In oxygenic phototrophs, the photosynthetic machinery is located in thylakoid membrane (TM), a specialized endogenous membrane system. How TM are synthesized remains however mostly unknown. The aim of this thesis was to clarify a link between the synthesis of chlorophyll (Chl)-binding proteins, the main protein component of TM, and the formation of TM system in the model cyanobacterium Synechocystis PCC 6803. During the project, the analysis of TM under various growth conditions and in Chl-deficient mutants has demonstrated that a sufficient amount of de novo produced Chl molecules is crucial for the TM biogenesis. Particularly, the synthesis of the photosystem II subunit CP47 and trimeric photosystem I appeared to be sensitive to a shortage in de novo made Chl molecules. Interestingly, a specialized ribosome-binding protein (Pam68) has been identified to facilitate the insertion of Chl molecules into CP47. The synthesis of Chl-proteins and the biogenesis of TM have been further explored in cells recovering from long-term nitrogen depletion. Using this approach, it was possible to identify a large structure in the cell cytosol, which is very likely the site of TM biogenesis, and to correlate the appearance of this structure with the restored biogenesis of Chl-binding proteins.

<p>Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex.</p><p>In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs.</p><p>The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.</p>

<p>This investigation addresses how photosynthetic efficiency is affected when pea (Pisum sativum) plants are restricted to a sole nitrogen source (i.e. ammonium or nitrate). The pea plants were watered with different nutrient solutions without NO3- or NH4+ for different time-periods in order to assay for nitrogen content. The soluble ammonium and nitrate content was measured throughout the entire growth period. No major differences were observed in nitrogen content during the starvation period up to 25 days. For technical reasons, cultivation of plants could not be extended beyond this time. The chloroplasts and thylakoids were isolated after 25 days and assayed for chlorophyll contents and photosynthetic activity.</p><p>The outcome of these tests indicates a small but unambiguous decrease in the photosynthesis activity for all treatments, relative the control.</p>

Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex. In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs. The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.

The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts.

This investigation addresses how photosynthetic efficiency is affected when pea (Pisum sativum) plants are restricted to a sole nitrogen source (i.e. ammonium or nitrate). The pea plants were watered with different nutrient solutions without NO3- or NH4+ for different time-periods in order to assay for nitrogen content. The soluble ammonium and nitrate content was measured throughout the entire growth period. No major differences were observed in nitrogen content during the starvation period up to 25 days. For technical reasons, cultivation of plants could not be extended beyond this time. The chloroplasts and thylakoids were isolated after 25 days and assayed for chlorophyll contents and photosynthetic activity. The outcome of these tests indicates a small but unambiguous decrease in the photosynthesis activity for all treatments, relative the control.

It has previously been stated that K+-ions in a plant cell have a counter-balancing role in which the efflux of K+-ions from the thylakoid lumen charge-balance the light-induced proton pumping that is known to occur across the thylakoid membrane, and this in turn stabilizes photosynthetic activity. In the present study, two different types of plants of the same ecotype (Col-0) of Arabidopsis thaliana have been studied: a wild-type and a T-DNA exon-mutant (tpk5-e) that has lost the expression of the protein known as Tandem-pore K+- channel (AtTPK5). The plants were grown in a hydroponic system under normal light conditions with 70% humidity. Homozygous (HM) tpk5-e mutant plants were screened using PCR and gene specific primers. Further, the photosynthetic activity was measured in 4 hour light-adapted plants and the photosynthetic activity of the tpk5-e mutant proved not to be significantly different in comparison to the wild-type when measuring the electron transport rate (ETR). Furthermore, the O2-evolution was also measured in 4 hour light-adapted plants and the tpk5-e mutant's O2-evolution proved to be significantly lower in the tpk5-e mutant in comparison to the wild-type under high light conditions. The plant fitness of the wild-type and tpk5-e mutant was also different judging from phenotypic traits such as chlorophyll expression. However, the measured chlorophyll amount of pigments chlorophyll a and b proved not to be significantly different in the tpk5-e mutant in comparison to the wild-type.

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.

Molecular dynamics simulations are a theoretical method enabling to trace the movement of atoms within a system. The system studied is usually treated on the atomistic level, however its overall properties can be also described satisfactory if several atoms are handled as one particle (coarse-grained molecular dynamics). This thesis presents molecular modeling and (coarse-grained) molecular dynamics as tools for the description of different biologically relevant systems. The coarse-grained force field parameters had to be developed prior to characterization of the thylakoid membrane from cyanobacterium Synechocystis PCC6803. Two different compositions of the membrane were studied in order to reveal differences in their behavior. The PsbI subunit of photosystem II was embedded into the thylakoid membrane and its behavior, both as an isolated protein and as a cluster of several units, was described. The last system examined was the C-type lectin-like domain of NKR-P1, a surface receptor of natural killer cells. Attention was payed to its structural characterization.

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