Most of the mitochondrial proteins in yeast are encoded in the nuclear genome, get synthesized by cytosolic ribosomes and are imported via TOM and TIM23 into the matrix or other subcompartments of mitochondria. The mitochondrial DNA in yeast however also encodes a small set of 8 proteins from which most are hydrophobic membrane proteins and build core components of the OXPHOS complexes. They get synthesized by mitochondrial ribosomes which are descendants of bacterial ribosomes and still have some similarities to them. On the other hand, mitochondrial ribosomes experienced various structural and functional changes during evolution that specialized them for the synthesis of the mitochondrial encoded membrane proteins. The mitoribosome contains mitochondria-specific ribosomal proteins and replaced the bacterial 5S rRNA by mitochondria-specific proteins and rRNA extensions. Furthermore, the mitoribosome is tethered to the inner mitochondrial membrane to facilitate a co-translational insertion of newly synthesized proteins. Thus, also the assembly process of mitoribosomes differs from that of bacteria and is to date not well understood. Therefore, the biogenesis of mitochondrial ribosomes in yeast should be investigated. To this end, a strain was generated in which the gene of the mitochondrial RNA-polymerase RPO41 is under control of an inducible GAL10-promoter. Since the scaffold of ribosomes is built by ribosomal RNAs, the depletion of the RNA-polymerase subsequently leads to a loss of mitochondrial ribosomes. Reinduction of Rpo41 initiates the assembly of new mitoribosomes, which makes this strain an attractive model to study mitoribosome biogenesis. Initially, the effects of Rpo41 depletion on cellular and mitochondrial physiology was investigated. Upon Rpo41 depletion, growth on respiratory glycerol medium was inhibited. Furthermore, mitochondrial ribosomal 21S and 15S rRNA was diminished and mitochondrial translation was almost completely absent. Also, mitochondrial DNA was strongly reduced due to the fact that mtDNA replication requires RNA primers that get synthesized by Rpo41. Next, the effect of reinduction of Rpo41 on mitochondria was tested. Time course experiments showed that mitochondrial translation can partially recover from 48h Rpo41 depletion within a timeframe of 4.5h. Sucrose gradient sedimentation experiments further showed that the mitoribosomal constitution was comparable to wildtype control samples during the time course of 4.5h of reinduction, suggesting that the ribosome assembly is not fundamentally altered in Gal-Rpo41 mitochondria. In addition, the depletion time was found to be critical for recovery of mitochondrial translation and mitochondrial RNA levels. It was observed that after 36h of Rpo41 depletion, the rRNA levels and mitochondrial translation recovered to almost 100%, but only within a time course of 10h. Finally, mitochondria from Gal-Rpo41 cells isolated after different timepoints of reinduction were used to perform complexome profiling and the assembly of mitochondrial protein complexes was investigated. First, the steady state conditions and the assembly process of mitochondrial respiratory chain complexes were monitored. The individual respiratory chain complexes and the super-complexes of complex III, complex IV and complex V were observed. Furthermore, it was seen that they recovered from Rpo41 depletion within 4.5h of reinduction. Complexome profiles of the mitoribosomal small and large subunit discovered subcomplexes of mitoribosomal proteins that were assumed to form prior to their incorporation into assembly intermediates. The complexome profiles after reinduction indeed showed the formation of these subcomplexes before formation of the fully assembled subunit. In the mitochondrial LSU one subcomplex builds the membrane facing protuberance and a second subcomplex forms the central protuberance. In contrast to the preassembled subcomplexes, proteins that were involved in early assembly steps were exclusively found in the fully assembled subunit. Proteins that assemble at the periphery of the mitoribosome during intermediate and late assembly steps where found in soluble form suggesting a pool of unassembled proteins that supply assembly intermediates with proteins. Taken together, the findings of this thesis suggest a so far unknow building-block model for mitoribosome assembly in which characteristic structures of the yeast mitochondrial ribosome form preassembled subcomplexes prior to their incorporation into the mitoribosome.

For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of quantification concatamers (QconCATs), which are synthetic proteins consisting of proteotypic peptides derived from the target proteins to be quantified. The QconCAT protein is labeled with a heavy isotope upon expression in E. coli and known amounts of the purified protein are spiked into a whole cell protein extract. Upon tryptic digestion, labeled and unlabeled peptides are released from the QconCAT and the native proteins, respectively, and both are quantified by LC-MS/MS. The labeled Q-peptides then serve as standards for determining the absolute quantity of the native peptides/proteins. Here we have applied the QconCAT approach to Chlamydomonas reinhardtii for the absolute quantification of the major proteins and protein complexes driving photosynthetic light reactions in the thylakoid membranes and carbon fixation in the pyrenoid. We found that with 25.2 attomol/cell the Rubisco large subunit makes up 6.6% of all proteins in a Chlamydomonas cell and with this exceeds the amount of the small subunit by a factor of 1.56. EPYC1, which links Rubisco to form the pyrenoid, is eight times less abundant than RBCS, and Rubisco activase is 32-times less abundant than RBCS. With 5.2 attomol/cell, photosystem II is the most abundant complex involved in the photosynthetic light reactions, followed by plastocyanin, photosystem I and the cytochrome b6/f complex, which range between 2.9 and 3.5 attomol/cell. The least abundant complex is the ATP synthase with 2 attomol/cell. While applying the QconCAT approach, we have been able to identify many potential pitfalls associated with this technique. We analyze and discuss these pitfalls in detail and provide an optimized workflow for future applications of this technique.

Biological soil crusts (biocrusts) are a common element of the Queensland (Australia) dry savannah ecosystem and are composed of cyanobacteria, algae, lichens, bryophytes, fungi and heterotrophic bacteria. Here we report how the CO2 gas exchange of the cyanobacteria-dominated biocrust type from Boodjamulla National Park in the north Queensland Gulf Savannah responds to the pronounced climatic seasonality and on their quality as a carbon sink using a semi-automatic cuvette system. The dominant cyanobacteria are the filamentous species Symplocastrum purpurascens together with Scytonema sp. Metabolic activity was recorded between 1 July 2010 and 30 June 2011, during which CO2 exchange was only evident from November 2010 until mid-April 2011, representative of 23.6 % of the 1-year recording period. In November at the onset of the wet season, the first month (November) and the last month (April) of activity had pronounced respiratory loss of CO2. The metabolic active period accounted for 25 % of the wet season and of that period 48.6 % was net photosynthesis (NP) and 51.4 % dark respiration (DR). During the time of NP, net photosynthetic uptake of CO2 during daylight hours was reduced by 32.6 % due to water supersaturation. In total, the biocrust fixed 229.09 mmol CO2 m−2 yr−1, corresponding to an annual carbon gain of 2.75 g m−2 yr−1. Due to malfunction of the automatic cuvette system, data from September and October 2010 together with some days in November and December 2010 could not be analysed for NP and DR. Based on climatic and gas exchange data from November 2010, an estimated loss of 88 mmol CO2 m−2 was found for the 2 months, resulting in corrected annual rates of 143.1 mmol CO2 m−2 yr−1, equivalent to a carbon gain of 1.7 g m−2 yr−1. The bulk of the net photosynthetic activity occurred above a relative humidity of 42 %, indicating a suitable climatic combination of temperature, water availability and light intensity well above 200 µmol photons m−2 s−1 photosynthetic active radiation. The Boodjamulla biocrust exhibited high seasonal variability in CO2 gas exchange pattern, clearly divided into metabolically inactive winter months and active summer months. The metabolic active period commences with a period (of up to 3 months) of carbon loss, likely due to reestablishment of the crust structure and restoration of NP prior to about a 4-month period of net carbon gain. In the Gulf Savannah biocrust system, seasonality over the year investigated showed that only a minority of the year is actually suitable for biocrust growth and thus has a small window for potential contribution to soil organic matter.

Grape powdery mildew, Erysiphe necator, is one of the most significant plant pathogens, which affects grape growing regions world-wide. Because of its short generation time and the production of large amounts of conidia throughout the season, E. necator is classified as a moderate to high risk pathogen with respect to the development of fungicide resistance. The number of fungicidal mode of actions available to control powdery mildew is limited and for some of them resistances are already known. Aryl-phenyl-ketones (APKs), represented by metrafenone and pyriofenone, and succinate-dehydrogenase inhibitors (SDHIs), composed of numerous active ingredients, are two important fungicide classes used for the control of E. necator. Over the period 2014 to 2016, the emergence and development of metrafenone and SDHI resistant E. necator isolates in Europe was followed and evaluated. The distribution of resistant isolates was thereby strongly dependent on the European region. Whereas the north-western part is still predominantly sensitive, samples from east European countries showed higher resistance frequencies. Classical sensitivity tests with obligate biotrophs can be challenging regarding sampling, transport and especially the maintenance of the living strains. Whenever possible, molecular genetic methods are preferred for a more efficient monitoring. Such methods require the knowledge of the resistance mechanisms. The exact molecular target and the resistance mechanism of metrafenone is still unknown. Whole genome sequencing of metrafenone sensitive and resistant wheat powdery mildew isolates, as well as adapted laboratory mutants of Aspergillus nidulans, where performed with the aim to identify proteins potentially linked to the mode of action or which contribute to metrafenone resistance. Based on comparative SNP analysis, four proteins potentially associated with metrafenone resistance were identified, but validation studies could not confirm their role in metrafenone resistance. In contrast to APKs, the mode of action of SDHIs is well understood. Sequencing of the sdh-genes of less sensitive E. necator isolates identified four different target-site mutations, the B-H242R, B-I244V, C-G169D and C-G169S, in sdhB and sdhC, respectively. Based on this information it was possible to develop molecular genetic monitoring methods for the mutations B-H242R and C-G169D. In 2016, the B-H242R was thereby identified as by far the most frequent mutation. Depending on the analysed SDH compound and the sdh-genotype, different sensitivities were observed and revealed a complex cross-resistance pattern. Growth competition assays without selection pressure, with mixtures of sensitive and resistant E. necator isolates, were performed to determine potential fitness costs associated with fungicide resistance. With the experimental setups used, a clear fitness disadvantage associated with metrafenone resistance was not identified, although a strong variability of fitness was observed among the tested resistant E. necator isolates. For isolates with a reduced sensitivity towards SDHIs, associated fitness costs were dependent on the sdh-genotype analysed. Competition tests with the B-H242R genotypes gave evidence that there are no fitness costs associated with this mutation. In contrast, the C-G169D genotypes were less competitive, indicating a restricted fitness compared to the tested sensitive partners. Competition assays of field isolates, which exhibited several resistances towards different fungicide classes, indicated that there are no fitness costs associated with a multiple resistant phenotype in E. necator. Overall, these results clearly indicate the importance to analyse a representative number of isolates with sensitive and resistant phenotypes.

Cells depend on the continuous renewal of their proteome composition during the cell cycle and in order to replace aberrant proteins or to react to changing environmental conditions. In higher eukaryotes, protein synthesis is achieved by up to five million ribosomes per cell. With the fast kinetics of translation, the large number of newly made proteins generates a substantial burden for protein homeostasis and requires a highly orchestrated cascade of factors promoting folding, sorting and final maturation. Several of the involved factors directly bind to translating ribosomes for the early processing of emerging nascent polypeptides and the translocation of ribosome nascent chain complexes to target membranes. In plant cells, protein synthesis also occurs in chloroplasts serving the expression of a relatively small set of 60–100 protein-coding genes. However, most of these proteins, together with nucleus-derived subunits, form central complexes majorly involved in the essential processes of photosynthetic light reaction, carbon fixation, metabolism and gene expression. Biogenesis of these heterogenic complexes adds an additional level of complexity for protein biogenesis. In this review, we summarize the current knowledge about co-translationally binding factors in chloroplasts and discuss their role in protein folding and ribosome translocation to thylakoid membranes.