Evolution of the APETALA2 Gene Lineage in Seed Plants
Cecilia Zumajo-Cardona1 and Natalia Pab
on-Mora*,1
1
Instituto de Biologıa, Universidad de Antioquia, Medellın, Colombia
*Corresponding author: Email: [email protected]
Associate editor: Michael Purugganan
Abstract
Gene duplication is a fundamental source of functional evolutionary change and has been associated with organismal diversification and the acquisition of novel features. The APETALA2/ETHYLENE RESPONSIVE ELEMENTBINDING FACTOR (AP2/ERF) genes are exclusive to vascular plants and have been classified into the AP2-like
and ERF-like clades. The AP2-like clade includes the AINTEGUMENTA (ANT) and the euAPETALA2 (euAP2) genes,
both regulated by miR172. Arabidopsis has two paralogs in the euAP2 clade, namely APETALA2 (AP2) and TARGET
OF EAT3 (TOE3) that control flowering time, meristem determinacy, sepal and petal identity and fruit development.
euAP2 genes are likely functionally divergent outside Brassicaceae, as they control fruit development in tomato, and
regulate inflorescence meristematic activity in maize. We studied the evolution and expression patterns of euAP2/
TOE3 genes to assess large scale and local duplications and evaluate protein motifs likely related with functional
changes across seed plants. We sampled euAP2/TOE3 genes from vascular plants and have found three major
duplications and a few taxon-specific duplications. Here, we report conserved and new motifs across euAP2/
TOE3 proteins and conclude that proteins predating the Brassicaceae duplication are more similar to AP2 than
TOE3. Expression data show a shift from restricted expression in leaves, carpels, and fruits in non-core eudicots and
asterids to a broader expression of euAP2 genes in leaves, all floral organs and fruits in rosids. Altogether, our data
show a functional trend where the canonical A-function (sepal and petal identity) is exclusive to Brassicaceae and it
is likely not maintained outside of rosids.
Key words: APETALA2, AP2, ERF, seed plants, gene duplication, gene evolution.
Introduction
Article
Flower development is genetically controlled by a complex
genetic circuitry that includes transcription factors controlling floral meristem identity followed by those that regulate
floral organ identity. Based on T-DNA floral homeotic
mutants in the model species Arabidopsis thaliana
(Brassicaceae), it was proposed that the overlapping expression of four classes of transcription factors (A, B, C, and E) was
sufficient to specify floral organ identity (Bowman et al. 1993;
Tissier et al. 1999; Pelaz et al. 2001). Thus, E class genes
(SEPALLATA) are responsible for the identity of all floral organs and interacting with them, A class genes (APETALA1/
APETALA2) specify sepals, A þ B (PISTILLATA and
APETALA3) regulate petal identity, B þ C (AGAMOUS) are
responsible for stamen identity and C class genes control
carpel identity (Coen and Meyerowitz 1991; Pelaz et al.
2000, 2001). For the most part, the genes included in the
ABCE floral model, belong to the superfamily of MADS-box
transcription factors (Theissen 2001; Kaufmann et al. 2005)
with the exception of the A-class gene APETALA2 (AP2),
which belongs to the APETALA2/ETHYLENE RESPONSIVE
ELEMENT-BINDING FACTOR (AP2/ERF) gene family
(Bowman et al. 1989; Theissen and Saedler 2001).
Studies on the evolution of different floral gene lineages
have provided evidence for the occurrence of gene duplication events during the evolution of seed plants. For the
most part large-scale duplications coincide with the
paleopolyploidy events that have been inferred at specific
evolutionary times, including the radiation of angiosperms,
monocots, and eudicots, as well as the diversification of
Brasssicaceae (Cui et al. 2006; Flagel and Wendel 2010; Jiao
et al. 2011). Duplication events are important as they are
thought to have originated the raw material for functional
diversification. Specifically, for those gene duplicates that result from polyploidy, redundancy followed by subfunctionalization has proven to be a regular trend, whereas
neofunctionalization and pseudogenization occur less often
(Conant et al. 2014). Comparative expression and functional
analyses outside Brassicaceae have concentrated largely on
the MADS-box genes from the model. In particular, they
have demonstrated that the role of B and C-class genes in
specifying stamen and carpel identity is conserved nearly
across all angiosperms investigated (Ambrose et al. 2000;
Zahn et al. 2006; Drea et al. 2007; Kramer et al. 2007;
Alvarez-Buylla et al. 2010; Yellina et al. 2010; Dreni et al.
2011; Hands et al. 2011; Lange et al. 2013). Conversely, the
role of A-class genes in non-core eudicots encompasses a
larger number of functions, as it is not restricted to the transition to the floral meristem or perianth identity (Murai et al.
2003; Litt 2007; Preston and Kellogg 2008; Pabon-Mora et al.
2012, 2013).
Comparative studies in A-class genes have focused on
APETALA1 (AP1) homologs. AP1 belongs to the APETALA1/
FRUITFULL gene lineage (Irish and Sussex 1990; Mandel et al.
ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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Mol. Biol. Evol. 33(7):1818–1832 doi:10.1093/molbev/msw059 Advance Access publication March 29, 2016
Evolution of the APETALA2 Gene Lineage . doi:10.1093/molbev/msw059
1992; Bowman et al. 1993). This lineage has undergone several
duplications in the angiosperms, with a major duplication
that coincides with the diversification of the core eudicots
resulting in the euAP1 and euFUL gene lineages (Litt and Irish
2003). In core eudicots, euAP1 orthologs determine floral
meristem and sepal identity, but their role in petal identity
is not conserved outside Brassicaceae (Huijser et al. 1992;
Ferrandiz et al. 2000; Berbel et al. 2001; Benlloch et al.
2006). On the other hand, the euFUL genes control the transition phase, leaf shape, and fruit development (Mandel and
Yanofsky 1995; Gu et al. 1998; Immink et al. 1999; Melzer et al.
2008; Burko et al. 2013). Non-core eudicots, which include basal eudicots, magnoliids, monocots, and basal angiosperms, have only one class of pre-duplication genes named
FUL-like genes, for their similarity in protein sequence to the
euFUL lineage (Litt and Irish 2003). FUL-like genes function in
leaf morphogenesis, inflorescence architecture, floral meristem, sepal and petal identity, and fruit development
(Pab
on-Mora et al. 2012, 2013). Thus, the AP1/FUL
genes seem to have undergone subfunctionalization in
the core eudicots, accompanied with the loss of function
in petal identity in most core eudicots investigated.
Therefore, the A-function (sepal and petal identity) from
the ABCE model is to date, the least conserved in early divergent flowering plants (Causier et al. 2010; Litt and Kramer,
2010).
APETALA2 (AP2), the other A-class gene in the model, is
one of the founding members of the AP2/ERF gene lineage
(Bowman et al. 1989; Theissen and Saedler 2001). AP2 was
included in the original model as an A-function gene (i.e.,
responsible for sepal and petal identity), based on the ap2
mutant phenotype that exhibits homeotic conversions of
sepals and petals to leaf-like organs with stigmatic surfaces
carrying ovules and stamens, respectively (Bowman et al.
1989; Kunst et al. 1989; Drews et al. 1991). In A. thaliana,
AP2 down-regulates the activity of AGAMOUS (a C-class
gene), by the binding of the AP2-R2 domain, to the noncanonical AT-rich target sequences in the second intron of
AG triggering the recruitment of the transcriptional corepressor TOPLESS (TPL) and the histone deacetylase HDA19 (Dinh
et al. 2012; Krogan et al. 2012). This interaction guarantees
that sterile and fertile organs have distinct nonoverlapping
regulation. In addition, AP2 also functions in embryo development by controlling size and cell number (Jofuku et al.
1994; Ohto et al. 2005). The AP2/ERF proteins are characterized by having two AP2 domains, AP2-R1 and AP2-R2, consisting of three b sheets and one a helix, important for DNA
binding. They have been divided into two classes based on the
number of domains (Jofuku et al. 1994). The AP2-like class
encodes proteins with two AP2 domains while the ERF-like
class proteins possess a single AP2 domain. Phylogenetic analyses of the AP2-like lineage indicate that it has undergone a
duplication event that coincides with the diversification of
the gymnosperms resulting in the euAPETALA2 (euAP2) and
AINTEGUMENTA (ANT) gene lineages (Kim et al. 2006). The
euAP2 lineage is characterized by possessing a microRNA172
(MIR172) binding site, which negatively regulates euAP2 gene
expression (Chen 2004; Kim et al. 2006).
MBE
Arabidopsis thaliana possesses six euAP2 paralogs:
€ TZE (SMZ), TARGET
SCHNARCHZAPFEN (SNZ), SCHLAFMU
OF EAT1 (TOE1), TOE2, TOE3, and AP2 (Kim et al. 2006)
(supplementary fig. S1, Supplementary Material online). Not
surprisingly, as they are all regulated by MIR172, the over
expression of MIR172 disrupts a number of processes during
flower development. 35S:MIR172 plants are similar to ap2
mutants, as they lose sepal and petal identity and instead
form carpels and stamens (Park et al. 2002; Aukerman and
Sakai 2003; Chen 2004; Yant et al. 2010). In addition, transgenic plants that express a MIR172-resistant AP2 gene exhibit
several floral defects such as indeterminate flowers with numerous reproductive organs and an enlarged floral meristem
(Zhao et al. 2007). This phenotype suggests that the repression of AP2 by miR172 is crucial for maintaining floral determinacy and the boundary between the outer sterile perianth
and the reproductive inner organs (Zhao et al. 2007;
Wollmann et al. 2010). miR172 also regulates the phase transition between vegetative and reproductive phases by repressing SNZ, SMZ, TOE1, TOE2, TOE3 (Aukerman and Sakai
2003; Mathieu et al. 2009; Yant et al. 2010). TOE3, the paralog
of AP2, is expressed and functions similarly to AP2, it also
regulates AG expression and it is involved in sepal and petal
identity (Jung et al. 2014). However, different from AP2, it is
directly regulated by SQUAMOSA PROMOTER BINDING
PROTEIN-LIKE 3 (SPL3) (Jung et al. 2014).
Functional data from AP2 orthologs in other core eudicots
is scarce. A partially conserved role in perianth identity has
been demonstrated in Antirrhinum majus, where the double
mutant lipless1/2, shows homeotic conversion from sepals to
leaves, leaving petals intact (Keck et al. 2003; Litt 2007). The
ortholog in Petunia sp., PhAP2A, is expressed similar to AP2
during flower development, but phap2a plants do not show
mutant phenotypes, suggesting that PhAP2A may be redundant with other AP2/ERF transcription factors in Petunia sp.
(Maes et al. 2001). In addition, AP2 as well as euAP2 orthologs
have been shown to play roles in fruit development and
maturation in both dry-dehiscent Arabidopsis fruits as well
as in fleshy-indehiscent tomato fruits (Chung et al. 2010;
Ripoll et al. 2011). Data regarding expression and function
of these genes outside eudicots are only available for some
monocots and gymnosperms. In maize there are two AP2-like
orthologs, INDETERMINATE SPIKELET1 (IDS1), which specifies
a determinate spikelet meristem fate and limits the number
of floral meristems produced (Chuck et al. 1998) and
GLOSSY15 (GL15) which controls the transition phase from
juvenile to adult leaves (Moose and Sisco 1994). In basal angiosperms expression of the Amborella tricopoda, AmtrAP2
was detected in tepals, staminodes, stamens, carpels, and
leaves (Kim et al. 2006). Expression of AP2-like genes has
also been studied in gymnosperms (Jofuku et al. 1994;
Chuck et al. 1998; Vahala et al. 2001; Shigyo and Ito 2004;
Kim et al. 2006), specifically during somatic embryogenesis, in
Larix marschilinsii and Picea abies. Each species has two paralogs, LmAP2L1 and LmAP2L2 (L. marschilinsii) and PtAP2L1
and PtAP2L2 (P. abies). LmAP2L1 and PtAP2L1 are constitutively expressed during somatic embryogenesis and LmAP2L2
and PtAP2L2 expression increase during the first stages of
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Zumajo-Cardona and Pabon-Mora . doi:10.1093/molbev/msw059
embryo development (Vahala et al. 2001; Shigyo and Ito 2004;
Guillaumot et al. 2008).
Available data regarding the evolution of AP2-like genes
are not sufficient to assess how these genes have functionally
changed over time, as the most recent phylogenies for AP2
genes have very limited sampling across flowering plants,
being mostly restricted to model species in the core eudicots
and the monocots (Kim et al. 2006). Here, we provide the
first phylogenetic hypothesis for euAP2 genes with sampling
from all representative major groups of seed plants. In addition, we identify two large-scale duplications in the
Monocots, one coinciding with the diversification of the
Papaveraceae, and an additional duplication coinciding
with the radiation of the Brassicaceae, the later resulting
in the APETALA2 and TOE3 clades. Furthermore, we present
novel conserved motifs in addition to the canonical AP2
motifs characterized in seed plants. Finally, we present expression analysis data from selected taxa across the seed
plant phylogeny that allows us to hypothesize about the
functional evolution of euAP2 genes in seed plants.
Materials and Methods
Identification of New Protein Motifs
To detect reported as well as new conserved motifs, 43 sequences of euAP2/TOE3 homologs (see “Phylogenetic analyses” section) were selected representing major seed plant
lineages (21 from core eudicots, two from basal eudicots,
six from monocots, four from basal angiosperms, and ten
from gymnosperms). Sequences were permanently translated
and uploaded as amino acids to the online MEME server
(http://meme.nbcr.net) (Bailey et al. 2006), and run with all
the default options.
Phylogenetic Analyses
To better understand the evolution of the AP2 gene lineage,
we extended our search of AP2-like genes to core eudicots
outside Arabidopsis, basal eudicots, monocots, basal angiosperms, gymnosperms, pteridophytes, lycophytes and bryophytes. Searches were performed using the A. thaliana
sequences (AP2, TOE1, TOE2, TOE3, SMZ, and SNZ) as a query
to identify homologs using BLAST tools (Altschul et al. 1990)
in the available genome database Phytozome (http://www.
phytozome.net) (Goodstein et al. 2012), as well as the plant
transcriptome repositories of the OneKP database (https://
www.bioinfodata.org/Blast4OneKP/), Phytometasyn (http://
www.phytometasyn.ca) and our own generated transcriptomes from Aristolochia fimbriata (Aristolochiaceae),
Bocconia frutescens (Papaveraceae), Cattleya trianae
(Orchidaceae), and Hypoxis decumbens (Hypoxidaceae).
Sequences in the genome and transcriptome databases
were compiled with Bioedit (http://www.mbio.ncsu.edu/bio
edit/bioedit.html) and manually edited to exclusively keep
the open reading frame (ORF) for all transcripts. Nucleotide
sequences were subsequently aligned using the online version of MAFFT (http://mafft.cbrc.jp/alignment/software/)
(Katoh et al. 2002) with a gap open penalty of 3.0, offset
value of 0.8, and all other default settings. The alignment was
then refined by hand using Bioedit. To find the nucleotide
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substitution model that best fit our data, we used the jmodeltest package implemented in MEGA6 (Tamura et al.
2013), which identified the GTRþ Gamma model as the
best-fit model for our dataset. Maximum likelihood (ML)
phylogenetic analyses using the nucleotide sequences were
performed with RaxML-HPC2 BlackBox (Stamatakis et al.
2008) through the CIPRES Science Gateway (https://www.
phylo.org/) (Miller et al. 2010). Bootstrapping was performed
according to the default criteria in RaxML where the bootstrapping stopped after 200–600 replicates. Trees were observed and edited using Mesquite (Maddison WP and
Maddison DR 2011) and FigTree v 1.4.2. (http://tree.bio.ed.
ac.uk/software/figtree/). Two analyses were made. The first
one included 420 sequences of euAP2 genes from across seed
plants and used SMZ and SNZ as outgroup sequences. This
analysis allowed us to discriminate the euAP2 sequences
closest to AP2, and exclude those that nested with TOE1
and TOE2 or those that had TOE1 and TOE2 conserved
motifs, which were named as “other euAP2” (supplementary
fig. 2, Supplementary Material online). For the second analysis only the 186 sequences that came out in the euAP2/
TOE3 clade in the first analysis were used (supplementary
table S1, Supplementary Material online). The selected
ingroup for the second analysis consisted of a total of 170
sequences of seed plants (i.e., 21 sequences from 11 species of
gymnosperms, 38 sequences from 26 species of basal angiosperms, 47 sequences from 21 species of monocots, nine
sequences from three species of basal eudicots, 12 sequences
from eight species of asterids, and 43 sequences from 22
species of rosids). SMZ, SNZ, and the Brassicaceae TOE1
and TOE2 genes were used as outgroup for the second analysis (supplementary table S1, Supplementary Material online). Newly isolated sequences from our own generated
transcriptomes from A. fimbriata (Aristolochiaceae), B.
frutescens (Papaveraceae), C. trianae (Orchidaceae), and H.
decumbens (Hypoxidaceae) can be found under Genbank
numbers KU898265–KU898272.
Rates of Evolution
To test for changes in selection constraints in the euAP2/
TOE3 Brassicaceae gene duplicates, we performed a series of
likelihood ratio tests using the branch-specific model implemented by the CodeML program of PAML package v.4.6
(Yang 2007). Using the nucleotide sequences, we compared
the one-ratio model that assumes a constant dN/dS ratio (per
site ratio of nonsynonymous—dN—to synonymous—dS—
substitutions) before and after the Brassicaceae duplication,
against a two-ratio model that assumes a different ratio for
each the AP2 clade and the TOE3 clade (foreground) relative
to the remaining sequences (background). The test was implemented only in the Brassicaceae duplicates as most protein motif changes were mapped to that particular
duplication. However, as the sequences have long spans
with difficult alignments, the test was conducted only for
the functional domains AP2-R1 and AP2-R2.
Expression Analyses by RT-PCR
euAP2 gene expression was assayed in dissected organs
from plants belonging to all major groups of seed plants
Evolution of the APETALA2 Gene Lineage . doi:10.1093/molbev/msw059
including: Ginkgo biloba (Ginkgoaceae; gymnosperm);
Houttuynia cordata (Saururaceae; basal angiosperm);
Aristolochia fimbriata (Aristolochiaceae; basal angiosperm);
Canna coccinea (Cannaceae; monocot), Bocconia frutescens,
Papaver somniferum (Papaveraceae; basal eudicots); Citrus
limonea (Rutaceae; rosid, core eudicot); Capsicum annum
and Nicotiana obtusifolia (Solanaceae; asterids, core eudicot).
G. biloba was dissected into leaves and ovules. The ovules
were further dissected into the sterile portion (collar) and
the fertile portion (megagametophyte and the nucellus
with the integuments). The angiosperms were separated
and dissected into: whole floral buds, sepals, petals, stamens,
carpels, fruits, and leaves. Exceptions include: (1) H. cordata
that was dissected into petaloid bracts, stamens, and carpels,
as it exhibits a perianth-less condition. (2) A. fimbriata that
has a sepal-derived petaloid perianth that forms a tubular
structure that was dissected into the basal (utricle), mid level
(tube), and distal (limb) portions, and a gynostemium formed
by the fusion of the stigmas and the anthers (Pabon-Mora
et al. 2015). (3) B. frutescens that is a natural homeotic mutant-lacking petals. (4) C. coccinea, where petaloid staminoids
and the only petaloid stamen were combined in the same
RNA extraction. (5) C. limonea in which we were able to
dissect floral organs at both preanthesis and anthesis. Total
RNA was prepared from dissected organs, using TRizol
Reagent (Invitrogen, Waltham, MA), was treated with
DNAseI (Roche, Basel, Switzerland) and quantified with a
nanoDrop 2000 (Thermo Scientific, Waltham, MA). 3 mg of
RNA were used as a template for cDNA synthesis
(SuperScriptIII RT, Invitrogen) using OligodT primers. For all
angiosperms two biological replicates were done, but in the
case of G. biloba, due to limited available material, only one
sample was processed. At least, three technical replicates for
all samples were run. Each amplification reaction incorporated 10 ml of Econotaq (Lucigen, Middleton, WI), 6 ml of nuclease free water, 1 ml of BSA (5 mg/ml), 1 ml fwd primer
(10 mM), 1 ml rev primer (10 mM), and 1 ml of template
DNA for a total of 20 ml. Primers used were designed specifically for each paralog in flanking sequences outside of the
AP2 domains (supplementary table 2, Supplementary
Material online). Thermal cycling profiles followed an initial
denaturation step (94 C for 5 min), 28–32 amplification cycles and a final extension step (72 C for 10 min). Each amplification cycle involved a denaturation step (94 C for 30 s),
an annealing step (50–55 C for 30 s), and an elongation step
(72 C for 40 s). ACTIN2 (A. thaliana ACT2, U37281) was used
as a load control. PCR products were run on a 1.5% agarose
gel stained with ethidium bromide and digitally photographed using a Whatman Biometra BioDoc Analyzer.
Results
Sequence Evolution Analyses
Our BLAST search resulted in the recovery of euAP2 sequences from lycophytes, ferns, and seed plants, but no clear
homologs from bryophytes were found. Sequences recovered
generally span the entire coding sequence, with the exception
of fern sequences that were mostly restricted to one or both
MBE
AP2-R1 and AP2-R2 domains (supplementary figs. S2, S3 and
table S1, Supplementary Material online). Seed plant sequences are for the most part complete coding sequences,
but some are missing 80–90 amino acids (AA) from the start
codon and others lack the stop codon. The average sequence
length is between 400 and 600 AA, but the smallest sequence
has 224 AA and the largest is 1,400-AA long. Our analyses
identified that the AP2-like sequence reported for Ceratopteris
thalictroides (CethaAP2) (Geuten and Coenen 2013) and the
maize homolog INDETERMINATE SPIKELET1 (IDS1) (Chuck
et al. 1998) do not belong to the euAP2/TOE3 clade (supple
mentary fig. S3, Supplementary Material online). Other fern
sequences do belong to the euAP2/TOE3 clade, but lycophyte
and fern sequences are all partial sequences, all of the analyses
hereafter described only include seed plant sequences (figs. 1–
3; supplementary fig. S3, Supplementary Material online). We
found that the two AP2 domains (repeated units 1 and 2,
abbreviated as AP2-R1 and AP2-R2) as well as the linker region between AP2-R1 and AP2-R2 are highly conserved in all
seed plant sequences (figs. 1 and 2; Kim et al. 2006). The
presence of the two AP2 domains suggests that all these
sequences have DNA binding capabilities. In our analysis
AP2-R1 corresponds with motifs 2, 3, and 4, while AP2-R2
is equivalent to motifs 1 and 5 and the linker region ([T/
A]GF[P/S/V]RGSS) corresponds to motif 10 (figs. 1 and 2).
The amino acidic motif that corresponds to the miR172 binding domain is conserved in most euAP2/TOE3 protein sequences, suggesting that they are likely, regulated by
miR172. The miR172-binding site (AAASSFG[S/P]) corresponds to motif 11 (figs. 1 and 2). However, a closer inspection on the RNA sequences in the miR172 motif shows
changes among paralogs especially in basal angiosperms
and gymnosperms, with copies changing 3–8 positions and
some completely lacking the motif (supplementary fig. S4,
Supplementary Material online).
In addition to the well-characterized R1 and R2, we detected additional conserved motifs (figs. 1 and 2). For instance, the first 8 AA of the ORF in all proteins correspond
to the amino acidic sequence MW[D/N]LNDSP (motif 8; figs.
1 and 2). This start motif has been recognized as an EAR
repression motif (DLNxxP) (Ohta et al. 2001; Hiratsu et al.
2003) and in our results is only absent from a few gymnosperm and basal angiosperm sequences (i.e., Canella winterana, and Encephalartos barteri; figs. 1 and 2). In the EAR motif
lacking sequences, no other repression motifs, like the alternative repression RLFGV motif, were found (Licausi et al.,
2013). Right after the start motif and before AP2-R1, three
additional motifs were identified. The first one close to the N
terminus of the protein is the PGKRVGSFSNSSSSAVVIEDGSD
DD/EEE motif (motif 12; figs. 1 and 2) with predominantly
polar uncharged residues and an ending with repeats of acidic
negatively charged residues. This motif is lacking from the
Brassicaceae TOE3 sequences, as well as from gymnosperm
and monocot euAP2/TOE3 sequences. The following second
and third motifs (corresponding to motifs 9 and 6 in figs. 1
and 2) are found right next to each other or close by in most
seed plant sequences; however, motif 9, VTR/HQ/NFFP, is
always present, whereas motif 6, FPRAHWVGKFCQSE is
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MBE
FIG. 1. Motifs conserved across euAP2/TOE3 proteins identified through a MEME analysis. Schematic representation of the conserved motifs, each
motif is represented by a colored box numbered at the top. The black lines represent unique sequences. The AP2-R1 (here represented by motifs 2–
4) and AP2-R2 (here represented by motifs 1 and 5) are highly conserved motifs across all seed plant proteins. Other conserved motifs include the
linker region between AP2-R1 and AP2-R2 (motif 10) and the miR172 binding site (motif 11). Scale bar indicates number of amino acids (AA).
lacking from all Brassicaceae TOE3 and most monocot sequences. All other conserved motifs are located toward the C
terminus of the protein downstream of theAP2-R2 domain.
Motif 7 (NLDLSLG; figs. 1 and 2) is a leucine-rich region found
consistently across seed plants that by sequence comparison
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we can identify as an ERF-associated amphiphilic repression
motif (EAR: LxLxLx) (Ohta et al. 2001; Hiratsu et al. 2003).
Motif 13 (WLQxNGFH/Q) is in the C terminus after the
miRNA binding site and it is also not present in the
Brassicaceae TOE3 sequences.
Evolution of the APETALA2 Gene Lineage . doi:10.1093/molbev/msw059
MBE
FIG. 2. Sequences of the conserved motifs detected on the euAP2/TOE3 homologs across seed plants. Black arrowheads indicate functionally
characterized amino acids important for DNA binding (according to Krizek 2003).
In general, while protein sequences are similar across
euAP2/TOE3 orthologs in all seed plants, the nucleotide composition is very different, suggesting that this gene lineage
exhibits predominantly synonymous substitutions. As an exception, the Brassicaceae TOE3 sequences are quite different,
both in nucleotide and protein composition as they have lost
at least four of the identified conserved motifs (6, 8, 12, and
13; figs. 1 and 2). Thus, in terms of protein sequence, seed
plant euAP2/TOE3 homologs are more similar to AP2 than
TOE3.
euAP2/TOE3 Gene Tree Reconstruction
Our first BLAST search using all of the euAP2 homologs from
A. thaliana as queries, resulted in 420 sequences, on which we
based our first ML analysis. The resulting topology (supple
mentary fig. S2, Supplementary Material online) points to a
number of taxon specific duplications with very low bootstrap (BS) values for the main clades. However, the analysis
showed a well-supported Brassicaceae-specific duplication
in the AP2 and TOE3 clades as well as another one in the
TOE1 and TOE2 clades (supplementary figs. S2 and S3,
Supplementary Material online). Based on this topology we
selected those sequences to proceed with our analyses that
belonged to the APETALA2/TARGET OF EAT3 containing
clade, hereafter referred as the euAP2/TOE3 clade, which
had representatives from all seed plants.
In the euAP2/TOE3 lineage, we were able to identify three
large-scale duplications. One such duplications was found in
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Zumajo-Cardona and Pabon-Mora . doi:10.1093/molbev/msw059
MBE
FIG. 3. ML tree of euAP2/TOE3 gene lineage in seed plants. Yellow stars indicate large-scale duplication events. These events coincide (from top to
bottom) with (1) the diversification of Brassicaceae, giving rise to AP2 and TOE3 clades; (2) the radiation of the Papaveraceae, and (3) the origin of
several monocot orders that include Arecales þ Asparagales þ Poales þ Zingiberales. Orange stars indicate species-specific duplications. BS values
>50% were placed at nodes; asterisks indicate bootstrap values (BS) of 100. Colors follow the top left conventions for each major group of seed
plants.
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Evolution of the APETALA2 Gene Lineage . doi:10.1093/molbev/msw059
the monocots, likely occurring previous to the radiation of
Arecales þ Asparagales þ Poales þ Zingiberales; this monocot-specific duplication results in two clades with low support, but with phylogenetic representation from all four
orders. However, the paralogous clades in Asparagales,
Poales, and Zingiberales have BS values ranging from 83 to
100. Our analysis also shows putative additional order specific
duplications within Zingiberales and Poales. A second duplication has likely occurred prior to the radiation of the
Papaveraceae, resulting in paralogous clades with 72 and 80
BS values. Finally, a third large-scale duplication was found
specific to the Brassicaceae, resulting in the AP2 and TOE3
clades with BS values of 100. In addition, our analysis showed
a large number of taxon-specific duplications (fig. 3). In gymnosperms species-specific paralogs were found in Stangeria
eriopus (Stangeriaceae) and E. barteri (Zamiaceae). In basal
angiosperms, taxon-specific duplications were shown to occur in C. winterana (Canellaceae), Drimis winteri
(Winteraceae), Idiospermum australiense (Calycanthaceae),
Gomortega keule (Gomortegaceae), Peperomia fraseri
(Piperaceae), and H. cordata (Saururaceae). Species-specific
duplicates were also identified in monocots, including
Typhonium blumei (Areceae), Panicum virgatum (Poaceae),
Heliconia spp. (Heliconiaceae), and Curcuma olena
(Zingiberaceae), some asterids, such as Mimulus gutatus
(Phrymaceae) and A. majus (Plantaginaceae), and, in rosids,
like Cucumis sativus (Curcubitaceae), Populus trichocarpa
(Salicaceae), Gossypium raimondii (Malvaceae), Glycine max
(Fabaceae), Phaseolus vulgaris (Fabaceae), and Linum usitatissimum (Linaceae). All species-specific duplications have a BS
of 100, and they coincide with the occurrence of polyploidization, both natural and driven by artificial selection.
In addition, we performed a PAML analysis to determine
changes in the selection rates in the Brassicaceae duplicates,
as most changes in protein motifs were mapped to that
particular duplication event. However, as the complete sequences had long spans difficult to align, our analysis was
restricted to the AP2-R1 and AP2-R2 domains. First, a one
ratio model was tested for all euAP2/TOE3 sequences resulting in a xo: 0.03904. Next a two-ratio model was implemented to test shifts in selection rates in each of the
Brassicaceae clades compared with the remaining sequences. The foreground value for the AP2 clade (xf:
0.01139) in comparison to the background value for the
TOE3 clade and the remaining euAP2/TOE3 sequences
(xb: 0.04059) suggests increased purifying selection for
the AP2 clade. In contrast the foreground value for the
TOE3 clade (xf: 0.04170) in comparison to the background
value for the AP2 clade and the remaining euAP2/TOE3
sequences (xb: 0.0388). This suggests relaxed purifying selection which correlates well with the loss of a number of
conserved motifs (i.e., motifs 6, 8, 10) in the TOE3 clade
when compared with the rest of the euAP2/TOE3 lineage.
Expression of euAP2/TOE3 Orthologs
In order to assess expression patterns of euAP2 homologs and
hypothesize functional roles we evaluated the expression of
selected euAP2/TOE3 genes in species representing each seed
MBE
plant group. For this, we selected the following plants based
on material availability: G. biloba (Ginkgoaceae), H. cordata
(Saururaceae), A. fimbriata (Aristolochiaceae), C. coccinea
(Zingiberaceae), B. frutescens, P. somniferum (Papaveraceae),
C. annuum, N. obtusifolia (Solanaceae) and C. limonea
(Rutaceae) (fig. 4).
In general, expression patterns of euAP2/TOE3 genes varied across taxa sampled. The only homolog evaluated for
expression patterns from rosids was CitrAP2 (C. limonea
AP2), and it was found to be broadly expressed in all floral
organs, fruits, and leaves. CitrAP2 is only absent in preanthethic sepals (fig. 4A). Asterid representatives included
the Nicotiana obtisifolia AP2 ortholog, NicAP2, which is
expressed in petals, carpels, and leaves (fig. 4B) and the
C. annuum AP2 ortholog, CapanAP2, detected in carpels
(at low levels), mature fruits and leaves (fig. 4C). The basal
eudicot, B. frutescens, has two copies: BofrAP2-1 and
BofrAP2-2 and each paralog shows different expression patterns: BofrAP2-1 is expressed in sepals, carpels, young, and
mature fruits as well as leaves, while BofrAP2-2 is found to be
expressed in carpels (at low levels), mature fruits and leaves
(fig. 4D). P. somniferum also has two copies, PsomAP2-1,
expressed in the floral bud and in carpels and PsomAP2-2
with the same expression pattern and also found in fruits (at
low levels, fig. 4E). Within Monocots we were able to explore
expression of the two copies of C. coccinea: CanAP2-1 and
CanAP2-2; CanAP2-1 is expressed mainly in fruits and detected (at low levels) in sepals and staminodes (and the
single fertile stamen), and CanAP2-2 was strongly expressed
in floral bud, staminodes, and detected (at low levels) in
carpel and fruit (fig. 4F). From basal angiosperms we evaluated the expression of HocAP2, one of the two copies found
in H. cordata, which was found broadly expressed in all floral
organs, except stamens, as well as in fruits and leaves (fig.
4G). In addition, we evaluated the expression of AfimAP2,
the single copy found in the transcriptome of A. fimbriata
and found that it is expressed in the floral bud, the upper
portions of the perianth, and in low levels in the fruits and
the leaves (fig. 4H). As a representative of the gymnosperms
we were able to test the expression of GbiAP2, the only copy
of G. biloba, which is expressed in the fertile portions of the
ovules and in leaves (fig. 4I).
Discussion
The AP2/ERF superfamily is one of the most complex gene
lineages with extensive duplications in vascular plants
(Sakuma et al. 2002; Kim et al. 2006). However, most studies
have focused on identifying the structural features of AP2/
ERF proteins, whereas phylogenetic studies have been done
with restricted sampling to model plants (i.e., Oryza,
Antirrhinum, Malus, Petunia, Solanum, and Arabidopsis) using
almost exclusively the AP2-R1 and AP2-R2 domains (Kim
et al. 2006; Shigyo et al. 2006; Tang et al. 2007). Our data,
which includes sampling from all seed plant genome and
transcriptome available databases, and a phylogenetic analysis
made with the full coding sequences, allowed us to identify
the euAP2/TOE3 clade within the euAP2 gene lineage, the
characteristic protein domains, a number of large scale as
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FIG. 4. Expression analyses of euAP2/TOE3 orthologs in different species of seed plants. ACTIN was used as loading control. (A) Expression of
CitrAP2 in C. limonea (Rutaceae, rosid). (B). Expression of NicAP2 in N. obtusifolia (Solanaceae, asterid). (C) Expression of CapanAP2 in C. annuum
(Solanaceae, asterid). (D) Expression pattern of BofrAP2-1 and BofrAP2-2 in B. frutescens (Papaveraceae, basal eudicot). (E) Expression of PsomAP2-1
and PsomAP2-2 in P. somniferum (Papaveraceae, basal eudicot). (F) Expression of CanAP2-1 and CanAP2-2 in C. coccinea (Cannaceae, monocot).
(G) Expression of HocAP2 in H. cordata (Saururaceae, basal angiosperm). (H). Expression of AfimAP2 in A. fimbriata (Aristolochiaceae, basal
angiosperm). (I) Expression pattern of GbiAP2 in G. biloba (Ginkgoaceae, gymnosperm). B: bract, C: carpel, Co: collar, F: fruit, F1: green fruit, F2:
yellowish fruit; Fb: Floral bud, Gy: gynostemium, I: integument, L: leaf, Li: limb, MG: megagametophyte, N: nucellus, O: ovary, Ov: ovule, P: petal, S:
sepal, St: stamen, T: tube, U: utricle. -C indicates the amplification reaction loaded without cDNA.
well as local duplications and the changes in protein sequences after such duplications. In addition, we report expression patterns in selected seed plants representing each
major group and present hypotheses on how these genes
may have acquired different roles during seed plant evolution.
The euAP2/TOE3 Sequences Exhibit Intact DNA
Binding Motifs, Linker Regions, miR172 Binding Sites
and Repression Domains (RD) Across Seed Plants
One of our goals was to determine if functional domains
present in AP2 and TOE3 were conserved across seed plants.
1826
Given that all sequences possess the invariable AP2-R1 and
AP2-R2 domains, it is likely that DNA binding capabilities are
maintained in all euAP2/TOE3 sequences (Shigyo and Ito
2004). Target sequences are likely to be those reported for
AP2-R1 and AP2-R2 that include TG- and AC-rich sequences,
that contrast with the conventional ANT-R1 and ANT-R2
GC-rich target sequences (figs. 1 and 2; Kagaya et al. 1999;
Dinh et al. 2012). Particular sites that have been detected
essential for DNA binding, like the C-terminus of the linker
and the N-terminus of AP2-R2, are invariant across seed
plants (Krizek 2003). One other persistent motif in all
Evolution of the APETALA2 Gene Lineage . doi:10.1093/molbev/msw059
euAP2 sequences identified is the miR172 binding site (figs. 1
and 2). In vivo assays showing direct miR172 binding and AP2
transcript reduction have only been done in Arabidopsis
(Aukerman and Sakai 2003; Chen 2004; Schwab et al. 2005;
Glazi
nska et al. 2009; Varkonyi-Gasic et al. 2012). However,
our results show that the same post-transcriptional regulation is likely to occur across angiosperms and even gymnosperms, where miR172 has been detected (Luo et al. 2013).
The miR172 domain is only lacking in some paralogs, including the ThehTOE3 Thellungiella halophila (Brassicaceae), and a
few euAP2/TOE3 basal angiosperm and gymnosperm sequences, suggesting that in a few instances the regulation
via miR172 has been lost (supplementary fig. 4,
Supplementary Material online). Two other motifs found
across seed plants exhibit the signatures of RD which function
through the recruitment of TOPLESS (TPL) and TPL-related
co-repressors (Ohta et al. 2001; Causier et al. 2012). These are
motifs 7 (LxLxL) present in both angiosperm and gymnosperm euAP2/TOE3 proteins, and motif 8 (LNDSP) found
exclusively in angiosperms. Thus, it is possible that conserved
roles for euAP2/TOE3 genes include the recruitment of TPL to
repress FLOWERING LOCUS T (FT) during transition to reproductive meristems, as well as AG during flower development,
and/or to repress other target genes in biotic and abiotic
stress responses (Causier et al. 2012). Protein interaction studies as well as ChIP assays will be critical to identify putative
partners and downstream genes regulated by euAP2/TOE3
proteins across seed plants (Mathieu et al. 2009; Luo et al.
2013).
The euAP2/TOE3 Lineage Has Undergone
Three Major Duplications and a Number of
Taxon-Specific Duplications
We focused our analysis on the euAP2/TOE3 gene sequences
from seed plants; however, we did identify sequences from
pteridophytes and lycophytes nesting among euAP2/TOE3
seed plant genes in very long branches (supplementary fig.
S3 and table S1, Supplementary Material online). Based on
our results, we can conclude that euAP2/TOE3 genes evolved
with the radiation of vascular plants as they are not present in
bryophyte genomes or transcriptomes available (Kim et al.
2006). We have been able to identify three major duplication
events in the euAP2/TOE3 gene lineage which coincide with
whole genome duplications (WGD) events previously identified. The Brassicaceae specific duplication resulting in the AP2
and TOE3 clades coincides with the a and b WGD in the
Brassicales (Barker et al. 2009; Donoghue et al. 2011). One
duplication was found in monocots, coinciding with the s
duplication in the radiation of commelinids (Zingiberales,
Poales, and Arecales; Jiao et al., 2014). Additional duplication
events in Poales coincide with order specific WGD events
(Jiao et al. 2011; D’Hont et al. 2012). Taxa-specific duplications
were found frequently, in particular in core-eudicot taxa corresponding to well-known polyploids, like Glycine (Fabaceae),
Gossypium (Malvaceae), Cucumis (Curcubitaceae), Populus
(Salicaceae), and Linum (Linaceae) (Dane and Tsuchiya
1976; Schmutz et al. 2010; Paterson et al. 2012;
MBE
Sveinsson et al. 2014). Other taxon-specific duplications
have occurred apparently with no link to reported polyploidyzation events, like in E. barteri (Zamiaceae), S. eriopus
(Stangeriaceae), C. winterana (Canellaceae), G. keule
(Gomortegaceae), and I. australiense (Calycanthaceae), pointing to an alternative origin of paralogs via tandem repeats or
retrotransposition (Lynch and Force 2000). Nevertheless, pinpointing taxa-specific duplications and losses will require a
broader search using more complete datasets in all publicly
available databases and including targeted cloning from desired phylogenetic key points.
Expression Analysis for euAP2/TOE3 Homologs
Reveals Broad Expression Patterns in Rosids and
More Restricted Patterns in Asterids
Expression patterns gathered from representative taxa across
seed plants allow us to propose hypotheses in terms of the
functional evolution of the euAP2/TOE3 gene lineage. In
rosids, the expression found for CitrAP2 (C. limonea) in all
floral organs as well as in fruits and leaves is very similar to the
expression of AP2 and TOE3 in A. thaliana (Brassicaceae; sup
plementary fig. S1, Supplementary Material online). Both AP2
and TOE3 can be detected in all floral organs during early
flower development and are also expressed in fruits and
leaves. Only at later floral developmental stages do the paralogs have divergent expression patterns, as AP2 becomes restricted to sepals and petals and TOE3 becomes restricted to
carpels (Kunst et al. 1989; Würschum et al. 2006; Winter et al.
2007; Wollmann et al. 2010; Ripoll et al. 2011; Dinh et al. 2012;
Jung et al. 2014). Additional expression patterns across rosids
are different; the only copy, identified in the fabid Medicago
truncatula (MetrAP2), is expressed in leaves, petals, and fruits
(eFP Browser, last accessed February 5, 2016; supplementary
fig. S4, Supplementary Material online). Finally, expression
identified for the four copies of Glycine max (GlyAP2,
GlyAP2-2, GlyAP2-3, and GlyAP2-4) show overlapping expression patterns in the shoot apical meristem, leaves, all floral
organs, and fruits (eFP Browser, last accessed February 5, 2016;
supplementary fig. S5, Supplementary Material online).
In asterids, broad expression patterns of euAP2 genes have
also been reported for PhAP2A, the Petunia ortholog that is
expressed in all floral organs as well as in fruits and leaves
(Maes et al. 2001). Similarly, LIPLESS1/2, the Antirrhinum species-specific copies, are expressed in all floral organs (Keck
et al. 2003). Nevertheless, the expression of other asterid
euAP2 orthologs we evaluated are very different. The
Solanaceae homologs CapanAP2 from C. annuum and
NicAP2 from N. obtusifolia have overlapping expression in
carpels and leaves (fig. 4B and C) but while NicAP2 is found
in petals and is absent in fruits, CapanAP2 is absent in petals
and present in the ripening fruit. The expression pattern in
C. annuum is more similar to that reported for SlAP2a in
tomato, with an increasing expression during fruit ripening
reaching a maximum during breaker stage (time at which
there is a shift from yellow to red) (Chung et al. 2010).
Nevertheless, tomato possesses two additional copies:
SlAP2b is expressed only in leaves and floral buds while
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Zumajo-Cardona and Pabon-Mora . doi:10.1093/molbev/msw059
SlAP2c is expressed in petals and immature green fruits (eFP
Browser, last accessed February 5, 2016; supplementary fig. S6,
Supplementary Material online). In the tetraploid genome of
Solanum tuberosum we were able to find three paralogs, all of
which have different expression patterns. SotAP2 is found in
sepals and fruits, in particular high levels are found in mature
fruits; SotAP2-2 is restricted to stamens and SotAP2-3 is expressed in leaves and petals (eFP Browser, last accessed
February 5, 2016; supplementary fig. S6, Supplementary
Material online). The orthologs, SlAP2a/SotAP2 and SlAP2b/
SotAP2-3, exhibit similar expression patterns while SlAP2c/
SotAP2-2, another ortholog, exhibits little overlap in expression patterns.
We can conclude that euAP2/TOE3 gene expression has
changed considerably between asterids and rosids, and while
a role on perianth identity for euAP2/TOE3 genes can still
occur in rosids, is unlikely to be maintained in asterids.
Instead, euAP2/TOE3 asterid homologs are likely functioning
in carpel patterning and fruit development (Chung et al. 2010;
Ripoll et al. 2011). Because perianth identity is controlled via
the transcriptional repression of AG via the recruitment of TPL
and HDA19, and the RD are intact in all core eudicot proteins,
we can hypothesize that changes in expression and function
are not linked to changes in coding sequences but to changes
in regulation and thus the acquisition of new downstream
targets or partners (Krogan et al., 2012; Licausi et al., 2013).
In this context, the euAP2–TPL interaction may be repressing
other targets. Regulation for euAP2/TOE3 genes will have to be
assessed by studying changes in the promoter sequences, as
well as post-transcriptional gene silencing via the miR172.
Our data show that the AP2/TOE3 function in perianth
identity is likely not conserved in core eudicots. It has been
determined that the other A-class genes, the AP1 homologs, like
SQUAMOSA (SQUA) in A. majus (Huijser et al. 1992), M.
truncatula PROLIFERATING INFLORESCENCE MERISTEM
(MtPIM) in Medicago (Benlloch et al. 2006), and
MACROCALYX (MC) in tomato (Vrebalov et al., 2002) do not
play roles in sepal and petal identity simultaneously, as AP1 does
in Arabidopsis. Thus, it is unlikely that AP1 or AP2 homologs fit
the canonical A-class function of the ABCE modelin core eudicots. Our expression data instead support the alternative (A)BC
model (Causier et al. 2010), where the (A)-function genes are
turned on to establish the floral meristem identity and by
default promote the identity of the first whorl organs, the sepals,
and later on are required for activation and regulation of the Band C-class genes. However, in situ hybridization and functional
studies will have to be done to test whether AP2/TOE3 homologs have dual roles early on in establishing floral meristem
identity and later in regulating fruit development as AP1/FUL
homologs do (Pab
on-Mora et al. 2012).
Outside Core Eudicots, euAP2/TOE3 Genes Are
Frequently Found in Carpels, Fruits, and Leaves
Our expression studies of euAP2 genes in basal eudicots show
that paralogous genes resulting from the Papaveraceaespecific duplication exhibit different expression patterns in
each species. Expression of the two paralogs in P. somniferum,
PsomAP2-1 and PsomAP2-2, is mostly coordinated and
1828
MBE
restricted to floral buds and carpels, and only PsomAP2-2
extends to the developing fruit. On the other hand, the B.
frutescens paralogs, BofrAP2-1 and BofrAP2-2 only overlap in
leaves and late stages of fruit development and BofrAP2-1 is
found also in sepals, carpels, and early stages of fruit development. These results show that paralogous copies have likely
maintained their function in carpel and fruit development,
but in parallel have lost their role during leaf development
and in early floral buds. Interestingly, functional data of the
A-class, AP1/FUL homologs in basal eudicots (known as the
FUL-like genes), also shows roles in leaf morphogenesis, floral
meristem and sepal identity, and fruit development, while
their contribution to petal identity is unclear (Pab
on-Mora
et al. 2012, 2013).
In petaloid monocots, like C. coccinea, the expression of
CanAP2-1 and CanAP2-2 paralogs are very different compared with those reported in grass monocots. While
CanAP2-1 is mostly expressed in fruits, CanAP2-2 is restricted
to floral buds and the petaloid staminodes (including the
fertile stamen; fig. 4F). On the other hand, in grasses, the floral
structures are remarkably divergent. Instead of petals, grass
flowers have two or three lodicules (petal homologs) outside
the stamens (Kellogg 2001). Most euAP2/TOE3 grass homologs are expressed in the lodicules. The AP2 ortholog in barley
(cleistogamy (Cly)/HvAP2) is normally downregulated by
miR172, and as a result lodicule development is promoted
pushing the opening of the outer floral organs and forming
noncleistogamous flowers (Nair et al. 2010). Synonymous
changes in the miR172 binding site in Cly result in over-expression of Cly and abnormal small lodicules that result in
cleistogamous flowers (Nair et al. 2010). There are two euAP2/
TOE3 paralogs in maize with divergent roles: GLOSSY15 controls the transition phase from juvenile to adult leaves (Moose
and Sisco 1994; Chuck et al. 1998) and ZemAP2 is expressed
mostly in the base of the leaf (eFP Browser, last accessed
February 5, 2016; supplementary fig. S6, Supplementary
Material online). Our analysis has revealed that IDS1 is not
part of the euAP2/TOE3 clade. In rice, there are two copies,
also likely having divergent roles: SHAT1 functions in determining the number, size, and identity of all floral organs, and
in particular stamens and carpels (Zhou et al. 2012), whereas
OrsaAP2 is expressed in the apical meristem and later stages
of the inflorescence (eFPBrowser, last accessed February 5,
2016; supplementary fig. S7, Supplementary Material online).
Our expression studies in petaloid monocots contrasts with
the data available in grasses, and suggest that functions of
euAP2/TOE3 genes in Poaceae and Zingiberaceae are likely
different.
In order to explore expression patterns in early diverging
angiosperms we further analyzed expression in H. cordata and
A. fimbriata (Piperales). Our results showed that the H. cordata HocAP2 expression occurs in all floral organs, leaves, and
fruits (fig. 4G). In addition, the A. fimbriata AfimAP2 is expressed in floral buds, the distal portion of the perianth, and
in low levels in leaves and fruits, but is not found in the
gynostemium (stamens fused congenitally with stigmas) or
in the inferior ovary (fig. 4H). Such broad expression especially
in H. cordata, is very similar to what was shown for the earliest
Evolution of the APETALA2 Gene Lineage . doi:10.1093/molbev/msw059
diverging flowering plant Amborella trichopoda (fig. 4G; Kim
et al. 2006). The expression of euAP2/TOE3 genes in gymnosperms is also broad. We showed that the G. biloba homolog,
GbiAP2, is detected in the fertile portions of the ovules and in
leaves. Interestingly, GbiAP2 is not expressed in the sterile
portion of the ovule, the collar, or the short branch subtending the ovules (fig. 4I; Jin et al. 2012; Douglas et al. 2015).
Altogether, the expression data gathered here suggest a
few alternative, not mutually exclusive scenarios, for the functional evolution of the euAP2/TOE3 genes. (1) As euAP2/TOE3
genes are consistently found in carpels and fruits in angiosperms one possibility is that the ancestral and maintained
role of euAP2 genes is the early patterning of carpel mediallateral structures and the regulation of fruit maturation, similar to what has been detected in Arabidopsis and tomato
(Chung et al. 2010; Ripoll et al. 2011). (2) Because ovules were
not removed from carpellary tissue, an alternative is that expression detected in carpels is the result of true expression in
ovules. Thus, it is possible that euAP2/TOE3 genes play the
ancestral roles of the sister gene lineage AINTEGUMENTA
(ANT) that include ovule integument patterning and female
gametophyte development (Jofuku et al. 1994; Elliott et al.
1996; Klucher et al. 1996; Mizukami and Fischer 2000; Krizek
2009). (3) As euAP2 genes are expressed in leaf tissue in most
seed plants studied, it is likely that besides scenarios (1) and/
or (2), the ancestral role of euAP2/TOE3 genes includes leaf
growth due to the regulation of cell division, a function reported for ANT genes (Mizukami and Fischer 2000).
Identifying roles of euAP2 homologs in different seed plant
lineages will require an exhaustive evaluation of spatiotemporal expression patterns coupled with extensive protein—
interaction studies, and with functional analysis in plants
amenable for transformation. The analyses presented in this
research provide a framework to investigate functional redundancy with close paralogs within the euAP2/TOE3 clade. In
addition, we hypothesize that putative roles of euAP2/TOE3
genes maintained across seed plants include a number of
developmental processes such as cell growth and cell division
during ovule and leaf patterning; whereas the role of euAP2/
TOE3 genes in sepal and petal identity is a derived role in
rosids and fixed in the Brassicaceae AP2 orthologs.
Acknowledgments
We thank Dr Barbara Ambrose (The New York Botanical
Garden and Henry Arenas-Castro (Instituto Humboldt)) for
helpful discussions on the results of this manuscript. We
thank Dr Juan Fernando Alzate (Centro de Secuenciaciœn
Genœmica Nacional) for the assembly and storage of local
non-model plant transcriptomes. We thank the OneKP repository staff and in particular Drs. Gane Ka-Shu Wong and
Dennis Stevenson for facilitating access to the online database. We also thank three anonymous reviewers for careful
reading of the manuscript and insightful comments. This
study was funded by the Committee for Research
Development (CODI), Convocatoria de Internacionalizacion
2015 at the Universidad de Antioquia (Medellın, Colombia)
and COLCIENCIAS (111565842812).
MBE
Supplementary Material
Supplementary figures S1–S7 and tables S1–S2 are available at
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
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