Proteomic Analysis of the Proplastid Envelope Membrane Provides Novel Insights into Small Molecule and Protein Transport across Proplastid Membranes

Bräutigam A, Weber APM. Proteomic Analysis of the Proplastid Envelope Membrane Provides Novel Insights into Small Molecule and Protein Transport across Proplastid Membranes. Molecular Plant. 2009;2(6):1247-1261.Proplastids are undifferentiated plastids of meristematic tissues that synthesize amino acids for protein synthesis, fatty acids for membrane lipid production, and purines and pyrimidines for DNA and RNA synthesis. Unlike chloroplasts, proplastids depend on supply, with reducing power, energy, and precursor metabolites from the remainder of the cell. Comparing proplastid and chloroplast envelope proteomes and the corresponding transcriptomes of leaves and shoot apex revealed a clearly distinct composition of the proplastid envelope. It is geared towards import of metabolic precursors and export of product metabolites for the rapidly dividing cell. The analysis also suggested a new role for the triosephosphate translocator in meristematic tissues, identified the route of organic nitrogen import into proplastids, and detected an adenine nucleotide exporter. The protein import complex contains the import receptors Toc120 and Toc132 and lacks the redox sensing complex subunits of Tic32, Tic55, and Tic62, which mirrors the expression patterns of the corresponding genes in leaves and the shoot apex. We further show that the protein composition of the internal membrane system is similar to etioplasts, as it is dominated by the ATP synthase complex and thus remarkably differs from that of chloroplast thylakoids

The role of membrane transport in metabolic engineering of plant primary metabolism

Weber APM, Bräutigam A. The role of membrane transport in metabolic engineering of plant primary metabolism. Current Opinion in Biotechnology. 2013;24(2):256-262.Plant cells are highly compartmentalized and so is their metabolism. Most metabolic pathways are distributed across several cellular compartments, which requires the activities of membrane transporters to catalyze the flux of precursors, intermediates, and end products between compartments. Metabolites such as sucrose and amino acids have to be transported between cells and tissues to supply, for example, metabolism in developing seeds or fruits with precursors and energy. Thus, rational engineering of plant primary metabolism requires a detailed and molecular understanding of the membrane transporters. This knowledge however still lags behind that of soluble enzymes. Recent advances include the molecular identification of pyruvate transporters at the chloroplast and mitochondrial membranes and of a new class of transporters called SWEET that are involved in the release of sugars to the apoplast

High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract

Bräutigam A, Gagneul D, Weber APM. High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract. Analytical Biochemistry. 2007;362(1):151-153

Comparative Proteomics of Chloroplast Envelopes from C-3 and C-4 Plants Reveals Specific Adaptations of the Plastid Envelope to C-4 Photosynthesis and Candidate Proteins Required for Maintaining C-4 Metabolite Fluxes (vol 148, pg 568, 2008)

Bräutigam A, Hoffmann-Benning S, Weber APM. Comparative Proteomics of Chloroplast Envelopes from C-3 and C-4 Plants Reveals Specific Adaptations of the Plastid Envelope to C-4 Photosynthesis and Candidate Proteins Required for Maintaining C-4 Metabolite Fluxes (vol 148, pg 568, 2008). Plant Physiology. 2008;148(3):1734

What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research

Bräutigam A, Gowik U. What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research. Plant Biology. 2010;12(6):831-841.Next generation sequencing (NGS) technologies have opened fascinating opportunities for the analysis of plants with and without a sequenced genome on a genomic scale. During the last few years, NGS methods have become widely available and cost effective. They can be applied to a wide variety of biological questions, from the sequencing of complete eukaryotic genomes and transcriptomes, to the genome-scale analysis of DNA-protein interactions. In this review, we focus on the use of NGS for plant transcriptomics, including gene discovery, transcript quantification and marker discovery for non-model plants, as well as transcript annotation and quantification, small RNA discovery and antisense transcription analysis for model plants. We discuss the experimental design for analysis of plants with and without a sequenced genome, including considerations on sampling, RNA preparation, sequencing platforms and bioinformatics tools for data analysis. NGS technologies offer exciting new opportunities for the plant sciences, especially for work on plants without a sequenced genome, since large sequence resources can be generated at moderate cost

The Plastid Outer Envelope – A Highly Dynamic Interface between Plastid and Cytoplasm

Plastids are the defining organelles of all photosynthetic eukaryotes. They are the site of photosynthesis and of a large number of other essential metabolic pathways, such as fatty acid and amino acid biosyntheses, sulfur and nitrogen assimilation, and aromatic and terpenoid compound production, to mention only a few examples. The metabolism of plastids is heavily intertwined and connected with that of the surrounding cytosol, thus causing massive traffic of metabolic precursors, intermediates, and products. Two layers of biological membranes that are called the inner (IE) and the outer (OE) plastid envelope membranes bound the plastids of Archaeplastida. While the IE is generally accepted as the osmo-regulatory barrier between cytosol and stroma, the OE was considered to represent an unspecific molecular sieve, permeable for molecules of up to 10 kDa. However, after the discovery of small substrate specific pores in the OE, this view has come under scrutiny. In addition to controlling metabolic fluxes between plastid and cytosol, the OE is also crucial for protein import into the chloroplast. It contains the receptors and translocation channel of the TOC complex that is required for the canonical post-translational import of nuclear-encoded, plastid-targeted proteins. Further, the OE is a metabolically active compartment of the chloroplast, being involved in, e.g., fatty acid metabolism and membrane lipid production. Also, recent findings hint on the OE as a defense platform against several biotic and abiotic stress conditions, such as cold acclimation, freezing tolerance, and phosphate deprivation. Moreover, dynamic non-covalent interactions between the OE and the endomembrane system are thought to play important roles in lipid and non-canonical protein trafficking between plastid and endoplasmic reticulum. While proteomics and bioinformatics has provided us with comprehensive but still incomplete information on proteins localized in the plastid IE, the stroma, and the thylakoids, our knowledge of the protein composition of the plastid OE is far from complete. In this article, we report on the recent progress in discovering novel OE proteins to draw a conclusive picture of the OE. A “parts list” of the plastid OE will be presented, using data generated by proteomics of plastids isolated from various plant sources

Understanding metabolite transport and metabolism in C-4 plants through RNA-seq

Schlueter U, Denton AK, Bräutigam A. Understanding metabolite transport and metabolism in C-4 plants through RNA-seq. Current Opinion in Plant Biology. 2016;31:83-90.RNA-seq, the measurement of steady-state RNA levels by next generation sequencing, has enabled quantitative transcriptome analyses of complex traits in many species without requiring the parallel sequencing of their genomes. The complex trait of C-4 photosynthesis, which increases photosynthetic efficiency via a biochemical pump that concentrates CO2 around RubisCO, has evolved convergently multiple times. Due to these interesting properties, C-4 photosynthesis has been analyzed in a series of comparative RNA-seq projects. These projects compared both species with and without the C-4 trait and different tissues or organs within a C-4 plant. The RNA-seq studies were evaluated by comparing to earlier single gene studies. The studies confirmed the marked changes expected for C-4 signature genes, but also revealed numerous new players in C-4 metabolism showing that the C-4 cycle is more complex than previously thought, and suggesting modes of integration into the underlying C-3 metabolism

Towards an integrative model of C4 photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C-4 species

C4 photosynthesis affords higher photosynthetic carbon conversion efficiency than C3 photosynthesis and it therefore represents an attractive target for engineering efforts aiming to improve crop productivity. To this end, blueprints are required that reflect C4 metabolism as closely as possible. Such blueprints have been derived from comparative transcriptome analyses of C3 species with related C4 species belonging to the NAD-malic enzyme (NAD-ME) and NADP-ME subgroups of C4 photosynthesis. However, a comparison between C3 and the phosphoenolpyruvate carboxykinase (PEP-CK) subtype of C4 photosynthesis is still missing. An integrative analysis of all three C4 subtypes has also not been possible to date, since no comparison has been available for closely related C3 and PEP-CK C4 species. To generate the data, the guinea grass Megathyrsus maximus, which represents a PEP-CK species, was analysed in comparison with a closely related C3 sister species, Dichanthelium clandestinum, and with publicly available sets of RNA-Seq data from C4 species belonging to the NAD-ME and NADP-ME subgroups. The data indicate that the core C4 cycle of the PEP-CK grass M. maximus is quite similar to that of NAD-ME species with only a few exceptions, such as the subcellular location of transfer acid production and the degree and pattern of up-regulation of genes encoding C4 enzymes. One additional mitochondrial transporter protein was associated with the core cycle. The broad comparison identified sucrose and starch synthesis, as well as the prevention of leakage of C4 cycle intermediates to other metabolic pathways, as critical components of C4 metabolism. Estimation of intercellular transport fluxes indicated that flux between cells is increased by at least two orders of magnitude in C4 species compared with C3 species. In contrast to NAD-ME and NADP-ME species, the transcription of photosynthetic electron transfer proteins was unchanged in PEP-CK. In summary, the PEP-CK blueprint of M. maximus appears to be simpler than those of NAD-ME and NADP-ME plants

RNA-seq assembly - are we there yet?

Schliesky S, Gowik U, Weber APM, Bräutigam A. RNA-seq assembly - are we there yet? Frontiers in Plant Science. 2012;3: 220.Transcriptomic sequence resources represent invaluable assets for research, in particular for non-model species without a sequenced genome. To date, the Next Generation Sequencing technologies 454/Roche and Illumina have been used to generate transcriptome sequence databases by mRNA-Seq for more than fifty different plant species. While some of the databases were successfully used for downstream applications, such as proteomics, the assembly parameters indicate that the assemblies do not yet accurately reflect the actual plant transcriptomes. Two different assembly strategies have been used, overlap consensus based assemblers for long reads and Eulerian path/de Bruijn graph assembler for short reads. In this review, we discuss the challenges and solutions to the transcriptome assembly problem. A list of quality control parameters and the necessary scripts to produce them are provided