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... Before the 2-D DIGE experiments, 2-D PAGE gels for each sample of soybean leaves (NT, NT-AgNO 3 , NT-AgNP, T, T-AgNO 3 , and T-AgNP) were performed (N = 3) as previously described [5] to evaluate the sample preparation and to obtain a proteomic profile for each sample. For 2-D DIGE analysis, the gels were run under the same conditions, and nine comparative groups were evaluated: (i) NT vs. NT-AgNO 3 , (ii) NT vs. NT-AgNP, (iii) NT-AgNO 3 vs. ...
... For LC-MS/MS analysis, the procedure was followed as Arruda et al. [5]. All files from the acquired mass spectra were converted into peaklist format using Mascot Distiller (Matrix Science, London, UK) and subjected to the NCBI and Glycine max database (checked in March 2018). ...
... All files from the acquired mass spectra were converted into peaklist format using Mascot Distiller (Matrix Science, London, UK) and subjected to the NCBI and Glycine max database (checked in March 2018). The MASCOT Server 2.3 MS/MS was used and search parameters were the same as Arruda et al. [5]. The significance threshold was set at p < 0.05, which corresponds to a minimum score of 32. ...
Article
Significance: Once nanoparticles have been employed for several applications in recent years and they can be released in the environmental matrices, this study highlights proteomic and enzymatic alterations in transgenic and non-transgenic soybeans, an important crop, after cultivation with silver nanoparticles. Such strategy employing proteomic and enzymatic approaches to evaluate soybeans exposed to silver nanoparticles has not yet been reported. Therefore, the results obtained in this study can.
... Although metallomics presents plenty of examples involving medical [8,12], biochemical [13][14][15], environmental [16], plant science [17,18], among other areas [19,20], scarce information are achieved involving transgenic (T) or genetically modified (GM) plants. In fact, some comparative studies of T x NT soybean or GM x N-GM Arabidopsis thaliana plants were carried out regarding possible alterations of metals, enzymes, proteins and metalloproteins in different plant compartments [21][22][23][24][25]. Due to its completely sequenced genome [26], A. thaliana is currently used as a plant model for a diversity of studies [27][28][29], and, recently, this specie was evaluated through an ionomic approach using laser ablation mass spectrometry (LA-ICP-MS) [25], where the authors put in evidence an imbalance regarding plant homeostasis involving selenium and sulfur transporters. ...
... hydroxyl radicals (OH • ). Excessive concentration of such molecules causes protein and DNA modifications, lipid peroxidation and even lead to death of cell, among others [23,24,51]. For this reason, the control of ROS concentration through some cellular pathways is of utmost importance to cells. ...
... to different conditions such as genetic modification [23,24,52,53] or metal irrigation [14][15][16]25,54,55] is also reported. The results of ROS for GM and N-GM leaves from selenium treated plants are present in Fig. 3. ...
Article
This work reports the evaluation of the Arabidopsis thaliana, genetically modified or not, cultivated in the absence or in the presence of different concentrations of sodium selenite, employing mass spectrometry-based platforms and metallomics and enzymatic approaches. The genetic modification is evidenced through PCR analysis. The total selenium determination in the leaves of such culture is performed through ICP-MS. The metalloproteins are identified through LC-ICP-MS and ESI-MS/MS, which are involved in a diversity of modifications observed in the genetically and non-genetically modified plants. Additionally, some enzymes are also evaluated, which are involved in the oxidative stress such as SOD and catalase, as well as the concentrations of MDA and H2O2. Results on Se accumulation in the genetically modified is higher than non-genetically modified leaves as 130%, 19% and 17% for control, Se-low and Se-high treatments, respectively. From the results achieved in this work, it was verified that the genetic modification itself is a stressful event to the plants, once while a decreased of 32% water-soluble protein content and ca. 48% on SOD activity were observed, an increase of 69% on the H2O2 production was noted in GM leaves, when the Se was added to the culture. Additionally, the genetic modification conferred plant resistance to Se oxidative stress, due to the decrease of both water-soluble protein content (33%) and SOD activity (66%), as well as the increment on H2O2 production (19%) in control GM leaves, when compared with N-GM leaves.
... Despite the importance of evaluating ROS and the enzymes involved in its combat (i.e., superoxide dismutase-SOD ( Fridovich, 1995), catalase-CAT (Scandalios et al., 1997), glutathione reductase-GR, among others), the few examples found in the literature for soybeans (Arruda et al., 2013a) are not focused in comparative studies involving T and NT soybeans. In fact, there are only two examples that were carried out by our research team and will be briefly reviewed in this section. ...
... Techniques based on modern biotechnology known as omics sciences, studying DNA, RNA, proteins, metabolites, and metals, among others, have been used in an attempt to answer some questions about the soybean culture and for evaluating the effects of the T soybean species at the molecular level (Arruda et al., 2013a;Barbosa et al., 2012;Clark et al., 2013). Despite more than a decade of use, no case of T food human consumption was reported as malicious. ...
... Much of the studies found in the literature are focused on these proteins (Natarajan et al., 2009). However, other studies involve a more thorough proteomic investigation of soybean proteins (Arruda et al., 2013a;Barbosa et al., 2012;Garcia et al., 2006;Natarajan et al., 2007Natarajan et al., , 2009) as well as studies involving food safety and the possible effects T organisms Krzyzowska et al., 2010). Krzyzowska et al. (2010) evaluated the possible effects of T triticale, which is a hybrid between wheat and rye, on animal health in five successive generations of rats. ...
Chapter
For a very long time, humanity has been searching for more productive cultivars, employing techniques such as grafting, cutting, or artificial pollination. However, with the increasing demand for food, genetic modification has become of utmost importance. The insertion of the cp4-EPSPS gene for producing a soybean tolerant to herbicides was one the pioneers in terms of genetically modified organisms. This chapter briefly comments about this history as well as emphasizes comparative studies involving transgenic and nontransgenic soybeans (seeds and plants), focusing on (metallo)proteins, metabolites, some enzymes involved in the reactive oxygen species combat, and ions, which are evaluated in different studies. Additionally, research trends are also presented.
... The cry gene, encoding Cry protein showing a broad insecticidal spectrum, has been widely used [23]. As for herbicide resistance genes, the epsps gene, encoding 5enolpyruvate shikimic acid-3-phosphate synthase (EPSPS), is most widely used in GM crops [24][25][26]. Previous studies have focused more on environmental factors and the impact of GM on crop growth processes. ...
... A total of 145, 178, and 88 DAMs were found in the BBL/ZH58, BFL-1/ZH58, and BFL-2/ZH58×CH72 comparisons, respectively. None of these DEPs and DAMs were identified as novel toxins or allergens, only changed in abundance, consistent with previous reports [4,24,43]. The GM maize varieties were not dramatically different to their non-GM parents based on seed proteomic profiles; only less than 2.5% of the total identified proteins were screened out as DEPs. ...
Article
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Label-free quantitative proteomic (LFQ) and widely targeted metabolomic analyses were applied in the safety evaluation of three genetically modified (GM) maize varieties, BBL, BFL-1, and BFL-2, in addition to their corresponding non-GM parent maize. A total of 76, 40, and 25 differentially expressed proteins (DEPs) were screened out in BBL, BFL-1, and BFL-2, respectively, and their abundance compared was with that in their non-GM parents. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most of the DEPs participate in biosynthesis of secondary metabolites, biosynthesis of amino acids, and metabolic pathways. Metabolomic analyses revealed 145, 178, and 88 differentially accumulated metabolites (DAMs) in the BBL/ZH58, BFL-1/ZH58, and BFL-2/ZH58×CH72 comparisons, respectively. KEGG pathway enrichment analysis showed that most of the DAMs are involved in biosynthesis of amino acids, and in arginine and proline metabolism. Three co-DEPs and 11 co-DAMs were identified in the seeds of these GM maize lines. The proteomic profiling of seeds showed that the GM maize varieties were not dramatically different from their non-GM control. Similarly, the metabolomic profiling of seeds showed no dramatic changes in the GM/non-GM maize varieties compared with the GM/GM and non-GM/non-GM maize varieties. The genetic background of the transgenic maize was found to have some influence on its proteomic and metabolomic profiles.
... A cascade of enzymes involved in combating reactive oxygen species, such as ascorbate peroxidase, glutathione reductase, and catalase, were also expressed at a higher level in transgenic soybean seeds in another investigation [18]. Unintended effects of the inserted EPSPS-CP4 transgene were linked to energy metabolic disturbances in other studies [18][19][20]. It can be hypothesized that the plant is searching for a new equilibrium to maintain heterologous EPSPS-CP4 metabolism within levels that can be tolerated by the plant [16]. ...
... In addition to ribosome and protein export metabolism, GM plants activate sulfur and purine metabolism, fatty acid biosynthesis and metabolism, biosynthesis of monoterpenoids, and glutathione metabolism. Most of these pathways have already been described in previous studies as being altered in response to drought and herbicide stress in GM plants [15,[17][18][19][20]. However, when adding the effect of genetic transformation to herbicide and drought stress factors, the present study detected the alteration of additional pathways, suggesting an accumulation of metabolic costs. ...
Article
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While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems. Therefore, the aim of this study was to analyze the metabolic cost by assessing the proteomic profiles of GM soybean varieties under glyphosate spraying and water deficit conditions compared to their non-transgenic conventional counterparts. We found evidence of cumulative adverse effects that resulted in the reduction of enzymes involved in carbohydrate metabolism, along with the expression of amino acids and nitrogen metabolic enzymes. Ribosomal metabolism was significantly enriched, particularly the protein families associated with ribosomal complexes L5 and L18. The interaction network map showed that the affected module representing the ribosome pathway interacts strongly with other important proteins, such as the chloro-plastic gamma ATP synthase subunit. Combined, these findings provide clear evidence for increasing the metabolic costs of GM soybean plants in response to the accumulation of stress factors. First, alterations in the ribosome pathway indicate that the GM plant itself carries a metabolic burden associated with the biosynthesis of proteins as effects of genetic transformation. GM plants also showed an imbalance in energy demand and production under controlled conditions, which was increased under drought conditions. Identifying the consequences of altered metabolism related to the interaction between plant transgene stress responses allows us to understand the possible effects on the ecology and evolution of plants in the medium and long term and the potential interactions with other organisms when these organisms are released in the environment.
... There are several studies investigating unintended effects in GM crops using different omics approaches and microarrays. In these studies, it was found that transgenic inserts of different species of plants might affect the overall expression of other endogenous genes [1,2,7,8,12,17,19,32,35,40,45,49,60,84,90]. However, several authors of these studies mention that the changes in gene expression and protein distribution caused by genetic modification were smaller than those caused by environmental factors or natural variations. ...
... In addition, the varieties containing either BT or NK603 were clustered separately and clearly different from the non-GM varieties [2]. Arruda et al. and Herrera-Agudelo et al. detected significant differences in proteins, metalloproteins, enzymes and metals between transgenic soybeans harboring a RR insert and non-transgenic soybeans [5][6][7]40]. ...
Article
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Background: The insertion of a transgene into a plant organism can, in addition to the intended effects, lead to unintended effects in the plants. To uncover such effects, we compared maize grains of two genetically modified varieties containing NK603 (AG8025RR2, AG9045RR2) to their non-transgenic counterparts (AG8025conv, AG9045conv) using high-throughput RNA sequencing. Moreover, in-depth analysis of these data was performed to reveal the biological meaning of detected differences. Results: Uniquely mapped reads corresponded to 29,146 and 33,420 counts in the AG8025 and AG9045 varieties, respectively. An analysis using the R-Bioconductor package EdgeR revealed 3534 and 694 DEGs (significant differentially expressed genes) between the varieties AG8025RR2 and AG9045RR2, respectively, and their non-transgenic counterparts. Furthermore, a Deseq2 package revealed 2477 and 440 DEGs between AG8025RR2 and AG9045RR2, respectively, and their counterparts. We were able to confirm the RNA-seq results by the analysis of two randomly selected genes using RT-qPCR (reverse transcription quantitative PCR). PCA and heatmap analysis confirmed a robust data set that differentiates the genotypes even by transgenic event. A detailed analysis of the DEGs was performed by the functional annotation of GO (Gene Ontology), annotation/enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) ontologies and functional classification of resulting key genes using the DAVID Bioinformatics Package. Several biological processes and metabolic pathways were found to be significantly different in both variety pairs. Conclusion: Overall, our data clearly demonstrate substantial differences between the analyzed transgenic varieties and their non-transgenic counterparts. These differences indicate that several unintended effects have occurred as a result of NK603 integration. Heatmap data imply that most of the transgenic insert effects are variety-dependent. However, identified key genes involved in affected pathways of both variety pairs show that transgenic independent effects cannot be excluded. Further research of different NK603 varieties is necessary to clarify the role of internal and external influences on gene expression. Nevertheless, our study suggests that RNA-seq analysis can be utilized as a tool to characterize unintended genetic effects in transgenic plants and may also be useful in the safety assessment and authorization of genetically modified (GM) plants.
... Cry insecticidal proteins exhibit high selectivity and a broad insecticidal spectrum and have been widely used in GM crops. Herbicide resistance genes include genes encoding 5-enolpyruvate shikimic acid-3-phosphate synthase (EPSPS) (Arruda et al., 2013;Garcia et al., 2019;He et al., 2001), phosphinothricin acetyltransferase (PAT/bar, PAT/pat) (Thompson et al., 1987;Wehrmann et al., 1996), and glyphosate N-acetyltransferase (Liang et al., 2017;Liu et al., 2015a) genes. Among these genes, the genes encoding 5-enolpyruvate shikimic acid-3-phosphate synthase and phosphinothricin acetyl transferase genes are the most widely used. ...
... Moreover, the DEPs were not identified as new proteins and showed only changes in abundance, indicating that the proteomic profiles of the 4 GM maize lines studied were not dramatically altered. These results are consistent with the results previously reported for some other GM crops (Albo et al., 2007;Arruda et al., 2013;Gong et al., 2012;Ren et al., 2009a;Ruebelt et al., 2006;Tan et al., 2016). ...
Article
Full-text available
Proteomics provides a powerful approach to evaluate the unintended effects of transgenic crops. In this study, iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics was applied to estimate the differences in the proteomic profiles of maize seeds from 3 natural genotypic varieties and 4 genetically modified (GM) lines. Compared with their isogenic controls, there were 108, 69, 180 and 204 differentially expressed proteins (DEPs) in GM maize C0030.2.4, C0030.3.5, C0010.1.1 and C0010.3.1 seeds. Molecular functional classification showed that these DEPs were mainly involved in catalytic activity and binding. KEGG pathway annotation showed that most of these DEPs participated in metabolic pathways, the biosynthesis of secondary metabolites and microbial metabolism in diverse environments. In addition to the foreign protein EPSPS, 4 maize proteins were simultaneously identified as being differentially expressed among the 4 GM maize lines. Nevertheless, the regulation trends of these DEPs were not exactly consistent among the different GM maize lines. The data also showed that the differences in protein expression among the different natural genotypic varieties were greater than those caused by genetic modification. These results provide new information regarding the unintended effects of GM maize seeds through iTRAQ-based quantitative proteomic analysis.
... Higher concentrations of hydrogen peroxide and malondialdehyde were also observed, clearly indicating an oxidative stress condition established in the transgenic genotype. In addition, 47 proteins were differentially abundant when comparing the leaves of both plants, 26 species were accurately identified, including proteins involved in genetic modification (CP4 EPSPS) [1]. ...
... Higher concentrations of hydrogen peroxide and malondialdehyde were also observed, clearly indicating an oxidative stress condition established in the transgenic genotype. In addition, 47 proteins were differentially abundant when comparing the leaves of both plants, 26 species were accurately identified, including proteins involved in genetic modification (CP4 EPSPS) [1]. ...
Article
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With the increase of glyphosate tolerant transgenic soybean cultivation areas, this substance has become the main herbicide for this crop, its use may interfere in the metabolism and nutritional aspects of transgenic plants. In this scenario, the objective of this work was to verify concentrations of amino acid valine and in glyphosate tolerant soybean cultivar. Comparing the averages between protein and total amino acid levels in transgenic soybean exposed and not exposed to glyphosate, it was found that there was no significant difference by Tukey test at 5% probability. In conclusion, the data indicate that although the transgenic event affects some routes, amino acid synthesis was not affected nor in nutritional terms at the glyphosate doses studied.
... However, it is logical to technically evaluate the risks1 of utilizing GM crops relative to their benefits and evaluate them with the conventional methods of1 genetic improvement [19]. The most successful case of public information is glyphosate resistant1 transgenic soybean [20], which has been commercializedc for over 20 years [21], and it is1 undoubtedly the most important genetic modification in soybeans [22]. ...
Chapter
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Legume crops are universally applicable for human and animal food and sustenance because of their relatively high protein and essential amino acid content. Furthermore, they have been linked to sustainable agriculture, noting their ability to bind to atmospheric nitrogen-fixing bacteria. Despite this, several technical limitations of leguminous crops keep their world production far behind that of cereals. This chapter of the book focuses on current developments in breeding and biotechnology of major legume crops. Conventional breeding has primarily set out to recover a number of vegetative and reproductive traits that are associated with different heritability values, which reflect how susceptible each character is to genetic improvement. In conclusion, legume breeding programs using classical breeding methods and biotechnological tools face a promising boost for further application of knowledge and information that may boost their overall production. In plant breeding, the development of improved crop varieties is limited by very long periods of cultivation. Therefore, to increase crop breeding efficiency, they are using new strategies such as high-throughput phenotyping and molecular breeding tools. In this chapter, recent findings on various aspects of crop improvement, plant breeding practices, to explain the development of conventional and molecular techniques.
... The Bacillus thuringiensis (Bt) cry gene (encoding the Cry protein exhibiting high selectivity and a broad insecticidal spectrum) has been widely used in GM crops [26]. Among herbicide-resistance genes, the epsps (encoding 5-enolpyruvate shikimic acid-3-phosphate synthase, EPSPS) [27][28][29] and bar/pat (encoding phosphinothricin acetyl transferase, PAT) [30,31] genes are most widely used. ...
Article
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Background Widely targeted metabolomics was applied to estimate the differences in the metabolite profiles of maize seeds from 3 natural genotypic varieties and 4 genetically modified (GM) lines. Results Pairwise comparison with their isogenic controls revealed 71, 121, 43 and 95 differentially accumulated metabolites (DAMs) in GM maize seeds of C0030.2.4, C0030.3.5, C0010.1.1 and C0010.3.1, respectively. KEGG pathway enrichment analysis showed that most of these DAMs participated in the biosynthesis of secondary metabolites and purine metabolism in GM maize C0030.2.4 and C0030.3.5, but participated in tryptophan metabolism and 2-oxocarboxylic acid metabolism in C0010.3.1 seeds and in metabolic pathways and the biosynthesis of secondary metabolites in C0010.1.1 seeds. The data also showed that the differences in metabolite accumulation, both total DAMs and co-DAMs, among the different natural genotypic varieties (418 DAMs and 39 co-DAMs) were greater than those caused by genetic modification (330 DAMs and 3 co-DAMs). Conclusions None of the DAMs were identified as new or unintended, showing only changes in abundance in the studied maize seeds. The metabolite profile differences among the 3 non-GM lines were more notable than those among GM lines. Different genetic backgrounds affect metabolite profiling more than gene modification itself. Graphic abstract
... The application of GBH in GM soybeans was also associated with phytotoxic responses, compromising essential biological processes and favoring the appearance of diseases [14]. Effects on secondary metabolism, oxidative, hormonal status, changes in photosynthesis were also observed for the same CP4 ESPSPS single cassette transgenic event [15][16][17][18]. ...
Article
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Background World agricultural production of genetically modified (GM) products, in particular, the combination of different traits/genes in the same plant has been a trend over the last decade. There have been concerns raised over stacking multiple herbicide and insect-resistant transgenes that could result in fitness costs depending on the type and strength of selection pressures exerted by the environment. Here, we report the results of transcriptomic analysis comparing the effect of glyphosate-based herbicide (GBH) in the single-transgene versus stacked, herbicide-resistant soybean varieties on various biological processes, metabolic pathways, and key shikimic enzymes. Results Gene expression data showed that defense metabolism and redox homeostasis were equally modulated in single-transgene and stacked-variety samples. Carbon accumulation and energy metabolisms were distinct between the varieties and photosynthesis metabolism was found negatively affected in the single-transgene variety only. In the stacked variety, the shikimate pathway was modulated by the accumulation of transcripts from phenylalanine gene and other cascade genes. As expected, the expression of native EPSPS was upregulated in both varieties when herbicide was applied. On the other hand, transgenic EPSPS expression was down-regulated in both GM varieties upon herbicide application which cannot be explained. Conclusion Glyphosate-based herbicides toxicity suggests its effects on carbon central metabolism and flux, redox metabolism, photosynthesis, and to hormone and defense response in plants. The observed unintended effects in GM herbicide-tolerant varieties unravel the deleterious effects previously observed on GM-tolerant varieties growth and production. The impact of GBH on shikimate and cascade pathways was observed in terms of both native and transgenic insensitive EPSPS modulation, alteration of jasmonic acid and lignin metabolism in both single-transgene and stacked variety. The energy metabolism and carbon flux were differently affected in these varieties. Oxidative stress, more specifically glutathione metabolism, induced by GBH, was also observed in this study. The stacked variety showed a more pronounced stress response (activation of specific stress defense proteins, Rboh, WRKY) and secondary compounds (β-glucosidase, isoflavone 7-O-methyltransferase). Omics profiling techniques, such as transcriptomics, can be considered tools to support risk assessment in detecting unintended effects due to the GBH application.
... Eukaryotic genes have many functional facets, due to translational and post translational processes, which form different types of proteins under various stages of development and environmental conditions from single gene 1 . One and two dimensional electrophoresis techniques are the basis of translational products analysis of complex biological processes that occur under any given condition [2][3][4] , such as abiotic stress [5][6][7][8] , wounding 9 , water deficiency [10][11][12] and peroxidase activity 13 . These are strong, cost effective and a valuable technique for studying gene expression at protein level [14][15][16][17] for species of which genome is not yet sequenced 18 . ...
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One and two dimensional electrophoresis is a powerful technique for separation of proteins from complex biological sources. These are effective techniques of gene expression analysis at translational level especially for the species of which genome is not yet sequenced. High resolution electrophoresis separation of proteins from complex biological systems is crucial in protein science. Three protein extraction methods, such as Phenol, Multi detergent and TCA acetone were analyzed in the present investigation to find out appropriate protein extraction method amenable to one dimensional (1-D) and two dimensional (2-D) protein profiling of Haloxylon recurvum and H. salicornicum. Plant species selected for the present investigation are major components of saline and arid ecosystem and valuable source of food additives, nutritional ingredients, herbal medicines and fuel in the region. Phenolic components present in these plants are major hindrances for extraction of pure proteins. High concentrations of good quality proteins represented by clear, intense and more bands/spots in 1-D and 2-D gels were extracted with Phenol extraction method in comparison to TCA acetone and Multi detergent methods. This is possibly the first report on extraction and purification of proteins amenable to 1-D and 2-D proteome profiling of Haloxylon species of Indian arid zone, and forms a basis for further comparative proteome analysis of these two contrasting species with respect to salinity and drought tolerant ability for prospects.
... Despite the appeal of GM, the insertion of an exogenous gene into the DNA of a given culture is expected to lead to some biochemical changes, as already demonstrated by some authors (Arruda et al., 2013;Barbosa et al., 2012;Fonseca et al., 2015;Leon et al., 2009;Vidal et al., 2015;Wang et al., 2015). For example, Barbosa et al. (2012) demonstrated some differences in proteomic levels between T (Roundup Ready®) and non-transgenic (NT) soybean seeds, with four differentials proteins found through the 2-D DIGE technique and mass spectrometry. ...
... Higher concentrations of hydrogen peroxide and malondialdehyde were also observed, clearly indicating an oxidative stress condition established in the transgenic genotype. In addition, 47 proteins were differentially abundant when comparing the leaves of both plants, 26 species were accurately identified, including proteins involved in genetic modification (CP4 EPSPS) [1]. ...
... Para entender o que foi realizado, é necessário comentar o modo de ação do glifosato. O glifosato, ingrediente ativo do herbicida RoundupÒ, (N-phosphonomethylglycine), se liga e bloqueia a atividade da enzima EPSP (5-enolpyruvylshikimate-3-phosphate) synthase, a qual participa da biossíntese de aminoácidos aromáticos nas plantas ( Arruda et al. 2013e Barbosa et al., 2012. Na ausência do glifosato, a enzima EPSPS atua catalizando a reação da S3P (ou, shikimate-3-phosphate), e da PEP (phosphoenolpyruvate), dando condições à produção de EPSP (5-enolpyruvylshikimate-3-phosphate) e fosfato orgânico, que são as substâncias, responsáveis pela síntese de aminoácidos aromáticos. ...
... Para entender o que foi realizado, é necessário comentar o modo de ação do glifosato. O glifosato, ingrediente ativo do herbicida RoundupÒ, (N-phosphonomethylglycine), se liga e bloqueia a atividade da enzima EPSP (5-enolpyruvylshikimate-3-phosphate) synthase, a qual participa da biossíntese de aminoácidos aromáticos nas plantas ( Arruda et al. 2013e Barbosa et al., 2012. Na ausência do glifosato, a enzima EPSPS atua catalizando a reação da S3P (ou, shikimate-3-phosphate), e da PEP (phosphoenolpyruvate), dando condições à produção de EPSP (5-enolpyruvylshikimate-3-phosphate) e fosfato orgânico, que são as substâncias, responsáveis pela síntese de aminoácidos aromáticos. ...
... Moreover, we assessed the consequences of Roundup herbicide application, which has not previously been undertaken 3 . Most GM vs non-GM omics investigations use one environmental and temporal replicate to test equivalence 4,[8][9][10][11][12][13] . We acknowledged further experiments are needed under different environmental conditions to determine the full range of GM process effects on this maize type. ...
Article
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The opinion expressed by Eriksson and colleagues' fails to recognise that there are no standard experimental designs for academic investigations involving omics analyses of genetically modified crops and that the only valid comparator to determine the effect of the process of transgenesis is a near isogenic variety grown at the same time and location, as was the case in our investigation of NK603 maize. Eriksson does not acknowledge that the quality of the rat liver tissues in our chronic Roundup toxicity study has neither been questioned nor branded as unsuitable for further investigation. In addition, Eriksson fails to appreciate that the statistical methods we used to analyse the liver metabolomics dataset are recognised as appropriate as some of a number of approaches that can be taken. Moreover, Eriksson neglects to mention that the proteomics analysis of the liver tissues highlights structural and functional damage from Roundup exposure. Thus our results are sound and the claims by Eriksson and colleagues of experimental flaws are unfounded.Replying to: Eriksson et al. Sci Rep 8 (2018); https://doi.org/10.1038/s41598-018-30440-7 .
... Yet, the main prolamin band identified in the present study with 22 kDa (Fig. 1) is in accordance with the results reported by Gomez-Martınez (2012). Future studies should perhaps employ more detailed analysis of Pearl Millet storage proteins by using 2D-PAGE, as has been done for a wide range of crops species (Arruda et al. 2013, Vilhena et al. 2015, Schmidt et al. 2016. ...
Article
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In this study, we are presenting recommendations to the best agricultural use as well as for plant breeding of three millet cultivars namely ENA1 and ENA2, which have African origin, and BRS1501 originally from India. These cultivars were evaluated for growth, yield and grain quality traits. The morphological traits evaluated in this study indicated that the African genotypes ENA1 and ENA2 are better than the Indian genotype BRS1501 for no-till farming or to produce forage with 15% of crude protein at flowering and at harvest to produce stover (around 7% of crude protein content) for livestock feeding. The BRS1501 cultivar exhibited the highest values for total crude protein, albumins and prolamins, phytate and mineral contents in grains. ENA1 and ENA2 exhibited the highest values of globulin and glutelin contents. The electrophoretic patterns for storage proteins were similar across the three millets cultivars, except for a higher intensity of two glutelin bands with 21 and 24 kDa in BRS1501. Together, the results allow us to recommend BRS1501 for grain production and ENA1 and ENA2 for biomass production.
... Regarding to genetically modified organisms, and soybean being emphasized, due to its commercial importance [14], some studies are indicating remarkable differences, when transgenic and non-transgenic are compared, not only in terms of assimilation of metals and their homeostasis, but also to (metallo)proteins, and enzymes [15][16][17]. ...
... Using 2-DE-and iTRAQ-based quantitative proteomics techniques, approximately 148 identified DEPs were detected. Compared with the total number of proteins identified using iTRAQ (6313), the number of DEPs identified was significantly smaller, and a limited number of proteins were found to be affected by gene insertion 38 . Moreover, the DEPs identified in the transgenic plants were not identified as new proteins but as proteins showing changes in abundance. ...
Article
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Proteomics has become a powerful technique for investigating unintended effects in genetically modified crops. In this study, we performed a comparative proteomics of the seeds of phytase-transgenic (PT) and non-transgenic (NT) maize using 2-DE and iTRAQ techniques. A total of 148 differentially expressed proteins (DEPs), including 106 down-regulated and 42 up-regulated proteins in PT, were identified. Of these proteins, 32 were identified through 2-DE and 116 were generated by iTRAQ. It is noteworthy that only three proteins could be detected via both iTRAQ and 2-DE, and most of the identified DEPs were not newly produced proteins but proteins with altered abundance. These results indicated that many DEPs could be detected in the proteome of PT maize seeds and the corresponding wild type after overexpression of the target gene, but the changes in these proteins were not substantial. Functional classification revealed many DEPs involved in posttranscriptional modifications and some ribosomal proteins and heat-shock proteins that may generate adaptive effects in response to the insertion of exogenous genes. Protein-protein interaction analysis demonstrated that the detected interacting proteins were mainly ribosomal proteins and heat-shock proteins. Our data provided new information on such unintended effects through a proteomic analysis of maize seeds.
... 2 GR plants are provided with an exogenous gene, cp4 5enolpyruvylshikimate-3-phosphate synthase (EPSPS), which encodes the bacterial version of the enzyme CP4EPSPS, conferring plants tolerance against glyphosate (N-phosphonomethyl glycine). 3 Non-GR plants, in contrast, are susceptible to the effects of glyphosate linked to the inhibition of EPSPS, which prevents the biosynthesis of aromatic amino acids. 4 The primary mode of action of glyphosate is the inhibition of EPSPS; however, the herbicide has several secondary effects on plant physiology, 5 which have even been observed in GR plants. ...
Article
We investigated the effects of different concentrations of glyphosate acid and one of its formulations (Roundup®) on seed germination of two glyphosate resistant (GR)- and one non-GR variety of soybean. As expected, the herbicide affected the shikimate pathway in non-GR seeds but not in GR seeds. We observed that glyphosate can disturb the mitochondrial electron transport chain leading to H2O2 accumulation in soybean seeds, which was, in turn, related to lower seed germination. In addition, GR-seeds showed increased activity of antioxidant systems when compared to non-GR seeds, making them less vulnerable to oxidative-stress induced by glyphosate. The differences in the responses of GR varieties to glyphosate exposure corresponded to their differences in enzymatic activity related to H2O2-sacavenging and mitochondrial Complex III (the proposed site of ROS induction by glyphosate). Our results shown that glyphosate ought to be used carefully as a pre-emergence herbicide in soybean field crop systems since this practice may reduce seed germination.
... Malondialdehyde (MDA) was monitored by measurements at 535 and 600 nm and the concentration calculated using an extinction coefficient of 1.55 × 10 −5 mol −1 cm −1 . MDA content was expressed in nmol g −1 fresh weight (Arruda et al. 2013). ...
... Typically, when cellular polyamine content increases, the levels of hydrogen peroxide also increases, activating antioxidant systems. Unintended effects of the inserted EPSPS-CP4 transgene was linked to energy metabolism disturbances in other studies [13][14][15] . It can be hypothesized that the plant is searching for a new equilibrium to maintain heterologous EPSPS-CP4 metabolism within levels that can be tolerated by the plant. ...
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Glyphosate tolerant genetically modified (GM) maize NK603 was assessed as ‘substantially equivalent’ to its isogenic counterpart by a nutrient composition analysis in order to be granted market approval. We have applied contemporary in depth molecular profiling methods of NK603 maize kernels (sprayed or unsprayed with Roundup) and the isogenic corn to reassess its substantial equivalence status. Proteome profiles of the maize kernels revealed alterations in the levels of enzymes of glycolysis and TCA cycle pathways, which were reflective of an imbalance in energy metabolism. Changes in proteins and metabolites of glutathione metabolism were indicative of increased oxidative stress. The most pronounced metabolome differences between NK603 and its isogenic counterpart consisted of an increase in polyamines including N-acetyl-cadaverine (2.9-fold), N-acetylputrescine (1.8-fold), putrescine (2.7-fold) and cadaverine (28-fold), which depending on context can be either protective or a cause of toxicity. Our molecular profiling results show that NK603 and its isogenic control are not substantially equivalent.
... Despite the completion of the soybean genome sequencing (Schmutz et al., 2010), functional genomics study of soybean is still challenging, and therefore proteomics approaches could alternatively be a powerful tool (Arruda et al., 2013;Komatsu and Ahsan, 2009). Hajduch et al. (2005) reported that both 2-DE and MALDI-TOF/TOF overall decrease in metabolism-related proteins and increase in proteins associated with destination and storage during seed filling. ...
Article
Vegetable soybean is an important economic and nutritious crop. In this study, 48 differentially expressed proteins were identified from filling seeds of soybean (Glycine max) cv. Mindou 6 by using two-dimensional electrophoresis (2-DE) combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/ MS). Among them, 25% were related to protein destination and storage, 42% to energy and metabolism, 15% to disease/defense, 6% to transporters, 4% to secondary metabolism, 4%to transcription,2%to protein synthesis, and2%to cell growth/division. Along with thematurity of seeds, the number of unchanged abundance proteins decreased, while that of both upregulated and downregulated proteins increased. Both downregulated expression of caffeic acid O-methyltransferase (COMT) and upregulated expression of sucrose-binding protein (SBP) 2 precursor may contribute to increase in digestibility, nutritional value, and eating quality of vegetative seeds at suitable picking period. The pattern of unchanged proteins during the whole seed-filling stage may be also beneficial to the quality of vegetable soybean. © 2016, American Society for Horticultural Science. All rights reserved.
... Malondialdehyde (MDA) was monitored by measurements at 535 and 600 nm and the concentration calculated using an extinction coefficient of 1.55 × 10 −5 mol −1 cm −1 . MDA content was expressed in nmol g −1 fresh weight (Arruda et al. 2013). ...
Article
Stress perception and signalling pathways between plant organs involve complex mechanisms and remain a major focus of interest. To further address the role of phytohormones in the modulation of stress perception from root-to-shoot signalling, we used ethylene-insensitive Never ripe (Nr) and auxin-insensitive diageotropica (dgt) tomato mutants combined with the grafting technique. Lipid peroxidation, H2O2, chlorophyll and proline contents, and activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in non-grafted and grafted tomato mutants subjected to cadmium (Cd) were analysed. The results revealed different responses according to genotype, grafting combination and Cd application. Non-grafted hormonal mutants exhibited higher Cd content in roots than MT plants, being 39.9% in Nr and 17.7% in dgt plants, whereas in leaves, the Cd content was higher in Nr plants. In grafted plants, where the rootstocks were exposed to Cd before grafting, the MT rootstock exhibited the highest Cd content. In non-grafted plants following Cd application, roots of Nr also exhibited a decrease in Ca concentration, whilst Mg, S, Cu and Zn decreased in Nr leaves. In grafted plants, it was possible to notice peculiar differences in nutrient concentration patterns according to grafting combination and Cd application. The proline and chlorophyll contents were less affected in the hormonal mutants. In the presence of Cd, the scions of grafted plants exhibited increased antioxidant enzymes activities in response to a signal from the rootstocks. However, it was possible to associate the involvement of ethylene and auxin with the antioxidant responses because the Nr and dgt genotypes were less affected by Cd stress than their wild-type counterpart, MT.
... From a proteomics viewpoint, the expected difference between PT and NT lines in the ideal case is the presence of transgene-induced proteins. Random insertion of exogenous genes into the plant genome could lead to disruption of endogenous genes and rearrangement of the genome and unintended effects may occur (Gong and Wang, 2013), but a limited number of DEPs are expected to be affected by a single gene insertion (Arruda et al., 2013). Moreover, in previous studies, 11.69% of protein spots were found to show differences in accumulation in seedling leaves between a hybrid and its parental lines and a similar magnitude was observed at the transcriptional level (Swanson-Wagner et al., 2006;Guo et al., 2014). ...
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To investigate unintended effects in genetically modified crops (GMCs), a comparative proteomic analysis between the leaves of the phytase-transgenic maize and the non-transgenic plants was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 57 differentially expressed proteins (DEPs) were successfully identified, which represents 44 unique proteins. Functional classification of the identified proteins showed that these DEPs were predominantly involved in carbohydrate transport and metabolism category, followed by post-translational modification. KEGG pathway analysis revealed that most of the DEPs participated in carbon fixation in photosynthesis. Among them, 15 proteins were found to show protein-protein interactions with each other, and these proteins were mainly participated in glycolysis and carbon fixation. Comparison of the changes in the protein and tanscript levels of the identified proteins showed that most proteins had a similar pattern of changes between proteins and transcripts. Our results suggested that although some significant differences were observed, the proteomic patterns were not substantially different between the leaves of the phytase-transgenic maize and the non-transgenic isogenic type. Moreover, none of the DEPs was identified as a new toxic protein or an allergenic protein. The differences between the leaf proteome might be attributed to both genetic modification and hybrid influence.
... Since water content remained above critical levels for the species, viability reduction seems to be a result of deteriorative metabolic processes arising from storage. Oxidative stress essentially occurs as a result of an imbalance between ROS production and antioxidant defences against ROS (Kibinza et al. 2011;Arruda et al. 2013). It is important to emphasize that ROS are naturally produced by the cell metabolism but must be maintained within certain levels to avoid the establishment of an oxidative stress condition. ...
Article
Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation of germination process, and both may result in viability loss. This study aimed to elucidate the biochemical basis of embryos survival of Araucaria angustifolia recalcitrant seeds during storage. After harvesting, seeds were stored at ambient conditions (without temperature and humidity control) and in a cold chamber (temperature of 10 ± 3 °C, and relative humidity of 45 ± 5 %). Moisture content, viability, H2O2 content, lipid peroxidation, protein content, and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), at 0, 15, 45 and 90 days of storage, were evaluated. Seed viability reduced about 40 % during the storage period accompanied by a reduction in soluble protein (about 64 % of reduction) in both storage conditions, and increased lipid peroxidation (about 115 % and 66 % for ambient and cold chamber conditions, respectively). H2O2 content used as a marker of oxidative stress was reduced during the period, possibly controlled by the action of CAT and APX, for which increased activities were observed. The results allowed the identification of seven SOD isoenzymes (one Mn-SOD, five Fe-SOD and one Cu/Zn-SOD), whose activities also increased in response to storage. Some biochemical damage resulting from storage was observed, but viability reduction was not due to failure of enzymatic protection mechanisms.
... Malondialdehyde (MDA) was monitored by measurements at 535 and 600 nm and the concentration calculated using an extinction coefficient of 1.55 × 10 −5 mol −1 cm −1 . MDA content was expressed in nmol g −1 fresh weight (Arruda et al. 2013). ...
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There is a very effective cross-talk between signals triggered by reactive oxygen species and hormonal responses in plants, activating proteins/enzymes likely to be involved in stress tolerance. Abscisic acid (ABA) is known as a stress hormone that takes part in the integration of signals. This work aimed to characterize the biochemical response and ultrastructural changes induced by cadmium (Cd) in the Micro-Tom (MT) sitiens ABA-deficient mutant (sit) and its wild-type (MT) counterpart. MT and sit plants were grown over a 96-h period in the presence of Cd (0, 10, and 100 μM CdCl2). The overall results indicated increases in lipid peroxidation, hydrogen peroxide content and in the activities of the key antioxidant enzymes such as catalase, glutathione reductase, and ascorbate peroxidase in both genotypes. On the other hand, no alteration was observed in chlorophyll content, while the activity of another antioxidant enzyme, superoxide dismutase, remained constant or even decreased in the presence of Cd. Roots and shoots of the sit mutant and MT were analyzed by light and transmission electron microscopy in order to characterize the structural changes caused by the exposure to this metal. Cd caused a decrease in intercellular spaces in shoots and a decrease in cell size in roots of both genotypes. In leaves, Cd affected organelle shape and internal organization of the thylakoid membranes, whereas noticeable increase in the number of mitochondria and vacuoles in MT and sit roots were observed. These results add new information that should help unravel the relative importance of ABA in regulating the cell responses to stressful conditions induced by Cd apart from providing the first characterization of this mutant to oxidative stress.
... Identifying the mechanisms of Al tolerance may make it possible to combine them to produce more tolerant genotypes. The efficiency of the plant antioxidant systems may thus be an important attribute for increasing tolerance of Al stress in plants, in a similar manner to what has been widely described in the literature for other heavy metals and even other abiotic stresses (Arruda and Azevedo 2009;Cia et al. 2012;Arruda et al. 2013;Boaretto et al. 2014;Bulbovas et al. 2014) and in interaction with microorganisms in the soil (Dourado et al. 2013(Dourado et al. , 2014. ...
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Over the last few decades, the concentration of cadmium (Cd) in the environment has increased considerably in many countries due to anthropogenic activities. Cd is one of the most toxic pollutants in the environment and affects many metabolic processes in plants. The main objective of this study was to evaluate the response of the production, nutritional, and enzymatic antioxidant system of two tomato genotypes (Calabash Rouge and CNPH 0082) grown in tropical soils that were treated with doses of Cd. Soil samples were collected from the layer of earth at a depth of 0–0.2 m in areas subjected to a minimum of human disturbance. The concentrations of Cd applied to the soil samples were 0, 1, 2, and 4 times (0, 3, 6, and 12 mg kg−1 of Cd) the agricultural intervention value adopted by current environmental legislation in the state of São Paulo, Brazil. Analysis of superoxide dismutase, catalase, glutathione reductase, guaiacol peroxidase, and ascorbate peroxidase activities, formation of stress indicator compound (malondialdehyde—MDA and hydrogen peroxide), parameters of production—dry mass of the shoot and root system (here in after “shoots” and “roots”), as well as nutrition, and both the bioavailable and total levels of this metal in the soil were performed. When the bioavailable content and total levels of Cd in the soil increased as a result of this metal doses applied, the biomass of both shoots and roots decreased in both genotypes (with the exception of the CNPH 0082 grown in clay soil) and displayed lower SPAD (relative chlorophyll index) values when exposed to contaminated environments with Cd concentrations. Cadmium treatment resulted in nutritional imbalances, mainly in terms of N, P, and Mn metabolism. Plants subjected to an elevated available content of metal in the soil exhibited increases in content of MDA and hydrogen peroxide and increased activity of catalase, ascorbate peroxidase, and guaiacol peroxidase in plant tissues when grown in both clay soil and sandy soil. Cadmium was phytotoxic to the plants causing a nutritional imbalance, especially on the metabolisms of N, P, and Mn. An oxidative stress condition was established in response to the Cd treatments applied, which led to changes in peroxidase activity.
... In general, the field of proteomics has been generating a large amount of valuable information (Arruda et al., 2013;Barbosa et al., 2012;Vidal et al., 2015). Some of these studies include protein accumulation during grain filling and maturation in the whole grain and in dissected tissues (aleurone layer, starch endosperm and embryo) (Finnie et al., 2002(Finnie et al., , 2004a, responses to environmental and genetic fluctuations (Eggert and Pawelzik, 2011;Finnie et al., 2004b;Yang et al., 2010), and the relationship between protein and grain quality for different end-use purposes (Bak-Jensen et al., 2004;Finnie et al., 2006;Jin et al., 2012;Østergaard et al., 2002. ...
Article
Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.
... In addition to cadmium, other elements such as Cu, Fe, P, Mg and Zn were also evaluated, allowing us to detect their imbalances in sunflower homeostasis due to the presence of cadmium; chlorosis was also detected. In terms of proteomics approaches, Arruda et al. (2013) evaluated the proteomic differences between transgenic (T) and non-transgenic (NT) soybean plants (modified by the insertion of the cp4EPSPS gene) and their correlations with possible oxidative stress. The activity of the enzymes evaluated, as well as the concentrations of malondialdehyde and hydrogen peroxide were found to be higher in T than in NT plants, and 47 proteins were found to have significantly different abundance. ...
Article
The present study evaluates the effects of different doses of cadmium (0, 50, 350 and 700 mg) on sunflower seeds, thus expanding the information available on this plant culture. For this task, the interaction between cadmium and key elements such as Cu, Fe, Mg, Mn, P, S and Zn is evaluated. An in vitro procedure is also performed to assess the cadmium bioavailability and bioaccessibility. Finally, metallobiomolecules are evaluated by SEC-ICP-MS in the seeds. The results indicated that cadmium is translocated to seeds such that the level of this element in the seeds was 130 times higher than the tolerable amount for the human body (0.1 mg kg−1). An imbalance of other metals is also detected. While 92%, 72% and 74% of cadmium is bioaccessible in the control, Cd-low (50 mg Cd) and Cd-high (700 mg Cd) groups, respectively, its bioavailability was markedly different, with only 6.5%, 2% and 1% bioavailable from the same groups, respectively. This behavior reflects on the bioaccessibility and bioavailability of other elements also evaluated. In terms of metallobiomolecules, cadmium greatly interferes with Fe-biomolecules synthesis. Finally, cadmium interferes with the seed germination (2nd generation of seeds), by increasing the dormancy time (ca. 50% higher) and by decreasing the germination rate (ca. 60% lower) compared with the control group.
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Following the submission of application EFSA-GMO-RX-023 under Regulation (EC) No 1829/2003 from Bayer Agriculture BV on behalf of Bayer CropScience LP, the Panel on Genetically Modified Organisms of the European Food Safety Authority was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide-tolerant genetically modified soybean 40-3-2, for food and feed uses, excluding cultivation within the European Union. The data received in the context of this renewal application contained post-market environmental monitoring reports, a systematic search and evaluation of literature, updated bioinformatic analyses, and additional documents or studies performed by or on behalf of the applicant. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the event in soybean 40-3-2 considered for renewal is identical to the sequence of the originally assessed event, the GMO Panel concludes that there is no evidence in renewal application EFSA-GMO-RX-023 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean 40-3-2.
Chapter
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Legume species have various applications in organism’s nutrition, medical, and conversion industries because of their high oil, high protein, and high value materials. These crops can prevent soil erosion and increase soil nitrogen for further crop cultivation by bacteria symbiosis as well. Concerning the benefits of these crops, there is a need for more breeding attempts to gain genetic achievements. Accelerated higher genetic gains are required to meet the demand of ever-increasing global population. In recent years, speedy developments have been witnessed in legume genomics due to advancements in next-generation sequencing (NGS) and high-throughput genotyping technologies. A fundamental change in current conventional breeding programs, combined with modern techniques, is of great importance. Thus, a combination of modern and conventional breeding techniques may conduct our goals to reach great achievement on legume breeding regarding industrial and medical uses, human and livestock nutrition faster.
Chapter
Pisum sativum L. is a winter season legume plant belonging to the Fabaceae family. It is cultivated in more than 95 countries primarily for its protein-rich seeds for food and feed purpose. Anthropogenic climate change has shown detrimental effects on field pea production because to many biotic as well as abiotic stresses. This chapter addresses extent and impact of abiotic stresses mainly heat, water scarcity, waterlogging, frost, saline soil, and soil nutrient deficiencies. Also, we discuss their management through genetic options for the development of climate resilient pea. To achieve this, the utilization of all gene pools (primary, secondary, and tertiary) of pea for genetic advancements aimed at abiotic stress tolerance has been emphasized. Various traditional breeding methodologies and recently developed technologies like, genome wide association mapping, genomic selection, gene editing, marker assisted breeding, and nano-biotechnology have been discussed for development of abiotic stress resilient cultivars. TILLING technology can be used to detect mutants from physical or chemical mutagenized populations of pea. As plant developed using this method is considered as non-GMO, it has wider public acceptance. Through the identification of molecular markers and genome-wide association studies, major quantitative trait loci associated with different abiotic stress tolerances have been identified which can be helpful to detect candidate genes. The recent accessibility of the pea draft genome can help to emphasize the molecular basis of agronomically essential traits. The development of transgenic abiotic stress resistant pea and the use of virus induced gene silencing (VIGS), as well as prospect of cisgenesis have also been reviewed. Bioinformatics tools like gene and genome database with comparative gene expression database provide necessary information about abiotic stress resistant candidate genes while protein and metabolome database give information about functional protein and pathways of genes. Brief accounts on social, political and regulatory issues are also discussed. This book chapter provides all information about pea with detailed genome sequence information and major innovation in various molecular technologies to develop abiotic stress resistant pea cultivars.
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With the increase of glyphosate tolerant transgenic soybean cultivation areas, this substance has become the main herbicide for this crop, its use may interfere in the metabolism and nutritional aspects of transgenic plants. In this scenario, the objective of this work was to verify the oil, protein, phytate and amino acid concentrations in glyphosate tolerant soybean cultivar. Comparing the averages between oil, protein and total amino acid levels in transgenic soybean exposed and not exposed to glyphosate, it was found that there was no significant difference by Tukey test at 5% probability. In conclusion, the data indicate that although the transgenic event affects some routes, amino acid synthesis was not affected nor in nutritional terms at the glyphosate doses studied.
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BACKGROUND An isobaric tags for relative and absolute quantitation (iTRAQ)‐based proteomic analysis was employed to study the seeds of two genetically modified (GM) rice lines, T2A‐1 and T1C‐19, and their nontransgenic isogenic variety, MH63, to investigate the unintended effects of genetic modification. RESULTS A total of 3398 proteins were quantitatively identified. Seventy‐seven differentially abundant proteins (DAPs) were identified in the T2A‐1/MH63 comparison, and 70 and 7 of these DAPs were upregulated and downregulated, respectively. A pathway enrichment analysis showed that most of these DAPs participated in metabolic pathways and protein processing in endoplasmic reticulum and were ribosome components. Some 181 DAPs were identified from the T1C‐19/MH63 comparison, and these included 115 upregulated proteins and 66 downregulated proteins. The subsequent pathway enrichment analysis showed that these DAPs mainly participated in protein processing in endoplasmic reticulum and carbon fixation in photosynthetic organisms and were ribosome components. None of these DAPs were identified as new unintended toxins or allergens, and only changes in abundance were detected. Fifty‐four co‐DAPs were identified in the seeds of the two GM rice lines, and protein–protein interaction analysis of these co‐DAPs demonstrated that some interacting proteins were involved in protein processing in endoplasmic reticulum and metabolic pathways, whereas others were identified as ribosome components. Representative co‐DAPs and proteins related to nutrients were analyzed using qRT‐PCR to determine their transcriptional levels. CONCLUSIONS The results suggested that the seed proteomic profiles of the two GM rice lines studied were not substantially altered from those of their natural isogenic control. © 2020 Society of Chemical Industry
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Background World agricultural production of genetically modified (GM) products, in particular, the combination of different traits/genes in the same plant has been a trend over the last decade. There has been concerns raised over stacking multiple herbicide and insect-resistant transgenes that could result in fitness costs depending on the type and strength of selection pressures exerted by the environment. Here, we report the results of transcriptomic analysis comparing the effect of glyphosate-based herbicide (GBH) in the single-transgene versus stacked, herbicide resistant soybean varieties on various biological processes, metabolic pathways, and key shikimic enzymes. Results Gene expression data showed that defense metabolism and redox homeostasis were equally modulated in single-transgene and stacked variety samples. Carbon accumulation and energy metabolisms were distinct between the varieties and photosynthesis metabolism was found negatively affected in the single-transgene variety only. In the stacked variety, the shikimate pathway was modulated by the accumulation of transcripts from phenylalanine gene and other cascade genes. As expected, the expression of native EPSPS was upregulated in both varieties when herbicide was applied. On the other hand, transgenic EPSPS expression was down regulated in both GM varieties upon herbicide application which cannot be explained. Conclusion Glyphosate-based herbicides toxicity suggests its effects on carbon central metabolism and flux, redox metabolism, photosynthesis, and to hormone and defense response in plants. The observed unintended effects in GM herbicide-tolerant varieties unravels the deleterious effects previously observed on GM tolerant varieties growth and production. The impact of GBH on shikimate and cascade pathways was observed in terms of both native and transgenic insensitive EPSPS modulation, alteration of jasmonic acid and lignin metabolism in both single-transgene and stacked variety. The energy metabolism and carbon flux were differently affected in these varieties. Oxidative stress, more specifically glutathione metabolism, induced by GBH, was also observed in this study. The stacked variety showed a more pronounced stress response (activation of specific stress defense proteins, Rboh, WRKY) and secondary compounds (β-glucosidase, isoflavone 7-O-methyltransferase). Omics profiling techniques, such as transcriptomics, can be considered tools to support risk assessment in detecting unintended effects due to the GBH application.
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Background: World agricultural production of genetically modified (GM) products, in particular, the combination of different traits/genes in the same plant has been a trend over the last decade. There has been concerns raised over stacking multiple herbicide and insect-resistant transgenes that could result in fitness costs depending on the type and strength of selection pressures exerted by the environment. Here, we report the results of transcriptomic analysis comparing the effect of glyphosate-based herbicide (GBH) in the single-transgene versus stacked, herbicide resistant soybeans varieties on various biological processes, metabolic pathways, and main shikimic enzymes. Results: Gene expression data were grouped according to the 'herbicide treatment factor'. Defense metabolism and redox homeostasis were equally modulated in single-transgene and stacked variety samples. Carbon accumulation and energy metabolisms were distinct between the varieties and photosynthesis metabolism was found negatively affected in the single-transgene variety only. In the stacked variety, the shikimate pathway was modulated by the accumulation of transcripts from phenylalanine gene and other cascade genes. As expected, the RT-PCR results confirmed that the expression of native EPSPS was upregulated in both varieties when herbicide was applied. On the other hand, transgenic EPSPS expression was down regulated in both GM varieties upon herbicide application which cannot be explained. Conclusion: Glyphosate-based herbicides toxicity suggests its effects on carbon central metabolism and flux, redox metabolism, photosynthesis, and to the plant’s hormone and defense response. The observed unintended effects in GM herbicide-tolerant varieties unravels the deleterious effects previously observed on GM tolerant varieties growth and production. The impact of GBH on shikimate and cascade pathways was observed in terms of both native and transgenic insensitive EPSPS modulation, alteration of jasmonic acid and lignin metabolism in both single-transgene and stacked variety. Whereas the energy metabolism and carbon flux were differently affected in these varieties. Oxidative stress, more specifically glutathione metabolism, induced by GBH, was observed in this study. The stacked variety showed a more pronounced stress response (activation of specific stress defense proteins, Rboh, WRKY) and secondary compounds (β-glucosidase, isoflavone 7-O-methyltransferase). Omics profiling techniques, such as transcriptomic, can be considered tools to support risk assessment based on detecting unintended effects due to the GBH application.
Preprint
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Background World agricultural production of genetically modified (GM) products in particular, the combination of different traits/genes in the same plant has been intense over the last decade. The stacking of herbicide and insect-resistant transgenic genes can result in fitness costs that rely on the type and strength of the selection pressure exerted by the environment. Here we report the results of transcriptomic analysis comparing the effect of glyphosate on various biological processes, metabolic pathways, and main shikimic enzymes in stacked versus single soybean resistant varieties. Results Gene expression data were grouped according to treatment, ie the herbicidal factor strongly influenced. Common physiological results between the single and established varieties were mainly in Redox metabolism, energy, and metabolism. Photosynthesis was only found negatively affected in the single variety. The defense components, although present in both varieties, show a more intense presence in staked pathways, that demonstrated pathways related to up-regulated secondary metabolites biosynthesis, a known response when plants are under various stress conditions. RT-PCR results confirmed that native EPSPS expression was up-regulated at the same level for single and stacked events. However, metabolic differences in expression were observed, suggesting a distinct cascade effect between simple and stacked, triggered by glyphosate application. Conclusion Changes in plant metabolism by glyphosate application have been observed in several pathways, particularly the shiquimate pathway, suggesting that event staking may promote a more intense defensive genetic response. Omics profiling techniques, such as transcriptome, can be considered tools to support risk assessment based on detecting unwanted effects, both on plant physiological changes and on the safety of foods and products from new genetic editing technologies.
Article
To investigate the unintended effects of GM crops, an iTRAQ-based comparative proteomic analysis was performed with seed cotyledons of 2 GM soybean lines, MON87705 and MON87701×MON89788, and the corresponding non-transgenic isogenic variety A3525. Thirty-five DAPs were identified in MON87705/A3525, 27 of which were upregulated and 8 downregulated. Thirty-eight DAPs were identified from the MON87701×MON89788/A3525 sample, including 29 upregulated proteins and 9 downregulated proteins. Pathway enrichment analysis showed that most of these DAPs participate in protein processing in endoplasmic reticulum and in metabolic pathways. Protein-protein interaction analysis of these DAPs demonstrated that the main interacting proteins are associated with post-translational modification, protein turnover, chaperones, and signal transduction mechanisms. Nevertheless, these DAPs were not identified as new unintended toxins or allergens and only showed changes in abundance. All these results suggest that the seed cotyledon proteomic profiles of the 2 GM soybean lines studied were not dramatically altered compared with that of their natural isogenic control.
Article
Amino acid profiles are useful to analyze the responses to glyphosate in susceptible and resistant soybean lines. Comparisons of profiles for 10 amino acids (Asp, Asn, Glu, Gln, Ser, His, Gly, Thr, Tyr, Leu) by HPLC in soybean roots were performed in two near isogenic pairs (four varieties). Foliar application of glyphosate was applied in soybean plants after 5 weeks of seeding. Roots of four varieties were collected at 0 and 72 h after glyphosate application (AGA) for amino acids analysis by HPLC. Univariate analysis showed a significant increasing of several amino acids in susceptible as well as resistant soybean lines; however, amino acids from carbon (C) and nitrogen (N) metabolism, such as Asp, Asn, Glu and Gln, and Ser, increased significantly in susceptible varieties at 72 h AGA. Multivariate analysis using principal components analysis (2D-PCA and 3D.PCA) allowed to identified and discriminated different groups based on the soybean genetic origin, showing the amino acid responses on susceptible and resistant varieties. Based on the results, it is possible to infer that the increase of Asn, Asp, Glu, Gln, and Ser in susceptible varieties would be related to the deregulation of C and N metabolism, as well as changes in the growth mechanisms regulated by Ser.
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ABSTRACT Both the scientific community and society have shown interest in improving the content of amino acids, carbohydrates and mineral nutrients in maize because it represents an important staple food in many developing countries. Earlier studies demonstrated that the treatment of seeds using ascorbic acid (AsA-seed priming) enhanced soluble carbohydrates, proteins and soluble amino acids for other species. AsA seed priming in maize showed the potential for reducing abiotic stresses. The effects on grain quality have not been previously demonstrated. This study investigated the impacts of AsA seed priming on maize kernel quality of seeds produced by the plants generated from the primed seeds, based on the amino acid profile and carbohydrate and mineral nutrient contents. AsA seed priming improved the maize kernel quality with respect to the ascorbate content, boron allocation, total carbohydrate content and increased soluble amino acid levels, including serine, tyrosine, alanine, valine, glutamate, arginine, proline, aspartate, lysine and isoleucine, whereas soluble methionine was decreased. Therefore, AsA seed priming can represent a potential technique for improving maize grain quality.
Article
In this paper, Cu, Fe, Mn and Zn contents in transgenic (T – MSOY7122RR) and non-transgenic (NT – MSOY8200) soybean seeds, sown at summer and winter cultivation periods are investigated using four microwave decomposition methods. Student’s t tests demonstrate significant differences (p = 0.05; n = 4), for Cu, Mn and Zn (namely, 8, 9 and 26% higher concentrations in T compared to NT seeds, respectively). Through principal component analysis, precursor and successor soybean seeds are identified. Cu is demonstrated to play an important role in the differentiation of the cultivars, whereas Fe and Zn are of particular relevance in the classification of seeds cultivated in winter against those in summer. Using in vitro extraction based on the Unified Bioaccessibility Method, the bioaccessibility of the above nutrients is proven to differ in both the gastric and gastrointestinal phases on the basis of the transgenesis and the cultivation periods.
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Edited by James E. Board, ISBN 978-953-51-0876-4, 624 pages, Publisher: InTech, Chapters published January 02, 2013 under CC BY 3.0 license DOI: 10.5772/45867
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Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.
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A catalase-deficient mutant (RPr 79/4) and the wild-type (cv. Maris Mink) barley (Hordeum vulgare L.) counterpart, were grown for 3 weeks in high CO2 (0.7%) and then transferred to air and ozone (120 nl 1−1) in the light and shade for a period of 4 days. Leaves and roots were analysed for catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and glutathione reductase (GR, EC 1.6.4.2) activities. CAT activity in the leaves of the RPr 79/4 catalase-deficient mutant was around 5-10% of that determined in Maris Mink, but in the roots, both genotypes contained approximately the same levels of activity. CAT activity in Maris Mink increased in the leaves after transferring plants from 0.7% CO2 to air or ozone, reaching a maximum of 5-fold, after 4 days in shade and ozone. For the catalase-deficient mutant, only small increases in CAT activity were observed in light/air and light/ozone treatments. In the roots, CAT activity decreased consistently in both genotypes, after plants were transferred from 0.7% CO2. The total soluble SOD activity in the leaves and roots of both genotypes increased after plants were transferred from 0.7% CO2. The analysis of SOD isolated from leaves following non-denaturing PAGE, revealed the presence of up to eight SOD isoenzymes classified as Mn-SOD or Cu/Zn-SODs; Fe-SOD was not detected. Significant changes in Mn- and Cu/Zn-SOD isoenzymes were observed; however, they could not account for the increase in total SOD activity. In leaves, GR activity also increased in Maris Mink and RPr 79/4, following transfer from 0.7% CO2; however, no constant pattern could be established, while in roots, GR activity was reduced after 4 days of the treatments. The data suggest that elevated CO2 decreases oxidative stress in barley leaves and that soluble CAT and SOD activities increased rapidly after plants were transferred from elevated CO2, irrespective of the treatment (light, shade, air or ozone).
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The aim of this study was to investigate the antioxidant responses of three bacteria (SD1, KD and K9) isolated from soil previously treated with the herbicides metolachlor and acetochlor. By 16S rRNA gene sequencing, we determined that SD1 is phylogenetically related to Enterobacter asburiae, while KD and K9 have divergent genomes that more closely resemble that of Enterobacter amnigenus. Decreased levels of lipid peroxidation were observed in SD1 and KD following treatment with 34 mM metolachlor or 62 mM acetochlor, respectively, indicating that both bacteria were able to adapt to an increase in ROS production. In the presence of 34 mM metolachlor or 62 mM acetochlor, all bacterial isolates exhibited increases in total catalase (CAT) activity (81% for SD1, 53% for KD and 59% for K9), whereas total SOD activity (assessed based on the profile and intensity of the bands) was slightly reduced when the bacteria were exposed to high concentrations of the herbicides (340 mM metolachlor or 620 mM acetochlor). This effect was due to a specific reduction in SOD IV (K9 and KD isolates) by 45% and 90%, respectively, and SOD V (SD1 isolate) isoenzymes by 60%. The most striking result was obtained in the SD1 isolate, where two novel isoenzymes of glutathione reductase (GR) that responded specifically to metolachlor were identified. In addition, acetochlor was shown to induce the expression of a new 57 kDa protein band in the K9 and KD isolates. The bacteria isolated from the herbicide-contaminated soil exhibited an efficient antioxidant system response at herbicide concentrations of up to 34 mM metolachlor or 62 mM acetochlor. These data suggest a mechanism for tolerance that may include the control of an imbalance in ROS production versus scavenging. The data suggest that specific isoenzymes of CAT and GR could be involved in this herbicide tolerance mechanism.
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Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase-inhibiting proteins (XIPs), and thaumatin-like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a ΔTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2-D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum ΔTri5. Some (iso)forms were up-regulated, whereas others were down-regulated. This pathogen-specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI- and XIP-type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non-glycosylated XIP forms increased more strongly than their glycosylated counterparts.
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In order to further address the modulation of signaling pathways of stress responses and their relation to hormones, we used the ethylene-insensitive Never ripe (Nr) and the auxin-insensitive diageotropica (dgt) tomato mutants. The two mutants and the control Micro-Tom (MT) cultivar were grown over a 40-day period in the presence of Cd (0.2 mM CdCl₂ and 1 mM CdCl₂). Lipid peroxidation, leaf chlorophyll, proline content, Cd content and antioxidant enzyme activities in roots, leaves and fruits were determined. The overall results indicated that the MT genotype had the most pronounced Cd damage effects while Nr and dgt genotypes might withstand or avoid stress imposed by Cd. This fact may be attributed, at least in part, to the fact that the known auxin-stimulated ethylene production is comprised in dgt plants. Conversely, the Nr genotype was more affected by the Cd imposed stress than dgt, which may be explained by the fact that Nr retains a partial sensitivity to ethylene. These results add further information that should help unraveling the relative importance of ethylene in regulating the cell responses to stressful conditions.
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Male and female poplar ( Populus cathayana Rehd.) cuttings respond differently to salinity stress. To understand these differences better, comparative morphological, physiological, and proteomics analyses were performed. Treatments with different concentrations of NaCl applied to male and female poplar cuttings for 4 weeks showed that females reacted more negatively at the morphological and physiological levels than did males, visible as shriveled leaves, decreased growth, lowered photosynthetic capacities, and greater Na(+) accumulation. The proteome analysis identified 73 proteins from 82 sexually related salt-responsive spots. They were involved in photosynthesis, protein folding and assembly, synthesis and degradation, carbon, energy and steroid metabolism, plant stress and defense, redox homeostasis, signal transduction, and so forth. The sex-related changes of these proteins were consistent with the different morphological and physiological responses in males and females. In conclusion, the higher salt resistance of male P. cathayana cuttings is related to higher expression and lower degradation of proteins in the photosynthetic apparatus, more effective metabolic mechanism and protective system, and greater capacity of hydrogen peroxide scavenging. This research allows us to further understand the possible different management strategies of cellular activities in male and female Populus when confronted by salt stress.
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Transgenic and non-transgenic soybean seeds were compared in terms of total element concentrations, behavior of elements during sequential extraction fractionation and element bioaccessibility using an in vitro simulated gastrointestinal digestion. The analysis were carried out by ICP-sector field-MS or size-exclusion ICP-MS (25 elements in concentrations varying from ng g⁻¹ to the % level). It seems that transgenic and non-transgenic soybean seeds exhibit statistically significant differences in concentrations of Cu, Fe and Sr, which are also reflected by element contents in water extracts and residues. Additionally, contributions of bioaccessible fractions of Cu, Fe and other elements (Mn, S, Zn) for transgenic soybean seeds appear to be larger than those found in non-transgenic soybean seeds.
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Two dimensional gel electrophoresis has been one of the techniques most used for protein separation in proteomics experiments and still continues to be so for some species such as plants. Despite the constant technical advances and continuous improvements in the field of 2-DE, the experimental design and analysis of protein abundance data continue to be ignored or not properly documented in the literature. An appropriate experimental design, followed by decisive statistical methods is mandatory to extract all the information that is concealed in the complexity of 2-DE data. In this work we review, in a biologist's language, all the experimental design and statistical tests to be considered while planning a 2-DE based proteomics experiment and for the correct analysis and interpretation of the data. We aim to provide the researcher with an up to date introduction to these areas, starting with the experimental design and ending with the application of multivariate statistical methodologies such as PCA, ICA or neural network-based self-organizing maps. In between we have described, in an understandable way, the current methodologies available to deal with all the stages of the experimental design, data processing and analysis.