Kazuo Ichimura

Effects of temperature, 8-hydroxyquinoline sulphate and sucrose on the vase life of cut rose flowers

Abstract Cut roses ( Rosa hybrida L.) cv. Sonia were continuously treated with water (control), 200 mg l −1 8-hydroxyquinoline sulphate (HQS) or 30 g l −1 sucrose plus 200 mg l −1 HQS (Suc+HQS) at 20, 25 and 30°C. Higher temperatures promoted flower opening and shortened vase life. HQS and Suc+HQS both extended the vase life at all temperatures, but Suc+HQS was the more effective treatment. Flower diameter and fresh weight were markedly increased by Suc+HQS, particularly at 20°C. Hydraulic conductance of stem segments from the control treatment decreased rapidly after harvest, but those for HQS and Suc+HQS treatments were maintained near their initial level. Concentrations of glucose, fructose and sucrose in petals were increased by the Suc+HQS treatment, compared with the control or HQS treatment. Correlations between sugar concentrations in petals and maximum flower diameter or vase life were positive. These results show that soluble carbohydrate concentration in petals is an important factor in determining the vase life of cut rose flowers at all temperatures examined and when hydraulic conductance is not reduced.

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoeanil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.

Extension of flower longevity in transgenic torenia plants incorporating ACC oxidase transgene

Abstract We demonstrated extension of flower longevity in torenia (Torenia fournieri Lind.) by introducing a fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene in sense or antisense orientation. Torenia was transformed by Agrobacterium-mediated gene transfer. Among primary transformants, 8 sense-gene-introduced plants (average flower longevity 2.7–7.1 days) and 3 antisense-gene-introduced plants (from 2.5–2.7 days) showed significantly greater longevity than did wild-type plants (2.0 days). Analysis of offspring suggested that the introduced gene had been inherited, and that the extended flower longevity was linked to the existence of the gene. Northern blot analysis and measurement of ethylene production demonstrated a reduction in mRNA level and endogenous ethylene production in plants with improved flower longevity.

Sucrose synthase and sucrose phosphate synthase, but not acid invertase, are regulated by cold acclimation and deacclimation in cabbage seedlings

Summary Cabbage seedlings acquire freezing tolerance during exposure to low temperature (acclimation), however, it is lost by returning the plants to moderate temperature (deacclimation). Soluble sugars, except myo -inositol, accumulated during cold acclimation in the leaves of cabbage seedlings. During deacclimation for 5 days, sucrose, glucose and fructose contents decreased rapidly to the same levels as before cold acclimation. To identify the enzymes responsible for changes in sugar concentrations, we investigated changes in activities of sucrose synthase (E.C. 2.4.1.13), sucrose phosphate synthase (E.C. 2.4.1.14) and acid invertase (E.C. 3.2.1.26) during cold acclimation and deacclimation. Activity of sucrose synthase during cold acclimation increased up to 3 times the activity before cold acclimation, but decreased to the level of activity before cold acclimation during deacclimation. The activity of sucrose phosphate synthase also increased during cold acclimation, but decreased to the level of activity before cold acclimation during deacclimation. However, the activity of acid invertase decreased gradually during cold acclimation and did not increase to the level of activity before cold acclimation during deacclimation. These results suggest that SS and SPS, but not acid invertase, are regulated by cold acclimation and deacclimation and play important roles in sugar accumulation and acquisition of freezing tolerance in the leaves of cabbage seedlings.

Effects of the time of sucrose treatment on vase life, soluble carbohydrate concentrations and ethylene production in cut sweet pea flowers

Cut sweet pea flowers were put in vase water containing 200 mg l−1 8-hydroxyquinoline sulfate (HQS), and 100 g l−1 sucrose was added to the solution during the first 24 h (initial treatment), from the 24th h on (late treatment) or throughout the experimental period (continuous treatment). The vase life of the florets in the control (with no sucrose added), initial-, late-, and continuous-treatment groups were 2.8, 6.0, 5.0 and 8.0 days, respectively. Climacteric ethylene production of the florets was the earliest in the control group followed by the late-, initial- and continuous-treatment groups, in this order. The concentrations of glucose, fructose and s ucrose at the 2nd day and later were the highest in the continuous-treatment group followed by the initial-treatment, late-treatment and control group, in this order. Thus, the correlation between sugar concentrations in petals and vase life was positive, whereas that between the sugar concentrations and ethylene production was negative. These results suggest that sugar concentration in petals affects the vase life of cut sweet pea flowers through ethylene production.

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.

Role of ethylene in senescence of cut Eustoma flowers

Abstract Flower senescence of Eustoma grandiflorum was investigated in relation to ethylene production and sensitivity to ethylene. Ethylene production of flowers increased with flower senescence. Ethylene was mainly produced in the pistil, in particular in the style, and production in the petal increased with flower senescence. Flowers were not sensitive to ethylene at anthesis, but became more sensitive with increasing senescence. Treatment with silver thiosulphate (STS), an ethylene action inhibitor, extended flower longevity. STS inhibited ethylene production from the whole flower, particularly that from the petal. Thus, ethylene is considered to be involved in the flower senescence of Eustoma.

Homologs of genes associated with programmed cell death in animal cells are differentially expressed during senescence of Ipomoea nil petals

In senescent petals of Ipomoea nil, we investigated the expression of genes showing homology to genes involved in animal programmed cell death (PCD). Three encoded proteins were homologous to apoptotic proteins in animals: Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE; homologous to caspases) and a monodehydroascorbate reductase [MDAR; homologous to apoptosis-inducing factor (AIF)]. AIFs harbor an oxidoreductase domain and an apoptotic domain. MDARs exhibit homology to the AIF oxidoreductase domain, not to the apoptotic domain. The three other genes studied relate to autophagy. They encode homologs to vacuolar protein sorting 34 (VPS34) and to the Arabidopsis autophagy-related proteins 4b and 8a (ATG4b and ATG8a). The transcript abundance of MDAR decreased continuously, whereas that of the other genes studies exhibited a transient increase, except ATG4b whose abundance stayed high after an increase. Treatment with ethylene advanced the time to visible petal senescence, and hastened the changes in expression of each of the genes studied. In order to assess the role of VPS34 in petal senescence, we studied the effect of its inhibitor 3-methyladenine (3-MA). 3-MA reduced the time to visible petal senescence, and also accelerated the time to DNA degradation. Remarkably, 3-MA increased the time to nuclear fragmentation, indicating that the time to visible petal senescence was independent of nuclear fragmentation. The data on 3-MA might suggest the idea that autophagy is not a cause of PCD, but part of the remobilization process.

Freezing tolerance and soluble sugar contents affected by water stress during cold-acclimation and de-acclimation in cabbage seedlings

The effect of water stress on freezing tolerance during cold-acclimation and de-acclimation in cabbage seedlings were studied. The seedlings were subjected to water stress by withholding water. The treatment wilted the seedlings and decreased the water content of their shoots. Exposure of seedlings to low temperatures (5°C) for 7 days induced freezing tolerance. Water stress promoted the increase in freezing tolerance during cold-acclimation. However, the interruption of water stress by rewatering decreased freezing tolerance rapidly to the same level as for continually watered seedlings. Exposure of cold-acclimated seedlings to normal growth temperature (20°C/15°C) reduced freezing tolerance within a day. Water stress during the de-acclimation period prevented the loss of freezing tolerance. Changes in glucose, fructose and sucrose contents were related to variations in freezing tolerance of seedlings. These results suggest that water stress affects freezing tolerance and is accompanied by changes in sugar contents during cold-acclimation and de-acclimation.

Pollination induces autophagy in petunia petals via ethylene

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.