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

Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells

In higher plants, secondary metabolites are often converted to their glycoconjugates by glycosyltransferases (GTases). We cloned a cDNA encoding GTase (NtGT2) from tobacco (Nicotiana tabacum L.). The recombinant enzyme expressed in Escherichia coli (rNTGT2) showed glucosylation activity against several kinds of phenolic compounds, particularly the 7-hydroxyl group of flavonoids and 3-hydroxycoumarin. The K(m) values of kaempferol and 3-hydroxycoumarin with rNTGT2 are 6.5 microM and 23.6 microM, respectively. The deduced amino acid sequence of NTGT2 shows 60-70% identity to that of anthocyanin 5-O-glucosyltransferase (A5GT); rNTGT2 did not show activity against the anthocyanins tested. NtGT2 gene expression was induced by treating tobacco cells with plant hormones such as salicylic acid. We consider that NtGT2 gene might have evolved from the same ancestral gene as the A5GT genes to the stress-inducible GTases that react on several phenolic compounds.     

Reciprocal regulation ofArabidopsis UGT78D2 and BANYULS is critical for regulation of the metabolic flux of anthocyanidins to condensed tannins in developing seed coats

Anthocyanins are major color pigments in plants. Their biosynthetic pathways are well established, and the majority of these biosynthetic enzymes have been identified in model plants such asArabidopsis, maize, and petunia. One exception inArabidopsis is UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT). This enzyme is known as Bronze-1 (Bz1 ) in maize, where it converts anthocyanidins to anthocyanins. Phylogenetic sequence analysis of theArabidopsis thaliana UDP-glycosyltransferase (UGT) family previously indicated that UGT78D1, UGT78D2, and UGT78D3 cluster together with UF3GTs from other species. Here, we report thatUGT78D2 encodes a cytosolic UGT that is functionally consistent with maize Bz-1. Biochemically, UGT78D2 catalyzes the glucosylation of both flavonols and anthocyanidins at the 3-OH position. A T-DNA-insertedugt78d2 mutant accumulates very little anthocyanin and lacks 3-O-glucosylated quercetin. Expression analysis indicated thatUGT78D2, in opposite toBANYULS, is highly expressed in anthocyanin-accumulating seedlings but repressed in condensed tannin-accumulating seed coats. This suggests that the reciprocal regulation of these two genes is important in directing the metabolic flux to either anthocyanins or condensed tannins. Consistent with this, the ectopic expression of UGT78D2 produces purple-colored seed coats due to the accumulation of anthocyanins. Taken together, our data indicate thatUGT78D2 encodes an enzyme equivalent to maize Bz1, and that the reciprocal regulation of UGT78D2 and BANYULS is critical for the regulation of metabolic flux of anthocyanidins inArabidopsis.     

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.     

Properties and genetic control of anthocyanin 5-O-glucosyltransferase in flowers of Petunia hybrida

An anthocyanin 5-O-glucosyltransferase from flowers of Petunia hybrida was purified about 30-fold. Using uridine 5-diphosphoglucose as glucose donor (K_m 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (K_m 3 M), isolated from pollen of Petunia. Delphinidin 3-rutinoside, cyanidin 3-rutinoside and delphinidin 3-glucoside did not serve as substrates. The glucosylation of petunidin 3-(p-coumaroyl)-rutinoside showed a pH-activity optimum at pH 8.3 and was neither stimulated by Mg²⁺ or Ca²⁺, nor inhibited by ethylenediaminetetraacetic acid. After separating the 5-O-glucosyltransferase from the anthocyanidin 3-O-glucosyltransferase by means of chromatofocusing, it was shown that both enzymes exhibit a high degree of positional specificity. The 5-O-glucosyltransferase activity was correlated with the gene An1, but not with the gene Gf.Abbreviations HPLC high performance liquid chromatography - 3GT 3-O-glucosyltransferase - 5GT 5-O-glucosyltransferase - 3RGac 3-(p-coumaroyl)-rutinoside - 3RGac5G 3-(p-coumaroyl)-rutinoside-5-glucoside - UDPGlc uridine 5-diphosphoglucose     

Biochemical and molecular characterization of a novel UDP-glucose:anthocyanin 3'-O-glucosyltransferase,a key enzyme for blue anthocyanin biosynthesis,from gentian

Gentian (Gentiana triflora) blue petals predominantly contain an unusually blue and stable anthocyanin, delphinidin 3-O-glucosyl-5-O-(6-O-caffeoyl-glucosyl)-3'-O-(6-O-caffeoyl-glucoside) (gentiodelphin). Glucosylation and the subsequent acylation of the 3'-hydroxy group of the B-ring of anthocyanins are important to the stabilization of and the imparting of bluer color to these anthocyanins. The enzymes and their genes involved in these modifications of the B-ring, however, have not been characterized, purified, or isolated to date. In this study, we purified a UDP-glucose (Glc):anthocyanin 3'-O-glucosyltransferase (3'GT) enzyme to homogeneity from gentian blue petals and isolated a cDNA encoding a 3'GT based on the internal amino acid sequences of the purified 3'GT. The deduced amino acid sequence indicates that 3'GT belongs to the same subfamily as a flavonoid 7-O-glucosyltransferase from Schutellaria baicalensis in the plant glucosyltransferase superfamily. Characterization of the enzymatic properties using the recombinant 3'GT protein revealed that, in contrast to most of flavonoid glucosyltransferases, it has strict substrate specificity: 3'GT specifically glucosylates the 3'-hydroxy group of delphinidin-type anthocyanins containing Glc groups at 3 and 5 positions. The enzyme specifically uses UDP-Glc as the sugar donor. The specificity was confirmed by expression of the 3'GT cDNA in transgenic petunia (Petunia hybrida). This is the first report of the gene isolation of a B-ring-specific glucosyltransferase of anthocyanins, which paves the way to modification of flower color by production of blue anthocyanins.     

Genetic control of UDP-glucose: anthocyanin 5-O-glucosyltransferase from flowers of Matthiola incana R.Br.

In flower extracts of defined genotypes of Matthiola incana, an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5-diphosphoglucose (UDPGlc) to the 5-hydroxyl group of pelargonidin and cyanidin 3-glycosides and acylated derivatives. The best substrate for 5-glucosylation is the 3-xylosylglucoside acylated with p-coumarate, followed by the 3-xylosylglucoside and by the acylated (p-coumarate) 3-glucoside. The 3-glucoside itself is a very poor substrate. Besides UDPGlc, thymine 5-diphosphoglucose is a suitable glucosyl-donor, but with a reduced reaction rate (42%). The anthocyanin 5-O-glucosyltransferase exhibits a pH optimum at 7.5 and is generally inhibited by divalent ions and by ethylenediaminetetraacetic acid and p-chloromercuribenzoate. Investigations on different genotypes showed that the 5-O-glucosyltransferase activity is clearly controlled by the gene l. In confirmation of earlier chemogenetic work, enzyme activity is only present in lines with the wild-type allele l⁺. The anthocyanin 5-O-glucosyltransferase activity is strictly correlated with the formation of 5-glucosylated anthocyanins during bud development.Abbreviations Cg 3,5-T-cyanidin 3-sambubioside-5-glucoside - EDTA ethylene diaminetetraacetic acid - 5GT UDP-glucose: anthocyanin 5-O-glucosyltransferase - 3GT UDP-glucose: anthocyanidin/flavonol 3-O-glucosyltransferase - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - UDPGlc uridine 5-diphospho-glucose     

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.     

RELATION OF FLOWER COLOR TO OPTICAL-DENSITY SPECTRA OF INTACT TISSUE AND OF ANTHOCYANIN EXTRACTS

Data from measurements of optical density of intact tissue and of anthocyanins in extracts resolved on cellulose thin layer plates were compared with visual evaluations of color quality and intensity in poinsettia, rose, and snapdragon. Visual evaluation was in good agreement with both instrumental and chemical determinations. However, the number or kinds of anthocyanins present could not be predicted from the visual evaluation or from the spectra of the fresh tissue. Data from the resolved extracts did not provide a basis for predicting the optical-density spectrum or the color of the intact tissue. In addition to the genetic factors which have been shown to control (1) the type of anthocyanin, (2) the amount of anthocyanin, and (3) the distribution of anthocyanins within the flower, we suggest another group of genes which apparently affect color through control of structural modification of individual anthocyanins in the living cell through shift in pH, metal chelation, and/or copigmentation. Such genes are apparently responsible for the modification of red color within the Wh Wh genotype of poinsettias containing both pelargonidin and cyanidin glycosides and for a very similar pink color in a snapdragon and a rose, each containing a single anthocyanin, a pelargonidin glycoside, and a cyanidin glycoside, respectively.     

Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin

UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa. Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT. Several biochemical properties (optimum pH, Km values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs. Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P. frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P. frutescens.     

Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum)

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T₁ plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.     

Acyl-glucose-dependent glucosyltransferase catalyzes the final step of anthocyanin formation in Arabidopsis

The biosynthetic pathways that produce anthocyanins, the principal pigments for flower and leaf coloration in plants, have been extensively investigated. As a result, many of the enzymes involved in these pathways have been identified. Here, we make use of an inducible Arabidopsis thaliana system and demonstrate that the final step in the formation of the major anthocyanin molecule occurs via a glucosylation step catalyzed by acyl-glucose-dependent anthocyanin glucosyltransferase (AAGT). The glucosylation occurs at the 4-coumarate moiety of the anthocyanin molecule cyanidin 3-O-[2″-O-(2′″-O-(sinapoyl) xylosyl) 6″-O-(p-coumaroyl) glucoside] 5-O-[6″″-O-(malonyl) glucoside] leading to completion of the main anthocyanin structure, a reaction that has not previously been identified in studies of Arabidopsis anthocyanins. Earlier studies on flower AAGTs showed that they conjugate a glucose directly to the basic skeleton of anthocyanin. The present study provides the first evidence that an AAGT of Arabidopsis can conjugate a glucose to an acyl moiety of an anthocyanin modified with sugars and organic acids. The results from analyses of gene expression and of anthocyanin composition in a knock-out (KO) mutant and from a complementation test indicate that AtBGLU10 might encode this AAGT.     

早红考密斯及其芽变果实中花青素含量与相关酶活性变化的研究

以西洋梨早红考密斯及其绿色芽变果实为材料,研究了果实发育期间果皮色泽、花青苷含量及其相关酶活性变化.结果显示:(1)早红考密斯果皮色泽从成熟前的暗红色逐渐变为成熟时的浅红色,并在色泽分布不均匀的地方显出黄色底色,色泽指数(a*)值从花后45 d的16.4降低到成熟时的7.4,降低54.9%;花青苷含量从成熟前的258.4μg?g-1降到成熟时的118.3μg?g-1;早红考密斯果皮色泽和果皮花青苷含量具有密切的相关性.(2)早红考密斯的绿色芽变在果实发育的前期检测不到花青苷,发育后期果实向阳部出现浅红晕,但花青苷含量极低,与亲本差异极显著.(3)果实发育期间,两品种间苯丙氨酸解氨酶(PAL)变化趋势相似,总体呈下降趋势,且早红考密斯的活性总体低于其绿色芽变;两品种查耳酮异构酶(CHI)活性总体变化趋势基本一致,均呈现缓慢上升的趋势,在前期绿色芽变的CHI活性高于其亲本,后期低于亲本;类黄酮3-O-葡(萄)糖基转移酶(UFGT)活性在两品种间的差异较大,在整个果实发育期间早红考密斯的UFGT活性远高于其绿色芽变.研究表明,早红考密斯果皮色泽变化主要由花青苷的含量不同引起;PAL和CHI不是绿色芽变的直接原因;UFGT与花青苷合成密切相关,绿色芽变果皮中UFGT活性显著降低.     

Isolation of an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase from the monocot Agapanthus africanus

A cDNA encoding an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AaAA7GT) was isolated from Agapanthus africanus petals; this is the first AAGT identified in a monocot. Peak expression of AaAA7GT in developing A. africanus petals occurred before the flowering stage, and was later than found previously for other anthocyanin biosynthetic genes. Analysis of recombinant proteins showed AaAA7GT had strict substrate preference for anthocyanidin 3-O-glycosides. The AaAA7GT amino acid had high sequence similarity to glycoside hydrolase family 1 (GH1) proteins, which typically act as β-glycosidases. A phylogenetic analysis of amino acid sequences suggested that AAGTs were derived from glycosidase early in the angiosperm lineage.     

蓝色花形成分子机理研究进展

花色是观赏植物的重要特征, 在自然界中蓝色花占比很少, 很多观赏植物都缺少蓝色种质。因此, 研究蓝色花形成的分子机理对于蓝色花定向育种具有重要意义。研究表明, 花色的形成主要是通过花青苷积累, 花青素通过糖基化形成花青苷, 再通过酰基、甲基化修饰及金属离子络合反应, 在特定的液泡pH环境中呈现出稳定的蓝色。该文从花青苷合成与代谢途径入手, 对蓝色花形成关键基因功能、花青苷各位点酰化的影响、金属离子的作用、液泡pH值相关基因研究及蓝色花分子育种等方面进行综述。     

Flavonoid composition related to petal color in different lines of Clitoria ternatea

Flavonoids in the petals of several C. ternatea lines with different petal colors were investigated with LC/MS/MS. Delphinidin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was newly isolated from the petals of a mauve line (wm) together with three known anthocyanins. They were identified structurally using UV, MS, and NMR spectroscopy. Although ternatins, a group of 15 (poly)acylated delphinidin glucosides, were identified in all the blue petal lines (WB, BM-1, 'Double Blue' and 'Albiflora'), WM accumulated delphinidin 3-O-(6"-O-malonyl)-beta-glucoside instead. The white petal line (WW) did not contain anthocyanins. Quantitative data showed that the total anthocyanin contents in WB and 'Double Blue' were ca. 8- and 10-fold higher than that in BM-1, a bud mutant of 'Double Blue', respectively. The total anthocyanin content in 'Albiflora' was less than 2 x 10(-3) times those in WB or 'Double Blue'. While all the lines contained the same set of 15 flavonol glycosides in similar relative ratios, the relative ratio of myricetin glycosides in ww and 'Albiflora' was ca. 30-70 times greater than those in the other lines. The change in flower color from blue to mauve was not due to a change in the structure of an anthocyanidin from delphinidin, but to the lack of (polyacylated) glucosyl group substitutions at both the 3'- and 5'-positions of ternatins. This implies that glucosylation at the 3'- and 5'-positions of anthocyanin is a critical step in producing blue petals in C. ternatea.     

The Differentiation of Pigmentation in Flower Parts, IV. Flavonoid Elaborating Enzymes From Petals of Impatiens balsamina s

Extracts of the flower petals of Impatiens balsamina L. contain enzymes which catalyze the glycosylation of phenolic compounds. Enzymes have been extracted which glycosylate hydroquinone to arbutin and at least 3 different flavonols to the 3-monoglucoside. The hydroquinone glucosylating enzyme is similar to enzymes previously described except that it requires an unidentified low molecular weight cofactor. The glucosylation of flavonols follows normal enzyme kinetics; it requires a nucleotide diphosphate glucose donor for activity, and is made more evident by the presence of glucono-1:5-lactone, an inhibitor of endogenous glucosidases. It is suggested that the flavonol glucosylating enzyme acts naturally to glucosylate a precursor of both flavonols and anthocyanins to the 3-monoglucoside. The only elaboration of an anthocyanin observed with petal extracts was an acylation of pelargonidin-3-monoglucoside.     

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.