Mutation in TERMINAL FLOWER1 Reverses the
Photoperiodic Requirement for Flowering in the Wild
Strawberry Fragaria vesca1[W]
Elli A. Koskela, Katriina Mouhu, Maria C. Albani, Takeshi Kurokura, Marja Rantanen, Daniel J. Sargent,
Nicholas H. Battey, George Coupland, Paula Elomaa, and Timo Hytönen*
Department of Agricultural Sciences, University of Helsinki, FIN–00014 Helsinki, Finland (E.A.K., K.M., T.K.,
M.R., P.E., T.H.); Max Planck Institute for Plant Breeding Research, D–50829 Cologne, Germany (M.C.A., G.C.);
School of Biological Sciences, University of Reading, RG6 6AS Reading, United Kingdom (T.K., N.H.B.); East
Malling Research, ME19 6BJ Kent, United Kingdom (D.J.S.); and Istituto Agrario San Michele all’Adige, 38010
San Michele all’Adige, Italy (D.J.S.)
Photoperiodic flowering has been extensively studied in the annual short-day and long-day plants rice (Oryza sativa) and
Arabidopsis (Arabidopsis thaliana), whereas less is known about the control of flowering in perennials. In the perennial wild
strawberry, Fragaria vesca (Rosaceae), short-day and perpetual flowering long-day accessions occur. Genetic analyses showed
that differences in their flowering responses are caused by a single gene, SEASONAL FLOWERING LOCUS, which may encode
the F. vesca homolog of TERMINAL FLOWER1 (FvTFL1). We show through high-resolution mapping and transgenic approaches
that FvTFL1 is the basis of this change in flowering behavior and demonstrate that FvTFL1 acts as a photoperiodically regulated
repressor. In short-day F. vesca, long photoperiods activate FvTFL1 mRNA expression and short days suppress it, promoting
flower induction. These seasonal cycles in FvTFL1 mRNA level confer seasonal cycling of vegetative and reproductive
development. Mutations in FvTFL1 prevent long-day suppression of flowering, and the early flowering that then occurs
under long days is dependent on the F. vesca homolog of FLOWERING LOCUS T. This photoperiodic response mechanism
differs from those described in model annual plants. We suggest that this mechanism controls flowering within the perennial
growth cycle in F. vesca and demonstrate that a change in a single gene reverses the photoperiodic requirements for flowering.
Plants must time their vegetative and reproductive
growth phases accurately in order to ensure their own
survival as well as the survival of their progeny. In
addition to endogenous cues such as their developmental stage and hormone levels, plants are able
to monitor environmental signals, most importantly
photoperiod and temperature, for developmental timing (Simpson, 2004; Samach and Wigge, 2005; Turck
et al., 2008; Kim et al., 2009; Mutasa-Göttgens and
Hedden, 2009). Appropriate developmental timing is
especially important for perennial species, which may
live for many years, undergoing repeated cycles of
vegetative and reproductive (flowering) development,
which are regulated by changes in the seasons through
the year. Photoperiod is the major environmental cue
for developmental timing in several perennial species
(Cooper and Calder, 1964; Böhlenius et al., 2006;
1
This work was supported by the Academy of Finland (grant no.
137439 to T.H.) and the University of Helsinki (grant no. DW–
4881545211 to T.H).
* Corresponding author; e-mail timo.hytonen@helsinki.fi.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Timo Hytönen (timo.hytonen@helsinki.fi).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.196659
Gyllenstrand et al., 2007; Heide and Sønsteby, 2007),
because it is the only constant environmental signal
that undergoes the same cyclical pattern every year,
with increasing amplitude toward higher latitudes.
Many perennial plants are economically important
crops. Therefore, knowledge of the molecular regulation of flowering and developmental cycling in these
species will enhance their cultivation as well as breeding. The woodland strawberry, Fragaria vesca, which
belongs to the most important family of fruit crops, the
Rosaceae, serves as a convenient perennial model
(Shulaev et al., 2008, 2011). The presence of different
accessions with opposite photoperiodic responses
(Heide and Sønsteby, 2007; Sønsteby and Heide, 2008;
Mouhu et al., 2009) makes this species a particularly
useful model for photoperiodic research among perennials. Short-day (SD) F. vesca has a perennial life
cycle characteristic of the Rosaceae; flower initiation
occurs at the apical meristem under SD and low temperatures during autumn, and flowers emerge the
following spring. Under the long days (LDs) of summer, new shoots that have emerged in the uppermost
nodes remain vegetative, then as the days become
sufficiently short and temperature declines, flower
primordia are again produced (Battey et al., 1998;
Battey, 2000; Heide and Sønsteby, 2007). Detailed
physiological analyses in SD F. vesca demonstrated
that a short photoperiod is obligatory for flower
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Koskela et al.
induction at intermediate temperatures; at temperatures above 20°C, floral development is inhibited; and
cool temperatures of about 10°C strongly induce
flowering in both SD and LD conditions (Heide and
Sønsteby, 2007). Similar to SD F. vesca, the photoperiod
sensitivity of several cultivars of octoploid garden
strawberry (Fragaria 3 ananassa) is strongly dependent
on temperature (Heide, 1977; Bradford et al., 2010),
suggesting that diploid F. vesca may be used as a
model for more complex cultivated species.
In contrast to the seasonal flowering SD F. vesca,
perpetual flowering accessions of the species (F. vesca f.
semperflorens) flower and bear fruits from early summer until late autumn (Brown and Wareing, 1965). In
three perpetual flowering accessions, reversed environmental responses compared with SD accessions
have been reported. They are LD plants, which flower
rapidly under LDs and high temperature, as opposed
to SDs and low temperature, which repress flowering
(Sønsteby and Heide, 2008; Mouhu et al., 2009). Classical genetic studies have shown that perpetual flowering is caused by recessive alleles of a single repressor
gene called SEASONAL FLOWERING LOCUS (SFL;
Brown and Wareing, 1965; Albani et al., 2004). Perpetual flowering cultivars (often called remontant or
everbearing) are known also in cultivated strawberry.
These cultivars have been considered as day-neutral or
temperature-dependent LD plants in different studies
(Sønsteby and Heide, 2007; Weebadde et al., 2008;
Bradford et al., 2010; Stewart and Folta, 2010).
Photoperiodic flowering has been explained by the
coincidence of CONSTANS (CO) diurnal expression
rhythm and external light signals in both SD and LD
model annuals, rice (Oryza sativa) and Arabidopsis
(Arabidopsis thaliana), respectively (Suárez-López et al.,
2001; Hayama et al., 2003). In both species, CO controls
flowering through the CETS (for CEN, TFL1, and FT)
family protein FLOWERING LOCUS T (FT), which is
thought to be a universal flowering signal (SuárezLópez et al., 2001; Hayama et al., 2003; Komiya et al.,
2009; Turnbull, 2011). In the LD plant Arabidopsis, CO
protein accumulates in the leaf phloem companion
cells and activates FT expression only in LDs when the
light period coincides with the CO expression peak in
the late afternoon (An et al., 2004; Valverde et al., 2004;
Corbesier et al., 2007). In contrast, in the SD plant rice,
an FT homolog, Heading date3a, is activated when the
CO homolog Heading date1 peaks after dusk in SDs
(Hayama et al., 2003). After the activation of FT expression, the FT protein moves through the phloem to
the shoot apex (Corbesier et al., 2007; Jaeger and
Wigge, 2007; Tamaki et al., 2007), where 14-3-3 proteins bridge its interaction with a bZIP transcription
factor, FLOWERING LOCUS D (FD; Taoka et al.,
2011). The FT/14-3-3/FD complex, in turn, induces
flowering by up-regulating the floral meristem identity
genes APETALA1 (AP1) and FRUITFULL (FUL; Abe
et al., 2005; Wigge et al., 2005). Another CETS family
protein, the floral repressor TERMINAL FLOWER1
(TFL1), also binds to FD and suppresses the expression
of LEAFY, AP1, and FUL (Ratcliffe et al., 1999; Hanano
and Goto, 2011). In Arabidopsis, TFL1 is developmentally regulated and has not been linked to the
photoperiodic pathway. At the vegetative stage, TFL1
mRNA is weakly expressed in the lower part of the
apical meristem, whereas the protein can move short
distances to repress flowering in the main apex. After
flower induction, TFL1 is strongly up-regulated to
maintain the indeterminate inflorescence meristem
(Shannon and Meeks-Wagner, 1991; Bradley et al.,
1997; Ratcliffe et al., 1999; Conti and Bradley, 2007).
The functions of key flowering genes seem to be at
least partially conserved between annual and perennial
species, but their regulation may vary in the perennial
context (Albani and Coupland, 2010). FT homologs have
been shown to promote flowering in many perennial
species (Endo et al., 2005; Böhlenius et al., 2006; Hsu
et al., 2006; Kotoda et al., 2010), but at least in Populus
trichocarpa, FT is also involved in the control of growth
cessation (Böhlenius et al., 2006). Detailed analyses of
two Populus FT paralogs have shown that seasonal
changes in their expression control the cycling of reproductive and vegetative growth. Winter cold (vernalization) activates FT1, which promotes flowering, whereas
LD and high temperature activate FT2, which promotes
vegetative growth and prevents bud set (Hsu et al.,
2011). In Arabis alpina, the return to vegetative development after flowering is regulated by transient silencing of
the FLOWERING LOCUS C (FLC) ortholog PERPETUAL
FLOWERING1 (PEP1) by vernalization (Wang et al.,
2009). pep1-1 mutants show reduced vegetative growth
but also flower perpetually, which is a trait reported in
other perennials such as rose (Rosa sp.) and F. vesca
(Brown and Wareing, 1965; Iwata et al., 2012). Furthermore, TFL1 homologs have been shown to repress
flowering and to control the length of the juvenile
phase in apple (Malus domestica), Populus, and A. alpina
(Kotoda et al., 2006; Mohamed et al., 2010; Wang et al.,
2011).
Recently, Iwata et al. (2012) identified a F. vesca
homolog of TFL1 (FvTFL1) as a candidate gene for SFL.
They showed that a 2-bp deletion in FvTFL1 is associated with perpetual flowering in F. vesca, but functional validation was lacking (Iwata et al., 2012). Here,
we provide functional evidence for FvTFL1 being SFL.
Our results further indicate that FvTFL1 is the key
component of the perennial photoperiodic pathway in
F. vesca: it confers an SD requirement for flowering and
controls cycling between vegetative and reproductive
phases. We also demonstrate how a 2-bp deletion in
FvTFL1 changes the regulation of seasonal life cycles
and leads to FvFT-dependent LD flowering.
RESULTS
Mutation in the Floral Repressor FvTFL1 Reverses the
Photoperiodic Requirement for Flower Induction
SFL, which causes seasonal flowering in F. vesca
(Brown and Wareing, 1965; Albani et al., 2004), was
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FvTFL1 Controls Photoperiodic Flowering
initially mapped to F. vesca linkage group 6 (Sargent
et al., 2004). In a recent study, association was found
between perpetual flowering habit and a 2-bp deletion
in the first exon of F. vesca TFL1 homolog, and the gene
was roughly mapped between two markers with a
physical distance of approximately 11 Mb in the same
linkage group (Iwata et al., 2012). We narrowed down
the location of SFL within the mapping window of 248
kb, and our extensive linkage analysis in two populations fully supports the association found by Iwata
et al. (2012; Supplemental Figs. S1 and S2).
To explore the function of FvTFL1, we overexpressed
the SD F. vesca (PI551792 [National Clonal Germplasm
Repository]; abbreviated to VES in the figures) FvTFL1
and the allele with a 2-bp deletion under the control of
the cauliflower mosaic virus P35S promoter in the LD
accession Hawaii-4 (PI551572; abbreviated to H4 in the
figures). Primary transgenic lines overexpressing the
wild-type FvTFL1 allele from SD F. vesca (P35S:FvTFL1)
remained vegetative for at least 10 months under inductive LD conditions, whereas plants overexpressing
the mutated FvTFL1 (P35S:FvmTFL1) flowered continuously (Fig. 1, A–C; Supplemental Fig. S3). We further
analyzed the flowering time by growing the plants propagated from single-leafed runner cuttings under LD.
Both nontransgenic Hawaii-4 and P35S:FvmTFL1 lines
flowered early, whereas P35S:FvTFL1 lines and SD F.
vesca were still vegetative after about 10 weeks (Fig. 1D).
These data indicate that FvTFL1 represses flowering
under LD and that the 2-bp deletion in FvTFL1 leads to a
nonfunctional FvTFL1 protein.
In order to further test the hypothesis that FvTFL1 is
SFL, we produced transgenic lines in the SD F. vesca
background and analyzed their flowering phenotypes
in LD or after a strong flower induction treatment
(4 weeks of SD at 11°C). FvTFL1 RNA interference
(RNAi) silencing lines flowered almost at the same
time in both treatments (Fig. 2, A and B). In contrast,
nontransgenic control plants remained vegetative in
LD and required SD induction treatment for flowering,
whereas P35S:FvTFL1 lines stayed vegetative in both
treatments (Fig. 2, A and B; Supplemental Fig. S4). In
line with the role of Arabidopsis TFL1 in the transcriptional repression of floral meristem identity genes
(Liljegren et al., 1999; Ratcliffe et al., 1999; Hanano and
Goto, 2011), the expression of putative F. vesca AP1/
FUL homologs correlated negatively with the expression of functional FvTFL1 in the transgenic F. vesca
lines (Figs. 1, E–H, and 2, C and D). In conclusion, our
results demonstrate that FvTFL1 is the major floral
repressor SFL, which prevents the activation of floral
meristem identity genes and flowering under LD
conditions in SD F. vesca. Moreover, the absence of
functional FvTFL1 reverses the photoperiodic requirements for flower induction.
We also expressed a P35S:FvTFL1 overexpression
construct in the Arabidopsis tfl1-2 mutant, which flowers early especially under SD and produces a determinate inflorescence with a few flowers (Shannon and
Meeks-Wagner, 1991; Supplemental Fig. S5B). We analyzed three independent P35S:FvTFL1 lines in the tfl1-2
mutant background and showed that FvTFL1 fully
complemented the inflorescence defects and the earlyflowering phenotype of the mutant in both SDs and
LDs (Supplemental Fig. S5), indicating that FvTFL1 is
an ortholog of TFL1.
Figure 1. FvTFL1 is the floral repressor SFL. A to C, Phenotypes of SD F. vesca (A), LD accession Hawaii-4 (H4; B), and a
transgenic line overexpressing functional FvTFL1 (P35S:FvTFL1) in the H4 background (C). Plants were grown under LD for 15
weeks. WT, Wild type. D, Flowering time of SD F. vesca (VES), H4, and transgenic lines overexpressing functional and nonfunctional FvTFL1 (FvTFL1-ox and FvmTFL1-ox, respectively) under the control of the P35S promoter. Plants were propagated
from runner cuttings. n = 3 (VES), n = 7 (H4), n = 8 (FvmTFL1-ox2), n = 2 (FvmTFL1-ox3), and n = 6 (FvTFL1-ox1 and FvTFL1ox2). E to H, Expression of FvTFL1 (E), FvAP1 (F), FvFUL1 (G), and FvFUL2 (H) in the primary shoot apices of H4 and transgenic
lines grown in LDs. n = 3 for H4 and n = 1 for each transgenic line. Results for two independent transgenic lines are shown as
biological replicates. nd, Not detected.
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Koskela et al.
Figure 2. Silencing of FvTFL1 leads to daylength-independent flowering. A, Phenotypes of FvTFL1 RNAi silencing and overexpression lines in the SD F. vesca background. Clonally propagated plants (runner cuttings) of SD F. vesca and P35S:FvTFL1RNAi-1 and P35S:FvTFL1-1 lines were subjected to SD induction treatment for 4 weeks followed by LDs (left) or grown
continuously under LDs (right). B, Flowering time of SD F. vesca and P35S:FvTFL1-RNAi and P35S:FvTFL1 plants (RNAi and
OX, respectively) in SDs and LDs. Flowering time is indicated as days to anthesis from the beginning of the treatments.
Treatments and plant materials were as described in A. Values indicate means 6 SD. n = 4 (OX-1), n = 5 (RNAi-2), n = 6 (RNAi-1
and OX-2), and n = 7 (SD F. vesca). C and D, Expression of FvTFL1 (C) and FvAP1 (D) in the apices of two independent P35S:
FvTFL1-RNAi (RNAi) lines. Values indicate means 6 SD. n = 3 (RNAi-1 and SD F. vesca) or n = 2 (RNAi-2).
FvTFL1 Is Not Involved in the Photoperiodic Control of
Vegetative Development
Under LDs, strawberry vegetative development
typically involves the formation of a single leaf rosette
and the differentiation of axillary buds to stolons (also
called runners), whereas flower inductive conditions
inhibit the emergence of stolons and activate the production of branch crowns (i.e. axillary leaf rosettes;
Hytönen et al., 2004, 2009; Heide and Sønsteby, 2007).
We analyzed the role of FvTFL1 in the control of
vegetative development by using transgenic lines in a
SD F. vesca background. Similar to wild-type plants,
both FvTFL1 overexpression and RNAi lines continuously produced runners under LD, whereas 3 weeks
of SD was enough to stop stolon formation in all
lines (Fig. 3, A and B). After the cessation of stolon
production in SD, the number of branch crowns started
to increase in both wild-type plants and transgenic
lines, whereas under LD no branch crowns were formed
(Fig. 3, C and D). These results suggest that although the
photoperiodic control of vegetative and floral development is tightly connected in strawberries (for review, see
Hytönen and Elomaa, 2011), FvTFL1 does not affect
vegetative development and is only involved in the
photoperiodic control of floral initiation.
Photoperiodic Repression of FvTFL1 Expression Correlates
with Floral Initiation
In Arabidopsis, TFL1 is weakly expressed in the
lower part of the vegetative apical meristem, but its
expression is activated in the inflorescence meristem
Figure 3. FvTFL1 is not involved in the photoperiodic
control of vegetative development. A and B, The
cumulative number of stolons formed from the axillary buds during photoperiodic treatments in the
FvTFL1 RNAi (A) and overexpression (B) lines in
comparison with SD F. vesca (VES). C and D, The
cumulative number of branch crowns (axillary leaf
rosettes) formed after the photoperiodic treatments in
the FvTFL1 RNAi (C) and overexpression (D) lines in
comparison with SD F. vesca. Experimental conditions and the number of plants used for each line are
described in Figure 2.
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FvTFL1 Controls Photoperiodic Flowering
after flower induction (Bradley et al., 1997; Ratcliffe et al.,
1999). We analyzed the relative expression of FvTFL1
normalized against the stable control gene FvMSI1
MULTICOPY SUPPRESSOR OF IRA1 (Mouhu et al.,
2009). FvTFL1 mRNA was detected in all tissues tested
(Supplemental Fig. S6A). Similar to TFL1, it was highly
expressed in the shoot apex compared with leaves in
SD F. vesca grown under LDs (Fig. 4A), but a more detailed analysis revealed a divergent expression pattern.
According to in situ hybridization, under LDs FvTFL1
mRNA was localized in the whole apical meristem, young
leaf initials, and vascular tissues (Fig. 4, B and C). Furthermore, strong photoperiodic regulation of FvTFL1 was
detected in the shoot apices but not in the leaves of SD F.
vesca seedlings (Fig. 4D; Supplemental Fig. S6B). Under
LDs, FvTFL1 was highly expressed in the shoot apex,
whereas only weak expression was found in plants grown
under SDs for 3 weeks (Fig. 4D). However, floral meristem identity genes were not yet activated in SD-grown
SD F. vesca at this time (Supplemental Fig. S7), indicating
that the plants were still vegetative. Because several
Norwegian F. vesca accessions have been shown to require
more than 1 month of SD treatment for flower induction
(Heide and Sønsteby, 2007), we grew our SD F. vesca
plants in SDs for 45 d followed by flowering-time analysis
under LDs. We observed flower buds about 1 month after
the SD treatment in all plants of SD F. vesca, whereas all
LD-grown plants remained vegetative (Fig. 4E).
In contrast to SD F. vesca, LD-grown Hawaii-4
seedlings flowered rapidly; seedlings raised under SDs
for 45 d flowered late, and the plants grown continuously under SDs remained vegetative for several months
(Fig. 4, E and F), indicating that Hawaii-4 is a LD plant.
In contrast to seedlings, plants propagated from the
stolons of LD-grown mother plants were day neutral
and flowered continuously under both LDs and SDs
(Supplemental Fig. S8). In Hawaii-4, FvTFL1 mRNA
expression was low in both photoperiods (Fig. 4D),
most likely because of nonsense-mediated mRNA decay, a mechanism that prevents the accumulation of
mRNA containing premature stop codons (Conti and
Izaurralde, 2005). In association with flowering, the
putative floral meristem identity genes FvAP1 and
FvFUL1 were up-regulated in Hawaii-4 seedlings
grown under LDs (Supplemental Fig. S6). Taken together, our data indicate that the SD requirement for
flower induction in SD F. vesca is based on the photoperiodic regulation of FvTFL1, and the lack of FvTFL1
function leads to the rapid activation of flowering in
LD-grown Hawaii-4.
Regulation of the Perennial Growth Cycle by FvTFL1
Figure 4. The expression of FvTFL1 correlates with photoperiodic
flowering. A, Expression of FvTFL1 in leaves and shoot apices of SD F.
vesca grown under LD. Values indicate means 6 SD (n = 4). B, Localization of the FvTFL1 mRNA in the shoot apex of SD F. vesca grown
under LDs. C, FvTFL1 sense probe control for B. Bars = 100 mm. D,
Expression of FvTFL1 in primary shoot apices of LD- and SD-grown SD
F. vesca (VES) and Hawaii-4 (H4) seedlings at the three-leaf stage.
Values indicate means 6 SD (n = 4). E, Flowering time of seedlings of
H4 and SD F. vesca shown as time to visible flower bud. Plants were
grown either under continuous LD or for 45 d under SD followed by
LDs. F, Flowering time of H4 seedlings shown as the number of leaves
in the main shoot before terminal inflorescence under continuous SDs
and LDs.
We further analyzed the expression patterns of
FvTFL1 as well as FvAP1 and FvFUL1 in SD F. vesca
plants grown under SDs followed by LDs and observed the flowering cycle of the plants. Our data
showed that FvTFL1 was gradually down-regulated in
the apices of the primary shoots grown under SDs,
with a concomitant up-regulation of floral meristem
identity genes 4 to 6 weeks after the beginning of the
SD treatment (Fig. 5, A–C). When SD-grown plants
were returned to LDs after 45 SDs, high FvTFL1 and
low FvAP1 and FvFUL1 mRNA levels were again
detected in the apices of axillary shoots that emerged
after the end of the SD treatment. The observed gene
expression patterns were clearly related to flowering,
which started about 5 weeks after the end of the SD
treatment and continued for about 1 month before
declining again (Fig. 5D). In contrast to SD F. vesca,
Hawaii-4 continuously produced new inflorescences
only under LDs (Fig. 5D). The observed expression
pattern of FvTFL1 in SD F. vesca indicates that the
photoperiodic regulation of FvTFL1 mRNA expression
is crucial for cycling between vegetative and reproductive phases in SD F. vesca.
FvFT1 Promotes Flowering under LDs in the
F. vesca LD Accession
Functional analysis of FT in several plant species
indicates its role as a florigen, a general floral activator
(Turck et al., 2008; Turnbull, 2011). To establish the
role of FT relative to that of SFL, we analyzed the
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Koskela et al.
expression patterns of F. vesca FT-like genes. As demonstrated in Figure 6, A and B, FvFT1 and FvFT2
showed contrasting spatial expression patterns in SD
F. vesca. FvFT1 had the highest expression level in old
leaves, and no expression or weak expression was
observed in other tissues, whereas FvFT2 was expressed
almost exclusively in flower buds. The tissue-specific
expression pattern of FvFT1 follows the consensus that
FT is a mobile signal originating in photosynthetically
active leaves (Corbesier et al., 2007; Tamaki et al.,
2007). Furthermore, genomic synteny is conserved
Figure 5. Photoperiod regulates the perennial growth cycle through
FvTFL1. A to C, Time-course analysis of FvTFL1 (A), FvAP1 (B), and
FvFUL1 (C) expression in the primary shoot apices of SD F. vesca. LDgrown plants were subjected to SDs for 6 weeks (wk 0–6) and returned
to LDs for 5 weeks (+5 LD). Values indicate means 6 SE (n = 3). D,
Cumulative number of inflorescences in clonally propagated plants
(runner cuttings) of Hawaii-4 (H4) and SD F. vesca (VES). Plants were
grown under flower-inductive (6 weeks of LD/SD starting from week 0
for H4/VES followed by LD) or noninductive (SD/LD for H4/VES)
conditions.
Figure 6. Spatial and temporal expression of F. vesca FT genes. A and
B, FvFT1 (A) is expressed in old leaves and FvFT2 (B) in flower buds in
SD F. vesca. Plants were grown under LD after flower induction.
Values indicate means 6 SD (n = 2). YL, Young unopened leaf; OL,
opened leaf; YP, young petiole; OP, petiole of the opened leaf; CR,
crown (stem including meristems); RT, runner tip; FB, flower bud; FL,
open flower; RO, root. C, Expression of FvFT1 in leaves of SD- and LDgrown seedlings of F. vesca SD accession (VES) and LD accession
Hawaii-4 (H4) at the three-leaf stage. Values indicate means 6 SD (n =
4). D, Diurnal rhythm of FvFT1 expression is present only under LDs in
the SD F. vesca. Runner-propagated plants were either grown under
LDs or transferred to SDs 8 d before sampling. Values indicate
means 6 SE (n = 3). ZT, Zeitgeiber time. E, Time-course analysis of
FvFT1 in leaves of SD F. vesca plants. Plants were moved to flowerinductive SDs at week 0. Values indicate means 6 SE (n = 3).
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FvTFL1 Controls Photoperiodic Flowering
only around Arabidopsis FT and FvFT1 (Shulaev et al.,
2011), suggesting that FvFT1 is a likely ortholog of FT.
The comparison of FvFT1 expression levels in seedlings of SD F. vesca and Hawaii-4 in different photoperiods revealed that FvFT1 was expressed under LD
in both genotypes, whereas no FvFT1 expression was
detected in SD-grown seedlings (Fig. 6C). The finding
that FvFT1 mRNA levels did not correlate positively
with flowering in the SD F. vesca contrasted with the
situation in other SD plants (Hayama et al., 2003, 2007;
Kong et al., 2010) and prompted us to perform diurnal
rhythm and time-course analyses in older plants of SD
F. vesca. As shown in Figure 6D, FvFT1 showed a clear
LD-specific expression peak during the night. Moreover, FvFT1 was strongly down-regulated before flower
induction under short photoperiods (Fig. 6E). Thus,
the activation of FvFT1 mRNA expression correlated
positively with flower induction only in the LD accession of F. vesca.
To explore the role of FT in the LD flowering response, we produced FvFT1 RNAi silencing (P35S:
FvFT1-RNAi) plants in the perpetual flowering LD accession Hawaii-4. We found down-regulation of floral
meristem identity genes in the shoot apex of three
independent transgenic lines grown under LD (Fig. 7,
A–C; Supplemental Fig. S7). Furthermore, all of these
RNAi lines were clearly late flowering under LD (Fig.
7D). These results suggest that in LD-grown Hawaii-4,
FvFT1 is required for the normal up-regulation of floral
meristem identity genes, which marks the beginning of
floral initiation at the apex (Mandel and Yanofsky,
1995; Ferrándiz et al., 2000; Mouhu et al., 2009).
DISCUSSION
Early physiological studies indicated the existence of
photoperiodically controlled flowering activating and
inhibitory signals in strawberries (Hartmann, 1947;
Guttridge, 1959; Vince-Prue and Guttridge, 1973). Our
results confirm that both signals are present in F. vesca
and that SFL is a major switch controlling photoperiodic responses. We provide functional evidence that
SFL encodes a Fragaria homolog of the floral repressor
TFL1 and demonstrate that FvTFL1 is photoperiodically regulated. Our results suggest that in the SD accessions of F. vesca, down-regulation of FvTFL1 under
SD allows flower induction to occur only in the autumn, which leads to seasonal flowering the next
spring. In contrast, a mutation in FvTFL1 causes rapid
FT-dependent LD flowering and continuous initiation
of inflorescences in the perpetual flowering LD accessions.
TFL1 Homologs Are Major Floral Repressors in Rosaceae
A previous study by Iwata et al. (2012) and our data
show that perpetual flowering is associated with a
2-bp deletion in F. vesca TFL1 homolog (Supplemental
Figs. S1 and S2). We conducted functional analysis of
Figure 7. FvFT1 is a LD-specific activator of flowering. A, Expression
of FvFT1 in the leaves of P35S:FvFT1-RNAi lines in the Hawaii-4 (H4)
background and in the H4 LD accession grown under LDs. Samples
were collected 16 h after dawn (n = 1). The same lines also were
analyzed 4 h after dawn (Supplemental Fig. S9). B and C, Expression of
FvAP1 (B) and FvFUL1 (C) in the primary shoot apices of P35S:FvFT1RNAi lines in the H4 background and in the H4 LD accession grown
under LDs. n = 3 for H4 and n = 1 for each transgenic line. Results for
three independent transgenic lines are shown as biological replicates.
D, Flowering time of P35S:FvFT1-RNAi lines in the H4 background
and in the H4 LD accession grown under LDs. Plants were propagated
from runner cuttings with a single leaf. Values indicate means 6 SD
(n = 6–11).
FvTFL1 by overexpression and RNAi approaches.
FvTFL1 strongly represses flowering in F. vesca, because the introduction of a P35S:FvTFL1 construct
containing functional FvTFL1 into a perpetual flowering LD accession prevented the activation of putative
floral meristem identity genes (FvAP1/FUL) and flower
initiation under LD. Furthermore, overexpression of
the mutated version of the gene did not change the
flowering phenotype, indicating that it does not encode a functional protein. As constitutive overexpression of TFL1 homologs from different species has been
shown to cause pleiotropic effects (Böhlenius et al.,
2006; Imamura et al., 2011), we used RNAi silencing of
FvTFL1 in SD F. vesca to provide further evidence that
FvTFL1 is SFL. Silencing of FvTFL1 in SD F. vesca removed the SD requirement for flower induction and
changed the plants to LD flowering. As TFL1 homologs prevent LD flowering and cause the seasonal
flowering habit in F. vesca (Figs. 1 and 2), control the
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Koskela et al.
length of the juvenile phase in apple (Kotoda et al.,
2006), and may also cause seasonal flowering in roses
(Iwata et al., 2012), they are possibly major floral repressors and regulators of the perennial growth cycle
in the Rosaceae family.
Earlier studies have shown that photoperiod acts in
an opposite manner to control flower initiation and
vegetative reproduction through stolons (Hytönen
et al., 2004; Heide and Sønsteby, 2007). The finding
that neither overexpression nor silencing of FvTFL1
affected the photoperiodic control of stolon/branch
crown formation from axillary buds showed that
FvTFL1 is not directly involved in the regulation of
vegetative development of the strawberry shoot. This
is consistent with the genetic evidence that two separate single loci, SFL and RUNNERING LOCUS, respectively, control flowering habit and the formation
of stolons in F. vesca (Brown and Wareing, 1965). However, FvTFL1 may indirectly affect vegetative growth,
because the development of a terminal inflorescence
promotes the outgrowth of the uppermost axillary buds
as branch crowns by reducing apical dominance
(Arney, 1953).
Regulation of FvTFL1 mRNA Expression Controls
Photoperiodic Flowering and Seasonal Growth Cycles
In Arabidopsis, TFL1 is developmentally regulated
(Bradley et al., 1997; Ratcliffe et al., 1999), and to our
knowledge, no photoperiodic control of TFL1 homologs has been previously reported. Our results suggest
that the photoperiodic control of FvTFL1 mRNA level
in the shoot apex is a primary mechanism to control
photoperiodic flowering in SD F. vesca. In SD-grown
plants, FvTFL1 was strongly down-regulated before
the activation of floral meristem identity genes and
flower initiation. The functionality of transcriptional
regulation was supported by the phenotype of FvTFL1
RNAi silencing lines in the SD F. vesca background. In
two independent lines, the reduction of FvTFL1 mRNA
levels was enough to induce flowering under noninductive LD conditions. In contrast, none of the P35S:
FvTFL1 lines flowered under flower-inductive conditions in either SD or LD F. vesca backgrounds, indicating
that plants cannot overcome constitutive overexpression of FvTFL1 mRNA. In conclusion, we have identified
FvTFL1 as a component of the perennial photoperiodic
pathway in F. vesca. We propose that down-regulation
of FvTFL1 mRNA expression in the shoot apex of SD F.
vesca under SD is required for flower induction and the
activation of FvAP1/FUL genes. In contrast, the lack of
functional FvTFL1 in LD accessions reverses the photoperiodic requirement for flowering.
The finding that nonfunctional FvTFL1 causes perpetual flowering of LD accessions suggests that in the
SD F. vesca, the regulation of FvTFL1 contributes to the
cycling of vegetative and reproductive phases, and our
gene expression results support this hypothesis. Whereas
FvTFL1 was down-regulated and FvAP1/FUL was up-
regulated in the apex of the primary shoot under SD,
subsequent LD conditions restored high FvTFL1 and
low FvAP1/FUL mRNA levels in the apices of new
vegetative axillary shoots. Earlier studies have shown
that strawberries are able to initiate flowers at the
apex of the primary shoot and axillary shoots, which
have reached competence to flower (Arney, 1953;
Hytönen et al., 2004; Hytönen and Elomaa, 2011).
Therefore, we hypothesize that the down-regulation
of FvTFL1 under SD in the autumn allows flower
induction to occur in the apex of the primary shoot
and older axillary shoots. However, its up-regulation
in the LDs of spring is crucial for perennialism, because it prevents further floral initiation in the newly
emerged axillary shoots until SD conditions return.
Our results on FvTFL1 and earlier findings that seasonal changes in the expression of two Populus FTlike genes regulate the perennial growth cycle in
Populus (Hsu et al., 2011) highlight the importance of
CETS proteins in the control of perennialism. Also, in
A. alpina, TFL1 contributes to perennialism by regulating the age-dependent sensitivity of meristems to
vernalization; but transient silencing of the FLC
ortholog, PEP1, by vernalization has a major role in
the cycling between vegetative and generative phases
in this species (Wang et al., 2009, 2011).
In contrast to FvTFL1, which is broadly expressed in
the shoot apex (Fig. 4B), Arabidopsis TFL1 is weakly
expressed in the axillary meristems and lower part of
the apical meristem during the vegetative phase and is
activated in the inflorescence meristem (Bradley et al.,
1997; Ratcliffe et al., 1999). This intriguing difference is
probably associated with differences in inflorescence
development. In Arabidopsis, TFL1 is activated in the
inflorescence meristem in order to allow indeterminate
Figure 8. Model showing the photoperiodic control of flowering in F.
vesca SD and LD accessions. Arrows indicate activation, and bars
indicate repression. The regulation of FvAP1/FUL by FvFT1 in SD F.
vesca is shown by a dashed line because the strong repressor FvTFL1
overrides the floral activator function of FvFT1.
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FvTFL1 Controls Photoperiodic Flowering
growth of the raceme (Bradley et al., 1997; Ratcliffe
et al., 1999), whereas the production of a cymose inflorescence with a terminal flower in strawberry (Jahn
and Dana, 1970) would be expected to require the silencing of FvTFL1 in the apical meristem.
FvTFL1 Overcomes the Function of the LD-Specific Floral
Activator FvFT1
The CO/FT module controls photoperiodic flowering in both annual and perennial plants (Suárez-López
et al., 2001; Hayama et al., 2003; Böhlenius et al., 2006),
and FT is thought to be a universal flowering signal
(Turck et al., 2008; Turnbull, 2011; Wigge, 2011). In
concordance with studies in other plants, we observed
that the expression of FvFT1, the likely ortholog of FT,
correlated with flowering in the LD accession Hawaii-4.
Furthermore, strong reduction in the expression of
FvAP1/FUL genes in FvFT1 RNAi plants as well as
their late-flowering phenotype suggested that FvFT1
controls flower induction in F. vesca LD accessions
(Fig. 7). However, in the SD F. vesca, FvFT1 was highly
expressed in noninductive LD, which contrasts with
findings in other SD plants (Hayama et al., 2003, 2007;
Kong et al., 2010). Our data suggest that in this genotype, FvTFL1 expression in the shoot apex may
overcome the function of FvFT1 as an activator of
flowering. FT and TFL1 are closely related proteins
whose opposite function is caused by differences in the
external loop (Ahn et al., 2006). A recent study showed
that FT forms a so-called “florigen activation complex”
together with FD and 14-3-3 proteins (Taoka et al.,
2011). Because TFL1 homologs in different species can
also bind FD and 14-3-3 (Pnueli et al., 2001; Purwestri
et al., 2009; Hanano and Goto, 2011), FvTFL1 may inhibit flowering by competing for binding partners with
FvFT1. This is consistent with the regulation of flowering in tomato (Solanum lycopersicum), in which the
balance between the expression of FT and TFL1 homologs controls flowering (Shalit et al., 2009). However,
in SD F. vesca, both FvFT1 and FvTFL1 are downregulated under flower-inductive conditions. Therefore,
we hypothesize that flower induction in SD F. vesca
takes place via an FvFT1-independent mechanism,
whereas in Hawaii-4, FvFT1 functions as a LD-specific
floral promoter in the absence of functional FvTFL1.
On the other hand, we cannot exclude the possibility
that FT is involved in the LD activation of FvTFL1,
which is photoperiodically regulated only in the shoot
apex (Fig. 4D; Supplemental Fig. S6B). As the perception of photoperiod is thought to occur in the leaves
(An et al., 2004; Corbesier et al., 2007), a systemic signal may be required for the photoperiodic control of
FvTFL1. FT is a good candidate for such a signal, because it is emerging as a general photoperiodic signaling molecule in diverse plant species (Wigge, 2011).
Furthermore, Hecht et al. (2011) have shown evidence
that leaf-expressed FT may mediate the photoperiodic
control of another CETS family gene in the pea (Pisum
sativum) shoot apex. Further studies are needed to resolve the function of FvFT1 in SD F. vesca and to establish which mechanism activates floral meristem
identity genes after the down-regulation of FvTFL1.
A Model of the Regulation of Flowering and Growth
Cycles by F. vesca TFL1 and FT Homologs
Based on our studies, we propose a model (Fig. 8) in
which the flowering of SD F. vesca accessions is repressed through LD-activated FvTFL1 expression,
which overrides the floral activator function of FvFT1.
When photoperiod drops below a critical level during
autumn, FvTFL1 mRNA levels decrease, floral meristem identity genes are up-regulated, probably through
an FvFT1-independent pathway, and floral development begins. In the following spring and summer,
FvTFL1 is activated again in new axillary shoots and
keeps them vegetative until inductive conditions
return. In contrast, the absence of functional FvTFL1 in
LD accessions leads to FvFT1-mediated flower induction and continuous flowering under LDs. More
studies are needed to explore how this model generated in the diploid Fragaria can be translated to the
more complex octoploid cultivated strawberry, which
shows similarities in the environmental control of flowering with F. vesca (Heide, 1977; Heide and Sønsteby,
2007; Sønsteby and Heide, 2007, 2008; Bradford et al.,
2010).
The timing and duration of flowering season are
traits of importance to crop production because they
are major factors contributing to yield potential and
the length of the harvest season. Natural variation in
TFL1 homologs has been important for the domestication and crop improvement of several annual crops
(Turnbull, 2011), and our study shows the importance
of TFL1 in the perennial context. As TFL1 homologs
exist in all angiosperms (Karlgren et al., 2011) and their
function as floral repressors is conserved between
plant families (Shannon and Meeks-Wagner, 1991;
Foucher et al., 2003; Kotoda et al., 2006; Mohamed
et al., 2010; Imamura et al., 2011), our findings are of
general significance for understanding the regulation
of flowering- and perennial-specific traits in a wide
range of economically important crops.
MATERIALS AND METHODS
Plant Material
Fragaria vesca accession (PI551792) and F. vesca f. semperflorens Hawaii-4
(PI551572) were obtained from the National Clonal Germplasm Repository.
The Arabidopsis (Arabidopsis thaliana) tfl1-2 mutant (Nottingham Arabidopsis
Stock Centre identifier N3091) in the Landsberg erecta background was used in
the genetic complementation test.
Growth Conditions and Phenotyping
F. vesca plants were grown in greenhouse rooms equipped with darkening
curtains. Standard LD growth conditions were 18 h of high-pressure sodium
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Koskela et al.
light (approximately 150 mmol m22 s21) at 18°C. In photoperiodic experiments
at 18°C, 12 h of high-pressure sodium light (approximately 150 mmol m22 s21)
was provided for all plants (SD), and under LD, 6 h of day extension was
given as low-intensity incandescent light (approximately 8 mmol m22 s21).
Arabidopsis plants were grown under LD (16 h of light at 22°C) or SD (10 h of
light at 22°C) in a growth chamber. Light was provided by high-pressure
sodium lamps (approximately 180 mmol m22 s21). Flowering time was measured either as the number of leaves initiated before the first inflorescence or
the number of days from sowing to the first open flower.
Statistics
The x 2 goodness-of-fit test was performed on the flowering data from the
F2 and BC1 mapping populations. The critical value at a = 0.05 was 3.841 for
all x 2 tests.
Genotyping and Mapping
were performed as described earlier (Oosumi et al., 2006). Plants of the T1 or
T0 generation were analyzed in Arabidopsis and F. vesca, respectively.
In Situ Hybridization
In situ hybridization was performed on the apex of the main shoot as
described previously (Kurokura et al., 2006). The probe template fragment was
amplified by reverse transciption-PCR using the primers shown in Supplemental
Table S1 and cloned into pDrive cloning vector (Qiagen). Sense and antisense
probes were synthesized from T7 or SP6 promoters.
Sequence data from this article can be found in the GenBank data library
under accession numbers JN172097 and JN172098.
Supplemental Data
DNA extraction was done using a previously described protocol (Albani
et al., 2004). The markers used for mapping are listed in Supplemental Table
S1. Linkage maps were constructed as described earlier (Sargent et al., 2004).
The F2 mapping population was characterized for FvTFL1 using TFL1-6FAM
primers (Supplemental Table S1).
Identification of Single-Nucleotide Polymorphism
Markers by Genome Resequencing
DNA of SD F. vesca (PI551792) was extracted (Albani et al., 2004) and
treated with RNase A. A genomic DNA library was generated using the
Genomic DNA Sample Prep Kit (Illumina), and 30-bp reads were sequenced
using the Illumina GAIIx flow cell. Image analysis and base calling were done
by OLB version 1.6.0 software and sequence alignment by CASAVA GERALD
version 1.6.0. A default chastity threshold of 0.6 was used for purity filtering.
All sequence reads with two or fewer mismatches were aligned with 801,610
bp of the genomic reference sequence of Hawaii-4 between markers V8p98
and V8p278 (Supplemental Fig. S1) using Bowtie version 0.12.2 and Readaligner version 2010_4rc2 software, and the resulting .sam files (sequence
alignment/map files) were viewed by Tablet version 1.10.05.21 in order to find
single-nucleotide polymorphisms. Resequencing and related bioinformatics
were carried out at Biomedicum Genomics, University of Helsinki.
The following materials are available in the online version of this article.
Supplemental Figure S1. Fine-mapping of SFL.
Supplemental Figure S2. Deletion in FvTFL1 cosegregates with SFL in the
SD F. vesca 3 F. vesca f. semperflorens BC1 population.
Supplemental Figure S3. Phenotypes of 10-month-old LD-grown Hawaii-4
and P35S:FvTFL1 plants.
Supplemental Figure S4. FvTFL1 expression in P35S:FvTFL1 transgenic
lines.
Supplemental Figure S5. FvTFL1 complements the Arabidopsis tfl1-2
mutant phenotype.
Supplemental Figure S6. Expression analysis of FvTFL1.
Supplemental Figure S7. Effect of photoperiod on the expression of floral
meristem identity genes.
Supplemental Figure S8. F. vesca LD accession Hawaii-4 flowers continuously after LD induction in both SD and LD conditions.
Supplemental Figure S9. Expression of FvFT1 in LD-grown P35S:FvFT1RNAi lines.
Supplemental Table S1. Primers used in this study.
Expression Analysis
Total RNA was extracted, cDNA was synthesized, and real-time PCR was
performed as described earlier (Mouhu et al., 2009). FvMSI1 was used as a
stable control gene (Mouhu et al., 2009) for normalization. The number of
biological replicates is indicated in each figure legend. Three technical replicates were performed for each sample. Real-time PCR primers are listed in
Supplemental Table S1.
ACKNOWLEDGMENTS
We thank L. Sarelainen for technical assistance in genetic transformation
and J.P.T. Valkonen, O. Junttila, and M. Mattsson for critical reading of the
manuscript.
Received March 5, 2012; accepted May 3, 2012; published May 7, 2012.
Plasmid Constructs
Plasmid constructs for overexpressing FvTFL1 or FvmTFL1 were created
according to Gateway Technology with Clonase II (Invitrogen). For overexpression of TFL1, the primers used to amplify cDNA from SD F. vesca (PI551792)
and sfl mutant Baron Solemacher (PI551507) were 59-AAAAAGCAGGCTCTGTACAACCTTTTCTCTTCTCCCTCT-39 (attB1) and 59-AGAAAGCTGGGTCCTCCCTGCAAGGTGCCTA-39 (attB2). For the double-stranded FvTFL1 RNAi
construct, the fragment was amplified from SD F. vesca cDNA with primers
59-AAAAAGCAGGCTTGTTTGGCCTTGGCATCTCG-39 (attB1) and 59-AGAAAGCTGGGTTCTGCAGTCACCGCCAAACC-39 (attB2). For creating FvFT1
RNAi constructs, cDNA from SD F. vesca (PI551792) was amplified with primers
59-AAAAAGCAGGCTTGTTTGGCCTTGGCATCTCG-39 (attB1) and 59-AGAAAGCTGGGTTCTGCAGTCACCGCCAAACC-39 (attB2). The destination vectors were p7WG2D for overexpression and PK7GWIWG2(II) for RNAi (Karimi
et al., 2002).
Transformation
Arabidopsis plants were transformed with Agrobacterium tumefaciens strain
GV3101 by the floral dip method (Zhang et al., 2006). Strawberry transformations
LITERATURE CITED
Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y,
Ichinoki H, Notaguchi M, Goto K, Araki T (2005) FD, a bZIP protein
mediating signals from the floral pathway integrator FT at the shoot
apex. Science 309: 1052–1056
Ahn JH, Miller D, Winter VJ, Banfield MJ, Lee JH, Yoo SY, Henz SR,
Brady RL, Weigel D (2006) A divergent external loop confers antagonistic activity on floral regulators FT and TFL1. EMBO J 25:
605–614
Albani MC, Battey NH, Wilkinson MJ (2004) The development of ISSRderived SCAR markers around the SEASONAL FLOWERING LOCUS
(SFL) in Fragaria vesca. Theor Appl Genet 109: 571–579
Albani MC, Coupland G (2010) Comparative analysis of flowering in annual and perennial plants. Curr Top Dev Biol 91: 323–348
An H, Roussot C, Suárez-López P, Corbesier L, Vincent C, Piñeiro M,
Hepworth S, Mouradov A, Justin S, Turnbull C, et al (2004)
CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 131: 3615–
3626
1052
Plant Physiol. Vol. 159, 2012
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
FvTFL1 Controls Photoperiodic Flowering
Arney SE (1953) Studies of the growth and development of the genus
Fragaria. II. The initiation, growth and emergence of leaf primordia in
Fragaria. Ann Bot (Lond) 17: 477–492
Battey NH (2000) Aspects of seasonality. J Exp Bot 51: 1769–1780
Battey NH, Le Mière P, Tehranifar A, Cekic C, Taylor S, Shrives KJ,
Hadley P, Greenland AJ, Darby J, Wilkinson MJ (1998) Genetic and
environmental control of flowering in strawberry. In KE Cockshull, D
Gray, GB Seymour, B Thomas, eds, Genetic and Environmental Manipulation of Horticultural Crops. CABI, Wallingford, UK, pp 111–131
Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S,
Strauss SH, Nilsson O (2006) CO/FT regulatory module controls timing
of flowering and seasonal growth cessation in trees. Science 312: 1040–1043
Bradford E, Hancock JF, Warner RM (2010) Interactions of temperature
and photoperiod determine expression of repeat flowering in strawberry. J Am Soc Hortic Sci 135: 102–107
Bradley D, Ratcliffe OJ, Vincent C, Carpenter R, Coen E (1997) Inflorescence commitment and architecture in Arabidopsis. Science 275: 80–83
Brown T, Wareing P (1965) Genetical control of everbearing habit and 3
other characters in varieties of Fragaria vesca. Euphytica 14: 97–112
Conti L, Bradley D (2007) TERMINAL FLOWER1 is a mobile signal controlling Arabidopsis architecture. Plant Cell 19: 767–778
Conti E, Izaurralde E (2005) Nonsense-mediated mRNA decay: molecular
insights and mechanistic variations across species. Curr Opin Cell Biol
17: 316–325
Cooper JP, Calder DM (1964) The inductive requirements for flowering of
some temperate grasses. Grass Forage Sci 19: 6–14
Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A,
Farrona S, Gissot L, Turnbull C, et al (2007) FT protein movement
contributes to long-distance signaling in floral induction of Arabidopsis.
Science 316: 1030–1033
Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M (2005)
Ectopic expression of an FT homolog from citrus confers an early
flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.).
Transgenic Res 14: 703–712
Ferrándiz C, Gu Q, Martienssen R, Yanofsky MF (2000) Redundant regulation of meristem identity and plant architecture by FRUITFULL,
APETALA1 and CAULIFLOWER. Development 127: 725–734
Foucher F, Morin J, Courtiade J, Cadioux S, Ellis N, Banfield MJ, Rameau C
(2003) DETERMINATE and LATE FLOWERING are two TERMINAL
FLOWER1/CENTRORADIALIS homologs that control two distinct phases
of flowering initiation and development in pea. Plant Cell 15: 2742–2754
Guttridge CG (1959) Further evidence for a growth-promoting and flowerinhibiting hormone in strawberry. Ann Bot (Lond) 23: 612–621
Gyllenstrand N, Clapham D, Källman T, Lagercrantz U (2007) A Norway
spruce FLOWERING LOCUS T homolog is implicated in control of
growth rhythm in conifers. Plant Physiol 144: 248–257
Hanano S, Goto K (2011) Arabidopsis TERMINAL FLOWER1 is involved in
the regulation of flowering time and inflorescence development through
transcriptional repression. Plant Cell 23: 3172–3184
Hartmann HT (1947) Some effects of temperature and photoperiod on
flower formation and runner production in the strawberry. Plant Physiol
22: 407–420
Hayama R, Agashe B, Luley E, King R, Coupland G (2007) A circadian
rhythm set by dusk determines the expression of FT homologs and the
short-day photoperiodic flowering response in Pharbitis. Plant Cell 19:
2988–3000
Hayama R, Yokoi S, Tamaki S, Yano M, Shimamoto K (2003) Adaptation
of photoperiodic control pathways produces short-day flowering in rice.
Nature 422: 719–722
Hecht V, Laurie RE, Vander Schoor JK, Ridge S, Knowles CL, Liew LC,
Sussmilch FC, Murfet IC, Macknight RC, Weller JL (2011) The pea
GIGAS gene is a FLOWERING LOCUS T homolog necessary for grafttransmissible specification of flowering but not for responsiveness to
photoperiod. Plant Cell 23: 147–161
Heide OM (1977) Photoperiod and temperature interactions in growth and
flowering of strawberry. Physiol Plant 40: 21–26
Heide OM, Sønsteby A (2007) Interactions of temperature and photoperiod
in the control of flowering of latitudinal and altitudinal populations of
wild strawberry (Fragaria vesca). Physiol Plant 130: 280–286
Hsu C-Y, Adams JP, Kim H, No K, Ma C, Strauss SH, Drnevich J,
Vandervelde L, Ellis JD, Rice BM, et al (2011) FLOWERING LOCUS T
duplication coordinates reproductive and vegetative growth in perennial poplar. Proc Natl Acad Sci USA 108: 10756–10761
Hsu C-Y, Liu YX, Luthe DS, Yuceer C (2006) Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell 18: 1846–1861
Hytönen T, Elomaa P (2011) Genetic and environmental regulation of
flowering and runnering in strawberries. Genes Genomes Genomics 5:
56–64
Hytönen T, Elomaa P, Moritz T, Junttila O (2009) Gibberellin mediates
daylength-controlled differentiation of vegetative meristems in strawberry (Fragaria 3 ananassa Duch). BMC Plant Biol 9: 18
Hytönen T, Palonen P, Mouhu K, Junttila O (2004) Crown branching and
cropping potential in strawberry (Fragaria 3 ananassa, Duch.) can be
enhanced by daylength treatments. J Hortic Sci Biotechnol 79: 466–471
Imamura T, Nakatsuka T, Higuchi A, Nishihara M, Takahashi H (2011)
The gentian orthologs of the FT/TFL1 gene family control floral initiation
in Gentiana. Plant Cell Physiol 52: 1031–1041
Iwata H, Gaston A, Remay A, Thouroude T, Jeauffre J, Kawamura K,
Oyant LH, Araki T, Denoyes B, Foucher F (2012) The TFL1 homologue
KSN is a regulator of continuous flowering in rose and strawberry. Plant
J 69: 116–125
Jaeger KE, Wigge PA (2007) FT protein acts as a long-range signal in
Arabidopsis. Curr Biol 17: 1050–1054
Jahn OL, Dana MN (1970) Crown and inflorescence development in the
strawberry, Fragaria ananassa. Am J Bot 57: 607–612
Komiya R, Yokoi S, Shimamoto K (2009) A gene network for long-day
flowering activates RFT1 encoding a mobile flowering signal in rice.
Development 136: 3443–3450
Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacteriummediated plant transformation. Trends Plant Sci 7: 193–195
Karlgren A, Gyllenstrand N, Källman T, Sundström JF, Moore D,
Lascoux M, Lagercrantz U (2011) Evolution of the PEBP gene family in
plants: functional diversification in seed plant evolution. Plant Physiol
156: 1967–1977
Kim DH, Doyle MR, Sung S, Amasino RM (2009) Vernalization: winter and
the timing of flowering in plants. Annu Rev Cell Dev Biol 25: 277–299
Kong F, Liu B, Xia Z, Sato S, Kim BM, Watanabe S, Yamada T, Tabata S,
Kanazawa A, Harada K, et al (2010) Two coordinately regulated homologs of FLOWERING LOCUS T are involved in the control of photoperiodic flowering in soybean. Plant Physiol 154: 1220–1231
Kotoda N, Hayashi H, Suzuki M, Igarashi M, Hatsuyama Y, Kidou S,
Igasaki T, Nishiguchi M, Yano K, Shimizu T, et al (2010) Molecular
characterization of FLOWERING LOCUS T-like genes of apple (Malus 3
domestica Borkh.). Plant Cell Physiol 51: 561–575
Kotoda N, Iwanami H, Takahashi S, Abe K (2006) Antisense expression of
MdTFL1, a TFL1-like gene, reduces the juvenile phase in apple. J Am Soc
Hortic Sci 131: 74–81
Kurokura T, Inaba Y, Sugiyama N (2006) Histone H4 gene expression and
morphological changes on shoot apices of strawberry (Fragaria 3 ananassa Duch.) during floral induction. Sci Hortic (Amsterdam) 110:
192–197
Liljegren SJ, Gustafson-Brown C, Pinyopich A, Ditta GS, Yanofsky MF
(1999) Interactions among APETALA1, LEAFY, and TERMINAL
FLOWER1 specify meristem fate. Plant Cell 11: 1007–1018
Mandel MA, Yanofsky MF (1995) A gene triggering flower formation in
Arabidopsis. Nature 377: 522–524
Mohamed R, Wang C-T, Ma C, Shevchenko O, Dye SJ, Puzey JR,
Etherington E, Sheng X, Meilan R, Strauss SH, et al (2010) Populus
CEN/TFL1 regulates first onset of flowering, axillary meristem identity
and dormancy release in Populus. Plant J 62: 674–688
Mouhu K, Hytönen T, Folta K, Rantanen M, Paulin L, Auvinen P, Elomaa
P (2009) Identification of flowering genes in strawberry, a perennial SD
plant. BMC Plant Biol 9: 122
Mutasa-Göttgens E, Hedden P (2009) Gibberellin as a factor in floral regulatory networks. J Exp Bot 60: 1979–1989
Oosumi T, Gruszewski HA, Blischak LA, Baxter AJ, Wadl PA, Shuman
JL, Veilleux RE, Shulaev V (2006) High-efficiency transformation of the
diploid strawberry (Fragaria vesca) for functional genomics. Planta 223:
1219–1230
Pnueli L, Gutfinger T, Hareven D, Ben-Naim O, Ron N, Adir N, Lifschitz
E (2001) Tomato SP-interacting proteins define a conserved signaling
system that regulates shoot architecture and flowering. Plant Cell 13:
2687–2702
Purwestri YA, Ogaki Y, Tamaki S, Tsuji H, Shimamoto K (2009) The 14-3-3
protein GF14c acts as a negative regulator of flowering in rice by interacting
with the florigen Hd3a. Plant Cell Physiol 50: 429–438
Plant Physiol. Vol. 159, 2012
1053
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Koskela et al.
Ratcliffe OJ, Bradley DJ, Coen ES (1999) Separation of shoot and floral
identity in Arabidopsis. Development 126: 1109–1120
Samach A, Wigge PA (2005) Ambient temperature perception in plants.
Curr Opin Plant Biol 8: 483–486
Sargent DJ, Davis TM, Tobutt KR, Wilkinson MJ, Battey NH, Simpson DW
(2004) A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria. Theor Appl Genet 109: 1385–1391
Shalit A, Rozman A, Goldshmidt A, Alvarez JP, Bowman JL, Eshed Y,
Lifschitz E (2009) The flowering hormone florigen functions as a general
systemic regulator of growth and termination. Proc Natl Acad Sci USA
106: 8392–8397
Shannon S, Meeks-Wagner DR (1991) A mutation in the Arabidopsis
TFL1 gene affects inflorescence meristem development. Plant Cell 3:
877–892
Shulaev V, Korban SS, Sosinski B, Abbott AG, Aldwinckle HS, Folta
KM, Iezzoni A, Main D, Arús P, Dandekar AM, et al (2008) Multiple
models for Rosaceae genomics. Plant Physiol 147: 985–1003
Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher
AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, et al (2011) The genome
of woodland strawberry (Fragaria vesca). Nat Genet 43: 109–116
Simpson GG (2004) The autonomous pathway: epigenetic and posttranscriptional gene regulation in the control of Arabidopsis flowering
time. Curr Opin Plant Biol 7: 570–574
Sønsteby A, Heide OM (2007) Long-day control of flowering in everbearing strawberries. J Hortic Sci Biotechnol 82: 875–884
Sønsteby A, Heide OM (2008) Long-day rather than autonomous control of
flowering in the diploid everbearing strawberry Fragaria vesca ssp semperflorens. J Hortic Sci Biotechnol 83: 360–366
Stewart PJ, Folta KM (2010) A review of photoperiodic flowering research
in strawberry (Fragaria spp.). Crit Rev Plant Sci 29: 1–13
Suárez-López P, Wheatley K, Robson F, Onouchi H, Valverde F,
Coupland G (2001) CONSTANS mediates between the circadian clock
and the control of flowering in Arabidopsis. Nature 410: 1116–1120
Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K (2007) Hd3a
protein is a mobile flowering signal in rice. Science 316: 1033–1036
Taoka K, Ohki I, Tsuji H, Furuita K, Hayashi K, Yanase T, Yamaguchi M,
Nakashima C, Purwestri YA, Tamaki S, et al (2011) 14-3-3 proteins act
as intracellular receptors for rice Hd3a florigen. Nature 476: 332–335
Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen:
FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 59: 573–594
Turnbull C (2011) Long-distance regulation of flowering time. J Exp Bot 62:
4399–4413
Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland
G (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303: 1003–1006
Vince-Prue D, Guttridge CG (1973) Floral initiation in strawberry: spectral
evidence for the regulation of flowering by long-day inhibition. Planta
110: 165–172
Wang R, Albani MC, Vincent C, Bergonzi S, Luan M, Bai Y, Kiefer C,
Castillo R, Coupland G (2011) Aa TFL1 confers an age-dependent response
to vernalization in perennial Arabis alpina. Plant Cell 23: 1307–1321
Wang R, Farrona S, Vincent C, Joecker A, Schoof H, Turck F, AlonsoBlanco C, Coupland G, Albani MC (2009) PEP1 regulates perennial
flowering in Arabis alpina. Nature 459: 423–427
Weebadde CK, Wang D, Finn CE, Lewers KS, Luby JJ, Bushacra JJ, Sjulin
TM, Hancock JF (2008) Using a linkage mapping approach to identify
QTL for day-neutrality in the octoploid strawberry. Plant Breed 127:
94–101
Wigge PA (2011) FT, a mobile developmental signal in plants. Curr Biol 21:
R374–R378
Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU,
Weigel D (2005) Integration of spatial and temporal information during
floral induction in Arabidopsis. Science 309: 1056–1059
Zhang XR, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacteriummediated transformation of Arabidopsis thaliana using the floral dip method.
Nat Protoc 1: 641–646
1054
Plant Physiol. Vol. 159, 2012
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.