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La régulation redox est un mécanisme ancien présent chez les organismes biologiques et impliquée dans diverses voies métaboliques. En particulier chez les organismes photosynthétiques elle est responsable des mécanismes d‘adaptation rapide dans un environnement constamment modifié. Dans les chloroplastes le système ferrédoxine/thiorédoxine est la cascade redox principale qui relie l‘activité de plusieurs enzymes plastidiales à la source lumineuse. Le rôle central dans ce système est joué par la ferrédoxine-thiorédoxine réductase (FTR), une protéine hétérodimérique qui récupère des électrons à partir de la ferrédoxine photoréduite et les transfère pour réduire des thiorédoxines plastidiales. Ces protéines peuvent alors réduire des enzymes cibles, requérant l‘accessibilité de paires de cystéines dans un disulfure dont la réduction résulte en une activation/ inactivation de la cible. Jusqu‘à présent des plantes viables n‘ont pu être obtenues en l‘absence de ce système de régulation. Dans cette thèse des secteurs du système redox ont été explorés chez la plante modèle Physcomitrella patens (une mousse). Par manipulation de gènes l‘influence de l‘enzyme FTR sur la croissance et le développement de la plante a été analysée suivant différents paramètres. De manière à impacter la fonction de la réductase des changements nucléotidiques simples ont été introduits au niveau des codons programmant les cystéines catalytiques et dans un deuxième temps le gène complet a été supprimé. De façon inattendue nous n‘avons observé aucun effet significatif sur la viabilité et le développement des plantes mutantes. De plus, nous avons détecté dans P. patens des thiorédoxines additionnelles absentes chez les plantes à graine qui sont fonctionnelles vis à vis des enzymes cibles mais non-réduites par la FTR. Ceci rend possible un scénario de compensation chez les mutants via un système de réduction FTR-indépendant qui reste à caractériser. Deux des cibles photorégulées, la fructose-1,6-bisphosphatase (FBPase) et la sédoheptulose-1,7-bisphosphatase (SBPase), fonctionnent dans la phase de régénération du cycle de Calvin-Benson cycle et elles possèdent plusieurs caractéristiques de catalyse et de régulation similaires. En combinant des approches biochimiques et structurales, une comparaison fonctionnelle et structurale des deux phosphatases de P. patens a été conduite. De plus l‘analyse phylogénétique a révélé une origine procaryotique indépendante des deux séquences en dépit de leurs similitudes structurales et catalytiques. De plus trois articles de revue résument la plasticité et la représentativité du modèle P. patens pour la recherche forestière, les principes généraux de la régulation redox relativement aux aspects évolutifs et fonctionnels chez les plantes ainsi que l‘ état de l‘art de la régulation redox chez les espèces ligneuses en utilisant principalement le peuplier comme modèle / Redox regulation is an ancient mechanism present in biological organisms and is involved in diverse cellular pathways. In particular in photosynthetic organisms it is responsible for fast adaption mechanisms to a constantly changing environment. In chloroplasts the ferredoxin/thioredoxin system represents the main redox regulatory cascade which links the activity of several plastid enzymes to the energy source, light. A central role in this system is played by the heterodimeric ferredoxin-thioredoxin reductase (FTR), which gains electrons from the photo-reduced ferredoxin and transfers those further on via reduction to plastidal thioredoxins. Those proteins in turn reduce their target enzymes and require therefore the availability of redox sensitive cysteine pairs whose reduction results in an inactivation/activation switch of the targets. So far no viable plants could be obtained in complete absence of this redox regulation system. In this thesis single sections of the system were explored in the model plant Physcomitrella patens. Through gene manipulation the influence of the FTR enzyme on plant growth and development was analysed. In order to impact on the function of the reductase, firstly single nucleotide exchange of the catalytic cysteines was performed and later on the gene was completely deleted. Surprisingly, no significant effect could be observed on the viability and development of mutant lines compared to WT plants. Furthermore we found that P. patens possesses in contrast to seed plants additional thioredoxins which are functional for reduction of FTR target enzymes but are most likely not supplied with electrons by this reductase. Thus a possible rescue scenario independent of FTR could be assumed for P. patens and also by other redox regulation systems present in chloroplasts. Two of the FTR target enzymes, fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase, are functional in the regeneration phase of the Calvin-Benson cycle and share similar characteristics in regulation and catalysis. By combining biochemical and structural approaches, a functional comparison of both phosphatases was conducted using cDNAs from P. patens. A stricter TRX-dependent regulation and catalytic cleavage ability for both substrates, FBP and SBP, could be observed for PpSBPase, whereas PpFBPase is only capable of cleaving FBP. By obtaining the oxidized X-ray structure of both enzymes these observations can be associated with the distinct positions of regulatory sites and the various sizes of the substrate binding pocket. In addition, the phylogenetic analysis revealed an independent prokaryotic origin for both phosphatases. Furthermore we summarized in three review articles the amenability of P. patens as model plant for forest research, the general principles of redox regulation in respect of evolution and functional mechanisms in plants, and the current state of the art in forest redox regulation using poplar as exemplary model

Thiazole biosynthetic protein (THI1) is involved in the synthesis of the thiazole ring, a thiamine (vitamin B1) component. Thiamine is an essential co-factor in several carbohydrate and amino acid metabolic pathways. Prokaryotes and a few eukaryotes, such as fungi and plants, are able to synthesize thiamine de novo. These organisms contain the genes that encode the corresponding enzymes (such as THI1) that perform this metabolic function. THI1 actually functions as a reagent rather than as a conventional catalytic enzyme, as the THI1 polypeptide itself serves as the sulfide donor for thiazole formation. This gene also plays a role in organelle DNA damage tolerance. Arabidopsis thaliana has only one copy of the thi1 gene (At-thi1). Transcripts derived from At-thi1 are targeted simultaneously to chloroplasts and mitochondria by differential usage of two in-frame initiation codons. The tz-201 A. thaliana thi1 mutant has been shown to accumulate more sucrose in its tissues than wild-type plants. This suggests that a better understanding of thi1 genes and the role they play in cellular sucrose accumulation may be relevant for improving commercially important crops such as sugarcane. Sugarcane (Saccharum spp.) is a C4 photosynthesis monocot. Unlike A. thaliana, sugarcane has at least two thi1 copies (sc-thi1.1 and sc-thi1.2), as do the other C4 grasses. This thesis concerns the molecular and functional analyses of sugarcane thi1 (sc-thi1) gene homologues. The identified alleles related to sc-thi1.2 have some differences in sequence and seems to be diverging into two subgroups (sc-thi1.2a and sc-thi1.2b), based on phylogenetic analyses. Expression analysis showed that each sc-thi1 copy is expressed differentially in individual tissues and in developing stages levels. Subcellular analysis showed that sc-thi1.1 and sc-thi1.2b have the same cellular distribution pattern, distinct from the observed for sc-thi1.2a. Sc-thi1.1 and sc-thi1.2b were also able to partially complement thiamine auxotrophy in a yeast mutant deficient in thiamine biosynthesis. A similar complementation assay is not possible in the A. thaliana tz-201 mutant owing to low transformation efficiencies. Thus, Physcomitrella patens was chosen to generate thi1 mutant lines for future functional complementation studies. P. patens is a moss used as a plant model, with a small size, short life cycle and a haploid dominant phase. Despite its simplicity, it has six thi1 homologues copies. Homologous Recombination was used to generate P. patens thi1 mutants. In each case, a target thi1 gene was disrupted by replacing its coding region with an antibiotic resistance gene cassette. Single mutants were obtained for all six thi1 gene copies. All the knockout lines were able to survive and grow with only minor effects on morphology and physiology. Deletion of one of the thi1 gene copies (PpThi1.20F) drastically affected protoplast survival and regeneration, suggesting a role for this gene in early (polar) cell division and differentiation. The experimental design, which permits recycling of the selectable marker cassettes, provides a research platform for the construction of double, triple, quadruple or quintuple mutants in the future. The individual mutants line generated in this work, as well as the possible multiple mutants, will be useful for thi1 functional complementation experiments and for discerning the specific functions of individual thi1 gene family members. / THI1 (proteína da biossíntese de tiazol) está envolvida na síntese do anel de tiazol, um componente de tiamina (vitamina B1). A tiamina é um cofator essencial em várias vias metabólicas de carboidratos e aminoácidos. Somente procariontes e alguns eucariontes, como fungos e plantas, são capazes de sintetizar a tiamina de novo. A proteína THI1 atua mais como um reagente do que como uma enzima catalítica convencional, pois usa a si mesmo como doador de sulfeto para a formação do anel de tiazol. Este gene também está envolvido na tolerância ao dano no DNA das organelas. A. thaliana apresenta apenas uma cópia do gene thi1. Seu transcrito primário é direcionado simultaneamente aos cloroplastos e mitocôndrias através do uso diferencial de dois códons de iniciação, presentes no mesmo quadro aberto de leitura. Além disso, o mutante tz-201 de A. thaliana acumula mais sacarose em seus tecidos do que a planta selvagem. Isso sugere que um melhor entendimento do gene thi1 e seu papel no acúmulo de sacarose podem ser importantes para o melhoramento comercial de cultivares, como cana-de-açúcar. Cana-de-açúcar (Saccharum spp.) é uma monocotiledônea de metabolismo fotossintético C4. Diferentemente do observado em A. thaliana, a cana-de-açúcar possui pelo menos duas cópias (sc-thi1.1 e sc-thi1.2) homólogas a thi1, como observado também para outras gramíneas C4. Nesta tese são discutidas análises moleculares e funcionais dos homólogos do gene thi1 (sc-thi1) de cana-de-açúcar. Os alelos identificados como relativos a sc-thi1.2 apresentam algumas diferenças em suas sequências e, baseado em análises filogenéticas, parecem estar divergindo em dois subgrupos (sc-thi1.2a e sc-thi1.2b). As análises de expressão mostraram que cada cópia de sc-thi1 é diferencialmente expressa em diferentes tecidos e estágios de desenvolvimento. A análise de localização subcelular mostrou sc-thi1.1 e sc-thi1.2b apresentam o mesmo padrão de distribuição, distinto do observado para sc-thi1.2a. Sc-thi1.1 e sc-thi1.2b também foram capazes de complementar parcialmente a auxotrofia para tiamina em leveduras mutantes, deficientes na via de biossíntese de tiamina. Um teste similar de complementação funcional mutante tz-201 de A. thaliana não é possível no devido à baixa eficiência de transformação. Assim, Physcomitrella patens foi escolhida para gerar linhagens mutantes de thi1 para futuros estudos de complementação funcional. P. patens é um musgo usado como planta modelo, apresenta tamanho pequeno, um ciclo de vida curto e uma fase dominante haploide. Apesar de sua simplicidade, possui seis cópias homólogas a thi1. A técnica de Recombinação Homóloga foi escolhida para gerar os mutantes thi1 de P. patens. Em cada mutante, uma das cópias de thi1 foi interrompida, substituindo sua região codificante por um cassete de gene de resistência. Mutantes individuais foram obtidos para as seis cópias do gene thi1. As linhagens knockouts foram capazes de sobreviver e crescer apenas com alguns pequenos efeitos em sua morfologia e fisiologia. A deleção de uma das cópias de thi1 (PpThi1.20F) afetou drasticamente a sobrevivência e regeneração dos protoplastos, sugerindo um papel deste cópia gênica no inicio da divisão e diferenciação celular. O desenho experimento utilizado para a geração destes mutantes permite a reciclagem dos cassetes de seleção, fornecendo uma plataforma para a construção de duplos, triplos, quádruplos, quíntuplos e sêxtuplos mutantes no futuro. Os mutantes individuais para cada cópia de thi1 gerados nesse trabalho, bem como os possíveis mutantes múltiplos, serão úteis para experimentos de complementação funcional e o discernimento de funções específicas de diferentes membros da família gênica thi1.

Thiazole biosynthetic protein (THI1) is involved in the synthesis of the thiazole ring, a thiamine (vitamin B1) component. Thiamine is an essential co-factor in several carbohydrate and amino acid metabolic pathways. Prokaryotes and a few eukaryotes, such as fungi and plants, are able to synthesize thiamine de novo. These organisms contain the genes that encode the corresponding enzymes (such as THI1) that perform this metabolic function. THI1 actually functions as a reagent rather than as a conventional catalytic enzyme, as the THI1 polypeptide itself serves as the sulfide donor for thiazole formation. This gene also plays a role in organelle DNA damage tolerance. Arabidopsis thaliana has only one copy of the thi1 gene (At-thi1). Transcripts derived from At-thi1 are targeted simultaneously to chloroplasts and mitochondria by differential usage of two in-frame initiation codons. The tz-201 A. thaliana thi1 mutant has been shown to accumulate more sucrose in its tissues than wild-type plants. This suggests that a better understanding of thi1 genes and the role they play in cellular sucrose accumulation may be relevant for improving commercially important crops such as sugarcane. Sugarcane (Saccharum spp.) is a C4 photosynthesis monocot. Unlike A. thaliana, sugarcane has at least two thi1 copies (sc-thi1.1 and sc-thi1.2), as do the other C4 grasses. This thesis concerns the molecular and functional analyses of sugarcane thi1 (sc-thi1) gene homologues. The identified alleles related to sc-thi1.2 have some differences in sequence and seems to be diverging into two subgroups (sc-thi1.2a and sc-thi1.2b), based on phylogenetic analyses. Expression analysis showed that each sc-thi1 copy is expressed differentially in individual tissues and in developing stages levels. Subcellular analysis showed that sc-thi1.1 and sc-thi1.2b have the same cellular distribution pattern, distinct from the observed for sc-thi1.2a. Sc-thi1.1 and sc-thi1.2b were also able to partially complement thiamine auxotrophy in a yeast mutant deficient in thiamine biosynthesis. A similar complementation assay is not possible in the A. thaliana tz-201 mutant owing to low transformation efficiencies. Thus, Physcomitrella patens was chosen to generate thi1 mutant lines for future functional complementation studies. P. patens is a moss used as a plant model, with a small size, short life cycle and a haploid dominant phase. Despite its simplicity, it has six thi1 homologues copies. Homologous Recombination was used to generate P. patens thi1 mutants. In each case, a target thi1 gene was disrupted by replacing its coding region with an antibiotic resistance gene cassette. Single mutants were obtained for all six thi1 gene copies. All the knockout lines were able to survive and grow with only minor effects on morphology and physiology. Deletion of one of the thi1 gene copies (PpThi1.20F) drastically affected protoplast survival and regeneration, suggesting a role for this gene in early (polar) cell division and differentiation. The experimental design, which permits recycling of the selectable marker cassettes, provides a research platform for the construction of double, triple, quadruple or quintuple mutants in the future. The individual mutants line generated in this work, as well as the possible multiple mutants, will be useful for thi1 functional complementation experiments and for discerning the specific functions of individual thi1 gene family members. / THI1 (proteína da biossíntese de tiazol) está envolvida na síntese do anel de tiazol, um componente de tiamina (vitamina B1). A tiamina é um cofator essencial em várias vias metabólicas de carboidratos e aminoácidos. Somente procariontes e alguns eucariontes, como fungos e plantas, são capazes de sintetizar a tiamina de novo. A proteína THI1 atua mais como um reagente do que como uma enzima catalítica convencional, pois usa a si mesmo como doador de sulfeto para a formação do anel de tiazol. Este gene também está envolvido na tolerância ao dano no DNA das organelas. A. thaliana apresenta apenas uma cópia do gene thi1. Seu transcrito primário é direcionado simultaneamente aos cloroplastos e mitocôndrias através do uso diferencial de dois códons de iniciação, presentes no mesmo quadro aberto de leitura. Além disso, o mutante tz-201 de A. thaliana acumula mais sacarose em seus tecidos do que a planta selvagem. Isso sugere que um melhor entendimento do gene thi1 e seu papel no acúmulo de sacarose podem ser importantes para o melhoramento comercial de cultivares, como cana-de-açúcar. Cana-de-açúcar (Saccharum spp.) é uma monocotiledônea de metabolismo fotossintético C4. Diferentemente do observado em A. thaliana, a cana-de-açúcar possui pelo menos duas cópias (sc-thi1.1 e sc-thi1.2) homólogas a thi1, como observado também para outras gramíneas C4. Nesta tese são discutidas análises moleculares e funcionais dos homólogos do gene thi1 (sc-thi1) de cana-de-açúcar. Os alelos identificados como relativos a sc-thi1.2 apresentam algumas diferenças em suas sequências e, baseado em análises filogenéticas, parecem estar divergindo em dois subgrupos (sc-thi1.2a e sc-thi1.2b). As análises de expressão mostraram que cada cópia de sc-thi1 é diferencialmente expressa em diferentes tecidos e estágios de desenvolvimento. A análise de localização subcelular mostrou sc-thi1.1 e sc-thi1.2b apresentam o mesmo padrão de distribuição, distinto do observado para sc-thi1.2a. Sc-thi1.1 e sc-thi1.2b também foram capazes de complementar parcialmente a auxotrofia para tiamina em leveduras mutantes, deficientes na via de biossíntese de tiamina. Um teste similar de complementação funcional mutante tz-201 de A. thaliana não é possível no devido à baixa eficiência de transformação. Assim, Physcomitrella patens foi escolhida para gerar linhagens mutantes de thi1 para futuros estudos de complementação funcional. P. patens é um musgo usado como planta modelo, apresenta tamanho pequeno, um ciclo de vida curto e uma fase dominante haploide. Apesar de sua simplicidade, possui seis cópias homólogas a thi1. A técnica de Recombinação Homóloga foi escolhida para gerar os mutantes thi1 de P. patens. Em cada mutante, uma das cópias de thi1 foi interrompida, substituindo sua região codificante por um cassete de gene de resistência. Mutantes individuais foram obtidos para as seis cópias do gene thi1. As linhagens knockouts foram capazes de sobreviver e crescer apenas com alguns pequenos efeitos em sua morfologia e fisiologia. A deleção de uma das cópias de thi1 (PpThi1.20F) afetou drasticamente a sobrevivência e regeneração dos protoplastos, sugerindo um papel deste cópia gênica no inicio da divisão e diferenciação celular. O desenho experimento utilizado para a geração destes mutantes permite a reciclagem dos cassetes de seleção, fornecendo uma plataforma para a construção de duplos, triplos, quádruplos, quíntuplos e sêxtuplos mutantes no futuro. Os mutantes individuais para cada cópia de thi1 gerados nesse trabalho, bem como os possíveis mutantes múltiplos, serão úteis para experimentos de complementação funcional e o discernimento de funções específicas de diferentes membros da família gênica thi1.

Made available in DSpace on 2014-08-20T13:32:58Z (GMT). No. of bitstreams: 1 tese_filipe_de_carvalho_victoria.pdf: 30767989 bytes, checksum: d4b2b8995ad70a6cbd4d6e6ff672fa95 (MD5) Previous issue date: 2011-05-27 / Iron is an essential element for plant development, involved in metabolic processes, such respiration and photosynthesis. However, data regarding the genotype by environment interaction are lacking. Comparative analysis with lower plant groups and crop plants can increase the understanding about these processes. The use of bryophytes as model plants rise as a promising strategy since they present simpler patterns of development. The present work aimed to identify the occurrence patterns of molecular markers in model plant species, as well as to infer about the phylogenetical relationships of gene families related with iron homoestasis in plants, allowing the development of tranfer strategies of genomic data across model and orphan species. Using bioinformatics tools, a survey analysis was performed to detect repetitive elements in EST banks of eleven plant species. To validate the SSR markers found, 100 primer pairs were developed on the microsatelite sequences obtained for Physcomitrella patens Brid. and tested against genomic DNA of Polytrichum juniperinum Hedw. Phylogenetic and divergence time analysis was performed for the gene families Iron Regulated Transporter (IRT), Ferric Redectase Oxidase (FRO), Nicotinamide synthase (NAS), Yellow Stripe-Like (YSL) and Natural Resistance-Associated Macrophage Protein (NRAMP), related to the iron homoestasis, with help of the Bayesian inference and using the rice, Arabidopsis and P. patens genes for the Blast search in distinct land plants species. Also, primers for transposable elements recognizably related to Ysl genes were developed and applied jointly with the SSR primers by the IRAP/REMAP technic searching to find microsatellite markers associated to copies of this gene family. A total of 13,133 SSR markers were discovered in non- redundant EST databases made for all eleven species chosen for this study. The dimer motifs are more frequent in lower plant species, such as green algae and mosses, and the trimer motifs are more frequent for the majority of higher plant groups, such as monocots and dicots. Thirty percent of EST-SSE were successfully transferred with a relative polimorphism information across Physcomitrella patens Brid. and P. juniperinum, being promising for mapping and comparative genome analyses in plants. A total of 243 iron uptake gene sequences for 30 plant species were found using rice and Arabidopsis thaliana (L.) Heynh. homologues as queries. The evolutionary fingerprinting analyses suggested a positive selective pressure on iron uptake genes for most of the plant homologues analyzed, enabling an optimization and maintenance of gene function. The divergence time analysis indicates IRT as the most ancient gene family and FRO as the most recent. NRAMP and YSL genes appear as a close branch in the evolution of iron uptake gene families. No recent duplication in grasses were found based in the bayesian inference, and paralogue copies were only observed for dicot species. The Nramp cis-acting homology search indicated an ancestral duplication hypothesis for this gene family in grasses. Using IRAP/REMAP techniques, it was observed that YSL homologues in Physcomitrella are surrounded by copia-like retrotransposons as occurs in the maize ZmYSL1 copy. Also Polytrchum juniperinum Hedw. in vitro cultures were estabilished using spores as explants. Protonemal and gametophyte development were obtained using a growth regulator free culture medium. / O ferro é um elemento essencial para o crescimento e desenvolvimento das plantas, envolvido em processos metabólicos essenciais, como fotossíntese e respiração. Porém, são poucos os dados relacionando a interação entre diferentes genótipos e ambientes. Análises comparativas entre plantas inferiores e plantas cultivadas podem possibilitar o melhor entendimento destes processos. O uso de briófitas como modelo para estudos de processos biológicos em plantas surge como uma estratégia promissora devido ao padrão relativamente simples de desenvolvimento destas plantas. O presente trabalho objetivou identificar padrões de ocorrência de marcadores moleculares em plantas modelo, bem como inferir acerca da filogenia das famílias gênicas envolvidas na homoestase do ferro em plantas, possibilitando a criação de estratégias de transferência de informação genômica entre espécies modelo e espécies órfãs. Utilizando ferramentas de bioinformática foram realizadas análises exploratórias para detectar as ocorrências de elementos repetitivos em bancos de ESTs de onze espécies de plantas. Para a validação destes marcadores moleculares foram desenvolvidos 100 conjuntos de iniciadores a partir das sequências contendo microssatélites obtidas para Physcomitrella patens Brid. e testadas contra o DNA genômico de Polytrichum juniperinum Hedw. Foram realizadas análises filogenéticas e de divergência das famílias gênicas Iron Regulated Transporter (IRT), Ferric Redectase Oxidase (FRO), Nicotinamide synthase (NAS), Yellow Stripe-Like (YSL) e Natural Resistance-Associated Macrophage Protein (NRAMP), envolvidas na homoestase de ferro por meio de inferência bayesiana, utilizando genes de arroz, Arabidopsis e Physcomitrella patens Brid. na busca de homólogos em diferentes espécies de plantas terrestres, com o auxílio da ferramenta Blast (NCBI). Também foram desenvolvidos iniciadores para elementos transponíveis reconhecidamente associados a genes Ysl de milho e utilizados conjuntamente com os iniciadores EST-SSR por meio da técnica IRAP/REMAP buscando encontrar marcadores microssatélites associados a cópias desta familia gênica. Como resultados foram identificados 13.133 marcadores microssatélites em bancos de dados não redundantes de regiões expressas (EST) de onze espécies de plantas. Os motivos dinucleotídeos foram mais frequentes em espécies basais, enquanto os motivos trinucleotídeos foram mais frequentes em espécies derivadas. Em 30% dos conjuntos de iniciadores EST SSR testados contra o DNA de P. juniperinum, foi obtido bandas polimórficas promissoras para estudos de mapeamento comparativo e de diversidade genética. Foram encontrados 243 homólogos de genes relacionados as famílias gênicas envolvidas com a homoestase de ferro em trinta espécies de plantas. A análise de fingerprinting realizada sugere que a maioria destes genes estão submetidos a seleção positiva, indicando acúmulo de mutações adaptativas, essencial para a manutenção e otimização da resposta gênica. A análise de tempo de divergência indica que os genes IRT são mais basais e os genes FRO os mais recentes entre as familias gênicas estudadas. As famílias NRAMP e YSL são evolutivamente próximas. A análise bayesiana das sequências e de regiões promotoras dos genes NRAMP não indica duplicações recentes em gramíneas, sendo as duplicações provenientes de divergência ancestral a origem do grupo. Parálogos foram identificados somente em dicotiledôneas. Por meio da transferência de marcadores IRAP/REMAP é observado que genes YSL de P. patens estão cercados por retroelementos do tipo cópia, a exemplo do que ocorre com o gene ZmYSL1 em milho. Também foi estabelecido o cultivo, em condições axênicas, de Polytrichum juniperinum Hedw. utilizando esporos como explantes, onde foi observado que protonemas são obtidos utilizando meio de cultura livre de fitorreguladores, regenerando gametófitos em cultivo in vitro.

Tip growth is a ubiquitous process throughout the plant kingdom in which a single cell elongates in one direction in a self-similar manner. To sustain tip growth in plants, the cell must regulate the extensibility of the wall to promote growth and avoid turgor-induced rupture. This process is heavily dependent on the cytoskeleton, which is thought to coordinate the delivery and recycling of vesicles containing cell wall materials at the cell tip. Although significant work has been done to elucidate the various molecular players in this process, there remains a need for a more mechanistic understanding of the cytoskeletonÂ’s role in tip growth. For this reason, specific emphasis should be placed on understanding the dynamics of the cytoskeleton, its associated motors, and their cargo. Since the advent of fluorescence fusion technology, various quantitative fluorescence dynamics techniques have emerged. Among the most prominent of these techniques is fluorescence recovery after photobleaching (FRAP). Despite its prominence, it is unclear how to interpret fluorescence recoveries in confined cellular geometries such as tip-growing cells. Here we developed a digital confocal microscope simulation of FRAP in tip-growing cells. With this simulation, we determined that fluorescence recoveries are significantly influenced by cell boundaries. With this FRAP simulation, we then measured the diffusion of VAMP72-labeled vesicles in the moss Physcomitrella patens. Using finite element modeling of polarized cell growth, and the measured VAMP72-labeled vesicle diffusion coefficient, we were able to show that diffusion alone cannot support the required transport of wall materials to the cell tip. This indicates that an actin-based active transport system is necessary for vesicle clustering at the cell tip to support growth. This provides one essential function of the actin cytoskeleton in polarized cell growth. After establishing the requirement for actin-based transport, we then sought to characterize the in vivo binding interactions of myosin XI, vesicles, and filamentous actin. Particle tracking evidence from P. patens protoplasts suggests that myosin XI and VAMP72-labeled vesicles exhibit fast transient interactions. Hidden Markov modeling of particle tracking indicates that myosin XI and VAMP72- labeled vesicles move along actin filaments in short-lived linear trajectories. These fast transient interactions may be necessary to achieve the rapid dynamics of the apical actin, important for growth. This work advances the fieldÂ’s understanding of fluorescence dynamics, elucidates a necessary function of the actin cytoskeleton, and provides insight into how the components of the cytoskeleton interact in vivo.

Photoperiodism refers to the organism’s ability to detect and respond to seasonal changes in the daily duration of light and dark and thus constitutes one of the most significant and complex examples of the interaction between the organism and its environment. This thesis attempts to describe the prevalence of variation in a photoperiodic response, its adaptive value, and its putative genetic basis in a common cruciferous weed, Capsella bursa-pastoris (Brassicaceae). Furthermore, the thesis presents a first comprehensive comparative overview of the circadian clock mechanism in an early land plant, Physcomitrella patens (Bryophyta), thus providing insights into the evolution of the plant circadian system. In an introductory survey of global gene expression changes among early- and late flowering accessions of C. bursa-pastoris we found an enrichment of genes involved in photoperiodic response and regulation of the circadian clock. Secondly, by phenotyping circadian rhythm variation in a worldwide sample of accessions with known flowering time, we detected robust latitudinal clines in flowering time and circadian period length, which constitute strong indications of local adaptation to photoperiod in the shaping of flowering time variation in this species. In an attempt to elucidate putative genetic causes for the correlated variation between circadian rhythm and flowering time, we found that sequence variation and diverged expression in components regulating light input to the clock, PHYTOCHROME B (PHYB) and DE-ETIOLATED 1 (DET1) make them strong candidate genes. Finally, we present a comparative study of circadian network topology in the moss P. patens. Phylogenetic analyses and time series expression studies of putative clock homologues indicated that several core clock genes present in vascular plants appeared to be lacking in the moss. Consequently, while the clock mechanism in higher plants constitutes at least a three-loop system of interacting components, the moss clock appears to comprise only a single loop. We conclude that C. bursa-pastoris is a highly suitable model system for the further elucidation of the molecular variation that influences adaptive change in natural plant populations. Furthermore, we believe that the continuing study of the seemingly less complex circadian network of P. patens not only can provide insights into the evolution of the plant circadian system, but also may help to clarify some of the remaining issues of the circadian clock mechanism in higher plants.

Non-specific lipid transfer proteins is a large and diverse protein family found in plants, with roles in biological systems ranging from long distance signaling to plant pathogen defense. Little is known about the roles of nsLTPs, but recent studies have cast some light on the issue, among other things proposing that they may be involved in the cutice formation on land-living liverworts, mosses and non-seedbearing plants. Increased cuticle formation is thought to be a part of a plants defense system against stress. In this experiment, the expression of nsLTPs type G in the moss Physcomitrella patens was examined by qRT-PCR on cDNA synthesized from already existing mRNA samples from moss under different abiotic stresses. The different stresses were UV-light, salt (ion toxicity), heavy metal, cold drought, plant hormone and osmosis. House-keeping gene P. patens beta-tubuline 1 was used as reference and relative expression analysis was performed. The study showed a general down-regulation of PpLTPg's in the abiotically stressed samples, and the possible coupled regulatory response of PpLTPg3 and PpLTPg5. The results imply that the PpLTPg's in Physcomitrella patens could be connected to biological processes that cease during stress, or that they worl through negative feedback to support plant defense against stress.

Plants are sessile organisms and thus more exposed to stressful environments. By changing the expression of stress related genes, plants are able to cope with stress. Alternative splicing (AS) of pre-mRNA is a major contributor to proteome diversity in eukaryotes. It has been shown that different abiotic stresses affect AS patterns, suggesting a functional role of AS in stress tolerance. The Serine/Arginine-rich proteins (SR proteins) are a conserved family of splicing regulators in eukaryotes. SR proteins are essential for AS and studies have shown that they are themselves subjects to AS after stress exposure which means that they can control their own splicing. In this study, the aim was to characterize the different SR-proteins in the SR subfamily in P. patens, analyze their phylogeny and measure the change in expression of the genes after exposure to five types of stress; osmotic, salinity, dehydration, cold and hormonal. The result showed both individual and overlapping changes in their expression profiles of the three genes. Furthermore, there was an alteration in the alternative splicing pattern for two genes during three of the stresses which resulted in intron retention and possibly a premature termination codon and subseqent non-sense mediated decay.

During the water-to-land transition, that occurred approximately 450 MYA, novel habitats wererevealed to the emerging plants. This terrestrial habitat was a harsh environment compared to theaquatic, with shifting substrate content, irregular supply of water, damaging UV-radiation andrapid fluctuating temperatures. Non-specific lipid transfer proteins (nsLTP) are today only foundin the land living plants and not in the green algae. This suggests that these genes might haveevolved to help the plants cope with the stressful conditions. In this study the expression patternhas been analysed of the nsLTPs in the moss Physcomitrella patens during the possible conditionsthat raised during the water-to-land transition. The moss was exposed to salt, UV-B, drought, copper, cold and osmotic stress. Quantitative real-time PCR was used to analyse the transcription levels. I found that six genes were upregulated during either cold, dehydration or UV-B stress. This suggest that these genes are involved in the plant defense against these abiotic stresse