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Exogenous mannitol treatment stimulates bud development and extends vase life of cut snapdragon flowers
Postharvest Biology and Technology 113 (2016) 20–28
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Exogenous mannitol treatment stimulates bud development and extends vase life of cut snapdragon flowers Kazuo Ichimura* , Satoshi Yoshioka, Tetsuya Yamada1 NARO Institute of Floricultural Science, Fujimoto, Tsukuba, Ibaraki 305-8519, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 April 2015 Received in revised form 26 October 2015 Accepted 28 October 2015 Available online xxx
With a view to understanding the role of soluble carbohydrates in flower development and vase life, cut snapdragon (Antirrhinum majus L.) cv. Yellow Butterfly spikes were treated with glucose, sucrose, sorbitol and mannitol, which is a major carbohydrate in them. Treatment with 10–500 mM mannitol markedly promoted flower bud development and stem growth of cut snapdragon. Stem growth accompanied with bud development was not observed in 250 mM glucose, sucrose and sorbitol treatments. Mannitol treatment extended the overall vase life of cut snapdragons more than the other carbohydrate due to the promotion of flower opening at upper spike part. A pulse-chase experiment with 14C-glucose, 14C-sucrose or 14C-mannitol showed that mannitol was metabolized slower than glucose and sucrose, suggesting that the different effects of carbohydrates on flower opening and stem growth may be due to different ability to be metabolized. The dry weight of flowers was greater in sucrose- or glucose-treated spikes than in mannitol-treated spikes, but the dry weight of upper stem parts including spike tips was gradually increased by mannitol treatment, suggesting that marked stem growth is due to applied mannitol. This explanation is supported by a tracer experiment with 14C-carbohydrates showing that accumulation of 14 C in spike tips was greater in the mannitol treatment than in the glucose treatment. Flow cytometry revealed that degradation of nuclei, a parameter of programmed cell death (PCD), was promoted by sucrose, glucose, or sorbitol treatment, but was suppressed by the mannitol treatment. Carbohydrate concentrations in spike tips were markedly increased by glucose, sucrose, and sorbitol treatments, but were only slightly increased by the mannitol treatment. Water potential and osmotic potential in the spike tips decreased rapidly with the sucrose or sorbitol treatments, but were only modestly decreased by the mannitol treatment. The results suggest that mannitol suppressed a decrease in osmotic potential of spike tips, resulting in the continuation of bud development. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Antirrhinum majus Fructose Glucose Osmotic potential Sorbitol Stem growth Sucrose
1. Introduction Cut flowers are usually placed under conditions below the light compensation point for photosynthesis, and thus, they do not assimilate much carbon by photosynthesis, and thereby lack reserve carbohydrate. Treating with the ubiquitous metabolic sugars glucose, fructose, or sucrose, and supplementing with antimicrobial compounds, extends the vase life of many cut flowers, including carnation (Paulin and Jamain, 1982), rose (Kuiper et al., 1995; Ichimura et al., 2006), and sweet pea (Ichimura and Hiraya, 1999). These metabolic sugars have generally been considered to be similarly effective in extending the vase life of cut
* Corresponding author. Fax: +81 29 838 6841. E-mail address: [email protected] (K. Ichimura). 1 Present address: Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. http://dx.doi.org/10.1016/j.postharvbio.2015.10.017 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
flowers (Halevy and Mayak, 1981). However, Ketsa and Boonrote (1990) reported that glucose extended the vase life of cut Dendrobium flowers more than sucrose. Similarly, continuous treatment with glucose or fructose was found to be more effective in extending the vase life of cut rose flowers than continuous treatment with sucrose (Ichimura et al., 2006). These findings indicate that exogenous sugars affect the vase life of cut flowers differently. Other than these ubiquitous metabolic sugars, the effects of some soluble carbohydrates occurring in plants on their flower opening and vase life have been studied. In rose, myo-inositol, which is present generally in higher plants (Anderson and Wolter, 1966; Loewus and Dickinson, 1982), is a major carbohydrate in leaves, whereas xylose and methyl glucoside are minor sugar constituents (Ichimura et al., 1997). Treatments with methyl glucoside and xylose promoted flower opening, but treatment with myo-inositol inhibited opening of cut rose flowers (Ichimura et al., 1999a,b). Mannitol is the major carbohydrate in Delphinium
K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
petals were counted as open flowers. Flowers were considered to have terminated their life when the petals had wilted or abscised. The length of the flower spike was measured every 5 days. When stem length, bud number, and open flower number did not further increase, the measurements were completed. The total number of flower buds was scored 30 days after the start of treatment when the stem growth had completely ceased. Under a stereoscopic microscope, flower spike tips were dissected, and the buds with bracts were scored and included in the total number of flower buds.
60
A
Spike length
55 Spike length (cm)
(Ichimura et al., 2000). Exogenous mannitol reduces sensitivity to ethylene, delays climacteric-like increases in ethylene production, and suppresses sepal abscission in cut Delphinium. These actions are observed with glucose, but are not observed with 3-o-methylglucoside, a non-metabolizable sugar (Ichimura et al., 2000). Snapdragon is an important cut flower because of its wide range of petal colors and good fragrance. Snapdragons have an indeterminate inflorescence with flower buds that open from the stem base to the apex. However, the vase life of cut snapdragons is relatively short and limited by flower abscission, wilting, or failure of buds to fully open (Larsen and Scholes, 1966; Wang et al., 1977; Nowak, 1981). Most snapdragon cultivars are sensitive to ethylene-induced flower abscission and wilting (Ichimura et al., 2008), but pulse treatment with silver thiosulfate complex (STS), an inhibitor of ethylene action, does not markedly extend the vase life of cut snapdragons (Ichimura et al., 2008; Nowak, 1981). In contrast, continuous treatments with sucrose and glucose markedly promote flower opening, thereby extending the overall vase life of cut snapdragon spikes (Ichimura and Hisamatsu, 1999, 2006). In addition to sucrose, analysis by high performance liquid chromatography (HPLC) detected glucose, fructose, and mannitol in snapdragon petals (Ichimura and Hisamatsu, 1999) as was reported in snapdragon leaves by Moore et al. (1997). Identification of mannitol was confirmed by Nuclear Magnetic Resonance analysis (Ichimura et al., 2005). Although treatment with mannitol somewhat extends the vase life of cut snapdragons, this treatment induces marked stem growth (Ichimura et al., 2005). In the present study, we investigated the effects of various carbohydrates including mannitol on the vase life of cut snapdragon flowers. To clarify how mannitol promotes bud development and stem growth, we investigated the accumulation and metabolism of soluble carbohydrates as well as water and osmotic potential of spike tips.
21
0 mM 1 mM 10 mM 100 mM 250 mM 500 mM
50 45 40 35 30 25 0
10
20
30
B
Bud number
40
2.1. Plant material Snapdragon (Antirrhinum majus L.) cv. Yellow Butterfly was grown under natural day-length conditions in a greenhouse (15 C minimum and 25 C maximum temperature). Flower spikes in which the most developed flower bud (3 cm in length) was expected to open the next day, were harvested in the morning and placed in tap water. They were immediately transported to a laboratory and used for experiments within 1 h.
2.3. Measurements of flower bud number and spike length The number of visible buds longer than 5 mm and open, wilted, and abscised flowers was scored daily. Flowers with fully unfolded
35 30 25 20 15 0
10
20
30
40
50
10
C
0 mM 1 mM 10 mM 100 mM 250 mM 500 mM
Open flower number Open flower number
2.2. Treatments with soluble carbohydrates The flower spikes were recut to 30 cm and individually placed in a 300-mL glass vessel containing 250 mL of carbohydrate solution. In the other experiments, three flower spikes were placed in a 500mL glass vessel containing 500 mL of carbohydrate solution. Mannitol concentration was set at 1, 10, 100, 250, or 500 mM, whereas glucose, sucrose, or sorbitol concentration was set at 250 mM because in a preliminary experiment, the most pronounced effect in mannitol treatment was observed at this concentration. All solutions including the control were supplemented with 200 mg L 1 8-hydroxyquinoline sulfate (8-HQS) to inhibit microbial proliferation. The spikes were kept at 23 C, 70% relative humidity, and 12-h light at 10 mmol m 2 s 1 from coolwhite fluorescence lamps.
50
0 mM 1 mM 10 mM 100 mM 250 mM 500 mM
Bud number
45
2. Materials and methods
40
50
8 6 4 2 0 0
10
20
30
40
50
Time (days) Fig. 1. Spike length (A), bud number (B), and open flower number (C) of cut snapdragons treated with mannitol at different concentrations. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. The number of flower buds was the sum of the flower buds longer than 5 mm, open flowers, and wilted flowers. Values are the mean of 8 replicates SE (A and B) or +SE (C).
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2.4. Determination of soluble carbohydrate concentration
2.7. Measurement of water and osmotic potential Spike tips of 1 cm length were cut from the flower spike. For measurement of osmotic potential, tips were placed in a vessel and kept at 30 C for 2 days. The vessel was then allowed to reach room temperature and the osmotic potential of the spike tips was measured using a cyclometer (Tru Psi, Decagon Devices, Pullman, Washington, USA) according to the manufacturer’s instructions. For measurement of water potential, tips were placed in the vessel without freezing and measured as described above. 2.8. Metabolism of glucose, sucrose and mannitol Stems below the position where first floret bud was generated were cut into disks with length of 5 mm. Disks (approximately 500 mg) were placed into a solution containing 250 mM
Spike length (cm)
50 Control Glucose Sucrose Mannitol Sorbitol
45 40 35 30 25 0
B
10
20
30
40
50
Bud number
45 40
Flower spikes were divided into four portions: basal 30-cm stem, flowers from the basal 30-cm stem, the remaining upper stem that was a newly grown portion after harvest, and flowers from the remaining upper stem. The tissues were dried at 80 C in an oven for three days and their dry weight (DW) was measured. Three spikes were used in each experiment, and experiments were repeated three times.
Control Glucose Sucrose Mannitol Sorbitol
35 30 25 20 15 0
10
20
30
40
16
C
Open flower number
14 Open flower number
Spike tips of 1 cm length were chopped, using a razor blade, in nuclear extraction buffer obtained from the High Resolution Kit for Plant DNA (Partec, Münster, Germany) provided by the manufacturer of the flow cytometer. The extraction buffer was a low pH solution containing Triton X-100. The extract was filtered through a 50-mm nylon mesh and the medium with the isolated nuclei was collected. A buffer with the fluorescent DNA stain 4,6-diamidino2-phenylindole (DAPI), also part of the standard reagent set (Partec) was added, and the solution was vortexed. The DNA content of the isolated nuclei was analyzed using a flow cytometer (Ploidy Analyzer, Partec). DNA levels were obtained from a total of 5000 nuclei. On the basis of histograms obtained from cytometry, the ratio of nuclei with decreased DNA was calculated as described in Yamada et al. (2006). Although it is unclear whether DNA masses detected by flow cytometry (FCM) have functions as nuclei, DNA masses were regarded as nuclei in the present study.
Spike length
55
2.5. Measurement of dry weight
2.6. Flow cytometry
60
A
Bud number
Spike tips of 1 cm length were collected and examined for the concentration of soluble carbohydrates. The organs were cut into small pieces and immersed in 10 mL of 80% ethanol at 75 C for 30 min. After cooling, 25 mL of 10% (w/v) galactose was added to the extract as an internal standard. The samples were then homogenized. After centrifugation, the residue was suspended in 5 mL of 80% ethanol and centrifuged twice. The three supernatants were combined and evaporated to dryness in vacuo below 50 C, and the residue was dissolved in 1 mL water. The solution was then passed through a Sep-Pak C-18 cartridge, followed by 2 mL water. An aliquot of the eluate was separated using an HPLC system (Jasco, Tokyo, Japan) equipped with a refractive index detector on a Shodex SUGAR SP0810 column (Showa Denko, Tokyo, Japan). The column was held at 80 C and water was eluted at a flow rate of 0.8 mL min 1. The peak identity was confirmed using authentic carbohydrates and the peak area was determined using an integrator. The concentration of each carbohydrate in the sample was calculated as previously described (Ichimura et al., 1999a).
Control Glucose Sucrose Mannitol Sorbitol
12 10 8 6 4 2 0 0
10
20
30
40
Time (days) Fig. 2. Spike length (A), bud number (B) and open flower number (C) of cut snapdragons treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. The number of flower buds was the sum of the flower buds longer than 5 mm, open flowers, and wilted flowers. Values are the mean of 8 replicates SE (A and B) or +SE (C).
14 C-glucose, 14C-sucrose or 14C-mannitol solution (61.79 kBq, 37 MBq mmol 1) in a Petri dish and kept at 23 C under darkness for 1 h. The disks were then transferred into 250 mM unlabeled glucose, sucrose, and mannitol solutions for 0, 6, 12 and 24 h and then immersed in 80% ethanol. The discs were homogenized in 80% ethanol and centrifuged. The resulting supernatant was combined and evaporated to dryness in vacuo below 50 C. The residue was dissolved in water and separated by HPLC equipped with a radioanalyzer (RLC-113, Aloka, Tokyo, Japan). The column and separation conditions were as described above.
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23
Fig. 3. Photographs of cut snapdragon flowers treated with various carbohydrates at 250 mM. All solutions including the control contained 200 mg L 1 8-HQS. Cut flower spikes treated with 250 mM glucose, sucrose, sorbitol, or mannitol at 23 C for 14 days (A). Far left, control; middle left, glucose; middle, sucrose; middle right, sorbitol; far right, mannitol. Cut flower spikes treated with 250 mM mannitol at 23 C for 25 days (B). Upper part of cut flower spikes treated with 250 mM mannitol at 23 C for 27 days (C).
Table 1 Effects of mannitol at various concentrations on the wilting of spike tips. Concentration (mM)
Time to wilting (days)
0 1 10 100 250 500
14.3 1.0 15.5 0.3 24.6 1.1*** 39.4 2.1*** 38.3 1.8*** 34.7 1.0***
Values are the mean of 8 spikes standard errors, and those with *** are significant at P < 0.001, compared with the control by the Dunnett’s test.
2.9. Distribution of carbon derived from
14
C-glucose or
14
C-mannitol
Cut spikes were held in 0.25 M glucose or mannitol solution for ten days as described above. The cut ends of spikes were 14 14 transferred into C-glucose or C-mannitol solution (23.135 kBq, 185 MBq mmol 1) for 1 h. The spikes were then transferred in to 250 mM glucose or 250 mM mannitol solution and kept at 23 C for 24 h in darkness. The spikes were divided into five portions: basal stem from cut end to 30 cm, flowers generated on the basal stem, remaining upper stem, flowers on remaining upper stem, and tips with length of 1 cm. These portions were dried at 80 C for more than 3 days. The dried samples were ground and 20 mg subsample was oxidized and dissolved in a liquid scintillator with a sample combustion system (ASC-113, Aloka).
The radioactivity of the solution was measured with a liquid scintillation counter (LSC-3500, Aloka). 2.10. Carbohydrate translocation Determination of carbohydrate translocation was based on the method of Gilbert et al. (1997). Flower stems with leaves were cut from the plants. To remove the upper parts with flowers and flower buds, the stems were re-cut to 20 cm and the cut ends of the stems were put into distilled water. The stems were held at 25 C under 250 mmol m 2 s 1 cool-white fluorescence lamps for about 1 h, and then individual leaves were cut from the stems and the cut ends were put into distilled water and placed in a 27-L acrylic chamber. Then, 14C-sodium bicarbonate (NaH14CO3) (9.25 MBq, 74 MBq mmol 1) was dissolved in water and lactic acid was added to the solution to evolve 14CO2. Leaves were allowed to photosynthesize for 30 min, after which the cut ends were placed in 20 mM EDTA solution and held at 20 C in darkness for 3 h. The resulting solutions were evaporated to dryness in vacuo below 50 C. The residue was dissolved in distilled water and separated with HPLC equipped with the radio-analyzer as described above. 2.11. Statistical analysis Data were analyzed by Tukey–Kramer multiple range test, Dunnett’s test, and Fisher’s Least Significant Difference test using KaleidaGraph software (v.4.1, Hulinks, Tokyo, Japan).
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3. Results
0.30 Dry weight (g/portion)
A
Upper stem
Control Glucose Sucrose Mannitol Sorbitol
0.25 0.20
3.1. Effects of mannitol at various concentrations on stem length and the number of total buds and open flowers
0.15 0.10 0.05 0.00 0
5
10
3.0 Dry weight (g/portion)
B
Control Glucose Sucrose Mannitol Sorbitol
2.5 2.0
15
20
25
Flowers on upper stem
1.5 1.0
3.2. Effects of various carbohydrates on stem length and the number of total buds and open flowers
0.5 0.0 0
5
10
15
3.0 Dry weight (g/portion)
C
2.0
25
Basal stem
Control Glucose Sucrose Mannitol Sorbitol
2.5
20
1.5 1.0 0.5 0.0 0 3.0
D
5
10
15
20
25
Control Flowers on basal stem Glucose Sucrose Mannitol Sorbitol
2.5 Dry weight (g/portion)
Mannitol at 10 mM or higher concentrations delayed the wilting of spike tips and stimulated stem growth, and the optimal concentration was 250 mM (Table 1, Fig. 1A). The stimulation of stem growth was accompanied by an increase in the number of buds. In control flower spikes, several flowers opened within the first few days (Fig. 1C). Mannitol at 1 and 10 mM slightly affected flower opening, whereas mannitol at 100 mM or higher concentrations markedly promoted flower opening. Mannitol at 100 mM or higher concentrations caused abortion of buds on about the 15th node from the base of spikes, but flowers on the upper part of the spikes opened almost fully. The time from harvest to the time when open flowers without wilting were not observed in each flower spike was 11.8 0.4 days at 0 mM, 11.8 0.6 days at 1 mM, 14.3 0.6 days at 10 mM, 27.8 1.5 days at 100 mM, 31.0 1.0 days at 200 mM, and 28.4 2.0 days at 500 mM (means of eight replicates SE).
2.0 1.5 1.0 0.5 0.0 0
5
10
15
20
25
Time (days) Fig. 4. Dry weight of upper stem (A), flowers on upper stem (B), basal stem (C), and flowers on basal stem (D) in cut snapdragon flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Values are the mean of 3 replicates SE.
In glucose and sucrose treatments, a considerable increase in spike length and an increase in bud number was only observed during the first 5 days; thereafter, spike length and bud number increased slightly (Figs. 2 and 3). In the sorbitol treatment, spike length and bud number slightly increased, suggesting that sorbitol treatment inhibits bud development. In contrast, in the mannitol treatment the spike length increased linearly until the 20th day, accompanied by a marked increase in bud number. Browning of spike tips, a senescent symptom, was not observed until about the 30th day in mannitol-treated spikes (Table 2). The treatments with glucose and sucrose promoted flower opening (Figs. 2 and 3), although the glucose treatment caused browning of the spike tips one week after the start of the treatment (personal observation), after which bud number did not increase. Treatment with sorbitol promoted flower opening. Flower buds at the middle part of the spikes aborted. Flower buds on the upper part opened, but were smaller than those on the basal part of the spikes (Fig. 3). However, spikes treated with mannitol excreted a white powder on the surface of flowers (Fig. 3), which was confirmed to be mannitol by HPLC analysis. The time from harvest to the time when open flowers without wilting were not observed in each flower spike was 11.0 0.4 days in the control, and 22.7 0.8, 15.4 0.4, 18.2 0.6, and 29.8 1.3 days (mean of eight replicates SE) in the spikes treated with glucose, sucrose, sorbitol, and mannitol, respectively. This indicates that mannitol treatment extended vase life of cut snapdragons more than the other carbohydrate treatments. We examined the total number of flower buds at the 30th day using a stereoscopic microscope to clarify whether development of flower buds was promoted by mannitol treatment. The total number of flower buds was the highest in the spikes treated with mannitol (Table 3). 3.3. Metabolism of glucose, sucrose and mannitol After labeling, radioactivity of 14C-glucose and 14C-sucrose were decreased to 14.3 1.6 and 14.0 0.5% of applied radioactivities, respectively, but that of 14C-mannitol was decreased to 21.1 2.9% of applied radioactivity (mean of three replicates SE). The differences between mannitol and glucose or sucrose were
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25
14
1.2e+6 Glucose Mannitol
Radioactivity (dpm)
1.0e+6 25000
8.0e+5
20000 15000
6.0e+5
10000
3.6. Degradation of nuclei in the tips of spikes treated with various carbohydrates
5000
4.0e+5
C-glucose and 14C-mannitol. Accumulation of 14C in flowers at both parts was greater in the glucose treatment than in the mannitol treatment (Fig. 5), although accumulation of 14C in the tip was greater in the mannitol treatment than in the glucose treatment, suggesting that distribution of carbon differed depending on the applied carbohydrate.
0
2.0e+5 0.0 B. Stem B. Flower U. Stem U. Flower
Tip
Fig. 5. Accumulation of 14C derived from 14C-glucose and 14C-mannitol in basal stem (B. Stem), flowers on basal stem (B. Flower), upper stem (U. Stem), flowers on upper stem (U. Flower) and tip of cut snapdragon flowers. The inset shows the data of tip. Values are the mean of 3 replicates SE.
significant at the 5% level by the Fisher’s Least Significant Difference test.
In the control, the ratio of nuclei with decreased DNA in the spike tip was constant until the 25th day and increased thereafter (Fig. 6). The ratio of nuclei with decreased DNA in spike tips was increased by sucrose and sorbitol treatments from the 5th day, although the increase was much more promoted by sucrose than by sorbitol. In the tips of spikes treated with glucose, the ratio of nuclei with decreased DNA was constant until the 15th day, and increased thereafter. Increases in the ratio of nuclei with decreased DNA were suppressed by the mannitol treatment. 3.7. Soluble carbohydrate concentrations in the tips of spikes treated with various carbohydrates
3.4. Changes in DW of various spike parts The dry weight of basal stems was increased by glucose and sucrose treatments more than by the mannitol treatment (Fig. 4C). DW of flowers on the basal stem was markedly increased by sucrose, followed by glucose, whereas DW of flowers on the basal stem was linearly increased by mannitol (Fig. 4D). DW of upper stems was increased by glucose and sucrose treatments until the 10th or 15th day, respectively, whereas DW of upper stems was linearly increased by mannitol treatment and was heaviest among treatments on the 20th day (Fig. 4A). DW of flowers on the upper stem was markedly increased by sucrose, followed by glucose, but was only slightly increased in the control and sorbitol treatments (Fig. 4B). In contrast, DW of flowers on the upper stem in the mannitol treatment linearly increased with time, although the maximum DW was less than that in glucose and sucrose treatments. 3.5. Distribution of carbon derived from applied glucose or mannitol To clarify the distribution of carbon derived from applied glucose or mannitol, we performed a tracer experiment using
Nuclei with decreased DNA (%)
100
Control Glucose Sucrose Mannitol Sorbitol
80 60
At the start of the experiment, glucose was the most abundant carbohydrate, followed by mannitol in the spike tips (Fig. 7). The carbohydrate concentrations in the control decreased during the first day after the start of treatment and remained very low thereafter. Treatments with glucose or sucrose markedly increased glucose, fructose and sucrose concentrations, and slightly increased mannitol concentrations in the tips. Treatment with mannitol markedly increased mannitol concentration in the spike tips on the 2nd day, but was relatively constant thereafter. Sorbitol treatment increased sorbitol and fructose concentrations, but only slightly increased other carbohydrate concentrations. 3.8. Water potential and osmotic potential in tips of spikes treated with various carbohydrates Osmotic potential in the spike tips treated with sucrose and sorbitol rapidly decreased with time (Fig. 8A). Glucose treatment also decreased osmotic potential on 15th day. In contrast, the mannitol treatment maintained the osmotic potential at relatively high levels compared with other carbohydrate treatments. Osmotic potential in the spike tips during the first 10 day decreased to 0.43, 3.76, and 1.92 kPa in glucose, sucrose, and sorbitol treatments, respectively. On the basis of data in Fig. 7, osmotic potential due to total soluble carbohydrates in spike tips during this time is calculated to decrease 0.64, 2.68, and 1.09 kPa in the abovementioned carbohydrate treatments, respectively. The water potential in the spike tips showed a trend similar to that for osmotic potential (Fig. 8B). 3.9. Translocated carbohydrate
40 20 0 0
5
10
15
20
25
30
35
Time (days) Fig. 6. Nuclei with decreased DNA in spike tips of cut flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Standard errors were calculated after arcsine transformation of data. Values are the mean of 3 replicates SE.
Leaves were exposed to 14CO2, and then phloem exudates were collected from the cut ends of the pedicel. The phloem exudates were separated with HPLC, and 14C-carbohydrate was detected. Only a peak corresponding to that of sucrose was detected in the phloem exudates (data not shown). 4. Discussion For flower opening, large amounts of soluble carbohydrates are required as substrates for respiration as well as synthetic materials and osmolytes to maintain osmotic potential (Halevy and Mayak, 1979). As observed in many cut flowers (Halevy and Mayak, 1981;
26
Concentration (mmol g-1FW)
0.5
Concentration (mmol g-1FW)
0.5
Concentration (mmol g-1FW)
0.5
Concentration (mmol g-1FW)
A
0.5
Concentration (mmol g-1FW)
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0.5
Glucose Fructose Sucrose Mannitol Sorbitol
0.4 0.3 0.2 0.1 0.0 0
B
0.3
15
Glucose
0.1 0.0 5
10
0.3 0.2
15
Sucrose
Glucose Fructose Sucrose Mannitol Sorbitol
0.4
0.1 0.0 5
10
0.3 0.2
15
Mannitol
Glucose Fructose Sucrose Mannitol Sorbitol
0.4
0.1 0.0 0
E
10
0.2
0
D
5 Glucose Fructose Sucrose Mannitol Sorbitol
0.4
0
C
Control
5
10
Sorbitol
Glucose Fructose Sucrose Mannitol Sorbitol
0.4 0.3 0.2
15
0.1 0.0 0
5
10
15
Time (days) Fig. 7. Carbohydrate concentrations in spike tips of cut flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Control (A), glucose (B), sucrose (C), mannitol (D), sorbitol (E). Values are the mean of 3 replicates SE.
Ichimura, 1998; Pun and Ichimura, 2003), sucrose treatment promotes flower opening in cut snapdragons (Ichimura and Hisamatsu, 1999). In the present study, glucose and sucrose treatments increased the number of open flowers more than mannitol and sorbitol treatments (Fig. 2). Mannitol was
metabolized less than glucose and sucrose in the tracer experiment with 14C-carbohydrates. Sorbitol is hardly metabolized in plants except for by some Roseaceae plants (Chong and Taper, 1972, 1974; Coffin et al., 1976; Paulin and Jamain, 1982). Thus, the effects of different carbohydrates on flower opening appear to be largely attributed to their ability to be metabolized. Mannitol treatment considerably promoted flower opening in cut snapdragons, although the effects of mannitol were less than those of glucose and sucrose (Figs. 1–3). However, flower opening of cut rose is completely inhibited by exogenous mannitol (Ichimura et al., 1999a,b). In addition, mannitol treatment inhibits flower growth and causes visible damage to leaves in cut chrysanthemum (Kofranek and Halevy, 1972). Because mannitol does not occur in rose and chrysanthemum, the deleterious effects of mannitol may be due to the absence of an enzyme that metabolizes mannitol. In higher plants, the key enzyme for mannitol catabolism is mannitol dehydrogenase, which catalyzes the conversion of mannitol to mannose (Stoop and Pharr, 1992; Stoop et al., 1995). Treatment with mannitol extended the overall vase life of cut snapdragons more than those with the other carbohydrates due to the promotion of flower opening at upper spike part (Figs. 2 and 3). The total DW of spikes did not increase in the control, but markedly increased due to the carbohydrate treatments (Fig. 4), suggesting that the increase in DW was mostly derived from applied carbohydrates. Trends of DW changes in flower parts varied with applied carbohydrates. In mannitol-treated flower spikes, DW of the upper stems linearly increased, suggesting that applied carbon derived from mannitol may accumulate in stems, leading to marked stem growth. Differences in 14C accumulation in the flower spikes were observed between 14C-glucose and 14C-mannitol treatments (Fig. 5), indicating the different effects of applied carbohydrates on carbohydrate distribution. This phenomenon has not yet been reported in other cut flowers. In cut roses, carbon derived from applied sucrose mainly accumulates in leaves, and then moves to flowers (Paulin and Jamain, 1982). Similarly, carbon derived from applied sucrose accumulates mainly in leaves in cut Eustoma (Shimizu-Yumoto et al., 2010). In contrast, carbon derived from applied sucrose mainly accumulates in the flowers in cut carnation (Sacalis and Durkin, 1972; Paulin and Jamain, 1982). These findings indicate that carbon accumulation varies with flower species as well as with the applied carbohydrate. Senescence of spike tips was suppressed by the mannitol treatment (Tables 1–3, Figs. 2 and 6). Nuclei with decreased DNA content, which were determined by FCM, are a parameter of PCD (Yamada et al., 2006). Nuclei with decreased DNA markedly increased in sucrose-, glucose-, and sorbitol-treated flowers, but the mannitol treatment suppressed this increase (Fig. 6), suggesting that mannitol suppresses progression of PCD in the spike tips. Mannitol acts as a radical scavenger in in vitro and in vivo studies and contributes to the reduction of oxidative stress (Shen et al., 1997a,b; Smirnoff and Cumbes, 1989), and oxidative stress has been found to induce senescence in Arabidopsis (Kurepa et al., 1988). In contrast, sucrose accelerated the senescence of tips, although it also acts as a radical scavenger in vitro (Smirnoff and Cumbes, 1989). Thus, the effect of mannitol on the inhibition of senescence may not be attributable to the inhibition of oxidative stress. To understand the mechanisms of carbohydrate-induced senescence of spike tips involved in PCD, we examined the water and osmotic potential of the spike tips. The mannitol treatment did not have much effect on the water and osmotic potential of the spike tips (Fig. 8). In contrast, wilting of spike tips was accompanied by an extreme increase in carbohydrate concentrations (Fig. 7) and a decrease in water and osmotic potential in glucose, sucrose and sorbitol treatments (Fig. 8). In these
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0
A
Water potential
Water potential (MPa)
-1 -2 -3 Control Glucose Sucrose Mannitol Sorbitol
-4 -5 -6 -7 0
2
4
6
8
10
12
14
16
0 Osmotic potential (MPa)
B
Osmotic potential
-1 -2 -3 Control Glucose Sucrose Mannitol Sorbitol
-4 -5 -6 -7 0
2
4
6
8
10
12
14
16
Time (days) Fig. 8. Water potential (A) and osmotic potential (B) in spike tips of cut flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Values are the mean of 3 replicates SE.
Table 2 Effects of various carbohydrates at 250 mM on the wilting of spike tips. Carbohydrate
Time to wilting (days)
Control Glucose Sucrose Sorbitol Mannitol
13.8 1.0c 11.9 1.2bc 7.6 0.8ab 6.1 0.6a 37.6 1.6d
Values are the mean of 8 spikes SE, and those with the same letters within columns are not significantly different (P < 0.05) by the Tukey–Kramer multiple range test.
Table 3 Effects of various carbohydrates on the increase in the number of flower buds. Treatment
Increase in bud numbera
Control Glucose Sucrose Sorbitol Mannitol
2.6 1.8a 2.6 1.9a 5.6 2.0a 5.8 2.0a 13.8 1.7b
Flower spikes were treated with various carbohydrates at 250 mM. Values are the mean of 12 spikes SE, and those with the same letters within columns are not significantly different (P < 0.05) by the Tukey–Kramer multiple range test. a The total number of flower buds including opened flowers were scored 30 days after the start of treatment. The buds with bracts observed by dissecting the terminal buds under a stereoscopic microscope were also included. Bud number at harvest was 38.7.
treatments, a decrease in osmotic potential can largely be attributed to an increase in soluble carbohydrate concentrations. When the osmotic potential of plant tissues decreases to very low
27
levels, high water influx is required to maintain normal levels of water potential. Thus, in glucose, sucrose, and sorbitol treatments, the extreme low osmotic potential in the spike tips due to the accumulation of carbohydrates caused water stress, leading to wilting of spike tips. Although glucose and sucrose are rapidly metabolized in plant tissues, the sucrose treatment markedly increased carbohydrate concentrations, decreased osmotic potential and accelerated senescence of spike tips more than glucose treatment (Figs. 7 and 8). Similarly, glucose treatment is more effective than sucrose treatment in extending vase life in cut Dendrobium (Ketsa and Boonrote, 1990) and rose (Ichimura et al., 2006). In the present experiments, the percent concentration of the sucrose solution was higher than that of the glucose solution because molar concentrations of both solutions were 250 mM. However, senescence of spike tips was promoted more by sucrose than by glucose at a same percent concentration of 3% (unpublished results). Thus, the different actions by these sugars cannot be attributed to different percent concentrations. Mannitol suppressed the senescence of spike tips more than glucose. Although the total soluble carbohydrate concentration in spike tips on day 10 was greater in the glucose treatment than in the mannitol treatment (Fig. 7), 14C accumulation of the spike tips was greater in mannitol treatment than in glucose treatment (Fig. 5), suggesting that mannitol promoted carbon supplement to spike tips more than glucose. The higher carbohydrate concentration of spike tips by the glucose treatment may be attributed to water loss associated with spike tip wilting. Sorbitol is hardly metabolized in many plants (Chong and Taper, 1972, 1974; Coffin et al., 1976; Paulin and Jamain, 1982), but fructose concentrations were increased by sorbitol treatment, suggesting that sorbitol is metabolized in snapdragons. In higher plants, sorbitol is metabolized to fructose by NAD-sorbitol dehydrogenase (Negm and Loescher, 1979) or NADP-sorbitol dehydrogenase (Yamaki, 1984). Production of fructose in snapdragon may be attributed to this enzyme. However, sorbitol rather inhibited stem growth. Marked stem growth was only observed in the mannitol treatment (Figs. 2 and 3). We found that mannitol was metabolized more slowly than glucose and sucrose in snapdragon. Thus, we propose that the suppression of spike tip senescence is specific to mannitol action. Different effects of mannitol and sorbitol on stem growth may alternatively be attributed to some difference in ability to be metabolized, although the mechanism remains unclear. In celery and lilac, mannitol is a translocated carbohydrate (Bieleski, 1982; Loescher, 1987). However, our results indicated that sucrose but not mannitol was translocated in snapdragon. Thus, the specific action of mannitol observed in the present study cannot be attributed to carbohydrate translocation. In the present study, mannitol suppressed the senescence of spike tips and promoted stem growth, but spikes treated with mannitol excreted a white mannitol powder on the surface of flowers (Fig. 3). This was due to the low solubility of mannitol, and the effect would markedly reduce the flower’s ornamental value. Ichimura and Hisamatsu (2006) previously reported that combined treatment with glucose and mannitol extends the vase life of cut snapdragons more than treatment with mannitol alone or glucose alone. However, the white mannitol powder appeared in spikes treated with all solutions containing mannitol. Thus, mannitol may not be suitable for the vase life of cut snapdragon. Other than snapdragons, there are various cut flowers with indeterminate inflorescences, including freesia, gladiolus, stock, and Tweedia caerulea. As observed in cut snapdragons treated with glucose or sucrose, spike tip growth in these cut flowers terminates during vase life, resulting in the failure of flower opening at upper positions, although the cut flowers are treated with these sugars
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(unpublished data). In contrast, the termination of spike tip growth was suppressed by mannitol treatment, which was associated with moderate accumulation of carbohydrate in spike tips (Fig. 7). Thus, we propose that the termination of spike growth in these flowers may be suppressed by treatments with some carbohydrates that maintain carbohydrate of spike tips at appropriate levels. However, mannitol treatment caused deleterious effects on cut snapdragon, despite being the major carbohydrate in this plant (Fig. 3). In addition, mannitol treatment causes chemical injury to cut chrysanthemum and rose in which mannitol is not present (Kofranek and Halevy 1972; Ichimura et al., 1999a). We are exploring carbohydrates that are metabolized to the same degree as mannitol. The vase life of cut flowers will be improved by treatments including these carbohydrates. 5. Conclusion Mannitol treatment delayed senescence of spike tips, resulting in marked stem growth and promotion of bud development in cut snapdragon flowers. This effect was not observed in glucose, sucrose, and sorbitol treatments, resulting in the marked extension of overall vase life. Pulse-chase experiments with 14C-carbohydrates showed that mannitol metabolized slower than glucose and sucrose. Degradation of nuclei, a parameter of PCD, in the spike tips was suppressed by mannitol treatment, compared with other carbohydrates. Treatment with mannitol suppressed an increase in soluble carbohydrate concentrations in spike tips more than the other carbohydrates. Water potential and osmotic potential in the spike tips was modestly decreased by the mannitol treatment, resulting in the suppression of senescence. The other carbohydrates increased the carbohydrate concentrations in spike tips and decreased osmotic potential, resulting in their senescence. Acknowledgement The authors thank Mrs. K. Matsuda for her technical assistance. References Anderson, L., Wolter, K.E., 1966. Cyclitols in plants: biochemistry and physiology. Annu. Rev. Plant Physiol. 17, 209–222. Bieleski, R.L., 1982. Sugar alcohols. In: Loewus, F.A., Tanner, W. (Eds.), Encyclopedia of Plant Physiology, New Series 13A. Springer-Verlag, Berlin, pp. 158–192. Chong, C., Taper, C.D., 1972. Malus tissue cultures. I. Sorbitol (D-glucitol) as a carbon source for callus initiation and growth. Can. J. Bot. 50, 1399–1404. Chong, C., Taper, C.D., 1974. Malus tissue cultures. II. Sorbitol metabolism and carbon nutrition. Can. J. Bot. 52, 2361–2364. Coffin, R., Taper, C.D., Chong, C., 1976. Sorbitol and sucrose as carbon source for callus culture of some species of the Rosaceae. Can. J. Bot. 54, 547–551. Gilbert, G.A., Wilson, C., Madore, M.A., 1997. Root-zone salinity alters raffinose oligosaccharide metabolism and transport in coleus. Plant Physiol. 115, 1267– 1276. Halevy, A.H., Mayak, S., 1979. Senescence and postharvest physiology of cut flowers, part 1. Hortic. Rev. 1, 204–236. Halevy, A.H., Mayak, S., 1981. Senescence and postharvest physiology of cut flowers, part 2. Hortic. Rev. 3, 59–143. Ichimura, K., 1998. Improvement of postharvest life in several cut flowers by the addition of sucrose. JARQ 32, 275–280. Ichimura, K., Hiraya, T., 1999. Effect of silver thiosulfate complex (STS) in combination with sucrose on the vase life of cut sweet pea flowers. J. Jpn. Soc. Hortic. Sci. 68, 23–27. Ichimura, K., Hisamatsu, T., 1999. Effects of continuous treatment with sucrose on the vase life, soluble carbohydrate concentrations, and ethylene production of cut snapdragon flowers. J. Jpn. Soc. Hortic. Sci. 68, 61–66. Ichimura, K., Hisamatsu, T., 2006. Effects of continuous treatments with glucose, sucrose and mannitol on the vase life of cut snapdragon flowers. Bull. Natl. Inst. For. Sci. 5, 45–53.
Ichimura, K., Kohata, K., Koketsu, M., Yamaguchi, Y., Yamaguchi, H., Suto, K., 1997. Identification of methyl glucopyranoside and xylose as soluble sugar constituents in roses (Rosa hybrida L.). Biosci. Biotechnol. Biochem. 61, 1734– 1735. Ichimura, K., Mukasa, Y., Fujiwara, T., Kohata, K., Goto, R., Suto, K., 1999a. Possible roles of methyl glucoside and myo-inositol in the opening of cut rose flowers. Ann. Bot. 83, 551–557. Ichimura, K., Ueyama, S., Goto, R., 1999b. Possible roles of soluble carbohydrate constituents in cut rose flowers. J. Jpn. Soc. Hortic. Sci. 68, 354–359. Ichimura, K., Kohata, K., Goto, R., 2000. Soluble carbohydrates in Delphinium and their influence on sepal abscission in cut flowers. Physiol. Plant. 108, 307–313. Ichimura, K., Kohata, K., Yamada, K., 2005. Promotion of spike elongation in cut snapdragons by mannitol. Acta Hortic. 669, 119–124. Ichimura, K., Taguchi, M., Norikoshi, R., 2006. Extension of the vase life of cut roses by treatment with glucose, isothiazolinonic germicide, citric acid and aluminum sulphate solution. JARQ 40, 263–269. Ichimura, K., Yoshioka, S., Shimizu-Yumoto, H., 2008. Effects of silver thiosulfate complex on the vase life of cut snapdragon flowers. Environ. Control Biol. 46, 155–162. Ketsa, S., Boonrote, A., 1990. Holding solutions for maximizing bud opening and vase-life of Dendrobium ‘Youppadeewan’ flowers. J. Hortic. Sci. 65, 41–47. Kofranek, A.M., Halevy, A.H., 1972. Conditions for opening cut chrysanthemum flower buds. J. Am. Soc. Hortic. Sci. 97, 578–584. Kuiper, D., Ribot, S., van Reenen, H.S., Marissen, N., 1995. The effect of sucrose on the flower bud opening of Madelon cut roses. Sci. Hortic. 60, 325–336. Kurepa, J., Smalle, J., Van Montagu, M., Inze, D., 1988. Oxidative stress tolerance and longevity in Arabidopsis: the late-flowering mutant gigantea is tolerant to paraquat. Plant J. 14, 759–764. Larsen, F.E., Scholes, J.F., 1966. Effects of 8-hydroxyquinoline citrate, N-dimethyl amino succinamic acid, and sucrose on vase-life and spike characteristics of cut snapdragons. Proc. Am. Soc. Hortic. Sci. 89, 694–701. Loescher, W.H., 1987. Physiology and metabolism of sugar alcohols in higher plants. Physiol. Plant. 70, 553–557. Loewus, F.A., Dickinson, D.B., 1982. Cyclitols. In: Loewus, F.A., Tanner, W. (Eds.), Encyclopedia of Plant Physiology, New Series 13A. Springer-Verlag, Berlin, pp. 193–216. Moore, Bd., Palmquist, D.E., Seemann, J.R., 1997. Influence of plant growth at high CO2 concentrations on leaf content of ribulose-1,5-bisphosphate carboxylase/ oxygenase and intracellular distribution of soluble carbohydrates in tobacco, snapdragon, and parsley. Plant Physiol. 115, 241–248. Negm, F.B., Loescher, W.H., 1979. Detection and characterization of sorbitol dehydrogenase from apple callus tissue. Plant Physiol. 64, 69–73. Nowak, J., 1981. Chemical pre-treatment of snapdragon spikes to increase cutflower longevity. Sci. Hortic. 15, 255–262. Paulin, A., Jamain, C., 1982. Development of flowers and changes in various sugars during opening of cut carnations. J. Am. Soc. Hortic. Sci. 107, 258–261. Pun, U.K., Ichimura, K., 2003. Role of sugars in senescence and biosynthesis of ethylene in cut flowers. JARQ 37, 219–224. Sacalis, J.N., Durkin, D., 1972. Movement of 14C in cut roses and carnations after uptake of 14C-sucorse. J. Am. Soc. Hortic. Sci. 97, 481–484. Shen, B., Jensen, R.G., Bohnert, H.J., 1997a. Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts. Plant Physiol. 113, 1177–1183. Shen, B., Jensen, R.G., Bohnert, H.J., 1997b. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiol. 115, 527–532. Shimizu-Yumoto, H., Kondo, M., Sanoh, Y., Ohsumi, A., Ichimura, K., 2010. Effect of abscisic acid on the distribution of exogenous carbon derived from sucrose applied to cut Eustoma flowers. J. Hortic. Sci. Biotechnol. 85, 83–87. Smirnoff, N., Cumbes, Q.J., 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–1060. Stoop, J.M.H., Pharr, D.M., 1992. Partial purification and characterization of mannitol: mannose 1-oxidoreductase from celeriac (Apium graveolens var. rapaceum) roots. Arch. Biochem. Biophys. 298, 612–619. Stoop, J.M.H., Williamson, J.D., Conkling, M.A., Pharr, D.M., 1995. Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures. Plant Physiol. 108, 1219–1225. Wang, C.Y., Baker, J.E., Hardenburg, R.E., Lieberman, M., 1977. Effects of two analogs of rhizobitoxine and sodium benzoate on senescence of snapdragons. J. Am. Soc. Hortic. Sci. 102, 517–520. Yamada, T., Takatsu, Y., Kasumi, M., Ichimura, K., van Doorn, W.G., 2006. Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomea nil). Planta 224, 1279–1290. Yamaki, S., 1984. Isolation of vacuoles from immatured apple fruit flesh and compartmentation of sugars, organic acids, phenolic compounds and amino acids. Plant Cell Physiol. 25, 151–166.
Contents lists available at ScienceDirect
Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Exogenous mannitol treatment stimulates bud development and extends vase life of cut snapdragon flowers Kazuo Ichimura* , Satoshi Yoshioka, Tetsuya Yamada1 NARO Institute of Floricultural Science, Fujimoto, Tsukuba, Ibaraki 305-8519, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 April 2015 Received in revised form 26 October 2015 Accepted 28 October 2015 Available online xxx
With a view to understanding the role of soluble carbohydrates in flower development and vase life, cut snapdragon (Antirrhinum majus L.) cv. Yellow Butterfly spikes were treated with glucose, sucrose, sorbitol and mannitol, which is a major carbohydrate in them. Treatment with 10–500 mM mannitol markedly promoted flower bud development and stem growth of cut snapdragon. Stem growth accompanied with bud development was not observed in 250 mM glucose, sucrose and sorbitol treatments. Mannitol treatment extended the overall vase life of cut snapdragons more than the other carbohydrate due to the promotion of flower opening at upper spike part. A pulse-chase experiment with 14C-glucose, 14C-sucrose or 14C-mannitol showed that mannitol was metabolized slower than glucose and sucrose, suggesting that the different effects of carbohydrates on flower opening and stem growth may be due to different ability to be metabolized. The dry weight of flowers was greater in sucrose- or glucose-treated spikes than in mannitol-treated spikes, but the dry weight of upper stem parts including spike tips was gradually increased by mannitol treatment, suggesting that marked stem growth is due to applied mannitol. This explanation is supported by a tracer experiment with 14C-carbohydrates showing that accumulation of 14 C in spike tips was greater in the mannitol treatment than in the glucose treatment. Flow cytometry revealed that degradation of nuclei, a parameter of programmed cell death (PCD), was promoted by sucrose, glucose, or sorbitol treatment, but was suppressed by the mannitol treatment. Carbohydrate concentrations in spike tips were markedly increased by glucose, sucrose, and sorbitol treatments, but were only slightly increased by the mannitol treatment. Water potential and osmotic potential in the spike tips decreased rapidly with the sucrose or sorbitol treatments, but were only modestly decreased by the mannitol treatment. The results suggest that mannitol suppressed a decrease in osmotic potential of spike tips, resulting in the continuation of bud development. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Antirrhinum majus Fructose Glucose Osmotic potential Sorbitol Stem growth Sucrose
1. Introduction Cut flowers are usually placed under conditions below the light compensation point for photosynthesis, and thus, they do not assimilate much carbon by photosynthesis, and thereby lack reserve carbohydrate. Treating with the ubiquitous metabolic sugars glucose, fructose, or sucrose, and supplementing with antimicrobial compounds, extends the vase life of many cut flowers, including carnation (Paulin and Jamain, 1982), rose (Kuiper et al., 1995; Ichimura et al., 2006), and sweet pea (Ichimura and Hiraya, 1999). These metabolic sugars have generally been considered to be similarly effective in extending the vase life of cut
* Corresponding author. Fax: +81 29 838 6841. E-mail address: [email protected] (K. Ichimura). 1 Present address: Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. http://dx.doi.org/10.1016/j.postharvbio.2015.10.017 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
flowers (Halevy and Mayak, 1981). However, Ketsa and Boonrote (1990) reported that glucose extended the vase life of cut Dendrobium flowers more than sucrose. Similarly, continuous treatment with glucose or fructose was found to be more effective in extending the vase life of cut rose flowers than continuous treatment with sucrose (Ichimura et al., 2006). These findings indicate that exogenous sugars affect the vase life of cut flowers differently. Other than these ubiquitous metabolic sugars, the effects of some soluble carbohydrates occurring in plants on their flower opening and vase life have been studied. In rose, myo-inositol, which is present generally in higher plants (Anderson and Wolter, 1966; Loewus and Dickinson, 1982), is a major carbohydrate in leaves, whereas xylose and methyl glucoside are minor sugar constituents (Ichimura et al., 1997). Treatments with methyl glucoside and xylose promoted flower opening, but treatment with myo-inositol inhibited opening of cut rose flowers (Ichimura et al., 1999a,b). Mannitol is the major carbohydrate in Delphinium
K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
petals were counted as open flowers. Flowers were considered to have terminated their life when the petals had wilted or abscised. The length of the flower spike was measured every 5 days. When stem length, bud number, and open flower number did not further increase, the measurements were completed. The total number of flower buds was scored 30 days after the start of treatment when the stem growth had completely ceased. Under a stereoscopic microscope, flower spike tips were dissected, and the buds with bracts were scored and included in the total number of flower buds.
60
A
Spike length
55 Spike length (cm)
(Ichimura et al., 2000). Exogenous mannitol reduces sensitivity to ethylene, delays climacteric-like increases in ethylene production, and suppresses sepal abscission in cut Delphinium. These actions are observed with glucose, but are not observed with 3-o-methylglucoside, a non-metabolizable sugar (Ichimura et al., 2000). Snapdragon is an important cut flower because of its wide range of petal colors and good fragrance. Snapdragons have an indeterminate inflorescence with flower buds that open from the stem base to the apex. However, the vase life of cut snapdragons is relatively short and limited by flower abscission, wilting, or failure of buds to fully open (Larsen and Scholes, 1966; Wang et al., 1977; Nowak, 1981). Most snapdragon cultivars are sensitive to ethylene-induced flower abscission and wilting (Ichimura et al., 2008), but pulse treatment with silver thiosulfate complex (STS), an inhibitor of ethylene action, does not markedly extend the vase life of cut snapdragons (Ichimura et al., 2008; Nowak, 1981). In contrast, continuous treatments with sucrose and glucose markedly promote flower opening, thereby extending the overall vase life of cut snapdragon spikes (Ichimura and Hisamatsu, 1999, 2006). In addition to sucrose, analysis by high performance liquid chromatography (HPLC) detected glucose, fructose, and mannitol in snapdragon petals (Ichimura and Hisamatsu, 1999) as was reported in snapdragon leaves by Moore et al. (1997). Identification of mannitol was confirmed by Nuclear Magnetic Resonance analysis (Ichimura et al., 2005). Although treatment with mannitol somewhat extends the vase life of cut snapdragons, this treatment induces marked stem growth (Ichimura et al., 2005). In the present study, we investigated the effects of various carbohydrates including mannitol on the vase life of cut snapdragon flowers. To clarify how mannitol promotes bud development and stem growth, we investigated the accumulation and metabolism of soluble carbohydrates as well as water and osmotic potential of spike tips.
21
0 mM 1 mM 10 mM 100 mM 250 mM 500 mM
50 45 40 35 30 25 0
10
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B
Bud number
40
2.1. Plant material Snapdragon (Antirrhinum majus L.) cv. Yellow Butterfly was grown under natural day-length conditions in a greenhouse (15 C minimum and 25 C maximum temperature). Flower spikes in which the most developed flower bud (3 cm in length) was expected to open the next day, were harvested in the morning and placed in tap water. They were immediately transported to a laboratory and used for experiments within 1 h.
2.3. Measurements of flower bud number and spike length The number of visible buds longer than 5 mm and open, wilted, and abscised flowers was scored daily. Flowers with fully unfolded
35 30 25 20 15 0
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C
0 mM 1 mM 10 mM 100 mM 250 mM 500 mM
Open flower number Open flower number
2.2. Treatments with soluble carbohydrates The flower spikes were recut to 30 cm and individually placed in a 300-mL glass vessel containing 250 mL of carbohydrate solution. In the other experiments, three flower spikes were placed in a 500mL glass vessel containing 500 mL of carbohydrate solution. Mannitol concentration was set at 1, 10, 100, 250, or 500 mM, whereas glucose, sucrose, or sorbitol concentration was set at 250 mM because in a preliminary experiment, the most pronounced effect in mannitol treatment was observed at this concentration. All solutions including the control were supplemented with 200 mg L 1 8-hydroxyquinoline sulfate (8-HQS) to inhibit microbial proliferation. The spikes were kept at 23 C, 70% relative humidity, and 12-h light at 10 mmol m 2 s 1 from coolwhite fluorescence lamps.
50
0 mM 1 mM 10 mM 100 mM 250 mM 500 mM
Bud number
45
2. Materials and methods
40
50
8 6 4 2 0 0
10
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50
Time (days) Fig. 1. Spike length (A), bud number (B), and open flower number (C) of cut snapdragons treated with mannitol at different concentrations. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. The number of flower buds was the sum of the flower buds longer than 5 mm, open flowers, and wilted flowers. Values are the mean of 8 replicates SE (A and B) or +SE (C).
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2.4. Determination of soluble carbohydrate concentration
2.7. Measurement of water and osmotic potential Spike tips of 1 cm length were cut from the flower spike. For measurement of osmotic potential, tips were placed in a vessel and kept at 30 C for 2 days. The vessel was then allowed to reach room temperature and the osmotic potential of the spike tips was measured using a cyclometer (Tru Psi, Decagon Devices, Pullman, Washington, USA) according to the manufacturer’s instructions. For measurement of water potential, tips were placed in the vessel without freezing and measured as described above. 2.8. Metabolism of glucose, sucrose and mannitol Stems below the position where first floret bud was generated were cut into disks with length of 5 mm. Disks (approximately 500 mg) were placed into a solution containing 250 mM
Spike length (cm)
50 Control Glucose Sucrose Mannitol Sorbitol
45 40 35 30 25 0
B
10
20
30
40
50
Bud number
45 40
Flower spikes were divided into four portions: basal 30-cm stem, flowers from the basal 30-cm stem, the remaining upper stem that was a newly grown portion after harvest, and flowers from the remaining upper stem. The tissues were dried at 80 C in an oven for three days and their dry weight (DW) was measured. Three spikes were used in each experiment, and experiments were repeated three times.
Control Glucose Sucrose Mannitol Sorbitol
35 30 25 20 15 0
10
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40
16
C
Open flower number
14 Open flower number
Spike tips of 1 cm length were chopped, using a razor blade, in nuclear extraction buffer obtained from the High Resolution Kit for Plant DNA (Partec, Münster, Germany) provided by the manufacturer of the flow cytometer. The extraction buffer was a low pH solution containing Triton X-100. The extract was filtered through a 50-mm nylon mesh and the medium with the isolated nuclei was collected. A buffer with the fluorescent DNA stain 4,6-diamidino2-phenylindole (DAPI), also part of the standard reagent set (Partec) was added, and the solution was vortexed. The DNA content of the isolated nuclei was analyzed using a flow cytometer (Ploidy Analyzer, Partec). DNA levels were obtained from a total of 5000 nuclei. On the basis of histograms obtained from cytometry, the ratio of nuclei with decreased DNA was calculated as described in Yamada et al. (2006). Although it is unclear whether DNA masses detected by flow cytometry (FCM) have functions as nuclei, DNA masses were regarded as nuclei in the present study.
Spike length
55
2.5. Measurement of dry weight
2.6. Flow cytometry
60
A
Bud number
Spike tips of 1 cm length were collected and examined for the concentration of soluble carbohydrates. The organs were cut into small pieces and immersed in 10 mL of 80% ethanol at 75 C for 30 min. After cooling, 25 mL of 10% (w/v) galactose was added to the extract as an internal standard. The samples were then homogenized. After centrifugation, the residue was suspended in 5 mL of 80% ethanol and centrifuged twice. The three supernatants were combined and evaporated to dryness in vacuo below 50 C, and the residue was dissolved in 1 mL water. The solution was then passed through a Sep-Pak C-18 cartridge, followed by 2 mL water. An aliquot of the eluate was separated using an HPLC system (Jasco, Tokyo, Japan) equipped with a refractive index detector on a Shodex SUGAR SP0810 column (Showa Denko, Tokyo, Japan). The column was held at 80 C and water was eluted at a flow rate of 0.8 mL min 1. The peak identity was confirmed using authentic carbohydrates and the peak area was determined using an integrator. The concentration of each carbohydrate in the sample was calculated as previously described (Ichimura et al., 1999a).
Control Glucose Sucrose Mannitol Sorbitol
12 10 8 6 4 2 0 0
10
20
30
40
Time (days) Fig. 2. Spike length (A), bud number (B) and open flower number (C) of cut snapdragons treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. The number of flower buds was the sum of the flower buds longer than 5 mm, open flowers, and wilted flowers. Values are the mean of 8 replicates SE (A and B) or +SE (C).
14 C-glucose, 14C-sucrose or 14C-mannitol solution (61.79 kBq, 37 MBq mmol 1) in a Petri dish and kept at 23 C under darkness for 1 h. The disks were then transferred into 250 mM unlabeled glucose, sucrose, and mannitol solutions for 0, 6, 12 and 24 h and then immersed in 80% ethanol. The discs were homogenized in 80% ethanol and centrifuged. The resulting supernatant was combined and evaporated to dryness in vacuo below 50 C. The residue was dissolved in water and separated by HPLC equipped with a radioanalyzer (RLC-113, Aloka, Tokyo, Japan). The column and separation conditions were as described above.
K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
23
Fig. 3. Photographs of cut snapdragon flowers treated with various carbohydrates at 250 mM. All solutions including the control contained 200 mg L 1 8-HQS. Cut flower spikes treated with 250 mM glucose, sucrose, sorbitol, or mannitol at 23 C for 14 days (A). Far left, control; middle left, glucose; middle, sucrose; middle right, sorbitol; far right, mannitol. Cut flower spikes treated with 250 mM mannitol at 23 C for 25 days (B). Upper part of cut flower spikes treated with 250 mM mannitol at 23 C for 27 days (C).
Table 1 Effects of mannitol at various concentrations on the wilting of spike tips. Concentration (mM)
Time to wilting (days)
0 1 10 100 250 500
14.3 1.0 15.5 0.3 24.6 1.1*** 39.4 2.1*** 38.3 1.8*** 34.7 1.0***
Values are the mean of 8 spikes standard errors, and those with *** are significant at P < 0.001, compared with the control by the Dunnett’s test.
2.9. Distribution of carbon derived from
14
C-glucose or
14
C-mannitol
Cut spikes were held in 0.25 M glucose or mannitol solution for ten days as described above. The cut ends of spikes were 14 14 transferred into C-glucose or C-mannitol solution (23.135 kBq, 185 MBq mmol 1) for 1 h. The spikes were then transferred in to 250 mM glucose or 250 mM mannitol solution and kept at 23 C for 24 h in darkness. The spikes were divided into five portions: basal stem from cut end to 30 cm, flowers generated on the basal stem, remaining upper stem, flowers on remaining upper stem, and tips with length of 1 cm. These portions were dried at 80 C for more than 3 days. The dried samples were ground and 20 mg subsample was oxidized and dissolved in a liquid scintillator with a sample combustion system (ASC-113, Aloka).
The radioactivity of the solution was measured with a liquid scintillation counter (LSC-3500, Aloka). 2.10. Carbohydrate translocation Determination of carbohydrate translocation was based on the method of Gilbert et al. (1997). Flower stems with leaves were cut from the plants. To remove the upper parts with flowers and flower buds, the stems were re-cut to 20 cm and the cut ends of the stems were put into distilled water. The stems were held at 25 C under 250 mmol m 2 s 1 cool-white fluorescence lamps for about 1 h, and then individual leaves were cut from the stems and the cut ends were put into distilled water and placed in a 27-L acrylic chamber. Then, 14C-sodium bicarbonate (NaH14CO3) (9.25 MBq, 74 MBq mmol 1) was dissolved in water and lactic acid was added to the solution to evolve 14CO2. Leaves were allowed to photosynthesize for 30 min, after which the cut ends were placed in 20 mM EDTA solution and held at 20 C in darkness for 3 h. The resulting solutions were evaporated to dryness in vacuo below 50 C. The residue was dissolved in distilled water and separated with HPLC equipped with the radio-analyzer as described above. 2.11. Statistical analysis Data were analyzed by Tukey–Kramer multiple range test, Dunnett’s test, and Fisher’s Least Significant Difference test using KaleidaGraph software (v.4.1, Hulinks, Tokyo, Japan).
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3. Results
0.30 Dry weight (g/portion)
A
Upper stem
Control Glucose Sucrose Mannitol Sorbitol
0.25 0.20
3.1. Effects of mannitol at various concentrations on stem length and the number of total buds and open flowers
0.15 0.10 0.05 0.00 0
5
10
3.0 Dry weight (g/portion)
B
Control Glucose Sucrose Mannitol Sorbitol
2.5 2.0
15
20
25
Flowers on upper stem
1.5 1.0
3.2. Effects of various carbohydrates on stem length and the number of total buds and open flowers
0.5 0.0 0
5
10
15
3.0 Dry weight (g/portion)
C
2.0
25
Basal stem
Control Glucose Sucrose Mannitol Sorbitol
2.5
20
1.5 1.0 0.5 0.0 0 3.0
D
5
10
15
20
25
Control Flowers on basal stem Glucose Sucrose Mannitol Sorbitol
2.5 Dry weight (g/portion)
Mannitol at 10 mM or higher concentrations delayed the wilting of spike tips and stimulated stem growth, and the optimal concentration was 250 mM (Table 1, Fig. 1A). The stimulation of stem growth was accompanied by an increase in the number of buds. In control flower spikes, several flowers opened within the first few days (Fig. 1C). Mannitol at 1 and 10 mM slightly affected flower opening, whereas mannitol at 100 mM or higher concentrations markedly promoted flower opening. Mannitol at 100 mM or higher concentrations caused abortion of buds on about the 15th node from the base of spikes, but flowers on the upper part of the spikes opened almost fully. The time from harvest to the time when open flowers without wilting were not observed in each flower spike was 11.8 0.4 days at 0 mM, 11.8 0.6 days at 1 mM, 14.3 0.6 days at 10 mM, 27.8 1.5 days at 100 mM, 31.0 1.0 days at 200 mM, and 28.4 2.0 days at 500 mM (means of eight replicates SE).
2.0 1.5 1.0 0.5 0.0 0
5
10
15
20
25
Time (days) Fig. 4. Dry weight of upper stem (A), flowers on upper stem (B), basal stem (C), and flowers on basal stem (D) in cut snapdragon flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Values are the mean of 3 replicates SE.
In glucose and sucrose treatments, a considerable increase in spike length and an increase in bud number was only observed during the first 5 days; thereafter, spike length and bud number increased slightly (Figs. 2 and 3). In the sorbitol treatment, spike length and bud number slightly increased, suggesting that sorbitol treatment inhibits bud development. In contrast, in the mannitol treatment the spike length increased linearly until the 20th day, accompanied by a marked increase in bud number. Browning of spike tips, a senescent symptom, was not observed until about the 30th day in mannitol-treated spikes (Table 2). The treatments with glucose and sucrose promoted flower opening (Figs. 2 and 3), although the glucose treatment caused browning of the spike tips one week after the start of the treatment (personal observation), after which bud number did not increase. Treatment with sorbitol promoted flower opening. Flower buds at the middle part of the spikes aborted. Flower buds on the upper part opened, but were smaller than those on the basal part of the spikes (Fig. 3). However, spikes treated with mannitol excreted a white powder on the surface of flowers (Fig. 3), which was confirmed to be mannitol by HPLC analysis. The time from harvest to the time when open flowers without wilting were not observed in each flower spike was 11.0 0.4 days in the control, and 22.7 0.8, 15.4 0.4, 18.2 0.6, and 29.8 1.3 days (mean of eight replicates SE) in the spikes treated with glucose, sucrose, sorbitol, and mannitol, respectively. This indicates that mannitol treatment extended vase life of cut snapdragons more than the other carbohydrate treatments. We examined the total number of flower buds at the 30th day using a stereoscopic microscope to clarify whether development of flower buds was promoted by mannitol treatment. The total number of flower buds was the highest in the spikes treated with mannitol (Table 3). 3.3. Metabolism of glucose, sucrose and mannitol After labeling, radioactivity of 14C-glucose and 14C-sucrose were decreased to 14.3 1.6 and 14.0 0.5% of applied radioactivities, respectively, but that of 14C-mannitol was decreased to 21.1 2.9% of applied radioactivity (mean of three replicates SE). The differences between mannitol and glucose or sucrose were
K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
25
14
1.2e+6 Glucose Mannitol
Radioactivity (dpm)
1.0e+6 25000
8.0e+5
20000 15000
6.0e+5
10000
3.6. Degradation of nuclei in the tips of spikes treated with various carbohydrates
5000
4.0e+5
C-glucose and 14C-mannitol. Accumulation of 14C in flowers at both parts was greater in the glucose treatment than in the mannitol treatment (Fig. 5), although accumulation of 14C in the tip was greater in the mannitol treatment than in the glucose treatment, suggesting that distribution of carbon differed depending on the applied carbohydrate.
0
2.0e+5 0.0 B. Stem B. Flower U. Stem U. Flower
Tip
Fig. 5. Accumulation of 14C derived from 14C-glucose and 14C-mannitol in basal stem (B. Stem), flowers on basal stem (B. Flower), upper stem (U. Stem), flowers on upper stem (U. Flower) and tip of cut snapdragon flowers. The inset shows the data of tip. Values are the mean of 3 replicates SE.
significant at the 5% level by the Fisher’s Least Significant Difference test.
In the control, the ratio of nuclei with decreased DNA in the spike tip was constant until the 25th day and increased thereafter (Fig. 6). The ratio of nuclei with decreased DNA in spike tips was increased by sucrose and sorbitol treatments from the 5th day, although the increase was much more promoted by sucrose than by sorbitol. In the tips of spikes treated with glucose, the ratio of nuclei with decreased DNA was constant until the 15th day, and increased thereafter. Increases in the ratio of nuclei with decreased DNA were suppressed by the mannitol treatment. 3.7. Soluble carbohydrate concentrations in the tips of spikes treated with various carbohydrates
3.4. Changes in DW of various spike parts The dry weight of basal stems was increased by glucose and sucrose treatments more than by the mannitol treatment (Fig. 4C). DW of flowers on the basal stem was markedly increased by sucrose, followed by glucose, whereas DW of flowers on the basal stem was linearly increased by mannitol (Fig. 4D). DW of upper stems was increased by glucose and sucrose treatments until the 10th or 15th day, respectively, whereas DW of upper stems was linearly increased by mannitol treatment and was heaviest among treatments on the 20th day (Fig. 4A). DW of flowers on the upper stem was markedly increased by sucrose, followed by glucose, but was only slightly increased in the control and sorbitol treatments (Fig. 4B). In contrast, DW of flowers on the upper stem in the mannitol treatment linearly increased with time, although the maximum DW was less than that in glucose and sucrose treatments. 3.5. Distribution of carbon derived from applied glucose or mannitol To clarify the distribution of carbon derived from applied glucose or mannitol, we performed a tracer experiment using
Nuclei with decreased DNA (%)
100
Control Glucose Sucrose Mannitol Sorbitol
80 60
At the start of the experiment, glucose was the most abundant carbohydrate, followed by mannitol in the spike tips (Fig. 7). The carbohydrate concentrations in the control decreased during the first day after the start of treatment and remained very low thereafter. Treatments with glucose or sucrose markedly increased glucose, fructose and sucrose concentrations, and slightly increased mannitol concentrations in the tips. Treatment with mannitol markedly increased mannitol concentration in the spike tips on the 2nd day, but was relatively constant thereafter. Sorbitol treatment increased sorbitol and fructose concentrations, but only slightly increased other carbohydrate concentrations. 3.8. Water potential and osmotic potential in tips of spikes treated with various carbohydrates Osmotic potential in the spike tips treated with sucrose and sorbitol rapidly decreased with time (Fig. 8A). Glucose treatment also decreased osmotic potential on 15th day. In contrast, the mannitol treatment maintained the osmotic potential at relatively high levels compared with other carbohydrate treatments. Osmotic potential in the spike tips during the first 10 day decreased to 0.43, 3.76, and 1.92 kPa in glucose, sucrose, and sorbitol treatments, respectively. On the basis of data in Fig. 7, osmotic potential due to total soluble carbohydrates in spike tips during this time is calculated to decrease 0.64, 2.68, and 1.09 kPa in the abovementioned carbohydrate treatments, respectively. The water potential in the spike tips showed a trend similar to that for osmotic potential (Fig. 8B). 3.9. Translocated carbohydrate
40 20 0 0
5
10
15
20
25
30
35
Time (days) Fig. 6. Nuclei with decreased DNA in spike tips of cut flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Standard errors were calculated after arcsine transformation of data. Values are the mean of 3 replicates SE.
Leaves were exposed to 14CO2, and then phloem exudates were collected from the cut ends of the pedicel. The phloem exudates were separated with HPLC, and 14C-carbohydrate was detected. Only a peak corresponding to that of sucrose was detected in the phloem exudates (data not shown). 4. Discussion For flower opening, large amounts of soluble carbohydrates are required as substrates for respiration as well as synthetic materials and osmolytes to maintain osmotic potential (Halevy and Mayak, 1979). As observed in many cut flowers (Halevy and Mayak, 1981;
26
Concentration (mmol g-1FW)
0.5
Concentration (mmol g-1FW)
0.5
Concentration (mmol g-1FW)
0.5
Concentration (mmol g-1FW)
A
0.5
Concentration (mmol g-1FW)
K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
0.5
Glucose Fructose Sucrose Mannitol Sorbitol
0.4 0.3 0.2 0.1 0.0 0
B
0.3
15
Glucose
0.1 0.0 5
10
0.3 0.2
15
Sucrose
Glucose Fructose Sucrose Mannitol Sorbitol
0.4
0.1 0.0 5
10
0.3 0.2
15
Mannitol
Glucose Fructose Sucrose Mannitol Sorbitol
0.4
0.1 0.0 0
E
10
0.2
0
D
5 Glucose Fructose Sucrose Mannitol Sorbitol
0.4
0
C
Control
5
10
Sorbitol
Glucose Fructose Sucrose Mannitol Sorbitol
0.4 0.3 0.2
15
0.1 0.0 0
5
10
15
Time (days) Fig. 7. Carbohydrate concentrations in spike tips of cut flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Control (A), glucose (B), sucrose (C), mannitol (D), sorbitol (E). Values are the mean of 3 replicates SE.
Ichimura, 1998; Pun and Ichimura, 2003), sucrose treatment promotes flower opening in cut snapdragons (Ichimura and Hisamatsu, 1999). In the present study, glucose and sucrose treatments increased the number of open flowers more than mannitol and sorbitol treatments (Fig. 2). Mannitol was
metabolized less than glucose and sucrose in the tracer experiment with 14C-carbohydrates. Sorbitol is hardly metabolized in plants except for by some Roseaceae plants (Chong and Taper, 1972, 1974; Coffin et al., 1976; Paulin and Jamain, 1982). Thus, the effects of different carbohydrates on flower opening appear to be largely attributed to their ability to be metabolized. Mannitol treatment considerably promoted flower opening in cut snapdragons, although the effects of mannitol were less than those of glucose and sucrose (Figs. 1–3). However, flower opening of cut rose is completely inhibited by exogenous mannitol (Ichimura et al., 1999a,b). In addition, mannitol treatment inhibits flower growth and causes visible damage to leaves in cut chrysanthemum (Kofranek and Halevy, 1972). Because mannitol does not occur in rose and chrysanthemum, the deleterious effects of mannitol may be due to the absence of an enzyme that metabolizes mannitol. In higher plants, the key enzyme for mannitol catabolism is mannitol dehydrogenase, which catalyzes the conversion of mannitol to mannose (Stoop and Pharr, 1992; Stoop et al., 1995). Treatment with mannitol extended the overall vase life of cut snapdragons more than those with the other carbohydrates due to the promotion of flower opening at upper spike part (Figs. 2 and 3). The total DW of spikes did not increase in the control, but markedly increased due to the carbohydrate treatments (Fig. 4), suggesting that the increase in DW was mostly derived from applied carbohydrates. Trends of DW changes in flower parts varied with applied carbohydrates. In mannitol-treated flower spikes, DW of the upper stems linearly increased, suggesting that applied carbon derived from mannitol may accumulate in stems, leading to marked stem growth. Differences in 14C accumulation in the flower spikes were observed between 14C-glucose and 14C-mannitol treatments (Fig. 5), indicating the different effects of applied carbohydrates on carbohydrate distribution. This phenomenon has not yet been reported in other cut flowers. In cut roses, carbon derived from applied sucrose mainly accumulates in leaves, and then moves to flowers (Paulin and Jamain, 1982). Similarly, carbon derived from applied sucrose accumulates mainly in leaves in cut Eustoma (Shimizu-Yumoto et al., 2010). In contrast, carbon derived from applied sucrose mainly accumulates in the flowers in cut carnation (Sacalis and Durkin, 1972; Paulin and Jamain, 1982). These findings indicate that carbon accumulation varies with flower species as well as with the applied carbohydrate. Senescence of spike tips was suppressed by the mannitol treatment (Tables 1–3, Figs. 2 and 6). Nuclei with decreased DNA content, which were determined by FCM, are a parameter of PCD (Yamada et al., 2006). Nuclei with decreased DNA markedly increased in sucrose-, glucose-, and sorbitol-treated flowers, but the mannitol treatment suppressed this increase (Fig. 6), suggesting that mannitol suppresses progression of PCD in the spike tips. Mannitol acts as a radical scavenger in in vitro and in vivo studies and contributes to the reduction of oxidative stress (Shen et al., 1997a,b; Smirnoff and Cumbes, 1989), and oxidative stress has been found to induce senescence in Arabidopsis (Kurepa et al., 1988). In contrast, sucrose accelerated the senescence of tips, although it also acts as a radical scavenger in vitro (Smirnoff and Cumbes, 1989). Thus, the effect of mannitol on the inhibition of senescence may not be attributable to the inhibition of oxidative stress. To understand the mechanisms of carbohydrate-induced senescence of spike tips involved in PCD, we examined the water and osmotic potential of the spike tips. The mannitol treatment did not have much effect on the water and osmotic potential of the spike tips (Fig. 8). In contrast, wilting of spike tips was accompanied by an extreme increase in carbohydrate concentrations (Fig. 7) and a decrease in water and osmotic potential in glucose, sucrose and sorbitol treatments (Fig. 8). In these
K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
0
A
Water potential
Water potential (MPa)
-1 -2 -3 Control Glucose Sucrose Mannitol Sorbitol
-4 -5 -6 -7 0
2
4
6
8
10
12
14
16
0 Osmotic potential (MPa)
B
Osmotic potential
-1 -2 -3 Control Glucose Sucrose Mannitol Sorbitol
-4 -5 -6 -7 0
2
4
6
8
10
12
14
16
Time (days) Fig. 8. Water potential (A) and osmotic potential (B) in spike tips of cut flowers treated with 250 mM glucose, sucrose, mannitol and sorbitol. All solutions including the control contained 200 mg L 1 8-HQS and cut flowers were held at 23 C. Values are the mean of 3 replicates SE.
Table 2 Effects of various carbohydrates at 250 mM on the wilting of spike tips. Carbohydrate
Time to wilting (days)
Control Glucose Sucrose Sorbitol Mannitol
13.8 1.0c 11.9 1.2bc 7.6 0.8ab 6.1 0.6a 37.6 1.6d
Values are the mean of 8 spikes SE, and those with the same letters within columns are not significantly different (P < 0.05) by the Tukey–Kramer multiple range test.
Table 3 Effects of various carbohydrates on the increase in the number of flower buds. Treatment
Increase in bud numbera
Control Glucose Sucrose Sorbitol Mannitol
2.6 1.8a 2.6 1.9a 5.6 2.0a 5.8 2.0a 13.8 1.7b
Flower spikes were treated with various carbohydrates at 250 mM. Values are the mean of 12 spikes SE, and those with the same letters within columns are not significantly different (P < 0.05) by the Tukey–Kramer multiple range test. a The total number of flower buds including opened flowers were scored 30 days after the start of treatment. The buds with bracts observed by dissecting the terminal buds under a stereoscopic microscope were also included. Bud number at harvest was 38.7.
treatments, a decrease in osmotic potential can largely be attributed to an increase in soluble carbohydrate concentrations. When the osmotic potential of plant tissues decreases to very low
27
levels, high water influx is required to maintain normal levels of water potential. Thus, in glucose, sucrose, and sorbitol treatments, the extreme low osmotic potential in the spike tips due to the accumulation of carbohydrates caused water stress, leading to wilting of spike tips. Although glucose and sucrose are rapidly metabolized in plant tissues, the sucrose treatment markedly increased carbohydrate concentrations, decreased osmotic potential and accelerated senescence of spike tips more than glucose treatment (Figs. 7 and 8). Similarly, glucose treatment is more effective than sucrose treatment in extending vase life in cut Dendrobium (Ketsa and Boonrote, 1990) and rose (Ichimura et al., 2006). In the present experiments, the percent concentration of the sucrose solution was higher than that of the glucose solution because molar concentrations of both solutions were 250 mM. However, senescence of spike tips was promoted more by sucrose than by glucose at a same percent concentration of 3% (unpublished results). Thus, the different actions by these sugars cannot be attributed to different percent concentrations. Mannitol suppressed the senescence of spike tips more than glucose. Although the total soluble carbohydrate concentration in spike tips on day 10 was greater in the glucose treatment than in the mannitol treatment (Fig. 7), 14C accumulation of the spike tips was greater in mannitol treatment than in glucose treatment (Fig. 5), suggesting that mannitol promoted carbon supplement to spike tips more than glucose. The higher carbohydrate concentration of spike tips by the glucose treatment may be attributed to water loss associated with spike tip wilting. Sorbitol is hardly metabolized in many plants (Chong and Taper, 1972, 1974; Coffin et al., 1976; Paulin and Jamain, 1982), but fructose concentrations were increased by sorbitol treatment, suggesting that sorbitol is metabolized in snapdragons. In higher plants, sorbitol is metabolized to fructose by NAD-sorbitol dehydrogenase (Negm and Loescher, 1979) or NADP-sorbitol dehydrogenase (Yamaki, 1984). Production of fructose in snapdragon may be attributed to this enzyme. However, sorbitol rather inhibited stem growth. Marked stem growth was only observed in the mannitol treatment (Figs. 2 and 3). We found that mannitol was metabolized more slowly than glucose and sucrose in snapdragon. Thus, we propose that the suppression of spike tip senescence is specific to mannitol action. Different effects of mannitol and sorbitol on stem growth may alternatively be attributed to some difference in ability to be metabolized, although the mechanism remains unclear. In celery and lilac, mannitol is a translocated carbohydrate (Bieleski, 1982; Loescher, 1987). However, our results indicated that sucrose but not mannitol was translocated in snapdragon. Thus, the specific action of mannitol observed in the present study cannot be attributed to carbohydrate translocation. In the present study, mannitol suppressed the senescence of spike tips and promoted stem growth, but spikes treated with mannitol excreted a white mannitol powder on the surface of flowers (Fig. 3). This was due to the low solubility of mannitol, and the effect would markedly reduce the flower’s ornamental value. Ichimura and Hisamatsu (2006) previously reported that combined treatment with glucose and mannitol extends the vase life of cut snapdragons more than treatment with mannitol alone or glucose alone. However, the white mannitol powder appeared in spikes treated with all solutions containing mannitol. Thus, mannitol may not be suitable for the vase life of cut snapdragon. Other than snapdragons, there are various cut flowers with indeterminate inflorescences, including freesia, gladiolus, stock, and Tweedia caerulea. As observed in cut snapdragons treated with glucose or sucrose, spike tip growth in these cut flowers terminates during vase life, resulting in the failure of flower opening at upper positions, although the cut flowers are treated with these sugars
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K. Ichimura et al. / Postharvest Biology and Technology 113 (2016) 20–28
(unpublished data). In contrast, the termination of spike tip growth was suppressed by mannitol treatment, which was associated with moderate accumulation of carbohydrate in spike tips (Fig. 7). Thus, we propose that the termination of spike growth in these flowers may be suppressed by treatments with some carbohydrates that maintain carbohydrate of spike tips at appropriate levels. However, mannitol treatment caused deleterious effects on cut snapdragon, despite being the major carbohydrate in this plant (Fig. 3). In addition, mannitol treatment causes chemical injury to cut chrysanthemum and rose in which mannitol is not present (Kofranek and Halevy 1972; Ichimura et al., 1999a). We are exploring carbohydrates that are metabolized to the same degree as mannitol. The vase life of cut flowers will be improved by treatments including these carbohydrates. 5. Conclusion Mannitol treatment delayed senescence of spike tips, resulting in marked stem growth and promotion of bud development in cut snapdragon flowers. This effect was not observed in glucose, sucrose, and sorbitol treatments, resulting in the marked extension of overall vase life. Pulse-chase experiments with 14C-carbohydrates showed that mannitol metabolized slower than glucose and sucrose. Degradation of nuclei, a parameter of PCD, in the spike tips was suppressed by mannitol treatment, compared with other carbohydrates. Treatment with mannitol suppressed an increase in soluble carbohydrate concentrations in spike tips more than the other carbohydrates. Water potential and osmotic potential in the spike tips was modestly decreased by the mannitol treatment, resulting in the suppression of senescence. The other carbohydrates increased the carbohydrate concentrations in spike tips and decreased osmotic potential, resulting in their senescence. Acknowledgement The authors thank Mrs. K. Matsuda for her technical assistance. References Anderson, L., Wolter, K.E., 1966. Cyclitols in plants: biochemistry and physiology. Annu. Rev. Plant Physiol. 17, 209–222. Bieleski, R.L., 1982. Sugar alcohols. In: Loewus, F.A., Tanner, W. (Eds.), Encyclopedia of Plant Physiology, New Series 13A. Springer-Verlag, Berlin, pp. 158–192. Chong, C., Taper, C.D., 1972. Malus tissue cultures. I. Sorbitol (D-glucitol) as a carbon source for callus initiation and growth. Can. J. Bot. 50, 1399–1404. Chong, C., Taper, C.D., 1974. Malus tissue cultures. II. Sorbitol metabolism and carbon nutrition. Can. J. Bot. 52, 2361–2364. Coffin, R., Taper, C.D., Chong, C., 1976. Sorbitol and sucrose as carbon source for callus culture of some species of the Rosaceae. Can. J. Bot. 54, 547–551. Gilbert, G.A., Wilson, C., Madore, M.A., 1997. Root-zone salinity alters raffinose oligosaccharide metabolism and transport in coleus. Plant Physiol. 115, 1267– 1276. Halevy, A.H., Mayak, S., 1979. Senescence and postharvest physiology of cut flowers, part 1. Hortic. Rev. 1, 204–236. Halevy, A.H., Mayak, S., 1981. Senescence and postharvest physiology of cut flowers, part 2. Hortic. Rev. 3, 59–143. Ichimura, K., 1998. Improvement of postharvest life in several cut flowers by the addition of sucrose. JARQ 32, 275–280. Ichimura, K., Hiraya, T., 1999. Effect of silver thiosulfate complex (STS) in combination with sucrose on the vase life of cut sweet pea flowers. J. Jpn. Soc. Hortic. Sci. 68, 23–27. Ichimura, K., Hisamatsu, T., 1999. Effects of continuous treatment with sucrose on the vase life, soluble carbohydrate concentrations, and ethylene production of cut snapdragon flowers. J. Jpn. Soc. Hortic. Sci. 68, 61–66. Ichimura, K., Hisamatsu, T., 2006. Effects of continuous treatments with glucose, sucrose and mannitol on the vase life of cut snapdragon flowers. Bull. Natl. Inst. For. Sci. 5, 45–53.
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