Formation of Flavonol 3-O-Diglycosides and Flavonol 3-O-Triglycosides by Enzyme Extracts from Anthers of Tulipa cv. Apeldoorn / Characterization and Activity of Three Different O -Glycosyltransferases during Anther Development

Abstract

Abstract Three distinct glycosyltransferases have been isolated and partially purified from anthers of Tulipa cv. Apeldoorn. The following designations are proposed; UDP-glucose: flavonol 3-O-glucosyltransferase (GT-I), UDP-rhamnose: flavonol 3-O-glucoside rhamnosyltransferase (GT-II) and UDP-xylose: flavonol 3-glycoside xylosyltransferase (GT-III). The three enzymes exhibited an identical pH optimum within the range of 8.5 -9.0. The estimated molecular weight of GT-I and GT-II was about 40 000, GT-III showed a molecular weight of 30 000. GT-III required ions like NH4 +or Ca2+ whereas these ions have almost no influence on GT-I and GT-II activity. The enzymes have a slight requirement for SH-reagents, particularly DTE. As opposed to GT-II activity of GT-I and GT-III is significantly influenced by SH reagents and PCMB. Sucrose enhanced GT-III activity but only slightly GT-I activity, GT-II activity is not influenced. Flavonol aglycones can function as glycosyl acceptor for the GT-I, whereas flavonol 3-O-glycosides, luteolin, dihydroquercetin, naringenin, cyanidin, p-coumaric acid and some other phenols were inactive as acceptor. The best acceptors were isorhamnetin and quercetin (Km: 0.9 × 10-6 ᴍ). GT-II did not accept aglycones as substrates. For this enzyme flavonol 3-O-glucosides were the most attractive substrates. GT-III did not have any affinity towards aglycones, too. This enzyme exhibited a high specificity for flavonol 3-O-glucosides as well as flavonol 3-O-galactosides. Both enzymes, the GT-II and GT-III, were able to glycosylate flavonol 3-O-diglycosides forming triglycosides. UDP-glucose (Km = 1.0 × 10-4 ᴍ), UDP-rhamnose and UDP-xylose where shown to be the best glycosyl donors for GT-I, GT-II or GT-III respectively. The glycosyl transfer catalysed by the GT-I was shown to be a freely reversible reaction. In the whole anthers, highest specific activities of GT-I and GT-II were found during late stages of anther development. Similar results were obtained using the contents of anthers or the tapetum fraction. In contrary, high GT-III activity can be detected already in young stages of anther devel­opment. The highest activities of the three glycosyltransferases were found in the tapetum fraction, whereas the pollen fraction exhibited only poor activities.