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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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A tracer experiment is carried out with transgenic T (variety M 7211 RR) and non-transgenic NT (variety MSOY 8200) soybean plants to evaluate if genetic modification can influence the uptake and translocation of Fe. A chelate of EDTA with enriched stable (57)Fe is applied to the plants cultivated in vermiculite plus substrate and the (57)Fe acts as a tracer. The exposure of plants to enriched (57)Fe causes the dilution of the natural previously existing Fe in the plant compartments and then the changed Fe isotopic ratio ((57)Fe/(56)Fe) is measured using a quadrupole-based inductively coupled plasma mass spectrometer equipped with a dynamic reaction cell (DRC). Mathematical calculations based on the isotope dilution methodology allow distinguishing the natural abundance Fe from the enriched Fe (incorporated during the experiment). The NT soybean plants acquire higher amounts of Fe from natural abundance (originally present in the soil) and from enriched Fe (coming from the (57)Fe-EDTA during the experiment) than T soybean ones, demonstrating that the NT soybean plants probably absorb higher amounts of Fe, independently of the source. The percentage of newly incorporated Fe (coming from the treatment) was approximately 2.0 and 1.1% for NT and T soybean plants, respectively. A higher fraction (90.1%) of enriched Fe is translocated to upper parts, and a slightly lower fraction (3.8%) is accumulated in the stems by NT plants than by T ones (85.1%; 5.1%). Moreover, in both plants, the Fe-EDTA facilitates the transport and translocation of Fe to the leaves. The genetic modification is probably responsible for differences observed between T and NT soybean plants.

This work reports on the use of a three dimensional separation system to enhance metalloprotein information when considering soybean seeds. Separations using size exclusion chromatography (SEC) allowed identification of three metal fractions. Following an anion exchange (AEX) chromatographic separation in the second dimension, the resultant sub-fractions were lyophilized and subjected to a third dimension of separation using a polyacrylamide gel electrophoresis (SDS-PAGE). After the separation, the bands were digested, and, in addition to others, the following proteins, previously associated with metals, were identified: 3-lipoxygenase A chain (soybean) complex with 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid, beta-amylase [Glycine max], seed lipoxygenase-1, lipoxygenase [G. max], seed lipoxygenase-2 (Pisum sativum) and beta-conglycinin., Biological Significance: Techniques presenting high resolution are desired due to their capability in resolving great amount of signals (responses) generated from hundreds of proteins generally found in different samples. To the best of our knowledge, this is the first time that bidimensional chromatographic system which allied to another separation dimension is applied for improving protein identification, so that higher number and different proteins were found when comparing 2D dimension with 3D dimension. In fact, this strategy is welcoming in proteomics studies, in order to improve the comprehension of those systems that present large number of proteins. This article is part of a Special Issue entitled: Environmental and structural proteomics., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This work evaluates the activity of a few key enzymes involved in combating reactive oxygen species (ROS), such as ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), glutathione reductase (EC 1.6.4.2), and superoxide dismutase (EC 1.15.1.1), as well as the concentration of malondialdehyde and hydrogen peroxide in transgenic and non-transgenic soybean leaves. Additionally, differential protein species from leaves of both genotypes were evaluated by applying a regulation factor of ≥1.8 to further corroborate the hypothesis that genetic modification itself can be a stress factor for these plants. For this task, transgenic soybean plants were obtained from seeds modified with the cp4EPSPS gene. The results revealed higher activities of all evaluated enzymes in transgenic than in non-transgenic soybean leaves (ranging from 13.8 to 70.1%), as well as higher concentrations of malondialdehyde and hydrogen peroxide in transgenic soybean leaves, clearly indicating a condition of oxidative stress established in the transgenic genotype. Additionally, 47 proteins were differentially abundant when comparing the leaves of both plants, with 26 species accurately identified, including the protein involved in the genetic modification (CP4EPSPS). From these results, it is possible to conclude that the plant is searching for a new equilibrium to maintain its metabolism because the stress condition is being maintained within levels that can be tolerated by the plant., Biological Significance: The present paper is the first one in the literature where are shown translational aspects involving plant stress and the genetic modification for soybean involving the cp4 EPSPS gene. The main biological importance of this work is to make possible the demystification of the genetic modification, allowing answers for some questions that still remain unknown, and enlarge our knowledge about genetically modified organisms. This article is part of a Special Issue entitled: Translational Plant Proteomics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This work reports the use of 2D-HPLC-ICP-MS to enlarge metallomics information when considering soybean seeds. Separations using size exclusion chromatography (SEC) allowed the identification of three metal fractions: the first corresponding to molecular weights from 38.1 to 181.1 kDa, the second from 8.2 to 17.2 kDa and the third from 0.4 to 3.8 kDa. In a second dimension, using anion exchange chromatography (AEX), three sub-fractions containing Fe, Mg and Mn, one containing Cu, and three containing Co, Cu, Mg, Mn and Zn were obtained. After these separations, 33 proteins were identified using the ESI-MS/MS technique, and divided into four functional categories: plant growth/cell division, protein destination and storage, metabolism and unclassified proteins. Among the identified proteins, proteins previously related to metals were found., (This journal is © The Royal Society of Chemistry 2012)

This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater.

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.