Green algae have evolved several photo-protective responses to cope with high-light stress. The present study examines the metabolic changes during photoacclimation to high-light in Chlamydomonas reinhardtii using nuclear magnetic resonance and mass spectrometry. Using principal component analysis, a clear metabolic response to highlight intensity was observed on global metabolite pools in Chlamydomonas, with major changes in the levels of amino acids and related nitrogen metabolites. Amino acid biosynthesis was induced during short-term photoacclimation presumably to alleviate excess excitation pressure in the plastid. An increase in mitochondrial metabolism through downstream photorespiratory and glyoxylate metabolism, pathways thought to act in a photo-protective capacity, was also observed. Long-term light stress resulted in a significant increase in antioxidant metabolites, ascorbate and dehydroascorbate. These results suggest that metabolism plays a direct role in coping with the imbalance in the excess excitation pressure generated during high-light stress; however, this metabolomics survey has generated additional questions about the roles of nitrogen assimilation associated metabolites in photoacclimatory responses to high-light in Chlamydomonas.

Acclimation of photosynthesis allows plants to adjust the composition of their photosynthetic apparatus to adapt to changes in the environment, and is important in maintaining fitness. Dynamic acclimation refers to acclimation of fully developed leaves, after developmental processes have ceased. Rates of photosynthesis fluctuate with environmental change, and this requires metabolic adjustments. It has previously been shown that acclimation requires the chloroplastic glucose 6-phosphate/ phosphate translocator GPT2. Using label-free proteomics we characterised the acclimation deficient gpt2 mutant. High light acclimation involves changes in the composition of the photosynthetic proteome and increases in many other metabolic enzymes, but in gpt2 plants, a reduced ability to alter protein composition, and enhanced stress responses were seen. Using a combined transcriptomics and proteomics approach we also analysed acclimation to low temperature. We show that photosynthetic acclimation requires the cytosolic fumarase, FUM2. In fum2 mutants, an enhanced transcriptional response to low temperature was seen, which was impaired at the level of the proteome, relative to the WT. We also identified a protein encoding a β-Amylase, BAM5, that strongly responded to high light acclimation. The role of this protein was further characterised, and we show a nonchloroplastic location. Furthermore, suppression of this gene resulted in plants that were unable to acclimate, and had a reduced sugar content. This research highlights novel and diverse roles for proteins in acclimation, and provides a comprehensive proteomic profiling of high light and low temperature acclimation that has previously been lacking.

Plants absorb light for photosynthesis, but not all is used. Excess light energy may lead to photoinhibition of photosynthesis and irreversible photooxidative damage. Plants have evolved mechanisms for energy dissipation under high light stress. One such response may involve production of ELIP. It is of interest to know what signal(s) may be involved in ELIP expression. My hypothesis is that redox status of the chloroplast photosynthetic electron transport Chain (PETC) and/or chlororespiration may induce ELIP expression. Using the Arabidopsis thaliana immutans (im) chlororespiratory mutant, this hypothesis was tested. Etiolated seedlings of this variegated mutant were subjected to various light intensities over 0-24 hr period and ELIP mRNA levels were analyzed. These were compared with the wild type plants treated in the same manner. It was found that mature thylakoids may not be required for ELIP expression, and that both photoreceptor-dependent and independent components may be involved in ELIP expression.

Nos últimos 100 anos, a temperatura média global aumentou aproximadamente 0,6ºC e deverá continuar a subir a uma taxa rápida podendo atingir 2ºC. No entanto, não se sabe como as plantas responderão a essa mudança climática, pois existem poucas informações sobre as respostas fisiológicas das plantas a incrementos de temperatura. Este trabalho tem por objetivo avaliar os efeitos de duas condições luminosas e duas condições de temperatura (com interação entre eles), em perspectiva futura, sobre o comportamento morfológico, fisiológico e bioquímico de quatro espécies da Floresta Mesófila Semidecídua: Aspidosperma cylindrocarpon e Aspidosperma polyneuron (Apocynaceae); Cariniana estrellensis e Cariniana legalis (Lecythidaceae). As plantas foram submetidas aos seguintes tratamentos: 100% de radiação e temperatura ambiente (100%RTA), 100% de radiação e temperatura elevada em +2ºC (100%RTE), 20% de radiação (por sombreamento) e temperatura ambiente (20%RTA), e 20% de radiação e temperatura elevada em +2ºC (20%RTE). Aspidosperma polyneuron apresentou-se como a espécie mais sensível a radiação intensa, seguida de C. legalis ambas já ameaçadas de extinção. Os parâmetros morfológicos confirmam a teoria de que plantas cronicamente fotoinibidas continuam crescendo, porém, em menores taxas. Os dados bioquímicos reforçam os fisiológicos mostrando fragilidade das espécies secundárias à radiação intensa e os efeitos principalmente negativos do aquecimento aplicado. / In the last 100 years the average global temperature increased by 0.6ºC and it might increase in a fast rate reaching up to 2ºC. However, little is known about how plants might respond to the climate change because there is not enough information about the physiological alterations plants might suffer due to increased temperature. We evaluated morphological, physiological and biochemical alterations under the effects of two light/temperature conditions and its interactions in four different plant species of the Semideciduous Mesophitic Forest: Aspidosperma cylindrocarpon and Aspidosperma polyneuron (Apocynaceae); Cariniana estrellensis and Cariniana legalis (Lecythidaceae). All plants suffered the following treatments: 100% radiation and environment temperature (100%RTA), 100% radiation and environment temperature +2ºC (100%RTE), 20% radiation (shade) and environment temperature (20%RTA), and 20% radiation (shade) and environment temperature +2ºC (20%RTE). Aspidosperma polyneuron was shown to be the most sensible specie to high radiation, followed by C. legalis, both threatened with extinction. Morphological parameters confirmed the theory that chronically photo-inhibited plants keep growing, but at lower rates. Biochemical findings also confirmed our physiological data showing increased fragility of the secondary species to high radiation and heating.

Chlamydomonas reinhardtii can utilize CO2 for autotrophic growth (HSM plus 5% CO2) or acetate for mixotrophic growth (TAP). This study was to elucidate the differential regulation of methionine sulfoxide reductase (MSR) gene expression between HSM plus 5% CO2 and TAP cultured cells, and also to determine the difference of gene expression in response to high light (1,000 £gE m-2 s-1). The role of photosynthetic electron transport (PET) in the regulation of MSR gene expression was also examined by the use of PET inhibitors. High light inhibited PSII activity (Fv/Fm and Fv'/Fm') of HSM plus 5% CO2 and TAP cultured cells., while the responses of CrMSR gene expression in mixotrophically grown cells were different from autotrophically grown cells, High light increased the expression of CrMSRA1, CrMSRA2, CrMSRA3, CrMSRA5, CrMSRB1.2, and CrMSRB2.1, but inhibited the expression of CrMSRA4 and CrMSRB2.2 in autotrophically grown cells. The expression of CrMSRA3, CrMSRA5, and CrMSRB2.1 in mixotrophically grown cells was increased by high light but that of CrMSRA1, CrMSRA4, and CrMSRB2.2 was inhbited. The number of MSR isoform that was up-regulated by high light was greater in autotrophically grown cell than that in mixotrophically grown cells. Using the PET inhibitors (3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)), most of the CrMSRA expression was regulated by reduced QA for autotrophically grown cells while reduced PQ was the main site for mixotrophically grown cells by high light. The expression of CrMSRB in autotrophically grown cells was mainy modulated by QA (-) or Cytb6f (-), while that was not affected by PET, except a role of Cytb6f (-) on the high light-induced CrMSRB2.2 expression. We fouind that CrMSRB gene expression in autotrophically grown cells was highly affected by PET but not for micotrophically grtown cells. The present result that H2O2 did not accumulate in autotrophically and mixotrophically grown cells suggests that H2O2 may be not involved in the regulation of high light regulation of CrMSR gene expression. The present study shows that the mRNA expression of CrMSR isoforms in Chlamydomonas was diffrerentially regulated between autotrophically and mixttrophically grown cells. The relationship between the utilization of different C source and CrMSR gene expression will be discussed.

The thesis focuses on regulation of the chlorophyll biosynthetic pathway and its coordination with synthesis of chlorophyll-binding proteins in the cyanobacterium Synechocystis sp. PCC 6803. One of the aims was to analyze correlation between syntheses of photosystems and chlorophyll in Synechocystis cells using radioactive labeling of proteins and chlorophyll by 35S and 14C, respectively. I also investigated the role of enzymes catalyzing protochlorophyllide reduction step in the chlorophyll biosynthesis by analyzing the synthesis and accumulation of photosynthetic proteins in Synechocystis mutants lacking one of the enzymes. Further, roles of Ycf54 and Psb27 proteins in stability and assembly of oxidative cyclase and CP43, respectively, are also described.

Nos últimos 100 anos, a temperatura média global aumentou aproximadamente 0,6ºC e deverá continuar a subir a uma taxa rápida podendo atingir 2ºC. No entanto, não se sabe como as plantas responderão a essa mudança climática, pois existem poucas informações sobre as respostas fisiológicas das plantas a incrementos de temperatura. Este trabalho tem por objetivo avaliar os efeitos de duas condições luminosas e duas condições de temperatura (com interação entre eles), em perspectiva futura, sobre o comportamento morfológico, fisiológico e bioquímico de quatro espécies da Floresta Mesófila Semidecídua: Aspidosperma cylindrocarpon e Aspidosperma polyneuron (Apocynaceae); Cariniana estrellensis e Cariniana legalis (Lecythidaceae). As plantas foram submetidas aos seguintes tratamentos: 100% de radiação e temperatura ambiente (100%RTA), 100% de radiação e temperatura elevada em +2ºC (100%RTE), 20% de radiação (por sombreamento) e temperatura ambiente (20%RTA), e 20% de radiação e temperatura elevada em +2ºC (20%RTE). Aspidosperma polyneuron apresentou-se como a espécie mais sensível a radiação intensa, seguida de C. legalis ambas já ameaçadas de extinção. Os parâmetros morfológicos confirmam a teoria de que plantas cronicamente fotoinibidas continuam crescendo, porém, em menores taxas. Os dados bioquímicos reforçam os fisiológicos mostrando fragilidade das espécies secundárias à radiação intensa e os efeitos principalmente negativos do aquecimento aplicado. / In the last 100 years the average global temperature increased by 0.6ºC and it might increase in a fast rate reaching up to 2ºC. However, little is known about how plants might respond to the climate change because there is not enough information about the physiological alterations plants might suffer due to increased temperature. We evaluated morphological, physiological and biochemical alterations under the effects of two light/temperature conditions and its interactions in four different plant species of the Semideciduous Mesophitic Forest: Aspidosperma cylindrocarpon and Aspidosperma polyneuron (Apocynaceae); Cariniana estrellensis and Cariniana legalis (Lecythidaceae). All plants suffered the following treatments: 100% radiation and environment temperature (100%RTA), 100% radiation and environment temperature +2ºC (100%RTE), 20% radiation (shade) and environment temperature (20%RTA), and 20% radiation (shade) and environment temperature +2ºC (20%RTE). Aspidosperma polyneuron was shown to be the most sensible specie to high radiation, followed by C. legalis, both threatened with extinction. Morphological parameters confirmed the theory that chronically photo-inhibited plants keep growing, but at lower rates. Biochemical findings also confirmed our physiological data showing increased fragility of the secondary species to high radiation and heating.

Adaptation to environmental changes is important for the survival of living organisms. Under extreme abiotic conditions, organic molecules (such as lipids, proteins and nucleic acids) are prone to damage. Under these conditions stress response mechanisms are activated, either to prevent the source of damage or to promote the rapid turnover of damaged molecules. Like all photoautotrophic organisms, cyanobacteria are sensitive to high light intensity and oxidative stress, which induces damage to the photosynthetic apparatus. My thesis is divided in two subjects related to particular stress responses in the cyanobacterium Synechocystis sp. PCC 6803: 1) the role of Deg/HtrA proteases and 2) investigations on the small CAB-like proteins. Deg/HtrA proteases are ATP-independent serine endopeptidases with a characteristic C-terminal PDZ domain. These proteases are largely dispersed among living organisms, with many different functions, mostly involved in protein quality control. The genome of Synechocystis sp. PCC 6803 contains three genes coding for Deg/HtrA proteases: HtrA, HhoA and HhoB. These proteases are essential for survival under high light and heat stress, and may overlap in their functions. During my Ph.D. studies I focused on the identification of the precise localization of the Deg/HtrA proteases in the cyanobacterial cell, analyzed the biochemical properties of recombinant Synechocystis Deg/HtrA proteases in vitro and adopted proteomic and metabolomic approaches to study the physiological importance of these proteases. My data show that Deg/HtrA proteases are not only important in stress response mechanisms under adverse conditions, but are also involved in the stabilization of important physiological processes, such as polysaccharides biosynthesis and peptidoglycan turnover. The small CAB-like proteins (SCPs) belong to the light harvesting-like family of stress induced proteins and are thought to be involved in the photoprotection of the photosynthetic apparatus. Five small CAB-like proteins where identified in Synechocystis sp. PCC 6803 (ScpA-E). In my studies I identified another relative to the SCPs, LilA, which I found to be co-transcribed with ScpD. I also focused on the subcellular localization and identification of potential interaction partners of the SCPs.