Plant Science 168 (2005) 95–104
www.elsevier.com/locate/plantsci

MdTFL1, a TFL1-like gene of apple, retards the transition from thevegetative to reproductive phase in transgenic Arabidopsis
Nobuhiro Kotoda*, Masato Wada
Department of Apple Research, National Institute of Fruit Tree Science, Incorporated Administrative Agency,
National Agriculture and Bio-oriented Research Organization (NARO), Shimo-kuriyagawa, Morioka 020-0123, Japan
Received 7 June 2004; accepted 20 July 2004
Available online 17 August 2004

Abstract
Unlike herbaceous plants, fruit trees such as the apple (Malus  domestica Borkh.) flower and set fruit only after an extended juvenile
phase lasting several years. While studying juvenility in apple trees, we cloned Malus domestica TFL1 (MdTFL1), a gene homologous to
TERMINAL FLOWER 1 (TFL1) that suppresses the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) and maintains the
inflorescence meristem in Arabidopsis. MdTFL1 mRNA was expressed preferentially in apple vegetative tissues such as apical buds, stems
and roots of seedlings, and expression peaked in early July in apical buds, about two weeks prior to floral bud differentiation. Transgenic
Arabidopsis expressing MdTFL1 flowered noticeably later than wild-type plants and exhibited a phenotype similar to that of transgenic
Arabidopsis overexpressing TFL1. These results suggest that MdTFL1 is involved in the maintenance of the vegetative phase in apple and that
it functions analogously to TFL1.
# 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: CETS; Juvenility; Malus  domestica; MdTFL1; TFL1

1. Introduction
The development of all woody plants from seed includes
a juvenile phase lasting up to 40 years in certain forest trees[1]. During the juvenile phase, flowering does not occur andcannot be induced under normal conditions [1]. Thus, thebreeding of fruit trees such as the apple (Malus  domestica
Borkh.) often takes more than 20 years including periods ofcross-pollination, seedling selection and regional trials to
produce varieties that meet consumer demands. Generally,
the juvenile period of M.  domestica lasts 7–8 years [2], buta certain Malus species used as root stock neither flowers nor
sets fruit for substantially more than 8 years. Hence, the long
juvenile phase is the primary factor that limits the efficient
breeding of fruit trees, and thus tree fruit production would
benefit from a better understanding of the mechanism ofapple flower induction and development. However, the* Corresponding author. Tel.: +81 19 641 3164; fax: +81 19 641 3819.
E-mail address: koto@affrc.go.jp (N. Kotoda).

genetic factors that control flower induction in apple trees
have yet to be investigated.
The most striking advances in our understanding of thegenetic control of flowering time come from studies in
Arabidopsis [3,4]. Three Arabidopsis genes that control
flowering time have been isolated, namely LEAFY (LFY) [5],APETALA1 (AP1) [6] and TERMINAL FLOWER 1 (TFL1)[7,8]. In transgenic Arabidopsis, over-expression of LFY or
AP1 shortens the juvenile period and causes early flowering[9,10] whereas TFL1 over-expression causes late flowering[11]. TFL1 plays a key role in the maintenance of the
inflorescence meristem by preventing the expression of LFY
and AP1 in the shoot apical meristem [11,12].
Juvenility in fruit trees remains largely undefined. As
such, there is keen interest in regulatory genes such as TFL1,
Antirrhinum CENTRORADIALIS (CEN) [13] and tomato
SELF-PRUNING (SP) [14], which maintain the vegetative
phase and promote the emergence of shoots rather thanflowers. There is also strong interest in genes LFY and AP1,
which promote flowering. CEN, TFL1 and SP are closely
related and belong to a small gene family (CETS) that0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2004.07.024

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encodes 23-kDa proteins. These three plant genes were thefirst to be assigned biological functions [15] and their aminoacid sequences exhibit similarity with a group of mammalian phosphatidylethanolamine binding proteins (PEPBs)
originally named for their ability to bind phospholipids invitro [16]. FLOWERING LOCUS T (FT) also belongs to the
CETS family and is a homologue of TFL1 [17,18]. However,FT antagonizes the TFL1 function and thereby promotes
flowering in concert with LFY [17,18].
In mammals, PEBPs are believed to be precursors of ahippocampal neurostimulatory peptide (HCNP), and PEBPs
are inhibitors of Raf-1 kinase activity [19]. The tomato
protein SP, a member of the CETS family, interacts withseveral proteins termed SIPs (SP-interacting proteins) that
include SPAK (SP-associated kinase), a NIMA-like kinase,and 14-3-3 isoforms [15]. Tomato SIPs bind to CETS
proteins such as Antirrhinum CEN, as well as to Arabidopsis
TFL1 and its functional antagonist FT, providing evidence
that SP/SIPs interactions in tomato are conserved in
distantly related plants [15].
Several types of apple genes that may be involved inflower development have been isolated and characterized.
Of the MADS-box genes from apple, MdMADS2 and
MdMADS5 have been analyzed in detail [20–22].
MdMADS2 and MdMADS5 promote flowering in transgenic
tobacco and Arabidopsis, respectively, and they function
analogously to AP1 [21,23]. Additionally, apple AFL1 andAFL2, two orthologues of LFY, are involved in flowering.
AFL1 and AFL2 share 90% homology within their coding
regions, and while these genes function similarly to LFYtheir expression patterns differ from that of LFY [24].
The genes mentioned above promote flower induction or
flower development in apple. However, progression from thejuvenile phase in perennial crops such as fruit trees also
requires that juvenility/vegetative maintenance factors becleared. The present work describes the isolation andcharacterization of a TFL1-like gene, termed MdTFL1,
which is involved in the maintenance of juvenile/vegetative
phase in apple.

2. Materials and methods
2.1. Plant materials
The apple (Malus  domestica Borkh.) cultivar‘Jonathan’ was used to isolate and characterize the MdTFL1
gene. Apple leaves and flowers were collected from theexperimental field at our research center in Morioka, Japan.
Jonathan apple seedlings were used for expression analysis.
Arabidopsis thaliana Columbia (Col) and tfl1-1 wereobtained from the Arabidopsis Biological Resource Centerat The Ohio State University and wild-type plants were
employed for Agrobacterium-mediated transformation.
Arabidopsis seeds were stratified for 3–4 days at 4 8C andthen grown on an agar plate containing 0.5  Murashige andSkoog (MS) medium (Wako Pure Chemicals Co. Ltd.,Tokyo, Japan) in growth chambers at 22 8C. Ten days aftersowing the agar plate, seedlings were transferred to soil andgrown in growth chambers under long-day (LD) conditions(16 h light/8 h dark).
2.2. Gene cloning
Full-length MdTFL1 cDNA was obtained by the 50 and 30
rapid amplified cDNA ends (RACE) method [25]. Cassetteligated cDNAs from the shoot apexes of apples wereprepared using the LA-PCR cloning kit (Takara Biomedicals, Tokyo, Japan). Primers used in this study are listed inTable 1. The first amplified apple cDNA contained 233 bp
between the 5S and 3A designed from TFL1, CEN and SP
cDNA sequences. The 30 RACE was carried out between

Table 1
Primer sequences used in PCR cloning of MdTFL1
PrimerOligonucleotidea

Primers for an internal fragment
5S sense primer
3A antisense primer

50 -ATTGTGACTGACATCCCAGGC-30
50 -CG/TT/CTGIGCA/GTTA/GAAA/GAAIAC-30

Cassette primers
C1 primer
C2 primer

50 -GTACATATTGTCGTTAGAACGCGTAATACGACTCA-30
50 -CGTTAGACGCGTAATACGACTCACTATAGGGAGA-30

50 RACE primers
R1A antisense primer
R2A antisense primer

50 -TTCGAGTGCTGAAGTGATCCCTC-30
50 -CAG CGA CAG GAA GAC CCA GGT CA-30

30 RACE primers
R1S sense primer
R2S sense primer

50 -CACCACAGATGCCACATTTGGAA-30
50 -GAGGTGGTGAGTTATGAGAT-30

MdTFL1 specific primers
MdTFL2S sense primer
MdTFL2A antisense primer

50 -CTCTTAAAATGAAAAGAGCC-30
50 -TTCTCACATGTCAATAAGTT-30

aAbbreviation: I, inosine.

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cassette primer C1 or C2 and either 5S, R1S or R2S. A 428bp DNA fragment amplified with C2/R2S primers wascloned into pBluescript II SK+ (Stratagene, La Jolla, CA,
USA). The 50 RACE was carried out between cassette primer
C1 or C2 and either 3A, R1A or R2A. A 469-bp DNA
fragment amplified with C2/R1A primers was cloned asindicated for the 30 RACE and sequenced. Full-length
cDNAs were amplified with the sense primer MdTFL2S andantisense primer MdTFL2A. Various fragments containing a
656-bp cDNA were obtained and cloned into pBluescript II
SK+ or pUC119 (Takara Biomedicals). Consequently, four
pBluescript II SK+ clones (pBMDTFL1-, 2-, 5-, and 12+)and 2 pUC119 clones (pUMDTFL1- and 3+) were obtained.
These clones were sequenced completely by the dideoxy
method using a Hitachi SQ5500S automated sequencer(Hitachi, Tokyo, Japan).
2.3. DNA blot analysis
Genomic DNA was obtained from young Jonathan apple
leaves. The DNA (10 mg) was digested individually with
BamHI, EcoRI, HindIII, NcoI, XbaI, or XhoI according to
the manufacturer’s instructions (Takara Biomedicals) andthen separated on a 0.8% agarose gel. The DNA bands weretransferred to Hybond-N+ (Amersham Pharmacia Biotech,
Buckinghamshire, UK) and hybridized with a digoxigenin(DIG)-labeled PCR probe encoding the MdTFL1 gene.
Hybridization was performed in a 0.5 M Na–Pi buffer

97(0.25 M Na2HPO4, pH 7.2) containing 7% SDS and 1 mM
EDTA at 65 8C for 16 h followed by three washes in a
40 mM Na–Pi buffer containing 1% SDS at 65 8C for
20 min. The washes and detection methods were performed
according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). The membrane was exposed to
X-ray film (Fuji Photo Film, Tokyo, Japan).
2.4. Expression analysis
RNA was isolated from Jonathan apples harvested atvarious stages of development. Detection of MdTFL1, AFL1
and AFL2 by RT-PCR was performed by using the RT-PCR
high (Toyobo, Tokyo, Japan). A specific primer (50 GTGGCATACATTGTAAATA-30 ) for MdTFL1 and random
primers for AFL1 and AFL2 were used in reverse
transcription reaction with 1 mg of total RNA as a template.
Then, MdTFL1, AFL1 and AFL2 specific transcripts wereidentified using the following primers: a sense primer 2S andan antisense primer 2A for MdTFL1 (Table 1), a sense
primer 6S (50 -CAGAGGGAGCACCCGTTCATTGTGAC30 ) and an antisense primer AFL1R (50 -TTCATTCAGTGTGCCCTAGCC-30 ) for ALF1 and a sense primer
6S and an antisense primer AFL2R (50 -TCAAACTCTCTCTGCAGAACTGGC-30 ) for AFL2. PCR reactions
were run for 40 cycles at 50 8C. The PCR products were runon 1.5% (w/v) agarose gel and then blotted on the HybondN+ (Amersham Pharmacia Biotech). Hybridizations were

Fig. 1. Nucleotide and deduced amino acid sequences (single-letter code) of MdTFL1 cDNA. The asterisk (*) indicates a stop codon. Arrows above thesequences indicate the primers used for RACE-PCR.

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Fig. 2. (a) Comparison of the amino acid sequences of MdTFL1 and MdFT with those of other CETS proteins. The proteins (translated from the cDNA sequence
where necessary) are ATC [28], BFT [18], BNTFL1-1, BNTFL1-3, BRTFL1-1, and BNTFL1-2 [39], CEN (accession no. S81193), CET1, CET2 and CET4[37], CiFT (accession no. AB027456), FT (accession no. AB027504), Hd3a (accession no. AB052944), and LpTFL1 [33], MdTFL1 and MdFT (accession nos.

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performed using DIG-labeled PCR probes specific for
MdTFL1, AFL1 or AFL2. The detection methods were
performed as for DNA blotting using the manufacturer’s
protocol (Roche Diagnostics). Chemiluminescence was
detected using the LAS-1000 image analyzer (Fuji Photo
Film).
2.5. Construction of transformation vectors
pBMDTFL12+ was cut with BamHI and KpnI to release
the MdTFL1 cDNA fragment, that was then ligated into
pUC119 (cut with the same enzymes) yielding
pUMDTFL12.1+. pUMDTFL12.1+ was then cut with
Xba1 and SacI to release the MdTFL1 cDNA fragmentthat was subsequently ligated in the sense orientation into
the binary vector pSMAK251 containing the CaMV 35S
promoter [26] cut with the same restriction enzymes.
The resulting plasmid was named pSMDTFL12.1.2+(35S::MdTFL1 construct). The TFL1 cDNA (EST129D7T7) was obtained from the Arabidopsis Biological
Resource Center at The Ohio State University. TFL1 cDNA
was amplified by LA-PCR using EST 129D7T7 as atemplate and cloned into a T-tailed SmaI site of pUC119,
producing pUTFL129.5. After confirming the TFL1
sequence using an automated DNA sequencer (Hitachi),pUTFL129.5 was cut with XbaI and SacI and the liberated
TFL1 fragment was then ligated into the binary vector
pSMAK251 cut with the same restriction enzymes, yielding
pSTFL129.5.1 (35S::TFL1 construct).

99

3. Results
3.1. Cloning the apple MdTFL1 gene
To investigate the genes that play a role in apple
juvenility, we cloned MdTFL1, a putative homologue ofArabidopsis TFL1. A cDNA library was constructed fromapple apical buds to facilitate the isolation of full-length
MdTFL1 cDNA using the RACE method. Primer sites andthe nucleotide sequences of primers used for cloning areshown in Fig. 1 and Table 1, respectively.
The cDNA and predicted amino acid sequences for
MdTFL1 are shown in Fig. 1. The MdTFL1 cDNA coding
region exhibits 76%, 73%, 71% and 58% sequence identity
to TFL1, CEN, SP and FT, respectively. The protein productpredicted from the MdTFL1 sequence comprises 172
residues and exhibits similarity to TFL1 (75%), CEN(74%), SP (72%) or FT (55%) (Fig. 2a). The gene wastherefore designated MdTFL1 for Malus domestica TFL1
homologue. A phylogenic comparison of MdTFL1 withother CETS family members showed that it is grouped with
FT-like, CEN-like (dicot), TFL1-like (dicot), and CEN/
TFL1-like (monocot) proteins (Fig. 2b). Apple genomic
DNA was digested individually either with BamHI, EcoRI,
HindIII, NcoI, XbaI or XhoI, and DNA blotting was
performed using an MdTFL1 probe. One to four major bands
were detected in each digest, suggesting that MdTFL1
homologue exists in apple (Fig. 3).
3.2. Expression pattern of MdTFL1 in apple

2.6. Transformation of Arabidopsis
A. tumefaciens strain EHA101 was used to transform
Arabidopsis thaliana (Col) plants by the floral-dip method[27]. For the selection of transformed plants, sterilized seeds
were suspended in 0.1% sterile agar, plated on kanamycin
selection plates, and then transferred to a growth chamber(BIOTRON, Nippon Medical and Chemical Instruments
Co., Ltd., Tokyo, Japan) set at 22 8C under LD conditions.
The selection plates contained a 0.5  MS medium (Wako
Pure Chemicals Co., Ltd.), 0.8% agar (Difco Laboratories,
Detroit, MI, USA), 500 mg/l cefotaxime (Wako Pure
Chemicals Co., Ltd.), and 25 mg/l kanamycin monosulfate(Meiji Seika Kaisha Ltd., Tokyo, Japan). Transformants
were identified as kanamycin-resistant when seedlings in themedium produced green leaves and well-established roots.
Resistant transformants were transplanted to moistened
potting soil composed of vermiculite and perlite [1:1 (v/v)]
after 2–5 adult leaves had developed. The day of sowing wascounted as day 0. Morphological analyses were performedon the primary (T0) and subsequent generations (T1 and T2).

MdTFL1 mRNA expression was analyzed in various
living tissues and in apices of current apple shoots during
different stages of development by RT-PCR southern blot.
Total RNA was isolated from reproductive tissues (sepals,
petals, stamens, and carpels) and vegetative tissues (mature
leaves and apical buds of vegetative shoots and cotyledons,
stems, roots, and apical buds of seedlings). MdTFL1 mRNAwas expressed in vegetative tissues, such as apical buds,
seedling stem, and roots, but not in reproductive tissues suchas floral organs (Fig. 4a). To elucidate seasonal changes in
MdTFL1 mRNA expression in the apical buds, apices werecollected from current apple shoots each month from June to
February. Floral buds at the balloon stage were also collectedin late April. Total RNA was isolated from each sample andsubjected to expression analysis. MdTFL1 mRNA wasexpressed strongly in early July, about two weeks before
floral bud differentiation with gradual decrease to late-July(Fig. 4b). It was consistent with the result of expression
analysis in different tissues that the MdTFL1 mRNA wasdetected in vegetative shoots (water sprout) in June (Fig. 4b,

AB052994 and AB161112) and MFT [18], PnFT1b, PnFT2a, and PnFTL1a (accession nos. AB109804, AB110009 and AB161110), RCN1, RCN2, and RCN3[34], SP (accession no. U84140), TFL1 (accession no. U77674), and TSF [18], and Vitis TFL1 (Vitis vinifera TFL1-like protein, accession no. AF378127).
Identical residues at each position are shown in black. (b) Phylogenic tree of MdTFL1, MdFT and other CETS family protein sequences in (a). The ClustalW
program was used to align 28 complete protein sequences. Bold characters represent apple proteins.

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number of rosette leaves, a measure of developmental time
to flowering, was higher in the 35S::MdTFL1 transgenic
plants (e.g., 14.3 for S10-2 seedlings compared to 9.3 and
6.4 for the wild-type and tfl1-1 control plants, respectively).
At 35 days after sowing, a typical 35S::MdTFL1 transformant had produced 12-15 rosette leaves but no flower buds
or bolting (Fig. 5b). In contrast, the wild-type plants
displayed many flowers and brown pods on day-35 (Fig. 5a).
In addition, there was little difference between wild-type
Arabidopsis control plants and those transformed with a
35S::MdTFL1 antisense construct with respect to days toflowering and number of rosette leaves at flowering (data notshown).
3.4. Comparison of 35S::MdTFL1 and 35S::TFL1
transgenic Arabidopsis

Fig. 3. DNA blot analysis. Genomic DNA was isolated from leaves of theapple cultivar ‘Jonathan’. DNA aliquots (10 mg) were digested with individual restriction enzymes and subjected to agarose gel electrophoresis. The
DNA blot was hybridized with a DIG-labeled MdTFL1 DNA probe.
Restriction enzymes used to digest apple DNA are shown at the top: B,
BamHI; E, EcoRI; H, HindIII; N, NcoI; Xb, XbaI; Xh, XhoI. Numbers to theleft indicate DNA size markers in kbp.

6.19 V). In contrast, the expression of AFL1, an apple
orthologue of FLO/LFY [24], was detected when theexpression level of MdTFL1 was relatively low (Fig. 4b).
On the other hand, the expression of AFL2, another apple
orthologue of FLO/LFY, was detected constantly during
flower development as previously reported [24].
3.3. MdTFL1 transgene delays Arabidopsis flowering
To determine whether the MdTFL1 gene functions
analogously to Arabidopsis TFL1, we constructed a binary
vector pSMDTFL12.1.2+ containing full-length MdTFL1
cDNA inserted in the sense-oriented direction under controlof the 35S CaMV promoter. pSMDTFL12.1.2+ wasintroduced into wild-type Arabidopsis plants (Col) by
Agrobacterium-mediated transformation. Thirty-one independent kanamycin-resistant transgenic plants were identified. Five of the 31 primary transformants (T0 generation)
exhibited significantly delayed flowering while 7 floweredslightly later than wild-type plants (data not shown).
The T1 generation inherited the phenotype of the T0
generation. Quantitative characteristics of six independent
kanamycin-selected segregating T2 transgenic lines areshown in Table 2. The earliest flowering occurred at 29.0
days after sowing in one line, while the latest flowering
occurred at 39.2 days compared to 24.3 days for the wildtype control plants under LD photoperiods (Table 2). The

35S::MdTFL1 transformants exhibited a phenotype
similar to that of 35S::TFL1 transformants. One
35S::MdTFL1 T1-transformant line with a strong phenotype(S10-2; Fig. 5d) did not flower until day-90. The 35S::TFL1
transformant (Fig. 5c, right side) had not yet flowered
whereas the wild-type control plant (Fig. 5c, left side) had
flowered and set fruits at 42 days after sowing. Each of thesetransformants displayed long primary inflorescences, theuppermost 5–6 of which displayed normal flower buds inappearance (Fig. 5e,f). Floral buds formed in the
35S::MdTFL1 and 35S::TFL1 transformants often failed
to flower or set seeds. In 35S::MdTFL1 and 35S: TFL1
transformants, the number of cauline leaves at flowering
increased relative to that of wild-type control plants(Table 2).

4. Discussion
Apple MdTFL1 exhibits a high degree of sequencehomology to TFL1, a member of the CETS family. A
comparison of MdTFL1 with other CETS family members
shows that it groups with TFL1 and four Brassica proteins(Fig. 2b). It is logical that MdTFL1 groups with proteinsfrom dicots rather than monocots because the apple belongs
to Rosaceae family, which consists of dicotyledonous plants.
Recent studies have revealed that CEN/TFL1/SP and FT are
the members of a small gene family. There are six members
in Arabidopsis and approximately six in tomato [28,29]. In
pea, two TFL1-like genes exist and function differently [30].
Poplar, which is a woody plant, such as the apple, contains atleast eight CETS genes in its genome (T. Igasaki, personalcommunication). The DNA blot analysis for MdTFL1
implied the existence of other TFL1-like gene(s) in apple.
Recently, an apple gene MdFT (GenBank Accession no.
AB161112) homologous to FT, which will be a member of
CETS family in apple, was isolated. Several regions ofnotable sequence homology have been described for
mammalian PEBPs. They include a D-P-D-x-P motif

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101

Fig. 4. RT-PCR analysis. 1 mg of total RNA was used for RT-PCR. Subsequent southern blots were performed using DIG-labeled DNA probes for MdTFL1,
AFL1 and AFL2. (a) PCR for MdTFL1 was performed on cDNAs obtained from reproductive and vegetative tissues, then PCR products were hybridized with a
DIG-labeled MdTFL1 DNA probe. Reproductive tissues: sepals, petals, stamens, and carpels. Vegetative tissues: apical bud (V), cotyledons, stems, roots, andapical buds. V, vegetative shoots. (b) PCRs for MdTFL1, AFL1 and AFL2 were performed on cDNAs obtained from apical buds of apple during flower
development, then PCR products were hybridized with DIG-labeled MdTFL1, AFL1 and AFL2 DNA probes, respectively. Numbers above the lanes show thedate of harvest (month, day). Arrows indicate the onset of floral bud differentiation and flowering. EtBr, ethidium bromide.

followed at some distance by a histidine residue and then a
G-x-H-R motif, all of which contribute to the conformationof the ligand-binding site [31]. In MdTFL1, a D-P-D-x-P
motif runs from residue 70 to 74, a histidine residue ispositioned at 86, and a G-x-H-R motif runs from residue 115
to 118. These motifs are conserved in other members of the
CETS family.
MdTFL1 mRNA was expressed in apical buds ofvegetative shoots (water sprout) and seedling stems, roots

and apical buds, but not in sepals, petals, stamens, or carpels.
These observations demonstrate that MdTFL1 is expressed
preferentially in vegetative tissues, although MdTFL1 is not
expressed in mature leaves like TFL1 in Arabidopsis [7,32].
In Antirrhinum, however, CEN mRNA is present not only inthe apical meristem but in other tissues [13], and tomato SP
is expressed throughout development in all the primordialorgans [14]. Unlike MdTFL1, other CETS family genes suchas LpTFL1 in ryegrass, RCN2 in rice and PsTFL1 genes inTable 2
Phenotype of T2-transformed Arabidopsis expressing the MdTFL1
Controls and transgenic linesa

Days to floweringb,dRosette leaves at floweringc,dCauline leaves at floweringc,dNo. of plants

wt
tfl1-1
35S::TFL1
S6-5
S10-2
S21-2
S22-2
S28-2

24.3  0.9
20.4  1.1
34.3  5.0
29.0  3.4
33.5  2.2
36.1  3.7
39.2  7.7
36.6  4.6

9.3  0.9
6.4  0.8
15.3  2.9
11.7  2.1
14.3  1.5
13.5  1.9
13.8  2.0
12.4  2.2

2.1  0.3
1.1  0.3
5.0  1.6
3.7  1.3
5.5  0.9
6.3  1.3
5.8  2.3
7.2  2.3

10
9
3
6
8
8
6
5

a

Seedlings from controls (wt, tfl1-1, 35S::TFL1) and secondary transformants (T1) carrying the MdTFL1 gene, were grown under long-day (16 h light/8 hdark) conditions. T2-seedlings were selected with kanamycin.
bDays to flowering indicates the time at which flower primordia first became visible to the naked eye.
cRosette and cauline leaves were counted on the day flower primordia became visible.
d
All values are mean  S.E.M.

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Fig. 5. Comparison of 35S::MdTFL1 and 35S::TFL1 transgenic Arabidopsis plants: (a) and (b) 35-day-old plant (ecotype Columbia) grown under LD
conditions (16 h light/8 h dark). (a) Wild-type (WT) control plant. (b) T1 transgenic line S21-1. No flower buds were present in line S21-1, although brown
siliques were developing in the control plant. (c) Wild-type (WT) control plant (left) and 35S::TFL1 transformant (right) at 42 days after sowing. (d)35S::MdTFL1 transformant (line S10-2) at 90 days after sowing, when a flower first opened. (e) Inflorescence of a 35S::TFL1 transformant. (f) Inflorescence of a
35S::MdTFL1 transformant. Scale bars, 3.0 cm.

pea are expressed in both vegetative and reproductive tissues[30,33,34]. MdTFL1 expression differed from that of SP,
LpTFL1, RCN2 and PsTFL1 in that MdTFL1 is expressed
preferentially in vegetative tissues. The expression pattern of
MdTFL1 in different tissues resembled that of SP9D, amember of the CETS family in tomato since both genes wereexpressed in shoot apices and roots, not in reproductive
tissues [29]. In fact, MdTFL1 exhibits greater similarity to
SP9D (77%) than to SP (72%) in amino acids sequences.
Regarding seasonal-dependent MdTFL1 expression in theapices of apple, the mRNAwas expressed strongly in early July(about 8 weeks after full bloom), approximately two weeks
prior to the initiation of floral bud formation. Thereafter,
expression decreased gradually to late July (Fig. 4b).

MdTFL1 is possibly involved in the regulation of flower
induction from late June to early July (6–8 WAFB) since thisperiod is thought to be critical for the determination ofmeristem identity in apple [20,35,36]. In Arabidopsis,
Antirrhinum and tobacco, the production of flowers or shoots
most likely depends on the relative expression patterns of
TFL1/CEN-like genes and floral meristem identity genes suchas LFYand AP1 [32,37]. Interestingly, the seasonal expression
level of MdTFL1 in apple apices appears to be complementary
to that of AFL1, which gradually increases from early July tolate August, although AFL2 expression is constant during
flower development (Fig. 4b). Thus, the induction of flowering
may also depend on the relative expression of MdTFL1 andAFL1 in apple. The genetic interaction of MdTFL1 and AFL1

N. Kotoda, M. Wada / Plant Science 168 (2005) 95–104

remains to be analyzed to understand the regulation of flower
induction in apple.
Over-expression of MdTFL1 retarded the transition fromthe vegetative to the reproductive phase in transgenic
Arabidopsis plants (Fig. 5 and Table 2). 35SS::MdTFL1transformants exhibited increased numbers of rosette andcauline leaves and an extended vegetative phase comparedto wild-type control plants. These results suggest that
MdTFL1 maintains the inflorescence meristem in transgenic
Arabidopsis, resulting in a delay of flowering. In addition,
35S::MdTFL1 transformants resembled 35S::TFL1 transformants not only in delayed flowering but also inmorphological characteristics, which may be related tothe similar expression pattern between MdTFL1 and TFL1
(Figs. 4 and 5).
In woody plants such as apple, the maintenance of thejuvenile phase is one of the most important early-stage
events during plant development. In apple, however, genesthat control the transition from the vegetative to thereproductive phase have not yet been determined. Our
work shows that MdTFL1 is a member of the CETS familyof apple and that over-expression of MdTFL1 causes delayed
flowering in transgenic Arabidopsis. Analysis of the
MdTFL1 sequence, expression pattern and function suggeststhat this gene may play a key role in maintaining the juvenile
and/or vegetative phase in apple. Recently, we confirmedthat several transgenic apples with antisense MdTFL1
flowered extremely early [38]. Future transgenic approaches
may suppress the expression of endogenous MdTFL1 so as
to reduce the generation time of apple trees that normallyexhibit a long juvenile period.

Acknowledgments
We thank Drs. F. Motoyoshi of Okayama University and
H. Ichikawa of the National Institute of Agrobiological
Sciences for providing the lambda vector with the TFL1
genomic clone and the binary vector pSMAK251, respectively. We also thank Ms. T. Sekita for technical assistance.
The EST clone 129D7T7 and tfl1-1 seeds were kindly
provided by the Arabidopsis Biological Resource Center at
Ohio State University. This work was supported by the‘‘Development of innovative plants and animals usingtransformation and cloning’’ project of MAFF and theProgram for Promotion of Basic Research Activities forInnovative Biosciences (PROBRAIN). This publication
constitutes the contribution number 1338 of the National
Institute of Fruit Tree Science. The accession number for the
MdTFL1 sequence reported in this manuscript is AB052994.

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