Made available in DSpace on 2014-08-20T14:33:00Z (GMT). No. of bitstreams: 1 Dissertacao_Tiago_ Pedo.pdf: 721394 bytes, checksum: 63af037478179a59f5249bef3336f8e7 (MD5) Previous issue date: 2012-03-02 / The experiment was conducted in the period between December 2010 and June 2011, at the Capão do Leão Campus of Universidade Federal of Pelotas. The study aimed to analyze the growth and physiological characteristics of the qualitative production of tomato grafted and ungrafted under a protected environment. The seedlings were produced by the method of cutting terminal and ungrafted in polystyrene trays. After the period of budding, the seedlings were transplanted to the greenhouse and evaluated for growth, assimilate partitioning, yield and fruit quality and randomized blocks. From the comparative analysis of growth, the plants grafted on the hybrid Kaguemusha® showed a higher accumulation of dry matter at the end of the cycle and net assimilation rate (Ea) for more than 2/3 of the crop cycle. The relative growth rate, net assimilation rate and Ra peaked in the early phase of the cycle, and decreasing from then until the end of the cycle regardless of treatment. Grafting resulted in higher early, dry matter content of fruits, average fruit weight, shoot/root ratio, harvest index and total phenols, however the grafted plants were less productive than those of ungrafted. It was also that of ungrafted plants had soluble solids content and total yield than the grafted, however the distribution of these classes had a higher percentage of unmarketable fruits compared plants subjected to grafting. / O experimento foi realizado no periodo compreendido entre dezembro de 2010 a junho de 2011, no Campus Capão do Leão da Universidade Federal de Pelotas. O trabalho teve por objetivo analisar as características fisiológicas de crescimento e as qualitativas produtivas do tomateiro enxertado e pé-franco em ambiente protegido. As mudas foram produzidas pelo método de estaca terminal e o pé-franco em bandejas de poliestireno expandido. Após o periodo da enxertia as mudas foram transplantadas para casa de vegetação, sendo realizadas avaliações de crescimento, partição de assimilados, produtividade e qualidade dos frutos em blocos casualizados. A partir da análise comparativa dos dados de crescimento, as plantas enxertadas sobre o híbrido Kaguemusha® apresentaram maior acumulo de matéria seca ao final do ciclo e taxa assimilatória líquida (Ea) superior durante 2/3 do ciclo de cultivo. As taxas de crescimento relativo, assimilatória líquida e de crescimento relativo de área foliar atingiram o pico na fase inicial do ciclo, sendo decrescentes a partir de então até o final do ciclo independente do tratamento. A enxertia resultou em maior precocidade, teor de matéria seca de frutos, massa média de frutos, razão parte aérea/raiz, e teor de fenóis totais, entretanto as plantas enxertadas foram menos produtivas que as de pé-franco. Verificou-se ainda que plantas de pé-franco apresentaram teor de sólidos solúveis totais e produção total de frutos superior as enxertadas, entretanto na distribuição destes em classes apresentou maior percentual de frutos não comerciais quando comparadas as plantas submetidas à enxertia. Palavras chave: Solanum lycopersicum, taxas, partição de assimilados, pós-colheita.

Développer des plantes résistantes aux pathogènes nécessite d’identifier des sources de résistance, de les introduire dans le fonds génétique souhaité et de s’assurer de leurs performance et durabilité. Par l’étude de 2 pathosystèmes distincts, ces travaux de thèse m’ont permis d’appréhender ces différentes étapes.Le pathosystème tomate-bégomovirus présente un intérêt économique important. Une étude par transgénèse suggère que la surexpression de SlNAC1 (codant un facteur de transcription) est bien impliquée dans des mécanismes de résistance au bégomovirus, probablement en activant les mécanismes de résistance systémique acquise. Cette surexpression semble s’accompagner d’un retard de croissance, incompatible avec une utilisation en amélioration des plantes. Le pathosystème tomate-potyvirus permet de comparer au sein d’une même espèce des allèles de résistance naturels et induits, reposant sur des mutations affectant un facteur d’initiation de la traduction eIF4E1. Les travaux menés sur ces allèles ont permis de mettre en lumière une redondance au sein de la petite famille multigénique eIF4E vis-à-vis de la résistance, impliquant eIF4E2 dans les mécanismes de sensibilité et sont en faveur de l’utilisation d’allèles de résistance fonctionnels. De plus, l’introgression de l’allèle naturel dans une lignée de tomate possédant déjà d’autres gènes de résistance aux pathogènes s’accompagne d’une érosion du spectre de résistance dû à un contournement massif par la souche PVY-LYE84.Nos travaux soulignent ainsi les précautions à prendre lors de la mise en place de résistances génétiques ainsi que la nécessité de concilier ces approches avec l’étude du développement de la plante. / The development of plants resistant to need to identify sources of resistance, to introduce them into the desired genetic background and subsequently to monitor their performance and durability. By studying two distinct pathosystems, this thesis work has allowed me to address those three stages.The tomato-begomoviruses pathosystem presents a significant economic interest. Transgenic approach suggests that SlNAC1 (coding for a transcription factor) is involved in resistance mechanisms to begomoviruses, probably by triggering systemic acquired resistance. Besides, the SlNAC1 overexpression seems to be associated with growth retardation, a side-effect incompatible with its use in plant breeding.The tomato-potyviruses pathosystem allowed the comparison within the same species of both natural and induced resistance alleles, associated with mutations affecting the translation initiation factor eIF4E1. Work on those alleles highlighted a redundancy effect within the eIF4E small multigenic family toward resistance, affecting eIF4E2 and favouring the use of functional resistance alleles in resistance. In parallel, the introgression of thebroad-spectrum resistance natural allele was introgressed into a tomato line that already possess other pathogen resistance genes is accompanied by an erosion of the resistance spectrum, due to a massive resistance breakdown by the PVY-LYE84 strain.Altogether, this current work highlight cautions to be taken for introducing resistance genes as well as stressing the importance of combining those approaches with the study of plant development.

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

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

Le traitement photodynamique antimicrobien (acronyme anglais APDT) est apparu comme une solution alternative pour lutter contre les microorganismes multi-résistants. Cette méthode basée sur l'utilisation de photosensibilisateurs dont les porphyrines, fonctionne aussi contre les agents pathogènes des plantes ce qui nous a conduit à proposer une approche de type APDT dans le domaine de l’agriculture. Au cours de ce travail de thèse, nous nous sommes intéressés au côté « vert » de la mise en place de cette approche en réalisant une étude approfondie sur des plantules de deux espèces végétales: Arabidopsis thaliana et Lycopersicum esculentum (tomate) et sur une suspension cellulaire de tabac (TBY-2). Des porphyrines anioniques et cationiques hydrosolubles ont été testées. Nous avons montré qu’aucune de ces porphyrines testées à forte concentration (⩾ 80 μM) n’était cytotoxique à l’obscurité sur les plantules ou la suspension. Par contre sous photopériode (16h), les porphyrines cationiques testées à faible concentration (3,5 μM) se sont révélées létales pour les plantules d’Arabidopsis alors qu’elles n’ont fait que ralentir la croissance des plantules de tomate. Etonnamment, les porphyrines anioniques même testées à forte concentration n’ont pas (ou très peu) induit d’altérations de croissance des plantules. Cette situation se trouve inversée dans les cellules TBY-2 qui sont beaucoup plus sensibles aux porphyrines anioniques photoactivées qui induisent leur mort par apoptose. Ce modèle cellulaire nous a permis de comprendre i) les mécanismes d'interaction porphyrines anioniques avec la paroi cellulaire et ii) quels mécanismes étaient mis en place dans les cellules en réponse au stress photodynamique. En conclusion, ces études préliminaires sur le végétal laissent sérieusement entrevoir la possibilité de développer l’APDT en agriculture ciblée aux pathogènes de plantes et sans effet notable sur les plantes d’intérêt agronomique et les microorganismes du sol. / Antimicrobial photodynamic treatment (APDT) has emerged as an alternative modality to strive against multidrug-resistant microorganisms. This method, based on the use of photosensitizers including porphyrins, has also shown substantial efficiency to kill plant pathogens. This led us to propose an APDT approach in the field of agriculture. During this thesis work, we were interested in the "green" side of this approach. Thus, we performed an in-depth study on two plant species: Arabidopsis thaliana and Lycopersicum esculentum (tomato) and on Tobacco Bright Yellow-2 suspension culture (TBY-2 cells) using anionic and cationic water-soluble porphyrins. We have shown that none of these porphyrins tested at high concentration (⩾ 80 μM) was cytotoxic on plantlets or suspension cells under dark conditions. On the other hand, the cationic porphyrins tested at low concentration (3.5 μM) were lethal for Arabidopsis while they only slowed the growth of tomato plantlets under 16 h photoperiod. Surprisingly, the same anionic porphyrins tested at high concentrations did not induce growth alterations of both plantlets. This situation is reversed in TBY-2 cells, which are much more sensitive to photoactivated anionic porphyrins that induce apoptosis-like cell death. TBY-2 cells allowed to understand i) the interactions of anionic porphyrins with the cell wall and ii) the induced cellular and molecular mechanisms in response to photodynamic stress. In conclusion, these preliminary studies on the plants and plant cells suggest the possibility of developing APDT in agriculture. It will be targeted to plant pathogens without any side effects on plants of agronomic interest and soil microorganisms.

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.

As modificações ambientais provocadas pela humanidade, em especial pela agricultura, têm sido motivo de preocupação e estudos por todo o mundo. Nesse sentido, os solos figuram como um dos compartimentos ambientais mais impactados pelo uso de agrotóxicos, produtos amplamente utilizados na agricultura moderna. O objetivo deste trabalho foi avaliar se e como os agrotóxicos Score® 250 EC (i.a. difenoconazol) e Kraft® 36 EC (i.a. abamectina), isolados e em mistura, afetam a flora (germinação e crescimento do tomate - Lycopersicon lycopersicum), as comunidades microbianas do solo (biomassa microbiana em carbono, respiração, quociente metabólico e enzima β-glucosidase) e os enquitreídeos (reprodução de Enchytraeus crypticus em diferentes densidades e a relação trófica entre estes e os microrganismos), ao longo do tempo após a contaminação do solo. Em relação aos parâmetros microbianos, a despeito de algumas variações em decorrência da presença dos agrotóxicos, os indicadores das funções gerais do sistema edáfico não foram grandemente prejudicados, expressando uma redundância funcional. Não obstante, o solo-controle apresentou maior estabilidade em relação às variações dos parâmetros, sugerindo que os microrganismos presentes neste solo apresentaram uma maior robustez e estabilidade ao longo do tempo. Em relação aos testes com E. crypticus, os índices de reprodução não foram significativamente alterados em função da ação dos agrotóxicos, mas ratificou-se a importância dos microrganismos na dieta dos mesmos, haja vista as correlações entre o tamanho das comunidades microbianas e as taxas de crescimento dos enquitreídeos. Com relação ao crescimento e germinação do tomate, puderam ser observados valores maiores de altura e massa seca das plântulas em relação ao controle. Em contrapartida, observou-se uma possível melhor tendência de crescimento das plântulas no solo controle ao longo do tempo. Por fim, considerando a complexa problemática brasileira em relação ao uso de agrotóxicos, reiterou-se a importância do cumprimento das recomendações de aplicação de ambos os agrotóxicos avaliados por parte dos agricultores e recomendou-se a iniciativa de diminuição progressiva da sua utilização. / Environmental changes caused by humanity, especially by agriculture, have been a cause for concern throughout the world. The soils are one of the environmental compartments most impacted by the use of pesticides, which are widely used in modern agriculture. This research aimed at evaluating whether and how the insecticide Kraft® 36 EC (a.i. abamectin) and the fungicide Score® 250 EC (a.i. difenoconazole), isolated and in mixture, affect: a) the soil microbial communities (microbial biomass carbon, respiration, metabolic quotient and β-glucosidase enzyme); b) enchytraeid worms (reproduction of Enchytraeus crypticus in different densities and their trophic relation with microorganisms); and c) the flora (emergence and growth of tomato - Lycopersicon lycopersicum), over 84 days after contamination. The microbial indicators were not greatly impaired and expressed a functional redundancy, despite some variations due to the presence of pesticides. Nevertheless, the control group presented a greater stability than the contaminated ones, suggesting a greater robustness and stability of microorganisms over time, which are important factors for a better quality of the soil. In relation to E. crypticus tests, the importance of microorganisms on their diet was ratified, since positive correlations between the size of microbial communities and the enchytraeids rate of increase were observed. The different densities of E. crypticus were not significantly influenced by the effects of pesticides on the microorganisms. With respect to the growth and germination of tomato, higher values of height and dry mass of the seedlings were observed in contaminated groups. On the other hand, a higher growth tendency of the seedlings in the control soil was observed over time. Finally, considering the complexity of the Brazilian pesticide issue, we emphasize the importance of compliance of the recommendations on pesticides application and reinforce the initiative of decreasing their use.

Die Tomate ist weltweit eines der am meist konsumierten Gemüse, doch der hohe Verzehr erhöht auch das Risiko einer allergischen Reaktion. Zu den Allergenen zählen viele Proteine der Pflanzenabwehr, welche durch verschiedenste biotische und abiotische Stressoren induziert werden. Allerdings ist bis heute der Einfluss biotischer Faktoren auf die Allergenität von Obst und Gemüse weitestgehend unbekannt. Diese Dissertation befasst sich mit den Auswirkungen der symbiontischen Mykorrhiza und des pathogenen Pepino Mosaikvirus auf die Allergenität von Tomaten. Früchte von mykorrhizierten Tomatenpflanzen wiesen eine höhere Expression allergenkodierender Gene auf als die nicht mykorrhizierten Kontrollen, allerdings übertrug sich dies nicht auf die Allergenität der Tomaten. Pepino Mosaikvirus-infizierte Tomatenpflanzen zeigten keine generelle Aktivierung der Allergenexpression und auch keine erhöhte Allergenität. Aufgrund großer individueller Reaktionsunterschiede der Probanden in den klinischen Allergietests konnten einzeln auftretende Unterschiede nicht über die Gesamtheit der getesteten Allergiker abgesichert werden. Die individuellen Reaktionen der Probanden konnten wiederum nicht auf die Anzahl und Identität der Allergene von Tomatenpflanzen mit unterschiedlichen genetischen Hintergründen oder aus verschiedenen Anbauweisen zurückgeführt werden. Während der Experimente wurden 13 neue putative Tomatenallergene identifiziert unter denen das Cyclophilin rekombinant in Escherichia coli hergestellt und seine allergene Aktivität bestätigt wurde. Schlussfolgernd nahmen biotische Faktoren keinen nennenswerten Einfluss auf die Allergenität von kommerziell angebauten Tomaten. Über dies hinaus wurden neue Erkenntnisse über persistente Virus-Pflanzen Interaktionen gewonnen. Schließlich konnte die Liste der putativen Tomatenallergene um einige Kandidaten erweitert werden, von denen das Cyclophilin als Allergen bestätigt wurde. / Tomatoes are now among most consumed vegetables worldwide; unfortunately accompanied by an increasing risk of allergic reactions. Pathogenesis-related proteins can act as allergens and are induced by various biotic and abiotic stresses. Up to now nearly nothing is known about the impact of biotic factors on allergenic potentials. This thesis investigates the allergenicity of symbiotically mycorrhized and Pepino mosaic virus-infected tomato fruits. Although induced allergen-encoding gene expression was detected in the fruits of mycorrhizal tomato plants, there was no impact on allergenicity. In contrast, general induction of defence-related allergens in Pepino mosaic virus-infected tomato fruits was not observed. Consequently, clinical allergy tests did not reveal any generally increased allergenic potential of Pepino mosaic virus-infected tomato fruits. High inter-individual differences in clinical allergy tests made it difficult to make statistically confirmed statements about the allergenicity of colonised tomato fruits. However, the hypothesis that such individual variability is based on differential reactions of individual subjects to particular allergens in tomato fruits, from plants with certain genetic backgrounds or cultivated under distinct conditions, had to be rejected. During these investigations, 13 new putative tomato allergens were identified. One of the candidates, cyclophilin, was recombinantly produced in Escherichia coli and its allergenic activity was confirmed in different clinical allergy tests with tomato-allergic subjects. In conclusion, this study demonstrates that biotic factors are only of minor importance for the allergenic potential of commercially produced tomatoes. Moreover, the experiments revealed new insights into persistent plant-virus interactions. In particular, these extended the list of putative tomato allergens to include new candidates, and confirmed the allergenic activity of one of these, namely cyclophilin.

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.