Tesis sobre el tema "Plants secretion"

A study of some aspects of essential oil secretion in plants was conducted. The first part of the study involved analysis of the volatile terpenoid content and composition of leaf extracts from a range of Australian native plants by gas chromatography and mass spectrometry. Secretory structures were studied by several microscopic imaging techniques including conventional bright and dark field optical microscopy, confocal microscopy, and scanning (SEM) and transmission (TEM) electron microscopy. Three methods were employed for scanning electron microscopy. Sample material was prepared for conventional SEM by chemical fixation and rapid freeze fixation, and fresh material was imaged by environmental SEM. These methods were compared, and the images acquired by environmental SEM were invariably of a superior standard as the biological integrity of the samples was retained throughout, and the samples were free of process-induced artefacts. Several other tests were conducted and results discussed in some detail. In the final part of the study, aspects of essential oil secretion were examined by histochemical methods. The first of these was a new method based on traditional approaches to histochemistry. The monoterpene phenols thymol and carvacrol were located in glandular trichomes of Lamiaceae species by means of a colour-change reaction of the phenols with a nitrosophenol/acid reagent. The second used magnetic resonance imaging by a chemical shift selective method to locate, non invasively, the aromatic monoterpenes thymol and anethole in secretory structures in the fruit of Carum copticum (Apiaceae) and the leaves of Backhousia anisata (Myrtaceae) respectively.
Doctor of Philosophy (PhD) (Science)

Thesis (Ph.D.) -- University of Western Sydney, 2001.
"This thesis is presented in fulfilment of the degree of Doctor of Philosophy in Science at the University of Western Sydney, Richmond, New South Wales, Australia" Bibliography : p. 145-163.

Doctor of Philosophy
Department of Plant Pathology
Barbara S. Valent
Rice blast caused by the ascomycetous fungus Magnaporthe oryzae remains a threat to global sustainable agriculture and food security. This pathogen infects staple cereal crops such as rice, wheat, barley and millets, as well as turf grasses, in a distinct way among fungal plant pathogens, which we described in the first chapter. In addition to economical importance, rice blast is a model pathosystem for difficult-to-study biotrophic fungi and fungal-plant interactions. We are studying proteins that fungi secrete inside living cells to block plant defenses and control host cell processes; these proteins are called effectors. To date mechanisms for secretion and delivery of effectors inside host cells during disease establishment remain unknown. This step is critical to ensure the successful infection. So far, the only commonality found among all unique small-secreted blast effector proteins is their accumulation in a novel in planta structure called the biotrophic-interfacial complex (BIC). Identifying effectors and understanding how they function inside rice cells are important for attaining durable disease control. In the second chapter, we presented one approach to address this challenge. We characterized four candidate effector genes that were highly expressed specifically during the rice cell invasion. Using transgenic fungi that secrete fluorescently-labeled versions of each protein allowed me to follow them during invasion in vivo by live cell imaging. These candidates show distinct secretion patterns suggesting a spatially-segregated secretion mechanism for effectors. Results revealed a BIC-located strong candidate cytoplasmic blast effector, two putative cell-to-cell movement proteins and a putative extrainvasive hyphal membrane (EIHM)-matrix protein, which has become a valuable tool for assessing successful infection sites. In the third chapter, we test if normal secretion components of filamentous fungi are involved in accumulation of effectors into BICs. We report localization studies with M. oryzae orthologs of conserved secretion machinery components to investigate secretion mechanisms for effectors showing preferential BIC accumulation and for non-BIC proteins such as BAS4. Especially bright fluorescence adjacent to BICs from Mlc1p (Myosin Light Chain, a Spitzenkörper marker), from Snc1p (a secretory vesicle marker), and from Yup1p (a putative t-SNARE endosomal protein) suggest secretion actively occurs in the BIC-associated cells. Localization of Spa2p (a polarisome marker), as a distinct spot at the tips of the bulbous invasive hyphae (IH) in planta, suggests the existence of two secretion complexes after the fungus switches growth from the polarized filamentous primary hyphae to bulbous IH. In the final chapter on future perspectives, we present some strategies towards the molecular understanding of the M. oryzae secretion mechanism during biotrophic invasion, which will lead to novel strategies for disease control.

The plant cell wall determines cell shape and is essential for plant growth during development. Pectin is an important component of cell walls and, like many wall polysaccharides, is synthesized in the Golgi apparatus and secreted by vesicles. In Arabidopsis thaliana seed coats, pectin-rich mucilage is secreted in a polarized fashion during a specific stage of development. How the Golgi apparatus in seed coat cells accommodates the large increase in pectin-rich mucilage provides a unique window into the cellular machinery that supports cell wall polysaccharide biosynthesis and secretion. Examination of seed coat cells, using cryo-fixation and transmission electron microscopy and electron tomography, showed that Golgi stacks undergo dramatic changes in structure during mucilage production. Initiation of mucilage biosynthesis also correlated with increased numbers of Golgi stacks per cell. To understand if these cellular changes were dependent on pectin biosynthesis, the cell structure of a reduced mucilage mutant, mum4, was studied by similar methods and revealed that, while the morphology of Golgi stacks was dependant on mucilage, the increased stack number was not. To determine what proportion of the scattered Golgi stacks were producing mucilage, immunogold labeling with the novel mucilage-specific antibody CCRC-M36 was used to detect the pectin cargo. The large percentage of labeled Golgi stacks found suggests that many stacks produce pectin synchronously, rather than a subset of specialist Golgi. To test if a pectin modifying enzyme, MUM2, is co-secreted with pectin, a tagged MUM2 was engineered and introduced into mum2 mutants, where it rescued the mutant phenotype. However, the tag was not detectable using antibodies in immunofluorescence. Although mucilage was secreted to the top of the cell, antibody label demonstrated that pectin-producing stacks were randomly distributed throughout the cytoplasm, indicating that the destination of cargo has little effect on location of the Golgi stack producing it. The mechanism of targeting of vesicles with the domain of the plasma membrane exclusively at the mucilage pocket is unknown, although the correlation of a population of densely staining vesicles and abundant cortical microtubules in the cell cortex at the site of secretion was documented.

Les protéines synthétisées et sécrétées par les bactéries jouent des rôles importants pour leur survie. Les bactéries à Gram négatif ont développé des voies de sécrétion en tant qu'armes principales pour transporter des facteurs de virulence dans l'environnement extracellulaire ou dans des cellules hôte. L'un de ces systèmes, le T9SS a été principalement étudié chez l'agent pathogène oral Porphyromonas gingivalis et chez la bactérie mobile Flavobacterium johnsoniae. Un autre complexe, le T2SS est le principal déterminant de la virulence de la bactérie Pseudomonas aeruginosa, un agent pathogène de la fibrose kystique. Dans le cadre de ma thèse, j'ai résolu la structure atomique de plusieurs composants centraux du T9SS et du T2SS. Concernant le projet T9SS, j'ai essayé de cristalliser le domaine cytoplasmique de GldL de F. johnsoniae. La co-cristallisation de GldL avec des Nbs a été réalisée sans succès. Néanmoins, les structures cristallines de deux nanobody contre GldL ont été résolues par remplacement moléculaire. De plus, j'ai également travaillé sur la protéine PG1058 de P. gingivalis. J'ai résolu sa structure par diffraction anomale à la longueur d’onde du selenium. Concernant le projet T2SS, je me suis concentré sur la partie N-terminale de XcpQ, une sous-unité de la sécrétine. J'ai résolu la structure cristalline de XcpQN012 seul et en complexe avec le nanobody vhh04 à une résolution de 2,98 Å et de 2,9 Å, respectivement. Enfin, j'ai participé à la détermination structurale de TssK, un composant de plaque de base du système de T6SS et déterminer la structure cristalline d'un nanobody contre le domaine périplasmique de PorM
Proteins synthesized and secreted by bacteria serve many important roles in their survival. In particular, Gram-negative bacteria have evolved secretion pathways as the main weapons for transporting virulence factors into target cells or into the extracellular environment. One of these systems, the type IX secretion system (T9SS) or the Por secretion system, has been studied mainly in the oral pathogen Porphyromonas gingivalis and the gliding bacterium Flavobacterium johnsoniae. Another complex, the type II secretion system (T2SS) is the main determinant of the virulence of Pseudomonas aeruginosa, a cystic fibrosis pathogen. In my PhD thesis, I solved the atomic structure of several core components of both T9SS and T2SS.For the T9SS project, I tried to crystallize the cytoplasmic domain of GldL from F. johnsoniae. The co-crystallization of GldL with Nbs was unsuccessfull. The crystal structures of two nanobodies against GldL were solved by molecular replacement. I also worked on the PG1058 protein of P. gingivalis. I obtained crystals of the selenomethionine-derivatized PG1058 OmpA_C-like domain that diffracted up to 1.55 Å, and solved its structure by single-wavelength anomalous diffraction. For the T2SS project, I focused on the N-terminal part of XcpQ, a subunit of the secretin. I solved the crystal structure of XcpQN012 alone and in complex with nanobody vhh04 at a resolution of 2.98 Å and 2.9 Å, respectively. In addition, I also took part in the structural determination of the base plate component TssK of the T6SS and determined the crystal structure of one nanobody (vhh19) against the periplasmic domain of PorM

Appressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae. Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.

Colletotrichum graminicola is a hemibiotrophic pathogen of maize that causes anthracnose leaf and stalk rot diseases. The pathogen penetrates the host and initially establishes an intracellular biotrophic infection, in which the hyphae are separated from the living host cell by a membrane that is elaborated by the host, apparently in response to pathogen signals. A nonpathogenic mutant (MT) of C. graminicola was generated that germinates and penetrates the host normally, but is incapable of establishing a normal biotrophic infection. The mutated gene is Cpr1, conserved in eukaryotes and predicted to encode a component of the signal peptidase complex. How can we explain why the MT is normal in culture and during early stages of pathogenicity, but is deficient specifically in the ability to establish biotrophy? To address this, first I characterized the insertion in the 3’ UTR of the MT strain in detail, something that had not been done before. The wild-type (WT) transcript did not differ from predictions, but the MT produced several aberrant transcript species, including truncated and non-spliced transcripts, and the normal one. Aberrant splicing of MT cpr1 was observed both in RNAseq transcriptome data and reverse-transcription polymerase chain reaction (RT-PCR), under different growth conditions and in planta. I also conducted a bioinformatic analysis of other conserved components of the secretory pathway in the MT and WT in planta. One explanation for nonpathogenicity of the MT is that it cannot cope with an increase in secretory activity during infection, and fails to produce necessary pathogenicity factors. With the transcriptome data, I was able to identify effector proteins that were expressed in the WT but not in the MT. Another possible explanation for the MT phenotype is that the MT can’t adapt to stress imposed by the plant. I developed a growth assay to characterize the effect of chemical stressors in vitro. The MT was more sensitive to most stressors, when compared to the WT. The transcriptome data indicates that the genes involved in different stress pathways are expressed in planta in both WT and MT, although very few genes are differentially expressed across the different growth stages.

Orientador: Simone de Pádua Teixeira
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Apesar de a família Leguminosae ser rica em espécies, exibir ampla distribuição geográfica e grande variação morfológica, seus representantes têm sido pouco estudados em termos de estruturas secretoras florais. Tais estruturas podem estar localizadas em diferentes partes da flor e estão associadas à atração de polinizadores e/ ou defesa. As flores polinizadas por animais exalam odores que são produzidos e liberados por meio de glândulas de odor ou osmóforos, estruturas que apresentam grande variedade morfológica. Estudos anatômicos de tais glândulas são importantes, pois além de fornecerem informações para o entendimento das interações ecológicas de plantas e seus polinizadores, podem fornecer dados que auxiliem na determinação de relações filogenéticas entre as espécies. Assim, este trabalho objetivou (1) identificar as estruturas secretoras presentes nas flores de espécies de Leguminosae com polinização noturna, (2) levantar caracteres anatômicos florais compartilhados por essas espécies, (3) determinar se existem relações entre a polinização diurna e noturna e a morfologia dos osmóforos em Leguminosae (4) e investigar se os tricomas secretores podem atuar como osmóforos. Botões florais e flores de 14 espécies zoófilas de Leguminosae foram fixados e processados para análises em microscopias de luz, eletrônica de varredura e eletrônica de transmissão. Análises em cromatografia gasosa e cromatografia líquida de alta eficiência foram feitas nos tricomas secretores foliares e florais de Bauhinia. As flores polinizadas à noite apresentam várias características condizentes a este tipo de polinização, como antese com duração de uma noite, coloração inconspícua e forte odor. A prefloração ou fusão do perianto e a presença de hipanto em forma de copo permite o acúmulo de néctar nestas flores. A grande quantidade de néctar produzido nas espécies quiropterófilas tem relação com a estrutura robusta de seus nectários. No perianto, especialmente nas pétalas, encontram-se estruturas responsáveis pela produção do odor nas espécies de Leguminosae com polinização zoófila. A presença de regiões secretoras não especializadas na epiderme e no mesofilo das sépalas e/ou pétalas corrobora a liberação de odor difusa na maioria das espécies. Osmóforos típicos, ou seja, glândulas de perfume com estrutura anatômica especializada na emissão de fragrâncias, restritas a certas regiões da flor, foram observados em quatro das 12 espécies estudadas. Os osmóforos estudados sempre exibiram algum tipo de célula ou tecido secretor de terpeno; porém, compostos fenólicos e proteínas também foram detectados. Outros tipos de estruturas secretoras, frequentemente associadas a mecanismos de defesa, como tricomas, cavidades e idioblastos secretores foram encontrados. Dentre elas, destacam-se os tricomas secretores cavitados em Bauhinia, os quais secretam as mesmas classes de compostos nas folhas e flores e, assim, apresentam função de defesa e não na atração dos polinizadores. Conclui-se que não existe relação entre o tipo de osmóforo (típico ou difuso) e o hábito do polinizador (diurno ou noturno). As estruturas secretoras florais encontradas (exceto os nectários) apresentam significado biológico e/ou taxonômico, mas não podem ser associadas à determinada síndrome. Já a anatomia dos nectários revelou correlações entre seu tamanho, a quantidade de néctar produzido e o tipo de polinizador demonstrando sua grande importância na relação flor-polinizador
Abstract: Although the Leguminosae family is rich in species showing wide geographic distribution and a large morphological variation, their representatives have been poorly studied in terms of floral secretory structures. Such structures can be located in different parts of the flower and are associated with attraction of pollinators and/ or defense. Flowers pollinated by animals emit scents that are produced and released by means of scent glands or osmophores, structures that show a great morphological variety. Anatomical studies of these glands are important because besides providing information for the understanding of ecological interactions of plants and their pollinators, they can provide data to assist in the determination of phylogenetic relationships among species. This study aimed (1) to identify the secretory structures present in the flowers of Leguminosae species with nocturnal pollination, (2) to reveal floral anatomical features shared by these species, (3) to determine whether relationships exist between the diurnal and nocturnal pollination and the morphology of osmophores in Leguminosae (4) and to investigate if trichomes can function as osmophores. Flower buds and flowers of 14 zoophilic species of Leguminosae were fixed and processed for analysis in light, scanning electron and transmission electron microscopy. Analysis in CG and HPLC were performed in leaf and floral trichomes in Bauhinia. Flowers pollinated at night have several characteristics consistent with this type of pollination, as anthesis with duration of one night, inconspicuous coloration and strong odor. The aestivation or fusion of the perianth and the presence of cup-shaped hypanthium allows the nectar accumulation in these flowers. The large amount of nectar produced in the chiropterophilous species is related to the robust structure of their nectaries. In the perianth, especially in petals, occur structures responsible for producing the scent in the Leguminosae species with zoophilic pollination. The presence of non-specialized secretory regions in the epidermis and mesophyll of sepals and/ or petals confirms the diffuse release of scent in most species. Typical osmophores, i.e., scent glands with anatomical structure specialized in the emission of fragrances, restricted to certain regions of the flower, were observed in four of the 12 species studied. The studied osmophores always exhibited some type of cell or tissue secreting terpene; but phenolic compounds and proteins were also detected. Other types of secretory structures often associated with defense mechanisms, such as secretory trichomes, idioblasts and cavities were found. Among these are the cavitated trichomes in Bauhinia, which secrete the same classes of compounds in the leaves and flowers and thus have no role in the pollinator attraction but in the plant defense. It is concluded that there is no relationship between the type of osmophore (typical or diffuse) and the habit of pollinator (diurnal or nocturnal). The floral secretory structures found (except nectaries), have biological and/ or taxonomic significance, but they cannot be associated with a particular syndrome. However, the anatomy of nectaries showed correlations among their size, the amount of nectar produced and the type of pollinator demonstrating their great importance in the flower-pollinator relationship
Doutorado
Biologia Vegetal
Doutora em Biologia Vegetal

Peu de protéines nécessaires à l’infection des plantes communes aux bactéries, aux champignons et aux oomycètes ont été identifiées à part les enzymes dégradant les parois des cellules végétales. Nous avons caractérisé la structure et les propriétés d’une protéine, IbpS, sécrétée par la bactérie phytopathogène nécrotrophe Dickeya dadantii. Des homologues de cette protéine sont présents non seulement chez des bactéries à Gram+ mais aussi chez des champignons, des oomycètes et quelques animaux. Ce gène d’origine bactérienne a été transféré une fois chez les oomycètes et probablement plusieurs fois chez les champignons. IbpS peut fixer le fer et le cuivre, des métaux à activité redox. Elle a une structure en Venus Fly Trap classique mais avec des caractéristiques originales : elle forme des dimères en solution et possède un nouveau mode de fixation du métal. IbpS est impliquée dans le processus infectieux de D. dadantii et de Botrytis cinerea, un champignon phytopathogène nécrotrophe. Nous proposons qu’IbpS, une fois sécrétée, fixe le fer et le cuivre exogène, réduisant ainsi leur concentration intracellulaire et la formation de ROS dans le microorganisme. La sécrétion de cette protéine fixant les métaux semble être un mécanisme de protection contre l’oxydation requis pendant l’infection partagé par des phytopathogènes nécrotrophes
Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Herein, we have characterized the structure and properties of a protein (IbpS) secreted by the plant pathogenic bacterial necrotroph Dickeya dadantii. Homologs of this protein are present in not only Gram+ bacteria but also in fungi, oomycetes, most phytopathogens, and some animals. The gene originating from bacteria was transferred once in oomycetes and most likely several times in fungi. IbpS is capable of binding the redox-active metals iron and copper and has a classical Venus Fly trap fold with some original characteristics: it forms dimers in solution and has a novel metal binding site. IbpS is involved in D. dadantii and of the Botrytis cinerea, a fungal necrotroph, infection process. We propose that secreted IbpS binds exogenous iron and copper, reducing their intracellular concentrations of these metals and ROS formation in the microorganisms. Secretion of this metal scavenging protein appears to be a common antioxidant protection mechanism shared by necrotrophic phytopathogens and required during infection

Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulence
Fungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O cancro cítrico está entre as principais doenças que afetam a produção de laranjas no Brasil e é causado pela bactéria fitopatogênica gram-negativa Xanthomonas citri subsp. citri (Xac). O presente trabalho teve por objetivo analisar a expressão diferencial de proteínas secretadas pela bactéria selvagem e por um mutante (02H02) assintomático, que teve a proteína HrpB4, que participa de seu sistema de secreção tipo III (SSTT) inativada, em condição de cultivo em meio rico CN e em meio XAM1 indutor de hipersensibilidade e patogenicidade (genes hrp). As proteínas secretadas em meio de cultura foram extraídas pela ação do ácido tricloroacético (TCA) e identificadas através de espectrometria de massas. Tais análises identificaram 55 proteínas diferentes secretadas em ambos os meios de cultura, tanto para Xac quanto para 02H02, de modo que 13 destas proteínas são comuns entre a Xac e seu mutante cultivados em XAM1 e 14 são exclusivas para Xac cultivada em XAM1, as quais deixaram de ser secretadas no 02H02. Proteínas relacionadas aos genes reguladores do SSTT foram detectadas em condição infectante para ambas as bactérias, demonstrando a eficácia do meio de cultura XAM1 em induzir Hrp. Foi observado que diversas proteínas secretadas pelo sistema de secreção tipo II (SSTD) em condição infectante para Xac e seu mutante possuem um papel ativo na degradação das paredes celulares do hospedeiro e podem ser reguladas por proteínas controladoras do SSTT. Fatores de sinalização difusíveis produzidos por Xac aparentemente sofreram alteração em sua secreção no mutante devido à inativação do pilus do SSTT, demonstrando a relação dessa molécula com o SSTT. A não detecção de proteínas secretadas diretamente pelo SSTT denota que as mesmas podem estar sendo secretadas no interior de vesículas lipídicas de membrana externa, assim como ocorre em Xanthomonas campestris
Citrus canker is among the major diseases which affect citrus production in Brazil and is caused by the gram-negative phytopathogenic bacterium Xanthomonas citri subsp. citri (Xac). This work aimed to analyze the differential expression of secreted proteins by the wild bacterium and by an asymptomatic mutant (02H02), lacking the type III secretion system (TTSS) protein HrpB4, in rich cultivation medium NB and in the hrp inducing medium XAM1. The proteins secreted in all culture media have been extracted by trichloroacetic acid based protocols (TCA) and identified using mass spectrometry. The analysis identified 55 different proteins secreted in both culture medium for Xac and 02H02, of which 13 are common among Xac and its mutant cultivated in XAM1 and 14 proteins are exclusively secreted by Xac cultivated in XAM1. Proteins related to the TTSS regulatory genes have been detected in infecting condition in both bacteria, showing the effectiveness of XAM1 hrp inducing medium. It has been observed that several type II secretion system’s secreted proteins showed an active role in host cell wall degradation and may be regulated by type III secretion system’s proteins in Xac and 02H02 in infecting condition. Diffusible signal factors produced by wild Xac apparently suffered an altered secretion in the mutant due the inactivation of the type three secretion system’s pilus, showing the relationship of this molecule with this secretion system. The lack of detection of proteins secreted by the TTSS denote that these proteins may be secreted in the interior of outer membrane lipid vesicles, just like it was verified in Xanthomonas campestris

Ralstonia solanacearum, l'agent causal du flétrissement bactérien, est considéré comme l'un des agents pathogènes bactériens les plus importants au monde. Le pouvoir pathogène de cette bactérie du sol est principalement basé sur son système de sécrétion de type III (SST3) et ses effecteurs de type III (ET3s), provoquant la maladie sur plus de 250 espèces végétales. R. solanacearum injecte ses ET3s à travers cette seringue moléculaire directement à l'intérieur de la plante hôte. Ces ET3s détournent les réponses de défense de la plante, soit dans le cytoplasme, soit dans le noyau, afin de supprimer l'immunité de la plante et de favoriser la multiplication bactérienne. La sécrétion des ET3s est finement contrôlée au niveau post-traductionnel par des protéines associées au contrôle de la sécrétion de type III, et par des protéines chaperonnes de type III.A ce jour, la fonction in planta de ces effecteurs et protéines chaperonnes, et la manière dont R. solanacearum module l’immunité de la plante en sa faveur restent mal comprises. Mon projet de thèse visait à mieux comprendre le rôle des déterminants de la pathogénicité de R. solanacearum en identifiant certaines des cibles d’A. thaliana, directes ou indirectes, modulées par la bactérie. Pour ce faire, j'ai utilisé des populations naturelles d'A. thaliana à deux échelles géographiques et adopté l'approche consistant à confronter des populations naturelles à des mutants de R. solanacearum dans lesquels des déterminants majeurs de pathogénie sont mutés. Cette approche est nouvelle puisque à ce jour les études d’association pangénomique (GWAS) menées sur les interactions plantes-agents pathogènes utilisent des souches sauvages de phytopathogènes. En outre, cette approche a permis de découvrir une diversité de réponses qui n'avaient pas été détectées auparavant. Dans la première partie de mon projet de thèse, j'ai identifié des QTLs impliqués dans la résistance quantitative à la maladie en réponse à des mutants simples de R. solanacearum, et j'ai validé fonctionnellement ces QTLs (Quantitative Trait Loci) comme facteurs de sensibilité. Dans la deuxième partie de ma thèse, nous avons étudié un gène codant pour une protéine de type NLR que nous avons appelé Bacterial Wilt Susceptibility 1 (BWS1). Nous avons montré que BWS1 agissait comme un facteur de sensibilité quantitatif, ayant un rôle de régulateur négatif dans une réponse immunitaire dépendante de SGT1.)
Ralstonia solanacearum, the causal agent of bacterial wilt, is considered one of the world’s most important bacterial pathogens. This soil-borne bacterium relies mainly on its type III secretion system (T3SS) and type III effectors (T3Es) in order to cause disease in more than 250 plant species. R. solanacearum injects its T3Es through this molecular syringe directly inside the host plant. These T3Es hijack plant defense responses in either the cytoplasm or the nucleus aiming to suppress plant immunity and promote bacterial multiplication. T3E secretion is finely controlled at the post-translational level by helper proteins, called T3SS control proteins, and type III chaperones.To date, the in planta function of these effectors and helper proteins and how R. solanacearum modulates plant genes to its favor remains poorly understood. My thesis project aimed to better understand the role of R. solanacearum pathogenicity determinants by identifying some of the direct or indirect plant targets of A. thaliana, modulated by the bacterium. For this purpose, I used natural populations of A. thaliana on two geographical scales and adopted the approach of challenging mapping populations to R. solanacearum mutants in which major pathogenic determinants are mutated. This approach is new since most of the GWAS (Genome-Wide Association Studies) in plant-pathogen interactions use wild-type strains of phytopathogens. Furthermore, it unveiled a previously undetected diversity of responses. In the first part of my Ph.D. project, I identified QTLs (Quantitative Trait Loci) involved in quantitative disease resistance to R. solanacearum single mutants and I validated these QTLs as susceptibility factors. In the second part of my thesis, we studied a gene encoding for a NLR protein that we called Bacterial Wilt Susceptibility 1 (BWS1). We showed that BWS1 was acting as quantitative susceptibility factor, mediating a negative regulation of an SGT1-dependent immune response

Resumo: O cancro cítrico está entre as principais doenças que afetam a produção de laranjas no Brasil e é causado pela bactéria fitopatogênica gram-negativa Xanthomonas citri subsp. citri (Xac). O presente trabalho teve por objetivo analisar a expressão diferencial de proteínas secretadas pela bactéria selvagem e por um mutante (02H02) assintomático, que teve a proteína HrpB4, que participa de seu sistema de secreção tipo III (SSTT) inativada, em condição de cultivo em meio rico CN e em meio XAM1 indutor de hipersensibilidade e patogenicidade (genes hrp). As proteínas secretadas em meio de cultura foram extraídas pela ação do ácido tricloroacético (TCA) e identificadas através de espectrometria de massas. Tais análises identificaram 55 proteínas diferentes secretadas em ambos os meios de cultura, tanto para Xac quanto para 02H02, de modo que 13 destas proteínas são comuns entre a Xac e seu mutante cultivados em XAM1 e 14 são exclusivas para Xac cultivada em XAM1, as quais deixaram de ser secretadas no 02H02. Proteínas relacionadas aos genes reguladores do SSTT foram detectadas em condição infectante para ambas as bactérias, demonstrando a eficácia do meio de cultura XAM1 em induzir Hrp. Foi observado que diversas proteínas secretadas pelo sistema de secreção tipo II (SSTD) em condição infectante para Xac e seu mutante possuem um papel ativo na degradação das paredes celulares do hospedeiro e podem ser reguladas por proteínas controladoras do SSTT. Fatores de sinalização difusíveis produzidos por Xac aparentemente sofreram alteração em sua secreção no mutante devido à inativação do pilus do SSTT, demonstrando a relação dessa molécula com o SSTT. A não detecção de proteínas secretadas diretamente pelo SSTT denota que as mesmas podem estar sendo secretadas no interior de vesículas lipídicas de membrana externa, assim como ocorre em Xanthomonas campestris
Abstract: Citrus canker is among the major diseases which affect citrus production in Brazil and is caused by the gram-negative phytopathogenic bacterium Xanthomonas citri subsp. citri (Xac). This work aimed to analyze the differential expression of secreted proteins by the wild bacterium and by an asymptomatic mutant (02H02), lacking the type III secretion system (TTSS) protein HrpB4, in rich cultivation medium NB and in the hrp inducing medium XAM1. The proteins secreted in all culture media have been extracted by trichloroacetic acid based protocols (TCA) and identified using mass spectrometry. The analysis identified 55 different proteins secreted in both culture medium for Xac and 02H02, of which 13 are common among Xac and its mutant cultivated in XAM1 and 14 proteins are exclusively secreted by Xac cultivated in XAM1. Proteins related to the TTSS regulatory genes have been detected in infecting condition in both bacteria, showing the effectiveness of XAM1 hrp inducing medium. It has been observed that several type II secretion system's secreted proteins showed an active role in host cell wall degradation and may be regulated by type III secretion system's proteins in Xac and 02H02 in infecting condition. Diffusible signal factors produced by wild Xac apparently suffered an altered secretion in the mutant due the inactivation of the type three secretion system's pilus, showing the relationship of this molecule with this secretion system. The lack of detection of proteins secreted by the TTSS denote that these proteins may be secreted in the interior of outer membrane lipid vesicles, just like it was verified in Xanthomonas campestris
Orientador: Jesus Aparecido Ferro
Coorientador: Julio Cezar Franco de Oliveira
Banca: Maria Teresa Marques Novo
Banca: Leandro Márcio Moreira
Mestre

Dans la cellule végétale, les protéines solubles vacuolaires et extracellulaires entrent dans le système endomembranaire de sécrétion au niveau du réticulum endoplasmique. Ces protéines sont ensuite transportées via des vésicules de transport jusqu'à leur compartiment de stockage, la vacuole et le compartiment extracellulaire, en passant par l'appareil de Golgi. Dès leur insertion dans la lumière du RE, ces protéines vont subir des modifications co-traductionnelles, en particulier la N-glycosylation et le repliement qui leur conféreront stabilité et activité biologique. Des suspensions cellulaires de tabac BY-2 exprimant simultanément plusieurs protéines-marqueur vacuolaires et extracellulaires nous ont permis d'étudier les implications des modifications co-traductionnelles dans le transport de protéines et de glycoprotéines à travers le système endomenbranaire de sécrétion. Nous avons montré qu'un defaut de repliement d'une glycoprotéine vacuolaire peut d'une part entraîner une modification de sa N-glycosylation et d'autre part sa réorientation vers la surface cellulaire. Les mécanismes de tri et la maturation des glycoprotéines vacuolaires ont été également étudiés en présence de drogues perturbant le fonctionnement de l'appareil de Golgi. Enfin, le transport protéique dans le système endomembranaire de sécrétion a été étudié dans des conditions où la N-glycosylation est inhibée par la tunicamycine. Ainsi, nous avons montré qu'en absence de N-glycosylation, le transport à la vacuole des protéines et des glycoprotéines a lieu mais se trouve fortement ralenti. En revanche, la sécrétion des glycoprotéines extracellulaires est totalement inhibée alors que, dans les mêmes conditions, les protéines sont transportées vers le compartiment extracellulaire. Nous avons pu préciser que les glycoprotéines extracellulaires non-glycosylées en presence de tunicamycine sont dégradées dans le système endomembranaire de sécrétion par un mécanisme pré-golgien et indépendant des protéasomes.

U okviru ove doktorske disertacije ispitan je hemijski sastav i biološke aktivnosti metanolnih i vodenih ekstrakata odabranih samoniklih vrsta tribusa Urticeae, rod  Urtica:  U. dioica  subsp.  dioica  var.  pubescens, U.  dioica  subsp.  dioica  var.  dioica  i  U. kioviensis  i tribusa Parietarieae, rod  Parietaria:  P. officinalis,  P.lusitanica L. subsp. lusitanica, P. judaica L. subsp. judaica i P. serbica. Cilj rada bio je da se odredi sadrţaj biološki aktivnih jedinjenja u ovim, do sada veoma malo ispitanim vrstama famijije Urticaceae, i utvrdi njihov potencijal primene kao pomoćnih lekovitih sredstava i dodataka ishrani.Hemijski sastav ekstrakata ispitivanih vrsta određen je primenom: tečnohromatografskih tehnika (LC-DAD-MS i LC-MS-MS) za kvalitativnu analizu metanolnih ekstrakata, dok je za kvantitativnu analizu odabranih fenolnih jedinjenja primenjena LC-MS-MS tehnika. Spektrofotometrijskim metodama je određen sadržajukupnih fenolnih komponenti i flavonoida. Ispitivanja bioloških aktivnosti ekstrakata obuhvatila su: određivanje antioksidantne i antiinflamatorne aktivnosti kao i sposobnost ekstrakata da inhibiraju acetilholinesterazu.  Određen je uticaj odabranih metanolnih ekstrakata na imuni odgovor i proliferaciju intestinalnih ćelijskih linija pacova (IEC18) i ĉoveka (Caco2).Dobijeni rezultati ukazuju da odabrane vrste tribusa Urticeae i Parietarieae, odnosno rodova  Urtica  i Parietaria  predstavljaju bogate izvore biološki aktivnih jedinjenja koja ispoljavaju raznovrsne biološke aktivnosti. Sa hemotaksonomskog aspekta izdvajaju se sledeća jedinjenja kao potencijalni taksonomski markeri: viši sadržaj 5-O-kafeoilhinske kiseline u ekstraktima herbi vrsta roda  Urtica, i visok sadrţajepikatehina u ekstraktima korena vrsta roda  Parietaria. Ekstrakt herbe vrste  U. kioviensis  se od ostalih izdvaja po tome što ne sadrži rutin a sadrži  C-glikozide, u najvećoj meri viteksin. Od svih ispitivanih ekstrakata, ekstrakti korena  Parietaria  vrsta su ispoljili najbolji antioksidantni potencijal u većini izvršenih testova. Najsnažniji antiinflamatorni potencijal je ispoljio ekstrakt korena vrste  P. officinalis  a prate ga ekstrakti korena vrsta roda  Urtica.  Veoma dobar antiinflamatorni potencijal su ispoljili infuzi herbi vrste U. dioica  (čajevi od koprive). Svi ispitani metanolni ekstrakti su ispoljili odličnu inhibiciju enzima acetilholinesteraze a kao najbolji se izdvajaju ekstrakti korena  Parietaria  vrsta i vrste  U. kioviensis. Povećanu sekreciju citokina rat MCP1 i GROα  izazivaju ekstrakti korena vrsta  P. officinalis  i  P. judaica  ubazalnim uslovima i uslovima LPS-stimulisane inlamacije, dok ekstrakti vrste  U. dioica  povećavaju bazalnu a smanjuju LPS-stimulisanu sekreciju. Stimulaciju sekrecije ova dva citokina, ispitivani ekstrakti vrše interakcijom sa adapternim proteinom MyD88 (ali ne intereaguju sa TLR4 receptorom) i NF -κB signalnimputem. Ekstrakt korena vrste  P. officinalis  povećava LPS-om indukovanu ekspresiju enzima COX-2 u IEC18 ćelijama, dok je ekstrakt korena vrste  U. dioica  smanjuje. Efekat epitelizacije ili zarastanja rane na monosloju IEC18 ćelija ispoljavaju ekstrakti herbe i korena vrste  P. officinalis. Ispitivani ekstrakti ne menjaju značajno seksreciju citokina hMCP1 i IL-8 u Caco2 ćelijama niti ispoljavaju značajan uticaj na           njihovu proliferaciju.
Within this doctoral thesis the chemical composition and biological activity of methanol and aqueous extracts of the selected plant species belonging to the Urticeae and Parietarieae tribe, more specifically to the  Urtica  and  Parietaria  genuses was evaluated (Urtica:  U. dioica  subsp. dioica  var.  pubescens,  U.  dioica  subsp.  dioica  var.  dioica  and  U. kioviensis;  Parietaria:  P. officinalis,  P. lusitanica  subsp.  lusitanica,  P. judaica  subsp.  judaica  and  P. serbica). The principal aim was to determine the content of biologically active  compounds in this, poorlyexamined species of the Urticaceae family, and determine their potential as additional remedy and dietary supplements.Qualitative analysis of methanol extracts was performed by LC-DAD-MS i LC-MS/MS analysis, and LC-MS/MS for quantitative analysis of selected phenolic compounds. Total phenolics and flavonoids were determined spectrophotometrically. In order to assess the biological potential, the antioxidant and anti-inflammatory activities of the extracts were studied as well as  their ability to inhibit acetylcholinesterase. The immuno-modulatory effects of the selected methanol extract on the immune response and proliferation of intestinal epithelial cells (IEC18 and Caco2)was determined.The obtained results suggest that the examined species of the Urticeae and Parietarieae tribe (genuses  Urtica  and  Parietaria) are abundant with the biologically active compounds that express a broad spectrum of biological activities. As a potential chemotaxonomic markers stand out the following  compounds: 5-O-caffeoilquinic acid (highly abundant in the herb extracts of the  Urtica  spp.) and epicatechin (highly abundant in the root extracts of the  Parietaria  spp.).  U.kioviensis  herb extracts differs from the rest by high content of vitexin and total lack of rutin. The best antioxidant potential have exhibited the root extracts of the  Parietaria  species. The strongest anti-inflammatory potential had the root extract of the  P. officinalis, followed by root extracts of the  Urtica  spp. Excellent anti-inflammatory activity have exhibited the aqueous  extracts of  U. dioica  herbs  –  stinging nettle teas. All tested methanol extracts have inhibited enzyme acetylcholinesterase, the best inhibitors being root extracts of  U. kioviensis  and Parietaria  species. Root extracts of  P. officinalis  and  P. judaica  have increased the basal and LPS-stimulated secretion of rat MCP1 and GROα, while  U. dioica  extracts increased the basal but decreased the LPS-stimulated secretion. The examined extracts interact with the MyD88 (but not the TLR4) and NF-κB signaling pathway. The root extract of  P. officinalis  increase LPS-stimulated expression of COX-2 in IEC18 cells, while the root extract of  U. dioica  decreases it.The herb and root extract of P. officinalis  exhibit the wound healing effect. Investigated extracts do not significantly alter the secretion of hMCP1 and IL-8 in Caco2 cells and exhibit no significant effect to their proliferation.
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A study of some aspects of essential oil secretion in plants was conducted. The first part of the study involved analysis of the volatile terpenoid content and composition of leaf extracts from a range of Australian native plants by gas chromatography and mass spectrometry. Secretory structures were studied by several microscopic imaging techniques including conventional bright and dark field optical microscopy, confocal microscopy, and scanning (SEM) and transmission (TEM) electron microscopy. Three methods were employed for scanning electron microscopy. Sample material was prepared for conventional SEM by chemical fixation and rapid freeze fixation, and fresh material was imaged by environmental SEM. These methods were compared, and the images acquired by environmental SEM were invariably of a superior standard as the biological integrity of the samples was retained throughout, and the samples were free of process-induced artefacts. Several other tests were conducted and results discussed in some detail. In the final part of the study, aspects of essential oil secretion were examined by histochemical methods. The first of these was a new method based on traditional approaches to histochemistry. The monoterpene phenols thymol and carvacrol were located in glandular trichomes of Lamiaceae species by means of a colour-change reaction of the phenols with a nitrosophenol/acid reagent. The second used magnetic resonance imaging by a chemical shift selective method to locate, non invasively, the aromatic monoterpenes thymol and anethole in secretory structures in the fruit of Carum copticum (Apiaceae) and the leaves of Backhousia anisata (Myrtaceae) respectively.

Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...

蛋白質分泌及胞吐作用是指蛋白質在內質網(ER)中合成後前往質膜(PM) ， 隨後， 被分泌到達細胞外的過程。 而細胞分泌路徑是指蛋白質途經數個含有膜包被的細胞器後運送到细胞之外。 這些細胞器，包括內質網， 高爾基體， 反式高爾基網絡(TGN) 及質膜。 分泌蛋白分泌出细胞之外後， 在细胞外基質中進行其功能。
為達到這項研究的目的，我們結合了細胞、分子和生物化學上的方法， 來對蛋白質運輸路徑及參與蛋白質分泌的細胞器進行研究。首先，通過MALDI-MS/MS對煙草懸浮BY-2細胞中的原態分泌性蛋白質進行分析。第二，把已識別的分泌蛋白包括陽離子過氧化物酶同工酶40K（40K）和N1過氧化物酶（N1），透過轉基因細胞的GFP融合表達方式、及應用特異性抗體於免疫螢光和膠體金免疫電鏡上的測定來對其特性作進一步分析。 第三，總合以上的研究，煙草懸浮BY-2細胞的典型蛋白質分泌路徑次序為質網 - 高爾基體 - 反式高爾基網絡 - 質膜。
Protein secretion or exocytosis is the process by which proteins synthesized in the endoplasmic reticulum (ER) travel to the plasma membrane (PM) for their subsequent secretion outside of the cell. The secretory pathway responsible for protein secretion contains several membrane-bounded organelles such as the ER, Golgi apparatus, trans-Golgi Network (TGN), and PM. The secreted proteins move outside of the cell and perform their functions in the extracellular matrix.
The general objective of this study was to examine the transport pathways and organelles involved in protein secretion in plant cells using a combination of cellular, molecular and biochemical approaches. First, major native secreted proteins in suspension cultures of tobacco BY-2 culture cells were identified via MALDI-MS/MS analysis. Second, the identified secreted proteins, cationic peroxidase Isozyme 40K (40K) and peroxidase N1 (N1), were further characterized by examining the GFP fusion expression of transgenic cell lines and by generating specific antibodies in immunofluorescent and immunogold electron microscope (EM) studies. Third, throughout all of these studies, a typical ER-Golgi-TGN-PM pathway was mapped for protein secretion in tobacco BY-2 cells.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Lam, Chun Kok.
"December 2012."
Thesis (M.Phil.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 79-84).
Abstracts also in Chinese.
Thesis /Assessment Committee --- p.i
Statement --- p.i
Abstract --- p.ii
摘要 --- p.iv
Acknowledgements --- p.v
List of Abbreviations --- p.xiii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1. --- Secreted protein --- p.1
Chapter 1.2. --- Secretory Pathway --- p.1
Chapter 1.3. --- Protein secretion --- p.2
Chapter 1.4. --- Plant Peroxidases --- p.3
Chapter 1.5. --- Project Objective --- p.4
Chapter 1.6. --- Significance --- p.4
Chapter Chapter 2 --- Materials and Methods --- p.6
Chapter 2.1. --- Mass spectrometry analysis --- p.6
Chapter 2.2. --- Generation of 40K/N1-GFP construct --- p.7
Chapter 2.2.1. --- For transient expression --- p.7
Chapter 2.2.2. --- For stable expressing constructs --- p.7
Chapter 2.3. --- Transient expression of 40K/N1-GFP --- p.7
Chapter 2.4. --- Generation of transgenic cell lines --- p.8
Chapter 2.5. --- Fluorescence microscopic screening --- p.9
Chapter 2.6. --- Generation and characterization of antibodies specific for 40K/N1 peroxidase --- p.9
Chapter 2.7. --- Confocal immunofluorescence studies --- p.10
Chapter 2.8. --- (TIRF) Total internal reflection fluorescence microscopy --- p.11
Chapter 2.9. --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.11
Chapter 2.10. --- Drug Treatment --- p.12
Chapter 2.10.1. --- Dexamethasone (dex) --- p.12
Chapter 2.10.2. --- Brefeldin A (BFA)/Concanamycin A (ConcA) --- p.12
Chapter 2.11. --- Salt treatment (plasmolysis) --- p.13
Chapter 2.12. --- EM (electron microscopy) study --- p.13
Chapter Chapter 3 --- Results --- p.14
Chapter 3.1. --- Protein secretion from tobacco BY2 cells --- p.14
Chapter 3.2. --- Western blot analysis --- p.15
Chapter 3.3. --- Protein expression in tobacco plant tissues --- p.15
Chapter 3.4. --- EM labeling on the wild type BY-2 cells --- p.16
Chapter 3.5. --- Localization in the tobacco root tip apoplast --- p.17
Chapter 3.6. --- 40K/N1 peroxidase transient/stable cell line expression --- p.18
Chapter 3.7. --- Time course study of 40K/N1 peroxidase-GFP cell line expression after induction --- p.18
Chapter 3.8. --- Plasmolysis (salt treatment analysis) --- p.20
Chapter 3.9. --- Brefeldin A (BFA) and concanamycin A (ConcA): Trafficking through the Golgi and TGN --- p.20
Chapter 3.10. --- Examining exocytosis by total internal reflectance fluorescence (TIRF) --- p.22
Chapter 3.11. --- Immunolabeling study --- p.23
Chapter 3.12. --- EM study on transgenic 40K & N1 peroxidase-GFP cell lines --- p.24
Chapter Chapter 4 --- Discussion --- p.26
Chapter 4.1. --- Trafficking from the ER to the extracellular matrix --- p.26
Chapter 4.2. --- Secretion through PM by exocytosis --- p.28
Chapter 4.3. --- Time required for the secretory pathway --- p.29
Chapter 4.4. --- Similarities of 40K and N1 --- p.30
Chapter 4.5. --- Future perspectives --- p.30
References --- p.79

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008.
Title from title screen (site viewed Jan. 13, 2009). PDF text: 227 p. : ill. (some col.) ; 2 Mb. UMI publication number: AAT 3323493. Includes bibliographical references. Also available in microfilm and microfiche formats.

Almost all conifer ovules produce a liquid secretion as part of reproduction. This secretion, termed an ovular secretion, is produced during ovule receptivity and is involved in pollen capture and transport. Historically, examinations of the ovular secretion have focused on how they are part of pollination mechanisms. As a result, the chemical composition of the ovular secretion has not been examined systematically. Investigations into the constituents of the ovular secretion were limited to analyses for simple water soluble compounds such as sugars, minerals, amino acids and organic acids. More recently, the protein component of the secretion has been investigated using mass spectrometry-based proteomics. Proteins involved in processes such as carbohydrate modification, proteolysis, and defence have been identified in conifer ovular secretions. This biochemical complexity suggests a broader view of the function of the ovular secretion is warranted. However, protein identifications only provide putative information on function. Functional characterization of these proteins is needed in order to fully understand how they contribute to ovular secretion function. The research outlined in this dissertation describes the first functional characterizations of proteins found in conifer ovular secretions. Three proteins - invertase, chitinase, and thaumatin-like protein - were characterized in the ovular secretions of Douglas-fir (Pseudotsuga menziesii) and hybrid yew (Taxus × media). The Douglas-fir ovular secretion is capable of converting sucrose to glucose and fructose, confirming that invertases present in the secretion are functional. The invertase activity was maximal at pH 4.0. Activity was 77% of maximal at pH 4.5, the physiological pH. This indicates that post-secretory hydrolysis of sucrose occurs in situ in the Douglas-fir ovular secretion. Invertases in the ovular secretion are likely involved in controlling the movement of carbohydrates to developing pollen and could facilitate pollen selection. Chitinases present in the Douglas-fir ovular secretion are functional at physiological conditions. All three modes of chitinolytic activity, i.e. endochitinase, chitobiosidase and β-N-acetylglucosaminidase, were detected at physiological pH. β-N-acetylglucosaminidase activity was 80 % of maximal at physiological pH. Chitinases are pathogenesis-related proteins capable of hydrolysing chitin in fungal cell walls. These results suggest the ovular secretion is capable of defending the ovule against infection by phytopathogens. Thaumatin-like protein was immunolocalized to the cell wall and amyloplasts in Douglas-fir and yew nucellar tissue in a pattern consistent with a defensive role. It was also localized to the cell wall of fungal spores and germinating hyphae that were present in the micropyle of a yew ovule. These results provide additional evidence for an antifungal role for the ovular secretion. Functioning enzymes involved in pollen-ovule interactions and ovule defence are present in the conifer ovular secretion. The ovular secretion has functions beyond pollen capture. A revised functional model for the conifer ovular secretion is proposed.
Graduate
2021-08-17

碩士
國立臺北藝術大學
美術學系美術創作碩士班
99
Being a practicer of artistic creations, I focus on digging the nature of the creation, and referring to sequence of ideas about images in the current trend while making my artistic creation statement. Through scanning what I have done, along the way of my tracks, it became a systematic language to present my pieces. Hopefully I can maintain certain openness and use it to be the model of my works, as well as accomplishment of the goal of writing. In this thesis, I will bring out two important ideas in my art works: one is the secretive spaces, the other one is so called image-archaeology in the space, in which we may learn that why the concepts of spaces and images are repeatedly shown in my works. Roughly speaking, the secretive spaces is opposite to the spaces could generally be seen. With this idea, I found a cleft to nail and address the unseen spaces and recreate a narrative image to reverse the way of observing places. As for the image-archaeology, it could be regarded with two parts, destroying and digging; which mainly discusses the reason that why I choose those places to shoot , also specify its relationships in my works. In short, the whole thesis, I will try to describe and analyze the turning point of my way to look on the images, especially in my practices, also the selection of spaces, the events in the images, and the layout of the devices in my recent works along with its each chapter.

Most gymnosperms produce a pollination drop that captures and transports pollen into the ovule. Pollination drops have other functions. These include influencing pollen germination and pollen tube growth, defending the ovule from pathogens and providing a food reward in insect-pollinated gymnosperms. Mineral and organic molecules, including proteins, are responsible for these additional functions. To date, pollination drops from a handful of conifers and one non-conifer gymnosperm, Welwitschia mirabilis, have been subjected to proteomic analysis. In the present study, tandem mass spectrometry was used to detect proteins in all gymnosperm lineages: cycads (Ceratozamia hildae, Cycas rumphii, Zamia furfuracea); Gnetales (Ephedra compacta, E. distachya, E. foeminea, E. likiangensis, E. minuta, E. monosperma, E. trifurca; Gnetum gnemon; Welwitschia mirabilis); Ginkgo biloba; conifers (Taxus x media). PEAKS 6 DB (Bioinformatics Solutions, Waterloo, ON, Canada) was used to make protein identifications. Proteins were detected in all gymnosperm species analyzed. The numbers of proteins identified varied between samples as follows: one protein in Welwitschia female; nine proteins in Cycas rumphii; 13 proteins on average in Ephedra spp.; 17 proteins in Gnetum gnemon; 38 proteins on average in Zamia furfuracea; 57 proteins in Ginkgo biloba; 61 proteins in Ceratozamia hildae; 63 in Taxus x media; 138 proteins in Welwitschia male. The types of proteins identified varied widely. Proteins involved in carbohydrate modification, e.g. galactosidase, chitinase, glycosyl hydrolase, glucosidase, were present in most gymnosperms. Similarly, defence proteins, e.g. reduction-oxidation proteins, lipid-transfer proteins and thaumatin-like proteins, were identified in many gymnosperms. Gymnosperms that develop a deep pollen chamber as the nucellus degrades, e.g., cycads, Ginkgo, Ephedra, generally contained higher proportions of proteins localized to intracellular spaces. These proteins represent the pollination drop degradome. Gymnosperms that either lack a pollen chamber, e.g. Taxus, or have a shallow pollen chamber, e.g. Gnetum, had greater proportions of extracellular proteins. These proteins represent the pollination drop secretome. Our proteomic analyses support the hypothesis that the pollination drops of all extant gymnosperms constitute complex reproductive secretions.
Graduate

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es.
Graduation date: 2012