RESUMEN

Leaves of tea (Camellia sinensis L.) contain extraordinary large amounts of (-)-epigallocatechin, (-)-epicatechin, (+)-gallocatechin, and (+)-catechin and derivatives of these compounds that show positive effects on human health. The health-promoting effects of flavan 3-ols, especially those of green tea, are of scientific and public interest. Furthermore, they play a crucial role in defense against pathogens of tea. Therefore, biosynthesis of these flavonoid compounds was investigated. The anthocyanidin reductase enzyme recently described from Arabidopsis and Medicago was shown to be present in tea with very high activity and produces epicatechin as well as epigallocatechin from the respective anthocyanidins, thus explaining the very high contents of these compounds. A strong combined dihydroflavonol 4-reductase/leucoanthocyanidin 4-reductase activity was demonstrated and catalyzes the key steps in catechin and gallocatechin formation. Together with the enzyme activities and substrate specificities of the preceding enzymatic reactions, the biosynthesis of the most prominent flavonoids of tea is elucidated.

Asunto(s)

RESUMEN

A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.

Asunto(s)

RESUMEN

Flavone synthase I, a soluble 2-oxoglutarate-dependent dioxygenase catalyzing the oxidation of flavanones to flavones in several Apiaceae species, was induced in parsley cell cultures by continuous irradiation with ultraviolet/blue light for 20 h. The enzyme was extracted from these cells and purified by a revised purification protocol including the fractionation on hydroxyapatite, Fractogel EMD DEAE, and Mono Q anion exchangers, which resulted in an apparently homogeneous flavone synthase at approximately 10-fold higher yield as compared to the previous report. The homogeneous enzyme was employed to raise an antiserum in rabbit for partial immunological characterization. The specificity of the polyclonal antibodies was demonstrated by immunotitration and Western blotting of the crude ammonium sulfate-fractionated enzyme as well as of the enzyme at various stages of the purification. High titer cross-reactivity was observed toward flavone synthase I, showing two bands in the crude extract corresponding to molecular weights of 44 and 41 kDa, respectively, while only the 41 kDa was detected on further purification. The polyclonal antiserum did not cross-react with recombinantly expressed flavanone 3beta-hydroxylase from Petunia hybrida or flavonol synthase from Citrus unshiu, two related 2-oxoglutarate-dependent dioxygenases involved in the flavonoid pathway.

Asunto(s)

RESUMEN

During the past decade, the increasing knowledge of flavonoid biosynthesis and the important function of flavonoid compounds in plants and in human and animal nutrition have made the biosynthetic pathways to flavonoids and isoflavonoids excellent targets for metabolic engineering. Recent strategies have included introducing novel structural or regulatory genes, and the antisense or sense suppression of genes in these pathways.

Asunto(s)

RESUMEN

The phenylpropanoid pathway results in the synthesis of thousands of compounds, including flavonoids like flavonols, anthocyanidins and tannins. In Arabidopsis thaliana, the lack of tannins in the seed coat (testa) causes the transparent testa (tt) phenotype. In the present study, we identified the gene responsible for the tt7 mutation. We show that TT7 encodes the enzyme flavonoid 3'-hydroxylase (F3'H), and demonstrate that this P450-dependent monooxygenase has F3'H activity. The availability of the AtF3'H gene and promoter sequence will allow us to study the coregulation of a complete set of flavonol and anthocyanidin biosynthesis genes in A. thaliana, and makes in vitro synthesis of hydroxylated flavonoids more feasible.

Asunto(s)

RESUMEN

In Gerbera hybrids, flavone synthesis is controlled by the locus Fns. The responsible enzyme, flavone synthase II, belongs to the NADPH-dependent cytochrome P450 monooxygenases. From two different chemogenetic defined Gerbera lines with the dominant (fns +.) or recessive (fns fns) alleles at the locus Fns, a cytochrome P450 fragment (CypDDd7a) was isolated using a differential display technique with upstream primers based on the conserved heme-binding region of cytochrome P450 proteins. The full-length cDNA (CYP93B2) which contained the open-reading frame and part of the CypDDd7a sequence was isolated via 5'-RACE and end-to-end PCR with gene specific primers. Northern blot analysis of total RNA of Gerbera hybrids indicated that the CYP93B2 gene was only transcribed in lines with the dominant allele fns + and that the transcript levels during flower development are in agreement with the measured enzyme activity of FNS II and flavone accumulation. Microsomes from yeast cells expressing CYP93B2 catalysed the direct formation of [14C]-flavones from the respective [14C]-flavanones. Thus, CYP93B2 was shown to encode flavone synthase II. This is the first report of the isolation and expression of a functional FNS II cDNA clone from any species. The comparison of amino acid sequences revealed that CYP93B2 had 54% identity with the sequence of CYP93B1, which has recently been reported as a (2S)-flavanone 2-hydroxylase of Glycyrrhiza echinata L.

Asunto(s)

RESUMEN

The yellow colour of Chrysanthemum segetum petals is due to the presence of the 7-O-glucosides of quercetin and particularly gossypetin (8-hydroxyquercetin). In petal extracts of C. segetum an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5'-diphosphoglucose (UDPG) to the 7-hydroxyl group of flavonols with gossypetin and quercetin as the best substrates. Besides flavonols flavanones and flavones were found to be glucosylated in the 7-position. The pH-optimum of the reaction highly depended on the substrate used. With quercetin as substrate, maximal enzyme activity occurred at a pH of 8.25 and a temperature of 25 degrees C, but 7-O-glucosylation also proceeded at low temperatures. Studies on temperature stability revealed, that there was no influence on the glucosylation reaction up to 40 degrees C. Higher temperatures led to a loss of enzyme activity. Using gossypetin as a substrate a similar course of temperature stability was observed. Addition of Mg2+, Ca2+ and KCN slightly stimulated 7-O-glucosylation, whereas Co2+, Cu2+, Fe2+, Hg2+, p-hydroxymercuribenzoate and N-ethylmaleimide showed a strong inhibitory effect. Additional enzymatic studies were performed with the commercial strain "Stern des Orients" where gossypetin 7-O-glucoside is restricted to the inner parts of the petals. For enzyme extracts from both parts of the petals gossypetin was found to be the most attractive substrate. In comparison to quercetin (133.4 mu kat/kg protein) an about three times higher specific activity of the 7-O-glucosyltransferase(s) was determined with gossypetin (382.1 mu kat/kg protein) as substrate, indicating that hydroxylation of quercetin in 8-position to gossypetin precedes 7-O-glucosylation.

Asunto(s)

RESUMEN

Using a cDNA encoding the flavanone 3ß-hydroxylase (FHT) from Dianthus caryophyllus (carnation) as a probe, we isolated the FHT gene from a genomic library. Sequence analysis revealed that the FHT gene consists of three exons and two introns. Two putative light-regulated elements were identified in the promoter region by sequence comparison. Southern blot analysis indicated that a single copy of the FHT gene is in the plant genome. Furthermore, a stable and an unstable FHT mutant of D. caryophyllus, both showing almost no FHT activity, were analyzed by Southern, Northern and Western blotting. It turned out that the FHT gene is present in both mutants, but no protein was detectable in the mutant flowers. FHT mRNA in amounts comparable to that found in the wildtype is present in flowers of the stable mutant, indicating a block in translation, but not in flowers of the unstable mutant, indicating a block in transcription. The translational block of the FHT mRNA of the stable mutant was demonstrated by in vitro translation of total flower mRNA followed by the specific measurement of FHT activity.

RESUMEN

A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.

Asunto(s)

RESUMEN

We have cloned and sequenced cDNAs for the glyceraldehyde-3-phosphate dehydrogenase of glycolysis, gapC, from a bryophyte, a gymnosperm, and three angiosperms. Phylogenetic analyses are presented for these data in the context of other gapC sequences and in parallel with published nucleotide sequences for the chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL). Relative-rate tests were performed for these genes in order to assess variation in substitution rate for coding regions, along individual plant lineages studied. The results of both gene analyses suggest that the deepest dichotomy within the angiosperms separates not magnoliids from remaining angiosperms, but monocotyledons from dicotyledons, in sharp contrast to prediction from the Euanthial theory for angiosperm evolution. Furthermore, these chloroplast and nuclear sequence data taken together suggest that the separation of monocotyledonous and dicotyledonous lineages took place in late Carboniferous times [approximately 300 Myr before the present (Mybp)]. This date would exceed but be compatible with the late-Triassic (approximately 220 Mybp) occurrence of fossil reproductive structures of the primitive angiosperm Sanmiguelia lewisii.

Asunto(s)

RESUMEN

A cDNA encoding flavanone 3 beta-hydroxylase was isolated from petals of Petunia hybrida. The open reading frame of the nearly full length cDNA coded for a 369-amino acid polypeptide with a calculated Mr of 41,466. The function of this nucleotide sequence was verified by comparison with amino acid sequence of the amino terminus and tryptic peptides from purified plant enzyme, by Northern blotting with RNA from wild type and mutant plants, and by prokaryotic expression yielding an enzymatically active hydroxylase. Computer-aided sequence analysis revealed high similarity (73.5%) to flavanone 3 beta-hydroxylase from barley. Genomic Southern blot analysis showed the presence of only one gene for flavanone 3 beta-hydroxylase in P. hybrida.

Asunto(s)

RESUMEN

Flavonoid analysis and supplementation experiments with dihydroflavonols and leucocyanidin on two cyanic, two acyanic and one white/red-variegated flowering strain of Dianthus caryophyllus (carnation) showed that in the acyanic strains recessive alleles (aa) of the gene A interrupt the anthocyanin pathway between dihydroflavonols and leucoanthocyanidins. The instability in the variegated strain involves the same step and is obviously caused by the multiple allele a (var) . In confirmation of these results, dihydroflavonol 4-reductase activity could be demonstrated in enzyme extracts from cyanic flowers and cyanic parts of variegated flowers but not in preparations from acyanic flowers or acyanic parts. The enzyme catalyzes the stereospecific reduction of (+)dihydrokaempferol to (+)-3,4-leucopelargonidin with NADPH as cofactor. A pH optimum around 7.0 and a temperature optimum at 30° C was determined, but the reduction reaction also proceeded at low temperatures. (+)Dihydroquercetin and (+)dihydromyricetin were also reduced to the respective flavan-3,4-cis-diols by the enzyme preparations from carnation flowers, and were even better substrates than dihydrokaempferol.

RESUMEN

Twelve loci have previously been identified in tomato (Lycopersicon esculentum) that control the intensity and distribution of anthocyanin pigmentation; these are useful genetic markers because they encode phenotypes that are readily visualized in the hypocotyls of emerging seedlings. In order to obtain molecular probes for tomato anthocyanin biosynthesis genes, we isolated two cDNAs which encode chalcone synthase (CHS), one of the key enzymes in anthocyanin biosynthesis, from a tomato hypocotyl cDNA library. By comparing their nucleic acid sequences, we determined that the two CHS cDNAs have an overall similarity of 76% at the nucleotide level and 88% at the amino acid level. We identified hybridization conditions that would distinguish the two clones and by Northern analysis showed that 1.5 kb mRNA species corresponding to each cDNA were expressed in cotyledons, hypocotyls and leaves of wild-type seedlings. Hybridization of the cDNAs at low stringency to genomic blots indicated that in tomato, CHS genes comprise a family of at least three individual members. The two genes that encode the CHS cDNAs were then placed onto the tomato genetic map at unique loci by restriction fragment length polymorphism mapping. We also assayed the activity of CHS and another enzyme in the anthocyanin pathway, flavone 3-hydroxylase, in hypocotyl extracts of wild-type tomato and a number of anthocyanin-deficient mutants. Five mutants had reduced CHS activity when compared to the wild-type controls. Of these, three were also reduce in flavone 3-hydroxylase activity, suggesting a regulatory role for these loci. The other two mutants were preferentially reduced in CHS activity, suggesting a more specific role for these loci in CHS expression.

Asunto(s)

RESUMEN

Petunia hybrida is one of the classical subjects of investigation in plants in which the pathway of anthocyanin biosynthesis has been analysed genetically and biochemically. In petunia cyanidin- and delphinidin-derivatives, but no pelargonidin-derivatives are produced as pigments. This is due to the substrate specificity of the dihydroflavonol 4-reductase of petunia, which cannot reduce dihydrokaempferol. The petunia mutant RL01, which accumulates dihydrokaempferol, shows no flower pigmentation. RL01 served as a recipient for the transfer of the A1 gene of Zea mays encoding dihydroquercetin 4-reductase, which can reduce dihydrokaempferol and thereby provided the intermediate for pelargonidin biosynthesis. Transformation of RL01 with a vector p35A1, containing the A1-complementary DNA behind the 35S promotor leads to red flowers of the pelargonidin-type. Thus a new flower pigmentation pathway has been established in these plants.

Asunto(s)

RESUMEN

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.

RESUMEN

Callus cultures were derived from stems and leaves of 3 anthocyanin producing and 3 acyanic lines of Callistephus chinensis (Compositae). The tissue cultures of the cyanic lines were shown to produce cyanidin whereas in the calli of the acyanic lines no anthocyanin synthesis occurred Culture conditions were improved in order to enhance both anthocyanin production and growth of the tissue cultures.

RESUMEN

A cell-free extract from flowers of Matthiola incana catalyzes a NADPH-dependent stereospecific reduction of (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4'-trihydroxyflavan-3,4-cis-diol). The pH-optimum of this reaction is around 6. The rate of reaction with NADH was about 50% of that found with NADPH. (+)-Dihydroquercetin and (+)-dihydromyricetin were also reduced by the enzyme preparation to the corresponding flavan-3,4-cis-diols. Correlation between the genotype of M. incana and the presence of dihydroflavonol 4-reductase is strong evidence that this enzyme is involved in anthocyanin biosynthesis.

RESUMEN

(+)Leucopelargonidin [(2R,3S,4R)-3,4,5,7,4'-pentahydroxyflavan] and (+)leucocyanidin [(2R,3S,4R)-3,4,5,7,3',4'-hexahydroxyflavan] were synthesized from (+)dihydrokaempferol and (+)dihydroquercetin, respectively, by sodium-borohydride reduction. The chemical and optical purity of these compounds was established by ultraviolet, proton-nuclear-magnetic-resonance, and optical-rotatory-dispersion spectroscopy. Supplementation experiments with these leucoanthocyanidins were carried out with genetically defined acyanic flowers of Matthiola incana. Feeding of leucopelargonidin and leucocyanidin to line 17 (blocked between dihydroflavonols and anthocyanins) and line 18 (blocked in the chalcone-synthase gene) led to formation of the corresponding anthocyanidin 3-O-glucosides, whereas supplementation of line 19 (blocked in a locus other than line 17 between dihydroflavonols and anthocyanins) did not result in anthocyanin synthesis. The conversion of leucopelargonidin into pelargonidin 3-O-glucoside was further confirmed by incorporation of [4-(3)H]leucopelargonidin into pelargonidin derivatives. The results are strong indications for the role of leucoanthocyanidins (flavan-3,4-diols) as intermediates in anthocyanin biosynthesis.

RESUMEN

In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3'-position. Similar to the flavonoid 3'-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3'-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3'-position in Petunia. Thus, the introduction of the 3'-hydroxyl group is clearly achieved by hydroxylation of C15-intermediates, and the concomitant occurrence of the 3',4'-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.