Submitted by (edna.saturno@ufrpe.br) on 2016-11-22T13:47:20Z No. of bitstreams: 1 Ana Elizabete Lopes Ribeiro.pdf: 647150 bytes, checksum: d6f6ac0c70b90999363b5fddfcd687c2 (MD5) / Made available in DSpace on 2016-11-22T13:47:20Z (GMT). No. of bitstreams: 1 Ana Elizabete Lopes Ribeiro.pdf: 647150 bytes, checksum: d6f6ac0c70b90999363b5fddfcd687c2 (MD5) Previous issue date: 2009-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Neozygites floridana (Weiser & Muma) (Zygomycetes: Entomophthorales) infects several species of Tetranychidae in different locations around the world. In Brazil, N. floridana was reported in Tetranychus evansi Baker & Pritchard and Tetranychus urticae Koch. Disease caused by this pathogen on tetranychids indicates its potential in biological control programs. In this study was tested the specificity of N. floridana isolates to different species of Tetranychidae, the epizootiology of this fungus in a greenhouse, in semi-field and in the field on T. evansi in Solanum americanum Mill., and evaluated the combination of some biotic and abiotic factors on the formation of resting spores. For all strains studied contamination, infection and mummification was always higher to the species of mites from which the strains were obtained. The main natural enemies of T. evansi were N. floridana and Phytoseiulus longipes Evans. The population increase of tetranychids was always followed by increases in the density of natural enemies. It was found that the population density of natural enemies is strongly influenced by climatic factors and the density of T. evansi. The occurrence of N. floridana appears to be restricted to periods of highest rainfall from May to September. The predator P. longipes had itshighest population density during the dry period of the year, from October to February. The mites with resting spores were only 0.03%, from 13,516 mites evaluated. All mites found with resting spores belonged to the species T. evansi and to isolate LQ4. During the dry season we did not observe N. floridana structures in adult females of T. evansi under conditions of semi-field. S. americanum is a natural host for maintenance of populations of T. evansi and also allows the development of natural enemies such as N. floridana and P. longipes in the field. / Neozygites floridana (Weiser & Muma) (Zygomycetes: Entomophthorales) tem sido relatado infectando diversas espécies de Tetranychidae em diferentes locais do mundo. No Brasil, N. floridana foi relatado em Tetranychus evansi Baker & Pritchard e Tetranychus urticae Koch. Epizootias causadas por este patógeno a populações de tetraniquídeos indicam um potencial para uso deste agente no controle biológico. Neste trabalho testou-se a especificidade de cinco isolados de N. floridana a diferentes espécies de Tetranychidae; estudou-se a epizootiologia deste fungo e a ação de Phytoseiulus longipes Evans (Acari: Phytoseiidae) em condições de casa-de-vegetação, semi-campo e campo sobre T. evansi em Solanum americanum Mill., além do efeito de alguns fatores bióticos e abióticos na formação de esporos de resistência de N. floridana. Os estudos revelaram que os isolados estudados apresentaram maior contaminação, infecção e mumificação para as espécies de ácaros das quais foram obtidos. Os principais inimigos naturais de T. evansi em S. americanum no Recife-PE foram N. floridana e P. longipes. O aumento populacional desse tetraniquídeo foi sempre seguido por aumentos na densidade dos inimigos naturais. Verificou-se que, além de estar diretamente ligada à densidade de T. evansi, a densidade populacional dosinimigos naturais é bastante influenciada por fatores climáticos. A época de ocorrência de N. floridana esta restrita ao período de maior precipitação, entre os meses de maio-setembro. O predador P. longipes teve sua densidade populacional elevada durante o período mais seco do ano, entre outubro e fevereiro. A proporção de ácaros com esporos de resistência foi 0,03%, em um total de 13.516 ácaros avaliados. Todos os ácaros encontrados com esporos de resistência pertenciam à espécie T. evansi e ao isolado LQ4. S. americanum é um hospedeiro natural para manutenção de populações de T. evansi e também permite o desenvolvimento de inimigos naturais como N. floridana e P. longipes em condições de campo.

No description available.

[EN] The reduction of agricultural biodiversity is a serious problem for our agriculture. Increasingly fewer species are cultivated, and these are also increasingly homogeneous. The introduction of new crops for the horticultural diversification is one of the activities that can be implemented to minimize the impact of this variability loss. In this regard, pepino (Solanum muricatum) is a crop that may be of interest to our horticulture and neighboring markets. It is a species of Andean origin, usually propagated vegetatively, and that can be grown in the Mediterranean area. The pepino is used for its juicy, sweet and aromatic fruits, which in addition presents significant amounts of beneficial compounds for health. The COMAV genebank has a collection of pepino and wild relatives accessions. At present, morphological descriptors are available for proper characterization of these materials, but it was necessary to develop phenological descriptors as the ones presented in this work. In particular, the BBCH scale developed in this thesis is a key that allow us to describe and define the various stages of phenological development in the species, displaying numerous applications. On the other hand, a correct characterization of the accessions preserved in the genebank was necessary. For this reason, and in addition to carrying out a morphological characterization, a molecular characterization using SSR markers derived from tomato was performed. In this case, the benefit of the genetic proximity between the two species allowed us to transfer the tomato markers (widely studied species) to pepino, allowing us to differentiate morphologically and molecularly the wild species from the cultivated one, and within the latter, differentiate modern and traditional types. A limitation in the study of the genetics of pepino was the small number of DNA sequences available in the databases. For this reason, we sequenced the transcriptome of a variety of pepino (Sweet Long) as well as the species that is considered the wild ancestor of the pepino, S. caripense. The sequencing and subsequent assembly of the transcriptome has allowed an initial comparative analysis between pepino and its closely related species, tomato and potato, a phylogenetic study of cultivated Solanaceae, a comparative analysis of some genes of agronomic interest, and the massive development of molecular markers. Because of the potential nutraceutical properties of the pepino fruit, we decided to perform a characterization using the previously studied collection. We have evaluated the dry matter, protein, antioxidants, pigments and minerals contents. On the other hand, considering that polyphenols are one of the most important antioxidant compounds, we conducted a study trying to elucidate the profile of polyphenols and their antioxidant activity in four pepino and one S. caripense entries. We also measured the effect of the pepino extracts on macrophage cells subjected to oxidative stress. The results obtained revealed a significant reduction in nitric oxide production, which indicates the existence of an anti-inflammatory effect. These beneficial properties of pepino are its main strength and, together with a high organoleptic quality and good promotion can encourage the introduction and development of this crop. In summary, in this thesis we have obtained relevant information about the diversity of pepino and we have studied its phenological, morphological, molecular, genomic, nutritional and nutraceutical characteristics. / [ES] La reducción en la diversidad agrícola es un problema grave en nuestra agricultura. Cada vez se cultiva un menor número de especies distintas, y estas además son cada vez más homogéneas. Una de las actividades que se pueden llevar a cabo para minimizar el impacto de esta pérdida de variabilidad es la introducción de nuevos cultivos para la diversificación hortícola. En este sentido, el pepino dulce (Solanum muricatum) es un cultivo que puede tener interés para nuestra horticultura y para los mercados vecinos. Se trata de una especie de origen andino que normalmente se propaga vegetativamente, y que se puede cultivar en el área mediterránea. El pepino dulce se aprovecha por sus frutos jugosos, dulces y muy aromáticos, además de ello presenta cantidades relevantes de compuestos beneficiosos para la salud. El banco de germoplasma del COMAV conserva una colección de entradas de pepino dulce y de especies silvestres relacionadas. En la actualidad se disponen de descriptores morfológicos para la correcta caracterización de estos materiales, pero se hacía necesario disponer descriptores fenológicos como los desarrollados en este trabajo. En particular, la escala BBCH desarrollada es una clave que permite describir y delimitar los distintos estadios de desarrollo fenológico en la especie, presentando numerosas aplicaciones. Por otro lado era necesaria una correcta caracterización de estas entradas conservadas en el banco de germoplasma, para ello y como complemento a la realización de una caracterización morfológica, se llevó a cabo una caracterización molecular empleando marcadores SSR derivados de tomate. En este caso, se sacó beneficio de la proximidad genética entre ambas especies para transferir los marcadores de tomate (especie ampliamente estudiada) a pepino dulce, permitiendo diferenciar tanto morfológicamente, como molecularmente las especies silvestres de la cultivada, y dentro de esta los tipos modernos de los tradicionales. Una limitación en el estudio de la genética del pepino dulce era el número reducido de secuencias de ADN disponibles en las bases de datos, razón por la cual se proyectó la secuenciación del transcriptoma de una variedad de pepino dulce (Sweet Long) y de una entrada de la especie que se considera el ancestro silvestre del pepino dulce S. caripense. Esta secuenciación y posterior ensamblaje del transcriptoma ha permitido realizar un estudio inicial donde se ha realizado un análisis comparativo entre pepino dulce y sus especies cercanas tomate y patata, un estudio filogenético entre Solanáceas cultivadas, un análisis comparativo de algunos genes de interés agronómico, así como el desarrollo masivo de marcadores moleculares. Debido a las potenciales propiedades nutracéuticas de los frutos de pepino dulce, se decidió realizar una caracterización de la misma en la colección de entradas estudiada anteriormente. Por un lado se ha evaluado el contenido en materia seca, proteínas, antioxidantes, pigmentos y minerales; por otro lado, teniendo en cuenta que los polifenoles son unos de los compuestos con mayor poder antioxidante, se llevó a cabo un estudio que pretendió dilucidar el perfil de polifenoles en cuatro entradas de pepino dulce y una entrada de S. caripense, así como su poder antioxidante. Como complemento a esto último se evaluó el efecto de extractos de pepino dulce sobre células de macrófagos sometidas a estrés oxidativo, observándose una reducción significativa en la producción de óxido nítrico, lo cual indica la existencia de un efecto antiinflamatorio. Estas propiedades beneficiosas del pepino dulce son su mayor virtud, y junto con una elevada calidad organoléptica y una buena promoción, pueden favorecer la introducción y desarrollo de este cultivo. En definitiva, esta tesis supone la obtención de información relevante sobre la diversidad del pepino dulce, además de un estudio en distintos aspectos, como el fenológico, morfol / [CAT] La reducció en la diversitat agrícola és un problema greu en la nostra agricultura. Cada vegada es cultiva un menor nombre d'espècies diferents, i aquestes a més són cada vegada més homogènies. Una de les activitats que es poden dur a terme per a minimitzar l'impacte d'aquesta pèrdua de variabilitat és la introducció de nous cultius per a diversificació hortícola. En aquest sentit, el cogombre dolç (Solanum muricatum) és un cultiu que pot tenir interès per a la nostra horticultura i per als mercats veïns. Es tracta d'una espècie d'origen andí que normalment es propaga vegetativament, i que es pot cultivar en l'àrea mediterrània. El cogombre dolç s'aprofita pels seus fruits sucosos, dolços i molt aromàtics, a més d'això presenta quantitats rellevants de compostos beneficiosos per a la salut. El banc de germoplasma del COMAV conserva una col·lecció d'entrades de cogombre dolç i d'espècies silvestres relacionades. En l'actualitat es disposen de descriptors morfològics per a la correcta caracterització d'aquests materials, però es feia necessari disposar descriptors fenològics com els desenvolupats en aquest treball. En particular, l'escala BBCH desenvolupada és una clau que permet descriure i delimitar els diferents estadis de desenvolupament fenològic en l'espècie, presentant nombroses aplicacions. D'altra banda era necessària una correcta caracterització d'aquestes entrades conservades en el banc de germoplasma, per a això i com a complement a la realització d'una caracterització morfològica es va dur a terme una caracterització molecular emprant marcadors SSR derivats de tomaca. En aquest cas, es va traure benefici de la proximitat genètica entre ambdues espècies per a transferir els marcadors de tomaca (espècie àmpliament estudiada) a cogombre dolç, permetent diferenciar tant morfològicament, com molecularment les espècies silvestres de la cultivada, i dins d'aquesta els tipus moderns dels tradicionals. Una limitació en l'estudi de la genètica del cogombre dolç era el nombre reduït de seqüències d'ADN disponibles en les bases de dades, raó per la qual es va projectar la seqüenciació del transcriptoma d'una varietat de cogombre dolç (Sweet Long) i d'una entrada de l'espècie que es considera l'ancestre silvestre del cogombre dolç, S. caripense. Aquesta seqüenciació i posterior assemblatge del transcriptoma ha permès realitzar un estudi inicial on s'ha realitzat una anàlisi comparativa entre cogombre dolç i les seues espècies properes tomaca i creïlla, un estudi filogenètic entre solanàcies cultivades, una anàlisi comparativa d'alguns gens d'interès agronòmic, així com el desenvolupament massiu de marcadors moleculars. A causa de les potencials propietats nutracèutiques dels fruits de cogombre dolç, es va decidir realitzar una caracterització de la mateixa en la col·lecció d'entrades estudiada anteriorment. D'una banda s'ha avaluat el contingut en matèria seca, proteïnes, antioxidants, pigments i minerals; d'altra banda, tenint en compte que els polifenols són uns dels compostos amb major poder antioxidant, es va dur a terme un estudi que va pretendre dilucidar el perfil de polifenols en quatre entrades de cogombre dolç i una entrada de S. caripense, així com el seu poder antioxidant. Com a complement a això últim es va avaluar l'efecte d'extractes de cogombre dolç sobre cèl·lules de macròfags sotmeses a estrès oxidatiu, observant-se una reducció significativa en la producció d'òxid nítric, la qual cosa indica l'existència d'un efecte antiinflamatori. Aquestes propietats beneficioses del cogombre dolç són la seua major virtut, i juntament amb una elevada qualitat organolèptica i una bona promoció, poden afavorir la introducció i desenvolupament d'aquest cultiu. En definitiva, aquesta tesi suposa l'obtenció d'informació rellevant sobre la diversitat del cogombre dolç, a més d'un estudi en diferents aspectes, com el feno / Herraiz García, FJ. (2016). Desarrollo de herramientas morfológicas y genómicas para el estudio del pepino dulce (Solanum muricatum) y especies relacionadas. Caracterización de su valor nutracéutico [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61962 / TESIS

La gale bactérienne des Solanées à graines est une maladie répandue dans la plupart des aires de production de tomate et des Capsicum spp. (piment, poivron) du monde. Elle est très sévère dans les régions tropicales et subtropicales et sa présence est récurrente dans la région Sud-Ouest de l’océan Indien. Cette maladie est complexe car cinq taxons sont actuellement reconnus comme agents causaux, X. vesicatoria , X. perforans , X. gardneri , X. euvesicatoria et X. campestris pv. raphani . Néanmoins certaines études récentes suggèrent des synonymies de certaines de ces espèces entre elles et également avec d’autres Xanthomonas. Les objectifs principaux de la thèse étaient (1) l’analyse de la diversité sur une collection mondiale à l’aide des deux techniques moléculaires à haut débit, AFLP et MLSA, avec un accent particulier sur la diversité génétique et pathologique régionale (2) la description des relations phylogénétiques entre ces taxons et les autres Xanthomonas (3) la mise au point d’un outil d’identification rapide qui tienne compte de la diversité de l’agent pathogène et basé sur des marqueurs SCAR identifiés par AFLP. Une absence de congruence entre les topologies d’arbres dérivées des séquences de 4 gènes de ménage étudiés a été mise en évidence, de même que plusieurs évènements de recombinaison sur trois d’entre eux. Un inventaire des espèces trouvées dans les îles SWIO a pu être dressé, mettant à jour une grande diversité dans cette région. Nos données ont confirmé de fortes similarités génétiques entre X. alfalfae , X. euvesicatoria et X. perforans d’une part et de X. cynarae et X. gardneri d’autre part, qui ont probablement le statut d’espèces-synonyme. / Bacterial spot of Solanaceae is present in most areas of the world where tomato and pepper are cultivated. Its incidence is especially high in tropical and subtropical regions, such as the islands of South West Indian Ocean. This desease can be caused by five taxa: X. vesicatoria , X. perforans , X. gardneri , X. euvesicatoria et X. campestris pv. raphani, but recent studies suggest that some of those species are synonymous or actually correspond to other species of Xanthomonas. The objectives of this work were (1) to assess the genetic diversity of a word collection of strains using two high throughput molecular techniques: AFLP and MLSA; (2) to describe the phylogenetic relationships between the different taxa caising bacterial spot and other Xanthomonas; (3) to develop a rapid identification method based on SCAR markers identified by AFLP, which would take into account the global diversity of the pathogen. Tree topologies derived from the sequences of four housekeeping genes were not congruent and recombination events could be detected in three of them. A survey of bacterial spot of tomato and pepper in the South West Indian Ocean showed a broad diversity of the species causing this disease in the region. Our data confirmed the strong genetic similarity between, X. alfalfae , X. euvesicatoria et X. perforans, as well as between X. cynarae and X. gardneri, which are probably synonymous species.

Neste trabalho foram desenvolvidos quatro métodos analíticos para a determinação de cádmio, cromo, cobre, níquel e ferro em vegetais da família Solanaceae, cultivados pelos métodos convencional e orgânico de produção, obtidos em feiras e mercados da cidade de Porto Alegre, Brasil. Estes vegetais são muito consumidos devido a grande quantidade de nutrientes, no entanto, podem ser contaminados por elementos potencialmente tóxicos, por meio da exposição a poluição, uso de pesticidas, fertilizantes e água contaminada. As determinações foram conduzidas pela técnica de espectrometria de absorção atômica de alta resolução com fonte contínua em forno de grafite por análise direta de sólidos. Para Cd, Cr e Cu foram desenvolvidos três métodos independentes. No caso da determinação de Ni e Fe foi realizada análise simultânea, empregando linhas analíticas alternativas. Temperaturas de pirólise e atomização foram otimizadas para todos os elementos analisados, visando sinais analíticos com elevada sensibilidade e livres de interferências. Os métodos otimizados foram aplicados em amostras de pimentão, batata, tomate, berinjela, pimenta e physalis. Várias amostras apresentaram concentrações superiores aos limites máximos estabelecidos por legislações específicas. Os métodos propostos envolveram mínimo preparo de amostra, reduzindo riscos de contaminação, tempo analítico e custos, além de demonstrarem-se simples, precisos, rápidos e sensíveis. / In this work, four methods have been developed for the determination of cadmium, chromium, copper, nickel and iron in vegetables of the Solanaceae family, cultivated in conventional and organic farming systems, obtained from local markets and local farms, located in the city of Porto Alegre, Brazil. These vegetables are much appreciate due to the large amount of nutrients present in it. However, vegetables can be contaminated by potentially toxic elements, through the exposure to pollution, use of pesticide, fertilizers and contaminated water. The assays were carried out using a high-resolution continuum source graphite furnace atomic absorption spectrometer and direct solid sample analysis. Cd, Cr and Cu had their methods individually developed. In the case of the determination of Ni and Fe, it was applied simultaneous analysis, employing alternative analytical lines. Pyrolysis and atomization temperatures were optimized for all target elements, aiming for analytical signals with high sensitivity and free from interferences. The optimized methods were applied in samples of bell peppers, potato, tomato, eggplant, pepper and physalis. Several samples presented concentrations above the maximum permitted by specific legislation. The proposed methods were accurate, simple, fast and sensitive; moreover, a minimum sample preparation was necessary, reducing contamination, analytical time and costs.

Neste trabalho foram desenvolvidos quatro métodos analíticos para a determinação de cádmio, cromo, cobre, níquel e ferro em vegetais da família Solanaceae, cultivados pelos métodos convencional e orgânico de produção, obtidos em feiras e mercados da cidade de Porto Alegre, Brasil. Estes vegetais são muito consumidos devido a grande quantidade de nutrientes, no entanto, podem ser contaminados por elementos potencialmente tóxicos, por meio da exposição a poluição, uso de pesticidas, fertilizantes e água contaminada. As determinações foram conduzidas pela técnica de espectrometria de absorção atômica de alta resolução com fonte contínua em forno de grafite por análise direta de sólidos. Para Cd, Cr e Cu foram desenvolvidos três métodos independentes. No caso da determinação de Ni e Fe foi realizada análise simultânea, empregando linhas analíticas alternativas. Temperaturas de pirólise e atomização foram otimizadas para todos os elementos analisados, visando sinais analíticos com elevada sensibilidade e livres de interferências. Os métodos otimizados foram aplicados em amostras de pimentão, batata, tomate, berinjela, pimenta e physalis. Várias amostras apresentaram concentrações superiores aos limites máximos estabelecidos por legislações específicas. Os métodos propostos envolveram mínimo preparo de amostra, reduzindo riscos de contaminação, tempo analítico e custos, além de demonstrarem-se simples, precisos, rápidos e sensíveis. / In this work, four methods have been developed for the determination of cadmium, chromium, copper, nickel and iron in vegetables of the Solanaceae family, cultivated in conventional and organic farming systems, obtained from local markets and local farms, located in the city of Porto Alegre, Brazil. These vegetables are much appreciate due to the large amount of nutrients present in it. However, vegetables can be contaminated by potentially toxic elements, through the exposure to pollution, use of pesticide, fertilizers and contaminated water. The assays were carried out using a high-resolution continuum source graphite furnace atomic absorption spectrometer and direct solid sample analysis. Cd, Cr and Cu had their methods individually developed. In the case of the determination of Ni and Fe, it was applied simultaneous analysis, employing alternative analytical lines. Pyrolysis and atomization temperatures were optimized for all target elements, aiming for analytical signals with high sensitivity and free from interferences. The optimized methods were applied in samples of bell peppers, potato, tomato, eggplant, pepper and physalis. Several samples presented concentrations above the maximum permitted by specific legislation. The proposed methods were accurate, simple, fast and sensitive; moreover, a minimum sample preparation was necessary, reducing contamination, analytical time and costs.

Neste trabalho foram desenvolvidos quatro métodos analíticos para a determinação de cádmio, cromo, cobre, níquel e ferro em vegetais da família Solanaceae, cultivados pelos métodos convencional e orgânico de produção, obtidos em feiras e mercados da cidade de Porto Alegre, Brasil. Estes vegetais são muito consumidos devido a grande quantidade de nutrientes, no entanto, podem ser contaminados por elementos potencialmente tóxicos, por meio da exposição a poluição, uso de pesticidas, fertilizantes e água contaminada. As determinações foram conduzidas pela técnica de espectrometria de absorção atômica de alta resolução com fonte contínua em forno de grafite por análise direta de sólidos. Para Cd, Cr e Cu foram desenvolvidos três métodos independentes. No caso da determinação de Ni e Fe foi realizada análise simultânea, empregando linhas analíticas alternativas. Temperaturas de pirólise e atomização foram otimizadas para todos os elementos analisados, visando sinais analíticos com elevada sensibilidade e livres de interferências. Os métodos otimizados foram aplicados em amostras de pimentão, batata, tomate, berinjela, pimenta e physalis. Várias amostras apresentaram concentrações superiores aos limites máximos estabelecidos por legislações específicas. Os métodos propostos envolveram mínimo preparo de amostra, reduzindo riscos de contaminação, tempo analítico e custos, além de demonstrarem-se simples, precisos, rápidos e sensíveis. / In this work, four methods have been developed for the determination of cadmium, chromium, copper, nickel and iron in vegetables of the Solanaceae family, cultivated in conventional and organic farming systems, obtained from local markets and local farms, located in the city of Porto Alegre, Brazil. These vegetables are much appreciate due to the large amount of nutrients present in it. However, vegetables can be contaminated by potentially toxic elements, through the exposure to pollution, use of pesticide, fertilizers and contaminated water. The assays were carried out using a high-resolution continuum source graphite furnace atomic absorption spectrometer and direct solid sample analysis. Cd, Cr and Cu had their methods individually developed. In the case of the determination of Ni and Fe, it was applied simultaneous analysis, employing alternative analytical lines. Pyrolysis and atomization temperatures were optimized for all target elements, aiming for analytical signals with high sensitivity and free from interferences. The optimized methods were applied in samples of bell peppers, potato, tomato, eggplant, pepper and physalis. Several samples presented concentrations above the maximum permitted by specific legislation. The proposed methods were accurate, simple, fast and sensitive; moreover, a minimum sample preparation was necessary, reducing contamination, analytical time and costs.

Les cascades de Mitogen-Activated Protein Kinases (MAPKs) sont présentes chez tous les eucaryotes et permettent la transduction des signaux de l’extérieur vers l’intérieur de la cellule. Chez les végétaux, elles sont très abondantes et actives dans une multitude de processus, autant lors de la réponse aux stress que lors du développement. Elles fonctionnent comme un système de phosphorelais, se transférant un groupement phosphate d’une protéine à l’autre, de la MAPKKK à la MAPKK (MKK), puis de la MKK à la MAPK (MPK) et finalement, de la MPK vers des facteurs de transcription ou toute autre protéine qui permettra un changement au niveau de la réponse cellulaire. Depuis quelques années, plusieurs membres de la grande famille des MAPKs ont été étudiés pour leur rôle dans la reproduction sexuée des végétaux. Des mutants ont été caractérisés, mais jusqu’à maintenant, peu de voies complètes ont été décelées. Des précédents travaux dans le laboratoire ont démontré que deux MAPKKKs, de la sous-famille des MEKKs, ScFRK1 et ScFRK2, sont importantes pour le développement normal de l’ovule et du pollen chez Solanum chacoense, une espèce de pomme de terre sauvage diploïde. Sachant que les mutants des gènes les plus orthologues chez Arabidopsis thaliana ne possèdent pas les mêmes phénotypes, nous avons émis l’hypothèse que les Solanacées, du moins S. chacoense, possèdent une famille de MAPKKKs différente, qui n’est pas présente chez A. thaliana. Nous avons donc analysé les génomes/transcriptomes/protéomes de 15 espèces issues de différents clades du règne végétal afin d’étudier les relations phylogénétiques à l’intérieur de la sous-famille des MEKKs. Cela nous a permis d’observer que ScFRK1 et ScFRK2 ne sont pas seuls, mais sont inclus dans un groupe monophylétique que nous avons nommé la classe des FRKs (FRK pour Fertilization-Related Kinase). De plus, nous avons observé une expansion considérable de cette classe chez les Solanacées, comparativement à d’autres dicotylédones comme le peuplier, la vigne ou le coton. La classe des FRKs est absente chez les monocotylédones étudiées (riz et maïs) et ne possède qu’un seul membre (une FRK primitive) chez l’angiosperme basal Amborella trichopoda. Cette analyse phylogénétique des MEKKs nous a poussés à nous poser des questions sur l’origine de la classe des FRKs ainsi que sur son rôle au sein des Solanacées. Dans un deuxième temps, nous avons fait la caractérisation fonctionnelle de ScFRK3, un troisième membre de la classe des FRKs chez S. chacoense, aussi impliqué dans le développement des gamétophytes mâle et femelle. Du patron d’expression jusqu’à l’établissement d’une voie de signalisation potentielle, en passant par la caractérisation phénotypique des mutants, plusieurs expériences ont été réalisées dans le but de comprendre le rôle de ScFRK3 au niveau de la reproduction chez S. chacoense. Dans un contexte plus global, il est important de se questionner sur les rôles semblables, mais forcément différents, des trois membres de la famille FRKs qui ont été caractérisés jusqu’à présent. / Mitogen-Activated Protein Kinases (MAPKs) signaling cascades are found in all Eucaryotes and allow signal transduction from the outside of the cell to the inside. In plants, they are particularly numerous and play roles in several signaling processes, including stress responses and response to developmental cues. Their system involves a phosphorelay: they interact with each other to transfer a phosphate group. It starts with an activated MAPKKK, which transfers the phosphate group to a MAPKK (MKK), then this MKK transfers the signal to a MAPK (MPK), which ends this relay by phosphorylating transcription factors or any other proteins that will, in a way or an other, change the cell response according to the signal. During the last few years, many MAPKs members have been studied for their role in plants sexual reproduction. Some mutants were characterized, but until now, our knowledge of complete signaling cascades is very limited. Previous studies in our lab have shown that two MAPKKKs from the MEKK subfamily, ScFRK1 and ScFRK2, are important for male and female gametophytes development in Solanum chacoense, a wild diploid potato species. Genes that are the most orthologous to ScFRK1 and ScFRK2 in Arabidopsis thaliana, AtMAPKKK19, 20 and 21, do not seem to play the same roles in reproduction, which led us to make the hypothesis that in solanaceous species, at least in S. chacoense, there is one MAPKKK family that is different and not present in A. thaliana. At first, we did analyze the genomes/transcriptomes/proteomes of 15 species from different clads of the plant kingdom to find all the members of the MEKK subfamily of MAPKKKs in order to study their phylogenetic relationship. We then observed that ScFRK1 and ScFRK2 are included in a large monophyletic group which was called the FRK class (Fertilization Related Kinase). Moreover, we also observed that this class has considerably expanded within the solanaceous species, compared to other species like A. thaliana, poplar, cotton or grape vine. The FRK class is totally absent in the monocot species studied (rice and maize) and only one member is found in the basal angiosperm Amborella trichopoda. This phylogenetic analysis led us to ask questions about the origins of the FRK class and its role inside the Solanaceae family. Secondly, we characterized ScFRK3, a third member of the FRK class in S. chacoense, which is also involved, as its two FRK sisters, in male and female gametophytes development. From its expression pattern to the establishment of a potential signaling cascade, analysis and phenotyping of ScFRK3 mutant lines, many experiments were realized in order to understand the role of ScFRK3 in S. chacoense sexual reproduction. Overall, the appearance of this new and expanded class of MEKKs questions its specific role in comparison to other species that have much lesser members, mainly when compared to the model plant A. thaliana, which harbor only a fifth of the FRKs found in solanaceous species.

Le gène Solanum chacoense Fertilization-Related Kinase 1 (ScFRK1) code pour une protéine de la famille des MAPKK kinases exprimée spécifiquement dans les ovules. Son transcrit s’accumule principalement dans la zone micropylaire du sac embryonnaire à l’anthèse et diminue rapidement après pollinisation. Ces résultats suggèrent un rôle possible avant ou pendant la fécondation. Bien qu'aucune expression ne soit détectée dans le pollen à maturité, la protéine est cependant présente dans les cellules mères de microspores. Des plantes transgéniques sous-exprimant ScFRK1 ne montrent aucun phénotype au niveau des tissus végétatifs, mais présentent de petits fruits dépourvus de graines. L’étude microscopique du gamétophyte femelle révèle que son développement ne progresse pas au-delà du stade de la mégaspore fonctionnelle et une grande proportion de sacs embryonnaires anormaux est corrélée avec une faible expression de ScFRK1. De plus, la production de pollen viable diminue en fonction de la baisse des niveaux d’expression du gène, ce qui pourrait s’expliquer par un problème au cours de la mitose I. Puisque l’intégrité du sac embryonnaire est essentielle au guidage des tubes polliniques, nous avons conçu un système de guidage semi-in vivo permettant d’évaluer la capacité des ovules du mutant ScFRK1 à les attirer. L’attraction est sévèrement affectée dans de telles conditions, ce qui confirme l'implication des cellules de la zone micropylaire comme source attractive. Notre système nous a également permis de démontrer que le guidage est très spécifique à l’espèce et que cette attraction constitue un mécanisme important favorisant la spéciation et la maintenance des barrières interspécifiques dans la reproduction sexuée des végétaux. / The Solanum chacoense Fertilization-Related Kinase 1 (ScFRK1) is a member of plant MAPKKK that is specifically expressed in ovules. ScFRK1 mRNA levels accumulate predominantly in the egg apparatus cells of the embryo sac at mature stage and decrease rapidly following pollination. These results suggest both pre- and post-fertilization roles in ovule development. Although no expression could be detected in mature pollen, FRK1 mRNAs could be detected in pollen mother cells. Transgenic plants expressing sense or antisense ScFRK1 showed no abnormal phenotype in vegetative tissues but produced seedless fruits upon pollination. A microscope-based examination of developing female gametophytes revealed that its formation did not progress further than the functional megaspore stage in affected transgenic plants and, as the levels of ScFRK1 mRNA decreased, the percentage of normal embryo sacs declined. Surprisingly, even in severely affected plants producing no or very few embryo sacs, pollination led to the production of parthenocarpic fruits. Similarly, viable pollen production declined with decreasing levels of ScFRK1 and this could be linked to affected mitosis I. Since embryo sac integrity is a prerequisite for pollen tube guidance, we devised a semi-in vivo pollen tube growth system to assess the ability of the ScFRK1 mutant ovules to attract pollen tubes. As expected, guidance was severely affected, confirming the involvement of the egg apparatus cells as the source of attracting molecules. Attraction was also determined to be highly species-specific and developmentally-regulated with the acquisition of attraction competence on anthesis day.

Le gène Solanum chacoense Fertilization-Related Kinase 1 (ScFRK1) code pour une protéine de la famille des MAPKK kinases exprimée spécifiquement dans les ovules. Son transcrit s’accumule principalement dans la zone micropylaire du sac embryonnaire à l’anthèse et diminue rapidement après pollinisation. Ces résultats suggèrent un rôle possible avant ou pendant la fécondation. Bien qu'aucune expression ne soit détectée dans le pollen à maturité, la protéine est cependant présente dans les cellules mères de microspores. Des plantes transgéniques sous-exprimant ScFRK1 ne montrent aucun phénotype au niveau des tissus végétatifs, mais présentent de petits fruits dépourvus de graines. L’étude microscopique du gamétophyte femelle révèle que son développement ne progresse pas au-delà du stade de la mégaspore fonctionnelle et une grande proportion de sacs embryonnaires anormaux est corrélée avec une faible expression de ScFRK1. De plus, la production de pollen viable diminue en fonction de la baisse des niveaux d’expression du gène, ce qui pourrait s’expliquer par un problème au cours de la mitose I. Puisque l’intégrité du sac embryonnaire est essentielle au guidage des tubes polliniques, nous avons conçu un système de guidage semi-in vivo permettant d’évaluer la capacité des ovules du mutant ScFRK1 à les attirer. L’attraction est sévèrement affectée dans de telles conditions, ce qui confirme l'implication des cellules de la zone micropylaire comme source attractive. Notre système nous a également permis de démontrer que le guidage est très spécifique à l’espèce et que cette attraction constitue un mécanisme important favorisant la spéciation et la maintenance des barrières interspécifiques dans la reproduction sexuée des végétaux. / The Solanum chacoense Fertilization-Related Kinase 1 (ScFRK1) is a member of plant MAPKKK that is specifically expressed in ovules. ScFRK1 mRNA levels accumulate predominantly in the egg apparatus cells of the embryo sac at mature stage and decrease rapidly following pollination. These results suggest both pre- and post-fertilization roles in ovule development. Although no expression could be detected in mature pollen, FRK1 mRNAs could be detected in pollen mother cells. Transgenic plants expressing sense or antisense ScFRK1 showed no abnormal phenotype in vegetative tissues but produced seedless fruits upon pollination. A microscope-based examination of developing female gametophytes revealed that its formation did not progress further than the functional megaspore stage in affected transgenic plants and, as the levels of ScFRK1 mRNA decreased, the percentage of normal embryo sacs declined. Surprisingly, even in severely affected plants producing no or very few embryo sacs, pollination led to the production of parthenocarpic fruits. Similarly, viable pollen production declined with decreasing levels of ScFRK1 and this could be linked to affected mitosis I. Since embryo sac integrity is a prerequisite for pollen tube guidance, we devised a semi-in vivo pollen tube growth system to assess the ability of the ScFRK1 mutant ovules to attract pollen tubes. As expected, guidance was severely affected, confirming the involvement of the egg apparatus cells as the source of attracting molecules. Attraction was also determined to be highly species-specific and developmentally-regulated with the acquisition of attraction competence on anthesis day.