Plant Growth Regul (2011) 64:75–81
DOI 10.1007/s10725-010-9538-x

ORIGINAL PAPER

Effects of fruit load, shading, and 9,10-ketol-octadecadienoic acid
(KODA) application on MdTFL1 and MdFT1 genes in apple buds
M. Kittikorn • K. Okawa • H. Ohara •N. Kotoda • M. Wada • M. Yokoyama •O. Ifuku • S. Yoshida • S. Kondo

Received: 8 June 2010 / Accepted: 18 October 2010 / Published online: 3 November 2010
Ó Springer Science+Business Media B.V. 2010

Abstract The effects of fruit load, shading, and 9, 10ketol-octadecadienoic acid (KODA) application on theexpression of MdTFL1 and MdFT1 genes were investigatedin apples (Malus domestica Borkh.). The expression of
MdTFL1 in apical buds from 21 to 63 days after full bloom(DAFB) in plants subjected to heavy crop treatment (HCT)
was higher than that in plants subjected to flower thinning
treatment (FTT). In contrast, the expression of MdFT1 did
not show a clear difference between HCT and FTT. The
shading treatment increased the expression of MdTFL1 at
35, 49, and 80 DAFB. However, MdFT1 did not show aclear difference between shading and non-shading treatments. KODA application decreased the expression of
MdTFL1 at 49 DAFB, but it did not have a clear effect on
the expression of MdFT1 from 21 to 91 DAFB. KODA
application did not influence endogenous gibberellic acid(GA) concentrations in apical buds. These results show that
KODA may be related to flower bud formation through its
influence on MdTFL1. The relationship between KODA
This research was supported by the Research and Development
Program for New Bio-industry Initiatives.
M. Kittikorn  K. Okawa  H. Ohara  S. Kondo (&)
Graduate School of Horticulture, Chiba University, Matsudo,
Chiba 271-8510, Japane-mail: s-kondo@faculty.chiba-u.jp
N. Kotoda  M. Wada
National Institute of Fruit Tree Science, Shimo-kuriyagawa,Morioka 020-0123, Japan
M. Yokoyama  O. Ifuku
Innovative Science Research and Development Center,
Shiseido Co, Yokohama, Kanagawa 236-8643, Japan
S. Yoshida
Plant Science Center, RIKEN, Yokohama 230-0045, Japan

and GA with regard to the flower bud formation of apples
was also discussed.
Keywords 9,10-Ketol-octadecadienoic acid (KODA) 
Gibberellic acid (GA)  Malus domestica  MdTFL1 
MdFT1Introduction
Flower bud formation is one of the most important physiological processes in higher plants, and flowering time iscontrolled by photoperiod, temperature, endogenous signals, and other factors. Many fruit trees have an extended
juvenile phase. For example, the juvenile period can last
2–10 years for citrus (Citrus sinensis L.), 5–8 years forapple (Malus domestica Borkh.), and even longer for pear
trees (Pyrus communis L) before these trees start to flower(Ryugo 1986). This characteristic is the primary factor that
limits the efficient breeding of fruit trees. Recently, it was
shown that 9,10-ketol-octadecadienoic acid (KODA) isrelated to flower induction of Lemna paucicostata andPharbitis nil (Yokoyama et al. 2000; Suzuki et al. 2003).
The transient elevation of the endogenous KODA level
during a dark period is also related to flower induction(Suzuki et al. 2003; Yokoyama et al. 2005). KODA isformed from 9-hydroxy-10-oxo-12 (Z), 15(Z)-octadecadienoic acid (9-HPOT) and is generated from linolenic acidby 9-lipoxygenase (9-LOX) (Fig. 1). The presence ofendogenous gibberellic acid (GA) from the seeds as well as
the application of GA are known to inhibit flowering inapples (Ramı́rez et al. 2004). In contrast to GA, endogenous KODA concentrations were high in the apical buds oftrees whose rate of flower bud formation was also high

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Plant Growth Regul (2011) 64:75–81

Fig. 1 The pathway for KODA
biosynthesis in higher plants

COOH

α-linolenic acid (LA)13-LOX

9-LOX

lipoxygenase (LOX)

COOH

COOH
OOH

OOH

13-hydroperoxy-LA (13-HPOT)

9-hydroperoxy-LA (9-HPOT)

allene oxide synthase (AOS)

COOH

COOH

O

O

12,13-allene oxide

9,10-allene oxide

allene oxide cyclase12-oxo-PDA
β-oxidation

COOH
O OH

9,10-ketol-octadecadienoic acid (KODA)

O

COOH
COOH

Jasmonic acid

(Kittikorn et al. 2010). Therefore, KODA may be associated with flower bud formation in apples.
Studies have established the molecular mechanisms offlowering in Arabidopsis (Bradley et al. 1997) and somefruit trees (Carmona et al. 2007; Esumi et al. 2007). In
Arabidopsis, genes such as FLOWERING LOCUS T (FT),SUPPRESSOR OF CONSTANS 1 (SOC1), LEAFY (LFY),APETALA 1 (AP1), and TERMINAL FLOWER 1 (TFL1)
play central roles in determining the inflorescence architecture (Ratcliffe et al. 1998; Henderson and Dean 2004).
TFL1 gene expression repressed flowering and maintained
the identity of inflorescence meristems by preventing theexpression of AP1 and LFY (Ratcliffe et al. 1998, 1999).
Over-expression of MdTFL1 generated late flowering
phenotypes in apples (Kotoda and Wada 2005). In addition,
MdTFL1 was detected in vegetative tissues such as apicalbuds, stems, and cotyledons, but not in reproductive tissues
such as floral organs (sepals, petals, and stamens), suggesting a key role in maintaining the juvenile and/or
vegetative phase in apples (Kotoda et al. 2006). Contrary to
the functional role of TFL1, FT1 promoted floweringby altering the expression of other endogenous genes(Kardailsky et al. 1999; Esumi et al. 2007). Environmental
factors such as fruit load and shading influence flower bud
formation in apples (Rom 1996; Kittikorn et al. 2010).
However, there is no report on the relationship between
environmental factors and the expression of genes that areassociated with flower bud formation. Also, it is unclear
whether KODA’s effect on these genes is related to flower
bud formation.

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OH O

12,13-α-ketol-LA

In the present study, we examined the effects of fruit
load, shading, and KODA application on the expression of
MdTFL1 and MdFT1 genes in apples.

Materials and methods
Chemicals
KODA (9,10-ketol-octadecadienoic acid) was prepared as
described previously (Kittikorn et al. 2010). New OsmacÒ
was purchased from Hayashi Chemical Co., Tokyo, Japan.
[2H0] GA1, [2H0] GA4, [2H2] GA1, and [2H2] GA4 werepurchased from the Research School of Chemistry ofAustralian National University (Canberra, Australia).
Plant materials
Experiments were conducted using ‘Fuji’ apple trees
grafted onto Malus prunitoria (Willd.) Borkh. rootstocks
growing in an open field at Chiba University, which islocated at 36°N, lat., 139°E, long., and at an alti. of 747 m.
Experiment 1: Effect of fruit load on the expressionof MdTFL1 and MdFT1 genes
Two groups of three 32-year-old trees were selected randomly: one that underwent flower thinning treatment(FTT), in which all flowers were removed at full bloom,
and one that underwent heavy crop treatment (HCT), inPlant Growth Regul (2011) 64:75–81

which the number of leaves per fruit was adjusted to 20 at
20 days after full bloom (DAFB). At 21, 49, 63, and 103
DAFB, ninety apical buds (30 buds per tree) were randomly collected for analysis of the expression of MdTFL1
and MdFT1 genes.
Experiment 2: Effect of shading on the expressionof MdTFL1 and MdFT1 genes
Three 18-year-old trees, randomly selected, were covered
with cheesecloth (50% shading) at 20 DAFB. The
untreated control was three non-shaded trees, also randomly selected. The number of leaves per fruit was
adjusted to 40 at 20 DAFB. This number of leaves per fruitis usual fruit load not causing biennial bearing. At 35, 49,
and 80 DAFB, ninety apical buds (30 buds per each tree)
were randomly collected for analysis of the expression of
MdTFL1 and MdFT1 genes.
Experiment 3: Effect of KODA applicationon the expression of MdTFL1 and MdFT1 genes,
and on the GA concentrations
Three 32-year-old trees were randomly selected. One-yearold shoots of 240 (720 apical buds in three trees), also
randomly selected at 7 DAFB, in each tree were sprayedwith 100 lM KODA with surfactant New Osmac at 7, 21,
35, and 49 DAFB. On each day of treatment, the tips of theshoots were sprayed once until runoff. The untreated controls were one-year-old shoots that were not sprayed with
KODA. The number of leaves per fruit was adjusted to 40
at 20 DAFB. At 7, 21, 35, 49, 63, and 91 DAFB, 120 apicalbuds (40 buds per tree) were randomly collected for analysis of the expression of MdTFL1 and MdFT1 genes and
GA concentrations.
To determine the rate of flower bud formation inexperiments 1, 2, and 3, we randomly collected 90 apicalbuds (three replications of 30 buds per tree) after defoliation. Each apical bud was longitudinally dissected, and therate of flower bud formation was determined with a stereomicroscope. In accordance with a previously reported
method (Kittikorn et al. 2010), buds in which the apicalmeristem was dome-shaped were counted as flower buds.
The rate of flower bud formation was decided by the
number of flower buds in 90 apical buds.
RNA extraction and cDNA synthesis
Total RNA from apical buds was extracted following theprotocol described by Kotoda et al. (2000). A 2-lg aliquotof total RNA of apical buds at several developmental
stages was treated with 5U of DNase I (Takara Bio,
Otsu, Japan) and used for first-strand cDNA synthesis.

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For reverse transcription-polymerase chain reaction(RT–PCR), cDNA was synthesized in 20-ll reaction
volume using ReverTraAce (Toyobo Co., Osaka, Japan)
according to the manufacturer’s instructions. RNA
extraction and cDNA synthesis were performed three times
on each sampling date. The apple cDNAs of MdTFL1 andMdFT1 used in this study were deposited in the DNA databank of Japan (DDBJ) with accession numbers AB052994
and AB161112, respectively (Kotoda and Wada 2005).
Analysis of gene expression in apical buds
RT–PCR analysis was performed by amplifying 1 lL of
cDNA with 15 pmol of specific primers for MdTFL1 (50 CTCTTAAAATGAAAAGAGCCTCGG-30 , forward; 50 TGTGGCATACATTGTAAATA-30 , reverse) and MdFT1(50 -ATGCCTAGGGATAGGGACCCC-30 , forward; 50 TTATCTTCTCCTTCCACCGGA-30 , reverse) (Kotoda andWada 2005). These primers amplified fragments of 699 and530 bp, respectively. The PCR conditions were: 35 cycles
at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s for
MdFT1, and 40 cycles at 94°C for 30 s, 50°C for 30 s, and72°C for 60 s for MdTFL1. As a positive control, theextracted RNA was confirmed by amplification of ACTIN
homologs with 35 cycles of RT–PCR (data not shown)(Gasic et al. 2004). For each sample, 5 lL of the amplification reaction was size-fractionated onto 1% agarose gel
and stained with ethidium bromide. Transcript levels of
MdFT1 and MdTFL1 in the samples were compareddirectly against an ACTIN homolog control by using theNIH Image Program (http://rsb.info.nih.gov/nih-image/).
The entire amplification process was replicated three times
with total RNA from other sample tissues, to evaluate theexpression of each gene.
Analysis of endogenous GAs
Endogenous gibberellins were extracted and quantified
according to the procedure described by Kittikorn et al.
(2010). As an internal standard, 200 ng each of [2H2] GA1
and [2H2] GA4 was added to the 80% MeOH extracts. GA1
and GA4 containing fractions from analytical reverse-phasehigh performance liquid chromatography (HPLC) were
methylated with ethereal diazomethane and trimethylsilylated with Deriva-sil (20 ll; Chrompak, Raritan, NJ, USA)at 70°C for 10 min before gas chromatography-mass
spectrometry (GC–MS) analysis. The derivatives wereanalyzed by GC–MS-SIM (selected ion monitoring)(QP 5000; Shimadzu, Japan) using a CP-Sil 5 CB column(Chrompack, Middelburg, Netherlands; 0.25-mm i.d. 925 m, 0.25-lm film thickness); linear He flow at
50.2 cmsec-1, column temperature step gradient, 60°C for
2 min, 60 to 270°C at 10°Cmin-1, and 270°C for 35 min;

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Statistical analysis
Data were presented as the means ? SE of three replications, subjected to analysis of variance procedures, andseparated by Fisher’s least significant difference, P B 0.05
(SAS Institute, Cary, NC, USA).

MdTFL1A

FTT
HCT

Expression level relative
to control

electron potential, 70 eV. The amount of endogenous GAs
was determined from the peak area ratios of non-deuterated
to deuterated GAs. The endogenous concentrations of GA1
and GA4 were calculated from the peak area ratios of m/z
286/284 and 508/506, respectively.

Plant Growth Regul (2011) 64:75–81

160
140
120
100
80
60
40
20
0

21

49

63

103
FTT

HCT

LSD 0.05= 3.37

21

49
63
Days after full bloom

103

MdFT1

B
FTT

Results

Effect of KODA application on the expressionof MdTFL1 and MdFT1 genes, rate of flower bud
formation, and GA concentration
The expression of MdTFL1 genes at 21 and 35 DAFB did
not differ greatly between the KODA-treated and untreatedtrees, but the expression of MdTFL1 at 49 DAFB in KODA
treatment was lower than the untreated control (Fig. 4a). In
contrast, the expression of MdFT1 genes was not greatly
different between the treatments, and the relationshipbetween the expression levels fluctuated at each date(Fig. 4b). The rate of flower bud formation in buds towhich KODA was applied was significantly higher thanthat in the untreated control (Fig. 4c). KODA application

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80

21

49

63

103
LSD 0.05= 3.26

60
40
20
0
21

49
63
Days after full bloom

103

C
Rate of flower bud formation(%)

Transcript levels of MdTFL1 genes in HCT were significantly higher than those in FTT (Fig. 2a). The expressionof MdTFL1 in HCT was strongly detected at 21 and 49
DAFB, but decreased after 63 DAFB in both HCT and
FTT. The expression of MdFT1 genes was detected
throughout the measuring dates in both HCT and FTT(Fig. 2b). However, the relationship between the expression levels of MdFT1 in HCT and FTT fluctuated at eachdate (Fig. 2b). The flower bud formation rate in FTT was
significantly higher than that in HCT (Fig. 2c).
In shading treatment, the expression of MdTFL1 washigher than that in non-shading treatment throughout themeasuring dates (Fig. 3a). The expression of MdFT1 at 35
and 49 DAFB in shading treatment was higher than that in
non-shading treatment, but the opposite result was found at
80 DAFB (Fig. 3b). The shading treatment significantly
decreased the flower bud formation rate (Fig. 3c).

Expression level relative
to control

HCT

Effects of fruit load and shading on the expressionof MdTFL1 and MdFT1 genes and the rate of flower
bud formation100
LSD 0.05= 21.94

80
60
40
20
0
FTT

HCT

Fig. 2 RT-PCR analysis of MdTFL1 (a) and MdFT1 (b) and the rateof flower bud formation (c) in apical buds of ‘Fuji’ apples subjected to
the flower thinning (FTT) and heavy crop treatments (HCT). The
experimental values were plotted compared to the control (Actin)value. Data are the means ± SE of three replications

did not significantly influence the total GA concentrations
throughout the measuring dates (Fig. 5).

Discussion
The function of TFL1 in Arabidopsis involves the determination of inflorescence meristem identity as well as
floral transition (Bradley et al. 1997). It delays floweringand regulates plant growth through the maintenance ofindeterminancy of the shoot apex (Ratcliffe et al. 1998).

Plant Growth Regul (2011) 64:75–81

79
MdTFL1A

A

Control

MdTFL1
Control

Shading

Expression level relative
to control

80

49

140

Control120

Shading1008060

LSD 0.05= 13.21

40
20

Expression level relative
to control

KODA

35

0
35

49

300

7

21

63

91

49

MdFT1

KODA

35

49

LSD 0.05= 4.07

50
40
30
20
10
0
49

7

100

80

Expression level relative
to control

Expression level relative
to control

35

Control

21

35

49

63

91

LSD 0.05= 18.61

80
60
40
20
0

80

7

21

Days after full bloom

35

49

63

91

Days after full bloom

100
LSD 0.05= 26.55

80
60
40
20
Control

Shading

Rate of flower bud formation
(%)

C

C
Rate of flower bud formation
(%)

KODA

50

B

60

0

91

Control

100

Shading3563

LSD 0.05= 15.93

Control

70

49

Days after full bloom

MdFT1

80

35

150

Days after full bloom
B

21

200

0

80

7

250

120
100

LSD0.05 =4.69

80
60
40
20
0

Control

KODA

Fig. 3 RT-PCR analysis of MdTFL1 (a) and MdFT1 (b) and the rateof flower bud formation (c) in apical buds of ‘Fuji’ apples subjected tothe shading and non-shading treatments. The experimental values
were plotted compared to the control (Actin) value. Data are themeans ± SE of three replications

Fig. 4 RT-PCR analysis of MdTFL1 (a) and MdFT1 (b) and the rateof flower bud formation (c) in KODA-treated apical buds. We
applied100 lM KODA at 7, 21, 35, and 49 days after full bloom. The
experimental values were plotted compared to the control (Actin)value. Data are the means ± SE of three replications

A homolog of the TFL1 gene has been isolated and characterized in fruit trees such as apples (Kotoda and Wada
2005; Kotoda et al. 2006), citrus (Pillitteri et al. 2004),
pears (Pyrus pyrifolia Nakai) and quinces (Cydonia oblonga Mill.) (Esumi et al. 2005). In our previous study, thetransition time from vegetative buds to floral buds was 70
DAFB (Kittikorn et al. 2010). The expression of MdTFL1
decreased sharply after 63 DAFB in our study. This finding
is compatible with a role of these genes in the timingfrom vegetative phase to reproductive phase and in maintaining meristem indeterminacy (lateral and inflorescencemeristems) in the bud (Pillitteri et al. 2004; Kotoda andWada 2005). The induction of flowering and floral development is coincident with the lack of MdTFL1 expression
in the late stages of flower development (Kotoda and Wada
2005).
We demonstrated that endogenous KODA may be
associated with flower bud formation that was influenced
by fruit load in apples (Kittikorn et al. 2010). That is, theendogenous KODA concentrations in HCT were lower
than those in FTT. The presence of fruit inhibits flower
initiation in the bourse bud, but this inhibition can be

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Plant Growth Regul (2011) 64:75–81

Total GA concentrations(GA1+GA4 μg / kg FW)

2000
Control
1600

KODA

1200
800
400
0

LSD 0.05 = 467.5

7

21

35

49

63

77

91

Days after full bloom

Fig. 5 Effect of KODA application (100 lM) on endogenous GA
concentrations in the apical buds. Data are the means ± SE of three
replications

relieved if the fruits are removed at an early stage (42–56
DAFB) of development (Dennis and Neilson 1999). The
presence of fruit decreases flower induction, and MdTFL1
is detected strongly in vegetative tissues (Kotoda et al.
2006). Therefore, the high expression of MdTFL1 in HCTmay be associated with the low rate of flower bud formation. Our assumption may be supported by the fact that theexpression of MdTFL1 in shading treatment, during which
the rate of flower bud formation was low, was also detected
more strongly than that in non-shaded trees.
MdFT1 and MdFT2 genes have been isolated, and theirfunctions were clarified in apples (Kotoda and Wada 2005;
Kotoda et al. 2010). The transcriptional activation of
MdFT1 is closely associated with the regulation of
flowering in apples, with expression of genes such as
MdMADS12 (Kotoda et al. 2010). In our study, theexpression of MdFT1 was observed throughout the measuring dates in each treatment. These results agree with theobservation that MdFT1 was highly detected in the apicalbuds of fruit-bearing shoots from the period of flower
induction to the early stage of flower development (Kotoda
et al. 2010). However, KODA applied to shoots in thepresent study did not affect the expression of MdFT1.
In contrast, the expression of MdTFL1 at 49 DAFB was
decreased by KODA application. We have observed thatthe degree of shoot elongation was the highest from 35 to
49 DAFB in our study (data not presented). In addition, aprevious study showed that the TFL1 homolog is associatedwith juvenility in fruit trees (Pillitteri et al. 2004). Therefore, it is possible that KODA application induces flower
bud formation in apples through the effect to MdTFL1transcription.
GA4 plays an important role in flower bud physiology inapples (Looney et al. 1985). Exogenous GA1, GA4, andiso-GA7 inhibited flower induction in apples (Ramı́rez
et al. 2004). In our study, KODA application to the buds
did not influence endogenous GA concentrations in thebuds. However, the KODA application increased therate of flower bud formation without affecting the GA

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concentrations. Therefore, KODA and GA may influence
the flower bud formation independently. It is unclear where
KODA is synthesized in apples. Further analysis will beneeded to determine the primary site where KODA that
accumulates in the bud is synthesized.

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