Arthritis - Oxidative stress in rheumatoid arthritis

Author

Oxidative stress in rheumatoid arthritis

ironjustice

2007-01-27, 9:31 pm

1 [The role of oxidative stress in the etiopathogenesis of rheumatoid
arthritis]
Matyska-Piekarska E, =C5=81uszczewski A, =C5=81acki J, Wawer I
Postepy Hig Med Dosw (Online). 2006; 60: 617-23

Numerous scientific investigations confirmed the occurrence of
oxidative stress in rheumatoid arthritis patients. There is evidence
demonstrating elevated levels of oxidative stress markers and oxidative
damage caused by reactive oxygen species (ROS) to lipids, proteins,
sugars, and DNA, as well as a significant decrease in total antioxidant
capacity, which protects the organism against ROS activity. Extensive
ROS production can significantly accelerate the process of articular
cartilage damage. It is believed that many disease-modifying
anti-rheumatic drugs (DMARDs) affect oxidative stress, although there
has been insufficient research to confirm such a relationship.

Who loves ya.
Tom

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DEAD PEOPLE WALKING=20
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Fire Chief

2007-01-27, 9:31 pm

numbnutz tom wrote:

> 1 [The role of oxidative stress in the etiopathogenesis of rheumatoid
> arthritis]

What is Oxidative Stress?

Oxidative Stress (OS) is a general term used to describe the steady
state level of oxidative damage in a cell, tissue, or organ, caused by
the reactive oxygen species (ROS). This damage can affect a specific
molecule or the entire organism. Reactive oxygen species, such as free
radicals and peroxides, represent a class of molecules that are derived
from the metabolism of oxygen and exist inherently in all aerobic
organisms. There are many different sources by which the reactive
oxygen species are generated. Most reactive oxygen species come from
the endogenous sources as by-products of normal and essential metabolic
reactions, such as energy generation from mitochondria or the
detoxification reactions involving the liver cytochrome P-450 enzyme
system. Exogenous sources include exposure to cigarette smoke,
environmental pollutants such as emission from automobiles and
industries, consumption of alcohol in excess, asbestos, exposure to
ionizing radiation, and bacterial, fungal or viral infections.

The level of oxidative stress is determined by the balance between the
rate at which oxidative damage is induced (input) and the rate at which
it is efficiently repaired and removed (output) (see Figure. 1). The
rate at which damage is caused is determined by how fast the reactive
oxygen species are generated and then inactivated by endogenous defense
agents called antioxidants. The rate at which damage is removed is
dependent on the level of repair enzymes. The determinants of oxidative
stress are regulated by an individual's unique hereditary factors, as
well as his/her environment and characteristic lifestyle.
Unfortunately, under the present day life-style conditions many people
run an abnormally high level of oxidative stress that could increase
their probability of early incidence of decline in optimum body
functions.

.... numbnutz tom: fertilizer of newsgroups.

Fire Chief

2007-01-27, 9:31 pm

numbnutz tom wrote:

> 1 [The role of oxidative stress in the etiopathogenesis of rheumatoid
> arthritis]

Oxidative Stress

Oxidative stress is imposed on cells as a result of one of three
factors: 1) an increase in oxidant generation, 2) a decrease in
antioxidant protection, or 3) a failure to repair oxidative damage.
Cell damage is induced by reactive oxygen species (ROS). ROS are either
free radicals, reactive anions containing oxygen atoms, or molecules
containing oxygen atoms that can either produce free radicals or are
chemically activated by them. Examples are hydroxyl radical,
superoxide, hydrogen peroxide, and peroxynitrite. The main source of
ROS in vivo is aerobic respiration, although ROS are also produced by
peroxisomal b-oxidation of fatty acids, microsomal cytochrome P450
metabolism of xenobiotic compounds, stimulation of phagocytosis by
pathogens or lipopolysaccharides, arginine metabolism, and tissue
specific enzymes. Under normal conditions, ROS are cleared from the
cell by the action of superoxide dismutase (SOD), catalase, or
glutathione (GSH) peroxidase. The main damage to cells results from the
ROS-induced alteration of macromolecules such as polyunsaturated fatty
acids in membrane lipids, essential proteins, and DNA. Additionally,
oxidative stress and ROS have been implicated in disease states, such
as Alzheimer's disease, Parkinson's disease, cancer, and aging.

References:

Fiers, W., et al., More than one way to die: apoptosis, necrosis and
reactive oxygen damage Oncogene., 18, 7719-7730 (1999).

Nicholls, D.G., and Budd, S.L., Mitochondria and neuronal survival.
Physiol. Rev., 80, 315-360 (2000).

Hayes, J.D., et al., Glutathione and glutathione-dependent enzymes
represent a co-ordinately regulated defense against oxidative stress.
Free Radic. Res., 31, 273-300 (1999).

.... Abortion, n: what numbnutz' mother should have had.

Fire Chief

2007-01-27, 9:31 pm

numbnutz tom spewed:

> 1 [The role of oxidative stress in the etiopathogenesis of rheumatoid
> arthritis]

OXIDATIVE STRESS
TABLE OF CONTENTS

Activation of Oxygen
Biological Reactions of Oxygen Radicals
Oxidative Damage to Lipids
Classical Peroxidation Reactions
Unique Reactions in Plant Membranes
Oxidative Damage to Proteins
Oxidative Damage to DNA
Sites of Activated Oxygen Production
Chloroplasts
Mitochondria
Endoplasmic Reticulum
Microbodies
Plasma membranes
Cell Walls
Defence Mechanisms
Superoxide Dismutase
Catalase
Ascorbic Acid
Glutathione
Tocopherol
Carotenoids
Herbicide tolerance
Paraquat
Photosensitizing Herbicides
Summary and Conclusions
References
Return to Main Table of Contents

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ACTIVATION OF OXYGEN
One of the paradoxes of life on this planet is that the molecule that
sustains aerobic life, oxygen, is not only fundamentally essential for
energy metabolism and respiration, but it has been implicated in many
diseases and degenerative conditions (Marx, 1985). A common element in
such diverse human disorders as ageing, arthritis, cancer, Lou Gehrig's
disease and many others is the involvement of partially reduced forms
of oxygen. Our realisation of the significance of oxygen in disorders
and stress-induced dysfunctions in cultivated plants is recent due in
no small part to the difficulty in detecting and tracing oxygen
molecules, to the multitude of forms and intermediates that oxygen can
assume, and to the extreme reactivity and rate of the chemical
reactions involved. As a consequence we often in our experiments can
only look for the "footprints" of oxygen reactions in our attempts to
determine cause-effect relationships in stress responses. The following
chapter describes our current understanding of the general principles
of activated oxygen.

Atmospheric oxygen in its ground-state is distinctive among the gaseous
elements because it is a biradical, or in other words it has two
unpaired electrons. This feature makes oxygen paramagnetic; it also
makes oxygen very unlikely to participate in reactions with organic
molecules unless it is "activated". The requirement for activation
occurs because the two unpaired electrons in oxygen have parallel
spins. According to Pauli's exclusion principle, this precludes
reactions with a divalent reductant, unless this reductant also has two
unpaired electrons with parallel spin opposite to that of the oxygen,
which is a very rare occurrence. Hence, oxygen is usually non-reactive
to organic molecules which have paired electrons with opposite spins.
This spin restriction means that the most common mechanisms of oxygen
reduction in biochemical reactions are those involving transfer of only
a single electron (monovalent reduction).

Activation of oxygen may occur by two different mechanisms: absorption
of sufficient energy to reverse the spin on one of the unpaired
electrons, or monovalent reduction. The biradical form of oxygen is in
a triplet ground state because the electrons have parallel spins. If
triplet oxygen absorbs sufficient energy to reverse the spin of one of
its unpaired electrons, it will form the singlet state, in which the
two electrons have opposite spins (Fig. 1). This activation overcomes
the spin restriction and singlet oxygen can consequently participate in
reactions involving the simultaneous transfer of two electrons
(divalent reduction). Since paired electrons are common in organic
molecules, singlet oxygen is much more reactive towards organic
molecules than its triplet counterpart.

Figure 1

Nomenclature of the various forms of oxygen

The second mechanism of activation is by the stepwise monovalent
reduction of oxygen to form superoxide (O 2), hydrogen peroxide (H2O2),
hydroxyl radical ( OH) and finally water according to the scheme shown
in figure 2. The first step in the reduction of oxygen forming
superoxide is endothermic but subsequent reductions are exothermic.

Superoxide can act as either an oxidant or a reductant; it can oxidise
sulphur, ascorbic acid or NADPH; it can reduce cytochrome C and metal
ions. A dismutation reaction leading to the formation of hydrogen
peroxide and oxygen can occur spontaneously or is catalysed by the
enzyme superoxide dismutase. In its protonated form (pKa =3D 4.8)
superoxide forms the perhydroxyl radical ( OOH) which is a powerful
oxidant (Gebicki and Bielski, 1981), but its biological relevance is
probably minor because of its low concentration at physiological pH.

Figure 2 The activation states of oxygen. Non-activated oxygen is a
biradical. From this triplet state it can be activated by either
reversing the spin on one of the unpaired electrons to form the singlet
state or by reduction. The first reduction reaction is endothermic
forming superoxide. Subsequent reductions form hydrogen peroxide,
hydroxyl radical and water. The electronic state for each activation
step is shown with the energy of the reaction in Kcal/mole.

The univalent reduction of superoxide produces hydrogen peroxide which
is not a free radical because all of its electrons are paired (Fig. 2).
Very often the reduction products of oxygen are referred to by
biologists as oxygen free radicals which is a misnomer because in
chemistry a free radical is defined as an atom or molecule with an
unpaired electron. It is more appropriate to refer to the intermediate
reduction products of oxygen as activated not as free radicals because
triplet oxygen (ground state) is a radical and hydrogen peroxide is
not.

Hydrogen peroxide is noteworthy because it readily permeates membranes
and it is therefore not compartmentalised in the cell. Numerous enzymes
(peroxidases) use hydrogen peroxide as a substrate in oxidation
reactions involving the synthesis of complex organic molecules. The
well-known reactivity of hydrogen peroxide is not due to its reactivity
per se, but requires the presence of a metal reductant to form the
highly reactive hydroxyl radical which is the strongest oxidizing agent
known and reacts with organic molecules at diffusion-limited rates.

Fenton described in the late nineteenth century (Fenton, 1894; 1899)
the oxidising potential of hydrogen peroxide mixed with ferrous salts.
Forty years later, Haber and Weiss (1934) identified the hydroxyl
radical as the oxidising species in these reactions:

(1)

In biological systems the availability of ferrous ions limits the rate
of reaction, but the recycling of iron from the ferric to the ferrous
form by a reducing agent can maintain an ongoing Fenton reaction
leading to the generation of hydroxyl radicals. One suitable reducing
agent is superoxide which participates in the overall reaction 2 as two
half reactions shown in reactions 3 and 4:

(2)

(3)

(4)

Therefore, in the presence of trace amounts of iron, the reaction of
superoxide and hydrogen peroxide will form the destructive hydroxyl
radical and initiate the oxidation of organic substrates. Metals other
than iron may also participate in these electron transfer reactions by
cycling between oxidised and reduced states.

The oxidation of organic substances may proceed by two possible
reactions =C4 addition of OH to the organic molecule, or abstraction of
a hydrogen atom from it. In the addition reaction (reaction 5), the
hydroxyl radical adds to an organic substrate forming a hydroxylated
product that is further oxidised by ferrous ions, oxygen or other
agents to a stable, oxidised product (reactions 6 and 7). The
hydroxylated products can also dismutate to form cross-linked products
(reaction 8).

(5)

(6)

(7)

(8)

In the abstraction reaction, the hydroxyl radical oxidises the organic
substrate forming water and an organic radical (reaction 9). The latter
product has a single unpaired electron and thus can react with oxygen
in the triplet ground-state (reaction 10). The addition of triplet
oxygen to the carbon radical can lead to the formation of a peroxyl
radical which can readily abstract hydrogen from another organic
molecule leading to the formation of a second carbon radical (reaction
11). This chain reaction is why oxygen free radicals cause damage far
in excess of their initial concentration.

(9)

(10)

(11)

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BIOLOGICAL REACTIONS OF OXYGEN RADICALS
The reactions of activated oxygen with organic substrates are complex
even in vitro with homogenous solutions, but in biological systems
there are even more complications due to the surface properties of
membranes, electrical charges, binding properties of macromolecules,
and compartmentalisation of enzymes, substrates and catalysts. Thus,
various sites even within a single cell differ in the nature and extent
of reactions with oxygen.

The nature of the oxidative injury that causes cell death is not always
obvious. The mechanisms by which oxygen radicals damage membrane lipids
are well accepted, and consequently oxidative damage is often
exclusively associated with these peroxidation reactions in membrane
lipids. What is sometimes overlooked in our research on environmental
stress in plants is that activated forms of oxygen also degrade
proteins and nucleic acids, reactions which can also be very lethal. In
this section some of the major reactions of activated oxygen with
lipids, protein, and nucleic acids are reviewed.

OXIDATIVE DAMAGE TO LIPIDS
Classical Peroxidation Reactions
The reactions of oxygen free radicals with polyunsaturated lipids have
been extensively researched because of their involvement in rancidity
and the development of undesirable odours and flavours in foods.
Historically these reactions are the most frequently cited consequence
of oxygen radical production in plant cells. Perhaps the mechanisms
were so well established by oil chemists long before the recognition of
their importance in biology that plant biologists applied these
mechanisms directly to their experimental systems, rarely questioning
their validity or transposability. This has delayed recognition of the
presence of free radical reactions in plant membranes. The complexity
of the biological membrane is well established and the reader is
referred elsewhere for more detailed considerations of its structure
(Leshem, 1992). The lipid bilayer membrane is composed of a mixture of
phospholipids and glycolipids that have fatty acid chains attached to
carbon 1 and 2 of the glycerol backbone by an ester linkage. The
peroxidation reactions differ among these fatty acids depending on the
number and position of the double bonds on the acyl chain and the
reader is referred to Frankel (1985) for a detailed review. The
following is a simplified summary of these reactions for a general
lipid, `R', and for a specific fatty acid, linoleate, which is common
in plant cell membranes.

The peroxidation of lipids involves three distinct steps: initiation,
propagation and termination. The initiation reaction between an
unsaturated fatty acid (e.g. linoleate) and the hydroxyl radical
involves the abstraction of an H atom from the methylvinyl group on the
fatty acid (reaction 9); in the case of linoleate this occurs at
carbon-11 (Fig. 3). The remaining carbon centred radical, forms a
resonance structure sharing this unpaired electron among carbons 9 to
13. In the propagation reactions, this resonance structure reacts with
triplet oxygen, which remember is a biradical having two unpaired
electrons and therefore reacts readily with other radicals. This
reaction forms a peroxy radical (reaction 10). In the case of
linoleate, addition occurs at either carbon-9 or -13 (Fig 3). The
peroxy radical then abstracts an H atom from a second fatty acid
forming a lipid hydroperoxide and leaving another carbon centred free
radical (reaction 11) that can participate in a second H abstraction
(reaction 10). Therefore, once one hydroxyl radical initiates the
peroxidation reaction by abstracting a single H atom, it creates a
carbon radical product (R) that is capable of reacting with ground
state oxygen in a chain reaction. The role of the hydroxyl radical is
analogous to a "spark" that starts a fire. The basis for the hydroxyl
radical's extreme reactivity in lipid systems is that at very low
concentrations it initiates a chain reaction involving triplet oxygen,
the most abundant form of oxygen in the cell.

The lipid hydroperoxide (ROOH) is unstable in the presence of Fe or
other metal catalysts because ROOH will participate in a Fenton
reaction leading to the formation of reactive alkoxy radicals:

(12)

Therefore, in the presence of Fe, the chain reactions are not only
propagated but amplified. Note that two radicals are produced by the
summation of reactions 9 to 12. Among the degradation products of ROOH
are aldehydes, such as malondialdehyde, and hydrocarbons, such as
ethane and ethylene, that are commonly measured end products of lipid
peroxidation.

Figure 3 The peroxidation of linoleic acid. The hydroxyl radical
abstracts a H atom from carbon-11 of the fatty acid between the two
double bonds forming water. The electron deficiency is shared among
carbons 9 to 13 in a resonance structure. Triplet oxygen that has two
unpaired electrons may attach to this structure at either carbon -9 or
-13 forming a peroxy radical. This peroxy radical will abstract another
hydrogen atom from a second linoleic acid molecule in a propagation
reaction forming a lipid hydroperoxide. Chain breakage and
cross-linkage reactions subsequently occur to produce aldehydes,
hydrocarbons, alcohols and cross-linked dimers.

The peroxidation reactions in membrane lipids are terminated when the
carbon or peroxy radicals cross-link to form conjugated products that
are not radicals, such as those shown in reactions 13 to 15:

(13)

(14)

(15)

Typically high molecular weight, cross-linked fatty acids and
phospholipids accumulate in peroxidised membrane lipid samples.

Singlet oxygen can react readily with unsaturated fatty acids producing
a complex mixture of hydroperoxides. Again, the chemistry of these
reactions is based on foods (Bradley and Minn, 1992). Oxidation of
unsaturated fatty acids by singlet oxygen produces distinctly different
products than the hydroxyl radical (Bradley and Minn, 1992). Once
formed the lipid hydroperoxides will decompose into a variety of
products, some of which can produce oxygen free radicals in the
presence of metal catalysts (reaction 12).

Unique Reactions in Plant Membranes
The above mechanisms predict that oxygen free radical or lipid
peroxidation reactions in plant membranes would selectively degrade
unsaturated fatty acids and accumulate aldehydes, hydrocarbons, and
cross-linked products. When examining the effects of environmental
stresses on plant membranes, many studies have measured the products of
lipid peroxidation, such as malondialdehyde and/or ethane and concluded
that oxygen free radicals are involved in these stress responses. When
the substrates of these reactions, the membrane fatty acids, have been
examined, it has been very often observed that the unsaturated fatty
acids are not selectively degraded, and therefore these reports have
concluded that oxygen free radicals are not involved in these stress
responses. This controversy has caused many to rule out the involvement
of oxygen free radicals in processes such as seed ageing (Wilson and
McDonald, 1986). However, in vitro experiments that have treated plant
membranes with Fenton reaction products have shown that degradation of
plant membrane lipids by oxygen free radicals does not involve
selective loss of unsaturated fatty acids. For example in Table 1,
microsomal membranes isolated from wheat (Triticum aestivum) crowns and
liposomes prepared from a commercial preparation of soybean asolecithin
were treated in vitro with oxygen radicals generated by Fe-ascorbate.
In both samples, there was destruction of fatty acids and their
recovery from solution was lower after the free radical treatment. In
the liposome sample there was selective degradation of unsaturated
fatty acids; the proportion of linoleic and linolenic acids relative to
the other fatty acids declined. In contrast, treatment of the wheat
microsomal membranes caused degradation og the phospholipids but no
change in the proportion of the fatty acids. Inother words, there was
not selective degradation of the unstaurated fatty acids. Clearly other
reactions to those described above were occurring in these plant
membranes.

An alternative to the classical mechanism of lipid peroxidation was
proposed by Niehaus (1978) based on his observation that most esters
react with superoxide by cleaving the C-O bond. Since the fatty acid
chains are attached to the glycerol backbone of the phospholipid
molecule by an ester bond (Fig. 4), superoxide attack on a phospholipid
bilayer would produce free fatty acids by de-esterification reactions.
This was experimentally observed by Senaratna et al. (1985) in
microsomal membranes from soybean seed axes treated in vitro with
superoxide from xanthine oxidase.

Table 1: Degradation of phospholipid and esterified fatty acid in two
membrane systems by Fenton reaction products.
Data for each fatty acid are expressed as its proportion (%) of all
fatty acids recovered (i.e.each sample totals 100%). Phospholipid (PL)
recovery is expressed as a % of the original PL before treatment.

Adapted from McKersie et al. (1990)

Fatty Acid Liposomes Wheat Microsomes
Before After Before After
16:0 21 34 29 30
18:0 3 5 1 1
18:1 7 10 6 7
18:2 61 47 28 29
18:3 8 4 37 35
PL Recovery 100 51 100 65

Kinetic analysis indicates that superoxide attack on esters occurs by a
nucleophilic addition mechanism (Afanas'ev, 1985).

The peroxy radical RC(O)OO would abstract an H atom to form a
hydroperoxide that would decompose into an acid RCOO=C4 (Afanas'ev,
1985). In the case of a phospholipid, RCOO=C4 would be a free fatty
acid.

These reactions would not be selective for unsaturated fatty acids and
therefore degradation of fatty acids attached to phospholipid molecules
would be random. Experimentally if these reactions occurred in plant
membranes, we would observe loss of phospholipids but no change in
fatty acid unsaturation. Although this is commonly observed in stress
conditions in plants, most researchers usually attribute these products
to the action of an enzyme, such as phospholipase or a non-specific
lipase.

Figure 4 The structure of a typical phospholipid molecule.
Phospholipids form the essential structural component of plant
membranes. Their amphipathic nature dicates that the phospholipid
headgroup is oriented toward the external aqueous phase and the fatty
acid tails are oriented towards the interior hydrophobic phase. The
fatty acids are attached by ester linkages to the sn-1 (palmitoyl;
16:0) and sn-2 (linoleoyl; 18-2) positions of glycerol. The phosphate
headgroup (R) is attached to the Sn-3 position. There are two common
sites of oxygen free radical attack on the phospholipid molecule - the
unsaturated double binds of the fatty acid and the ester linkage
between glycerol and the fatty acid.

Enzymatic and chemical reactions can clearly give the same products and
the distinction between these mechanisms is clouded even further by the
realisation that the formation of superoxide is the result of a
dysfunctioning enzyme (see later section). Therefore, the debate is
somewhat philosophical. Are there enzymes whose primary function is to
degrade (turnover) phospholipid molecules during periods of stress? or
are there enzymes that primarily function in redox transfer of
electrons; but that dysfunction to form superoxide during stress?

It is not at all clear why some plant membranes such as the microsomal
membranes from wheat crowns, exhibit de-esterification reactions
instead of peroxidation reactions. Presumably, it is due to differences
in composition, possibly the presence of specific lipid-soluble,
membrane antioxidants (phenols, flavonoids, quinones), whose effect on
free radical reactions are poorly understood.

OXIDATIVE DAMAGE TO PROTEINS
Oxidative attack on proteins results in site-specific amino acid
modifications, fragmentation of the peptide chain, aggregation of
cross-linked reaction products, altered electrical charge and increased
susceptibility to proteolysis. The amino acids in a peptide differ in
their susceptibility to attack, and the various forms of activated
oxygen differ in their potential reactivity. Primary, secondary, and
tertiary protein structures alter the relative susceptibility of
certain amino acids. In spite of this complexity, generalisations can
be made. Sulphur containing amino acids, and thiol groups specifically,
are very susceptible sites. Activated oxygen can abstract an H atom
from cysteine residues to form a thiyl radical that will cross-link to
a second thiyl radical to form disulphide bridges. Alternatively,
oxygen can add to a methionine residue to form methionine sulphoxide
derivatives. Reduction of both of these may be accomplished in
microbial systems by thioredoxin and thioredoxin reductase (Farr and
Kogama, 1991). A protein-methionine-S-oxide reductase has been measured
in pea chloroplasts (Ferguson and Burke, 1992). This enzyme reduces the
methionyl sulfoxide back to methionyl residues in the presence of
thioredoxin (Brot and Weissbach, 1982). In some instances this enzyme
has restored the biological activity of a protein, but this function in
plants has not been described.

Other forms of free radical attack on proteins are not reversible. For
example, the oxidation of iron-sulphur centres by superoxide destroys
enzymatic function (Gardner and Fridovich, 1991). Many amino acids
undergo specific irreversible modifications when a protein is oxidised.
For example, tryptophan is readily cross-linked to form bityrosine
products (Davies, 1987). Histidine, lysine, proline, arginine, and
serine form carbonyl groups on oxidation (Stadtman, 1986). The
oxidative degradation of protein is enhanced in the presence of metal
cofactors that are capable of redox cycling, such as Fe. In these
cases, the metal binds to a divalent cation binding site on the
protein. The metal then reacts with hydrogen peroxide in a Fenton
reaction to form a hydroxyl radical that rapidly oxidises an amino acid
residue at or near the cation binding site of the protein (Stadtman,
1986). This site-specific alteration of an amino acid usually
inactivates the enzyme by destruction of the cation binding site.

Oxidative modification of specific amino acids is one mechanism of
marking a protein for proteolysis (Stadtman, 1986). In E. coli there
are specific proteases that degrade oxidised proteins (Farr and Kogoma,
1991) and similar specificity is expected in plants. It is well
documented that the various peptide components of photosystem II
turnover at different frequencies; the D1 protein specifically is noted
for its high rate of turnover, and it is assumed that this is a
consequence of oxidative attack at specific sites on the protein
(Barber and Andersson, 1992).

Return to the Oxidative Stress Table of Contents

OXIDATIVE DAMAGE TO DNA
Activated oxygen and agents that generate oxygen free radicals, such as
ionising radiation, induce numerous lesions in DNA that cause
deletions, mutations and other lethal genetic effects. Characterisation
of this damage to DNA has indicated that both the sugar and the base
moieties are susceptible to oxidation, causing base degradation, single
strand breakage, and cross-linking to protein (Imlay and Linn, 1986).
Degradation of the base will produce numerous products, including
8-hydroxyguanine, hydroxymethyl urea, urea, thymine glycol, thymine and
adenine ring-opened and -saturated products.

The principle cause of single strand breaks is oxidation of the sugar
moiety by the hydroxyl radical. In vitro neither hydrogen peroxide
alone nor superoxide cause strand breaks under physiological
conditions, and therefore, their toxicity in vivo is most likely the
result of Fenton reactions with a metal catalyst. At least in E. coli
these Fenton reactions can be driven by NADH. For example, the ndh
mutant in E. coli accumulates NADH as a result of the mutant's
inability to donate electrons from NADH to respiratory pathways; as a
result, the mutant is hypersensitive to hydrogen peroxide. Studies of
other E. coli mutants have lead to the conclusion that a Fenton active
metal is bound to DNA, probably chelated to phosphodiester linkage. If
the bound metal is reduced by a small diffusible molecule, such as
NAD(P)H or superoxide, it will react with hydrogen peroxide to form the
hydroxyl radical (Imlay and Linn, 1986). The short-lived hydroxyl
radical then oxidises an adjacent sugar or base causing breakage of the
DNA chain.

Cross-linking of DNA to protein is another consequence of hydroxyl
radical attack on either DNA or its associated proteins (Oleinick et
al., 1986). Treatment with ionising radiation or other hydroxyl radical
generating agents causes covalent leakages such as thymine-cysteine
addicts, between DNA and protein. When these cross-linkages exist,
separation of protein from DNA by various extraction methods is
ineffective. Although DNA-protein cross-links are about an order of
magnitude less abundant than single strand breaks, they are not as
readily repaired, and may be lethal if replication or transcription
precedes repair.

DNA is an obvious weak link in a cell's ability to tolerate oxygen free
radical attack. First, it seems that DNA is effective in binding metals
that are involved in Fenton reactions, and secondly less damage can be
tolerated in DNA than other macromolecules. As a consequence, the cell
has a number of DNA repair enzymes (Beyer et al., 1991). One reason why
eukaryotic organisms have compartmentalised DNA in the nucleus, away
from sites of redox cycling that are high in NAD(P)H and other
reductants, may be to avoid oxidative damage.

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SITES OF ACTIVATED OXYGEN PRODUCTION
As indicated above, there are two forms of activated oxygen that are
formed by distinctly different mechanisms. The reduction of oxygen to
form superoxide, hydrogen peroxide and hydroxyl radicals is the
principle mechanism of oxygen activation in most biological systems.
However in photosynthetic plants, the formation of singlet oxygen by
the photosystems has importance. Activated oxygen is often formed as a
component of metabolism to enable "complex" chemical reactions, such as
the oxidation of xenobiotics or the polymerisation of lignin, but in
other instances activated oxygen is formed by the dysfunctioning of
enzymes or electron transport systems, as a result of perturbations in
metabolism caused by chemical or environmental stress.

CHLOROPLASTS
As described by Elstner (1991), there are at least four sites within
the chloroplast that can activate oxygen (Fig.6).

(1) PSI can reduce oxygen by the Mehler reaction which is an important
mechanism of oxygen activation in the chloroplast. The reducing side of
PSI is thought to contribute significantly to the monovalent reduction
of oxygen under conditions where NADP is limiting. This would occur,
for example, if the Calvin cycle did not oxidise NADPH as rapidly as
PSI supplied electrons.

(2) Photoactivated chlorophyll normally transfers its excitation energy
to the PS reaction centres, but under conditions that prevent the
captured light energy from being utilised in the electron transport
systems, this energy can excite oxygen from the triplet to singlet
form. These conditions include stomatal closure caused by drought,
damage to the membrane transport systems, lack of specific nutrients,
or the presence of xenobiotic chemicals such as pollutants or
herbicides.

(3) The oxidising side of PSII facilitates four single electron
transfers from water to the PSII reaction centre releasing triplet or
ground-state oxygen. Leaks of electrons from this site to molecular
oxygen, or release of partially reduced oxygen products are thought to
make a relatively minor contribution to activated oxygen production,
but nonetheless it has been demonstrated that certain alcohols can be
reduced by PSII.

Photorespiration is the most obvious oxygenation pathway in the
chloroplast. Rubisco catalyses the addition of oxygen to carbon 2 of
RuBP forming phosphoglycolate and phosphoglycerate. Although this does
not generate activated oxygen in the chloroplast, the subsequent
metabolism of glycolate in the peroxisomes does.

Figure 5 Schematic representation of the electron transport system in
the thylakoid membrane showing three possible sites of activated oxygen
production.

a) Singlet oxygen may be produced from triplet chlorophyll in the light
harvesting complex.

b) Superoxide and hydrogen peroxide may "leak" from the oxidizing
(water-splitting) side of PSII.

c) Triplet oxygen may be reduced to superoxide by ferredoxin on the
reducing side of PSI.

MITOCHONDRIA
Most oxygen is consumed by the cytochrome oxidase enzyme in the
mitochondrial electron transport system, and involves the sequential
transfer of four electrons to oxygen, releasing water. Plant
mitochondria have an additional site of oxygen reduction at the
alternative oxidase, distinguished from cytochrome oxidase by its
resistance to cyanide. However, neither of these sites produce
significant quantities of superoxide (Rich and Bonner, 1978). However,
isolated mitochondria produce H2O2 and O 2 in the presence of NADH
(Loschen et al., 1973; 1974). Antimycin A, which blocks electron flow
after ubiquinone (Fig. 7) enhances oxygen reduction. Presumably other
conditions which also increase the reduction of ubiquinone favour
reduction of oxygen in the ubiquinone =C4 cytochrome b region of the
chain (Rich and Bonner, 1978). The various Fe-S proteins and NADH
dehydrogenase have also been implicated as possible sites of superoxide
and hydrogen peroxide formation (Turrens et al., 1982).

Figure 6 Schematic representation of the electron transport system in
the mitochondrial membrane showing a possible site of superoxide
production by reduced ubiquinones.

..

ENDOPLASMIC RETICULUM
Various oxidative processes, including oxidation, hydroxylations,
dealkylations, deaminations, dehalogenation and desaturation, occur on
the smooth endoplasmic reticulum. Mixed function oxygenases that
contain a heme moiety add an oxygen atom into an organic substrate
using NAD(P)H as the electron donor. The generalised reaction catalysed
by cytochrome P450 is:

(17)

The best characterised cytochrome P450 in plants is
cinnamate-4-hydroxylase which functions in flavonoid and lignin
biosynthesis, but other mixed function oxidases function in other
biochemical pathways including gibberellin and sterol biosynthesis.
Activation of oxygen by these systems is an essential prerequisite to
oxygen addition reactions in the synthesis of these "complex"
metabolites. Superoxide is produced by microsomal NAD(P)H dependent
electron transport involving cytochrome P450 (Winston and Cederbaum,
1983). One possible site at which this may occur is shown in figure 7.
After the univalent reduction of the substrate (RH) and the addition of
triplet oxygen to form the complex P450 - RHOO the complex may
decompose to P450-RH and release superoxide.

Figure 7 Schematic representation of the cytochrome P450 electron
transport system on the endoplasmic reticulum showing one possible site
of superoxide production.

Cytochrome P450 reacts first with its organic substrate, RH. The
complex is reduced by a flavoprotein to form a radical intermediate
that can readily react with triplet oxygen because each has one
unpaired electron. This oxygenated complex may be reduced by cytochrome
b or occasionally the comples may decompose releasing superoxide

Return to the Oxidative Stress Table of Contents

MICROBODIES
Peroxisomes and glyoxysomes are organelles with a single membrane that
compartmentalises enzymes involved in the =DF-oxidation of fatty acids,
and the glyoxylic acid cycle including glycolate oxidase, catalase and
various peroxidases. Glycolate oxidase produces H2O2 in a two electron
transfer from glycolate to oxygen (Lindqvist et al., 1991). Xanthine
oxidase, urate oxidase and NADH oxidase generate superoxide as a
consequence of the oxidation of their substrates. The xanthine oxidase
reaction is often used in vitro as a source of superoxide producing one
mole of superoxide during the conversion of xanthine to uric acid
(Fridovich, 1970).

PLASMA MEMBRANES
A superoxide-generating NAD(P)H oxidase activity has been clearly
identified in plasmalemma enriched fractions (Vianello and Macri,
1991). These flavoproteins may produce superoxide by the redox cycling
of certain quinones or nitrogenous compounds. In the root, NAD(P)H
oxidase reduces Fe3+ to Fe2+ converting it to a form that can be
transported. Dysfunction of this root enzyme will produce superoxide
(Cakmak and Marschner, 1988). An auxin-activated NADH oxidase has been
associated with acidification of the cell wall and auxin-stimulated
cell elongation (Morr=E9 et al., 1988).

The plant NAD(P)H oxidase may have an analogous function to the animal
enzyme. Leucocytes contain an NADH oxidase on the outer membrane
surface which is activated in response to a foreign agent, generating
superoxide that initiates oxidative reactions that destroy the
potential pathogen (Hohn and Lehere, 1975). In plants, fungal elicitors
cause a similar formation of superoxide that has been linked to the
hypersensitive response to some pathogenic fungi (Doke and Ohashi,
1988; Doke et al., 1991). Wounding, heat shock and xenobiotics
transiently activate this superoxide generating reaction, and
consequently, it has been proposed that these superoxide generating
reactions may serve as a signal in plant cells to elicit responses to
biological, physical or chemical stress (Doke et al., 1991).

CELL WALLS
Although it is not immediately obvious, cell walls are active sites of
metabolism, and also oxygen activation. Some of these reactions may be
involved in the defense reactions against pathogens as described above.
Others may involve the degradation or compartmentation of xenobiotic
chemicals. However, the most common reactions are biosynthetic. For
example, the phenylpropanoid precursors of lignin are crosslinked by
H2O2 dependent reactions, that randomly link the subunits to form
lignin (Gross, 1980). NADH is generated by a cell wall malate
dehydrogenase, and then used to form H2O2 (Gross et al., 1977),
possibly by the NADH oxidase on the plasmalemma (Vianello and Macri,
1991). Diamine oxidases are also involved in production of activated
oxygen in the cell wall using diamines or polyamines (putrescine,
spermidine, cadaverine, etc.) to reduce a quinone that will autoxidize,
forming peroxides (Vianello and Macri, 1991; Elstner, 1991).

---------------------------------------------------------------------------=
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DEFENCE MECHANISMS
SUPEROXIDE DISMUTASE
Superoxide dismutase (SOD) was first isolated by Mann and Keilis (1938)
and thought to be a copper storage protein. Subsequently, the enzyme
was identified by a number of names, erythrocuprein, indophenol
oxidase, and tetrazolium oxidase until its catalytic function was
discovered by McCord and Fridovitch (1969). SOD is now known to
catalyse the dismutation of superoxide to hydrogen peroxide and oxygen:

(18)

Therefore, the activity of this enzyme determines the relative
proportions of the two constituents of the Haber-Weiss reaction that
generates hydroxyl radicals (reaction 2). Since SOD is present in all
aerobic organisms and most (if not all) subcellular compartments that
generate activated oxygen, it has been assumed that SOD has a central
role in the defence against oxidative stress (Beyer, et al., 1991;
Bowler et al., 1992; Scandalias, 1993). There are three distinct types
of SOD classified on the basis of the metal cofactor: the copper/zinc
(Cu/Zn - SOD), the manganese (Mn-SOD) and the iron (Fe-SOD) isozymes
(Bannister et al., 1987). These isozymes can be separated by native
polyacrylamide gel electrophoresis, their activity detected by negative
staining and identified on the basis of their sensitivity to KCN and
H2O2. The Mn-SOD is resistant to both inhibitors, whereas the Cu/Zn-SOD
is sensitive to both inhibitors; Fe-SOD is resistant to KCN, and
sensitive to H2O2. The subcellular distribution of these isozymes is
also distinctive. The Mn-SOD is found in the mitochondria of eukaryotic
cells; some Cu/Zn-SOD isozymes are found in the cytosol, others in the
chloroplasts of higher plants. The Fe-SOD isozymes are often not
detected in plants, but when detected, Fe-SOD is usually associated
with the chloroplast compartment (Bowler et al., 1992). The prokaryotic
Mn-SOD and Fe-SOD, and the eukaryotic Cu/Zn-SOD enzymes are dimers,
whereas the Mn-SOD of mitochondria are tetramers (Scandalias, 1993).
Peroxisomes and glyoxysomes of watermelons (Citrillus vulgaris) have
been shown to contain both Cu/Zn- and Mn-SOD activity (Sandalio and Del
Rio, 1988), but there are no reports of extracellular SOD enzymes in
plants. All forms of the SOD are nuclear-encoded and are targeted to
their respective subcellular compartments by an amino terminal
targeting sequence. Several forms of SOD have been cloned from a
variety of plants (Scandalias, 1990; Bowler, 1992).

Prokaryotic cells, and many eukaryotic algae contain only the Mn-SOD
and Fe-SOD isozymes which are believed to be more ancient forms. In the
bacteria E. coli, SOD activity is transcriptionally regulated by the
SOX RS operon (Farr and Kogoma, 1991) but investigations into the
regulatory mechanism of SOD expression in plants are only beginning
(Bowler et al., 1992). To date it has been shown that SOD activity is
increased in cells in response to diverse environmental and xenobiotic
stresses including paraquat, high light, waterlogging and drought.
Apparently, each of the SOD isozymes are independently regulated
according to the degree of oxidative stress experienced in the
respective subcellular compartments, but how this is communicated at
the molecular level is unknown. Bowler et al (1992) have suggested that
this role may be served by unique lipid peroxidation products from each
organelle that diffuse from the site of oxidative damage to the nucleus
where they would enhance transcription of specific SOD genes.

Several reviews on superoxide dismutase have recently been published
which describe the characteristics of the enzymes, the cloned cDNA
sequences and genes, and the effects of overexpression in transgenic
plants (Bowler, et al., 1994, Doke, et al., 1994, Foyer, et al., 1994,
Gressel and Galun, 1994, Scandalios, 1993, Van Camp, et al., 1994)

CATALASE
Catalase is a heme-containing enzyme that catalyses the dismutation of
hydrogen peroxide into water and oxygen. The enzyme is found in all
aerobic eukaryotes and is important in the removal of hydrogen peroxide
generated in peroxisomes (microbodies) by oxidases involved in
=DF-oxidation of fatty acids, the glyoxylate cycle (photorespiration)
and purine catabolism. Catalase was one of the first enzymes to be
isolated in a highly purified state. All forms of the enzyme are
tetramers in excess of 220,000 molecular weight. Multiple forms of
catalase have been described in many plants. These forms have been
cloned from maize (Redinbaugh et al., 1988; Scandalias, 1990) and
homologous genes has been cloned from several other plants. Maize has
three isoforms termed cat-1, cat-2 and cat-3, that are on separate
chromosomes and are differentially expressed and independently
regulated (Scandalias, 1990). Cat-1 and cat-2 are localised in
peroxisomes and the cytosol, whereas cat-3 is mitochondrial. Careful
examination of the structure of beef liver catalase has shown four
NADPH binding sites per catalase tetramer (Fita and Rossmann, 1985),
but these sites were not in close association with the hydrogen
peroxide reaction centre. Instead, NADPH functions in animal catalase
to protect against inactivation by hydrogen peroxide (Kirkman et al.,
1987). The only plant catalase examined, potato, does not contain NADPH
(Beaumont et al., 1990). It is interesting in this regard to note that
catalase is very sensitive to light and has a rapid turnover rate
similar to that of the D1 protein of PSII (Hertwig et al., 1992). This
may be a result of light absorption by the heme group or perhaps
hydrogen peroxide inactivation. Regardless, stress conditions which
reduce the rate of protein turnover, such as salinity, heat shock or
cold, cause the depletion of catalase activity (Hertwig et al., 1992;
Feirabend et al., 1992). This may have significance in the plant's
ability to tolerate the oxidative components of these environmental
stresses.

ASCORBIC ACID
L-ascorbic acid (vitamin C) is an important vitamin in the human diet
and is abundant in plant tissues. Green leaves have the same amount of
ascorbate as chlorophyll. Because of its nutritional importance, the
distribution of ascorbate has been extensively quantified in plants;
however, relatively little consideration has been given to its function
in the plant. Ascorbate has been shown to have an essential role in
several physiological processes in plants, including growth,
differentiation and metabolism (Foyer, 1993). Ascorbate functions as a
reductant for many free radicals, thereby minimising the damage caused
by oxidative stress but ascorbate may have other functions which remain
undefined.

Figure 8. Structure of ascorbic acid and its metabolites

Apparently synthesis of ascorbate occurs in the cytosol because a
specific ascorbate translocator has been identified on the chloroplast
envelope. L-ascorbic acid is synthesised from hexose sugars in higher
plants but controversy remains concerning some steps in its synthesis
(Loewus, 1988). Although two distinct pathways are possible (Foyer,
1993), higher plants primarily convert D-glucose to ascorbate by a
direct conversion that maintains the carbon chain in the same sequence.
The pathway involves the oxidation of carbon-1 of D-glucose and enediol
formation between carbons 2 and 3:

(19) D-glucose D-glucosone L-sorbosone L-ascorbic acid

Ascorbate can directly scavenge oxygen free radicals with and without
enzyme catalysts and can indirectly scavenge them by recycling
tocopherol to the reduced form. By reacting with activated oxygen more
readily than any other aqueous component, ascorbate protects critical
macromolecules from oxidative damage. The reaction with the hydroxyl
radical is limited only by diffusion.

Figure 9 Synthesis and degradation of L-ascorbic acid in plant tissues.

Ascorbic acid is synthesized from D-glucose. As an antioxidant,
ascorbate will react with superoxide, hydrogen peroxide or the
tocopheroxyl radical to form monodehydroascorbic acid and/or
dehydroascorbic acid. The reduced forms are recycled back to ascorbic
acid by monodehydroascorbate reductase and dehydroascorbate reductase
using reducing equivalents from NAD(P)H or glutathione, respectively.
Dehydroascorbate may decompose into tartrate and oxalate.

The reaction with superoxide may serve a physiologically similar role
to SOD:

(20)

2 O 2 + 2H+ + ascorbate 2H2O2 + dehydroascorbate

The reaction with hydrogen peroxide is catalysed by ascorbate
peroxidase (Asada, 1992):

(21)

H2O2 + 2 ascorbate 2H2O + 2 monodehydroascorbate

The indirect role of ascorbate as an antioxidant is to regenerate
membrane-bound antioxidants, such as a-tocopherol, that scavenge
peroxyl radicals and singlet oxygen, respectively:

(22)

tocopheroxyl radical + ascorbate -tocopherol + monodehydroascorbate

The above reactions indicate that their are two different products of
ascorbate oxidation, monodehydroascorbate and dehydroascorbate, that
represent one and two electron transfers, respectively (Fig. 9). The
monodehydroascorbate can either spontaneously dismutate (reaction 23)
or is reduced to ascorbate by NAD(P)H monodehydroascorbate reductase
(reaction 24):

(23) 2 monodehydroascorbate ascorbate + dehydroascorbate

(24) monodehydroascorbate + NAD(P)H ascorbate + NAD(P)

The dehydroascorbate is unstable at pH greater than 6 decomposing into
tartrate and oxalate. To prevent this, dehydroascorbate is rapidly
reduced to ascorbate by dehydroascorbate reductase using reducing
equivalents from glutathione (GSH):

(25)

2 GSH + dehydroascorbate GSSG + ascorbate

Ascorbate has been found in the chloroplast, cytosol, vacuole and
extra-cellular compartments of the cell. About 20-40% of the ascorbate
in the mesophyll leaf cell is in the chloroplast. The chloroplast
contains all the enzymes to regenerate reduced ascorbate from its
oxidised products. Foyer and Halliwell (1976) proposed that hydrogen
peroxide was dissipated in the chloroplast by the coupling of ascorbate
and glutathione redox cycling as shown in figure 10. Many of the
details of this pathway and characterisation of the enzymes has been
conducted by K. Asada in Japan. Consequently this sequence of reactions
is referred to as the Halliwell-Asada pathway. Illuminated chloroplasts
produce superoxide and hydrogen peroxide from sites on the thylakoids,
most commonly PSI. Superoxide is converted into hydrogen peroxide by
either spontaneous dismutation or by the SOD enzyme. Hydrogen peroxide
is scavenged by ascorbate and the enzyme ascorbate peroxidase (Asada,
1992). The monodehydroascorbate has two routes of regeneration, one via
monodehydroascorbate reductase, the other via dehydroascorbate
reductase and glutathione. The terminal electron donor is NADPH. This
pathway serves two functions. One is the detoxification of hydrogen
peroxide that might otherwise participate is Fenton reactions, and the
second in the oxidation of NADPH. The latter function is an apparently
energy-consuming, wasteful process analogous to photorespiration. It
might at first seem more logical that the chloroplast contain catalase
because it would allow the dissipation of hydrogen peroxide without
"wasting" NADPH. However, it should be realised that conditions
favouring electron transfer from PSI to oxygen generally cause a high
redox potential, i.e. high NADPH/NADP ratio. By reducing this redox
potential through the Halliwell-Asada pathway, the tendency of PSI to
reduce oxygen is minimised.

Although ascorbate metabolism has been studied in most detail in the
chloroplast, it is likely that all enzymes for its regeneration also
exist in the cytosol of both photosynthetic and non-photosynthetic
cells. For example different isozymes for ascorbate peroxidase have
been shown to exist in the cytosol and chloroplast compartments (Chen
and Asada, 1989). The cell wall is also an important site of ascorbate
metabolism because it contains mM concentrations of ascorbate. Here,
ascorbate may have a role in cell wall biosynthesis (Polle et al.,
1990). The cell wall does not contain ascorbate peroxidase, but
contains ascorbate oxidase (Chichiricco et al., 1989). This enzyme
contains 8-12 copper molecules per enzyme and catalyses the reaction:

(26)

2 ascorbate + O2 + 2H+ 2 dehydroascorbate + 2H2O

Since the enzymes to recycle oxidised forms of ascorbate are not
present in the cell wall, it has been proposed that the plasmalemma may
have an ascorbate translocator to shuttle oxidised and reduced forms
between the cytosol and cell wall (Foyer, 1993).

Figure 10 The redox cycling of ascorbate in the chloroplast often
referred to as the Halliwell-Asada pathway.

GLUTATHIONE
Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) whose antioxidant
function is facilitated by the sulphydryl group of cysteine
(Rennenberg, 1982). On oxidation, the sulphur forms a thiyl radical
that reacts with a second oxidised glutathione forming a disulphide
bond (GSSG). Some legumes contain homoglutathione (hGSH) that is a
homologous tripeptide of Glu-Cys-Ala (Klapheck, 1988). Detailed reviews
of GSH chemistry are available elsewhere (Alscher, 1989; Hausladen and
Alscher, 1993). GSH has a redox potential of -340 mV that enables GSH
to reduce dehydroascorbate to ascorbate or to reduce the disulphide
bonds of proteins.

GSH is found in most tissues, cells and subcellular compartments of
higher plants. Its levels declines with tissue age and vary among
growth environments; for example, glutathione levels are higher in the
light than in the dark. At the subcellular level, GSH concentration is
highest in the chloroplast, averaging between 1 and 4 mM, but
significant quantities also accumulate in the cytosol. GSH exists
predominantly in the reduced form with estimates varying from 70% in
barley chloroplasts (Smith et al., 1985) to 90% in pea chloroplasts
(Bielawski and Joy, 1986).

GSH can function as an antioxidant in many ways. It can react
chemically with singlet oxygen, superoxide and hydroxyl radicals and
therefore function directly as a free radical scavenger. GSH may
stabilise membrane structure by removing acyl peroxides formed by lipid
peroxidation reactions (Price et al., 1990). As detailed in section
2=2E4.3, GSH is the reducing agent that recycles ascorbic acid from its
oxidised to its reduced form by the enzyme dehydroascorbate reductase
(Loewus, 1988). GSH can also reduce dehydroascorbate by a non-enzymatic
mechanism at pH > 7 and GSH concentrations greater than 1 mM. This may
be a significant pathway in chloroplasts whose stromal pH in the light
is about 8 and GSH concentrations may be as high as 5 mM (Foyer and
Halliwell, 1976).

There are alternative functions for GSH in cellular metabolism
independent of its antioxidant properties. It may have a significant
role in the transport of reduced sulphur from leaves to sink tissues
such as the root (Rennenberg, 1982). GSH also participates in the
detoxification of xenobiotics as a substrate for the enzyme
glutathione-S-transferase. The well documented tolerance of maize to
the triazine herbicides is the result of conjugation of GSH to the
herbicide (Timmerman, 1989). GSH is also the precursor of the
phytochelatins that act as heavy metal binding peptides in plants
(R=FCegsegger et al., 1990).

The synthesis and degradation of GSH occurs continuously through the
glutamyl cycle that has been well characterised in animals (Meister,
1988) and at least portions have been confirmed in plants (Hell and
Bergman, 1988; 1990). The first step in GSH synthesis (reaction 27) is
the combination of glutamate and cysteine to form glutamylcysteine by
the enzyme glutamylcysteine synthetase. The subsequent step involves
the addition of glycine by the enzyme glutathione synthetase (reaction
28). In the legumes that accumulate hGSH, this second step involves the
addition of alanine by the enzyme homoglutathione synthetase (reaction
29).

(27) Glu + Cys Glu-Cys

(28) Glu-Cys + Gly Glu-Cys-Gly

(29) Glu-Cys + Ala Glu-Cys-Ala

The degradation of GSH involves first the cleavage of the bond between
glutamate and cysteine by glutamyl transpeptidase and the transfer of
the glutamate residues to an acceptor amino acid:

Subsequently the Cys-Gly dipeptide is degraded by dipeptidases and
Glu-aa by glutamylcyclotransferase:

(30) Glu-Cys-Gly + aa Glu-aa + Cys-Gly

(31) Cys-Gly Cys + Gly

(32) Glu-aa 5-oxoproline + aa

Alternatively, plants may contain an additional pathway to that
described in animals for degradation of GSH involving a GSH-specific
carboxypeptidase and glutamylcyclotransferase (Steinkamp and
Rennenberg, 1984):

(33) Glu-Cys-Gly Glu-Cys + Gly

(34) Glu-Cys 5-oxoproline + Cys

The presence of the alternative degradative pathway in plants is
significant, because Glu-Cys is a common intermediate in the synthesis
and degradation of GSH and thus may be a site for regulating GSH
content in plant cells (Hausladen and Alscher, 1993). The final step in
the degradation is the conversion of 5-oxoproline to glutamate by
5-oxoprolinase:

(35) 5-oxoproline Glu

The enzymes that catalyse the synthesis and degradation of GSH have
been characterised in both the chloroplastic and cytosolic compartments
(Hausladen and Alscher, 1993). The numerous inhibitors of these enzymes
have been used in animal systems to characterise the function of GSH in
cellular metabolism but their use in plant systems has not been
extensive. Similarly, although a number of environmental stresses have
been shown to cause GSH accumulation (Alscher, 1989), the mechanism
involved has not been defined.

The reduction of GSSG to GSH is catalysed by the enzyme glutathione
reductase (GR) which has been purified from a number of plant tissues
(Smith et al., 1989), and cDNA for GR has been cloned from pea
(Creisson et al., 1992). The amino acid sequences and structures as
determined by x-ray crystallography are very similar among the human
and E. coli GR so far examined, suggesting that this is a very highly
conserved enzyme (Hausladen and Alscher, 1993). Nonetheless, the plant
literature has conflicting reports of the subunit composition of GR;
heterotetramer or homodimer forms have been reported (Hausladen and
Alscher, 1993). This is not totally unexpected. Although animals have
only one form of GR, plants have multiple forms of the enzyme. For
example, pea leaf GR has been resolved into eight isoenzymes by two
dimensional gel electrophoresis (Edwards et al., 1990). These isozymes
are most likely associated with different subcellular compartments; GR
is associated mainly with the chloroplast but significant activity is
also found in the cytosol and a lesser amount in the mitochondria. The
different isozymes quite probably represent the products of different
genes with slightly or perhaps significantly different sequences, and
with differing regulation and responses to environmental signals.

Animals contain a selenium containing enzyme, glutathione peroxidase,
that reduces hydrogen peroxide forming GSSG and thereby serves as an
alternative means of detoxifying activated oxygen. This enzyme was
thought to be absent from higher plants (Smith and Shrift, 1979) but
recently there have been reports of glutathione peroxidase in cultured
cells ((Drotar et al., 1985; et al. 1991). A plant cDNA showing
homology to animal glutathione peroxidase has been isolated from
Nicotiana sylvestris (Criqui et al., 1992).

Return to the Oxidative Stress Table of Contents

TOCOPHEROL
The tocopherols, specifically a-tocopherol (vitamin E), have been
studied extensively in mammalian research as membrane stabilisers and
multifaceted antioxidants, that scavenge oxygen free radicals, lipid
peroxy radicals, and singlet oxygen (Diplock et al., 1989). This role
is related to its fully substituted benzoquinone ring and fully reduced
phytyl chain (Fig. 11). Its hydrophobic nature dictates that
a-tocopherol is located exclusively in cell membranes and is oriented
with the benzoquinone ring in close association with the carbonyl of
the glycerol component of the phospholipid, and with the phytyl chain
associated with the fatty acids in the hydrophobic inner regions of the
membrane bilayer. The ring oxygen is near the surface of the membrane
bilayer but remains exposed to the lipid environment.

Because of its dietary importance tocopherol levels have been
documented extensively in plant tissue (Hess, 1993). Tocopherol
concentrations vary among plant tissues from 200 ng g-1 fresh weight
(fw) in potato tubers to 5 mg g-1 fw in oil palm leaflets. Tocopherol
has been found in all higher plants, in both photosynthetic and
non-photosynthetic tissues. Although tocopherol is a well documented
component of chloroplast membranes, there are no quantitative estimates
of its distribution among other plant cell membranes.

Tocopherol is actually a family of antioxidants (Hess, 1993) that
includes four tocols that have a phytyl chain and analogous
tocotrienols that have a geranylgeranyl chain (Fig. 11j). a-Tocopherol
is generally considered to be the most active form of the tocols; the
other forms may be biosynthetic precursors. The relative synthesis of
the tocols and tocotrienols may depend on the relative availability of
phytyl and geranylgeranyl precursors. a-Tocopherol synthesis occurs in
plastids with the aromatic ring formed by the shikimic acid pathway and
the phytyl chain synthesized from geranylgeranyl pyrophosphate through
the terpenoid pathway on the plastid envelope (Fryer, 1992). The latter
forms a common link among the synthesis of chlorophyll, tocopherol, and
carotenoids.

a-Tocopherol is well established as a membrane stabilising agent.
Although some of this activity is due to its influence on membrane
lipid organisation, at least part of this activity is the result of its
ability to complex free fatty acids (Fryer, 1992). Free fatty acids act
as detergents in membranes causing disruption of the bilayer, membrane
aggregation and fusion. The association between the carboxyl group of
the fatty acid and the ring of tocopherol reduces this destabilisation.

The antioxidant properties of tocopherol are the result of its ability
to quench both singlet oxygen and peroxides (Fryer, 1992). Tocopherol
is a less efficient scavenger of singlet oxygen than =DF=ADcarotene and
therefore in the thylakoid membrane it may function to break carbon
radical chain reactions by trapping peroxyl radicals:

(36) ROO + tocopherol ROOH + tocopherol

The tocopheroxyl radical is stabilised by the fully substituted
benzoquinone ring and therefore does not propagate the radical
reaction. This is in effect a termination reaction making tocopherol an
effective free radical trap.

Because the active oxygen of the a-tocopherol is located near the
surface of the bilayer and because it readily diffuses laterally in the
plane of the bilayer, tocopherol can react with peroxyl radicals formed
in the bilayer as they diffuse to the aqueous phase. This position also
allows the tocopheroxyl radical to be reduced by ascorbate in the
aqueous phase to regenerate a-tocopherol (reaction 22). Glutathione
reduces tocopheroxyl radicals in alcohol solutions, but there is only
limited evidence for this in biological systems (Hess, 1993).

Figure 11 Structure of tocopherols and tocotrienols commonly found in
plants.

CAROTENOIDS
Carotenoids are C40 isoprenoids and tetraterpenes that are located in
the plastids of both photosynthetic and non-photosynthetic plant
tissues. In chloroplasts, the carotenoids function act as accessory
pigments in light harvesting, but perhaps a more important role is
their ability to detoxify various forms of activated oxygen and triplet
chlorophyll that are produced as a result of excitation of the
photosynthetic complexes by light.

There are two classes of carotenoids. The carotenes are hydrocarbons;
the xanthophylls are carotene derivatives that contain one or more
oxygen atoms. Carotenoids are synthesised from geranylpyrophosphate
from the isoprenoid pathway in plastids, and thus have common
precursors to chlorophyll and tocopherol. =DF-carotene is formed by the
cyclisation of lycopene and the xanthophylls are formed by mixed
function oxidases that introduce hydroxyl groups to the carotene
molecule. Details of these biosynthetic pathways have been recently
reviewed (Young, 1991a; Pallett and Young, 1993). Certain classes of
herbicides function by the inhibition of carotenoid biosynthesis
(Young, 1991a).

The carotenoids can exist in a ground state or in one of two excited
states after the absorption of light energy. Details of carotenoid
photochemistry are given elsewhere (Young, 1991b). In terms of its
antioxidant properties carotenoids can protect the photosystems in one
of four ways: by reacting with lipid peroxidation products to terminate
chain reactions (Burton and Ingold, 1984); by scavenging singlet oxygen
and dissipating the energy as heat (Mathis and Kleo, 1973); by reacting
with triplet or excited chlorophyll molecules to prevent formation of
singlet oxygen; or by the dissipation of excess excitation energy
through the xanthophyll cycle.

Carotenoids may augment a-tocopherol in scavenging peroxy radicals
(Burton and Ingold, 1984):

(37) =DF-car + ROO =DF-car + ROOH

(38) =DF-car + ROO inactive products

These reactions therefore act as chain terminations but unlike
tocopherol a mechanism to recycle =DF-carotene to the reduced form has
not been described.

The main protective role of =DF-carotene in photosynthetic tissue may be
through its direct quenching of triplet chlorophyll, which would
prevent the generation of singlet oxygen, and therefore completely
avoid oxidative stress.

(39) 3Chl* + 1=DF-car 1Chl + 3=DF-car*

(40) 3=DF-car* 1=DF-car + heat

In reactions 39 and 40, energy is transferred from chlorophyll to a
carotenoid which subsequently dissipates the energy in a non-radiative
form (heat). Thus, the carotenoids act as a competitive inhibitor for
the formation of singlet oxygen, and this is aided considerably by
their proximity to chlorophyll in the light harvesting complex. This
method of protection is especially critical as light intensity
increases above saturating levels (Demming-Adams and Adams, 1993).
Another carotenoid, zeaxanthin, has been implicated in the dissipation
of thermal energy, but the precise mechanism has not been resolved.
Zeaxanthin apparently facilitates the conversion of triplet to singlet
chlorophyll in more efficient manner than =DF-carotene. The xanthophyll
cycle involves the reversible conversion of the xanthophylls between
two forms, violaxanthin and zeaxanthin. A de-epoxidase enzyme catalyses
the de-epoxidation of violaxanthin to zeaxanthin in the presence of
excess light, and an epoxidase catalyses the reverse reaction in
darkness or low light. Zeaxanthin therefore accumulates under light
intensities that exceed photosynthetic capacity. The de-epoxidase has a
low pH optimum (5.1), whereas the epoxidase has a high pH optimum
(7.5). Reduced ascorbate serves as the electron donor for the
de-epoxidase, whereas NADPH supplies reducing equivalents for the
epoxidase (Fig. 13).

Figure 12 Structure of two common carotenoids found in plants,
=DF-carotene and zeaxanthinin.

Figure 13: The Xanthophyll cycle for the cycling of violaxanthin and
zeaxanthin.

The de-epoxidation reaction converting violaxanthin to zeaxanthin is
favoured under high light and pH 5.1, whereas the epoxidation reaction
is favoured under low light and pH 7.5. The enzymes for both reactions
are in the chloroplast thylakoid lumen and therefore during periods of
photosynthesis the lumen is acidified, and zeaxanthin accumulates. The
reverse reactions occur in the dark and violaxanthin accumulates.

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HERBICIDE TOLERANCE
A number of herbicides that are widely used on many agricultural crops
act by the production of oxygen free radicals. A few plants have
evolved or have been selected for tolerance to these herbicides. The
mechanism of tolerance in some plants is related to the plants' ability
to scavenge activated oxygen and thereby avoid oxidative stress. Two
examples will be discussed in detail.

PARAQUAT
Paraquat and diquat are bipyridylium (viologen) herbicides that are
effective as non-selective herbicides when applied to leaves. Symptoms
include rapid wilting and desiccation of the leaf, followed by necrosis
with 24 hours. Both light and chlorophyll are required for rapid
response to the herbicide, which suggested to the early researchers
that the herbicide was acting on photosynthesis. In the dark and in
etiolated seedlings the symptoms appear more slowly (Mees, 1960). At
the ultrastructural level, the first injury symptoms are chloroplast
swelling, followed by breakdown of the tonoplast and the plasmalemma,
and then breakdown of the thylakoids and chloroplast envelope (Dodge
and Lawes, 1974). These observations indicated that cellular organelles
differ in their sensitivity to the toxic agent and that the toxic agent
is able to diffuse within the cell from its site of production in the
chloroplast to the tonoplast and plasmalemma where it has its action
promotes the visible symptoms of wilting and necrosis.

That the toxic agent is some form of activated oxygen was suggested by
the first experiments of Mees (1960) who postulated that hydrogen
peroxide was produced from the photosynthetic electron transport
system. This was based on the observations that monuron, an inhibitor
of the Hill reaction, slowed the phytotoxic symptoms. Calderbank (1968)
proposed that the hydroxyl radical was produced by these reactions, and
subsequently Farrington et al. (1973) suggested the involvement of
superoxide.

Paraquat has a redox potential of -446 mV, which is critical to its
function as a free radical generator. Any reducing agent with
sufficient energy can denote an electron to the bipyridylium divalent
cation, paraquat2+, to form a free radical, paraquat1+. The oxidation
of the bipyridylium radical to form the original paraquat2+ results in
the transfer of the electron to oxygen and the formation of superoxide
(Fig. 14). Subsequent Haber-Weiss and Fenton reactions yield toxic
hydroxyl radicals. The paraquat2+ ion is again ready for another cycle,
and thus the herbicide functions as a catalyst to transfer reducing
equivalents to oxygen. In the leaf, the major reducing agent with
sufficient energy to reduce paraquat is the primary electron acceptor
in PSI, ferredoxin, but other reducing agents may be effective.
Consequently, bipyridylium herbicides are also quite toxic to non
photosynthetic organisms, including humans.

Resistance to paraquat has appeared in a number of weedy species after
prolonged use of the herbicide (Fuerst and Vaughn, 1990), in Lolium
perenne after breeding and selection (Harper and Harvey, 1978), and in
genetically engineered plants, animal cells, and microbes (see below).
In paraquat-sensitive plants that have been selected in the field by
prolonged exposure paraquat, the mechanism(s) of resistance is
ambiguous. In biotypes of Conyza baraciensis (Shaaltiel and Gressel,
1986) and in perennial ryegrass, Lolium perenne (Harper and Harvey,
1978), resistance was correlated with increased activity of superoxide
dismutase and other free radical scavenging enzymes. However, in other
resistant plants there is reduced translocation of the herbicide to its
site of action in the chloroplast (Fuerst et al., 1985; Preston et al.,
1992), and in other instances, a correlation with superoxide dismutase
activity was not observed (Vaughn and Fuerst, 1985).

Unfortunately, the situation is just as ambiguous in genetically
engineered organisms that have modified expression of superoxide
dismutase or glutathione reductase. Gruber et al. (1990) expressed the
E=2E coli Mn-superoxide dismutase in the cyanobacterium Anacystis
nidulans, and observed that the transformants had less damage when
treated with paraquat. Similarly, Bowler et al. (1991) expressed a
Mn-superoxide dismutase cDNA from Nicotiana plumbiginfolia in N.
tabacum and showed reduced cellular damage when the transformed plants
were treated with paraquat. However, tomato and tobacco plants that
were transformed with a Cu/Zn-superoxide dismutase cDNA from Petunia
did not have enhanced paraquat resistance (Tepperman and Dunsmuir,
1990).

Superoxide dismutase is unlikely to be the only enzyme involved in
providing protection against activated oxygen produced by paraquat.
Glutathione peroxidase has been associated with paraquat resistance in
a human cell line, HL60 (Kelner and Bagnell, 1990). A transgenic N.
tabacum plant that expresses the glutathione reductase gene from E.
coli had reduced susceptibility to paraquat compared to the control
plants based on visual leaf damage (Aono et al., 1991). Malan et al.
(1990) observed that in maize inbreds that tolerance of paraquat (and
also drought) was not significantly correlated with the activity of
either superoxide dismutase or glutathione reductase individually, but
it was correlated with the two enzyme activities collectively.
Glutathione reductase reduces production of reduced oxygen by isolated
chloroplasts in the presence of paraquat presumably by restoration of
the NADP pool (H=E4rtl et al., 1992).

There are also developmental changes in the plant's tolerance to
paraquat. For example, in winter wheat, mesophyll protoplasts, excised
leaves, and seedlings have increased tolerance to paraquat after
acclimation of the seedlings at 2=B0C (Kendall and McKersie, 1989;
Bridger et al., 1994). This was associated with an increased quantity
of lipid soluble antioxidants that would minimise the deleterious chain
reactions associated with lipid peroxidation (Kendall and McKersie,
1989).

The above discussion illustrates a generalisation which will be a
common theme in the chapters to follow. Most often our experimental
research involves simple experiments that have focused on individual
components of stress tolerance, in this case herbicide tolerance. The
plant is not so simple and as a general rule will not develop a single
method of surviving a potentially catastrophic stress, if two or more
can be developed in parallel. In some instances discussed later these
mechanisms of tolerance may be mutually exclusive, but very often they
are complimentary and serve as backup systems, insurance or additive
protection. Looking at the case in point, tolerance of the free radical
generating herbicide paraquat, it is obvious that a number of
mechanisms may be involved: (a) reduced translocation of the herbicide
to its site of action; (b) altered redox potential of PSI primary
electron acceptor so that the paraquat is not an efficient electron
acceptor and will therefore not generate superoxide; (c) enhanced
scavenging of superoxide produced by PSI; and (d) enhanced protection
of membrane components from lipid peroxidation reactions. All the above
mechanisms except (b) have been described in tolerant plants.
Presumably mechanism (b) is not viable because changes in redox
potential would also change the efficiency of electron transfer between
ferredoxin and NADP, and this would reduce photosynthetic efficiency
and competitiveness or yield. The actual mechanism(s) selected will
undoubtedly vary among plants.

Figure 14 The redox cycling reaction of paraquat that causes superoxide
production.

PHOTOSENSITISING HERBICIDES
Several herbicide formulations promote the accumulation of metabolic
intermediates of chlorophyll, namely tetrapyrroles. In the light,
photons are absorbed by the tetrapyroles and the energy used to create
singlet oxygen that kills the plant. Two classes of photobleaching
herbicides, the p-nitrodiphenyl ethers and aminolevulinic acid-based
modulator, differ in the precise mechanisms of tetrapyrrole
accumulation.

The p-nitrodiphenyl ethers, exemplified by acifluorfen, are used as
selective herbicides in soybeans (Johnson et al., 1978) and require
light for herbicidal activity. Early symptoms of injury include
wilting, desiccation, and necrosis. Ultrastructural studies have shown
that the plasmalemma and tonoplast are the first organelles to be
disrupted, not the chloroplast, which explains why enhanced electrolyte
leakage is one of the first indicators of injury from a
photosensitising herbicide. Acifluorfen selectively inhibits
protoporphyrinogen oxidase (Protox) which is the penultimate step in
heme and Mg-protoporphyrin IX synthesis on the chloroplast envelope
(Fig. 15) (Matrinage et al., 1989; Witkowski and Halling, 1989). This
blockage results in the accumulation of its substrate
protoporphyrinogen which at the time was thought to oxidise to its
product protoporphyrin IX by a "spontaneous" reaction (Witkowski and
Halling, 1989). The loss of enzymatic control of protoporphyrin IX
synthesis was associated with lower heme levels and increased carbon
flow into the pathway (Kouji et al., 1989). Because heme is a feedback
inhibitor of aminolevulinic acid synthesis, this change in regulation
of the pathway was thought to lead to the 100 fold accumulation of
protoporphyrin IX (Matsumoto and Duke, 1990) but a detailed evaluation
of the data from these early experiments on acifluorfen action were
inconsistent with the herbicide acting solely via tetrapyrrole
accumulation and other mechanisms have been proposed (Mayasick et al.,
1990; Rebeiz et al., 1990). Subsequently, in vivo localisation studies
have shown that tetrapyrroles accumulate at sites outside of the
chloroplast such as the plasmalemma, which may explain why
photosensitising damage is not seen initially in the chloroplast
(Lehnen et al., 1990).

The presumed "spontaneous" oxidation of protoporphyrinogen IX to
protoporphyrin IX is apparently accomplished by a protoporphyrinogen
oxidising enzyme outside of the chloroplast, probably on the
plasmalemma (Jacobs et al., 1991). This enzyme is presumably less
sensitive than the chloroplast enzyme to the nitrodiphenyl ether
inhibitors, and the presence of this second enzyme would explain many
of the inconsistencies of the earlier data (Robeiz et al., 1990), the
observed accumulation of protoporphyrin IX outside of the chloroplast,
and the observed depletion of heme in the chloroplast. The major
mechanism of action of acifluorfen and other diphenyl either herbicides
is now thought to involve a blockage of the chloroplastic pathway
shunting the protoporphyrinogen to an extra-chloroplastic pathway that
first uncouples the regulation of the tetrapyrrole pathway on the
chloroplast envelope by low heme levels, and secondly causes the
accumulation of a photosensitising agent, protoporphrin IX in the
cellular membranes.

The mode of action of the aminolevulinic acid-based herbicides is more
clearly established because unlike other herbicides they were designed
based on a knowledge of chlorophyll synthesis and free radical
chemistry (Rebeiz et al., 1990). All formulations are based on the
concept of supplying aminolevulinic acid to the plant as a precursor of
tetrapyrrole synthesis (Fig. 15), in conjunction with a modulator that
qualitatively and quantitatively alters the pattern of tetrapyrrole
accumulation by selectively stimulating or inhibiting various steps in
the biosynthetic pathway. The accumulated tetrapyrroles serve as
photosensitizers that produce singlet oxygen in the light. The
herbicide formulations are most effective if the sprayed plants remain
in darkness for a few hours to allow tetrapyrrole accumulation before
exposure to the light. After about 20 min illumination, the leaves
develop isolated bleached areas that expand followed by wilting,
desiccation and necrosis.

Although the photosensitising herbicides were assumed to be
non-selective, laboratory and field trials have indicated that
significant selectivity can be achieved by modifications of the
formulations. This is caused by the different capacities of tissues and
plants of accumulate tetrapyrroles and presumably to sequester or
translocate the herbicides. Details of the mechanism of selectivity are
given elsewhere (Rebeiz et al., 1990).

Some plants have apparently achieved increased resistance to
acifluorfen by an enhanced scavenging capacity of oxygen free radicals
(Gullner et al., 1991). Bean leaves treated with acifluorfen responded
by higher levels of reduced glutathione and higher activity of
glutathione reductase (Schmidt and Kunert, 1986). A tobacco line that
was resistant to paraquat was also co-tolerant of acifluorfen (Gullner
et al., 1991). This tolerance was associated with a greater
inducibility of several components of the oxygen free radical
scavenging system. Winter wheat (Triticum aestivum) seedlings that
develop tolerance of freezing and of paraquat as they acclimate at
2=B0C, also develop increased resistance to acifluorfen (Bridger et al.,
1994). Alfalfa plants that express a transgene of the Mn-SOD cDNA from
Nicotiana have an increased LD50 for acifluorfen (McKersie et al.,
1993).

Figure 15 The biosynthetic pathway of chlorophyll and heme.

The proposed sites of action of diphenylether herbicides is the
protoporphyrinogen oxidase on the chloroplast envelope. Inhibition of
this enzyme has two effects:

a) reduces heme levels, which increases carbon flow into the pathway by
reducing its feedback inhibition of the pathway.

b) diverts protoporphyrinogen IX to the plasmalemma where a second
oxidase converts it to protoporphyrin IX.

Aminolevulinic acid-based herbicides stimulate the pathway by supplying
excess substrate and modulators promote the accumulation of chlorophyll
precursors that act as photosensitizers.
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SUMMARY AND CONCLUSIONS
Oxygen free radicals or activated oxygen has been implicated in diverse
environmental stresses in plants and animals and appears to be a common
participation in most, if not all, degenerative conditions in
eukaryotic cells. The peroxidation of lipids, the cross-linking and
inactivation of proteins and mutations in DNA are typical consequences
of free radicals, but because the reactions occur quickly and often are
components of complex chain reactions, we usually can only detect their
"footprints". Activated forms of oxygen are important in the
biosynthesis of "complex" organic molecules, in the polymerisation of
cell wall constituents, in the detoxification of xenobiotic chemicals
and in the defence against pathogens. Thus, the plant's dilemma is not
how to eliminate the activation of oxygen, but how to control and
manage the potential reactions of activated oxygen. Complex systems of
scavenging activated oxygen therefore exist in plant cells with
complimentary and interdependent strategies. Some components such as
the carotenoids prevent the formation of activated oxygen by competing
for the energy leaked from the photosystems. Other components are lipid
soluble and reside in the membrane bilayer to terminate the lipid
peroxidation chain reactions. Still others, ascorbate and glutathione,
are aqueous scavenger that detoxify activated oxygen directly or serve
to recycle other protective components back to their reduced state. The
enzymes that catalyse the synthesis, degradation and recycling of these
antioxidants are essential to viability. Consequently they are highly
conserved among plants, and exist in multiple forms in different
subcellular compartments and different tissues to allow precise
regulation.

There are numerous sites of oxygen activation in the plant cell, which
are highly controlled and tightly coupled to prevent release of
intermediate products. Under stress situations, it is likely that this
control or coupling breaks down and the process "dysfunctions" leaking
activated oxygen. This is probably a common occurrence in plants
especially when we consider that a plant has minimal mobility and
control of its environment. These uncoupling events are not detrimental
provided that they are short in duration and that the oxygen scavenging
systems are able to detoxify the various forms of activated oxygen. If
the production of activated oxygen exceeds the plant's capacity to
detoxify it, deleterious degenerative reactions occur, the typical
symptoms being loss of osmotic responsiveness, wilting, and necrosis.
At the subcellular level, disintegration of membranes and aggregation
of proteins are typical symptoms. Therefore it is the balance between
the production and the scavenging of activated oxygen that is critical
to the maintenance of active growth and metabolism of the plant and
overall environmental stress tolerance.
Return to the Oxidative Stress Table of Contents

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Bryan D. McKersie
Dept of Crop Science, university of Guelph
December, 1996

..=2E. Ambition for numbnutz is to make a fool of himself.