Supramolecular structures of thylakoid membrane protein complexes:supercomplex organization under different environmental conditions

In this dissertation electron microscopy has been used as a main technique for structural characterization of the photosynthetic protein complexes isolated from different organisms. The photosynthetic processes are conducted by membrane-embedded protein complexes in the thylakoid membrane, known as photosystem I and II (PSI and PSII). The structural organization and architecture of the photosystems varies depending on species and the photic environment. In the first study, knock-out mutants of the moss Physcomitrella patens reveal the role of subunit Lhcb9 in regulation of the antenna size under low light conditions. Similarly, in a following study of the isolated supercomplexes from Norway spruce we show the presence of large PSII supercomplexes and PSII megacomplexes, while PSI binds multiple LHCII trimers under varying light adaptation conditions. Another study represents the organization and size of light-harvesting antenna of PSI from the colonial green alga Botryococcus braunii. Based on electron microscopy analysis, we concluded that the larger light-harvesting antenna in Botryococcus braunii is important for the cells in the interior of a colony. Our study on the diatom Thalassiosira pseudonana revealed the presence of a larger PSI and a unique PSII structure which might be necessary for capturing and regulation of the excitation energy. The structural characterization of Chlorella ohadii PSI shows an additional core subunit PsaM and also pigments bound to the antenna proteins which enable a high photosynthetic performance under extreme light intensities in the desert

Revealing the architecture of the photosynthetic apparatus in the diatom Thalassiosira pseudonana

Diatoms are a large group of marine algae that are responsible for about one-quarter of global carbon fixation. Light-harvesting complexes of diatoms are formed by the fucoxanthin chlorophyll a/c proteins and their overall organization around core complexes of photosystems (PSs) I and II is unique in the plant kingdom. Using cryo-electron tomography, we have elucidated the structural organization of PSII and PSI supercomplexes and their spatial segregation in the thylakoid membrane of the model diatom species Thalassiosira pseudonana. 3D sub-volume averaging revealed that the PSII supercomplex of T. pseudonana incorporates a trimeric form of light-harvesting antenna, which differs from the tetrameric antenna observed previously in another diatom, Chaetoceros gracilis. Surprisingly, the organization of the PSI supercomplex is conserved in both diatom species. These results strongly suggest that different diatom classes have various architectures of PSII as an adaptation strategy, whilst a convergent evolution occurred concerning PSI and the overall plastid structure

Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens

Phosphorylation-dependent formation of photosystem I supercomplexes provides both short- and long-term acclimation of moss photosynthetic apparatus to changing environmental cues.Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments

IDENTIFYING AND TRACKING MARINE PROTEIN AND ITS IMPORTANCE IN THE NITROGEN CYCLE USING PROTEOMICS

Protein comprises the largest compartment of organic nitrogen in the ocean, and makes up a major portion of organic carbon in phytoplankton. Protein has long been thought to be highly labile in the environment and rapidly lost during diagenesis. However, the analysis of dissolved and particulate organic matter with NMR has revealed that much of dissolved and particulate marine organic nitrogen is linked by amide bonds, the very bonds that join amino acids in proteins. Throughout the global ocean, total hydrolysable amino acids (THAAs, the building blocks of proteins) can be measured in the water column and sediments, yet their biosynthetic source has remained elusive. Here, analytical techniques were developed combining protein solubilizing buffer extractions, gel electrophoresis, and proteomic mass spectrometry in order to investigate the biogeochemical significance of marine protein from primary production during transport and incorporation in sediments. These techniques enabled the detection and classification of previously unidentified marine sedimentary proteins. Specific proteins were tracked through the water column to continental shelf and deeper basin (3490 m) sediments of the Bering Sea, one of the world's most productive ecosystems. Diatoms were observed to be the principal source of identifiable protein in sediments. In situ shipboard phytoplankton degradation experiments were conducted to follow protein degradation, and it was observed that individual proteins remained identifiable even after 53 days of microbial recycling. These studies show that proteins can be identified from complex environmental matrices, and the methods developed here can be applied to investigate and identify proteins in degraded organic matter from a broad range of sources. The longevity of some fraction of algal proteins indicates that carbon and nitrogen sources can be tracked down the marine water column to sediments in diatom dominated systems as well as other types of phytoplankton. Using proteomic techniques to understand the marine carbon and nitrogen cycles will become increasingly important as climate change influences the timing, location, and phylogeny of those organisms responsible for oceanic primary production