PPR-Proteine bilden die größte Familie von RNA-Bindeproteinen in Pflanzen und sie werden fast ausschließlich in die Mitochondrien oder Plastiden importiert, wo sie eine wesentliche Rolle im RNA-Metabolismus spielen (Lurin et al., 2004). Doch die Funktionsweise der Proteine ist noch weitgehend unbekannt. In dieser Arbeit wurde das plastidäre PPR-Protein PPR5 in Zea mays funktionell charakterisiert, dessen Ortholog in Arabidopsis thaliana essentiell für die Embryogenese ist (Cushing et al., 2005). Mittels PPR5-Immunopräzipitation und einer Analyse der kopräzipitierten RNA konnte in vivo eine spezifische Assoziation mit der ungespleißten tRNA-Glycin (UCC) nachgewiesen werden. Analysen von ppr5-Mais-Mutanten offenbarten einen Stabilitätsverlust dieser RNA. Es wurde gefolgert, dass PPR5 das Transkript vor einem endonukleolytischen Abbau schützt. Die weiteren Projekte der Arbeit widmeten sich der Evolution der Familie. Um Erkenntnisse zur Funktion und Spezifität nahe verwandter PPR-Proteine zu erhalten, wurden die drei nächsten Verwandten von PPR5 identifiziert und Mais-Mutanten isoliert. Weiterhin wurde PPR54 untersucht. Es konnte gezeigt werden, dass PPR54 in Mais – wie in Arabidopsis (Tillich, nicht publiziert) – für das Spleißen des ndhA-Introns benötigt wird. Damit wurden erstmalig orthologe PPR-Proteine in einer Mono- und einer Dikotylen funktionell analysiert. Die vorgelegten Analysen mündeten in drei allgemeingültigen Schlussfolgerungen zur Funktion der PPR-Proteine. 1) Plastidäre PPR-Proteine, die in Dikotylen wie Arabidopsis für die Embryogenese notwendig sind, üben eine Funktion in der plastidären Translation aus. 2) Die vorgeschlagene Funktionsweise von PPR5 erfordert nicht die Rekrutierung anderer, katalytisch aktiver Proteine, sondern ihr liegt ein passiver, auf der Bindung einer RNA beruhender Mechanismus zugrunde. 3) Die Funktion orthologer PPR-Proteine ist in Mono- und Dikotylen konserviert, wie am Beispiel von PPR54 experimentell nachgewiesen wurde. / PPR proteins are the largest family of RNA binding proteins in plants and the vast majority of them is localized to mitochondria or chloroplasts, where they are major players in the RNA metabolism of defined transcripts (Lurin et al., 2004). However, the mechanistic function of these proteins is still not clear. In this study, the plastid PPR protein PPR5, whose ortholog in Arabidopsis thaliana is embryo-essential (Cushing et al., 2005), was functionally characterized in Zea mays. By PPR5 immunoprecipitation and analyses of the coimmunoprecipitated RNA, a specific association to the unspliced tRNA glycine (UCC) was shown in vivo. The analysis of ppr5 maize mutants demonstrated a loss of stability of the tRNA precursor in mutants. It was concluded that the interaction with PPR5 protects the unspliced tRNA from endonucleolytic decay. In addition, close relatives of PPR5 were identified in maize (PPR2, PPR50, and PPR51) by phylogenetic means and maize mutants were isolated. A future characterization of four paralogous PPR proteins might answer whether closely related PPR proteins have similar functions or RNA targets. The analysis of PPR54 in maize demonstrated that PPR5 is needed for the splicing of the ndhA intron in maize as it is in Arabidopsis (Tillich, not published). Three important conclusions concerning the function of PPR proteins in general were drawn from the studies of chosen PPR proteins presented here. First, plastid PPR proteins that are essential in embryo development in eudicots like Arabidopsis should be necessary for plastid translation in most cases. Second, the characterization of PPR5 revealed a possibly ancient functional mechanism of PPR proteins which does not invoke the recruitment of additional catalytic factors but relies on the passive binding of RNA elements. Last, the conservation of function of orthologous PPR proteins in monocots and eudicots, which was shown in the case of PPR54, was demonstrated experimentally for the first time.

Die chloroplastidäre Genexpression ist durch charakteristische posttranskriptionelle Ereignisse, wie RNA-Prozessierung, RNA-Stabilität, RNA-Edierung oder RNA-Spleißen gekennzeichnet. Diese Prozesse werden fast ausnahmslos durch kernkodierte Proteine realisiert. PPR-Proteine (Pentatricopeptid repeat) stellen unter diesen kernkodierten Faktoren die größte Proteinfamilie dar. Das plastidäre Protein P67 gehört zur kleinen Untergruppe der PPR-Proteine mit SMR-Domäne (small MutS-related), deren molekulare Funktion im organellären Nukleinsäuremetabolismus bislang unverstanden ist. P67 zeigt eine nahe Verwandtschaft zu GUN1, einem zentralen Bestandteil retrograder Signalwege. Der hier analysierte P67-Knockout in Mais verursacht hellgrüne Phänotypen, eine drastische Reduktion der plastidären ATPase und Keimlingsletalität, was die essentielle Beteiligung von P67 an den Prozessen der Chloroplastenbiogenese und der Expression der plastidär kodierten ATPase-Untereinheiten vermuten lässt. Mögliche Implikationen eines fehlenden Phänotyps von Mutanten des P67-Orthologs aus Arabidopsis thaliana werden diskutiert. Eine Ausnahmestellung unter den Proteinen des chloroplastidären RNA-Metabolismus nimmt der einzige plastidär kodierte RNA-Reifungsfaktor MatK ein. Die genomische Position des matK-Gens im Intron der trnK-UUU ist in allen grünen Landpflanzen konserviert. MatK ist mit bakteriellen Maturasen verwandt, die spezifisch den Spleißprozess ihres Heimatintrons unterstützen. Dagegen deuten genetische und phylogenetische Studien zusätzliche MatK-Funktionen in trans an. In der vorliegenden Arbeit wird die spezifische Interaktion von MatK mit sieben Gruppe-IIA-Intron enthaltenden Transkripten in vivo gezeigt. Darunter befinden sich vier tRNA-Vorläufer (trnK-UUU mit dem matK-Heimatintron sowie trnV-UAC, trnI-GAU, trnA-UGC) und drei proteinkodierende Vorläufertranskripte (rpl2, rps12, atpF). Die Feinkartierung der MatK-Bindung im trnK-Heimatintron zeigt eine Assoziation mit multiplen Regionen. Organelläre Gruppe-II-Introns gelten als Vorläufer der spleißosomalen Introns. Die Assoziation mit multiplen Gruppe-II-Introns macht MatK somit zu einem interessanten Modell für die Evolution der transaktiven Spleißaktivität im Kern. Analysen der Expression von MatK und seinen Zielen deuten auf ein komplexes Muster möglicher regulativer Interaktionen hin. / Chloroplast gene expression is characterized by posttranscriptional events including RNA cleavage, RNA stability, RNA editing, and RNA splicing. The underlying processing machinery is almost exclusively encoded in the nucleus. PPR proteins (pentatricopeptide repeat) form the biggest protein family among these factors and are major players of the aforementioned posttranscriptional processes. The plastidial protein P67 is a member of a small subgroup of PPR proteins with SMR domain (small MutS-related). Molecular functions of this protein family in organellar nucleic acid metabolism are yet unknown. P67 is a close relative of GUN1, an essential component of the chloroplast to nucleus retrograde signalling pathway. It is shown here that a P67 knockout in maize causes pale green phenotypes, a dramatic reduction in ATPase levels, and seedling lethality. This indicates an essential role of P67 for chloroplast biogenesis and expression of the plastid encoded ATPase. The finding that mutants of the P67-orthologe in Arabidopsis lack a phenotype is discussed against the background of physiological differences between maize and Arabidopsis. A special case among proteins involved in plastid RNA metabolism is MatK - the only plastid encoded RNA maturation factor. The genomic position of the matK gene in the trnK-UUU intron is conserved throughout autotrophic land plants. MatK is related to bacterial maturases - highly specific splice factors supporting splice processes of their respective home introns. There is, however, indirect genetic and phylogenetic evidence that MatK acts also in trans as a common plastidial splice factor serving various group II introns. This study shows that MatK interacts specifically with seven group IIA introns in vivo. Among them are four tRNA precursor transcripts (trnK-UUU including the matK home intron as well as trnV-UAC, trnI-GAU, trnA-UGC) and three protein-coding precursors (rpl2, rps12, atpF). Fine mapping of MatK binding sites within the trnK home intron uncovers protein RNA interactions with diverse intron regions. Organellar introns have been suggested as evolutionary ancestors of nuclear spliceosomal introns. Consequently, association of MatK with multiple group II intron ligands makes the plastidial maturase an attractive model for an early trans-acting nuclear splice activity. Analyses of the expression of MatK and its targets revealed a complex pattern of possible regulatory interactions.

La stabilité génomique des organelles de plantes suscite un grand intérêt dans le domaine de la biologie végétale. En effet, plusieurs études récentes suggèrent que ce type d’instabilité génomique pourrait mener à l’isolation de traits intéressants en l’agronomie. Plusieurs protéines sont d’ailleurs déjà été identifiés comme étant impliqués dans le maintien de la stabilité de ces génomes, tels que MSH1, la famille des POLI, OSB1, les protéines Whirly et les Recombinases A (RECA). Le génome nucléaire d’Arabidopsis thaliana encode trois protéines s’apparentant à la Recombinase A bactérienne et qui sont ciblées à la mitochondrie et/ou au chloroplaste, soit RECA1, RECA2 et RECA3. Globalement, ces gènes partagent une similarité de séquence de 61% avec leur homologue bactérien chez Escherichia coli. Chez les bactéries ces protéines jouent un rôle essentiel dans la recombinaison homologue et sont impliquées dans la réparation de l’ADN. Chez Arabidopsis, il a été démontré que RECA2 et RECA3 sont nécessaires au maintien de l’intégrité du génome mitochondriale. Toutefois leur contribution à la stabilité du génome chloroplastique ainsi que le rôle de RECA1 restent obscures. Le but de ce projet est donc de déterminer la contribution éventuelle des protéines RECA d’Arabidopsis dans la réparation de l’ADN chloroplastique et plus précisément le rôle du gène RECA1. Nous énonçons l’hypothèse que les RECA de plantes se comportent effectivement comme leurs orthologues bactériens en étant impliqués dans la recombinaison homologue. Dans le cadre de ce projet, nous avons tenté d’isoler des lignées mutantes pour chacun des gènes RECA d’Arabidopsis. En somme, nous avons pu obtenir des lignées convenables pour notre étude que dans le cas du gène RECA1. Ces lignées ont été utilisées pour évaluer la contribution de ce gène à la stabilité du génome du chloroplaste. Ensuite, pour étudier la relation épistatique des gènes RECA1, WHY1 et WHY3, un croisement des différentes lignées mutantes pour ces gènes a été réalisé. Nous avons ensuite étudié la sensibilité de toutes ces lignées mutantes à la ciprofloxacine, un agent causant des bris double brin exclusivement dans les organelles de plantes. Finalement, iii nous avons testé la présence de réarrangements dans le génome du chloroplaste en condition normal ou en présence de stress génotoxique. Nos résultats démontrent que les protéines Whirly et RECA1 sont impliquées dans deux voies de réparation de l’ADN différentes et que les Whirly sont suffisantes pour s’occuper des bris d’ADN double brin en l’absence de RECA1. Nous démontrons également que l’absence de Whirly et RECA1 entraine une forte augmentation de la quantité de réarrangements dans le génome du chloroplaste. De plus nous proposons que la polymérase POLIB est impliquée dans la même voie de réparation que RECA1. Finalement nous proposons un modèle pour expliquer nos résultats et impliquons RECA1 dans un mécanisme de réparation d’ADN et aussi un rôle potentiel dans la réplication. / The stability of plant organelles genomes elicits a great interest in the domain of plant biology. In fact, numerous studies suggest that genomic instability can lead to the isolation of interesting traits in the field of agronomy. Some factors such as MSH1, the POLI family, OSB1, the Whirly proteins and the Recombinase A (RECA), have already been identified has being implicated in the maintenance of genome stability. The nuclear genome of Arabidopsis thaliana encodes three proteins, RECA1, RECA2 and RECA3, that shares a high resemblance with bacterial Recombinase A. They are targeted to the mitochondria and/or to the chloroplast. Globally, these genes share a similarity of sequence of 61% with their bacterial homologue in Escherichia coli. In bacteria these proteins play an essential part in homologous recombination and are implicated in DNA repair. In Arabidopsis, RECA2 and RECA3 have been shown as being essential to maintain the integrity of the mitochondrial genome but their contribution to the stability of the chloroplast as well as the role of RECA1 remains obscure. The goal of this project is to establish the eventual contribution of the Arabidopsis RECA proteins in the repair of chloroplast DNA and more precisely the role of the RECA1 gene. We propose the hypothesis that plants RECA act in the same fashion as their bacterial orthologues by being implicated in homologous recombination. Within the framework of this project, we have attempted to isolate mutant lines for each RECA gene of Arabidopsis. In the end, we were able to obtain appropriate lines for our study only for the RECA1 gene. These lines were then used to evaluate the contribution of the gene to chloroplast genome stability. Afterwards, in order to study the epistatic relationship between the RECA1, WHY1 and WHY3 genes, a cross between different mutant lines of these genes was realised. We then studied the sensitivity of all of those mutant lines to ciprofloxacine, an agent causing double stranded breaks exclusively in plant organelles. Finally, we evaluated the presence of rearrangements in the chloroplast genome under normal conditions and under the presence of a genotoxic stress. Our v results show that the Whirly and RECA1 proteins are implicated in two separate pathways of DNA reparation and that the Whirly proteins are sufficient to take in charge DNA double strand breaks generated by the absence of RECA1. We also demonstrate that the absence of Whirly and RECA1 causes an increasein the quantity of rearrangements in the chloroplast genome. Furthermore, we propose that the polymerase POLIB is implicated in the same repair pathway as RECA1. Finally we propose a model to explain our results and implicate RECA1 in a DNA repair mechanism and propose a role for RECA1 in DNA replication.

No description available.

Das plastidäre Genexpressionssystem höherer Pflanzen ist hochkomplex und differiert beträchtlich von dem prokaryotischer Vorfahren. Jeder einzelne Schritt der RNA-Prozessierung und Translation ist abhängig von nukleär kodierten, posttranslational in den Chloroplasten importierten Proteinen, insbesondere RNA-Bindeproteinen, wie den chloroplastidären Ribonukleoproteinen (cpRNP). Ein wichtiger und im Fokus dieser Arbeit stehender Vertreter ist CP29A, welcher ein breites Bindespektrum aufweist und in vivo mit einer Vielzahl von Transkripten interagiert. Frühere Studien belegen dennoch, dass ein Knockout von CP29A unter Standardkultivierungsbedingungen nicht in einem makroskopisch vom Wildtyp differierenden Phänotyp resultiert. Unter Kältestress hingegen ist CP29A für die Entwicklung photosynthetisch aktiver Chloroplasten essenziell. Zur tiefergehenden Charakterisierung der molekularen Funktion von CP29A wurde in der vorliegenden Arbeit eine genomweite Transkriptomanalyse der cp29a-Mutante durchgeführt. Es konnte erstmals gezeigt werden, dass CP29A bereits unter Standardkultivierungsbedingungen das Spleißen vieler plastidärer Introns unterstützt. Kälteinduziert weist das phänotypisch auffällige Gewebe der cp29a-Mutante eine globale Beeinträchtigung der Genexpression sowie massive Defekte der plastidären mRNA-Prozessierung auf. Da die Funktionalität von Proteinen substanziell von ihrer Lokalisation abhängig ist, wurden antikörperbasierte mikroskopische Lokalisationsstudien durchgeführt. Diese dokumentieren, dass CP29A kältestressinduziert zu dynamischen Granula lokalisiert, welche separiert von den plastidären Nukleoiden vorliegen und mit einem häufig in stressinduzierten Granula detektierten Protein kolokalisieren. / The plastid gene expression system in higher plants is highly complex and differs significantly from the gene expression system of the prokaryotic ancestors. Each individual step of RNA-processing and translation is dependent on nuclear-encoded RNA-binding proteins, such as chloroplast ribonucleoproteins (cpRNP), which are imported post-translationally into the chloroplast. An important representative is CP29A, which has a broad binding spectrum and interacts with a large number of transcripts in vivo. Earlier studies show that, while a knockout of CP29A under standard-cultivation-conditions does not result in a macroscopically different phenotype, under cold stress conditions CP29A is essential for the development of photosynthetically active chloroplasts. For a more detailed characterization of the molecular function of CP29A, a genome-wide transcriptome analysis of the cp29a mutant was carried out. It could be shown for the first time that CP29A already supports the splicing of many chloroplast introns under standard-cultivation-conditions. Cold-induced the phenotypically noticeable mutant tissue shows a global impairment of gene expression and massive defects in plastid mRNA processing. Since the functionality of proteins is substantially dependent on their localization, antibody-based microscopic localization studies were carried out. They reveal that cold stress-induced CP29A localizes to dynamic granules, which are separate from the plastid nucleoids and colocalize with a protein commonly detected in stress-induced granules.

Die chloroplastidäre und mitochondriale Genexpression ist abhängig von einer großen Anzahl an RNA-Bindeproteinen (RBPs). Eine besonders abundante Familie sind die chloroplastidären Ribonukleoproteine (cpRNPs). Während Ziel-RNAs mehrerer cpRNPs und die Phänotypen entsprechender Mutanten beschrieben wurden, bleibt ihre molekulare Funktion weitgehend ungeklärt. In dieser Arbeit wurden Studien der cp29a Mutante durch genomweite Analysen erweitert. Diese legen nahe, dass die eigentliche Rolle von CP29A in phänotypisch erkennbarem Mutanten-Gewebe durch sekundäre Defekte maskiert wird. Um primäre Defekte zu identifizieren, wurden in vivo Bindestellen von CP29A mit einer neuen Chloroplasten-adaptierten Methode, die UV-Licht zur Quervernetzungen nutzt, bestimmt. Transkripte, die für Untereinheiten des Photosystem II und des Cytochrom-b6f-Komplexes kodieren, waren unter den Zielen von CP29A überrepräsentiert. Weiterhin wurden mehrere Bindestellen in Nachbarschaft zu Bindestellen von PPR-Proteinen identifiziert. Mit einer alternativen Methode, die chemische Quervernetzung nutzt, wurden Ziel-RNAs eines weiteren cpRNP, CP31A, identifiziert. Transkripte, die für Untereinheiten des NADH-Dehydrogenase Komplexes kodieren, waren überrepräsentiert. Diese Daten führten zu einer neuen Hypothese, die die Funktion von cpRNPs im Zusammenspiel mit PPR-Proteinen in der Prozessierung funktionell verwandter RNAs postuliert. Ein weiterer für die Genexpression relevanter Mechanismus ist die Bildung membranloser Kompartimente durch flüssig-flüssig Phasentrennung. Es wurde eine in silico Analyse durchgeführt, um organelläre Proteine mit Domänen, die auf flüssig-flüssig Phasentrennung hindeuten, zu identifizieren. Funktionen mit Bezug zu Genexpression, insbesondere RNA-Edierung, waren bei diesen Proteinen mit Prionen-ähnlichen Domänen (PLDs) überrepräsentiert. Zwei Kandidaten wurden auf ihre Neigung zur flüssig-flüssig Phasentrennung durch in vitro Experimente und in vivo Mikroskopie untersucht. / Gene expression in chloroplasts and mitochondria relies on a large number of RNA-binding proteins (RBPs), which are involved in the processing of polycistronic precursor transcripts. A particular abundant family are the chloroplast ribonucleoproteins (cpRNPs). While target RNAs and mutant phenotypes of several cpRNPs were described, insights on their molecular function remained sparse. In this thesis, analyses of cp29a mutants were extended by genome-wide transcriptome data, which suggest that in phenotypically noticeable mutant tissue the actual role of CP29A might be masked by secondary effects. To identify primary defects, in vivo binding sites of CP29A on its target transcripts were determined using a novel chloroplast-adapted approach using crosslinking by UV-light. Identified targets of CP29A are functionally enriched in mRNAs encoding subunits of the photosystem II and the cytochrome b6f complex. Moreover, several binding sites were identified in close proximity to characterized binding sites of PPR proteins. Using an alternative approach, employing chemical crosslinking, targets of another cpRNP, CP31A, were identified. Targets are enriched in genes encoding subunits of the NADH-like dehydrogenase complex. In combination, these data led to a novel hypothesis on the molecular function of cpRNPs working together with PPR proteins in the processing of functionally related RNAs. Another increasingly recognized mechanism in gene expression is the formation of membraneless organelles by liquid-liquid phase separation. An in silico screen for organellar proteins containing domains indicative of phase separation was performed. The identified set of proteins with prion-like domains (PLDs) is enriched in functions related to gene expression, particularly RNA-editing. Two selected candidate proteins were characterized for their propensity to undergo phase separation by in vitro phase separation assays and in vivo microscopy.

L'un des phénomènes les plus importants survenus pendant la maturation du fruit de tomate est le changement de couleur du vert au rouge. Ce changement a lieu dans les plastes et correspond à la différenciation des plastes photosynthétiques, les chloroplastes, en plastes non-photosynthétiques qui accumulent des caroténoïdes, les chromoplastes. Dans cette thèse, nous présentons d'abord une introduction bibliographique sur le domaine de la transition chloroplaste-chromoplaste, en décrivant les modifications structurales et physiologiques qui se produisent pendant la transition. Puis, dans le premier chapitre, nous présentons des observations microscopiques de plastes isolés à trois stades de mûrissement, puis des enregistrements en temps réel de la fluorescence des pigments sur les tranches de fruits de tomate. Il a été possible de montrer que la transition chloroplaste-chromoplaste était synchrone pour tous les plastes d'une seule cellule et que tous les chromoplastes proviennent de chloroplastes préexistants. Dans le deuxième chapitre, une approche protéomique quantitative de la transition chloroplaste-chromoplaste est présentée, pour identifier les protéines différentiellement exprimées. Le traitement des données a identifié 1932 protéines parmi lesquelles 1529 ont été quantifiées par spectrométrie de masse. Les procédures de quantification ont ensuite été validées par WESTERN blot de certaines protéines. La chromoplastogénèse comprend les changements métaboliques suivants : diminution de l'abondance des protéines de réaction à la lumière et du métabolisme des glucide, et l'augmentation de la biosynthèse des terpénoïdes et des protéines de stress. Ces changements sont couplés à la rupture de la biogenèse des thylakoïdes, des photosystèmes et des composants de production d'énergie, et l'arrêt de la division des plastes. Dans le dernier chapitre nous avons utilisé la lincomycine, un inhibiteur spécifique de la traduction à l'intérieur des plastes, afin d'étudier les effets sur la maturation des fruits et sur l'expression de gènes nucléaires impliqués dans la maturation. Les résultats préliminaires indiquent que l'inhibition de la traduction des protéines dans les plastes affecte la maturation du fruit en réduisant l'accumulation de caroténoides. L'expression de plusieurs gènes nucléaires a été modifiée mais une relation claire avec le phénotype altéré de maturation n'a pas pu être établie. Au total, notre travail donne de nouveaux aperçus sur le processus de différenciation chromoplaste et fournit des données nouvelles ressources sur le protéome plaste / One of the most important phenomenons occurring during tomato fruit ripening is the color change from green to red. This change takes place in the plastids and corresponds to the differentiation of photosynthetic plastids, chloroplasts, into non photosynthetic plastids that accumulate carotenoids, chromoplasts. In this thesis we first present a bibliographic introduction reviewing the state of the art in the field of chloroplast to chromoplast transition and describing the structural and physiological changes occurring during the transition. Then, in the first chapter we present an in situ real-time recording of pigment fluorescence on live tomato fruit slices at three ripening stages. By viewing individual plastids it was possible to show that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell and that all chromoplasts derived from pre-existing chloroplasts. In chapter two, a quantitative proteomic approach of the chloroplast-to-chromoplast transition is presented that identifies differentially expressed proteins. Stringent curation and processing of the data identified 1932 proteins among which 1529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immune-blot evaluation of some proteins. Chromoplastogenesis appears to comprise major metabolic shifts (decrease in abundance of proteins of light reactions and carbohydrate metabolism and increase in terpenoid biosynthesis and stress-related protein) that are coupled to the disruption of the thylakoid and photosystems biogenesis machinery, elevated energy production components and loss of plastid division machinery. In the last chapter, we have used lincomycin, a specific inhibitor of protein translation within the plastids, in order to study the effects on fruit ripening and on the expression of some ripening-related nuclear genes. Preliminary results indicate that inhibiting protein translation in the plastids affects fruit ripening by reducing the accumulation of carotenoids. The expression of several nuclear genes has been affected but a clear relationship with the altered ripening phenotype could not be established. Altogether, our work gives new insights on the chromoplast differentiation process and provides novel resource data on the plastid proteome

Gracilariaceae is a worldwide distributed family that includes numerous economically important species. Currently, five genera are recognized in the Gracilariaceae: Gracilariophila (parasitic), Curdiea, Melanthalia, Gracilaria, and Gracilariopsis. Some species of Gracilaria were taxonomically transferred to Hydropuntia. However, this genus is quite controversial. High-throughput sequencing (HTS) techniques has led to an increase in studies using complete organellar genomes, which have been used to infer phylogenetic relationships in Rhodophyta and the investigation of other aspects of red algal genomes, including gene synteny and horizontal gene transfers (HGT). HTS also facilitated the search for extrachromosomal plasmids and its influence in the organellar genomes by HGT. We applied HTS to assemble and annotate organellar genomes (mitochondria and chloroplast) from seven species of Gracilariaceae using Illumina HiSeq 2500 platform. We also received raw reads of 31 samples of Gracilariaceae from Dr. Goia Lyra that were analysed and included in our work. We used these data, combined with published genomes, to infer phylogenies and compare the genome architecture of these species representing the main lineages in Gracilariaceae. The mitochondrial and chloroplast genomes were highly conserved in gene synteny among the species, and variation mainly occurred in regions where insertions of plasmid-derived sequences (PDSs) were found, which were similar to known red algae extrachromosomal plasmids. In mitochondrial genomes, the PDS insertions were in two regions where the transcription direction changes: between cob and trnL genes, and trnA and trnN genes. PDS insertions in chloroplast genome were in different positions, but generally found between psdD and rrs genes. The bacterial leuC/leuD operon was found in Gracilaria tenuistipitata, G. chilensis, M. intermedia chloroplasts genomes, and also in G. vermiculophylla extrachromosomal plasmid. Phylogenetic trees show two different origins of leuC/leuD: genes found in chloroplasts and plasmids were close to proteobacteria, and genes encoded in the nucleus are close to Viridiplantae and cyanobacteria. Gracilariaceae may be a good model to study the impact of PDS in genome evolution due to the frequent presence of these sequences inserted in organellar genomes. Our phylogenetic analyses demonstrated similar evolutionary histories between the chloroplast and mitochondrial genomes. However, chloroplast phylogeny was better resolved with full support. Our taxonomical sampling supports the presence of three main lineages: Melanthalia/Curdiea, Gracilariopsis and Gracilaria. Melanthalia intermedia was sister to a monophyletic clade including Gracilaria and Gracilariopsis, which were resolved as monophyletic genera. Furthermore, the characteristics of organellar genome architecture, Gracilariopsis and Gracilaria genera are also supported by the loss of the plastid gene petP in Gracilaria and the rearrangement position of the gene trnH in the mitochondrial genome. Beside this, we found no support for the genus Hydropuntia as originally proposed / A família Gracilariaceae está globalmente distribuída e inclui várias espécies economicamente importantes. Atualmente, cinco gêneros são reconhecidos em Gracilariaceae: Gracilariophila (parasita), Curdiea, Melanthalia, Gracilaria e Gracilariopsis. Algumas espécies de Gracilaria foram taxonomicamente transferidas para Hydropuntia. Entretanto, esse gênero é bastante controverso. Técnicas de sequenciamento de alta performance (HTS) levaram a um aumento de estudos usando genomas organelares completos, que têm sido usados para inferir relações filogenéticas em Rhodophyta e na investigação de outros aspectos dos genomas de algas vermelhas, incluindo sintenia gênica e transferências horizontal de genes (HGT). O HTS também facilitou a busca por plasmídeos extracromossômicos e sua influência nos genomas organelares por HGT. Nós utilizamos HTS para montar e anotar genomas organelares (mitocôndrias e cloroplastos) de sete espécies de Gracilariaceae usando a plataforma Illumina HiSeq 2500 e recebemos sequências de 31 amostras Gracilariaceae da Dr. Goia Lyra que foram montadas, anotadas e incluídas em nossas análises. Utilizamos esses dados, combinados com genomas publicados, para inferir filogenias e comparar a arquitetura do genoma dessas espécies representando as principais linhagens em Gracilariaceae. Os genomas mitocondrial e plastidial são altamente conservados na sintenia gênica e a variação ocorreu principalmente em regiões onde foram encontradas inserções de sequências derivadas de plasmídeos (PDS), similares aos plasmídeos extracromossômicos conhecidos de algas vermelhas. Nos genomas mitocondriais, as inserções de PDS estavam em duas regiões onde a direção da transcrição muda: entre os genes cob e trnL e os genes trnA e trnN. As inserções de PDS no genoma do cloroplasto estavam em posições diferentes, mas geralmente encontradas entre os genes psdD e rrs. O operon bacteriano leu/leuD foi encontrado nos genomas dos cloroplastos de Gracilaria tenuistipitata, G. chilensis, M. intermedia e também no plasmídeo de G. vermiculophylla. As árvores filogenéticas mostram duas origens diferentes de leuC/leuD: os genes encontrados no cloroplasto e no plasmídeo estavam próximos de proteobactérias, e os genes codificados no núcleo estavam próximos de Viridiplantae e cianobactérias. Gracilariaceae pode ser um bom modelo para estudar o impacto de PDS na evolução de genomas devido à presença frequente de inserções PDS em genomas organelares. Nossas análises filogenéticas demonstraram histórias evolutivas similares entre cloroplasto e mitocondria. No entanto, a filogenia de cloroplasto foi melhor resolvida com valores máximos de Bootstrap em todos os ramos. Nossa amostragem taxonômica corrobora a presença de três linhagens principais: Melanthalia/Curdiea, Gracilariopsis e Gracilaria. Melanthalia intermedia aparece como grupo-irmão do clado monofilético incluindo Gracilaria e Gracilariopsis, que foram resolvidos como gêneros monofiléticos. Além disso, baseado nas características da arquitetura do genoma organelar, os gêneros Gracilariopsis e Gracilaria se distinguem pela perda do gene plastidial petP em Gracilaria e pela posição de rearranjo do gene trnH no genoma mitocondrial. Nós não encontramos evidências para a permanencia o gênero Hydropuntia como originalmente proposto

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.

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.