Download Effect of extending the photoperiod with low

Tree Physiology 34, 534–546
doi:10.1093/treephys/tpu033
Research paper
Effect of extending the photoperiod with low-intensity red or ­
far-red light on the timing of shoot elongation and flower-bud
formation of 1-year-old Japanese pear (Pyrus pyrifolia)
Akiko Ito1,4, Takanori Saito1,2, Takaaki Nishijima3 and Takaya Moriguchi1,2
1Plant
Physiology and Fruit Chemistry Division, NARO Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan; 2Graduate School of Life and
Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan; 3Ornamental Plants Research Division, NARO Institute of Floricultural
Science, Tsukuba, Ibaraki 305-8519, Japan; 4Corresponding author ([email protected])
Received September 23, 2013; accepted April 3, 2014; published online May 29, 2014; handling Editor Chung-Jui Tsai
To investigate the effects of light quality (wavelength) on shoot elongation and flower-bud formation in Japanese pear
(Pyrus pyrifolia (Burm. f.) Nakai), we treated 1-year-old trees with the following: (i) 8 h sunlight + 16 h dark (SD); (ii) 8 h
sunlight + 16 h red light (LD(SD + R)); or (iii) 8 h sunlight + 16 h far-red (FR) light (LD(SD + FR)) daily for 4 months from
early April (before the spring flush) until early August in 2009 and 2010. In both years, shoot elongation stopped earlier in the LD(SD + FR) treatment than in the SD and LD(SD + R) treatments. After 4 months of treatments, 21% (2009) or
40% (2010) of LD(SD + FR)-treated trees formed flower buds in the shoot apices, whereas all the shoot apices from SD or
LD(SD + R)-treated plants remained vegetative. With an additional experiment conducted in 2012, we confirmed that FR
light at 730 nm was the most efficacious wavelength to induce flower-bud formation. Reverse transcription–quantitative
polymerase chain reaction revealed that the expression of two floral meristem identity gene orthologues, LEAFY (PpLFY2a)
and APETALA1 (PpMADS2-1a), were up-regulated in the shoot apex of LD(SD + FR). In contrast, the expression of a flowering
repressor gene, TERMINAL FLOWER 1 (PpTFL1-1a, PpTFL1-2a), was down-regulated. In addition, expression of an orthologue
of the flower-promoting gene FLOWERING LOCUS T (PpFT1a) was positively correlated with flower-bud formation, although
the expression of another orthologue, PpFT2a, was negatively correlated with shoot growth. Biologically active cytokinin
and gibberellic acid concentrations in shoot apices were reduced with LD(SD + FR) treatment. Taken together, our results
indicate that pear plants are able to regulate flowering in response to the R : FR ratio. Furthermore, LD(SD + FR) treatment
terminated shoot elongation and subsequent flower-bud formation in the shoot apex at an earlier time, possibly by influencing the expression of flowering-related genes and modifying plant hormone concentrations.
Keywords: cytokinin, flowering-related genes, fluorescence lamp, gibberellic acid, light-emitting diodes, phase transition.
Introduction
The regulation of flowering in fruit trees, including Japanese
pear (Pyrus pyrifolia (Burm. f.) Nakai), has long been of interest to fruit tree growers. Japanese pear buds flush in April, and
the emerged shoots continue to elongate until May (for spurs)
or late June (for long shoots) and then form terminal buds on
their apices. The terminal buds sometimes flush repeatedly
(two or three times) during the summer season, with a higher
frequency in years with abundant rainfall or with high temperatures. Japanese pear tree flower buds are usually the terminal buds on spurs and the terminal and axillary buds on long
shoots. Trees in the juvenile phase rarely produce flower buds;
however, the terminal buds of spurs often bear flowers even in
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Light effects on pear shoot growth/flowering 535
juvenile trees. Usually, fewer flowers are borne on either the
terminal or the axillary buds of long shoots.
Cessation of shoot elongation is a prerequisite event for
flower-bud induction and development (Ito et al. 1999). Floral
induction of Japanese pear occurs in late June, when day length
is at its maximum and temperatures are high in the northern
hemisphere; however, most temperate deciduous trees, including Japanese pear, are considered insensitive to photoperiod and temperature for flower induction. Therefore, flower
induction is assumed to be entirely under endogenous control (reviewed by Jackson and Sweet 1972, Westwood 1978,
Kurokura et al. 2013). High temperature, high light intensity
and sometimes a long photoperiod also can accelerate flower
initiation and development in Japanese pear (Rakngan et al.
1995), but these effects may be related to differences in photosynthetic activity and daily carbon assimilation rather than to
a phase transition from vegetative to reproductive as in other
temperate deciduous trees (reviewed by Wilkie et al. 2008).
Owing to their potential use to control flowering in deciduous fruit trees, the effects of hormones on flower-bud formation
have long been studied (reviewed by Jackson and Sweet 1972,
Westwood 1978, Owens 1991). Gibberellins (GAs) generally
inhibit flowering of fruit trees (Looney et al. 1985, Horvath
2009), whereas applications of synthetic cytokinins (CKs) generally increase flower-bud number in apple and pear (Luckwill
1970, McLaughlin and Greene 1984, Banno et al. 1985, Ito
et al. 2000). Similar trends were observed in studies comparing
endogenous hormone concentrations in Japanese pear; ‘Hosui’,
a cultivar that flowers abundantly, contains higher levels of CKs
and lower levels of GAs in the buds than ‘Kosui’, a cultivar that
produces fewer flowers (Banno et al. 1985). In addition, shoots
that bend from the vertical to a horizontal position have higher
levels of CKs and lower levels of GA4 in their buds, which favour
flower bud production on that shoot (Ito et al. 1999). These
studies suggest the involvement of GAs and CKs in the regulation of flower-bud formation; however, the precise roles of these
phytohormones remain obscure. In addition, the effects of plant
hormone application strongly depend on the timing and the
developmental condition of the trees and, thus, show very large
year-to-year fluctuations (Wilkie et al. 2008).
In contrast, flower induction by photoperiod is a common
mechanism to control flowering in herbaceous species (e.g.,
Simpson and Dean 2002, Zeevaart 2008). Photoperiod is
perceived by photoreceptors, such as phytochromes (PHYs:
red (R) and far-red (FR) light receptors) and cryptochromes
(CRYs: blue (B) light receptors). Thus, light quality (wavelength) is extremely important in photoperiodic perception and
responses. In the long-day model plant Arabidopsis thaliana
(L.) Heynh., light signals perceived by photoreceptors regulate CONSTANS (CO) activity together with the circadian clock
(Yanovsky and Kay 2002). Post-transcriptional activation of
CO rapidly activates FLOWERING LOCUS T (FT) transcription
(Valverde et al. 2004) when plants are growing in inductive
long-day conditions (Kardailsky et al. 1999, Kobayashi et al.
1999, Corbesier et al. 2007). The promotion of flowering
by FR and B is perceived and transduced by PHYA and CRY,
respectively, whereas the inhibition of flowering by R is relayed
by PHYB (Mockler et al. 2003). However, the B light floweringinductive pathway may sometimes not function in some
plant species (e.g., Gypsophila paniculata L., Hori et al. 2011,
Nishidate et al. 2012) partly because of the coaction between
photoreceptors (Elmlinger et al. 1994).
The different effects of light quality on flowering regulation
have recently been described in a wide range of plant species.
For example, a low R : FR ratio promotes flowering in longday plants, such as Arabidopsis (Hori et al. 2011), Campanula
carpatica Jacq. (Kristiansen 1988), Eustoma grandiflorum (Raf.)
Shinn. (Yamada et al. 2008, Sato et al. 2009), G. paniculata
(Hori et al. 2011, Nishidate et al. 2012) and Matthiola incana
(L.) R. Br. (Yoshimura et al. 2006). Conversely, night breaks
with R light retard flowering of short-day plants, such as rice
(Oryza sativa L.) (Ishikawa et al. 2009) and chrysanthemum
(Chrysanthemum × morifolium Ramat.) (Sumitomo et al. 2012).
The promotive effect of FR on flowering is generally observed
in long-day plants (Runkle and Heins 2003, Kurepin et al.
2007b) but may also be found in short-day plants (Collins
1966, Sumitomo et al. 2009). As mentioned above, flowering
regulation in pear appears to be photo-insensitive; however,
considering the presence of photoreceptors such as PHYA,
PHYB and CRY1 in the pear genome (Bai et al. 2013), the
involvement of photoperiod and/or light quality cannot be ruled
out completely.
In order to investigate the effects of extending photoperiods
with lights of different qualities on Japanese pear flower-bud
formation, we irradiated pear plants daily with R or FR light
during the artificial 16 h dark period. We found that light quality
influenced shoot growth and flower abundance and that these
morphological changes were accompanied by changes in flowering-related gene expression and GA and CK concentrations.
We discuss these changes in the context of light-sensitive seasonal phase transition in pear. In this study, we show that pear
plants are able to respond to light quality by regulating the
cessation of shoot elongation and the formation of flower buds.
Materials and methods
Plant material
Experiments were conducted at the orchard of the NARO
Institute of Fruit Tree Science, Tsukuba, Japan (lat. 36°N, long.
140°E) in 2009, 2010 and 2012. For each year, 1-year-old
trees of Japanese pear (P. pyrifolia ‘Kosui’) grafted on Pyrus
calleryana Decne. were purchased from a commercial nursery
(Komachi En, Nagano, Japan) and cut to 120 cm height. ‘Kosui’
is the leading pear cultivar in Japan, with a vigorous shoot
Tree Physiology Online at http://www.treephys.oxfordjournals.org
536 Ito et al.
growth habit and few flower buds on its long shoots. Each tree
was transplanted into a 20 l plastic pot containing a peat-based
potting compost on 24, 11 and 15 March in 2009, 2010 and
2012, respectively, and then immediately moved into a greenhouse. Trees were grown in the greenhouse without control of
environmental conditions (temperature and light) until the light
treatments started. Twenty-five, 32 and 16 plants were used
for each light treatment in 2009, 2010 and 2012, respectively.
Experimental conditions
During the light treatments, all plants received natural sunlight for
8 h per day (08.30–16.30 h) and were shaded for the remaining 16 h using blackout screens (shading >95%, Howaitosiruba,
Tokankosan Co., Ltd, Tokyo, Japan). During the 16 h shading
period, plants were subjected to different light treatments. In
2009 and 2010, the following three treatments were conducted
(SD, short day; LD, long day): dark (SD); lit using red (R)-rich
light (LD(SD + R)); or lit using FR-rich light (LD(SD + FR)). The
R-rich light was supplied with bulb-type light-emitting diodes
(LEDs; Ekonoraito AG, ELM Inc., Kagoshima, Japan), and FR-rich
light was supplied with FR-rich fluorescent tubes (FL20SFR-74,
Toshiba Lighting & Technology Corporation, Tokyo, Japan) covered with red filters (RGB color pipe, Panasonic Electric Works
Co., Ltd, Tokyo, Japan) to ensure that only light with wavelengths
>500 nm could penetrate the filter. In 2012, in order to identify the most effective wavelength of FR light, FR-rich light was
supplied either with FR-rich fluorescent tubes (same as used in
2009 and 2010) or with bulb-type LEDs having a main peak
of emission at 700, 730 or 760 nm (provided by Shibasaki,
Inc., Saitama, Japan) and was compared with a dark treatment. Treated plants received R or FR light at a low intensity of
~2 µmol m−2 s−1 (600–700 nm for R and 650–900 nm for FR,
respectively) at the base of the plant. The spectral photon distributions of the light sources are shown in Figure 1.
Measurements
Plants were treated between 6 April and 14 August in 2009,
between 5 April and 6 August in 2010 and between 6 April
and 7 August in 2012. In all years, light treatments were started
before the spring flush. Growth (length and leaf number) of the
uppermost shoot of each plant was recorded approximately
every 10 days. The analyses described below were conducted
only in 2010 but not in 2009 and 2012, except for the measurement of the light effect on flower-bud formation.
Length and leaf number of the uppermost shoot of each plant
were recorded every 10 days. The increments of shoot growth
(shoot length and leaf number) per day and relative to the size
of the tree (relative growth rate; RGR) were calculated according to the following equation for shoot length and leaf number:
RGR n =
(ln (S
n +1
)
– Sn + 1) / (Tn +1 – Tn ) ,
Tree Physiology Volume 34, 2014
Figure 1. ​Spectral distribution of photon fluxes of R (LED) and FR
lights (fluorescent tubes and LEDs) used in these experiments. Plants
received R or FR light at ~2 µmol m−2 s−1 (600–700 nm for R and
650–900 nm for FR) at the base of the plants.
where S expresses shoot growth (length in centimetres or leaf
number) and T is the number of days after treatment. In this
equation, +1 was added to (Sn+1 − Sn) before natural logarithmic transformation in order to obtain an answer from the equation even in the case where Sn+1 − Sn = 0.
To investigate the changes in mRNA levels, the apical portion
of the uppermost shoots (3 cm long, including unfolding leaves
<1 cm, referred to as shoot apex hereafter) and mature leaves
from three individual trees were sampled between 08.45 and
09.30 h on Days 10 (15 April), 31 (6 May), 51 (26 May), 73
(17 June) and 93 (7 July) after initiating the light treatments.
For plant hormone analyses, shoot apices of six other shoots
(two shoots × three replicates) were sampled in a similar manner on Days 10 (15 April), 31 (6 May) and 73 (17 June) after
the treatment. In order to determine whether the apical buds
were dormant, 6–10 plants were moved outside of the greenhouse on 17 June, 7 July and 6 August in 2010 and kept in natural environmental conditions. The high temperatures and long
days between 17 June and 31 August in Tsukuba induced bud
burst in the Japanese pear trees. After 3 weeks in natural (forcing) conditions, the number of apical buds that had burst was
recorded. For the plants evaluated on 6 August, the number of
apical buds containing a flower was also recorded. Frequencies
of flower-bud formation after light treatments were also recorded
for plants treated in 2009 and 2012 by forcing bud burst.
Total RNA extraction and reverse transcription–
quantitative polymerase chain reaction
Total RNA was extracted from shoot apices and mature leaves of
‘Kosui’ collected according to the method described by Ito et al.
Light effects on pear shoot growth/flowering 537
(2013). The synthesis of first-strand complementary DNA was
carried out using the SuperScript™ III first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). Five-microgram aliquots of
total RNA used in the reaction were first treated with RNA-free
DNaseI (Promega, Madison, WI, USA) and reverse transcribed
using SuperScript III oligo (dT) 20 primers according to the
manufacturer’s instructions (Invitrogen). Reverse transcription–
quantitative polymerase chain reaction (RT-qPCR) was performed in a 7500 Real-Time PCR System (Applied Biosystems,
Foster City, CA, USA) with a SYBR Premix Ex Taq kit (TaKaRa,
Kyoto, Japan) as described in the manufacturer’s protocol.
HistoneH3 and SAND were used as internal references in
all experiments, as reported by Imai et al. (2014). Specific
primers for PpFT1a, PpFT2a, PpTFL1-1a, PpTFL1-2a, PpLFY2a,
PpMADS2-1a, HistoneH3 and SAND are shown in Table 1.
RT-qPCR was performed two times using different extracted
total RNA (two biological replicates), and each replicate was
tested three times (technical replicates) for a total of six measures for each reference gene. Given that the expression
trends were similar irrespective of the reference genes used,
one data set using HistoneH3 was used to produce the ­figures.
Plant hormone analyses
Shoot apices were analysed for CKs and GAs by liquid chromatography–tandem mass spectrometry (LC/MS/MS). Extraction
and purification of CKs were performed as described by
Nishijima et al. (2011) and of GAs as described by Horimoto
et al. (2011). Target molecules in this study are N6 -(Δ2isopentenyl)adenine (iP), trans-zeatin (tZ), N6 -(Δ2-isopentenyl)
adenosine (iPR), tZ riboside (tZR), GA4 and GA1, and their
stable isotope-labelled compounds were used as internal standards ([2H6]-iP, [2H5]-tZ, [2H6]-iPR, [2H5]-tZR, [2H2]-GA4 and
[2H2]-GA1, OlchemIm Ltd, Olomouc, Czech Republic).
Frozen samples (0.5–1.0 g) were homogenized in liquid
nitrogen with a mortar and pestle, mixed with a solution of
methanol/water/formic acid (15/4/1) containing internal standards, and then extracted at −20 °C overnight. The extract was
purified following the method of Dobrev and Kaminek (2002).
The basic fractions that contained CKs and the acidic fractions that contained GAs were evaporated separately to dryness. The dried samples for CKs were then dissolved in 10%
aqueous methanol containing 0.05% acetic acid and analysed
by LC/MS/MS (model 2695/TSQ7000, Waters/Thermo Fisher
Scientific, Waltham, MA, USA) using positive ion electrospray
ionization. Cytokinins were separated using an ODS column
(MD, 5 µm, 2.5 mm × 250 mm, Shiseido Fine Chemicals,
Tokyo, Japan) at a flow rate of 0.2 ml min−1, with gradients
of methanol/water containing 0.05% acetic acid. The ionization voltage was 4.7 kV, capillary temperature was 200 °C,
and collision energy was −22 to −34 V depending on the CK
species. For GAs, the dried samples were subjected to highperformance liquid chromatography on a Senshu Pak N(CH3)24151-N column (150 mm × 10 mm i.d., Senshu Scientific,
Tokyo, Japan) that was eluted with methanol containing 0.1%
acetic acid at a flow rate of 2 ml min−1 at 40 °C. Fractions with
retention times of 26–30 min for GA1 and 18–26 min for GA4
were separately evaporated, redissolved in 3% aqueous acetonitrile and injected into an LC/MS/MS system consisting of an
UPLC (AQUITY UPLC, Waters Corporation, Milford, MA, USA)
and a tandem quadrupole mass detector (AQUITY TQ Detector,
Waters Corporation). Gibberellins were separated using an ODS
column (AQUITY UPLC BEH C18, 1.7 µm, 2.1 mm × 100 mm,
Waters Corporation) at a flow rate of 0.2 ml min−1 with gradients of acetonitrile/water containing 0.05% acetic acid.
The ionization voltage was 2.9 kV, capillary temperature was
150 °C, and collision energy was −14 to −22 V depending on
Table 1. ​Target genes and primers used in SYBR Green RT-qPCR analyses.
Target gene (accession no.)
Primer sequence
Product size (bp)
PpFT1a (AB524587)
5′-CTAGAGTTGATATTGGTGGC-3′
5′-CCAATTGGCGAAACACCACC-3′
5′-CTAGAGTTGATACCGGTGGT-3′
5′-CCAATTGGCGAAACAGCACA-3′
5′-TCAACGCGCAGAGAGAAAC-3′
5′-GGGAAACAAGAGTTACAATAGCCTATC-3′
5′-TGTCACAGCCAAACCTAGAGTTG-3′
5′-GGTCACTAGGGCCAGGACAA-3′
5′-GAGTGGCAACTGAGAGTCCA-3′
5′-TTTTCAGCAGCCCAGTTCTT-3′
5′-GTTCAAGTGGGGCCTACGAG-3′
5′-CGGCCGCAAAGTACAACCAG-3′
5′-GTCAAGAAGCCCCACAGATAC-3′
5′-CTGGAAACGCAGATCAGTCTTG-3′
5′-CCCAGGACTTTGAGCTTTATGC-3′
5′-TATCACCATGAAAAGGGGCTTG-3′
231
PpFT2a (AB571595)
PpTFL1-1a (AB601157)
PpTFL1-2a (AB524588)
PpMADS2-1a (AB504717)
PpLFY2a (AB853872)
HistoneH3 (AB623164)
SAND (AB795982)
231
132
98
124
115
153
136
Tree Physiology Online at http://www.treephys.oxfordjournals.org
538 Ito et al.
the GA species. Cytokinins and GAs were quantified in selected
ion recording mode with the internal standard method using
the corresponding deuterated standards.
Statistical analyses
We conducted multiple regression analyses to explain the
relationships between shoot growth (response variable) and
gene expression (explanatory variables) and also to explain
the interrelationships between each of the gene expression
patterns. A set of explanatory variables was selected by a
stepwise method, and the set possessing the smallest value of
Akaike’s information criterion was employed.
For comparing the plant hormone concentrations, we conducted a two-way analysis of variance (ANOVA) to show the
independent and interactive effects of the two factors, ‘light
treatment’ and ‘date’. When the interactive effects (‘light treatment’ × ‘date’) were significant, means were separated with
the Tukey–Kramer test. We also conducted a regression analysis between shoot growth and plant hormone concentrations.
Results
Shoot growth
In the 2009 experiment, we observed that leaf production
ceased in mid-May in all three treatments, SD, LD(SD + R)
and LD(SD + FR), and some of the shoots treated with SD
or LD(SD + R) flushed again (second flush) and elongated
continuously, reaching a greater length in LD(SD + R) than
in SD (Figure 2a). When growth stopped initially (middle of
May), shoot length was longer in LD(SD + FR) than in SD and
LD(SD + R) (Figure 2b).
In 2010, leaf production ceased initially in mid-May and
resumed subsequently (second flush) in all three treatments
(Figure 2c). In the SD and LD(SD + R) treatments, third flushes
occurred, and leaf production continued until the end of the
light treatments, with a larger intensity in LD(SD + R) than
in SD. In contrast, in LD(SD + FR), leaf production ceased in
June and did not resume thereafter. Thus, shoot elongation in
LD(SD + FR) ceased earlier than in the other two treatments.
Shoot elongation in some plants treated with LD(SD + FR)
ceased with the death of the apical portion, including the apical meristem, in a fashion similar to the ‘self-pruning’ phenomenon (see Figure S1 available as Supplementary Data at Tree
Physiology Online) that is observed, albeit rarely, in field-grown
pear trees. In these plants, the most distal lateral bud became
a terminal bud instead of the apical meristem, as observed in
plants having sympodial growth habits.
Shoot elongation until mid-May was most enhanced in
LD(SD + FR) compared with SD and LD(SD + R) (Figure 2d).
During this period of the first elongation stage, leaf number
increased to a similar extent for all three of the light treatments.
Thus, the longer shoot length in LD(SD + FR) is attributed to
Tree Physiology Volume 34, 2014
its longer internodes compared with the other two treatments;
however, the final shoot length was longer in LD(SD + R) than
in LD(SD + FR) because plants receiving the latter treatment
ceased leaf (node) production earlier than plants subjected to
the former treatment. Shoots in SD elongated throughout the
treatment but weakly; thus, their final shoot length was the
shortest among the three treatments.
In order to identify the most effective wavelength to regulate shoot growth, we irradiated pear trees in 2012 with LEDs
having emission peaks of 700, 730 or 760 nm in addition to
the FR-rich fluorescent tubes as used in 2009 and 2010. We
observed that leaf production ceased once, in mid-May, and
resumed again (second flush) in all five treatments (Figure 2e).
In LD(SD + FR), LD(SD + 700 nm LED) and LD(SD + 730 nm
LED), leaf production ceased in June and did not flush again
(no third flush). In contrast, in the SD and LD(SD + 760 nm
LED) treatments, a third flush was observed, and the associated leaf production continued until July. The shoot lengths
were similar among all the treatments with the exception of
LD(SD + 760 nm LED)-treated trees, which had longer shoots
than the others, probably due to their higher number of leaves
(nodes) and the longer internodes (Figure 2f).
In the experiments conducted over 3 years, shoot growth
was more vigorous in 2010 and 2012 than in 2009. Shoots
in SD-treated plants flushed three times during the experiments in 2010 and 2012, whereas plants flushed only twice in
2009. However, the following results concerning shoot growth
were consistently obtained: (i) leaf production ceased earlier,
but (ii) shoot length in the earlier growth stages was larger in
LD(SD + FR) than in SD and LD(SD + R). The LD(SD + R) treatment was not conducted in 2012.
Flower-bud formation in shoot apices
In 2010, all of the apical buds treated with SD, LD(SD + R) or
LD(SD + FR) flushed again when moved out and forced in natural conditions on 17 June, 7 July and 6 August (n = 7–10, data
not shown), indicating that apical buds were not in an endodormant state. All of the apical buds remained in the vegetative
state when forced on 17 June and 7 July. Most importantly,
apical buds of four of the 10 (40% in 2010) uppermost shoots
of plants treated with LD(SD + FR) flowered when forced on 6
August in 2010, whereas apical buds of the shoots treated with
SD and LD(SD + R) remained vegetative (n = 7–10, Table 2).
Likewise in 2009, 21% (three of 14) of the apical buds of
uppermost shoots of plants treated with LD(SD + FR) flowered,
whereas the shoots treated with SD and LD(SD + R) remained
vegetative (n = 14–16, Table 2) when forced.
In 2012, flower-bud formation was observed only when
trees received photoperiods extended with FR-rich fluorescent
lamps (LD(SD + FR)) or 730 nm LED (LD(SD + 730 nm LED);
n = 14–16, Table 2). No other treatments produced flower buds
on the shoot apices. This result shows that FR light at 730 nm
Light effects on pear shoot growth/flowering 539
Figure 2. ​Effect of an 8 h photoperiod (SD) and of extending the photoperiod with red light (LD(SD + R)) or far-red light (LD(SD + FR),
LD(SD + 700 nm LED), LD(SD + 730 nm LED) and LD(SD + 760 nm LED)) on leaf number (a, c and e) and lengths of annual shoots (b, d and f)
of Japanese pear (n = 10, mean ± SEM) in 2009 (a and b), 2010 (c and d) and 2012 (e and f). Sampling times in 2010 for flowering-related gene
expression ( ) and for plant hormone analyses ( ) are also shown.
is the most effective wavelength for inducing flower-bud formation, compared with the 700 or 760 nm wavelengths.
Expression of flowering-related genes in the shoot apices
RT-qPCR revealed that the expression of flowering-related
genes in leaves and shoot apices were differently affected both
by the light treatments and by the seasons, and the changes in
response to light treatments were complex (Figure 3). In leaves,
no significant expression of PpTFL1-1a, PpTFL1-2a, PpLFY2a
and PpMADS2-1a was observed throughout the experiments
(data not shown), and expression levels of PpFT1a and PpFT2a
were very low in leaves compared with shoot apices (Figure 3).
Therefore, only the data for PpFT1a and PpFT2a expression in
leaves is shown here; expression in leaves was not included
in the subsequent multiple regression analyses. These results
are in accordance with reports showing that the expression of
FTs in mature apple leaves is much lower than in floral buds
(meristems; Kotoda et al. 2010). Likewise, expression of LFYs
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540 Ito et al.
and TFLs (Esumi et al. 2005) and AP1s (Ubi et al. 2013, and
unpublished data by T. Moriguchi) is lower in leaves than in
floral buds in Japanese pear.
In shoot apices, significant changes were found in PpLFY2a
and PpMADS2-1a, the orthologues of LFY and AP1; expression increased in LD(SD + FR) from early May to early July
and from mid-June to early July, respectively (Figure 3). In
contrast, PpTFL1-1a was highly expressed with LD(SD + R)
treatment, especially at later stages. Expression of PpTFL1-2a
declined as shoots matured in LD(SD + FR), whereas in SD
and LD(SD + R) treatments, expression increased at later
stages and peaked near 7 July or 17 June, respectively. The
Table 2. ​Effects of extending the photoperiod with R or FR light irradiation on the percentage of flower-bud formation on the apical buds
of Japanese pear shoots.
Treatment
Light source
2009 2010
n
SD
LD(SD + R)
LD(SD + FR)
LD(SD + 700 nm)
LD(SD + 730 nm)
LD(SD + 760 nm)
7–10
0
640 nm LED
0
FR-rich fluorescent lamp 21.0
700 mm LED
–
730 nm LED
–
760 nm LED
–
14–16
0
0
40.0
–
–
–
2012
14–16
0
–
14.3
0
18.8
0
expression patterns of PpLFY2a, PpMADS2-1a, PpTFL1-2a and
PpTFL1-2a coincided with the promotion of flower-bud formation in LD(SD + FR). Although the expression levels were
much lower in leaves than in shoot apices, both PpFT1a and
PpFT2a were expressed at higher levels in LD(SD + R)-treated
plants than in plants subjected to the other two treatments
(Figure 3).
In order to elucidate further the roles of these genes in the
transition from vegetative to reproductive phases, we carried out multiple regression analyses of shoot growth using
the expression of six selected genes as explanatory variables
(Table 3). Relative increments of shoot length and leaf number
correlated negatively with the expression of PpFT2a.
LFY and AP1 are known as the meristem identity genes in
Arabidopsis (Simon et al. 1996), and their functions in initiating the developmental cascade that leads to flowering is well
characterized (Horvath 2009). In order to understand the
complex roles of flowering-related genes in the transition from
the vegetative to the reproductive phase, we conducted multiple regression analyses of PpLFY2a or PpMADS2-1a expression in Japanese pear compared with other flowering-related
genes (Table 3). Expression of PpLFY2a correlated negatively with the expression of PpTFL1-1a, whereas expression
of PpMADS2-1a correlated positively with the expression of
Figure 3. ​Effects of an 8 h photoperiod (SD) and of extending the photoperiod with red light (LD(SD + R)) or far-red light (LD(SD + FR)) on the
relative expression levels of flowering-related genes in the apices and leaves of Japanese pear annual shoots. Gene expression was measured by
RT-qPCR using the HistoneH3 gene as a reference (n = 2, mean ± measurement range).
Tree Physiology Volume 34, 2014
Light effects on pear shoot growth/flowering 541
Table 3. ​Sets of explanatory variables resulting from multiple regression analyses between flowering-related gene expression vs increments of
shoot growth, and flowering-related gene expression vs meristem identity genes (LEAFY and AP1 homologues). ns and ** indicate non-significant
and significant at P < 0.01, respectively.
Response
variable
Explanatory variables
PpFT1a
RGR (shoot
length)
PRC2
SPRC3
P
RGR (leaf no.)
PRC
SPRC
P
ln(PpLFY2a)
PRC
SPRC
P
ln(PpMADS2-1a)
PRC
SPRC
P
PpFT2a
PpTFL1-1a
PpTFL1-2a
PpLFY2a
−0.049
−0.645
0.009
**
−0.027
−0.739
0.002
**
−0.266
−0.656
0.008
**
1.588
0.537
0.007
**
0.550
0.384
0.076
ns
−1.040
−0.700
0.004
**
R2 1
P
Intercept
0.502
0.015
−0.187
0.547
0.002
−0.011
0.430
0.0080
−4.727
0.774
0.0007
−8.511
PpMADS2-1a
1Multiple
correlation coefficient adjusted for the degrees of freedom.
regression coefficient.
3Standardized partial regression coefficient.
2Partial
PpFT1a and negatively with PpTFL1-2a expression. Expression
of PpFT2a was also positively correlated with PpMADS2-1a
expression, but its contribution was insignificant (P ≥ 0.05).
Plant hormone contents in the shoot apex
Concentrations of both CKs and GA4 in the shoot apex
decreased as the shoots were maturing (Figure 4). Although
the RGR of shoot length did not correlate with either plant hormone, the RGR of leaf number was positively correlated with
the concentration of tZR, iP, iPR and GA4 (Table 4). These
observations suggest that both CKs and GA4 are involved in
regulating the seasonal termination of shoot elongation rather
than regulating shoot internode elongation.
Analyses of variance revealed that the effects of the light
treatments were significant for tZ and iPR. The tZ concentration was lower in LD(SD + FR)-treated shoot apices in
comparison with SD, and the iPR concentration was lower
after LD(SD + FR) treatment than after LD(SD + R) treatment
(Figure 4). The effect of light treatments on GA4 concentrations varied depending on the shoot developmental stage;
light effects were not significant on 15 April, but became
more pronounced in LD(SD + R) than SD and LD(SD + FR)
on 6 May, and were lower in LD(SD + FR) than in SD and
LD(SD + R) on 17 June.
In this experiment, the GA4 concentration was 6–40 times
higher than that of GA1. This finding was in accordance with
reports that the most abundant bioactive GA in Japanese pear
is GA4 (Banno et al. 1985, Ito et al. 1999). Therefore, only the
data for GA4 are shown here.
Discussion
The involvement of photoperiod in flower induction has
not been clearly demonstrated in Rosaceae fruit trees (e.g.,
Kurokura et al. 2013), although photoperiodic control of flowering is well known in herbaceous species (e.g., Simpson and
Dean 2002, Zeevaart 2008). So far, it has been assumed that
floral bud formation in Rosaceae fruit trees is not driven by specific environmental stimuli, such as photoperiod and temperature, but by autonomous seasonal developmental processes
(Wilkie et al. 2008). However, the presence of orthologous
photoperiod-related genes, such as PHY and CO in the pear
(Bai et al. 2013) and apple genomes (Velasco et al. 2010),
supports the possible existence of photoperiodic signalling
pathways in these species (Mimida et al. 2013). Indeed, our
study shows that extending the photoperiod from 8 to 24 h
with FR light caused shoot growth to terminate early and subsequent flower-bud formation to begin early (Figure 2 and
Table 2). These developmental changes were accompanied by
a decrease in PpTFL1-2a expression and increases in PpLFY2a
and PpMADS2-1a transcript abundance (Figure 3). The percentage of apical buds successfully producing flower buds
was not high but was observed repeatedly (ranging from 14
to 40% over 3 years; Table 2). We hypothesize that extending the photoperiod with FR does not reduce apical meristem
activity or induce dormancy because (i) the apical buds of all
of the trees flushed during the outdoor forcing conditions after
4 months of light treatments, and (ii) leaves remained green
and healthy during all of the light treatments. The reports that
Tree Physiology Online at http://www.treephys.oxfordjournals.org
542 Ito et al.
Figure 4. ​Effects of an 8 h photoperiod (SD) and of extending the photoperiod with red light (LD(SD + R)) or far-red light (LD(SD + FR)) on concentrations of N6 -(Δ2-isopentenyl)adenine (isopentenyl adenine), trans-zeatin (t-Zeatin), N6 -(Δ2-isopentenyl)adenosine (isopentenyl adenosine),
t-Zeatin riboside and GA4 in the apices of annual shoots of Japanese pear (n = 3, mean ± SEM). Statistical analyses of the effects of light treatments
and dates on these plant hormone concentrations (two-way ANOVA, with factors ‘treatment’ (T) and ‘date’ (D)) are also presented. ns, *, ** and ***
indicate non-significant or significant at P < 0.05, 0.01 and 0.001, respectively. Different letters indicate a significant difference at P ≤ 0.05 with
the Tukey–Kramer test FW, fresh weight.
Table 4. ​Regression analyses between increments of shoot growth
vs plant hormone concentrations in shoot apices. ns, *, ** and ***
indicate non-significant or significant at P < 0.05, 0.01 and 0.001,
respectively.
vs RGR (shoot
length)
R2
R
P
vs RGR (leaf
no.)
R2
R
P
tZ
tZR
iP
iPR
GA4
0.049
−0.220
0.569
ns
0.348
0.590
0.094
ns
0.334
0.578
0.103
ns
0.665
0.816
0.007
**
0.175
0.418
0.263
ns
0.762
0.873
0.002
**
0.266
0.515
0.155
ns
0.499
0.706
0.033
*
0.252
0.502
0.168
ns
0.832
0.912
0.001
***
photoperiod does not influence the induction and progression
of endodormancy in Japanese pear (Takemura et al. 2011), as
in apple and European pear (Heide and Prestrud 2005), also
are in accordance with our assumption. In addition, extending the photoperiod with R light increased leaf number and
shoot length compared with the SD treatment (Figure 2). The
elongation patterns in these treatments were similar in that the
numbers (frequencies) of additional flushes were the same.
Thus, we infer that the stronger inductive effect of R light on
Tree Physiology Volume 34, 2014
shoot growth compared with the SD treatment is related to
the photosynthesis-promoting activity of R light (Wilkie et al.
2008) rather than to morphogenesis-inducing effects.
The R and FR lights are perceived and transduced by the PHY
family of photoreceptors (Franklin and Whitelam 2007). In our
2012 experiment, we showed that 730 nm FR light is the most
effective wavelength for stopping shoot growth and inducing
flower-bud formation (Figure 2 and Table 2). This result is in
accordance with the absorbance maximum of the FR-absorbing
form of PHY (705–740 nm of Avena PHY, Vierstra and Quail
1983). In Arabidopsis, five PHY genes (PHYA–PHYE) have been
identified (Sharrock and Quail 1989, Clack et al. 1994). PHYA is
involved in the promotion of flowering by FR, whereas PHYB is
involved in the inhibition of flowering by R (Mockler et al. 2003).
On the other hand, light-stable PHYB detects a low R : FR ratio,
and plants evoke a shade-avoidance response, including the
elongation of internodes/petioles and increased apical dominance, in addition to early flowering (Halliday et al. 2003, Salter
et al. 2003). Similar observations were seen in strawberry,
also a member of the Rosaceae family (e.g., Folta and Childers
2008 and literature therein), where flowering of the facultative
short-day (annual (once)-flowering) variety is inhibited with R
Light effects on pear shoot growth/flowering 543
light (Takeda et al. 2008) and that of the day-neutral or facultative long-day (everbearing (perpetual)) variety is induced
with 735 nm FR light (Yanagi et al. 2006). To date, no information is available on the exact number of PHY genes in the pear
genome and their respective roles, but the enhanced internode
elongation by FR irradiation may be one of the shade-avoidance
responses by FR irradiation mediated by PHYB. At this time, we
are unable to show whether flower induction is mediated via
PHYA or PHYB. Future research in strawberry as a model plant
of the Rosaceae family, in addition to pear, will give us further
insights into the light-detecting mechanism in these plants.
The promotive effect of flowering by flowering-inductive
photoperiod in long-day and short-day plants is accompanied by the upregulation of FT expression (e.g., Ishikawa et al.
2009, Hori et al. 2011, Sumitomo et al. 2012). We also found a
positive correlation between PpFT1a and PpMADS2-1a expression in shoot apices (Table 3), suggesting a significant role
of PpFT1a in pear flower induction. In addition, we assumed
that the negative correlation of PpFT2a expression with shoot
growth (RGRs of both shoot length and leaf number) may relate
to its promoting effect on flower induction, because flowerbud induction of pear plants occurs only after shoot elongation
ceases (Ito et al. 1999). In apple, two FT orthologues, namely
MdFT1 and MdFT2, have been identified. It is possible that
MdFT1 is involved in flower initiation, whereas MdFT2 functions in reproductive organ development (Kotoda et al. 2010).
Interestingly, two distinct roles for Populous FT orthologues are
reported (Böhlenius et al. 2006); one is responsible for reproductive onset in response to winter temperatures and the other
is responsible for vegetative growth in response to warm temperatures and long days (Hsu et al. 2011). Relationship analysis based on a phylogenetic tree constructed using deduced
amino-acid sequences demonstrated that PpFT1a and PpFT2a
were more closely related to the Rosaceae genes than those
of other taxa (unpublished data by T. Saito, S. Bai, A. Ito and T.
Moriguchi). The highest identity of 99% was observed between
PpFT1a and apple MdFT1, whereas PpFT2a was highly identical (99%) to apple MdFT2. Although our experiment lacks
direct evidence, we hypothesize that both FT orthologues in
Japanese pear may be involved in accelerating flower formation in a manner similar to apple FT orthologues. In addition,
we hypothesize that PpFT1a and PpFT2a expressed in leaves
may have less significant roles in flower induction in shoot apices in Japanese pear because (i) these FT orthologues were
expressed at lower levels in leaves than in shoot apices, and
(ii) FT protein is not transported from leaves to the apical meristem in apple (Tränkner et al. 2010).
A prominent role of PpTFL1-1a and PpTFL1-2a in the determination of seasonal flowering time may be suggested by their
high (negative) correlation with PpLFY2a and PpMADS2-1a
expression, respectively (Table 3). TFL1 is a key gene involved
in repression of flowering by preventing the expression of AP1
and LFY (Bradley et al. 1996, Ratcliffe et al. 1998, 1999, Boss
et al. 2004) by competing with FT for binding with FLOWERING
LOCUS D (FD) (Hanano and Goto 2011, McGarry and Ayre
2012). In apple, the TFL1-like gene MdTFL1 is assumed to be
involved in the seasonal regulation of flower induction (Kotoda
and Wada 2005, Kotoda et al. 2006, Hattasch et al. 2008,
Mimida et al. 2009, 2011b). Likewise, the perpetual flowering nature of strawberries and roses, also members of the
Rosaceae family, was ascribed to a mutation in TFL1 (Iwata
et al. 2012). In addition, TFL1 expression changes in response
to photoperiod and decreases under a flower-inducing short
photoperiod in a short-day accession of wild strawberry,
whereas TFL1 expression is activated under a non-inducible
long photoperiod (Koskela et al. 2012). We propose that FR
irradiation accelerated the flowering cascade by lowering the
expression of PpTFL1-1a/2a and led to flower induction in a
manner similar to what is observed in other Rosaceae plants.
In our experiment, concentrations of CKs and GAs in shoot
apices declined as the shoot matured (Figure 4); thus, we presume that their primary roles are related to the regulation of
shoot growth. Additional decreases in tZ and GA4 concentrations
in shoot apices were found when extending the photoperiod
with FR. While this finding may be related to the early flowering, the magnitude of the decreases in tZ and GA4 levels were
small in comparison to the decline in shoot development. Far-red
irradiation is reported to increase GA biosynthesis in elongating
petioles of Arabidopsis (Hisamatsu et al. 2005, Kurepin et al.
2007a, Mutasa-Gottgens and Hedden 2009), although the FR
effects on GA production in shoot meristems have not yet been
shown to our knowledge. As for CKs, FR irradiation promotes
CK catabolism in Stellaria longipes (Kurepin et al. 2007a), while
in Arabidopsis the breakdown is accompanied by up-regulation
of cytokinin oxidase genes (Carabelli et al. 2007). Increases in
TFL1 orthologue expression by applications of GAs and CKs were
reported in annual (once) flowering rose (Randoux et al. 2012)
and apple (Mimida et al. 2011a), respectively. Both reports are
in accordance with our results indicating that PpTFL1-1a and
PpTFL1-2a expression decreased in FR-irradiated shoot apices
with lower GA4 and tZ concentrations. However, in field-grown
apples and pears, GA applications inhibit flowering and treatments with CKs promote flowering in general (McLaughlin and
Greene 1984, Looney et al. 1985), although their effects vary
depending on the developmental stage (Ito et al. 2000, Wilkie
et al. 2008) and may relate to flower-bud development rather
than to flower initiation (Banno et al. 1985, Ito et al. 1999).
Further elucidation of the relationship between light, plant hormones and flower induction are needed in the future.
Conclusions
Extending the photoperiod with FR light led to early termination of
shoot elongation and early flower initiation by the terminal buds
Tree Physiology Online at http://www.treephys.oxfordjournals.org
544 Ito et al.
of Japanese pear. Expression of genes that regulate flowering
was affected by the light conditions, and multiple regression analyses suggested that TFL1 orthologues expressed in shoot apices
may have important roles in response to light conditions for the
promotion of flower induction. FLOWERING LOCUS T orthologues
may also have a significant role in the regulation of flower initiation but with a lesser magnitude than the TFL1 orthologues.
Extending the photoperiod with FR light also decreased the concentrations of CKs and GAs in shoot apices. We presume that
these changes in flowering-related genes and plant hormones
are involved in the early termination of shoot elongation and early
initiation of flowering induced by extending the photoperiod with
FR light, but this issue needs to be investigated further.
Supplementary data
Supplementary data are available at Tree Physiology online.
Acknowledgments
The authors thank Dr S. Kubota for helpful suggestions in
establishing protocols for plant hormone analyses and Ms N. Ito
for her technical assistance with molecular biological analyses.
Conflict of interest
None declared.
Funding
This work was partially supported by a Research Grant from
NARO Gender Equality Program and a Grant-In-Aid ‘Elucidation
of biological mechanisms of photoresponse and development
of advanced technologies utilizing light’ from the Ministry of
Agriculture, Forestry and Fishery, Japan.
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