Activities of antioxidants in plants under environmental stress.pdf

Activities of antioxidants in plants Akram Ali and fahad Alqurainy

Activities of antioxidants in plants under environmental stress

Akram Ali* Fahad Alqurainy Department of Botany and Microbiology, Faculty of Science, King Saud University, P.O. box 2455, Riyadh 11451, Kingdom of Saudi Arabia. *Corresponding author Permanent address: Department of Botany, Faculty of Science, Zagazig University, Zagazig, Egypt. E-mail: [email protected] Abstract

There is growing evidence that in plants subjected to environmental stress. It causes significant crop losses. The stresses are numerous and often crop- or location-specific. They include increased UV-B radiation, water, high salinity, metal toxicity, herbicides, fungicides, air pollutants, light, temperatue, topography and hypoxia (restricted oxygen supply in waterlogged and compacted soil),. Research in this area is driven by the hope of improving crop yield in afflicted areas. The balance between the production of activated oxygen species and the quenching activity of antioxidant is upset which often results in oxidative damage. Many metabolic processes produce active oxygen species. Among the four major active oxygen species [superoxide radical O-

2, hydrogen peroxide H2O2, hydroxyl radical OH and singlet oxygen 1O2] H2O2 and the hydroxyl radical are most active, toxic and destructive. High salt concentrations normally impair the cellular electron transport within the different subcellular compartments and lead to the generation of reactive oxygen species (ROS) such as singlet oxygen superoxide, hydrogen peroxide and hydroxyl radicals. Excess of ROS triggers phytotoxic reactions such as lipid peroxidation, protein degradation and DNA mutation. Since higher plants are immobile they cannot escape environmental stresses. The ability of higher plants to scavenge the toxic active oxygen seems to be a very important determinant of their tolerance to environmental stress. Many enzymes and secondary compounds of higher plants have been demonstrated in vitro experiments to protect against oxidative damage by inhibition or quenching free radicals and reactive oxygen species. The roles of many other compounds as potential antioxidants can be inferred by similarity to synthetic antioxidants of related structure. The evidence supports at least a partial antioxidant role in vivo for many classes of plant metabolite. Key words: Environmental stresses, crop losses, plant adaptation, antioxidant defense.

Most environmental stresses are affecting on the production of active oxygen species in plants, causing oxidative stress (1,2,3). Also, there is growing evidence that in plants subjected to environmental stress. The balance between the production of activated oxygen species and the quenching activity of antioxidant is upset, which often results in oxidative damage (4,5,1,6).

Environmental stress causes significant crop losses. The stresses are numerous and often crop- or location-specific. They include increased UV-B radiation, water, high salinity, temperature extremes, hypoxia (restricted oxygen supply in waterlogged and compacted soil), mineral nutrient deficiency, metal toxicity, herbicides, fungicides, air pollutants, light, temperature and topography. Research in this area is driven by the hope of improving crop yield in afflicted areas. Currently, real, but slow advances are being made by crop breeders

Activities of antioxidants in plants Akram Ali 2

and agronomists using tried-and-tested methodology; however, biotechnology will increasingly have a role as genes involved in stress resistance arc cloned and their mode of action elucidated (7).

It is apparent that many environmental stresses exert at least part of their effect by causing oxidative damage (8). Consequently, the antioxidant defense system of plants has been attracting considerable interest (9). Characterization of mutants and transgenic plants with altered expression of antioxidant is a potentially powerful approach to understanding the functioning of the antioxidant system and its role in protecting plants against stress, and significant progress is now being made in this area.

Atmospheric oxygen has been recognized for more than 100 years as the agent responsible for the deterioration of organic materials exposed to air. The parallel role of oxygen, a molecule essential form many forms of life, as a destructive (toxic) agent for living tissues has been discovered much more recently. Even under optimal conditions many metabolic processes produce active oxygen species. Among the four major active oxygen species [superoxide radical O-

2, hydrogen peroxide H2O2, hydroxyl radical OH and singlet oxygen 1O2] H2O2 and the hydroxyl radical are most active, toxic and destructive (1). In plants the most important of these are driven by or associated with light dependent events. Photosynthetic cells are prone to oxidative stress because they contain an array of photo-sensitizing pigments and they both produce and consume oxygen. The photosynthetic electron transport system is the major source of active oxygen species in plant tissues (10), have the potential to generate singlet oxygen 1O2 and superoxide O-

2.

Olga et al. (11) concluded that generation of reactive oxygen species (ROS) is characteristic for hypoxia and especially for reoxygenation. Of the ROS, hydrogen peroxide (H2O2) and superoxide (O2

--) are both produced in a number of cellular reactions, including the iron-catalysed Fenton reaction, and by various enzymes such as lipoxygenases, peroxidases, NADPH oxidase and xanthine oxidase. The main cellular components susceptible to damage by free radicals are lipids (peroxidation of unsaturated fatty acids in membranes), proteins (denaturation), carbohydrates and nucleic acids. Consequences of hypoxia-induced oxidative stress depend on tissue and/or species (i.e. their tolerance to anoxia), on membrane properties, on endogenous antioxidant content and on the ability to induce the response in the antioxidant system. Effective utilization of energy resources (starch, sugars) and the switch to anaerobic metabolism and the preservation of the redox status of the cell are vital for survival (12). The formation of ROS is prevented by an antioxidant system: low molecular mass antioxidants (ascorbic acid, glutathione, and tocopherols), enzymes regenerating the reduced forms of antioxidants, and ROS-interacting enzymes such as SOD, peroxidases and catalases (12).

In plant tissues many phenolic compounds (in addition to tocopherols) are potential antioxidants: flavonoids, tannins and lignin precursors may work as ROS-scavenging compounds (11). Antioxidants act as a cooperative network, employing a series of redox reactions. Interactions between ascorbic acid and glutathione, and ascorbic acid and phenolic compounds are well known. Under oxygen deprivation stress some contradictory results on the antioxidant status have been obtained. Experiments on overexpression of antioxidant production do not always result in the enhancement of the antioxidative defense, and hence increased antioxidative capacity does not always correlate positively with the degree of protection (12). Here we present a consideration of factors which possibly affect the effectiveness of antioxidant protection under oxygen deprivation as well as under other environmental stresses. Such aspects as compartmentalization of ROS formation and antioxidant localization, synthesis and transport of antioxidants, the ability to induce the antioxidant defense and cooperation (and/or compensation) between different antioxidant systems are the determinants of the competence of the antioxidant system (11).

In this review my aim is to concentrate on current investigations of the environmental stresses, basis of stress resistance and on the potential of plants to improve functions by making stress resistance.


1. Environmental stresses impact 1.1. Ultraviolet stress:

Sunlight contains energetic short wavelength ultraviolet (UV) photons which are potentially detrimental because of their destructive interactions with many cellular molecules, such as the amino acids of essential proteins, nucleic acids bases or membrane lipids (1). Intense light has long been known to disrupt metabolic processes in plants, including photosynthesis, respiration glucose assimilation, and phosphorylation (13). Approximately 4% of the total energy contained in sunlight occurs in the ultraviolet region (wavelengths shorter than 400 nm). The intensity of UV irradiance at the earth's surface varies greatly with season, time of day, latitude) ozone layer thickness, altitude, and cloud cover. Distinctions are sometimes made between the UV-A (400-320nm) and the UV-B (320-290nm) regions. In both cases, however, the fundamental mechanisms of photochemical damage are similar although different receptor molecules (chromophores) may be involved.

UV-B damages DNA by causing oxidative cross linking between adjacent pyrimidine bases forming cyclobutane pyrimidine dimers and pyrimidine pyrimidone dimmers (14). Unless repaired, these block transcription and replication. Repair is achieved by light-activated photolyases which reduce the dimers in a light-dependent manner. Mutants deficient in photolyase activity have been isolated in rice (15) and Arabidopsis (uvr2) (16). Both are UV-B sensitive and unable to repair cyclobutane pyrimidine dimers. The photolyase (PHR1) from Arabidopsis has been cloned by PCR using primers based on animal type II photolyases. Furthermore, PHR1 and uvr2 were shown to be the same by PCR, the mutant having a single base pair deletion (17). Identification of the photolyase gene opens the way for investigating the consequences of its overexpression on UV-B resistance.

There have been many reports on deleterious physiological effects on plants exposed to high levels of UV-B which may increase if stratospheric ozone concentrations decrease. The destructive action of UV irradiation results from both direct and indirect mechanisms involving endogenous sensitizers and the generation of active oxygen species. Physiological and biochemical effects of UV-B radiation include effects on enzymes, stomatal, resistance concentrations of chlorophyll, protein and lipid, reduction in leaf area, and tissue damage (18

,19). Some plants, however, appears to be quite resistant to increased UV irradiation. The

differential susceptibility of plants to UV stress is clearly an important factor in their competitive relationships in terrestrial ecosystems (18); experiments with agriculturally important species pairs grown in pots have indicated that significant effects on biomass production took place when UV-B was present either at ambient or artificial increased levels. Photochemical damaging events in cells are initiated by the uptake of the electronic energy of a photon by a UV absorbing molecule (19). In the UV region of the electromagnetic spectrum, the energy of such photons is sufficient to break covalent bonds, although it is unusual for their energy converts the target molecules in its ground state to an electronically excited state whose excess energy manifests itself in a different and often quite unstable electron configuration. The initial excited state ,a short -a short- lived singlet haning, fully paired electrons, may be deactivated by fluorescence (emission of a photon having a longer wavelength than the exciting radiation) and return to the ground state ,it nay react with neighboring molecules (although this is not common with singlet since their lifetime are normally too short for them to diffuse over very many molecular diameters) or it may undergo internal rearrangement to a longer lived excited state .The triplet state is much more likely to react chemically with surrounding molecules (18 ,19). 1.2. Water stress:

Water stress is perhaps the most prevalent cause of crop yield loss but also the most difficult to tackle because of the strong link between transpiration and photosynthesis. Gene


expression and signal transduction in water stressed plants has been recently reviewed (20). There is evidence for a mitogen-activated protein kinase type system in plants analogous to that involved in yeast osmoregulation. In support of such a system, a protein kinase is rapidly activated in maize roots exposed to low water potential (21). The role of dehydrins, late embryogenesis proteins and related proteins, which accumulate in seeds and water-stressed vegetative tissues, has been reviewed (22). Transgenic rice expressing HVA1, a gene encoding a late embryogenesis abundant protein from barley, has increased tolerance to drought and NaC1 as shown by simple growth analysis (23). Aquaporins (water channel proteins) are clearly involved in controlling water movement between cells (24) and may be a target for manipulating water flow through the plant with potential for improving water relations and water use efficiency.

On exposure to osmotic stress as a result of drought, high salinity and low temperature plants accumulate a range of metabolically benign solutes, collectively known as compatible solutes or osmolytes. Their primary function is turgor maintenance but they may have other protective effects on macromolecules in dehydrating cells. The solutes accumulated vary between species and include proline, betaines, dimethylsulfonioproprionate (DMSP), polyols (mannitol, sorbitol, and pinitol), trehalose, and fructans. Over the past five years, a number of transgenic plants have been produced in which overaccumulation occurs (e.g. proline) or in which the ability to accumulate osmolytes not previously present has been introduced. The results suggest that they can improve plant growth during osmotic stress even at osmotically-insignificant levels (8). Glycine betaine (GB) is accumulated by a taxonomically restricted range of species. In higher plants, it is synthesised from choline via betaine aldehyde using choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BALDH) in chloroplasts (25). Some bacteria, however, convert choline to betaine in a one step reaction catalysed by choline oxidase. CodA, which encodes choline oxidase from Arthrobacter globiformis, has been expressed in Arabidopsis, a non-betaine accumulator. The gene had a chloroplast targetting sequence. The average leaf concentration was low but, if chloroplast localised, would be 50 mM, which approaches an osmotically-significant concentration. The transgenic plants, judged by photographs and measurement of plant length, were more tolerant to NaC1 and continuous light at 5°C (26). Cytoplasmic targeting had less effect on tolerance. The same group also showed that the cyanobacterium Synechococcus, transformed with the same gene, was also more tolerant to low temperature-induced photo-inhibition and provided evidence that GB affected membrane phase transitions and accelerated recovery of photo-inhibition (27). There is a report that expression of Atriplex hortensis BALDH in rice increases GB content and salinity tolerance (28). Application of GB in foliar sprays is reported to improve the growth of water-stressed tobacco in laboratory experiments and maize, sorghum and soybean crops in the field (29,30,31). These interesting observations need more investigation, perhaps, to rule out the possibility that improved growth is caused by improved nitrogen supply. Contrary to this, exogenous GB is apparently toxic to Brassica napus, a non accumulator (32). Higher plant BALDH has been cloned and this has now been followed by cloning of CMO. Like BALDH, CMO expression is upregulated by NaCI. It has a Rieske-type [2Fe-2S] cluster and it is ferredoxin-dependent and, therefore, it represents a novel type of plant oxidase (25). It is suggested that metabolic engineering of GB synthesis with plant BALDH and CMO would be preferable to using choline oxidase because the plant-derived genes may also have promotors, which could drive increased expression during water stress (25). Furthermore, CMO uses chloroplast ferredoxin as reductant, thus linking timing of high stress in the light to betaine synthesis (25). BALDH is equally efficient at catalysing oxidation of 3-dimethylsulfonioproprionaldehyde to DMSE a compatible solute of even narrower distribution than GB (33) and ability to synthesise DMSP could have evolved by co-opting this enzyme. Transgenic tobacco expressing beet BALDH also had enhanced dehydrogenase activity towards 3-dimethylsulfonioproprionaldehyde and two other aldehydes, confirming the view that BALDH is a multisubstrate enzyme (34). BALDI-I expression may be more widespread than GB accumulation. BALDH is expressed in rice (non-GB accumulator) and the rice gene


has high homology to barley BALDH. Unlike BALDH in betaine accumulating plants, the rice enzyme is located in peroxisomes. Feeding betaine aldehyde dehydroganse to rice plants increases their GB content, and on the basis of photographic evidence, improves the growth of the plants at high salinity and low water content (35).

Evidence suggests that drought causes oxidative damage through generation of oxygen radicals or inhibition of antioxidant systems in plant (36,37,38). Drought related physiological changes such as a decrease in leaf water and stomatal closure, result in limited CO2 availability to the channeling of reducing equivalents to the production of active oxygen species rather CO2 fixation (39). Among the four major active oxygen species [superoxide radical O-

2, hydrogen peroxide H2O2, hydroxyl radical OH and singlet oxygen 1O2] H2O2 and the hydroxyl radical are most active toxic and destructive (1). Hydrogen peroxide can be produced by either dismutation of O-

2 by SOD or photorespiration. 1.3. Salinity stress:

Height salt concentration normally impair the cellular electron transport within the different subcellular compartments and lead to the generation of reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radicals (40,45,46). Excess of ROS triggers phytotoxic reactions such as lipid peroxidation, protein degradation and DNA mutation (47,48,49).

Mannitol is accumulated by a wide range of species in response to salinity (50). Mannitol synthesizing ability was introduced into transgenic tobacco by the E. coli mtlD gene encoding Mannitol dehydrogenase. These plants accumulated modest amounts of mannitol but were said to be more salt-tolerant (51). Bohnert and co-workers (52) have now produced transgenic tobacco in which mtlD expression is targeted to the chloroplast. A concentration of 100mM was estimated. It has been suggested that compatible solutes, including mannitol, could be antioxidants by scavenging hydroxyl radicals (OH) (56,58). This might be significant for plants exposed to drought and high salinity as there is strong evidence that oxidative generation of active oxygen species increases under such conditions (59). The chloroplast-targetted mannitol accumulating tobacco has been used to test this hypothesis. Mannitol accumulation did not affect photosynthesis. The transgenic plants were more tolerant to OH" generated in chloroplasts by methyl viologen treatment. The OH* content of transgenic plants was also lower (52) suggesting that the protection could result from OH* scavenging by mannitol. In a further paper (53), the same group have provided convincing evidence that a key target for OH* produced by illuminated thylakoids is the Calvin cycle enzyme phosphoribulokinasc (regulated by thiol-disulfide interconversion). This inactivation, in mixtures containing thylakoids and phosphoribulokinase, was prevented by mannitol and could explain the in vivo protective effect of mannitol. Further support for the efficiency of mannitol acting as an OH* scavenger in vivo has been provided by transformation of Saccaromyces cerevisiae with the same mtlD gene. A mutant unable to grow at high osmolarity because of inability to synthesize glycerol, the normal osmoregulatory solute of yeast, had this ability restored by introduction of mannitol synthesis capacity. Furthermore, the transgenic yeast was also more tolerant to chemically-generated OH" in the growth medium (60). These studies suggest osmolytes could indeed have multiple functions and could explain the protective effects observed at osmotically insignificant concentrations (61). 1.4. Metals stress:

Accumulation of phytotoxic metal results from industrial and agricultural practices. The Zn, Cu, and Cd are widespread pollutants resulting in stunted growth, chlorosis and necrosis (63,65). Copper Cu2 ions cause light mediated lipid peroxidation and pigment bleaching (66,67). Prolonged exposure to CuSO4 resulted in chlorophyll bleaching in rye and the endogenous CAT level declined (70). Thus enhanced susceptibility to photooxidative damage was related to the rapid loss of CAT activity, Cu2 ions are redox active and catalyze fentonperoxides also originate from the induction of lipoxygenase in the presence of Cu2. This


enzyme is known to initiate lipid peroxidation. Cadmium treatment, decrease the chlorophyll and heme levels of germinating mung

bean seedlings by induction of lipoxgenase with the simultaneous inhibition of the antioxidative enzyme SOD and CAT (71). Such inhibition results from binding of the metal to the important sulfhydryl groups of enzymes, which exacerbates the phytotoxic action of metals (72).

Pang et al. (74) studied improving the plant ability to resist lead stress, effect of 0.05 mg/L La (NO3)3 on the activities of catalase (CAT), superoxide dismutase (SOD), the level of malondialdehyde (MDA) in wheat seedlings under lead stress were studied. The effect of La+3 on plant growth, chlorophyll content in wheat seedlings after adding 0, 50, 100 mg/l Pb (NO3)3 to the nutrient solution for 12 days was observed. The plants were grown in nutrient solution in a strictly controlled climate growth room. Effects of La+3 (with La treatment) compared with check groups was evidently observed. The activities of SOD and CAT in root were enhanced 0.45–1.69 times and 33.20–77.77% respectively and MDA content was reduced 11.05–27.49% in root after treatments from the second day till the end of the experiment. The activities of SOD and CAT was found to be increased slightly (P < 0:05) and MDA content decreased in shoot and root of wheat seedlings by La+3 under lead stress within five days after treatments compared with Pb1 and Pb2 groups. It was assumed that antioxidant enzymes was found to be increased by La (NO3)3, the antioxidant potential of the wheat seedlings to resist lead stress enhanced. It is suggested that La+3 could be used to resist lead stress at the beginning under stress while the stress was not so serious. 1.5. Herbicides stress:

Several herbicides have been found to generate active oxygen species, either by direct involvement in radical production or by inhibition of biosynthetic pathways. The generation of the hydrocarbon gas ethane, the production of malonaldehyde and changes in electrolytic conductivity has frequently been used as sensitive markers for herbicide action in plants (75,76). The bipyridinium and diphenyl ether herbicides have been the most insensitively investigated in terms of their oxidative action in plants.

The bipyridinium herbicides generate oxygen radicals directly in the light. Compounds such as paraquat (also known as methyl viologen) induce light dependent oxidative damage in plants. Members of this group are called total kill herbicids (77). The di-cationic nature of these compounds facilitates their reduction to radical cation. The PSI-mediated reduction of the paraquat di-cation results in the formation of a mono-cation radical which then reacts with molecular oxygen to produce O-

2 with the subsequent production of other toxic species, such as H2O2 and OH (44). The diphenyl ethers, cylic imides and lutidine derivative, act by inhibition biosynthetic pathways with the subsequent accumulation of reactive radical-forming intermediates. These compounds cause severe toxicological problems and results in peroxidation of membrane lipids and general cellular oxidation.

The mode of action of these herbicides is based on the ability to induce the abnormal accumulation of photosensitizing tetrapyrroles specifically protoporphyrin IX (67). This pigment is able to cause light dependent generation. These herbicides can also catalyze the oxidation of protoporphyrinogen to protoporphyrin IX. The penultimate step of both heme and chlorophyll biosynthesis is recorded (79). It is some what anomalous that the reaction product protoporphyrin IX accumulates in condition where the enzyme which catalyses its formation is expected to be inhibited.

Other compounds such as diuron, that block photosynthetic electron transport and inhibitors of cartenoids biosynthesis, such as norflurazon, initiate photooxidative processes most probably via the generation of 1O2 (78,80). Herbicides which block photosynthesis cause increased excitation energy transfer from triplet chlorophyll to oxygen while those inhibit carotenoid biosynthesis eliminate important quenchers of the triplet chlorophyll and 1O2.


1.6. Fungicides stress:

A number of agricultural chemicals such as fungicides (91) and herbicides (92) have been shown to possess antiozonant properties. However, most of these investigations have concentrated on the effectiveness of agrochemicals in preventing plants from ozone injury, and relatively little work has focused on the physiological and biochemical modes of action.

The strobilurins are an important new class of fungicides with a unique mode of action which targets mitochondrial respiration in fungi. Few studies on the physiologic effects of strobilurins on and in plants showed that strobilurins increased grain yields, dry matter, chlorophyll and protein contents and delayed senescence (95). 1.7. Air pollutant stress:

Atmospheric pollutants such as ozone (O3) and sulfur dioxide (SO2) have been implicated in free radical formation (96,99) and are considered to be one of the major factors influencing modem forest decline. Ozone, which originates from a natural photochemical degradation of nitrous oxides (NO2), seems to be a greater threat to plants than pollution (102). Mehlhorn et al. (99) suggested that the phytotoxicity of O2 is due to its oxidizing potential and the consequent formation of radicals that induce free radical chain reactions. The O3 concentration in the intercellular air spaces of leaves is close to ozone (103). Ozone is, thus unlikely to reach the chloroplast but it nevertheless, causes pigment bleaching and lipid peroxidation (104,105). Stimulation of both synthesis and degradation of the PSII-DI protein occurs in spruce trees following O3 treatment (106,107) and a decrease in the activity and quantity of rubisco has been found in poplar following exposure to O3 (108).

Exposure to SO2 results in tissue damage and release of stress ethylene from both photosynthetic and non-photosynthetic tissues (110,111). Fumigation with SO2 causes a shift in cytoplasmic pH. The prodon concentration of the cytoplasm is one of the most important factors regulating cellulase activity When cells are exposed to SO2 an appreciable acidification of the cytoplasm occur because this gas reacts with water to form sulfurous acid which may then be converted into sulphuric acid (112,113,114). These results, in loss of photosynthetic function caused by inhibition of the activity of SH-containing light-activated enzymes of the inhibition of the activity of SH- containing light-activated enzymes of the chloroplast (115,116,117).

The oxidation of sulfite to sulfate in the chloroplast also gives rise to the formation of O-

2 (96). The oxidation of sulfite is initiated by light and is mediated by photosynthetic electron transport. Navari-Izzo et al. (120) reported that the degradation of membrane lipid components possibly by de-esterification rather than peroxidation with SO2. They found no evidence to support the view that free radical attack on polyunsaturated fatty acids occurred at low pollutant concentrations.

1.8. Light stress:

A wealth of evidence shows that antioxidants are responsive to photooxidative stress (9,83). In bacteria, signal transduction systems involved in responses to oxidative stress have been identified (121); however, very little is known in plants. An important step forward has been made in identifying a possible mechanism of detecting oxidative stress in chloroplasts of leaves exposed to high levels of light. Transfer of Arabidopsis leaves from low light to high light causes rapid induction of mRNA for two nuclear-encoded cytosolic ascorbate peroxidase genes (APX1 and APX2). Also, within this 15 minute period the ratio of reduced to oxidized glutathione decreases, indicating that the leaves are under oxidative stress as a result of exposure to excess excitation energy. The induction of the APX genes was prevented by


treatment with 3-(3,4-dichlorophenyl)-l,l-dimethylurea, which blocks photosynthetic electron transport before plastoquinone (PQ). Conversely, 2,5-dibromo-3-methyl-6- isopropyl-p-benzoquinone, which blocks electron transport at the cytochrome b/f complex, causes higher APX expression in low light. The results can be interpreted as suggesting that the redox state of PQ has a role in acting as a sensor of excess light. Interestingly, glutathione feeding also blocked the response and it was suggested that extra glutathione swamps a signal generated by the redox state of the endogenous pool (6). These observations require further investigation because it is clear that there is a rapid signal transduction process leading to induction of APX when leaves are exposed to excessive light.

Evidence of similar redox signaling in controlling light-mediated phosphorylation of light harvesting complexes and expression of photosynthesis enzymes exists (122,123). Signal transduction via increased cytosolic Ca2+ has been suggested (124) and this observation has been extended by demonstration of elevated cytosolic Ca2+ in stomatal guard cells by methyl viologen and hydrogen peroxide treatment, which then causes closure (127). 1.9. Temperature stress:

Various tolerance mechanisms have been suggested on the basis of the biochemical and physiological changes related to chilling injury (63,42,128). Levitt has suggested that a major target of chilling injury is cell membranes (130). As temperature is reduced, a specific temperature determined by the ratio of saturated to unsaturated fatty acids accelerates the conversion of lipids of a liquid-crystalline condition into that of a solid condition in plant cell membranes (131). The conversion of fatty acid may give rise to chilling resistance at lower temperatures in the plant cells. However some plants, which show a similar fatty acid ratio under chilling conditions, are very sensitive to chilling injury compared to others; thus other mechanisms may also be necessary for chilling injury. In previous studies it has been suggested that oxidative stress induced by chilling stress may play a pivotal role for chilling injury in plant cells (132,133).

Dong and Chin (134) were investigated in the following: the antioxidant defense system and chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber (Cucumis sati6us L.). Chilling stress preferentially enhanced the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. In order to analyze the changes of antioxidant enzyme isoforms against chilling stress, foliar extracts were subjected to native PAGE. Leaves of cucumber had four isoforms of Mn-SOD and two isoforms of Cu: Zn-SOD. Fe-SOD isoform was not observed in this plant. Expression of Cu: Zn-SOD and Mn-SOD was preferentially enhanced by chilling stress. Expression of Mn-SOD-2 and -4 was enhanced after 48 h of the poststress period. Five APX isoforms were presented in the leaves of cucumber. The intensities of APX-4 and -5 were enhanced by chilling stress, whereas that of APX-3 was significantly increased in the poststress periods after chilling stress. Gel stained for GR activity revealed six isoforms in the plant. Activation levels for most of GR isoforms were higher in the stressed-plants than the control and poststressed-plants, but that of GR-1 isoform was significantly higher in the poststressed-plants than chilling stressed-plants. Dong and Chin (134) results collectively suggest that chilling stress activates the enzymes of an SOD: ascorbate-glutathione cycle under catalase deactivation in the leaves of cucumber, but the response timing of enzyme isoforms against various environmental stresses is not the same for all isoforms of antioxidant enzymes. 1.10. Topography stress:

High mountain plants must have a very effective carbon assimilation mechanism due to a very short growing period. The extreme climatic conditions of high mountain zone, high irradiance, low temperature, rapid temperature change and a reduced CO2 partial pressure


creates unfavorable conditions for photosynthesis (64,69,135). Previous studies revealed that alpine plants are highly efficient in photosynthesis at low temperatures and are also adapted to high irradiance (135). The biggest damage caused by the high light intensity in plants is the inactivation of D1 protein (located on PSII) and the catalase (CAT) enzyme. Low temperature stress has a similar effect upon PSII and CAT (69). The alpine plants have a much more effective protection mechanism against oxidative damage compared with the plants growing in lower altitude regions (69,136,137). The steppe plants are also affected by the combination of high light intensity, high temperature and drought stresses.

The steppe regions have a very poor vegetation and a very short vegetative period since

these conditions limit plant growth (68,138). The high light intensity, high temperature and the temperature difference between night and day increases the generation of reactive oxygen species and thus the risk of oxidative damage. The plants may have developed two strategies to adapt to these severe conditions: antioxidant protection and avoidance from oxidative stress (68). The damage from the electron transfer system results in the formation of free radicals (singlet oxygen, superoxide radical and hydroxyl radicals). It is necessary to activate the biochemical protection mechanism of the plant in order to eliminate these extremely hazardous radicals (140).

The antioxidant protection requires high amounts of carotenoids, ascorbic acid, a-

tocopherol, glutathione, phenolics and flavinoids (139) and the increased activities of CAT, superoxide dismutase (SOD) ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase (GR) enzymes (80,141). The antioxidant defense mechanism protects the unsaturated membrane lipids, nucleic acids, enzymes and other cellular structures from the harmful effects of free radicals (84,140,142). The tolerance of Homogyne alpina, an alpine plant, to light stress is explained by the presence of a stable CAT enzyme. Ranunculus glacialis growing at the same altitude has a weak antioxidant system (69,137). The activity of antioxidant enzymes and amount of carotenoids in Retama raetam, a desert plant, has been found to be higher than in non-desert plants (68). 1.11. Lack of oxygen stress:

Lack of oxygen or anoxia is a common environmental challenge which plants have to face throughout their life. Winter ice encasement, seed imbibitions, spring floods and excess of rainfall are examples of natural conditions leading to root hypoxia or anoxia. Low oxygen concentration can also be a normal attribute of a plants' natural environment. Wetland species and aquatic plants have developed adaptative structural and metabolic features to combat oxygen deficiency. A decrease in adenylate energy charge, cytoplasmic acidification, anaerobic fermentation, elevation in cytosolic Ca2+ concentration, changes in the redox state and a decrease in the membrane barrier function, are the main features caused by lack of oxygen (143,144,147,148,149,150). Regulation of anoxic metabolism is complex and not all the features are well established. In the recent paper by Gout et al. (12) a cytoplasmic acidification process has been temporally resolved in sycamore (Acer pseudoplatanus) cell culture by NMR (nuclear magnetic resonance). The immediate response of cytoplasmic pH was solely dependent on proton-releasing metabolization of the nucleoside triphosphate pool; the long-term regulation (after 20 min of anoxia) involves lactate synthesis, succinate, malate, amino acid metabolism and ethanolic fermentation (12).

Under natural conditions anoxic stress includes several transition states (hypoxia, anoxia and reoxygenation) characterized by different O2 concentrations (Table 1). Excessive generation of reactive oxygen species (ROS), i.e. under oxidative stress, is an integral part of many stress situations, including hypoxia. Hydrogen peroxide accumulation under hypoxic conditions has been shown in the roots and leaves of Hordeum vulgare (151) and in wheat roots (153). The presence of H2O2 in the apoplast and in association with the plasma membrane has


been visualized by transmission electron microscopy under hypoxic conditions in four plant species (154).

Table 1: ROS scavenging and detoxifying enzymes

Enzyme EC umber Reaction catalyzed

Superoxide dismutase 1.15.1.1 O2.- + O2

.- + 2H+ ↔ 2H2O2 + O2

Catalase 1.11.1.6 2H2O2 ↔ O2 + 2H2O

Glutathione peroxidase 1.11.1.12 2GSH + PUFA-OOH ↔ GSSG + PUFA + 2H2O

Glutathione S-transferases 2.5.1.18 RX + GSH ↔ HX + R-S-GSH*

Phospholipid-hydroperoxide glutathione peroxidase

1.11.1.9 2GSH + PUFA-OOH (H2O2) ↔ GSSG + 2H2O**

Ascorbate peroxidase 1.11.1.11 AA + H2O2 ↔ DHA + 2H2O

Guaiacol type peroxidase 1.11.1.7 Donor + H2O2 ↔ oxidized donor + 2H2O***

Monodehydroascorbate reductase 1.6.5.4 NADH + 2MDHA ↔ NAD+ + 2AA

Dehydroascorbate reductase 1.8.5.1 2GSH + DHA ↔ GSSG + AA

Glutathione reductase 1.6.4.2 NADPH + GSSG ↔ NADP+ + 2GSH * R may be an aliphatic, aromatic or heterocyclic group; X may be a sulfate, nitrite or halide group. **Reaction with H2O2 is slow. *** AA acts as an electron donor (100).

In these experiments H2O2 was probably of enzymatic origin considering the low oxygen concentration in the system and the positive effects of the various inhibitors of H2O-producing enzymes. Indirect evidence of ROS formation (i.e. lipid peroxidation products) under low oxygen has been detected (145,155,157,158,159).

The phenomenon of cross-tolerance to various environmental stresses suggests the existence of a common factor, which provides crosstalk between different signalling pathways. ROS have recently been considered as possible signaling molecules in the detection of the surrounding oxygen concentration (167). It has been suggested also that ROS and oxygen concentration (including hypoxia) can be sensed via the same mechanism. Several models employ direct sensing of oxygen (via haemoglobin or protein SH oxidation) or ROS sensing. There are two models which suggest either a decrease in ROS under oxygen deprivation (low NADPH oxidase activity) or an increase in ROS due to the inhibition of the mitochondrial electron transport chain.

Molecular oxygen is relatively unreactive (41) due to its electron configuration. Activation of oxygen (i.e. the first univalent reduction step) is energy dependent and requires an electron donation. The subsequent one-electron reduction steps are not energy dependent and can occur spontaneously or require appropriate e-/H+ donors. In biological systems transition metal ions (Fe2+, Cu+) and semiquinones can act as e- donors. Four-electron reduction of oxygen in the respiratory electron transport chain (ETC) is always accompanied with a partial one- to three-electron reduction, yielding the formation of ROS. This term includes not only free radicals (superoxide radical, O2

.-, and hydroxyl radical, OH), but also molecules such as hydrogen peroxide (H2O2), singlet oxygen (1O2) and ozone (O3), Both O2

.-- and the hydroperoxyl radical HO2

- undergo spontaneous dismutation to produce H2O2. Although H2O2 is less reactive than O2

.-, in the presence of reduced transition metals such as Fe2+ in a chelated form (which is the case in biological systems), the formation of OH can occur in the Fenton reaction.

A potential route for the formation of a damaging species from a photochemical activated triplet state is the transfer of triplet energy to molecular oxygen. The product of the energy transfer reaction is singlet oxygen 1O2. The chlorophyll pigments associated with the electron transport system are the primary source of 1O2. The 1O2 may also arise as a by product of lipoxygenase activity, like the hydroxyl radical, 1O2 is highly destructive reacting


with most biological molecules at near diffusion controlled rate (78,168). Several classes of biological molecules are susceptible to attack by 1O2 including several protein amino acids (cycteine, methionine, tryptophan and histidine) which react with it at quit rapid rate (85). Polyunsaturated fatty acids also react at much slower rate, increasing with the number of double bonds in the molecule, to form lipid hydoperoxides and O-

2 radicals (169,170) this take place by enzyme lipoxygenase. Primary radical (R') formed as a result of UV irradiation lead to formation of lipid radicals (L'). Lipid radicals react with O2 (a reaction limited only by the rate of diffusion) to produce lipid peroxy radicals (LOO'). This peroxide is likely contributors to damage and dysfunction of cell and organelle membranes. The subject of singlet oxygen and plants has been reviewed by Knox & Dodge, (78).

Nonphotochemical routes for oxidative damage in plants usually involve the interaction of molecular oxygen with free radicals to produce new, potentially harmful free radical species containing oxygen. This type of reaction may occur directly, or it may be promoted by enzyme catalysts normally present in the plant cell such as the enzyme lipoxygenase (171). Atmospheric oxygen is unusual in that its ground state has two unpaired electrons; it is a triplet state with considerable diradical character. These penults are to enter into energetically favorable chain reactions with many organic free radicals.

The formation of organic (usually carbon centered) free radicals R' from non radical precursors is called the "initiation phase" of the autooxidation. This process, which is often quite slow, results in the characteristic lag period of a radical chain reaction. In the propagation phase of the reaction there is a build up of peroxy radicals, ROO-, and the subsequent reaction of peroxy radicals with compounds (R'H) having extractable hydrogen atoms. The new radicals are then available for further reaction with molecular oxygen. Finally when all the oxygen or active hydrogen species are used up the 'termination phase' begins. In this phase, the radicals recombine with each other to produce inactive, nonradical products. Synthetic organic chemists have created many effective inhibitors of oxidative damage for rubber, hydrocarbon fuels, plastics foodstuffs and many other materials. 2. Role of antioxidants systems in plant defense Since higher plants are immobile, they can't escape from environmental stresses. The ability of higher plants to scavenge the toxic effects of active oxygen seems to be very important determinant of their tolerance to these stresses. Antioxidants are the first line of defense against free radical damage. They are critical for maintaining optimum health of plant cells. There are several antioxidant enzymes, peptides and metabolites involved in the scavenging of active oxygen in plants, and their activation are known to increase upon exposure to oxidative stress (172). Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroscorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). Antioxidant metabolites include phenolic and nitrogen compounds. 2.1. Enzymatic and peptide defense:

Data on antioxidant levels and the activity of antioxidant regenerating 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 II 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 (146). An investigation on the antioxidative defense system in the


roots of wheat seedlings under root hypoxia or whole plant anoxia (152) 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 MDHAR, DHAR and glutathione reductase (GR) decreased slightly or remained unaltered under hypoxia, while anoxia caused a significant inhibition of enzyme activities (152). Inhibition of GR, ascorbate peroxidase (APX), CAT and SOD activities has been shown also by Yan et al. (158) in com leaves under prolonged flooding, while a short-term treatment led to an increase in the activities. Induction of enzymes involved in the ascorbate-dependent antioxidative system (APX, MDHAR, DHAR) has been shown for anaerobically germinated rice seedlings after transfer to air. In submerged seedlings (i.e. under hypoxic conditions) the activities of antioxidative enzymes were lower compared with airgerminated controls (measured as changes in the protein levels of enzymes) (173). The imposition of anoxia and subsequent reoxygenation caused a decrease both in the content of ascorbate and in its reduction state in the roots of cereals and the rhizomes of Iris spp. (156). Prolongation of the anoxic treatment led to a decline in the antioxidant level, both reduced and oxidized forms, in all plants tested. A decrease in the AAJDHA ratio indicated a shift in the reduction state of the ascorbate pool under oxygen deprivation.

The phytotoxicity of O3 is due to its high oxidative capacity through the induction of active oxygen species (AOS) in exposed plant tissue, such as super oxide (O2

.-), hydrogen peroxide (H2O2), hydroxyl radical (*OH) and singlet oxygen (1O2) (177,178). Plants have evolved protective scavenging systems in response to these AOS. Antioxidant enzymes, such as super oxide dismutase (SOD), catalase (CAT), peroxidase (POX), as well as the enzymes of the ascorbate-glutathione cycle (Halliwell-Asada cycle): ascorbate peroxidase (APX), glutathione reductase (GR), monodehydro-ascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) provide endogenous defense against the accumulation of harmful concentrations of AOS, however, they also have repeatedly been shown to be affected by O3

(179). The data published on the role of antioxidant enzymes in protecting plants from

ozone damage are contradictory. Lee and Bennett (180) found EDU-enhanced snapbean tolerance to O3 injury always correlated with the increase in SOD, CAT and POX in the leaves. Levels of APX and GR activities in pea exposed to O3 were approximately twice of those found in control plants (101). In red spruce, loblolly pine, and Scotch pine, all SOD isozymes were induced by ozone (182). In contrast to these reports, other workers have shown that SOD in french bean (Phaseolus vulgaris) (183), soybean (184) and Norway spruce (185) were largely unaffected by O3, Ozone susceptibility of P. vulgaris cultivars tested by McKersie et al. (129) was not correlated with SOD activity and the amount of lipid soluble antioxidants. In spinach leaves exposed to ozone SOD and CAT levels were decreased (186). Therefore, the role of these antioxidant enzymes in the O3 detoxification in plants is still contradictory.

It has been suggested that O2- and H2O2 play an important role in the mechanism of mediating ozone injury (141). Most of the evidence for a causal relationship of AOS in ozone phytotoxicity is indirect and derives from the observed changes of the enzymes scavenging the two main AOS, such as SOD, POX, CAT, APX and GR. In fact, the increase of these enzymes was induced by the accumulation of O2- and H2O2 in plants exposed to ozone (187). Mehlhorn et al. (99) for the first time directly detected elevated levels of free radicals in plants exposed to ozone using electron spin resonance detection (ESR). It is still unclear how the dynamic balance between the AOS levels and the scavenging enzymes is disturbed in plants exposed to ozone. 2.1.1. CATALASE AND PEROXIDASE

Plant catalases are tetrameric homoproteins that exist as multiple isoezymes encoded by nuclear genes. They are located mostly in peroxisomes and glyoxysomes, although a specific isozyme Cat3 is present in maize mitochondria (188). The catalase of soybean nodules is a typical homotetramer of 220 kDa (190). This enzyme may be especially abundant in the peroxisomes of determinate nodules, by urease and possible other oxidases (191). A long-known metalloenzyme, catalase is one of the most efficient protein catalysis known, it


promotes the redox reaction. 2H2O2→2H2O+O2

Hydrogen peroxide itself is not particularly reactive with most biologically precursor for more reactive oxidant such as HO. Although catalase is rather specific for H2O2, it reacts with a limited number of organic hydroperoxides such as MeOOH, using them to carry out oxidative reactions on the acceptor molecules while simultaneously reducing the peroxidic substrate.

Catalase (Cat) is a high capacity but low affinity enzyme which destroys hydrogen peroxide. In Nicotiana plumbaginifolia leaves, the major part of the Cat activity is due to peroxisomal Cat which detoxifies hydrogen peroxide produced by photorespiration. Reduction of Cat activity by introduction of an antisense construct resulted in plants with 10% of wild-type activity (192). The plants had an apparently normal phenotype at low light intensity but developed white necrotic lesions upon exposure to high light. These symptoms were observed some time ago in a catalase-deficient barley mutant (194) and as in barley; symptom development depends on production of photorespiratory hydrogen peroxide, because symptoms are less pronounced in low light. The antisense plants have a number of interesting characteristics. Glutathione pool size increases, presumably because its synthesis is induced by oxidative stress, although a large proportion is oxidised, again as seen in the barley mutant or after catalase inhibition by aminotriazole treatment (8,192). The ability of leaves containing the antisense construct to destroy exogenous H2O2 was lowered, presumably because the lower catalase activity in the leaf resulted in a smaller concentration gradient for H2O2 diffusion into the leaf discs. The conclusion that catalase is a sink for H2O2 and that higher affinity peroxidases, such as ascorbate peroxidase (APX), deal with lower concentrations (192) is justified, although it is reassuring that their results and conclusions are those which could have been reached by biochemical reasoning. The low catalase plants were more sensitive to stresses such as ozone and high salinity, as well hydrogen peroxide and methyl viologen. Catalase suppression by antisense has also been used to test the proposed role of salicylate inhibition of catalase in induction of pathogen defense responses. Two groups have shown that low catalase plants have increased expression of pathogenesis-related proteins and increased pathogen resistance (14,195).

Other important plant enzymes, the peroxidases, also function in this mode. In addition defense against active oxygen compounds plants peroxidases have other important cellular roles. However, in different cases endogenous auxin levels are regulated by the enzymes auxin oxidase and peroxidase (196). The activities of some antioxidant enzymes increase during stress treatment, and the types of enzymatic activities that increase are dependent on the form stress imposed. The enzymes whose activities increase during stress treatment may play an important role in defense against that particular stress.

The intracellular level of H2O2 is regulated by a wide range of enzymes, the most important being catalase (193) and peroxidases. Catalase functions through an intermediate catalase- H2O2 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 peroxidatic reaction. Compound I is a much more effective oxidant than H2O2 itself, thus the reaction of Compound I with another H2O2 molecule (catalase action) represents a one-electron transfer, which splits peroxide and produces another strong oxidant, the hydroxyl radical (OH') (41). OH' is a very strong oxidant and can initiate radical chain reactions with organic molecules, particularly with PUPA in membrane lipids.

Under anoxia a differential response of the peroxidase system has been observed in coleoptiles and roots of rice seedlings. There was a decrease in activity of cell wallbound guaiacol and syringaldazine peroxidase activities, while soluble peroxidase activity was not affected in coleoptiles. In contrast anoxia-grown roots showed an increase in the cell wall-bound peroxidases (181). Acclimation to anoxia has been shown to be dependent, at least partly, on peroxidases, which have been up-regulated by anoxic stress (197). In rice seedlings ADH and SOD activities responded nonsignificantly to submergence, while catalase activity increased upon re-admission of oxygen (174). However, under strict anoxia in bakers yeast (Saccharomyces cerevisiae) the expression of peroxisomal catalase A was down-regulated by


anoxia (198). 2.1.2. DEHYDROASCORBALE REDUCTASE (DHAR)

DHAR is thought to play an important role in the oxidative stress tolerance of plants by regenerating ascorbate from dehyroascorbate (86,199). In some plants, DHAR activity has also been reported to increase upon exposure to high temperature, high light intensity and water deficiency (38,200,201), respectively. However, Tanaka et al. (118) have reported that DHAR activities show little change under stresses. Thus drought stress does not necessarily induce DHAR activity. 2.1.3. ASCORBATE PEROXIDASE (APX) AND GLUTATHIONE REDUCTASE (GR)

APX and GR are the major scavengers of hydrogen peroxide in plant cells (10) and their activities increase in response to various environmental stressors. In leaves Arabidopsis thaliana APX activity increased during exposure of plants to ozone, sulfur dioxide (202) chilling and UV-B (109,203). Ascorbate peroxidase (APX) and glutathione reductase (GR) activities are increased in water-stressed spinach leaves. In Arabidopsis leaves, the decrease in CAT activity when exposed to high temperature, high light intensity and water deficiency preceded the increase of APX and GR activity. This decrease in CAT activity might trigger the induction of APX and GR activities by reducing the ability of cells to scavenge hydrogen peroxide (201). Under conditions of salt stress, the salt tolerance cultivar exhibited increased total superoxide disumutase (SOD) and ascorbate peroxidase APX activity, whereas both enzyme activities decreased in acutely salt stressed seedling of the sensitive cultivar (204).

2.1.4. PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is a key enzyme in the

protection of the membranes exposed to oxidative stress and it is inducible under various stress conditions. The enzyme catalyses the regeneration of phospholipid hydroperoxides at the expense of GSH and is localized in the cytosol and the inner membrane of mitochondria of animal cells. PHGPX can also react with H2O2 but this is a very slow process. Until now, 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 (205). The PHGPX protein and its encoding gene csa have been isolated and characterized in citrus. It has been shown that csa is directly induced by the substrate of PHGPX under heat, cold and salt stresses, and that this induction occurs mainly via the production of ROS (206). 2.1.5. THE ASCORBATE- GLUTATHIONE (ASC-GSH CYCLE)

It is one of the main antioxidant defenses in plants. This pathway has been reviewed extensively elsewhere (82,97,207) and is most widely recognized for its role in the scavenging of H2O2 in chloroplasts. However, all the components of this pathway are also present in the mitochondria and peroxisomes of leaves (210) and in the cytosol of nodule. 2.1.6. CHLOROPLAST

Chloroplasts are equipped with effective antioxidative defense systems to withstand peroxidative attack of toxic O2 species. One such system is the operative mechanism of ascorbate-dependent H2O2. Scavenging enzymes, which by removing H2O2, play a critical role against the generation of the potent oxidant OH (87,98). The enzyme system involves detoxification of H2O2 to H2O by ascorbate (ASA) peroxidase; regeneration of ascorbate (ASA) is catalyzed either by monodehydroascorbate (MDASA) or dehydroascorbate (DHASA) reductase at the expense of NADH or reduced glutathione (GSH), respectively. For the regeneration of GSH, glutathione (GSSG) reductase functions using NADPH.

Catalase reacts with H2O2 directly to form water and oxygen (212). Catalase activities declined with progress of water stress thus favoring the accumulation of' H2O2. Peroxidases catalyze hydrogen peroxide dependent oxidation of substrates (RH2) according to the general


equation. RH2+ H2O2→2 H2O+R

A significant increase in peroxidase activity using guaiacal as an artificial substrate was brought about by water stress (37). An increase of peroxidase activity was observed in other studies under drought (213) and other stress condition such as salt (214).

Elevated H2O2 concentrations could release peroxidase from membrane, structures, with which it is normally associated, as in the case of the insoluble ascorbate peroxidase of spinach chloroplasts (216). Peroxidase could be synthesized de novo at least in some cases (215).

Water stress could increase the accumulation of peroxidase substrates such as glutathione, ascorbate, and phenolic compounds, which, in turn are scavenger of activated oxygen species (212). 2.1.7. SUPEROXIDE DISMUTASE (SOD):

The SOD family is composed of metalloprotein that catalyze the dismutation of O-2

radical to O2 and H2O2. Three classes of SODs are known in plants, depending on the active site metal cofactor (Mn, Fe, or Cu plus Zn). The MnSODs and FeSODs are structurally related, whereas Cu ZnSODs show no structural relationship to the other and are thought to have evolved independently. The three enzymes exhibit distinct molecular properties, including differential sensitivity to inhibitor and they are located in different subcellular compartments.

A large number of electron processes have been described that convert O2 to its radical anion reduction product, O-

2, superoxide. Superoxide dismutases catalyze the conversion of O-

2 to H2O2 and oxygen catalysis at ordinary physiological pH, although O-2 is

quite stable above pH 11 nevertheless, virtually all aerobic organisms that have been examined contain SOD. SOD is a powerful enough catalyst to increase the rate of the reaction by several orders of magnitude at physiological pH.

Superoxide, like H2O2, is not directly reactive toward most organic compounds (at least not as an oxidant), but it probably gives rise to more reactive oxygen species of higher potential toxicity have been shown to decline in the older leaves of tobacco plants, which revealed signs of membrane damage . There were clear correlation between the activity of these two enzymes and the degree of lipid peroxidation in leaves. The authors suggested that both enzymes are important agents for protecting leaves from the deleterious effects of membrane lipid destruction. Sreannivasulu et al. (204) reported that under conditions of salt stress the salt-tolerant cultivar (Setaria italica) exhibited increased total superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity, whereas both stressed seedlings of the sensitive cultivar.

Enhanced fonnation of ROS under stress conditions induces both protective responses and cellular damage. The scavenging of O2

.- is achieved through an upstream enzyme, SOD, which catalyses the dismutation of superoxide to H2O2. This reaction has a 10 000-fold faster rate than spontaneous dismutation (141). The enzyme is present in all aerobic organisms and in all subcellular compartments susceptible of oxidative stress (141). Recently, a new type of SOD with Ni in the active centre has been described in Streptomyces (217). The other three types of this enzyme, classified by their metal cofactor, can be found in living organisms, and they are the structurally similar FeSOD (prokaryotic organisms, chloroplast stroma) and MnSOD (prokaryotic organisms and the mitochondrion of eukaryotes); and the structurally unrelated Cu/ZnSOD (cytosolic and chloroplast enzyme, gram-negative bacteria). These isoenzymes differ in their sensitivity to H2O2 and KCN (218). 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 com (Zea mays), where a 7-d 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 (158). An excessive accumulation of superoxide due to the reduced activity of SOD under flooding stress was shown also (158). In anoxically treated wheat and rice roots the activity of SOD has been determined without a prolonged re-


oxygenation period, immediately after termination of the anoxic treatment. In the course of this experiment the activity decreased in wheat under both aeration and anoxia, but in the anoxic samples this decline was slower. As a result, after 3 d of anoxia the activity was 65 % higher than in the control roots. In the more anoxia-tolerant rice, anoxia did not affect SOD activity (159). Similar results have been reported by Pavelic et al. (219) for potato cell culture during the post-anoxic period: only 60 % of initial specific SOD activity remained after 3 h reoxygenation. In cereals the activity of SOD has been found to decline depending on the duration of the anoxic treatment, while in Iris pseudacorus a 14-fold increase was observed during a reoxygenation period (220). An increase in total SOD activity has been also detected in wheat roots under anoxia but not under hypoxia. The degree of increase positively correlated with duration of anoxia (153). Induction of SOD activity under hypoxia by 40-60 % in the roots and leaves of Hordeum vulgare has been shown by Kalashnikov et al. (151).

Hence, investigations of SOD activity in different plant species under hypoxia (submergence) and/or anoxia have resulted in contradictory observations (Table 2). The explanation can be found in different tolerance to anoxia between species and experimental set-up (e.g. a prolonged reoxygenation period in the case of Iris spp., while in cereal roots activity of the enzyme was determined immediately after anoxia). The formation of ROS already under hypoxic conditions and during the oxidative burst after re-admission of oxygen could cause rapid substrate overload of constitutive SOD, while induction was hindered probably by other factors [e.g. time, activity of downstream enzymes in the ROS-detoxification cascade, inhibition by the end product (H2O2) and consequences of anoxic metabolism]. Observations on SOD activity in different plant species under several stress conditions (drought, salinity and high/ low temperature) suggest that different mechanisms may be involved in oxidative stress injury (222,223). Activation of oxygen may proceed through different mechanisms, not necessarily producing a substrate for SOD. Changes in O2 electronic configuration can lead to the formation of highly reactive singlet oxygen (1O2). Comparison of drought and water stress effects on tolerant and intolerant wheat genotypes suggests that different mechanisms can participate in ROS detoxification. For example, water stress did not affect SOD activity, while under drought conditions a significant increase was detected (224). In another experiment, oxidative stress conditions combined with cold acclimation of cold-resistant and unresistant wheat cultivars, SOD activity in the leaves and in the roots was unaffected by the low temperature treatment but plants exhibited higher guaiacol peroxidase activity (225). Inefficiency of ROSdetoxifying enzymes (SOD, CAT, ascorbate peroxidase and non-specific peroxidase) has been shown under water deficit-induced oxidative stress in rice (226). In this paper a decrease in enzymatic activity was accompanied by LP, chlorophyll bleaching, loss of ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol and carotenoids in stressed plants. The authors suggested the formation of a certain strong pro-oxidant, which is neither superoxide nor H2O2 under the conditions of water deficit (226). The ability of plants to overcome oxidative stress only partly relies on the induction of SOD activity and other factors can regulate the availability of the substrate for SOD. Diversification of the pathways of ROS formation, compartmentalization of oxidative processes (charged ROS cannot penetrate the membrane) and compartmentalization of SOD isozymes. It is also possible that in different plant species and tissues different mechanisms are involved in the protection against oxidative stress. 2.1.8. GLUTATHIONE: (THIOLS)

The thiol tripeptide GSH (yGlu-Gys-Gly) is a versatile antioxidant that can directly scavenge ROS and participate in the ASC-GSH cycle for H2O2 removal in the chloroplasts and nodule cytosol (207). It is also involved in many other functions of plants, including the transport and storage of sulfur, stress tolerance and the detoxification of heavy metals (82). A tripeptide bearing a thiol group, glutathione (GSH) is found in very high concentrations in many cells. It reacts with many oxidants such as H2O2 to form the oxidized form, a disulphide known as GSSG.

ROOH +2GSH →ROH +GSSG+H2O


The above reaction is catalysed in mammalian cells by selenium containing enzyme glutathione peroxidase. Glutathione also reacts without enzyme catalysis with many other potentially damaging intracellular oxidants such as 1O2, O-

2 and HO-. Tanaka et al. (119) have reported that APX and GR activities are increased in water stressed spinach leaves.

A tripeptide glutathione (γ-glutamylcysteinylglycine) is an abundant compound in plant tissues. It has been found virtually in all cell compartments: cytosol, endoplasmic reticulum, vacuole and mitochondria (211), where GSH executes multiple functions. GSH is the main storage form of sulfur, and it acts as a potent detoxifier of xenobiotics through GSH-conjugation, and can serve as a precursor of phytochelatins (90,227). Together with its oxidized form (GSSG) glutathione maintains a redox balance in the cellular compartments. The latter property is of great biological importance, since it allows fine-tuning of the cellular redox environment under normal conditions and upon the onset of stress, and provides the basis for GSH stress signalling. Indeed, the role for GSH in redox regulation of gene expression has been described in many papers (228,229). Due to redox properties of the GSH/GSSG pair and reduced SH-group of GSH, it can participate in the regulation of the cell cycle (230).

Functioning of GSH as antioxidant under oxidative stress has received much attention during the last decade. A central nucleophilic cysteine residue is responsible for high reductive potential of GSH. It scavenges cytotoxic H2O2, and reacts non-enzymatically with other ROS: singlet oxygen, superoxide radical and hydroxyl radical (140). The central role of GSH in the antioxidative defense is due to its ability to regenerate another powerful water-soluble antioxidant, ascorbic acid, via the ascorbate-glutathione cycle (81,82). 2.1.9. OTHER PROTEINS

Some soybean proteins have been shown to inhibit lipid oxidation (231). There are many scattered observation particularly in the food science literature that peptide or protein hydrolysates protect lipids from oxidation. It is possible that these may be due to the metal complexion capacity of these substances. In addition to metabolic changes and accumulation of low-molecular weight protective compounds a large set of plant genes is transcriptional activated, which lead to accumulation of new proteins in vegetative tissue of plants under osmotic stress conditions (23,232). It is generally assumed that stress induced proteins might play a role in tolerance, but direct evidence is still lacking and the function of many stress responsive genes are unknown. It has been hypothesized, based on the correlation of late embryogenesis abundant (LEA) gene expression with physiological and environmental stresses and the prediction novel structure of the LEA proteins, that LEA protein may play a protective role in plant cells under various stresses condition. Moreover, this protective role may be essential for the survival of the plant under extreme stress condition (232,233). 2.2. Metabolic compounds defense

Antioxidants, is designing chemicals, when added in small quantities to a materials, react rapidly with the free radical intermediates of an autooxidation chain and stop it from progressing. An excellent example of this type of inhibitor is the synthetic hindered phenol 2,6-di-tert-butyl-4 methyl phenols often called BHT which react with mol- of peroxy radical and converts them to much less active products. It has been recognized for some time that naturally occurring substances including those found in higher plants, also have antioxidant activity. Recently, there has been increasing interest in oxygen-containing free radicals in biological systems and their implied roles as causative agents in the etiology of a variety of chronic disorders. Accordingly attention is being focused on the protective biochemical functions of naturally occurring antioxidants in the cells of the organisms containing them, and on the mechanisms of their action.

It has also been reported that plants with high levels of antioxidants, whether constitutive or induced have a greater resistance to such oxidative damage (84,189,234,235,236). The primary components of this antioxidant system include carotenoids, ascorbate, glutathione, vitamin E (α-tocopherols) flavonoids, phenolic acids, other phenols, alkaloids, polyamines,


chlorophyll derivatives, amino acids and amines and miscellaneous compounds. A number of studies indicated that the degree of oxidative cellular damage in plants exposed to a biotic stress is controlled by the capacity of antioxidative systems (37,82,129,237,238,239). 2.2.1. PHENOLIC COMPOUNDS

Phenolics are diverse secondary metabolites (flavonoids, tannins, hydroxycinnamate esters and lignin) abundant in plant tissues (240). 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 delocalize the unpaired electron (chain-breaking function), and from their ability to chelate transition metal ions (termination of the Fenton reaction) (242). 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 (244). 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 (245). According to our unpublished results the content of condensed tannins (flavonols), as measured by high performance liquid chromatography, was 100 times higher in I. pseudacorus rhizomes than in those of I. germanica. The effect of anoxia on the flavonol content (a decrease after 35 d of treatment) suggests their participation in the antioxidative defense in I. pseudacorus rhizomes. 2.2.1.1. Vitamin E (α-tocopherol):

Tocopherols and tocotrienols are essential components of biological membranes where they have both antioxidant and non-antioxidant functions (246). There are four tocopherol and tocotrienol isomers (α-, β-, γ-, δ-) which structurally consist of a chroman head group and a phytyl side chain giving vitamin E compounds amphipathic character (248). Relative antioxidant activity of the tocopherol isomers in vivo is α > β > γ > δ which is due to the methylation pattern and the amount of methy I groups attached to the phenolic ring of the polar head structure. Hence, α-tocopherol with its three methyl substituents has the highest antioxidant activity of tocopherols (248). Though antioxidant activity of tocotrienols vs. tocopherols is far less studied, α-tocotrienol is proven to be a better antioxidant than α-tocopherol in a membrane environment (249). Tocopherols, synthesized only by plants and algae, are found in all parts of plants (251). Chloroplast membranes of higher plants contain α-tocopherol as the predominant tocopherol isomer, and are hence well protected against photooxidative damage (253). There is also evidence that α-tocopherol quinone, existing solely in chloroplast membranes, shows antioxidant properties similar to those of α-tocopherol (255).

Vitamin E is a chain-breaking antioxidant, i.e. it is able to repair oxidizing radicals directly, preventing the chain propagation step during lipid autoxidation (250). It reacts with alkoxy radicals (LO'), lipid peroxyl radicals (LOO') and with alkyl radicals (L'), derived from PUPA oxidation (248,257). The reaction between vitamin E and lipid radical occurs in the membrane-water interphase where vitamin E donates a hydrogen ion to lipid radical with consequent tocopheroxyl radical (TOH') formation (257). Regeneration of the TOH' back to its reduced form can be achieved by vitamin C (ascorbate), reduced glutathione (253) or coenzyme Q (247). In addition, tocopherols act as chemical scavengers of oxygen radicals, especially singlet oxygen (via irreversible oxidation of tocopherol), and as physical deactivators of singlet oxygen by charge transfer mechanism (253). TOH' formation sustains prooxidant action of tocopherol. At high concentration tocopherols act as prooxidant synergists with transition metal ions, lipid peroxides or other oxidizing agents (248). It has been clearly shown, that prooxidant function of tocopherol on low density lipoprotein was clearly inhibited in vitro by antioxidants (ascorbate or ubiquinol) (258).

In addition to antioxidant functions vitamin E has several non-antioxidant functions in membranes. Tocopherols have been suggested to stabilize membrane structures. Earlier


studies have shown that α-tocopherol modulates membrane fluidity in a similar manner to cholesterol, and also membrane permeability to small ions and molecules (253). In recent studies α-tocopherol has been shown to decrease the permeability of digalactosyldiacyl-glycerol vesicles for glucose and protons (259). There is also recent evidence of interaction between PS II with α-tocopherol and α-tocopherol quinone (256). Complexation of tocopherol with free fatty acids and lysophospholipids protects membrane structures against their deleterious effects. The process is of great physiological relevance, since phospholipid hydrolysis products are characteristics of pathological events such as hypoxia, ischemia or stress damage (246). In addition, several other non-antioxidant functions of a-tocopherol have been described such as protein kinase C inhibition, inhibition of cell proliferation, etc. as reviewed by Azzi and Stocker (260).

Naturally occurring compounds with vitamin E activity are the tocopherols, a group of closely related phenolic benzochroman derivatives having extensive ring alkylation. These compounds occur not only in plant but also in mammalian tissues. Among the latter, antioxidant compounds of low molecular weight such as α-tocopherols, play an important role in protecting chloroplastic membranes from the deleterious effects of lipid peroxy radicals and singlet oxygen (253,262,266).

The α-tocopherols are usually recycled back by ascorbic acid or reduced glutathione following oxidation by lipid peroxy radicals. However, it can be irreversible converted to the corresponding quinone and quinone epoxide after reacting with singlet oxygen (253), (261). Besides, an increased synthesis of antioxidant such as α-tocopherols has been correlated with a higher tolerance to drought (263) and other environmental stresses (254). Munne-Bosch et al. (266) reported that α-tocopherols progressively decreased in sage during the drought. Therefore, the leaves contained smaller pools of antioxidant defenses to counteract oxygen toxicity during the drought and this explain among other biochemical and structural feature, the susceptibility of this species to stress.

The most biologically active of the four major tocopherols is α-tocopherols. The peroxy radical derived from α-tocopherols is also stabilized because the unpaired electrons of the chroman ring oxygen are held nearly perpendicular to the plane the phenyl ring calculations suggest the stabilization is on the order of 3 kcal/mol (264). Vitamin E is also one of the best quenchers for 1O2 yet test, with a quenching rate constant of approximately 6x 108 (in methanol) and it also appears to react with O-

2 to gave a phenoxy radical 15 mg g-1 fresh weight of α-tocopherols (267). This compound may serve to protect symbiosome membranes and other nodule membranes against lipid peroxidation. 2.2.1.2. Flavonoids:

It has been recognized that several classes of flavonoid showed antioxidant activity toward a variety of easily oxidizable compounds. Flavanoids occur widely in the plant kingdom, and are especially common in leaves flowering tissues, and pollens. They are also abundant in woody parts such as stems and barks. Flavanoids are usually accumulated in the plant vacuole as glycosides. The concentration of flavonoid in plant cells often exceeds 1 mM, with concentrations 3 to 10 mM being reported in the epidermal cells of Vicia faba (268).

The synthesis of many flavonoids and other phenolic compounds is greatly affected by light; for example tobacco plants grown under supplemental levels of UV contained about twice the concentration of total soluble phenolic compounds compared to the control plants. Plants grown in full sun have also been shown to contain higher levels of flavonoids than shade grown plants (245).

Flavonoids are not only accumulated in the plant vacuole as glycosides but also found as exudates on the leaves and other aerial surfaces of some species of plants. Their physiological functions have long remained unknown except for a rot as a protective filter against harmful UV radiation. Additional physiological functions of flavonoids have been discovered; for example, their role as antioxidants (243) as factor inducing pollen germination and pollen-tube elongation (269,270) and as antifungus agents (phytoalexins). Phenylpropanoid compounds might alleviate oxidative stress by shading visible light or UV. Furthermore, some


flavonoids and anthocyanins have been shown to work as antioxidants in vitro (201). Flavonoids and other phenolics are abundant in nodules, where, despite their obvious role as signal molecules during nodule initiation-they can inhibit lipid peroxidation by intercepting the peroxyl radicals formed in nodule membranes (271). Several flavonoids were shown to be potent inhibitors of the enzymes lipoxygenase and prostaglandin synthetase, which convert polyunsaturated fatty acids to oxygen-containing derivatives. Highest activity against both enzymes was shown by luteolin (5,7,3,4 -tetrahydroxyflavone) and 3,4 -dihydroxy flavone; at about 3x 10-5, they inhibited 50% of lipoxygenase activity. The author did not speculate on possible mechanisms for the inhibition of these enzymes by these particular flavonoids.

Caldwell et al. (272) have poineered a concept that flavonoids, with strong absorption in the 300-400 UV region, are acting as internal light filters the protection of chloroplasts and other organelles from UV damage .The light-filtering ability of these compounds may reinforce their powerful antioxidant effects to provide a high level of protection against damaging oxidants generate either thermally or by light.

Phenolic compounds and flavonoids are among the most influential and wide distributed secondary products in the plant kingdom. Many of these play important physiological and ecological roles, being involved in resistance to different of stress (243,273). 2.2.1.3. Phenolic acids:

Acidic compounds incorporating phenolic groups have been repeatedly implicated as active antioxidants (274). Caffeic acid, chlorogenic acid and its isomers including 4-O-caffeoylquinic acid were isolated from sweet potatoes. Chlorogenic acid was found to be the most abundant phenolic acid in the plant extract and also the most active antioxidant; a 1.2 x l05 M solution inhibited over 80% of peroxide formation in a linoleic acid test system (274).

Esters of caffeic acid with sterols and triterpene alcohols have been isolate from the seed of the grass Phalaris canariensis. The fatty acids of the seed were predominantly unsaturated, suggesting that the esters were acting to protect then from oxidation (275). The lipid soluble esters were effective antioxidants in tests with lard or sardine oil heated at 60°C. In the tests, the esters were added as mixtures but at least some components appeared to have activity approaching or exceeding that of BHT2, 6-di-tert-buty;-4-methylphenol.

Plant phenolics have often been referred to secondary metabolites and many of these compounds play an essential role in the regulation of plant growth development and interaction with other organisms. In higher pants, most secondary phenolics are derived at least in part from phenylalanine, a product of the shikimic acid pathway. The shikimic acid pathway begins from simple carbohydrates and proceeds to amino acids such phenylalanine and tyrosine (274,275).

Most researches showed that the levels of total and free amino acids increase remarkably during water stress. Gzik (276) reported that the total of 18 amino acids including phenylalanine and tyrosine increased in sugar beet leaf during water stress. In addition, a remarkable increase in free amino acid content in some plants subjected to water stress was also reported (277,278 ).

Ayaz et al. (273) reported that, an increase in the content of phenolic acids in rolled leaves could be related with increasing level of amino acids synthesis induced during water stress. However, the increasing levels of amino acids (mainly phenylalanine and tyrosine) may trigger the production of phenolic acids (cinnamic acid pathway) leading to lignin biosynthesis. Lignin is such a key component of water transporting tissue, the ability to make lignin most have been one of the most important adaptation permitting primitive plants to colonize dry land (274). So, the increase of some phenolic acids in rolled leaves of Cotenant may be explained by lignin biosynthesis in cell wall for preventing water loss (273). On the other hand, most of the increased phenolic acids analyzed in this work are insoluble and semi-soluble state and play a role as cell solute as well as sugars for osmotic adjustment (273). The increase of phenolic acids content may be linked to the lignifications of cell walls and, in part, the synthesis of certain amino acids maintaining osmotic adjustment in cell.


2.2.1.4. Other phenols: Rosmaridiphenol, a diterpene derivative with adjacent OH groups was isolated from

Rosmarinus officinalis (rosemary). Its antioxidant activity in heated lard exceeded that of BHA and approached that of BHT. Related phenolic diterpenes with antioxidant activity have also been isolated from this plant (262,264).

Carnosic acid is a diterpene that displays high antioxidant activity and which protects biological membranes from lipid peroxidation (279,280). Previous studies, it has been shown that carnosic acid may protect chloroplasts from oxidative stress, but this mechanism has only been tested in rosemary, a drought- tolerant species. A group of potent antioxidant for the air oxidation linoleic acid was isolated from the methanol extract of the rhizome of Curcuma langal (turmeric). The abundant and most active constituent of the extract was the orange pigment, curcumin. Its 50 % inhibitory concentration for the linoleic acid test system was about 5 x 10-4 M; it was more active then vitamin E in the procedure used by the authors and the synthetic antioxidants, BHA and BHT. The mechanism of curcumin activity may include metal ion chelation by central B-diketone group. The lignin isolated from sesame (Sesamum indicum) seed, significantly inhibited the autooxidation of linoleic acid at 40°C when added at 5.8 x 105 M. Polyhydroxylated chalcones such as butein which are biosynthetic intermediates between cinnarnic acids and flavonoids, also show considerable antioxidant activity for lard (279,280). 2.2.2. NITROGEN COMPOUNDS 2.2.2.1. Alkaloids:

Increasingly evidence from a variety of sources is indicating that the basic nitrogen compounds of higher plants include many representatives that are potent inhibitors of various oxidative induced by radioactive cobalt irradiation (in soybean lecithin liposomes) was inhibited by the bisbenzylisoquinoline alkaloid cepharanthine (281). Caffeine, from the leaves of tea (Thea sinensis) and coffee (Coffea arabica) was shown to have antioxidative activity (in a linoleic acid oxidation test) comparable to that of BHA and BHT. Several alkaloids of various structural types have been found to be potent inhibitors of 1O2. Particularly effective are indole alkaloids such as strychnine and brucine that have a basic nitrogen atom in a rigid, cage like structure. Such alkaloids appear to be strictly physical quenchers and are not destroyed chemically by the process of quenching. Thus, in principle, they could inactivate many molecules of singlet oxygen per molecule of alkaloid. Polyamines such as spermine are related simple cyclic alkaloids found in legumes. This compound was shown in electron spin resonance experiments to scavenge the superoxide radical at rather high spermine concentration (0.01- 0.03 M). This finding may have some bearing on the observed effects of polyamines as membrane stabilizing substance (281).

Alkaloids of quinolizidine type, for example sparteine have been found to be stored principally in the epidermal cells of four plants in the genus lipinus. Ahmed et al. (282) and Ali (283) suggested that this storage pattern was consistent with a phytochemical role for these substances as antifeedant chemical defense compounds, but it would also be consistent with an antioxidant role. It is unlikely that these alkaloids are acting as UV light filtering agents because their absorption in the solar UV range would be minimal. Alkaloids are commonly accumulated in the tissue of plants subjected to different types of stress (282,283). 2.2.2.2. Polyamines:

Polyamines (spermidine and spermine) play a variety of physiological roles in plant growth and development (283,285). They are also potent ROS scavengers and inhibitors of lipid peroxidation (286). Furthermore, exogenous application of polyamines has been shown to protect against various stress conditions such as cold, wilting, pollution and salinity (284,287). Among the common polyamines putrescine appears to be the most sensitive external stress. Accumulation of putrescine has been observed in response to low pH, high salt concentration, K and Mg deficiency, NH4 treatment exposure to SO2 and ozone, water stress, heat stress, chilling and lake of oxygen (283,288,289,290,291).


The protection of plant against ozone damage (292,293) by an exogenous supply of polyamines is believed to be cause by the free radical scavenge property of the polyamines (286). Also, the protection of plant against stress damage by an exogenous supply of polyamines is believed to be cause by the free radical scavenge of the polyamines (283). 2.2.2.3. Chlorophyll derivatives:

Although both chlorophyll (1) and pheophytin (2) promote the oxidation of lipids in the light, they are inhibitors of autooxidation under dark conditions (264). At 30°C, 2 x 10-5 M chlorophyll A was superior by a factor of Ca two to BHT (66).The compounds appear to be unreactive toward lipid hydroperoxides, but do react with peroxy radicals; electron spin resonance data indicate the presence of tetrapyrrole radical cation (264). 2.2.2.4. Amino acids and amines:

Many amino acids have been tested for their antioxidant activity especially in food, based system. Among the amino acids for which antioxidant activity has been claimed are arginine, histidine, cysteine, tryptophane, lysine, methionine and threonine (294). The literature reports are often very confusing with data suggesting that some amino acids may exhibit antioxidant potential under some conditions of temperature or pH or oxygen concentration but have no effect or actually promote oxidation in others. For example, alanine and histidine were reported to inhibit the oxidation of linoleic acid at pH 9.5 and to promote it at pH 7.5 (294). 2.2.2.5. Carotenoids:

Carotenoids fulfill two major roles in photosynthetic organisms. Their first role is to act as light harvesting pigments, extending the range of the light spectrum available for use in the photosynthetic process. They absorb light in the region from 450-570 nm, where the chlorophyll molecules do not, and pass the captured energy on the chlorophylls. Secondly, carotenoids provide photosynthetic systems with methods of photo-protection. O-

2 has been detected in chloroplasts of water stressed wheat (125).

Singlet oxygen is an extremely powerful oxidant, powerful enough to cause the death of the organism in question. Carotenoids prevent the formation of singlet oxygen by quenching the triplet state of the chlorophyll molecules as they arise (295). 2.2.3. OTHER COMPOUNDS 2.2.3.1. Vitamin C (ascorbic acid):

Ascorbic acid (AA) has been proposed for a long time as a biological antioxidant. It exists in rather high concentrations in many cellular environments, such as the stroma of chloroplasts where its level is 2.3 x 10-3 M. Ascorbate has been demonstrated in many qualitative studies to possess significant antioxidant activity (54). For example l03 M ascorbate inhibited the photooxidation of a kampferol by illuminated spinach chloroplasts. Ascorbate reduces two equivalents of O-

2 produce H2O2 and triketo derivative dehydroascorbic acid. Ascorbate also reacts with 1O2 at a relatively fast rate (82).

AA is one of the most studied and powerful antioxidants (54,55,82,296,298). It has been detected in the majority of plant cell types, organelles and in the apoplast. Under physiological conditions AA exists mostly in the reduced form (90 % of the ascorbate pool) in leaves and chloroplasts (55); and its intracellular concentration can build up to millimolar range (e.g. 20 mM in the cytosol and 20-300 mM in the chloroplast stroma (88). The ability to donate electrons in a wide range of enzymatic and non-enzymatic reactions makes AA the main ROS-detoxifying compound in the aqueous phase. AA can directly scavenge superoxide, hydroxyl radicals and singlet oxygen and reduce H2O2 to water via ascorbate peroxidase reaction (82). In chloroplasts, AA acts as a cofactor of violaxantin de-epoxidase thus sustaining dissipation of excess exitation energy (55). AA regenerates tocopherol from tocopheroxyl radical providing membrane protection (299). In addition, AA carries out a number of non-antioxidant functions in the cell. It has been implicated in the regulation of the cell division, cell cycle progression


from G1 to S phase (54,301) and cell elongation (302). Vitamin C is a universal reductant and antioxidant of plants. It is found at

concentration of 1-2 Mm in legume nodules (208,303) and is positively correlated with nodule effectiveness (209). It is an essential metabolite for the operation of the ASC- GSH pathways, but it also has beneficial effects that do not require the presence of APX. ASC can directly scavenge ROS and reduce ferric Lb and LbIV (304). It is also involved in hydroxylation of proline, regulation of the cell cycle and numerous fundamental processes of plant growth and development (82,305).

Some reports concerning the exogenous application of the vitamins to salinized plants and their role in stimulation of their growth are scarce (306). These compounds were also scarcely tried to counteract some of the adverse effects of salinity stress (307). Thus exogenous addition of such substances to the test organism could lead to growth stimulation through the activation of some enzymatic reactions (308,309).

Shalate and Neumann (310) reported that salt stress increased the accumulation in roots, stems and leaves of lipid peroxidation products produced by interactions with damaging active oxygen species. Additional ascorbic acid partially inhibited this response but did not significantly reduce sodium uptake or plasma membrane leakiness.

Ascorbate is a key soluble antioxidant (54). Isolation of an Arabidopsis mutant containing 30% of the wild type ascorbate concentration has provided the first genetic evidence for its importance in stress resistance. It was selected by hypersensitivity to ozone and is also hypersensitive to UV-B and sulfur dioxide (311). The mutation has no pleiotropic effects on other parts of the antioxidant system with the exception of reduced APX activity, possibly because ascorbate is required to stabilize APX (312). Reduction of cytosolic APX activity by expression of APX antisense mRNA also causes increased sensitivity to ozone damage, suggesting that intracellular, as well as extracellular, ozone detoxification by ascorbate is required (315). Overexpression of APX in tobacco chloroplasts had no effect on ozone resistance (316); either it is in the wrong subcellular compartment or it is not a limiting factor. 2.1. Lack of oxygen defense:

Mechanisms for the generation of ROS in biological systems are represented by both non-enzymatic and enzymatic reactions. The partition between these two pathways under oxygen deprivation stress can be regulated by the oxygen concentration in the system. Non-enzymatic one electron O2 reduction can occur at about 10-4 M and higher oxygen concentrations (316), while in very low O2 concentrations plant terminal oxidases (Km 10-6 M for oxygen) and the formation of ROS via mitochondrial ETC still remain functional.

Among enzymatic sources of ROS, xanthine oxidase (XO), an enzyme responsible for the initial activation of dioxygen should be mentioned. As electron donors XO can use xanthine, hypoxanthine or acetaldehyde (318). The latter has been shown to accumulate under oxygen deprivation (319) and can represent a possible source for hypoxiastimulated ROS production. The next enzymatic step is the dismutation of the superoxide anion by superoxide dismutase (SOD, EC.1.l5.1.l) to yield H2O2. Due to its relative stability the level of H2O2 is regulated enzymatically by an array of catalases (CAT) and peroxidases localized in almost all compartments of the plant cell. Peroxidases, besides their main function in H2O2 elimination, can also catalyse O2

.- and H2O2 formation by a complex reaction in which NADH is oxidized using trace amounts of H2O2 first produced by the non-enzymatic breakdown of NADH. Next, the NAD' radical formed reduces O2 to O2

.-, some of which dismutates to H2O2 and O2 (93). Thus, peroxidases and catalases play an important role in the fine regulation of ROS concentration in the cell through activation and deactivation of H2O2 (41). Lipoxygenase (LOX, linoleate:oxygen oxidoreductase, EC.1.13.11.l2) reaction is another possible source of ROS and other radicals. It catalyses the hydroperoxidation of polyunsaturated fatty acids (PUPA) (320).


Table 2. Differential response of SOD to oxygen deprivation stress

Plant* Site Stress SOD activity Reference

Iris pseudacorus L. (t) Rhizomes Anoxia + reaeration Increase Monk et al. (221)

Lotus (Nelumbo nucifera

Gaertn.) (t) Seedlings Hypoxia +

reoxygenation Increase Ushimaru et al. (175)

Rice (Oryza sativa L.) (t) Roots Anoxia Decline Chirkova et al. (159)

Rice (Oryza sativa L.) (t) Seedlings Hypoxia Plastidic SOD decline; Ushimaru et al. (174)

(submerged plants) mitochondrial SOD decline

Iris germanica L. (i) Rhizomes Anoxia + reaeration Decline Monk et al. (221)

Soybean (Glycine max (L.)

Merr.) (i) Seedlings Anoxia Increase Van Toai and Bolles (73)

Barley (Hordeum vulgare L.) (i) Roots Hypoxia Increase Kalashnikov et al. (151)

Narrow-leaved lupin (Lupinus Waterlogging + FeSOD and Cu/Zn SOD, Yu and Rengel (222)

angustifolius L.) reoxygenation increase; MnSOD, decline

Wheat (Triticum aestivum L.) (i) Roots Hypoxia Unaffected Biemelt et al. (153)

Anoxia Increase

Wheat (Triticum aestivum L.) (i) Roots Anoxia Decline Chirkova et al. (159)

Maize (Zea mays L.) (i) Hypoxia Decline Yan et al. (158)

(submerged plants)

Potato (Solanum tuberosum L.) (i) Cell culture Anoxia + reoxygenation Decline Pavelic et al. (219)

* (t), Plants tolerant to oxygen deprivation stress; (i), plants intolerant to oxygen deprivation stress.

The hydroperoxyderivatives of PUF A can undergo autocatalytic degradation, producing radicals and thus initiating the chain reaction of lipid peroxidation (LP). In addition LOX-mediated formation of singlet oxygen (259) or superoxide (169) has been shown. A specific LOX activity increase and its positive correlation with the duration of anoxia have been detected in potato cells (219).

Several apoplastic enzymes may also 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 H2O2 accumulation. Also an extracellular germin-like oxalate oxidase catalyses the formation of H2O2 and CO2 from oxalate in the presence of oxygen (318). Amine oxidases catalyse the oxidation of biogenic amines to the corresponding aldehyde with a release of NH3 and H2O2. Data on polyamine (putrescine) accumulation under anoxia in rice and wheat shoots (321) and predominant localization of amine oxidase in the apoplast, suggest amine oxidase participation in H2O2 production under oxygen deprivation.

ROS can be also formed as by-products in the electron transport chains of chloroplasts (97), mitochondria and the plasma membrane (cytochrome b-mediated electron transfer) (41). Plant mitochondrial ETC, with its redox-active electron carriers, is considered as the most probable candidate for intracellular ROS formation. Mitochondria have been shown to produce ROS (superoxide anion O2

.- and the succeeding H2O2) due to the electron leakage at the ubiquinone site-the ubiquinone:cytochrome b region (322) and at the matrix side of complex I (NADH dehydrogenase) (323,324). Hydrogen peroxide generation by higher plant mitochondria and its regulation by uncoupling of ETC and oxidative phosphorylation have


been demonstrated by Braidot et al. (325).

Lipid peroxidation is a natural metabolic process under normal aerobic conditions and it is one of the most investigated consequences of ROS action on membrane structure and function. PUFA, the main components of membrane lipids, is susceptible to peroxidation. The initiation phase of LP includes activation of O2 which is rate limiting. Hydroxyl radicals and singlet oxygen can react with the methylene groups of PUFA forming conjugated dienes, lipid peroxy radicals and hydroperoxides (Smimoff, 1995):

PUFA-H + X. →_ PUFA. + X-H

PUFA. + O2 →_ PUFA-OO.

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

PUFA-OO. + PUFA-H → PUFA-OOH + PUFA

The formation of conjugated dienes occurs when free radicals attack the hydrogens of methylene groups separating double bonds and leading to a rearrangement of the bonds (326). The lipid hydroperoxides produced (PUFA-OOH) can undergo reductive cleavage by reduced metals, such as Fe2+, according to the following equation:

Fe2+ complex + PUFA-OOH → Fe3+ complex + OH- + PUFA-O.

The lipid alkoxy I radical produced, PUFA-O., can initiate additional chain reactions (257):

PUFA-O.+ PUFA-H → PUFA-OH + PUFA'

The multi-stage character of the process, i.e. branching of chain reactions, allows several ways of regulation (327). Among the regulated properties are the structure of the membranes: composition and organization of lipids inside the bilayer in a way which prevents LP (328), the degree of PUFA unsaturation, mobility of lipids within the bilayer, localization of the peroxidative process in a particular membrane and the preventive antioxidant system (ROS scavenging and LP product detoxification). 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 (329).

Lipid and membrane integrity during oxygen deprivation are among the key factors in the survival of plants. Under anoxia a decrease in membrane integrity is a symptom of injury, and it can be measured as changes in the lipid content and composition (160), as activation of lipid peroxidation (145,146,155,159), as enhanced electrolyte leakage (161,162) and as a decrease in adenylate energy charge (163330,331). 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. In previous studies it has been shown that anoxia-tolerant plant species such as Acorus calamus and Schoenoplectus lacustris are able to preserve their polar lipids during anoxia and in post-anoxia, while in anoxia-sensitive plants (e.g. Iris germanica) a significant decrease in polar lipids and a simultaneous increase in free fatty acids (FFA) occur during anoxic stress with markedly enhanced lipid peroxidation during reoxygenation (332).

A decrease in unsaturated to saturated fatty acid ratio under anoxia may represent a result of LP and, at the same time sets limits for substrates of LP, the PUFA. This is the case in the anoxia-tolerant Acorus calamus, where 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 2 d of reaeration (319). Similar results have been obtained for the anoxia-tolerant and -intolerant cereals rice and wheat, respectively (160). On the other hand, no significant qualitative and quantitative changes have been detected in the composition offatty acids in anaerobically treated rice seedlings (333). 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 energy metabolism (333). Indeed, a correlation exists between the leakage of electrolytes (i.e. membrane damage) under low ATP and a release of FFA from anoxic tissue (140,147). The role of ATP in the maintenance of membrane lipid integrity under anoxia has been confirmed by Rawyler et al. (334) in potato cell culture. It has been shown that, when the rate of ATP synthesis falls below 10 µmol g-l fresh weight h-l, the integrity of membranes cannot be preserved and FFA are liberated via lipolytic acyl hydrolase (334). In general, lipids of anoxia-tolerant plants are more preserved during oxygen deprivation in respect to the composition and the degree of unsaturation. During recent years evidence has accumulated on the importance of lipid metabolism, and especially on unsaturated fatty acids, in the induction of defense reactions under biotic and abiotic stresses. Linolenic acid (18:3) has been shown to be a precursor of jasmonic acid, a signal transducer in defense reactions in plant-pathogen interactions (335). FFA, liberated during membrane breakdown under stress conditions, is not only the substrates for LP, but can act also as uncouplers in mitochondrial ETC (317).

Lipid hydroperoxides, formed as a result of LP, can affect membrane properties, i.e. increase hydrophilicity of the internal side of the bilayer (336). This phenomenon is very important for the termination of LP, since increased hydrophilicity of the membrane favours the regeneration of tocopherol by ascorbate.

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 transition metal ions), and low energy supply are factors favourable for ROS generation (219). Formation of free radicals within minutes after restoration of the oxygen supply has been shown by electron paramagnetic resonance (EPR) spectroscopy in the rhizodermis of the anoxia-intolerant I. germanica, while in the tolerant I. pseudacorus no signal was detected (145). An investigation on the dynamics of LP [changes in conjugated dienes, trienes and thiobarbituric acid reactive susbstances (TBARS)] in the same plant species confirmed this observation: neither dienes nor TBARS production was detected in the anoxia-tolerant I. pseudacorus, with the exception of a 45-d anoxic treatment (155). Accumulation of various LP products 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 after several days of anoxia (155,159). The length of the anoxic/ hypoxic treatment has been shown to affect the intensity of LP in post-anoxia. Cultured potato cells are known to exhibit a two-phase response to anoxia in respect to lipid hydrolysis: no FFA release has been detected up to 12 h under oxygen deprivation, while after 12 h intensive liberation of FF A sustained by lipolytic acid hydrolase has been observed. This behaviour was mirrored by post-anoxic LP: negligible after short-term anoxia and elevated after lipid hydrolysis had occurred (219).

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)+ (143). Impairment of membrane structure and function under anoxia contribute to ROS-induced post-anoxic injury. This causes peroxidation of lipid membranes, depletion of reduced glutathione, an increase in cytosolic Ca2+ concentration, oxidation of protein thiol groups and membrane depolarization.

Hypoxic pretreatment of plants prior to anoxia leads to increased survival (148,149,337). The minimal duration of hypoxia required for the acclimation has been estimated at 2-4 h for the root tips of maize seedlings (338). The biochemical and physiological features induced by this pretreatment suggest the involvement of several systems for increased stress tolerance. Of these, one is aimed at the maintenance of energy resources through the support of sugar utilization and ATP formation via the glycolytic pathway, while avoiding lactate accumulation and cytoplasmic acidosis. The majority of the genes induced codes for enzymes involved in starch and glucose mobilization, glycolysis and ethanol fermentation (164,339). For example, 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 phosphoenolpyruvic acid (PEP), has been reported in maize (340). Some other glycolytic and fermentation pathway enzymes, such as alcohol dehydrogenase (ADH, EC 1.1.1.1), glucose phosphate isomerase, pyruvate decarboxylase (pDC, EC 4.1.1.1), and sucrose synthase have been characterized as hypoxic ally induced in maize. ADH and PDC, enzymes of ethanolic fermentation, were induced by hypoxic pretreatment in rice cultivars with different tolerance to anoxia (341,342). Interestingly, both abscisic acid (ABA) and hypoxic pretreatment of Lactuca sativa L. seedlings have resulted in increased survival of roots and elevated ADH activity (343). However, endogenous ABA level did not respond to hypoxic pretreatment, suggesting that ABA was not involved in hypoxia-induced anoxia tolerance. The crucial role of protein synthesis under hypoxic conditions, but not under anoxia, has been shown in root tips of maize seedlings (338). Among 46 individual proteins analysed, four anaerobic proteins have been identified: ADHl, enolase, glyceraldehyde-3-phosphate dehydrogenase and PDC. The rate of their synthesis under hypoxia was enhanced (or comparable) under normoxic conditions. As expected, cycloheximide treatment during hypoxic acclimation (but not under anoxia) resulted in decreased anoxia tolerance (338). Interestingly, low oxygen (5 %) treatment of arabidopsis plants resulted in higher tolerance to hypoxia (0.1 % O2) but not anoxia (341,342). In these experiments differential response of shoots and roots was observed. In conclusion, early (hypoxic) induction of the ethanolic fermentation pathway and sugar utilization allows the maintenance of the energy status through regeneration of NADH and, hence, improves anoxia tolerance. Under natural conditions oxygen concentration would decrease gradually, and hence anoxia is always preceeded by hypoxia.

Another metabolic feature that has been shown to be upregulated (though not always) under lack of oxygen is the antioxidant system. In an investigation on SOD activity and expression under hypoxia, anoxia and subsequent reaeration, the appearance of additional isozymes has been shown under anoxia. Judged by a cycloheximide treatment, this activity could not be attributed to de novo synthesis (153). It has been shown also that anoxic pretreatment protected soybean cells from H2O2 induced cell death. Such resistance was associated with up-regulation of peroxidases and alternative oxidase (197). The beneficial effect of alternative oxidase protein accumulation under anoxia is due to electron flow bifurcation and reduced probability of ROS formation under subsequent reoxygenation. It has been known for a long time that the main damage caused by anoxic stress occurs during re-admission of oxygen. Some ROS formation can take place in hypoxic tissues as a result of over reduction of redox chains. Hence, anoxic stress is always accompanied to some extent by oxidative stress (generation of ROS) and its consequences. Induction of some components of the antioxidant system by hypoxic pretreatment can be due to such ROS accumulation and signalling (167,344).

Less information is available on tocopherol status under oxygen deprivation. Since oxidative stress is non-specific and many diverse environmental stress factors, e.g. light, drought, chilling temperature and flooding, affect plant tissues enhancing production of ROS in chloroplasts and inducing photo-oxidation of thylakoid membranes (43), the response of tocopherols to other abiotic stresses will be discussed. In an experiment where isolated spinach thylakoids and thylakoids with an exogenously added high concentration of α-tocopherol were exposed either to photosynthetically active radiation (PAR) or to UV-B light, lipid peroxidation occurred only in normal thylakoids while no peroxidation was detected in membranes with high amounts of a-tocopherol. According to the results, there was no decrease in endogenous α -tocopherol in normal thylakoids, while in artificially treated thylakoids α -tocopherol contents decreased though no significant lipid peroxidation could be detected (345). The latter results contradict previous studies on lipid peroxidation since increased peroxidation of membranes has been described to occur only after significant amounts of membrane α -tocopherol have been depleted (300,327).

During drought, plants show a general response to stress by increasing tocopherol and carotenoid contents in photosynthetic tissues (264) which is accompanied by a similar sized


rise in total glutathione pool and a depletion of ascorbate at least in many grass species (126). Substantial increases in a-tocopherol during water-stress have been detected in leaves of Rosmarinus officinalis L. (264), Melissa officinalis L. (265) and Fagus sylvatica L. (62). Enhanced activity of the xanthophyll cycle measured as increases in de-epoxidized xanthophylls (antheraxanthin and zeaxanthin) during drought is also a feature shared in water-stressed plant species. Rosemary plants also have species-specific antioxidants, abietane diterpenes, known for their function in inhibiting lipid peroxidation and superoxide generation in chloroplasts and micro somes, which are consumed during drought-stress in scavenging oxygen radicals (261).

There is evidence that the chilling-tolerance of plants is correlated with increasing amounts of antioxidants and increasing activity of radical scavenging enzymes. A chilling-tolerant maize genotype has been shown to contain higher amounts of both α -tocopherol and glutathione and higher GR activity than a chilling-sensitive maize genotype (346). It is known that ascorbate regenerates tocopherols from their radical forms (257). However, artificially increased ascorbate content in maize leaves did not improve the preservation of endogenous tocopherol during high light and chilling stress, but the high ascorbate content increased the usage of glutathione (347).

Studies on vitamin E in the underground parts of plants during stress are scarce, which might in part be a consequence of the fact that generally the predominating tocopherol isomer of plant tissues, a-tocopherol, is mainly localized in chloroplasts (248), and tocopherol synthesis is described to take place only in chloroplasts and chromoplasts (348). During long-term anoxic stress vitamin E contents in the rhizomes of two iris species, highly anoxia-tolerant Iris pseudacorus and anoxia-sensitive I. germanica, have been determined. Tocopherols (α- and β-) were identified in both iris species, l3-tocopherol being the predominant tocopherol isomer especially in rhizomes of I. germanica which also possessed markedly higher total tocopherol content than I. pseudacorus. Anoxia caused a decrease in tocopherol isomers in both iris species (156).

The vitamin E composition in rhizomes of the iris species is unique since there are no previous reports of plant species having β -tocopherol as the main tocopherol isomer in vegetative tissues. In addition, according to mass spectrometry the identified isomer is β -dehydrotocopherol with one double bond in its phytyl side chain (156), while tocopherols have saturated phytyl chains. Dehydrotocopherols have been found previously in etiolated shoots of maize and barley (349). There is evidence that tocopherol isomers differ from each other in their functional properties. When the effectiveness of tocopherol isomers in quenching of singlet oxygen was studied, α -and β -tocopherols were equally effective in quenching singlet oxygen physically, but β-tocopherol showed almost no chemical reactivity with singlet oxygen, while α-tocopherol had the highest chemical reactivity of tocopherol isomers (350). Inhibition of protein kinase C activity and cell proliferation is a specific non-antioxidant function of α -tocopherol in animal cells. 13- Tocopherol lacks this ability but when the two isomers are present together β -tocopherol prevents the inhibitory effect of α -tocopherol (260).

Though the total tocopherol content was higher in I. germanica than in the more anoxia-tolerant I. pseudacorus (156), this could not prevent the massive lipid degradation in I. germanica during anoxia found by Henzi and Braendle (332). There are also earlier reports suggesting that anoxia causes more pronounced lipid peroxidation in the rhizomes of I. germanica than in I. pseudacorus during reaeration (155,157). In anaerobically germinated rice seedlings a three-fold increase in tocopherol and low TBARS formation have been observed (176). However, an anoxia-induced elevation in the tocopherol level observed in the anoxia-intolerant wheat and oat seedlings could not be detected in rice seedlings subjected to anoxia (159). There are in vitro studies suggesting that under anaerobic conditions a radicalinitiated reaction between linoleic acid hydroperoxide or methyl linoleate hydroperoxide and α-tocopherol occurs, forming an addition compound of the two reactants, the reaction being


terminated in the presence of air (351). Carbon-centred radicals are also formed during anaerobic conditions, tending to add to the oxygen of tocopheroxyl radicals forming 6-0-lipid alkyl-chromanol adducts (248). 3. Factors affecting the antioxidant defense system

Considering the experimental data discussed above, it is difficult to delineate a universal mechanism for the whole antioxidant system response to anoxia. It is necessary to discuss other factors involved in the protective machinery of plants under oxygen deprivation with a particular emphasis on the antioxidant system.

3.1. Oxygen deprivation stress-specific factors Metabolic changes specifically induced by anoxia (a drop in cytosolic pH, a decrease

in adenyl ate energy charge, membrane lipid peroxidation, excess of NADH) may alter the antioxidant status of the tissue. One of the most important consequences of energy limitation under anoxia is the altered redox state of the cell. When oxygen-the terminal electron acceptor of ETC-is unavailable, intermediate electron 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 rice (165) and is considered important for plant survival under anoxia. A decrease in NADH/NAD+ has been observed in the anoxia-intolerant wheat and bean (165). 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) and peroxidation of lipids. If oxygen deprivation persists, the need for oxidized NAD+ and ATP leads to the fermentation pathway, where both LDH and ADH can regenerate NAD+. Among the possible targets of oxygen deprivation stress in respect of the antioxidant system are denovo antioxidant synthesis, intra- and intercellular transport, recycling of antioxidants and impaired cooperation of the antioxidant network.

3.2. Compartmentalization of lipophilic antioxidants ROS (with the exception of H2O2) are charged species and cannot penetrate

biological membranes; hence local antioxidant protection is more important than an overall increase in antioxidants. In a model of lipid peroxidation in tissue disorders, Shewfelt and Purvis (327) emphasize the importance of compartmentalization within the cell. The fate of the tissue may rely on the antioxidant capacity of a specific membrane structure (327). In previous studies on the compartmentalization of tocopherols in photosynthetic tissues tocopherol has been localized in chloroplasts and plastids, while other tocopherol isomers have been found in chloroplasts, mitochondria and microsomes (251). Some evidence exists on the importance of compartmentalization of other lipophilic antioxidants. In a recent study on the chloroplast localized antioxidant camosic acid of rosemary (Rosmarinus officinalis) leaves, it was shown that, after ROS scavenging, the camosic acid metabolites are transferred to the plasma membrane (263). Identification of the cell structures affected primarily in oxidative stress, as well as the localization pattern of different antioxidants especially in non-photosynthesizing plant organs, are still poorly studied areas. Accumulation of antioxidants and ROS in different cell compartments could lead to lowered antioxidant defense, and hence would require fine tuning of cellular metabolism to achieve protection. Under severe stress conditions such a regulatory mechanism can be impaired.

3.3. Possibility for de novo synthesis of antioxidants under particular stress conditions

Limitations for GSH biosynthesis under oxygen deprivation mainly arise from the


restriction of the energy supply. Two ATP-dependent steps represent the GSH biosynthetic pathway: synthesis of γ-glutamylcysteine catalysed by γ-ECS (γ-glutamylcysteine synthase, EC 6.3.2.2), and glycine addition to y-glutamylcysteine catalysed by glutathione synthetase (227). GSH is synthesized in both the chloroplasts and the cytosol (90). Besides ATP availability, several other factors affect GSH biosynthesis: cysteine supply, GSH turnover (since GSH is a feedback inhibitor of γ-ECS), GSH conjugation (see below) and environmental factors. A decline in ATP content observed under anoxia (166,334) Hanhijarvi and Fagerstedt, 1995; increases the probability of ROS formation in the ETC of mitochondria and, at the same time, inhibits an energy-dependent step in GSH biosynthesis. The key enzyme in GSH biosynthesis, γ-ECS, requires ATP as a cofactor and has an alkaline pH optimum of 8-8.4 (89).

The AA biosynthetic pathway in plants has been elucidated recently (57,313,352). The pathway proceeds through D-glucose ↔ GDP-D-mannose ↔ GDP-L-galactose → L-galactose → L-galactono-l,4-lactone, the latter being an immediate precursor of L-ascorbate (non-inversion pathway, i.e. no inversion of glucose carbon skeleton occurs); while in animals AA biosynthesis involves the conversion of derivatives of D-glucose (353). Another possible non-inversion route involves the following reactions: D-glucose → D-glucosone → L-sorbosone → AA (354). Nevertheless, evidence exists on the possibility of AA biosynthesis through other pathways (355). The final step in AA biosynthesis occurs in the inner mitochondrial membrane and is catalysed by L-galactono-y-lactone dehydrogenase (GAL, EC 1.3.2.3), an enzyme with specificity to cytochrome c as an electron acceptor (356). Association of AA biosynthesis with the functional activity of mitochondrial ETC sets a limit to AA synthesis under lack of oxygen due to saturation of ETC and reduction of cytochrome-c. Another factor, unfavourable for AA synthesis, is anoxiainduced cytoplasmic acidosis, which can affect the activity of GAL (357).

3.4. Efficient transport of antioxidants Under physiological pH the reduced form of AA is negatively charged, and therefore

cannot freely diffuse through the biological membranes. In contrast, dehydroascorbic acid (DHA) is more likely to penetrate the membrane. In plants, the AA biosynthetic site is localized on the inner mitochondrial membrane (314,352) and, hence, AA should be transported out from the mitochondria to the cytosol, chloroplast, and across the plasma membrane to the apoplast to provide antioxidative defense. Until now, a mitochondrial transporter has not been characterized. Evidence has been accumulating on the existence of both AA and DHA specific transporters on the plant plasma membrane. DHA appears to be the preferred form of transport from the apoplast to the cytosol in Phaseolus vulgaris (297) and in Nicotiana tabacum (298). The Km values for intercellular transport of AA (90 µM) and DHA (20 µM) through high-affinity carriers also suggest that DHA is more readily taken up by the cell (54). Recently, the existence of an AAJDHA exchanger on the plant plasma membrane has been suggested (297). The mechanism of exchange employs the proton-electrochemical gradient across the plasma membrane, as shown with uncoupler [carbony lcyanide- 3-chloropheny Ihydrazone (CCCP)] experiments, while DHA uptake occurs via facilitated diffusion and shows no dependence on proton and ion gradients. The pathways of AA (DHA) transport are of crucial importance under anoxia, since the inner mitochondrial membrane potential dissipates after a short lag phase, sustained by ATP hydrolysis via F1F0-ATPase, and hence, only proton gradient-independent transport is possible. In our experiments (156) DHA was the main form in the ascorbate pool of cereal roots (AA/DHA ratios between 0.2 and 0.8), a fact that can be partly explained by the preferred transport of DHA from shoots to roots. It is not clear whether AA biosynthesis occurs in the plant root mitochondria to the same extent as it does in green tissues, where more precursors are available. However, expression of L-galactono-γ-lactone dehydrogenase mRNA has been found in tobacco leaves, shoots and roots in almost equal quantities (358). Besides, high-level irradiance has been shown to have a stimulating effect on ascorbate accumulation in leaves and the chloroplasts (87), and dark-induced ascorbate deficiency has been described in leaf cell walls of Phaseolus vulgaris (359). It is also possible


that intercellular transport of DHA can act as a signal of redox imbalance under stress, a condition that is known to induce defense responses.

Less is known about the glutathione transport mechanism in plant tissues. Most of the studies are focused on the function of glutathione-S-transferases (GST, EC 2.5.U8) in herbicide detoxification, conjugation of GSH to cytotoxic compounds arising from oxidative stress, pathogen attack and heavy metals (360). The onset of hypoxia and subsequent reoxygenation is manifested by enhanced ROS formation and LP. Peroxidation products such as membrane lipid hydroperoxides (e.g. 4-hydroxyalkenals), epoxides, organic hydroperoxides (361) and oxidative products of DNA degradation (base propanols) are the substrates of GST; they can be conjugated to GSH and detoxified (94,360). In plant tissues GSH-conjugates are transported from the cytosol into the vacuole for storage via ATP-binding cassette transporters (362), including the GS-X pump. Conjugation to GSH serves as a specific 'tag' for recognition, transport and sequestration of endogenous and stressspecific metabolites. ATP-dependent transport of GSSG, but not GSH into barley vacuoles via glutathione Sconjugate ATPase has been described (363). However, under energy limitation during hypoxia! anoxia the translocation to vacuole can be inhibited, while GSH conjugation can still occur. In addition, ascorbate peroxidase-mediated conjugation of GSH to unsaturated phenylpropanoids (trans-cinnamic and para-coumaric acids) has been shown in plants. GSH-conjugate is presumably formed via peroxidase-dependent formation of thiyl free radicals that react with the alkyl double bond (364). Conjugation of GSH to LP products can lead to the depletion of the total glutathione pool, since glutathione turnover will be repressed. Indeed, exhaustion of the glutathione pool under anoxia and reoxygenation was not accompanied with concurrent increase in GSSG (156).

Other pathways for glutathione transport have been described in animal tissues and yeast. In rabbit kidney mitochondria (365) and in yeast (366) uptake of GSH by dicarboxylate and 2oxoglutarate carriers in the inner mitochondrial membrane has been demonstrated. The first high affinity plasma membrane GSH transporter (Km 54 µM) different from glutathione-conjugate pumps and dicarboxylate transporters has been identified in the yeast Saccharomyces cerevisiae. The transporter protein shares homology with S. pombe and with five proteins from Arabidopsis thaliana (367).

3.5. Cooperation between different antioxidant systems It is very important for plant survival under stress conditions that antioxidants can

work in co-operation, thus providing better defense and regeneration of the active reduced forms. The most studied example of the antioxidant network is the ascorbate-glutathione (Halliwell-Asada) pathway in the chloroplasts, where it provides photoprotection (82) by removing H2O2. Recently, components of this cycle have been detected in other cell compartments (211).

Ascorbate works in co-operation not only with glutathione, but also takes part in the regeneration of (αtocopherol, providing synergetic protection of the membranes (299). Tocopherol has been reported to be in direct interaction also with reduced glutathione (253) and reduced coenzyme Q (257). In a recent study, Kagan et al. (247) suggested that tocopherol and reduced coenzyme Q, when present together in a membrane, show combined antioxidant activity which is markedly synergetic.

Recently, redox coupling of plant phenolics with ascorbate in the H2O2-peroxidase system has been shown (241,245). It takes place in the vacuole, where H2O2 diffuses and can be reduced by peroxidases using phenolics as primary electron donors. Both AA and the monodehydroascorbic acid radical can reduce phenoxy I radicals generated by this oxidation. If regeneration of AA is performed in the cytosol and AA is supplied back to the vacuole, a peroxidase/phenolics/ AA system could function in vacuoles and scavenge H2O2 (241). This mechanism is specific for plant tissues and can improve stress tolerance under oxidative stress.

Species and tissue specificity adds to the already complex antioxidant response. It is also important to carry out experiments under strictly controlled conditions with respect to


oxygen concentration and to distinguish between hypoxia and anoxia. Accumulating data suggest that low oxygen concentration plays a crucial role in the induction of anoxic metabolism, i.e. triggers the expression of genes responsible for anaerobic fermentation, sugar utilization (338) and antioxidant defense. Another important point for the experimental set-up is the unavoidable reoxygenation period, when most of anoxia-induced damage has been shown to occur (145). To clarify the situation ROS-levels should be detected (e.g. by electron spin resonance spectrometry) in similar conditions.

Hypoxic tissues exhibit possibilities for enhanced ROS production, accumulation of LP substrates (FFA) and LP itself. These possibilities rise from mitochondria-dependent ROS generation, acetaldehyde dependent OT formation via XO, lipoxygenase action on membrane lipids and finally from lipolytic acyl hydrolase-catalysed liberation of FFA, which underpins a burst in LP on return to normoxia. Shortterm oxygen deprivation stress possibly causes limited accumulation of ROS and peroxidized lipids. At this stage the rate of ROS formation and the degree of lipid peroxidation can be regulated by constitutive endogenous antioxidants. This in part can explain the lack of antioxidant system induction under oxygen deprivation in some experiments. It is noteworthy, that accumulation of ROS and LP products already under hypoxic conditions can bear a signal for low oxygen concentration in the tissue.

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