Made available in DSpace on 2017-07-10T17:40:52Z (GMT). No. of bitstreams: 1 Sidiane_Coltro_Roncato.pdf: 1950234 bytes, checksum: ab588be5ce6968a641dc1b750a09907e (MD5) Previous issue date: 2015-08-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this work was to study the effect of Crambe abyssinica leaf extract on Meloidogyne incognita and Meloidogyne javanica control in tomato plants, and the possible effect of allyl isothiocyanate component in this interaction. Five experiments were conducted. The first experiment was carried out with both Meloidogyne species in vitro: dried leaves of crambe (200 mg L-1) were added in the solvents: water for infusion and grinding, acetone, water + ethanol, methyl alcohol, hexane and chloroform. After the evaporation rotative, the aqueous extracts were analyzed in vitro for nematicide activity (hatching, mortality and motility of second stage juveniles (J2)). After the extracts with increased activity were tested at doses of 200, 300, 400 and 500 mg L-1 of crambe dry leaves. To quantify the allyl isothiocyanate in the extracts was used high performance liquid chromatography (HPLC). The second and third experiment were with M. incognita and hydroalcoholic extract to 250 mg L-1 was used, and each experiment was repeated twice, being the second time with the two best times of the first. The first repetition of the second experiment was conducted in factorial design (3x4+1) in a greenhouse, with three extract application routes: leaf; soil; leaf+soil, in four periods: before inoculation; on inoculation; after inoculation; and weekly up to 45 days and an additional treatment (inoculated and untreated control). In the second repetition, the factorial design was (3x2+1), three routes of application as mentioned above, and two periods (after inoculation and weekly). The third experiment includes the same treatments, however, to the application routes is added the immersion of the tomato roots. The fourth and fifth experiments were with M. javanica, using the same methodology described for the experiments with M. incognita. The results derived from the extracts of hydroalcoholic solution, methanol and trituration were more effective in the action against nematodes, however, the hydroalcoholic one caused higher mortality, with 93% and 64% for M. incognita and M. javanica, respectively. The greatest effect was on the dose of 250 mg L-1, and only the extracts obtained by methanol solution and the hydroalcoholic presented allyl isothiocyanate. When the hydroalcoholic extract was tested in a greenhouse in tomato plants, to M. incognita the applications via soil and weekly did not differ statistically from the applications soil+leaf for the analyzed variables. When the immersion of the tomato plants rooting system was added, weekly applications routes via root+soil and root+soil+leaf stood out, and nematode population in the soil was reduced by 75.55% and 71.95%, respectively, however, the second experiment confirmed the control only to the application via soil+root+leaf. For M. javanica, applications of the extract via soil+leaf weekly caused reduction of total galls on 46.15%. When added the immersion of the root system, the weekly application via root+soil+leaf reduced the egg masses and total galls on 60.95% and 27.95%, respectively. Therefore, crambe extract has potential to make up the integrated management of M. javanica and M. incognita on tomato plants / O objetivo deste trabalho foi estudar o efeito do extrato de folhas de Crambe abyssinica no controle de Meloidogyne incognita e Meloidogyne javanica em tomateiro e, possível atuação do componente alil isotiocianato nesta interação. Foram conduzidos cinco experimentos. O primeiro experimento foi realizado com ambas as espécies de Meloidogyne in vitro: folhas secas de crambe (200 mg L-1) foram adicionadas nos solventes: água para infusão e trituração, acetona, água+álcool etílico, álcool metílico, hexano e clorofórmio. Após a evaporação rotativa, os extratos aquosos foram analisados in vitro para atividade nematicida (eclosão, mortalidade e motilidade de juvenis de segundo estádio (J2)). Após, os extratos com maior atividade foram testados nas doses 200, 300, 400 e 500 mg L-1 de folhas secas de crambe. Para quantificar o alil isotiocianato nos extratos foi utilizada cromatografia líquida de alta eficiência (HPLC). O segundo e terceiro experimento foram com M. incognita e extrato hidroalcoólico à 250 mg L-1 foi utilizado e, cada experimento foi repetido duas vezes, sendo a segunda vez com as duas melhores épocas da primeira. A primeira repetição do segundo experimento foi conduzido em esquema fatorial (3x4+1) em casa de vegetação, com três vias de aplicação do extrato: foliar; solo; foliar+solo, em quatro épocas: antes da inoculação; na inoculação; após a inoculação; e semanalmente até 45 dias e um tratamento adicional (testemunha inoculada e não tratada). Na segunda repetição, o esquema fatorial foi (3x2+1), três vias de aplicação como citado anteriormente, e duas épocas (após a inoculação e semanalmente). O terceiro experimento contempla os mesmos tratamentos, porém, nas vias de aplicação é adicionado a imersão das raízes de tomateiro. O quarto e quinto experimentos foram com M. javanica, com a mesma metodologia descrita para os experimentos com M. incognita. Os resultados dos extratos originados da solução hidroalcoólica, metanólica e por trituração apresentaram-se mais efetivos na ação contra os nematoides, porém, o hidroalcoólico ocasionou maior mortalidade, com 93% e 64% para M. incognita e M. javanica, respectivamente. O maior efeito foi na dose de 250 mg L-1, e somente os extratos obtidos por solução metanólica e o hidroalcoólica apresentaram alil isotiocianato. Quando o extrato hidroalcoólico foi testado em casa de vegetação em plantas de tomateiro, para M. incognita as aplicações via solo e semanalmente não diferiram estatisticamente das aplicações via solo+folha para as variáveis analisadas. Quando foi adicionada a imersão do sistema radicular do tomateiro, aplicações semanais com as vias raiz+solo e raiz+solo+folha se destacaram, e a população do nematoide no solo foi reduzida em 75,55% e 71,95%, respectivamente, porém, o segundo experimento confirmou o controle apenas para a aplicação via raiz+solo+folha. Para M. javanica, aplicações do extrato via solo+folha semanalmente ocasionaram redução de galhas totais em 46,15%. Quando adicionada a imersão do sistema radicular, a aplicação semanal via raiz+solo+folha reduziu massa de ovos e galhas totais em 60,95% e 27,95%, respectivamente. Portanto, o extrato de crambe tem potencial para compor o manejo integrado de M. javanica e M. incognita em tomateiro

Atualmente, o HLB é considerado a principal doença que acomete as plantas cítricas. Diante desse fator, pesquisas por cultivares resistentes a esta doença são necessárias. A transformação genética via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verificação do funcionamento dos genes em resposta a patógenos, isto porque as plantas modelos possuem como característica ciclo de vida curto e alto poder de regeneração. Assim sendo, objetivou-se com o presente trabalho, a transformação genética via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as proteínas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotilédones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transformação genética com os genes PR-8 e CDR-1. Esses explantes foram subcultivados até o aparecimento de brotos regenerantes e posteriormente plantas transgênicas, as quais foram aclimatizadas e levadas a casa-de-vegetação. A transgenia foi confirmada por PCR e o número de inserções do gene por Southern blot. As plantas foram cultivadas até a obtenção da geração T1. Simultaneamente, foram realizados experimentos de transformação genética em segmentos de epicótilo, provenientes de sementes de laranja ‘Hamlin’ germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transgênicas e sua caracterização. Paralelamente, foi realizada a construção da curva padrão pela análise de qPCR para identificação de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transgênicas de tomate Micro-Tom com o gene PR-8 e três com o gene CDR-1. As eficiências de transformação foram em torno de 0,38 a 1,98%. Três plantas de tomate Micro-Tom transgênicas com o gene PR-8 foram caracterizadas por Southern blot e o número de inserções variou de 1 a 3. Dezenove plantas transgênicas de laranja ‘Hamlin’ com o gene CDR-1 foram obtidas através dos experimentos de transformação genética. A eficiência de transformação foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 número de cópia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

Tomato (Solanum lycopersicum L.) consumption has increased every year due to the fruit attractiveness, several utilizations, and beneficial effects for human health. However, tomato fruits can accumulate a Cd concentration that exceeds the safety threshold for human consumption of vegetables, even when plants are grown in soil with acceptable Cd level. Cd is a non-essential, hazardous element to biological systems, triggering several diseases in humans. In plants, Cd disturbs the antioxidant machinery, changes the nutritional status, and impairs the photoassimilate production and/or partitioning, hence reducing fruit yield and quality. However, distinct tomato accessions can present contrasting tolerance degree to Cd toxicity, as detected by our group in previous studies. The use of these accessions is a powerful approach to identify strategies employed by plants to cope with Cdinduced challenges, and the acknowledgement of such strategies can be potentially used in breeding and biotechnological programs to improve fruit yield and quality in crops that were cultivated in contaminated fields. The set of studies that compose the present thesis aimed (i) to identify the main mechanisms for the contrasting tolerance degree to Cd-induced toxicity in tomato accessions after short and long-term Cd exposure; (ii) to evaluate the relationship among tolerance degree and fruits attributes in plants that were grown in Cd-containing soil, and (iii) to determine the transgenerational effects of Cd-induced stress. In the first experiment, nine tomato accessions with a varied tolerance degree, which was based on biomass accumulation, to Cd exposure were grown in hydroponic solution containing CdCl2 35 μM for 6 days. Avoidance of high Mg concentration in roots was identified as a plant strategy to mitigate Cd toxicity by preventing formation of root hairs. Regarding the mode of action of Cd toxicity, Mn excess in leaves, in addition to the high Cd concentration per se, seems to be coupled to leaf damages that are enhanced by the increased Zn and B concentrations in the photosynthetic tissues. In the second experiment, tolerant (Yoshimatsu) and sensitive (Tropic Two Orders) genotypes were grown in Cd-containing soil, in order to evaluated production parameters. After plant exposure to Cd, the tolerant genotype presented an increased fruit diameter, height and weight, when compared to the control plants. In both cultivars, Cd concentration varied according to the following descending order: roots = leaf blade > (floral receptacle, peduncle and sepals) > stem = fruit peel = fruit pulp. Moreover, data suggested that floral receptacle and its related-structures acted as a barrier to the Cd transportation to the fruits, but it was not enough to avoid Cd reaching the fruits. Furthermore, Cd exposure provoked remarkable reductions in the Mg concentration in roots of sensitive and tolerant genotypes, revealing that both tomato cultivars are able to employ this mechanism for plant acclimation to long-term Cd exposure. Considering such information, it is possible that, under the short-term Cd exposure, tolerant accessions activate this mechanism either early or faster than sensitive genotypes. In addition, positive transgenerational effects on seed germination and vigor of the tolerant genotype were triggered by the plant-mother cultivation in Cd-containing media, despite of the increased chromosomal abnormality. This work reported new insights about the effects of Cd exposure on tomato development, tolerance mechanisms, fruit quality and yield of tomato, as well as Cd distribution in the plants. / O consumo de tomate (Solanum lycopersicum L.) tem aumentado a cada ano devido a atratividade dos frutos, suas diversas utilizações e efeitos benéficos para a saúde humana. No entanto, os frutos de tomate podem acumular uma concentração de cádmio (Cd) que excede o limiar de segurança para o consumo humano, mesmo quando as plantas são cultivadas em solo com níveis aceitáveis de Cd. Cádmio e um elemento não-essencial, extremamente perigoso para os sistemas biológicos, desencadeando varias doenças em seres humanos. Nas plantas, o Cd perturba a maquinaria antioxidante, altera o estado nutricional e prejudica a produção e /ou o particionamento de fotoassimilados, frequentemente reduzindo a produtividade e qualidade de frutos. No entanto, diferentes acessos de tomateiros podem apresentar contrastantes graus de tolerância a toxicidade gerada pela exposição ao Cd, como detectado em estudos anteriores de nosso grupo. O uso desses acessos e uma abordagem poderosa para identificar as estratégias empregadas pelas plantas para lidar com os desafios induzidos pelo Cd; e o conhecimento de tais estratégias pode ser potencialmente utilizado em programas biotecnológicos e de melhoramento genético. Deste modo, o conjunto de estudos que compõem a presente tese objetivou (i) identificar os principais mecanismos que suportam o grau de tolerância contrastante a toxicidade induzida por Cd em acessos de tomate após exposição a curto e longo prazos a este metal pesado; (ii) avaliar a relação entre o grau de tolerância e os atributos físico-químico de frutos oriundos de tomateiros cultivados em solo contendo Cd, e (iii) determinar os efeitos transgeracionais do estresse induzido por Cd. No primeiro experimento, nove acessos de tomateiro com graus variados de tolerância a exposição ao Cd, baseado na acumulação de biomassa, foram cultivados em solução hidropônica contendo 35 μM de CdCl2 durante 6 dias. O impedimento de elevada concentração de magnésio (Mg) em raízes foi identificado como possível estratégia da planta para mitigar a toxicidade de Cd, por meio da evitação da formação de pelos radiculares. Em relação ao modo de ação da toxicidade induzida por Cd, o excesso de Mn, em adição a elevada concentração de Cd, parece estar acoplado aos danos foliares que são acentuados ainda mais pelas altas concentrações de zinco (Zn) e boro (B) nos tecidos fotossintéticos de plantas sob exposição ao Cd. No segundo experimento, os genótipos tolerantes (Yoshimatsu) e sensíveis (Tropic Two Orders) foram cultivados em solo contendo Cd, a fim de avaliar os parâmetros de produção. O genótipo tolerante apresentou frutos com maior diâmetro, altura e peso após o cultivo em solo contendo Cd, quando comparado as plantas controle. Em ambas as cultivares, a concentração de Cd variou de acordo com a seguinte ordem descendente: raízes = folíolos> (receptáculo floral, pedúnculo e sépalas) > caule = casca de fruta = polpa de fruta. Alem disso, dados sugerem que o receptáculo floral e suas estruturas atuaram como uma barreira ao transporte de Cd para os frutos, entretanto, ela não foi suficiente para evitar que o Cd atingisse os frutos. Em adição, a exposição ao Cd provocou notáveis reduções na concentração de Mg nas raízes de genótipos sensíveis e tolerantes, revelando que a aclimatação das plantas depende do baixo status de Mg em tecidos radiculares. Desde que ambas as cultivares são capazes de empregar este mecanismo, os dados sugerem que, durante a exposição a curto prazo ao Cd, acessos tolerantes são capazes de ativa-lo ou mais cedo ou mais rápido do que acessos sensíveis. Ademais, efeitos transgeracionais positivos na germinação e vigor das sementes do genótipo tolerante foram desencadeados pelo cultivo planta-mãe em solo com Cd, apesar do aumento de anormalidades cromossômicas. Este trabalho reportou novos conhecimentos sobre os efeitos da exposição ao Cd sobre o desenvolvimento do tomateiro, mecanismos de tolerância, qualidade e rendimento de frutos, bem como a distribuição de Cd dentro da planta.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this work were determine the optimum plot size and the uniformity assay size for estimating the optimum plot size in order to evaluate the fresh fitomass of lettuce plants and the mass of fruits of pepper, tomato, snap bean and zucchini. Production data were collected in uniformity assay with lettuce, pepper, tomato, snap bean and zucchini conducted at the Departamento de Fitotecnia da Universidade Federal de Santa Maria. Inside the planting row were planned different uniformity assay sizes. For each uniformity assay size planned were performed 3.000 resamples with replacement and were estimated for each sample the coefficient of spatial autocorrelation of the first order (p), the variance (s2), the mean (m) and the optimum plot size (Xo) by the method of maximum curvature of the coefficient of variation model (CMCV). Posteriorly, were also calculated the minimum values, percentile 2,5%, mean, percentile 97,5% and maximum values of statistics p, s2, m and Xo. For Xo statistic, even was calculated the amplitude of the confidence interval of 95%. Finally, were determined the uniformity assay size, starting from the smaller uniformity assay size planned (three basic units) and considering as uniformity assay size the number of basic units from which the amplitude of the confidence interval of 95% of optimum plot size was minor or equal to two basic experimental units. The uniformity assay size influences the estimation of the optimum plot size for evaluate the fresh fitomass of lettuce plants and the mass of pepper, tomato, snap bean and zucchini fruits. Uniformity assay with 27 basic units (27 plants) are enough for estimate the optimum plot size for evaluate the fresh fitomass of lettuce plants with a confidence interval of 95% minor or equal to two basic experimental units. Uniformity assay with pepper with 29 basic units (29 plants) in plastic greenhouse, with tomato with 12 basic units (12 plants) in plastic tunnel and with snap bean with 21 basic units (42 plants) in plastic tunnel, are enough for estimate the optimum plot size for evaluate the mass of fruits with a confidence interval of 95% minor or equal to two basic experimental units. Uniformity assay with snap bean with 18 basic units (36 plants) and with zucchini with ten basic units (ten plants) in plastic greenhouse are enough for estimate the optimum plot size for evaluate the mass of fruits with a confidence interval of 95% minor or equal to three basic experimental units. It is recommended the use of parcels with six basic experimental units for evaluate the fresh fitomass of lettuce plants and parcels with seven, five, six and eight basic experimental units, respectively, for evaluate the mass of fruits of pepper, tomato, snap bean and zucchini. / O objetivo deste trabalho foi determinar o tamanho de parcela e o tamanho de ensaio de uniformidade para estimar o tamanho de parcela a fim de avaliar a fitomassa fresca de plantas de alface e a massa de frutos de pimentão, de tomateiro, de feijão-vagem e de abobrinha italiana. Dados de produção de alface, de pimentão, de tomateiro, de feijão-vagem e de abobrinha italiana foram coletados em ensaios de uniformidade realizados no Departamento de Fitotecnia da Universidade Federal de Santa Maria. Dentro de cada linha de cultivo foram planejados diferentes tamanhos de ensaios de uniformidade. Para cada tamanho de ensaio de uniformidade planejado foram realizadas 3.000 reamostragens com reposição e estimados para cada amostra o coeficiente de autocorrelação espacial de primeira ordem (p), a variância (s2), a média (m) e o tamanho de parcela (Xo) pelo método da curvatura máxima do modelo do coeficiente de variação (CMCV). Posteriormente, também foram calculados os valores mínimo, percentil 2,5%, média, percentil 97,5% e máximo das estatísticas p, s2, m, e Xo. Ainda, para a estatística Xo, calculou-se a amplitude do intervalo de confiança de 95%. Finalmente, determinou-se o tamanho de ensaio de uniformidade, partindo-se do menor tamanho de ensaio planejado (três unidades básicas) e considerando como tamanho de ensaio de uniformidade o número de unidades básicas a partir do qual a amplitude do intervalo de confiança de 95% foi menor ou igual a duas unidades experimentais básicas. O tamanho do ensaio de uniformidade influencia a estimativa do tamanho de parcela para avaliar a fitomassa fresca de plantas de alface e a massa de frutos de pimentão, de tomateiro, de feijão-vagem e de abobrinha italiana. Ensaios de uniformidade com 27 unidades básicas (27 plantas) são suficientes para estimar o tamanho de parcela para avaliar a fitomassa fresca de plantas de alface com amplitude do intervalo de confiança de 95% menor ou igual a duas unidades experimentais básicas. Ensaios de uniformidade de pimentão com 29 unidades básicas (29 plantas) em estufa plástica, de tomateiro com 12 unidades básicas (12 plantas) em túnel plástico e de feijão-vagem com 21 unidades básicas (42 plantas) em túnel plástico, são suficientes para estimar o tamanho de parcela para avaliar a massa de frutos com amplitude do intervalo de confiança de 95% menor ou igual a duas unidades experimentais básicas. Ensaios de uniformidade de feijão-vagem com 18 unidades básicas (36 plantas) e de abobrinha italiana com dez unidades básicas (dez plantas) em estufa plástica são suficientes para estimar o tamanho de parcela para avaliar a massa de frutos com amplitude do intervalo de confiança de 95% menor ou igual a três unidades experimentais básicas. Recomenda-se o uso de parcelas com seis unidades experimentais básicas para avaliar a fitomassa fresca de plantas de alface e com sete, cinco, seis e oito unidades experimentais básicas, respectivamente, para avaliar a massa de frutos de pimentão, de tomateiro, de feijão-vagem e de abobrinha italiana.

Les derniers acteurs de la voie de signalisation à l’éthylène sont des facteurs de transcription appelés ERF (Ethylene Response Factors). La connaissance de leur rôle spécifique dans la régulation des processus développementaux dépendant de l’éthylène reste limitée. Les travaux présentés dans la thèse concernent la caractérisation fonctionnelle du gène Sl-ERF.B3, un membre de cette grande famille de régulateurs transcriptonnels dans la tomate (Solanum lycopersicum). Utilisant une stratégie répresseur dominant ; il est montré en particulier que ce gène intervient dans la mise en place de la réponse à l’éthylène et dans le contrôle de la maturation du fruit. L’expression d’une construction ERF.B3-SRDX, une version chimérique de Sl-ERF.B3 fusionné à un domaine répresseur de type EAR, entraine des phénotypes pléotropiques aussi bien dans la signalisation de l’éthylène que dans le développement des parties végétatives et des organes reproducteurs. Ainsi, une altération de la triple réponse à l’éthylène est constatée chez les lignées transgéniques et au stade adulte, les plantes présentent des phénotypes d’épinastie des feuilles, de sénescence prématurée des fleurs et d’abscission accélérée des fruits. L’ensemble de ces observations est corrélée avec une modification de l’expression de gènes impliqués dans la biosynthèse et la réponse à l’éthylène. Ces données suggèrent que ERF.B3 intervient dans un mécanisme de rétro-control de la réponse à l’éthylène en agissant à la fois sur les gènes de biosynthèse et de signalisation de l’hormone. Au niveau du fruit, la sur-expression d’ERF.B3-SRDX entraine une modification du processus de maturation avec un retard notable de l’avènement de l’acquisition de la compétence à murir. Cependant, une fois la maturation initiée, elle s’accompagne d’une forte production d’éthylène et d’une accélération du ramollissement du fruit. A l’inverse, l’accumulation de pigment est inhibée par altération de la voie de biosynthèse des caroténoïdes. Ces données phénotypiques sont corrélées avec le niveau d’expression des gènes clés impliqués dans ces processus. Les résultats indiquent que dans les lignées transgéniques, il y a découplage de certaines caractéristiques de la maturation du fruit et permettent de mettre en lumière le rôle d’ERF.B3 dans la régulation des processus de développement dépendant de l’éthylène chez la tomate. / Ethylene Response Factors (ERFs) are known to be the last transcription factors of the ethylene transduction pathway. Their specific role in ethylene-dependent developmental processes remains poorly understood. This work demonstrated a specific role of Sl- ERF.B3, a member of the ERF gene family in tomato (Solanum lycopersicum), in mediating ethylene response and fruit ripening through a dominant repressor strategy. ERF.B3-SRDX dominant repressor etiolated seedlings displayed partial constitutive ethylene-response in the absence of ethylene and adult plants exhibited typical ethylenerelated alterations such as leaf epinasty, premature flower senescence and accelerated fruit abscission. The multiple symptoms related to enhanced ethylene sensitivity correlate with the altered expression of ethylene biosynthesis and signaling genes, suggesting the involvement of Sl-ERF.B3 in a feedback mechanism regulating components of ethylene production and response. In addition, over-expression of ERF.B3-SRDX in tomato results in alterations in both fruit morphology and ripening process. The attainment of competence to ripen is dramatically delayed in ERF.B3-SRDX fruits but once ripening proceeds it is associated with high climacteric ethylene production and enhanced fruit softening while pigment accumulation is strongly reduced. Moreover, a number of genes involved in the fruit ripening process showed expression pattern deviating from that of wild type. These data suggest a putative role of Sl-ERF.B3 in the transcriptional network underlying the ripening process and uncover a mean for uncoupling some of the main features of fruit ripening such as fruit softening and pigment accumulation. Overall, the study highlighted the importance of an ERF gene in ethylene-mediated developmental processes such as plant growth and fruit ripening.

Analyse fonctionnelle de la protéine Enhancer of Zeste, SlEZ2, chez la tomate, Solanum lycopersicumLes protéines Polycomb, initialement découvertes chez la drosophile, ont récemment caractérisées chez les plantes où elles remplissent des fonctions essentielles au cours du développement de la plante. Chez la drosophile, les protéines polycomb (PcG) agissent sous forme de trois complexes multi-protéiques : PRC1, PRC2 et PhoRC. Seulement, deux de ces complexes ont été identifiés chez les plantes : un orthologue fonctionnel du complexe PRC1 (PRC1-like) et PRC2. Le complexe PRC2 maintien la chromatine dans un état condensé et intervient dans le contrôle du développement des fleurs, des graines, des fruits et des feuilles. Chez la tomate Solanum lycopersicum, le complexe PRC2 est composé de trois protéines polycomb : SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste). Les protéines SlE(Z) portent l’activité histone méthyl transférase qui permet la mise en place de la marque répressive H3K27me3. Chez la plante modèle, Arabidopsis thaliana, cette marque joue un rôle essentiel au cours du développement de la plante Afin d’étudier le rôle du complexe PRC2 dans le développement du fruit et de la plante de tomate, et plus particulièrement de la protéine SlE(Z), nous avons identifié trois gènes codant les protéines SlE(Z) : SlEZ1, SlEZ2 et SlEZ3. Au laboratoire, il a récemment été montré que la protéine SlEZ1 intervient au cours du développement floral (How Kit et al., 2010). L’objectif de ce travail est de déterminer la fonction de la protéine SlEZ2 au cours du développement du fruit et de la plante de tomate. Pour cela, nous avons analysé des plantes transgéniques sous exprimant le gène SlEZ2, orthologue au gène CURLY LEAF d’A. thaliana, par stratégie RNAi. Ce travail indique que la protéine SlEZ2 est impliquée dans la croissance de la plante de tomate, ainsi que dans le développement des feuilles, des fleurs et des fruits. Les plantes transgéniques présentent des phénotypes pléiotropes tels que des fleurs et des feuilles modifiées, un fort taux d’avortement des fruits, des fruits de texture et de couleur altérées ainsi qu’une réduction de la taille des plantes. De plus, nous avons identifiés quatre gènes ciblés par la protéine SlEZ2 dont l’expression est dérégulée dans les feuilles. Il s’agit de deux gènes à MADS box, TAG1 et TAGL1, ainsi que de deux gènes KNOX, LeT6 et TKN4. / Functional analysis SlEZ2, a tomato Enhancer of zeste proteinPolycomb proteins, first discovered in Drosophila, have been identified in plants and play essential functions in plant development. In Drosophila, polycomb proteins (PcG) acts as a complex and three have been identified: PRC1, PRC2 and PhoRC. However, only two polycomb complexes have been identified in plants: like-PCR1 and PRC2. The PCR2 complex maintain chromatin in a closed state and control flower, seed, fruit and leaf development.In tomato Solanum lycopersicum, PRC2 is composed by three polycomb proteins SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste)(Enhancer of Zeste). SlE(Z) proteins have a methyltransferase activity that puts in place an repressive epigenetic mark a trimethylation of lysine 27 histone 3. In plant model, Arabidopsis thaliana, this mark plays an essential role in plant development but little is known about PRC2 role in plant and fruit development of tomato. In order to unravel the function of the E(z) protein in the control of tomato fruit and plant development, we have characterized three E(z) encoding genes, namely SlEz1, SlEz2 and SlEZ3. In a recent work, we reported that SlEZ1 protein plays a role in flower development (How Kit at al., 2010). The aim of this present study was to determine the function of the SlEZ2 protein in plant and fruit development. We present our results focusing on RNAi transgenic plants which underexpressed SlEZ2 gene, homologue of Curly Leaf Arabidopsis gene. This analysis indicates that SlEZ2 protein is implicated in tomato plant growth and affects also leaf, flower and fruit development. Phenotypes include abnormal flowers and leafs, fruit development abortion, altered fruit colour and texture and plant of reduced size. Moreover, we characterize four target genes of SlEZ2 genes in leaves which present a deregulated expression : TAG1, TAGL1, LeT6 and TKN4.

Au sein de l’unité mixte de recherche 619 de l’Institut National de Recherche Agronomique, le groupe « Organogénèse du Fruit et Endoréduplication » étudie les acteurs moléculaires prenant part au contrôle du cycle cellulaire dans le fruit de tomate. L’objet de la présente thèse est l’étude de l’inhibiteur du cycle cellulaire Kip-Related Protein, et son rôle durant le développement du fruit. Identification de motifs protéiques fonctionnels chez l’Inhibiteur de Kinase Cycline-Dependent SlKRP1 chez Solanum lycopersicum : Leur rôle dans les interactions avec des partenaires du cycle cellulaire Les Kip-related proteins (KRPs) jouent un rôle majeur dans la régulation du cycle cellulaire. Il a été montré qu’ils inhibent les complexes CDK/Cyclin et ainsi bloquent la progression du cycle cellulaire. Malgré leur manque d’homologie avec leurs homologues animaux au delà de leur motif de liaison CDK/Cyclin, localisé à l’extrémité C-terminal de la protéine dans les séquences de plante, des études antérieurs ont montré la présence de motifs conservés spécifiques aux plantes chez certaines KRPs. Nous n’avons cependant que peu d’information concernant leur fonction. Nous montrons ici que les KRPs sont distribués en deux sous groupes phylogénétiques, et que chaque sous-groupe dispose de courts motifs spécifiques conservés. Les KRPs du sous-groupe 1 disposent ainsi de six motifs conservés entre eux. Utilisant SlKRP1, qui appartient au sous-groupe 1, nous avons identifié des motifs responsables de la localisation de la protéine et de ses interactions protéine-protéine. Nous montrons que le motif 2 est responsable de l’interaction avec CSN5, une sous-unité du complexe signalosome, et que le motif 5 a un effet redondant avec le motif 3 pour ce qui est de la localisation sub-cellulaire de la protéine. Nous montrons de plus que SlKRP1 est capable de guider SlCDKA1 et SlCycD3;1 vers le noyau, et ce même en l’absence du motif de liaison CDK/Cycline précédemment référencé. Ce nouveau site d’interaction est probablement localisé dans la partie centrale de la séquence de SlKRP1. Ces résultats apportent de nouveaux indices quant au rôle de la partie encore méconnue de cette protéine. La surexpression de SlKRP1 dans le mésocarpe de tomate détruit la proportionnalité entre endoréduplication et taille cellulaire Le fruit est un organe spécialisé résultant du développement de l’ovaire après pollinisation et fertilisation, et qui offre un environnement adéquat pour la maturation des graines et leur dispersion. De part leur importance en nutrition humaine et leur importance économique, les espèces à fruit charnu ont été le sujet d’étude développementales principalement orientée vers la formation de l’ovaire, la mise à fruit et la maturation du fruit. La phase de croissance du fruit a été beaucoup moins étudiée, bien que la division cellulaire et la croissance cellulaire prenant place durant cette période soient cruciales à la détermination de la taille finale du fruit, ainsi que de sa masse et sa forme. Le développement du mésocarpe du fruit de tomate se déroule par la succession d’une phase de division cellulaire suivie d’une phase d’expansion cellulaire associée à l’endoréduplication, menant à la formation de cellules géantes (jusqu’à 0,5mm) avec des niveaux de ploïdie pouvant atteindre 256C. Bien qu’une relation évidente entre endoréduplication et croissance cellulaire ait été montrée par de nombreux exemples chez les plantes, le rôle exact de l’endoréduplication n’a toujours pas été élucidé, étant donné que la plupart des expériences induisant une modification du niveau d’endoréduplication dans la plante affectaient aussi la division cellulaire. Nous avons étudié la cinétique du dévelopement du mésocarpe de tomate au niveau morphologique et cytologique et avons étudié l’effet de la diminution du niveau d’endoréduplication sur le dévelopement du fruit en sur-exprimant l’inhibiteur du cycle cellulaire Kip-Related Protein 1 (SlKRP1) spécifiquement dans les cellules en croissance du mésocarpe de tomate. Nous montrons une proportionnalité directe entre endoréduplication et taille cellulaire durant le développement normal du fruit, ce qui nous a permis de construire un modèle de développement du mésocarpe définissant l’épaisseur du péricarpe en ne prenant en compte que le nombre de divisions cellulaires et le nombre de tours d’endoréduplication. De façon surprenante, les mésocarpes de tomate affectés dans leur niveau d’endoréduplication par la sur-expression de SlKRP1 ne sont pas affectés au niveau de la taille des cellules ou du fruit, ni dans leur contenu métabolique. Nos résultats démontrent pour la première fois qu’alors que le niveau de ploïdie est étroitement lié avec la taille des cellules et du fruit, l’endoréduplication n’est pas responsable de la croissance cellulaire du mésocarpe de tomate. / Within the Joint Research Unit 619 of the National Institute of Agronomic Research (INRA), the group "Organogenesis of the Fruit and endoreduplication" examines the molecular players involved in cell cycle control in tomato fruit. The purpose of this thesis is the study of the cell cycle inhibitor Kip-Related Protein and its role during fruit development. Identification of protein motifs in the functional inhibitor of Cyclin-Dependent Kinase in Solanum lycopersicum SlKRP1: Their role in interactions with partners in the cell cycle The Kip-related proteins (KRPs) play a major role in the regulation of cell cycle. It has been shown to inhibit the CDK / Cyclin and thus block cell cycle progression. Despite their lack of homology with their counterparts in animals beyond their binding motif CDK / Cyclin, located at the C-terminal protein sequences in the plant, previous studies have shown the presence of conserved motifs plant specific in some KRPs, but there is little information about their function. We show here that the KRPs are distributed into two phylogenetic groups, and that each subgroup has specific short conserved motifs. The KRPs from subgroup 1 have six conserved motifs. Using SlKRP1, which belongs to subgroup 1, we have identified the motifs responsible for the localization of the protein and protein-protein interactions. We demonstrate that the pattern 2 is responsible for the interaction with CSN5, a subunit of the signalosome complex, and that the motif 5 is redundant with motif 3 with respect to the sub-cellular localization of the protein. We also show that SlKRP1 is capable of guiding SlCDKA1 and SlCycD3; 1 to the nucleus, even in the absence of CDK / cyclin binding motif previously referenced. This new site of interaction is probably located in the central part of the sequence of SlKRP1. These results provide new clues about the role of the little-known part of this protein. Overexpression of SlKRP1 in tomato mesocarp disrupts the proportionality between endoreduplication and cell size The fruit is a specialized organ which results from the ovary after pollination and fertilization, and provides a suitable environment for seed maturation and dispersal. Because of their importance in human nutrition and economic importance, fleshy fruit species have been the subject of study mainly focused on the developmental formation of the ovary, fruit set and fruit ripening. The stage of fruit growth has been much less studied, although cell division and cell growth taking place during this period are crucial to determining the final size of the fruit, as well as its mass and shape. The development of tomato fruit mesocarp occurs by the estate of a phase of cell division followed by a phase of cell expansion associated with endoreduplication, leading to the formation of giant cells (up to 0.5 mm) with ploidy levels of up to 256C. Although a clear relationship between endoreduplication and cell growth has been shown by many examples in plants, the exact role of endoreduplication has still not been elucidated, since most of the experiments leading to a change in the level of endoreduplication in plants also affected cell division. We studied the kinetics of the development of tomato mesocarp morphologically and cytologically and studied the effect of the reduced level of endoreduplication in the development of the fruit over-expressing the cell cycle inhibitor Kip-Related Protein 1 (SlKRP1) specifically in the growing cells of the tomato mesocarp. We show a direct proportionality between endoreduplication and cell size during normal development of the fruit, which allowed us to build a model for development of mesocarp defining the thickness of the pericarp by taking into account the number of cell divisions and the number of rounds of endoreduplication. Surprisingly, the tomato mesocarps affected in their level of endoreduplication by over-expression of SlKRP1 are not affected in terms of cell size and fruit, or on their metabolic content. Our results demonstrate for the first time that while the level of ploidy is closely linked with cell size and fruit, endoreduplication is not responsible for the cell growth of tomato mesocarp.

No description available.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Cary L. Rivard / Grafting with inter-specific hybrid rootstock is effective for tomato (Solanum lycopersicum) growers looking to reduce soilborne disease organically and increase fruit yield in the Southeastern US. However, production with grafted tomatoes has not been tested in the Great Plains region of the US. Small-acreage growers would like to produce grafted plants themselves, but many have difficulty with propagation due to water stress in the scion post-grafting and/or high temperatures within healing chambers. Growers may be able to reduce water stress post-grafting by removing the upper portion of the shoot to reduce leaf surface area, but no data exist on the potential effects of this practice on mature plant yield. Five high tunnel and one open-field study were conducted in 2011 and 2012 to investigate yield effects related to the use of two rootstocks and shoot removal during the grafting procedure. Grafting significantly increased fruit yield in five of the six trials (P<0.05). The average yield increases by Maxifort and ‘Trooper Lite’ rootstocks were 53% and 51%, respectively, across all trials. In some trials shoot removal during the grafting process reduced yield and could depend upon rootstock vigor. Another series of experiments were performed testing the efficacy of shoot removal for graft survival during the healing period prior to field planting. Five healing chambers designs were evaluated, and no significant effects of treatment design were observed upon grafted seedling survival. Plants grafted with no chamber had success rates of 81% to 91%. Additionally, three grafting leaf removal techniques were studied, and a partial leaf removal method had significantly higher success rates as compared to fully foliated and defoliated plants (P<0.05). Partial leaf removal may be recommended as a way to reduce water stress in the plant, and could potentially be a way to simplify the grafting process for small-scale producers.

Realizaram-se dois experimentos, em condições de campo, com os objetivos de (a) determinar o efeito de sistemas de condução de plantas e de tutoramento sobre a produção e seus componentes em híbridos de tomate de mesa de hábito de crescimento indeterminado dos segmentos Salada Longa Vida (SLV) e Santa Cruz (SC) (Experimento 1), e (b) avaliar o efeito do espaçamento entre plantas no sistema de condução com uma única haste tutorada com fitilho sobre a produção e seus componentes para híbridos do segmento SLV (Experimento 2). No experimento 1 adotou-se o delineamento em blocos ao acaso com os tratamentos em esquema fatorial 5 x 2, e quatro repetições, sendo avaliados três híbridos SLV e dois SC em dois sistemas de condução de plantas. No experimento 2, o delineamento utilizado também foi em blocos ao acaso, com os tratamentos em esquema fatorial 2 x 4, e quatro repetições, avaliando-se dois híbridos SLV. Os experimentos foram conduzidos em Itatiba, SP, de julho de 2007 a janeiro de 2008, e de janeiro a julho de 2009. Foram consideradas todas as plantas de cada parcela e avaliaram-se o número total frutos (NTF), a produção total por parcela (PTP), a massa de frutos descartados (MFD), o número de frutos comerciais (NFC), a produção comercial (PCO), a produção por planta (PPP), a massa média dos frutos (MMF), a massa de frutos comercializáveis nas classes AAA, AA e A, para ambos os grupos varietais, bem com o número de frutos com rachaduras radiais (RAD), com rachaduras concêntricas (CON), com lóculo aberto (LAB), manchados (MAN), ocos (OCO) e deformados (DEF). O sistema de condução de plantas com duas hastes tutoradas com bambu proporcionou incremento de NTF, NFC e PPP, para ambos os segmentos em comparação à condução de uma única planta por cova com uma haste tutorada com fitilho. O sistema de condução com bambu, para ambos os segmentos, proporcionou menor MMF e a massa de frutos das classes AAA e A também foi menor em relação ao sistema com fitilho. O híbrido Forty mostrou menor MFD devido ao menor MAN no sistema de condução com bambu. Não houve diferença entre os dois sistemas de condução para PTP, PCO, LAB e a massa de frutos da classe AA, para os dois segmentos de mercado. No experimento 2, não houve diferença entre os híbridos estudados nas diferentes densidades de plantio para produção e para a maioria de seus componentes, exceto para PPP, onde Forty superou Valenty, e para MFD, em que Valenty superou Forty devido a maior freqüência de LAB. O menor espaçamento entre plantas proporcionou maior NTF, PTP, NFC, PCO e massa de frutos das classes AA e A para ambos os híbridos, bem como menor MFD, PPP, MMF, massa de frutos da classe AAA e número de frutos com RAD e MAN para ambos os híbridos. O maior espaçamento entre plantas contribuiu para menor ocorrência de frutos com LAB para ambos os híbridos. / Two experiments were conducted under field conditions with the objectives of (a) determining the effect of training and staking systems of plants on yield potential and yield components of fresh market tomato hybrids of indeterminate growth habit of varietal segments Salad Extended Shelf Life (SLV) and Santa Cruz (SC) (Experiment 1), and (b) evaluating the effect of spacing between plants in the single-stem training system with polypropylene wire on yield and yield components for hybrids of segment SLV (Experiment 2). In experiment 1 it was adopted the randomized block design with the treatments in a 5 x 2 factorial scheme, and four replications, being evaluated three SLV hybrids and two SC hybrids in two systems of plant training. In experiment 2, the experimental design used was also randomized blocks with the treatments in a 2 x 4 factorial scheme, and four replications, being evaluated two SLV hybrids. The experiments were conducted in Itatiba, SP, from July 2007 to January 2008, and from January to July 2009. All plants of each plot were considered and the total number of fruits (NTF), the total production per plot (PTP), the mass of discarded fruits (MFD), the number of commercial fruits (NFC), the commercial production (PCO), the production per plant (PPP), the average mass of fruits (MMF) and the marketable fruit mass of classes AAA, AA and A, for both segments were evaluated, as well as the number of fruits with radial cracking (RAD), concentric cracking (CON), open locules with exposed seed (LAB), blotchy appearance (MAN), puffiness (OCO), and deformed (DEF). It was found that, the training system with one plant per hole, conducted with two stems staked with bamboo, provided increment of NTF, NFC and PPP, for both segments in comparison to the system of a single plant per hole with one stem trained with a propylene wire. The staking system with bamboo, for both segments, provided a smaller MMF and the mass of fruits of classes AAA and A was also lower in relation to the system with a propylene wire. The hybrid Forty showed lower MFD due to lower MAN in the staking system with bamboo. It was not found any difference between the two handling systems for PTP, PCO, LAB and for the mass of the fruits of class AA, for the two market segments. In experiment 2, there was no difference between the hybrids studied in different planting densities for production and for the majority of its components, except for PPP, in which Forty was shown to be superior than Valenty, and for MFD, in which Valenty exceeded Forty due to the greater frequency of LAB. The smaller spacing between plants provided larger NTF, PTP, NFC, PCO, and a mass of the fruits of classes AA and A for both hybrids, as well as a smaller MFD, PPP, MMF, a mass of fruits class AAA, and also a lower number of fruits with RAD and MAN for both hybrids. It was also found that, for both hybrids, under wider spacing between plants the occurrence of fruits with LAB significantly decreased.