The chloroplast originates from an endosymbiotic event 1.5 billion years ago, when a free living photosynthetic bacteria was engulfed by a eukaryotic host. The chloroplastic genome has through evolution lost many genes to the nuclear genome of the host. To coordinate the gene expression between the two genomes, plants have evolved two types of communication, nucleus-to-plastid (anterograde) and plastid-to-nucleus (retrograde) signalling. This thesis will focus on retrograde communication with emphasis on redox and tetrapyrrole mediated signalling. In this thesis, we establish the tetrapyrrole Mg-ProtoIX as an important retrograde negative regulator of nuclear encoded plastid proteins. We show that Mg-ProtoIX accumulates in both artificial and natural stress conditions, and that the accumulation is tightly correlated to regulation of nuclear gene expression. Using confocal microscopy, we could visualize Mg-ProtoIX in the cytosol during stress conditions. In addition, exogenously applied Mg-ProtoIX stayed in the cytosol and was enough to trigger a signal to the nucleus. The results presented here indicate that Mg-ProtoIX is transported out of the chloroplast to control nuclear gene expression. Mg-ProtoIX mediated repression of the nuclear gene, COR15a, occurs via the transcription factor HY5. HY5 is influenced by both plastid signals and the photoreceptors. Here, we show that photoreceptors are part of Mg-ProtoIX mediated signalling as well as excess light adaptation. We identified the blue light receptor, CRY1, as a light intensity sensor that partly utilizes HY5 in the high light response. To further understand the high light regulation of nuclear genes, we isolated a mutant with redox insensitive (rin) high light response. The rin2 mutant has a mutated plastid protein with unknown function. Characterization of the rin2 mutant revealed that the protein is important in regulating plastid gene expression as well as nuclear gene expression. The rin2 mutant is the first characterized rin mutant and could prove important in elucidating the cross-talk between redox mediated coordination between the plastid and the nuclear genome.

Polyphenol oxidases (PPOs) are ubiquitous enzymes that oxidize phenols to quinones in the presence of molecular oxygen, often leading to tissue discolouration. They are sometimes considered as defense proteins but other functions, for example in phenolic compound biosynthesis, have also been found. In this thesis, bioinformatic searches were conducted to identify putative PPO genes from available genomes representing five Viridiplantae lineages: chlorophytes, bryophytes, lycophytes, monocotyledonous anthophytes and eudicotyledonous anthophytes. Duplicated PPO genes were found in most land plant genomes. A detailed investigation of the poplar (Populus trichocarpa) PPO gene family found nine genes that exhibit differential expression profiles during development and following stress, of which PtrPPO1 was the only significant wound-inducible PPO gene. A phylogenetic reconstruction of the poplar PPOs identified PtrPPO13 to be an unusual PPO homolog and it was studied in detail. Experimental evidence indicated that PtrPPO13 is expressed in most organs, and unlike most PPOs, is localized to the vacuole. Together, the phylogeny, gene expression and subcellular localization studies suggest that PPOs are likely to have variable physiological functions in plants and that PtrPPO13 is distinct from most typical PPOs. / Graduate / 0309

A complete proteome analysis of the chloroplast stroma, using 2D-PAGE, from spinach and Arabidopsis was performed. To improve the identification of proteins a computer program named SPECLUST was used. In SPECLUST, peak masses that are similar in many spots cluster together because they originate from the same protein with different locations on the gel. Within this program peaks in a cluster can be investigated in detail by peaks-in-common, and the unidentified masses that differ between spots in a cluster could be caused by protein modifications, which was analysed further by MS/MS. The thylakoid is an internal membrane system in the chloroplast where protein complexes involved in photosynthesis are housed. Enclosed in the thylakoid membrane is the chloroplast lumen, with a proteome estimated to contain 80-200 different proteins. Because the chloroplast lumen is close to the photosynthesis machinery in the plant, one can expect that the lumen proteome will change depending on if the plant is dark or light adapted. DIGE analysis of lumen proteins found that 15 lumen proteins show increased relative abundance in light-adapted plants. In addition co-expression analysis of lumen protein genes suggests that the lumen protein genes are uniformly transcriptionally regulated, not only by light but in a general manner. Plastocyanin is one of the proteins involved in the electron transfer in photosynthesis. Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis, where PETE2 is the more abundant isoform. Knockout mutants of each of the plastocyanin isoforms shows that a 90% reduction of plastocyanin levels affects rates of photosynthesis and growth only slightly. A corresponding over-expression of plastocyanin in each of the two knockout mutants results in essentially wild-type photosynthetic performance. Reduced plastocyanin levels make the plant sensitive to Cu stress and therefore plastocyanin plays a major role as a Cu sink. A by-product of photosynthesis is hydrogen peroxide, which may be harmful for the plant. The discovery that an abundant protein found in the chloroplast lumen, TL29, shared sequence homology to Ascorbate Peroxidase (APX) was therefore of interest. We have evidence that TL29 is not an APX protein; it lacks the heme-binding active site and shows no activity. TL29 is located in the grana region and is electrostaticaly attached to the thylakoid membrane. It has four isoforms, with different pIs, both in the native and denatured form. It has no interaction with ascorbate, when compared to raAPX1. TL29 has two cysteine residues and one of them seems to have redox-regulated function, proposing that it may interact with other proteins close to PSII.

Dunaliella, a genus of unicellular, biflagellate green algae, is one of the most studied microalgae for mass culture and is of commercial importance as a source of natural -carotene. Dunaliella species have the desirable properties of halotolerance and photoautotrophy that makes their large-scale culture simple and cheap using resources unsuitable for conventional agriculture. The ease and cost-effectiveness of culture makes Dunaliella a desirable target for increased production of natural compounds by metabolic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds. However, the lack of efficient genetic transformation systems has been a major limitation in the manipulation of these microalgae. In chapter four we describe the development of a nuclear transformation system for Dunaliella tertiolecta. The gene encoding the phleomycin-binding protein from Streptoalloteichus hindustanus, was chosen as the selectable marker as this protein retains activity at high salt concentrations. To drive expression of the chosen selectable marker, two highly expressed Dunaliella tertiolecta RbcS genes and their associated 5' and 3' regulatory regions were isolated and characterised (chapter three). Dunaliella transformation cassettes containing the RbcS promoter and terminator regions flanking the ble antibiotic resistance gene were constructed. These expression cassettes were tested in Chlamydomonas reinhardtii cells and found to drive expression of the ble gene in this heterologous system. This study also demonstrated that truncation of both the D. tertiolecta RbcS1 and RbcS2 regulatory regions significantly increases the expression of the ble gene in C. reinhardtii cells. To determine if the foreign DNA could stably integrate into the Dunaliella genome, four transformation methods: microprojectile bombardment, glass bead-mediated transformation, PEG-mediated transformation and electroporation were tested and a number of parameters varied. Southern blot analysis revealed that the plasmid DNA transiently entered the Dunaliella cells following electroporation but was rapidly degraded. Following electroporation, one stably transformed Dunaliella line was recovered. This is the first demonstration of the stable transformation of this alga. Chloroplast transformation is becoming a favoured method for the production of recombinant proteins in plants, as levels of heterologous protein are often higher than those achieved by transforming the nucleus. The Dunaliella chloroplast genome has not been genetically characterised, and thus there were no existing promoter and terminator sequences or sequences of intergenic regions that could be used for vectors in transformation of the chloroplast. Therefore, this study aimed to isolate and characterise promoters of highly expressed genes and matching terminators capable of driving transgene expression, and also to characterise intergenic regions that would be suitable insertion sites for the vector construct (chapter five). The complete gene sequence of two highly expressed Dunaliella chloroplast genes psbB and rbcL including the promoter and terminator regions as well as the coding sequence of the psbA gene were cloned and sequenced. In addition, the psbA gene is useful as a selectable marker as introduced mutations confer resistance to the herbicide 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU). Two homologous transformation constructs based on mutated psbA genes were developed and tested using microprojectile bombardment. A number of parameters were tested including: the size of the gold microprojectile particle, the distance of the plates from the point of discharge, plating onto membranes or filter paper, helium pressure, addition of an osmoticum to the medium and recovery time. Although no chloroplast transformants were recovered in this study, these homologous recombination constructs should prove useful in the development of a chloroplast transformation protocol. The other major component of this study was to investigate the use of microalgae as an expression system for the production of recombinant proteins. Transformation of Chlamydomonas reinhardtii, a species related to Dunaliella, is well developed. In chapter six, this study examined the expression of two human proteins, -lactalbumin and IGF-1 in Chlamydomonas reinhardtii. Plasmids containing the C. reinhardtii RbcS2 promoter upstream of the cDNAs of these two proteins were introduced into C. reinhardtii cells using glass-bead mediated transformation. Transgenic C. reinhardtii lines were generated and shown to contain the transgenes by PCR and Southern hybridisation. RT- PCR and northern hybridisation were subsequently used to demonstrate that the transgenes were transcriptionally active. The transcripts however, could only be detected by RT-PCR indicating that the genes were transcribed at low levels. Accumulation of the -lactalbumin protein could not be demonstrated, suggesting that although the transgenes were transcribed, they were either not translated or translated at levels below the sensitivity of western blot analysis or that any protein produced was rapidly degraded. Previous studies have indicated that in microalgae codon usage is vital in translation of the foreign protein. Codon modification of the IGF-I and -lactalbumin genes should lead to higher levels of protein accumulation. This study reports the first successful stable nuclear transformation of Dunaliella tertiolecta. Therefore it is now feasible that Dunaliella can be examined as a bioreactor for the expression of recombinant proteins. In addition, two chloroplast genes (psbB and rbcL) and their corresponding promoters and terminators have been characterised and a selectable marker cassette based on the mutated psbA gene constructed.

Il serait opportun de débuter cette introduction en donnant une définition claire du processus d'édition des ARN, mais c'est aussi un exercice périlleux car le terme d'édition des ARN a été utilisé dans la littérature pour décrire une multitude de processus biochimiques différents et la distinction entre les processus d'édition ou de modification est parfois confuse. Le terme d’édition des ARN a été utilisé pour la première fois en 1986 pour décrire l’insertion de 4 résidus uridines dans le transcrit mitochondrial coxII chez le trypanosome (Benne et al., 1986). La communauté scientifique était sceptique et on a alors pensé que ce mécanisme était sans doute spécifique à ce « drôle » de protozoaire. Puis, rapidement, l'édition d'ARNm a été décrite chez de nombreux organismes eucaryotes, soit pour expliquer des processus d'insertions ou de délétions de nucléotides (qui altèrent le nombre de nucléotides contenus dans la molécule d'ARN) soit pour décrire des conversions ou des remplacements de nucléotides (qui altèrent l'identité des nucléotides contenus dans la molécule d'ARN). Plus tard, le terme d'édition des ARN a été utilisé pour décrire des désaminations (le plus fréquemment C-en-U, et A-en-I) survenant dans les ARNt et les ARNr d'organismes eucaryotes et procaryotes, mais aussi des modifications mineures des résidus (comme l'ajout de groupement méthyl). De même la polyadénylation de la partie 3' de certains ARNt est aussi communément appelée processus d'édition des ARN. Enfin, un phénomène d'édition cotranscriptionnel des ARN lié au « patinage » de l'ARN polymérase a également été mis en évidence chez certains virus.

This thesis examined aspects of the reproductive and molecular biology of Eucalyptus marginata (jarrah). The aims were to develop protocols for controlled pollination, that could be used in clonal orchard trees to breed jarrah seedlings that have a known genetic resistance to Phytophthora cinnamomi (dieback), for use in rehabilitation after mining and logging. An intimate knowledge of the breeding biology of jarrah was necessary to achieve this aim. The project also aimed to increase knowledge of the genetic diversity and structure of jarrah, in order to make informed decisions regarding the collection of material to be used for clonal propagation. Previous research has had little success in producing viable seed from any controlled pollinations, but clonal material resistant to P. cinnamomi has been produced using tissue culture. The question posed in this thesis was ‘Can we improve breeding and propagation techniques of jarrah?’ Techniques were developed for testing of in vitro pollen viability and pollen storage, pollination and fertilisation success after controlled pollinations, including determination of stigma receptivity and development of bud isolation techniques using alfoil. The variation in female fertility between genotypes was examined. The use of paclobutrazol was explored as a method of increasing the level of viable seed production in clonal orchard trees. The use of fertiliser as well as the growth retardant was also explored to see if it increased the level of seed production even more. Genetic diversity, genetic differentiation and phylogeny within Eucalyptus marginata were examined using nuclear and chloroplast DNA analysis with Restricted Fragment Length Polymorphisms. While it was first thought that the fertilisation rate was quite low, it was confirmed that the fertilisation rate is similar to other eucalypt species. The zygote abortion rate was quite high in one clone, but one wild tree had a similar seed production rate to other eucalypt species. The zygote and endosperm appeared to be different in the clone and the wild tree observed. The level of seed production was examined in clones and wild trees and it was found that the level was often quite low, particularly in the clones (0 – 13% in clones, 0 – 18% in wild trees) in comparison with other Eucalyptus species, and varied between genotypes. The use of a growth retardant such as paclobutrazol may increase the production of viable seed, if it is applied during autumn. The results were inconclusive for the fertiliser/paclobutrazol experiment, since the paclobutrazol was applied during spring which was the worst time of year for increasing seed production. There were differences between genotypes in reaction to both the paclobutrazol and the fertiliser/paclobutrazol. Genetic diversity was moderate in comparison with other Eucalyptus species, and there was a low level of genetic differentiation between populations in the nuclear genome. No differentiation was observed between the morphologically recognised subspecies in the nuclear genome, but differentiation between the populations on the Swan Coastal Plain and populations on the Darling Plateau was seen in the chloroplast genome, indicating that there was historical separation of these two areas. The conclusions arising from this work are that while controlled pollinations are possible in Eucalyptus marginata the clones that were used in these experiments have often behaved differently to the wild trees in the time of anthesis and levels of viable seed production, and in one clone (5J119) the zygote and endosperm nuclei appeared to be very different to the zygote and endosperm nuclei of a wild tree. Further investigation is necessary to see if these differences are related to the low level of seed production observed in the clonal populations. Paclobutrazol may be worth exploring further as a means of increasing seed production. Material to be used for rehabilitation and seed orchards can be collected from a wide area in the main distribution of the species, although trees on the Swan Coastal Plain are distinct from the trees in the main forest area in the chloroplast genome.

Thesis (M.S.)--Wake Forest University. Dept. of Biology, 2003. / Vita. Includes bibliographical references (leaves 20-22).

Chez les eucaryotes, la photosynthèse a lieu dans le chloroplaste, un organite spécifique de la cellule végétale et caractérisé par différents compartiments : (i) l'enveloppe, la double membrane qui délimite le chloroplaste ; (ii) le stroma, phase aqueuse principalement composée de protéines solubles et (iii) un système membranaire interne, les thylacoïdes, qui contiennent les complexes photosynthétiques. Les thylakoïdes forment un réseau tridimensionnel continu et sont différenciés en deux domaines physiques distincts : des empilements de vésicules de membrane (appelés granas ou BBY) et des extensions de membrane simple (lamelles stromales). Les complexes majeurs de la photosynthèse ne sont pas distribués de manière égale dans cette membrane à cause de contraintes électrostatiques et stériques. Ainsi, le photosystème I et l'ATP-synthétase sont enrichis dans les granas, le photosystème II dans les lamelles stromales alors que d'autres complexes, comme le cytochrome b6f, auraient une répartition équivalente entre granas et lamelles stromales. Pour faire face aux variations environnementales de lumière (en qualité et quantité), les plantes ont développé des processus pour moduler leur capacité d'absorption et d'utilisation de la lumière par les photosystèmes, processus regroupés sous le terme de « quenching non photosynthétique ou NPQ ». Dans le cadre de ma thèse, je me suis intéressé à deux composants du NPQ, les états de transition et la dissipation sous forme de chaleur (partie qE).Le premier objectif de ma thèse a été d'identifier de nouveaux acteurs impliqués dans les transitions d'état et ceci en étudiant la relocalisation de protéines au sein des sous-compartiments des thylacoïdes par une approche protéomique. En effet, il a été montré que certaines antennes collectrices de lumière sont réorganisées dans les membranes photosynthétiques lors des transitions d'état. Jusqu'à présent, aucune description exhaustive de la composition et distribution des protéines dans les sous-compartiments de thylacoïdes n'avait été réalisée. J'ai donc dans un premier temps développé des protocoles de purification des sous-compartiments des thylakoïdes (granas et lamelles stromales) à partir de chloroplastes de plantes sauvage d'Arabidopsis thaliana. Ensuite, grâce à une approche d'analyse protéomique semi-quantitative, nous avons pu déterminer la localisation d'environ 300 protéines des thylacoïdes. Les résultats suggèrent que la localisation de complexes photosynthétiques est beaucoup plus dynamique que celle jusqu'à lors proposée. En effet, même s'ils sont préférentiellement identifiés dans un sous-compartiment, certains complexes photosynthétiques présentent une double localisation qui était inattendue. De plus, la composition en sous-unités de ces complexes diffère selon leur localisation, dans les granas et dans les lamelles stromales, suggérant l'existence de processus de régulation de la photosynthèse jusqu'à lors insoupçonnés. Cette approche a ensuite été appliquée sur des plantes mutantes d'Arabidopsis affectées dans les transitions d'état afin d'identifier des protéines pouvant être impliquées dans ce processus d'adaptation. En parallèle, je me suis intéressé au qE . L'activation de ce mécanisme n'est pas constitutive et nécessite la formation d'un gradient de pH entre le stroma et le lumen des thylacoïdes (ΔpH). L'objectif de l'étude a été d'identifier des acteurs pouvant contrôler la formation de ce gradient de pH. Pour cela, nous nous somme focalisés sur le rôle d'un transporteur de potassium récemment caractérisé, TPK3. Grâce à des approches biophysiques et biochimiques, nous avons démontré que TPK3 est impliqué, in vivo, dans la modulation des deux composantes de la force proton motrice (pmf), le gradient de pH (ΔpH) et la différence de potentiel (Δψ). En contrôlant la répartition de la force proton motrice,TPK3, permet une utilisation correcte de la lumière en dissipant l'excès d'énergie. / Within higher plants and algae, photosynthesis is carried out in the chloroplast. Structurally, chloroplasts are organized in (i) the envelope, a double membrane system surrounding the chloroplast (ii) the stroma, the aqueous space which mainly contains soluble proteins and the (iii) thylakoids, a three-dimensional membrane network where photosynthetic electron transport reactions occur. Thylakoids are non-homogeneously folded, and comprise two major domains: (i) the grana-BBY, which are stacks of thylakoids particularly enriched in photosystem II, LHCII (the antenna-protein complex responsible for light harvesting) and (ii) the stroma lamellae, which are unstacked thylakoids connecting grana stacks enriched in photosystem I and ATP synthase. Plants can respond to changes in the environmental light conditions by several means as those which are collectively called non-photochemical quenching or NPQ. During my thesis, I mainly focused on two components of the NPQ: state transition (qT) and high-energy state quenching (qE).State transitions is the process by which PSII-antenna proteins are re-organized between stroma-lamellae and grana-BBY following changes in ambient light both of intensity and spectral composition. State transitions play a key role in the plant adaptation but many aspects of this process remain unclear. The main objective of my thesis was to study the thylakoid protein re-localization between stroma-lamellae and grana-BBY during state transitions using a proteomic-based approach. At this aim I firstly focused on the sub-thylakoid protein localization in Arabidopsis WT and I developed different protocols for the purification of the two sub-compartments (stroma-lamellae and grana-BBY) starting from intact chloroplasts. Later, thanks to a semi-quantitative proteomic approach, I determined the precise localization of around 300 thylakoid proteins in Arabidopsis WT. Results suggested that the localization of the different photosynthetic complexes is much more dynamics than previously hypothesized. In fact, even if characterized by a preferential localization, some photosynthetic complexes displayed an unexpected double localization. Moreover the subunit composition of these complexes was found to vary according to their localization (BBY or stroma-lamellae) suggesting the existence of mechanisms of regulation which have never been evidenced before. Later, we used the same mass-spectrometry-based approach on two different Arabidopsis mutants unable to perform state transitions. The objective was to highlight the involvement of other proteins (other than LHCII) which could possibly be re-localized within the photosynthetic membrane during state transitions. In the second part of my thesis, I focused on the high-energy state quenching component of the NPQ. qE allows the plant to dissipate excessive light energy as heat. This process it's not constitutive but need to be activated by the formation of a difference in the pH between the stroma and the thylakoid lumen (ΔpH). The objective of the study was to identify new possible actors in the regulation of the ΔpH formation. At this purpose I focused on a recently characterized potassium channel, TPK3. Thanks to a biophysical and biochemical approach, we demonstrated that TPK3 is involved, in vivo, in the modulation of the two components of the proton motive force (pmf), the ΔpH and the difference in the electric field Δψ. By controlling the repartition of the pmf, TPK3, controls also the formation of the NPQ and directly affects light utilization and dissipation in vivo. This avoids serious damages to the photosynthetic chain when plants are exposed to high-light conditions

L'auteur n'a pas fourni de résumé en français / L'auteur n'a pas fourni de résumé en anglais

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.