TY - JOUR
AU - Satoh, Shigeru
AB - Abstract Ethylene production and expression of ethylene biosynthetic genes was investigated in senescing flowers of carnation (Dianthus caryophyllus L.) cultivars ‘White Candle (WC)’ and ‘Light Pink Barbara (LPB)’, with long and short vase‐lives, respectively. Ethylene production from the gynoecium and petals of senescing ‘WC’ flowers was below the limit of detection, in agreement with the repressed ethylene production from the whole flowers. However, exogenous ethylene treatment caused the accumulation of transcripts for DC‐ACS1 and DC‐ACO1 genes in both the gynoecium and petals, resulting in ethylene production from the flowers. Moreover, application of ABA or IAA, which are known to exhibit their action through the induction of ethylene synthesis in the gynoecium, to ‘WC’ flowers from their cut stem‐end induced ethylene production and wilting in the flowers. These findings suggested that, in ‘WC’ flowers the mechanism of ethylene biosynthesis, i.e. the induction of expression of genes for ethylene biosynthesis and the action of resulting enzymes, was not defective, but that its function was repressed during natural senescence. Transcripts of DC‐ACO1, DC‐ACS3, and DC‐ACS1 were present in the gynoecium of senescing ‘LPB’ flowers. In the gynoecium of senescing ‘WC’ flowers, however, the DC‐ACO1 transcript was present, but the DC‐ACS1 transcript was absent and the DC‐ACS3 transcript was detected only in a small amount; the latter two were associated with the low rate of ethylene production in the gynoecium of ‘WC’ flowers. These findings indicated that the repressed ethylene production in ‘WC’ flowers during natural senescence is caused by the repressed ethylene production in the gynoecium, giving further support for the role of the gynoecium in regulating petal senescence in carnation flowers. Carnation, Dianthus caryophyllus, ethylene production, flower senescence, gynoecium, long‐lasting flowers. Received 24 November 2003; Accepted 4 December 2003 Introduction Ethylene is a primary plant hormone involved in the senescence of cut carnation flowers (Abeles et al., 1992; Borochov and Woodson, 1989; Reid and Wu, 1992). A large amount of ethylene is synthesized several days after full opening of the flower during natural senescence (Manning, 1985; Peiser, 1986; Woodson et al., 1992), or several hours after compatible pollination (Nichols, 1977; Nichols et al., 1983; Larsen et al., 1995) or treatment with exogenous ethylene (Borochov and Woodson, 1989; Wang and Woodson, 1989). The increased ethylene production accelerates in‐rolling of petals resulting in wilting of the flower. Ethylene is synthesized through the following pathway: l‐methionine → S‐adenosyl‐l‐methionine → 1‐aminocyclopropane‐1‐carboxylate (ACC) → ethylene. ACC synthase and ACC oxidase catalyse the last two reactions (Yang and Hoffman, 1984; Kende, 1993). So far, three genes encoding ACC synthase (DC‐ACS1, DC‐ACS2, and DC‐ACS3) and one gene encoding ACC oxidase (DC‐ACO1) have been identified from carnation (Park et al., 1992; Henskens et al., 1994; Jones and Woodson, 1999; Wang and Woodson, 1991). These genes are regulated in a tissue‐specific manner during flower senescence; DC‐ACO1 is expressed in both the gynoecium and petals of carnation flowers that are undergoing senescence, and DC‐ACS1 is also expressed in both the gynoecium and petals, but mainly in the latter, whereas DC‐ACS2 and DC‐ACS3 occur in the gynoecium (Henskens et al., 1994; ten Have and Woltering, 1997; Jones and Woodson, 1999). In carnation flowers, it has been revealed that ethylene is first produced from the pistil and the evolved ethylene induces autocatalytic ethylene production in petals, resulting in wilting of the petals, during the natural senescence of carnation flowers (ten Have and Woltering, 1997; Shibuya et al., 2000). This observation suggests the role of the gynoecium in controlling the senescence of petals in the flowers. In the carnation flowers, if the gynoecium could not produce a sufficient amount of ethylene to induce ethylene production in petals, the whole flower would not suffer from ethylene‐dependent wilting in their petals and have a prolonged vase‐life. Carnation plants with such characteristics may be present among cultivars or variants that have been shown to have flowers with a prolonged vase‐life. The characterization of ethylene production in those flowers should help to determine the role of the gynoecium in the senescence of carnation flowers, and to elucidate the regulation of genes for ethylene biosynthesis in the gynoecium and petals during senescence of the flower. In the early 1990s, carnation cultivars and strains with unusual ethylene‐related behaviour were reported: cvs Killer (Serrano et al., 1991), Sandra (Wu et al., 1991), Chinera (Reid and Wu, 1992), and Sandorosa (Mayak and Tirosoh, 1993), and strains 87‐37G‐2, 81‐2, and 799 (Brandt and Woodson, 1992). A common feature of these cultivars and strains except for ‘Chinera’ is the low rate of ethylene production during the senescence of flowers. So far, however, there have been no reports on the regulation of expression of ACC synthase and ACC oxidase genes in flowers of those carnation plants. Unfortunately, many of the carnation cultivars and strains reported in the early days are not available in Japan. In the present study, carnation plants were found whose flowers produce little ethylene and have a prolonged vase‐life. The production of ethylene and the expression of genes for ethylene biosynthesis in flower tissues of those carnations were examined in order to determine the role of ethylene production from the gynoecium in the senescence of carnation flowers. Materials and methods Plant materials Flowers of carnations (Dianthus caryophyllus L.) that belong to both the standard and the spray categories of carnation flower were used. Carnation flowers at the usual commercial stage of flowering, the paintbrush stage for carnation flowers of the standard category and at the stage when the first flower out of six to eight flower buds on a stem was almost fully open for carnation flowers of the spray category, were harvested in the morning at nurseries of commercial growers in Miyagi, Chiba, Ibaraki, or Nagano prefectures. The flowers harvested in the Miyagi prefecture were transported dry (without dipping the cut stem end in water) to the laboratory on the day of harvest, but those from other prefectures arrived on the day after harvest. Stems were trimmed to 30 cm, placed with their cut end in distilled water and held for 1 or 2 d under 14 h d–1 white fluorescent light (15 µmol m–2 s–1) at 23 °C. Flowers of the standard‐category carnations were used when they became fully open, and those of the spray category were used when the second and the third flowers on a stem became fully open. The full opening stage of flowers was when their outermost petals were at right angles to the stem of the flower. This time point was designated as day 0. Depending on the number of fully opened flowers available on day 0, each experiment was conducted with five or three flowers. Respective experiments with these flowers were repeated twice or more with similar results, and so only the results of one typical experiment are shown. Analysis of the vase‐life of cut flowers Flowers at their full opening stage (day 0) were trimmed to 3 cm in stem length and placed with their stem end in 20 ml distilled water in 50 ml glass vials (one flower per vial). Five flowers were used per treatment. The flowers were left under the conditions described above, and water was replaced daily. Flowers were observed and photographed daily to record senescence symptoms, i.e. in‐rolling and subsequent wilting of petals, desiccation and discoloration of the petal margins, and ethylene production in flowers were examined once a day. Vase‐life in days is expressed as the mean ±SE of the five flowers. Treatment of flowers with ethylene, ABA or IAA For ethylene treatment, the vials with flowers on day 0 were placed in a 60 l glass chamber with ethylene at 2 µl l–1 for 0–18 h under the same conditions as above but under continuous light. Five flowers were collected at 6 h intervals from the glass chamber. The flowers were held in the open air for 1 h prior to the measurement of ethylene production in order to let the ethylene accumulated in flower tissues diffuse out. Ethylene production from the whole flower, gynoecium, and petals was measured as described in the following section. For treatments with ABA or IAA, the stem end of carnation flowers (day 0 and 3 cm in stem length) was placed in 20 ml of 1 mM ABA or 1 mM IAA solutions in 50 ml glass vials (one flower per vial). The concentrations of ABA and IAA were sufficient to induce senescence of carnation flowers (Shibuya et al., 2000). The flowers were left for 24 h under continuous light as described above. The symptoms of petal senescence were recorded daily, and ethylene production from the whole flower and then from the detached gynoecium and petals was measured as described below. Measurement of ethylene production Ethylene production from the whole flower was measured by enclosing the flowers in 350 ml glass containers (one flower per container) for 1 h at 23 °C (Kosugi et al., 1997). A 1 ml gas sample was taken into a hypodermic syringe from the container through a rubber septum in the lid of the container, and analysed for ethylene with a gas chromatograph (263‐30, Hitachi, Tokyo) equipped with an alumina column and a flame ionization detector. Then the flowers were separated by hand into the gynoecium (ovary plus styles), petals, and the remaining sepals plus stem. The respective parts of the flower were enclosed in glass containers of appropriate sizes (inner volumes; 5 ml for gynoecium, 180 ml for petals and the remaining sepals plus stem) for 1 h, and the ethylene evolved was measured as described above. A preliminary experiment with flowers on day 0 showed that wound‐induced ethylene production from detached flower parts was below the limit of detection. After the assay of ethylene production, petals, gynoecium, and the remaining sepals and stem were weighed, immediately frozen in liquid N2, and stored at –80 °C until isolation of RNA. RNA gel blot analysis of transcripts of ACC synthase and ACC oxidase genes In the experiment shown in Fig. 2 of the Results, the levels of transcripts of ACC oxidase and ACC synthase genes in total RNA fractions from the gynoecium and the petals of carnation flowers treated with ethylene were determined by gel blot analysis. Total RNA was isolated by the SDS‐phenol method (Palmiter, 1974) from the respective tissues of the flowers. Ten micrograms of total RNA was denatured at 65 °C for 15 min in 10 mM MOPS, pH 7.0, containing 2.2 M formaldehyde and 50% (v/v) formamide. The denatured RNA was separated on a 1.0% agarose gel containing 2.2 M formaldehyde and transferred onto nylon membranes (Hybond N+, Amersham Pharmacia Biotech, Tokyo). The cDNA fragments amplified by RT‐PCR, as described in the following section, were used as the probes for gel blot analysis. The DNA probes were labelled with HRP and hybridized with the blot using ECL Direct™ (Amersham Pharmacia Biotech) according to the manufacturer’s instruction. Hybridization signals were detected using X‐ray film (RX‐U, Fuji Photo Film, Tokyo). RT‐PCR analysis of transcripts of ACC synthase and ACC oxidase genes In the experiment shown in Fig. 4 of the Results, the levels (actually, the presence or absence) of transcripts of ACC oxidase gene (DC‐ACO1) and ACC synthase genes (DC‐ACS1, DC‐ACS2, and DC‐ACS3) in the total RNA fractions obtained from the petals and the gynoecium of carnation flowers undergoing natural senescence were determined by amplification by RT‐PCR. RT‐PCR was performed according to standard procedures with necessary optimization. Briefly, the RT reaction was conducted in a reaction mixture containing 500 ng total RNA, 50 pmol oligo dT, 50 mM TRIS‐HCl, 100 mM KCl, 4 mM DTT, 10 mM MgCl2, 0.5 mM dNTP mixture, and AMV reverse transcriptase XL (Takara, Kyoto, Japan) in a total volume of 20 µl at pH 8.3, for 1 h at 42 °C then for 30 min at 51 °C. The reaction was stopped by heating at 70 °C for 15 min. PCR was conducted in a reaction mixture containing 0.5 µl of the RT reaction product, 5 pmol each of the upstream and downstream primers, 10 mM TRIS (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 200 µM dNTP mixture, and 2.5 units of Taq DNA polymerase (Sigma‐Aldrich, Inc.) in a total volume of 25 µl. PCR was carried out for 5 min at 94 °C, followed by 30 cycles of 50 s at 94 °C, 45 s at 55 °C, 90 s at 72 °C, and supplemental incubation for 7 min at 72 °C with an automatic thermal cycler (GeneAmp PCR System 2400, Perkin‐Elmer/Cetus). The PCR products were separated on a 2.0% agarose gel and visualized by ethidium bromide staining. The upstream and downstream primers for PCR were: 5′‐ATG GCA AAC ATT GTC AAC TT‐3′ and 5′‐TTT CCA CCA ATG TTG GCG CC‐3′, respectively, for DC‐ACO1 cDNA; 5′‐GTC TAA AAT CAT GAG TTA TTA A‐3′ and 5′‐CTA TAT CAA TGA TCT TAC ACA T‐3′ for DC‐ACS1 cDNA; 5′‐GCT AAG CCT GTC GTG GCG TCT ACT AA‐3′ and 5′‐CCA ATT ACA ATG CCT TGA AA‐3′ for DC‐ACS2 cDNA, and 5′‐TGA ATT CAG TAT GGA GAT TAG AT‐3′ and 5′‐CCT ACA ATG AGT TTG GTC AT‐3′ for DC‐ACS3 cDNA. The sizes of amplified cDNA fragments and their positions in the original cDNAs were 820 bp and 40–859 bp, respectively, in DC‐ACO1 cDNA (accession number M62380), 220 bp and 1688–1907 bp, respectively, in DC‐ACS1 cDNA (accession number M66619), 370 bp and 315–684 bp, respectively, in DC‐ACS2 cDNA (accession number AF049138), and 228 bp and 1267–1494 bp, respectively, in DC‐ACS3 cDNA (accession number AF049137). To examine the identities after PCR and the amount of template RNA, the fragment of actin (DC‐ACT1, Waki et al., 2001) was amplified using the upstream primer and downstream primers, 5′‐CGT CAC CAA CTG GGA TGA CA‐3′ and 5′‐CGA TGG CTG GAA GAG GAC TT‐3′, respectively. This gave a cDNA fragment of 571 bp, which corresponded to the position 51–621 bp in DC‐ACT1 cDNA (accession number AY007315). Results Carnation cultivars having flowers with a long vase‐life Carnation plants having flowers with a long‐vase life were sought by assaying ethylene production from the flowers and observing the morphological changes of the flowers during natural senescence. After testing flowers of 30 cultivars, which were chosen randomly at first and then following the recommendation for a long‐lasting trait of flowers from several carnation‐breeding companies, five cultivars were found whose flowers did not produce ethylene in large amounts during senescence and had a vase‐life longer than that of ordinal commercial carnation varieties. These were cultivars ‘White Candle (WC)’, ‘Cream Candle’, ‘Royal Green’, ‘Le Noirs’ and ‘Shion’. Table 1 summarizes the characteristics of flowers of these cultivars compared with those of ‘Nora’ and ‘Light Pink Barbara (LPB)’, which were chosen as controls. ‘Nora’ carnation belongs to the standard category, but the other six cultivars to the spray category of carnation flowers. Flowers of the low‐ethylene‐producing (LE) cultivars did not show a significant climacteric rise of ethylene production, and the maximum ethylene production rates detected during the senescence period were very low; less than 0.1 nmol g–1 h–1 (Table 1). The flowers of LE cultivars did not show petal in‐rolling or rapid wilting during senescence, which are typical symptoms of flower senescence in carnations. Instead, the first symptoms of petal senescence in the LE cultivars were desiccation, discoloration, and necrosis of the petal margins, which gradually spread to the remaining petal portions. By contrast, flowers of ‘Nora’ and ‘LPB’ produced ethylene in large amounts, showing a climacteric rise of ethylene production; the maximum rate of ethylene production was 4.04 nmol g–1 h–1 and 1.87 nmol g–1 h–1 in ‘Nora’ and ‘LPB’, respectively. Maximum ethylene production was detected just before or at the onset of in‐rolling of petals in these two carnation cultivars. Flowers of the LE cultivars had vase‐lives longer than those of ‘Nora’ and ‘LPB’. The vase‐life of ‘WC’ flowers was three and two times longer than that of ‘Nora’ and ‘LPB’ flowers, respectively (Table 1). Since ‘WC’ flowers had the longest vase‐life (c. 18 d), this cultivar was chosen for further characterization of ethylene production at the molecular level and compared with ‘LPB’ flowers (vase‐life was c. 9 d) as a control. In the following experiments, however, the vase‐life (that roughly corresponds to time of maximum ethylene production) of ‘LPB’ flowers was shortened to c. 6 d compared with that shown in Table 1 (c. 9 d). There was a tendency that the vase life of ‘LPB’ flowers harvested in the winter (December–March) was longer than that of those harvested in the summer (late May–September) (data not shown). Regardless of the difference in harvest season, however, ‘LPB’ flowers always produced ethylene in a climacteric manner and in similar amounts during natural senescence. Ethylene production in the whole flowers, gynoecium and petals of ‘WC’ flowers during natural senescence Figure 1 compares ethylene production from whole flowers, petals, and gynoecium between ‘WC’ and ‘LPB’ flowers during natural senescence. Ethylene production of whole ‘WC’ flowers was below the limit of detection during the senescence period (19 d), except for the ethylene production rate of below 0.2 nmol g–1 h–1 on day 17. When petals and gynoecium were detached from whole flowers and subjected to ethylene production assay, their ethylene production level was again below or around the limit of detection during the senescence period. By contrast, whole ‘LPB’ flowers produced ethylene in a large amount during natural senescence, attaining a maximal rate (2.70 nmol g–1 h–1) on day 5. The detached gynoecium and petals produced ethylene with a time‐course similar to that of whole flowers, attaining the maximum rates of ethylene production on day 5. The ethylene production in the petals of carnation flowers undergoing natural senescence is induced by ethylene produced from the gynoecium of the flowers (Shibuya et al., 2000). Therefore, it was suspected that the absence of ethylene production in the ‘WC’ flowers resulted from the malfunction of the mechanism of ethylene biosynthesis in the gynoecium and/or petals of the flowers. This possibility was checked by treating ‘WC’ flowers with exogenous ethylene in the next experiment. Effects of exogenous ethylene treatment on ethylene production from the whole flowers and flower parts and the transcript levels for ACC synthase and ACC oxidase in the petals and gynoecium of ‘WC’ flowers The flowers of ‘WC’ and ‘LPB’ on day 0 were treated with ethylene at 2 µl l–1 for 18 h, and senescence profiles, ethylene production from the whole flowers and isolated flower parts, and the accumulation of transcripts for ACC oxidase and ACC synthases in the gynoecium and petals were investigated (Fig. 2). Flowers of both cultivars exhibited in‐rolling of the petal rim 6 h after the start of ethylene treatment and wilting of whole petals at 12 h (data not shown). Ethylene production was detected 6 h after the start of ethylene treatment and it increased until 18 h in both ‘WC’ and ‘LPB’ flowers (Fig. 2A). At 12 h after the start of ethylene treatment, the rate of ethylene production was higher in ‘WC’ flowers than in ‘LPB’ flowers. In ‘LPB’ flowers, ethylene was produced almost solely in the petals, but in ‘WC’ flowers it was produced in all the tissues, although the rate of ethylene production was much higher in the petals than in the gynoecium and other tissues (sepals plus stem tissue) (Fig. 2). The accumulation of transcripts of genes for ACC synthase and ACC oxidase was investigated by RNA gel blot analysis with total RNAs extracted from the gynoecium and petals of flowers treated with ethylene for 0–24 h (Fig. 2C, D). As described in the Introduction, three genes for ACC synthase (DC‐ACS1, DC‐ACS2, and DC‐ACS3) and one gene for ACC oxidase (DC‐ACO1) have been identified in carnation plants to date. However, since transcripts for DC‐ACS2 and DC‐ACS3 were undetectable by RNA gel blot analysis in the present study, transcripts of DC‐ACS2 and DC‐ACS3 were omitted from Fig. 2C, D. In the gynoecium of both ‘WC’ and ‘LPB’ flowers, the DC‐ACO1 transcript was detected 6 h after the start of ethylene treatment and its level increased up to 18 h, except for a decline in its level in the ‘LPB’ gynoecium at 12 h (Fig. 2C). The DC‐ACS1 transcript was detected in a small amount at 12–18 h in the gynoecium of ‘WC’ flowers, but not in the gynoecium of ‘LPB’ flowers. In the petals of both cultivars, the DC‐ACO1 transcript was detected at 6 h of ethylene treatment and increased up to 18 h. The DC‐ACS1 transcript was detected at 12 h and increased abundantly at 18 h in ‘WC’ petals, whereas it was only detected at 18 h in ‘LPB’ petals (Fig. 2D). Induction of ethylene production in ‘WC’ flowers by treatment with ABA or IAA To obtain further evidence for ethylene production in ‘WC’ flowers, the effects of exogenously applied ABA and IAA on ethylene production in the flowers were examined (Fig. 3). ‘WC’ flowers on day 0 produced hardly any ethylene (<0.05 nmol g–1 h–1) at 0 h (before treatment with the chemicals). Corresponding to the repressed ethylene production in the whole flowers, ethylene production from the gynoecium and petals was below the limit of detection on day 0. Treatment with either 1 mM ABA or 1 mM IAA for 24 h caused abundant ethylene production in the gynoecium and petals (Fig. 3). Ethylene production rates were higher in both the gynoecium and petals of IAA‐treated flowers than in those of ABA‐treated flowers; they were more than 1 nmol g–1 h–1 in the former and less than 1 nmol g–1 h–1 in the latter. Petal wilting was observed 24 h after the start of treatment with ABA or IAA. PCR analysis of transcripts for ACC oxidase and ACC synthase genes in the gynoecium and petals of ‘WC’ flowers during natural senescence As described previously, the transcripts for DC‐ACS2 and DC‐ACS3 were not detected by RNA gel blot analysis of total RNAs extracted from the tissues of carnation flowers, which produced ethylene at a rate as high as 4 nmol g–1 h–1 or more after exogenous ethylene treatment (Fig. 2A). Thus, instead of RNA gel blot analysis, PCR analysis was carried out to increase the sensitivity of the assay, which seemed suitable for the analysis of total RNAs extracted from flowers which produce ethylene at a moderate rate of 2–3 nmol g–1 h–1 (‘LPB’ flowers) or a rate near the limit of detection (‘WC’ flowers) during natural senescence. For the PCR analysis, combinations of upstream and downstream primers were used as described in the Materials and methods; in particular, the upstream and downstream primers for DC‐ACS1, 2, and 3 were designed from the nucleotide sequence of the 3′‐peripheral region of cDNAs in order to amplify specifically the nucleotide fragments of respective ACC synthase transcripts (Jones and Woodson, 1999). It was confirmed that a given combination of the upstream and downstream primers amplified the nucleotide fragment of expected size when the plasmid harbouring the corresponding cDNA was used as a template (data not shown). Figure 4A shows ethylene production from whole flowers during natural senescence; the ‘LPB’ flowers produced ethylene, attaining a maximal rate (1.92± 1.02 nmol g–1 h–1) on day 5, whereas ethylene production from ‘WC’ flowers was below the limit of detection during the senescence period of 21 d. In the gynoecium of ‘LPB’ flowers, the PCR amplification product of the DC‐ACO1 transcript was detected from day 0 through day 9; the amount of PCR product was small on day 0 and maximal on day 6 in association with the increase in ethylene production. By contrast, in the gynoecium of ‘WC’, the DC‐ACO1 PCR product was not detected on days 0 and 3, and was only detected from day 6 through day 15, the amount of the PCR product being most abundant on day 6. The PCR product of DC‐ACS3 was detected from day 3 through day 9, and that of DC‐ACS1 on days 5 and 6 in the gynoecium of ‘LPB’. The PCR product of DC‐ACS2 was also detected in a small amount on day 5 in the gynoecium of ‘LPB’ (although its signal is not seen in the photograph shown in Fig. 4B). On the other hand, in the gynoecium of ‘WC’ only the DC‐ACS3 PCR product was detected in a small amount from day 9 through day 15. The presence of DC‐ACO1 PCR product from day 5 through day 9 and that of DC‐ACS1 PCR product from day 6 through day 9 were obvious in the petals of ‘LPB’. The DC‐ACS1 PCR product was not detected in ‘WC’ petals during the senescing period of 21 d, although the DC‐ACO1 PCR product was detected from day 9 through day 21, except for day 15. Discussion Previous studies on ethylene production in the carnation flower revealed that ethylene is produced first in the gynoecium during natural and pollination‐induced senescence (Nichols, 1977; Woodson et al., 1992; Jones and Woodson, 1999; ten Have and Woltering, 1997) and it was suggested that the produced ethylene, acting as a diffusible signal, is perceived by petals and induces autocatalytic ethylene production in the petals. Jones and Woodson (1997) showed that in the pollination‐induced senescence of carnation flowers, when styles are treated with diazocylopentadiene, an inhibitor of ethylene action, pollination no longer accelerated either ethylene production or senescence of the petals. Shibuya et al. (2000) showed that the removal of the gynoecium from carnation flowers prevented the onset of ethylene production and markedly prolonged the vase‐life of the flowers undergoing natural senescence. They also showed that the removal of the gynoecium negated the promoting effects of ABA, IAA, and ACC, applied through the cut stem end from the flowers, on ethylene production in the flowers. All of the previous results suggested the pivotal role of the gynoecium in controlling the senescence of carnation flowers, in which ethylene produced in the gynoecium triggers the onset of ethylene production in the petals, resulting in the wilting of the petals. In the present study, further evidence was sought for the role of the gynoecium in carnation flower senescence by investigating the regulation of ethylene production in long‐lasting carnation flowers with repressed ethylene production. In the present study, five cultivars of carnation (Dianthus caryophyllus L.) plants were found whose cut flowers produced little or no ethylene during natural senescence. Since ‘WC’ flowers had the longest vase‐life, this cultivar was chosen for further characterization of ethylene production at the molecular level, compared with ‘LPB’ flowers as a control. The repressed ethylene production in ‘WC’ whole flowers resulted from the absence of ethylene production (non‐detectable) in their gynoecium and petals during the natural senescence period of about 20 d (Fig. 1). The previous notion that ethylene production in the gynoecium plays a triggering role in the senescence of carnation flowers suggests that the repressed ethylene production in the gynoecium was the primary cause of the repressed ethylene production in ‘WC’ flowers undergoing natural senescence. How is ethylene production repressed in the gynoecium of ‘WC’ flowers? It was first suspected that there was a defect in the mechanism of ethylene biosynthesis in the gynoecium, and also in the petals, of ‘WC’. However, treatment of ‘WC’ flowers with exogenous ethylene caused ethylene production, that is, the activation of enzymes for ethylene biosynthesis, in both the gynoecium and petals (Fig. 2). The induced ethylene production was accompanied by the accumulation of transcripts of DC‐ACS1 and DC‐ACO1 in both the gynoecium and petals (Fig. 2). Moreover, application of exogenous ABA or IAA to ‘WC’ flowers from their cut stem end induced ethylene production in both the gynoecium and petals, resulting in wilting of the flowers (Fig. 3). ABA and IAA are known to act through the induction of ethylene synthesis in the gynoecium (Shibuya et al., 2000). These findings indicated that neither the gynoecium nor the petals of ‘WC’ were defective in the mechanism of ethylene synthesis. The expression of genes for ethylene biosynthesis was examined in the gynoecium and petals of ‘WC’ compared with the control ‘LPB’ (Fig. 4). DC‐ACS1 transcripts were detected in the petals of ‘LPB’ flowers, but not in the petals of ‘WC’, which is consistent with the finding of ethylene production in senescing ‘LPB’ but not in ‘WC’ flowers, since most of the ethylene produced from senescing carnation flowers comes from their petals. The presence of the DC‐ACO1 transcript in the petals of senescing ‘WC’ flowers suggested that ethylene was actually evolved from the gynoecium, although its amount was too low to be measured (Fig. 2). Therefore, it was probable that the absence of DC‐ACS1 transcript and the presence of DC‐ACO1 transcript in the petals of ‘WC’ flowers resulted from ethylene production at a threshold level from the gynoecium, which was insufficient to induce the expression of the DC‐ACS1 gene, but sufficient to induce the expression of the DC‐ACO1 gene in the petals. By contrast, in the ‘LPB’ flowers undergoing natural senescence, the amount of ethylene evolved from the gynoecium was enough to induce the expression of both DC‐ACO1 and DC‐ACS1 genes in the petals. Previously, Jones and Woodson (1997) presented the idea that there is a threshold level of ethylene production from the style to induce senescence of petals in the pollination‐induced senescence of carnation flowers. The DC‐ACO1 transcript was accumulated in the gynoecium of both ‘WC’ and ‘LPB’ flowers undergoing natural senescence, indicating that the expression of the DC‐ACO1 gene was not the cause of the difference in ethylene production between ‘WC’ and ‘LPB’ flowers. Transcripts of DC‐ACS3 and DC‐ACS1 accumulated to a significant amount in the gynoecium of senescing ‘LPB’ flowers, whereas only the DC‐ACS3 transcript was detected and in a small amount from day 9 through day 12 in the gynoecium of senescing ‘WC’ flowers. Thus, the absence of the DC‐ACS1 transcript and the presence of only a small amount of DC‐ACS3 transcript were associated with low ethylene production in the gynoecium of ‘WC’. Treatment with exogenous ethylene of ‘WC’ flowers at the full opening stage (day 0) induced ethylene production in all tissues, i.e. petals, gynoecium, and the remaining organs (sepals+stem) of the flowers (Fig. 2). On the other hand, in ‘LPB’ flowers treated with exogenous ethylene, ethylene was produced in the petals and in a small amount in the remaining tissues, but was hardly produced in the gynoecium. These findings suggested a distinct difference in the sensitivity to exogenous ethylene between the gynoecia of ‘LPB’ and ‘WC’ flowers. However, ethylene was produced in the gynoecium of naturally senescing flowers of both cultivars without the action of ethylene. Thus an unknown factor(s) was suggested to be involved in the induction of ethylene production in the gynoecium of carnation flowers undergoing natural senescence. In conclusion, it is thought that, in ‘LPB’ flowers which produce a considerable amount of ethylene during natural senescence, genes for ACC synthase (DC‐ACS3, DC‐ACS1, and DC‐ACS2 in this order regarding the extent of expression) as well as ACC oxidase (DC‐ACO1) are expressed in the gynoecium and ethylene is produced in a sufficient amount to induce the autocatalytic ethylene production in the petals. By contrast, in ‘WC’ flowers, the expression of ACC synthase genes in the gynoecium is limited and only the DC‐ACS3 is slightly expressed resulting in a very low activity of ACC synthase and undetectable ethylene production although the DC‐ACO1 gene is expressed, resulting in the production of ACC oxidase. Acknowledgements We are grateful to Mr K Kanematsu of Daiichi Seeds Co., Tokyo, Japan for supplying cv. White Candle flowers. This study was supported by a Grant‐in‐Aid (No. 14360013) to SS from the Japan Society for the Promotion of Science. View largeDownload slide Fig. 1. Changes in ethylene production from whole flowers, gynoecium, and petals of ‘White Candle (WC)’ (A) and ‘Light Pink Barbara (LPB)’ (B) flowers during natural senescence. Ethylene production from whole flowers, petals, and the gynoecium (ovary plus styles) was examined at a given time of senescence. Day 0, the time of full‐opening of the flowers. Each point is the mean ±SE of three flowers. View largeDownload slide Fig. 1. Changes in ethylene production from whole flowers, gynoecium, and petals of ‘White Candle (WC)’ (A) and ‘Light Pink Barbara (LPB)’ (B) flowers during natural senescence. Ethylene production from whole flowers, petals, and the gynoecium (ovary plus styles) was examined at a given time of senescence. Day 0, the time of full‐opening of the flowers. Each point is the mean ±SE of three flowers. View largeDownload slide Fig. 2. Effects of ethylene treatment on ethylene production from whole flowers (A) and respective tissues of the flower (B), and transcript levels of DC‐ACS1 and DC‐ACO1 genes in the gynoecium (C) and the petals (D) of ‘WC’ and ‘LPB’ flowers. (A) Ethylene production from the whole flowers. Carnation flowers on day 0 were treated with ethylene at 2 µl l–1 for 0–18 h under continuous light. Samples of five flowers were collected at 6 h intervals from the glass chamber. The flowers were held in open air for 1 h prior to measurement of ethylene production to let the ethylene accumulated in flower tissues diffuse out. Each point is the mean ±SE of five flowers. (B) Ethylene production in respective tissues of the flower. Five flowers in another set were separated into the petals, the gynoecium (ovary plus styles) and others (sepals plus stem tissues) and subjected to assay for ethylene production. (C) Transcript levels in the gynoecium. After ethylene measurement, petals and the gynoecium were detached from the five flowers used in (A), combined to make respective samples, and used for the isolation of total RNA. For gel blot analysis, 10 µg of total RNA was applied to each lane and separated by electrophoresis. The blot was hybridized with HRP‐labelled DNA probes several times; first with that for DC‐ACS1, then with that for DC‐ACS2 and that for DC‐ACS3 and, finally, with that for DC‐ACO1, in that order. Uniform loading of total RNAs was confirmed by visualizing ribosomal RNAs by ethidium bromide staining. Since transcripts for DC‐ACS2 and DC‐ACS3 were undetectable by RNA gel blotting, the photographs for the transcripts of DC‐ACS2 and DC‐ACS3 were omitted from the figure. (D) Transcript levels in the petals. View largeDownload slide Fig. 2. Effects of ethylene treatment on ethylene production from whole flowers (A) and respective tissues of the flower (B), and transcript levels of DC‐ACS1 and DC‐ACO1 genes in the gynoecium (C) and the petals (D) of ‘WC’ and ‘LPB’ flowers. (A) Ethylene production from the whole flowers. Carnation flowers on day 0 were treated with ethylene at 2 µl l–1 for 0–18 h under continuous light. Samples of five flowers were collected at 6 h intervals from the glass chamber. The flowers were held in open air for 1 h prior to measurement of ethylene production to let the ethylene accumulated in flower tissues diffuse out. Each point is the mean ±SE of five flowers. (B) Ethylene production in respective tissues of the flower. Five flowers in another set were separated into the petals, the gynoecium (ovary plus styles) and others (sepals plus stem tissues) and subjected to assay for ethylene production. (C) Transcript levels in the gynoecium. After ethylene measurement, petals and the gynoecium were detached from the five flowers used in (A), combined to make respective samples, and used for the isolation of total RNA. For gel blot analysis, 10 µg of total RNA was applied to each lane and separated by electrophoresis. The blot was hybridized with HRP‐labelled DNA probes several times; first with that for DC‐ACS1, then with that for DC‐ACS2 and that for DC‐ACS3 and, finally, with that for DC‐ACO1, in that order. Uniform loading of total RNAs was confirmed by visualizing ribosomal RNAs by ethidium bromide staining. Since transcripts for DC‐ACS2 and DC‐ACS3 were undetectable by RNA gel blotting, the photographs for the transcripts of DC‐ACS2 and DC‐ACS3 were omitted from the figure. (D) Transcript levels in the petals. View largeDownload slide Fig. 3. Ethylene production from the whole flower, gynoecium, and petals of ‘WC’ flowers before and after treatment with ABA (A) or IAA (B). Two sets of three ‘WC’ flowers at the full‐opening stage (day 0) were prepared. One set of the flowers was used immediately (0 h) for the measurement of ethylene production, and another set after treatment with 1 mM ABA or 1 mM IAA for 24 h. Experiments with ABA or IAA treatment were conducted independently. After ethylene production from the whole flower was determined, the flowers were separated into the petals, the gynoecium (ovary plus styles), and others (sepals plus stem tissues), and the former two tissues were subjected to assay for ethylene production. Each value is the mean ±SE of three flowers. View largeDownload slide Fig. 3. Ethylene production from the whole flower, gynoecium, and petals of ‘WC’ flowers before and after treatment with ABA (A) or IAA (B). Two sets of three ‘WC’ flowers at the full‐opening stage (day 0) were prepared. One set of the flowers was used immediately (0 h) for the measurement of ethylene production, and another set after treatment with 1 mM ABA or 1 mM IAA for 24 h. Experiments with ABA or IAA treatment were conducted independently. After ethylene production from the whole flower was determined, the flowers were separated into the petals, the gynoecium (ovary plus styles), and others (sepals plus stem tissues), and the former two tissues were subjected to assay for ethylene production. Each value is the mean ±SE of three flowers. View largeDownload slide Fig. 4. Changes in ethylene production from whole flowers (A) and transcript levels of ACC synthase genes (DC‐ACS1, DC‐ACS2, and DC‐ACS3) and an ACC oxidase gene (DC‐ACO1) in the gynoecium (B) and the petals (C) of ‘WC’ and ‘LPB’ flowers during natural senescence. (A) Ethylene production. Carnation flowers at a given time of senescence were examined for ethylene production from whole flowers. Day 0, the time of full‐opening of the flowers. Each point is the mean ±SE of three flowers. (B) Transcript levels in the gynoecium. After ethylene measurement, the petals and the gynoecium (ovary plus styles) were detached from three flowers, combined to make respective samples, and used for the isolation of total RNA. For RT‐PCR analysis, 500 ng of total RNA was used as the template for making 1st strand cDNA in a 20 µl reaction mixture, and a 0.5 µl portion of the reaction mixture was used for PCR of 30 cycles in a total reaction mixture of 25 µl. The whole amount of PCR products was applied to each lane and separated by electrophoresis. The PCR product of actin (DC‐ACT1) was included to show the uniform sampling of total RNAs and loading of the PCR products. (C) Transcript levels in the petals. View largeDownload slide Fig. 4. Changes in ethylene production from whole flowers (A) and transcript levels of ACC synthase genes (DC‐ACS1, DC‐ACS2, and DC‐ACS3) and an ACC oxidase gene (DC‐ACO1) in the gynoecium (B) and the petals (C) of ‘WC’ and ‘LPB’ flowers during natural senescence. (A) Ethylene production. Carnation flowers at a given time of senescence were examined for ethylene production from whole flowers. Day 0, the time of full‐opening of the flowers. Each point is the mean ±SE of three flowers. (B) Transcript levels in the gynoecium. After ethylene measurement, the petals and the gynoecium (ovary plus styles) were detached from three flowers, combined to make respective samples, and used for the isolation of total RNA. For RT‐PCR analysis, 500 ng of total RNA was used as the template for making 1st strand cDNA in a 20 µl reaction mixture, and a 0.5 µl portion of the reaction mixture was used for PCR of 30 cycles in a total reaction mixture of 25 µl. The whole amount of PCR products was applied to each lane and separated by electrophoresis. The PCR product of actin (DC‐ACT1) was included to show the uniform sampling of total RNAs and loading of the PCR products. (C) Transcript levels in the petals. Table 1. Vase‐life, ethylene production and senescence pattern of carnation cultivars Cultivar  Vase‐lifea (d)  Ethylene productionb (nmol h–1 g–1 fr wt)  Senescence patternc  Nora  5.8±0.4  4.04±0.30  W  Light Pink Barbara  9.6±0.2  1.87±0.12  W  White Candle  18.6±0.4  0.08±0.05  N  Cream Candle   15.8±1.4  0.03±0.03  N  Royal Green  13.4±1.3  0.01±0.03  N  Shion  12.3±0.2  0.03±0.03  N  Le Noir  12.2±1.5  0.00±0.00  N  Cultivar  Vase‐lifea (d)  Ethylene productionb (nmol h–1 g–1 fr wt)  Senescence patternc  Nora  5.8±0.4  4.04±0.30  W  Light Pink Barbara  9.6±0.2  1.87±0.12  W  White Candle  18.6±0.4  0.08±0.05  N  Cream Candle   15.8±1.4  0.03±0.03  N  Royal Green  13.4±1.3  0.01±0.03  N  Shion  12.3±0.2  0.03±0.03  N  Le Noir  12.2±1.5  0.00±0.00  N  a Each value is the mean ±SE of five flowers. b Ethylene production rates are shown by the maximum values observed just before the onset of petal wilting in ‘Nora’ and ‘Light Pink Barbara’, which showed a climacteric rise of ethylene production, but by the maximum values found during the senescence period in the remaining cultivars, which did not show a climacteric rise of ethylene production. c W, in‐rolling and wilting of the petals; N, desiccation, discoloration and necrosis of the petals. 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TI - Repressed ethylene production in the gynoecium of long‐lasting flowers of the carnation ‘White Candle’: role of the gynoecium in carnation flower senescence
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erh081
DA - 2004-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/repressed-ethylene-production-in-the-gynoecium-of-long-lasting-flowers-Ll38fFuNCb
SP - 641
EP - 650
VL - 55
IS - 397
DP - DeepDyve
ER -