Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

Deep Proteomics reveals incorporation of unedited proteins into mitochondrial protein complexes in Arabidopsis.

The mitochondrial proteome consists of numerous types of proteins which either are encoded and synthesized in the mitochondria, or encoded in the cell nucleus, synthesized in the cytoplasm and imported into the mitochondria. Their synthesis in the mitochondria, but not in the nucleus, relies on the editing of the primary transcripts of their genes at defined sites. Here, we present an in-depth investigation of the mitochondrial proteome of Arabidopsis (Arabidopsis thaliana) and a public online platform for the exploration of the data. For the analysis of our shotgun proteomic data, an Arabidopsis sequence database was created comprising all available protein sequences from the TAIR10 and Araport11 databases, supplemented with sequences of proteins translated from edited and non-edited transcripts of mitochondria. Amino acid sequences derived from partially edited transcripts were also added to analyze proteins encoded by the mitochondrial genome. Proteins were digested in parallel with six different endoproteases to obtain maximum proteome coverage. The resulting peptide fractions were finally analyzed using liquid chromatography (LC) coupled to ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS). We generated a 'deep mitochondrial proteome' of 4,692 proteins. 1,339 proteins assigned to mitochondria by the SUBA5 database (https://suba.live) accounted for >80% of the total protein mass of our fractions. The coverage of proteins by identified peptides was particularly high compared to single-protease digests, allowing the exploration of differential splicing and RNA editing events at the protein level. We show that proteins translated from non-edited transcripts can be incorporated into native mitoribosomes and the ATP synthase complex. We present a portal for the use of our data, based on 'proteomaps' with directly linked protein data. The portal is available at www.proteomeexplorer.de.

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics.

Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses.

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

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.

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.

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.

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).

Protein interaction patterns in Arabidopsis thaliana leaf mitochondria change in dependence to light.

Mitochondrial biology is underpinned by the presence and activity of large protein assemblies participating in the organelle-located steps of respiration, TCA-cycle, glycine oxidation, and oxidative phosphorylation. While the enzymatic roles of these complexes are undisputed, little is known about the interactions of the subunits beyond their presence in these protein complexes and their functions in regulating mitochondrial metabolism. By applying one of the most important regulatory cues for plant metabolism, the presence or absence of light, we here assess changes in the composition and molecular mass of protein assemblies involved in NADH-production in the mitochondrial matrix and in oxidative phosphorylation by employing a differential complexome profiling strategy. Covering a mass up to 25 MDa, we demonstrate dynamic associations of matrix enzymes and of components involved in oxidative phosphorylation. The data presented here form the basis for future studies aiming to advance our understanding of the role of protein:protein interactions in regulating plant mitochondrial functions.

Absence of Complex I Implicates Rearrangement of the Respiratory Chain in European Mistletoe.

The mitochondrial oxidative phosphorylation (OXPHOS) system, which is based on the presence of five protein complexes, is in the very center of cellular ATP production. Complexes I to IV are components of the respiratory electron transport chain that drives proton translocation across the inner mitochondrial membrane. The resulting proton gradient is used by complex V (the ATP synthase complex) for the phosphorylation of ADP. Occurrence of complexes I to V is highly conserved in eukaryotes, with exceptions being restricted to unicellular parasites that take up energy-rich compounds from their hosts. Here we present biochemical evidence that the European mistletoe (Viscum album), an obligate semi-parasite living on branches of trees, has a highly unusual OXPHOS system. V. album mitochondria completely lack complex I and have greatly reduced amounts of complexes II and V. At the same time, the complexes III and IV form remarkably stable respiratory supercomplexes. Furthermore, complexome profiling revealed the presence of 150 kDa complexes that include type II NAD(P)H dehydrogenases and an alternative oxidase. Although the absence of complex I genes in mitochondrial genomes of mistletoe species has recently been reported, this is the first biochemical proof that these genes have not been transferred to the nuclear genome and that this respiratory complex indeed is not assembled. As a consequence, the whole respiratory chain is remodeled. Our results demonstrate that, in the context of parasitism, multicellular life can cope with lack of one of the OXPHOS complexes and give new insights into the life strategy of mistletoe species.