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Interactions among LOX metabolites regulate temperature-mediated flower bud formation in morning glory (Pharbitis nil)
Journal of Plant Physiology 169 (2012) 1815–1820
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Interactions among LOX metabolites regulate temperature-mediated flower bud formation in morning glory (Pharbitis nil) Kyong-Hee Nam a,b , Teruhiko Yoshihara a,∗ a b
Laboratory of Bio-organic Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kitaku, Sapporo 060-8589, Japan Bio-Evaluation Center, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon, Chungbuk 363-883, Republic of Korea
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Article history: Received 16 May 2012 Received in revised form 27 July 2012 Accepted 27 July 2012 Keywords: Hydroperoxy linolenic acid Jasmonic acid 9,10-Ketol-octadecadienoic acid Lipoxygenase Theobroxide
a b s t r a c t We examined the relationship between temperature (15–35 ◦ C) and flower induction as it is influenced by linolenic acid (LA) cascade products, lipoxygenase (LOX; EC 1.13.11.12), allene oxide synthase (AOS; EC 4.2.1.92), and allene oxide cyclase (AOC; EC 5.3.99.6) generated in morning glory (Pharbitis nil Choisy). The maximum amount of LOX protein was detected when plants were grown at 30 ◦ C, whereas endogenous AOS and AOC proteins were markedly accumulated at 15 ◦ C. Although both test levels of 9(S)- and 13(S)hydroperoxy linolenic acid (HPOT) showed similar temperature dependencies, reflecting the profile of LOX, the relative amount of 13(S)-HPOT was much higher than that of 9(S)-HPOT, regardless of temperature regime. This implied a faster reaction pathway to 9,10-␣-ketol octadecadienoic acid (KODA) in the LA cascade. In the 13(S)-HPOT pathway, the highest level of endogenous jasmonic acid (JA) was observed at 15 ◦ C. Our results suggest that at a high temperature (30 ◦ C), 9(S)-HPOT may be readily metabolized into KODA to promote flower bud formation. By contrast, at a low temperature, high levels of AOS and AOC result in an accumulation of JA that inhibits this developmental process. Accordingly, depending on the growing temperature, flower bud formation in P. nil is possibly regulated by the interactions among LOX metabolites, with KODA serving as a promoter and JA as an inhibitor. © 2012 Elsevier GmbH. All rights reserved.
Introduction Photoperiod and temperature are major environmental factors that influence flower induction and development. Morning glory (Pharbitis nil) is a very sensitive short day (SD) plant that can be induced to produce flower buds after exposure to a single 16-h dark period (Imamura et al., 1966). However, even under SD conditions, flowering is stimulated at 24–30 ◦ C, but not at 12–18 ◦ C (Reese and Erwin, 1997; Nam et al., 2005). One of the 9-lipoxygenase (LOX; EC 1.13.11.12) derived metabolites, 9,10-␣-ketol octadecadienoic acid (KODA), has been isolated as a flower-inducing factor from Lemna paucicostata (Fig. 1; Takimoto et al., 1989, 1991, 1994; Yokoyama et al., 2000; Yamaguchi et al., 2001). During a critical photoperiod for flower induction in P. nil, KODA levels are also transiently increased in the cotyledons and immature floral buds but not in the foliar buds (Suzuki et al., 2003; Yokoyama et al., 2005).
Abbreviations: AOC, allene oxide cyclase; AOS, allene oxide synthase; CO, CONSTANS; FT, FLOWERING LOCUS T; GC–SIM-MS, gas chromatography–selected ion monitoring-mass spectrometry; HPOT, hydroperoxy linolenic acid; JA, jasmonic acid; KODA, 9,10-ketol-octadecadienoic acid; LA, linolenic acid; LD, long day; LOX, lipoxygenase; Me-JA, methyl jasmonate; PHYB, phytochrome B; SD, short day; TA, tuberonic acid; TAG, tuberonic acid glucoside. ∗ Corresponding author. Tel.: +81 166 48 3121; fax: +81 166 48 8718. E-mail address: [email protected] (T. Yoshihara). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.07.009
Jasmonic acid (JA), which is derived from 13(S)-hydroperoxy linolenic acid (HPOT) by 13-LOX catalysis, might be involved in potato tuberization (Pelacho and Mingo-Castel, 1991; Pruski et al., 2002; Sarkar et al., 2006). Several researchers have suggested that LOX is associated with substances that participate in the formation of potato tubers, especially tuberonic acid glucoside (TAG), a derivative of JA (Yoshihara et al., 1989; Kolomiets et al., 2001; Farmaki et al., 2007). In P. nil, exogenously applied methyl jasmonate (Me-JA) inhibits flowering, with its influence being dependent on ˛ photoperiod conditions (Maciejewska and Kopcewicz, 2002; Kesy et al., 2011). In addition, substances that block jasmonate biosynthesis, such as aspirin, phenidone, and ibuprofen, can partially recover the impeding effect of far-red light on flowering in P. nil ˛ et al., 2011). (Maciejewska et al., 2004; Kesy Results from experiments with Arabidopsis thaliana have suggested that LOX mediates a photoperiodic signal in the transition from vegetative to reproductive growth (Ye et al., 2000). We have previously proposed that this protein may directly interact with light and temperature to regulate both flower induction and potato tuberization (Nam et al., 2005). LOX is activated at tuber-inducing low temperatures, and large amounts of JA, tuberonic acid (TA), and TAG are metabolized in potato (Nam et al., 2005, 2008). It is also possible that LOX-derived metabolites promote tuberization by antagonizing the effect of gibberellin (GA) (Sarkar, 2008). Theobroxide, a natural product isolated from culture filtrates of the fungus Lasiodiplodia theobromae, can induce potato tubers
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Fig. 1. Linolenic acid cascade.
and P. nil flower buds under non-inductive photoperiods (Yoshihara et al., 2000). This compound also induces the development of both tissue types even at non-inductive high temperatures (Nam and Yoshihara, 2011). Application of theobroxide to the leaf surfaces of potato and P. nil enables developmental transformations under unfavorable environmental conditions by enhancing both the activity of LOX and endogenous levels of JA (Gao et al., 2003; Yang et al.,
2004; Kong et al., 2005). Consequently, LOX-derived metabolites are involved not only in potato tuberization, but also in flower bud formation by morning glory. Both of these processes are controlled by signals generated in the leaves, which then travel throughout the plant to the shoot apical meristems for flowering or to the underground stolons for tuberization (Suárez-López, 2005). A. thaliana CONSTANS (AtCO)
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and phytochrome B (PHYB) produce inductive and inhibitory signals that balance flowering and tuberization (Sarkar, 2008, 2010). Under non-inductive long days, FLOWERING LOCUS T (FT) acts as a key mobile signal that controls not only flowering transitions but also the induction of potato tuberization (Navarro et al., 2011). Although it is somewhat clear that a certain mechanism exists to balance flowering and tuberization, depending on environmental cues, little is known about how that mechanism operates. We previously assessed the relationship between LOX activity and temperature on flower bud formation and proposed that this protein might modulate temperature-dependent induction (Nam et al., 2005). Here, we report on our investigation of LOX-derived metabolites and various enzymes in the linolenic acid (LA) cascade. We again used leaves of P. nil to examine how flower induction may be a function of temperature. Materials and methods Plant materials and temperature treatments Seeds of dwarf-type morning glory, Pharbitis nil Choisy cv. Sun Smile, were obtained from Takii Seed Co. (Kyoto, Japan). They were sown in pots filled with a mixture of peat moss and perlite (1:1, v/v). The resultant plants were cultivated in a growth chamber (NK System, Biotron NC 350, Japan) at 25 ◦ C under long day (LD) conditions (18 h light/6 h dark). Growth and temperature treatments were applied as previously described (Nam et al., 2005). When the seedlings were three weeks old, they were transferred to growth chambers and cultured under SD conditions (10 h light/14 h dark) at 15, 20, 25, 30, or 35 ◦ C. Leaves were collected after two and three weeks of this temperature treatment, when flower buds began to appear. Determining levels of endogenous 9(S)- and 13(S)-HPOT Hydroperoxy linolenic acid (HPOT) was purified and quantified as described by Göbel et al. (2002), with simple modifications. One gram of frozen leaf tissue was extracted in 10 mL of extraction buffer (3:2 hexane:2-propanol). After (6Z,9Z,11E,13S)-3-Hydroxy6,9,11-octadeca-trienoic acid (␥-13-HOT) was added as an internal standard, the extract was centrifuged at 4500 × g for 10 min at 4 ◦ C. The clear upper phase was collected and the pellet was extracted three times with 3 mL of extraction buffer. A 6.7% (w/v) solution of potassium sulfate was then added to the combined organic phases. After thorough mixing, the upper layer containing the oxylipin metabolites was dried and dissolved in CH3 CN/H2 O/CH3 COOH (55:45:0.02, v/v/v). The reaction products were analyzed by reverse phase-HPLC, using a Wakosil PTH column (4.6 mm × 250 mm). The mobile phase consisted of CH3 CN/H2 O/CH3 COOH (55:45:0.02, v/v/v) at a flow rate of 1.4 mL min−1 , and absorbance at 234 nm was recorded. Retention times for 9(S)-HPOT (16.1 min) and 13(S)-HPOT (17.0 min) were monitored by authentic standards. The 13(S)HPOT and ␥-13-HOT were prepared by incubating either ␣-LA or ␥-LA with soybean LOX (Hamberg and Gotthammar, 1973). The 9(S)-HPOT was obtained by incubating ␣-LA with tomato fruit homogenates (Matthew et al., 1977). Determining levels of endogenous JA The JA content was analyzed according to the method of Matsuura et al. (2002). Leaf tissues (1 g) were first soaked in 10 mL of 80% aqueous MeOH (10 mL) for 48 h and then filtered. As an internal standard, [10,11,11,12,12,12-2 H6 ]-JA ([d]-JA) was added at a final concentration of 0.5 g g−1 fresh weight (FW). The filtrate was evaporated under reduced pressure, re-dissolved in H2 O, adjusted to pH 2–3 with 4 M HCl, and extracted with EtOAc
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(15 × 3 mL). The combined organic layers were extracted with saturated NaHCO3 (15 × 3 mL). The aqueous layer was again adjusted to pH 2–3 with 4 M HCl and extracted with EtOAc (15 × 3 mL). The combined organic layers were concentrated, and the residue was dissolved in 1 mL of H2 O and applied to a Bond Elut C18 cartridge (Varian; CA, USA). This cartridge was successively washed with H2 O (1 × 2 mL) and MeOH/H2 O (4:1, v/v; 1.3 × 3 mL). The 80% MeOH eluate was concentrated in vacuo, and the residue was applied to a Bond Elut DEA cartridge (Varian). After the cartridge was successively washed with MeOH (1 × 2 mL) and 1 M AcOH/MeOH (1 × 4 mL), the AcOH/MeOH eluate was concentrated and dissolved in 1 mL of MeOH. Diazomethane, in diethyl ether, was then added to a MeOH solution for methylation. The concentrated residue was purified on an HPLC system (Hitachi L7100; HITACHI, Japan) equipped with a YMC-Pack ODS column (300 mm × 10 mm; YMC Co. Ltd., Japan), at a flow rate of 2.5 mL min−1 . The solvent system was MeOH/H2 O/AcOH (90:10:0.1, v/v/v). The JA fractions were evaporated, and the resultant residue was analyzed by gas chromatography–selected ion monitoring-mass spectrometry (GC–SIM-MS) (QP5000 System; Shimadzu, Japan), using a ZB-1 column (0.25 mm × 30 m, 0.5-m phase thickness). Our GC temperature program followed 80 ◦ C for 1 min, 80–290 ◦ C at 20 ◦ C/min, and 290 ◦ C for 5 min. The injector and detector temperatures were 200 ◦ C and 280 ◦ C, respectively. Amounts of endogenous JA were calculated by comparing peak areas with corresponding internal standards. Measurement of LOX activity The assay for LOX activity was performed as we previously described (Nam et al., 2005). Briefly, 0.1 g of frozen leaf powder was homogenized in 1 mL of 0.1 M phosphate buffer (pH 7.3) and centrifuged at 15,000 × g for 20 min at 4 ◦ C. The crude enzyme that was obtained was then added to LA and 0.1 M sodium acetate buffer solution (pH 4.4), and was analyzed with a HITACHI U2800 spectrophotometer. The increase in absorbance at 234 nm was measured to monitor the formation of conjugated-diene compounds, e.g. 9-HPOT and 13-HPOT. One unit of LOX activity was defined as 1 mol of product formed per milligram of protein per minute. Protein contents were determined by the method of Bradford (1976), using bovine serum albumin (BSA) as a standard. Protein extraction We added 0.1 g samples of leaf tissue to 1 mL of 0.1 M phosphate buffer (pH 7.3) and centrifuged at 15,000 × g for 20 min at 4 ◦ C. The supernatant was used as the enzyme extract. Protein contents, with BSA as a standard, were evaluated by the method of Bradford (1976). Electrophoresis and immunoblot analysis Proteins were separated on a 10% SDS-polyacrylamide gel according to the method of Laemmli (1970). Electrophoretically separated proteins were transferred from those gels to nitrocellulose sheets per the protocol of Towbin et al. (1979). Blots were developed using chemiluminescence reagent (Perkin-Elmer Life Sciences) according to the manufacturer’s instructions. We used rabbit polyclonal antibodies raised against potato LOX (Royo et al., 1996), plus antibodies against the recombinant allene oxide synthase (AOS; EC 4.2.1.92) of Arabidopsis thaliana (Laudert and Weiler, 1998) and the recombinant allene oxide cyclase (AOC; EC 5.3.99.6) of A. thaliana (Ziegler et al., 2000). All antibodies were used at 1:5000 dilutions.
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Fig. 3. Endogenous levels of jasmonic acid in leaves of dwarf-type Pharbitis nil ‘Sun Smile’ after 2 and 3 weeks of temperature treatment. Values represent means of 3 independent measurements ± SE. **Significantly different from control temperature (15 ◦ C) at p < 0.01, according to Bonferroni t-test.
Endogenous levels of JA in leaves of P. nil grown at various temperatures The endogenous level of JA was influenced by temperature (Fig. 3). After both two and three weeks of treatment, JA levels were greatest at 15 ◦ C but decreased at higher temperatures. A detailed profile of JA content showed a rapid decline between 15 ◦ C and 30 ◦ C, and a moderate decrement between 30 ◦ C and 35 ◦ C. Regardless of temperature, endogenous JA levels were always higher after two weeks than after three weeks. Fig. 2. Endogenous levels of 9(S)-HPOT (A) and 13(S)-HPOT (B) in leaves of dwarftype Pharbitis nil ‘Sun Smile’ after 2 and 3 weeks of temperature treatment. Values represent means of 3 independent measurements ± SE. *Significantly different from control temperature (15 ◦ C) at p < 0.05, according to Bonferroni t-test. 9(S)- and 13(S)-HPOT are 9(S)- and 13(S)-hydroperoxy linolenic acids, respectively.
Statistical analysis To compare differences among treatments, we performed statistical analyses with ANOVA and Bonferroni t-tests at p < 0.05 and p < 0.01, respectively. Data were averaged from three independent experiments, with 10 seedlings examined for each temperature treatment.
Correlation between temperature and LOX activity Similar to the findings from our previous experiments (Nam et al., 2005), LOX activities, as measured by UV absorption (A234 nm) of the resulting diene, were dependent upon temperature (Fig. 4). The highest levels were recorded in seedlings grown at 30 ◦ C. Activity increased gradually for plants between 15 ◦ C and 30 ◦ C but was slightly decreased when they were exposed to 30 ◦ C or 35 ◦ C. At all temperatures tested, LOX activity was higher in the second week than in the third week.
Results Endogenous levels of 9(S)- and 13(S)-HPOT in leaves of P. nil grown at various temperatures To examine the effect of temperature on peroxidation from LA, we measured the endogenous contents of 9(S)- and 13(S)-HPOT from the leaves of P. nil after seedlings were grown under SD conditions for two or three weeks at 15, 20, 25, 30, or 35 ◦ C (Fig. 2). At all temperatures tested, levels of 9(S)-HPOT were relatively lower than those of 13(S)-HPOT. For 9(S)-HPOT, that level gradually increased with temperature, peaking at 30–35 ◦ C after both two and three weeks (Fig. 2A). Levels of 13(S)-HPOT were significantly temperature-dependent (Fig. 2B). After two weeks, the amount of 13(S)-HPOT increased with temperature, peaking at 30 ◦ C but declining somewhat at 35 ◦ C. Contents of 13(S)-HPOT were lower after three weeks than those measured after two weeks.
Fig. 4. Effect of temperature on lipoxygenase activity in leaves of dwarf-type Pharbitis nil ‘Sun Smile’ collected 2 (䊉) and 3 () weeks after temperature treatment. LOX activity was measured at 20–25 ◦ C. Values represent means of 3 independent measurements ± SE. **Significantly different from control temperature (15 ◦ C) at p < 0.01, according to Bonferroni t-test.
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Fig. 5. Immunoblot analysis of LOX (lipoxygenase), AOS (allene oxide synthase), and AOC (allene oxide cyclase) proteins in leaves from 3-week-old seedlings of dwarf-type Pharbitis nil ‘Sun Smile’. Leaves were sampled 2 weeks after temperature treatments. A total of 20 g of protein was applied to each lane. Immunoblots were probed with antibodies for anti-LOX, anti-AOS, and anti-AOC, at 1:5000 dilutions. Equivalent experiments were repeated at least 3 times.
Accumulations of LOX, AOS, and AOC proteins in the leaves of P. nil grown at different temperatures Because AOS and AOC proteins are important in the reactions of LOX metabolites, as demonstrated within the LA cascade, we examined the relationship between temperature and the amounts of AOS and AOC produced. Immunoblot analyses revealed that endogenous LOX accumulations were low between 15 ◦ C and 25 ◦ C but high between 30 ◦ C and 35 ◦ C, with the maximum accumulation recorded at 30 ◦ C (Fig. 5A). Levels of endogenous AOS protein were markedly higher at 15 ◦ C, but were at minimal amounts at 30 ◦ C and 35 ◦ C (Fig. 5B). Similarly, the levels of AOC protein were relatively large between 15 ◦ C and 25 ◦ C, with lower levels being measured in leaves exposed to either 30 ◦ C or 35 ◦ C (Fig. 5C).
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9(S)-HPOT detected during P. nil flowering would suggest facile reaction pathways to KODA. However, this assumption still remains controversial. For example, in an earlier study by Royo et al. (1996), the expression level of 9-LOX was always higher than that of 13LOX in developing potato tubers, which is the opposite of what we found here during floral bud formation. Even after its consumption by the JA biosynthesis pathway via the AOS/AOC branch, the amount of 13(S)-HPOT substrate remained at a much higher level than that of 9(S)-HPOT at all temperatures. To convert the unstable allene oxide to the first cyclic precursor of JA, the association between AOC and AOS must be maintained in close proximity (Farmaki et al., 2007). Here, in response to low temperature, that AOS/AOC branch to JA biosynthesis was stimulated, resulting in high endogenous amounts of both enzymes. The endogenous level of JA was high when plants were exposed to 15 ◦ C, but it declined as the temperature rose. JA possibly functions in the inhibition of flowering in P. nil (Maciejewska ˛ et al., 2011). Consequently, that high and Kopcewicz, 2002; Kesy amount of endogenous JA, which was due to the accumulation of AOS and AOC at non-inductive low temperatures, suggests that this phytohormone may prevent the formation of flower buds in that species. In conclusion, the elevated level of LOX and the low amount of 9(S)-HPOT at 30 ◦ C may have led to a greater accumulation of KODA, a flower-inducing promoter in P. nil. Furthermore, the large quantity of JA measured in leaves at 15 ◦ C probably blocked flower bud formation. These results suggest that this developmental process in P. nil depends on growing temperature, possibly being regulated by interactions among LOX metabolites in which KODA acts as a promoter and JA is the inhibitor.
Discussion We demonstrated earlier (Nam et al., 2005) that 30 ◦ C is the optimum temperature for flower induction in P. nil, and that lower temperatures are associated with delayed flowering. In the study presented here, LOX levels and extent of activity, as determined by UV absorption and immunoblotting analysis, peaked at 30 ◦ C but were slightly decreased at 35 ◦ C. These results indicate that temperature regulates the formation of flower buds through the action of LOX. In plants, there are wide variations in the ratios between 9(S)and 13(S)-HPOT (Royo et al., 1996; Kim et al., 2003). Here, the endogenous content of 13(S)-HPOT was much higher than that of 9(S)-HPOT. We might expect that the profile depicting temperature dependence of 9(S)- and 13(S)-HPOT would be equivalent to that for LOX because both are catalyzed by LOX enzymes. The amount of 9(S)-HPOT was very low, and its profile corresponded to the behavior of LOX. Its level relative to that of 13(S)-HPOT also was low, with only a negligible amount measured at the cooler temperature. By contrast, the 13-LOX pathway was characterized by a high level of 13(S)-HPOT, which showed a temperature dependence similar to that of the LOX protein. High levels of AOS accumulated in leaves when plants were grown at 15 ◦ C, perhaps because of the low level of the substrate 9(S)-HPOT. To acclimate to low and unfavorable temperature conditions, P. nil may produce a large amount of this protein in order to facilitate KODA production. By contrast, we had earlier noted that, for potato tuberization, the level of 9(S)-HPOT was instead much higher than that of 13(S)-HPOT (Nam et al., 2008). In a series of reactions, the presence of a small amount of an intermediate might possibly be attributed to asymmetric production and consumption of the substance. That is, if the successive reaction kinetics is slow, the substance will be accumulated, and vice versa. Thus, the low level of 13(S)-HPOT measured during tuberization implied a fast reaction mechanism to JA. Likewise, the low level of
Acknowledgments The authors thank Prof. C. Wasternack for supplying the antiAOC antibodies, Dr. F. Schaller and Prof. E.W. Weiler for the anti-AOS antibodies, and Prof. J.J. Sanchez-Serrano for the anti-LOX antibodies.
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Journal of Plant Physiology journal homepage: www.elsevier.de/jplph
Interactions among LOX metabolites regulate temperature-mediated flower bud formation in morning glory (Pharbitis nil) Kyong-Hee Nam a,b , Teruhiko Yoshihara a,∗ a b
Laboratory of Bio-organic Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kitaku, Sapporo 060-8589, Japan Bio-Evaluation Center, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon, Chungbuk 363-883, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 May 2012 Received in revised form 27 July 2012 Accepted 27 July 2012 Keywords: Hydroperoxy linolenic acid Jasmonic acid 9,10-Ketol-octadecadienoic acid Lipoxygenase Theobroxide
a b s t r a c t We examined the relationship between temperature (15–35 ◦ C) and flower induction as it is influenced by linolenic acid (LA) cascade products, lipoxygenase (LOX; EC 1.13.11.12), allene oxide synthase (AOS; EC 4.2.1.92), and allene oxide cyclase (AOC; EC 5.3.99.6) generated in morning glory (Pharbitis nil Choisy). The maximum amount of LOX protein was detected when plants were grown at 30 ◦ C, whereas endogenous AOS and AOC proteins were markedly accumulated at 15 ◦ C. Although both test levels of 9(S)- and 13(S)hydroperoxy linolenic acid (HPOT) showed similar temperature dependencies, reflecting the profile of LOX, the relative amount of 13(S)-HPOT was much higher than that of 9(S)-HPOT, regardless of temperature regime. This implied a faster reaction pathway to 9,10-␣-ketol octadecadienoic acid (KODA) in the LA cascade. In the 13(S)-HPOT pathway, the highest level of endogenous jasmonic acid (JA) was observed at 15 ◦ C. Our results suggest that at a high temperature (30 ◦ C), 9(S)-HPOT may be readily metabolized into KODA to promote flower bud formation. By contrast, at a low temperature, high levels of AOS and AOC result in an accumulation of JA that inhibits this developmental process. Accordingly, depending on the growing temperature, flower bud formation in P. nil is possibly regulated by the interactions among LOX metabolites, with KODA serving as a promoter and JA as an inhibitor. © 2012 Elsevier GmbH. All rights reserved.
Introduction Photoperiod and temperature are major environmental factors that influence flower induction and development. Morning glory (Pharbitis nil) is a very sensitive short day (SD) plant that can be induced to produce flower buds after exposure to a single 16-h dark period (Imamura et al., 1966). However, even under SD conditions, flowering is stimulated at 24–30 ◦ C, but not at 12–18 ◦ C (Reese and Erwin, 1997; Nam et al., 2005). One of the 9-lipoxygenase (LOX; EC 1.13.11.12) derived metabolites, 9,10-␣-ketol octadecadienoic acid (KODA), has been isolated as a flower-inducing factor from Lemna paucicostata (Fig. 1; Takimoto et al., 1989, 1991, 1994; Yokoyama et al., 2000; Yamaguchi et al., 2001). During a critical photoperiod for flower induction in P. nil, KODA levels are also transiently increased in the cotyledons and immature floral buds but not in the foliar buds (Suzuki et al., 2003; Yokoyama et al., 2005).
Abbreviations: AOC, allene oxide cyclase; AOS, allene oxide synthase; CO, CONSTANS; FT, FLOWERING LOCUS T; GC–SIM-MS, gas chromatography–selected ion monitoring-mass spectrometry; HPOT, hydroperoxy linolenic acid; JA, jasmonic acid; KODA, 9,10-ketol-octadecadienoic acid; LA, linolenic acid; LD, long day; LOX, lipoxygenase; Me-JA, methyl jasmonate; PHYB, phytochrome B; SD, short day; TA, tuberonic acid; TAG, tuberonic acid glucoside. ∗ Corresponding author. Tel.: +81 166 48 3121; fax: +81 166 48 8718. E-mail address: [email protected] (T. Yoshihara). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.07.009
Jasmonic acid (JA), which is derived from 13(S)-hydroperoxy linolenic acid (HPOT) by 13-LOX catalysis, might be involved in potato tuberization (Pelacho and Mingo-Castel, 1991; Pruski et al., 2002; Sarkar et al., 2006). Several researchers have suggested that LOX is associated with substances that participate in the formation of potato tubers, especially tuberonic acid glucoside (TAG), a derivative of JA (Yoshihara et al., 1989; Kolomiets et al., 2001; Farmaki et al., 2007). In P. nil, exogenously applied methyl jasmonate (Me-JA) inhibits flowering, with its influence being dependent on ˛ photoperiod conditions (Maciejewska and Kopcewicz, 2002; Kesy et al., 2011). In addition, substances that block jasmonate biosynthesis, such as aspirin, phenidone, and ibuprofen, can partially recover the impeding effect of far-red light on flowering in P. nil ˛ et al., 2011). (Maciejewska et al., 2004; Kesy Results from experiments with Arabidopsis thaliana have suggested that LOX mediates a photoperiodic signal in the transition from vegetative to reproductive growth (Ye et al., 2000). We have previously proposed that this protein may directly interact with light and temperature to regulate both flower induction and potato tuberization (Nam et al., 2005). LOX is activated at tuber-inducing low temperatures, and large amounts of JA, tuberonic acid (TA), and TAG are metabolized in potato (Nam et al., 2005, 2008). It is also possible that LOX-derived metabolites promote tuberization by antagonizing the effect of gibberellin (GA) (Sarkar, 2008). Theobroxide, a natural product isolated from culture filtrates of the fungus Lasiodiplodia theobromae, can induce potato tubers
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Fig. 1. Linolenic acid cascade.
and P. nil flower buds under non-inductive photoperiods (Yoshihara et al., 2000). This compound also induces the development of both tissue types even at non-inductive high temperatures (Nam and Yoshihara, 2011). Application of theobroxide to the leaf surfaces of potato and P. nil enables developmental transformations under unfavorable environmental conditions by enhancing both the activity of LOX and endogenous levels of JA (Gao et al., 2003; Yang et al.,
2004; Kong et al., 2005). Consequently, LOX-derived metabolites are involved not only in potato tuberization, but also in flower bud formation by morning glory. Both of these processes are controlled by signals generated in the leaves, which then travel throughout the plant to the shoot apical meristems for flowering or to the underground stolons for tuberization (Suárez-López, 2005). A. thaliana CONSTANS (AtCO)
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and phytochrome B (PHYB) produce inductive and inhibitory signals that balance flowering and tuberization (Sarkar, 2008, 2010). Under non-inductive long days, FLOWERING LOCUS T (FT) acts as a key mobile signal that controls not only flowering transitions but also the induction of potato tuberization (Navarro et al., 2011). Although it is somewhat clear that a certain mechanism exists to balance flowering and tuberization, depending on environmental cues, little is known about how that mechanism operates. We previously assessed the relationship between LOX activity and temperature on flower bud formation and proposed that this protein might modulate temperature-dependent induction (Nam et al., 2005). Here, we report on our investigation of LOX-derived metabolites and various enzymes in the linolenic acid (LA) cascade. We again used leaves of P. nil to examine how flower induction may be a function of temperature. Materials and methods Plant materials and temperature treatments Seeds of dwarf-type morning glory, Pharbitis nil Choisy cv. Sun Smile, were obtained from Takii Seed Co. (Kyoto, Japan). They were sown in pots filled with a mixture of peat moss and perlite (1:1, v/v). The resultant plants were cultivated in a growth chamber (NK System, Biotron NC 350, Japan) at 25 ◦ C under long day (LD) conditions (18 h light/6 h dark). Growth and temperature treatments were applied as previously described (Nam et al., 2005). When the seedlings were three weeks old, they were transferred to growth chambers and cultured under SD conditions (10 h light/14 h dark) at 15, 20, 25, 30, or 35 ◦ C. Leaves were collected after two and three weeks of this temperature treatment, when flower buds began to appear. Determining levels of endogenous 9(S)- and 13(S)-HPOT Hydroperoxy linolenic acid (HPOT) was purified and quantified as described by Göbel et al. (2002), with simple modifications. One gram of frozen leaf tissue was extracted in 10 mL of extraction buffer (3:2 hexane:2-propanol). After (6Z,9Z,11E,13S)-3-Hydroxy6,9,11-octadeca-trienoic acid (␥-13-HOT) was added as an internal standard, the extract was centrifuged at 4500 × g for 10 min at 4 ◦ C. The clear upper phase was collected and the pellet was extracted three times with 3 mL of extraction buffer. A 6.7% (w/v) solution of potassium sulfate was then added to the combined organic phases. After thorough mixing, the upper layer containing the oxylipin metabolites was dried and dissolved in CH3 CN/H2 O/CH3 COOH (55:45:0.02, v/v/v). The reaction products were analyzed by reverse phase-HPLC, using a Wakosil PTH column (4.6 mm × 250 mm). The mobile phase consisted of CH3 CN/H2 O/CH3 COOH (55:45:0.02, v/v/v) at a flow rate of 1.4 mL min−1 , and absorbance at 234 nm was recorded. Retention times for 9(S)-HPOT (16.1 min) and 13(S)-HPOT (17.0 min) were monitored by authentic standards. The 13(S)HPOT and ␥-13-HOT were prepared by incubating either ␣-LA or ␥-LA with soybean LOX (Hamberg and Gotthammar, 1973). The 9(S)-HPOT was obtained by incubating ␣-LA with tomato fruit homogenates (Matthew et al., 1977). Determining levels of endogenous JA The JA content was analyzed according to the method of Matsuura et al. (2002). Leaf tissues (1 g) were first soaked in 10 mL of 80% aqueous MeOH (10 mL) for 48 h and then filtered. As an internal standard, [10,11,11,12,12,12-2 H6 ]-JA ([d]-JA) was added at a final concentration of 0.5 g g−1 fresh weight (FW). The filtrate was evaporated under reduced pressure, re-dissolved in H2 O, adjusted to pH 2–3 with 4 M HCl, and extracted with EtOAc
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(15 × 3 mL). The combined organic layers were extracted with saturated NaHCO3 (15 × 3 mL). The aqueous layer was again adjusted to pH 2–3 with 4 M HCl and extracted with EtOAc (15 × 3 mL). The combined organic layers were concentrated, and the residue was dissolved in 1 mL of H2 O and applied to a Bond Elut C18 cartridge (Varian; CA, USA). This cartridge was successively washed with H2 O (1 × 2 mL) and MeOH/H2 O (4:1, v/v; 1.3 × 3 mL). The 80% MeOH eluate was concentrated in vacuo, and the residue was applied to a Bond Elut DEA cartridge (Varian). After the cartridge was successively washed with MeOH (1 × 2 mL) and 1 M AcOH/MeOH (1 × 4 mL), the AcOH/MeOH eluate was concentrated and dissolved in 1 mL of MeOH. Diazomethane, in diethyl ether, was then added to a MeOH solution for methylation. The concentrated residue was purified on an HPLC system (Hitachi L7100; HITACHI, Japan) equipped with a YMC-Pack ODS column (300 mm × 10 mm; YMC Co. Ltd., Japan), at a flow rate of 2.5 mL min−1 . The solvent system was MeOH/H2 O/AcOH (90:10:0.1, v/v/v). The JA fractions were evaporated, and the resultant residue was analyzed by gas chromatography–selected ion monitoring-mass spectrometry (GC–SIM-MS) (QP5000 System; Shimadzu, Japan), using a ZB-1 column (0.25 mm × 30 m, 0.5-m phase thickness). Our GC temperature program followed 80 ◦ C for 1 min, 80–290 ◦ C at 20 ◦ C/min, and 290 ◦ C for 5 min. The injector and detector temperatures were 200 ◦ C and 280 ◦ C, respectively. Amounts of endogenous JA were calculated by comparing peak areas with corresponding internal standards. Measurement of LOX activity The assay for LOX activity was performed as we previously described (Nam et al., 2005). Briefly, 0.1 g of frozen leaf powder was homogenized in 1 mL of 0.1 M phosphate buffer (pH 7.3) and centrifuged at 15,000 × g for 20 min at 4 ◦ C. The crude enzyme that was obtained was then added to LA and 0.1 M sodium acetate buffer solution (pH 4.4), and was analyzed with a HITACHI U2800 spectrophotometer. The increase in absorbance at 234 nm was measured to monitor the formation of conjugated-diene compounds, e.g. 9-HPOT and 13-HPOT. One unit of LOX activity was defined as 1 mol of product formed per milligram of protein per minute. Protein contents were determined by the method of Bradford (1976), using bovine serum albumin (BSA) as a standard. Protein extraction We added 0.1 g samples of leaf tissue to 1 mL of 0.1 M phosphate buffer (pH 7.3) and centrifuged at 15,000 × g for 20 min at 4 ◦ C. The supernatant was used as the enzyme extract. Protein contents, with BSA as a standard, were evaluated by the method of Bradford (1976). Electrophoresis and immunoblot analysis Proteins were separated on a 10% SDS-polyacrylamide gel according to the method of Laemmli (1970). Electrophoretically separated proteins were transferred from those gels to nitrocellulose sheets per the protocol of Towbin et al. (1979). Blots were developed using chemiluminescence reagent (Perkin-Elmer Life Sciences) according to the manufacturer’s instructions. We used rabbit polyclonal antibodies raised against potato LOX (Royo et al., 1996), plus antibodies against the recombinant allene oxide synthase (AOS; EC 4.2.1.92) of Arabidopsis thaliana (Laudert and Weiler, 1998) and the recombinant allene oxide cyclase (AOC; EC 5.3.99.6) of A. thaliana (Ziegler et al., 2000). All antibodies were used at 1:5000 dilutions.
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Fig. 3. Endogenous levels of jasmonic acid in leaves of dwarf-type Pharbitis nil ‘Sun Smile’ after 2 and 3 weeks of temperature treatment. Values represent means of 3 independent measurements ± SE. **Significantly different from control temperature (15 ◦ C) at p < 0.01, according to Bonferroni t-test.
Endogenous levels of JA in leaves of P. nil grown at various temperatures The endogenous level of JA was influenced by temperature (Fig. 3). After both two and three weeks of treatment, JA levels were greatest at 15 ◦ C but decreased at higher temperatures. A detailed profile of JA content showed a rapid decline between 15 ◦ C and 30 ◦ C, and a moderate decrement between 30 ◦ C and 35 ◦ C. Regardless of temperature, endogenous JA levels were always higher after two weeks than after three weeks. Fig. 2. Endogenous levels of 9(S)-HPOT (A) and 13(S)-HPOT (B) in leaves of dwarftype Pharbitis nil ‘Sun Smile’ after 2 and 3 weeks of temperature treatment. Values represent means of 3 independent measurements ± SE. *Significantly different from control temperature (15 ◦ C) at p < 0.05, according to Bonferroni t-test. 9(S)- and 13(S)-HPOT are 9(S)- and 13(S)-hydroperoxy linolenic acids, respectively.
Statistical analysis To compare differences among treatments, we performed statistical analyses with ANOVA and Bonferroni t-tests at p < 0.05 and p < 0.01, respectively. Data were averaged from three independent experiments, with 10 seedlings examined for each temperature treatment.
Correlation between temperature and LOX activity Similar to the findings from our previous experiments (Nam et al., 2005), LOX activities, as measured by UV absorption (A234 nm) of the resulting diene, were dependent upon temperature (Fig. 4). The highest levels were recorded in seedlings grown at 30 ◦ C. Activity increased gradually for plants between 15 ◦ C and 30 ◦ C but was slightly decreased when they were exposed to 30 ◦ C or 35 ◦ C. At all temperatures tested, LOX activity was higher in the second week than in the third week.
Results Endogenous levels of 9(S)- and 13(S)-HPOT in leaves of P. nil grown at various temperatures To examine the effect of temperature on peroxidation from LA, we measured the endogenous contents of 9(S)- and 13(S)-HPOT from the leaves of P. nil after seedlings were grown under SD conditions for two or three weeks at 15, 20, 25, 30, or 35 ◦ C (Fig. 2). At all temperatures tested, levels of 9(S)-HPOT were relatively lower than those of 13(S)-HPOT. For 9(S)-HPOT, that level gradually increased with temperature, peaking at 30–35 ◦ C after both two and three weeks (Fig. 2A). Levels of 13(S)-HPOT were significantly temperature-dependent (Fig. 2B). After two weeks, the amount of 13(S)-HPOT increased with temperature, peaking at 30 ◦ C but declining somewhat at 35 ◦ C. Contents of 13(S)-HPOT were lower after three weeks than those measured after two weeks.
Fig. 4. Effect of temperature on lipoxygenase activity in leaves of dwarf-type Pharbitis nil ‘Sun Smile’ collected 2 (䊉) and 3 () weeks after temperature treatment. LOX activity was measured at 20–25 ◦ C. Values represent means of 3 independent measurements ± SE. **Significantly different from control temperature (15 ◦ C) at p < 0.01, according to Bonferroni t-test.
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Fig. 5. Immunoblot analysis of LOX (lipoxygenase), AOS (allene oxide synthase), and AOC (allene oxide cyclase) proteins in leaves from 3-week-old seedlings of dwarf-type Pharbitis nil ‘Sun Smile’. Leaves were sampled 2 weeks after temperature treatments. A total of 20 g of protein was applied to each lane. Immunoblots were probed with antibodies for anti-LOX, anti-AOS, and anti-AOC, at 1:5000 dilutions. Equivalent experiments were repeated at least 3 times.
Accumulations of LOX, AOS, and AOC proteins in the leaves of P. nil grown at different temperatures Because AOS and AOC proteins are important in the reactions of LOX metabolites, as demonstrated within the LA cascade, we examined the relationship between temperature and the amounts of AOS and AOC produced. Immunoblot analyses revealed that endogenous LOX accumulations were low between 15 ◦ C and 25 ◦ C but high between 30 ◦ C and 35 ◦ C, with the maximum accumulation recorded at 30 ◦ C (Fig. 5A). Levels of endogenous AOS protein were markedly higher at 15 ◦ C, but were at minimal amounts at 30 ◦ C and 35 ◦ C (Fig. 5B). Similarly, the levels of AOC protein were relatively large between 15 ◦ C and 25 ◦ C, with lower levels being measured in leaves exposed to either 30 ◦ C or 35 ◦ C (Fig. 5C).
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9(S)-HPOT detected during P. nil flowering would suggest facile reaction pathways to KODA. However, this assumption still remains controversial. For example, in an earlier study by Royo et al. (1996), the expression level of 9-LOX was always higher than that of 13LOX in developing potato tubers, which is the opposite of what we found here during floral bud formation. Even after its consumption by the JA biosynthesis pathway via the AOS/AOC branch, the amount of 13(S)-HPOT substrate remained at a much higher level than that of 9(S)-HPOT at all temperatures. To convert the unstable allene oxide to the first cyclic precursor of JA, the association between AOC and AOS must be maintained in close proximity (Farmaki et al., 2007). Here, in response to low temperature, that AOS/AOC branch to JA biosynthesis was stimulated, resulting in high endogenous amounts of both enzymes. The endogenous level of JA was high when plants were exposed to 15 ◦ C, but it declined as the temperature rose. JA possibly functions in the inhibition of flowering in P. nil (Maciejewska ˛ et al., 2011). Consequently, that high and Kopcewicz, 2002; Kesy amount of endogenous JA, which was due to the accumulation of AOS and AOC at non-inductive low temperatures, suggests that this phytohormone may prevent the formation of flower buds in that species. In conclusion, the elevated level of LOX and the low amount of 9(S)-HPOT at 30 ◦ C may have led to a greater accumulation of KODA, a flower-inducing promoter in P. nil. Furthermore, the large quantity of JA measured in leaves at 15 ◦ C probably blocked flower bud formation. These results suggest that this developmental process in P. nil depends on growing temperature, possibly being regulated by interactions among LOX metabolites in which KODA acts as a promoter and JA is the inhibitor.
Discussion We demonstrated earlier (Nam et al., 2005) that 30 ◦ C is the optimum temperature for flower induction in P. nil, and that lower temperatures are associated with delayed flowering. In the study presented here, LOX levels and extent of activity, as determined by UV absorption and immunoblotting analysis, peaked at 30 ◦ C but were slightly decreased at 35 ◦ C. These results indicate that temperature regulates the formation of flower buds through the action of LOX. In plants, there are wide variations in the ratios between 9(S)and 13(S)-HPOT (Royo et al., 1996; Kim et al., 2003). Here, the endogenous content of 13(S)-HPOT was much higher than that of 9(S)-HPOT. We might expect that the profile depicting temperature dependence of 9(S)- and 13(S)-HPOT would be equivalent to that for LOX because both are catalyzed by LOX enzymes. The amount of 9(S)-HPOT was very low, and its profile corresponded to the behavior of LOX. Its level relative to that of 13(S)-HPOT also was low, with only a negligible amount measured at the cooler temperature. By contrast, the 13-LOX pathway was characterized by a high level of 13(S)-HPOT, which showed a temperature dependence similar to that of the LOX protein. High levels of AOS accumulated in leaves when plants were grown at 15 ◦ C, perhaps because of the low level of the substrate 9(S)-HPOT. To acclimate to low and unfavorable temperature conditions, P. nil may produce a large amount of this protein in order to facilitate KODA production. By contrast, we had earlier noted that, for potato tuberization, the level of 9(S)-HPOT was instead much higher than that of 13(S)-HPOT (Nam et al., 2008). In a series of reactions, the presence of a small amount of an intermediate might possibly be attributed to asymmetric production and consumption of the substance. That is, if the successive reaction kinetics is slow, the substance will be accumulated, and vice versa. Thus, the low level of 13(S)-HPOT measured during tuberization implied a fast reaction mechanism to JA. Likewise, the low level of
Acknowledgments The authors thank Prof. C. Wasternack for supplying the antiAOC antibodies, Dr. F. Schaller and Prof. E.W. Weiler for the anti-AOS antibodies, and Prof. J.J. Sanchez-Serrano for the anti-LOX antibodies.
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