Atmospheric CO₂ enrichment is employed by greenhouse tomato growers to increase fruit yields, and CO₂ applications are managed according to atmospheric set points or CO₂ injection rates. These methods do not immediately focus on the targets of CO₂ applications: plant performance and the regulation of plant carbon status. This thesis explores several plant-based approaches that may have potential for use in the management of CO₂ in greenhouse tomato production. Three plant-based approaches to CO₂ management were explored in commercial and experimental tomato greenhouses. These were: (1) simulation modeling, (2) non-destructive analysis of growth and (3) the status of plant carbon reserves. A cost and benefit analysis (c/b) using simulation modeling was carried out using grower-collected greenhouse environment and yield data. Simulation modeling was useful for retrospectively determining c/b of several CO₂ scenarios. The model was effective in predicting long term yields, but not short term yield variations, which limits its application for CO₂ management. Non-destructive measures of growth: stem length and diameter, leaf area and fruit load were found to be too sluggish for daily CO₂ dosing decision-making. Finally, plants growing under CO₂ enrichment can deposit substantial carbon as starch in their leaves. Plant carbon status was evaluated by determining the spatial distribution of leaf starch in the shoot and by following its variation diurnally and after the onset of CO₂ enrichment. As starch is difficult to measure by a grower, leaf mass per unit area (LMA) was also monitored for assessment as a surrogate measure for starch. Leaves in positions 7 to 9 were identified as the most meaningful in the shoot to sample. Diurnal profiles indicated these leaves carryover substantial starch from one day to the next. Monitoring starch at its peak time of accumulation (14 h to 16 h), at sunset and sunrise will indicate how much the peak starch reserves are used overnight. If starch remains high between peak and sunrise the following day, then the plants are in a carbon-surplus state and CO₂ enrichment could be postponed. For upper canopy leaves LMA is substantially influenced by starch and thus is a promising surrogate.

Tomatoes (Lycopersicon esculentum Mill) are one of the most popular vegetable crops grown for fresh market and processing in the U.S. Grafting involves the uniting of a shoot or bud scion with a rootstock to form a compound plant, mainly for managing soil-borne diseases and increasing crop yield. The objectives were to examine the effects of reciprocal and self grafts on tomato fruits, number of fruits, weight, and quality of the cultivars, ‘Cherokee Purple’, ‘Mister Stripey’, ‘Crista’, and ‘Maxifort’. Grafted seedlings were planted at WKU Farm on raised beds, protected with red or black plastic mulch under drip irrigation system with regular supply of water. Matured fruits were harvested, weighed, and number of fruits from each plant recorded. The highest yielding combination was the scion ‘Cherokee purple’ on ‘Maxifort’ rootstock, which produced 304g and 745g heavier fruits than ‘Crista’ and ‘Mister Stripey’, respectively. The quality grade of ‘Crista’ was superior to ‘Cherokee Purple’ and ‘Mister Stripey’ while ‘Mister Stripey’ produced the greatest number of fruits but were of lower quality. Fruits from plants grown on red plastic mulch were significantly larger, heavier, and were of higher quality than those grown on black plastic mulch. However, plants grown on black plastic mulch produced significantly more fruits per plant. There was little advantage for self-grafting of ‘Cherokee Purple’ and ‘Crista’. However, ‘Mister Stripey’ was responsive to self-grafting and merits further investigation. The best rootstock was ‘Maxifort’ which produced the biggest, heaviest fruits of the best quality. ‘Cherokee Purple’ as a scion produced the largest and heaviest fruits, while ‘Crista’ produced the highest quality fruits. ‘Mister Stripey’ was the most prolific in terms of number of fruits per plant.

Atmospheric CO₂ enrichment is employed by greenhouse tomato growers to increase fruit yields, and CO₂ applications are managed according to atmospheric set points or CO₂ injection rates. These methods do not immediately focus on the targets of CO₂ applications: plant performance and the regulation of plant carbon status. This thesis explores several plant-based approaches that may have potential for use in the management of CO₂ in greenhouse tomato production. Three plant-based approaches to CO₂ management were explored in commercial and experimental tomato greenhouses. These were: (1) simulation modeling, (2) non-destructive analysis of growth and (3) the status of plant carbon reserves. A cost and benefit analysis (c/b) using simulation modeling was carried out using grower-collected greenhouse environment and yield data. Simulation modeling was useful for retrospectively determining c/b of several CO₂ scenarios. The model was effective in predicting long term yields, but not short term yield variations, which limits its application for CO₂ management. Non-destructive measures of growth: stem length and diameter, leaf area and fruit load were found to be too sluggish for daily CO₂ dosing decision-making. Finally, plants growing under CO₂ enrichment can deposit substantial carbon as starch in their leaves. Plant carbon status was evaluated by determining the spatial distribution of leaf starch in the shoot and by following its variation diurnally and after the onset of CO₂ enrichment. As starch is difficult to measure by a grower, leaf mass per unit area (LMA) was also monitored for assessment as a surrogate measure for starch. Leaves in positions 7 to 9 were identified as the most meaningful in the shoot to sample. Diurnal profiles indicated these leaves carryover substantial starch from one day to the next. Monitoring starch at its peak time of accumulation (14 h to 16 h), at sunset and sunrise will indicate how much the peak starch reserves are used overnight. If starch remains high between peak and sunrise the following day, then the plants are in a carbon-surplus state and CO₂ enrichment could be postponed. For upper canopy leaves LMA is substantially influenced by starch and thus is a promising surrogate.

Atmospheric CO₂ enrichment is employed by greenhouse tomato growers to increase fruit yields, and CO₂ applications are managed according to atmospheric set points or CO₂ injection rates. These methods do not immediately focus on the targets of CO₂ applications: plant performance and the regulation of plant carbon status. This thesis explores several plant-based approaches that may have potential for use in the management of CO₂ in greenhouse tomato production. Three plant-based approaches to CO₂ management were explored in commercial and experimental tomato greenhouses. These were: (1) simulation modeling, (2) non-destructive analysis of growth and (3) the status of plant carbon reserves. A cost and benefit analysis (c/b) using simulation modeling was carried out using grower-collected greenhouse environment and yield data. Simulation modeling was useful for retrospectively determining c/b of several CO₂ scenarios. The model was effective in predicting long term yields, but not short term yield variations, which limits its application for CO₂ management. Non-destructive measures of growth: stem length and diameter, leaf area and fruit load were found to be too sluggish for daily CO₂ dosing decision-making. Finally, plants growing under CO₂ enrichment can deposit substantial carbon as starch in their leaves. Plant carbon status was evaluated by determining the spatial distribution of leaf starch in the shoot and by following its variation diurnally and after the onset of CO₂ enrichment. As starch is difficult to measure by a grower, leaf mass per unit area (LMA) was also monitored for assessment as a surrogate measure for starch. Leaves in positions 7 to 9 were identified as the most meaningful in the shoot to sample. Diurnal profiles indicated these leaves carryover substantial starch from one day to the next. Monitoring starch at its peak time of accumulation (14 h to 16 h), at sunset and sunrise will indicate how much the peak starch reserves are used overnight. If starch remains high between peak and sunrise the following day, then the plants are in a carbon-surplus state and CO₂ enrichment could be postponed. For upper canopy leaves LMA is substantially influenced by starch and thus is a promising surrogate. / Land and Food Systems, Faculty of / Graduate

Les fruits et légumes constituent la principale source de vitamine C dans l’alimentation humaine, mais leur concentration en vitamine C varie fortement en fonction de la saison et des conditions de culture. Au cours de cette thèse, nous avons testé successivement différentes hypothèses afin de mettre en évidence le rôle de la lumière dans ces variations, en prenant comme modèle le fruit de tomate. Nous avons supposé i) un effet direct du rayonnement intercepté par les fruits sur le métabolisme de la vitamine C (synthèse, recyclage, dégradation), ou ii) un effet du rayonnement sur les feuilles augmentant le transport de molécules (sucres, vitamine C, …) favorisant l’accumulation de vitamine C dans le fruit. Nos résultats ont souligné la complexité de la régulation de la teneur en vitamine C dans les fruits par la lumière, qui dépend principalement du microclimat radiatif du fruit et dans une moindre mesure du rayonnement intercepté par les feuilles, en interaction avec le stade de développement du fruit. L’étude de la relation sucres/vitamine C dans les fruits a montré que les sucres n’étaient pas limitants pour la synthèse de vitamine C. L’impact du rayonnement sur le métabolisme de la vitamine C a été étudié, en interaction avec la température, sur fruits détachés. Le rayonnement augmente les teneurs en vitamine C des fruits pour les températures inférieures ou égales à 23°C en liaison avec l’augmentation des expressions des gènes de la voie de biosynthèse et des activités des enzymes de recyclage, particulièrement à 12°C. À forte température (31°C), la lumière ne modifie pas la teneur en vitamine C du fruit malgré l’augmentation de l’expression de certains gènes de la voie de synthèse, mais on observe une diminution du recyclage de la vitamine C (DHAR) et une augmentation d’un produit de dégradation de la vitamine C (thréonate). Les données recueillies ont permis d’initier un modèle d’accumulation de la vitamine C au cours du développement du fruit qui dans le futur prendra en compte les facteurs de l’environnement / Fruits and vegetables are the major source of vitamin C in human diet; however, their vitamin C content varies with environmental conditions and agricultural practices. In this work, we successively tested different hypotheses concerning light impact on these variations, using tomato fruit as model. We hypothesized that i) light reaching fruit could have a direct impact on vitamin C metabolism (synthesis, recycling and degradation) or that ii) light reaching leaves could increase the transport of molecules triggering vitamin C accumulation in fruit (sugars, vitamin C…). Our results showed that vitamin C variations with light are complex and depend mostly on light reaching the fruit and to a lesser extent on light reaching leaves,according to fruit developmental stage. The study of vitamin C/sugars relationship in fruit showed that sugars were not determinant in explaining variations in vitamin C. Light impact on vitamin C metabolism were studied, in interaction with temperature, on off-vine fruit ripening. Light increased fruit vitamin C content for temperature lower or equal to 23°C byincreasing transcripts of vitamin C biosynthetic pathway and activity of vitamin C recycling enzyme, particularly at low temperature (12°C). At high temperature (31°C), light did not increase fruit vitamin C content but it decreased DHAR activity and increased threonate content likely produced from vitamin C degradation. The data obtained were used to initiate the building of a model describing vitamin C content during fruit development that will integrate environmental factors in the future

Las proteínas pequeñas de choque térmico (sHSP) responden a una amplia variedad de estreses ambientales, estabilizando proteínas parcialmente desplegadas y evitando su agregación irreversible, en forma independiente de ATP. En plantas, las sHSPs son especialmente diversas siendo las sHSPs de organelas una característica única de las plantas. La estructura primaria de las sHSP incluye una secuencia N-terminal no conservada de longitud variable, un dominio α-cristalino conservado (ACD) y una secuencia C-terminal corta no conservada. El dominio ACD representa una característica conservada presente en todas las sHSPs, sin embargo, no todas las proteínas que contienen un dominio ACD son sHSP. Las sHSPs pertenecen a una gran superfamilia, siendo su importancia funcional y fisiológica en gran parte desconocida. El objetivo de este trabajo fue dilucidar el rol de sHSPs de localización mitocondrial (sHSPs-M) en Arabidopsis thaliana y Solanum lycopersicum en situaciones de estrés ambiental, y caracterizar probables promotores bidireccionales que regulan la expresión de genes codificantes de proteínas con dominio ACD con orientación cabeza a cabeza en el genoma de Arabidopsis. Este trabajo cubre aspectos desde la organización genómica y la función de sHSPs-M en Arabidopsis hasta el rol de las sHSPs-M en la respuesta al estrés por frío de frutos de tomate. Para ello, se generaron plantas mutantes de Arabidopsis y de tomate utilizando la tecnología de silenciamiento génico por micro ARNs artificiales. Las plantas mutantes fueron analizadas en su proteoma, metaboloma y lipidoma en distintas condiciones de estrés. A continuación, se presenta un resumen de los resultados obtenidos. En la primera parte de este trabajo, se realizó la caracterización funcional de genes con orientación cabeza a cabeza en el genoma de Arabidopsis, que codifican para proteínas con ACD y las regiones intergénicas correspondientes. Se lograron identificar y caracterizar cuatro distintos promotores bidireccionales, entre ellos el promotor del gen At5g51440 que codifica una sHSP de localización mitocondrial (sHSP23.5). Los resultados obtenidos sugieren que el promotor bidireccional contenido en el par At5g51430-At5g51440 es fuertemente inducido por altas temperaturas en una dirección, pero no así en la dirección opuesta. El promotor contenido en el par At1g06460-At1g06470 mostró una actividad alta en ambas direcciones, teniendo por ello, un alto potencial de aplicación en ingeniería genética. Los dos promotores restantes mostraron mayor actividad en una dirección y, por lo tanto, pueden ser considerados como promotores bidireccionales asimétricos. El estudio funcional de los promotores seleccionados reveló el potencial biotecnológico de los mismos ya que pueden ser inducidos específicamente en una determinada condición (como altas temperaturas) en una o en ambas direcciones cuando sea necesario. En la segunda parte, se presenta la caracterización funcional de sHSP mitocondriales en condiciones de estrés y durante el desarrollo de Arabidopsis. Se identificaron tres genes parálogos en el genoma de Arabidopsis (At5g51440, At4g25200, y At1g52560), y se diseñaron microARN artificiales específicos para la obtención de plantas mutantes por silenciamiento (amiR simple, doble y triple). Las plantas amiR simples y dobles (para sHSP23.5 y sHSP23.6) no mostraron un fenotipo evidentemente afectado, probablemente debido a la compensación o redundancia funcional de las sHSP mitocondriales. Por otro lado, las plantas triple mutante amiR23.5/23.6/26.5 muestran un fenotipo alterado en las etapas vegetativa y reproductiva. Estas plantas presentan hojas pequeñas, células epidérmicas con áreas reducidas, pero no reducción en el número de células epidérmicas por hoja. Además, exhiben hojas cloróticas, raíces cortas y menor producción de semillas en comparación con las plantas Col-0. Las plantas triple amiR son considerablemente pequeñas debido a una alteración en el proceso de expansión celular, pero no en la proliferación celular, lo que indica una profunda alteración en el programa de desarrollo de la planta. En el análisis proteómico de las mutantes amiR se observó un aumento significativo de proteínas implicadas en el metabolismo, y alteración en la abundancia de varias proteínas relacionadas con la traducción, y con el funcionamiento y la estructura de los ribosomas. La triple mutante exhibió un mayor número de proteínas con abundancia alterada involucradas en estos procesos en comparación con las mutantes simples y doble amiR23.5/23.6. Estos cambios tan amplios observados en proteínas relacionadas con el funcionamiento de los ribosomas sugieren una posible alteración en la función normal de los mismos. Los resultados presentados en este trabajo proporcionan evidencias sobre el importante rol de las sHSPs-M no sólo en la respuesta a las altas temperaturas, sino también durante el desarrollo de la planta de Arabidopsis. Los resultados indican que una compensación funcional podría ser responsable del fenotipo observado en las plantas mutantes con niveles reducidos de cada sHSPs-M individual. Sin embargo, la reducción simultanea de las tres sHSPs-M causó una profunda alteración en la función normal de mitocondrias y ribosomas, afectando gravemente el metabolismo energético y la homeostasis celular, lo que llevó a alteraciones en el desarrollo correcto de la planta. En la última parte de este trabajo, las consecuencias de la disminución de la proteína sHSP23.8 de localización mitocondrial en frutos de tomate fueron investigadas. Los frutos fueron analizados en su fenotipo y en la susceptibilidad al daño por frío. Los frutos de las plantas mutantes amiR23.8 que fueron conservados en frío mostraron mayor pérdida de agua y de electrolitos en el tejido pericárpico en comparación con frutos WT. El deterioro observado en los frutos amiR23.8 indica que estos frutos mutantes son mayormente susceptibles al estrés por frío desarrollando síntomas de daño por frío. El lipidoma de los frutos amiR23.8 frigoconservados mostró cantidades alteradas de glicerolípidos, y los niveles de lípidos saturados en amiR23.8 disminuyeron luego del tratamiento con frío, pero no por debajo de los niveles encontrados en frutos WT en condiciones normales. Lo opuesto se encontró en el porcentaje relativo de lípidos insaturados, con niveles significativamente más bajos de insaturaciones en frutos amiR23.8 en condiciones normales y después del enfriamiento. Los resultados presentados indican una degradación diferencial de lípidos extraplastídicos y plastídicos en frutos amiR23.8, y alteraciones en la remodelación del lipidoma luego del estrés por frío, lo que podría conducir a una mayor sensibilidad al daño por frío. Los resultados discutidos aquí indican que la proteína sHSP23.8 podría estar directamente involucrada en los mecanismos de protección contra el estrés por frío en frutos de tomate.

Made available in DSpace on 2015-03-26T13:30:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 406033 bytes, checksum: e60f5f4a8043be1ed0638012130590c6 (MD5) Previous issue date: 2006-02-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The tomato fruit borer Neoleucinodes elegantalis (Guenée) (Lepidoptera: Crambidae) is considered a key pest in tomato crops in Brazil. However, there is not a sampling plan for this insect to compose decision-making systems of IPM programs. Thus, the objective of this work was to determine the Conventional sampling plan for N. elegalis. This work was carried out in twenty commercial tomato crops from march to april 2005 in Coimbra, MG state. The crops were divided in two phenological stages: plants with up to three tomato clusters and plants with more than three tomato clusters. The studied variables were eggs/fruit, egg mass/fruit, fruits with eggs (%), eggs/fruit with diameter [< or =] 4,0 cm, egg mass/fruit with diameter [< or =] 4,0 cm, percentage of fruits with eggs with diameter [< or =] 4,0 cm and clusters with eggs (%). It was studied the frequency distribution of the data for each variable. The choice of the variable to be used in the sampling plan was based on the cost and time required by the sampling plan. Once, the best variable was chosen, the numbers of samples to compose the sampling plan was determined. The data of the variable eggs/fruit adjusted to the negative binomial distribution and egg mass/fruit adjusted to the Poisson distribution in plants with up to three clusters. The data of the variable eggs/fruit in plants with more than three clusters adjusted to the Poisson distribution. The others data did not adjusted to any of the tested frequency distributions. The more adequate variable for the egg sampling of N. elegantalis was the percentage of clusters with eggs since it enables sampling plans of lesser cost and requires less sampling time. The sampling of N. elegantalis in plants with up to three clusters must be carried through evaluating the percentage of clusters with eggs in the basal cluster of 42 plants. This sampling plan requires a time of 0,15 hours and cost of R$ 1,83/sampling. Nevertheless, in crops where the plants possess more than three clusters, the sampling of this pest must be carried through evaluating the percentage of clusters with eggs in the 2º and 3º apicals clusters of 36 plants. This sampling plan requires a time of 0,24 hours and cost of R$2,03/sampling. / A broca pequena do tomateiro Neoleucinodes elegantalis (Guenée) (Lepidoptera: Crambidae) é considerada uma praga-chave desta cultura. Entretanto, não existe até o momento, plano de amostragem para este inseto a compor sistemas de tomada de decisão de programas de manejo integrado de pragas. Assim, o objetivo desta pesquisa foi determinar o plano de amostragem convencional para N. elegalis. Este trabalho foi realizado em vinte lavouras comerciais de tomate de março a abril de 2005 em Coimbra, MG. As lavouras foram divididas em dois estádios fenólogicos: plantas com até três cachos e com mais de três cachos. As variáveis estudadas foram ovos/fruto, massa de ovos/fruto, frutos com ovos (%), ovos/fruto com diâmetro [< ou = ]4,0 cm, massa de ovos/fruto com diâmetro [< ou =] 4,0 cm, percentagem de frutos com diâmetro [< ou =] 4,0 cm com ovos e cachos com ovos (%). Estudou-se a distribuição de freqüência dos dados de cada variável. A seleção da variável a ser usada no plano de amostragem foi baseada no custo e tempo requeridos pelo plano de amostragem. De posse da melhor variável, determinou-se os números de amostras a compor os planos de amostragem. Os dados da variável ovos/fruto ajustaram-se à distribuição binomial negativa e massa de ovos/fruto, à distribuição de Poisson em plantas com até três cachos. Os dados da variável número de ovos/fruto em plantas com mais de três cachos ajustaram-se a distribuição de Poisson. Os dados das demais variáveis não se ajustaram a nenhuma das distribuições de freqüência testadas. A variável mais adequada para a amostragem de ovos N. elegantalis foi a percentagem de cachos com ovos, por gerar planos de amostragem de menor custo e que requerem menor tempo de amostragem. A amostragem de N. elegantalis em plantas com até três cachos deve ser realizada avaliando-se a percentagem de cachos com ovos no cacho mais baixeiro de 42 plantas. Este plano de amostragem requer um tempo de 0,15 h e custo de R$ 1,83/amostragem. Já em lavouras em que as plantas possuam mais de três cachos a amostragem deste inseto-praga deve ser realizada avaliando-se a percentagem de cachos com ovos no 2º e 3º cachos mais apicais de 36 plantas. Este plano de amostragem requer um tempo de 0,24 h e custo de R$ 2,03/amostragem.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-02T13:08:37Z No. of bitstreams: 1 Wendel Jose Teles Pontes.pdf: 931365 bytes, checksum: 3be37ee2d65cbbb102520601b95203cb (MD5) / Made available in DSpace on 2016-12-02T13:08:37Z (GMT). No. of bitstreams: 1 Wendel Jose Teles Pontes.pdf: 931365 bytes, checksum: 3be37ee2d65cbbb102520601b95203cb (MD5) Previous issue date: 2010-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / he reproduction in insects is affected by a wide array of factors that acts directly on the reproductive output, related with offspring quality and quantity: the interaction, by physical, chemical and visual cues, between insects and host plants; the larval growth rate affecting adult size and mating rate, as a mechanism of reproductive sucess. Thus, studies on insect reproduction are recommended to help estimate and to understand population dynamics, for both endangered species and to control crop pests. The tomato fruit borer Neoleucinodes elegantalis Guenée (Lepidoptera: Crambidae) is one of the most important pest species on Solanaceae in Brazil, and the aims of this works is to study this species regarding to: (i) the role of physical, chemical and visual cues on oviposition; (ii) the causes of sexual dimorphism and (iii) how male mating history affects his ability to obtain new matings. This work also propose an equation that can help to estimate the initial size of fragmented spermatophores found inside the reproductive tracts of females, based on measures of the fragments. The results showed: (i) that physical and chemical cues increase significantly the oviposition of N. elegantalis, as well showed that light intensityalso affect oviposition; (ii) that differences in dayly growth rate is the cause of the observedsexual dimorphism in this species, and (iii) that recently mated males are equally able to achieve new matings as virgin ones, within at least 24-h period. / A reprodução em insetos depende de diversos fatores que afetam diretamente o sucesso reprodutivo, em relação à qualidade e quantidade da progênie: a interação mediada por sinais químicos, físicos e visuais, entre o inseto e seu hospedeiro; taxa de crescimento larval relacionado com o tamanho final do adulto e a frequência de cópulas, como mecanismo de sucesso reprodutivo. Portanto, o estudo da reprodução de insetos é recomendado para se estimar e compreender a dinâmica de populações, tanto para a preservação de espécies em extinção, como para o controle de pragas agrícolas. A broca-pequena-do-tomateiro Neoleucinodes elegantalis Guenée (Lepidoptera: Crambidae) é uma das pragas mais importantes das solanáceas no Brasil, o que estimula o desenvolvimento de qualquer estudo voltado para a melhor compreensão de sua biologia e dinâmica populacional. Assim o objetivo deste trabalho é estudar (i) o efeito dos sinais físicos, químicos e visuais que afetam sua oviposição; (ii) as causas do seu dimorfismo sexual e (iii) o efeito da experiência de cópula de machos na sua capacidade de obter novosacasalamentos. Este trabalho ainda propõe uma equação que possa auxiliar em estimar o tamanho inicial de um espermatóforo já fragmentado, baseado nas medidas do fragmento encontrado no trato reprodutivo das fêmeas. Os resultados demonstram que: (i) os sinais físicos e químicos oferecidas estimulam significativamente a oviposição de N. elegantalis, bem como revela quepistas visuais afetam a oviposição; (ii) que a diferença na taxa diária de crescimento larval é responsável pelo dimorfismo sexual nesta espécie e (iii) que machos recém-copulados têm a mesma chance de conseguir uma nova cópula que um macho virgem, num período de 24 horas.

L’acide ascorbique (AsA) est un antioxydant essentiel à la fois pour l’homme et les végétaux. L’AsA provenant des plantes représente la source principale de vitamine C dans l’alimentation quotidienne. Au-delà de son impact nutritionnel, augmenter la teneur en AsA dans le fruit de tomate serait susceptible d’influencer la qualité des fruits après la récolte, en termes de conservation mais également de résistance à des pathogènes. Bien que le métabolisme de l’AsA soit bien caractérisé, les mécanismes impliqués dans sa régulation restent jusqu'à présent peu compris. Des études récentes menées sur des feuilles d’Arabisdopsis thaliana montrent que certaines protéines seraient capables de réguler la teneur en AsA, en agissant au niveau transcriptionnel ou post-transcriptionnel. A ce jour, ce type de régulation n’a pas été encore décrit chez les fruits. Dans ce but, une approche de génétique directe a été développée afin d’étudier les mécanismes impliqués dans la régulation de la teneur en AsA et ceci dans le fruit de tomate (Solanum lycopersicum). L’analyse d’une population de mutants EMS de tomate Micro-Tom a permis l’identification de lignées de mutants présentant des teneurs en AsA de 2,5 à 4 fois plus importantes que celles observées dans les fruits de tomate sauvage. La caractérisation de ces lignées a conduit à des résultats prometteurs pour l’étude de la qualité des fruits après la récolte. Une stratégie de NGS-mapping a permis l’identification des mutations causales responsables du phénotype AsA observé. Ainsi, le criblage de mutants EMS a permis la découverte de nouvelles protéines inattendues, permettant de confirmer au niveau moléculaire l’existence d’une interaction directe en la signalisation lumineuse et la régulation de la voie de biosynthèse de l’AsA. / The ascorbic acid (AsA) is an essential antioxidant in both plants and humans. Plant-derived AsA is the major source of vitamin C in the human diet. In addition to its effect on tomato nutritional value, increasing tomato AsA content would likely affect postharvest storage and resistance to pathogens of the fruit. While AsA metabolism is well characterized, the mechanisms involved in its regulation remain poorly understood. Recent studies in Arabidopsis leaves indicate that few regulatory proteins can regulate this pathway at transcriptional and post-transcriptional levels. Still nothing equivalent has been described in fruits. In that aim, a forward genetic approach has been carried out to investigate the regulation of AsA in tomato (Solanum lycopersicum) fruit. The screening of an EMS tomato mutant population in the miniature cultivar Micro-Tom for identifying mutant lines with AsA-enriched fruits was done. Among the 500 M2 mutant families screened, four mutant lines with higher AsA content ranging from 2.5 to 4 fold were selected. These mutant lines have been characterized for postharvest traits quality and showed promising results. A method based on NGS-mapping allowed the identification of the putative AsA-enriched related gene. Thus, the screening of EMS mutants led to original findings such as the discovery of new unexpected proteins regulating AsA in plants, and particularly in fruits. Our work confirms at the molecular level the direct interaction between light signaling component and the regulation of the AsA biosynthesis pathway.

Les interactions entre l’auxine et l’éthylène sont complexes et contrôlent divers processus de développement des plantes tels que l’élongation racinaire ou la différentiation des racines secondaires. Mais, il existe peu d’études montrant le rôle des interactions entre ces deux hormones au cours du développement et de la maturation des fruits. Le changement de couleur des fruits chez la tomate est une caractéristique de la maturation qui est associée à la fois à la dégradation des chlorophylles et à l’accumulation des caroténoïdes. Dans ce travail, l’application exogène d’auxine et d’éthylène a montré l’impact de ces deux hormones sur la maturation de la tomate et en particulier sur le changement de couleur des fruits. Nous avons montré que l’acide indol-acétique (IAA) retarde la transition du vert à l’orange/rouge, alors que l’éthylène, apporté sous la forme d’acide 1-aminocyclopropane-1-carboxylique (ACC), son précurseur, accélère la coloration des fruits. Par contre, l’inhibition de l’auxine par le PCIB, un antagoniste de l’auxine, provoque les mêmes effets que l’éthylène. L’analyse des caroténoïdes montre que l’ACC comme le PCIB augmente la teneur en lycopène et diminue la teneur en carotène alors que l’IAA provoque l’effet inverse. L’étude de l’accumulation des ARNs messagers de plusieurs gènes clés de la voie de biosynthèse des caroténoïdes a montré que le gène β-lcy codant pour la lycopène cyclase joue un rôle clé dans le contrôle de la biosynthèse et de l’accumulation des pigments et que son expression est fortement dépendante de l’équilibre auxine-éthylène. D’autre part, nos résultats ont montré que le gène rin joue un rôle important dans le contrôle de l’expression des gènes clés de la voie de biosynthèse des caroténoïdes. Pour avoir une meilleure vision des gènes différentiellement exprimés par l’auxine et l’éthylène au cours de la maturation, l’analyse du transcriptome des fruits traités par de l’ACC et de l’IAA a été réalisée par RNA-Seq au laboratoire. Parmi les facteurs de transcriptions étudiés, le gène Sl-IAA17, un membre de la famille des AUX/IAA, est fortement affecté par l’auxine et l’éthylène. La caractérisation fonctionnelle du gène Sl-IAA17 pendant le développement du fruit a été réalisée en créant des lignées transgéniques sous exprimant ce gène en mettant en œuvre la stratégie des ARNs interférents. Ces lignées présentent un phénotype caractéristique produisant des fruits de plus gros calibre que celui des fruits sauvages. Les analyses histologiques des tissus des fruits ont montré que ce phénotype est associé à un péricarpe plus épais. En microscopie, nous avons constaté que l’augmentation de l’épaisseur du péricarpe dans les lignées transgéniques n’était pas due à un plus grand nombre de cellules mais à l’augmentation de la taille des cellules. Enfin, nous avons observé que l’expansion des cellules dans les fruits transgéniques est étroitement couplée avec des niveaux de ploïdie plus élevés que dans les fruits sauvages, ce qui suggère une stimulation du processus endoréduplication. Ces résultats démontrent très clairement l’existence d’une étroite relation entre la signalisation de l’auxine, le contrôle de la taille du volume cellulaire et le processus d’endoréduplication. En conclusion, les résultats présentés fournissent des connaissances nouvelles sur les interactions entre l’auxine et l’éthylène au cours du développement du fruit et en particulier au cours de la transition fruit immature - fruit mature. De plus, ils apportent des éléments nouveaux sur la connaissance du rôle de la voie de signalisation de l’auxine dans le contrôle du développement des fruits charnus et en particulier sur la fonction de certains membres des AUX/IAA sur la détermination du volume et du poids des fruits. / The interaction between auxin and ethylene are complex and control various processes of plant development, such as root elongation or differentiation of secondary roots. But there are few studies showing the role of interactions between these two hormones during development and maturation of the fruit. The color change in the tomato fruit is a feature of the maturation that is associated with the degradation of the chlorophyll and carotenoid accumulation. In this work, the application of exogenous auxin and ethylene showed the impact of these two hormones in the tomato ripening and in particular the change of fruit color. We have shown that indole-acetic acid (IAA) delays the transition from green to orange / red, while ethylene, supplied as 1-aminocyclopropane-1-carboxylic acid form (ACC), its precursor, accelerated this transition. However the auxin inhibition by p-chlorophenoxy isobutyic acid (PCIB), an auxin antagonist, caused the same effects similar to ethylene. The carotenoid analysis showed that the ACC and PCIB increase the lycopene content and reduced the carotene content while IAA causes the opposite effect. The study of the accumulation of mRNAs for several key genes of the carotenoid biosynthetic pathway has shown that the gene β-lcy encoding lycopene cyclase plays a key role in the control of biosynthesis and accumulation of pigments and that its expression is highly dependent on the auxin-ethylene balance. In addition, our results showed that the rin gene plays an important role in controlling the expression of the key carotenoid biosynthetic pathway genes. To get a better view of differentially expressed genes by auxin and ethylene during ripening, transcriptome analysis of fruits treated with ACC and IAA was performed by a preliminary RNA-Seq approach. Among the transcription factors studied in the laboratory, the gene Sl-IAA17, a member of the family of Aux/IAA was affected by auxin and ethylene. Functional characterization of Sl-IAA17 gene during fruit development was performed by creating transgenic lines under-expressing this gene by RNAi. These lines display a phenotype producing bigger fruit than wild type. Histological analysis of the tissues showed that fruit phenotype is associated with a thicker pericarp. By microscopy, we observed that increasing the thickness of the pericarp in the transgenic lines was not due to a greater number of cells but to the increase in cell size. Finally, we observed that cell expansion in transgenic fruit is tightly coupled with higher ploidy levels than wild fruits, suggesting a stimulation of the endoreduplication process. These results clearly demonstrate the existence of a close relationship between the auxin signal, the control cell size, fruit volume and the endoreduplication process. In conclusion, the results provide new insights into the interactions between auxin and ethylene during fruit development and in particular during the transition immature fruit, mature fruit. In addition, they provide new information on the understanding of the role of the signaling pathway of auxin in controlling the development of fleshy fruits and in particular on the basis of certain members of the AUX/IAA on regulating volume and fruit weight.