Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5 (7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as b-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acylglucose-dependent glucosyltransferases.

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In general, carnations (Dianthus caryophyllus) have each of four kinds of anthocyanins acylated by malic acid. A few carnation cultivars are known to display a peculiar dusky color supposedly caused by anthocyanic vacuolar inclusions (AVIs). The hereditary pattern suggests that the peculiar color is controlled by a single recessive factor tightly linked with existence of AVIs containing non-acylated anthocyanins. To diversify the peculiar color carnation, we produced a bluish purple line displaying a highly novel metallic appearance by crossbreeding. By subjecting the line to ion-beam irradiation, we generated metallic reddish purple, metallic crimson and metallic red lines. The major anthocyanin of the metallic bluish purple and reddish purple lines was pelargonidin 3,5-diglucoside, whereas that of the metallic crimson and red lines was pelargonidin 3-glucoside. All four metallic lines did not have transcripts for anthocyanin malyltransferase. Metallic crimson and red lines did not express the acyl-glucosedependent anthocyanin 5-O-glucosyltransferase gene. In contrast to the dusky color types, metallic lines have highly condensed AVIs and water-clear vacuolar sap in the petal adaxial epidermal cells. Differences in the number of AVIs on the abaxial side were observed within mutants containing the same anthocyanin, thereby affecting their shade and hue. We demonstrated that (1) a factor generating the AVIs is inactivated anthocyanin malyltransferase gene, (2) AVIs in water-clear vacuolar sap in the adaxial epidermal cells generate the novel metallic appearance, and (3) ion beam breeding is a useful tool for increasing metallic colors by changing anthocyanin structure and the level of AVIs.

The pink, red and crimson petal colors of carnations (Dianthus caryophyllus) are produced by anthocyanins. The anthocyanins, pelargonidin and cyanidin can be modified by two glucoses at the 3 and 5 positions, and by a single malic acid. Petal color variation can result from failure of such modification, for example, the lack of a glucose at the 5 position is responsible for the color variants of some commercial varieties. With respect to this variation, modification by 5-O-glucosyltransferase plays the most important role in glucosylation at the 5 position. Recently, we identified a novel acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase (AA5GT), that uses acyl-glucoses, but not UDP-glucose, as the glucose donor. Although we showed that loss of AA5GT expression was responsible for loss of glucosylation at the 5 position of anthocyanin in some varieties, the cause of this repression of AA5GT expression could not be determined. Here, we have succeeded in isolating the AA5GT gene and found that it consists of 12 exons and 11 introns. In carnation varieties lacking a glucose at the 5 position, we identified the insertion of two different retrotransposons, Ty1dic1 and Retdic1, into AA5GT. Ty1dic1, which belongs to the class I long terminal repeat (LTR)-retrotransposons of Ty1/copia families, was inserted into exon 10. Retdic1, which includes a long interspersed nuclear element (LINE)-like sequence, was inserted into intron 5. Thus, insertion of either Ty1dic1 or Retdic1 can disrupt AA5GT and result in the lack of glucosylation at the 5 position in anthocyanins.