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The Plant Cell, Vol. 13, 229–244, February 2001, www.plantcell.org © 2001 American Society of Plant Physiologists
RESEARCH ARTICLE
Petunia Ap2-like Genes and Their Role in Flower and
Seed Development
Tamara Maes,1 Nancy Van de Steene, Jan Zethof, Mansour Karimi, Mariëlla D’Hauw, Gwenny Mares,
Marc Van Montagu, and Tom Gerats2
Laboratorium of Genetics, University of Ghent, Flemish Institute for Biotechnology, 9000 Ghent, Belgium
We have isolated three Apetala2 (Ap2)-like genes from petunia and studied their expression patterns by in situ hybridization. PhAp2A has a high sequence similarity to the A function gene Ap2 from Arabidopsis and a similar expression
pattern during flower development, suggesting that they are cognate orthologs. PhAp2B and PhAp2C encode for AP2like proteins that belong to a different subgroup of the AP2 family of transcription factors and exhibit divergent, nearly
complementary expression patterns during flower development compared with PhAp2A. In contrast, all three PhAp2
genes are strongly expressed in endosperm. The phenotype of the petunia A-type mutant blind cannot be attributed to
mutations in the petunia Ap2 homologs identified in this study, and reverse genetics strategies applied to identify
phap2a mutants indicate that PhAp2A might not be essential for normal perianth development in petunia. Nevertheless,
we show that PhAp2A is capable of restoring the homeotic transformations observed in flowers and seed of the ap2-1
mutant of Arabidopsis. Although the interspecific complementation proves that PhAp2A encodes a genuine Ap2
ortholog from petunia, additional factors may be involved in the control of perianth identity in this species.
INTRODUCTION
The overall conservation of flower developmental mechanisms in different species is illustrated by several observations. First, analogous homeotic mutants exist in different
species, and generally their phenotypes result from mutations in homologous genes. Analysis of homeotic flower organ mutants has resulted in the ABC model, which
proposes that three types of functions (A, B, and C) exist
that determine the identity of developing primordia in a
combinatorial manner (Coen and Meyerowitz, 1991).
Second, changes in the expression pattern of a C function
gene in a given species often have phenotypic consequences
comparable to similar alterations in the expression pattern of
its homolog in other species (Mizukami and Ma, 1992; Bradley
et al., 1993; Tsuchimoto et al., 1993; Pnueli et al., 1994).
Third, flower developmental genes can be conserved to the
extent that their products are functional across species barri-
1 Current
address: Centro de Investigacion y Desarrollo, Consejo
Superior de Investigacion Científica, Jordi-Girona 18-26, 08034 Barcelona, Spain.
2 To whom correspondence should be addressed. E-mail toger@
gengenp.rug.ac.be; fax 32-9-2645349.
ers. Overexpression of flower meristem and organ development genes can cause similar phenotypic changes in various
species (Mandel et al., 1992; Weigel and Nilsson, 1995), and
B-type mutants have been complemented by the introduction
of B-type genes from other species (Samach et al., 1997).
No data have been reported that allow a comparison of
the function of the homologs of the A-class gene Apetala2
(Ap2) among the different model systems.
At an early stage of flower development, the Ap2 gene of
Arabidopsis promotes the establishment of the floral meristem, in cooperation with Apetala1 (Ap1), Leafy (Lfy), and
Cauliflower1 (CAL1; Irish and Sussex, 1990; Bowman et al.,
1993). During floral development, Ap2 is essential for the determination of the identity of sepals and petals. In the weak
ap2-1 mutant, leaf-like organs replace sepals, and petals exhibit antheroid characteristics; in strong ap2 mutants, carpels
are formed in the outer whorl of the flower, petals are absent,
and the number of stamens is reduced (Komaki et al., 1988;
Kunst et al., 1989). The homeotic transformations found in
ap2 mutants can be explained in part by ectopic expression
of Agamous (Ag), as observed in the two outer whorls of their
flowers (Drews et al., 1991). Apart from Ap2, two other genes,
Leunig (Lug) and Curly Leaf (Clf), are involved in the repression of Ag in the perianth (Liu and Meyerowitz, 1995;
Goodrich et al., 1997). Ap2 also is required for normal seed
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The Plant Cell
Figure 1. DNA Gel Blot Analysis of PhAp2 Genes in Petunia (Line V23) and Amino Acid Alignments of Petunia PhAp2 genes and Arabidopsis Ap2.
Petunia Apetala2 Genes
coat development, and ap2 mutants lack the epidermal plateau of the epidermis of wild-type seed (Jofuku et al., 1994;
Leon-Kloosterziel et al., 1994; Modrusan et al., 1994).
Ap2 was isolated by T-DNA tagging. It encodes a protein
that belongs to a rapidly growing family of plant-specific
transcription factors that contains the AP2 domain, a conserved ⵑ60–amino acid region involved in DNA binding
(Okamuro et al., 1997; Riechman and Meyerowitz, 1998).
The AP2 family of proteins consists of two main branches.
One branch contains proteins that have a single copy of the
AP2 domain. These are involved in different kinds of stress
responses and include the ethylene responsive element
binding proteins and the C repeat binding factor CBF1
(Ohme-Takagi and Shinshi, 1995; Stockinger et al., 1997).
Ap2, Aintegumenta (Ant), Glossy15 (Gl15), and Indeterminate spikelet 1 (Ids1) all contain two AP2 domains and belong
to the second branch of the Ap2-like transcription factor family that plays a role in plant development. Ant is required for
ovule and female gametophyte development (Elliott et al.,
1996; Klucher et al., 1996), Gl15 is required for the maintenance of juvenile traits of epidermal cells in maize leaves from
node 2 to node 6 (Moose and Sisco, 1996), and Ids1 controls
maize spikelet meristem fate (Chuck et al., 1998).
In this study, we report the isolation and partial characterization of three Ap2-like genes of petunia: PhAp2A, PhAp2B,
and PhAp2C. To determine which of these three genes represents the Ap2 ortholog in petunia, we compared their sequences with Ap2, analyzed their expression patterns during
plant development by in situ hybridization, and applied a reverse genetics approach to identify mutant alleles by transposon insertion mutagenesis. We also mapped the three gene
copies to determine if one of them could encode Blind (Bl),
which appeared not to be the case. This A-type mutant from
petunia has been described previously and has been suggested to be caused by the loss of activity of the petunia Ap2
ortholog (Vallade et al., 1987; Tsuchimoto et al., 1993). The
phenotype of flowers of the bl mutant is similar to that seen in
ap2 and clf mutants in Arabidopsis and in fistulata (fis) mutants in Antirrhinum species (Motte et al., 1998).
In addition, we have demonstrated the functional conservation of the A-type genes PhAp2A and Ap2 by complementing
the ap2-1 mutant of Arabidopsis with the petunia PhAp2A gene.
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RESULTS
PhAp2A Is the Closest Ortholog of Ap2; PhAp2B and
PhAp2C Belong to a Different Subfamily of Ap2-like
Genes in Petunia
An 800-bp PhAp2A-1 cDNA insert was used to probe petunia genomic DNA gel blots at medium stringency. Under
these conditions, three hybridizing bands can be observed,
one of which has a higher intensity than the other two (Figure 1A). When the blot was stripped and rehybridized with a
4-kb PhAp2A genomic subclone, only the strongest hybridizing band was observed (Figure 1B). These results indicate
that PhAp2A belongs to a gene family and that at least two
other PhAp2A homologous gene copies exist in the petunia
genome, which were termed PhAp2B and PhAp2C.
The PhAp2A cDNA encodes a protein of 519 amino acid
residues (Figure 1C). Similarity searches using tBlastn revealed the Arabidopsis AP2 protein as the most homologous entry in the GenBank database. PHAP2A shows
64% overall amino acid identity and 77% similarity with
AP2. The homology of PHAP2A and AP2 is not limited to
the AP2 domains but extends through the reading frame
of the two proteins. A serine-rich putative transcription activation domain (amino acids 18 to 60) and a putative nuclear localization signal (amino acids 148 to 158; Jofuku et
al., 1994) are conserved, as are the N and C termini of the
proteins (Figure 1C). The homology between the two proteins is especially high in the two AP2 domains (63 of 67
and 66 of 68 residues identical), whereas the linker that
connects them is completely conserved at the amino acid
level (Figure 1C).
The PhAp2B cDNA encodes a 457–amino acid protein
(Figure 1C). The AP2 domains and the linker of PHAP2A
and AP2 are more closely related than those of the two
petunia proteins PHAP2A and PHAP2B. Interestingly, of
the 21 amino acid substitutions that occur in this region
between PHAP2A and PHAP2B, five amino acids are similarly substituted in the two domains and two more in the
linker of the predicted Arabidopsis RAP2.7 sequence (Figure 1C).
Figure 1. (continued).
(A) Blot probed with the 800-bp PhAp2A-1 cDNA fragment that contains the Ap2 domains (medium stringency).
(B) Blot probed with the 4-kb genomic XbaI fragment of PhAp2A (high stringency).
(C) Comparison of the overall amino acid sequences, the AP2 domains (shaded), linkers, and putative nuclear localization signals (in boldface) of
PhAp2A (GenBank accession number AF132001), Ap2 (ATU12546), PhAp2B (AF132002), PhAp2C (partial sequence), RAP2.7 (AF003100), and
ANT (U40256). Amino acids in the AP2 domains, which are conserved in all mentioned sequences, are in white on a shaded background; the five
conserved amino acids in the domains of PhAp2B and RAP2.7 are white on black. Gaps are indicated by dashes. No putative nuclear localization signal could be identified in front of the first Ap2 domain in the Ant gene; the overall homology of Rap2.7 and Ant with the other Ap2-(like)
genes outside the domain regions was too low to allow a meaningful comparison.
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The Plant Cell
Figure 2. In Situ Analysis of Expression of PhAp2A, PhAp2B, PhAp2C, and pMADS3 during Flower Development.
Each row represents a set of consecutive 10-␮m sections that were hybridized with the different 35S-CTP–labeled antisense riboprobes (except
for [E], which represents a similar section).
Petunia Apetala2 Genes
Outside of the AP2 domains, there is very little sequence
conservation between PHAP2A and PHAP2B. Two conserved motifs (F/VDF/ILKT/V and VTKEF/LFPV/LS) could be
identified in the N-terminal part of PHAP2B and PHAP2C
and in the Arabidopsis RAP2.7 (GenBank accession number
T20443; Okamuro et al., 1997), and another one could be
identified in the C-terminal part of PHAP2B and RAP2.7 (PL/
MFSV/TAAASSGF). These data suggest that PHAP2B,
PHAP2C, and RAP2.7 belong to a different subgroup than
AP2 and PHAP2A (Figure 1C).
No PhAp2C cDNA clone could be retrieved from the
inflorescence or ovary cDNA library, although reverse
transcription–polymerse chain reaction (RT-PCR) using two
PhAp2C-specific primers on young petunia inflorescences
and flowers suggested that the gene is expressed.
Chromosomal Localization of PhAp2A, PhAp2B, and
PhAp2C Shows That They Do Not Coincide with the
Bl Locus
Flowers of the bl mutant of petunia have patches of stigmoid tissue at the tips of their sepals, and antheroids replace the flower limb. These homeotic transformations are
caused by ectopic expression of the C-type gene pMADS3
(pMADS3 ⫽ FBP14; Kater et al., 1998) in bl leaves, sepals,
and petals (Tsuchimoto et al., 1993). It has been proposed
that Bl is a negative regulator of pMADS3 and that Bl determines the expression boundaries of pMADS3 (Tsuchimoto
et al., 1993). Because Ap2 is a negative regulator of the
C-type gene Ag in Arabidopsis, we wanted to determine if
one of its homologs in petunia coincides with the Bl locus.
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We performed a linkage analysis for the three PhAp2
genes by analyzing restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism
(AFLP; Vos et al., 1995), and phenotypic markers in different
backcrosses. According to the (V26 ⫻ W137) ⫻ W137 progeny analysis, PhAp2A is located on chromosome 4, PhAp2B
is located on chromosome 2, and PhAp2C is located on
chromosome 6. PhAp2A and the Bl locus, both on chromosome 4, appear to be loosely linked (see Methods for details).
Thus, none of the PhAp2 loci coincides with the Bl locus.
Expression of PhAp2 Genes during Plant Development
The spatial and temporal distribution of the PhAp2A,
PhAp2B, and PhAp2C mRNA and the mRNA expression of
the petunia C-type gene pMADS3 were analyzed by in situ
hybridization of consecutive sections of wild-type inflorescences and flowers or flower parts (Figures 2A, 2E, 2I, and
2M) to 35S-labeled PhAp2A, PhAp2B, PhAp2C, and pMADS3
antisense riboprobes. The specificity of the probes used for
the in situ hybridization experiments was tested by RNA gel
blot analysis (not shown). The different stages of flower development referred to in the text are described in Table 1.
Spatiotemporal Expression Pattern of PhAp2A Is
Complementary to That of PhAp2B and PhAp2C during
Flower Development and Overlaps Partially with That
of pMADS3
In the wild-type petunia inflorescence, uniform PhAp2A expression could be observed in stage 1 to 2 inflorescence
Figure 2. (continued).
(A) Bright-field photograph of the top of the inflorescence with a transverse section through the inflorescence meristem and a stage 4 to 5 flower
and an oblique section through a stage 6 flower and part of a stage 7 flower.
(B) Dark-field photograph of a section like that shown in (A) with PhAp2A expression.
(C) Dark-field photograph of a section like that shown in (A) with PhAp2B expression.
(D) Dark-field photograph of a section like that shown in (A) with pMADS3 expression.
(E) Bright-field photograph of a longitudinal section through a stage 7 flower.
(F) Dark-field photograph of a section like that shown in (E) with PhAp2A expression.
(G) Dark-field photograph of a section like that shown in (E) with PhAp2B expression.
(H) Dark-field photograph of a section like that shown in (E) with pMADS3 expression.
(I) Bright-field photograph of a transverse section through a stage 8 to 9 flower.
(J) Dark-field photograph of a section like that shown in (I) with PhAp2A expression.
(K) Dark-field photograph of a section like that shown in (I) with PhAp2C expression.
(L) Dark-field photograph of a section like that shown in (I) with pMADS3 expression.
(M) Bright-field photograph of a longitudinal section through an inflorescence with an early stage 9 flower and a stage 6 flower.
(N) Dark-field photograph of a section like that shown in (M) with PhAp2A expression.
(O) Dark-field photograph of a section like that shown in (M) with PhAp2C expression.
(P) Dark-field photograph of a section like that shown in (M) with pMADS3 expression.
ac, connective tissue of the anther; ap, anther primordium; br, bract; ca, carpel; fp, flower primordium; ov, ovary; ow, ovary wall; pc, procambium; pe, petal; pl, placenta; se, sepal; st, stamen; vb, vascular bundle. Bars in (A), (E), (I), and (M) ⫽ 100 ␮m.
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The Plant Cell
Table 1. Stages of Flower Development in Petunia hybrida Line W138
Estimated Size (␮m)b
Stage Description
1
2
3
4
5
6
7
8
9
10
11
Apical meristem is loaf shaped
Two bract primordia (B1 and B2) arise on the apical meristem under an angle of ⵑ150⬚C and
give it a bean-shaped appearance
Flower primordium becomes distinct of the inflorescence meristem when the growth rate of
approximately half the cells in the apical meristem increases
Floral meristem enlarges, and the first sepal primordium arises
The rest of the five sepals primordia arise sequentially in a spiral; bract 1 overlies the flowerbud
Sepals elongate to enclose the bud, trichomes start to differentiate on the first sepal, petal and
stamen primordia arise; no carpel primordia visible
Stamens become lobed; two carpel primordia arise and unite to form a tube
Stamens become stalked at their base, petals elongate to the size of the stamens, the
gynoecium fuses at the top
Stamen filaments and style start to elongate, degeneration of anther tissue starts, ovule
primordia arise
Carpel transmitting tissue and papillae form, ovule primordia become stalked, integument
growth initiates
Endothecial cell walls show band thickening, merging of two pollen sacs into single locule,
integument covers nucellus completely
IM1a
IM1
60 ⫻ 100
100 ⫻ 170
Total
IM2
FP1
FB1
FB1
FB1
FP2
FB
FB
130 ⫻ 240
(stage 1)
Ø 120
Ø 140
Ø 200
Ø 350
(stage 4)
Ø 400
Ø 600
FB
Ø 800
NM
NM
a Subscript numbers reflect the temporal order of initiation; lower numbers are initiated first (sizes are indicative and were estimated from SEM for
stage 1 to 6, and from paraffin sections for stages 7 and 8, in both cases from fixed and dehydrated material). IM, inflorescence meristem; FP,
flower primordium; FB, flower bud.
b NM, not measured; Ø, diameter.
meristems and in young stage 4 to 5 flower buds. A weaker
signal was observed in the procambial cells of the developing stem. In bracts subtending stage 4 to 5 flower buds and
in stage 6 sepals, the PhAp2A mRNA was not distributed
uniformly but localized to a layer of ground parenchyma
cells (Figure 2B). The expression of PhAp2A in sepals and
bracts decreased after stage 7 to 8 (Figures 2F, 2J, and 2N).
Petals exhibited strong PhAp2A expression throughout their
development. In young petal primordia until stage 5 to 6, the
signal had a uniform appearance (Figure 2B). In later stages,
PhAp2A was expressed in a layer of cells in the ground parenchyma, as in sepals and bracts. This layer runs through
the center of the flower tube tissue, assumes a more adaxial
position in the flower limb (Figures 2J and 2N), and passes
around the dorsal side of the vascular bundle of the main
vein of the petal (not shown).
Stamen primordia of stage 6 flower buds showed uniform
expression that decreased by stage 7 and became concentrated in the region where the vascular bundles of the filaments develop (Figures 2B, 2F, and 2J). At stage 9, weak
expression also was detected in the anther wall and in the
connective tissue (not shown). A hybridizing signal also was
detected over mature pollen grains (Figure 3H). Young carpel primordia of stage 7 flower buds exhibited strong
PhAp2A expression (Figure 2F). In stage 7 to 9 flower buds,
the signal was strong in the central cell layers of the ovary
wall, in the placenta, and in ovule primordia (Figures 2J and
2N). When the ovules became stalked, the signal in the
ovary wall and the placenta decreased. PhAp2A mRNA then
could be detected mainly in the vascular bundle of the placenta, and a weak signal also could be seen in the vascular
bundle of the style and stigma (Figure 3B). By stage 11, the
hybridization signal in the ovules became restricted to the
funiculus and the integument (Figures 3E and 3K).
PhAp2B and PhAp2C expression was detected in the
outer cell layers of young bracts subtending stage 1 to 2 inflorescence meristems. Stage 7 flowers expressed PhAp2B
and PhAp2C in the epidermis of the sepals (Figures 2C and
2G). Expression was maintained in sepals at least until stage
9 to 10, by which time the signal was no longer localized
specifically in the outer cell layers and acquired a spotty appearance (Figures 2K and 2O). Weaker PhAp2B and PhAp2C
expression also was detected in the outer cell layers around
the main veins of stage 9 petals (Figure 2K). The two genes
were strongly expressed in the connective tissue of stage 8
to 9 stamens and around stage 10 to 11 as well in the anther
wall. No signal was observed over the vascular bundles and
the filaments or, at this stage, over the developing microspores (Figures 2K, 2O, 3C, and 3D). However, in mature
anther filaments, a spotty PhAp2C signal was observed (not
shown), and although the connective tissue degenerated
and the hybridization signal disappeared from the anther
wall, PhAp2B and PhAp2C signals could be observed over
the pollen grains (Figure 3I). The fourth whorl gynoecia of
Petunia Apetala2 Genes
stage 9 flowers exhibited low PhAp2B and PhAp2C expression. As the style elongated, the signal increased in intensity
and became restricted to the epidermis of the ovary wall
(Figures 2K, 3C, 3D, 3F, and 3L). We did not detect any
PhAp2B or PhAp2C expression in the placenta or in the developing ovules during the stages of flower development
described in this article.
The expression patterns of the PhAp2 genes were compared with the pattern of the C function gene pMADS3.
pMADS3 expression first was detected in the center of
stage 5 flowers, before any stamen primordia development
was visible. The expression in developing stamen primordia
remained uniform until stage 7, when the anthers and filaments start to differentiate (Figures 2D and 2H). Stage 8 to 9
stamens showed some pMADS3 expression in the anther
wall and in the connective tissue, but the strongest signal
was observed over the vascular bundle in the filament (Figures 2L and 2P). No signal was detected over the developing microspores (Figure 3G). pMADS3 was expressed
strongly in carpel primordia. By stage 8, when the gynoecia
fuse at the top, expression decreased in the ovary wall but
became very strong in the vascular bundles of the placenta
and at the positions where the ovule primordia will arise
(Figures 2L and 2P). By stage 9, pMADS3 was expressed in
the vascular bundle of the style and the placenta and in the
epidermal layer of the style and stigma. Ovule primordia
showed strong and uniform expression that persisted until
the ovules were mature (Figures 2L, 3A, and 3J). The expression pattern observed for pMADS3 was in agreement
with that reported by others (Kater et al., 1998).
Expression Patterns of PhAp2A, PhAp2B, and PhAp2C
Coincide in Mature Seed
The expression of the PhAp2 genes was investigated further
in mature seed, germinating seedlings, and young plants.
Strong expression was detected for PhAp2A, PhAp2B, and
PhAp2C in the endosperm of mature seed. The hybridizing
signal in endosperm was distributed asymmetrically, in that
the side where the radicle emerges during germination did not
show any expression (Figures 4A to 4D). No expression could
be detected in the mature embryo. Leaf primordia of young
seedlings showed a clear signal for PhAp2A, as did the leaf
primordia and the leaves of young plantlets (Figures 4E to 4H).
As was observed for the developing floral organs, the PhAp2A
signal appeared to be distributed homogeneously in leaf primordia and to concentrate subsequently in a layer of ground
parenchyma cells of the developing leaves (Figure 4F).
The expression of the PhAp2A gene thus differs markedly from that of PhAp2B/PhAp2C during vegetative and
floral development; in contrast, the expression patterns of
the three genes coincide in mature seed. Mutants in which
the functions of these genes have been abolished are required to reveal the roles of the PhAp2 genes during plant
development.
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Transposon Insertion Mutagenesis of PhAp2A
Based on the results of in situ gene expression analysis and
sequence comparisons, PhAp2A was the only likely candidate
to perform the orthologous Ap2 function in petunia. To prove
that PhAp2A functions as an A-type gene, we screened for
loss-of-function alleles by transposon insertion mutagenesis.
A PCR-based screening method (Ballinger and Benzer, 1989;
Kaiser and Goodwin, 1990) was used to select for insertions of
the nonautonomous transposable element dTph1 in PhAp2A
by using three available petunia populations (Koes et al., 1995;
Figure 5A). A set of PhAp2A-specific primers and a primer
complementary to the terminal inverted repeat of the transposon were used; five heterozygous insertion mutants were detected in a population of 3456 W138 plants (Figure 5B).
The positions of the dTph1 elements were determined by
cloning and sequencing of the fragments flanking the transposon insertions in PhAp2A. Three plants contained a
dTph1 insertion in intron sequences (T2194-14, intron VIII;
V2047-10, intron I; V2193-7, intron III). The two other plants
harbored a dTph1 element in the first exon, after amino acids 17 (V2116-10) and 141 (V2025-6) (Figure 5B). None of
the homozygous insertion mutants segregating in the progeny of the selfed heterozygous primary insertion mutants
had an altered phenotype.
We used RT-PCR with two gene-specific primers flanking
the exon 1 insertion site to analyze the PhAp2A mRNA in the
exon 1 insertion mutants V2025-6 and V2116-10. The mutant cDNA fragments are ⵑ300 bp longer than the wild-type
PhAp2A cDNA fragments. This suggests that there is no
premature termination of the messenger due to transcription
termination signals in the element and that dTph1 is
cotranscribed with the PhAp2A messenger (shown for the
V2025-6 insertion allele in Figure 5C). Also, wild-type and
mutant cDNA fragments exhibited a comparable abundance
in plants heterozygous for the insertion. Thus, the dTph1 insertions did not seem to affect the level of PhAp2A transcription. The dTph1 element is 284 bp long and induces a
target site duplication of 8 bp upon insertion. The translated
nucleotide sequence of the elements inserted in the PhAp2A
gene contains stop codons in the three reading frames.
Thus, theoretically, only 17 amino acids of the PHAP2A protein of V2116-10 and 141 amino acids of the PHAP2A protein of V2025-6 would be translated, which should result in a
functional knockout of the PhAp2A gene.
In plants homozygous for the V2025-6 insertion, a low level
of wild-type mRNA sometimes was detected by RT-PCR. To
determine whether this product was formed after excision of
dTph1 or by alternative splicing, we crossed this insertion
allele into an activator minus background to stabilize the insertion. The Activator element controls transposition of
dTph1 elements (Gerats et al., 1990). Stabilization of the
dTph1 system was monitored by means of a dTph1-tagged
reporter gene that controls anthocyanin synthesis (see
Methods). Plants homozygous for the PhAp2A(V2025-6)
allele and lacking the active Activator produced only the
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The Plant Cell
Figure 3. Expression of PhAp2 Genes and pMADS3 in Third and Fourth Whorl Organs.
Petunia Apetala2 Genes
mutant mRNA and, again, exhibited a wild-type phenotype.
The expression patterns of PhAp2A and pMADS3 in plants
homozygous for the PhAp2A(V2025-6) insertion allele were
analyzed by in situ hybridization and did not differ from the
patterns found in wild-type petunia (results not shown).
PhAp2A Complements the ap2-1 Mutant of Arabidopsis
Because the phap2a insertion mutants we obtained did not
exhibit a mutant phenotype, we decided to express PhAp2A
in an ap2-1 background in Arabidopsis to determine whether
this might result in complementation. The ap2-1 mutant is
temperature sensitive. At 15⬚C, the sepals have leaf-like characteristics, and the petals are slightly phylloid; at 25⬚C, the
sepals are stigmoid leaves, and the petals are staminoid; and
at 29⬚C, the sepals are carpelloid, and the petals are absent,
resembling the phenotype of the strong ap2 mutants
(Bowman et al., 1989). The binary vector pBIN19-35S PhAp2A
was used to transform the ap2-1 mutant (see Methods). Kanamycin-resistant calli and shoots were selected at 23⬚C, but
rooted transformants were allowed to develop at 26⬚C to induce a more extreme ap2-1 phenotype.
All shoots from four of five independent transformants
generated phenotypically wild-type flowers that produced
phenotypically wild-type seed (Figure 6). Together with the
sequence and expression data, the complementation data
prove that PhAp2A encodes the Ap2 ortholog of petunia.
DISCUSSION
In this study, we report the isolation and partial characterization of three Ap2-like genes from petunia: PhAp2A,
PhAp2B, and PhAp2C. The PHAP2A protein is the closest
AP2 homolog described so far. The 18–amino acid cores of
the AP2 domain, which theoretically are capable of forming
237
an amphipathic ␣ helix (Jofuku et al., 1994), are identical in
the two proteins. If the DNA binding specificity of the AP2like proteins is determined by the amphipathic ␣-helix core
itself, PHAP2A should be able to bind the target sequences
of AP2. In contrast with what has been observed for other
AP2-like proteins, the homology between PHAP2A and AP2
extends throughout the whole protein, suggesting that these
genes are orthologs.
Two closely related Ap2-like gene copies, PhAp2B and
PhAp2C, detected by DNA gel blot hybridization were isolated. Based on the occurrence of similar amino acid substitutions in their AP2 domains, PHAP2B and PHAP2C, like RAP2.7,
and IDS1 belong to a different subgroup of AP2-like transcription factors than AP2 and PHAP2A.
Expression Patterns of PhAp2A and PhAp2B/PhAp2C
Suggest That These Genes May Have Both Overlapping
and Unique Functions
Like Ap2 and Ant in Arabidopsis, PhAp2A is expressed uniformly in young lateral organ primordia. As the organs mature,
the signal in developing bracts, sepals, petals, and the ovary
wall gradually decreases in the outer layers of these organs
and eventually disappears.
During flower development, PhAp2B and PhAp2C expression patterns are remarkably complementary to that of
PhAp2A. PhAp2B and PhAp2C expression begins to appear
in bracts, sepals, petals, and the ovary wall when PhAp2A
expression is decreasing. In general, we observed little if any
overlap between the expression of PhAp2A and that of PhAp2B
and PhAp2C. Only some of the Ap2-like genes that have been
described have been well characterized in terms of both expression and function. The Gl15 gene has been described to
control the identity of epidermal cells in maize (Moose and
Sisco, 1996). The expression patterns of PhAp2B and PhAp2C
are compatible with a function in the development of the
Figure 3. (continued).
(A) Dark-field photograph of a section through carpels and part of the anthers of a stage 10 flower with pMADS3 expression.
(B) Dark-field photograph of a section like that shown in (A) with PhAp2A expression.
(C) Dark-field photograph of a section like that shown in (A) with PhAp2B expression.
(D) Dark-field photograph of a section like that shown in (A) with PhAp2C expression.
(E) Dark-field photograph of a section through carpels and part of the anthers of a stage 11 flower with PhAp2A expression.
(F) Dark-field photograph of a section like that shown in (E) with PhAp2C expression.
(G) Dark-field photograph of a section through merged pollen sacs, just before dehiscence, with pMADS3 expression.
(H) Dark-field photograph of a section like that shown in (G) with PhAp2A expression.
(I) Dark-field photograph of a section like that shown in (G) with PhAp2C expression.
(J) Dark-field photograph of a section through ovary of stage 11 flower (detail) with pMADS3 expression.
(K) Dark-field photograph of a section like that shown in (J) with PhAp2A expression.
(L) Dark-field photograph of a section like that shown in (J) with PhAp2C expression.
ac, connective tissue of the anther; aw, anther wall; fu, funiculus; in, integument; ov, ovule; ow, ovary wall; pe, petal; po, pollen grain; vb, vascular bundle. Bars in (A) to (L) ⫽ 100 ␮m.
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The Plant Cell
Figure 4. Expression of PhAp2 Genes in Germinating Seed and Expression of PhAp2A in Young Seedlings and Young Vegetative Plants.
(A) Phase-contrast photograph of a stained longitudinal section through a germinating seedling.
(B) Dark-field photograph of a section like that shown in (A) with PhAp2A expression.
(C) Dark-field photograph of a section like that shown in (A) with PhAp2B expression.
(D) Dark-field photograph of a section like that shown in (A) with PhAp2C expression.
(E) Bright-field photograph of a longitudinal section through a seedling with emerging leaf primordia.
(F) Dark-field photograph of a section like that shown in (E) with PhAp2A expression.
(G) Bright-field photograph of a longitudinal section through the apex of a young vegetative plantlet.
(H) Dark-field photograph of a section like that shown in (G) with PhAp2A expression.
co, cotyledon; en, endosperm; le, leaf; lp, leaf primordium; r, radicle. Bars in (A) to (H) ⫽ 100 ␮m.
epidermis. The divergence of the amino acid sequences
between PHAP2B/PHAP2C and GL15, however, suggests
that they are not orthologous.
PhAp2A also is expressed during vegetative development. A strong signal can be observed in leaf primordia of
young seedlings that at later stages decreases and concentrates in the inner layers of the developing leaves. Thus, the
progression of PhAp2A expression during floral development is the same as that during vegetative development,
supporting the idea that the floral organs are nothing but
specialized, modified leaves (von Goethe, 1790).
The homology between the AP2 protein of Arabidopsis
and the PHAP2A protein of petunia, together with the resemblance of their expression patterns, strongly suggests
that the two genes are genuine orthologs.
Like Ap2 and Ant of Arabidopsis, PhAp2A also is expressed during ovule development. The PhAp2A expression
in integuments is in accordance with the role of Ap2 in seed
Petunia Apetala2 Genes
coat development observed in Arabidopsis. PhAp2A, PhAp2B,
and PhAp2C show an identical expression pattern in mature
and germinating seed. A strong, asymmetrical signal is observed in the endosperm, in which it is excluded from the
side of radicle emergence. This expression pattern resembles that of prolamin seed storage proteins of maize (also
called zeins), which start to be expressed coordinately in the
endosperm of maize 8 to 10 days after pollination. The promoters of these zein genes share a common regulatory element, the prolamin box (5⬘-TGTAAAG-3⬘), that is a good
candidate for being the cis-acting regulatory element controlling this expression pattern (Forde et al., 1985). Recently,
an endosperm-specific factor belonging to the Dof family of
zinc finger proteins that binds the prolamin box and interacts with Opaque 2, a known transcriptional activator of
zein gene expression, was isolated from maize (VicenteCarbajosa et al., 1997). A perfect prolamin box (TGTAAAG)
and four related elements (TGHAAARK) were detected in the
promoter of the PhAp2A gene (T. Maes, results not shown).
At least one other Ap2-like gene, an Ant homolog of maize
(GenBank accession number Z47554), also is expressed in
postpollination maize endosperm (Elliott et al., 1996; Daniell
et al., 1996).
PhAp2A and the Petunia Class A Function: Isolation of
PhAp2A Insertion Alleles
In Arabidopsis, the class A gene Ap2 fulfills two roles in the
process of floral organ identity determination: a cadastral
function consisting of repression of the class C gene Agamous and an organ specification function in the perianth
(sepal identity and determination of meristems in the second
whorl).
To determine whether PhAp2A performs the same function in petunia that Ap2 does in Arabidopsis, we screened
for dTph1 insertions in PhAp2A. Two of the five isolated
PhAp2A insertion alleles had an insertion in the first exon of
the gene. Surprisingly, W138 plants homozygous for these
exon 1 insertions in PhAp2A do not exhibit a mutant phenotype in floral development.
239
homologs of the Ap2 gene of Arabidopsis play a crucial role
as the class A function genes in the development of all angiosperm flowers.
Alternatively, the lack of a phenotype could be due to
functional redundancy caused by the activity of another
gene without any sequence homology with the Ap2 genes.
This would imply that Arabidopsis would lack such a gene.
Because the bl mutant shows analogous, albeit less pronounced, homeotic transformations reminiscent of A-type
mutants of Arabidopsis, Bl is an obvious candidate for such
a function. In Arabidopsis, at least two other mutants have
been described that exhibit flowers displaying homeotic
transformations similar to those in ap2 mutants: lug and clf
(Liu and Meyerowitz, 1995; Goodrich et al., 1997). clf mutations have pleiotropic effects on flowering time, leaf shape,
and flower morphology. In clf flowers, sepals and petals
show partial transformation toward carpels and stamens.
CLF seems to act later in flower development than AP2 and
LUG (Liu and Meyerowitz, 1995; Goodrich et al., 1997).
Another possibility is that the A function, as encoded by
Ap2 of Arabidopsis, does not exist as such in petunia. The
changes in the identity of perianth organs of A-type mutants
found in petunia (bl) and in Antirrhinum species (fistulata and
stylosa) can be attributed completely to the lack of cadastral
activity, that is, to the lack of control of the expression domain of the C-type genes (Tsuchimoto et al., 1993; Motte et
al., 1998). Until now, none of the A function mutants in these
two species has been attributed to a mutation in an Ap2-like
gene. In addition, Arabidopsis is the only species in which a
gene has been identified that controls both sepal and petal
specification and the expression of the class C gene Agamous. In Arabidopsis, Ap2 is required to establish the morphological features of the sepal epidermis, which differ from
those of leaves in that the sepal epidermis does not have
stellate trichomes (Bowman et al., 1989). In petunia, the inflorescence leaf epidermis and the sepal epidermis are indistinguishable, suggesting that the Ap2 ortholog of petunia
has no function in the specification of sepal cell identity. If
the only role of the PhAp2A gene in petunia consists of directly or indirectly negatively regulating the C function in the
perianth, mutations that exclusively affect the transcription
activation domain may not interfere with its function in flower
development. This brings us to our second hypothesis.
A Complete PhAp2A Knockout?
Because attempts to obtain phenotypic phap2a mutants
through cosuppression or antisense strategies have been
equally unsuccessful (T. Maes, unpublished results), our first
hypothesis is based on the assumption that the dTph1 insertions in exon 1 of PhAp2A knock out all of the potential
functions of PHAP2A. We showed that the sequences of
PhAp2B/PhAp2C, and their expression patterns during
flower development, are very distinct from those of PhAp2A,
making the hypothesis that they exert the same function improbable. Thus, the absence of a phenotype in the PhAp2A
insertion mutants seems to contradict the hypothesis that
A Partial PhAp2A Knockout?
Theoretically, truncated versions of the PHAP2A protein
could be translated from the mRNA of the exon 1 insertion
mutants we have obtained. In both cases, the truncated
protein would be missing the putative transcription activation domain but would retain the putative nuclear localization signal and the AP2 domains. Such a truncated protein
presumably still could function as a repressor and possibly
regulate pMADS3 expression. Because PhAp2A overexpression can complement the ap2-1 mutation, the easiest
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The Plant Cell
Figure 5. Insertion Mutagenesis of the PhAp2A Gene.
(A) Screening for insertion mutants. DNA pools of 1080 plants, organized in pools of 10 block samples, 12 row samples, and 9 column samples,
were subjected to PCR using PhAp2A primer 002 in combination with the dTph1 inverted repeat primer. The PCR products were analyzed by
DNA gel blot analysis using a PhAp2A-specific probe. Two positive plants were identified with the following coordinates: block 5, row 6, column
1 ⫽ V2025-6 and block 9, row 10, column 9 ⫽ V2047-10. The column signal of the second positive plant was missing in this screen but was
identified in a similar experiment using PhAp2A primer 003. Marks indicate positive signals.
(B) Positions of the dTph1 insertions in PhAp2A and the positions of the primers used in the screening experiment. UTR, untranslated region.
(C) RT-PCR analysis of wild-type mRNA and PhAp2A(V2025-6) mRNA in the transposition-stabilized act⫺/act⫺ background. Products were amplified from first strand cDNA by using PhAp2A primers 001 and 002. The wild-type product (WT; lane 1) is ⵑ300 bp smaller than the product
from the homozygous insertion mutant (INS; lane 2), which contains the dTph1 element (lane 3; PstI size marker).
Petunia Apetala2 Genes
way to verify whether we have obtained loss-of-function
mutants, or if a truncated PhAp2A gene retains the ability to
regulate Ag, would be to express a similarly truncated
PhAp2A gene in the ap2-1 mutant of Arabidopsis.
PhAp2A Behaves as a Functional A-Type Gene
in Arabidopsis
Promoter 35S-driven PhAp2A gene expression in Arabidopsis complements all mutant aspects of flower development
of the ap2-1 mutation. Ectopic expression of Ag and the
consequent homeotic changes in the perianth are inhibited,
and the expression of genes required for the determination
of sepal versus leaf identity is regulated correctly, in that
241
normal sepals develop in the complemented flowers. In
conclusion, the complementation of the ap2-1 mutant unequivocally proves our hypothesis that PhAp2A represents
the functional ortholog of Ap2. Therefore, additional factors
must be involved in the control of perianth identity in petunia. Although the general conservation of developmental
control systems is widely accepted, there are examples in
which divergence has occurred. A case in point seems to be
the essential role played by sonic hedgehog (Shh) in the
formation of body asymmetries in chick but not in mouse
(Pagan-Westphal and Tabin, 1998). Here, we might have another example in which two orthologs have greatly diverged
in at least some aspects of their function. Thus, either petunia PhAp2A has lost its perianth identity control function or
Arabidopsis Ap2 has gained such a function. Further analyses involving a range of species are needed to clarify this
point in more detail.
METHODS
Plant Material
Petunia hybrida line W138 contains a high copy number of the nonautonomous transposable element dTph1, which requires the presence of an (active) Activator, located on chromosome 1, to undergo
transposition (Gerats et al., 1990; Huits et al., 1995).
Petunia lines W138 and W162 are homozygous for an unstable recessive mutation caused by an insertion of the nonautonomous element dTph1 in the An1 gene. An1 controls the expression of several
structural genes in the anthocyanin pathway, and an1 mutants have
a white corolla. The formation of red or pink revertant sectors and
spots in line W138 results from excision of the dTph1 element and
can be used as a reporter system for transposition activity. Line
W162 does not contain the Activator of the two-element system, and
hence this line has white flowers (Huits et al., 1995). The bl mutant
was in the R51 background.
DNA Gel Blot Analysis
Figure 6. Complementation of the Arabidopsis ap2-1 Mutant by
PhAp2A.
(A) Arabidopsis ap2-1 flower (grown at 26⬚C). The flower is from a
transformant that did not exhibit complementation.
(B) Arabidopsis ap2-1::35S PhAp2A flower (grown at 26⬚C) exhibiting full complementation toward the wild type.
(C) Scanning electron micrograph of the seed coat of a noncomplementing ap2-1 transformant.
(D) Scanning electron micrograph of the seed coat of an ap2-1::35S
PhAp2A transformant.
(E) Detail of (C).
(F) Detail of (D).
Bar in (C) and (D) ⫽ 100 ␮m; bar in (E) and (F) ⫽ 10 ␮m.
DNA was extracted from petunia leaves as described by Souer et al.
(1995). Twenty micrograms of DNA was digested and separated by
agarose gel electrophoresis. Blotting and the composition of the
hybridization buffer were as described by Amersham. ␣-32P-dCTP–
labeled DNA probes were synthesized using the Megaprime DNA labeling kit (Amersham). Hybridizations and washes were performed at
60⬚C with 2 ⫻ SSC, 0.1% SDS and either 0.5 ⫻ SSC, 0.1% SDS (intermediate stringency) or 0.1 ⫻ SSC, 0.1% SDS (high stringency; 1 ⫻
SSC is 0.15 M NaCl and 0.015 M sodium citrate).
Isolation and Sequencing of the PhAp2 Genes
The full-length Arabidopsis Ap2 cDNA was used to screen 105
plaque-forming units (PFUs) of a petunia ␭GEM4 cDNA library made
from flower buds at low stringency. Initially, four positive clones were
isolated that contained an 800-bp partial cDNA insert. This partial
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The Plant Cell
cDNA clone was used to screen 3 ⫻ 106 PFUs of a Uni-ZAP XR cDNA
library from inflorescence meristems at medium stringency. The 15
positive clones that were isolated were analyzed by polymerase chain
reaction (PCR) using two cPhAP2A-1–specific primers (p001 and
p002). All clones corresponded to the same gene copy. Four fulllength PhAp2B cDNA clones were isolated by screening 3 ⫻ 106 PFUs
of a HybriZAP ovary cDNA library using a 300-bp PCR fragment amplified from a 1.1-kb PhAp2B genomic subclone as a probe.
The cPhAP2A-1 insert (a kind gift from Bart den Boer, Aventis Crop
Sciences, Gent) was used to screen a V26 genomic library in ␭ at
high stringency. Positive clones were analyzed by restriction analysis
and represented the same gene. A 4.0-kb XbaI fragment cross-hybridizing with the cPhAP2-1 insert and a 3.2-kb flanking fragment were
subcloned in pUC18, and deletion clones were generated using the
method of Henikoff (1984). The PhAp2A gene contains nine introns;
the positions of introns I to VI are conserved compared with those of
the introns of the Arabidopsis Ap2 gene.
To isolate PhAp2B and PhAp2C, we constructed a new genomic library from line W138 plants in ␭Gem11 (Promega). The library was
screened with the cPhAP2A-1 cDNA insert, and washes were at intermediate stringency (see DNA Gel Blot Analysis above). Twenty-nine
clones were isolated in the primary screen, 15 of which were analyzed
further. They could be divided based on their restriction patterns into
three groups that corresponded to the three gene copies observed in
DNA gel blots. A 1.2-kb SstI-XbaI PhAp2B fragment and a 1.1-kb
EcoRI-HindIII PhAp2C fragment were subcloned and sequenced.
Sequences were determined by the dideoxy chain termination
method (Sanger et al., 1977) using fluorescent dye terminators and
AmpliTaq FS in a cycle-sequencing protocol as described by the
manufacturer (Roche Molecular Systems Inc., Branchburg, NJ) on an
AB1373A or an ABI1377 automatic sequencer. Gaps in the sequence
were closed using a primer-based strategy.
insertion, showed that seven of 20 bl F2 plants tested carried the
PhAp2A(T2194-14) allele. Because PhAp2A(T2194-14) homozygotes
are wild type, and bl segregated as a normal recessive mutation, and
because seven of 20 bl mutants contain the insertion allele, we conclude that PhAp2A does not coincide with Bl. Moreover, bl mutants
that are homozygous for the PhAp2A(T2194-14) insertion allele exhibit a phenotype similar to that of bl mutants that carry wild-type
PhAp2A alleles.
In Situ Hybridization Studies
Petunia tissue (line W138) was fixed in 3.7% formaldehyde, 5% acetic acid, and 50% ethanol, cleared in Histo-clear (National Diagnostics, Atlanta, GA), and embedded in paraffin. Consecutive 10-␮m
tissue sections were placed on sets of dimethylsilane-treated
(Sigma) microscope slides so that comparable sections could be hybridized with different probes. The cDNA inserts of cPhAp2A-2 and
pMADS3 were amplified from the plasmid using the T3 and T7 sequencing primers. PhAp2A and pMADS3 antisense mRNA and
sense mRNA (control) probes were 35S-labeled mRNA probes synthesized from this PCR fragment by using the T7 and T3 RNA polymerase (Promega). An ⵑ300-bp fragment (nucleotides 198 to 596 of
the PhAp2B cDNA sequence shown in Figure 3) and a 300-bp fragment were amplified from genomic subclones of PhAp2B and
PhAp2C by using the T7 primer and a degenerate, chimeric T3-exon
2 PhAp2 primer (5⬘-AATAACCCTCACTAAAGCAG/TATG/ATGAGATTCCC-3⬘). 35S-labeled PhAp2B and PhAp2C antisense mRNA and
sense mRNA (control) probes were synthesized from these fragments
by using the T3 and T7 RNA polymerase. The in situ hybridization itself
was performed as described by Angerer and Angerer (1992). Hybridized slides were washed for 1 hr at room temperature in 2 ⫻ SSC and
0.01 M DTT and for 1 hr at 48⬚C in 0.1 ⫻ SSC, 0.01 M DTT, and 50%
formamide. Dehydrated slides were dipped into photographic emulsion and exposed for 6 to 10 days before development.
Chromosomal Localization of PhAp2A, PhAp2B, and PhAp2C
We detected HindIII restriction fragment length polymorphisms
(RFLPs) for the three PhAp2 copies between lines V26 and W137. To
determine the map positions of PhAp2A, PhAp2B, and PhAp2C, we
crossed line V26 to line W137, and the F1 hybrid was backcrossed to
line W137. A population of 61 individuals was used to construct a
genetic map. Linkage between 183 amplified fragment length polymorphism (AFLP) markers, obtained from 14 different primer combinations (EcoRI plus three selective nucleotides, MseI plus three
selective nucleotides), four phenotypic markers (Hf1 on chromosome
1, Fl on chromosome 2, An11 on chromosome 3, and Rt on chromosome 6), and the PhAp2 RFLPs in this backcross population, was analyzed by the JoinMap program (Stam, 1993). The AFLP linkage
groups identified in the progeny of the (V26 ⫻ W137) ⫻ W137 test
cross could be anchored to their specific chromosomes by linkage to
the phenotypic markers and by comparison with the locations of 40
AFLP markers that had been located previously in a different set of
crosses (Gerats et al., 1995).
The linkage between PhAp2A and the Bl locus, both on chromosome 4, was investigated further using a mutant PhAp2A allele as a
segregating marker. Plant T2194-14 is heterozygous for a transposon insertion in intron VIII (see Transposon Insertion Mutagenesis of
PhAp2A). T2194-14 was crossed to line R51 (bl/bl), and F1 hybrids
that contained the PhAp2A insertion allele were selfed. All of the F1
plants were wild type. Of 96 F2 plants, 26 exhibited a bl phenotype.
PCR analysis, using gene-specific primers flanking the transposon
Transposon Insertion Mutagenesis
The screening for dTph1 insertion mutants was performed as described
by Koes et al. (1995). The primers used to screen for dTph1 insertions in
PhAp2A were as follows: p002, 5⬘-GTGCCGTGTCAAATC-3⬘; p003, 5⬘CCGGAATTCACAATATCGTGGAGTTACCTT-3⬘; p004, 5⬘-CGGGATCCCTGATCTGCCCCATTGTCTGTA-3⬘; and p005, 5⬘-CGGGATCCCTTCAAAGAGACTGGAGAATGT-3⬘. The presence of a dTph1 element in
one or both alleles of individual plants was determined by PCR analysis
using a set of two gene-specific primers. p001 (5⬘-GGTAAACGAGTCGGATCTTTTTCGAC-3⬘) and p010 (5⬘-CGGTACCAATGTGGGATCTAAATGATTC-3⬘) were used in combination with p002 to analyze the
progeny of the primary V2025-6 and V2116-10 mutants, respectively.
The PCRs were performed in 50 ␮L of 1 ⫻ PCR buffer (Perkin-Elmer)
supplied with MgCl2 (1.5 mM) and 1 unit of Taq polymerase (PerkinElmer) and consisted of 35 cycles of 30 sec denaturing at 94⬚C, 30 sec
annealing at 55⬚C, and 1.5 min polymerization at 72⬚C. DNA fragments
flanking the dTph1 elements in PhAp2A were amplified by PCR, cloned
in pGEM-T, and sequenced to determine their exact insertion positions.
Reverse Transcription–PCR
Total RNA from flower buds was treated with DNase, extracted with
phenol/chloroform, and precipitated, and 1 ␮g was reverse tran-
Petunia Apetala2 Genes
scribed from an oligo-dT primer by using the Superscript preamplification system for first-strand synthesis (Gibco BRL). The first-strand
cDNA was used as a template for PCR amplification with either
p001/p002 (V2025-6) or p010/p002 (V2116-10).
Overexpression of PhAp2A in Arabidopsis thaliana
Primers naste22 (5⬘-GAGATCTCAATGTGGGATCTAAATGATTC-3⬘)
and naste23 (5⬘-CGAATTCGGTCATGAGGGTCTCATAAGAGA-3⬘)
were used to amplify the PhAp2A coding sequences. The fragment
obtained was cloned in pGEM-T (Promega), and the sequence was
verified. An ⵑ1600-bp PhAp2A fragment was cut from this plasmid
by using the BglII site in primer naste22 and the HincII site from the
polylinker of pGEM-T. The binary vector pBIN m-gfp5-ER (kindly provided by James Haseloff, Cambridge, UK) was digested with SacI,
blunted, digested with BamHI, and ligated to the ⵑ1600-bp BglIIHincII PhAp2A fragment to generate pBIN19-35S-PhAp2A.
The resulting overexpression vector pBIN19-35S-PhAp2A was introduced into Agrobacterium tumefaciens C58C1(Rif) (pMP90[Gent])
by triparental mating. The Arabidopsis ap2-1 mutant (kindly provided
by Maarten Koornneef, LUW, Wageningen) was transformed as described by Clarke et al. (1992). Kanamycin-resistant calli and shoots
were selected at 23⬚C, and shoots and selfed progeny of primary
transformants were grown at 26⬚C under long-day conditions (16 hr
light and 8 hr dark).
ACKNOWLEDGMENTS
We thank Bart den Boer for starting the petunia work on Ap2 by isolating cPhAp2A-1, Wilson Ardiles and Raimundo Villaroel for
sequencing, Gerco Angenent for the kind gift of the pMADS3 plasmid, Ronald Koes and Erik Souer for making their 3D screening
libraries and the inflorescence cDNA library available, the people of
the microscopy group of Gilbert Engler for advice concerning in situ
hybridizations, and Mario Pezzotti for the carpel-specific cDNA library. T.M. and N.V.d.S. were supported by the Instituut voor
Wetenschap en Technologie.
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Petunia Ap2-like Genes and Their Role in Flower and Seed Development
Tamara Maes, Nancy Van de Steene, Jan Zethof, Mansour Karimi, Mariëlla D'Hauw, Gwenny Mares,
Marc Van Montagu and Tom Gerats
Plant Cell 2001;13;229-244
DOI 10.1105/tpc.13.2.229
This information is current as of August 3, 2017
References
This article cites 44 articles, 27 of which can be accessed free at:
/content/13/2/229.full.html#ref-list-1
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