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1.
J Exp Bot ; 74(8): 2653-2666, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-36629279

RESUMEN

In a previous study we used asymmetric-flow field-flow fractionation to determine the polymer mass (Mw), gyration radius (Rw) and the polydispersity index of glutenin polymers (GPs) in wheat (Triticum aestivum). Here, using the same multi-location trials (4 years, 11 locations, and 192 cultivars), we report the factors that are associated with the conformation (Conf) of the polymers, which is the slope of Log(Rw) versus a function of Log(Mw). We found that Conf varied between 0.285 and 0.740, it had low broad-sense heritability (H2=16.8), and it was significantly influenced by the temperature occurring over the last month of grain filling. Higher temperatures were found to increase Rw and the compactness and sphericity of GPs. Alleles for both high- and low-molecular-weight glutenin subunits had a significant influence on the Conf value. Assuming a Gaussian distribution for Mw, the number of polymers present in wheat grains was computed for different kernel weights and protein concentrations, and it was found to exceed 1012 GPs per grain. Using atomic force microscopy and cryo-TEM, images of GPs were obtained for the first time. Under higher average temperature, GPs became larger and more spherical and consequently less prone to rapid hydrolysis. We propose some orientations that could be aimed at potentially reducing the impact of numerous GPs on people suffering from non-celiac gluten sensitivity.


Asunto(s)
Polímeros , Triticum , Triticum/genética , Triticum/metabolismo , Polímeros/metabolismo , Glútenes/genética , Glútenes/metabolismo , Grano Comestible/genética , Grano Comestible/metabolismo
2.
Theor Appl Genet ; 133(3): 751-770, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31907562

RESUMEN

KEY MESSAGE: A set of eight SNP markers was developed to facilitate the early selection of HMW-GS alleles in breeding programmes. In bread wheat (Triticum aestivum), the high molecular weight glutenin subunits (HMW-GSs) are the most important determinants of technological quality. Known to be very diverse, HMW-GSs are encoded by the tightly linked genes Glu-1-1 and Glu-1-2. Alleles that improve the quality of dough have been identified. Up to now, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of grain proteins is the most widely used for their identification. To facilitate the early selection of HMW-GS alleles in breeding programmes, we developed DNA-based molecular markers. For each accession of a core collection (n = 364 lines) representative of worldwide bread wheat diversity, HMW-GSs were characterized by both genotyping and SDS-PAGE. Based on electrophoresis, we observed at least 8, 22 and 9 different alleles at the Glu-A1, Glu-B1 and Glu-D1 loci, respectively, including new variants. We designed a set of 17 single-nucleotide polymorphism (SNP) markers that were representative of the most frequent SDS-PAGE alleles at each locus. At Glu-A1 and Glu-D1, two and three marker-based haplotypes, respectively, captured the diversity of the SDS-PAGE alleles rather well. Discrepancies were found mainly for the Glu-B1 locus. However, statistical tests revealed that two markers at each Glu-B1 gene and their corresponding haplotypes were more significantly associated with the rheological properties of the dough than were the relevant SDS-PAGE alleles. To conclude, this study demonstrates that the SNP markers developed provide additional information on HMW-GS diversity. Two markers at Glu-A1, four at Glu-B1 and two at Glu-D1 constitute a useful toolbox for breeding wheat to improve end-use value.


Asunto(s)
Glútenes/genética , Glútenes/metabolismo , Fitomejoramiento/métodos , Triticum/genética , Alelos , Electroforesis en Gel de Poliacrilamida , Genes de Plantas , Marcadores Genéticos , Haplotipos , Peso Molecular , Polimorfismo de Nucleótido Simple , Triticum/metabolismo
3.
Mol Biol Rep ; 47(7): 5439-5449, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32627139

RESUMEN

Grain development of Triticum aestivum is being studied extensively using individual OMICS tools. However, integrated transcriptome and proteome studies are limited mainly due to complexity of genome. Current study focused to unravel the transcriptome-proteome coordination of key mechanisms underlying carbohydrate metabolism during whole wheat grain development. Wheat grains were manually dissected to obtain grain tissues for proteomics and transcriptomics analyses. Differentially expressed proteins and transcripts at the 11 stages of grain development were compared. Computational workflow for integration of two datasets related to carbohydrate metabolism was designed. For CM proteins, output peptide sequences of proteomic analyses (via LC-MS/MS) were used as source to search corresponding transcripts. The transcript that turned out with higher number of peptides was selected as bona fide ribonucleotide sequence for respective protein synthesis. More than 90% of hits resulted in successful identification of respective transcripts. Comparative analysis of protein and transcript expression profiles resulted in overall 32% concordance between these two series of data. However, during grain development correlation of two datasets gradually increased up to ~ tenfold from 152 to 655 °Cd and then dropped down. Proteins involved in carbohydrate metabolism were divided in five categories in accordance with their functions. Enzymes involved in starch and sucrose biosynthesis showed the highest correlations between proteome-transcriptome profiles. High percentage of identification and validation of protein-transcript hits highlighted the power of omics data integration approach over existing gene functional annotation tools. We found that correlation of two datasets is highly influenced by stage of grain development. Further, gene regulatory networks would be helpful in unraveling the mechanisms underlying the complex and significant traits such as grain weight and yield.


Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Triticum/genética , Triticum/metabolismo , Carbohidratos/genética , Cromatografía Liquida/métodos , Grano Comestible/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Plantas/genética , Proteoma/genética , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Transcriptoma/genética
4.
J Proteome Res ; 14(10): 4432-9, 2015 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-26228564

RESUMEN

The nuclear proteome of the grain of the two cultivated wheat species Triticum aestivum (hexaploid wheat; genomes A, B, and D) and T. monococcum (diploid wheat; genome A) was analyzed in two early stages of development using shotgun-based proteomics. A procedure was optimized to purify nuclei, and an improved protein sample preparation was developed to efficiently remove nonprotein substances (starch and nucleic acids). A total of 797 proteins corresponding to 528 unique proteins were identified, 36% of which were classified in functional groups related to DNA and RNA metabolism. A large number (107 proteins) of unknown functions and hypothetical proteins were also found. Some identified proteins may be multifunctional and may present multiple localizations. On the basis of the MS/MS analysis, 368 proteins were present in the two species, and in two stages of development, some qualitative differences between species and stages of development were also found. All of these data illustrate the dynamic function of the grain nucleus in the early stages of development.


Asunto(s)
Grano Comestible/química , Genoma de Planta , Proteínas Nucleares/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Proteoma/aislamiento & purificación , Triticum/genética , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatografía Liquida , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Espectrometría de Masas , Redes y Vías Metabólicas/genética , Anotación de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Proteómica , Especificidad de la Especie , Triticum/clasificación , Triticum/crecimiento & desarrollo , Triticum/metabolismo
5.
Anal Bioanal Chem ; 407(12): 3471-9, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25701425

RESUMEN

Wheat is the second largest crop cultivated around the world and constitutes a major part of the daily diet in Europe. It is therefore important to determine the content of micronutrient in wheat and wheat-based food products to define the contribution of wheat-based foods to the nutrition of the consumers. The aim of the present work was to develop a simple and rapid method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of seven water-soluble vitamins in various wheat-based food materials. The vitamins present in the test material were separated in less than 15 min by using a reverse-phase C18 column, and analyzed by positive ion electrospray selected reaction monitoring MS/MS. The MS response for all the vitamins was linear over the working range (0.05 to 9 µg/mL) with correlation coefficients ranging between 0.991 and 1. Limits of quantification in the different food materials ranged from 0.09 to 3.5 µg/g. Intra-day and inter-day precision were found satisfactory. The developed method was applied for the simultaneous analysis of the water-soluble vitamin natural content of different semi-coarse wheat flours and in their corresponding baking products.


Asunto(s)
Cromatografía de Fase Inversa/métodos , Harina/análisis , Análisis de los Alimentos/métodos , Espectrometría de Masas en Tándem/métodos , Vitaminas/análisis , Isótopos de Carbono , Hidrólisis , Límite de Detección , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Solubilidad , Espectrometría de Masa por Ionización de Electrospray , Triticum/química , Vitaminas/química
6.
J Proteome Res ; 12(11): 4702-16, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24032428

RESUMEN

Glutens, the storage proteins in wheat grains, are a major source of protein in human nutrition. The protein composition of wheat has therefore been an important focus of cereal research. Proteomic tools have been used to describe the genetic diversity of wheat germplasms from different origins at the level of polymorphisms in alleles encoding glutenin and gliadin, the two main proteins of gluten. More recently, proteomics has been used to understand the impact of specific gluten proteins on wheat quality. Here we review the impact of proteomics on the study of gluten proteins as it has evolved from fractionation and electrophoretic techniques to advanced mass spectrometry. In the postgenome era, proteomics is proving to be essential in the effort to identify and understand the interactions between different gluten proteins. This is helping to fill in gaps in our knowledge of how the technological quality of wheat is determined by the interaction between genotype and environment. We also collate information on the various storage protein alleles identified and their prevalence, which makes it possible to infer the effects of wheat selection on grain protein content. We conclude by reviewing the more recent use of transgenesis aimed at improving the quality of gluten.


Asunto(s)
Alelos , Variación Genética , Glútenes/genética , Glútenes/metabolismo , Proteómica/métodos , Triticum/química , Biología Computacional , Técnicas de Transferencia de Gen , Genómica/métodos , Glútenes/química , Proteómica/tendencias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
7.
Int J Mol Sci ; 14(3): 5650-67, 2013 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-23478438

RESUMEN

Analysis of Portuguese wheat (Triticum aestivum L.) landrace 'Barbela' revealed the existence of a new x-type high molecular weight-glutenin subunit (HMW-GS) encoded at the Glu-A1 locus, which we named 1Ax1.1. Using one-dimensional and two-dimensional electrophoresis and mass spectrometry, we compared subunit 1Ax1.1 with other subunits encoded at the Glu-A1 locus. Subunit 1Ax1.1 has a theoretical molecular weight of 93,648 Da (or 91,508 Da for the mature protein) and an isoelectric point (pI) of about 5.7, making it the largest and most acidic HMW-GS known to be encoded at Glu-A1. Specific primers were designed to amplify and sequence 2601 bp of the Glu-A1 locus from the 'Barbela 28' wheat genome. A very high level of identity was found between the sequence encoding 1Ax1.1 and those encoding other alleles of the locus. The major difference found was an insertion of 36 amino acids in the central repetitive domain.

8.
J Proteome Res ; 11(5): 2754-73, 2012 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-22394196

RESUMEN

Proteomic analysis of albumins and globulins (alg) present in starchy endosperm of wheat (Triticum aestivum cv Récital), at 21 stages of grain development, led to the identification of 487 proteins. Four main developmental phases of these metabolic proteins, with three subphases in phase three and two in phase four, were shown. Hierarchical cluster analysis revealed nine major expression profiles throughout grain development. Classification of identified proteins in 17 different biochemical functions provided a uniform picture of temporal coordination among cellular processes. Proteins involved in cell division, transcription/translation, ATP interconversion, protein synthesis, protein transport, along with amino acid, lipid, carbohydrate and nucleotide metabolisms were highly expressed in early and early mid stages of development. Protein folding, cytoskeleton, and storage proteins peaked during the middle of grain development, while in later stages stress/defense, folic acid metabolism, and protein turn over were the abundant functional categories. Detailed analysis of stress/defense enzymes revealed three different evolutionary profiles. A global map with their predicted subcellular localizations and placement in grain developmental scale was constructed. The present study of complete grain development enriched our knowledge on proteome expression of alg, successively from endosperm cell division and differentiation to programmed cell death.


Asunto(s)
Endospermo/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Almidón/metabolismo , Triticum/crecimiento & desarrollo , Albúminas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Muerte Celular , Diferenciación Celular , División Celular , Endospermo/crecimiento & desarrollo , Perfilación de la Expresión Génica , Globulinas/metabolismo , Espectrometría de Masas , Biosíntesis de Proteínas , Pliegue de Proteína , Transporte de Proteínas , Proteoma/clasificación , Proteínas de Almacenamiento de Semillas/clasificación , Proteínas de Almacenamiento de Semillas/metabolismo , Triticum/metabolismo
9.
J Exp Bot ; 63(2): 1001-11, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22080980

RESUMEN

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.


Asunto(s)
Endospermo/fisiología , Proteínas de Plantas/metabolismo , Proteoma , Estrés Fisiológico/fisiología , Triticum/fisiología , Respuesta de Proteína Desplegada/fisiología , Alelos , Muerte Celular , Retículo Endoplásmico/metabolismo , Endospermo/genética , Endospermo/crecimiento & desarrollo , Endospermo/ultraestructura , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Genotipo , Dureza , Estrés Oxidativo/fisiología , Fenotipo , Proteínas de Plantas/genética , Proteínas de Almacenamiento de Semillas/genética , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/fisiología , Semillas/ultraestructura , Factores de Tiempo , Triticum/genética , Triticum/crecimiento & desarrollo , Triticum/ultraestructura
10.
Theor Appl Genet ; 125(7): 1433-48, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22751952

RESUMEN

Albumins and globulins from the endosperm of Triticum aestivum L. cv Chinese Spring (CS) were analysed to establish a proteome reference map for this standard wheat cultivar. Approximately, 1,145 Coomassie-stained spots were detected by two-dimensional gel electrophoresis (2DE), 410 of which were identified using mass spectrometry and data mining. Salt-soluble endosperm proteins from 67 CS deletion lines were also separated by 2DE (four gels per line). Image analysis of the 268 2DE gels as compared to the CS reference proteome allowed the detection of qualitative and quantitative variations in endosperm proteins due to chromosomal deletions. This differential analysis of spots allowed structural or regulatory genes, encoding 211 proteins, to be located on segments of the 21 wheat chromosomes. In addition, variance analysis of quantitative variations in spot volume showed that the expression of 391 proteins is controlled by one or more chromosome bins with 262 significant increases and 196 significant decreases in spot volume. The spot volume of several proteins was increased or decreased by numerous chromosomal regions and homoeologous-like regulation was revealed for some proteins. Quantitative or qualitative variation in a total of 386 proteins was influenced by genes assigned to at least one chromosomal region, while 66 % of all stained proteins were not found to be influenced by chromosome bins. Proteomics of deletion lines can, therefore, be used to simultaneously analyse the composition and genetics of a complex tissue, such as the wheat endosperm.


Asunto(s)
Albúminas/genética , Ecotipo , Endospermo/genética , Eliminación de Gen , Globulinas/genética , Proteómica/métodos , Triticum/genética , Albúminas/metabolismo , Cromosomas de las Plantas/genética , Electroforesis en Gel Bidimensional , Endospermo/metabolismo , Globulinas/metabolismo , Proteoma/metabolismo , Triticum/metabolismo
11.
Proteomics ; 11(3): 371-9, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21268267

RESUMEN

Grains of hexaploid wheat, Triticum aestivum (cv. Récital), were collected at 15 stages of development, from anthesis to physiological maturity, 0-700°C days (degree days after anthesis). Two hundred and seven proteins of grain peripheral layers (inner pericarp, hyaline, testa and aleurone layer) were identified by 2-DE, MALDI-TOF MS and data mining, then were classified in 16 different functional categories. Study of the protein expression over time allowed identification of five main profiles and four distinct phases of development. Composite expression curves indicated that there was a shift from metabolic processes, translation, transcription and ATP interconversion towards storage and defence processes. Protein synthesis, protein turnover, signal transduction, membrane transport and biosynthesis of secondary metabolites were the mediating functions of this shift. A picture of the dynamic processes taking place in peripheral layers during grain development was obtained in this study. It should further help in the construction of proteome reference maps for the developing peripheral layers.


Asunto(s)
Grano Comestible/crecimiento & desarrollo , Grano Comestible/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteómica , Semillas/metabolismo , Triticum/metabolismo , Electroforesis en Gel Bidimensional , Proteínas de Plantas/análisis , Proteoma/análisis , Semillas/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triticum/crecimiento & desarrollo
12.
Foods ; 10(2)2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33498368

RESUMEN

This study investigates genetic and environmental variation in starch content and characteristics of 14 French bread cultivars. Understanding the impact of these factors on wheat quality is important for processors and especially bakers to maintain and meet the requirements of industrial specifications. Different traits were evaluated: starch content, distribution of starch granules, percentage of amylose and amylopectin and their molecular characteristics (weight-average molar mass, number-average molar mass, polydispersity and gyration radius). Genetic, environment and their interaction had significant effects on all parameters. The relative magnitude of variance attributed to growth conditions, for most traits, was substantially higher (21% to 95%) than that attributed to either genotype (2% to 73%) or G × E interaction (2% to 17%). The largest environmental contribution (95%) to total variance was found for starch dispersity. The highest genetic influence was found for the percentage of A-type starch granules. G × E interaction had relatively little influence (≈7%) on total phenotypic variance. All molecular characteristics were much more influenced by environment than the respective percentages of amylose and amylopectin were. This huge difference in variance between factors obviously revealed the importance of the effect of growing conditions on characteristics of cultivars.

13.
J Agric Food Chem ; 69(14): 4307-4318, 2021 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-33784092

RESUMEN

The metabolomic profiling analyses of 11 vitamins' statuses of wheat grain in a subsample of 167 accessions from the INRAE worldwide bread wheat core collection planted in two contrasting environments in France (Le Moulon and Clermont-Ferrand) have been evaluated using a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure. This has allowed us to perform a genome-wide association study (GWAS) for these nutritional traits of interest combining the phenotypic data with the genotypic data derived from the TaBW280K SNP chip. Considering both thresholds (P < 0.0003 and R2 ≥ 8%), the GWAS identified between 1 and 22 marker-trait associations (MTAs) for the individual vitamins at the individual locations, and 12 SNP markers were stable and associated with vitamin contents across two environments. Desirable alleles and superior genotypes identified in the current analysis provide novel genetic data that can be used for future research on the genetics of vitamins and their application in wheat breeding.


Asunto(s)
Pan , Triticum , Cromatografía Liquida , Francia , Estudio de Asociación del Genoma Completo , Genotipo , Fenotipo , Fitomejoramiento , Polimorfismo de Nucleótido Simple , Espectrometría de Masas en Tándem , Triticum/genética , Vitaminas
14.
Proteomics ; 10(16): 2901-10, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20641138

RESUMEN

The identification of 249 proteins in the first 2 wks of wheat grain development enabled the chronological description of the early processes of grain formation. Cell division involved expression of the enzymes and proteins of the cytoskeleton and structure, DNA repair and replication enzymes and cellular metabolism enzymes (synthesis of amino acids, cell wall initiation, carbon fixation and energy production, cofactors and vitamins) with a peak expression at 125 degrees C day (degrees day after anthesis). After the first synthesis of amino acids, protein transport mechanisms, translation signals, sugar metabolism (polymerization of protein) and stress/defence proteins were activated with stable expression between 150 and 280 degrees C day. Proteins responsible for folding and degradation, including different subunits of proteasome, were highly expressed at 195 degrees C day. Proteins associated with starch granules (GBSS type 1) were present at the beginning of grain formation and increased regularly up to 280 degrees C day. Heat shock proteins (HSP70, 80, 90) were expressed throughout the early grain development stages.


Asunto(s)
Proteínas de Plantas/clasificación , Proteómica/métodos , Semillas/química , Triticum , Recuento de Células , Electroforesis en Gel Bidimensional , Procesamiento de Imagen Asistido por Computador , Cinética , Redes y Vías Metabólicas , Fragmentos de Péptidos/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Transducción de Señal , Triticum/crecimiento & desarrollo , Triticum/metabolismo , Triticum/fisiología , Tripsina
15.
J Proteome Res ; 9(6): 3299-310, 2010 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-20481496

RESUMEN

Starch consists of the two glucose polymers, amylose and amylopectin, and is deposited as semicrystalline granules inside plastids. The starch granule proteome is particularly challenging to study due to the amount of interfering compounds (sugars, storage proteins), the very low starch granule-associated protein content and also the dynamic range of abundant proteins. Here we present the protocol for extraction and 2-DE of wheat starch granule-associated proteins whose most important steps are: (i) washing and sonication to remove interfering compounds (storage proteins) from the surface of the granules, (ii) scanning electron microscopy (SEM) observations to monitor purification and granules swelling, (iii) appropriate protein extraction and solubilization to obtain enough proteins for Coomassie blue staining and proteomic analysis. Our objective was to minimize the amount of contamination by storage proteins and to preserve the structure of the starch and of starch-associated proteins and to maximize the number of polypeptides that can be resolved. For quantitative proteomic analysis of proteins associated with wheat starch granules, we developed a two-step protein extraction protocol including TCA/acetone precipitation and phenol extraction. With this protocol, proteins were extracted from wheat starch granules and solubilized and satisfactory blue-stained 2-DE protein maps were obtained. The majority of the spots associated with starch granules were identified by peptide mass fingerprinting and MS/MS and functionally classified into carbohydrate metabolism and stress defense.


Asunto(s)
Endospermo/química , Proteínas de Plantas/química , Proteoma/química , Almidón/química , Triticum/química , Electroforesis en Gel Bidimensional , Microscopía Electrónica de Rastreo , Proteínas de Plantas/clasificación , Proteínas de Plantas/aislamiento & purificación , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem
16.
BMC Genomics ; 11: 185, 2010 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-20298594

RESUMEN

BACKGROUND: Transcription factors (TFs) regulate gene expression by interacting with promoters of their target genes and are classified into families based on their DNA-binding domains. Genes coding for TFs have been identified in the sequences of model plant genomes. The rice (Oryza sativa spp. japonica) genome contains 2,384 TF gene models, which represent the mRNA transcript of a locus, classed into 63 families. RESULTS: We have created an extensive list of wheat (Triticum aestivum L) TF sequences based on sequence homology with rice TFs identified and classified in the Database of Rice Transcription Factors (DRTF). We have identified 7,112 wheat sequences (contigs and singletons) from a dataset of 1,033,960 expressed sequence tag and mRNA (ET) sequences available. This number is about three times the number of TFs in rice so proportionally is very similar if allowance is made for the hexaploidy of wheat. Of these sequences 3,820 encode gene products with a DNA-binding domain and thus were confirmed as potential regulators. These 3,820 sequences were classified into 40 families and 84 subfamilies and some members defined orphan families. The results were compiled in the Database of Wheat Transcription Factor (wDBTF), an inventory available on the web http://wwwappli.nantes.inra.fr:8180/wDBFT/. For each accession, a link to its library source and its Affymetrix identification number is provided. The positions of Pfam (protein family database) motifs were given when known. CONCLUSIONS: wDBTF collates 3,820 wheat TF sequences validated by the presence of a DNA-binding domain out of 7,112 potential TF sequences identified from publicly available gene expression data. We also incorporated in silico expression data on these TFs into the database. Thus this database provides a major resource for systematic studies of TF families and their expression in wheat as illustrated here in a study of DOF family members expressed during seed development.


Asunto(s)
Bases de Datos de Ácidos Nucleicos , Proteínas de Plantas/genética , Factores de Transcripción/genética , Triticum/genética , Biología Computacional , Regulación de la Expresión Génica de las Plantas , Internet , Oryza/genética , Filogenia
17.
BMC Plant Biol ; 10: 124, 2010 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-20573275

RESUMEN

BACKGROUND: Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF x SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. RESULTS: At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. CONCLUSIONS: PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of which is available from the CIMMYT and INRA Clermont-Ferrand germplasm collections, should also promote information sharing in the identification of individual LMW-GS and thus provide useful information for quality improvement in common wheat.


Asunto(s)
Glútenes/química , Triticum/química , Alelos , ADN de Plantas/genética , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Glútenes/genética , Glútenes/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triticum/genética
18.
Int Arch Allergy Immunol ; 153(1): 35-45, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20357483

RESUMEN

BACKGROUND: Adults suffering from wheat-dependant, exercise-induced anaphylaxis (WDEIA) develop IgE directed against wheat omega5-gliadins (major allergens for this allergy) and against wheat low-molecular weight glutenin subunits (LMW-GS). However, the ability of LMW-GS to trigger an inflammatory response is still unknown. It also remains to be determined if IgE from these patients bind the same epitopes on LMW-GS and omega5-gliadins or if the epitopes are independent. METHODS: WDEIA patients were selected and skin prick tests (SPTs) were performed on them using commercial gluten, wheat flour extracts, prolamin fractions and a purified natural LMW-GS P42. The IgE-binding ability of natural and recombinant wheat prolamins was verified by immunoblot experiments. Cross-reactivity between LMW-GS and omega5-gliadins was studied by immunoblot inhibition experiments, using purified natural omega5-gliadin as an inhibitor. RESULTS: Patients developed positive SPTs with natural LMW-GS fractions and/or with the purified LMW-GS P42. Natural and recombinant LMW-GS were highly reactive with patient IgE in immunoblot experiments, as was omega5-gliadin. However, differences in reactivity were evident within the LMW-GS group. Except for one recombinant LMW-GS (P73), IgE cross-reactivity between LMW-GS and natural omega5-gliadin was only partial. CONCLUSION: LMW-GS are able to promote local inflammation and they share common epitopes with omega5-gliadins. The nature of these epitopes is discussed. LMW-GS also carried specific epitopes, completely independent from the omega5-gliadin epitopes. Thus, LMW-GS behaved partly as independent allergens.


Asunto(s)
Anafilaxia , Ejercicio Físico , Gliadina/inmunología , Glútenes/inmunología , Hipersensibilidad al Trigo/inmunología , Adulto , Alérgenos/efectos adversos , Alérgenos/inmunología , Secuencia de Aminoácidos , Anafilaxia/etiología , Anafilaxia/inmunología , Antígenos/inmunología , Compuestos Bicíclicos Heterocíclicos con Puentes , Reacciones Cruzadas , Gliadina/efectos adversos , Gliadina/química , Gliadina/genética , Glútenes/efectos adversos , Glútenes/química , Glútenes/genética , Humanos , Inmunoglobulina E/metabolismo , Persona de Mediana Edad , Piperidinas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Pruebas Cutáneas , Triticum/inmunología , Adulto Joven
19.
Foods ; 9(11)2020 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-33207650

RESUMEN

We characterized the molecular weight distribution of polymeric proteins (PP) of bread wheat grains using asymmetric flow field flow fractionation (A4F). The experiment, involving six environmental conditions and 130 cultivars, offered the opportunity to approach the phenotypic values of the polymer characteristics and their contribution of the rheological properties of flours and/or doughs. The contents of high-molecular-weight polymers (MW > 2 × 106 g·mol-1) that can be considered as "rheologically active polymers" (RAPP) for their major contribution to dough baking strength and mixing tolerance were mainly controlled by environmental factors. Under the influence of the growing conditions, at the cellular level, the redox status of non-protein free thiol, such as glutathione, is modified and leads to the formation of polymeric protein-bound glutathione conjugates (PPSSG). The accumulation of these conjugates reduces the formation of the RAPP by limiting the intermolecular interactions between PP in the grain during desiccation. This phenomenon is, therefore, potentially responsible for decreases in the technological properties of the wheat genotypes concerned. These first results invite us to continue our investigations to fully confirm this phenomenon, with emphasis on the behavior of wheat genotypes under various growing conditions.

20.
Foods ; 9(5)2020 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-32466243

RESUMEN

The polymers of wheat glutenins are studied here using asymmetric flow field flow fractionation (A4F). Molecular mass (Mw), gyration radius (Rw), and the polydispersity index (PI) of polymers were measured over a four-year, multi-local wheat trial in France. The experiment, involving 11 locations and 192 cultivars, offered the opportunity to approach the genetic and environmental factors associated with the phenotypic values of the polymer characteristics. These characteristics, which were all highly influenced by environmental factors, exhibited low broad-sense heritability coefficients and were not influenced by grain protein content and grain hardness. The 31 alleles encoding the glutenin subunits explained only 17.1, 25.4, and 16.8% of the phenotypic values of Mw, Rw, and PI, respectively. The climatic data revealed that a 3.5 °C increase between locations of the daily average temperature, during the last month of the grain development, caused an increase of more than 189%, 242%, and 434% of the Mw, Rw, and PI, respectively. These findings have to be considered in regard to possible consequences of global warming and health concerns assigned to gluten. It is suggested that the molecular characteristics of glutenins be measured today, especially for research addressing non-celiac gluten sensitivity (NCGS).

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