1. INTRODUCTION

1.1. Physiology of anoxic stress

Oxygen status of cells and tissues varies significantly during organism ontogenesis, and depends on environmental conditions of oxygen supply. Under flooding the root system is the most susceptible plant organ to suffer from oxygen deprivation. Since under natural conditions transient hypoxia occurs prior to strict anoxia, and because of accumulating evidence that low oxygen concentration may trigger metabolic responses, it is necessary to define anoxia here. According to Drew (1997) tissues or cells are hypoxic when the O ₂ partial pressure limits the production of ATP by mitochondria. Anoxia occurs when the production of ATP by oxidative phosphorylation is negligible relative to that generated by glycolysis and fermentation. To prolong survival under unfavourable conditions plants develop structural and metabolic adaptations, which are genetically controlled. Since the problem of tolerance to flooding and anoxia is of great economical importance, numerous investigations have been undertaken to elucidate the mechanisms underlying high resistance to oxygen deprivation. Despite the fact that the response of a particular organism can be very specific, some general changes brought about by anoxic stress can be described.

1.2. Structural adaptations to anoxia

Constitutive aerenchyma formation in the stems and roots of aquatic and flooding tolerant species provides long distance oxygenation of hypoxic tissues. Two distinct pathways lead to the formation of interconnected and gas-filled spaces: Cell separation during development and programmed cell death (and/or necrosis), the latter being widespread even among dry-land species (Drew 1997). Non-constitutive induction of aerenchyma via cell death implies ethylene signalling in sunflower stems (Jackson 1985). Low concentrations of ethylene in air (0.1 - 1.0 m l/L ^-1 ) promote cell death selectively in normoxic roots, while hypoxic roots have been shown to contain higher concentrations of ethylene and its precursor, and increased activity of enzymes responsible for ethylene biosynthesis (He et al. 1994). Anoxic stress also causes changes in cell ultrastructure, most of them leading to cell injury and death. Plasmolysis of the cells, mitochondrial elongation and swelling are detectable at early stages of anoxia (Aldrich 1985, Andreev et al. 1991).

1.3. Anoxia induced metabolic changes

No fundamental differences in metabolic pathways have been detected between anoxia tolerant and intolerant plant species. Probably a complex of minor adaptations underlies better survival of anoxia-resistant plants (Pfister-Sieber and Braendle 1994).

Under oxygen deprivation mitochondrial electron transport chain (ETC) is suppressed and, hence, the synthesis of ATP is inhibited. Cells overcome energy shortage by switching to anaerobic glycolytic ATP production, and the availability of sugars as respirable substrates becomes extremely important. However, roots have been shown to suffer from sugar starvation under anoxia, because of inhibition of phloem transport, where the unloading step is affected (Saglio 1985). Mobilisation of starch is also affected under anoxia: The anoxia-intolerant wheat seeds fail to degrade starch of the endosperm under anoxia, while in the more tolerant rice a -amylases, responsible for starch breakdown, are induced under anaerobiosis (Perata et al. 1992). In general, anoxia causes a significant decrease in ATP levels especially in the anoxia-intolerant species but also in the tolerant species, and the ability of the tolerant plant species to maintain their energy supply for a longer time is considered to be the key factor for survival under anoxia (Chirkova et al. 1984, Hanhijärvi and Fagerstedt 1994, 1995, Crawford 1992).

Another important feature of anoxic metabolism is acidification of the cytoplasm and accumulation of fermentation products. A decrease in cytoplasmic pH from 7.3 - 7.4 to 6.8 is attributed to lactic acid production. Inhibition of lactate dehydrogenase and activation of pyruvate decarboxylase by the acidic pH leads to accumulation of acetaldehyde, which is converted to ethanol (by alcohol dehydrogenase, ADH, EC 1.1.1.1.) - one of the end products of anaerobic metabolism. However, the degree of acidification cannot be attributed solely to lactic acid accumulation. Another possibility is passive H ⁺ leakage from the vacuole under limited ATP availability and inhibition of vacuolar H ⁺ -ATPase (Ratcliffe 1995).

Under prolonged anoxia ethanol is the most abundant end product of fermentation, even in anoxia-tolerant plants, which are able to release it into the surrounding medium. It has been shown that the ability to remove volatile end products of fermentation (i.e. ethanol and acetaldehyde) leads into increased anoxia tolerance in the anoxia sensitive chickpea ( Cicer arietinum L.) (Crawford and Zochowski 1984). Under anaerobic fermentation pyruvate can be converted into products other than ethanol and lactate. Increased amounts of alanine (by transamination of aspartate), malate and succinate can be detected at the beginning of anoxia. It seems that diversification of the glycolytic end products may be helpful in anoxia-tolerance, however the exact physiological mechanism to avoid injury is not fully known (Pfister-Sieber and Braendle 1994)

One of the most important consequences of energy limitation under anoxia is altered redox state of the cell. When oxygen - the terminal electron acceptor of ETC - is unavailable, the intermediate e ^- carriers become reduced. This process in turn affects redox active metabolic reactions. Indeed, the ability to maintain redox characteristics of the cell (i.e. NADH/NAD ⁺ -ratio) unaltered for a prolonged period has been shown for the anoxia-tolerant species rice and Glyceria ( Chirkova 1992) and is considered important for plant survival under anoxia. A decrease in NADH/NAD ⁺ has been observed in the anoxia - intolerant wheat and bean (Chirkova et al. 1992). Ascorbic acid (AA) and glutathione (GSH) metabolism (reduction to an active antioxidant form) may provide an additional sink mechanism for excess protons and NADH production during the first stages of anaerobiosis. Participation of NADH in monodehydroascorbic acid (MDHA) reduction to ascorbate has been demonstrated (Noctor and Foyer 1998) as a process, which can play an adaptive role supplying reducing equivalents for antioxidant turnover. The redox changes can affect other redox-dependent reactions i.e. the oxidation state of ferrous ions - the promoters of ROS generation (through the Fenton reaction, eq. 4) and peroxidation of lipids. If oxygen deprivation persists, the need for oxidised NAD ⁺ and ATP leads to the fermentation pathway, where both LDH and ADH can regenerate NAD ⁺ .

The signalling mechanism leading to anoxia-specific metabolic responses is not yet understood. Several investigations indicate that cytosolic Ca ²⁺ may play the role of a second messenger. A rapid biphasic elevation of cytosolic Ca ²⁺ has been observed in Arabidopsis thaliana seedlings (Sedbrook et al. 1996) and the inhibition of organellar Ca ²⁺ channels by ruthenium red has been reported to cause inhibition of anoxic gene expression and post-stress survival of plants. Anoxia is known to induce a rapid elevation of cytosolic Ca ²⁺ in maize suspension-cultured cells (Subbaiah et al. 1998). This rise was attributed to the release of the ion from intracellular stores, particularly from the ER and mitochondria. Together with the experiments performed on animal cells, these data suggest Ca ²⁺ participation in anoxic signalling (Sedbrook et al. 1996).

1.4. Anoxia and gene expression

Anoxia-induced metabolic changes are associated with changes in gene expression: A decrease in general mRNA translation and an activation of expression of 'anoxic genes' has been observed. Normal protein synthesis is inhibited under anoxia, and only 10-20 anaerobically induced proteins (ANPs) appear. However, they account for more than 70% of total translation (Sachs et al. 1980, 1996). The majority of the genes induced code for enzymes involved in starch and glucose mobilisation, glycolysis and ethanol fermentation (Russel and Sachs 1991, Chirkova and Voitzekovskaya 1999). E.g. anaerobic induction of enolase (2-phospho-D-glycerate hydratase, EC 4.2.1.11), an integral enzyme in glycolysis, which catalyses the interconversion of 2-phosphoglycerate to PEP, has been reported in maize (Lal et al. 1998). Some other glycolytic and fermentation pathway enzymes, such as alcohol gehydrogenase, glucose phosphate isomerase, pyruvate decarboxylase (PDC) and sucrose synthase have been characterised as ANPs in maize. However, two genes not related to sugar metabolism and inducible under oxygen deprivation have been found. They may be involved in aerenchyma formation (Sachs et al. 1996).

The patterns of ADH and PDC expression in Arabidopsis thaliana includes two sets of alcoholic fermentation pathway genes, each of which may play a different role in the adaptive response to anoxia. One set is strongly induced in roots, while the other is expressed constitutively in both roots and leaves (Dolferus et al. 1997). Differential transcript levels of genes associated with glycolysis and alcohol fermentation suggest that corresponding genes may be differently regulated under submergence stress. A stimulating effect of low oxygen concentration (0 to 4%) on the induction of adh1 transcripts and ADH activity has been observed in roots and shoots of maize (Andrews et al. 1993). Anaerobiosis-specific transcriptional and translational changes have been detected in rice coleoptiles and roots: Plants responded to anoxia within 1 hour by synthesising low molecular weight proteins (c. 33 kD) (Breviario et al. 1994), the transition peptides. However, the early induced genes, those responding after 1-2 h of anoxia, have not been studied extensively. A novel gene family aie (anaerobically inducible early) has been identified recently in a very flooding-tolerant variety of rice. mRNA levels of aie genes peaked after 1.5 to 3 h of anoxia and were at high levels after 72 h of anoxia (Huq and Hodges 1999). Sequence analysis of one of the aie genes did not show any significant homology to any known genes or proteins.

1.5. Membrane function and structure under anoxia

Changes in the physical properties of membranes and their function and composition under oxygen deprivation are non-specific stress reactions, which have been reported in a number of other stresses (Chirkova 1988, Shewfelt and Purvis 1995). Under anoxia a decrease in membrane integrity is a symptom of injury, measured as changes in lipid content and composition (Chirkova et al. 1989, Hetherington 1982), activation of lipid peroxidation (Crawford 1994, Crawford and Braendle 1996, Chirkova et al. 1998, Blokhina et al. 1999), enhanced electrolyte leakage (Chirkova et al. 1991a, 1991b) and as a decrease in adenylate energy charge (Hanhijärvi and Fagerstedt 1994, 1995, Chirkova et al. 1984). Since de novo lipid synthesis is energy dependent, and could hardly occur under anoxia, the preservation of membrane lipids is the most efficient way to maintain functional membranes. A decrease in unsaturated to saturated fatty acid ratio under anoxia may represent a result of lipid peroxidation (LP), and at the same time sets limits for substrates of LP, the polyunsaturated fatty acids (PUFA). In the anoxia tolerant A. calamus a decrease in linolenic acid (18:3) is compensated by linoleic (18:2) and oleic (18:0) acids under oxygen deprivation. The original lipid composition is recovered during two days of re-aeration (Pfister-Sieber and Brändle 1994). In general, lipids of anoxia-tolerant plants are more preserved during oxygen deprivation in respect to composition and the degree of unsaturation. Similar results have been obtained for the anoxia tolerant and intolerant cereals rice and wheat, respectively (Chirkova et al. 1989). In the rhizomes of the anoxia resistant Iris pseudacorus imposition of anoxia is known to result in a significant decrease in the ratio of saturated to unsaturated fatty acids in polar lipids. In contrast, no changes in either lipid classes or fatty acid composition have been observed in Iris germanica rhizomes, although these Iris -species have been shown to possess a highly similar lipid profile (Hetherington 1982). On the other hand, there are no significant qualitative and quantitative changes in the composition of fatty acids in anaerobically treated rice seedlings (Generosova et al. 1998). In that study it was postulated that the reduction of unsaturated fatty acids esterified in lipids was of no significance as a mechanism of plant adaptation to anaerobic conditions. The key role in survival was assigned to the energy metabolism (Generosova et al. 1998). However, during the recent years evidence has accumulated on the importance of lipid metabolism, and especially on unsaturated fatty acids, in the induction of defence reactions under biotic and abiotic stresses. Linolenic acid (18:3) has been shown to be a precursor of jasmonic acid, the latter being a signal transducer in defence reactions in plant-pathogen interactions (Rickauer et al. 1997). Free fatty acids, liberated during membrane breakdown under stress conditions, are not only the substrates for LP, but also can act as uncouplers in mitochondrial ETC (Skulachev 1998).

Alterations in the degree of fatty acid unsaturation have been observed also under abiotic stress conditions: A four-fold increase in the ratio of unsaturated to saturated fatty acids in a cold-tolerant cultivar of bermudagrass has been observed under low temperature (Samala et al. 1998). Also low irradiance and spectral composition of light are known to affect C-18 fatty acid desaturation in soybean leaves (Burkey et al. 1997). Lipid hydroperoxides, formed as a result of LP, can affect the membrane properties, i.e. increase hydrophilicity of the internal side of the bilayer (Frenkel 1991). This phenomenon is very important for the termination of LP, since increased hydrophilicity of the membrane favours the regeneration of tocopherol by ascorbate.

Hence, membrane lipids undergo changes under anoxia, which may be considered as adaptive, and which may result in the acceleration of lipid peroxidation after restoration of the oxygen supply. However, preliminary steps may occur under anoxic conditions, i.e. maintenance of the high level of fatty acid unsaturation, appearance of free fatty acids and the production of low amounts of ROS due to membrane associated electron transport.

Reoxygenation injury is a well-documented fact for both animal and plant tissues. Indeed, under anoxia saturated electron transport components, the highly reduced intracellular environment (including ions of transition metals) and low energy supply are factors favourable for ROS generation. Formation of free radicals within minutes after restoration of the oxygen supply has been shown by electron paramagnetic resonance spectroscopy (EPR) in the rhizodermis of the anoxia-intolerant I. germanica , while in the tolerant I. pseudacorus no signal was detected (Crawford et al. 1994). Readmission of oxygen most probably causes the generation of superoxide radicals measured as an induction of SOD activity during re-aeration (Monk et al. 1989). Accumulation of various products of LP as a result of reoxygenation has been observed in the roots of the anoxia-intolerant wheat and tolerant rice, the latter showing higher membrane stability and lower level of LP (Chirkova et al. 1998, Blokhina et al. 1999).

The existence of anoxia inducible changes in plant metabolism implies that plant cells sense anoxic conditions and respond to them quickly by glycolytic production of ATP and the regeneration of NAD(P) ⁺ (Richard et al. 1994). Anoxic and especially postanoxic injury is the result of the formation of ROS. This causes peroxidation of lipid membranes, depletion of reduced glutathione, an increase in cytosolic Ca ²⁺ concentration, oxidation of protein thiol groups and membrane depolarisation.

1.6. Role of ROS in the stress response

The generation of reactive oxygen species (ROS) is considered to be a primary event under a variety of stress conditions (Noctor and Foyer 1998). The consequences of ROS formation depend on the intensity of the stress and on the physicochemical conditions in the cell (i.e. antioxidant status, redox state and pH). It has been generally accepted that active oxygen produced under stress is a detrimental factor, which causes lipid peroxidation, enzyme inactivation, and oxidative damage to DNA (Shewfelt and Purvis 1995). However, during the recent years evidence has accumulated on the participation of ROS and their oxygenated products in a signal transduction cascade (Tarchevskii 1992, Lander 1997). Antioxidant status and redox state of the cell are the main components in the fine regulatory mechanism of ROS signal specificity (Lander 1997). ROS seem to affect the cell through a combination of the following factors: the amount of ROS produced (correlates with the severity of the stress) and biochemical status of the cell (i.e. activity of antioxidative and other enzymes, antioxidant content, pH, energy resources, integrity of membranes, redox characteristics etc.). The particular mechanisms and the place of ROS in the signal transduction cascade are not yet known. Recently, it has been proposed that sensing of the O ₂ -concentration and of ROS shares the same mechanism (Semenza 1999). Several models of sensing oxygen concentration have been proposed for animal cells and bacteria.

A. Direct oxygen sensing:

The sensor, heme, binds O ₂ directly and adopts an inactive oxygenated state; when oxygen concentration declines, deoxygenation of heme occurs. This process is considered as the first step in oxygen sensing. There is an increasing amount of evidence that low concentrations of haemoglobin are present in roots of many plant species, although the physiological function is described only for the roots of plants capable of nodulation, i.e. facilitation of the oxygen supply. An alternative function for plant haemoglobin may be the indication of O ₂ -concentration and switching of metabolism from oxidative to fermentative pathway (Crawford 1992). Two haemoglobin genes have been identified in Arabidopsis thaliana , one of which is induced by hypoxia (Dolferus et al. 1997). In flooding-stressed barley roots and in isolated barley aleurone layers exposed to anaerobic conditions, induction of the haemoglobin gene has been observed and was considered an integral part of the normal anaerobic response (Taylor et al. 1994). Besides its possible function as an oxygen sensor, another two physiological functions have been suggested for haemoglobin under low oxygen tension: Oxygen carrier and terminal oxidase or oxygenase (Hill 1998).

B. Redox cycling of iron-sulphur clusters represents another possibility for oxygen signalling.

Sensing via O ₂ -metabolites (ROS) suggests several mechanisms:

C. NAD(P)H oxidase converts O ₂ to superoxide , which is dismutated by SOD to hydrogen peroxide. This model predicts that under oxygen deprivation the production of ROS declines, thus providing a redox signal for hypoxia.

D. Complex IV (cytochrome c oxidase) of the mitochondrial electron transport chain (ETC) is inhibited under hypoxic conditions and upstream electron leakage leads to ROS formation at complex III. (from Semenza 1999).

The experimental data obtained until now are contradictory and partly support all proposed models. Evaluation of the models in animal cells mostly employs the effect of O ₂ /ROS on inhibition or induction of expression of the hypoxia-inducible factor 1 (HIF-1), a transcription factor stimulating O ₂ delivery, glucose transporters and glycolytic enzymes, which facilitate ATP production (Semenza 1998). In plants the involvement of ROS in signalling and in the induction of the stress response is well characterized for the hypersensitive response, although the sensor of oxygen metabolites is unclear (Goodman 1994, Lamb and Dixon 1997). Participation of ROS in the stress response has been described phenomenologically under a wide range of environmental conditions: dehydration, high salinity, low temperature, anoxia, senescence and high ozone concentrations. Altogether these observations suggest a fundamental role for reactive oxygen species in non-specific stress adaptation and signalling.

1.7. Chemistry of reactive oxygen species: Types of ROS

Four-electron reduction of oxygen in the respiratory chain is always accompanied with a partial one- to three-electron reduction, yielding the formation of ROS. This term includes not only free radicals (O ₂ ^{- Ÿ} , HO ^· ), but also the molecules H ₂ O ₂ , singlet oxygen ¹ O ₂ and ozone O ₃ .

Molecular oxygen is relatively unreactive (Elstner 1987) due to its electron configuration (two unpaired electrons with parallel spins). Activation of oxygen (i.e. first univalent reduction step) is energy dependent and requires an electron donation. The following one- electron reduction steps are not energy dependent and can occur spontaneously or require appropriate e ^- /H ⁺ donors. In biological systems transition metal ions (Fe ²⁺ , Cu ⁺ ) and semiquinones can act as e ^- donors. The superoxide anion O ₂ ^-· can be protonated at a low pH to yield the perhydroperoxyl radical ^· HO ₂ (1). Both O ₂ ^- and ^· HO ₂ undergo spontaneous dismutation to produce H ₂ O ₂ (2, 3).

Although H ₂ O ₂ is less reactive than O ₂ ^-· in the presence of reduced transition metals such as Fe ²⁺ in a chelated form (which is the case in biological systems), the formation of OH ^· can occur in the Fenton reaction:

Fe ³⁺ complex can be efficiently reduced by O ₂ ^-· (5), the product of one-electron oxygen reduction, thus providing the cycling of Fenton reaction:

Mechanisms for the generation of ROS in biological systems are represented by both non-enzymatic and enzymatic reactions. The partition between two pathways under oxygen deprivation stress can be regulated by oxygen concentration in the system. Non-enzymatic one electron O ₂ reduction can occur at about 10 ^-4 M and higher oxygen concentrations (Skulachev 1997). Even in very low O ₂ concentrations plant terminal oxidases (K _m 10 ^-6 M for oxygen) and the formation of ROS via mitochondrial ETC still remain functional.

Among other enzymatic sources of ROS, xanthine oxidase (XO), an enzyme responsible for initial activation of dioxygen should be mentioned. As electron donors XO can use xanthine, hypoxanthine or acetaldehyde (Bolwell and Wojtaszek 1997). The latter has been shown to accumulate under oxygen deprivation (Pfister-Sieber and Braendle 1994) and can represent a possible source for hypoxia-stimulated ROS production.

The next enzymatic step is the dismutation of the superoxide anion by SOD:

The reaction catalysed by SOD has a 10 000-fold faster rate than spontaneous dismutation (Bowler et al. 1992). The intracellular level of H ₂ O ₂ is regulated by a wide range of enzymatic reactions and those catalysed by catalase and peroxidases are the most important ones. Catalase functions through an intermediate catalase-H ₂ O ₂ complex (Compound I) and produces water and dioxygen (catalase action), or can decay to the inactive Compound II. In the presence of an appropriate substrate compound I drives the perioxidatic reaction. Compound I is a much more effective oxidant than H ₂ O ₂ itself, thus the reaction of Compound I with another H ₂ O ₂ molecule (catalase action) represents a one-electron transfer, which splits peroxide and produces another strong oxidant, the hydroxyl radical OH ^Ÿ (Elstner 1987). OH ^· is a very strong oxidant and can initiate radical chain reactions with organic molecules, particularly with polyunsaturated fatty acids (PUFA) in membrane lipids.

Lipoxygenase (LOX, linoleate:oxygen oxidoreductase, EC 1.13.11.12) reaction is another possible source of ROS and other radicals. It catalases the hydroperoxidation of PUFA (Rosahl 1995). The hydroperoxyderivatives of PUFA can undergo autocatalytic degradation, producing radicals and thus initiating a chain reaction of LP. In addition LOX-mediated formation of singlet oxygen (Kanofsky and Axelrod 1986) or superoxide (Lynch and Thompson 1984) has been shown.

Peroxidases, besides their main function in H ₂ O ₂ elimination, can also catalyse O ₂ ^{- Ÿ} and H ₂ O ₂ formation by a complex reaction in which NADH is oxidized using trace amounts of H ₂ O ₂ first produced by non-enzymatic breakdown of NADH. Next the NAD ^Ÿ radical formed reduces O ₂ to O ₂ ^{- Ÿ} , some of which dismutates to H ₂ O ₂ and O ₂ (Lamb and Dixon 1997). Thus, peroxidases and catalase play an important role in the fine regulation of ROS concentration in the cell through activating and deactivating H ₂ O ₂ (Elstner 1987).

Several other apoplastic enzymes may lead to ROS production under normal and stress conditions. Other oxidases, responsible for the two-electron transfer to dioxygen (amino acid oxidases and glucose oxidase) can contribute to H ₂ O ₂ accumulation. Also an extracellular germin-like oxalate oxidase catalyses the formation of H ₂ O ₂ and CO ₂ from oxalate in the presence of oxygen (Bolwell and Wojtaszek 1997). In barley roots the induction of the germin gene expression has been shown to occur in response to salt stress and after treatments with salicylate, methyl jasmonate and plant growth regulators (Hurkman and Tanaka 1996). Amine oxidases catalyse the oxidation of biogenic amines to the corresponding aldehyde with a release of NH ₃ and H ₂ O ₂ . Data on polyamine (putrescine) accumulation under anoxia in rice and wheat shoots (Reggiani and Bertani 1989) and predominant localisation of amine oxidase in the apoplast, suggest amine oxidase participation in H ₂ O ₂ production under oxygen deprivation.

H ₂ O ₂ is the first stable compound among ROS produced in the plant cell under normal conditions and as a result of stress. Hence, it is the most probable candidate for ROS-mediated signal transduction. This compound is relatively stable, is able to penetrate the plasma membrane as an uncharged molecule, and, therefore can be transported to the site of action (Foyer et al. 1997). Until now it is not clear how the organism senses ROS (see section 1.6.), but all models proposed discuss redox modification of cellular components and consider changes in O ₂ /ROS concentration as a primary event in the signalling cascade. To achieve signal specificity three important components of the signalling pathway via ROS have to be considered: 1. The source of the signal, 2. Target susceptibility (e.g. exposed SH groups of a protein or orientation of unsaturated fatty acids in the membrane), and 3. The antioxidant status of the cell (terminates the signal or allows it to proceed) (Alscher et al. 1997, Lander 1997). Thus, ROS action and the development of the stress response (adaptation) rely on a dynamic equilibrium between the rate of ROS production (concentration) and their utilisation. When the concentration of ROS exceeds the antioxidative capacity of the system oxidative stress occurs. The imposition of stress results in the elevation of ROS levels (Foyer et al. 1994, Alscher et al. 1997) and causes changes in the redox balance through the oxidation of metabolically active compounds leading to lipid peroxidation and degradation.

1.8. Lipid peroxidation

LP is a natural metabolic process under normal conditions. It can be divided into three stages: initiation, propagation and termination (Shewfelt and Purvis 1995). The initiation phase includes activation of O ₂ (see section 1.7.) and is rate limiting. Polyunsaturated fatty acids (PUFA, the main components of membrane lipids) are susceptible to peroxidation. LP is one of the most investigated consequences of ROS action on the membrane structure and function. The idea of LP as a solely destructive process has changed during the last decade. It has been shown that lipid hydroperoxides and oxygenated products of lipid degradation as well as LP initiators (i.e. ROS) can participate in the signal transduction cascade (Tarchevskii 1992).

Hydroxyl radicals and singlet oxygen can react with the methylene groups of PUFA forming conjugated dienes, lipid peroxy radicals and hydroperoxides (Smirnoff 1995):

The peroxyl radical formed is highly reactive and is able to propagate the chain reaction:

The formation of conjugated dienes occurs when free radicals attack the hydrogens of methylene groups separating double bonds and leading to rearrangement of the bonds (Fig. 3) (Recknagel 1984). The lipid hydroperoxides produced (PUFA- OOH) can undergo reductive cleavage by reduced metals, such as Fe ²⁺ , according to the following equation:

The lipid alkoxyl radical produced, PUFA- O ^· , can initiate additional chain reactions (Buettner 1993).

However, until now it is not quite clear whether peroxidation can be considered a cause of membrane damage and metabolic disorders, or a secondary effect of these processes. This problem arises from controversial observations concerning the mechanisms and products of LP in plant tissues. A comprehensive model for lipid peroxidation in plant tissues (Shewfelt and Purvis 1995) emphasizes the importance of chemical, rather than biochemical, processes in the oxidative stress. However, phospholipid hydroperoxides can be formed enzymatically via LOX reaction (Rosahl 1995). The multistage character of the process i.e. branching of chain reactions, allows several ways of regulation (Schwelt and Purvis 1995).

Among the regulated properties are the constitutive properties of the membranes (composition and organisation of lipids inside the bilayer in a way which prevents LP (Merzlyak 1989), the degree of PUFA unsaturation, mobility of lipids within a bilayer, localization of the peroxidative process in a particular membrane and the preventive antioxidant system (ROS scavenging and LP product detoxification) (Fig. 4).

1.9. Antioxidant system

To control the level of ROS and to protect cells under stress conditions, plant tissues contain several enzymes scavenging ROS (SOD, catalase, peroxidases) and a network of low molecular mass antioxidants (ascorbate, glutathione, phenolic compounds, tocopherols). In addition, a whole array of enzymes is needed for the regeneration of the active forms of the antioxidants (ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase). In the following chapters they are all presented separately .

1.9.1. Superoxide dismutase (SOD)

Because of high reactivity of OH ^· radicals (the main cause of cellular damage under oxidative stress), it is difficult to control their concentration enzymatically. Living organisms avoid the presence of this radical by controlling the upstream reaction of superoxide dismutation via SOD (eq. 7). The enzyme is present in all aerobic organisms and in all subcellular compartments susceptible of oxidative stress (Bowler et al. 1992). The three types of this enzyme can be identified by their metal cofactor: The structurally similar FeSOD (procaryotic organisms, chloroplast stroma) and MnSOD (procaryotic organisms and the mitochondrion of eucaryots); and structurally unrelated Cu/ZnSOD (cytosolic and chloroplast enzyme). Apart from their localisation, these isoenzymes differ in their sensitivity to H ₂ O ₂ and KCN (Bannister et al. 1987). All three enzymes are nuclear encoded, and SOD genes have been shown to be sensitive to environmental stresses, presumably as a consequence of increased ROS formation. This has been shown in an experiment with corn ( Zea mays ), where a 7-day flooding treatment resulted in a significant increase in TBARS content, membrane permeability and the production of superoxide anion-radical and hydrogen peroxide in the leaves (Yan et al. 1996). An excessive accumulation of superoxide due to the reduced activity of SOD under flooding stress was shown also (Yan et al. 1996). Similar results on SOD activity have been obtained for wheat and rice roots under anoxia (Chirkova et al. 1998).

1.9.2. Catalase and peroxidases

These enzymes execute the next step in the detoxification of ROS: The elimination of excess H ₂ O ₂ , and, as discussed above (see section 1.7), participate in the fine regulation of the H ₂ O ₂ -concentration in the cell.

1.9.3. Phospholipid hydroperoxide glutathione peroxidase

Phospholipid hydroperoxide glutathione peroxidase (PXGPX) is a key enzyme in the protection of the membranes exposed to oxidative stress and is inducible under various stress conditions. The enzyme catalyses the regeneration of phospholipid hydroperoxides at the expense of GSH (13) and is localised in the cytosol and the inner membrane of mitochondria of animal cells. PXGPX can also react with H ₂ O ₂ but this is a very slow process.

Until know, most of the investigations have been performed on animal tissues. Recently, a cDNA clone homologous to PHGPX has been isolated from tobacco, maize, soybean, and Arabidopsis (Sugimoto et al. 1997). The PHGPX protein and its encoding gene csa have been isolated and characterised in citrus. It has been shown that csa is directly induced by the substrate of PHGPX under the conditions of heat, cold and salt stresses, and that this induction occurs mainly via the production of ROS (Avsian-Kretchmer et al. 1999).

1.9.4. Enzymes regenerating active forms of ascorbate and glutathione

To prevent LP, H ₂ O ₂ can also be removed by the Halliwell-Asada pathway (Fig. 5), originally described in the chloroplasts (Nakano and Asada 1980). In a recent investigation on the relationship between H ₂ O ₂ metabolism and the senescence process in mitochondria and peroxisomes Jumenez et al. (1998) have indicated the presence of ascorbate-glutathione cycle components in both organelles. Ascorbate-specific peroxidase, which actually reacts with H ₂ O ₂ , works in cooperation with dehydroascorbate reductase, glutathione and glutathione reductase with NADPH as a donor of reducing equivalents. The reaction may be of particular importance under hypoxic conditions, since it allows NADP ⁺ regeneration. The latter is implicated in ATP production via glycolysis.

1.9.5. Redox active compounds: ascorbate and glutathione

Ascorbic acid has an ability to scavenge a wide range of ROS: Superoxide anion, singlet oxygen and H ₂ O ₂ , and acts as a chain-breaking antioxidant (Beyer 1994). However, in the presence of metal ions and at high concentrations of ascorbate, it can act as a pro-oxidant (Foyer et al. 1991). Under these conditions hydroperoxyl and superoxide radicals can be formed in vitro (Halliwell and Gutteridge 1989). The autooxidation of ascorbate proceeds with the formation of an intermediate superoxide anion:

As a water soluble compound ascorbic acid functions most efficiently in the aqueous phase of the cell, and is able to carry out the non-enzymatic regeneration of -tocopherol (TOH) from the a -tocopheroxyl radical (TO ^Ÿ ) in the hydrophobic surroundings (Beyer 1994). Besides, ascorbate takes part in the regulation of the cell cycle by affecting the progression from G1 to S phase, and it has been implicated in the regulation of cell elongation (Smirnoff 1995).

Glutathione is a potent cellular reductant with a broad redox potential. It acts as a scavenger of peroxides and serves as a storage and transport form of reduced sulphur (May et al. 1998). It has been shown also that glutathione acts as a regulator of gene expression (Alscher 1989, Baier and Dietz 1997), and is a precursor of phytochelatins (Grill et al. 1989). Due to the redox active thiol group GSH may be involved in the regulation of the cell cycle and can act as a defence compound against oxidative stress. GSH has been shown to participate in the regeneration of the reduced form of ascorbate through non-enzymatic reduction of DHA at an alkaline pH (Noctor et al. 1998).

1.9.6. Phenolic compounds

Polyphenols possess ideal structural chemistry for free radical scavenging activity, and they have been shown to be more effective antioxidants in vitro than tocopherols and ascorbate. Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors, and from the ability of the polyphenol-derived radical to stabilize and delocalise the unpaired electron (chain-breaking function), and from their ability to chelate transition metal ions (termination of the Fenton reaction) (Rice-Evans et al. 1997). Another mechanism underlying the antioxidative properties of phenolics is the ability of flavonoids to alter peroxidation kinetics by modification of the lipid packing order and to decrease fluidity of the membranes (Arora et al. 2000). These changes could sterically hinder diffusion of free radicals and restrict peroxidative reactions. Moreover, it has been shown recently that phenolic compounds can be involved in the hydrogen peroxide scavenging cascade in plant cells (Takahama and Oniki 1997). According to our unpublished results (R. Tegelberg) the content of condensed tannins (flavonols) as measured by HPLC, was 100 times higher in I. pseudacorus rhizomes in comparison with that of I. germanica . The effect of anoxia on the flavonol content (a decrease after 35 days of treatment) suggests their participation in the antioxidative defence in I. pseudacorus rhizomes.

1.9.7. Tocopherols

Tocopherols (Fig. 5) are unique antioxidants in carrying antioxidant functions for the detoxification of several types of ROS: quenching of singlet oxygen, direct reaction with OH ^· radical, interaction with other free radicals i.e. termination of the chain via production of the unreactive tocopheryl radical (Kamal-Eldin and Appelqvist 1996, Kagan 1989). Due to the low rate constant of chain propagation (equation 10), tocopherol can compete with this reaction to "repair" PUFA- OO ^· , forming lipid hydroperoxides, PUFA- OOH:

The optimal concentration ratio of TOH to PUFA- H in the membranes is estimated at 1:1000, thus one tocopherol molecule is able to protect 1000 lipid molecules from the chain propagation step in equations 10 and 14 (Buettner 1993). Since tocopherol functions in the lipid phase of the cell protecting membrane structures; its ability to synergetically interact with the water-soluble ascorbate provides the basis for overall cellular defence (Fig. 4). Ascorbate recycles tocopherol via TO ^· , producing the ascorbate radical:

AA ^-· can be removed by dismutation, yielding AA- H ^- and dehydroascorbate (DHA). Both DHA and AA ^-· can be reduced by enzyme systems, which use NADH or NADPH as sources of reducing equivalents (Buettner 1993).

Tocopherols are not evenly distributed in cell membranes. There are membrane domains in biological membranes where lipid composition and fluidity differ from the other parts of the membrane. It has been suggested that tocopherols are accumulated in the most fluid membrane domains containing most of the unsaturated fatty acids of the membrane. There are two proposed mechanisms, which compensate the low tocopherol concentration in cell membranes: First, the accumulation of tocopherols into the most fluid membrane domains supports the antioxidant function of tocopherols in protecting the PUFAs against lipid peroxidation. Secondly, studies have revealed that tocopherols move rapidly in the lateral plane of the lipid bilayer, hence being able to move to parts of the membrane where they are needed (Gomez-Fernandez et al. 1989).

In addition, tocopherols have non-antioxidant functions in cell membranes which are less known. They seem to regulate membrane structures by modifying membrane permeability and phase transition (Wassal et al. 1986). It has been demonstrated that tocopherols can protect biological membranes against phospholipases and their hydrolysis products, free fatty acids and lysophospholipids, which are characteristically produced in large amounts in several stress situations such as hypoxia and ischemia (Kagan 1989). a -Tocopherol forms stable complexes with free fatty acids and lysophospholipids which stabilize the membrane structure. There are differences in complex-formation efficiency between tocopherol isomers. The efficiency in complex-formation is in order of a > b> g> d , which is suggested to explain the differing biological activities of tocopherol-isomers in vivo (Fryer 1992). It is noteworthy that a -tocopherol is a more lipophilic tocopherol isomer than b -, g - or d -tocopherol due to its three methyl substituents attached to the phenolic ring (Kamal-Eldin and Appelqvist 1996). a -Tocopherol is localised deeper in the membrane core than other tocopherol isomers, and it is possible that the different localisation in membrane has some role in the efficiency of a -tocopherol to form complexes with free fatty acids or lysophospholipids. It has been suggested that a -tocopherol can prevent or even abolish the disordering effects of free fatty acids and lysophospholipids due to the formation of complexes within the membrane. Presence of the double bonds in the acyl chain of free fatty acids or lysophospholipids enhanced the interaction between the chromanol head group of a -tocopherol and the acyl chain.

Membrane hydrolysis products disturb also membrane proteins. Studies have revealed that a -tocopherol is also able to eliminate the modifying effects of free fatty acids on intrinsic membrane proteins (Kagan 1989). It has been suggested that a fundamental part of the biological action of tocopherols is due to their ability to physically stabilise membrane structures (Fryer 1992).

1.10. Antioxidative network

It is very important for plant survival under stress conditions that antioxidants can work in co-operation, thus providing better defence and regeneration of the active reduced forms. Ascorbate and glutathione remove H ₂ O ₂ via the Halliwell-Asada pathway (Fig. 6). Ascorbate works in co-operation not only with glutathione, but also maintains the regeneration of a -tocopherol, providing synergetic protection of the membranes (Thomas et al. 1992). Recently, redox coupling of plant phenolics with ascorbate in the H ₂ O ₂ -peroxidase system has been shown. This acts in the vacuole, where H ₂ O ₂ diffuses and can be reduced by peroxidases using phenolics as primary electron donors. Phenoxyl radicals generated by this oxidation can be reduced by both AA and the monodehydroascorbic acid radical. If regeneration of AA is performed in the cytosol and AA is supplied back to the vacuole, a peroxidase/phenolics/AA system in vacuoles could function to scavenge H ₂ O ₂ (Yamasaki and Grace 1998). This mechanism is specific for plant tissues and can improve stress tolerance under oxidative stress.

1.11. Antioxidant status of the cell under stress conditions

Data on antioxidant levels and the activity of antioxidant enzymes are somewhat contradictory, both decreases and increases in antioxidative capacity of the tissues have been reported. Such diversification partly arises from the response specificity of a particular plant species and from different experimental conditions (stress treatment, duration of stress, assay procedure and parameters measured). A large-scale investigation on monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) activities, and AA and GSH contents in 11 species with contrasting tolerance to anoxia has revealed an increase in MDHAR and/or DHAR in the anoxia tolerant plants after several days of anoxic treatment. In the intolerant plants activities were very low or without any changes. GSH decreased significantly during the post-anoxic period, while AA showed increased values in the tolerant species (Wollenweber-Ratzer and Crawford 1994). In anaerobically germinated rice seedlings a 3-fold increase in tocopherol and low TBARS formation has been observed (Ushimaru et al. 1994). However, an anoxia-induced elevation in the tocopherol level observed in the anoxia-intolerant wheat seedlings could not be detected in rice seedlings subjected to anoxia ( I ). In Iris spp. both a - and b -tocopherol levels decreased only after long-term anoxia ( III ). An investigation on the antioxidative defence system in the roots of wheat seedlings under root hypoxia or whole plant anoxia (Biemelt et al. 1998) has revealed a significant increase in the reduced forms of ascorbate and glutathione. Nevertheless, a rapid decrease in the redox state of both antioxidants was observed during reaeration. The activities of monodehydroascorbate reductase, dehydroascorbate reductase and GR decreased slightly or remained unaltered under hypoxia, while anoxia caused a significant inhibition of enzyme activities (Biemelt et al. 1998). Inhibition of glutathione reductase (GR), ascorbate peroxidase, catalase and superoxide dismutase (SOD) activities has been shown also by Yan et al. (1996) in corn leaves under prolonged flooding, while a short term treatment led to an increase in the activities. Considering the above-mentioned data, it is difficult to delineate a universal mechanism for the whole antioxidant system response to anoxia. Of course, it may be probable that there is no such mechanism, and other factors are involved in the protective machinery of plants. Metabolic changes specifically induced by anoxia may alter the antioxidant status of the tissue.

1.12. Role of mitochondria in stress response

Dependence of plant survival on energy metabolism under environmental stress and the central role of mitochondria are well established. However, mitochondrial functions in the stress response are not limited only by the energy supply. The phenomenon of permeability transition in the inner membrane of mitochondria may be a possible link between the perception of the stress signal and the adaptive response.

Mammalian mitochondria contain an inner membrane channel, the permeability transition pore (PTP), that, when fully open, permits free diffusion of solutes with a molecular mass of up to 1500 Da (Bernardi et al. 1994, Zoratti and Szabo 1991). The PTP is controlled by several ligands as well as by the electrical potential across the membrane; high values favour the closed state, while dissipation of the potential increases pore opening probability (Bernardi 1992). The same effect on PTP has been observed under alkaline pH and elevated levels of reactive oxygen species (ROS). High matrix Ca ²⁺ -concentrations, inorganic phosphate and oxidation of intramitochondrial pyridine nucleotides promote pore opening, whereas ADP, H ⁺ and cyclosporin A (a cyclic immunosuppressant peptide) cause inhibition. In mammalian mitochondria oxygen depletion has been shown to open the PTP. Such factors as a decrease in the inner membrane potential, formation of ROS in ETC and Ca ²⁺ uptake by mitochondria have been shown to promote pore opening. Anoxia-induced metabolic changes in plant cells create similar conditions, and hence provide an opportunity for PTP induction.

The physiological function of the permeability transition pore is not fully understood, but circumstantial evidence suggests that it is involved in Ca ²⁺ homeostasis (Bernardi and Petronilli 1996, Ichas et al. 1997) and linked to stress sensing through the programmed cell death. There is some evidence that a similar pore, which is regulated partly in a different manner, is to be found in yeast cell mitochondria (Jung et al. 1997). There is also some evidence that a similar pore is present in plant mitochondria (Vianello et al. 1995).

1.13. Aims of the present study

Data accumulated on the generation of ROS under various stress conditions, and their involvement in the regulation of the adaptive response and effects on metabolically active macromolecules and cell structures, are important for understanding stress physiology and the fine mechanisms underlying stress tolerance. Oxygen deprivation, by its nature, seems to occupy a special place in the general scheme for ROS participation in the stress response. However, changes in membrane lipid structure and function, disturbances in membrane integrity and other anoxia induced changes suggest peroxidative damage (LP), especially after re-admission of oxygen. The hypothesis that oxygen concentration and ROS may be recognised by the cell through the same sensing mechanism (Semenza 1999), makes anoxic stress an important model for the investigation of this mechanism.

The main goals of the present study are to delineate the development of stress reaction in the time-course of anoxia and during reoxygenation, and to elucidate the protective mechanisms underlying higher tolerance to anoxia. Special emphasis is placed on the evidence of ROS generation under oxygen deprivation and under reoxygenation, on the development of membrane LP and on the role of antioxidant systems in a range of plant species with different tolerance to anoxia. An attempt is undertaken to characterise mitochondrial functions during the transition to anoxia and to identify a permeability transition pore in the inner mitochondrial membrane. To complete the study the following experimental approaches were chosen:

-direct visualisation of H ₂ O ₂ under oxygen deprivation and reoxygenation by means of transmission electron microscopy for the localisation of the sites of ROS generation.

-investigation of membrane LP at different stages of the process: Propagation phase (conjugated diene and triene formation) and termination phase (TBARS accumulation) in order to characterise the functional state of the membranes.

-estimation of the impact of post- anoxic reaeration on peroxidative processes by the determination of TBARS content in anoxically treated samples under a nitrogen atmosphere in an air-tight chamber; H ₂ O ₂ visualisation under the same experimental conditions.

-evaluation of the role of the antioxidant system in LP regulation and anoxia-tolerance by the measurement of SOD activity, changes in the redox state and content of the hydrophilic antioxidants ascorbate and glutathione, and by determination of the hydrophobic tocopherol concentration.

-characterisation of mitochondrial functions under anoxic conditions, and of the possible involvement of PTP in the stress response by measurement of O ₂ consumption, Ca ²⁺ transport, changes in the membrane potential and alterations in mitochondrial volume.

The parameters studied are discussed in a framework of anoxia-induced metabolic changes in order to integrate the data into a general sequence of events representing stress response to provide better understanding of anoxia tolerance.