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Protein quality is important for patients needing medical nutrition, especially those dependent on tube feeding. A blend of dairy and vegetable proteins (35% whey, 25% casein, 20% soy, 20% pea, P4) developed to obtain a more balanced amino acid profile with higher chemical scores, was compared to its constituent single proteins. Fourteen healthy elderly subjects received P4, whey, casein, soy, and pea (18 g/360 mL bolus) on five separate visits. Blood samples were collected at baseline until 240 min after intake. Amino acid availability was calculated using incremental maximal concentration (iCmax) and area under the curve (iAUC). Availability for P4 as a sum of all amino acids was similar to casein (iCmax and iAUC) and whey (iCmax) and higher vs. soy (iCmax and iAUC) and pea (iCmax). Individual amino acid availability (iCmax and iAUC) showed different profiles reflecting the composition of the protein sources: availability of leucine and methionine was higher for P4 vs. soy and pea, availability of arginine was higher for P4 vs. casein and whey. Conclusions: The P4 amino acid profile was reflected in post-prandial plasma levels and may be regarded as more balanced compared to the constituent single proteins.

The extraction of bioactive compounds in a biorefinery context could be a way to valorize agri-food byproducts, but there is a remaining part that also requires attention. Therefore, in this work the integrated extraction of phenolic compounds, including the bioactive oleuropein, and proteins from olive mill leaves was addressed following three schemes, including the use of ultrasound. This affected the total phenolic content (4475.5&ndash, 6166.9 mg gallic acid equivalents/100 g), oleuropein content (675.3&ndash, 1790.0 mg/100 g), and antioxidant activity (18,234.3&ndash, 25,459.0 &micro, mol trolox equivalents/100 g). No effect was observed on either the protein recovery or the content of sugars and lignin in the extraction residues. Concerning the recovery of proteins, three operational parameters were evaluated by response surface methodology. The optimum (63.1%) was achieved using NaOH 0.7 M at 100 &deg, C for 240 min. Then, the selected scheme was applied to olive leaves from the field, observing differences in the content of some of the studied components. It also changed the lignocellulosic profile of the extraction residues of both leaf types, which were enriched in cellulose. Overall, these results could be useful to diversify the valorization chain in the olive sector.

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300–390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space. Background: Cruciferin represents the most abundant protein in Brassica napus seeds where its efficient storage is essential under minimized space conditions. Results: The cruciferin complex has an octameric barrel-like structure of ∼420 kDa. Conclusion: The barrel-like structure represents a compact building block optimized for maximal storage of amino acids. Significance: Novel insights into structure and packing of seed storage proteins.

Sarcopenia, highly linked with fall, frailty, and disease burden, is an emerging problem in aging society. Higher protein intake has been suggested to maintain nitrogen balance. Our objective was to investigate whether pre-sarcopenia status was associated with lower protein intake. A total of 327 community-dwelling elderly people were recruited for a cross-sectional study. We adopted the multivariate nutrient density model to identify associations between low muscle mass and dietary protein intake. The general linear regression models were applied to estimate skeletal muscle mass index across the quartiles of total protein and vegetable protein density. Participants with diets in the lowest quartile of total protein density (

The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/ Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Salem, T. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mazzella, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wengier, D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Muschietti, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The protein complexes of the mitochondrial respiratory chain associate in defined ways forming supramolecular structures called respiratory supercomplexes or respirasomes. In plants, additional oxidoreductases participate in respiratory electron transport, e.g. the so-called "alternative NAD(P)H dehydrogenases" or an extra terminal oxidase called "alternative oxidase" (AOX). These additional enzymes were previously reported not to form part of respiratory supercomplexes. However, formation of respiratory supercomplexes might indirectly affect "alternative respiration" because electrons can be channeled within the supercomplexes which reduces access of the alternative enzymes towards their electron donating substrates. Here we report an investigation on the supramolecular organization of the respiratory chain in thermogenic Arum maculatum appendix mitochondria, which are known to have a highly active AOX for heat production. Investigations based on mild membrane solubilization by digitonin and protein separation by blue native PAGE revealed a very special organization of the respiratory chain in A. maculatum, which strikingly differs to the one described for the model plant Arabidopsis thaliana: (i) complex I is not present in monomeric form but exclusively forms part of a I + III2 supercomplex, (ii) the III2 + IV and I + III2 + IV supercomplexes are detectable but of low abundance, (iii) complex II has fewer subunits than in A. thaliana, and (iv) complex IV is mainly present as a monomer in a larger form termed "complex IVa". Since thermogenic tissue of A. maculatum at the same time has high AOX and I + III2 supercomplex abundance and activity, negative regulation of the alternative oxidase by supercomplex formation seems not to occur. Functional implications are discussed. © 2010 Elsevier Masson SAS. All rights reserved.

Mildly extracted peanut allergen Ara h 1 was previously reported to occur as an oligomeric complex. In this paper we describe how the protein in this oligomeric complex interacts noncovalently with phenolic compounds of the proanthocyanidin type. These interactions are being disrupted during anion exchange chromatography, resulting in the dissociation of the oligomeric Ara h 1 complex into protein trimers. By use of the known three-dimensional structure of ß-conglycinin, a soy protein homologous to Ara h 1, proline-rich regions were observed in silico on both faces of its trimeric structure, which are conserved in Ara h 1. These proline-rich regions could explain the binding of proanthocyanidins to Ara h 1 and the formation of multiple Ara h 1 trimer complexes. This was supported by the observation that the addition of peanut proanthocyanidins to trimeric Ara h 1 and to ß-conglycinin resulted in the formation of soluble oligomeric protein complexes. The structurally related legumin proteins do not contain such proline-rich regions on both sides of the protein, and proanthocyanidins were shown to have a lower affinity for legumin proteins from peanuts and soybeans (peanut allergen Ara h 3 and soy glycinin, respectively). Ara h 1 present as the oligomeric complex is assumed to be the representative form of the allergen in which it is consumed by humans. Keywords: Peanut allergy; Ara h 1; protein-polyphenol interaction; proanthocyanidins.

Background: Allergies to grass pollen are the number one cause of outdoor hay fever. The human immune system reacts with symptoms to allergen from pollen. Objective: We investigated the natural variability in release of the major group 5 allergen from grass pollen across Europe. Methods: Airborne pollen and allergens were simultaneously collected daily with a volumetric spore trap and a high-volume cascade impactor at 10 sites across Europe for 3 consecutive years. Group 5 allergen levels were determined with a Phl p 5-specific ELISA in 2 fractions of ambient air: particulate matter of greater than 10 mu m in diameter and particulate matter greater than 2.5 mu m and less than 10 mu m in diameter. Mediator release by ambient air was determined in Fc epsilon RI-humanized basophils. The origin of pollen was modeled and condensed to pollen potency maps. Results: On average, grass pollen released 2.3 pg of Phl p 5 per pollen. Allergen release per pollen (potency) varied substantially, ranging from less than 1 to 9 pg of Phl p 5 per pollen (5% to 95% percentile). The main variation was locally day to day. Average potency maps across Europe varied between years. Mediator release from basophilic granulocytes correlated better with allergen levels per cubic meter (r(2) = 0.80, P < .001) than with pollen grains per cubic meter (r(2) = 0.61, P < .001). In addition, pollen released different amounts of allergen in the non-pollenbearing fraction of ambient air, depending on humidity. Conclusion: Across Europe, the same amount of pollen released substantially different amounts of group 5 grass pollen allergen. This variation in allergen release is in addition to variations in pollen counts. Molecular aerobiology (ie, determining allergen in ambient air) might be a valuable addition to pollen counting. European Commission HIALINE

Background Foams are high porosity and low density materials. In nature, they are a common architecture. Some of their relevant technological applications include heat and sound insulation, lightweight materials, and tissue engineering scaffolds. Foams derived from natural polymers are particularly attractive for tissue culture due to their biodegradability and bio-compatibility. Here, the foaming potential of an extensive list of materials was assayed, including slabs elaborated from whole flour, the starch component only, or the protein fraction only of maize seeds. Methodology/Principal Findings We used supercritical CO2 to produce foams from thermoplasticized maize derived materials. Polyethylene-glycol, sorbitol/glycerol, or urea/formamide were used as plasticizers. We report expansion ratios, porosities, average pore sizes, pore morphologies, and pore size distributions for these materials. High porosity foams were obtained from zein thermoplasticized with polyethylene glycol, and from starch thermoplasticized with urea/formamide. Zein foams had a higher porosity than starch foams (88% and 85%, respectively) and a narrower and more evenly distributed pore size. Starch foams exhibited a wider span of pore sizes and a larger average pore size than zein (208.84 vs. 55.43 μm2, respectively). Proof-of-concept cell culture experiments confirmed that mouse fibroblasts (NIH 3T3) and two different prostate cancer cell lines (22RV1, DU145) attached to and proliferated on zein foams. Conclusions/Significance We conducted screening and proof-of-concept experiments on the fabrication of foams from cereal-based bioplastics. We propose that a key indicator of foamability is the strain at break of the materials to be foamed (as calculated from stress vs. strain rate curves). Zein foams exhibit attractive properties (average pore size, pore size distribution, and porosity) for cell culture applications; we were able to establish and sustain mammalian cell cultures on zein foams for extended time periods. © 2015 Trujillo-de Santiago et al.

Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described. © 2006 American Chemical Society.

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Bere 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA.

The intercellular washing fluid (IWF) of Malus domestica cv. Holsteiner Cox before and after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the leaves was investigated in a comparative manner. SDS-PAGE in combination with ESI Q-ToF mass spectrometry, and homology search in relevant data bases revealed the highly up-regulated expression of several pathogenesis-related plant proteins in the apoplast of the leaves treated with P. fluorescens. These proteins were β-1,3-glucanase, chitinase, thaumatin-like protein, ribonuclease-like protein, and a hevein-like protein. Moreover, a 9 kDa non-specific lipid transfer protein was significantly reduced after the application of P. fluorescens. The possible relevance of a pre-treatment of apple cultivars with the non-pathogenic bacterium P. fluorescens Bk3, as an alternative method to the treatment with fungicides, for increasing the resistance of susceptible apple cultivars against an infection with the fungus Venturia inaequalis is discussed.

Gel network formation of pea legumin (8.4% on a protein basis, pH 7.6) was monitored via dynamic rheological measurements. Gelation was performed in the absence and presence of the thiol-blocking reagent N-ethylmaleimide, at different rates of heating and cooling. Overall, it was shown that pea legumin gel formation was not effected by changes in the heating rate, and the two differently heated samples were unaffected by the addition of 20 mM NEM, which indicated that disulfide bonds were not essential within the network strands of these legumin gels. However, slowly cooling the legumin samples caused disulfide bonds to become involved within the network; this was observed by a large increase in gel strength that was then substantially reduced when repeating the sample in the presence of NEM. These experiments were repeated with soybean glycinin in order to determine whether a common model for gel formation of legumin-like proteins could be built, based upon molecular reasoning. The two proteins were affected in the same way by changes in the conditions used, but when applying a procedure of reheating and recooling the gel networks responded differently. Pea legumin gel networks were susceptible to rearrangements that caused the gels to become stronger after reheating/recooling, yet glycinin gel networks were not. It was concluded that the same physical and chemical forces drove the processes of denaturation, aggregation, and network formation. Each process can therefore be readily targeted for modification based upon molecular reasoning. Pea legumin and soybean glycinin gel networks had structurally different building blocks, however. A model of gelation aimed at texture control therefore requires additional information.

International audience; Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

This study consisted of two experiments, conducted to assess the impact of phase at which vegetable protein (VP) diets are introduced to broiler chicks, and preference of birds for diets based on soybean or canola meal (CM). Two hundred and ten day-old Cobb 500 chicks were randomly distributed into five dietary groups in the main experiment. One group was fed on animal protein (AP) diet all through to 21 days of age; two other groups were started on AP diet for 7 days and then switched to diets containing soybean meal (AP-SBM) or AP-CM, while two other diets (SBM-AP and CM-AP) were started on one of the VP diets for 7 days and then switched to AP diet. A sub-experiment on thirty birds raised on a commercial diet to 7 days was used in a feed selection test to quantify the preference of birds for the diets containing mainly CM or SBM. Chicks were reared under similar care and management conditions and the diets were iso-caloric and iso-nitrogenous. Results of the main experiment showed that chicks on CM-AP diet ate more (p

Background: Prosopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus.Results: Next generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads.Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic.Conclusions: This study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data.The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera. © 2013 Torales et al.; licensee BioMed Central Ltd. Fil:Rivarola, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Acuña, C.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Fernández, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Hopp, H.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Poltri, S.N.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.

Snakin-1 (SN1) is an antimicrobial cysteine-rich peptide isolated from potato (Solanum tuberosum) that was classified as a member of the Snakin/Gibberellic Acid Stimulated in Arabidopsis protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1, potato lines did not show remarkable morphological differences from the wild type; SN1 silencing resulted in reduced height, which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of the adaxial epidermis of mature leaves revealed that silenced lines had 70% to 90% increases in mean cell size with respect to wild-type leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-green fluorescent protein fusion protein was localized in plasma membrane, and bimolecular fluorescence complementation assays revealed that SN1 self-interacted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry, Fourier transform infrared spectroscopy, and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in seven cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism, and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. © 2011 American Society of Plant Biologists. All Rights Reserved. Fil:Almasia, N.I. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Fernandez, P.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Hopp, H.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Estevez, J.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Carrari, F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Vazquez-Rovere, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

In situPCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues.In situPCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants,in situRT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by usingin situRT-PCR inRosa hybridaflower buds and leaves. As target genes, we used the abundantβ-actinandRhFULgene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg2+ (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5mMATP. Remarkably, the withdrawal of ATP or Mg2+ brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ∼30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg129 and Arg152, are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Aran, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mora-García, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wolosiuk, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The bulbs of tulip (Tulipa gesneriana L.) evs Negrita and Cassini and the corms of freesia (Freesia refracta Klatt.) cvs Polaris and Aladin were used in the study to investigate molecular changes during the storage and sprouting stage besides phenologic observations. One part of the bulbs and corms were directly planted (DP) into greenhouse without any treatment. Another part of the bulbs and corms were either stored in room condition (RC; 18-25 degrees C and 55-65% RH) or in normal atmosphere (NA; 5+/-1 degrees C and 70-85% RH) for 120 days before planting. In both storage treatments, the bulbs and corms were transplanted to the greenhouse in three periods with 40 days intervals. In every period, a part of the bulbs and corms were taken for the molecular analyses before planting. In addition, some of DP bulbs and corms were removed from soil at the same periods for molecular analyses. The storage of the bulbs and corms reduced the sprouting durations in comparison to the DP treatment. However, NA storage either reduced (in tulip) or inhibit (in freesia) to proportions of plant formation. The highest and the lowest ascorbate peroxidase (APX) activity were found during NA storage and DP treatment, respectively, in both species. In addition APX activity was higher in tulip than in freesia. Sharp decrease in total soluble protein (TSP) content particularly after 40 and 60 days of almost all treatments can be an evidence of breakdown of storage proteins. TSP profiles of tulip bulbs showed a major polypeptide, 18 kDa, with different band intensities according to the treatments. In freesia, 66 kDa protein accumulated especially in last two sampling periods of NA storage, whereas 58 kDa protein became invisible in last two sampling periods of NA storage. Since prolonged NA storage inhibits the sprouting and proportions of plant formation, 66 kDa and 58 kDa bands might be related to dormancy or sprouting in freesia.

Background: Eukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.Results: We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region.Conclusions: These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants. © 2011 González et al; licensee BioMed Central Ltd. Fil:González, R.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ricardi, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Iusem, N.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2 2, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses., This work was made possible by funds to JPM from the Swiss National Science Foundation (grant 125370). Jose´ Le´on was supported by Ministerio de Ciencia e Innovacion of Spain (BIO2008-00839) and Ricardo Mir by a FPU Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Plants have evolved sophisticated mechanisms to sense and respond to pathogen attacks. Resistance against necrotrophic pathogens generally requires the activation of the jasmonic acid (JA) signaling pathway, whereas the salicylic acid (SA) signaling pathway is mainly activated against biotrophic pathogens. SA can antagonize JA signaling and vice versa. Here, we report that the necrotrophic pathogen Botrytis cinerea exploits this antagonism as a strategy to cause disease development. We show that B. cinerea produces an exopolysaccharide, which acts as an elicitor of the SA pathway. In turn, the SA pathway antagonizes the JA signaling pathway, thereby allowing the fungus to develop its disease in tomato (Solanum lycopersicum). SA-promoted disease development occurs through Nonexpressed Pathogen Related1. We also show that the JA signaling pathway required for tomato resistance against B. cinerea is mediated by the systemin elicitor. These data highlight a new strategy used by B. cinerea to overcome the plant's defense system and to spread within the host. © 2011 American Society of Plant Biologists. All rights reserved. Fil:Rigano, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rodriguez, M.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Vojnov, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation. Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lisi, Christian Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Alleva, Karina Edith. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Pagano, María Elba. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Ardila, Fernando. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Mozzicafreddo, Matteo. Universita Degli Di Camerino; Italia Fil: Cuccioloni, Massimiliano. Universita Degli Di Camerino; Italia Fil: Angeletti, Mauro. Universita Degli Di Camerino; Italia Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina

In strawberry, the putative participation of aquaporins should be considered during fruit ripening. Furthermore, the availability of different firmness cultivars in this non-climacteric fruit is a very useful tool to determine their involvement in softening. In a previous work, the cloning of a strawberry fruit-specific aquaporin, FaPIP1;1, which showed an expression profile associated with fruit ripening was reported. Here, FaPIP2;1, an aquaporin subtype of PIP2 was cloned and its functional characterization in Xenopus oocytes determined. The FaPIP2;1 gene encodes a water channel with high water permeability (Pf) that is regulated by cytosolic pH. Interestingly, the co-expression of both FaPIP subtypes resulted in an enhancement of water permeability, showing Pf values that exceeds their individual contribution. The expression pattern of both aquaporin subtypes in two cultivars with contrasting fruit firmness showed that the firmer cultivar (Camarosa) has a higher accumulation of FaPIP1 and FaPIP2 mRNAs during fruit ripening when compared with the softer cultivar (Toyonoka). In conclusion, not only FaPIP aquaporins showed an expression pattern associated with fruit firmness but it was also shown that the enhancement of water transfer through the plasma membrane is coupled to the presence/absence of the co-expression of both subtypes. © 2010 The Author(s). Fil:Mut, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

A cytoplasmically inherited chlorophyll-deficient mutant of barley (Hordeum vulgare) termed cytoplasmic line 3 (CL3), displaying a viridis (homogeneously light-green colored) phenotype, has been previously shown to be affected by elevated temperatures. In this article, biochemical, biophysical, and molecular approaches were used to study the CL3 mutant under different temperature and light conditions. The results lead to the conclusion that an impaired assembly of photosystem I (PSI) under higher temperatures and certain light conditions is the primary cause of the CL3 phenotype. Compromised splicing of ycf3 transcripts, particularly at elevated temperature, resulting from a mutation in a noncoding region (intron 1) in the mutant ycf3 gene results in a defective synthesis of Ycf3, which is a chaperone involved in PSI assembly. The defective PSI assembly causes severe photoinhibition and degradation of PSII. © 2009 American Society of Plant Biologists. Fil:Landau, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Lainez, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Background and AimsPre-harvest sprouting susceptibility in grain sorghum (Sorghum bicolor) is related to low seed dormancy and reduced embryo sensitivity to inhibition of germination by abscisic acid (ABA). Intra-specific variability for pre-harvest sprouting might involve differential regulation of ABA signalling genes.MethodsSorghum genes encoding homologues for ABA signalling components from other species (ABI5, ABI4, VP1, ABI1 and PKABA1) were studied at the transcriptional and protein level (ABI5) during grain imbibition for two sorghum lines with contrasting sprouting phenotypes and in response to hormones.Key ResultsTranscript levels of these genes and protein levels of ABI5 were higher in imbibed immature caryopses of the more dormant line. Dormancy loss was related to lower transcript levels of these genes and lower ABI5 protein levels in both genotypes. Exogenous ABA inhibited germination of isolated embryos but failed to prevent ABI5 rapid decrease supporting a role for the seed coat in regulating ABI5 levels.ConclusionsSeveral genes involved in ABA signalling are regulated differently in imbibed caryopses from two sorghum lines with contrasting pre-harvest sprouting response before - but not after - physiological maturity. A role for ABI5 in the expression of dormancy during grain development is discussed. Fil:Rodríguez, M.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mendiondo, G.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Maskin, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Gudesblat, G.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Iusem, N.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Previous work suggested that the apoplastic phenol composition and its interaction with apoplastic class III peroxidases (PODs) are decisive in the development or avoidance of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.). This study characterizes apoplastic PODs with particular emphasis on the activities of specific isoenzymes and their modulation by phenols in the Mn-sensitive cowpea cultivar TVu 91 as affected by Mn and silicon (Si) supply. Si reduced Mn-induced toxicity symptoms without affecting the Mn uptake. Blue Native-PAGE combined with Nano-LC-MS/MS allowed identification of a range of POD isoenzymes in the apoplastic washing fluid (AWF). In Si-treated plants Mn-mediated induction of POD activity was delayed. Four POD isoenzymes eluted from the BN gels catalysed both H(2)O(2)-consuming and H(2)O(2)-producing activity with pH optima at 6.5 and 5.5, respectively. Four phenols enhanced NADH-peroxidase activity of these isoenzymes in the presence of Mn(2+) (p-coumaric=vanillic>>benzoic>ferulic acid). p-Coumaric acid-enhanced NADH-peroxidase activity was inhibited by ferulic acid (50%) and five other phenols (50-90%). An independent component analysis (ICA) of the total and apoplastic GC-MS-based metabolome profile showed that Mn, Si supply, and the AWF fraction (AWF(H(2)O), AWF(NaCl)) significantly changed the metabolite composition. Extracting non-polar metabolites from the AWF allowed the identification of phenols. Predominantly NADH-peroxidase activity-inhibiting ferulic acid appeared to be down-regulated in Mn-sensitive (+Mn, -Si) and up-regulated in Mn-tolerant (+Si) leaf tissue. The results presented here support the previously hypothesized role of apoplastic NADH-peroxidase and its activity-modulating phenols in Mn toxicity and Si-enhanced Mn tolerance.

The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H2O2, ·O2-, and HO· production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis- (ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 μM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 μM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl2 (an ·O2- dismutating agent) or the HO· scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25-30% of the maize leaf blade elongation. Fil:Rodríguez, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Menéndez, A.B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (

This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (

Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92°C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92°C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Monosaccharides transporters play important roles in assimilate supply for sink tissue development. In this study, a new monosaccharide transporter gene OsMST6 was identified from rice (Oryza sativa L.). The predicted OsMST6 protein shows typical features of sugar transporters and shares 79.6% identity with the rice monosaccharide transporter OsMST3. Heterologous expression in yeast (Saccharomyces cerevisiae) demonstrated that OsMST6 is a broad-spectrum monosaccharide transporter, with a K m of 266.1 μΜ for glucose. OsMST6-green fluorescent protein fusion protein is localized to the plasma membrane in plant. Semi-quantitative RT-PCR analysis exhibited that OsMST6 is expressed in all tested organs/tissues. In developing seeds, OsMST6 expression level is high at the early and middle grain filling stages and gradually declines later. Further analysis detected its expression in both maternal and filial tissues. RNA in situ hybridization analysis indicated that OsMST6 is predominantly expressed in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm of young seeds, in mesophyll cells of source leaf blades, and in pollens and the connective vein of anthers. In addition, OsMST6 expression is up-regulated by salt stress and sugars. The physiological role of OsMST6 for seed development and its roles in other sink and source tissues are discussed. © 2008 Springer-Verlag.

This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (

Monosaccharides transporters play important roles in assimilate supply for sink tissue development. In this study, a new monosaccharide transporter gene OsMST6 was identified from rice (Oryza sativa L.). The predicted OsMST6 protein shows typical features of sugar transporters and shares 79.6% identity with the rice monosaccharide transporter OsMST3. Heterologous expression in yeast (Saccharomyces cerevisiae) demonstrated that OsMST6 is a broad-spectrum monosaccharide transporter, with a K m of 266.1 μΜ for glucose. OsMST6-green fluorescent protein fusion protein is localized to the plasma membrane in plant. Semi-quantitative RT-PCR analysis exhibited that OsMST6 is expressed in all tested organs/tissues. In developing seeds, OsMST6 expression level is high at the early and middle grain filling stages and gradually declines later. Further analysis detected its expression in both maternal and filial tissues. RNA in situ hybridization analysis indicated that OsMST6 is predominantly expressed in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm of young seeds, in mesophyll cells of source leaf blades, and in pollens and the connective vein of anthers. In addition, OsMST6 expression is up-regulated by salt stress and sugars. The physiological role of OsMST6 for seed development and its roles in other sink and source tissues are discussed. © 2008 Springer-Verlag.

An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the detection of traces of mustard protein in mustard seed-derived flavoring ingredients. Limited cross-reactivity testing showed that no other plant proteins reacted significantly. From the animal proteins tested, only milk showed some cross-reactivity. With this sensitive assay, it was shown that refined mustard seed oil produced by steam distillation does not contain detectable amounts of mustard protein. Mustard seed oil is used as a flavoring in very low quantities, typically between 40 and 200 mg/kg. Thus, 100 g of a food product flavored with 200 mg of mustard seed oil per kg containing

Mildly extracted peanut allergen Ara h 1 was previously reported to occur as an oligomeric complex. In this paper we describe how the protein in this oligomeric complex interacts noncovalently with phenolic compounds of the proanthocyanidin type. These interactions are being disrupted during anion exchange chromatography, resulting in the dissociation of the oligomeric Ara h 1 complex into protein trimers. By use of the known three-dimensional structure of β-conglycinin, a soy protein homologous to Ara h 1, proline-rich regions were observed in silico on both faces of its trimeric structure, which are conserved in Ara h 1. These proline-rich regions could explain the binding of proanthocyanidins to Ara h 1 and the formation of multiple Ara h 1 trimer complexes. This was supported by the observation that the addition of peanut proanthocyanidins to trimeric Ara h 1 and to β-conglycinin resulted in the formation of soluble oligomeric protein complexes. The structurally related legumin proteins do not contain such proline-rich regions on both sides of the protein, and proanthocyanidins were shown to have a lower affinity for legumin proteins from peanuts and soybeans (peanut allergen Ara h 3 and soy glycinin, respectively). Ara h 1 present as the oligomeric complex is assumed to be the representative form of the allergen in which it is consumed by humans. © 2007 American Chemical Society.

Monosaccharide transporters mediate the membrane transport of a variable range of monosaccharides, which plays a crucial role in sugar distribution throughout the plant. To investigate the significance of monosaccharide transporters for rice (Oryza sativa L.) seed development, cDNA of a new putative monosaccharide transporter gene OsMST4 was isolated. The deduced OsMST4 protein shows typical features of monosaccharide transporters, and shares high homology with other plant homologues. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that OsMST4 is a functional monosaccharide transporter capable of transporting glucose, fructose, mannose and galactose. Transcriptional analysis revealed that OsMST4 is expressed in all tested organs/tissues. In developing caryopses, its expression is high at the early and middle grain filling stages, and declines gradually to low levels after that. Further analysis revealed that it is expressed in both the maternal tissue and the filial tissue, with its highest expression in embryo. Cellular location in young caryopses through RNA in situ hybridization showed that OsMST4 mRNA mainly accumulates in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm. The expression pattern of OsMST4 was further confirmed by histochemical analysis of the OsMST4-promoter-β-glucuronidase (GUS) transgenic rice plants. These data indicate that OsMST4 is actively involved in monosaccharides supply for seed development during the course of grain filling. In addition, the cell type-specific expression patterns of OsMST4 in other sink and source tissues were also investigated, and its corresponding physiological roles were discussed. © 2007 Springer Science+Business Media B.V.

Monosaccharide transporters mediate the membrane transport of a variable range of monosaccharides, which plays a crucial role in sugar distribution throughout the plant. To investigate the significance of monosaccharide transporters for rice (Oryza sativa L.) seed development, cDNA of a new putative monosaccharide transporter gene OsMST4 was isolated. The deduced OsMST4 protein shows typical features of monosaccharide transporters, and shares high homology with other plant homologues. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that OsMST4 is a functional monosaccharide transporter capable of transporting glucose, fructose, mannose and galactose. Transcriptional analysis revealed that OsMST4 is expressed in all tested organs/tissues. In developing caryopses, its expression is high at the early and middle grain filling stages, and declines gradually to low levels after that. Further analysis revealed that it is expressed in both the maternal tissue and the filial tissue, with its highest expression in embryo. Cellular location in young caryopses through RNA in situ hybridization showed that OsMST4 mRNA mainly accumulates in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm. The expression pattern of OsMST4 was further confirmed by histochemical analysis of the OsMST4-promoter-β-glucuronidase (GUS) transgenic rice plants. These data indicate that OsMST4 is actively involved in monosaccharides supply for seed development during the course of grain filling. In addition, the cell type-specific expression patterns of OsMST4 in other sink and source tissues were also investigated, and its corresponding physiological roles were discussed. © 2007 Springer Science+Business Media B.V.

An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the detection of traces of mustard protein in mustard seed-derived flavoring ingredients. Limited cross-reactivity testing showed that no other plant proteins reacted significantly. From the animal proteins tested, only milk showed some cross-reactivity. With this sensitive assay, it was shown that refined mustard seed oil produced by steam distillation does not contain detectable amounts of mustard protein. Mustard seed oil is used as a flavoring in very low quantities, typically between 40 and 200 mg/kg. Thus, 100 g of a food product flavored with 200 mg of mustard seed oil per kg containing

Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described. © 2006 American Chemical Society.

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Bere 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA.

Gel network formation of pea legumin (8.4% on a protein basis, pH 7.6) was monitored via dynamic rheological measurements. Gelation was performed in the absence and presence of the thiol-blocking reagent N-ethylmaleimide, at different rates of heating and cooling. Overall, it was shown that pea legumin gel formation was not effected by changes in the heating rate, and the two differently heated samples were unaffected by the addition of 20 mM NEM, which indicated that disulfide bonds were not essential within the network strands of these legumin gels. However, slowly cooling the legumin samples caused disulfide bonds to become involved within the network; this was observed by a large increase in gel strength that was then substantially reduced when repeating the sample in the presence of NEM. These experiments were repeated with soybean glycinin in order to determine whether a common model for gel formation of legumin-like proteins could be built, based upon molecular reasoning. The two proteins were affected in the same way by changes in the conditions used, but when applying a procedure of reheating and recooling the gel networks responded differently. Pea legumin gel networks were susceptible to rearrangements that caused the gels to become stronger after reheating/recooling, yet glycinin gel networks were not. It was concluded that the same physical and chemical forces drove the processes of denaturation, aggregation, and network formation. Each process can therefore be readily targeted for modification based upon molecular reasoning. Pea legumin and soybean glycinin gel networks had structurally different building blocks, however. A model of gelation aimed at texture control therefore requires additional information.

Objective: To study differences in dietary intake between adults with different socioeconomic status (SES) and trends over time. Design: Cross-sectional study based on data of three Dutch National Food Consumption Surveys (DNFCS-1 1987/88; DNFCS-2 1992; DNFCS-3 1997/98), obtained from a panel by a stratified probability sample of the non-institutionalized Dutch population. Subjects: A total of 6008 men and 6957 women aged 19 y and over. Methods: Dietary intake was assessed with a 2 day dietary record. Background information was obtained by structured questionnaire. Sociodemographic variables were available from panel information. SES, based on educational level, occupation and occupational position was categorized into (very) low, middle and high. Analysis of variance with age as covariable was used to explore the effects of SES on dietary intake and anthropometry. Statistical tests for trend were carried out with models in which week-weekend-day effects and an interaction term of time with SES were also included. Results: The prevalence of obesity and skipping of breakfast was higher among people with a low SES. In all three surveys, subjects in the (very) low SES group reported having a higher consumption of potatoes, meat and meat products, visible fats, coffee and soft drinks (men only). Subjects with a high SES reported consuming more vegetables, cheese and alcohol. As regards nutrients, in all surveys a higher SES was associated with higher intake of vegetable protein, dietary fibre and most micronutrients. A higher SES was also associated with a lower fat intake but the differences between social classes were rather small and not consistent when the contribution of alcohol to energy intake was taken into account. Conclusion: In general, dietary intake among subjects in higher SES groups tended to be closer to the recommendations of the Netherlands Food and Nutrition Council and this phenomenon was quite stable over a period of 10y.

Objective: To study differences in dietary intake between adults with different socioeconomic status (SES) and trends over time. Design: Cross-sectional study based on data of three Dutch National Food Consumption Surveys (DNFCS-1 1987/88; DNFCS-2 1992; DNFCS-3 1997/98), obtained from a panel by a stratified probability sample of the non-institutionalized Dutch population. Subjects: A total of 6008 men and 6957 women aged 19 y and over. Methods: Dietary intake was assessed with a 2 day dietary record. Background information was obtained by structured questionnaire. Sociodemographic variables were available from panel information. SES, based on educational level, occupation and occupational position was categorized into (very) low, middle and high. Analysis of variance with age as covariable was used to explore the effects of SES on dietary intake and anthropometry. Statistical tests for trend were carried out with models in which week-weekend-day effects and an interaction term of time with SES were also included. Results: The prevalence of obesity and skipping of breakfast was higher among people with a low SES. In all three surveys, subjects in the (very) low SES group reported having a higher consumption of potatoes, meat and meat products, visible fats, coffee and soft drinks (men only). Subjects with a high SES reported consuming more vegetables, cheese and alcohol. As regards nutrients, in all surveys a higher SES was associated with higher intake of vegetable protein, dietary fibre and most micronutrients. A higher SES was also associated with a lower fat intake but the differences between social classes were rather small and not consistent when the contribution of alcohol to energy intake was taken into account. Conclusion: In general, dietary intake among subjects in higher SES groups tended to be closer to the recommendations of the Netherlands Food and Nutrition Council and this phenomenon was quite stable over a period of 10y.

We have suggested a model for the electronic excited states of the minor plant antenna, CP29, by incorporating a considerable part of the current information offered by structure determination, site-directed mutagenesis, and spectroscopy in the modeling. We have assumed that the electronic excited states of the complex have been decided by the chlorophyll-chlorophyll (Chl) and Chl-protein interactions and have modeled the Coulombic interaction between a pair of Chls in the point-dipole approximation and the Chl-protein interactions are treated as empirical fit parameters. We have suggested the Qy dipole moment orientations and the site energies for all the chlorophylls in the complex through a simultaneous simulation of the absorption and linear dichroism spectra. The assignments proposed have been discussed to yield a satisfactory reproduction of all prominent features of the absorption, linear and circular dichroism spectra as well as the key spectral and temporal characteristics of the energy transfer processes among the chlorophylls. The orientations and the spectral assignments obtained by relatively simple exciton calculations have been necessary to provide a good point of departure for more detailed treatments of structure-function relationship in CP29. Moreover, it has been discussed that the CP29 model suggested can guide the studies for a better understanding of the structure-function relationship in the major plant antenna, LHCII.