Submitted by AMANDA CRISTINA BALDASSI null (amanda_baldassi@hotmail.com) on 2018-03-12T13:58:08Z No. of bitstreams: 1 AmandaBaldassi_final_corrigida.pdf: 1978602 bytes, checksum: 10397a5b59a6e3e263d85dd4e9677785 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-03-12T18:57:53Z (GMT) No. of bitstreams: 1 baldassi_ac_me_jabo.pdf: 1978602 bytes, checksum: 10397a5b59a6e3e263d85dd4e9677785 (MD5) / Made available in DSpace on 2018-03-12T18:57:53Z (GMT). No. of bitstreams: 1 baldassi_ac_me_jabo.pdf: 1978602 bytes, checksum: 10397a5b59a6e3e263d85dd4e9677785 (MD5) Previous issue date: 2018-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As mudanças climáticas globais podem alterar significativamente o metabolismo das células vegetais. Um cenário com alta concentração de CO2 atmosférico pode ser benéfico para plantas do tipo C3 devido ao estímulo à fixação de carbono. No entanto, esse aumento esperado na taxa de assimilação de carbono também pode aumentar a atividade e a expressão das enzimas envolvidas na cadeia de transporte de elétrons, resultando em maior acúmulo de espécies reativas de oxigênio. Nesse trabalho, foi estudada a resposta do sistema antioxidante em cloroplastos de Eucalyptus urophylla cultivados sob altas concentrações de CO2. As plantas de E. urophylla expostas a alta concentração de CO2 (980 ppm) mostraram um aumento no fechamento estomático e pequena, mas significativa, indução do estresse oxidativo em relação as plantas cultivadas em concentração atmosférica de CO2. Entretanto, essas respostas não foram observadas quando as plantas foram cultivadas em concentração de CO2 igual a 680 ppm, cujo tratamento induziu abertura estomática. Através de uma abordagem de proteômica de descoberta, foram identificadas e detectadas 19 proteoformas antioxidantes cloroplastidiais e 2 proteoformas diferencialmente reguladas. Dentre essas, destacam-se uma proteoforma de ascorbato peroxidase e uma de superóxido dismutase, as quais estão possivelmente envolvidas na neutralização de espécies reativas de oxigênio após cultivo em atmosfera enriquecida com CO2, e que podem ser utilizadas como marcadores bioquímicos de estresse abiótico ou como alvos em programas de engenharia genética. / Global climate change can significantly alter plant cell metabolism. A higher atmospheric CO2 scenario may be beneficial for C3 plants through the stimulation of carbon fixation. However, this predicted increase in the rate of carbon assimilation may also increase the activity and expression of enzymes involved in the electron transport chain, resulting in higher accumulation of reactive oxygen species. Here, we studied the responses of the chloroplast antioxidant system of Eucalyptus urophylla plants cultivated under high-CO2 conditions. E. urophylla plants exposed to a high concentration (980 ppm) of CO2 showed an increase in stomatal closure and a small, but significant, induction of oxidative stress in relation to plants grown at atmospheric CO2 concentration. However, these responses were not observed at a CO2 concentration equal to 680 ppm, which induced stomatal aperture. With the discovery proteomics approach used herein, we identified 19 chloroplast antioxidant proteoforms and pinpointed 2 differentially regulated antioxidants proteoforms. We highlight an isoform of ascorbate peroxidase and one of superoxide dismutase, which are possibly involved in the neutralization of reactive oxygen species upon cultivation in a high CO2 atmosphere and could be used as biochemical markers of abiotic stress or as targets in genetic engineering programs. / FAPESP: 2015/23354-8

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13360号 / 論農博第2891号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5299(農学部図書室) / (主査)教授 中﨑 鉄也, 教授 白岩 立彦, 教授 土井 元章 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Witsenia is a monospecific genus of the putatively basal group, the woody Iridaceae. This upright iris has extremely long black and yellow flowers ( see fig. 1) that are thought to have been pollinated by an extinct Sunbird. The role of the unusual black floral colouration is investigated as this colour is seldom associated with bird pollination. This plant typically exists in discreet and restricted populations in wet habitats in the South Western Cape (South Africa). The restricted nature of the plant is peculiar as it occurs in either low or high altitudes, yet appears to be extremely sensitive to altitude. Popular belief suggests that Witsenia maura occurs in the Peninsula only, and results from this study show the Peninsula population to be genetically separate from other populations, reflecting a long term separation. Samples from nine populations are sequenced to investigate haplotypic variation within the species, and dispersal of ancestral populations. This thesis investigates the current knowledge of Witsenia, its ecology, history and distribution. An examination of flowers under UV light reveals the first evidence of UV nectar guides in an ornithophilous flower. Conservation issues are also addressed, and it is established that although small and apparently shrinking due to global warming, populations are nonetheless viable if managed properly. A molecular study of the species and examinination of its variation revealed exceptional haplotype diversity. This diversity can best be explained by swamps acting as refugia during interglacial periods.

The mechanisms behind the high level of plant diversity and endemism observed in the Cape Floral Region (CFR) of South Africa have been the focus of many studies. Recently developed methods that employ DNA sequence data are making major contributions in reconstructing evolutionary histories of CFR species. Concurrently, palaeoenvironmental evidence is used increasingly to explain the impact of past climates on species ranges. This paper combines these two approaches by analysing the distribution of genetic diversity of the Afro-montane liverwort Jamesoniella colorata and associating its inferred evolutionary history with major palaeoclimatic trends in South Africa. Liverworts are generally well-suited for phylogeographical studies because they often have low dispersal rates, broad geographical ranges and long evolutionary persistence. In addition, the high among-population diversity observed in J colorata is conducive to the interpretation of significant historical events. The GIS-based bioclimatic envelope shows a strong correlation between potential habitat and the known distribution of J colorata and indicates that sampling in this study was sufficient to make accurate phylogeographical inferences. A combination of phylogeographical data and population genetics evidence suggests that populations of J colorata in the Western Cape Province have experienced range contractions into upper-montane refugia and range expansions into lower altitudes in response to warming and cooling climatic trends, respectively. These range shifts have probably taken place throughout the Quaternary glacial-interglacials cycles, , which are thought to have been influential in shaping modem patterns of diversity. In lV an attempt to assign approximate dates to the two expansion events inferred for J. colorata, an average chloroplast mutation rate was applied to the trnL-F cpDNA mismatch distribution. The results roughly place the expansions within the last glacial period, demonstrating the general accordance of the phylogeographical and palaeoclimatic data. The molecular work in this study also brought into question the taxonomic status of several specimens that showed distinctly divergent DNA sequences. Preliminary morphological inspection of the specimens revealed subtle but clear differences in leaf and stem anatomy that were once associated with J. oenops, a species synonymised with J. colorata in 1971.

Les membranes des chloroplastes sont riches en monogalactosyldiacylglycérol (MGDG) et digalactosyldiacylglycérol (DGDG), deux galactolipides essentiels pour la biogenèse de ces organites et le fonctionnement de la machinerie photosynthétique. MGD1 est la galactolipide synthase majeure chez Arabidopsis thaliana. C’est une protéine monotopique, localisée dans l’enveloppe interne des membranes des chloroplastes, qui transfère un résidu galactose à partir d’UDP-galactose sur le diacylglycérol (DAG) pour former le MGDG. MGD1 a besoin de lipides anioniques tels que le phosphatidylglycérol (PG) pour être active, mais le mécanisme d’activation reste à ce jour inconnu. Des études antérieures ont permis d’identifier le résidu P189 comme étant potentiellement impliqué dans la liaison au PG, et le résidu H155 comme base catalytique potentielle. Le but de ma thèse a été d’obtenir plus d’informations sur le mécanisme de régulation de la MGD1, enzyme clé de la biogenèse des chloroplastes. Mon étude s’est portée sur la protéine native MGD1 et deux de ses mutants, H155A et P189A. Ces protéines ont été exprimées chez Escherichia coli et purifiées jusqu’à homogénéité. Une nouvelle méthode de dosage de l’activité MGDG synthase, adaptée de la technique de bioluminescence UDP-GloTM de Promega, a été mise au point. Cette méthode présente de nombreux avantages en termes de rapidité et de sensibilité comparée à la technique classiquement utilisée qui nécessite l’utilisation d’un UDP-Galactose radiomarqué. L’étude des propriétés d’association de MGD1 et des mutants à des membranes modèles, à l’aide de la technique des monocouches de Langmuir, a permis de proposer un mécanisme réactionnel inédit, impliquant une dyade catalytique de type PG-His, qui permet d’expliquer le rôle d’activateur du PG. Un nouveau test d’activité, basé sur cette même technique de Langmuir, qui présente l’avantage de pouvoir discriminer la capacité de liaison de MGD1 à la membrane et sa capacité à transférer un galactose sur son accepteur DAG a été mis en place. Il s’agit du premier exemple de synthèse d’un galactolipide sur une membrane biomimétique constituée d’un mélange DAG-PG. / The membranes of chloroplasts are enriched in monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two essential galactolipids to the biogenesis of these organelles and the functioning of photosynthetic machinery. MGD1 is the major galactolipid synthase in Arabidopsis thaliana. It is a monotopic protein, located in the inner envelope of chloroplast membranes, which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG) to form MGDG. MGD1 needs anionic lipids such as phosphatidylglycerol (PG) to be active, but the activation mechanism is still unknown. Previous studies identified the P189 residue as potentially involved in PG binding, and H155 as the putative catalytic base. The aim of my thesis was to progress in our understanding of the regulation of MGD1 activity, a key enzyme in the biogenesis of chloroplasts. My study focused on the native protein MGD1 and two mutants, H155A and P189A. These proteins were expressed in Escherichia coli and purified to homogeneity. A new method for assaying MGDG synthase activity, adapted from the Promega UDP-GloTM bioluminescent technique has been developed. This method has many advantages in terms of speed and sensitivity compared to the conventionally used technique which requires the use of a radiolabeled UDP-Galactose. Furthermore, the study of the association properties of MGD1 and mutants with model membranes, using the Langmuir monolayer technique, led us to propose a novel reaction mechanism, involving a PG-His type catalytic dyad, which may explain the role of PG as activator. A new activity test, based on the Langmuir technique, which has the advantage to discriminate the MGD1 binding capacity to the membrane and its ability to transfer a galactose to its acceptor DAG has been set up. This is the first example of galactolipid synthesis on a biomimetic membrane formed of a DAG-PG mixture.

Le chloroplaste est un composant majeur de la cellule végétale. Cet organite est le fruit d’une endosymbiose, survenue entre une cellule eucaryote et une cyanobactérie. Ainsi, 95% des gènes codant pour les protéines plastidiales ont été transférés vers le génome nucléaire au cours de l’évolution. En conséquence, la plupart des protéines chloroplastiques sont aujourd’hui codées par le noyau, synthétisées dans le cytosol sous forme de précurseurs dotés d’une une extension N-terminale clivable (le "peptide de transit") et ensuite importées sans les chloroplastes via le système TOC/TIC (Translocons localisés au niveau des membranes externe et interne de l'enveloppe des chloroplastes). Jusqu'à récemment, toutes les protéines destinées aux compartiments chloroplastiques internes étaient censées posséder une séquence d’adressage N-terminale clivable et engager la machinerie d’import général TOC/TIC. Cependant, des études récentes reposant sur des approches protéomiques ont révélé l’existence de plusieurs protéines chloroplastiques dépourvues de la séquence additionnelle clivable. La première évidence de telles protéines dites non canoniques a été fournie par notre équipe, étudiant le protéome de l’enveloppe du chloroplaste d’Arabidopsis, qui a conduit à l’identification d’une protéine quinone oxidoréductase homologue nommée « ceQORH ». Bien que dépourvues de peptide de transit clivable, il s’est avéré que ces protéines sont capables de rejoindre les compartiments chloroplastiques internes. D’autre part, il a été également montré que l’import de ces protéines dans le chloroplaste n’est pas médiée par la machinerie de translocation générale TOC/TIC. De plus, il s’est avéré que ces protéines ont la particularité d’être multilocalisées dans les cellules de différents tissus de la feuille. Cependant, les mécanismes moléculaires qui contrôlent la localisation sub-cellulaire de telles protéines chloroplastiques non canoniques demeurent encore inconnus. Pour mieux caractériser fonctionnellement les composantes des systèmes d’import alternatifs de protéines chloroplastiques non canoniques, nous avons adopté une approche directe qui reposait sur des techniques biochimiques combinant le crosslink chimique, la purification par affinité et la spectrométrie de masse. Cette stratégie nous a permis d’identifier un partenaire, impliqué dans le contrôle de l’adressage de la protéine ceQORH dans le chloroplaste. Alternativement, nous avons réalisé une bio-analyse du protéome de l’enveloppe du chloroplaste et qui nous a permis de revisiter la composition du protéome de l’enveloppe du chloroplaste. Afin d’expliquer la localisation sub-cellulaire variable de la protéine ceQORH, les membres de l’équipe ont émis l’hypothèse d’une interaction probable de cette protéine avec un partenaire cytosolique. Dans la dernière partie de cette étude, nous avons validé l’interaction, in planta, entre ceQORH et son partenaire par une approche génétique qui portait sur l’analyse de l’impact de l’absence de ce dernier sur la régulation de la localisation sub-cellulaire de la protéine ceQORH. / Chloroplasts are a major component of plant cells. Their origin traces back to a cyanobacterial ancestor that was engulfed by an ancient eukaryotic cell and eventually integrated as an organelle during evolution. As a result, more than 95% of the ancestral cyanobacterial genes were transferred to the host cell nucleus. Proteins encoded by these relocated genes need to return to internal chloroplast compartments. This import is mainly achieved by the general TOC/TIC machinery located at the chloroplast surface. Until recently, all proteins destined to chloroplast were believed to possess an N-terminal and cleavable chloroplast targeting peptide, and to engage the TOC/TIC machinery. However, recent studies have revealed the existence of several non-canonical preproteins, lacking cleavable transit peptides. The first evidence for such ‘non-canonical’ chloroplast proteins was provided by our team studying the Arabidopsis chloroplast envelope proteome, leading to the identification of a quinone oxidoreductase homologue termed « ceQORH ». Furthermore, a few such proteins were demonstrated to use alternative targeting pathways, independent of the TOC/TIC machinery. To better characterize components of such alternative targeting machineries, a targeted study combining affinity purification and mass spectrometry aiming to identify alternative receptors at the chloroplast surface has been performed. This study allowed us to identify new “partner” involved in the control of chloroplast targeting of ceQORH protein. Alternatively, we also revisited the chloroplast envelope proteome composition and initiated a gene candidate approach. In addition, some non-canonical proteins are shared by plastids and other cell compartments. However, molecular mechanisms controlling subcellular localization of these non-canonical plastid proteins remain unknown. In order to explain the variable subcellular localization of ceQORH protein, our team hypothesized a probable interaction of ceQORH with a cytosolic partner. In the last part of this study, we validated the interaction between ceQORH and its partner in planta by a genetic approach analyzing the impact of the absence of the cytosolic partner on the regulation of the sub-cellular localization of ceQORH protein.

No description available.

Drought and temperature stress are the two most important factors limiting crop productivity. A physiological and proteomic analysis of soybean was performed to investigate the negative impact of these variables. The previous studies reported the beneficial effect of silicon under stress conditions. Silicon supplementation may promote plant growth under drought stress, although the mechanism to alleviate drought stress is uncertain. The proteomic analysis of chloroplast proteins was carried out to study the response of silicon supplementation under drought stress. Similarly, temperature stress, mainly heat stress, has an adverse effect on soybean plants. The comparative analysis of two soybean cultivars, DS25-1 and DS31-243 was examined under ambient and elevated CO2 levels. The proteomic study employed two-dimensional gel analysis and mass spectrometry techniques to identify differentially expressed proteins under drought and temperature stress. The identified proteins are involved in metabolic processes, photosynthesis, signaling, redox homeostasis, and other cellular pathways. Most of these proteins were involved in metabolism, response to heat and photosynthesis showing significant cross-tolerance mechanisms. The physiological parameters like stomatal conductance, transpiration, photosynthesis, and chlorophyll pigments were also studied. Silicon application improved plant growth under drought stress. However, at the elevated CO2 level, plants showed better growth under heat stress. Together, these findings suggested the role of silicon and elevated CO2 concentration in drought and temperature stress in soybean. The differentially expressed proteins helped mitigate the negative impact of these stress, mainly the photosynthetic, antioxidant and heat shock proteins, and enhance crop growth and productivity under drought and heat stress.

<p>  </p> <p>Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all plants and certain algae. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their function. The translocon at the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components in plants: Toc75, Toc33/34 and Toc159. Our overall goal is to elucidate the architecture of the TOC complex to gain mechanistic insights into protein translocation into chloroplasts. However, the major bottleneck preventing structure determination of the TOC complex has been the inability to produce or isolate the complex to sufficient yields and purity for structural studies. We began by using phage display to screen for synthetic antigen-binding fragments (sABs) that bind to the soluble POTRA domains of Toc75 from both <em>Arabidopsis thaliana</em> and <em>Pisum sativum</em>. We then characterized the POTRA-sAB interactions using size-exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS), isothermal titration calorimetry (ITC), and X-ray crystallography. Finally, we show that we can use an affinity tagged sAB to isolate the TOC complex directly from pea biomass. This study has paved the way for high-resolution structural studies of the TOC complex from plants to understand protein translocation mechanisms. </p>

Hierarchical sampling from populations, incipient and recognized varieties within Phacelia dubia and P. maculata has revealed high levels of intraspecific polymorphism in chloroplast DNA. Much of the variation is partitioned between populations as evidenced by population-specific variants at fixation in all three populations of P. dubia var. interior and in both populations of P. maculata. Nine of 16 populations were polymorphic for cpDNA haplotypes. A total of 16 haplotypes was found in a sample of 106 individuals; the most common occurred in eight of the 16 populations and in 31 per cent of the individuals in the entire sample. A phylogenetic analysis revealed four basic plastome types. The two major groups of plastomes were separated by four independent base-pair mutations which suggests an ancient split in the evolution of plastid genomes. Representatives from each major plastome division were found in each of five populations spanning two allopatric varieties of P. dubia. The geographical distribution of haplotypes and lack of evidence for recent admixture argue against migration as a source of the polymorphism. It is more likely that the current taxonomic varieties are descendants of a polymorphic common ancestor.