of amino acids in the deduced polypeptides. Corresponding full-length cDNAs were ESTs
b
c
The nearest chromosomal marker to the ZCN genes.
Localization of the
or generated in RT-PCR.
ZCN genes to the chromosome bins.
evidently because no expressing tissues were found
(Table I). The intron/exon structures of the ZCN genes
were determined from the alignments of the cDNA
and genomic sequences (Fig. 2). If a cDNA was not
obtained, the genomic sequence was used to determine the probable gene structure by alignment to the
conserved coding regions and intron positions exhibited by other gene family members. Of the ZCN genes
found, 22 of 24 have a conserved genomic structure
consisting of four exons. The exceptions, ZCN2 and
ZCN20, have three exons that apparently resulted
from fusion of exons 1 and 2. Most ZCN genes have
a compact gene structure in the range of 1 to 3 kb. The
sizes of exon 2 and exon 3 are generally 63 and 42 bp,
respectively, which are also the conserved exon sizes
among other plant PEBP-related genes (Carmel-Goren
et al., 2003; Zhang et al., 2005; Carmona et al., 2007).
ZCN14, ZCN18, ZCN21, and ZCN24 are the largest
genes, with sizes between 5 to 9 kb, as a result of
expanded introns. The complete amino acid sequence
for each gene was deduced from its cDNA sequence
and in a few cases its genomic sequence (Table I).
Protein Phylogenetic Analysis and Rice Synteny
Phylogenetic analysis was used to infer the evolutionary relationship between the 24 maize and 19 rice
PEBP proteins. The six Arabidopsis PEBP proteins and
the wheat and barley FT functionally homologous
proteins corresponding to VRN3 (Yan et al., 2006), were
included as phylogenetic references. Protein alignments
(ClustalW algorithm) revealed dispersed protein conservation, but with higher conservation over the ligandbinding site (Banfield and Brady, 2000). The most
conserved protein segments beginning with EDL/DPL
and ending with RRR/GGR residues (PEPB domain
pfam01161; Supplemental Table S3) were used for the
phylogenetic analysis (Kumar et al., 2004). The PEBP
proteins form three well-marked subfamilies, which
we designate after the three Arabidopsis reference genes,
TFL1-like, MFT-like, and FT-like (Fig. 3). The dendogram topology of the plant PEBP proteins is consistent
with a previous report (Chardon and Damerval, 2005).
The rice proteins, with the exception of OsTFL8,
formed monophyletic subgroups with one or two maize
proteins. The previously reported rice-specific OsFTL5
and OsFTL11 (Chardon and Damerval, 2005) are now
associated with ZCN16 and ZCN17, respectively. Among
the 24 maize ZCN proteins, 14 ZCN proteins appeared
as seven paired monophyletic subgroups: ZCN1-ZCN3,
ZCN4-ZCN5, ZCN7-ZCN8, ZCN9-ZCN10, ZCN13ZCN21, ZCN18-ZCN24, and ZCN19-ZCN25. These
gene pairs likely represent paralogs, a product of the
tetraploid ancestry of maize (Gaut, 2001; Bruggmann
et al., 2006).
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PEBP Gene Family in Maize
Figure 1. ZCN gene positions on maize IBM2
genetic map and syntenic rice genes. ZCN
positions are inferred from the nearest markers
of the corresponding BAC fingerprinting contigs
(Table I). Centromeric regions are shown as
black rectangles. Pseudogene, ZCN23, is
marked by an asterisk (*). Seven duplicated pairs
of 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 the
highest probability of synteny are shown on
the left side of the maize chromosome.
The TFL1-like subfamily is composed of six maize
and four rice proteins. Maize ZCN1-ZCN3-ZCN6 proteins group with rice proteins RCN1 and RCN3. ZCN1
and ZCN3 genes seem to be a recent duplication,
because they share 89.3% nucleotide identity within
exons and 79.4% nucleotide identity within introns
(Supplemental Fig. S2). The other monophyletic group
is composed of maize proteins ZCN2-ZCN4-ZCN5
and rice RCN2-RCN4, the latter thought to represent a
duplicated rice chromosomal segment (Chardon and
Damerval, 2005). The ZCN4-ZCN5 gene pair does not
share much intron homology, suggesting a less recent
duplication. The MFT-like subfamily is the smallest,
composed of three maize and two rice genes: ZCN9ZCN10 grouped with OsMFT2, and ZCN11 grouped
with OsMFT1.
The FT-like subfamily is the largest, composed of 15
maize and 13 rice genes. This subfamily forms two
large monophyletic groups that we named FT-like I
and FT-like II. The FT-like I group is of particular
interest because it includes the key floral activators
from Arabidopsis, FT, and TSF (Huang et al., 2005;
Yamaguchi et al., 2005). The FT-like I group is divided
into two main subgroups. One subgroup is composed
of the Arabidopsis FT and TSF plus seven maize and
five rice proteins, including maize gene pairs ZCN18ZCN24 and ZCN19-ZCN25 and rice OsFTL4 and
OsFTL6. The other subgroup contains maize ZCN14
and ZCN15 within the well-supported monophyletic
grouping of known monocot floral activators, including rice OsFTL2 (Hd3a) and OsFTL3 (Hd3b; Kojima
et al., 2002), wheat and barley VRN3, which are named
TaFT and HvFT (Yan et al., 2006). The FT-like II group
is composed of six maize and five rice proteins, but no
Arabidopsis proteins, which suggests diversification
and expansion of this subgroup after the monocoteudicot radiation. The ZCN7-ZCN8 pair forms a
grouping with ZCN12 and OsFTL9. ZCN13-ZCN21
and ZCN26 are similar to OsFTL13 and OsFTL12,
respectively. Only OsFTL8 does not directly group
with any of the maize proteins.
Synteny analysis was performed to assess the correspondence between the phylogenetic groupings of the
PEBP proteins and the prevailing rice-maize syntenic
context for their gene locations, recognizing that synteny
or departures therefrom can exist between these complex cereal genomes. The maize gene map positions and
their syntenic rice counterpart(s) are summarized in
Figure 1. In cases of a single maize-to-rice gene phylogenetic correspondence, the synteny was present and
unambiguous. These examples are ZCN11-OsMFT1,
ZCN2-RCN2, ZCN16-OsFTL5, ZCN17-OsFTL11, ZCN20OsFTL7, ZCN14-OsFTL1, ZCN12-OsFTL9, and ZCN26OsFTL12. In cases where duplicated maize genes
corresponded to a single rice gene, both maize duplicated
segments might be considered syntenic to a single rice
locus. These examples are ZCN9/ZCN10-OsMFT2,
ZCN13/ZCN21-OsFTL13, ZCN18/ZCN24-OsFTL4, and
ZCN19/ZCN25-OsFTL6. In one case, a single maize
gene (ZCN15) corresponded to tandemly duplicated
rice genes (Hd3a/Hd3b), and the syntenic context was
apparent. More challenging for syntenic interpretation
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Danilevskaya et al.
ment. The critical structural variations among distinct
members occur at the loops connecting these secondary structure elements and at the C-terminal regions.
To build 3-D models for the maize ZCN proteins,
we used homology modeling with the Arabidopsis
TFL1 (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 two
representative candidates (ZCN2 and ZCN14) based
on the highest sequence similarity to their Arabidopsis
Figure 2. ZCN exon/intron structures. Genomic structure of the ZCN
genes is represented by black boxes as exons and spaces between the
black boxes corresponds to introns. The sizes of the exons and introns
can be estimated using the vertical lines. Genes are arranged into
phylogenetic subfamilies. The major subfamilies are denoted on the right.
are genes involved in apparently ancient segmental duplications prior to the rice-maize divergence. The large
duplicated segments of rice chromosomes 2 and 4,
carrying RCN2-OsFTL5 and RCN4-OsFTL6 genes, respectively, appeared to be syntenic to maize genes
ZCN4-ZCN21 and ZCN5-ZCN19 on chromosomes 2
and 10, but this synteny remains somewhat ambiguous.
A similar situation exists for the rice RCN1-RCN3 genes
that reside in duplicated segments of rice chromosomes
11 and 12. The closely related maize genes ZCN1, ZCN3,
and ZCN6 mapped to duplicated segments of chromosomes 3, 10, and 4, and synteny was difficult to assign.
We were also not able to determine clear rice synteny
with the maize pair ZCN7/ZCN8. The complete maize
genome sequence may help clarify these and other
remaining inconsistencies.
Maize ZCN Protein Structural Analysis
All PEBP proteins share a common structure, with a
dominant feature being a central antiparallel b-sheet
flanked by a small b-sheet on one side and two a-helices
on the other. All family members also possess a highly
conserved putative anion-binding site located at one
end of the central b-sheet, supporting the idea that
the proteins are functioning in part through forming
complexes with a phosphorylated ligand (Banfield
et al., 1998; Serre et al., 1998; Banfield and Brady, 2000;
Ahn et al., 2006). The three-dimensional (3-D) structure of the CEN proteins suggested a role as a kinase
regulator (Banfield and Brady, 2000), hinting that plant
PEBP proteins may function as modulators of signaling cascades controlling plant growth and develop-
Figure 3. Phylogenetic analysis of the maize, rice, and Arabidopsis
PEBP proteins. The protein sequences of 51 plant PEBP gene products
including 45 Poaceae monocot (black) and six Arabidopsis (red) were
limited to the conserved EDL/DPL...RRR/GGR region encompassing
most of the complete proteins. The phylogenetic test used was the
Bootstrap test by minimum evolution method, as implemented by
MEGA version 3.1 (Kumar et al., 2004). Dendogram branches are
labeled with percentage of 1,000 iterations supporting each branch.
The scale bar at bottom reflects the frequency of amino acid substitutions between sequences as determined by the Poisson corrective
distance model.
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PEBP Gene Family in Maize
counterparts. ZCN2 has 58% amino acid sequence
identity to TFL1, and ZCN14 has 72% sequence identity
to FT. The models were constructed with MODELER,
and refined further by guidance from stereochemistry
violation penalty rules. The overall refined models
abided to their Arabidopsis templates with Ca RMSDs
(root mean square deviations of Ca atomic coordinates) less than 1 Å (Fig. 4, A and B). Comparison
between ZCN2 and TFL1 at the putative phosphatebinding 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, His85/88, Asp-140/144, His-118/121, and Phe-120/123
(Fig. 4A). Similarly, ZCN14 and FT were also highly
conserved 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 identified structural features for distinguishing TFL1 and
FT specificity, including the interacting triads, Glu-112His-88-Asp-144 of TFL1 corresponding to Glu-109-His85-Asp-140 of ZCN2 and Glu-109-Tyr-85-Gln-140 of FT
corresponding to Glu-107-Tyr-83-Gln-138 of ZCN14 at
the binding site entrance, were all well preserved between maize and Arabidopsis (Fig. 4, A and B). To investigate whether the binding site could accommodate
a phosphorylated ligand, the bovine PEBP structure
(PDB:1b7a) was superimposed, and then its cocrystallized ligand phosphoric acid mono-(2-amino-ethyl)
ester (OPE in PDB structure) was transferred into the
ZCN2 model. Further docking analysis regarding
geometric complementarity and binding energy suggested that the OPE could fit well into the cavity without serious stereo conflict (Supplemental Fig. S3).
Conservation of ZCN protein primary structures was
evaluated from the alignment of the ligand-binding
motif and the external loop (Fig. 4C). All maize proteins
possess invariable residues in the critical positions
forming the ligand-binding pocket corresponding to
Arabidopsis TFL1/FT residues Asp-74/71, Asp-75/73,
Pro-78/75, His-90/87, and His-121/118 (Fig. 4C). The
critical His-88 in TFL1 that defines the opposing repressor/activator activities of TFL1 and FT, respectively, 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 the
exception of ZCN11 (MFT-like) and ZCN17 (FT-like I),
that have Leu and Asn, respectively. The distinct feature of MFT-like proteins is substitution of conserved
Glu-112/109 for Met in ZCN9 and ZCN10 or for Val in
ZCN11. All the other maize proteins have Glu in this
position.
The external loop encoded by exon 4 is important
for the opposing activities of Arabidopsis TFL1/FT
proteins (Ahn et al., 2006). The external loop is clearly
defined in the alignment due to its variation in size
between subfamilies (Fig. 4C). The loops of TFL1-like
proteins are variable in size (14–17 residues) and have
only two conserved residues despite the high amino
acid conservation over the remainder of the proteins
(100 invariant out of 180 total residues). The external
loop of the MFT clade is also variable, ranging from 14
to 20 residues and having three invariant amino acids.
The ZCN11 protein is the most diverged from the
consensus with the longest (20 residues) external loop.
The external loop of the FT-like subfamily has a
uniform size of 14 amino acids with significant conservation of amino acids between members of the
group. For instance, nine out of 14 residues are invariant in the FT-like I group, and eight out of 14/15 in the
FT-like II group. This is consistent with the previous
observation for the FT-like subfamily in other species
(Ahn et al., 2006). In conclusion, the conservation of
primary, secondary, and tertiary protein structures of
maize ZCN proteins suggest their biochemical functions might also be preserved.
Expression Patterns of the ZCN Genes
As a step toward elucidating maize ZCN gene
function, we surveyed ZCN transcript accumulation
across a wide range of organs and tissues using the
massively parallel signature sequence (MPSS) RNA
profiling database (Fig. 5). MPSS technology is an
open-ended platform that quantifies the level of transcript accumulation of virtually all genes in a sample
by counting the number of individual mRNA molecules produced from each gene in the form of a 17-mer
tag signature sequence (Brenner et al., 2000). The 17mer tag distributions confirmed the low level of ZCN
gene expression across all tissues, as predicted from
their low EST abundance. ZCN14 and ZCN11 appeared to be the only highly expressed genes in the
broad set of tissues queried. The MFT-like subfamily
showed preferred expression in kernel tissues. The FTlike II group was expressed mainly in leaves. To refine
our analysis of gene expression patterns depicted by
MPSS, we performed RT-PCR in nine different tissues:
root, stem, immature leaf, mature leaf blade, shoot
apex, immature tassel, immature ear, embryo, and
endosperm. For selected ZCN genes, a more detailed
developmental analysis was performed. Gene expression was detected using RT-PCR with pairs of genespecific primers corresponding to the first and last
exon of each gene (Supplemental Table S2). Thus, this
primer design allowed us to detect alternative transcript splicing patterns.
The maize TFL1-like subfamiliy showed similar
tissue expression patterns with expression in roots,
young stems, immature ear, and tassel (Fig. 6A).
Because the Arabidopsis TFL1 gene maintains shoot
apical and inflorescence meristem indeterminacy, we
focused on the expression of TFL1-like genes in developing shoot apices, tassels, and ears (Fig. 6B). The
closely related ZCN1 and ZCN3 genes are expressed in
shoot apices during vegetative development and in
both tassel and ear primordia during reproductive
development (Fig. 6B). ZCN1 and ZCN3 accumulated
significant amounts of unspliced pre-mRNA at early
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Danilevskaya et al.
Figure 4. Structural models of two
ZCN proteins and alignment of
maize and Arabidopsis PEBP proteins. Protein structure ribbon presentation of ZCN2 (A) and ZCN14
(B). The secondary structures are
assigned by Pymol and colored as
orange. The external loop is highlighted as magenta. The conserved
residue side chains in the binding
pocket are shown as sticks and the
triad residue side chains are represented as stick and balls with atoms
color coded: green for carbon, blue
for nitrogen, and red for oxygen. C,
Multiple sequence alignment of the
conserved ligand-binding motif
and the external loop of maize
ZCN and the Arabidopsis TFL1,
MFT, FT, and TSF proteins. The
external loop is boxed. Invariant
amino acids are highlighted yellow,
similar amino acids are highlighted
green or blue. Intron positions are
marked by arrows above the alignment. Asterisks (*) indicate invariable and pound signs (#) variable,
but critical amino acids forming the
ligand-binding pocket. Invariable
residues correspond to the following position in TFL1/FT proteins:
Asp-74/71, Asp-75/73, Pro-78/75,
His-90/87, His-121/118. Variable
His-88/Tyr-85 residues define the
opposing activity of TFL1 and FT,
respectively. Ampersand (&) signs
mark Arg-133/130 and Arg-143/
139 in the external loop that is
conserved in most proteins, with
the exceptions of ZCN2, ZCN19,
and ZCN25.
stages. ZCN3-spliced mRNA appeared in significant
proportions after the floral transition in the V9 stage
tassel and ear primordia, whereas ZCN1 mRNA remained mostly unspliced. None of the other TFL1-like
genes accumulated unspliced transcript.
ZCN6 mRNA was detected at lower levels in shoot
apices and tassel primordia, but its expression is upregulated in developing ears. ZCN2 and ZCN6 were
expressed before and after the floral transition like
ZCN1 and ZCN3, but they show a distinct pattern of
accumulation (Fig. 6B). ZCN2 mRNA accumulation
gradually increased in shoot apices around the floral
transition, peaking in V7 and V8 tassels and decreasing in later stages. ZCN6 had a complementary pattern, 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 stages
tested whereas ZCN6 was expressed at the V7 to V9
stages and decreased later. ZCN4 and ZCN5 transcripts accumulated after the floral transition in both
tassel and ear primordia (Fig. 6B), suggesting they act
specifically in the developing inflorescence after the
floral transition, similar to snapdragon (Antirrhinum
majus) CEN (Bradley et al., 1996) and pea DET (Foucher
et al., 2003). Five out of the six TFL1-like maize genes
were expressed in roots (Fig. 6A).
MFT-like ZCN transcripts accumulated predominantly in kernels with the exception of ZCN11, which
accumulated in both kernel and seedling tissues (Fig.
6C). Developmental profiling of the MFT-like genes
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PEBP Gene Family in Maize
Figure 5. Maize ZCN family transcript
survey across various tissues using
MPSS technology. Relative ZCN gene
transcript abundance measured as
mean frequency of 17-mer tags in part
per million in varies tissues. ZCN13
was omitted from the survey due to
lack of a gene-specific tag. ZCN2,
ZCN20, and ZCN21 do not produce
expressed 17-mer tags.
was performed on whole kernels starting from 2 to 8 d
after pollination (DAP) and on dissected embryo and
endosperm tissues from kernels at 10 to 26 DAP. ZCN9
mRNA was detected in the embryo after 10 DAP and
faint expression was found in the endosperm after 14
DAP. ZCN10 mRNA was detected only in the embryo
after 14 DAP with a significant proportion of unspliced
transcript. ZCN11 transcript accumulated in ovules
before pollination and continued to accumulate in
both embryo and endosperm at all postpollination
stages (Fig. 6D).
The FT-like subfamily is a complex group composed
of 15 maize ZCN genes in two groups named FT-like I
and FT-like II. The FT-like I group is composed of
seven maize genes that are further divided into two
subgroups, one with Arabidopsis FT-TSF, and the
other formed by the FT functionally conserved monocot homologs (Fig. 3). The duplicated ZCN19-ZCN25
pair of genes was expressed in roots. ZCN16 and
ZCN20 produced unspliced mRNA in many tissues.
ZCN17 is actively transcribed in roots and stems.
ZCN18 is expressed in the stem and leaves producing
a mixture of spliced and unspliced transcript. ZCN24
mRNA was not detected in this set of tissues (Fig. 6E).
ZCN14 and ZCN15 are grouped tightly with FT homologous monocot floral activators (Fig. 3), thus being
the most favorable candidates for possessing FT function. However, their expression patterns do not support their function as floral activators. In the broad set
of tissues surveyed, ZCN14 mRNA was detected in the
tassel and ear primordia and the endosperm, whereas
ZCN15 expression was not detected in these samples
(Fig. 6E). The original ZCN15 EST was found in a
pedicel cDNA library. In developing kernels ZCN15
mRNA was detected in whole kernels between 4 and 8
DAP and in the dissected pedicels from later stage
kernels (Fig. 6F). Dissected pedicels often contain the
basal endosperm. Therefore, ZCN15 transcript accumulates in kernels after fertilization, most likely in the
basal layer of the endosperm. This expression pattern
is inconsistent with ZNC15 as a floral activator. ZCN14
transcript was detected in ovules and early developing
kernels, but its level gradually decreased after 12 DAP
(Fig. 6F). Because ZCN14 is the only FT-like gene whose
transcript accumulates in the ear and tassel primordia,
we investigated its expression in these tissues as well
(Fig. 6F). In shoot apices, weak transcript accumulation
is detected at the V5 stage, coinciding with the floral
transition. ZCN14 mRNA levels increased in developing tassels at the V7 stage and remained constant up to
the V9 stage. ZCN14 transcript accumulated in V7 stage
ear buds and later developing ear stages. The ZCN14
expression pattern is quite different from Arabidopsis
FT and rice Hd3a, which are transcribed exclusively in
leaf blades (Corbesier et al., 2007; Jaeger and Wigge,
2007; Mathieu et al., 2007; Tamaki et al., 2007). Thus
ZCN14 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, and
ZCN26, are expressed predominantly in leaf blades
(Fig. 6G). Three other genes seem to be nonfunctional
or with a limited function because they produce either
unspliced mRNA (ZCN7) or their expression was not
detected in any tissues tested (ZCN13 and ZCN21). We
investigated further the developmental expression
pattern of ZCN8, ZCN12, and ZCN26 from FT-like II
and ZCN14 and ZCN18 from FT-like I in stems, immature leaves, and leaf blades at the V2 to V9 stages
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Danilevskaya et al.
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 developing
kernels (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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PEBP Gene Family in Maize
(Fig. 6H). We also included ZCN7 to test mRNA
splicing during development; however, only unspliced ZCN7 RNA was detected in stems and immature leaves and no RNA was found in leaf blades at
various developmental stages. Its paralog ZCN8 also
produced unspliced transcript in stems and immature
leaves, but in expanded leaf blades, ZCN8 mRNA was
completely spliced (Fig. 6H). This finding indicates
that ZCN8 activity may be regulated by mRNA splicing. Importantly, the spliced ZCN8 transcript was detected in leaf blades before the floral transition at the
V3 stage. Its level increased at the V4 stages just before
the floral transition and stayed high afterward. This
expression pattern makes ZCN8 an attractive candidate as a floral activator. Another leaf-specific gene,
ZCN12, was found to be activated in leaf blades at the
V7 stage after the floral transition and continued to be
expressed at later stages (Fig. 6H). ZCN26 is expressed
predominantly in leaf blades and produced a significant amount of unspliced pre-mRNA. ZCN18 was expressed mainly in the stem with weaker expression in
leaf blades. ZCN14 mRNA was barely detected by RTPCR in immature leaves and in leaf blades at vegetative V4 and V5 stages, but after the floral transition, the
ZCN14 mRNA level increased and was reliably detected in leaf blades (Fig. 6H).
Yeast Two-Hybrid Interactions between DLF1 and Maize
FT-Like Proteins
In Arabidopsis the floral activator FT interacts with
the bZIP transcription factor FD to activate the floral
identity genes inducing flower development (Abe et al.,
2005). FT and FD protein interaction was demonstrated
genetically 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 likely
candidate for FT function is ZCN8. Its expression pattern is consistent with a role as the floral activator. In
maize the bZIP transcription factor DLF1 is an activator
of the floral transition and has function similar to the
Arabidopsis FD (Muszynski et al., 2006). We tested
interactions between DLF1 and ZCN8, ZCN12 and
ZNC14 proteins in the yeast two-hybrid system (Table
II). The DLF1 protein shows self interaction that is
typical for bZIP transcription factors that form dimers
through Leu zippers. None of the ZCN proteins showed
self interaction. Of three ZCN proteins tested, only
ZCN8 interacts with DLF1 in both combinations as bait
and prey (Table II; Supplemental Fig. S4). ZCN14
interacts with DLF1 as bait but not as prey, indicating
a weaker or ambiguous interaction. This result strongly
supports the hypothesis that the ZCN8 gene functions
as a floral activator in maize similar to the Arabidopsis
FT and rice Hd3a.
DISCUSSION
Maize Contains an Expanded PEBP Gene Family
Growing evidence indicates that PEBP-related proteins are potent and sometimes mobile regulators of
plant architecture, development, and seasonal growth
adaptation (Bohlenius et al., 2006; Yan et al., 2006; Conti
and Bradley, 2007; Corbesier et al., 2007; Tamaki et al.,
2007). A survey study on this important gene family in
cereals was published earlier, but maize genes were
represented largely by raw sequences available from
public EST and GSS (genome sample survey) databases
(Chardon and Damerval, 2005). In this article we redress this gap by identifying and curating the entire
maize PEBP gene family, including complete gene and
protein structures, evolutionary relationships with rice
and Arabidopsis, and spatiotemporal specific gene
expression patterns.
We identified 25 ZCN genes, although one is likely a
pseudogene. Eudicot genomes such as Arabidopsis and
poplar have about a half-dozen PEBP genes (Mimida
et al., 2001; Carmel-Goren et al., 2003; Carmona et al.,
2007). Monocots are trending higher, with at least
19 known in rice (Chardon and Damerval, 2005). The
phylogenetic analysis revealed three major ZCN subfamilies, the TFL1-like, MFT-like, and FT-like, as in other
plant genomes, reinforcing the idea that the monocoteudicot last common ancestor had at least three PEBP
members (Chardon and Damerval, 2005). Gene distribution between these rice subfamilies is 4:2:13
(TFL1-like:MFT-like:FT-like). The ZCN subfamily distribution is 6:3:15, which is nearly double the number
relative to the presumed monocot ancestor. The larger
FT-like subfamily fell into two groups with the FT-like
II group remaining monocot specific.
The maize PEBP gene family expansion (to 25 members) compared to rice (19 members) could be explained by the ancient tetraploid ancestry of maize, in
which a genome duplication occurred after divergence
from the rice and maize common ancestor, followed by
subsequent 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 of
tissues (G) and in developing stems and leaves (H). Horizontal arrows indicate spliced mRNA. White stars indicate the floral
transition. Asterisks (*) mark genes with no detectable expression. V3 to V10 indicates the vegetative stages of plant development
defined according to the appearance of the leaf collar of the uppermost leaf. The superscript numbers 1 and 2, mark the early and
later stages of V4 and V7, respectively, as judged by the shorter internode length at the early stages. Beginning of the floral
transition was determined by the elongation of the shoot apical meristem followed by the appearance of the spikelet pair
meristem that typically takes 1 to 2 d. Tissues are abbreviated as: EM, Embryo; EN, endosperm; IL, immature leaves; IT, immature
tassel; IE, immature ear; LB, leaf blade; SAM, shoot apical meristem; R, root; S, stem.
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Danilevskaya et al.
Table II. Yeast two-hybrid interactions between DLF1 and FT-like
ZCN8, ZCN12, and ZCN14 proteins
Interactions were assessed by transformed yeast colony growth on
the SD/-Leu/-Trp/-His dropout media (Supplemental Fig. S4) using the
reciprocal combinations of the bait pGBKT7 vector and the prey
pGADT7 vector with insertions of the tested cDNAs. 1, Interaction; 2,
no interaction; 6, a weak interaction only in one vector combination.
Proteins
DLF1
DLF1
ZCN8
ZCN12
ZCN14
1
1
2
6
ZCN8
ZCN12
ZCN14
2
2
2
(Paterson et al., 2004; Swigonova et al., 2004; Bruggmann
et al., 2006). This diploidization process is thought to
have shed at least 50% of the duplicated genes (Lai
et al., 2004). The seven duplicated pairs of ZCN genes
may have survived this diploidization contraction,
contributing to the higher overall family gene count.
Syntenic relationships between rice and maize PEBP
genes were sometimes confusing due to gene duplications and deletions during evolution of each species;
however, for 13 rice genes their syntenic maize ZCN
counterparts were found (Fig. 1).
Most of the ZCN Genes Have Distinct
Expression Patterns
Most of the ZCN genes appear to be functional,
because all but one gene predicts a credible complete
open reading frame, and all but three genes (ZCN13,
ZCN21, and ZCN24) have transcripts detected in at
least one tissue of 13 tested by RT-PCR. Although the
three poorly expressed genes may be heading toward
evolutionary degeneracy, their open reading frames
are complete, and important cryptic expression cannot
be ruled out. Further, the duplicated ZCN13 and
ZCN21 genes are located near centromeres 5 and 2,
where expression may be suppressed due to epigenetic silencing spreading from the heterochromatin.
ZCN20 is also centromeric and shows very low expression.
The presence of duplicated genes raises the question
about their functional redundancy. According to evolutionary models, duplicated genes may undergo different selection processes: nonfunctionalization where
one copy loses function, hypofunctionalization where
one copy decreases in expression or function, neofunctionalization where one copy gains a novel function, or subfunctionalization where the two copies
partition or specialize into distinct functions (Lynch
and Conery, 2000; Otto and Yong, 2002; Duarte et al.,
2006). These evolutionary fates may be indicated with
divergence in expression patterns or protein structure.
We compared the expression patterns of the seven
duplicate ZCN gene pairs that were not duplicated in
the 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 relatively high level whereas ZCN25 transcript is barely
detected. ZCN18 is expressed in stems and leaves, but
a ZCN24 transcript was not found in any tissue tested.
The ZCN8/ZCN7 pair could be nonfunctionalization
involving differential splicing. Both ZCN8 and ZCN7
are transcribed, but ZCN7 transcript remains unspliced
in all tissues tested, whereas ZCN8 mRNA is completely spliced in the leaf blade where function is
anticipated.
Possible subfunctionalization trends are inferred by
expression patterns for gene pairs ZCN1/ZCN3,
ZCN4/ZCN5, and ZCN9/ZCN10, which showed clear
expression pattern shifts. ZCN1 and ZCN3 are both
expressed in the shoot apices before the floral transition
and in the ear and tassel primordia after the floral
transition, but they produce different levels of fully
spliced mRNA. ZCN1 transcripts are mostly unspliced,
whereas ZCN3 produces mostly spliced mRNA, and
accumulates this fully spliced mRNA in ears and
tassels at later developmental stages. ZCN4 and ZCN5
are expressed in developing ears and tassels after the
floral transition, but their expression is activated at
somewhat different stages of development. The duplicated genes ZCN9 and ZCN10 are expressed in kernels,
but ZCN9 is expressed in the embryo and endosperm,
whereas ZCN10 is embryo specific. The other duplicated pair ZCN13/ZCN21 may have undergone bilateral nonfunctionalization, as apparently neither gene is
expressed.
Nine ZCN Genes Display Regulated RNA Processing
In plants regulated RNA processing and alternative
splicing are common mechanisms of the posttranscriptional regulation of gene expression (Reddy, 2007).
Nine ZCN genes show distinct patterns of transcript
processing that appeared to be development and tissue dependent. The first pattern is displayed by
ZCN16 and ZCN20 that produce low levels of unspliced pre-mRNA in many tissues accompanied by
properly spliced transcript in one tissue, leaf for
ZCN16, and tassel for ZCN20. The second pattern is
displayed by ZCN10, ZCN18, and ZCN26 that are
expressed at relatively high levels predominantly in
one tissue type, embryo, stem, and leaf blade, respectively. They produced unspliced pre-mRNAs and two
to three alternatively spliced transcripts per gene. The
third pattern is displayed by the ZCN1/ZCN3 genes
that showed developmentally regulated alternative
splicing. ZCN1 pre-mRNA is mostly unspliced in the
shoot apices before the floral transition and at early
stages of ear and tassel development, whereas ZCN3
produces proportionally more fully spliced mRNA in
these tissues, and fully spliced mRNA in ear and tassel
at later stages that suggests a more prominent role for
ZCN3 relative to ZCN1. The fourth and most intrigu-
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PEBP Gene Family in Maize
ing pattern is displayed by ZCN8, which apparently
demonstrates tissue-specific RNA maturation. A low
level of unspliced ZCN8 pre-mRNA is detected in
stem and immature leaves at V3 to V9 stage seedlings,
but fully spliced ZCN8 transcripts capable of producing a functional protein appeared in expanded leaf
blades at the same seedling stages. Its paralog, ZCN7,
produced unspliced pre-mRNA in stems and immature leaves, but no detectable transcripts in leaf blades.
This indicates that ZCN8 may play a larger role
relative to its duplicate ZCN7.
ZCN8 Is a Candidate Gene for the Floral Activator
FT in Maize
The Arabidopsis FT protein and its orthologs in other
plant species serve as long-range developmental signals in activation of the floral transition (Lifschitz and
Eshed, 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 ZCN
genes in the FT-like subfamily, only ZCN8 and ZCN26
are expressed in leaf blades before and after the floral
transition, and neither is expressed in the shoot apical
meristem as would be expected for FT function (Fig. 6,
G and H). ZCN8 is favored because it produces fully
spliced mRNA in leaves, whereas ZCN26 produces
several splicing variants. However, there are inconsistencies that warrant discussion. First, the ZCN8 locus
has no clear syntenic counterpart in rice. Second, ZCN8
protein is not phylogenetically subgrouped with the FT
homologous proteins that are floral activators in rice,
barley, and wheat (Fig. 3). Third, ZCN8 has two substitutions in the putative ligand-binding domain compared to Arabidopsis FT: Val-120(FT)/Leu-118(ZCN8)
and Gln-140(FT)/His-138(ZCN8). Based on structural
analysis, it is unlikely that these two substitutions
dramatically alter the ZCN8’s binding capability. Val120 of FT is at the bottom of the binding site and its side
chain is approximately 6 Å away from the modeled
OPE according to the bovine protein (PDB: 1b7a), thus
leaving ample space to accommodate Leu-118 in ZCN8
without blocking the ligand binding. Gln-140 of FT is at
the binding cavity rim and fully exposed to solvent
(PDB: 1wkp). The change to His-138 in ZCN8 seemingly maintains the Gln’s hydrophilic property, and
might even preserve the potential capability to hydrogen bond to the incoming ligand through its nitrogen
atom on the imidazolium ring, ND1 or NE2.
However, the yeast two-hybrid assay provides
strong evidence that ZCN8 likely functions as the FT
floral activator because only the ZCN8 protein interacts
with the DLF1 bZIP transcription factor similar to
interaction of the FT and the bZIP transcription factor
FD in Arabidopsis (Abe et al., 2005). Moreover, ZCN8
is linked to marker umc2075 on chromosome 8 that
maps in the vicinity of the flowering QTL vegetative-togenerative 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 maize
floral activator will require a genetic study of mutants
and/or transgenic plants. It is also important to point out
that none of the FT-like ZCN genes were expressed in
immature leaves, the tissue where the major floral regulator indeterminate1 is expressed (Colasanti et al., 1998).
Maize Candidate Genes for the Floral Repressor TFL1
The Arabidopsis TFL1 gene plays an opposing role to
the FT gene, being a repressor of the floral transition
(Kardailsky et al., 1999; Kobayashi et al., 1999). The
function of TFL1 in meristem indeterminacy maintenance is conserved in diverse species (Pnueli et al.,
1998; Amaya et al., 1999; Nakagawa et al., 2002; Foucher
et al., 2003; Pillitteri et al., 2004; Kotoda and Wada, 2005;
Zhang et al., 2005; Boss et al., 2006). TFL1 seems to play
a key role in the evolution of inflorescence architecture
(Prusinkiewicz et al., 2007).
We identified six maize genes whose proteins
formed a well-supported monophyletic subfamily
with Arabidopsis TFL1. The important TFL1 protein
structures are all preserved in the six maize homologous proteins including His-88, which is critical for
repressing flowering (Hanzawa et al., 2005), and the
divergent external loop (Ahn et al., 2006). Modeling
3-D structure of maize ZCN2 protein revealed that its
overall fold structure, putative phosphate-binding site,
and features distinguishing its floral repressive activities are all preserved in the maize TFL1-like proteins.
Thus, all six maize homologous proteins may potentially function similar to TFL1. The expression patterns
of all six genes are also consistent with their putative
role in meristem maintenance, as all are expressed in
the shoot apices or the developing inflorescence. According to their expression patterns, TFL1-like ZCN
genes can be divided into two groups. One group of
genes (ZCN1, ZCN3, ZCN4, and ZCN6) is expressed
before and after the floral transition, suggesting functions in both vegetative and reproductive meristems.
The second group, ZCN4 and ZCN5, is expressed only
in the developing inflorescence after the floral transition, suggesting functions in maintenance of the inflorescence meristem. This suggestive partitioning of
function between several genes for the independent
maintenance of the vegetative and inflorescence meristem 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, and
Flower Development
Despite their significant protein similarity, each ZCN
family member may have partially overlapping or even
distinct biological functions based on their developmental expression differences. Among the maize ZCN
genes, we have ascribed eight with putative functions
relating to floral transition and meristem determinacy,
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Danilevskaya et al.
guided by the examples of the Arabidopsis FT/TSF and
TFL1 families, respectively (Kardailsky et al., 1999;
Kobayashi et al., 1999; Yamaguchi et al., 2005).
The expression patterns of maize ZCN genes hint at
roles in root development. The TFL1-like maize genes
ZCN1, ZCN3, ZCN2, and ZCN5 are expressed in the
root tips as fully spliced mRNA, suggesting a possible
function in the root apical meristem. In addition, among
the FT-like subfamily, ZCN19, ZCN25, and ZCN17 are
expressed mainly in the roots. These could also be involved in root development perhaps as a mobile signal akin to FT.
Another likely role for some of the ZCN genes is
in kernel development. The seed-specific expression
of the maize MFT-like genes ZCN9 and ZCN10, and
broader kernel plus other tissues expression of ZCN11,
suggests these MFT-like genes function during seed
development. Indeed, the Arabidopsis MFT gene is
highly expressed in early developing seeds (Supplemental Table S4). This finding supports the hypothesis
that Arabidopsis MFT may act during seed development in addition to its role as a redundant floral inducer
(Yoo et al., 2004). Moreover, rice OsMFT1 and OsMFT2
display high level expression (by MPSS) in developing
and germinating seeds (Supplemental Table S5). The
MFT-like genes’ EST distributions from barley, rice, and
wheat are apparently derived from spike or kernel
cDNA libraries (Chardon and Damerval, 2005), supporting the hypothesis of a kernel-preferred function
for the MFT-like genes in the Poaceae. Two genes from
the FT-like subfamily, ZCN14 and ZCN15, are also
expressed in kernels. The highly restrictive expression
of ZCN15 in the basal layer of the endosperm after
fertilization indicates a possible novel function in developing endosperm. In short, genes from both MFT-like
and FT-like subfamilies are expressed in developing
kernels and may function in distinct aspects of kernel
development.
ZCN14 expression in various tissues suggests its
involvement in other developmental processes beyond
flowering time. ZCN14 is activated after the floral
transition in the tassel and ear primordia and expressed
during flower development and later in ovules and
kernels. To date, ZCN14 is the only FT-like gene with
apparent expression in the inflorescence meristem. Rice
OsFTL1 syntenic to ZCN14 has a similar pattern of
expression in spikes and kernels (Izawa et al., 2002;
Chardon and Damerval, 2005). Barley HvFT2 and a
putative wheat ortholog appear to have similar expression in spikes and kernels (Chardon and Damerval,
2005; Faure et al., 2007). This raises the possibility of a
role for ZCN14 and related orthologs in plant seed and
flower development well after the floral transition.
In conclusion, this study of the maize PEBP gene
family affirms the conserved protein structures and
function in flowering time, but the gene expression
results, including alternative splicing, point to likely
adaptation and specialization of physiological roles,
further investigation of which this study may help
guide.
MATERIALS AND METHODS
Maize PEBP Gene Identification
A local implementation of the National Center for Biotechnology Information 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 potential 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 transcript 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 manufacturer’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 (Clontech) 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.
262
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
PEBP Gene Family in Maize
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 tissuedevelopmental 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 homologous modeling were taken from the x-ray structures PDB:1wko A chain at
1.80 Å resolution and PDB:1wpb A chain at 2.60 Å 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 Brugière for pedicel RNA samples, and Pooja Patel for help with yeast two-hybrid vector construction.
Received September 21, 2007; accepted November 3, 2007; published November 9, 2007.
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