Stefanie Wienkoop

An improved protocol to study the plant cell wall proteome

Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927.

Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules

The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry. Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected. Several ATPases and aquaporins were present, indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM.

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.

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).

d-GLYCERATE 3-KINASE, the Last Unknown Enzyme in the Photorespiratory Cycle in Arabidopsis, Belongs to a Novel Kinase Family

d-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.

Integration of Metabolomic and Proteomic Phenotypes Analysis of Data Covariance Dissects Starch and RFO Metabolism from Low and High Temperature Compensation Response in Arabidopsis Thaliana

Statistical mining and integration of complex molecular data including metabolites, proteins, and transcripts is one of the critical goals of systems biology (Ideker, T., Galitski, T., and Hood, L. (2001) A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2, 343–372). A number of studies have demonstrated the parallel analysis of metabolites and large scale transcript expression. Protein analysis has been ignored in these studies, although a clear correlation between transcript and protein levels is shown only in rare cases, necessitating that actual protein levels have to be determined for protein function analysis. Here, we present an approach to investigate the combined covariance structure of metabolite and protein dynamics in a systemic response to abiotic temperature stress in Arabidopsis thaliana wild-type and a corresponding starch-deficient mutant (phosphoglucomutase-deficient). Independent component analysis revealed phenotype classification resolving genotype-dependent response effects to temperature treatment and genotype-independent general temperature compensation mechanisms. An observation is the stress-induced increase of raffinose-family-oligosaccharide levels in the absence of transitory starch storage/mobilization in temperature-treated phosphoglucomutase plants indicating that sucrose synthesis and storage in these mutant plants is sufficient to bypass the typical starch storage/mobilization pathways under abiotic stress. Eventually, sample pattern recognition and correlation network topology analysis allowed for the detection of specific metabolite-protein co-regulation and assignment of a circadian output regulated RNA-binding protein to these processes. The whole concept of high-dimensional profiling data integration from many replicates, subsequent multivariate statistics for dimensionality reduction, and covariance structure analysis is proposed to be a major strategy for revealing central responses of the biological system under study.

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes.

The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane. The PBM and the space between the PBM and the bacteroid membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts. Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS. A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation from pea root nodules. Proteins in the PBM and PS fractions obtained from the symbiosomes were separated by two‐dimensional gel electrophoresis, and 89 spots were analysed by tandem mass spectrometry. The proteins of 46 spots could be identified by database search. The results showed that PS and even PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate. Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V‐ATPase, BIP, and an integral membrane protein known from COPI‐coated vesicles, were found in the PBM fraction, supporting the role of the endomembrane system in PBM biogenesis.

ProMEX: a mass spectral reference database for proteins and protein phosphorylation sites

BackgroundIn the last decade, techniques were established for the large scale genome-wide analysis of proteins, RNA, and metabolites, and database solutions have been developed to manage the generated data sets. The Golm Metabolome Database for metabolite data (GMD) represents one such effort to make these data broadly available and to interconnect the different molecular levels of a biological system [1]. As data interpretation in the light of already existing data becomes increasingly important, these initiatives are an essential part of current and future systems biology.ResultsA mass spectral library consisting of experimentally derived tryptic peptide product ion spectra was generated based on liquid chromatography coupled to ion trap mass spectrometry (LC-IT-MS). Protein samples derived from Arabidopsis thaliana, Chlamydomonas reinhardii, Medicago truncatula, and Sinorhizobium meliloti were analysed. With currently 4,557 manually validated spectra associated with 4,226 unique peptides from 1,367 proteins, the database serves as a continuously growing reference data set and can be used for protein identification and quantification in uncharacterized biological samples. For peptide identification, several algorithms were implemented based on a recently published study for peptide mass fingerprinting [2] and tested for false positive and negative rates. An algorithm which considers intensity distribution for match correlation scores was found to yield best results. For proof of concept, an LC-IT-MS analysis of a tryptic leaf protein digest was converted to mzData format and searched against the mass spectral library. The utility of the mass spectral library was also tested for the identification of phosphorylated tryptic peptides. We included in vivo phosphorylation sites of Arabidopsis thaliana proteins and the identification performance was found to be improved compared to genome-based search algorithms. Protein identification by ProMEX is linked to other levels of biological organization such as metabolite, pathway, and transcript data. The database is further connected to annotation and classification services via BioMoby.ConclusionThe ProMEX protein/peptide database represents a mass spectral reference library with the capability of matching unknown samples for protein identification. The database allows text searches based on metadata such as experimental information of the samples, mass spectrometric instrument parameters or unique protein identifier like AGI codes. ProMEX integrates proteomics data with other levels of molecular organization including metabolite, pathway, and transcript information and may thus become a useful resource for plant systems biology studies. The ProMEX mass spectral library is available at http://promex.mpimp-golm.mpg.de/.

MASCP Gator: An Aggregation Portal for the Visualization of Arabidopsis Proteomics Data

Proteomics has become a critical tool in the functional understanding of plant processes at the molecular level. Proteomics-based studies have also contributed to the ever-expanding array of data in modern biology, with many generating Web portals and online resources that contain incrementally expanding and updated information. Many of these resources reflect specialist research areas with significant and novel information that is not currently captured by centralized repositories. The Arabidopsis (Arabidopsis thaliana) community is well served by a number of online proteomics resources that hold an abundance of functional information. These sites can be difficult to locate among a multitude of online resources. Furthermore, they can be difficult to navigate in order to identify specific features of interest without significant technical knowledge. Recently, members of the Arabidopsis proteomics community involved in developing many of these resources decided to develop a summary aggregation portal that is capable of retrieving proteomics data from a series of online resources on the fly. The Web portal is known as the MASCP Gator and can be accessed at the following address: http://gator.masc-proteomics.org/. Significantly, proteomics data displayed at this site retrieve information from the data repositories upon each request. This means that information is always up to date and displays the latest data sets. The site also provides hyperlinks back to the source information hosted at each of the curated databases to facilitate more in-depth analysis of the primary data.