Colette Larré

Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance

A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting α helices and β sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.

Biochemical Analysis and Rheological Properties of Gluten Modified by Transglutaminase

ABSTRACT A transglutaminase from Streptoverticillium sp. was used to create new covalent intermolecular cross-links between proteins in gluten. This modification induced drastic changes in its physicochemical properties as well as in its rheological behavior. To understand these changes, we characterized the gluten extractability in acetic acid and identified the proteins of supernatant and pellet by immunoblotting using antibodies specific for each prolamin class. The proportion of soluble proteins decreased drastically after transglutaminase treatment due to the formation of large insoluble polymers as shown by SDS-PAGE. Among the constitutive proteins of gluten, the high molecular weight glutenin subunits were the most affected in the transglutaminase reaction. The rheological behavior of gluten after 18 hr of incubation with transglutaminase was studied in shear by dynamic measurements over 10-3 – 101 Hz frequency range and by creep and recovery tests. The behavior of treated glutens remained that of ...

Protein composition of oil bodies from mature Brassica napus seeds.

Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies and/or myrosin cells, they must be purified step by step using floatation technique in order to remove non‐specifically trapped proteins. An exhaustive description of the protein composition of rapeseed OBs from two double‐zero varieties was achieved by a combination of proteomic and genomic tools. Genomic analysis led to the identification of sequences coding for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of these proteins were also identified through proteomic analysis and displayed a high level of sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin orthologs appeared acetylated on their N‐terminal alanine residue and both caleosins and steroleosins displayed a low level of phosphorylation.

Brachypodium distachyon grain: characterization of endosperm cell walls

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ∼52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.

Exploring the nuclear proteome of Medicago truncatula at the switch towards seed filling

Despite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome of Medicago truncatula at the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano-liquid chromatography-tandem mass spectrometry analysis of nuclear protein bands excised from one-dimensional SDS-PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO-like) or nucleocytoplasmic trafficking (dynamin-like GTPase). The results revealed that nuclei of 12-dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing-related ARGONAUTE proteins). This study also revealed the presence of chromatin-modifying enzymes and RNA interference proteins that have roles in RNA-directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.

Brachypodium distachyon grain: identification and subcellular localization of storage proteins

Seed storage proteins are of great importance in nutrition and in industrial transformation because of their functional properties. Brachypodium distachyon has been proposed as a new model plant to study temperate cereals. The protein composition of Brachypodium grain was investigated by separating the proteins on the basis of their solubility combined with a proteomic approach. Salt-soluble proteins as well as salt-insoluble proteins separated by two-dimensional gel electrophoresis revealed 284 and 120 spots, respectively. Proteins from the major spots were sequenced by mass spectrometry and identified by searching against a Brachypodium putative protein database. Our analysis detected globulins and prolamins but no albumins. Globulins were represented mainly by the 11S type and their solubility properties corresponded to the glutelin found in rice. An in silico search for storage proteins returned more translated genes than expressed products identified by mass spectrometry, particularly in the case of prolamin type proteins, reflecting a strong expression of globulins at the expense of prolamins. Microscopic examination of endosperm cells revealed scarce small-size starch granules surrounded by protein bodies containing 11S globulins. The presence of protein bodies containing glutelins makes B. distachyon closer to rice or oat than to wheat endosperm.

A comprehensive overview of grain development in Brachypodium distachyon variety Bd21

A detailed and comprehensive understanding of seed reserve accumulation is of great importance for agriculture and crop improvement strategies. This work is part of a research programme aimed at using Brachypodium distachyon as a model plant for cereal grain development and filling. The focus was on the Bd21-3 accession, gathering morphological, cytological, and biochemical data, including protein, lipid, sugars, starch, and cell-wall analyses during grain development. This study highlighted the existence of three main developmental phases in Brachypodium caryopsis and provided an extensive description of Brachypodium grain development. In the first phase, namely morphogenesis, the embryo developed rapidly reaching its final morphology about 18 d after fertilization (DAF). Over the same period the endosperm enlarged, finally to occupy 80% of the grain volume. During the maturation phase, carbohydrates were continuously stored, mainly in the endosperm, switching from sucrose to starch accumulation. Large quantities of β-glucans accumulated in the endosperm with local variations in the deposition pattern. Interestingly, new β-glucans were found in Brachypodium compared with other cereals. Proteins (i.e. globulins and prolamins) were found in large quantities from 15 DAF onwards. These proteins were stored in two different sub-cellular structures which are also found in rice, but are unusual for the Pooideae. During the late stage of development, the grain desiccated while the dry matter remained fairly constant. Brachypodium exhibits some significant differences with domesticated cereals. Beta-glucan accumulates during grain development and this cell wall polysaccharide is the main storage carbohydrate at the expense of starch.

Allergy to deamidated gluten in patients tolerant to wheat: specific epitopes linked to deamidation

Gluten proteins can be modified by deamidation to enhance their solubility and technological applications. However, severe allergic reactions have been reported after the consumption of food products containing deamidated gluten (DG) in subjects tolerant to wheat. This work aimed to characterize allergen profiles for these patients in comparison with those of patients allergic to wheat and to identify IgE‐binding epitopes.

Assessment of allergenicity of diploid and hexaploid wheat genotypes: Identification of allergens in the albumin/globulin fraction

Wheat is an important part of the daily diet of millions of people. However, this staple food is also responsible for food allergies. Ancient cultivars of wheat are gaining interest today but nothing is known about their allergenicity. Many wheat proteins have been reported as causative food allergens, including some prolamin-type gluten proteins, and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work is to obtain information about the allergenicity of the salt soluble A/G fraction of an ancient diploid cultivar compared with a standard hexaploid bread wheat cultivar using 20 sera from patients with wheat allergy. Differences in the IgE reactivity of sera towards the two genotypes were quantified by ELISA. Qualitative differences in IgE-binding proteins were searched after 1D or 2D electrophoresis. For most of the sera, the concentration in A/G specific IgE was higher for the hexaploid T. aestivum (cv Récital) than for the diploid T. monococcum (cv Engrain). The analysis of 2D spots revealed by immunoblotting leads to the identification by mass spectrometry of 39 IgE-binding proteins, some of them unknown until now as wheat allergens. Numerous allergens were identified, differences observed between Engrain and Récital will be discussed.

Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens.

SCOPE Wheat gluten proteins such as gliadins constitute major food allergens. Gluten can be modified industrially by deamidation which increases its solubility and enhances its use as a food ingredient. Sensitization to deamidated gluten has been reported to cause severe allergic reactions with anaphylaxis. The aim of this study was therefore to compare the sensitization and elicitation potentials of native (NG) and deamidated (DG) gliadins. The reactivity pattern of mice IgE was also compared with that of DG-allergic patients. METHODS AND RESULTS The ability of DG to sensitize Balb/c mice using intra-peritoneal administration with aluminium hydroxide as an adjuvant, and to elicit an allergic response after a challenge, was tested in comparison with NG. Mice sensitized with DG secreted higher levels of total IgE, IL-4, gliadin-specific IgE and IgG1 than mice sensitized with NG. By contrast, mice sensitized with NG produced higher levels of gliadin-specific IgG2a and INFγ. After a challenge, histamine levels were higher in mice sensitised with DG. CONCLUSIONS DG can sensitize mice much more efficiently than NG. Moreover, this mouse model of allergy to DG revealed an IgE reactivity pattern against purified gliadins which was very similar to that of DG-allergic patients.