A physiological, biochemical and proteomic characterization of Saccharomyces cerevisiae trk1,2 transport mutants grown under limiting potassium conditions.

Saccharomyces cerevisiae mutants lacking both isoforms of the main plasma membrane potassium transporter display impaired potassium transport and defective growth at limiting concentrations of the cation. Moreover, they are hyperpolarized and have a lower intracellular pH than wild-type. In order to unravel global physiological processes altered in trk1,2 mutants, we have established conditions at which both wild-type and mutants can grow at different rates. Using a combination of physiological, biochemical and proteomic approaches, we show that during growth at suboptimal potassium concentrations, double trk1,2 mutants accumulate less potassium and reach lower yields. In contrast, the mutants maintain increased viability in the stationary phase and retain more potassium. Moreover, the mutants show increased expression of stress-related proteins such as catalase T, thioredoxin peroxidase or hexokinase 2, suggesting that they are better adapted to the additional stress factors associated with entry into stationary growth phase.

Systems biology of monovalent cation homeostasis in yeast: the translucent contribution.

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.

Adaptation to potassium starvation of wild-type and K(+)-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach.

Saccharomyces cerevisiae wild-type (BY4741) and the corresponding mutant lacking the plasma membrane main potassium uptake systems (trk1,trk2) were used to analyze the consequences of K(+) starvation following a proteomic approach. In order to trigger high-affinity mode of potassium transport, cells were transferred to potassium-free medium. Protein profile was followed by two-dimensional (2-D) gels in samples taken at 0, 30, 60, 120, 180, and 300 min during starvation. We observed a general decrease of protein content during starvation that was especially drastic in the mutant strain as it was the case of an important number of proteins involved in glycolysis. On the contrary, we identified proteins related to stress response and alternative energetic metabolism that remained clearly present. Neural network-based analysis indicated that wild type was able to adapt much faster than the mutant to the stress process. We conclude that complete potassium starvation is a stressful process for yeast cells, especially for potassium transport mutants, and we propose that less stressing conditions should be used in order to study potassium homeostasis in yeast.

Back to the basics: Maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses.

Two dimensional gel electrophoresis has been one of the techniques most used for protein separation in proteomics experiments and still continues to be so for some species such as plants. Despite the constant technical advances and continuous improvements in the field of 2-DE, the experimental design and analysis of protein abundance data continue to be ignored or not properly documented in the literature. An appropriate experimental design, followed by decisive statistical methods is mandatory to extract all the information that is concealed in the complexity of 2-DE data. In this work we review, in a biologist's language, all the experimental design and statistical tests to be considered while planning a 2-DE based proteomics experiment and for the correct analysis and interpretation of the data. We aim to provide the researcher with an up to date introduction to these areas, starting with the experimental design and ending with the application of multivariate statistical methodologies such as PCA, ICA or neural network-based self-organizing maps. In between we have described, in an understandable way, the current methodologies available to deal with all the stages of the experimental design, data processing and analysis.

Two-dimensional electrophoresis based proteomic analysis of the pea (Pisum sativum) in response to Mycosphaerella pinodes.

Responses to Mycosphaerella pinodes in pea were studied by using a proteomics approach. Two-dimensional electrophoresis (2-DE) was used in order to compare the leaf proteome of two pea cultivars displaying different phenotypes (susceptible and partial resistance to the fungus), as well as in response to the inoculation. Multivariate statistical analysis identified 84 differential protein spots under the experimental conditions (cultivars/treatments). All of these 84 protein spots were subjected to MALDI-TOF/TOF mass spectrometry to deduce their possible functions. A total of 31 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the identified proteins corresponded to enzymes belonging to photosynthesis, metabolism, transcription/translation and defense and stress categories. Results are discussed in terms of responses to pathogens.

2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases.

By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20mM) than for the wild (3-6mM) cells. Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5-8 pH range. More differences in protein content (37-64mgg(-1) cell dry weight) and number of resolved spots (178-307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species.

Combined proteomic and transcriptomic analysis identifies differentially expressed pathways associated to Pinus radiata needle maturation.

Needle differentiation is a very complex process that leads to the formation of a mature photosynthetic organ from pluripotent needle primordia. The proteome and transcriptome of immature and fully developed needles of Pinus radiata D. Don were compared to described changes in mRNA and protein species that characterize the needle maturation developmental process. A total of 856 protein spots were analyzed, defining a total of 280 spots as differential between developmental stages, from which 127 were confidently identified. A suppressive subtractive library (2048 clones, 274 non redundant contigs) was built, and 176 genes showed to be differentially expressed. The Joint data analysis of proteomic and transcriptomic results provided a broad overview of differentially expressed pathways associated with needle maturation and stress-related pathways. Proteins and genes related to energy metabolism pathways, photosynthesis, and oxidative phosphorylation were overexpressed in mature needles. Amino acid metabolism, transcription, and translation pathways were overexpressed in immature needles. Interestingly, stress related proteins were characteristic of immature tissues, a fact that may be linked to defense mechanisms and the higher growth rate and morphogenetic competence exhibited by these needles. Thus, this work provides an overview of the molecular changes affecting proteomes and transcriptomes during P. radiata needle maturation, having an integrative vision of the functioning and physiology of this process.

Differences in the triticale (X Triticosecale Wittmack) flag leaf 2-DE protein profile between varieties and nitrogen fertilization levels.

Nitrogen nutrition is one of the major factors limiting the growth and production of crop plants. Limited information on proteome changes occurring in response to nitrogen amount have been available up to now. We used 2-DE to investigate proteome differences between two triticale varieties and the changes caused by nitrogen nutrition deficit in the flag leaf tissue. Some physiological features, such as the number of tillers per plant, SPAD index, dry weight, and protein content were measured previous to the proteomic analysis. Statistical analysis identified 29 differential protein spots in the selected pairwise comparisons of experimental conditions and correlated with the expression cluster revealed by the principal component analysis. The 29 protein spots were subjected to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) to deduce their possible functions. Many of these changes referred to enzymes involved in photosynthesis, metabolic pathways implicated in the balance of the energy, and redox status of the cell. This work provides a first characterization of the proteome changes that occur in response to nitrogen deficit in flag leaves of triticale plants.

Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins.

Based on the presence of phospholipids in the extracellular fluids (EFs) of sunflower seeds, we have hypothesized on the existence of vesicles in the apoplastic compartment of plants. Ultracentrifugation of sunflower EF allowed the isolation of particles of 50-200 nm with apparent membrane organization. A small GTPase Rab was putatively identified in this vesicular fraction. Since Rab proteins are involved in vesicular traffic and their presence in exosomes from animal fluids has been demonstrated, evidence presented here supports the existence of exosome-like vesicles in apoplastic fluids of sunflower. Their putative contribution to intercellular communication in plants is discussed.

Differential expression proteomics to investigate responses and resistance to Orobanche crenata in Medicago truncatula.

BACKGROUND: Parasitic angiosperm Orobanche crenata infection represents a major constraint for the cultivation of legumes worldwide. The level of protection achieved to date is either incomplete or ephemeral. Hence, an efficient control of the parasite requires a better understanding of its interaction and associated resistance mechanisms at molecular levels. RESULTS: In order to study the plant response to this parasitic plant and the molecular basis of the resistance we have used a proteomic approach. The root proteome of two accessions of the model legume Medicago truncatula displaying differences in their resistance phenotype, in control as well as in inoculated plants, over two time points (21 and 25 days post infection), has been compared. We report quantitative as well as qualitative differences in the 2-DE maps between early- (SA 27774) and late-resistant (SA 4087) genotypes after Coomassie and silver-staining: 69 differential spots were observed between non-inoculated genotypes, and 42 and 25 spots for SA 4087 and SA 27774 non-inoculated and inoculated plants, respectively. In all, 49 differential spots were identified by peptide mass fingerprinting (PMF) following MALDI-TOF/TOF mass spectrometry. Many of the proteins showing significant differences between genotypes and after parasitic infection belong to the functional category of defense and stress-related proteins. A number of spots correspond to proteins with the same function, and might represent members of a multigenic family or post-transcriptional forms of the same protein. CONCLUSION: The results obtained suggest the existence of a generic defense mechanism operating during the early stages of infection and differing in both genotypes. The faster response to the infection observed in the SA 27774 genotype might be due to the action of proteins targeted against key elements needed for the parasite's successful infection, such as protease inhibitors. Our data are discussed and compared with those previously obtained with pea 1 and transcriptomic analysis of other plant-pathogen and plant-parasitic plant systems.

Changes in the protein profile of Quercus ilex leaves in response to drought stress and recovery.

To characterize the molecular response of holm oak to drought stress and its capacity to recover 9-month-old Quercus ilex seedlings were subjected to three treatments for a 14-d period: (i) continuous watering to field capacity (control plants, W), (ii) no irrigation (drought treatment, D), and (iii) no irrigation for 7d followed by a watering period of 7d (recovery treatment, R). In drought plants, leaf water potential decreased from -0.72 (day 0) to -0.99MPa (day 7), and -1.50MPa (day 14). Shoot relative water content decreased from 49.3% (day 0) to 47.7% (day 7) and 40.8% (day 14). Photosystem II quantum yield decreased from 0.80 (day 0) to 0.72 (day 7) and 0.73 (day 14). Plants subjected to water withholding for 7d reached, after a 7-d rewatering period, values similar to those of continuously irrigated control plants. Changes in the leaf protein pattern in response to drought and recovery treatments were analyzed by using a proteomic approach. Twenty-three different spots were observed when comparing the two-dimensional electrophoresis profile of control to both drought and recovered plants. From these, 14 proteins were identified from tryptic peptides tandem mass spectra by using the new Paragon algorithm present in the ProteinPilot software. The proteins identified belong to the photosynthesis, carbohydrate and nitrogen metabolism, and stress-related protein functional categories.

Proteomic analysis of Pinus radiata needles: 2-DE map and protein identification by LC/MS/MS and substitution-tolerant database searching.

Pinus radiata is one of the most economically important forest tree species, with a worldwide production of around 370 million m (3) of wood per year. Current selection of elite trees to be used in conservation and breeding programes requires the physiological and molecular characterization of available populations. To identify key proteins related to tree growth, productivity and responses to environmental factors, a proteomic approach is being utilized. In this paper, we present the first report of the 2-DE protein reference map of physiologically mature P. radiata needles, as a basis for subsequent differential expression proteomic studies related to growth, development, biomass production and responses to stresses. After TCA/acetone protein extraction of needle tissue, 549 +/- 21 well-resolved spots were detected in Coommassie-stained gels within the 5-8 pH and 10-100 kDa M(r) ranges. The analytical and biological variance determined for 450 spots were of 31 and 42%, respectively. After LC/MS/MS analysis of in-gel tryptic digested spots, proteins were identified by using the novel Paragon algorithm that tolerates amino acid substitution in the first-pass search. It allowed the confident identification of 115 out of the 150 protein spots subjected to MS, quite unusual high percentage for a poor sequence database, as is the case of P. radiata. Proteins were classified into 12 or 18 groups based on their corresponding cell component or biological process/pathway categories, respectively. Carbohydrate metabolism and photosynthetic enzymes predominate in the 2-DE protein profile of P. radiata needles.

Antifungal activity of a new phenolic compound from capitulum of a head rot-resistant sunflower genotype.

In a previous study, we observed that bract and corolla extracts from a Sclerotinia sclerotiorum-resistant sunflower contained high amounts of the known coumarins scopoletin, scopolin, and ayapin. There was a correlation between coumarin concentration and disease resistance. Thin layer chromatography showed higher concentrations of three other compounds in the resistant genotype when compared to the susceptible. A bioassay-directed purification that used column chromatography and HPLC allowed the isolation of a new compound, 3-acetyl-4-acetoxyacetophenone, and known compounds, demethoxyencecalin and 3-acetyl-4-hydroxyacetophenone. Structures were assigned from spectral data, and bioactivities were characterized by in vitro bioassays against S. sclerotiorum. The new compound, 3-acetyl-4-acetoxyacetophenone, had an antifungal activity similar to the coumarin ayapin, previously described as a potent Sclerotinia inhibitor. The speed and simplicity by which these compounds can be detected make them suitable for use in screening procedures that may identify genotypes with valuable levels of resistance. A screening of seven sunflower genotypes in a field experiment showed a correlation between these compounds and resistance to Sclerotinia.

Plant proteome analysis: a 2006 update.

This 2006 'Plant Proteomics Update' is a continuation of the two previously published in 'Proteomics' by 2004 (Canovas et al., Proteomics 2004, 4, 285-298) and 2006 (Rossignol et al., Proteomics 2006, 6, 5529-5548) and it aims to bring up-to-date the contribution of proteomics to plant biology on the basis of the original research papers published throughout 2006, with references to those appearing last year. According to the published papers and topics addressed, we can conclude that, as observed for the three previous years, there has been a quantitative, but not qualitative leap in plant proteomics. The full potential of proteomics is far from being exploited in plant biology research, especially if compared to other organisms, mainly yeast and humans, and a number of challenges, mainly technological, remain to be tackled. The original papers published last year numbered nearly 100 and deal with the proteome of at least 26 plant species, with a high percentage for Arabidopsis thaliana (28) and rice (11). Scientific objectives ranged from proteomic analysis of organs/tissues/cell suspensions (57) or subcellular fractions (29), to the study of plant development (12), the effect of hormones and signalling molecules (8) and response to symbionts (4) and stresses (27). A small number of contributions have covered PTMs (8) and protein interactions (4). 2-DE (specifically IEF-SDS-PAGE) coupled to MS still constitutes the almost unique platform utilized in plant proteome analysis. The application of gel-free protein separation methods and 'second generation' proteomic techniques such as multidimensional protein identification technology (MudPIT), and those for quantitative proteomics including DIGE, isotope-coded affinity tags (ICAT), iTRAQ and stable isotope labelling by amino acids in cell culture (SILAC) still remains anecdotal. This review is divided into seven sections: Introduction, Methodology, Subcellular proteomes, Development, Responses to biotic and abiotic stresses, PTMs and Protein interactions. Section 8 summarizes the major pitfalls and challenges of plant proteomics.

Towards a global analysis of porcine alveolar macrophages proteins through two-dimensional electrophoresis and mass spectrometry.

Alveolar macrophages (AM) are the primary phagocytes of the innate immune systems, constituting a link between innate and adaptive immunity. With the aim of studying the porcine AM biology and the dynamics of pig-pathogen cell interactions, we have obtained a reference 2-DE map of the porcine AM proteins. The proteins were separated by 2-DE using a 5-8 range pH gradient in isoelectric focusing and over 800 spots were detected. A set of proteins, covering the pI 5.2-7.4 and M(W) 19 to 106kDa ranges, was subjected to MS analysis and 106 proteins were assigned identification by PMF, this identification being confirmed by MS/MS. An important number of proteins is involved in immunological functions, signalling process, transport or apoptosis, confirming that macrophages are involved in a wide range of biological functions. This reference map provides a useful tool for identifying protein pattern changes as a result of inflammation, exposure to infectious agents or genetic diseases.

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

Pre-haustorial resistance to broomrape (Orobanche cumana) in sunflower (Helianthus annuus): cytochemical studies.

Sunflower broomrape (Orobanche cumana Wallr.) is a root holoparasitic angiosperm considered as one of the major constraints for sunflower production in Mediterranean areas. Breeding for resistance is regarded as the most effective, feasible, and environmentally friendly solution to control this parasite. However, the existing sources of genetic resistance are defeated by the continuous emergence of new more virulent races of the parasite. In this work, the interaction between sunflower and O. cumana has been analysed in order to gain insights into the mechanisms involved in resistance. Two sunflower genotypes were selected showing different behaviour against the new race F of O. cumana, HE-39998 (susceptible) and HE-39999 (resistant), and both compatible and incompatible interactions were compared. Pot and Petri dish bioassays revealed that only HE-39998 plants were severely affected, supporting a high number of successfully established broomrapes to mature flowering, whereas in HE-39999 root tubercles were never observed, resistance being associated with browning symptoms of both parasite and host tissues. Histological aspects of the resistance were further investigated. Suberization and protein cross-linking at the cell wall were seen in the resistant sunflower cells in contact with the parasite, preventing parasite penetration and connection to the host vascular system. In addition, fluorescence and confocal laser microscopy (CLM) observations revealed accumulation of phenolic compounds during the incompatible reaction, which is in agreement with these metabolites playing a defensive role during H. annuus-O. cumana interaction.

Plant proteome analysis: a 2004-2006 update.

Since the appearance of the review entitled "Plant Proteome Analysis" in Proteomics in February 2004 (Cánovas, F. M., Dumas-Gaudot, E., Recorbert, G., Jorrín, J. et al., Proteomics 2004, 4, 285-298), about 200 original articles focusing on plant proteomics have been published. Although this represents less than 1% of the global proteomics output during this period, it nevertheless reflects an increase in activity over the period 1999-2004. These papers concern the proteome of at least 35 plant species but have concentrated mainly on thale cress (Arabidopsis thaliana) and rice (Oryza sativa). The scientific objectives have ranged from a proteomic analysis of organs, tissues, cell suspensions, or subcellular fractions to the study of plant development and response to various stresses. A number of contributions have covered PTMs and protein interactions. The dominant analytical platform has been 2-DE coupled to MS, but "second generation" techniques such as DIGE, multidimensional protein identification technology, isotope-coded affinity tags, and stable isotope labeling by amino acids in cell culture have begun to make an impact. This review aims to provide an update of the contribution of proteomics to plant biology during the period 2004-2006, and is divided into six sections: introduction, subcellular proteomes, plant development, responses to biotic and abiotic stresses, PTMs, and protein interactions. The conclusions summarize a view of the major pitfalls and challenges of plant proteomics.

Variation in the holm oak leaf proteome at different plant developmental stages, between provenances and in response to drought stress.

Major proteins of the holm oak leaf proteome have been previously identified using a combination of 2-DE, MS analysis and BLAST similarity search (Jorge et al., Proteomics 2005, 5, 222-234). That study, conducted with field samples from mature trees, revealed the existence of a great variability in the 2-DE protein map, with qualitative as well as quantitative changes, both analytical and biological. A similar study has been carried out with 2-year-old seedlings to analyze and study: (i) changes in the 2-DE protein profile at different tree developmental stages; (ii) the 2-DE protein map variability between three different Spanish provenances; and (iii) variations in the 2-DE protein profile in response to drought stress. Although the protein profile of leaves from seedlings and mature trees was fairly similar, the biological variance found was lower in the former. In the present study, new proteins have been identified. At least four different protein spots differentiated Spanish provenances, two of them identified as an ATP synthase alpha chain, and a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Fourteen different protein spots were qualitatively variable between well-watered and drought-stressed seedlings, with some of them corresponding to enzymes of carbohydrate and protein metabolism. Data presented indicated the mobilization of storage proteins and carbohydrates, as well as photosynthesis inhibition under drought conditions.

A proteomic approach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi).

As a global approach to gain a better understanding of the mechanisms involved in pea resistance to Erysiphe pisi, changes in the leaf proteome of two pea genotypes differing in their resistance phenotype were analyzed by a combination of 2-DE and MALDI-TOF/TOF MS. Leaf proteins from control non-inoculated and inoculated susceptible (Messire) and resistant (JI2480) plants were resolved by 2-DE, with IEF in the 5-8 pH range and SDS-PAGE on 12% gels. CBB-stained gels revealed the existence of quantitative and qualitative differences between extracts from: (i) non-inoculated leaves of both genotypes (77 spots); (ii) inoculated and non-inoculated Messire leaves (19 spots); and (iii) inoculated and non-inoculated JI2480 leaves (12 spots). Some of the differential spots have been identified, after MALDI-TOF/TOF analysis and database searching, as proteins belonging to several functional categories, including photosynthesis and carbon metabolism, energy production, stress and defense, protein synthesis and degradation and signal transduction. Results are discussed in terms of constitutive and induced elements involved in pea resistance against Erysiphe pisi.