Transcriptional regulation of ethylene receptor and CTR genes involved in ethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha

Abstract

RESEARCH PAPER

Transcriptional regulation of ethylene receptor and CTR genes involved in ethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha

Nan Ma *, Hui Tan *, Xiaohui Liu, Jingqi Xue, Yunhui Li and Junping Gao{dagger}

Department of Ornamental Horticulture, China Agricultural University, Beijing 100094, China

{dagger}To whom correspondence should be addressed. E-mail: gaojp@cau.edu.cn

Received 28 September 2005; Accepted 26 April 2006

  

In this work, the effect of ethylene on flower opening of cut rose (Rosa hybrida) cv. Samantha was studied. However, although ethylene hastened the process of flower opening, 1-MCP (1-methylcyclopropene), an ethylene action inhibitor, impeded it. Ethylene promoted ethylene production in petals, but 1-MCP did not inhibit this process. Of the four ethylene biosynthetic genes tested, Rh-ACS1 and Rh-ACS2 were undetectable; Rh-ACS3 and Rh-ACO1 expression was enhanced by ethylene slightly and greatly, respectively. However, their mRNA amounts were not inhibited by 1-MCP compared with controls. Expression of seven signalling component genes was also studied, including three ethylene receptors (Rh-ETR1, Rh-ETR3, and Rh-ETR5), two CTRs (Rh-CTR1 and Rh-CTR2), and two transcription factors (Rh-EIN3-1 and Rh-EIN3-2). Transcripts of Rh-ETR5, Rh-EIN3-1, and Rh-EIN3-2 were accumulated in a constitutive manner and had no or little response to ethylene or 1-MCP, while transcript levels of Rh-ETR1 and Rh-CTR1 were substantially elevated by ethylene, and those of Rh-ETR3 and Rh-CTR2 were greatly enhanced by ethylene; 1-MCP reduced all the four genes to levels much less than those in control flowers. These results show that ethylene triggers physiological responses related to flower opening in cut rose cv. Samantha, and that continued ethylene perception results in flower opening. Ethylene may regulate flower opening mainly through expression of two ethylene receptor genes (Rh-ETR1 and Rh-ETR3) and two CTR (Rh-CTR1 and Rh-CTR2) genes.

Key words: Cut roses, ethylene biosynthesis, ethylene signalling, flower opening, gene expression, Rosa hybrida

Journal of Experimental Botany 2006 57(11):2763-2773. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/)


Introduction

 

 
The gaseous phytohormone ethylene is involved in many aspects of plant growth and development, including seed germination, flower and leaf senescence and abscission, and fruit ripening. It also functions as an important modulator in plant responses to biotic and abiotic stimuli, like pathogen attack, flooding, chilling, and mechanical damage (Johnson and Ecker, 1998; Bleecker and Kende, 2000).

In higher plants, the ethylene biosynthesis pathway has been well defined as Yang's Cycle (Yang and Hoffman, 1984). In this pathway, AdoMet (S-adenosylmethionine) is converted to ACC (1-aminocyclopropane-1-carboxylic acid) by ACS (ACC synthase), and then ACC is oxidized to ethylene catalysed by ACO (ACC oxidase) (Yang and Hoffman, 1984; Kende, 1993). ACS is encoded by a medium-sized multigene family and expression of ACS genes is regulated by internal developmental and external stress cues (Barry et al., 2000; Nakano et al., 2003). ACO is encoded by a small multigene family and ACO genes are also regulated differently in response to developmental and environmental events (Clark et al., 1997; Nakatsuka et al., 1997, 1998). ACS is the rate-limiting enzyme in the biosynthetic pathway and ethylene production is modulated mainly via regulation of expression of ACS genes (Kende, 1993; Wang et al., 2002). However, in some cases, ACO is also the key regulator restricting plant ethylene production (Vriezen et al., 1999; Wagstaff et al., 2005).

During past decades, much effort has been made to understand the ethylene signal transduction pathway, and a framework of ethylene perception and signal transduction has been established through molecular genetic research in Arabidopsis (Guo and Ecker, 2004). Ethylene is perceived by a membrane-associated receptor family (the ETR/ERS genes) which is similar to the bacterial two-component histidine kinase receptors (Bleecker, 1999; Stepanova and Ecker, 2000). CTR1 is down-stream of these receptors and encodes a protein whose sequence is similar to the Raf family of Ser/Thr protein kinases (Kieber et al., 1993). The receptors and CTR1 all act as negative regulators, and binding of ethylene results in inactivation of the receptors and CTR1 sequentially (Hua and Meyerowitz, 1998). Receptor and CTR1 proteins form a complex via protein–protein interaction, and the complex is located in the endoplasmic reticulum; this association and location are required for CTR1 function (Chen et al., 2002; Gao et al., 2003; Huang et al., 2003). EIN2, an Nramp-like protein, acts as a down-stream component of CTR1 and is activated by inactivation of CTR1 (Alonso et al., 1999). The activated EIN2 signals to the nucleus and activates EIN3/EIL. As transcription factors, EIN3/EIL trigger transcription of down-stream genes, and induce the ethylene response (Wang et al., 2002).

Flower opening is a crucial developmental event for phanerogams. Ethylene has been demonstrated to influence several aspects of flower development, including flower sex determination (Rudich et al., 1972; Yamasaki et al., 2001), flower opening (Reid et al., 1989; Yamamoto et al., 1994), pollination-induced petal senescence (O'Neill et al., 1993; Tang and Woodson, 1996; Clark et al., 1997; Jones and Woodson, 1997; Bui and O'Neill, 1998; Llop-Tous et al., 2000), and petal abscission (van Doorn and Stead, 1997; van Doorn, 2002). Ethylene affects pollination-induced petal senescence primarily through temporal- and spatial-specific expression of ACS and ACO genes, while ethylene receptor gene expression is also involved in this event (Shibuya et al., 2002). However, to date, little is known about the function of ethylene in flower opening pre-pollination.

In modern cut roses, flower opening is a gradual and slow process. Previous studies showed that flower opening of cut roses was mostly sensitive to ethylene, but the response to ethylene varied among cultivars (Reid et al., 1989; Yamamoto et al., 1994; Cai et al., 2002). Wang et al. (2004) reported isolation of an ACS gene, RKacc7, and its expression increased at the onset of petal senescence in cut rose. In miniature potted roses, seven genes—four ethylene receptor (RhETR1, 2, 3, 4), two CTR1-like (RhCTR1 and 2), and one EIN3 (RhEIN3)—have been isolated. The expression of RhETR3, and RhCTR1 and 2 were up-regulated by exogenous ethylene, and expression of RhETR3 and RhCTR1 increased during flower senescence (Müller et al., 2000a, b, 2002, 2003). Until now, however, an important question remained unanswered: does ethylene regulate flower opening of roses through its biosynthesis, or signalling pathway, or both?

In the present work an attempt was made to understand the effect of ethylene on flower pre-pollination opening, and to identify key regulatory components in ethylene biosynthesis and signalling pathways in cut roses. For this purpose, flowers of cut rose cv. Samantha were treated at stage 2 (completely opened bud; Wang et al., 2004; Ma et al., 2005) with ethylene and 1-MCP (1-methylcyclopropene), an ethylene action inhibitor (Sisler et al., 1999). Morphological changes were observed and ethylene production and expression of ethylene biosynthesis and signal transduction genes were determined after treatment. The results demonstrate that ethylene is involved in the induction of full flower opening; transcriptional regulation of an ethylene receptor and CTR genes were involved in this induction process.


Materials and Methods

 

 
Plant materials
Cut rose (Rosa hybrida) cv. Samantha, a cultivar whose opening is accelerated by ethylene treatment (Cai et al., 2002; Tan et al., 2006), was obtained from a commercial grower in Beijing. It was harvested at flower opening stage 2, and then placed immediately in tap water. Flower opening stages were described previously in Wang et al. (2004) and Ma et al. (2005): stage 0, unopened bud; stage 1, partially opened bud; stage 2, completely opened bud; stages 3 and 4, partially opened flower; stage 5, fully opened flower with anther appearance (yellow); stage 6, fully opened flower with anther appearance (black). Within 1 h of harvest, the flowers were delivered to the laboratory; after the stems were cut to a length of 25 cm under water, the flowers were placed in deionized water.

Ethylene and 1-MCP treatments
The effects of different ethylene (2–20 ppm) and 1-MCP (0.5–2 ppm) concentrations on rose flower opening were tested previously, and stable and repeatable results were obtained with 10 ppm ethylene and 2 ppm 1-MCP treatment. Therefore, for ethylene and 1-MCP treatments, flowers were sealed in chambers (64 l) with 10 ppm ethylene or 2 ppm 1-MCP at 25 °C for different times. For competitive experiments, flowers were treated by ethylene for 12 h or 24 h prior to 24 h 1-MCP treatment, or treated by 1-MCP for 12 h or 24 h prior to 24 h ethylene treatment. Control flowers were sealed with an air atmosphere. After treatments, flowers were placed in a vase with deionized water and under controlled conditions at 23–25 °C, 30–40% relative humidity, and a 12/12 h light/dark photoperiod at an illumination of ~40 µmol m–2 s–1. For each treatment, 10 flowers were randomly chosen for morphological observation.

Ethylene measurements
Petals of each individual flower were collected and placed in an airtight container (0.3 l). The containers were capped and incubated for 20 min at 25 °C. Then a head space gas sample of 1 ml was withdrawn, using a gas-tight hypodermic syringe, and injected into a gas chromatograph (GC 17A, Shimadzu, Kyoto, Japan) for ethylene concentration measurement. The gas chromatograph is equipped with a flame ionization detector and an activated alumina column. All measurements were performed with five replicates.

RNA extraction and northern blot analysis
Total RNA from petals was extracted using the hot borate method based on the method of Wan and Wilkins (1994) and with a modification. Briefly, petals were ground with liquid nitrogen and were homogenized with the preheated extraction buffer (200 mM sodium tetraborate decahydrate, 30 mM EGTA, 1% deoxycholic acid sodium salt, 10 mM DTT, 2% PVP 40, 1% NP-40) at a rate of 2–2.5 ml g–1 FW. After addition of Protease K (Merck), the extract was incubated at 42 °C for 1.5 h; then the extract was centrifuged at 12 000 g, 4 °C for 15 min. RNA was precipitated overnight with 2 M LiCl, washed by 2 M LiCl, dissolved in 1 M TRIS-Cl (pH 7.5). Then the RNA was precipitated with 2.5x volumes of 100% ethanol at –80 °C for 2 h.

Ten micrograms of total RNA was fractionated on 1.2% agarose gel containing 2.5% formaldehyde (v/v). Then the RNA was transferred onto a nylon membrane and fixed with a UV cross-linker (Spectroline, USA). DIG-labelled probes were generated by PCR using a PCR DIG probe synthesis kit (Boeringer Mannheim, Germany) with specific primers. The sequences of these primers have been described previously: Rh-ACS1, 2, 3 and Rh-ACO1 in Ma et al. (2005); RhETR1, RhETR3, RhETR5, RhCTR1, RhCTR2, RhEIN3-1, and RhEIN3-2 in Tan et al. (2006). The accession numbers of the genes studied are as follows: Rh-ACS1, AY061946; Rh-ACS2, AY803737; Rh-ACS3, AY803738; Rh-ACO1, AF441282; Rh-ETR1, AY953869; Rh-ETR3, AY953392; Rh-ETR5, AF441283; Rh-CTR1, AY032953; Rh-CTR2, AY029067; Rh-EIN3-1, AF443783; Rh-EIN3-2, AY919867.

Hybridization was carried out overnight at 46 °C for ACO, ethylene receptors, and EIN3; and at 43 °C for ACS and CTRs. The membranes were washed twice in 2x SSC at 37 °C and twice in 0.1x SSC for 30 min at 59 °C for ACO, ethylene receptors, and EIN3; and at 56 °C for ACS and CTR transcripts. The membranes were subjected to a chemiluminescent reaction with CDP-StarTM according to the manufacturer's protocol (DIG-Detection System, Boeringer Mannheim), and then exposed to Fuji Medical X-ray film.

In this work, one flower was regarded as an independent sample. Three flowers were taken as three replicates at each time point, and total RNA was extracted from the petals of each flower separately. The three RNA samples from each time point were then subjected to northern hybridization independently. Representative results are demonstrated here. All experiments were performed twice in 2003 and 2004.



Results

  

Effects of ethylene and 1-MCP on flower opening
The opening process of cut rose flowers can be divided into three periods: the bud period including stages 0 and 1; the partially opened flower period including stages 2–4; and the fully opened flower period including stages 5 and 6. Pre-pollination opening for cut rose flowers refers to the second period—the partially opened period—in this work.

To understand how the flower opening process in cut rose cv. Samantha was affected by different timings of ethylene treatment, firstly, flowers at stage 2 were treated with 10 ppm ethylene or 2 ppm 1-MCP for 24, 48, and 72 h, respectively. As shown in Fig. 1A and Table 1A, control flowers opened normally. Ethylene treatment caused flowers to open quickly and show anthers much earlier than the control; the partially opened period was shortened from 5 d in control flowers to 3.0–3.4 d. Ethylene treatment also resulted in irregularly shaped flowers and petals, and even abscission of petals, and longer treatment time resulted in more severe symptoms. By contrast, 1-MCP inhibited full opening of flowers by impeding the unfurling of middle and inner layer petals, and causing flowers to maintain partially opened status at stage 4 until wilting. The partially opened period was prolonged significantly from 5 d in control flowers to 7.6–8.1 d in 1-MCP treatment (Fig. 1A; Table 1A). In addition, different 1-MCP treatment times did not result in any observable difference.

Secondly, the flowers were treated with ethylene and 1-MCP for 6, 12, 18, and 24 h. Figure 1B and Table 1B show that, compared with control flowers, 12 h treatment with ethylene resulted in obvious morphological changes, including unfurling of the outer layer petals and curly edges to the petals; and, more importantly, flowers showed anthers more obviously, although the process of flower opening was not shortened significantly. 1-MCP treatment for 12 h or longer inhibited full flower opening and prolonged the partially opened period significantly.

To confirm that ethylene was essential in promoting flower opening, competitive experiments between ethylene and 1-MCP were performed. As shown in Fig. 1C, 24 h of ethylene treatment followed by 1-MCP treatment completely negated the accelerating effect on full opening caused by 12 h or 24 h ethylene pretreatment: it counteracted ethylene-induced shortening of the partially opened period. On the contrary, 24 h ethylene treatment failed to influence the opening process of flowers when they were pretreated by 1-MCP, even when the treatment time was just 12 h (Table 1C).

The results above indicate that ethylene treatment was able to accelerate the flower opening process, and continuous ethylene perception was required for full opening of flowers in cut rose cv. Samantha.

Effects of ethylene and 1-MCP on ethylene production in petals
Previous work revealed that the petal is the primary ethylene-releasing organ of the flower (Shen et al., 2004). To understand the relationship between ethylene treatment and flower opening and ethylene production in petals, ethylene production in petals was determined after a range of treatments.

For control flowers, ethylene production in petals was 0.52–0.73 nl C2H4 g–1 FW h–1 during the partially opened period, increased slowly and reached 1.68 nl C2H4 g–1 FW h–1 when flowers were fully opened, and then decreased slightly (Fig. 2A-1). For ethylene-treated flowers, ethylene production was enhanced and longer treatment resulted in greater enhancement. Immediately after 24 h ethylene treatment, ethylene production was 1.31 nl C2H4 g–1 FW h–1, about twice that in the control, and increased sharply after 48 h or 72 h ethylene treatment (Fig. 2A-2). Interestingly, 1-MCP did not reduce ethylene production of petals, especially towards the end of the period in the vase compared with the control. Also no significant difference in ethylene production was found between different 1-MCP treatments (Fig. 2A-3).

As shown in Fig. 2B, at 12 h, ethylene production in ethylene-treated petals was enhanced and reached 1.41 nl C2H4 g–1 FW h–1, about 1.5-fold greater than in the control, and longer treatment time resulted in higher production within 24 h; by contrast, 1-MCP treatment did not affect ethylene production in any significant way.

The effects of ethylene and 1-MCP on ethylene production were further confirmed experimentally through competitive treatments. As shown in Fig. 2C, for the flowers pretreated with ethylene for 24 h, ethylene production in petals was reduced to a level close to that in the control by subsequent 24 h 1-MCP treatment. Additionally, ethylene could not elevate ethylene production in flowers pre-treated with 1-MCP. These results indicate that the effect of 1-MCP cannot be reversed by ethylene, while the effect of ethylene can be reversed by 1-MCP.

Effects of ethylene and 1-MCP on gene expression
To identify the genes involved in accelerated flower opening of cut rose cv. Samantha when treated by ethylene, first the expression of four ethylene biosynthetic enzymes was determined. As shown in Fig. 3, expression of Rh-ACS1 and Rh-ACS2 was undetectable in control or in ethylene- or 1-MCP-treated petals. The transcript level of Rh-ACS3 was increased slightly by ethylene, and not decreased by 1-MCP treatment. Rh-ACO1 expression was elevated greatly by ethylene and a longer treatment time resulted in a higher Rh-ACO1 transcript level, which was slightly inhibited by 1-MCP (Fig. 3A, B). In competitive experiments, the amount of Rh-ACS3 transcript in petals pretreated by ethylene was not changed by subsequent 1-MCP treatment, and vice versa. Follow-up treatment by 1-MCP after ethylene treatment effectively reduced the Rh-ACO1 transcript level to that of the control, although expression of the gene was increased greatly by ethylene pretreatment. Ethylene was incapable of influencing the Rh-ACO1 expression level in petals of 1-MCP pretreated flowers (Fig. 3C).

These results are consistent with those related to ethylene production above, and indicate that the inhibitory effect of 1-MCP on full flower opening is most likely to be independent of ethylene biosynthesis in petals. Therefore, the expression level of seven ethylene signalling pathway component genes in petals was determined. These genes were three ethylene receptors (Rh-ETR1, Rh-ETR3, and Rh-ETR5), two CTRs (Rh-CTR1 and Rh-CTR2), and two EIN3s (Rh-EIN3-1 and Rh-EIN3-2). The level of Rh-ETR5 in petals was not affected by either ethylene or 1-MCP treatment when compared with the control. Rh-ETR1 and Rh-ETR3 transcripts were markedly strengthened by ethylene and weakened by 1-MCP; and the expression level of Rh-ETR1 was always lower than that of Rh-ETR3 in the same experiment (Fig. 4A, B). Additionally, 12 h ethylene treatment effectively increased Rh-ETR1 and Rh-ETR3 transcript levels, and 1-MCP decreased them substantially (Fig. 4B); these effects were consistent with the morphological results from Fig. 1B. Similar patterns of change were observed in the expression of Rh-CTR1 and Rh-CTR2 after ethylene and 1-MCP treatment. However, the transcripts of Rh-EIN3-1 and Rh-EIN3-2 all accumulated in a constitutive manner and did not respond to either ethylene or 1-MCP treatment (Fig. 4).

 
The results from experiments on competition between ethylene and 1-MCP showed that Rh-ETR5, Rh-EIN3-1, and Rh-EIN3-2 all exhibited constitutive expression patterns (Fig. 4C). But the ethylene-induced increase of Rh-ETR1, Rh-ETR3, Rh-CTR1, and Rh-CTR2 transcripts was completely reversed by subsequent 1-MCP treatment, and the expressions of these genes was even decreased to levels lower than those in the control. However, 1-MCP-caused expression inhibition of these genes was irreversible by subsequent ethylene treatment (Fig. 4C).

The above results suggest that the expression changes of ethylene receptor and CTR genes caused by ethylene or 1-MCP treatment may contribute to the regulation of the full flower opening process, and Rh-ETR1, Rh-ETR3, Rh-CTR1, and Rh-CTR2 may be pivotal genes responsible for the influence of ethylene in regulating flower opening in cut rose cv. Samantha.


Discussion

 

 
Although the regulatory role of ethylene in flower petal senescence has been well documented in many plants, like orchid (Bui and O' Neill, 1998), petunia (Tang et al., 1994, 1996), carnation (Jones and Woodson, 1997, 1999), and tomato (Llop-Tous et al., 2000), little is known about ethylene's regulatory role in flower opening. In this study, the effect of ethylene in regulating flower opening was investigated in cut rose cv. Samantha and an attempt was made to identify the key genetic components whose expression contributes to this regulation. Using NBD (norbornadiene), an inhibitor of ethylene action, Wang and Woodson (1989) found that flower opening of carnation was tightly dependent on ethylene, and continuous ethylene perception was necessary for flower opening and senescence. The present results (Fig. 1A, B) showed that ethylene treatment accelerated full flower opening, and 1-MCP inhibited this process effectively in cut roses. Competitive experiments confirmed these points; after being exposed to ethylene for 24 h and starting to exhibit typical characteristics of ethylene treatment, when transferred to 1-MCP atmosphere, flowers failed to show anthers and stayed in the partially opened state (Fig. 1C; Table 1C). These results suggest that ethylene plays a critical role in regulating the progression of flower opening in cut rose cv. Samantha, and continuous perception of ethylene is required for flowers to open fully.

Using probes from Pelargonium in miniature potted roses, Müller et al. (2000a) determined the level of ACS and ACO transcripts and found that the expression of ACO increased at a late stage of flower development in both long-lasting cv. Vanilla and short-lasting cv. Bronze, whereas ACS transcript increased in ‘Vanilla’ and remained constant at a low level in ‘Bronze’. Recently, an ACS gene, Rkacc7, was isolated by screening a cDNA library of senescent petals, and its expression shows an increase at the onset of petal senescence in cut rose cv. Kardinal (Wang et al., 2004). Previous work showed that there were at least three ACS genes and one ACO gene in cut roses and their expressions occurred during different developmental events (Ma et al., 2005). However, whether ethylene modulates flower opening through ethylene biosynthesis was unknown.

In this study, ethylene-induced elevation of ethylene production in petals was accompanied by an acceleration of full flower opening in cut rose, but 1-MCP treatment did not decrease ethylene production, although it did succeed in impeding full flower opening (see Figs 1, 2). These results indicate that the inhibitory role of 1-MCP in flower opening is unlikely to be played through the regulation of ethylene production.

In this work, transcripts of Rh-ACS1 and Rh-ACS2 were undetectable. These results are consistent with previous work which showed that Rh-ACS1 was transcribed specifically in response to wounding, and Rh-ACS2 was detectable only in senescent petals (Ma et al., 2005). Expression of Rh-ACS3, the same gene as Rkacc7 (Wang et al., 2004), was up-regulated slightly by ethylene, but was not down-regulated by 1-MCP (Fig. 3). The Rh-ACO1 transcript level was elevated substantially by ethylene; however, like Rh-ACS3, it was not inhibited by 1-MCP compared with the control (Fig. 3). Taken together, these data suggest that a feedback regulation of ethylene biosynthesis is unlikely to be involved in the regulation of full flower opening, though full flower opening needs continuous ethylene perception in cut rose cv. Samantha.

In Arabidopsis, expression of AtETR2, AtERS1, and AtERS2 was enhanced by exogenous ethylene (Hua and Meyerowitz, 1998). In tomato, LeETR1 and LeETR2 showed constitutive expression, Nr, LeETR4, and LeETR5 expression levels increased as fruit ripened, and Nr was ethylene-inducible in mature fruit (Wilkinson et al., 1995; Lashbrook et al., 1998; Tieman and Klee, 1999). In carnation petals, DC-ERS2 expression level decreased at the late stage of petal senescence; DC-ETR1 was almost unchanged and DC-ERS1 was undetectable throughout senescence (Shibuya et al., 2002). Müller et al. (2000a, b) reported that different ethylene receptors had different expression patterns in miniature potted roses. In the present study, Rh-ETR5 exhibited a constant expression and was not influenced by ethylene or 1-MCP. However, Rh-ETR1 and Rh-ETR3 mRNA levels were enhanced by ethylene, and weakened by 1-MCP, and Rh-ETR3 had a higher expression level (Fig. 4).

CTR was isolated first from Arabidopsis (Kieber et al., 1993). The expression of AtCTR1, the sole CTR1 in Arabidopsis, was constitutive and not affected by external stimuli, including ethylene (Gao et al., 2003). In tomato, the expression of LeCTR1 increased during flower senescence and fruit ripening, and was highly up-regulated by ethylene (Leclercq et al., 2002; Zegzouti et al., 1999), while the expression pattern of LeCTR2 was the same as AtCTR1 (Alexander and Grierson, 2002). In the present work, Rh-CTR1 and Rh-CTR2 were expressed in an ethylene-dependent manner in cut rose cv. Samantha, and this was consistent with previous observations in miniature potted roses (Müller et al., 2002a, b). A recent study by Huang et al. (2003) showed that ethylene receptor and CTR1 protein formed a complex which was located in endoplasmic reticulum, and the function of CTR1 was regulated via association/dissociation with the ethylene receptor proteins. Based on this view, it is considered that Rh-ETR1 and Rh-ETR3 may well be more important modulators in the regulation of full flower opening than Rh-CTR1 or Rh-CTR2.

EIN3 is a positive regulator in the ethylene signalling pathway. However, there is no evidence to indicate that EIN3 is up-regulated by ethylene or ACC in Arabidopsis (Chao et al., 1997), tomato (Tieman et al., 2001), tobacco (Kosugi and Ohashi, 2000; Rieu et al., 2003), or miniature potted roses (Müller et al., 2003). In the present work, Rh-EIN3-1 and Rh-EIN3-2 showed constitutive expression in cut rose (Fig. 4), a result consistent with previous reports.

Although a recent study reported that the transcriptional regulation of an EIN3-like gene, DC-EIL3, played an important role in growth and development of carnation (Iordachescu and Verlinden, 2005), several experiments have demonstrated that EIN3 is regulated tightly at a post-transcriptional, rather than a transcriptional level (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004).

 

Acknowledgements

This work was supported by a grant (no. 30471220) from the National Nature Science Foundation of China.


Footnotes

* These authors contributed equally to this work.


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Figures

Fig. 1 Effects of ethylene and 1-MCP on flower opening in cut rose cv. Samantha: (A) flowers treated by exposure to ethylene or 1-MCP for 24, 48, and 72 h; (B) flowers treated by exposure to ethylene or 1-MCP for 0, 6, 12, 18, and 24 h; (C) flowers treated competitively with ethylene and 1-MCP. BT, Before treatment; 0AT, 1AT, and 3AT, 0, 1, 3 d after treatment, respectively; E, ethylene; M, 1-MCP.

figure 1

 

 

 


Fig. 2 Changes in ethylene production in petals during and after ethylene or 1-MCP treatment: (A) petals treated by ethylene or 1-MCP for 24–72 h; (B) petals treated by ethylene or 1-MCP for 0–24 h; (C) petals treated competitively by ethylene and 1-MCP. In (A-2) and (A-3), the three arrows indicate end-points of ethylene or 1-MCP treatment for 24, 48, and 72 h, respectively. A, Air; E, ethylene; M, 1-MCP. The number following A, E, or M is hours exposed to air, ethylene, or 1-MCP. Each bar represents the standard error, n=5.

transcriptional%20f02.gif

 

 

 


Fig. 3 Expression of Rh-ACS and Rh-ACO genes in petals treated by ethylene and 1-MCP: (A) petals treated by ethylene or 1-MCP for 24–72 h; (B) petals treated by ethylene or 1-MCP for 0–24 h; (C) petals treated competitively with ethylene and 1-MCP. A, Air; E, ethylene treatment; M, 1-MCP treatment. The number following A, E, or M is hours exposed to air, ethylene, or 1-MCP. Total RNA was prepared from petals immediately after the determination of ethylene production as shown in Fig. 2. Each lane contains 10 µg total RNA. EtBr-stained rRNA was used as an internal control to normalize the amount of total RNA. Total RNA was isolated from three individual samples at each time point and all of the hybridizations were repeated at least three times. Representative results are shown here.

transcriptional%20f03.gif

 

 

 


Fig. 4 Expression of Rh-ETR, Rh-CTR, and Rh-EIN3 genes in petals treated by ethylene and 1-MCP: (A) petals treated by ethylene or 1-MCP for 24–72 h; (B) petals treated by ethylene or 1-MCP for 0–24 h; (C) petals treated alternatively by ethylene and 1-MCP. A, Air; E, ethylene treatment; M, 1-MCP treatment. The number following A, E, or M is hours exposed to air, ethylene, or 1-MCP. Total RNA was prepared from petals immediately after the determination of ethylene production, as shown in Fig. 2. Each lane contains 10 µg total RNA. EtBr-stained rRNA was used as an internal control to normalize the amount of total RNA. Total RNA was isolated from three individual samples at each time point and all of the hybridizations were repeated at least three times. Representative results are shown here.

transcriptional%20f04.gif

 


 


Table

 


Table 1 The process of flower opening after treatment with ethylene or 1-MCP in cut rose cv. Samantha

Treatment (h)   Partially opened flower

Fully opened flower

Vase life

    Stage 3 Stage 4 Stages 5 and 6 Stages 3–6

(A) Ethylene or 1-MCP for 24–72 h
Control (air)   1.8 b 3.2 b 1.7 a 6.7 b
Ethylene 24 1.2 c 2.2 c 1.6 a 5.0 c
  48 1.0 c 2.2 c 1.0 ab 4.2 d
  72 1.0 c 2.0 c 0.5 b 3.5 e
1-MCP 24 2.0 ab 5.6 a 0.0 b 7.6 a
  48 2.1 a 6.0 a 0.0 b 8.1 a
  72 1.8 b 5.8 a 0.0 b 7.6 a
(B) Ethylene or 1-MCP for 0–24 h
Control (air)   1.6 a 3.3 c 1.6 a 6.5 ab
Ethylene 6 1.7 a 3.4 c 1.5 a 6.6 a
  12 1.7 a 3.0 cd 1.5 a 6.2 ab
  18 1.4 a 2.5 de 1.4 a 5.3 bc
  24 1.0 b 1.8 e 1.8 a 4.6 c
1-MCP 6 1.7 a 3.3 c 1.3 a 6.3 ab
  12 1.6 a 4.9 b 0.0 b 6.5 ab
  18 1.8 a 5.4 a 0.0 b 7.2 a
  24 1.8 a 5.4 a 0.0 b 7.2 a
(C) Competitive treatment
Control (air)   1.7 a 3.8 c 1.5 b 7.0 bcd
Ethylene-12 Air-24 1.5 ab 3.0 d 2.3 a 6.8 cd
  1-MCP-24 1.7 a 4.9 b 1.3 bc 7.9 ab
1-MCP-12 Air-24 1.7 a 4.8 b 1.0 c 7.5 abc
  Ethylene-24 1.8 a 5.0 b 1.0 c 7.8 abc
Ethylene-24 Air-24 1.0 c 1.8 e 2.0 a 4.8 e
  1-MCP-24 1.3 bc 4.8 b 0.4 d 6.5 d
1-MCP-24 Air-24 1.8 a 6.3 a 0.0 d 8.1 a
  Ethylene-24 1.7 a 6.0 a 0.2 d 7.9 ab

Each value represents means of 10 replicates. Different letters indicate significant differences between treatments according to Duncan's multiple range tests (P



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