Plant Molecular Biology 49: 567–577, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

567

Apple has two orthologues of FLORICAULA/LEAFY involved in flowering
Masato Wada1,∗ , Qiu-fen Cao2 , Nobuhiro Kotoda1, Jun-ichi Soejima1 and Tetsuo Masuda1
1 Apple Research Center, National Institute of Fruit Tree Science, Shimokuriyagawa, Morioka, Iwate 020-0123

Japan; (∗ author for correspondence; e-mail: mwada@affrc.go.jp); 2 Faculty of Agriculture, Iwate University,
Morioka, Iwate 020-8550, Japan.
Received 20 March 2001; accepted in revised form 28 November 2001

Key words: AFL1, AFL2, apple, floral meristem identity gene, FLORICAULA/LEAFY

Abstract
Two orthologues of FLORICAULA/LEAFY, AFL1 and AFL2 (apple FLO/LFY), were isolated from the floral budsof apple trees. Their expression was detected in various tissues and during differentiation of the floral buds.
Furthermore, the flowering effectiveness of each gene was assessed with transgenic Arabidopsis. Both AFL1 andAFL2 showed high homology to each other (90%) and a high degree of similarity to PTLF and PEAFLO (70%),
which are homologues of FLO/LFY from poplar and pea, respectively. RNA blot analysis showed that AFL1 was
expressed only in the floral bud during the transition from vegetative to reproductive growth, whereas AFL2 wasexpressed in vegetative shoot apex, floral buds, floral organs and root. Genomic Southern analysis showed thatapple had other homologues in addition to AFL1 and AFL2. The transgenic Arabidopsis with over-expressed AFL2
showed accelerated flowering and gave rise to several solitary flowers from rosette axils directly. AFL1 had similareffects, but the phenotypes of the transgenic Arabidopsis with AFL1 were weaker than those with AFL2. These
results suggest that both genes are involved in flower differentiation in apple.

Introduction
Increasing knowledge of the flowering mechanism in
Arabidopsis has opened the floodgate for the identification of several key genes that control floral development (Okamuro et al., 1993; Levy and Dean 1998;
Pidkowich et al., 1999). One of such genes, LEAFY(LFY), has been reported to be necessary for the transition from vegetative to reproductive development. It
has been defined as a floral meristem identity gene like
FLORICAULA (FLO) from Antirrhinum majus (Coenet al., 1990). Mutations in these genes result in theconversion of flowers into indeterminate secondary
shoots (Weigel et al., 1992). The over-expression of
LFY under the 35S promoter in Arabidopsis caused
early flowering and converted all lateral shoots info
solitary flowers. In heterologous plants such as aspen, LFY could have effects similar to acceleration
The nucleotide/amino acid sequence data reported will appear in the
EMBL, GenBank and DDBJ databases under the accession numbers
AB056158 (AFL1) and AB056159 (AFL2).

of flowering and induction of ectopic flowers (Weigel
and Nilsson 1995). These reports strongly suggest thatthe ability to control the expression of LFY, or of orthologues from other plants, could make it possible toinduce various plants to blossom whenever and however possible. Consequently, these studies should beconsidered a contribution to agriculture and forestry.
Apple is one of the most commercially valuable
fruit trees, with production second only to grape in thewhole world. Apple has an extended juvenile phase,during which vegetative growth is maintained. This
characteristic is recognized as a disadvantage in breeding and in stable annual production. Thus, it will
be necessary to understand the genetic mechanism
underlying transition from vegetative to reproductive
phase. Only a few studies have reported on the mechanism underlying the development of apple flowers.
Recently, MADS-box genes have been correlated toapple fruit set, and they have been cloned and characterized (Sung et al., 1999, 2000). These apple MADS
genes seem to be involved in floral organ and fruit

568
development rather than in the transition from thevegetative to the reproductive meristem. We have undertaken this study in apple to identify the homologuesof FLO/LFY involved in floral development. In thispaper, we describe the isolation and characterizationof LFY homologues from apple floral apex during
the vegetative-to-reproductive phase transition. In addition, the isolated genes were determined to havefunctional activity in Arabidopsis flowering.

Materials and methods
Plant materials
The apple (Malus × domestica Borkh.) cv. Jonathan,grown in our orchard at the Apple Research Center of
the National Institute of Fruit Tree Science (Morioka,
Japan), was used in this study. Developing floral shootapexes 0.5–1.0 cm from the tip were cut and collectedfrom early June to late August at two-week intervals.
The cut apexes, including meristems, were frozen inliquid nitrogen immediately and stored at −80 ◦ C.
RNA extraction and cDNA cloning
Total RNA was extracted from the cut apexes using
a method based on cetyltrimethylammonium bromide(CTAB), and the cDNA was constructed by oligo(dT)primer with reverse transcriptase. Partial cDNA fragments homologous to LFY were amplified by RT-PCR(Kotoda et al., 2000). Full-length cDNA was obtainedby using 5 and 3 ’ RACE methods. The first amplified
cDNA of apple was 440 bp between 6S (sense primer
5 -CAGAGGGAGCACCCGTTCATTGTGAC-3) and
10A (antisense primer 5 -GACGC/AAGCTTT/GGTT/
GGGA/GACATACCA-3 ) designed from Arabidopsis
thaliana LFY (Weigel et al., 1992) and Antirrhinum
majus FLO cDNA sequences (Coen et al., 1990). Thisfragment was named AFL400. Specific primers forboth 5 and 3 RACE methods were designed from this
AFL400. The cassette-ligated cDNAs from the shootapexes were prepared using the LA-PCR cloning kit(Takara, Tokyo). The amplification of the 3 end of thecDNAs was carried out between cassette primer 1 or 2
and 6S or 3SP2 (5 -TCCAGAACATTGCCAAGGAG3 ) (see Figure 1). A 600 bp DNA fragment amplified C2-3SP2 was cloned at the EcoRV site using pBlueScript SKII+ (Stratagene, La Jolla, CA).
Ten clones were completely sequenced in both directions and showed a division into two kinds ofcDNA homologues. The amplification of the 5 end

was carried out between cassette primer 1 or 2
and 10A, 5SP2 (5 -CAATGACCACAAGAGGCTTG3 ), 5SP3 (complement sequence of 3SP2) or
5SP4 (complement sequence of 6S) (see Figure 1).
An almost 750 bp band amplified C2-5SP4 wascloned and sequenced in the same way as in
the 3 RACE method. The resultant sequences
also showed two kinds of cDNA. The full-length
cDNAs were amplified by the shared sense primer
AFLF, 5 -GTGGAAAATATGGATCCAGATGCC-3,and either one of two antisense primers AFL1R, 5 TTCATTCAGTCTGCCCTAGCC-3 , or AFL2R, 5 TCAAACTCTCTCTGCAGAACTGGC-3 (see Figure 1) from the cDNA library of floral shoot apexes.
Fragments of about 1.4 kbp cDNA were obtained andsubcloned into pBlueScript SKII+ (EcoRV site) andsequenced completely in both directions. Two kinds ofcDNA were named AFL1 and AFL2, respectively. The
sequencing was performed by dideoxy methods using a Hitachi SQ5500S automated sequencer (Hitachi,
Tokyo, Japan) and analyzed by the Genetyx computer
analysis program (Software Development Co., Tokyo,
Japan).
Southern blot and expression analyses
Genomic DNA was extracted by the method of Murray and colleagues (Murray and Thompson, 1980;
Bousquet et al., 1990). The Southern analysis was
carried out using a 440 bp DIG-labeled AFL400 PCR
fragment amplified between 6S and 10A primers.
Genomic DNA was digested by restriction enzyme
EcoRI, BamHI or HindIII, respectively, and blotted onto a nylon membrane (Hybond N+, Amersham). Hybridization was performed in DIG EASYHYB solution (Roche Biochemicals, Tokyo, Japan)
overnight at 50 ◦ C. The filter was rinsed twice in
2× SSC, 0.1% SDS at room temperature, and washedin 0.5× SSC, 0.1% SDS for 15 min, twice, at 65 ◦ C.
Chemiluminescence was detected according to the
manufacturer’s instructions, and the resulting images
were analyzed with a LAS-1000 image analyzer (FujiFilm, Tokyo, Japan). RNA extraction from each tissue and organ was carried out by the same method
as described above. First-strand cDNA synthesis was
performed with 1.0 µg of total RNA using the RTPCR HIGH kit (Toyobo, Tokyo, Japan) as directedby the manufacturer. The common sense primer was
3SP2 and the each antisense primer was AFL1R or
AFL2R, the pair of 3SP2-AFL1R primers amplified a
532 bp fragment and the 3SP2-AFL2R primers ampli-

569

Figure 1. Alignment of nucleotide sequences of AFL1 and AFL2 cDNA. The upper line shows the nucleotide sequence of AFL1 cDNA, andthe lower line shows that of AFL2 cDNA. Each primer used for RT-PCR or the RACE method is shown under each arrow. The 5SP4 and 6Sprimers, or 5SP3 and 3SP2, have the same position, but opposite directions; the arrowheads indicate their directions (5SP4 and 5SP3 wereantisense, 6S and 3SP2 were sense primers). The box marked M shows the possible start point of translation, and the box marked STOP shows
the possible end of translation of AFL1 and AFL2. The ∗ indicates an identical nucleotide between AFL1 and AFL2 cDNA.

fied a 468 bp fragment. The PCR was performed with
AmpliTaq Gold (PE Biosystems, Japan) and was first
incubated at 95 ◦ C for 10 min, then incubated by astepped program (94 ◦ C, 60 s; 62 ◦ C, 60 s; 72 ◦ C,
120 s) for 40 cycles. A 5 µl portion of the reaction mixture was separated on a 1.5% agarose gel andstained by ethidium bromide.

Plant transformation
The AFL1 and AFL2 cDNA were amplified between
AFLF and AFL1R or AFL2R, respectively (see Figure 1). Then each amplified cDNA was blunted and
XbaI linkers were ligated both ends, cloned into a
pIG121Hm binary vector at the XbaI site. The vec-

570
tor contained the 35S promoter and terminator, as
well as the neomycine phosphotransferase and hygromycin gene as selective markers (Ohta et al.,1990). Then sense or anti-sense directional insertion
clones were selected. Sense directional clones werenamed AFL1S and AFL2S; on the contrary, AFL2
inserted at antisense direction was named AFL2AS.
From our EST project on apple flower development,
the C045 clone, which is 1.6 kb in length with ahomeobox domain, has high homology with the carrot gene (Kawahara et al., 1995). The C045 wasinserted into pIG121Hm vector, as with AFL genes.
An Agrobacterium tumifaciens GV3101 strain (Van
Larebeke et al., 1974) was used for the transformation of the A. thaliana Columbia ecotype. A. thaliana
plants greenhouse-grown for two weeks were transformed by the in planta method (Clough and Bent,
1998). Resultant seeds were planted on a 1/2MS culture medium containing kanamycin (20 µg/ml) andhygromycin (20 µg/ml) as selective antibiotics. The
antibiotic-resistant plants were maintained as transgenic lines, and their flower phenotypes were observed
under long-day conditions (16 h light/8 h dark).
Northern analysis of transgenic plants
Total RNA was isolated from rosette leaves of transgenic Arabidopsis grown for 3–4 weeks under the
long-day conditions. The isolation method was thesame as described above. An equal amount (1 µg) of
total RNA from each plant line was electrophoresedwith 0.8% agarose gel and blotted onto a nylon membrane (Hybond N+, Amersham). Hybridization was
performed with a DIG-labeled RNA probe using themethod of Kotoda et al. (2000). The resultant images
were detected and analyzed with the LAS-1000 image
analyzer.

Results
Cloning and sequence analyses of AFL1 and AFL2
A comparison of Arabidopsis LFY (Weigel et al.,1992), snapdragon FLO (Coen et al., 1990) and otherhomologues (poplar, PTLF; pea, PEAFLO; petunia,
ALF; and tomato, TOFL) (Hofer et al., 1997; Soueret al., 1998; Molinero-Rosales et al., 1999; Rottmann
et al., 2000) showed the presence of several conserved
regions. Two of these regions, QREHPFIV (6S) and
WYVPTKLR (10A), were used to design degenerateoligonucleotide primers for RT-PCR (Kotoda et al.,2000). Using these primers with cDNA prepared froma floral shoot apex of apple, we obtained a PCRamplified fragment (440 bp) containing both primers
and other conserved sequences. This 440 bp fragment
was named AFL400 (apple FLORICAULA LEAFY homologue). To obtain a full-length AFL cDNA, several
primers for 5 /3 RACE were designed (Figure 1). Twokinds of AFL cDNA were obtained by this RACE
method. The 5 common primer (AFLF) was designedin the 5 non-coding region, and the 3 primer (AFL1R
or AFL2R) was designed in the 3 non-coding region;
each region had a specific sequence. The two kindsof amplified 1.4 kb fragments were sequenced andnamed AFL1 and AFL2. The coding regions of AFL1
and AFL2 showed 90% homology to each other, but
the 3 non-coding regions had less than 60% homology. AFL1 had a 9 bp 5 non-coding region and a
176 bp 3 non-coding region upstream of the poly(A)tail, then a 1230 bp coding region encoding 410 aminoacids potentially. AFL2 had a 23 bp 5 non-coding region and a 181 bp 3 non-coding region, and AFL2
cDNA also had a 1230 bp region encoding 410 aminoacids. Their deduced amino acid sequences indicatedthe same length, but two gaps existed (Figure 2).
The deduced amino acid sequence of AFL1 or AFL2
cDNA has, respectively, 71.5% or 72.9% homology
with PTLF, 71.1% or 72.3% with PEAFLO, 71.9% or
72.9% with ALF, 69.7% or 70.1% with TOFL, 68.8%
or 69.3% with FLORICAULA, 62.0% or 62.8% withLEAFY, 63.1% or 64.4% with ELF1 (Southerton et al.,1998), 53.2% or 55.6% with RFL (Kyouzuka et al.,1998), and 46.9% or 46.5% with NEEDLY (Mouradov
et al., 1998). LFY, RFL, ALF and TOFL each had aproline-rich region (roughly the first 40 amino acids),
but only AFL2 had an alanine-rich region instead, andboth AFL1 and AFL2 lacked a proline-rich region.
Genomic analyses and expression patterns of AFL1
and AFL2
Genomic DNA extracted from apple was digested withrarely cutting enzymes (HindIII, BamHI, EcoRI), andprobed at high stringency with a DIG-labeled AFL400
amplified PCR method. Both AFL1 and AFL2 cDNA
sequences corresponding to AFL400 had no sites ofthese restriction enzymes. However, only the genomic
sequence (including a roughly 900 bp intron, we estimated the length by genome PCR) corresponding
to AFL2 had a site of EcoRI. Figure 3 shows that
there were four bands with HindIII digestion, threebands with BamHI and six with EcoRI. So, these re-571

Figure 2. Alignment of deduced apple amino acid sequences corresponding to the AFL1 and AFL2 cDNA with LFY (A. thaliana), FLO(snap dragon) and homologues from poplar (PTLF), pea (PEAFLO), petunia (ALF), tomato (TOFL), Eucalyptus (ELF1), rice (RFL) and pine
(NEEDLY). Identical residues of the alignments are boxed (white letters on black background). Arrows indicate the positions of primers used
to obtain the AFL400 fragment. Open arrowheads show the gaps. One is at 217N of AFL2, the other is at 402G of AFL1.

sults indicated that there was at least one homologous
gene in addition to AFL1 and AFL2. We cannot detect bands of northern hybridization with an AFL1- or
AFL2-specific probe. Therefore, PCR primers specificto each of AFL1 and AFL2 were used to detect theexpression patterns in several tissues. One µg of total
RNA was inoculated from each flower organ (sepal,
petal, stamen and carpel), floral shoot apex (F.S.A.),
vegetative shoot apex (V.S.A.), root, stem and leaf.
The RT-PCR of AFL1-specific primers resulted in amplification of a single band at only F.S.A.; on the other
hand, the RT-PCR of AFL2-specific primers resulted
in a single band in most floral organs, as well as in
F.S.A., V.S.A. and root (Figure 4). Increasing the PCR

cycle did not change these expression patterns. Bothamplified DNA fragments were the expected lengths.
Figure 5 shows the expression time course of AFL1
and AFL2 on developing floral apexes by RT-PCR as
in Figure 4. The numbers on the top picture indicate sampling date (month/day). Jonathan is one of
the apple cultivars we used in this experiment. This
cultivar’s floral apexes vegetatively developed until
around the end of June, and typical changes to domed
apical meristems were observed microscopically fromaround the end of August. The transition period, i.e.,
the period in which no phenotypic changes were observed compared with vegetative growth, appeared to
be about a month. AFL1 began to express clearly at572

Figure 3. DNA blot analysis of apple genome. Genomic DNA(10 µg) from apples were digested with EcoRI (E), BamHI (B) or
HindIII (H), and hybridized with a probe prepared from a PCR-DIG
AFL400 (6S-10A primers). The left numbers indicate molecular size(kb).

that interval, but AFL2 expression was found on all
searched days. These results indicated that AFL1 expression was restricted in a highly regulated mannerand the expression occurred just before the phenotypic change of the floral apexes. On the other hand,
AFL2 expression was constitutive and showed no signs
coupled with the shift.

Figure 4. RT-PCR analysis of AFL1 or AFL2 RNA in different tissues. The top picture shows PCR amplification with
AFL1-specific primers. The middle one shows PCR amplification
with AFL2-specific primers. The bottom one shows agarose gel(1.5%) electrophoresis of RNA samples stained by ethidium bromide from each apple tissue. F.S.V.: floral shoot apex. V.S.A.:
vegetative shoot apex.

Ectopic expression in Arabidopsis
Although the functions of AFL1 and AFL2 should bedetermined by the development of transgenic apple
trees, this approach would be actually difficult because
to develop transgenic apple trees with flowers would
take several years. We therefore used Arabidopsis(Columbia) to generate transgenic plants. Both AFL1
and AFL2 cDNAs were placed under the control ofa CaMV 35S promoter and introduced to Arabidopsis
by the in planta method (Clough et al., 1998). Twentyfive independent transformants by AFL1 were selectedby antibiotics medium, and three lines (1S-1, 1S-2 and
1S-23) showed clear phenotypes of early floweringand solitary flowers arising from rosette axils. In thecase of AFL2 transformation, 19 independent transformants were selected, and four lines (2S-2, 2S-6,
2S-9 and 2S-18) showed more severe phenotypes than
did the AFL1 transformants (Figure 6). The typicalphenotypes of AFL2 transformants were several solitary flowers arising from rosette axils (Figure 6b, c).

Figure 5. RT-PCR analysis of AFL1 and AFL2 RNA at different
times. The number on the top picture shows the month/day wheneach floral shoot apex was collected, from 5 June to 10 October.
The picture order is the same as in Figure 4 (top, AFL1; middle,
AFL2; bottom, total RNA). The top bar shows the transition patternfrom vegetative to reproductive phase. The left white area shows
vegetative state, and the right shaded area shows reproductive state.
The middle area with the check pattern shows transition state.

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Table 1. Effects on leaf and solitary flower numbers on 35S::AFL1 or AFL2 transgenic plants
under long-day conditions (16 h L/8 h D).
Plant

Rosette leaves

Range ofrosette leaves

Solitary flowers

Range ofsolitary flowers

n

Exp.1
WT
AFL1S1
1S2
2S2
2S9

9.60 ± 1.12
8.50 ± 0.75
8.78 ± 0.89
5.84 ± 0.80
7.00 ± 0.92

8–12
8–10
7–10
4–7
6–9

0
0.37 ± 0.74
0.28 ± 0.72
4.84 ± 0.68
5.50 ± 1.06

0
0–2
0–2
4–6
4–7

15
8
14
13
8

Exp.2
WT
AFL1S2
2S2
2AS2
C045

9.86 ± 1.12
9.36 ± 1.09
6.00 ± 0.77
9.60 ± 0.96
9.56 ± 0.85

9–12
7–12
5–7
8–11
8–11

0
0.81 ± 1.46
4.45 ± 0.68
0
0

0
0–5
3–5
0
0

14
22
10
10
30

WT; wild type, AFL1S; 35S::AFL1 transgenic plants lines, AFL2S; 35S::AFL2 transgenic
plants lines, AFL2AS2; 35S::anti-sense directional AFL2 transgenic plants line, C045; transgenic plants line with homeotic gene from apple under 35S promoter, n, plant number.
All values are given as the mean and standard deviation. Each experiment was performed
independently.

In the case of the 35S::LFY transformed Arabidopsis(Weigel and Nilsson, 1995), the primary shoot immediately terminated with the formation of a solitary
flower. But, the primary shoot of the AFL2 transformed Arabidopsis never terminated and had lateral
flowers from axils of cauline leaf instead of secondary
shoots (Figure 6c). The solitary flowers often showedabnormal flower phenotypes (Figure 6f–h), and were
often observed with five petals and five sepals (Figure 6g). These flowers had fewer stamens instead of
more sepals and petals. Other aberrant flowers wereobserved to have naked embryos and open carpels(Figure 6f), and an apetala1-like phenotype that subtended secondary flowers in flower organs (Figure 6g).
But most of the aberrant flowers were self-pollinatedand normally grown siliques, and the resultant seeds
also had the ability to germinate. These transgenic
plant lines were used to determine the expression of
induced genes. Northern hybridization showed eachspecific band of AFL1 and AFL2 (data not shown).
The expression band of AFL1 was slightly bigger thanthat of AFL2. For each gene, the level of expression
had no relationship with the severity of its phenotypes. AFL1- or AFL2-transformed Arabidopsis wereself-pollinated, and the phenotypes of offspring wereanalyzed. The early-flowering and solitary flowers
from rosette axils were inherited in the next generation and co-segregated with the kanamycin- andhygromycin-resistant genes. The phenotypes of thesetransgenic lines grown under long-day conditions aredescribed in Table 1. Both AFL1 and AFL2 lines
showed early flowering compared to the wild typeand control (AFL2AS or C045) because of their lower
numbers of rosette leaves. Further, the AFL2 line had
fewer rosette leaves than the AFL1 line. Under shortday conditions (8 h light/16 h dark), these tendencies
did not change (data not shown). The AFL2 line had
about five solitary flowers per plant, but the AFL1line often had no solitary flowers. The numbers ofsolitary flowers were affected by day length: shortday conditions resulted in far fewer solitary flowers
in both the AFL2 and AFL1 lines. On the other hand,as the control of AFL1 and AFL2 transgenic plants,
AFL24S (AFL2 inserted anti-sense direction) into thesame vector was introduced to Arabidopsis. A total of
18 transgenic plants were obtained and all transgeniclines showed no early flowering and excess solitary
flowers. Another control C045 obtained 16 transgeniclines which showed no differences with wild type.

Discussion
We obtained cDNA clones, AFL1 and AFL2, as apple
homologues of FLORICAULA/LEAFY by the RACE
method. These clones were amplified in a cDNA

574
library of developing floral buds. RT-PCR analysis
showed that AFL1 was expressed at the floral bud only,
and that AFL2 was expressed at the floral bud andat floral organs, vegetative apex and root (Figure 4).
The genomic region hybridized by AFL400 had one
intron-corresponding site, which existed in the LFY
genome (Weigel et al., 1992). Genomic blot analysis showed more than 3 bands with each restriction
enzyme (Figure 3). These findings suggested the apple genome has at least three genes homologous to
AFL400. We reported in a previous paper (Kotoda
et al., 2000) that northern hybridization analysis by an
AFL400 probe showed highly hybridized nearly 1.4 kb
bands at sepals and leaves. These 1.4 kb bands did
not express AFL1 or AFL2, because RT-PCR analysis
showed no amplification of leaf tissues by each specific primer (Figure 4). This indicated that at least one
other AFL family, in addition to AFL1 and AFL2, exists in the apple genome. In addition, there were highlevels of homologues expressed in leaves and sepals,
yet we had no PCR amplification bands on leaf cDNA
by 6S and 10A primers. This suggested that other homologues had less similarity to each other than was
found between AFL1 and AFL2. In most plants forwhich LFY homologues were determined, these LFYhomologous genes had a single copy in each genome(Arabidopsis, snapdragon, pea, tomato, petunia andpoplar; Weigel et al., 1992; Hofer et al., 1997; Soueret al., 1998; Molinero-Rosales et al., 1999; Rottman
et al., 2000). On the other hand, Eucalyptus has threehomologous genes in the genome, but two of themwere found not to be expressed and had stop codons
in their coding regions. Hence, only one gene seems
to function actually in Eucalyptus (Southerton et al.,1998). In gymnosperms, Pinus radiata has two homologous genes, NEEDLY and PRFLL, in the genome(Mouradov et al., 1998; Mellerowicz et al., 1998).
NEEDLY was expressed during vegetative development, while PRFLL was expressed in male cones andnot in the females during reproductive development.
A search for LFY homologues in basal angiosperms
and Gnetales showed that the LFY family is not always
single-copy in diploids (Frohlich and Meyerowitz,
1997). Frohlich further proposed that there is a possibility that angiosperms had a decreased copy numberof LFY genes from ancestral plants (Frohlich andParker, 2000). This was consistent with our findingsthat apple has more than three LFY homologues, andEucalyptus had a couple of pseudogenes of possible
LFY homologues. These plants may have retained theancestral character of LFY genes.

It is believed that apple originated in western Asiaand that cultivated apples are complex polyploids(Korban and Chen, 1992). Compared with reported
plant species, apple appeared to have more LFY homologues in their genomes because of their complex
polyploid origin. The origin of this complexity has notbeen fully understood. Figure 1 shows that AFL1 andAFL2 had high homology (90%) in their coding regions, but they were expressed in distinct tissues andtimes in floral and vegetative development (Figures 4
and 5). To determine the respective functions of AFL1
and AFL2, transgenic Arabidopsis by 35S::AFL1 or
35S::AFL2 were constructed (Figure 6). Ectopic expression of these genes in Arabidopsis caused early
flowering and the growth of solitary flowers fromrosette axils as with 35S::LFY transgenic Arabidopsis
under long-day conditions (Table 1) (Weigel and Nillson, 1995; Ratcliffe et al., 1999). 35S::AFL2 transgenic Arabidopsis had solitary flowers from cauline
leaf axils instead of secondary shoots (Figure 6c) andmore solitary flowers than AFL1 transgenic lines (Table 1). All 35S::AFL2 transgenic Arabidopsis had solitary flowers from axils of cauline leaves on primaryshoot (lf in Figure 6c). But, 35S::AFL1 transgenic
plants had fewer solitary flowers from cauline leaves.
Ten transgenic lines by 35S::AFL1 were observed for
50–60 days under long-day conditions, most lines had
secondary shoots from cauline leaves. But, a solitary flower was observed on one axil per couple oftransgenic plants (Figure 6i). After longer culture, secondary shoot-like structure arose from the same axil(s in Figure 6i). Such shoot like structure had neverbeen observed with 35S::AFL2 transgenic plants. Thissuggested that AFL1 also had the transition abilityof vegetative to reproductive growth like AFL2 qualitatively, but the effects were much weaker than for
AFL2. In short-day conditions (8 h light/14 h dark),both transgenic plants showed earlier flowering than
the wild type, but there were 20–26 rosette leaves forbolting under the AFL1 lines, 13–18 rosette leaves
under AFL2, and 30–35 rosette leaves under the nontransgenic ones (wild type). The numbers of solitary flowers arising from rosette axils decreased withboth transgenic lines (data not shown). So the earlyflowering and solitary flowers from rosette axils wereaffected by the photoperiod condition. It was guessedthat AFL1 and AFL2 worked as orthologues of LFY in
Arabidopsis plants (Weigel and Nilsson, 1995). AFL2transgenic lines showed various aberrant flowers (Figure 6f–h), in which solitary flowers had severe phenotypes. Unclosed carpels, higher numbers of petals and575

Figure 6. Transgenic Arabidopsis plants transformed with 35S::AFL1 or 35S::AFL2 grown under a 16 h light/8 h dark photoperiod. a. A
phenotype of wild-type Arabidopsis (ecotype: Columbia). b. 35S::AFL2 transformed plant with several solitary flowers (s) arising from rosette
axils directly. c. 35S::AFL2 transformed plant, same as b. The primary shoot grows in an indeterminate manner normally, but the lateral flowers(lf) with axils of cauline leaves were observed. d. 35S::AFL1 transformed plant. Compared with 35S::AFL2 plants, the arising solitary flowers
were fewer or non-existent, and the flowers tended to arise from lower axils of rosette leaves. e. A normal flower of wild type. f–h. Aberrant
solitary flowers observed on 35S::AFL2 plants. f. The naked embryos (white-head arrow) in unclosed carpels. g. Flower with five sepals andfive petals. h. Flower subtends secondary flowers in their axils (white-head arrow) like the apetala1 mutant of Arabidopsis (Alejandra et al.,1992). i. A solitary flower(s) and shoot-like structure (sl) from axil of a cauline leaf (cl) on primary shoot of 35S::AFL1 transgenic plants. j,
k. Fasciated and bending primary shoot apexes (sa) of 35S::AFL2 transgenic plants.

576
sepals, and an apetala1 (Alejandra et al., 1992) likephenotype suggested that ectopic expression of AFL2
had several effects on internal genes controlling thedevelopment of the flower organs. A few reports havemade transgenic Arabidopsis by LFY homologues
from other plants (Mouradov et al., 1998; Southerton
et al., 1998; Kyouzuka et al., 1998). NEEDLY from radiata pine and ELF1 from Eucalyptus transgenic lines
showed early flowering and produced solitary flowers
from rosette axils and terminal flowers from primaryshoots. Those authors mentioned that these phenomena were dependent on photoperiod, as we found the
AFL transgenic lines to be. However, AFL transgeniclines never terminated a primary shoot with a flower.
Both AFL lines showed indeterminate floral development on their shoots. The 2S-2, AFL2 transgenic line,
which had severe phenotypes (Table 1), was sometimes observed to have primary shoots twisting backon themselves to form a fasciated loop (Figure 6j,k) before recovering and continuing their indeterminate growth. A similar phenomenon was reported on
double-transgenic Arabidopsis by LFY and Terminal
Flower 1 (TFL1) (Bradley et al., 1997; Ohshima et al.,
1997; Ratcliffe et al., 1999). The two genes have
opposite functions in floral development, so their expression under the 35S promoter appeared to resultin such aberrant fasciations of the primary shoot because of a lack of harmony between the transition
to florescent meristem (LFY) and the maintenance ofinflorescent meristem (TFL1). Thus, it was demonstrated that AFL2 had a function similar to LFY in
suppressing TFL1. These results have given a clear indication that apple LFY homologues AFL1 and AFL2
worked as LFY orthologues.
AFL1 and AFL2 had high homology to each other,
but their transgenic lines of Arabidopsis showed different phenomena (Figures 1 and 6, Table 1). AFL1
seemed to have weaker effects in Arabidopsis. The
alignments of AFL1 and AFL2 showed there were no
partial differences greater than four serial amino acids(Figure 1). This suggested that there is a possibility that such differences in phenomena between theirtransgenic lines were caused by one amino acid replacement, which might cause dissociation to specificproteins or DNA sequences with the product of AFL1
or AFL2. So, investigation into the effects of suchdifference on these phenomena could clarify the functional domain of LFY orthologues. Lately, MADS-box
genes from Arabidopsis, SEPALLATA1, 2, 3 (SEP1, 2,3), were demonstrated to function as a class of organ
identity genes that are required for the developmentof petals, stamens and carpels. Interestingly, no single mutant ever showed abnormal flowers or abnormal
development. But the triple mutant alone showed all
flower organs changed to sepaloid ones (Pelaz et al.,2000). Apple AFL1 and AFL2 also could work like
SEP genes in floral development, for which both AFL
genes may be needed. Unlike the case with SEP genes,
the temporal expressions of AFL1 and AFL2 differed(Figure 5). AFL2 expressed constantly at all stages,
but AFL1 seemed to express in accordance with floral
development. It is possible that the AFL1 plays a keyrole in floral development. To test this hypothesis, itwill be necessary to determine in what cells the two
AFL genes are expressed in apple flower bud, or what
effects are observed, in transgenic apple flowers by the
AFL genes under the control of the 35S promoter. The
results of such an investigation will clarify the role ofeach gene in apple floral developments.

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