Mutational analysis of substrate specificity in a <Emphasis Type="Italic">Citrus paradisi</Emphasis> flavonol 3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.     

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities.     

Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 μM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.     

Flavonoids from the flowers of Adenium obesum (Forssk.) Roem. & Schult., Mandevilla sanderi (Hemsl.) Woodson,and Nerium oleander L. (Apocynaceae)

Twenty-two ornamental flowers from different Adenium obesum, Mandevilla sanderi, and Nerium oleander cultivars/seedlings were analyzed for the presence of anthocyanins, flavonols, and chlorogenic acid using nuclear magnetic resonance (NMR) and mass spectrometry (MS). Cyanidin 3-O-[6-O-(rhamnosyl)-galactoside] and cyanidin 3-O-(galactoside) were identified as the major and minor anthocyanins, respectively, in three A. obesum seedlings that had red and red-purple flowers.Cyanidin 3-O-[2-O-(xylosyl)-galactoside] was identified as the major anthocyanin, whereas cyanidin 3-O-[6-O-(rhamnosyl)-galactoside] and cyanidin 3-O-(galactoside) were identified as the minor anthocyanins in 8 M. sanderi cultivars that had red and red-purple flowers. Cyanidin 3-O-[6-O-(rhamnosyl)-galactoside] and cyanidin 3-O-(galactoside) were identified as the major anthocyanins, whereas cyanidin 3-O-[2-O-(xylosyl)-galactoside] was identified as the minor anthocyanin in 8 N. oleander cultivars with red and red-purple flowers. Low levels of anthocyanins were detected in the N. oleander and M. sanderi cultivars that had white flowers, and there were no anthocyanins detected in the N. oleander cultivars with yellow flowers. Chlorogenic acid and four flavonols, quercetin 3-O-[6-O-(rhamnosyl)-galactoside], quercetin 3-O-[6-O-(rhamnosyl)-glucoside], kaempferol 3-O-(galactoside), and kaempferol 3-O-[6-O-(rhamnosyl)-galactoside], were identified in the flowers from all 22 cultivars/seedlings investigated.     

Characterization of 5-O-glucosyltransferase involved in anthocyanin biosynthesis in Cyclamen purpurascens

Wild cyclamen (Cyclamen purpurascens) is considered as a precious breeding material for the development of new cultivars. Malvidin 3,5-diglucoside is the main anthocyanin in the petals of C. purpurascens, whereas the F1 progeny of the C. persicum × C. purpurascens cultivars cross contains 3,5-diglucoside-type anthocyanins as the main pigment. The anthocyanin 5-O-glucosyltransferase (A5GT) enzyme is responsible for the glycosylation of the A ring of anthocyanin at the 5-O-position, which implies that the expression of A5GT is dominant in the petals of C. purpurascens × C. persicum cultivars. Here, we isolated the complete open reading frame of the A5GT gene from C. purpurascens (Cpur5GT). Results of qRT-PCR revealed that Cpur5GT shows tissue-specific expression, with strong expression in fully opened petals and weak expression in young petals. In vitro enzyme assay showed that when uridine diphosphate glucose was used as the sugar donor, recombinant Cpur5GT could catalyze the glycosylation of 3-glucoside-type anthocyanidins at the 5-O-position, but when uridine diphosphate galactose was served as glycosyl donor, the reaction could not be performed. These results demonstrate that Cpur5GT exhibits valid anthocyanin glucosylation activity and could be used to analyze the mechanism of A5GT-mediated flower coloration in cyclamen in future studies.     

O-methylation of flavonoids by cell-free extracts of calamondin orange

Cell-free extracts of calamondin orange (Citrus mitis) catalysed the O-methylation of almost all hydroxyls of a number of flavonoids, indicating the existence in citrus tissues of ortho, meta, para and 3-O-methyltransferases. The latter, hitherto unreported enzyme, catalysed the formation of 3-O-methyl ethers of galangin and quercetin. The stepwise O-methylation of a number of compounds, especially quercetin and quercetagetin, tends to suggest a coordinated sequence of O-methylations on the surface of a multienzyme complex. The methyl acceptor abilities of the flavonoid substrates used are discussed in relation to their hydroxyl substitution patterns and their negative electron density distribution.     

Biologically-active flavonoids from Gossypium arboreum

A new flavonol glycoside, gossypetin 8-O-rhamnoside, was isolated from flower petals of Gossypium arboreum along with quercetin 7-O-glucoside, quercetin 3-O-glucoside and quercetin 3′-O-glucoside. These compounds showed antibacterial activity against Pseudomonas maltophilia and Enterobacter cloacae.     

UDP-Glucose: Cyanidin 3-O-glucosyltransferase from red cabbage seedlings

An enzyme, catalysing the glucosylation of cyanidin at the 3-position using uridine diphosphate-D-glucose (UDPG) as glucosyl-donor, has been isolated and purified about 50-fold from young red cabbage (Brassica oleracea) seedlings. The pH optimum for this reaction was ca 8 and no additional cofactors were required. The reaction was inhibited by cyanidin (above 0.25 mM) and by very low concentrations of the reaction product cyanidin-3-glucoside (5 μM). The K_m values for UDPG and cyanidin were 0.51 and 0.4 mM respectively. In addition to cyanidin the enzyme could also glucosylate the following compounds at the 3-position: pelargonidin, peonidin, malvidin, kaempferol, quercetin, isorhamnetin, myricetin and fisetin. In contrast, cyanidin-3-glucoside, cyanidin-3-sophoroside, cyanidin-3,5-diglucoside, apigenin, luteolin, naringenin and dihydroquercetin were not glucosylated.     

Phenolic compounds from parasitic Sapria himalayana f. albovinosa and Sapria myanmarensis (Rafflesiaceae) in Myanmar

Three anthocyanins, four flavonols, three aromatic acids and five gallotannins were isolated from Sapria himalayana f. albovinosa in Myanmar. They were identified as cyanidin 3-O-glucoside (1), cyanidin 3-O-xyloside (2) and peonidin 3-O-glucoside (3) (anthocyanins), quercetin 3-O-glucoside (4), quercetin 7-O-glucoside (5), quercetin 3-O-glucuronide (6) and isorhamnetin 3-O-glucoside (7) (flavonols), ellagic acid (8), gallic acid (9) and ethyl gallate (10) (aromatic acids), and 1,2,4,6-tetragalloylglucose (11), 1,4,6-trigalloylglucose (12), 1,2,6-trigalloylglucose (13), 1,2,4-trigalloylglucose (14) and 1,6-digalloylglucose (15) (gallotannins) by UV, LC-MS, acid hydrolysis, NMR and/or HPLC comparisons with authentic samples. The chemical composition of S. myanmarensis was qualitatively the same with that of S. himalayana f. albovinosa. Phenolic compounds of the Rafflesiaceae species including Sapria, Rafflesia and Rhizanthes were isolated and identified in this survey for the first time.     

Anthocyanins and flavonols from the blue flowers of six Meconopsis species in Bhutan

Blue flowers of six Bhutani Meconopsis species, M. bhutanica, M. bella, M. horridula, M. simplicifolia, M. primulina and M. polygonoides, were surveyed for anthocyanins and other flavonoids. Four anthocyanins were isolated and identified as cyanidin 3-O-sambubioside-7-O-glucoside (1), cyanidin 3-O-[xylosyl-(1 → 2)-(6″-malonylglucoside)]-7-O-glucoside (2), cyanidin 3-O-sambubioside (4) and cyanidin 3-O-[xylosyl-(1 → 2)-(6″-malonylglucoside)] (5). On the other hand, 12 flavonols were isolated from their Meconopsis species with various combination and characterized as kaempferol 3-O-glycosides (8–12), kaempferol 3,7-O-glycosides (13–16), quercetin 3-O-glycosides (17 and 18) and isorhamnetin 3-O-glycoside (19). Of six Meconopsis species which were surveyed in this experiment, anthocyanin and flavonol composition of five species except for M. horridula was clarified for the first time. Their Meconopsis species showed the different flavonoid profiles, respectively, and flavonoid diversity within the glycosylation level of Meconopsis flowers were indicated.     

Isolation and characterization of anthocyanin 5-O-glucosyltransferase from flowers of Dahlia variabilis

In the cyanic flowers ofDahlia variabilis (Asteraceae), an enzyme was demonstrated which catalyzes a glucosyl group transfer from UDP-glucose to the 5 position of anthocyanidin 3-O-glucoside and 3-O-malonylglucoside. The anthocyanin 5-O-glucosyltransferase (5GT) was purified 88-fold at 8 percnt; yield by (NH₄)₂SO₄ precipitation followed by successive chromatography on DEAE-cellulose, Sephacryl S-200 and Mono P. 5GT exhibited a pH optimum at 8.0 and a pI of 4. 2. Its apparent molecular weight calculated from Sephacryl S-200 was 53 kDa. Its activity was stimulated by 2-ME and DTE but strongly inhibited by PCMB and NEM. It was slightly activated by Mg²⁺ and Ca²⁺ but strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Fe³⁺ and Al³⁺. No effect of EDTA was observed. The apparent Km values for cyanidin 3-O-glucoside, cyanidin 3-O-(6′′-O-malonyl)glucoside and UDP-glucose were 120 μmol/L, 75 μmol/L and 250 μmol/L, respectively. Pelargonidin 3-O-glucoside and malonylglucoside were also considerable substrates, but low relative activity was observed for delphinidin 3-O-glucoside which has yet not been found inDahlia flowers.Dahlia 5GT showed substrate specificities different from those reported forSilene, Petunia, Matthiola andPerilla. Neither ADP-glucose nor UDP-galactose could serve as glycosyl donor.     

Characterization of three distinct flavonol O-methyltransferases from Chrysosplenium americanum

The partially purified O-methyltransferase (OMT) system of Chrysosplenium americanum was found to catalyse the stepwise O-methylation of quercetin to its mono-, di- and trimethyl derivatives. It also utilized the partially methylated flavonol intermediates to form the next higher order of O-methylated products; thus indicating the involvement of several OMTs. The latter were resolved by chromatofocusing into three distinct peaks of enzyme activity which focused at pI values 4.8, 5.4 and 5.7. The former enzyme O-methylated quercetin at the 3-position, whereas the latter two O-methylated 3, 7-di-O-methyl quercetagetin at the 3′- and 6-positions, respectively. None of the focused enzymes accepted caffeic acid, or other flavonoids such as kaempferol or luteolin, as substrates; thus indicating specificity towards flavonols with 3′, 4′- substitution. The three OMTs had similar MWs and the K_m values for their substrates were of the same order of magnitude. The biochemical role of these novel enzymes is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.     

Isolation and characterization of UDP-glycose: Cyanidin 3-O-glucosyltransferase from the flower buds ofSenecio x hybridus

An UDPG: cyanidin 3-O-glucosyltransferase was isolated and purified about 260-fold from the flower buds ofSenecio x hybridus. The enzyme showed a pH optimum of 7.5 and no additional cofactors were required. The Km values for cyanidin and UDPG were 0.33 and 0.20 mM, respectively. Its molecular mass estimated by Sephacryl S-200 chromatography was 52 kDa.     

Anthocyanins with unusual furanose sugar (apiose) from leaves of Synadeniumgrantii (Euphorbiaceae)

Four anthocyanins, cyanidin 3-O-(2″-(5?-(E-p-coumaroyl)-β-apiofuranosyl)-β-xylopyranoside)-5-O-β-glucopyranoside, cyanidin 3-O-(2″-(5?-(E-p-coumaroyl)-β-apiofuranosyl)-β-xylopyranoside), cyanidin 3-O-(2″-(5?-(E-caffeoyl)-β-apiofuranosyl)-β-xylopyranoside) and cyanidin 3-O-(2″-(5?-(E-feroyl)-β-apiofuranosyl)-β-xylopyranoside) were isolated from leaves of African milk bush, (Synadeniumgrantii Hook, Euphorbiaceae) together with the known cyanidin 3-O-β-xylopyranoside-5-O-β-glucopyranoside and cyanidin 3-O-β-xyloside. The four former pigments are the first reported anthocyanins containing the monosaccharide apiose, and the three 5?-cinnamoyl derivative-2″-(β-apiosyl)-β-xyloside subunits have previously not been reported for any compound.     

Acylated cyanidin 3-sambubioside-5-glucosides from the purple-violet flowers of Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (Brassicaceae)

A novel acylated cyanidin 3-sambubioside-5-glucoside was isolated from the purple-violet flowers of Matthiola longipetala subsp. bicornis (Sm) P. W. Ball. (family: Brassicaceae), and determined to be cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] by chemical and spectroscopic methods. In addition, two known acylated cyanidin 3-sambubioside-5-glucosides, cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] and cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(trans-feruloyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside] were identified in the flowers.     

Anthocyanins from red flowers of Camellia cultivar 'Dalicha'

Five anthocyanins, cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(Z)-p-coumaroyl)-β-galactopyranoside (2), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(E)-p-coumaroyl)-β-galactopyranoside (3), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(E)-caffeoyl)-β-galactopyranoside (4), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-galactopyranoside (5), and cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-glucopyranoside (6), together with the known cyanidin 3-O-(2-O-β-xylopyranosyl)-β-galactopyranoside (1), were isolated from red flowers of Camellia cultivar ‘Dalicha’ (Camellia reticulata) by chromatography using open columns. Their structures were subsequently determined on the basis of spectroscopic analyses, i.e., ¹H NMR, ¹³C NMR, HMQC, HMBC, HR ESI-MS and UV-vis.     

Are the characteristics of betanidin glucosyltransferases from cell-suspension cultures of Dorotheanthus bellidiformis indicative of their phylogenetic relationship with flavonoid glucosyltransferases?

Uridine 5′-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-β-glucoside) and gomphrenin I (betanidin 6-O-β-glucoside), respectively. Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as eluent. Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual hydroxyl groups of the respective acceptor compounds. The 5-GT catalyzes the transfer of glucose to the C-4′ hydroxyl group of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate. Both enzymes also catalyze the formation of the respective 7-O-glucosides, but to a minor extent. Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e. 5-GT and 6-GT. The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose:flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta. In addition to the 5-GT and 6-GT, we isolated a UF3GT from D. bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin. The same result was obtained with a UF3GT from Antirrhinum majus and a flavonol 4′-O-glucosyltransferase from Allium cepa. Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative of their phylogenetic relationship with flavonoid glucosyltransferases? Received: 11 February 1997 / Accepted: 18 April 1997     

Effects of Macroposthonia xenoplax on the Growth of Concord Grape

Concord grape (Vitis labrusca) plants were inoculated with Macroposthonia xenoplax at levels of 100, 1,000, and 10,000 nematodes. After 4 months, plants inoculated with 10,000 M. xenoplax were stunted, and root systems were darker and had fewer feeder roots than those in other treatments. The lower nematode inoculation levels suppressed top growth but did not affect root growth. M. xenoplax reproduced well on Concord grapes.