A double knockout mutant of acyl-glucose-dependent anthocyanin glucosyltransferase genes in Delphinium grandiflorum.

Blue coloration in delphinium flowers arises from 7-polyacylated anthocyanins which are modified alternately with acyl and glucosyl residues at the 7 position of the aglycone. Previously, we identified two independent genes for acyl-glucose-dependent anthocyanin 7-(6-(p-hydroxybenzoyl)-glucoside) glucosyltransferases (AA7BG-GT); recombinant proteins from the two cDNAs were produced in Escherichia coli and showed AA7BG-GT activity in vitro. Here, a double knockout mutant of both genes was found to lack modification of the second glucosyl residue following further acyl and glucosyl modifications. Both genes in the double mutant had nucleotide sequence changes and deletions that disrupted their transcripts and caused loss of AA7BG-GT activity in sepals. These results provide genetic confirmation that both genes are responsible for AA7BG-GT enzyme activity.

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.

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.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

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