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.     

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.     

芜菁UF3GT基因的克隆及表达特性

目的:克隆‘津田’芜菁和‘赤丸’芜菁的UDP-葡萄糖∶类黄酮3-O-葡萄糖基转移酶(UF3GT)基因并研究其表达特性。方法:利用RT-PCR方法克隆‘津田’芜菁BrUF3GT1基因和‘赤丸’芜菁BrUF3GT2基因,并进行生物信息学分析;通过Northern杂交检测BrUF3GT1和BrUF3GT2基因的UV-A诱导表达特性;对BrUF3GT1和BrUF3GT2基因进行原核诱导表达。结果:BrUF3GT1和BrUF3GT2的开放读框为1407 bp,编码468个氨基酸残基;氨基酸序列分析显示,BrUF3GT1和BrUF3GT2与拟南芥UF3GT的同源性为87%,从第16~453位氨基酸残基的肽段具有糖基转移酶家族成员的结构域;BrUF3GT1和BrUF3GT2基因具有高度同源性,核苷酸序列在7个位点存在差异,推导的氨基酸序列在1个位点存在差异;Northern杂交结果显示,UV-A可以诱导BrUF3GT1和BrUF3GT2基因表达,基因的表达量与处理时间相关;原核诱导表达及纯化后可以获得相对分子质量分别约为51.88×103和51.89×103的BrUF3GT1和BrUF3GT2蛋白。结论:克隆了‘津田’芜菁和‘赤丸’芜菁的UF3GT基因,为初步阐明2种芜菁的花青素生物合成机理奠定了实验基础。     

箭叶淫羊藿EsUF3GT基因的克隆及表达分析

类黄酮3-O-糖基转移酶(flavonoid 3-O-glucosyltransferase,UF3GT)可以把不稳定的花色素催化成花色素苷。本研究采用同源基因克隆技术获得箭叶淫羊藿Epimedium sagittatum(Sieb.and Zucc.)Maxim.UF3GT基因c DNA开放阅读框(Open Reading Frame,ORF)序列,命名为Es UF3GT(Gen Bank注册号为KJ648620)。序列分析表明,该基因ORF全长为1356 bp,编码451个氨基酸,与其它植物中UF3GT蛋白序列的相似性为40%~50%。进化树分析发现,Es UF3GT同催化类黄酮3-O糖基化的糖基转移酶聚为一枝。qRTPCR分析结果显示,Es UF3GT在花中的表达量最高,约为叶片、花蕾中表达水平的2.3倍,果实及根中表达水平的19倍。花青素含量检测表明,花蕾中的含量最高(130.4 mg/100 g),分别是叶片、花、果实及根中含量的3.5、5.2、72、87倍。我们推测Es UF3GT参与了箭叶淫羊藿花色素苷的生物合成,此结果为深入开展EsUF3GT的生化功能研究奠定了基础。     

cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora

Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less understood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) ( EC 2.3.1.153 ) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52 736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multifunctional acyltransferases ( St-Pierre et al . (1998 ) Plant J. 14, 703–713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.     

Effect of polyamines on ethylene production

The polyamines putrescine, cadaverine, spermidine and spermine reduced the amount of ethylene produced by senescing petals of Tradescantia but they did not prevent anthocyanin leakage from these same petals. These polyamines also inhibited auxin-mediated ethylene production by etiolated soybean hypocotyls to less than 7 % of the control. The basic amino acids lysine and histidine reduced the amount of auxin-induced ethylene produced by soybean hypocotyls by ca 50 %. In the hypocotyls, methionine was unable to overcome the inhibition caused by the polyamines. The polyamines spermidine and spermine inhibited ethylene production induced by the application of 1-aminocyclopropane-1-carboxylic acid and they also reduced the endogenous content of this amino acid in the treated tissues.     

丽格海棠花青素苷成分及分布对花色的影响

以红色、红心白边、粉红、玫红、黄色、黄心红边、浅粉和白色8种花色丽的格海棠花瓣为试验材料,采用目视测色法、RHSCC比色法和色差仪测定花瓣表型,通过组织切片法观察花瓣色素细胞的显微结构和分布特点,采用双光束紫外-可见光分光光度计和高效液相色谱-电喷雾离子化-质谱连用技术(HPLC-ESI-MS)测定分析花瓣中花青素苷的成分和含量,为探讨丽格海棠花色的呈色机理和花色育种提供参考。结果显示:(1)丽格海棠的明度L*随花瓣颜色变深而降低,红度a*则表现出相反趋势,红度(a*)和彩度(C*)值与明度(L*)呈显著负相关关系,且a*和C*是影响L*的主要因素。(2)红花品种花瓣色素主要分布于上表皮细胞和海绵组织中;红白花品种花瓣色素主要分布于上下表皮中,且下表皮积累量更多;粉色花和玫红花品种花瓣色素主要分布于上下表皮细胞;黄红花和粉白色花品种花瓣上表皮中含有少量色素,而黄花和白花品种花瓣几乎没有色素积累。各花色丽格海棠花瓣上表皮细胞均为圆锥形,且红花和红白花品种锥形化程度最高,它们花瓣下表皮细胞均呈扁平的长方形。(3)8个丽格海棠品种花瓣中共检测出15种花青素苷,其中10种为芍药素苷,3种为矢车菊素苷,1种为锦葵素苷,1种为飞燕草素苷,酰化花青素苷占多数;红花品种花瓣中总花青素苷含量最高,玫红花品种次之,黄花和白花品种中未检出;除粉红花品种外,其余含花青素苷的品种中芍药素苷含量最高,均占总花青素苷含量的50%以上,是花瓣的主要呈色物质。(4)丽格海棠花瓣中总花青素苷含量与其红度(a*)、彩度(C*)值呈正相关关系、与其L*值呈负相关关系。研究表明,花青素苷的积累有利于丽格海棠花瓣红色化,并影响其花瓣彩度(C*)及明度(L*);色素分布细胞数量和上表皮细胞锥形化明显影响花瓣呈色,且花瓣主要的呈色物质为芍药素苷,酰基化修饰可能影响其明度。     

Isolation and characterization of the fragrant cyclamen O-methyltransferase involved in flower coloration

Anthocyanin O-methyltransferase (OMT) is one of the key enzymes for anthocyanin modification and flower pigmentation. We previously bred a novel red-purple-flowered fragrant cyclamen (KMrp) from the purple-flowered fragrant cyclamen 'Kaori-no-mai' (KM) by ion-beam irradiation. Since the major anthocyanins in KMrp and KM petals were delphinidin 3,5-diglucoside and malvidin 3,5-diglucoside, respectively, inactivation of a methylation step in the anthocyanin biosynthetic pathway was indicated in KMrp. We isolated and compared OMT genes expressed in KM and KMrp petals. RT-PCR analysis revealed that CkmOMT2 was expressed in the petals of KM but not in KMrp. Three additional CkmOMTs with identical sequences were expressed in petals of both KM and KMrp. Genomic PCR analysis revealed that CkmOMT2 was not amplified from the KMrp genome, indicating that ion-beam irradiation caused a loss of the entire CkmOMT2 region in KMrp. In vitro enzyme assay demonstrated that CkmOMT2 catalyzes the 3' or 3',5' O-methylation of the B-ring of anthocyanin substrates. These results suggest that CkmOMT2 is functional for anthocyanin methylation, and defective expression of CkmOMT2 is responsible for changes in anthocyanin composition and flower coloration in KMrp.     

Cyanidin glycosides in flowers of genus Corydalis (Fumariaceae)

Nine taxa of Corydalis were surveyed for their floral anthocyanins. Five cyanidin glycosides: cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside, cyanidin 3-(2^G-xylosylrutinoside) and cyanidin 3-(2^G-xylosylrutinoside)-7-glucoside were isolated from these taxa and identified by chemical and spectroscopic techniques. A novel anthocyanin was found in the flowers of Corydalis elata and Corydalis flexuosa cultivars, and identified to be cyanidin 3-(2^G-xylosylrutinoside)-7-glucoside. Two anthocyanins, cyanidin 3-sambubioside and cyanidin 3-(2^G-xylosylrutinoside), were also found for the first time in Corydalis flowers. Furthermore, the major anthocyanin constituent of the flowers was cyanidin 3-sambubioside in the outer petals of Corydalis ambigua and Corydalis lineariloba, and cyanidin 3-rutinoside in those of Corydalis decumbens, Corydalis curvicalcarata and Corydalis speciosa. Similarly, Corydalis incisa contained cyanidin 3-(2^G-xylosylrutinoside), and C. flexuosa ‘China Blue’ and ‘Blue Panda’, and C. elata contained the most complex structural pigment, cyanidin 3-(2^G-xylosylrutinoside)-7-glucoside, as their dominant anthocyanin in their outer petals. According to the results of anthocyanin analyses, these nine plants were classified into four groups: groups A (three taxa), B (two taxa), C (one taxa) and D (three taxa). On the other hand, the anthocyanin constituent of their inner petals was composed of cyanidin 3-rutinoside as only one dominant anthocyanin.     

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.     

The expression level of anthocyanidin synthase determines the anthocyanin content of crabapple (<Emphasis Type="Italic">Malus</Emphasis> sp.) petals

In addition to contributing to the coloration of plant organs and their defense against herbivores, the consumption of anthocyanins in the human diet has a number of health benefits. Crabapple (Malus sp.) represents a valuable experimental model system to research the mechanisms and regulation of anthocyanin accumulation, in part due to the often vivid and varied petal and leaf coloration that is exhibited by various cultivars. The enzyme anthocyanidin synthase (ANS) plays a pivotal role in anthocyanin biosynthesis; however, the relationship between ANS expression and petal pigmentation has yet to be established in crabapple. To illuminate the mechanism of anthocyanin accumulation in crabapple petals, we evaluated the expression of two crabapple ANS allelic genes (McANS-1 and McANS-2) and the levels of anthocyanins in petals from cultivars with dark red (‘Royalty’) and white (‘Flame’) petals, as well as another (‘Radiant’) whose petals have an intermediate pink color. We determined that the expression of McANS in the three cultivars correlated with the variation of anthocyanin accumulation during different petal developmental stages. Furthermore, transgenic tobacco plants constitutively overexpressing one of the two McANS genes, McANS-1, had showed elevated anthocyanin accumulation and a deeper red coloration in their petals than those from untransformed control lines. In conclusion, we propose that McANS are responsible for anthocyanin accumulation during petal coloration in different crabapple cultivars.     

An ankyrin repeat protein is involved in anthocyanin biosynthesis in Arabidopsis

The ankyrin domain is one of the most common protein motifs in eukaryotic proteins. Repeated ankyrin domains are ubiquitous and their mediation of protein-protein interactions is involved in a number of physiological and developmental responses such as the cell cycle, signal transduction and cell differentiation. A novel putative phytochrome-interacting ankyrin repeat protein 2 (PIA2) containing three repeated ankyrin domains was identified in Arabidopsis. An in vitro pull-down and phosphorylation assay revealed that PIA2 is phosphorylated and interacts directly with oat phytochrome A. The N-terminal domain of PIA2 was specifically phosphorylated, whereas interactions between the domains of PIA2 and phytochrome A had no Pr/Pfr preference. PIA2 was ubiquitously expressed in most tissues and was localized in both the nucleus and the cytoplasm independent of treatment with light of specific wavelengths. Anthocyanin accumulation in seedlings grown under far-red light, a typical phenotype of wild-type plants, was reduced in a loss-of-function mutant of PIA2 (pia2), whereas anthocyanin accumulation was increased in an overexpressing plant (PIA2-OX). The gene expression of UDP-flavonoid-3'-glucosyl-transferase (UF3GT), a major enzyme in the anthocyanin biosynthesis processes, was decreased in pia2 knockout plants suggesting that decreased anthocyanin was because of the decreased expression of UF3GT. Our results suggest that PIA2 plays a role in the anthocyanin biosynthesis during seedling development as a novel phytochrome-interacting protein.     

The effect of prolonged exposure of pregnant rats to high ambient temperature on several constituents of the genital tract's fluids

1. Various constituents of the genital tract's (GT) fluids were measured in heat-exposed rats (kept for at least 30 days at 35 +/- 1 degree C) and control rats (maintained at 22 +/- 2 degrees C). 2. There were no differences between the groups in the GT fluid volume, protein, free amino acids and glucose contents. 3. Arterial and venous blood glucose levels, pO2 and pH values were similar in both groups. 4. GT fluid protein hydrolysate from heat-exposed rats showed significantly reduced contents of glycine and alanine and elevated contents of valine and lysine as compared with controls. 5. The GT fluid's free amino acid components showed reduced glycine and elevated valine and isoleucine in the heat-exposed group as compared with controls. 6. Progesterone levels in GT fluid of heat-exposed rats was 60% higher than that of controls. 7. It is suggested that the higher progesterone concentration and the altered relative contents of several free amino acids with a possible change in the proteins of the GT fluid may affect the development of the embryo in heat-exposed rats.     

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.