Bioquímica y Proteómica Vegetal y Agrícola

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Publicaciones Resumen

Two-dimensional gel electrophoresis-based proteomic analysis of the Medicago truncatula–rust (Uromyces striatus) interaction.

M.A. Castillejo, R. Susın, E. Madrid, M. Fernandez-Aparicio, J.V. Jorrın, D. Rubiales

A two-dimensional gel electrophoresis (2-DE) based proteomic approach has been used to study the Medicago truncatula–Uromyces striatus interaction. The 2-DE leaf protein profile of three M. truncatula genotypes displaying different phenotypes (susceptible and showing prehaustorial and posthaustorial resistance) in both noninoculated and inoculated plants have been compared. Multivariate statistical analysis identified 63 differential protein spots under the experimental conditions (genotypes/treatments). Variable spots were subjected to tandem mass spectrometry (MS, matrix-assisted laser desorption ionisation time of flight, MALDI-TOF/TOF) analysis to identify their possible functions. A total of 27 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of these observed changes correspond to enzymes involved in photosynthesis, energy metabolic pathways and stress related, whose pattern expression was different in relation to susceptibility/resistance of the genotypes studied. Results obtained in this work suggest that differences observed could be related to efficiency in energy utilisation and the induction of proteins involved in defence mechanism operating during early stages of infection.

Review Article Proteomics of Plant Pathogenic Fungi.

Raquel Gonzalez-Fernandez, Elena Prats, and Jesus V. Jorrın-Novo

Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

Proteomics of fungal plant pathogens: the case of Botrytis cinerea.

R. González Fernández, and J.V. Jorrín Novo

Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environment-friendly crop protection strategies, molecular studies of the fungal biological cycle and interaction with its host are necessary. For that reason, several approaches have been made using both classical genetic, cell biology and biochemistry and modern, holistic and high-throughput, omic techniques. Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection. In this chapter we review how proteomics can contribute to such studies and objectives, with examples of our current research with Botrytis cinerea, and reference to the technical platform used, based on electrophoresis-MS-based proteomics, and specific protocols.

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

Miguel Curto, Luis Valledor, Clara Navarrete, Dolores Gutiérrez, Hana Sychrova, José Ramos, Jesús Jorrin

By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50 mM) 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 (20 mM) than for the wild (3–6 mM) 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 (37–64 mg g−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 accumulatior of low Mr protein species.

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

Luis Valledor, Jesús Jorrín

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.

Proteomics research on forest trees, the most recalcitrant and orphan plant species

Nieves Abril, Jean-Marc Gion, René Kerner, Gerhard Müller-Starck, Rafael M. Navarro Cerrillo,
Christophe Plomion, Jenny Renaut, Luis Valledor, Jesús V. Jorrin-Novo

The contribution of proteomics to the knowledge of forest tree (the most recalcitrant and almost forgotten plant species) biology is being reviewed and discussed, based on the author's own research work and papers published up to November 2010. This review is organized in four introductory sections starting with the definition of forest trees (1), the description of the environmental and economic importance (2) and its derived current priorities and research lines for breeding and conservation (3) including forest tree genomics (4). These precede the main body of this review: a general overview to proteomics (5) for introducing the forest tree proteomics section (6). Proteomics, defined as scientific discipline or experimental approach, it will be discussed both from a conceptual and methodological point of view, commenting on realities, challenges and limitations. Proteomics research in woody plants is limited to a reduced number of genera, including Pinus, Picea, Populus, Eucalyptus, and Fagus, mainly using first-generation approaches, e.g., those based on two-dimensional electrophoresis coupled to mass spectrometry. This area joins the own limitations of the technique and the difficulty and recalcitrance of the plant species as an experimental system. Furthermore, it contributes to a deeper knowledge of some biological processes, namely growth, development, organogenesis, and responses to stresses, as it is also used in the characterization and cataloguing of natural populations and biodiversity (proteotyping) and in assisting breeding programmes.

Differences in the Triticale (X Triticosecale Wittmack) Flag Leaf 2-DE Protein Profile between Varieties and Nitrogen Fertilization Levels

Mari Angeles Castillejo, Hristofor K. Kirchev y Jesus Jorrin Novo

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.

Application of proteomics to the assessment of the response to ionising radiation in Arabidopsis thaliana

Morgane Gicquel, Marie-André Esnaulta, JesùsV. Jorrín-Novo, Francisco Cabello-Hurtado

Ionising radiation (IR) affects cellular and tissue function. However, the biological effects and interactions induced by IR are unclear. The aim of this study was to decipher the proteomic patterns that influence these pathways. The proteomes of Arabidopsis thaliana roots and rosettes were analysed in response to sub-lethal IR doses (0, 10, and 40 Gy). For each dose, the dynamic response was observed at different time points (2, 24 and 72 h). To quantitatively measure the effect of IR on the proteome, total proteins were extracted and subjected to 2-DE, and the changes in the 2-DE protein profiles were analysed. Statistical analysis revealed a total of 172 proteins (145 in leaves and 27 in roots) that were differentially expressed. These proteins were subsequently analysed by MALDI-TOF/TOF MS and comparative database analysis, and 144 (118 in leaves and 26 in roots) proteins were identified. The changes in the protein profile were quantitatively more significant for the 40 Gy dose than for the 10 Gy dose. In addition, specific functional groups of proteins were identified based on the consistency of the dose- and time-responses. The molecular mechanisms involved in the response to IR and a comparison of the observed responses are discussed.

Facing challenges in Proteomics today and in the coming decade: Report of Roundtable Discussions at the 4th EuPA Scientific Meeting, Portugal, Estoril 2010 Jürgen Coxa, Ron M.A.Heerenb, Peter Jamesc, Jesús V. Jorrin-Novod, Eugene Kolkere,f, Fredrik Levanderg, Nicholas Morriceh, Paola Picottii, Pier Giorgio Righettij, Jean-Charles Sánchezk, Christoph W. Turckl, Roman Zubarevm, Bruno M. Alexandren, Fernando J. Corraleso, György Marko-Vargap, Sinead O'Donovanq, Serena O'Neilr, Jozsef Prechls, Tânia Simõesn, Wolfram Weckwertht, Deborah Penquen,

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with Pseudomonas syringae

Ana M. Maldonado-Alconada, Sira Echevarrıa-Zomeño, Christian Lindermayr, Inmaculada Redondo-Lopez, Jorg Durner, Jesus V. Jorrin-Novo

Nitric oxide (NO) is a key signaling molecule in plants, being its biological effects mainly mediated through S-nitrosylation of cysteine thiols. Using the biotin switch method combined with mass spectrometry analysis we have identified 127 targets of S-nitrosylation in Arabidopsis cell suspension cultures and leaves challenged with virulent and avirulent isolates of Pseudomonas syringae pv. tomato. The NO targets are proteins associated with carbon, nitrogen, and sulpfur metabolism, photosynthesis, the cytoskeleton, stress-, pathogen- and redox-related and signaling proteins. Some proteins were previously identified in plants and mammals, while others (63%) represent novel targets of S-nitrosylation. Our data suggest that NO might be orchestrating the whole plant physiology, presumably through covalent modification of proteins.

Combined Proteomic and Transcriptomic Analysis Identifies Differentially Expressed Pathways Associated to Pinus radiata Needle Maturation

Luis Valledor, Jesus V. Jorrın, Jose Luis Rodrıguez, Christof Lenz, Monica Meijon, Roberto Rodrıguez, and Maria Jesus Cañal

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.

Abscisic acid and sucrose increase the protein content in date palm somatic embryos, causing changes in 2-DE profile Besma

Besma Sghaier-Hammami, Jesús V. Jorrín-Novo, Radhia Gargouri-Bouzid, Noureddine Drira

Various supplements (abscisic acid (ABA) or sucrose) were added to the initial embryo culture medium (M3) with the aim of improving the vigour of vitroplants deriving from date palm somatic embryogenesis. ABA (20 and 40 lM) and sucrose (90 g/l) applied for 4 and 2 weeks respectively increased embryo thickness, with no apparent difference in length. ABA (5–40 lM) increased embryo proliferation rate. Somatic embryos maintained in modified M3 (M3 supplemented with ABA and an increased sucrose concentration) contained a higher amount of protein than those maintained in initial M3 (no ABA, 30 g/l of sucrose), with a 1.5–1.7-fold increase depending on the compound and concentration assayed. The 1-D and 2-DE protein profiles showed qualitative and quantitative differences between the somatic embryos cultured in initial M3 (control) and in modified M3. Statistical analysis of spot intensity was performed by principal component analysis, yielding two accurate groups of samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with an average linkage algorithm. Thirty-four variable spots were identified using mass spectrometry analysis. Identified proteins were classified into the following functional categories: energy metabolism (five proteins); protein translation, folding and degradation (9); redox maintenance (5); cytoskeleton (3); storage protein (2); and with no assigned function as (10). While ''up-regulation" of stress-related proteins and ''down-regulation" of energy metabolism proteins were observed in somatic embryos matured in M3 supplemented with ABA, storage proteins (legumin) were ''up-regulated" in somatic embryos matured in M3 supplemented with increased sucrose.

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) through acorn protein profile analysis

José Valero Galván, Luis Valledor, Rafael Ma. Navarro Cerrillo,
Eustaquio Gil Pelegrín, Jesus V. Jorrín-Novo

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.), the dominant tree species in the typical Mediterranean forest, have been carried out by using electrophoresis-based proteomic analysis of acorns. Ten populations distributed throughout the Andalusia region have been surveyed. Acorns were sampled from individual trees and proteins extracted from seed flour by using the TCA–acetone precipitation protocol. Extracts were subjected to SDS-PAGE and 2-DE for protein separation, gel images captured, spot or bands quantified, and subjected to statistical analysis (ANOVA, SOM and clustering). Variable bands or spots among populations were subjected to MALDI-TOF/TOF and LC-MS/MS for identification. The protein yield of the used protocol varied among populations, and it was in the 2.92–5.92 mg/g dry weight range. A total of 23 bands were resolved by SDS-PAGE in the 3–35 kDa Mr range, with 8 and 12, out of the total, showing respectively qualitative and quantitative statistically significant differences among populations. Data allowed grouping populations, with groups being correlated according to geographical location and climate conditions, to northern and southern, as well as the discrimination of both mesic and xeric groups. Acorn flour extracts from the most distant populations were analyzed by 2-DE, and 56 differential spots were proposed as markers of variability. Identified proteins were classified into two principal categories; storage and stress/defense protein. Besides providing the first reference map of mature acorn seeds, the use of SDS-PAGE and proteomics in characterizing natural biodiversity in forest trees will be discussed.

Publicaciones	Resumen
Two-dimensional gel electrophoresis-based proteomic analysis of the Medicago truncatula–rust (Uromyces striatus) interaction. M.A. Castillejo, R. Susın, E. Madrid, M. Fernandez-Aparicio, J.V. Jorrın, D. Rubiales	A two-dimensional gel electrophoresis (2-DE) based proteomic approach has been used to study the Medicago truncatula–Uromyces striatus interaction. The 2-DE leaf protein profile of three M. truncatula genotypes displaying different phenotypes (susceptible and showing prehaustorial and posthaustorial resistance) in both noninoculated and inoculated plants have been compared. Multivariate statistical analysis identified 63 differential protein spots under the experimental conditions (genotypes/treatments). Variable spots were subjected to tandem mass spectrometry (MS, matrix-assisted laser desorption ionisation time of flight, MALDI-TOF/TOF) analysis to identify their possible functions. A total of 27 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of these observed changes correspond to enzymes involved in photosynthesis, energy metabolic pathways and stress related, whose pattern expression was different in relation to susceptibility/resistance of the genotypes studied. Results obtained in this work suggest that differences observed could be related to efficiency in energy utilisation and the induction of proteins involved in defence mechanism operating during early stages of infection.
Review Article Proteomics of Plant Pathogenic Fungi. Raquel Gonzalez-Fernandez, Elena Prats, and Jesus V. Jorrın-Novo	Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.
Proteomics of fungal plant pathogens: the case of Botrytis cinerea. R. González Fernández, and J.V. Jorrín Novo	Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environment-friendly crop protection strategies, molecular studies of the fungal biological cycle and interaction with its host are necessary. For that reason, several approaches have been made using both classical genetic, cell biology and biochemistry and modern, holistic and high-throughput, omic techniques. Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection. In this chapter we review how proteomics can contribute to such studies and objectives, with examples of our current research with Botrytis cinerea, and reference to the technical platform used, based on electrophoresis-MS-based proteomics, and specific protocols.
2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases. Miguel Curto, Luis Valledor, Clara Navarrete, Dolores Gutiérrez, Hana Sychrova, José Ramos, Jesús Jorrin	By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50 mM) 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 (20 mM) than for the wild (3–6 mM) 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 (37–64 mg g−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 accumulatior of low Mr protein species.
Back to the basics: Maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses. Luis Valledor, Jesús Jorrín	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.
Proteomics research on forest trees, the most recalcitrant and orphan plant species Nieves Abril, Jean-Marc Gion, René Kerner, Gerhard Müller-Starck, Rafael M. Navarro Cerrillo, Christophe Plomion, Jenny Renaut, Luis Valledor, Jesús V. Jorrin-Novo	The contribution of proteomics to the knowledge of forest tree (the most recalcitrant and almost forgotten plant species) biology is being reviewed and discussed, based on the author's own research work and papers published up to November 2010. This review is organized in four introductory sections starting with the definition of forest trees (1), the description of the environmental and economic importance (2) and its derived current priorities and research lines for breeding and conservation (3) including forest tree genomics (4). These precede the main body of this review: a general overview to proteomics (5) for introducing the forest tree proteomics section (6). Proteomics, defined as scientific discipline or experimental approach, it will be discussed both from a conceptual and methodological point of view, commenting on realities, challenges and limitations. Proteomics research in woody plants is limited to a reduced number of genera, including Pinus, Picea, Populus, Eucalyptus, and Fagus, mainly using first-generation approaches, e.g., those based on two-dimensional electrophoresis coupled to mass spectrometry. This area joins the own limitations of the technique and the difficulty and recalcitrance of the plant species as an experimental system. Furthermore, it contributes to a deeper knowledge of some biological processes, namely growth, development, organogenesis, and responses to stresses, as it is also used in the characterization and cataloguing of natural populations and biodiversity (proteotyping) and in assisting breeding programmes.
Differences in the Triticale (X Triticosecale Wittmack) Flag Leaf 2-DE Protein Profile between Varieties and Nitrogen Fertilization Levels Mari Angeles Castillejo, Hristofor K. Kirchev y Jesus Jorrin Novo	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.
Application of proteomics to the assessment of the response to ionising radiation in Arabidopsis thaliana Morgane Gicquel, Marie-André Esnaulta, JesùsV. Jorrín-Novo, Francisco Cabello-Hurtado	Ionising radiation (IR) affects cellular and tissue function. However, the biological effects and interactions induced by IR are unclear. The aim of this study was to decipher the proteomic patterns that influence these pathways. The proteomes of Arabidopsis thaliana roots and rosettes were analysed in response to sub-lethal IR doses (0, 10, and 40 Gy). For each dose, the dynamic response was observed at different time points (2, 24 and 72 h). To quantitatively measure the effect of IR on the proteome, total proteins were extracted and subjected to 2-DE, and the changes in the 2-DE protein profiles were analysed. Statistical analysis revealed a total of 172 proteins (145 in leaves and 27 in roots) that were differentially expressed. These proteins were subsequently analysed by MALDI-TOF/TOF MS and comparative database analysis, and 144 (118 in leaves and 26 in roots) proteins were identified. The changes in the protein profile were quantitatively more significant for the 40 Gy dose than for the 10 Gy dose. In addition, specific functional groups of proteins were identified based on the consistency of the dose- and time-responses. The molecular mechanisms involved in the response to IR and a comparison of the observed responses are discussed.
Facing challenges in Proteomics today and in the coming decade: Report of Roundtable Discussions at the 4th EuPA Scientific Meeting, Portugal, Estoril 2010 Jürgen Coxa, Ron M.A.Heerenb, Peter Jamesc, Jesús V. Jorrin-Novod, Eugene Kolkere,f, Fredrik Levanderg, Nicholas Morriceh, Paola Picottii, Pier Giorgio Righettij, Jean-Charles Sánchezk, Christoph W. Turckl, Roman Zubarevm, Bruno M. Alexandren, Fernando J. Corraleso, György Marko-Vargap, Sinead O'Donovanq, Serena O'Neilr, Jozsef Prechls, Tânia Simõesn, Wolfram Weckwertht, Deborah Penquen,
Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with Pseudomonas syringae Ana M. Maldonado-Alconada, Sira Echevarrıa-Zomeño, Christian Lindermayr, Inmaculada Redondo-Lopez, Jorg Durner, Jesus V. Jorrin-Novo	Nitric oxide (NO) is a key signaling molecule in plants, being its biological effects mainly mediated through S-nitrosylation of cysteine thiols. Using the biotin switch method combined with mass spectrometry analysis we have identified 127 targets of S-nitrosylation in Arabidopsis cell suspension cultures and leaves challenged with virulent and avirulent isolates of Pseudomonas syringae pv. tomato. The NO targets are proteins associated with carbon, nitrogen, and sulpfur metabolism, photosynthesis, the cytoskeleton, stress-, pathogen- and redox-related and signaling proteins. Some proteins were previously identified in plants and mammals, while others (63%) represent novel targets of S-nitrosylation. Our data suggest that NO might be orchestrating the whole plant physiology, presumably through covalent modification of proteins.
Combined Proteomic and Transcriptomic Analysis Identifies Differentially Expressed Pathways Associated to Pinus radiata Needle Maturation Luis Valledor, Jesus V. Jorrın, Jose Luis Rodrıguez, Christof Lenz, Monica Meijon, Roberto Rodrıguez, and Maria Jesus Cañal	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.
Abscisic acid and sucrose increase the protein content in date palm somatic embryos, causing changes in 2-DE profile Besma Besma Sghaier-Hammami, Jesús V. Jorrín-Novo, Radhia Gargouri-Bouzid, Noureddine Drira	Various supplements (abscisic acid (ABA) or sucrose) were added to the initial embryo culture medium (M3) with the aim of improving the vigour of vitroplants deriving from date palm somatic embryogenesis. ABA (20 and 40 lM) and sucrose (90 g/l) applied for 4 and 2 weeks respectively increased embryo thickness, with no apparent difference in length. ABA (5–40 lM) increased embryo proliferation rate. Somatic embryos maintained in modified M3 (M3 supplemented with ABA and an increased sucrose concentration) contained a higher amount of protein than those maintained in initial M3 (no ABA, 30 g/l of sucrose), with a 1.5–1.7-fold increase depending on the compound and concentration assayed. The 1-D and 2-DE protein profiles showed qualitative and quantitative differences between the somatic embryos cultured in initial M3 (control) and in modified M3. Statistical analysis of spot intensity was performed by principal component analysis, yielding two accurate groups of samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with an average linkage algorithm. Thirty-four variable spots were identified using mass spectrometry analysis. Identified proteins were classified into the following functional categories: energy metabolism (five proteins); protein translation, folding and degradation (9); redox maintenance (5); cytoskeleton (3); storage protein (2); and with no assigned function as (10). While ''up-regulation" of stress-related proteins and ''down-regulation" of energy metabolism proteins were observed in somatic embryos matured in M3 supplemented with ABA, storage proteins (legumin) were ''up-regulated" in somatic embryos matured in M3 supplemented with increased sucrose.
Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) through acorn protein profile analysis José Valero Galván, Luis Valledor, Rafael Ma. Navarro Cerrillo, Eustaquio Gil Pelegrín, Jesus V. Jorrín-Novo	Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.), the dominant tree species in the typical Mediterranean forest, have been carried out by using electrophoresis-based proteomic analysis of acorns. Ten populations distributed throughout the Andalusia region have been surveyed. Acorns were sampled from individual trees and proteins extracted from seed flour by using the TCA–acetone precipitation protocol. Extracts were subjected to SDS-PAGE and 2-DE for protein separation, gel images captured, spot or bands quantified, and subjected to statistical analysis (ANOVA, SOM and clustering). Variable bands or spots among populations were subjected to MALDI-TOF/TOF and LC-MS/MS for identification. The protein yield of the used protocol varied among populations, and it was in the 2.92–5.92 mg/g dry weight range. A total of 23 bands were resolved by SDS-PAGE in the 3–35 kDa Mr range, with 8 and 12, out of the total, showing respectively qualitative and quantitative statistically significant differences among populations. Data allowed grouping populations, with groups being correlated according to geographical location and climate conditions, to northern and southern, as well as the discrimination of both mesic and xeric groups. Acorn flour extracts from the most distant populations were analyzed by 2-DE, and 56 differential spots were proposed as markers of variability. Identified proteins were classified into two principal categories; storage and stress/defense protein. Besides providing the first reference map of mature acorn seeds, the use of SDS-PAGE and proteomics in characterizing natural biodiversity in forest trees will be discussed.