Many economically important crop systems exhibit an Age-Related Resistance (ARR) response whereby mature plants become resistant to pathogens they were susceptible to when younger. The signaling pathways and mechanisms of ARR have not been well studied. Arabidopsis displays ARR in response to P. syringae pv tomato (Pst). Several studies provide evidence that intercellular salicylic acid (SA) accumulation is required for ARR and SA acts as a direct antimicrobial agent to limit bacterial growth and biofilm-like aggregate formation. SA accumulation mutants are ARR defective; however, a modest level of resistance is occasionally observed leading to the hypothesis that other compounds contribute to ARR as antimicrobial agents. Previous studies demonstrated that CYP71A13 (a key enzyme in indolic biosynthesis) is expressed during the ARR response. I demonstrated that CYP71A12 functionally compensated for CYP71A13 during ARR, as cyp71a12/cyp71a13-1 mutants were consistently ARR-defective compared to their respective single mutants. I demonstrated that dihydrocamalexic acid (DHCA) accumulated in intercellular washing fluids (IWFs) collected from plants during the ARR response using high resolution mass spectrometry-based profiling. DHCA was detected in IWFs collected from wild-type ARR-competent plants and, was absent in IWFs from ARR-incompetent cyp71a12/cyp71a13 mutants. In vitro DHCA antimicrobial activity against P. syringae was not observed, but exogenous infiltration of DHCA into the leaf intercellular space restored ARR in cyp71a12/cyp71a13 mutants Unlike SA which exhibits direct antimicrobial activity against P. syringae, DHCA does not and instead may affect pathogen virulence in other ways. My research provides evidence that intercellular DHCA contributes to ARR in response to P. syringae in Arabidopsis. Understanding the genes and metabolites contributing to ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease. / Thesis / Master of Science (MSc) / During Age-Related Resistance (ARR), mature plants including some crop plants become resistant to pathogens they were susceptible to when younger. How ARR works is poorly understood. My objective was to identify potential antimicrobial metabolites contributing to ARR in Arabidopsis against the bacterial pathogen Pseudomonas syringae. Genetic analyses combined with mass-spectrometry based metabolite profiling demonstrates that two cytochromes P450, CYP71A12 and CYP71A13 contribute to ARR. My research provides evidence that DHCA accumulates in the leaf intercellular space in ARR-competent plants, where it may act to inhibit the bacterial infection process. DHCA has low antimicrobial activity against P. syringae suggesting its mechanism of action is not directly antimicrobial. Importantly, application of DHCA to the leaf intercellular space of cyp71a12/cyp71a13 restored ARR, confirming that DHCA contributes to ARR in Arabidopsis. Understanding ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease.

<p dir="ltr">Plants emit an amazing diversity of volatile organic compounds (VOCs) that in addition to being utilized by humans for a multitude of applications, allow plants to communicate with their environment, and play numerous roles in plant growth and development. Plants must be able to perceive and distinguish between VOC cues mediating plant-plant, plant-insect, and plant-microbe interactions to appropriately respond to stimuli. Due to the plethora of biological processes dependent on VOCs, significant progress has been made towards understanding the biosynthesis of plant VOCs and their regulation, and, in recent years, the molecular mechanisms involved in VOC emission. However, to date, little is known about how VOCs are perceived by plants and trigger cellular response(s). In animals, VOCs are recognized by odorant receptors known as G-protein-coupled receptor (GPCR) proteins. However, the few GPCR genes identified in plants appear to have different functions and the lack of a reliable marker for VOC perception has hampered research in this field.</p><p dir="ltr">The discovery of natural fumigation of terpenoids in petunias provides a means of studying VOC perception and the downstream signaling pathways by providing a visual indicator of perception. Transcriptomic analysis of wild-type and transgenic petunias deficient in terpenoid synthesis revealed a link between terpene perception in pistils with the karrikin-like signaling pathway. By utilizing biochemical, computational, and in planta experiments, we demonstrate that of the four petunia karrikin-insensitive receptors (PhKAI2), one of the Lamiid-specific KAI2 intermediate clade receptors, PhKAI2ia, can stereo-specifically perceive the (−)-germacrene D signal emitted from the floral tubes, triggering a KAI2-mediated signaling cascade and affecting plant fitness. Downregulation of PhKAI2ia results in significantly smaller stigmas compared to wild-type, and the phenotype cannot be complemented by the treatment of pistils with (−)-germacrene D, indicating that PhKAI2ia transgenic plants are acting as deaf receptors. We also show that the binding of (−)-germacrene D to PhKAI2ia is sufficient to induce complex formation with more axillary growth 2 (PhMAX2) and the subsequent degradation of suppressor of MAX2 (PhSMAX1a).</p><p dir="ltr">Altogether, our research uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).</p>

Este trabalho estabelece uma comparação entre amêndoas de Iryanthera Grandis Ducke (Myristicaceae) atacadas por insetos e amêndoas inteiras através do isolamento, identificação e determinação estrutural de alguns de seus constituintes químicos. Os extratos hexânicos foram fracionados por técnicas cromatográficas e forneceram a neolignana (8R,7\'S,8\'S)-2,3-dimetil-4-(p-hidroxifenil)-6-hidroxitetralina e a lactona (2S,3S,4S)-2-(heptadecil-17\'-fenil)-3-hidroxi-4-metilbutanolido, além de dois tocotrienóis: 2,8-dimetil-2-(4,8,12-trimetil-3,7,11-tridecatrienil)-6-cromanol e 2,8-dimetil-2-(4,12-dimetil-8-carbóxi-3,7,11-tridecatrienil)-6-cromanol, sendo o primeiro encontrado apenas nas amêndoas atacadas por insetos e o segundo, apenas nas amêndoas inteiras. Estas 4 substâncias já haviam sido isoladas de I. grandis em trabalho anterior. Um estudo quantitativo revelou a presença de 25% em massa a mais de metabólitos especiais nas amêndoas atacadas por insetos, quando comparadas às amêndoas inteiras, (provavelmente devido a uma maior concentração de triglicerídeos nas amêndoas inteiras). A investigação fitoquímica dos extratos etanólicos forneceu, após partição com solventes e fracionamentos cromatográficos, incluindo o uso de cromatografia líquida de alta eficiência, 4 flavonolignóides inéditos designados de iryantherinas G, H, I e J. A biossíntese de flavonolignóides envolve provavelmente o ataque eletrofilico de álcool cinamílico ao anel floroglucinólico de uma diidrochalcona. No caso destas iryantherinas, o álcool cinamílico é representado pela unidade 1,4-diaril-2,3-dimetilbutílica. As propostas estruturais das substâncias isoladas foram baseadas no exame de espectros dos produtos naturais e/ou dos derivados acetilados. As técnicas espectrométricas utilizadas foram EM, RMN de 1H e de 13C, RMN bidimensional HOMOCOSY 1H-1H, experimentos de DEPT 135° e para a observação do Efeito Nuclear Overhauser. / This work describes the comparison between unspoiled fruits of Iryanthera Grandis (Ducke) and fruits which had been attacked by insects by means of isolation, identification and structural elucidation of some of its chemical constituents. Hexanic extracts submitted to chromatographic techniques afforded neolignan (8R,7\'S,8\'S)-2,3-dimethyl-4-(p-hydroxyphenil)-6-hydroxytetraline and lactone (2S,3S,4R)-2-(heptadecyl-17\'-phenyl)-3-hydroxy-4-methyl\'butanolide, besides two tocotrienols: 2,8-dimethyl-2-(4,8,12-trimethyl-3,7,11-tridecatrienyl)-6-cromanol and 2,8-dimethyl-2-(4,12-dimethyl-8-carboxyl-3,7,11-tridecatrienyl)-6-cromanol. Ethanolic extracts submitted to partition and chromatografic analysis including HPLC techmques afforded 4 new flavonolignoids, iryantherins G, H, I and J. Biosynthesis of flavonolignoids results from eletrophyllic attack of cinnamyl alcohol to the phloroglucinolic ring of a dihydrochalcone. In this work, the cinnamyl alcohol is represented by the 1,4-diaryl-2,3-dimethyl unit. Structural proposals of isolated compounds were based on MS, 1H and 13C NMR, bidimensional HOMOCOSY 1H-1H, DEPT 135&#176; and NOE experiments.

A cana-de-açúcar do ponto de vista socioeconômico e do agronegócio é atualmente uma das culturas mais importantes e em ascensão em regiões dos trópicos e subtrópicos do mundo pela sua produção de biomassa e seu potencial como biocombustível. Para expandir o seu cultivo é necessário uma série de estudos, sobretudo no que se refere ao levantamento de características ecofisiológicas e agroclimáticas envolvidas na produção. A carência de estudos, juntamente com a baixa diversidade genética dos genótipos utilizados nos programas de melhoramento, limita a oferta de variedades adaptadas com alto potencial de rendimento. Desde a revolução verde, é reconhecido o papel da nutrição mineral no aumento da produtividade das culturas. Notadamente, a carência de nitrogênio é um dos principais fatores limitantes da produtividade canavieira. Assim, os objetivos gerais desta tese visam contribuir para a compreensão dos mecanismos de regulação da nutrição nitrogenada (eficiência no uso do nitrogênio), a partir dos processos envolvidos no transporte e redução do nitrato e seus efeitos no balanço carbono - nitrogênio, nas fases iniciais de desenvolvimento da cana-deaçúcar. Para isto foi feito um estudo nutricional nas variedades RB83-5089 e SP80- 3280 por até 60 dias, num sistema hidropônico. Foi utilizada a solução de Hoagland e Arnon (1950) modificada como base nutricional na qual foram adicionadas doses baixas (0.05; 0.1; 0.25; 0.5 mM) e altas (1.0; 2.5; 5.0; 10.0; 15.0 e 25.0 mM) de nitrato para medir os efeitos bioquímicos e fisiológicos na atividade da nitrato redutase em folhas e raízes. Foram utilizados como parâmetros o índice de massa e massa seca da parte aérea e raízes, o conteúdo e a razão clorofila \"a\" e \"b\", a taxa de assimilação fotossintética, a condutância estomática e a transpiração. Os resultados mostraram que a variedade SP80-3280 foi mais responsiva às aplicações de baixas e altas doses de nitrato e, de forma geral, a atividade da nitrato redutase não apresentou limitação na mobilização de nitrato via xilema e se confirmou que a cinética do processo foi dependente da dose fornecida para as duas variedades. Nas análises dos teores de clorofila \"a\", as duas variedades foram responsivas ao aumento das doses de N, entretanto, os teores de clorofila \"b\" foram direta e inversamente proporcionais às doses de N nas variedades SP80-3280 e RB83-5089, respectivamente. A análise de índice de massa junto com a massa seca permitiu estabelecer que não houveram diferenças significativas entre estas variedades no acúmulo de matéria seca. / The sugarcane from the standpoint of socioeconomic and agribusiness is currently one of the most important crops in the tropics and subtropics areas of the world and it is in rising growth for its production of biomass and potential as biofuel. In order to expand the cultivation it is necessary a series of studies, especially with regard to agroclimatical ecophysiological characteristics involved in the production. The lack of studies, along with low genetic diversity of the genotypes used in breeding programs, limits the supply of adapted varieties with high yield potential. Since the green revolution, it is recognized the importance of nutrition to increase crop productivity. Notably, the deficiency of nitrogen is one of the main limiting factors affecting sugarcane yield. Thus, the overall objectives of this thesis aim to contribute to understand the regulatory mechanisms of nitrogen nutrition (nitrogen use efficiency), by the processes involved in the transport, nitrate reduction and its effects on the carbon - nitrogen balance, in early stages of development of sugarcane. To this, a nutritional study was prepared using sugarcane varieties RB83-5089 and SP80-3280 for 60 days in a hydroponic system. It was used Hoagland and Arnon (1950) modified solution as the nutritional basis on which was added low (0.05, 0.1, 0.25; 0.5 mM) and high (1.0, 2.5, 5.0, 10.0, 15.0 and 25.0 mM) doses of nitrate to measure the biochemical and physiological effects on the activity of nitrate reductase in leaves and roots. The parameters analysed were, the index of mass and dry weight of shoots and roots, chlorophyll \"a\" and \"b\" content and its ratio, photosynthetic assimilation rate, stomatal conductance and transpiration. The results showed that the variety SP80-3280 was more responsive to low and high levels of nitrate and, in general, the activity of nitrate reductase showed no limitation in the mobility of nitrate via xylem and it confirmed that the kinetics of the process was dependent on the doses provided to the two varieties. In the analysis of chlorophyll \"a\" content, the two varieties were responsive to the increase of N doses. However, the level of chlorophyll \"b\" was directly and inversely proportional to N rates in SP80- 3280 and RB83-5089, respectively. The analysis of mass index with dry weight allowed establishing that there were no significant differences between these varieties regarding to dry tissue accumulation.

Chicory (Cichorium intybus L.), a valuable forage for ruminant livestock in temperate regions, appears highly degradable in the rumen. Fundamental reasons for the rapid breakdown of chicory cell walls in the rumen were studied. Cell walls were isolated from laminae and midribs of chicory (cv. Grasslands Puna II) leaves. The walls, which, except for the walls of xylem tracheary elements in vascular bundles, were non-lignified, were fractionated progressively with 50 mM CDTA, 50 mM Na2CO3, 1 M KOH, 4 M KOH, 4 M KOH + 3.5% H3BO3, and hot water. The polysaccharides were similar to those in nonlignified walls of other dicotyledons, but with high proportions of pectic polysaccharides (67% of the total wall polysaccharides in the laminae). These included homogalacturonans (HGs, 50% of the total wall polysaccharides in laminae) and rhamnogalacturonan I (RG I). In contrast, the proportions of cellulose, xyloglucans, heteroxylans and glucomannans were low. The locations of different pectic polysaccharides were determined using the monoclonal antibodies JIM5 and JIM7 against HGs with low and high degrees of methyl esterification, respectively, LM6 against arabinan and LM5 against galactan. All primary walls were labelled with all the antibodies used. However, the middle lamella, tricellular junctions and the corners of intercellular spaces were labelled with JIM5 and JIM7, but not with LM5. The middle lamella was labelled with LM6, but not the corners of intercellular spaces. These results support the involvement in cell adhesion of HGs with low degrees of methyl esterification. A preparation of endopolygalacturonase (endo-PG) was used to investigate cell adhesion, and its effect on forage particle breakdown was determined using weight loss, chemical analysis and immunofluorescence labelling. The preparation dramatically reduced particle size. Cell separation was accompanied by a loss of HGs with low degrees of methyl esterifcation from the middle lamella and corners of intercellular spaces. A consequential loss of cell adhesion evidently caused leaf breakdown. The degradation of fresh chicory leaves by rumen bacteria was investigated by measuring weight loss, monosaccharide release and immunocytolabelling. Two bacteria, the pectolytic Lachnospira multiparus D32 and the cellulolytic Fibrobacter succinogenes S85, effectively degraded chicory. Pectic polysaccharides were degraded faster than other wall polysaccharides, with uronic acid released faster and more completely than neutral monosaccharides. The preponderance of non-lignified primary walls and abundance of pectic polysaccharides may account, in part, for the rapid degradation of forage chicory in the rumen. The HGs in the middle lamellae and corners of intercellular spaces probably have a role in cell adhesion, and their degradation is probably responsible for the rapid reduction in the particle size of chicory leaves in the rumen.

Este trabalho estabelece uma comparação entre amêndoas de Iryanthera Grandis Ducke (Myristicaceae) atacadas por insetos e amêndoas inteiras através do isolamento, identificação e determinação estrutural de alguns de seus constituintes químicos. Os extratos hexânicos foram fracionados por técnicas cromatográficas e forneceram a neolignana (8R,7\'S,8\'S)-2,3-dimetil-4-(p-hidroxifenil)-6-hidroxitetralina e a lactona (2S,3S,4S)-2-(heptadecil-17\'-fenil)-3-hidroxi-4-metilbutanolido, além de dois tocotrienóis: 2,8-dimetil-2-(4,8,12-trimetil-3,7,11-tridecatrienil)-6-cromanol e 2,8-dimetil-2-(4,12-dimetil-8-carbóxi-3,7,11-tridecatrienil)-6-cromanol, sendo o primeiro encontrado apenas nas amêndoas atacadas por insetos e o segundo, apenas nas amêndoas inteiras. Estas 4 substâncias já haviam sido isoladas de I. grandis em trabalho anterior. Um estudo quantitativo revelou a presença de 25% em massa a mais de metabólitos especiais nas amêndoas atacadas por insetos, quando comparadas às amêndoas inteiras, (provavelmente devido a uma maior concentração de triglicerídeos nas amêndoas inteiras). A investigação fitoquímica dos extratos etanólicos forneceu, após partição com solventes e fracionamentos cromatográficos, incluindo o uso de cromatografia líquida de alta eficiência, 4 flavonolignóides inéditos designados de iryantherinas G, H, I e J. A biossíntese de flavonolignóides envolve provavelmente o ataque eletrofilico de álcool cinamílico ao anel floroglucinólico de uma diidrochalcona. No caso destas iryantherinas, o álcool cinamílico é representado pela unidade 1,4-diaril-2,3-dimetilbutílica. As propostas estruturais das substâncias isoladas foram baseadas no exame de espectros dos produtos naturais e/ou dos derivados acetilados. As técnicas espectrométricas utilizadas foram EM, RMN de 1H e de 13C, RMN bidimensional HOMOCOSY 1H-1H, experimentos de DEPT 135° e para a observação do Efeito Nuclear Overhauser. / This work describes the comparison between unspoiled fruits of Iryanthera Grandis (Ducke) and fruits which had been attacked by insects by means of isolation, identification and structural elucidation of some of its chemical constituents. Hexanic extracts submitted to chromatographic techniques afforded neolignan (8R,7\'S,8\'S)-2,3-dimethyl-4-(p-hydroxyphenil)-6-hydroxytetraline and lactone (2S,3S,4R)-2-(heptadecyl-17\'-phenyl)-3-hydroxy-4-methyl\'butanolide, besides two tocotrienols: 2,8-dimethyl-2-(4,8,12-trimethyl-3,7,11-tridecatrienyl)-6-cromanol and 2,8-dimethyl-2-(4,12-dimethyl-8-carboxyl-3,7,11-tridecatrienyl)-6-cromanol. Ethanolic extracts submitted to partition and chromatografic analysis including HPLC techmques afforded 4 new flavonolignoids, iryantherins G, H, I and J. Biosynthesis of flavonolignoids results from eletrophyllic attack of cinnamyl alcohol to the phloroglucinolic ring of a dihydrochalcone. In this work, the cinnamyl alcohol is represented by the 1,4-diaryl-2,3-dimethyl unit. Structural proposals of isolated compounds were based on MS, 1H and 13C NMR, bidimensional HOMOCOSY 1H-1H, DEPT 135&#176; and NOE experiments.

A cana-de-açúcar do ponto de vista socioeconômico e do agronegócio é atualmente uma das culturas mais importantes e em ascensão em regiões dos trópicos e subtrópicos do mundo pela sua produção de biomassa e seu potencial como biocombustível. Para expandir o seu cultivo é necessário uma série de estudos, sobretudo no que se refere ao levantamento de características ecofisiológicas e agroclimáticas envolvidas na produção. A carência de estudos, juntamente com a baixa diversidade genética dos genótipos utilizados nos programas de melhoramento, limita a oferta de variedades adaptadas com alto potencial de rendimento. Desde a revolução verde, é reconhecido o papel da nutrição mineral no aumento da produtividade das culturas. Notadamente, a carência de nitrogênio é um dos principais fatores limitantes da produtividade canavieira. Assim, os objetivos gerais desta tese visam contribuir para a compreensão dos mecanismos de regulação da nutrição nitrogenada (eficiência no uso do nitrogênio), a partir dos processos envolvidos no transporte e redução do nitrato e seus efeitos no balanço carbono - nitrogênio, nas fases iniciais de desenvolvimento da cana-deaçúcar. Para isto foi feito um estudo nutricional nas variedades RB83-5089 e SP80- 3280 por até 60 dias, num sistema hidropônico. Foi utilizada a solução de Hoagland e Arnon (1950) modificada como base nutricional na qual foram adicionadas doses baixas (0.05; 0.1; 0.25; 0.5 mM) e altas (1.0; 2.5; 5.0; 10.0; 15.0 e 25.0 mM) de nitrato para medir os efeitos bioquímicos e fisiológicos na atividade da nitrato redutase em folhas e raízes. Foram utilizados como parâmetros o índice de massa e massa seca da parte aérea e raízes, o conteúdo e a razão clorofila \"a\" e \"b\", a taxa de assimilação fotossintética, a condutância estomática e a transpiração. Os resultados mostraram que a variedade SP80-3280 foi mais responsiva às aplicações de baixas e altas doses de nitrato e, de forma geral, a atividade da nitrato redutase não apresentou limitação na mobilização de nitrato via xilema e se confirmou que a cinética do processo foi dependente da dose fornecida para as duas variedades. Nas análises dos teores de clorofila \"a\", as duas variedades foram responsivas ao aumento das doses de N, entretanto, os teores de clorofila \"b\" foram direta e inversamente proporcionais às doses de N nas variedades SP80-3280 e RB83-5089, respectivamente. A análise de índice de massa junto com a massa seca permitiu estabelecer que não houveram diferenças significativas entre estas variedades no acúmulo de matéria seca. / The sugarcane from the standpoint of socioeconomic and agribusiness is currently one of the most important crops in the tropics and subtropics areas of the world and it is in rising growth for its production of biomass and potential as biofuel. In order to expand the cultivation it is necessary a series of studies, especially with regard to agroclimatical ecophysiological characteristics involved in the production. The lack of studies, along with low genetic diversity of the genotypes used in breeding programs, limits the supply of adapted varieties with high yield potential. Since the green revolution, it is recognized the importance of nutrition to increase crop productivity. Notably, the deficiency of nitrogen is one of the main limiting factors affecting sugarcane yield. Thus, the overall objectives of this thesis aim to contribute to understand the regulatory mechanisms of nitrogen nutrition (nitrogen use efficiency), by the processes involved in the transport, nitrate reduction and its effects on the carbon - nitrogen balance, in early stages of development of sugarcane. To this, a nutritional study was prepared using sugarcane varieties RB83-5089 and SP80-3280 for 60 days in a hydroponic system. It was used Hoagland and Arnon (1950) modified solution as the nutritional basis on which was added low (0.05, 0.1, 0.25; 0.5 mM) and high (1.0, 2.5, 5.0, 10.0, 15.0 and 25.0 mM) doses of nitrate to measure the biochemical and physiological effects on the activity of nitrate reductase in leaves and roots. The parameters analysed were, the index of mass and dry weight of shoots and roots, chlorophyll \"a\" and \"b\" content and its ratio, photosynthetic assimilation rate, stomatal conductance and transpiration. The results showed that the variety SP80-3280 was more responsive to low and high levels of nitrate and, in general, the activity of nitrate reductase showed no limitation in the mobility of nitrate via xylem and it confirmed that the kinetics of the process was dependent on the doses provided to the two varieties. In the analysis of chlorophyll \"a\" content, the two varieties were responsive to the increase of N doses. However, the level of chlorophyll \"b\" was directly and inversely proportional to N rates in SP80- 3280 and RB83-5089, respectively. The analysis of mass index with dry weight allowed establishing that there were no significant differences between these varieties regarding to dry tissue accumulation.

<p dir="ltr">Photosynthesis is the basis of primary productivity on Earth. Oxygenic photosynthesis utilizes the nearly inexhaustible energy of radiant solar light to fix atmospheric carbon dioxide into usable forms of chemical energy and produces dioxygen as a product. Central to this process are several large hetero-oligomeric protein complexes that comprise the photosynthetic electron transport chain. Photosystem II (PSII) initiates electron transport through the light-driven oxidation of water, in-turn relinquishing protons and oxygen. Through this reaction, electrons are used to form the reductant NADPH, while protons form a proton-motive gradient that is used to drive synthesis of ATP. As a result of this highly energetic reaction, PSII is often subject to oxidative photodamage due to the production of reactive oxygen species. Inevitably, accumulation of oxidative photodamage disrupts the catalytic activity of PSII, resulting in a loss of photosynthetic activity. To deal with the nearly constant incurred photodamage to PSII, oxygenic photoautotrophs undergo a disassembly and repair cycle that results in the complete turnover of the damaged D1 subunit of PSII. Due to its high tendency for damage, the D1 subunit has a half-life of under one hour in high light intensity. Despite our current understanding of photoinhibition and PSII repair, it is still unclear how D1 is replaced so rapidly in response to damaging conditions. Previous research has indicated a role for phosphorylation of PSII in D1 turnover, however the mechanism has not been totally resolved. In the first chapter of this thesis, our current understanding of PSII phosphorylation and oxidative damage is reviewed in the context of PSII repair. In the second chapter, the role of protein phosphorylation in the PSII repair cycle is investigated in the model organism <i>Arabidopsis</i>. Using several PSII phosphorylation mutants, we demonstrate that phosphorylation seems to mediate disassembly of large PSII supercomplexes and dimers into smaller subcomplexes. In the third chapter, the role of oxidative photodamage is investigated in mediating PSII disassembly. Here, we use several <i>in vitro</i> assays to demonstrate that photodamage is sufficient to induce the disassembly of smaller PSII subcomplexes. In the fourth chapter, a technique for determining the stoichiometry of photosynthetic complexes is examined, with implications for understanding PSII repair. Finally, in the fifth chapter, several conclusions and unanswered questions from this thesis are discussed.</p>

Estudos sobre agroquímico geralmente reportam sobre sua eficiência no controle de pragas. Como no caso do tiametoxam, um inseticida sistêmico do grupo dos neonicotinóides que atua inibindo a ação do receptor nicotínico acetilcolina (nAChR) dos insetos levando-os à morte. Porém, essa molécula apresenta ações fisiológicas nas plantas capazes de influenciar o desenvolvimento das culturas, e quando um agroquímico apresenta essa atividade é comumente chamado de bioativador, devido ao aumento do vigor e da produtividade das plantas tratadas. O objetivo deste trabalho foi avaliar os efeitos bioquímicos, fisiológicos e produtivos de doses crescentes do tiametoxam, aplicado via tratamento de sementes, nos cultivos de trigo, braquiária, arroz e milho. Experimentos com trigo, braquiária e arroz foram conduzidos em vasos, sob casade- vegetação, no Horto Experimental do Departamento de Ciências Biológicas da ESALQ/USP, Piracicaba, SP. Enquanto a condução da cultura do milho ocorreu em casa-de-vegetação, do Departamento de Agronomia no CEDETEG/UNICENTRO, e em área de plantio comercial, ambos no município de Guarapuava, PR. Durante a realização dos experimentos foram mensuradas características biométricas de: índice de emergência; altura de plantas; índice de clorofilas (SPAD); massa seca da parte aérea; comprimento, área e volume radicular. Parâmetros bioquímicos como: atividade de nitrato redutase; fenilalanina amônia-liase; conteúdo de proteína total; pigmentos fotossintéticos e conteúdo de nutrientes foliares. Parâmetros sobre a qualidade nutricional da braquiária também foram analisadas. E ao final do ciclo reprodutivo dos cereais foram determinados alguns parâmetros relacionados à produção. Foi constatado que o tiametoxam interferiu sobre diversos parâmetros bioquímicos, em todas as culturas testadas, com destaque para a potencial ação dessa molécula sobre atividades de nitrato redutase e fenilalanina amônia-liase, bem como sobre o conteúdo de proteína total em trigo e braquiária, além de alterar o teor de pigmentos fotossintéticos foliares em arroz e milho. Para as culturas do trigo e da braquiária foram notadas modificações severas sobre a fisiologia e produção. Concluiu-se que a aplicação de tiametoxam, via tratamento de sementes, foi responsável por moderar o metabolismo e o desenvolvimento vegetal, até o final do ciclo da planta, de maneira que as plantas submetidas a essa molécula expressaram maior vigor e, consequentemente, apresentaram maior aptidão para incrementar sua produção. / Studies on agrochemicals generally report on their effectiveness in pests control. As with thiamethoxam, a systemic insecticide of the neonicotinoid group that acts by inhibiting the action of acetylcholine nicotinic receptor (nAChR) of insects, leading to death. But this molecule which has physiological effect in plants, can influence crop development; and when an agrochemical presents this activity, it is commonly called bioactivator due to increased vigor and productivity of the treated plants. The aim of this study was to evaluate: biochemical, physiological and productive effects of increasing doses of thiamethoxam applied as seed treatment in wheat, Brachiaria, rice and corn crops. Experiments with wheat, rice and Brachiaria were conducted in pots under greenhouse, in the Horto Experimental of Biological Sciences Department of ESALQ/USP, Piracicaba, SP. While one corn crop was conducted in a greenhouse of the Department of Agronomy at CEDETEG / UNICENTRO, the other one was conducted in the field, both in Guarapuava, PR. We measure the biometric characteristics: emergence index, plant height, chlorophyll index (SPAD), shoot dry weight, length, root area and volume. Biochemical parameters such as: nitrate reductase activity, phenylalanine ammonia-lyase, total protein content, photosynthetic pigments and leaf nutrient content. Parameters on the nutritional quality of Brachiaria were also analyzed. And at the end of the reproductive cycle of cereals some parameters related to production were determined. It was found that thiamethoxam interfered in various biochemical parameters in all tested crops, highlighting the potential action of this molecule on activities of nitrate reductase and phenylalanine ammonia-lyase, as well as the total protein content in wheat and pasture, besides changing the content of photosynthetic pigments in rice and corn leaves. For wheat and Brachiaria crops severe changes were noted on the plant physiology and production. It is concluded that the application of thiamethoxam, via seed treatment, was responsible for moderating metabolism and plant development, until the end of the life cycle of the plant, so that the plants under this molecule expressed greater vigor and thus had higher ability to increase production.

O presente trabalho descreve o isolamento, identificação e determinação estrutural dos constituintes químicos das inflorescências e folhas de Iryanthera sagotiana e das inflorescências e pericarpos de I. lancifolia, espécies da família Myristicaceae, que ocorrem na Amazônia. A investigação fitoquímica das inflorescências de I. sagotiana (Benth.) Warb. forneceu, após extração, partição com solventes orgânicos e fracionamentos cromatográficos, quatro diidrochalconas: 2\',4\'-diidroxi-4,6\'- dimetoxidiidrochalcona (I) ; 2\',4\',6\'-triidróxi-4-metoxidiidrochalcona; 2\',4,4\'-triidróxi-6\'-metoxidiidrochalcona (II) e 2\',4\'-diidroxi-4-metóxi-6\'-glicopiranosil-diidrochalcona, sendo as duas últimas, inéditas. Foram também isolados e identificados o benzaldeído, o ácido diidro-p-cumárico, dois flavonóis glicosilados: afzelina e quercitrina e quatro flavanonóis glicosilados: engeletina, isoengeletina, astilbina e isoastilbina. Das folhas de I. sagotiana foram isoladas as diidrochalconas I, II e o dímero 3\',3\"\'-bis-2\', 4\',6\'-triidróxi-4-metoxidiidrochalcona, inédito na literatura, além da afzelina, quercitrina, engeletina e astilbina. Os pericarpos de I. lancifolia Ducke forneceram, após procedimentos cromatográficos, as diidrochalconas I e 2\',4-diidróxi-4\',6\'dimetoxidiidrochalcona, inédita, e três flavonolignóides também inéditos: rel (1\"R,2\"R,3\"S)-3\'-(1\",4\"-di-p-hidroxifenil-2\",3\"-dimetilbutil)-2\',4\'-diidróxi-4,6\'-dimetoxidiidrochalcona (III); rel (1\"R,2\"S,3\"R)-3\'-(1\",4\"-di-p-hidroxifenil-2\",3\"-dimetilbutil)-2\',4\'-diidróxi-4,6\'-dimetoxidiidrochalcona (IV) e 5\'-(1\",4\"-di-p-hidroxifenil-2\",3\"-dimetilbutil)-2\',6\'-diidróxi-4,4\'dimetoxidiidrochalcona. Foram isoladas também três lactonas policetídicas inéditas: (2S,3S,4S)-2-(7-dodecenil)-3-hidróxi-4-metil-butanolido, rel (2S,3R,4S)-2-(7\'-dodecenil)-3-hidróxi-4-metil-butanolido e 2-dodecil-3-hidróxi-4-metil-but-2-enolido, além do tocotrienol 2,8-dimetil-2-(4,8,12-trimetil-3,7,11-tridecatrienil)-6-cromanol e da lignana (8R,TS,8\'S)-4,4\'diidróxi-2,7\'-ciclolignana. Das inflorescências de I. lancifolia foram isolados a diidrochalcona I, os flavonolignóides III e IV, o tocotrienol e o esteróide 3-&#946;-O-&#946;-D-galactopiranosilsitosterol. A elucidação estrutural das substâncias isoladas foi baseada em métodos espectrométricos: EM, RMN de 1H e 13C (PND e DEPT 135°), técnicas de RMN bidimensionais (HOMOCOSY e HETERO-COSY) e experimentos para a observação de NOE. Reações de acetilação e epoxidação foram efetuadas para se obterem derivados mais informativos. As atividades antioxidantes da afzelina, da quercitrina, do flavonolignóide IV e do tocotrienol foram avaliadas em comparação com a atividade antioxidante da vitamina E, empregando-se a inibição da autooxidação de homogenato de cérebro como modelo experimental. A capacidade antioxidante foi medida pela produção de malonildialdeído (MDA) e a concentração necessária para inibir 50% da autoxidação (Ql/2) foi calculada. Os valores de Ql/2 obtidos para afzelina, quercitrina, flavonolignóide, tocotrienol e vitamina E foram, respectivamente: 62,30; 2,29; 4,83; 1,08 e 11,20 &#181;M. Estes ensaios foram realizados pela pesquisadora Dra. Solange C. Davino e pela professora Dra. Sílvia B. M. Barros, da Faculdade de Ciências FarmacêutIcas da USP. / This work describes the isolation, identification and structural elucidation of chemical constituents from inflorescences and leaves of Iryanthera sagotiana (Myristicaceae) and from inflorescences and pericarps of I. lancifolia, collected in Amazon region. After extraction and partition with organic solvents and chromatographic fractionation, phytochemical analysis of inflorescences of I. sagotiana (Benth.) Warb. afforded four dihydrochalcones: 2\',4\'-dihydroxy-4,6\'dimethoxydihydrochalcone (I); 2\',4\',6\'-trihydroxy-4-methoxy-dihydrochalcone; and the new 2\',4,4\'-trihydroxy-6\'-methoxy-dihydrochalcone (II) and 2\',4\'-dihydroxy- 4-methoxy-6\'-glucopyranosyl-dihydrochalcone. Benzaldehyde, dihydro-p-cumaric acid besides two glycosylated flavonols: afzelin and quercitrin and four glycosylated flavanonols: engeletin, isoengeletin, astilbin and isoastilbin were also isolated and identified. Leaves of I. sagotiana yielded dihydrochalcones I, II and the new dimer 3\',3\"\'-bis-2\',4\',6\'-trihydroxy-4-methoxydihydrochalcone in addition to afzelin, quercitrin, engeletin and astilbin. Pericarps of I. lancifolia Ducke afforded, after fractionation procedures, dihydrochalcones I e 2\',4-dihydroxy-4\',6\'-dimethoxy-dihydrochalcone, which has not been reported in literature yet, as well as three new flavonolignoids: rel (1\"R,2\"R,3\"S)-3\'-(1\",4\"-di-p-hydroxyphenyl-2\",3\"dimethylbutyl)-2\',4\'-dihydroxy-4,6\'-dimethoxydihydrochalcone (III); rel (1\"R,2\"S,3\"R)-3\'-(1\",4\"-di-p-hydroxyphenyl-2\",3\"-dimethylbutyl)-2\',4\'-dihydroxy-4,6\'-dimethoxydihydrochalcone (IV) e 5\'-(1\",4\"-di-p-hydroxyphenyl-2\",3\"dimethylbutyl)-2\',6\'-dihydroxy-4,4\'-dimethoxy-dihydrochalcone. Three new polyketide lactones were also isolated: (2S,3S,4S)-2-(7-dodecenyl)-3-hydroxy-4-methyl-butanolide, rel (2S,3R,4S)-2-(7\'-dodecenyl)-3-hydroxy-4-methylbutanolide e 2-dodecyl-3-hydroxy-4-methyl-but-2-enolide, besides tocotrienol 2,8-dimethyl-2-(4,8,12-trimethyl-3,7,11-tridecatrienyl)-6-cromanol and lignan (8R,T\'S,8\'S)-4,4\'-dihydroxy-2, 7\'-cyclo1ignan. From inflorescences of I. lancifolia, dihydrochalcone I, flavonolignoids III e IV, tocotrienol and the steroid 3-&#946;-O-&#946;-D-galactopyranosylsitosterol were isolated. Structural elucidation of the isolated compounds were based on spectrometric methods: MS, 1H and 13C NMR (PND and DEPT 135°), bidimensional NMR techniques (HOMOCOSY e HETERO-COSY) as well as NOE experiments. Acetylation and epoxidation reactions were carried out in order to get more informative derivatives. Antioxidant activities of afzelin, quercitrin, flavonolignoid IV and tocotrienol were evaluated in comparison with the antioxidant activity of vitamin E, using the inhibition of autoxidation of brain homogenates as a experimental model. The antioxidant capacity was measured by malondialdehyde (MDA) production and the necessary concentration to inhibit 50% of autoxidation (Q12) was calculated. The following values of Q1/2 were obtained for afzelin, quercitrin, flavonolignoid, tocotrienol and vitamin E, respectively: 62,30; 2,29; 4,83; 1,08 e 11,20 &#181;M.