Journal of Experimental Botany, Vol. 57, No. 11, pp. 2763–2773, 2006
doi:10.1093/jxb/erl033 Advance Access publication 14 July, 2006
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
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†
Department of Ornamental Horticulture, China Agricultural University, Beijing 100094, China
Abstract
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 RhETR3 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.
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
* These authors contributed equally to this work.
y
To whom correspondence should be addressed. E-mail: [email protected]
ª 2006 The Author(s).
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/) which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Received 28 September 2005; Accepted 26 April 2006
2764 Ma et al.
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 8C 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 8C, 30–40% relative humidity, and a 12/12 h light/dark photoperiod at an illumination of ;40 lmol mÿ2 sÿ1. 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 8C. 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
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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?
Ethylene receptor and CTR gene expression in rose flower opening 2765
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 1MCP 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.
Results
Effects of ethylene and 1-MCP on ethylene
production in petals
Effects of ethylene and 1-MCP on flower opening
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 gÿ1 FW hÿ1 during the partially opened
period, increased slowly and reached 1.68 nl C2H4 gÿ1 FW
hÿ1 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 gÿ1 FW
hÿ1, 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
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. Prepollination 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
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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 gÿ1 FW. After addition of Protease K (Merck), the extract was
incubated at 42 8C for 1.5 h; then the extract was centrifuged at
12 000 g, 4 8C 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 2.53 volumes of 100% ethanol at
ÿ80 8C 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 8C for ACO, ethylene
receptors, and EIN3; and at 43 8C for ACS and CTRs. The membranes
were washed twice in 23 SSC at 37 8C and twice in 0.13 SSC
for 30 min at 59 8C for ACO, ethylene receptors, and EIN3; and at
56 8C for ACS and CTR transcripts. The membranes were subjected
to a chemiluminescent reaction with CDP-Star 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.
2766 Ma et al.
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 gÿ1 FW hÿ1, 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 pretreated 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 RhACS1 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
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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.
Ethylene receptor and CTR gene expression in rose flower opening 2767
Table 1. The process of flower opening after treatment with ethylene or 1-MCP in cut rose cv. Samantha
Each value represents means of 10 replicates. Different letters indicate significant differences between treatments according to Duncan’s multiple range
tests (P <0.05).
Treatment (h)
Fully opened flower
Vase life
Stage 3
Stage 4
Stages 5 and 6
Stages 3–6
1.8
1.2
1.0
1.0
2.0
2.1
1.8
b
c
c
c
ab
a
b
3.2
2.2
2.2
2.0
5.6
6.0
5.8
b
c
c
c
a
a
a
1.7
1.6
1.0
0.5
0.0
0.0
0.0
a
a
ab
b
b
b
b
6.7
5.0
4.2
3.5
7.6
8.1
7.6
b
c
d
e
a
a
a
1.6
1.7
1.7
1.4
1.0
1.7
1.6
1.8
1.8
a
a
a
a
b
a
a
a
a
3.3
3.4
3.0
2.5
1.8
3.3
4.9
5.4
5.4
c
c
cd
de
e
c
b
a
a
1.6
1.5
1.5
1.4
1.8
1.3
0.0
0.0
0.0
a
a
a
a
a
a
b
b
b
6.5
6.6
6.2
5.3
4.6
6.3
6.5
7.2
7.2
ab
a
ab
bc
c
ab
ab
a
a
1.7
1.5
1.7
1.7
1.8
1.0
1.3
1.8
1.7
a
ab
a
a
a
c
bc
a
a
3.8
3.0
4.9
4.8
5.0
1.8
4.8
6.3
6.0
c
d
b
b
b
e
b
a
a
1.5
2.3
1.3
1.0
1.0
2.0
0.4
0.0
0.2
b
a
bc
c
c
a
d
d
d
7.0
6.8
7.9
7.5
7.8
4.8
6.5
8.1
7.9
bcd
cd
ab
abc
abc
e
d
a
ab
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-MCPcaused 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.
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
(A) Ethylene or 1-MCP for 24–72 h
Control (air)
Ethylene
24
48
72
1-MCP
24
48
72
(B) Ethylene or 1-MCP for 0–24 h
Control (air)
Ethylene
6
12
18
24
1-MCP
6
12
18
24
(C) Competitive treatment
Control (air)
Ethylene-12
Air-24
1-MCP-24
1-MCP-12
Air-24
Ethylene-24
Ethylene-24
Air-24
1-MCP-24
1-MCP-24
Air-24
Ethylene-24
Partially opened flower
2768 Ma et al.
4
Control
Ethylene production (nL
C2H4/gFW*h)
Ethylene production (nL
C2H4/gFW*h)
12
10
8
Control
6
4
2
Control
Ethylene
1-MCP
3
2
1
0
0
0
1
2
3
4
5
6
7
0
6
Vase (day)
12
18
24
Treatment time (hour)
10
Ethylene production (nL
C2H4/gFW*h)
Ethylene 24
Ethylene 48
Ethylene 72
8
6
4
2
0
2
1
M24E24
Vase (day)
M24A24
7
E24M24
6
E24A24
5
Cont 48
4
M12E24
3
M12A24
2
E12M24
1
E12A24
0
0
Cont 36
Ethylene production (nL
C2H4/gFW*h)
Ethylene
Treatment
1-MCP
Ethylene production (nL
C2H4/gFW*h)
12
10
1-MCP 24
1-MCP 48
1-MCP 72
8
6
4
2
0
0
1
2
3
4
5
6
7
Vase (day)
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.
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
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3
12
Ethylene receptor and CTR gene expression in rose flower opening 2769
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 longlasting 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
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
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 lg 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.
2770 Ma et al.
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
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
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 lg 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.
Ethylene receptor and CTR gene expression in rose flower opening 2771
have demonstrated that EIN3 is regulated tightly at a posttranscriptional, rather than a transcriptional level (Guo and
Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004).
Acknowledgement
This work was supported by a grant (no. 30471220) from the
National Nature Science Foundation of China.
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