**2. Plant stress**

In this section we have decided to focus our attention on the antioxidant responses trigged by some key biotic and abiotic stresses that have more significant information available, based on recent publications.

When oxidative stress is taken into account, it is interesting to mention firstly, the role of molecular oxygen (O2) in our environment. Due to the presence of oxygen and its reactions, both positive and negative aspects inherent to the process can occur, which is called oxida‐ tive stress.

synthesis of aromatic amino acids. Its inhibition provokes delay in the development of plants, amino acids unbalance, and death of plants [5]. As a consequence it is easy to ration‐ alize that plants genetically modified can normally develop in the presence of this herbicide, being the excellent result obtained in terms of soybean crop production also attributed to the transgenic cultures used. Exemplifying, *ca*. 49 million hectares (60% of the cultivated world

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

In this way, our hypothesis is that the genetic modification itself is contributing for changing a variety of characteristics of this organism, producing alterations, in a cascade manner, to the metabolism. As a result of these modifications, the genetically modified soybean is appa‐

In order to evaluate this hypothesis, our research group has been, for the last eight years, carry‐ ing out some comparative studies taking into account alterations in proteins, metalloproteins, metals and enzymes. Since the content of proteins in soybean seeds is high (*ca*. 40%) [7], hun‐ dreds of proteins are expected to be found after a separation process, making our proposal a hard task. Then, the utilization of the most up-to-date analytical techniques presenting high resolu‐

In this chapter, the concept of plant stress is mainly one related to oxidative stress, followed by a variety of examples regarding soybean. Additionally, basic concepts of proteomics and metallomics will be described, followed by a compilation of the results from all strategies and techniques that we have been adopting along the period devoted to the study of trans‐ genic soybean, which were utilized for corroborating our previous hypothesis. Other exam‐ ples in the literature are also presented in order to support our data. Then, techniques based on bidimensional chromatographic and non-chromatographic protein separations (*i.e.* 2D-HPLC, 2D PAGE), image analysis for protein expression evaluations (*i.e.* 2D DIGE), inorgan‐ ic mass spectrometry for identification/quantification of metals (*i.e.* HR-SF-ICP-MS, ICP-MS, LA-ICP-MS), organic mass spectrometry for characterization of proteins (*i.e.* MALDI-QTOF-MS, ESI-LC-MS-MS), and hyphenated techniques for improving the quality on protein infor‐ mation (*i.e.* 2D-HPLC-ICP-MS) will be also emphasized. In the end of this chapter, a section of future Trends is provided, putting in evidence, in our point of view, some other strategies

Finally, it is important to stress that the main goal of this chapter, and also of our studies, is only to present those results found within a series of projects developed by our research group concerning transgenic soybean. Despite the awareness of a public disagreement about the cultivation and commercialization of transgenic soybean, this chapter does not have the

In this section we have decided to focus our attention on the antioxidant responses trigged by some key biotic and abiotic stresses that have more significant information available,

intention neither to defend genetic modification nor to make any criticisms to it.

tion in terms of enzymes, proteomics and metallomics approaches is almost imperative.

area) are occupied nowadays by transgenic soybean culture [6].

rently searching a new equilibrium as a living organism in nature.

to be adopted for an in-depth investigation of this transgenic crop.

**2. Plant stress**

Relationships

584

based on recent publications.

Among all planets in our solar system, Earth is the only one that contains O2, and the only one able to support aerobic life as the way that we understand its meaning. According to [8], the concentration of 21% (v/v) O2 on Earth's atmosphere is derived from the photosynthetic activities of cyanobacterias and plants. The reference [9] commented that by an estimate, the total amount of O2 in the Earth is about 410 x 103 Erda moles and from this value, 38.4 x 103 Erda moles is in the water form. When the aerobic life is concerned, these authors comment‐ ed that this specific style of life is responsible for the major portion of O2 turnover: photo‐ synthesis is the main input of O2, and respiration the main output.

Oxygen is relatively non reactive, but in some situations (as normal metabolic activity or when under environmental disturbance), it is able to switch to an excited state, producing free radicals and similar forms [9-10]. Then, it is clear in this scenario that adaptation proc‐ esses to environmental changes are crucial for plant growth and survival. In view of its im‐ portance, it is interesting to remember the processes which lead to the reduction of molecular O2. According to [11] such processes occur following four steps and generate sev‐ eral O2 species. The first one requires an extra energy but the subsequent steps are exother‐ mic, occurring spontaneously. The reaction products (H2O2; O2 •; HO2 - ; OH- ) can act in different ways in the cellular environment.

Hydrogen peroxide is a relatively long-lived molecule and can diffuse from its site of pro‐ duction [12]. Beside this, its toxicity has long been known. The O2 • radical half-life is short (2-4 µs), but it is highly reactive and can form hydroperoxides and can oxidize histidine, me‐ thionine and tryptophan. When this radical is in the cellular environment, it causes lipid peroxidation as a consequence of oxidative deterioration of membrane polyunsaturated lip‐ ids. So, the hydrogen peroxide is not only toxic to cells, but in an extracellular medium it may react with transition metals, such as iron and copper, generating hydroxyls, which can cause cell damage. Beside this, when the levels of lipid peroxidation are higher (normally lipid peroxidation values are estimated by the concentration of malondialdehyde in sam‐ ples) it suggests indirectly the establishment of a condition of oxidative stress. The hydroxyl radical (OH- ) has a very strong potential and half-life of less than 1µs, and as a consequence, it has very high affinity for biological molecules [13]. What is particularly interesting about these species is that all of them can be generated by molecular oxygen reduction and they may play roles as toxic molecules or they can be excellent candidates for events/studies in‐ volving plant cell signaling [8, 13-16].

In these terms, the production of reactive oxygen species (ROS) is generally described as harmful due to their potential to cause irreversible damage to photosynthetic components in plants. However, despite this potential in causing harmful oxidation, modulation of ROSantioxidant interaction plays a role in many stresses, as well as other responses to the envi‐ ronment. Additionally, this system can be considered as a powerful signaling process to molecules involved in the control of plant growth and development as well as priming accli‐

matory responses to stress stimuli [17-18]. In these terms, oxidative stress can be described as a central factor in abiotic and biotic stress that occurs due to imbalances in any cell com‐ partment between the production of ROS and antioxidant defense [16, 19].

As indicated in [18] it is possible to verify that the pathways of ROS signaling are made by homeostatic regulation which can be achieved by the antioxidant redox buffering, making possible the determination of lifetime and the specificity of the ROS signal. It is interesting to emphasize that plants which demonstrate low activities for catalase (CAT) and cytosolic ascorbate peroxidase (APX), two key enzymes involved in the breakdown of H2O2, show less severe stress symptoms when compared to the ones where one of these enzymes is missing [20].

Talking about antioxidant defense systems, it can be attested that, in plants, the first line of defense against oxidative stress is the avoidance of ROS production [17] and once formed, ROS must be detoxified in order to either avoid or minimize eventual damages. In this way, the detoxification mechanisms can be considered as a second line of defense against the det‐ rimental effects of ROS [21]. Beside this, some antioxidant enzymes can be considered as a second defense line against oxidative stress, since they act either as a catalyzer in ROS reac‐ tion or are involved in directing ROS processing [22]. The repair of oxidatively damaged proteins can be considered as the third line of defense against ROS [23].

According to [24] ROS species are commonly generated under stress conditions and due to its strong oxidative capacity, it acts on all types of biomolecules. In terms of the interactive effects of these species, it is possible to say that it can react with each other and with other molecules. For example, O2 •- may react with lipids peroxides or nitric oxide, leading to the formation of peroxynitrite, which is less reactive than peroxides. In the same context, [13] pointed out that plants may favor the formation of one or other reactive species by preferen‐ tially scavenging peroxide (H2O2) with antioxidants or, in contrast, accumulating peroxide by the activation of superoxide dismutase (SOD).

The oxidative response in plants can be exacerbated by stressful conditions [16]. At the mo‐ lecular level, the extent and nature of this response can differ among species and even among those closely related varieties of the same species. For example, 24 differentially ex‐ pressed genes in soybean leaves were observed after glyphosate treatment when comparing tolerant and non tolerant soybean lines [25]. Therefore, oxidative responses are not only linked to the genetic expression. The reference [26] shows that some biochemical parameters (such as total soluble amino acid content and CAT activity in soybean roots) were also al‐ tered as a response to differential glyphosate application. The increase in the enzyme activi‐ ties indicates ROS generation and a subsequent antioxidant response. Alterations in the antioxidative system of suspension-cultured soybean cells were observed [27], which were induced by oxidative stress using a peroxidizing herbicide (oxyfluorfen). Ascorbate and glu‐ tathione (non-enzymatic cellular antioxidants) showed different responses and the activities of some enzymes involved in cellular defense were also altered. For instance, peroxidase and catalase increased by 40 – 70% while glutathione S-transferase (GST) exhibited a 6-fold increase under oxyfluorfen stress.

Stress-induced ROS accumulation is counteracted either by enzymatic oxidant systems that include a variety of scavengers, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), and catalase (CAT), or by non enzymatic low molecular weight metabolites, such as carotenoids and flavonoids [16, 28-29]. As an example related to the influence of the enzymatic machinery under a stress situation, the reference [30] pointed out that as a response to stress, plants may increase the activities of some enzymes such as glutathione S-transferase (GST), involved in the detoxifi‐ cation of xenobiotics. These authors also investigated in detail the mechanisms of interaction between the GST enzyme and its substrates, indicating that the information might help in the engineering of new GSTs with improved detoxification efficiency [30].

matory responses to stress stimuli [17-18]. In these terms, oxidative stress can be described as a central factor in abiotic and biotic stress that occurs due to imbalances in any cell com‐

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

As indicated in [18] it is possible to verify that the pathways of ROS signaling are made by homeostatic regulation which can be achieved by the antioxidant redox buffering, making possible the determination of lifetime and the specificity of the ROS signal. It is interesting to emphasize that plants which demonstrate low activities for catalase (CAT) and cytosolic ascorbate peroxidase (APX), two key enzymes involved in the breakdown of H2O2, show less severe stress symptoms when compared to the ones where one of these enzymes is

Talking about antioxidant defense systems, it can be attested that, in plants, the first line of defense against oxidative stress is the avoidance of ROS production [17] and once formed, ROS must be detoxified in order to either avoid or minimize eventual damages. In this way, the detoxification mechanisms can be considered as a second line of defense against the det‐ rimental effects of ROS [21]. Beside this, some antioxidant enzymes can be considered as a second defense line against oxidative stress, since they act either as a catalyzer in ROS reac‐ tion or are involved in directing ROS processing [22]. The repair of oxidatively damaged

According to [24] ROS species are commonly generated under stress conditions and due to its strong oxidative capacity, it acts on all types of biomolecules. In terms of the interactive effects of these species, it is possible to say that it can react with each other and with other

formation of peroxynitrite, which is less reactive than peroxides. In the same context, [13] pointed out that plants may favor the formation of one or other reactive species by preferen‐ tially scavenging peroxide (H2O2) with antioxidants or, in contrast, accumulating peroxide

The oxidative response in plants can be exacerbated by stressful conditions [16]. At the mo‐ lecular level, the extent and nature of this response can differ among species and even among those closely related varieties of the same species. For example, 24 differentially ex‐ pressed genes in soybean leaves were observed after glyphosate treatment when comparing tolerant and non tolerant soybean lines [25]. Therefore, oxidative responses are not only linked to the genetic expression. The reference [26] shows that some biochemical parameters (such as total soluble amino acid content and CAT activity in soybean roots) were also al‐ tered as a response to differential glyphosate application. The increase in the enzyme activi‐ ties indicates ROS generation and a subsequent antioxidant response. Alterations in the antioxidative system of suspension-cultured soybean cells were observed [27], which were induced by oxidative stress using a peroxidizing herbicide (oxyfluorfen). Ascorbate and glu‐ tathione (non-enzymatic cellular antioxidants) showed different responses and the activities of some enzymes involved in cellular defense were also altered. For instance, peroxidase and catalase increased by 40 – 70% while glutathione S-transferase (GST) exhibited a 6-fold

•- may react with lipids peroxides or nitric oxide, leading to the

partment between the production of ROS and antioxidant defense [16, 19].

proteins can be considered as the third line of defense against ROS [23].

missing [20].

Relationships

586

molecules. For example, O2

increase under oxyfluorfen stress.

by the activation of superoxide dismutase (SOD).

In the context so far referred, and particularly with soybean, which is the main focus of this chapter, molecular and biochemical studies have explored several aspects related to the ma‐ nipulation of metabolic pathways towards adaptation responses which can help to mitigate oxidative stress.

If ROS scavenging pathways in plants are the main focus, the involvement of at least 3 cy‐ cles have to be considered: a) the water-water cycle in chloroplasts, including SOD; b) the ascorbate-glutathione cycle in chloroplasts, cytosol, mitochondria, appoplast and peroxi‐ somes; and c) GPX and CAT in the peroxisomes [31]. The equilibrium between the produc‐ tion and the scavenging of ROS may be altered by biotic and abiotic stress factors such as UV radiation, temperature, air pollution, pathogen attack, heavy metals, nutrient deficiency, and herbicides, among others [32].

The clear understanding of the mechanisms by which some endogenous or exogenous agents can lead to plant toxicity and how plants answer to this specific situation, is essential. Besides this, understanding how toxicity occurs, what kind of alterations occur in plant structure and metabolism among other situations are important steps for genetic breeding programs, when searching for new varieties susceptible or tolerant to stress factors and even for bioremediation/phytoremediation programs [32].

Although there has been a rapid progress in recent years in the field of plant stress studies, there is a consensus among researches that there are still many uncertainties in understand‐ ing how effectively ROS affects the stress response of plants [32-33].

A short list of examples that will emphasize the detoxification mechanism involved in plant stress defense and a diversity of enzymes that can be involved in the dismutation of ROS is then presented. For example, soybean has been shown to be highly sensitive to ozone (O3) and the oxidation of some proteins may cause alterations in the activities of enzymes across nitrogen and sulfur nutrient assimilation pathways linked to stress responses [34]. The chronic exposure to high O3 may lead to increased expression or oxidation of proteins, in‐ cluding APX, GSTs [34] and decrease the activities of monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) [35], indicating a fundamental role of these en‐ zymes in stress response when soybean is subjected to O3. Furthermore, soybean submitted to chronic high O3 concentration and then exposed to an acute O3 stress provided evidence that there was an immediate transcriptional reprogramming that allowed for maintained or

increased ascorbate (AA) content in plants grown at high O3 [36]. In another study using two tropical soybean varieties (PK 472 and Bragg) exhibiting differential sensitivity to O3, reference [37] showed that the CAT activity decreased whereas peroxidase increased in both varieties upon exposure to O3, but reflecting the greatest sensitivity of PK 472 in relation to the high magnitude of the reductions in the levels of antioxidants, metabolites and nutrients. Besides this, the damage O3 effects produced were found to be more prominent during the reproductive than the vegetative growth stage.

Although soybean plants have shown a positive and significant correlation between activity of antioxidant enzymes and the osmolyte proline (Pro) content to water deficit stress [38], the metabolic reasons associated with the differential sensitivity of soybean cultivars to wa‐ ter deficit stress are not well understood [39]. According to the authors, water deficit stress increased antioxidant enzyme activities of SOD, CAT and GPX more at mild than at high water deficit stress [39]. In addition, soybean plants have shown protective mechanisms as‐ sociated to proline concentration and GR, APX, and CAT activities under salt stress [40].

The increase in soybean productivity has been also accounted to the development and wide‐ spread use of improved cultivars with increased resistance to stressful conditions. A promis‐ ing technique for agricultural improvement in arid and semiarid areas is the use of a pretreatment of soybean dry seeds with a low dose of gamma rays (20 Gy) before planting, enhancing drought tolerance and minimizing the yield losses caused by a water deficit con‐ dition [41].Overall, application of a low dose of gamma irradiation (20 Gy) increased the ac‐ tivities of phosphoenol pyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase/ oxygenase (RUBISCO) under drought stress, avoiding the destructive effects of water defi‐ cits on chloroplasts [41]. Furthermore, the manipulation of Pro can affect the (h)GSH, amino acids concentrations and APX activity, contributing to the detoxification of ROS in soybean subjected to simultaneous drought and heat stresses [42].

The regulation of thiol metabolism has become important for optimizing crop yield and quality of soybean [43]. The sulfur assimilatory pathway in soybean metabolism can be me‐ tabolized into molecules that protect plants against oxidative stress. The genetic manipula‐ tion of the cytosolic isoform of O-acetylserine sulfhydrylase (OASS), an enzyme involved in the sulfur assimilatory pathway, resulted in high levels of thiols and increased tolerance of plants to metal toxicity [44].

It is also important that information concerning changes in antioxidant capacity in immature seeds harvested at different reproductive stages [45], exhibited decreases in free radical scavenging activity and total antioxidant capacity with the advancement of maturity. This occurred concomitant with increased concentration of tocopherol and isoflavone isomers. Therefore, it is important to take into consideration that not only organs or tissues may present distinct responses to stress, but the plant stage of development is also important.

Reference [46] reported that different metals may act and induce different levels of copperzinc superoxide dismutase (Cu-Zn/SOD) expression in soybean plants exposed to Cd and Pb. Also, Cd caused the induction of Cu-Zn/SOD mRNA accumulation for all Cd concentra‐ tions and Pb-treated roots showed induction of these isoenzymes only at medium metal concentrations.

increased ascorbate (AA) content in plants grown at high O3 [36]. In another study using two tropical soybean varieties (PK 472 and Bragg) exhibiting differential sensitivity to O3, reference [37] showed that the CAT activity decreased whereas peroxidase increased in both varieties upon exposure to O3, but reflecting the greatest sensitivity of PK 472 in relation to the high magnitude of the reductions in the levels of antioxidants, metabolites and nutrients. Besides this, the damage O3 effects produced were found to be more prominent during the

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

Although soybean plants have shown a positive and significant correlation between activity of antioxidant enzymes and the osmolyte proline (Pro) content to water deficit stress [38], the metabolic reasons associated with the differential sensitivity of soybean cultivars to wa‐ ter deficit stress are not well understood [39]. According to the authors, water deficit stress increased antioxidant enzyme activities of SOD, CAT and GPX more at mild than at high water deficit stress [39]. In addition, soybean plants have shown protective mechanisms as‐ sociated to proline concentration and GR, APX, and CAT activities under salt stress [40].

The increase in soybean productivity has been also accounted to the development and wide‐ spread use of improved cultivars with increased resistance to stressful conditions. A promis‐ ing technique for agricultural improvement in arid and semiarid areas is the use of a pretreatment of soybean dry seeds with a low dose of gamma rays (20 Gy) before planting, enhancing drought tolerance and minimizing the yield losses caused by a water deficit con‐ dition [41].Overall, application of a low dose of gamma irradiation (20 Gy) increased the ac‐ tivities of phosphoenol pyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase/ oxygenase (RUBISCO) under drought stress, avoiding the destructive effects of water defi‐ cits on chloroplasts [41]. Furthermore, the manipulation of Pro can affect the (h)GSH, amino acids concentrations and APX activity, contributing to the detoxification of ROS in soybean

The regulation of thiol metabolism has become important for optimizing crop yield and quality of soybean [43]. The sulfur assimilatory pathway in soybean metabolism can be me‐ tabolized into molecules that protect plants against oxidative stress. The genetic manipula‐ tion of the cytosolic isoform of O-acetylserine sulfhydrylase (OASS), an enzyme involved in the sulfur assimilatory pathway, resulted in high levels of thiols and increased tolerance of

It is also important that information concerning changes in antioxidant capacity in immature seeds harvested at different reproductive stages [45], exhibited decreases in free radical scavenging activity and total antioxidant capacity with the advancement of maturity. This occurred concomitant with increased concentration of tocopherol and isoflavone isomers. Therefore, it is important to take into consideration that not only organs or tissues may present distinct responses to stress, but the plant stage of development is also important.

Reference [46] reported that different metals may act and induce different levels of copperzinc superoxide dismutase (Cu-Zn/SOD) expression in soybean plants exposed to Cd and Pb. Also, Cd caused the induction of Cu-Zn/SOD mRNA accumulation for all Cd concentra‐

reproductive than the vegetative growth stage.

Relationships

588

subjected to simultaneous drought and heat stresses [42].

plants to metal toxicity [44].

It is also important to bring into the scene of stressful condition for plants, the soil type used. In a recent work soybean exposed to Cd and Ba [47] showed that the activity of antioxidant enzymes can change depending on the soil type, time-length of exposure and metal concen‐ tration [47]. For instance, GR and SOD activities in the leaves of soybean plants grown in an Oxisol soil contaminated with Cd decreased over time, whilst remaining high in an Entisol soil. The changes of enzyme activities were mainly dependent on buffering capacity of the soils with the Entisol exhibiting a lower capacity, with the plants suffering higher oxidative stress than those plants grown in a clay soil such as presented by an Oxisol soil [47].

Moreover, it is also important to investigate the effect of stressful conditions in soybean pro‐ ductivity taking into consideration more than one environmental contaminant or stress fac‐ tor in the same agricultural region. In this context, the combination of Cd and acid rain pollution damaged the cell membrane, decreased the activities of POX and CAT, showing a higher potential threat to soybean seed germination than the single separate effect of each contaminant [48]. In another study, a correlation between the rate of ROS generation and an‐ tioxidant enzyme activities was established under hypoxia and high CO2 concentration [49]. The CAT activity in soybean plants increased during the first hours of hypoxia whereas per‐ oxidase activity started to play a more key role in cell defense only after a longer exposure to hypoxia. In this study, the processes of ROS accumulation and antioxidant enzymes were induced by the higher CO2 content, indicating that CO2 can switch on plant adaptation to hypoxic stress [49].

Another interesting study involving the combination of distinct stressor agents was carried out by combining Al and Cd with both leading to synergistic effects on plant growth and antioxidant responses in two soybean cultivars with different Al tolerance levels [50]. Ac‐ cording to the authors, the Al treatments and low pH value (4.0) caused reduction in chloro‐ phyll content and net photosynthetic rate, leading to growth reduction. The increased SOD and peroxidase activities were detected in the plants submitted to both metals, especially in the Al-sensitive cv. Zhechun 2, which also exhibited significantly higher Al and Cd contents than the Al tolerant cv. Liao-1. Moreover, Cd supplementation increased Al content in the plants exposed to Al+Cd stress [50]. Such an observation confirms another key aspect that should receive attention which is how the elements of the soil interact and can define an up‐ take profile by the plant root system possibly resulting in an induction of stress condition. Such studies are also of the upmost importance when considering the use of phytoremedia‐ tion as a technique to recover a contaminated soil.

Similarly, studies about the interactions between plant roots and beneficial metal-tolerant microorganisms are gaining importance and may be an important approach to be consid‐ ered in studies about plant adaptation and alleviation to a variety of environmental stresses [51]. For example, soybean plants inoculated with arbuscular mycorrhizal (AM) fungi showed reduced MDA content and increased APX activity to the oxidative stress generated by paraquat (PQ) [52]. In another study, activities of SOD and peroxidase were increased in the shoots of soybean plants with mycorrhizal (M) fungi grown under

NaCl salinity [53]. Once again, a more integrated view is needed and deserves attention. These two studies commented above indicate the importance of mycorrhizal fungi regu‐ lation as a general strategy to protect plants from stress. If soil type is added to this equation, a much more complex situation is created and such an integrated study re‐ flects the reality of many agronomic situations. This also raises the question over the use of hydroponic systems to study oxidative stress in plants, particularly when induced by non-essential elements, since it is not necessarily the real field condition. Yet, is not our intention to say that such studies under hydroponic conditions are not important. On the contrary, they also have advantages. However, a more dynamic or integrated type of study should be considered in our point of view.

Curiously, grafting, which is a well-known agronomic technique largely used in agriculture, has not been used much in studies of stress in plants. The grafting technique has a tremen‐ dous potential to add further important understanding about stress signaling, assimilation, transport and accumulation of metals, opening a new perspective to study these grafted plants at the biochemical and molecular levels. Unfortunately, very few examples are availa‐ ble in the literature focusing on the investigation of plant stress responses. An example of such a study is the one carried out by [54] who showed that Cd seed concentration can be influenced by the difference in translocation of Cd from soil to the seed and in Cd accumula‐ tion capacity of roots among soybean cultivars by the use of grafting.

Nowadays, the development of plant manipulation techniques, for example the production and use of transgenic plants, has contributed to studies involving plant antioxidant respons‐ es induced either by exogenous or endogenous factors (such as herbicide, metals, pollution). Studies involving the mechanisms leading to stress-tolerant plants are important and need‐ ed, since they can aid understanding and create new possibilities for the use of these kinds of plants. The knowledge provided by the "omics studies" such as proteomics, metabolo‐ mics, metallomics and genomics, added to enzymatic evaluation, can provide information that can decisively help in answering many questions related to oxidative stress and ROS control [32].

Taking into account the importance of "omics" platforms, as well as their use for corroborat‐ ing our initial hypothesis, the following sections will focus on these important strategies. They will be divided into proteomics and metallomics, with brief descriptions of each one, as well as some discussions and examples regarding transgenic cultures, but always concen‐ trating the focus on soybean.
