Proteomic analysis of young sugarcane plants with contrasting salt tolerance.

Soil salinity affects sugarcane (Saccharum officinale L.) production in arid and semiarid climates, severely reducing productivity. This study aimed to identify differentially regulated proteins in two cultivars that differ markedly in tolerance of saline soil. Plants were grown for 30 days and then subjected to treatments of 0 and 160 mM NaCl for 15 days. The tolerant cultivar showed a 3-fold upregulation of lipid metabolising enzymes, GDSL-motif lipases, which are associated with defence to abiotic stress, and which were not upregulated in the sensitive cultivar. Lipoxygenase was 2-fold upregulated in the tolerant cultivar but not in the sensitive cultivar, as were Type III chlorophyll a/b binding proteins. Other differences were that in the sensitive cultivar, the key enzyme of C4 photosynthesis, phosphoenolpyruvate carboxylase was downregulated, along with other chloroplast enzymes. Na+ concentrations had not reached toxic concentrations in either cultivar by this time of exposure to salt, so these changes would be in response to the osmotic effect of the soil salinity, and likely be in common with plants undergoing drought stress.

Comparison of generational effect on proteins and metabolites in non-transgenic and transgenic soybean seeds through the insertion of the cp4-EPSPS gene assessed by omics-based platforms.

This work evaluates different generations of transgenic (cp4-EPSPS gene) and non-transgenic soybean plants through proteomics and metabolomics. For proteomics purpose, 24 differentially abundant protein spots were found through 2-D DIGE, being 4 belonging to transgenic plants. From this total, 19 were successfully identified, storage proteins as predominant class. Some identified proteins are involved in growing and cell division, and stress response, such as LEA and dehydrin. For metabolomics, 17 compounds were putatively annotated, mainly belonging to the secondary metabolism, such as flavonoids. From these analyzes, all generations and varieties of the soybean are prone to be differentiate by PLS-DA. According to our results, transgenic plants appear to be more stable than non-transgenic ones. In addition, the omics-based approaches allowed access some relations between those differential spot proteins and metabolites, mainly those storage proteins and flavonoid.

Validation of IgY for the diagnosis of Streptococcus agalactiae-caused endocarditis and bacterial meningitis in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae (Sta), which belongs to Lancefield group B, causes sepsis, endocarditis and bacterial meningitis in human neonates and Nile tilapia. Because the pathophysiology of Sta infection is partially similar in both species, the identification of biomarkers for the diagnosis and study of this disease is of importance for human and animal health. Therefore, in the present study, we produced an immunoglobulin Y (IgY) by immunizing laying hens with Sta proteins and evaluated its ability to detect Sta in paraffinized tilapia brain and cardiac tissue by direct immunofluorescence (IMF) and indirect immunohistochemistry (IHC). The IgY produced was effective in the diagnosis of Sta infection in Nile tilapia, justifying the use of this species as a biomodel for the study of this disease.

Protein identification before and after glyphosate exposure in Lolium multiflorum genotypes.

BACKGROUND: Weeds reduce crop yields, and among the methods used to control these plants, the use of chemicals is preferred. However, the repeated application of herbicides with the same mechanism of action selects for resistant populations. The aim of this study was to evaluate glyphosate resistance in Lolium multiflorum (Lam.) and relate the resistance to protein expression in the absence and presence of the herbicide using a metabolic-proteomic approach. RESULTS: Glyphosate resistance was confirmed, with a sevenfold difference in resistance between susceptible and resistant genotypes. Among the possible mechanisms affecting resistance, mutations in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), herbicide differential translocation and overexpression of EPSPS are suggested. Susceptible plants had higher growth than did resistant plants in the absence of the herbicide, in addition to greater expression of protein groups related to photosynthesis and to tolerance to biotic and abiotic stresses. With application of glyphosate, resistant plants maintained their metabolism and began to express EPSPS and other candidate proteins related to herbicide resistance. CONCLUSIONS: In the absence of glyphosate, the susceptible plants would replace the resistant plants over time, and abiotic or biotic stresses would accelerate this process. Resistance in plants resulted from a combination of target-site and non-target-site resistance mechanisms. We identified several candidate proteins that could be investigated in future studies on glyphosate resistance. © 2017 Society of Chemical Industry.

Luxurious Nitrogen Fertilization of Two Sugar Cane Genotypes Contrasting for Lignin Composition Causes Changes in the Stem Proteome Related to Carbon, Nitrogen, and Oxidant Metabolism but Does Not Alter Lignin Content.

Sugar cane is an important crop for sugar and biofuel production. Its lignocellulosic biomass represents a promising option as feedstock for second-generation ethanol production. Nitrogen fertilization can affect differently tissues and its biopolymers, including the cell-wall polysaccharides and lignin. Lignin content and composition are the most important factors associated with biomass recalcitrance to convert cell-wall polysaccharides into fermentable sugars. Thus it is important to understand the metabolic relationship between nitrogen fertilization and lignin in this feedstock. In this study, a large-scale proteomics approach based on GeLC-MS/MS was employed to identify and relatively quantify proteins differently accumulated in two contrasting genotypes for lignin composition after excessive nitrogen fertilization. From the ∼1000 nonredundant proteins identified, 28 and 177 were differentially accumulated in response to nitrogen from IACSP04-065 and IACSP04-627 lines, respectively. These proteins were associated with several functional categories, including carbon metabolism, amino acid metabolism, protein turnover, and oxidative stress. Although nitrogen fertilization has not changed lignin content, phenolic acids and lignin composition were changed in both species but not in the same way. Sucrose and reducing sugars increased in plants of the genotype IACSP04-065 receiving nitrogen.

Sunflower HaGPAT9-1 is the predominant GPAT during seed development.

In oil crops, triacylglycerol biosynthesis is an important metabolic pathway in which glycerol-3-phosphate acyltransferase (GPAT) performs the first acylation step. Mass spectrometry analysis of developing sunflower (Helianthus annuus) seed membrane fractions identified an abundant GPAT, HaGPAT9 isoform 1, with a N-terminal peptide that possessed two phosphorylated residues with possible regulatory function. HaGPAT9-1 belongs to a broad eukaryotic GPAT family, similar to mammalian GPAT3, and it represents one of the two sunflower GPAT9 isoforms, sharing 90% identity with HaGPAT9-2. Both sunflower genes are expressed during seed development and in vegetative tissues, with HaGPAT9-1 transcripts accumulating at relatively higher levels than those for HaGPAT9-2. Green fluorescent protein tagging of HaGPAT9-1 confirmed its subcellular accumulation in the endoplasmic reticulum. Despite their overall sequence similarities, the two sunflower isoforms displayed significant differences in their enzymatic activities. For instance, HaGPAT9-1 possesses in vivo GPAT activity that rescues the lethal phenotype of the cmy228 yeast strain, while in vitro assays revealed a preference of HaGPAT9-1 for palmitoyl-, oleoyl- and linoleoyl-CoAs of one order of magnitude, with the highest increase in yield for oleoyl- and linoleoyl-CoAs. By contrast, no enzymatic activity could be detected for HaGPAT9-2, even though its over-expression modified the TAG profile of yeast.

Heat Shock Protein 90 kDa (Hsp90) Has a Second Functional Interaction Site with the Mitochondrial Import Receptor Tom70.

To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom70·Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria.

SIM-XL: A powerful and user-friendly tool for peptide cross-linking analysis.

Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity. Moreover, our search engine is the first to capitalize on reporter ions for selecting tandem mass spectra derived from cross-linked peptides. It also makes available a 2D interaction map and a spectrum-annotation tool unmatched by any of its kind. We show SIM-XL to be more sensitive and faster than a competing tool when analyzing a data set obtained from the human HSP90. The software is freely available for academic use at http://patternlabforproteomics.org/sim-xl. A video demonstrating the tool is available at http://patternlabforproteomics.org/sim-xl/video. SIM-XL is the first tool to support XL data in the mzIdentML format; all data are thus available from the ProteomeXchange consortium (identifier PXD001677). This article is part of a Special Issue entitled: Computational Proteomics.

Comparative proteomic analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale and different monocot species.

The rhizome is responsible for the invasiveness and competitiveness of many plants with great economic and agricultural impact worldwide. Besides its value as an invasive organ, the rhizome plays a role in the establishment and massive growth of forage, providing biomass for biofuel production. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development, and function in plants. In this work, we characterized the proteome of rhizome apical tips and elongation zones from different species using a GeLC-MS/MS (one-dimensional electrophoresis in combination with liquid chromatography coupled online with tandem mass spectrometry) spectral-counting proteomics strategy. Five rhizomatous grasses and an ancient species were compared to study the protein regulation in rhizomes. An average of 2200 rhizome proteins per species were confidently identified and quantified. Rhizome-characteristic proteins showed similar functional distributions across all species analyzed. The over-representation of proteins associated with central roles in cellular, metabolic, and developmental processes indicated accelerated metabolism in growing rhizomes. Moreover, 61 rhizome-characteristic proteins appeared to be regulated similarly among analyzed plants. In addition, 36 showed conserved regulation between rhizome apical tips and elongation zones across species. These proteins were preferentially expressed in rhizome tissues regardless of the species analyzed, making them interesting candidates for more detailed investigative studies about their roles in rhizome development.

A systems-wide comparison of red rice (Oryza longistaminata) tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement.

BACKGROUND: The rhizome, the original stem of land plants, enables species to invade new territory and is a critical component of perenniality, especially in grasses. Red rice (Oryza longistaminata) is a perennial wild rice species with many valuable traits that could be used to improve cultivated rice cultivars, including rhizomatousness, disease resistance and drought tolerance. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development and function in this plant. RESULTS: We used an integrated approach to compare the transcriptome, proteome and metabolome of the rhizome to other tissues of red rice. 116 Gb of transcriptome sequence was obtained from various tissues and used to identify rhizome-specific and preferentially expressed genes, including transcription factors and hormone metabolism and stress response-related genes. Proteomics and metabolomics approaches identified 41 proteins and more than 100 primary metabolites and plant hormones with rhizome preferential accumulation. Of particular interest was the identification of a large number of gene transcripts from Magnaportha oryzae, the fungus that causes rice blast disease in cultivated rice, even though the red rice plants showed no sign of disease. CONCLUSIONS: A significant set of genes, proteins and metabolites appear to be specifically or preferentially expressed in the rhizome of O. longistaminata. The presence of M. oryzae gene transcripts at a high level in apparently healthy plants suggests that red rice is resistant to this pathogen, and may be able to provide genes to cultivated rice that will enable resistance to rice blast disease.

Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays.

Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. BIOLOGICAL SIGNIFICANCE: Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Global quantitative proteomics using spectral counting: an inexpensive experimental and bioinformatics workflow for deep proteome coverage.

As the field of proteomics shifts from qualitative identification of protein "subfractions" to quantitative comparison of proteins from complex biological samples, it is apparent that the number of approaches for quantitation can be daunting for the result-oriented biologist. There have been many recent reviews on quantitative proteomic approaches, discussing the strengths and limitations of each. Unfortunately, there are few detailed methodological descriptions of any one of these quantitative approaches. Here we present a detailed bioinformatics workflow for one of the simplest, most pervasive quantitative approach-spectral counting. The informatics and statistical workflow detailed here includes newly available freeware, such as SePro and PatternLab which post-process data according to false discovery rate parameters, and statistically model the data to detect differences and trends.

The response of Asterochloris erici (Ahmadjian) Skaloud et Peksa to desiccation: a proteomic approach.

The study of desiccation tolerance of lichens, and of their chlorobionts in particular, has frequently focused on the antioxidant system that protects the cell against photo-oxidative stress during dehydration/rehydration cycles. In this study, we used proteomic and transcript analyses to assess the changes associated with desiccation in the isolated phycobiont Asterochloris erici. Algae were dried either slowly (5-6 h) or rapidly (<60 min), and rehydrated after 24 h in the desiccated state. To identify proteins that accumulated during the drying or rehydration processes, we employed two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with individual protein identification using trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analyses revealed that desiccation caused an increase in relative abundance of only 11-13 proteins, regardless of drying rate, involved in glycolysis, cellular protection, cytoskeleton, cell cycle, and targeting and degradation. Transcripts of five Hsp90 and two ß-tubulin genes accumulated primarily at the end of the dehydration process. In addition, transmission electron microscopy (TEM) images indicate that ultrastructural cell injuries, perhaps resulting from physical or mechanical stress rather than metabolic damage, were more intense after rapid dehydration. This occurred with no major change in the proteome. These results suggest that desiccation tolerance of A. erici is achieved by constitutive mechanisms.

Next-generation sequencing-based transcriptomic and proteomic analysis of the common reed, Phragmites australis (Poaceae), reveals genes involved in invasiveness and rhizome specificity.

PREMISE OF THE STUDY: The common reed (Phragmites australis), one of the most widely distributed of all angiosperms, uses its rhizomes (underground stems) to invade new territory, making it one of the most successful weedy species worldwide. Characterization of the rhizome transcriptome and proteome is needed to identify candidate genes and proteins involved in rhizome growth, development, metabolism, and invasiveness. METHODS: We employed next-generation sequencing technologies including 454 and Illumina platforms to characterize the reed rhizome transcriptome and used quantitative proteomics techniques to identify the rhizome proteome. KEY RESULTS: Combining 336514 Roche 454 Titanium reads and 103350802 Illumina paired-end reads in a de novo hybrid assembly yielded 124450 unique transcripts with an average length of 549 bp, of which 54317 were annotated. Rhizome-specific and differentially expressed transcripts were identified between rhizome apical tips (apical meristematic region) and rhizome elongation zones. A total of 1280 nonredundant proteins were identified and quantified using GeLC-MS/MS based label-free proteomics, where 174 and 77 proteins were preferentially expressed in the rhizome elongation zone and apical tip tissues, respectively. Genes involved in allelopathy and in controlling development and potentially invasiveness were identified. CONCLUSIONS: In addition to being a valuable sequence and protein data resource for studying plant rhizome species, our results provide useful insights into identifying specific genes and proteins with potential roles in rhizome differentiation, development, and function.

Search engine processor: Filtering and organizing peptide spectrum matches.

The search engine processor (SEPro) is a tool for filtering, organizing, sharing, and displaying peptide spectrum matches. It employs a novel three-tier Bayesian approach that uses layers of spectrum, peptide, and protein logic to lead the data to converge to a single list of reliable protein identifications. SEPro is integrated into the PatternLab for proteomics environment, where an arsenal of tools for analyzing shotgun proteomic data is provided. By using the semi-labeled decoy approach for benchmarking, we show that SEPro significantly outperforms a commercially available competitor.

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm).

BACKGROUND AND AIMS: Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination. METHODS: Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC. KEY RESULTS: During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied. CONCLUSIONS: The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia.

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes, oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed.

Proteome analysis of cold acclimation in sunflower.

Cold acclimation is the phenomenon in which plants are exposed to low, but nonfreezing, temperatures before exposure to drastic temperatures. To investigate how sunflower plants adjust their metabolism during cold treatment, a comparative proteomic approach, based on spectral counting data, was adopted to identify differentially expressed proteins in leaves of freezing susceptible (Hopi) and tolerant (PI 543006 and BSD-2-691) lines after cold acclimation. In total 718, 675, and 769 proteins were confidently identified by tandem mass spectrometry in Hopi, PI 543006, and BSD-2-691 sunflower lines. Tolerant lines PI 543006 and BSD-2-691 showed the highest number of differentially expressed proteins, as 43, 72, and 168 proteins changed their expression in Hopi, PI 543006, and BSD-2-691 sunflower lines, respectively, at 95% confidence. Cold-responsive proteins were mostly involved in metabolism, protein synthesis, energy, and defense processes in all sunflower lines studied. Hierarchical clustering of all differentially expressed proteins resulted in the characterization of 14 different patterns of expression across Hopi, PI 543006, and BSD-2-691 and indicated that tolerant lines showed different proteome responses to cold acclimation.

Proteome analysis of leaves of the desiccation-tolerant grass, Sporobolus stapfianus, in response to dehydration.

Drought and its affects on agricultural production is a serious issue facing global efforts to increase food supplies and ensure food security for the growing world population. Understanding how plants respond to dehydration is an important prerequisite for developing strategies for crop improvement in drought tolerance. This has proved to be a difficult task as all of the current research plant models do not tolerate cellular dehydration well and, like all crops, they succumb to the effects of a relatively small water deficit of -4MPa or less. For these reasons many researchers have started to investigate the usefulness of resurrection plants, plants that can survive extremes of dehydration to the point of desiccation, to provide answers as to how plants tolerate water loss. We have chosen to investigate the leaf proteome response of the desiccation-tolerant grass Sporobolus stapfianus Gandoger to dehydration to a water content that encompasses the initiation of the cellular protection response evident in these plants. We used a combination of two-dimensional Difference Gel Electrophoresis (2D-DIGE) and liquid chromatography-tandem-mass spectrometry to compare the proteomes of young leaves from hydrated plants to those dehydrated to approximately 30% relative water content. High-resolution 2D-DIGE revealed 96 significantly different proteins and 82 of these spots yielded high-quality protein assignments by tandem-mass spectrometry. Inferences from the bioinformatic annotations of these proteins revealed the possible involvement of protein kinase-based signaling cascades and brassinosteroid involvement in the regulation of the cellular protection response. Enzymes of glycolysis, both cytoplasmic and plastidic, as well as five enzymes of the Calvin cycle increased in abundance. However, the RuBisCO large subunit and associated proteins were reduced, indicating a loss of carbon fixation but a continued need to supply the necessary carbon skeletons for the constituents involved in cell protection. Changes in abundance of several proteins that appear to have a function in chromatin structure and function indicate that these structures undergo significant changes as a result of dehydration. These observations give a unique "snap-shot" of the proteome of S. stapfianus at a critical point in the passage towards desiccation.