Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

Exploration of the Diversity of Vicine and Convicine Derivatives in Faba Bean (Vicia faba L.) Cultivars: Insights from LC-MS/MS Spectra.

While numerous Fabaceae seeds are a good nutritional source of high-quality protein, the use of some species is hampered by toxic effects caused by exposure to metabolites that accumulate in the seeds. One such species is the faba or broad bean (Vicia faba L.), which accumulates vicine and convicine. These two glycoalkaloids cause favism, the breakdown of red blood cells in persons with a glucose-6-phosphate dehydrogenase deficiency. Because this is the most common enzyme deficiency worldwide, faba bean breeding efforts have focused on developing cultivars with low levels of these alkaloids. Consequently, quantification methods have been developed; however, they quantify vicine and convicine only and not the derivatives of these compounds that potentially generate the same bio-active molecules. Based on the recognition of previously unknown (con)vicine-containing compounds, we screened the fragmentation spectra of LC-MS/MS data from five faba bean cultivars using the characteristic fragments generated by (con)vicine. This resulted in the recognition of more than a hundred derivatives, of which 89 were tentatively identified. (Con)vicine was mainly derivatized through the addition of sugars, hydroxycinnamic acids, and dicarboxylic acids, with a group of compounds composed of two (con)vicine residues linked by dicarboxyl fatty acids. In general, the abundance profiles of the different derivatives in the five cultivars mimicked that of vicine and convicine, but some showed a derivative-specific profile. The description of the (con)vicine diversity will impact the interpretation of future studies on the biosynthesis of (con)vicine, and the content in potentially bio-active alkaloids in faba beans may be higher than that represented by the quantification of vicine and convicine alone.

Deep into the Apoplast: Grapevine and Plasmopara viticola Proteomes Reveal the Secret Beneath Host and Pathogen Communication at 6 h After Contact.

The apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors, thus activating signal transduction pathways. As a result of this first contact, the host triggers a defense response that involves the modulation of extra- and intracellular proteins. In grapevine-pathogen interactions, little is known about the trafficking between extra- and intracellular spaces. Grapevine is an economically important crop that relies on heavy fungicide use to control several diseases, and a deeper knowledge on the activation of its immune response is crucial to define new control strategies. In this study, we focused on the first 6 h postinoculation with Plasmopara viticola to evaluate grapevine proteome modulation in the apoplast. The in planta P. viticola proteome was also assessed to enable a deeper understanding of plant-pathogen communication. Our results showed that several plant mechanisms are triggered in the tolerant grapevine cultivar Regent after inoculation, such as oomycete recognition, plant cell wall modifications, reactive oxygen species signaling, and secretion of proteins to disrupt oomycete structures. On the other hand, P. viticola proteins related to development and virulence were the most predominant. This pioneer study highlights the early dynamics of cellular communication in grapevine defense that leads to the successful establishment of an incompatible interaction.

Molecular insights into plant desiccation tolerance: transcriptomics, proteomics and targeted metabolite profiling in Craterostigma plantagineum.

The resurrection plant Craterostigma plantagineum possesses an extraordinary capacity to survive long-term desiccation. To enhance our understanding of this phenomenon, complementary transcriptome, soluble proteome and targeted metabolite profiling was carried out on leaves collected from different stages during a dehydration and rehydration cycle. A total of 7348 contigs, 611 proteins and 39 metabolites were differentially abundant across the different sampling points. Dynamic changes in transcript, protein and metabolite levels revealed a unique signature characterizing each stage. An overall low correlation between transcript and protein abundance suggests a prominent role for post-transcriptional modification in metabolic reprogramming to prepare plants for desiccation and recovery. The integrative analysis of all three data sets was performed with an emphasis on photosynthesis, photorespiration, energy metabolism and amino acid metabolism. The results revealed a set of precise changes that modulate primary metabolism to confer plasticity to metabolic pathways, thus optimizing plant performance under stress. The maintenance of cyclic electron flow and photorespiration, and the switch from C3 to crassulacean acid metabolism photosynthesis, may contribute to partially sustain photosynthesis and minimize oxidative damage during dehydration. Transcripts with a delayed translation, ATP-independent bypasses, alternative respiratory pathway and 4-aminobutyric acid shunt may all play a role in energy management, together conferring bioenergetic advantages to meet energy demands upon rehydration. This study provides a high-resolution map of the changes occurring in primary metabolism during dehydration and rehydration and enriches our understanding of the molecular mechanisms underpinning plant desiccation tolerance. The data sets provided here will ultimately inspire biotechnological strategies for drought tolerance improvement in crops.

Proteomics Evidence of a Systemic Response to Desiccation in the Resurrection Plant Haberlea rhodopensis.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.

An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample.

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics and more specialized 'OMICS' such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and a wider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltration-centrifugation method that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv 'Trincadeira' and 'Regent', was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteins were identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes.

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites, Proteins and Lipids with Perspectives on Their Potential and Sources.

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-ß-glucosidases, pectinesterases, polygalacturonases, ß-galactosidases and ß-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Distribution of cell-wall polysaccharides and proteins during growth of the hemp hypocotyl.

MAIN CONCLUSION: The immuno-ultrastructural investigation localized cell-wall polysaccharides of bast fibers during hemp hypocotyl growth. Moreover, for the first time, the localization of a peroxidase and laccase is provided in textile hemp. In the hypocotyl of textile hemp, elongation and girth increase are separated in time. This organ is therefore ideal for time-course analyses. Here, we follow the ultrastructural rearrangement of cell-wall components during the development of the hemp hypocotyl. An expression analysis of genes involved in the biosynthesis of cellulose, the chief polysaccharide of bast fiber cell walls and xylan, the main hemicellulose of secondary cell walls, is also provided. The analysis shows a higher expression of cellulose and xylan-related genes at 15 and 20 days after sowing, as compared to 9 days. In the young hypocotyl, the cell walls of bast fibers show cellulose microfibrils that are not yet compacted to form a mature G-layer. Crystalline cellulose is detected abundantly in the S1-layer, together with unsubstituted/low-substituted xylan and, to a lesser extent, in the G-layer. The LM5 galactan epitope is confined to the walls of parenchymatic cells. LM6-specific arabinans are detected at the interface between the cytoplasm and the gelatinous cell wall of bast fibers. The class III peroxidase antibody shows localization in the G-layer only at older developmental stages. The laccase antibody shows a distinctive labelling of the G-layer region closest to the S1-layer; the signal becomes more homogeneous as the hypocotyl matures. The data provide important insights on the cell wall distribution of polysaccharide and protein components in bast fibers during the hypocotyl growth of textile hemp.

A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax.

Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes.

Insights into Lignan Composition and Biosynthesis in Stinging Nettle (Urtica dioica L.).

Stinging nettle (Urtica dioica L.) has been used as herbal medicine to treat various ailments since ancient times. The biological activity of nettle is chiefly attributed to a large group of phenylpropanoid dimers, namely lignans. Despite the pharmacological importance of nettle lignans, there are no studies addressing lignan biosynthesis in this plant. We herein identified 14 genes encoding dirigent proteins (UdDIRs) and 3 pinoresinol-lariciresinol reductase genes (UdPLRs) in nettle, which are two gene families known to be associated with lignan biosynthesis. Expression profiling of these genes on different organs/tissues revealed a specific expression pattern. Particularly, UdDIR7, 12 and 13 displayed a remarkable high expression in the top internode, fibre tissues of bottom internodes and roots, respectively. The relatively high expression of UdPLR1 and UdPLR2 in the young internodes, core tissue of bottom internode and roots is consistent with the high accumulation of lariciresinol and secoisolariciresinol in these tissues. Lignan quantification showed a high abundance of pinoresinol in roots and pinoresinol diglucosides in young internodes and leaves. This study sheds light on lignan composition and biosynthesis in nettle, providing a good basis for further functional analysis of DIRs and PLRs and, ultimately, engineering lignan metabolism in planta and in cell cultures.

Genetical genomics of quality related traits in potato tubers using proteomics.

BACKGROUND: Recent advances in ~omics technologies such as transcriptomics, metabolomics and proteomics along with genotypic profiling have permitted the genetic dissection of complex traits such as quality traits in non-model species. To get more insight into the genetic factors underlying variation in quality traits related to carbohydrate and starch metabolism and cold sweetening, we determined the protein content and composition in potato tubers using 2D-gel electrophoresis in a diploid potato mapping population. Upon analyzing we made sure that the proteins from the patatin family were excluded to ensure a better representation of the other proteins. RESULTS: We subsequently performed pQTL analyses for all other proteins with a sufficient representation in the population and established a relationship between proteins and 26 potato tuber quality traits (e.g. flesh colour, enzymatic discoloration) by co-localization on the genetic map and a direct correlation study of protein abundances and phenotypic traits. Over 1643 unique protein spots were detected in total over the two harvests. We were able to map pQTLs for over 300 different protein spots some of which co-localized with traits such as starch content and cold sweetening. pQTLs were observed on every chromosome although not evenly distributed over the chromosomes. The largest number of pQTLs was found for chromosome 8 and the lowest for chromosome number 10. For some 20 protein spots multiple QTLs were observed. CONCLUSIONS: From this analysis, hotspot areas for protein QTLs were identified on chromosomes three, five, eight and nine. The hotspot on chromosome 3 coincided with a QTL previously identified for total protein content and had more than 23 pQTLs in the region from 70 to 80 cM. Some of the co-localizing protein spots associated with some of the most interesting tuber quality traits were identified, albeit far less than we had anticipated at the onset of the experiments.

Insights into the molecular regulation of monolignol-derived product biosynthesis in the growing hemp hypocotyl.

BACKGROUND: Lignin and lignans are both derived from the monolignol pathway. Despite the similarity of their building blocks, they fulfil different functions in planta. Lignin strengthens the tissues of the plant, while lignans are involved in plant defence and growth regulation. Their biosyntheses are tuned both spatially and temporally to suit the development of the plant (water conduction, reaction to stresses). We propose to study the general molecular events related to monolignol-derived product biosynthesis, especially lignin. It was previously shown that the growing hemp hypocotyl (between 6 and 20 days after sowing) is a valid system to study secondary growth and the molecular events accompanying lignification. The present work confirms the validity of this system, by using it to study the regulation of lignin and lignan biosynthesis. Microscopic observations, lignin analysis, proteomics, together with in situ laccase and peroxidase activity assays were carried out to understand the dynamics of lignin synthesis during the development of the hemp hypocotyl. RESULTS: Based on phylogenetic analysis and targeted gene expression, we suggest a role for the hemp dirigent and dirigent-like proteins in lignan biosynthesis. The transdisciplinary approach adopted resulted in the gene- and protein-level quantification of the main enzymes involved in the biosynthesis of monolignols and their oxidative coupling (laccases and class III peroxidases), in lignin deposition (dirigent-like proteins) and in the determination of the stereoconformation of lignans (dirigent proteins). CONCLUSIONS: Our work sheds light on how, in the growing hemp hypocotyl, the provision of the precursors needed to synthesize the aromatic biomolecules lignin and lignans is regulated at the transcriptional and proteomic level.

Changes in the Proteome of Medicago sativa Leaves in Response to Long-Term Cadmium Exposure Using a Cell-Wall Targeted Approach.

Accumulation of cadmium (Cd) shows a serious problem for the environment and poses a threat to plants. Plants employing various cellular and molecular mechanisms to limit Cd toxicity and alterations of the cell wall structure were observed upon Cd exposure. This study focuses on changes in the cell wall protein-enriched subproteome of alfalfa (Medicago sativa) leaves during long-term Cd exposure. Plants grew on Cd-contaminated soil (10 mg/kg dry weight (DW)) for an entire season. A targeted approach was used to sequentially extract cell wall protein-enriched fractions from the leaves and quantitative analyses were conducted with two-dimensional difference gel electrophoresis (2D DIGE) followed by protein identification with matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time of flight (TOF/TOF) mass spectrometry. In 212 spots that showed a significant change in intensity upon Cd exposure a single protein was identified. Of these, 163 proteins are predicted to be secreted and involved in various physiological processes. Proteins of other subcellular localization were mainly chloroplastic and decreased in response to Cd, which confirms the Cd-induced disturbance of the photosynthesis. The observed changes indicate an active defence response against a Cd-induced oxidative burst and a restructuring of the cell wall, which is, however, different to what is observed in M. sativa stems and will be discussed.

Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

Combining -Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago sativa) Stem Metabolism.

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a non-targeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

A proteome analysis of freezing tolerance in red clover (Trifolium pratense L.).

BACKGROUND: Improvement of freezing tolerance of red clover (Trifolium pratense L.) would increase its persistence under cold climate. In this study, we assessed the freezing tolerance and compared the proteome composition of non-acclimated and cold-acclimated plants of two initial cultivars of red clover: Endure (E-TF0) and Christie (C-TF0) and of populations issued from these cultivars after three (TF3) and four (TF4) cycles of phenotypic recurrent selection for superior freezing tolerance. Through this approach, we wanted to identify proteins that are associated with the improvement of freezing tolerance in red clover. RESULTS: Freezing tolerance expressed as the lethal temperature for 50 % of the plants (LT50) increased markedly from approximately -2 to -16 °C following cold acclimation. Recurrent selection allowed a significant 2 to 3 °C increase of the LT50 after four cycles of recurrent selection. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to study variations in protein abundance. Principal component analysis based on 2D-DIGE revealed that the largest variability in the protein data set was attributable to the cold acclimation treatment and that the two genetic backgrounds had differential protein composition in the acclimated state only. Vegetative storage proteins (VSP), which are essential nitrogen reserves for plant regrowth, and dehydrins were among the most striking changes in proteome composition of cold acclimated crowns of red clovers. A subset of proteins varied in abundance in response to selection including a dehydrin that increased in abundance in TF3 and TF4 populations as compared to TF0 in the Endure background. CONCLUSION: Recurrent selection performed indoor is an effective approach to improve the freezing tolerance of red clover. Significant improvement of freezing tolerance by recurrent selection was associated with differential accumulation of a small number of cold-regulated proteins that may play an important role in the determination of the level of freezing tolerance.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

BACKGROUND: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. METHODS: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. RESULTS: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. CONCLUSIONS: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

A proteomics study of colostrum and milk from the two major small ruminant dairy breeds from the Canary Islands: a bovine milk comparison perspective.

Colostrum and milk feeding are key factors for the newborn ruminant survival, affecting the future performance of the animal. Nowadays, there is an increasing interest in the potential of feeding newborn ruminants (mainly goat kids and lambs) with colostrum and milk from other more productive ruminant species (mainly cows). Although some studies regarding differences between colostrum and milk from these three species have been performed, herein we conduct for the first time a comparison using a proteomics 2-Dimensional Electrophoresis gel-based approach between these three ruminant species. In this study colostrum and milk samples from six Holstein cows, six Canarian sheep and six Majorera goats were used to determine the chemical composition, immunoglobulin G (IgG) and M (IgM) concentrations and proteomics profiles. Results showed that in general sheep colostrum and milk contained higher fat, protein and lactose percentages compared to bovine and goat samples. Additionally, no differences in the IgG or IgM concentrations were found among any of the three studied species, with the exception of sheep colostrum that showed the highest IgM concentration. With reference to the proteomics-based approach, some high abundant proteins such as serum albumin precursor, beta-caseins or different immunoglobulins components were found in colostrum, milk or even both. Nevertheless, differences in other proteins with immune function such as serotransferrin or lactoperoxidase were detected. This study shows that despite the similar immunoglobulin concentrations in colostrum and milk from the three studied species, differences in several immune components can be detected when these samples are studied using a proteomics approach. Finally, this study also provides a base for future investigation in colostrum and milk proteomics and metabolomics.

INPPO2014, First INPPO World Congress on "Plant Proteomics: Methodology to Biology"-A global platform for involving, gathering and disseminating knowledge.

The International Plant Proteomics Organization (INPPO) is a global platform of the plant proteomics community or, more generally, the scientific community that uses proteomics to address plant biology. Organizing an international conference is one of its initiatives to promote plant proteomics by involving and gathering scientists/researchers/students and by disseminating the acquired knowledge. In this fourth INPPO Highlights, the first INPPO World Congress 2014 (INPPO2014) is described and discussed. The INPPO2014 was held at the University of Hamburg (Germany) with the title "Plant Proteomics: Methodology to Biology" under the leadership of Sabine Lüthje (Germany). Participants (around 150) from 38 nations attended this congress covering all continents. The four-day scientific program comprised 52 lectures and 61 poster presentations in a highly professional and friendly atmosphere on mass spectrometry and gel-based proteomics. Two round-table open discussions deliberated on plant proteomics, its associated international organizations/initiatives and future INPPO perspectives. The Second INPPO World Congress 2016 (INPPO2016) "The Quest for Tolerant Varieties-Phenotyping at Plant and Cellular Level" is planned to be organized in Bratislava (Slovakia) under the leadership of Martin Hajduch (Slovak Republic) and Sébastien Carpentier (Belgium) and cosponsored by the COST action FA1306.