J. AMER. SOC. HORT. SCI. 138(2):102–107. 2013.

Application of an Analog of 9, 10-ketoloctadecadienoic acid (KODA), Affected Flower Bud
Formation and MdTFL1 and MdFT1 Gene
Expressions in Apple Buds under Heavy-crop
and Shade Conditions
Monrudee Kittikorn, Katsuya Okawa, Hitoshi Ohara, and Satoru Kondo1
Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan
Nobuhiro Kotoda and Masato Wada
National Institute of Fruit Tree Science, Morioka 020-0123, Japan
Mineyuki Yokoyama and Ohji Ifuku
Technology and Engineering Center, Shiseido Co., Yokohama 236-8643, Japan
Ariake Murata and Naoharu Watanabe
Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan
ADDITIONAL INDEX WORDS. alternate bearing, flowering, gibberellic acid, juvenility, Malus domestica, reproductive phase,vegetative phase
ABSTRACT. Changes of endogenous 9, 10-ketol-octadecadienoic acid (KODA) concentrations, which is synthesizedfrom linolenic acid by 9-lipoxygenase, were analyzed in apple [Malus ·sylvestris (L.) Mill. var. domestica (Borkh.)
Mansf.] buds. In addition, the effects of 15, 16-chloro, hydroxy-9-hydroxy-10-oxo-12(Z)-octadecenoic acid (CKODA)
application, which is an analog of KODA, on flower bud formation and the expression of MdTFL1 (terminal flower 1)
and MdFT1 (flowering locus t 1) genes in apple buds were investigated in heavy-crop treatment (HCT) and undershade. An increase of endogenous KODA in the buds in the fruit-thinning treatment, which resulted in a higher
proportion of flower bud formation than in HCT, was observed at 63 days after full bloom, but no such increase wasfound in HCT. In the shade-treated and heavy-crop trees, the expression of MdTFL1 in the buds to which CKODA wasapplied was lower than that in untreated buds. In contrast, under shade, the expression of MdFT1 in the CKODAtreated buds was higher than that in untreated buds. These results suggest that endogenous KODA may be associatedwith flower bud formation, and its application may be effective at improving the proportion of flower bud formation
through its effect on MdTFL1 and MdFT1.

Most fruit trees such as apple trees have a long vegetative
period, which is undesirable for growers (Foster et al., 2003;
Mimida et al., 2009). In the adult phase, alternate bearing is
induced by growing conditions such as heavy fruit load thatis associated with gibberellins (Ramirez et al., 2001, 2004).
Therefore, the clarification of the physiological mechanism forregulation of flower bud formation is indispensable for stableapple production.
Environmental conditions such as light intensity also influence floral initiation in apple. For instance, low irradiancehas been shown to inhibit floral initiation in apical buds of‘Fuji’ apple (Kittikorn et al., 2011). The flower inductions of
duckweed (Lemna paucicostata L.) and japanese morning glory(Pharbitis nil Choisy) were shown to be associated with KODA
levels (Suzuki et al., 2003; Yokoyama et al., 2000). Furthermore, it was shown that endogenous KODA concentrationsin apical buds were higher in trees with a higher flower bud
formation, and that KODA spraying of one-year-old shoots
Received for publication 21 Dec. 2012. Accepted for publication 4 Feb. 2013.
We thank Dr. Haruo Shimada, Shiseido Co., for help in a determination of
structure of CKODA used in this work.
1
Corresponding author. E-mail: s-kondo@faculty.chiba-u.jp.

102

increased the proportion of flower bud formation in trees withnormal fruit load [40 leaves per fruit (Kittikorn et al., 2010,2011)]. These facts show that KODA may be associated withflower bud formation in apple.
Studies on the molecular mechanisms of flowering havebeen well established in arabidopsis [Arabidopsis thaliana (L.)
Heynh. (Bradley et al., 1997)]. Numerous floral meristem
identity genes have been isolated from arabidopsis, and several
models for the genetic pathways of floral induction anddifferentiation such as FT, suppressor of constans 1 (SOC1),
leafy (LFY), apetala 1 (AP1), and TFL1 have been reported(Henderson and Dean, 2004; Kobayashi et al., 1999; Ratcliffe
et al., 1998). Overexpression of LFY, FT, or AP1 has beenshown to shorten the vegetative phase, causing early flowering(Kardailsky et al., 1999), whereas overexpression of TFL1 has
been shown to cause late flowering in transgenic arabidopsis(Ratcliffe et al., 1998). TFL1 is a key gene involved in repression
flowering and maintains the identity of inflorescence meristems
by preventing the expression of AP1 and LFY (Ratcliffe et al.,1998). Arabidopsis plants in which the MdTFL1 gene wastransferred showed delayed flowering (Kotoda and Wada, 2005),
but ‘Orin’ apple plants with transferred antisense MdTFL1 RNA
flowered after eight months compared with the untreated control
J. AMER. SOC. HORT. SCI. 138(2):102–107. 2013.

plants, which took six years to flower. In addition, MdTFL1 wasdetected in vegetative tissues such as apical buds, stems, andcotyledon, but not in reproductive tissues such as floral organs(sepal, petal, and stamen), suggesting its key role in maintaining the juvenile and/or vegetative phase in apple (Kotoda et al.,2006). Contrary to the functional role of TFL1, FT promoted
flowering by altering the expression of other endogenous genes(Esumi et al., 2007; Kardailsky et al., 1999). Invariably, overexpression of FT-like genes has led to premature flowering under
non-inductive conditions in species such as arabidopsis (Abeet al., 2005), rice [Oriza sativa L. (Tamaki et al., 2007)], tomato[Solanum lycopersicum L. (Lifschitz et al., 2006)], and grape[Vitis vinifera (Carmona et al., 2007)]. KODA application
decreased MdTFL1 expression in treated buds (Kittikorn et al.,2011). Therefore, it is possible that KODA application induces
flower bud formation in apple through an effect on MdTFL1transcription. An investigation of the relationship among KODA,
MdTFL1, and MdFT1 under various growing conditions may
clarify the roles of KODA. However, the effect of KODA application under non-inductive conditions has not been examined.
We have found that CKODA (Fig. 1), an analog of KODA,
was more stable than KODA at high temperatures (Kondo et al.,2012). This study examined the effect of CKODA applicationon the transcription of MdTFL1 and MdFT1 and flower bud
formation in apple under heavy cropload and shade conditions.

(three replications of 30 buds per tree) were randomly
collected for analysis of endogenous KODA from eachtreatment. The sampled buds were from non-fruiting spurs ineach treatment.
ANALYSIS OF ENDOGENOUS KODA. The buds, including
whole apical tissues (100 mg fresh weight per 30 buds in one
tree from each treatment), were ground in liquid nitrogenand extracted with methanol (10 mL) using [U-13C] KODA
(1 pmolmL–1) as an internal standard at 4 C. The filtrate wasconcentrated in vacuo and the concentrates were re-dissolved inmethanol (5 mL) and washed with hexane (10 mL) to remove
pigments. The methanol layer thus obtained was evaporated invacuo to yield solid materials. The resultant solid materials
were re-dissolved in 100 mL ethanol, and 5 mL of the solution
was subjected to high-performance liquid chromatographymass spectrometry (HPLC-MS/MS) (LTQ-Orbitrap Discovery;
Thermo Fisher Scientific, Bremen, Germany) equipped with aCapcell Pak C18 UG 120 column (2 mm i.d. · 7.5 cm; Shiseido,
Tokyo, Japan) that was maintained at 4 C . The samples wereanalyzed at a flow rate of 0.2 mLmin–1 using a chromatographic
gradient of two mobile phases (A = 0.05% aqueous formic acid,B = acetonitrile). Solvent B was linearly increased from 15% to100% over 15 min.
The MS conditions were set as follows: ionization mode,
electron spray ionization (negative); capillary temperature,
330 C; capillary voltage, –27.50 V; collision energy, 35.0 eV;
and full mass range, m/z 50 to m/z 350. MS2 values wereMaterials and Methods
acquired from a parent ion at m/z 309 [M-H]– for KODA with
PLANT MATERIALS. Experiments were conducted in 2009 and a scan range of m/z 85 to m/z 310. The MS2 spectra of [U-13C]2010 on ‘Fuji’ apple trees grafted onto ‘Marubakaido’ root- KODA were determined based on a selection reaction monstock (Malus prunifolia Borkh. var. ringo Asami) growing in an itoring an ion at m/z 327 [M-H]– with a scan range of m/z 263.5
open field of Chiba University located at lat. 36 N and long. to m/z 264.5. [U-13C]KODA was eluted at tR = 10.47 min with
an ion peak at m/z 327 [M-H]–. The MS2 spectrum from the ion
139 E at an altitude of 747 m.
EXPT. 1: EFFECT OF CROP LOAD ON THE CHANGES OF ENDOGENOUS at m/z 327 exhibited ion peaks at m/z 309 (100%) [M-H2O]–
KODA. Two groups of three 32-year-old trees (three replica- and m/z 264 (68.0%) [M-H2O-H13COO]–. The recovery oftions of one tree per each treatment) were created randomly in KODA was estimated based on the ion intensity at m/z 309.
2009: one that underwent flower thinning treatment (FTT), in Similarly, KODA was detected at tR = 10.40 min with an ion
which all flowers were removed at full bloom, and one that peak at m/z 309. The MS2 spectrum from the ion at m/z 309
underwent HCT, in which the number of leaves per fruit was showed ion peaks at m/z 291 (100%) [M-H2O] and m/z 247
adjusted to 20 at 20 d after full bloom (DAFB). At 21, 49, (72.3%) [M-H2O-HCOO]–. Although 13-a-ketol had the same
63, and 103 DAFB, 90 terminal buds from shoot meristems retention time and ion peak at m/z 309 [M-H]– as those of
KODA, the MS2 spectrum of 13-a-ketol wasrecorded with ion peaks at m/z 291 (100%)[M-H2O]– and m/z 247 (3.1%) [M-H2OHCOO]–. Furthermore, the ionization efficacy for m/z 291 of 13-a-ketol was 3.56 times
higher than that of KODA. Quantification of
KODA was based on the ion intensities of m/z
247 and m/z 291 of KODA and 13-a-ketol.
EXPT. 2: EFFECT OF CKODA APPLICATIONON THE EXPRESSION OF MDTFL1 AND MDFT1
GENES UNDER HEAVY-CROP AND SHADE CONDITIONS.
In the 2010 HCT, four 33-year-old trees wererandomly selected. The number of leaves
per fruit was adjusted to 20 at 20 DAFB asin Expt. 1. One hundred forty one-year-old
shoots (560 apical buds in four trees), also
randomly selected at 7 DAFB, in each tree
were sprayed with 100 mM CKODA withÒ
Fig. 1. Structure of 9, 10-ketol-octadecadienoic acid (KODA) and 15, 16-chloro, hydroxyl-9-hydroxy- surfactant 0.1% (v/v) (New Osmac ; Hayashi
10-oxo-12(Z)-octadecenoic acid (CKODA). CKODA was formed by introducing a chlorine atom Chemical, Tokyo, Japan) at 7, 21, 35, and
49 DAFB (HCT+CKODA+). On each day ofand a hydroxyl group at the C-15 olefin of KODA.
J. AMER. SOC. HORT. SCI. 138(2):102–107. 2013.

103

treatment, the tips of the shoots with Table 1. Gene-specific primer sets used for quantitative real-time reverse transcription–polymerase
chain reaction analyses of MdTFL1 and MdFT1 in apical buds of ‘Fuji’ apple.
three nodes were sprayed once until
runoff. The untreated controls Gene
Forward primer (5#–3#)
Reverse primer (5#–3#)(HCT+CKODA–) were one-year-old MdTFL1
TGTTGTCACAGCCAAACCTAG
ACAATCCAGTGCAGGTGCTCC
shoots that were not sprayed with MdFT1
CTTGTTGTTGGACGAGTGG
GAGTGTAGAAAGTCCTGAGATCKODA. At 7, 21, 35, 49, 63, 77, Actin
TTCAGGCTGTTCTTTCTCTATATGC
GACTTCTGGACAACGGAATCTC
and 96 DAFB, 90 apical buds (18buds per each tree) were randomly
sampled. Apical buds were frozen immediately in liquid nitrogen were diluted (1:10 v/v) and amplified using the gene-specificand then stored at –80 C until analysis of MdTFL1 and MdFT1 primers shown in Table 1. The quantitative reverse transcription–
gene expression.
polymerase chain reaction (qRT-PCR) was performed using the
In the shade treatment, three randomly selected 19-year-old SYBR master mix (KAPA SYBRÒ FAST qPCR kit; NIPPON
trees were covered with cheesecloth (50% shading) at 20 Genetics, Tokyo, Japan) with ABI StepOnePlus (Applied BioDAFB. The number of leaves per fruit was adjusted to 40 at systems, Foster City, CA). The steps involved in amplification
20 DAFB. This number of leaves per fruit is the usual fruit were performed as follows: an initial denaturation at 95 C forload not causing alternate bearing. One hundred twenty one- 10 s followed by 40 cycles of amplification at 95 C for 3 s andyear-old shoots in each tree (360 apical buds in three trees), also annealing at 60 C for 32 s. The specificity of the amplification
randomly selected at 7 DAFB, were sprayed with 100 mM was verified on the basis of a melting curve obtained at the end
CKODA, like in the crop load experiment (Shade+CKODA+). of the PCR cycle. The relative expression level was normalAt 7, 21, 35, 49, 63, and 77 DAFB, 60 apical buds (20 buds ized to that of the actin gene (Yooyongwech et al., 2008).
per tree) were randomly sampled for analysis of the expresSTATISTICAL ANALYSIS. The SAS analysis of variance prosion of MdTFL1 and MdFT1 genes. The untreated controls cedure (Version 8.02; SAS Institute, Cary, NC) was used to(Shade+CKODA–) were one-year-old shoots that were not determine the treatment effects, and the mean separation was
sprayed with CKODA.
analyzed by Fisher’s least significant difference (P # 0.05). The
To determine the proportion of flower bud formation in the data are presented as the mean values of three replications ± SE.
cropload and shade experiments, 90 apical buds (three replications of 30 buds per tree) were randomly collected after
Results
defoliation. Each apical bud was longitudinally dissected, andThe proportion of flower bud formation of FTT was
the proportion of flower bud formation was determined witha stereomicroscope. Buds that apical meristems showed to be significantly higher than that of HCT (Table 2). A significant
dome-shaped were counted as flower buds according to a pre- increase in endogenous KODA in FTT was observed at 63 andvious report (Kittikorn et al., 2010). The proportion of flower 103 DAFB, although there was no significant difference ofbud formation was calculated by the number of flower buds in KODA levels at 21 and 49 DAFB between HCT and FTT(Fig. 2). In HCT trees, endogenous KODA levels did not show
90 apical buds.
PREPARATION OF CKODA. CKODA (C18H31ClO5) was significant changes from 21 to 103 DAFB.
formed by introducing a chlorine atom and a hydroxyl group
The relative expression levels of MdTFL1 in HCT+CKODA+at the C-15 olefin of KODA. KODA (32 mM) of 250 mL was were significantly lower than those in HCT+CKODA– at 21, 35,
mixed with 690 mL distilled water followed by the addition of and 49 DAFB (Fig. 3A). In both treatments, the expression of
60 mL dilute aqueous solution [10% of commercial sodium
hypochlorite solution (active chlorine 8.5% to 13.5%; Nacalai
Tesque, Kyoto, Japan)] and vigorously mixed. The conver- Table 2. Proportion of flower bud formation in apical buds of ‘Fuji’sion rate of CKODA from KODA was more than 60%.
apple treated with 15, 16-chloro, hydroxyl-9-hydroxy-10-oxoCKODA was extracted by ethyl acetate and isolated by HPLC
12(Z)-octadecenoic acid (CKODA) under heavy-crop treatment(NANOSPACE SI-1; Shiseido) using an ultraviolet detector(HCT) or shade.
at 210 nm with a Capcell Pak C18 column. The mobile phase
Proportion of flower bud formation
was 50% acetonitrile in 0.05% trifluoroacetic acid, and the Treatmentz[mean ± SE (%)]
column temperature and flow rate were 30 C and 1 mLmin–1, HCT+CKODA–
60.3 ± 0.58y
respectively. The retention time of CKODA was 5.4 min. The HCT+CKODA+
67.6 ± 0.60
product was identified with HPLC-MS/MS. The fragment FTT
81.3 ± 3.84
ions of CKODA were m/z 363 [M-H]– and 345 [M-H-H2O]–. LSD
2.30
0.05
RNA EXTRACTION, CDNA SYNTHESIS, AND QUANTITATIVE REAL22.0 ± 2.16
TIME RT-PCR. Total RNA from apical buds was extracted
Shade+CKODA–
61.6 ± 0.74
following the protocol mentioned in a previous report (Kittikorn Shade+CKODA+
6.35
et al., 2010). DNase treatment (5 units; Takara Bio, Otsu, Japan) LSD
0.05
at 37 C for 20 min was applied to 2 mg of total RNA to z
eliminate any contaminating DNA in the samples. The re- HCT = number of leaves per fruit was adjusted to 20 at 20 d after fullbloom (DAFB). Shade = trees covered with cheesecloth (50% shading)action was stopped and then incubated at 70 C for 10 min.
at 20 DAFB; the number of leaves per fruit was adjusted to 40 at 20
The treated RNA was used as a template for cDNA synthesis. DAFB. FTT = flower thinning treatment, in which all flowers were
The cDNA was synthesized in a 20-mL reaction volume using removed at full bloom.
ReverTraAce (Toyobo, Osaka, Japan) according to the man- yThe proportion of flower bud formation was calculated by the number
ufacturer’s instructions. RNA extraction and cDNA synthesis of flower buds in 90 apical buds. Mean separation was analyzed bywere repeated twice on each sampling date. The cDNA samples Fisher’s least significant difference (LSD) (P# 0.05).
104

J. AMER. SOC. HORT. SCI. 138(2):102–107. 2013.

Fig. 2. Changes in endogenous 9, 10-ketol-octadecadienoic acid (KODA) concentrations in the apical buds of ‘Fuji’ apple subjected to the flower thinning
treatment (FTT), in which all flowers were removed at full bloom and heavycrop treatment (HCT), in which the number of leaves per fruit was adjusted
to 20 at 20 d after full bloom. Data are mean ± SE of three replications.

MdTFL1 decreased sharply after 63 DAFB and leveled off at
77 DAFB in HCT+CKODA+ and HCT+CKODA– at 98 DAFB.
The expression patterns of MdFT1 were similar in both CKODAtreated and -untreated buds under HCT conditions, although thetimes of the increases and decreases differed (Fig. 3B). That is,
the expression of MdFT1 in HCT+CKODA– increased toward
49 DAFB and then decreased at 63 DAFB, whereas that in
HCT+CKODA+ increased toward 35 DAFB and then decreased at 49 DAFB. The proportion of flower bud formationin HCT +CKODA + was significantly higher than that in
HCT+CKODA– (Table 2).
In the shade treatment, the application of CKODA significantly increased the proportion of flower bud formation compared with that of untreated control (Table 2). The expression ofFig. 3. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis of (A) MdTFL1 and (B) MdFT1 in apical buds of‘Fuji’ apple treated with 15, 16-chloro, hydroxyl-9-hydroxy-10-oxo-12(Z)octadecenoic acid (CKODA) under heavy-crop treatment (HCT), in which thenumber of leaves per fruit was adjusted to 20 at 20 d after full bloom. The
quantitative RT-PCR was performed using the SYBR master mix. The steps
involved in amplification were performed as follows: an initial denaturation at
95 C for 10 s followed by 40 cycles of amplification at 95 C for 3 s andannealing at 60 C for 32 s. The relative expression level was normalized tothat of the actin gene. Data are mean ± SE of three replications.

J. AMER. SOC. HORT. SCI. 138(2):102–107. 2013.

MdTFL1 genes at 7, 21, and 35 DAFB did not differ significantly between Shade+CKODA+ and Shade+CKODA–, but theexpression of MdTFL1 in Shade+CKODA– was higher thanthat in Shade+CKODA+ at 49, 63, and 77 DAFB (Fig. 4A). In
contrast, the expression in Shade+CKODA+ did not show muchchange.
In general, the patterns of change of MdFT1 expression inboth CKODA-treated and -untreated buds were similar undershade conditions (Fig. 4B). The MdFT1 expressions increased
toward 35 DAFB and then decreased at 49 DAFB. However, theexpressions of MdFT1 in Shade+CKODA+ were significantly
higher than those in Shade+CKODA–.
Discussion
In a previous report, we reported the changes of endogenous
KODA in apple buds with 15-hydroxypentadecanoic acid as
the internal standard using a fluorescence detector in HPLC
(Kittikorn et al., 2010). In the current study, endogenous KODA
was analyzed using liquid chromatography–MS/MS for moreaccurate determination. The endogenous KODA concentrations
in FTT were significantly higher than those of HCT. Furthermore, these concentrations increased significantly at 63 DAFB
and before flower bud formation, as shown in a previous report(Kittikorn et al., 2010). This fact suggests that endogenous
KODA may play a positive role in flower bud formation inapple. Either endogenous gibberellins (GAs) from the seeds or
GA spray is known to inhibit flowering in apple (Ramirez et al.,2001, 2004). Therefore, it is possible that low endogenous
KODA concentrations in the buds of HCT are associated with

Fig. 4. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis of (A) MdTFL1 and (B) MdFT1 in apical buds of‘Fuji’ apple treated with 15, 16-chloro, hydroxyl-9-hydroxy-10-oxo-12(Z)octadecenoic acid (CKODA) under shade, in which the trees were covered
with cheesecloth (50% shading) at 20 d after full bloom (DAFB). The numberof leaves per fruit was adjusted to 40 at 20 DAFB. The quantitative RT-PCR
was performed using the SYBR master mix. The steps involved in amplification were performed as follows: an initial denaturation at 95 C for 10 sfollowed by 40 cycles of amplification at 95 C for 3 s and annealing at 60 C
for 32 s. The relative expression level was normalized to that of the actin gene.
Data are mean ± SE of three replications.

105

high GA concentrations compared with those in FTT (Kittikorn
et al., 2010). However, KODA application to the buds did not
influence endogenous GA concentrations in the buds (Kittikorn
et al., 2011). Therefore, the relationship between auxin andcytokinin, which influence flower bud formation (Sanyal andBangerth, 1998), and KODA should also be examined. In addition,
the origin of endogenous KODA in the buds of apple requires
further investigation.
It has been shown that FT and TFL1 homologs are importantin regulating the flowering time and maintaining the inflorescence meristem in several plant species (Hanke et al., 2007).
A homolog of the FT and TFL1 gene family has been isolated
and characterized in fruit trees such as apple (Kotoda andWada, 2005; Kotoda et al., 2006), grape (Carmona et al., 2007),sweet orange [Citrus sinensis L. Osbeck (Pillitteri et al., 2004)],
and pear and quince [Pyrus pyrifolia Nakai and Cydonia
oblonga Mill. (Esumi et al., 2005)]. MdTFL1 gene in apple,
which is a TFL1-like gene, functions equivalently to TFL1
expressed constitutively in transgenic arabidopsis plants, suggesting that it has the potential to complement the TFL1
function (Kotoda et al., 2006). It could function redundantly
as a flowering repressor and a regulator of vegetative meristem
identity because MdTFL1 is detected strongly in vegetative
tissues (Mimida et al., 2009). Our previous report (Kittikorn
et al., 2011) showed that the accumulation of MdTFL1 mRNAtranscript levels was found in buds under both heavy-crop andshade treatments that related to the decrease of flower bud
formation. The proportion of flower bud formation in apicalbuds of apple increased after 70 DAFB (Kittikorn et al., 2010).
In our study, CKODA application in HCT decreased MdTFL1mRNA transcript levels at 21, 35, and 49 DAFB, although theexpression of MdTFL1 in HCT+CKODA– decreased sharply
after 63 DAFB. It has been shown that MdTFL1 mRNA wasexpressed strongly in June, which is 7 weeks after full bloom,
which occurs 1 to 2 weeks after the initiation of floral bud
formation; from June, the expression decreased gradually
toward late July (Kotoda and Wada, 2005). In our study, theproportion of flower bud formation in Shade+CKODA– waslower than that in HCT+CKODA–. In addition, the expression of MdTFL1 in Shade+CKODA– was higher than that in
HCT+CKODA– at 63 DAFB. Although the MdTFL1 expression
in Shade+CKODA– decreased at 77 DAFB, the expression level
was significantly higher than that of Shade+CKODA+. These
results support that the induction of flower bud formation andfloral development may be coincident with the interruption of
MdTFL1 expression (Kotoda and Wada, 2005; Mimida et al.,2009). Furthermore, MdTFL1 expression in our study showsthat low light intensity may have a more negative effect on
flower bud formation than a heavy cropload.
Contrary to the functional role of TFL1, FT promoted
flowering by altering the expression of other endogenous genes(Esumi et al., 2007; Kardailsky et al., 1999). The mobile FT
protein has been identified as a main component of the florigen
or flowering hormone in arabidopsis, tomato, and rice (Lifschitz
et al., 2006; Turck et al., 2008; Zeevaart, 2008). In apple, MdFT1
and MdFT2 genes have been isolated and their functions
clarified (Kotoda and Wada, 2005; Kotoda et al., 2010). A
transgenic experiment revealed that both MdFT1 and MdFT2
had the potential to function as floral promoters in apple.
However, the transcriptional activation of MdFT1 is more closely
associated with the regulation of flower induction in apple thanthat of MdFT2. Therefore, we examined the transcription
106

level of MdFT1. In our study, the expression of MdFT1 in
HCT+CKODA+ reached a peak earlier and higher than in
HCT+CKODA–. It has been reported that the increase of
MdFT1 (Hättasch et al., 2009; Kotoda et al., 2010) and AFL1
and AFL2 transcription levels (apple orthologs of LFY) (Wadaet al., 2002) in the apical meristems of one-year-old apple shootsbegan 1 to 2 weeks earlier than flower initiation. In our study,
MdFT1 expression levels in HCT+CKODA– were higher thanthose in Shade+CKODA– at 21 to 49 DAFB. The expressions of
MdFT1 were increased earlier by CKODA application in both
HCT and shade treatment. The increase of the proportion of
flower bud formation at 70 DAFB (Kittikorn et al., 2010), which
did not occur after the increase of MdFT1 but did occur after
the decrease of MdTFL1 expression, suggests that the role of
MdTFL1 may be downstream of MdFT1. It was demonstrated
that the dynamic state of MdFT1 and MdTFL1 genes observedin our study may be associated with flower bud formation.
In summary, endogenous KODA in the buds of trees inwhich the proportion of flower bud formation was high increased before the initiation of flower bud formation. In heavycrop and shade-treated trees, the application of CKODA, aderivative of KODA, increased the proportion of flower bud
formation and the expression of MdFT1 but decreased theexpression of MdTFL1.
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