Ryo Norikoshi’s research while affiliated with Tokyo University of Agriculture and other places

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.

Lily bulbs of Oriental hybrid ‘Casa Blanca’ produced in Hokkaido or Niigata were packed in polyethylene bags with wet peat moss and subjected to pre-chilling at various temperatures for various durations from 0 to 20 weeks before storing at –2.0°C. On moving to –2.0°C, bulbs were dissected and the shoot length and Brix of shoot tip juice were measured. Soluble carbohydrate concentrations of the shoot tip were analyzed by a conventional extraction method using hot ethanol with HPLC. After storage at –2.0°C for more than 7 months, they were grown in a glass house kept at a minimum temperature of 15°C to check for the occurrence of black shoots, non-flowering plants, and damaged leaves, which might be caused by long-term storage at –2.0°C. Rates of black shoots, non-flowered plants, and damaged leaves became high when bulbs were subjected to pre-chilling at 1°C for more than 12 weeks. The rise in rates and decrease of the sucrose content occurred simultaneously. Furthermore, changes in the sucrose content were closely related to changes of Brix. When bulbs were pre-chilled at 1, 6, 8, or 12°C for 8 weeks followed by 1°C for 10 weeks, those pre-chilled at 1 or 6°C showed a decreased sucrose content or Brix at the end of pre-chilling and higher rates of non-flowering plants and damaged leaves after storage at –2.0°C for 24 weeks. An extended duration of storage at –2.0°C for 8 weeks induced black shoots and non-flowering plants when bulbs were pre-chilled at 1°C for 18 weeks before moving to –2.0°C. From these results, it was concluded that excessively long pre-chilling of bulbs at low temperatures below 6°C induces black shoots and leaf damage after long-term storage at a subzero temperature.

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.

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.

Treatment with sucrose promoted petal growth associated with flower opening in cut roses. We investigated the effect of sucrose treatment on cell size and subcellular concentration of soluble carbohydrates in petals of cut rose cv. Sonia flowers. Petals of sucrose-treated flowers, but not control flowers, markedly curved outward, resulting in complete reflection. Petal fresh weight (FW), petal area, and adaxial epidermal cell size in the control flowers increased with time, and treatment with sucrose accelerated this increase, indicating that sucrose promotes petal cell expansion. Glucose, fructose, sucrose, methyl glucoside, and xylose were detected in the petals. In the petals of control flowers, concentration of these carbohydrates, except fructose, decreased. Sucrose treatment markedly increased glucose and fructose concentrations in petals. Estimation of subcellular volumes based on transmission electron micrographs showed that volume of cell walls and vacuoles in the petals of control flowers increased in response to sucrose treatment. Sucrose treatment increased glucose and fructose concentrations in the vacuole and glucose, fructose, and xylose concentrations in the apoplast. We concluded that sucrose treatment increases glucose and fructose concentrations in the vacuole, which may reduce the osmotic potential of the symplast and increase water uptake leading to cell expansion during flower opening.

Petal growth associated with flower opening is due to cell expansion. To elucidate the role of soluble carbohydrates in expansion of petal cells in Eustoma grandiflorum, its soluble carbohydrates were identified, and changes in their subcellular concentrations during flower opening were investigated. In addition to glucose, fructose, sucrose, and myo-inositol, d-bornesitol was identified using 1H-NMR. d-Bornesitol was the major soluble carbohydrate in leaves and stems. Given that cyclitols are known to be the translocated carbohydrates in alfalfa, phloem exudate was analyzed. However, the translocated carbohydrate was suggested to be sucrose, and not d-bornesitol. In the petals, glucose and sucrose content increased whereas d-bornesitol and myo-inositol contents were almost constant during flower opening. The fructose content in petals was very low. Glucose, sucrose, myo-inositol, and d-bornesitol were found mainly in the vacuole, although sucrose was also found in the cytoplasm. In the petals of open flowers, glucose and sucrose concentrations in the vacuole increased to 60 and 53 mM. Inorganic ion concentrations in the symplast and apoplast did not increase during flower opening. The osmotic potential of the symplast and apoplast in the petals was lower at the open stage than the potential of those at the bud stage, and this difference was mainly attributed to increases in glucose and sucrose concentrations. The results suggest that the accumulation of glucose and sucrose in the vacuole reduces the symplastic osmotic potential, which appears to be involved in the cell expansion associated with flower opening, but that the contribution of d-bornesitol as an osmoticum to cell expansion is limited in Eustoma.

Supplementary material 1 . Scanning electron micrographs of adaxial and abaxial epidermal cells in the sepals during flower opening. Adaxial epidermal cells at stage 1 (a), stage 2 (b) and stage 3 (c). Abaxial epidermal cells at stage 1 (d), stage 2 (e) and stage 3 (f). The scale bars represent 50 μm

We investigated morphological changes in petal cells during flower development and opening in Eustoma grandiflorum. The morphology of petal epidermal cells was observed by scanning electron microscopy, and their number was determined. The numbers of adaxial and abaxial epidermal cells increased during flower development. Increase in these numbers terminated before flower opening earlier in abaxial than in adaxial epidermal cells. Measurements of cell number and area showed that the petal growing stage during flower development and opening can be divided into four phases: cell division and expansion, cell division, cell division and expansion, and cell expansion. Adaxial epidermal cells in the petal blade showed a conical-papillate shape whereas adaxial epidermal cells in the petal claw were longitudinally elongated in shape. Abaxial epidermal cells were longitudinally elongated in both petal blade and claw. The ultrastructure of petal cells at the bud stage and the open stage was observed by transmission electron microscopy. In the petal cells at the bud stage, nuclei and several plastids were observed, although the cells were mainly occupied with vacuoles. Relatively large spherical electron-dense bodies were observed only in the vacuoles of adaxial epidermal cells at the bud stage. The petal cells were largely occupied with enlarged vacuoles at the open stage. We conclude that petal growth in Eustoma is divided into four phases, based on the activities of cell division and expansion, and that petal growth in the final phase is mainly due to cell expansion with marked enlargement of vacuoles.