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Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata
Journal of Plant Physiology 169 (2012) 987–991
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Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata Aya Shimakawa a , Takeshi Shiraya b , Yuta Ishizuka c , Kaede C. Wada a , Toshiaki Mitsui a,b , Kiyotoshi Takeno a,c,∗ a
Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan c Department of Biology, Faculty of Science, Niigata University, Ikarashi, Niigata 950-2181, Japan b
a r t i c l e
i n f o
Article history: Received 23 January 2012 Received in revised form 24 February 2012 Accepted 25 February 2012 Keywords: Flowering Lemna paucicostata Salicylic acid Stress
a b s t r a c t The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering. © 2012 Elsevier GmbH. All rights reserved.
Introduction Flowering in many plant species is regulated by environmental cues, such as the length of the night in photoperiodic flowering and the temperature in vernalization (Thomas and Vince-Prue, 1997), and recent studies indicate that stress also regulates flowering (Wada and Takeno, 2010; Takeno, 2012). The short-day (SD) plant, Pharbitis nil (synonym Ipomoea nil), can be induced to flower under long days (LD) when grown under poor-nutrition or lowtemperature stress conditions (Hatayama and Takeno, 2003; Wada et al., 2010b). The SD plant, Perilla frutescens var. crispa, can flower under LD when grown under low-intensity light stress (Wada et al., 2010a). Ultraviolet radiation stress (Martínez et al., 2004) ˇ and nutrition stress (Koláˇr and Senková, 2008) also induced early flowering in Arabidopsis thaliana. Non-photoperiodic flowering has been sporadically reported in several plant species other than those
Abbreviations: AOA, aminooxyacetic acid; AOPP, l-2-aminooxy-3phenylpropionic acid; FT, FLOWERING LOCUS T; KODA, ␣-ketol of octadecadienoic acid; LD, long days; PAL, phenylalanine ammonia-lyase; SA, salicylic acid; SD, short days. ∗ Corresponding author at: Department of Biology, Faculty of Science, Niigata University, Ikarashi, Niigata 950-2181, Japan. Tel.: +81 25 262 6369; fax: +81 25 262 6369. E-mail address: [email protected] (K. Takeno). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2012.02.009
mentioned above, and a review of those reports suggested that most of the factors responsible for flowering can be regarded as stresses (Wada and Takeno, 2010; Takeno, 2012), including high- or low-intensity light, high or low temperature, drought, poor nutrition and mechanical stimulation. Moreover, the plants induced to flower by stresses produced fertile seeds, and the progeny developed normally (Wada et al., 2010a, b). Plants can modify their developmental processes to adapt to stress conditions, and stressinduced flowering is one of such adaptations: plants flower as an emergency response when stressed, thus ensuring their ability to produce the next generation. Through this mechanism, plants can preserve the species, even under unfavorable environmental conditions. Therefore, stress-induced flowering can be considered as universal and as important as photoperiodic flowering and vernalization (Wada and Takeno, 2010; Takeno, 2012). A transmissible flowering stimulus, such as florigen in photoperiodic flowering, is involved in the stress-induced flowering of P. nil (Wada et al., 2010b). The P. nil ortholog of FLOWERING LOCUS T (FT), PnFT2, is involved in the stress-induced flowering of P. nil. However, because the activity of phenylalanine ammonialyase (PAL) and the salicylic acid (SA) content increase when plants are stressed (Dixon and Paiva, 1995; Scott et al., 2004), it is also possible that SA functions as the flowering stimulus in stressinduced flowering. PAL catalyzes the conversion of phenylalanine to t-cinnamic acid, and SA is one of the metabolic intermediates derived from t-cinnamic acid. Aminooxyacetic acid (AOA) and
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A. Shimakawa et al. / Journal of Plant Physiology 169 (2012) 987–991
l-2-aminooxy-3-phenylpropionic acid (AOPP), which function as PAL inhibitors (Kessmann et al., 1990; Appert et al., 2003), inhibited stress-induced flowering in P. nil and P. frutescens (Hatayama and Takeno, 2003; Wada et al., 2010a, b). In addition, the inhibitory effect of AOA and AOPP was negated by t-cinnamic acid, SA and benzoic acid (a precursor of SA) in P. nil under stress conditions (Hatayama and Takeno, 2003; Wada et al., 2010b). These facts suggest that SA is involved in the regulatory mechanism of stressinduced flowering and that this flowering response may be regulated by either PnFT2 or SA in P. nil. However, no evidence has yet demonstrated that the endogenous SA levels increase when plants are induced to flower through the application of stress factors. Recently, we found that the SD plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower under noninductive photoperiodic conditions when grown under conditions of starvation stress, results that are reminiscent of the seminal article by Cleland and Ajami (1974), who found that exogenously applied SA can induce flowering in Lemna gibba. Since the publication of this pioneering study, many articles have reported that SA and its related compounds, including benzoic acid, induce flowering in L. gibba, L. paucicostata and some other Lemnaceous species under non-inductive photoperiodic conditions (reviewed by Kandeler, 1985). However, no positive correlation was found between the endogenous level of benzoic acid and photoperiodic conditions in L. gibba and L. paucicostata (Fujioka et al., 1983). SA is therefore not considered to be an endogenous regulatory factor in photoperiodic flowering. In the present study, we found that flowering in L. paucicostata was induced by both photoperiodic cues and stress factors. Thus, we postulated that SA is an endogenous regulating factor in stress-induced flowering but not in photoperiodic flowering. Accordingly, this study examined whether the endogenous SA level would increase once flowering was induced by starvation stress in L. paucicostata. Materials and methods Plant materials and growth conditions SD plant Lemna paucicostata Hegelm. (synonym Lemna aequinoctialis Welwitsch) strains 151, 441 and 6746 (provided by Prof. Emeritus A. Takimoto, Kyoto University, Kyoto, Japan) were used. The fronds of each strain were aseptically cultured in 1/2 strength Hutner’s medium supplemented with 1% sucrose (Takimoto et al., 1989). The number of fronds increased through the vegetative multiplication in this medium, and the younger generations were routinely transplanted to new media to maintain the strains. Unless otherwise specified, the assay medium used was 1/10 strength M medium supplemented with 1% sucrose and 1 M benzylaminopurine (pH 4.0) (Fujioka et al., 1986). Uniformly grown three-frond colonies were chosen from the stock culture and aseptically transferred to a 100 mL Erlenmeyer flask containing 20 mL of assay medium (1 colony/flask). In general, the fronds were grown at 25 ◦ C under a 16-h light and 8-h dark period (LD) conditions. White light of approximately 100 mol m−2 s−1 at the frond level was provided by fluorescent lamps (Toshiba FL40SSW/37, Toshiba Corporation, Tokyo, Japan). Incandescent lamps (30 mol m−2 s−1 , Toshiba RF100V57WM, Toshiba Corporation, Tokyo, Japan) were used when far-red light was added to white light. Treatment with stress or SD As the stress treatment, the fronds were cultured in tap water without any additional components instead of the usual assay medium. The tap water used was drinking water supplied by public water supply system and contains 0.6–0.9 mg/L nitrite plus
nitrate whereas the 1/2 strength Hutner’s medium used as control medium contains 100 mg/L NH4 NO3 . As the SD treatment, the fronds were cultured under 8-h light and 16-h dark period (SD) conditions.
Treatment with chemicals Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (AOPP) were used as inhibitors of the PAL enzyme. Both of the compounds were obtained from Wako Pure Chemicals Industries (Osaka, Japan). Either AOA or AOPP was added to the tap water in which the fronds were cultured; SA (Wako Pure Chemicals Industries, Osaka, Japan) was also added to the tap water.
Scoring of the flowering response All of the fronds were dissected using a binocular microscope to determine whether flowers had formed. A flowering response was defined as the percentage of fronds with flowers of all of the fronds cultured in a flask (% flowering). The number of fronds per flask was presented as an indicator of the vegetative growth. Three flasks were used for each treatment, and the mean and standard error (SE) of the percent flowering and the number of fronds per flask were calculated. Each experiment was repeated several times.
Analysis of SA The fronds were harvested, frozen in liquid nitrogen and stored at −80 ◦ C prior to the analysis. The frozen fronds were homogenized in 70% methanol (5 mL/g fresh weight of tissue) using a mortar and pestle with 100 ng deuterium-labeled 2-hydroxybenzoic-3,4,5,6d4 acid (CDN Isotopes, Quebec, Canada) as an internal standard. The homogenate was hand-centrifuged to precipitate the cellular debris, and the supernatant was evaporated in vacuo at 37 ◦ C. The pH of the resulting aqueous phase was adjusted to 3.0 and partitioned with an equal volume of ethyl acetate three times. The resulting acidic ethyl acetate-soluble fraction was concentrated in vacuo at 37 ◦ C and redissolved in 2 mL of 60% methanol. The methanol solution was separated using a Sep-Pak Plus C18 cartridge (Waters Corporation, Milford, USA). The eluate was evaporated in vacuo to dryness and redissolved in 50 L of 70% methanol containing 20 mM sodium acetate (pH 2.5). The methanol solution was injected into an ODS column (3 mm × 250 mm, Imtakt Cadenza CD-C18, Imtakt Corp., Kyoto, Japan) connected to a high performance liquid chromatograph (Hitachi L-6200, Hitachi Co., Tokyo, Japan) and separated using 70% methanol with 20 mM sodium acetate (pH 2.5) at a flow rate of 0.15 mL min−1 . The column temperature was 40 ◦ C. The eluate with a retention time of the authentic SA was collected as the SA fraction. The collected sample was subjected to liquid chromatography–mass spectrometry using an LTQ Orbitrap XL (Thermo Fischer Scientific, Bremen, Germany) at a flow rate of 5–10 L min−1 for 3 min, and the ESI electron spray injection method was used. The ionization voltage was set to 5 kV, and the MS scan range was m/z 200–400. The data processing was performed using Xcalibur Qual Browser (version 2.1, Thermo Fisher Scientific, Bremen, Germany). The amount of endogenous SA was estimated using the detected peak intensities of the internal standard (m/z 141.05) and the endogenous SA (m/z 137.02). The analysis of each sample was repeated 3–6 times, and the mean and SEs were calculated. The experiment was repeated 3 times, with similar results.
A. Shimakawa et al. / Journal of Plant Physiology 169 (2012) 987–991 Table 1 Flowering induced by poor-nutrition conditions in Lemna paucicostata. Strain
Conditions
Flowering (%)
Fronds/flask
151
SD • Nutrition LD • Nutrition LD • Poor nutrition
15.6 ± 3.15a 0.521 ± 0.114c 6.3 ± 1.0b
1283 ± 125.8a 1639 ± 59.88a 47 ± 3.2b
441
SD • Nutrition LD • Nutrition LD • Poor nutrition
90.0 ± 3.60a 0 ± 0c 7.2 ± 1.7b
1472 ± 185.2a 863 ± 49.9b 37 ± 1.9c
6746
SD • Nutrition LD • Nutrition LD • Poor nutrition
84.7 ± 3.41a 1.39 ± 0.188c 27 ± 0.42b
341 ± 45.7b 830 ± 78.8a 33 ± 3.7c
Three strains of L. paucicostata were cultured in tap water (poor nutrition) or 1/10 strength M medium supplemented with 1% sucrose and 1 M benzylaminopurine (nutrition) at 25 ◦ C under conditions of a 16-h light and 8-h dark period (LD) or conditions of an 8-h light and 16-h dark period (SD) for 4 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed within the same strain.
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Table 3 Effects of PAL inhibitors on the flowering in Lemna paucicostata strain 6746 cultured under poor-nutrition conditions. PAL inhibitor
Concentration (M)
Flowering (%)
AOA
0 10−7 10−6 10−5
11 13 4.4 0
± ± ± ±
0.61a 3.0a 2.6ab 0b
30 43 31 5.0
± ± ± ±
1.7b 1.9a 3.3b 0.58c
AOPP
0 10−6 10−5 10−4
8.3 6.5 12 0
± ± ± ±
2.8b 2.3b 5.5ab 0a
60 48 47 61
± ± ± ±
0.88a 5.2ab 1.7b 5.2ab
Fronds/flask
L. paucicostata strain 6746 was cultured in tap water (poor-nutrition conditions) supplemented with aminooxyacetic acid (AOA) or l-2-aminooxy-3-phenylpropionic acid (AOPP) at 25 ◦ C under conditions of a 16-h light and 8-h dark period for 3 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed using the same inhibitor.
The results indicate that cultivation under poor-nutrition conditions functioned as a stress factor and induced flowering under non-inductive photoperiodic conditions in L. paucicostata.
Results Induction of flowering by poor nutrition Three strains of L. paucicostata were cultured in tap water under non-inductive LD conditions for 4 weeks, and flowering occurred in each of the strains (Table 1). The control plants cultured in a nutrient medium showed no or almost no flowering response under the LD conditions, whereas the flowering response under the poornutrition conditions was the strongest in strain 6746. All three of the strains cultured under the SD conditions as a positive control flowered, and their flowering percentages were higher than those under the poor-nutrition treatment. The number of fronds per flask cultured in tap water was much smaller than that cultured in the nutrient medium for all of the tested strains. The three strains were cultured in tap water under LD conditions for 2–4 weeks to examine the timing of the flowering response. A weak flowering response was detected after 2 weeks of the stress treatment in strains 151 and 6746, and an apparent flowering response was detected after 3 weeks of the stress treatment in all of the strains (Table 2). The flowering percentage did not change or rather decreased from the third week to the fourth week. The number of fronds per flask did not significantly increase during the cultivation from the second week to the fourth week, with the exception of strain 441. The fronds were healthy and green in color when observed after 2 weeks in culture but turned to a whitishyellow after the third week in all of the strains (data not shown). These observations indicate that vegetative growth was suppressed when the plants were grown in the tap water. Table 2 Flowering in Lemna paucicostata cultured under poor-nutrition conditions for different lengths of time. Strain
Culture period (weeks)
Flowering (%)
Fronds/flask
151
2 3 4
1.1 ± 1.1b 7.4 ± 1.8a 2.2 ± 1.2ab
34 ± 4.8a 44 ± 1.5a 44 ± 2.7a
441
2 3 4
0 ± 0b 5.7 ± 0.092a 1.9 ± 0.96b
27 ± 0.67b 35 ± 0.58a 34 ± 2.3a
6746
2 3 4
4.4 ± 3.1b 23 ± 0.79a 17 ± 2.3a
40 ± 3.0a 47 ± 2.7a 49 ± 2.4a
Three strains of L. paucicostata were cultured in tap water (poor-nutrition conditions) at 25 ◦ C under conditions of a 16-h light and 8-h dark period for 2–4 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed within the same strain.
PAL-inhibitor inhibition of the flowering induced by poor nutrition Strain 6746, the most sensitive strain to the starvation stress, was cultured in tap water under LD conditions to induce flowering, and PAL inhibitors were added to the tap water to examine their effects on the starvation stress-induced flowering. The flowering was completely inhibited by AOA at 10−5 M and by AOPP at 10−4 M (Table 3). The number of fronds per flask was decreased by the AOA treatment at 10−5 M, whereas the multiplication of fronds was not suppressed by AOPP at 10−4 M. Endogenous SA contents Strain 6746 was cultured in tap water for 3 weeks to induce flowering, and SA was extracted from the fronds for quantification. Significantly higher amounts of SA were detected in those fronds that flowered under the poor-nutrition conditions when compared to the vegetative fronds cultured under nutrient conditions (Table 4). This result was reproduced in three independent experiments. Promotion of flowering by exogenously applied SA Strain 6746 was cultured in tap water with added SA and grown under non-inductive LD conditions for 3 weeks. The SA promoted flowering when supplied at 3 × 10−5 M (Table 5); in contrast, flowering was inhibited by SA at 10−4 M. Discussion Three strains of the SD plant, L. paucicostata, were induced to flower when cultured in tap water under non-inductive LD conditions (Table 1). The vegetative multiplication of the fronds was restricted when cultured in tap water, which indicates that the vegetative growth of the plants was suppressed in tap water, implying that the plants were stressed (Hatayama and Takeno, 2003). In comparison with the flowering response under SD conditions, which was 15–90% (depending on the strain), the poor-nutrition conditions produced a weaker flowering response in each strain examined. However, virtually no flowering occurred under the nutrient conditions. Therefore, L. paucicostata is induced to flower by starvation stress. The time-course study showed that flowering occurred after two (strains 151 and 6746) or three (strain 441) weeks of the
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Table 4 Flowering and the endogenous salicylic acid (SA) content in Lemna paucicostata strain 6746 cultured under poor-nutrition conditions. Experiment
Conditions
Flowering (%)
Fronds/flask
I
Nutrition Poor nutrition
0.21 ± 0.1a 18 ± 2.8b
722 ± 106a 43 ± 4.9b
SA content (ng/g fw) (relative value) 66 ± 8.3a (100) 953 ± 214b (1443)
II
Nutrition Poor nutrition
0 ± 0a 5.7 ± 3.3a
620 ± 32a 36 ± 1.3b
215 ± 29.9a (100) 2280 ± 586b (1060)
III
Nutrition Poor nutrition
0 ± 0a 6.5 ± 2.1b
665 ± 152a 37 ± 6.7b
511 ± 109a (100) 1530 ± 333b (299)
L. paucicostata strain 6746 was cultured in tap water (poor nutrition) for 3 weeks or in 1/10 strength M medium supplemented with 1% sucrose and 1 M benzylaminopurine (nutrition) for 1 week. Far-red light was supplied with white light, and the plants were grown at 25 ◦ C for 3 weeks under nutrition conditions or at 23 ◦ C for 2 weeks followed by 25 ◦ C for 1 week under poor-nutrition conditions in experiment I. The plants were grown at 23 ◦ C and 25 ◦ C in experiments II and III, respectively. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed within the same experiment.
stress treatment and that the flowering response was the strongest at the third week after the start of the stress treatment in each strain (Table 2). The number of fronds per flask did not significantly increase after flowering was induced. Furthermore, the fronds became whitish-yellow in color when cultured in tap water for over 3 weeks, indicating that the fronds were withering under the prolonged stress conditions and may not have been able to continue to produce flowers. The flower of L. paucicostata is quite small and ephemeral and therefore easily decays and disappears soon after anthesis. Thus, the flowering percentages decreased from the third week to the fourth week in all of the strains. It has been reported that L. paucicostata flowers in nitrogendeficient or nitrogen-free media independently of the daylength (Tanaka, 1986; Tanaka et al., 1988, 1991). Experiments on such daylength-independent flowering were performed using a culture medium containing mineral nutrient salts and 1% sucrose with reduced or no nitrogen. In our study, however, we cultured the plants in tap water without any mineral salts or energy source. This culture condition induced the suppression of growth, indicating that the plants were stressed. Therefore, the flowering response found in the present study may be different from that under nitrogen-deficient conditions. Yamaguchi et al. (2001) isolated norepinephrine and ␣-ketol of octadecadienoic acid (KODA), from which flower-inducing factors are derived, from L. paucicostata. KODA was found to be induced by drought, heat or osmotic stress conditions (Yokoyama et al., 2000). These results are consistent with our finding that L. paucicostata has a mechanism that responds to stress and promotes flowering. However, the clarification of the relationship between these flower-promoting factors and SA requires further investigation. It has long been supposed that SA is involved in the regulation of flowering in Lemna spp.; however, the supporting evidence presented to date is merely that exogenously applied SA can induce flowering. Therefore, we attempted to reduce the endogenous SA level by inhibiting its biosynthesis to ascertain whether this inhibits flowering. SA is synthesized from t-cinnamic acid, which Table 5 Effects of salicylic acid on flowering in Lemna paucicostata strain 6746 cultured under poor-nutrition conditions. Concentration (M)
Flowering (%)
0 10−6 3 × 10−6 10−5 3 × 10−5 10−4
4.9 3.8 4.6 11 42 0.74
± ± ± ± ± ±
1.7bcd 0.17c 2.3bcd 2.5b 1.9a 0.38d
Fronds/flask 63 62 60 56 61 95
± ± ± ± ± ±
7.5b 10ab 6.7b 3.5b 2.0b 8.7a
L. paucicostata strain 6746 was cultured in tap water (poor-nutrition conditions) supplemented with salicylic acid at 25 ◦ C under conditions of a 16-h light and 8-h dark period for 3 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test.
is converted from phenylalanine by PAL in the majority of plant species (Yalpani et al., 1993); SA is also derived from isochorismate in bacteria and some plant species, including A. thaliana (Chen et al., 2009). Generally, stress induces PAL activity, resulting in the accumulation of SA and anthocyanin (Dixon and Paiva, 1995; Scott et al., 2004). Indeed, when L. gibba is induced to flower by stress, the fronds turn red in color due to the accumulation of anthocyanin, suggesting the activation of PAL (unpublished data). PAL inhibitors suppressed the stress-induced flowering in P. nil and P. frutescens, an inhibition that was overcome by SA (Wada et al., 2010a, b). These findings led us to speculate that stress-induced flowering is regulated by SA, which is synthesized by PAL. Accordingly, we applied PAL inhibitors to L. paucicostata that was cultured under stress conditions and found that AOA and AOPP completely inhibited flowering at concentrations of 10−5 M and 10−4 M, respectively (Table 3). These results suggest that SA is involved in the regulation of stress-induced flowering in L. paucicostata. We then quantified the endogenous SA content in those fronds that flowered due to the starvation stress and the control fronds without flowers finding that the SA content increased when L. paucicostata was stressed and flowered (Table 4). This is the first evidence showing that the SA content increases when flowering is induced in Lemnaceous species. Lastly, we found that exogenously applied SA promoted flowering (Table 5), and the inhibiting effect of SA at a high concentration is consistent with previous reports (Cleland and Ajami, 1974; Cleland and Tanaka, 1979). Taken together, the results presented here suggest that endogenous SA plays a role in the promotion of stress-induced flowering and not photoperiodic flowering. SA has been the focus of intensive research due to its function as an endogenous signal (Rivas-San Vicente and Plasencia, 2011), and the induction of flowering is one of the functions of SA. It was previously suggested that SA is involved in stress-induced flowering in P. nil and P. frutescens (Wada et al., 2010a, b), and the ultraviolet light stress-induced flowering response of A. thaliana was weaker in the SA-deficient NahG transgenic lines than in the wild type (Martínez et al., 2004). These facts support our conclusion that SA is involved in stress-induced flowering. The proteins derived from FT and its orthologs have been reported to be florigens (Corbesier et al., 2007; Lin et al., 2007; Tamaki et al., 2007). The expression of A. thaliana FT and sunflower HAFT, the FT homolog, were induced by SA (Martínez et al., 2004; Dezar et al., 2010), and the expression of PnFT2, an ortholog of FT in P. nil, was induced by stress conditions (Wada et al., 2010b). These findings indicate that FT, SA and stress may collaborate to regulate flowering. It is now apparent that flowering in L. paucicostata is mediated by both stress and SA. Because the L. paucicostata FT homolog has already been registered in a public database (although it remains unpublished), this plant is thus an ideal experimental material for examining the interaction between FT expression, SA synthesis and stress responses.
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Acknowledgments We are grateful to Prof. Emeritus Atsushi Takimoto, Kyoto University, for providing L. paucicostata. We thank Kazuyoshi Hasegawa for his technical assistance. References Appert C, Zon´ J, Amrhein N. Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues. Phytochemistry 2003;62:415–22. Chen Z, Zheng Z, Huang J, Lai Z, Fan B. Biosynthesis of salicylic acid in plants. Plant Signal Behav 2009;4:493–6. Cleland CF, Ajami A. Identification of the flower-inducing factor isolated from aphid honeydew as being salicylic acid. Plant Physiol 1974;54:904–6. Cleland CF, Tanaka O. Effect of daylength on the ability of salicylic acid to induce flowering in the long-day plant Lemna gibba G3 and the short-day plant Lemna paucicostata 6746. Plant Physiol 1979;64:421–4. Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, et al. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 2007;316:1030–3. Dezar CA, Giacomelli J, Manavella PA, Ré DA, Alves-Ferreira M, Baldwin IT, et al. HAHB10, a sunflower HD-Zip II transcription factor, participates in the induction of flowering and in the control of phytohormone-mediated responses to biotic stress. J Exp Bot 2010;62:1061–76. Dixon RA, Paiva L. Stress-induced phenylpropanoid metabolism. Plant Cell 1995;7:1085–97. Fujioka S, Sakurai A, Sugimoto N, Yamaguchi I, Murofushi N, Takahashi N, et al. Isolation and identification of lutein from Lemna paucicostata 381 as an inhibitor of benzoic acid-induced flowering in Lemna paucicostata 151. Agric Biol Chem 1986;50:2053–9. Fujioka S, Yamaguchi I, Murofushi N, Takahashi N, Kaihara S, Takimoto A. Flowering and endogenous levels of benzoic acid in Lemna species. Plant Cell Physiol 1983;24:235–9. Hatayama T, Takeno T. The metabolic pathway of salicylic acid rather than of chlorogenic acid is involved in the stress-induced flowering of Pharbitis nil. J Plant Physiol 2003;160:461–7. Kandeler R. Lemnaceae. In: Halevy AH, editor. CRC Handbook of Flowering, vol. 3. Boca Raton, FL: CRC Press, Inc; 1985. p. 251–79. Kessmann H, Edwards R, Geno PW, Dixon RA. Stress responses in alfalfa (Medicago sativa L.): V. Constitutive and elicitor-induced accumulation of isoflavonoid conjugates in cell suspension cultures. Plant Physiol 1990;94:227–32.
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ˇ Koláˇr J, Senková J. Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana. J Plant Physiol 2008;165:1601–9. Lin MK, Belanger H, Lee YJ, Varkonyi-Gasic E, Taoka K, Miura E, et al. FLOWERING LOCUS T protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell 2007;19:1488–506. Martínez C, Pons E, Prats G, León J. Salicylic acid regulates flowering time and links defence responses and reproductive development. Plant J 2004;37: 209–17. Rivas-San Vicente M, Plasencia J. Salicylic acid beyond defence: its role in plant growth and development. J Exp Bot 2011;62:3321–38. Scott IM, Clarke SM, Wood JE, Mur LAJ. Salicylate accumulation inhibits growth at chilling temperature in Arabidopsis. Plant Physiol 2004;135:1040–9. Takeno K. Stress-induced flowering. In: Ahmad P, Prasad MNV, editors. Abiotic Stress Responses in Plants: Metabolism, Productivity and Sustainability. New York: Springer; 2012. p. 331–45. Takimoto A, Kaihara S, Hirai N, Koshimizu K, Hosoi Y, Oda Y, et al. Flower-inducing activity of water extracts of Lemna. Plant Cell Physiol 1989;30:1018–21. Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K. Hd3a protein is a mobile flowering signal in rice. Science 2007;316:1033–6. Tanaka O. Flower induction by nitrogen deficiency in Lemna paucicostata 6746. Plant Cell Physiol 1986;27:875–80. Tanaka O, Nagase H, Kitazume Y, Mizoguchi F, Izawa K. Induction of flowering by nitrogen deficiency in Lemna paucicostata 441. Plant Cell Physiol 1988;29:1379–84. Tanaka O, Yamamoto T, Nakayama Y, Ozaki T, Takeba G. Flowering induced by nitrogen deficiency in Lemna paucicostata 151. Plant Cell Physiol 1991;32: 1173–7. Thomas B, Vince-Prue D. Photoperiodism in Plants. San Diego: Academic Press; 1997. Wada KC, Takeno K. Stress-induced flowering. Plant Signal Behav 2010;5:1–4. Wada KC, Kondo H, Takeno K. Obligatory short-day plant, Perilla frutescens var. crispa can flower in response to low-intensity light stress under long-day conditions. Physiol Plant 2010a;138:339–45. Wada KC, Yamada M, Shiraya T, Takeno K. Salicylic acid and the flowering gene FLOWERING LOCUS T homolog are involved in poor-nutrition stress-induced flowering of Pharbitis nil. J Plant Physiol 2010b;167:447–52. Yalpani N, Leon´ J, Lawton MA, Raskin I. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiol 1993;103:315–21. Yamaguchi S, Yokoyama M, Iida T, Okai M, Tanaka O, Takimoto A. Identification of a component that induces flowering of Lemna among the reaction products of ␣-ketol linolenic acid (FIF) and norepinephrine. Plant Cell Physiol 2001;42: 1201–9. Yokoyama M, Yamaguchi S, Inomata S, Komatsu K, Yoshida S, Iida T, et al. Stressinduced factor involved in flower formation of Lemna is an ␣-ketol derivative of linolenic acid. Plant Cell Physiol 2000;41:110–3.
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Journal of Plant Physiology journal homepage: www.elsevier.de/jplph
Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata Aya Shimakawa a , Takeshi Shiraya b , Yuta Ishizuka c , Kaede C. Wada a , Toshiaki Mitsui a,b , Kiyotoshi Takeno a,c,∗ a
Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan c Department of Biology, Faculty of Science, Niigata University, Ikarashi, Niigata 950-2181, Japan b
a r t i c l e
i n f o
Article history: Received 23 January 2012 Received in revised form 24 February 2012 Accepted 25 February 2012 Keywords: Flowering Lemna paucicostata Salicylic acid Stress
a b s t r a c t The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering. © 2012 Elsevier GmbH. All rights reserved.
Introduction Flowering in many plant species is regulated by environmental cues, such as the length of the night in photoperiodic flowering and the temperature in vernalization (Thomas and Vince-Prue, 1997), and recent studies indicate that stress also regulates flowering (Wada and Takeno, 2010; Takeno, 2012). The short-day (SD) plant, Pharbitis nil (synonym Ipomoea nil), can be induced to flower under long days (LD) when grown under poor-nutrition or lowtemperature stress conditions (Hatayama and Takeno, 2003; Wada et al., 2010b). The SD plant, Perilla frutescens var. crispa, can flower under LD when grown under low-intensity light stress (Wada et al., 2010a). Ultraviolet radiation stress (Martínez et al., 2004) ˇ and nutrition stress (Koláˇr and Senková, 2008) also induced early flowering in Arabidopsis thaliana. Non-photoperiodic flowering has been sporadically reported in several plant species other than those
Abbreviations: AOA, aminooxyacetic acid; AOPP, l-2-aminooxy-3phenylpropionic acid; FT, FLOWERING LOCUS T; KODA, ␣-ketol of octadecadienoic acid; LD, long days; PAL, phenylalanine ammonia-lyase; SA, salicylic acid; SD, short days. ∗ Corresponding author at: Department of Biology, Faculty of Science, Niigata University, Ikarashi, Niigata 950-2181, Japan. Tel.: +81 25 262 6369; fax: +81 25 262 6369. E-mail address: [email protected] (K. Takeno). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2012.02.009
mentioned above, and a review of those reports suggested that most of the factors responsible for flowering can be regarded as stresses (Wada and Takeno, 2010; Takeno, 2012), including high- or low-intensity light, high or low temperature, drought, poor nutrition and mechanical stimulation. Moreover, the plants induced to flower by stresses produced fertile seeds, and the progeny developed normally (Wada et al., 2010a, b). Plants can modify their developmental processes to adapt to stress conditions, and stressinduced flowering is one of such adaptations: plants flower as an emergency response when stressed, thus ensuring their ability to produce the next generation. Through this mechanism, plants can preserve the species, even under unfavorable environmental conditions. Therefore, stress-induced flowering can be considered as universal and as important as photoperiodic flowering and vernalization (Wada and Takeno, 2010; Takeno, 2012). A transmissible flowering stimulus, such as florigen in photoperiodic flowering, is involved in the stress-induced flowering of P. nil (Wada et al., 2010b). The P. nil ortholog of FLOWERING LOCUS T (FT), PnFT2, is involved in the stress-induced flowering of P. nil. However, because the activity of phenylalanine ammonialyase (PAL) and the salicylic acid (SA) content increase when plants are stressed (Dixon and Paiva, 1995; Scott et al., 2004), it is also possible that SA functions as the flowering stimulus in stressinduced flowering. PAL catalyzes the conversion of phenylalanine to t-cinnamic acid, and SA is one of the metabolic intermediates derived from t-cinnamic acid. Aminooxyacetic acid (AOA) and
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l-2-aminooxy-3-phenylpropionic acid (AOPP), which function as PAL inhibitors (Kessmann et al., 1990; Appert et al., 2003), inhibited stress-induced flowering in P. nil and P. frutescens (Hatayama and Takeno, 2003; Wada et al., 2010a, b). In addition, the inhibitory effect of AOA and AOPP was negated by t-cinnamic acid, SA and benzoic acid (a precursor of SA) in P. nil under stress conditions (Hatayama and Takeno, 2003; Wada et al., 2010b). These facts suggest that SA is involved in the regulatory mechanism of stressinduced flowering and that this flowering response may be regulated by either PnFT2 or SA in P. nil. However, no evidence has yet demonstrated that the endogenous SA levels increase when plants are induced to flower through the application of stress factors. Recently, we found that the SD plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower under noninductive photoperiodic conditions when grown under conditions of starvation stress, results that are reminiscent of the seminal article by Cleland and Ajami (1974), who found that exogenously applied SA can induce flowering in Lemna gibba. Since the publication of this pioneering study, many articles have reported that SA and its related compounds, including benzoic acid, induce flowering in L. gibba, L. paucicostata and some other Lemnaceous species under non-inductive photoperiodic conditions (reviewed by Kandeler, 1985). However, no positive correlation was found between the endogenous level of benzoic acid and photoperiodic conditions in L. gibba and L. paucicostata (Fujioka et al., 1983). SA is therefore not considered to be an endogenous regulatory factor in photoperiodic flowering. In the present study, we found that flowering in L. paucicostata was induced by both photoperiodic cues and stress factors. Thus, we postulated that SA is an endogenous regulating factor in stress-induced flowering but not in photoperiodic flowering. Accordingly, this study examined whether the endogenous SA level would increase once flowering was induced by starvation stress in L. paucicostata. Materials and methods Plant materials and growth conditions SD plant Lemna paucicostata Hegelm. (synonym Lemna aequinoctialis Welwitsch) strains 151, 441 and 6746 (provided by Prof. Emeritus A. Takimoto, Kyoto University, Kyoto, Japan) were used. The fronds of each strain were aseptically cultured in 1/2 strength Hutner’s medium supplemented with 1% sucrose (Takimoto et al., 1989). The number of fronds increased through the vegetative multiplication in this medium, and the younger generations were routinely transplanted to new media to maintain the strains. Unless otherwise specified, the assay medium used was 1/10 strength M medium supplemented with 1% sucrose and 1 M benzylaminopurine (pH 4.0) (Fujioka et al., 1986). Uniformly grown three-frond colonies were chosen from the stock culture and aseptically transferred to a 100 mL Erlenmeyer flask containing 20 mL of assay medium (1 colony/flask). In general, the fronds were grown at 25 ◦ C under a 16-h light and 8-h dark period (LD) conditions. White light of approximately 100 mol m−2 s−1 at the frond level was provided by fluorescent lamps (Toshiba FL40SSW/37, Toshiba Corporation, Tokyo, Japan). Incandescent lamps (30 mol m−2 s−1 , Toshiba RF100V57WM, Toshiba Corporation, Tokyo, Japan) were used when far-red light was added to white light. Treatment with stress or SD As the stress treatment, the fronds were cultured in tap water without any additional components instead of the usual assay medium. The tap water used was drinking water supplied by public water supply system and contains 0.6–0.9 mg/L nitrite plus
nitrate whereas the 1/2 strength Hutner’s medium used as control medium contains 100 mg/L NH4 NO3 . As the SD treatment, the fronds were cultured under 8-h light and 16-h dark period (SD) conditions.
Treatment with chemicals Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (AOPP) were used as inhibitors of the PAL enzyme. Both of the compounds were obtained from Wako Pure Chemicals Industries (Osaka, Japan). Either AOA or AOPP was added to the tap water in which the fronds were cultured; SA (Wako Pure Chemicals Industries, Osaka, Japan) was also added to the tap water.
Scoring of the flowering response All of the fronds were dissected using a binocular microscope to determine whether flowers had formed. A flowering response was defined as the percentage of fronds with flowers of all of the fronds cultured in a flask (% flowering). The number of fronds per flask was presented as an indicator of the vegetative growth. Three flasks were used for each treatment, and the mean and standard error (SE) of the percent flowering and the number of fronds per flask were calculated. Each experiment was repeated several times.
Analysis of SA The fronds were harvested, frozen in liquid nitrogen and stored at −80 ◦ C prior to the analysis. The frozen fronds were homogenized in 70% methanol (5 mL/g fresh weight of tissue) using a mortar and pestle with 100 ng deuterium-labeled 2-hydroxybenzoic-3,4,5,6d4 acid (CDN Isotopes, Quebec, Canada) as an internal standard. The homogenate was hand-centrifuged to precipitate the cellular debris, and the supernatant was evaporated in vacuo at 37 ◦ C. The pH of the resulting aqueous phase was adjusted to 3.0 and partitioned with an equal volume of ethyl acetate three times. The resulting acidic ethyl acetate-soluble fraction was concentrated in vacuo at 37 ◦ C and redissolved in 2 mL of 60% methanol. The methanol solution was separated using a Sep-Pak Plus C18 cartridge (Waters Corporation, Milford, USA). The eluate was evaporated in vacuo to dryness and redissolved in 50 L of 70% methanol containing 20 mM sodium acetate (pH 2.5). The methanol solution was injected into an ODS column (3 mm × 250 mm, Imtakt Cadenza CD-C18, Imtakt Corp., Kyoto, Japan) connected to a high performance liquid chromatograph (Hitachi L-6200, Hitachi Co., Tokyo, Japan) and separated using 70% methanol with 20 mM sodium acetate (pH 2.5) at a flow rate of 0.15 mL min−1 . The column temperature was 40 ◦ C. The eluate with a retention time of the authentic SA was collected as the SA fraction. The collected sample was subjected to liquid chromatography–mass spectrometry using an LTQ Orbitrap XL (Thermo Fischer Scientific, Bremen, Germany) at a flow rate of 5–10 L min−1 for 3 min, and the ESI electron spray injection method was used. The ionization voltage was set to 5 kV, and the MS scan range was m/z 200–400. The data processing was performed using Xcalibur Qual Browser (version 2.1, Thermo Fisher Scientific, Bremen, Germany). The amount of endogenous SA was estimated using the detected peak intensities of the internal standard (m/z 141.05) and the endogenous SA (m/z 137.02). The analysis of each sample was repeated 3–6 times, and the mean and SEs were calculated. The experiment was repeated 3 times, with similar results.
A. Shimakawa et al. / Journal of Plant Physiology 169 (2012) 987–991 Table 1 Flowering induced by poor-nutrition conditions in Lemna paucicostata. Strain
Conditions
Flowering (%)
Fronds/flask
151
SD • Nutrition LD • Nutrition LD • Poor nutrition
15.6 ± 3.15a 0.521 ± 0.114c 6.3 ± 1.0b
1283 ± 125.8a 1639 ± 59.88a 47 ± 3.2b
441
SD • Nutrition LD • Nutrition LD • Poor nutrition
90.0 ± 3.60a 0 ± 0c 7.2 ± 1.7b
1472 ± 185.2a 863 ± 49.9b 37 ± 1.9c
6746
SD • Nutrition LD • Nutrition LD • Poor nutrition
84.7 ± 3.41a 1.39 ± 0.188c 27 ± 0.42b
341 ± 45.7b 830 ± 78.8a 33 ± 3.7c
Three strains of L. paucicostata were cultured in tap water (poor nutrition) or 1/10 strength M medium supplemented with 1% sucrose and 1 M benzylaminopurine (nutrition) at 25 ◦ C under conditions of a 16-h light and 8-h dark period (LD) or conditions of an 8-h light and 16-h dark period (SD) for 4 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed within the same strain.
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Table 3 Effects of PAL inhibitors on the flowering in Lemna paucicostata strain 6746 cultured under poor-nutrition conditions. PAL inhibitor
Concentration (M)
Flowering (%)
AOA
0 10−7 10−6 10−5
11 13 4.4 0
± ± ± ±
0.61a 3.0a 2.6ab 0b
30 43 31 5.0
± ± ± ±
1.7b 1.9a 3.3b 0.58c
AOPP
0 10−6 10−5 10−4
8.3 6.5 12 0
± ± ± ±
2.8b 2.3b 5.5ab 0a
60 48 47 61
± ± ± ±
0.88a 5.2ab 1.7b 5.2ab
Fronds/flask
L. paucicostata strain 6746 was cultured in tap water (poor-nutrition conditions) supplemented with aminooxyacetic acid (AOA) or l-2-aminooxy-3-phenylpropionic acid (AOPP) at 25 ◦ C under conditions of a 16-h light and 8-h dark period for 3 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed using the same inhibitor.
The results indicate that cultivation under poor-nutrition conditions functioned as a stress factor and induced flowering under non-inductive photoperiodic conditions in L. paucicostata.
Results Induction of flowering by poor nutrition Three strains of L. paucicostata were cultured in tap water under non-inductive LD conditions for 4 weeks, and flowering occurred in each of the strains (Table 1). The control plants cultured in a nutrient medium showed no or almost no flowering response under the LD conditions, whereas the flowering response under the poornutrition conditions was the strongest in strain 6746. All three of the strains cultured under the SD conditions as a positive control flowered, and their flowering percentages were higher than those under the poor-nutrition treatment. The number of fronds per flask cultured in tap water was much smaller than that cultured in the nutrient medium for all of the tested strains. The three strains were cultured in tap water under LD conditions for 2–4 weeks to examine the timing of the flowering response. A weak flowering response was detected after 2 weeks of the stress treatment in strains 151 and 6746, and an apparent flowering response was detected after 3 weeks of the stress treatment in all of the strains (Table 2). The flowering percentage did not change or rather decreased from the third week to the fourth week. The number of fronds per flask did not significantly increase during the cultivation from the second week to the fourth week, with the exception of strain 441. The fronds were healthy and green in color when observed after 2 weeks in culture but turned to a whitishyellow after the third week in all of the strains (data not shown). These observations indicate that vegetative growth was suppressed when the plants were grown in the tap water. Table 2 Flowering in Lemna paucicostata cultured under poor-nutrition conditions for different lengths of time. Strain
Culture period (weeks)
Flowering (%)
Fronds/flask
151
2 3 4
1.1 ± 1.1b 7.4 ± 1.8a 2.2 ± 1.2ab
34 ± 4.8a 44 ± 1.5a 44 ± 2.7a
441
2 3 4
0 ± 0b 5.7 ± 0.092a 1.9 ± 0.96b
27 ± 0.67b 35 ± 0.58a 34 ± 2.3a
6746
2 3 4
4.4 ± 3.1b 23 ± 0.79a 17 ± 2.3a
40 ± 3.0a 47 ± 2.7a 49 ± 2.4a
Three strains of L. paucicostata were cultured in tap water (poor-nutrition conditions) at 25 ◦ C under conditions of a 16-h light and 8-h dark period for 2–4 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed within the same strain.
PAL-inhibitor inhibition of the flowering induced by poor nutrition Strain 6746, the most sensitive strain to the starvation stress, was cultured in tap water under LD conditions to induce flowering, and PAL inhibitors were added to the tap water to examine their effects on the starvation stress-induced flowering. The flowering was completely inhibited by AOA at 10−5 M and by AOPP at 10−4 M (Table 3). The number of fronds per flask was decreased by the AOA treatment at 10−5 M, whereas the multiplication of fronds was not suppressed by AOPP at 10−4 M. Endogenous SA contents Strain 6746 was cultured in tap water for 3 weeks to induce flowering, and SA was extracted from the fronds for quantification. Significantly higher amounts of SA were detected in those fronds that flowered under the poor-nutrition conditions when compared to the vegetative fronds cultured under nutrient conditions (Table 4). This result was reproduced in three independent experiments. Promotion of flowering by exogenously applied SA Strain 6746 was cultured in tap water with added SA and grown under non-inductive LD conditions for 3 weeks. The SA promoted flowering when supplied at 3 × 10−5 M (Table 5); in contrast, flowering was inhibited by SA at 10−4 M. Discussion Three strains of the SD plant, L. paucicostata, were induced to flower when cultured in tap water under non-inductive LD conditions (Table 1). The vegetative multiplication of the fronds was restricted when cultured in tap water, which indicates that the vegetative growth of the plants was suppressed in tap water, implying that the plants were stressed (Hatayama and Takeno, 2003). In comparison with the flowering response under SD conditions, which was 15–90% (depending on the strain), the poor-nutrition conditions produced a weaker flowering response in each strain examined. However, virtually no flowering occurred under the nutrient conditions. Therefore, L. paucicostata is induced to flower by starvation stress. The time-course study showed that flowering occurred after two (strains 151 and 6746) or three (strain 441) weeks of the
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Table 4 Flowering and the endogenous salicylic acid (SA) content in Lemna paucicostata strain 6746 cultured under poor-nutrition conditions. Experiment
Conditions
Flowering (%)
Fronds/flask
I
Nutrition Poor nutrition
0.21 ± 0.1a 18 ± 2.8b
722 ± 106a 43 ± 4.9b
SA content (ng/g fw) (relative value) 66 ± 8.3a (100) 953 ± 214b (1443)
II
Nutrition Poor nutrition
0 ± 0a 5.7 ± 3.3a
620 ± 32a 36 ± 1.3b
215 ± 29.9a (100) 2280 ± 586b (1060)
III
Nutrition Poor nutrition
0 ± 0a 6.5 ± 2.1b
665 ± 152a 37 ± 6.7b
511 ± 109a (100) 1530 ± 333b (299)
L. paucicostata strain 6746 was cultured in tap water (poor nutrition) for 3 weeks or in 1/10 strength M medium supplemented with 1% sucrose and 1 M benzylaminopurine (nutrition) for 1 week. Far-red light was supplied with white light, and the plants were grown at 25 ◦ C for 3 weeks under nutrition conditions or at 23 ◦ C for 2 weeks followed by 25 ◦ C for 1 week under poor-nutrition conditions in experiment I. The plants were grown at 23 ◦ C and 25 ◦ C in experiments II and III, respectively. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test performed within the same experiment.
stress treatment and that the flowering response was the strongest at the third week after the start of the stress treatment in each strain (Table 2). The number of fronds per flask did not significantly increase after flowering was induced. Furthermore, the fronds became whitish-yellow in color when cultured in tap water for over 3 weeks, indicating that the fronds were withering under the prolonged stress conditions and may not have been able to continue to produce flowers. The flower of L. paucicostata is quite small and ephemeral and therefore easily decays and disappears soon after anthesis. Thus, the flowering percentages decreased from the third week to the fourth week in all of the strains. It has been reported that L. paucicostata flowers in nitrogendeficient or nitrogen-free media independently of the daylength (Tanaka, 1986; Tanaka et al., 1988, 1991). Experiments on such daylength-independent flowering were performed using a culture medium containing mineral nutrient salts and 1% sucrose with reduced or no nitrogen. In our study, however, we cultured the plants in tap water without any mineral salts or energy source. This culture condition induced the suppression of growth, indicating that the plants were stressed. Therefore, the flowering response found in the present study may be different from that under nitrogen-deficient conditions. Yamaguchi et al. (2001) isolated norepinephrine and ␣-ketol of octadecadienoic acid (KODA), from which flower-inducing factors are derived, from L. paucicostata. KODA was found to be induced by drought, heat or osmotic stress conditions (Yokoyama et al., 2000). These results are consistent with our finding that L. paucicostata has a mechanism that responds to stress and promotes flowering. However, the clarification of the relationship between these flower-promoting factors and SA requires further investigation. It has long been supposed that SA is involved in the regulation of flowering in Lemna spp.; however, the supporting evidence presented to date is merely that exogenously applied SA can induce flowering. Therefore, we attempted to reduce the endogenous SA level by inhibiting its biosynthesis to ascertain whether this inhibits flowering. SA is synthesized from t-cinnamic acid, which Table 5 Effects of salicylic acid on flowering in Lemna paucicostata strain 6746 cultured under poor-nutrition conditions. Concentration (M)
Flowering (%)
0 10−6 3 × 10−6 10−5 3 × 10−5 10−4
4.9 3.8 4.6 11 42 0.74
± ± ± ± ± ±
1.7bcd 0.17c 2.3bcd 2.5b 1.9a 0.38d
Fronds/flask 63 62 60 56 61 95
± ± ± ± ± ±
7.5b 10ab 6.7b 3.5b 2.0b 8.7a
L. paucicostata strain 6746 was cultured in tap water (poor-nutrition conditions) supplemented with salicylic acid at 25 ◦ C under conditions of a 16-h light and 8-h dark period for 3 weeks. The values are the means ± SEs. The values followed by same letters do not differ significantly at the 1% level, as determined by the t-test.
is converted from phenylalanine by PAL in the majority of plant species (Yalpani et al., 1993); SA is also derived from isochorismate in bacteria and some plant species, including A. thaliana (Chen et al., 2009). Generally, stress induces PAL activity, resulting in the accumulation of SA and anthocyanin (Dixon and Paiva, 1995; Scott et al., 2004). Indeed, when L. gibba is induced to flower by stress, the fronds turn red in color due to the accumulation of anthocyanin, suggesting the activation of PAL (unpublished data). PAL inhibitors suppressed the stress-induced flowering in P. nil and P. frutescens, an inhibition that was overcome by SA (Wada et al., 2010a, b). These findings led us to speculate that stress-induced flowering is regulated by SA, which is synthesized by PAL. Accordingly, we applied PAL inhibitors to L. paucicostata that was cultured under stress conditions and found that AOA and AOPP completely inhibited flowering at concentrations of 10−5 M and 10−4 M, respectively (Table 3). These results suggest that SA is involved in the regulation of stress-induced flowering in L. paucicostata. We then quantified the endogenous SA content in those fronds that flowered due to the starvation stress and the control fronds without flowers finding that the SA content increased when L. paucicostata was stressed and flowered (Table 4). This is the first evidence showing that the SA content increases when flowering is induced in Lemnaceous species. Lastly, we found that exogenously applied SA promoted flowering (Table 5), and the inhibiting effect of SA at a high concentration is consistent with previous reports (Cleland and Ajami, 1974; Cleland and Tanaka, 1979). Taken together, the results presented here suggest that endogenous SA plays a role in the promotion of stress-induced flowering and not photoperiodic flowering. SA has been the focus of intensive research due to its function as an endogenous signal (Rivas-San Vicente and Plasencia, 2011), and the induction of flowering is one of the functions of SA. It was previously suggested that SA is involved in stress-induced flowering in P. nil and P. frutescens (Wada et al., 2010a, b), and the ultraviolet light stress-induced flowering response of A. thaliana was weaker in the SA-deficient NahG transgenic lines than in the wild type (Martínez et al., 2004). These facts support our conclusion that SA is involved in stress-induced flowering. The proteins derived from FT and its orthologs have been reported to be florigens (Corbesier et al., 2007; Lin et al., 2007; Tamaki et al., 2007). The expression of A. thaliana FT and sunflower HAFT, the FT homolog, were induced by SA (Martínez et al., 2004; Dezar et al., 2010), and the expression of PnFT2, an ortholog of FT in P. nil, was induced by stress conditions (Wada et al., 2010b). These findings indicate that FT, SA and stress may collaborate to regulate flowering. It is now apparent that flowering in L. paucicostata is mediated by both stress and SA. Because the L. paucicostata FT homolog has already been registered in a public database (although it remains unpublished), this plant is thus an ideal experimental material for examining the interaction between FT expression, SA synthesis and stress responses.
A. Shimakawa et al. / Journal of Plant Physiology 169 (2012) 987–991
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