Expression of Ethylene Biosynthetic Genes in the Gynoecium and Receptacle Associated With Sepal Abscission During Senescence in Delphinium Grandiorum

Delphinium owers are highly sensitive to ethylene and its sepals abscise during senescence, which is associated with increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) activities and ethylene production in gynoecium and receptacle. Three ACS genes (DgACS1, DgACS2, and DgACS3) and three ACO genes (DgACO1, DgACO2, and DgACO3) were cloned from Delphinium grandiorum cv. Super Grand Blue. To investigate the contribution of these genes to ethylene production, their expression was analyzed in these genes in the gynoecium and receptacle during natural senescence and following ethylene exposure and pollination. Ethylene production in the gynoecium and receptacle increased during natural ower senescence. The transcript levels of the ACS and ACO genes in these organs, excluding DgACS2 in the receptacle, increased during senescence. Exposure to ethylene accelerated sepal abscission and more strongly increased ethylene production in the receptacle than in the gynoecium. DgACS1 transcript levels in the gynoecium and DgACS2 and DgACO3 transcript levels in the receptacle were increased by ethylene exposure. Pollination accelerated sepal abscission and increased ethylene production in the gynoecium and receptacle. Pollination slightly affected ACS and ACO transcript levels in the gynoecium, whereas DgACO3 transcript level in the receptacle were markedly increased. These results reveal that ACS and ACO gene expression is differently regulated in the gynoecium and receptacle, and some of these genes are more strongly upregulated by ethylene exposure and pollination in the receptacle than in the gynoecium, suggesting the signicance of the receptacle to sepal abscission. three Total RNA was isolated using RNeasy plant Genomic DNA in the RNA sample was digested with the RNase-free DNase set. Quality and quantity of RNA were checked using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientic). Synthesis of cDNA was performed with random hexamer primers using the Invitrogen SuperScriptIII rst-strand system for RT-PCR. The following primers were used for real-time RT-PCR: for DgACS1, 5(cid:0)-TGGACAACGAGACGATGGAG-3(cid:0) (forward) and 5(cid:0)-GAAGCTGAGACGGAGGTTGTT-3(cid:0) (reverse); for DgACS2, 5(cid:0)-GACTCCTCATACTCCTATCCCACAG-3(cid:0) (forward) and 5(cid:0)-ACCTCCTGGTGTTTATTTCACAGTC-3(cid:0) (reverse); for DgACS3, 5(cid:0)-TTTTGCCAACATGAGCCAAC-3(cid:0) (forward) and 5(cid:0)-TTGTGGCATCTTCTTTTCTCCA-3(cid:0) (reverse); for DgACO1, 5(cid:0)-CCCAAATTTGTTTTCGAGGA-3(cid:0) (forward) and 5(cid:0)-TAATAAGCCGTGGCGATAGG-3(cid:0) (reverse); for DgACO2, 5(cid:0)-GGTGAAGTTTCAAGCCAAGG-3(cid:0) (forward) and 5(cid:0)-TTCTTCACTTGTTTGGGCACT-3(cid:0)


Introduction
The phytohormone ethylene plays an important role in various stages of plant growth and development, including senescence. Exposure to ethylene accelerates the senescence of owers in many species, and the primary symptom of senescence induced by ethylene is petal wilting or petal (sepal) abscission (Woltering and van Doorn 1988). In many ethylene-sensitive owers, such as carnation (Nichols 1968) and Eustoma grandi orum (Ichimura et al. 1998), the petals wilt in response to ethylene exposure. In Digitalis (Stead and Moore 1983), geranium (Hilioti et al. 2000), and torenia owers (Goto et al. 1999), petals abscise upon exposure to ethylene. In these owers, ethylene production in the gynoecium or pistil increases with the progression of ower senescence. However, this nding is not observed in the petals (Stead and Moore 1983;Goto et al. 1999;Hilioti et al. 2000). Therefore, ethylene produced by the gynoecium has been considered responsible for petal abscission.
The biosynthetic pathway for ethylene in higher plants has been established. 1-Aminocyclopropane-1carboxylic acid (ACC), the precursor of ethylene, is produced by the conversion of S-adenosyl-l-methionine (SAM) by ACC synthase (ACS). ACC is converted to ethylene, carbon dioxide, and HCN by ACC oxidase (ACO). ACS is generally considered to be a rate-limiting enzyme for ethylene biosynthesis in plants because ACO activity is constitutive in many species (Yang and Hoffman 1984;Kende 1993). However, ACO is important for ethylene biosynthesis in some examples, including fruit maturation (Nakatsuka et al. 1998) and ower senescence (Tang et al. 1994). In cut carnation owers, the biosynthesis of ethylene is regulated by the activities of ACS and ACO (Woodson et al. 1992; Woltering et al. 1993; Lee et al. 1997).
ACS and ACO proteins are encoded by multi-gene families (Argueso et al. 2007;Lin et al. 2009), and these genes are differently regulated in response to developmental and environmental factors (Lin et al. 2009).
Delphinium has long spikes with owers of various colors, such as white, blue, and purple. The genus Delphinium consists of more than 300 species, and D. elatum, D. grandi orum and D. × belladonna, which is a hybrid of D. elatum and D. grandi orum, are widely produced as ornamental plants. Instead of D. × belladonna, which was mainly produced approximately a decade ago, D. grandi orum is widely produced for cut owers. Delphinium owers are sensitive to ethylene, and their sepals rapidly abscise upon exposure (Woltering and van Doorn 1988;, which is accompanied by signs of programmed cell death (Yamada et al. 2007). In Delphinium, sepals and petals are directly connected to the receptacle, but not to the gynoecium. Thus, ethylene produced by the receptacle should directly induce the abscission of the sepals (Ichimura et al. 2009). Ethylene production in the gynoecium and receptacle increases during ower senescence, but that in the other oral organs including petals and sepals, does not increase (Ichimura et al. 2009). ACS and ACO activity in the gynoecium and receptacle increases during ower senescence (Ichimura et al. 2009). Although genes for ethylene receptors and signal transduction components have been identi ed in D. elatum (Kuroda et al. 2003(Kuroda et al. , 2004 and D. × belladonna (Tanase and Ichimura 2006), ACS and ACO genes have not yet been cloned in Delphinium. The quanti cation of ACS and ACO gene expression will help to clarify the effects of ethylene on sepal abscission.
In the present study, we cloned ACS and ACO genes and determined their nucleotide sequences in D. grandi orum. Furthermore, we investigated their expression of ACS and ACO genes in the gynoecium and receptacle during natural senescence, following exposure to ethylene, and after pollination in cut D. grandi orum owers.

Plant materials
Delphinium grandi orum cv. Super Grand Blue (Miyoshi, Tokyo, Japan) was grown in a greenhouse under natural day length conditions with temperatures ranging from 15°C to 25°C. On the day of anthesis, owers with a peduncles of 3 cm in length were cut from the middle region of the ower spikes. Cut peduncles were placed in a vessel with distilled water within 1 h after harvest and used for all subsequent experiments. Cut owers were held at 23°C, 70 % relative humidity, and 10 µmol m −2 s −1 irradiance using cool-white uorescence lamps under a 12-h photoperiod.
The morphology of D. grandi orum owers is shown in Figure 1. The spike features approximately 10 owers (Fig. 1A), whereas petals lack in the ower, unlike other Delphinium species (Fig. 1B). Its gynoecium is covered with stamens (Fig. 1C). The receptacle lies below the gynoecium, and sepals are attached to the receptacle (Fig. 1D).

Ethylene treatment
The cut owers were placed in individual vessels containing distilled water and then transferred to a 70-L transparent acrylic box tted with a septum, through which ethylene was introduced to achieve a concentration of 10 µl L −1 . The box was kept at 23°C under a 12 h-photoperiod with 10 µmol m −2 s −1 irradiance from cool-white uorescent lamps for 24 h. After exposure to ethylene, owers were removed from the chamber and kept under the same temperature and lighting conditions as above.

Pollination
In the greenhouse, stamens were removed on the day of anthesis, and the individual owers were enveloped with paper bags to prevent pollination. The stigma matured approximately 4 days after emasculation. Then, the owers were cut from the plant, transported to the laboratory, and placed in water. The owers were pollinated with fresh pollen collected from the same cultivar. The owers were held under the aforementioned environmental conditions as above.
Observation of the pollen tube growth

Measurement of ethylene production
Gynoecia and receptacles were individually placed in test tubes (14.8 mL). They were sealed and maintained at 23°C. One hour later, a 1-mL gas sample was withdrawn using a syringe and used to determine the ethylene concentration using a gas-chromatograph (GC-14B, Shimadzu, Kyoto, Japan) equipped with an alumina column and a ame ionization detector. The results of ethylene production were expressed on a fresh weight basis.

RNA extraction
Frozen gynoecium and receptacle (0.1 g FW) were powdered under liquid nitrogen by a Shake Master Auto (Bio medical science, Tokyo, Japan) and added to 450 µL of RLT buffer including 1 % 2mercaptoethanol. The mixture was incubated at 56°C for 1 min. Total RNA was extracted using RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) according to the manufacture's manual with some modi cation.
To clone the 3′ and 5′ ends of the target cDNA, RACE-PCR was performed using 3′-Full RACE Core Set (Takara bio) and CapFishing Full-Length cDNA Premix Kit (Seegene, Seoul, Korea). Speci c primers were designed based on the sequence data obtained from the partial-length of the target cDNA (Table S1). PCR products were puri ed and sub-cloned into the pGEM-TeasyVecter. The full length cDNA was cloned and the DNA sequence was con rmed using speci c primers.

Determination of nucleotide sequence
Sequencing of DNA was performed using an Applied Biosystems ABI Prism DNA sequencer (Model 377; Thermo Fisher Scienti c) and the Applied Biosystems Big Dye Terminator v.3.1 Cycle Sequencing Kit (Thermo Fisher Scienti c), employed with both T7HT and M3RP universal primers. Sequence analysis was performed using ClustalW2 (European Bioinformatics Institute, http://www.ebi.ac.uk/Tools/msa/clustalw2/). Homology was obtained by comparing the sequenced clones with the existing translated DNA in the database of the National Center for Biotechnology Information BLAST algorithm (http://www.ncbi.nlm.nih.gov/nucleotide/). Phylogenic analysis was performed with Phylogeny.fr (http://www.phylogeny.fr/).
PCR reactions were performed with Thermal Cycler Dice Real Time System (Takara bio), using SYBR Premix Ex Taq II (Perfect Real Time, Takara bio). Each reaction was performed using 0.8 µL aliquot of 20 µL cDNA solution derived from 1 µg of total RNA. The following program was applied: initial polymerase activation at 95°C for 30 s; then 40 cycles at 95°C for 5 s, 60°C for 30 s. The speci city of the PCR was checked using a heat dissociation protocol (from 95°C to 60°C) after the nal cycle.
The absolute transcript level was determined by the second derivative maximum method (Luu-The et al., 2005) using a dilution series of plasmid DNA containing target sequence as external standards. To standardize the data, the ratio between the absolute transcript level of the target gene and the control gene was calculated for each sample.

Results
Cloning and nucleotide sequences of ACS genes Three ACS genes were cloned and designated DgACS1, DgACS2 and DgACS3 and deposited in the DDBJ with accession nos LC603027, LC603028, and LC603029, respectively. The cDNAs of these genes were 1901, 1739, and 1721 bp in length, respectively. The predicted proteins consisted of 486 amino acids for DgACS1, 485 amino acids for DgACS2, and 444 amino acids for DgACS3. The amino acid homologies were 80.9 % between DgACS1 and DgACS2, 55.8 % between DgACS1 and DgACS3, and 53.2 % between DgACS2 and DgACS3. Seven conserved domains were observed in the three genes ( Supplementary Fig.   S1).
ACS proteins have been categorized into three types based on the sequences of C-terminal region (Lin et al. 2009). Type 1 ACS proteins have been revealed to be phosphorylated by the mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK), whereas type 2 ACS proteins only have CDPK site. Type 3 proteins lack these two sites for phosphorylation. Based on these criteria, the predicted proteins of DgACS1 and DgACS2 belong to type 1, whereas that of DgACS3 belongs to type 3 ( Supplementary Fig. S2).
Cloning and nucleotide sequences of ACO genes Three ACO genes were cloned and designated DgACO1, DgACO2 and DgACO3, and deposited in the DDBJ with accession nos LC603030, LC603031, and LC603032, respectively. The cDNAs of these genes were 1182, 1159, and 1072 bp in length, respectively. The predicted proteins consisted of 317 amino acids for DgACO1, 317 amino acids for DgACO2 and 319 amino acids for DgACO3 (Supplementary Fig. S3). The amino acid homologies were 78.6 % between DgACO1 and DgACO2, 78.6 % between DgACO1 and The phylogenic analysis revealed that ACO proteins were clustered into three groups (Houben and Van de Poel, 2019). The amino acid sequences of the three genes were extremely similar, and they clustered into type 1 (Supplementary Fig. S4).
Ethylene production and ACS and ACO transcript levels in the gynoecium and receptacle during natural senescence Ethylene production in the gynoecium was relatively high on day 0, and they decreased over the rst 3 days before increasing on day 4 (Fig. 2). Ethylene production in receptacle changed similarly to that in gynoecium.
In the gynoecium, DgACS1 had the highest transcript levels among the three ACS genes at harvest, whereas its levels decreased during the rst 3 days and increased thereafter (Fig. 3). DgACS2 transcript level in gynoecium gradually increased during senescence, and DgACS3 transcript level markedly increased from day 4. In receptacle, DgACS2 transcript level was highest on day 0 among the three ACS transcripts, but its level decreased during senescence. DgACS1 transcript level was relatively high on day 0, and it decreased over the rst 3 days before increasing on day 4. DgACS3 transcript level increased during senescence.
In the gynoecium, DgACO1 transcript level was much higher than those of the other two ACO genes on day 0, and it increased during senescence (Fig. 4). DgACO2 transcript level in the gynoecium gradually increased during senescence, whereas DgACO3 transcript level decreased during the rst 5 days before increasing thereafter. In receptacle, DgACO1 had much higher transcript level than the other two ACO genes on day 0, and its level increased over the rst 5 days and decreased thereafter. DgACO2 level was almost constant during the rst 4 days before increasing thereafter. DgACO3 level increased during senescence.
Ethylene production and ACS and ACO transcript levels of gynoecium and receptacle after exposure to ethylene Exposure to ethylene accelerated sepal abscission, and the sepals abscised within 24 h. Ethylene production was more strongly increased in receptacle than in gynoecium after 8 h of ethylene exposure (Fig. 5).
In gynoecium, exposure to ethylene increased DgACS1 and DgACS2 transcript levels at most time points (Fig. 6). However, ethylene exposure only slightly affected DgACS3 transcript level. In receptacle, DgACS2 transcript level was highest among the three genes and higher after ethylene treatment than in the control at all time points. DgACS1 transcript level was not noticeably increased by ethylene treatment versus control. DgACS3 transcript level was decreased by ethylene treatment at all time points, and its level was lowest among the three transcripts.
In gynoecium, DgACO1 transcript levels were highest among the three transcripts, but ethylene exposure only slightly increased its level (Fig. 7). Ethylene exposure did not increase DgACO2 and DgACO3 transcript levels. In receptacle, DgACO1 transcript level was increased by ethylene treatment at all time points. DgACO2 transcript level was not increased by ethylene treatment, whereas DgACO3 transcript level was markedly increased by ethylene exposure.
Effect of pollination on sepal abscission and pollen tube growth D. grandi orum owers are protandrous, and the gynoecium matured 3 or 4 days after anthesis under the present experimental conditions. Pollination accelerated sepal abscission, which was observed within 24 h after pollination. Pollen tube growth in gynoecia was observed via uorescent microscopy. Many pollen tubes reached the ovary 6 h after pollination (Supplementary Fig. S5).

Ethylene production and ACS and ACO transcript levels in gynoecium and receptacle in unpollinated and pollinated owers
Ethylene production was extremely low in unpollinated whole owers throughout the experimental period. Similarly, ethylene production in gynoecium and receptacle was also extremely low in unpollinated owers. Pollination increased ethylene production in the whole ower. Similarly, pollination markedly increased ethylene production in gynoecium, and the two ethylene peaks were observed after pollination (Fig. 8). Pollination increased ethylene production in receptacles, with production peaking at 8 h after pollination.
In gynoecium, DgACS1 transcript level was slightly increased by pollination, whereas those of the other ACS genes were not increased (Fig. 9). In the receptacle, DgACS2 transcript level was increased by pollination, but DgACS1 and DgACS3 transcript levels were not altered.
In gynoecium, DgACO1 transcript level, which was highest among the three transcripts, was increased by pollination (Fig. 10). However, the levels of the other two ACO transcript levels were not increased by pollination. In receptacle, DgACO1 transcript level was higher in pollinated owers than in unpollinated owers at all time points. Although DgACO2 transcript level was slightly changed by pollination, DgACO3 transcript level was markedly increased by pollination.

Discussion
Delphinium owers are highly sensitive to ethylene and the sepals abscise during senescence (Ichimura et al. 2009). In this study, we isolated three ACS and three ACO genes from D. grandi orum, and the expression analysis in gynoecium and receptacle was performed during natural senescence and following ethylene exposure and pollination. ACS and ACO gene expression was differently regulated in the gynoecium and receptacle.

Ethylene production of gynoecium and receptacle
Although ethylene production in gynoecium and receptacle at harvest was relatively high, its production increased in these organs during senescence (Fig. 2). This result somewhat differed from that in D. belladonna, in which ethylene production is relatively low at harvest in gynoecium and receptacle (Ichimura et al. 2009). The difference of ethylene production at harvest may be attributable to the different physiological properties of these species.
The trends of ethylene production in whole owers following ethylene exposure and pollination differed from those in gynoecium and receptacle (Figs. 5 and 8). The sum of the gynoecium and receptacle fresh weight was one-tenth of that of the whole ower. Although ethylene production in oral organs other than gynoecium and receptacle was extremely low during natural senescence (Ichimura et al. 2009), ethylene production may decrease in these organs following ethylene exposure and pollination, resulting in differences in ethylene production between the whole ower and the two organs.

ACS and ACO gene expression during natural ower senescence
In the carnation gynoecium, DcACS2 and DcACO1 transcript levels increase during senescence (ten Have and Woltering 1997; Jones and Woodson 1999). Similarly, SlACS1A transcript levels increased during senescence in the tomato (Llop-Tous et al. 2000). In petals displaying abscission, changes in ACS and ACO transcript levels in gynoecium during natural senescence have only been reported for geranium, but no substantial increases in their transcript levels were observed (Clark et al. 1997). In the present study, the peak DgACS3 transcript level was much higher than those of the other ACS transcripts (Fig. 3). DgACO1 transcript levels were higher than those of the other ACO transcripts (Fig. 4). These results suggest that DgACS3 and DgACO1 expression appears to largely contribute to increased ethylene production in gynoecium.
To date, the expression of ACS and ACO genes in receptacle has only been investigated in carnation (Jones and Woodson 1999) and rose (Xue et al. 2008), but no obvious changes in these transcript levels were observed during senescence. In receptacle, DgACS2 transcript level was much higher than those of the other ACS transcripts on day 0, but its level decreased during senescence (Fig. 3). Conversely, DgACS1 and DgACS3 transcript levels increased during senescence. Similarly, the levels of the three ACO transcripts increased during senescence, although some differences were observed (Fig. 4). Thus, the expression of these ACS and ACO genes appears to contribute to ethylene production in receptacle.
ACS and ACO gene expression following ethylene exposure Exposure to ethylene accelerated sepal abscission, which is consistent with previous ndings (Ichimura et al. , 2009. Ethylene exposure more strongly increased ethylene production in receptacle than in gynoecium (Fig. 5), suggesting that the responsiveness of ethylene production to ethylene exposure is higher in receptacle than in gynoecium. The receptacle, but not the gynoecium, is connected to sepals in D. grandi orum owers (Fig. 1). Thus, sepal abscission following ethylene exposure may be induced by increased ethylene production of the receptacle.
ACS and ACO gene expression in receptacle following ethylene exposure has only been reported in carnation (Jones and Woodson 1999) and rose (Xue et al. 2008). Among these genes, only RhACS2 expression was slightly increased by ethylene exposure (Xue et al. 2008). DgACS2 and DgACO3 transcript levels in the receptacle were relatively high, and they were markedly increased by ethylene exposure (Figs. 6 and 7). This nding suggests that DgACS2 and DgACO3 may contribute to increased ethylene production in receptacle associated with sepal abscission.
DgACS1 and DgACS2 transcript levels in gynoecium were increased by ethylene exposure, whereas the expression of DgACS3 and the three ACO genes did not increase (Figs. 6 and 7). These results suggest that DgACS1 and DgACS2 contribute to increased ethylene production in gynoecium in response to ethylene exposure. To date, the expression of ACS and ACO genes in pistil or gynoecium following ethylene exposure has been reported in carnation (Jones andWoodson 1997, 1999), petunia (Tang et al. 1994

ACS and ACO gene expression in pollinated owers
As reported in many plants, including Digitalis (Stead and Moore 1979), geranium (Clark et al. 1997) and torenia (Goto et al. 1999), pollination accelerated sepal abscission in D. grandi orum. Ethylene production in gynoecium markedly increased 4 h after pollination (Fig. 8). However, only DgACO1 transcript level in the gynoecium was increased by pollination (Fig. 10). In Delphinium gynoecium, ACO activity at anthesis is relatively high (Ichimura et al. 2009). The pollen of some plants, including petunia, contains large amounts of ACC (Whitehead et al. 1983). In petunia owers, an early increase in ethylene production is associated with the ACC content in pollen (Singh et al. 1992). We con rmed that pollen tubes reached ovary 6 h after pollination ( Supplementary Fig. S5). Thus, marked increase in ethylene production in gynoecium may be attributed to pollen-born ACC, which is converted to ethylene by ACO activity. Alternatively, other ACS and ACO genes may be speci cally induced by pollination, although these types of ACS and ACO genes have not yet been identi ed in other plant species.
The coordination of senescence processes in pollinated owers is involved in interorgan signaling (Jones and Woodson 1997). The transmissible factor for this signaling has been proposed to be ethylene (Woltering et al. 1995) or ACC . Ethylene production in receptacle increased 4 h after pollination (Fig. 8), suggesting that pollination generates some transmissible factor that induces ethylene production of the receptacles because receptacle lies below the gynoecium. Increased ethylene production was associated with increases in DgACO1 and DgACO3 transcript levels in receptacle (Fig. 10). Thus, ethylene produced by the gynoecium in response to pollination may increase the expression of these genes, resulting in increased ethylene production in receptacle.
Signi cance of the receptacle and possible model for sepal abscission The signi cance of gynoecium to petal senescence has been emphasized for petal wilting in petunia ) and carnation (Shibuya et al. 2000) and for petal abscission in Digitalis (Stead and Moore 1983) and geranium (Clark et al. 1997). Although changes in some ACS and ACO transcript levels in receptacle were reported in carnation (Jones and Woodson 1999) and rose (Xue et al. 2008), the signi cance of this organ to petal wilting was only mentioned by Hsieh and Sacalis (1986), who reported ACC levels in receptacle during senescence in cut carnation. The signi cance of receptacle to sepal abscission has been described only for Delphinium owers (Ichimura et al. 2009). In our study, ethylene exposure and pollination increased ethylene production more strongly in receptacle than in gynoecium, and this nding was associated with marked increases in some ACS and ACO transcript levels in receptacle. These results suggest the signi cance of receptacle to sepal abscission. Thus, the receptacle is likely to be a primarily organ that regulates sepal abscission in Delphinium.
We thus propose a model for sepal abscission based on the present results (Fig. 11). During natural ower senescence, increases in DgACS1, DgACS3, DgACO1 and DgACO3 transcript levels are primarily responsible for ethylene production in receptacle, leading to sepal abscission. However, whether increase in ethylene production in gynoecium affects ethylene biosynthesis is unclear. Exposure to ethylene increases DgACS2 and DgACO3 transcript levels in receptacle, leading to sepal abscission. Receptacle should directly respond to exogenous ethylene. Pollination increases ethylene production in gynoecium without inducing ACS and ACO gene expression. The increase in ethylene production in gynoecium may lead to increased DgACS2 and DgACO3 transcript levels in receptacle, resulting in sepal abscission.

Conclusion
Ethylene production in gynoecium and receptacle in D. grandi orum owers somewhat increased during natural senescence. Three ACS genes (DgACS1, DgACS2, and DgACS3) and three ACO genes (DgACO1, DgACO2, and DgACO3) were cloned. The transcript levels of the ACS and ACO genes in these organs increased during senescence, excluding DgACS2 in receptacle. Exposure to ethylene more strongly increased ethylene production in receptacle than in gynoecium and induced sepal abscission. DgACS1 transcript level in gynoecium and DgACS2 and DgACO3 transcript levels in receptacle were increased by ethylene exposure. Pollination increased ethylene production of gynoecium and receptacle and markedly accelerated sepal abscission. However, pollination only slightly affected ACS and ACO transcript levels in gynoecium, whereas DgACO3 transcript level in receptacle was markedly increased. These results suggest that increases in the transcript levels of some ACS and ACO genes in gynoecium and receptacle may contribute to the increases in ethylene production associated with sepal abscission. Ethylene exposure and pollination more strongly increased the expression of these genes in receptacle than in gynoecium, suggesting the signi cance of receptacle to sepal abscission.