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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.

To understand the biochemical mechanism underlying flower opening, the manner 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.

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.

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.

The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

Autophagy is one of the main mechanisms of degradation and remobilization of macromolecules, and it appears to play an important role in petal senescence. However, little is known about the regulatory mechanisms of autophagy in petal senescence. Autophagic processes were observed by electron microscopy and monodansylcadaverine staining of senescing petals of petunia (Petunia hybrida); autophagy-related gene 8 (ATG8) homologues were isolated from petunia and the regulation of expression was analysed. Nutrient remobilization was also examined during pollination-induced petal senescence. Active autophagic processes were observed in the mesophyll cells of senescing petunia petals. Pollination induced the expression of PhATG8 homologues and was accompanied by an increase in ethylene production. Ethylene inhibitor treatment in pollinated flowers delayed the induction of PhATG8 homologues, and ethylene treatment rapidly upregulated PhATG8 homologues in petunia petals. Dry weight and nitrogen content were decreased in the petals and increased in the ovaries after pollination in detached flowers. These results indicated that pollination induces autophagy and that ethylene is a key regulator of autophagy in petal senescence of petunia. The data also demonstrated the translocation of nutrients from the petals to the ovaries during pollination-induced petal senescence.

Tweedia caerulea flowers are sensitive to ethylene and the closing of the flowers, a characteristic of senescence, is accelerated by exposure to ethylene. T. caerulea flowers were continuously treated with ethanol at concentrations of 0, 2, 4, 6, 8, 10 or 12 %, and treatment levels at 4 % and above showed delayed closing. Ethanol accelerated climacteric increase in ethylene production from flowers. Although ethylene production was higher in gynoecium than in petals, ethanol treatment accelerated ethylene production by both organs. Exposure to ethylene increased autocatalytic ethylene production, and production was further accelerated by ethanol treatment. When flowers treated with ethanol were exposed to ethylene, senescence was delayed compared to that for untreated flowers, suggesting that ethanol reduces the sensitivity of flowers to ethylene. These results indicate that treatment with ethanol delays petal senescence in cut T. caerulea flowers, possibly through reduced sensitivity to ethylene.

Application of forchlorfenuron (CPPU) to flower buds induces morphologically different paracorollas, i.e., wide and narrow type, in torenia (Torenia fournieri L.). We investigated the morphological properties and the role of floral homeotic genes in the formation of these two types of paracorolla. The morphology of epidermal cells and distribution pattern of vascular bundles in the wide paracorolla was the same as in the petal; however, in the narrow paracorolla, the morphology of epidermal cells was either petal-like or stamen-like, and the distribution pattern of vascular bundles was stamen-like. In situ hybridization analysis of the floral homeotic genes showed that a class A gene, T. fournieri SQUAMOSA (TfSQUA), and class B genes, TfDEFICIENS (TfDEF) and TfGLOBOSA (TfGLO), were expressed in the broad part of the primordia of the wide paracorolla, as in the petal. Class C genes, TfPLENA1 (TfPLE1) and TfFARINELLI (TfFAR), were only expressed at the margin of the primordia. However, in the primordia of the narrow paracorolla, TfSQUA was only expressed at the margin, but the class B genes and one of the class C genes (TfPLE1) were expressed in a broad section, as in the primordia of the wide paracorolla. This expression pattern in the narrow paracorolla was intermediate between that of the petal and the stamen. In later developmental stages, quantitative real-time PCR analyses showed that, in the wide paracorolla, TfSQUA and class B genes were highly expressed but the expression of class C genes, as in the petal, was low. In the narrow paracorolla, class B genes were also highly expressed as in the petal; however, the expression of both TfSQUA and the class C genes was low, as in the stamen and the petal, respectively. This expression pattern probably reflects the unstable floral organ identity of the narrow paracorolla, and the expression pattern in paracorollas is determined by the site where the paracorolla is formed.

We previously reported that cytokinin (CK) application enlarged the corolla of petunia (Petunia hybrida Vilm.). In this paper, the effect of the large-flowered trait of petunia caused by the Grandiflora (G) allele to CK biosynthesis was investigated. Concentrations of endogenous CK nucleobases (i.e., N6-(Δ2-isopentenyl)adenine (iP) and trans-Zeatin (tZ)), CK nucleosides (iP riboside and tZ riboside), and a CK glucoside (iP-7-glucoside) in the developing corolla were markedly lower in large-flowered cultivars with a Gg genotype than small- and medium-flowered cultivars with a gg genotype. We isolated the cDNAs encoding CK biosynthesis enzymes expressed in the young corolla, i.e., cDNAs of an adenosine phosphate-isopentenyltransferase gene (SHO), a CK riboside 5'-monophosphate phosphoribohydrolase gene (PhLOG), and two CK oxidase/dehydrogenase genes (PhCKX1 and PhCKX2). Expression levels of SHO and PhLOG in the developing corolla were not markedly different among small-, medium-, and large-flowered cultivars. However, expression levels of PhCKX1 and PhCKX2, which catalyze CK degradation through removal of the isoprenoid side chain, were much higher in large-flowered cultivars than in small- and medium-flowered cultivars. Therefore, low CK concentrations in large-flowered cultivars are very probably induced by the promoted expressions of PhCKX1 and PhCKX2, which may partially repress corolla enlargement.

Expressions of the genes involved in the cytokinin (CK) phosphorelay signaling circuitry in petunia (Petunia hybrida Vilm.) were investigated with regard to the large-flowered trait induced by the semidominant Grandiflora (G) gene. We isolated three cDNAs encoding type-A response regulators (RRs, PhRR1-3) and one cDNA encoding CK receptor histidine protein kinase (PhHK). Expression levels of PhRR1-3 and PhHK in the developing corolla were markedly higher in large-flowered cultivars with the Gg genotype than in small- and medium-flowered cultivars with the gg genotype. Higher expression levels of PhRR1-3 and PhHK in the Gg than in the gg genotypes was also consistent in BC4 plants in which the genetic background except for G allele was mostly uniform. These results suggest that Gg genotype induces a high expression level of PhRR1-3 and PhHK. These changes in expression caused by the G allele were the same as those caused by the CK response. Since type-A RRs repress CK signaling, the G-allele-induced high expression levels of PhRR1-3 presumably have partial inhibitory effects on G-allele-induced corolla enlargement.

This study aimed to clarify the morphological properties and the role of homeotic genes in the development of paracorolla, a petaloid organ fused with stamens at the base in Antirrhinum majus. Two primordia of paracorolla initiated at the lateral and slightly outer (i.e., basal) position of a stamen primordium, suggesting that the paracorolla is a stipule of the stamen. In the developed paracorolla, expression of the A-class gene SQUAMOSA (SQUA) and the B-class genes DEFICIENS (DEF) and GLOBOSA (GLO) were high, while those of the C-class genes PLENA (PLE) and FARINELLI (FAR) were very low. In contrast, in the paracorolla primordia, expressions of SQUA and PLE were not detected. Thus, the expression patterns of homeotic genes in the paracorolla were basically the same as those of the petal, and this pattern was established during paracorolla development. In the stamen, which fused with the paracorolla at the base, DEF, GLO, PLE, and FAR were expressed in the anther. However, in the filament, SQUA was also expressed at a higher level in addition to these genes, indicating that the expression pattern shifted from the stamen type in the anther, to the petal type in the paracorolla, through the intermediate type in the filament. This gradient of expression pattern among these organs, which apparently belong to the same whorl of the flower, may be essential for the development of the paracorolla into a petaloid organ.

The effects of gellan gum gels and Murashige and Skoog (MS) inorganic salts at various strengths on the vase life of ‘Rote Rose’ rose cut flowers were investigated. To form the gels, gellan gum was dissolved with MS salts at various concentrations. To simulate the transport of cut rose flowers, stems were placed in gellan gum gels and held at 23°C for 24 h or 48 h. Cut flowers were then transferred into distilled water and their fresh weight (FW) and vase life were determined. According to the observed vase life and changes in FW, gels formed with 0.2% gellan gum and standard-strength MS salts appear to be the most suitable for cut roses. Although the hardness of gellan gum gel increases with increased concentrations of gellan gum and MS salts, no correlation between vase life and gel hardness was observed. To determine the potential water availability of gellan gum gel, gels were placed in a centrifuge filter device, centrifuged at 3000 × g for 10 min and the amount of water released from the gel was measured. A highly positive correlation between the amount of water released from the gel and water uptake of flowers placed in gellan gum gels was observed. A positive correlation between the amount of water released and vase life was also observed. Thus, using centrifugation to measure the volume of water released from gellan gum gels appears to be a simple and rapid method for evaluating the performance of gellan gum gels as wetting agents.

To clarify whether climacteric-like increases in ethylene production of senescing petals are also induced in the absence of the gynoecium in cut carnation (Dianthus caryophyllus cv. Barbara) flowers, we compared ethylene production and expression of ethylene-biosynthesis genes in detached petals and in petals, which remained on flowers (attached petals). No significant difference in longevity was observed between the attached and detached petals when held in distilled water, and both showed the inward rolling typical of senescing flowers. Treatment with silver thiosulfate complex (STS), an ethylene inhibitor, similarly delayed senescence of attached and detached petals. Climacteric-like increases in ethylene production of petals and gynoecium started on the same day, with similar bursts in attached and detached petals. Transcript levels of DcACS1 and DcACO1 were very low at harvest and increased similarly during senescence in both petal groups. Removal of the gynoecium did not significantly delay wilting of attached petals. In flowers with the gynoecium removed, the petals produced most of the ethylene while production by the other floral organs was very low, suggesting that wound-induced ethylene is not the reason for the ineffectiveness of gynoecium-removal in inhibiting flower senescence. These results indicate that ethylene biosynthesis is induced in carnation petals irrespective of the gynoecium.

Summary In flowering plants, floral longevity is species-specific and is closely linked to reproductive strategy; petal senescence, a type of programmed cell death (PCD), is a highly regulated developmental process. However, little is known about regulatory pathways for cell death in petal senescence, which is developmentally controlled in an age-dependent manner. Here, we show that a NAC transcription factor, designated EPHEMERAL1 (EPH1), positively regulates PCD during petal senescence in the ephemeral flowers of Japanese morning glory (Ipomoea nil). EPH1 expression is induced independently of ethylene signaling, and suppression of EPH1 resulted in Japanese morning glory flowers that are in bloom until the second day. The suppressed expression of EPH1 delays progression of PCD, possibly through suppression of the expression of PCD-related genes, including genes for plant caspase and autophagy in the petals. Our data further suggest that EPH1 is involved in the regulation of ethylene-accelerated petal senescence. In this study, we identified a key regulator of PCD in petal senescence, which will facilitate further elucidation of the regulatory network of petal senescence.

Inositol 1,4,5-trisphosphate receptor types 1 (IP 3 -R1) and 3 (IP 3 -R3) were found in the rat suprachiasmatic nucleus (SCN) and the levels of both of IP 3 -R1 and IP 3 -R3 mRNA showed a reciprocal circadian rhythm. IP 3 -R1 immunoreactive cells were localized in neuronal cells in the SCN. In contrast, IP 3 -R3 immunoreactive cells were mainly localized in the astrocytes in the ventrolateral region of the SCN. These results suggest that IP 3 -R1 and IP 3 -R3 may have differential roles in the SCN in the mammalian circadian rhythm.

To investigate the molecular mechanism of the melatonin rhythm in the mammalian retina, we examined the temporal mRNA expression pattern of arylalkylamine (serotonin) N -acetyltransferase (AA-NAT), the rate-limiting enzyme in melatonin synthesis in the rat retina. Rat AA-NAT mRNA was detected exclusively in the retinal photoreceptors in the outer nuclear layer—low during the day and increased more than threefold at night. The nocturnal AA-NAT expression in rat retina was also confirmed by RNase protection and the AA-NAT enzymatic activity. This is the first report to localize the site of AA-NAT mRNA circadian expression in mammalian photoreceptor cells.

p53 protein plays a role for control of cell proliferation and the induction of apoptosis in normal cells. However, its role in the circadian rhythms that control many physiological functions including locomotor behavior remains unknown. The present study examined the locomotors activity rhythms of mice which have homozygous mutations of p53 gene. The period of drinking activity rhythms in p53 knockout (p53 KO) mice became unstable under constant dark. Light pulse causes a big phase shifts at CT15.5-17, when p53 mRNA expression peaks in the suprachiasmatic nucleus (SCN). Furthermore, photic entrainment of p53 KO mice is unusual under light-dark conditions. These findings suggest that p53 is involved in entrainment of the circadian behavioral rhythm.

The hormone melatonin, secreted primarily from the pineal gland, plays an important physiological role in synchronizing biological rhythms and neuroendocrine. Presently, we find the expression of the serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT) mRNA, the rate-limiting enzyme in the conversion of serotonin to melatonin, in the rat suprachiasmatic nucleus (SCN) which contains the biological circadian clock in mammals. AA-NAT mRNA content in rat SCN did not show a significant circadian rhythm. However, AA-NAT enzyme activity was lowest at midday and highest at early night, and the rhythm persisted under constant dark conditions. These results indicate that the rat SCN is capable of synthesizing melatonin and suggest that melatonin synthesis in the SCN may be regulated by the circadian clock at the post transcriptional level.

Electrophoretical mobility shift assay (EMSA) is a simple, rapid, and highly sensitive technique for detection of single- or double-stranded DNA-binding proteins such as transcription factors in crude nuclear extracts (F.M. Ausubel, R. Brent, R.E. Kingston, D. D. Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, 1989, pp. 12.0.1-12.2.10 [1]; J. Carey, Gel Retardation. Methods Enzymol., 208 (1991) 103-117 [2]). By using this technique, it is possible to quantify the abundance, relative affinity and binding specificity of DNA-binding proteins. Since proteins which bind specifically to radiolabeled DNA probes retard the mobility of the probe during electrophoresis (it also called gel retardation assay), discrete bands correspond to the individual DNA-protein complexes. Furthermore, EMSA allows one to determine which member(s) of a certain protein family are included in the DNA-protein complex by means of specific antibodies raised against the DNA-binding protein (supershift assay).