Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-27T18:33:44Z No. of bitstreams: 1 texto completo.pdf: 878289 bytes, checksum: 70c4ac466fdd682cb943dce260aec8df (MD5) / Made available in DSpace on 2017-04-27T18:33:44Z (GMT). No. of bitstreams: 1 texto completo.pdf: 878289 bytes, checksum: 70c4ac466fdd682cb943dce260aec8df (MD5) Previous issue date: 2004-08-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Experimentos foram conduzidos em laboratório e casa de vegetação objetivando o aprimoramento do método clássico de multiplicação de Pasteuria penetrans ‘in vivo’, proposto em 1980 por Stirling e Wachtel. No primeiro experimento comparou-se a produção de endósporos da P. penetrans em raízes de tomateiro de crescimento indeterminado cv. Santa Clara e determinado cv. TRural 1. Maiores pesos de matéria fresca e seca e número de endósporos foram observados em tomateiro cv. Santa Clara. O segundo experimento foi realizado para determinar a concentração ideal de endósporos de P. penetrans na suspensão e o tempo de agitação necessário para adesão adequada de endósporos aos nematóides para multiplicação da bactéria. Para que se obtenham seis endósporos, em média, por juvenil de segundo estádio (J2) de Meloidogyne javanica, verificou-se serem necessárias suspensões contendo 3,3 x 10 5 endósporos/mL, agitadas por 10 a 20 minutos, ou 3,3 x 10 4 endósporos/mL por 50 minutos. Em outro ensaio, comparou-se a multiplicação de P. penetrans em população pura de M. incognita ou em população composta de M. incognita e M. javanica oriunda de um campo de cultivo de tomate. A produção de endósporos de P. penetrans em tomateiro inoculado com M. incognita foi aproximadamente três vezes superior àquela em plantas inoculadas com população mista. Realizou-se também um teste de adesão de P. penetrans às duas populações do nematóide. Foi observado maior número de endósporos aderidos aos J2 de M. incognita. A produção de endósporos da P. penetrans em plantas de tomateiro cv. Santa Clara com 15, 30, 45 ou 60 dias de idade e inoculadas com 5.000, 15.000 ou 25.000 J2 foi avaliada. As plantas com 30 e 45 dias de idade inoculadas com 25.000 J2 permitiram a multiplicação de P. penetrans cerca de 19 vezes maior àquela obtida em plantas inoculadas com 5.000 J2. Com o objetivo de determinar se maiores níveis de matéria orgânica adicionada ao substrato provocavam alterações fisiológicas nos nematóides ou nas plantas de tomate, estudou-se a influência de diferentes proporções de solo, areia e esterco de curral (1:1:0, 2:2:1, 1:1:1 ou 1:1:2 (V:V:V), respectivamente) e três níveis de inóculo de espécies de Meloidogyne (3.000, 6.000 e 9.000 J2) sobre a concentração de fenóis em raízes de tomateiro, no teor de lipídios de espécies de Meloidogyne, e em possíveis alterações em células gigantes induzidas por esses nematóides. Não se observou efeito dos tratamentos no teor de lipídios dos nematóides. A concentração de fenóis nas raízes aumentou à medida que se acrescentou mais esterco de curral ao substrato ou quando as plantas foram inoculadas com mais nematóides (9.000 J2). As células gigantes em raízes de plantas cultivadas nos substratos 1:1:0 e 2:2:1 (solo:areia:esterco) foram mais numerosas, maiores e com maior número de núcleos. Por outro lado, as células gigantes de plantas cultivadas no substrato 1:1:1 e 1:1:2, além de menos numerosas, apresentaram alterações no tamanho e formato, demonstrando o efeito deletério das maiores doses de esterco sobre esses sítios de alimentação. O último ensaio foi conduzido para avaliar o efeito de crescentes quantidades de esterco de curral nos substratos e níveis de inóculo de espécies de Meloidogyne na reprodução de P. penetrans. Maior percentual de fêmeas infectadas por P. penetrans foi observado quando se utilizou o substrato 1:1:0 em relação aos substratos 1:1:1 e 1:1:2 ou quando as plantas foram inoculadas com 3.000 J2. O experimento foi repetido uma vez e na primeira condução do experimento, plantas cultivadas no substrato 1:1:0 ou inoculadas com 9.000 J2 apresentaram maior número de endósporos; entretanto, na segunda condução do experimento as plantas inoculadas com 9.000 J2 e cultivadas no substrato 2:2:1 foram as que permitiram maior reprodução de P. penetrans. / Greenhouse and laboratory experiments were conducted to improve the classical method of multiplying P. penetrans ‘in vivo’, proposed by Stirling & Wachtel in 1980. In the first experiment, the mass production of P. penetrans in tomatoes of indeterminated and determinated growth, cv. Santa Clara and Trural I, respectively, was compared. Higher fresh and dry root weight and endospore number were observed in ‘Santa Clara’ tomato. The second experiment was conducted to determine the best endospore concentration of P. penetrans in the aqueous suspension and the time of shaking necessary to obtain adequate attachment of endospores on the nematodes, as the first step for P. penetrans multiplication. To obtain an average of six endospores per second stage juvenile (J2) of M. javanica, it is necessary to shake suspension of 3,3 x 10 5 endospores/mL per 10 to 20 minutes or to shake 3,3 x 10 4 endospores/mL for 50 minutes. In another experiment, the reproduction of P. penetrans in pure population of M. incognita and in a mixed population of M. incognita and M. javanica, originated from the field, was studied. The endospore production of P. penetrans in tomato plants inoculated with M. incognita was approximately three times higher than in plants inoculated with the field population. An attachment test of P. penetrans on the two populations was performed and higher number of endospores attached to J2 of M. incognita was observed. The multiplication of P. penetrans in 15, 30, 45 or 60 day-old ‘Santa Clara’ tomato plants inoculated with 5,000, 15,000 or 25,000 J2, was also evaluated. The 30 and 45 days old plants inoculated with 25,000 J2 provided P. penetrans multiplication up to nineteen times more than plants inoculated with 5,000 J2 in the first experimental run. To determine if high cow manure levels added to substrate promote physiological changes on the nematodes or on the tomato plants, the influence of cow manure amendment to mixtures of soil and sand giving rates of 1:1:0; 2:2:1; 1:1:1 and 1:1:2 (V:V:V) of soil, sand and manure, respectively, and three inoculum levels of Meloidogyne species, i.e., 3,000; 6,000 and 9,000 J2 on the phenolic content in the tomato roots, changes in nematode lipid content and possible alterations in the giant cells induced by the nematodes, were studied. No conclusion could be drawn about the effect of manure on the nematode lipid content. The phenolic content in the roots increased as more cow manure was added to the substrate or when the plants were inoculated with more nematodes (9,000 J2). The giant cells in the roots of plants cultivated in the substrates 1:1:0 and 2:2:1 were more numerous, bigger and with more nuclei. On the other hand, the giant cells of plants cultivated on 1:1:1 and 1:1:2 substrates were less numerous, showed changes on their format and were smaller, demonstrating the deleterious effect of organic amendments to these feeding sites. A subsequent experiment was carried out to evaluate the effect of the interaction of increasing rates of cow manure in the substrates and of nematode levels on the reproduction of Pasteuria penetrans. Higher perceptual of infected females by P. penetrans was observed when the plants grew in the substrate 1:1:0 than in the substrates 1:1:1 and 1:1:2, or when plants were inoculated with 3,000 J2. The experiment was repeated once and in the first run, plants cultivated on substrate 1:1:0 or inoculated with 9,000 J2 had higher endospore number. However, in the second run, plants inoculated with 9,000 J2 and cultivated on substrate 2:2:1 yielded more endospores per root system. / Tese importada do Alexandria

Thèse ou mémoire avec insertion d'articles. / Cette thèse présente une vue d'ensemble complète de trois études qui ont évalué le potentiel d'utilisation du frass, matériau résiduel résultant de la bioconversion de la matière organique par les larves de la mouche soldat noire (LMSN), comme amendement organique en horticulture. Au cours de la première étude, des essais in vitro de superposition de cultures ont révélé la présence de microorganismes produisant des composés inhibant la croissance mycélienne des sept agents phytopathogènes (champignons/oomycètes) importants dans les extraits du frass et les extraits du régime de référence Gainesville (GV). Parmi les bactéries/champignons isolé(e)s du régime GV et du frass, la bactérie Bacillus velezensis, utilisée comme agent de lutte biologique, a montré une forte activité antifongique/anti-oomycète suggérant que cette bactérie présente dans le régime GV survive au processus d'élevage de LMSN et soit l'un des facteurs clés contribuant à l'activité antifongique/anti-oomycète du frass. Cela souligne l'importance des caractéristiques microbiennes de la matière première dans les propriétés antifongiques et anti-oomycètes du frass. La deuxième étude a comparé les caractéristiques du frass dérivé de deux régimes alimentaires différents : un régime à base de déchets de fruits/légumes/boulangerie complété par des déchets de brasserie (FVBB) et un régime GV. Les résultats ont démontré que les deux frass contenaient des éléments nutritifs essentiels, comparables aux engrais organiques disponibles commercialement. De plus, le frass provenant du régime FVBB ont présenté des effets d'antibiose, inhibant la croissance des pathogènes des plantes, tout en favorisant de meilleures performances des larves, une composition nutritionnelle et une réduction efficace des déchets. Dans la troisième étude, le potentiel du frass de LMSN à limiter le développement du flétrissement causé par Fusarium oxysporum f. sp. lycopersici (FOL) chez les plants de tomate a été étudié. Le frass provenant des régimes GV et FVBB a été pasteurisé à 70°C (pendant 1 heure) ou non avant d'être incorporé dans le milieu de culture des plants de tomate. Les résultats ont révélé que le frass provenant du régime GV, pasteurisé ou non, réduisait significativement la gravité du flétrissement causé par FOL et la colonisation des racines par le champignon pathogène comparativement au frass provenant du régime FVBB et au groupe témoin sans frass, tout en étant comparables au compost. De plus, l'étude a identifié des différences dans les communautés microbiennes de la rhizosphère associées au type de frass et à la pasteurisation, certaines familles bénéfiques étant associées à une réduction de la gravité de la maladie. Les trois études démontrent collectivement le potentiel du frass de LMSN comme amendement organique efficace pour la gestion des agents pathogènes des cultures horticoles. Le choix du régime d'élevage des larves influence considérablement l'activité antifongique du frass et son efficacité globale dans la lutte contre les maladies des plantes. / This thesis presents a comprehensive overview of three studies evaluating the potential use of frass, the residual material resulting from the bioconversion of organic matter by black soldier fly larvae (BSFL), as an organic amendment in horticulture. In the first study, in vitro culture overlay assays revealed the presence of microorganisms producing compounds inhibiting mycelial growth of seven important plant pathogens (fungi/oomycetes) in frass extracts and Gainesville diet (GV) extracts, a widely used reference diet. Among the bacteria/fungi isolated from the GV diet and frass, the bacterium Bacillus velezensis, used as a biological control agent, showed strong antifungal/anti- oomycete activity suggesting that this bacterium is present in the GV diet survives the BSFL rearing process and is one of the key factors contributing to the antifungal/anti-oomycete activity of frass. This highlights the significance of feedstock microbial characteristics in the frass antifungal and anti- oomycete properties and suggests the possibility of exploiting frass to control horticultural plant pathogens. The second study compared the characteristics of frass derived from two different diets: a fruit/vegetable/bakery waste-based diet supplemented with brewery waste (FVBB) and a GV reference diet. Results demonstrated that frass derived from both diets contained essential nutrients and elements, comparable to commercially available organic fertilizers. Moreover, frass from the FVBB diet also exhibited antibiosis effects, inhibiting the growth of plant pathogens, while also promoting better larval performance, nutritional composition, and efficient waste reduction. In the third study, the potential of BSFL frass to limit the development of Fusarium oxysporum f. sp. lycopersici (FOL) in tomato plants was investigated. Frass derived from the GV and FVBB diets underwent pasteurization at 70°C (for 1 hour) and not before being incorporated into the growing medium of tomato plants. The results revealed that frass derived from GV diet, regardless of pasteurization, significantly reduced the severity of wilt caused by FOL and pathogen root colonization by the pathogenic fungus compared to frass derived from FVBB diet and the control group with no frass while being comparable with compost. Additionally, the study identified differences in rhizosphere microbial communities associated with the type of frass and pasteurization, with certain beneficial orders associated with the reduced disease severity. The three studies collectively demonstrate the potential of BSFL frass as an efficient organic amendment for plant pathogens management in horticultural crops. The choice of larval rearing diet significantly influences the frass antifungal activity and overall effectiveness in controlling plant diseases.

Le développement du fruit de tomate s’accompagne d’un phénomène d’endopolyploïdisation(amplification de l’ADN en l'absence de mitose) associé à la croissance cellulaire. Au stade vert mature huit niveaux de ploïdie sont présents (2C à 256C) dans le péricarpe.Une première partie du travail a porté sur l’étude de la distribution spatiale des niveaux de ploïdie dans ce tissu. Cet objectif a nécessité la mise au point d’une méthode originale de détermination de la ploïdie in situ reposant sur la technique de BAC-FISH. Nous avons montré que les cellules les plus polyploïdes se situent dans les assises internes du péricarpe, et qu’elles sont aussi les plus grandes. Ces cellules semblent déjà formées au moment de l’anthèse. Cette cartographie de la ploïdie associée à une analyse de la taille cellulaire a également montré que la taille finale des cellules ne dépend pas uniquement de leur niveau de ploïdie mais également de leur position dans le péricarpe. Enfin, nos résultats suggèrent que l’endopolyploïdisation précède la croissance cellulaire.Dans une deuxième partie du travail, nous avons étudié la structure des noyaux en microscopie à fluorescence et électronique. L’endopolyploïdisation affecte profondément la taille et la forme des noyaux, qui acquièrent un volume important et une forme complexe avec de profondes invaginations. La taille du nucléole augmente avec celle du noyau, ce qui suggère une activité de transcription accrue. De plus, la présence de nombreuses mitochondries à proximité des noyaux polyploïdes suggère une forte activité métabolique en lien avec l’endopolyploïdisation. L’utilisation de la méthode BAC-FISH a permis également de montrer que la polyploïdie se faisait par endoreduplication avec la formation de chromosomes polytènes.Dans une troisième partie nous avons cherché, en criblant une banque de mutants Micro-Tom, à identifier des lignées affectées dans l’endoreduplication afin d’étudier l’impact de ce phénomène sur la vitesse de croissance du fruit. Nous avons caractérisé plusieurs familles dont les niveaux moyens de ploïdie variaient par rapport à la lignée de référence. Une de ces familles présente un phénotype stable au cours de deux générations, avec une augmentation d’au moins 30 % de la ploïdie moyenne et une augmentation de la taille des cellules du péricarpe. Cependant cette famille présentant aussi un développement relativement parthénocarpique de ses fruits, sa caractérisation n’a pas pu être poursuivie dans le cadre de ce travail. / Tomato fruit development includes massive endopolyploidisation events (DNA duplication inthe absence of mitoses) within pericarp cells, in which 8 DNA levels from 2 C to 256 C are detected atmature green stage.The first part of this work dealt with the study of the spatial distribution of ploidy levels inpericarp. To achieve this purpose, a new method for in situ ploidy assessment was set up using aBAC-FISH protocol. The main results are 1/ the most polyploid cells are located in central mesocarpcell layers; 2/ the most polyploid cells are also the largest cells; 3/ these cells are likely to be alreadypresent in ovary at anthesis. Ploidy mapping has also shown that the final cell size does not dependonly on ploidy level but also on cell location in pericarp, and that endopolyploidization is likely set up intissues before cell expansion.The structure of the polyploid nucleus was studied by using fluorescence microscopy andelectron microscopy. Endopolyploidization profoundly modifies the size and shape of nuclei, whichbecome much larger and acquire a complex shape with deep invaginations. Nucleolus size increases,which is likely related to transcriptional increase. Moreover, the presence of numerous mitochondria inthe close vicinity of the nuclear membrane reinforces the hypothesis of increased nuclear andmetabolic activity in polyploid cells. The BAC-FISH in situ method for ploidy assessment also revealedthat endopolyploidization proceeded through polyteny.In the last part of this work, we screened a tomato Micro-Tom tilling bank for mutants affectedin endopolyploidization. The aim was to use tomato lines with distinct ploidy levels to check theinfluence of ploidy on fruit growth rate. Several mutant families were identified with moderatelyincreased ploidy levels. One of these families exhibited transmissible phenotype through 2generations, with ploidy increased by ca. 30 % and increased pericarp cell size. As these mutants hadalso a strongly pronounced parthenocarpic phenotype, their characterization could not be furtheradvanced in the frame of this work.

L'un des phénomènes les plus importants survenus pendant la maturation du fruit de tomate est le changement de couleur du vert au rouge. Ce changement a lieu dans les plastes et correspond à la différenciation des plastes photosynthétiques, les chloroplastes, en plastes non-photosynthétiques qui accumulent des caroténoïdes, les chromoplastes. Dans cette thèse, nous présentons d'abord une introduction bibliographique sur le domaine de la transition chloroplaste-chromoplaste, en décrivant les modifications structurales et physiologiques qui se produisent pendant la transition. Puis, dans le premier chapitre, nous présentons des observations microscopiques de plastes isolés à trois stades de mûrissement, puis des enregistrements en temps réel de la fluorescence des pigments sur les tranches de fruits de tomate. Il a été possible de montrer que la transition chloroplaste-chromoplaste était synchrone pour tous les plastes d'une seule cellule et que tous les chromoplastes proviennent de chloroplastes préexistants. Dans le deuxième chapitre, une approche protéomique quantitative de la transition chloroplaste-chromoplaste est présentée, pour identifier les protéines différentiellement exprimées. Le traitement des données a identifié 1932 protéines parmi lesquelles 1529 ont été quantifiées par spectrométrie de masse. Les procédures de quantification ont ensuite été validées par WESTERN blot de certaines protéines. La chromoplastogénèse comprend les changements métaboliques suivants : diminution de l'abondance des protéines de réaction à la lumière et du métabolisme des glucide, et l'augmentation de la biosynthèse des terpénoïdes et des protéines de stress. Ces changements sont couplés à la rupture de la biogenèse des thylakoïdes, des photosystèmes et des composants de production d'énergie, et l'arrêt de la division des plastes. Dans le dernier chapitre nous avons utilisé la lincomycine, un inhibiteur spécifique de la traduction à l'intérieur des plastes, afin d'étudier les effets sur la maturation des fruits et sur l'expression de gènes nucléaires impliqués dans la maturation. Les résultats préliminaires indiquent que l'inhibition de la traduction des protéines dans les plastes affecte la maturation du fruit en réduisant l'accumulation de caroténoides. L'expression de plusieurs gènes nucléaires a été modifiée mais une relation claire avec le phénotype altéré de maturation n'a pas pu être établie. Au total, notre travail donne de nouveaux aperçus sur le processus de différenciation chromoplaste et fournit des données nouvelles ressources sur le protéome plaste / One of the most important phenomenons occurring during tomato fruit ripening is the color change from green to red. This change takes place in the plastids and corresponds to the differentiation of photosynthetic plastids, chloroplasts, into non photosynthetic plastids that accumulate carotenoids, chromoplasts. In this thesis we first present a bibliographic introduction reviewing the state of the art in the field of chloroplast to chromoplast transition and describing the structural and physiological changes occurring during the transition. Then, in the first chapter we present an in situ real-time recording of pigment fluorescence on live tomato fruit slices at three ripening stages. By viewing individual plastids it was possible to show that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell and that all chromoplasts derived from pre-existing chloroplasts. In chapter two, a quantitative proteomic approach of the chloroplast-to-chromoplast transition is presented that identifies differentially expressed proteins. Stringent curation and processing of the data identified 1932 proteins among which 1529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immune-blot evaluation of some proteins. Chromoplastogenesis appears to comprise major metabolic shifts (decrease in abundance of proteins of light reactions and carbohydrate metabolism and increase in terpenoid biosynthesis and stress-related protein) that are coupled to the disruption of the thylakoid and photosystems biogenesis machinery, elevated energy production components and loss of plastid division machinery. In the last chapter, we have used lincomycin, a specific inhibitor of protein translation within the plastids, in order to study the effects on fruit ripening and on the expression of some ripening-related nuclear genes. Preliminary results indicate that inhibiting protein translation in the plastids affects fruit ripening by reducing the accumulation of carotenoids. The expression of several nuclear genes has been affected but a clear relationship with the altered ripening phenotype could not be established. Altogether, our work gives new insights on the chromoplast differentiation process and provides novel resource data on the plastid proteome

La cuticule, une matrice lipidique extracellulaire constituée de cires et d’un squelette de cutine, est la barrière de défense la plus externe des plantes face à leur environnement. Elle intervient dans de nombreuses propriétés agronomiques comme la conservation post récolte, les propriétés mécaniques ou bien l’aspect du fruit, dont la brillance. Afin d’isoler des mutants de cuticule, le criblage d’une collection de mutants EMS de tomate a été entrepris, en se basant sur la brillance des fruits, conduisant à la sélection de 24 mutants. Chez ceux-ci, des analyses biochimiques ont montré de fortes variations de charge et de composition de la cuticule, notamment chez les mutants de cutine. La caractérisation de 4 mutants remarquables a été entreprise afin d’identifier les mutations responsables des phénotypes de brillance. Le mutant le plus affecté, présentant une charge en cutine réduite de 85% par rapport au type sauvage, a révélé une mutation du gène SlGDSL2 codant pour une acylhydrolase à motif GDSL, responsable de la polymérisation de la cutine. Afin d’étudier la formation et la régulation de la cutine, la suite du travail a consisté à obtenir et à caractériser des simples et des doubles mutants affectés dans la synthèse des monomères de cutine, le transport apoplastique et la polymérisation de la cutine. / The cuticle, an extracellular lipid matrix consisting of waxes and of a cutin skeleton is the outermost plants protection barrier against their environment. The cuticle is involved in many agronomic traits such as post-harvest storage, biomechanical or fruit appearance properties like surface brightness. In order to isolate cuticle mutants, the screening of an EMS tomato mutants collection has been undertaken, based on fruit brightness, leading to the selection of 24 mutants. Biochemical analyzes have shown wide variations in cuticle loads and compositions, especially in cutin mutants. The characterization of 4 remarkable mutants was undertaken to identify the mutations responsible for brightness phenotypes. The most affected mutant shows a cutin load reduced by 85% compared to the wild type, and is due to a mutation in the SlGDSL2 gene, encoding a GDSL-motive acylhydrolase enzyme, responsible for the cutin polymerization. In order to further study the cutin formation and regulation, the next work was to obtain and characterize single and double mutants affected in cutin monomer synthesis, apoplastic transport and cutin polymerization.

Le gène FW2.2 correspond au locus de caractère quantitatif (QTL) majeur impliqué dans le contrôle de la taille finale du fruit de tomate. FW2.2 appartient à une famille multigénique et code une protéine transmembranaire de 163 acides aminés dont la fonction demeure de nos jours inconnue. Pourtant décrite comme un régulateur négatif des mitoses, par conséquent comme un régulateur de la taille du fruit et cloné plus de 12 ans auparavant, aucune fonction biochimique, physiologique ni même développementale n’a été déterminée concernant cette protéine. Ce qui est d’autant plus étonnant car aucun lien n’a été révélé entre sa fonction protéique et sa capacité à influencer le cycle cellulaire. L’analyse d’une nouvelle version du génome de la tomate nous a permis d’identifier 17 nouvelles séquences homologues à FW2.2 (que nous avons nommé FW2.2-like) et l’alignement de ces séquences nous a permis d’observer une importante conservation du motif PLAC8 commun à cette famille multigénique. L’étude phylogénétique que nous avons réalisée ne nous a donné aucune indication quant à la fonction potentielle de transporteur de métaux lourds de la protéine FW2.2 malgré le fait que sa séquence protéique présente les mêmes caractéristiques que celles décrites chez des transporteurs de métaux lourds. Des expériences d’électrophysiologie ne nous ont pas permis de confirmer son rôle de transporteur, mais des dosages de contenu minéral réalisés sur des péricarpes de fruits de tomate présentant des niveaux d’expression différents pour FW2.2 nous ont permis d’observer une différence de stockage du cadmium dans le péricarpe de ces fruits. Nous avons également étudié le rôle de la protéine FW2.2 dans le développement des plantes en utilisant des lignées de plantes et des lignées cellulaires surexprimant le gène FW2.2. Ceci nous a mené à l’hypothèse que la protéine FW2.2 pouvait être impliquée dans la voie de signalisation des brassinostéroïdes. Pour terminer, nous avons tenté de comprendre quels mécanismes de régulation étaient déclenchés par FW2.2 en recherchant ses partenaires potentiels par le biais de l’application de la technique du Split-Ubiquitin. / The FW2.2 gene corresponds to the major Quantitative Trait Locus (QTL) governing fruit size in tomato. FW2.2 belongs to a multigene family and encodes a transmembrane protein of 163 amino acids whose actual function remains unknown. Although described as a negative regulator of cell divisions and consequently as a regulator of fruit size, any definitive biochemical, physiological and developmental function assigned to FW2.2 is still lacking although the gene was cloned more than twelve years ago. Especially the fundamental question of what kind of link is there between the FW2.2 protein function and cell cycle regulation is all even more relevant. The analysis of the recently released genome of tomato identified 17 new sequences related to FW2.2 (SlFW2.2-like genes) and the protein sequence alignments showed the conservation of the PLAC8 motif common to this multigene family. Our phylogenetic studies did not give any clues relative to the FW2.2 function even though it presents sequence characteristics described for heavy metal transporters. Electrophysiology experiments did not allow the confirmation of the ion transporter function but a total ion content measurement on tomato fruit pericarps differing by their levels of FW2.2 expression showed a difference in the fruit pericarp cadmium content. We also investigated the role of the FW2.2 protein on the plant development using plant and cell lines that overexpress this gene and it appeared that this protein may be involved in the brassinosteroid signal pathway. The regulatory mechanisms mediated by the action of FW2.2 on mitotic activity during fruit development have also been analyzed by looking for potential partners interacting with the FW2.2 protein using the technique of split-ubiquitin.

La domestication des plantes a débuté il y a quelques milliers d’années quand les hommes se sont sédentarisés. Ils ont sélectionné les plantes sauvages portant des caractères phénotypiques d’intérêt pour la consommation et production humaine. Ce processus évolutif a par conséquent modifié le patrimoine génétique des espèces domestiquées. Cette thèse se penche sur les traces génétiques induites par la domestication chez trois espèces de Solanacées : l’aubergine (Solanummelongena), le piment (Capsicum annuum) et la tomate (S. lycopersicum). En effet, si les caractères phénotypiques des plantes cultivées ont été sélectionnés depuis des milliers d’années, les conséquences moléculaires d’une telle sélection restent peu étudiées à l'échelle du génome. Cette étude est basée sur des données de diversité et d’expression de gènes (RNAseq). En utilisant des méthodes comparatives entre des variétés cultivées et leurs espèces sauvages apparentées, j’a iétudié, à l’échelle intra-spécifique, d’une part les histoires démographiques de chacune des espèces,et d’autre part les changements de diversité nucléotidique et d’expression des gènes dus à la domestication. La comparaison de ces trois événements indépendants de domestication, offre l’opportunité de décrypter les changements génétiques qui convergent chez ces trois espèces lors du processus de sélection humaine.Suite à une introduction qui pose le cadre de cette étude et présente l’état de l’art, le premier chapitre, s’inscrit dans un ouvrage portant sur la génomique des populations d’espèces modèles. Il propose une synthèse des connaissances accumulées en plus d’un siècle de recherche sur l’espèce modèle qu’est la tomate (S. lycopersicum). Ce chapitre permet également de compléter le contexte scientifique dans lequel cette thèse s’inscrit, notamment, en retraçant l’importance que les espèces sauvages apparentées ont eu dans l’amélioration de l’adaptabilité des variétés cultivées actuelles.L’hypothèse du deuxième chapitre révèle la convergence des changements démographiques entre les trois espèces malgré leurs événements indépendants de domestication. L’étude comparée d’inférences de scénarios démographiques a permis de reconstruire l’histoire démographique de chaque espèce cultivée. Ces inférences ont aussi facilité l’estimation des paramètres tels que les flux migratoires entre les espèces sauvages et cultivées, la force des goulots d’étranglement liés à l’intensité de la sélection humaine et la durée des événements de domestication. Ce chapitre permet de démontrer que les changements démographiques liés à la domestication dépendent de l’état de sympatrie ou d’allopatrie des variétés cultivées avec leurs sauvages apparentées. Les connaissances quant à la datation des événements de domestication de nos trois espèces restent très faibles, et les inférences ont permis d’établir des estimations de durée de domestication relativement précise. Ces nouvelles connaissances apportent une plus-value à cette étude pour nos trois espèces et nous invitent à s’interroger sur les différents compartiments du génome qui ont été sélectionnées et modifiées lors de la domestication.Le troisième chapitre teste l’hypothèse d’une convergence évolutive des changements moléculaires, notamment transcriptionnels, induits par la domestication et l’amélioration moderne.La comparaison des variétés cultivées à leurs espèces sauvages apparentées permet d’évaluer la convergence des mécanismes de régulation et d’adaptation liés à la domestication. C’est en testant la corrélation entre les traces génétiques (diversité nucléotidique) de sélection et les changements d’expression des gènes observés chez les variétés cultivées que l’hypothèse de départ a été validée.Cette analyse montre que la domestication, au-delà même de changements nucléotidiques, a modifié l’expression des gènes chez les trois espèces. / Domestication started thousand years ago when human shifted from hunter-gatherer to agrarian societies. They started selecting wild plants for phenotypes related to consumption andyield. This evolutionary process induced changes in the gene pool of domesticated plants. This thesis focuses on genetic footprints induced by domestication within a trio of Solanaceae species: the eggplant (Solanum melongena), the pepper (Capsicum annuum) and the tomato (S. lycopersicum).Crop plants have been selected for thousand years on phenotypic traits, but the molecularconsequences of such selection remain unknown at the genome-wide scale. The study was performed on a RNAseq data set; using comparative methods between crops and their wild relatives,I studied, at the intra-specific scale, the demographic history, and, both the nucleotide diversity and the gene expression changes due to domestication. Comparing these three independent events ofdomestication, is a great opportunity to decipher the interspecific genetic changes, converging for the three species, during the human selection process.The first chapter is a book chapter about population genomics in model species. It details thestate of art of hundred years of research on tomato as model species (S. lycopersicum). Tomato is amodel species in genetics, as well as in population genomics thanks to the important collection of genomic data that have been accumulating over years. Tomato has the strongest economic importance within the trio of studied species. By highlighting the importance of crop wild relative species for adaptability improvement of modern cultivars, this chapter describes the scientific context of this thesis work.The two next chapters are following these researches and show the importance to both conserve and study the crop wild relative species.In the second chapter, I hypothesize that demographic changes within the three species experience a convergence, despite their independent domestication events. The comparative studyof demographic inferences allows the reconstruction of each domesticated species demographichistory. Theses inferences facilitate the parameter estimations such as the migration rate between crop and wild, the bottleneck strength paired with the human selection and the duration of thedomestication events. This chapter reveals a common bottleneck phenomenon as well as migration rate dependent to the allopatric or sympatric state of the crops with their wild relatives. Knowledge concerning the domestication events dating, for each of the three species, remain poorly studied and this thesis work discloses relative domestication time durations.These new insights bring valuable knowledge to the three species and induce a questioning on thedifferent genome parts that are selected and modified through domestication.The third chapter, test the hypothesis of a convergent evolution of molecular changes,especially transcriptional, induced by domestication and modern breeding. The comparative analysis of crop plants and their wild relatives assesses the convergence of regulation and adaptation mechanisms due to domestication. By testing the correlation between the selection footprints on genes and the gene expression changes in crop compared to their wild relative species, the previous hypothesis was confirmed. This analysis implies that domestication modified gene expression in the three species beyond only nucleotide polymorphisms. The ortholog analysis of our species genes, confirmed that domestication facilitated the fruit development and plant growth but relaxed selective pressure on genes of plant defense and environmental stresses tolerance. Demonstrating demographic changes and molecular footprints of domestication, my PhD thesis highlights several proofs of convergence.

Orientador: Pedro Luis da Costa Aguiar Alves / Coorientador: Rogério Falleiros Carvalho / Banca: Priscila Lupino Gratão / Banca: Tiago Pereira Salgado / Banca: Mariluce Pascoina Nepomuceno / Resumo: As plantas emitem sinais a fim de acionar respostas de defesa ao estresse sofrido pela baixa radiação solar (sombreamento), que pode ser causado pela presença de outras plantas. O fitormônio ácido jasmônico (AJ) pode fazer parte das respostas das plantas ao sombreamento. Os objetivos deste trabalho foram avaliar a participação do AJ nas respostas do tomateiro (Solanum lycopersicum L.), híbrido Heinz 9553, ao sombreamento artificial e à competição com a planta daninha Raphanus raphanistrum L. O primeiro experimento foi realizado em duas épocas do ano (outono-inverno e primavera-verão). O tomateiro foi cultivado sob sombreamento artificial (sombrite) e luz natural (ambiente sem cobertura), e aplicou-se metil-jasmonato (MeJA - 0; 0,15; 0,25 e 0,50 mM) e ibuprofeno (IBU - 0; 1,25; 2,5 e 5,0 mM) aos 7, 14, 21 e 28 dias após o transplante das mudas. Observou-se que a condição luminosa teve maior efeito nas respostas apresentadas pelas plantas do que o MeJA e o IBU, independente da concentração aplicada. Os efeitos nas análises bioquímicas foram mais pronunciados, mas não houve diferenças nas análises de crescimento. O segundo experimento consistiu em manter o tomateiro H9553 sob sombreamento promovido pela planta daninha R. raphanistrum L. (nabiça). Estudou-se a competição sob diferentes densidades da planta daninha (0, 11, 16 e 22 plantas m-2), sendo aplicado o MeJA (0,50 mM) no tomateiro. Os resultados mostraram que em condições de alto nível de sombreamento, o aumento da quantid... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Plants emit signals in order to trigger defense responses to the stress of low solar radiation (shading), which may be caused by the presence of other plants. Jasmonic acid phormonium (JA) may be part of the plants responses to shading. The aims of this work were to evaluate the participation of JA in tomato responses (Solanum lycopersicum L.), Heinz 9553 hybrid, to artificial shading and competition with the weed Raphanus raphanistrum L. The first experiment was carried out at two seasons of the year (autumn-winter and spring-summer). Tomatos were cultivated under artificial shading (sombrite) and natural light (uncovered environment), and methyl jasmonate (MeJA: 0, 0.15, 0.25 and 0.50 mM) and ibuprofen (IBU: 0, 1.25, 2.5 and 5.0 mM) were applied at 7, 14, 21 and 28 days after transplanting the seedlings. It was observed that the light condition had greater effect on the responses presented by the plants than the MeJA and the IBU, regardless of the applied concentration. The effects on biochemical analyzes were more pronounced, but there were no differences in growth analyzes. The second experiment consisted in keeping tomato H9553 under shading promoted by the weed R. raphanistrum L. (radish). Competition was studied under different densities of R. raphanistrum (0, 11, 16 and 22 plants m-2), and the MeJA (0.50 mM) was applied to the tomato. The results showed that in conditions of high shading, increasing the amount of JA did not interfere in the development of the plants and did not affect productivity, but the greatest effect on the crop was due to the low luminosity. Thus, in general, it can be concluded that the accumulation of JA is involved with conditions of low R:FR ratio, such as shading, but its effects on the plant due to this condition are not accentuated. However, further studies need to be performed on non-model plants to further assess JA participation in shaded plants. / Doutor

Com o objetivo de avaliar as mudas e a produção de tomate (Lycopersicon esculentum Mill), tipo italiano \'Netuno\', no campo desenvolvidas em diferentes tipos de volume de recipiente com a combinação de varias idades de transplante foi conduzido este experimento no Departamento de Produção Vegetal da Escola Superior de Agricultura ¨Luiz de Queiroz\", Universidade de São Paulo, no município de Piracicaba - Estado de São Paulo, entre abril a agosto de 2005. Foram utilizadas bandejas de 72, 128, 288 e 450 células, com volumes celulares de 121,2; 34,6; 12,0 e 14,0 cm3 respectivamente. As idades de avaliação foram aos 19, 24, 29 e 34 dias após a semeadura. O delineamento experimental utilizado foi o de blocos casualizados, no esquema fatorial de 4 x 4. Um dos fatores foi representado pelas diferentes bandejas, e o segundo fator, pelas diferentes idades de avaliação. Na produção de mudas foram avaliadas, número de folhas, área foliar, altura da muda, massa fresca e seca de parte aérea e raiz e qualidade visual das mudas. Logo após o transplante foram avaliadas características de desenvolvimento vegetativo: área foliar, número de folhas, altura das plantas, diâmetro da haste, massa fresca de folhas e raiz e qualidade visual das plantas. Posteriormente durante a colheita foram avaliadas características de produção; precocidade, massa e número comercial de frutos, massa e número total de frutos. Também foi realizada a seleção dos frutos em pequenos, médios e grandes. Na comparação entre os tratamentos foram observadas algumas diferenças em algumas características. Os volumes maiores apresentaram melhor qualidade de mudas (121,2 e 34,6 cm³) e os volumes menores apresentaram mudas estioladas, raquíticas, desuniformes. Os volumes maiores apresentaram maior área foliar, massa fresca e seca de raiz. A melhor idade para o transplante foi de 24 e 29 dias para os volumes maiores, recomendando-se o volume de célula de 34,6 cm³. Com relação aos resultados de produção de frutos, não houve diferencia entre os tratamentos utilizados, porém, obteve se precocidade na colheita de frutos com volumes maiores. A muda de 24 dias teve o maior número e massa media de frutos por planta nas colheitas precoces. Número comercial e total de frutos foi maior nas idades de 19, 24 e 29 dias. As idades mais precoces e os volumes maiores apresentaram maior número e massa de frutos médios por planta. Não houve diferença para frutos grandes em todos os tratamentos estudados. / This study was carried out from April to August, 2005, Piracicaba, Sao Paulo State, Brazil. The objective of this work was to evaluate the effect of the cell recipient and seedlings age on the quality of transplants and tomato (Lycopersicon esculentum Mill) production \'Netuno\' in open field. The treatments resulted from the combination of for tray cell sizes 121,2; 34,6; 12,0 e 14,0 cm3 and four transplanting ages (19, 24, 29 e 34 days). It done in a randomized block design ( 4 x 4 factorial). One of the factors was represented by different cell volume and the second factor was transplants age. In the transplant production was evaluated, number of leaves, leaf area, fresh and dry leaves and root mass, seedlings quality (before production was evaluated the same vegetative characteristics) and after transplanting, the earliness, and total yield fruits (number and mass of fruits commercial fruits). The fruits were classified in small, medium and large sizes. The larger volumes (121, 2 e 34, 6 cm³) presented better quality of seedlings than of smaller one (12, 0 e 14, 0 cm³). There was etiolated, stunted, desuniformity, and the cell volume of 12,0 cm³ seedlings was yellows and small. The larger volumes presented higher fresh and dry mass. The better age for transplanting was 24 to 29 days for the larges volumes, being recommended the volume of cell of 34,6 cm³. No significant differences were observed between all treatments for fruits production, however, earliness in larger volumes was observed. Transplants with 24 days age were most productive for earliness. Commercial and total fruits number per plant increment at 19, 24 and also 29 days age. Transplants with 19, 24 days age and larger cell volumes had increased the number and mass of early fruits. The earliness age and the larger cell volume presented higher number and mass of medium fruits. There were not differences for larger fruits for all treatments.

Plant domestication led to a loss of genetic variation in many crops, due to the excessive emphasis in the selection of edible organs (root, leaf, stem or fruit) and the low selection pressure for other traits in the cultivated environment. This \'genetic erosion\' led to loss of alleles associated with resistance to environmental stresses, such as drought and salinity, which can in turn culminate in productivity losses. In tomato (Solanum lycopersicum L.), it is possible to tap into a reservoir of valuable genetic variation in its wild relatives. Identification of genetic variants associated with tomato domestication, and with stress resistance mechanisms which may have been lost during domestication, could be used to aid in breeding programs. In the present work, which was divided into two chapters, we carried out crosses between the wild species S. pennellii and the miniature tomato cultivar Micro-Tom (MT) and created two introgression lines (ILs), one with reduced organ size and another with increased drought tolerance. In the first chapter, we report the characterization and mapping of the IL denominated as Tiny organs and reduced yield (Toy). Toy harbors a S. pennellii genome segment on chromosome 7 and presents a considerable reduction in both vegetative (leaves) and reproductive (fruit) organs. We discuss how this could be a relevant trait underpinning tomato domestication. In the second chapter, we describe the drought tolerance mechanism of the IL Water Economy Locus in Lycopersicon (Well). Well harbors a S. pennellii genome segment on chromosome 1 and shows lower hydraulic conductance, possibly related to decreased xylem vessel size. The results shown suggest that this lower hydraulic conductance promotes a disturbance in the soil-plant-atmosphere hydraulic continuum leading to changes in stomatal behavior, which, in turn, are probably related to the delayed wilting of Well under conditions of water deficit. / A domesticação das plantas levou a uma perda de variação genética em muitas culturas, devido à ênfase excessiva na seleção de órgãos comestíveis (raiz, folha, caule ou fruto) e a baixa pressão de seleção para outras características no ambiente cultivado. Essa \"erosão genética\" levou à perda de alelos associados à resistência de diversos estresses ambientais, como seca e salinidade, os quais, por sua vez, podem conduzir a perdas significativas na produtividade das plantas. Entretanto, no tomate (Solanum lycopersicum L.), é possível acessar um banco valioso de variação genética nas espécies selvagens relacionadas. Assim, a identificação de variantes genéticas associadas ao processo de domesticação do tomateiro e a mecanismos de resistência a estresses ambientais, os quais podem ter sido perdidos durante a domesticação, pode auxiliar em programas de melhoramento do tomateiro e de outras culturas de interesse comercial. Diante disso, no presente trabalho, o qual foi dividido em dois capítulos, realizamos cruzamentos entre a espécie selvagem, S. pennellii, e a cultivar miniatura de tomateiro Micro-Tom (MT) para criamos duas linhas de introgressão (ILs), uma com tamanho de órgão reduzido e outra com maior tolerância à seca. No primeiro capítulo, relatamos a caracterização e mapeamento da IL denominada como Tiny organs and reduced yield (Toy). O genótipo Toy carrega um segmento do genoma de S. pennellii no cromossomo 7 e apresenta uma considerável redução em órgãos vegetativos (folhas) e reprodutivos (frutos). Os resultados obtidos conduziram a uma discussão de como esse genótipo pode ser relevante para a domesticação do tomateiro, devido ao seu impacto no tamanho de diversos orgão. Por outro lado, no segundo capítulo, descrevemos o mecanismo de tolerância à seca da IL Water Economy Locus em Lycopersicon (Well). Plantas Well carregam um segmento do genoma de S. pennellii no cromossomo 1 e exibem uma menor condutância hidráulica, possivelmente relacionada ao tamanho reduzido do vaso xilemático. A menor condutância hidráulica do genótipo Well conduz a perturbações no contínuo solo/planta/atmosfera levando a mudança no comportamento estomático, que, por sua vez, provavelmente está relacionado a maior resistência ao murchamento apresentada por esse material em condições de déficit hídrico.