**3. Implication of proteomics in understanding soybean stress**

Soybean is grown worldwide with an average protein content of 40% (highest protein con‐ tent with respect to other food crops) and oil content of 20% (which is second only to that of groundnut among the leguminous foods). Furthermore, soybean improves soil fertility by fixing nitrogen from the atmosphere in symbiosis with nitrogen fixing bacteria. It is, howev‐ er, susceptible to various types of stresses (abiotic and biotic). Tolerance and susceptibility to stresses are complex phenomena because they are quantitatively inherited and can occur during different stages of plant growth and development. Extrinsic stress is regarded as the most important stress agent, which results from changes in abiotic factors such as tempera‐ ture, climatic factors and chemical components, either naturally occurring or manmade. Fur‐ ther, biotic stresses (occurs as a result of damage done to plants by other living organisms, such as bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds, bacterial, fungal, algal and viral diseases) can also cause huge deterioration in plant growth and yield. Plants have developed adaptive features against these stresses. The genome remains un‐ changed to a large extent in any particular cell while proteins change dramatically as genes are turned on or off in response to stress. The proteome determines the cellular phenotype and its plasticity in response to external signals. It is proteins that are directly involved in both normal and stress-associated biochemical processes. Therefore, a more complete under‐ standing of stress in soybean may be gained by looking directly into the proteins within a stressed cell or tissue. Proteomic based techniques that allow large-scale protein profiling

are powerful tools for the identification of proteins involved in stress-responses in plants. Extensive studies have evaluated changes in protein levels in plant tissues in response to stresses. Unfortunately, these studies have been mainly focused on non-legume species such as *Arabidopsis* and rice, and only recently have been enlarged to include some legumes. As a result only a handful of studies have been carried out in legumes, although in the next few years there should be a significant increase in the number of legume species and stresses would be analyzed. Recently, proteomic approaches have been applied to various legumes like *M. truncatula*, lentils, lupin, common bean, cowpea and soybean to identify proteins in‐ volved in the response to different stresses. Interestingly, many of the induced proteins from these different stresses were common or belonged to overlapping pathways [13].

Considerable amount of research has been carried out during the last decade to find the ef‐ fect of stress under extreme . These include chloroplast membrane, cell wall and nuclear en‐ velope, while some researchers have focused on individual tissues *viz*. seeds, mitochondria, root tips, vacuoles, chloroplasts and thylakoids. To date, lots of reports have come which emphasize changes in protein expression levels during a particular or integrative stress con‐ sequently affecting cellular metabolism. Proteomics provides direct assessment of the bio‐ chemical processes of monitoring the actual proteins performing signaling, enzymatic, regulatory and structural functions encoded by the genome and transcriptome.

Following are the different categories of proteins with important properties, which have been shown to play a crucial role against abiotic environmental stress as well as biotic stress. The data so collected from various plants including soybean is based on 2-DE, mass spec‐ trometry and bioinformatics tools.

#### (a) *Antioxidants Enzymes*

Reactive oxygen species (ROS) in plant cellulars are produced as a consequence of myriad stimuli ranging from abiotic and biotic stress, production of hormonal regulators, as well as cell processes such as polar growth and programmed cell death [14]. These reactive mole‐ cules are generated at a number of cellular sites, including mitochondria, chloroplasts, per‐ oxisomes, and at the extracellular side of the plasma membrane. ROS trigger signal transduction events, such as mitogen-activated protein kinase cascades eliciting specific cel‐ lular response.s. The influence of these molecules on cellular processes is mediated by both the perpetuation of their production and their amelioration by scavenging enzymes such as superoxide dismutase, ascorbate peroxidase, and catalase. The location, amplitude, and du‐ ration of production of these molecules are determined by the specificity of the responses [15]. Accumulation of ROS as a result of various environmental stresses is a major cause of loss of crop productivity worldwide. ROS affect many cellular functions by damaging nucle‐ ic acids, oxidizing proteins, and causing lipid peroxidation. It is important to note that whether ROS will act as damaging, protective or signaling factors depends on the delicate equilibrium between ROS production and scavenging at the proper site and time. ROS can damage cells as well as initiate responses such as new gene expression. The cell response evoked is strongly dependent on several factors. The subcellular location for formation of ROS may be especially important for a highly reactive ROS, because it diffuses only a very short distance before reacting with a cellular molecule. Stress-induced ROS accumulation is counteracted by enzymatic antioxidant systems that include a variety of scavengers, such as superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, glutathione S-transfer‐ ase, catalase and non-enzymatic low molecular metabolites, such as ascorbate, glutathione (red.), α-tocopherol, carotenoids and flavonoids. In addition, proline can now be added to an elite list of non-enzymatic antioxidants that microbes, animals, and plants need to coun‐ teract the inhibitory effects of ROS [16]. Plant stress tolerance may therefore be improved by the enhancement of *in vivo* levels of antioxidant enzymes. The antioxidants as described are found in almost all cellular compartments which signify the importance of ROS detoxifica‐ tion for cellular survival. It has also been shown that ROS influence the expression of a num‐ ber of genes and signal transduction pathways which suggest that cells have evolved strategies to use ROS as biological stimuli and signals that activate and control various ge‐ netic stress-response programs. Control of plant pathogens by genetic engineering has tar‐ geted ROS for development of pathogen resistant crop varieties [17]. Antisense technology has been used to reduce the capability to scavenge H2O2 in case of model plants like *Arabi‐ dopsis thaliana* and *Nicotiana tabacum*. In these plants, antioxidant enzymes like catalase and ascorbate peroxidase are under-expressed and it has been found that they were hyper-re‐ sponsive to pathogen attack. This further confirms that the ability of plant cells to regulate the efficiency in their ROS-removal strategies is a key point in their resistance against patho‐ gens. The technology is yet to be implemented in case of legumes including soybean as in‐ tensive research is going on future prospects of the technology.

#### (b) *Abscissic acid signaling and related protein*

are powerful tools for the identification of proteins involved in stress-responses in plants. Extensive studies have evaluated changes in protein levels in plant tissues in response to stresses. Unfortunately, these studies have been mainly focused on non-legume species such as *Arabidopsis* and rice, and only recently have been enlarged to include some legumes. As a result only a handful of studies have been carried out in legumes, although in the next few years there should be a significant increase in the number of legume species and stresses would be analyzed. Recently, proteomic approaches have been applied to various legumes like *M. truncatula*, lentils, lupin, common bean, cowpea and soybean to identify proteins in‐ volved in the response to different stresses. Interestingly, many of the induced proteins from

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

Considerable amount of research has been carried out during the last decade to find the ef‐ fect of stress under extreme . These include chloroplast membrane, cell wall and nuclear en‐ velope, while some researchers have focused on individual tissues *viz*. seeds, mitochondria, root tips, vacuoles, chloroplasts and thylakoids. To date, lots of reports have come which emphasize changes in protein expression levels during a particular or integrative stress con‐ sequently affecting cellular metabolism. Proteomics provides direct assessment of the bio‐ chemical processes of monitoring the actual proteins performing signaling, enzymatic,

Following are the different categories of proteins with important properties, which have been shown to play a crucial role against abiotic environmental stress as well as biotic stress. The data so collected from various plants including soybean is based on 2-DE, mass spec‐

Reactive oxygen species (ROS) in plant cellulars are produced as a consequence of myriad stimuli ranging from abiotic and biotic stress, production of hormonal regulators, as well as cell processes such as polar growth and programmed cell death [14]. These reactive mole‐ cules are generated at a number of cellular sites, including mitochondria, chloroplasts, per‐ oxisomes, and at the extracellular side of the plasma membrane. ROS trigger signal transduction events, such as mitogen-activated protein kinase cascades eliciting specific cel‐ lular response.s. The influence of these molecules on cellular processes is mediated by both the perpetuation of their production and their amelioration by scavenging enzymes such as superoxide dismutase, ascorbate peroxidase, and catalase. The location, amplitude, and du‐ ration of production of these molecules are determined by the specificity of the responses [15]. Accumulation of ROS as a result of various environmental stresses is a major cause of loss of crop productivity worldwide. ROS affect many cellular functions by damaging nucle‐ ic acids, oxidizing proteins, and causing lipid peroxidation. It is important to note that whether ROS will act as damaging, protective or signaling factors depends on the delicate equilibrium between ROS production and scavenging at the proper site and time. ROS can damage cells as well as initiate responses such as new gene expression. The cell response evoked is strongly dependent on several factors. The subcellular location for formation of ROS may be especially important for a highly reactive ROS, because it diffuses only a very short distance before reacting with a cellular molecule. Stress-induced ROS accumulation is

these different stresses were common or belonged to overlapping pathways [13].

regulatory and structural functions encoded by the genome and transcriptome.

trometry and bioinformatics tools.

(a) *Antioxidants Enzymes*

Relationships

400

Abscissic acid (ABA) has been implicated in plant response to environmental stress by inter‐ fering at different levels with signaling. Its level increases under stress conditions to trigger metabolic and physiological changes [18]. It has become increasingly clear that the isolated abiotic signaling network is controlled by ABA and the biotic network is controlled by salicyl‐ ic acid, jasmonic acid and ethylene are interconnected at various levels [19]. The concept of marker genes whose expression is believed to be regulated by individual hormones does not do justice to the nature of the network. The apparent cross-talk in stress-hormone signaling makes it difficult to assign a marker gene or a mutant phenotype to a specific hormonecontrolled pathway. The signaling network into which the four stress hormones and other signals feed is apparently designed to allow plants to adapt optimally to specific situations by integrating possibly conflicting information from environmental conditions, biotic stress, and developmental as well as nutritional status. Promoter analyses of ABA/stress-responsive genes revealed that a DNA sequence element consisting of ACGTGGC is important for ABA regu‐ lation. For the past several years, researchers have been trying to identify transcription fac‐ tors that regulate the expression of ABA/stress-responsive genes *via* the consensus element, which is generally known as 'Abscisic Acid Response Element' (ABRE). Many basic leucine zipper class DNA-binding proteins that interact with the element have been reported [20]. Researchers have focused on the small subfamily of *Arabidopsis* basic leucine zipper proteins referred to as ABFs (ABRE-binding factors), whose expression is induced by ABA and by various abiotic stresses (i.e., cold, high salt and drought). ABA is involved in responses to environmental stress such as salinity, and is required by the plant for stress tolerance as found recently on soybean studies. The leaf ABA content in salt-tolerant soybean increased signifi‐

cantly under salt stress, while in case of salt sensitive soybean has almost negligible increase in ABA. It is thus possible that ABA enhances salt tolerance in soybean [21].

### (c) *GABA-related protein*

*γ*-Aminobutyric acid (GABA) is a non-protein amino acid that is conserved from bacteria through yeast to vertebrates and was discovered in plants over half-a-century ago. It is mainly metabolized through a short pathway called the GABA shunt, because it bypasses two steps of the tricarboxylic-acid (TCA) cycle. The pathway is composed of three enzymes: the cytosolic and mitochondrial glutamate decarboxylase (GAD), GABA transaminase (GA‐ BA-T) and succinic semialdehyde dehydrogenase (SSADH). Although there are differences in the subcellular localization of GABA-shunt enzymes in different organisms have been re‐ ported (for e.g. in yeast, SSADH is present inside cytosol) [22]. In an alternative reaction, succinic semialdehyde can be converted to GHB (*γ*-hydroxybutyric acid) through a GHB de‐ hydrogenase (GHBDH) present in animals and recently identified in plants [23]. Interesting‐ ly, research of GABA in vertebrates has focused mainly on its role in the context of plant responses to stress, because of its rapid and dramatic production in response to biotic and abiotic stresses. For example, disruption of the unique SSADH gene in *Arabidopsis* results in plants undergoing necrotic cell death caused by the accumulation of reactive oxygen inter‐ mediates (ROIs) when they are exposed to environmental stresses [24]. A recent article re‐ ports that a gradient of GABA concentration is essential for the growth and guidance of pollen tubes and suggests that this amino acid plays a role in intercellular signaling in plants, possibly similar to its role in animals. The main question raised by these recent find‐ ings is whether GABA itself serves as a signaling molecule in plants. If so, this would imply that GABA is capable of mediating developmental changes and cell guidance by interacting with specialized plant receptors [25].

### (d) *Mitogen-activated protein kinase signaling and related proteins*

Like other eukaryotes, plants use mitogen-activated protein kinase (MAPK) cascades to reg‐ ulate various cellular processes in response to a broad range of biotic and abiotic stress. These cascades promote the transient activation of MAPKs by a dual phosphorylation of Thr and Tyr within the activation loop of the MAPK. Recent studies indicate that MAPKs are not only regulated through phosphorylation by upstream kinases, but also by direct binding of different protein factors [26]. The constitutive activation of MAPKs was found to result in detrimental effects, underlining the importance of a negative regulation of MAPK signaling. MAPK phosphatases (MKPs) are negative regulators of MAPKs. Recent progress in analyz‐ ing plant MKP mutants has revealed their important role in fine-tuning MAPK signaling. In particular, the dual-specificity phosphatase MKP1 and the protein tyrosine phosphatase (PTP1) negatively regulate defense responses and resistance to a bacterial pathogen by counter balancing the activation of two MAPKs (MPK3 and MPK6). Interestingly, MKP1 and PTP1 bind CaM, and the phosphatase activity of MKP1 is increased by CaM in a Ca2+ dependent manner. Thus, Ca2+ and MAPK signaling pathways appear to be connected through the regulation of plant MAPKs and MKPs by CaM [27].

(e) *Calcium signaling and related proteins*

Plant cells are equipped with highly efficient mechanisms to perceive, transduce and re‐ spond to a wide variety of internal and external signals during their growth and develop‐ ment. Perception of signals *via* receptors results in generation or synthesis of nonproteinaceous molecules which are termed as messengers. The messengers include Ca2+ ions, small organic molecules such as cyclic nucleotide monophosphates, inositol triphosphates and inorganic molecules such as H2O2 and NO. The elements of receptors, messengers, sensors and targets vary depending on the signal received. Identification and functional assignment of these elements in a stimulus-specific signal transduction pathway is a challenging area for plant biologists. With the completion of genome sequences of various organisms, including *Arabidopsis thaliana*, *Oryza sativa*, *Medicago trunculata*, *Glycine max* etc. it has become evident that plants have a large number of motifs containing helix-loop-helix which binds to Ca2+ [28]. Further, Ca2+ has been implicated in mediating various developmental processes (pollen tube growth, root-hair and lateral root development and nodulation), hormone regulated cellular activities (cell division and elongation, stomatal closure/opening), pathogen- and elicitorinduced defense related processes, and a variety of abiotic stress signal induced gene expres‐ sion. However, the identity and functions of downstream transducers and mechanisms by which Ca2+ mediates a variety of cellular responses are just begin to unravel in plants. In plants, spatially and temporally distinct changes in cellular Ca2+ concentrations, designated as "Ca2+ signatures" that are evoked in response to different stimuli like drought, salt or osmotic stresses, temperature, light and plant hormones represent a central mechanistic principle to present defined stimulus-specific information [29]. These specific "Ca2+ signatures" are formed by the tightly regulated activities of channels and transporters at different membranes and cell organelles. While the identity and function of components of the Ca2+ extrusion system are rather well understood in plant cells, the molecular identity of Ca2+ specific influx channels has remained unknown. However, non-specific influx of Ca2+ mediated by ligand gated cation channels like cyclic nucleotide gated channels and glutamate receptor-like proteins contrib‐ ute to different Ca2+ mediated cellular functions like the response to pathogens, pollen tube growth and abiotic stress. The unique structural composition of Ca2+ binding proteins and the complexity of the target proteins regulated by the Ca2+ sensors allow the plant to tightly control the appropriate adaptation to its ever changing environment. It is actually still not well understood about interface of information presentation by a specific Ca2+ signal and initia‐ tion of information decoding by Ca2+ sensors that represent a most critical step in specific information processing [30].

## **4. Significance of proteomics in soybean allergenicity**

cantly under salt stress, while in case of salt sensitive soybean has almost negligible increase

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

*γ*-Aminobutyric acid (GABA) is a non-protein amino acid that is conserved from bacteria through yeast to vertebrates and was discovered in plants over half-a-century ago. It is mainly metabolized through a short pathway called the GABA shunt, because it bypasses two steps of the tricarboxylic-acid (TCA) cycle. The pathway is composed of three enzymes: the cytosolic and mitochondrial glutamate decarboxylase (GAD), GABA transaminase (GA‐ BA-T) and succinic semialdehyde dehydrogenase (SSADH). Although there are differences in the subcellular localization of GABA-shunt enzymes in different organisms have been re‐ ported (for e.g. in yeast, SSADH is present inside cytosol) [22]. In an alternative reaction, succinic semialdehyde can be converted to GHB (*γ*-hydroxybutyric acid) through a GHB de‐ hydrogenase (GHBDH) present in animals and recently identified in plants [23]. Interesting‐ ly, research of GABA in vertebrates has focused mainly on its role in the context of plant responses to stress, because of its rapid and dramatic production in response to biotic and abiotic stresses. For example, disruption of the unique SSADH gene in *Arabidopsis* results in plants undergoing necrotic cell death caused by the accumulation of reactive oxygen inter‐ mediates (ROIs) when they are exposed to environmental stresses [24]. A recent article re‐ ports that a gradient of GABA concentration is essential for the growth and guidance of pollen tubes and suggests that this amino acid plays a role in intercellular signaling in plants, possibly similar to its role in animals. The main question raised by these recent find‐ ings is whether GABA itself serves as a signaling molecule in plants. If so, this would imply that GABA is capable of mediating developmental changes and cell guidance by interacting

Like other eukaryotes, plants use mitogen-activated protein kinase (MAPK) cascades to reg‐ ulate various cellular processes in response to a broad range of biotic and abiotic stress. These cascades promote the transient activation of MAPKs by a dual phosphorylation of Thr and Tyr within the activation loop of the MAPK. Recent studies indicate that MAPKs are not only regulated through phosphorylation by upstream kinases, but also by direct binding of different protein factors [26]. The constitutive activation of MAPKs was found to result in detrimental effects, underlining the importance of a negative regulation of MAPK signaling. MAPK phosphatases (MKPs) are negative regulators of MAPKs. Recent progress in analyz‐ ing plant MKP mutants has revealed their important role in fine-tuning MAPK signaling. In particular, the dual-specificity phosphatase MKP1 and the protein tyrosine phosphatase (PTP1) negatively regulate defense responses and resistance to a bacterial pathogen by counter balancing the activation of two MAPKs (MPK3 and MPK6). Interestingly, MKP1 and PTP1 bind CaM, and the phosphatase activity of MKP1 is increased by CaM in a Ca2+ dependent manner. Thus, Ca2+ and MAPK signaling pathways appear to be connected

in ABA. It is thus possible that ABA enhances salt tolerance in soybean [21].

(c) *GABA-related protein*

Relationships

402

with specialized plant receptors [25].

(e) *Calcium signaling and related proteins*

(d) *Mitogen-activated protein kinase signaling and related proteins*

through the regulation of plant MAPKs and MKPs by CaM [27].

Soybeans have played a central role in concerns about GM introduced allergens and in us‐ ing GM to remove intrinsic allergens. Soybean is a rich and inexpensive source of proteins for humans and animals. Soybean milk and dairy product replacement is growing in accept‐ ance, not only by people sensitive to lactose and/or milk proteins, but also for health consid‐ erations. Soybean protein is widely used in thousands of processed foods throughout the industrialized world and is a staple crop in Asia. Soybean ranks among the eight most sig‐ nificant food allergens. Soybean sensitivity is estimated to occur in 5-8% of children and

1-2% of adults. The allergic reaction is only rarely life-threatening with the primary adverse reactions to consumption being atopic (skin) reactions and gastric distress. Symptoms of soy allergy usually appear within a few minutes to two hours of eating soy ingredients. People with soy allergies may cross-react with peanuts or other legumes, such as beans or peas. Soy is one of the most common allergens for infants who have not yet begun eating solid foods, because they may be fed soy-based infant formula. It is rare for babies to have a traditional IgE mediated food allergy to soy, but some babies may develop milk-soy protein intolerance [31-34] or food protein induced enterocolitis syndrome [http://foodallergies.about.com/od/ soyallergies/a/Soy-Allergy-Overview.htm]. Infants will usually develop these sensitivities within a few months of birth, and most will outgrow them by the age of two. Most people with soy allergies can tolerate the small amount of soy protein that remains in refined soy‐ bean oil and soy lecithin. Both of these ingredients may cause allergic reactions in highly sensitized people. There are some data available that describe the natural variation in aller‐ gen proteins that occur in soybean. For a better understanding of the variation of allergen proteins that might be expected to occur in GM soybeans, it is important to determine the natural variation of protein composition both in wild and GM soybeans. "Proteomics" ap‐ proach is the foremost one which allows protein identification and quantification with ut‐ most accuracy.

Biotechnology critics have claimed that an apparent rise in the number of soybean allergic individuals in the UK is correlated with the development of GM soybeans in the American market. GM-soybeans that have been developed in the US include herbicide-resistance (glyphosate) and seeds with higher percentage of essential amino acids, *esp*. methionine. Ex‐ periments have directly tested the allergenicity of herbicide-tolerant soybeans using immu‐ nological tests with samples from soybean-sensitive people. These assays have shown that herbicide-resistant GM soybeans do not present any measurable differences in allergenicity compared with non-GM soybeans and are, therefore, substantially equivalent by allergenic criteria. Sensitive people remain allergic to GM soybeans, but there is no additional allergen‐ ic risk to others. According to some reports protein expressed corresponding to transgene responsible for herbicide-resistance in soybeans has allergenic motifs [35]. On ingestion a portion of the transgene along with the promoter get transferred to human gut bacteria. The transformed bacteria containing transgene continues to produce herbicide-resistance aller‐ genic protein even when the individual is not eating GM soy. Therefore an individual is constantly exposed to potentially allergenic protein, being created within his gut. Further, herbicide-resistant protein is made more allergenic due to its misfolding brought by rear‐ rangement of unstable transgenes. Some reports emphasize the fact that protein allergenicity is due to suppression of pancreatic-enzymes due to which protein remains in the gut for lon‐ ger duration contributing to allergies. There is insufficient data to support *in vivo* toxicity of herbicide-resistant protein either due to transformation or enzyme suppression [36]. GMsoybeans with enhanced methionine content such as prolamines and 2S albumins were test‐ ed for its allergenicity before its commercialization. It was found that allergenicity was much higher with respect to wild soybeans [37]. Consequently the development of GM soy‐ bean with enhanced methionine has been abandoned and no product was released, thus no‐ body was harmed by its adverse reactions. Recently, one of the interesting analyses has been done on GM-soy irrespective of herbicide resistance or enhanced methionine content. It has found that GM transformation process may lead to increment in natural allergens in soy‐ beans. The level of one known allergen is trypsin inhibitor which is 27% higher in raw GM soy varieties with respect to natural varieties [38]. Further, it has also been found that cooked GM soy has sevenfold higher amount of trypsin inhibitor as compared to cooked non-GM soy due to its extreme heat stability. There are several reports including both sup‐ portive as well unsupportive towards effects of GM-soy on humankind as well as on other flora and fauna of the environment. It will require intensive research including proteomics before their release into the commercial markets.

1-2% of adults. The allergic reaction is only rarely life-threatening with the primary adverse reactions to consumption being atopic (skin) reactions and gastric distress. Symptoms of soy allergy usually appear within a few minutes to two hours of eating soy ingredients. People with soy allergies may cross-react with peanuts or other legumes, such as beans or peas. Soy is one of the most common allergens for infants who have not yet begun eating solid foods, because they may be fed soy-based infant formula. It is rare for babies to have a traditional IgE mediated food allergy to soy, but some babies may develop milk-soy protein intolerance [31-34] or food protein induced enterocolitis syndrome [http://foodallergies.about.com/od/ soyallergies/a/Soy-Allergy-Overview.htm]. Infants will usually develop these sensitivities within a few months of birth, and most will outgrow them by the age of two. Most people with soy allergies can tolerate the small amount of soy protein that remains in refined soy‐ bean oil and soy lecithin. Both of these ingredients may cause allergic reactions in highly sensitized people. There are some data available that describe the natural variation in aller‐ gen proteins that occur in soybean. For a better understanding of the variation of allergen proteins that might be expected to occur in GM soybeans, it is important to determine the natural variation of protein composition both in wild and GM soybeans. "Proteomics" ap‐ proach is the foremost one which allows protein identification and quantification with ut‐

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

Biotechnology critics have claimed that an apparent rise in the number of soybean allergic individuals in the UK is correlated with the development of GM soybeans in the American market. GM-soybeans that have been developed in the US include herbicide-resistance (glyphosate) and seeds with higher percentage of essential amino acids, *esp*. methionine. Ex‐ periments have directly tested the allergenicity of herbicide-tolerant soybeans using immu‐ nological tests with samples from soybean-sensitive people. These assays have shown that herbicide-resistant GM soybeans do not present any measurable differences in allergenicity compared with non-GM soybeans and are, therefore, substantially equivalent by allergenic criteria. Sensitive people remain allergic to GM soybeans, but there is no additional allergen‐ ic risk to others. According to some reports protein expressed corresponding to transgene responsible for herbicide-resistance in soybeans has allergenic motifs [35]. On ingestion a portion of the transgene along with the promoter get transferred to human gut bacteria. The transformed bacteria containing transgene continues to produce herbicide-resistance aller‐ genic protein even when the individual is not eating GM soy. Therefore an individual is constantly exposed to potentially allergenic protein, being created within his gut. Further, herbicide-resistant protein is made more allergenic due to its misfolding brought by rear‐ rangement of unstable transgenes. Some reports emphasize the fact that protein allergenicity is due to suppression of pancreatic-enzymes due to which protein remains in the gut for lon‐ ger duration contributing to allergies. There is insufficient data to support *in vivo* toxicity of herbicide-resistant protein either due to transformation or enzyme suppression [36]. GMsoybeans with enhanced methionine content such as prolamines and 2S albumins were test‐ ed for its allergenicity before its commercialization. It was found that allergenicity was much higher with respect to wild soybeans [37]. Consequently the development of GM soy‐ bean with enhanced methionine has been abandoned and no product was released, thus no‐ body was harmed by its adverse reactions. Recently, one of the interesting analyses has been

most accuracy.

Relationships

404

Plant biotechnology has not only tried to produce GM-soy which is herbicide resistance or with enhanced methionine content but also aimed to remove naturally occurring allergens in native soy varieties. Presently primary treatment for food allergies is avoidance, but it is unavoidable in case of soybean protein which is present in thousands of products. There‐ fore, it is very difficult to avoid soybean and its derived products. Research is going on to produce hypoallergenic variants of soybean which has potential to reduce the risk of ad‐ verse reactions. Soybeans possess as many as 15 proteins recognized by IgEs from soybeansensitive people [39]. The immunodominant soybean allergens are the *β*-subunit of conglycinin and P34 or Gly m Bd 30k (cysteine proteases from papain family). The P34/Gly m Bd 30k protein is a unique member of the papain superfamily lacking the catalytic cys‐ teine residue that is replaced by a glycine which is 70% more allergenic with respect to con‐ glycinin. There are several approaches that have been taken to produce a hypoallergenic soybean. One approach was to search cultivars which lack allergens and then crossing its germplasm to elite germplasm. This approach could not be implemented as there was no soybean cultivar (either domesticated or wild) present which lack P34/Gly m Bd 30k. Immu‐ nological assays of P34/Gly m Bd 30k with antibodies from soybean-sensitive people result‐ ed in the identification of 14 contiguous and non-contiguous linear epitopes. The presence of so many distinct linear epitopes means that the probability of a naturally occurring variant with a sufficient number of alterations to disrupt the allergenicity is extremely small. Protein engineering could be performed to alter amino acid sequence by disrupting allergenic se‐ quences. Using linear peptides to test possible modifications, it is straightforward to assay numerous variants and pick one that is not recognized by the IgE population. The epitope modification approach is not feasible to produce an essentially hypoallergenic variant. The problem with this technology is to remove completely the intrinsic allergen and substitute the `hypoallergenic' variant in its place. Further, the modification of the protein to remove the allergenic epitopes may alter the protein's folding, that, in turn, may affect the protein's intracellular targeting, stability and accumulation. All these possibilities will need to be test‐ ed for experimentally and, finally; the newly produced hypoallergenic variant will need to be tested to ensure that it too is not a new allergen. For these reasons, substituting a hypoal‐ lergenic variant of a plant still has a high technological threshold and has yet to be achieved. The alternative GM approach is to eliminate the allergen by suppression. There have been several attempts to reduce and/or eliminate allergens using gene suppression technology. Gene-silencing techniques involve transgenic soybeans with eliminated immunodominant human allergen P34/Gly m Bd 30k. It involves complete elimination of the P34/Gly m Bd 30k

allergen from the initial somatic embryos through the third generation homozygous soy‐ beans. Suppression of the allergen did not introduce any changes in the pattern of growth and development of the plant or seed at both the gross and subcellular level. In order to compare the P34-suppressed soybeans with the wild type, large-scale proteomic analysis was performed. Imaging of the 2D gels identified over 1400 individual elements. Mass spec‐ trometry analysis of about 140 of these spots confirmed that the only overt changes in com‐ position in the transgenic soybeans was the suppression of the P34/Gly m Bd 30k protein with no other proteins induced or suppressed [40]. Further analysis with sera samples from soybean-sensitive people confirmed a loss of the P34 allergen and no induction of any new allergens. The proteome and immunological analysis together confirms that it is feasible to suppress an endogenous allergen without introducing adverse effects on the plant or chang‐ ing the composition of the soybean seed in any way other than the removal of the targeted protein. This result meets the test of `substantial equivalence' where the GM soybean seed is essentially identical except for the change in the single desired characteristic. Suppressing P34/Gly m Bd 30k in GM soybeans is a first step and a demonstration in addressing the growing concerns about food allergies and its relationship to the development of GM crops. More detailed studies and approaches should provide the tests needed to gain regulatory approval in nations that are currently cautious about this technology. Natarajan *et al*. [41] have compared the profiles of allergen and anti-nutritional proteins both in wild and GM soybean seeds. 2D-PAGE was used for the separation of proteins at two different pH ranges and applied a combined MALDI-TOF-MS and LC-MS analysis for the identification of pro‐ teins. Although overall distribution patterns of the allergen and anti-nutritional proteins Gly m Bd 60K (conglycinin), Gly m Bd 30K, Gly m Bd 28K, trypsin inhibitors, and lectin ap‐ peared similar, there was remarkable variation in the number and intensity of the protein spots between wild and GM soybean. The wild soybean showed fifteen polypeptides of Gly m Bd 60K and three polypeptides of trypsin inhibitors. GM soybean showed twelve poly‐ peptides of Gly m Bd 60K and two polypeptides of trypsin inhibitors. In contrast, the GM soybean showed two polypeptides of Gly m Bd 30K and three polypeptides of lectin and the wild type showed two and one polypeptides of Gly m Bd 30K and lectin, respectively. The same number of Gly m Bd 28K spots was observed in both wild and GM soybean [41].

The fear of allergic reactions has produced much of the concern about the risks of GM crops. In order to broadly apply genetic modification to crops, there is an urgent need for better biochemical and molecular methods, including animal models, to test for food allergens ex‐ perimentally so that the supporting data can be provided to evaluate newly proposed and actual GM products. In order to design transgenes, it would be useful to predict allergenici‐ ty but, currently, there are no models that would permit accurate assessment of allergenic potential of proteins unrelated to known allergens. Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Pre‐ vious studies on hepatocytes from young female mice fed on GM soybean demonstrated nu‐ clear modifications involving transcription and splicing pathways [42, 43]. The morphofunctional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to hepatocyte metabolism, stress response, calcium signaling and mitochondria were differen‐ tially expressed in GM-fed mice, indicating a more marked expression of senescence mark‐ ers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. This study demonstrates that GM soybean intake can influence some liver features, although the mechanisms remain unknown. Therefore, it is required to investigate the long-term consequences of GM-diets, further studies are required for potential synergistic effects with other factors like ageing, stress etc.
