Novel Proteins Involved in Dynamics of The Photosynthetic Apparatus

During the past few years, a considerable number of research articles have been published relating to the structure and function of the major photosynthetic protein complexes, photosystem (PS) I, PSII, cytochrome (Cyt) b6f, and adenosine triphosphate (ATP) synthase. Sequencing of the Arabidopsis thaliana (Arabidopsis) genome together with several high-quality proteomics studies has, however, revealed that the thylakoid membrane network of plant chloroplasts still contains a number of functionally unknown proteins. These proteins may have a role as auxiliary proteins guiding the assembly, maintenance, and turnover of the thylakoid protein complexes, or they may be as yet unknown subunits of the photosynthetic complexes. Novel subunits are most likely to be found in the NAD(P)H dehydrogenase (NDH) complex, the structure and function of which have remained obscure in the absence of detailed crystallographic data, thus making this thylakoid protein complex a particularly interesting target of investigation. In this thesis, several novel thylakoid-associated proteins were identified by proteomics-based methods. The major goal of characterization of the stroma thylakoid associated polysome-nascent chain complexes was to determine the proteins that guide the dynamic life cycle of PSII. In addition, a large protein complex of ≥ 1,000 kDa, residing in the stroma thylakoid, was characterized in greater depth and it was found to be a supercomplex composed of the PSI and NDH complexes. A set of newly identified proteins from Arabidopsis thylakoids was subjected to detailed characterization using the reverse genetics approach and extensive biochemical and biophysical analysis. The role of the novel proteins, either as auxiliary proteins or subunits of the photosynthetic protein complexes, was revealed. Two novel thylakoid lumen proteins, TLP18.3 and AtCYP38, function as auxiliary proteins assisting specific steps of the assembly/repair of PSII. The role of the 10-kDa thylakoid lumen protein PsbR is related to the optimization of oxygen evolution of PSII by assisting the assembly of the PsbP protein. Two integral thylakoid membrane proteins, NDH45 and NDH48, are novel subunits of the chloroplast NDH complex. Finally, the thylakoid lumen immunophilin AtCYP20-2 is suggested to interact with the NDH complex, instead of PSII as was hypothesized earlier.

NADPH-Dependent Thioredoxin System In Regulation of Chloroplast Functions

Photosynthesis, the process in which carbon dioxide is converted into sugars using the energy of sunlight, is vital for heterotrophic life on Earth. In plants, photosynthesis takes place in specific organelles called chloroplasts. During chloroplast biogenesis, light is a prerequisite for the development of functional photosynthetic structures. In addition to photosynthesis, a number of other metabolic processes such as nitrogen assimilation, the biosynthesis of fatty acids, amino acids, vitamins, and hormones are localized to plant chloroplasts. The biosynthetic pathways in chloroplasts are tightly regulated, and especially the reduction/oxidation (redox) signals play important roles in controlling many developmental and metabolic processes in chloroplasts. Thioredoxins are universal regulatory proteins that mediate redox signals in chloroplasts. They are able to modify the structure and function of their target proteins by reduction of disulfide bonds. Oxidized thioredoxins are restored via the action of thioredoxin reductases. Two thioredoxin reductase systems exist in plant chloroplasts, the NADPHdependent thioredoxin reductase C (NTRC) and ferredoxin-thioredoxin reductase (FTR). The ferredoxin-thioredoxin system that is linked to photosynthetic light reactions is involved in light-activation of chloroplast proteins. NADPH can be produced via both the photosynthetic electron transfer reactions in light, and in darkness via the pentose phosphate pathway. These different pathways of NADPH production enable the regulation of diverse metabolic pathways in chloroplasts by the NADPH-dependent thioredoxin system. In this thesis, the role of NADPH-dependent thioredoxin system in the redox-control of chloroplast development and metabolism was studied by characterization of Arabidopsis thaliana T-DNA insertion lines of NTRC gene (ntrc) and by identification of chloroplast proteins regulated by NTRC. The ntrc plants showed the strongest visible phenotypes when grown under short 8-h photoperiod. This indicates that i) chloroplast NADPH-dependent thioredoxin system is non-redundant to ferredoxinthioredoxin system and that ii) NTRC particularly controls the chloroplast processes that are easily imbalanced in daily light/dark rhythms with short day and long night. I identified four processes and the redox-regulated proteins therein that are potentially regulated by NTRC; i) chloroplast development, ii) starch biosynthesis, iii) aromatic amino acid biosynthesis and iv) detoxification of H₂O₂. Such regulation can be achieved directly by modulating the redox state of intramolecular or intermolecular disulfide bridges of enzymes, or by protecting enzymes from oxidation in conjunction with 2-cysteine peroxiredoxins. This thesis work also demonstrated that the enzymatic antioxidant systems in chloroplasts, ascorbate peroxidases, superoxide dismutase and NTRC-dependent 2-cysteine peroxiredoxins are tightly linked up to prevent the detrimental accumulation of reactive oxygen species in plants.

Internet-based breast cancer patient’s pathway as an empowering patient educational tool

This three-phase study was conducted to examine the effect of the Breast Cancer Patient’s Pathway program (BCPP) on breast cancer patients’ empowering process from the viewpoint of the difference between knowledge expectations and perceptions of received knowledge, knowledge level, quality of life, anxiety and treatment-related side effects during the breast cancer treatment process. The BCPP is an Internet-based patient education tool describing a flow chart of the patient pathway during the breast treatment process, from breast cancer diagnostic tests to the follow-up after treatments. The ultimate goal of this study was to evaluate the effect of the BCPP to the breast cancer patient’s empowerment by using the patient pathway as a patient education tool. In phase I, a systematic literature review was carried out to chart the solutions and outcomes of Internet-based educational programs for breast cancer patients. In phase II, a Delphi study was conducted to evaluate the usability of web pages and adequacy of their content. In phase III, the BCPP program was piloted with 10 patients and patients were randomised to an intervention group (n=50) and control group (n=48). According to the results of this study, the Internet is an effective patient education tool for increasing knowledge, and BCPP can be used as a patient education method supporting other education methods. However, breast cancer patients’ perceptions of received knowledge were not fulfilled; their knowledge expectations exceed the perceived amount of received knowledge. Although control group patients’ knowledge expectations were met better with the knowledge they received in hospital compared to the patients in the intervention group, no statistical differences were found between the groups in terms of quality of life, anxiety and treatment-related side effects. However, anxiety decreased faster in the intervention group when looking at internal differences between the groups at different measurement times. In the intervention group the relationship between the difference between knowledge expectations and perceptions of received knowledge correlated significantly with quality of life and anxiety. Their knowledge level was also significant higher than in the control group. These results support the theory that the empowering process requires patient’s awareness of knowledge expectations and perceptions of received knowledge. There is a need to develop patient education to meet patients’ perceptions of received knowledge, including oral and written education and BCPP, to fulfil patient’s knowledge expectations and facilitate the empowering process. Further research is needed on the process of cognitive empowerment with breast cancer patients. There is a need for new patient education methods to increase breast cancer patients’ awareness of knowing.

Biosynthesis of pyranonaphthoquinone polyketides:characterization of the alnumycin pathway

Alnumycin A is an aromatic pyranonaphthoquinone (PNQ) polyketide closely related to the model compound actinorhodin. While some PNQ polyketides are glycosylated, alnumycin A contains a unique sugar-like dioxane moiety. This unusual structural feature made alnumycin A an interesting research target, since no information was available about its biosynthesis. Thus, the main objective of the thesis work became to identify the steps and the enzymes responsible for the biosynthesis of the dioxane moiety. Cloning, sequencing and heterologous expression of the complete alnumycin gene cluster from Streptomyces sp. CM020 enabled the inactivation of several alnumycin biosynthetic genes and preliminary identification of the gene products responsible for pyran ring formation, quinone formation and dioxane biosynthesis. The individual deletions of the genes resulted in the production of several novel metabolites, which in many cases turned out to be pathway intermediates and could be used for stepwise enzymatic reconstruction of the complete dioxane biosynthetic pathway in vitro. Furthermore, the in vitro reactions with purified alnumycin biosynthetic enzymes resulted in the production of other novel compounds, both pathway intermediates and side products. Identification and molecular level studies of the enzymes AlnA and AlnB catalyzing the first step of dioxane biosynthesis – an unusual C-ribosylation step – led to a mechanistic proposal for the C-ribosylation of the polyketide aglycone. The next step on the dioxane biosynthetic pathway was found to be the oxidative conversion of the attached ribose into a highly unusual dioxolane unit by Aln6 belonging to an uncharacterized protein family, which unexpectedly occurred without any apparent cofactors. Finally, the last step of the pathway was found to be catalyzed by the NADPH-dependent reductase Aln4, which is able to catalyze the conversion of the formed dioxolane into a dioxane moiety. The work presented here and the knowledge gained of the enzymes involved in dioxane biosynthesis enables their use in the rational design of novel compounds containing C–C bound ribose, dioxolane and dioxane moieties.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

Regulation of photosynthetic electron transfer in plant chloroplasts

This thesis focuses on the molecular mechanisms regulating the photosynthetic electron transfer reactions upon changes in light intensity. To investigate these mechanisms, I used mutants of the model plant Arabidopsis thaliana impaired in various aspects of regulation of the photosynthetic light reactions. These included mutants of photosystem II (PSII) and light harvesting complex II (LHCII) phosphorylation (stn7 and stn8), mutants of energy-dependent non-photochemical quenching (NPQ) (npq1 and npq4) and of regulation of photosynthetic electron transfer (pgr5). All of these processes have been extensively investigated during the past decades, mainly on plants growing under steady-state conditions, and therefore many aspects of acclimation processes may have been neglected. In this study, plants were grown under fluctuating light, i.e. the alternation of low and high intensities of light, in order to maximally challenge the photosynthetic regulatory mechanisms. In pgr5 and stn7 mutants, the growth in fluctuating light condition mainly damaged PSI while PSII was rather unaffected. It is shown that the PGR5 protein regulates the linear electron transfer: it is essential for the induction of transthylakoid ΔpH that, in turn, activates energy-dependent NPQ and downregulates the activity of cytochrome b6f. This regulation was shown to be essential for the photoprotection of PSI under fluctuations in light intensity. The stn7 mutants were able to acclimate under constant growth light conditions by modulating the PSII/PSI ratio, while under fluctuating growth light they failed in implementing this acclimation strategy. LHCII phosphorylation ensures the balance of the excitation energy distribution between PSII and PSI by increasing the probability for excitons to be trapped by PSI. LHCII can be phosphorylated over all of the thylakoid membrane (grana cores as well as stroma lamellae) and when phosphorylated it constitutes a common antenna for PSII and PSI. Moreover, LHCII was shown to work as a functional bridge that allows the energy transfer between PSII units in grana cores and between PSII and PSI centers in grana margins. Consequently, PSI can function as a quencher of excitation energy. Eventually, the LHCII phosphorylation, NPQ and the photosynthetic control of linear electron transfer via cytochrome b6f work in concert to maintain the redox poise of the electron transfer chain. This is a prerequisite for successful plant growth upon changing natural light conditions, both in short- and long-term.

Molecular Mechanisms and Interactions in Regulation of Photosynthetic Reactions

In photosynthesis, light energy is converted to chemical energy, which is consumed for carbon assimilation in the Calvin-Benson-Bassham (CBB) cycle. Intensive research has significantly advanced the understanding of how photosynthesis can survive in the ever-changing light conditions. However, precise details concerning the dynamic regulation of photosynthetic processes have remained elusive. The aim of my thesis was to specify some molecular mechanisms and interactions behind the regulation of photosynthetic reactions under environmental fluctuations. A genetic approach was employed, whereby Arabidopsis thaliana mutants deficient in specific photosynthetic protein components were subjected to adverse light conditions and assessed for functional deficiencies in the photosynthetic machinery. I examined three interconnected mechanisms: (i) auxiliary functions of PsbO1 and PsbO2 isoforms in the oxygen evolving complex of photosystem II (PSII), (ii) the regulatory function of PGR5 in photosynthetic electron transfer and (iii) the involvement of the Calcium Sensing Receptor CaS in photosynthetic performance. Analysis of photosynthetic properties in psbo1 and psbo2 mutants demonstrated that PSII is sensitive to light induced damage when PsbO2, rather than PsbO1, is present in the oxygen evolving complex. PsbO1 stabilizes PSII more efficiently compared to PsbO2 under light stress. However, PsbO2 shows a higher GTPase activity compared to PsbO1, and plants may partially compensate the lack of PsbO1 by increasing the rate of the PSII repair cycle. PGR5 proved vital in the protection of photosystem I (PSI) under fluctuating light conditions. Biophysical characterization of photosynthetic electron transfer reactions revealed that PGR5 regulates linear electron transfer by controlling proton motive force, which is crucial for the induction of the photoprotective non-photochemical quenching and the control of electron flow from PSII to PSI. I conclude that PGR5 controls linear electron transfer to protect PSI against light induced oxidative damage. I also found that PGR5 physically interacts with CaS, which is not needed for photoprotection of PSII or PSI in higher plants. Rather, transcript profiling and quantitative proteomic analysis suggested that CaS is functionally connected with the CBB cycle. This conclusion was supported by lowered amounts of specific calciumregulated CBB enzymes in cas mutant chloroplasts and by slow electron flow to PSI electron acceptors when leaves were reilluminated after an extended dark period. I propose that CaS is required for calcium regulation of the CBB cycle during periods of darkness. Moreover, CaS may also have a regulatory role in the activation of chloroplast ATPase. Through their diverse interactions, components of the photosynthetic machinery ensure optimization of light-driven electron transport and efficient basic production, while minimizing the harm caused by light induced photodamage.

Pp2a: At The Crossroads Between Growth and Defence In Plants

Living organisms manage their resources in well evolutionary-preserved manner to grow and reproduce. Plants are no exceptions, beginning from their seed stage they have to perceive environmental conditions to avoid germination at wrong time or rough soil. Under favourable conditions, plants invest photosynthetic end products in cell and organ growth to provide best possible conditions for generation of offspring. Under natural conditions, however, plants are exposed to a multitude of environmental stress factors, including high light and insufficient light, drought and flooding, various bacteria and viruses, herbivores, and other plants that compete for nutrients and light. To survive under environmental challenges, plants have evolved signaling mechanisms that recognise environmental changes and perform fine-tuned actions that maintain cellular homeostasis. Controlled phosphorylation and dephosphorylation of proteins plays an important role in maintaining balanced flow of information within cells. In this study, I examined the role of protein phosphatase 2A (PP2A) on plant growth and acclimation under optimal and stressful conditions. To this aim, I studied gene expression profiles, proteomes and protein interactions, and their impacts on plant health and survival, taking advantage of the model plant Arabidopsis thaliana and the mutant approach. Special emphasis was made on two highly similar PP2A-B regulatory subunits, B’γ and B’ζ. Promoters of B’γ and B’ζ were found to be similarly active in the developing tissues of the plant. In mature leaves, however, the promoter of B’γ was active in patches in leaf periphery, while the activity of B’ζ promoter was evident in leaf edges. The partially overlapping expression patterns, together with computational models of B’γ and B’ζ within trimeric PP2A holoenzymes suggested that B’γ and B’ζ may competitively bind into similar PP2A trimmers and thus influence each other’s actions. Arabidopsis thaliana pp2a-b’γ and pp2a-b’γζ double mutants showed dwarfish phenotypes, indicating that B’γ and B’ζ are needed for appropriate growth regulation under favorable conditions. However, while pp2a-b’γ displayed constitutive immune responses and appearance of premature yellowings on leaves, the pp2a-b’γζ double mutant supressed these yellowings. More detailed analysis of defense responses revealed that B’γ and B’ζ mediate counteracting effects on salicylic acid dependent defense signalling. Associated with this, B’γ and B’ζ were both found to interact in vivo with CALCIUM DEPENDENT PROTEIN KINASE 1 (CPK1), a crucial element of salicylic acid signalling pathway against pathogens in plants. In addition, B’γ was shown to modulate cellular reactive oxygen species (ROS) metabolism by controlling the abundance of ALTERNATIVE OXIDASE 1A and 1D in mitochondria. PP2A B’γ and B’ζ subunits turned out to play crucial roles in the optimization of plant choices during their development. Taken together, PP2A allows fluent responses to environmental changes, maintenance of plant homeostasis, and grant survivability with minimised cost of redirection of resources from growth to defence.

Alternative electron transfer routes involved in photoprotection of cyanobacteria

In oxygenic photosynthesis, the highly oxidizing reactions of water splitting produce reactive oxygen species (ROS) and other radicals that could damage the photosynthetic apparatus and affect cell viability. Under particular environmental conditions, more electrons are produced in water oxidation than can be harmlessly used by photochemical processes for the reduction of metabolic electron sinks. In these circumstances, the excess of electrons can be delivered, for instance, to O2, resulting in the production of ROS. To prevent detrimental reactions, a diversified assortment of photoprotection mechanisms has evolved in oxygenic photosynthetic organisms. In this thesis, I focus on the role of alternative electron transfer routes in photoprotection of the cyanobacterium Synechocystis sp. PCC 6803. Firstly, I discovered a novel subunit of the NDH-1 complex, NdhS, which is necessary for cyclic electron transfer around Photosystem I, and provides tolerance to high light intensities. Cyclic electron transfer is important in modulating the ATP/NADPH ratio under stressful environmental conditions. The NdhS subunit is conserved in many oxygenic phototrophs, such as cyanobacteria and higher plants. NdhS has been shown to link linear electron transfer to cyclic electron transfer by forming a bridge for electrons accumulating in the Ferredoxin pool to reach the NDH-1 complexes. Secondly, I thoroughly investigated the role of the entire flv4-2 operon in the photoprotection of Photosystem II under air level CO2 conditions and varying light intensities. The operon encodes three proteins: two flavodiiron proteins Flv2 and Flv4 and a small Sll0218 protein. Flv2 and Flv4 are involved in a novel electron transport pathway diverting electrons from the QB pocket of Photosystem II to electron acceptors, which still remain unknown. In my work, it is shown that the flv4-2 operon-encoded proteins safeguard Photosystem II activity by sequestering electrons and maintaining the oxidized state of the PQ pool. Further, Flv2/Flv4 was shown to boost Photosystem II activity by accelerating forward electron flow, triggered by an increased redox potential of QB. The Sll0218 protein was shown to be differentially regulated as compared to Flv2 and Flv4. Sll0218 appeared to be essential for Photosystem II accumulation and was assigned a stabilizing role for Photosystem II assembly/repair. It was also shown to be responsible for optimized light-harvesting. Thus, Sll0218 and Flv2/Flv4 cooperate to protect and enhance Photosystem II activity. Sll0218 ensures an increased number of active Photosystem II centers that efficiently capture light energy from antennae, whilst the Flv2/Flv4 heterodimer provides a higher electron sink availability, in turn, promoting a safer and enhanced activity of Photosystem II. This intertwined function was shown to result in lowered singlet oxygen production. The flv4-2 operon-encoded photoprotective mechanism disperses excess excitation pressure in a complimentary manner with the Orange Carotenoid Protein-mediated non-photochemical quenching. Bioinformatics analyses provided evidence for the loss of the flv4-2 operon in the genomes of cyanobacteria that have developed a stress inducible D1 form. However, the occurrence of various mechanisms, which dissipate excitation pressure at the acceptor side of Photosystem II was revealed in evolutionarily distant clades of organisms, i.e. cyanobacteria, algae and plants.