Application of an Analog of 9, 10-ketol-octadecadienoic acid (KODA), Affected Flower Bud Formation and MdTFL1 and MdFT1 Gene Expressions in Apple Buds under Heavy-crop and Shade Conditions

in Journal of the American Society for Horticultural Science
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  • 1 Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan
  • | 2 National Institute of Fruit Tree Science, Morioka 020-0123, Japan
  • | 3 Technology and Engineering Center, Shiseido Co., Yokohama 236-8643, Japan
  • | 4 Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan

Changes of endogenous 9, 10-ketol-octadecadienoic acid (KODA) concentrations, which is synthesized from 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 under shade. 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 was found in HCT. In the shade-treated and heavy-crop trees, the expression of MdTFL1 in the buds to which CKODA was applied was lower than that in untreated buds. In contrast, under shade, the expression of MdFT1 in the CKODA-treated buds was higher than that in untreated buds. These results suggest that endogenous KODA may be associated with flower bud formation, and its application may be effective at improving the proportion of flower bud formation through its effect on MdTFL1 and MdFT1.

Abstract

Changes of endogenous 9, 10-ketol-octadecadienoic acid (KODA) concentrations, which is synthesized from 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 under shade. 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 was found in HCT. In the shade-treated and heavy-crop trees, the expression of MdTFL1 in the buds to which CKODA was applied was lower than that in untreated buds. In contrast, under shade, the expression of MdFT1 in the CKODA-treated buds was higher than that in untreated buds. These results suggest that endogenous KODA may be associated with 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 that is associated with gibberellins (Ramirez et al., 2001, 2004). Therefore, the clarification of the physiological mechanism for regulation of flower bud formation is indispensable for stable apple production.

Environmental conditions such as light intensity also influence floral initiation in apple. For instance, low irradiance has 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 concentrations in apical buds were higher in trees with a higher flower bud formation, and that KODA spraying of one-year-old shoots increased the proportion of flower bud formation in trees with normal fruit load [40 leaves per fruit (Kittikorn et al., 2010, 2011)]. These facts show that KODA may be associated with flower bud formation in apple.

Studies on the molecular mechanisms of flowering have been 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 and differentiation 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 been shown 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 was transferred 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 plants, which took six years to flower. In addition, MdTFL1 was detected in vegetative tissues such as apical buds, stems, and cotyledon, 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 (Abe et 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 MdTFL1 transcription. 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 application on the transcription of MdTFL1 and MdFT1 and flower bud formation in apple under heavy cropload and shade conditions.

Fig. 1.
Fig. 1.

Structure of 9, 10-ketol-octadecadienoic acid (KODA) and 15, 16-chloro, hydroxyl-9-hydroxy-10-oxo-12(Z)-octadecenoic acid (CKODA). CKODA was formed by introducing a chlorine atom and a hydroxyl group at the C-15 olefin of KODA.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 2; 10.21273/JASHS.138.2.102

Materials and Methods

Plant materials.

Experiments were conducted in 2009 and 2010 on ‘Fuji’ apple trees grafted onto ‘Marubakaido’ rootstock (Malus prunifolia Borkh. var. ringo Asami) growing in an open field of Chiba University located at lat. 36° N and long. 139° E at an altitude of 747 m.

Expt. 1: effect of crop load on the changes of endogenous KODA.

Two groups of three 32-year-old trees (three replications of one tree per each treatment) were created randomly in 2009: one that underwent flower thinning treatment (FTT), in which all flowers were removed at full bloom, and one that underwent HCT, in which the number of leaves per fruit was adjusted to 20 at 20 d after full bloom (DAFB). At 21, 49, 63, and 103 DAFB, 90 terminal buds from shoot meristems (three replications of 30 buds per tree) were randomly collected for analysis of endogenous KODA from each treatment. The sampled buds were from non-fruiting spurs in each 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 nitrogen and extracted with methanol (10 mL) using [U-13C] KODA (1 pmol·μL−1) as an internal standard at 4 °C. The filtrate was concentrated in vacuo and the concentrates were re-dissolved in methanol (5 mL) and washed with hexane (10 mL) to remove pigments. The methanol layer thus obtained was evaporated in vacuo to yield solid materials. The resultant solid materials were re-dissolved in 100 μL ethanol, and 5 μL of the solution was subjected to high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) (LTQ-Orbitrap Discovery; Thermo Fisher Scientific, Bremen, Germany) equipped with a Capcell Pak C18 UG 120 column (2 mm i.d. × 7.5 cm; Shiseido, Tokyo, Japan) that was maintained at 4 °C . The samples were analyzed at a flow rate of 0.2 mL·min−1 using a chromatographic gradient of two mobile phases (A = 0.05% aqueous formic acid, B = acetonitrile). Solvent B was linearly increased from 15% to 100% 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 were acquired from a parent ion at m/z 309 [M-H] for KODA with a scan range of m/z 85 to m/z 310. The MS2 spectra of [U-13C] KODA were determined based on a selection reaction monitoring an ion at m/z 327 [M-H] with a scan range of m/z 263.5 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 at m/z 327 exhibited ion peaks at m/z 309 (100%) [M-H2O] and m/z 264 (68.0%) [M-H2O-H13COO]. The recovery of KODA was estimated based on the ion intensity at m/z 309. Similarly, KODA was detected at tR = 10.40 min with an ion peak at m/z 309. The MS2 spectrum from the ion at m/z 309 showed ion peaks at m/z 291 (100%) [M-H2O] and m/z 247 (72.3%) [M-H2O-HCOO]. Although 13-α-ketol had the same retention time and ion peak at m/z 309 [M-H] as those of KODA, the MS2 spectrum of 13-α-ketol was recorded with ion peaks at m/z 291 (100%) [M-H2O] and m/z 247 (3.1%) [M-H2O-HCOO]. Furthermore, the ionization efficacy for m/z 291 of 13-α-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-α-ketol.

Expt. 2: Effect of CKODA application on the expression of MDTFL1 and MDFT1 genes under heavy-crop and shade conditions.

In the 2010 HCT, four 33-year-old trees were randomly selected. The number of leaves per fruit was adjusted to 20 at 20 DAFB as in 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 μM CKODA with surfactant 0.1% (v/v) (New Osmac®; Hayashi Chemical, Tokyo, Japan) at 7, 21, 35, and 49 DAFB (HCT+CKODA+). On each day of treatment, the tips of the shoots with three nodes were sprayed once until runoff. The untreated controls (HCT+CKODA) were one-year-old shoots that were not sprayed with CKODA. At 7, 21, 35, 49, 63, 77, and 96 DAFB, 90 apical buds (18 buds per each tree) were randomly sampled. Apical buds were frozen immediately in liquid nitrogen and then stored at –80 °C until analysis of MdTFL1 and MdFT1 gene expression.

In the shade treatment, three randomly selected 19-year-old trees were covered with cheesecloth (50% shading) at 20 DAFB. The number of leaves per fruit was adjusted to 40 at 20 DAFB. This number of leaves per fruit is the usual fruit load not causing alternate bearing. One hundred twenty one-year-old shoots in each tree (360 apical buds in three trees), also randomly selected at 7 DAFB, were sprayed with 100 μM CKODA, like in the crop load experiment (Shade+CKODA+). At 7, 21, 35, 49, 63, and 77 DAFB, 60 apical buds (20 buds per tree) were randomly sampled for analysis of the expression of MdTFL1 and MdFT1 genes. The untreated controls (Shade+CKODA) were one-year-old shoots that were not sprayed with CKODA.

To determine the proportion of flower bud formation in the cropload and shade experiments, 90 apical buds (three replications of 30 buds per tree) were randomly collected after defoliation. Each apical bud was longitudinally dissected, and the proportion of flower bud formation was determined with a stereomicroscope. Buds that apical meristems showed to be dome-shaped were counted as flower buds according to a previous report (Kittikorn et al., 2010). The proportion of flower bud formation was calculated by the number of flower buds in 90 apical buds.

Preparation of CKODA.

CKODA (C18H31ClO5) was formed by introducing a chlorine atom and a hydroxyl group at the C-15 olefin of KODA. KODA (32 mm) of 250 mL was mixed with 690 mL distilled water followed by the addition 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 conversion rate of CKODA from KODA was more than 60%. CKODA was extracted by ethyl acetate and isolated by HPLC (NANOSPACE SI-1; Shiseido) using an ultraviolet detector at 210 nm with a Capcell Pak C18 column. The mobile phase was 50% acetonitrile in 0.05% trifluoroacetic acid, and the column temperature and flow rate were 30 °C and 1 mL·min−1, respectively. The retention time of CKODA was 5.4 min. The product was identified with HPLC-MS/MS. The fragment ions of CKODA were m/z 363 [M-H] and 345 [M-H-H2O].

RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR.

Total RNA from apical buds was extracted following the protocol mentioned in a previous report (Kittikorn et al., 2010). DNase treatment (5 units; Takara Bio, Otsu, Japan) at 37 °C for 20 min was applied to 2 μg of total RNA to eliminate any contaminating DNA in the samples. The reaction was stopped and then incubated at 70 °C for 10 min. The treated RNA was used as a template for cDNA synthesis. The cDNA was synthesized in a 20-μL reaction volume using ReverTraAce (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. RNA extraction and cDNA synthesis were repeated twice on each sampling date. The cDNA samples were diluted (1:10 v/v) and amplified using the gene-specific primers shown in Table 1. The quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed using the SYBR master mix (KAPA SYBR® FAST qPCR kit; NIPPON Genetics, Tokyo, Japan) with ABI StepOnePlus (Applied Biosystems, Foster City, CA). 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 and annealing at 60 °C for 32 s. The specificity of the amplification was verified on the basis of a melting curve obtained at the end of the PCR cycle. The relative expression level was normalized to that of the actin gene (Yooyongwech et al., 2008).

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.

Table 1.

Statistical analysis.

The SAS analysis of variance procedure (Version 8.02; SAS Institute, Cary, NC) was used to determine the treatment effects, and the mean separation was analyzed by Fisher’s least significant difference (P ≤ 0.05). The data are presented as the mean values of three replications ± se.

Results

The proportion of flower bud formation of FTT was significantly higher than that of HCT (Table 2). A significant increase in endogenous KODA in FTT was observed at 63 and 103 DAFB, although there was no significant difference of KODA levels at 21 and 49 DAFB between HCT and FTT (Fig. 2). In HCT trees, endogenous KODA levels did not show significant changes from 21 to 103 DAFB.

Table 2.

Proportion of flower bud formation 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) or shade.

Table 2.
Fig. 2.
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 heavy-crop 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.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 2; 10.21273/JASHS.138.2.102

The relative expression levels of MdTFL1 in HCT+CKODA+ were significantly lower than those in HCT+CKODA at 21, 35, and 49 DAFB (Fig. 3A). In both treatments, the expression of 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 CKODA-treated and -untreated buds under HCT conditions, although the times 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 formation in HCT+CKODA+ was significantly higher than that in HCT+CKODA (Table 2).

Fig. 3.
Fig. 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 the number 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 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.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 2; 10.21273/JASHS.138.2.102

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 of MdTFL1 genes at 7, 21, and 35 DAFB did not differ significantly between Shade+CKODA+ and Shade+CKODA, but the expression of MdTFL1 in Shade+CKODA was higher than that in Shade+CKODA+ at 49, 63, and 77 DAFB (Fig. 4A). In contrast, the expression in Shade+CKODA+ did not show much change.

Fig. 4.
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 number of 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 s followed 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.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 2; 10.21273/JASHS.138.2.102

In general, the patterns of change of MdFT1 expression in both CKODA-treated and -untreated buds were similar under shade conditions (Fig. 4B). The MdFT1 expressions increased toward 35 DAFB and then decreased at 49 DAFB. However, the expressions 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 more accurate 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 in apple. 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 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 and cytokinin, which influence flower bud formation (Sanyal and Bangerth, 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 important in 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 and Wada, 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 mRNA transcript levels was found in buds under both heavy-crop and shade treatments that related to the decrease of flower bud formation. The proportion of flower bud formation in apical buds of apple increased after 70 DAFB (Kittikorn et al., 2010). In our study, CKODA application in HCT decreased MdTFL1 mRNA transcript levels at 21, 35, and 49 DAFB, although the expression of MdTFL1 in HCT+CKODA decreased sharply after 63 DAFB. It has been shown that MdTFL1 mRNA was expressed 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, the proportion of flower bud formation in Shade+CKODA was lower 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 and floral development may be coincident with the interruption of MdTFL1 expression (Kotoda and Wada, 2005; Mimida et al., 2009). Furthermore, MdTFL1 expression in our study shows that 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 than that of MdFT2. Therefore, we examined the transcription 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) (Wada et al., 2002) in the apical meristems of one-year-old apple shoots began 1 to 2 weeks earlier than flower initiation. In our study, MdFT1 expression levels in HCT+CKODA were higher than those 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 observed in our study may be associated with flower bud formation.

In summary, endogenous KODA in the buds of trees in which the proportion of flower bud formation was high increased before the initiation of flower bud formation. In heavy-crop and shade-treated trees, the application of CKODA, a derivative of KODA, increased the proportion of flower bud formation and the expression of MdFT1 but decreased the expression of MdTFL1.

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  • Kotoda, N., Iwanami, H., Takahashi, S. & Abe, K. 2006 Antisense expression of MdTFL1, a TFL1-like gene, reduces the juvenile phase in apple J. Amer. Soc. Hort. Sci. 131 74 81

    • Search Google Scholar
    • Export Citation
  • Kotoda, N. & Wada, M. 2005 MdTFL1, a TFL1-like gene of apple, retards the transition from the vegetative to reproductive phase in transgenic arabidopsis Plant Sci. 168 95 104

    • Search Google Scholar
    • Export Citation
  • Lifschitz, E., Eviatar, T., Rozman, A., Shalit, A., Goldshmidt, A. & Amsellem, Z. 2006 The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli Proc. Natl. Acad. Sci. USA 103 6398 6403

    • Search Google Scholar
    • Export Citation
  • Mimida, N., Kotoda, N., Ueda, T., Igarashi, M., Hatsuyama, Y., Iwanami, H., Moriya, S. & Abe, K. 2009 Four TFL1/CEN-like genes on distinct linkage groups show different expression patterns to regulate vegetative and reproductive development in apple (Malus × domestica Borkh.) Plant Cell Physiol. 50 394 412

    • Search Google Scholar
    • Export Citation
  • Pillitteri, L.J., Lovatt, C.J. & Walling, L.L. 2004 Isolation and characterization of a terminal flower homolog and its correlation with juvenility in citrus Plant Physiol. 135 1540 1551

    • Search Google Scholar
    • Export Citation
  • Ramirez, H., Hoad, G.V., Benavides, A. & Rangel, E. 2001 Gibberellins in apple seeds and the transport of [3H]-GA4 J. Mexican Chem. Soc. 45 47 50

  • Ramirez, H., Torres, J., Benavides, A., Hernandes, J. & Robledo, V. 2004 Fruit bud initiation in apple cv Red Delicious linked to gibberellins and cytokinins J. Mexican Chem. Soc. 48 7 10

    • Search Google Scholar
    • Export Citation
  • Ratcliffe, O.J., Amaya, I., Vincent, C.A., Rothstein, S., Carpenter, R., Coen, E.S. & Bradley, D.J. 1998 A common mechanism controls the life cycle and architecture of plants Development 125 1609 1615

    • Search Google Scholar
    • Export Citation
  • Sanyal, D. & Bangerth, F. 1998 Stress induced ethylene evolution and its possible relationship to auxin-transport, cytokinin levels, and flower bud induction in shoots of apple seedlings and bearing apple trees Plant Growth Regulat. 24 127 134

    • Search Google Scholar
    • Export Citation
  • Suzuki, M., Yamaguchi, S., Iida, T., Hashimoto, I., Teranishi, H., Mizoguchi, M., Yano, F., Todoroki, Y., Watanabe, N. & Yokoyama, M. 2003 Endogenous α-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil Plant Cell Physiol. 44 35 43

    • Search Google Scholar
    • Export Citation
  • Tamaki, S., Matsuo, S., Wong, H.L., Yukoi, S. & Shimamoto, K. 2007 Hd3a protein is a mobile flowering signal in rice Science 316 1033 1036

  • Turck, F., Fornara, F. & Coupland, G. 2008 Regulation and identity of florigen: flowering locus t moves center stage Annu. Rev. Plant Biol. 59 573 594

    • Search Google Scholar
    • Export Citation
  • Wada, M., Cao, Q., Kotoda, N., Soejima, J. & Masuda, T. 2002 Apple has two orthologues of floricaula/leafy involved in flowering Plant Mol. Biol. 49 567 577

    • Search Google Scholar
    • Export Citation
  • Yokoyama, M., Yamaguchi, S., Inomata, S., Komatsu, K., Yoshida, S., Iida, T., Yokokawa, Y., Yamaguchi, M., Kaihara, S. & Takimoto, A. 2000 Stress-induced factor involved in flower formation of lemna is an α-ketol derivative of linolenic acid Plant Cell Physiol. 41 110 113

    • Search Google Scholar
    • Export Citation
  • Yooyongwech, S., Horigane, A.K., Yoshida, M., Yamaguchi, M., Sekozawa, Y., Sugaya, S. & Gemma, H. 2008 Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy Physiol. Plant. 134 522 523

    • Search Google Scholar
    • Export Citation
  • Zeevaart, J.A. 2008 Leaf-produced floral signals Curr. Opin. Plant Biol. 11 541 547

Contributor Notes

We thank Dr. Haruo Shimada, Shiseido Co., for help in a determination of structure of CKODA used in this work.

Corresponding author. E-mail: s-kondo@faculty.chiba-u.jp.

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    Structure of 9, 10-ketol-octadecadienoic acid (KODA) and 15, 16-chloro, hydroxyl-9-hydroxy-10-oxo-12(Z)-octadecenoic acid (CKODA). CKODA was formed by introducing a chlorine atom and a hydroxyl group at the C-15 olefin of KODA.

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    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 heavy-crop 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.

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    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 the number 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 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.

  • View in gallery

    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 number of 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 s followed 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.

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  • Kotoda, N., Hayashi, H., Suzuki, M., Igarashi, M., Hatsuyama, Y., Kidou, S., Igasaki, T., Nishiguchi, M., Yano, K., Shimizu, T., Takahashi, S., Iwanami, H., Moriya, S. & Abe, K. 2010 Molecular characterization of flowering locus t-like genes of apple (Malus × domestica Borkh.) Plant Cell Physiol. 51 561 575

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    • Export Citation
  • Kotoda, N., Iwanami, H., Takahashi, S. & Abe, K. 2006 Antisense expression of MdTFL1, a TFL1-like gene, reduces the juvenile phase in apple J. Amer. Soc. Hort. Sci. 131 74 81

    • Search Google Scholar
    • Export Citation
  • Kotoda, N. & Wada, M. 2005 MdTFL1, a TFL1-like gene of apple, retards the transition from the vegetative to reproductive phase in transgenic arabidopsis Plant Sci. 168 95 104

    • Search Google Scholar
    • Export Citation
  • Lifschitz, E., Eviatar, T., Rozman, A., Shalit, A., Goldshmidt, A. & Amsellem, Z. 2006 The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli Proc. Natl. Acad. Sci. USA 103 6398 6403

    • Search Google Scholar
    • Export Citation
  • Mimida, N., Kotoda, N., Ueda, T., Igarashi, M., Hatsuyama, Y., Iwanami, H., Moriya, S. & Abe, K. 2009 Four TFL1/CEN-like genes on distinct linkage groups show different expression patterns to regulate vegetative and reproductive development in apple (Malus × domestica Borkh.) Plant Cell Physiol. 50 394 412

    • Search Google Scholar
    • Export Citation
  • Pillitteri, L.J., Lovatt, C.J. & Walling, L.L. 2004 Isolation and characterization of a terminal flower homolog and its correlation with juvenility in citrus Plant Physiol. 135 1540 1551

    • Search Google Scholar
    • Export Citation
  • Ramirez, H., Hoad, G.V., Benavides, A. & Rangel, E. 2001 Gibberellins in apple seeds and the transport of [3H]-GA4 J. Mexican Chem. Soc. 45 47 50

  • Ramirez, H., Torres, J., Benavides, A., Hernandes, J. & Robledo, V. 2004 Fruit bud initiation in apple cv Red Delicious linked to gibberellins and cytokinins J. Mexican Chem. Soc. 48 7 10

    • Search Google Scholar
    • Export Citation
  • Ratcliffe, O.J., Amaya, I., Vincent, C.A., Rothstein, S., Carpenter, R., Coen, E.S. & Bradley, D.J. 1998 A common mechanism controls the life cycle and architecture of plants Development 125 1609 1615

    • Search Google Scholar
    • Export Citation
  • Sanyal, D. & Bangerth, F. 1998 Stress induced ethylene evolution and its possible relationship to auxin-transport, cytokinin levels, and flower bud induction in shoots of apple seedlings and bearing apple trees Plant Growth Regulat. 24 127 134

    • Search Google Scholar
    • Export Citation
  • Suzuki, M., Yamaguchi, S., Iida, T., Hashimoto, I., Teranishi, H., Mizoguchi, M., Yano, F., Todoroki, Y., Watanabe, N. & Yokoyama, M. 2003 Endogenous α-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil Plant Cell Physiol. 44 35 43

    • Search Google Scholar
    • Export Citation
  • Tamaki, S., Matsuo, S., Wong, H.L., Yukoi, S. & Shimamoto, K. 2007 Hd3a protein is a mobile flowering signal in rice Science 316 1033 1036

  • Turck, F., Fornara, F. & Coupland, G. 2008 Regulation and identity of florigen: flowering locus t moves center stage Annu. Rev. Plant Biol. 59 573 594

    • Search Google Scholar
    • Export Citation
  • Wada, M., Cao, Q., Kotoda, N., Soejima, J. & Masuda, T. 2002 Apple has two orthologues of floricaula/leafy involved in flowering Plant Mol. Biol. 49 567 577

    • Search Google Scholar
    • Export Citation
  • Yokoyama, M., Yamaguchi, S., Inomata, S., Komatsu, K., Yoshida, S., Iida, T., Yokokawa, Y., Yamaguchi, M., Kaihara, S. & Takimoto, A. 2000 Stress-induced factor involved in flower formation of lemna is an α-ketol derivative of linolenic acid Plant Cell Physiol. 41 110 113

    • Search Google Scholar
    • Export Citation
  • Yooyongwech, S., Horigane, A.K., Yoshida, M., Yamaguchi, M., Sekozawa, Y., Sugaya, S. & Gemma, H. 2008 Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy Physiol. Plant. 134 522 523

    • Search Google Scholar
    • Export Citation
  • Zeevaart, J.A. 2008 Leaf-produced floral signals Curr. Opin. Plant Biol. 11 541 547

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