Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling

A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.     

Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C₄ plants.     

Construction of a proteome reference map and response of Gaeumannomyces graminis var. tritici to 2,4-diacetylphloroglucinol

Take-all disease, caused by Gaeumannomyces graminis var. tritici (Ggt), is one of the most serious root diseases in wheat production. In this study, a proteomic platform based on 2-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Tandem Mass Spectrometry (MALDI-TOF/TOF MS) was used to construct the first proteome database reference map of G. graminis var. tritici and to identify the response of the pathogen to 2,4-diacetylphloroglucinol (DAPG), which is a natural antibiotic produced by antagonistic Pseudomonas spp. in take-all suppressive soils. For mapping, a total of 240 spots was identified that represented 209 different proteins. The most abundant biological function categories in the Ggt proteome were related to carbohydrate metabolism (21%), amino acid metabolism (15%), protein folding and degradation (12%), translation (11%), and stress response (10%). In total, 51 Ggt proteins were affected by DAPG treatment. Based on gene ontology, carbohydrate metabolism, amino acid metabolism, stress response, and protein folding and degradation proteins were the ones most modulated by DAPG treatment. This study provides the first extensive proteomic reference map constructed for Ggt and represents the first time that the response of Ggt to DAPG has been characterized at the proteomic level.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Proteomic analysis of Citrus sinensis roots and leaves in response to long-term magnesium-deficiency

Background

Magnesium (Mg)-deficiency is frequently observed in Citrus plantations and is responsible for the loss of productivity and poor fruit quality. Knowledge on the effects of Mg-deficiency on upstream targets is scarce. Seedlings of ‘Xuegan’ [Citrus sinensis (L.) Osbeck] were irrigated with Mg-deficient (0 mM MgSO₄) or Mg-sufficient (1 mM MgSO₄) nutrient solution for 16 weeks. Thereafter, we first investigated the proteomic responses of C. sinensis roots and leaves to Mg-deficiency using two-dimensional electrophoresis (2-DE) in order to (a) enrich our understanding of the molecular mechanisms of plants to deal with Mg-deficiency and (b) understand the molecular mechanisms by which Mg-deficiency lead to a decrease in photosynthesis.

Results

Fifty-nine upregulated and 31 downregulated protein spots were isolated in Mg-deficient leaves, while only 19 upregulated and 12 downregulated protein spots in Mg-deficient roots. Many Mg-deficiency-responsive proteins were involved in carbohydrate and energy metabolism, followed by protein metabolism, stress responses, nucleic acid metabolism, cell wall and cytoskeleton metabolism, lipid metabolism and cell transport. The larger changes in leaf proteome versus root one in response to Mg-deficiency was further supported by our observation that total soluble protein concentration was decreased by Mg-deficiency in leaves, but unaffected in roots. Mg-deficiency had decreased levels of proteins [i.e. ribulose-1,5-bisphosphate carboxylase (Rubisco), rubisco activase, oxygen evolving enhancer protein 1, photosynthetic electron transfer-like protein, ferredoxin-NADP reductase (FNR), aldolase] involved in photosynthesis, thus decreasing leaf photosynthesis. To cope with Mg-deficiency, C. sinensis leaves and roots might respond adaptively to Mg-deficiency through: improving leaf respiration and lowering root respiration, but increasing (decreasing) the levels of proteins related to ATP synthase in roots (leaves); enhancing the levels of proteins involved in reactive oxygen species (ROS) scavenging and other stress-responsive proteins; accelerating proteolytic cleavage of proteins by proteases, protein transport and amino acid metabolism; and upregulating the levels of proteins involved in cell wall and cytoskeleton metabolism.

Conclusions

Our results demonstrated that proteomics were more affected by long-term Mg-deficiency in leaves than in roots, and that the adaptive responses differed between roots and leaves when exposed to long-term Mg-deficiency. Mg-deficiency decreased the levels of many proteins involved in photosynthesis, thus decreasing leaf photosynthesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1462-z) contains supplementary material, which is available to authorized users.     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases

Articular cartilage is composed of cells and an extracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. There is currently a great lack of knowledge about the chondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the human normal articular chondrocyte. Cells were isolated from cartilages obtained from autopsies without history of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DE and visualized by silver nitrate or CBB staining. Almost 200 spots were excised from the gels and analyzed using MALDI-TOF or MALDI-TOF/TOF MS. The analysis leads to the identification of 136 spots that represent 93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy (16%), protein fate (14%), metabolism (12%), and cell stress (12%). From all the identified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, heat shock protein 47, and mitochondrial superoxide dismutase were more abundant in chondrocytes than in other types of mesenchymal cells such as Jurkat-T cells. As metabolic program of chondrocytes is altered in osteoarthritis and other rheumatic diseases, this proteomic map is an important tool for future studies on these pathologies.     

Proteomic changes of Citrus roots in response to long-term manganese toxicity

Key message

Fifty-three and thirty-nine differentially expressed protein spots were isolated from Mn-toxic Citrus sinensis and Citrus grandis roots, respectively. Mn-toxicity-induced changes in protein profiles greatly differed between the two species.

Abstract

Limited information is available on the manganese (Mn)-toxicity-responsive proteins in plant roots. ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) seedlings were irrigated for 17 weeks with 2 (control) or 600 μM (Mn-toxic) MnSO₄. C. sinensis displayed more tolerance to Mn-toxicity than C. grandis, which may be related to more Mn accumulation in roots and less Mn distribution in shoots. Using two-dimensional electrophoresis (2-DE), we isolated 11 up-regulated and 42 down-regulated protein spots from Mn-toxic C. sinensis roots, and 25 up-regulated and 14 down-regulated protein spots from Mn-toxic C. grandis roots. This indicated more metabolic flexibility in C. sinensis roots, thus contributing to the Mn-tolerance of C. sinensis. According to the biological functional properties, these differentially expressed proteins in the two species were classified into the following categories: protein metabolism, nucleic acid metabolism, carbohydrate and energy metabolism, stress responses, cell wall and cytoskeleton, cell transport, signal transduction and fatty acid metabolism. Under Mn-toxicity, proteins involved in nucleic acid metabolism, glycolysis and cell transport were up-regulated in nontolerant C. grandis roots, and down-regulated in tolerant C. sinensis roots. The notable down-regulation of proteins in Mn-toxic C. sinensis roots with less accumulation of carbohydrates may provide an advantage to the net carbon balance by lowering related metabolic processes, and enhancing the Mn-tolerance of C. sinensis. To conclude, there are many important differences in Mn-toxicity-induced changes in protein profiles and metabolic responses between the two species.     

The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Proteome analysis of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae

The plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, which is one of the most serious diseases of rice. Xoo has been studied for over one century, and much has been learned about it, but proteomic investigation has been neglected. In this study, proteome reference maps of Xoo were constructed by two-dimensional gel electrophoresis, and 628 spots in the gels representing 469 different protein species were identified with MALDI-TOF/TOF MS. The identified spots were assigned to 15 functional categories according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the annotations from the National Center for Biotechnology Information (NCBI) database. The data set has been deposited in the World-2DPAGE database (Database ID: 0044). In addition, comparative proteomic analysis revealed that proteins related to the TonB-dependent transportation system and energy metabolism are involved in the phenazine-1-carboxylic acid resistance in Xoo. In conclusion, we have established a proteome database for Xoo and have used this database in a comparative proteomic analysis that identified proteins potentially contributing to phenazine-1-carboxylic acid resistance in Xoo.     

Proteomics Analyses and Morphological Structure of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Inactivated by Pulsed Magnetic Field

Pulsed magnetic field (PMF) technology has emerged as a non-thermal method for inhibition of spoilage microorganism in food. In this study, we evaluate the effect of PMF treatment on the inactivation of Bacillus subtilis. The mechanisms responsible for cell death were also studied using transmission electron microscopy (TEM) and proteome approaches. Results showed that the survival rate of B. subtilis generally decreased with an increase of pulse numbers at the intensity of 3.30 T. The observation of TEM showed damage in cell cytoplasm and cytoplasmic membrane after PMF treatment. Additionally, 18 differentially expressed protein spots were identified by two dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis. The down-regulated outer membrane protein A (OmpA) illustrated that PMF destroyed the cell membrane. Furthermore, Gene ontology (GO) analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were used to characterize the functions of those proteins. That PMF treatment damaged the membrane component, depressed cellular molecular functions and biological process, and decreased the carbohydrate metabolism and energy metabolism, which explain the death of cells. The presented results give the better view into the proteome of food microorganism and provide insight into the nature of PMF inactivation mechanisms.     

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal Findings

Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions

The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.     

A proteome study of the proliferation of cultured Medicago truncatula protoplasts

A proteome study of the first five days of Medicago truncatula protoplast cultures was done to investigate molecular changes taking place during protoplast proliferation. A total of 1556 protein spots were analysed, of which 886 protein spots showed significant (p<0.005) changes in abundance at some time during the first five days of protoplast culture. Of the 886 significantly changing protein spots, 89 proteins were identified by MALDI-TOF MS. The majority of the identified proteins were part of four main cellular processes that may be involved in protoplast proliferation: energy metabolism, defence or stress response, secondary metabolism and protein synthesis and folding. The accumulation pattern of these proteins indicates extensive changes in the energy metabolism of the cells, accompanied by the activation of stress response pathways and modifications of the cell wall. In addition, seven PR10-like (pathogenesis related) proteins were identified. The accumulation pattern of these seven PR10-like proteins suggests that they could have a developmental role during protoplast proliferation.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.     

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.