Plant Cell Physiol. 49(3): 291–300 (2008)
doi:10.1093/pcp/pcn010, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Rapid paper

The FLOWERING LOCUS T/TERMINAL FLOWER 1 Family
in Lombardy Poplar
Tomohiro Igasaki 1, *, Yumiko Watanabe 1, Mitsuru Nishiguchi 1 and Nobuhiro Kotoda 2
1

Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute (FFPRI), Tsukuba, 305-8687 Japan
Department of Apple Research, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization (NARO),
Morioka, 020-0123 Japan2

genomic DNA), AB106111 (PnFT1 mRNA), AB369069(PnFT1 genomic DNA), AB109804 (PnFT2 mRNA), AB369070(PnFT2 genomic DNA), AB110612 (PnFT3 mRNA),
AB369071 (PnFT3 genomic DNA), AB110612 (PnFT4 mRNA),
AB369073 (PnFT4 genomic DNA) and AB369072 (PnFTL9
genomic DNA).

Genes in the FLOWERING LOCUS T (FT) and
TERMINAL FLOWER 1 (TFL1) family have been shown
to be important in the control of the switch between vegetativeand reproductive growth in several plant species. We isolatednine members of the FT/TFL1 family from Lombardy poplar(Populus nigra var. italica Koehne). Sequence analysis of themembers of the FT/TFL1 family revealed considerable
homology within their coding regions both among family
members and to the members of the same family inArabidopsis, tomato and grapevine. Moreover, members of
this family in all four species examined display a common
exon–intron organization. Phylogenetic analysis revealed thatthe genes fall into four different clades: two into the TFL1
clade; five into the FT clade; and one each into the MOTHER
OF FT AND TFL1 and BROTHER OF FT AND TFL1
clades. One gene in the TFL1 clade, PnTFL1, is expressed invegetative meristems, and transgenic Arabidopsis that ectopically expressed PnTFL1 had a late-flowering phenotype. The
expression patterns of two genes in the FT clade, PnFT1 and
PnFT2, suggested a role for them in the promotion of
flowering, and transgenic Arabidopsis that ectopically
expressed either PnFT1 or PnFT2 had an early-flowering
phenotype.

Introduction
The features of the growth and development of woody
plants differ from those of annual herbaceous plants, suchas Arabidopsis thaliana. In particular, woody plants have avery long juvenile phase. In some species, the juvenile phase
lasts as long as 40 years before flowering occurs for the firsttime (Hackett 1985, Greenwood 1987). This long juvenile
phase has been an obstacle in the breeding of woody plants,
including fruit trees. Some efforts at accelerating flowering
by application of plant hormones have been made (Mullins
et al. 1989, Meilan 1997), but the techniques are applicable
to only limited species of woody plants and the mechanisms
involved remain unknown. Therefore, it is important tocharacterize the molecular mechanisms of flowering inwoody plants and to develop new breeding technologies forthe acceleration of flowering by genetic engineering.
The genus Populus is an important resource, serving, inparticular, as a source of raw materials for pulp, paper,
board and lumber products (Mitchell et al. 1992). Due tothis importance, an international team of researchers,
funded by the US Department of Energy, determined thenucleotide sequence of the entire genome of black cottonwood (P. trichocarpa) one of the Populus species (Tuskan
et al. 2006). The Lombardy poplar (P. nigra var. italica
Koehne) is one of the most common species in the same
genus and it has been planted worldwide. Furthermore,a system for transformation and a library of full-length
enriched expressed sequence tags (ESTs) in this species havebeen established (Mohri et al. 1996, Nanjo et al. 2004,
Nishiguchi et al. 2006). Therefore, the Populus species is

Keywords: Flowering control — FLOWERING LOCUS T(FT) — Poplar — Populus nigra — TERMINALFLOWER 1 (TFL1).
Abbreviations: BFT, BROTHER OF FT AND TFL1; EST,
expressed sequence tag; FT, FLOWERING LOCUS T; LD, longday; MFT, MOTHER OF FT AND TFL1; N-J, Neighbor-Joining;
ORF, open reading frame; PAR, photosynthetically active radiation; P35S, cauliflower mosaic virus 35S promoter; PnFTL,
Populus nigra FT/TFL1-like; RACE-PCR, rapid amplification of
cDNA ends-PCR; RT–PCR, reverse transcription–PCR; TFL1,TERMINAL FLOWER 1.
The nucleotide sequences reported in this paper havebeen submitted to the DDBJ database under accession
numbers AB161110 (PnFTL1 mRNA), AB183451 (PnFTL1 genomic DNA), AB181183 (PnTFL1 mRNA), AB369067 (PnTFL1
genomic DNA), AB181185 (PnFTL3 mRNA), AB181186 (PnFTL3
genomic DNA), AB181241 (PnFTL4 mRNA), AB369068 (PnFTL4*Corresponding author: E-mail, iga@ffpri.affrc.go.jp; Fax, þ81-29-873-0507.
291

292

FT/TFLI family in poplar

generally chosen by researchers as the model genus ofwoody plants, and we selected the Lombardy poplar for thepresent study.
The genes that control flowering time in Arabidopsis
have been isolated and characterized (for reviews, see
Mouradov et al. 2002, Komeda 2004). In our efforts tounderstand the long juvenile phase of woody plants, we havefocused on a family that includes the flowering-promoter
gene, FLOWERING LOCUS T (FT) and the repressor gene
TERMINAL FLOWER1 (TFL1). In Arabidopsis, thisfamily consists of six members: FT; TFL1; MOTHER
OF FT AND TFL1 (MFT); ARABIDOPSIS THALIANACENTRORADIALIS HOMOLOG (ATC); TWIN SISTEROF FT (TSF); and BROTHER OF FT AND TFL1 (BFT)(Kobayashi et al. 1999). The FT and TFL1 genes have
opposing effects on flowering time (Kardailsky et al. 1999,
Kobayashi et al. 1999), and their effects have been related
to the presence, in the gene products, of critical amino acidresidues, namely Tyr85/Gln140 in FT and His88/Asp144 in
TFL1 (Hanzawa et al. 2005, Ahn et al. 2006). Comparativeand functional genomic research in several species of woody
plants is starting to provide information on the conservation of flowering-regulatory pathways in both woody andherbaceous plants (Brunner and Nilsson 2004, Böhlenius
et al. 2006, Hsu et al. 2006, Kotoda et al. 2006, Carmona
et al. 2007). Böhlenius et al. (2006) and Hsu et al. (2006) eachisolated an ortholog of FT from Populus species on the basisof information provided by the sequence of the entire
genome of black cottonwood, and they showed that theorthologs function similarly in the induction of flowering.
However, there are likely to be many more members of the
FT/TFL1 family in Populus, as well as in Arabidopsis. The
isolation and functional analysis of such genes are now
required for a full understanding of the molecular mechanisms of flowering in Populus.
We report here the cloning and identification of nine
members of the FT/TFL1 family from Lombardy poplar.
We describe the genomic structure, phylogenetic relationships, patterns of expression of the FT/TFL1 family, as wellas the phenotypes of transgenic Arabidopsis that ectopically
expressed individual genes in the FT/TFL1 family of
Lombardy poplar.

Results
Cloning of genes in the FT/TFL1 family from Lombardy
poplarSince preliminary Southern blotting analysis revealed
the presence of several members of the FT/TFL1 family in
Lombardy poplar, we performed degenerate reverse transcription–PCR (RT–PCR) with degenerate primers derived
from the strongly conserved regions of plant members of thisfamily (Fig. 1). We obtained partial cDNAs with sevenunique sequences and designated them PnFTL1 to PnFTL7(P. nigra FT/TFL1-like). The 50 end and the 30 end of open
reading frames (ORFs) of each corresponding gene werepredicted after rapid amplification of cDNA ends-PCR(RACE-PCR) and inverse PCR with the partial cDNA
sequences and specific pairs of primers for each gene, as
shown in Supplementary Table 1. We obtained nine cDNA
clones and 17 genomic DNA clones, including full-length
ORFs, from an individual female clone of Lombardy poplar(Fig. 1). Careful analysis of the sequences of these genes
revealed that several of them were allelic variants and thatthe genes were derived from at least nine loci in the genomeof Lombardy poplar. Comparison of the cDNA sequenceswith the genomic sequences showed that PnFTL7 mRNA
was generated from two loci. Therefore, for convenience, we
designated the gene with the longer genomic sequence
PnFTL8. Moreover, a novel gene, PnFTL9, which was
amplified with the same set of primers as used to amplify
PnFTL5/PnFTL6, was found among the genomic sequences.
However, we failed to generate a cDNA clone of PnFTL9,
even though we designed several specific sets of primers andextensively analyzed various organs from Lombardy poplar.
Identification of PnFTL genes
At both the nucleotide and the amino acid level, thePnFTL genes were strongly homologous to one anotheralong their entire length (Fig. 1). The PnFTL2 and PnFTL3
genes were very similar to TFL1, while the PnFTL5,
PnFTL6, PnFTL7, PnFTL8 and PnFTL9 genes were very
similar to FT (Fig. 1 and Table 1).
To resolve the evolutionary relationships among
members of the FT/TFL1 family, we performed phylogenetic analysis of this family from Lombardy poplar,
Arabidopsis, tomato (Lycopersicon esculentum), apple(Malus  domestica), citrus (Citrus sinensis and C. unshiu)
and grapevine (Vitis vinifera). Neighbor-Joining (N-J)
distance analysis, with the alignment of entire amino acidsequences, generated an unrooted tree with four major
clades. PnFTL2 and PnFTL3 were in the TFL1 clade;
PnFTL5, PnFTL6, PnFTL7, PnFTL8 and PnFTL9 were in
the FT clade; PnFTL1 was in the BFT clade; and PnFTL4
was in the MFT clade (Fig. 2).
Later, given the results of this phylogenetic analysis andsubsequent analyses, we shall refer to several of the abovementioned genes by their functional designations, as follows:
the PnFTL2, PnFTL5, PnFTL6, PnFTL7 and PnFTL8 genes
will be designated the PnTFL1, PnFT1, PnFT2, PnFT3 and
PnFT4 genes, respectively (see, for example, Table 2).
The genomic clones of the PnFTL genes allowed
identification of four exons and three introns (Fig. 3A).
All genes showed evidence of conserved genomic organization and the exons were located in identical positions
relative to the amino acid sequences of members of theFT/TFLI family in poplar

293

MFT
PnFTL4

63
63

FT
TSF
PnFT3/4 (PnFTL7/8)
PnFT1 (PnFTL5)
PnFT2 (PnFTL6)
PnFTL9

65
65

TFL1
ATC
PnTFL1 (PnFTL2)
PnFTL3

68
66
66
65

BFT
PnFTL1

65
65

64
64
64
64

*

MFT
PnFTL4

133
133

FT
TSF
PnFT3/4 (PnFTL7/8)
PnFT1 (PnFTL5)
PnFT2 (PnFTL6)
PnFTL9

135
135
134
134
134
126

TFL1
ATC
PnTFL1 (PnFTL2)
PnFTL3

138
136
136
135

BFT
PnFTL1

135
135

MFT
PnFTL4

*

173
173

FT
TSF
PnFT3/4 (PnFTL7/8)
PnFT1 (PnFTL5)
PnFT2 (PnFTL6)
PnFTL9

175
175
174
174
174
166

TFL1
ATC
PnTFL1 (PnFTL2)
PnFTL3

177
175
174
173

BFT
PnFTL1177
173

Fig. 1 Alignment of the deduced amino acid sequences of the products of the FT/TFL1 family in Lombardy poplar and Arabidopsis
thaliana [FT, AGI (Arabidopsis Genome Initiative; http://www.arabidopsis.org) code At1g65480; TFL1, AGI code At5g03840; MFT, AGI
code At1g18100; ATC, AGI code At2g27550; TSF, AGI code At4g20370; and BFT, AGI code At5g62040]. All poplar sequences were
derived from an individual female clone of Lombardy poplar (PnF-1). Amino acids in blue and in yellow are identical and similar,
respectively, in at least 10 of the 14 members of the FT/TFL1 family. Amino acids common to genes in the FT clade are shown in pink andthose in the TFL1 clade are shown in green. Dots indicate gaps introduced to maximize the extent of homology among sequences. The
arabic numerals in the sequences represent the positions of amino acid residues from the site of initiation of translation. The multiplealignments were generated with ClustalW software. Half-arrows indicate the positions of degenerate primers for PCR that we used for the
first cloning of the partial cDNAs. Arrowheads indicate the positions of introns. Asterisks indicate amino acids that are critical to thedefinition of proteins in the FT and TFL1 clade. Two red boxes indicate the regions that are essential for FT-like activity in exon IV.

Arabidopsis FT/TFL1 family (Figs. 1, 3A). In Lombardy
poplar, exons I and IV varied in length from 195 to 204 bp
and from 218 to 224 bp, respectively. In the case of exon II,
that of PnFTL9 was 38 bp long, whereas exon II of othergenes was 62 bp long in every case. The length of exon III
was 41 bp in all genes examined. However, the length of

introns varied. As in Arabidopsis, the genes in the TFL1 and
BFT clades, namely PnFTL1, PnTFL1 and PnFTL3,
had short introns of 81–353 bp, while the genes in the FT
and MFT clades, PnFTL4, PnFT1, PnFT2, PnFT3,
PnFT4 and PnFTL9, had long introns of 112–1,603 bp(Fig. 3A).

294

FT/TFLI family in poplar

The genes in the FT clade of Lombardy poplar could
be classified into two subgroups, with subgroup I consistingof PnFT3 and PnFT4, and subgroup II consisting of
PnFT1, PnFT2 and PnFTL9 (Fig. 3B). The sequences of
introns were 485% homologous within each subgroup, but
550% homologous between subgroups. We also found that
PnFTL9 had a deletion of 393 bp in the region that
corresponded to exon II and intron II of PnFTL6 (Fig. 3B).
To compare the genes of Lombardy poplar and black
cottonwood, we searched for PnFTL genes in the black
cottonwood genome (http://genome.jgi-psf.org/Poptr1/
Poptr1.home.html). We found homologs of each gene in
the non-FT clade, namely the counterparts of PnFTL1,
PnTFL1, PnFTL3 and PnFTL4 in black cottonwood(Table 2). In the case of genes in the FT clade, two genes
in subgroup I were found in Lombardy poplar, whereas
only one corresponding locus was found in black cottonwood (Table 2). In contrast, three genes in subgroup II were
found in both species (Table 2).
We excluded PnFTL9 from subsequent studies because
the deletion and extensive substitutions in the regions
essential for FT-like function suggested that the gene was
not expressed and that its product would not be functional(Figs. 1, 3B).

expression of PnFTL3 in winter lateral buds, but levelsof expression were low and only one-thousandth of thoseof PnTFL1 (Fig. 4C).
In the FT clade, we detected the expression of PnFT1 inalmost all organs, with particularly high levels of expression

to TFL1

to FT

PnFTL1
PnTFL1 (PnFTL2)
PnFTL3
PnFTL4
PnFT1 (PnFTL5)
PnFT2 (PnFTL6)

62.4/76.4
70.9/82.1
71.3/83.7
48.9/62.4
55.9/66.5
56.4/66.5

58.5/68.9
56.5/68.9
56.8/67.6
44.6/58.2
76.6/85.1
77.1/85.1

PnFT3 (PnFTL7)/
PnFT4 (PnFTL8)
PnFTL955.3/68.2

78.3/85.7

48.6/59.2

65.7/74.3

Identity and similarity were calculated by ClustalW software(Thompson et al. 1994).

FT
TSF
CiFT
SP3D
SP6A
FT
VvFT
clade
MdFT
100 PnFT3 (PnFTL7)
PnFT4 (PnFTL8)
PnFT1 (PnFTL5)
99 PnFT2 (PnFTL6)
44
PnFTL9

38
31
11

31

16
20
54

51

94

28
22
98

41
82
71
95

51
0.05

Gene

97

100

Organ-specific patterns of expression of PnFTL genes
To explore the expression of PnFTL genes in Lombardy
poplar, we performed an analysis by real-time RT–PCR
using primers specific for each gene (Supplementary
Table 2). In the TFL1 clade, PnTFL1 was expressed in
hypocotyls, epicotyls and winter lateral buds of both juvenile
and reproductive-phase plants (Fig. 4B), while PnFTL3 wasexpressed mainly in the seeds (Fig. 4C). We also detected theIdentity/similarity (%)

MFTPnFTL4 MFT
clade
SP2I
VvMFT
SP5G

97

72

Table 1 Identity and similarity of the deduced amino acidsequences encoded by PnFTL genes in comparison withthose encoded by FT and TFL1

99
100

94

TFL1
SP9D
VvTFL1B
CiTFL1
MdTFL1
TFL1
MdTFL1–2
clade
SP
ATC
VvTFL1A
PnTFL1 (PnFTL2)
PnFTL3BFT
BFT
VvTFL1C
clade
PnFTL1

Fig. 2 Phylogenetic analysis of the members in the FT/TFL1
family. The tree was constructed by the Neighbor-Joining (N-J)
method for members of the FT/TFL1 family in Lombardy poplar(PnFTL1, PnTFL1, PnFTL3, PnFTL4, PnFT1, PnFT2, PnFT3, PnFT4
and PnFTL9), A. thaliana (FT, TFL1, MFT, ATC, FT, TSF and BFT),
tomato (SP, accession No. U84140; SP2I, accession No.
AY186734; SP3D, accession No. AY186735; SP5G, accession
No. AY186736; SP6A, accession No. AY186737; and SP9D,
accession No. AY186738), grapevine (VvFT, accession No.
DQ871590; VvTFL1A, accession No. DQ871591; VvTFL1B,
accession No. DQ871592; VvTFL1C, accession No. DQ871593;
and VvMFT, accession No. DQ871594), apple (MdTFL1, accession
No. AB162040; MdTFL1-2, accession No. AB162046; and MdFT,
accession No. AB161112) and citrus (CiFT, accession No.
AB027456; and CiTFL1, accession No. AY344244). The N-J
unrooted dendrograms were generated from alignment of deduced
amino acids with the ClustalW program, and the phylogenetic tree
was displayed by the MEGA4 software package (Tamura et al.
2007). Bootstrap values for 100 resamplings are shown on eachbranch. The scale indicates the average number of substitutions persite. Genes in Lombardy poplar are in red.

FT/TFLI family in poplar

in early-summer leaves and capsules of reproductive trees(Fig. 4E). Similarly, we detected strong expression of PnFT2
in early-summer leaves and capsules of reproductive trees,
while levels of expression in several vegetative organs, such as
roots, stems and winter lateral (vegetative) buds, were very
low compared with those of PnFT1 (Fig. 4F). The pattern ofTable 2 Location of the counterparts of PnFTL genes in
the genome of black cottonwood
Lombardy
poplar gene

Location of counterpart inblack cottonwood

PnFTL1
PnTFL1 (PnFTL2)
PnFTL3
PnFTL4
PnFT1 (PnFTL5)
PnFT2 (PnFTL6)
PnFTL9oChromosome XV
Scaffold_66a
Chromosome IX
Chromosome XV
Chromosome X
Scaffold_1409a
Scaffold_1444an

PnFT3 (PnFTL7)
PnFT4 (PnFTL8)

Chromosome VIIIa

A scaffold is a portion of the genomic sequence that has beenreconstructed from end-sequenced whole-genome shotgun clones;
the actual location of each scaffold within the genome is unknown.

A

295

expression of PnFT3/4 was rather different. We detected
strong expression of PnFT3/4 in stems and winter lateralbuds, whereas expression in early-summer leaves during the
reproductive phase was barely detectable (Fig. 4G).
The PnFTL1 gene, the ortholog of BFT, was expressed
mainly in the organs of juvenile-phase trees (Fig. 4A), and
PnFTL4, the ortholog of MFT, was expressed mainly in theseeds (Fig. 4D).
Seasonal changes in expression of PnFTL genes
To examine detailed patterns of expression of genes in
the FT clade, namely PnFT1, PnFT2 and PnFT3/4, wecollected the leaves and lateral buds of branches fromjuvenile-phase trees and from healthy branches and probable floral regions of fruit-bearing branches ofreproductive-phase trees. The result of monitoring the fateof lateral buds in probable floral region of fruit-bearing
branches showed that approximately 86% of lateral buds inthe region would differentiate into floral buds (data notshown). At the end of May, we detected the strong expressionof PnFT1 and of PnFT2 in leaves of healthy branches andfruit-bearing branches from reproductive-phase trees; levelsof expression were almost 1,000 times higher than thosein branches of juvenile-phase trees. These high levels fell tobasal levels by the middle of August (Fig. 5A, B). The levels

ATG

Stop

MFT

195

PnFTL4

195

FT

201

TSF

201

PnFT3 (PnFTL7)

198

PnFT4 (PnFTL8)

198

PnFT1 (PnFTL5)

198

PnFT2 (PnFTL6)

198

PnFTL9

198

TFL1

210

ATC

204

PnTFL1 (PnFTL2)

204

PnFTL3

201

562
181

62

126

224

852

41

112

1419

815

713

645

124

524

156

258

224

224

1125

176

MFT
clade

224

224

1391

1141

224

1391

123

1601

891

123

1581

892

126

1290

38

210

206

353
83

118

277

135

94

111

309

221

105

218

224
224
224

892

B

221

87

FT
clade

TFL1
clade

PnFT3 (PnFTL7 )PnFT4 (PnFTL8)

218

87.5

98.4

99.6

44.0

<40

<40

100

97.9

99.6

89.7

91.6

99.3

Poplar FT
subgroup I

PnFT1 (PnFTL5)
BFT

201

PnFTL1

201

103
89

87
122

176
81

230
218

BFT
clade

PnFT2 (PnFTL6)
PnFTL9100 bpPoplar FT
subgroup II

393 bpdeletionFig. 3 (A) Genomic organization of members of the FT/TFL1 family in Lombardy poplar and Arabidopsis. Boxes represent exons and lines
represent introns. Numbers indicate the lengths of exons and introns (bp). (B) Comparison of the introns in genes in the FT calde. The
similarities between introns (as percentages) are shown.

296

FT/TFLI family in poplar

1.0E−2

A

Bud
flush

PnFTL1

5.0E−3

2E−2

Leaf
abcission

Floralinitiation

A

PnFT1

B

PnFT2

C

PnFT3/4

D

PnFT3/4

E

PnTFL1

0
PnTFL1B

1E−2

1.0E+00

5.0E−10
4.0E−4

1E−1

PnFTL3C

5E−2

2.0E−40

0

D

PnFTL4

1.0E+05.0E−10
3.0E−1PnFT1E

2.0E−1

Relative expression

Relative expression

1.5E+0

2E−3

1E−3

0

1.0E−16E−20
3.0E−1

PnFT2

F

4E−2

2.0E−12E−2

1.0E−100

1.5E−1

PnFT3/4

G

1E+1

1.0E−1
5.0E−25E+0

Fig. 4 Expression of genes in the FT/TFL1 family in various organs
and cells of Lombardy poplar. An aliquot of first-strand cDNA
mixture, corresponding to 50 ng of total RNA, was amplified by
PCR with gene-specific primers, as listed in Supplementary
Table S1. Levels of detected amplicons were normalized byreference to amplified products that corresponded to the gene forpolyubiquitin in Lombardy poplar (PnUB1; accession No.
AB182939). Values are means  SE from the results of 3–6
replicates. Sd, seeds; RS, H, Co and Ep, roots, hypocotyls,
cotyledons and epicotyls of 2-week-old seedlings; RJ, SJ, LJ and
BJ, roots, stems, early-summer leaves and winter lateral buds ofjuvenile-phase plants; RR, SR, LR, BR, MB, FB, MF, FF and Cp,
roots, stems, early-summer leaves, winter lateral buds, male flower
buds, female flower buds, male flowers, female flowers andcapsules of reproductive phase plants; and SC, cells of Lombardy
poplar in suspension culture.

6
ov
N

27
p
Se

9

30

18
Au
g

Reproductive
phase

ay

Juvenile
phase

M

Seedling

0

Vegatative Reproductive
organsorgans

Ju
l

Sd
RS
H
Co
Ep
RJ
SJ
LJ
BJ
RR
SR
LR
BR
MB
FB
MF
FF
Cp
SC

0

Fig. 5 Seasonal variations in the expression of genes in the FT/
TFL1 family of Lombardy poplar. (A–E) The expression of PnFT1(A), PnFT2 (B) and PnFT3/4 (C) in the leaves and the expression of
PnFT3/4 (D) and PnTFL1 (E) in the lateral buds of Lombardy poplar
trees at the juvenile phase (5 years old; diamonds) and on healthybranches (triangles) and fruit-bearing branches (circles) of trees in
the reproductive phase (430 years old). Shaded areas indicate theprobable period for initiation of flowers, and arrowheads indicate
the periods of bud flush and leaf abscission. Five mature leaves andthree sets of lateral buds (approximately 200 mg each) werecollected and pooled at 2 p.m. on the date indicated. An aliquot of
first-strand cDNA mixture, corresponding to 0.5 mg of total RNA,
was amplified by PCR with gene-specific primers. Levels ofdetected amplicons were normalized by reference to amplified
products that corresponded to PnUB1. Values are means  SE of
results from five replicates. Each symbol without a bar indicatesthat the SE fell within the symbol.

FT/TFLI family in poplar

A

B

D

E

F

C

G

297

H

I

K

J

L

Fig. 6 Phenotypes of transgenic Arabidopsis ectopically expressed genes in the FT/TFL1 family from Lombardy poplar under long-day
conditions. (A–H) A 21-day-old wild-type plant (A; control), 35-day-old transgenic plants that harbored P35S::PnTFL1 (B) andP35S::PnFTL3 (C), a 14-day-old wild-type plant (D; control) and 14-day-old transgenic plants that harbored P35S::FT (E), P35S::PnFT1 (F),P35S::PnFT2 (G) and P35S:: PnFT3/4 (H). (I–L) Flowers of wild-type (I) and transgenic P35S::PnTFL1 plants (J–L). Bars in (A)–(H) represent
1 cm and those in (I)–(L) represent 1 mm.

Table 3 Comparison of flowering time and number ofrosette leaves in transgenic Arabidopsis plants that
ectopically expressed individual PnFTL genes
Construct

Days to No. of rosette
floweringleavesWS (control)
22.7  0.5b
P35S::FT
14.5  0.5a
P35S::PnFT1 (PnFTL5)
12.0  0.4a
P35S::PnFT2 (PnFTL6)
11.7  0.5a
P35S::PnFT3/4 (PnFTL7/8) 13.0  0.5a
P35S::TFL1
31.7  1.9c
P35S::PnTFL1 (PnFTL2)
40.3  5.7c
P35S::PnFTL3
36.8  4.4c
P35S::PnFTL1
20.3  0.4b
P35S::PnFTL4
22.8  0.4b6.7  0.5b
3.5  0.4a
2.1  0.1a
2.4  0.2a
2.9  0.2a
8.5  0.6c
10.6  0.4c
10.0  0.6c
6.4  0.1b6.8  0.4b

Plants were grown under LD. Values are means  SE of the resultsfrom 12 independent lines of transgenic plants. Scheffé’s multiple
comparison test was used to compare the effects of treatments.
Superscripts letters indicate a statistically significant difference
from results with other letters in the same column (P50.05).

in leaves of branches at the juvenile phase did not change
significantly during this period (Fig. 5A, B). However, thelevels of expression of PnFT1 and of PnFT2 were quite
low in the lateral buds of all branches, and we did notobserve any seasonal changes (data not shown).
The patterns of expression of PnFT3/4 were different
from those of PnFT1 and PnFT2 (Fig. 5C). Levels of
expression of PnFT3/4 had risen 20- to 100-fold in the leavesof each branch examined by the beginning of November,when senescence was induced in leaves of Lombardy poplar(Fig. 5C). Similarly, in the lateral buds of each branch, levelsof expression of PnFT3/4 had risen almost 10-fold by the
beginning of November (Fig. 5D).
We also analyzed expression of PnTFL1 in the lateralbuds of each branch. The level of expression of PnTFL1 hadrisen 10-fold in the lateral buds of healthy branches

examined by the beginning of July, and fell to basal levelsby the middle of August (Fig. 5E). A similar pattern wasobserved in lateral buds of branches from juvenile-phasetrees (data not shown). On the other hand, in the lateralbuds from fruit-bearing branches, the level of expressionof PnTFL1 had risen approximately 2-fold by the beginningof July (Fig. 5E).

Phenotypes of transgenic Arabidopsis plants that expressed
ectopic PnFTL genes
To determine the functions of PnFTL genes, we
introduced the ORF of each gene under the control of thecauliflower mosaic virus 35S promoter (P35S) into
Arabidopsis. Then, we selected 12 independent homozygouslines, in the third generation for each construct, forphenotypic analyses (i.e. n ¼ 12 in all cases below).
Transgenic Arabidopsis that ectopically expressed the
P35S::PnTFL1 and P35S::PnFTL3 constructs (TFL1 clade),required 40.3  5.7 and 36.8  4.4 d, respectively, for flowering, whereas wild-type plants flowered within 22.7  0.5 dunder long-day (LD; 16 h light/8 h dark) conditions(Fig. 6A–C and Table 3). In addition, in the case of thefirst generation of P35S::PnTFL1 and P35S::PnFTL3
plants, two lines in each case did not flower during 45
months of growth and failed to produce any seeds (data notshown). Each gene affected the vegetative and the earlyinflorescence stage of Arabidopsis, as revealed by theincreased numbers of nodes produced before and after
bolting. During the juvenile phase, wild-type Arabidopsis
plants produced 6.7  0.5 rosette leaves under LD conditions, whereas the P35S::PnTFL1 and P35S::PnFTL3 plants
produced 10.6  0.4 and 10.0  0.6 rosette leaves,respectively (Table 3). The numbers of cauline leaves on
the main stems were also higher in both types of transgenic
plant than in wild-type plants after the plants had bolted(data not shown). In addition, we frequently observed
abnormal flowers with leaf-like sepals (Fig. 6J–L) and298

FT/TFLI family in poplar

occasionally inflorescences that were developing on flowers
themselves.
In contrast, transgenic Arabidopsis that expressed
P35S::PnFT1, P35S::PnFT2 and P35S::PnFT3/4 constructs(FT clade) had a dramatic early-flowering phenotype(Fig. 6F–H and Table 3). Flowering of P35S::PnFT1,
P35S::PnFT2 and P35S::PnFT3/4 plants occurred after
12.0  0.4, 11.7  0.5 and 13.0  0.5 d, respectively, andeach plant had 1.9  0.1, 2.2  0.2 and 2.6  0.2 rosetteleaves, respectively (Table 3).
PnFTL1 and PnFTL4 did not affect the flowering,flower morphologies and inflorescences of transgenic
Arabidopsis under LD conditions. Flowering of
P35S::PnFTL1 and P35S::PnFTL4 plants occurred after
20.3  0.4 and 22.8  0.4 d, respectively, and each plant had
6.4  0.1 and 6.8  0.4 rosette leaves, respectively (Table 3).

Discussion
Collectively, we have identified nine members of the
PnFTL gene family, as revealed by comparisons of sequencesand genomic organization (Table 1 and Fig. 3). We thoughtthat we had isolated and identified all members of the FT/
TFL1 gene family in Lombardy poplar for the following
reasons. Enough numbers of genes in the FT/TFL1 family
were isolated with the comparison of the numbers of those in
Arabidopsis, tomato, rice (Oryza sativa) and grapevine(Kobayashi et al. 1999, Carmel-Goren et al. 2003, Chardon
and Damerval 2005, Carmona et al. 2007), and with theresult of the comparison with the genome of black cottonwood (Table 2). Our phylogenetic analysis showed that thenine genes fall into four clades: two into the TFL1 clade; fiveinto the FT clade; and one each into the MFT and BFT clades(Fig. 2). Our results provide the first comprehensive
description of the FT/TFL1 family in Populus species, andto our knowledge this is the first report to demonstrate thefunction of genes in the TFL1 clade in poplar.
The expression of PnTFL1 indicated that this gene isprobably associated with meristematic regions, such aslateral buds (Fig. 4B), and the levels of expression of thisgene increased remarkably in vegetative buds during theprobable period for initiation of flowers (Fig. 5E). The
pattern of expression of PnFTL3 resembled that of PnTFL1,
although the patterns did not overlap completely(Fig. 4B, C). Both genes, however, prevented determinationof the floral meristem in transgenic Arabidopsis (Fig. 6B, C
and Table 3). Moreover, we observed abnormal flowers with
leaf-like sepals on both P35S::PnTFL1 and P35S::PnFTL3transgenic Arabidopsis plants (Fig. 6J–L). Similar observations have been reported in Arabidopsis plants that
ectopically expressed genes in the TFL1 clade, namely
TFL1 of Arabidopsis (Ratcliffe et al. 1998), LpTFL1 ofryegrass (Jensen et al. 2001) and VvTFL1A of grapevine(Carmona et al. 2007). Thus, our results suggest that thesegenes might play a role in the maintenance of meristematicfunctions during vegetative development in Lombardy
poplar, as well as in other plants. Moreover, the TFL1-likegenes in other woody plants, citrus and apple appear to be
involved in the regulation of the juvenile phase (Pillitteri et al.
2004, Kotoda et al. 2006). We plan to examine whether
suppression of the expression of PnTFL1 might reduce thelength of the juvenile phase in Lombardy poplar. Further
analyses of these genes, using transgenic poplar, mightprovide information about their involvement in the transition from the juvenile to the reproductive phase.
Although each of the genes in the FT clade had asimilar FT-like activity in transgenic Arabidopsis (Fig. 6F–H
and Table 3), the patterns of expression differed between
subgroup I (PnFT3 and PnFT4) and subgroup II (PnFT1
and PnFT2). The genes in subgroup II were expressed
mainly in the early-summer leaves of Lombardy poplar
during the reproductive phase and they were not expressed
during the juvenile phase, although the patterns ofexpression of PnFT1 and PnFT2 did not overlap completely(Fig. 5A, B). These observations resemble those reported
for one of the counterparts of these genes in eastern
cottonwood (P. deltoides) by Hsu et al. (2006) that anincrease in the expression of this gene might be important
for transition from the juvenile to the reproductive
developmental stage in poplar. Thus, in poplar, as in
Arabidopsis and rice, the FT protein produced in the leaves
might move to the meristem and promote flowering(Corbesier et al. 2007, Tamaki et al. 2007). However, wefailed to find any differences in terms of the patterns andlevels of expression of these genes between healthybranches, which seldom produce flowers, and fruit-bearing
branches (Fig. 5A, B). It seems likely, therefore, that thestate of branches (vegetative or reproductive) might becontrolled by other factors in addition to the patterns andlevels of the expression of FT and related genes, and we hadthought that the patterns and levels of expression of
PnTFL1 indicated that it is one of the candidates for thefactors of floral initiation. In contrast, the expression of
PnFT3/4, which was classified into another subgroup,
increased in senescent leaves of both reproductive andjuvenile-phase plants late in the year (Fig. 5C). These results
support the suggestion by Böhlenius et al. (2006) that anincrease in the expression of the counterpart of PnFT3/4 in
European aspen (P. tremula) might be important for shortday-induced cessation of growth. However, this latter gene isalso expressed in early-summer leaves, an observation thatsuggests that floral initiation might also be controlled by thisgene in European aspen (Böhlenius et al. 2006). Although theEuropean aspen is very different in this respect from
Lombardy poplar, differences among species or sections
within the genus Populus would not be unexpected.

FT/TFLI family in poplarBöhlenius et al. (2006) and Hsu et al. (2006) demonstrated
that the acceleration of flowering is simply dependent on
increasing levels of endogenous functional FT protein by
the analyses of the transgenic Populus that overexpressed
an FT gene in each subgroup.
Neither the PnFTL1 nor the PnFTL4 gene influenced
flowering time in transgenic Arabidopsis that expressed each
respective gene ectopically (Table 3). However, since bothgenes exhibited restricted patterns of expression andincluded sequences conserved in each clade that had been
isolated from several other species, we can assume that thesegenes are functional. Further analysis is now necessary tovalidate this assumption.
In Lombardy poplar and black cottonwood, we foundfive and four genes in the FT clade (Table 2). In other species,
the number of genes in the FT clade was 1–3; namely, in
Arabidopsis, tomato, rice and grapevine, there were two,
three, two and one genes, respectively (Kobayashi et al. 1999,
Carmel-Goren et al. 2003, Chardon and Damerval 2005,
Carmona et al. 2007). This considerable redundancy of genes
in the FT clade suggests that these genes might play
important roles in the life cycle in the genus Populus.
In summary, we isolated nine members of the FT/TFL1
family from Lombardy poplar and identified the functionsof the some of their products. Our goal is to clarify not only
mechanisms of flowering but also phenomena that control
flowering in woody plants in order to exploit them forbreeding purposes.

Materials and Methods
Plant materials
Lombardy poplar trees that had been planted on the groundsof Hokkaido University (Sapporo, Japan) were used as source
material. A clonal strain of Lombardy poplar, propagated by
cutting, was grown in a phytotron at 258C and at 70% relative
humidity under metal halide lamps [500 mmol m2 s1 of photosynthetically active radiation (PAR); 16 h photoperiod].
Arabidopsis thaliana seeds were stratified for 3–4 d at 48C andthen grown on a solid medium that contained Murashige andSkoog’s basal salts (Murashige and Skoog 1962), Gamborg’s B5
vitamins (Gamborg et al. 1968) and 1% sucrose, supplemented
with 0.4% gellan gum (MSB5 medium), in a growth chamber at
228C under cool white fluorescent light (50 mmol m2 s1 of PAR,16 h photoperiod). A 7–10 d after sowing, seedlings were transferred to soil and grown under the same conditions.

Isolation of total RNA
Total RNA was extracted from various organs and tissues ofLombardy poplar with hexadecyltrimethylammonium bromide(Shinohara and Murakami 1996), and purified with a WizardÕ
SV Total RNA Isolation kit (Promega, Madison, WI, USA). Firststrand cDNA was synthesized from 5 mg of total RNA by reverse
transcriptase with an oligo(dT)20 primer according to the instructions supplied with ReverTra AceÕ (Toyobo, Tokyo, Japan).

299

Isolation and sequencing of FT/TFL1 genes from Lombardy poplar
We performed PCR using the sets of primers shown in
Supplementary Table S1. Partial PnFTL genes were amplified withdegenerate primers whose design was based on conserved regions ofplant FT/TFL1 genes. The ends of the ORFs of genes were
determined by 30 and 50 RACE-PCR (Frohman et al. 1988) or
inverse PCR (Ochman et al. 1988). All products of PCR were
subcloned and sequenced (ABI Prism 3100 and 3100-Avant DNA
sequencer; Applied Biosystems, Foster City, CA, USA). The
sequences of full-length ORFs and genomic sequences were
determined by sequencing of subcloned products of PCR that had
been amplified with first-strand cDNA or genomic DNA derived
from an individual female clone as template. To exclude thepossibility of recombination or mutation by PCR, we performed
triplicate experiments and sequenced 4100 clones per gene.

Sequence alignment and construction of a phylogenetic tree
Sequences were aligned by the N-J method with ClustalW
software (Saitou and Nei 1987, Thompson et al. 1994). An N-J tree
was produced from the results of 1,000 bootstrap replicates using
ClustalW, and the phylogenetic tree was displayed with the
MEGA4 software package (Tamura et al. 2007).

Analysis of gene expression
We performed real-time PCR for detection of the expressionof genes in the FT/TFL1 family in Lombardy poplar using theLightCyclerTM system (Roche, Penzberg, Germany) with a‘LightCyclerTM FastStart DNA Master SYBR Green I’ kit(Roche). The specific transcript of each Lombardy poplar FT/
TFL1 family member was identified with gene-specific primers, as
shown in Supplementary Table S1, and primers specific for the
Lombardy poplar gene for polyubiquitin (PnUB1) were used forthe control. Reactions were allowed to proceed for 45 cycles, as
follows: 10 s at 958C and 16 s at 668C, with each set of PnFTL genespecific primers (Supplementary Table S2). Values were normalized on the basis of the amount of PnUB1 cDNA.

Transformation of Arabidopsis
Agrobacterium tumefaciens GV3101 (pMP90), harboring abinary vector, was used to transform Arabidopsis plants by thefloral dip method (Clough and Bent 1998). All binary vectors usedin this study were constructed by introducing FT/TFL1 cDNA into
the hygromycin-selectable binary vector pSMAH621 (Igasaki et al.
2000). For selection of transformed plants, sterilized seeds wereplated on MSB5 medium supplemented with 20 mg l1 hygromycin(Wako Pure Chemical Industries, Osaka, Japan) and allowed
to germinate in the growth chamber. Hygromycin-resistanttransformants were transplanted to soil on the tenth day aftersowing. The expression of introduced genes in selected transformants was confirmed by RT–PCR. The day of sowing was taken
as day 0. We analyzed the morphology of plants in the third
generation.

Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.
oxfordjournals.org.

300

FT/TFLI family in poplar

Funding
The Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN);
Grant-in-Aid for Young Scientists [no. 18780246 (B)] of
the Ministry of Education, Sports, Science and Technologyof Japan.

Acknowledgments
The authors thank Dr. Hideyuki Saito (Hokkaido University,
Japan) for the generous gift of samples of the various organs ofLombardy poplar that were used in this study. The authors also
thank Ms. Yoko Ooi, Ms. Mami Aizawa, Ms. Ai Hagiwara andMs. Michiyo Hirota for their skilled technical assistance, and Dr.
Kiyoshi Tazaki (Forestry Science and Technology Institute) for
critical reading of this manuscript.

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(Received December 4, 2007; Accepted January 11, 2008)