Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Biochemical and genetic characterization of Arabidopsis flavanone 3beta-hydroxylase

Flavanone 3beta-hydroxylase (F3H; EC 1.14.11.9) is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC 1.14.11.23) and anthocyanidin synthase (EC 1.14.11.19), can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.     

Metabolic engineering of anthocyanin biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3beta-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.     

Anthocyanin pathway in rice (Oryza sativa L): identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanidins in pericarp

The present study has surveyed a collection of indica rice (Oryza sativa) lines for tissue-specific anthocyanin pigmentation pattern, which has also been used for a genetically meaningful classification. This classification helped predict probable genotypes of rice lines and, in the process, a leaf blade-specific dominant inhibitor of pigmentation (Ilb) was predicted and its presence later confirmed in two lines. We ascribe most tissue-specific accumulation of anthocyanins to the presence of a different set of Pl alleles. Cyanidin, as a major pigment, and peonidin, as a minor pigment, were detected in purple-pigmented tissues. Further, the floral organ-derived tissues always contained a higher level of anthocyanins and, correspondingly, a relatively increased proportion of peonidin. One line, N22B, with a brown pericarp was identified and shown to accumulate proanthocyanidins, but with no anthocyanins, in the pericarp. We propose that the accumulation of proanthocyanidins is due to a block in the anthocyanin biosynthetic pathway in rice at the anthocyanidin synthase-mediated conversion of leucoanthocyanidin to anthocyanidin.     

Flavonoid Biosynthetic Pathway and Cereal Defence Response: An Emerging Trend in Crop Biotechnoloy

The flavonoid pathway, derived from the phenylpropanoid and acetyl CoA and malonyl CoA pathways, gives rise to a diverse array of compounds such as isoflavonoids, anthocyanins, proanthocyanidins, etc. that are attributed with a multitude of biological functions. There is an increasing evidence on the role of flavonoids in defence response in many plant species though the exact mechanism is yet to be defined unequivocally. On the contrary, there is very little information on the role of flavonoids in cereal defence response. A novel strategy to improve disease resistance in cereals is by the engineering of the flavonoid pathway for enhancement of specific flavonoids in selected tissue under pathogen attack. Recent developments in molecular biology and genetics of flavonoid pathway in several plants would allow one to test its potential in improving disease resistance. Further, the powerful molecular techniques and reproducible transformation protocols would enable the manipulation of the flavonoid pathway in cereals. Transferring allied pathways, or a part of pathways to target plants seem to be within the reach of many groups. It is therefore, time for a second look at this secondary pathway particularly in the context of disease resistance phenomenon.     

Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Activation of flavonoid biosynthesis by solar radiation in bilberry (<Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> L.) leaves

The effect of solar radiation on flavonoid biosynthesis was studied in bilberry (Vaccinium myrtillus L.) leaves. Expression of flavonoid pathway genes of bilberry was studied in the upper leaves of bilberry, exposed to direct sunlight, in the shaded leaves growing lower in the same plants and in fruits. Bilberry-specific digoxigenin–dUTP-labeled cDNA fragments of five genes from the general phenylpropanoid pathway coding phenylalanine ammonia-lyase and from the flavonoid pathway coding chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase were used as probes in gene expression analysis. Anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids from the leaves and fruits were identified and quantified using high-performance liquid chromatography combined with a diode array detector. An increase in the expression of the studied flavonoid pathway genes was observed in leaves growing under direct sun exposure. Also, the concentrations of anthocyanins, catechins, flavonols and hydroxycinnamic acids were higher in the leaves exposed to direct sunlight. However, the concentration of polymeric procyanidins was lower in sun-exposed leaves, whereas that of prodelphinidins was slightly increased. The results give further support for the protective role of flavonoids and hydroxy cinnamic acids against high solar radiation in plants. Also, the roles of different flavonoid compounds as a defense against stress caused by sun exposure is discussed.Abbreviations ANS Anthocyanidin synthase - CHS Chalcone synthase - DFR Dihydroflavonol 4-reductase - F3H Flavanone 3-hydroxylase - GPD Glyceraldehyde-3-phosphate dehydrogenase - PAL Phenylalanine ammonia-lyase     

Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria xananassa)

The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria xananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3',4'-hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4'- and 3',4'-hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3',4'-hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products.     

Effects of methyl jasmonate on accumulation of flavonoids in seedlings of common buckwheat (Fagopyrum esculentum Moench)

The jasmonates, which include jasmonic acid and its methyl ester (MJ), play a central role in regulating the biosynthesis of many secondary metabolites, including flavonoids, and also are signaling molecules in environmental stresses. Synthesis of anthocyanins pigments is a final part of flavonoids pathway route. Accumulation of the pigments in young seedlings is stimulated by various environmental stresses, such as high-intensity light, wounding, pathogen attack, drought, sugar and nutrient deficiency. The anthocyanins take part in defense system against excess of light and UV-B light, and therefore it is probably main reason why young plant tissues accumulate enlarged levels of the pigments. The effects of exogenously applied MJ on level of anthocyanins, glycosides of apigenin, luteolin, quercetin and proanthocyanidins in seedlings of common buckwheat (Fagopyrum esculentum Moench) were studied. MJ decreased contents of all the found cyanidin glycosides and its aglycone in hypocotyls of buckwheat seedlings. However contents of particular anthocyanins in cotyledons of buckwheat seedlings treated with the plant hormone were not significantly different from the control. Applied doses of MJ did not affect levels of quercetin, apigenin and luteolin glycosides in the analyzed parts of buckwheat seedlings: cotyledons and hypocotyls. On the other hand, treatment of buckwheat seedlings with MJ clearly stimulated of proanthocyanidins biosynthesis in hypocotyls. We suggest that methyl jasmonate induces in hypocotyls of buckwheat seedlings the leucocyanidin reductase or anthocyanidin reductase, possible enzymes in proanthocyanidins synthesis, and/or inhibits anthocyanidin synthase, which transforms leucocyanidin into cyanidin. According to our knowledge this is the first report regarding the effect of methyl jasmonate on enhancing the accumulation of proanthocyanidins in cultivated plants.     

转CiCHS基因拟南芥的黄酮代谢及抗氧化能力分析

该研究克隆了中间锦鸡儿的查尔酮合成酶基因(CiCHS)并转入野生型拟南芥和tt4突变体,用qRT-PCR检测了转基因拟南芥中内源AtCHS基因的表达量,用分光光度法分析了转基因拟南芥的总黄酮、丙二醛含量及DPPH自由基清除能力,用HPLC法检测了转基因拟南芥的柚皮苷含量。结果显示:(1)转基因拟南芥中,内源AtCHS基因的表达量约为野生型的十分之一,总黄酮含量明显高于野生型;HPLC测得转基因株系中柚皮苷含量高于野生型;紫外照射处理前后转基因拟南芥中丙二醛积累量明显少于野生型。(2)转基因株系提取物对DPPH自由基清除能力显著高于野生型。(3)CiCHS基因互补拟南芥tt4突变体,转基因株系的种皮呈现浅棕色。研究表明,中间锦鸡儿CiCHS基因异源表达后生成了柚皮苷,使转基因植物的抗氧化性增强,部分恢复了tt4突变体的种皮颜色。     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Expressing the maize anthocyanin regulatory gene Lc increased flavonoid content in the seed of white pericarp rice and purple pericarp rice

The colour of red, purple, brown and white occurs in pericarp of rice. Here, the maize anthocyanin regulatory gene Lc under control of the promoter of the rice glutelin gene Gt1 was introduced in the white pericarp rice “Chao2-10” and purple pericarp rice “Qingjiaozidao”. The results demonstrated that some transgenic “Chao2-10” rice pericarps became brown, and the total flavonoid contents in the unpolished rice of the two transgenic rices increased significantly compared with their respective controls. Unpolished rice kernel thickness and weight in the two transgenic rices decreased slightly.     

Flavonoid genes of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>)

Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3β-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.     

Transgenic rice lines expressing maize C1 and R-S regulatory genes produce various flavonoids in the endosperm

Flavonoids, compounds that possess diverse health-promoting benefits, are lacking in the endosperm of rice. Therefore, to develop transgenic lines that produce flavonoids, we transformed a white rice cultivar, Oryza sativa japonica cv. Hwa-Young, with maize C1 and R-S regulatory genes. Expression of these transgenes was restricted to the endosperm using the promoter of a rice prolamin gene. The pericarp of the C1/R-S homozygous lines became dark brown in accordance with their maternal genotype, whereas the endosperm turned chalky, similar to the opaque kernel phenotype. Analysis via high-performance liquid chromatography (HPLC) revealed that numerous kinds of flavonoids were produced in these transgenic kernels. To identify individual flavonoids, the number of HPLC peaks was reduced through moderate acid hydrolysis, followed by ethyl acetate partitioning. Amongst the major flavonoids, dihydroquercetin (taxifolin), dihydroisorhamnetin (3'-O-methyl taxifolin) and 3'-O-methyl quercetin were identified through liquid chromatography/mass spectrometry/mass spectrometry and nuclear magnetic resonance analyses. Fluorescence labelling with diphenylboric acid showed that the flavonoids were highly concentrated in the cells of four to five outer endosperm layers. More importantly, a high fluorescence signal was present in the cytosol of the inner endosperm layers. However, the overall signal in the inner layers was significantly lower because starch granules and protein bodies occupied most of the cytosolic space. Our estimate of the total flavonoid content in the transgenic kernels suggests that C1/R-S rice has the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.