Orientador: Marco Aurélio Zezzi Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T00:56:12Z (GMT). No. of bitstreams: 1 Silva_MarceloAnselmoOseasda_D.pdf: 21762318 bytes, checksum: 0f395a27c9b28a3d7414579ff560bd4a (MD5) Previous issue date: 2012 / Resumo: Inúmeros trabalhos envolvendo uma nova área de estudos denominada metalômica destacam a necessidade de melhor compreender a interação entre íons metálicos e proteínas. Esta Tese empregou o girassol (Helianthus annuus L.), uma planta da família das oleaginosas que apresenta uma relevante importância econômica na produção de óleo vegetal, bem como em processos de fitorremediação, em um estudo metalômico para avaliação de possíveis alterações na expressão de proteínas mediante o desenvolvimento das plantas irrigadas com íons selenito ou zinco. Inicialmente, o estudo avaliou quatro diferentes métodos para a adequada para extração e separação de proteínas das folhas de girassol empregando a técnica de eletroforese bidimensional em gel de poliacrilamida (2DPAGE), sendo que o melhor procedimento consistiu na extração empregando tampão na presença de fenol. A posterior caracterização das proteínas, nos spots proteicos, empregando espectrometria de massas também foi conduzida evidenciando, pela primeira vez para o organismo estudado, a expressão de 12 novas proteínas. Um estudo ionômico foi desenvolvido para avaliar a distribuição destes elementos nas diferentes estruturas das plantas, buscando correlação com os processos biológicos que estariam ocorrendo nos girassóis. O estudo revelou uma correlação entre o nível de selênio e enxofre nas plantas sugerindo a incorporação do selênio em aminoácidos utilizados para síntese de proteínas. O emprego da técnica de ablação a laser acoplada ao espectrômetro de massas com plasma acoplado indutivamente (LA-ICP-MS) também apresenta destaque na pesquisa, complementando os dados obtidos no estudo ionômico, por meio da identificação de selênio nos spots proteicos extraídos das folhas dos girassóis e separadas por 2D-PAGE. Desenvolveu-se também desenvolver um método para análise quantitativa direta e simultânea de selênio e enxofre nas folhas das plantas empregando a hifenação LA-ICP-MS, buscando correlação entre os dois elementos / Abstract: Several papers highlight a new research area called metallomic, and points out the necessity to better understand the relationship between metallic ions and proteins. This Thesis has employed the sunflower (Helianthus annuus L.), which is considered an oilseed plant with significant economical importance for the production of vegetal oil, and also used in fitoremediation processes, to carry out a metallomic study focused on the evaluation of possible changes in proteins expression due to the irrigation of the plants with selenite or zinc ions. Initially, the study evaluated four different methods for the adequate extraction and separation of sunflower leaf proteins using the two-dimensional gel electrophoresis (2DPAGE) technique and the best result was obtained using a phenol based extraction procedure. Further protein characterization in protein spots were carried out using mass spectrometry and revealed for the first time for the studied organism, the expression of 12 new proteins. An ionomic study was also developed to investigate the distribution of these elements in the different structures of the plants, looking for the correlation of obtained data with biological processes that could be occurring in the sunflowers. The study showed a correlation between selenium and sulfur levels, suggesting the incorporation of selenium into amino acids used for the synthesis of proteins. The application of laser ablation technique coupled to the inductively coupled plasma mass spectrometer (LA-ICP-MS) has also been highlighted in this research, complementing the results obtained in the ionomic study through the identification of selenium in sunflower leaves protein spots. In addition, it was evaluated an analytical procedure for the direct and simultaneous quantitative determination of selenium and sulfur in the leaves of the plants using LA-ICP-MS, searching for a correlation between both elements / Doutorado / Quimica Analitica / Doutor em Ciências

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / The fate and bioavailability of organic contaminants in plants is a major ecological and human health concern. Current wet chemistry techniques that employ strong chemical treatments and extractions with volatile solvents, such as GC-MS, HPLC, and radiolabeling, although helpful, degrade plant tissue resulting in the loss of spatial distribution and the production of artifacts. Synchrotron radiation infrared microspectroscopy (SR-IMS) permits direct analysis of plant cell wall architecture at the cellular level in situ, combining spatially localized information and chemical information from the IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers such as cellulose, lignin, and proteins in the root tissue of hydroponically grown sunflower and maize plants as well as to determine the fate and effect of several organic contaminants in those root tissues. Principal components analysis (PCA), a data compression technique, was employed to reveal the major spectral variances between untreated and organic contaminant treated root tissues. Treatment with 1H-benzotriazole (BT) caused alterations to the lignin component in the root tissue of plants. The BT was found in xylem and epidermal tissue of sunflower plants but not associated with any particular tissue in maize roots. 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) altered the pectin and polysaccharide structure in both maize and sunflower. SR-IMS revealed the reduction of DNTs to their aromatic amine form in the vascular and epidermal tissues at low concentration. At high concentration, DNTs appeared to be associated with all the plant tissues in maize and sunflower. Exposure of sunflower and maize to 2,6-dichlorophenol (2,6-DCP) caused alterations to the polysaccharide and protein component of the root tissue. In some cases, phenolic compounds were observed in the epidermal tissue of maize and sunflower roots. The results of this research indicate that SR-IMS has the potential to become an important analytical tool for determining the fate and effect of organic contaminants in plants.

Purple nutsedge (Cyperus rotundus) is a troublesome weed that outcompetes crops and contributes to poor yields. In the past, agriculturalists controlled purple nutsedge by fumigating soil with methyl bromide but the fumigant has since been classified as a controlled substance under the Montreal Protocol. This study evaluated the effectiveness of several alternative purple nutsedge control techniques and compared them with results obtained from the application of Roundup. Concentration treatment effects for the allelopathic seed powders of watercress and turnip were tested in a field trial while seed powders of yellow mustard and sunflower were tested in a potted trial. The allelopathic amendments significantly delayed weed emergence but several factors interfered with long-term effectiveness. Roundup was determined to be the most effective season-long weed control among the treatments consistently leaving the least amount of surviving weeds and underground organs.

L’efficience d’utilisation de l’eau (WUE), rapport entre la biomasse produite et l’eau consommée, est un trait essentiel à étudier en agronomie pour améliorer la production des cultures soumises à la sécheresse. Cependant, mesurer l’eau consommée par un couvert végétal est difficile à réaliser. L’objectif général de ce travail de thèse est de répondre à trois principales questions : (i) peut-on déterminer WUE en utilisant la discrimination isotopique du carbone (CID) facile à mesurer? (ii) comment l’analyse de la variabilité de WUE et CID peut contribuer à sélectionner des génotypes de tournesol soumis à la sécheresse? (iii) et les variations de WUE peuvent-elles être révélées par les variations de relations hydriques? Quatre expériences ont été conduites en serre pendant deux années : (i) avec deux scenario de sécheresse, l’une progressive, l’autre stable, et (ii) avec cinq niveaux de contenu en eau du sol stables. Les principaux traits mesurés sont WUE, CID et d’autres traits représentant les relations hydriques tels que le contrôle de la transpiration (FTSWt), la capacité d’extraction de l’eau (TTSW) et la tolérance à la déshydratation (OA). Une forte corrélation négative a été mise en évidence entre WUE et CID. Une large variabilité a également été observée pour FTSWt, TTSW et OA. Ces résultats permettent de connaitre le contrôle génétique de WUE et CID, ainsi que des traits associés, lesquels n’ont jamais été relatés dans la littérature. De plus, l’analyse QTL pour FTSWt n’a pas non plus été réalisée, chez aucune plante. Des QTL pour WUE et CID ont été identifiés pour différents scenario de sécheresse. Les QTL pour CID sont considérés comme ‘constitutifs’, parce qu’ils sont détectés dans les différents scenarios. Les QTL pour CID co-localisent avec ceux pour WUE, pour la biomasse et pour la transpiration cumulée. Des co-localisations de QTL ont également été observées entre FTSWt et TTSW, entre TTSW et WUE-CID-Biomasse, et entre FTSWt-TTSW et Biomasse. Cette étude met en évidence que WUE est physiologiquement et génétiquement associée à CID. De plus, CID représente un excellent substitue à la mesure de WUE et permet d’améliorer l’efficience d’utilisation de l’eau par sélection assistée par marqueurs. / Water use efficiency (WUE), measured as the ratio of plant biomass to water consumption, is an essential agronomical trait for enhancing crop production under drought. Measuring water consumption is logistically difficult, especially in field conditions. The general objective of the present Thesis is to respond to three main questions: (i) can WUE be determined by using carbon isotope discrimination (CID), easy to measure?, (ii) how WUE and CID variation analysis can contribute to the genotypic selection of sunflower subjected to drought?, and (iii) can WUE variation be revealed by the variation of plant-water relation traits. Four experiments were carried out in greenhouse across two different years: (i) on two drought scenarios, progressive soil drying and stable water-stress, and (ii) on five levels of soil water content. The main traits that have been measured include WUE, CID, as well as plant-water relation traits, i.e. control of transpiration (FTSWt), water extraction capacity (TTSW), and dehydration tolerance (OA). A highly significant negative correlation was observed between WUE and CID, and a wide phenotypic variability was observed for both WUE and CID. A wide variability was also observed for FTSWt, TTSW and OA. The results provide new insight into the genetic control of WUE and CID related-traits, which, unlike to other crops, genetic control of WUE, CID, and TTSW in sunflower have never been reported in the literature. Further, quantitative trait loci (QTL) mapping for FTSWt was never reported in any plant species. The QTL for WUE and CID were identified across different drought scenarios. The QTL for CID is considered as a ‘‘constitutive’’ QTL, because it is consistently detected across different drought scenarios. The QTL for CID co-localized with the QTL for WUE, biomass and cumulative water transpired. Co-localization was also observed between the QTL for FTSWt and TTSW, between the QTL for TTSW and WUE-CID-biomass, as well as between the QTL for FTSWt-TTSW and biomass. This study highlights that WUE is physiologically and genetically associated with CID. CID is an excellent surrogate for WUE measurement, and can be used to improve WUE by using marker-assisted selection (MAS) to achieve the ultimate goal of plant breeding at genomic level.

Thesis (MSc. Agriculture (Agricultural Economics)) -- University of Limpopo, 2016 / The study focused on the market participation of the smallholder sunflower farmers of Sekhukhune District in Limpopo Province of South Africa. A total of 100 smallholder sunflower farmers of Sekhukhune were randomly selected for the study. Data was gathered through the use of structured questionnaires with open-ended questions. The data was analysed using the Logistic Regression model on SPSS 26.0 (statistical package for social sciences). The probability of the smallholder sunflower farmers participating in markets was determined significantly by gender of household head, farm size, level of education, marketing experience, distance to the market, ownership of vehicle and stakeholder availability. Recommendations were made with regard to that of training, monitoring, funding, Government support, access to production land and the involvement of organizations with the smallholder sunflower farmers. Training should be provided for the smallholder sunflower farmers because farmers are always faced with new technology and market changes, there should be a budget that is put aside for the funding of the smallholder farmers, monitoring programs should be implemented in order to monitor the progress of the smallholder farmers, increased access to production land for farming, government support through financial and input support, smallholder farmers should get involved with organizations that can help them with training and funding, and the government should implement policies that are commodity based. Key words: smallholder farmers, market participation, Logistic Regression Model, sunflower, Limpopo province

Please read the abstract (Summary) in the section, 09summary and aknowledgements, of this document / Dissertation (MSc (Entomology))--University of Pretoria, 1988. / Zoology and Entomology / MSc / unrestricted

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used e.g. as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l-1 sucrose, 0.5 mg l-1 of the auxin 1-naphthalene acetic acid and 0.5 mg l-1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.

Wirkstoffgewinnung mittels chemischer Synthese führt oft zu Stereoisomeren, welche aufwendig getrennt werden müssen und manche Moleküle sind nur sehr kostenintensiv oder gar nicht darstellbar. Landwirtschaftliche Gewinnung bedeutet Nachteile wie Schadstoffeinsatz und großer Flächenbedarf. Der Einsatz von pflanzlichen Zell- und Gewebekulturen überwindet die genannten Hürden [1, 2]: mit Methoden der Pflanzenbiotechnologie ist es möglich, pflanzliche Inhaltsstoffe in ihrer natürlichen, bioaktiven Form das ganze Jahr über unabhängig von biotischen/abiotischen Umweltfaktoren bei gleichbleibender Qualität und Quantität zu produzieren [3, 4]. Suspensionskulturen und hairy roots gelten momentan als die in vitro-Kulturtypen mit dem größten biotechnologischen Potential. Erstere sind in Flüssigmedium kultivierte Kalluszellen. Bei Kallus handelt es sich um undifferenzierte Pflanzenzellen, welche tumorartig wachsen und durch Zugabe von Pflanzenhormonen an der Differenzierung gehindert werden. Hairy roots entstehen durch Infektion eines Pflanzenteils mit dem Bodenbakterium Agrobacterium rhizogenes. Die so erhaltene Haarwurzelkultur kann ohne Hormonzusatz vermehrt werden, ihre Morphologie erfordert aber häufig eine Anpassung bestehender Kultivierungsgefäße [2, 5]. / In advance of industrial applications of in vitro plant cell or tissue cultures e.g., as bioactive ingredients for pharmaceuticals, an intense characterization concerning growth and productivity has to be performed. Innovative respiration measurement techniques in shake flask scale were applied to investigate and compare heterotrophic, photomixotrophic and hairy root cultures of sunflower. Furthermore, the qualification of RAMOS for screening of plant in vitro cultures is discussed.

Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits for plants, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. In order to have a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) by phenotyping a diversity panel of cultivated sunflower (Helianthus annuus) for root colonization under inoculation with the AM fungus Rhizophagus intraradices. This mapping panel consists of 261 inbred lines that capture approximately 90% of the genetic diversity present in the cultivated sunflower germplasm. Using a mixed linear model approach with a high-density genetic map, we determined regions of the genome that are likely associated with AM colonization in sunflower. Additionally, we used a ‘core 12' set of twelve diverse lines (representing approximately 50% of the genetic diversity in the cultivated germplasm) to assess the effect that inoculation with AM fungi has on dried shoot biomass and macronutrient uptake. Colonization rate among lines in the mapping panel ranged from 0 to 70% and was not correlated with mycorrhizal growth response, shoot P response, or shoot K response among the core 12 lines. Association mapping yielded three SNPs that were significantly associated with AM colonization rate. These SNPs explained 19.0%, 14.4%, and 27.9% of the variance in three different metrics used to measure the degree of root colonization. Three genes of interest identified from the significant regions that contained these SNPs are potentially related to plant defense. Overall, our data suggests that candidate genes involved in plant defense may affect AM colonization rates within cultivated sunflower, and that these genes have a large effect size.

Los mercados internacionales de aceites comestibles se caracterizan por la competitividad y las exigencias de calidad, lo que localmente demanda mejores métodos para la caracterización y el control de calidad de materias primas y productos, la optimización de las tecnologías y condiciones de procesamiento y almacenamiento, y el desarrollo de productos o procesos alternativos que permitan el mejor aprovechamiento de los subproductos. El complejo aceitero argentino es uno de los sectores de la industria alimenticia que ha evidenciado un gran crecimiento durante los últimos años, habiendo incrementado significativamente su capacidad de procesamiento. El aceite de girasol es, en nuestro país, el segundo en importancia después del de soja, teniendo en cuenta sus características de sabor suave y su tenue color amarillo, resulta uno de los preferidos para su empleo doméstico o industrial. La extracción por solvente es la etapa fundamental en la obtención de aceites vegetales a partir de semillas oleaginosas, es un proceso complejo debido a las características de la estructura celular del vegetal y a que junto a los triglicéridos son extraídos los componentes minoritarios. En nuestro país la industria oleaginosa destina la mayor parte de su producción a la exportación, por lo que las exigencias de calidad que enfrentan requieren de la optimización de los procesos de fabricación y un conocimiento detallado de las variables que los afectan. Es por ello que los estudios realizados en esta área tienen una aplicación inmediata en la industria local, y son de gran importancia tanto científica como técnica. Los objetivos de esta tesis son: 1. Estudiar el proceso de extracción por solvente de aceites vegetales, específicamente aceite de girasol, considerando la influencia de los compuestos minoritarios en el procesamiento de la materia prima y la calidad del aceite obtenido. 2. Estudiar la extracción de ceras de girasol mediante el lavado de la semilla evaluando la conveniencia del uso de distintos solventes y el efecto del tiempo de contacto y la temperatura. A continuación se detalla la organización del trabajo realizado. En cada uno de los capítulos se describen resultados, conclusiones y bibliografía consultada. En el capítulo 1 se realiza una descripción del panorama mundial de la demanda de aceite de girasol y de los subproductos del sistema oleaginoso. Se analiza brevemente la producción de aceite de girasol en Argentina y se plantean los objetivos específicos de la tesis. En el capítulo 2 se describe al Helianthus annuus L. y la composición de aceite analizando compuestos mayoritarios y minoritarios (tocoferoles, fosfolípidos y ceras). Se exhibe una breve reseña del procesamiento de las semillas de girasol para la extracción del aceite y de los procesos de refinamiento del aceite crudo. El capítulo 3 describe las materias primas utilizadas y presenta la metodología experimental empleada. Se detallan las técnicas utilizadas para la cuantificación de los compuestos minoritarios de interés que contribuyen a la calidad y estabilidad del aceite (fosfolípidos, tocoferoles y ceras) En el capítulo 4 se caracteriza la materia prima. Se analizan las características generales y distribución de tamaño de las semillas utilizadas en el estudio de extractabilidad de ceras, así como la distribución en la semilla de los compuestos menores (fosfolípidos, tocoferoles y ceras). En cuanto a las muestras procedentes del proceso de extracción de aceite se presenta un análisis del contenido de humedad, aceite y compuestos minoritarios. En el capítulo 5 se estudia la extracción de ceras de girasol mediante el lavado de las semillas, evaluando la conveniencia del uso de distintos solventes y el efecto del tiempo de contacto y la temperatura. Se analiza también el efecto de este método de eliminación de ceras sobre la aptitud al descascarado que presentan las semillas. En el capítulo 6 se exponen las curvas de extracción del aceite de expellers de girasol y de los compuestos minoritarios (tocoferoles, fosfolípidos y ceras), obtenidas en un sistema batch, a diferentes temperaturas. El objetivo fue obtener curvas de extracción y la aplicación de un modelo para la determinación de los coeficientes de difusividad efectivos. En el capítulo 7 se presenta un análisis de la variación de la composición del aceite y calidad del mismo simulando las distintas etapas de un extractor industrial en estado estacionario. La información obtenida de las curvas cinéticas del aceite y de los compuestos menores se incorporó en un programa de simulación teniendo en cuenta varias etapas de extracción. El capítulo 8 resume las conclusiones generales y propone algunas futuras líneas de investigación. / The international markets of edible oils are characterized by the competitiveness and the demands of quality. This fact locally requires of improved methods for the characterization and the quality control of raw material and products, the optimization of the technologies and the processing and storage conditions, and the development of products or alternative processes that allow the best by-products use. The Argentinean oily complex is one of the sectors of the food industry that has evidenced a huge growth during the last years, having increased its processing capacity significantly. The sunflower oil is, in our country, the second in importance after soya oil. Due to its soft flavor and delicate yellow color, it is preferred domestic or industrial use. The solvent extraction is the fundamental stage in the vegetable-oil production from oilseeds. It is a complex process due to both the characteristics of the cellular structure and the extraction of minor compounds together with the triglycerides. In our country, oil enterprises assign a high percentage of its production for export. In this way, they confront a quality demand that needs of the process optimization together with a depth knowledge of the variables that affect the oil extraction. That is the reason of why the studies carried out in this area have an immediate application in the local industry, and they are of great scientifical and technical relevance. The objectives of this thesis are: 1. Study the solvent extraction process of vegetable oils, specifically sunflower oil, considering the influence of the minor compounds in the processing of the raw material and the quality of the obtained oil. 2. Study the extraction of sunflower waxes through the seed washing, evaluating the suitability of the use of different solvents and the effect of contact time and temperature. The organization of the work is given below. Results, conclusions and reviewed bibliography are described in each chapter. Chapter 1 shows the world demand of sunflower oil and their by-products. The production of sunflower oil in Argentina is analyzed shortly and the specific objectives of the thesis are outlined. Chapter 2 describes the Helianthus annuus L. specie, and the composition of its oil analyzing majority and minority compounds (tocopherols, phospholipids and waxes). A brief review about the oil extraction process and its refining is exhibited. Chapter 3 gives a general description of the raw materials and describes the experimental methodology used in this thesis. In addition, the techniques used for the quantification of minor compounds that contribute to the quality and stability of the oil (phospholipids, tocopherols and waxes) are detailed. Chapter 4 characterizes the raw materials. The main characteristics and size distribution of the seeds used in the waxes extractability study as well as the distribution of the minor compounds (phospholipids, tocopherols and waxes) in the seed are analyzed, an analysis of the humidity, oil and minor-compounds contents of the samples coming from the oil extraction process is presented. Chapter 5 studies the extraction of sunflower waxes through the washing of the seeds, evaluating the convenience of the use of different solvents and the effect of contact time and temperature. It is also analyzed the solvent washing effect on both the wax removal and the seed dehulling ability. Chapter 6 gives the extraction curves of major (triglycerides) and minor compounds (tocopherols, phospholipids and waxes) obtained from sunflower expellers at different temperatures and batch system. A model is applied for the determination of effective-diffusion coefficients. Chapter 7 presents an analysis of the variation of oil composition and quality, simulating the different stages of an industrial extractor in stationary state. The obtained information of the kinetic curves of the oil and of the minor compounds was implemented in a simulation software that takes into account several extraction stages. Chapter 8 summarizes the main conclusions and proposes some future investigation lines.