Activities of Antioxidants in Plants Under Environmental Stress

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Activities of antioxidants in plants Akram Ali and fahad Alqurainy

Activities of antioxidants in plants under

environmental stress

Akram Ali* Fahad AlqurainyDepartment 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, ZagazigUniversity, Zagazig, Egypt.E-mail: [email protected]

AbstractThere 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. Theyinclude 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 improvingcrop yield in afflicted areas.The balance between the production of activated oxygen speciesand the quenching activity of antioxidant is upset which often results in oxidative damage.Many metabolic processes produce active oxygen species. Among the four major activeoxygen species [superoxide radical O-2, hydrogen peroxide H2O2, hydroxyl radical OH andsinglet oxygen 1O2] H2O2and the hydroxyl radical are most active, toxic and destructive. High

salt concentrations normally impair the cellular electron transport within the differentsubcellular compartments and lead to the generation of reactive oxygen species (ROS) such assinglet oxygen superoxide, hydrogen peroxide and hydroxyl radicals. Excess of ROS triggersphytotoxic reactions such as lipid peroxidation, protein degradation and DNA mutation. Sincehigher plants are immobile they cannot escape environmental stresses. The ability of higherplants 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 plantshave been demonstrated in vitro experiments to protect against oxidative damage byinhibition or quenching free radicals and reactive oxygen species. The roles of many othercompounds as potential antioxidants can be inferred by similarity to synthetic antioxidants ofrelated structure. The evidence supports at least a partial antioxidant role in vivo for manyclasses of plant metabolite.

Key words: Environmental stresses, crop losses, plant adaptation, antioxidant defense.

Most environmental stresses are affecting on the production of active oxygen speciesin 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 oxygenspecies and the quenching activity of antioxidant is upset, which often results in oxidativedamage (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 cropyield in afflicted areas. Currently, real, but slow advances are being made by crop breeders


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Activities of antioxidants in plants Akram Ali 2

and agronomists using tried-and-tested methodology; however, biotechnology willincreasingly have a role as genes involved in stress resistance arc cloned and their mode ofaction 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 beenattracting considerable interest

(9). Characterization of mutants and transgenic plants with

altered expression of antioxidant is a potentially powerful approach to understanding thefunctioning of the antioxidant system and its role in protecting plants against stress, andsignificant progress is now being made in this area.

Atmospheric oxygen has been recognized for more than 100 years as the agentresponsible 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 livingtissues has been discovered much more recently. Even under optimal conditions many

metabolic processes produce active oxygen species. Among the four major active oxygenspecies [superoxide radical O-2, hydrogen peroxide H2O2, hydroxyl radical OH and singlet

oxygen1

O2] H2O2and the hydroxyl radical are most active, toxic and destructive(1)

. In plantsthe 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 thepotential to generate singlet oxygen 1O2and superoxide O

-2.

Olga et al.(11)

concluded that generation of reactive oxygen species (ROS) ischaracteristic 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 damageby free radicals are lipids (peroxidation of unsaturated fatty acids in membranes), proteins(denaturation), carbohydrates and nucleic acids. Consequences of hypoxia-induced oxidativestress 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 anaerobicmetabolism 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 ofantioxidants, and ROS-interacting enzymes such as SOD, peroxidases and catalases (12).

In plant tissues many phenolic compounds (in addition to tocopherols) are potentialantioxidants: flavonoids, tannins and lignin precursors may work as ROS-scavengingcompounds (11). Antioxidants act as a cooperative network, employing a series of redox

reactions. Interactions between ascorbic acid and glutathione, and ascorbic acid and phenoliccompounds are well known. Under oxygen deprivation stress some contradictory results on

the antioxidant status have been obtained. Experiments on overexpression of antioxidantproduction do not always result in the enhancement of the antioxidative defense, and henceincreased antioxidative capacity does not always correlate positively with the degree ofprotection (12). Here we present a consideration of factors which possibly affect the

effectiveness of antioxidant protection under oxygen deprivation as well as under otherenvironmental stresses. Such aspects as compartmentalization of ROS formation andantioxidant localization, synthesis and transport of antioxidants, the ability to induce theantioxidant defense and cooperation (and/or compensation) between different antioxidantsystems are the determinants of the competence of the antioxidant system (11).

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

making stress resistance.


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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, includingphotosynthesis, respiration glucose assimilation, and phosphorylation (13). Approximately 4%of the total energy contained in sunlight occurs in the ultraviolet region (wavelengths shorterthan 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. Inboth cases, however, the fundamental mechanisms of photochemical damage are similaralthough different receptor molecules (chromophores) may be involved.

UV-B damages DNA by causing oxidative cross linking between adjacent pyrimidinebases forming cyclobutane pyrimidine dimers and pyrimidine pyrimidone dimmers (14). Unlessrepaired, these block transcription and replication. Repair is achieved by light-activated

photolyases which reduce the dimers in a light-dependent manner. Mutants deficient inphotolyase 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) fromArabidopsis 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 singlebase 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 thegeneration of active oxygen species. Physiologicaland biochemical effects of UV-B radiation include effects on enzymes, stomatal, resistance

concentrationsofchlorophyll, protein and lipid, reduction in leaf area, and tissue damage(18

,19).

Some plants, however, appears to be quite resistant to increased UV irradiation. Thedifferential susceptibility of plants to UV stress is clearly an important factor in theircompetitive 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 fortheir 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 electronconfiguration. The initial excited state ,a short -a short- lived singlet haning, fully pairedelectrons, 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 withneighboring molecules (although this is not common with singlet since their lifetime arenormally too short for them to diffuse over very many molecular diameters) or it may undergointernal 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


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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 tothat 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, lateembryogenesis proteins and related proteins, which accumulate in seeds and water-stressedvegetative tissues, has been reviewed

(22). Transgenic rice expressing HVA1, a gene encoding

a late embryogenesis abundant protein from barley, has increased tolerance to drought andNaC1 as shown by simple growth analysis (23). Aquaporins (water channel proteins) are clearlyinvolved in controlling water movement between cells (24) and may be a target formanipulating 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 ascompatible solutes or osmolytes. Their primary function is turgor maintenance but they mayhave other protective effects on macromolecules in dehydrating cells. The solutes accumulatedvary between species and include proline, betaines, dimethylsulfonioproprionate (DMSP),

polyols (mannitol, sorbitol, and pinitol), trehalose, and fructans. Over the past five years, anumber 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 beenintroduced. The results suggest that they can improve plant growth during osmotic stress evenat 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 cholinemonooxygenase (CMO) and betaine aldehyde dehydrogenase (BALDH) in chloroplasts

(25).

Some bacteria, however, convert choline to betaine in a one step reaction catalysed by cholineoxidase. CodA, which encodes choline oxidase from Arthrobacter globiformis, has beenexpressed in Arabidopsis, a non-betaine accumulator. The gene had a chloroplast targettingsequence. 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 andcontinuous light at 5C

(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 evidencethat GB affected membrane phase transitions and accelerated recovery of photo-inhibition (27).

There is a report that expression of AtriplexhortensisBALDH in rice increases GBcontent 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 soybeancrops in the field (29,30,31). These interesting observations need more investigation, perhaps, torule out the possibility that improved growth is caused by improved nitrogen supply. Contrary

to this, exogenous GB is apparently toxic to Brassicanapus, a non accumulator(32)

. Higherplant BALDH has been cloned and this has now been followed by cloning of CMO. LikeBALDH, CMO expression is upregulated by NaCI. It has a Rieske-type [2Fe-2S] cluster andit is ferredoxin-dependent and, therefore, it represents a novel type of plant oxidase (25). It issuggested that metabolic engineering of GB synthesis with plant BALDH and CMO would bepreferable to using choline oxidase because the plant-derived genes may also have promotors,

which could drive increased expression during water stress(25)

. Furthermore, CMO useschloroplast ferredoxin as reductant, thus linking timing of high stress in the light to betainesynthesis

(25). BALDH is equally efficient at catalysing oxidation of 3-

dimethylsulfonioproprionaldehyde to DMSE a compatible solute of even narrower distributionthan 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 viewthat 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


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has high homology to barley BALDH. Unlike BALDH in betaine accumulating plants, therice enzyme is located in peroxisomes. Feeding betaine aldehyde dehydroganse to rice plantsincreases 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 relatedphysiological changes such as a decrease in leaf water and stomatal closure, result in limitedCO2availability to the channeling of reducing equivalents to the production of active oxygenspecies rather CO2 fixation

(39). Among the four major active oxygen species [superoxideradical O-2, hydrogen peroxide H2O2, hydroxyl radical OH and singlet oxygen

1O2] H2O2and

the hydroxyl radical are most active toxic and destructive(1)

. Hydrogen peroxide can beproduced by either dismutation of O

-2by 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 degradationandDNA 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. colimtlD geneencoding Mannitol dehydrogenase. These plants accumulated modest amounts of mannitol butwere 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 of100mM 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 oxidativegeneration of active oxygen species increases under such conditions (59).

The chloroplast-targetted mannitol accumulating tobacco has been used to test thishypothesis. Mannitol accumulation did not affect photosynthesis. The transgenic plants weremore 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 fromOH* scavenging by mannitol. In a further paper (53), the same group have provided convincingevidence 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 andcould explain the in vivo protective effect of mannitol. Further support for the efficiency ofmannitol acting as an OH* scavenger in vivo has been provided by transformation ofSaccaromyces cerevisiae with the same mtlD gene. A mutant unable to grow at highosmolarity 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 growthmedium

(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 andnecrosis

(63,65). Copper Cu

2ions 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 damagewas related to the rapid loss of CAT activity, Cu

2 ions are redox active and catalyze

fentonperoxides also originate from the induction of lipoxygenase in the presence of Cu2. This


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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 tothe important sulfhydryl groups of enzymes, which exacerbates the phytotoxic action ofmetals

(72).

Pang et al.(74)

studied improving the plant ability to resist lead stress, effect of 0.05mg/L La (NO3)3on the activities of catalase (CAT), superoxide dismutase (SOD), the level ofmalondialdehyde (MDA) in wheat seedlings under lead stress were studied. The effect of La+3on plant growth, chlorophyll content in wheat seedlings after adding 0, 50, 100 mg/l Pb

(NO3)3to the nutrient solution for 12 days was observed. The plants were grown in nutrientsolution 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 rootwere enhanced 0.451.69 times and 33.2077.77% respectively and MDA content wasreduced 11.0527.49% in root after treatments from the second day till the end of theexperiment. 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+3under lead stress withinfive days after treatments compared with Pb1 and Pb2 groups. It was assumed that antioxidantenzymes was found to be increased by La (NO3)3, the antioxidant potential of the wheatseedlings to resist lead stress enhanced. It is suggested that La

+3could 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. Thegeneration of the hydrocarbon gas ethane, the production of malonaldehyde and changes inelectrolytic conductivity has frequently been used as sensitive markers for herbicide action inplants (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 dependentoxidative 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 radicalwhich then reacts with molecular oxygen to produce O-2with the subsequent production ofother 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 ofreactive radical-forming intermediates. These compounds cause severe toxicological problemsand 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 abnormalaccumulation of photosensitizing tetrapyrroles specifically protoporphyrin IX (67). This

pigment is able to cause light dependent generation. These herbicides can also catalyze theoxidation of protoporphyrinogen to protoporphyrin IX. The penultimate step of both heme andchlorophyll biosynthesis is recorded

(79). It is some what anomalous that the reaction product

protoporphyrin IX accumulates in condition where the enzyme which catalyses its formationis expected to be inhibited.

Other compounds such as diuron, that block photosynthetic electron transport andinhibitors of cartenoids biosynthesis, such as norflurazon, initiate photooxidative processes

most probably via the generation of1O2

(78,80). Herbicides which block photosynthesis cause

increased excitation energy transfer from triplet chlorophyll to oxygen while those inhibitcarotenoid biosynthesis eliminate important quenchers of the triplet chlorophyll and

1O2.


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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 factorsinfluencing modem forest decline. Ozone, which originates from a natural photochemicaldegradation of nitrous oxides (NO2), seems to be a greater threat to plants than pollution

(102).Mehlhornet al. (99)suggested that the phytotoxicity of O2is due to its oxidizing potential and

the consequent formation of radicals that induce free radical chain reactions. The O3concentration 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 lipidperoxidation (104,105). Stimulation of both synthesis and degradation of the PSII-DI proteinoccurs in spruce trees following O3 treatment

(106,107) and a decrease in the activity andquantity 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 bothphotosynthetic and non-photosynthetic tissues (110,111). Fumigation with SO2causes 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 SO2an appreciable acidificationof the cytoplasm occur because this gas reacts with water to form sulfurous acid which maythen 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 theinhibition 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 tosupport 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 havebeen 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 ofleaves exposed to high levels of light. Transfer of Arabidopsis leaves from low light to highlight causes rapid induction of mRNA for two nuclear-encoded cytosolic ascorbate peroxidasegenes (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 ofexposure to excess excitation energy. The induction of the APX genes was prevented by


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treatment with 3-(3,4-dichlorophenyl)-l,l-dimethylurea, which blocks photosynthetic electrontransport 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 stateof PQ has a role in acting as a sensor of excess light. Interestingly, glutathione feeding alsoblocked the response and it was suggested that extra glutathione swamps a signal generated bythe 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 APXwhen 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)

. Signaltransduction via increased cytosolic Ca

2+ has been suggested

(124) and this observation has

been extended by demonstration of elevated cytosolic Ca2+

in stomatal guard cells by methylviologen 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 andphysiological 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 specifictemperature determined by the ratio of saturated to unsaturated fatty acids accelerates theconversion of lipids of a liquid-crystalline condition into that of a solid condition in plant cellmembranes (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 ratiounder chilling conditions, are very sensitive to chilling injury compared to others; thus othermechanisms may also be necessary for chilling injury. In previous studies it has beensuggested that oxidative stress induced by chilling stress may play a pivotal role for chillinginjury in plant cells (132,133).

Dong and Chin

(134)

were investigated in the following: the antioxidant defense systemand chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber(Cucumis sati6us L.). Chilling stress preferentially enhanced the activities of the superoxidedismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidasespecific to guaiacol, whereas it induced the decrease of catalase activity. In order to analyzethe changes of antioxidant enzyme isoforms against chilling stress, foliar extracts weresubjected to native PAGE. Leaves of cucumber had four isoforms of Mn-SOD and twoisoforms 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 werepresented in the leaves of cucumber. The intensities of APX-4 and -5 were enhanced bychilling stress, whereas that of APX-3 was significantly increased in the poststress periodsafter 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 andpoststressed-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 catalasedeactivation in the leaves of cucumber, but the response timing of enzyme isoforms againstvarious 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 toa very short growing period. The extreme climatic conditions of high mountain zone, highirradiance, low temperature, rapid temperature change and a reduced CO2 partial pressure


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creates unfavorable conditions for photosynthesis(64,69,135)

. Previous studies revealed thatalpine plants are highly efficient in photosynthesis at low temperatures and are also adapted tohigh 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 temperaturestress 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 growingin 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 sincethese conditions limit plant growth (68,138). The high light intensity, high temperature and thetemperature difference between night and day increases the generation of reactive oxygenspecies 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 extremelyhazardous 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 andglutathione reductase (GR) enzymes (80,141). The antioxidant defense mechanism protects theunsaturated membrane lipids, nucleic acids, enzymes and other cellular structures from theharmful 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 glacialisgrowing 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 behigher 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 toface throughout their life. Winter ice encasement, seed imbibitions, spring floods and excessof rainfall are examples of natural conditions leading to root hypoxia or anoxia. Low oxygenconcentration can also be a normal attribute of a plants' natural environment. Wetland species

and aquatic plants have developed adaptative structural and metabolic features to combatoxygen deficiency. A decrease in adenylate energy charge, cytoplasmic acidification,anaerobic fermentation, elevation in cytosolic Ca

2+concentration, changes in the redox state

and a decrease in the membrane barrier function, are the main features caused by lack ofoxygen (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 acidificationprocess has been temporally resolved in sycamore (Acer pseudoplatanus) cell culture by NMR(nuclear magnetic resonance). The immediate response of cytoplasmic pH was solelydependent on proton-releasing metabolization of the nucleoside triphosphate pool; the long-term regulation (after 20 min of anoxia) involves lactate synthesis, succinate, malate, aminoacid metabolism and ethanolic fermentation (12).

Under natural conditions anoxic stress includes several transition states (hypoxia,anoxia and reoxygenation) characterized by different O2concentrations (Table 1). Excessive

generation of reactive oxygen species (ROS), i.e. under oxidative stress, is an integral part ofmany stress situations, including hypoxia. Hydrogen peroxide accumulation under hypoxicconditions has been shown in the roots and leaves ofHordeum vulgare

(151)and in wheat roots

(153). The presence of H2O2in the apoplast and in association with the plasma membrane has


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been visualized by transmission electron microscopy under hypoxic conditions in four plantspecies (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 2H2O2O2 + 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-hydroperoxideglutathione peroxidase

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

Ascorbate peroxidase 1.11.1.11 AA + H2O2DHA + 2H2O

Guaiacol type peroxidase 1.11.1.7 Donor + H2O2oxidized donor + 2H2O***

Monodehydroascorbate reductase 1.6.5.4 NADH + 2MDHA NAD+ + 2AADehydroascorbate 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 H2O2is slow.

*** AA acts as an electron donor(100)

.

In these experiments H2O2 was probably of enzymatic origin considering the lowoxygen 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 theexistence of a common factor, which provides crosstalk between different signalling

pathways. ROS have recently been considered as possible signaling molecules in the detectionof the surrounding oxygen concentration (167). It has been suggested also that ROS and oxygenconcentration (including hypoxia) can be sensed via the same mechanism. Several modelsemploy 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 (lowNADPH 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 requiresan electron donation. The subsequent one-electron reduction steps are not energy dependentand can occur spontaneously or require appropriate e-/H+ donors. In biological systemstransition 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 termincludes 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 H2O2is 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 canoccur in the Fenton reaction.

A potential route for the formation of a damaging species from a photochemicalactivated triplet state is the transfer of triplet energy to molecular oxygen. The product of theenergy transfer reaction is singlet oxygen 1O2. The chlorophyll pigments associated with theelectron transport system are theprimary source of

1O2. The1O2 may also arise as a by

product of lipoxygenase activity, like the hydroxyl radical,1O2is highly destructive reacting


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with most biological molecules at near diffusion controlled rate(78,168)

. Several classes ofbiological molecules are susceptible to attackby

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 ofdouble bonds in the molecule, to form lipid hydoperoxides and O

-2radicals

(169,170) this take

place by enzyme lipoxygenase. Primary radical (R') formed as a result of UV irradiation leadto formation of lipid radicals (L'). Lipid radicals react with O2(a reaction limited only by therate of diffusion) to produce lipid peroxy radicals (LOO'). This peroxide is likely contributorsto damage and dysfunction of cell and organelle membranes. The subject of singlet oxygenand plants has been reviewed by Knox&Dodge,

(78).

Nonphotochemical routes for oxidative damage in plants usually involve theinteraction of molecular oxygen with free radicals to produce new, potentially harmful freeradical species containing oxygen. This type of reaction may occur directly, or it may bepromoted by enzyme catalysts normally present in the plant cell such as the enzymelipoxygenase (171). Atmospheric oxygen is unusual in that its ground state has two unpairedelectrons; 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 oftenquite slow, results in the characteristic lag period of a radical chain reaction. In thepropagation phase of the reaction there is a build up of peroxy radicals, ROO-, and thesubsequent reaction of peroxy radicals with compounds (R'H) having extractable hydrogenatoms. 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. Inthis phase, the radicals recombine with each other to produce inactive, nonradical products.Synthetic organic chemists have created many effective inhibitors of oxidative damage forrubber, 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. Theability 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 ofdefense against free radical damage. They are critical for maintaining optimum health of plantcells. There are several antioxidant enzymes, peptides and metabolites involved in thescavenging 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 metabolitesinclude 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 aparticular plant species and from different experimental conditions (stress treatment, durationof stress, assay procedure and parameters measured). A large-scale investigation onmonodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR)activities, and AA and GSH contents in II species with contrasting tolerance to anoxia hasrevealed 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 increasedvalues in the tolerant species (146). An investigation on the antioxidative defense system in the


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roots of wheat seedlings under root hypoxia or whole plant anoxia(152)

has revealed asignificant increase in the reduced forms of ascorbate and glutathione. Nevertheless, a rapiddecrease in the redox state of both antioxidants was observed during reaeration. The activities

of MDHAR, DHAR and glutathione reductase (GR) decreased slightly or remained unalteredunder hypoxia, while anoxia caused a significant inhibition of enzyme activities

(152).

Inhibition of GR, ascorbate peroxidase (APX), CAT and SOD activities has been shown alsoby 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-dependentantioxidative system (APX, MDHAR, DHAR) has been shown for anaerobically germinatedrice 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 itsreduction 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, inall 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 superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), as well as the enzymes of theascorbate-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 ofAOS, 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 fromozone damage are contradictory. Lee and Bennett (180) found EDU-enhanced snapbeantolerance 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 O 3 were approximately twice ofthose found in control plants (101). In red spruce, loblolly pine, and Scotch pine, all SODisozymes 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)werelargely unaffected by O3, Ozone susceptibility of P. vulgaris cultivars tested by McKersie etal.(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 roleof these antioxidant enzymes in the O3 detoxification in plants is still contradictory.

It has been suggested that O2- and H2O2play an important role in the mechanism ofmediating ozone injury (141). Most of the evidence for a causal relationship of AOS in ozonephytotoxicity is indirect and derives from the observed changes of the enzymes scavenging thetwo 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 H2O2in plants exposed to ozone(187)

.

Mehlhorn et al. (99)for the first time directly detected elevated levels of free radicals in plantsexposed to ozone using electron spin resonance detection (ESR). It is still unclear how thedynamic balance between the AOS levels and the scavenging enzymes is disturbed in plantsexposed to ozone.

2.1.1. CATALASE AND PEROXIDASE

Plant catalases are tetrameric homoproteins that exist as multiple isoezymes encodedby nuclear genes. They are located mostly in peroxisomes and glyoxysomes, although aspecific isozyme Cat3is present in maize mitochondria

(188). The catalase of soybean nodulesis a typical homotetramer of 220 kDa (190). This enzyme may be especially abundant in theperoxisomes 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


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promotes the redox reaction.2H2O22H2O+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 alimited number of organic hydroperoxides such as MeOOH, using them to carry out oxidativereactions on the acceptor molecules while simultaneously reducing the peroxidic substrate.

Catalase (Cat) is a high capacity but low affinity enzyme which destroys hydrogenperoxide. In Nicotianaplumbaginifolia leaves, the major part of the Cat activity is due toperoxisomal Cat which detoxifies hydrogen peroxide produced by photorespiration. Reductionof 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 butdeveloped white necrotic lesions upon exposure to high light. These symptoms were observedsome time ago in a catalase-deficient barley mutant

(194) and as in barley; symptom

development depends on production of photorespiratory hydrogen peroxide, becausesymptoms are less pronounced in low light. The antisense plants have a number of interestingcharacteristics. 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 mutantor after catalase inhibition by aminotriazole treatment

(8,192). The ability of leaves containing

the antisense construct to destroy exogenous H2O2 was lowered, presumably because thelower catalase activity in the leaf resulted in a smaller concentration gradient for H 2O2diffusion into the leaf discs. The conclusion that catalase is a sink for H2O2and that higheraffinity 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 tostresses 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 salicylateinhibition of catalase in induction of pathogen defense responses. Two groups have shownthat low catalase plants have increased expression of pathogenesis-related proteins andincreased pathogen resistance (14,195).

Other important plant enzymes, the peroxidases, also function in this mode. Inaddition defense against active oxygen compounds plants peroxidases have other importantcellular roles. However, in different cases endogenous auxin levels are regulated by theenzymes auxin oxidase and peroxidase (196). The activities of some antioxidant enzymesincrease during stress treatment, and the types of enzymatic activities that increase aredependent 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 intermediatecatalase- H2O2complex (Compound I) and produces water and dioxygen (catalase action) orcan decay to the inactive Compound II. In the presence of an appropriate substrate CompoundI 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 notaffected 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 ADHand SOD activities responded nonsignificantly to submergence, while catalase activityincreased 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


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anoxia(198)

.

2.1.2. DEHYDROASCORBALE REDUCTASE (DHAR)

DHAR is thought to play an important role in the oxidative stress tolerance of plantsby 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 andwater 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 induceDHAR 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)

andtheir activities increase in response to various environmental stressors. In leaves Arabidopsisthaliana 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. InArabidopsisleaves, the decrease in

CAT activity when exposed to high temperature, high light intensity and water deficiencypreceded the increase of APX and GR activity. This decrease in CAT activity might triggerthe induction of APX and GR activities by reducing the ability of cells to scavenge hydrogenperoxide

(201).Under conditions of salt stress, the salt tolerance cultivar exhibited increased

total superoxide disumutase (SOD) and ascorbate peroxidase APX activity, whereas bothenzyme 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 variousstress conditions. The enzyme catalyses the regeneration of phospholipid hydroperoxides atthe expense of GSH and is localized in the cytosol and the inner membrane of mitochondria of

animal cells. PHGPX can also react with H2O2but this is a very slow process. Until now, mostof the investigations have been performed on animal tissues. Recently, a cDNA clonehomologous 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 andsalt stresses, and that this induction occurs mainly via the production of ROS

(206).

2.1.5. THE ASCORBATE- GLUTATHIONE (ASC-GSH CYCLE)

Itis 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 ofH2O2 in chloroplasts. However, all the components of this pathway are also present in themitochondria and peroxisomes of leaves

(210)and in the cytosol of nodule.

2.1.6. CHLOROPLASTChloroplasts are equipped with effective antioxidative defense systems to withstand

peroxidative attack of toxic O2 species. One such system is the operative mechanism ofascorbate-dependent H2O2. Scavenging enzymes, which by removing H2O2, play a critical roleagainst the generation of the potent oxidant OH (87,98). The enzyme system involvesdetoxification 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. Forthe regeneration of GSH, glutathione (GSSG) reductase functions using NADPH.

Catalase reacts with H2O2directly to form water and oxygen(212). Catalase activities

declined with progress of water stress thus favoring the accumulation of' H2O2. Peroxidasescatalyze hydrogen peroxide dependent oxidation of substrates (RH2) according to the general


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equation.RH2+ H2O22 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 inother studies under drought

(213)and other stress condition such as salt

(214).

Elevated H2O2concentrations could release peroxidase from membrane, structures,with which it is normally associated, as in the case of the insoluble ascorbate peroxidase ofspinach chloroplasts (216). Peroxidase could be synthesized de novo at least in some cases (215).

Water stress could increase the accumulation of peroxidase substrates such asglutathione, 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 O2and 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 tohave 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-2to H2O2and oxygen catalysis at ordinary physiological pH, although O-2is

quite stable above pH 11 nevertheless, virtually all aerobic organisms that have beenexamined 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 higherpotential toxicity have been shown to decline in the older leaves of tobacco plants, whichrevealed signs of membrane damage . There were clear correlation between the activity ofthese two enzymes and the degree of lipid peroxidation in leaves. The authors suggested thatboth enzymes are important agents for protecting leaves from the deleterious effects ofmembrane lipid destruction. Sreannivasulu et al. (204) reported that under conditions of salt

stress thesalt-tolerant cultivar (Setaria italica) exhibited increased total superoxide dismutase(SOD) and ascorbate peroxidase (APX) activity, whereas both stressed seedlings of thesensitive cultivar.

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

.- is achieved through an upstreamenzyme, 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 aerobicorganisms 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)

. Theother three types of this enzyme, classified by their metal cofactor, can be found in livingorganisms, and they are the structurally similar FeSOD (prokaryotic organisms, chloroplaststroma) 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 anexperiment with com (Zea mays), where a 7-d flooding treatment resulted in a significantincrease in TBARS content, membrane permeability and the production of superoxide anion-

radical and hydrogen peroxide in the leaves(158)

. An excessive accumulation of superoxidedue 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-


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oxygenation period, immediately after termination of the anoxic treatment. In the course ofthis experiment the activity decreased in wheat under both aeration and anoxia, but in theanoxic 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 SODactivity

(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 hreoxygenation. In cereals the activity of SOD has been found to decline depending on theduration of the anoxic treatment, while in Iris pseudacorus a 14-fold increase was observedduring a reoxygenation period (220). An increase in total SOD activity has been also detected inwheat 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 rootsand leaves ofHordeum 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). Theexplanation can be found in different tolerance to anoxia between species and experimentalset-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 ROSalready under hypoxic conditions and during the oxidative burst after re-admission of oxygencould cause rapid substrate overload of constitutive SOD, while induction was hinderedprobably by other factors [e.g. time, activity of downstream enzymes in the ROS-detoxification cascade, inhibition by the end product (H2O2) and consequences of anoxicmetabolism]. Observations on SOD activity in different plant species under several stressconditions (drought, salinity and high/ low temperature) suggest that different mechanisms

may be involved in oxidative stress injury(222,223)

. Activation of oxygen may proceed throughdifferent mechanisms, not necessarily producing a substrate for SOD. Changes in O2electronic configuration can lead to the formation of highly reactive singlet oxygen (

1O2).

Comparison of drought and water stress effects on tolerant and intolerant wheat genotypessuggests that different mechanisms can participate in ROS detoxification. For example, waterstress did not affect SOD activity, while under drought conditions a significant increase was

detected (224). In another experiment, oxidative stress conditions combined with coldacclimation of cold-resistant and unresistant wheat cultivars, SOD activity in the leaves and inthe roots was unaffected by the low temperature treatment but plants exhibited higher guaiacolperoxidase activity (225). Inefficiency of ROSdetoxifying enzymes (SOD, CAT, ascorbateperoxidase and non-specific peroxidase) has been shown under water deficit-inducedoxidative 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), -tocopherol and carotenoids in stressed plants. The authors suggested the formation of a certainstrong pro-oxidant, which is neither superoxide nor H2O2under the conditions of water deficit(226). The ability of plants to overcome oxidative stress only partly relies on the induction ofSOD 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 mechanismsare 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 chloroplastsand 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 inmany cells. It reacts with many oxidants such as H2O2to form the oxidized form, a disulphideknown as GSSG.

ROOH +2GSH ROH +GSSG+H2O


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

-2and HO

-. Tanaka et al. (119)have

reported that APX and GR activities are increased in water stressed spinach leaves.

A tripeptide glutathione (-glutamylcysteinylglycine) is an abundant compound inplant tissues. It has been found virtually in all cell compartments: cytosol, endoplasmicreticulum, 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 latterproperty is of great biological importance, since it allows fine-tuning of the cellular redoxenvironment under normal conditions and upon the onset of stress, and provides the basis forGSH stress signalling. Indeed, the role for GSH in redox regulation of gene expression hasbeen 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 highreductive potential of GSH. It scavenges cytotoxic H2O2, and reacts non-enzymatically withother ROS: singlet oxygen, superoxide radical and hydroxyl radical (140). The central role ofGSH 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 proteinhydrolysates protect lipids from oxidation. It is possible that these may be due to the metalcomplexion capacity of these substances. In addition to metabolic changes and accumulationof 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 underosmotic 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 stressresponsive genes are unknown. It has been hypothesized, based on the correlation of lateembryogenesis abundant (LEA) gene expression with physiological and environmentalstresses and the prediction novel structure of the LEA proteins, that LEA protein may play aprotective role in plant cells under various stresses condition. Moreover, this protective rolemay be essential for the survival of the plant under extreme stress condition (232,233).

2.2. Metabolic compounds defenseAntioxidants, 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 fromprogressing. 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 radicaland 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 antioxidantactivity. Recently, there has been increasing interest in oxygen-containing free radicals inbiological systems and their implied roles as causative agents in the etiology of a variety ofchronic disorders. Accordingly attention is being focused on the protective biochemicalfunctions 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, whetherconstitutive 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 (-tocopherols) flavonoids, phenolic acids, other phenols, alkaloids, polyamines,


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chlorophyll derivatives, amino acids and amines and miscellaneous compounds. A number ofstudies indicated that the degree of oxidative cellular damage in plants exposed to a bioticstress 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, hydroxycinnamateesters 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 effectiveantioxidants in vitro than tocopherols and ascorbate. Antioxidative properties of polyphenolsarise from their high reactivity as hydrogen or electron donors, and from the ability of thepolyphenol-derived radical to stabilize and delocalize the unpaired electron (chain-breakingfunction), and from their ability to chelate transition metal ions (termination of the Fentonreaction) (242). Another mechanism underlying the antioxidative properties of phenolics is the

ability of flavonoids to alter peroxidation kinetics by modification of the lipid packing orderand 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 plantcells

(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 flavonolcontent (a decrease after 35 d of treatment) suggests their participation in the antioxidative

defense inI. pseudacorus rhizomes.

2.2.1.1. Vitamin E (-tocopherol):Tocopherols and tocotrienols are essential components of biological membranes

where they have both antioxidant and non-antioxidant functions(246)

. There are fourtocopherol and tocotrienol isomers (-, -, -, -) 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 > > > which is due tothe methylation pattern and the amount of methy I groups attached to the phenolic ring of thepolar head structure. Hence, -tocopherol with its three methyl substituents has the highestantioxidant activity of tocopherols (248). Though antioxidant activity of tocotrienols vs.tocopherols is far less studied, -tocotrienol is proven to be a better antioxidant than -

tocopherol in a membrane environment(249)

. Tocopherols, synthesized only by plants andalgae, are found in all parts of plants

(251). Chloroplast membranes of higher plants contain -

tocopherol as the predominant tocopherol isomer, and are hence well protected againstphotooxidative damage (253). There is also evidence that -tocopherol quinone, existing solelyin chloroplast membranes, shows antioxidant properties similar to those of -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 withalkoxy radicals (LO'), lipid peroxyl radicals (LOO') and with alkyl radicals (L'), derived fromPUPA 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 withconsequent tocopheroxyl radical (TOH') formation (257). Regeneration of the TOH' back to itsreduced 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, especiallysinglet oxygen (via irreversible oxidation of tocopherol), and as physical deactivators ofsinglet oxygen by charge transfer mechanism

(253). TOH' formation sustains prooxidant action

of tocopherol. At high concentration tocopherols act as prooxidant synergists with transitionmetal ions, lipid peroxides or other oxidizing agents (248). It has been clearly shown, thatprooxidant function of tocopherol on low density lipoprotein was clearly inhibited in vitroby

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


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studies have shown that -tocopherol modulates membrane fluidity in a similar manner tocholesterol, and also membrane permeability to small ions and molecules (253). In recentstudies -tocopherol has been shown to decrease the permeability of digalactosyldiacyl-

glycerol vesicles for glucose and protons(259)

. There is also recent evidence of interactionbetween PS II with -tocopherol and -tocopherol quinone

(256). Complexation of tocopherol

with free fatty acids and lysophospholipids protects membrane structures against theirdeleterious effects. The process is of great physiological relevance, since phospholipidhydrolysis products are characteristics of pathological events such as hypoxia, ischemia orstress damage (246). In addition, several other non-antioxidant functions of a-tocopherol havebeen 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. Thesecompounds occur not only in plant but also in mammalian tissues. Among the latter,antioxidant compounds of low molecular weight such as -tocopherols, play an important rolein protecting chloroplastic membranes from the deleterious effects of lipid peroxy radicals and

singlet oxygen (253,262,266).The -tocopherols are usually recycled back by ascorbic acid or reduced glutathione

following oxidation by lipid peroxy radicals. However, it can be irreversible converted to thecorresponding quinone and quinone epoxide after reacting with singlet oxygen

(253),

(261).

Besides, an increased synthesis of antioxidant such as -tocopherols has been correlated witha higher tolerance to drought (263)and other environmental stresses (254). Munne-Bosch et al.(266)reported that -tocopherols progressively decreased in sage during the drought. Therefore,

the leaves contained smaller pools of antioxidant defenses to counteract oxygen toxicityduring the drought and this explain among other biochemical and structural feature, thesusceptibility of this species to stress.

The most biologically active of the four major tocopherols is -tocopherols. Theperoxy radical derived from -tocopherols is also stabilized because the unpaired electrons ofthe 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 oneof the best quenchers for 1O2yet test, with a quenching rate constant of approximately 6x 10

8(in methanol) and it also appears toreact with O

-2 to gave a phenoxy radical 15 mg g

-1fresh

weight of -tocopherols (267). This compound may serve to protect symbiosome membranesandother 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 plantkingdom, 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 theplant 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 isgreatly affectedby light; for example tobacco plants grown under supplementallevels ofUV contained abouttwice the concentration of total soluble phenoliccompounds compared to the control plants.

Plants grown in full sun have also been shown to contain higher levels of flavonoids thanshade grown plants

(245).

Flavonoids are not only accumulated in the plant vacuole as glycosides but alsofound as exudates on the leaves and other aerial surfaces of some species of plants. Theirphysiological functions have long remained unknown except for a rot as a protective filteragainst harmful UV radiation. Additional physiological functions of flavonoids have been

discovered; for example, their role as antioxidants(243)

as factor inducing pollen germinationand pollen-tube elongation

(269,270)and as antifungus agents (phytoalexins). Phenylpropanoid

compounds might alleviate oxidative stress by shading visible light or UV. Furthermore, some


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flavonoids and anthocyanins have been shown to work as antioxidants in vitro(201)

. Flavonoidsand other phenolics are abundant in nodules, where, despite their obvious role as signalmolecules during nodule initiation-they can inhibit lipidperoxidation by intercepting the

peroxyl radicals formed in nodule membranes(271)

. Several flavonoids were shown to bepotent inhibitors of the enzymes lipoxygenase and prostaglandin synthetase, which convertpolyunsaturated fatty acids to oxygen-containing derivatives. Highest activity against bothenzymes was shown by luteolin (5,7,3,4 -tetrahydroxyflavone) and 3,4 -dihydroxy flavone; atabout 3x 10-5, they inhibited 50% of lipoxygenase activity. Theauthordid not speculate onpossible mechanisms for the inhibition of theseenzymesbythese 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 andother organelles from UV damage .The light-filtering ability of these compounds mayreinforce their powerful antioxidant effects to provide a high level of protection againstdamaging oxidants generate either thermally or by light.

Phenolic compounds and flavonoids are among the most influential and widedistributed 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 themost abundant phenolic acid in the plant extract and also the most active antioxidant; a 1.2 x

l05M 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 theseed of the grass Phalaris canariensis. The fatty acids of the seed were predominantlyunsaturated, suggesting that the esters were acting to protect then from oxidation (275). Thelipid soluble esters were effective antioxidants in tests with lard or sardine oil heated at 60C.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 andinteraction with other organisms. In higher pants, most secondary phenolics are derived atleast in part from phenylalanine, a productofthe 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 includingphenylalanine and tyrosine increased in sugar beet leaf during water stress. In addition, aremarkable increase in free amino acid content in some plants subjected to water stress wasalso reported (277,278 ).

Ayaz et al.(273)

reported that, an increase in the content of phenolic acids inrolled

leaves could be related with increasing level of amino acids synthesis i

Documents

Activities of Antioxidants in Plants Under Environmental Stress