La ppGpp et la pppGpp, qui sont synthétisées/hydrolysées par les RelA/Spot homologs (RSH), jouent un rôle centrale dans l’adaptation des bactéries contre la privation des nutriments et les stress environnementaux. Les enzymes RSH et ppGpp ont été découverts dans le chloroplaste. Il a été récemment démontré que ppGpp joue un rôle comme répresseur globale de l’expression de gènes chloroplastiques. Certains stresses environnementaux et hormones induisent l’accumulation de ppGpp chez les plantes, cependant le mécanisme moléculaire n’est pas encore connu. Ici nous nous sommes intéressés à découvrir les facteurs qui interagissent avec les RSH, et qui donc sont susceptible de réguler le métabolisme du ppGpp. En utilisant un crible double-hybridation de levure nous avons identifiées des proteines qui interagissent avec les RSH y compris l’acyl carrier protein (ACP) et des GTPases associées au ribosome. ACP et RSH1 semblent être indispensables pour l’accumulation de ppGpp induite par la carence de la biosynthèse des acides gras, tandis que le ppGpp et un GTPase associé au ribosome contribuent à la résistance contre le heat-shock. Nous avons aussi effectué du co-immunoprécipitation spectrométrie de masse avec RSH1. Plusieurs protéines ont été identifiées y compris des protéines associées au nucléoid et des protéines liées à la signalisation chloroplastique, indiquant que RSH1 pourrait etre impliqué dans la machinerie de transcription chloroplastique. Nos résultats montrent que chez les plantes le ppGpp joue un rôle non seulement comme chez les bacteriés mais aussi participe à de nombreux processus biologiques qui sont spécifiques aux plantes. / Guanosine tetra-phosphate and penta-phosphate (ppGpp and pppGpp), which are synthesized/hydrolyzed by RelA/Spot homolog (RSH) enzymes, play a central role in the adaptation of bacteria to nutrient limitation or other stresses. Both RSH enzymes and ppGpp are present in the chloroplasts of plants. Recent studies have shown that ppGpp acts as a global repressor of chloroplast gene expression. Certain environmental stresses and hormones induce ppGpp accumulation in chloroplasts, however the molecular mechanisms underlying the activation of ppGpp signalling in response to such stimuli is essentially unknown. We searched for factors that interact with RSH enzymes and so could play a role in activating ppGpp signalling. Using a targeted yeast two hybrid screen several proteins were identified that interact with RSH enzymes including acyl carrier protein (ACP) and ribosome associated GTPases. ACP and RSH1 appear to be required for ppGpp induction in response to fatty acid biosynthesis depletion, while ppGpp and an RSH-interacting GTPase contribute to the resistance of plants to heat shock. We also performed non-targeted co-immunoprecipitation mass spectrometry (CoIP-MS) of RSH1. New RSH interaction candidates were identified, including plastid nucleoid associated proteins and chloroplast signalling proteins, suggesting that RSH1 may be associated with the plastid transcription machinery. Our results give new insights into ppGpp signalling, and show that some elements are conserved between plants and bacteria, while others are implicated in plant-specific biological processes.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv & Cons Biol) / Salicornia tegetaria is an endemic salt marsh macrophyte that is widely distributed in estuaries along the South African coast. The aims of the study were to understand the phylogeography of the species, compare the biomass allocation in two regions and to determine phenological patterns of S. tegetaria between the warm and cool temperate biogeographical regions. The phylogeography of S. tegetaria was studied using the noncoding chloroplast DNA region rpS16 and nuclear rDNA ITS region. Five samples each were collected from eighteen estuaries stretching from Orange River in the Northern Cape to Mngazana Estuary in the Eastern Cape. Above- and belowground biomass was collected and physico-chemical conditions measured at Olifants, Berg and Langebaan Estuaries in the cool temperate, and Heuningnes, Nahoon and Kwelera Estuaries in the warm temperate biogeographical regions. The growth and flowering phenology of S. tegetaria in relation to environmental conditions was investigated in the cool temperate Langebaan Estuarine Embayment and compared to findings in the warm temperate, permanently open Kowie Estuary. The physico-chemical gradient found between the cool and warm temperate biogeographical regions may be useful to study climate change effects on plant species. The comparison of similar habitats in each region may provide insight into how different climate regimes may affect biomass allocation and phenology.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-26T11:03:36Z No. of bitstreams: 2 Dissertação - Mayara Stefany da Silva Mariano Feitosa - 2017.pdf: 5065674 bytes, checksum: 310307efca7b4a8f1475e4e8accf0214 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-29T10:36:46Z (GMT) No. of bitstreams: 2 Dissertação - Mayara Stefany da Silva Mariano Feitosa - 2017.pdf: 5065674 bytes, checksum: 310307efca7b4a8f1475e4e8accf0214 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-10-29T10:36:46Z (GMT). No. of bitstreams: 2 Dissertação - Mayara Stefany da Silva Mariano Feitosa - 2017.pdf: 5065674 bytes, checksum: 310307efca7b4a8f1475e4e8accf0214 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Sugarcane (Saccharum spp.) is one of the most important crops in tropical and subtropical regions of the world. It is cultivated in more than 100 countries, where it is used as raw material to obtain sugar and bioethanol. Given its importance, many efforts have been carried out to characterize the genome of sugarcane cultivars. The eukaryotic genomes are confined in different cellular compartments that present different inheritance patterns. Plastids and mitochondria have their own genetic system, comprising DNA, RNA and all the demanded components for replication, transcription and protein synthesis, that occur inside these organelles. The primary function of chloroplasts and mitochondria is energy transduction. Chloroplasts are responsible to convert light into chemical energy during photosynthesis, while mitochondria provides energy to the cell in form of ATP molecules during respiration. In this work, the chloroplast and mitochondrial genomes of sugarcane cultivar RB867515 are assembled and characterized, using data from two next generation sequencing technologies – Illumina and PacBio. In chloroplasts, we sought to identify evidences of heteroplasmy, by using long reads from PacBio technology in the assembly process. In mitochondria, we investigated the occurrence of genetic and structural genomic variations. The assemblies were carried out using screened reads for the organellar genomes. These reads were selected by mapping whole genome shotgun reads to reference genome sequences of chloroplast and mitochondria, that are publicly available. The organellar reads were assembled using SPAdes and Organelle_PBA. Gene annotation was obtained using DOGMA, GeSeq and Mitofy tools. Two chloroplast haplotypes (isoforms) were identified in the cultivar RB867515. These isoforms are different from each other because they present a distinct orientation of the SSC (small single copy) region, confirming the hypothesis of chloroplast heteroplasmy in sugarcane. The genome of each chloroplast isoform comprises 141,181 bp, and shows a typic quadripartite structure, that includes a long single copy region (LSC) of 83,047 bp, which is flanked by two inverted repeat regions (IRs) of 22,795 bp and a small-single copy region (SSC), between IRs, of 12,544 bp. The assembled mitochondrial genome comprised two chromosomes of 300,765 bp and 194,383 bp. The estimates of GC (~44%) and AT (~56%) contents were similar to those obtained for other angiosperms. A total of 39 CDSs, 5hypothetical conserved genes, 5 rRNAs, 18 tRNAs and 9 gene fragments transferred from chloroplast were annotated. The RB867515 mitochondrial chromosomes showed differences when compared to those from S. officinarum, including single nucleotide polymorphisms (SNPs), genetic duplications and genomic expansions. / A cana-de-açúcar (Saccharum spp.) é uma das mais importantes culturas das regiões tropicais e subtropicais do mundo. A cana é cultivada em mais de 100 países, fornecendo matéria-prima para a obtenção de produtos como açúcar e bioetanol. Dada sua importância, diversos esforços vêm sendo realizados com o objetivo de se realizar a caracterização genômica de cultivares de cana-de-açúcar. Os genomas eucarióticos são distribuídos em diferentes compartimentos celulares que apresentam padrões distintos de herança. Plastídeose mitocôndrias possuem sistema genético próprio, contendo DNA, RNA e todos os componentes necessários para os processos de replicação, transcrição e síntese de proteínas, que ocorrem nestas organelas. Cloroplastos e mitocôndrias são organelas que têm como função principal a transdução de energia. Os cloroplastos são responsáveis pela conversão de energia luminosa em energia química, durante a fotossíntese. As mitocôndrias fornecem energia em forma de ATP, por meio da respiração celular. O presente trabalho foi desenvolvido com o objetivo de se realizar a montagem e a caracterização dos genomas cloroplastidial e mitocondrial da cultivar de cana-de-açúcar RB867515, utilizando dados de sequenciamento de nova geração Illumina e PacBio. Em cloroplastos, buscou-se identificar, pela utilização de reads longos obtidos pela tecnologia PacBio no processo de montagem, evidências de ocorrência de heteroplasmia cloroplastidial em cultivares modernas de cana-de- açúcar. No genoma mitocondrial investigou-se a ocorrência de variações genéticas e genômicas estruturais. Os assemblies foram obtidos pela utilização de reads organelares, selecionados através do mapeamento a sequências de referência de cloroplastos e mitocôndrias, disponíveis publicamente. Os assemblies obtidos foram realizados com os softwares SPAdes e Organelle_PBA. A anotação gênica foi realizada utilizando as ferramentas DOGMA, GeSeq e Mitofy. Foram identificados dois haplótipos (isoformas) de cloroplastos na cultivar RB867515. Estas isoformas diferem entre si pela ocorrência de orientações distintas da região SSC (small single copy), confirmando a hipótese de heteroplasmia cloroplastidial em cana-de-açúcar. Cada haplótipo é constituído por 141.181 pb e exibe uma estrutura quadripartida típica, que inclui uma região longa de cópia única (LSC) de 83.047 pb flanqueada por duas regiões de repetições invertidas (IRs) de 22.795 pb e uma pequena região de cópia única (SSC) entre as IRs de 12.544 pb. O genoma mitocondrial montado foi constituído por dois cromossomos: o cromossomo 1 de comprimento total de 300.765 pb e o cromossomo 2, de 194.383 pb. As estimativas obtidas para os conteúdos GC (~44%) e AT (~56%) foram concordantes com as de outras angiospermas. Foram anotados 39 CDSs, 5 genes hipotéticos conservados, 5 rRNAs, 18 tRNAs e 9 fragmentos de genes transferidos de cloroplastos. A comparação dos cromossomos mitocondriais da cultivar RB867515 com aqueles de S. officinarum permitiu a identificação de polimorfismos de bases únicas (SNPs), duplicações gênicas e expansões genômicas.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-12-20T16:03:47Z No. of bitstreams: 2 Tese - Luciana Cristina Vitorino - 2015.pdf: 8226538 bytes, checksum: 818b91ae152e61751eb80f72e40d2ef6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-12-26T13:21:49Z (GMT) No. of bitstreams: 2 Tese - Luciana Cristina Vitorino - 2015.pdf: 8226538 bytes, checksum: 818b91ae152e61751eb80f72e40d2ef6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-12-26T13:21:49Z (GMT). No. of bitstreams: 2 Tese - Luciana Cristina Vitorino - 2015.pdf: 8226538 bytes, checksum: 818b91ae152e61751eb80f72e40d2ef6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / There is strong evidence that the Neotropical vegetation has been influenced by climate changes at the end of the Tertiary and Quaternary. The response of vegetation to the cold weather and dry these periods, consistent with the occurrence of the last glacial maximum, still remains in debate. It is suggested that the areas currently occupied by the Cerrado and seasonal forests (SDTFs) are remnants of a continuous vegetation that existed in the past. From the perspective of that species widely distributed in savannas and Brazilian SDTFs could help unravel the role of climate change Pleistocene on the current distribution pattern of genetic diversity in these vegetation types, we sampled 17 populations of Tabebuia serratifolia in SDTFs and 24 populations Tabebuia ochracea in savannas and sequenced intergenic non-coding regions of chloroplast (psbA-trnH, trnG-trnS e trnC-ycf6) as well as the nuclear region ITS (nrDNA). Later, we used coalescent analysis, Ecological Niche Modeling techniques (ENM) and simulations of demographic hypothesis for these species in an attempt to broaden the understanding of the changes undergone by neotropical landscape during the last ice age cycles. The sampled populations for both species showed high genetic diversity for both markers (hcpDNA = 0.8731 and hnrDNA = 0.7723 - T. serratifolia and hcpDNA = 0.927 and hnrDNA = 0.637 - T. ochracea), and large structure (Fst(cpDNA) = 0.528, P < 0.001 and Fst(ITS) = 0.742, P < 0.001 - T. serratifolia and Fst(cpDNA) = 0.742, P < 0.001 and Fst(ITS) = 0.544, P < 0.001 - T. ochracea). The coalescing analysis showed the time to the most recent common ancestor between haplotypes of the sampled populations, oldest to T. serratifolia (~ 3.4 Ma - 95% CI 1.9 - 6.8), which for T. ochracea (~ 1.9 Ma - 95% CI 0.1 - 2.3). The two species show standard recent population expansion and the niche modeling revealed for the T. serratifolia a higher potential distribution area during Holoceno medium while for T. ochracea the highest suitability area was predicted for maximum glacial last (LGM - 21ka), going in favor the hypothesis that the savannas and STDFs have submitted in the past, a wider distribution than currently known. / Há fortes evidências de que a vegetação Neotropical tenha sido influenciada por alterações climáticas ocorridas no final do período Terciário e no Quaternário. A resposta da vegetação ao clima mais frio e seco desses períodos, condizente com a ocorrência do último máximo glacial, ainda permanece em debate. Sugere-se que as áreas atualmente ocupadas pelo Cerrado e florestas estacionais (SDTFs) sejam remanescentes de uma vegetação contínua que existia no passado. Sob a perspectiva de que espécies amplamente distribuídas em savanas e SDTFs brasileiras pudessem ajudar a desvendar o papel das mudanças climáticas do Pleistoceno sobre o padrão atual de distribuição da diversidade genética nessas formações vegetais, nós amostramos 17 populações de Tabebuia serratifolia em SDTFs e 24 populações de Tabebuia ochracea em savanas e sequenciamos regiões intergênicas não codificantes de cloroplasto (psbA-trnH, trnG-trnS e trnC-ycf6), bem como a região nuclear ITS (rDNA). Posteriormente, utilizamos análises coalescentes, técnicas de Modelagem de Nicho Ecológico (ENM) e simulações de hipóteses demográficas para essas espécies, na tentativa de ampliar o entendimento das mudanças sofridas pela paisagem neotropical durante os últimos ciclos de glaciação. As populações amostradas para as duas espécies apresentaram alta diversidade genética para ambos os marcadores (hcpDNA = 0.8731 e hnrDNA = 0.7723 – T. serratifolia e hcpDNA = 0.927 e hnrDNA = 0.637 – T. ochracea), e grande estruturação (Fst(cpDNA) = 0.528, P < 0.001 e Fst(ITS) = 0.742, P < 0.001 – T. serratifolia e Fst(cpDNA) = 0.742, P < 0.001 e Fst(ITS) = 0.544, P < 0.001 – T. ochracea). As análises coalescentes mostraram um tempo para o ancestral comum mais recente, entre os haplótipos das populações amostradas, mais antigo para T. serratifolia (~3.4 Ma - 95% CI 1.9 - 6.8), que para T. ochracea (~1.9 Ma - 95% CI 0.1 - 2.3). As duas espécies apresentaram padrão de expansão populacional recente sendo que a modelagem de nicho revelou, para T. serratifolia, maior área de distribuição potencial durante o Holoceno médio enquanto que para T. ochracea a maior área de adequabilidade foi predita para o último glacial máximo (LGM – 21ka), indo de encontro à hipótese de que as savanas e as STDFs possam ter apresentado, no passado, uma distribuição mais ampla que a conhecida atualmente.

Na célula eucariota, os processos metabólicos estão compartimentalizados em organelas e proteínas sintetizadas no citosol são endereçadas para elas por meio de sistema celular específico. Devido a sobreposições funcionais entre organelas, uma dada proteína pode ser requerida em mais de um compartimento. É o caso de proteínas com duplo direcionamento, em que um gene nuclear é capaz de gerar produtos protéicos direcionados para mais de uma organela. Cerca de 60 proteínas de plantas já tiveram seu duplo direcionamento demonstrado, a maioria para mitocôndrias e cloroplastos, portanto um fenômeno não tão raro como imaginado. Estudos recentes procuram esclarecer os mecanismos que permitem esse duplo direcionamento, mesmo existindo fatores celulares que garantem a especificidade do transporte para cada organela. O presente trabalho está dividido em três partes. Na primeira, foram investigados aspectos evolutivos do duplo direcionamento. Na análise comparativa de famílias gênicas com membros cujos produtos protéicos apresentam duplo direcionamento, em Arabidopsis thaliana e Oryza sativa, foi demonstrada a conservação do duplo direcionamento de monodeidroascorbato redutase, metionina aminopeptidase e, provavelmente, de THI1 (enzima da biossíntese de tiazol), entre as duas espécies. Os dados sugeriram um mesmo padrão de evolução em famílias incluindo membros com duplo direcionamento. Na segunda parte, o foco foi a seqüência de duplo direcionamento. Documentado o duplo direcionamento, para mitocôndrias e cloroplastos, de proteínas de ligação ao RNA, RBP1a, RBP1b e RPS19, mutações sítiodirigidas foram introduzidas na seqüência de direcionamento de RBP1b. A importância de aminoácidos positivos para o direcionamento de proteínas para mitocôndrias foi confirmada. Demonstrou-se que a mutação da alanina na posição 2, conservada em seqüências de direcionamento ambíguas para mitocôdrias e cloroplastos, não afeta o duplo direcionamento de RBP1b. A informação para o duplo direcionamento foi localizada entre os 17 primeiros aminoácidos da região amino-terminal. Os sinais de direcionamento para cloroplastos se distribuíram ao longo da seqüência. Enquanto a metade amino-terminal da seqüência foi suficiente para determinar o duplo direcionamento, a seqüência compreendendo os 13 aminoácidos seguintes afetaram a eficiência do transporte. Nessas análises, mostrou-se a adequação de método quantitativo na medida dos sinais de fluorescência da GFP, para ser aplicado em estudos quantitativos do direcionamento de proteínas para mitocôndrias e cloroplastos, in vivo. Na terceira parte, o foco foi o mecanismo traducional do duplo direcionamento de THI1, em A. thaliana. Entretanto, não foi possível testar a hipótese da presença de um sítio interno de entrada do ribossomo (IRES) no mRNA de thi1, pois não se encontrou um sistema de expressão transiente capaz de reproduzir dados da literatura que mostraram o duplo direcionamento de THI1. Nos sistemas testados, a proteína foi encontrada somente em cloroplastos, o que inviabilizou o prosseguimento da investigação. / Compartimentalization of the metabolic processes in organelles, each one having a characteristic protein pool and distinct functions, is a property of eukaryote cells. A highly specific cellular system directs proteins, which are synthesized in the cytosol, to the proper organelles. However, due to functional overlaps between organelles, a given protein may be needed in different compartments. This is the case of dual-targeted proteins, which are the product of single nuclear genes, but are somehow directed to different organelles. About 60 plant proteins have had their dual targeting demonstrated, most of them to mitochondria and cloroplasts, the phenomenon being not so rare as previously supposed. Investigations have focused on the mechanisms, which enable protein dual targeting, even in the presence of other cell mechanisms that guarantee the specific protein transport to each organelle. The present work on the subject can be divided in three parts. In the first part, evolutionary aspects of dual targeting were investigated. A comparative analysis of gene families that included members encoding dual-targeted proteins in Arabidopsis thaliana and Oryza sativa demonstrated that the dual targeting of monodehidro-ascorbate reductase, methyonine aminopeptidase and, problably, of the thiazole biosynthetic enzyme THI1 was evolutionary conserved between the two species. In addition, the data suggested the same pattern of evolution for families with members presenting dual targeting. The focus of the second part was the ambiguous sequence for dual targeting. After showing that the RNA-binding proteins RBP1a, RBP1b and RPS19 were dual-targeted to mitochondria and cloroplasts, sitedirected mutations were introduced in the targeting sequence of RBP1b. The importance of positive-charged amino acids for directing the protein to mitochondria was confirmed. Mutation of alanine at position 2, which is conserved in ambiguous sequences, was shown not to affect RBP1b dual targeting. Information for dual targeting was localized among the 17 first amino acids in the amino-terminal region. The signals for directing the protein to cloroplasts appeared distributed along the targeting sequence. While the amino-terminal half of the sequence was sufficient for RBP1b dual targeting, the sequence comprising the next 13 amino acids appeared to affect the efficiency of the transport. In these analyses, a quantitative method to measure the intensity of fluorescent signals of GFP had its efficacy demonstrated to be adopted for in vivo quantitative analysis of dual targeting to mitochondria and cloroplasts. In the third part, the focus was the translational mechanism enabling THI1 dual targeting to mitochondria and cloroplasts, in A. thaliana. It was hypothesized that an internal ribosomal entry site (IRES) was present in thi1 mRNA. However, a transient expression system could not be found that reproduced the literature data demonstrating THI1 dual targeting. In the tested systems the protein was addressed solely to cloroplasts, thus preventing the objective to be pursued.

Sacoglossan sea slugs are one of the best known examples of specialist herbivores in the marine environment and can be found strongly associated with their algal hosts and food sources. Perhaps the most intriguing characteristic of many sacoglossans is their ability to sequester functional chloroplasts from their algal food sources in a process called kleptoplasty. Despite this, there continues to be issues regarding taxonomic identification of species. In turn, the ecological characteristics of many of these slugs, such as algal host and food source preference, as well as their behavioral aspects, have received little attention. A prime example of these issues arises in one such kleptoplastic sacoglossan found at Sunset Beach, Tarpon Springs, USA. The slug had previously been identified as Elysia patina based on a recent description, but later evidence suggested this was incorrect. Furthermore, despite the evidence found for the slug’s photosynthetic capabilities, little was known of its ecological relationships and behavior. The purposes of this study were to: 1) correct the uncertain taxonomy of the Tarpon Springs slug previously identified as Elysia patina, and 2) explore the ecological and behavioral characteristics of the slug. First, a comparative study was performed between the Tarpon Springs slug and its original description, as well as descriptions for the superficially-similar congener slug, Elysia papillosa. The gross anatomy, dorsal surface vascular morphology, radular morphology, egg mass morphology, and developmental timeline of the Tarpon Springs slug were used as means of comparison with the previous descriptions. The results of the comparison show that the Tarpon Springs slug was in fact E. papillosa rather than E. patina, and that the most recent description used to identify the slug as E. patina was incorrect and should not be used. Second, a descriptive study of the ecological and behavioral aspects of E. papillosa at the Sunset Beach site were performed. From October 2014 to September 2015, bi-weekly algal collections were made to determine the seasonal abundance of the slug and a possible relationship between slug abundance and algal abundance. Next, a second collection study was performed bi-monthly from April to July 2016 to identify the preferred algal host of E. papillosa between the three most abundant rhizophytic algae at the site, Penicillus capitatus, Penicillus lamourouxii, and Halimeda incrassata, all of which have been previously reported as being hosts of E. papillosa. The results of these studies showed no relationship between slug abundance and algal abundance, however E. papillosa was found to have a seasonal population fluctuation, with the fewest slugs found in winter and summer months and the most slugs found in the spring and fall months, especially in April and May. Elysia papillosa was also found in significantly higher numbers on the alga P. capitatus compared to the other two algal species, although some slugs were found on P. lamourouxii. Only one slug was found on H. incrassata, indicating it is not being used as a host despite previous reports. Further exploration into the genetics of sequestered chloroplasts would provide critical details into E. papillosa’s feeding behavior in situ. Lastly, because of E. papillosa’s photosynthetic abilities, an experiment was performed to determine if E. papillosa exhibited phototactic behavior. Fully-fed slugs were found to have no preference for either light or dark conditions, indicating their behavior was not being influenced by their photosynthetic abilities. This information provides a means of comparison with future studies of the phototactic behavior of kleptoplastic slugs, and could provide insight into how the longevity of functional chloroplasts in each species of slug could influence their behavior.

Following their endosymbiotic uptake, plastids undergo profound changes to genome content and to their associated biochemistry. I have investigated how evolutionary transitions in plastid genomes may impact on biochemical pathways associated with plastid gene expression, focusing on the highly unusual plastids found in one group of eukaryotes, the alveolates. The principal photosynthetic alveolate lineage is the dinoflagellate algae. Most dinoflagellate species harbour unusual plastids derived from red algae. The genome of this plastid has been fragmented into small, plasmid-like elements termed “minicircles”. Transcripts of this genome receive a 3’ poly(U) tail and, in some species, undergo extensive sequence editing. Some dinoflagellates have replaced their original plastids with others, in a process termed “serial endosymbiosis”. The major non-photosynthetic alveolates are the apicomplexans, which include the malaria parasite Plasmodium. Apicomplexans are descended from free-living algae and possess a vestigial plastid, which originated through the same endosymbiosis as the ancestral red dinoflagellate plastid. This plastid has lost all genes involved in photosynthesis and does not possess a poly(U) tail addition pathway. I have investigated the consequences of the fragmentation of the red algal dinoflagellate plastid genome on plastid transcription. I have characterised non-coding transcripts in plastids of the dinoflagellate Amphidinium carterae, including the first evidence for antisense transcripts in an algal plastid. Antisense transcripts in dinoflagellate plastids do not receive poly(U) tails, suggesting that poly(U) tail addition may play a role in strand discrimination during transcript processing. I have additionally characterised transcript processing in dinoflagellate plastids that were acquired through serial endosymbiosis. I have shown that poly(U) tail addition and editing occur in the haptophyte-derived serial endosymbionts of the fucoxanthin-containing dinoflagellates Karenia mikimotoi and Karlodinium veneficum. This is the first evidence that plastids acquired through serial endosymbiosis may be supported by pathways retained from previous symbioses. Transcript editing constrains the phenotypic consequences of divergent mutations in fucoxanthin plastid genomes, whereas poly(U) tail addition plays a central role in recognising and processing translationally functional fucoxanthin plastid mRNAs. I have additionally shown that certain genes within fucoxanthin plastids are located on minicircles. This demonstrates convergent evolution in the organisation of the fucoxanthin and red algal dinoflagellate plastid genomes since their endosymbiotic acquisition. Finally, I have investigated transcript processing in the algae Chromera velia and Vitrella brassicaformis. These species are closely related to apicomplexans but are still photosynthetic and apply poly(U) tails to plastid transcripts, as with dinoflagellates. I have shown that poly(U) tails in these species are preferentially associated with translationally functional mRNAs of photosynthesis genes. This is the first plastid transcript processing pathway documented to target a specific functional gene category. Poly(U) tail addition may direct transcript cleavage and allow photosynthesis gene transcripts to accumulate to high levels. The loss of this pathway from ancestors of apicomplexans may have contributed to their transition from photosynthesis to parasitism.

Phytochromes (PHYs) and plant hormones have been emerging as important regulators of fleshy fruit physiology and quality traits; however, the relevance of PHY-hormonal signaling crosstalk in controlling fruit development and metabolism remains elusive. This Thesis assesses the role of PHYs and their interplay with auxins, cytokinins and ethylene during the regulation of tomato (Solanum lycopersicum) fruit development and ripening, with a focus on the control of the plastid biogenesis, sugar metabolism and carotenoid accumulation. In Chapter I, we present evidence that the deficiency in PHY chromophore phytochromobilin (P&Phi;B) biosynthesis, which leads to a global deficiency in functional PHYs, represses fruit chloroplast biogenesis in immature fruits and inhibits fruit sugar accumulation by transcriptionally downregulating sink- and starch biosynthesis-related enzymes. Genetic and physiological evidence suggested the involvement of both auxins and cytokinins as mediators of the negative impact of P&Phi;B deficiency on fruit sink strength and chloroplast formation. During the ripening phase, P&Phi;B deficiency was shown to delay the rise in climacteric ethylene production, affecting the ripening initiation rather than its progression. PHY-hormonal signaling crosstalk was shown to be active not only in the more externally positioned fruit tissues (i.e., pericarp) but also in the most inner fruit regions (i.e., columella). We, therefore, concluded that the global deficiency in functional PHY drastically affects fruit sugar metabolism, chloroplast formation as well as the timing of ripening via an intricate interplay involving phytochromes, auxins, cytokinins and ethylene. In Chapter II, we employed fruit-specific RNAi-mediated silencing of PHY genes to shed light on the specific role played by fruit-localized PHYs and their downstream signaling cascades on tomato fruit physiology and quality traits. Data revealed that fruit-localized SlPHYB2 negatively regulates chlorophyll accumulation in immature fruits whereas SlPHYA positively influences the plastid division machinery. Both SlPHYA and SlPHYB2 were shown to play overlapping, yet distinct, roles in controlling fruit starch metabolism and carotenoid biosynthesis. Our data implicated cytokinin signaling-related proteins as mediators of the SlPHYA-dependent regulation of plastid division machinery, and specific AUXIN RESPONSE FACTORs as intermediates in the PHY-mediated regulation of fruit sugar and carotenoid metabolisms. We concluded that fruit-localized SlPHYA- and SlPHYB2-mediated light perception regulate fruit plastid biogenesis as well as sugar and carotenoid metabolisms via coordinated changes in key components of both auxin and cytokinin signaling cascades. Altogether, this study brings important insights into the combined action of PHYs and hormones in the control of fruit plastid biogenesis and highlights that the interplay between PHY-hormonal signaling cascades influences essential features of tomato fruit quality, such as the sugar and carotenoid accumulation / Fitocromos (PHYs) e fitormônios têm sido caracterizados como importantes reguladores da fisiologia e qualidade de frutos carnosos; todavia, a importância de interações entre a sinalização hormonal e dos PHYs no controle do desenvolvimento e metabolismo de frutos ainda permanece pouco elucidada. Este trabalho de Tese avaliou o papel dos PHYs e das suas interações com as auxinas, as citocininas e o etileno sobre a regulação do desenvolvimento e amadurecimento de frutos de tomateiro (Solanum lycopersicum), particularmente no que tange ao controle da biogênese plastidial e metabolismos de açúcares e de carotenoides. No Capítulo I são apresentadas evidências de que a deficiência na produção de fitocromobilina (P&Phi;B), a qual resulta numa deficiência global in PHYs funcionais, impacta negativamente a biogênese de cloroplastos em frutos imaturos e inibe o acúmulo de açúcares por meio da repressão transcricional de enzimas relacionadas a biossíntese de amido e força de dreno nos frutos. Evidências genéticas e fisiológicas indicaram o envolvimento tanto das auxinas quanto das citocininas como mediadoras do impacto negativo da deficiência de P&Phi;B sobre a força de dreno dos frutos bem como na formação de cloroplastos. Durante a fase de amadurecimento, a deficiência em P&Phi;B atrasou a produção climatérica de etileno, afetando o início do amadurecimento mas não a sua progressão. As interações entre PHYs e hormônios mostraram-se ativas não apenas nos tecidos posicionados mais externamente (i.e., pericarpo) mas também nas regiões mais internas do fruto (i.e., columela). Conclui-se, portanto, que a deficiência global em PHYs funcionais afeta drasticamente o metabolismo de açucares, formação de cloroplastos, bem como o tempo de amadurecimento através de uma interação complexa envolvendo fitocromos, auxinas, citocininas e etileno. No Capítulo II utilizamos o silenciamento fruto-específico de PHYs a fim de desvendar de que forma a fisiologia e parâmetros de qualidade do tomate seriam regulados por PHYs presentes no próprio fruto. Os dados obtidos revelaram que moléculas de SlPHYB2 presentes no próprio fruto regulam negativamente o acúmulo de clorofilas nos frutos imaturos, já as de SlPHYA influenciam positivamente a maquinaria de divisão plastidial, e tanto SlPHYA quanto SlPHYB2 desempenham papel sobrepostos, porém distintos, no controle do metabolismo de amido e acúmulo de carotenoides em frutos de tomateiro. Evidências sugerem que proteínas relacionadas à sinalização de citocininas atuariam como mediadoras do impacto de SlPHYA sobre a maquinaria de divisão plastidial, e que AUXIN RESPONSE FACTORs específicos seriam intermediários no controle dos PHYs sobre os metabolismos de açúcares e carotenoides. Conclui-se, dessa forma, que a percepção de luz mediada por moléculas de SlPHYA e SlPHYB2 presentes no próprio fruto regulam a biogênese plastidial e os metabolismos de açúcares e carotenoides por meio de alterações coordenadas em componentes chaves das cascatas de sinalização de auxinas e citocininas. Quando combinados, os dados obtidos neste estudo apresentam novidades importantes sobre a ação conjunta de PHYs e fitormônios no controle da biogênese plastidial e demonstram que a interação entre esses sinalizadores influencia características essenciais da qualidade de frutos de tomateiro, tais como o acúmulo de açúcares e de carotenoides

A síndrome Kranz é um conjunto de características anatômicas, ultra-estruturais e bioquímicas que culminam na realização da fotossíntese C4. Tal síndrome apresenta grande diversidade dentre as Angiospermas, tornando-se conveniente seu estudo em todos os níveis acima citados para a completa caracterização da mesma. No presente trabalho foi investigada a síndrome Kranz de Gomphrena arborescens e G. scapigera (Amaranthaceae) com ênfase na origem ontogenética da bainha Kranz, na descrição ultra-estrutural e na confirmação bioquímica sobre o tipo de fotossíntese C4. O desenvolvimento foliar dessas espécies indica que a bainha Kranz é originada da camada mais interna do mesofilo, a endoderme foliar. Uma discussão sobre os termos presentes na literatura para a descrição dessa bainha, todos eles focados em sua função na fotossíntese C4, demonstra a importância de se utilizar termos que informem a origem ontogenética dessa bainha, como endoderme e periciclo. Na análise ultra-estrutural, foram identificados possíveis fatores que interferem na fotossíntese de ambas as espécies, como o espessamento e a composição da parede da bainha Kranz, o posicionamento centrípeto dos cloroplastos e a presença de retículo periférico nos mesmos. Embora a análise bioquímica tenha resultado em informações ainda não conclusivas, o dimorfismo dos cloroplastos sugere a realização da fotossíntese C4 do tipo NADP-ME. O presente trabalho, de uma forma geral, contribui ao conhecimento da síndrome Kranz dentre as Amaranthaceae s.s., um grupo em que a ultra-estrutura e a bioquímica ainda são pouco conhecidas, e ressalta a importância dos estudos anatômicos, principalmente com enfoque ontogenético, para o melhor conhecimento da diversidade da síndrome Kranz dentre as Angiospermas. / The Kranz syndrome is a set of anatomical, ultrastructural and biochemical features that culminate in the C4 photosynthesis. This syndrome has a huge diversity among Angiosperms, so it became suitable to survey all the levels above cited for its complete characterization. In the present work the Kranz syndrome of Gomphrena arborescens and G. scapigera (Amaranthaceae) is studied, with emphasis on the ontogenetic origin of the Kranz sheath, on the ultrastructural description, and on the biochemical confirmation about the C4 photosynthesis kind. The foliar development of these species shows that the Kranz sheath is originated from the inner layer of the mesophyll, the foliar endodermis. A discussion about the literature terms used to describe the Kranz sheath, all of them referring to the function of this layer in C4 photosynthesis, demonstrates the importance of using terms that inform the ontogenetic origin of this layer, such as endodermis and perycicle. The ultrastructural analysis identified possible factors that interfere on the C4 photosynthesis of both species, such as wall thickening and composition of Kranz sheath cells, the centripetal position of chloroplasts and the peripheral reticulum in chloroplasts. Although biochemical analysis has resulted in no conclusive information, the chloroplast dimorphism suggests the NADP-ME C4 photosynthesis. This work, in a general way, contributes to the knowledge of the Kranz syndrome among Amaranthaceae s.s., a group that has the ultrastructure and the biochemistry of C4 photosynthesis poorly known. It also draws attention to the importance of anatomical surveys concerning the ontogenetic origin of Kranz sheath for a better understanding on the diversity of Kranz syndrome among Angiosperms.

Zuzana Kubínová: Study of Quantitative Parameters of Norway Spruce Needle Structure under the Effect of Elevated CO2 Concentration and Different Irradiance, Doctoral Thesis, Prague 2019 Abstract Atmospheric concentration of CO2 is increasing, while its influence on plants is still not fully elucidated. Norway spruce (Picea abies L. Karst.) is an abundant conifer tree in European temperate and boreal forests, which behave as carbon sink in the global carbon cycle. The physiological response to elevated CO2 concentration may be interconnected with changes in leaf anatomy and morphology. Needle structure is also determined by other factors in addition to CO2 concentration, irradiance being the most important one. Thus, effect of irradiance was also included in our studies. The effects of elevated CO2 concentration and irradiance on Norway spruce needle structure were studied using new applications of well-established quantitative methods and novel methods enabling effective and unbiased analysis of needle structural traits. The General Procrustes analysis showed to be effective for needle shape on cross section comparison and the disector method proved to be suitable for chloroplast number estimates. The influence of elevated CO2 concentration and different irradiance on needle structure was studied at two...