From single proteins to supercomplexes : a proteomic view on plant mitochondria

The primary function of plant mitochondria is respiration, which is why they are often referred to as “powerhouses of the cell”. Besides their central role in energy metabolism, plant mitochondria are also involved in the photorespiratory C2 cycle and in the provision of carbon skeletons to support efficient nitrogen assimilation. All these functions are catalyzed by mitochondrial proteins. Their composition, abundance and interactions in plant mitochondria are the subject of this thesis. In yeast, Trypanosomes, and several mammalian cell types, mitochondria are organized as extensive mitochondrial networks, resulting in a situation where a cell only hosts few but large mitochondria. In plants, hundreds of small mitochondria are only connected by fusion and fission over time but not physically. Hence, the organelles form individual, functional units. Paradoxically, their biochemical and physiological characterization focuses on large organelle populations and thereby disregards the properties of the individual mitochondrion. This partially is based on the fact that cell biological approaches capturing structural features of plant mitochondria often are of limited value for understanding their physiological properties. Chapter 2.1 of this thesis models the protein content of a single mitochondrion by combining proteomics with classical cell biology. Besides other insights into the function of a single plant mitochondrion, it could be shown that proteins involved in ATP synthesis and transport make up nearly half of the plant mitochondrial proteome. The five protein complexes of the OXPHOS system contribute most to this segment of the mitochondrial proteome, underlining the overall importance of mitochondrial ATP synthesis for the entire plant cell. Despite the central function of OXHPOS components in plants, certain unicellular parasites and yeasts apparently do not need a complete OXPHOS system. Intriguingly, it recently has been reported that the mitochondrial genome of the multicellular parasitic flowering plant Viscum album (European mistletoe) is reduced and lacks the genes encoding the mitochondrially encoded subunits of complex I. This implies that the corresponding genes either have been lost or, alternatively, were transferred to the nuclear genome. The consequences for the mitochondrial respiratory chain were so far unknown. Chapter 2.2 presents data suggesting that V. album indeed lacks mitochondrial complex I. The absence of complex I is accompanied by a rearrangement of the respiratory chain including (i) stable supercomplexes composed of complexes III2 and IV, and (ii) the occurrence of numerous alternative oxidoreductases. Mitochondria of V. album also possess less cristae than mitochondria from non-parasitic plants, which can be explained by low amounts of ATP synthase dimers. The mitochondrial proteome consists of proteins encoded in the nucleus or in the rudimentary mitochondrial genome. The few proteins encoded on the mitochondrial genome are translated by mitochondrial ribosomes. While structure and composition of these mitoribosomes are well established in yeast and mammals, the current knowledge of plant mitoribosomes is negligible. Isolation of plant mitoribosomes is difficult due to their sedimentation coefficient, which is very close to that of cytosolic ribosomes, their interaction with the inner mitochondrial membrane, and the attachment of cytosolic ribosomes to the mitochondrial surface. As part of this dissertation, plant mitoribosomes were analyzed via a novel complexome profiling strategy (chapter 2.3). This revealed an unconventional molecular mass of the small ribosomal subunit of plants. In addition, several pentatricopeptide repeat (PPR) proteins were discovered to form part of both, the large and the small mitoribosomal subunit

Complexome profiling reveals association of PPR proteins with ribosomes in the mitochondria of plants

Mitochondrial transcripts are subject to a wealth of processing mechanisms including cis- and trans-splicing events, as well as base modifications (RNA editing). HUndreds of proteins are required for these processes in plant mitochondria, many of which belong to the pentatricopeptide repeat (PPR) protein superfamily. The structure, localization, and function of these proteins is only poorly Understood. Here we present evidence that several PPR proteins are boUnd to mitoribosomes in plants. A novel complexome profiling strategy in combination with chemical crosslinking has been employed to systematically define the protein constituents of the large and the small ribosomal subunits in the mitochondria of plants. We identified more than 80 ribosomal proteins, which include several PPR proteins and other non-conventional ribosomal proteins. These findings reveal a potential coupling of transcriptional and translational events in the mitochondria of plants. Furthermore, the data indicate an extremely high molecular mass of the “small” subunit, even exceeding that of the “large” subunit

Cryo-EM structure of the respiratory I + III2 supercomplex from Arabidopsis thaliana at 2 Å resolution

Protein complexes of the mitochondrial respiratory chain assemble into respiratory supercomplexes. Here we present the high-resolution electron cryo-microscopy structure of the Arabidopsis respiratory supercomplex consisting of complex I and a complex III dimer, with a total of 68 protein subunits and numerous bound cofactors. A complex I-ferredoxin, subunit B14.7 and P9, a newly defined subunit of plant complex I, mediate supercomplex formation. The component complexes stabilize one another, enabling new detailed insights into their structure. We describe (1) an interrupted aqueous passage for proton translocation in the membrane arm of complex I; (2) a new coenzyme A within the carbonic anhydrase module of plant complex I defining a second catalytic centre; and (3) the water structure at the proton exit pathway of complex III2 with a co-purified ubiquinone in the QO site. We propose that the main role of the plant supercomplex is to stabilize its components in the membrane

Getting Ready for Large-Scale Proteomics in Crop Plants

Plants are an indispensable cornerstone of sustainable global food supply. While immense progress has been made in decoding the genomes of crops in recent decades, the composition of their proteomes, the entirety of all expressed proteins of a species, is virtually unknown. In contrast to the model plant Arabidopsis thaliana, proteomic analyses of crop plants have often been hindered by the presence of extreme concentrations of secondary metabolites such as pigments, phenolic compounds, lipids, carbohydrates or terpenes. As a consequence, crop proteomic experiments have, thus far, required individually optimized protein extraction protocols to obtain samples of acceptable quality for downstream analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). In this article, we present a universal protein extraction protocol originally developed for gel-based experiments and combined it with an automated single-pot solid-phase-enhanced sample preparation (SP3) protocol on a liquid handling robot to prepare high-quality samples for proteomic analysis of crop plants. We also report an automated offline peptide separation protocol and optimized micro-LC-MS/MS conditions that enables the identification and quantification of ~10,000 proteins from plant tissue within 6 h of instrument time. We illustrate the utility of the workflow by analyzing the proteomes of mature tomato fruits to an unprecedented depth. The data demonstrate the robustness of the approach which we propose for use in upcoming large-scale projects that aim to map crop tissue proteomes

The Viscum album Gene Space database

The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on ‘Benchmarking Universal Single-Copy Orthologs’ (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA)

The Viscum album Gene Space database

The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on ‘Benchmarking Universal Single-Copy Orthologs’ (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA)

Unravelling post-transcriptional PrmC-dependent regulatory mechanisms in Pseudomonas aeruginosa.

Transcriptional regulation has a central role in cellular adaptation processes and is well investigated. In contrast, the importance of the post-transcriptional regulation on these processes is less well defined. The technological advancements have been critical to precisely quantify protein and mRNA level changes and hold promise to provide more insights into how post-transcriptional regulation determines phenotypes. In Pseudomonas aeruginosa the methyltransferase PrmC methylates peptide chain release factors to facilitate translation termination. Loss of PrmC activity abolishes anaerobic growth and leads to reduced production of quorum sensing-associated virulence factors. Here, by applying SILAC technology in combination with mRNA-sequencing, they provide evidence that the P. aeruginosa phenotype can be attributed to a change in protein to mRNA ratios of selected protein groups. The UAG-dependent translation termination was more dependent on PrmC activity than the UAA- and UGA-dependent translation termination. Additionally, a bias toward UAG stop codons in global transcriptional regulators was found. The finding that this bias in stop codon usage determines the P. aeruginosa phenotype is unexpected and adds complexity to regulatory circuits. Via modulation of PrmC activity the bacterial cell can cross-regulate targets independently of transcriptional signals, a process with an underestimated impact on the bacterial phenotype

The role of amino acid metabolism during abiotic stress release

Plant responses to abiotic stress include various modifications in amino acid metabolism. By using a hydroponic culture system, we systematically investigate modification in amino acid profiles and the proteome of Arabidopsis thaliana leaves during initial recovery from low water potential or high salinity. Both treatments elicited oxidative stress leading to a biphasic stress response during recovery. Degradation of highly abundant proteins such as subunits of photosystems and ribosomes contributed to an accumulation of free amino acids. Catabolic pathways for several low abundant amino acids were induced indicating their usage as an alternative respiratory substrate to compensate for the decreased photosynthesis. Our results demonstrate that rapid detoxification of potentially detrimental amino acids such as Lys is a priority during the initial stress recovery period. The content of Pro, which acts as a compatible osmolyte during stress, was adjusted by balancing its synthesis and catabolism both of which were induced both during and after stress treatments. The production of amino acid derived secondary metabolites was up‐regulated specifically during the recovery period, and our dataset also indicates increased synthesis rates of the precursor amino acids. Overall, our results support a tight relationship between amino acid metabolism and stress responses