Primary Metabolism Is Distinctly Modulated by Plant Resistance Inducers in Coffea arabica Leaves Infected by Hemileia vastatrix.

Epidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.

Phellem Cell-Wall Components Are Discriminants of Cork Quality in Quercus suber.

Cork is a renewable, non-wood high valued forest product, with relevant ecological and economic impact in the Mediterranean-type ecosystems. Currently, cork is ranked according to its commercial quality. The most valuable planks are chosen for cork stoppers production. Cork planks with adequate thickness and porosity are classified as stoppable quality cork (SQC). The chemical composition of cork is known, but the regulation of metabolic pathways responsible of cork production and composition, hence of cork quality, is largely unknown. Here, we tested the hypothesis that post-genomic events may be responsible for the development of SQC and N-SQC (non-stoppable quality cork). Here, we show that combined proteomics and targeted metabolomics (namely soluble and cell wall bound phenolics) analyzed on recently formed phellem allows discriminate cork planks of different quality. Phellem cells of SQC and N-SQC displayed different reducing capacity, with consequential impact on both enzymatic pathways (e.g., glycolysis) and other cellular functions, including cell wall assembly and suberization. Glycolysis and respiration related proteins were abundant in both cork quality groups, whereas the level of several proteins associated to mitochondrial metabolism was higher in N-SQC. The soluble and cell wall-bound phenolics in recently formed phellem clearly discriminated SQC from N-SCQ. In our study, SQC was characterized by a high incorporation of aromatic components of the phenylpropanoid pathway in the cell wall, together with a lower content of hydrolysable tannins. Here, we propose that the level of hydrolysable tannins may represent a valuable diagnostic tool for screening recently formed phellem, and used as a proxy for the quality grade of cork plank produced by each tree.

Maize IgE binding proteins: each plant a different profile?

BACKGROUND: Allergies are nearly always triggered by protein molecules and the majority of individuals with documented immunologic reactions to foods exhibit IgE hypersensitivity reactions. In this study we aimed to understand if natural differences, at proteomic level, between maize populations, may induce different IgE binding proteins profiles among maize-allergic individuals. We also intended to deepen our knowledge on maize IgE binding proteins. RESULTS: In order to accomplish this goal we have used proteomic tools (SDS-PAGE and 2-D gel electrophoresis followed by western blot) and tested plasma IgE reactivity from four maize-allergic individuals against four different protein fractions (albumins, globulins, glutelins and prolamins) of three different maize cultivars. We have observed that maize cultivars have different proteomes that result in different IgE binding proteins profiles when tested against plasma from maize-allergic individuals. We could identify 19 different maize IgE binding proteins, 11 of which were unknown to date. Moreover, we found that most (89.5%) of the 19 identified potential maize allergens could be related to plant stress. CONCLUSIONS: These results lead us to conclude that, within each species, plant allergenic potential varies with genotype. Moreover, considering the stress-related IgE binding proteins identified, we hypothesise that the environment, particularly stress conditions, may alter IgE binding protein profiles of plant components.

A proteomics study of the induction of somatic embryogenesis in Medicago truncatula using 2DE and MALDI-TOF/TOF.

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.

Initial water deficit effects on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance: metabolic reorganization prior to early stress responses.

The early (2-4 d) effects of slowly imposed soil water deficit on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance in different organs (leaf blade, stem stele, stem cortex, and root) were evaluated on 23-d-old plants (growth chamber assay). Our work shows that several metabolic adjustments occurred prior to alteration of the plant water status, implying that water deficit is perceived before the change in plant water status. The slow, progressive decline in soil water content started to be visible 3 d after withholding water (3 DAW). The earliest plant changes were associated with organ-specific metabolic responses (particularly in the leaves) and with leaf conductance and only later with plant water status and photosynthetic rate (4 DAW) or photosynthetic capacity (according to the Farquhar model; 6 DAW). Principal component analysis (PCA) of the physiological parameters, the carbohydrate and the hormone levels and their relative values, as well as leaf water-soluble metabolites full scan data (LC-MS/MS), showed separation of the different sampling dates. At 6 DAW classically described stress responses are observed, with plant water status, ABA level, and root hormonal balance contributing to the separation of these samples. Discrimination of earlier stress stages (3 and 4 DAW) is only achieved when the relative levels of indole-3-acetic acid (IAA), cytokinins (Cks), and carbon metabolism (glucose, sucrose, raffinose, and starch levels) are taken into account. Our working hypothesis is that, in addition to single responses (e.g. ABA increase), the combined alterations in hormone and carbohydrate levels play an important role in the stress response mechanism. Response to more advanced stress appears to be associated with a combination of cumulative changes, occurring in several plant organs. The carbohydrate and hormonal balance in the leaf (IAA to bioactive-Cks; soluble sugars to IAA and starch to IAA; relative abundances of the different soluble sugars) flag the initial responses to the slight decrease in soil water availability (10-15% decrease). Further alterations in sucrose to ABA and in raffinose to ABA relative values (in all organs) indicate that soil water availability continues to decrease. Such alterations when associated with changes in the root hormone balance indicate that the stress response is initiated. It is concluded that metabolic balance (e.g. IAA/bioactive Cks, carbohydrates/IAA, sucrose/ABA, raffinose/ABA, ABA/IAA) is relevant in triggering adjustment mechanisms.

Metabolic analysis revealed altered amino acid profiles in Lupinus albus organs as a result of boron deficiency.

We analysed the changes in the metabolites of Lupinus albus organs (leaf-blades, petioles, apexes, hypocotyls and roots) as a consequence of B deficiency. The deficiency did not affect malate concentration and induced only minor changes in the sugar content, suggesting that the carbohydrate metabolism is little affected by the deficiency. Contrarily, marked changes in the content of free amino acids were observed, with some specific variations associated with the different organs. These changes indicate that various aspects of metabolism implicated in the amino acid accumulation were affected by B deficiency. Most of the detected changes appear to have implications with some stress responses or signalling processes. Asparagine and proline that increase in many stresses also accumulated in petioles, apexes and hypocotyls. Accumulation of γ-aminobutyric acid shunt amino acids, indicative of production of reactive oxygen species, occurs in the same three organs and also the roots. The increase in the branched-chain amino acids, observed in all organs, suggests the involvement of B with the cytoskeleton, whereas glycine decrease in leaf-blades and active growing organs (apexes and roots) could be associated with the proposed role of this amino acids in plant signalling in processes that might be associated with the decreased growth rates observed in B deficiency. Despite the admitted importance of free amino acids in plant metabolism, the available information on this matter is scarce. So our results bring new information concerning the effects of B deficiency in the metabolism of the several L. albus organs.

Detection of novel trypsin inhibitors in the cotyledons of Phaseolus vulgaris seeds.

Protease inhibitors play important roles in plants in association with stress. Trypsin inhibitors (TIs) in particular are known to act as protective agents against insect and pathogen attacks. The growing relevance of these inhibitors requires expedited techniques for their detection. By using the two-dimensional electrophoresis (2-DE) reverse zymography technique, we identified, from the crude extract of bean seeds, nine novel polypeptides that showed trypsin inhibitor activity. One of these polypeptide inhibitors yielded no homology in the database, which can be an indication that we are found a new protein with unique TI properties. The remaining showed homology with proteins annotated in the UniProt database and form, together with a Kunitz type inhibitor, a new TI cluster for Phaseolus spp. Three of these polypeptides showed additional high homology with lectins, likely indicating that they have lectin properties, while the other five showed high homology with alpha-amylase inhibitors, indicating that they probably have a dual inhibitory effect against trypsin and the alpha-amylase enzyme. These bifunctional inhibitors can be highly useful for crop management, since the two inhibitory activities are important for plants when coping with pathogen and pest attacks.

Taking advantage of nonspecific trypsin cleavages for the identification of seed storage proteins in cereals.

The lack of basic amino acids in seed storage proteins has resulted in the proposal to use chymotrypsin in their study. A comparative study of trypsin and chymotrypsin digestion initially confirmed this preference; however, reanalysis of the trypsin data set defining the specificity as 'semitrypsin' provided enough extra data to bridge the gap between both proteases. Rationale as to why numerous semitryptic peptides are observed in the study of these proteins is provided.

Associating wound-related changes in the apoplast proteome of Medicago with early steps in the ROS signal-transduction pathway.

Early wound-related changes in the leaf apoplast proteome of Medicago truncatula have been characterized by 2-DE and MALDI-TOF/TOF and the differential expression of 28/110 extracellular proteins could be reproducibly observed 6 h after wounding. Wounding induced an initial (0-30 min) burst of O2-, followed by a later (3-6 h) production of O2- and H2O2. The infiltration of 5 microM DPI

Analysis of carbohydrates in Lupinus albus stems on imposition of water deficit, using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry.

This work reports the development and application of a negative ion mode online LC-ESI-MS method for studying the effect of water deficit on the carbohydrate content of Lupinus albus stems, using a porous graphitic carbon (PGC) stationary phase and an ion trap mass spectrometer. Using this method, separation and detection of several water soluble carbohydrates, ranging from mono-, di-, and oligosaccharides (raffinose, stachyose, and verbascose) to sugar alcohols was achieved in approximately 10 min. This on-line PGC-LC-ESI-MS method shows good linearity with correlation coefficients R(2)>0.99, selectivity, short analysis time, and limits of detection (LOD) ranging from 0.4 to 9 pmol for sugars and 4-20 pmol for sugar alcohols. This PGC-LC-ESI-MS method is sensitive and allowed us to detect even small alterations in carbohydrate levels in L. albus stems that resulted from a mild/early water deficit (nmol g(-1)DW). This paper describes details of our method and its application to the quantitative analysis of water soluble underivatised carbohydrates extracted from L. albus stem tissues that have been subjected to early and severe water deficit conditions, followed by a rewatering period.

Dehydrins in Lupinus albus: pattern of protein accumulation in response to drought.

Dehydrins (DHNs) are proteins that accumulate abundantly in various plant tissues in response to environmental stresses and during seed maturation, possibly assisting cells in tolerating dehydration. White lupins (Lupinus albus L.) are able to withstand periods of severe water deficit (WD) and previous work suggested that the stem plays a central role as a survival structure. To investigate DHNs involvement in this strategy, we studied tissue specific protein accumulation of a RAB16-like DHN in lupin during a progressive WD and early recovery. Differences were found between leaves, stems and roots. In leaves and roots, the accumulation of the RAB16-like DHN was independent of the water status whereas in the stem (cortex and stele), DHNs were only detected under severe plant WD (stele relative water content, RWC, reduction of 6-7% and cortex RWC reduction of 20%). DHN mRNA analysis by RT-PCR, showed the presence of one DHN mRNA regardless of the tissue or the plant water status.

A proteomic study to identify soya allergens--the human response to transgenic versus non-transgenic soya samples.

BACKGROUND: In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya versus its non-transgenic control. METHODS: We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples. RESULTS: We identified 2 new potential soybean allergens--one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study. CONCLUSION: Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment.

Immunolocalization of a class III chitinase in two muskmelon cultivars reacting differently to Fusarium oxysporum f. sp. melonis.

Fusarium oxysporum f. sp. melonis is a highly specialized fungus that attacks the root system of melon (Cucumis melo L.). In this work the presence of a class III chitinase was examined by immunological techniques in the root and stem base of a susceptible (cv. Galia) and a resistant (cv. Bredor) melon during the infection process. By immunolocalization it was not possible to detect the constitutive presence of class III chitinase in any of the cultivars. However, the immunolabelling appeared in the root tissues of both cultivars as a consequence of wounding and of infection by F. oxysporum f. sp. melonis. Distinct patterns of chitinase detection were observed in the roots of the two cultivars as the infection progressed. Furthermore, by western blotting distinct class III chitinase isoforms were detected, which responded differently to the F. oxysporum f. sp. melonis infection. Our results strongly indicate that a relationship exists between class III chitinase and melon resistance to Fusarium infection, and that the resistance is associated with certain isoforms of this enzyme.

Lack of detectable allergenicity of transgenic maize and soya samples.

BACKGROUND: The safety issues regarding foods derived from genetically modified (GM) plants are central to their acceptance into the food supply. The potential allergenicity of proteins newly introduced in GM foods is a major safety concern. OBJECTIVE: We sought to monitor, in potentially sensitive human populations, the allergenicity effects of 5 GM materials obtained from sources with no allergenic potential and already under commercialization in the European Union. METHODS: We have performed skin prick tests with protein extracts prepared from transgenic maize (MON810, Bt11, T25, Bt176) and soya (Roundup Ready) samples and from nontransgenic control samples in 2 sensitive groups: children with food and inhalant allergy and individuals with asthma-rhinitis. We have also tested IgE immunoblot reactivity of sera from patients with food allergy to soya (Roundup Ready) and maize (MON810, Bt11, Bt176) samples, as well as to the pure transgenic proteins (CryIA[b] and CP4 5-enolpyruvylshikimate-3-phosphate synthase). RESULTS: None of the individuals undergoing tests reacted differentially to the transgenic and nontransgenic samples under study. None of the volunteers tested presented detectable IgE antibodies against pure transgenic proteins. CONCLUSION: The transgenic products under testing seem to be safe in terms of allergenic potential. We propose postmarket testing as an important screening strategy for putative allergic sensitization to proteins introduced in transgenic plants.

Sugar metabolism in developing lupin seeds is affected by a short-term water deficit.

A short-term water deficit (WD) imposed during the pre-storage phase of lupin seed development [15-22 d after anthesis (DAA)] accelerated seed maturation and led to smaller and lighter seeds. During seed development, neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) have a central role in carbohydrate metabolism. Neutral invertase is predominant during early seed development (up to 40 DAA) and sucrose synthase during the growing and storage phase (40-70 DAA). The contribution of acid invertase is marginal. WD decreased sucrose synthase activity by 2-fold and neutral invertase activity by 5-6-fold. These changes were linked to a large decrease in sucrose ( approximately 60%) and an increase of the hexose:sucrose ratio. Rewatering restored sucrose synthase activity to control levels while neutral invertase activity remained depressed (30-60%). A transient accumulation of starch observed in control seeds was abolished by WD. Despite the several metabolic changes the final seed composition was largely unaltered by WD except for approximately 60% increase in stachyose and raffinose (raffinose family oligosaccharides). This increase in raffinose family oligosaccharides appears as the WD imprinting on mature seeds.

Effect of drought and rewatering on the metabolism of Lupinus albus organs.

Alterations in the metabolism of Lupinus albus organs that result from and subsequently follow a period of severe water deficit (WD) are described. By means of 13C-nuclear magnetic resonance (NMR), changes in the major metabolites were monitored in several plant organs (leaflets and petiole, roots, stem stele and cortex). During the stress, most of the leaves were lost and the stem functioned as a storage repository of sugars (glucose and sucrose) and amino acids (asparagine and proline). Upon rewatering, lupin plants rapidly re-established the relative water content (RWC) and produced new leaves. However, at the metabolic level, the events seem to be more complex, since proline (a stress related metabolite) disappeared rapidly while sugars and asparagine reached the initial pattern more slowly, particularly in the stem.

A biochemical and molecular characterization of LEP1, an extensin peroxidase from lupin.

An analysis of apoplastic extensin cross-linking activity in vegetative organs of Lupinus albus indicated that leaves contained the highest specific activity. Assays of peroxidases fractionated from this material demonstrated that this activity could be largely attributed to a soluble and apoplastic 51-kDa peroxidase, denoted LEP1. Relative to other purified peroxidases, LEP1 demonstrates high extensin cross-linking activity and can be classified as an extensin peroxidase (EP). Optimal conditions for the in vitro oxidation of other phenolic substrates included 1.5-3.0 mm peroxide at pH 5.0. EP activity of LEP1 was low under these conditions but optimal and substantially higher with 100 microm peroxide and neutral pH, suggesting that physiological changes in pH and peroxide in muro could heavily influence the extensin cross-linking activity of LEP1 in vivo. Analysis of LEP1 glycans indicated 11-12 N-linked glycans, predominantly the heptasaccharide Man3XylFucGlcNAc2, but also larger structures showing substantial heterogeneity. Comparative assays with horseradish peroxidase isoform C and peanut peroxidases suggested the high level of glycosylation in LEP1 may be responsible for the high solubility of this EP in the apoplastic space. A full-length cDNA corresponding to LEP1 was cloned. Quantitative reverse transcriptase-PCR demonstrated LEP1 induction in apical portions of etiolated hypocotyls 30-60 min after exposure to white light, prior to the onset of growth inhibition. Comparative modeling of the translated sequence indicated an unusually unobstructed equatorial cleft across the substrate access channel, which might facilitate interaction with extensin and confer higher EP activity.