A Novel Glucosylation Reaction on Anthocyanins Catalyzed by Acyl-Glucose-Dependent Glucosyltransferase in the Petals of Carnation and Delphinium(C)(W)

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 β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose- dependent glucosyltransferases.

INTRODUCTION

Anthocyanins are one of the colored secondary metabolites responsible for the colorful variations in flowers. Only six types of anthocyanin aglycone are produced in the plant kingdom; the rich variety of flower colors is principally the result of differences in the modification of these compounds with glycosyl and acyl moieties to generate thousands of molecular variants (Tanaka et al., 2008). One of the simplest anthocyanins is 3-mono-Oglycopyranosyl anthochanidin (anthocyanidin 3-glycoside), which is synthesized using UDP-glucose as a donor molecule and anthocyanidin aglycone as an acceptor by UDP-glucose-dependent anthocyanin 3-O-glucosyltransferase (UA3GT). UA3GT activity has been detected in several plant species, and the gene encoding the enzyme was first identified as the Bz1 allele in maize (Zea mays; Furtek et al., 1988). This identification was rapidly followed by isolation of homologous genes and cDNAs from many other species. In some plant species, other sugar moieties, such as galactose or arabinose, are conjugated at the 3 position of anthocyanidins (Yonekurα-Sakakibara et al., 2009). The other typical glycosylation site is the 5 position of anthocyanidins to give 3,5-di-O-glycopyranosyl anthocyanins. UDP-glucose- dependent 5-O-glucosyltransferase was first identified in...