Classification of the antioxidant defense system as the ground for ...

ISSN 10214437, Russian Journal of Plant Physiology, 2011, Vol. 58, No. 2, pp. 210–217. © Pleiades Publishing, Ltd., 2011. Original Russian Text © E.V. Pradedova, O.D. Isheeva, R.K. Salyaev, 2011, published in Fiziologiya Rastenii, 2011, Vol. 58, No. 2, pp. 177–185.

REVIEWS

Classification of the Antioxidant Defense System as the Ground for Reasonable Organization of Experimental Studies of the Oxidative Stress in Plants E. V. Pradedova, O. D. Isheeva, and R. K. Salyaev Siberian Institute of Plant Physiology and Biochemistry, Siberian Division, Russian Academy of Sciences, ul. Lermontova 132, 664033 Irkutsk, Russia; fax: 7 (3952) 510754; email: [email protected] Received May, 24, 2010

Abstract—A specialized system for antioxidant defense prevents damaging effects of reactive oxygen species (ROS) in the cells of plants and other organisms. The components of this system are numerous and diverse. Repeated attempts were taken to classify these components, which, however, did not result in the creation of unique and commonly accepted classification. In the last few decades, a great body of information concern ing antioxidants (AO) has been accumulated, and it demands classification. Today this demand is especially urgent: such classification of the system of AO defense would generalize and systematize the available knowl edge of AO and the system of AO defense as a whole and also helps in more efficient and purposeful studying the mechanisms of organism defense against unfavorable factors accompanied by ROS generation. This review discusses the purposeful of the reasonable classification of the AO defense system; we present several examples of current classifications and suggest our resolution of this problem. Keywords: plants, antioxidants, the system of antioxidant defense and its classification. DOI: 10.1134/S1021443711020166

INTRODUCTION In recent years, a hypothesis, according to which the cause for the oxidative stress is a disturbance in the balance between ROS generation and elimination rather than the intense ROS generation per se, becomes increasingly popular [1]. It is suggested that the main reason for oxidative stress could be the failure of defense systems. The systems of the antioxidant defense counteract the damaging ROS effects. The main components of these systems are antioxidants (AO). This term was initially applied to compounds inhibiting free radical oxidation (FRO) [2]. Later, the application of the terms “antioxidant system” and “antioxidants” was widened, especially in the biologi cal literature [2, 3]. Thus, currently it is often implied that the AO defense system includes not only the sys tems eliminating ROS and preventing their generation but also the systems of detoxification, e.g., eliminating compounds damaged due to their spontaneous oxida tion by oxygen. Another definition of AO is also met: AO are natural or synthetic substances retarding or preventing oxidation of organic compounds [4]. Among them are substances capable of neutralization Abbreviations: AA—ascorbic acid; AO—antioxidants; FRO— free radical oxidation; GPO—glutathione peroxidase; GSH— glutathione reduced; GST—glutathione Stransferase; MDAR— monodehydroascorbate reductase; SOD—superoxide dismutase.

of free radicals and suppression of FRO and also those protecting biological structures [1–4]. To provide for the most efficient defense, all ele ments of the system, i.e., antioxidants, are located in different subcellular structures [1]. In spite of a sub stantial progress made in the understanding functions of particular or local components of the AO defense, a complex intracellular net of differently compartmen tated AO hampers the understanding the system func tioning as a whole. Classification of the antioxidant system helps much in the development of a general notion about functioning of the intracellular antioxi dant net. On the basis of reasonable classification of the system components, taking into account the spec ificity of their structure, function, localization, and interaction, more purposeful and efficient studying of defense against unfavorable factors accompanied by ROS generation becomes possible. PRINCIPLES OF ANTIOXIDANT DEFENSE SYSTEM CLASSIFICATION The classification system, taking into account AO specific features, needs in definite grounds. The unique principles of classification of the AO defense systems are not yet elaborated and demand a compre hensive examination. Therefore, until now there is no universal classification of these systems but there are

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numerous attempts to classify them after various traits: molecular weight, action mechanism, including cata lytic activity, hydrophobicity, and hydrophily. Below we present diverse published attempts of AO system classifications and principles of their creation. All these classifications have some advantages and drawbacks. Advantages of some of them are their sim ple and convenient structure, whereas drawbacks are the incomplete characterization of all AO functional peculiarities. AO Classification after Their Catalytic Activity At present, most often AO are divided into enzy matic and nonenzymatic after their catalytic activity [1–8]. Enzymatic AO are characterized by a high spec ificity of their action directed against a definite ROS form, by a specificity of their cell and organ localiza tion, and by usage of metals (Cu, Zn, Mn, Fe, and Se) as catalyzers [3]. The typical members of these AO are superoxide dismutase (SOD), catalase, glutathione peroxidase (GPO), glutathione reductase, all enzymes of the ascorbate–glutathione cycle, and transferases [3, 9]. Nonenzymatic defense system is represented by such AO as carotenoids, ascorbic acid (AA), reduced glutathione (GSH), αtocopherol, flavonoids, and others. All above compounds could neutralize ROS nonenzymatically or enzymatically. AO Classification after Their Molecular Weights Chesnokova et al. [10] raised the problem of the absence of unique AO classification and analyzed it. They noted that AO are often divided on the basis of their molecular weights. The first group comprises highmolecularweight compounds: enzymes of AO defense and also proteins binding ions, Fe2+ and Cu2+, which catalyze free radi cal processes (SOD, ceruloplasmin, catalase, glu tathionedependent enzymes, albumins, ferritins, lactoferrin, metallothioneins, etc.). The second group comprises lowmolecularweight AO: amino acids, polyamines, GSH, AA, αtoco pherol, vitamins of the groups A, K, and P [3–11]. Classification of AO after catalytic activity and molecular weight are very close to each other. They differ only in the features underlying them: in the first case, it is the structure, e.g., distinguishing between protein and nonprotein nature; and in the second case, it is the molecular weights of compounds. AO Classification after Their Localization One more type of classification is based on the peculiarities of AO chemical nature, which are divided taking into account their lipophility [12]. In this case, AO are subdivided into water and lipidsoluble com pounds. The group of watersoluble AO includes high molecularweight (SOD, catalase, GPO, albumins) RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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and lowmolecularweight (AA, citric acid, nicotinic acid, cysteine, lipoic and benzoic acids, ceruloplas min, polyphens, flavonoids, transferrins, lactoferrin, urea, and uric acid) [10]. Lipidsoluble AO include phospholipids, tocopherols, vitamin K3, ubiquinone, steroid hormones, carotenoids, etc. [12–14]. Water soluble AO are localized and efficiently function in the cell cytosol, in the water phases of cell structures, and also in apoplastic fluids. Lipidsoluble AO are local ized in biological membranes and protect them from spontaneous FRO [3, 9–15]. This classification allow us to assess where, in which compartments (lipid or water) will be localized and function one or another AO [12]. Therefore, it is believed that hydrophobicity and hydrophily determine AO localization rather than their structural features, i.e., lipid and watersoluble AO are subdivided in dependence on the site of their localization [15]. In dependence on localization in the tissue, AO could be subdivided into three groups. The first group: intracellular AO functioning inside the cells (SOD, catalase, peroxidases, etc.). The second group: AO in the cell membranes (α tocopherol, βcarotene, glutathione transferase, etc.) The third group: extracellular AO present in the extracellular liquids (transferrin, lactoferrin, albumin, extracellular SOD, extracellular GPO, AA, GSH, tocopherols, urate, ceruloplasmin) [16]. AO Classification after the Levels of Defense Protection of biological structures against FRO is known to be provided by various mechanisms, which function on different levels. In this connection, some researchers subdivide AO after the levels of defense. The first level is an elimination of oxygen radicals and hydrogen peroxide and also quenching of singlet oxygen, and this is provided by enzymes and low molecularweight AO. The second level is a prevention of hydroxyl radical (referred to secondary radicals) and other active initi ators of FRO generation. At this level, the main role is played by chelators of transition metals (Fe, Cu) [2]. Chesnokova et al. [10] attracted attention to another point of view: protection of multicellular organisms against ROS occurs at least on five levels. The first one is cell systemic defense due to a decrease in the O2 concentration in tissues; The second level is provided in the process of four electron reduction of the bulk of intracellular O2 with the involvement of cytochrome oxidase and without the release of free radicals; The third level is an enzymatic elimination of pro duced superoxide anionradical and H2O2; The fourth level is free radical scavenging by AO; The fifth level is an enzymatic reduction of hydrop eroxides of polyunsaturated FAs [10]. No. 2

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These authors described the occurrence of another system of defense levels: The first line of defense is enzymes suppressing ini tiation of lipid peroxidation (POL) and preventing oxidative destruction of nonlipid components; The second line of defense are lowmolecular AO; The third line of defense are enzymes metabolizing final POL products (aldehydes, alkenes, alcohol). Among them are aldehyde reductase, cytochrome P450, etc; The fourth line of defense provides for reparative regeneration of injured molecules, in particular, the reduction of disulfide bonds in proteins, regeneration of AO; The fifth line of defense is the system of peroxida tion and free radical processes including cyclic nucle otides, prostaglandins, leukotrienes (cyt. after [10]). Lyu and Efimov [17] distinguish only three levels of defense: The first level of defense is antioxygen one. It func tions due to respiratory enzymes and a special group of compounds depositing excess oxygen. This mecha nism maintains a relatively low oxygen level in the cell. This line of defense could not prevent peroxidation because free radicals required for it are produced in the normal metabolic processes and induced by diverse agents. The second level of defense is antiradical one des tined for free radical scavenging and suppression of such processes as, for example, POL. The third level of defense is antiperoxide one. At this level, produced peroxides are degraded by corre sponding enzymes and inactivated by the interaction with definite compounds. AO Classification after Mechanisms of Their Action In the ground of AO defense separation after the mechanisms of their action or the specificity of their •– targets: ROS (O 2 , H2O2, HO•, 1О2, and others); nonradical initiators of FRO; radicals, intermediates of FRO, etc. Therefore, classifications after the levels and mechanisms of defense coincide often. Thus, AO could be subdivided after the mechanism of their action as follows: (1) enzymatic and nonenzymatic; chela tors and Fe2+ oxidants; and (3) compounds exerting membraneacting effect, socalled structural AO [15]. As was mentioned above, a direct destination of AO in the cells of all organisms is not only ROS elimina tion but also the removal of toxic products produced due to interaction with them. In dependence on spe cialization, diverse AO fulfill their functions differ ently; therefore, they are subdivided after the mecha nism of their action as follows: (1) “Scavengers” purifying the organism from all free radicals, most frequently by reduction them to stable inactive products;

(2) “Traps”, i.e., AO manifesting affinity for a def inite free radical (singlet oxygen, hydroxyl radical, etc.); (3) AO breaking the chains. The molecules of these compounds are more reactive than their radicals. They are often phenols, which give easily their electrons to radicals, converting them into molecular products and converting themselves into weak phenol radicals inca pable of the involvement in the chain reactions [18]. AO could be subdivided after the mechanism of action and molecular weight: (1) Enzymes eliminating ROS (SOD, catalase, per oxidase, FPO); (2) Enzymes of lipid detoxification (glutathione S transferase (GST), phospholipid hydroperoxide GPO, and ascorbate peroxidase). (3) Lowmolecularweight AO (AA, GSH, phe nolic compounds, tocopherols). (4) Compounds regenerating AO active forms (monodehydroascorbatereductase (MDAR), dehy droascorbate reductase, glutathione reductase, etc.) [19]. The mechanism of compound antioxidant action is determined primarily by its chemical nature. There fore, it is believed that AO subdivision after the pres ence of some functional groups related to their antiox idant activity in their molecules is most convenient. In this case, the relation between the presence of func tional groups and the mechanism of AO action is taken into account. Thus, AO are subdivided after their indi rect (mediated) and direct action: (1) AO of indirect action could suppress FRO only in biological material (from cell organelles to the whole organism), but they are not effective in vitro. The diverse mechanisms could be used for their action: activation (reactivation) of antioxidant enzymes; suppression of reactions resulting in the ROS generation in the organism; the shift of FRO toward the formation of less reactive ROS; selective induction of the genes encoding the systems of AO defense and damage reparation; metabolism normal ization, etc. (2) AO of direct action, in contrast, manifest direct antioxidant properties, which are determined by the presence of definite functional groups. They are subdi vided into the five main types: (a) Proton donors, i.e., compounds with easily movable hydrogen atom, which are capable of free radical scavenging. Among them, phenols (toco pherols, phenol and naphthol derivatives, flavonoids, catechins, and others), nitrogencontaining heterocy clic compounds (melatonin, derivatives of 1,4dihy dropyridine, derivatives of pyrrolopyrimidine), thiols (GSH, cysteine, homocysteine, Nacetylcysteine, dihydrolipoic acid); (b) Polyenes, i.e., compounds with several unsatur ated bonds. They are easily oxidized, thus competing for ROS and radicals with other biological molecules and thus protecting the latter against oxidation. They

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are capable of interaction with diverse free radicals, using double bonds as binding sites. The main repre sentatives of these AO are retinoids (retinal, retinoic acid, retinol, and its esters) and carotenoids (car otenes, lycopin, spirylloxanthine, astaxanthin, and others); (c) Catalyzers, i.e., compounds capable of cata lyzing ROS and FRO intermediate elimination with out the formation of new free radicals. These com pounds are also known as “enzyme mimetics.” As dis tinct from above AO groups, AOcatalyzers are efficient at lower concentrations. The typical repre sentatives of catalyzers are SOD and GPO; (d) Traps of radicals, i.e., compounds interacting with free radicals with the formation of adducts of rad ical nature with a limited reactivity. Typical represen tatives are nitrons, in particular, phenyltertbutyl nitron. They bind efficiently superoxide and hydroxyl radicals; (e) Chelators, i.e., compounds suppressing metal dependent FRO reactions due to binding transition metal cations; the latter catalyze reactions of ROS generation [12]. It is believed that AO are capable of suppression of free radical redox processes using a single or several mechanisms. Therefore, preventive AO and ROS inhib itors [3] are distinguished. The mechanism of antioxi dant action of preventive AO is the change in the sub strate structure retarding its oxidation, reduction in the О2 concentration, binding or oxidation of transi tion metals, or peroxide conversion into stable prod ucts of oxidation (alcohols, aldehydes, and ketones). ROS inhibitors eliminate oxygen radicals and break the oxidative chain via interaction with organic radicals. Among them are AO enzymes with a narrow specializa tion, which break the chain oxygen reduction at vari ous stages, and nonenzymatic AO in water and lipid phases; they scavenge organic radicals. Since POL activity in membranes depends markedly on their structure, it was suggested to separate one more group of structural AO (tocopherols, steroids, eicosanoids), which suppress POL, thus stabilizing membranes [3]. Some other views on the AO action mechanisms exist. Thus, defense against oxidative stress occurs using the two different mechanisms: (1) Suppression of superoxide radical generation because of a decrease in the О2 concentration in the cell due to its more rapid utilization by the mitochon drial respiratore chain (“nonohmic” proton leak, pores in the mitochondrial membrane, and uncou pling of oxidative phosphorylation, oxidases cata lyzing fourelectron О2 reduction, which function was uncoupled from ATP synthesis) [20–22]. (2) Functioning of the antioxidant system involv ing: (A) lowmolecular AO subdivided into hydrophilic, which protect components of hyaloplasm, mitochon drial matrix, etc. (GSH, AA, and others) and hydro RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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phobic AO protecting membranes (tocopherols, caro tenoids, and others); (B) AO enzymes provide for metabolism of not only ROS but also active oxidized compounds, supporting the following lines of defense: •–

(a) against O 2 SOD; (b) against Н2О2 – seleniumdependent GPO and catalase; (c) against lipid peroxides – GPO, GST, and phos pholipidhydroperoxide GPO; (d) neutralization of secondary metabolites—GST, formaldehyde dehydrogenase, glyoxalase, aldehyde dehydrogenase, quinone reductase, epoxide hydro lase, etc. [20, 22]. In the physiological and biochemical studies of animals and bacteria, a comparative classification of the AO defense systems described by Davies [23] is often mentioned. The scheme suggested by this author is not complex and multicomponent; the mechanisms of AO action are in its ground. (1) Primary system of AO defense. This system is in a direct contact with ROS and eliminates them; it includes vitamins (A, E, and C), GSH, and uric acid, and also enzymes utilizing ROS, such as SOD and peroxidases; (2) Secondary system of AO defense. This system provides for the reparation of ROSinduced injuries of physiologically important molecules; it includes enzymes of the catabolism (lipases, proteases, pepti dases, etc.) and also the enzymes of DNA reparation. This second line of defense is switched on under con ditions of extreme activation of oxidative stress or when the systems of ROS detoxification are sup pressed or damaged [2, 23]. This scheme is believed to be most flexible and convenient, although some its components require detail description and explanation. Classification on the Basis of the Struggle against Toxic O2 Action Other researchers also believe that organisms used the two main strategies in the struggle against O2 toxic effects. One of them is directed to the decrease in the O2 level and blockage of its conversion into ROS (alterna tive oxidase, transferrin, ferritin, metallothioneins, etc.), whereas another one is the removal of appeared ROS (SOD, glutathione peroxidases, ascorbate perox idases, peroxiredoxins, thioredoxins, AA, tocopherol, etc. [21, 24]. Keeping in mind these strategies, another division of all AO defense systems is described, e.g., into the three main groups. The first group is the prevention of ROS generation. ROSinduced damages are localized predominantly in the sites of transition metal localization, primarily Fe and Cu. Therefore, an important component of No. 2

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defense against ROS, preventing free radical pro cesses, is chelating such metals by specialized and nonspecialized proteins, for example, by ferritin, transferrin, albumins, and others. The second group is breaking the free radical chain and neutralization of radicals by antioxidant enzymes and quenchers. Antioxidant enzymes include SOD, catabolyzing superoxide anion, catalase, and GPO, which degrade hydrogen peroxide and hydroperox ides, respectively. Lowmolecularweight AO (GSH, αtocopherol, AA, uric acid, and others) also could break free radical chains, preventing its spreading. GSH is also referred to quenchers; it neutralizes hydroxyl radical and singlet oxygen. However, it is a substrate for some enzymes and the element for regen eration of vitamins E and C. The third group is reparation of damages. This group includes secondary enzymes of the AO defense, which are functionally connected with glutathione. Glutathione Stransferases catalyze conjugation of GSH with nucleophilic xenobiotics or cell compo nents damaged by ROS. As a result, the ROS loose their toxic properties. NADPHdependent glu tathione reductase reduces oxidized glutathione due to NADPH oxidation. The latter, in its turn, is reduced by glucose6phosphate dehydrogenase [25]. Different researchers have different notions about the components of the secondary system of the antiox idant defense. In one case, this is enzymes of DNA reparation and utilization of damaged molecules by hydrolytic enzymes of various specializations. In another case, the secondary system includes enzymes neutralizing xenobiotics and metabolic drugs and also enzymes synthesizing and repairing the components of the antioxidant system and their substrates [23, 25]. Classification on the Basis of the Multilevel Defense Organization Subdivision of the antioxidant system into two or three groups on the basis of the mechanisms of their action is simple and convenient. Therefore, such clas sifications are met in publications most often [2, 9, 13]. However, as was repeatedly mentioned, AO defense is not limited by only ROS elimination. Therefore, those of abovelisted classifications, which included only these AO properties, did not embrace other important AO functions. It is evident that defense against ROS starts from preventive measures and finished by reparation processes and detoxifica tion of secondary metabolites [2, 8, 20, 21, 24]. There fore, we think that to embrace all basic AO functions, recent classifications should be combined and some what widen, keeping in mind specific features of AO structure [23, 25]. When classification is based on the mechanism of AO action, we think that four main groups should be distinguished. First group – preven tion of ROS generation; the second group – primary defense system, i.e., ROS neutralization; the third

group – secondary defense system or synthesis and rep aration of antioxidants; and the fourth group – removal of damages and detoxification of secondary metabolites (table). This is used as a basis for the system of AO defense we suggest. In this classification, unambiguous definition of components in each group allows a correct AO distri bution. For example, the first group is characterized by processes preventing ROS generation: depositing excess oxygen, transition metal chelating, neutraliza tion or utilization of xenobiotics. The second group is the system of primary defense; it includes all known AO directly contacting with ROS and eliminating them or breaking free radical chains. The third group is the secondary defense systems, which components are closely related to reparation and synthesis of low molecular AO, maintaining their pool. The fourth group comprises components providing for the elimi nation of damages arising due to oxidation of physio logically important molecules. The systems neutraliz ing secondary metabolic products and capable of ROS generation [2, 21, 25] could be also referred to this group. We think that separation of this group in the antioxidant system classification seems justified when enzymes referred to this group manifest a narrow spe cialization, i.e., hydrolyze molecules damaged only as a result of contact with ROS. The example is a multi catalytic protease complex found in Arabidopsis thaliana, which is capable of protein hydrolysis when the latter are damaged by oxidation [26]. Most ele ments of this group, which are listed in above described classifications, could function also in nor mal metabolism, providing for reparation and elimi nation of “errors” arising due to other types of dam ages, not induced by ROS [2]. The suggested classification of the antioxidant sys tem does not comprise new components or new sec tions but combines the main levels of defense described in abovedescribed classifications. The presented material demonstrated once more the hierarchy within the antioxidant defense system, indicated its multilevel organization [17]. It becomes evident that the mechanisms of AO defense are the part of the total system of the homeostasis in the organism internal medium and the defense system per se, as distinct from traditional notions about it, could be somewhat wider [27]. Currently, it is believed that the main way for organism surviving under aerobic conditions is the usage of mechanisms preventing ROS generation in the О2 metabolism rather than phylogenetically developed mechanisms of the strug gle against ROS produced [27]. Classification of anti oxidant systems after the levels of defense or AO action mechanisms combines and systematizes all knowledge of the organism AO defense. Like each other system atization of accumulated knowledge, classification of the antioxidant defense systems permit the building of a definite chain of events occurring at the development of oxidative stress and counteracting it. In this built


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Classification of the antioxidant defense systems First group

Second group

Third group

Fourth group

Prevention of ROS genera Primary defense systems tion (inactivation and neu (ROS neutralization and tralization of potential break of free radical chain) prooxidants)

Secondary defense systems (syn thesis and reparation of AO, elec tron donors, and substrated for an tioxidant enzymes)

Damage removal and detoxifi cation (reparation or hydrolysis of damaged molecules, neutral ization of secondary metabolites)

O2 depositing: CoQH2oxi dase, cytochrome oxidase, and other terminal oxidas es with a high affinity for oxygen

Highmolecularweight compounds: enzymes and Fecontaining proteins (SOD, catalase, peroxi dases, GST, peroxiredox ins, etc.)

Highmolecularweight com pounds: glutamate reductase, ascorbate reductase, MDAR, thioredoxins, thioredoxin reduc tase, glutathione reductase, γ glutamylcysteine synthetase, glu cose6phosphate dehydrogena se, etc.

Damage removal: enzymes of DNA reparation, degradation of lipids, proteins, etc. (endo nucleases, exonucleases, ligas es, proteases, peptidases, phos pholipases,and others)

Transition metal chelates: ferritin, transferring, albu mins, metallothioneins, and others

Lowmolecularweight Lowmolecularweight com compounds; GSH, AA, pounds: AA, GSH, etc. tocopherol, lipoic acid, ubiquinone, phenol de rivatives, carotenoids, etc

Secondary metabolite neutral ization: GST, GSH, glucuronyl transferase, sulfo and acetyl transferases, quinone reduc tase, formaldehyde dehydrogenase, glyoxylase, etc.

Xenobiotic neutralization: GST, GSH, etc.

chain of successive or simultaneous events, we can easily see “weak” (incompletely studied) links deserved a researcher attention. Therefore, classifica tion, as more or less systemized information, could help approaches and plant further studies on the better ground. At present, numerous facts were obtained indicat ing that plant acclimation to unfavorable or stress con ditions was correlated with the enhanced AO defense [9]. It was noted that the AO defense system of differ ent plant species respond differently to one and the same stress. Such conclusion resulted from a compar ison of some enzyme activities (SOD, catalase, perox idases, MDAR, and glutathione reductases) ([9] and references therein). It was also established that the response of the antioxidant system depended on the severity, duration, and the type of the stress. Changes in SOD, catalase, peroxidases, and glutathione reduc tase activity depended on the peculiarity of stress fac tor action [9]. When a narrow set of primary enzymes is studied and other levels of defense are not consid ered, which is met frequently, some difficulties arise in explanation why enzyme systems of tolerant and sen sitive plants respond similarly or, on the contrary, tol erant plants responded differently ([9] and references therein). We believe that classification based on the AO action mechanisms could help in the choice of the level of AO defense and its group, which deserves to be studied, when, for example, the task is to study oxida tive stress at different stages of stressor action in plants differing in tolerance. To determine the primary target of stressor action is very complex or impossible. Although effects of differ RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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ent factors are integrated in the cell in such a way that they promote ROS overproduction and development of oxidative stress [9], different stresses induce differ ent organism responses and could induce different pathological states. It is especially often met in ani mals and humans. It is believed that different types of FRO could play a definite role. The contribution of different FRO stages in definite human pathologies is believed to vary substantially: in one cases, POL is most important; in other cases, protein oxidative inju ries; and in the third case, modification of nucleic acids [12]. Certainly, in all cases, there is no damage to only a single type of macromolecules. Nevertheless, in different deceases different stages of FRO play the key roles in the development of pathological tissue changes (cited after [12]). It is possible to suppose that, at different pathology stages, most efficient AO will be those, which targets are the products of a “dominating” process of FRO. Similar phenomena could evidently occur in plant organisms. This is indi rectly confirmed by the fact that, under conditions of oxidative stress developing under the influence of such stressors as bacteria and fungi, primary enzymatic defense exerts less efficient action than protection from lowmolecularweight AO [1, 28]. At the action of biotic and abiotic factors, activation of different FRO stages and corresponding links of corresponding defense chains in plant cells is expected, and classifi cation of the antioxidant systems presents a general notion about components of these chains and AO functional defense systems. Development of organism tolerance to ROS is determined by synchronous functioning of the main No. 2

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components of the antioxidant system. Antioxidant defense is mainly determined by the state of coupled “antioxidant structures,” which provide for continu ous production of continuously spent AO [29]. We believe that AO classification helps to choose func tionally coupled links of the antioxidant system (for example, ROS elimination and AO reparation or some others). Since the level of intracellular AO is under genetic control [3], systematic studying of function ally connected AO from different groups of the antiox idant system could help in some cases to reveal rapidly the weak points in the genomes of sensitive organisms. Such approach, when several AO functional groups or defense levels are taken into account, is rarely used in the works performed with plants, as a rule, only when definite redox cycles are studied, for example, the ascorbate–glutathione cycle, etc. [9, 30]. The mechanism of the antioxidant system action is complex and multistage; it includes additive, synergis tic, and antagonistic interactions between AO [3]. Diverse AO structure, close interrelation between AO of different nature, their polyfunctionality, coupled or differently directed functioning are the factors creat ing definite difficulties for AO systematization. Returning to classification presented in the table, it should be noted that AO could function not only in different sites of the antioxidant system but also using different mechanisms. Therefore, one and the same AO could be included in different groups. For exam ple, GST fulfills conjugating and thiolyase functions. Due to the first function, this enzyme could be referred simultaneously to the groups of “preventing ROS generation” and “damage removal and detoxifi cation.” Due to the second function, GST could be referred to primary AO. AA also could be referred to the two groups, of primary and secondary defense; it is directly involved in ROS elimination in enzymatic and nonenzymatic reactions and is an important compo nent during reduction of oxidized tocopherol. Similar situation is observed for GSH. Difficulties in AA and GSH classification could be partially explained by the fact that lowmolecular AO in the system of AO defense function not autonomically but in mutual cooperation, within the enzymatic redoxcycles [27]. In these cycles, they can be redoxcofactors. Among such cycles, the ascorbate–glutathione cycle and dehydroascorbate reductase cycle are most studied [9, 27]. In some sense, reduction of αtocopherol is also the cyclic process [29]. Degradation of peroxides could occur in the enzymatic redox cycle of glu tathione (GSH, GPO, glutathione reductase); the hexosomonophosphate pathway generates the reduc tion equivalent, NADPH, which is used for above cycle functioning [27]. In general, the antioxidant system functioning in the cells is determined by “cyclic” and “cascade” AO interactions within the cell or its compartments. The occurrence of peculiar “antioxidant” chains of elec tron transport is suggested; the efficiency of these

chains is determined by simultaneous functioning of various components of the antioxidant system [3]. One of the known examples of such chains is a corre lation between activities of catalase and SOD, which, as it is believed, is connected with switching over the electron flow [1]. These enzymes function as the com ponents of a single chain of ROS utilization, although they can be located in different cell regions. With the revealing and studying new “cycles” and close synergistic interactions or redoxinteractions between AO of different nature, a requirement will appear later to classify AO not after action mechanism but after “cyclic” and “cascade” interactions (or “chains of electron transport”) like it is made for the cell signaling systems. In this case, evidently, new sec tions in AO classification, like in classification of sig naling system, are expected, such as “the antioxidant system of the ascorbate–glutathione cycle,” “the anti oxidant system of AA,” “the antioxidant system of glutathione,” “the antioxidant system of phenolic compounds,” “the antioxidant system of specialized enzymes (SOD, catalase), and so on. Similarly as dif ferent cell signaling pathways are functionally con nected, different elements of the antioxidant system are arranged in the net of close interactions and mutual conversions. In conclusion, we should once more call attention to the fact that a vast body of information is accumu lated concerning AO, and this material not only dem onstrates their important role in cell defense against ROS excess and their damaging effects but also per mits systematization of various levels and mechanisms of antioxidant defense. REFERENCES 1. Kolupaev, Yu.E., Reactive Oxygen Species in Plants as Affected by Stressors: Formation and Possible Func tions, Vestn. Khar’kovskogo Nats. Agrarn. Unta, Ser. Biologiya, 2007, no. 3, pp. 6–26. 2. Merzlyak, M.N., Activated Oxygen and Oxidative Pro cesses in Plant Cell Membranes, Itogi Nauki i Tekhniki, Ser. Fiziol. Rast., 1989, vol. 6. 3. Men’shchikova, E.B. and Zenkov, N.K., Antioxidants and Inhibitors of Radical Oxidative Processes, Usp. Sovrem. Biol., 1993, vol. 113, pp. 442–455. 4. Kuznetsov, Vl.V. and Dmitrieva, G.A., Fiziologiya ras tenii (Plant Physiology), Moscow: Vysshaya Shkola, 2005. 5. Apel, K. and Hirt, H., Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal Transduc tion, Annu. Rev. Plant Biol., 2004, vol. 55, pp. 373–399. 6. Ahmad, P., Sarwat, M., and Sharma, S., Reactive Oxy gen Species, Antioxidants and Signaling in Plants, J. Plant Biol., 2008, vol. 51, pp. 167–173. 7. PhamHuy, L.A., He, H., and PhamHuy, C., Free Radicals, Antioxidants in Disease and Health, J. Biomed. Sci., 2008, vol. 4, pp. 89–96. 8. Shao, H.B., Chu, L.Y., Lu, Z.H., and Kang, C.M., Primary Antioxidant Free Radical Scavenging and


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