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Quantification of the Production of Dihydrokaempferol by Flavanone 3-Hydroxytransferase Using Capillary ElectrophoresisOwens, Daniel K., Hale, Tracy, Wilson, Lori J., McIntosh, Cecilia A. 17 April 2002 (has links)
A sensitive method using capillary electrophoresis for the separation, detection, and quantification of dihydrokaempferol (1) is reported. Well-resolved, sharp symmetrical peaks were obtained in grapefruit leaf extracts for 1, naringenin (2), and the internal standard, naringin (3). Long columns were required to resolve 1 from 2 in crude enzyme reactions and this resulted in run times of 60 min. The limit of detection for 1 was found to be 1.44 ng/μL (4.2 pg). The method showed excellent linearity and reproducibility. The method was used to determine the activity of flavanone 3-hydroxytransferase (F3H) in leaf tissue of grapefruit by quantification of the production of dihydrokaempferol in controlled time course reactions. The sensitivity of the method makes it adaptable to assaying F3H activity in individual young seedlings and/ or in small tissue samples and requires only 100 mg of tissue.
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Polyphénols d’agrumes (flavanones) : extraction de glycosides de la peau d’orange, synthèse de métabolites chez l’homme (glucuronides) et étude physico-chimique de leur interaction avec la sérum albumine / Citrus polyphenols (flavanones) : extraction of glycosides from orange peel, synthesis of metabolites (glucuronides) found in human and a physico-chemical study to investigate their interaction with human serum albuminKhan, Muhammad Kamran 15 November 2010 (has links)
Un groupe d'études épidémiologiques fournit une bonne preuve de la relation inverse associé à la consommation de fruits et légumes et les maladies chroniques important comme maladies cardiovasculaires et certains types de cancers. Après les longues années d'études sur phytomacronutrients, le rôle de phytomicronutrients tels que les polyphénols est désormais très étudiée et appréciée dans le contrôle de ces maladies dégénératives. La présente étude combine les études d'extraction, de synthèse et d'analyse sur les principaux polyphénols des fruits d'agrumes, FLAVANONES. Connaissance de nutritionnels et de santé a augmenté la production d'agrumes en provenance des dernières décennies. Ces productions plus générer des bye-produits. Pour leur utilisation alternative à des antioxydants extraits riches, l'extraction assistée par ultrasons (UAE) des polyphénols en particulier flavanones de l'orange (Citrus sinensis L.) par son peau en utilisant l'éthanol comme solvant de qualité alimentaire a été prouvé son efficacité en comparaison avec la méthode conventionnelle . Un plan composite central (CCD) a révélé que l'approche des conditions optimisées pour UAE ont une température de 40 ° C, une puissance de 150W sonication et un 4:1 (v / v) d'éthanol: ratio de l'eau. En outre, l'activité antioxydante déterminée par les tests DPPH et ORAC a confirmé la pertinence des UAE pour la préparation d'extraits de plantes riches en antioxydants.Les glucuronides de flavanone sont les principaux métabolites phénoliques détectés dans le plasma humain après la consommation d'agrumes. Jusqu'à maintenant, toutes les études sur les cellules liées au cancer ou les maladies cardiovasculaires ont été réalisées soit sur les aglycones ou sur leurs glycosides. Par conséquent, il ya grand besoin de glucuronides flavanone pure pour démontrer le potentiel réel de flavanones dans la prévention de ces maladies. Dans ce travail, glucuronides de naringénine (4'- et 7-O-β-D-glucuronides) et de hespérétine (3'- et 7-O-β-D-glucuronides), les aglycones flavanone majeur dans le pamplemousse et d'orange, respectivement, ont été synthétisés chimiquement par une protection et la déprotection sélective des groupements d'acide glucuronique et de flavanone. La caractérisation structurale complète de composés purifiés a été réalisée par résonance magnétique nucléaire et spectrométrie de masse.L'affinité des quatre glucuronides pour l'albumine sérum d’humaine (HSA) a été testée par leur capacité à éteindre la fluorescence intrinsèque de HSA (Trp, seul résidu de sous-domaine IIA). Leurs constantes de fixation (K) ont été estimées de l'ordre de 30 à 60 × 103 M-1 et comparées à celles de l'aglycones (70 à 90 × 103 M-1). Les enquêtes de la liaison compétitive ou non compétitive de la glucuronides dans la présence de sondes fluorescentes (sarcosine dansyl) nous a permis d'obtenir un aperçu dans les sites de liaison. L'étude a également été étendue aux chalcones hespérétine et naringénine (synthétisés en utilisant des conditions alcalines optimisée), qui sont les précurseurs de biosynthèse des flavanones / A bunch of epidemiological studies provides good evidence on the inverse relationship associated with the consumption of fruits and vegetables and the chronic diseases importantly cardiovascular diseases and some types of cancers. After the long years of study on phytomacronutrients, the role of phytomicronutrients such as polyphenols is now highly studied and appreciated in the control of such degenerative diseases. The present study combines the extraction, synthetic and analytical studies on the major polyphenols of citrus fruits, FLAVANONES.Awareness of nutritional and health facts has increased the production of citrus fruits from last few decades. These higher productions generate higher by-products. For their alternative utilisation to have antioxidants rich extracts, the ultrasound-assisted extraction (UAE) of polyphenols especially flavanones from orange (Citrus sinensis L.) peel by using ethanol as afood grade solvent has been proved its efficiency when compared with the conventional method. A central composite design (CCD) approach revealed that the optimized conditions for UAE were a temperature of 40°C, a sonication power of 150W and a 4:1 (v/v) ethanol:water ratio. Furthermore, the antioxidant activity determined by the DPPH and ORAC tests confirmed the suitability of UAE for the preparation of antioxidant-rich plant extracts. Flavanone glucuronides are the major phenolic metabolites detected in human plasma after consumption of citrus fruits. Up to now all cell studies related to cancer or cardiovascular diseases were conducted either on the aglycones or on their glycosides. Hence, there is great need of pure flavanone glucuronides to demonstrate the real potential of flavanones in the prevention of these diseases. In this work, glucuronides of naringenin (4′- and 7-O-β-D-glucuronides) and hesperetin (3′- and 7-O-β-D-glucuronides), the major flavanone aglycones in grapefruit and orange respectively, have been chemically synthesized by selective protection and deprotection of flavanone and glucuronic acid moieties. The complete structural characterisation of purified compounds were realised by nuclear magnetic resonance and mass spectrometry.The affinity of the four glucuronides for human serum albumin (HSA) was tested via their ability to quench the intrinsic fluorescence of HSA (single Trp residue in sub-domain IIA). Their binding constants (K) were estimated in the range of 30 – 60 × 103 M-1 and compared with those of the aglycones (70 – 90 × 103 M-1). Investigations of competitive or noncompetitive binding of the glucuronides in the presence of fluorescent probes (dansyl sarcosine) allowed us to get some insight in the binding sites. The study was also extended to the hesperetin and naringenin chalcones (synthesised using optimized alkaline conditions), which are the biosynthetic precursors of flavanones
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Polyphénols d'agrumes (flavanones) : extraction de glycosides de la peau d'orange, synthèse de métabolites chez l'homme (glucuronides) et étude physico-chimique de leur interaction avec la sérum albumineKhan, Muhammad Kamran 15 November 2010 (has links) (PDF)
Un groupe d'études épidémiologiques fournit une bonne preuve de la relation inverse associé à la consommation de fruits et légumes et les maladies chroniques important comme maladies cardiovasculaires et certains types de cancers. Après les longues années d'études sur phytomacronutrients, le rôle de phytomicronutrients tels que les polyphénols est désormais très étudiée et appréciée dans le contrôle de ces maladies dégénératives. La présente étude combine les études d'extraction, de synthèse et d'analyse sur les principaux polyphénols des fruits d'agrumes, FLAVANONES. Connaissance de nutritionnels et de santé a augmenté la production d'agrumes en provenance des dernières décennies. Ces productions plus générer des bye-produits. Pour leur utilisation alternative à des antioxydants extraits riches, l'extraction assistée par ultrasons (UAE) des polyphénols en particulier flavanones de l'orange (Citrus sinensis L.) par son peau en utilisant l'éthanol comme solvant de qualité alimentaire a été prouvé son efficacité en comparaison avec la méthode conventionnelle . Un plan composite central (CCD) a révélé que l'approche des conditions optimisées pour UAE ont une température de 40 ° C, une puissance de 150W sonication et un 4:1 (v / v) d'éthanol: ratio de l'eau. En outre, l'activité antioxydante déterminée par les tests DPPH et ORAC a confirmé la pertinence des UAE pour la préparation d'extraits de plantes riches en antioxydants.Les glucuronides de flavanone sont les principaux métabolites phénoliques détectés dans le plasma humain après la consommation d'agrumes. Jusqu'à maintenant, toutes les études sur les cellules liées au cancer ou les maladies cardiovasculaires ont été réalisées soit sur les aglycones ou sur leurs glycosides. Par conséquent, il ya grand besoin de glucuronides flavanone pure pour démontrer le potentiel réel de flavanones dans la prévention de ces maladies. Dans ce travail, glucuronides de naringénine (4'- et 7-O-β-D-glucuronides) et de hespérétine (3'- et 7-O-β-D-glucuronides), les aglycones flavanone majeur dans le pamplemousse et d'orange, respectivement, ont été synthétisés chimiquement par une protection et la déprotection sélective des groupements d'acide glucuronique et de flavanone. La caractérisation structurale complète de composés purifiés a été réalisée par résonance magnétique nucléaire et spectrométrie de masse.L'affinité des quatre glucuronides pour l'albumine sérum d'humaine (HSA) a été testée par leur capacité à éteindre la fluorescence intrinsèque de HSA (Trp, seul résidu de sous-domaine IIA). Leurs constantes de fixation (K) ont été estimées de l'ordre de 30 à 60 × 103 M-1 et comparées à celles de l'aglycones (70 à 90 × 103 M-1). Les enquêtes de la liaison compétitive ou non compétitive de la glucuronides dans la présence de sondes fluorescentes (sarcosine dansyl) nous a permis d'obtenir un aperçu dans les sites de liaison. L'étude a également été étendue aux chalcones hespérétine et naringénine (synthétisés en utilisant des conditions alcalines optimisée), qui sont les précurseurs de biosynthèse des flavanones
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Antiplasmodial and larvicidal flavonoids from Derris trifoliataPeter, Martin G., Yenesew, Abiy, Twinomuhwezi, Hannington, Kabaru, Jacques M., Akala, Hoseah M., Kiremire, Bernard T., Heydenreich, Matthias, Eyase, Fredrick, Waters, Norman C., Walsh, Douglas S. January 2009 (has links)
From the dichloromethane-methanol (1:1) extract of the seed pods of Derris trifoliata, a new flavanone derivative (S)-lupinifolin 4´-methyl ether was isolated. In addition, the known flavonoids lupinifolin and rotenone were identified. The structures were determined on the basis of spectroscopic evidence. Lupinfolin showed moderate in vitro antiplasmodial activity against the D6 (chloroquine-sensitive) and W2 (chloroquineresistant)
strains of Plasmodium falciparum. The different parts of this plant showed larvicidal activities against Aedes aegypti and rotenoids were identified as the active principles.
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Cyclopia maculata : source of flavanone glycosides as precursors for taste modulating aglyconesDu Preez, Brigitte Von Pressentin 04 1900 (has links)
Thesis (MScFoodSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The flavanone aglycones, hesperetin and eriodictyol, have been identified as potential taste modulators
with reported sweetness-enhancing and bitterness-masking properties, respectively. Reduction of the
sugar content of food products has become important in view of the global obesity epidemic. Taste
modulators have shown potential to enhance the sweet taste of reduced-sugar foods without
unfavourably affecting their flavour profile. On the other hand, bitterness-masking taste modulators are
useful to mask the bitter taste of functional phytochemical ingredients. In the current study, Cyclopia
maculata (honeybush) was investigated as potential source of hesperetin- and eriodictyol-enriched
extracts. Hesperetin and eriodictyol were present mainly below the quantification limit in C. maculata
plant material, including unfermented leaf and stem material, unfermented and fermented tea, as well as
the fermented by-product (< 40 mesh and > 12 mesh). Conversely, their rutinoside and modulatinginactive
derivatives, hesperidin and eriocitrin were present at substantially higher concentrations in the
plant material. The stems and by-product were shown to be good sources of hesperidin, but not
eriocitrin. The qualitative and quantitative phenolic profile of the by-product was similar to that of the
stems. The tea processing by-product was therefore selected to optimise extraction of flavanone
glycosides for subsequent de-glycosylation of the flavanone glycosides to aglycones.
The by-product was subjected to ultrasound-assisted extraction to investigate its potential as
renewable source of the flavanone glycosides. Response surface methodology (RSM) was employed to
optimise and study the individual and interactive effects of the process variables, namely ethanol
concentration (% v/v), time (min), temperature (°C), and solvent:solid ratio (mL/g), on flavanone
glycoside extraction. The hesperidin yield and content (of extract), as well as extract yield, increased with
an increase in extraction time, temperature and solvent:solid ratio. Practical process restrictions limited
global optimisation and only an optimum of 52.8% (v/v) ethanol for extract and hesperidin yield could be
reached. Temperature was the parameter with the most significant effect (p < 0.05) on extraction
efficiency among those studied. Practical process parameter values that were feasible for industrial
application (52.8% (v/v) ethanol, 20 mL/g solvent:solid ratio, 60°C and 30 min) were selected for the
preparation of a flavanone glycoside-enriched extract from the tea processing by-product. The flavanone glycoside-enriched extract was subjected to acid-catalysed hydrolysis to deglycosylate
hesperidin and eriocitrin to hesperetin and eriodictyol, respectively. RSM was employed to
optimise the acid hydrolysis process and to study the effect of the hydrolysis parameters (temperature
(°C) and time (min)) on hydrolysis efficiency. At the maximum temperature (92.1°C) and corresponding
optimum time (98.4 min) ca 80% conversion of hesperidin to hesperetin was achieved. Substantially more
eriodictyol formed during acid hydrolysis than eriocitrin present in the initial extract owing to the deglycosylation
of unidentified glycosides with the same aglycone. Unidentified breakdown products
imparting a red colour to the acid-hydrolysed extract were also observed. The total phenolic content of the acid-hydrolysed extract was significantly higher (p < 0.05) than that of the unhydrolysed extract,
indicating the formation of unidentified compounds with the ability to reduce the Folin-Ciocalteau
reagent, although no significant difference (p ≥ 0.05) between the antioxidant activities of these extracts,
as assessed with the DPPH radical scavenging and ORAC assays, was observed. The potential of enzymatic
bioconversion as an alternative to acid-catalysed hydrolysis was investigated using commercial
hesperidinase. Bioconversion resulted only in de-rhamnosylation with ca 100% conversion of hesperidin
to hesperetin-7-O-glucoside in an aqueous C. maculata extract at pH 4.0 and 40°C. / AFRIKAANSE OPSOMMING: Die flavanoon aglikone, hesperetien and eriodiktiol, is geïdentifiseer as potensiële smaakmoduleerders
met berigte soetheid-versterkende en bitter-maskerende eienskappe, onderskeidelik. Vermindering van
die suikerinhoud van voedselprodukte het belangrik geword in die lig van die wêreldwye vetsugepidemie.
Smaakmoduleerders het die potensiaal getoon om die soet smaak van voedsel met verlaagde
suikerinhoud te versterk sonder om hul geurprofiel ongunstig te beïnvloed. Andersyds is bittermaskerende
smaakmoduleerders nuttig om die bitter smaak van funksionele fitochemiese bestanddele te
maskeer. In die huidige studie is Cyclopia maculata (heuningbos) ondersoek as ‘n potensiële bron van
hesperetien- and eriodiktiol-verrykte ekstrakte. Hesperetien and eriodiktiol was hoofsaaklik teenwoordig
onder die kwantifiseringsperk in C. maculata plantmateriaal, insluitend ongefermenteerde blaar- en
stokmateriaal, ongefermenteerde en gefermenteerde tee, asook die gefermenteerde byproduk (< 40
maas en > 12 maas). Hierteenoor was hul rutinosiedes en modulerend-onaktiewe derivate, hesperidien
and eriositrien, teenwoordig in aansienlik hoër konsentrasies in die plantmateriaal. Die stokmateriaal en
byproduk is getoon om goeie bronne van hesperidien, maar nie eriositrien nie, te wees. Die kwalitatiewe
en kwantitatiewe fenoliese profiel van die byproduk was soortgelyk aan dié van die stokke. Die teeprosesseringsbyproduk
is dus geselekteer om die ekstraksie van flavanoonglikosiede, voorafgaande hul
de-glikosilering na aglikone, te optimeer.
Die byproduk is aan ekstraksie met behulp van ultrasoniese klank onderwerp om die potensiaal
daarvan as hernubare bron van flavanoonglikosiede te ondersoek. Respons-oppervlak Metodologie
(ROM) is gebruik om die individuele en wisselwerking effekte van die proses veranderlikes, naamlik
etanolkonsentrasie (% v/v), tyd (min), temperatuur (°C), en oplosmiddel:vastestof verhouding (mL/g), op
flavanoonglikosied ekstraksie te optimiseer en te bestudeer. Die hesperidienopbrengs en -inhoud (van
ekstrak), sowel as die ekstrakopbrengs, het toegeneem met ‘n toename in die ekstraksietyd, -
temperatuur en oplosmiddel:vastestof verhouding. Praktiese prosesbeperkings het die globale
optimisering beperk en slegs ‘n optimum van 52.8% (v/v) etanol vir ekstrak- en hesperidienopbrengs kon
bereik word. Temperatuur was die parameter met die mees beduidende effek (p < 0.05) op ekstraksie
doeltreffendheid van dié wat bestudeer is. Praktiese prosesparameterwaardes wat haalbaar is vir
industriële toepassing (52.8% (v/v) etanol, 20 mL/g oplosmiddel:vastestof verhouding, 60°C en 30 min) is
geselekteer vir die voorbereiding van 'n flavanoonglikosied-verrykte ekstrak uit die teeprosesseringsbyproduk. Die flavanoonglikosied-verrykte ekstrak is aan suur-gekataliseerde hidrolise onderwerp om
hesperidien en eriositrien na hesperetien en eriodiktiol, onderskeidelik, te de-glikosileer. ROM is gebruik
om die suurhidrolise proses te optimeer en die effek van die hidrolise parameters (temperatuur (°C) en
tyd (min)) op hidrolise doeltreffendheid te bestudeer. Ongeveer 80% omskakeling van hesperidien na
hesperetien is behaal teen die maksimum temperatuur (92.1 °C) en ooreenstemmende optimum tyd (98.4 min). Aansienlik meer eriodiktiol is tydens suurhidrolise gevorm as eriositrien wat in die
oorspronklike ekstrak teenwoordig was, as gevolg van de-glikosilering van ongeïdentifiseerde glikosiede
met dieselfde aglikoon. Ongeïdentifiseerde afbreekprodukte, wat 'n rooi kleur aan die suurgehidroliseerde
ekstrak gegee het, is ook waargeneem. Die totale fenoliese inhoud van die suurgehidroliseerde
ekstrak was beduidend hoër (p < 0.05) as dié van die ongehidroliseerde ekstrak, wat die
vorming van onbekende verbindings met die vermoeë om die Folin-Ciocalteau reagens te reduseer
aandui, hoewel daar geen beduidende verskil (p ≥ 0.05) tussen die antioksidant-aktiwiteite van hierdie
ekstrakte, soos bepaal met die DPPH radikaal blussings- en ORAC toetse, waargeneem is nie. Die
potensiaal van ensiematiese bio-omskakeling as 'n alternatief vir suur-gekataliseerde hidrolise is
ondersoek met behulp van kommersiële hesperidinase. Bio-omskakeling het slegs tot de-ramnosilering
gelei met ca 100% omskakeling van hesperidien na hesperetien-7-O-glukosied in 'n C. maculata
waterekstrak by pH 4.0 en 40°C.
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Farmakokinetika flavanonů / Pharmacokinetics of flavanonesUramová, Daniela January 2017 (has links)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Daniela Uramová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of diploma thesis: Pharmacokinetics of flavanones The aim of the work was to summarize the available information regarding the fate of the flavanones in the human organism. These flavonoids are a common part of human diet, and therefore oral administration is the most relevant and examined. There are many obstacles in the digestive tract which are lowering their absorption. Flavanones in human food occur mainly in the form of glycosides, and therefore must be deglycosylated by the β- glucosidase enzyme family. Aglycones are absorbed mainly in the small intestine. Flavonoids in the form of non-cleavable glycosides (e.g., rutinosides) are absorbed in the distal parts of the digestive system, after cleavage of the sugar component by intestinal bacteria. They also decompose the flavanone ring. This leads to substances with a phenylpropionic structure which can be absorbed. In general, flavanones are subject to extensive metabolism by cytochrome P450, not only in the liver but also in the enterocytes, which greatly limits their bioavailability. They are also rapidly conjugated with glucuronic or sulfuric acid. The...
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Flavanone-7-O-Glucosyltransferase Activity From Petunia hybridaDurren, Randy L., McIntosh, Cecilia A. 01 November 1999 (has links)
Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 μM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavonoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.
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Flavanone 3-Hydroxylase Expression in Citrus Paradisi and Petunia Hybrida SeedlingsPelt, Jennifer L., Downes, W. Andrew, Schoborg, Robert V., McIntosh, Cecilia A. 01 January 2003 (has links)
Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.
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Molecular and Biochemical Genetics of 2-Oxoglutarate-Dependent Dioxygenases Required for Flavonoid Biosynthesis in Arabidopsis thalianaPelletier, Matthew K. 24 April 1997 (has links)
Three 2-oxoglutarate-dependent dioxygenases required for flavonoid biosynthesis were characterized in Arabidopsis thaliana. Genes encoding flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and leucoanthocyanidin dioxygenase (LDOX) were cloned and sequenced. The predicted proteins encoded by each of these Arabidopsis genes shared high homology with all F3H, FLS, or LDOX sequences available in Genbank. Low-stringency DNA blot analysis indicated that F3H and LDOX are encoded by a single gene in Arabidopsis, while FLS may be encoded by two or three genes.
RNA blot analysis was performed to determine the expression patterns of these three genes relative to previously-cloned genes encoding flavonoid biosynthetic enzymes. Light-induction experiments and analysis of regulatory mutants showed that the CHS, CHI, F3H, and FLS1 are coordinately regulated in Arabidopsis seedlings, encode enzymes acting near the beginning of the pathway, and are therefore referred to as "early" genes. The same experiments showed that DFR and LDOX are regulated distinctly from "early" genes, share similar expression patterns in response to light, and are not expressed in the ttg mutant. DFR and LDOX are therefore referred to as "late" genes due to the timing of expression in response to light and the fact that they encode enzymes acting late in flavonoid biosynthesis.
To determine whether any of the previously-identified transparent testa mutants were defective in F3H, FLS, or LDOX, the chromosomal locations of these genes in the Arabidopsis genome were determined. The positions of these genes suggested that no previously-identified tt mutant was defective in the cloned FLS or LDOX structural genes, while tt6 was potentially the F3H locus. The coding region of F3H was amplified by PCR from tt6 genomic DNA and sequenced, and several point mutations were found in the coding region of this allele, three of which are predicted to result in amino acid substitutions.
Polyclonal antibodies were also developed using four different purified, recombinant flavonoid enzymes as antigens. These antibodies were used to determine the pattern of accumulation of flavonoid enzymes in developing seedlings. Immunoblot analysis was also performed to determine whether mutations in genes encoding specific flavonoid enzymes or an enzyme in pathways that compete for or provide substrate for flavonoid biosynthesis (mutants defective in tryptophan or ferulic acid biosynthesis) affect the levels of flavonoid enzymes. These analyses showed that mutant seedlings which lacked specific flavonoid or tryptophan biosynthetic enzymes accumulated higher steady-state levels of other enzymes in the pathway. These results suggest that the accumulation of specific flavonoid intermediates or indole can lead directly or indirectly to higher levels of flavonoid enzymes. / Ph. D.
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Examination of 2-Oxoglutarate Dependant Dioxygenases Leading to the Production of Flavonols in <i>Arabidopsis thaliana</i>Owens, Daniel Kenneth 21 October 2005 (has links)
The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis.
To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology.
Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis. / Ph. D.
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