Tree Physiology 31, 555–566
doi:10.1093/treephys/tpr028

Research paper

Apple FLOWERING LOCUS T proteins interact with transcription
factors implicated in cell growth and organ development
Naozumi Mimida1,4,7, Shin-Ichiro Kidou2,5, Hiroshi Iwanami1, Shigeki Moriya1, Kazuyuki Abe1,
Charlotte Voogd3, Erika Varkonyi-Gasic3 and Nobuhiro Kotoda1,6,7
1Apple Breeding and Physiology Research Team, National Institute of Fruit Tree Science, Morioka 020-0123, Japan; 2Cryobiosystem Research Center, Iwate University,

Received June 13, 2010; accepted March 16, 2011; published online May 12, 2011; handling Editor Torgny Näsholm

Understanding the flowering process in apple (Malus × domestica Borkh.) is essential for developing methods to shorten thebreeding period and regulate fruit yield. It is known that FLOWERING LOCUS T (FT) acts as a transmissible floral inducer inthe Arabidopsis flowering network system. To clarify the molecular network of two apple FT orthologues, MdFT1 and MdFT2,
we performed a yeast two-hybrid screen to identify proteins that interact with MdFT1. We identified several transcriptionf­actors, including two members of the TCP (TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORs) family,
designated MdTCP2 and MdTCP4, and an Arabidopsis thaliana VOZ1 (Vascular plant One Zinc finger protein1)-like protein,
designated MdVOZ1. MdTCP2 and MdVOZ1 also interacted with MdFT2 in yeast. The expression domain of MdTCP2 and
MdVOZ1 partially overlapped with that of MdFT1 and MdFT2, most strikingly in apple fruit tissue, further suggesting a potential interaction in vivo. Constitutive expression of MdTCP2, MdTCP4 and MdVOZ1 in Arabidopsis affected plant size, leaf
morphology and the formation of leaf primordia on the adaxial side of cotyledons. On the other hand, chimeric MdTCP2,
MdTCP4 and MdVOZ1 repressors that included the ethylene-responsive transcription factors (ERF)-associated amphiphilic
repression (EAR) domain motif influenced reproduction and inflorescence architecture in transgenic Arabidopsis. These results
suggest that MdFT1 and/or MdFT2 might be involved in the regulation of cellular proliferation and the formation of new tissues and that they might affect leaf and fruit development by interacting with TCP- and VOZ-family proteins.
DDBJ accession nos. AB531019 (MdTCP2a mRNA), AB531020 (MdTCP2b mRNA), AB531021 (MdTCP4a mRNA), AB531022(MdTCP4b mRNA) and AB531023 (MdVOZ1a mRNA).
Keywords: apple (Malus × domestica Borkh.), FLOWERING LOCUS T, TCP family, Vascular plant One Zinc finger protein(VOZ), yeast two-hybrid.

Introduction
Apple (Malus × domestica Borkh.) is one of the most importantcommercial crops with over 60 million tonnes of production peryear worldwide (FAOSTAT; Food and Agriculture Organizationof the United Nations 2007 http://faostat.fao.org/). Commercial
apple breeding programmes have been focused on a range ofdesired traits, such as texture, fruit size, fruit skin colour, ­disease

resistance and taste, but the relatively long juvenile period,
which can last 4–8 years (Zimmerman 1972, Hackett 1985),
has been and still is a hindrance to fast and efficient development of new cultivars. Therefore, shortening the juvenile period
for earlier flowering is an important component of apple breeding. Elucidation of the molecular mechanism of the transition
from the juvenile to adult phase would provide means to© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Morioka 020-8550, Japan; 3The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research), Mt Albert, Private Bag 92169, Auckland Mail Centre,
Auckland 1142, New Zealand; 4Present address: Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan; 5Present address: Graduate School of Natural Sciences,
Nagoya City University, Nagoya 467-8501, Japan; 6Present address: Fruit Genome Research Team, National Institute of Fruit Tree Science, Shizuoka 424-0292, Japan;
7Corresponding authors (mmd@iwate-u.ac.jp, koto@affrc.go.jp)

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Mimida et al.

Tree Physiology Volume 31, 2011

(Kotoda et al. 2010). Similar results were obtained in other tree
species, such as Satsuma mandarin (Citrus unshiu) and poplar(Populus deltoides), showing that constitutive expression of
FT-like genes in these plants induces precocious flowering (Endoet al. 2005, Böhlenius et al. 2006, Hsu et al. 2006). However,
the true function of endogenous FT orthologues remains unclear
in woody plants. Because the growth phase of most woody
plants involves a relatively long juvenile period as compared withthat of other perennial plants, knock-out trees lacking the expression of FT-like genes have not been reported.
The aim of this study was to identify proteins that bind to
MdFT1 and MdFT2 using a yeast two-hybrid system in order toelucidate the molecular network of FT orthologues in apple.
Further, we functionally characterized the transcription factors
shown to interact with MdFT1 and MdFT2 proteins, two members of the apple TCP (TEOSINTE BRANCHED1, CYCLOIDEA
and PROLIFERATING CELL FACTORs)-like family and one­ ember of the apple VOZ (Vascular plant One Zinc fingerm
protein)-like family.

Materials and methods
Plant materials
Tissue samples of apple (Malus × domestica Borkh.) cultivar‘Jonathan’ were collected from the experimental field at the
National Institute of Fruit Tree Science in Morioka, Japan.
Seedlings obtained from the cross ‘Fuji’ and ‘Orin’ were used
for expression analysis in the juvenile phase.

Yeast two-hybrid assays
Construction of a bait vector
To construct the DNA binding domain (BD) vector for screening proteins that interact with MdFT1, the full coding region of
MdFT1 was amplified by polymerase chain reaction (PCR) andthe resulting product was cloned in-frame in the pBD-GAL4
Cam vector (designated MdFT1/BD).
Construction of a GAL4 AD vector library Total RNA wasextracted from flowers, fruit and leaves of the apple cv.
‘Jonathan’ according to the cetyltrimethyl ammonium bromide
method as described by Kotoda et al. (2000). Poly (A)+ RNA
was isolated using a PolyATract mRNA Purification Kit (Promega,
Madison, WI, USA). The GAL4 AD vector library was constructed using a GAL4 Two-Hybrid Phagemid Vector Kit(Stratagene, La Jolla, CA, USA) and a Lambda DNA Packaging
System (Promega).
Screening using the yeast two-hybrid system After converting
the phage GAL4 AD vector library into a plasmid library, theyeast strain YRG-2 (Mata ura3-52 his3-200 ade2-101 lys2801 trp1-901 leu2-3 112 gal4-542 gal80-538 LYS2::UASGAL1TATA GAL1-HIS3 URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ)
harbouring MdFT1/BD was co-transformed with the AD cDNA
plasmid library (~5.5 × 106 independent AD plasmids) by the

Downloaded from treephys.oxfordjournals.org at Kajukenkyujo on June 15, 2011a­ ccelerate breeding in fruit trees. In addition, elucidation of thetransition process from the vegetative to the reproductive phase
could provide an insight for understanding the alternate fruit
bearing (biennial bearing) of apple and other perennial woody
fruit trees, which is a significant problem for breeding and­cultivation (Chan and Cain 1967).
In the Arabidopsis flowering network system, it is known thattransition from the vegetative to the reproductive phase is initiated by four signalling pathways, namely gibberellin, autonomous, vernalization and light-dependent signalling pathways(Simpson and Dean 2002, Boss et al. 2004). The FLOWERING
LOCUS T (FT) gene acts as the floral integrator of both thephotoperiod and vernalization pathways. It is promoted by photoperiodic induction through the zinc finger protein, CONSTANS(CO), and is repressed by the vernalization integrator of
FLOWERING LOCUS C (FLC) and LIKE HETEROCHROMATIN
PROTEIN1 (LHP1)/TERMINAL FLOWER2 (TFL2), a homologueof HETEROCHROMATIN PROTEIN1 (HP1) (Michaels andAmasino 1999, Kotake et al. 2003, Takada and Goto 2003). FT
has been identified as a causative gene for a late-floweringmutant and encodes a protein similar to phosphatidylethanolamine-binding protein (PEBP) and Raf-1 kinase inhibitor protein (Kardailsky et al. 1999, Kobayashi et al. 1999). Studies in
Arabidopsis demonstrated that the FT protein travels from theleaves to the shoot apex. It has been demonstrated that the FT
protein acts as a transmissible floral inducer, such as a florigen(Corbesier et al. 2007, Jaeger and Wigge 2007, Lin et al. 2007,
Mathieu et al. 2007, Notaguchi et al. 2008). TERMINALFLOWER1 (TFL1), another member of the PEBP-like gene family,acts as a floral repressor (Bradley et al. 1997). Analyses of thecrystal structure of FT and TFL1 and transgenic plants with thechimeric fusion of FT and TFL1 revealed that specific regions
within their fourth exons function as a potential ligand-binding
pocket, and that replacing these regions allows the opposite
effects on flowering time (Ahn et al. 2006). These results suggest that there are fundamental differences in the interactive
partners or protein-bound conformations between TFL1 and FT
that selectively regulate their downstream signal transduction
pathways in the active or inactive state (Ahn et al. 2006).
We have already identified and characterized two apple FT-likegenes, MdFT1 and MdFT2, which had 95% identity to eachother at the nucleotide level (Kotoda et al. 2010). MdFT1 was
expressed highly in the apical buds of fruit-bearing shoots (FBS)
during the transition period from vegetative to reproductive
phase. On the other hand, MdFT2 was highly abundant in the
reproductive organs, such as young fruit (Kotoda et al. 2010).
This differential expression suggests distinct roles, and the patterns of transcript accumulation imply that MdFT1 possibly accelerates flowering and MdFT2 functions in reproductive organ
development. Therefore, two FT-like genes may have functionally diverged in apple. Moreover, the transgenic apple overexpressing MdFT1 showed an extreme early-flowering phenotype

The interacting genes with apple FT orthologues 557

Screening an apple cDNA library
To isolate the full-length cDNAs of apple TCP and VOZ1 homologues MdTCP2, MdTCP4 and MdVOZ1, we screened an apple
cDNA library that was constructed using a ZAP-cDNA Synthesis
Kit (Stratagene) and a Packagene® Lambda DNA Packaging
System (Promega). Approximately 5.0 × 105 plaques werescreened with MdTCP2, MdTCP4 or MdVOZ1 fragments asprobes obtained by the two-hybrid screening. Plasmids were
rescued from the phage by in vivo excision with the ExAssist
helper phage (Stratagene) for sequence analysis.

Hybridization analysis
Southern blot analysis was performed as described bySambrook et al. (1989). Total DNA (15 µg) isolated from theleaves of apple ‘Fuji’ was digested with EcoRI, HindIII or XbaIand then separated on a 0.8% agarose gel by electrophoresis.
The DNA bands were blotted onto a Hybond-N+ nylon membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA)and hybridized with digoxigenin (DIG)-labelled full-length
cDNA probes of MdTCP2a, MdTCP4a or MdVOZ1a. Hybridization
was performed in DIG Easy Hyb (Roche) at 42 °C for 16 h followed by two rinses in 2× sodium saline citrate (SSC) containing 1% (w/v) sodium dodecyl sulphate (SDS) at room
temperature for 10 min and two washes in 0.5× SSC containing 1% (w/v) SDS at 68 °C for 10 min. Chemiluminescent signals were visualized using the LAS1000 image analyser (Fuji
Photo Film, Aichi, Japan).

Nucleotide and amino acid sequence analyses
Nucleotide sequences were 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). Sequence database searching
was performed using BLAST2 of National Center for
Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.
gov/), Genome Database for Rosaceae (GDR, http://www.
rosaceae.org/) and Plant transcription factor database version
2.0 (Zhang et al. 2011, Center for Bioinformatics, Peking
University, China, http://planttfdb.cbi.pku.edu.cn/). Amino acidsequences were analysed using the Clustal X multiple
sequence alignment program ver. 1.83 (Jeanmougin et al.
1998) and the phylogenetic tree was displayed using the
NJplot program (Perrière and Gouy 1996).

Expression analysis by reverse transcriptase (RT)–PCR
and quantitative real-time RT–PCR
As described in our previous study (Mimida et al. 2009), total
RNA samples were obtained from calyxes, petals, stamens,
carpels, peduncles, fruit receptacles (FRs), seeds, fruit, mature
leaves (MLs) and apical buds of FBS from June to the following
April in the adult phase. Apical buds of vegetative shoots (VS),stems and roots were obtained from 2-week-old seedlings in
the juvenile phase. First-strand cDNAs were synthesized from3 µg of total RNA in 60 µl of reaction mixture using the ReverTra
Ace Kit (Toyobo, Japan). Apple Histone H3 was used as aninternal control for gene expression (Mimida et al. 2009,
Kotoda et al. 2010).
Subsequent PCR was performed with 1 µl of the first-strand
cDNA mixture and EX-Taq polymerase (Takara Biomedical,
Otsu, Japan). Thermal cycler programmes were as follows:
96 °C for 3 min followed by 32 cycles of 96 °C for 20 s,
58 °C for 30 s and 72 °C for 30 s for MdTCP2a and MdTCP2b;
30 cycles of 96 °C for 20 s, 56 °C for 30 s and 72 °C for
30 s for MdTCP4a/MdTCP4b (the primer set of MdTCP4a/b F
and MdTCP4a/b R amplifies both genes); and 32 cycles of
96 °C for 20 s, 56 °C for 30 s and 72 °C for 30 s for
MdVOZ1a.
Quantitative real-time RT–PCR (qRT–PCR) analysis was carried out to validate the result of RT–PCR analysis following themanual for the Applied Biosystems 7300 Real-Time PCR System(Applied Biosystems, Foster City, CA, USA). Quantitative realtime RT–PCR reactions were performed with 0.15 µg of thefirst-strand cDNA mixture as a template by using the power
SYBR Green Master mix (Applied Biosystems). Thermal cycler
programmes were as follows: 50 °C for 2 min and 95 °C for
10 s followed by 40 cycles of 95 °C for 15 s and 60 °C for
15 s for each gene. Software analysis of ­absolute quantification was performed with sequence detection software version
1.3.1 (Applied Biosystems). The primer sets used in RT–PCR
and qRT–PCR analysis are shown in Table 1.

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Downloaded from treephys.oxfordjournals.org at Kajukenkyujo on June 15, 2011lithium method (Gietz and Woods 2002). The yeast transformants were incubated on selective agar medium with Minimal
SD Base, -His/-Leu/-Trp DO supplement (Clontech Laboratories
Inc., Mountain View, CA, USA) and 3 mM 3-amino-1,2,4-­triazole(3-AT) containing 4% glucose as a carbon source at 30 °C for
4–5 days. The AD plasmids were extracted from the transformants grown on a triple dropout (-His/-Leu/-Trp) medium plate,
and then re-transformed in Escherichia coli for sequence
analysis.
A selection test for histidine (HIS3) reporter gene activity: toconfirm positive candidates that interact with MdFT1 and alsowith MdFT2, we generated the bait vector construct of MdFT2
and the yeast was transformed with a pair of bait (MdFT1/BD
and MdFT2/BD) and prey (MdTCP2a/AD, MdTCP4a/AD and
MdVOZ1a/AD) plasmids. The yeast transformants were tested
for HIS3 reporter gene activity on selective agar medium with
Minimal SD Base, -His/-Leu/-Trp DO supplement and 1 mM
3-AT containing 4% glucose at 30 °C for 3 days or 0 mM 3-AT
containing 2% glucose at 20 °C for 7 days.
5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal)filter assay: the assay was performed for lacZ reporter gene
expression by detecting β-galactosidase activity. A filter assay
was performed according to protocols described in the GAL4
Two-Hybrid Phagemid Vector Kit instruction manual (Stratagene).

558 Mimida et al.
Table 1. PCR primer sequences for gene expression analysis
PrimerOligonucleotide sequence

MdTCP2a F
MdTCP2a R

5′-GAA TCA GAG CCA GCA CCT TTC-3′
5′-GTT GTT GTT TAT GCT CTG TTG GTA
ACT-3′
5′-GGG AAC CAA AGT CAG CAC C-3′5′-GGA AAT GTT GTG CGG GTA AGC-3′
5′-CAG CAG CAGC CGT CGT TGT TCG G-3′5′-GAG ATG AGA TGA GAG CTT GAG CCC
TTT TGA AGA G-3′5′-AGA GAA TGG TGG CTT GGA ACA-3′5′-GAT GAT CCA CCG CCG TTA TC-3′
5′-GCA CCT CTA TGA GTA CGA GAT C-3′5′-GGA GCC CAT CAA ACT TCT ATC AG-3′

MdTCP2b F
MdTCP2b R
MdTCP4a/b F
MdTCP4a/b R
MdTCP4a real-time F
MdTCP4a real-time R
MdVOZ1 F
MdVOZ1 R

Vector construction

Arabidopsis transformation
Arabidopsis (Columbia) was transformed using theAgrobacterium tumefaciens strain EHA101 by floral dipping(Clough and Bent 1998). Transformed Arabidopsis plants wereselected on MS plates (Murashige and Skoog 1962) containing carbenicillin (250 mg−1 l) and kanamycin (35 mg−1 l). T1 or
T2 plants (primary transformants were defined as T1 plants)
were grown on MS plates containing kanamycin for 5 or 10days, and then transferred to the soil and grown under longday conditions (16 h light/8 h darkness) at 22 °C.

Results
Identification of proteins that interact with MdFT1 by the
yeast two-hybrid system
A yeast two-hybrid system with the full-length MdFT1 as abait was used for screening ~5.5 × 106 plasmids from theTree Physiology Volume 31, 2011

Comparison of amino acid sequences and phylogenetic
analysis of TCP2, TCP4 and VOZ1 homologues
To assess the number of TCP2, TCP4 or VOZ1 homologues in
the apple genome, genomic Southern hybridization was performed using full-length cDNAs of MdTCP2a, MdTCP4a or
MdVOZ1a as probe, respectively. One to four major hybridizing bands were detected in each lane of DNA digested with
EcoRI, HindIII or XbaI, suggesting that at least two distinct locifor each gene exist in the apple genome (Figure 2). In an
attempt to isolate apple MdTCP2a, MdTCP4a or MdVOZ1ahomologues, ~5.0 × 105 plaques were screened from acDNA library with an MdTCP2a, MdTCP4a or MdVOZ1a probe,
respectively.
We obtained a total of 22 positive plaques by screening a
cDNA library with an MdTCP2a probe and then classified theminto two different cDNAs, MdTCP2a and another gene that wedesignated MdTCP2b. The amino acid sequence alignment
showed that MdTCP2a had 91.8 and 44.2% similarity to
MdTCP2b and TCP2, respectively, and that MdTCP2b had
43.0% similarity to TCP2.
Nine positive plaques were obtained with an MdTCP4aprobe, and subsequent sequence analysis revealed that they
could be classified as two different cDNAs, namely MdTCP4a
and another gene that we designated MdTCP4b. The amino

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To construct transgenes of 35SΩ:MdTCP2a, 35SΩ:MdTCP4aand 35SΩ:MdVOZ1a, the full-length cDNAs of MdTCP2a,
MdTCP4a and MdVOZ1a were amplified by PCR with a pair ofprimers containing XbaI or KpnI. The PCR products were subsequently digested with XbaI and KpnI, and then cloned in the
XbaI–KpnI sites of the modified pSMAK193E binary vector(35SΩ/pSMAK193E) (Mimida et al. 2009) to place it between
the cauliflower mosaic virus (CaMV) 35S promoter fused withthe Ω sequence (Gallie and Walbot 1992) and rubisco terminator (TrbcS).
To generate 35SΩ:MdTCP2a-SRDX, 35SΩ:MdTCP4a-SRDX
and 35SΩ:MdVOZ1a-SRDX, the full-length cDNAs of MdTCP2a,
MdTCP4a and MdVOZ1a were amplified by PCR with a pair ofprimers containing XbaI or SmaI. The PCR products were subsequently digested with XbaI and SmaI and then cloned in the
XbaI–SmaI sites of the modified pSMAK193E binary vector(35SΩ-SRDX/pSMAK193E) to clone it in-frame with the 12
amino acid repression domain (SRDX) (Hiratsu et al. 2003).

cDNA AD library prepared from the flowers, fruit and leavesof ‘Jonathan’ apple. As a result, 26 clones were obtained andsequence analysis revealed that seven of the clones represented transcription factors, one represented the kinase/
receptor class, 11 were classified as others and seven fell
into the junk DNA category (Table 2). The transcription factors ­isolated in this study were homologues of the Arabidopsis
TCP and VOZ family of genes. Three candidate clones of the
TCP-family genes carried cDNA inserts of different sizes
derived from a single gene, designated MdTCP4a, and one
candidate carried an insert of another gene, designated
MdTCP2a. Both MdTCP2a and MdTCP4a contained a TCP
domain with a proposed basic helix–loop–helix motif (datanot shown). All candidate clones of AtVOZ1 homologues carried cDNA inserts with different sizes derived from a single
gene, designated MdVOZ1a. It contains three conserved
regions: acidic stretch region, zinc finger motif and basic
region (data not shown).
To confirm that the candidate clones interact with MdFT1,
the growth test for HIS3 reporter gene activity and an X-gal
filter assay were performed using the yeast transformants­harbouring a pair of bait and prey plasmids (MdFT1/BD and
MdTCP2a/AD, MdTCP4a/AD or MdVOZ1a/AD). As a result, theactivities of HIS3 and ß-galactosidase were detected inyeast cells with all combinations analysed (Figure 1a and b). In­addition, it was confirmed that MdFT2 also interacted with
MdTCP2a and MdVOZ1a (Figure 1c).

The interacting genes with apple FT orthologues 559
Table 2. Summary of screening for MdFT1 interacting proteins using the yeast two-hybrid system
Number of AD
clones obtainedConsensus gene set number­(chromosome number)

Arabidopsis homologue(accession number)

Annotation

2, 21, 283

ath:AT1G28520

Transcription factor, AtVOZ1 homologue.

4, 11, 343

ath:AT3G15030

Transcription factor, TCP4 homologue.

36

1

ath:AT4G18390

Transcription factor, TCP2 homologue.

5
9

1
1

MDP0000729316 (chr 5, MdVOZ1a)[MDP0000879912(chr10)]
MDP0000243495 (chr11, MdTCP4a)[MDP0000184743 (chr10, MdTCP4b)]
MDP0000763497 (chr 5, MdTCP2a)[MDP0000287069 (chr 5, MdTCP2b)]
MDP0000608985 (chr 4)
MDP0000181717 (chr 9)

ath:AT1G04280
ath:AT3G04680

10
17
18
22
26

1
1
1
1
1

MDP0000270976 (chr 1)
MDP0000135679 (chr 1)
MDP0000870498 (chr 2)
MDP0000340981 (chr13)
MDP0000299813 (chr13)

ath:AT2G46080
ath:AT5G10780
ath:AT5G66230
ath:AT1G28120
ath:AT3G10700

30, 382

ath:AT1G71230

33

1

MDP0000145618 (chr 8),
MDP0000184474 (chr 5)
MDP0000896759 (chr13)35

1

MDP0000924612 (chr 5)

ath:AT4G17190

44

1

MDP0000608985 (chr 4)

ath:AT5G36230

Junk DNA

7--

REV3C homologue.
ATP/GTP-binding/pre-mRNA cleavage
complex family protein.
Zinc ion binding protein.
Transmembrane protein.
Unknown protein.
Ubiquitin thioesterase Otubain family protein.
ATP binding/galactokinase/ kinase/phosphotransferase.
COP9 signalosome subunit protein
homologue.
Two-component sensor molecule, TWIN LOVPROTEIN 1 (TLP1) homologue.
FARNESYL DIPHOSPHATE SYNTHASE 2(FPS2) homologue.
Translation initiation factor containing basic
leucine zipper and W2 domains 2.
Frame-shift or non-coding DNA

ath:AT2G02710

Sequence database similarity searching was performed using the Genome Database for Rosaceae (GDR, http://www.rosaceae.org/). The con­sensus gene set number between brackets shows the homologue of the isolated gene.

acid sequence alignment showed that MdTCP4a had 88.7 and56.1% similarity to MdTCP4b and TCP4, respectively, and that
MdTCP4b had 55.6% similarity to TCP4.
Recently, the apple genome project has progressed (Velasco
et al. 2010) and expression sequence tag (EST) data arealso available (Newcomb et al. 2006, Zhang et al. 2011).
Interrogation of the DNA database identified numerous TCPfamily homologues in apple (Figure 3a). The TCP-family proteins were grouped as three distinct clades: TEOSINTEBRANCHED1 (TB1) and CYCLOIDEA (CYC) form the TB1/CYC
clade, while CINCINNATA (CIN) and PROLIFERATING CELL
FACTORs (PCF) form individual clades (Aguilar-Martínez et al.
2007). The four TCP-family homologues isolated from apple
belonged to the CIN clade in the phylogenetic tree constructedusing the amino acid sequences (Figure 3a).
Hybridization with an MdVOZ1a probe resulted in a total ofeight positive plaques. However, all of them carried a cDNA
insert derived from a single gene, MdVOZ1a. The alignment ofamino acid sequence and phylogenetic analysis showed that
MdVOZ1a belonged to the VOZ1 clade in the VOZ family, andhad 76.4 and 47.1% similarity to AtVOZ1 and AtVOZ2, respectively (Figure 3b). As a result of database searching, five homologues belonging to VOZ1 and VOZ2 clades were found as
EST clones and in the apple genome (Figure 3b).

Expression patterns of MdTCP2, MdTCP4 and MdVOZ1Tissue-specific expression patterns of MdTCP2a, MdTCP2b,
MdTCP4a/MdTCP4b (or MdTCP4a) and MdVOZ1a were­investigated by RT–PCR and qRT–PCR analyses. The MdTCP2a­transcript accumulated to relatively high levels in petals andfruit, and was also detected in calyxes, stamens, seeds, MLs
and FBS in the adult phase, and in VS in the juvenile phase of­seedlings (Figure 4a). The expression pattern of MdTCP2b
was similar to that of MdTCP2a in the highly expressing­tissues, but was not detected in juvenile VS. Reverse
transcriptase–­polymerase chain reaction analysis showed­relatively high levels of MdTCP4a/MdTCP4b expression in petals and MLs, lower levels of expression in calyxes, stamens,
seeds and FBS in the adult phase, and low levels in MLs andVS in the juvenile phase (Figure 4a). Quantitative real-time
RT–PCR analysis confirmed high levels of MdTCP4a transcripts
in petals (Figure 4a). MdVOZ1a transcript accumulation wasobserved in multiple tissues, and was detected strongly infruit, calyxes and petals, but its expression was considerably
lower in sexual organs, such as stamens and carpels, and MLsof the seedlings (Figure 4a).
Floral bud differentiation and development occur fromsummer through to spring in the apical buds of FBS (Kotoda
et al. 2000, Mimida et al. 2009). We have previously ­analysed

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Clone (no.)

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Mimida et al.
the seasonal expression patterns of genes related to flowering, such as MdFT1, MdFT2, MdTFL1, MdCEN and MdAP1, in
apical buds (Mimida et al. 2009, Kotoda et al. 2010).
Therefore, RT–PCR and qRT–PCR analyses were ­performed
to establish seasonal expression patterns of MdTCP2a,
MdTCP2b, MdTCP4 and MdVOZ1a using total RNA isolated
from the apical buds of FBS in each month from June to thefollowing April. Similar levels of MdTCP2a transcript weredetected across all seasons (Figure 4b). In contrast,
MdTCP2b expression was significantly higher in the laterstage of flower bud development. The expression levels of
MdTCP4a/MdTCP4b or MdTCP4a were low from June to thefollowing February but increased in March and April. The
expression of MdVOZ1a decreased from June to September
and increased from January to April (Figure 4b).

Analysis of apple TCP4, TCP2 or VOZ1 homologues intransgenic Arabidopsis

Figure 2. ​Genomic Southern blot probed with MdTCP2a (left),
MdTCP4a (middle) and MdVOZ1a (right), respectively. Genomic DNA(15 µg) of ‘Fuji’ apple was digested with EcoRI (EI), HindIII (H) or XbaI
(XI) and subjected to hybridization analyses.

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Figure 1. Interactions with MdFT1 and MdFT2 proteins detected inyeast. (a) Interaction of MdFT1 with MdTCP2a, MdTCP4a and
MdVOZ1a. A selection test was performed for the HIS3 reporter
gene of yeast transformants on the selective plates with Minimal SD
Base and -His/-Leu/-Trp DO supplement containing 1 mM 3-AT and
4% glucose at 30 °C for 3 days. (b) X-gal filter lift assay forß-­galactosidase activity using yeast transformant cells. (c) Interactionof MdFT1 and MdFT2 with MdTCP2a and MdVOZ1a. A selection test
was performed for the HIS3 reporter gene of yeast t­ransformants on
the selective plates (-His/-Leu/-Trp) with 0 mM 3-AT and 2% glucose
at 20 °C for 7 days. Wild-type cells were transformed with a pair ofbait and prey plasmids: MdFT1/BD or MdFT2/BD and MdFT1/AD,
MdTCP2a/AD, MdTCP4a/AD or MdVOZ1a/AD by the lithium method.

To establish the possible roles of MdTCP2a, MdTCP4a or
MdVOZ1a, the gain-of-function and loss-of-function approach
was used in transgenic Arabidopsis plants. Constitutive­expression constructs, 35SΩ:MdTCP2a, 35SΩ:MdTCP4aand 35SΩ:MdVOZ1a, and chimera repressor constructs,
35SΩ:MdTCP2a-SRDX, 35SΩ:MdTCP4a-SRDX and 35SΩ:MdVOZ1a-SRDX (Figure 5a), were introduced into the
Arabidopsis plants. Over 50 independent lines were obtainedfor each transgene. The T1 or T2 generation of these transgenic
plants were grown in long-day conditions and their phenotypes
were examined.
As compared with the wild-type plants (Figure 5b and h),most transgenic plants with 35SΩ:MdTCP2a and 35SΩ:MdTCP4a displayed a phenotype with elongated hypocotyls
in green (Figure 5c, d, f, i, j and l). A few of them also had
fused cotyledons similar to those of double mutants of cupshaped cotyledon1 (cuc1) and cuc2 (Aida et al. 1997, Figure 5d
and j). In addition, plants expressing MdTCP4a had narrowleaves and reduced plant size (Figure 5e, l and p). A few plants
showed a phenotype with increased number of shoots (datanot shown).
Transgenic plants with 35SΩ:MdTCP2a-SRDX or 35SΩ:MdTCP4a-SRDX showed similar phenotypes to each other.
Newly emerging leaves on the adaxial side of cotyledons wereobserved in some transgenic plants (Figure 5e, g, k, m and o).
Others had wavy leaves similar to those of the jaw mutant(Palatnik et al. 2003, Figure 5n) and were sterile.
The 35SΩ:MdVOZ1a construct induced sterile flowers withenhanced elongation of flower stalks, abnormal inflorescences
and terminus by a flower (Figure 5q, r, s and t) at a frequencyof <10%. In <10% of all transformants, 35SΩ:MdVOZ1a-SRDX
induced sterile flowers and abnormal inflorescences with three
to five flowers, resulting in plants with terminal flowers or
dwarfed shape (Figure 5u, v, w, x and y).

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Figure 3. ​Relationship of the predicted TCP- or VOZ-family proteins between apple and other plant species. (a) Phylogenetic tree of TCP-family
proteins of Arabidopsis and apple. (b) Phylogenetic tree of VOZ-family proteins of Arabidopsis, rice and apple. These trees were constructed using
Arabidopsis TCP-family proteins described by Aguilar-Martínez et al. (2007), Arabidopsis VOZ-family proteins described by Mitsuda et al. (2004),
Os01g0753000, Os05g0515700 and other amino acid sequences obtained from Plant transcription factor database version 2.0 (Mdo), Genome
Database for Rosaceae (HDP) and the New Zealand Institute for Plant & Food Research Ltd (HM) in addition to MdTCP2a, MdTCP2b, MdTCP4a,
MdTCP4b and MdVOZ1a shown in bold in this paper. Numbers along branches are bootstrap values (1000 replicates). The unit for the scale bars
displays branch lengths (0.05 substitutions/site).

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Figure 4. Expression analyses of MdTCP2, MdTCP4 and MdVOZ1 by RT–PCR and qRT–PCR. (a) Expression pattern of MdTCP2a, MdTCP2b,
MdTCP4a/MdTCP4b (RT–PCR), MdTCP4a (qRT–PCR) and MdVOZ1a in various tissues. Samples from left to right are as follows: calyxes, petals,
stamens, carpels, peduncles, fruit receptacles (FRs), fruit, seeds, mature leaves (MLs) and apical buds of fruit-bearing shoots (FBS) on September
12 [FBS (Sep)] in the adult phase; MLs, apical buds of vegetative shoots (VS), stems and roots of 2-week-old seedlings in the juvenile phase.
(b) Expression pattern of MdTCP2a, MdTCP2b, MdTCP4a/MdTCP4b (RT–PCR), MdTCP4a (qRT–PCR) and MdVOZ1a genes in apical buds of FBS
from June to April. Values are means ± standard deviation of the results from three replicates. The units on the vertical axis show 1.0 × 10−9 g ofgene transcripts/1.5 × 10−4 g of total RNA. It is noteworthy that in Morioka, Japan, the timing of the flowering of the floral buds of ‘Jonathan’ is early
May, the timing of floral differentiation and fruit ripening is the beginning of July and mid-October, respectively, and the dormancy period starts from
November. The number of PCR cycles is indicated at the left of each panel.

Discussion
Over the past decade, studies of the flowering process in
Arabidopsis have provided us with the important findings of FT(reviewed in Turck et al. 2008). An existing model proposes

Tree Physiology Volume 31, 2011that the formation of a protein–protein complex between FTand FLOWERING LOCUS D (FD) is implicated in the activationof the APETALA1 (AP1) gene promoter (Abe et al. 2005, Wigge
et al. 2005). Taking into consideration that FT acts as a trans-­

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Figure 5. ​Arabidopsis transgenic plants expressing apple TCP2, TCP4 and VOZ1 homologues. (a) Schematic representation of the vector construction of transgenes, MdTCP2a, MdTCP4a and MdVOZ1a. (b–m) Typical lines of the wild-type (b, h), 35SΩ:MdTCP2a (c, d, i, j), 35SΩ:MdTCP4a (f, l),35SΩ:MdTCP2a-SRDX (e, k) and 35SΩ:MdTCP4a-SRDX (g, m) plants. (n) 35SΩ:MdTCP2a-SRDX plant (no. 1). (o) Rosette leaves of a
35SΩ:MdTCP2a-SRDX plant (no. 2). (p) 35SΩ:MdTCP4a plant (no. 1). (q) 35SΩ:MdVOZ1a plant (no. 1). (r) Inflorescence of a 35SΩ:MdVOZ1aplant (no. 1). (s) 35SΩ:MdVOZ1a plant (no. 2). (t) Inflorescence of a 35SΩ:MdVOZ1a plant (no. 2). (u) 35SΩ:MdVOZ1a-SRDX plant (no. 1).
(v) 35SΩ:MdVOZ1a-SRDX plant (no. 2). (w) 35SΩ:MdVOZ1a-SRDX plant (no. 3). (x) Close-up of the inflorescence in (w). (y) Developed­inflorescence in the 35SΩ:MdVOZ1a-SRDX plant (no. 3) in (w). Arrowheads in (e), (g), (k) and (m) indicate leaves formed on the adaxial side of
the cotyledons. These plants were grown under long-day conditions (16 h light/8 h dark). Scale bars are 1 cm in (o) and (p).

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Tree Physiology Volume 31, 201135SΩ:TCP3-SRDX (Palatnik et al. 2003, Koyama et al. 2007,
Figure 5). In addition, MdFT1/MdFT2 and MdTCP2a/MdTCP2b
exhibited overlapping expression profiles in reproductive
organs and particularly MdFT2 and MdTCP2a/MdTCP2b werehighly expressed in the developing fruit of apple (Kotoda et al.
2010, Figure 4). Moreover, the Arabidopsis CIN class genes are
expressed in immature siliques at a high level (Koyama et al.
2007). Our findings suggest that the formation of protein–protein complex by MdFT1 or MdFT2 and CIN class proteins, suchas MdTCP2a and MdTCP2b, might play a role in cell growthand proliferation and affect leaf development, meristem formation and fruit development. Krieger et al. (2010) reported thatheterozygosity for loss-of-function alleles of SINGLE FLOWER
TRUSS (SFT), a tomato FT orthologue, increases fruit yield by
up to 60%, suggesting the involvement of FT orthologues in
the fruit development of dicotyledonous crops. On the other
hand, MdTCP4a was expressed specifically in petals and shootapices in April during a period of flower development after dormancy (Figure 4). Therefore, MdTCP4a may affect floral organ
development by interaction with MdFT2, the expression ofwhich increases rapidly in apical buds of FBS after dormancy(Kotoda et al. 2010).
MdFT1 and MdFT2 interacted with MdVOZ1a more strongly
than with TCP-family proteins (Figure 1a and c). MdVOZ1a wasexpressed in calyxes, petals and fruit at a relatively high level(Figure 4a). Because a DNA database search revealed that
there were at least four VOZ1 homologues including MdVOZ1a,these homologues should also be investigated in future work.
Both 35SΩ:MdVOZ1a and 35SΩ:MdVOZ1a-SRDX transgenes
induced a sterile phenotype in Arabidopsis, suggesting that
MdVOZ1a is also involved in the development of reproductive
organs (Figure 5). AtVOZ1, a zinc-finger transcription factor,
was identified as a V-PPase promoter-binding protein by ayeast one-hybrid system (Mitsuda et al. 2004). V-PPase acts
as a vacuolar proton pump and is expressed in the young fruitof Japanese pear (Shiratake et al. 1997). AtVOZ1 is expressedin vascular bundles of various tissues, stamens, and the junction of silique and pedicel, which is the position likely corresponding to apple fruit (Mitsuda et al. 2004). Expression of FT
homologues has also been detected in the fruit of apple andcitrus (Nishikawa et al. 2007, Kotoda et al. 2010). These results
suggest that the protein complex of MdFT1–MdVOZ1a and/or
MdFT2–MdVOZ1a might play an important role in the vacuole
during fruit development, although MdFT2 fused with the green
fluorescent protein is reported to be localized in the cytoplasmic membrane (Li et al. 2010).
In grasses, vegetative meristem is not maintained under theconstitutive expression of FT, because FT induces precocious
flowering and represses vegetative growth. In a perennial
woody plant like Norway spruce, however, FT orthologues haveadditional functions. They play a role in regulating seasonal
growth and bud outgrowth in the juvenile phase (Gyllenstrand

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element of gene ­promoters, it could interact with several transcription factors and control multiple genes. In other words, it
could be inferred that FT has other functions in addition toflowering control (Lifschitz et al. 2006, Shalit et al. 2009).
Recently, it was demonstrated that MdFT2, one of two apple
FT orthologues, was highly expressed in developing fruit(Kotoda et al. 2010). Thus, MdFT2 might be involved in reproductive organ development as well as in flowering.
In this study, we performed a large-scale yeast two-hybrid
screen to identify proteins that interact with MdFT1. We haveobtained several candidates that interact with MdFT1. They
include the homologue of two-component sensor molecule
TWIN LOV PROTEIN 1 (TLP1, AT2G02710). TLP1 is a photoreceptor protein for blue light and contains the light, oxygenor voltage (LOV) domain (Crosson et al. 2003). Apple TLP1like genes might play a role in controlling flowering in
response to photoperiod and light quality via interaction with
MdFT1 and/or MdFT2. However, the molecular mechanismsof flowering in the light-dependent pathway remain largely
unknown in apple. The protein in the kinase/receptor classshowed no sequence homology to SPAK [SELF PRUNING(SP)-associated kinase], which interacts with SP, a memberof the PEBP-like protein family in tomato (Pnueli et al. 2001).
Other candidate genes include several transcription factors(Table 1). Among them, TCP and VOZ1 homologues wereidentified that interacted strongly with both MdFT1 and
MdFT2 in yeast (Figure 1). Transgenic Arabidopsis plants
expressing either MdFT1 or MdFT2 showed an early-flowering
phenotype (Kotoda et al. 2010, Li et al. 2010). Taking thesefacts into consideration, it was suggested that MdFT1 and
MdFT2 could function equivalently at the protein level. TCPfamily genes play crucial roles in cell growth and proliferation (Cubas et al. 1999), whereas AtVOZ is possibly involvedin the regulation of vacuolar pyrophosphatase (V-PPase)gene expression during pollen development (Mitsuda et al.
2004). These reports suggest that MdTCP2a, MdTCP4a and
MdVOZ1a might also play important roles in regulating cell
division and differentiation in apple.
The Arabidopsis TCP family consists of 24 paralogue genes(Koyama et al. 2007). Interestingly, the apple genome contains
most homologues of Arabidopsis TCP genes (Figure 3a). CIN
class genes, which might be functionally redundant, areinvolved in regulating CUC gene expression to promote meristem formation and to determine the correct position of organ
primordia and boundaries (Koyama et al. 2007). Five of the
CIN class genes are regulated at the transcriptional levelby miRNAs (Palatnik et al. 2003). The phenotype of
Arabidopsis plants transformed with the 35SΩ:MdTCP2aand 35SΩ:MdTCP4a constructs was similar to that of plants­carrying 35S:miRNA-resistant TCP2 (mTCP2), 35S:mTCP4 and35S:mTCP3. The phenotype of Arabidopsis with 35SΩ:MdTCP2aSRDX and 35SΩ:MdTCP4a-SRDX was also similar to that of

The interacting genes with apple FT orthologues 565

Acknowledgments
We thank Dr H. Ichikawa of the National Institute ofAgrobiological Sciences for providing the binary vector
pSMAK193E.

Funding
This work was supported by the Bio-oriented Technology
Research Advancement Institution (BRAIN) for the Promotionof Basic Research Activities for Innovative Biosciences(PROBRAIN) to N.K.

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