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Ethylene production by gynoecium and receptacle is associated with sepal abscission in cut Delphinium flowers
Postharvest Biology and Technology 52 (2009) 267–272
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Ethylene production by gynoecium and receptacle is associated with sepal abscission in cut Delphinium flowers Kazuo Ichimura ∗ , Hiroko Shimizu-Yumoto, Rie Goto National Institute of Floricultural Science, Tsukuba, Ibaraki 305-8519, Japan
a r t i c l e
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Article history: Received 25 September 2008 Accepted 23 December 2008 Keywords: 1-Aminocyclopropane-1-carboxylic acid (ACC) ACC synthase ACC oxidase Delphinium Ethylene Wounding
a b s t r a c t Delphinium flowers are sensitive to ethylene, and exposure to ethylene is known to accelerate sepal abscission. The relationship of ethylene to sepal abscission in cut Delphinium flowers was investigated. The gynoecium and receptacle each contributed to climacteric-like increases in ethylene production whereas the sepals, petals and stamens did not. 1-Aminocyclopropane-1-carboxylic acid (ACC) concentration, ACC synthase and ACC oxidase activities in the gynoecium and receptacle increased in the senescing flowers. Wounding of the gynoecium or receptacle accelerated abscission of sepals, which was accompanied by a marked increase in ethylene production. Accelerated sepal abscission was counteracted by treatment with silver thiosulphate complex (STS), an inhibitor of ethylene action. The results of this study show that ethylene produced by the gynoecium and receptacle is closely associated with sepal abscission in cut Delphinium flowers. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ethylene plays an important role in various stages of plant growth and development, including senescence, and exposure to ethylene accelerates senescence of flowers in many species (Woltering and van Doorn, 1988). Primary senescence induced by ethylene produces petal wilting or petal (sepal) abscission, and in many ethylene-sensitive flowers, such as carnation (Nichols, 1968), Eustoma (Ichimura et al., 1998) and orchid (Porat et al., 1994), the petals wilt when exposed to ethylene. In cut carnations, ethylene production in petals increases during natural flower senescence. Additionally, the gynoecium produces a significant amount of ethylene prior to the onset of the climacteric-like increase in ethylene production from the petals (Woodson et al., 1992; ten Have and Woltering, 1997). Shibuya et al. (2000) reported that ethylene production in petals did not increase following the removal of gynoecia, indicating that gynoecia trigger the climacteric-like increase in ethylene production in petals of carnation flowers. In Digitalis and torenia flowers, petals abscise upon exposure to ethylene (Stead and Moore, 1983; Goto et al., 1999). In these flowers, ethylene production in gynoecia increases with the progression of flower senescence, but ethylene production in petals does not (Stead and Moore, 1983; Goto et al., 1999). Therefore, gynoecium-produced
∗ Corresponding author. Fax: +81 29 838 6841. E-mail address: [email protected] (K. Ichimura). 0925-5214/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2008.12.008
ethylene appears to act at the abscission zone, leading to petal abscission. Wounding of the stigma has been found to stimulate petal wilting in petunia (Gillisen and Hoekstra, 1984; Whitehead et al., 1984) and Eustoma (Ichimura and Goto, 2000) and petal abscission in torenia (Goto et al., 1999). These detrimental effects observed following stigma wounding have been associated with ethylene (Whitehead et al., 1984; Goto et al., 1999; Ichimura and Goto, 2000). In Digitalis, by contrast, wounding of stigma and style did not increase ethylene production and nor accelerated petal abscission (Stead and Moore, 1979, 1983). The biosynthetic pathway for ethylene in higher plants has been elucidated as follows: methionine → S-adenosyl-l-methionine (SAM) → ACC → ethylene. ACC, the precursor of ethylene, is produced by the conversion of SAM by ACC synthase and is converted to ethylene, carbon dioxide and HCN by ACC oxidase. In carnation petals, both ACC synthase and ACC oxidase contribute to the climacteric-like increase of ethylene production (Woodson et al., 1992; Woltering et al., 1993; Lee et al., 1997). Delphinium, which is a popular ornamental plant and is often used for cut flowers, has long spikes with flowers of various colors, such as white, blue, and purple. Delphinium flowers are sensitive to ethylene, and their sepals rapidly abscise upon exposure (Woltering and van Doorn, 1988; Ichimura et al., 2000). Ethylene production from flowers increases during flower senescence (Ichimura et al., 2000), which is accompanied by signs of programmed cell death (Yamada et al., 2007). Treatment with silver thiosulphate complex
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(STS), an inhibitor of ethylene action, delays the abscission of sepals (Uda et al., 1997). These findings indicate that sepal abscission in Delphinium is regulated by ethylene. Genes for ethylene receptors and signal transduction components have been identified in Delphinium flowers (Kuroda et al., 2003, 2004; Tanase and Ichimura, 2006). However, which organs and enzymes contribute to the regulation of ethylene biosynthesis remains to be elucidated. In the present paper, we showed that gynoecia and receptacles have high activities of ethylene production in cut Delphinium flowers. Furthermore, we studied changes in ACC, ACC synthase and ACC oxidase in these organs and the effect of wounding on sepal abscission. 2. Materials and methods 2.1. Plant materials Delphinium hybrid cv. Bellamosum were grown in a greenhouse under natural day length conditions with temperatures ranging between a minimum of 15 ◦ C and maximum of 25 ◦ C. On the day of anthesis, flowers with a peduncle of 3 cm in length were cut from the middle region of the flower spikes (Fig. 1A and B). Cut peduncles were placed in a vessel with distilled water within 1 h after harvest and used for all subsequent experiments. Unless otherwise stated, cut flowers were kept at 23 ◦ C, 70% relative humidity, and 10 mol m−2 s−1 irradiance using cool-white fluorescence lamps under a 12 h photoperiod. 2.2. Evaluation of ethylene sensitivity The cut flowers (12 for each treatment) were placed in individual vessels containing distilled water and placed in a 70 L transparent acrylic box fitted with a septum through which ethylene was introduced to achieve a concentration of 10 L L−1 . The box was kept at 23 ◦ C under a 12 h photoperiod with 10 mol m−2 s−1 irradiance from cool-white fluorescent lamps for 24 h. After exposure to ethylene, flowers
were removed from the chamber and kept under the same temperature and lighting conditions as above. Sepals were gently touched once per day to evaluate sepal abscission. Sensitivity to ethylene was evaluated using the time elapsed from the end of the ethylene treatment to the time of sepal abscission. 2.3. Measurement of ethylene production Whole flower or sepals were placed individually in 30 mL Erlenmeyer flasks (38 mL) whereas various other parts were individually placed in test tubes (14.8 mL). They were sealed and maintained at 23 ◦ C. Two hours later, a 1 mL gas sample was withdrawn using a syringe and used to determine ethylene concentration by a Shimadzu gas-chromatograph model GC-7A equipped with an alumina column and a flame ionization detector. 2.4. Determination of ACC Gynoecia and receptacles collected from 10 flowers were immersed in 5 mL of boiling 80% ethanol for 20 min. The extract was homogenized and centrifuged at 3000 × g for 5 min. The supernatant was evaporated to dryness in vacuo below 45 ◦ C. The dry residue was rehydrated in 0.8 mL of distilled water. ACC was oxidized in vitro to produce ethylene according to Lizada and Yang (1979). Known amounts of ACC were converted to ethylene to produce a standard curve for the quantification of ACC levels in each tissue type. 2.5. Extraction and determination of ACC synthase ACC synthase was extracted by the method of Mor et al. (1985) with modifications. Gynoecia and receptacles collected from 10 flowers were frozen in liquid nitrogen and ground with a mortar and pestle. The frozen powder was then mixed with 10 volumes of extraction buffer containing 100 mM HEPES-KOH (pH 8.5), 5 mM DTT, 1 mM Na2 -EDTA and 10 M pyridoxal 5-phosphate (PLP). The homogenate was filtered through 8 layers of gauze and centrifuged at 20,000 g for 15 min. The supernatant was desalted using Sephadex G-25. All extraction procedures were carried out below 4 ◦ C. A standard reaction mixture (final volume, 1.3 mL) containing 0.8 mL of the enzyme preparation, 4 mM HEPES-KOH (pH8.5), 0.2 mM SAM and 0.4 M PLP was incubated at 30 ◦ C for 1 h. The reaction was terminated by the addition of 0.1 mL
Fig. 1. . Morphology of Delphinium flower. (A) Morphology of flower spike. (B) Morphology of flower with petals and sepals. Stamens cover and obscure the gynoecium. (C) Morphology of gynoecium and receptacle. Bar indicates 10 mm.
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of 20 mM HgCl2 . Reaction mixtures containing enzyme samples but lacking SAM were used as blanks for ACC formed in the assay. The amount of ACC formed was assayed according to Lizada and Yang (1979). 2.6. Extraction and determination of ACC oxidase ACC oxidase was extracted by the method of Dupille et al. (1993) with modifications. Gynoecia and receptacles collected from 10 flowers were frozen in liquid nitrogen and ground with a mortar and pestle. The frozen powder was then mixed with 10 volumes of extraction buffer containing 100 mM Tris–HCl (pH 7.5), 10% (v/v) glycerol, 30 mM Na ascorbate and 5 mM DTT. The homogenate was filtered through 8 layers of gauze and centrifuged at 20,000 g for 15 min. The resulting supernatant was used as an enzyme preparation. All extraction procedures were carried out below 4 ◦ C. Enzyme activity was assayed at 30 ◦ C for 30 min in a sealed 15 mL tube that contained 2.15 mL reaction mixture consisting of 80 mM Tris-HCl (pH 7.5), 8% (v/v) glycerol, 24 mM Na ascorbate, 0.1 mM FeSO4 , 47 mM NaHCO3 , 0.5 mM ACC and crude extract. Ethylene accumulation in the headspace was determined by gas chromatography as described above. 2.7. Chemical treatments Stamens were removed from flowers with forceps on the day of harvest. The base of each gynoecium was wetted with 10 L of either 0.5 mM ACC, 10 mM l-˛(2-aminoethoxyvinyl)glycine (AVG), 100 or 400 mM ˛-aminoisobutyric acid (AIB), 5 mM CoCl2 or 0.5 mM STS solutions. STS solution was freshly prepared by mixing equal volumes of AgNO3 and Na2 S2 O3 ·5H2 O in a 1 to 8 molar ratio. The concentrations of inhibitors were based on previous reports (Woltering et al., 1995; Clark et al., 1997; Uda et al., 1997). Distilled water was used as the control. After treatment, cut flowers were kept under the same environmental conditions as described above. 2.8. Wounding of various organs To wound the gynoecia and receptacles, stamens were removed by forceps on the day of harvest because the gynoecia are fully covered with stamens (Fig. 1C). The gynoecia, receptacles, petals and sepals were crushed 5 times with a small pair of tweezers. In some experiments, the cut peduncles were placed for 30 min in 0.5 mM STS solution, and then transferred to distilled water. Immediately after STS treatment, the gynoecium or receptacle was wounded. After these wounding treatments, cut flowers were kept under the same environmental conditions as described above.
3. Results 3.1. Effect of exposure to ethylene on sepal abscission Abscission of sepals was not accelerated in flowers exposed to ethylene at 10 L L−1 for 24 h on the day of harvest, but exposure to 10 L L−1 ethylene accelerated abscission at one or two days after harvest (Table 1). Thus, although sensitivity to ethylene was not high on the day of harvest, it increased one day after harvest. 3.2. Changes in ethylene production from various organs The ethylene production by flowers was low during the first two days and sharply increased thereafter (Fig. 2 A). To determine the contribution of each organ to ethylene production, we examined ethylene production in the petal, sepal, stamen, gynoecium and receptacle (Fig. 1). Ethylene production from the receptacle was almost constant during the first two days, but sharply increased thereafter. Similarly, ethylene production in the gynoecium was low during the first three days and sharply increased thereafter. On Table 1 Effects of exposure to ethylene on sepal abscission of cut Delphinium flowers. Ethylene treatment
Control 10 L L−1 for 24 h
Time after harvest (days)
Time to sepal abscission after ethylene treatment (days)
0 0 1 2
3.3 ± 0.1 3.2 ± 0.2 0.6 ± 0.2** 0.1 ± 0.1**
Values are means of 16 flowers ± SE ** Values are significant at P < 0.01, compared with the control by Dunnett’s test.
Fig. 2. Changes in ethylene production by whole flowers (A) and various organs (B) during vase life. Values are means of 4 replicates ± SE.
the contrary, ethylene production from petals and sepals remained low throughout. Ethylene production in the stamen also did not increase with flower senescence (Fig. 2B). Fresh weights of gynoecium and receptacle at harvest were 24 and 49 mg, respectively. Fresh weight of the gynoecium increased with time, whereas fresh weight of the receptacle was almost constant. Accordingly, ethylene production on an organ basis was greater in the receptacle than in the gynoecium at harvest, and this difference decreased during flower senescence (data not shown). 3.3. Changes in ACC concentration, ACC synthase activity and ACC oxidase activity ACC concentration of the gynoecium and the receptacle was very low until one day after harvest and increased thereafter (Fig. 3). ACC synthase activity in the gynoecium was relatively high on the day of harvest, decreased over three days but increased thereafter. In the receptacle, ACC synthase activity was low during the first two days and increased thereafter. ACC oxidase activity in the gynoecium was relatively high during first one day and then decreased, and in the receptacle, ACC oxidase activity was low during first two days and increased thereafter. 3.4. Effect of ethylene inhibitors and ACC on the abscission of sepals Treatment with AVG, an inhibitor of ACC synthase, significantly delayed the abscission of sepals more than that with STS (Table 2).
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K. Ichimura et al. / Postharvest Biology and Technology 52 (2009) 267–272 Table 3 Effects of wounding to gynoecium and receptacle on the abscission of sepals of cut Delphinium flowers. Treatment
Time to sepal abscission (days)
Untreated Stamen removal Receptacle wounding Gynoecium wounding and stamen removal Receptacle wounding and stamen removal
4.5 ± 0.2a 3.9 ± 0.1a 4.1 ± 0.1a 2.6 ± 0.2b 2.4 ± 0.3b
Values are means of 8 flowers ± SE and those with the same letters within columns are not significantly different (P < 0.05) by Tukey–Kramer’s multiple range test.
In contrast, treatments with AIB and CoCl2 , inhibitors of ACC oxidase, did not delay sepal abscission. Treatment with ACC did not accelerate sepal abscission. 3.5. Effect of wounding the gynoecium and receptacle on the abscission of sepals Removal of the stamens alone and wounding of the receptacle alone did not significantly accelerate the abscission of sepals. However, wounding the gynoecium in combination with stamen removal accelerated the abscission of sepals (Table 3). Similarly, wounding the receptacle in combination with stamen removal accelerated the abscission. In contrast, stigma, sepal and petal wounding alone did not promote abscission of flowers (data not shown). When stamens were removed, ethylene production by gynoecia and receptacles increased (Fig. 4). Two days after wounding of the gynoecium or the receptacle, ethylene production was the highest in the receptacle followed by the gynoecium, regardless of the treatment. Treatment with STS completely nullified the acceleration of sepal abscission induced by gynoecium or receptacle wounding (Table 4). 4. Discussion
Fig. 3. Changes in ACC concentration (A), ACC synthase activity (B) and ACC oxidase activity (C) in gynoecium and receptacle during vase life. Values are means of 3 replicates ± SE.
Sensitivity to ethylene seems to be low in Delphinium flowers on the day of anthesis as abscission of sepals was not accelerated by exposure to 10 L L−1 ethylene. However, with aging, the abscission of sepals was accelerated by exposure to ethylene (Table 1), indicating that sensitivity to ethylene increases as the flower ages. In Eustoma (Ichimura et al., 1998), Pelargonium (Evensen, 1991) and torenia (Goto et al., 1999), sensitivity to ethylene likewise increased as flowers aged. Days to sepal abscission after exposure to 10 L L−1 ethylene were more than one day in Delphinium flow-
Table 2 Effects of various compounds on the abscission of sepals of cut Delphinium flowers. Treatment
Time to sepal abscission (days)
Control STS (0.5 mM) AVG (10 mM) AIB (100 mM) AIB (400 mM) CoCl2 (5 mM) ACC (0.5 mM)
4.8 9.6 13.2 3.9 4.1 4.6 4.7
± ± ± ± ± ± ±
0.2a 0.5b 0.3c 0.1a 0.3a 0.2a 0.3a
Values are means of 10 flowers ± SE and those with the same letters within columns are not significantly different (P < 0.05) by Tukey–Kramer’s multiple range test.
Fig. 4. Effects of gynoecium and receptacle wounding on ethylene production from gynoecium and receptacle. Values are means of 4 replicates ± SE.
K. Ichimura et al. / Postharvest Biology and Technology 52 (2009) 267–272 Table 4 Effects of gynoecium or receptacle wounding with or without STS on sepal abscission of cut Delphinium flowers. Treatment
STS concentration (mM)
Time to sepal abscission (days)
Control
0 0.5 0 0.5 0 0.5
4.1 12.8 3.2 12.4 3.2 11.9
Gynoecium wounding Receptacle wounding
± ± ± ± ± ±
0.2b 0.4c 0.2a 0.4c 0.1a 0.4c
Cut flowers were treated with 0.5 mM STS for 30 min. After STS treatment, the stamens were removed and the gynoecium or receptacle wounded. Values are means of 8 flowers ± SE and those with the same letters within columns are not significantly different (P < 0.05) by Tukey–Kramer’s multiple range test.
ers (Table 1). In contrast, petals of Pelargonium (Evensen, 1991) and Digitalis (Stead and Moore, 1983) abscised by exposure to ethylene lower than 1 L L−1 within one day, indicating that sensitivity to ethylene in Delphinium is not as high as in these flowers. In our study, Delphinium flowers showed a climacteric-like increase in ethylene production (Fig. 1), but treatment with STS or AVG, which inhibit ethylene production, markedly delayed sepal abscission (Table 2). These findings suggest that sepal abscission in Delphinium is induced by an increase in endogenous ethylene production when sensitivity to ethylene has increased. Similar findings have been reported in Digitalis (Stead and Moore, 1983) and torenia (Goto et al., 1999). The coordination of the senescence processes within the flower has been suggested to involve inter-organ signaling (ten Have and Woltering, 1997; Shibuya et al., 2000). In carnation flowers, removal of the gynoecium suppresses increase in ethylene production and wilting of petals, suggesting that the gynoecium induces petal wilting (Shibuya et al., 2000). The mobile factor for interorgan signaling has been proposed to be ethylene (Woltering et al., 1995) or ACC (Reid et al., 1984). In Digitalis (Stead and Moore, 1983) and torenia flowers (Goto et al., 1999), the gynoecium shows a climacteric-like increase in ethylene production, but petals do not. It has been hypothesized that in these flowers, ethylene produced by the gynoecium acts on the abscission zone, leading to petal abscission. The gynoecium and receptacle in Delphinium flowers showed climacteric-like increases in ethylene production with flower senescence, but petals and sepals did not (Fig. 2). ACC synthase and ACC oxidase activity were much higher in the gynoecium than in the receptacle (Fig. 3), suggesting that ethylene production is higher in the gynoecium than in the receptacle. From these findings, we hypothesize that ethylene produced by the gynoecium induces ethylene production from the receptacle. The sepals are directly connected with the receptacle, but not with the gynoecium, in Delphinium flowers. Thus, ethylene produced by the receptacle should directly act on abscission of the sepals. ACC synthase is generally considered to be a rate-limiting step for ethylene biosynthesis in plants because ACC oxidase activity is constitutive in many instances (Yang and Hoffman, 1984; Kende, 1993). However, ACC oxidase is important for ethylene biosynthesis in some situations, including fruit maturation (Nakatsuka et al., 1998) and flower senescence (Tang et al., 1993). Similarly, the biosynthesis of ethylene is regulated by ACC synthase and ACC oxidase activities in cut carnation flowers (Woodson et al., 1992; Woltering et al., 1993; Lee et al., 1997). Likewise, ACC concentration, ACC synthase and ACC oxidase activities increased in the gynoecium and the receptacle of senesced Delphinium flowers (Fig. 3). In addition, AVG markedly delayed sepal abscission and exogenous ACC did not accelerate it (Table 2). Thus, an increase in both ACC synthase and ACC oxidase contributes to ethylene biosynthesis in the gynoecium and receptacle, and this increase may be required for sepal abscission in Delphinium.
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In our study, AIB and CoCl2 did not delay sepal abscission (Table 2). Uda et al. (1997) also reported that AIB did not delay sepal abscission in another Delphinium cultivar. The ineffectiveness of these compounds may be due to the insufficient accumulation of these compounds at a sufficient concentration at active sites in Delphinium. AIB and CoCl2 were also shown to not completely suppress the conversion of ACC to ethylene in cocklebur (Satoh and Esashi, 1980, 1983). ACC synthase and ACC oxidase activities in the receptacle were low during the first two days, while those in the gynoecium were relatively high (Fig. 3). Although ACC concentration was low, the ethylene production was not (Figs. 2 and 3), suggesting that ACC produced by ACC synthase may have been converted to ethylene by the high activity of ACC oxidase, thus preventing the accumulation of ACC. In the carnation gynoecium, relatively high ACC synthase activity was observed prior to the accumulation of ACC (Woodson et al., 1992). The wounding of the gynoecium and receptacle in combination with stamen removal accelerated the abscission of sepals (Table 3), which was accompanied by a marked increase in ethylene production (Fig. 4). This effect was completely nullified by STS treatment (Table 4). These findings clearly show that acceleration of sepal abscission through wounding is regulated by ethylene. Wounding of organs has been shown to increase ethylene production in various plants (Yang and Hoffman, 1984). In petunia (Whitehead et al., 1984), Eustoma (Ichimura and Goto, 2000) and torenia (Goto et al., 1999), wounding of the stigma increases ethylene production, leading to accelerated flower senescence. Similarly, wounding of filaments accelerated flower senescence in Portulaca flowers (Ichimura and Suto, 1998). In addition to these organs, the gynoecium and the receptacle of Delphinium have high levels of ethylene production and ethylene has been shown to be mobile in plants (Jackson and Campbell, 1975; Zeroni et al., 1977). Woltering et al. (1997) also showed that ethylene diffused after stigma wounding in Petunia. Thus, we propose that ethylene produced in the gynoecium due to wounding induces ethylene production of the gynoecium, which moves to the abscission zone and consequently accelerating sepal abscission while ethylene produced by wounding of the receptacle acts on abscission zone, leading to sepal abscission. In our study, removal of stamens alone increased ethylene production by the gynoecium and receptacle. Since stamens are directly connected to the receptacle, removal of stamens likely wounds the receptacle, leading to increased ethylene production. Pulse treatment with STS markedly delayed sepal abscission even when the gynoecium or the receptacle was wounded (Table 4). Pulse treatment with AVG also markedly delayed sepal abscission (Table 2), but severely inhibits sepal growth in cut Delphinium spikes (unpublished results). In addition, pulse treatments with AIB and aminooxyacetic acid, inhibitors of ethylene biosynthesis, and 1methylcyclopropene, an inhibitor of ethylene action, do not delay sepal abscission in cut Delphinium (Uda et al., 1997; Ichimura et al., 2002). Thus, pulse treatment with STS should be useful for extending the vase life of cut Delphinium flowers. In a comparison of several treatments shown in Table 2, STS showed marked effects on delaying sepal abscission as shown in Table 4, although STS was applied in a concentration of 0.5 mM in both experiments. The difference in effectiveness is possibly due to a difference in application methods. In the test of chemical effects shown in Table 2, the flower parts were wetted with STS solution, while STS solution was absorbed to inhibit wound-induced abscission in the data shown in Table 4. Although the amount of absorbed STS has not been confirmed, sufficient amounts of STS solution would be absorbed in the experimental results shown in Table 4. Pollination accelerates flower senescence in many flowers, such as dendrobium (Porat et al., 1994), petunia (Whitehead et al., 1984),
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torenia (Goto et al., 1999), Digitalis (Stead and Moore, 1979) and Eustoma (Ichimura and Goto, 2000). In the present study, the stamens were not removed, except for in the experiment on wounding effects because the removal of stamens would accelerate ethylene production (Table 4). Delphinium flowers are protandrous and the gynoecium matures three or four days after anthesis under the present experimental conditions. Indeed, artificial pollination on the day of anthesis does not accelerate the sepal abscission in Delphinium (unpublished results). In the present study, the flowers may have been naturally pollinated, but ethylene production had started to increase on day 2, a point at which the stigma would not be matured (Fig. 1). Thus, the effect of natural pollination on ethylene production appears to be negligible in cut Delphinium under the present experimental conditions. In conclusion, sensitivity to ethylene of Delphinium flowers increased with aging. The gynoecium and receptacle showed a climacteric-like increase in ethylene production, but other organs did not. ACC concentration, ACC synthase and ACC oxidase activities were relatively high in the senesced gynoecium and receptacle. Wounding of the gynoecium or receptacle accelerated the abscission of sepals, and this was completely suppressed by STS. Thus, ethylene produced by the gynoecium and receptacle is responsible for sepal abscission in cut Delphinium flowers. Acknowledgements We thank Drs. W. G. van Doorn and U. K. Pun for their critical reading of this manuscript and Mrs. K. Matsuda for her assistance. References Clark, D.G., Richards, C., Hilioti, Z., Lind-Iversen, S., Brown, K., 1997. Effect of pollination on accumulation of ACC synthase and ACC oxidase transcripts, ethylene produciton and flower petal abscission in geranium (Pelargonium × hortorum L H. Bailey). Plant Mol. Biol. 34, 855–865. Dupille, E., Rombaldi, C., Lelievre, J.M., Cleyet-Marel, J.C., Pech, J.C., Latche, A., 1993. Purification, properties and partial amino-acid sequence of 1aminocyclopropane-1-carboxylate oxidase from apple fruit. Planta 190,65–70. Evensen, K.B., 1991. Ethylene responsiveness changes in Pelargonium × domesticum florets. Physiol. Plant. 82, 409–412. Gillisen, L.J.W., Hoekstra, F.A., 1984. Pollination-induced corolla wilting in Petunia hybrida rapid transfer through the style of a wilting-inducing substance. Plant Physiol. 75, 496–498. Goto, R., Aida, R., Shibata, M., Ichimura, K., 1999. Role of ethylene on flower senescence of Torenia. J. Japan. Soc. Hort. Sci. 68, 263–268. Ichimura, K., Goto, R., 2000. Acceleration of senescence by pollination of cut Asukano-nami Eustoma flowers. J. Japan. Soc. Hort. Sci. 69, 166–170. Ichimura, K., Kohata, K., Goto, R., 2000. Soluble carbohydrate in Delphinium and their influence on sepal abscission in cut flowers. Physiol. Plant. 108, 307–313. Ichimura, K., Shimamura, M., Hisamatsu, T., 1998. Role of ethylene in senescence of cut Eustoma flowers. Postharvest Biol. Technol. 14, 193–198. Ichimura, K., Shimizu, H., Hiraya, T., Hisamatsu, T., 2002. Effect of 1methylcyclopropene (1-MCP) on the vase life of cut carnation Delphinium and sweet pea flowers. Bull. Natl. Inst. Flor. Sci. 2, 1–8. Ichimura, K., Suto, K., 1998. Role of ethylene in acceleration of flower senescence by filament wounding in Portulaca hybrid. Physiol. Plant. 104, 603–607. Jackson, M.B., Campbell, D.J., 1975. Movement of ethylene from roots to shoots, a factor in the responses of tomato plants to waterlogged soil conditions. New Phytol. 74, 397–406. Kende, H., 1993. Ethylene biosynthesis. Annu. Rev. Plant. Physiol. Mol. Biol. 44, 283–307. Kuroda, S., Hakata, M., Hirose, Y., Shiraishi, M., Abe, S., 2003. Ethylene production and enhanced transcription of an ethylene receptor gene, ERS1, in Delphinium during abscission of florets. Plant Physiol. Biochem. 41, 812–820.
Kuroda, S., Hirose, Y., Shiraishi, M., Davies, E., Abe, S., 2004. Co-expression of an ethylene receptor gene, ERS1, and ethylene signaling regulator gene, CTR1, in Delphinium during abscission of florets. Plant Physiol. Biochem. 42, 745–751. Lee, M.M., Lee, S.H., Park, K.Y., 1997. Effects of spermine on ethylene biosynthesis in cut carnation (Dianthus caryophyllus L) flowers during senescence. J. Plant Physiol. 151, 68–73. Lizada, M.C.C., Yang, S.F., 1979. A simple and sensitive assay for 1aminocyclopropane-1-carboxylic acid. Anal. Biochem. 100, 140–145. Mor, Y., Halevy, A.H., Spiegelstein, H., Mayak, S., 1985. The site of 1aminocyclopropane-1-carboxylic acid synthesis in senescing carnation petals. Physiol. Plant. 65, 196–202. Nakatsuka, A., Murachi, S., Okunishi, H., Shiomi, S., Nakano, R., Kubo, Y., Inaba, A., 1998. Differential expression and internal feedback regulation of 1-aminocyclopropene-1-carboxylate synthase, 1-aminocyclopropene-1carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiol. 118, 1295–1305. Nichols, R., 1968. The response of carnations (Dianthus caryophyllus) to ethylene. J. Hort. Sci. 43, 335–349. Porat, R., Borochov, A., Halevy, A.H., 1994. Pollination-induced changes in ethylene production and sensitivity to ethylene in cut dendrobium orchid flower. Sci. Hort. 58, 215–221. Reid, M.S., Fujino, D.W., Hoffman, N.E., Whitehead, C.S., 1984. 1-Aminocyclopropane1-carboxylic acid (ACC)—The transmitted stimulus in pollinated flowers? J Plant Growth Regul. 3, 189–196. Satoh, S., Esashi, Y., 1980. ˛-Aminoisobutyric acid: a probable competitive inhibitor of conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene. Plant Cell Physiol. 21, 939–949. Satoh, S., Esashi, Y., 1983. ˛-Aminoisobutyric acid, propyl gallate and cobalt ion and the mode of inhibition of ethylene production by cotyledonary segments of cocklebur seeds. Physiol. Plant. 57, 521–526. Shibuya, K., Yoshioka, T., Hashiba, T., Satoh, S., 2000. Role of the gynoecium in natural senescence of carnation (Dianthus caryophyllus L) flowers. J. Exp. Bot. 51, 2067–2073. Stead, A.D., Moore, K.G., 1979. Studies on flower longevity in Digitalis: Pollination induced corolla abscission in Digitalis flowers. Planta 146, 409–414. Stead, A.D., Moore, K.G., 1983. Studies on flower longevity in Digitalis: The role of ethylene in corolla abscission. Planta 157, 15–21. Tanase, K., Ichimura, K., 2006. Expression of ethylene receptors Dl-ERS1-3 and DlERS2, and ethylene response during flower senescence in Delphinium. J. Plant Physiol. 163, 1159–1166. Tang, X., Wang, H., Brandt, A.S., Woodson, W.R., 1993. Organization and structure of the 1-aminocyclopropene-1-carboxylate oxidase gene family from Petunia hybrida. Plant Mol. Biol. 23, 1151–1164. ten Have, A., Woltering, E.J., 1997. Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L) flower senescence. Plant Mol. Biol. 34, 89–97. Uda, A., Yamanaka, M., Fukushima, K., 1997. Pretreatment effect of novel ethylene inhibitors on extending longevity of carnation, larkspur and sweet pea cut flowers. Kinki Chugoku Agric. Res. 93, 65–70. Whitehead, C.S., Halevy, A.H., Reid, M.S., 1984. Roles of ethylene and 1aminocyclopropane-1-carboxylic acid in pollination and wound-induced senescence of Petunia hybrida flowers. Physiol. Plant. 61, 643–648. Woltering, E.J., De Vrije, T., Harren, F., Hoekstra, F.A., 1997. Pollination and stigma wounding: Same response, different signal? J. Exp. Bot. 48., 1027–1033. Woltering, E.J., Somhorst, D., de Beer, C.A., 1993. Roles of ethylene production and sensitivity in senescence of carnation flower (Dianthus caryophyllus) cultivars White Sim Chinera and Epomeo. J. Plant Physiol. 141, 329–335. Woltering, E.J., Somhorst, D., van der Veer, P., 1995. The role of ethylene in interorgan signaling during flower senescence. Plant Physiol. 109, 1219–1225. Woltering, E.J., van Doorn, W.G., 1988. Role of ethylene in senescence of petals-morphological and taxonomical relationships. J. Exp. Bot. 39, 1605–1616. Woodson, W.R., Park, K.Y., Drory, A., Larsen, P.B., Wang, H., 1992. Expression of ethylene biosynthetic pathway transcripts in senescing carnation flowers. Plant Physiol. 99, 526–532. Yamada, T., Ichimura, K., van Doorn, W.G., 2007. Relationship between petal abscission and programmed cell death in Prunus yedoensis and Delphinium belladonna. Planta 226, 1195–1205. Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant Physiol. 35, 155–189. Zeroni, M., Jerie, P.H., Hall, M.A., 1977. Studies on the movement and distribution of ethylene in Vicia faba L. Planta 134, 119–125.
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Ethylene production by gynoecium and receptacle is associated with sepal abscission in cut Delphinium flowers Kazuo Ichimura ∗ , Hiroko Shimizu-Yumoto, Rie Goto National Institute of Floricultural Science, Tsukuba, Ibaraki 305-8519, Japan
a r t i c l e
i n f o
Article history: Received 25 September 2008 Accepted 23 December 2008 Keywords: 1-Aminocyclopropane-1-carboxylic acid (ACC) ACC synthase ACC oxidase Delphinium Ethylene Wounding
a b s t r a c t Delphinium flowers are sensitive to ethylene, and exposure to ethylene is known to accelerate sepal abscission. The relationship of ethylene to sepal abscission in cut Delphinium flowers was investigated. The gynoecium and receptacle each contributed to climacteric-like increases in ethylene production whereas the sepals, petals and stamens did not. 1-Aminocyclopropane-1-carboxylic acid (ACC) concentration, ACC synthase and ACC oxidase activities in the gynoecium and receptacle increased in the senescing flowers. Wounding of the gynoecium or receptacle accelerated abscission of sepals, which was accompanied by a marked increase in ethylene production. Accelerated sepal abscission was counteracted by treatment with silver thiosulphate complex (STS), an inhibitor of ethylene action. The results of this study show that ethylene produced by the gynoecium and receptacle is closely associated with sepal abscission in cut Delphinium flowers. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ethylene plays an important role in various stages of plant growth and development, including senescence, and exposure to ethylene accelerates senescence of flowers in many species (Woltering and van Doorn, 1988). Primary senescence induced by ethylene produces petal wilting or petal (sepal) abscission, and in many ethylene-sensitive flowers, such as carnation (Nichols, 1968), Eustoma (Ichimura et al., 1998) and orchid (Porat et al., 1994), the petals wilt when exposed to ethylene. In cut carnations, ethylene production in petals increases during natural flower senescence. Additionally, the gynoecium produces a significant amount of ethylene prior to the onset of the climacteric-like increase in ethylene production from the petals (Woodson et al., 1992; ten Have and Woltering, 1997). Shibuya et al. (2000) reported that ethylene production in petals did not increase following the removal of gynoecia, indicating that gynoecia trigger the climacteric-like increase in ethylene production in petals of carnation flowers. In Digitalis and torenia flowers, petals abscise upon exposure to ethylene (Stead and Moore, 1983; Goto et al., 1999). In these flowers, ethylene production in gynoecia increases with the progression of flower senescence, but ethylene production in petals does not (Stead and Moore, 1983; Goto et al., 1999). Therefore, gynoecium-produced
∗ Corresponding author. Fax: +81 29 838 6841. E-mail address: [email protected] (K. Ichimura). 0925-5214/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2008.12.008
ethylene appears to act at the abscission zone, leading to petal abscission. Wounding of the stigma has been found to stimulate petal wilting in petunia (Gillisen and Hoekstra, 1984; Whitehead et al., 1984) and Eustoma (Ichimura and Goto, 2000) and petal abscission in torenia (Goto et al., 1999). These detrimental effects observed following stigma wounding have been associated with ethylene (Whitehead et al., 1984; Goto et al., 1999; Ichimura and Goto, 2000). In Digitalis, by contrast, wounding of stigma and style did not increase ethylene production and nor accelerated petal abscission (Stead and Moore, 1979, 1983). The biosynthetic pathway for ethylene in higher plants has been elucidated as follows: methionine → S-adenosyl-l-methionine (SAM) → ACC → ethylene. ACC, the precursor of ethylene, is produced by the conversion of SAM by ACC synthase and is converted to ethylene, carbon dioxide and HCN by ACC oxidase. In carnation petals, both ACC synthase and ACC oxidase contribute to the climacteric-like increase of ethylene production (Woodson et al., 1992; Woltering et al., 1993; Lee et al., 1997). Delphinium, which is a popular ornamental plant and is often used for cut flowers, has long spikes with flowers of various colors, such as white, blue, and purple. Delphinium flowers are sensitive to ethylene, and their sepals rapidly abscise upon exposure (Woltering and van Doorn, 1988; Ichimura et al., 2000). Ethylene production from flowers increases during flower senescence (Ichimura et al., 2000), which is accompanied by signs of programmed cell death (Yamada et al., 2007). Treatment with silver thiosulphate complex
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(STS), an inhibitor of ethylene action, delays the abscission of sepals (Uda et al., 1997). These findings indicate that sepal abscission in Delphinium is regulated by ethylene. Genes for ethylene receptors and signal transduction components have been identified in Delphinium flowers (Kuroda et al., 2003, 2004; Tanase and Ichimura, 2006). However, which organs and enzymes contribute to the regulation of ethylene biosynthesis remains to be elucidated. In the present paper, we showed that gynoecia and receptacles have high activities of ethylene production in cut Delphinium flowers. Furthermore, we studied changes in ACC, ACC synthase and ACC oxidase in these organs and the effect of wounding on sepal abscission. 2. Materials and methods 2.1. Plant materials Delphinium hybrid cv. Bellamosum were grown in a greenhouse under natural day length conditions with temperatures ranging between a minimum of 15 ◦ C and maximum of 25 ◦ C. On the day of anthesis, flowers with a peduncle of 3 cm in length were cut from the middle region of the flower spikes (Fig. 1A and B). Cut peduncles were placed in a vessel with distilled water within 1 h after harvest and used for all subsequent experiments. Unless otherwise stated, cut flowers were kept at 23 ◦ C, 70% relative humidity, and 10 mol m−2 s−1 irradiance using cool-white fluorescence lamps under a 12 h photoperiod. 2.2. Evaluation of ethylene sensitivity The cut flowers (12 for each treatment) were placed in individual vessels containing distilled water and placed in a 70 L transparent acrylic box fitted with a septum through which ethylene was introduced to achieve a concentration of 10 L L−1 . The box was kept at 23 ◦ C under a 12 h photoperiod with 10 mol m−2 s−1 irradiance from cool-white fluorescent lamps for 24 h. After exposure to ethylene, flowers
were removed from the chamber and kept under the same temperature and lighting conditions as above. Sepals were gently touched once per day to evaluate sepal abscission. Sensitivity to ethylene was evaluated using the time elapsed from the end of the ethylene treatment to the time of sepal abscission. 2.3. Measurement of ethylene production Whole flower or sepals were placed individually in 30 mL Erlenmeyer flasks (38 mL) whereas various other parts were individually placed in test tubes (14.8 mL). They were sealed and maintained at 23 ◦ C. Two hours later, a 1 mL gas sample was withdrawn using a syringe and used to determine ethylene concentration by a Shimadzu gas-chromatograph model GC-7A equipped with an alumina column and a flame ionization detector. 2.4. Determination of ACC Gynoecia and receptacles collected from 10 flowers were immersed in 5 mL of boiling 80% ethanol for 20 min. The extract was homogenized and centrifuged at 3000 × g for 5 min. The supernatant was evaporated to dryness in vacuo below 45 ◦ C. The dry residue was rehydrated in 0.8 mL of distilled water. ACC was oxidized in vitro to produce ethylene according to Lizada and Yang (1979). Known amounts of ACC were converted to ethylene to produce a standard curve for the quantification of ACC levels in each tissue type. 2.5. Extraction and determination of ACC synthase ACC synthase was extracted by the method of Mor et al. (1985) with modifications. Gynoecia and receptacles collected from 10 flowers were frozen in liquid nitrogen and ground with a mortar and pestle. The frozen powder was then mixed with 10 volumes of extraction buffer containing 100 mM HEPES-KOH (pH 8.5), 5 mM DTT, 1 mM Na2 -EDTA and 10 M pyridoxal 5-phosphate (PLP). The homogenate was filtered through 8 layers of gauze and centrifuged at 20,000 g for 15 min. The supernatant was desalted using Sephadex G-25. All extraction procedures were carried out below 4 ◦ C. A standard reaction mixture (final volume, 1.3 mL) containing 0.8 mL of the enzyme preparation, 4 mM HEPES-KOH (pH8.5), 0.2 mM SAM and 0.4 M PLP was incubated at 30 ◦ C for 1 h. The reaction was terminated by the addition of 0.1 mL
Fig. 1. . Morphology of Delphinium flower. (A) Morphology of flower spike. (B) Morphology of flower with petals and sepals. Stamens cover and obscure the gynoecium. (C) Morphology of gynoecium and receptacle. Bar indicates 10 mm.
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of 20 mM HgCl2 . Reaction mixtures containing enzyme samples but lacking SAM were used as blanks for ACC formed in the assay. The amount of ACC formed was assayed according to Lizada and Yang (1979). 2.6. Extraction and determination of ACC oxidase ACC oxidase was extracted by the method of Dupille et al. (1993) with modifications. Gynoecia and receptacles collected from 10 flowers were frozen in liquid nitrogen and ground with a mortar and pestle. The frozen powder was then mixed with 10 volumes of extraction buffer containing 100 mM Tris–HCl (pH 7.5), 10% (v/v) glycerol, 30 mM Na ascorbate and 5 mM DTT. The homogenate was filtered through 8 layers of gauze and centrifuged at 20,000 g for 15 min. The resulting supernatant was used as an enzyme preparation. All extraction procedures were carried out below 4 ◦ C. Enzyme activity was assayed at 30 ◦ C for 30 min in a sealed 15 mL tube that contained 2.15 mL reaction mixture consisting of 80 mM Tris-HCl (pH 7.5), 8% (v/v) glycerol, 24 mM Na ascorbate, 0.1 mM FeSO4 , 47 mM NaHCO3 , 0.5 mM ACC and crude extract. Ethylene accumulation in the headspace was determined by gas chromatography as described above. 2.7. Chemical treatments Stamens were removed from flowers with forceps on the day of harvest. The base of each gynoecium was wetted with 10 L of either 0.5 mM ACC, 10 mM l-˛(2-aminoethoxyvinyl)glycine (AVG), 100 or 400 mM ˛-aminoisobutyric acid (AIB), 5 mM CoCl2 or 0.5 mM STS solutions. STS solution was freshly prepared by mixing equal volumes of AgNO3 and Na2 S2 O3 ·5H2 O in a 1 to 8 molar ratio. The concentrations of inhibitors were based on previous reports (Woltering et al., 1995; Clark et al., 1997; Uda et al., 1997). Distilled water was used as the control. After treatment, cut flowers were kept under the same environmental conditions as described above. 2.8. Wounding of various organs To wound the gynoecia and receptacles, stamens were removed by forceps on the day of harvest because the gynoecia are fully covered with stamens (Fig. 1C). The gynoecia, receptacles, petals and sepals were crushed 5 times with a small pair of tweezers. In some experiments, the cut peduncles were placed for 30 min in 0.5 mM STS solution, and then transferred to distilled water. Immediately after STS treatment, the gynoecium or receptacle was wounded. After these wounding treatments, cut flowers were kept under the same environmental conditions as described above.
3. Results 3.1. Effect of exposure to ethylene on sepal abscission Abscission of sepals was not accelerated in flowers exposed to ethylene at 10 L L−1 for 24 h on the day of harvest, but exposure to 10 L L−1 ethylene accelerated abscission at one or two days after harvest (Table 1). Thus, although sensitivity to ethylene was not high on the day of harvest, it increased one day after harvest. 3.2. Changes in ethylene production from various organs The ethylene production by flowers was low during the first two days and sharply increased thereafter (Fig. 2 A). To determine the contribution of each organ to ethylene production, we examined ethylene production in the petal, sepal, stamen, gynoecium and receptacle (Fig. 1). Ethylene production from the receptacle was almost constant during the first two days, but sharply increased thereafter. Similarly, ethylene production in the gynoecium was low during the first three days and sharply increased thereafter. On Table 1 Effects of exposure to ethylene on sepal abscission of cut Delphinium flowers. Ethylene treatment
Control 10 L L−1 for 24 h
Time after harvest (days)
Time to sepal abscission after ethylene treatment (days)
0 0 1 2
3.3 ± 0.1 3.2 ± 0.2 0.6 ± 0.2** 0.1 ± 0.1**
Values are means of 16 flowers ± SE ** Values are significant at P < 0.01, compared with the control by Dunnett’s test.
Fig. 2. Changes in ethylene production by whole flowers (A) and various organs (B) during vase life. Values are means of 4 replicates ± SE.
the contrary, ethylene production from petals and sepals remained low throughout. Ethylene production in the stamen also did not increase with flower senescence (Fig. 2B). Fresh weights of gynoecium and receptacle at harvest were 24 and 49 mg, respectively. Fresh weight of the gynoecium increased with time, whereas fresh weight of the receptacle was almost constant. Accordingly, ethylene production on an organ basis was greater in the receptacle than in the gynoecium at harvest, and this difference decreased during flower senescence (data not shown). 3.3. Changes in ACC concentration, ACC synthase activity and ACC oxidase activity ACC concentration of the gynoecium and the receptacle was very low until one day after harvest and increased thereafter (Fig. 3). ACC synthase activity in the gynoecium was relatively high on the day of harvest, decreased over three days but increased thereafter. In the receptacle, ACC synthase activity was low during the first two days and increased thereafter. ACC oxidase activity in the gynoecium was relatively high during first one day and then decreased, and in the receptacle, ACC oxidase activity was low during first two days and increased thereafter. 3.4. Effect of ethylene inhibitors and ACC on the abscission of sepals Treatment with AVG, an inhibitor of ACC synthase, significantly delayed the abscission of sepals more than that with STS (Table 2).
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K. Ichimura et al. / Postharvest Biology and Technology 52 (2009) 267–272 Table 3 Effects of wounding to gynoecium and receptacle on the abscission of sepals of cut Delphinium flowers. Treatment
Time to sepal abscission (days)
Untreated Stamen removal Receptacle wounding Gynoecium wounding and stamen removal Receptacle wounding and stamen removal
4.5 ± 0.2a 3.9 ± 0.1a 4.1 ± 0.1a 2.6 ± 0.2b 2.4 ± 0.3b
Values are means of 8 flowers ± SE and those with the same letters within columns are not significantly different (P < 0.05) by Tukey–Kramer’s multiple range test.
In contrast, treatments with AIB and CoCl2 , inhibitors of ACC oxidase, did not delay sepal abscission. Treatment with ACC did not accelerate sepal abscission. 3.5. Effect of wounding the gynoecium and receptacle on the abscission of sepals Removal of the stamens alone and wounding of the receptacle alone did not significantly accelerate the abscission of sepals. However, wounding the gynoecium in combination with stamen removal accelerated the abscission of sepals (Table 3). Similarly, wounding the receptacle in combination with stamen removal accelerated the abscission. In contrast, stigma, sepal and petal wounding alone did not promote abscission of flowers (data not shown). When stamens were removed, ethylene production by gynoecia and receptacles increased (Fig. 4). Two days after wounding of the gynoecium or the receptacle, ethylene production was the highest in the receptacle followed by the gynoecium, regardless of the treatment. Treatment with STS completely nullified the acceleration of sepal abscission induced by gynoecium or receptacle wounding (Table 4). 4. Discussion
Fig. 3. Changes in ACC concentration (A), ACC synthase activity (B) and ACC oxidase activity (C) in gynoecium and receptacle during vase life. Values are means of 3 replicates ± SE.
Sensitivity to ethylene seems to be low in Delphinium flowers on the day of anthesis as abscission of sepals was not accelerated by exposure to 10 L L−1 ethylene. However, with aging, the abscission of sepals was accelerated by exposure to ethylene (Table 1), indicating that sensitivity to ethylene increases as the flower ages. In Eustoma (Ichimura et al., 1998), Pelargonium (Evensen, 1991) and torenia (Goto et al., 1999), sensitivity to ethylene likewise increased as flowers aged. Days to sepal abscission after exposure to 10 L L−1 ethylene were more than one day in Delphinium flow-
Table 2 Effects of various compounds on the abscission of sepals of cut Delphinium flowers. Treatment
Time to sepal abscission (days)
Control STS (0.5 mM) AVG (10 mM) AIB (100 mM) AIB (400 mM) CoCl2 (5 mM) ACC (0.5 mM)
4.8 9.6 13.2 3.9 4.1 4.6 4.7
± ± ± ± ± ± ±
0.2a 0.5b 0.3c 0.1a 0.3a 0.2a 0.3a
Values are means of 10 flowers ± SE and those with the same letters within columns are not significantly different (P < 0.05) by Tukey–Kramer’s multiple range test.
Fig. 4. Effects of gynoecium and receptacle wounding on ethylene production from gynoecium and receptacle. Values are means of 4 replicates ± SE.
K. Ichimura et al. / Postharvest Biology and Technology 52 (2009) 267–272 Table 4 Effects of gynoecium or receptacle wounding with or without STS on sepal abscission of cut Delphinium flowers. Treatment
STS concentration (mM)
Time to sepal abscission (days)
Control
0 0.5 0 0.5 0 0.5
4.1 12.8 3.2 12.4 3.2 11.9
Gynoecium wounding Receptacle wounding
± ± ± ± ± ±
0.2b 0.4c 0.2a 0.4c 0.1a 0.4c
Cut flowers were treated with 0.5 mM STS for 30 min. After STS treatment, the stamens were removed and the gynoecium or receptacle wounded. Values are means of 8 flowers ± SE and those with the same letters within columns are not significantly different (P < 0.05) by Tukey–Kramer’s multiple range test.
ers (Table 1). In contrast, petals of Pelargonium (Evensen, 1991) and Digitalis (Stead and Moore, 1983) abscised by exposure to ethylene lower than 1 L L−1 within one day, indicating that sensitivity to ethylene in Delphinium is not as high as in these flowers. In our study, Delphinium flowers showed a climacteric-like increase in ethylene production (Fig. 1), but treatment with STS or AVG, which inhibit ethylene production, markedly delayed sepal abscission (Table 2). These findings suggest that sepal abscission in Delphinium is induced by an increase in endogenous ethylene production when sensitivity to ethylene has increased. Similar findings have been reported in Digitalis (Stead and Moore, 1983) and torenia (Goto et al., 1999). The coordination of the senescence processes within the flower has been suggested to involve inter-organ signaling (ten Have and Woltering, 1997; Shibuya et al., 2000). In carnation flowers, removal of the gynoecium suppresses increase in ethylene production and wilting of petals, suggesting that the gynoecium induces petal wilting (Shibuya et al., 2000). The mobile factor for interorgan signaling has been proposed to be ethylene (Woltering et al., 1995) or ACC (Reid et al., 1984). In Digitalis (Stead and Moore, 1983) and torenia flowers (Goto et al., 1999), the gynoecium shows a climacteric-like increase in ethylene production, but petals do not. It has been hypothesized that in these flowers, ethylene produced by the gynoecium acts on the abscission zone, leading to petal abscission. The gynoecium and receptacle in Delphinium flowers showed climacteric-like increases in ethylene production with flower senescence, but petals and sepals did not (Fig. 2). ACC synthase and ACC oxidase activity were much higher in the gynoecium than in the receptacle (Fig. 3), suggesting that ethylene production is higher in the gynoecium than in the receptacle. From these findings, we hypothesize that ethylene produced by the gynoecium induces ethylene production from the receptacle. The sepals are directly connected with the receptacle, but not with the gynoecium, in Delphinium flowers. Thus, ethylene produced by the receptacle should directly act on abscission of the sepals. ACC synthase is generally considered to be a rate-limiting step for ethylene biosynthesis in plants because ACC oxidase activity is constitutive in many instances (Yang and Hoffman, 1984; Kende, 1993). However, ACC oxidase is important for ethylene biosynthesis in some situations, including fruit maturation (Nakatsuka et al., 1998) and flower senescence (Tang et al., 1993). Similarly, the biosynthesis of ethylene is regulated by ACC synthase and ACC oxidase activities in cut carnation flowers (Woodson et al., 1992; Woltering et al., 1993; Lee et al., 1997). Likewise, ACC concentration, ACC synthase and ACC oxidase activities increased in the gynoecium and the receptacle of senesced Delphinium flowers (Fig. 3). In addition, AVG markedly delayed sepal abscission and exogenous ACC did not accelerate it (Table 2). Thus, an increase in both ACC synthase and ACC oxidase contributes to ethylene biosynthesis in the gynoecium and receptacle, and this increase may be required for sepal abscission in Delphinium.
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In our study, AIB and CoCl2 did not delay sepal abscission (Table 2). Uda et al. (1997) also reported that AIB did not delay sepal abscission in another Delphinium cultivar. The ineffectiveness of these compounds may be due to the insufficient accumulation of these compounds at a sufficient concentration at active sites in Delphinium. AIB and CoCl2 were also shown to not completely suppress the conversion of ACC to ethylene in cocklebur (Satoh and Esashi, 1980, 1983). ACC synthase and ACC oxidase activities in the receptacle were low during the first two days, while those in the gynoecium were relatively high (Fig. 3). Although ACC concentration was low, the ethylene production was not (Figs. 2 and 3), suggesting that ACC produced by ACC synthase may have been converted to ethylene by the high activity of ACC oxidase, thus preventing the accumulation of ACC. In the carnation gynoecium, relatively high ACC synthase activity was observed prior to the accumulation of ACC (Woodson et al., 1992). The wounding of the gynoecium and receptacle in combination with stamen removal accelerated the abscission of sepals (Table 3), which was accompanied by a marked increase in ethylene production (Fig. 4). This effect was completely nullified by STS treatment (Table 4). These findings clearly show that acceleration of sepal abscission through wounding is regulated by ethylene. Wounding of organs has been shown to increase ethylene production in various plants (Yang and Hoffman, 1984). In petunia (Whitehead et al., 1984), Eustoma (Ichimura and Goto, 2000) and torenia (Goto et al., 1999), wounding of the stigma increases ethylene production, leading to accelerated flower senescence. Similarly, wounding of filaments accelerated flower senescence in Portulaca flowers (Ichimura and Suto, 1998). In addition to these organs, the gynoecium and the receptacle of Delphinium have high levels of ethylene production and ethylene has been shown to be mobile in plants (Jackson and Campbell, 1975; Zeroni et al., 1977). Woltering et al. (1997) also showed that ethylene diffused after stigma wounding in Petunia. Thus, we propose that ethylene produced in the gynoecium due to wounding induces ethylene production of the gynoecium, which moves to the abscission zone and consequently accelerating sepal abscission while ethylene produced by wounding of the receptacle acts on abscission zone, leading to sepal abscission. In our study, removal of stamens alone increased ethylene production by the gynoecium and receptacle. Since stamens are directly connected to the receptacle, removal of stamens likely wounds the receptacle, leading to increased ethylene production. Pulse treatment with STS markedly delayed sepal abscission even when the gynoecium or the receptacle was wounded (Table 4). Pulse treatment with AVG also markedly delayed sepal abscission (Table 2), but severely inhibits sepal growth in cut Delphinium spikes (unpublished results). In addition, pulse treatments with AIB and aminooxyacetic acid, inhibitors of ethylene biosynthesis, and 1methylcyclopropene, an inhibitor of ethylene action, do not delay sepal abscission in cut Delphinium (Uda et al., 1997; Ichimura et al., 2002). Thus, pulse treatment with STS should be useful for extending the vase life of cut Delphinium flowers. In a comparison of several treatments shown in Table 2, STS showed marked effects on delaying sepal abscission as shown in Table 4, although STS was applied in a concentration of 0.5 mM in both experiments. The difference in effectiveness is possibly due to a difference in application methods. In the test of chemical effects shown in Table 2, the flower parts were wetted with STS solution, while STS solution was absorbed to inhibit wound-induced abscission in the data shown in Table 4. Although the amount of absorbed STS has not been confirmed, sufficient amounts of STS solution would be absorbed in the experimental results shown in Table 4. Pollination accelerates flower senescence in many flowers, such as dendrobium (Porat et al., 1994), petunia (Whitehead et al., 1984),
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torenia (Goto et al., 1999), Digitalis (Stead and Moore, 1979) and Eustoma (Ichimura and Goto, 2000). In the present study, the stamens were not removed, except for in the experiment on wounding effects because the removal of stamens would accelerate ethylene production (Table 4). Delphinium flowers are protandrous and the gynoecium matures three or four days after anthesis under the present experimental conditions. Indeed, artificial pollination on the day of anthesis does not accelerate the sepal abscission in Delphinium (unpublished results). In the present study, the flowers may have been naturally pollinated, but ethylene production had started to increase on day 2, a point at which the stigma would not be matured (Fig. 1). Thus, the effect of natural pollination on ethylene production appears to be negligible in cut Delphinium under the present experimental conditions. In conclusion, sensitivity to ethylene of Delphinium flowers increased with aging. The gynoecium and receptacle showed a climacteric-like increase in ethylene production, but other organs did not. ACC concentration, ACC synthase and ACC oxidase activities were relatively high in the senesced gynoecium and receptacle. Wounding of the gynoecium or receptacle accelerated the abscission of sepals, and this was completely suppressed by STS. Thus, ethylene produced by the gynoecium and receptacle is responsible for sepal abscission in cut Delphinium flowers. Acknowledgements We thank Drs. W. G. van Doorn and U. K. Pun for their critical reading of this manuscript and Mrs. K. Matsuda for her assistance. References Clark, D.G., Richards, C., Hilioti, Z., Lind-Iversen, S., Brown, K., 1997. Effect of pollination on accumulation of ACC synthase and ACC oxidase transcripts, ethylene produciton and flower petal abscission in geranium (Pelargonium × hortorum L H. Bailey). Plant Mol. Biol. 34, 855–865. Dupille, E., Rombaldi, C., Lelievre, J.M., Cleyet-Marel, J.C., Pech, J.C., Latche, A., 1993. Purification, properties and partial amino-acid sequence of 1aminocyclopropane-1-carboxylate oxidase from apple fruit. Planta 190,65–70. Evensen, K.B., 1991. Ethylene responsiveness changes in Pelargonium × domesticum florets. Physiol. Plant. 82, 409–412. Gillisen, L.J.W., Hoekstra, F.A., 1984. Pollination-induced corolla wilting in Petunia hybrida rapid transfer through the style of a wilting-inducing substance. Plant Physiol. 75, 496–498. Goto, R., Aida, R., Shibata, M., Ichimura, K., 1999. Role of ethylene on flower senescence of Torenia. J. Japan. Soc. Hort. Sci. 68, 263–268. Ichimura, K., Goto, R., 2000. Acceleration of senescence by pollination of cut Asukano-nami Eustoma flowers. J. Japan. Soc. Hort. Sci. 69, 166–170. Ichimura, K., Kohata, K., Goto, R., 2000. Soluble carbohydrate in Delphinium and their influence on sepal abscission in cut flowers. Physiol. Plant. 108, 307–313. Ichimura, K., Shimamura, M., Hisamatsu, T., 1998. Role of ethylene in senescence of cut Eustoma flowers. Postharvest Biol. Technol. 14, 193–198. Ichimura, K., Shimizu, H., Hiraya, T., Hisamatsu, T., 2002. Effect of 1methylcyclopropene (1-MCP) on the vase life of cut carnation Delphinium and sweet pea flowers. Bull. Natl. Inst. Flor. Sci. 2, 1–8. Ichimura, K., Suto, K., 1998. Role of ethylene in acceleration of flower senescence by filament wounding in Portulaca hybrid. Physiol. Plant. 104, 603–607. Jackson, M.B., Campbell, D.J., 1975. Movement of ethylene from roots to shoots, a factor in the responses of tomato plants to waterlogged soil conditions. New Phytol. 74, 397–406. Kende, H., 1993. Ethylene biosynthesis. Annu. Rev. Plant. Physiol. Mol. Biol. 44, 283–307. Kuroda, S., Hakata, M., Hirose, Y., Shiraishi, M., Abe, S., 2003. Ethylene production and enhanced transcription of an ethylene receptor gene, ERS1, in Delphinium during abscission of florets. Plant Physiol. Biochem. 41, 812–820.
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