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.

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.

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (Kmapp = 67 μ M; Vmax = 20.45 pKat/μg), kaempferol (Kmapp = 12 μ M; Vmax = 11.63 pKat/μg), and myricetin (Kmapp = 33 μ M; Vmax = 12.21 pKat/μg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu2+, Fe2+, and Zn2+ as well as UDP (Kiapp = 69.5 μ M), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

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Pour s’adapter à leur environnement, les plantes ont acquis la capacité de produire une grande quantité de métabolites spécialisés à partir d’un nombre limité de structures de base. Parmi les modifications apportées à ces structures de base, la réaction de glycosylation permet d’augmenter la solubilité du composé, de réduire sa toxicité et de contribuer à une meilleure stabilité de certaines molécules ayant pour conséquence la modification de leur transport et/ou de leur stockage. Les UDP-glycosyltransférases (UGT) forment une vaste famille de glycosyltransférases chez les plantes. Elles regroupent des enzymes glycosylant principalement des hormones et des phénylpropanoides en utilisant l’UDP-sucre comme donneur de sucre. L’objectif de ce travail consiste à contribuer à la caractérisation fonctionnelle de la famille des UGT72 chez le peuplier afin d’identifier le rôle de ses membres dans les processus développementaux liés aux arbres et dans leurs réponses au stress. Plusieurs membres de cette famille ont déjà été caractérisés chez d’autres espèces comme A. thaliana, M. truncatula et C. sinensis, mettant en évidence la capacité de certaines UGT72s à glycosyler les monolignols, une implication dans le processus de lignification, un rôle dans des processus de défense contre les pathogènes ou encore une fonction de détoxification de certains polluants. Parmi les 8 UGT72s identifiées chez le peuplier, nous avons montré qu’in vitro UGT72AZ2 glycosyle l’acide férulique et l’acide sinapique, UGT72B37 le p-coumaraldéhyde, le coniféraldéhyde, le sinapaldéhyde, le coniferyl alcool et le sinapyl alcool, UGT72B39 le coniféryl alcool et UGT72A2 la naringénine. Tous les membres de la famille UGT72 sont exprimés dans les tissus vasculaires, suggérant un rôle dans le développement vasculaire. La surexpression de UGT72AZ1 ou UGT72AZ2 provoque l’accumulation de glucosides de monolignols (respectivement coniférine et syringine ou coniférine seulement), sans toutefois affecter la quantité totale de lignine. Concernant la localisation subcellulaire, excepté pour UGT72A2, les UGT72s du peuplier sont localisées dans le réticulum endoplasmique et le noyau, suggérant respectivement, un rôle dans la régulation de la voie des phénylpropanoides et dans la maintenance de l’ADN. UGT72A2 se démarque des autres membres de cette famille, car elle est localisée dans les chloroplastes et dans des vésicules associées aux chloroplastes, suggérant un rôle dans la régulation des phénylpropanoides dans le chloroplaste et/ou dans la maintenance du chloroplaste. En appui de ces hypothèses, nous avons constaté que la photosynthèse est affectée dans les lignées sous-exprimant UGT72A2, provoquant un jaunissement des feuilles. De plus, les feuilles de lignées sous-exprimant UGT72A2 développent un stress oxydatif associé à une réduction de l’accumulation des flavonoïdes et de l’activité des enzymes antioxydantes, suggérant un rôle de UGT72A2 dans l’homéostasie des formes réactives de l’oxygène (ROS). / In order to adapt to their environment, plants have developed the capacity to produce a diversified range of specialized metabolites by modifying a core set of molecules. Among those modifications, glycosylation allows to increase the solubility, to reduce the toxicity and to stabilize compounds in order to modify their transport and/or allow their storage. The UDP-glycosyltransferases (UGT) forming the largest glycosyltransferase superfamily in plants, combine enzymes which glycosylate mainly hormones and phenylpropanoids by using UDP-sugar as sugar donor. The purpose of this dissertation is to contribute to the functional characterization of the UGT72 family in poplar to unravel the role of its members in tree developmental processes and in stress response. Members of this family already characterized in other species (e.g. Arabidopsis thaliana, Medicago truncatula and Camellia sinensis) have been found to glycosylate monolignols and some of them have been associated with lignification, defence against pathogens and detoxification of pollutants. Among the 8 UGT72s identified in poplar, we have shown that UGT72AZ2 glycosylates in vitro ferulic acid and sinapic acid, UGT72B37 p-coumaraldehyde, coniferaldehyde, sinapaldehyde, coniferyl alcohol and sinapyl alcohol, UGT72B39 coniferyl alcohol and UGT72A2 naringenin. All the UGT72 members are expressed in vascular tissues suggesting a role in vascular development. The overexpression of UGT72AZ1 or UGT72AZ2 in poplar triggers the accumulation of monolignol glucosides (both coniferin and syringin or only coniferin, respectively) but has no impact on lignin content. With respect to the subcellular localization, except for UGT72A2, poplar UGT72s are localized in the endoplasmic reticulum and in the nucleus suggesting a possible role in the phenylpropanoid pathway regulation and in DNA maintenance, respectively. UGT72A2 stands out from the other poplar UGT72s by being localized in the chloroplast and chloroplast associated bodies, suggesting a role in the phenylpropanoid regulation in chloroplasts and/or in chloroplast maintenance. Moreover, supporting these hypotheses, photosynthesis was affected in lines downregulated for UGT72A2, as shown by a leaf yellowing phenotype and an oxidative stress in these lines as compared to the wild type. The flavonoid biosynthesis and the activity of enzymes involved into the reactive oxygen species (ROS) scavenging seem to be reduced by the downregulation of UGT72A2 suggesting a role of this UGT in the ROS homeostasis. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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.