Initially, a study concerning characterization and quantification of silver nanoparticles (AgNP) was performed to evaluate silver (Ag) concentration in AgNP after the synthesis, as well as in its fractions (AgNP-res and Ag-sup ). According to results, approximately 50% of Ag is in the form of nanoparticles (NPs) in the AgNP solution after the synthesis, while the other 50% of Ag is in the ionic free form. These results suggest that, for studies related to AgNP application, is necessary to purify them by centrifugation and removal of the supernatant (Ag-sup), so that only the AgNP-res fraction containing NPs will be used. If this strategy is not considered and the solution after the synthesis is directly employed, possibly the effects observed in a study will be synergistic due to the presence of both NPs and free Ag in the solution after the synthesis. After the evaluation of AgNP to be used in this study, a cultivation test was first carried out with a final concentration of 250 mg kg-1 of Ag in the substrate. Under these conditions, both non-transgenic (NT) and transgenic (T) soybean plants did not survive to the cultivation with Ag (AgNO3) nitrate, indicating that the plants interacted differently with the different forms of Ag. Then, for a comparative evaluation, cultivations with final concentration of 50 mg kg-1 Ag on the substrate were carried out. Then, a proteomic study by 2-D DIGE was performed and several differentially abundant spots were found between NT and T soybeans from control groups and those plants which grown in the presence of AgNP or AgNO3. NT and T plants showed a different behavior in terms of differential spots, since NT soybeans were more affected by the cultivation with AgNO3, whereas T plants were more sensitive to the treatment with AgNP, including a significant increase (Tukey test - p < 0.05) in the total amount of proteins in soybean leaves. Then, the proteomic evaluation was finalized with the identification, by mass spectrometry, of those proteins present in the differential spots between the control groups and those which were exposed to AgNP or AgNO3. In general, regardless of the Ag form used in the cultivation, both soybean varieties are under stress condition, since some proteins related to such condition have been identified, among them ribulose. In addition to ribulose, species such as superoxide dismutase, ATP synthase, triosephosphate isomerase, among others, were identified in treated NT and T soybean groups, suggesting that the cultivation with AgNP and AgNO3 possibly resulted in an oxidative stress condition. Additionally, an enzymatic study was performed to increase the understanding regarding oxidative stress caused by AgNP and AgNO3 cultivations, previously observed in the proteomic evaluation. Enzymes such as catalase (CAT) and superoxide dismutase (SOD), as well as the content of H2O2 and malondialdehyde (MDA) were assessed, confirming that NT and T plants exposed to AgNP and AgNO3 are under stress conditions. All the results obtained through a proteomic and enzymatic approaches suggest that, in fact, the response of a plant, in terms of reactive oxygen species, content and activity of antioxidant enzymes, under oxidative stress conditions, is dependent on several factors, among them the plant species studied (in our case the variety) and the type of metal/metalloid or NPs used during cultivation. Regarding the uptake and assimilation of Ag, in general, T soybeans translocated a greater amount of Ag when compared to NT plants. Considering the different treatments, regardless of the soybean variety, the cultivation with AgNP resulted in a higher translocation rate. Through ionomic evaluation, significant differences (p ? 0.05) in terms of macro and micronutrients were found in all compartments, especially in the roots. Therefore, the presence of Ag, a non-essential element for the plant biochemistry, in its different forms (AgNP and AgNO3) interferes with the absorption of some nutrients. This fact may be related to stress conditions and consequent formation of chlorosis in the leaves of those treated plants, as observed in our study. Additionally, necrosis formation in the basal part of stems was observed for NT and T plants exposed to AgNP or AgNO3. Finally, the utilization of mild extractor buffers and hyphenated strategies based on liquid chromatography coupled to both ICP-MS and ESI-Orbitrap techniques were considered in the evaluation of metal species that may be altered in NT and T soybean leaves due to the cultivation with AgNP or AgNO3. After obtaining SEC-ICP-MS profiles, changes in the signal intensity were observed for Mn, Fe, Zn and Cu between control and treated groups. Then, from the four fractions found in SEC-ICP-MS, fraction three (F3) was only considered and analyzed by HILIC-ICP-MS. Profiles for Fe, Zn and Cu, previously monitored by SEC-ICP-MS, were also obtained. Then, the same F3 fraction was evaluated by HILIC-ESI-Orbitrap and some ions with isotopic patterns, mainly Cu and Zn, were found. These ions were selected for future HILIC-ESI-Orbitrap fragmentation experiments in tandem mode. With such results, it is expected to identify metal species and, consequently, provide additional information regarding possible pathways that may be altered in NT and T soybean plants due to AgNP and AgNO3 cultivations (AU) |
