Embora o cultivo de tomate seja muito antigo e amplamente distribuído, ainda enfrenta desafios para o melhoramento dos níveis de produção e, da qualidade para o processamento e consumo fresco. A grande maioria das características de interesse agronômico está determinada por loci de caracteres quantitativos (QTL), dificultando ainda mais a identificação e transferência gênica. Diversas características tornam o tomateiro um bom modelo para estudos de dissecação dos determinantes genéticos de QTL. Primeiro, a disponibilidade de fontes de germoplasma selvagens ainda inexploradas que podem aumentar a variabilidade genética, somada à possibilidade de cruzamento entre espécies não simpátricas e à autogamia. Segundo, a grande quantidade de informação genética como mapas, coleções de ESTs, QTL, e populações de mapeamento. Terceiro, o genoma de S. lycopersicum está completamente sequenciado. Por fim, devido a suas diferenças morfogenéticas em relação à espécie modelo Arabidopsis thaliana, o tomate se torna uma alternativa para estudos de eudicotiledonias, principalmente em estudos relacionadas a metabolismo de frutos carnosos. A partir da abundante plataforma de dados disponível e por meio de ferramentas de genômica, e genética reversa, este trabalho aborda o estudo de duas regiões do genoma do tomateiro envolvidas no metabolismo de aminoácidos e na determinação da estrutura da parede celular. O estudo de genômica comparativa de uma região do cromossomo 7 permitiu revelar a perfeita sintenia existente entre S. lycopersicum e a espécies selvagem S. pennellii e estimar o tempo de divergência em 2,7 MAA. Complementarmente, foi possível determinar que as diferenças fenotípicas entre as espécies são maiormente devidas a mudanças nas regiões regulatórias e à presença de SNPs. O estudo funcional do gene LFP (Low Free Putrescine) permitiu caracterizar uma proteína plastidial, até o momento desconhecida em tomateiro, que participa do metabolismo de poliaminas. O silenciamento de LFP resultou na redução da disponibilidade de putrescina livre e no aumento da biomassa vegetativa. Por outro lado, os resultados obtidos do estudo do gene GAUT4 (galacturonosiltransferase 4) demonstraram que a enzima codificada se localiza em Golgi e participa do metabolismo das pectinas. Em frutos, a redução dos níveis de GAUT4 resultou na diminuição de pectina e na alteração da sua composição, porém estes se apresentaram mais firmes. Adicionalmente, o silenciamento do gene GAUT4 modificou o particionamento de açúcares provocando o aumento de massa vegetativa em detrimento do índice de colheita, revelando assim um mecanismo regulatório que comunica o metabolismo da parede celular ao controle da relação fonte-dreno. Desta forma, os resultados obtidos aportam dados fundamentais para a melhor compreensão de caracteres de interesse agronômico, assim como de processos fisiológicos complexos e pouco explorados até o momento no tomateiro / Although tomato is an old and widely distributed culture, it still faces challenges to improve production levels and quality for processing and consumption. The vast majority of agronomical important characteristics are determine by quantitative trait loci (QTL), further hindering the gene identification and transfer. Several features make tomato a good model for studying the genetic determinants underneath QTL. First, the availability of unexploited sources of wild germplasm that can increase genetic variability, coupled with the possibility of interbreeding between no sympatric species and autogamy. Second, the large amount of available genetic information as maps, EST collections, QTL, and an extensive collection of mapping populations. Third, the genome of S. lycopersicum is completely sequenced. Finally, due to their morphogenetic differences in relation to model species Arabidopsis thaliana, tomato becomes an alternative to eudicotyledons studies, especially in studies related to fleshy fruits metabolism. From the abundant data platform available and by means of genomic tools, and reverse genetics, this work addresses the study of two tomato genomic regions involved in amino acids and cell wall metabolisms. The comparative genomic study in a region of chromosome 7 has revealed the perfect synteny between S. lycopersicum and the wild species S. pennellii, and estimated the time of divergence between both species in 2.7 MYA. Additionally, it was possible to determine that the phenotypic differences between species are mostly due to changes in regulatory regions and the presence of SNPs. The functional study of LFP gene (Low Free Putrescine) allowed us to characterize a plastid protein, yet unknown in tomato, which participates in the metabolism of polyamines. The LFP silencing resulted in reduced availability of free putrescine and increased vegetative biomass. Furthermore, the functional characterization of the GAUT4 (galacturonosyltransferase 4) gene demonstrated that the encoded enzyme is located in the Golgi apparatus and participates in the pectin metabolism. In fruits, the reduced levels of GAUT4 resulted in decreased pectin and in the change of its composition. Additionally, GAUT4 gene silencing modified sugars partitioning leading to an increased vegetative biomass together with a drastic reduction of the harvest index. Thus, revealing a regulatory mechanism that communicates the cell wall metabolism to source-sink relationship control. Concluding, the results obtained contribute to a better understanding of agronomical important traits, as well as of complex physiological processes little explored in tomato so far

[EN] When addressing the genetic dissection of a complex trait, what really matters is the identification of the genes with major effects, because their modification may result in qualitative changes in the phenotype. For this purpose, a mutagenesis-based approach has two advantages: first, the identification of a mutant reveals that the altered gene has a key effect on the trait; secondly, the phenotypic characterization of the mutant allows making an inference about the gene function. Insertion mutagenesis by T-DNA provides an additional advantage: a gene tagged by a T-DNA insert can be easily identified using PCR-based techniques (e.g. Anchor- PCR). In order to identify genes that control developmental traits and abiotic stress tolerance in tomato and wild related species, we are performing a program of insertion mutagenesis in collaboration with the groups of Dr. Rafael Lozano (University of Almeria) and Dr. MªCarmen Bolarín (CEBAS-Murcia). The objectives of the present Doctoral Thesis are framed in the context of this insertion mutagenesis program in tomato and wild relatives. First, in order to expand the collections of T-DNA lines previously generated in our group, 952 T-DNA lines of tomato, 405 of Solanum pimpinellifolium and 550 of S. cheesmaniae have been obtained. Secondly we performed the evaluation of progenies from 1545 T-DNA lines of tomato, 194 T-DNA lines of S. pimpinellifolium and 149 T-DNA lines of S. cheesmaniae. The screening in vitro of those progenies allowed us to identify 43 mutants altered in early developmental traits. In addition, we were able to detect three mutants of tomato and another one of S. cheesmaniae which are hypersensitive to salt stress. The phenotypic and genetic characterization of selected mutants has been carried out. Finally, we performed the functional analysis of the PMS (PROTECTING MERISTEMS AGAINST SALINITY) gene tagged in the pms-916 tomato mutant. Our results suggest that the PMS gene plays an essential role in the protection of the shoot apical meristem and young tissues of the tomato plant under salinity stress conditions. / [ES] Cuando se aborda la disección genética de un carácter complejo, lo que realmente importa es identificar los genes con efectos principales, ya que su alteración puede provocar cambios cualitativos en el fenotipo. Para este propósito, el uso de una aproximación basada en la generación de mutantes tiene dos ventajas: la identificación de un mutante revela que el gen alterado tiene un efecto clave sobre el carácter y, en segundo lugar, el fenotipo del mutante permite hacer una inferencia sobre la función del gen. La mutagénesis insercional con T-DNA aporta una ventaja adicional ya que si el gen queda etiquetado por un inserto su identificación es relativamente fácil, porque basta con amplificar a partir de una secuencia conocida del T-DNA mediante Anchor-PCR. Con el fin de identificar genes que controlan caracteres del desarrollo y tolerancia a estrés abiótico en tomate y especies silvestres relacionadas, en nuestro laboratorio se está llevando a cabo un programa de mutagénesis insercional en colaboración con los grupos del Dr. Lozano (Universidad de Almería) y la Dra. Bolarín (CEBAS-Murcia). Los objetivos de esta Tesis Doctoral se enmarcan en el contexto de este programa de mutagénesis insercional en tomate y especies relacionadas. En primer lugar, con el fin de ampliar la colección de líneas T-DNA que previamente se generó en nuestro laboratorio, se han obtenido 952 líneas T-DNA de tomate, 405 de Solanum pimpinellifolium y 550 de S. cheesmaniae. Se ha realizado el escrutinio de las progenies de 1545 líneas T-DNA de tomate, 194 líneas T-DNA de S. pimpinellifolium y 149 líneas T-DNA de S. cheesmaniae. La evaluación in vitro de estas progenies ha permitido detectar 43 mutantes alterados en caracteres del desarrollo temprano. Además, se han identificado tres mutantes de tomate y uno de S. cheesmaniae hipersensibles a estrés salino. El tercer objetivo de la Tesis ha consistido en la caracterización fenotípica y genética de los mutantes seleccionados. Por último, se ha realizado el análisis funcional del gen PMS (PROTECTING MERISTEMS AGAINST SALINITY) etiquetado en el mutante de inserción pms-916. Nuestros resultados sugieren que el gen PMS desempeña un papel esencial en la protección del meristemo apical y las hojas jóvenes de la planta de tomate en condiciones de estrés salino. / [CA] Quan s'aborda la dissecció genètica d'un caràcter complex, el que realment importa és identificar els gens amb efectes principals, ja que la seua alteració pot provocar canvis qualitatius en el fenotip. Per a este propòsit, l'ús d'una aproximació basada en la generació de mutants té dos avantatges: la identificació d'un mutant revela que el gen alterat té un efecte clau sobre el caràcter i, en segon lloc, el fenotip del mutant permet fer una inferència sobre la funció del gen. La mutagènesi insercional amb T-DNA aporta un avantatge addicional ja que si el gen queda etiquetat per un insert la seua identificació és relativament fàcil, perquè n'hi ha prou amb amplificar a partir d'una seqüència coneguda del T-DNA per mitjà d'Anchor-PCR. Per tal d'identificar gens que controlen caràcters del desenvolupament i la tolerància a estrés de tipus abiòtic en tomaca i espècies silvestres relacionades, en el nostre laboratori s'està duent a terme un programa de mutagènesi insercional en col¿laboració amb els grups del Dr. Lozano (Universidad de Almería) i la Dra. Bolarín (CEBAS-Murcia). Els objectius d'esta Tesi Doctoral s'emmarquen en el context d'este programa de mutagènesi insercional en tomaca i espècies relacionades. En primer lloc, per tal d'ampliar la col¿lecció de línies T-DNA que prèviament es va generar en el nostre laboratori, s'han obtingut 952 línies T-DNA de tomaca, 405 de Solanum pimpinellifolium i 550 de S. cheesmaniae. S'ha realitzat l'escrutini de les progènies de 1545 línies T-DNA de tomaca, 194 línies T-DNA de S. pimpinellifolium i 149 línies T-DNA de S. cheesmaniae. L'avaluació in vitro d'estes progènies ha permés detectar 43 mutants alterats en caràcters del desenvolupament primerenc. A més, s'han identificat tres mutants de tomaca i un de S. cheesmaniae hipersensibles a estrés salí. El tercer objectiu de la Tesi ha consistit en la caracterització fenotípica i genètica dels mutants seleccionats. Finalment, s'ha realitzat l'anàlisi funcional del gen PMS (PROTECTING MERISTEMS AGAINST SALINITY) etiquetat en el mutant d'inserció pms-916. Els nostres resultats suggerixen que el gen PMS exercix un paper essencial en la protecció del meristem apical i els fulls jóvens de la planta de tomaca en condicions d'estrés salí. / Sánchez Martín-Sauceda, S. (2016). Generación de mutantes de inserción de tomate cultivado y silvestre e identificación de genes implicados en procesos de desarrollo y tolerancia a estrés abiótico [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/63148

Li Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 146-161). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES AND TABLES --- p.XIII / LIST OF ABBREVIATIONS --- p.XX / Chapter CHAPTER ONE --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Authentication of Chinese medicines --- p.1 / Chapter 1.1.1 --- The need for authentication of Chinese medicines --- p.1 / Chapter 1.1.2 --- Traditional methods for authentication --- p.2 / Chapter 1.1.3 --- Molecular methods for authentication --- p.4 / Chapter 1.1.3.1 --- DNA fingerprinting --- p.5 / Chapter 1.1.3.2 --- DNA sequencing --- p.6 / Chapter 1.1.3.2.1 --- Choosing a suitable region for DNA sequencing --- p.7 / Chapter 1.1.3.2.2 --- Chloroplast irnL-trnF region --- p.9 / Chapter 1.1.3.2.3 --- Complete sequence of ITS rDNA region --- p.10 / Chapter 1.1.3.2.4 --- 5S rDNA intergenic spacer --- p.11 / Chapter 1.1.3.2.5 --- Calculation of similarities among sequences --- p.12 / Chapter 1.1.3.2.6 --- Construction methods of phylograms --- p.12 / Chapter 1.2 --- The need for molecular authentication of three medicinal herbs --- p.14 / Chapter 1.2.1 --- The herb Baiying --- p.14 / Chapter 1.2.1.1 --- The poisoning case reported in Hong Kong --- p.14 / Chapter 1.2.1.2 --- The identity of genuine Baiying --- p.15 / Chapter 1.2.1.3 --- Morphological characters of the herb Baiying --- p.15 / Chapter 1.2.1.4 --- Medicinal values of Baiying --- p.17 / Chapter 1.2.1.5 --- Xungufeng as the adulterant of Baiying --- p.17 / Chapter 1.2.1.5.1 --- The toxic chemicals aristolochic acids --- p.18 / Chapter 1.2.1.6 --- The need for molecular authentication of Baiying --- p.19 / Chapter 1.2.2 --- The herb Baihuasheshecao --- p.19 / Chapter 1.2.2.1 --- The identity of Baihuasheshecao --- p.19 / Chapter 1.2.2.2 --- Morphological characters of the herb Baihuasheshecao --- p.20 / Chapter 1.2.2.3 --- Medicinal uses --- p.23 / Chapter 1.2.2.4 --- Chemical profile --- p.24 / Chapter 1.2.2.5 --- Adulterants of Baihuasheshecao --- p.24 / Chapter 1.2.2.6 --- Chemical studies of H. diffusa and H. corymbosa --- p.25 / Chapter 1.2.2.7 --- Existing methods for authentication --- p.26 / Chapter 1.2.2.8 --- The need for molecular authentication of Baihuasheshecao --- p.28 / Chapter 1.2.3 --- The herb Chuanlianzi --- p.28 / Chapter 1.2.3.1 --- The identity of Chuanlianzi --- p.28 / Chapter 1.2.3.2 --- Medicinal values --- p.29 / Chapter 1.2.3.3. --- The bioactive chemical --- p.31 / Chapter 1.2.3.4 --- Kulianzi as the substitute of Chuanlianzi --- p.31 / Chapter 1.2.3.5 --- Poisoning cases reported due to ingestion of Kulianzi --- p.32 / Chapter 1.2.3.6 --- Comparative studies of Chuanlianzi and Kulianzi --- p.32 / Chapter 1.2.3.7 --- The need for molecular authentication of Chuanlianzi --- p.33 / Chapter CHAPTER TWO --- OBJECTIVE --- p.35 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.36 / Chapter 3.1 --- Plant and herb samples --- p.36 / Chapter 3.2 --- Total DNA extraction --- p.48 / Chapter 3.2.1 --- Cetyltriethylammonium bromide extraction --- p.48 / Chapter 3.2.2 --- Commercial kit extraction --- p.49 / Chapter 3.3 --- DNA amplification --- p.50 / Chapter 3.4 --- DNA fingerprinting --- p.51 / Chapter 3.4.1 --- DNA concentration determination --- p.51 / Chapter 3.4.2 --- ISSR fingerprinting --- p.52 / Chapter 3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 3.6 --- Purification of PCR product --- p.53 / Chapter 3.7 --- Cloning of PCR product --- p.54 / Chapter 3.7.1 --- Ligation --- p.54 / Chapter 3.7.2 --- Transformation --- p.55 / Chapter 3.7.3 --- Cell cultivation --- p.55 / Chapter 3.7.4 --- Plasmid extraction --- p.55 / Chapter 3.7.5 --- Insert confirmation --- p.56 / Chapter 3.8 --- Determination of DNA concentration --- p.56 / Chapter 3.9 --- DNA sequencing --- p.57 / Chapter 3.9.1 --- Cycle sequencing --- p.57 / Chapter 3.9.2 --- Purification of cycle sequencing products --- p.57 / Chapter 3.9.3 --- DNA analysis --- p.58 / Chapter 3.10 --- Sequence analysis --- p.58 / Chapter CHAPTER FOUR --- MOLECULAR AUTHENTICATION OF BAIYING --- p.59 / Chapter 4.1 --- Results --- p.59 / Chapter 4.1.1 --- Sequence alignment --- p.59 / Chapter 4.1.2 --- Percentage similarity analysis --- p.68 / Chapter 4.1.3 --- Phylogram study --- p.71 / Chapter 4.2 --- Discussion --- p.79 / Chapter 4.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Baiying and Xungufeng --- p.79 / Chapter 4.2.2 --- Molecular authentication of Baiying --- p.80 / Chapter 4.3 --- Conclusion --- p.81 / Chapter CHAPTER FIVE --- MOLECULAR AUTHENTICATION OF BAIHUASHESHECAO --- p.82 / Chapter 5.1 --- Results --- p.82 / Chapter 5.1.1 --- ITS region used for DNA sequencing --- p.82 / Chapter 5.1.2 --- Sequence alignment --- p.82 / Chapter 5.1.3 --- Percentage similarity analysis --- p.88 / Chapter 5.1.4 --- Phylogram study --- p.90 / Chapter 5.2 --- Discussion --- p.98 / Chapter 5.2.1 --- Evaluation of complete sequence of ITS region in differentiation of Hedyotis species --- p.98 / Chapter 5.2.2 --- Molecular authentication of retailed Baihuasheshecao --- p.99 / Chapter 5.2.3 --- Analysis of conflicting data between this study and published results --- p.99 / Chapter 5.2.3.1 --- Comparison of ITS-1 region --- p.101 / Chapter 5.2.3.2 --- Comparison of ITS-2 region --- p.104 / Chapter 5.2.3.3 --- Proposed reasons for the conflicting data --- p.108 / Chapter 5.3 --- Conclusion --- p.109 / Chapter CHAPTER SIX --- MOLECULAR AUTHENTICATION OF CHUANLIANZI --- p.110 / Chapter 6.1 --- Results --- p.110 / Chapter 6.1.1 --- DNA sequencing --- p.110 / Chapter 6.1.1.1 --- Complete sequence of ITS region used for DNA sequencing --- p.110 / Chapter 6.1.1.1.1 --- Sequence alignment --- p.111 / Chapter 6.1.1.1.2 --- Percentage similarity analysis --- p.113 / Chapter 6.1.1.2 --- 5S rDNA intergenic spacer used for DNA sequencing --- p.113 / Chapter 6.1.1.2.1 --- Sequencing alignment --- p.114 / Chapter 6.1.1.2.2 --- Percentage similarity analysis --- p.122 / Chapter 6.1.1.2.3 --- Phylogram study --- p.128 / Chapter 6.1.2 --- ISSR fingerprinting --- p.136 / Chapter 6.2 --- Discussion --- p.138 / Chapter 6.2.1 --- DNA sequencing results --- p.138 / Chapter 6.2.2. --- ISSR fingerprinting results --- p.139 / Chapter 6.2.3 --- Investigation of the identity of retailed Chuanlianzi --- p.140 / Chapter 6.2.4 --- Taxonomic interpretation for Melia species --- p.141 / Chapter 6.2.5 --- Kulianzi involved in this study --- p.141 / Chapter 6.3 --- Conclusion --- p.141 / Chapter CHAPTER SEVEN --- CONCLUSION --- p.143 / BILBIOGRAPHY --- p.146 / APPDENDIX - MATERIAL PREPARATION --- p.162

Made available in DSpace on 2014-07-29T15:16:34Z (GMT). No. of bitstreams: 1 Geraldo V Jr.pdf: 2978413 bytes, checksum: 9291dd7d1942c2ad5aa0b98f24c1ea57 (MD5) Previous issue date: 2004-09-10 / Solanum lycocarpum A. St.-Hil., popularly known as lobeira (wolf-fruit), is easily found in the Brazilian savanna. It is characterized as a bushy plant with average height of about 5 meters (16 feet), fragile branches, featuring a nearly round berry type of fruit, with diameter that ranges from 8 to 15 cm (3.1 to 5.9 inches), yellowish green color even when ripe, fleshy and juicy pulp. The lobeira is used in folk medicine for several therapeutic purposes such as bronchitis, worm diseases, diabetes and ulcer. A study with ethanolic extract of fruit (EEF) of the lobeira showed analgesic and antidermatogenic activity in the experimental samples of acetic acid-induced abdominal writhing and ear edema. In the present work were performed the morphoanatomic study of the fruit and the antiinflammatory evaluation of the alkaloidic fraction (FA) acquired through the EEF. The morphoanatomic analysis of the fruit pointed out the existence of elements that allow its characterization, like the stellate trichomes, present in leaves of the same species as well. The FA was obtained by acid extraction of the EEF, being defined by thin-layer chromatography the presence of the glycoalkaloid solamargine, found in other species of the Solanum genus. The antiinflamatory activity was estimated in vivo through the ear edema method induced by the croton oil and by the leukocyte migration in the peritonitis induced by carrageenin. It was estimated in vitro the FA ability to restrain the phospholipase A2 action in snake poison. The prior treatment of animals with FA in doses of 50 to 100mg/kg (s.c.) reduced the ear edema formation, and in doses of 30 a 300mg/kg (s.c. and p.o.) decreased the leukocyte migration. The experimental models in vivo demonstrated that the FA displayed characteristic results of an antiinflamatory activity similar to the one shown by the glycocorticoids. The results obtained with the FA could be outcome of the glycoalkaloid presence and also indicate that the FA has the active principles responsible for the biological activity verified in EEF. The FA has not presented inhibitive activity of phospholipase A2 that could be detected by in vitro method that was applied. It becomes necessary the purification of the glycoalkaloid and the completion of specific trials aiming to confirm the antiinflamatory action as well as the participating mechanisms. / Solanum lycocarpum A. St.-Hil., popularmente conhecida como lobeira, é facilmente encontrada no cerrado. Caracteriza-se como uma planta arbustiva com até cinco metros de altura, ramos frágeis, apresentando fruto tipo baga globosa, de oito a quinze centímetros de diâmetro, cor verde-amarelada mesmo quando maduro, polpa carnosa e suculenta. A lobeira é usada na medicina popular para diversos fins terapêuticos, como por exemplo, bronquite, verminose, diabete e úlcera. Estudo com o extrato etanólico do fruto (EEF) da lobeira demonstrou atividade analgésica e antiedematogênica nos modelos experimentais de contorção abdominal e edema de orelha. No presente trabalho foram realizados o estudo morfoanatômico do fruto e a avaliação da atividade antiinflamatória da fração alcaloídica (FA) obtida a partir do EEF. A avaliação morfoanatômica do fruto indicou a presença de elementos que permitem sua caracterização, como os tricomas estrelados, presentes também nas folhas da mesma espécie. A FA foi obtida por extração ácida do EEF, sendo caracterizada, mediante cromatografia de camada delgada, a presença do glicoalcalóide solamargina, encontrado também em outras espécies do gênero Solanum. A atividade antiinflamatória foi avaliada in vivo através do método do edema de orelha induzido pelo óleo de cróton e pela migração celular na peritonite induzida por carragenina. Foi avaliada in vitro a capacidade da FA em inibir a ação da fosfolipase A2 presente em veneno de cobra. O tratamento prévio dos animais com FA nas doses de 50 e 100mg/kg (s.c.) reduziu a formação do edema de orelha, e nas doses de 30 a 300mg/kg (s.c. e p.o.) reduziu a migração leucocitária. Os modelos experimentais in vivo demonstraram que a FA apresentou resultados característicos de uma atividade antiinflamatória semelhante à apresentada pelos glicocorticóides. Estes resultados poderiam ser decorrentes da presença do glicoalcalóide e também indicar que a FA possui os princípios ativos responsáveis pela atividade biológica verificada no EEF. A FA não apresentou atividade inibitória da fosfolipase A2 que pudesse ser detectada pelo método in vitro utilizado. Faz-se necessário a purificação do glicoalcalóide e a realização de ensaios específicos visando confirmar a ação antiinflamatória assim como os mecanismos envolvidos.

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Recientemente se ha demostrado la importancia de las vitaminas y carotenoides del tomate en la prevención de enfermedades degenerativas, por lo que resultaría de gran interés mejorar el contenido de estos compuestos en dicha hortaliza. Pero para iniciar un programa de mejora se deberían cumplir los siguientes objetivos: (i) estudiar la variabilidad presente en germoplasma de Solanum, (ii) ver la influencia del ambiente en estos caracteres, (iii) estudio del control genético de dichos caracteres en las entradas seleccionadas y (iv) aumentar la rapidez y precisión de la cuantificación de los carotenoides. En esta tesis se ha puesto de manifiesto la gran variabilidad presente en germoplasma de tomate, que podría usarse tanto como parentales donantes de alta acumulación de compuestos funcionales (la entrada BGV8166), como directamente en campo aumentando la agrobiodiversidad (unas 20 entradas entre tomate común y cherry con un mayor contenido que el considerado normal para tomate cultivado). Además, se evaluaron 10 entradas preseleccionadas como potencialmente interesantes en 3 ambientes de cultivo, cuyas diferencias tuvieron una influencia destacable en la expresión fenotípica de los caracteres analizados. Sin embargo, el efecto genotípico tuvo la mayor contribución al fenotipo junto a una considerable interacción con el ambiente. Entre las entradas evaluadas, destacaron: CDP1568, CDP7090 y CDP9822 de S. pimpinellifolium que podrán usarse como parentales donantes en la mejora del contenido de carotenoides del tomate cultivado y la entrada CDP4777 (S.lycopersicon var cerasifome) con alto potencial genotípico y estabilidad para acumular (beta)-caroteno y ácido ascórbico. De estas entradas, se seleccionó la CDP4777 por ser de la misma especie que el tomate cultivado, para analizar con detalle su control genético en la acumulación de (beta)-caroteno y ácido ascórbico. Los resultados indicaron que la acumulación de (beta)-caroteno derivada de CDP4777 fue principalmente de carácter aditivo. / Adalid Martínez, AM. (2011). Mejora de la calidad nutritiva del tomate: búsqueda de fuentes de variabilidad, estudio de la influencia del ambiente y determinación del control genético [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/12265

La tomate (Solanum lycopersicum) est l’espèce modèle pour l’étude du développement des fruits charnus. Il a notamment été montré que la signalisation hormonale était un élément central d’un réseau complexe de régulations gouvernant ce processus. Le but de ce travail de thèse était de réaliser la validation fonctionnelle de nouveaux gènes candidats potentiellement impliqués dans la régulation du développement du fruit. Pour cela, le travail s’est découpé autour de trois axes : 1) la validation de l’utilisation de quatre promoteurs de tomate et d’un promoteur d’Arabidopsis thaliana dans des constructions permettant la surexpression ou le silencing de gènes dans le fruit de tomate, à des phases particulières de son développement ou dans des tissus spécifiques du fruit, 2) l’analyse fonctionnelle de gènes codant pour des protéines à F-Box chez la tomate et 3) l’analyse fonctionnelle du gène SlGEM1.Le premier axe de ce travail a porté sur la caractérisation des promoteurs des gènes de tomate IMA (INHIBITOR OF MERISTEM ACTIVITY), TPRP (TOMATO PROLIN-RICH PROTEIN), PPC2 (PHOSPHOENOLPYRUVATE CARBOXYLASE 2) et PG (POLYGALACTURONASE), ainsi que du promoteur du gène CRC (CRABS-CLAW) d’Arabidopsis thaliana. Des plantes transgéniques ont été générées permettant d’exprimer, sous le contrôle de ces différents promoteurs, le gène rapporteur GUS seul ou fusionné à la GFP (Green fluorescent protein) et à un signal d’adressage au noyau (NLS). L’étude de ces plantes a permis de mettre en évidence la spécificité spatio-temporelle de l’expression de ces différents promoteurs lors du développement du fruit. Dans le cas du promoteur LePPC2, la fusion transcriptionnelle avec la GFP et un signal NLS a permis de montrer une activité spécifique dans les cellules du péricarpe des fruits de tomate en phase d’expansion cellulaire. Cette partie du travail de thèse a validé l’utilisation de ces différents promoteurs pour de futures analyses fonctionnelles de gènes régulateurs du développement du fruit chez la tomate.Le deuxième axe de ce travail a porté sur l’identification de protéines à F-Box impliquées dans la régulation du développement du fruit chez la tomate. En effet, il a été montré que ces protéines à F-Box jouent un rôle particulièrement important dans les processus de régulation chez les plantes grâce à leur fonction de reconnaissance de protéines régulatrices cibles par les complexes SCF (SKP1-Cullin-F-Box), qui sont alors marquées par ubiquitinylation, pour leur dégradation par le protéasome 26S. Parmi les 95 séquences de protéines à F-Box disponibles dans les bases de données d’EST au début de ce travail, quatre gènes candidats ont été retenus pour leur expression tissu-spécifique lors du développement précoce du fruit. Des plantes transgéniques (RNAi et sur-expression) ont été générées pour chacun de ces gènes candidats et leur analyse préliminaire a permis de proposer un rôle dans le développement chez la tomate pour une d’entre elles.Le troisième axe de ce travail a porté sur la caractérisation fonctionnelle du gène SlGEM1, l’orthologue du gène AtGEM (GLABRA2-EXPRESSION MODULATOR), dont le niveau d’expression semblait corrélé à la taille des cellules dans le fruit de tomate. En effet, les données disponibles sur AtGEM montrant son implication dans la coordination de la prolifération et la différenciation des cellules en faisaient un candidat intéressant au contrôle de la taille et du développement du fruit. Des plantes transgéniques (RNAi et sur-expression) SlGEM1 ont été générées, et des mutants de ce gène ont été identifiés par TILLING dans la banque de mutants EMS de la variété Microtom de tomate disponible au laboratoire. Le phénotypage de ces plantes a permis de mettre en évidence une potentielle implication de SlGEM1 dans le développement des fleurs et dans la fertilité des grains de pollen. / Tomato (Solanum lycopersicum) is a model species for studying fleshy fruit development. It has been shown that hormone signaling plays a crucial role in the complex regulatory network governing this developmental process. The aim of this thesis was to perform the functional validation of new candidate genes potentially involved in the regulation of fruit development. For this, the work was divided into three parts: 1) validate the use of four tomato promoters and one promoter of Arabidopsis thaliana in recombinant vectors for the overexpression or silencing of genes in tomato fruit at particular phases of development or in specific fruit tissues, 2) functional analysis of genes encoding F-Box proteins in tomato and 3) functional analysis of SlGEM1 gene.The first part of this work has focused on the characterization of tomato promoters from IMA (INHIBITOR ACTIVITY OF MERISTEM), TPRP (TOMATO Proline-Rich Protein), PPC2 (PHOSPHOENOLPYRUVATE CARBOXYLASE 2) and PG (polygalacturonase), as well as the promoter of Arabidopsis thaliana CRC gene (CRABS-CLAW). Transgenic plants were generated to express the GUS reporter gene alone or fused to GFP and a NLS signal for nucleus targeting, under the control of the different promoters. The study of these transgenic plants highlighted the specific spatio-temporal expression of these promoters during fruit development. In the case of LePPC2 promoter, the transcriptional fusion with NLS-GFP revealed a specific activity in the large cells of tomato fruit pericarp. This part of the thesis work validated the use of the five different promoters for future functional analysis of genes regulating fruit development in tomato.The second part of this work focused on the identification of F-Box proteins involved in the regulation of tomato fruit development. In plants, it has been shown that F-Box proteins play an important role in regulating processes, due to their specific interaction with regulatory proteins targeted by the SCF complex (SKP1-Cullin-F-Box), leading to their ubiquitination and degradation by the 26S proteasome. Among the 95 sequences of F-Box proteins available in tomato databases at the beginning of this work, four candidate genes were selected for their tissue-specific expression during tomato fruit early development. Transgenic plants (RNAi and over-expression) were generated for each of these candidate genes and their preliminary analysis allowed to propose a role in tomato vegetative development for one of them.The third part of this work focused on the functional characterization of SlGEM1, orthologous to AtGEM (GLABRA2-EXPRESSION MODULATOR). Indeed, a previous work revealed that the expression level of SlGEM1 was correlated with tomato fruit cell size. Available data on AtGEM showing its involvement in the coordination of cell proliferation and differentiation made it an attractive candidate to the control of fruit size and development. Transgenic plants (SlGEM1 RNAi and over-expression) were generated, and mutants of this gene were identified by TILLING in the Microtom mutant collection available in the laboratory. Phenotyping of these plants suggested the involvement of SlGEM1 in flower development and in the fertility of pollen grains.

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

A produção de hortaliças em substratos é uma técnica de cultivo que pode aumentar a produtividade, economizar recursos e facilitar o manejo nutricional das plantas. Contudo, para que isso seja possível, deve-se conhecer os fatores que afetam o crescimento das plantas cultivadas nesse sistema. O objetivo deste trabalho foi avaliar os efeitos de diferentes doses de nitrogênio da solução nutritiva (SN) (60, 80 100, 120 e 140% da dose padrão) em dois tipos de substratos (S1: fibra de coco e S2: a base de casca de pinus) no cultivo de minitomate Sweet Grape. Foram avaliadas ao longo do ciclo de cultivo o pH, a condutividade elétrica (CE) e o teor de nitrato (NO3-drenado) e potássio (K+drenado) na solução drenada dos vasos, o índice SPAD e o teor de nitrato (NO3- pecíolo) e potássio (K+ pecíolo) da seiva do pecíolo das folhas e o teor de nitrogênio da massa seca das folhas (Nfolha). Foram determinados o número de cacho por planta (NCPP) e o comprimento médio das hastes (CMH). Os frutos colhidos foram classificados, contados e pesados para a obtenção da massa média dos frutos grandes (MMFG), médios (MMFM) e pequenos (MMFP), massa total de frutos por planta (MTFPP), massa total de frutos grandes (MTFG), médios (MTFM), pequenos (MTFP) e rachados (MTFRA), número total de frutos por planta (NTFPP), número total de frutos grandes (NTFG), médios (NTFM), pequenos (NTFP) e rachados (NTFRA). Amostras de frutos foram coletadas em três períodos e avaliadas quanto ao teor de sólidos solúveis (TSS), acidez titulável (AT) e concentração de ácido ascórbico (AA). Os valores de pHdrenado diminuíram com o aumento das doses de N da solução nutritiva. Os efeitos do N no pHdrenado se intensificaram ao longo do ciclo de cultivo em razão do acúmulo de sais no meio radicular, principalmente no S1. A CEdrenada aumentou com dose de N e foi maior no S2. O aumento da dose de N elevou os teores de NO3-drenado em ambos os substratos. O K+drenado foi superior na dose de 140% de N no S1. Os valores de índice SPAD e Nfolha aumentaram com a elevação das doses de N da solução nutritiva e reduziram ao longo do ciclo de cultivo. O NO3-pecíolo elevou com o aumento da dose de N da SN. A MMFG foi superior com a adição de 140% de N em relação à dose de 60%. O aumento da dose de N elevou o NTFG, NTFM, NTFP, NTRA e NTFPP. O maior NTFG foi obtido no S2 enquanto que o maior NTFPP, NTFP e NTFRA ocorreu no S1. A MTFG, MTFM, MTFP, MTFRA e MTFPP aumentaram com o aumento da dose de N. Os valores mais elevados de MTFG e MTFP foram obtidos na dose de 140% de N, 331,05 e 1.249,82 g planta-1, respectivamente. A maior MTFM foi obtida na dose de 120% de N, não diferindo, porém, das doses de 100 e 140% de N. O TSS, a AT e o AA dos frutos não foram influenciados pelas doses de N e tipos de substrato. / Vegetables production in soilless media is a technique that can increase yield, saving resources and facilitate the nutritional management of plants. However, for that to be possible, its necessary to know how the factors affect the plants growth in this system. The aim of this study was to evaluate the effects of different nitrogen rates of nutrient solution (NS) (60, 80 100, 120 and 140% of the standard) in two type of soilless media (S1: coconut fiber and S2: pine bark as base) for the mini tomato Sweet Grape growth. Were evaluated during the growing season pH, electrical conductivity (EC) and the nitrate content (NO3-drained) and potassium (K+ drained) in the solution drained from the vessels, SPAD index and the nitrate (NO3-pecíolo) and potassium (K+ petiole) content on petiole sap from the leaves and the nitrogen content in leaf dry mater (Nleaf). Were determined the number of fruit per plant (NFPP) and the average length of stems (ALS). The harvested fruits were sorted, counted and weighed to obtain the average mass of large fruits (AMLF), medium fruits (AMMF) and small fruits (AMSF), total weight of fruits per plant (TWFPP), total weight of large fruits (TWLF) medium fruits (TWMF), small fruits (TWSF) and cracked fruits (TWCF), total number of fruits per plant (TNFPP), total number of large fruits (TNLF), medium fruits (TNMF), small frutis (TNSF) and cracked fruits (TNCF). Fruit samples were collected in three periods and evaluated for soluble solids (SS), titratable acidity (TA) and concentration of ascorbic acid (AA). The pHdrained values decreased with increasing N levels in nutrient solution. The effects of N on pHdrained intensified throughout the growing season due to the accumulation of salts in the root medium, especially in S1. The CEdrained increased with N rate and was higher in S2. The increase in N rate increased the levels of NO3-drained on both substrates. The K+ drained was higher at 140% N dose in S1. SPAD index and Nleaf values increased with higher N rates in nutrient solution and reduced throughout the growing season. The NO3 - petiole increased with increasing dose of N in NS. The MMFG was higher with the addition of 140% N in relation to the dose of 60%. The increase in N rate increased the TNLF, TNMF, TNSF, TNCF and TNFPP. The biggest TNLF was obtained in S2 while the largest TNFPP, TNSF and TNCF occurred in S1. The TWLF, TWMF, TWSF, TWCF and TWFPP increased with increasing N levels in NS. The highest values of TWLF and TWSF were obtained at 140% N level, 331.05 and 1249.82 g plant-1, respectively. The biggest TWMF was obtained at 120% N level in NS, however, not differing of the 100 and 140% N levels. The SS, TA and AA fruits were not influenced by N and substrate types.

Usualmente a bataticultura no Brasil migra para novas áreas devido à alta ocorrência de doenças de solo, onde seu cultivo é realizado anualmente após a cultura do milho (Zea mays) com preparo de solo feito com grade aradora, implemento cujo uso frequente gera impedimentos físicos em subsuperfície. Atualmente, é limitado o esforço de pesquisa sobre sistemas sustentáveis baseados no baixo uso de insumos externos que minimizem impactos prejudiciais ao ambiente. Desse modo, o preparo profundo de solo em sucessão a poáceas, constitui uma alternativa viável a qual pode levar os produtores a manter altos níveis de produção e boa qualidade dos tubérculos, sem que seja necessária a mudança da área plantada. O presente estudo teve o objetivo de comparar dois sistemas de preparo de solo (preparo convencional e preparo profundo) para a cultura da batata (Solanum tuberosum L.) cv. Atlantic, em sucessão a poáceas, quanto aos atributos do solo, produtividade e dinâmica do crescimento da batateira. O experimento foi conduzido por um ano com inicio em setembro de 2011 em área do Departamento de Produção Vegetal, ESALQ/USP, situada na latitude 22°42\'09\'\' S e na longitude 47°38\'01\'\' W a 569 m de altitude. Foram estudadas quatro sucessões de poáceas com batata, sendo os tratamentos: T1: sucessão Panicum maximum cv. Tanzânia - batata, sob preparo profundo (Tanzânia PP), T2: sucessão Brachiaria brizantha cv. Marandu - batata, sob preparo profundo (Braquiária PP) e T3: sucessão milho (Zea mays cv. AG 6080) - batata, sob preparo profundo (Milho PP). Para efeito de comparação, foi utilizado como tratamento controle T4: a sucessão milho - batata em preparo convencional de solo (Milho PC), com uso de grade aradora a 0,20 m de profundidade antes do plantio. Para a análise estatística foi adotado o delineamento em blocos casualizados, com quatro tratamentos e seis repetições. As unidades experimentais foram constituídas por três linhas duplas de 10,0 m de comprimento. A linha dupla central teve 1,5 m de suas extremidades desconsideradas e os 7,0 m centrais, correspondente a 12,6 m² em área, definidos como unidade experimental útil ou unidade de observação. Foram avaliadas as características: produção de matéria seca da parte aérea das poáceas, resistência do solo à penetração, macroporosidade e densidade do solo, estande, número de hastes e sanidade da parte aérea da batateira, produtividade e danos aos tubérculos e dinâmica do crescimento. Os dados obtidos foram submetidos à análise de variância (ANOVA, p<=0,05). Posteriormente, as médias foram comparadas pelo teste de Tukey ao nível de 5% de probabilidade de erro, pelos programas estatísticos SAS e Sisvar. O preparo profundo de solo, em relação ao preparo convencional, independentemente da poácea utilizada em sucessão promoveu melhorias nos atributos físicos do solo, nos horizontes entre 0,20 e 0,60 m, propiciando redução da resistência à penetração, aumento da macroporosidade e diminuição da densidade, incrementou em 35,9% a produtividade da cv. Atlantic e proporcionou maior acúmulo de MS em todos os órgãos da planta. / Potato cropping in Brazil is marked by a constant need to migrate to new areas due to a high incidence of soil diseases, where the crop is annually planted after corn (Zea mays) and soil preparation is done by conventional tillage, using plow and harrow, a technique that when performed frequently can cause physical impediments in the soil\'s subsurface. There is limited research done on sustainable systems for potato production in Brazil, and these are characterized by lower use of chemical products, resulting in less detrimental impacts to the environment. Therefore, deep soil preparation for potato cropping succeeding grasses may be a viable alternative to producers attempting to reach and maintain high levels of production and quality of potato tubers without having to relocate to new cropping areas. The objective of the present study was to compare two types of soil preparation (i.e. conventional tillage and deep tillage) for potato crops (Solanum tuberosum) cv. Atlantic, succeeding the cultivation of a grass type, evaluating grass and soil characteristics, and productivity and growth dynamics of the potato crop. The experiment was conducted for one year starting in September 2011 in an area belonging to the Crop Science Department, ESALQ/USP , located at 22°42\'09\'\' South Latitude and 47°38\'01\'\' West Longitude at 569 m of altitude. It was studied the use of four grass types succeeded by potato cropping distributed in treatments: T1: Panicum maximum cv. Tanzania and deep tillage soil preparation (Tanzânia PP), T2: Brachiaria brizantha cv. Marandu and deep tillage soil preparation (Braquiária PP) and T3: corn \'AG 6080\', deep tillage soil preparation (Milho PP). A fourth treatment was used as control treatment, T4: corn \'AG 6080\', using conventional tillage soil preparation (Milho PC), preparing the soil at 0.20 m depth before planting the potato crop. For statistical analysis it was adopted a randomized block design with four treatments and six replications. Each experimental unit consisted of three double rows of 10.0 m in length. Plants located in the final 1.5 m ends of each row were discarded and the 7.0 m located in the center were used, which corresponded to 12.6 m² in total area. The grass characteristic evaluated was the dry matter (DM) production above ground level, the soil characteristics evaluated were: resistance to penetration, macroporosity, and density, and finally the potato characteristics studied were: bulk density, number and sanity of potato shoots, and tubers production and occurrence of damage to tubers and the growth dynamics of the potato crop. Data were subjected to analysis of variance (ANOVA p<=0.05). Tukey test was used to compare means adopting a 5% level to assess statistical significance using the softwares SAS and Sisvar. Deep soil tillage compared to conventional tillage promoted improvements in soil physical properties, for soil horizons between 0.20 and 0.60 m, providing lower resistance to penetration, increased macroporosity and lower soil density, regardless of the grass type, what resulted in increases of 35.9% in productivity of potato crop cv. Atlantic, with a greater DM accumulation in all plant organs evaluated.