Towards understanding the evolution and functional diversification of DNA-containing plant organelles:[version 1; referees: 3 approved]

Plastids and mitochondria derive from prokaryotic symbionts that lost most of their genes after the establishment of endosymbiosis. In consequence, relatively few of the thousands of different proteins in these organelles are actually encoded there. Most are now specified by nuclear genes. The most direct way to reconstruct the evolutionary history of plastids and mitochondria is to sequence and analyze their relatively small genomes. However, understanding the functional diversification of these organelles requires the identification of their complete protein repertoires – which is the ultimate goal of organellar proteomics. In the meantime, judicious combination of proteomics-based data with analyses of nuclear genes that include interspecies comparisons and/or predictions of subcellular location is the method of choice. Such genome-wide approaches can now make use of the entire sequences of plant nuclear genomes that have emerged since 2000. Here I review the results of these attempts to reconstruct the evolution and functions of plant DNA-containing organelles, focusing in particular on data from nuclear genomes. In addition, I discuss proteomic approaches to the direct identification of organellar proteins and briefly refer to ongoing research on non-coding nuclear DNAs of organellar origin (specifically, nuclear mitochondrial DNA and nuclear plastid DNA)

Chloroplast Proteins without Cleavable Transit Peptides

Most chloroplast proteins (cp proteins) are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence (cTP), and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs (non-canonical cp proteins) have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as approximately 30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein (RFP)-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein (PDII), and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others ('potential cp proteins') were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies

An interdisciplinary approach to data management

Many scientific issues involve interdisciplinary approaches that demand scientists with diverse skills and research fields. For the design and fabrication of new materials, this is especially true since new materials with macroscopically observable properties must be proposed based on changes at the molecular level. Research projects of this kind pose particular challenges for efficient execution and documentation, as research data management (RDM) tools usually fit very well to a specific research area, but cannot provide solutions for interdisciplinary topics. In order to guarantee consistent research and its documentation across disciplines, different tools, which may be used in several groups, must be used cooperatively. In the context of the Science Data Center MoMaF, among other things, strategies are being developed to enable research data management across scales. The RDM tools used for this are Chemotion and Kadi4Mat. The systems cover research at the molecular level (chemotion ELN) as well as simulation activities on the meso- and macroscopic scale (Kadi4Mat), and will be extended within the Science Data Center to enable cooperative use of the systems for work across scales. A first use case shows how Chemotion ELN can be used to document necessary parameters at the molecular level, in order to then be able to manage simulations of phase separation processes on their basis in a further step with the help of Kadi4Mat. For this purpose, the procedure and documentation method of already completed projects were first analysed in order to be able to propose a concept for future processes. Chemotion ELN is used in the presented procedure to document molecular descriptions, the performance of polymerization reactions and their outcome, as well as the properties obtained experimentally and from the literature. Kadi4Mat manages and transfers the parameters from the molecular description as input for mesoscopic simulations that describe the phase separation process in a time-dependent manner. Finally, by applying analysis tools on the time-dependent data via Kadi4Mat, macroscopic properties can be derived across scales as a function of the molecular composition

A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed