Protein analysis reveals differential accumulation of late embryogenesis abundant and storage proteins in seeds of wild and cultivated amaranth species.

BACKGROUND: Amaranth is a plant naturally resistant to various types of stresses that produces seeds of excellent nutritional quality, so amaranth is a promising system for food production. Amaranth wild relatives have survived climate changes and grow under harsh conditions, however no studies about morphological and molecular characteristics of their seeds are known. Therefore, we carried out a detailed morphological and molecular characterization of wild species A. powellii and A. hybridus, and compared them with the cultivated amaranth species A. hypochondriacus (waxy and non-waxy seeds) and A. cruentus. RESULTS: Seed proteins were fractionated according to their polarity properties and were analysed in one-dimensional gel electrophoresis (1-DE) followed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 34 differentially accumulated protein bands were detected and 105 proteins were successfully identified. Late embryogenesis abundant proteins were detected as species-specific. Oleosins and oil bodies associated proteins were observed preferentially in A. cruentus. Different isoforms of the granule-bound starch synthase I, and several paralogs of 7S and 11S globulins were also identified. The in silico structural analysis from different isoforms of 11S globulins was carried out, including new types of 11S globulin not reported so far. CONCLUSIONS: The results provide novel information about 11S globulins and proteins related in seed protection, which could play important roles in the nutritional value and adaptive tolerance to stress in amaranth species.

Molecular characterization and in vitro interaction analysis of Op14-3-3 µ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway.

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 µ, was amplified by 3'-RACE methodology. Op14-3-3 µ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 µ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 µ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 µ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 µ protein was observed. The putative interaction of 14-3-3 µ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 µ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SIGNIFICANCE: Opuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

The proteome map of the escamolera ant (Liometopum apiculatum Mayr) larvae reveals immunogenic proteins and several hexamerin proteoforms.

The larvae of escamolera ant (Liometopum apiculatum Mayr) have been considered a delicacy since Pre-Hispanic times. The increased demand for this stew has led to massive collection of ant nests. Yet biological aspects of L. apiculatum larvae remain unknown, and mapping the proteome of this species is important for understanding its biological characteristics. Two-dimensional gel electrophoresis (2-DE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to characterize the larvae proteome profile. From 380 protein spots analyzed, 174 were identified by LC-MS/MS and homology search against the Hymenoptera subset of the NCBInr protein database using the Mascot search engine. Peptide de novo sequencing and homology-based alignment allowed the identification of 36 additional protein spots. Identified proteins were classified by cellular location, molecular function, and biological process according to the Gene Ontology annotation. Immunity- and defense-related proteins were identified including PPIases, FK506, PEBP, and chitinases. Several hexamerin proteoforms were identified and the cDNA of the most abundant protein detected in the 2-DE map was isolated and characterized. L. apiculatum hexamerin (LaHEX, GeneBank accession no. MH256667) contains an open reading frame of 2199 bp encoding a polypeptide of 733 amino acid residues with a calculated molecular mass of 82.41 kDa. LaHEX protein is more similar to HEX110 than HEX70 from Apis mellifera. Down-regulation of LaHEX was observed throughout ant development. This work represents the first proteome map as well as the first hexamerin characterized from L. apiculatum larvae.

Consumption of Amaranth Induces the Accumulation of the Antioxidant Protein Paraoxonase/Arylesterase 1 and Modulates Dipeptidyl Peptidase IV Activity in Plasma of Streptozotocin-Induced Hyperglycemic Rats.

BACKGROUND/AIM: Amaranth is a source of several bioactive compounds, among which peptides with inhibitory activity upon dipeptidyl peptidase IV (DPP-IV) have been reported. However, there is no information about the action of amaranth DPP-IV-inhibitory peptides using in vivo models. The aim of this work was to evaluate the effect of amaranth consumption on plasma and kidney DPP-IV activity as well the changes in plasma proteome profile of streptozotocin (STZ)-induced hyperglycemic rats. METHODS: Rats were fed for 12 weeks with a diet containing 20% popped amaranth grain. Kidneys and blood samples were collected for lipid profile, DPP-IV activity and expression, and proteomic analysis. RESULTS: Total cholesterol and DPP-IV activity in plasma was increased in hyperglycemic rats, but this effect was reverted by amaranth consumption. Triacylglycerols were increased in the hyperglycemic group fed amaranth, and the highest levels of high-density lipoproteins were also observed in this group. These data correlated with the accumulation of apolipoprotein A-II in plasma. Accumulation of the antioxidant protein paraoxonase/arylesterase 1 in STZ-induced hyperglycemic rats was observed when amaranth was supplied in the diet. CONCLUSION: This study provides new insights into the molecular mechanisms by which amaranth exerts its beneficial health action in a hyperglycemic state.

Purification and biochemical characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds.

Chan (Hyptis suaveolens) is a Mesoamerican crop highly appreciated since the pre-Hispanic cultures. Its proteins are a good source of essential amino acids; however, there are no reports on the properties of its individual proteins. In this study, the 11S globulin (Hs11S) was purified and biochemically characterized. The molecular weight of native Hs11S was about 150-300 kDa with isoelectric points of 5.0-5.3, composed by four monomers of 53.5, 52, 51.1 and 49.5 kDa, each formed by one acidic subunit and one basic subunit linked by a disulfide bond. Dynamic light scattering, size exclusion chromatography and native PAGE show that Hs11S is assembled in different oligomeric forms. LC-MS/MS analysis confirmed its identity. Hs11S presents antigenic determinants in common with lupin 11S globulin. Carbohydrate moieties or phosphate groups linked to Hs11S were not detected. This information is very useful in order to exploit and utilize rationally chan 11S globulin in food systems.

Identification of Ligustrum lucidum pollen allergens using a proteomics approach.

BACKGROUND: Ligustrum spp. are members of the Oleaceae family, one of the most prominent allergic families worldwide. The genus Ligustrum contains approximately fifty species, including Ligustrum lucidum, which have been widely cultivated as ornamental plants, and its pollen is a source of inhalant allergens associated with respiratory allergic diseases. Little is known about the presence of allergenic proteins in L. lucidum. METHODS: The L. lucidum pollen proteins were extracted by a modified phenolic extraction method. A pool of four sera from mono sensitive patients was analyzed by 2DE immunoblotting and mass spectrometric analysis was performed on 6 immunoreactive protein spots. RESULTS: SDS-PAGE of L. lucidum pollen extract revealed proteins in ranges of 15-150 kDa. The 2DE gel profile of the L. lucidum pollen protein extract showed approximately 180 spots, and the 2DE immunoblots obtained using sera from Ligustrum monosensitive patients as the source of IgE antibodies revealed six allergen protein spots, corresponding to Profilin, Enolase, Fra e 9.01 (ß-1,3-glucanase), Pollen-specific Polygalacturonases, Alanine aminotransferase, and two ATP synthase beta subunits. CONCLUSION: We report for the first time the identification of IgE-reactive proteins from L. lucidum.

Low temperature conditioning of garlic (Allium sativum L.) "seed" cloves induces alterations in sprouts proteome.

Low-temperature conditioning of garlic "seed" cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that "seed" bulbs from "Coreano" variety conditioned at 5°C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic "seed" cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23°C), and the other was conditioned at low temperature (5°C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic "seed" cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies.

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress.

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC-MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C3-C4 photosynthetic plant.

Salt stress-induced alterations in the root proteome of Amaranthus cruentus L.

Salt stress is one of the major factors limiting crop productivity worldwide. Amaranth is a highly nutritious pseudocereal with remarkable nutraceutical properties; it is also a stress-tolerant plant, making it an alternative crop for sustainable food production in semiarid conditions. A two-dimensional electrophoresis gel coupled with a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approach was applied in order to analyze the changes in amaranth root protein accumulation in plants subjected to salt stress under hydroponic conditions during the osmotic phase (1 h), after recovery (24 h), and during the ionic phase of salt stress (168 h). A total of 101 protein spots were differentially accumulated in response to stress, in which 77 were successfully identified by LC-MS/MS and a database search against public and amaranth transcriptome databases. The resulting proteins were grouped into different categories of biological processes according to Gene Ontology. The identification of several protein isoforms with a change in pI and/or molecular weight reveals the importance of the salt-stress-induced posttranslational modifications in stress tolerance. Interestingly stress-responsive proteins unique to amaranth, for example, Ah24, were identified. Amaranth is a stress-tolerant alternative crop for sustainable food production, and the understanding of amaranth's stress tolerance mechanisms will provide valuable input to improve stress tolerance of other crop plants.

In vitro inhibition of dipeptidyl peptidase IV by peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins.

Bioactive compounds present in foods could potentially have beneficial effects on human health. In this study we report the in vitro inhibitory capacity of peptides released from amaranth seed proteins after enzymatic digestion, against dipeptidyl peptidase IV (DPPIV); an enzyme known to deactivate incretins, hormones involved in insulin secretion. Other seeds, such as soybean, black bean, and wheat were also tested. The highest inhibition of DPPIV was observed with amaranth peptides released after simulated gastrointestinal digestion, showing an IC(50) of 1.1mg/mL in a dose-dependent manner. In silico tryptic digestion of amaranth globulins was carried out releasing peptides larger than 13 residues. Some of these peptides were used for the in silico prediction of their binding modes with DPPIV. Docking models showed that the possible mechanism of globulin peptides to inhibit DPPIV was through blocking the active dimer formation. Peptides were also found inside the major cavity where the natural substrates reach the catalytic site of the enzyme. This is the first report of the identification of inhibitory DPPIV peptides from amaranth hydrolysates and the prediction of their binding modes at the molecular level, leading to their possible use as functional food ingredients in the prevention of diabetes.

Proteomic analysis of differentially accumulated proteins during ripening and in response to 1-MCP in papaya fruit.

Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.

Identification of calcium stress induced genes in amaranth leaves through suppression subtractive hybridization.

Calcium (Ca(2+)) is a critical ion for the growth and development of plants and plays an important role in signal transduction pathways in response to biotic and abiotic stresses. We investigated the Ca(2+) stress responsive-genes in amaranth leaves by using the suppression subtractive hybridization technique. Screening of the libraries generated 420 up-regulated transcripts and 199 down-regulated transcripts. The differentially expressed transcripts were associated with general stress response, transcription factors, gene regulation, signal transduction, and some other with unknown function. Selected genes were used to study their differential regulation by sqRT-PCR. Among the up-regulated transcripts, a fragment containing the motif of C3HC4-type RING-Zinc family was further characterized. The ORF of amaranth zinc finger protein (AhZnf) has a closer relationship with its ortholog from Ricinus communis while is distantly related to the Arabidopsis thaliana C3HC4-type ortholog. We have identified a novel putative zinc finger protein along with other novel proteins such as the wall associated kinase, phosphoinositide binding protein, and rhomboid protease involved in response to Ca(2+) stress in amaranth leaves.

Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.

Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits.