Identification of flavonoid 3'-hydroxylase in the yellow flower of Delphinium zalil.

The flowers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or pelargonidin derivatives. To date, no delphinium cultivars have been found with red flowers due to the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis ability because of the loss of function of flavonoid 3' hydroxylase (F3'H). Here, we show that the wild delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides in its sepals, presumably through F3'H activity. We isolated F3'H cDNA from D. zalil (DzF3'H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3'H protein could convert naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and quercetin, respectively. An expression analysis confirmed that blue flowered D. grandiflorum does not express F3'H, and also showed that flavonoid 3',5'-hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3'H can act as a flavonoid hydroxylase to produce cyanidin accumulation. The introduction of the DzF3'H gene into other delphinium species by conventional breeding may enable development of cultivars with novel flower colors.

Identification of the glucosyltransferase gene that supplies the p-hydroxybenzoyl-glucose for 7-polyacylation of anthocyanin in delphinium.

In delphiniums (Delphinium grandiflorum), blue flowers are produced by the presence of 7-polyacylated anthocyanins. The polyacyl moiety is composed of glucose and p-hydroxybenzoic acid (pHBA). The 7-polyacylation of anthocyanin has been shown to be catalysed by two different enzymes, a glucosyltransferase and an acyltransferase; both enzymes utilize p-hydroxybenzoyl-glucose (pHBG) as a bi-functional (Zwitter) donor. To date, however, the enzyme that synthesizes pHBG and the gene that encodes it have not been elucidated. Here, five delphinium cultivars were investigated and found to show reduced or undetectable 7-polyacylation activity; these cultivars synthesized delphinidin 3-O-rutinoside (Dp3R) to produce mauve sepals. One cultivar showed a deficiency for the acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AA7GT) necessary for mediating the first step of 7-polyacylation. The other four cultivars showed both AA7GT activity and DgAA7GT expression; nevertheless, pHBG accumulation was significantly reduced compared with wild-type cultivars, whereas p-glucosyl-oxybenzoic acid (pGBA) was accumulated. Three candidate cDNAs encoding a UDP-glucose-dependent pHBA glucosyltransferase (pHBAGT) were identified. A phylogenetic analysis of DgpHBAGT amino acid sequences showed a close relationship with UGTs that act in acyl-glucose synthesis in other plant species. Recombinant DgpHBAGT protein synthesized pHBG and had a high preference for pHBA in vitro. Mutant cultivars accumulating pGBA had very low expression of DgpHBAGT, whereas expression during the development of sepals and tissues in a wild cultivar showed a close correlation to the level of accumulation of pHBG. These results support the conclusion that DgpHBAGT is responsible for in vivo synthesis of pHBG in delphiniums.

[Consumer's psychological processes of hoarding and avoidant purchasing after the Tohoku earthquake].

This study examined psychological processes of consumers that had determined hoarding and avoidant purchasing behaviors after the Tohoku earthquake within a dual-process model. The model hypothesized that both intentional motivation based on reflective decision and reactive motivation based on non-reflective decision predicted the behaviors. This study assumed that attitude, subjective norm and descriptive norm in relation to hoarding and avoidant purchasing were determinants of motivations. Residents in the Tokyo metropolitan area (n = 667) completed internet longitudinal surveys at three times (April, June, and November, 2011). The results indicated that intentional and reactive motivation determined avoidant purchasing behaviors in June; only intentional motivation determined the behaviors in November. Attitude was a main determinant of the motivations each time. Moreover, previous behaviors predicted future behaviors. In conclusion, purchasing behaviors were intentional rather than reactive behaviors. Furthermore, attitude and previous behaviors were important determinants in the dual-process model. Attitude and behaviors formed in April continued to strengthen the subsequent decisions of purchasing behavior.

p-Hydroxybenzoyl-glucose is a zwitter donor for the biosynthesis of 7-polyacylated anthocyanin in Delphinium.

The blue color of delphinium (Delphinium grandiflorum) flowers is produced by two 7-polyacylated anthocyanins, violdelphin and cyanodelphin. Violdelphin is derived from the chromophore delphinidin that has been modified at the 7-position by Glc and p-hydroxybenzoic acid (pHBA) molecules. Modification of violdelphin by linear conjugation of Glc and pHBA molecules to a Glc moiety at the 7-position produces cyanodelphin. We recently showed that anthocyanin 7-O-glucosylation in delphinium is catalyzed by the acyl-Glc-dependent anthocyanin glucosyltransferase (AAGT). Here, we sought to answer the question of which enzyme activities are necessary for catalyzing the transfer of Glc and pHBA moieties to 7-glucosylated anthocyanin. We found that these transfers were catalyzed by enzymes that use p-hydroxybenzoyl-Glc (pHBG) as a bifunctional acyl and glucosyl donor. In addition, we determined that violdelphin is synthesized via step-by-step enzymatic reactions catalyzed by two enzymes that use pHBG as an acyl or glucosyl donor. We also isolated a cDNA encoding a protein that has the potential for p-hydroxybenzoylation activity and two AAGT cDNAs that encode a protein capable of adding Glc to delphinidin 3-O-rutinoside-7-O-(6-O-[p-hydroxybenzoyl]-glucoside) to form violdelphin.

Co-expression of an ethylene receptor gene, ERS1, and ethylene signaling regulator gene, CTR1, in Delphinium during abscission of florets.

We are trying to determine the mechanisms responsible for ethylene-induced floret abscission in cut flowers of Delphinium and recently identified an ethylene receptor gene, ERS1, and studied its response to ethylene treatment. In order to identify additional components of the ethylene response network in Delphinium, we performed 3' and 5' rapid amplification of cDNA ends (RACE) using the consensus sequence of the serine/threonine kinase domain of the ethylene signaling regulator gene (CTR1) involved in the constitutive triple response (CTR) to ethylene. The full-length cDNA (2754 nt) encoded a protein of 800 amino acids, which contained the expected serine/threonine kinase domain, the consensus ATP-binding site, and the serine/threonine kinase catalytic site. The protein had quite high (>50%) overall identity to CTR1 from Arabidopsis and tomato, and 70-75% identity in the catalytic site. The amount of mRNA encoding both CTR1 and ERS1 more than doubled within 6 h in cut florets incubated in the presence of exogenous ethylene. Similarly, the amount of ERS1 transcript doubled in florets within 6 d of harvesting, presumably in response to endogenous ethylene, while CTR1 mRNA increased to about 40% over the same period. However, in the presence of silver thiosulfate (STS), an ethylene inhibitor, the level of both transcripts remained essentially unchanged for the first 8 d before declining to very low levels. Florets on the control plants had almost completely abscised by 6 d, but the florets on STS-treated plants had not abscised by 20 d, by which time the flowers were almost dead. The data are consistent with the hypothesis that endogenous ethylene evokes the accumulation of both these transcripts (and their encoded proteins), thereby speeding up abscission and reducing the useful shelf life of the cut flowers.

A method for predicting critical load evaluating adhesion of coatings in scratch testing.

In this paper based on the experiment principle of evaluating adhesion property by scratch testing, the peeling mechanism of thin films is discussed by applying contact theory and surface physics theory. A mathematical model predicting the critical load is proposed for calculating critical load as determined by scratch testing. The factors for correctly evaluating adhesion of coatings according to the experimental data are discussed.