p-Hydroxybenzoyl-Glucose Is a Zwitter Donor for the Biosynthesis of 7-Polyacylated Anthocyanin in Delphinium

Abstract

ORCID IDs: 0000-0002-3002-368X (Y.O.); 0000-0001-7474-9493 (N.S.).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-Orutinoside-7-O-(6-O-[ p-hydroxybenzoyl]-glucoside) to form violdelphin.

Isolation of anthocyanin 7-<i>O</i>-glucosyltransferase from Canterbury bells (<i>Campanula medium</i>)

“…The retention time of the new peak was consistent with that of authentic delphinidin 3-O-rutinoside-7-O-glucoside (Dp3R7G) ( Figure 1E) prepared as described previously (Nishizaki et al 2013). This analysis confirmed that glucosylation at the 7 position of anthocyanin was catalyzed by an acyl-glucose dependent glucosyltransferase in C. medium.…”

“…Thus, transcription of CmAA7GT was greatest just before flower opening. This pattern of expression has also been found for other AAGTs that are expressed specifically in flower tissues (Matsuba et al 2010;Miyahara et al 2012;Nishizaki et al 2013).…”

“…Blue Get Mee, precipitated with ammonium sulfate, and desalted as previously described (Nishizaki et al 2013). A standard reaction mixture (20 µl) consisted of 3.7 µg of crude protein, 45 nmol p-hydroxybenzoylglucose (pHBG) and 2 nmol delphinidin 3-Orutinoside (Dp3R) in 1.6 µmol sodium citrate buffer (pH 5.5); the mixture was incubated at 30°C for 1 h. The reaction was terminated by addition of 1.5 µl 20% phosphoric acid and precipitates were removed after centrifugation.…”

Dihydroflavonol 4-reductase activity is associated with the intensity of flower colors in delphinium

“…Differences in modification of the major anthocyanin caused differences in maximum absorption, and gave rise to the orange, red and pale pink coloration groups. Although there were clear phenotypic differences between the 'CRD' (red flowers) and 'SKH' and 'SHP' (pale pink flowers) varieties, the anthocyanin structures in the three varieties were similar; in particular, the 7-position of Pg was modified with the same residues that are believed to play an important role in determining flower color (Nishizaki et al 2013). The HPLC analysis and ESI-MS analysis suggested that the structure of the main anthocyanin had less effect on color quality in varieties with red, orange and pale pink flowers.…”

Transcriptional regulators of flavonoid biosynthesis and their application to flower color modification in Japanese gentians

“…This result implies that both acylations are carried out during gentiodelphin biosynthesis by the same enzyme, which was accordingly renamed as anthocyanin 5,3′-aromatic acyltransferase (A5/3′AT). Recently, vacuolar-type glucosyltransferases (GTs) and ATs, which utilize acyl-glucose as the glucosyl and acyl donors, respectively, have been identified to be involved in anthocyanin modification steps in several species, including Arabidopsis, carnation, agapanthus, and delphinium (Matsuba et al 2010;Miyahara et al 2013;Miyahara et al 2012;Nishizaki et al 2013). These GTs and ATs belong to a completely distinct protein family of cytosolic UGTs and acyl-CoA dependent ATs.…”