**3. Research dealing with proteomics in soybeans.**

### **3.1. Food safety**

of a chemical group to one or more amino acids [39]. The main goals of PTM studies in pro‐ teomics are identifying the proteins that have them, mapping the sites where these modifi‐ cations occur, quantifying their occurrence at the different sites and characterizing

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

The fact that covalent modifications result in changes in the protein molecular masses makes it possible for these modifications and the amino acids that carry them to be identified by mass spectrometry, allowing more than 300 different types of PTM's to be identified until now with the aid of this technique. Nevertheless, according to Mann and Jensen (2003) [41], mass spectrometry has reduced power of resolution of PTM's because they occur at low stoi‐ chiometric levels. This problem may be resolved by adopting fractioning methods prior to sequencing that allow enrichment of the sample for the proteins that have a certain type of PTM. Large scale modified protein enrichment systems are generally carried out by means

One example is the IMAC system – a column of immobilization through affinity to a metal for isolation of phosphorylated proteins in which metal ions of Fe(III) are joined to the ma‐

cooperative PTM's [40].

Relationships

540

**Figure 8.** General outline of the SILAC technique [36].

of affinity chromatography.

In the case of food, proteins are especially important for evaluation of food safety because they may place consumer health at risk. That is because proteins may be involved in synthesis of toxins and antinutrients, as well as being a toxin, an antinutrient or even an allergenic [43].

Soybeans are an important source of food throughout the world, being consumed in daily meals of all types. It has also been widely used as a food substitute by people that have in‐ tolerance to lactose or other milk proteins [44]. Nevertheless, in this species are also found proteins considered allergenic. Thus, knowledge regarding the proteins with toxic/antinutri‐ tional potential present in this grain becomes fundamental for development of biotechnolog‐ ical strategies that would have the target of elimination or inactivation in the genome of these species of genes that codify for these proteins.

Therefore, application of proteomic analysis in this type of study has been widely discussed. In relation to products derived from genetically modified (GM) foods, proteomic techniques have been applied because they allow a wide-ranging approach and analysis of many varia‐ bles simultaneously in the same sample [45]. Ocana et al. (2007) [46], studying GM proteins present in soybean and maize samples using proteomic analysis, identified the protein CP4 EPSPS, which confers tolerance to glyphosate herbicide. These samples were submitted to specific separation techniques followed by two-dimensional electrophoresis and mass spec‐ trometry for detection and characterization of the proteome.

Related to allergies, various allergens belonging to the superfamily of cupins and prolamins have been identified in soybeans [47]. Research has suggested that a heterogeneous group of soybean proteins bond to the IgE antibody and are potential allergens as, for example, Gly

m Bd 30k, β-conglycinin, Gly m Bd 28k, glycinin, Kunitz type protease inhibitor, some pro‐ teins present in the hull (Gly m 1.0101, Gly m 1.0102 e Gly m 2), profilin (Gly m 3), SAM 22 (Gly m 4), and other allergens like lectin and lipoxygenase [47,48]. According to Wilson et al. (2005) [49], in spite of the allergens identified in soybeans, the challenge of food researchers is developing a process for eradicating the immunodominant allergens, maintaining the functionality, nutritional value and effectiveness in the subsequent products derived from soybeans. For that reason, research has been developed using genetic engineering for silenc‐ ing the soybean gene responsible for synthesis of the protein Gly m Bd 30K, one of the main soybean proteins that develop allergic reactions with serums of sensitive patients [44].

### **3.2. Biotic and abiotic factors**

In a similar manner, various studies have shown that the proteomic approach is highly use‐ ful for investigation of crop response to environmental stresses because it compares the way the proteome is affected by different physiological conditions.

Saline stress is one of the many types of abiotic stresses that affect plants and compromise their yield. Salinity is a common agricultural problem in arid and semiarid regions and cre‐ ates large unproductive areas. There has been an ever greater search for cultivars adaptable to this condition. Sobhanian et al. (2010) [10], used proteomic techniques to evaluate the me‐ tabolism of proteins in leaves, hypocotyls and roots submitted to different NaCl concentra‐ tions (Figure 9), thus leading to saline stress.

Results in soybeans suggest that, in adaptation to saline conditions, proteins perform different roles in each organ, and the proteins most affected by saline stress are those related to photo‐ synthesis. Therefore, there is less energy production, and, consequently, reduction in plant growth. The conclusion suggests that the gene Glyceraldehyde-3-phosphate dehydrogenase may be, in the future, one of the target genes to improve tolerance to saline stress in this species.

Another type of abiotic stress studied in soybeans in which a proteomic approach is used is flooding stress [50,51]. Growing this species in areas subject to flooding makes the root envi‐ ronment anoxic, affecting nodulation or root growth. That way, plants respond with greater or less efficiency, allowing the distinction between cultivars which are tolerant and intoler‐ ant to this stress.

Proteomic analyses of soybean seedlings in response to flooding were undertaken by Shi et al. (2008) [52] to identify the key proteins involved in this process. To identify the first pro‐ teins produced in response to flooding, the roots of the seedlings were used for extraction of the proteins. The two-dimensional gel results suggest that cytosolic ascorbate peroxidase 2 (cAPX 2) is involved in response to flooding stress in young soybean seedlings.

In the case of drought stress, up-regulation of reactive oxygen species (ROS) scavengers such as superoxide dismutase (SOD) was reported in soybean seedlings [53]. The proteome analysis of two-day-old soybean seedlings subjected to drought stress by withholding of water for two days revealed a variety of responsive proteins involved in metabolism, dis‐ ease/defense and energy including protease inhibitors [53]. The major reason for loss of crop yields under drought stress is a decrease in carbon gain through photosynthesis. Proteome analysis of soybean root under drought condition showed that two key enzymes involved in carbohydrate metabolism, UDP- glucose pyrophosphorylase and 2,3-bisphosphoglycerate independent phosphoglycerate mutase, were down-regulated upon exposure to drought [54]. The identification of proteins such as UDP-glucose pyrophosphorylase and 2,3- bi‐ sphosphoglycerate has provided new insights that may lead to a better understanding of the molecular basis of responses to drought stress in soybean

**Figure 9.** Soybean seedlings submitted to different concentrations of NaCl [10].

m Bd 30k, β-conglycinin, Gly m Bd 28k, glycinin, Kunitz type protease inhibitor, some pro‐ teins present in the hull (Gly m 1.0101, Gly m 1.0102 e Gly m 2), profilin (Gly m 3), SAM 22 (Gly m 4), and other allergens like lectin and lipoxygenase [47,48]. According to Wilson et al. (2005) [49], in spite of the allergens identified in soybeans, the challenge of food researchers is developing a process for eradicating the immunodominant allergens, maintaining the functionality, nutritional value and effectiveness in the subsequent products derived from soybeans. For that reason, research has been developed using genetic engineering for silenc‐ ing the soybean gene responsible for synthesis of the protein Gly m Bd 30K, one of the main soybean proteins that develop allergic reactions with serums of sensitive patients [44].

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

In a similar manner, various studies have shown that the proteomic approach is highly use‐ ful for investigation of crop response to environmental stresses because it compares the way

Saline stress is one of the many types of abiotic stresses that affect plants and compromise their yield. Salinity is a common agricultural problem in arid and semiarid regions and cre‐ ates large unproductive areas. There has been an ever greater search for cultivars adaptable to this condition. Sobhanian et al. (2010) [10], used proteomic techniques to evaluate the me‐ tabolism of proteins in leaves, hypocotyls and roots submitted to different NaCl concentra‐

Results in soybeans suggest that, in adaptation to saline conditions, proteins perform different roles in each organ, and the proteins most affected by saline stress are those related to photo‐ synthesis. Therefore, there is less energy production, and, consequently, reduction in plant growth. The conclusion suggests that the gene Glyceraldehyde-3-phosphate dehydrogenase may be, in the future, one of the target genes to improve tolerance to saline stress in this species. Another type of abiotic stress studied in soybeans in which a proteomic approach is used is flooding stress [50,51]. Growing this species in areas subject to flooding makes the root envi‐ ronment anoxic, affecting nodulation or root growth. That way, plants respond with greater or less efficiency, allowing the distinction between cultivars which are tolerant and intoler‐

Proteomic analyses of soybean seedlings in response to flooding were undertaken by Shi et al. (2008) [52] to identify the key proteins involved in this process. To identify the first pro‐ teins produced in response to flooding, the roots of the seedlings were used for extraction of the proteins. The two-dimensional gel results suggest that cytosolic ascorbate peroxidase 2

In the case of drought stress, up-regulation of reactive oxygen species (ROS) scavengers such as superoxide dismutase (SOD) was reported in soybean seedlings [53]. The proteome analysis of two-day-old soybean seedlings subjected to drought stress by withholding of water for two days revealed a variety of responsive proteins involved in metabolism, dis‐ ease/defense and energy including protease inhibitors [53]. The major reason for loss of crop yields under drought stress is a decrease in carbon gain through photosynthesis. Proteome

(cAPX 2) is involved in response to flooding stress in young soybean seedlings.

the proteome is affected by different physiological conditions.

tions (Figure 9), thus leading to saline stress.

**3.2. Biotic and abiotic factors**

Relationships

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ant to this stress.

Stress by toxicity caused by the presence of high quantities of aluminum in the soil has also been investigated in soybeans from the perspective of proteomics [55,56]. Duressa et al. (2011) [56], studying cultivars tolerant and susceptible to high doses of aluminum, made proteomic analyses of roots, arriving at the conclusion that the greatest expression of en‐ zymes involved with citrate synthesis would be a good strategy in the search for cultivars tolerant to this mineral (Figure 10).

Another focus of the study within the context of selection of superior soybean genotypes using the proteomic approach is exposure to ultraviolet radiation, which has gained importance with the prominent worldwide concern for global warming and the consequent degradation of the ozone layer. Xu et al. (2007) [57], studied the proteome of soybean leaves to investigate the pro‐ tective role of flavonoids against the incidence of UV-B radiation. The authors suggest that high levels of flavonoid reduce the sensitivity of the plant to this radiation.

In relation to biotic stresses caused by pathogens like fungi, bacteria, nematodes and virus‐ es, proteomic tools are also greatly used because they allow understanding of the plantpathogen relationship [11,58,59,60] and also how the nodulation process occurs by means of symbiosis between the soybean roots and rhizobia [61]. In these cases, proteomic analysis

provides the information that will be used by genetic breeding in the search for cultivars re‐ sistant to various diseases.

**Figure 10.** profile of aluminum regulated-proteins in PI 416937 72 h posttreatment [56].

Zhang et al*.* (2011) [58] evaluated the responses of cultivars tolerant and susceptible to the fun‐ gus *Phytophthora sojae* by means of two-dimensional electrophoresis. The authors observed 46 proteins being expressed (Figure 11), among which only 11% were related to plant defense.

In addition, proteomic studies that deal with seed development also play an essential role [62]. The data obtained may help to interpret the function of genes that determine protein concentration, considered as a key characteristic for genetic breeding of soybeans. Moreover, differential proteomic analyses designed to describe the changes that occur from maturation to senescence in organs and organelles have been reported. There is also already a soybean proteome database, providing information on the proteins involved in the soybean response to stress caused by drought, salinity and, principally, flooding [63].

**Figure 11.** Identification of 26 and 20 protein spots from Yudou25 (A) and NG6255 (B), respectively. The numbers with arrows indicate the differentially expressed protein spots. Ip and Mr are shown on the gels [58].
