New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins.

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.

Evaluation of silicon influence on the mitigation of cadmium-stress in the development of Arabidopsis thaliana through total metal content, proteomic and enzymatic approaches.

The mitigation of Cd-stress through Si addition to Arabidopsis thaliana cultivation is evaluated in terms of total metal content, proteomic and enzymatic approaches. Four different treatment are evaluated: TC (control, without Si or Cd addition), T1 (with Si addition), T2 (with Cd addition), and T3 (with Si and Cd addition). Through the total determination of Cd and Si in Arabidopsis leaves, the Cd concentration decreased by half when T2 is compared with T3 treatment. In terms of proteomic approach, some differential protein species are achieved by comparative proteomics through 2-D DIGE of all treatments evaluated. Fifty six differential abundant proteins spots (abundance factor ≥1.5) are detected, and 32 of them accurately characterized and identified through nESI-LC-MS/MS. These proteins are differentially produced due to Cd and/or Si treatments, which mainly include proteins associated with disease/defense, energy and metabolism. The most difference in the abundance of proteins is found due to the presence or absence of Si in plants treated with Cd. Regarding the enzymatic approaches, a major increase is found on APX, CAT and GR activities (5.0, 3.5, and 1.5-fold, respectively). The same is observed for the MDA concentration because an increase of 3-fold is found when TC are compared to those treated with T2. However, when T3 plants are evaluated, the enzymes activities are similar to TC plants. Differences ranging from 6.5 to 21% are detected considering the activity of SOD in the treatments (T1-T3 x TC). The decreased activities of CAT, APX and GR and lower MDA concentration indicate a lower reactive oxygen species production in plants treated with Cd and Si. Based on a proteomics point of view it is possible to conclude that Si-Cd interactions occur at protein level and allow plants to respond effectively to the Cd toxicity, revealing the active involvement of Si on mechanisms involved in Si-induced Cd tolerance in Arabidopsis plants. Additionally, from an enzymatic point of view, it is possible to conclude that Si positively interferes diminishing the negative effects of Cd in Arabidopsis by decreasing the reactive oxygen species generation and increasing the antioxidative enzyme activity.

Comparative studies focusing on transgenic through cp4EPSPS gene and non-transgenic soybean plants: an analysis of protein species and enzymes.

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.