Regular Paper

Four TFL1/CEN-Like Genes on Distinct Linkage Groups
Show Different Expression Patterns to Regulate
Vegetative and Reproductive Development in Apple(Malus×domestica Borkh.)
Naozumi Mimida1, Nobuhiro Kotoda1,3,*, Takanori Ueda1, Megumi Igarashi2, Yoshimichi Hatsuyama2,
Hiroshi Iwanami1, Shigeki Moriya1, and Kazuyuki Abe1
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

Recent molecular analyses in several plant species revealed
that TERMINAL FLOWER1 (TFL1) and CENTRORADIALIS(CEN) homologs are involved in regulating the ﬂowering
time and/or maintaining the inﬂorescence meristem. In
apple (Malus×domestica Borkh.), four TFL1/CEN-likegenes, MdTFL1, MdTFL1a, MdCENa and MdCENb, were
found and mapped by a similar position on putatively
homoeologous linkage groups. Apple TFL1/CEN-like genes
functioned equivalently to TFL1 when expressed
constitutively in transgenic Arabidopsis plants, suggestingthat they have a potential to complement the TFL1
function. Because MdTFL1 and MdTFL1a were expressedin the vegetative tissues in both the adult and juvenile
phases, they could function redundantly as a ﬂowering
repressor and a regulator of vegetative meristem identity.
On the other hand, MdCENa was mainly expressed in fruit
receptacles, cultured tissues and roots, suggesting that it
is involved in the development of proliferating tissues but
not in the control of the transition from the juvenile to theadult phase. In contrast, MdCENb was silenced in mostorgans probably due to gene duplication by the polyploidorigin of apple. The expression patterns of MdTFL1 and
MdCENa in apple were also supported by the heterologous
expression of β-glucuronidase fused with their promoter
regions in transgenic Arabidopsis. Our results suggest thatfunctional divergence of the roles in the regulation ofvegetative meristem identity may have occurred amongfour TFL1/CEN-like genes during evolution in apple.

Keywords: Apple • CENTRORADIALIS (CEN) • Flowering
time • Gene family • Malus×domestica Borkh. • TERMINALFLOWER1 (TFL1).
Abbreviations: AP1, APETALA1; ATC, ARABIDOPSISTHALIANA CENTRORADIALIS; BFT, BROTHER OF FT AND
TFL1; CaMV, cauliﬂower mosaic virus; CEN, CENTRORADIALIS;
DIG, digoxigenin; EST, expressed sequence tag; FBS, fruitbearing shoot; FLC, FLOWERING LOCUS C; FT, FLOWERING
LOCUS T; GUS, β-glucuronidase; I-PCR, inverse PCR; IS,inverted sequence; LD, long-day; LFY, LEAFY; LG, linkage
group; MFT, MOTHER OF FT AND TFL1; ncRNA, non-coding
RNA; RT–PCR, reverse transcription–PCR; SOC1, SUPPRESSOROF OVEREXPRESSION OF CONSTANS1; SS, succulent shoots;
TFL1, TERMINAL FLOWER1; TSF, TWIN SISTER OF FT.
The nucleotide sequences reported in this paper havebeen submitted to the DDBJ under accession numbers
AB366637 (MdCENa genomic DNA), AB366638 (MdCENb
genomic DNA), AB366639 (MdTFL1 genomic DNA),
AB366640 (MdTFL1a genomic DNA), AB366641 (MdCENa
mRNA), AB366642 (MdCENb mRNA), AB366643 (MdTFL1amRNA), AB458503 (MdAP1a mRNA).

Introduction
Apple (Malus×domestica Borkh.), which belongs to the
Rosaceae subfamily, Maloideae, is one of the most important
fruit tree crops in the world from an economic standpoint.
However, most fruit crops, such as apple, have a long generation

3Present address: Fruit Genome Research Team, National Institute of Fruit Tree Science, 485-6 Okitsu-nakacho, Shimizu, Shizuoka

424-0292 Japan.
*Corresponding author: E-mail, koto@affrc.go.jp; Fax, +81-54-369-2115.

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001, available online at www.pcp.oxfordjournals.org© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

394

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

Four TFL1/CEN-like genes in apple

time, which makes their breeding cycle slower (Sax 1957,
Hackett 1985). Thus, understanding the phase transition ofapple at a molecular level would provide an insight into themethods of acceleration of breeding in fruit trees. The shootapex of higher plants passes through three more or lessdistinct phases during its post-embryonic development(Poethig 1990). The apple passes through a relatively long
juvenile phase, during which the vegetative shoot elongatesvigorously and does not bear ﬂoral buds for 4–8 years.
In woody plants, such as apple, attainment of the ability
to ﬂower indicates the end of the juvenile phase (Fig. 1).
When actual ﬂowering does not coincide with the end of thejuvenile phase, the intervening period can be referred to as
an adult vegetative phase or transition phase (Zimmerman
1972, Hanke et al. 2007). In the adult phase, reproductive
parts of the apple tree produce fruit-bearing shoots (FBS)and enter the seasonal ﬂower/fruit formation cycle (reproductive growth cycle) (Fig. 1). On the other hand, vegetative

FlowerAttainment of
inductionability to flower

parts produce vegetative shoots, such as succulent shoots(SS) (Supplementary Fig. S1A), and enter the vegetative
growth cycle (Fig. 1). After ﬂowers bloom on FBS in thespring, a new budded shoot (bourse shoot) emerges from theaxillary meristem on the bourse (Supplementary Fig. S1B).
Approximately 95% of vegetative meristems on the bourseshoots become committed to ﬂoral development, which consequently convert into inﬂorescence meristems and thendevelop into ﬂoral meristems (Foster et al. 2003). The remaining 5% of vegetative meristems produce vegetative shoots. The
morphology of SS, which emerge directly form the branch or
trunk, is similar to that of juvenile shoots with vigorous growth,and SS do not bear ﬂoral buds for several years. Fruit ripens in
the autumn and the apple tree enters the dormant period
from late autumn. When the seeds from the ripened fruit germinate, they enter the ontogenetic growth cycle (Fig. 1).
In Arabidopsis [Arabidopsis thaliana (L.) Heynh.], thetransition from the vegetative to the reproductive phase
1st flowering(4-8 years after
germination)

2nd~ flowering

Vegetative growth
Adultapple tree
WinterJuvenille
shootDormancy

Fruit-bearing
shoot
Spring

Dormant budof fruit-bearing
shoot

Ontogenetic
growth cycle
Seedling(1 month old)

Seasonal flower/fruit
formation cycle
Reproductive
growth cycle

AutumnFlower cluster
Floralmeristem

Fruit ripening

Dormant bud

Fruit

Seed
Seed
Germination

Juvenile phase (vegetative)

Inflorescencemeristem

Floral bud
differentiation(Flower initiation)
SummerVegetative
meristem

Vegetative
growth cycle
Flowerinduction

Vegetative
meristemCommitted toflowering

Vegetative(succulent)shoot
Flower cluster
Fertilization
Young fruit

Bourse shoot

Fruit development

Adult vegetative phase (transition phase)
Adult phase (reproductive)
Adult phase (vegetative)

Fig. 1 Schematic representation of the apple growth cycle based on the studies of Hackett (1985), Zimmerman (1972), Poethig (1990), Kotoda
et al. (2000), Foster et al. (2003) and Hanke et al. (2007).

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

395

N. Mimida et al.

is initiated by four independent pathways of signal transduction, i.e. the gibberellin-dependent, autonomous, vernalizationdependent and light-dependent pathways (for reviews, see
Levy and Dean 1998, Mouradov et al. 2002, Simpson and
Dean 2002, Boss et al. 2004, Komeda 2004). These signals aretransmitted via integrator genes, such as FLOWERING LOCUS
T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1) and FLOWERING LOCUS C (FLC), to the ﬂoral meristem identity genes APETALA1 (AP1) and LEAFY (LFY) at
the apical meristems. TERMINAL FLOWER1 (TFL1) is a key
gene involved in repressing ﬂowering and maintaining theinﬂorescence meristem by preventing the expression of AP1
and LFY (Bradley et al. 1997, Ratcliffe et al. 1998, Ratcliffe
et al. 1999, Boss et al. 2004).
In apple, twin ﬂowering genes, such as MdAP1 (MdMADS5)(Yao et al. 1999, Kotoda et al. 2000, 2002) and MdAP1a(accession no. AB458503) of AP1-like genes, MdTFL1 and
MdTFL1a (MdTFL1-2) of TFL1-like genes (Esumi et al. 2005,
Kotoda and Wada 2005, Kotoda et al. 2006, this study) and
MdLHP1a and MdLHP1b of LHP1-like genes (Mimida et al.
2007), have been isolated and characterized. The abundanceof MdTFL1 and AFL1 (a LFY-like gene of apple) transcripts inthe apical buds of FBS varies considerably through the various developmental stages; MdTFL1 is signiﬁcantly downregulated, whereas AFL1 is up-regulated after ﬂoral bud
differentiation (Kotoda and Wada 2005). These results suggest that AFL1 and MdTFL1 play antagonistic roles in regulating the phase transition and could potentially affect ﬂoral
development throughout the growing season in apple. However, the expression patterns of homolog genes are not necessarily equivalent between apple and Arabidopsis. For
example, MdAP1 expression is prominent in sepals but is notdetected in petals (Kotoda et al. 2000). The ﬂoral pathway
genes in individual plant species are unique in their expression patterns and, possibly, in their protein–protein interaction networks. These differences are thought to be importantin the variations of ﬂowering time and in the distinctive
morphological characteristics among plant species (Andersen et al. 2004). In particular, the TFL1 homolog genes in the
ﬂoral pathway are shown to have unique features in ﬂowering time and plant architecture in each plant species.
The Arabidopsis genome contains six genes in the FT/TFL1
family, including the MOTHER OF FT AND TFL1 (MFT), FT,
TWIN SISTER OF FT (TSF), BROTHER OF FT AND TFL1 (BFT),
ARABIDOPSIS THALIANA CENTRORADIALIS (ATC) and
TFL1 (Bradley et al. 1997, Ohshima et al. 1997, Kardailsky
et al. 1999, Kobayashi et al. 1999, Mimida et al. 2001, Yooet al. 2004, Yamaguchi et al. 2005). The analyses of TFL1/
CENTROLADIALIS (CEN; Bradley et al. 1996)-like genes indicot plants suggested that the functional divergence among
these groups occurred selectively in each plant species. For
example, the tﬂ1 mutant of Arabidopsis shows early ﬂowering,and its apex of inﬂorescence is terminated by fused ﬂowers

396(Bradley et al. 1997, Ohshima et al. 1997). On the other hand,
the T-DNA-tagged mutant of the ATC of the CEN group(CEN-like genes) shows no novel phenotypes (Mimida et al.
2001, Yoo et al. 2005). In snapdragon (Antirrhinum majus L.),the centroradialis (cen) mutant forms a determinate inﬂorescence, but shows no early ﬂowering (Bradley et al. 1996). In
pea (Pisum sativum L.), two different mutants defective in
the PsTFL1a and PsTFL1c gene of the TFL1 group, determinate (det) and late ﬂowering (lf), lead to terminal ﬂower andearly ﬂowering, respectively (Foucher et al. 2003). This diversityof the function of the TFL1/CEN-like genes appears to berelated to the indication by Ahn et al. (2006) that a particular region in the fourth exon of the TFL1 gene has evolved
more rapidly than that in the FT gene. Therefore, the analyses
of TFL1/CEN-like genes will provide a novel explanation forthe manner in which higher plants have come to gain diversity in morphology and variation in ﬂowering time in avariety of species as well as provide useful information forthe molecular breeding of crops.
We previously reported that apple transgenic plants
expressing the antisense RNA of MdTFL1 showed precocious
ﬂowering (Kotoda et al. 2003, Kotoda et al. 2006), revealing that
MdTFL1 controls the transition from the juvenile/vegetative tothe reproductive phase in apple. However, it is suggested
that several TFL1/CEN-like genes would also be present in
the apple genome and might have different functions among
them. Therefore, we isolated and characterized other
TFL1/CEN-like genes from apple and elucidated the functional relationships among them.

Results
Four TFL1/CEN-like genes are present in the apple
genome
We attempted to isolate apple TFL1/CEN-like genes byscreening a genomic library and using a PCR strategy. As aresult, four TFL1/CEN-like genes were obtained, and they
were designated MdTFL1, MdTFL1a, MdCENa and MdCENb.
MdTFL1 and MdTFL1a corresponded to previously reported
cDNAs (Esumi et al. 2005, Kotoda et al. 2005), whereas
MdCENa and MdCENb were novel genes of the FT/TFL1
family in apple. To assess the number of TFL1/CEN-like genes
in the apple genome, genomic Southern hybridization was
performed using the partial sequence of MdTFL1 or MdCENa
as a probe. Two to four major hybridizing bands weredetected in each lane of DNA digested with EcoRI, HindIII or
XbaI (Fig. 2A). The genomic regions to be hybridized withthe probes for MdTFL1 and MdTFL1a contained no sites of
the restriction enzyme used for digestion. On the other
hand, that for MdCENa had a HindIII site, and that for MdCENb
had a HindIII site and an EcoRI site (Fig. 2B). In view of these
considerations, there are at least two distinct TFL1-like genes

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

Four TFL1/CEN-like genes in apple

A
(kb)

EI

H

XI

EI

H

XI

23.1
9.42
6.56
4.36
2.32
2.02

0.56

MdTFL1

MdCENaBtc (14 repeats)+1

C1 C2 C3500 (bp)

C4

MdTFL1EI

H

XI

H
Probe site

MdTFL1a

XI

EI

EI

ag (12 repeats)+1

C1C2C3
EI

HIS1

H H -IS1′

C4

IS1″

H

Probe siteag (21 repeats)+1
MdCENa
H

XI

EI

H

ct (5 repeats)+1
MdCENb
XI

H EI

H XI

C
Consensus sequence
C 1 GCTWCTTTGAGYSATTGAGATGCCCTCTCWGCACKACTAGCAGCTTGGCAGSAGGCACTCAGAGAGCAGGTGGAGYTCCTTTATGCTCTTGKGMCTCMTC
C 2 ACAGTATATTTCCATTTCAAAATTTKYRTTACCCTAAMTGCMAAAGATRAGAKTTTCCYTKATTAGATTGTGAMTGTTCAGGAAKAATTCTAGGCAWTTTGYTGTTTTT
C 3 GRATTATATAATGATTTTCTGGCCTAWCTAAGGAAAAACATCCCCCTTWWTTTTTGAWMAAAAWAWTCAATAATTAGGTTTACAACCAATCAAGAACTAATTCCAAGGRKCC
R (A/G), Y (C/T), M (A/C), K (G/T), S (G/C), W (A/T)

Fig. 2 The apple genome has four TFL1- and CEN-like genes. (A) Genomic Southern blot analysis for MdTFL1 (left) or MdCENa (right). The
genomic DNA (15 µg) of the ‘Fuji’ apple was digested with EcoRI (EI), HindIII (H) or XbaI (XI) and subjected to hybridization. The molecular size
markers are shown in kb on the left. (B) Schematic representation of the gene structures and restriction maps of four apple TFL1/CEN-like genes.
The genomic sequences determined in this study are indicated by bars. The black boxes on the bars show exons. The relative positions of theinitiation codon (ATG) are shown as +1. The positions of the highly conserved sequence between MdTFL1 and MdTFL1a are shown by C1, C2, C3
and C4. The inverted repeat sequences in the MdTFL1a gene are indicated by IS1, -IS1′ and IS1″ (for sequences, see Supplementary Fig. S2B). The
position and sequence direction of IS1, -IS1′ and IS1″ are indicated by arrows. The SSR shown by tc (14 repeats), ag (12 repeats), ag (21 repeats)
and ct (5 repeats) was used as a marker for the genome mapping of MdTFL1, MdTFL1a, MdCENa and MdCENb, respectively. The probe regions
used in the Southern blot are shown as bars indicated by vertical arrows on MdTFL1 and MdCENa. The scale bar on the map represents
approximately 500 bp. (C) The consensus sequence of C1, C2 or C3 (for the consensus sequence of C4, see Supplementary Fig. S2A).

(MdTFL1 and MdTFL1a) and two CEN-like genes (MdCENa
and MdCENb) in the apple genome.
The coding region of MdTFL1 exhibited 93.6% identity tothat of MdTFL1a at the nucleotide level, and these genes
consisted of four exons, 198, 62, 41 and 218 bp, encoding aputative protein of 172 amino acids. However, the length of
the second and third introns was considerably different

between MdTFL1 and MdTFL1a (Fig. 2B). To examine
whether conserved sequences exist in the non-coding
regions of MdTFL1 and MdTFL1a, we searched the regions
using MEME (the Multiple Em for Motif Elicitation), revealing that three conserved sequences were found in the putative promoter regions, which were represented as C1, C2 andC3 (100–112 bp in length, respectively). C1, C2 and C3 werePlant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

397

N. Mimida et al.

localized tandemly in the region between –2,075 and–1,436 in MdTFL1 and between –2,075 and –1,436 in MdTFL1a, exhibiting 90.0, 88.1 and 90.2% identity, respectively,
with the corresponding motifs of the two genes (Fig. 2B, C).
In addition, there were conserved sequences in the
5′-ﬂanking regions of MdTFL1 and MdTFL1a, designated C4(–450 to –72 of MdTFL1 and –396 to –1 of MdTFL1a, respectively), which had 79% identity (Supplementary Fig. S2A).
Furthermore, inverted sequences were found in MdTFL1a,
represented as IS1, -IS1′ and IS1″ (Fig. 2B). The sequences ofIS1, -IS1′ and IS1″ were present in the putative promoter
region between –1,600 and –1,440, in the third intron
between +1,187 and +1,339, and in the third intron between+1,858 and +2,013, respectively. They exhibited 66.9–70%
identity at the nucleotide level among them (Supplementary Fig. S2B). The MdTFL1 gene contained a simple
sequence repeat (SSR) of tc (14 repeats) in the 5′-non-coding
region and the MdTFL1a gene contained a SSR of ag(12 repeats) in the 3′-non-coding region (Fig. 2B). On theother hand, the coding region of MdCENa exhibited 95.6%
identity to that of MdCENb at the nucleotide level. These
genes consisted of four exons, 204, 62, 41 and 218 bp, encoding a putative protein of 174 amino acids. The MdCENa genecontained an SSR of ag (21 repeats) in the third intron,
whereas a similar SSR was not found in the third intron of
MdCENb (Fig. 2B).

Four TFL1/CEN-like genes are localized in a different
linkage group
To assign the TFL/CEN-like genes in the apple linkage group(LG), genetic mapping was carried out using the SSRs present in each gene as DNA markers. The position of the target
SSR sequence in each gene is shown in Fig. 2B. By analyzing
two sets of seedlings from the cross between ‘Ralls Janet’(Ra) and Mitsubakaido (Malus sieboldii Rehder) and between‘Delicious’ (De) and Mitsubakaido as the mapping populations (Igarashi et al. 2008), we assigned MdTFL1 to the northend of LG 12 of ‘Ralls Janet’ (Ra12) and to a region close
to markers rD05A20-0.9 and E35M48-350 on LG 12 of‘Delicious’ (De12), and MdTFL1a to the north end of LG 14(De14) and to a region between markers E38M61-180 andE37M62-195d on LG 14 (Ra14), respectively (Fig 3A). On theother hand, MdCENa was assigned to the middle of LG 03(Ra03 and De03), and MdCENb was assigned between
E37M49-900 and CH02d08-L/S on LG 11 (Ra11) and aroundE32M59-180, rD11D66-0.85 and CH02d08-L/S on LG 11(De11) (Fig. 3B).

Relationship among the genes of the TFL1/CEN
group in dicot plants
We carried out the alignment and phylogenetic tree analysis
using the deduced amino acid sequences of the TFL1/CEN-like398

gene together with our isolated genes. As a result, MdTFL1
and MdTFL1a were more similar to TFL1 (84 and 83% similarity, respectively) than to CEN (82 and 81%, respectively).
On the other hand, MdCENa and MdCENb were more similar to CEN (85 and 86%, respectively) than to TFL1 (83%).
Alignment of the deduced amino acid sequences of the
TFL1/CEN-like gene showed that the His88 and Asp139 inapple TFL1/CEN-like proteins, which are amino acids characteristic of TFL1/CEN-like proteins, were conserved among
other plant species (Fig. 4A, see asterisks). The substitutionsof valine (V) with phenylalanine (F) or cysteine (C) at position 73 in MdTFL1 or MdTFL1a, respectively, were characteristic of TFL1 group members in the Maloideae species of the
Rosaceae family (Fig. 4A, see an arrow). The apple TFL1/
CEN-like proteins were classiﬁed into two distinct groups,
called ‘TFL1’ and ‘CEN’ on the phylogenetic tree (Fig. 4B).
MdTFL1 and MdTFL1a belonged to the TFL1 group members in the Maloideae of the Rosaceae. MdCENa and MdCENb
showed the highest similarity (91%) to VvTFL1A, a TFL1/
CEN ortholog of grapevine (Vitis vinifera L.), among TFL1/
CEN-like proteins in the phylogenic tree (Fig. 4B).

Four TFL1/CEN-like genes show different expression
patterns in various tissues and apical buds
throughout the growing season in apple
Expression analysis by reverse transcription–PCR (RT–PCR)
was performed in various tissues for four apple TFL1/CENlike genes. MdTFL1 was expressed mainly in apical buds of
FBS and cultured tissues (shoots) derived from adult trees
and apical buds of vegetative shoots, stems and roots of
14-day-old seedlings in the juvenile phase, whereas MdTFL1a
was expressed mainly in stamens, peduncles and apical budsof FBS and apical buds of vegetative shoots, stems and rootsof seedlings (Fig. 5A). On the other hand, the transcripts of
MdCENa were detected in petals, fruit receptacles, cultured
tissues and roots whereas those of MdCENb could not be
detected in any tissues at the 35 cycles of PCR (Fig. 5A)
although RT–PCR Southern analysis revealed that theexpression pattern of MdCENb was similar to that of MdCENa
with a considerably lower expression level. To conﬁrm theexpression of MdCEN (MdCENa and MdCENb), an additional
RT–PCR was performed using common PCR primers for
MdCENa and MdCENb, reﬂecting the expression pattern of
MdCENa (Fig. 5A). These expression patterns examined
were conﬁrmed by RT–PCR Southern analysis, where transcripts at the lower expression level could be detected.
To examine whether the expression levels of the TFL1/
CEN-like genes change in the apical buds of SS and FBS
throughout the growing season, RT–PCR analysis was
carried out at different PCR cycles using total RNAs extracted
from the apical buds of SS and FBS of the apple cultivar‘Jonathan’ from June to October in 2004. It is noteworthy

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

Four TFL1/CEN-like genes in apple

A
Ra12

De12
−
−
+/−
+
+
−
+/−
−
+
+
+
−
+

0.0
3.8
9.5
12.0
13.2
14.4
15.6
17.9
22.9
27.7

MdTFL1
E36M50-135
NZ28f04-L/S
E41M48-395
rD12D77-0.8
E32M49-900CH05g07-L2/S2
E35M48-295
E35M48-190
rD12D79-0.1
rD09A01-0.8
E35M48-195
E37M62-300

Ra14
0.0
1.8

rD05A20-0.9 +E35M48-350 +MdTFL1 +0.0
4.8

NZ28f04-L/S +/−E41M48-395 +

12.4
13.9

E35M48-195 −E40M49-200 +

23.6
26.3

CH01f02-L/S −/+33.4

rD09D49-1.0 +E33M61-100 +

41.6
43.7

OPD20-0.25 −

57.1

De14
+
−
+
+
−
+
+
+
−/+
+
−
+
+/−
+/−
+
+
+
−
−/+
+
+
+
−
+
+
−

E36M50-140
E38M61-180 *MdTFL1aE37M62-195d *E36M48-300 **E40M61-110 *E36M50-850 *E38M50-160ud *CH05g07-L1/S1 *rD14Z06-1.2
E32M48-1000
E33M48-650
CH05g11-L/N
CH05e05-L/S
rD15D10-0.2
rD15C42-0.8
E38M61-560
E32M60-350u
CH05d03-L/S
E40M49-160
rD12C87-0.5
rD12D77-0.5
rD03C96-1.5
E41M61-230
rD07B01-1.7
rD05B45-0.5

3.7
4.3
6.2

+/−
−/+
+
−
+
−
+

CH04h02-L1/S1
CH04h02-L2/S2
E33M60-210d
E33M60-210u
rD14B76-1.4
rD06A55-0.75
E37M49-900

15.2

−

MdCENb

22.0
25.0
27.8
30.2
32.2
35.2
38.2

+/−
+/−
+
+
+/−
+
+
−
−
+
+
+
+
+
+
+
−

CH02d08-L/S
CH02d12-L/S
rD14D44-0.7
E41M48-90
CH04a12-L/S
rD15B09-0.3
E40M48-490
E41M61-290
E40M47-520
rD11C34-0.3
rD11D21-1.0
rD12D79-0.5
rD14D85-0.8
E36M48-170
E35M49-490d **E35M49-210 **E32M59-240u

2.7
4.0
5.2
7.7
8.9
12.1
18.6
25.4
26.0
28.8
30.9
37.4
38.3
41.8
43.6
48.6

49.8

MdTFL1a +E35M50-350 +E40M48-500u +E40M61-110 −rD15F09-0.5 −rD14B37-1.2 +CH05g07-L/S +/−E36M50-160 −CH01g05-L/S +/−rD14D64-0.7 −

0.0
4.6
5.9
6.9

CH05e05-L/S +/−CH03g04-L/S −/+CH05g11-L/S +/−S1115He0.65 +CH03a02-L/S +/−CH05d03-L/S +/−

29.4
30.9

rD12C87-0.5 −
MdMADS3-L/S −/+E41M61-230 −

42.5
46.9
47.0

E38M48-350 +

54.2

9.4
12.3
18.9

32.3
33.7

B
Ra03
0.0
2.0
3.0
10.5
11.4
15.1
17.5
18.0
19.2
20.4
24.2
26.8
29.3
33.0
35.6
44.8
46.0
48.5

De03
+/−
+
−
+
−
−
−
−
+
+
+
−
−
−
+
+
+
−
−
+
+/−
+
−
−/+

CH03e03-L/S
E38M49-270
E35M49-270u
E35M61-160
E33M48-700
rD14D85-1.1
S1416RI0.5
E36M50-375
E32M62-420
E40M62-600
MdCENa
E32M59-500
S0845Pt1.2
E33M48-350
E37M47-210
rD14D00-1.2
E41M61-350
E36M50-290
E36M48-110
E36M48-1000
MS14h03-L/S
E35M48-700
F201RV0.75
CH03g12-L1/S1

Ra11
0.0

CH03e03-L/S −/+E35M61-160 +CH03g07-L/S −/+E38M47-110 +E35M49-270u −E38M49-270 +E33M60-250 +

0.0

14.6
16.0
17.4

−
−
+
+
+

31.8

MS14h03-L/S +/−44.8

MdCENaS0845Pt1.2
E33M48-170
E37M47-210
rD14D00-1.2

F201RV0.75 −CH03g12-L1/S1 −/+rD14C76-0.6 +

1.2

42.7

55.3
58.1

43.1
43.5
50.4
51.7
57.2

De11

69.0

−

rD04B66-1.3

74.3

+

E38M50-230 *

80.6
81.7
84.2

+ E37M50-105+/− CH04d07-L/N+ E37M47-250

CH04h02-L1/S1 +/−CH04h02-L2/S2 −/+E33M60-210u −E33M61-265 +rD13D52-0.7 +rD11D97-0.8 +E37M61-410 +rD13C73-0.5 +rD05B66-1.3 +E32M59-180 +MdCENb −rD11D66-0.85 +CH02d08-L/S −/+rD08D72-1.0 +CH02d12-L/S +/−E40M48-490 −* E38M50-90 +* rD12A56-0.7 +* E36M50-140 +* E38M47-190 +* rD14D58-0.6 −* E41M62-95 −* E41M61-290 −* rD11C34-0.3 +* rD11D21-0.9 +* rD15B09-0.8 −E40M47-520 −F108Bm0.2 +rD05D66-0.7 −rD13B66-0.5 −CH04g07-L/S +/−rD04B66-1.3 −rD12B19-0.5 −* E37M50-105 +* E37M50-95 −* CH04d07-L/S +/−E40M47-130 +

0.0
1.4
2.8
3.4
6.1
9.6
13.7
15.2
17.6
23.7
24.7
27.8
30.1
45.6

56.6

56.9
58.0
64.9
69.1
70.5
73.3
77.6

Fig. 3 Location of the TFL1/CEN-like genes on the linkage maps of the apple cultivars ‘Ralls Janet’ (Ra) and ‘Delicious’ (De). (A) Location of
MdTFL1 and MdTFL1a on linkage groups 12 (Ra12, De12) and 14 (Ra14, De14), respectively. (B) Location of MdCENa and MdCENb on linkage
groups 3 (Ra03, De03) and 11 (Ra11, De11), respectively. The DNA markers on each linkage group are described in detail by Igarashi et al. (2008).
Segregation ratios deviating signiﬁcantly from the expected ratios (P < 0.05, 0.01) are indicated with one or two asterisks, respectively. The linkage
phase information is provided as + or –, indicating on which of the homologous chromosomes the marker/allele is located.

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

399

N. Mimida et al.

A

MdTFL1
MdTFL1a
MdCENaMdCENbVvTFL1A
VvTFL1B
TFL1
ATC
CEN

1 : ...MKRAS.EPLVVGRVIGDVLDSFTATTKMSVTYNTK....LVCNGLELFPSVVTAKPRV :1 : ...MARVP.EPLVVGRVIGDVLDSFTPTTHMSVTYNAK....LVCNGLELFPSVVTAKPRV :1 : .....MLSSDPLVVGRVIGDVVDYFSPSVKMTVSYNSN...KKVYNGHELFPSSVTIKPKV :1 : .....MMSSDPLVVGRVIGDVVDYFSPSVRMTVTYNSN...KKVYNGHELFPSSVTIKPKV :1 : ...MARMS.DPLIVGRVIGDVVDSFCSTVKMTVTYNSN...KQVYNGHELFPSSVTIKPKI :1 : ...MARIL.EPLIVGRVIGDVLDPFPPTIKMTVTYHNK....QICNGYELYPSSITVKPRV :1 : MENMGTRVIEPLIMGRVVGDVLDFFTPTTKMNVSYNKK....QVSNGHELFPSSVSSKPRV :1 : ...MARISSDPLMVGRVIGDVVDNCLQAVKMTVTYNSD...KQVYNGHELFPSVVTYKPKV :1 : ..MAAKVSSDPLVIGRVIGDVVDHFTSTVKMSVIYNSNNSIKHVYNGHELFPSAVTSTPRV :

53
53
53
53
54
53
57
55
59

MdTFL1
MdTFL1a
MdCENaMdCENbVvTFL1A
VvTFL1B
TFL1
ATC
CEN

54 : EIQGGDMRSFFTLVMTDPDFPGPSDPYLREHLHWIVTDIPGTTDATFGREVVSYEMPKPNI : 114
54 : EIQGGELRSFFTLVMTDPDCPGPSDPYLREHLHWIVTDIPGTTDAAFGREALSYEMPRPNI : 114
54 : EVHGGDLRSFFTLVMTDPDVPGPSDPYLKEHLHWIVTDIPGTTDNTFGMEVVKYEMPRPNI : 114
54 : EVHGGDLRSFFTLVMTDPDVPGPSDPYLKEHLHWIVTDIPGTTDNTFGKEVVKYEMPRPNI : 114
55 : EVEGGDMRSFFTLIMTDPDVPGPSDPYLREHLHWIVTDIPGTTDSTFGREIVNYEMPRPNI : 115
54 : EVQGGDLRSFFTLVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGREVLSYEIPKPNI : 114
58 : EIHGGDLRSFFTLVMIDPDVPGPSDPFLKEHLHWIVTNIPGTTDATFGKEVVSYELPRPSI : 118
56 : EVHGGDMRSFFTLVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDVSFGKEIIGYEMPRPNI : 116
60 : EVHGGDMRSFFTLIMTDPDVPGPSDPYLREHLHWIVTDIPGTTDSSFGKEVVSYEMPRPNI : 120**

MdTFL1 115 : GIHRFVFVLFKQNQR.QSINTP..SSRDHFSTRSFAAENDLGLPVAAVYFNAQRETAARRR : 172
MdTFL1a 115 : GIHRFVFVLFKQKRR.QSINIP..SSRDCFSTRSFAAENDLGLPVAAVYFNAQRESAARRR : 172
MdCENa 115 : GIHRFVFLLFKQKGR.QTVIPP..ASKDHFNTRKFAEANDLGLPVTAVFFNAQRETAARRR : 172
MdCENb 115 : GIHRFVFLLFKQKAR.QTVIPP..ASKDHFNTRKFAEANDLGLPVTAVFFNAQRETAARRR : 172
VvTFL1A 116 : GIHRFVFLLFKQKRR.QTVNPP..SSRDRFSTRNFAEENELGPPVAAVFFNAQRETAARKR : 173
VvTFL1B 115 : GIHRFVFVLFKQKRR.QTVNTP..TSRDHFNTRSFAAENDLGLPVAAVFFNAQRETAARRR : 172
TFL1
119 : GIHRFVFVLFRQKQR..RVIFPNIPSRDHFNTRKFAVEYDLGLPVAAVFFNAQRETAARKR : 177
ATC
117 : GIHRFVYLLFKQTRRGSVVSVP..SYRDQFNTREFAHENDLGLPVAAVFFNCQRETAARRR : 175
CEN
121 : GIHRFVFLLFKQKKRGQAMLSPPVVCRDGFNTRKFTQENELGLPVAAVFFNCQRETAARRR : 181

Norway spruce
PaFT4

B
Maloideae

TFL1 group(TFL1-like)tomatoSP9D

MdTFL1apple
PpTFL1-1
MdTFL1a
CoTFL1-1 EjTFL1-2 PpTFL1-2
orangeCsTFL1-1
CsTFL1-2
CsTFL
grapevine
CoTFL1-2
VvTFL1B EjTFL1-1
tobacco
CET5

pea
PsTFL1cPsTFL1bpea
ATCArabidopsis

pea
PsTFL1aBrassicaceae
VvTFL1A
grapevine

BrTFL1-2
BrTFL1-1
BnTFL1-2
BnTFL1-1
BoTFL1-2
Brassica
BoTFL1-1

MdCENaMdCENb apple

TFL1
Arabidopsis
CET4 CET2
tobacco0.05

Fig. 4400

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

CEN
Arabidopsis
SP
tomatoCEN group(CEN-like)Four TFL1/CEN-like genes in apple

that the ﬂowering stage of apple was mid-May, and thetiming of ﬂoral bud differentiation for the following spring
was early July in Morioka, Japan (Kotoda et al. 2000). In theapical buds of SS, the expression of MdTFL1 was maintained
throughout the actively growing season and gradually
decreased after mid-September (Fig. 5B). MdTFL1a was also
expressed between June and October, with the transcriptional signals showing a broad monophasic peak (Fig. 5B). In
contrast, both the transcripts of MdCENa and MdCENb wereundetectable even by Southern blot after RT–PCR ampliﬁcation (data not shown). In the apical buds of FBS, strong
expression of MdTFL1 was observed in mid-June at no morethan 30 cycles of PCR, with the signals gradually decreasingfrom late June (Fig. 5C). The expression pattern of MdTFL1ashowed three peaks in early June, late August and October(Fig. 5C), although the expression level of MdTFL1a seemed
to be relatively lower than that of MdTFL1. On the other
hand, MdCENa expression signals were considerably lower
even at 35 cycles of PCR, although the expression pattern of
MdCENa was somewhat similar to that of MdTFL1a exceptthat the second peak of the expression of MdCENa was inlate July (Fig. 5C). With regard to MdCENb, the expression
could not be detected even by Southern blot after RT–PCR
ampliﬁcation (data not shown).

Spatial expression patterns of MdTFL1 and MdCENa
with the β-glucuronidase (GUS) reporter gene in
Arabidopsis
Because apple is a woody perennial plant, it is difﬁcult toobserve a spatial gene expression pattern in the whole plant
throughout its life cycle. Therefore, to examine the spatial
expression patterns of MdTFL1 and MdCENa, we generated
four types of transgenic Arabidopsis plants with the GUS
reporter gene driven by the regulatory sequences of putative
promoters (designated pMdTFL1:GUS and pMdCENa:GUS)or genomic fragments of those genes (designatedgMdTFL1:GUS and gMdCENa:GUS) (Fig. 6A). Consequently,
we obtained >30 independent transgenic lines for each GUS
fusion transgene. Among them, representative transgeniclines, pMdTFL1:GUS (#3), gMdTFL1:GUS (#9), pMdCENa:GUS(#2) and (#3) and gMdCENa:GUS (#11), were selected, and
the GUS staining patterns of these plants were observed
throughout the developmental stages from seed to adultplant. In pMdTFL1:GUS (#3), no GUS expression was observedin the mature seed (Fig. 6B), 36-h-old germinating seed
(Fig. 6C), and 52-h-old seedling (Fig. 6D). After 82 h, GUS
expression was ﬁrst detected weakly in the root–hypocotyltransition zone (Fig. 6E). In 5-day-old seedlings, strong GUS
expression was observed in the hypocotyl and vascular tissueof the primary root but not in the elongation andmeristematic zone (Fig. 6F). In a 16-day-old plant, GUS
expression was clearly detected in the vegetative apicalmeristem, hypocotyl and roots developed in the early stage
but rarely in the lateral roots (Fig. 6G, I). Upon progressionof development, GUS expression was not detected in thewhole inﬂorescence, including the reproductive organs (datanot shown). In gMdTFL1:GUS lines, the spatial patterns of
GUS expression were similar to those in pMdTFL1:GUS lines,
although GUS was rarely expressed in the vegetative
meristems (Fig. 6H, J).
On the other hand, in the pMdCENa:GUS lines, there were
two types showing relatively different levels of GUS expression (higher or intermediate). In pMdCENa:GUS (#2), whichshowed a higher level of GUS expression as a whole, no GUS
expression was observed in the mature seed (Fig. 6K). GUS
staining was ﬁrst detected weakly in cotyledon tips immediately after germination (Fig. 6L), and then weak GUS expression was detected in the root–hypocotyl transition zone
52 h after sowing (Fig. 6M). At 82 h after sowing, GUS expression appeared in the specialization zone of the primary root
(Fig. 6N). In 5-day-old seedlings, strong GUS expression wasobserved in the primary root and the tips of expanded
cotyledons (Fig. 6O). In a 16-day-old plant, GUS expression
was also strongly detected in the tips of true leaves and bothprimary and lateral roots (Fig. 6P). The GUS expression
patterns of pMdCENa:GUS (#3) which showed a intermediate level of GUS expression and gMdCENa:GUS (#11) weresimilar to that of pMdCENa:GUS (#2). However, the GUS

Fig. 4 Relationship among TFL1/CEN-like proteins found in dicot plants. (A) Comparison of the deduced protein sequence of MdTFL1, MdTFL1a,
MdCENa and MdCENb (accession Nos. AB052994, AB366643, AB366641 and AB366642, respectively) with those of TFL1 (Arabidopsis; Bradley
et al. 1997, Ohshima et al. 1997), CEN (snapdragon, Bradley et al. 1996), and VvTFL1A (VvTFL1) and VvTFL1B (grapevine; Boss et al. 2006, Carmona
et al. 2007). Sequence identities among TFL1- and CEN-like proteins are shown on a black background. 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). The bracket represents the region of a potential ligand-binding pocket in TFL1/CEN-like proteins (Ahn et al. 2006). The triangles show the
intron positions, which are conserved in TFL1/CEN-like proteins. The arrow indicates the position of the conserved amino acids in the membersof the TFL1 group in the subfamily Maloideae of the Rosaceae. (B) Phylogenetic analysis of TFL1- and CEN-like proteins from apple, Arabidopsis,
Brassica spp. (Brassica napus L., Brassica oleracea L. and Brassica rapa L.), Chinese quince (Chaenomeles sinensis Koehne.), grapevine, Japanesepear [Pyrus pyrifolia (Burm.) Nak.], loquat [Eriobotrya japonica (Thunb.) Lindl.], orange (Citrus sinensis L. Osbeck), pea, quince (Cydonia oblonga
Mill.), tobacco and tomato (Solanum lycopersicum L.). The unit for the scale bar displays branch lengths (0.05 substitutions/site). The protein
sequence data were obtained from the DNA database (DDBJ/EMBL/GenBank). For PsTFL1b, the sequence information of partial cDNA was used.
The conifer Norway spruce (Picea abies L. Karst.) FT homolog, PaFT4, was used as an outgroup.

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

401

N. Mimida et al.

Roots

Stems

VS

MLs

CTs

FBS(Aug)

MLs

FruitsJuvenile

Peels

FRs

Carpels

Stamens

Petals

SepalsSeeds

Peduncles

Adult

A

35 cycles

MdTFL1blot
35 cycles
MdTFL1ablot
35 cycles

MdCEN

blot
35 cycles

MdCENablot
35 cycles

MdCENbblot

Histone H3

30 cycles

Succulent shoot apices (Jonathan)
Jun/6
Jun/20Jun/27
Jul/18
Jul/24
Jul/31
Aug/7
Aug/18Aug/22
Aug/29Sep/4
Sep/12
Sep/19Sep/26
Oct/3
Oct/10Oct/24B

30 cycles
MdTFL135 cycles
30 cycles

MdTFL1a35 cycles
32 cycles

Histone H3
Bearing shoot apices (Jonathan)Floral bud
differentiation
Jun/6
Jun/20Jun/27
Jul/18
Jul/24
Jul/31
Aug/7
Aug/18Aug/22
Aug/29Sep/4
Sep/12
Sep/19Sep/26
Oct/3
Oct/10Oct/24C

30 cycles
MdTFL135 cycles
30 cycles

MdTFL1a35 cycles
35 cycles

MdCENablot
Histone H3

32 cycles

Fig. 5 Expression analyses of apple TFL1/CEN-like genes. (A) Expression patterns of four apple TFL1/CEN-like genes in various tissues of ‘Fuji’ apple
and seedlings (‘Fuji’ × ‘Orin’). The samples from left to right are as follows: seeds, sepals, petals, stamens, carpels, peduncles, fruit receptacles (FRs),
fruit peels, fruits, mature leaves (MLs) and apical buds of FBS on August 7 [FBS(Aug)] in the adult phase; cultured tissues (CTs); and MLs, apicalbuds of vegetative shoots (VS), stems and roots of 2-week-old seedlings in the juvenile phase. For the CTs, shoots in the culture box 1 month after
subculture were collected and used for the experiment. (B) RT–PCR products of MdTFL1 or MdTFL1a in apical buds of SS of ‘Jonathan’ apple from
June to October in 2004. (C) RT–PCR products of MdTFL1, MdTFL1a or MdCENa in apical buds of FBS of ‘Jonathan’ apple from June to Octoberin 2004. The number of PCR cycles is indicated at the right of each panel. RT–PCR Southern blot (indicated as ‘blot’ at the right of A and C) was
carried out to conﬁrm the results of RT–PCR analysis. Vertical arrows indicate the timing of ﬂoral bud differentiation of ‘Jonathan’ apple in (C).
Apple Histone H3 was used as an internal control of gene expression.

402

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

Four TFL1/CEN-like genes in apple

A −2kb+1b ATG

MdTFL1 genepMdTFL1 GUS

GUS

pSMAK312Blue

gMdTFL1 GUS

GUS

pBI101
MdCENa gene
pMdCENa GUS

GUS

pSMAK312Blue

gMdCENa GUS

GUS

pBI101B

C

pMdTFL1 #3

F

G

pMdTFL1 #3

D

E

pMdTFL1 #3

pMdTFL1 #3

pMdTFL1 #3

H

I

pMdTFL1 #3

K

pMdTFL1 #3

J

gMdTFL1 #9

O

pMdCENa #2

M

gMdTFL1 #9

P

pMdCENa #2

L

pMdCENa #2

N

pMdCENa #2

Q

pMdCENa #2

pMdCENa #2 pMdCENa #3

S

R

U

pMdCENa #3

T
gMdCENa #11
gMdCENa #11

gMdCENa #11

Fig. 6 Histochemical analysis of transgenic Arabidopsis plants carrying the β-glucuronidase (GUS) gene fused with MdTFL1 or MdCENa. (A)Schematic representation of vector constructs for pMdTFL1:GUS, gMdTFL1:GUS, pMdCENa:GUS and gMdCENa:GUS; (B) mature seed of pMdTFL1(#3); (C) 36-h-old germinating seed of pMdTFL1 (#3); (D) 52-h-old seedling of pMdTFL1 (#3); (E) 82-h-old seedling of pMdTFL1 (#3); (F) 5-day-old
seedling of pMdTFL1 (#3); (G) 16-day-old seedling of pMdTFL1 (#3); (H) 16-day-old seedling of gMdTFL1 (#9); (I) vegetative meristem (VM) in a
16-day-old seedling of pMdTFL1 (#3); (J) VM in a 16-day-old seedling of gMdTFL1 (#9); (K) mature seed of pMdCENa (#2); (L) 36-h-old germinating
seed of pMdCENa (#2); (M) 52-h-old seedling of pMdCENa (#2); (N) 82-h-old seedling of pMdCENa (#2); (O) 5-day-old seedling of pMdCENa (#2);(P) 16-day-old seedling of pMdCENa (#2); (Q) 16-day-old seedling of pMdCENa (#3); closed-up roots of (Q) are shown at the bottom on the right;(R) 18-day-old seedling of gMdCENa (#11); (S) VM in a 16-day-old seedling of pMdCENa (#3); (T) cauline inﬂorescence of gMdCENa (#11); (U)inﬂorescence of gMdCENa (#11). Transgenic plants in B–U were stained in a staining solution [1 mM 5-bromo-4-chloro-3-indolyl glucuronide(X-Gluc), 0.1 M sodium phosphate buffer at pH 7.0, 5% methanol, and 0.01% Silwet L-77 (Nippon Unicar)] at 37°C for 24 h.

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

403

A

Wild-type

404
35S:MdTFL1/pSMAK251

35S Ω:MdTFL1a/pSMAK193E
35S

35S

B

F

tfl1-1

MdTFL1

Ω

35S

5 mm

Wild-type

35S:MdTFL1/
tfl1-1(#2)

TrbcS

MdTFL1a
TrbcS

35S:MdCENa/pSMAK251

MdCENa
TrbcS

C
5 mm

D

G

35S Ω:MdTFL1a/
tfl1-1(#8)

1 mm

tfl1-1

E

20 mm
5 mm

H
5 mm

35S Ω:MdTFL1a/
wt (#17)
35S:MdTFL1/
tfl1-1(#2)
35S Ω:MdTFL1a/
tfl1-1(#8)

35S:MdCENa/
tfl1-1(#12)

35S:MdTFL1/
wt(#22)

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.
0.5 mm35S:
35S:MdCENa/
35S:MdCENa/wt(#18)
MdCENa/wt(
wt(#18)18)
35S:MdCENa/
35S:
35S:MdCENa/wt(#18)
MdCENa/wt(
wt(#18)
18)

I
5 mm

35S:MdCENa/t
fl1-1(#12)

J
5 cm

35S Ω:MdTFL1a/wt(#2)

35S:MdCENa/
wt(#5)

Fig. 7 Plant architecture of transgenic Arabidopsis with apple TFL1/CEN-like genes. (A) Schematic representation of the transformation vectors, 35S:MdTFL1, 35SΩ:MdTFL1a and
35S:MdCENa, used in this study. (B) Inﬂorescence of the wild type. (C) Inﬂorescence of tﬂ1-1. (D) Inﬂorescences of 35S:MdCENa/wt (#18). (E) Close-up view of the apical meristem of (D).
(F) Cauline leaves of 35SΩ:MdTFL1a/wt (#17). (G) Inﬂorescence of 35S:MdTFL1/tﬂ1-1 (#2). (H) Inﬂorescence of 35SΩ:MdTFL1a/tﬂ1-1 (#8). (I) Inﬂorescence of 35S:MdCENa/tﬂ1-1 (#12).
(J) Appearance of control wild-type and transgenic Arabidopsis plants. From left to right, wt, tﬂ1-1, 35S:MdTFL1/tﬂ1-1 (#2), 35SΩ:MdTFL1a/tﬂ1-1 (#8), 35S:MdCENa/tﬂ1-1 (#12),
35S:MdTFL1/wt (#22), 35SΩ:MdTFL1a/wt (#2) and 35S:MdCENa/wt (#5). Photographs of B, C, G–J at 35 d, D and E at 50 d, and F at 90 d after sowing. These plants were grown at 22°C
under LD conditions (16 h light/8 h dark).

N. Mimida et al.

Four TFL1/CEN-like genes in apple

expression of pMdCENa:GUS (#3) was detected speciﬁcallyin the bases and tips of the secondary roots (Fig. 6Q) and the
GUS expression in the roots of gMdCENa:GUS (#11) was too
weak to be detected (Fig. 6R). In aerial tissues, the GUS
expression of pMdCENa:GUS and gMdCENa:GUS wasdetected strongly in the leaf tips, developing leaf primordia,
stipules, axillary meristems on the base of rosette andcauline leaves, the junctions of siliques and pedicels (silique
receptacles) and the base of ﬂower stalks (Fig. 6R–U).

Constitutive expression of the apple TFL1/CEN-likegenes affect ﬂowering time and plant morphology
in Arabidopsis
To determine the effects of apple TFL1/CEN-like genes on
ﬂowering time and inﬂorescence morphology, we generated
transgenic Arabidopsis plants with MdCENa under the control of the cauliﬂower mosaic virus (CaMV) 35S promoter(35S) or with MdTFL1a under the control of the CaMV 35S
promoter fused with the Ω sequence (35SΩ) (Fig. 7A). Consequently, we obtained >20 independent transgenic lines for
35S:MdCENa and 35SΩ:MdTFL1a (identiﬁed as 35S:MdCENa/
wt and 35SΩ:MdTFL1a/wt, respectively). Transformantswith 35S:MdTFL1 (35S:MdTFL1/wt), as described in ourprevious paper, were used as a control for comparison
Table 1 The flowering time of transgenic plants (wild-type background) with 35S:MdTFL1, 35SΩ:MdTFL1a and 35S:MdCENa
StrainWt (Col)
35S:MdTFL1/wt (T3)
#21
#22
#28
35SΩ:MdTFL1a/wt (T2)
#2
#4
#13
#15
#17
35S:MdCENa/wt (T2)
#1
#2
#3
#5
#8
#10

No. ofplants20

No. of rosetteleavesa, b
6.8 ± 0.2

No. of cauline
leavesa, b
2.2 ± 0.1

99

10.9 ± 0.6
13.0 ± 0.6

3.0 ± 0.3
3.3 ± 0.2

10

15.8 ± 1.3

6.7 ± 0.3

10
7
10
10
15

9.3 ± 0.2
9.4 ± 0.4
9.5 ± 0.3
9.4 ± 0.2
8.3 ± 0.1

2.5 ± 0.2
2.6 ± 0.2
2.9 ± 0.2
2.7 ± 0.2
2.2 ± 0.2

10
10
6
10
8
5

10.4 ± 0.2
9.5 ± 0.3
9.0 ± 0.6
18.1 ± 1.5
8.5 ± 0.4
8.6 ± 0.4

2.6 ± 0.2
2.8 ± 0.2
2.7 ± 0.2
5.8 ± 0.6
2.4 ± 0.2
2.2 ± 0.2

Plants were grown on the potted soil at 22°C under the day length conditions of
16 h of light/8 h of darkness (long-day conditions).
aThe numbers of rosette and cauline leaves were counted as an indicator of
ﬂowering time.
bValues are the mean ± SE.

(Kotoda and Wada 2005). Five lines of 35S:MdCENa/wt and
35SΩ:MdTFL1a/wt in the T2 or T3 generation were grownunder long-day (LD) conditions (16 h light/8 h dark),
and their phenotypes were examined. The morphology
and ﬂowering time of the inﬂorescence were affectedin the 35S:MdCENa/wt and 35SΩ:MdTFL1a/wt lines.
35S:MdCENa/wt (#5) plants produced 18.1 ± 1.5 rosetteleaves, and 35SΩ:MdTFL1a/wt (#2) plants produced 9.3 ± 0.2
rosette leaves. In contrast, the wild-type plants produced
6.8 ± 0.2 rosette leaves (Table 1). In 35S:MdCENa/wt lines
with a strong phenotype, the inﬂorescences continued todevelop after bolting. The coﬂorescences were generated fromthe main axis by the conversion of ﬂoral organs into inﬂorescences (Fig. 7D). The inﬂorescences of 35SΩ:MdTFL1a/
wt with a strong phenotype were similar to those of
35S:MdCENa/wt and 35S:MdTFL1/wt, whereas the cauline
leaves of 35Ω:MdTFL1a/wt became more rounded thanthose of 35S:MdCENa/wt or 35S:MdTFL1/wt (Fig. 7F). The
three transgenic lines of 35SΩ:MdTFL1a/wt never ﬂowered
for >9 months (data not shown).
To investigate whether apple TFL1 homologs function
equivalently to Arabidopsis TFL1, we introduced the
35S:MdTFL1 (Kotoda and Wada 2005), 35SΩ:MdTFL1a or
35S:MdCENa transgene into Arabidopsis tﬂ1-1 mutant plantsthat show early ﬂowering and determinate terminal ﬂowering
under LD conditions (Fig. 7C, J and Table 2). Consequently,>20 kanamycin-resistant lines with 35S:MdTFL1,
Table 2 The ﬂowering time of transgenic plants (tﬂ1-1 background)with 35S:MdTFL1, 35SΩ:MdTFL1a, and 35S:MdCENa
StrainNo. ofplantsNo. of rosetteleavesa, b

No. of cauline
leavesa, b

Wt (Col)tﬂ1-1
35S:MdTFL1/tﬂ1-1 (T3)
#1
#2
#4
#5
35SΩ:MdTFL1a/tﬂ1-1 (T2)
#4
#7

20
20

6.8 ± 0.2
6.2 ± 0.2

2.2 ± 0.1
0.9 ± 0.1

15
15
14
15

12.5 ± 0.4
12.6 ± 0.3
12.6 ± 0.3
12.8 ± 0.6

4.7 ± 0.4
3.6 ± 0.2
5.4 ± 0.2
4.7 ± 0.315159.7 ± 0.3
7.5 ± 0.3

2.6 ± 0.1
2.0 ± 0.1

#8
35S:MdCENa/tﬂ1-1 (T2)
#10
#12
#13
#15

20

11.2 ± 0.3

4.3 ± 0.3

15
15
15
14

10.3 ± 0.3
11.7 ± 0.4
12.3 ± 0.6
14.7 ± 0.6

1.7 ± 0.2
3.7 ± 0.1
3.7 ± 0.4
6.1 ± 0.4

Plants were grown on the potted soil at 22°C under the day length conditions of
16 h of light/8 h of darkness (long-day conditions).
aThe number of rosette and cauline leaves were counted as an indicator of ﬂoweringtime.
bValues are mean ± SE.

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405

N. Mimida et al.

35SΩ:MdTFL1a or 35S:MdCENa were obtained (identiﬁed as
35S:MdTFL1/tﬂ1-1, 35SΩ:MdTFL1a/tﬂ1-1 and 35S:MdCENa/tﬂ1-1, respectively). Among them, four lines of 35S:MdTFL1/
tﬂ1-1, three lines of 35SΩ:MdTFL1a/tﬂ1-1 and four lines of
35S:MdCENa/tﬂ1-1 in the T2 or T3 generation were selected
and grown under LD conditions. In these transgenic lines, allinﬂorescences, including coﬂorescences, were basically maintained (Fig. 7G–I), and the ﬂowering time was more delayed
than that of tﬂ1 mutant and wild-type plants (Fig. 7J andTable 2). 35S:MdTFL1/tﬂ1-1 (#5) plants produced 12.8 ± 0.6
rosette leaves, 35SΩ:MdTFL1a/tﬂ1-1 (#8) plants produced
11.2 ± 0.3 rosette leaves and 35S:MdCENa/tﬂ1-1 (#12) plants
produced 11.7 ± 0.4 rosette leaves. In contrast, wild-type andtﬂ1-1 plants produced 6.8 ± 0.2 and 6.2 ± 0.2 rosette leaves,respectively (Table 2).

Discussion
Because the sequences in the coding regions of MdTFL1 and
MdTFL1a were too similar to clarify the functional difference
between the two genes by the knockdown strategy, we
inferred the function from their expression patterns. As aresult, relatively high levels of accumulation of both MdTFL1
and MdTFL1a transcripts were observed in tissues of apple
seedlings in the juvenile phase (Fig. 5A). In addition, both
MdTFL1 and MdTFL1a were expressed throughout thegrowing season in the apical buds of SS, which do not
produce ﬂower buds (Fig. 5B). These results suggested that
not only MdTFL1 but also MdTFL1a participates in the maintenance of the juvenile phase and vegetative shoot growththroughout the growing season, although the expression of
MdTFL1a was maintained longer in SS. This is also supportedby the phenotype of transgenic Arabidopsis expressing
MdTFL1a, which showed prolonged vegetative (V) and ﬁrstinﬂorescence (I1) phases (V phase, rosette leaves are produced; I1 phase, shoots with a subtending cauline leaf areproduced; phases deﬁned by Ratcliffe et al. 1998) similar tothat of transgenic Arabidopsis with MdTFL1 (Fig. 7J andTables 1, 2). In apical buds of FBS, MdTFL1 and MdTFL1awere both expressed in June, which is the period of vegetative growth, and the expression of these genes decreased
dramatically after late June, which is the transition period
from vegetative growth to reproductive growth in the adultphase, suggesting that these genes regulate the fate of theinﬂorescence meristems coordinately at the appropriate
time (Fig. 5C). These results are consistent with the fact thatthe shoot apical meristems stop producing any novel leaf
primordia or axillary meristems at the onset of ﬂoral transition in early summer (Foster et al. 2003). Since ﬂower buds ofapple are mixed, containing both vegetative and reproductive parts, the two peaks of the accumulation of MdTFL1atranscripts in late summer and autumn might be associatedwith the growth of vegetative or ﬂoral primordia inside the406

ﬂoral buds after the onset of ﬂoral transition (Fig. 5C).
Regarding MdTFL1, the expression patterns of MdTFL1 were
further supported by those of the GUS reporter gene intransgenic Arabidopsis lines with pMdTFL1:GUS andgMdTFL1:GUS (Fig. 6F–J). Heterologous GUS expression in
the vegetative apical meristem of transgenic Arabidopsis(Fig. 6I) was consistent with the result of RT–PCR (Fig. 5A).
In future studies, GUS expression analysis using transgenic
apple with pMdTFL1:GUS or gMdTFL1:GUS will be required.
The result of DNA blot and RT–PCR analysis and the phenotype of 35S:MdTFL1/wt obtained in this study on MdTFL1,
which was used as a comparison with the other apple
TFL1/CEN-like genes, was consistent with that of a previous
study (Kotoda and Wada 2005).
On the basis of comparisons of MdTFL1 with MdTFL1a,four conserved sequences were found in the putative
promoter regions (C1, C2 and C3) and ﬂanking regions close
to the initiation codon (C4) (Fig. 2B, C). These common
motifs may reﬂect the similar expression proﬁles of MdTFL1
and MdTFL1a in the vegetative tissues. Interestingly, thethird intron in the MdTFL1a gene was 10-fold longer thanthat of MdTFL1 and contained two inverted sequences (-IS1′
and IS″, Fig. 2B). Moreover, a sequence similar to -IS1′ andIS1″ was also found in the putative MdTFL1a promoter
region (IS1, Fig. 2B). The IS1 sequence was found to be wellaligned with several expressed sequence tag (EST) clones inthe DNA database derived from various tissues of apple, suggesting that active IS1-like sequences are present widely inthe apple genome (data not shown). Recent studies revealed
that small non-coding RNAs (ncRNAs) play a major role inregulating gene expression and that ncRNA genes coding
functional RNA molecules are often found in the introns(Brown et al. 2003). It is possible that inverted sequences,such as the IS1 element in the MdTFL1a gene, become target
sequences for the transcriptional regulator or are related tothe intronic ncRNA and play a role in regulating gene
expression.
The exon–intron organization, coding regions and expression patterns of MdCENa and MdCENb in the CEN groupwere highly similar. In addition, MdCENa and MdCENb werelocated at a similar position on LG 3 and LG 11 of apple linkage groups, respectively (Fig. 3B). However, the upstream
putative promoter sequences of these genes were considerably different, and the expression level of MdCENb was much
lower than that of MdCENa (Fig. 5A). Based on these results,
the function of MdCENb may have been lost during evolution, probably due to gene duplication by the polyploidorigin of the Maloideae (Sax 1933). On the other hand,
MdCENa was expressed in reproductive organ tissues such as
fruit receptacles, as well as in vegetative tissues, such as roots
and cultured tissues, unlike MdTFL1 and MdTFL1a. In addition, the expression of MdCENa could not be detected in theapices of SS and vegetative shoots of seedlings (Fig. 5A, B).

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

Four TFL1/CEN-like genes in apple

These results suggest that MdCENa would function in a different manner from MdTFL1 and MdTFL1a, although
MdCENa seemed to have the potential to complement thefunction of TFL1-like genes and maintain shoot growth fromthe analysis of the transgenic Arabidopsis expressing MdCENa
in the wild-type and tﬂ1 mutant background (Fig. 7 andTables 1, 2). Transgenic tobacco plants constitutively
expressing CEN from Antirrhinum showed a prolonged vegetative phase, but those expressing TFL1 from Arabidopsis
did not show an altered phenotype (Amaya et al. 1999).
Additionally, Ahn et al. (2006) demonstrated that a chimeric
fusion protein of TFL1 replaced with a 17 amino acidsegment (called segment B) in the fourth exon of CEN could
function like CEN in tobacco, suggesting that segment B isessential for the expression of CEN function. It is interestingthat VvTFL1 (VvTFL1A) from grapevine, showing high similarity to MdCENa and MdCENb (Fig. 4), delayed ﬂowering in
tobacco, suggesting a CEN function for VvTFL1 (Boss et al.
2006). MdCENa also may have a CEN function, since apple
CEN and TFL1 group genes shared only six identical residues
in this segment B (Fig. 4A). The GUS expression in the transgenic Arabidopsis lines of pMdCENa:GUS and gMdCENa:GUS
also supported the results of RT–PCR analysis in that it
was detected in the silique receptacles, roots and axillary
meristems but not in the apical meristems (Fig. 6O–U). As awhole, the activity of the 2.15 kb promoter and 3.45 kb
genomic regions of MdCENa seemed to reﬂect the actual
gene expression in apple. The GUS expression patterns in
the lines with pMdCENa:GUS and gMdCENa:GUS are reminiscent of that in transgenic Arabidopsis with GUS under thecontrol of the LpTFL1 (a perennial ryegrass TFL1 homolog)
promoter (pLpTFL1:GUS) (Jensen et al. 2001). LpTFL1
controls ﬂoral transition in perennial ryegrass, and GUS
expression of transgenic Arabidopsis with pLpTFL1:GUS is
restricted to the vegetative axillary meristems in Arabidopsis(Jensen et al. 2001). Thus, MdCENa may be involved in thedevelopment of the proliferating tissues or organs including
fruit receptacles, which develop into edible parts, but not in
the control of the transition from the juvenile to the adultphase, as demonstrated by the fact that it was completely
silenced in the apical buds of vegetative shoots of juvenile
seedlings; however, it might inﬂuence the regulation of ﬂowering in apple indirectly. The expression pattern of GUS intransgenic Arabidopsis with pMdCENa:GUS or gMdCENa:GUS
is different from that of ATC in Arabidopsis, where ATCtranscripts are detected in the tissues surrounding vascularbundles in the hypocotyl of 14-day-old plants (Mimida et al.
2001), suggesting that the function of MdCENa is different
from that of ATC. The function of ATC in Arabidopsis,
however, remains to be investigated.
The number of exons/introns and the sequence of coding
regions in TFL1/CEN-like genes were basically conserved
between apple and Arabidopsis, and the phenotypes of

Arabidopsis plants constitutively expressing apple TFL1/
CEN-like genes (Fig. 7 and Tables 1, 2) closely resembled
those exhibited by Arabidopsis expressing TFL1/CEN groupmembers from other plant species (Mimida et al. 2001,
Jensen et al. 2001, Pillitteri et al. 2004, Ordidge et al. 2005,
Guo et al. 2006, Boss et al. 2006, Carmona et al. 2007, Igasaki
et al. 2008). However, apple TFL1/CEN-like genes showed
expression patterns characteristic of each gene. These results
suggest that each TFL1/CEN-like gene of apple has developed
the role to specialize by changing its expression pattern inthe process of evolution. It is widely known that nearly all
higher eukaryotes, including ﬂowering plants, have genomes
that exhibit extensive gene redundancy due to genome doubling or polyploidy (Wendel 2000, Adams and Wendel 2005).
In polyploid plants, there is a certain level of duplication
within the genome and the expression of the duplicatedgenes (homoeologs) is retained or lost on an evolutionary
timescale. The origin of the Maloideae subfamily of the Rosaceae, which includes apple, has also been studied for a longtime (Sax 1933, Chevreau et al. 1985, Morgan et al. 1994,
Evans and Campbell 2002, Luby 2003). Maliepaard et al.
(1998) ﬁrst identiﬁed the duplication of LG5 and LG10 in its
entirety by examining the positions of EST-RFLP (restriction
fragment length polymorphism) markers on an apple linkage map of the cross ‘Prima’ × ‘Fiesta’, which are domestic
apple cultivars. In addition, LGs 3 and 11 and LGs 12 and
14 seem to be partly homoeologous to each other, respectively(Maliepaard et al. 1998, Gardiner et al. 2007). The homoeology of those linkage groups was also supported by our results
showing that MdTFL1 and MdTFL1a were mapped on LGs 12
and 14, respectively (Fig. 3A), and MdCENa and MdCENb, on
LGs 3 and 11, respectively (Fig. 3B). Adams et al. (2003) showthat a high percentage of genes exhibit silencing or biased
expression that is developmentally regulated and that, in a
few case, alternative homoeologs have been reciprocallysilenced in different organs, suggesting subfunctionalization.
Accordingly, TFL1-like genes might also have been subfunctionalized by altering the expression in some organs or
tissues, whereas one of the CEN-like genes might have beensilenced in most organs during evolution.
In this study, we collectively described four TFL1/CEN-likegenes of apple for the ﬁrst time and elucidated the functionof each gene from the viewpoint of their different expression
pattern, transformation of a heterologous plant and the
hypothesis of a polyploidy origin of apple. However, molecular studies on the mechanism of ﬂowering of apple havebeen made complicated by the fact that, in most cases, there
are pairs of homologous genes in the apple genome (Mimida
et al 2007). Our results suggest that MdTFL1a functions
mainly in vegetative tissues, redundantly with MdTFL1,
whereas MdCENa may function in the proliferating tissues
whether they are vegetative or reproductive. Further studies
will be required to clarify the function of TFL1/CEN-like genes

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

407

N. Mimida et al.

by analyzing transgenic apples, for example with an RNAi(RNA interference) construct, and by investigating their
spatial and temporal expression patterns by in situ hybridization in order to develop the technology to control theﬂowering of apple in the juvenile and adult phase.

Materials and Methods
Plant materials
The tissue samples of the apple (Malus×domestica Borkh.)
cvs. ‘Fuji’ (age: 20 years) and ‘Jonathan’ (age: 19–20 years)
were collected from the experimental ﬁeld at the National
Institute of Fruit Tree Science in Morioka, Japan. The seedlings from a cross between the apple cvs. ‘Fuji’ and ‘Orin’
were used for the expression analysis of apple TFL1/CEN-likegenes in the juvenile phase. The A. thaliana tﬂ1-1 mutant(Columbia background) was obtained from the Arabidopsis
Biological Resource Center at Ohio State University andemployed for mutant rescue experiments.

Isolation of the genomic clones
For construction of the genomic library, the genomic DNA
from leaves of ‘Fuji’ apple was extracted according to the
CTAB (cetyltrimethylammonium bromide) method as
described by Kotoda et al. (2000). The λ phage genomic
library was constructed using a Lambda FIXII/XhoI Partial
Fill-in Vector Kit (Stratagene, La Jolla, CA, USA) and a Lambda
DNA Packaging System (Promega, Madison, WI, USA). The
average size of the inserted genomic DNAs was about 10,000
bp. To obtain a CEN-like genomic sequence from apple, PCR
ampliﬁcation of DNA fragments was performed with a pairof degenerate primers 3F and 4R. The PCR-ampliﬁed fragments were cloned into the pT7Blue T-Vector (Novagen,Darmstadt, Germany), and these clones were then sequenced(designated gMdCENa34 and gMdCENb34). The gMdCENa34 or the ﬁrst to second exon-coding region of MdTFL1
cDNA labeled with a PCR digoxigenin (DIG) mixture (Roche
Diagnostics, Mannheim, Germany) was used as a probe toscreen 2.0 × 105 p.f.u. of the apple genomic library to obtain
TFL1/CEN-like genes. The hybridization signals were detectedin the same manner as described in the section on hybridization analysis. The λ clone DNAs from positive plaques weredigested with the restriction enzymes NotI or XbaI andligated into the corresponding site of pBluescriptII SK (+).
These clones were then sequenced. For inverse PCR (I-PCR)of MdCENb, 15 µg of genomic DNA was digested with XbaIand self-ligated with T4 DNA ligase (TAKARA BIO INC.,
Otsu, Japan). I-PCR was carried out with primers
MdCENb480R, MdCENbT8F and MdCENb3′UTRF. The
ampliﬁed PCR fragments were cloned into the pT7Blue
T-Vector, and these clones were then sequenced. Primer
sequences and positions are shown in Supplementary Table
S1 and Supplementary Fig. S3A, respectively.
408

Cloning of cDNAs
To obtain the cDNAs of MdTFL1a and MdCENa, the ﬁrststrand cDNAs were synthesized from 1 µg of total RNAs
from apical buds of FBS in 20 µl of a reaction mixture using
the ReverTra Ace kit (Toyobo, Tokyo, Japan). A PCR
fragment of MdTFL1a was ampliﬁed with the primers,
MdTFL1aRTF1-XbaI and MdTFL1aRTR1, followed by anested PCR with a pair of primers, MdTFL1aRTF1-XbaI and
MdTFL1aRTR2, for MdTFL1a. A PCR fragment of MdCENa
was ampliﬁed with the primers, MdCENaF1 and MdCENaR1,
followed by a nested PCR with MdCENaF2-XbaI and MdCENaR2-SacI for MdCENa. The ampliﬁed PCR products werecloned into the pT7Blue T-Vector. Primer sequences andpositions are shown in Supplementary Table S1 andSupplementary Fig. S3A, respectively.

Mapping of apple TFL/CEN-like genes
SSR markers for the four genes were developed, and SSR
marker detection was performed using a post-PCR ﬂuorescence labeling method (Kukita et al. 2002) for an F1 mapping
population with 83 seedlings from the cross between ‘Ralls
Janet’ and Mitsubakaido (Malus sieboldii Rehder) andanother one with 72 seedlings from the cross between ‘Delicious’ and Mitsubakaido (Igarashi et al. 2008). The PCR primers used are as follows: MdTFL1P-SSR2-U and MdTFL1P-SSR2-L
for MdTFL1; MdTFL1A-SSR1-U and MdTFL1A-SSR2-L for
MdTFL1a; MdCENa-SSR3-U and MdCENa-SSR2-L for
MdCENa; and MdCENb-SSR-U and MdCENb-SSR-L for
MdCENb (for primer sequences, see Supplementary Table
S1). For ﬂuorescence labeling, some nucleotides were added
to the 5′ end of the forward primers to obtain the sequence
ATT. A 2 µl aliquot of the PCR product was added to 4 µl of
the post-PCR labeling reaction buffer [15mM Tris–HCl,
pH 8.3, 15 mM MgCl2, 0.1 µM R110-ddUTP, 0.006 U µl–1
Klenow fragment and 0.0288 U µl–1 Thermo Sequenase DNA
Polymerase. The mixed solution was incubated at 37°C for
5 min, followed by 57°C for 15 min. The reaction was stopped
by the addition of 6 µl of 20 mM EDTA. The appropriate
amounts of labeling products were puriﬁed by ethanol precipitation before loading onto an ABI3100 Genetic Analyzer(Applied Biosystem, Foster City, CA, USA). The resulting
electropherogram was analyzed using GeneScan Analysis
Software and Genotyper Software (Applied Biosystem). The
linkage map was constructed using JoinMap ver. 3.0 (Van
Ooijen and Voorrips 2001).

Sequence analysis
The nucleotide sequence was determined 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). Searching for theconserved sequence regions in the genomic MdTFL1 and
MdTFL1a was performed with the MEME system ver. 3.0

Plant Cell Physiol. 50(2): 394–412 (2009) doi:10.1093/pcp/pcp001 © The Author 2009.

Four TFL1/CEN-like genes in apple(http://meme.sdsc.edu/meme/website/intro.html). Aminoacid sequences were analyzed using the CLUSTAL X multiple
sequence alignment program ver. 1.83 (Jeanmougin et al.
1998) and GeneDoc (Nicholas et al. 1997). The phylogenetic
tree was displayed using the Njplot-unrooted (Perrière andGouy 1996). A homology search was performed with BLAST
2 through the GenomeNet (Bioinformatics Center, Kyoto
University, http://www.genome.jp/).

Nucleic acid extraction and hybridization analysis
Southern hybridization was carried out according to themethod described by Sambrook et al. (1989). The genomic
DNA (15 µg) was isolated from mature leaves of ‘Fuji’,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 membranes were hybridized with the
DIG-labeled ﬁrst to second exon-coding region of MdTFL1
cDNA or gMdCENa34. The hybridization was performed in
DIG Easy Hyb (Roche) at 42°C for 16 h followed by two rinses
in 2× SSC containing 1% (w/v) SDS at room temperature for10 min and two washes in 0.5× SSC containing 1% (w/v) SDS
at 68°C for 10 min. Chemiluminescent signals were visualizedusing the LAS1000 image analyzer (Fuji Photo Film, Tokyo,
Japan).

Expression analysis by RT–PCR
For RT–PCR analysis, total RNAs were extracted from sepals,
petals, stamens, carpels, peduncles, fruit receptacles, fruit
peels, whole young fruits, mature leaves and apical buds of
FBS in ‘Fuji’ apple trees, and apical buds of FBS and SS (fromJune to October in 2004) in ‘Jonathan’ apple trees and leaves,
apices of vegetative shoots, stems, and roots of 14-day-old
seedling and in vitro-cultured tissues (shoots) of ‘Fuji’ apple.
The ﬁrst-strand cDNAs were synthesized from 3 µg of total
RNAs in 60 µl of a reaction mixture using a ReverTra Ace kit(Toyobo). The subsequent PCRs were performed with 1 µl of
the ﬁrst-strand cDNA mix as templates. A DNA fragment ofeach transcript of TFL1/CEN-like genes was ampliﬁed withthe following primers: MdTFL(286→307) and MdTFL1(575→598) for MdTFL1; MdTFL(286→307) and MdTFL1a(585←608) for MdTFL1a; MdCEN-F and MdCEN-R for
MdCEN (MdCENa/MdCENb); MdCEN-F and MdCENa-R
for MdCENa; and MdCENb-F and MdCENb-R for MdCENb(for primer sequences and positions, see Supplementary
Table S1 and Supplementary Fig. S3B, respectively). As aninternal control, a fragment of apple Histone H3 was ampliﬁed with a pair of primers: 5′-TGA AGA AGC CCC ACA GAT
A-3′ and 5′-ACA CAA GAA ACT ATA AAC C-3′ as describedby Kotoda et al. (2006). The thermal cycle programs were as
follows: 96°C for 3 min, followed by 30–35 cycles of 9°C for
20 s, 60°C for 30 s, and 72°C for 30 s for MdTFL1, MdTFL1a,
MdCEN, MdCENa and MdCENb, and 96°C for 3 min, followed

by 30–32 cycles of at 96°C for 20 s, 56°C for 30 s, and 72°C for
30 s for Histone H3. The electrophoresed and blotted RT–PCR
products were hybridized with the DIG-labeled full-length
cDNA of MdTFL1 or MdCENa.

Construction of the transformation vector for theconstitutive expression of MdTFL1a and MdCENa
To construct a vector for the constitutive expression of MdTFL1a, the coding region of MdTFL1a was ampliﬁed by PCR witha pair of primers, MdTFL1aRTF1-XbaI and MdTFL1aR-KpnI.
An ampliﬁed PCR product was subsequently digested withXbaI and KpnI and then cloned into the XbaI–KpnI sites ofthe modiﬁed pSMAK193E binary vector (35SΩ/pSMAK193E)
to be placed between the CaMV 35S promoter fused withthe Ω sequence (Gallie and Walbot, 1992) and the terminator of rubisco (TrbcS). For MdCENa, the coding region of
MdCENa was ampliﬁed by PCR with a pair of primers,
MdCENaF2-XbaI and MdCENaR2-SacI. An ampliﬁed PCR
product was subsequently digested with XbaI and SacI andthen cloned into the XbaI–SacI sites of pSMAK251 to beplaced between the promoter 35S and the nopaline synthaseterminator (Tnos). The transformation vector of MdTFL1 forconstitutive expression was used as described previously(Kotoda et al. 2005). Primer sets used in vector construction
are listed in Supplementary Table S1.

Construction of the transformation vector for the
MdTFL1 and MdCENa promoters with the GUS
reporter gene
For the pMdTFL1:GUS chimeric gene, the genomic MdTFL1
sequence from –2,129 to +9 was ampliﬁed by PCR withprimers (–2kMdTFL1BEIF-BamHI and MdTFL19bR-EcoRV).
The PCR products were digested with BamHI and EcoRV andthen cloned in-frame into the BamHI–SmaI sites of the
pBI221 vector to be placed upstream of GUS (designatedpMdTFL1:GUS:Tnos/pBI221). After being digested with
EcoRI, the DNA fragment containing MdTFL1 (–1,800 to+9):GUS:Tnos was cloned into the EcoRI site of thepSMAK312Blue binary vector. For the gMdTFL1:GUS chimeric gene, the genomic MdTFL1 sequence from –2,129 to+1,832 (before the stop codon) was ampliﬁed by PCR withprimers (–2kMdTFL1BEIF-BamHI and +1.8kMdTFL1R)
designed on the basis of the sequences for the cDNA and λphage clone of MdTFL1. An ampliﬁed PCR product wascloned into the SmaI site of pBI101 (Clontech Laboratories
Inc., Mountainview, CA, USA) in-frame by blunt end ligation.
For the pMdCENa:GUS chimeric gene, the genomic MdCENa
sequence from –2,129 to +20 was ampliﬁed by PCR withprimers (–2kMdCENaF-EcoRI and +20MdCENaR-EcoRV).
An ampliﬁed PCR product was digested with EcoRI and
EcoRV and then cloned into the EcoRI–EcoRV sites of the
pBI221 vector to be placed upstream of GUS in-frame (designated as pMdCENa:GUS:Tnos/pBI221). After being digested

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with EcoRI, the cassette of pMdCENa:GUS:Tnos was cloned
into the EcoRI site of the pSMAK312Blue binary vector. For
the gMdCENa:GUS chimeric gene, the genomic MdCENa
sequence from –2,129 to +1,349 (before the stop codon)
was ampliﬁed by PCR with primers (–2kMdCENaF-EcoRI and+1.3kMdCENaR). An ampliﬁed PCR product was cloned into
the SmaI site of pBI101 (Clontech) in-frame by blunt end
ligation. Primer sets used in vector construction are listed in
Supplementary Table S1.

Arabidopsis transformation
Arabidopsis was transformed with the constructs used inthis study via the Agrobacterium tumefaciens strains, EHA101(for the constructs based on the pSMAK series) or GV3126(for the constructs based on the pBI101), by the inﬁltration
method as described by Bechtold et al. (1993). To select thetransgenic plants, bleach-sterilized seeds from infected
plants were plated on an MS plate (Murashige and Skoog
1962) containing carbenicillin (250 mg l–1) and kanamycin(35 mg l–1). The obtained kanamycin-resistant seeds for the
T1, T2 or T3 generation (primary transformants were deﬁned
as T1 plants) were sown on agar plates followed by stratiﬁcation
at 4°C for 3 d, and grown in a growth chamber
controlled at 22°C under LD conditions (16 h light/8 h dark).

Histochemical analysis in transgenic Arabidopsis
with the GUS geneTo detect GUS activity, GUS staining was performed according to the method described by Jefferson (1987). Whole
plants of transgenic Arabidopsis with the GUS gene were
stained in a staining solution [1 mM 5-bromo-4-chloro-3indolyl glucuronide (X-Gluc), a 0.1 M sodium phosphatebuffer at pH 7.0, 5% methanol, and 0.01% Silwet L-77 (Nippon
Unicar, Tokyo, Japan)] at 37°C for 24 h. The stained plants
were bleached with 70% ethanol.

Supplementary data
Supplementary data are available at PCP online.

Funding
The Bio-oriented Technology Research Advancement Institution (BRAIN) Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN) to N.K.

Acknowledgments
We thank Dr. H. Ichikawa, National Institute of Agrobiological Sciences, Tsukuba, Japan, for kindly providing the binary
vectors pSMAK251, pSMAK193E and pSMAK312Blue, andDr. E. E. Hood, Texas A&M University College Station, forproviding Agrobacterium tumefaciens EHA101. We also
thank Dr. M.G. Meluttu, Union Christian College, India, for410

critical reading of this manuscript. Arabidopsis thaliana tﬂ1-1
seeds were provided by the Arabidopsis Biological Resource
Center at Ohio State University.

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(Received September 1, 2008; Accepted January 2, 2009)

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