Dans les conditions naturelles, les plantes sont soumises à des conditions environnementales très variées qui peuvent conduire à un excès d'énergie dans les chloroplastes, résultant en une production d'espèces réactives de l'oxygène (ERA). Pour faire face à ces ERA, les plantes ont développé différents mécanismes de protection, comme des alkénal réductases, des peroxyrédoxines et des lipocalines. Le travail présenté dans cette thèse a pour but de caractériser et de déterminer leur importance dans la protection des contre les stress oxydants. Une analyse préliminaire de mutants d'Arabidopsis a mis en évidence le rôle important des peroxyrédoxines à 2 cystéines et des lipocalines dans la tolérance au stress photooxydant. Cette thèse s'est surtout concentrée sur les lipocalines. Deux lipocalines ont été récemment identifiées chez les plantes, TIL (lipocaline thermoinduite) et CHL (chloroplastique), toutes les deux induites par des conditions de stress. Chez Arabidopsis, chaque lipocaline semble être spécialisée dans la réponse à des conditions différentes: chaleur (AtTIL) et fortes lumières (AtCHL). Le double mutant AtCHL KO × AtTIL KO est plus sensible à la chaleur et la forte lumière que les simples mutants. La mutation de AtCHL a augmenté fortement la photosensibilité de mutants (vte1, npq1) affectés dans des mécanismes de protection des lipides (tocopherols, zéaxanthine), confirmant ainsi le rôle des lipocalines dans la protection contre la peroxydation lipidique. Les résultats obtenus dans cette thèse montrent que les lipocalines AtTIL and AtCHL ont des fonctions redondantes dans la protection des lipides qui sont essentielles à la résistance des plantes au stress. / Under field conditions, plants are exposed to various environmental conditions that can lead to an excess of energy in the chloroplasts, resulting in the production of toxic reactive oxygen species (ROS). To cope with the harmful effects of ROS, plants have developed various protection mechanisms, such as alkenal-reductases, peroxiredoxins and lipocalins. The work performed in this thesis aimed at understanding their importance in the protection against lipid peroxidation. A first screening of Arabidopsis mutants lacking one of those mechanisms brought into light that 2-Cys PRX and lipocalins are important for the tolerance against photooxidative stress. This thesis is focused mainly on lipocalins, a group of proteins recognized as carriers of small lipophilic molecules. However, two true lipocalins have been recently identified in plants, the temperature-induced lipocalin (TIL) and the chloroplastic lipocalin (CHL), their expression beeing induced by various abiotic stresses. Each lipocalin appeared to be specialized in the responses to specific stress conditions in Arabidopsis, with AtTIL and AtCHL playing a protective role against heat and high light, respectively. The double mutant AtCHL KO × AtTIL KO deficient in both lipocalins was more sensitive to temperature, drought and light stresses than the single mutants. Seeds of the AtCHL KO × AtTIL KO double mutant were very sensitive to natural and artificial aging, and again this phenomenon was associated with the oxidation of polyunsaturated lipids. The results obtained in this thesis show that AtTIL and AtCHL have overlapping functions in lipid protection which are essential for stress resistance and survival.

A transformação plastidial oferece uma série de vantagens em relação à transformação nuclear, como: altos níveis de expressão de proteínas, capacidade de expressar múltiplos transgenes em operons e contenção gênica pela ausência de transmissão pelo pólen. Devido ao alto número de cópias do genoma plastidial por cloroplasto e ao alto número de cloroplastos por células vegetais, são necessários ciclos de regeneração sob condições seletivas para obter transformantes homoplásmicos. A análise de homoplasmia é realizada pela metodologia de Southern blot ou pelo teste de herança do transgene pela germinação de sementes em meio seletivo. O Southern blot é trabalhoso, demorado e para maior sensibilidade envolve o uso de radioisótopos, enquanto o teste de germinação é realizado somente após a produção de sementes necessitando de um ciclo de reprodução da planta. Assim, o objetivo deste trabalho foi desenvolver um método rápido, sensível e eficaz para determinar o grau de homoplasmia de plantas transplastômicas, baseado na técnica de PCR em tempo real. Folhas de fumo foram transformadas com vetores compostos pelos genes 9 dessaturase (pMR1), 15 dessaturase (pMR3), -3 elongase (pMR5) e 12/3 dessaturase (pMR10), todos contendo o gene de seleção aadA. No total, 44 plantas foram obtidas, sendo 21 plantas positivas para a inserção do transgene. O grau de homoplasmia foi determinado pela proporção entre o número de cópias do transgene e o número de cópias do gene endógeno. Inicialmente, misturas de DNA de plantas transplastômicas homoplásmicas (pMR1 e pMR3) com DNA de planta tipo selvagem foram preparadas para simular diferentes graus de homoplasmia. DNA da planta transplastômica ou do plasmídeo foi diluído em série para construção das curvaspadrão, com a quantidade dos genes sendo estimada por meio da plotagem nessas curvas. Os índices de homoplasmia detectados na PCR em tempo real foram compatíveis com os resultados do teste de germinação com valores abaixo de 1 para plantas heteroplásmicas, 1 para a planta homoplásmica e 0 para as plantas sem a inserção do transgene. Os resultados das análises de amostras coletadas após o primeiro ciclo de regeneração mostraram que 13 das 21 plantas já se apresentavam em estado homoplásmico não sendo necessários mais ciclos de regeneração. A PCR em tempo real mostrou ser um método eficiente para análise do grau de homoplasmia de plantas transplastômicas. / Plastid transformation offers several advantages in relation to nuclear transformation, such as high-level of protein expression, the feasibility of expressing multiple transgenes in operons and gene containment through the lack of pollen transmission. Due to the high copy number of plastidial genome in chloroplasts and the high number of chloroplasts per plant cells, regeneration cycles under selective conditions are necessary to obtain homoplasmic transformants. Homoplasmy analysis is performed by Southern blot methodology or transgene inheritance test through seed germination in selective medium. Southern blot is laborious, time consuming and for more sensitivity it would require the use of radioisotopes, while germination test can be performed only after seed production which require a plant reproduction cycle. The objective of this study was to develop a fast, sensitive and effective method to determine the homoplasmy degree of transplastomic plants, based on real-time PCR. Tobacco leaves were transformed with vectors containing the 9 desaturase (pMR1), 15 desaturase (pMR3), -3 elongase (pMR5) and 12/3 desaturase (pMR10) each one with the aadA selection gene. In total, 44 plants were obtained, of which 21 were positive for the insertion of the transgene. The homoplasmy degree was determined by the proportion between the number of transgene copies and the number of endogenous gene copies. Initially, mixtures of homoplastomic plants DNA (pMR1 and pMR3) with wild-type plant DNA were prepared to simulate different degrees of homoplasmy. Transplastomic plant DNA or plasmid DNA was diluted to construct the standard curves and the gene amount was detected by plotting in this curves. The homoplasmy rate detected in real-time PCR were consistent with the results of germination test with values below 1 for heteroplasmic plants, 1 for homoplasmic plants and 0 for plants without the transgene insertion. The results obtained from the samples collected after the first regeneration cycle showed that 13 of the 21 plants were already in a homoplasmic state and did not require more cycles of regeneration. The real-time PCR proved to be an effective method for analyzing the homoplasmy degree of transplastomic plants.

Os cloroplastos encontrados nas folhas de plantas e algas pertencem a uma classe de organelas subcelulares denominadas de plastídios. Os plastídios possuem seu próprio genoma (plastoma), o qual contêm genes que participam de funções essenciais no metabolismo vegetal, como fotossíntese, síntese de aminoácidos e outras rotas biossintéticas. O plastoma da maioria das plantas superiores tem tamanho entre 120 a 180 kb. Em angiospermas é caracterizado pela presença de duas regiões repetidas invertidas (IRs) separadas por duas regiões de cópia única; uma longa (LSC) e outra curta (SSC). Para o sequenciamento completo da molécula do DNA (cpDNA) de Eucalyptus grandis, foram preparadas bibliotecas que geraram 9.033 seqüências de DNA. A seqüência completa foi determinada e possui o tamanho de 160.292 pb. As regiões IRs apresentam ter 26.400 pb; a SSC, 18.501 pb e; a LSC, 88.991 pb. As regiões codificadoras foram anotadas por análise de similaridade ao plastoma de Eucalyptus globulus. Todas as categorias gênicas presentes neste plastoma foram encontradas no plastoma de E. grandis. Estas duas espécies possuem 99,57% de similaridade na seqüência de nucleotídeos e apresentam total similaridade na organização dos seus genes. A disponibilidade de plastomas completos de diferentes espécies de Eucalyptus, torna possível realizar estudos comparativos para a identificação de polimorfismos espécie-específicos, importantes para serem utilizados como marcadores moleculares no melhoramento genético de Eucalyptus. / The chloroplasts found in plant leaves and algae belong to a class of subcellular organelle called plastids. The plastids has its own genome (plastome) which contain genes that participate in mainly functions on plant metabolism like photosynthesis, amino acids synthesis and other biosynthetic pathways. The plastome of most of higher plants are between 120-180 kb in size. It is characterized in angiosperms by two inverted repeat regions (IRs) separated by two regions of unique single copies; one large (LSC) and another small (SSC). For the complete sequencing of the DNA (cpDNA) molecule of chloroplast from Eucalyptus grandis, libraries were prepareted and generated 9,033 DNA sequences. The complete sequence was determined and it is 160,292 bp in size. The IRs showed to have 26,400 bp; the SSC, 18,502 bp and; the LSC, 88.991 bp in size. The gene coding regions were annotated by similarity analysis against the Eucalyptus globulus plastome. All types of gene categories present in E. globulus were found in E. grandis plastome. These two species show 99.57% of similarity on nucleotide sequence and they both present total similarity in their gene organization. The availability of complete plastomes from different Eucalyptus species make possible to perform comparative studies to identify species-specific polymorphisms, wich are important to be used as molecular markers in Eucalyptus breeding programs.

Há controvérsia sobre os processos responsáveis pela atual distribuição de Florestas Tropicais Sazonalmente Secas (FTSS) na América do Sul. Este tipo de vegetação compreende uma grande proporção de todas as espécies neotropicais. Entender o que modela a sua distribuição pode fornecer novas perspectivas para a evolução deste bioma e contribuirpara os aspectos de sua conservação. O trabalho avaliou a evolução deste bioma no sul do Brasil, onde as FTSS e as Florestas Tropicais (FT) são amplamente intercaladas. Para isso, foi reconstruídaa história filogeográfica do cacto, Cereus hildmannianus, uma espécie característica e abundante das FTSS. Métodos de datação molecular, estrutura populacional e filogeografia foram realizadas para avaliar os eventos histórico-demográficospor meio de uma amostragem densa que compreendeu 24 populações e cerca de 150 amostrasde, pelo menos, uma dentre as seis regiões genômicas nuclear e cloroplastidiais selecionadas. A partir disso, foi investigado um possível cenário da dinâmica populacional de C. hildmannianus. Os resultados indicam uma separação da espécie em dois grupos principais (ST: 0,788) com eventos de expansão populacional: umem regiões costeiras e o outro no interior do sul do Brasil, concondante com a distribuição dos núcleos das FTSS. O tempo do ancestral comum mais recente de C. hildmannianus, há 2,56 milhões de anos, remete a especiação deste ao período pré-Glacial. Os resultados do padrão de distribuição de C. hildmannianus foram concordantes com as áreas de endemismo para outros táxons das FTSS. Os eventos de dispersão e de vicariância entre as FTSS e as FT podem estar associados às mudanças paleoclimáticas durante os períodos glaciais do Quaternário, promovendo eventos de retração/expansão nestas florestas. A compreensão desses padrões na história biogeográfica de populações naturais podem auxiliar futuros planos de conservação deste bioma, na América do Sul. / There is controversy about the processes responsible for the current distribution of Seasonally Dry Tropical Forests (SDTF) in South America. This vegetation type comprises a large proportion of all Neotropical species. Understanding what shapes your distribution may provide new insights into the evolution of this ecosystem and contribute to aspects of conservation. The study evaluated the evolution of this biome in southern Brazil, where SDTF and Rainforests are widely interspersed. For this, we reconstructed the phylogeographic history of the cactus, Cereus hildmannianus, a kind of characteristic and abundant SDTF. Molecular dating methods, population structure and phylogeography were performed to evaluate the historical and demographic events through a dense sampling which comprised 24 populations and about 150 samples of at least one among the six nuclear and chloroplast genomic regions selected. From this, we investigated a possible scenario of population dynamics of C. hildmannianus. The results indicate a separation of the species into two main groups (ST: 0.788) with events of population expansion: one in coastal regions and the other inside the south of Brazil, concondante with the distribution of the nuclei of SDTF. The time of the most recent common ancestor of C. hildmannianuswere 2.56 million years ago, this speciation refers to the pre-Glacial. The results of the distribution pattern C. hildmannianus were consistent with areas of endemism for other taxa of SDTF. The events of dispersal and vicariance between SDTF and Rainforests may be related to paleoclimatic changes during glacial periods of the Quaternary, promoting events shrinkage /expansion in these forests. Understanding these patterns in the biogeographic history of natural populations may aid future conservation plans this biome in South America.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2016-11-07T16:30:36Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Evelise Carvalho.pdf: 2321912 bytes, checksum: 3957e800afc8b54ee2d2bc427c9dffd6 (MD5) / Made available in DSpace on 2016-11-07T16:30:36Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Evelise Carvalho.pdf: 2321912 bytes, checksum: 3957e800afc8b54ee2d2bc427c9dffd6 (MD5) Previous issue date: 2015-05-19 / Algas verdes da classe Trebouxiphyceae estão entre os organismos presentes no continente Antártico, onde a espécie mais relatada é a macroalga verde Prasiola crispa (Lightfoot) Kützing. Considerada um organismo extremófilo, pois se desenvolve com muito sucesso no habitat extremo da Antártica, ainda são raros na literatura dados moleculares sobre esta espécie, o que impede uma avaliação sobre sua taxonomia e posição filogenética. Com o advento das tecnologias de sequenciamento de nova geração, os genomas de organelas tornaram-se uma grande ferramenta para estudos de filogenia, pois fornecem inúmeros dados filogenéticos, sequências de proteínas e nucleotídeos e também informações sobre conteúdo gênico e arquitetura. Neste trabalho, foi determinada a sequência dos genomas do cloroplasto (cpDNA) e mitocondrial (mtDNA) de P. crispa, com o intuito de inferir as relações evolutivas deste organismos com outras espécies de plantas verdes, bem como uma análise estrutural. Os genomas plastidial e mitocondrial foram sequenciados por Macrogen Service (SolexaIllumina Hi-Seq 2500). A montagem, anotação, alinhamento, construção da filogenia e análise sintênica foram realizados in silico com softwares específicos. O cpDNA e mtDNA P. crispa apresentam 196.502 pb e 89.819 pb, respectivamente. Estes genomas acessórios apresentam 21 genes putativos relacionados com a fotossíntese e 18 genes relacionados com o metabolismo oxidativo. A análise filogenômica baseada no cpDNA demonstrou que P. crispa agrupou com alga trebouxiophyceae Prasiolopsis sp. formando o clado Prasiola juntamente com Stichococcus bacilaris. Nossos resultados para filogenômica embasada no mtDNA revelam que P. crispa agrupa com as outras espécies da classe Trebouxiphyceae. A análise de sintenia do cpDNA e mtDNA de P. crispa com a espécies de plantas verdes relacionadas evolutivamente demonstram que estes organismos apresentam poucos blocos gênicos sintênicos. Este trabalho pioneiro com a alga P. crispa, demonstra que os genomas acessórios suprem uma gama de dados moleculares que podem ser utilizados para estudos filogenômicos. Além disto, as informações geradas a partir do sequenciamento do cpDNA e mtDNA de P. crispa fornecem um aporte para estudos futuros mais aprofundados / Green algae from Trebouxiophyceae class are among the organisms in the Antarctic continent, where the most reported species is the green macroalga Prasiola crispa (Lightfoot) Kützing. This algae is considered an extremophile organism because develops successfully in the harsh Antarctic habitat, however studies reporting molecular data of this species are still lacking in the literature, which prevents an assessment of their correct taxonomy and phylogenetic position. With the advent of next generation sequencing technologies, it because easier to obtain molecular information as for example from organelle genomes making them a great tool for taxonomic studies because they provide a great number of, phylogenetic data, nucleotides, protein sequences, gene content and architecture information. In this study, we determined the sequence of the chloroplast (cpDNA) and mitochondrial (mtDNA) genome of P. crispa in order to infer the evolutionary relationships of the organisms with other species of green plants, as well as a structural analysis. Plastid and mitochondrial genome was sequenced by Macrogen Service (Illumina Solexa Hi-Seq 2500). The genome assembly, annotation, sequences alignment, phylogeny construction, and structural analyses were performed in silico with specific softwares. Plastid and Mitochondrial genomes have a total length of 196,502 bp and 89,819 bp, respectively. These genomes presented 21 putative photosynthesis related genes and 18 oxidative metabolism related genes, respectively. Phylogenetic analysis based on the cpDNA demonstrated that P. crispa grouped with Trebouxiophyceae algae Prasiolopsis sp. forming the Prasiola clade along with Stichococcus bacilaris. Our results for phylogenetic analysis grounded in mtDNA show that P. crispa groups with other species of Trebouxiphyceaen alga. Synteny analysis of P. crispa cpDNA and mtDNA with evolutionarily related species of green plants shows that these organisms have few syntenic gene blocks. This pioneering work with P. crispa provided the accessories genomes which suppled a range of molecular data that can be employed to taxonomic studies. In addition, the information generated from the sequencing of cpDNA and mtDNA of P. crispa provide a contribution for further studies.

L'expression du génome plastidial, un des trois génomes (nucléaire, mitochondrial et plastidial) qui coexistent dans les cellules végétales, est assurée par trois ARN polymérases. Deux NEP (Nuclear-Encoded RNA Polymerase) transcrivent la plupart des gènes de ménage tandis que la PEP (Plastid-Encoded RNA Polymerase) transcrit principalement les gènes liés à la fonction photosynthétique en s'associant à des facteurs de transcription d'origine nucléaire (facteurs sigma) importés dans le plaste. De précédents travaux dans l'équipe ont montré que, contrairement aux observations généralement admises, les trois ARN polymérases sont nécessaires pour assurer une germination efficace des graines d'Arabidopsis. L'objectif de notre travail est de comprendre comment ces enzymes ont été mises en place au cours de la formation de la graine. Pour cela, nous avons analysé l'expression de l'appareil transcriptionnel et du transcriptome plastidial durant les trois phases de formation de la graine d'Arabidopsis thaliana, c'est à dire l'embryogenèse, la maturation (phase photosynthétique) et la dessiccation. L'expression globale du transcriptome plastidial montre que les changements quantitatifs des transcrits sont les plus élevés pour les transcrits des gènes liés à la fonction photosynthétique. Ils sont très fortement exprimés pendant la phase de la maturation et diminuent ensuite, comme leurs protéines correspondantes. Nous observons également une forte accumulation des protéines codant les NEP et les sous unités de la PEP pendant la période de maturation des graines, suivie d'une forte diminution pendant la dessiccation. Cependant, les ARNm correspondants augmentent pendant la dessiccation. Le stockage de ces ARNm codant l'appareil transcriptionnel constitue une étape cruciale pour l'efficacité de la germination de la graine. La quantité de matériel biologique disponible pour ces études étant très limitée, nous avons développé une nouvelle technique de détection des ADNc sur lame de quartz, utilisant la microscopie TIRF. Cette méthode augmente la résolution (elle permet la détection de molécules uniques) et diminue considérablement la quantité de matériel nécessaire à l'hybridation. Finalement, nous avons analysé les conditions sous lesquelles se déroule la photosynthèse embryonnaire. Ces études ont montré que la photosynthèse dans l'embryon se déroule dans un environnement particulier, hypoxique, et sous un éclairement enrichi en longueurs d'onde vertes. Cependant, la structure et le fonctionnement de l'appareil photosynthétique sont semblables à ceux d'une feuille. Nous avons également montré que l'étape transitoire de la photosynthèse embryonnaire est indispensable à la vigueur germinative des graines. Les résultats obtenus lors de ce travail apportent de nouvelles informations sur le fonctionnement de la transcription plastidiale au cours de la formation de la graine. L'importance de l'accumulation d'ARNm, de certaines protéines ainsi que celle de la photosynthèse embryonnaire dans la vigueur germinative ont été soulignées. Ces données permettent de comprendre comment l'efficacité de la germination est conditionnée par la phase de formation de la graine. / Transcription of the plastid genome, one of the three genomes (nuclear, mitochondrial and plastidial) that co-exist in the plant cell, is performed by three ARN polymerases. Two NEPs (Nucleus-Encoded Plastid RNA polymerases) transcribe mainly housekeeping genes and one PEP (plastid-encoded RNA polymerase) transcribes principally photosynthesis related genes. PEP needs transcription factors of the sigma type that are nucleus-encoded. We have previously shown that all three RNA polymerases are present in dry seeds and are necessary for efficient germination. These findings raised the question of how theses RNA polymerases come into the dry seeds and what is the importance of plastid gene expression during seed formation. To answer this question my work consisted in the characterization of plastid gene expression profiles and the expression of the components of the plastid transcriptional machinery during the three phases of seed formation, i. e. embryogenesis, maturation (embryonic photosynthesis) and desiccation. The analysis of global plastid transcriptome patterns shows that mRNAs encoding proteins engaged in photosynthesis show the highest quantitative changes during seed formation. Highest mRNA levels are observed during maturation. During desiccation, photosynthesis related mRNA levels as well as the levels of the corresponding proteins strongly decrease. Concerning the expression of NEP and PEP components, we observe also a peak of protein accumulation during maturation that is followed by a strong diminution of the protein levels. On the other hand, the corresponding mRNAs increase continuously during desiccation. This means that these mRNAs accumulate without being translated. We conclude that the storage of mRNAs coding components of the plastid transcriptional machinery in dry seeds is important for efficient germination. Regarding the limited amount of biological material that is available for these types of studies, we have developed a new method for cDNA analyses on microchips that utilises quartz plates and TIRF microscopy. In this way we can visualise single molecules and the amount of necessary material is considerable diminished. Finally, we have also partially characterized the conditions under which embryonic photosynthesis is performed. These studies show that photosynthesis occurs in a special environment that is characterized by hypoxic atmosphere and green enriched light. However, the structure and functioning of the photosynthetic apparatus in seed chloroplasts seems to be very similar to that of chloroplasts in green leaves. This opens the question of how seed photosynthesis can be efficient. On the other hand we have shown that embryonic photosynthesis is indeed very important for efficient germination. Altogether, results provide new information on the functioning of plastid photosynthesis and transcription during seed formation. They underline the importance of the accumulation of NEP and PEP coding mRNAs in dry seeds. We suggest that embryonic photosynthesis influences seed germination not only by providing reserve compounds but also by producing NEP and PEP proteins. Although the majority of these proteins are degraded during desiccation, traces persist and are stored in dry seeds thus assuring immediate transcription of the plastid genome during imbibition/stratification. Our results explain how efficiency of germination is conditioned during seed formation.

Evolutionary biology is dependent on accurate phylogenies. In this thesis two branch support methods, Bayesian posterior probablities and bootstrap frequencies, were evaluated with simulated data and empirical data from the chloroplast genome. Bayesian inference was found to be more powerful and less conservative than maximum likelihood bootstrapping, but considerably more sensitive to choice of parameters. Bayesian inference increased in power when data were underparameterized, but the associated increase in type I error was comparatively larger. The chloroplast DNA phylogeny of the tribe Sileneae (Caryophyllaceae) was inferred by analysis of 33,149 aligned nucleotide bases representing 24 taxa. The position of the SW Anatolian taxa Silene cryptoneura and S. sordida strongly disagreed with previous studies on nuclear DNA sequence data, and indicate a possible case of homoploid hybrid origin. Silene atocioides and S. aegyptiaca formed a sister group to Lychnis and remaining Silene, thus suggesting that Silene may be paraphyletic, despite recent revisions based on molecular data. Several nodes in the phylogeny remained poorly supported, despite large amounts of data. Additional sequence sampling is not expected to solve this problem. The main reason for poor resolution is probably a combination of rapid radiation and substitution rate hererogeneity. Apparent incongruent patterns between different regions of the chloroplast genome are evaluated with ancient interspecific chloroplast recombination as explanatory model. Extremely elevated substitution rates in the exons of the plastid clpP gene was documented in Oenothera and three separate lineages of Sileneae. Introns have been lost in some of the lineages, but where present, intron sequences have a markedly slower substitution rate, similar to the rates found in other introns of their genomes. Three branches in the phylogeny show significant whole gene positive selection. In two of the lineages multiple partial copies of the gene were found.

The Sun is the ultimate energy source on Earth. Photosynthetic organisms are able to catalyze the conversion of solar energy to chemical energy by a reaction called photosynthesis. In plants, this process occurs inside a green organelle called the chloroplast. The protein complexes involved in the photosynthetic light reactions are situated in the thylakoid membrane, which encloses a tiny space called lumen. The Peptidyl-Prolyl cis-trans Isomerase (PPIase) family is the most abundant protein family in the thylakoid lumen. The three PPIase subfamilies, cyclophilins, FKBPs (FK506 binding proteins) and parvulins form a group by their enzymatic activity despite lack of sequence similarity between the subfamilies. Cyclophilins and FKBPs, collectively called immunophilins, were originally discovered as the targets of the immunosuppressive drugs cyclosporine A and FK506, respectively. By suppressing the immune response in humans, these immunophilin-drug complexes revolutionized the field of organ transplantation by preventing graft rejection. Cis-trans isomerization of peptide bonds preceding the amino acid proline is the rate-limiting step of protein folding and several immunophilins have been shown to be important for catalysis of protein folding in vivo. PPIases have been found to be part of large protein complexes as well as in functions such as signalling, protein secretion, RNA processing and cell cycle control. A picture is therefore emerging in which the actual interaction between the PPIase and its target is perhaps more important than the PPIase activity. In the present work, PPIases have been characterized in the chloroplast thylakoid lumen of Spinacia oleracea (spinach) and Arabidopsis thaliana (Arabidopsis). The most active PPIase in the spinach lumen was identified as the cyclophilin TLP20. AtCYP20-2, the Arabidopsis homologue of TLP20, was found to be upregulated at high light and attached to the thylakoid membrane, more precisely to the outer regions of photosystem II supercomplexes. In Arabidopsis, up to 5 cyclophilins and 11 FKBPs were predicted to reside in the lumen. Of these 16 immunophilins, only 2 were identified as active PPIases and significant differences were observed between the two plant species. AtCYP20-2, like TLP20, is an active isomerase although AtFKBP13 is the most active PPIase in the lumen of Arabidopsis. Mutant Arabidopsis plants deficient in AtCYP20-2 displayed no phenothypical changes or decrease in total lumenal PPIase activity. Being the only active PPIase in the mutants, the redox sensitive AtFKBP13 is proposed to compensate for the lack of AtCYP20-2 by oxidative activation. In agreement with the experimental data, the sequence analyses of catalytic domains of lumenal immunophilins demonstrate that only AtCYP20-2 and AtFKBP13 possess the amino acids found essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. It is concluded that with the exception of AtCYP20-2 and AtFKBP13 most immunophilins in the lumen of Arabidopsis lost their PPIase activity on peptide substrates and developed other specialized functions.

Les chloroplastes, responsables de la photosynthèse chez les organismes autotrophes, possèdent un génome plastidial codant de 100 à 130 gènes dont environ 80 pour des protéines principalement impliquées dans la photosynthèse, la transcription et la traduction. L'expression de ces gènes, coordonnée entre le plaste et le noyau, implique deux types d'ARN polymérases, la NEP (Nucleus Encoded RNA Polymerase) et la PEP (plastid Encoded RNA Polymerase) laquelle s‟associe à l‟un des 6 facteurs sigma (SIG), codés dans le noyau pour la reconnaissance spécifique de promoteurs de transcription. Nous avons tout d‟abord analysé le rôle de ces facteurs sigma dans la régulation transcriptionnelle des deux opérons codant des sous-unités de l‟ATP synthase, atpI/H/F/A et atpB/E, en précisant le rôle particulier de SIG3 dans la reconnaissance spécifique du promoteur (-418) de l‟atpH. Nous avons identifié les promoteurs des transcrits polycistronique et ceux situés en amont des gènes atpH et atpE, et avons montré (1) que les gènes des deux opérons sont co-régulés par SIG3 et SIG2 sauf atpI régulé par SIG2 seul et (2), que SIG3 jouerait un rôle essentiel dans la surexpression monocistronique d‟atpH par la reconnaissance d‟un promoteur (-418) en amont de atpH. L‟analyse systématique des transcrits plastidiaux accumulés en fonction de l‟éclairement des plantes nous a permis de corréler cette surexpression à un éclairement élevé (1300 μE) de plantes matures. SIG3 reconnaît aussi spécifiquement le promoteur de psbN, gène localisé sur le brin opposé de l‟opéron psbB/T/H/petB/petD, produisant un ARN anti-sens de psbT et de la région intergénique psbT/psbH. Nos résultats montrent que l‟anti-sens de psbT couvre la région codante, le 5'UTR et la quasi-totalité 3' UTR du transcrit sens psbT, pouvant ainsi réguler la production de PSBT en interférant dans la traduction par la formation d‟un duplex ARN. L‟anti-sens pourrait aussi intervenir dans le processing dans la région 5‟ UTR de psbH.

The sacoglossan sea slug Elysia clarki is able to photosynthesize for three to four months using chloroplasts sequestered from its algal food sources. Furthermore, the slug is able to store multiple chloroplasts from different algal species within the same cell. This research, consisting of several related studies, explores the role that provision of organic nutrients via photosynthesis plays in the biology of the slug. The first chapter demonstrates that, under conditions of starvation, photosynthetic activity in E. clarki remains fully functional for one month after which it then declines. During the first month of starvation the slug exhibits similar feeding behavior as slugs provided a continuous supply of food, suggesting that photosynthesis delays the onset of starvation-induced behavioral changes. The second chapter explores E. clarki's spatial relationships with algae known to be food sources in the field. In areas with high slug density, edible algal populations were very low. DNA barcoding was employed to demonstrate that the algae found near slugs were poor predictors of which foods were actually consumed by slugs. Generally, there was a mismatch between algae available in the field and slug diets. The third chapter explores how E. clarki is able to maintain photosynthesis. After labeling with a C14 ALA incubation process, then chlorophyll was extracted from slugs and purified using HPLC. Results indicate that recently collected E. clarki are able to synthesize chlorophyll, whereas slugs starved for 3 months were not. Photosynthesis plays a very important role for E. clarki and its relationships with food algae.