Molecular Characterization of FLOWERING LOCUS T-Like
Genes of Apple (Malus × domestica Borkh.)

1Apple Breeding and Physiology Research Team, National Institute of Fruit Tree Science, 92-24 Nabe-yashiki, Shimo-kuriyagawa, Morioka,

020-0123 Japan
2Aomori Green Biocenter, Aomori Prefectural Agriculture and Forestry Research Center, 221-10 Yamaguchi, Nogi, Aomori, 030-0142

Japan
3Cryobiofrontier Research Center, Iwate University, 3-18-8 Ueda, Morioka, 020-8550 Japan
4Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute (FFPRI), 1 Matsunosato, Tsukuba, 305-8687
Japan
5Fruit Genome Research Team, National Institute of Fruit Tree Science, 485-6 Okitsu-nakacho, Shimizu, Shizuoka, 424-0292 Japan
6Present address: Fruit Genome Research Team, National Institute of Fruit Tree Science, 485-6 Okitsu-nakacho, Shimizu, Shizuoka,
424-0292 Japan
7Present address: Hirosaki Industrial Research Institute, Aomori Prefectural Technological Research Center, 80 Fukuromachi, Hirosaki,
036-8363 Japan
8Present address: Graduate School of Natural Science, Nagoya City University, 1 Yamanohata, Mizuho-cho, Mizuho-ku, Nagoya, 467-8501
Japan∗Corresponding author: E-mail, koto@affrc.go.jp or ringoya_daisuki@yahoo.co.jp; Fax, +81-54-369-2115(Received January 14, 2010; Accepted February 20, 2010)

The two FLOWERING LOCUS T (FT)-like genes of apple(Malus × domestica Borkh.), MdFT1 and MdFT2, have been
isolated and characterized. MdFT1 and MdFT2 were mapped,
respectively, on distinct linkage groups (LGs) with partial
homoeology, LG 12 and LG 4. The expression pattern of
MdFT1 and MdFT2 differed in that MdFT1 was expressed
mainly in apical buds of fruit-bearing shoots in the adultphase, with little expression in the juvenile tissues, whereas
MdFT2 was expressed mainly in reproductive organs,including ﬂower buds and young fruit. On the other hand,both genes had the potential to induce early ﬂowering sincetransgenic Arabidopsis, which ectopically expressed MdFT1 or
MdFT2, ﬂowered earlier than wild-type plants. Furthermore,
overexpression of MdFT1 conferred precocious ﬂowering inapple, with altered expression of other endogenous genes,
such as MdMADS12. These results suggest that MdFT1 could
function to promote ﬂowering by altering the expression of
those genes and that, at least, other genes may play animportant role as well in the regulation of ﬂowering in apple.
The long juvenile period of fruit trees prevents early croppingand efﬁcient breeding. Our ﬁndings will be useful information
to unveil the molecular mechanism of ﬂowering and todevelop methods to shorten the juvenile period in various
fruit trees, including apple.
Keywords: Apple • FLOWERING LOCUS T (FT) • Flowering time
• Juvenility • Malus × domestica Borkh.

Regular Paper

Nobuhiro Kotoda1,6,∗, Hidehiro Hayashi1, Motoko Suzuki1, Megumi Igarashi2,7, Yoshimichi Hatsuyama2,7,
Shin-ichiro Kidou3,8, Tomohiro Igasaki4, Mitsuru Nishiguchi4, Kanako Yano5, Tokurou Shimizu5,
Sae Takahashi1, Hiroshi Iwanami1, Shigeki Moriya1 and Kazuyuki Abe1

Abbreviations: AP1, APETALA1; ATC, ARABIDOPSIS THALIANACENTRORADIALIS; BFT, BROTHER OF FT AND TFL1; CaMV,
cauliﬂower mosaic virus; CAPS, cleaved ampliﬁed polymorphic
sequence; CEN, CENTRORADIALIS; DAF, days after ﬂowering;
DIG, digoxigenin; FBS, fruit-bearing shoots; FLC, FLOWERING
LOCUS C; FT, FLOWERING LOCUS T; FUL, FRUITFULL; LD, longday; LFY, LEAFY; LG, linkage group; MFT, MOTHER OF FT AND
TFL1; N–J, Neighbor–Joining; RT–PCR, reverse transcription–
PCR; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1;
SS, succulent shoots; SSR, simple sequence repeat; TFL1,TERMINAL FLOWER1; TSF, TWIN SISTER OF FT.
The nucleotide sequences reported in this paper have been
submitted to the DDBJ under accession numbers AB458504(MdFT2 mRNA), AB458505 (MdFT2 genomic DNA), AB458506(MdFT1 genomic DNA), AB501124 (MdSOC1a mRNA), andAB501125 (MdSOC1b mRNA).

Introduction
Apple (Malus spp.), which belongs to the Rosaceae subfamily
Maloideae, is one of the most important fruit tree crops in theworld. However, the long juvenile phase characteristic of woody
plants, including apple, makes their breeding cycle slower(Zimmerman 1972, Hackett 1985). In apple, the juvenile phase
generally lasts from 4 to 8 years or more. For example, the ‘Fuji’
apple (Malus × domestica Borkh.) from ‘Ralls Janet’ × ‘Delicious’,

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021, available online at www.pcp.oxfordjournals.org© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

561

N. Kotoda et al.

one of the most popular apple cultivars in the world (O’Rourke
et al. 2003), set fruit for the ﬁrst time 12 years after sowing(Sadamori et al. 1963), whereas the ‘Sansa’ apple from
‘Gala’ × ‘Akane’, top-grafted on fruit-bearing trees for early
selection, set fruit after 7 years (Yoshida et al. 1988). In general,
selection efﬁciency is limited until the seedlings set fruit. In thecase of fruit breeding, the most important traits for selection,such as texture, ﬂavor, ripening time and shelf life, are related
to the fruit itself. Therefore, various practical techniques toaccelerate ﬂowering and fruiting of seedlings in the juvenile
phase have been considered for years. In apple, grafting theseedling onto fruit-bearing trees (top grafting) or onto dwarfing rootstocks, such as ‘Malling 9 (M. 9)’ (Pearl 1932) and ‘JM1’(Soejima et al. 1998), results in earlier ﬂowering by 1 or 2 years.
After the end of the juvenile phase, ﬂower induction occurs inlate June, and ﬂower initiation with morphological changes
at the apical meristem starts to form ﬂoral primordia in mid
July in Morioka, Japan (Kotoda et al. 2000). However, little is
known about the physiological and genetic factors involving
the transition to ﬂowering in apple.
On the other hand, studies on Arabidopsis (Arabidopsis
thaliana) and snapdragon (Antirrhinum majus) have led tothe identiﬁcation of many genes involved in ﬂowering. In
Arabidopsis, the transition from the vegetative to the reproductive phase is initiated by four independent pathways ofsignal transduction, i.e. the autonomous, and gibberellin-,vernalization- and light-dependent pathways (for reviews, see
Koornneef 1998, Levy and Dean 1998). These signals aretransmitted via integrator genes, such as FLOWERING LOCUS T(FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1)/AGAMOUS LIKE20 and FLOWERING LOCUS C (FLC),
to the ﬂoral meristem identity genes APETALA1 (AP1) and
LEAFY (LFY) at the apical meristems (for reviews, see Araki 2001,
Jack 2004, Michaels 2009). In addition to the integrator genes,
TERMINAL FLOWER1 (TFL1; Bradley et al. 1997, Oshima et al.
1997) is also a key gene that represses ﬂowering and maintains
the inﬂorescence meristem by preventing the expression of
AP1 and LFY (Ratcliffe et al. 1998, Ratcliffe et al. 1999). TFL1 was
the ﬁrst member of the TFL1/FT family to be identiﬁed asa homolog of an animal phosphatidylethanolamine-binding
protein (PEBP), considered to be a Raf-1 kinase inhibitor (Yeunget al. 1999). Thereafter, the Arabidopsis genome was foundto contain six members of the TFL1/FT family, including
ARABIDOPSIS THALIANA CENTRORADIALIS (ATC), BROTHER
OF FT AND TFL1 (BFT), FT, MOTHER OF FT AND TFL1 (MFT)and TWIN SISTER OF FT (TSF) (Kardailsky et al. 1999, Kobayashi
et al. 1999, Mimida et al. 2001, Yoo et al. 2004, Yamaguchi
et al. 2005).
FT was identiﬁed as a causative gene for a typical lateﬂowering mutant ft, the phenotype of which was opposite
to that of tﬂ1 (Kardailsky et al. 1999, Kobayashi et al. 1999).
Hanzawa et al. (2005) reported that the antagonistic functionsof a ﬂoral activator and a repressor encoded by FT and TFL1,
respectively, could be converted by a single amino acid substitution. Recently, it was found that the FT protein moves from562

the leaf to the shoot apex, where FT interacts with the bZIP
transcription factor, FD, to activate AP1 in Arabidopsis (Abeet al. 2005, Wigge et al. 2005, Corbesier et al. 2007). In rice
(Oryza sativa), it was demonstrated that the protein encoded
by Hd3a (Kojima et al. 2002), a rice equivalent of the FT gene,similarly travels from the leaf to the shoot apex (Tamaki
et al. 2007), probably in the absence of GF14c (a G-box factor
14-3-3c protein), a direct inhibitor of Hd3a (Purwestri et al.
2009). Furthermore, grafting experiments in cucurbit species(Cucurbita maxima and C. moschata) demonstrated that theFT protein, but not FT mRNA crossed the graft union in thephloem translocation stream (Lin et al. 2007). Experiments
in Arabidopsis also revealed that the FT protein is graft transmissible (Notaguchi et al. 2008). The results of these studies
suggest that the FT protein acts as a mobile ﬂower-inducing
signal, such as a ﬂorigen, as noted by Chailahyan (for reviews,
see Chailahyan 1968, Giakountis and Coupland 2008, Zeevaart2008).
Based on studies of Arabidopsis and other plant species,
much effort has been devoted to unveiling the molecularmechanism of ﬂowering and to manipulate the ﬂowering
time in horticultural trees since the last decade (for reviews,
see Benlloch et al. 2007, Hanke et al. 2007, Wilkie et al. 2008).
Apple genes related to ﬂowering have been investigated byanalyzing its expression patterns (Yao et al. 1999, Kotoda et al.
2000, van der Linden et al. 2002, Esumi et al. 2005, Hättasch
et al. 2008) and heterologous transgenic plants (Sung et al. 1999,
Kotoda et al. 2002, Wada et al. 2002). In this context, we havesearched for the genes that maintain the juvenile/vegetative
growth or induce the transition to ﬂowering in apple. As
a result, transgenic apple expressing the antisense RNA of
MdTFL1 (an apple ortholog of TFL1) showed precocious
ﬂowering (Kotoda et al. 2003). Therefore, we further investigated the genes of the TFL1/FT family as candidates that canshorten the juvenile/vegetative growth phase and manipulate
the ﬂowering time in apple (Kotoda and Wada 2005, Kotoda
et al. 2006, Mimida et al. 2009). In this study, we have isolated
and characterized the two FT-like genes of apple and examined
their function using transgenic Arabidopsis. Furthermore, weinvestigated transgenic apple overexpressing the FT-like geneto clarify its function in apple.

Results
Identiﬁcation of FT-like genes from apple
We previously isolated MdFT, an apple homolog of FT, andclassiﬁed it as an FT-like gene by phylogenetic analysis (Kotoda
and Wada 2005). However, the result of DNA blot analysis
suggested the existence of another MdFT-like gene in apple(Fig. 1A). Therefore, we screened the cDNA library derived
from ﬂower buds of ‘Fuji’ apple to obtain FT-like genes homologous to MdFT. As a result, positive clones converged to two
kinds of cDNA. One corresponded to MdFT, which we renamed
MdFT1, but the other seemed to be a novel gene homologous

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

FT-like genes of apple

A

B

C

D

Fig. 1 (A) DNA blot analysis for FT-like genes of apple. The genomic DNA (15 µg) of the ‘Fuji’ apple was digested individually with EcoRI, HindIII or
XbaI and then separated on a 0.8% (w/v) agarose gel. DNA bands were transferred to Hybond N+ and hybridized with a digoxigenin (DIG)labeled MdFT1 cDNA. Hybridization was performed in DIG Easy Hyb (Roche Diagnostics) at 42°C for 16 h followed by two washes in 0.5 × SSC
containing 1% (w/v) SDS at 68°C for 20 min. The molecular size markers are shown in kb on the left. (B) Schematic representation of the genomic
organization of FT-like genes of the apple and Arabidopsis FT gene. Boxes represent exons and lines represent introns. Numbers represent thelengths (bp) of exons (above the boxes) and introns (below the lines). The scale bar on the map represents approximately 200 bp. H represents
a restriction enzyme site of HindIII in MdFT1 and MdFT2 genes. (C) Comparison of the deduced protein sequence of MdFT1 and MdFT2 (accession
Nos. AB161112 and AB458504, respectively) with those of the TFL1/FT family from apple, Arabidopsis, tomato, grapevine, Lombardy poplar andpea. The AGI (Arabidopsis Genome Initiative; http://www.arabidopsis.org) code or accession number of each gene, as pertinent, is as follows: FT(AGI code At1g65480), TSF (AGI code At4g20370), SFT (tomato, accession No. AY186735), VvFT (grapevine, accession No. DQ871590), PnFT1(Lombardy poplar, accession No. AB106111), TFL1 (AGI code At5g03840), ATC (AGI code At2g27550), SP (tomato, accession No. U84140),
PsTFL1a (pea, accession No. AY340579), VvTFL1A (VvTFL1) (grapevine, accession No. DQ871591), MdTFL1 (apple, accession No. AB052994) and
MdCENa (apple, accession No. AB366641). Amino acids in black and in gray are identical and similar, respectively, in at least seven of the 14
members of the TFL1/FT family. Amino acids common to genes in the FT clade are shown in pink and those in the TFL1 clade are shown in green.
The gaps indicated by dashes are attributed to the lack of amino acids. The asterisks indicate the amino acid positions related to antagonistic
functions between TFL1 and FT (Hanzawa et al. 2005, Ahn et al. 2006). The ﬁrst square in red, called segment B, represents the region of
a potential ligand-binding pocket in TFL1/FT family proteins, and the second square in red represents a region more conserved in FT than in
TFL1 (Ahn et al. 2006). The triangles show the intron positions, which are conserved in the TFL1/FT family proteins. (D) Phylogenetic analysis of
TFL1/FT family proteins. The tree was constructed by the Neighbor–Joining (N–J) method for the deduced amino acid sequence of the membersof the TFL1/FT family from apple (MdFT1, MdFT2, MdTFL1, MdTFL1a, accession No. AB366643; MdCENa and MdCENb, accession No. AB366642),
Arabidopsis (ATC, BFT, FT, MFT, TFL1 and TSF), grapevine [VvFT and VvMFT, accession No. DQ871594; VvTFL1A (VvTFL1) and VvTFL1B,
accession No. DQ871592; VvTFL1C, accession No. DQ871593], Lombardy poplar (PnFT1 and PnFT3, accession No. AB110612; PnFTL1,
accession No. AB369067; PnFTL4, accession No. AB181241), morning glory (InFT1, accession No. ABW73562), orange (CsTFL, accession No.
AY344244), pea (PsTFL1a and PsTFL1c, accession No. AY343326), Satsuma mandarin (CiFT1, accession No. AB027456), snapdragon[CENTRORADIALIS (CEN), accession No. S81193) and tomato (SP, SFT and SP9D, accession No. AY186738). The protein sequence data wereobtained from the DNA database (DDBJ/EMBL/GenBank). The N–J unrooted dendrograms were generated from the alignment of
deduced amino acids with the Clustal X program, and the phylogenetic tree was displayed using the N–J plot unrooted program(Perrière and Gouy, 1996). Bootstrap values for 1,000 resamplings are shown in each branch. The unit for the scale bar displays branch lengths(0.02 substitutions/site).

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to MdFT, which we designated MdFT2. The MdFT1 and MdFT2
genes consisted of four exons of 198, 62, 41 and 224 bp encoding a putative protein of 174 amino acids, which resembled the
genomic structure of the FT gene (Fig. 1B). The coding sequenceof MdFT1 cDNA exhibited 94.2% identity to that of MdFT2
cDNA at the nucleotide level, although the sequence of eachexon showed slightly different identities for MdFT1 and
MdFT2 (93.4, 100.0, 95.1 and 95.1% for the ﬁrst, second, thirdand fourth exons, respectively). The genomic sequence including the coding region of MdFT2 (1,963 bp) was longer thanthat of MdFT1 (1,620 bp) due to the extended introns of
MdFT2 (Fig. 1B).
Sequence analysis using the deduced amino acid sequencesof TFL1/FT homologs revealed that MdFT1 and MdFT2 werethe most similar to each other (94.3% identity), and they also
had a high identity with FT-like members, such as VvFT, PnFT1,
PnFT3, SFT, CiFT1, TSF and FT (86.7, 85.5, 85.1, 84.5, 83.6, 76.3
and 74.6%, respectively) at the amino acid level (for accession
numbers, see the legend of Fig. 1C). The alignment of thededuced amino acid sequences of the TFL1/FT family showedthat there were 19 amino acid residues speciﬁc to FT-likemembers and 15 residues speciﬁc to TFL1/CEN-like members(Fig. 1C). In particular, both MdFT1 and MdFT2 had Tyr84
and Gln139 in the positions corresponding to Tyr85 and Gln140
of Arabidopsis FT (Fig. 1C, see asterisks). Tyr85/His88 and
Gln140/Asp144 are likely to be the most critical residues for
distinguishing FT and TFL1 in Arabidopsis (Hanzawa et al.
2005, Ahn et al. 2006).
In order to clarify the relationships among the TFL1/FT
family members, we constructed a phylogenetic tree by
Neighbor–Joining (N–J) distance analysis of apple, Arabidopsis,
grapevine (Vitis vinifera), morning glory (Ipomoea nil), orange(Citrus sinensis), pea (Pisum sativum), Lombardy poplar(Populus nigra), Satsuma mandarin (Citrus unshiu), snapdragon
and tomato (Solanum esculentum) as shown in Fig. 1D.
The phylogenetic tree was divided into four major clades,
represented by BFT, FT, MFT and TFL1. As expected, MdFT2,
together with MdFT1, was classiﬁed into the FT clade. FT-likemembers of apple were more closely related to those of
deciduous woody plants, such as grapevine and poplar.

Localization of MdFT1 and MdFT2 on a linkage map
The Rosaceae subfamily Maloideae, which includes the genus
Malus, has a higher haploid base chromosome number, x = 17,
than other members of the family Rosaceae, probably due tothe hypothesized polyploid origin (Sax 1933, Chevreau et al.
1985). Since the DNA blot and sequence analysis suggested that
MdFT1 and MdFT2 were not allelic (Fig. 1A), we attempted toshow clearly on which linkage group MdFT1 and MdFT2 werelocated and to consider the evolutionary origin of those genes.
Therefore, genetic mapping was performed for the genes using
two kinds of F1 mapping populations from crosses between‘Delicious’ (De) and Mitsubakaido (Malus sieboldii Rehder) andbetween ‘Ralls Janet’ (Ra) and Mitsubakaido (Igarashi et al.
2008). As a result, MdFT1 was assigned to the south end of564

linkage group 12 (LG 12) of ‘Delicious’ (De12), with 70.7 cM
from the markers rD05A20–0.9 and E35M48–350 at the northend, whereas MdFT2 was assigned to the locus close to themarker E38M61–395 at the south end of LG 4 of ‘Ralls Janet’(Ra04) (Fig. 2).

Expression patterns of MdFT1 and MdFT2 in apple
To identify when and where MdFT1 and MdFT2 were expressed,
quantitative real-time reverse transcription–PCR (qRT–PCR)
was performed on various tissues of apple in the adult phase,juvenile phase or tissue culture using primers speciﬁc to eachgene (Supplementary Table S1). The transcript of MdFT1
accumulated mainly in apical buds of fruit-bearing shoots (FBS)(AB1, on June 24; AB2, on August 7), ﬂower buds (at the balloon
stage), ﬂoral organs, such as stamens, and whole young fruits.
The level of MdFT1 transcript was very low in the tissues of
1-month-old seedlings in the juvenile phase, such as roots,
stems, mature leaves and apical buds of vegetative shoots, withlittle detection in seeds and cultured shoots, which included
apical buds, stems and leaves. The level of MdFT1 transcript in
the mature leaves was relatively low in both the juvenile andthe adult phase as compared with that detected in the apicalbuds and reproductive organs in the adult phase, although theexpression of MdFT1 in mature leaves was higher in the adultphase than in the juvenile phase. The MdFT1 transcript level inmature fruit and peel was below the detection limit (Fig. 3A).
On the other hand, the transcript of MdFT2 was detected
mainly in reproductive organs, such as ﬂower buds, sepals,
petals, stamens, carpels, receptacles, peduncles and whole
young fruits, with some expression also detected in mature fruit(Fig. 3B). In apical buds of FBS, we also detected the expressionof MdFT2, but the expression level of MdFT2 was relatively
lower than that of MdFT1. In the tissues of 1-month-old seedlings, MdFT2 was expressed at relatively higher levels than
MdFT1; however, the transcript of MdFT2 in mature leaves was
low in both the juvenile and the adult phases, similarly to
MdFT1. We could not detect the expression of MdFT2 in seeds
and peel (Fig. 3B). The transcripts of MdFT2 accumulated inshoots of cultured tissues more than those of MdFT1, although
the transcription level of both was much lower than that in theapical buds of FBS.
Seasonal expression pattern in apical buds of apple in theadult phase showed that MdFT1 was expressed to higher levels
than MdFT2 in FBS from late June to August. In addition, MdFT1
was expressed relatively more highly in FBS than in succulent
shoots (SS), which are vegetative shoots that do not produce
ﬂower buds, while the expression level of MdFT2 was similar
between FBS and SS, from early June to mid August (Fig. 3C, D).
The expression of MdFT2 peaked in mid September and thereafter decreased during the dormant period with a rapid increase
from late March (Fig. 3D). On the other hand, the transcript of
MdTFL1 decreased rapidly toward mid July before the peak of
MdFT1 expression in apical buds of FBS, with the signal in thoseof SS showing a delayed decrease at late September (Fig. 3E).
For MdAP1, it started to increase from late August after the

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

FT-like genes of apple

De12

Ra04
0.0
5.2
12.8
19.0
20.2
23.1
23.6

24.2

24.3
25.2
31.5
34.0

57.4
58.6
61.1

–/+
–
–
+
–
–/+
+
+
+
–
+
+
+
–
–
+
–
–
+

–
+
+
–

NZ05g08-L/S
rD09D12-1.2
E37M61-130
rD14C55-0.3
E37M62-195u
CH02h11a-L/S
E38M48-170
rD13A31-0.4
E32M62-500u
E36M60-450
E38M50-520
rD09C84-1.1
E41M48-320d
E36M50-350
E37M60-155
rD11F42-0.7
rD06A55-0.8
rD15D02-0.4
rD09C84-0.9

E37M47-500
E38M61-395
MdFT2-Dr0.44
E37M60-145u

0.0

+
+

rD05A20-0.9
E35M48-350

4.8+

MdTFL1-139

12.4
13.9

+/– NZ28f04-L/S+ E41M48-395

23.6
26.3

–
+

33.4

–/+ CH01f02-L/S

41.6
43.7

+
+

rD09D49-1.0
E33M61-100

57.4

–

OPD20-0.25

70.7

+

MdFT1-Hf0.63

E35M48-195
E40M49-200

period of ﬂower induction, consistent with the onset of thedevelopment of ﬂoral organ primordia (Fig. 3F).

Ectopic expressions of the apple FT-like genes
in Arabidopsis
To determine the effects of apple FT-like genes on ﬂowering
time and inﬂorescence morphology, we generated two kindsof transgenic Arabidopsis plants with MdFT1 or MdFT2
under the control of the cauliﬂower mosaic virus (CaMV) 35S
promoter fused with the Ω sequence (35SΩ) (Fig. 4A).
Consequently, we obtained >20 independent transgenic lines
for 35SΩ : MdFT1 and 35SΩ:MdFT2 (designated 35SΩ : MdFT1/wt
and 35SΩ : MdFT2/wt, respectively). Among them, 10 independent lines per construct were used for further analysis. Eight to
10 plants in the T2 generation per line were grown under longday (LD, 16 h light/8 h dark) conditions, and their phenotypes
were examined.
Seven of 10 transgenic Arabidopsis lines with 35SΩ : MdFT1
ﬂowered signiﬁcantly earlier than wild-type plants under LD
conditions at a P-value of <0.01 for the number of rosetteleaves (Fig. 4B, Table 1). For example, 35SΩ : MdFT1/wt (#7)
ﬂowered with 3.9 ± 0.2 rosette and 2.1 ± 0.1 cauline leaves, while

Fig. 2 Location of the FT-like genes on the linkage map of
the apple cultivars ‘Ralls Janet’ (Ra) and ‘Delicious’ (De).
MdFT1 and MdFT2 were located on LG 12 (De12) and LG 4
(Ra04), respectively. Linkage groups (LGs) of ‘Ralls Janet’(Ra04) and ‘Delicious’ (De12) are derived from segregation
data of ‘Ralls Janet’ and ‘Delicious’ populations, respectively.
LG numbers are according to Maliepaard et al. (1998). Loci
are listed on the right side of the linkage maps. The left sideshows genetic distances (cM). The linkage phase information
is provided as + or −, indicating on which of the homologous
chromosomes the marker/allele is located. In the case of
SSR markers, the sizes of SSR bands are shown by threeﬁgure numbers or the relative sizes of the SSR bands areshown by the letter ‘L (long)’ or ‘S (short)’. CAPS markers
are shown with the gene name, the restriction enzyme(Dr, DraI; Hf, HinfI) and the length of the scored fragment(kbp). For a detailed explanation of molecular markers, see
Supplementary Table S2 of this paper and the materials
and methods of Igarashi et al. (2008).

wild-type plants ﬂowered with 6.7 ± 0.3 rosette and 3.6 ± 0.2
cauline leaves (Table 1). Nine out of 10 transgenic lines with
35SΩ : MdFT2 also ﬂowered signiﬁcantly earlier than wild-type
plants under LD conditions at a P-value of <0.01 for the numberof rosette leaves (Fig. 4B, Table 1). For example, 35SΩ : MdFT2/
wt (#3) exhibiting a strong phenotype ﬂowered with 4.0 ± 0.2
rosette and 1.9 ± 0.2 cauline leaves, whereas wild-type plants
ﬂowered with 6.7 ± 0.3 rosette and 3.6 ± 0.2 cauline leaves(Table 1). No difference in the appearance of ﬂowers and inﬂorescences was observed among 35SΩ : MdFT1/wt, 35SΩ : MdFT2/
wt and wild-type plants (Fig. 4C). An early ﬂowering phenotype
was also observed in 35SΩ : MdFT1/wt and 35SΩ : MdFT2/wt
under short-day conditions (data not shown).

Overexpression of MdFT1 in apple
Transgenic experiments using Arabidopsis revealed that both
MdFT1 and MdFT2 had the potential to function as ﬂoral promoters in apple. Expression analysis by qRT–PCR suggested
that MdFT1 played a role during ﬂoral transition, at least in tissues such as apical buds. Therefore, to clarify whether MdFT1
confers an early-ﬂowering phenotype in apple, we produced
transgenic apples carrying the 35SΩ : MdFT1 already used forPlant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

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N. Kotoda et al.

0.25

0.20

0.20

0.15

0.15

0.10

0.10

0.05

0.05

0

0

Mature Leaves

Apical buds of FBS

0.06

Floral organs

D 0.07

MdFT1

Fruit

MdFT2

0.06

FBS
SS

Jan 23

Feb 20Mar 19Apr 07

Feb 20Mar 19Apr 07

Dec 19Jan 23

Nov 07

Sep 19Oct 24

Aug 22

Nov 07

Sep 19Oct 24

Aug 22

FBS
SS

Jul 24

Apr 07

Mar 19Feb 200

Jan 230.2

0Dec 190.4

0.05
Nov 070.10

Oct 24

0.6

Sep 190.15

Aug 22

0.8

Jun 27

Dec 191.0

0.20

Jun 27

0.25

MdAP1

1.2

Jun 06

FBS
SS

Jul 24

Jul 24

F 1.4

MdTFL10.30

Jun 06

Jun 27

Apr 07

Mar 19Feb 200

Dec 190

Jan 230.01
Nov 070.02

0.01
Oct 240.02

Sep 190.03

Jul 240.03

Aug 22

0.04

Jun 270.04

E 0.35

FBS
SS

0.05

Jun 060.05

Jun 06

Relative expression

C 0.07

MdFT2

Sd
Rt
Sm
MLJ
ABJ
ML
AB1
AB2
FB
Se
Pe
St
Ca
Re
Pd
YF
MF
Pl
CS

0.25

Seedlings

Relative expression

B 0.30

MdFT1

Sd
Rt
Sm
MLJ
ABJ
ML
AB1
AB2
FB
Se
Pe
St
Ca
Re
Pd
YF
MF
Pl
CS

Relative expression

A 0.30Fig. 3 Expression patterns of MdFT1 (A) and MdFT2 (B) in various tissues, and seasonal expression patterns of MdFT1 (C), MdFT2 (D), MdTFL1(E) and MdAP1 (F) in apple by quantitative real-time RT–PCR. The samples for (A) and (B) from left to right are as follows: seeds (Sd), roots (Rt),stems (Sm), mature leaves (MLJ) and apical buds (ABJ) of 1-month-old seedlings (from a cross of cv. ‘Fuji’ × cv. ‘Orin’) in the juvenile phase;mature leaves (ML, collected on June 24) in the adult phase; apical buds of FBS on June 24 (AB1) and on August 7 (AB2) in the adult vegetative/
reproductive phase; ﬂower buds at the balloon stage (FB), sepals (Se), petals (Pe), stamens (St), carpels (Ca), receptacles (Re), peduncles (Pd),young fruit on June 15 (YF), mature fruit on November 18 (MF) and peel (Pl) from ‘Fuji’ apple in the adult reproductive phase; and shoots oftissue culture (CS). The samples for (C) to (F) from left to right are as follows: apical buds of fruit-bearing shoots (FBS) on June 6, June 27, July 24,
August 22, September 19, October 24, November 7, December 19, January 23, Feburuary 20, March 19 and April 7; or succulent shoots (SS) on
June 6, June 27, July 24, August 22, September 19 and October 24. Levels of detected amplicons were normalized by reference to ampliﬁed
products that corresponded to apple HistoneH3. Values are means ± SD of results from three replicates. Each symbol without a bar indicates thatthe standard deviation fell within the symbol. The primer sets used in quantitative real-time RT–PCR analysis and the PCR conditions aredescribed in Supplementary Table S1.

Arabidopsis transformation, using micropropagated tissues ofan apple rootstock cv. ‘JM2’.
As a result, we obtained six independent transgenic lines
designated MdFT1#1-1, MdFT1#1-2, MdFT1#3, MdFT1#5,
MdFT1#6, and MdFT1#8. Five of six transgenic lines (MdFT1#1-1,

566

MdFT1#1-2, MdFT1#3, MdFT1#6 and MdFT1#8) produced
ﬂower buds or opened ﬂowers in vitro 8–12 months after
Agrobacterium infection (2–6 months after regeneration).
For example, MdFT1#3 produced terminal ﬂower buds at
the top of each shoot, and some of them came into ﬂower

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

FT-like genes of apple

RB

LB

A

Pnos
BglII

Tnos

nptII

BamHI

P35S

Tnos

HindIII
XbaI

T-DNA Region

SalI

MdFT1 or MdFT2

B
35S :MdFT1

35S :MdFT2

WT (Col)C

WT (Col)

35S :MdFT1

35S :MdFT2

Fig. 4 Phenotypes of transgenic Arabidopsis with apple FT-like genes under long-day conditions. (A) Schematic representation of the transformation vectors, 35SΩ:MdFT1 and 35SΩ:MdFT2. LB, left border; nptII, neomycin phosphotransferase II gene; P35S, promoter region of cauliﬂower
mosaic virus 35S; Pnos, nos promoter; RB, right border; Tnos, 3′ region of nos. (B) Appearance of wild-type Arabidopsis (ecotype Columbia, left),transgenic Arabidopsis with 35SΩ:MdFT1 (middle) and transgenic Arabidopsis with 35SΩ:MdFT2 (right) 26 d after transfer to the growth chamber. (C) Inﬂorescence and ﬂower of wild-type Arabidopsis (left), transgenic Arabidopsis with 35SΩ:MdFT1 (middle) and transgenic Arabidopsis
with 35SΩ:MdFT2 (right). The photographs were taken after ﬂowering for each plant. For details on the ﬂowering time of transgenic lines, see
Table 1. The wild type (Col) was used for Arabidopsis transformation. The plants in (B) and (C) were grown under LD conditions (16 h light/8 hdark) at 22°C.

(Fig. 5A, B), whereas the control plant never produced ﬂower
buds under the same culture conditions (Fig. 5C). In addition,
the shoots of MdFT1#1-1 were grafted onto the apple rootstock‘JM7’ and transferred to the greenhouse (Fig. 5D). The potted
MdFT1#1-1 also produced a ﬂower bud and opened a solitary
ﬂower; however, the ﬂower had >8 petals (Fig. 5E), in contrast
to the normal ﬂower, which had ﬁve sepals, petals, carpels
and approximately 20 stamens (Fig. 5F). Importantly, thein vitro ﬂowering lines, such as MdFT1#3 and MdFT1#8,
which produced in vitro ﬂower buds more frequently thanthe others, showed a much less vigorous growth of shoots.
Several lines with such severe phenotypes senesced, probably
due to their inability to form new vegetative shoot meristems.

The result of DNA blot analysis for transgenic lines MdFT1#1-1,
MdFT1#5 and MdFT1#8 indicated that MdFT#1-1 had
two copies and the other lines had one copy of the transgene(Fig. 5G).

Inﬂuence of overexpression of MdFT1 on the otherapple genes
We examined the inﬂuence of the overexpression of MdFT1
on the expression of other endogenous genes, which had beenshown or expected to play a role in ﬂowering in apple. To avoid
the ﬂuctuation of the gene expression by artiﬁcial and otherenvironmental factors, we used the cultured shoots of each line
grown in a growth chamber under the same conditions.

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

567

N. Kotoda et al.

Table 1 Flowering time of transgenic lines ectopically expressing MdFT1 or MdFT2
Line

LD conditions (16 h light/8 h dark)
No. of plants

Rosette leaves

Cauline leaves

Total leaves

Wt (Col)

10

6.7 ± 0.3

3.6 ± 0.2

10.3 ± 0.4

Vector/Wt (Col)87.3 ± 0.3

3.3 ± 0.2

10.5 ± 0.2#194.0 ± 0.3∗∗

2.6 ± 0.2∗∗

6.6 ± 0.4∗∗#210

3.9 ± 0.2∗∗

2.6 ± 0.2∗∗

6.5 ± 0.2∗∗#3

10

6.2 ± 0.2

3.1 ± 0.1∗

9.3 ± 0.2∗#410

4.0 ± 0.2∗∗

2.2 ± 0.1∗∗

6.2 ± 0.2∗∗#510

4.3 ± 0.2∗∗

2.1 ± 0.1∗∗

6.4 ± 0.2∗∗#610

6.7 ± 0.3

2.6 ± 0.2∗∗

9.3 ± 0.4#710

3.9 ± 0.2∗∗

2.1 ± 0.1∗∗

6.0 ± 0.2∗∗#897.9 ± 0.5

2.3 ± 0.2∗∗

10.2 ± 0.4#9104.0 ± 0.2∗∗

2.8 ± 0.2∗∗

6.8 ± 0.3∗∗

10

4.3 ± 0.3∗∗

2.3 ± 0.1∗∗

6.6 ± 0.4∗∗#194.9 ± 0.3∗∗

2.1 ± 0.1∗∗

7.0 ± 0.3∗∗#210

5.0 ± 0.3∗∗

3.0 ± 0.2∗

8.0 ± 0.2∗∗#310

4.0 ± 0.2∗∗

1.9 ± 0.2∗∗

5.9 ± 0.3∗∗#410

4.5 ± 0.3∗∗

2.3 ± 0.1∗∗

6.8 ± 0.3∗∗#510

4.3 ± 0.2∗∗

2.2 ± 0.1∗∗

6.5 ± 0.2∗∗#610

4.1 ± 0.2∗∗

3.3 ± 0.2

7.4 ± 0.4∗∗#795.3 ± 0.3∗

2.3 ± 0.2∗∗

7.7 ± 0.4∗∗#88

4.4 ± 0.4∗∗

2.8 ± 0.3∗

7.1 ± 0.3∗∗#910

4.9 ± 0.1∗∗

2.1 ± 0.1∗∗

7.0 ± 0.0∗∗

10

4.3 ± 0.1∗∗

2.0 ± 0.0∗∗

6.3 ± 0.1∗∗

35SΩ:MdFT1/wt#10
35SΩ:MdFT2/wt#10

Plants in the second generation (T2) were grown under long-day (LD) conditions. Numbers of rosette and cauline leaves
were counted on the day ﬂoral organs became visible. Values are means ± SE. Student’s t-test was performed to compare theeffects of treatments. One or two asterisks indicate a statistically signiﬁcant difference from the wild-type plants in the same
column (P <0.05, 0.01, respectively).

The result of qRT–PCR analysis showed that MdMADS12[a FRUITFULL (FUL)-like gene of apple; van der Linden et al.
2002] was signiﬁcantly upregulated in the in vitro ﬂoweringlines MdFT1#1-1 and MdFT1#8, where the MdFT1 transcripts
accumulated in large numbers, whereas the expression level of
MdMADS12 in a line not ﬂowering in vitro, MdFT1#5, wasalmost the same as that in the control (Fig. 6A, B). In addition,
MdSOC1a and MdSOC1b (putative apple orthologs of SOC1;
accession Nos. AB501124 and AB501125, respectively), and
MdAP1 (equivalent to MdMADS5, an apple ortholog ofAP1) were more up-regulated in the lines MdFT1#1-1 andMdFT1#8, than in line MdFT1#5 and the non-transgenic
control (Fig. 6C–E). The expression of MdLHP1a/MdLHP1b(MdLHP1a and MdLHP1b, apple homologs of LHP1/TFL2;
Mimida et al. 2007) was not affected by the overexpression of
MdFT1 (Fig. 6F).

568

Discussion
MdFT1 and MdFT2 are located in distinct linkage
groups with duplicated segments
MdFT1 and MdFT2 were mapped, respectively, on loci in distinct linkage groups, LG 12 and LG 4 (Fig. 3). Because there is ahomoeology between the distal parts of LG 12 and LG 4 (Gardiner et al. 2007), MdFT1 and MdFT2 might have been derived
from an ancient gene that ancestors of the original hybrid
would have had. Coincidentally, MdFT1 was located on the
distal part of LG 12, the same linkage group as MdTFL1. Thisﬁnding suggests that MdFT1 and MdTFL1 have evolved in close
relation to each other and that at least one chromosome, corresponding to LG 12, plays an important role in the regulationof ﬂowering in apple. To date, it has been revealed that apple

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

MdFT1#3

DCont

MdFT1#3

E

Control(kbp)

MdFT1#8

G

MdFT1#5

C

B

A

MdFT1#1-1

FT-like genes of apple

23.1
9.4
6.5
4.3
2.3
2.0

F

MdFT1#1-1

0.5
MdFT1#1-1

Cont

Fig. 5 Precocious ﬂowering of transgenic apples overexpressing MdFT1. (A) Floral buds (indicated as an open triangle) formed at the top of theshoots of transgenic apple ‘JM2’ (line MdFT1#3) in the culture box. (B) In vitro ﬂowering of a solitary ﬂower (indicated as a ﬁlled triangle) at the
top of the transgenic shoots in the culture box. (C) Cultured shoots of an apple ‘JM2’ control plant. A white bar indicates 1 cm. (D) Opened ﬂowerof transgenic apple ‘JM2’ (line MdFT1#1-1) in the greenhouse. A white bar indicates 3 cm. (E) Close-up view of (D). The number of petals increased
compared with a normal ﬂower with ﬁve petals. (F) Flower of an apple cv. ‘JM2’ control plant grown in the ﬁeld. (G) DNA blot analysis oftransgenic apples ‘JM2’ with 35SΩ:MdFT1 (MdFT1#1-1, MdFT1#5 and MdFT1#8). Genomic DNA (15 µg) was isolated from the cultured tissues ofeach transgenic line and a control plant, digested with HindIII, and then separated on a 0.8% (w/v) agarose gel. A blotted membrane was
hybridized with a DIG-labeled nptII gene.

0.012

2.0

0.008

1.5

0.006

1.0

0.004

0.5

0.002

0

0

0.10

0.10

0.08

C

MdSOC1a
0.08

0.06

0.06

0.04

0.04

0.02

0.02

0

0
1.2

0.006
0.005

E

MdAP1

1.0

0.002

0.4

0.001

0.2

0

0

In vitro
floweringlines

Not in vitro
floweringlines

Control

0.6

MdFT1#5

0.8

0.003

MdFT1#8

0.004

B

MdMADS12D

MdSOC1bF

MdLHP1a/
MdLHP1b

In vitro
floweringlines

Not in vitro
floweringlines

Control

0.010

MdFT1#5

MdFT1

MdFT1#8

A

MdFT1#1-1

2.5

MdFT1#1-1

Relative expression

Relative expression

Relative expression

3.0

Fig. 6 Expression analysis for apple genes inthe shoots of transgenic lines. (A–F)
Expression of MdFT1 (A), MdMADS12 (B),
MdSOC1a (C), MdSOC1b (D), MdAP1 (E) and
MdLHP1a/MdLHP1b (F) in the transgenic
shoots of in vitro ﬂowering lines (MdFT1#1-1
and MdFT1#8) and a line not floweringin vitro (MdFT1#5), and in non-transgenic
shoots as a control. Shoots were collectedfrom each line cultured in a proliferating
medium under LD conditions for 4 weeks tosynchronize the growth state of each line.
MdMADS12 , MdSOC1a , MdSOC1b and
MdAP1 are genes encoding a MADS-box
transcription factor from apple; andMdLHP1a / MdLHP1b ( MdLHP1a and
MdLHP1b ) are apple twin homologs of
LHP1. Primers for MdLHP1a/MdLHP1b were
designed to detect both MdLHP1a and
MdLHP1b. HistoneH3 was used as an internal
control of gene expression. In A–F, nontransgenic apple cv. ‘JM2’ was used as acontrol plant (control). Transcript levels werenormalized against apple HistoneH3. Values
are means ± SD of results from three replicates,and each box without a bar indicates that thestandard deviation fell within the box. The
primer sets used in quantitative real-time
RT–PCR analysis and the PCR conditions aredescribed in Supplementary Table S1.

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569

N. Kotoda et al.

has at least six members of the TFL1/FT family in its genome(Kotoda et al. 2005, Mimida et al. 2009, this study), althoughmembers that belong to the BFT or MFT clade have not beenreported. Other eudicots, such as Arabidopsis and tomato,
have about six TFL1/FT family genes (Mimida et al. 2001,
Carmel-Goren et al. 2003). However, the Lombardy poplar
genome has nine (Igasaki et al. 2008), probably due to a recent
gene duplication event 8–13 million years ago (Tuskan et al.
2006). Because apple has duplicated chromosomal regions as
well, further study might reveal additional members falling into
the clade of MFT or BFT in the TFL1/FT family in apple.

MdFT1 and MdFT2 show different expression
proﬁles in apple
The expression pattern of MdFT1 and MdFT2 differed in that
MdFT1 was expressed mainly in apical buds of FBS in the adultphase, whereas MdFT2 was expressed mainly in reproductive
organs, including ﬂower buds and young fruit (Fig. 3A, B).
In addition, it was found that the transcripts of MdFT1 could
hardly be detected in the tissues of 1-month-old seedlings in
the juvenile phase, in contrast to the transcripts of MdFT2. In
Satsuma mandarin, two citrus homologs of FT, CiFT1 and CiFT2,
which are highly and speciﬁcally expressed in young fruit, were
not involved in the transition to ﬂowering, and only the third
citrus homolog of FT, CiFT3, expressed at a relatively low level inleaves and stems but with no expression in reproductive organs,turned out to play a key role in regulating ﬂower induction(Nishikawa et al. 2007). Considering that MdFT2 was highly
expressed in the reproductive organs and that it was expressedsimilarly in the apical buds of both FBS and SS during theperiod of ﬂower induction, MdFT1 might play a key role in thetransition to ﬂowering in apple (Fig. 3), although Arabidopsis
FT and its paralog TSF are both highly expressed in ﬂowers anddeveloping siliques as well as in leaves (Kobayashi et al. 1999,
Takada and Goto, 2003, Yamaguchi et al. 2005). The expression
pattern of MdFT1 in various tissues was somewhat similar tothat of tomato SFT (formerly SPD3, a tomato ortholog of FT;Carmel-Goren et al. 2003, Lifshitz et al. 2006) rather than that of
Arabidopsis.
In apple, ﬂower induction occurs in late June [about
40–50 days after ﬂowering (DAF)] and ﬂower initiation withmorphological changes at the apical meristem starts to form
ﬂoral primordia in mid July (about 60 DAF) in Morioka, Japan(Kotoda et al. 2000). MdFT1 was expressed to relatively higher
levels in the apical buds of FBS than in those of SS from theperiod of ﬂower induction to the early stage of ﬂower development. This result was consistent with the observation that theexpression of MdTFL1 decreased rapidly from early June,
although it is unclear where in apical buds MdFT1 was expressed(Fig. 3C, E). Recently, Hättasch et al. (2008) reported that theincrease in transcription of MdFT (MdFT1 in this study), AFL1
and AFL2 (apple orthologs of LFY; Wada et al. 2002) in the apicalmeristems of current-year shoots of apple began 1–2 weeks
earlier than ﬂower initiation, suggesting that ﬂower induction is
initiated by these genes. The higher expression level of MdFT2

570

in mid September and early April might be related to thedevelopment of ﬂoral organs in apple (Fig. 3D), because theexpression pattern of MdAP1 reﬂected the development of
ﬂoral organs, such as sepals and receptacles (Fig. 3F, Kotoda
et al. 2000).

MdFT1 and MdFT2 have the potential to act as
ﬂoral promoters and overexpression of MdFT1
confers precocious ﬂowering in apple
Transgenic Arabidopsis overexpressing MdFT1 and MdFT2
showed early ﬂowering as compared with the control plant(Fig. 4, Table 1). Similar early-ﬂowering phenotypes of transgenic Arabidopsis had been reported upon the ectopic expression of FT-like genes from heterologous plant species, such as
citrus (Kobayashi et al. 1999), tomato (Teper-Bamnolker andSamach 2005, Lifschitz et al. 2006), grapevine (Sreekantan andThomas 2006, Carmona et al. 2007), poplar (Hsu et al. 2006,
Igasaki et al. 2008) and cucurbits (Lin et al. 2007), as well as theoverexpression of FT and TSF. No differences in the extent of
ﬂower-promoting activity between MdFT1 and MdFT2 in transgenic Arabidopsis suggested that MdFT1 and MdFT2 bothhave the potential to act as ﬂoral promoters in apple (Table 1).
However, expression analysis by qRT–PCR implied that, during
ﬂoral transition, MdFT1 plays a more important role, at leastin tissues within the apical buds of FBS, where transition to
ﬂowering occurs.
Consequently, ﬁve out of six transgenic apples overexpressing MdFT1 ﬂowered in vitro 8–12 months after Agrobacterium
infection (Fig. 5). As a whole, the precocity of ﬂowering in
MdFT1-overexpressing lines was much higher than that inpreviously reported MdTFL1 down-regulated apples (Kotoda
et al. 2003, Kotoda et al. 2006). The transgenic lines with
35SΩ : MdFT1 that produced ﬂower buds in vitro had a tendency to have small, rounded leaves and show an extremely
weak growth habit. In the transgenic lines MdFT1#1-1 andMdFT1#8, which showed in vitro ﬂowering, MdFT1 was abundantly expressed, while the expression of MdFT1 was extremely
low in line MdFT1#5, possibly due to co-suppression (Fig. 6A).
As expected, line MdFT#5 has not ﬂowered in vitro or in pots
for at least 3 years after regeneration. These results suggest that
there is a positive correlation between MdFT1 expression andﬂower induction in transgenic plants. Besides MdFT1, the heterologous gene PnFT3 (a poplar ortholog of FT; Igasaki et al. 2008)
also promoted the transition to ﬂowering when overexpressedin the apple cultivar ‘Greensleeves’ (Supplementary Fig. S2).
Apple trees normally produce ﬁve ﬂowers per cluster, and each
ﬂower has ﬁve sepals, petals and carpels and approximately 20
stamens. The changes in the number of ﬂoral organs in thetransgenic plant might result from disturbance of the expression of ﬂoral organ identity genes, such as MADS-box genes,
due to the overexpression of MdFT1 (Fig. 5E). Contrary to our
results, Hättasch et al. (2009) reported that transgenic apples
with MdFT did not ﬂower for at least a year in vitro and in thegreenhouse. This observation might be due to the fact that theregeneration competence of the transformed calli from some

Plant Cell Physiol. 51(4): 561–575 (2010) doi:10.1093/pcp/pcq021 © The Author 2010.

FT-like genes of apple

scion cultivars was severely impaired when FT-like genes were
overexpressed, resulting in the selection of transgenic lines withlittle expression of those genes.

Up-regulation of MADS-box genes could resultin in vitro ﬂowering in the transgenic lines
overexpressing MdFT1
QRT–PCR analysis revealed that MdMADS12 and MdAP1
were signiﬁcantly more up-regulated in the cultured shoots oftransgenic lines that highly expressed MdFT1 than in those ofline MdFT1#5, which had little expression of MdFT1, and a control (Fig. 6). These results seem to be consistent with the ﬁnding by Teper-Bamnolker and Samach (2005) that FUL and AP1
were highly misexpressed in the young seedlings and the older
rosette leaves of Arabidopsis overexpressing FT. Interestingly,
Flachowsky et al. (2007) reported that the overexpression of
BpMADS4, a birch (Betula pendula) FUL-like MADS-box gene
(Elo et al. 2001), induced early ﬂowering in the apple cv. ‘Pinova’,with frequent in vitro ﬂowering. In addition, the phenotype ofthe transgenic apple with 35S : BpMADS4 resembled that with
35SΩ : MdFT1 in that it produced solitary ﬂower buds androunded leaves in tissue culture. Considering that MdMADS12
is a putative ortholog of FUL, MdMADS12 might play an important role in ﬂower induction of apple downstream of MdFT1.
In Arabidopsis, FUL is required in several developmental processes, including silique and leaf development, and in thetransition to ﬂowering (Gu et al. 1998, Ferrandiz et al. 2000,
Teper-Bamnolker and Samach, 2005). On the other hand, theup-regulation of MdAP1 in the transgenic lines indicated thatthe shoots had been ﬂorally induced because the expression of
MdAP1 starts to increase in the apical buds of FBS after ﬂower
initiation (Figs. 3F and 6E; Kotoda et al. 2000). In addition,
MdSOC1a and MdSOC1b were also up-regulated in the in vitro
ﬂowering lines with a similar expression pattern to MdAP1(Fig. 6C–E), implicating them as the common targets of MdFT1,
as SOC1 and AP1 are activated by the FT–FD complex in
Arabidopsis (Abe et al. 2005, Wigge et al. 2005, Michaels 2009).
Our results suggest that MdFT1 could function upstream ofthose genes to regulate ﬂowering in apple, although whether
or not the regulation is direct remains to be demonstrated.
In future studies, the interaction of partner genes, such astranscription factors, with MdFT1 and/or MdFT2 will be
investigated.

Materials and Methods
Plant materials
The tissue samples of an apple (Malus × domestica Borkh.) cvs.
‘Fuji’ and ‘Jonathan’ in adult phase (age: 19–20 years) were collected from the experimental ﬁeld at the National Institute ofFruit Tree Science in Morioka, Japan. One-month-old juvenile
seedlings from a cross between apple cvs. ‘Fuji’ and ‘Orin’ wereused for the expression analysis of apple FT-like genes in thejuvenile phase. Micropropagated tissues of an apple rootstock

cv. ‘JM2’ [Marubakaido ‘Seishi’ (Malus prunifolia Borkh. var.
ringo Asami) × ‘Malling 9’ (‘M. 9’)] (Soejima et al. 1998) and an
apple cv. ‘Greensleeves’ were kept at 24°C under LD conditions(16 h photoperiod; cool white ﬂuorescent light, 50 µmol m−2 s−1)
in the proliferating medium (MS medium containing B5 vitamin, 1 mg l−1 6-benzyl-aminopurine, 0.1 mg l−1 indole-3-butyricacid) in the culture box and subcultured every 4 weeks. The leaf
explants of those apple cultivars were used for Agrobacteriummediated transformation. For Arabidopsis transformation,
wild-type plants of ecotype Columbia (Col) were used.

Nucleic acid extraction and hybridization analysis
The genomic DNA was isolated by a cetyltrimethylammonium
bromide (CTAB)-based method modiﬁed by Yamamoto andMukai as described in Kotoda et al. (2002). The genomic DNA(15 µg) was digested with EcoRI, HindIII or XbaI, electrophoresed on a 0.8% agarose gel, and then blotted onto Hybond-N+
nylon membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The blotted membrane was hybridized withdigoxigenin- (DIG; Roche Diagnostics, Mannheim, Germany)labeled MdFT1 cDNA, which was ampliﬁed by PCR with a pairof primers, MdFT1(1→24) and MdFT1(508←528), and a DIG
mixture (Roche Diagnostics). For DNA blot analysis of transformants, each genomic DNA of transgenic lines and a control
plant was digested with HindIII and the blotted membrane was
hybridized with a DIG-labeled nptII probe. The hybridization
and washing was performed as described in Kotoda et al. (2002).
Chemiluminescent signals were visualized using the LAS1000
image analyzer (Fuji Photo Film, Tokyo, Japan). The primer set islisted in Supplementary Table S1.

Isolation of FT-like genes from apple
The cDNA library derived from ﬂower buds of ‘Fuji’ apple
was constructed as described by Mimida et al. (2007). MdFT1(accession No. AB161112; Kotoda and Wada 2005) labeled with
[α-32P]dCTP (GE Healthcare Bio-Sciences Corp.) was used asa probe to screen 2.0 × 105 plaque-forming units (pfu) of thecDNA library. Positive plaques were excised to the pBluescript
SK(−) phagemid (Stratagene, La Jolla, CA, USA), and approximately 20 clones were then sequenced using a DTCS Quick
Start Kit for Dye Terminator Cycle Sequencing (Beckman
Coulter, Fullerton, CA, USA) and an automated DNA sequencer
CEQ 8000 (Beckman Coulter).
To obtain genomic DNA of MdFT1, the cDNA of MdFT1
labeled with DIG was used as a probe to screen 2.0 × 105 pfu of
the genomic library of ‘Fuji’ apple. The lambda clone DNAs
from positive plaques were digested with restriction enzyme
NotI and ligated into the corresponding site of pBluescriptII
SK (+) (Stratagene). To obtain a MdFT2 genomic sequence
from apple, on the other hand, PCR ampliﬁcation of DNA
fragments was performed in a mixture of a pair of primers
MdFT2-EcoRI(−171→−148) and MdFT2(842←859)-XhoI, ahigh-ﬁdelity DNA polymerase (KOD plus; Toyobo, Osaka,
Japan) and 250–300 ng of genomic DNA of ‘Fuji’ apple. PCR wasprogrammed for pre-heating at 94°C for 2 min followed by

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N. Kotoda et al.

30 cycles of 94°C for 30 s, 50°C for 30 s and 68°C for 2 min.
The PCR-ampliﬁed fragments digested with EcoRI and XhoI
were cloned into the corresponding site of pBluescriptII SK (+)(Stratagene), and then four clones were sequenced. The primer
sets used in gene cloning are listed in Supplementary Table S1.

Mapping of FT-like genes on the linkage
map of apple
Cleaved ampliﬁed polymorphic sequence (CAPS) markers for
the MdFT1 and MdFT2 genes were developed, and genotyping
was performed using an F1 mapping population with 72 seedlings from the cross between ‘Delicious’ and Mitsubakaido(Malus sieboldii Rehder) and another population with 83 seedlings from the cross between ‘Ralls Janet’ and Mitsubakaido(Igarashi et al. 2008). A population from ‘Delicious’ and Mitsubakaido was used for the mapping of MdFT1. PCR was performed
with a pair of primers MdFT1-5f and MdFT1-4r. A 632 bp product was digested with HinfI, and the resultant 632 bp fragment
speciﬁc to ‘Delicious’ was used for segregation analysis. On theother hand, a population from ‘Ralls Janet’ and Mitsubakaido
was used for the mapping of MdFT2. PCR was performed witha pair of primers MdFT2-7f and MdFT2-8rm. A 1,064 bp product was digested with DraI, giving a 435 bp fragment speciﬁc
to ‘Ralls Janet’, which was used for segregation analysis. The
linkage map was constructed using JoinMap ver. 3.0 (Van Ooijen
and Voorrips 2001). Primer sets, restriction enzymes and PCR
conditions for the CAPS markers are described in Supplementary Table S2.

Sequence analysis
Amino acid sequences were analyzed using the Clustal X
multiple sequence alignment program ver. 1.83 (Jeanmougin
et al. 1998) and BioEdit ver. 7.7.0 (H. Hall, http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The phylogenetic tree was displayed using the N–J plot unrooted (Perrière and Gouy, 1996)
with bootstrap values for 1,000 resamplings in each branch.

Expression analysis by q RT–PCR
For analysis, total RNA was extracted from ﬂower buds (at theballoon stage), sepals, petals, stamens, carpels, receptacles,
peduncles, young fruit, mature fruit, peel, mature leaves(collected on June 24) and apical buds of FBS (collected on June
24 and August 7) from adult ‘Fuji’ apple trees; leaves, apicalbuds of vegetative shoots, stems and roots from 1-month-old
juvenile seedlings and seeds from a cross between ‘Fuji’ and‘Orin’ apples; apical buds of FBS (from June to the following
April in 2004) and SS (from June to October in 2003) in‘Jonathan’ apple trees; in vitro cultured shoots of ‘Fuji’ apple;
and transgenic and non-transgenic lines of ‘JM2’ apple by usinga PolyA Tract mRNA puriﬁcation kit (Promega, Madison, WI,
USA). For seasonal expression analysis, apical buds of all
samples from the orchard were collected between 10 and 14 h.
The ﬁrst-strand cDNAs were synthesized from 1 µg of total
RNAs in 20 µl of a reaction mixture using a QuantiTect Reverse

572

Transcription kit (Qiagen GmbH, Hilden, Germany). The subsequent PCRs were performed with 1 µl of the ﬁrst-strand cDNA
as templates in a total volume of 12.5 µl by using ABI PRISM
7000 (Applied Biosystems, Foster City, CA, USA). Transcriptsof MdFT1, MdFT2, MdLHP1a/MdLHP1b (MdLHP1a and
MdLHP1b), MdTFL1, MdMADS12, MdSOC1a, MdSOC1b, MdAP1
and HistoneH3 were identiﬁed with the speciﬁc primers(the primer sets used in qRT–PCR analysis and the PCR conditions are described in Supplementary Table S1). An apple
HistoneH3 gene (Kotoda et al. 2006) was used as an internal
control. QRT–PCR was performed three times and transcriptlevels were normalized against apple HistoneH3.

Construction of the transformation vector
To construct a vector for the constitutive expression of MdFT1
and MdFT2, the coding region of each gene was ampliﬁed by
PCR with thr pair of primers MdFT1-XbaI and MdFT1-SalI for
MdFT1, and with thr pair of primers MdFT2-XbaI and MdFTaSalI for MdFT2 (Supplementary Table S1). An ampliﬁed PCR
product was subsequently digested with XbaI and SalI andthen cloned into the XbaI/SalI sites of the modiﬁed pBI221(pBI9526Ω) to be placed between the CaMV 35S promoter
fused with the Ω sequence (Gallie and Walbot 1992) and theterminator of the nopaline synthase (nos) (Supplementary
Fig. S1). The resultant plasmid was cut with ApaI, and then the
fragment containing MdFT1 or MdFT2 was ligated into thesame restriction enzyme site of the pSMAK312Blue binary
vector (pSMAK312Blue; H. Ichikawa, in preparation). For vector
construction of 35SΩ::PnFT3S, poplar PnFT3S ampliﬁed by PCR
was inserted into the modiﬁed pSMAK193E (pSMAK193E;
H. Ichikawa, in preparation) to be placed between 35SΩ andthe 3′ region of the Arabidopsis rbcS-2B gene (TrbcS).

Arabidopsis transformation
Agrobacterium tumefaciens strain EHA101 was used totransform A. thaliana (Col) plants by the ﬂoral-dip method(Clough and Bent 1998). Kanamycin-resistant transformants
were transplanted from the plate to moistened potting soil
composed of vermiculite and perlite [1 : 1 (v/v)] after 2–5 adultleaves had developed and grown in the growth chamber(Biotron, Nippon Medical and Chemical Instruments Co., Ltd.,Tokyo, Japan) set at 22°C under LD conditions (16 h photoperiod; cool white ﬂuorescent light, 50 µmol m−2 s−1). Morphological analyses were performed on the second generation (T2).

Apple transformation
The apple cvs. ‘JM2’ and ‘Greensleeves’ were transformed
with A. tumefaciens strain EHA101 bearing 35SΩ:MdFT1 and
35SΩ:PnFT3 (PnFT3; a poplar ortholog of FT), respectively.
Agrobacterium tumefaciens was cultured overnight on a shaker
in 20 ml of liquid LB medium with 100 mg l−1 trobicin (Pﬁzer
Japan, Tokyo, Japan) at 28°C. After centrifugation, the pellet
was resuspended with MS medium (Murashige and Skoog
1962) and further diluted to an optical density (OD) of 0.5–0.8
at 600 nm. Leaf explants were infected with the inoculum for

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FT-like genes of apple

30 min. The selection of transgenic shoots for ‘Greensleeves’
was performed according to the procedure described byJames et al. (1989) and Yao et al. (1995) and the procedure for‘JM2’ will be published elsewhere (S. Takahashi, in preparation).
Transformed shoots were multiplied in the proliferating
medium supplemented with 50 mg l−1 kanamycin. Two to
three shoots per line were grafted to apple rootstock ‘JM7’[Marubakaido ‘Seishi’ (Malus prunifolia Borkh. var. ringo
Asami) × ‘Malling 9’ (‘M. 9’)] (Soejima et al. 1998). Grafted
apples were grown in an isolated greenhouse under natural
day-length with the temperature set at 20–25°C during thegrowing season as described in Kotoda et al. (2006).

Supplementary data
Supplementary data are available at PCP online.

Funding
The Bio-oriented Technology Research Advancement Institution[Program for Promotion of Basic Research Activities forInnovative Biosciences (to N.K.)]; the Ministry of Agriculture,
Forestry and Fisheries of Japan [‘Development of innovativeplants and animals using transformation and cloning’ (to N.K.)];
the Ministry of Education, Sports, Science, and Technology ofJapan [grant-in-aid for scientiﬁc research (to N.K.)].

Acknowledgments
We thank Dr. H. Ichikawa for providing the binary vectors
pSMAK312B and pSMAK193E, Dr. E. E. Hood for providing
Agrobacterium tumefaciens EHA101, Ms. T. Sekita and A. Igarashi
for their technical assistance, and Dr. E. Varkonyi-Gasic for
critical reading of the manuscript.

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