Palatinose-hydrolyzing Activity and Its Relation to Modulation of Flower Opening in Response to the Sugar in Dianthus Species

Abstract

Palatinose (isomaltulose) is an analog of sucrose and was regarded as non-metabolizable in plant tissues until recently. In the present study, we found that crude extracts from carnation petals had activity to hydrolyze palatinose. Preliminary characterization of this activity using a crude enzyme extract from ‘Lillian’ carnation petals revealed that hydrolyzing activity was exhibited by α-glucosidase, which uses isomaltose and palatinose, both being α-1,6-glucosides, as substrates. Exogenous application of palatinose stimulated flower opening of carnation cultivars (Dianthus caryophyllus ‘Lillian’, ‘Pure Red’, and ‘Light Pink Barbara’), but suppressed it in D. barbatus ‘Shin-higuruma’. Palatinose-hydrolyzing activity was much higher in the extract from carnation than that from D. barbatus. These observations suggested that palatinose stimulated flower opening in carnation by supplying glucose and fructose, but suppressed it in D. barbatus, probably through the inhibition of general metabolism, similar to the action of α-glucosidase, caused by its excess accumulation.

Introduction

The vase life of cut ornamental flowers is the period from flower opening to senescence. It is necessary to slow down both processes to prolong the display time of the flowers. To achieve this final goal in the flower-opening process, we have recently studied the molecular mechanism of flower opening using cut carnation flowers as a model ornamental (Harada et al., 2010; Morita et al., 2011). Flower opening involves elongation, expansion, and outward bending of petals, which result from the enlargement of petal cells (Evans and Reid, 1988; Kenis et al., 1985; Koning, 1984). Sugar accumulation in petal cells reduces the petal water potential and promotes water influx into the petal cells, resulting in cell enlargement (Evans and Reid, 1988; Ho and Nichols, 1977; Ichimura et al., 2003). In general, sucrose is one of the prevailing sugars involved in these processes and is metabolized by invertase and sucrose synthase. Morita et al. (2011) showed the involvement of sucrose synthase gene (DcSUS1) expression in petal cell growth during the opening of carnation flowers.

Sucrose is cleaved into glucose and fructose by invertase and into UDP-glucose and fructose by the reverse reaction of sucrose synthase (Giegenberger and Stitt, 1993). These reactions are thought to be the initial step of sucrose metabolism, leading to flower opening. Therefore, if we could disrupt or inhibit sucrose metabolism in the petals of opening flowers, the flower-opening process could be slowed down, resulting in prolonged display time of the flowers. With this aim in mind, we preliminary tested the effects of some disaccharides analogous to sucrose on the flower opening of Dianthus species, and found that palatinose (isomaltulose, Fig. 1) promoted flower opening in carnation (Dianthus caryophyllus L.) but suppressed it in D. barbatus. Palatinose was regarded as a non-metabolizable sugar in plants for a long period (Fernie et al., 2001; Sinha et al., 2002). However, we found that petals of carnation and D. barbatus flowers have activity to hydrolyze palatinose to glucose and fructose. This paper shows our findings on the hydrolyzing activity of palatinose in carnation petals, the opposite action of palatinose on flower opening between carnation and D. barbatus, and its possible mechanism in flower opening.

Fig. 1.

Structures of sucrose, palatinose, and isomaltose. Sucrose is α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside (β-d-fructofuranosyl-(2→1)-α-d-glucopyranoside), palatinose α-d-glucopyranosyl-(1→6)-α-d-fructofuranose, and isomaltose α-d-glucopyranosyl-(1→6)-α-d-glucopyranose.

Materials and Methods

Plant materials and determination of flower-opening profiles

Cut flowers of carnation cultivars (Dianthus caryophyllus L. ‘Lillian’, ‘Light Pink Barbara’, and ‘Pure Red’), which belong to the spray category of carnation flowers, were harvested when the first flower (floret) out of five to six flower buds on a stem was nearly open at the nursery of commercial growers in Kagawa prefecture (‘Lillian’) and Miyagi prefecture (‘Light Pink Barbara’ and ‘Pure Red’). The flowers were transported dry to the laboratory at Kyoto Prefectural Institute of Agricultural Biotechnology in Kyoto Prefecture the day after harvest. They were placed in plastic containers with their cut stem end in water under continuous light from white fluorescent lamps (14 μmol·m⁻²·s⁻¹ PPFD) at 23°C. The flower-opening process was separated into 6 stages according to Harada et al. (2010). Details of the respective stages are shown in Morita et al. (2011).

For examination of the flowering process affected by palatinose application, ‘Lillian’ flowers at opening stage 1 (Os 1), when petals have just emerged from buds (Harada et al., 2010), were used. A bunch of 20 florets with 15-cm stems was placed with their cut stem ends in a 900-mL glass jar with 500 mL test solution. Test solutions were water (control) and 1% (w/v) palatinose. When flowering profiles were compared between 1% palatinose and 1% (w/v) sucrose, florets with 10-cm stems were detached from ‘Lillian’, ‘Light Pink Barbara’, and ‘Pure Red’ flowers at Os 1–2 and bunches of 10 florets each were used per treatment.

The 1% (w/v) solution of the disaccharides corresponded to 29.3 mM. Test solutions contained a bactericide (Legend MK, a synonym of Kathon biocide; a mixture of 5-chloro-2-methyl-1,2-thiazol-3-one and 2-methyl-1,2-thiazol-3-one; Rohm and Haas Co., Philadelphia, PA, USA) at 100 μL·L⁻¹. The flowers were left under the same conditions as described above, and the flower-opening process was checked every day and photographed.

Flowers of D. barbatus ‘Shin-higuruma’ were harvested at the nursery of the Research Institute of Environment, Agriculture and Fisheries, Osaka Prefecture, and transported dry to Kyoto Prefectural Institute of Agricultural Biotechnology on the day of harvest. After arrival, the flowers were treated as above for carnation flowers. For determination of flower (bud) opening, a bunch of 5 flowers, each composed of more than 10 clusters of florets (actually closed buds), was treated as described above for carnation. Determinations of flower-opening profiles after treatment with disaccharides were repeated more than three times, and typical results are shown.

Preparation of crude enzyme extracts

For enzyme extraction, petals were detached from fully opened flowers (Os 6) of three carnation cultivars and D. barbatus, and petals of ‘Pure Red’ carnation at Os 3, to make sub-samples of 5 g each, and were stored at −80°C until extraction of enzymes. To obtain crude enzyme extracts, a 5-g sample of frozen petals was pulverized with a chilled mortar and pestle. After being thawed, the sample was homogenized for 10 min in 20 mL extraction buffer [100 mM HEPES-NaOH (pH 7.5) containing 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol]. The supernatant was recovered by centrifugation at 17,400 × g for 15 min and subjected to ammonium sulfate fractionation. The precipitate between 30–70% (w/w) saturation was obtained and dissolved in 2.5 mL elution buffer (10% glycerol was omitted from the extraction buffer). The sample was used as the crude enzyme preparation after the removal of contaminated ammonium sulfate by passing through Disposable PD-10 Desalting Columns (GE-Healthcare UK Ltd, Buckinghamshire, England). Protein contents were determined according to the method of the Bio-Rad Bradford protein assay kit (Bio-Rad Lab., Hercules, CA, USA) with BSA as a standard. Enzyme preparation was carried out at 4°C, and the obtained crude enzyme preparation was stored at −80°C until use.

Enzyme assays

For determination of the hydrolyzing activities of palatinose, a sample of 50 μL enzyme extract was incubated with 100 mM Na-acetate buffer (pH 4.5) and 25 mM palatinose in a total volume of 0.2 mL. The reaction was conducted at 30°C for 1 h. The reaction time was lengthened to 2 or 3 h when necessary, and the pH of the reaction mixture was changed from pH 4.0 to pH 5.7. To see the effects of α-glucosidase inhibitors on the hydrolysis of palatinose by the protein extracts, voglibose or miglitol was added at 10 mM to the reaction mixture. On the other hand, when isomaltase activity in the crude enzyme extract was measured, palatinose was replaced with isomaltose for the substrate. Moreover, to determine the effect of isomaltose on palatinose-hydrolyzing activity in the enzyme extracts, isomaltose at 0, 25, 50 mM was added to the reaction mixture. In this case, only the amount of fructose formed was determined. The control reaction was carried out with the enzyme samples preheated to 100°C for 3 min. The enzyme reactions were conducted with two or three replicates.

The enzyme reaction was stopped by heating at 100°C for 3 min. Then the mixture was immediately cooled in ice-water and centrifuged at 18,600 × g for 5 min to spin down the denatured proteins. Aliquots of the supernatant of the reaction mixture were used for the enzymatic assay of produced glucose or glucose + fructose using F-kit (TC d-glucose/d-fructose, Cat. No. 139106; Roche Diagnostics K.K., Tokyo, Japan).

To determine the effect of palatinose on α-glucosidase activity, p-nitrophenyl-α-d-glucopyranoside, a synthetic substrate for α-glucosidase, at 10 mM was included as the substrate in the above reaction mixture and palatinose at 0, 20, and 50 mM as an inhibitor. To stop the enzyme reaction, 1 mL of Na₂CO₄ was added to the reaction mixture. After measuring A₄₀₅, the amount of p-nitrophenol formed was calculated using its molecular coefficient, ɛ = 1.78 × 10⁴ M⁻¹·cm⁻¹.

Chemicals

Palatinose monohydrate and isomaltose were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), voglibose and miglitol from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and sucralose from Sigma-Aldrich Japan K.K. (Tokyo, Japan). Isopyranosucrose was obtained from Dr. S. Onodera (Rakuno Gakuen University, Ebetsu, Japan) and Dr. H. Okada (Ohtaka Kohso K.K., Otaru, Japan). The purity of palatinose monohydrate was >98% by HPLC analysis.

Results and Discussion

Palatinose-hydrolyzing activity in the crude extracts from carnation petals

We found that crude extracts from carnation petals had activity to hydrolyze palatinose. Therefore, we characterized this activity using the crude extract from ‘Lillian’ carnation petals. The crude enzyme extract hydrolyzed palatinose, producing glucose and fructose in the ratio of about 1 : 1.2 (n = 3). The ratio should be 1 : 1 when palatinose is cleaved. The biased yield of fructose might have been caused by the unknown metabolism of produced glucose or by the unequal quantitation of glucose and fructose by enzymatic determination using the F-kit, but we did not investigate further. The hydrolyzing reaction progressed linearly until at least 3 h when the reaction was monitored by measuring the amount of glucose formed. The crude enzyme extract also had activity to hydrolyze isomaltose, producing glucose. The optimum pH for the hydrolysis reaction was pH 4.5 for both palatinose and isomaltose (Fig. 2A). The hydrolyzing activity to isomaltose was 3.1±0.3 (n=3) times higher than that to palatinose when both substrates were used at 25 mM. The α-glucosidase inhibitors, miglitol and voglibose, at 10 mM inhibited severely the hydrolysis of isomaltose and palatinose (Fig. 2B): inhibition of isomaltose hydrolysis by miglitol and voglibose was 99.5% and 97.2%, respectively, and 94.2% and 89.5%, respectively, of palatinose hydrolysis.

Fig. 2.

Some characteristics of palatinose-hydrolyzing activity in the crude extract from petals of ‘Lillian’ carnation. A. Optimum pH for the reaction. The initial palatinose concentration was 20 mM. Data for isomaltase at 20 mM are shown for comparison. ●, palatinose; ○, isomaltose. B. Inhibition by voglibose and migtitol, both inhibitors of α-glucosidase. The reactions were conducted with 20 mM palatinose or isomaltose and 10 mM of either inhibitor. C, control; V, voglibose; M, miglitol. C. Inhibition of palatinose-hydrolyzing activity by isomaltose. Production of fructose from 20 mM palatinose was assayed in the absence or presence of 25 and 50 mM isomaltose. The graph was made by combining two separate experiments. The control activity for 25 mM palatinose was 0.196 μmol·h⁻¹·mg⁻¹ protein, and that for 50 mM palatinose 0.148 μmol·h⁻¹·mg⁻¹ protein. D. Inhibition of α-glucosidase activity by palatinose. α-Glucosidase activity was assayed by the formation of p-nitrophenol from p-nitrophenyl-α-d-glucopyranoside in the absence or presence of 20 and 50 mM palatinose. Control activity = 0.130 μmol·h⁻¹·mg⁻¹ protein. In all experiments, each determination was conducted with 2 or 3 replications. In B, data are shown by the mean ± SE of 3 replicated determinations.

Interestingly, addition of isomaltose to the enzyme reaction mixture using palatinose as the substrate reduced the amount of fructose produced from palatinose. The reduction of the amount of fructose formed was 19.6% and 34.6% at 25 and 50 mM isomaltose, respectively (Fig. 2C), when the concentration of palatinose was 25 mM. Moreover, palatinose inhibited α-glucosidase activity in the crude extract when activity was measured using p-nitrophenyl-α-d-glucopyranoside (pNPGP) as a substrate. When the substrate pNPGP concentration was 10 mM, 20 and 50 mM palatinose inhibited the hydrolysis of pNPGP by 17.1% and 30.1%, respectively (Fig. 2D).

The hydrolysis reaction showed Michaelis-Menten kinetics on each of three substrates, palatinose, isomaltose, and pNPGP (data not shown), and Lineweaver-Burk plots gave Km and Vmax as follows: 3.6 mM and 0.161 μmol·h⁻¹·mg⁻¹ protein for palatinose, 11.4 mM and 0.685 μmol·h⁻¹·mg⁻¹ protein for isomaltose, and 8.0 mM and 0.500 μmol·h⁻¹·mg⁻¹ protein for pNPGP. Palatinose had Km and Vmax lower than those of isomaltose and pNPGP.

Overall, based on these findings, we suggest that palatinose was hydrolyzed by α-glucosidase, which also uses as substrates isomaltose and palatinose, both being α-1,6-glucosides (Fig. 1). This situation is different from that in mammals. Apart from its use as a signal molecule in plant science, palatinose has been used widely as a sweetener alternative to sucrose, which is metabolized slowly and alleviates diabetes in humans. Goda and Hosoya (1983) showed that palatinose was hydrolyzed by a distinct enzyme, isomaltase, in a sucrase-isomaltase complex from the rat small intestine.

As described in the Introduction, palatinose was regarded as non-metabolizable in plant cells (Fernie et al., 2001; Sinha et al., 2002). Just recently, however, it has been reported that palatinose can be metabolized in cultured cells and stalk tissues of sugarcane, and fruit tissues of grape and nectarine, although the precise enzymatic nature responsible for its hydrolysis has not been fully characterized (Wu and Birch, 2011). Wu and Birch (2011) documented that palatinose [α-d-glucopy-ranosyl-(1→6)-α-d-fructofuranose] was hydrolyzed by sucrose synthase (SuSy) and soluble acidic invertase (SAI) under SAI assay conditions, extracted from cultured sugar cane cells and grape and nectarine fruit tissues. They also mentioned that sugar cane SAI hydrolyzes leucrose [α-d-glucopyranosyl-(1→5)-α-d-fructopyranose] and turanose [α-d-glucopyranosyl-(1→3)-α-d-fructopyranose] in addition to palatinose. Invertase (EC 3.2.1.26) is β-d-fructofuranoside fructohydrolase (accepted name: β-fructofuranosidase) whose activity is defined as hydrolysis of terminal non-reducing β-d-fructofuranoside residues in β-d-fructofuranosides, such as sucrose [α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside], raffinose [α-d-galactopyranosyl-(1→6)-α-d-glucopyranosyl-(1→2)- β-d-fructofuranoside], and stachyose [α-d-galactopyranosyl-(1→6)-α-d-galactopyranosyl-(1→6)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside].

Palatinose, leucrose, and turanose do not have non-reducing β-d-fructofuranoside residues, but have reducing residues. Therefore, palatinose-hydrolyzing activity in the extract of cultured sugar cane cells, and nectarine and grape tissues does not fit the definition of invertase, suggesting the involvement of another hydrolyzing enzyme(s); this was possible since Wu and Birch (2011) used crude enzyme extracts that were prepared by passing the homogenate of plant tissue through a gel-filtration column for desalting (Wu and Birch, 2011).

SuSy requires UDP for hydrolysis of sucrose and probably palatinose. In the present study, UDP was not included in the reaction mixture for the palatinose hydrolysis assay using an extract of carnation petals. This indicated that the palatinose-hydrolyzing activity detected in the present study did not involve SuSy.

Promoting or suppressing action of palatinose on flower opening of Dianthus species

In a preliminary experiment, we examined the effects of several disaccharides analogous to sucrose, i.e., trehalose, isomaltose, isopyranosucrose, sucralose, and palatinose, at 0.3–3% (w/v) on flower opening of ‘Lillian’ carnation. Trehalose, isomaltose, and isopyranosucrose had no notable effects on flower opening as compared with the control (water). Sucralose, which is a sucrose derivative containing three Cl atoms in its molecule, had detrimental effects, browning of petals and leaves, and spoiled ornamental value of the flowers. Only palatinose was found to have a promoting effect on the flower opening of ‘Lillian’ carnation. Therefore, we concentrated our study on the effect of palatinose in the following experiments.

Continuous application of palatinose at 1% (w/v) enhanced the flower opening of ‘Lillian’ carnation (Fig. 3A). Its promoting effect was seen 5 days after the start of the experiment. The maximum effect was observed on day 10, and at that time the diameter and volume of opened flowers were bigger in flowers treated with palatinose than in control flowers. The magnitude of promotion by 1% (w/v) palatinose was similar to that by 1% (w/v) sucrose 6 days after the start of treatment (Fig. 3B). In addition, 1% (w/v) palatinose promoted flower opening in ‘Light Pink Barbara’ carnation, but to a lesser extent than ‘Lillian’ carnation (data not shown). Moreover, 1% (w/v) palatinose tended to stimulate flower opening in ‘Pure Red’ carnation even 2 days after the start of treatment when sucrose and glucose had no effect on flower opening. On day 5, the promoting effects on flower opening in ‘Pure Red’ carnation were similar among palatinose, sucrose and glucose (Fig. 3C).

Fig. 3.

Flowering opening profiles of ‘Lillian’ and ‘Pure Red’ carnation as stimulated by 1% palatinose. A. Bunches of ‘Lillian’ carnation flowers, 20 detached florets with 15-cm stems at Os 1, were left for given periods at 23°C, with their cut stem ends in water (control) or 1% (w/v) palatinose solution. B. Bunches of 8 ‘Lillian’ flowers at Os 2 were treated for 6 days without and with 1% (w/v) palatinose or 1% (w/v) sucrose. C. Comparison among palatinose, sucrose and glucose in the promotion of flower opening of ‘Pure Red’ carnation. ‘Pure Red’ carnation flowers at Os 1 were treated without or with the respective sugars (all at 1%) for the given days. Data are the mean of 4 replications, each with 5 flowers. Data for the control, glucose and sucrose are the same as those shown previously (Satoh et al., 2013). Data for each day were not significant by Steel’s multiple range test (p <0.05). ●, control (H₂O); ○, 1% (w/v) palatinose; □, 1% (w/v) glucose; ▵, 1% (w/v) sucrose.

By contrast, 1% (w/v) palatinose suppressed the flower opening of D. barbatus ‘Shin-higuruma’ when the chemical was applied continuously (Fig. 4). The inhibitory effect of palatinose was seen 5 days after the start of incubation, and thereafter a small number of buds opened during the incubation period until day 15. Most of the unopened buds were dead and dried-up on day 15.

Fig. 4.

Suppression by palatinose of flower opening in D. barbatus ‘Shin-higuruma’. Bunches of 8 flowers with 15-cm stems and almost closed buds (Os 1) were left with their cut stem ends in water (control) or 1% (w/v) palatinose solution for the given days at 23°C.

The magnitude of the promoting effect of palatinose was similar to that of sucrose in ‘Lillian’ carnation 6 days, and in ‘Pure Red’ carnation 5 days after the start of incubation (Fig. 3B, C). Sucrose was shown to lengthen the vase life of cut carnation flowers (Mayak and Dilley, 1976; Mayak and Kofranek, 1976), when it was applied to fully opened carnation flowers. On the other hand, sucrose has been shown to promote bud opening (flower opening) in many flowers, such as rose (Kuiper et al., 1995), liatris (Borochov and Keren-Paz, 1984), Gipsophyla (Downs et al., 1988), and carnation (Koyama and Uda, 1994; Minakuchi et al., 2007; Satoh et al., 2005).

Possible mechanism of promotion and inhibition by palatinose of flower opening in Dianthus species

In the course of our study, we first assumed that palatinose might affect (inhibit) sucrose metabolism mediated by invertase or by the reverse reaction of sucrose synthase in carnation and D. barbatus petals undergoing flower opening. However, separately from the work by Wu and Birch (2011), we found that crude protein fractions extracted from carnation and D. barbatus petals could hydrolyze palatinose, producing glucose and fructose. After this observation, we tried to reveal the nature of palatinose-hydrolyzing activity as described above, then tried to relate the activity to its effects on flower opening of carnation and D. barbatus.

We compared the activity of palatinose hydrolysis among crude extracts from petals of carnations and D. barbatus (Fig. 5). All of the extracts from petals of ‘Lillian’, ‘Light Pink Barbara’, and ‘Pure Red’ carnation and D. barbatus ‘Shin-higuruma’ had hydrolyzing activities on palatinose. However, the hydrolyzing activities were 2.8–5.4 times higher in the extracts from carnation petals than from D. barbatus petals when activity was measured with 25 mM palatinose.

Fig. 5.

Comparison among carnation cultivars and B. barbatus ‘Shin-higuruma’ of palatinose-hydrolyzing activity in crude extracts from petals. Os 3 and Os 6 are flowers at opening stage 3 and 6 (fully open flowers). Histograms with different letters are significantly different by Tukey’s multiple range test (p < 0.05). LPB, ‘Light Pink Barbara’.

Based on these findings, we speculated the mechanism of action of palatinose on the promotion of flower opening in carnation and suppression in D. barbatus. In carnation petals, in which palatinose hydrolysis is high, exogenously applied palatinose is hydrolyzed forming glucose and fructose, which in turn have promoting effects on flower opening. This situation is probably similar to that of exogenously applied sucrose, which is hydrolyzed by invertase, forming glucose and fructose, resulting in the promotion of flower opening (Fig. 3B, C). We actually observed the presence of high invertase activity in carnation petals (data not shown). In contrast, in D. barbatus, in which there is less palatinose-hydrolyzing activity, exogenously applied palatinose might accumulate without hydrolysis and inhibit α-glucosidase activity, resulting in malfunction of basic sugar metabolism in petal cells and eventually repression of flower opening. This can be anticipated since Kashimura et al. (2008) showed that palatinose competed with substrate α-glucosyldisaccharides and inhibited their hydrolysis in the rat small intestine, and we actually observed the suppression of α-glucosidase activity to hydrolyze a synthetic substrate, p-nitrophenyl-α-d-glucopyranoside, by palatinose (Fig. 2D).

In conclusion, the present study showed that palatinose promoted flower opening in carnation, whereas suppressed it in D. barbatus. Petals of carnation and D. barbatus had activity to hydrolyze palatinose, which was previously thought not to be metabolized in plants. The opposite action of palatinose in carnation and D. barbatus may be caused by differences in its hydrolyzing activity.

Acknowledgment

We thank Dr. S. Onodera (Rakuno Gakuen University) and Dr. H. Okada (Ohtaka Kohso K.K.) for providing isopyranosucrose.

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