Wolfram Weckwerth

Proteome Analysis of Arabidopsis Leaf Peroxisomes Reveals Novel Targeting Peptides, Metabolic Pathways, and Defense Mechanisms

We have established a protocol for the isolation of highly purified peroxisomes from mature Arabidopsis thaliana leaves and analyzed the proteome by complementary gel-based and gel-free approaches. Seventy-eight nonredundant proteins were identified, of which 42 novel proteins had previously not been associated with plant peroxisomes. Seventeen novel proteins carried predicted peroxisomal targeting signals (PTS) type 1 or type 2; 11 proteins contained PTS-related peptides. Peroxisome targeting was supported for many novel proteins by in silico analyses and confirmed for 11 representative full-length fusion proteins by fluorescence microscopy. The targeting function of predicted and unpredicted signals was investigated and SSL>, SSI>, and ASL> were established as novel functional PTS1 peptides. In contrast with the generally accepted confinement of PTS2 peptides to the N-terminal domain, the bifunctional transthyretin-like protein was demonstrated to carry internally a functional PTS2. The novel enzymes include numerous enoyl-CoA hydratases, short-chain dehydrogenases, and several enzymes involved in NADP and glutathione metabolism. Seven proteins, including β-glucosidases and myrosinases, support the currently emerging evidence for an important role of leaf peroxisomes in defense against pathogens and herbivores. The data provide new insights into the biology of plant peroxisomes and improve the prediction accuracy of peroxisome-targeted proteins from genome sequences.

PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor

The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27 782 predicted phosphoserine sites distributed across 17 035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link http://phosphat.mpimp-golm.mpg.de

Metabolomics: from pattern recognition to biological interpretation.

Metabolomics is a technology that aims to identify and quantify the metabolome -- the dynamic set of all small molecules present in an organism or a biological sample. In this sense, the technique is distinct from metabolic profiling, which looks for target compounds and their biochemical transformation. The combination of both approaches is an emerging technique for the characterization of biological samples and for drug treatment. Metabolomics has proven to be very rapid and superior to any other post-genomics technology for pattern-recognition analyses of biological samples. Changing steady state concentrations and fluctuations of metabolites that occur within milliseconds are a result of biochemical processes such as signalling cascades: metabolomic techniques are instrumental in measuring these changes rapidly and sensitively. Metabolite data can be complemented by protein, transcript and external (environmental) data, thereby leading to the identification of multiple physiological biomarkers embedded in correlative molecular networks that are not approachable with targeted studies.

1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana

Summary Arabidopsis thaliana serves as a model organism for the study of fundamental physiological, cellular, and molecular processes. It has also greatly advanced our understanding of intraspecific genome variation. We present a detailed map of variation in 1,135 high-quality re-sequenced natural inbred lines representing the native Eurasian and North African range and recently colonized North America. We identify relict populations that continue to inhabit ancestral habitats, primarily in the Iberian Peninsula. They have mixed with a lineage that has spread to northern latitudes from an unknown glacial refugium and is now found in a much broader spectrum of habitats. Insights into the history of the species and the fine-scale distribution of genetic diversity provide the basis for full exploitation of A. thaliana natural variation through integration of genomes and epigenomes with molecular and non-molecular phenotypes.

Metabolite profiling in plant biology: platforms and destinations

Optimal use of genome sequences and gene-expression resources requires powerful phenotyping platforms, including those for systematic analysis of metabolite composition. The most used technologies for metabolite profiling, including mass spectral, nuclear magnetic resonance and enzyme-based approaches, have various advantages and disadvantages, and problems can arise with reliability and the interpretation of the huge datasets produced. These techniques will be useful for answering important biological questions in the future.

Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen

Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methyl-coenzyme M reductase (mcr) transcripts decreased concomitantly with mRNAs of enzymes involved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated.

A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis.

Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis.

Medicago truncatula Root Nodule Proteome Analysis Reveals Differential Plant and Bacteroid Responses to Drought Stress

Drought is one of the environmental factors most affecting crop production. Under drought, symbiotic nitrogen fixation is one of the physiological processes to first show stress responses in nodulated legumes. This inhibition process involves a number of factors whose interactions are not yet understood. This work aims to further understand changes occurring in nodules under drought stress from a proteomic perspective. Drought was imposed on Medicago truncatula ‘Jemalong A17’ plants grown in symbiosis with Sinorhizobium meliloti strain 2011. Changes at the protein level were analyzed using a nongel approach based on liquid chromatography coupled to tandem mass spectrometry. Due to the complexity of nodule tissue, the separation of plant and bacteroid fractions in M. truncatula root nodules was first checked with the aim of minimizing cross contamination between the fractions. Second, the protein plant fraction of M. truncatula nodules was profiled, leading to the identification of 377 plant proteins, the largest description of the plant nodule proteome so far. Third, both symbiotic partners were independently analyzed for quantitative differences at the protein level during drought stress. Multivariate data mining allowed for the classification of proteins sets that were involved in drought stress responses. The isolation of the nodule plant and bacteroid protein fractions enabled the independent analysis of the response of both counterparts, gaining further understanding of how each symbiotic member is distinctly affected at the protein level under a water-deficit situation.

Can we discover novel pathways using metabolomic analysis

Metabolomic analysis aims at the identification and quantitation of all metabolites in a given biological sample. Current data acquisition and network analysis strategies are classified on the basis of pathway elucidation and characteristics of theoretical networks. The development of metabolomic methods and tools is progressing rapidly, but an understanding of the resulting data is limited owing to a fundamental lack of biochemical and physiological knowledge about network organization in plants.

Metabolomics- and proteomics-assisted genome annotation and analysis of the draft metabolic network of Chlamydomonas reinhardtii

We present an integrated analysis of the molecular repertoire of Chlamydomonas reinhardtii under reference conditions. Bioinformatics annotation methods combined with GCxGC/MS-based metabolomics and LC/MS-based shotgun proteomics profiling technologies have been applied to characterize abundant proteins and metabolites, resulting in the detection of 1069 proteins and 159 metabolites. Of the measured proteins, 204 currently do not have EST sequence support; thus a significant portion of the proteomics-detected proteins provide evidence for the validity of in silico gene models. Furthermore, the generated peptide data lend support to the validity of a number of proteins currently in the proposed model stage. By integrating genomic annotation information with experimentally identified metabolites and proteins, we constructed a draft metabolic network for Chlamydomonas. Computational metabolic modeling allowed an identification of missing enzymatic links. Some experimentally detected metabolites are not producible by the currently known and annotated enzyme set, thus suggesting entry points for further targeted gene discovery or biochemical pathway research. All data sets are made available as supplementary material as well as web-accessible databases and within the functional context via the Chlamydomonas-adapted MapMan annotation platform. Information of identified peptides is also available directly via the JGI-Chlamydomonas genomic resource database (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html).