The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Gerard Branlard - One of the best experts on this subject based on the ideXlab platform.
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proteome evolution of wheat triticum aestivum l Aleurone Layer at fifteen stages of grain development
Journal of Proteomics, 2015Co-Authors: I. Nadaud, Christophe Chambon, Ayesha Tasleemtahir, Annelaure Chateignerboutin, Didier Viala, Gerard BranlardAbstract:Abstract The Aleurone Layer (AL) is the grain peripheral tissue; it is rich in micronutrients, vitamins, antioxidants, and essential amino acids. This highly nutritive part of the grain has been less studied partly because its isolation is so laborious. In the present study, the ALs of Triticum aestivum (variety Recital) were separated manually at 15 stages of grain development. A total of 327 proteins were identified using 2-DE LC-MS/MS. They were classified in six main groups and 26 sub-groups according to their biochemical function. Proteomic analysis revealed seven different profiles distributed among three main development stages: (i) early AL development, with proteins involved in intense metabolic activities in the growth and development of the cell wall compounds; (ii) the intermediate stage, characterized by oxidative stress and defense proteins (65%) linked with loss of water in peripheral Layers during grain filling; and (iii) AL maturation, involving the production of amino acids and the control of reactive oxidative species to enable the accumulation and maturation of globulins within the AL. The present study provides the first insights into developing proteome in the AL. We describe the numerous AL enzymes involved in the accumulation of storage protein and in the protection of the endosperm over time. Biological significance The hand dissection of wheat Aleurone Layer (AL) was carried in this study for the first time on fifteen developmental stages from cell differentiation to grain maturity. Three major phases were revealed over AL development: cell division activities, globulins storage, and grain protection. Enzymes related to metabolites and vitamins were abundantly expressed during the two first phases. In parallel to the progressive globulins accumulation, the final phase was characterized by key enzyme synthesis involved in energy production, amino-acids and antioxidant synthesis plus others to face hypoxia and dehydration of grain tissues.
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proteomic analysis of the mature kernel Aleurone Layer in common and durum wheat
Journal of Cereal Science, 2012Co-Authors: Smail Meziani, I. Nadaud, Marzouk Benali, Christophe Chambon, Brigitte Gaillardmartinie, Gerard BranlardAbstract:Abstract The Aleurone Layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties ( T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed.
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Proteomic analysis of the mature kernel Aleurone Layer in common and durum wheat
Journal of Cereal Science, 2012Co-Authors: Smail Meziani, I. Nadaud, Marzouk Benali, Christophe Chambon, Brigitte Gaillard-martinie, Gerard BranlardAbstract:The Aleurone Layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties (T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed. © 2012 Elsevier Ltd.
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isolation of the wheat Aleurone Layer for 2d electrophoresis and proteomics analysis
Journal of Cereal Science, 2008Co-Authors: B. Laubin, I. Nadaud, Christophe Chambon, Valerie Lullienpellerin, Brigitte Gaillardmartinie, Gerard BranlardAbstract:Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the Aleurone Layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral Layers including the Aleurone Layer or only the Aleurone Layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell Layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the Aleurone Layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral Layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat.
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Isolation of the wheat Aleurone Layer for 2D electrophoresis and proteomics analysis
Journal of Cereal Science, 2008Co-Authors: B. Laubin, I. Nadaud, Valerie Lullien-pellerin, Christophe Chambon, Brigitte Gaillard-martinie, Gerard BranlardAbstract:Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the Aleurone Layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral Layers including the Aleurone Layer or only the Aleurone Layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell Layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the Aleurone Layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral Layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat. © 2008 Elsevier Ltd. All rights reserved.
Christine Finnie - One of the best experts on this subject based on the ideXlab platform.
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Immobilisation of barley Aleurone Layers enables parallelisation of assays and analysis of transient gene expression in single cells.
Plant Physiology and Biochemistry, 2017Co-Authors: Christina Mark, Arto Heiskanen, Claus Krogh Madsen, Martin Dufva, Jenny Emnéus, Henrik Brinch-pedersen, Christine FinnieAbstract:Abstract The barley Aleurone Layer is an established model system for studying phytohormone signalling, enzyme secretion and programmed cell death during seed germination. Most analyses performed on the Aleurone Layer are end-point assays based on cell extracts, meaning each sample is only analysed at a single time point. By immobilising barley Aleurone Layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same Aleurone Layer samples over time, in the presence or absence of plant hormones and other effectors. The system is also amenable to transient gene expression by particle bombardment, with simultaneous monitoring of cell death. In conclusion, the easy to handle and efficient experimental setup developed here enables continuous monitoring of tissue samples, parallelisation of assays and single cell analysis, with potential for time course studies using any plant tissue that can be immobilised, for example leaves or epidermal peels.
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spatio temporal appearance of α amylase and limit dextrinase in barley Aleurone Layer in response to gibberellic acid abscisic acid and salicylic acid
Journal of the Science of Food and Agriculture, 2015Co-Authors: Azar Shahpiri, Nasim Talaei, Christine FinnieAbstract:BACKGROUND Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the Aleurone Layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley Aleurone Layers is presented for the important enzymes in starch degradation: α-amylase and limit dextrinase (LD). RESULTS Proteins were visualized by immunoblotting in Aleurone Layers and culture supernatants from dissected Aleurone Layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from Aleurone Layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from Aleurone Layers. CONCLUSIONS Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated Aleurone Layers or Aleurone Layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from Aleurone Layers treated with GA+ABA or GA+SA. © 2014 Society of Chemical Industry
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Spatio‐temporal appearance of α‐amylase and limit dextrinase in barley Aleurone Layer in response to gibberellic acid, abscisic acid and salicylic acid
Journal of the Science of Food and Agriculture, 2014Co-Authors: Azar Shahpiri, Nasim Talaei, Christine FinnieAbstract:BACKGROUND Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the Aleurone Layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley Aleurone Layers is presented for the important enzymes in starch degradation: α-amylase and limit dextrinase (LD). RESULTS Proteins were visualized by immunoblotting in Aleurone Layers and culture supernatants from dissected Aleurone Layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from Aleurone Layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from Aleurone Layers. CONCLUSIONS Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated Aleurone Layers or Aleurone Layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from Aleurone Layers treated with GA+ABA or GA+SA. © 2014 Society of Chemical Industry
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Gibberellic acid-induced Aleurone Layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress.
Plant Physiology, 2013Co-Authors: Gregorio Barba-espín, Birte Svensson, Plaipol Dedvisitsakul, Per Hägglund, Christine FinnieAbstract:The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) Aleurone Layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced Aleurone Layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked Aleurone Layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 Aleurone Layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the Aleurone Layer as a model system for studying plant protein secretion.
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proteomes of the barley Aleurone Layer a model system for plant signalling and protein secretion
Proteomics, 2011Co-Authors: Christine Finnie, Azar Shahpiri, Birgit Andersen, Birte SvenssonAbstract:The cereal Aleurone Layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the Aleurone Layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley Aleurone Layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the Aleurone Layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the Aleurone Layer system that makes it promising for investigations of plant protein secretion mechanisms.
I. Nadaud - One of the best experts on this subject based on the ideXlab platform.
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proteome evolution of wheat triticum aestivum l Aleurone Layer at fifteen stages of grain development
Journal of Proteomics, 2015Co-Authors: I. Nadaud, Christophe Chambon, Ayesha Tasleemtahir, Annelaure Chateignerboutin, Didier Viala, Gerard BranlardAbstract:Abstract The Aleurone Layer (AL) is the grain peripheral tissue; it is rich in micronutrients, vitamins, antioxidants, and essential amino acids. This highly nutritive part of the grain has been less studied partly because its isolation is so laborious. In the present study, the ALs of Triticum aestivum (variety Recital) were separated manually at 15 stages of grain development. A total of 327 proteins were identified using 2-DE LC-MS/MS. They were classified in six main groups and 26 sub-groups according to their biochemical function. Proteomic analysis revealed seven different profiles distributed among three main development stages: (i) early AL development, with proteins involved in intense metabolic activities in the growth and development of the cell wall compounds; (ii) the intermediate stage, characterized by oxidative stress and defense proteins (65%) linked with loss of water in peripheral Layers during grain filling; and (iii) AL maturation, involving the production of amino acids and the control of reactive oxidative species to enable the accumulation and maturation of globulins within the AL. The present study provides the first insights into developing proteome in the AL. We describe the numerous AL enzymes involved in the accumulation of storage protein and in the protection of the endosperm over time. Biological significance The hand dissection of wheat Aleurone Layer (AL) was carried in this study for the first time on fifteen developmental stages from cell differentiation to grain maturity. Three major phases were revealed over AL development: cell division activities, globulins storage, and grain protection. Enzymes related to metabolites and vitamins were abundantly expressed during the two first phases. In parallel to the progressive globulins accumulation, the final phase was characterized by key enzyme synthesis involved in energy production, amino-acids and antioxidant synthesis plus others to face hypoxia and dehydration of grain tissues.
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proteomic analysis of the mature kernel Aleurone Layer in common and durum wheat
Journal of Cereal Science, 2012Co-Authors: Smail Meziani, I. Nadaud, Marzouk Benali, Christophe Chambon, Brigitte Gaillardmartinie, Gerard BranlardAbstract:Abstract The Aleurone Layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties ( T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed.
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Proteomic analysis of the mature kernel Aleurone Layer in common and durum wheat
Journal of Cereal Science, 2012Co-Authors: Smail Meziani, I. Nadaud, Marzouk Benali, Christophe Chambon, Brigitte Gaillard-martinie, Gerard BranlardAbstract:The Aleurone Layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties (T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed. © 2012 Elsevier Ltd.
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isolation of the wheat Aleurone Layer for 2d electrophoresis and proteomics analysis
Journal of Cereal Science, 2008Co-Authors: B. Laubin, I. Nadaud, Christophe Chambon, Valerie Lullienpellerin, Brigitte Gaillardmartinie, Gerard BranlardAbstract:Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the Aleurone Layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral Layers including the Aleurone Layer or only the Aleurone Layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell Layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the Aleurone Layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral Layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat.
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Isolation of the wheat Aleurone Layer for 2D electrophoresis and proteomics analysis
Journal of Cereal Science, 2008Co-Authors: B. Laubin, I. Nadaud, Valerie Lullien-pellerin, Christophe Chambon, Brigitte Gaillard-martinie, Gerard BranlardAbstract:Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the Aleurone Layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral Layers including the Aleurone Layer or only the Aleurone Layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell Layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the Aleurone Layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral Layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat. © 2008 Elsevier Ltd. All rights reserved.
Jean Daussant - One of the best experts on this subject based on the ideXlab platform.
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contribution of Aleurone Layer and scutellum to α amylase synthesis and secretion in wheat and rice grains
Physiologia Plantarum, 1991Co-Authors: Cluadine Thevenot, Elizabeth Simondcote, Jean DaussantAbstract:Intact Aleurone Layers from wheat (Triticum aestivum L. cv Camp Remy) and rice (Oryza sativa L. cv Cigalon) grains both contained and secreted more α-amylase (EC 3.2.1.1.) than did the corresponding scutellar tissues. This discrimination was already evident at the earliest stages of germination at which the tissues could be isolated, and became more pronounced upon subsequent germination and growth. Isoenzyme patterns obtained upon isoelectric focusing showed a considerable polymorphism of the α-amylases of each cereal. The enzyme polymorphism pattern was the same in the Aleurone Layer and in the scutellum, but some secondary constituents appeared to be more specific for the one or the other of the tissues. Moreover, the isozymes found in the tissues were the same as those found to be secreted. A third α-amylase antigen which differs from the well established α1 and α11 forms was identified in the germinating wheat grains. The presence of Ca2+ in the secretion medium favoured maximum secretion of α-amylases from the wheat scutellum and Aleurone Layers, whereas it inhibited the secretion of the enzymes from the rice Aleurone Layer.
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Contribution of Aleurone Layer and scutellum to α‐amylase synthesis and secretion in wheat and rice grains
Physiologia Plantarum, 1991Co-Authors: Cluadine Thevenot, Elizabeth Simond‐côte, Jean DaussantAbstract:Intact Aleurone Layers from wheat (Triticum aestivum L. cv Camp Remy) and rice (Oryza sativa L. cv Cigalon) grains both contained and secreted more α-amylase (EC 3.2.1.1.) than did the corresponding scutellar tissues. This discrimination was already evident at the earliest stages of germination at which the tissues could be isolated, and became more pronounced upon subsequent germination and growth. Isoenzyme patterns obtained upon isoelectric focusing showed a considerable polymorphism of the α-amylases of each cereal. The enzyme polymorphism pattern was the same in the Aleurone Layer and in the scutellum, but some secondary constituents appeared to be more specific for the one or the other of the tissues. Moreover, the isozymes found in the tissues were the same as those found to be secreted. A third α-amylase antigen which differs from the well established α1 and α11 forms was identified in the germinating wheat grains. The presence of Ca2+ in the secretion medium favoured maximum secretion of α-amylases from the wheat scutellum and Aleurone Layers, whereas it inhibited the secretion of the enzymes from the rice Aleurone Layer.
Birte Svensson - One of the best experts on this subject based on the ideXlab platform.
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Gibberellic acid-induced Aleurone Layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress.
Plant Physiology, 2013Co-Authors: Gregorio Barba-espín, Birte Svensson, Plaipol Dedvisitsakul, Per Hägglund, Christine FinnieAbstract:The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) Aleurone Layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced Aleurone Layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked Aleurone Layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 Aleurone Layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the Aleurone Layer as a model system for studying plant protein secretion.
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proteomes of the barley Aleurone Layer a model system for plant signalling and protein secretion
Proteomics, 2011Co-Authors: Christine Finnie, Azar Shahpiri, Birgit Andersen, Birte SvenssonAbstract:The cereal Aleurone Layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the Aleurone Layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley Aleurone Layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the Aleurone Layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the Aleurone Layer system that makes it promising for investigations of plant protein secretion mechanisms.
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feasibility study of a tissue specific approach to barley proteome analysis Aleurone Layer endosperm embryo and single seeds
Journal of Cereal Science, 2003Co-Authors: Christine Finnie, Birte SvenssonAbstract:Abstract Proteome analysis in the pI 4–7 range of aqueous extracts from whole barley seeds results in complex 2D-gel spot patterns that represent proteins from all seed tissues. Proteins were extracted from dissected Aleurone Layer, embryo and endosperm for separate analysis of these components and comparison with single seed extracts. The analysis showed that while the starchy endosperm comprises approximately 85% of the mature seed dry weight, it contributes less than 50% of the soluble protein to seed extracts. Although the Aleurone Layer and embryo are minor components of the seed in terms of dry weight, their proteins contribute significantly to the 2D-gel pattern. Distinct but overlapping patterns were observed for the dissected tissues. While whole seed gels contained about 850 spots, endosperm, Aleurone Layer and embryo gels had about 575, 850 and 1000 spots, respectively, representing a total increase of at least 15%. Protein patterns were also reduced in complexity and this enrichment of subsets of proteins facilitated identification by mass spectrometry of proteins of interest in mature seed tissues and developing endosperm. These techniques can therefore be used with advantage to describe the proteomes of dissected seed tissues.
