Journal of Plant Physiology 173 (2015) 116–119
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Pollination increases ethylene production in Lilium hybrida cv. Brindisi flowers but does not affect the time to tepal senescence or tepal abscission Silvia Pacifici a , Domenico Prisa a , Gianluca Burchi a , Wouter G. van Doorn b,∗ a b
Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA-VIV), Via dei Fiori 8, 51012 Pescia, Italy Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 25 August 2014 Accepted 27 August 2014 Available online 6 September 2014 Keywords: Abscission Ethylene Pollination Senescence Tepal
a b s t r a c t In many species, pollination induces a rapid increase in ethylene production, which induces early petal senescence, petal abscission, or flower closure. Cross-pollination in Lilium hybrida cv. Brindisi resulted in a small increase in flower ethylene production. In intact plants and in isolated flowers, pollination had no effect on the time to tepal senescence or tepal abscission. When applied to closed buds of unpollinated flowers, exogenous ethylene slightly hastened the time to tepal senescence and abscission. However, exogenous ethylene had no effect when the flowers had just opened, i.e. at the time of pollination. Experiments with silver thiosulphate, which blocks the ethylene receptor, indicated that endogenous ethylene had a slight effect on the regulation of tepal senescence and tepal abscission, although only at the time the tepals were still inside buds and not in open flowers. Low ethylene-sensitivity after anthesis therefore explains why pollination had no effect on the processes studied. © 2014 Elsevier GmbH. All rights reserved.
Introduction In many species, flower senescence is hastened by pollination. Depending on the species, pollination can also induce early abscission of petals/tepals. Flower closure is another species-specific response to pollination. In other species, however, pollination does not hasten these symptoms (O’Neill, 1997; van Doorn, 1997). Pollen grains placed on the stigma can emit a signal that increases the expression of genes involved in ethylene synthesis. This results in a local increase in ethylene production. The emitted ethylene stimulates ethylene production in more distant parts of the style and subsequently in the ovary. An autocatalytic increase in ethylene synthesis takes place in the ovary. This ethylene diffuses into the petals, where it induces early senescence and/or abscission (ten Have and Woltering, 1997; O’Neill, 1997; Jones and Woodson, 1999; Jones, 2008; Klee and Clark, 2010). Petal senescence in many flowers is regulated by endogenous ethylene, while in many other flowers such ethylene regulation is absent. By contrast, almost all examples of petal abscission are regulated by endogenous ethylene (Woltering and van Doorn,
Abbreviation: STS, silver thiosulphate. ∗ Corresponding author. Tel.: +1 530 867 3684; fax: +1 530 752 4554. E-mail address: [email protected] (W.G. van Doorn). http://dx.doi.org/10.1016/j.jplph.2014.08.014 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
1988; van Doorn, 2001). Ethylene can also induce early closure of turgid flowers (van Doorn, 2002). The regulation by ethylene is apparently an adaptation that allows pollination to cease the attraction of pollinators to the flower. This increases pollinator efficiency (van Doorn, 1996). After treatment with ethylene the tepals of Erythronium americanum and Lilium martagon showed advanced senescence symptoms as well as advanced abscission, which occurred at about the same time (van Doorn, 2001). Pollination also hastened the time to the end of flower life span, both in E. americanum and in L. martagon (van Doorn, 1997). In lily, preliminary data suggested that ethylene had an effect on tepal senescence in some cultivars, but no effect in others (Elgar et al., 1999; van Doorn, 2001). This suggests that pollination might have a similar effect in some cultivars but not in others. Using lily cv. Brindisi, we tested if pollination hastened the time to tepal senescence and abscission. We determined the effect of pollination on the rate of ethylene production by the flowers. We also treated the flowers with ethylene. To test the possible role of endogenous ethylene we treated the flowers with silver thiosulphate (STS), which blocks the ethylene receptor (Veen and van de Geijn, 1978). As the onset of tepal senescence in some species takes place before flower opening (Bancher, 1938; van Doorn et al., 2003; Wagstaff et al., 2003), we applied ethylene and STS in one series of experiments before flower opening. As the time of pollination is
S. Pacifici et al. / Journal of Plant Physiology 173 (2015) 116–119
just after flower opening, we also applied ethylene and STS just after anthesis. It was hypothesized that in the cultivar tested (a) tepal senescence and tepal abscission is not affected by pollination, (b) pollination does not increase the rate of ethylene production, and (c) endogenous ethylene is not be a regulator of tepal senescence or tepal abscission.
Materials and methods Plants, pollination Lily plants (Lilium hybrid, Longiflorum – Asiatic group, cv. Brindisi) were produced by a grower in Pescia, Italy. Eighty plants that were about to be harvested were uprooted and brought to the laboratory greenhouse where they were planted in sandy soil containing about 50% organic matter. Individual flowers were tagged just after opening, when the anthers were still green. Flowers at this stage were used for pollination. Pollination occurred by moving mature, dehiscent anthers of lily cv. Pavia (Longiflorum – Asiatic group) over the stigma, until the whole stigma was coloured. In commercial lily cultivars, self-pollination does not result in fertilization and ovary development, but cross-pollination with other cultivars results in normal development (B. Nesi, personal communication). Control flowers remained unpollinated. Pollination was also carried out using cut flowers. Open flowers were severed from cut inflorescences which were held at 20 ◦ C until the lowermost buds had opened. One flower was used per inflorescence. The flower pedicel (2 cm long) was placed in water, one flower per vial. The cut flowers were held in climate-controlled room at 20 ◦ C and about 60% RH. Photosynthetically active photon flux was about 10 mol m−2 s−1 (Philips TDL 36 W/84 cool white fluorescent tubes), from 07:00 to 19:00. Pollination was carried out using fresh anthers from open flowers of cv. Pavia. Pollination thus occurred immediately after full flower opening. Controls remained unpollinated. Emasculation, i.e. the removal of the anthers, can result in a higher rate of ethylene production than pollination (Woltering, 1990). In some experiments we therefore emasculated flowers by severing the anthers at their junction with the filaments. This treatment was in some tests combined with pollination. The time to tepal senescence was defined as the time between the onset of the experiment and the first visible tepal senescence symptoms. Tepals were defined to be senescent when their tips showed discoloration to white, or (more rarely) when such discoloration occurred at another part of the tepal surface. The time to petal abscission was the time between the onset of the experiment and abscission, which was defined by the fall of more than two tepals. Observations were made daily. In experiments with floral buds the time to full flower opening was assessed by daily examination. The time between full flower opening and the time to the onset of tepal senescence or to the onset of tepal abscission was calculated.
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Using a syringe, ethylene (Alltech, Deerfield, IL, USA) was injected trough a rubber septum into closed 0.6 L vials at 0.1 L L−1 , or 0.1 and 0.5 L L−1 , depending on the experiment. Controls did not receive ethylene. In experiments in which ethylene was applied before flower opening, the lids remained closed until the flowers had fully opened. In experiments where ethylene was applied after flower opening the ethylene levels in the vials were maintained until the flowers showed tepal abscission. After opening of the lids the flowers were left in the vials. Both in the controls and in the ethylene treatments, excess carbon dioxide was removed using calcium hydroxide, 2 g per vial. In the controls ethylene was removed by potassium permanganate, using Ethysorb (a mixture of aluminium oxide and potassium permanganate; Stay Fresh, London, UK), also 2 g per vial. The experiment was conducted at 20 ◦ C. The ethylene concentration in the vials was checked daily, using gas chromatography, as described below, and was adjusted if required. Treatments with silver thiosulphate (STS) were also carried out before flower opening or just after opening. STS was prepared by slowly adding an aqueous silver nitrate (Carlo Erba, Milano, Italy) solution to an eightfold higher concentration of an aqueous sodium thiosulfate (Sigma) solution. In experiments in which STS was applied to floral buds, the treatments started 5 days prior to flower opening. STS was placed in the vase solution and was not replenished (continuous treatment), or it was given for 4 h after which flowers were placed in water. A concentration range of STS was used for both types of treatments whereby the continuous treatment used a tenfold lower concentration (0.1 mM range) than the 4 h pulse treatment (1.0 mM range). The concentrations used are mentioned in the text and Supplement Table. Ethylene production Ethylene production was determined using the lowermost severed flowers from lily inflorescences. The flowers had just opened. The rate of ethylene production was determined by placing individual open flowers with 2 cm pedicels in 0.6 L glass vials with a 1 cm layer of water (20 mL), at 20 ◦ C. The lids which contained a septum were closed for 1 h prior to taking a 1 mL gas sample from the head space, using a syringe. After taking the gas sample the vials were opened again. The samples were injected in a Thermo Electron Corp gas chromatograph (Trace GC Ultra, Thermo Fisher, Waltham, MA, USA) using flame ionization detection. Treatments were (a) controls (not pollinated, not emasculated), (b) pollination with cv. Pavia pollen, and (c) pollination as well as emasculation. Statistics Results were compared by T-test or by one-way analysis of variance and F-test using Graphpad Prism (La Jolla, CA, USA). Data on the time to senescence and abscission were based on 7 replicate floral buds or open flowers. Ethylene production measurements used 10 replications. All experiments were repeated at a later date, in the same form or with slight variation.
Chemicals
Results
Experiments with chemicals were carried out with individual floral buds or open flowers, placed in water in the climatecontrolled room. When using buds, the lowermost bud on a freshly cut inflorescence was severed and placed, with 2 cm stems (peduncles), in 0.6 L glass vials containing 1 cm of water. In tests in which treatments started after flower opening, the lowermost flower had opened on an intact plant in the greenhouse. Flowers were severed and then treated as the closed buds.
Effects of pollination on the time to tepal senescence and tepal abscission Visible tepal senescence in intact lily cv. Brindisi started with discoloration in a small area at the tepal tips. These areas subsequently desiccated. Slightly afterwards, discoloration and dehydration symptoms were observed at several other areas on the tepal surface. By the time of abscission the distal parts of the
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S. Pacifici et al. / Journal of Plant Physiology 173 (2015) 116–119
Table 1 Effects of pollination on the time from full flower opening to tepal abscission and tepal senescence in lily flowers cv. Brindisi. Flowers were either attached to intact plants in the greenhouse at the end of winter (experiment 1), or were cut from inflorescences when open and individually placed in water (experiment 2). In the emasculation treatment the anthers were removed by cutting at their junction with the filaments. Time to tepal senescence (d)a
Time to tepal abscission (d)a
Experiment 1 Intact plants in the greenhouse Lowermost (first) flowers on the inflorescence Control 7.0 a 9.5 a Pollinated 6.9 a 9.9 a 7.1 a 9.6 a Emasculated 7.0 a 9.7 a Pollinated and emasculated Third and fourth flowers on the inflorescence Control 5.8 b 7.7 b 5.6 b 7.5 b Pollinated 5.8 b 7.6 b Emasculated 5.8 b 7.5 b Pollinated and emasculated Experiment 2 Cut flowers placed in water in a temperature-controlled room Lowermost (first) flowers on the inflorescence 5.2 a 7.5 a Control 5.5 a 7.5 a Pollinated 5.4 a 7.3 a Pollinated and emasculated a
Data are means of 10 replicate flowers. Results per experiment and within each column with the same letter are not statistically different (P < 0.05).
tepals showed advanced dehydration symptoms. In a few rare flowers in the greenhouse the tepals did not fall but showed complete desiccation. Pollination experiments using pollen of cv. Pavia were carried out with flowers on intact plants in the greenhouse. Inflorescences had 4–6 flower buds. Generally, the most proximal (basal) flower on an inflorescence opened first, and showed senescence symptoms first. Visible tepal senescence symptoms were followed by tepal abscission (Table 1). Compared to these proximal flowers, the more distal flowers exhibited a shorter time between opening and visible tepal senescence, between opening and tepal abscission, and between senescence and abscission (Table 1). In intact plants pollination did not hasten the time to visible tepal senescence or tepal abscission (Table 1). Emasculation (removal of all anthers) and those of pollination together with emasculation were also tested. These treatments had no effect on the time to tepal senescence or tepal abscission (Table 1). Pollination experiments with cv. Pavia pollen were also carried out in the laboratory, using only the lowermost open flower from an inflorescence. Flowers were severed after they had just opened. Pollination had no effect on the time to tepal senescence or tepal abscission (Table 1). Emasculation also did not affect on the time to tepal senescence or tepal abscission, nor did emasculation plus pollination (Table 1). Ethylene production The rate of ethylene production in the controls slightly increased during the 3 days of measurement. By day 3 is was about 0.7 nL mg−1 FW h−1 (Fig. 1). Pollination at t = 0 h did not affect the rate of ethylene production at t = 6 h, but increased production after about one day (Fig. 1). The difference had further increased by day 2 after pollination, but by day 3 the effect of pollination had subsided (Fig. 1). The treatment in which the flowers were pollinated and at the same time emasculated resulted in a higher rate of ethylene production on day 2, compared to pollination alone (Fig. 1). Emasculation alone resulted in rates of ethylene production that were similar to those after pollination (data not shown).
Fig. 1. Ethylene production by isolated cv. Brindisi lily flowers after pollination or pollination plus emasculation. The oldest bud on an inflorescence was allowed to open and was then severed and its pedicel placed in water at 20 ◦ C. Open flowers were controls, were pollinated with pollen of cv. Pavia lilies, or were emasculated (pollinia were removed). Data are means of 10 replicate flowers ±SD.
Effects of exogenous ethylene Floral buds or open flowers were exposed to ethylene. In one series of experiments the onset of 0.5 l L−1 ethylene application started prior to flower opening and the treatment ended 4 days later. In the other series ethylene exposure started when the flowers had just opened. One example is shown of each of these types of experiments. In the first type of experiments only the lowermost (oldest) bud on an inflorescence was used. Buds were severed and placed in water. One group of buds had a reddish hue, while another group was still uniformly green but about to get the reddish hue. In untreated controls the time between the onset of the experiment and the onset of opening of the green buds was 6 days while the time to opening of the reddish buds was 4 days. Compared to untreated controls, ethylene applied continuously at 0.1 L L−1 hastened opening by two days in the green buds and one day in the reddish buds (both statistically significant at P < 0.05; Supplement Table 1). The time between full flower opening and the onset of senescence or abscission was not affected by the ethylene treatment (Supplement Table 1). In the second series of tests (starting ethylene exposure just after bud opening) the uppermost (oldest) flowers had opened on intact plants in the greenhouse. Flowers were severed and treated with ethylene at 0.1 and 5.0 L L−1 until the time of tepal abscission. The treatments had no effect on the time to tepal senescence or abscission (Supplement Table 1). Effects of STS STS, if applied continuously in the vase solution at 0.05, 0.1 or 0.2 mM, starting 5 days prior to bud opening, had no effect on the time to bud opening (Supplement Table 1). The 0.1 or 0.2 mM STS treatment increased the time between full flower opening and the onset of tepal senescence (Supplement Table 1; P < 0.01). It also increased the time between full flower opening and the onset of tepal abscission (Supplement Table 1; P < 0.01).
S. Pacifici et al. / Journal of Plant Physiology 173 (2015) 116–119
In tests in which STS was applied for only 4 h, 5 days prior to flower opening, at 0.5, 1.0 and 2.0 mM, the time to floral bud opening was not affected, but a longer time to tepal senescence and tepal abscission was found at all three concentrations tested (Supplement Table 1; P < 0.01). STS, applied when the flowers had just fully opened, had no effect on the time to tepal senescence or the time to tepal abscission. In these tests, STS was applied continuously at 0.1, 0.2 and 0.4 mM, or for 4 h at 0.5, 1.0 and 2.0 mM (Supplement Table 1). Discussion Pollination resulted in an increase in ethylene production by the flowers, but it had no effect on the time to tepal senescence or tepal abscission. The maximum rate of ethylene production (1.2 nL mg−1 FW h−1 ) was relatively low compared to that in other pollinated flowers. For example, in whole Dendrobium flowers the maximum was 100 nL mg−1 FW h−1 (Ketsa and Languwalai, 1996), in carnation more than 55 nL (Nichols, 1977), in Petunia about 17 nL (Shibuya et al., 2013), and in Phalaenopsis about 6 nL (Tsai et al., 2005). In these examples pollination induced early petal/tepal senescence. However, in Gentiana scabra the rate of ethylene production of whole flowers after pollination was less than 2 nL mg−1 FW h−1 . This also was accompanied by hastened senescence (Shimizu-Yumoto and Ichimura, 2012). The rate of 1.2 nL mg−1 FW h−1 in whole lily flowers reported here is similar to that in G. scabra, but in lily it did not result in early petal/tepal senescence or abscission. Emasculation together with pollination resulted in a slight further increase in ethylene production, but this was still not associated with hastening of tepal senescence or tepal abscission. The data suggest that the processes leading to tepal senescence or tepal abscission were insensitive to the relatively small amount of ethylene produced by pollination or pollination plus emasculation. This was confirmed in tests showing that tepal senescence and abscission were also insensitive to a relatively high dose of exogenously applied ethylene, at the time of pollination. Experiments with STS, which inhibits the ethylene receptor, further confirmed lack regulation by endogenous ethylene of tepal senescence and tepal abscission, at the time of pollination. It was observed however that at an earlier stage, when the buds were still closed, tepal senescence and tepal abscission were slightly delayed by STS, while treatment with exogenous ethylene had no effect. This suggests that tepal senescence and abscission were to a small degree regulated by endogenous ethylene at that early stage. The data also indicate that processes leading to senescence and abscission start early in lily. This would agree with the very early onset of senescence processes in other monocotyledonous flowers such as Iris, Gladiolus (Bancher, 1938; van Doorn et al., 2003) and Alstroemeria (Wagstaff et al., 2003). It should be noted that exogenous ethylene hastened bud opening. By contrast, STS did not affect bud opening. This suggests that floral bud opening normally is not regulated by endogenous ethylene. The effect of exogenous ethylene might be similar to a stress-induced increase in ethylene production by the plant. In previous work on the effect of ethylene in lily cultivars, ethylene was applied to closed buds. Elgar et al. (1999) observed little
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effect of exogenous ethylene on tepal senescence in the lily cultivars studied, but van Doorn (2001) found an effect in some of the cultivars tested. In the present experiments it was again found that exogenous ethylene, applied before flower opening, hastened tepal senescence and abscission. In cut Alstroemeria peruviana flowers, in which vase life is ended by tepal abscission rather than visible tepal senescence, Wagstaff et al. (2005) reported that STS applied for 1 h on day 4 prior to flower opening, delayed abscission by one to three days, depending on the experiment. This is similar to the present data on lily cv. Brindisi. We hypothesized that pollination would not affect the time to tepal senescence or tepal abscission. This hypothesis was confirmed. We also hypothesized that pollination would not result in an increase in ethylene production, which was falsified. We furthermore hypothesized that senescence and abscission would not be affected by exogenous ethylene. This was also falsified, as it depended on the stage of development whether STS had an effect or not. It is concluded that (a) pollination did not hasten tepal senescence or tepal abscission in the lily hybrid studied, (b) that pollination increased the rate of ethylene production by the flower, (c) that by the time of pollination endogenous ethylene had no effect on tepal senescence and abscission, but it had a small effect on these processes during a period before flower opening. The ethylene produced by pollination thus was without effect because the systems inducing senescence and abscission were apparently no longer responsive to the hormone. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2014. 08.014. References Bancher E. Österr Bot Zeitschr 1938;87:221–8. Elgar HJ, Woolf AB, Bieleski RL. Postharvest Biol Technol 1999;16:257–67. Jones ML, Woodson WR. J Am Soc Hort Sci 1999;124:598–604. Jones ML. Plant Sci 2008;175:190–6. Ketsa S, Languwalai K. Postharvest Biol Technol 1996;8:57–64. Klee HJ, Clark DG. Davies PJ, editor. Plant hormones: physiology, biochemistry and molecular biology. Dordrecht: Springer; 2010. p. 377–98. Nichols R. Planta 1977;135:155–9. O’Neill SD. Annu Rev Plant Biol 1997;48:547–74. Shibuya K, Niki T, Ichimura K. J Exp Bot 2013;64:1111–20. Shimizu-Yumoto H, Ichimura K. Postharvest Biol Technol 2012;63:111–5. ten Have A, Woltering EJ. Plant Mol Biol 1997;34:89–97. Tsai WC, Lee PF, Chen HI, Hsiao YY, Wei WJ, Pan ZJ, et al. Plant Cell Physiol 2005;46:1125–39. van Doorn WG. Nature 1996;379:779–80. van Doorn WG. J Exp Bot 1997;48:1615–22. van Doorn WG. Ann Bot 2001;87:447–56. van Doorn WG. Ann Bot 2002;89:375–83. van Doorn WG, Balk PA, van Houwelingen AM, Hoeberichts FA, Hall RD, Vorst O, et al. Plant Mol Biol 2003;53:845–63. Veen H, van de Geijn SC. Planta 1978;140:93–6. Wagstaff C, Chanasut U, Harren FJM, Laarhoven LJ, Thomas B, Rogers HJ, et al. J Exp Bot 2005;56:1007–16. Wagstaff C, Malcolm P, Rafiq A, Leverentz M, Griffiths G, Thomas B, et al. New Phytol 2003;160:49–59. Woltering EJ. J Exp Bot 1990;41:1021–9. Woltering EJ, van Doorn WG. J Exp Bot 1988;39:1605–16.
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Pollination increases ethylene production in Lilium hybrida cv. Brindisi flowers but does not affect the time to tepal senescence or tepal abscission Silvia Pacifici a , Domenico Prisa a , Gianluca Burchi a , Wouter G. van Doorn b,∗ a b
Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA-VIV), Via dei Fiori 8, 51012 Pescia, Italy Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 25 August 2014 Accepted 27 August 2014 Available online 6 September 2014 Keywords: Abscission Ethylene Pollination Senescence Tepal
a b s t r a c t In many species, pollination induces a rapid increase in ethylene production, which induces early petal senescence, petal abscission, or flower closure. Cross-pollination in Lilium hybrida cv. Brindisi resulted in a small increase in flower ethylene production. In intact plants and in isolated flowers, pollination had no effect on the time to tepal senescence or tepal abscission. When applied to closed buds of unpollinated flowers, exogenous ethylene slightly hastened the time to tepal senescence and abscission. However, exogenous ethylene had no effect when the flowers had just opened, i.e. at the time of pollination. Experiments with silver thiosulphate, which blocks the ethylene receptor, indicated that endogenous ethylene had a slight effect on the regulation of tepal senescence and tepal abscission, although only at the time the tepals were still inside buds and not in open flowers. Low ethylene-sensitivity after anthesis therefore explains why pollination had no effect on the processes studied. © 2014 Elsevier GmbH. All rights reserved.
Introduction In many species, flower senescence is hastened by pollination. Depending on the species, pollination can also induce early abscission of petals/tepals. Flower closure is another species-specific response to pollination. In other species, however, pollination does not hasten these symptoms (O’Neill, 1997; van Doorn, 1997). Pollen grains placed on the stigma can emit a signal that increases the expression of genes involved in ethylene synthesis. This results in a local increase in ethylene production. The emitted ethylene stimulates ethylene production in more distant parts of the style and subsequently in the ovary. An autocatalytic increase in ethylene synthesis takes place in the ovary. This ethylene diffuses into the petals, where it induces early senescence and/or abscission (ten Have and Woltering, 1997; O’Neill, 1997; Jones and Woodson, 1999; Jones, 2008; Klee and Clark, 2010). Petal senescence in many flowers is regulated by endogenous ethylene, while in many other flowers such ethylene regulation is absent. By contrast, almost all examples of petal abscission are regulated by endogenous ethylene (Woltering and van Doorn,
Abbreviation: STS, silver thiosulphate. ∗ Corresponding author. Tel.: +1 530 867 3684; fax: +1 530 752 4554. E-mail address: [email protected] (W.G. van Doorn). http://dx.doi.org/10.1016/j.jplph.2014.08.014 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
1988; van Doorn, 2001). Ethylene can also induce early closure of turgid flowers (van Doorn, 2002). The regulation by ethylene is apparently an adaptation that allows pollination to cease the attraction of pollinators to the flower. This increases pollinator efficiency (van Doorn, 1996). After treatment with ethylene the tepals of Erythronium americanum and Lilium martagon showed advanced senescence symptoms as well as advanced abscission, which occurred at about the same time (van Doorn, 2001). Pollination also hastened the time to the end of flower life span, both in E. americanum and in L. martagon (van Doorn, 1997). In lily, preliminary data suggested that ethylene had an effect on tepal senescence in some cultivars, but no effect in others (Elgar et al., 1999; van Doorn, 2001). This suggests that pollination might have a similar effect in some cultivars but not in others. Using lily cv. Brindisi, we tested if pollination hastened the time to tepal senescence and abscission. We determined the effect of pollination on the rate of ethylene production by the flowers. We also treated the flowers with ethylene. To test the possible role of endogenous ethylene we treated the flowers with silver thiosulphate (STS), which blocks the ethylene receptor (Veen and van de Geijn, 1978). As the onset of tepal senescence in some species takes place before flower opening (Bancher, 1938; van Doorn et al., 2003; Wagstaff et al., 2003), we applied ethylene and STS in one series of experiments before flower opening. As the time of pollination is
S. Pacifici et al. / Journal of Plant Physiology 173 (2015) 116–119
just after flower opening, we also applied ethylene and STS just after anthesis. It was hypothesized that in the cultivar tested (a) tepal senescence and tepal abscission is not affected by pollination, (b) pollination does not increase the rate of ethylene production, and (c) endogenous ethylene is not be a regulator of tepal senescence or tepal abscission.
Materials and methods Plants, pollination Lily plants (Lilium hybrid, Longiflorum – Asiatic group, cv. Brindisi) were produced by a grower in Pescia, Italy. Eighty plants that were about to be harvested were uprooted and brought to the laboratory greenhouse where they were planted in sandy soil containing about 50% organic matter. Individual flowers were tagged just after opening, when the anthers were still green. Flowers at this stage were used for pollination. Pollination occurred by moving mature, dehiscent anthers of lily cv. Pavia (Longiflorum – Asiatic group) over the stigma, until the whole stigma was coloured. In commercial lily cultivars, self-pollination does not result in fertilization and ovary development, but cross-pollination with other cultivars results in normal development (B. Nesi, personal communication). Control flowers remained unpollinated. Pollination was also carried out using cut flowers. Open flowers were severed from cut inflorescences which were held at 20 ◦ C until the lowermost buds had opened. One flower was used per inflorescence. The flower pedicel (2 cm long) was placed in water, one flower per vial. The cut flowers were held in climate-controlled room at 20 ◦ C and about 60% RH. Photosynthetically active photon flux was about 10 mol m−2 s−1 (Philips TDL 36 W/84 cool white fluorescent tubes), from 07:00 to 19:00. Pollination was carried out using fresh anthers from open flowers of cv. Pavia. Pollination thus occurred immediately after full flower opening. Controls remained unpollinated. Emasculation, i.e. the removal of the anthers, can result in a higher rate of ethylene production than pollination (Woltering, 1990). In some experiments we therefore emasculated flowers by severing the anthers at their junction with the filaments. This treatment was in some tests combined with pollination. The time to tepal senescence was defined as the time between the onset of the experiment and the first visible tepal senescence symptoms. Tepals were defined to be senescent when their tips showed discoloration to white, or (more rarely) when such discoloration occurred at another part of the tepal surface. The time to petal abscission was the time between the onset of the experiment and abscission, which was defined by the fall of more than two tepals. Observations were made daily. In experiments with floral buds the time to full flower opening was assessed by daily examination. The time between full flower opening and the time to the onset of tepal senescence or to the onset of tepal abscission was calculated.
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Using a syringe, ethylene (Alltech, Deerfield, IL, USA) was injected trough a rubber septum into closed 0.6 L vials at 0.1 L L−1 , or 0.1 and 0.5 L L−1 , depending on the experiment. Controls did not receive ethylene. In experiments in which ethylene was applied before flower opening, the lids remained closed until the flowers had fully opened. In experiments where ethylene was applied after flower opening the ethylene levels in the vials were maintained until the flowers showed tepal abscission. After opening of the lids the flowers were left in the vials. Both in the controls and in the ethylene treatments, excess carbon dioxide was removed using calcium hydroxide, 2 g per vial. In the controls ethylene was removed by potassium permanganate, using Ethysorb (a mixture of aluminium oxide and potassium permanganate; Stay Fresh, London, UK), also 2 g per vial. The experiment was conducted at 20 ◦ C. The ethylene concentration in the vials was checked daily, using gas chromatography, as described below, and was adjusted if required. Treatments with silver thiosulphate (STS) were also carried out before flower opening or just after opening. STS was prepared by slowly adding an aqueous silver nitrate (Carlo Erba, Milano, Italy) solution to an eightfold higher concentration of an aqueous sodium thiosulfate (Sigma) solution. In experiments in which STS was applied to floral buds, the treatments started 5 days prior to flower opening. STS was placed in the vase solution and was not replenished (continuous treatment), or it was given for 4 h after which flowers were placed in water. A concentration range of STS was used for both types of treatments whereby the continuous treatment used a tenfold lower concentration (0.1 mM range) than the 4 h pulse treatment (1.0 mM range). The concentrations used are mentioned in the text and Supplement Table. Ethylene production Ethylene production was determined using the lowermost severed flowers from lily inflorescences. The flowers had just opened. The rate of ethylene production was determined by placing individual open flowers with 2 cm pedicels in 0.6 L glass vials with a 1 cm layer of water (20 mL), at 20 ◦ C. The lids which contained a septum were closed for 1 h prior to taking a 1 mL gas sample from the head space, using a syringe. After taking the gas sample the vials were opened again. The samples were injected in a Thermo Electron Corp gas chromatograph (Trace GC Ultra, Thermo Fisher, Waltham, MA, USA) using flame ionization detection. Treatments were (a) controls (not pollinated, not emasculated), (b) pollination with cv. Pavia pollen, and (c) pollination as well as emasculation. Statistics Results were compared by T-test or by one-way analysis of variance and F-test using Graphpad Prism (La Jolla, CA, USA). Data on the time to senescence and abscission were based on 7 replicate floral buds or open flowers. Ethylene production measurements used 10 replications. All experiments were repeated at a later date, in the same form or with slight variation.
Chemicals
Results
Experiments with chemicals were carried out with individual floral buds or open flowers, placed in water in the climatecontrolled room. When using buds, the lowermost bud on a freshly cut inflorescence was severed and placed, with 2 cm stems (peduncles), in 0.6 L glass vials containing 1 cm of water. In tests in which treatments started after flower opening, the lowermost flower had opened on an intact plant in the greenhouse. Flowers were severed and then treated as the closed buds.
Effects of pollination on the time to tepal senescence and tepal abscission Visible tepal senescence in intact lily cv. Brindisi started with discoloration in a small area at the tepal tips. These areas subsequently desiccated. Slightly afterwards, discoloration and dehydration symptoms were observed at several other areas on the tepal surface. By the time of abscission the distal parts of the
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Table 1 Effects of pollination on the time from full flower opening to tepal abscission and tepal senescence in lily flowers cv. Brindisi. Flowers were either attached to intact plants in the greenhouse at the end of winter (experiment 1), or were cut from inflorescences when open and individually placed in water (experiment 2). In the emasculation treatment the anthers were removed by cutting at their junction with the filaments. Time to tepal senescence (d)a
Time to tepal abscission (d)a
Experiment 1 Intact plants in the greenhouse Lowermost (first) flowers on the inflorescence Control 7.0 a 9.5 a Pollinated 6.9 a 9.9 a 7.1 a 9.6 a Emasculated 7.0 a 9.7 a Pollinated and emasculated Third and fourth flowers on the inflorescence Control 5.8 b 7.7 b 5.6 b 7.5 b Pollinated 5.8 b 7.6 b Emasculated 5.8 b 7.5 b Pollinated and emasculated Experiment 2 Cut flowers placed in water in a temperature-controlled room Lowermost (first) flowers on the inflorescence 5.2 a 7.5 a Control 5.5 a 7.5 a Pollinated 5.4 a 7.3 a Pollinated and emasculated a
Data are means of 10 replicate flowers. Results per experiment and within each column with the same letter are not statistically different (P < 0.05).
tepals showed advanced dehydration symptoms. In a few rare flowers in the greenhouse the tepals did not fall but showed complete desiccation. Pollination experiments using pollen of cv. Pavia were carried out with flowers on intact plants in the greenhouse. Inflorescences had 4–6 flower buds. Generally, the most proximal (basal) flower on an inflorescence opened first, and showed senescence symptoms first. Visible tepal senescence symptoms were followed by tepal abscission (Table 1). Compared to these proximal flowers, the more distal flowers exhibited a shorter time between opening and visible tepal senescence, between opening and tepal abscission, and between senescence and abscission (Table 1). In intact plants pollination did not hasten the time to visible tepal senescence or tepal abscission (Table 1). Emasculation (removal of all anthers) and those of pollination together with emasculation were also tested. These treatments had no effect on the time to tepal senescence or tepal abscission (Table 1). Pollination experiments with cv. Pavia pollen were also carried out in the laboratory, using only the lowermost open flower from an inflorescence. Flowers were severed after they had just opened. Pollination had no effect on the time to tepal senescence or tepal abscission (Table 1). Emasculation also did not affect on the time to tepal senescence or tepal abscission, nor did emasculation plus pollination (Table 1). Ethylene production The rate of ethylene production in the controls slightly increased during the 3 days of measurement. By day 3 is was about 0.7 nL mg−1 FW h−1 (Fig. 1). Pollination at t = 0 h did not affect the rate of ethylene production at t = 6 h, but increased production after about one day (Fig. 1). The difference had further increased by day 2 after pollination, but by day 3 the effect of pollination had subsided (Fig. 1). The treatment in which the flowers were pollinated and at the same time emasculated resulted in a higher rate of ethylene production on day 2, compared to pollination alone (Fig. 1). Emasculation alone resulted in rates of ethylene production that were similar to those after pollination (data not shown).
Fig. 1. Ethylene production by isolated cv. Brindisi lily flowers after pollination or pollination plus emasculation. The oldest bud on an inflorescence was allowed to open and was then severed and its pedicel placed in water at 20 ◦ C. Open flowers were controls, were pollinated with pollen of cv. Pavia lilies, or were emasculated (pollinia were removed). Data are means of 10 replicate flowers ±SD.
Effects of exogenous ethylene Floral buds or open flowers were exposed to ethylene. In one series of experiments the onset of 0.5 l L−1 ethylene application started prior to flower opening and the treatment ended 4 days later. In the other series ethylene exposure started when the flowers had just opened. One example is shown of each of these types of experiments. In the first type of experiments only the lowermost (oldest) bud on an inflorescence was used. Buds were severed and placed in water. One group of buds had a reddish hue, while another group was still uniformly green but about to get the reddish hue. In untreated controls the time between the onset of the experiment and the onset of opening of the green buds was 6 days while the time to opening of the reddish buds was 4 days. Compared to untreated controls, ethylene applied continuously at 0.1 L L−1 hastened opening by two days in the green buds and one day in the reddish buds (both statistically significant at P < 0.05; Supplement Table 1). The time between full flower opening and the onset of senescence or abscission was not affected by the ethylene treatment (Supplement Table 1). In the second series of tests (starting ethylene exposure just after bud opening) the uppermost (oldest) flowers had opened on intact plants in the greenhouse. Flowers were severed and treated with ethylene at 0.1 and 5.0 L L−1 until the time of tepal abscission. The treatments had no effect on the time to tepal senescence or abscission (Supplement Table 1). Effects of STS STS, if applied continuously in the vase solution at 0.05, 0.1 or 0.2 mM, starting 5 days prior to bud opening, had no effect on the time to bud opening (Supplement Table 1). The 0.1 or 0.2 mM STS treatment increased the time between full flower opening and the onset of tepal senescence (Supplement Table 1; P < 0.01). It also increased the time between full flower opening and the onset of tepal abscission (Supplement Table 1; P < 0.01).
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In tests in which STS was applied for only 4 h, 5 days prior to flower opening, at 0.5, 1.0 and 2.0 mM, the time to floral bud opening was not affected, but a longer time to tepal senescence and tepal abscission was found at all three concentrations tested (Supplement Table 1; P < 0.01). STS, applied when the flowers had just fully opened, had no effect on the time to tepal senescence or the time to tepal abscission. In these tests, STS was applied continuously at 0.1, 0.2 and 0.4 mM, or for 4 h at 0.5, 1.0 and 2.0 mM (Supplement Table 1). Discussion Pollination resulted in an increase in ethylene production by the flowers, but it had no effect on the time to tepal senescence or tepal abscission. The maximum rate of ethylene production (1.2 nL mg−1 FW h−1 ) was relatively low compared to that in other pollinated flowers. For example, in whole Dendrobium flowers the maximum was 100 nL mg−1 FW h−1 (Ketsa and Languwalai, 1996), in carnation more than 55 nL (Nichols, 1977), in Petunia about 17 nL (Shibuya et al., 2013), and in Phalaenopsis about 6 nL (Tsai et al., 2005). In these examples pollination induced early petal/tepal senescence. However, in Gentiana scabra the rate of ethylene production of whole flowers after pollination was less than 2 nL mg−1 FW h−1 . This also was accompanied by hastened senescence (Shimizu-Yumoto and Ichimura, 2012). The rate of 1.2 nL mg−1 FW h−1 in whole lily flowers reported here is similar to that in G. scabra, but in lily it did not result in early petal/tepal senescence or abscission. Emasculation together with pollination resulted in a slight further increase in ethylene production, but this was still not associated with hastening of tepal senescence or tepal abscission. The data suggest that the processes leading to tepal senescence or tepal abscission were insensitive to the relatively small amount of ethylene produced by pollination or pollination plus emasculation. This was confirmed in tests showing that tepal senescence and abscission were also insensitive to a relatively high dose of exogenously applied ethylene, at the time of pollination. Experiments with STS, which inhibits the ethylene receptor, further confirmed lack regulation by endogenous ethylene of tepal senescence and tepal abscission, at the time of pollination. It was observed however that at an earlier stage, when the buds were still closed, tepal senescence and tepal abscission were slightly delayed by STS, while treatment with exogenous ethylene had no effect. This suggests that tepal senescence and abscission were to a small degree regulated by endogenous ethylene at that early stage. The data also indicate that processes leading to senescence and abscission start early in lily. This would agree with the very early onset of senescence processes in other monocotyledonous flowers such as Iris, Gladiolus (Bancher, 1938; van Doorn et al., 2003) and Alstroemeria (Wagstaff et al., 2003). It should be noted that exogenous ethylene hastened bud opening. By contrast, STS did not affect bud opening. This suggests that floral bud opening normally is not regulated by endogenous ethylene. The effect of exogenous ethylene might be similar to a stress-induced increase in ethylene production by the plant. In previous work on the effect of ethylene in lily cultivars, ethylene was applied to closed buds. Elgar et al. (1999) observed little
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effect of exogenous ethylene on tepal senescence in the lily cultivars studied, but van Doorn (2001) found an effect in some of the cultivars tested. In the present experiments it was again found that exogenous ethylene, applied before flower opening, hastened tepal senescence and abscission. In cut Alstroemeria peruviana flowers, in which vase life is ended by tepal abscission rather than visible tepal senescence, Wagstaff et al. (2005) reported that STS applied for 1 h on day 4 prior to flower opening, delayed abscission by one to three days, depending on the experiment. This is similar to the present data on lily cv. Brindisi. We hypothesized that pollination would not affect the time to tepal senescence or tepal abscission. This hypothesis was confirmed. We also hypothesized that pollination would not result in an increase in ethylene production, which was falsified. We furthermore hypothesized that senescence and abscission would not be affected by exogenous ethylene. This was also falsified, as it depended on the stage of development whether STS had an effect or not. It is concluded that (a) pollination did not hasten tepal senescence or tepal abscission in the lily hybrid studied, (b) that pollination increased the rate of ethylene production by the flower, (c) that by the time of pollination endogenous ethylene had no effect on tepal senescence and abscission, but it had a small effect on these processes during a period before flower opening. The ethylene produced by pollination thus was without effect because the systems inducing senescence and abscission were apparently no longer responsive to the hormone. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2014. 08.014. References Bancher E. Österr Bot Zeitschr 1938;87:221–8. Elgar HJ, Woolf AB, Bieleski RL. Postharvest Biol Technol 1999;16:257–67. Jones ML, Woodson WR. J Am Soc Hort Sci 1999;124:598–604. Jones ML. Plant Sci 2008;175:190–6. Ketsa S, Languwalai K. Postharvest Biol Technol 1996;8:57–64. Klee HJ, Clark DG. Davies PJ, editor. Plant hormones: physiology, biochemistry and molecular biology. Dordrecht: Springer; 2010. p. 377–98. Nichols R. Planta 1977;135:155–9. O’Neill SD. Annu Rev Plant Biol 1997;48:547–74. Shibuya K, Niki T, Ichimura K. J Exp Bot 2013;64:1111–20. Shimizu-Yumoto H, Ichimura K. Postharvest Biol Technol 2012;63:111–5. ten Have A, Woltering EJ. Plant Mol Biol 1997;34:89–97. Tsai WC, Lee PF, Chen HI, Hsiao YY, Wei WJ, Pan ZJ, et al. Plant Cell Physiol 2005;46:1125–39. van Doorn WG. Nature 1996;379:779–80. van Doorn WG. J Exp Bot 1997;48:1615–22. van Doorn WG. Ann Bot 2001;87:447–56. van Doorn WG. Ann Bot 2002;89:375–83. van Doorn WG, Balk PA, van Houwelingen AM, Hoeberichts FA, Hall RD, Vorst O, et al. Plant Mol Biol 2003;53:845–63. Veen H, van de Geijn SC. Planta 1978;140:93–6. Wagstaff C, Chanasut U, Harren FJM, Laarhoven LJ, Thomas B, Rogers HJ, et al. J Exp Bot 2005;56:1007–16. Wagstaff C, Malcolm P, Rafiq A, Leverentz M, Griffiths G, Thomas B, et al. New Phytol 2003;160:49–59. Woltering EJ. J Exp Bot 1990;41:1021–9. Woltering EJ, van Doorn WG. J Exp Bot 1988;39:1605–16.
