Journal of Plant Physiology 171 (2014) 1679–1684

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Physiology

Ethylene production associated with petal senescence in carnation

flowers is induced irrespective of the gynoecium
Kazuo Ichimura ∗ , Tomoko Niki
NARO Institute of Floricultural Science, Fujimoto, Tsukuba, Ibaraki 305-8519, Japan

a r t i c l e i n f o s u m m a r y

Article history: To clarify whether climacteric-like increases in ethylene production of senescing petals are also induced
Received 20 March 2014 in the absence of the gynoecium in cut carnation (Dianthus caryophyllus cv. Barbara) flowers, we com-
Received in revised form 13 August 2014 pared ethylene production and expression of ethylene-biosynthesis genes in detached petals and in
Accepted 13 August 2014
petals, which remained on flowers (attached petals). No significant difference in longevity was observed
Available online 21 August 2014
between the attached and detached petals when held in distilled water, and both showed the inward
rolling typical of senescing flowers. Treatment with silver thiosulfate complex (STS), an ethylene inhibitor,
Keywords:
similarly delayed senescence of attached and detached petals. Climacteric-like increases in ethylene pro-
1-Aminocyclopropane-1-carboxylic acid
oxidase
duction of petals and gynoecium started on the same day, with similar bursts in attached and detached
1-Aminocyclopropane-1-carboxylic acid petals. Transcript levels of DcACS1 and DcACO1 were very low at harvest and increased similarly during
synthase senescence in both petal groups. Removal of the gynoecium did not significantly delay wilting of attached
Carnation petals. In flowers with the gynoecium removed, the petals produced most of the ethylene while produc-
Ethylene tion by the other floral organs was very low, suggesting that wound-induced ethylene is not the reason
Petal senescence for the ineffectiveness of gynoecium-removal in inhibiting flower senescence. These results indicate that
ethylene biosynthesis is induced in carnation petals irrespective of the gynoecium.
© 2014 Elsevier GmbH. All rights reserved.

Introduction the climacteric increase in ethylene production of the gynoecium

Carnation flowers are highly sensitive to ethylene (Wu et al.,
1991; Onozaki et al., 2004) and ethylene production increases in
flowers during senescence (Nichols, 1966). The application of eth-
ylene inhibitors including silver thiosulfate complex (STS) (Veen,
1979), aminoethoxyvinylglycine (Baker et al., 1977) and aminooxy-
acetic acid (Broun and Mayak, 1981) delays senescence of carnation
flowers, indicating that the senescence is regulated by endogenous
ethylene.
Following pollination, ethylene production in carnation
increases sequentially in the order style, ovary and petals (Jones
and Woodson, 1997, 1999a). This suggests that some signals gen-
erated by pollination induce ethylene production. For unpollinated
carnation flowers, it has been postulated that ethylene produced
by the gynoecium regulates petal senescence (ten Have and
Woltering, 1997). This assumption is based on data showing that
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACO,
1-aminocyclopropane-1-carboxylic acid oxidase; ACS, 1-aminocyclopropane-
1-carboxylic acid synthase; DW, distilled water; STS, silver thiosulfate complex.
∗ Corresponding author. Tel.: +81 29 838 6801; fax: +81 29 838 6841.
E-mail address: ichimu@affrc.go.jp (K. Ichimura).
is preceded by an ethylene burst in petals (Woodson and Brandt,
1991) and that a climacteric increase is absent in detached petals
(ten Have and Woltering, 1997). Furthermore, removal of the
gynoecium, which is accompanied by suppression of ethylene
production, markedly delayed petal senescence (Shibuya et al.,
2000). These findings suggest that ethylene produced by the
gynoecium may trigger ethylene production in petals, thereby
inducing petal wilting in unpollinated flowers. However, whether
petal senescence of carnation flowers occurs independently or is
influenced by other organs remains controversial. Mor et al. (1980)
reported no difference in longevity of attached and detached
carnation petals. In addition, removal of the gynoecium did not
delay senescence of attached petals (Mor et al., 1980; Sacalis and
Lee, 1987).
The biosynthetic pathway of ethylene has been eluci-
dated in higher plants as follows: methionine → S-adenosyl-
l-methionine (SAM) → 1-aminocyclopropane-1-carboxylic acid
(ACC) → ethylene. ACC is produced from SAM by ACC synthase
(ACS). The precursor of ethylene, ACC, is converted to ethylene,
carbon dioxide and HCN by ACC oxidase (ACO). In general, the con-
version by ACS was identified as the rate-limiting step because ACO
activity is constitutive in many plant tissues (Kende, 1993). Sim-
ilarly, ACS and ACO were found to contribute to the climacteric

http://dx.doi.org/10.1016/j.jplph.2014.08.006
0176-1617/© 2014 Elsevier GmbH. All rights reserved.
1680 K. Ichimura, T. Niki / Journal of Plant Physiology 171 (2014) 1679–1684
increase in ethylene production of carnation petals (Woodson et al.,
1992; Woltering et al., 1993; Lee et al., 1997).
ACS has been shown to be encoded by a multi-gene family
(Kende, 1993). In carnation, three genes encoding ACS have been
isolated and are designated DcACS1, DcACS2 and DcACS3 (Park et al.,
1992; Jones and Woodson, 1999b). The expression of DcACS1 in
petals increased during senescence of carnation flowers (Woodson
et al., 1992). ACO is known to be encoded by a multi-gene family, but
only one ACO gene, DcACO1, has been cloned in carnation (Wang
and Woodson, 1991). In cut carnation flowers, the expression of
DcACO1 is very low in petals and ovary at harvest, but increases
during senescence (Jones and Woodson, 1997).
In the present study, we investigated the longevity, ethylene
production and transcript levels of DcACS1 and DcACO1 of attached
and detached petals to clarify whether a climacteric-like increase
in senescence-associated ethylene production is induced in the
absence of the gynoecium in cut carnations.
Materials and methods
Plant materials
Carnation (Dianthus caryophyllus L.) cv. Barbara was grown in a
greenhouse under natural sunlight. When outer petals were per-
pendicular to the stem axis in the morning, flowers were harvested
and transported immediately to a controlled-environment room.
The peduncles were trimmed to 50 mm, and treatment was started
within 1 h.
Attached and detached petals
Five outer petals were detached from an individual flower
(detached petals) on the day of harvest and were compared to petals
remaining intact on flowers (attached petals).
STS treatment
A 0.2 mM silver thiosulfate complex (STS) solution was prepared
by mixing 0.1 M AgNO3 and 0.1 M Na2 S2 O3 at a volume ratio of 1:8
and dilution of the mix with distilled water (DW) at 1–55. Harvested
flowers (n = 8) were treated with STS or DW (control) at 23 ◦ C, 70%
relative humidity at 10 ␮mol m−2 sec−1 light intensity for 4 h prior
to petal detachment and the start of observation.
Removal of gynoecium
In a separate examination of intact flowers (attached petals), the
gynoecium was left intact (gynoecium intact, n = 20) or removed
on the day of harvest (gynoecium removed, n = 20) as described
by Shibuya et al. (2000). Briefly, the calyx was stripped, and the
gynoecium was snapped off from each flower by hand.
Incubation conditions and evaluation of petal longevity
Following manipulations and/or STS treatment, flowers and
detached petals were transferred to DW and held at 23 ◦ C, 70% rela-
tive humidity with a photoperiod of 12 h at 10 ␮mol m−2 sec−1 light
from cool-white fluorescent lamps. Petal longevity was determined
as the duration from the start of incubation to the petals showing
inward rolling, wilting or necrosis. For the evaluation of detached
petal longevity, the appearance of senescence symptoms in three
of five petals from an individual flower was taken as the endpoint.
Ethylene production
Petals, gynoecium, stamen, receptacle and calyx were collected
at 0, 2, 3, 4, 5, 6, 7 and 8 d after harvest. All of the petals removed
from the attached petal group were enclosed in a glass bottle
(148 mL), and detached petals and the other organs were sepa-
rately enclosed in test tubes (15 mL). After incubation at 23 ◦ C for
1 h, an aliquot of gas (1 mL) was withdrawn with a hypodermic
syringe and injected into a gas chromatograph (GC-14B, Shimadzu,
Kyoto, Japan) equipped with an alumina column and flame ion-
ization detector. The GC conditions used were: 100 ◦ C injection
temperature, 80 ◦ C column temperature, and 40 mL min−1 carrier
gas flow rate.
Preparation of total RNA and real-time RT-PCR
Two petals were collected from each of three flowers at 0, 4, 5, 6,
7 and 8 d after harvest. Petals from each time point were pooled and
total RNA was isolated using ISOGEN (Nippongene, Toyama, Japan).
Extractions were digested with an RNase-free DNase kit (Qiagen,
Hilden, Germany) and purified using an RNeasy mini kit (Qiagen).
The quality and quantity of RNA were checked using a DU640 spec-
trophotometer (Beckman Coulter, Fullerton, CA, USA) and agarose
gel electrophoresis.
Total RNA samples (1 ␮g) were reverse-transcribed using the
PrimeScript RT Master Mix (Takara Bio, Otsu, Japan) in 20 ␮L reac-
tions. Synthesized cDNA (1 ␮L aliquot) was amplified using SYBR
Premix Ex Taq II (Takara Bio) and the following primer pairs: DcACS1
(forward, 5 -CAG TGT GAC GCC ATT GAA AC-3 and reverse, 5 -TTA
AGT TCA ATG TTT TGT TGA GAA GA-3 ); DcACO1 (forward, 5 -GCC
AAC ATT GGT GGA AAA AG-3 and reverse, 5 -CAA TCC ATA GGA
CAT GGA ACA-3 ); and DcUbq3-7, a control gene (forward, 5 -GTT
GTT GGT TTC AGG GCT GGT TTG-3 and reverse, 5 -CTA CGG TAA
TTG AGA ATT CAC ACC GAA ATG-3 ). Amplification was performed
using the Thermal Cycler Dice Real Time System (TP800, Takara
Bio) with an initial polymerase activation at 95 ◦ C for 30 s followed
by the following gene-specific programs: DcUbq3-7 and DcACS1, 40
cycles at 95 ◦ C for 5 s and 60 ◦ C for 30 s; DcACO1, 40 cycles at 95 ◦ C
for 5 s, 60 ◦ C for 30 s and 72 ◦ C for 30 s. The specificity of the PCR was
checked using a heat dissociation protocol (from 60 to 95 ◦ C) after
the final cycle. In addition, the PCR products were run on agarose gel
electrophoresis to confirm that the amplification products were of
the target length. The specificity of each primer pair was confirmed
by direct DNA sequencing of the PCR products.
The absolute transcript level was determined by the second
derivative maximum method (Luu-The et al., 2005) using a dilu-
tion series of plasmid DNA containing target sequences as external
standards. The transcript levels of DcUbq3-7 were almost constant
during petal senescence as reported by Nomura et al. (2012). To
standardize the data, the ratio between the absolute transcript level
of the target gene and the control gene was calculated for each sam-
ple. The mean absolute transcript level and ratios were obtained for
three independent experiments.
Statistical analysis
The significance of pairwise differences in means was analyzed
by the t-test using Stat View software (v.5.0, SAS Institute Inc., Cary,
NC, USA).
Results
Longevity of attached and detached petals
Longevity was 26.5 h longer for the detached petal group than
the attached petal group, but the difference in longevity was not
 K. Ichimura, T. Niki / Journal of Plant Physiology 171 (2014) 1679–1684 1681

Table 1 160

Ethylene production (nL g-1FW h-1)

Longevity of attached and detached petals pre-treated with silver thiosulfate com-
plex (STS). 140 A
Treatment Longevity (d) Attached
120
Control STS
Detached
100
Attached petals 4.9 ± 0.4 13.9 ± 0.7
Detached petals 6.0 ± 0.5 12.9 ± 0.6 80
Significance (P value) 0.0867 0.2769

Cut flowers were treated with 0.2 mM STS for 4 h prior to petal detachment.
60
Values are means of 8 replicates ± SE. Significance was calculated by t-test.
40

significant (Table 1). Detached petals showed the same typical 20

inward rolling as attached petals. Treatment with STS markedly 0
and uniformly delayed senescence of attached and detached petal 0 2 4 6 8
groups. For STS-treated flowers, both attached and detached petal
0.5
groups showed no inward rolling, but necrosis was observed
(Table 1, Fig. 1). B
0.4 Attached

Relative expression
Ethylene production of attached and detached petals
Detached
Ethylene production remained low for both attached and 0.3
detached petals to day 4, and increased thereafter. Ethylene
0.2

0.1

0.0
0 2 4 6 8
50
C
40
Attached
Relative expression

Detached
30

20

10

0
0 2 4 6 8

Time after harvest (days)

Fig. 2. Time course of ethylene production (A), DcACS1 (B) and DcACO1 (C) expres-
sion for attached and detached carnation petals. DcACS1 and DcACO1 transcript levels
are presented relative to the transcript level of DcUbq3-7. Values are the means of
three independent experiments ± SE.

production of attached petals peaked on day 5, which was 1 d ear-

lier than the peak in ethylene production observed for detached
petals (Fig. 2A).

DcACS1 and DcACO1 transcript levels of attached and detached

petals

DcACS1 transcript levels were very low in attached and detached

Fig. 1. Carnation petals following treatment with silver thiosulfate complex (STS)
for 4 h (DW = distilled water control). Photographs were taken 10 d after harvest.
(A) Attached petals treated with DW (left) or STS (right). (B) Detached petals treated
with DW (left) or STS (right). Bar = 5 cm.
petals until day 4, then increased and peaked on day 6. The DcACS1
transcript level decreased more rapidly in attached petals than in
detached petals (Fig. 2B).
Trends of DcACO1 transcript levels were similar to those of
DcACS1, but peak expression was lower for detached petals (Fig. 2C).
1682 K. Ichimura, T. Niki / Journal of Plant Physiology 171 (2014) 1679–1684

Table 2 250

Ethylene production (nL flower h )

-1
Effect of gynoecium removal on the longevity of attached carnation petals.
A

-1
Treatment Longevity (d)
200
Gynoecium intact 6.4 ± 0.5
Gynoecium removed 7.0 ± 0.6 Petal
Significance (P value) 0.4241
150 Stamen
Values are means of 20 replicates ± SE. Significance was calculated by t-test. Gynoecium
Receptacle
100 Sepal
Effect of gynoecium removal on the longevity of petals and
ethylene production of various organs 50

Flowers with the gynoecium removed had slightly delayed petal

senescence, but this effect was not significant (Table 2). 0
Ethylene production of various organs on a fresh weight basis 0 2 4 6 8
shows that in flowers with the gynoecium intact, ethylene produc- 250

Ethylene production (nL flower h )

-1
tion of all organs was very low until day 3, and ethylene production B

-1
of all organs except for sepals increased thereafter and peaked
200
on day 6 (Fig. 3). Ethylene production was highest for the gynoe-
Petal
cium among the organs examined. In flowers with the gynoecium
removed, ethylene production of petals, stamens and receptacle
Stamen
150
increased during senescence. Increased ethylene production of the Receptacle
stamens was somewhat accelerated by gynoecium removal. Sepal
On a per flower basis, ethylene production of petals and gynoe- 100
cium increased during senescence in flowers with the gynoecium
intact whereas ethylene production of the other organs was very 50
low (Fig. 4). In flowers with the gynoecium removed, ethylene
production of petals increased during senescence but ethylene pro-
duction of the other organs remained very low during senescence. 0
0 2 4 6 8

Time after harvest (days)

300
Ethylene production (nL g-1FW h-1)

Fig. 4. Time course of ethylene production per flower of petal, stamen, gynoecium,
A receptacle and sepal in cut carnation flowers with intact (A) and removed gynoecium
250
(B). Values are the means of four independent experiments ± SE.
Petal
200 Stamen
Gynoecium
Discussion
150 Receptacle
Sepal The timing of the ethylene increase was almost identical in
100 attached and detached petals, but with slight differences in DcACS1
and DcACO1 transcript levels (Fig. 2). Thus, although the genes
50 responsible for ethylene biosynthesis can be induced in detached
petals in the absence of a gynoecium, DcACS1 and DcACO1 transcript
0 levels decreased more slowly in detached petals than attached
0 2 4 6 8 petals. We observed that petal wilting progressed somewhat more
300 slowly in detached petals than in attached petals (data not shown),
Ethylene production (nL g-1FW h-1)

which may be related to the slight differences in the gene expres-

B
250 sion.
In the present study, the basal parts of detached petals were
Petal
placed directly in DW, which could be expected to have a deleteri-
200 Stamen
ous effect on longevity. However, STS treatment produced similar
Receptacle
delays in senescence of attached and detached petals (Table 1),
150 Sepal
and detached petals in the control DW treatment showed typical
inward rolling while those in the STS treatment did not. Wulster
100 et al. (1982b) reported that inward rolling is accelerated by expo-
sure to ethylene in detached petals. These morphological changes
50 were similar to those observed for attached petals. Thus, detach-
ment of the outer petals did not have a considerable deleterious
0 effect on longevity in this experimental system. Mor and Reid
0 2 4 6 8 (1980) also reported that an experimental system using detached
petals similar to our system was useful for investigating the senes-
Time after harvest (days) cence of petals.
In carnation cv. White Sim, ten Have and Woltering (1997)
Fig. 3. Time course of ethylene production per fresh weight of petal, stamen, gynoe-
cium, receptacle and sepal in cut carnation flowers with intact (A) and removed showed that ethylene production and DcACS1 and DcACO1 tran-
gynoecium (B). Values are the means of four independent experiments ± SE. script levels did not increase in detached petals. Ethylene
 K. Ichimura, T. Niki / Journal of Plant Physiology 171 (2014) 1679–1684
production was previously shown to be higher in the basal part
than in the upper part of carnation petals (Mor et al., 1985), and
Wulster et al. (1982a) reported that ethylene production of the
upper part of the petals does not increase during senescence. In
the present study, whole petals were detached from the flowers
for evaluation. However, in the study of ten Have and Woltering
(1997), the method for detaching petals was not described in detail,
and we suspect that the basal part of petals was not included and
this exclusion may explain the differences in the results between
the two studies.
There are several reports showing no delay in senescence of cv.
White Sim petals after removal of the gynoecium (Mor et al., 1980;
Sacalis and Lee, 1987; Woodson and Brandt, 1991), which contra-
dicts findings for cv. Reiko flowers by Shibuya et al. (2000). In the
study of Sacalis and Lee (1987), the gynoecium was removed by
forceps, while the gynoecium was removed by hand in the study
of Shibuya et al. (2000). Petal senescence was not suppressed in
cv. Reiko flowers for which the gynoecium was removed by for-
ceps (Shibuya, personal communication). Thus, we removed the
gynoecium by hand, following the method of Shibuya et al. (2000).
However, petal senescence was only slightly delayed in cv. Bar-
bara (Table 2), and climacteric-like increase in ethylene production
of gynoecium started at the same time point as for the petals
(Fig. 3A). In contrast, the onset of the increase in ethylene produc-
tion occurred earlier in the ovary than in the petals in cv. White Sim
carnations (Woodson and Brandt, 1991; ten Have and Woltering,
1997), suggesting that the effect of the gynoecium on ethylene pro-
duction of petals associated with petal senescence varies among the
cultivars. In cv. Barbara, the role of the gynoecium on ethylene pro-
duction associated with petal wilting may be weaker than that in
cv. White Sim. As reported by Shibuya et al. (2000), the ovary of cv.
Reiko is relatively large, compared with those of general cultivars
(Shibuya, personal communication). Thus, cv. Reiko might be a spe-
cific cultivar in which petal senescence is markedly suppressed by
the removal of gynoecium.
Wounding of the floral organ increases ethylene production and
accelerates petal senescence in many flowers, including petunia
(Whitehead et al., 1984), Portulaca (Ichimura and Suto, 1998) and
Eustoma (Ichimura and Goto, 2000). The ineffectiveness of gynoe-
cium removal in delaying senescence (Table 2) may be due to
wounding effects. In this study, time course evaluation of ethyl-
ene production of all floral organs showed that ethylene in flowers
with the gynoecium removed was mainly produced by the petals
and increased in the receptacle and stamens during senescence,
but was much lower on a per flower basis in the receptacle and
stamens than in the petals. Further, ethylene production of the
stamens and receptacle started to increase at the same time as in
the petals (Figs. 3 and 4). Thus, the ineffectiveness of gynoecium
removal on inhibiting petal senescence appears not to be due to
wounding ethylene.
Pollination accelerates ethylene production and petal senes-
cence in carnation (Jones and Woodson, 1997, 1999a). Thus, it can
be assumed that senescence of detached petals may be accelerated
by pollination-induced ethylene before the petals were detached.
However, carnation flowers are known to be protandrous: the style
with stigma is immature at anthesis (Jones and Woodson, 1997;
Jones, 2002), and we indeed confirmed that the style with stigma
does not mature for a minimum of 3 d after anthesis in cv. Bar-
bara. In the present study, petals were detached from the flowers
on the day of anthesis, suggesting that pollination does not occur
in flowers. Moreover, ethylene production of the gynoecium has
been reported to increase within 1 d after pollination (Jones and
Woodson, 1997, 1999a). We did not carry out emasculation due
to the difficulty of doing so, but most of the anthers in this culti-
var were aborted. As ethylene production of the gynoecium did not
increase within the 3 d after harvest (Figs. 3 and 4), we conclude
1683
that effects of pollination on the results obtained in the present
study are negligible.
The gynoecium has been regarded as an important organ in
the regulation of ethylene production of petals in pollinated car-
nation flowers because an increase in ethylene production of the
gynoecium is observed prior to the onset of the climacteric-like
increase of ethylene production in the petals (Jones and Woodson,
1997, 1999a). Longevity of carnation detached petals tended to
be longer than for attached petals (Table 1), suggesting that the
gynoecium may induce ethylene production of petals. However,
climacteric-like increases in ethylene production associated with
petal senescence are induced even in the absence of the gynoecium.
In carnation flowers, the petals are directly connected to the
receptacle but not to the gynoecium. There have been few studies
on the role of the receptacle on petal senescence in flowers show-
ing petal wilting during senescence. Although ethylene production
of the receptacle was not investigated, Hsieh and Sacalis (1986)
reported that ACC concentration is higher in the receptacle of cut
carnations than in other floral organs and that it increases dur-
ing senescence. In the present study, ethylene production of the
receptacle increased during senescence (Fig. 3A), suggesting that
ethylene production of the receptacle may affect petal ethylene
production. Understanding ethylene biosynthesis in the receptacle
will be important for understanding the role of inter-organ com-
munication in petal senescence.
In conclusion, little difference in longevity was observed
between attached and detached petals in carnation cv. Barbara.
Treatment with STS delayed senescence of both petal samples,
suggesting that the experimental system in the present study is
valid. Ethylene production and DcACS1 and DcACO1 transcript lev-
els in attached and detached petals similarly increased during
flower senescence. Removal of the gynoecium did not significantly
delay petal senescence, and ethylene production was only slightly
increased by the removal of the gynoecium. Thus, senescence of
petals can be induced in the absence of the gynoecium.
Acknowledgment
We thank Dr. Shibuya of NIFS for valuable discussion on this
study.
References
Baker JE, Wang CY, Lieberman M, Hardenburg R. Delay of senescence in carnations
by a rhizobitoxine analog and sodium benzoate. HortScience 1977;12:38–9.
Broun R, Mayak S. Aminooxyacetic acid as an inhibitor of ethylene-synthesis and
senescence in carnation flowers. Sci Hort 1981;15:277–82.
Hsieh Y-C, Sacalis J. Levels of ACC in various floral portions during aging of cut
carnations. J Am Soc Hort Sci 1986;111:942–4.
Ichimura K, Goto R. Acceleration of senescence by pollination of cut ‘Asuka-no-nami’
Eustoma flowers. J Jpn Soc Hort Sci 2000;69:166–70.
Ichimura K, Suto K. Role of ethylene in acceleration of flower senescence by filament
wounding in Portulaca hybrid. Physiol Plant 1998;104:603–7.
Jones ML. Ethylene responsiveness in carnation styles is associated with stigma
receptivity. Sex Plant Reprod 2002;15:107–12.
Jones ML, Woodson WR. Pollination-induced ethylene in carnation. Role of stylar
ethylene in corolla senescence. Plant Physiol 1997;115:205–12.
Jones ML, Woodson WR. Interorgan signaling following pollination in carnations. J
Am Soc Hort Sci 1999a;124:598–604.
Jones ML, Woodson WR. Differential expression of three members of the 1-
aminocyclopropane-1-carboxylate synthase gene family in carnation. Plant
Physiol 1999b;119:755–64.
Kende H. Ethylene biosynthesis. Annu Rev Plant Physiol Mol Biol 1993;44:283–307.
Lee MM, Lee SH, Park KY. Effects of spermine on ethylene biosynthesis in cut car-
nation (Dianthus caryophyllus L.) flowers during senescence. J Plant Physiol
1997;151:68–73.
Luu-The V, Paquet N, Calvo E, Cumps J. Improved real-time RT-PCR method for
high-throughput measurements using second derivative calculation and double
correction. Biotechniques 2005;38:287–93.
Mor Y, Reid MS. Isolated petals – a useful system from studying flower senescence.
Acta Hort 1980;113:19–24.
Mor Y, Reid MS, Kofranek AM. Role of the ovary in carnation senescence. Sci Hort
1980;13:377–83.
1684 K. Ichimura, T. Niki / Journal of Plant Physiology 171 (2014) 1679–1684
Mor Y, Halevy AH, Spiegelstein H, Mayak S. The site of 1-aminocyclopropane-
1-carboxylic acid synthesis in senescing carnation petals. Physiol Plant
1985;65:196–202.
Nichols R. Ethylene production during senescence of flowers. J Hort Sci
1966;41:279–90.
Nomura Y, Morita S, Harada T, Satoh S. Cloning, characterization and expression of
carnation (Dianthus caryophyllus L.) ubiquitin genes and their use as a normal-
ization standard for gene expression analysis in senescing petals. J Jpn Soc Hort
Sci 2012;81:357–65.
Onozaki T, Ikeda H, Shibata M. Video evaluation of ethylene sensitivity after anthesis
in carnation (Dianthus caryophyllus L.) flowers. Sci Hort 2004;99:187–97.
Park KY, Drory A, Woodson WR. Molecular cloning of an 1-aminocyclopropane-
1-carboxylate synthase from senescing carnation flower petals. Plant Mol Biol
1992;18:377–86.
Sacalis JN, Lee JS. Promotion of floral longevity by the ovary in carnation flowers. J
Am Soc Hort Sci 1987;112:118–21.
Shibuya K, Yoshioka T, Hashiba T, Satoh S. Role of the gynoecium in natural senes-
cence of carnation (Dianthus caryophyllus L.) flowers. J Exp Bot 2000;51:2067–73.
ten Have A, Woltering EJ. Ethylene biosynthetic genes are differentially expressed
during carnation (Dianthus caryophyllus L.) flower senescence. Plant Mol Biol
1997;34:89–97.
Veen H. Effects of silver on ethylene synthesis and action in cut carnations. Planta
1979;145:467–70.
Wang H, Woodson WR. A flower senescence-related mRNA from carnation shares
sequence similarity with fruit ripening-related mRNAs involved in ethylene
biosynthesis. Plant Physiol 1991;96:1000–1.
Whitehead CS, Halevy AH, Reid MS. Roles of ethylene and 1-aminocyclopropane-1-
carboxylic acid in pollination and wound-induced senescence of Petunia hybrida.
Physiol Plant 1984;61:643–8.
Woltering EJ, Somhorst D, de Beer CA. Roles of ethylene production and sensitivity
in senescence of carnation flower (Dianthus caryophyllus) cultivars White Sim,
Chinera and Epomea. J Plant Physiol 1993;141:329–35.
Woodson WR, Brandt AS. Role of the gynoecium in cytokinin-induced carnation
petal senescence. J Am Soc Hort Sci 1991;116:676–9.
Woodson WR, Park KY, Drory A, Larsen PB, Wang H. Expression of ethylene
biosynthetic pathway transcripts in senescing carnation flowers. Plant Physiol
1992;99:526–32.
Wu MJ, Zacarias L, Reid MS. Variation in the senescence of carnation (Dianthus
caryophyllus L.) cultivars. II. Comparison of sensitivity to exogenous ethylene
and of ethylene binding. Sci Hort 1991;48:109–16.
Wulster G, Sacalis J, Janes HW. Senescence in isolated carnation petals. Effects
of indoleacetic acid and inhibitors of protein synthesis. Plant Physiol
1982a;70:1039–43.
Wulster G, Sacalis J, Janes HW. The effect of inhibitors of protein synthesis on
ethylene-induced senescence in isolated carnation petals. J Am Soc Hort Sci
1982b;107:112–5.