原著論文
2,4- ピリジンジカルボン酸のスプレーカーネーション切り花の観賞期間延長作用
2014 年 83 巻 1 号 p. 72-80
詳細
2014 年 83 巻 1 号 p. 72-80
2,4-Pyridinedicarboxylic acid (PDCA) is a structural analog of 2-oxoglutarate and has been shown to inhibit 2-oxoglutarate-dependent dioxygenases by competing with 2-oxoglutarate, and ethylene production in detached carnation flowers by competing with ascorbate on 1-aminocyclopropane-1-carboxylate (ACC) oxidase action. In the present study, the inhibition of ACC oxidase action by PDCA was confirmed with a recombinant enzyme produced in Escherichia coli from carnation DcACO1 cDNA. PDCA had various effects on ethylene production in cut ‘Light Pink Barbara (LPB)’ carnation flowers; ethylene production was accelerated or delayed in some flowers, whereas it did not change in others as compared to untreated control flowers. This varied action of PDCA may be caused by its possible combined actions; that is, inhibition of ACC oxidase action as well as its action on unidentified biochemical processes which use 2-oxoglutarate as a co-substrate, such as the biosynthesis and inactivation of gibberellins. Meanwhile, PDCA treatment significantly prolonged the vase life of bunches of cut ‘LPB’ carnation flowers; the magnitude of the extension of vase life was 53, 111, and 135% at 0.3, 1, and 2 mM PDCA, respectively, as compared with the non-treated control. Also, PDCA lengthened the vase life of ‘Mule’ carnation flowers. The present findings suggest the potential of PDCA as a preservative for cut flowers of spray carnations.
During senescence of carnation flowers, a climacteric increase in ethylene production occurs, and the evolved ethylene induces in-rolling of petals, resulting in wilting of whole flowers. In accordance with a sharp increase in ethylene production, there is a surge in activities of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase (ten Have and Woltering, 1997; Woodson et al., 1992). ACC synthase requires 5′-pyridoxal phosphate (PLP) as an essential cofactor and converts S-adenosyl-l-methionine to ACC, whereas ACC oxidase degrades ACC to ethylene by requiring O2, Fe2+, ascorbic acid, and CO2 (Iturriagagoittia-Bueno et al., 1996; Smith et al., 1992; Yang and Hoffman, 1984).
The effect of ethylene on flower senescence can be diminished by treating flowers with inhibitors of ethylene biosynthesis or action. Treatment with these inhibitors prolongs the vase life of cut ethylene-sensitive ornamental flowers, including carnation. So far, there have been several inhibitors of ethylene biosynthesis, which have been tested in trials or used practically in the flower industry; that is, aminooxyacetic acid (Fujino et al., 1980), aminoethoxyvinylglycine (Baker et al., 1977), 2-aminoisobutyric acid (Onozaki and Yamaguchi, 1992; Satoh and Esahi, 1980, 1983; Serrano et al., 1990), 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (Midoh et al., 1996), and so on. On the other hand, silverthiosulfate complex (STS) blocks the action of ethylene receptor and inhibits autocatalytic ethylene production, leading to prolongation of the vase life of carnation flowers (Altman and Solomos, 1995; Reid et al., 1980; Veen, 1979). Presently, STS is an essential tool for the delay of senescence and preservation of some ornamental flowers that exhibit climacteric ethylene production or are sensitive to ethylene.
2,4-Pyridinedicarboxylic acid (PDCA) is a structural analog of 2-oxoglutarate (OxoGA) and acts as a competitive inhibitor of OxoGA-dependent dioxygenases with respect to OxoGA (Kivirikko and Myllyharju, 1998; Kivirikko and Pihlajaniemi, 1998). Such OxoGA-dependent dioxygenases include proline-4-hydroxylase (Kivirikko and Myllyharju, 1998; Kivirikko and Pihlajaniemi, 1998; Vlad et al., 2010) and enzymes involved in gibberellin biosythesis and metabolism, such as gibberellin 3β-dioxygenase (gibberellin 3β-hydroxylase), gibberellin-44 dioxygenase, gibberellin 2β-dioxygenase (gibberellin 2β-hydroxylase) (Hedden and Kamiya, 1997; Lange et al., 1994a, b; Smith and MacMillan, 1984, 1986).
Iturriagagoittia-Bueno et al. (1996) revealed that OxoGA competitively inhibited ACC oxidase with respect to ascorbate. Vlad et al. (2010) reported that PDCA inhibited ethylene production in detached carnation flowers and delayed senescence of the flowers. Just recently, Fragkostefanakis et al. (2013) proved that PDCA actually inhibited the in vitro activity of ACC oxidase prepared from tomato pericarp tissues. These results suggested that PDCA inhibits ACC oxidase by competing with ascorbate.
In the work of Vlad et al. (2010), control flowers of ‘White Sim’ carnation started to senesce on day 6 and senescence was almost completed on day 7, whereas flowers treated with 2 mM PDCA showed two-phase senescence profiles; that is, 60% of the treated flowers had senesced by 7 days, whereas the remaining 40% senesced gradually until 14 days after the start of treatment. This two-phase senescence profile caused by PDCA treatment appeared to be unusual since PDCA inhibited ethylene production completely (Fig. 3 in Vlad et al., 2010). The authors did not explain why this unusual two-phase senescence had occurred. On the other hand, although PDCA could inhibit ethylene production in detached carnation flowers, it remains unclear whether PDCA could be used practically as a flower preservative to improve the vase life of cut carnations.
We aimed to obtain further knowledge of PDCA action on ethylene production and senescence in carnation flowers, and to examine whether PDCA can be used as a preservative to prolong the vase life of carnation flowers, which had not been dealt with in previous papers. In the present study, we examined the action of PDCA on recombinant ACC oxidase, which was synthesized in Eschericha coli from carnation ACC oxidase cDNA, ethylene production and senescence in detached carnation flowers, and the senescence of bunches of cut carnation flowers.
For the construction of an expression plasmid for carnation DcACO1, the entire coding region with NdeI and BamHI recognition sites at the 5′- and the 3′ ends, respectively, was amplified from DcACO1 cDNA (Kosugi et al., 2000) by PCR with the upstream and down-stream primers, 5′-CCCCCATATGGCAAACATTGTC AACTTCCC-3′ and 5′-CCCCGGATCCTCAAGCAGTT GGAATGGGAC-3′, respectively. The amplified product was digested with NdeI and BamHI. The resultant DcACO1 fragments were inserted into the corresponding site of pET-15b (Novagen/MERK-Japan, Tokyo, Japan).
The constructed plasmid with the DcACO1 coding region was introduced into E. coli BL21(DE3). The E. coli cells were cultured in 0.2 L of LB medium (2 g polypeptone, 1 g yeast extract, and 2 g NaCl in 0.2 L, pH 7) supplemented with 1% glucose at 27°C for 3 h until the A600 reached about 0.5. Thereafter, isopropylthio-β-d-galactoside (IPTG) was added to the culture at 1 mM. The culture was further incubated for 8 h at 27°C, and cells were collected. The soluble extract from cells was subjected to purification with a nickel chelate matrix Protino Ni-IDA 1000 packed columns (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. The obtained DcACO1 protein was checked for purity by SDS-PAGE and detection with dye-staining. Protein contents were determined according to the method of Bradford (1976) with bovine serum albumin (BSA) as a standard.
Assay of ACC oxidase activityACC oxidase activity was assayed by incubating a 50 μL enzyme sample (50 μg protein) with 50 mM (N-morpholino)propanesulfonic acid (MOPS)-NaOH (pH 7.5), 7% glycerol, 3.5 mM dithiothreitol, 25 mM Na-ascorbate, 50 μM FeSO4, 25 mM NaHCO3, l mM ACC and PDCA or 2-oxoglutaric acid (OxoGA) at given concentrations in a total volume of l mL. The reaction was carried out in a 5 mL glass test-tube with a serum rubber cap. After 15 min of incubation at 30°C, a 1 mL head-gas sample was taken and subjected to ethylene analysis with a gas chromatograph (GC-8; Shimadzu, Kyoto, Japan), equipped with an alumina column (operating temperature, 80°C) and a flame ionization detector. Assays of enzyme activities were repeated three times with similar results, but typical results are shown in the text.
Carnation flowersTwo carnation cultivars, Dianthus caryophyllus L. ‘Light Pink Barbara (LPB)’ and ‘Mule’, which belong to the spray type of carnation flowers, were used, and their flowers were harvested in the spring (March–May) of 2013. Flowers at the usual commercial stage of flowering, at which the first flower out of six to eight flower buds on a stem was almost fully open, were harvested with 80-cm long stems at the nursery of a commercial grower in Miyagi Prefecture. The harvested flowers were transported dry to Kyoto Prefectural Institute of Agricultural Biotechnology in Kyoto prefecture the day after harvest. After arrival, they were placed in plastic buckets with their cut stem end in tap water under continuous light from white fluorescent lamps (14 μmol·m−2·s−1 PPFD) at 23°C and 40–70% relative humidity.
Ethylene production and senescence profile of single flowers undergoing senescence as treated with PDCAThe flower-opening process in carnation was separated into 6 stages (Os 1 to Os 6) as described by Harada et al. (2010), and the flower senescence process into 4 stages (Ss 1 to Ss 4) as described previously (Morita et al., 2011). Os 6 is the stage at which their outermost petals had just reached right angles to the stems (just fully-open stage). Ss1 is the last phase of fully-open flowers, and Ss 2 the stage when petal in-rolling and wilting started.
In the experiments with single flowers (Figs. 2 and 3), the second or third flower was detached from the stem when it reached Os 6. This time point was designated as day 0. Carnation flowers with 1-cm stems were detached from stems and their fresh weights were measured. The flowers were placed with their basal ends in 5 mL vials (one flower per vial) containing 2 mL de-ionized water (control) or test solutions, and held for 2 weeks under the conditions described above. The test solutions were 0, 0.3, 1, and 2 mM PDCA (pH was not adjusted). The test solutions were replenished every 5 days after the start of the experiment. Ethylene production was monitored daily by enclosing individual flowers in 150 mL plastic containers for l h at 23°C. A l mL head-gas sample was taken with a hypodermic syringe from the container and assayed for ethylene as described above. Ethylene production rates were normalized by the initial fresh weight of the flowers. Flower senescence stage was monitored daily after the ethylene assay, and flowers that had reached Ss 2 were regarded as senescent flowers that had lost ornamental value.
Change in the ethylene production rate of single ‘Light Pink Barbara’ flowers during senescence as affected by PDCA treatment at the given concentrations. Ethylene production was determined individually using 5 flowers per treatment, and is shown by different symbols (○, □, △, ▽, ◇). Closed circles in the figure show flowers (one flower per dot) that reached Ss 2 on the given day. Open circles in C and D show flowers which did not senesce on day 14. A, 0 mM PDCA (control); B, 0.3 mM PDCA; C, 1 mM PDCA; D, 2 mM PDCA.
Change in the number of fully-open and non-senescent flowers during senescence as affected by PDCA treatment. Data are taken from Figure 2 and shown as a function of the incubation period (day). ●, control; ○, 0.3 mM PDCA; □, 1 mM PDCA; △, 2 mM PDCA.
In the experiment with carnation plants with multiple flowers on a stem (Figs. 4 and 6), the cut flowers were used immediately after they were delivered. For ‘LPB’ carnation, each flower stem was prepared to have five flower buds by removing open flowers and immature tight buds. Stems of cut flowers were trimmed to 60 cm and bunches of 5 stems, each having 5 flower buds (25 buds in total per bunch), were put in 0.9 L glass jars with their stem end in 300 mL of the test solution (one bunch per glass jar). The cut flowers were left under the conditions described above for 24 days, and during this period the test solutions were renewed every week. The test solutions were distilled water (control), 0.3, 1, and 2 mM PDCA. The pH of the test solution was not adjusted and no germicide was added to the solutions. Cut flowers were observed daily to record flower opening and senescence symptoms as described above. Fully-open non-senescent (not wilted and turgid) flowers, which were regarded as flowers ranging from Os 6 to Ss 2, were counted daily and the percentage of these flowers to the total number (25) of initial flower buds per bunch was calculated. The experiment was conducted with 3 replicated bunches, a bunch of flowers per glass jar. The vase life of the cut flowers in days is expressed by the number of days during which the percentage of fully-open non-senescent flowers was 40% or more.
Changes in the percentage of fully-open and non-senescent flowers in cut ‘Light Pink Barbara’ flowers treated continuously with or without PDCA at the given concentrations. This carnation cultivar has multiple flowers on a stem (spray type). The percentage of fully-open flowers (Os 6–Ss 2) was calculated from the number of those flowers to the total number of initial flower buds (25 buds per 5 flowers). Data are from 3 replicates, each with 5 flowers and shown by different symbols (○, △, □), at the given PDCA concentrations. A, 0 mM PDCA (control); B, 0.3 mM PDCA; C, 1 mM PDCA; D, 2 mM PDCA.
Changes in the percentage of fully-open and non-senescent flowers in cut ‘Mule’ flowers treated with 0 (control; A) or 2 mM PDCA (B). Two bunches each, with 24 and 26 initial buds per bunch for the control and 25 and 30 initial buds per bunch for PDCA treatment, were used for the respective treatment. Different symbols (○, □) show individual bunches. The percentage of fully-open and non-senescent flowers was obtained as described in the legend to Figure 4.
The experiment with ‘Mule’ carnation was conducted similarly to that with ‘LPB’ carnation but with slight modification. PDCA treatment was carried out at 0 mM (control) and 2 mM for 28 days. The control consisted of 2 bunches of 5 flower stems, each bunch having 24 or 26 initial buds, and PDCA treatment was the same, but with 25 or 30 initial buds. Because of the low percentages of fully-open and non-senescent flowers in this cultivar, we redefined the vase life of flowers as the period when 20% or more flowers were fully open and non-senescent.
Statistical analysesStatistical analyses were carried out by Dunnett’s (Fig. 1) or Williams’ (Table 1) multiple range tests using an on-line statistical analysis program MEPHAS (http://www.gen-info.osaka-u.ac.jp/testdocs/tomocom/, October 16, 2013).
Inhibition of ACC oxidase activity by 2,4-pyridinedicarboxylic acid (PDCA) and 2-oxoglutaric acid (OxoGA). ACC oxidase was prepared by the expression of carnation DcACO1 cDNA in E. coli. ACC oxidase activity was determined at l mM ACC in the absence or presence of OxoGA or PDCA at the given concentrations. Data are the means ± SE of triplicate determinations. * shows significant difference, whereas NS is not significant, from the control (0 mM) by Dunnett’s test (p < 0.05).
Comparison of vase life among cut ‘Light Pink Barbara’ flowers treated with PDCA.
The recombinant ACC oxidase expressed from a carnation ACC oxidase cDNA, DcACO1, had a Km of 118 μM for ACC. This Km value was comparable to that reported previously for ACC oxidase from carnation petals; 30–425 μM depending on CO2 concentration (Nijenhuis-de Vries et al., 1994) and 111–125 μM in the presence of NaHCO3 (Kosugi et al., 1997).
Figure 1 shows the inhibitory effects of PDCA and OxoGA on the activity of the recombinant ACC oxidase. PDCA inhibited enzyme activity by 47% at 0.5 mM, and the magnitude of inhibition increased gradually as its concentration increased, attaining 85% inhibition at 10 mM. OxoGA tended to inhibit enzyme activity until 5 mM, and significantly inhibited it by 51% at 10 mM. PDCA was stronger than OxoGA in the inhibition of carnation recombinant ACC oxidase. The present results confirmed that PDCA inhibited the activity of ACC oxidase, which was produced in E. coli from DcACO1 cDNA. Iturriagagoittia-Bueno et al. (1996) showed that OxoGA inhibited ACC oxidase by competing with ascorbate. PDCA was shown to compete with OxoGA in the inhibition of vertebrate collagen proline-4-hydroxylase, an OxoGA-dependent dioxygenase (Kivirikko and Myllyharju, 1998; Kivirikko and Pihlajaniemi, 1998). Therefore, it is speculated that PDCA inhibits ACC oxidase activity by competing with ascorbate, although there is a possibility that PDCA inhibited ACC oxidase activity by chelating Fe2+ (Smith et al., 1992).
Effect of PDCA on ethylene production and senescence of single detached senescing flowerThen we examined the ethylene production from carnation flowers treated without or with PDCA at the given concentrations (Fig. 2). Five flowers each were used for the respective treatments. In four control flowers, ethylene production started on day 5, reached maximum, ranging from 0.89 to 2.18 nmol·h−1·g−1 FW, on day 6, and rapidly declined on day 7, but it was delayed by one day in the remaining one out of the tested 5 flowers. By treatment with 0.3 mM PDCA, ethylene production started on day 4 and attained the maximum on day 5 in three flowers, whereas it was delayed by 2 days in another flower, and little ethylene production was detected in the remaining flower for 14 days. In 4 out of 5 flowers treated with 1 mM PDCA, ethylene production started on day 5 or day 6, and reached maxima the next day, similarly to that in the control. On the other hand, no ethylene production was detected in the remaining flower treated with 1 mM PDCA for 14 days. The maximum ethylene production of 5 flowers treated with 1 mM PDCA was 0.88 ± 0.26 nmol·h−1·g−1 FW, whereas that of 5 control flowers was 1.56 ± 0.22 nmol·h−1·g−1 FW. Ethylene production tended to be less in the flowers treated with 1 mM PDCA than in the control flowers, although the difference was not significant by Student’s t-test at p < 0.05. In flowers treated with 2 mM PDCA, ethylene production varied with time; that is, ethylene production peaked on day 3, 5, 8, and 11 for four flowers, respectively, whereas no ethylene production was observed in the remaining flower during 14 days.
Vlad et al. (2010) showed that PDCA at 2 mM completely inhibited ethylene production in detached flowers of ‘White Sim’ carnation, but their data showed only one flower each for the control and PDCA treatment. The present experiment confirmed that complete inhibition of ethylene production by PDCA treatment occurred in some flowers by treatment with 1 and 2 mM PDCA.
In Figure 3, the time course of flower senescence is shown by the decrease of non-senescent flowers, which was determined from the flowers used for the assay of ethylene production in Figure 2. Closed circles in Figure 2 indicate the individual flowers senesced, that is, flowers reached Ss 2 by the given time, because carnation flowers before Ss 2 are acceptable for display. It was shown that the onset of petal in-rolling leading to flower wilting usually starts almost simultaneously or just one day after the peak of ethylene production (Kosugi et al., 2002; Nukui et al., 2004; Onoue et al., 2000). In the control flowers, senescence started on day 6 and was completed on day 9. In PDCA-treated flowers, senescence started on day 5 or day 6 and progressed until the next day depending on the concentration of PDCA, but it was not completed. Three out of 5 flowers treated with 2 mM PDCA remained sound on day 6 and gradually decreased to one flower on day 14, and one flower each remained sound until day 13 or day 14 in the flowers treated with 0.3 or 1 mM PDCA, respectively.
This overall profile of senescence in the present study was similar to the two-phase senescence profile reported by Vlad et al. (2010), who showed that treatment of ‘White Sim’ flowers with 2 mM PDCA caused senescence of 60% of flowers by 7 days, whereas the remaining 40% senesced gradually until 14 days. The reason why PDCA caused two-phase senescence in carnation flowers is unclear, but it might have been caused, as discussed later, by side effects of PDCA on some OxoGA-dependent dioxygenases involved in important biochemical processes such as gibberellin (GA) biosynthesis and metabolism.
Determination of the vase life by observing the number of open flowersTo examine the effect of PDCA treatment on the vase life of cut carnation flowers, we employed a method to determine the vase life of spray carnation flowers by observing the change in the percentage of open flowers to the total number of initial flower buds (Satoh et al., 2005). Figure 4 shows the changes in the percentage of open flowers in cut ‘LPB’ carnation flowers treated continuously with 0 (control), 0.3, 1, and 2 mM PDCA. In the control flowers, the number of fully-open flowers increased rapidly from day 3 of incubation and reached maximum on day 8–9 (72–88% flowers were fully open depending on bunches), then declined rapidly. Similarly, flowers treated with 0.3 mM PDCA attained the maximum percentage (84–88%) of fully-open flowers on day 8–9, but thereafter exhibited the delay of decrease in the percentage of fully-open and non-senescent flowers. Flowers treated with 1 mM PDCA attained the maximum percentage of fully-open flowers of 88–100% on day 9, and with 2 mM PDCA, 88–92% on day 9. The decrease in the percentage of fully-open flowers was delayed in flowers treated with 1 mM PDCA and, especially with 2 mM PDCA. Moreover, treatment with 1 and 2 mM PDCA seemed to accelerate flower opening from day 4 to day 9 as compared with the control.
Figure 5 shows flower opening profiles of the control and flowers treated with 2 mM PDCA, which were chosen as typical specimens out of 3 replicates, respectively, 15 days after the start of the experiment. The control flowers showed in-rolling and wilting of petals, which are typical symptoms of senescence induced by ethylene. On the other hand, in the flowers treated with PDCA, a small number of flowers senesced similarly to the control flowers in the early phase of senescence from day 10 to day 15, whereas thereafter, especially after 20 days in the case of flowers treated with 2 mM PDCA, the flowers withered with browning at the petal margins as well as with jumbled, but turgid, fading petals. These senescence symptoms were typical of senescence in carnation flowers and occurred independent of ethylene action (Otsu et al., 2007; Satoh, 2011).
Flower-opening profiles of cut ‘Light Pink Barbara’ flowers treated with 0 (control) or 2 mM PDCA. Typical profiles for each treatment out of 3 replicated bunches were chosen 15 days after the start of the experiment shown in Figure 4, and photographed. (See online article for color version of the figure.)
The vase life of cut flowers was defined as the period when 40% or more flowers were fully open and non-senescent (Satoh et al., 2005), and was obtained for respective bunches of treated flowers from Figure 4A–D, and the data are summarized in Table 1. Vase life was 8.3 days for the control, and 12.7, 17.5, and 19.5 days for the flowers treated with 0.3, 1, and 2 mM PDCA, respectively. Therefore, the vase life of ‘LPB’ carnation flowers was significantly lengthened by 53, 111, and 135% by treatment with 0.3, 1, and 2 mM PDCA in this order.
Figure 6 shows the changes in the percentage of open flowers in cut ‘Mule’ carnation flowers. In this experiment, ca. 25% of the initial buds were already fully open and non-senescent on day 0, and the percentage of fully-open and non-senescent flowers increased rapidly until day 4 both in the control and PDCA-treated flowers. In the control, the average percentage rapidly declined to 26% on day 7 and 12% on day 9, and then remained minimal until day 28. However, in the PDCA-treated flowers the percentage gradually increased to day 12, remained at that level to day 16, then declined to less than 10% on day 28. The maximum average percentages were 46% on day 4 in the control and 58% on day 16 in the PDCA-treated flowers. These relatively low maximum percentages in ‘Mule’ flowers, as compared with those obtained with ‘LPB’ flowers (Fig. 4), could be caused by the low vigor to open fully in cut flowers as well as the presence of some tight buds, which did not open during the experiment, in bunches of these flowers. As described in Materials and Methods, the vase life of ‘Mule’ flowers was redefined as the period when 20% or more flowers were fully open and non-senescent. Vase life was determined as 7 and 9 days for the control, and 23 and 25 days for the PDCA-treated flowers. Figure 7 shows flower-opening profiles of the control and PDCA-treated flowers 11 days after the start of the experiment.
Flower-opening profiles of cut ‘Mule’ flowers treated with 0 (control) or 2 mM PDCA. Typical profiles for each treatment out of duplicated bunches were chosen 11 days after the start of the experiment shown in Figure 6, and photographed. (See online article for color version of the figure.)
In the present study, we tried to determine the effect of PDCA on the vase life of cut carnation flowers in the spray category of flowering by observing the number of open flowers, i.e., the percentage of open flowers to the total number of initial flower buds. The vase life determined by this method was shown to be similar to that determined by measuring ethylene production and observing senescence symptoms of carnation flowers (Satoh et al., 2005). PDCA extended the vase life of cut carnation flowers, which had 60-cm stems and were in bunches, as shown in Figures 4 and 6, and Table 1. The increase in numbers of fully-open and non-senescent flowers may have been caused by both acceleration of flower (bud) opening and delayed senescence of fully-open flowers. The acceleration of flower opening was suggested by the data shown in Figure 4C and D, in which PDCA at 1 and 2 mM tended to accelerate flower opening, and by those in Figure 6. The delayed senescence of fully-open flowers was probably caused by the inhibition of flower senescence caused by ethylene produced by the flowers themselves, since PDCA was actually shown to inhibit the in vitro action of ACC oxidase (Fig. 1).
However, with detached ‘LPB’ flowers with a 1-cm stem, ethylene production and the delay of flower senescence were unusual (Figs. 2 and 3), and different from the expected results if PDCA had only inhibitory action on ACC oxidase. Saks et al. (1992) and Saks and van Staden (1993a, b) showed that gibberellin A3 (GA3) delayed the senescence of cut carnation flowers when applied exogenously via the stem to flowers at pre- and fully-open stages, by repressing the climacteric rise of ethylene production. Moreover, they showed that pacrobutrazol, an inhibitor of GA biosynthesis, prevented buds from opening fully and shortened the vase life of partially open flowers. These previous investigations suggested the involvement of gibberellins (GAs) in the flower opening and senescence of carnation flowers. Apart from its direct action on ethylene biosynthesis through the inhibition of ACC oxidase, PDCA might affect ethylene production in carnation flowers indirectly through the modulation of OxoGA-dependent dioxygenases, those responsible for GA biosynthesis and metabolism, such as GA 3β-dioxygenase (GA 3β-hydroxylase), GA-44 dioxygenase, GA 2β-dioxygenase (GA 2β-hydroxylase) (Hedden and Kamiya, 1997; Lange et al., 1994a, b; Smith and MacMillan, 1984, 1986). The inhibition by PDCA of GA 2β-dioxygenase, which converts active GA to the inactive form, may maintain GA content high, resulting in the extension of the vase life of carnation flowers. It is necessary in the near future to determine the internal GA content in PDCA-treated flowers and correlate GA to ethylene production and senescence in carnation flowers. This indirect action may be the cause of its unusual action on ethylene production and senescence of carnation flowers with short stems (Fig. 2) although its main action seems to be the inhibition of ACC oxidase.
The present study showed that PDCA could prolong the vase life of spray carnations (Fig. 4; Table 1), which have several buds and flowers on a stem, but not that of carnations which have a single flower on a stem (Fig. 2). The promotive effect of PDCA on the vase life could be dependent on the stage(s) of flowers and it was probably restricted only to buds. Cut flowers of spray-type carnations had buds on a stem at the start of the experiment. Such buds eventually opened later in the experiment and PDCA might prolong their vase life. Thus, the total number of days of the vase life of each flower on a stem was larger for a stem treated with PDCA, which could be attributable to the promotive effects of PDCA on buds ab initio. In overall, the present study suggested that PDCA will be used as a preservative for cut flowers of spray carnations.
In cut flowers of tulip, stem growth during display time would destroy the ornamental value of the flowers. As discussed above, OxoGA acts as a co-substrate for dioxgenases involved in GA biosynthesis, such as GA 20-oxidase and GA 3-oxidase (Hedden and Kamiya, 1997; Lange et al., 1994a, b). Therefore, PDCA and its analogs may be used for the repression of stem growth by inhibiting GA biosynthesis, which remains an interesting research area for the future. It remains to be elucidated whether PDCA would prolong the vase life of cut flowers of ornamental species other than carnation.
