Nicolas Sommerer

Major Proteome Variations Associated with Cherry Tomato Pericarp Development and Ripening

Tomato (Solanum lycopersicum) is a model plant for studying fleshy fruit development. Several genetic and molecular approaches have been developed to increase our knowledge about the physiological basis of fruit growth, but very few data are yet available at the proteomic level. The main stages of fruit development were first determined through the dynamics of fruit diameter and pericarp cell number. Then, total proteins were extracted from pericarp tissue at six relevant developmental stages and separated by two-dimensional gel electrophoresis. Protein patterns were markedly different between stages. Proteins showing major variations were monitored. We identified 90 of 1,791 well-resolved spots either by matrix-assisted laser-desorption ionization time-of-flight peptide mass fingerprinting or liquid chromatography-mass spectrometry sequencing and expressed sequence tag database searching. Clustered correlation analysis results pointed out groups of proteins with similar expression profiles during fruit development. In young fruit, spots linked to amino acid metabolism or protein synthesis were mainly expressed during the cell division stage and down-regulated later. Some spots linked to cell division processes could be identified. During the cell expansion phase, spots linked to photosynthesis and proteins linked to cell wall formation transiently increased. In contrast, the major part of the spots related to C compounds and carbohydrate metabolism or oxidative processes were up-regulated during fruit development, showing an increase in spot intensity during development and maximal abundance in mature fruit. This was also the case for spots linked to stress responses and fruit senescence. We discuss protein variations, taking into account their potential role during fruit growth and comparing our results with already known variations at mRNA and metabolite-profiling levels.

Multiple Phosphorylations in the C-terminal Tail of Plant Plasma Membrane Aquaporins Role in Subcellular Trafficking of AtPIP2;1 in Response to Salt Stress

Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO2 columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser280 and Ser283) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser280 and Ser283 phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser283 is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser283 to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization.

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.

Nitrate Efflux at the Root Plasma Membrane: Identification of an Arabidopsis Excretion Transporter

Root NO3− efflux to the outer medium is a component of NO3− net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1–Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO3− transport activity (Km = 5 mM) in isolated root PM, electrically coupled to the ATP-dependant H+-pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO3− contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO3− efflux markedly increased in wild-type plants, leading to a prolonged NO3− excretion regime concomitant with a decrease in root NO3− content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO3− (but not of Cl−) was strongly impaired in naxt1 mutant PM. This identification of NO3− efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO3− efflux in stressed or unstressed plants.

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post‐translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

Complexity of the human whole saliva proteome

Recent characterization of the whole saliva proteome led to contradictory pictures concerning the complexity of its proteome. In this work, 110 proteins were analysed by mass spectrometry allowing the identification of 10 accessions previously not detected on protein two-dimensional maps, including myosin heavy chain (fast skeletal muscle, IIA and IIB), phosphatidylethanolamine binding protein, secretory actin-binding protein precursor and triosephosphate isomerase. Further comparison with available data demonstrated simultaneously a low diversity in terms of variety of accessions and a high complexity in terms of number of protein spots identifying the same accession, the two thirds of identified spots corresponding to amylases, cystatins and immunoglobulins. This diversity may be of interest in the development of non invasive diagnostic tool for several disease.ResumenLas recientes caracterizaciones del proteoma salival completo han llevado a resultados contradictorios. En este trabajo, se han analizado 110 proteínas por espectrometría de masas, lo que ha permitido la identificación de 10 nuevas no detectadas anteriormente en los mapas proteínicos bi-dimensionales. Incluyen cadena pesada de miosina (músculo esquelético rápido, IIa y IIb); proteína de unión a fosfatidiletanolamina, precursor de la proteína secretora de la unión a la actina y triosafosfato isomerasa. Una comparación más precisa con los datos de los estudios precedentes demuestra una baja diversidad en la variedad de accesos y una alta complejidad en el número de bandas correspondientes al mismo acceso. Los dos tercios de las bandas identificadas corresponden a amilasas, cistatinas e inmunoglobulinas. Esta diversidad puede ser de interés en el desarrollo de técnicas de diagnósticos no invasivas.

Use of reducing/nonreducing two-dimensional electrophoresis for the study of disulfide-mediated interactions between proteins in raw and heated bovine milk.

The composition and interactions of proteins in bovine milk, and modifications resulting from milk storage and processing, are complex and incompletely understood. Analysis of the milk proteome can elucidate milk protein expression, structure, interaction, and modifications. Raw milk was analyzed by two-dimensional electrophoresis (isolelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under reducing and nonreducing, or combined, conditions, followed by mass spectrometry of separated protein spots; a small number of high-abundance proteins, that is, caseins (alpha(S1)-, alpha(S2)-, beta-, kappa-, and gamma-), beta-lactoglobulin, alpha-lactalbumin, and serum albumin, represented the vast majority of protein spots on the two-dimensional electrophoretograms of raw milk samples, but some cross-linked protein complexes (mainly homopolymers of kappa-casein and alpha(S2)-casein but also some heteropolymeric complexes) were resolved under native/unheated conditions. When skim milk was heated to 90 degrees C for up to 10 min, the level of native whey proteins decreased in parallel with an increase in disulfide-linked complexes, including very complex heteropolymers, for example, casein/whey protein polymers containing multiple species. The analysis strategy used in this study reveals numerous disulfide-mediated interactions and can be proposed to analyze reduction/oxidation of milk and dairy product proteins following processing treatments applied for processing and storage.

A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines

A rapid, sensitive and selective analysis method using Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS) has been developed for the quantification of polyphenols in rosé wines. The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, the present method allows the selective quantification of up to 152 phenolic and two additional non-phenolic wine compounds in 30 min without sample purification or pre-concentration, even at low concentration levels. This method was repeatably applied to a set of 12 rosé wines and thus proved to be suitable for high-throughput and large-scale metabolomics studies.

Excreted/Secreted Proteins from Trypanosome Procyclic Strains

Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families) because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission.