Download The Florigen Genes FT and TSF Modulate

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The Florigen Genes FT and TSF Modulate Lateral Shoot
Outgrowth in Arabidopsis thaliana
Kazuhisa Hiraoka1, Ayako Yamaguchi1, Mitsutomo Abe1,2 and Takashi Araki1,*
1
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto,
606-8501 Japan
2
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-0033 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-75-753-6470.
(Received October 4, 2012; Accepted November 27, 2012)
Successful sexual reproduction of a plant with prolific seed
production requires appropriate timing of flowering and
concomitant change of architecture (e.g. internode elongation and branching) to facilitate production of the optimal
number of flowers while enabling continued resource production through photosynthesis. Florigen is the prime candidate for a signal linking the two processes. Growth analysis
of lateral shoots in mutants of FLOWERING LOCUS T (FT)
and TWIN SISTER OF FT (TSF) revealed a delay in the onset of
outgrowth and a reduction of the growth rate in ft plants in
long-day (LD) conditions and in tsf plants in short-day (SD)
conditions. Thus, as in the case of floral transition, FT and
TSF play dominant roles in LD and SD conditions, respectively, in the promotion of lateral shoot development.
Differential expression patterns of the two genes were in
good agreement with their differential roles both in the
floral transition and in lateral shoot development under
contrasting photoperiod conditions. By manipulating florigen production after bolting of the primary shoot, it was
shown that florigen promotes lateral shoot growth independently of its effect on the floral transition of the primary
shoot. Analysis of growth and gene expression in lateral
shoots of the mutants suggests that the loss of florigen
leads to a reduced rate of flower formation on lateral
shoots. Together, we propose that the two florigen genes
are an important key to linking the floral transition and
lateral shoot development to maximize the reproductive
success of a plant.
Keywords: Arabidopsis Branching Florigen FT
(FLOWERING LOCUS T) Lateral shoot outgrowth TSF
(TWIN SISTER OF FT).
Abbreviations: ACT2, ACTIN2; AP1, APETALA1; Col,
Columbia; CYCD, CYCLIN D; CK, cytokinin; DAS, days
after sowing; FLC, FLOWERING LOCUS C; FT, FLOWERING
LOCUS T; FUL, FRUITFULL; GA20ox, GIBBERELLIN
20-OXIDASE; GUS, b-glucuronidase; HSP, HEAT SHOCK
PROTEIN 18.2; Ler, Landsberg erecta; LFY, LEAFY; LD, long
day; No, Nossen; NRQ, normalized relative quantitiy; PEBP,
phosphatidylethanolamine-binding protein; PIN, PINFORMED; RT–PCR, reverse transcription–PCR; RT–qPCR,
reverse transcription–quantitative real-time PCR; STM,
SHOOT MERISTEMLESS; SD, short day; SAM, shoot apical
meristem; SFT, SINGLE FLOWER TRUSS; SOC1, SUPPRESSOR
OF OVEREXPRESSION OF CO1; TSF, TWIN SISTER OF FT; WUS,
WUSCHEL; ZT, Zeitgeber time
Introduction
In flowering plants, successful sexual reproduction leading to
prolific seed production requires appropriate timing of flowering. In annual plants such as Arabidopsis, also important is
appropriate remodeling of the plant architecture (e.g. internode elongation and branching) to facilitate production
of the optimal number of flowers while enabling continued
resource production through photosynthesis for seed formation. Therefore, a tight link between the floral transition and
the concomitant architectural change should somehow be
achieved. A systemic signaling mechanism will be the prime
candidate for the regulatory basis of linking the two processes.
In regulation of the timing of the floral transition, the environmental cues (photoperiod, light quality and quantity, temperature, and water and nutrient availability) and the internal
cues (age, size and phytohormone dynamics) converge on the
‘hubs’ known as the floral pathway integrator genes (Levy and
Dean 1998, Araki 2000, Srikanth and Schmid 2011, Yamaguchi
and Abe 2012). In Arabidopsis, the pathways responsive to
the appropriate environmental conditions (photoperiod and
vernalization) and those which reflect the internal status
of plants (autonomous, gibberellin and aging) intricately regulate the four integrator genes. Among them, FLOWERING
LOCUS T (FT) and its paralog TWIN SISTER OF FT (TSF)
encode approximately 20 kDa globular proteins of the
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168, available online at www.pcp.oxfordjournals.org
! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Role of FT and TSF beyond flowering
phosphatidylethanolamine-binding protein (PEBP) family
(Kardailsky et al. 1999, Kobayashi et al. 1999). In young plants,
long-day (LD) photoperiods up-regulate their expression in
phloem companion cells of cotyledons and leaves (FT and
TSF) and hypocotyl (TSF), and the autonomous and vernalization pathways negatively regulate both of the genes
(Suárez-López et al. 2001, Yanovsky and Kay 2002, Takada
and Goto 2003, Michaels et al. 2005, Yamaguchi et al. 2005).
As systemic long-distance signaling molecules called florigen,
FT and TSF proteins are transported to the shoot apical meristems (SAMs) (Corbesier et al. 2007, Jaeger and Wigge 2007,
Mathieu et al. 2007, Notaguchi et al. 2008), where they form a
complex involving a bZIP transcription factor FD to up-regulate
the expression of MADS-box genes such as APETALA1 (AP1),
which then orchestrates floral initiation and development (Abe
et al. 2005, Wigge et al. 2005, Kaufmann et al. 2010). In rice, and
possibly in Arabidopsis as well, formation of the florigen complex is mediated by 14-3-3 proteins (Purwestri et al. 2009, Taoka
et al. 2011). Of the remaining two integrator genes, a
MADS-box gene, SUPPRESSOR OF OVEREXPRESSION OF CO1
(SOC1), is involved in floral induction and commitment,
flower organ formation, meristem determinacy, and prevention
of secondary growth and shoot longevity (Melzer et al. 2008, Liu
et al. 2009, Lee and Lee 2010, Torti et al. 2012). The fourth
integrator gene LEAFY (LFY), which encodes a plant-specific
transcription factor, also plays multifaceted roles in floral competency, establishment and maintenance of inflorescence and
floral meristem identities, and floral organ formation (Moyroud
et al. 2010).
Flowering in many species is generally accompanied by various changes including increased leaf growth rate and change in
phyllotaxis (Bernier et al. 1981). As a rosette-forming plant,
Arabidopsis shows increased internode elongation of the primary shoot axis (bolting) and initiation and outgrowth of lateral buds to develop a racemose inflorescence with complex
lateral shoot systems. Of these changes, development of lateral
shoot systems strengthens the reproductive success of the
plant by increasing the number of flowers and the amount of
photosynthetic tissues available for resource production. It has
been shown that, as in the case of flowering, plants sense the
environmental and internal cues to decide whether to allow
outgrowth of each of their lateral buds (Domagalska and Leyser
2011). The central mechanism involved in the regulation of
lateral shoot outgrowth is called correlative inhibition, in
which each shoot including the main shoot competitively inhibits outgrowth of other shoots through each other’s polar
auxin transport stream (Ongaro et al. 2008, Prusinkiewicz et al.
2009). Besides auxin, cytokinins (CKs) and strigolactones are
involved in this mechanism (Tanaka et al. 2006, Hayward
et al. 2009, Crawford et al. 2010, Minakuchi et al. 2010). Inside
the buds, cell cycle re-progression is thought to be important
(Shimizu and Mori 1998, Tatematsu et al. 2005, Müller and
Leyser 2011). However, in spite of these advances in our understanding, the regulatory link between flowering and lateral
shoot development still remains to be investigated.
Recent studies showed that the florigen genes in Arabidopsis
and other plants play important roles in various physiological
aspects other than flowering. In Arabidopsis, FT regulates
stomatal opening (Kinoshita et al. 2011), meristem maintenance in cooperation with SHOOT MERISTEMLESS (STM) during
inflorescence development (Smith et al. 2011), and prevention
of indeterminate growth, floral reversion and aerial rosette formation (Melzer et al. 2008). FT is also implicated in FLCmediated regulation of seed germination (Chiang et al. 2009)
and some traits of vegetative phase change (Willmann
and Poethig 2011). In tomato, SINGLE FLOWER TRUSS (SFT)
regulates reiterative growth of shoots and influences leaf
maturation, compound leaf architecture, stem growth, and
abscission zone formation (Shalit et al. 2009). Also, allelic variation at the SFT locus is implicated in heterosis for yield (Krieger
et al. 2010). In rice, Heading date1, encoding a CONSTANS
ortholog, and Early heading date1, encoding a type-B response
regulator unique to Poaceae, regulate spikelet number per
panicle in concert through the up-regulation of two rice
florigen genes Heading date3a (Hd3a) and Rice Flowering-locus
T1 (Endo-Higashi and Izawa 2011). Increased tillering was
observed in transgenic rice plants expressing green fluorescent
protein-fused Hd3a under the phloem-specific promoters,
as was accelerated flowering (Tamaki et al. 2007). In a potato
cultivar which tuberizes only in short-day (SD) conditions,
overexpression of rice Hd3a induced flowering and tuberization
in non-inductive LD conditions (Navarro et al. 2011). The
potato genome contains nine PEBP family genes, of which
StSP3D acts as florigen, while StSP6A acts as ‘tuberigen’.
In Populus trees, SD-induced growth cessation and bud set in
autumn involve the regulation of PtFT1 (Böhlenius et al. 2006).
Thus, there emerged a view that FT proteins act as universal
long-distance systemic signals mainly to regulate shoot
meristem activities.
In this study, we investigated the roles of the two florigen
genes, FT and TSF, in the regulation of branch outgrowth to
uncover the regulatory link between flowering and lateral shoot
development in Arabidopsis. A comparison among triple and
quadruple mutants of the four floral pathway integrator genes
clearly showed that a defect in florigen genes causes retardation
of lateral shoot outgrowth. Then, detailed growth analyses of
lateral shoots in single and double mutants revealed the delay
in outgrowth onset and the reduction of growth rate in ft plants
under LD conditions and in tsf plants under SD conditions.
Thus, as in the case of the floral transition, FT and TSF play
dominant roles in LD and SD, respectively, in the promotion of
lateral shoot development. Differential patterns and profiles of
expression of the two genes revealed in this study were in good
agreement with their differential roles both in the floral transition and in lateral shoot development under contrasting
photoperiod conditions. By employing daylength shifts and
an inducible FT transgene to manipulate florigen production
after the bolting of the primary shoot, the role of florigen in
promoting lateral shoot growth independently of its effect
on the floral transition of the primary shoot was clearly
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demonstrated. A delay in the first flower anthesis and a reduced
rate of flower production as well as reduced expression of some
genes involved in flower development were observed in lateral
shoots of the mutants. These observations may suggest a hypothesis to be tested in the future that the two florigen genes
may regulate lateral shoot outgrowth and elongation via acceleration of flower formation. Taken together, we propose that
the two florigen genes are an important key to linking the floral
transition and lateral shoot development to maximize the reproductive success of a plant.
Results
Retardation of lateral shoot elongation caused by
loss-of-function mutations of FT and TSF
During the course of study of the four floral pathway integrator
genes, ft-1; tsf-1; lfy-1; soc1-2 quadruple mutants were generated. Under LD photoperiods, unlike the previously reported co;
ga1; fca triple mutants which neither bolted nor flowered without vernalization (Reeves and Coupland 2001), the quadruple
mutants eventually bolted like ft; lfy double (Ruiz-Garcı́a et al.
1997) and ft; soc1; lfy triple (Moon et al. 2005) mutants.
However, they never produced organs with flower-like characteristics, such as stigmatic papillae, which eventually appeared
in ft; lfy double and ft; soc1; lfy triple mutants (Ruiz-Garcı́a et al.
1997; data not shown). Intriguingly, the quadruple mutants
showed severe growth retardation of the secondary and tertiary
lateral shoots (Fig. 1A, D), resulting in a much simpler appearance than the bushy phenotype of the ft; lfy double mutants
(see Ruiz-Garcı́a et al. 1997). Whereas the absence of the LFY
function occasionally resulted in an empty axil phenotype, the
above-mentioned simple appearance was observed even in the
presence of the wild-type LFY allele (Fig. 1E). In contrast,
the presence of the wild-type TSF allele and, to some extent,
the wild-type SOC1 allele did recover the growth of secondary
and tertiary lateral shoots (Fig. 1B–D). The observed effect of
SOC1 on lateral shoot development is consistent with recent
reports (Melzer et al. 2008, Dorca-Fornell et al. 2011). However,
the effect of TSF was more drastic than that of SOC1, implying
that the two florigen genes have hitherto unexplored roles in
lateral shoot growth in Arabidopsis.
Based on these observations, the role of FT and TSF in lateral
shoot growth was examined in more detail. Because few axillary
shoots showed consistent vigorous outgrowth from the rosette
leaf axils of LD-grown plants carrying the defective ft mutation
and SD-grown plants (Supplementary Fig. S1; data not
shown), our analysis mainly focused on the lateral shoots
formed at the cauline leaf axils on the primary inflorescence
stem. When growth of the lateral shoot from the cauline leaf
axil was compared among the various nodes along the stem, a
similar tendency was observed (Supplementary Fig. S1).
Therefore, the longest lateral shoot (for definition, see the
Materials and Methods) was chosen as a representative example hereafter and designated simply as ‘lateral shoot’.
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Single and double mutants (ft-2, tsf-1 and ft-2; tsf-1) as well as
their wild-type plants [accession Columbia (Col)] were grown
under inductive LD or non-inductive SD conditions, and the
main and lateral shoot lengths were measured on a daily basis.
‘Day 0’ was defined as the day on which the main shoot of a
plant reached a height of 5 cm. The first flower of the main
shoot was at anthesis around Day 0 under our growth conditions. LD-grown wild-type and tsf-1 plants and SD-grown
wild-type plants ceased producing flowers around Day 14 and
Day 21, respectively, under our growth conditions. Therefore,
shoot lengths were measured until Day 14 for LD-grown
wild-type and single mutant plants, until Day 21 for
SD-grown wild-type plants, and until Day 28 for LD-grown
double mutants and SD-grown single and double mutants. As
explained above, the lengths of the longest lateral shoots were
compared among the four tested lines.
Under LD conditions, wild-type and tsf-1 plants showed
well-grown main and lateral shoots (Fig. 2A, B, E, G), as
expected from their flowering phenotypes (Supplementary
Table S1; Michaels et al. 2005, Yamaguchi et al. 2005, Jang
et al. 2009). However, ft-2 plants showed retarded growth of
lateral shoots with well-grown main shoots, and ft-2; tsf-1
double mutants showed more drastic retardation of lateral
shoot growth (Fig. 2C–E, G). To analyze this lateral shoot
phenotype in ft; tsf double and ft single mutants in detail,
the elongation rate of the lateral shoots (for definition, see
the Materials and Methods) was calculated. As shown in
Fig. 2H, the retardation phenotype was characterized by
two components: the delay in outgrowth initiation and the
slower elongation rate. Moreover, the delay in the onset of
outgrowth was accompanied by the delayed timing of first
flower anthesis and a reduced rate of flower formation on
the lateral shoots (Fig. 2F). Under SD conditions, wild-type
and ft-2 plants showed similar main and lateral shoot growth
(Fig. 3A, C, F, H, I). Surprisingly, tsf-1 plants showed apparent
retardation of lateral shoot growth compared with wild-type
and ft-2 plants (Fig. 3A–C, H, I). ft-2; tsf-1 plants showed more
drastic retardation of lateral shoot growth, like those grown
under LD conditions (Fig. 3D, H, I). tsf-1 and ft-2; tsf-1 plants
sometimes developed aerial rosettes at the axil of lower cauline
leaves even on Day 7 (Fig. 3E). This is consistent with the suggested role of FT in inhibition of aerial rosette formation
in concert with SOC1 and FRUITFULL (FUL) (Melzer et al.
2008). As in the case of LD conditions, a delay in anthesis of
the first flower and a reduced flower formation rate were
observed on the lateral shoots of tsf-1 and ft-2; tsf-1 mutants
under SD conditions (Fig. 3G).
Next, we utilized ft-2 and ft-2; tsf-1 plants in accession
Landsberg erecta (Ler) and ft-4, ft-5 and ft-6 plants in accession
Nossen (No) to examine the effect of various ft alleles and
genetic backgrounds. LD-grown ft-2 and ft; tsf plants in the
Ler background (Fig. 4A, C, E) and various ft mutants in the
No background (Fig. 4B, D, F) showed a phenotype similar to
that of LD-grown ft-2 and ft-2; tsf-1 plants in the Col background (Fig. 2). Therefore, we conclude that the effect of
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Role of FT and TSF beyond flowering
Fig. 1 Branching phenotype of triple and quadruple combinations of ft, tsf, soc1 and lfy mutations. The plants were grown under LD conditions.
(A) An ft-1; tsf-1; soc1-2; lfy-1 quadruple mutant at 124 DAS (days after sowing). (B) An ft-1; tsf-1; lfy-1 triple mutant at 124 DAS. (C) An ft-1;
soc1-2; lfy-1 triple mutant at 124 DAS. (D) An ft-1; soc1-2; lfy-1 triple mutant (left) and an ft-1; tsf-1; soc1-2; lfy-1 quadruple mutant (right) at
98 DAS. (E) The effect of a strong lfy mutation in the ft; tsf; soc1 background. A lfy-1 homozygous plant (quadruple (lfy); left), a lfy-1 heterozygous
plant (LFY/lfy; center) and a wild-type homozygous plant (LFY+; right), all in the ft-1; tsf-1; soc1-2 background at 83 DAS, are shown. Arrowheads
indicate axils devoid of a lateral bud. Scale bars: (A–C) 10 cm; (D) 5 cm; (E) 1 cm.
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
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Fig. 2 Effect of mutations in the florigen genes on lateral shoot growth under LD conditions. (A–D) Photographs of plants on Day 7 (Day 0
was defined as the date when the main stem reached 5 cm height). A representative plant of the wild type (Col) (A), tsf-1 (B), ft-2 (C) and ft-2;
tsf-1 (D). Scale bars: 5 cm. (E) Growth of the main shoot. (F) Number of open flowers on the longest lateral shoot per day. (G) Growth of
the longest lateral shoot. (H) Elongation rate of the longest lateral shoot. The data in E–H represent the mean ± SD (n = 7 to 9), except for
F (mean ± SEM).
loss-of-function ft and/or tsf mutations is neither allele- nor
genetic background-specific.
The expression of FT and TSF in young SD-grown plants is
much lower than in LD-grown plants (Yanovsky and Kay 2002,
Cerdán and Chory 2003, Yamaguchi et al. 2005; see Fig. 6), and
the LD to SD shift attenuates FT expression in 2-week-old plants
(Corbesier et al. 2007, Osnato et al. 2012). Therefore, we expected that the LD to SD shift resulted in rapid reduction in the
available amount of FT and TSF proteins followed by the decrement of lateral shoot growth. To test this, we grew wild-type
(Col) and tsf-1 plants under LD conditions until their main
shoot length exceeded 2 or 5 cm, and then moved batches of
plants to SD conditions. As expected, LD-grown wild-type and
tsf-1 plants showed no obvious difference in flowering time
(Supplementary Table S1), while SD-shifted plants showed
reduced lateral and main shoot growth irrespective of tsf-1
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mutation (Fig. 5). Taken together, these results suggest that
the two florigen genes promote lateral shoot outgrowth: FT
mainly in LD and TSF mainly in SD.
Spatio-temporal expression patterns of TSF and
FT in SD-grown plants
The observation of SD-grown plants (Fig. 3) suggests that TSF
plays a greater role than FT in the promotion of lateral shoot
outgrowth under SD conditions. However, the expression of
TSF under SD conditions in mature plants has not been well
investigated (cf. Yamaguchi et al. 2005). Therefore, we observed
the temporal and spatial profiles of TSF and FT expression
under SD conditions using reverse transcription–quantitative
PCR (RT–qPCR) and semi-quantitative reverse transcription–
PCR (RT–PCR) techniques and GUS (b-glucuronidase) staining
of gTSF::GUS and gFT::GUS lines. In both LD and SD conditions,
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Role of FT and TSF beyond flowering
Fig. 3 Effect of mutations in the florigen genes on lateral shoot growth under SD conditions. (A–D) Photographs of plants on Day 7. A
representative plant of the wild type (Col) (A), tsf-1 (B), ft-2 (C) and ft-2; tsf-1 (D). (E) Aerial rosette formation observed in ft-2; tsf-1 on Day 7. Parts
of the same plant are shown in the upper and lower photographs. Arrowheads indicate aerial rosettes. Scale bars: (A–D) 5 cm; (E) 1 cm. (F)
Growth of the main shoot. (G) Number of open flowers on the longest lateral shoot per day. (H) Growth of the longest lateral shoot. (I)
Elongation rate of the longest lateral shoot. The data in F–I represent the mean ± SD (n = 8 or 9), except for G (mean ± SEM).
the peak of FT and TSF expression was observed around the end
of the light period (Yamaguchi et al. 2005). Hence, to detect the
possible peak levels of expression, aerial parts of the plants were
harvested at Zeitgeber time (ZT) 7 for plants grown under SD
conditions [20, 40 and 60 days after sowing (DAS)] and at ZT15
for 60-day-old plants shifted from SD to LD conditions on
57 DAS.
TSF expression in the aerial parts of wild-type plants
increased as the plants matured (Fig. 6A, B). In 20-day-old
plants, the TSF expression level was very low. In 40-day-old
plants, however, TSF expression started to increase, and in
60-day-old plants its expression level increased to approximately 80% compared with the plants of the same age transferred to LD conditions for 4 d. In fact, GUS staining of
20-day-old gTSF::GUS plants revealed that the TSF expression
site was restricted to the vicinity of the SAM and to the vascular
tissues of hypocotyls (Fig. 6D, F, H, J), as reported previously
(Yamaguchi et al. 2005). In 40-day-old plants, TSF expression
was detected in the midvein of some fully expanded rosette
leaves (Fig. 6M, N) and in 60-day-old plants, some of which
already bolted in this experiment, TSF expression was detected
in the midvein and secondary veins of older rosette leaves,
irrespective of photoperiod (Fig. 6Q, R, U, V, Y, Z;
Supplementary Fig. S3). The results from these GUS staining
analyses are consistent with the RT–PCR and RT–qPCR results.
In contrast, FT was not up-regulated in the aerial parts of
SD-grown wild-type plants, and its expression in the 60-day-old
plants was much lower than TSF expression (Fig. 6A, B). GUS
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Fig. 4 Effect of various ft mutant alleles and genetic backgrounds. ft-2 and ft-2; tsf-1 in the Ler background (A, C, E) and ft-4, ft-5 and ft-6 in the No
background (B, D, F) and their respective wild-type plants were used. (A, B) Growth of the main shoot. (C, D) Growth of the longest lateral shoot.
(E, F) Elongation rate of the longest lateral shoot. The data represent the mean ± SD (n = 8 or 9).
Fig. 5 Effect of LD to SD shift treatment on shoot growth. Lengths of
the main shoot (A) and the longest lateral shoot (B) were measured
on Day 7. The data represent the mean ± SD (n = 7 or 8). The double
asterisks and the double daggers indicate P < 0.01 compared with the
LD-grown wild-type (Col) plants and tsf-1 plants, respectively.
stains of SD-grown gFT::GUS plants were faintly detected in the
tertiary vein of older rosette leaves (Fig. 6C, E, G, I, K, L, P, T, X;
Supplementary Fig. S2B). Daylength shift to LDs caused the
60-day-old plants to up-regulate the FT expression strongly,
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mainly in the tertiary veins (Fig. 6A, B, O, S, W;
Supplementary Fig. S2A). Thus, in our growth conditions,
FT expression in rosette leaves was regulated largely by photoperiod, while TSF expression in leaves was regulated largely in an
age-dependent manner. It is to be noted that a much higher
expression level of TSF than that of FT in leaves in SD is
accountable for its greater role in the lateral shoot growth in
this photoperiod condition.
The spatial expression pattern of FT and TSF after floral
transition was further investigated with SD-grown or SD to
LD-transferred gFT::GUS and gTSF::GUS plants. The expression
patterns in the cauline leaves were basically similar to those in
the rosette leaves (especially younger rosette leaves, Fig. 7I–L);
FT expression in the tertiary veins of cauline leaves was LDdependent (compare Fig. 7E, I with Fig. 7F, J), and TSF expression in the main vein tended to be observed in cauline leaves at
lower nodes (see Fig. 7L). No or faint signals were detected in
the apical regions of lateral shoots which were approximately
0.5–1 cm long (Fig. 7E–H). However, in developed inflorescences, FT expression was increased in the veins of pedicels
approximately from floral stage 11 (defined by Smyth et al.
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Role of FT and TSF beyond flowering
Fig. 6 Spatio-temporal expression patterns of FT and TSF in SD-grown plants. (A, B) Temporal profiles of FT and TSF expression in SD-grown
wild-type (Col) plants. (A) RT–qPCR analyses of FT (left) and TSF (right) expression. The data represent the means of five biological
replicates ± SD. (B) Relative comparison between FT and TSF expression levels through a modified RT–PCR analysis. Two of five cDNA samples
per group used in the RT–qPCR analyses in A were used as templates for digestion with EcoT14I (a site in TSF) or PstI (a site in FT). PCR products
of the full-length cDNA clones of FT and TSF were used as controls for digestion. EcoT14I-digested products (upper) and PstI-digested products
(middle) were electrophoresed on agarose gels. ACT2 (lower) was used as a reference. (C–J) GUS staining patterns of a gFT::GUS plant (C, E, G, I)
and a gTSF::GUS plant (D, F, H, J) grown under SD conditions at 20 DAS. (C, D) Hypocotyls and shoot apical regions. (E, F) Rosette leaves. (G, H)
Enlarged views of the apical regions in C and D. (I, J) Enlargements of part of the leaves in E and F. The arrowheads in H and I indicate faint signals.
(continued)
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1990) and in the medial vascular bundles and funiculus veins of
styles at later stages in a photoperiod-independent manner,
and only the veins of sepals showed photoperiod-dependent
FT induction (Fig. 7A, B). In contrast, faint TSF signals were
observed in the veins around receptacles from floral stage 12
(Fig. 7C, D). Thus, post-transitional expression of FT and TSF
was also observed in veins under SD conditions, while their
expression profiles in inflorescences were different from those
in rosette and cauline leaves.
Recovery from retardation of lateral shoot
elongation by induced FT and TSF expression
We then investigated whether recovery of FT and/or TSF
function from their deficient states can rescue the outgrowth
Fig. 7 Expression patterns of FT and TSF in the inflorescences of SD-grown plants. A gFT::GUS plant (A, E, I) and a gTSF::GUS plant (C, G, K) were
transferred from SD to LD conditions at 57 DAS, while another gFT::GUS plant (B, F, J) and gTSF::GUS plant (D, H, L) were kept under SD
conditions for 60 d. (A–D) Inflorescences of the main shoot. (E–H) Apical regions of the lateral shoots. (I–L) Cauline leaves and their nodal
regions. Scale bars: (A–D, I–L) 2 mm; (E–H) 1 mm.
Fig. 6 Continued
Scale bars: (C, D) 1 mm; (E, F) 2 mm; (G, H) 200 mm; (I, J) 400 mm. (K–N) GUS staining patterns of a gFT::GUS plant (K, L) and a gTSF::GUS plant (M, N)
grown under SD conditions at 40 DAS. (K, N) Whole aerial parts. (L, M) Enlargements of part of the leaves in K and N. The arrowheads in L and N indicate
faint signals. Scale bars: (K, N) 1 cm; (L, M) 500 mm. (O–Z) Spatial expression patterns of FT and TSF in SD-grown plants at 60 DAS. A gFT::GUS plant
(O, S, W) and a gTSF::GUS plant (Q, U, Y) were transferred from SD to LD conditions at 57 DAS, while another gFT::GUS plant (P, T, X) and gTSF::GUS plant
(R, V, Z) were kept under SD conditions. (O–R) Immature rosette leaves. (S–V) Mature rosette leaves. (W–Z) Enlarged views of the mature rosette leaves
in S–V. Scale bars: (O–R) 2 mm; (S–V) 5 mm; (W–Z) 1 mm.
360
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
Role of FT and TSF beyond flowering
arrest of lateral shoots. First, we took advantage of the inducible
system for FT activity, in which the FT-T7 gene is driven by the
HEAT SHOCK PROTEIN 18.2 promoter in the ft-1 background
(Notaguchi et al. 2008; hereafter designated HSP:FT-T7; ft-1).
HSP:FT-T7; ft-1 plants and non-transgenic ft-1 plants were
grown under LD conditions until floral transition occurred
and bolting began, and then the half batches of each line
were heat-treated twice [on the days when they were at
2-cm-tall bolting and at 5-cm-tall bolting (Day 0), respectively].
The main and lateral shoot lengths were measured on Day 7,
and were compared among heat-treated or untreated
and transgenic or non-transgenic plants. The heat-treated
HSP:FT-T7; ft-1 plants showed longer lateral shoots than the
heat-treated ft-1 and untreated plants (Fig. 8B), while
the main shoot growth was not affected by the heat shock
treatment (Fig. 8A).
Next, an SD to LD shift experiment was conducted. Under
SD conditions, tsf-1 and ft-2; tsf-1 plants showed retardation of
lateral shoot growth compared with wild-type and ft-2 plants,
while the differences in flowering time among the four tested
lines were much smaller than that among the lines grown under
LD conditions (Fig. 3; Supplementary Table S1). Also, shift
to LD conditions triggered strong induction of FT and much
weaker induction of TSF in leaves (Fig. 6). If the florigen genes
are involved in promotion of lateral shoot outgrowth, an SD to
LD shift triggers genotype-dependent growth acceleration.
Hence, the single and double mutant plants (ft-2, tsf-1 and
ft-2; tsf-1) as well as wild-type (Col) plants were grown under
SD conditions until Day 0, and then the half batches of each line
were transferred to LD conditions. In wild-type and ft-2 plants,
carrying the functional TSF gene with a high expression level in
SDs and weaker induction by LDs, the lateral shoots showed
modest or weak acceleration of outgrowth in response to an
LD shift (Fig. 8D). In contrast, tsf-1 plants, carrying the defective
TSF gene and the functional FT gene with strong induction
by LDs, showed more drastic LD-induced acceleration of lateral shoot growth, and the ft-2; tsf-1 plants completely lost
LD-dependent growth acceleration. Thus, these two experiments further support the idea that the florigen genes promote
lateral shoot growth in Arabidopsis.
Gene expression in lateral shoots of wild-type
and ft plants
Fig. 8 Effect of induction of florigen production on shoot growth.
(A, B) Effect of FT induction in HSP:FT-T7; ft-1 plants. Lengths of the
main shoot (A) and the longest lateral shoot (B) were measured on
Day 7 (Day 0 was defined as the date when the main stem reached
5 cm height). The data represent the mean ± SD (n = 8 or 9). The
double asterisk in B indicates P < 0.01 compared with each of the
three control groups. (C, D) Effect of an SD to LD shift on lateral
shoots of wild-type (Col), tsf-1, ft-2 and ft-2; tsf-1 plants. Lengths of
the main shoot (C) and the longest lateral shoot (D) were measured
on Day 7. The data represent the mean ± SD (n = 5 or 6). The values
indicate the differences between the mean of SD-grown plants (white
bars) and that of SD to LD-shifted plants (gray bars) of each line. The
asterisk and the double asterisks with which the values were labeled
indicate P < 0.05 and P < 0.01 when compared with SD-grown plants
and SD to LD-shifted plants, respectively. The double daggers with
which the white bars were labeled indicate P < 0.01 when compared
with SD-grown ft-2; tsf-1 plants.
To gain some insight into the effect of a compromised level of
florigen, we conducted RT–qPCR analyses for selected genes in
lateral shoots of wild-type (Col) and ft-2 plants grown under LD
conditions. The apical regions (hereafter simply called ‘buds’)
and the internodes of the lateral shoot at the axil of the lowest
cauline leaves (the first lateral shoot) were sampled on the day
when each plant was 2 cm tall and on Days 0, 3, 7 and 12. The
collected buds contained shoot apices and flower buds at floral
stage 12 or younger. The buds and internodes of the longest
lateral shoots in ft plants were also sampled on Days 7 and 12 in
order to distinguish the effect of FT from that of dormancy, as
the first lateral shoots tended to remain in the dormant state in
ft plants (Supplementary Fig. S1). DRM1 is an Arabidopsis
ortholog of a dormancy-associated gene originally isolated
from garden pea (Pisum sativum) and is down-regulated by
decapitation (Stafstrom et al. 1998, Tatematsu et al. 2005,
Aguilar-Martı́nez et al. 2007). Throughout the experimental
period, we observed a higher level of DRM1 accumulation in
the buds of the first lateral shoots of ft than in the buds of the
longest lateral shoots of ft or in wild-type buds (Fig. 9A), suggesting that the sampled first lateral shoots in the ft plants
remain in the dormant state. In internodes, DRM1 accumulation was increased over time, which may reflect the cessation of
their elongation (Fig. 9B).
Expression of SOC1 and AP1, two MADS-box genes which
are related to the floral transition and/or flower development
and are regulated by FT and TSF (Abe et al. 2005, Wigge et al.
2005, Moon et al. 2005, Yamaguchi et al. 2005, Yoo et al. 2005),
was next investigated. At 2-cm-tall bolting, both SOC1 and AP1
expression in the buds was detected in wild-type and ft-2 plants
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
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K. Hiraoka et al.
Fig. 9 Temporal expression profiles of selected genes. Buds and internodes of the lateral shoots in the axils of the lowest cauline leaves in
wild-type (Col) plants (filled squares) or ft-2 plants (filled triangles) were harvested at ZT1 on the day when their main shoots reached 2 cm
height and on Days 0, 3, 7 and 12. Those of the longest lateral shoot in ft-2 plants (open triangles) were also harvested on Days 7 and 12. The data
which represent the means of three biological replicates were plotted as a function of time (Day after 5-cm-tall bolting). (A) Expression data of
the buds. From left to right and top to bottom, DRM1, SOC1, AP1, STM, WUS, CYCD3;1, PIN1, PIN3, GA20ox1 and GA20ox2. (B) Expression data of
the internodes. From left to right and top to bottom, DRM1, SOC1, STM, CYCD3;1, GA20ox1, PIN1 and PIN3. Error bars represent SDs.
(Fig. 9A); the SOC1 expression level in the buds was similar in
both genotypes, while the AP1 level in the buds was lower in ft-2
than in the wild type. These findings suggest that ‘floral transition’ of lateral shoots in ft-2 plants occurred by the time of
2-cm-tall bolting, but that subsequent development was
delayed or somewhat altered. Consistently, AP1 expression
was detected in all the lateral buds of the 1-cm-tall AP1pro:
GUS transgenic (POP40) plants irrespective of ft-2 mutation
(Supplementary Fig. S4). Subsequent SOC1 expression in the
buds was not severely altered by ft-2 mutation. In contrast, AP1
expression in the wild-type buds showed a rapid increase to
362
reach a maximum on Day 3, while it showed an apparently
slower increase toward Day 7 in the ft-2 buds. In the internodes,
the SOC1 expression level in wild-type plants was much higher
than that in ft-2 plants (Fig. 9B). The AP1 level in the internodes
was below the limit of quantification (data not shown).
Then, the expression of genes associated with meristem maintenance [STM and WUSCHEL (WUS); Lenhard et al. 2002], cell
cycle [CYCLIN D3;1 (CYCD3;1); Dewitte et al. 2007], polar auxin
transport (PIN1 and PIN3; Zažı́malová et al. 2010) and
gibberellic acid synthesis [GIBBERELLIN 20-OXIDASE 1
(GA20ox1) and GA20ox2; Plackett et al. 2012, Rieu et al. 2008],
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
Role of FT and TSF beyond flowering
all of which can potentially affect lateral shoot outgrowth, was
analyzed. The expression levels of STM, CYCD3;1, PIN1 and PIN3
were not significantly affected in ft-2 plants (Fig. 9A, B). In contrast, WUS and GA20ox2 in the buds showed a somewhat similar
expression profile to that of AP1 (Fig. 9A); the expression in the
wild-type lateral buds showed a rapid increase, whereas that in
the ft-2 buds showed a gradual increase until Day 3, followed by a
steep induction. The GA20ox1 expression level in the lateral buds
was not affected by ft-2 mutation, while its expression level in the
ft-2 internodes was higher than that in the wild-type internodes
on Day 7 (Fig. 9A, B). Thus, expression of some of the selected
genes examined was affected by ft mutation in lateral buds or
internodes.
Discussion
Regulation of lateral shoot growth by FT and
TSF in Arabidopsis
We have shown that ft and tsf loss-of-function mutations
resulted in retardation of lateral shoot growth (Figs. 1–4)
and that the recovery of FT and/or TSF expression from their
deficient states led to the attenuation of the retardation
(Fig. 8). The impact of the loss of the genes (i.e. the extent of
retardation) in the mutants seems to reflect expression levels of
the respective genes mainly in leaves (Figs. 6,7). In the experiments using LD-grown plants (Figs. 2,4), simple comparison
could be difficult owing to the large differences in growth
states (e.g. age, number of rosette and cauline leaves, and
root density in the pot) among different genotypes to be
tested. In contrast, in the experiments using plants grown
under SD conditions (Fig. 3) and those carrying the inducible
FT transgene (Fig. 8), and in the SD to LD and LD to SD daylength shift experiments (Figs. 5,8), plants of a similar age
with similar numbers of rosette and cauline leaves could be
employed so that direct comparison among different genotypes was feasible. More importantly, these experimental settings (except for that under SD conditions), which generated
different activity states of the florigen genes well after the bolting of the plants, enabled us to examine the effect of florigen on
lateral shoot growth independently of its effect on the floral
transition of the primary shoot. Therefore, the results of these
experiments led us to the unequivocal conclusion that FT and
TSF regulate lateral shoot growth. It is to be noted, however,
that the effect of ft single mutation on lateral shoot growth
under LD conditions was not always observed (e.g. Fig. 4), and
that lateral shoots of ft; tsf double mutants eventually grew out
(Figs. 2,4). These findings may be because FT and TSF are not
the sole crucial regulators but represent two of the important
modulators of lateral shoot growth.
Expression of FT and TSF in lateral shoots was detected
only after they elongated, and was restricted in floral buds
(Fig. 7A–H). Our recent study showed that FT is not expressed
in axillary buds but is expressed in the distal part of the leaf
blade of the subtending leaf (M. Niwa, M. Endo and T. Araki,
unpublished observation). By using a local transient expression
system, it was also demonstrated that FT protein can move into
an axillary bud from the blade of the subtending leaf (M. Niwa,
M. Endo and T. Araki, unpublished observation). Expression of
FD, which encodes a partner of FT and TSF proteins, was clearly
detected in axillary buds before starting outgrowth (M. Niwa,
M. Endo and T. Araki, unpublished observation). Thus, florigen
can act mainly as a non-autonomous mobile signal from leaves
in the regulation of lateral shoot development as well.
Interestingly, a photoperiod-independent, rather high level of
expression of FT (and, to a much lesser extent, of TSF) in developing floral buds and flowers of the primary inflorescences
does not seem to play a significant role in lateral shoot outgrowth (Fig. 7A–D; see also Kobayashi et al. 1999, Adrian et al.
2010). For example, lateral shoots of SD-grown ft-2 plants
showed similar elongation profile to those of SD-grown
wild-type plants (Fig. 3). It is likely that the utilization of inflorescence (sink)-generated florigen is much less efficient than
that of leaf (source)-generated florigen.
Among the genes examined in this study (Fig. 9), the expression of CYCD3;1, STM, GA20ox1, PIN1 and PIN3 was not
affected by ft mutation in the apical region of the lateral shoots.
In contrast, DRM1 was more strongly expressed in ft-2 plants,
indicating that the lateral shoots of the mutants remained in
the dormant state at least during the early stages. Consistently,
the expression levels of AP1, WUS and GA20ox2 were apparently lower (less than half) in ft-2 plants. It has been well documented that the latter three genes are more strongly expressed
in developing flower buds than in other tissues including
internodes and leaves. AP1 is expressed in pedicels and developing flower buds from stage 1 to 10 (Mandel et al. 1992,
Gustafson-Brown et al. 1994), while WUS, also involved in
floral organ development, is expressed in inflorescences, with
the highest expression in developing anthers (Wellmer et al.
2004, Deyhle et al. 2007). GA20ox2 is strongly expressed in
inflorescences and distinctly detected in anthers and pistils
of stage 12 flowers (Rieu et al. 2008, Plackett et al. 2012).
Therefore, the observed difference in the expression of these
three genes in the lateral buds between the wild type and
ft mutant may reflect a difference in the rate of flower formation and development between the two genotypes. Actually, in
addition to slower elongation rates, delay of the first flower
anthesis and reduction in the number of open flowers per
day were observed in lateral shoots of LD-grown ft and ft; tsf
plants (Fig. 2F). Tsukaya et al. (1993) showed that elongation
growth of the main shoot is regulated by the developing first
flower buds. Also, Lohmann et al. (2010) recently reported that
the slow motion mutation caused a slower flower formation
rate and retardation of lateral shoot growth. Taking these findings together, one attractive hypothesis to be tested in the
future is that the two florigen genes may regulate lateral
shoot outgrowth and elongation via acceleration of flower formation. In the internodes of lateral shoots, SOC1 expression was
decreased in ft plants (Fig. 9B). Melzer et al. (2008) reported
that SOC1 suppressed secondary growth of the inflorescence
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
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K. Hiraoka et al.
stem together with FUL. Involvement of FT and TSF in
secondary growth and the relationship of lateral shoot development and secondary growth are interesting problems to
be addressed.
Differential contribution of FT and TSF to lateral
shoot development under different photoperiods
Differential effects of ft-2 and tsf-1 single mutations in lateral
shoot outgrowth under LD and SD conditions, respectively,
suggest that FT plays a more dominant role under LD conditions and TSF under SD conditions. A similar differential contribution of the two genes was previously reported in the case
of floral transition (Yamaguchi et al. 2005), although the effect
of the loss of TSF under SD conditions is difficult to demonstrate clearly in some experimental conditions (Michaels et al.
2005, Jang et al. 2009, this study). Therefore, our previous proposal that FT and TSF may fine-tune the timing of the floral
transition and provide robustness of the process (Yamaguchi
et al. 2005) seems also to be true for the regulation of lateral
shoot growth by the two genes.
As shown in this study, the differential contribution of the
two genes is due to differential expression profiles of the respective genes mainly in leaves. Under LD conditions, FT is
up-regulated in leaves much more robustly than TSF
(Kobayashi et al. 1999, Yamaguchi et al. 2005), while TSF (but
not FT) in leaves was gradually up-regulated in an agedependent manner under SD conditions (Fig. 6). The nature
of the age-dependent cues which trigger TSF expression is currently unknown. Recently, D’Aloia et al. (2011) reported that
CK applied to root was able to induce TSF expression in the
shoot. Therefore, CK synthesis and/or mobilization are likely
to be important components of this induction pathway.
Another candidate of apparent importance is the aging pathway mediated via the miRNA156–SQUAMOSA PROMOTOR
BINDING PROTEIN LIKE module (Wang et al. 2009,
Yamaguchi et al. 2009).
Significance of the regulation of lateral shoot
outgrowth by the florigen genes
Arabidopsis produces branched raceme inflorescences, with
flowers being sequentially produced. The annual racemes
may show high fitness, for example, if the length of the
growth seasons varies from year to year (Prusinkiewicz et al.
2007). Under favorable conditions, Arabidopsis plants may exploit abundant resources (space, nutrients, water, assimilates,
etc.) and hasten to produce flowers and fruits at one stretch
because the best conditions will not last for a long time. In
contrast, under semi-favorable conditions, the plants may
keep producing flowers more slowly to ‘hedge its bets’. In this
context, FT and TSF may play important roles to maximize the
number of seeds under given conditions, as postulated for SFT
in tomato (Krieger et al. 2010). Under LD conditions, FT plays
crucial roles not only to induce floral transition synchronously
(compare the standard deviations of Experiment 1 (LD) and
2 (SD) in Supplementary Table S1) but also to synchronize
364
flowering and several traits accompanying flowering such as
bolting (main shoot ‘outgrowth’) and lateral shoot outgrowth
and its subsequent elongation (Pouteau and Albertini 2009;
Fig. 2). Therefore, FT (and TSF) may vigorously synchronize
main and lateral shoot growth to guarantee fitness under
favorable LD conditions. Under non-inductive SD photoperiods, their lateral shoot outgrowth was delayed, and their
elongation rate and flower production were also slowed
down. However, other external or internal cues also regulate
lateral shoot development (Domagalska and Leyser 2011).
In well-fertilized plants under SD conditions, for example,
age-dependent TSF expression may support the other regulation pathways and guarantee relatively vigorous outgrowth of
lateral shoots.
In summary, we have shown that FT and TSF modulate the
lateral shoot growth under inductive and non-inductive photoperiods, respectively, via differential induction of expression.
Taken together with the findings in other plant species
(Tamaki et al. 2007, Shalit et al. 2009), it is likely that the regulation of lateral shoot development is an integral part of the
florigen action. To elucidate the molecular mechanism by
which the florigen genes modulate lateral shoot outgrowth
will be an important future challenge. In addition, differential
regulation of the two florigen genes has been reported in
several species (Komiya et al. 2009, Wada et al. 2010, Higuchi
et al. 2011). The fine-tuning regulation of expression of the
two florigen genes in Arabidopsis and other plants will be
another interesting problem to be addressed.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana (L.) Heynh. accession Col was mainly
used as the wild type. ft-1 and ft-2 introgressed into the Col
background were described in Notaguchi et al. (2008) and
Imura et al. (2012), respectively. tsf-1 was described in
Yamaguchi et al. (2005). ft-2, tsf-1, ft-1; soc1-2; lfy-1, ft-1; tsf-1;
lfy-1 and ft-1; tsf-1; soc1-2 with or without the lfy-1 allele were
newly generated in our laboratory. gFT::GUS (line AY#1) and
HSP::FT-T7 (MA#2); ft-1 were described in Notaguchi et al.
(2008), and gTSF::GUS (line #21-1) was described in
Yamaguchi et al. (2005). POP40 was kindly provided by
Dr. M.F. Yanofsky (University of California at San Diego,
USA), and the POP40 line introgressed into ft-2 was generated
in this study. Accessions Ler and No were also used as wild types
in some experiments. ft-2 in the Ler background and ft-4, ft-5
and ft-6 in the No background were described in Kobayashi
et al. (1999). ft-2; tsf-1 in the Ler background was newly generated in this study.
Seeds were imbibed in water for 3 or 4 d at 4 C, and were
sown on a piece of tap water-soaked Jiffy-7 peat pellet (Jiffy
Products) on a pot of vermiculite. Plants were grown in growth
chambers at 22 C under LD (16 h light/8 h dark) conditions
with white fluorescent lights (80 mmol m–2 s–1) or under SD
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
Role of FT and TSF beyond flowering
(8 h light/16 h dark) conditions with white fluorescent lights
(160 mmol m–2 s–1).
Measurement of shoot development
In all measurements, ‘Day 0’ was defined as the day on which
the main shoot of a plant reached a height of 5 cm. Shoot length
was measured at a fixed time each day from when the plants
started to bolt (for Figs. 2–4). The number of open flowers per
day was counted at the same time. ‘The longest lateral shoot’
was defined as the lateral shoot that has been the longest
during the most part of the experimental period. The elongation rate on Day X after the main shoot of a plant reached a
height of 5 cm [Rday(X)] was defined as
RdayðXÞ ¼ 0:5 LdayðX+1Þ LdayðX1Þ ,
where Lday(X) is the shoot length on Day X. ‘Open flowers’
were defined as flowers approximately at floral stages 13 and
14 (Smyth et al. 1990). Especially in ft-2; tsf-1 double mutants,
flower buds with no petals (or petal-like organs) or those which
never opened were observed among the early-arising buds of the
inflorescences. These flower buds were not counted. To avoid
double counting, one sepal of a counted flower was marked with
a marker pen. Statistical analyses were performed with SPSS 19 or
20 (IBM). First, Levene’s test was used to assess the assumption of
homogeneity of variances. Then, where the distribution of data
met the assumptions, one-way analysis of variance followed by
Tukey’s multiple comparison test was used. Otherwise, Welch’s
test followed by the Games–Howell test was used.
Heat shock treatment
HSP::FT-T7 (MA#2); ft-1 and ft-1 plants were grown under LD
conditions as described above. The height of the main shoot
was measured daily at ZT7, and the plants to be treated were
incubated at 40 C for 2 h (from ZT8 to ZT10) in the dark on the
day when the plants reached 2 cm in height and on Day 0.
GUS staining
reverse-transcribed in a 20 ml reaction mixture using
SuperScript III (Life Technologies; on 0.5 mg of total RNA) or
Transcriptor (Roche Applied Science; on 3 mg of total RNA).
After the reaction, the mixtures were diluted with 80 ml of TE
buffer, and 1 ml (RT–qPCR) or 2 ml (RT–PCR) of the aliquots were
used for one reaction.
RT–qPCR analyses were carried out in 96-well plates with
the CFX96 system (Bio-Rad Laboratories) using SYBR Green I.
The primers, the reaction programs and the reaction mixture
(20 ml) used for RT–qPCR are described in Supplementary
Table S2. Samples were run in technical triplicates,
except for the samples for the standard curve (technical
duplicates). Melting curve analyses were performed from
65 to 95 C to assess the specificity of the RT–qPCR products.
Quantification cycle values were converted into normalized
relative quantities (NRQs) according to Hellemans et al.
(2007). At4g34270 and At2g28390, the two reference genes
identified by Czechowski et al. (2005), were used. Relative
expression levels of each sample were calculated as the ratio
of the NRQ of the sample to the NRQ of the sample from
the wild-type lateral shoot buds harvested on Day 0 of the
experiments, except for Fig. 6A, in which the relative expression
values of FT and TSF in wild-type (Col) plants were obtained as
the ratio to the level in the SD to LD shifted plants.
For comparison of FT and TSF expression levels, a modified
RT–PCR analysis using two restriction enzymes was performed.
The primers, the reaction programs and the reaction mixture (30 ml) used in this RT–PCR analysis are listed
in Supplementary Table S2. The amplified 351-bp products
(a mixture of FT- and TSF-derived products) were digested by
EcoT14I or PstI (FT, 243- and 108-bp fragments by PstI; TSF,
256- and 95-bp fragments by EcoT14I). Then, the digested fragments and the PCR product of ACTIN2 (as a reference gene)
were electrophoresed on agarose gels and visualized by ethidium bromide staining.
Supplementary data
Plants were fixed for 15–30 min in 90% acetone on ice, and
then incubated at 37 C for about 24 h in the dark with
staining solution [0.5 mg ml–1 5-bromo-4-chloro-3-indolyl-bD-glucuronide, phosphate-buffered saline (pH 7.0; 130 mM
NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4), 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide, 0.1% (v/v) Triton
X-100]. After staining, plant materials were treated with 70%
ethanol to remove Chl. Then, for whole-mount observations,
the samples were cleared in an 8 : 1 : 2 (w/v/v) mixture of chloral
hydrate, glycerol and water. All photographs of GUS-stained
samples were processed with Adobe Photoshop CS6.
RT-qPCR and RT-PCR
RNA was extracted using TRIzol reagent (Life Technologies) or
Sepasol RNA I Super G (Nacalai Tesque), and was treated with
RNase-free DNase I (Life Technologies) according to the manufacturer’s instructions. Total RNA (0.5 or 3 mg) was
Supplementary data are available at PCP online.
Funding
This work was supported by the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan [Grant-in-Aid
for Scientific Research on Priority Areas (19060012, 19060016
to T.A.)].
Acknowledgments
We thank Dr. M.F. Yanofsky (University of California at San
Diego, USA) for POP40 seeds, Dr. T. Kohchi (Kyoto
University) for apparatus, Dr. Y. Tomita of our laboratory for
excellent technical assistance, and the past and present members of the Araki lab for helpful discussions and comments.
Plant Cell Physiol. 54(3): 352–368 (2013) doi:10.1093/pcp/pcs168 ! The Author 2012.
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K. Hiraoka et al.
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