Kazuo Ichimura’s research while affiliated with National Agriculture and Food Research Organization and other places

The vase life of cut dahlia (Dahlia × hortensis Guillaumin) flowers is generally short. Petal senescence is known to be divided into petal wilting, withering, and abscission. Petals with ongoing wilting were isolated from the inflorescence and supplied with water using 10 cultivars to clarify the type of petal senescence of dahlia. Based on the recovery of fresh weight and appearance of petals, senescent types could be categorized into three types: petal senescence in two, six, and two cultivars were categorized into abscission, abscission with withering, and wilting types, respectively. The pulse treatment with silver thiosulfate complex (STS) significantly extended the vase life of 7 out of 10 cultivars. Moreover, pulse treatment with STS followed by continuous treatment with GLA, which was comprised of glucose, isothiazolinone germicide, and aluminum sulfate, increased the relative fresh weight of the five dahlia cultivars more than pulse treatment with STS. Therefore, this combined treatment is available to extend the vase life of cut dahlia, irrespective of the senescent types.

We investigated the effects of light, darkness, and temperature during storage assuming transportation on flower wilting and the flowering period of potted carnation (Dianthus caryophyllus L.). In the two cultivars ‘Grand Rouge’ and ‘Mahalo’, the flowering period was shortened by more than 2 days and the rate of flower wilting after 7 days was increased to 30 percent under the dark condition at 20°C compared with the light condition (PPFD; 110–214 μmol・m–2・sec–1・12 hours). Ethylene production was observed in the wilted flowers of ‘Mahalo’. In the two cultivars, treatment with 1-methylcyclopropene (1-MCP) at 1000 ppb for 12 hours extended the flowering period by more than 1 day and reduced the rate of flower wilting after 7 days by more than 30 percent under the dark condition at 20°C compared with the control. The sugar (sucrose, glucose, and fructose) contents in the leaf of ‘Grand Rouge’ stored for 16 days in the dark at 10°C was 1.75 mg・g–1 FW, which was more than twice as high as at 20°C. In the two cultivars, the flowering period of the plants stored at 10°C was more than 2 days longer than that of those stored at 20°C, and flower wilting was not observed even after 14 days in the dark. In addition, the flower color of plants stored at 10°C for 14 days was almost the same as that before storage.

Delphinium flowers are highly sensitive to ethylene and its sepals abscise during senescence in association with an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) activities and ethylene production in gynoecium and receptacle. Three ACS genes (DgACS1, DgACS2, and DgACS3) and three ACO genes (DgACO1, DgACO2, and DgACO3) of D. grandiflorum cv. Super Grand Blue were cloned. To investigate the contribution of these genes to ethylene production, their expression in the gynoecium and receptacle was analyzed during natural senescence and after ethylene exposure and pollination. Ethylene production in the receptacle significantly increased, whereas that in the gynoecium exhibited only a slight increase during natural senescence. The transcript levels of ACS and ACO in these organs, excluding those of DgACS2 in the receptacle, increased during senescence. Exposure to ethylene accelerated sepal abscission and increased ethylene production more markedly in the receptacle than in the gynoecium. DgACS1 transcript levels in the gynoecium and DgACS2 and DgACO3 transcript levels in the receptacle increased after ethylene exposure. Pollination accelerated sepal abscission and increased ethylene production in the gynoecium and receptacle. Additionally, it slightly affected ACS and ACO transcript levels in the gynoecium, whereas DgACO3 transcript levels in the receptacle markedly increased. These results reveal that ACS and ACO expression is differently regulated in the gynoecium and receptacle. Furthermore, some of these genes are upregulated by pollination only in the receptacle, indicating the significance of the receptacle in ethylene biosynthesis associated with sepal abscission.

Flower opening is associated with the expansion of petal cells. To understand the role played by soluble carbohydrates during cell expansion associated with petal growth, changes in soluble carbohydrate concentrations in petal limbs during flower opening in Phlox drummondii were investigated. The size of adaxial and abaxial epidermal cells in the petal limbs gradually increased during flower opening. 2-C-Methyl-d-erythritol (2-C-ME) was identified using ¹H-NMR in P. drummondii petals. 2-C-ME was the most abundant carbohydrate in the petal limbs at five developmental stages, with the concentration of glucose the second highest, although the concentration of the latter was half of that of the 2-C-ME concentration in all five stages. The concentrations of 2-C-ME and glucose increased during flower opening. In contrast, inorganic ion concentrations did not increase during flower opening. The osmotic potential of petal limbs decreased considerably during the final stage of flower opening; this decrease could in part be attributed to the increasing 2-C-ME concentration. Transmission electron microscopic observations showed that the petal limb cells in open flowers were occupied primarily by the vacuole. The concentration of 2-C-ME in the vacuole was estimated to be 131 mM, which was much higher than the concentrations of the other carbohydrates. We conclude that the accumulation of 2-C-ME in the vacuole at a high concentration acts as an osmoticum, decreasing the osmotic potential of petal limbs and thereby increasing turgor pressure, which is thought to be involved in cell expansion of petal limbs during flower opening.

To understand the biochemical mechanism underlying flower opening, the mechanism of cell expansion, soluble carbohydrate concentration, and expression of expansin and xyloglucan endotransglucosylase/hydrolase (XTH) genes were investigated in the petals of Oriental lily (Lilium ‘Sorbonne’). Microscopic observation revealed that petal growth during flower opening mainly depended on cell expansion, which was accompanied by increases in glucose and fructose concentrations in the petals. The adaxial and abaxial sides of the petals grew at different rates during flower opening with petal reflection. To determine the concentration of soluble carbohydrates and the expression of expansin and XTH genes in adaxial and abaxial epidermal cells and parenchyma cells, these cells were separated using tweezers. We confirmed that these cells could be sufficiently separated. Glucose and fructose concentrations were higher in adaxial epidermal cells than in abaxial epidermal cells at the stage immediately preceding flower opening, but these differences diminished during flower opening. Three expansin genes, LhEXPA1, LhEXPA2, and LhEXPA3, and two XTH genes, LhXTH1 and LhXTH2 were isolated. LhXTH1 transcript levels in the petals markedly increased during flower opening and were higher in adaxial epidermal cells than in other types of cells. Conversely, the levels of the three EXPA transcripts decreased during flower opening and there were slight differences in their levels among different cell types, with a few exceptions. In conclusion, differences in glucose and fructose concentrations between adaxial and abaxial epidermal cells, together with the expression of LhXTH1, may contribute to cell expansion associated with flower opening.

Nigerosylmaltooligosaccharide (Nmo) has different degrees of polymerarization (DP) of nigerosyl oligosaccharides and maltooligosaccharides. We investigated the effects of treatments with Nmo, glucose, sucrose, and their combinations on the vase life of cut snapdragon (Antirrhinum majus) flowers. To apply these sugars, the cut ends of flower spikes were dipped into these sugar solutions. Treatment with 30 g L⁻¹ Nmo promoted bud development, resulting in flower opening at the upper part of the spikes. The Nmo treatment also extended the vase life of cut snapdragons more than glucose and sucrose treatments when vase life was evaluated as the time to when all flowers wilted. However, there were few open flowers, flowers were smaller and aurone levels were lower in the Nmo treatment, suggesting that treatment with Nmo alone is not sufficient for improving the vase life of cut snapdragons. Combined treatments of Nmo with glucose and sucrose increased the number of open flowers and significantly extended vase life more than treatment with glucose or sucrose alone. The deleterious effects of Nmo, such as the reduction in flower size and suppression of pigmentation, were alleviated by these combined treatments. Nmo was fractionated using a carbon celite column into DP2, DP3, DP4, and DP> 4. Spike length was significantly increased by fractions DP4 and DP>4, suggesting that oligosaccharides containing these fractions largely contribute to the development of spikes. In conclusion, Nmo is a readily available oligosaccharide that could be used in the manufacture of preservatives to improve the vase life of cut snapdragons.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) catalyzes the final step of ethylene biosynthesis. ACO proteins are also reportedly encoded by a multigene family. Although the presence of several ACO genes is suggested in carnation (Dianthus caryophyllus L.), DcACO1 is the only ACO gene in which full-length cDNA has been isolated. This study aimed to clone another ACO gene in carnation flowers and investigate changes the expression of the two ACO genes in floral organs during flower senescence. We cloned a homolog of the ACO gene from carnation gynoecium and designated it DcACO2. Transcript levels of DcACO2 were higher in the style and ovary than in the petals at harvest, although DcACO2 transcript levels in these organs increased during flower senescence. The ACO activity in the style was very high at harvest, suggesting that this high activity could be attributed to the translation of the DcACO2 transcript. However, DcACO2 transcript was only detected at very low levels in the petals of senesced flowers. Ethylene and ACC treatments accelerated petal wilting and increased ethylene production of the petals, style, and ovary. In the style and ovary, the DcACO1 transcript level was markedly higher than the DcACO2 transcript level as a result of ethylene and ACC treatments, and DcACO1 transcript levels in the petals were also increased by ethylene and ACC treatments. These results indicate that the expression of DcACO1 and DcACO2 are differently regulated among floral organs during senescence in carnation flowers.

Anthocyanin accumulation of petals is suppressed at high temperatures. We therefore investigated the effects of temperature and light intensity on anthocyanin biosynthesis in snapdragons (Antirrhinum majus). Snapdragon plants were placed under four environmental conditions, combining two temperatures, 20°C /14°C (moderate temperature) and 36°C /36°C (high temperature) and two photosynthetic flux density (PPFD), 50 μmol m⁻² s⁻¹ (low light intensity) and 200 μmol m⁻² s⁻¹ (high light intensity), and held for 4 days. Pelargonigin-3-rutinoside was only found in petals as an anthocyanin. Anthocyanin accumulation associated with pigmentation of petals was suppressed at high temperature, and this suppression was enhanced under low light. Transcript levels of AmCHS1, AmCHI, AmF3H, AmDFR, AmANS and AmUFGT, which are involved in anthocyanin biosynthesis, in petals were significantly lower at the high temperature than at the moderate temperature. Sugar contents in petals also were significantly lower at the high temperature than at the moderate temperature. To clarify whether sugar is involved in suppression of pigmentation at a high temperature, cut flowers were treated with 0.15 M sucrose. Sucrose treatment increased anthocyanin accumulation, and the sugar content in the petals of cut flowers held at the high temperature. Sucrose treatment also significantly increased AmCHS1, AmF3H, AmDFR, AmANS and AmUFGT transcript levels in the petals at the high temperature. The results suggest that suppression of anthocyanin accumulation at high temperatures under low light is involved in the suppression of anthocyanin biosynthetic genes, which may be partially mediated with decreased sugar contents in petals.

Delphinium flowers are highly sensitive to ethylene and its sepals abscise during senescence, which is associated with increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) activities and ethylene production in gynoecium and receptacle. Three ACS genes ( DgACS1 , DgACS2 , and DgACS3 ) and three ACO genes ( DgACO1 , DgACO2 , and DgACO3 ) were cloned from Delphinium grandiflorum cv. Super Grand Blue. To investigate the contribution of these genes to ethylene production, their expression was analyzed in these genes in the gynoecium and receptacle during natural senescence and following ethylene exposure and pollination. Ethylene production in the gynoecium and receptacle increased during natural flower senescence. The transcript levels of the ACS and ACO genes in these organs, excluding DgACS2 in the receptacle, increased during senescence. Exposure to ethylene accelerated sepal abscission and more strongly increased ethylene production in the receptacle than in the gynoecium. DgACS1 transcript levels in the gynoecium and DgACS2 and DgACO3 transcript levels in the receptacle were increased by ethylene exposure. Pollination accelerated sepal abscission and increased ethylene production in the gynoecium and receptacle. Pollination slightly affected ACS and ACO transcript levels in the gynoecium, whereas DgACO3 transcript level in the receptacle were markedly increased. These results reveal that ACS and ACO gene expression is differently regulated in the gynoecium and receptacle, and some of these genes are more strongly upregulated by ethylene exposure and pollination in the receptacle than in the gynoecium, suggesting the significance of the receptacle to sepal abscission.

Cut dahlia flowers have a short vase life of 3–7 days, and some cultivars exhibit petal abscission, even inside of cardboard boxes during transport. This petal abscission is induced by ethylene in many cases. In the present study, we investigated the role of ethylene in dahlia flower senescence by comparing the responses to ethylene, ethylene inhibitors, and ethylene production among cultivars. Exogenous ethylene significantly accelerated petal abscission in seven cultivars and petal wilting in other five cultivars out of 12 cultivars. Whole florets and detached receptacles (with bracts) produced different amounts of ethylene; ethylene production was higher in ‘Carnelian’ and ‘Port Light Pair Beauty’ than ‘Heavenly Peace’ and ‘Purple Stone’. Onset of senescence was delayed in detached petals compared with attached petals, suggesting that petal abscission was induced by ethylene produced by ovary and receptacles. The ethylene action inhibitor 1-methylcyclopropene inhibited petal abscission and delayed petal wilting in eight cultivars. Moreover, the silver thiosulfate complex delayed petal wilting of ‘Carnelian’. Together, our findings suggested that ethylene plays a role in senescence of cut dahlia flowers and ethylene inhibitors can extend their vase life.