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Comparative Proteome Analysis of the Tuberous Roots of Six Cassava (Manihot esculenta) Varieties Reveals Proteins Related to Phenotypic Traits.
Schmitz, GJ, de Magalhães Andrade, J, Valle, TL, Labate, CA, do Nascimento, JR
Journal of agricultural and food chemistry. 2016;(16):3293-301
Abstract
Cassava (Manihot esculenta Crantz) is a staple food and an important source of starch, and the attributes of its tuberous root largely depend on the variety. The proteome of cassava has been investigated; however, to date, no study has focused on varieties that reveal the molecular basis of phenotypical characteristics. Therefore, we aimed to compare the proteome of the tuberous roots of six cassava varieties that differed in carbohydrates, carotenoids, and resistance to diseases, among other attributes. Two-dimensional gels showed 146 differential spots between the varieties, and the functional roles of some differential proteins were correlated to phenotypic characteristics of the varieties, such as the amount of carbohydrates or carotenoids and the resistance to biotic or abiotic stresses. The results obtained here highlight elements that might help to direct the improvement of new cultivars of cassava, which is an economically and socially relevant crop worldwide.
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2.
CoffeebEST: an integrated resource for Coffea spp expressed sequence tags.
Paschoal, AR, Fernandes, ED, Silva, JC, Lopes, FM, Pereira, LF, Domingues, DS
Genetics and molecular research : GMR. 2014;(4):10913-20
Abstract
Coffee is one of the most important commodities in the world, and its production relies mainly on two species, Coffea arabica and Coffea canephora. Although there are diverse transcriptome datasets available for coffee trees, few research groups have exploited the potential knowledge contained in these data, especially with respect to fruit and seed development. Here, we present a comparative analysis of the transcriptomes of Coffea arabica and Coffea canephora with a focus on fruit development using publicly available expressed sequence tags (ESTs). Most of the fruit and seed EST data has been obtained from C. canephora. Therefore, we performed a fruit EST analysis of the 5 developmental stages of this species (18, 22, 30, 42, and 46 weeks after flowering) comprising 29,009 sequences. We compared C. canephora fruit ESTs to reference unigenes of C. canephora (7710 contigs and 8955 singletons) and C. arabica (15,656 contigs and 16,351 singletons). Additional analyses included functional annotation based on Gene Onthology, as well as an annotation using PlantCyc, a curated plant protein database. The Coffee Bean EST (CoffeebEST) is a public database available at http://bioinfo-02.cp.utfpr.edu.br/. This database represents an additional resource for the coffee scientific community, offering a user-friendly collection of information for non-specialists in coffee molecular biology to support experimental research on comparative and functional genomics.
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3.
Comparative proteomics of seed maturation in oilseeds reveals differences in intermediary metabolism.
Hajduch, M, Matusova, R, Houston, NL, Thelen, JJ
Proteomics. 2011;(9):1619-29
Abstract
Proteomics is increasingly being used to understand enzyme expression and regulatory mechanisms involved in the accumulation of storage reserves in crops with sequenced genomes. During the past six years, considerable progress has been made to characterize proteomes of both mature and developing seeds, particularly oilseeds - plants which accumulate principally oil and protein as storage reserves. This review summarizes the emerging proteomics data, with emphasis on seed filling in soy, rapeseed, castor and Arabidopsis as each of these oilseeds were analyzed using very similar proteomic strategies. These parallel studies provide a comprehensive view of source-sink relationships, specifically sucrose assimilation into organic acid intermediates for de novo amino acid and fatty acid synthesis. We map these biochemical processes for seed maturation and illustrate the differences and similarities among the four oilseeds. For example, while the four oilseeds appear capable of producing cytosolic phosphoenolpyruvate as the principal carbon intermediate, soybean and castor also express malic enzymes and malate dehydrogenase, together capable of producing malate that has been previously proposed to be the major intermediate for fatty acid synthesis in castor. We discuss these and other differences in the context of intermediary metabolism for developing oilseeds.
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4.
High-intensity pulsed electric fields processing parameters affecting polyphenoloxidase activity of strawberry juice.
Aguiló-Aguayo, I, Soliva-Fortuny, R, Martín-Belloso, O
Journal of food science. 2010;(7):C641-6
Abstract
High-intensity pulsed electric fields (HIPEF) were applied to strawberry juice to study the feasibility of inactivating polyphenoloxidase (PPO). Response surface methodology was used to evaluate the effect of HIPEF processing, in which total treatment time (1000 to 2000 μs), pulse frequency (50 to 250 Hz), pulse width (1.0 to 7.0 μs), and polarity (monopolar or bipolar) were the controlled variables at a constant electric field of 35 kV/cm. The proposed 2nd-order response functions were accurate enough to fit experimental results. Strawberry juice PPO was strongly reduced within the range of assayed conditions. HIPEF treatments were more effective in bipolar than in monopolar mode in inactivating PPO. Treatments of longer duration resulted in reductions of the enzyme activity. Moreover, it was feasible to minimize residual PPO activity (down to 2.5%) by selecting bipolar treatments at frequencies higher than 229 Hz and pulse widths between 3.23 and 4.23 μs for a constant total treatment time of 2000 μs.
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5.
Quantitative proteome analysis of barley seeds using ruthenium(II)-tris-(bathophenanthroline-disulphonate) staining.
Witzel, K, Surabhi, GK, Jyothsnakumari, G, Sudhakar, C, Matros, A, Mock, HP
Journal of proteome research. 2007;(4):1325-33
Abstract
This paper describes the application of the recently introduced fluorescence stain Ruthenium(II)-tris-(bathophenanthroline-disulphonate) (RuBP) on a comparative proteome analysis of two phenotypically different barley lines. We carried out an analysis of protein patterns from 2-D gels of the parental lines of the Oregon Wolfe Barley mapping population DOM and REC and stained with either the conventional colloidal Coomassie Brilliant Blue (cCBB) or with the novel RuBP solution. We wished to experimentally verify the usefulness of such a stain in evaluating the complex pattern of a seed proteome, in comparison to the previously used cCBB staining technique. To validate the efficiency of visualization by both stains, we first compared the overall number of detected protein spots. On average, 790 spots were visible by cCBB staining and 1200 spots by RuBP staining. Then, the intensity of a set of spots was assessed, and changes in relative abundance were determined using image analysis software. As expected, staining with RuBP performed better in quantitation in terms of sensitivity and dynamic range. Furthermore, spots from a cultivar-specific region in the protein map were chosen for identification to asses the gain of biological information due to the staining procedure. From this particular region, eight spots were visualized exclusively by RuBP and identification was successful for all spots, proving the ability to identify even very low abundant proteins. Performance in MS analysis was comparable for both protein stains. Proteins were identified by MALDI-TOF MS peptide mass fingerprinting. This approach was not successful for all spots, due to the restricted entry number for barley in the database. Therefore, we subsequently used LC-ESI-Q-TOF MS/MS and de novo sequencing for identification. Because only an insufficient number of proteins from barley is annotated, an EST-based identification strategy was chosen for our experiment. We wished to test whether under these limitations the application of a more sensitive stain would lead to a more advanced proteome approach. In summary, we demonstrate here that the application of RuBP as an economical but reliable and sensitive fluorescence stain is highly suitable for quantitative proteome analysis of plant seeds.
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6.
The major grass pollen group 5 allergen from Dactylis glomerata and its C-terminal split product both behave as dimers: implications for allergen standardization.
van Oort, E, Dieker, MC, de Heer, PG, Peltre, G, Aalberse, RC, van Ree, R
International archives of allergy and immunology. 2005;(2):113-22
Abstract
BACKGROUND On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M(r)) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M(r). The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays. METHODS Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity. RESULTS The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD(L)/(A)GY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE. CONCLUSIONS These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.
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7.
Environmental and transgene expression effects on the barley seed proteome.
Finnie, C, Steenholdt, T, Roda Noguera, O, Knudsen, S, Larsen, J, Brinch-Pedersen, H, Bach Holm, P, Olsen, O, Svensson, B
Phytochemistry. 2004;(11):1619-27
Abstract
The barley (Hordeum vulgare) cultivar Golden Promise is no longer widely used for malting, but is amenable to transformation and is therefore a valuable experimental cultivar. Its characteristics include high salt tolerance, however it is also susceptible to several fungal pathogens. Proteome analysis was used to describe the water-soluble protein fraction of Golden Promise seeds in comparison with the modern malting cultivar Barke. Using 2D-gel electrophoresis to visualise several hundred proteins in the pH ranges 4-7 and 6-11, 16 protein spots were found to differ between the two cultivars. Eleven of these were identified by mass spectrometric peptide mass mapping, including an abundant chitinase implicated in defence against fungal pathogens and a small heat-shock protein. To enable a comparison with transgenic seed protein patterns, differences in spot patterns between field and greenhouse-grown seeds were analysed. Four spots were observed to be increased in intensity in the proteome of greenhouse-grown seeds, three of which may be related to nitrogen availability during grain filling and total protein content of the seeds, since they also increased in field grown seeds supplied with extra nitrogen. Finally, the fate of transgene products in barley seeds was followed. Spots containing two green fluorescent protein constructs and the herbicide resistance marker phosphinothricin acetyltransferase were observed in 2D-gel patterns of transgenic seeds and identified by mass spectrometry. Phosphinothricin acetyltransferase was observed in three spots differing in pI suggesting that post-translational modification of the transgene product had occurred.
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8.
Allergies to cross-reactive plant proteins. Latex-fruit syndrome is comparable with pollen-food allergy syndrome.
Yagami, T
International archives of allergy and immunology. 2002;(4):271-9
Abstract
Both latex-fruit syndrome and oral allergy syndrome concomitant with pollinosis (pollen-food allergy syndrome) are considered to be caused by cross-reactivity between sensitizers and symptom elicitors. The cross-reactive food allergens relevant to these syndromes are mostly sensitive to heat and digestive enzymes. Such a vulnerable antigen cannot sensitize people perorally but provokes allergic reactions in already sensitized patients based on its cross-reactivity to the corresponding sensitizer. These types of food allergens are often called incomplete food allergens or nonsensitizing elicitors. Their features contrast with those of complete food allergens that have the capacity for peroral sensitization as well as symptom elicitation. Although highly antigenic and cross-reactive, carbohydrate epitopes do not generally elicit allergic reactions and often disturb in vitro IgE tests. Recent research has revealed that some of the cross-reactive allergens responsible for the two syndromes are proteins related to the defense responses of higher plants. Plant defense-related proteins are relatively conserved in the course of evolution and can supply cross-reactive epitopes. It is important to note that various stresses can stimulate the expression of these proteins, which implies that allergens increase in plants under stressful conditions like severe growing situations and exposure to some kinds of chemicals. Because defense-related proteins usually provide a plant with resistance to stresses, varieties that are apt to intensively induce such proteins are agriculturally valuable. Less toxic substances that cause crops to express defensive proteins are being investigated as a new type of agrochemical. Moreover, some defense-related proteins are going to be constantly produced in genetically modified plants. Even though these proteins can be useful agriculturally, their allergenicity should be evaluated carefully.