Presented diploma thesis is dedicated to the determination of selected chemical parameters of fruits of new currant varieties. The theoretical part is focused on the characteristics of currant, the biologically active substances of its fruits and also the utilization of current in the food industry. Further part of the theory deals with the main chemical parameters which were examined: polyphenols, anthocyanins, ascorbic acid and antioxidant activity. In the practical part there were determined selected chemical parameters in six white, twelve red and thirteen black varieties of currant. Anthocyanins and polyphenols have been determined spectrophotometrically. The content of ascorbic acid in selected varieties of currant was determined by HPLC. As a technique for the determinationof the antioxidant activity was selected the method of free radical DPPH.

No description available.

No description available.

Current fruit-zone management recommendation in the eastern US aims for 1-2 basal shoot leaf layers after fruit set to limit fungal disease and sunburn incidence, and prevent extreme heating of grapes. The goal of this work was to assess if fruit-zone leaf removal to an uncommonly greater extent, and/or at an earlier phenological stage, would favorably alter yield components or fruit composition in three popularly grown, red-fruited, Bordeaux varieties – Cabernet franc, Petit Verdot, and Cabernet Sauvignon. Pre-bloom leaf removal to various extents reduced crop yield by 41-78% when compared to no leaf removal across seasons and varieties. Pre-bloom leaf removal implementation in consecutive seasons tended to further reduce crop yield components compared to implementation in the first year. Pre-bloom leaf removal tended to reduce cluster compactness and bunch rot incidence when compared to post-fruit set and no leaf removal. Basal leaf removal to the greatest extents inconsistently reduced soluble solids and titratable acidity across varieties and seasons. Pre-bloom and post-fruit set leaf removal to the greatest extent consistently increased total grape phenolics and anthocyanins compared to no leaf removal in Cabernet Sauvignon, but inconsistently increased total grape phenolics compared to no leaf removal in Cabernet franc and Petit Verdot. Basal leaf removal to the greatest extents tended to increase the synthesis and degradation of carotenoids more consistently than no leaf removal, and this was particularly true for zeaxanthin. Petit Verdot and Cabernet franc wine color and aroma were inconsistently distinguishable between leaf removal treatments, and color intensity was rated higher in wines made with fruit from pre-bloom leaf removal compared to modest post fruit-set leaf removal plots. Waiting until after fruit set to remove fruit-zone leaves maintained crop yield and offered comparable improvements in fruit composition to pre-bloom leaf removal. Pre-bloom leaf removal of no more than four leaves is recommended to limit crop yield reduction, and modestly improve fruit composition. This work showed that fruit-zone leaf removal does not need to be conservative in the eastern US, particularly because the climate does not appear to be detrimental to fruit composition, and open fruit-zones reduce grape fungal disease incidence. / Ph. D.

Tesis por compendio / [ES] Los virus de plantas poseen la capacidad de parasitar células vegetales y poner a su disposición la maquinaria celular de la planta para la síntesis de sus propias proteínas. Aprovechando esta capacidad, la ingeniería genética ha dirigido muchos de sus esfuerzos en la modificación del genoma viral para utilizar los virus de plantas como vectores de expresión de proteínas de interés humano. Los potyvirus son un grupo de virus de plantas ampliamente estudiado y utilizado en biotecnología. Su genoma está formado por un RNA de cadena sencilla que codifica, principalmente, una poliproteína que se procesa para dar aproximadamente 10 proteínas maduras. En diferentes posiciones intercistrónicas se pueden insertar cDNAs que codifican proteínas de interés, las cuales se producen junto al resto de productos de la poliproteína. Si además estos cDNAs se flanquean por secuencias que codifican los sitios de procesamiento específicos de las proteasas virales, las proteínas heterólogas se liberan eficientemente de la poliproteína viral. Así, la inserción de un cDNA correspondiente al factor de transcripción Rosea1 de la ruta de las antocianinas en distintos potyvirus resulta en la biosíntesis de estos compuestos en las células vegetales infectadas. Las antocianinas son compuestos flavonoides coloreados que se pueden observar a simple vista, por lo que la expresión de Rosea1 es una herramienta biotecnológica muy útil para seguir la infección de virus de plantas. En esta Tesis se han investigado diferentes aplicaciones biotecnológicas basadas en la expresión del factor de transcripción Rosea1 y la consecuente acumulación de antocianinas en el contexto de la biología de los potyvirus. Primero, se construyó un clon del virus del mosaico amarillo del calabacín (ZYMV) etiquetado con Rosea1 (ZYMV-Ros1). El ZYMV es capaz de replicarse a nivel local pero no de moverse a larga distancia en plantas de Nicotiana benthamiana. Un análisis de la infección por ZYMV-Ros1 en una serie de plantas transgénicas de N. benthamiana silenciadas en distintas RNasas de tipo Dicer (DCL) permitió profundizar en el conocimiento de los mecanismos defensivos de la planta frente a este virus. Los resultados indicaron que DCL4 está implicada en restringir el movimiento sistémico del virus, ya que en plantas con este gen silenciado el virus es capaz de moverse a larga distancia. Además, las antocianinas tienen un gran interés nutricional, farmacéutico e incluso industrial, por su gran actividad antioxidante. Un clon viral derivado del virus Y de la patata (PVY) que expresaba Rosea1 (PVY-Ros1) indujo la acumulación de más antocianinas que las que contienen frutas y verduras que son fuente rica de estos valiosos antioxidantes. Este mismo clon PVY-Ros1 también se utilizó para estudiar la transmisión del virus mediante áfidos vectores, observándose como estos provocan frecuentemente el inicio de la infección en el tejido vascular. Este mismo clon viral permitió mostrar visualmente el efecto antiviral que tienen las nanopartículas de plata en plantas. Por último, en esta Tesis se combinó el factor de transcripción Rosea1 con distintas proteasas de los potyvirus para crear circuitos lógicos de regulación génica en plantas. Se observó que la fusión de distintas proteínas virales, como la proteína de inclusión nuclear b (NIb) de los potyvirus o fragmentos de ella, al extremo carboxilo terminal de Rosea1 inhibía la actividad del factor de transcripción. Sin embargo, la acumulación de antocianinas se pudo restablecer insertando sitios de reconocimiento de proteasas virales entre ambas partes y coexpresando tales proteasas. La especificidad de corte y la eficiente actividad catalítica de las proteasas de inclusión nuclear a (NIaPro) del virus del grabado del tabaco (TEV) y del virus de las venas moteadas del tabaco (TVMV) permitió construir circuitos genéticos basados en regulación postranscripcional que son capaces de realizar algunas operaciones lógicas básicas (YES, OR y AND) en tejidos vegetales. / [CA] Els virus de plantes posseeixen la capacitat de parasitar cèl·lules vegetals i posar a la seva disposició la maquinària cel·lular de la planta per a la síntesi de les seves pròpies proteïnes. Aprofitant aquesta capacitat, l'enginyeria genètica ha dirigit molts dels seus esforços a la modificació del genoma viral per utilitzar els virus de plantes com a vectors d'expressió de proteïnes d'interès humà. Els potyvirus són un grup de virus de plantes àmpliament estudiat i utilitzat en biotecnologia. El seu genoma està format per un RNA de cadena senzilla que codifica, principalment, una poliproteïna que es processa per donar aproximadament 10 proteïnes madures. En diferents posicions intercistróniques es poden inserir cDNAs que codifiquen proteïnes d'interès, les quals es produeixen alhora amb la resta de productes de la poliproteïna. Si a més aquests cDNAs es flanquegen per seqüències que codifiquen els llocs de processament específics de les proteases virals, les proteïnes heteròlogues s'alliberen eficientment de la poliproteïna viral. Així, la inserció d'un cDNA corresponent al factor de transcripció Rosea1 de la ruta de les antocianines en diferents potyvirus resulta en la biosíntesi d'aquests compostos en les cèl·lules vegetals infectades. Les antocianines són compostos flavonoides colorits que es poden observar a simple vista, de manera que l'expressió de Rosea1 és una eina biotecnològica molt útil per seguir la infecció de virus de plantes. En aquesta Tesi s'han investigat diferents aplicacions biotecnològiques basades en l'expressió del factor de transcripció Rosea1 i la conseqüent acumulació d'antocianines en el context de la biologia dels potyvirus. Primer, es va construir un clon del virus del mosaic groc del carbassó (ZYMV) etiquetat amb Rosea1 (ZYMV-Ros1). El ZYMV és capaç de replicar-se a nivell local però no de moure's a llarga distància en plantes de Nicotiana benthamiana. Un anàlisi de la infecció per ZYMV-Ros1 en una sèrie de plantes transgèniques de N. benthamiana silenciades en diferents RNases de tipus Dicer (DCL) va permetre aprofundir en el coneixement dels mecanismes defensius de la planta front aquest virus. Els resultats van indicar que DCL4 està implicada en restringir el moviment sistèmic del virus, ja que en plantes amb aquest gen silenciat el virus és capaç de moure's a llarga distància. A més, les antocianines són objecte d'un gran interès nutricional, farmacèutic i fins i tot industrial, per la seva gran activitat antioxidant. Un clon viral derivat del virus Y de la patata (PVY) que expressava Rosea1 (PVY-Ros1) va induir l'acumulació de més antocianines que les que contenen fruites i verdures que són font rica d'aquests valuosos antioxidants. Aquest mateix clon PVY-Ros1 es va utilitzar per estudiar la transmissió del virus mitjançant àfids vectors, observant com aquests provoquen freqüentment l'inici de la infecció en el teixit vascular. Aquest mateix clon viral també va permetre mostrar visualment l'efecte antiviral que tenen les nanopartícules de plata en plantes. Finalment, en aquesta Tesi es va combinar el factor de transcripció Rosea1 amb diferents proteases dels potyvirus per crear circuits lògics de regulació gènica en plantes. Es va observar que la fusió de diferents proteïnes virals, com la proteïna d'inclusió nuclear b (NIb) dels potyvirus o fragments d'ella, a l'extrem carboxil terminal de Rosea1 inhibia l'activitat del factor de transcripció. No obstant això, l'acumulació d'antocianines es va poder restablir inserint llocs de reconeixement de les proteases virals entre les dues parts i coexpressant aquestes proteases. L'especificitat de tall i l'eficient activitat catalítica de les proteases d'inclusió nuclear a (NIaPro) del virus del gravat del tabac (TEV) i del virus de les venes clapejades del tabac (TVMV) va permetre construir circuits genètics basats en regulació postranscripcional que són capaços de realitzar algunes operacions lògiques bàsiques (YES, OR i AND) en teixits vegetals. / [EN] Plant viruses have the ability to parasitize plant cells and use the plant cellular machinery for the synthesis of their own proteins. Taking advantage of this capacity, genetic engineering has focused many of its efforts in modifying the viral genome in order to use modified plant viruses as expression vectors of proteins of human interest. These proteins are stored in host plants that act as low-cost and highly secure biofactories. Potyviruses are a group of plant viruses widely studied and used in biotechnology. Their genome consist of a single-stranded RNA that mainly encodes a polyprotein that is processed in approximately 10 mature proteins. cDNAs flanked by protease specific processing sequences can be inserted in different intercistronic positions and efficiently processed by the viral proteases to produce proteins of interest together with the rest of the viral polyprotein products. Thus, the insertion of the cDNA corresponding to the transcription factor Rosea1 of the anthocyanin pathway in different potyviruses results in the biosynthesis of these compounds in infected plant cells. Anthocyanins are colored flavonoid compounds that can be observed with the naked eye, and thus, the expression of Rosea1 is a very useful biotechnological tool to follow the infection of plant viruses. In this work, we provided different biotechnological applications based on the expression of this transcription factor and the consequent accumulation of anthocyanins in the context of potyvirus biology. First, a zucchini yellow mosaic virus (ZYMV, genus Potyvirus) clone tagged with Rosea1 (ZYMV-Ros1) was constructed. ZYMV is able to replicate locally but not to move long distances in Nicotiana benthamiana plants. We analyzed the infection by ZYMV-Ros1 in a series of N. benthamiana transgenic plants in which the different Dicer-like (DCL) RNases were slilenced. The results showed that DCL4 is involved in restricting the systemic movement of the virus in N. benthamiana plants. This study allowed to deepen the knowledge of the defense mechanisms of the plant against this virus. Besides their biotechnological potential as markers of biological activities, anthocyanins have great nutritional, pharmaceutical and even industrial interest due to their high antioxidant activity. We found that a potato virus Y clone (PVY; genus Potyvirus) expressing Rosea1 (PVY-Ros1) induced the accumulation of a higher amount of anthocyanins than those contained in fruits and vegetables that are a rich source of these valuable antioxidants. The PVY-Ros1 clone was also used to study the transmission of the virus by aphid vectors. We observed that aphids frequently initiate infection in vascular tissue. This viral clone also allowed to visually show the antiviral effect of silver nanoparticles in plants. Finally, in this work, the Rosea1 transcription factor was combined with different potyvirus proteases to create genetic circuits in plants. We observed that the fusion of the nuclear inclusion protein b (NIb) of potyviruses, or fragments of it, to the carboxyl terminal end of Rosea1 inhibited the activity of the transcription factor. However, the accumulation of anthocyanins could be restored by inserting viral protease recognition sites in between NIb and Rosea1 and co-expressing those proteases. The cleavage specificity and the efficient catalytic activity of nuclear inclusion a proteases (NIaPro) of tobacco etch virus (TEV; genus Potyvirus) and tobacco spotted vein virus (TVMV; genus Potyvirus) allowed the construction of genetic circuits based on post-transcriptional regulation that are capable of performing some basic logical operations (YES, OR and AND) in plant tissues. / This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) through grants BIO2014-54269-R and AGL2013-49919-EXP, and by the Greek Ministry for Education and Religious Affairs (Program Aristeia II, 4499, ViroidmiR; ESPA 2007-2013). This research was supported by the Ministerio de Ciencia, Innovación y Universidades (Spain) grants AGL2013-49919-EXP, BIO2014-54269-R, BFU2015-66894-P and BIO2017-83184-R (co-financed FEDER funds) and by the Engineering and Physical Sciences Research Council and the Biotechnology and Biological Sciences Research Council (UK) grant BB/M017982/1. / Cordero Cucart, MT. (2021). Aplicaciones del factor de transcripción Rosea1 de la ruta de las antocianinas como marcador visual en virología molecular y biotecnología de plantas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171455 / Compendio

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2006. / The assessments of grape and wine quality are complex issues and the wine industry needs more objective analysis of grape and wine quality. The standard quality assessment protocol for grading grapes at most wine cellars in South Africa is based on viticultural practices and the determination of chemical parameters such as ºBrix, pH and titratable acidity (TA). Grape juice indices calculated by formulae such as ºBrix/pH, TA/pH, ºBrix/TA, ºBrix x (pH)2 have been used in the past but these approaches have had limited success. It was shown that the total anthocyanins and total phenolics of red grapes correlate with wine quality and provide additional objective measures of grape quality. Most methods for the quantification of total anthocyanins and total phenolics are complex and time consuming and therefore not easily implemented in the routine laboratory environment. Fourier transform infrared spectroscopy (FT-IR) is widely used in South African laboratories for the routine quantification of wine and grape parameters but the commercial calibration models supplied for the quantification of grape total anthocyanins and phenolics are not satisfactory. The focus of this study was to develop new FT-IR calibration models for the quantification of total anthocyanins and phenolics of grapes and to use the generated data during a preliminary evaluation of the implementation of these parameters as part of the grape quality control protocol at a commercial winery in South Africa. The potential of Fourier transform infrared spectroscopy (FT-IR) for the rapid quantification of total anthocyanins and total phenolics in red grapes was investigated and evaluated for prediction accuracy with independent validation sets. The design of calibration sets aimed at capturing most of the variation due to vintage (2004 and 2005), cultivar (Cabernet Sauvignon, Merlot, Pinotage, and Shiraz) and sugar concentration. Best prediction accuracies were obtained for calibration sets using grapes from a single vintage or cultivar or approximately the same sugar concentration. The highest prediction accuracies were obtained for total anthocyanins calibration sets of grapes with sugar concentrations ≥ 23.5ºBrix (SEP = 0.13 mg/g; R2 validation set = 0.77) and for total phenolics calibration sets of grapes with sugar concentrations < 23.5ºBrix (SEP = 0.13 OD280/g; R2 validation set = 0.74). Strong correlations were found between the spectral data and the total anthocyanins (SEP = 0.12 mg/g; R2 validation set = 0.84) and total phenolics concentration data (SEP = 0.10 OD280/g; R2 validation set = 0.76) for 2005 Merlot calibration sets indicating that the FT-IR spectra captured most of the variation. Overall the RPD (ratio of the standard deviation of the reference data to the standard error of prediction) values of all calibration models were below 3 indicating that calibration models are fit for screening purposes. Spectroscopic absorbance at 280 nm is not specific enough for the quantification of total phenolics and the use of an alternative reference method such as high performance liquid chromatography (HPLC) will be considered in the future. Principal component analysis (PCA) revealed that the major sources of variation in the FT-IR spectra of grapes could be ascribed to vintage and grape sugar concentration and this had an effect on the accuracy of the analytical data generated when using FT-IR spectroscopy. This report is the first to our knowledge where FT-IR has been used for the quantification of total anthocyanins and phenolics of grapes. The evaluation of the reference laboratory protocol for the quantification of total anthocyanins and total phenolics in grapes were evaluated in Chapter 4 and emphasized the importance of meticulous laboratory practices to obtain reliable reference data for calibration purposes. This large scale investigation of the total anthocyanins and phenolics concentrations in grapes is the first of its kind in South Africa and a quantitative database containing analytical data of the anthocyanins and total phenolics concentrations of 692 grape samples representing a wide range of grape maturities of Vitis vinifera cultivars Cabernet Sauvignon, Merlot, Pinotage and Shiraz from the 2004 and 2005 vintages was established based on the reference values. The data were used in a preliminary investigation of the implementation of total anthocyanins and total phenolics concentrations as part of grape quality classification at a commercial South African winery (Chapter 5). The results showed that the total anthocyanins and total phenolics concentration in grapes increased with increasing grape maturity (measured as sugar concentration). ANOVA and post-hoc analysis (Bonferroni testing) revealed significant differences between the total anthocyanins and total phenolics concentrations of the four Vitis vinifera cultivars investigated. Grapes harvested earlier in the season had significantly higher (p≤0.05) total anthocyanins and total phenolics concentrations than grapes harvested later in the season. This implies that grapes harvested earlier in the harvest season could produce wines with higher quality. Grapes from regularly irrigated vineyards had lower total anthocyanins and total phenolics concentrations compared to dryland vineyards. The current grape grading system in use at the industrial cellar did not correlate well with the total anthocyanins and total phenolics concentrations of grapes which highlighted the need for the inclusion of more objective measures during grape grading. The information captured in the database can be used as a starting point to establish profiles of the typical anthocyanins and total phenolics of South African grapes and data from more vintages should be included and continually updated. These findings highlight the important contribution of the results obtained in this preliminary study for the incorporation of total anthocyanins and phenolics concentrations as objective parameters of grape quality. Finally multivariate data analysis of the FT-IR spectra revealed important information regarding factors (both physical and chemical) that contribute to the variation of the spectra. The main variation between the 2004 and 2005 samples can probably be interpreted in terms of the water content of the samples.

Flavonoids are plant secondary metabolites, which belong to several groups varying in their chemical structure. Anthocyanins and tannins are important flavonoid components of wine that are responsible for its color, taste and other sensory properties. The concentration of anthocyanins in wine is affected by grape variety, processing technology, and climatic conditions. In this Thesis, we studied the changes in color and in related chemical composition, using three non-commercial samples of red wine: Svatovavřinecké (year 2010 and 2012), and home-made wine (prepared without addition of SO2). These changes in color were determined using standard colorimetric method (CIELab) and also a simplified two-parametric spectrophotometric method (tint/color density). The content of anthocyanins was followed using analytical RP-HPLC method. In paralel, simplified oenologic methods for estimation of phenolic compounds were used. Generally the wine samples changed color to darker tint. Chemically, this was caused by polymerisation reactions between anthocyanins and phenolic compounds. This led to the formation of stable pigments characterised by a higher absorption maximum at longetr wavelength, hence a darker tint. Key words: anthocyanins, color, red wine, phenolic compounds, malvidin-3-glucosid, polymeric reactions,...

Les anthocyanes sont une famille de polyphénols très répandus chez les végétaux. Chez la vigne, elles sont responsables de la coloration des baies des cépages rouges, et sont impliquées dans les propriétés organoleptiques des vins. Une nutrition azotée faible induit la production des anthocyanes dans les cellules de la pellicule de raisin des cépages rouges via des mécanismes de régulation qui ne sont pas encore totalement élucidés. Dans ce contexte, nous avons étudié les mécanismes moléculaires impliqués dans la réponse de l’accumulation des anthocyanes pour différents niveaux d’apports azotés. Deux matériels biologiques complémentaires ont été utilisés : des suspensions cellulaires de vigne (lignée GT3) et des plants de Cabernet-Sauvignon, cultivés au vignoble.L’augmentation de la synthèse d’anthocyanes en réponse à la diminution de la nutrition azotée a été confirmée dans les baies et les cellules de vigne en culture. Les analyses transcriptomiques globales (génome complet) et ciblées (qPCR) ont mis en lumière des modifications de l’expression génique, notamment de gènes liés au métabolisme des flavonoïdes, en réponse à la nutrition azotée. L’expression de nombreux gènes structuraux impliqués dans la voie de biosynthèse des anthocyanes est induite par une faible nutrition azotée. La variation de l’apport azoté influence également de façon coordonnée l’expression des gènes régulateurs positifs (facteurs de transcription de type MYB) et négatifs (protéine de type Lateral organ Boundary Domain (LBD)) des gènes de la biosynthèse des flavonoïdes chez la Vigne. L’expression de gènes liés à la production d’énergie (NADH, NADPH), est également affectée.En parallèle, une approche intégrative a été développée sur les suspensions cellulaires, en combinant des mesures d’activités enzymatiques, des dosages de métabolites primaires et secondaires, avec un modèle de balance de flux (Flux Balance Analysis, FBA). Les cartes de flux obtenues prédisent que la diminution de l’apport azoté entraîne une augmentation des flux métaboliques dans la voie du shikimate et des phénylpropanoïdes ; ainsi qu’une répression de la majorité des flux dans les différentes voies du métabolisme primaire, à l’exception de la voie des pentoses phosphates, dont le flux est maintenu, et de la voie de synthèse de l’amidon qui est accrue. Les résultats obtenus plaident en faveur d’un lien fort entre synthèse des anthocyanes et statut énergétique (ATP, NADPH) des cellules vigne. / Anthocyanins are polyphenol compounds very abundant in most of the plants. In grapevine, they give color to red berries and they improve red wine quality and increase the organoleptic properties of the wine. Low nitrogen supply stimulates anthocyanin production in berry skin cells of red grape varieties through regulation mechanisms that are far from being fully understood. In this context, we worked on the molecular mechanisms involved in anthocyanin biosynthesis response to nitrogen supply. Two complementary biological materials were used: grapevine cell suspensions (GT3 line) that originate from a teinturier cultivar and produce anthocyanins under normal conditions; and red grape berries of cv. Cabernet-Sauvignon cultivated in a commercial vineyard. Increases of anthocyanins synthesis in response to low nitrogen levels were confirmed in the field-grown berries and the cells suspensions. Both comparative global (microarrays) and targeted (qPCR) transcriptomic analysis showed different regulations on the expression of the genes involved in the secondary (especially the anthocyanin) and nitrogen metabolisms. The expression of most structural genes of the anthocyanin biosynthesis pathway was induced by a low nitrogen supply. Nitrogen controls also the expression of the positive (MYB transcription factors) and negative (Lateral organ Boundary Domain family protein LBD39) regulatory genes of the flavonoid pathway in grapevine. Furthermore, some genes improved in energy production (ATP, NADPH) were affected. In parallel, an integrative approach combining enzymatic activities and primary and secondary metabolites measurements with developing a Flux Balance Analysis (FBA) modeling approach was used on cells suspensions GT3. The flux maps deciphered that low nitrogen increases metabolic fluxes in shikimate and phenylpropanoid pathways and represses the majority metabolic fluxes in different pathways of primary metabolism. The two exceptions included the pentose phosphate pathway, which the flux metabolism was maintained, and the starch synthesis pathway, which was enhanced. The results obtained showed a strong link between anthocyanin synthesis and energy status (ATP, NADPH) in the berry cell suspensions.

Le contrôle combinatoire de l’ expression des gènes est une caractéristique importante du profil spatio-temporel de l'accumulation des flavonoïdes chez les plantes. Des résultats précédents avaient démontré chez Arabidopsis thaliana, que la régulation de l’accumulation des anthocyanes et des proanthocyanidines repose sur l'activité de facteurs de régulation de la transcription de type R2R3-MYB et bHLH qui forment des complexes ternaires ("MBW") avec la protéine TTG1 (WDR). L'objectif de la thèse était de caractériser les complexes MBW impliqués et leurs cibles, pour avoir une compréhension globale des mécanismes transcriptionnels qui contrôlent la biosynthèse des flavonoïdes.En utilisant différentes approches génétiques et moléculaires, nous avons montré que seuls les gènes « tardifs » (c’est à dire DFR, LDOX, BAN, TT19, TT12 et AHA10) sont des cibles directes des complexes MBW. Bien que le complexe de régulation impliquant les protéines TT2-TT8-TTG1 ait un rôle majeur dans la régulation de ces gènes structuraux dans la graine d’Arabidopsis, trois autres complexes contenant MYB5, GL3 ou EGL3 sont également impliqués de façon tissu-spécifique. Comme l’expression du gène TT8 joue un rôle clef dans ces régulations, une dissection fonctionnelle de son promoteur a été entreprise. Elle a montré la nature modulaire de ce promoteur avec deux domaines impliqués dans l’expression tissu-spécifique et un troisième dans la force du promoteur. Les résultats obtenus suggèrent également l’existence d’autres régulateurs qui restent à caractériser. Enfin, nous avons développé une nouvelle technique de caractérisation des interactions entre les facteurs de transcription et les promoteurs, basée sur l’expression transitoire dans des protoplastes de mousse (i.e. Physcomitrella patens). Nous avons ainsi pu identifier les éléments cis des promoteurs impliqués dans la régulation de l’expression de TT8 et de chacun des promoteurs cibles des complexes MBW.L’ensemble de ces travaux permet de fournir un modèle plus complet du réseau de régulations transcriptionnelles qui contrôle la biosynthèse des proanthocyanidines et des anthocyanes, ainsi que des outils et de nouvelles pistes pour poursuivre ces études chez Arabidopsis et d’autres plantes. / The combinatorial control of gene expression is a key feature of the spatio-temporal pattern of flavonoid accumulation in plants. Previous results have shown that the regulation of anthocyanins and proanthocyanidins (PAs or tannins) pigmentation relies on the transcriptional activity of R2R3-MYB and bHLH proteins that form “MBW” ternary complexes with TTG1 (WD-Repeats), in Arabidopsis thaliana. The purpose of the thesis was to figure out the nature and spatio-temporal activity of these MBW complexes and to identify their direct targets, which were essential steps toward a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. Using different molecular and genetic approaches this thesis has demonstrated that only late biosynthetic genes (namely DFR, LDOX, BAN, TT19, TT12 and AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex was shown to play the major role in regulating the expression of these structural genes in developing seeds, three additional MBW complexes that contain MYB5, GL3 or EGL3 are also involved, in a tissue-specific manner. Because the expression of TT8 is largely involved in these regulations, a functional dissection of its promoter was carried out. Two modules drive the tissue-specific activity of the TT8 promoter in PA- and anthocyanin-accumulating cells, and a third module is responsible for the strength of the promoter. Interestingly, this regulation involves at least six different MBW complexes. Our results also suggest that some putative new regulators remain to be discovered. Last, use of a newly developed fast and sensitive transient expression system that relies on protoplasts of the moss Physcomitrella patens has allowed the identification of both, specific cis-regulatory elements through which TT8 expression is regulated and the minimal promoter for each of the genes that are targeted by the MBW complexes.Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, we have provided a new and comprehensive view of the regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis, as well as new clues and tools for further investigation of this pathway in Arabidopsis and other plant species.

Neste trabalho, estudou-se o efeito da adição de acetonitrila na reatividade química em soluções micelares do detergente aniônico dodecil sulfato de sódio (SDS) e do detergente catiônico brometo de hexadeciltrimetilamônio (CTAB) e em agregados supramoleculares do sal biliar, colato de sódio (NaCh). Os sistemas utilizados para estudar a reatividade química na presença de acetonitrila foram: (a) fotorreatividade do estado excitado triplete de n-butil-3-nitrofenil éter (em SDS); (b) reação de hidrólise ácida do 2-(p-octoxifenil)-1,3-dioxolano no estado fundamental (em SDS); (c) constante de incorporação do co-íon N-dodecil-4-cianopiridínio (em CTAB); (d) a protonação/desprotonação do ácido fraco 4-metil-7-hidroxiflavílio, tanto no estado excitado como no estado fundamental (em SDS); e (e) a supressão dos estados excitados de 1-etilnaftaleno e 1-(1-naftil)-etanol (em NaCh). As modificações na estruturação do agregado micelar e nas propriedades do meio aquoso, provocadas pela presença de acetonitrila, fazem com que as moléculas orgânicas e os íons saiam mais rapidamente do agregado para a fase aquosa. As mudanças que ocorrem com a micela e com a partição de solutos diminuem o efeito catalítico da micela sobre a reatividade química. Os agregados de sal biliar possuem dois sítios distintos de ligação de solutos e a concentração mínima de sal biliar necessária para formar esses dois sítios depende da concentração de acetonitrila. Em concentrações de acetonitrila acima de 10 %, ocorre somente a formação de agregados com sítio primário, enquanto que acima de 30 % os agregados se desfazem por completo. Em baixas concentrações de acetonitrila (até aproximadamente 10 %), a dinâmica de dissociação de solutos do sítio secundário do agregado de sal biliar não modifica muito, enquanto que a velocidade de saída de solutos do sítio primário aumenta com a presença da acetonitrila. Desse modo, a acetonitrila pode ser empregada para acelerar a saída de uma espécie, tal como um intermediário reativo, do agregado primário, facilitando a aplicação de agregados de sal biliar como micro-reatores com dois sítios distintos de reação. / This work reports a study of the effect of the addition of acetonitrile on chemical reactivity in micellar solutions of the anionic detergent sodium dodecyl sulfate (SDS) and the cationic detergent hexadecyltrimethylammonium bromide (CTAB) and in supramolecular aggregates of the bile salt sodium cholate (NaCh). The systems employed in the study of chemical reactivity in the presence of acetonitrile were: (a) photoreactivity of the excited triplet state of N-butyl-3-nitrophenyl ether in SDS; (b) the acid catalyzed of 2-(p-octoxy-nitrophenyl)-1,3-dioxolane in the ground state in SDS; (c) incorporation of the coion N-dodecyl-4-cyanopyridine in CTAB; (d) protonation/deprotonation of the weak acid of the 4-methyl-7-hydroxyflavilium ion in the ground and excited state in SDS; and (e) quenching of the excited state of 1-ethylnaphthalene and 1-(1-naphthyl)-ethanol in NaCh. The modifications of the structure of the micellar aggregate and of the properties of the bulk aqueous phase induced by acetonitrile result in a faster rate of exit of organic molecules and ions from the aggregate into the aqueous phase. The changes that occur in the micelle and in the partitioning of solutes tend to diminish the catalytic effect of micelles on chemical reactivity. Bile salt aggregates possess two distinct sites for solubilization of solutes and the minimum concentration of bile salt necessary for the formation of these two sites depends on the concentration of acetonitrile. At acetonitrile concentrations above 10 %, only aggregates with the primary solubilization site are formed and, above 30 %, the aggregates are completely disrupted. At low acetonitrile concentrations (below 10 %), the dynamics of dissociation of solutes from the secondary site of bile salt aggregates changes very little, while the rate of exit of solutes from the primary site increases in the presence of acetonitrile. Thus, acetonitrile can be employed to accelerate the rate of exit of species such as reactive intermediates, facilitating the application of bile salt aggregates as two-reaction-site microreactors.