**8. Future trends**

Currently, the comparative studies concerning alterations in proteins, metalloproteins, met‐ als and enzymes have demonstrated significant differences among transgenic and nontransgenic soybean. These differences have indicated that these genetic modifications provide not only tolerance to herbicide but also cause many changes in the whole metabo‐ lism of the transgenic plants.

Carefully taking into account all the results presented, it is possible to raise the following question: what are the future trends in comparative studies involving transgenic and nontransgenic soybean? Since the whole metabolism of transgenic soybean plants seems to be different to the non-transgenic one, this is a promising research area, and too much work is still needed. Much more information is still ahead of us for a better comprehension of the specific aspects of the transgenic soybean plant metabolism. In this way, investigations into techniques and novel approaches, quantitative proteomics, imaging and mapping of ele‐ mental distribution, tracer experiments employing stable isotopes and also in natural varia‐ tion in the isotopic composition of the elements may possibly be the future trends in this topic. This will contribute to elucidation and expansion of our knowledge about transgenic soybean.

Since the proper functioning of life depends on the elements in a variety of processes, the understanding of molecular mechanisms of the elements and information on its chemical forms present in a living organism are very important. In this context, studies about identifi‐ cation and/or quantification of one or more chemical species of elements in transgenic and non-transgenic soybean samples are able to generate valuable information about their me‐ tabolisms. Therefore, it would be useful if more efforts were devoted to this topic. A novel technique that has an unexplored potential for speciation analysis is travelling wave ion mo‐ bility spectrometry coupled to mass spectrometry (TWIMS-MS) [116]. The use of this techni‐ que in speciation analysis of metals associated with biomolecules should increase due to its capability of differentiation of ions by shape and size, besides mass and charge. Until now, the studies employing ion mobility are concentrated in proof-of-the-concept using isolated species commercially available and its application to complex matrices certainly will be a big challenge, but very helpful to elucidate many questions.

The main objective of quantitative proteomics is to quantify protein expression alterations in response to a variety of changes, and, nowadays, one of the most challenging and emerging area of proteomics involves the developments of accurate quantitative methods for proteins. The quantitative proteomics is divided in absolute and relative subjects. In the absolute quantification, changes in protein expression are determined in exact amount or concentra‐ tion of each protein present. The relative one determines the up- or down-regulation of a protein relative to the control sample, and the results are presented as 'fold' increases or de‐ creases. The 2-D DIGE is an example of relative quantification technique that is applied to intact proteins and the differential expression determination is based on fluorescence as commented earlier in this chapter. Taking into account the soybean comparative studies the application of quantitative proteomics by 2-D DIGE or by other technique could continue es‐ tablishing the differences in protein expressions accurately [117].

than 20 proteins were identified, encompassing 4 different functional categories. Among them, βconglycinin, a protein previously associated to metals, was identified in three fractions, and one metalloprotein that binds Fe, lypoxigenase 1, was found in a high molecular weight fraction, the on‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Currently, the comparative studies concerning alterations in proteins, metalloproteins, met‐ als and enzymes have demonstrated significant differences among transgenic and nontransgenic soybean. These differences have indicated that these genetic modifications provide not only tolerance to herbicide but also cause many changes in the whole metabo‐

Carefully taking into account all the results presented, it is possible to raise the following question: what are the future trends in comparative studies involving transgenic and nontransgenic soybean? Since the whole metabolism of transgenic soybean plants seems to be different to the non-transgenic one, this is a promising research area, and too much work is still needed. Much more information is still ahead of us for a better comprehension of the specific aspects of the transgenic soybean plant metabolism. In this way, investigations into techniques and novel approaches, quantitative proteomics, imaging and mapping of ele‐ mental distribution, tracer experiments employing stable isotopes and also in natural varia‐ tion in the isotopic composition of the elements may possibly be the future trends in this topic. This will contribute to elucidation and expansion of our knowledge about transgenic

Since the proper functioning of life depends on the elements in a variety of processes, the understanding of molecular mechanisms of the elements and information on its chemical forms present in a living organism are very important. In this context, studies about identifi‐ cation and/or quantification of one or more chemical species of elements in transgenic and non-transgenic soybean samples are able to generate valuable information about their me‐ tabolisms. Therefore, it would be useful if more efforts were devoted to this topic. A novel technique that has an unexplored potential for speciation analysis is travelling wave ion mo‐ bility spectrometry coupled to mass spectrometry (TWIMS-MS) [116]. The use of this techni‐ que in speciation analysis of metals associated with biomolecules should increase due to its capability of differentiation of ions by shape and size, besides mass and charge. Until now, the studies employing ion mobility are concentrated in proof-of-the-concept using isolated species commercially available and its application to complex matrices certainly will be a

The main objective of quantitative proteomics is to quantify protein expression alterations in response to a variety of changes, and, nowadays, one of the most challenging and emerging area of proteomics involves the developments of accurate quantitative methods for proteins. The quantitative proteomics is divided in absolute and relative subjects. In the absolute quantification, changes in protein expression are determined in exact amount or concentra‐

big challenge, but very helpful to elucidate many questions.

ly fraction where an Fe peak was separated.

**8. Future trends**

Relationships

600

soybean.

lism of the transgenic plants.

According to the results presented earlier, some elements are present at higher concentrations in transgenic soybean seeds than in non-transgenic ones [66,94,105]. The transgenic seed seems to have ability to take up higher amounts of some metals from the soil and this is a sign that the process‐ es involved in intake, transport and storage of essential and toxic metals and metalloids probably are suffering changes due to genetic modification. Various new queries take place with this infor‐ mation, such as: The other transgenic plant parts (roots, stems and leaves), are also taking up, trans‐ porting and storing higher amounts of these metals? Other plant parts try to eliminate some excess of these metals? Are these higher amounts really an excess for a transgenic plant or not? Are there differences in the distributions of these metals among transgenic and non-transgenic soybean? A potential tool for obtaining a better insight in these processes can be to use tracer experiments em‐ ploying stable isotopes. In the last few years the use of stable isotopes and their isotope ratio meas‐ urements have gained importance for tracer experiments in biological and medical research [118]. In these studies stable isotopic tracers with an isotopic composition sufficiently different from the corresponding natural one is added to the studied system and changes in the selected isotope ratio monitored. The absorption or bioavailability of an element can be determined with this approach as well as information about element redistribution over various compartments of an organism [118-119]. According to our knowledge, no tracer study for essential or toxic metals evaluating transgenic and non-transgenic soybean is found in the literature and therefore there is a great amount of work to perform in this challenging area.

LA-ICP-MS offers *in situ* analysis of solid samples with respect to metals and nonmetals at trace concentration level mostly without sample preparation and without charging effects during the measurements. This technique can also be applied to the imaging of soft tissues such as plant leaves with relatively high spatial resolution and good sensitivity [120] and therefore, some investigations involving metals distribution by LA-ICP-MS in transgenic and non-transgenic soybean would also be a future trend.

Another challenging issue that can provide evidences supporting the hypothesis that genetic modification is affecting the metabolism of soybean plants involves the investigation of nat‐ ural variation in the isotopic composition of the elements. Even though isotopic abundances are assumed to be almost constant in nature, small isotopic or mass fractionation effects oc‐ cur in both natural and industrial processes [118]. Since the isotopes present the same num‐ ber of electrons, they show basically the same chemical behavior. However, there is a small discrepancy in their physicochemical behavior due to the mass difference, which may leave isotopes of the same element to take part with slightly different efficiencies in physical proc‐ esses or (bio)chemical reactions, and, consequently, to result in variations in the isotopic composition [118-119]. These differences in efficiency are associated to a minor distinction in

equilibrium for each different isotopic molecule - thermodynamic effect or in the rate with which the isotopes participate in a process or reaction - kinetic effect. Lighter elements, such as H, C, N, O and S suffer more pronounced isotopic variations because of the high relative mass difference between their isotopes. Nevertheless, heavier elements are subject to isotope fractionation, even though the change is minor [121].

As relative abundances cannot be measured directly, these studies are based on measuring the isotope ratio of an element because it is experimental accessible. The isotope ratio meas‐ ured in a particular sample (Rx) is compared to the corresponding one in another sample, frequently a reference sample (RRF) [118]. The differences found are frequently very small and thus high reproducibility/repeatability is required. Thus, the ICP-MS technique is be‐ coming the more advantageous choice for most applications employing isotope ratios, main‐ ly considering the recent instrumental developments. As the elements are subject to isotope fractionation in nature, the genetic modification could also provoke or intensify this effect.

In view of that comment here, it is easy to rationalize that many aspects can be explored when focusing on studies related to transgenic soybean.
