**6. Two-dimensional difference gel electrophoresis: 2-D DIGE**

A promising alternative for circumventing possible variations in the technique already de‐ scribed (2-D PAGE) is the two-dimensional difference gel electrophoresis (2-D DIGE). This technique, which is based on fluorescent cyanine dyes, allows comparisons between two ex‐ act quantitative proteomic samples, which are resolved on the same gel, minimizing the problems previously mentioned [82]. Moreover, there is the advantage of the high sensitivi‐ ty of these dyes (*ca*. 1 fmol of protein), which enables the detection of low abundance pro‐ teins when compared to other dyes used in the detection of protein spots, such as Coomassie Brilliant Blue (CBB) and silver staining [82]. Frequently, three samples are la‐ beled in 2-D DIGE: two of them are experimental samples whereas the third one is com‐ posed of a mixture of equal amounts of all experimental samples (*i.e*., a pooled internal

standard). This creates a standard for each protein during analysis. After 2D separation, dif‐ ferent protein samples labeled can be visualized separately by exciting the different dyes at their specific excitation wavelengths. Therefore, from the images generated for each dye, the signals from labeled protein spots are determined and the normalized intensities or spot volumes for each spot from different dyes (Cy2, Cy3, Cy5) are compared in order to identify differentially expressed proteins between the samples [82-83].

Once this technique is finely developed for finding possible biomarkers, reference [79] applied the 2-D DIGE technique and mass spectrometry (ESI-QTOF MS/MS) to assess differences in proteomic profiles of transgenic and non-transgenic soybean seeds. Three biological replicates were analyzed. A regulation factor of 1.5 (50% variation) was chosen as determined by the image analysis program and statistically significant differences in expression were determined (p≤0.05, according to the Student *t* test). The program of im‐ age analysis uses the automatic detection of the spots, and does not require any manual editing, either in adding or in altering the area defining the spots, in contrast to other programs for 2-D PAGE image.

As a result, a total of four proteins were differentially expressed between transgenic and non-transgenic soybean seeds, where two were overexpressed, being more highly ex‐ pressed in transgenic soybean, and two were underexpressed, being less expressed in transgenic soybean. Thus, these four spots were selected for identification by mass spec‐ trometry. As results, the spots were identified as: Actin (fragment) (*Glycine max*), in‐ volved in various types of cell motility, widely expressed in all eukaryotic cells and binds to ATP and other proteins [84]; cytosolic glutamine synthetase (*Cucumis melo*) (Fig‐ ure 1a), considered as a ligand enzyme, being highly expressed in many types of roots, binds ATP molecules and is responsible for the primary assimilation of ammonia in all living organisms, participates in nitrogen fixation [84]; Glycinin subunit G1 (*Glycine max*), responsible for the nutritional, physicochemical, and physiological characteristics of soy‐ bean seeds [85] and Glycine-rich RNA-binding protein (*Glycine max*) (Figure 1b), in‐ volved in cellular response to environmental and developmental conditions [84]. It is noteworthy that the actin protein was also detected by Brandão et al. [66] working with 2-D PAGE and image analysis, and with the same sample.

The results obtained in reference [79], comments about some differential proteins found, es‐ tablishing a relationship between oxidative stress (ROS production) and genetic modifica‐ tion. In this way, spectrophotometric enzymatic assays demonstrate that soybean transgenic seeds (for glyphosate resistance) exhibited higher activities for APX, CAT and GR enzymes compared to non-transgenic. Considering these results, the authors concluded that the oxi‐ dative stressful condition in transgenic seeds resulted in an increase of H2O2, which is proba‐ bly controlled by the action of APX and CAT and even GR. Related to SOD, reference [79] showed the results for SOD activity in nondenaturing polyacrylamide gel electrophoresis, and it was possible to observe eight SOD isoenzymes detected in both transgenic and nontransgenic soybean seeds, one as Mn-SOD, two as Fe-SOD and five as Cu/Zn-SOD. The au‐ thors commented that the reduction in SOD activity in transgenic seeds was much more a result of a reduction in the Fe-SOD isoenzymes activities. Finally, and as a conclusion, the genetic modification itself might have induced extra ROS generation.

standard). This creates a standard for each protein during analysis. After 2D separation, dif‐ ferent protein samples labeled can be visualized separately by exciting the different dyes at their specific excitation wavelengths. Therefore, from the images generated for each dye, the signals from labeled protein spots are determined and the normalized intensities or spot volumes for each spot from different dyes (Cy2, Cy3, Cy5) are compared in order to identify

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

Once this technique is finely developed for finding possible biomarkers, reference [79] applied the 2-D DIGE technique and mass spectrometry (ESI-QTOF MS/MS) to assess differences in proteomic profiles of transgenic and non-transgenic soybean seeds. Three biological replicates were analyzed. A regulation factor of 1.5 (50% variation) was chosen as determined by the image analysis program and statistically significant differences in expression were determined (p≤0.05, according to the Student *t* test). The program of im‐ age analysis uses the automatic detection of the spots, and does not require any manual editing, either in adding or in altering the area defining the spots, in contrast to other

As a result, a total of four proteins were differentially expressed between transgenic and non-transgenic soybean seeds, where two were overexpressed, being more highly ex‐ pressed in transgenic soybean, and two were underexpressed, being less expressed in transgenic soybean. Thus, these four spots were selected for identification by mass spec‐ trometry. As results, the spots were identified as: Actin (fragment) (*Glycine max*), in‐ volved in various types of cell motility, widely expressed in all eukaryotic cells and binds to ATP and other proteins [84]; cytosolic glutamine synthetase (*Cucumis melo*) (Fig‐ ure 1a), considered as a ligand enzyme, being highly expressed in many types of roots, binds ATP molecules and is responsible for the primary assimilation of ammonia in all living organisms, participates in nitrogen fixation [84]; Glycinin subunit G1 (*Glycine max*), responsible for the nutritional, physicochemical, and physiological characteristics of soy‐ bean seeds [85] and Glycine-rich RNA-binding protein (*Glycine max*) (Figure 1b), in‐ volved in cellular response to environmental and developmental conditions [84]. It is noteworthy that the actin protein was also detected by Brandão et al. [66] working with

The results obtained in reference [79], comments about some differential proteins found, es‐ tablishing a relationship between oxidative stress (ROS production) and genetic modifica‐ tion. In this way, spectrophotometric enzymatic assays demonstrate that soybean transgenic seeds (for glyphosate resistance) exhibited higher activities for APX, CAT and GR enzymes compared to non-transgenic. Considering these results, the authors concluded that the oxi‐ dative stressful condition in transgenic seeds resulted in an increase of H2O2, which is proba‐ bly controlled by the action of APX and CAT and even GR. Related to SOD, reference [79] showed the results for SOD activity in nondenaturing polyacrylamide gel electrophoresis, and it was possible to observe eight SOD isoenzymes detected in both transgenic and nontransgenic soybean seeds, one as Mn-SOD, two as Fe-SOD and five as Cu/Zn-SOD. The au‐ thors commented that the reduction in SOD activity in transgenic seeds was much more a

differentially expressed proteins between the samples [82-83].

2-D PAGE and image analysis, and with the same sample.

programs for 2-D PAGE image.

Relationships

594

Proteins involved in the RNA processing and alternative splicing, RNA transport, messen‐ ger RNA (mRNA) translation, mRNA stability, and mRNA silencing mechanisms have been shown to be required for normal plant development and the responses of plants to altered environments [86-87]. In our case, just the glycine-rich RNA-binding protein was differen‐ tially found after DIGE analysis, and this protein correlated to ROS production according to different articles [88, 89]. As already mentioned, the cytosolic glutamine synthetase is in‐ volved in nitrogen fixation. Oxidative stress can also control the expression of nitrogen-me‐ tabolism genes as recently demonstrated [90], demonstrating that cytosolic glutamine synthetase can be altered because of the oxidative stress observed in the transgenic soybean line [90].

**Figure 1.** Examples of spots with expression variation among samples of transgenic (T) and non-transgenic (NT) soy‐ bean seeds: a - cytosolic glutamine synthetase (*Cucumis melo*) and b - Glycine-rich RNA-binding protein (*Glycine max*) [modified from reference 79].
